Handbook of Low-Level

Laser Therapy
edited by
Michael R. Hamblin
Marcelo Victor Pires de Sousa
Tanupriya Agrawal
Published by
Pan Stanford Publishing Pte. Ltd.
Penthouse Level, Suntec Tower 3
8 Temasek Boulevard
Singapore 038988

Email: editorial@panstanford.com
Web: www.panstanford.com

British Library Cataloguing-in-Publication Data

A catalogue record for this book is available from the British Library.

Handbook of Low-Level Laser Therapy

Copyright 
c 2017 Pan Stanford Publishing Pte. Ltd.

ISBN 978-981-4669-60-3 (Hardcover)

ISBN 978-981-4669-61-0 (eBook)

Printed in the USA

 Contents

Preface xxxiii

1 What is Low-Level Laser (Light) Therapy? 1

Marcelo Victor Pires de Sousa
1.1 Introduction 1
1.2 Fundamental Science: Optics, Photochemistry, and
Photobiology 5
1.2.1 Tissue Optics 6
1.2.2 Photochemistry of Chromophores 7
1.2.3 Photobiology: Mechanisms of LLLT Effects 8
1.3 Research in LLLT 9
1.4 Clinical and Biomedical Applications of LLLT 11

2 History of Low-Level Laser (Light) Therapy 17

Michael R. Hamblin

3 Lasers, LEDs, and Other Light Sources 35

James Carroll
3.1 Introduction 35
3.2 State of the Art 37
3.3 History of Devices 38
3.4 Nomenclature 38
3.5 Laser Classification 39
3.6 Light Sources and Properties 40
3.6.1 Different Properties of Laser and LED Light
Sources 40
3.6.1.1 Wavelength 40
3.6.1.2 Coherence 42
3.6.1.3 Power 42
 3.6.1.4 Irradiance 42
3.6.1.5 Penetration 43
3.6.1.6 Pulses 44
3.6.1.7 Collimation 46
3.6.1.8 Spectral Width (Monochromaticity) 47
3.6.1.9 Stability 47
3.6.1.10 Polarization 47
3.6.1.11 Beam Area 48
3.6.1.12 Scanning vs. Contact Method 48
3.7 Summary on Light Sources and Properties 49

4 Is Coherence Important in Photobiomodulation? 51

Tomas Hode
4.1 Introduction 51
4.2 Is Coherence Lost Upon Entering Tissue? 53
4.2.1 How Fast is Too Fast? 55
4.3 What Biological Significance Could Speckles Have? 56
4.3.1 Intensity Thresholds 56
4.3.2 Polarization 58
4.3.3 Dynamic Environment 60
4.4 Summary 61

5 Tissue Optics 67
Bryan James Stephens and Linda Ramball Jones
5.1 Optical Properties of Tissues 67
5.1.1 Tissue with Weak Scattering 68
5.1.2 Tissue with Strong (Multiple) Scattering 68
5.1.3 Full Picture of Penetration 70
5.1.4 Optical Properties of Water 70
5.1.5 Optical Properties of Blood 73
5.1.6 Spectral Variation of Optical Properties 74
5.2 Methods and Algorithms for the Measurement of
Optical Parameters of Tissues 77
5.2.1 Integrating Sphere Technique 78
5.2.2 Kubelka–Munk Model 78
5.2.3 Inverse Methods 79
5.3 Methods and Algorithms for the Simulation of the
Light Interactions within Tissues 79
 5.3.1 Monte Carlo Simulation 80
5.3.2 Optical Tissue Phantoms 81
5.4 Practical Implementation 83

6 Light–Tissue Interaction and Light Dosimetry 87

Ana Carolina de Magalhães and Elisabeth Mateus Yoshimura
6.1 Light–Tissue Interactions 87
6.2 Light Dosimetry 96

7 Mitochondrial Light Absorption and Its Effect on ATP

Production 101
Nicolette Houreld
7.1 Mitochondria 102
7.1.1 Adenosine Triphosphate (ATP) Synthesis 102
7.2 Phototherapy 104
7.2.1 Effect of Phototherapy on Mitochondria and
ATP Synthesis 105
7.3 Conclusion 113

8 Water as a Photoacceptor, Energy Transducer, and

Rechargeable Electrolytic Bio-battery in
Photobiomodulation 119
Luis Santana-Blank, Elizabeth Rodrı́guez-Santana,
Jesús A. Santana-Rodrı́guez, Karin E. Santana-Rodrı́guez,
and Heberto Reyes-Barrios
8.1 Introduction 120
8.2 Absorption and Transport of Light Energy by Water 122
8.3 Photo-Infrared Pulsed Biomodulation 125
8.4 Water Oscillator Paradox 126
8.4.1 Bulk Water 127
8.4.1.1 Application I: Light energy absorption
and enhanced ATP 127
8.4.1.2 Application II: Light-modulated
biomolecular motors and pumps in
aqueous media 128
8.4.2 Confined-Space Water 129
8.4.3 Interfacial Water: What is EZ? 130
8.5 Metabolism and Scaling Laws 134
8.6 Conclusion 134
9 Role of Reactive Oxygen Species in Low-Level Laser
Therapy 141
Vikrant Rai
9.1 Mitochondrial Response to LLLT 142
9.2 LLLT-Induced Production of ROS 143
9.2.1 Cytochrome 143
9.2.2 Flavins 144
9.2.3 Porphyrins 145
9.3 Role of Reactive Oxygen Species 148
9.3.1 Oxidative Stress at Cellular Level Due to ROS 148
9.3.2 Antioxidant Effect of LLLT 149
9.3.3 Cellular Response to Increased ROS (Oxidative
Stress) 149
9.3.4 Response of Various Transcription Factors to
Oxidative Stress 151
9.3.5 ROS-Mediated Effect of LLLT on Nervous
System 153
9.3.6 ROS-Mediated Apoptosis 155
9.3.7 ROS Effect on Thrombosis and Hemostasis 156
9.3.8 ROS-Mediated Effect of LLLT on
Musculoskeletal System 157
9.3.9 ROS-Mediated Effect of LLLT on Oral Cavity 158
9.3.10 ROS-Mediated Effect of LLLT on Lungs 158

10 Molecular Basis for Photobiomodulation: Light-Induced

Nitric Oxide Synthesis by Cytochrome c Oxidase in
Low-Level Laser Therapy 165
Robert Oliver Poyton and Marina Hendrickson
10.1 Introduction 165
10.2 Cytochrome c Oxidase: A Photoreceptor for LLLT 166
10.3 Structure/Function of Mitochondrial Cytochrome c
Oxidase 167
10.4 Enzymatic Activities of Cytochrome c Oxidase 169
10.4.1 Regulation of Cox/H2 O Activity 170
10.4.2 Regulation of Cox/NO Activity 171
10.5 Low-Intensity Light Stimulates Cox/NO but Not
Cox/H2 O Activity 173
 10.5.1 Possible Mechanism for Light Stimulation
of Cox/NO 173
10.6 Cox/NO, NO, and LLLT 174
10.7 Summary 177

11 Cytoprotective Effect of Low-Level Light Therapy using LEDs

on Neurons 185
Margaret Wong-Riley and Huan Ling Liang
11.1 Introduction 186
11.2 Role of Cytochrome c Oxidase in
Photobiomodulation of Cultured Visual Cortical
Neurons 186
11.3 Neuroprotective Effect of 670 nm LED on Primary
Neurons Inactivated by Cyanide 190
11.4 Neuroprotective Effect of 670 nm LED on Primary
Neurons Poisoned by MPP+ and Rotenone:
Implications for Parkinson’s Disease 193
11.5 Neuroprotective Effect of Pretreatment with
670 nm LED on Primary Neurons Exposed to KCN,
Rotenone, or MPP+ 196
11.6 Neuroprotective Effect of 670 nm LED on
Cytochrome c Oxidase Activity of Deprived Visual
Cortex of Monocularly Enucleated Rats 199
11.7 cDNA Microarray Analysis of Genes Up- and
Down-Regulated by 670 nm LED in Deprived Visual
Cortex of Monocularly Enucleated Rats 201
11.8 Conclusion 203

12 Low-Level Laser and Cultured Neural Tissue 207

Patricia J. Armati and Roberta T. Chow
12.1 Why Use Cell or Tissue Culture Models? 207
12.2 Cell Lines 208
12.3 Specific Characteristics of Nervous System Cells 209
12.4 LLL, Cell Culture, and Peripheral Nervous
System 211
12.5 Delivery of LLL to Neural Tissue in Culture 215
12.6 LLL Irradiation of Cultured Sensory Neurons in
Pain-Related Studies 215
 12.7 Low-Level Laser: Excitatory or Inhibitory 216
12.8 Dorsal Root Ganglion Cultures of Nociceptor
Neurons 218
12.9 LLL Effects on Sympathetic Nervous System
Neurons 224
12.10 Central Nervous System in Culture 225

13 Shining a Light on Parkinson’s Disease 237

Daniel McKenzie Johnstone, Cécile Moro, Jonathan Stone,
Alim-Louis Benabid, and John Mitrofanis
13.1 Introduction 238
13.2 Overview of Parkinson’s Disease 238
13.3 Evidence for Neuroprotection by NIR Treatment in
Parkinson’s Disease 239
13.4 How Does NIR Work to Neuroprotect: Two
Mechanisms of Action? 244
13.5 NIR Treatment in Parkinson’s Disease Patients: Can
It Work? 245
13.6 Developing Methods for Intracranial NIR Delivery 247
13.7 Conclusion 247

14 Low-Level Laser Therapy and Stem Cells 253

Qi Zhang, Chang Zhou, and Tingting Dong
14.1 Mechanisms of LLLT Action in Stem Cells 254
14.1.1 Low-Level Laser Irradiation 254
14.1.2 Mechanisms of LLLT 254
14.1.3 Effects of LLLT 256
14.2 Effects of LLLT on Stem Cells 258
14.2.1 Hematopoietic Stem Cells 258
14.2.2 Mesenchymal Stem Cells 259
14.2.3 Adipose-Derived Stem Cells 261
14.3 Clinical Applications of LLLT on Stem Cells 261
14.3.1 LLLT for Stem Cell Transplantation 261
14.3.2 LLLT for Wound Healing and Skin
Restoring 262
14.3.3 LLLT for Neural Regeneration 263
14.3.4 LLLT for Treatment of Hair Loss 264
15 Antimicrobial Photodynamic Therapy 273
Vanderlei Salvador Bagnato, Cristina Kurachi,
Kate Cristina Blanco, and Natalia Mayumi Inada

16 Low-Level Laser (Light) Therapy for Wound Healing in

Animal Models 285
André Luiz Oliveira Ramos, Felipe Scholz Ramos, and
Marcelo Victor Pires de Sousa
16.1 Physiology of Wound Healing 286
16.1.1 Mechanisms of Wound Healing with LLLT 286
16.1.2 Types of Wound Healed by LIB 293
16.2 Thrombosis 295
16.3 LLLT Influence on Infected Wounds 296

17 Low-Level Laser Therapy for Arthritis in Animal Models:

Beneficial Effect and Action Mechanism 303
Flavio Aimbire and Paulo de Tarso Camilo de Carvalho

18 Low-Level Laser Therapy for Lung Diseases: From the Bench

to the Bed 317
Flavio Aimbire
18.1 Introduction 317
18.2 Asthma 319
18.2.1 Clinical Studies 319
18.2.2 Experimental Studies 323
18.3 Acute Respiratory Distress Syndrome 327
18.3.1 Clinical Studies 327
18.3.2 Experimental Studies 328
18.4 Chronic Obstructive Pulmonary Disease 331
18.4.1 Clinical Studies 331
18.4.2 Experimental Studies 332
18.5 Pneumonia 332
18.5.1 Clinical Studies 332
18.6 Tuberculosis 333
18.6.1 Clinical Studies 333

19 Low-Level Laser (Light) Therapy in Tendon Healing in

in Vitro and in Vivo Models 339
Lucas F. de Freitas and Michael R. Hamblin
19.1 Introduction 340
 19.2 Low-Level Light Therapy and Inflammation 344
19.3 Applications of Low-Level Light in Tendon Healing 346
19.3.1 In Vitro Studies 346
19.3.2 In Vivo Studies 347
19.3.3 LEDs Used in Tendon Healing 351
19.4 Conclusion 352

20 Bone Repair in Animal Models 357

Antonio Luiz B. Pinheiro, Luiz G. Pinheiro Soares,
and Aparecida Maria C. Marques
20.1 Introduction 357
20.1.1 Bone Tissue 357
20.1.2 Autologous Bone Grafting and Biomaterials 359
20.1.3 Guided Bone Regeneration 360
20.1.4 Phototherapy 361
20.2 Light Therapies in the Bone Repair of Animal
Models 361
20.3 Closing Remarks 364

21 Transcranial Low-Level Laser (Light) Therapy for Stroke and

Traumatic Brain Injury in Animal Models 371
Michael R. Hamblin, Luis De Taboada,
and Ying-Ying Huang
21.1 Introduction 372
21.2 Photobiology of Low-Level Laser Therapy 373
21.3 LLLT on Neuronal Cells 375
21.4 Human Skull Transmission Measurements 376
21.5 Epidemiology of Stroke 378
21.6 Mechanisms of Brain Injury after Stroke 379
21.7 Thrombolytic Therapy of Stroke 381
21.8 Investigational Neuroprotectants and
Pharmacological Intervention 382
21.9 Transcranial LLLT for Stroke 382
21.9.1 Transcranial LLLT in Animal Models for
Stroke 382
21.10 Traumatic Brain Injury 385
21.10.1 Transcranial LLLT Studies for TBI in
Animal Models 386
 21.10.2 Effect of Different Laser Wavelengths in
tLLLT in Closed-Head TBI Model in Mice 388
21.10.3 Effect of Pulsing in LLLT for CCI-TBI in Mice 389
21.10.4 Effects of tLLLT-Repetition Regimen in
CCI-TBI in Mice 391
21.10.5 Transcranial tLLLT in Mice with TBI
Stimulates the Brain to Repair Itself 393
21.11 Conclusion 395

22 Phototherapy in Peripheral Nerve Repair and Muscle

Preservation 403
Shimon Rochkind
22.1 Incomplete Peripheral Nerve Injury 405
22.2 Complete Peripheral Nerve Injury 407
22.3 Nerve Cells 409
22.4 Clinical Trial 410
22.5 Denervated Muscle 410
22.6 Conclusion 412

23 Low-Level Laser Therapy for Spinal Cord Repair 415

Takahiro Ando and Michael R. Hamblin
23.1 Introduction 415
23.2 Therapeutic Strategies for Spinal Injury 416
23.3 LLLT for Spinal Cord Repair 418
23.3.1 Laser Irradiation in Spinal Cord for
Therapy of Injured Peripheral Nerves 418
23.3.1.1 Animal studies 418
23.3.1.2 Clinical studies 420
23.3.2 LLLT for Nerve Transplantation of Spinal
Injured Animals 421
23.3.3 Effects of NIR Laser Irradiation Alone for
SCI Model 423
23.3.3.1 Experimental SCI model 423
23.3.3.2 Transmittance of transcutaneous
NIR laser to spinal cord 423
23.3.3.3 LLLT for injured spinal cord in
rats 425
 23.3.4 Clinical Study: Intravascular LLLT for
Chronic SCI Patients 427
23.4 Mechanism Studies of LLLT for SCI 428
23.5 LLLT for Other Spinal Cord Diseases 429
23.6 Conclusion 429

24 Low-Level Laser (Light) Therapy for the Treatment of Visual

System Injury and Disease 435
Janis T. Eells, Sandeep Gopalakrishnan, Michele M. Salzman,
Krisztina Valter, Jan Provis, Ricardo Natoli, John Mitrofanis,
Jonathan Stone, and Melinda Fitzgerald
24.1 Introduction 435
24.2 LLLT in Animal Models of Retinal and Optic Nerve
Injury 437
24.2.1 Methanol Intoxication 437
24.2.2 Light-Induced Retinal Damage 438
24.2.3 Optic Nerve Injury 440
24.3 LLLT in Animal Models of Retinal and Optic Nerve
Disease 441
24.3.1 Retinopathy of Prematurity 441
24.3.2 Diabetic Retinopathy 442
24.3.3 Retinitis Pigmentosa 443
24.3.4 Aging and Age-Related Macular
Degeneration 444
24.3.5 Parkinson’s Disease 444
24.4 LLLT in Clinical Investigations of Retinal Disease 445
24.4.1 Age-Related Macular Degeneration 445
24.4.2 Diabetic Retinopathy 446
24.5 Conclusion 447

25 Protection from Cardiac Ischemia and Reperfusion Injury 453

Agnes Keszler, Svjetlana Dosenovic, and
Martin Bienengraeber
25.1 Introduction 453
25.2 Repair of the Infarcted Heart 454
25.2.1 Underlying Mechanisms of Light-Induced
Repair after Myocardial Infarction 455
25.2.2 Induction of Stem Cells by Phototherapy 457
 25.3 Protection Against Acute Ischemia and Reperfusion
Injury 457
25.3.1 Alternative Sources of Nitric Oxide in
Light-Induced Cardioprotection 458
25.3.2 Cytochrome c Oxidase and NO 460
25.4 Discussion of Potential Clinical Applications 462
25.5 Conclusion 464

26 Low-Level Laser and Experimental Aortic Aneurysm:

Mechanisms and Therapeutic Implications 471
Lilach Gavish and S. David Gertz
26.1 Introduction 471
26.1.1 Aortic Elasticity and Resilience 472
26.1.2 Smooth Muscle Cells 473
26.1.3 Activated Monocytes/Macrophages 473
26.2 Effect of LLL on Experimental AAA 474
26.2.1 LLL Promotes SMC Proliferation and
Augments Collagen Synthesis in Vitro 474
26.2.1.1 Proliferation 474
26.2.1.2 Collagens I and III trihelix
formation 475
26.2.1.3 Collagen secretion 476
26.2.1.4 MMP activity 476
26.2.2 LLL Attenuates LPS-Induced Secretion of
Inflammatory Factors 476
26.2.2.1 Chemokine/cytokine expression 477
26.2.3 LLLI Prevents de Novo Formation and Halts
Further Progression of Pre-Induced AAA
in Vivo 477
26.2.3.1 De novo aneurysm formation 478
26.2.3.2 Progression of pre-existing
aneurysm 479
26.2.4 LLL Increases SMC Size and Collagen
Deposition 479
26.2.4.1 Medial SMC size 479
26.2.4.2 Collagen reinforcement 479
26.2.5 LLL Attenuates the Number of
Macrophages in Transmedial Aortic
Defects 481
 26.2.5.1 Macrophages in area of
transmedial defect 481
26.3 Therapeutic Approaches 483
26.3.1 Current Treatments and Early Detection 483
26.3.2 How Can LLL be used for Treatment of
AAA? 483
26.3.2.1 Noninvasive LLL 484
26.3.2.2 Minimally invasive intravascular
LLL 484
26.3.2.3 Minimally invasive laparoscopic
LLL 484
26.4 Conclusion 484

27 Low-Level Laser Therapy: A Treatment Modality for

Multiple Sclerosis Targeting Autoimmunity and Oxidative
Stress 491
Zenas George, Miguel A. Tolentino, and Jeri-Anne Lyons
27.1 Introduction 492
27.1.1 Multiple Sclerosis 492
27.1.2 Pathogenesis of Multiple Sclerosis 493
27.1.3 Animal model for Multiple Sclerosis 494
27.2 LLLT as an Emerging Treatment Modality for
Multiple Sclerosis 494
27.2.1 Efficacy of Phototherapy in Animal Model
for Multiple Sclerosis 494
27.2.2 LLLT for Treatment of MS 496
27.3 Future Directions 497
27.4 Conclusion 498

28 Low-Level Laser Therapy as an Alternative Treatment for

Snake Envenomation 503
Camila Squarzoni Dale and Stella Regina Zamuner
28.1 Introduction 503
28.2 Snake Envenomation of the Brothrops Genus 504
28.2.1 Local Manifestations 506
28.2.2 Systemic Manifestations 506
28.2.3 Anti-Venom Treatment 507
 28.3 Low-Level Laser Therapy for Treatment of Local
Manifestations of Bothrops Envenomation 508
28.3.1 Myonecrosis and LLLT 508
28.3.2 Local Inflammation and LLLT 511
28.3.3 Hyperalgesia and LLLT 512
28.4 Conclusion 513

29 Veterinary Low-Level Laser (Light) Therapy Applications for

Companion Animals 519
Richard L. Godine
29.1 Introduction: Finding Common Ground 519
29.2 Treatment Parameters 521
29.3 Musculoskeletal Conditions 522
29.3.1 Degenerative Joint Disease and
Osteoarthritis 522
29.3.1.1 DJD of the hip 522
29.3.1.2 DJD of stifle 526
29.3.1.3 DJD of elbow 526
29.3.2 Acute Musculoskeletal Injuries 527
29.3.2.1 Iliopsoas strain 527
29.3.2.2 Biceps and supraspinatus tendon
strain of the shoulder 529
29.3.2.3 Fractures 529
29.4 Dermatological Conditions 530
29.4.1 Surgical Wounds and Lacerations 530
29.4.2 Infected Wounds 530
29.4.3 Hot Spots and Otitis Externa 531
29.4.4 Snake and Insect Bites 531
29.5 Neurological Conditions 533
29.5.1 Intervertebral Disk Disease 533
29.5.2 Dementia 534
29.6 Renal Conditions 536
29.6.1 Feline Lower Urinary Tract Disease 536
29.6.2 Chronic Renal Failure 537
29.7 Other Internal Organs 537
29.8 Other Miscellaneous Applications for Light Therapy 538
29.8.1 Dental Applications 538
29.8.2 Ophthalmic Disorders 538
 29.8.3 Neoplasia 538
29.9 Conclusion 539

30 Emergence of Low-Level Laser (Light) Therapy in Clinical

Veterinary Practice 543
Ronald E. Hirschberg
30.1 Introduction: Factors Influencing Adaptation of
LLLT to Clinical Practice 544
30.1.1 Clinical Applications 545
30.1.1.1 Soft tissue, wound healing, and
ophthalmological applications 545
30.1.1.2 Spinal cord disease 546
30.1.1.3 Orthopedic conditions 548
30.1.1.4 Dermatology and light therapy 549
30.1.1.5 LLLT and metabolic disease 550
30.1.2 Treatment Parameters 551
30.1.3 Therapeutic Outline 553
30.1.4 Safety and Contraindications 554
30.1.5 Clinical and Practical Benefits of LLLT 555
30.1.6 Future of Photobiomodulation in
Veterinary Practice 557

31 Photomedicine for Exotic Animals: A Case-Based Discussion 559

Narda G. Robinson
31.1 Introduction 559
31.2 Hurdles 559
31.3 Clinical Applications 560
31.3.1 Traumatic Brain Injury 560
31.3.2 Spinal Cord Injury 562
31.3.3 Neuropathic and Orthopedic Pain 564
31.3.4 Wound Healing and Infection 565
31.3.5 Laser Acupuncture 569
31.4 Conclusion 574

32 Recalcitrant Wound: Using Low-Level Laser (Light) Therapy

to Manage Non-Healing Wounds and Ulcers 581
Raymond J. Lanzafame and Istvan Stadler
32.1 Introduction: An Overview of Normal Wound
Healing 582
 32.2 Photobiomodulation and Wound Healing 583
32.2.1 Photobiomodulation and Its Mechanisms 583
32.2.2 Applying Phototherapy to Wounds:
Wavelengths and Energy Density 584
32.2.3 Applying Phototherapy to Wounds:
Irradiance, Exposure Time, and Dose and
Treatment Frequency 585
32.2.4 Applying Phototherapy to Wounds: Skin
Pigmentation and Other Considerations 586
32.3 Bacterial Contamination and Wound Infection:
Antimicrobial Effects of LLLT 586
32.4 General Considerations for Wound Management 587
32.4.1 Initial Evaluation and Management 587
32.4.2 Evaluation of the Wound or Wounds 588
32.4.3 Wound Evaluation: Initial Documentation
and Management 588
32.4.4 Wound Evaluation: Photodocumentation 589
32.5 Clinical Applications and Considerations 591
32.5.1 Patient Selection 592
32.5.2 Device Selection and Use 592
32.6 Summary 593

33 Clinical Applications with Low-Level Laser Therapy in

Arthritis 597
Jan M. Bjordal
33.1 Introduction 597
33.2 Pathoanatomy and Inflammation in Early-Stage OA
and Avenues for LLLT Irradiation 600
33.2.1 Synovia 600
33.2.2 Bone 601
33.2.3 Cartilage and Meniscii 601
33.2.4 Peripheral Nerves and Pain Receptors 601
33.3 Complex Relationship between Inflammation,
Tissue Interaction, and Structural Chondral Matrix
Degeneration in OA 602
33.4 Why LLLT Works in OA? 602
33.5 Recommended Doses of LLLT in Arthritis 603
34 Use of Low-Level Laser Therapy and Light-Emitting Diode
Therapy to Improve Muscle Performance and Prevent
Damage: From Animal Models to Clinical Trials 609
Cleber Ferraresi, Nivaldo Parizotto, Vanderlei Bagnato, and
Michael R. Hamblin
34.1 Introduction 609
34.2 Experimental Models Using LLLT to Enhance
Muscle Performance and Prevent Damage 610
34.3 Experimental Models Using LEDT to Enhance
Muscle Performance and Damage Prevention 613
34.4 Clinical Trials Using LLLT to Increase Muscle
Performance and Prevent Damage: Acute
Responses 619
34.5 Clinical Trials Using LLLT to Enhance Muscle
Performance and Damage Prevention: Chronic
Responses 623
34.6 Clinical Trials Using LEDT to Improve Muscle
Performance and Prevent Damage: Acute
Responses 623
34.7 Clinical Trials Using LEDT to Improve Muscle
Performance and Prevent Damage: Chronic
Responses 634
34.8 Conclusion 634

35 Low-Level Laser Therapy of Pain: Clinical Applications 641

Roberta T. Chow
35.1 Background 641
35.2 What is Pain? 642
35.3 Types of Pain and Mechanisms 642
35.4 Mechanisms Underlying Pain Relief 644
35.4.1 Neural Blockade 644
35.4.2 Reduce Inflammation 645
35.4.3 Reduce Edema 646
35.4.4 Reduce Muscle Spasm 647
35.4.5 Tissue Repair 647
35.4.6 Release of Neurotransmitters 648
35.5 Conditions in Which LLLT is Used and Evidence 648
35.5.1 Reviews of LLLT and Pain 648
 35.5.2 Evidence for Specific Conditions 649
35.5.2.1 Arthridities 649
35.5.2.2 Neck pain 651
35.5.2.3 Back pain 651
35.5.2.4 Shoulder pain 653
35.5.2.5 Tendinopathy and enthesitis 653
35.5.2.6 Lateral epicondylitis 653
35.5.2.7 Trigger point and myofascial
pain 654
35.5.2.8 Neuropathic pain 655
35.5.2.9 Lymphedema 656
35.5.2.10 Post-operative pain 657
35.6 Pretreatment Pain Relief 657
35.6.1 Unique Effects of LLLT in Pain 658
35.7 Practical Considerations 658
35.7.1 Treating Knee Osteoarthritis as an
Example 659
35.8 Factors Influencing Outcomes 660
35.8.1 Laser Factors 660
35.8.1.1 Wavelength 660
35.8.1.2 What is the correct dose? 663
35.8.1.3 Application technique 663
35.8.2 Treatment Protocol 664
35.8.2.1 How long should a course of
treatment be? 664
35.8.3 Patient Factors 665
35.8.4 Disease Factors 665
35.9 What Are the Goals of Treatment with LLLT? 666
35.9.1 Monotherapy versus Adjunctive
Treatment 666
35.9.2 Why Some Patients Do Not Respond to
LLLT? 666
35.10 Practice Points 667
35.11 “Tip of the Iceberg” Principle 668
35.12 Prognostic Factors 669
35.13 Side Effects of Treatment 669
35.14 Conclusion 669
36 Low-Level Laser Therapy and Its Application in Tinnitus 685
Alessandra Nara de Souza Rastelli,
Emanuelle Teixeira Carrera, Gustavo Nicolodelli,
and Michael R. Hamblin
36.1 Introduction 686
36.2 Symptoms of Tinnitus Ringing in Ears 688
36.3 Types of Tinnitus 688
36.3.1 Subjective Tinnitus 688
36.3.2 Objective Tinnitus 689
36.3.3 Function and Dysfunction of Inner Ear 690
36.4 Causes of Tinnitus 691
36.5 Diagnosis of Tinnitus 692
36.6 Mechanisms of LLLT on Tinnitus 693
36.7 LLLT for Tinnitus 695
36.8 Conclusion 703

37 Laser Therapy for the Treatment of Radiculopathy 711

Jerome M. True and Luis C. Vera
37.1 Introduction 711
37.2 Pathomechanisms of Radiculopathy 713
37.3 Complex Spinal Pain Patient with Radiculopathy 715
37.4 Common Levels of Radiculopathy 716
37.4.1 Lumbar Radiculopathy 716
37.4.2 Cervical Radiculopathy 716
37.4.3 Thoracic Radiculopathy 718
37.5 Proposed Mechanisms of Laser Therapy on
Radiculopathy 721
37.6 Clinically Useful Treatment Protocols 723
37.6.1 Pulsed or Continuous Laser Therapy 723
37.6.2 Contact or Coupled Technique 725
37.6.3 Treatment of Associated Guarding Spasm 725
37.6.4 Treatment of Segmentally Innervated
Musculature 726
37.6.5 Treatment of L5 and S1 Radiculopathies 728
37.6.6 Treatment of C6 and C7 Radiculopathies 730
37.6.7 Treatment of Thoracic Radiculopathies 732
38 Difficult Path to Treating Acute Ischemic Stroke Patients
with Transcranial Near-Infrared Laser Therapy 741
Paul A. Lapchak, Pramod Butte, and Padmesh S. Rajput
38.1 Introduction 742
38.2 NILT Penetration Profiles in Animals and Humans 743
38.3 Translational NILT Studies in Stroke Models 746
38.3.1 Is There a Correlation between NILT Power
Density and Improved Behavioral Function
in Animal Models? 746
38.4 NILT Safety Trials 749
38.5 NILT Stroke Clinical Trial Development 750
38.5.1 NEST-1 750
38.5.2 NEST-2 752
38.5.3 NEST-3 753
38.6 Need to Optimize NILT in a Standardized
Translational Model 753
38.7 Conclusion 754

39 Low-Level Laser (Light) Therapy for Rehabilitation in

Traumatic Brain Injury and Stroke, including Chronic
Aphasia 761
Margaret A. Naeser, Paula I. Martin, Michael D. Ho,
Maxine H. Krengel, Yelena Bogdanova, Jeffrey A. Knight,
Megan K. Yee, Ross Zafonte, Bang-Bon Koo, John G. Roubil,
and Michael R. Hamblin
39.1 Introduction 762
39.2 Mechanisms of LLLT 762
39.3 Traumatic Brain Injury 763
39.3.1 Introduction to TBI in Humans 763
39.3.2 Brain Imaging Studies in TBI 765
39.3.3 Cognitive Dysfunction in TBI 766
39.3.4 Poor Sleep in TBI 767
39.3.5 Pharmacologic Treatments for TBI 767
39.3.6 Cognitive Rehabilitation Therapies for TBI 768
39.3.7 Transcranial LED Treatments to Improve
Cognition and Sleep in Chronic mTBI 769
39.3.8 Intranasal LED Treatments to Improve
Cognition and Sleep in mTBI 771
 39.4 Stroke 773
39.4.1 Transcranial LLLT to Treat Acute Stroke 773
39.4.1.1 Transcranial LLLT studies to treat
acute stroke: Small-animal
studies 773
39.4.1.2 Transcranial LLLT to treat acute
stroke: Human studies 775
39.4.2 Transcranial LLLT to Treat Chronic Stroke 776
39.4.2.1 Transcranial LLLT to treat
chronic stroke: Human studies 776
39.4.3 Transcranial LLLT to Improve Language in
Chronic Aphasia Due to Stroke 778
39.4.3.1 Aphasia 778
39.4.3.2 Importance of specific LED
placement areas on the scalp to
treat aphasia in chronic stroke 779
39.4.3.3 Bilateral tLED treatment method 780
39.4.3.4 Left-hemisphere-only tLED
treatment method 781
39.4.3.5 Transcranial LLLT to treat
primary progressive aphasia,
neurodegenerative disease 783
39.4.3.6 Additional tLED treatment
studies with chronic aphasia due
to stroke 785
39.5 Other Noninvasive Brain Stimulation Therapies to
Treat TBI or Stroke 786
39.5.1 Transcranial Magnetic Brain Stimulation 786
39.5.2 Transcranial Direct Current Stimulation 788
39.6 Conclusion 791

40 Transcranial Near-Infrared Light for Major Depressive

Disorder: Targeting the Brain Metabolism 809
Paolo Cassano, Abigail R. Archibald, and Dan V. Iosifescu
40.1 Introduction 809
40.2 Transcranial Near-Infrared Light: Biological
Properties and Safety 810
 40.3 Depression, Antidepressant Treatment, and Brain
Energy Metabolism 812
40.4 Near-Infrared Light: Mood Effects in Healthy
Volunteers 813
40.5 Near-Infrared Light: Effect on Mood in TBI and
PTSD Patients 814
40.6 Near-Infrared Light For Depression 816
40.6.1 Near-Infrared Light for Depression and
Anxiety: Single Session 816
40.6.2 Near-Infrared Light for Depression:
Multiple Sessions 817
40.6.3 Near-Infrared Light for Depression:
Multiple Sessions and Pulse Light 818
40.7 Conclusion 819

41 Low-Level Laser Therapy: A Corner Stone in the

Management of Cancer Therapy–Induced Mucositis 825
René-Jean Bensadoun, Idriss Troussier, and Raj G. Nair
41.1 Introduction 825
41.2 What is Mucositis? 826
41.3 Low-Level Laser Therapy 826
41.4 Clinical Trials 827
41.5 Recommendations and Future Directions 829
41.6 Conclusion 829

42 Photobiomodulation in Dentistry: Manipulating

Biostimulation and Bioinhibition for Clinical Success 833
Gerry Ross and Alana Ross
42.1 Introduction 834
42.1.1 Keys to Successful Use of PBM in Dentistry 835
42.1.2 Determining the Appropriate Dose 835
42.2 Dental Procedures Using Laser Therapy 837
42.2.1 Surgical Extractions 837
42.2.2 Alveolar Osteitis (Dry Socket) 839
42.2.3 Dental Infection 839
42.2.4 Restorations 841
42.2.4.1 Cementing crowns 843
42.2.5 Nausea and Gagging 843
 42.2.6 Dentin Hypersensitivity 844
42.2.7 Soft Tissue Lesions 846
42.2.7.1 Herpes lesions 847
42.2.7.2 Aphthous ulcers 848
42.2.7.3 Appliance irritation mucosal
lesions 849
42.2.8 Oral Mucositis 849
42.2.9 Gingivitis 850
42.2.10 Periodontitis 851
42.2.11 Endodontics 852
42.2.11.1 Pulpotomies 852
42.2.12 Nerve Regeneration 852
42.2.13 Orthodontics 854
42.2.14 Implants 855
42.2.15 Sinusitis 857
42.2.16 Temporomandibular Joint Pain 857
42.3 Conclusion 860

43 Photobiomodulation for the Clinical Treatment of

Age-Related Macular Degeneration 867
Graham Merry and Robert Dotson

44 Laser (Light) Therapy for Postherpetic Neuralgia 891

Kevin C. Moore and R. Glen Calderhead
44.1 Overview of Postherpetic Neuralgia 891
44.1.1 Aetiology 891
44.1.2 Incidence 892
44.1.3 Signs and Symptoms 892
44.1.4 Treatment Options 893
44.1.5 Prognosis 893
44.2 Laser (Light) Therapy 893
44.2.1 History 893
44.2.2 Clinical Research 895
44.2.3 Mechanisms of Action of LLLT 897
44.3 Enter the Light-Emitting Diode 898
44.3.1 Background 898
44.3.2 The “NASA LED” 898
44.3.3 Efficacy of LED Sources 900
 44.3.4 Clinical Evidence 900
44.4 Conclusion 902

45 Laser Acupuncture 907

Lucas F. de Freitas and Michael R. Hamblin
45.1 Introduction 908
45.2 Laser Acupuncture in Pain Reduction 911
45.3 Laser Acupuncture in Wound Healing 914
45.4 Laser Acupuncture in Respiratory Diseases 915
45.5 Laser Acupuncture in Heart Rate and Heart Rate
Variation 915
45.6 Laser Acupuncture and Brain Activity 917
45.7 Auricular Laser Acupuncture 922
45.8 Other Applications for Laser Acupuncture 923
45.9 Conclusion 927

46 Intravascular Laser Irradiation of Blood 933

Daiane Thais Meneguzzo, Leila Soares Ferreira,
Eduardo Machado de Carvalho,
and Cássia Fukuda Nakashima
46.1 Introduction 933
46.2 History of ILIB 934
46.3 Antioxidant Action of ILIB 936
46.4 Modified ILIB Techniques 943
46.4.1 Intranasal Irradiation 944
46.4.2 Wrist Skin Irradiation 945
46.5 Side Effects and Contraindications of ILIB 946

47 Nonsurgical Laser Therapy for Type 1 and Type 2 Diabetes 953

Leonardo Longo
47.1 Epidemiology 953
47.2 History 955
47.3 Background and Objectives 959
47.4 Study Design 961
47.5 Results and Discussion 967
47.6 Conclusion 972

48 Laser Therapy of Traumatic Central Nervous System Injuries 977

Leonardo Longo and Diego Longo
48.1 State of the Art and Objectives 977
 48.2 Study Design and Methodology 979
48.3 Results and Discussion 984
48.4 Conclusion 986

49 Low-Level Laser (Light) Therapy: Aesthetic Applications for

Hair 989
Felipe Scholz Ramos, André Luiz de Oliveira Ramos,
and Marcelo Victor Pires de Sousa
49.1 Physiology of Hair Growth (Phases) 990
49.2 Types of Hair Loss and Some Treatments 993
49.2.1 Androgenetic Alopecia 993
49.2.2 Alopecia Areata 994
49.2.3 Chemotherapy-Induced Alopecia 995
49.2.4 Telogen Effluvium 995
49.2.5 Scarring Alopecia 996
49.3 Treatments 996
49.3.1 Finasteride 997
49.3.2 Minoxidil 997
49.4 In Vivo Studies of LLLT 998
49.5 LLLT for Hair Growth: Clinical Trials 1000
49.6 LLLT for Hair Growth and Hair Loss
(Proposed Mechanisms) 1003
49.7 Phototherapy Devices for Hair Aesthetics 1008
49.8 Future Perspectives 1011
49.9 Glossary 1011

50 Low-Level Laser (Light) Therapy for Cosmetics and

Dermatology 1017
Mossum K. Sawhney and Michael R. Hamblin
50.1 Introduction 1017
50.2 LLLT in Dermatology 1018
50.2.1 LLLT for Skin Rejuvenation 1018
50.2.2 LLLT for Acne 1023
50.2.3 LLLT for Photoprotection 1026
50.2.4 LLLT for Herpes Virus 1028
50.2.5 LLLT for Vitiligo 1030
50.2.6 LLLT for Reduction of Pigmented Lesions 1032
50.2.7 LLLT for Hypertrophic Scars and Keloids 1033
 50.2.8 LLLT for Healing of Burns 1035
50.2.9 LLLT for Psoriasis 1037
50.3 Conclusion 1039

51 Low-Level Laser Therapy for Body Contouring and Fat

Reduction 1049
Gaurav K. Gupta
51.1 Background 1049
51.2 LLLT in Lipoplasty 1050
51.3 LLLT in Cellulite Treatment 1052
51.4 LLLT Mechanism of Action 1053
51.5 Future Directions 1054

52 Transcranial Low-Level Laser (Light) Therapy for

Neurocognitive Enhancement 1057
Julio C. Rojas and F. Gonzalez-Lima
52.1 Introduction 1057
52.2 Primary LLLT Neurochemical Effects: Cytochrome
Oxidase Effects on Oxygen and Nitric Oxide 1058
52.3 Secondary LLLT Neurobiological Effects:
Cytochrome Oxidase Induction and Cerebral
Hemodynamic Response 1060
52.4 Brain Network Mechanisms of LLLT Relevant to
Cognitive Function 1063
52.5 Dosimetry Parameters Relevant for Transcranial
LLLT and Cognitive Enhancement 1065
52.6 Cognitive Effects of Transcranial LLLT 1068
52.7 Conclusion 1071

53 Post-Operative Uses of Low-Level Laser Therapy 1077

Maria Cristina Chavantes, Nathali Cordeiro Pinto, and
Vanessa Milanesi Holanda
53.1 LLLT in Post-Cardiovascular Surgery 1078
53.1.1 LLLT in Thoracic-Cardiovascular Surgery 1081
53.2 LLLT in Neurosurgery Procedures 1082
53.2.1 Stroke 1082
53.2.2 Pain 1083
53.2.3 Spinal Cord: Trauma and Pain Problems 1083
 53.2.4 Application of LLLT in Pediatric and Adult
Neurosurgical Procedures 1084
53.2.5 LLLT in Neurosurgery Procedures 1087
53.3 Final Remarks on Post-Operative Surgeries 1089

54 Bright New World: Future Directions of Low-Level Laser

(Light) Therapy 1093
Marcelo Victor Pires de Sousa and Maria Cristina Chavantes
54.1 Introduction 1094
54.2 New Clinical Indications for LLLT 1094
54.2.1 Stem Cells 1095
54.2.2 Transcranial LLLT for Brain Disorders 1096
54.2.3 Ophthalmology 1097
54.2.4 Autoimmune Diseases 1098
54.2.5 Lung Disease and Tracheal Stenosis 1098
54.2.6 Hemodynamic Effect 1099
54.2.7 Performance Enhancement 1099
54.2.8 Optimizing Treatment 1100
54.3 Novel Light Sources for LLLT 1100
54.3.1 Wearable LLLT Devices: Bandages and
Clothing 1101
54.3.2 Implantable LEDs for Brain and Spine 1102
54.3.3 Swallowable LED Source Capsule 1102
54.4 A Bright New World with Photobiomodulation 1103

Index 1107
 Preface

Low-level laser (light) therapy (LLLT) has in recent years become

one of the fastest growing fields of medicine. Originally considered
to be firmly and enduringly sequestered in the arena of “alternative
and complementary medicine”, LLLT has staged something of a
breakout. The reasons for this remarkable change in perception by
both the medical profession and the general public are interesting to
consider.
Firstly we have the substantial advances in knowledge that
have been made in understanding the underlying mechanisms
of action. No longer do we have to rely on hand-waving and
vague comments about the cells “feeding on light” in an analogous
manner to photosynthesis in the plant kingdom. Now we understand
many of the molecular mechanisms of photon absorption, we
know which subcellular organelles respond to light, and appreciate
some of the signaling pathways and transcription factors that are
activated, and the tissue responses that occur including activation
and mobilization of stem cells.
The second big sea change has been the realization that we do not
necessarily need lasers to carry out LLLT. In the old days much laser
therapy was carried out by “practitioners” and therapists of various
types and was considered to be a specialty for which significant
training was required. This was reasonable in light of the real
concerns for laser eye safety and protecting against other possible
hazards. Now, however, the use of light-emitting diode (LED) arrays
is rapidly taking off, and these devices are readily available on
online shopping websites and are also sold on late-night television.
Although some knowledge is still required to understand the best
parameters to use for each different indication, and which can be
achieved relatively easily considering the broad dissemination of
information over the Internet.
Thirdly we have the growing realization that LLLT has a broad
range of systemic and regional effects in addition to the local effects
that were initially the main focus of everyone’s attention. Since LED
arrays by definition have a broad area illumination spot, significant
amounts of tissue are exposed to light, and light is absorbed by blood
flowing within the skin and other tissues that are exposed to light.
Light can be applied to nerves and lymph nodes to give regional
effects, as well as to the actual lesion that is being treated.
Fourthly we have seen an impressive increase in the number
of applications of LLLT to the brain. LLLT was originally tested as
a treatment for acute ischemic stroke and has been used for the
same over the last ten years. However, now its sphere has widened
and is being applied to other instances of brain trauma including
chronic stroke, acute traumatic brain injury (TBI), and chronic
TBI. A number of chronic neurodegenerative diseases including
Alzheimer’s disease and Parkinson’s disease have shown to be
benefited by LLLT. A wide range of psychiatric disorders including
depression, anxiety, post-traumatic stress disorder, and autism
spectrum disorder have been found to be susceptible to treatment
with LLLT.
Fifthly we are beginning to see significant progress in the use
of LLLT for enhancement of performance in normal people. The
most developed area of this application is the enhancement of
muscle performance in athletes and competitors in a wide range
of sports. Not only can LLLT increase the amount of work and
power that can be produced by muscles, but it can also increase the
speed of recovery after exercise and can be a great help in training
regimens. A less developed area is that of enhancement of cognitive
performance, and improvement in memory and mood using LLLT.
We expect that efforts toward realizing these goals will be emerging
soon.
Lastly, but worth mentioning, is the use of LLLT for cosmetic
and aesthetic improvements. Stimulation of hair regrowth is now
well established, and improvement of fine lines and wrinkles in
the face is also growing in popularity. The use of LLLT to combat
one of the biggest problems in the modern age, obesity and excess
fat deposits is also starting to take off. These applications address
many of the issues driving the home-use market for LLLT devices, as
consumers are generally prepared to spend their disposable income
on aesthetic improvements.
This handbook represents the most comprehensive edited book in
the field of LLLT [now called photobiomodulation (PBM) therapy]
that has been published to date. With 54 chapters spread over more
than 1100 pages it provides broad coverage of all the multitudinous
topics that comprise this most fascinating of medical therapies. The
reader will find chapters on the basic principles, mechanisms of
action, dosimetry, devices, in vitro studies, a large range of animal
models, clinical applications in veterinary medicine, and broad
coverage of a wide range of human clinical studies and uses. We
expect it to become the gold-standard reference book for some
considerable time to come.

Michael R. Hamblin
Marcelo Victor Pires de Sousa
Tanupriya Agrawal
Summer 2016
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Chapter 1

What is Low-Level Laser (Light) Therapy?

Marcelo Victor Pires de Sousa

Laboratory of Radiation Dosimetry and Medical Physics, Institute of Physics, Matão
Street, Alley R, 187, University of São Paulo, São Paulo, São Paulo 05508-900, Brazil
Bright Photomedicine Inc., Prof. Linnaeus Prestes Avenue, No. 2242,
Butantã - University City, IPEN - São Paulo, São Paulo 05508-000, Brazil
marcelovictor@usp.br

Low-level laser (light) therapy (LLLT) is a general name to refer to

many types of therapy based on photobiomodulation, a process that
cause biological alterations in organisms due to photon interaction
with atoms or molecules.

1.1 Introduction

The most usual LLLT procedures are carried out by irradiation of

low-level or low-powered lasers to sites of injury in order to speed
up cellular processes leading to better healing and decrease of
inflammation and pain. Almost all LLLT treatments are conducted
with red or near-infrared (NIR) light (600–1100 nm), with an
output power of 1–1000 mW in a non-heating energy density (0.1–
100 J/cm2 ). These LLLT procedures are non-invasive, since light can
go through tissues to reach the target tissue; non-thermal, just like

Handbook of Low-Level Laser Therapy

Edited by Michael R. Hamblin, Marcelo Victor Pires de Sousa, and Tanupriya Agrawal
Copyright c 2017 Pan Stanford Publishing Pte. Ltd.
ISBN 978-981-4669-60-3 (Hardcover), 978-981-4669-61-0 (eBook)
www.panstanford.com
July 6, 2016 17:5 PSP Book - 9in x 6in 01-Hamblin-c01

2 What is Low-Level Laser (Light) Therapy?

photosynthesis it deals with photochemical reactions; and it has a

vast scope of clinical applications.
Notwithstanding, the whole possibilities for LLLT are not covered
inside the over cited boundaries. The vast mounting LLLT literature
gifts us with many creative ways of light sources and ways to
deliver the light to the target cells or tissues. In contrast with
the past established name (low-level laser therapy), LLLT can be
performed with varied sources of light like light-emitting diodes
(LEDs), organic LEDs (OLEDs), and even, lamp or sun light filtered
by monochromators. Some biological processes can be modulated
by photochemical reactions triggered by photons of wavelength out
of the optical window of 600–1100 nm, like blue, green and far-
infrared. There are optimal parameters for LLLT, they usually stay
somewhere between 1–1000 mW for power and (0.1–100 J/cm2 )
for energy density and few minutes for time exposure, however,
not rarely one can find good results with parameters out of these
standards. Interestingly, there are many ways to deliver light in a
specific tissue, it can be as usual as pointing laser to the skin, in
contact or not; It can be by introduction of the light source in a
body cavity (mouth, ear, nose, vagina, etc.); or even, intravenous
or interstitial irradiation using an optical fiber inside a needle
(or catheter) to go through tissues. Taking all these possibilities
into account, we can realize that LLLT have even more clinical
applications than one suppose at a first glance to these therapies and
that we are dealing with a still developing field of knowledge. Some
devices currently used for LLLT procedures are shown in Fig. 1.1.

Figure 1.1 Some examples of devices and applications. (a) Laser cap
(Transdermal Cap Inc, Gates Mills, OH) for hair regrowth. (b) Intravascular
laser therapy. (c) Laserneedle acupuncture system (Laserneedle GmbH,
Glienicke-Nordbahn, Germany).
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Introduction 3

The LLLT is more than an alternative kind of treatment; it is

a whole new method to control cells and organisms by precise
alterations in molecules. The LLLT empower the contemporary
clinicians with a modern and transdisciplinary way to fight against
diseases and other undesired conditions in humans and other
animals. Moreover, it is a way to stimulate or inhibit some biological
processes in all kind of known living creature, since LLLT acts in
cell respiration. Letting our minds opened we can imagine that the
photobiomodulation processes are as old as life itself!
LLLT do not cause any visible or tangible change to the tissue
right at the moment of the treatment and some patients are
unable to believe that a real effect has been evoked by that
“little light” as they might say. Sometimes it is a hard task to
make them understand, or even accept, the photochemical and
photobiological events going on during the therapy. Moreover,
LLLT remains controversial even among researchers and clinicians;
sometimes this incredibility is related to lack of knowledge, other
times based on previous distrust in general non-conventional
medicine, and for these cases the proved effectiveness of LLLT can
dismiss the controversy. On the other hand, there are three more
fundamental reasons of controversy: the poor understanding about
the biochemical mechanisms behind LLLT, the large number of
parameters related to LLLT, and the fact that the LLLT parameters
must be personally adapted. Today it is almost generally accepted
that LLLT is triggered by the light absorption by the cytochrome c
oxidase inside mitochondria or in cell membrane but the whole
chain of reactions related to each case of ailment treatment remains
unsolved to many cases. To define a specific LLLT procedure it
is necessary to specify many parameters such as the wavelength,
fluence, power density, pulse structure, and timing. The choice of
such parameters must be adapted to each patient since the skin
color, age, gender, amount of hair and state of the tissue influence
the light absorption and scattering through the tissues. A mistake
in the choice of the parameters to each patient can lead to a less
effective or even negative outcome of the therapy.
The photobiomodulation effect with therapeutic purposes re-
ceives many names, as the one in the title of this handbook:
low-level laser (light) therapy and many other such as low-level
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4 What is Low-Level Laser (Light) Therapy?

laser therapy, low-level light therapy, low-intensity laser therapy,

low-reactive laser therapy, cold laser, non-thermal laser, soft laser,
biostimulation laser, and photobiomodulation laser, or even, light-
emitting diode therapy and organic light-emitting diode therapy.
For all these cases the adjectives “low-level”, “low-intensity”, “low-
reactive”, “soft”, “cold” makes an implicit comparison of LLLT light
sources with surgical laser which can cut, ablate and coagulate
biological tissues due to thermal effect. LLLT, in contrast with
surgical laser procedures does not increase macroscopic tissue
temperature since the energy density used is much smaller.
The first LLLT procedures were carried out using lasers, and
in the initial days of this, field researchers were not sure if the
biomodulation processes triggered by light were dependent on the
special properties of laser light such as monochromaticity (narrow
bandwidth), coherence, or polarization or if could be achieved
by other sources of light. Evidences have been raised in the last
decades that other light sources can produce photobiomodulation
and the major determining parameter for effectiveness of LLLT
is the wavelength, which must be one capable to be absorbed
by a photoreceptor molecule in the organism. In recent years
many researches and clinical procedures have been performed with
noncoherent light sources like LED, or halogen lamps connected to
monochromator filters. Even more recent are the use of OLED which
extend possibilities of LLLT since OLED can emit light uniformly
from a flexible surface.
The term “laser” is an acronym that stands for light amplification
by stimulated emission of radiation. The physical concept of
stimulated emission was proposed by Einstein in 1916, but it was
only in 1960 that the first working laser was built by Maiman. The
laser has interesting properties, like the high degree of spatial and
temporal coherence that makes this light source unique. Spatial
coherence allows a laser to be focused in a small spot or keep as a
narrow beam for long distances. In LLLT this characteristic increases
the average penetration of light through the biological tissues. In
addition, the temporal coherence allows lasers to have a very narrow
spectral emission (that can reach approximately 10−3 nm) and can
be used to produce pulses of light with few attoseconds (10−18 s).
July 6, 2016 17:5 PSP Book - 9in x 6in 01-Hamblin-c01

Fundamental Science 5

In fact, today, the shortest controlled time achieved by humans is a

pulse of 12 attoseconds from an attosecond laser.
An LED consists of a semiconducting material doped with some
impurities to create a gap of energy between the valence and
conduction bands. When the LED is switched on, a voltage is applied
to the electrons from conduction band and holes from the valence
band making them to recombine and to release energy in form
of photons, this phenomena is known as electroluminescence. The
emitting color (wavelength) of the LED is determined by the energy
band gap, and the LED intensity (brightness) depends exponentially
of the applied voltage while it is not high enough to damage the
device. Although the phenomena of electroluminescence is known
since 1907, it was only in 1962 that the first practical LEDs were de-
veloped emitting infrared and red light. Today, LED can be made with
a variety of inorganic semiconductor materials, for this reason, they
can emit many different colors, from the ultraviolet (λ < 400 nm)
to infrared (λ > 760 nm) or combined to emit white light, the
usual spectral bandwidth is approximately 30 nm and LED emitting
surface are usually smaller than 1 mm2 . A recent advancement is the
OLED which uses small organic molecules as the electroluminescent
material. Among other advantages OLED can emit from a large
(> 1 cm2 ) and flexible surface and they are more efficient.

1.2 Fundamental Science: Optics, Photochemistry, and

Photobiology

The electromagnetic spectrum of radiation ranges from gamma rays

(λ < 10−12 m) to radio waves (λ between 10−1 and 108 m). The
light visible to humans is a small portion of the electromagnetic
spectrum comprehended between 400–700 nm and together with
ultraviolet (100–400 nm) and infrared (700 nm−1 mm) light makes
the optical region of the electromagnetic spectrum of radiation.
Electromagnetic waves are created by the oscillation of electric and
magnetic fields which are described by the Maxwell’s equations.
Light, interpreted as wave, can be characterized by amplitude,
wavelength and polarization which determine, respectively the
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6 What is Low-Level Laser (Light) Therapy?

intensity (or brightness), the energy and the orientation of their

oscillation. With the advent of the quantum theory, light was better
understood and can be interpreted as a particle complementary
to the wave; thus, light can behave either as wave or particle.
The particles of light (photons) are massless packets (“quanta”) of
energy moving at approximately 3 × 108 m/s. The wavelength of
light is determined by the energy of single photon and the number
of photons in a single direction determines the intensity in that
direction.

1.2.1 Tissue Optics

Light crossing the interior of biological tissue interacts, basically,
in two ways: absorption and scattering [2]. The absorption occurs
when a photon interacts with an atom or molecule and the entire
energy of the photon is transferred to the atom or molecule. The
scattering interactions can change both direction and energy of
photons (inelastic), or only the direction (elastic scattering). Visible
and near IR light interacting with biological tissue give rise mainly
to elastic scattering. The scattering depends on size, shape, and
refraction index of the scattering center and on the wavelength of the
incident light. Knowledge of the penetration and distribution of light
inside biological tissues is a hard problem because absorption and
scattering depend on wavelength, tissue biochemistry, and anatomy
[3].
The effectiveness of LLLT is closely connected with the amount
of light reaching the target tissue. However, in many cases, a precise
and direct measurements in an intern tissue are not possible, for
this reason, the best description of the LLLT procedure requires
explaination of the irradiation and dose parameters in the output of
the device or in the surface of the skin (or other biological medium).
The LLLT dosimetry is the description of those parameters which
can be conveniently divided in two parts: irradiation parameters,
“the medicine”; and how light is delivered, “the dose”. The irradiation
parameters, such as wavelength (nm), power (W), beam area (cm2 ),
pulse structure, are related with the light source options. On the
other hand, “the dose” parameters, like energy (J), energy density
(J/cm2 ), treatment chronology and irradiation time (s) and area
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Fundamental Science 7

(cm2 ), are operator-controlled. In addition, the dosimetry depends

on specific characteristics of the patient and of the physiological
tissue conditions. All these interrelated parameters and patient-
specific conditions make the LLLT dosimetry a pitfall in many
research and clinical approach.

1.2.2 Photochemistry of Chromophores

Low-intensity light can induce biomodulation due to photochemical
reaction in the cell, a process referred as photobiomodulation,
which occurs only when light is absorbed by a photoacceptor, or
chromophore. A chromophore is a molecule, or part thereof, which
possesses electrons in a low-energy orbit which can absorb a photon
to get excited and go over the energy band gap to a high-energy
orbit [4]. The varieties of effects of LLLT are credited to light
absorption by one (or many) chromophores leading to physiological
changes, and even optogenetic modulation. The approach used,
majorly by T. I. Karu and collaborators on the past decades, to
identify the connection of specific chromophores and theirs effects,
was attempt to experimentally match the absorption spectrum of
a specific chromophore with the action spectra of physiological
alterations. This kind of experiment indicates that LLLT enhances
ATP production due to absorption of red and near infrared light
by cytochrome c oxidase in the interior of mitochondria and it
can be the base of the mechanisms of many photobiological effects
[5].
In addition to ATP, other mitochondrial products such as NADH,
protein, and RNA are enhanced by light as well as the oxygen
consumption [6]. There is a region of the electromagnetic spectrum
known as therapeutic window that consists of red and infrared
light (600–1100 nm), in which light penetrates deeper in biological
tissue. It is explained by the fact that tissue scattering, in optical
region, is higher for shorter wavelength and chromophores like
hemoglobin and melanin have absorption peaks at wavelength
shorter than 600 nm. In the other extreme of optical therapeutic
window, water is a major absorber at wavelengths greater than
1150 nm. In addition, flavoproteins and cytochromes can act like
chromophores for blue and green light respectively [7].
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8 What is Low-Level Laser (Light) Therapy?

1.2.3 Photobiology: Mechanisms of LLLT Effects

The mechanisms of action of LLLT are one of the most exciting
research topics in the field of photomedicine. Although, a unified
explanation seems not close yet, a number of theories for each case
have been developed, many of which were a result of light (600–
1100 nm) absorption due to chromophores inside mitochondria.
The basic concept behind majority of mechanistic explanations
is the photon energy being absorbed by the mitochondrial chro-
mophores and being converted to chemical energy that is used to
power up cells, it is supported by the many times described ATP
increasing after LLLT. Other interesting point is that LLLT might be
exerting its effects regulating cellular redox systems, it is evidenced
by the fact that pro-oxidant cells are more sensitive to LLLT than
normal cells [8].
Every specific LLLT application has an optimal dose; it means
that all parameters can be tuned to produce the most effective
treatment. These optimal parameters depend on the condition to be
treated and even the personal characteristics of the patient. When
irradiance or the irradiation time is too low, it is not enough to
diminish the treated condition [9]. On the other hand, too high
irradiance or irradiation time can induce over responses and re-
bound effects that can inhibit the treatment or even create undesired
outcomes [10]. These characteristics are in accordance with the
well-established idea that LLLT has a biphasic dose response that is a
generalization of “Arndt–Schulz Law”, a pharmacological rule for the
biological effects of drugs and poisons [11]. This law was established
in 1887 when Hugo Schulz described the stimulatory effect of low
concentration of poisons on yeast metabolism and later, together
with Rudolf Arndt, claimed that weak stimuli accelerate metabolism,
higher stimuli produce a maximum outcome, but strong stimuli
inhibit the biological activity. There are attempts to generalize the
Arndt–Schulz law for all types of stimuli, it receives the name
of “hormesis” [12] and many times appears in LLLT literature
[13].
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Research in LLLT 9

1.3 Research in LLLT

Transdisciplinarity refers to a research method which crosses

many disciplinary boundaries to create a holistic and deeper
understanding of a specific subject. With this concept in mind we
easily recognize photomedicine, in particular LLLT, is an absolutely
transdisciplinar and collaborative field of research. In order to
achieve new findings in LLLT mechanisms the researching team
should have some expertise and knowledge about biophysics and
biochemistry; new biomedical applications are developed not only
by physicians, but, many times is necessary for scientists and
engineers to collaborate; on the other hand the improvement
and development of new devices usually have insights from
scientists and clinicians together. Therefore, in general, laboratory
teams researching LLLT are composed by clinicians, scientists, and
engineers from the most diverse backgrounds.
Translational research might refer to the “bench-to-bedside”
enterprise of gearing knowledge from basic sciences to produce new
drugs, devices, and treatment options for patients. Complementarily,
translational research, specially according with public health inves-
tigators, attempts to bring new treatments and research knowledge
into practice for patients for whom the treatments are intended [14].
For this reason, the contemporary photomedicine research perfectly
fits in the paradigm of translational research since it fits in both
translational objectives. While considerable efforts are dedicated to
create new devices and treatment options in order to improve LLLT;
other important task is to make health professionals and patients
know about it.
An interesting observation is that the biostimulation effect of
light was discovered unintentionally by Endre Mester, in 1967, when
he was testing if laser irradiation could induce cancer in mice. Laser
did not induce cancer, but, surprisingly, it stimulated the regrowth of
hair. This result was incommensurably important, and was quickly
recognized; for this reason, Dr. Mester received the title “Doctor of
Sciences” from the Hungarian Academy of Sciences in 1971. By this
time, he started to treat patients with non-healing skin ulcers and
his low intensity laser irradiation protocol became well known all
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10 What is Low-Level Laser (Light) Therapy?

Figure 1.2 Number of LLLT publications per year according to a search for
low-level light therapy in the PubMed site in June of 2014.

over the world. Since this bright beginning, over 4000 basic science
or translational research, and approximately 400 clinical trials, were
published in the field of photobiomodulation (Fig. 1.2).
Basic science research of LLLT usually takes place with the
physics of light-tissue interactions, tissue optics, and the light
source properties; the chemistry of light absorption, chromophores,
photomodulation of reactive oxygen species, and photochemical
reactions; the biology of cell proliferation and migration in culture,
and the action spectra of cells photobiomodulation.
The translational research of LLLT concentrates in animal models
of a broad range of diseases and conditions. A special interest is
the condition which can have strong variations between patients, so
animal models can produce a controlled environment to research.
Some examples of conditions studied with animal models and
treated with LLLT are: wound healing, pain, arthritis, inflammation,
microbial infections, bone and tendon regeneration, traumatic brain
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Clinical and Biomedical Applications of LLLT 11

injury (TBI); and a sort of neurological diseases like Alzheimer,

Parkinson, and strokes.

1.4 Clinical and Biomedical Applications of LLLT

Light irradiation with low power density has been reported as a non-
invasive, non-carcinogenic, atraumatic, with no known side effect
therapy to many diseases and undesired conditions. In addition, it
is used to improve human wellness by aesthetical applications and
has diverse veterinary applications. The biomodulation achieved
by LLLT allows its application in situations apparently paradoxical,
since it sometimes can stimulate and in other situations can inhibit
the same biological effect. For this reason, LLLT is referred by many
researches as a regulator, leading the organisms to homeostasis.
Moreover, there are evidences of systemic effects of LLLT, it means
that application in one site of the body can produce an improvement
of condition in another body part. This can be explained by local
alterations which can be delivered to other sites through the blood
or the lymphatic system, the blockage of axonal flow can explain the
decreasing in pain sensation after LLLT in specific points in the pain
pathway from the painful site to the central nervous system.
The stimulatory effect of LLLT had as one of their first clinical
applications the wound healing since LLLT promotes beneficial
effects during the four phases of the wound healing process
(coagulation, inflammation, migration, and remodeling [15]). These
processes can be regulated by many growth factors and are
connected with nitric oxide (NO) signaling, which release and
production can be modulated by LLLT [11].
An interesting example of systemic effect was proved by Hopkins
et al. when they conducted a randomized, triple-blind, placebo-
controlled experiment in which 22 healthy subjects had induced two
standardized 1.27 cm2 abrasion wounds in their anterior forearms.
LLLT (820 nm, 8 J/cm2 , for 125 s, pulse rate of 700 Hz) was applied
in only one of the two randomly chosen wounds. Evidences for a
systemic blood (or carried by blood) effect of laser irradiation were
obtained in follow-up testing (in days 6, 8, and 10) since it revealed
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12 What is Low-Level Laser (Light) Therapy?

that the laser group had smaller wounds than the sham group for
both the treated and the untreated wounds [16].
Reduction of inflammation due to light therapy is one of the
most accepted LLLT effects. It is evidenced by decreasing in chemical
inflammatory mediators such as prostaglandin E2, leucocytes, and
tumor necrosis factor (TNFα) [17]. LLLT can modulate the pro-
inflammatory response increasing both the mRNA expression and
the protein concentration of anti-inflammatory mediators, such as
IL-10 (related to tendinitis) and heat shock protein-72 (HSP72)
(related to rheumatoid arthritis), these are processes similar to
the ones promoted by the treatment with glucocorticoid. The anti-
inflammatory and pro-inflammatory effects promoted by LLLT are
strong evidences that LLLT acts as a homeostasis regulator in
order to maintain balance between the anti- and pro-inflammatory
responses.
There are many ways LLLT acts to decrease pain, including
anti-inflammatory effects, neural blockade, stimulation of lymphatic
activity, tissue repair, and reduction of muscle spasm. Each of
these mechanisms has been studied in a translational manner
from subcellular levels to clinical application. Laser can relieve
nociceptive and neuropathic pain by partially inhibiting nerve
conduction and reducing afferent stimulation, mimicking some
functions of local anesthetic injections [18]. In addition, LLLT can
produce long lasting pain decreasing due to neuroplasticity, which
is the capacity of neurons in both the peripheral and central
nervous systems, to be modulated by increased or decreased
afferent activities from the somatosensory nerves [19]. LLLT has
been successfully used for pain decreasing in various situations
like arthritis, crystallopathies, tendinopathies, lateral epicondylitis,
post-operative and myofascial pain as well as musculoskeletal pain
in neck, back, and shoulder.
Photobiomodulation with red and NIR light have been applied
successfully to ameliorate cardio-vascular and respiratory system.
Oron et al. showed that laser irradiation caused increase in
newly formed blood vessels six days post infarction in rats [20].
Many functions in vascular walls are regulated by NO including
suppression of inflammatory response, vasodilatation, angiogenesis,
inhibition of apoptosis, and cell migration [21]. Beneficial effect of
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Clinical and Biomedical Applications of LLLT 13

LLLT on lung function and the reduction of clinical symptom have

been demonstrated by blood irradiation or transcutaneous lung
irradiation [22].
Recently, lasers and LED irradiation in the central nervous
system have been reported to bring positive outcomes for acute
and chronicle strokes, traumatic brain injury, memory and mood
disorders, various neurodegenerative diseases such as Parkinson,
Alzheimer, and retinal diseases [23–25]. Importantly, LLLT shows no
side effects in both animals and humans, which is a much desired
characteristic for a therapy carried out in brain. In addition, red
and NIR laser irradiation to the spinal cord, aiming a restoration of
traumatically injured peripheral nerves [26], and the systemic effect
of LLLT on the crushed sciatic nerves were proved [27].
LLLT application associated with operative procedure is rising
in importance. The previous application of LLLT decreases cell
death and dehiscence, LLLT during the operation decreases the
inflammatory process and in the post-operative care can reduce
the recovery time. These benefits can be reached even in large and
complicated surgeries such as saphenectomy in diabetic patients.
Special attention must be given to the applications of LLLT to
dentistry since there is virtually no procedure in dentistry that won’t
respond positively to photobiomodulation; using a drill to prepare
a restoration or an instrument for mechanical debridement starts
an inflammatory response within the tissue and pulp. LLLT helps
modulate the inflammatory response while reducing pain making
it a tool in every dental practitioner’s armamentarium that can
both ease the stress of the practitioner and improve the patient
experience.
Wellness can be provided by LLLT for aesthetic applications.
The regeneration and stimulation effect of LLLT promotes the
resurfacing and rejuvenating of the skin. Moreover, it is a very
well established treatment for hair regrowth and evidences are
accumulating for fat reduction.
Sports medicine will benefit from LLLT since professionals and
amateur athletes can highly recover from intense sports injuries.
In near future sports agencies will use “laser doping” since the
aforementioned beneficial effects and the pre-conditioning achieved
by laser and LED irradiation will highly improve athlete’s capacity.
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14 What is Low-Level Laser (Light) Therapy?

Not less impressive is the use of LLLT for veterinary medicine.

LLLT has been used for pets, basically, in same applications as
mentioned for humans. In addition, it can be used to improve
reproduction of farm animals and extinction threatened species.
Moreover, it is a side-effect free treatment for wild animals.
Despite many positive outcomes from clinical trials and labora-
tory studies, as shown above, yet LLLT has not been integrated by
mainstream medicine. Many possible explanations can be addressed
by scientists, such as lack of mechanisms and dose evidence and
large clinical trials. Other explanation of economical order are lack
of cost and reimbursement. These facts allied to the misinformation
of the population and the action of pseudo-specialists creates an
atmosphere of distrust surrounding the LLLT.

References

1. Chung, H., et al., The nuts and bolts of low-level laser (light) therapy.
Annals of Biomedical Engineering, 2012. 40(2): pp. 516–533.
2. Sousa, M.V., et al., Laser scattering by transcranial rat brain illumination.
In SPIE Photonics Europe. 2012: International Society for Optics and
Photonics.
3. Cheong, W.-F., Prahl, S.A., and Welch, A.J., A review of the optical
properties of biological tissues. IEEE Journal of Quantum Electronics,
1990. 26(12): pp. 2166–2185.
4. Karu, T., Photobiological fundamentals of low-power laser therapy. IEEE
Journal of Quantum Electronics, 1987. 23(10): pp. 1703–1717.
5. Karu, T., Primary and secondary mechanisms of action of visible to
near-IR radiation on cells. Journal of Photochemistry and Photobiology
B: Biology, 1999. 49(1): pp. 1–17.
6. Passarella, S., et al., Increase of proton electrochemical potential and
ATP synthesis in rat liver mitochondria irradiated in vitro by helium-
neon laser. FEBS Letters, 1984. 175(1): pp. 95–99.
7. Hamblin, M.R., and Demidova-Rice, T.N., Cellular chromophores and
signaling in low level light therapy. In Biomedical Optics (BiOS). 2007:
International Society for Optics and Photonics.
8. Tafur, J., and Mills, P.J., Low-intensity light therapy: Exploring the role of
redox mechanisms. Photomedicine and Laser Surgery, 2008. 26(4): pp.
323–328.
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References 15

9. Haxsen, V., et al., Relevance of laser irradiance threshold in the induction

of alkaline phosphatase in human osteoblast cultures. Lasers in Medical
Science, 2008. 23(4): pp. 381–384.
10. Lanzafame, R.J., et al., Reciprocity of exposure time and irradiance on
energy density during photoradiation on wound healing in a murine
pressure ulcer model. Lasers in Surgery and Medicine, 2007. 39(6): pp.
534–542.
11. Huang, Y.-Y., et al., Biphasic dose response in low level light therapy—an
update. Dose-Response, 2011. 9(4): pp. 602–618.
12. Stebbing, A., Hormesis—the stimulation of growth by low levels of
inhibitors. Science of the Total Environment, 1982. 22(3): pp. 213–234.
13. Hayworth, C.R., et al., In vivo low-level light therapy increases cy-
tochrome oxidase in skeletal muscle. Photochemistry and Photobiology,
2010. 86(3): pp. 673–680.
14. Woolf, S.H., The meaning of translational research and why it matters.
Jama, 2008. 299(2): pp. 211–213.
15. Diegelmann, R.F., and Evans, M.C., Wound healing: An overview of acute,
fibrotic and delayed healing. Front Biosci, 2004. 9(1): pp. 283–289.
16. Hopkins, J.T., et al., Low-level laser therapy facilitates superficial wound
healing in humans: A triple-blind, sham-controlled study. Journal of
Athletic Training, 2004. 39(3): p. 223.
17. Aimbire, F., et al., Low-level laser therapy induces dose-dependent
reduction of TNFα levels in acute inflammation. Photomedicine and
Laser surgery, 2006. 24(1): pp. 33–37.
18. Chow, R., et al., Inhibitory effects of laser irradiation on peripheral
mammalian nerves and relevance to analgesic effects: a systematic
review. Photomedicine and Laser Surgery, 2011. 29(6): pp. 365–381.
19. Chow, R.T., et al., Efficacy of low-level laser therapy in the man-
agement of neck pain: A systematic review and meta-analysis of
randomised placebo or active-treatment controlled trials. The Lancet,
2009. 374(9705): pp. 1897–1908.
20. Mirsky, N., et al., Promotion of angiogenesis by low energy laser
irradiation. Antioxidants and Redox Signaling, 2002. 4(5): pp. 785–790.
21. Kimura, H., and Esumi, H., Reciprocal regulation between nitric oxide
and vascular endothelial growth factor in angiogenesis. Acta Biochimica
Polonica—English Edition, 2003. 50(1): pp. 49–60.
22. Aimbire, F., et al., Effect of LLLT Ga–Al–As (685 nm) on LPS-induced
inflammation of the airway and lung in the rat. Lasers in Medical Science,
2005. 20(1): pp. 11–20.
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16 What is Low-Level Laser (Light) Therapy?

23. Hamblin, M., et al., Low-level light therapy aids traumatic brain injury.
SPIE Newroonm, 2011.
24. Lampl, Y., et al., Infrared laser therapy for ischemic stroke: A new
treatment strategy results of the neurothera effectiveness and safety
trial-1 (NEST-1). Stroke, 2007. 38(6): pp. 1843–1849.
25. Moges, H., et al., Light therapy and supplementary riboflavin in the SOD1
transgenic mouse model of familial amyotrophic lateral sclerosis (FALS).
Lasers in Surgery and Medicine, 2009. 41(1): pp. 52–59.
26. Gigo-Benato, D., Geuna, S., and Rochkind, S., Phototherapy for enhancing
peripheral nerve repair: A review of the literature. Muscle & Nerve, 2005.
31(6): pp. 694–701.
27. Rochkind, S., et al., Systemic effects of low-power laser irradiation on the
peripheral and central nervous system, cutaneous wounds, and burns.
Lasers in Surgery and Medicine, 1989. 9(2): pp. 174–182.
July 6, 2016 17:5 PSP Book - 9in x 6in 02-Hamblin-c02

Chapter 2

History of Low-Level Laser (Light)

Therapy

Michael R. Hamblin
Wellman Center for Photomedicine, Massachusetts General Hospital, 50 Blossom Street,
Boston, MA 02114, USA
Department of Dermatology, Harvard Medical School, Massachusetts General Hospital,
55 Fruit Street, Boston, MA 02114, USA
Harvard–Massachusetts Institute of Technology (MIT) Division of Health Sciences and
Technology, 77 Massachusetts Avenue, E25–518, Cambridge, MA 02139, USA
hamblin@helix.mgh.harvard.edu

Tracing the history of, what came to be known as low-level laser

therapy (LLLT), is an interesting challenge. More interesting perhaps
because there are two distinct timelines or historical strands to
be teased apart, namely the history of light therapy and the
development of the laser. The oldest of these is light therapy or
photomedicine, so we will address that first.
The history of photomedicine goes back over three thousand
years to India where sunlight was employed for therapeutic
purposes as recorded in the sacred Hindu text Atharva Veda dating
from 1400 BC. Sufferers from vitiligo (a patchy depigmentation of
the skin then thought to be a form of leprosy) were given certain
plant extracts to eat and then exposed to the sun [1]. Starting in
the 18th century, sporadic reports began to appear in the medical

Handbook of Low-Level Laser Therapy

Edited by Michael R. Hamblin, Marcelo Victor Pires de Sousa, and Tanupriya Agrawal
Copyright c 2017 Pan Stanford Publishing Pte. Ltd.
ISBN 978-981-4669-60-3 (Hardcover), 978-981-4669-61-0 (eBook)
www.panstanford.com
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18 History of Low-Level Laser (Light) Therapy

literature indicating that sunlight could be used to treat a wide

variety of different diseases. In 1735, Fiennius [2] described a
case in which he cured a cancerous growth on the lip using a
sunbath. In 1774, Faure [3] reported that he successfully treated
skin ulcers with sunlight, and in 1776 LePeyre and LeConte [4]
found that sunlight concentrated through a lens accelerated wound
healing and destroyed tumors. There were also reports that sunlight
had beneficial effects on internal maladies. In 1782, Harris [2]
used sunlight-exposed mollusk shells to improve a case of rickets
(fragile bones due to vitamin D deficiency), In 1845, Bonnet [5] first
reported that sunlight could be used to treat tuberculous arthritis (a
bacterial infection of the joints).
In the second half of the 19th century, the therapeutic application
of sunlight known as heliotherapy gradually became popular. In
1855, Rikli from Switzerland opened a clinic in Veldes in Slovenia
for the provision of heliotherapy [6]. In 1877, Downes and Blunt [7]
discovered by chance that sunlight could kill bacteria. They noted
that sugar water placed on a window-sill turned cloudy in the shade
but remained clear while in the sun. Upon microscopic examination
of the two solutions, they realized that bacteria were growing in the
shaded solution but not in the one exposed to sunlight.
Theobald Adrian Palm (1848–1928) (Fig. 2.1) discovered the role
of sunlight in the prevention of rickets [8]. He had worked as a
doctor both in Edinburgh in Scotland where rickets was rife, and
also as a missionary in Japan where rickets was rare. He deduced
that the constant pall of smoke that overhung Edinburgh (a city
known colloquially in the British Isles as “Auld Reekie”), and which
created gloom that effectively blocked the sun from reaching the
population, was the key difference between the two countries. Many
years later the role of sunlight exposure to the skin, in mediating the
biosynthesis of vitamin D, eventually explained these observations
[9].
Nils Ryberg Finsen (1860–1904) (Fig. 2.2) was born in the Faroe
Islands and studied medicine at the University of Copenhagen,
qualifying in 1890. He suffered from an illness, which later would
become known as Niemann–Pick disease, and is characterized by
progressive thickening of the connective tissue of the liver, the heart
and the spleen. His discovery that sun exposure improved his own
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History of Low-Level Laser (Light) Therapy 19

Figure 2.1 Theobald Adrian Palm (1848–1928).

symptoms encouraged him to treat his patients with light [10]. He

had particular success in 1893 when treating smallpox with red
light [11], and in 1895 when treating lupus vulgaris (also known
as scrofula or cutaneous tuberculosis) with what he thought was
ultra-violet light from an arc-lamp [12], (but in fact was probably
blue light [13]). The Finsen Institute was formed in Copenhagen
in 1896 and is still there today (Fig. 2.3), but it now concentrates
on cancer research rather than photomedicine. Finsen received the
Nobel Prize for Physiology or Medicine in 1903 [10], sadly just
before his death in 1904.
Two pioneering Swiss physicians, Oskar Bernhard (1861–1939)
at St. Moritz and Auguste Rollier (1874–1954) at Leysin were
responsible for extending the use of heliotherapy [14]. Solar therapy
as practiced by these practitioners included increasing graduated
exposures of parts of the body to sunlight, and the beneficial effects
were considered to be accentuated by the fresh and cold mountain
air in the Alps.
Bernhard (Fig. 2.4) obtained an impressive initial success
treating a large non-healing abdominal wound (from a knife attack)
that had resisted all other accepted healing approaches, and which
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20 History of Low-Level Laser (Light) Therapy

Figure 2.2 Nils Ryberg Finsen (1860–1904).

Figure 2.3 The Finsen Institute at Rigshospitalet in Copenhagen, Denmark.

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History of Low-Level Laser (Light) Therapy 21

Figure 2.4 Oskar Bernhard (1861–1939).

he decided to expose to the sun as a last desperate measure.

Thereafter he treated all non-healing and infected wounds with
sunlight. He then began to treat open tuberculous cavities and, soon
after, closed tuberculous foci of the bones, joints and glands with
sunlight. In 1905, Bernhard had established his own small private
clinic for sunlight therapy at St. Moritz that could accommodate
some thirty-three patients and had south-facing balconies on two of
the upper floors for convenient sun exposure.
Rollier (Fig. 2.5) was born at St. Aubin in the Swiss Canton
of Neuchatel and graduated in medicine from Zurich and Berne.
He became deeply disillusioned with the poor results obtained by
surgery in the treatment of skeletal tuberculosis and went into
a rural general practice at Leysin in the Alpes Vaudoises, where,
at Bernhard’s encouragement, he began to treat non-pulmonary
tuberculosis with sunshine and fresh air. Over the next 40 years the
technique Rollier devised for exposing the body to sunlight (Rollier’s
Sunlight Therapy or Heliotherapy) came to be broadly accepted
in Europe [15]. His clinic called “Les Frênes” was the first large
purpose-built sunlight therapy facility to be constructed in the world
(Fig. 2.6).
We will now “switch gears” in order to consider the other histo-
rical strand—the discovery of lasers—that eventually came together
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22 History of Low-Level Laser (Light) Therapy

Figure 2.5 Auguste Rollier (center) treating a patient with heliotherapy.

Figure 2.6 Rollier’s clinic “Les Frênes” in the Alps at Valais, Switzerland.
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History of Low-Level Laser (Light) Therapy 23

Figure 2.7 Albert Einstein (1879–1955).

to coalesce with light therapy to form what came to be known as

low-level laser (light) therapy (LLLT).
H. G. Wells in the War of the Worlds (1898) described the use
of “rays of light that carry grand and mysterious powers” [16]. The
end of the 19th century was seen as the age of “rays or waves” with
the discovery of radio, X-rays, and radioactivity. A whole spectrum
of electromagnetic radiation opened up, with wavelengths longer or
shorter than visible light, and it was constantly speculated as to what
amazing new applications might be discovered for this radiation
in medicine, communications, scientific research, or warfare. Albert
Einstein (1879–1955) (Fig. 2.7) in 1916 while considering the
implications of the newly discovered quantum physics, predicted
that electromagnetic rays could stimulate atoms to emit more rays
of the same wavelength [17]. However, at that time engineers had
no idea how to accomplish this interesting trick, and for decades the
idea seemed merely a theoretical curiosity of no practical interest.
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24 History of Low-Level Laser (Light) Therapy

The triumphal celebration of the science of physics that came

about in the late 1940s, after two ground-breaking advances (the
discovery of radar, and the use of the two atomic bombs) had
been popularly concluded to have helped the United States to
win World War II, had a big influence both on scientists and
on the general public. As the Cold War against the Soviet Union
emerged in the 1950s, the U. S. government made available
ever-larger funds for basic and applied physics research. With
the possibility of not only military, but also civilian applications,
corporations and entrepreneurs added their own funds and their
research personnel to the national effort. Industrial and university
laboratories proliferated and the tantalizing opportunity to make
further advances in physics was their main concern.
In the 1930s before the war it would have been theoretically
possible for scientists to have built a laser, as Einstein had provided
the theory, and furthermore the necessary optical tools were
already in existence. However at that time there was no over-
riding reason for them to do so. This need only arrived in 1950
from an unexpected direction. Short-wavelength radio waves, called
microwaves, had been used to make molecules vibrate in ways
that revealed valuable structural information (a technique called
microwave spectroscopy). Radar equipment left over from World
War II was re-jigged to provide the source of radiation.
Charles Hard Townes (1915–2015) (Fig. 2.8) who was working
at Columbia University in New York, had studied molecular
spectroscopy as a physicist in the 1930s, and during the war he
had worked on radar as an electronics engineer. The Office of
Naval Research encouraged him and other physicists to devise
a way to make powerful beams of microwave radiation at ever-
shorter wavelengths. In 1951 he found a solution. He reasoned that
inside a resonating cavity similar to the devices used to generate
radar waves, the right type of atoms or molecules might generate
shorter wavelength radiation if suitably stimulated. Townes gave
the problem to Herbert Zeiger, a postdoctoral student, and James
P. Gordon, a graduate student. By 1954 they had the device
working. Townes called it a MASER for “microwave amplification by
stimulated emission of radiation” (Fig. 2.9). As predicted by theory
the radiation was at a single wavelength, that is, monochromatic. In
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History of Low-Level Laser (Light) Therapy 25

Figure 2.8 Charles Hard Townes (1915–2015).

Moscow, A. M. Prokhorov and N. G. Basov were working in the same

direction, and they built a maser in 1955.
However the microwaves from masers had turned out to be only
moderately useful, more for scientific research than for military or
industrial applications. A few scientists thought an infrared maser
might be more important for applications and considered how to
make one. Infrared rays could not be easily manipulated like radar,
and indeed infrared optical components were largely unavailable.
Townes had been thinking about the problem intensively. In
1957, while studying the equations for amplifying radiation, he
realized that much shorter visible wavelengths would be much
easier to deal with than longer-wavelength infrared waves. He
could “skip over” the far-infrared region that had no available
optical components, to the visible region where well-established
optical techniques and components for manipulating light were
readily available. Townes discussed the problem with his colleague
and brother-in-law Arthur Schawlow who worked at Bell Labs.
Schawlow realized that the key feature was to contain the atoms
to be stimulated in a long, narrow cavity with mirrors at each
end. The light would bounce back and forth inside so that there
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26 History of Low-Level Laser (Light) Therapy

Figure 2.9 The ammonia gas maser demonstrated in New York in 1955.
Townes is on left and his graduate student James Power Gordon (1928–) is
on right.

would be increasing chances for stimulated atoms to emit more

light. One of the mirrors would be only partly silvered so that some
of the radiation could escape. This arrangement (the Fabry–Pérot
resonator or etalon) was already familiar to optics researchers [18].
The same key arrangement had also occurred to Gordon Gould
(1920–2005) (Fig. 2.10), who was a graduate student at Columbia

Figure 2.10 Gordon Gould (1920–2005).

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History of Low-Level Laser (Light) Therapy 27

University who had discussed the problem with Townes. For his
thesis research with the Nobel laureate Prof. I. I. Rabi, Gould had
already been working with “pumping” atoms to higher-energy states
so they would emit light. Gould realized that he was onto something
far beyond the much-discussed “infrared maser”. In his notebook
he confidently named the yet-to-be-invented device a LASER (for
light amplification by stimulated emission of radiation). In April
1959 Gould filed patent applications with his employer, the high-
tech research firm TRG. Nine months earlier Schawlow and Townes
had applied for a patent on behalf of Bell Labs, which employed
Schawlow on staff and Townes as a consultant. When the Bell patent
was granted, Gould sued, claiming he was first to conceive of the
device. Legal battles raged for the next thirty years. In 1987 Gould
and his backers began to win settlements and one of the greatest
patent wars in history was over.
Although in 1958 Gould, Schawlow, and Townes understood in
principle how to build a laser, the actual construction of a working
example would require a lot more work to define the correct lasing
medium and the best way to pump energy into it. Over the next two
years the race to make the first operating laser was truly in full swing
[19].
Townes at Columbia was working on potassium gas that was
theoretically predicted to be an excellent lasing medium, but its
corrosive properties attacked the seals, glass, and mirrors. Ali Javan
at Bell Labs was trying to use a mixture of helium and neon in
a long tube (an electrical discharge would excite the helium that
would transfer its energy to the neon). At Westinghouse Research
Labs and at the IBM Thomas J. Watson Research Center, the idea
was to use polished crystals as the resonator instead of a tube
filled with gas. Peter Sorokin at IBM tried a uranium-doped calcium
fluoride crystal polished to have square sides, while Irwin Wieder at
Westinghouse tried to pump a synthetic ruby crystal with a tungsten
lamp, but concluded “it was impossible to pump sufficient energy
into a ruby”. However Theodore Maiman (1927–2007) (Fig. 2.11)
working at Hughes Labs realized one did not necessarily need to use
a continuous wave pumping source, but instead a bright flash-lamp
could have the necessary peak optical power. On May 16, 1960 after
assembling a ruby crystal inside a coiled flash-lamp (Figs. 2.12A
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28 History of Low-Level Laser (Light) Therapy

Figure 2.11 Theodore Harold Maiman (1927–2007).

(A)

(B)

Figure 2.12 (A) Photograph of Maiman’s ruby laser; (B) Schematic drawing
of Maiman’s ruby laser.
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History of Low-Level Laser (Light) Therapy 29

Figure 2.13 Leon Goldman (1906–1997).

and 2.12B), the first ever pulses of red laser light were observed
[20]. Within two weeks of the press conference that announced
Maiman’s discovery in July 1960, the groups at Bell Labs and TRG
had also obtained flash-lamps like the one shown in Maiman’s
publicity photograph, and had replicated his device. In November,
1960 Sorokin recut his calcium fluoride crystals into cylinders, and
after also exciting them with a flash-lamp he obtained laser light,
while in December, Javan also finally succeeded with his He–Ne laser.
So after years of struggle, by the end of 1960 there were suddenly
three completely different types of laser systems operating in the
United States.
In the 1960s the two previously separate themes of (a)
photomedicine and (b) the newly discovered lasers gradually came
together. In 1961 Leon Goldman (1906–1997) (Fig. 2.13), while
working at the University of Cincinnati, started to experiment
with the effect that laser beams had on the skin [21], and asked
whether they could be used to remove tattoos [22]. He realized that
lasers could be used to perform “bloodless surgery” and believed
that high-power lasers were superior to cold steel for repairing
damaged livers and for debriding burn wounds [23]. Goldman was
officially designated the father of laser medicine in 1979 at the Opto-
Elektronic Conference in Munich, Germany [24]. In the early 1960s
Paul McGuff (1916–2002) while working at Tufts New England
Medical Center in Boston [25], made medical history by using a laser
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30 History of Low-Level Laser (Light) Therapy

Figure 2.14 Endre Mester (1903–1984).

beam to vaporize human cancer cells that had been transplanted

into a hamster [26].
The discovery of LLLT can be attributed to Endre Mester
(1903–1984) in Hungary, who has been called “the father of
photobiomodulation” [27] (Fig. 2.14). Mester had qualified in
medicine from the University of Budapest and went on to become
full professor and director of the 2nd Department of Surgery at
Semmelweis University in Budapest and was elected as President of
the Society of Hungarian Surgeons [27]. In 1965 Mester started laser
research and tried to repeat McGuff’s experiments by implanting
tumor cells beneath the skin of laboratory rats and exposing them
to the beam from a customized ruby laser. However the tumor
cells were not destroyed by doses of what was presumed to be
high-power laser energy, but instead, the skin incisions made to
implant the cancer cells appeared to heal faster in laser-treated
animals, compared to incisions of control animals that were not
treated with light [28]. Moreover the regrowth of hair on depilated
rat skin, was observed to be faster after exposure to his ruby laser
[29]. After being initially puzzled by these contradictory findings, he
realized that his custom-designed ruby laser was much weaker than
he originally thought it to be, and instead of being photo-ablative
against the tumor tissue, the low-power laser light stimulated the
skin to heal faster and caused the hair to regrow. This fortuitous
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References 31

observation (that would not have occurred if Mester had possessed a

modern laser power meter) led him to make a series of experiments
that showed that treatment with red light indeed produced faster
healing of skin wounds (Mester et al. 1971). In 1968 he published
the first indication of the biphasic dose response or Arndt–Schulz
effect in a study entitled “Studies on the inhibiting and activating
effects of laser beams” [30]. Mester continued research into laser
biostimulation until 1985 with many papers on wound healing both
pre-clinical and clinical [31–34], regeneration of muscle fibers [35]
and clinical treatment of skin necrosis [36]. In 1971 he was awarded
a Scientific Doctorate by the Hungarian Academy of Sciences in
recognition of his work. He had several collaborators during his laser
research and clinical work, particularly his two sons Adam Mester,
M.D., a radiologist, and Andrew Mester, M.D., an otolaryngologist.
They worked and published together [37] and after Prof. Mester’s
death, Adam and Andrew continued to carry out laser research and
clinical studies.
This brief outline of the early history of LLLT has we hope served
as a “lead-in” to the main subject matter of the Handbook. Some of
the other renowned figures in the field not mentioned here, are sill
alive and a few have contributed chapters to the present textbook.

Acknowledgements

Research in the Hamblin laboratory is supported by the United

States NIH grant R01AI050875.

References

1. Fitzpatrick, T. B., Pathak, M. A. Historical aspects of methoxsalen and

other furocoumarins. J Invest Dermatol 32:229–231; 1959.
2. Giese, A. C. Historical introduction. In: Giese, A. C., ed. Photophysiology.
New York: Academic Press; 1964.
3. Russell, E. H., Russell, W. K. Ultraviolet Radiation and Actinotherapy. New
York: William Wood; 1927.
4. Rollier, A. Heliotherapy. London: Oxford Medical Publishers; 1923.
July 6, 2016 17:5 PSP Book - 9in x 6in 02-Hamblin-c02

32 History of Low-Level Laser (Light) Therapy

5. Bonnet, A. Traite des Maladies des Articulations. Paris: Bailliere;

1845.
6. Barth, J., Kohler, U. Photodermatologie in Dresden-ein historischer Abriss.
Festschrift anlasslich des 75. Geburtstages von Prof. Dr. h.c. H.-E. Kleine-
Natrop (1917–1985). Dresden; 1992.
7. Downes, A., Blunt, T. P. Researches on the effect of light upon
bacteria and other organisms. Proc Royal Soc London 26:488–500;
1877.
8. Palm, T. A. Letter to the editor. Br Med J 2:1247; 1888.
9. DeLuca, H. F. The vitamin D story: A collaborative effort of basic
science and clinical medicine. FASEB journal : Official publication of the
Federation of American Societies for Experimental Biology 2:224–236;
1988.
10. Finsen, N. R. Nobel Lectures, Physiology or Medicine 1901–1921.
Amsterdam: Elsevier Publishing Company; 1967.
11. Finsen, N. R. The Red Light Treatment of Small-Pox. Br Med J 2:1412–
1414; 1895.
12. Finsen, N. R. Om Anvendelse i Medicinen af Koncentrerede Kemiske
Lysstraaler. Copenhagen, Denmark: Gyldendalske Boghandels Forlag;
1896.
13. Moller, K. I., Kongshoj, B., Philipsen, P. A., Thomsen, V. O., Wulf,
H. C. How Finsen’s light cured lupus vulgaris. Photodermatology,
Photoimmunology & Photomedicine 21:118–124; 2005.
14. Hobday, R. A. Sunlight therapy and solar architecture. Med Hist 41:455–
472; 1997.
15. Rollier, A. Heliotherapy: With Special Consideration of Surgical Tubercu-
losis. London: Frowde and Hodder & Stoughton; 1923.
16. Hughes, D. Y., Geduld, H. M. A Critical Edition of the War of the Worlds: H.G.
Wells’s Scientific Romance. Indianapolis, IN: Indiana University Press;
1993.
17. Einstein, A. Zur Quantentheorie der Strahlung. Physikalische Zeitschrift
18:121–128; 1917.
18. Townes, C. H. How the Laser Happened: Adventures of a Scientist. Oxford,
UK: Oxford University Press; 1999.
19. Hecht, J. Beam: The Race to Make the Laser. Oxford, UK: Oxford University
Press; 2005.
20. Maiman, T. H. Stimulated optical radiation in ruby. Nature 187:493–494;
1960.
July 6, 2016 17:5 PSP Book - 9in x 6in 02-Hamblin-c02

References 33

21. Goldman, L., Blaney, D. J., Kindel, D. J., Jr., Franke, E. K. Effect of the laser
beam on the skin. Preliminary report. J Invest Dermatol 40:121–122;
1963.
22. Goldman, L., Wilson, R. G., Hornby, P., Meyer, R. G. Radiation from a Q-
switched ruby laser. Effect of repeated impacts of power output of 10
megawatts on a tattoo of man. J Invest Dermatol 44:69–71; 1965.
23. Goldman, L., Rockwell, R. J., Jr. Laser systems and their applications in
medicine and biology. Advances in Biomedical Engineering and Medical
Physics 1:317–382; 1968.
24. Geiges, M. L. History of lasers in dermatology. Current Problems in
Dermatology 42:1–6; 2011.
25. McGuff, P. E., Bushnell, D., Soroff, H. S., Deterling, R. A., Jr. Studies of the
surgical applications of laser (light amplification by stimulated emission
of radiation). Surg Forum 14:143–145; 1963.
26. McGuff, P. E., Deterling, R. A., Jr., Gottlieb, L. S. Tumoricidal effect of laser
energy on experimental and human malignant tumors. N Engl J Med
273:490–492; 1965.
27. Gaspar, L. Professor Endre Mester, the father of photobiomodulation. J
Laser Dentistry 17:146–148; 2009.
28. Mester, E., Ludány, G., Sellyei, M., Szende, B., Tota, J. The simulating
effect of low power laser rays on biological systems. Laser Rev 1:3;
1968.
29. Mester, E., Szende, B., Gartner, P. The effect of laser beams on the growth
of hair in mice. Radiobiol Radiother (Berl) 9:621–626; 1968.
30. Mester, E., Ludany, G., Sellyei, M., Szende, B., Gyenes, G., Tota, G. J. Studies
on the inhibiting and activating effects of laser beams. Langenbecks
Archiv fur Chirurgie 322:1022–1027; 1968.
31. Mester, E., Nagylucskay, S., Doklen, A., Tisza, S. Laser stimulation of
wound healing. Acta chirurgica Academiae Scientiarum Hungaricae
17:49–55; 1976.
32. Kovacs, I. B., Mester, E., Gorog, P. Stimulation of wound healing with laser
beam in the rat. Experientia 30:1275–1276; 1974.
33. Mester, E., Spiry, T., Szende, B. Effect of laser rays on wound healing.
Bulletin de la Societe internationale de chirurgie 32:169–173; 1973.
34. Mester, E., Korenyi-Both, A., Spiry, T., Scher, A., Tisza, S. Stimulation of
wound healing by means of laser rays. (Clinical and electron microscop-
ical study). Acta chirurgica Academiae Scientiarum Hungaricae 14:347–
356; 1973.
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34 History of Low-Level Laser (Light) Therapy

35. Mester, E., Korenyi-Both, A., Spiry, T., Tisza, S. The effect of laser
irradiation on the regeneration of muscle fibers (preliminary report).
Zeitschrift fur experimentelle Chirurgie 8:258–262; 1975.
36. Mester, E., Tisza, S., Csillag, L., Mester, A. Laser treatment of coumarin-
induced skin necrosis. Acta Chirurgica Academiae Scientiarum Hungari-
cae 18:141–148; 1977.
37. Mester, E., Mester, A. F., Mester, A. The biomedical effects of laser
application. Lasers in Surgery and Medicine 5:31–39; 1985.
July 11, 2016 10:15 PSP Book - 9in x 6in 03-Hamblin-c03

Chapter 3

Lasers, LEDs, and Other Light Sources

James Carroll
THOR Photomedicine Ltd., Water Meadow House, Chesham, HP5 1LF, United Kingdom
james.carroll@thorlaser.com

3.1 Introduction

Most physicians have never heard of low-level light therapy (LLLT)

or the recently accepted term photobiomodulation (PBM), and yet
there are already over 1,000 devices on the world market to choose
from (Alibaba, 2015). Device prices range for $100 to $100,000,
and many claim to have been appointed the European CE Mark, a
few dozen have FDA clearance, some have TGA for Australia, Health
Canada, and JPAL clearance for Japan, but relatively few of those
devices been tested in published clinical trials.
There are single laser beam devices with power outputs ranging
from as little 1 mW to 30 W and also LED clusters comprising a few
red emitters to arrays with hundreds of LEDs, some delivering up to
480 W to treat the whole body.
Wavelengths used are typically in the red or the near-infrared
spectrum, though occasionally blue, green, yellow, or infrared
wavelengths up to 2.9 μm are being used. The light emission is

Handbook of Low-Level Laser Therapy

Edited by Michael R. Hamblin, Marcelo Victor Pires de Sousa, and Tanupriya Agrawal
Copyright c 2017 Pan Stanford Publishing Pte. Ltd.
ISBN 978-981-4669-60-3 (Hardcover), 978-981-4669-61-0 (eBook)
www.panstanford.com
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36 Lasers, LEDs, and Other Light Sources

typically continuous but some beams are pulsed (gated) or “super

pulsed” (see below for more details). The beam intensity (more
correctly called irradiance or power density) ranges significantly
from 1 mW/cm2 to 6 W/cm2 .
Some devices are applied in contact (hand-held or strapped-on),
others project light from a distance. Most devices are designed to be
used in contact and held stationary in one position, some advocate
a non-contact “scanning” or “painting” method, and a few systems
have projected scanning beam; there is even one which has rotating
beams projected from five octopus style arms suspended above the
patient. See Fig. 3.1 for images of these devices.

Figure 3.1 Low-level light therapy/photobiomodulation devices.

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State of the Art 37

Unfortunately many devices fail to deliver the power, penetra-

tion, and clinical benefits claimed by the manufacturers. Indepen-
dent tests of commercial devices in routine clinical use showed that
most commercial LLLT/PBM equipment perform significantly below
the claimed specification. Nussbaum et al. reported that on average
laser systems emitted only 60% of the specified power and LED
systems emitted only 31% of the claimed power (Nussbaum et al.,
1999).
It is conceivable that some skilled LLLT/PBM practitioners could
successfully treat a wide range of clinical conditions with almost
any of these devices, but most users (and many manufacturers
and researchers) will have little idea how well their LLLT/PBM
system is performing because they have inadequate knowledge
about the necessary irradiation parameters, dose, and the number
of treatments or intervals between treatments required.

3.2 State of the Art

The state of the art for LLLT/PBM devices is analogous to that of the
personal computer (PC) market in the early 1980s. There were many
hardware manufactures with different operating systems, each
trying to be unique and own a segment of the market. This approach
had the undesired effect of causing early adopter hesitation because
nobody wanted to buy the wrong PC and therefore waited until a
market winner emerged. Most of the early PC businesses failed and
only a few remained to catch the wave that followed.
The breakthrough in PC adoption was the invention of the
spreadsheet that inspired businesses to purchase multiple ma-
chines. Later, home computing became popular, thanks to computer
games and the CD-ROM encyclopaedia. Ultimately, the Internet led
to global mass adoption of PCs for home and business use.
The spreadsheet is often described as the “killer app” that
launched the PC revolution in the 80s, but at the time of writing
LLLT/PBM has yet to have its first significant commercial success
with such a blockbuster medical application. Once LLLT/PBM
establishes itself at standard care for its first medical indication then
other applications should follow more easily.
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38 Lasers, LEDs, and Other Light Sources

Older readers may recall famous computing brand names that

have now vanished such as Wang, DEC, Honeywell Bull, Commodore
64, Atari, Apricot, BBC Micro, and the ZX spectrum; LLLT/PBM has
to go through the same growing pains as other technologies before
it is accepted as a mainstream therapy and it seems likely that many
manufacturers will fail in the process. Markets like winners and
customers like industry standards, and where these will come from
is anybody’s guess at this point.

3.3 History of Devices

Prof. Andre Mester is often credited as the father of LLLT/PBM when

he reported stimulation of hair regrowth in mice and described the
effect as “laser biostimulation” although other forms of light healing
preceded laser biostimulation.
The first light source used as a phototherapy is of course the
sun. It was used by many ancient civilisations (Greeks, Romans,
Egyptians, and Incas) and subsequently came to be known as
heliotherapy (Honigsmann, 2013).
• 1774: Faure et al. reported on the cure of leg ulcers by
exposure to the sun’s rays.
• 1897: Fubini reported that red light filtered from the sun
altered cellular respiration.
• 1903: Finsen was awarded a Nobel Prize for his light
therapy for lupus vulgaris.
• 1927: Losev created the first light-emitting diode (LED).
• 1960: Maiman made and patented the first working laser
(Ruby).
• 1962: First laser diode was developed.
• 1962: First commercial LED was developed.
• 1967: Mester reported that red laser stimulates hair
regrowth in mice.

3.4 Nomenclature

There have been at least 70 different terms used to describe

LLLT/PBM and the various devices used. Here are just a few:
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Laser Classification 39

• Low-level laser therapy (LLLT) • Laser therapy

• Low-level light therapy (lllt) • Monochromatic light therapy
• Photobiomodulation (PBM) • Monochromatic phototherapy
• Class IV laser therapy • Near-infrared light therapy
• Cold laser therapy • LED phototherapy
• Laser biostimulation • MID laser therapy
• Low-energy laser therapy • Photobioactivation
• Low-intensity laser therapy • Photobiostimulation
• Low-energy photon therapy • Photoradiation
• Low-power laser therapy • Photostimulation
• Low-reactive level laser • Physiotherapy laser
• He–Ne laser therapy • Soft laser

The diverse nomenclature makes finding LLLT/PBM for research

difficult. This has been worsened by manufacturers who have
invented proprietary names, perhaps in the hope of achieving some
exclusive commercial benefit.

3.5 Laser Classification

Lasers are categorized according to their risk of injury, damage, or

fire. They are defined in the internationally agreed standard IEC
60825 or the USA standard ANSI Z136. The document is complex
and intended for physicists; however, the following brief summary
will give the reader a simplified summary of the five classes that all
lasers fall into.
Class 1/1M Considered non-hazardous. Hazard increases if viewed
with optical aids, including magnifiers, binoculars, or
telescopes. Used in laser printers and DVD players.
Class 2/2M Hazardous when viewed directly for long periods of
time and if viewed with optical aids. Used in bar code
readers.
Class 3R Can be momentarily hazardous when directly viewed
or when staring directly at the beam. Risk of injury
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40 Lasers, LEDs, and Other Light Sources

increases when viewed with optical aids. Used in laser

pointers.

Class 3B Lasers emitting 5 mW to 500 mW. Hazardous under

direct beam viewing conditions, but are normally safe
when viewing diffuse reflections. Commonly used for
LLLT/PBM.

Class 4 Lasers emitting typically over 500 mW. Can cut, burn,
and start fires. Used in surgery.

There are LLLT devices branded as Class IV, which do not cut because
the beam is defocused and the intensity is much lower just like the
3B lasers.
Readers from the United States may be more familiar with the old
international system using roman numerals such as “Class IIIA” and
“Class IV” as these are still in use by some institutions. It is expected
that the revised international standard will be adopted at point.

3.6 Light Sources and Properties

There are a wide variety of LLLT/PBM products on the market and

much marketing hype/misinformation on the internet is making
it very difficult for the doctor, therapist, or patient to make an
adequately informed choice about which product is best for their
needs. This section will discuss the common questions asked by the
newcomer about various types of devices, their properties, and their
relative merits.

3.6.1 Different Properties of Laser and LED Light Sources

3.6.1.1 Wavelength
The wavelengths most commonly used are red or near infrared
(600 nm to 1000 nm), though occasionally blue, green, yellow, or
infrared wavelengths up to 2.9 μm have also been used. An optimal
effective wavelength has not been established so far, and it seems
unlikely that there would be just one; there will be ranges of effective
wavelengths, each range having different advantages.
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Light Sources and Properties 41

(1) Optimal Effect: Light comprises packets of energy called pho-

tons. These photons have an energy expressed in electron volts
(eV). The energy of each photon depends on its wavelength.
For example, red photon has ∼2 eV energy while a blue
photon has ∼3 eV. Photons have to be absorbed to have an
effect. They are absorbed in the region of a molecule called
a chromophore. Different chromophores will absorb different
wavelength photons. When the energy of a photon is close to
the energy difference between two different molecular orbitals
(electrons) of an atom, then it is absorbed and causes a
conformation change (a momentary distortion) that may lead
to a chemical change. If say cytochrome c oxidase (Cox) is our
target molecule, and if say increased ATP production is our
criteria for optimal effect, then the wavelength that achieves this
with the least amount of energy may be considered “optimal”.
But what about release of nitric oxide or reduced ROS? Is that
occurring at the same wavelength that increases ATP? Which
wavelengths are best absorbed when Cox is oxidised and which
wavelengths are best absorbed when Cox is reduced? There are
many other other light-absorbing elements (e.g., flavins, heme,
myoglobin, or even water), so what are the optimal wavelengths
for each of these? There may not be one wavelength, but
there may be a range or may be several ranges, one for each
chromophore. Despite hundreds of laboratory studies with red
and near-infrared light (600 nm to 1000 nm) there appears
to be no consistently clear optimal wavelength for effect. By
one mechanistic route or the other, they produce good effects
although some penetrate better than others.
(2) Optimal Penetration: Best penetration is achieved when light
is not being absorbed or scattered. This presents a problem
because absorption is essential for physiological effect. If
nothing absorbs the light then no photon energy is transferred
and nothing changes. Visible light does not penetrate well in the
UV, blue, green, or yellow range (400 nm to 600 nm), but far-red
light (particularly 660 nm and 670 nm) penetrates better and
has shown good results in laboratory experiments and clinical
studies. Optimal penetration in humans seems to be in the range
690 nm and 860 nm and there is another peak in the range
1060 nm and 1100 nm (Smith, 1991).
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42 Lasers, LEDs, and Other Light Sources

3.6.1.2 Coherence
Coherent laser light is not a requirement for successful LLLT/PBM.
There are hundreds of studies using non-coherent light to good
effect (mostly LEDs). It is not true that coherence is lost in tissue;
it has been amply demonstrated that laser speckles remain in
deep tissue and this fact is an indication that coherence remains
(Hode et al., 2011). It is widely accepted that the coherent
properties of laser light are unimportant when treating superficial
tissues; however, it has been hypothesized that some additional
(therapeutic) effects from coherent and polarized radiation can
occur when treating deep anatomical targets (i.e., for low-back pain)
but has not yet been adequately explored in clinical trials. A few
attempts have been made to compare coherent laser light vs. non-
coherent LED light; some have shown coherent laser light to be
superior while others have shown non-coherent LED light to be
superior, but as not all parameters were identical the results are
unreliable.

3.6.1.3 Power
LLLT/PBM systems range from single laser beams of 1 mW to as
much as 95 W and LED arrays of up to 480 W that can treat an entire
body. What is important is that the light reaches the target (injury),
that the whole pathology is treated, and that the density of light at
the target is sufficient. It is argued (by sales and marketing people)
that more power means the required “dose” is achieved in less time,
and mathematically that is true; however, it has been shown many
times that there is a “dose-rate effect” and if the dose is delivered
too quickly the beneficial effects are diminished. This is because
the intensity (irradiance/power density) is too high. See section on
irradiance.

3.6.1.4 Irradiance
Also known as “power density”, “intensity”, or “fluence rate”,
irradiance is calculated as follows: power (W) ÷ beam area (cm2 )
at the tissue surface (not the size of the aperture).
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Light Sources and Properties 43

(1) Lasers are usually more powerful than LEDs (though not
always) and laser beams usually cover a smaller area than
LEDs (though not always). The combination of more power and
a smaller beam area means that most clinical laser systems
usually deliver a higher irradiance than LEDs. The advantage
of high irradiance is deeper penetration and faster analgesic
effects, the risk is that they are more likely to overtreat
superficial wounds and tendinopathies.
(2) As stated previously, a common piece of misinformation is that
more powerful/higher irradiance lasers achieve the desired
dose more quickly. It has been shown many times that there is
a “dose-rate effect” and if the dose is delivered too quickly the
beneficial effects are diminished. The ideal “effective” irradiance
has not been unequivocally established but it is probably in
the range of 0.15 mW/cm2 to 10 mW/cm2 at the tissue target
(Baratto et al., 2011, Tedford et al., 2015). The ideal “effective”
irradiance may vary with wavelength, tissue type, redox state,
and the desired effect.
(3) Measuring a laser beam area is not easy considering the follow-
ing factors: Diode laser beams are not homogeneous; the beam
is usually Gaussian (more intense in the middle and gets weaker
towards the periphery of the beam unless they have lenses
or diffusers to make the beam “flat top”), diode laser beams
are likely to be elliptical (unless they are emitted from a fiber
optic cable). So this is not a simple measurement/calculation. To
accurately establish the area, a beam profiler must be employed.
This is an expensive instrument that requires knowledge of
engineering or physics. Very few researchers or manufacturers
have their beams properly characterized with a beam profiler
so published reports on irradiance are unreliable (Jenkins and
Carroll, 2011). It is suggested that the 1/e2 point is used that is
the area in which 86.5% of the beam power occurs.

3.6.1.5 Penetration
The “effective” penetration of light (within a certain range of
wavelengths and power) is 3.5 cm or 4 cm (Hudson et al., 2013,
Tedford et al., 2015), although a firm contact technique with
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44 Lasers, LEDs, and Other Light Sources

8.0

7.0
O.D.

6.0

5.0

4.0

3.0
500 600 700 800 900 1000 1100
WAVELENGTH (NM)

Figure 3.2 The absorption spectrum of a human hand. The spectrum was
recorded with a very sensitive spectrophotometer with the palm of the hand
in the close juxtaposition to the photocathode (Unpublished data of Karl H.
Norris).

some pressure on soft tissues can improve this, possibly to 5 cm.

Wavelengths in the range 690 nm to 860 nm penetrate best and
another peak exists around 1064 nm (Smith, 1991) (Fig. 3.2).

3.6.1.6 Pulses
There is some evidence that pulsed beams can have effects that
are different from a continuous beams (Hashmi et al., 2010). Pulses
have five parameters: (i) peak power expressed in Watts (W), (ii)
pulse frequency (also known as pulse-repetition rate) meaning the
number of pulses per second expressed in Hertz (Hz), (iii) pulse
width meaning how long the pulse is on and is expressed in seconds
(s), (iv) duty cycle which is the ratio between the time the pulse is on
and off and is expressed as a percentage (%) or a ratio (e.g., 50:50),
and (v) the average power calculated as follows:
Peak power (W)·Pulse width (s)·Pulse frequency = Average power
There are two type of pulsing, modulated (sometime called gated
or chopped) and high-peak pulsed beams (sometimes called super
pulsed). See Fig. 3.3.
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Light Sources and Properties 45

10 Watt 10 Watts

1 Watt 1 Watt

0.5 Watt 0.5 Watt

1 1
Continuous Wave Second Chopped Second

10 Watt

1 Watt

0.5 Watt

“Super” pulsed 1
Second

Figure 3.3 Average power of continuous, modulated, and high-peak pulsed

beams.

(1) Modulated: A modulated pulse is a continuous beam that is

switched on and off. The rate at which they are switched on and
off is expressed in Hertz (Hz), which is the number of pulses per
second (sometimes called the pulse repetition rate). Ten pulses
a second would be 10 Hz. Often the ratio of time on/off is 50:50
(50% on and 50% off); therefore, a 200 mW continuous laser
that is modulated with a 50:50 duty cycle would lose half of
its power because it spends half the time off. Thus, we would
say the peak power is 200 mW but the average power is 100
mW. Sometimes the duty cycle may not be symmetrical, for
example, 90:10, that is, 90% on 10% off. Therefore, a 100 mW
continuous laser that is modulated 90:10 would have a peak
power of 200 mW but the average power would be 180 mW.
However, there are products on the market that increase the
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46 Lasers, LEDs, and Other Light Sources

peak power to compensate for duty cycle by increasing the

peak power so that 200 mW continuous is also 200 mW when
modulated. Which pulse frequencies and duty cycles work best
have not yet been adequately established.
(2) Super Pulsed: Super pulsed is a marketing term used to
distinguish high- peak pulsed lasers from modulated continuous
wave. This is common with lasers with a wavelength of 904 nm
or 905 nm. The peak power of each pulse of these devices is
typically in the range of 10 W to 200 W, the pulse width is
typically ∼200 ns, but the average power ranges from 10 mW
to 100 mW, which is relatively low when compared with other
LLLT/PBM products on the market. Interestingly the World
Association for Laser Therapy (WALT) dose guidelines suggest
lower doses when using 904 nm laser than for continuous wave
lasers. It is unknown if this is due to the wavelength or pulse
regime or just because researchers never tried larger doses.
There are other high-peak power lasers often used by dentists
such as Nd:Yag and Er:YAG (1064 nm and 2940 nm) that have
also shown effects on pain, inflammation, and tissue repair.
These are outside the popular range of wavelengths normally
associated with LLLT/PBM but still seem to have some good
clinical effects.

3.6.1.7 Collimation
Lasers can be collimated with lenses to form a straight, pencil-
shaped beam, though most commercial LLLT/PBM devices do not
collimate the beam. A notable exception are laser systems on
a pedestal that have a mechanical scanning feature that moves
the collimated laser beam to-and-fro across the treatment area.
Collimated lasers are a potentially greater ocular risk than the
more common divergent LLLT/PBM devices because if the beam
enters the eye, the observer may easily focus the beam to the retina
potentially causing an injury. Collimation offers no therapeutic
advantages other than the beam can be projected from a distance
with no diffusion of the beam until beam enters tissue when it
rapidly becomes scattered.
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Light Sources and Properties 47

3.6.1.8 Spectral Width (Monochromaticity)

Monochromatic means one coloured as contrasted with the broad
spectrum of “white” light. Lasers are more monochromatic than
any other light source. Diode lasers are typically ±0.5 nm (FWHM)
whereas LEDs are in the range of ±5 nm to ±20 nm FWHM. FWHM
means full width half maximum, which refers to the spectral width
at half the peak power (see Fig. 3.1). If an optimal wavelength for
LLLT/PBM is ever established, it will also be subject to spectral
width of the target chromaphore. At the time of writing, the
absorbing absorption spectra for Cox (and other possible useful
chromaphores for LLLT/PBM) are in question, but it seems likely
that these are tens of nanometers wide. So if or when an optimal
wavelength is established then an LED should be equally as effective
as a laser (assuming all other parameters are the same such as
irradiance and time).

3.6.1.9 Stability
Historically, gas and solid-state lasers were used but now most
systems employ diode lasers in the red–near-infrared spectrum
(600 nm to 1000 nm).

(1) Stability Wavelength: Diode lasers are rarely the exact wave-
length they claim to be. Firstly the diodes produced by
manufacturers may be ±10 nm of their claimed wavelength and
then the wavelength shifts as the diode warms up (∼0.4 nm per
1◦ C). Diode laser temperature may increase as much as 20◦ C
(depending on heat management built into the product) and the
wavelength would shift by 8 nm in that circumstance. Therefore,
diode lasers are rarely the wavelength they claim to be.
(2) Stability Power: Laser diodes can get hot and power decreases
as the temperature increases, so again, it depends how well the
heat is managed.

3.6.1.10 Polarization
Light is polarized when the waves oscillate in one orientation (one
plane). Some lasers emit polarized light (either linear or circular),
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48 Lasers, LEDs, and Other Light Sources

other light sources can be projected through a filter to polarize them.

Polarized light may have an effect on superficial birefringent protein
structures such as collagen. Several authors have demonstrated
effects on wound healing and burns with broad spectrum polarized
light (Durovic et al., 2008, Oliveira et al., 2011) but the polarization
is soon lost due to scattering and has not shown to be a significant
LLLT/PBM parameter (Demura et al., 2006, Karu et al., 2008).

3.6.1.11 Beam Area

A common misconception is that a beam is the size of the laser
probe or fiber aperture, but it is not. Measuring the area of a
beam requires special instruments; laser beams are often not
round and the distribution of light is not homogeneous. Usually
the distribution is Gaussian therefore it is hard to determine
where the edge of the beam is and to reliably calculate irradiance
unless you have a beam profiler. You may need to rely on the
manufacturer’s documentation though it may not be dependable.
If you are performing research, read Jenkins (2011) on how to
measure beams and report parameters (Jenkins and Carroll, 2011).

3.6.1.12 Scanning vs. Contact Method

Most devices are used in contact with the skin (you cannot treat
through clothing, bandages, dressings, etc.); however, some devices
project beams from a distance. The following considerations should
be kept in mind when using using these devices:

(1) If trying to get light to deep anatomical targets then using a

device with a lens in contact allows for better coupling of the
light source to the tissue and gives the opportunity to compress
soft tissues and get closer to the target.
(2) When using a manual scanning device then the beam area for
calculating dose should be power/the total area treated·time
but because it has been performed manually it is hard to be sure
about the area exactly covered and thus the dose is unclear.
(3) When using a mechanical scanning device with a collimated
beam controlled by electromechanical mirrors to automate the
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References 49

scanning pattern then the beam area for calculating dose should
be power/the total area treated·time.
(4) When using a scanning device with spinning circular stripes
of laser then finding out the beam area for calculating dose
is almost impossible. You would think it would be power/the
total area treated·time but because there is far more light
toward the centre of the beam, a higher dose is delivered than
at the periphery of the beam, which covers a larger area.

3.7 Summary on Light Sources and Properties

The scientists are not aligned on which wavelength, power,

irradiance, or treatment time is best; so how do you find out what
parameters are best? Read the systematic reviews that identify a
dose-rate effect for pathologies you intend to treat and follow those
guidelines. WALT publishes guidelines on musculoskeletal pain so
their web site (www.walt.nu) is worth a visit .
Product advertising is full of exaggeration and misinformation;
so how do you choose a product? Somehow you have to decide who
you trust.
Despite all this uncertainty, the clinical and laboratory data is
convincing, tens of thousands of doctors, dentists, veterinarians,
nurses, various therapists, and technicians across the world are us-
ing LLLT/PBM every day delivering an estimated million treatments
a month.

References

Alibaba.com (2015). Search query LLLT. www.alibaba.com/trade/search?

fsb=&SearchText=LLLT.
Baratto, L., Calza, L., Capra, R., Gallamini, M., Giardino, L., Giuliani, A.,
Lorenzini, L. and Traverso, S. (2011). Ultra-low-level laser therapy.
Lasers Med Sci, 26, 103–112.
Demura, S., Noguchi, T. and Matsuzawa, J. (2006) Comparison in the effect
of linear polarized near-infrared light irradiation and light exercise on
shoulder joint flexibility. Clin J Sport Med, 16, 293–297.
July 11, 2016 10:15 PSP Book - 9in x 6in 03-Hamblin-c03

50 Lasers, LEDs, and Other Light Sources

Durovic, A., Maric, D., Brdareski, Z., Jevtic, M. and Durdevic, S. (2008). The
effects of polarized light therapy in pressure ulcer healing. Vojnosanit
Pregl, 65, 906–912.
Hashmi, J. T., Huang, Y. Y., Sharma, S. K., Kurup, D. B., De Taboada, L., Carroll, J.
D. and Hamblin, M. R. (2010). Effect of pulsing in low-level light therapy.
Lasers Surg Med, 42, 450–466.
Hode, T., Jenkins, P., Jordison, S. and Hode, L. (2011). To what extent is
coherence lost in tissue? Proc. SPIE 7887, Mechanisms for Low-Light
Therapy VI
Honigsmann, H. (2013). History of phototherapy in dermatology. Photochem
Photobiol Sci, 12, 16–21.
Hudson, D. E., Hudson, D. O., Wininger, J. M. and Richardson, B. D. (2013).
Penetration of laser light at 808 and 980 nm in bovine tissue samples.
Photomed Laser Surg, 31, 163–168.
Jenkins, P. A. and Carroll, J. D. (2011). How to report low-level laser
therapy (LLLT)/photomedicine dose and beam parameters in clinical
and laboratory studies. Photomed Laser Surg, 29, 785–787.
Karu, T. I., Pyatibrat, L. V., Moskvin, S. V., Andreev, S. and Letokhov, V. S. (2008)
Elementary processes in cells after light absorption do not depend on
the degree of polarization: implications for the mechanisms of laser
phototherapy. Photomed Laser Surg, 26, 77–82.
Nussbaum, E. L., Yanzuylen, J. and Baxter, G. D. (1999). Specification
of Treatment Dosage in Laser Therapy: Unreliable Equipment and
Radiant Power Determination asGonfounding Factors. Physiotherapy
Canada, 5.
Oliveira, P. C., Pinheiro, A. L., De Castro, I. C., Reis, J. A., Jr., Noia, M. P.,
Gurgel, C., Teixeira Cangussu M. C. and Pedreira Ramalho, L. M. (2011).
Evaluation of the effects of polarized light (lambda400-200 nm) on the
healing of third-degree burns in induced diabetic and nondiabetic rats.
Photomed Laser Surg, 29, 619–25.
Smith, K. (1991). The Photobiological Basis of Low Level Laser Radiation
Therapy. Laser Therapy, 3, 6.
Tedford, C. E., Delapp, S., Jacques, S. and Anders, J. (2015). Quantitative
analysis of transcranial and intraparenchymal light penetration in
human cadaver brain tissue. Lasers Surg Med, 47, 312–322.
July 12, 2016 11:42 PSP Book - 9in x 6in 04-Hamblin-c04

Chapter 4

Is Coherence Important in
Photobiomodulation?

Tomas Hode
Immunophotonics Inc., 4320 Forest Park Ave. #303, St. Louis, MO 63108, USA
tomas@immunophotonics.com

4.1 Introduction

Coherence [1] may be defined as the property of wave-like states

that enables it to exhibit interference (i.e., addition of wave
functions). In the case of laser light, this interference gives rise
to a speckle field consisting of areas/volumes of higher and lower
intensity, which gives the field a distinct “grainy” pattern (Fig. 4.1).
A speckle pattern can be subjective or objective: If the visible laser
light illuminates a wallpaper or some other rough surface, the
viewer will see laser speckles in the image plane [2]. If either the
viewer or the illuminated target moves, the speckles will shift, and
the direction of the perceived movement depends on the viewer’s
eyes. This is called a subjective speckle pattern since the details
of the pattern depend on the parameters of the viewing system
(for example the shape of the eye). An objective speckle pattern is

Handbook of Low-Level Laser Therapy

Edited by Michael R. Hamblin, Marcelo Victor Pires de Sousa, and Tanupriya Agrawal
Copyright c 2017 Pan Stanford Publishing Pte. Ltd.
ISBN 978-981-4669-60-3 (Hardcover), 978-981-4669-61-0 (eBook)
www.panstanford.com
July 12, 2016 11:42 PSP Book - 9in x 6in 04-Hamblin-c04

52 Is Coherence Important in Photobiomodulation?

Figure 4.1 A black and white photograph of a speckle intensity pattern.

Note that the areas of higher intensity represent constructive interference,
and dark areas represent destructive interference. The average speckle size
is of the order of a few micrometers. Photo by L. Hode.

independent of the parameters of the viewing system and can be

imaged by using, for example, a photographic plate or optical sensor
without an objective.
The importance of coherence in photobiomodulation has been
debated over the last 30 years. The question is not purely academic
since the choice of phototherapeutic device may have practical
consequences for the healthcare practitioner, both financially (cost
of instrument) and therapeutically. If lasers are not needed to
acquire an optimized therapeutic effect, light-emitting diodes
(LEDs) or halogen lamps with band-pass filters could be used with a
lower expense for the practitioner.
It is occasionally purported that coherence is lost as soon as laser
light enters tissue and that coherence, therefore, cannot have any
therapeutic significance in photobiomodulation. Alternatively, it is
stated that “light is light” [3, 4]. Hode [5] and Enwemeka [6] have
argued that even if coherence is not lost in tissue, it is biologically
irrelevant. The basic premise of the argument is that the principal
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Is Coherence Lost Upon Entering Tissue? 53

mechanism of photobiomodulation is photon absorption and that it

makes no difference (in the fundamental photobiological process)
whether the light is coherent or not [7]. These arguments have been
supported by in vitro [8, 9] and in vivo studies on wound healing
[10] and by other superficial indications [11] that LED phototherapy
is as effective as laser phototherapy. On the other hand, several
comparative studies have shown that laser phototherapy appears to
be more effective than LED phototherapy in the treatment of both
deeper indications [12, 13], and in early in vitro [14] and wound-
healing studies [15].
While it can be reasonably argued that coherence, per se, is
unlikely to have any biological significance, it is more difficult to
dismiss the possibility that laser speckles may be a contributing
factor in a successful therapeutic outcome.

4.2 Is Coherence Lost Upon Entering Tissue?

It is easy to experimentally demonstrate that coherence is not

immediately lost, only reduced, in scattering media (such as tissue)
[16], which was first demonstrated in 1991 (Fig. 4.2) by L. Hode at
the Ninth Congress of the International Society for Laser Surgery
and Medicine in Los Angeles in response to claims that coherence
is entirely lost upon being diffusely spread in tissue [17–19].
It is also well known that if a volume of tissue is filled with
scattered laser light, a three-dimensional speckle pattern is formed
in the volume of tissue to which the scattered coherent light
reaches. Importantly, the speckle pattern will fluctuate if the target
surface/volume moves (such as surface deformation) or contains
moving particles (such as fluids in tissue) as a function of the
rate of the movement, a phenomenon quite frequently utilized
in various types of analytical and diagnostic applications. For
example, by analyzing the shifting configuration of speckles on a
surface illuminated by a laser, it is possible to describe the surface
deformation in real time [20]. Other practical uses of fluctuating
laser speckles include the monitoring of velocity of flow fields,
such as retinal blood-flow visualization [21], or transmissive laser
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54 Is Coherence Important in Photobiomodulation?

Figure 4.2 Light spots on the rear of a slab of mincemeat through which
light from a He–Ne laser and a flashlight has penetrated. The upper left spot
originates from the He–Ne laser, and the lower right spot originates from
the flashlight. Both spots are red after their passage through the meat, which
shows that red light has the best penetration of the visible light wavelengths.
Infrared radiation penetrates even better. The figure shows that coherence
of the laser light is not lost as the light penetrates the meat. The laser
speckles can be clearly seen, and it is obvious that there is a difference
between laser light and the light from a flashlight.

speckle imaging, which is proposed as a diagnostic for rheumatoid

arthritis [22].
In another application, under the same premise, laser speckle
contrast is utilized to quantify blood perfusion [23]: The lower the
speckle contrast (as captured by a camera with a fixed exposure
time), the higher the blood-flow velocity, essentially capturing
the time-integrated speckle pattern at a given exposure time. In
other words, since the speckle pattern changes during the time of
exposure, a time integration will occur, and the faster the speckle
pattern changes, the lower the speckle contrast will be for the same
exposure time.
An example of such changes in the configuration of a speckle pat-
tern, as a function of time and movement in an illuminated medium,
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Is Coherence Lost Upon Entering Tissue? 55

was demonstrated at the Mechanisms of Low-Light Therapy VI

Conference at SPIE in 2011 [24]. In this presentation, a film was
shown, which was captured after shining laser light through a slab
of ground meat at room temperature (www.laser.nu/speckl.MOV).
As can be seen in the film, the speckle pattern fluctuates over time
(due to slight movements of cells, fluids, etc., in the slab of meat),
and because the movements in the slab are much slower than, for
example, blood perfusion in living tissue, it is easy to capture the
fluctuations with a normal camera. This also explains why a speckle
pattern is not visually perceived when a laser shines through (for
example) a finger: The reason is not that coherence is lost due to
blood flow, but because the speckle pattern fluctuates so rapidly that
our eyes cannot register the fluctuations. The experimental set up
can be easily repeated in the laboratory [24].
Contrary to the conclusion that speckle contrast is reduced in
living tissue [25] (due to fluid movement) to such a degree that
it would render a speckle field biologically irrelevant [26], it is
evident that the rate of speckle fluctuations is influenced by fluid
movement [22], not the intensity gradients within the speckle field
(i.e., contrast).
The question that should be asked, instead, is how fast a
fluctuation of the speckle field is too fast to be biologically relevant?

4.2.1 How Fast is Too Fast?

Li and Champion [27] investigated the vibrational excitation and
relaxation of laser-excited chromophores and found, among other
things, that (a) the average photoexcitation rate under photosta-
tionary conditions (for a 420 nm laser with average power of 15
mW/cm2 , giving a photon flux of ∼1027 photons/s and cm2 , and
a photon absorption cross section of 4 × 1016 cm2 for the heme
chromophore) corresponded to a photon absorption event every
8–9 ps; (b) thermal saturation of both the chromophore and the
protein occurred after ∼100 ps; and (c) the relaxation times for a
chromophore–protein–solvent system were on the order of 10 ns
once the influx of photons stopped. Since the lifetime of an individual
speckle is on the order of a few milliseconds when living tissue is
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56 Is Coherence Important in Photobiomodulation?

illuminated with a laser, the speckle lifetime is clearly longer (orders

of magnitude) than reaction times in photoexcitation events.
Having demonstrated that speckle patterns indeed occur when
living tissue is illuminated with a laser and that the lifetime of
individual speckles is typically much longer than the reaction
times in photoexcitation events, then the question is why would
the presence of laser speckles in tissue have a different or
improved biological (therapeutic) impact than uniformly distributed
(noncoherent) light?

4.3 What Biological Significance Could Speckles Have?

4.3.1 Intensity Thresholds

Several studies have indicated that the effects of phototherapy
depend not only on energy density [5, 28 and references therein],
exposure time [29], and wavelength [28], but also on intensity [28,
30, 31].
The intensity thresholds have been suggested to be on the order
of 5–15 mW/cm2 for monochromatic light sources and the biological
responses either below or above these intensities appear to be
limited. The exact reason why such intensity thresholds exist in
phototherapy is not well investigated. The thresholds can be viewed
as the minimum rate (flux) at which photons need to be absorbed
by the target molecules to reach a certain photobiological effect.
The photon absorption rate depends on photon density (intensity)
and absorption cross section of the target molecule. The photon
absorption cross section, in turn, mainly depends on the wavelength
and polarization of the incident light, and the redox state and, to
some extent, the temperature of the chromophore.
Hode et al. [33] performed a Monte Carlo simulation on the
speckle intensity distribution in a scattering media (such as tissue)
and found that intensities of up to five times the mean can be
expected in tissue (Fig. 4.3). As a consequence, volumes of higher
intensities will occur in the tissue at greater depths in the tissue
than the average intensity otherwise would allow, thus potentially
increasing the effective depth of penetration of a laser as compared
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What Biological Significance Could Speckles Have? 57

Figure 4.3 Simulation of intensity distribution of a speckle field in a highly

scattering media (such as tissue). (a) Speckle pattern with exponential
intensity statistics. (b) Regions with intensity greater than mean. (c)
Regions with intensity greater than 2 × mean. (d) Regions with intensity
greater than 5 × mean.

to a noncoherent light source with otherwise the same parameters

(wavelength, power, etc.).
It is also worth noting that volumes of intensities lower than
mean will occur, which could be equally important for a successful
therapeutic outcome. In cases where the selected mean intensity is
too high, for example during treatments of superficial indications
with a device that is producing light of relatively high output power,
the volumes of lower intensity within the speckle field (if the
said device is a laser) might still be appropriate for the desired
photobiological effect, which would not be the case if a noncoherent
light source is used.
While not directly related to coherence, another factor related
to intensity is worth mentioning, which is typical for lasers (rather
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58 Is Coherence Important in Photobiomodulation?

Figure 4.4 Diagram of a superpulsed GaAs laser pulse profile. Typical pulse
widths range between 100 ns and 200 ns, with peak powers up to 100
W. The average output power is significantly lower and generally range
between 5 mW and 100 mW.

than LEDs and many other noncoherent sources), namely the ability
to produce pulses with very high peak power. By the so-called
superpulsing, it is possible to simultaneously have high momentary
intensity and low average power, thus giving low thermal influence.
A typical superpulsed laser is the GaAs-type laser (904 nm) with
pulse lengths around 100–200 ns and peak powers up to 25–
100 W (Fig. 4.4). As a consequence of the high peak powers, the
intensity in the tissue increases with an additional 1–2 orders of
magnitude compared to a continuous-wave light source (without
causing thermal damage), thus potentially increasing the effective
depth of penetration.

4.3.2 Polarization
It is not only the intensity that differs between coherent and
noncoherent light that enters tissue. The laser speckles so formed
also differ from noncoherent light of the same intensity; it is locally
polarized or, at least, partially polarized. Speckles are a result of
interference, and interference only occurs if the interfering light
is co-polarized and has some degree of coherence. If the E -fields
contributing to the intensity at a single point are not aligned, there
will be no constructive or destructive interference, i.e., no speckle.
The laser speckles are also formed if the penetrating light from the
illuminating laser is not polarized.
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What Biological Significance Could Speckles Have? 59

1 1
50 actual
0.8 0.9 theory
100 0.6 0.8
150
0.4 0.7
200
0.2

PDF, p(d)
0.6
250 0 0.5
300
-0.2 0.4
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-0.4 0.3
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500
-1 0
100 200 300 400 500 -1 -0.8 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8 1
degree of local polarization, d

Figure 4.5 Simulation of degree of polarization in a speckle field. (a)

Complex spatial distribution of the degree of polarization somewhat similar
to a speckle pattern. (b) Distribution of the theoretical versus actual degree
of polarization.

The simulations by Hode et al. [32] explored the statistics

of the superposition of two orthogonally polarized statistically
independent speckle patterns, specifically with regard to the
degree of polarization. The simulation showed that the degree of
polarization has a complex spatial distribution that looks somewhat
like a speckle pattern, and the first-order statistics are uniform.
The results of the simulations suggest that even though an incident
polarized beam becomes depolarized by the time it reaches a few
transport mean-free paths as it penetrates the tissue, there still
remains a complex spatial distribution of polarized light deep within
the tissue (Fig. 4.5).
Interestingly, there may be a polarization dependence for the
chromophore photon absorption cross section. Tolkachev [33]
compared chromophore excitation in nonpolarized versus polarized
light and found that the maximum excitation occurred when the
polarization vector was parallel to the direction of the dipole
moment of the chromophore (or close to it). The author concluded
that on an orientationally stable, fixed individual chromophore, the
excitation effect was two times larger than the effect of nonpolarized
light. In other words, the probability for photon absorption (cross
section) is influenced by polarization and may increase if the
direction of the polarized light is aligned with the dipole moment
of the molecule.
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60 Is Coherence Important in Photobiomodulation?

Combining the effects of the intensity distribution and polar-

ization patterns in tissue, it can be suggested that the rate of
photon absorption may increase with up to an order of magnitude
compared to noncoherent light sources. In scattering media such as
tissue, these effects could have a significant impact on the effective
penetration depth, i.e., the maximum depth at which direct and
desired photobiological effects take place. In a clinical setting, this
increased effective depth of penetration can make the difference
between having to rely on systemic effects versus getting direct
effects in the target tissue.

4.3.3 Dynamic Environment

As previously discussed, a speckle field in tissue will change
continuously due to, for example, blood flow. In an arbitrary point,
the optical field will vary in intensity, temperature, degree of
polarization, and direction of the Stokes vector in a random way,
which may have clinical significance. For example, Horvath and
Donko [34] measured the intensity differences in a speckle field and
concluded that although the actual temperature differences were
low (on the order of micro-degrees), the thermal micro-gradients
were very steep, which had the effect of increasing the rate of
diffusion in accordance with Fick’s equations. Furthermore, Rubinov
[35] showed in a series of experiments that illumination of biological
tissue by coherent laser light led to intensity gradients within the
speckle field:

“[Laser light] unavoidably leads to strong intensity gradients of the

radiation in the tissue due to speckle formation. This causes the
appearance of inter- and intracellular gradient forces whose action
may significantly influence the paths and speeds of biological
processes. In contrast to the photochemical action of light, which is
accompanied by absorption of quanta and has a specific character
(i.e. is characterized by a specific spectrum of action), the action
of the gradient field is of non-resonant type. It is not accompanied
by photon absorption and has a universal character - it depends
weakly on the radiation wavelength, but requires a high degree of
coherence.”
July 12, 2016 11:42 PSP Book - 9in x 6in 04-Hamblin-c04

Summary 61

These effects will not take place with illumination by noncoher-

ent monochromatic light of the same intensity.

4.4 Summary

Coherence is a unique property of laser light, and it gives rise to a

speckle field when an object is illuminated. It can be demonstrated
that coherence is not lost in scattering media, such as tissue, which
means that a three-dimensional speckle field will occur inside the
tissue when illuminated by a laser. Furthermore, studies indicate
that intensities of up to five times the mean intensity may occur
in a speckle field. Together with the fact that polarization patterns
occur in speckle fields (independent of whether the light was
originally polarized), and that the photon absorption cross section
is polarization dependent and potentially up to two times more
effective if the polarization vector is aligned with the dipole moment
of the target photoreceptor molecule, the rate of photon absorption
may locally be up to an order of magnitude higher if a coherent light
source is used instead of a noncoherent light source.
Because of these intensity and polarization distributions, the
speckle pattern may have a significant impact on the effective
penetration depth of in vivo laser phototherapy (Fig. 4.6) since it may
become easier to reach the required intensity thresholds in deeper
tissue.
Also if a superpulsed laser is used (e.g., GaAs laser, with peak
powers between 5 W and 100 W), intensities of additional 1–2
orders of magnitude may occur in the tissue. These observations
may be important for the clinical applications of phototherapy.
In deeper tissue, the intensity is lost to such an extent (as the
light passes the tissue above) that it falls under any necessary
intensity thresholds [28, 30, 31]. With coherent light sources, and
superpulsed lasers in particular, the photon absorption rate may be
increased by up to 2–3 orders of magnitude, which translated into
a clinical setting could result in an increased effective penetration
compared to noncoherent light sources.
Another aspect that may be of importance is the dynamic
environment created due to the speckle field, including intensity
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62 Is Coherence Important in Photobiomodulation?

Figure 4.6 Illustration of the potential difference in effective penetration

depth between coherent and noncoherent light sources. For any given
power density threshold (for example 5 mW/cm2 ), there will be a depth at
which the average intensity will be lower than the required power density
for phototherapeutic effects to take place. However, in the case of coherent
light (where a speckle field is present), there will be individual speckles
with intensities of up to 5× the average intensity, which means that tissue
situated deeper than the average power density threshold cutoff will be
exposed to power densities above the phototherapeutic threshold. In other
words, the effective penetration depth of coherent light sources (i.e., lasers)
may be greater than that for noncoherent light sources (e.g., LEDs).

gradients that could increase rates of diffusion over cell membranes.

Furthermore, formation of volumes with lower-than-mean inten-
sities may be important in cases when the average intensity is
too high for the optimal intensity window, for example during the
treatment of a superficial indication with a device producing high
output powers.
These observations could also explain why LED phototherapy
in some cases appears to be comparable to laser phototherapy for
superficial indications such as wound healing [10], but often not as
effective as laser phototherapy in cases of deep-seated indications
[12, 13]. In LED phototherapy of superficial tissue (e.g., wounds,
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References 63

in vitro, etc.), the intensity loss is not so much of a factor, and as

a consequence it becomes easier to reach any required intensity
thresholds than it is to reach the given thresholds in deeper tissue.
However, even for superficial indications, it may become more
important for the practitioner to select parameters that are optimal
for that indication at the target depth if a noncoherent light source
is used. If a coherent light source is used, an environment with
both higher and lower intensities than mean will be produced
in the tissue due to the speckle field, thus potentially making it
easier to reach the required intensity thresholds for the desired
photobiological effects at different output powers of the device.
Whether these effects truly are clinically relevant will require
further research, but as discussed earlier, it is very clear that
speckles, as a result of coherence, cannot readily be dismissed as a
potentially important factor in the overall efficacy of photobiomod-
ulation, particularly in a clinical setting.

References

1. Wolf, E. (2007). Introduction to the Theory of Coherence and Polarization

of Light (Cambridge University Press, UK).
2. Goodman, J. W. (2007). Speckle Phenomenon in Optics: Theory and
Applications (Roberts & Company Publishers, Colorado, USA).
3. Smith, K. C. (1991). The photobiological basis of low level laser radiation
therapy, Laser Therapy, 3(1), pp. 19–24.
4. Smith, K. C. (2005). Laser (and LED) therapy is phototherapy,
Photomedicine and Laser Surgery, 23(1), pp. 78–80.
5. Hode, L. (2005). The importance of coherence, Photomedicine and Laser
Surgery, 23(4), 431–234.
6. Enwemeka, C. S. (2005). Light is light, Photomedicine and Laser Surgery,
23(2), pp. 159–160.
7. Nussbaum, E., Baxter, G., and Lilge, L. (2003). A review of laser
technology and light–tissue interactions as a background to therapeutic
applications of low intensity lasers and other light sources, Physical
Therapy Reviews, 8(1), pp. 31–44.
8. Bertoloni, G., Sacchetto, R., Baroa, E., Ceccherellib, F., and Jori, G. (1993).
Biochemical and morphological changes in Escherichia coli irradiated by
July 12, 2016 11:42 PSP Book - 9in x 6in 04-Hamblin-c04

64 Is Coherence Important in Photobiomodulation?

coherent and non-coherent 632.8 nm light, Journal of Photochemistry

and Photobiology B, 18, pp. 191–196.
9. Vinck, E. M., Cagnie, B. J., Cornelissen, M. J., Declercq, H. A., and Cambier,
D. C. (2003). Increased fibroblast proliferation induced by light emitting
diode and low power laser irradiation, Lasers in Medical Science, 18(3),
pp. 95–99.
10. Whelan, H. T., Smits, R. L., Buchmann, E. V., Whelan, N. T., Turner, S. G.,
Margolis, D. A., Cevenini, V., Stinson, H., Ignatius, R., Martin, T., Cwiklinski,
J., Philippi, A. F., Graf, W. R., Hodgson, B., Gould, L., Kane, M., Chen, G., and
Caviness, J. (2001). Effect of light-emitting diode (LED) irradiation on
wound healing, Journal of Clinical Laser Medicine and Surgery, 19, pp.
305–314.
11. Whelan, H. T., Connelly, J. F., Hodgson, B. D., Barbeau, L., Post, A. C.,
Bullard, G., Buchmann, E. V., Kane, M., Whelan, N. T., Warwick, A., and
Margolis, D. (2002). NASA light-emitting diodes for the prevention of
oral mucositis in pediatric bone marrow transplant patients, Journal of
Clinical Laser Medicine and Surgery, 20(6), pp. 319–324.
12. Laakso, E. L., Cramond, T., Richardson, C., and Galligan, J. P. (1994).
Plasma ACTH and beta-endorphin levels in response to low level laser
therapy for myofascial trigger points, Laser Therapy, 3(6), pp. 133–
142.
13. Simunovic, Z., and Trobonjaca, T. (2001). Comparison between low level
laser therapy and visible incoherent polarised light in the treatment of
lateral epicondylitis — tennis elbow [Supplement], Lasers in Surgery
and Medicine, 13, no. 9.
14. Lederer, H., Stünkel, K., Denk, R., and Waidelich, W. (1982). Influence
of light on human immunocompetent cells in vitro, Laser 81 -
Optoelectronics in Medicine, Proceedings 5th International Congress, pp.
170–184.
15. Mester, E., Nagylucskay, S., Waidelich, W., Tisza, S., Greguss, P., Haina,
D., and Mester, A. (1978). Auswirkungen direkter Laserbestrahlung auf
meschliche Lymphocyten, Archives of Dermatological Research, 263, pp.
241–245.
16. Hode, L., and Tuner, J. (2014). Laser Phototherapy: Clinical Practice and
Scientific Background (Prima Books AB, Grangesberg, Sweden).
17. Greguss, P. (1985). Interaction of optical radiation with living matter,
Optics and Laser Technology, 3, pp. 151–158.
18. Greguss, P. (1990). Biostimulation of tissue by laser radiation, Proceed-
ings of SPIE 1353, pp. 79–91.
July 12, 2016 11:42 PSP Book - 9in x 6in 04-Hamblin-c04

References 65

19. King, P. (1989). Low level laser therapy: A review, Lasers in Medical
Science, 4(3), pp. 141–150.
20. Hode, L., and Biedermann, K. (1972). Observation of surface deforma-
tion in real time using laser speckles, Proceedings of the Conference in
Physics, Lund, Sweden, June 12–14.
21. Briers, J. D., and Fercher, A. F. (1982). Retinal blood-flow visualization by
means of single-exposure speckle photography, Investigative Ophthal-
mology and Visual Science, 22, pp. 255–259.
22. Dunn, J., Forrester, K., Martin, L., Tulip, J., and Bray, R. (2011). A
transmissive laser speckle imaging technique for measuring deep tissue
blood flow: An example application in finger joints, Lasers in Surgery and
Medicine, 43, pp. 21–28.
23. Yuan, S., Devor, A., Boas, D., and Dunn, A. (2005). Determination of
optimal exposure time for imaging of blood flow changes with laser
speckle contrast imaging, Applied Optics, 44(10), pp. 1823–1830.
24. Hode, T., Jenkins, P., Jordison, S., and Hode, L. (2011). To what extent is
coherence lost in tissue? Proceedings of SPIE 7887, pp. 788703.
25. Fixler, D., Duadi, H., Ankri, R., and Zalevsky, Z. (2011). Determination of
coherence length in biological tissues, Lasers in Surgery and Medicine,
43, pp. 339–343.
26. Zalevsky, Z., and Belkin, M. (2011). Coherence and speckle in pho-
tomedicine and photobiology, Photomedicine and Laser Surgery, 29(10),
pp. 655–656.
27. Li, P., and Champion, P. M. (1994). Investigations of the thermal
response of laser-excited biomolecules, Biophysical Journal, 66, pp. 430–
436.
28. Karu, T. (2007). Ten Lectures on Basic Science of Laser Phototherapy
(Prima Books AB, Grangesberg, Sweden).
29. van Breugel, H. H. F. I., and Bär, P. R. D. (1992). Power density and
exposure time of HeNe laser irradiation are more important than total
energy dose in photo-biomodulation of human fibroblasts in vitro,
Lasers in Surgery and Medicine, 12, pp. 528–537.
30. Lubart, R., Friedmann, H., Peled, I., and Grossman, N. (1993). Light effect
on fibroblast proliferation, Laser Therapy, 5, pp. 55–57.
31. Sommer, A. P., Pinheiro, A. L., Mester, A. R., Franke, R. P., and Whelan,
H. T. (2001). Biostimulatory windows in low-intensity laser activation:
Lasers, scanners, and NASA’s light-emitting diode array system, Journal
of Clinical Laser Medicine and Surgery, 19(1), pp. 29–33.
July 12, 2016 11:42 PSP Book - 9in x 6in 04-Hamblin-c04

66 Is Coherence Important in Photobiomodulation?

32. Hode, T., Duncan, D., Kirkpatrick, S., Jenkins, P., and Hode, L. (2009). The
importance of coherence in phototherapy, Proceedings of SPIE 7165, pp.
716507.
33. Tolkachev, V. A. (2004). Role of light polarization in the optothermal
effect, Journal of Applied Spectroscopy, 71(1), pp. 139–142.
34. Horvath, Z. G., and Donko, Z. (1992). Possible ab-initio explanation
of laser “biostimulation” effects, Proceedings of 3rd World Congress—
International Society for Low Power Laser Application in Medicine, pp.
57–60.
35. Rubinov, A. N. (2003). Physical grounds for biological effect of laser
radiation, Journal of Physics D: Applied Physics, 36, pp. 2317–2330.
July 14, 2016 17:46 PSP Book - 9in x 6in 05-Hamblin-c05

Chapter 5

Tissue Optics

Bryan James Stephensa and Linda Ramball Jonesb

a SOUNDTM, 265 King Arthur Circle, Franklin, TN 37067, USA
b Department of Physics, College of Charleston, 66 George Street, Charleston,

SC 29424, USA
b.stephens.wfu@gmail.com

Now that you have been exposed to the relevant parameters

in optical interactions with tissue, we can identify the principal
absorbers and scatterers as well as deal with some of the methods
of their optical characterization. With these data in hand, we need
some methods to quantify the dose distribution within the tissues
we wish to treat. In doing so, we will be on the path to uncovering
some of the fundamental mechanisms of laser (light) therapy, which
will open the door to subsequent clinical discussions.

5.1 Optical Properties of Tissues

Biological tissues are completely characterized for a given wave-

length of light with three microscopic optical parameters: absorp-
tion coefficient, scattering coefficient, and scattering anisotropy (μa ,
μs , and g) [2]. Absorption and scattering coefficients represent
the probability of either absorption or scattering occurring per

Handbook of Low-Level Laser Therapy

Edited by Michael R. Hamblin, Marcelo Victor Pires de Sousa, and Tanupriya Agrawal
Copyright c 2017 Pan Stanford Publishing Pte. Ltd.
ISBN 978-981-4669-60-3 (Hardcover), 978-981-4669-61-0 (eBook)
www.panstanford.com
July 14, 2016 17:46 PSP Book - 9in x 6in 05-Hamblin-c05

68 Tissue Optics

pathlength traveled by the light. The anisotropy factor is defined

as the average cosine of the scattering angle and so takes on
a value from −1 (complete backward scattering) to 1 (complete
forward scattering), where a value of 0 would indicate isotropic
scattering, on average. As light scatters through the tissue, the actual
pathlength is virtually always larger (and often much larger) than
the physical thickness of the tissue. The further from 1 (or −1) the
anisotropy factor for a given tissue (or set of tissues), the longer
the pathlength. In fact, for thicker or multilayer optical models,
the microscopic input parameters for specific tissues are often
replaced with more macroscopic characteristics such as attenuation
coefficients, refractive index, and thickness for each layer. These
macroscopic quantities are often useful when quicker calculations
(lower processing time) are needed or when dealing with tissue that
involve “weaker” scattering.

5.1.1 Tissue with Weak Scattering

A tissue with slight refractive-index changes, composed of very small
particles, scatters light “weakly.” Recall that the refractive index
represents how much slower light travels in the medium compared
to vacuum. Examples of weakly scattering tissues are cornea and
lens of the eye with μs ∼9.0 cm−1 and 1.0 cm−1 , respectively, at 514
nm [18]. The normal lens is composed of scattering particles with
diameter 20–200 nm and index of refraction 1.38. The human lens is
highly transparent to visible and near-infrared (NIR) light with some
absorption due to water above 900 nm. Scattering centers in the
cataract lens have diameters from 200 to 2000 nm with fluctuations
in the index of refraction between 1.40 and 1.48, greatly increasing
light scattering and opacity [20].
Here is a quick side note on “weakness.” Scattering, in general,
is highly dependent on the incident angle. Thus, while some tissues
are relatively weak scatterers of normally (perpendicular) incident
light, at strong angles, the effects may not be negligible.

5.1.2 Tissue with Strong (Multiple) Scattering

Epidermis and dermis tissues are composed of particles ranging
from 10 nm to 10 μm in size, resulting in multiple scattering of
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Optical Properties of Tissues 69

Table 5.1 Experimentally determined optical properties of

some biological tissues

Wavelength Scattering Coefficient Anisotropy

Tissue Type (nm) (cm−1 ) Factor g Source

Dermis 633 0.79 [20]

694 200 [1]
755 175 [1]
1064 130 [1]
Epidermis 633 0.82 [20]
694 400 [1]
755 350 [1]
1064 260 [1]
Fat 600 6.54* [22]
1087 3.70* [22]

*Reduced scattering coefficient (cm−1 )

visible and NIR light. Examples of “strongly” scattering tissues are

dermis, epidermis, bone, smooth muscle, and adipose tissue. The
optical properties of some of these are given in Table 5.1.
To clarify, strong scattering does not necessarily mean poor
penetration. Because there are often multiple scattering events
along the path of an incident photon of light, the result could be a
forward scattering event (leading to relatively deeper penetration),
even if the initial scattering direction was lateral. Plus, to get the
full picture of penetration, we cannot forget about absorption. For
example, Mie scattering (i.e., scattering of light by a collection of
spheres) in mammalian fat has been assumed, which would lead to
forward directed scattering and deep penetration in the NIR (optical
penetration depth of 19.6 mm at 760 nm). However, fat tissue
has a significant absorption peak at 930 nm, limiting the effective
penetration depth to 7.6 mm.
We can use strong scattering to our advantage in a diagnostic
sense. Scattering-dominated skin tissue yields useful reflectance
spectra that probe deep into the adipose layers (depending on
the detector geometry). NIR reflectance spectra are useful for the
characterization of blood and quantifying fat and water content in a
variety of tissues. More to follow on this.
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70 Tissue Optics

5.1.3 Full Picture of Penetration

What we “mean” by penetration, then, is how these three micro-
scopic quantities (the probability of absorption versus scattering
and the direction of scattering) average out to some “attenuation”
coefficient. But because of the probabilistic nature of light’s
interaction with biological tissue, we have to talk about penetration
in terms of probabilities as well. Some of the light from the bulb
above your head travels all the way through your body. If you have
a sensitive-enough detector and enough time, you can measure
the buildup of light under the soles of your feet. So to say that
any particular wavelength of light penetrates X cm deep is to say
nothing at all, since some amount (however miniscule) of virtually
all wavelengths of light can penetrate to virtually any depth of your
body.
When we say “penetration depth,” we mean the depth at which
100% of incident beam decreases to Y %, where Y is usually the
natural logarithm base e. So an effective penetration depth of 1 cm
means at 1 cm depth, the beam intensity has dropped to 36.8%.
In general, penetration depends on the constituent scattering/
absorbing materials of the tissue (both variety and quantity/
proportions) as well as the variation of their optical properties over
a range of wavelengths.

5.1.4 Optical Properties of Water

Water is the primary constituent of most tissues in our bodies, and
so most of the discussion that follows depends greatly on how light
interacts with water. Across the full range of the electromagnetic
spectrum, this can be a tricky situation. For example, when ionizing
radiations such as X-rays are incident on water, a significantly
possible event is that the absorption event breaks one of the O-H
bonds to create, what is called, a hydroxyl radical. This is a very
chemically reactive molecule that can lead to other, more dangerous
reactions that can lead to cell death. Radiation oncology takes special
advantage of free radicals as they are potent DNA breakers; in fact,
the hydroxyl radical that comes as a by-product of ionized water
accounts for about two-thirds of all radiation-induced mammalian
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Optical Properties of Tissues 71

12
10
8
LN Absorpon (1/cm)

6
4
2
0
-2 200 2200 4200 6200 8200 10200
-4
-6
-8
-10
Wavelength (nm)

Figure 5.1 Optical absorption of water [5]. Tabulated data are found at
http://omlc.org.

DNA damage [6]. In lower levels, however, these and other reactive
oxygen species serve as cell-signal carriers as well as to induce an
endogenous response that leads to an increased long-term defense
capacity against exogenous radicals and other foreign toxins.
But in the visible and infrared regions of the spectrum (Fig. 5.1),
where the light is non-ionizing and, therefore, does not have the
ability to break chemical bonds directly, water is a very simple
molecule. The two bonds that connect the H’s and O act like simple
springs. When a photon of light is absorbed, the energy stimulates
those springs to vibrate and/or twist, and as a result, the photon’s
energy is converted into heat. Just like any spring, there are certain
resonant frequencies, and if corresponding energies of light are
absorbed, the amount of vibrational energy converted to heat is
higher.
You use this idea every day when heating food in a microwave.
Microwave photons used in your house are ∼250,000 times less
energetic than visible light, but they excite certain resonance
frequencies in organic materials. Therefore, they cook your food (by
boiling the water within it) much better than a lamp (of visible light)
of the same power output would.
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72 Tissue Optics

NOTE: Remember that the energy E and frequency f of a photon are

related by E = hf, where h is Plank’s constant. Also the wavelength
of light λ and its frequency are related by λ = cf, where c is the speed
of light.
These resonances are of particular importance in the application
of infrared light. In surgical applications, where ablation or
vaporization is the goal, the water resonances above 1 micron
(where many of the Er:YAG and Ho:YAG lasers live) and at 10.6
microns in particular (CO2 laser) are popular choices.
The relatively weaker resonance at 965 nm is used therapeuti-
cally to increase tissue temperature more gently for the purpose
of temporary increase in circulation and pain relief associated
with musculoskeletal pain. In fact, the FDA clearances for some
commercially available lasers are based primarily on this effect
(www.fda.gov; e.g., K070400, K103511, K091497, K120604).
But more often in laser therapy, we aim to avoid these resonances
for the increased penetration it affords us. In the visible and NIR
range below 900 nm, water is relatively transparent, meaning its
absorption is low. For example, at 500 nm water absorbs 1800 times
less than at 970 nm, and over 3,000,000 times less than it does at
10,600 nm (absorption coefficients of 0.00025 cm−1 @ 500 nm, 0.45
cm−1 @ 970 nm, and 792.0 cm−1 @ 10,600 nm [5, 23]).
Water serves as the basis of our biological bodies, and so
these data are extremely important to consider. In fact, the
vascularization and, therefore, hydration status of any particular
tissue often not only dictate changes to the optical properties,
but also provide guidance for parameter optimization from a
therapeutic standpoint. An example comes with regard to pulsing
prescriptions. Different concentrations of water lead to tissues with
different heat dissipation characteristics [13]. More on this later,
but varying the pulse profile (both the on- and off-time of the
pulses) can lead to a variety of desirable/undesirable effects that can
help/impede the therapeutic application.
Water is not the whole story, however. It just provides the
backdrop to all the calculations. The other components of any given
tissue/fluid must also be addressed. Blood is a good example.
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Optical Properties of Tissues 73

extinction coeff (M-1 cm-1) 600

thousands
500 100

hundreds
400
50
300
200
0
100 600 800 1000
0
400 500 600 700 800
wavelength (nm)

Figure 5.2 Hemoglobin extinction coefficient (M−1 cm−1 ). Data compiled by

Scott Prahl (http://omlc.org) from W. B. Gratzer, Med. Res. Council Labs,
Holly Hill, London and N. Kollias, Wellman Laboratories, Harvard Medical
School, Boston.

5.1.5 Optical Properties of Blood

Though blood is mostly water, its other components lead to
strong absorption in the blue and green spectral regions (Fig. 5.2).
Consequently, the effective penetration depths of blue and green
light are less than 0.1 mm. The same property allows green laser
light to be quite useful to target superficial hypervascular lesions
such as port wine stains and varicose veins. Red blood cells
display Mie scattering with a scattering anisotropy approximately
0.97 from 650 to 1050 nm. The absorption of hemoglobin is
relatively smaller in the NIR with a deoxyhemoglobin absorption
peak at 960 nm, and oxyhemoglobin at 900 nm where the effective
penetration depth within blood vessels is approximately 0.5 mm
[3]. In fact, it is this difference in absorption spectra of oxy- versus
deoxyhemoglobin that stimulated the use of NIR light in the first
case. Neurologists began using this wavelength range of light to
monitor the location and amount of oxygenated brain tissue [24]
with particular applications in post-stroke victims.
One of the frontiers of modern laser research (including
contributions from this author as well as the editors of this book) has
taken these ideas to the next level with therapeutic applications in
mind. Capitalizing on the absorption peak of oxyhemoglobin may, in
fact, enable us to develop ways to enhance local tissue oxygenation.
Though the mechanisms of the effect are still being uncovered,
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74 Tissue Optics

preliminary results have shown that when oxyhemoglobin absorbs

light, it can be stimulated to reduce (or release its oxygen). The
increase in oxygen supply to ischemic (or otherwise “ailing”) tissues
has been known to have beneficial effects for decades, but only
recently have we been able to monitor this effect and quantify the
proportions of bound versus diffuse oxygen in living tissue in real
time.
Whether triggered by a temperature increase caused by the
oxyhemoglobin absorption or a temporary change in local pH or an
actual morphology change of the hemoglobin complex, this effect
could shed some light on the clinical success we have already seen.
Naeser et al. [11, 12] as well as Rochkind et al. [15–17], among
others, have studied the clinical benefit of transcranial laser therapy
in laboratory animals after inducing ischemic stroke as well as in
human patients with traumatic brain injury. A common hypothesis
throughout these findings is the increase in local ATP production,
and so climbing higher in the chain of events, a localized increase
in oxygen could certainly have a positive effect downstream in the
metabolic process.

5.1.6 Spectral Variation of Optical Properties

It is relatively simple to acquire an in vivo fiber-optic reflectance
spectrum of biological tissue from 400 to 1100 nm, and the
hemoglobin absorption creates a typical signature throughout the
entire range. The NIR portion is characterized by a deoxyhe-
moglobin absorption peak seen at 960 nm, oxyhemoglobin at
900 nm, and a water absorption peak at 980 nm (Fig. 5.3). Adipose
tissue has absorption peaks at 929 and 1040 nm [22].
However, one must be aware that the scan does not probe the
same depth throughout the range because the various spectral
regions have different penetration depths (Fig. 5.4).
Reflectance spectra are useful for non-invasive oximetry to
determine the local oxygenation level. Amelink et al. [1] reported a
mathematical fit to obtain the SpO2 using diffuse reflectance spectra,
as shown in Fig. 5.5.
A calculation advantage in the NIR is the lack of significant fluo-
rescence or inelastic scattering in natural living tissue. Fluorescence
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Optical Properties of Tissues 75

14
Absorption coefficient 12
10
8
(1/m)

6
4
2
0
400 600 800 1000
Wavelength (nm)

Figure 5.3 Absorption coefficient (m−1 ) of pig fat [21].

40
35
Percent Reflectance

30
25
20
15
10
5
0
400 500 600 700 800 900 1000
wavelength (nm)

Figure 5.4 In vivo fingertip reflectance collected with a bifurcated fiber

bundle, halogen lamp, and fiber-optic spectrometer. The lower wavelengths
often suffer from low signal to noise due to the lower intensity of the lamps
in that region as well as strong absorption of the tissue. Notice the subtle
absorption of hemoglobin and water around 960–980 nm compared to the
dramatic signature of hemoglobin in the visible spectrum (Graph developed
by LR Jones).

in this context refers to the process of absorption of light followed

by a re-emission of light of a different (lower) energy. Compton
(inelastic) scattering also involves a change in energy (wavelength)
in the incident versus outgoing light. In X-ray photon transport
calculations, both these effects (as well as others: photoelectric or
pair-production) are very important and not only add several steps
in the calculation process, but introduce the fact that the spectrum
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76 Tissue Optics

45
40
35
Percent reflectance

30
25
20
15
10
5
0
400 500 600 700 800
wavelength (nm)

Figure 5.5 Canine in vivo reflectance spectrum taken below the tongue
during surgery to monitor oxygenation level. The blue fitted line is
Amelink’s equation (Graph developed by LR Jones).

of light (distribution of wavelengths) changes as the beam of light

penetrates into the tissue. But in the NIR, these effects are negligible
and so an incident beam of, say, 800 nm light can be assumed to
consist of 800 nm light throughout the entire series of interactions.
In this respect, any particular photon of light is either absorbed
completely or bounced around (scattered) until it is absorbed.
So the wavelength of light does not change, but the tissue might. A
more recently identified effect involves changes in optical properties
of skin with respect to temperature (which is related to incident
power density) [8, 9]. In cases where higher power densities are
employed, a lot of energy is delivered and a significant proportion
of it (depending on the wavelength) is absorbed in the various
layers of skin and converted to heat. If the rate at which this
heat is dissipated is slower than the rate at which it is delivered,
the temperature may rise in such a way as to affect the optical
properties of the tissue. One of these effects is to increase the
“transparency” of the tissue (i.e., researchers have measured an
increase in fluence penetration to several depths). This effect has
been shown in both continuous wave (CW) delivery as well as
with superpulsed lasers, which deliver very short and sometimes
powerful bursts of light. In both cases, the time profiles show
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Methods and Algorithms for the Measurement of Optical Parameters of Tissues 77

that the longer the irradiation, the higher the penetrating fluence.
There is undoubtedly a threshold where this effect plateaus, not
only because it would be nonsensical to think that the tissue will
become completely transparent, but more importantly because this
increase in transparency over time coincides with an increase in
skin temperature. Clearly then, there are several opposing goals
involved in capitalizing on this phenomenon: we want to increase
penetration while mitigating any risks of tissue damage due to
superficial thermal accumulation and avoid any dose saturation that
may diminish the therapeutic efficacy.
Unlike with CW delivery, output parameters of superpulsed
lasers can be modified to account for this thermal saturation. Peak
power, pulse width, and dark time between pulses can all be varied
to get a variety of effects optically (let alone therapeutically). For
example, to take advantage of this (for a lack of a better term)
“photobleaching,” one might increase the pulse width to be longer
than the dissipation constant of the irradiated tissue. At the same
time, one might want to increase the dark time to allow the tissue to
thermally “relax” between pulses to avoid any risks of damage. In the
meantime, the peak power would also be adjusted to not only deliver
an appropriate therapeutic dose (to whichever depth is needed for
a particular condition/anatomy), but also to fine-tune the amount of
dose per pulse so that these other two parameters are optimized.

5.2 Methods and Algorithms for the Measurement of

Optical Parameters of Tissues

The first step toward being able to predict light’s interactions

within any clinical (or experimental) environment is compiling the
optical characteristics of all relevant tissue types involved ([19], for
example). Here we cover a few methods to do so.
Beer’s law can be used to calculate the absorption for a
transparent sample in a conventional spectrophotometer. However,
turbid samples cause light to scatter away from the optical
detector, yielding an apparent transmittance that overestimates the
absorption. Placing the detector as close as possible to the sample
will improve the result, but some light will still be lost. One solution
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78 Tissue Optics

70

60

50
muscle R
40 muscle T
mucosa R
30
mucosa T
20

10

0
400 500 600 700 800

Figure 5.6 Diffuse reflectance and transmittance spectra collected with

a single integrating sphere system for pig esophagus mucosa and muscle
samples (Graph developed by LR Jones).

is to employ an integrating sphere to collect the light that is scattered

out of the original optical path.

5.2.1 Integrating Sphere Technique

Diffuse reflectance and transmittance can be measured with an inte-
grating sphere: a spherical light collector that couples to an optical
detector. A single sphere can be used in which case the sample is
placed first in front of the sphere to collect the transmitted light
and then behind the sphere to collect the reflected light (Fig. 5.6).
The sample holder must have a small pathlength to minimize the
loss of light that is scattered to the side. A double sphere setup
can also be employed. In this case, the sample is sandwiched
between both spheres to measure the transmitted and reflected
light simultaneously. The diffuse reflectance and transmittance
measurements are processed to obtain absorption and reduced
scattering coefficients. If unscattered transmittance can also be
collected, one can resolve the scattering coefficient and anisotropy.

5.2.2 Kubelka–Munk Model

The Kubelka–Munk model was developed for diffuse incident light
and isotropic scattering. The model breaks the internal flux within
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Methods and Algorithms for the Simulation of the Light Interactions within Tissues 79

a material into forward and backward scattered streams, I (z) and

J (z). The light scatters and absorbs as a function of constants K and
S. For a tissue of thickness d, reflectance is equivalent to I(d)/I(0)
and transmittance is equivalent to J(0)/I(0). A solution to two
simultaneous first-order equations gives the following expressions
[4]:
1 + R2 − T 2
K/S =
2R
1 + T 2 − R2
S = (1/db) coth−1
 2Rb
b ≡ (K/S)(K/S + 2) (5.1)
The ratio K/S may be found from a diffuse reflectance measure-
ment for a very thick material where T approaches zero, as follows:
R = 1 + (K/S) − b (5.2)
The Kubelka–Munk model has been extended to collimated
incident beams and also to stacked layers. The constants K and S
are approximately related to absorption and scattering coefficients.

5.2.3 Inverse Methods

The inverse method involves measurement of macroscopic optical
parameters to derive the microscopic absorption and scattering
coefficients ([10], for example). Determination of three microscopic
optical constants (μa , μs , and g) requires three macroscopic
(or directly measureable) optical measurements such as diffuse
reflectance, diffuse transmittance, and unscattered transmittance
([7], for example). It is often the case that only two measurements
are available, so the optical constants are limited to the absorption
coefficient and “reduced” scattering coefficient [μs = μs (1 −g)]
(Fig. 5.7).

5.3 Methods and Algorithms for the Simulation of the

Light Interactions within Tissues

Now that we have some techniques to experimentally characterize

the optical properties of whichever tissues, we need a way to use
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80 Tissue Optics

Figure 5.7 Mucosa absorption (pink) and reduced scattering coefficients

(blue) derived with the inverse adding doubling method. http://omlc.org/
software/iad/index.html (Graph developed by Scott Prahl).

these data to make useful predictions. In general, our aim is to

understand what percentage of the incident beam gets absorbed
where (i.e., at different depths and radial distances from the central
beam axis).

5.3.1 Monte Carlo Simulation

“Monte Carlo” refers to the technique where random numbers are
generated to make decisions based on probabilities. For example,
it may be desired to determine whether a photon is absorbed or
scattered when it encounters a particle. The albedo of the tissue
is μs /(μa + μs ), which gives the fractional probability of a photon
being scattered in such a situation. In a Monte Carlo simulation, a
random number is generated with a value between 0 and 1 and
then compared to the albedo. If the random number is less than or
equal to the albedo, the photon is scattered; if the random number
is greater than the albedo, the photon is absorbed. In a similar way,
the scattering angle and other factors are determined.
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Methods and Algorithms for the Simulation of the Light Interactions within Tissues 81

Monte Carlo simulation is referred to as an “exact method”

because it involves the individual microscopic interactions of
photons with absorbing and scattering particles. The microscopic
interactions are based on the laws of optics, including the law
of reflection, Fresnel’s equations for reflection and refraction,
and Snell’s law for changes in refractive index. A bundle of
photons is generally injected into the tissue with regard to the
shape and characteristics of the incident light. The photon bundle
steps through the tissue and encounters scattering and absorbing
particles. Part of the bundle is absorbed and part is scattered at each
encounter, depending on the absorption and scattering coefficients.
The angle of the scattered light further depends on the anisotropy
factor and a specific scattering phase function.
The tissue may have a number of layers, each defined with
optical coefficients, refractive index, and thickness. The simulation
may keep track of time. It may adjust the absorption coefficient to
account for photobleaching, heating, or various other photoinduced
processes. The output of the simulation is typically a two- or three-
dimensional array of absorbed light energy.
Practical applications of Monte Carlo simulations include the
determination of the volume sampled for a given fiber-optic probe
geometry (Fig. 5.8).

5.3.2 Optical Tissue Phantoms

Though Monte Carlo simulation is an “exact method” of calculating
the distribution of absorbed dose throughout the exposed area, we
often need to check these simulations in a more “hands on” way:
tissue phantoms [14].
A tissue phantom is a liquid, solid, or semi-solid mixture of
scattering and absorbing materials. The proportions of the phantom
“ingredients” can be varied so that the optical properties match
specifically to model a particular tissue in a particular part of the
optical spectrum (Fig. 5.9). Typical scattering materials include
polystyrene microspheres and Intralipid.
Powdered non-dairy creamer has also been used successfully.
Whole blood, extracted hemoglobin, serum, and India ink are typical
absorbers.
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82 Tissue Optics

I mm

Figure 5.8 Monte Carlo simulation of a fiber held at three distances from
the surface of a tissue phantom: 0, 2.5, and 7.0 mm, left to right (Image
courtesy: Norris Preyer, College of Charleston). The top row in Fig. 5.6 looks
down on the tissue and shows where light that entered the fiber emerged.
The second row shows a cross section of the tissue, showing where detected
light traveled, and the bottom row shows the actual illuminated volume.

A tissue phantom provides a realistic light scattering environ-

ment for biochemical experiments or calibration of spectroscopic
methods. In fact, these phantoms can be used dynamically to
simulate some very interesting real-world applications. For example,
blood vessels can be modeled as tubes that circumvent the phantom
in different geometries, and things such as blood flow and oxygen
content of the tissues can be simulated and varied in real time (by
changing the “ingredients” of the blood phantom as well as the rate
at which it is pumped through the tubes). Similarly, things such as
respiratory gating can be simulated by inflating/deflating bags of air
placed within the phantom (Fig. 5.10).
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Practical Implementation 83

80
70 30
percent reflectance

60 25

percent reflectance
50 20
40 15
30
10
20
5
10
0
0 400 500 600 700
400 500 600 700
wavelength (nm) wavelength (nm)

Figure 5.9 Fiber-optic reflectance spectra of phantoms composed of

hemoglobin and either microspheres (left) or Intralipid (right) (Image
courtesy: LR Jones).

10000 3500
3000
8000
2500
FI A.U.

6000 2000
FI A.U.

4000 1500
1000
2000
500
0 0
500 600 700 500 600 700
wavelength (nm) wavelength (nm)

Figure 5.10 Fluorescence emission (405 nm excitation) for a Photofrin-

sensitized tissue phantom composed of Intralipid, serum, whole blood, and
saline. The serum provides a realistic autofluorescence background as seen
in the esophageal in vivo emission spectrum (right) of a patient 48 h after
Photofrin injection (Graph developed by LR Jones).

5.4 Practical Implementation

In general, the clinical effect of laser (light) therapy is only as good

as the accuracy and precision of its delivery. It is clear that although
in vitro experimentation is highly necessary to isolate individual
chromophore absorption characteristics and cellular mechanisms of
action, the Petri dish environment is quite different from our bodies.
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84 Tissue Optics

Internal dosimetry of laser (light) therapy is far too often

overlooked or “guesstimated” but is crucial information for the
design of treatment protocols and prediction of biological efficacy.
In vitro studies have given us a general idea of the range of
biostimulatory doses, but their results do not and should not be
directly extrapolated to form conclusions in vivo.
The science of dosimetry has been extensively developed in other
wavelength ranges of the electromagnetic spectrum to different
degrees of precision based on the danger of exposure of each.
Though we do not need the sub-millimeter accuracy of the
radiation oncologist who delivers ionizing radiation that can destroy
individual cells, the techniques they have developed offer a sensible
guide to understanding the photon transport in biological tissue. In
this chapter, we have reviewed some of these tools as we aim to
bridge this gap and understand exactly how dose is distributed at
depth in the body.
Here we have identified the different categories of tissues with
respect to their scattering strengths, discussed the techniques to
accurately measure the microscopic optical parameters of tissue,
and covered some methods of using these data to make useful
predictions. Much more work remains to be done in quantitative
internal dosimetry of laser therapy. This methodology outlined in
this chapter, however, is a necessary step in the right direction
on the path of understanding the delivery of light to our bodies.
Once further enlightened, we will be able to review both existing
and future studies to better understand the biological effect of the
delivered dose that came from the reported treatment prescriptions
and eventually converge on the optimal treatment parameters for
clinical success.

References

1. M. A. Ansari and E. Mohajerani. (2011). Mechanisms of laser–tissue

interaction: I. Optical properties of tissue. J Lasers Med Sci, 2(3), pp.
119–125.
2. W. F. Cheong, S. A. Prahl, and A. J. Welch. (1990). A review of the optical
properties of biological tissues. IEEE J Quantum Electron, 26, pp. 2166–
2185.
July 14, 2016 17:46 PSP Book - 9in x 6in 05-Hamblin-c05

References 85

3. M. Friebel, A. Roggan, G. Muller, M. Meinke. (2006). Determination of

optical properties of human blood in the spectral range 250 to 1100
nm using Monte Carlo simulations with hematocrit-dependent effective
scattering phase functions. J Biomed Opt, 11(3), pp. 034021.
4. L. I. Grossweiner. (1994). The Science of Photobiology (CRC Press), pp.
93–95.
5. G. M. Hale and M. R. Querry. (1973). Optical constants of water in the
200 nm to 200 μm wavelength region. Appl Opt, 12, pp. 555–563.
6. E. J. Hall. (1988). Radiobiology for the Radiologist, 3rd ed. (JB. Lippincott
Company, USA).
7. S. L. Jacques, C. A. Alter, and S. A. Prahl. (1987). Angular dependence of
He-Ne laser light scattering by human dermis. Lasers Life Sci, 1, pp. 309–
333.
8. J. Joensen, K. Øvsthus, R. K. Reed, S. Hummelsund, V. V. Iversen, R. A. L. B.
Lopes-Martins, and J. M. Bjorda. (2012). Skin penetration time-profiles
for continuous 810 nm and superpulsed 904 nm lasers in a rat model.
Photomed Laser Surg, 30(20), pp. 688–694.
9. S. Kim and S. Jeong. (2014). Effects of temperature-dependent optical
properties on the fluence rate and temperature of biological tissue
during low-level laser therapy. Lasers Med Sci, 29, pp. 637–644.
10. R. Marchesini, C. Clemente, E. Pignoli, and M. Brambilla. (1992). Optical
properties of in vitro epidermis and their possible relationship with
optical properties of in vivo skin. J Photochem Photobiol B: Biol, 16(2),
pp. 127–140.
11. M. A. Naeser, A. Saltmarche, M. H. Krengel, M. R. Hamblin, and J. A. Knight.
(2011). Improved cognitive function after transcranial, light-emitting
diode treatments in chronic, traumatic brain injury: Two case reports.
Photomed Laser Surg, 29(5), pp. 351–358.
12. M. A. Naeser and M. R. Hamblin. (2011). Potential for transcranial
laser or LED therapy to treat stroke, traumatic brain injury, and
neurodegenerative disease. Photomed Laser Surg, 29(7), pp. 443–446.
13. M. H. Niemz. (2007). Laser–Tissue Interactions: Fundamentals and
Applications (Biological and Medical Physics, Biomedical Engineering),
3rd ed. (Springer, USA).
14. B. W. Pogue and M. S. Patterson. (2006). Review of tissue simulating
phantoms for optical spectroscopy, imaging and dosimetry. J Biomed
Opt, 11(4), pp. 041102-1-16.
15. S. Rochkind, M. Nissan, M. Alon, M. Shamir, and K. Salame. (2001). Effects
of laser irradiation on the spinal cord for the regeneration of crushed
peripheral nerve in rats. Lasers Surg Med, 28, pp. 216–219.
July 14, 2016 17:46 PSP Book - 9in x 6in 05-Hamblin-c05

86 Tissue Optics

16. S. Rochkind. (2006). Photoengineering of neural tissue repair processes

in peripheral nerves and the spinal cord: Research development with
clinical applications. Photomed Laser Surg, 24(2), pp. 151–157.
17. S. Rochkind, D. El-Ani, Z. Nevo, and A. Shahar. (2009). Increase of
neuronal sprouting and migration using 780 nm laser phototherapy as
procedure for cell therapy. Lasers Surg Med, 41, pp. 277–281.
18. D. Sardar, B. Yust, F. Barrera, L. Minum, and A. Tsin. (2009). Optical
absorption and scattering of bovine cornea, lens and retina in the visible
region. Lasers Med Sci, 24(6), pp. 839–847.
19. S. H. Tseng, P. Bargo, A. Durkin, and N. Kollias. (2009). Chromophore
concentrations, absorption and scattering properties of human skin in-
vivo. Opt Express, 17(17), pp. 14599–14617.
20. V. Tuchin. (2000). Tissue Optics: Light Scattering Methods and Instru-
ments for Medical Diagnosis, Vol TT38 (SPIE Press, USA), pp. 109–133.
21. R. L. P. van Veen and H. J. C. M. Sterenborg, A. Pifferi, A. Torricelli, and
R. Cubeddu. (2004). Determination of VIS-NIR absorption coefficients
of mammalian fat, with time- and spatially resolved diffuse reflectance
and transmission spectroscopy. OSA Annual BIOMED Topical Meeting.
22. R. L. P. van Veen, H. J. C. M. Sterenborg, A. Pifferi, A. Torricelli, E.
Chikoidze, and R. Cubeddu. (2005). Determination of visible near-IR
absorption coefficients of mammalian fat using time- and spatially
resolved diffuse reflectance and transmission spectroscopy. J Biomed
Opt, 10(5), pp. 054004.
23. A. J. Welch, M. J. C. van Gemert, and W. M. Star. (2011). Definitions
and overview of tissue optics, in Optical-Thermal Response of Laser-
Irradiated Tissue, 2nd ed., A. J. Welch and M. J. C. van Gemert, ed.
(Springer Science, USA), p. 45.
24. S. Wray, M. Cope, D. Delpy, J. S. Wyatt, and E. O. R. Reynolds. (1988).
Characterization of the near infrared absorption spectra of cytochrome
aa3 and haemoglobin for the non-invasive monitoring of cerebral
oxygenation. Biochimica et Biophysica Acta, 933, pp. 184–192.
July 6, 2016 17:15 PSP Book - 9in x 6in 06-Hamblin-c06

Chapter 6

Light–Tissue Interaction and Light

Dosimetry

Ana Carolina de Magalhães and Elisabeth Mateus Yoshimura

Nuclear Physics Department, University of São Paulo, Rua do Matão, 187-Travessa R,
São Paulo 05508-090, Brazil
anamagalhaes@usp.br

Light–tissue interactions are an important aspect of low-level laser

therapy (LLLT). The biological effect, the depth to which light
reaches, and other issues result from these interactions. Light
dosimetry is also important because it is how we can compare
different treatments and optimize the parameters. This chapter
intends to present an overview of the main topics related to light–
tissue interactions and light dosimetry.

6.1 Light–Tissue Interactions

Light–tissue interactions have two main actors: optical radiation

and tissue. Optical radiation is the region of the electromagnetic
spectrum with wavelengths between 0.1 μm and 1000 μm, which
comprises ultraviolet, visible, and infrared radiations. Ultraviolet
radiation is the portion of spectrum between 100 nm and 400 nm,

Handbook of Low-Level Laser Therapy

Edited by Michael R. Hamblin, Marcelo Victor Pires de Sousa, and Tanupriya Agrawal
Copyright c 2017 Pan Stanford Publishing Pte. Ltd.
ISBN 978-981-4669-60-3 (Hardcover), 978-981-4669-61-0 (eBook)
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88 Light–Tissue Interaction and Light Dosimetry

which is usually divided into three ranges: UV-A (from 400 nm to

315 nm), UV-B (from 315 nm to 280 nm), and UV-C (from 280 nm
to 100 nm) [3]. Ultraviolet is responsible for tanning but is also
classified as carcinogenic for humans by the International Agency
for Research on Cancer (IARC) [4]. Visible radiation is the portion of
the optical radiation that is detected by our eyes, generating a direct
visual sensation, in other words that we can see [3]. Due to this
definition, it is not possible to define precisely the range of visible
light, because it depends on light quantity reaching the retina and
observer responsivity. However, its wavelengths are usually defined
between 400 nm and 780 nm [3]. The word light, specifically, has
two definitions: The first one is related to the vision sensations
and perceptions, or perceived light; the second one is related to
the visible radiation [3]. Last, but not less important, there is the
infrared radiation, with wavelengths between 780 nm and 1 mm.
It is also usually divided into three ranges: IR-A (from 780 nm
to 1400 nm), IR-B (from 1.4 μm to 3 μm), and IR-C (from 3 μm
to 1 mm) [3]. Objects, including people, emit thermal radiation,
whose spectrum depends on temperature. When those objects are
at around room temperature, this spectrum is almost all infrared.
Sometimes, light is used as synonym of optical radiation. CIE does
not recommend this meaning, but for simplicity, we will use this
sense of light forward. The second actor of light–tissue interactions
is the tissue, which is a specific kind of medium that the light
reaches. Regarding LLLT, this medium is a biological tissue where the
therapy is being applied. Cells, lipids, proteins, and other molecules
compose those tissues and interact with light. This interaction can
be separated into two steps: the interaction that occurs outside the
medium and the interaction that occurs inside the medium.
Outside the medium, reflection happens when light hits the
tissue surface. Reflection always occurs when a light beam hits
the surface between two media; usually all media reflect at least a
fraction of the incident beam. Depending on the type of the surface,
different beam fractions will be reflected. For smooth surfaces, the
beam will be reflected regularly (the reflection angle is equal to the
incident angle), and this is called specular reflection. Mirrors will
reflect the entire beam, and transparent materials, such as window
glass, will also produce specular reflection, but only a fraction of the
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Light–Tissue Interactions 89

light will be reflected. On the other hand, a rough surface, such as

skin, will irregularly reflect a fraction of the beam in all directions,
which is called diffuse reflection.
When we talk about media in light interaction, there are
three types of media: transparent, turbid, and opaque. Transparent
medium transmits almost all incident light, for example glass.
Opaque medium practically does not transmit light; an example
of opaque medium is coal. Otherwise, light that enters a turbid
media suffers, essentially, absorption and scattering, causing at-
tenuation, reduction of the light beam. Scattering is responsible
for the turbid appearance of those materials. Almost all kinds of
biological tissues are turbid media, and light inside them suffers
absorption and scattering; consequently light is attenuated [2].
The light–medium interaction depends on two factors: medium
composition and wavelength. Opacity and transparency also depend
on wavelength; therefore, one material might be opaque to a
wavelength and transparent to another. All known materials are
transparent to some wavelengths and opaque to other wavelengths
of the electromagnetic spectrum [9].
The beam fraction that is not reflected in the surface enters the
medium. This beam will suffer a shift in its propagation direction
because light speed changes between media. This phenomenon is
known as refraction, which is more apparent in transparent media.
The refraction index measures the change in the light’s speed in
a medium. It is defined as the ratio between the speed of light in
vacuum, c, and the speed of light in the medium, v (see Eq. 6.1). The
smaller the speed of light in the medium, the larger the refraction
index; consequently, the bigger the change in the propagation
direction. Refraction index depends on the medium and wavelength
[2].
c
n= (6.1)
v
The light entering a medium suffers attenuation, that is, its intensity
is reduced. Intensity is related to the power emitted by a light source
in a specific direction; it is the power per unit area. Depending
on the medium type, either absorption or scattering may occur,
which are the two causes of attenuation. In transparent media,
scattering almost does not occur; the only direction change is due
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90 Light–Tissue Interaction and Light Dosimetry

to refraction. For these media, a very small fraction of light is

absorbed, so almost all the light that enters the medium passes
through it without interacting. On the other hand, for an opaque
medium, the main interaction is absorption. Almost all light entering
an opaque medium is absorbed, so the intensity of light beam is
practically reduced to zero. For both cases, scattering almost does
not occur. Nevertheless, for turbid media, light interacts by either
absorption or scattering, mainly multiple scattering, due to medium
microscopic components, which scatter light to all directions. Both
interactions cause light attenuation, in other words reduce light
intensity.
In biological tissue, absorption is responsible for reaction to
stimulus. If absorption does not occur, there is no effect, as the
first law of photochemistry states: Light must be absorbed for
photochemistry to occur [11]. When a light photon is absorbed, its
energy might be used to excite atoms or molecules, inducing changes
in electronic or vibrational energy levels. Energy can also be used to
promote chemical reactions, causing photochemical response. These
chemical reactions will stimulate or even produce biological effects,
which can be either beneficial or prejudicial, depending on where
and how much light was absorbed by tissue. In photochemistry, as
light is similar to any drug, effects depend on doses [2].
In biological tissue, the chromophores are the molecules re-
sponsible for light absorption [2]. The chromophores are divided
in two groups: photoreceptors and photoaceptors. Photoreceptors
are molecules specialized in light absorption, and their function
depends on that. An example is the chlorophyll present in plants,
which has to absorb light to realize photosynthesis. On the
other hand, photoaceptors are molecules not specialized in light
absorption, but their capability to absorb light helps to accomplish
their functions. In other words, photoaceptors can execute their
functions without absorbing light, but doing this improves the
process. In biological tissue, most chromophores are photoaceptors,
and the main biological chromophores are water (which absorbs
mainly in the infrared range), macromolecules such as proteins,
and pigments such as melanin and hemoglobin [9]. A range of
wavelengths in the electromagnetic spectrum are less absorbed by
biological tissue. This range is in red and near infrared, between
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Light–Tissue Interactions 91

600 nm and 1200 nm, and is called therapeutic window [9].

Light with these wavelengths suffers relatively low absorption and
scattering, although it is more scattered than absorbed, so it can
reach deeper tissue layers and consequently treat those areas.
The scattering happens due to material microscopic components.
For biological tissue, these components are mainly macromolecules
and cells. We can model scattering inside tissue by two different
ways. In the first one, light interacts with macroscopic components
of the medium; for biological tissue, these might be cells and their
membranes or organelles, which work as media, with different
refraction index (n). Thus, light reflects or refracts on the medium
component surface, changing its propagation direction, that is, it
gets scattered. The other way is when the incident light interacts
with microscopic components of the medium, such as molecules or
atoms. When this occurs, these components may oscillate with the
incident frequency. If there is resonance, the photon is absorbed;
otherwise, if the oscillation frequency is far from resonance, the
photon is re-emitted with the same frequency but in the other
direction. Those atoms and molecules might be either inside or
outside cells.
The scattering might be either elastic or inelastic. For elastic
scattering, the wavelength is the same before and after scattering.
Otherwise, for inelastic scattering, the wavelength of the scattered
light is different from that of the incident light. Consequently,
frequency and energy are also different after scattering. For
biological tissue submitted to LLLT, the scattering is elastic.
There are two different models for light elastic scattering:
Rayleigh and Mie. The first one occurs when light hits a particle
of much smaller size than the light wavelength. Lord Rayleigh, in
1871, proposed that in this case, the electric field of the incident
wave makes the electrons of the scattering particle vibrate [7].
Those electrons emit light in the same wavelength but in different
direction. On the other hand, Mie scattering, discovered by Gustave
Mie in 1908, occurs when particles have size similar to the
wavelength. Actually, the theory developed by Mie describes light
scattering by spheres, and other particles with simple geometry
such as cylinders. However, as this theory is very complex, usually
for small particles Rayleigh theory is preferred because it is less
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92 Light–Tissue Interaction and Light Dosimetry

complicated. Therefore, when the particle is much smaller than the

wavelength Rayleigh theory is used and the scattering is referred
as Rayleigh scattering, and when the particle has size similar to
the wavelength Mie theory is used and the scattering is referred
as Mie scattering. The dependence on wavelength is different for
both scattering types: For Rayleigh scattering, scattered intensity is
inversely proportional to the fourth power of wavelength, λ−4 [7];
for Mie scattering, intensity has a weak dependence on wavelength.
Mie scattering happens preferentially in frontal direction unlike
Rayleigh scattering, which is almost isotropic, without preferential
direction [9]. As examples of both scattering models, Figs. 6.1 and 6.2
represent the angular distribution of red light, 0.6328 μm, scattered
by a spherical particle. In Fig. 6.1, the particle diameter is 0.05 μm
(much smaller than the wavelength), and Rayleigh scattering is used
for modelling; changing the diameter to 0.6 μm (similar to the
wavelength) results in Mie scattering, with the angular distribution
seen in Fig. 6.2.
As said earlier, in turbid media, both scattering and absorption
occur. Biological tissues in therapeutic window are almost all
considered turbid media. Furthermore, the proportion between
scattering and absorption is variable, depending on tissue and
wavelength. The scattering coefficient μs is related to the probability
of a photon being scattered when crossing a known thickness of
material. Similarly, the absorption coefficient μa is related to the
probability of a photon being absorbed in the same thickness of
material. Both coefficients depend on the material and incident
light wavelength. The attenuation coefficient μt is the sum of the
scattering and absorption coefficients, and the intensity for turbid
media is represented by Eq. 6.2, where I0 is the incident beam
intensity that enters the tissue, I is the transmitted beam intensity,
which has not suffered interaction after passing through a thickness
x of the material, and e is the neperian number. This equation is
valid only if photons that did not suffer any interaction are added
to the intensity I . For thin materials and small measuring areas, it
may work, but for thick materials, where multiple scattering takes
place, this measurement is not practicable; thus, the attenuation
coefficient is not used.
I = I0 e−μt x (6.2)
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Light–Tissue Interactions 93

Figure 6.1 Polar graph of the scattering pattern, for a small particle. In
the center of the plot is the spherical element (of diameter 0.05 μm)
that scatters light (of wavelength 0.6328 μm) incident from left. Three
polarizations are represented: natural, red line; perpendicular, green line;
and parallel, blue line. It is possible to see that the scattering is almost
isotropic. The data was calculated with the software Mie Theory, publicly
available software, available from the Oregeon Medical Laser Center [8].

A particular case of this law in Eq. 6.2 is the Beer–Lambert law,

which is valid when μa  μs . So the light intensity attenuation is
mainly due to absorption, as shown in Eq. 6.3. This equation can be
applied to the media where the main interaction is absorption, and
scattering practically does not occur for the wavelength considered.
I = I0 e−μa x (6.3)
In turbid media, multiple scattering usually occurs, and both kinds
of elastic scattering, Mie and Rayleigh, occur because there are
July 6, 2016 17:15 PSP Book - 9in x 6in 06-Hamblin-c06

94 Light–Tissue Interaction and Light Dosimetry

Figure 6.2 Polar graph of the scattering pattern for a large particle. In the
center of the plot is the spherical element (of diameter 0.6 μm) that scatters
light (of wavelength 0.6328 μm) incident from left. Just one polarization is
represented, because the scattering pattern does not vary much between
natural, perpendicular, and parallel polarizations. It is possible to see
that scattering is, preferentially, frontal. The data was calculated with the
software Mie Theory, publicly available software, available from the Oregeon
Medical Laser Center [8].

structures of different sizes in biological tissue, such as macromole-

cules, cells, and organelles. Since scattering occurs at many angles
with different probabilities and each type of scattering gives rise
to preferential scattering angles, as seen in Figs. 6.1 and 6.2, a
probability distribution function p (θ ) is defined as the probability
of a photon being scattered to a specific angle θ after crossing a
medium. This function is obtained through experimental data and
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Light–Tissue Interactions 95

is also known as the phase function and expressed depending on

propagation direction and scattered direction p (ŝ, ŝ  ). The quantity
used for describing the angular distribution of the scattered light is
the anisotropy factor g, defined as the mean value of θ. For isotropic
scattering, g = 0; for preferentially frontal scattering, g = 1; and
for backscattering, g = −1. For biological tissue, g is between
0.7 and 0.99, which means scattering angles between 8◦ and 45◦
[9].
The anisotropy factor is used to determine reduced scattering
and attenuation coefficients, μs and μt , where μs = μs (1 − g) and
μt = μa + μs . Reduced coefficients are used to simplify the diffusion
equation, but also because the product of μs by (1 − g) is higher
(and more significant) for smaller g values: The more efficient the
removal of light from the beam by scattering, the smaller the value
of g.
The problem of light propagation through turbid media can be
solved analytically with Maxwell laws and tissue optical properties;
unfortunately, it is very complex, mainly because biological tissue
has too many inhomogeneities [1]. An alternative to this process is
the photon transport theory, which takes a heuristic approach to the
problem. The photon transport equation models radiance through
a specific material. The radiance L(r , ŝ) is defined as the average
power flux density that passes through an area da, located in r and
direction ŝ. The unit of radiance is W/m2 sr.
In a cylindrical element, with unitary base and height ds,
composed by absorbing and scattering particles, with μa and μs
representing absorption and scattering coefficients, radiance is
reduced due to photons that were absorbed or scattered by this
element. On the other hand, photons scattered from directions ŝ 
to ŝ—photons scattered from outside to inside of the volume—
increase the radiance. Combining both contributions, the radiative
transport equation is represented by Eq. 6.4, where d is the
differential solid angle in direction ŝ  and p (ŝ, ŝ  ) is the phase
function, which describes the angular distribution for a unique
scattering and is related to the probability density function of
a photon being scattered from direction ŝ  to direction ŝ. As a
probability density function, p (ŝ, ŝ  ) is normalized to one, as shown
in Eq. 6.5. It is important to say that those equations consider a
July 6, 2016 17:15 PSP Book - 9in x 6in 06-Hamblin-c06

96 Light–Tissue Interaction and Light Dosimetry

volume without light sources [1, 5].


d L(r , ŝ)    
= − (μa + μs ) L(r , ŝ) + μs p ŝ, ŝ  L r, ŝ  d (6.4)
ds 4π


 
p ŝ, ŝ  d = 1 (6.5)
4π

6.2 Light Dosimetry

There is no consensus in the LLLT community about the best

quantity to quantify a “light dose.” Other applications of electro-
magnetic field define and use such quantities. The first example
is radiotherapy: In this medical field, absorbed dose is thoroughly
used. This quantity, in a rough definition, is the amount of energy
deposited by the ionizing radiation in a defined volume, divided
by the mass of this volume, with the unit gray (1 Gy = 1 J/kg).
There is a specialized branch of metrology in order to guarantee
that the value of the absorbed dose to water is traceable to a
primary standard internationally accepted. The second example
relies on the radiofrequency range of the electromagnetic spectrum.
In this case, the quantity SAR (specific energy absorption rate,
in the frequency range 100 kHz–10 GHz, with unit W/kg) is
defined for radiation protection reasons. We can say, in a simplified
manner, that SAR evaluates the time variation of the electromagnetic
energy dissipated in a defined volume, divided by the mass of this
volume. Both definitions (absorbed dose and SAR) consider the
volume where the radiation energy is absorbed and the mass of
this volume as the region of interest. Possibly, behind the use of
volumetric quantities is the fact that in both these regions of the
electromagnetic spectrum, the beam penetration is high. Table 6.1
shows some examples of penetration depth for some ranges of
the electromagnetic spectrum. The penetration depth (δ) of the
electromagnetic radiation is defined as the distance traveled by
the radiation in the material, which is necessary to reduce its
intensity to 37% of the initial intensity. Alternatively, δ is the depth
in the material where 63% of the energy of the electromagnetic
radiation is absorbed. The variation of δ in the range of frequencies
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Light Dosimetry 97

Table 6.1 Examples of penetration depths for various energy ranges of

electromagnetic radiation

Frequency f, Approximate
Wavelength λ or Main penetration
Photon Energy hf Application Tissue or tissue simulator depth δ

f = 2.45 GHz Microwave Lowwater content tissue 10 cm

oven High-water content tissue 2 cm
f = 1.2 or 1.6 GHz Navigation Low-water content tissue 15 cm
High-water content tissue 2.5 cm
1 < f <11 GHz Communication Low-water content tissue 20 >δ >3 cm
High-water content tissue 3 >δ >0.3 cm
λ = 820 nm LLLT Lipovenos 10% (fat 1 cm
emulsion used to simulate
tissue)
315 <λ< 400 Forensics; black Skin 50 μm
(UVA) - light
hf = 17 keV Breast image Muscle 1 cm
(Mo Kα line) (mammogram)
hf = 662 keV Radiotherapy Muscle 11 cm
(137 Cs gamma rays)
hf = 20 MeV Radiotherapy Muscle 50 cm
(X-rays produced in
electron accelerators)

(wavelengths) considered in Table 6.1 is conspicuous and is much

more extended if other electromagnetic bands are included.
Most of the quantities usually reported for LLLT practices are
related more with the optical source characteristics than with the
absorption of light in tissue. For this reason, we include in Table 6.2
a summary of the main radiometric quantities used to characterize
and measure radiation beam, and in particular light radiation.
Although several of these quantities are important to characterize
the light beam, most of them are not enough to assist light dosimetry.
The main reason for that is the scattering characteristic of biological
tissue: A parallel coherent laser beam incident on a small tissue
area spreads out rapidly to a broad illuminated region due to the
interaction with the large variety of structures inside the tissue. Any
internal point in the tissue will be illuminated with incoherent light
coming from various directions.
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98 Light–Tissue Interaction and Light Dosimetry

Table 6.2 Radiometric quantities

Quantity Definition Unit

Radiant power or radiant Power emitted by a source, transmitted or received, watt
flux (symbol: P or ) in the form of radiation. (W)
Radiant energy Time integral of the radiant power over a given joule (J)
(symbol: Q) duration, t. For constant power, Q = P t.
Irradiance (at a point of a Quotient of the radiant power incident on an W/m2
surface) element of the surface containing the point of
(symbol: E) interest, by the area of that element.
Radiance (in a given direc- On an illuminated surface, the radiance is given by W/m2 sr
tion, at a given point of a the following equation, where dE is the irradiance
surface) produced by the beam at the element of surface, with
(symbol: L) solid angle d, and θ is the angle between the surface
element and the beam direction.
L= dE
dcos θ
Radiant exposure (at a point Quotient of dQ, radiant energy incident on an J/m2
of a surface) element of the surface containing the point of
(symbol: H) interest, by the area dA of that element.
Fluence or radiant fluence For an infinitely small sphere surrounding the point J/m2
(symbol: ) of interest (usually inside the irradiated medium),
fluence is the quotient of the radiant energy that
enters the surface of the sphere (any direction) by
the area of the diametrical cross section of that
sphere.

Source: From Ref. [10]

Among the quantities listed in Table 6.2, the one that should
be related to any possible biological effect, and thus be considered
a dosimetric quantity, is the fluence. It is so because the light
energy included in the definition comprises all the light that
enters the volume of interest, regardless of the direction. It is
important to notice the difference between fluence and radiant
exposure: As both are expressed with the same units, sometimes
they are misinterpreted. Radiant exposure measures the light
energy that arrives at a surface (for instance, at the skin in an
LLLT procedure) coming from the source; fluence, at the same
point, includes the energy of the light that backscatters from the
interior of the illuminated tissue and from the surface. Radiant
exposure characterizes the beam and the irradiation time; fluence
July 6, 2016 17:15 PSP Book - 9in x 6in 06-Hamblin-c06

References 99

characterizes the interaction of the light beam with the illuminated

material.
Usually, fluence is not a constant in the volume or area
considered in LLLT, and evaluating it experimentally is difficult.
Calculations with Monte Carlo codes or experimental simulations
with phantoms with appropriate composition and shape [12] are
methods used for fluence estimation. Another possibility is the
calculation of the quantity SAEF (spatial average energy fluence)
obtained by the ratio of the radiant energy delivered to the tissue
in an area and where most of the light is scattered.
LLLT has innumerous applications, which will be discussed
in various chapters of this book. Depending on the body region
and on the pathological condition, the rigor with which the light
dose has to be determined can be different. Local superficial
injuries, illuminated in the injured region, are frequently treated
with the prescription of the total energy delivered by the light
source in the surface of the body; an interval of radiant energy
is enough to guarantee that biomodulation is obtained. Systemic
conditions mostly require the same kind of prescription (a range
of radiant energy values) and are performed with a transcutaneous
illumination of the whole blood in a fixed point of the body. However,
for some special treatments, like transcranial brain illumination,
or transcutaneous illumination of deep organs, a minimum value
of radiant energy must reach the target of the treatment so that
the effect is noticeable. For these situations, light dosimetry is very
important and the knowledge of the beam characteristics and the
tissue optical properties [6] is essential.

References

1. Cheong, W., Prahl, S., and Welch, A. (1990). A review of the optical
properties of biological tissues, Journal of Quantum Electronics, 26, pp.
2166–2185.
2. Grossweiner, L. (2005). The Science of Phototherapy: An Introduction
(Springer, Netherlands).
3. International Light Vocabulary, Commission Internationale de
l’Eclairage (International Commission on Illumination) http://
eilv.cie.co.at/
July 6, 2016 17:15 PSP Book - 9in x 6in 06-Hamblin-c06

100 Light–Tissue Interaction and Light Dosimetry

4. International Agency for Research on Cancer Monographs on the

Evaluation of Carcinogenic Risks to Humans, http://monographs.
iarc.fr/ENG/Classification/index.php.
5. Ishimaru, A. (1978). Wave Propagation and Scattering in Random Media
(IEEE Press, USA).
6. Jacques, S. L. (2013). Optical properties of biological tissues: a review,
Physics in Medicine and Biology, 58, pp. R37–R61.
7. Kerker, M. (1969). The Scattering of Light and Other Electromagnetic
Radiation (Academic Press, USA).
8. Mie Scattering by Scott Prahl. http://omlc.org/software/mie/
9. Niemz, M. (2004). Laser-Tissue Interactions: Fundamentals and Applica-
tions (Springer).
10. Sliney, D. H. (2007). Radiometric quantities and units used in photo-
biology and photochemistry: Recommendations of the Commission In-
ternationale de l’Eclairage (International Commission on Illumination),
Photochemistry and Photobiology, 83, pp. 1–8.
11. Smith, K. (1999). The first law of photochemistry and lasers, Official
Journal of the World Association for Laser Therapy, 11, pp. 162–163.
12. Sousa, M. V. P., Yoshimura, E. M., Ramos, A. L. O., Magalhães, A. C., Saito,
M. T., Santos, L. R., and Chavantes, M. C. (2011). Phantoms of fingers with
various tones of skin for LLLT dosimetry, Proceedings of SPIE, 7906,
79060U-1.
July 6, 2016 17:15 PSP Book - 9in x 6in 07-Hamblin-c07

Chapter 7

Mitochondrial Light Absorption and Its

Effect on ATP Production

Nicolette Houreld
Laser Research Centre, Faculty of Health Sciences, University of Johannesburg,
PO Box 17011, Doornfontein 2028, South Africa
nhoureld@uj.ac.za

Low-level laser therapy (LLLT), or phototherapy, has received a

lot of attention over the last decade. It has been used with great
success in a variety of in vitro and in vivo studies to promote
wound healing. The exact mechanism of action is, however, not
well understood, and a number of theories exist. Increasingly, many
researchers have studied these underlying mechanisms, and there is
growing evidence and a general consensus that mitochondria are the
absorbing chromophore for red and near-infrared (NIR) laser light
(600–1000 nm). Photon energy is absorbed by the mitochondrial
chromophore and is converted to chemical energy and in doing so
stimulates the cell. This chapter focuses on the effects of LLLT on
cells and explores the influence on mitochondria and adenosine
triphosphate (ATP).

Handbook of Low-Level Laser Therapy

Edited by Michael R. Hamblin, Marcelo Victor Pires de Sousa, and Tanupriya Agrawal
Copyright c 2017 Pan Stanford Publishing Pte. Ltd.
ISBN 978-981-4669-60-3 (Hardcover), 978-981-4669-61-0 (eBook)
www.panstanford.com
July 6, 2016 17:15 PSP Book - 9in x 6in 07-Hamblin-c07

102 Mitochondrial Light Absorption and Its Effect on ATP Production

7.1 Mitochondria

Mitochondria are double membranous organelles found in nu-

merous quantities in eukaryotic cells. These organelles are often
referred to as the “power house” of the cell as their main function is
to generate energy in the form of ATP. Mitochondria are involved not
only in energy production, but also in other cellular activities such as
apoptosis, cell signaling and cell cycling, calcium homeostasis, and
the generation of oxygen radicals.
Mitochondria consist of an outer membrane and an inner
membrane, separated by an intermembrane space. The inner
membrane is composed of numerous folds known as cristae, which
increase the surface area of the inner membrane. The area enclosed
by the inner membrane is referred to as the matrix (Fig. 7.1).
Contained within the inner membrane are a series of enzyme
complexes, known as the electron transport chain (ETC), which is
involved in ATP synthesis.

7.1.1 Adenosine Triphosphate (ATP) Synthesis

The main source of cellular ATP is generated via the ETC. The ETC
is a series of enzymes involved in the transfer of electrons from an
electron donor to an electron acceptor. This occurs via a series of
redox reactions and involves the transfer of protons (hydrogen ions,
H+ ) across the inner membrane, thereby creating a proton gradient
(), also known as the mitochondrial membrane potential (MMP).
The ETC is made up of four enzyme complexes, known as complex I,
complex II, complex III, and complex IV. Some regard the last enzyme
in the series, ATP synthase, as complex V.
The mitochondria synthesize ATP by oxidizing nicotinamide
adenine dinucleotide (NADH) during cellular respiration (Fig. 7.1).
NADH enters the ETC and is oxidized by complex I, NADH:
ubiquinone oxidoreductase (NADH-dehydrogenase), to NAD+ . Elec-
trons are transferred to coenzyme Q (ubiquinone), which is
reduced to ubiquinol (QH2 ). Four H+ molecules are translocated
to the intermembrane space. Succinate is oxidized to fumerate
by complex II, succinate: ubiquinone oxidoreductase (succinate
dehydrogenase). Electrons are accepted by ubiquinone, which is
July 6, 2016 17:15 PSP Book - 9in x 6in 07-Hamblin-c07

Mitochondria 103

Figure 7.1 Mitochondrion is a double membranous organelle found in

eukaryotic cells and is involved in the production of adenosine triphosphate
(ATP). During glycolysis, glucose is broken down to pyruvate, which is
translocated to the mitochondria where it enters the citric acid cycle.
Nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide
(FADH2 ) are produced in the citric acid cycle and are sources of electrons
for the electron transport chain (ETC). NADH enters the ETC and is oxidized
by complex I, reducing coenzyme Q (CoQ) to ubiquinol (CoQH2 ). Succinate is
oxidized to fumerate by complex II, as is FADH2 . CoQ is reduced to CoQH2 . In
complex III, CoQH2 passes electrons onto cytochrome c (cyt c). Cytochrome
c is oxidized by complex IV, and electrons are passed onto molecular oxygen
(O2 ) creating water (H2 O). In complex V, protons (H+ ) are actively pumped
back into the mitochondrial matrix, which releases free energy and is used
to phosphorylate adenosine diphosphate (ADP) to ATP.
July 6, 2016 17:15 PSP Book - 9in x 6in 07-Hamblin-c07

104 Mitochondrial Light Absorption and Its Effect on ATP Production

reduced to ubiquinol. No H+ molecules are translocated to the

intermembrane space. FADH2 , produced in the citric acid cycle,
is covalently attached to complex II and facilitates the transfer
of electrons to ubiquinone. In complex III (cytochrome bc1),
ubiquinol passes electrons onto cytochrome c (cyt c) and is
reduced to quinol. Six H+ molecules are translocated in the process.
Cytochrome c is oxidized by complex IV, cytochrome c oxidase
(Cox). The electrons are passed onto molecular oxygen (O2 ), creating
water (H2 O). Two H+ molecules are translocated. As all these H+
molecules are translocated across the inner membrane and into the
intermembrane space, a strong electrochemical gradient is created
(). Complex V, ATP synthase, acts as an ion channel and actively
pumps H+ back into the mitochondrial matrix. This releases free
energy, which is used to phosphorylate adenosine diphosphate
(ADP) to the energy-rich molecule, ATP. This process is referred to
as oxidative phosphorylation (Fig. 7.1). In total, 32 ATP molecules
are created.

7.2 Phototherapy

Phototherapy involves the application of low-level or low-powered

lasers to sites of injury to speed up cellular processes, and more
notably to speed up healing and reduce inflammation and pain.
It is a noninvasive, nonthermal treatment and has the ability to
modulate a wide variety of biological processes. Photon energy
must be absorbed by the cell and is absorbed by photoacceptors,
or chromophores, which results in a photochemical effect. A chro-
mophore is a molecule, or part thereof, which imparts some decided
color to the compound of which it is an ingredient [1]. In 1999,
Karu suggested that localized transient heating of the photoacceptor
may cause structural changes and trigger mechanisms such as
activation or inhibition of enzymes [2]. At 710–790 nm, the recorded
absorption spectra of HeLa cells were characteristic of reduced Cox,
while the region at 650–680 nm characterized the oxidized state
[3]. Later, in 2008, Karu et al. demonstrated that the redox state of
Cox was influenced by red light, and that this was dependent on the
initial redox state of the molecule at the time of irradiation [4]. Laser
July 6, 2016 17:15 PSP Book - 9in x 6in 07-Hamblin-c07

Phototherapy 105

irradiation at 632.8 nm resulted in relative reduction or relative

oxidation of Cox. It would appear that cellular redox state plays a
key role in the sensitivity of cells to phototherapy and may help to
explain variability in patient responsiveness [5]. This could explain
why pro-oxidant cells, such as diabetic cells, are more sensitive to
phototherapy as these conditions may result in oxidation of Cox
and lead to increased mitochondrial photosensitivity [6]. It was also
postulated by Karu [7] that irradiation intensified the transfer of
electrons in Cox by making more electrons available, resulting in
accelerated oxidative phosphorylation. A number of in vitro studies
on cells of different origin using a wavelength spectra of red to NIR
(600–1000 nm) have shown to modulate cellular processes such as
ATP production [8–18], cyclic AMP (cAMP) [10, 11, 19], and MMP
[10, 11, 13, 17, 20–23].

7.2.1 Effect of Phototherapy on Mitochondria and ATP

Synthesis
For phototherapy to have an effect, light has to be absorbed by
a chromophore. It is now widely accepted that the mitochondria
are such molecules for red and NIR lights, and there is mounting
evidence to support this.
ATP is synthesized by the mitochondrial ETC during the process
of oxidative phosphorylation. ATP provides energy needed by
the cell and drives many biochemical processes such as protein
synthesis. A number of studies on isolated mitochondria or cells
in culture have shown that visible red and NIR lights influence
the mitochondria and ATP synthesis (Fig. 7.2). Osteoblastic cells
irradiated at 830 nm (50 mW, 3 J/cm2 ) showed intense grouping
of granular mitochondria in the perinuclear region, suggesting high
mitochondrial activity solicited for cell division and the cells’ need
for energy [24]. Pires-Oliveira et al. exposed mouse conjunctive
tissue (L929 cells) to a 904 nm gallium arsenide (GaAs) diode laser
(repetition rate 10 KHz, pulse width 100 ns, 50 mW, 6 J/cm2 , and
50 mJ/cm2 ) [25]. Cell staining also showed increased mitochondrial
concentration in the perinuclear region, suggestive of increased
mitochondrial activity and increased ATP synthesis. Perinuclear
mitochondria may be associated with the endoplasmic reticulum
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106 Mitochondrial Light Absorption and Its Effect on ATP Production

Figure 7.2 Photons from red and near-infrared lights are absorbed by
cytochrome c oxidase (complex IV) of the electron transport chain. This
leads to an increase in protons (hydrogen ions, H+ ) moving across the inner
membrane, thereby increasing the proton gradient () or mitochondrial
membrane potential. This leads to an increase in adenosine triphosphate
(ATP), reactive oxygen species (ROS), intracellular calcium (Ca2+ ), and
photodissociation of nitric oxide (NO) from cytochrome c oxidase. There is
also an indirect increase in cyclic AMP (cAMP), which is derived from ATP.
There molecules lead to activation of signal transduction leading to gene
transcription. This, in turn, leads to observable effects such as increased
wound healing and decreased pain and inflammation.

with bidirectional calcium flow as well as acidic vesicles. This may

play an important role in mitochondrial proton gradient regulation.
DioC6-staining confirmed intense reticular activity from increased
protein synthesis [25].
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Phototherapy 107

Three photoacceptor molecules are known to absorb light in the

NIR range in mammalian tissues, namely hemoglobin, myoglobin,
and Cox, of which Cox is associated with energy production [26].
Cox, the terminal protein complex (complex IV) in the ETC, transfers
electrons from cytochrome c to molecular oxygen. The influence
of laser irradiation on Cox has been extensively studied, and it is
widely thought that it is the mitochondrial photoacceptor for red
and NIR lights. Karu et al. irradiated HeLa cells and identified the
action spectra for several processes, namely DNA, RNA synthesis,
and cellular adhesion and noted that they were all similar [3]. These
action spectra were then compared with the absorption spectra of
Cox in response to visible and NIR lights. The spectral absorbance
and the action spectra were very similar, and based on this Karu
suggested that Cox was the primary photoacceptor in mammalian
cells [3, 27]. Cox showed absorption peaks in the visible and NIR
range, and modification in its chemical behavior after exposure to
visible and NIR lights [28].
The photoreactivity of Cox is due to its four metal centers: two
heme moieties (heme a and heme a3) and two redox-active copper
sites (CuA and CuB) [7]. However, it is unclear which one of these
metals is the primary photoacceptor; CuA accepts electrons from
cytochrome c, and the transfer of electrons between CuA, heme
a, and heme a3-CuB results in the reduction of molecular oxygen
at seven intermediates [29]. Hu et al. [11] found an increase in
Cox in human melanoma cells post-irradiation at 632.8 nm with
1 and 2 J/cm2 (10 mW). They showed that the effect on Cox was
not secondary due to reactive oxygen species (ROS) generation
as a result of activation of another chromophore. Irradiation of
basophilic leukemia (RBL-2H3) cells at 405 nm resulted in the
rise of MMP and the release of Cox into the cytosol [30]. The
levels of Cox were not high enough to induce cellular apoptosis.
These results supported the notion that Cox is the photoacceptor
of laser irradiation [30]. NIR light-emitting diodes (NIR-LED) at
a wavelength of 670 nm have also been found to increase the
production of Cox in primary neuron cells. Three treatments with
NIR-LED were able to reverse the inhibitory effect of formic acid on
Cox [31, 32]. Enzyme kinetics was done on Cox post-irradiation (660
nm, 11 mW/cm2 , 5 and 15 J/cm2 ) in mitochondria isolated from
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108 Mitochondrial Light Absorption and Its Effect on ATP Production

normal and diabetic fibroblast cells. A significant increase in enzyme

activity was seen post-irradiation [9]. In a similar experiment, an
up-regulation was seen in complex IV [33]. Thus, laser irradiation
increases not only the levels of Cox, but also its catalytic ability.
An increase in activity of the other ETC protein complexes has
also been found following laser irradiation. In 1997, Yu et al. found
an increase in complex I, III, and IV activity in irradiated isolated
mitochondria (660 nm, 10 mW/cm2 , 0.6, 1.2, 2.4, and 4.8 J/cm2 )
[34]. The activities of complex II and complex V (ATPase), and lactate
dehydrogenase, were not affected by photoirradiation. Silveira et al.
irradiated injured male Wister rats at 904 nm (15–30 mW, 3 J/cm2
per session) and found an increase in complexes I, II, III, and IV and
succinate dehydrogenase [35, 36]. Irradiation at 660 nm with 5 or 15
J/cm2 of isolated mitochondria from normal, diabetic, and ischemic
cells influenced ETC activity [9]. Irradiation produced no significant
changes in complexes I and II. Irradiation with 15 J/cm2 produced a
significant decrease in complex III activity in diabetic mitochondria,
and an increase in ischemic mitochondria, as did 5 J/cm2 [9]. An
up-regulation in genes coding for complexes I, IV, and V has also
been found in stressed cells following irradiation at 660 nm [33].
It is evident that irradiation not only results in increased activity of
enzymes involved in the ETC, but also influences these enzymes at a
transcriptional level.
Studies on irradiated ATP have also been performed. Amat et al.
studied the behavior of ATP (0.025, 0.05, 0.25, 0.5, 2.5, and 5 mM)
following irradiation at 655 ± 10 nm or 830 ± 10 nm (100 mW,
70 mW/cm2 ) [37]. Irradiated ATP was added to the hexokinase
reaction. The hexokinase reaction marks the start of glycolysis,
where glucose is metabolized into pyruvate and ATP and provides
a source of energy under anaerobic conditions. It was observed
that irradiation of ATP resulted in ATP cleavage from hexokinase
in less time than in nonirradiated ATP, and that these changes were
wavelength and dose dependent. A threshold was found at 6 J/cm2 ,
with saturation at 12 J/cm2 . It was postulated that ATP underwent
conformational changes that altered its chemical behavior [37]. Karu
et al. [8] irradiated HeLa cells in the exponential phase of growth
with a He–Ne laser (632.8 nm, 10 W/m2 , 100 J/m2 ) and found
a significant increase in ATP 15 min post-irradiation, reaching a
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Phototherapy 109

maximum at 20 min. This increase was not evident in the lag phase
and at the beginning of the exponential phase. The increase in ATP
was dependent on the phase of growth of the culture [8]. Chen et
al. [15] found an increase in ATP in irradiated mouse embryonic
fibroblasts at 810 nm (0.3, 3, and 30 J/cm2 ). ATP levels peaked
immediately after a 5 min irradiation and declined to baseline levels
over 6 h. This increase was no longer evident after the addition of
mitochondrial inhibitors and inducer of hypoxia sodium azide and
deoxyglucose [15]. Hypoxia results in decreased synthesis of ATP
[12]. However, when hypoxic cells are irradiated, there is an increase
in ATP [10, 12, 14], showing without a doubt that LLLT increases ATP
production. In view of the fact that ATP is the key energy molecule
in cells, the consequence of visible and NIR laser irradiation on
ATP synthesis is of biological importance, especially in hypoxia and
oxidative stress [37]. An increase in ATP was also found in irradiated
normal and diabetic WS1 fibroblast cells irradiated at 660 nm [9],
human melanoma cells (A2058) at 632.8 nm [11], irradiated isolated
rat mitochondria at 632.8 nm (15 mW, 5 J/cm2 ) [13] and 670 nm
(25 mW/cm2 , 7.5 J/cm2 ) [14], and human neural progenitor (NHNP)
cells at 808 nm (50 mW/cm2 , 0.05 J/cm2 ) [16].
cAMP, derived from ATP by adenylate cyclase, acts as a secondary
messenger and is involved in a number of biological processes such
as proliferation, differentiation, and DNA and RNA synthesis [11].
Zungu et al. found an increase in cAMP in WS1 fibroblast cells
following irradiation at 632.8 nm (5 J/cm2 , 3 mW) [10]. Hu et al.
also found an increase in cAMP following irradiation at 632.8 nm (1
and 2 J/cm2 , 10 mW) [11]. An increase in cAMP was seen in human
adipose-derived stem cells irradiated at 660 nm with 4 and 8 J/cm2
(15.17 mW/cm2 ) [19]. This increase was no longer evident when
irradiated cells were treated with the adenylyl cyclase inhibitor
SQ22536.
MMP is directly related to ATP synthesis. As electrons are passed
from donor to acceptor in the ETC, protons are pumped across the
inner mitochondrial membrane creating a proton gradient ().
Irradiation of a variety of cell types has led to an increase in MMP.
Bortoletto et al. irradiated human Hep-2 cells at 635 nm (100,
150, and 200 s) [21]. They concluded that all irradiated cells had
increased MMP, as determined by JC1 staining, and hence increased
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110 Mitochondrial Light Absorption and Its Effect on ATP Production

ATP synthesis. An increase in MMP was also found in HaCaT cells

irradiated at 780 nm (200 mW, 2 J/cm2 ) [20], and neuronal cells
(PC12) irradiated at 670 nm (20 mW/cm2 ) [22]. Zungu et al. found
an increase in both MMP and ATP in WS1 fibroblast cells irradiated
at 632.8 nm (3 mW/cm2 , 5 J/cm2 ) [10]. Hu et al. irradiated human
melanoma cells (A2058) at 632.8 nm (10 mW, 0, 0.5, 1, and 2 J/cm2 )
and found an increase in MMP with a fluence of 1 and 2 J/cm2 [11].
They too found an increase in ATP as well as proliferation. These
increases were no longer evident when A2058 cells were treated
with a mitochondrial inhibitor. Irradiation of isolated mitochondria
(632.8 nm, 15 mW, 5 J/cm2 ) exhibited an increase in both MMP and
ATP, and the effects were directly related to the ETC as this increase
was no longer evident in the presence of inhibitors rotenone and
antimycin A [13]. It was further concluded that the increase in
ATP was as a direct result of laser-induced proton-motive force
(pmf). This was because in the presence of oligomycin, an antibiotic
that blocks ATP synthesis and reduces oxygen uptake but does
not interfere with electron carriers in the ETC, laser irradiation
resulted in an increase in MMP and had no effect on ATP levels
[13]. Irradiation of murine primary cortical neurons at 810 nm
(25 mW/cm2 ) resulted in an increase in mitochondrial function as
determined by an increase in MMP as well as ATP [17]. This increase
was seen at lower fluences, reaching a significant peak at 3 J/cm2 ,
and a decrease was seen at higher fluences. Mitochondria have also
been found to be photosensitive to green light. Chinese hamster
ovarian cells (B-14) were irradiated with an Nd:YAG laser (532 nm,
30 mW, 36 J), and an increase in MMP and in mitochondrial oxidative
phosphorylation was observed [23].
Mitochondrial respiration is inhibited by nitric oxide (NO)
binding to the heme iron:copper binuclear center (a3 /CuB ) of Cox,
and thus displacing oxygen [1, 38]. It has been suggested that laser
irradiation results in the photodissociation of NO from Cox, allowing
for an immediate influx of oxygen and resumption of respiration
and generation of ROS, and thus reversing mitochondrial inhibition
due to excessive NO binding [1, 39]. Irradiation of human skin
fibroblast cells, WS1, at 830 nm (5 J/cm2 , 4.4 mW/cm2 ) resulted in
an increase in ROS and NO 15 min post-irradiation in what would
appear to be a direct photochemical effect. Chen et al. irradiated
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Phototherapy 111

murine embryonic fibroblasts at 810 nm (0.3, 3, and 30 J/cm2 )

and showed an increase in mitochondrial ROS fluorescence [15].
This increase was confirmed via a quantitative assay. Interestingly,
Lan et al. showed an increase in Cox, but no increase in ROS
in melanoblast cells post-irradiation (632.8 nm, 2 J/cm2 ) [40]. In
these experimental conditions, ROS was not involved as a mediator
in response to laser irradiation. Pal et al. irradiated whole cell
fibroblast populations, as well as single fibroblast cells using a fiber-
optic based nanosensor probe from an He–Ne laser to a variety of
laser intensities (0.64–1.16 mW/cm2 for whole cultures and 330
mW/cm2 to 20 W/cm2 for single cells), exposure times, and total
energy densities (0.5–16 J/cm2 ) [41]. Time-dependent fluorescence
signals emitted due to the generation of ROS was evaluated. ROS
generation was dependent on laser intensity and dose [41]. Yu
et al. demonstrated that irradiation of isolated rat liver mitochondria
significantly increased the consumption of oxygen (600 nm, 10 mW/
cm2 , 0.6 and 1.2 J/cm2 ) [34]. Exposure of cardiocytes to broadband
visible light (tungsten halogen lamp) using blue, green, and red
filters resulted in an increase in ROS. Red light led to increased redox
reactivity and ROS production; however, blue light produced more
ROS than green and red light [42]. Blue light (470 nm) has been
found to kill methicillin-resistant Staphylococcus aureus (MRSA) [43]
and, may be due to this, increased ROS production.
Zhang et al. irradiated cardiac cells at 670 nm (25 mW/cm2 , 7.5
J/cm2 ) and showed that NIR treatment increased intracellular NO
[14]. This effect was abolished when NO scavengers were added,
and partially stopped by nitric oxide synthase (NOS) inhibitors. They
came to the conclusion that the increase in NO was due to NOS and
a second unidentified source, possibly Cox. NO-induced inhibition
of oxygen consumption with substrates for complex I or complex IV
was reversed by exposure to NIR [14]. Irradiation of mononuclear
cells to a 640 nm, 52 mm LED cluster (35.7 W/cm2 ) for 5 min
resulted in elevated levels of NO; however, the irradiation had no
effect on iNOS or eNOS, and there was a decrease in intracellular
ROS, and no change in ATP [44]. The increase in NO was likely
due to preformed stores as there was no increase in mRNA levels
of iNOS or eNOS following incubation for 4 or 24 h. A wavelength
of 810 nm (25 mW/cm2 ) resulted in an increase in both NO and
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112 Mitochondrial Light Absorption and Its Effect on ATP Production

ROS in murine primary neuronal cells [17]. There was an increase

in ROS at a fluence of 0.03 and 0.3 J/cm2 , peaking at 3 J/cm2 . A
higher fluence of 10 J/cm2 resulted in a decrease, and interestingly
a fluence of 30 J/cm2 resulted in an increase. A similar pattern
was seen in NO. It was suggested that this increase in ROS and
NO at higher fluencies may be responsible for the damaging effects
of high-fluence light and the overall biphasic dose response [17].
Sharma et al. proposed that laser irradiation of neuronal cells at
810 nm at a low fluence stimulated the release of NO from the
mitochondria and increased ATP and moderately increased ROS
synthesis, which activated signaling pathways [17]. They further
stated that a higher fluence damaged the mitochondria as seen by a
decrease in MMP and larger increase in ROS [17]. Laser irradiation,
as well as mitochondrial inhibitors, results in the production of ROS
and activation of NF-κB [15]. Phototherapy also caused an increase
in ATP, while the inhibitors decreased ATP levels. Antioxidants did
not abolish the production of ATP, which suggests that phototherapy
increases electron transport, which can cause increased electron
leakage in the absence of antioxidants, producing superoxides.
Mitochondria also play a role in the storage and homeostasis of
cellular calcium (Ca2+ ). The concentration of cellular Ca2+ regulates
a number of reactions and is important for signal transduction. An
increase in Ca2+ was seen in wounded, hypoxic, and acidic WS1
fibroblast cells irradiated at 632.8 nm with 5 J/cm2 (3 mW/cm2 ),
along with an increase in MMP, ATP, and cAMP [10]. Lan et al.
showed an increase in Ca2+ post-irradiation of melanoblast cells
(632.8 nm, 2 J/cm2 ) [40]. This increase, together with an increase
in Cox, is likely important in intermediary events in phototherapy.
Sharma et al. found an increase in Ca2+ in murine neuronal cells
irradiated at 810 nm (25 mW/cm2 ), reaching a peak at 3 J/cm2
[17]. A decrease was seen at a higher fluence of 10 and 30 J/cm2 .
Ca2+ is a physiological stimulus for ATP synthesis and has been
linked with the generation of ROS in the mitochondria. An increase
in MMP, ATP, and ROS was also noted [17]. Wu et al. showed that in
response to laser irradiation (405 nm, 0.6 mW, 60 s), mitochondria
are stimulated, which leads to signals being transferred from
the mitochondria to the cytosol [30]. This results in cytosolic
alkalinization, which causes the opening of Ca2+ channels [30].
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Conclusion 113

7.3 Conclusion

Numerous biostimulatory effects of LLLT have been demonstrated

over the last two decades, and the question is no longer whether
it has a biological effect, but rather what is the mechanism and
molecular basis for these effects? Having said that, there is an
increase in the amount of research done on the influence of red
and NIR laser lights on the mitochondria, and there is a growing
consensus that the mitochondria, more specifically Cox, is the
absorbing chromophore and is responsible for further down-stream
effects. Cox is considered the principal absorbing chromophore,
although other cytochromes, porphyrins, and heme proteins could
also be involved [17]. The absorption of photons released from red
to NIR light by mitochondrial Cox leads to an increase in electron
transport, and thus an increase in MMP, ATP, cAMP, NO, ROS, and
Ca2+ , which results in an increase in the amount of available energy
and signal transduction (Fig. 7.2). These biochemical changes lead to
macroscopic effects and promote cellular proliferation and wound
healing [29].
Despite the fact that blue and green lights have been shown
to have an effect on cells, phototherapy largely makes use of red
and NIR lights [45]. Reasons for this are that tissue absorption
and scattering are dependent on wavelength and are higher in the
blue region. Another reason is that hemoglobin and melanin, which
are principal tissue chromophores, have high absorption peaks at
wavelengths shorter than 600 nm, and water absorbs at wavelengths
greater than 1150 nm [45]. In the blue spectral region, flavoproteins
such as NADH-dehydogenase (complex I) can act as chromophores,
while green light can be absorbed by the prosthetic group of heme
in cytochromes b, c1 and c (iron protoporphyrin IX, PpIX), the same
heme as in myoglobin and hemoglobin [29].
It is known that Cox oxidation and the production of ATP are
dependent on MMP. The absorption of photons by the ETC leads
to an increase in MMP, ATP, cAMP, ROS, NO, and Ca2+ , and their
stimulation in vitro may explain the positive effects of phototherapy
seen in vivo [17]. The biostimulation process is initiated, and an
increase in proliferation and wound healing in a variety of different
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114 Mitochondrial Light Absorption and Its Effect on ATP Production

clinical situations is seen [20]. Phototherapy might be exerting its

effect on cells through its interaction with cellular redox systems,
and this could explain why pro-oxidant cells are more sensitive to
the effects of LLLT than normal cells [5, 6]. As ATP is the cells’
key energy molecule, the effect of visible and NIR lights on this
molecule and its production is of biological significance. Further,
well-controlled studies are needed to investigate the connection
between mitochondrial signaling and cellular events following
phototherapy.

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37. Amat, A., Rigau, J., Waynant, R. W., Ilev, I. K., Tomas, J., and Anders, J. J.
(2005). Modification of the intrinsic fluorescence and the biochemical
behaviour of ATP after irradiation with visible and near-infrared laser
light, J. Photochem. Photobiol. B: Biol, 85, pp. 26–32.
38. Ferraresi, C., Hamblin, M. R., and Parizotto, N. A. (2012). Low-level laser
(light) therapy (LLLT) on muscle tissue: Performance, fatigue and repair
benefited by the power of light, Photonics Lasers Med., 1(4), pp. 267–
286.
39. Lane, N. (2006). Cell biology: Power games Nature, 443, pp. 901–903.
40. Lan, C.-C. E., Wu, S.-B., Wu, C.-S., Shen, Y.-C., Chiang, T.-Y., Wei, Y.-H.,
and Yu, H.-S. (2012). Induction of primitive pigment cell differentiation
by visible light (helium-neon): A photoacceptor-specific response not
replicable by UVB irradiation, J. Mol. Med., 90, pp. 321–330.
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118 Mitochondrial Light Absorption and Its Effect on ATP Production

41. Pal, G., Dutta, A., Mitra, K., Grace, M. S., Amat, A., Romanczyk, T. B., Wu,
X., Chakrabarti, K., Anders, J., Gorman, E., Waynant, R. W., and Tata, D.
B. (2007). Effect of low intensity laser interaction with human skin
fibroblast cells using fiber-optic nano-probes, J. Photochem. Photobiol.
B: Biol., 86, pp. 252–261.
42. Eichler, M., Lavi, R., Friedmann, H., Shainberg, A., and Lubart, R. (2007).
Red light-induced redox reactions in cells observed with TEMPO,
Photomed. Laser Surg., 25, pp. 170–174.
43. Enwemeka, C. S., Williams, D., Enwemeka, S. K., Hollosi, S., and Yens,
D. (2009). Blue 470-nm light kills methicillin-resistant Staphylococcus
aureus (MRSA) in vitro, Photomed. Laser Surg., 27, pp. 221–226.
44. Saygun, I., Karacay, S., Serdar, M., and Ural, A. U. (2008). Effects of
laser irradiation on the release of basic fibroblast growth factor (bFGF),
insulin like growth factor-1 (IGF-1), and receptor of IGF-1 (IGFBP3)
from gingival fibroblasts, Lasers Med. Sci., 23, pp. 211–215.
45. Hamblin, M. R., and Demidova-Rice, T. N. (2007). Cellular chromophores
and signaling in low level light therapy, In: Hamblin, M. R., Waynant, R.
W., Anders, A. eds., Proceedings of the International Society for Optical
Engineering, San Jose, California, USA, 6428, pp. 642801-1–642801-14.
July 6, 2016 17:16 PSP Book - 9in x 6in 08-Hamblin-c08

Chapter 8

Water as a Photoacceptor, Energy

Transducer, and Rechargeable
Electrolytic Bio-battery in
Photobiomodulation

Luis Santana-Blank, Elizabeth Rodrı́guez-Santana,

Jesús A. Santana-Rodrı́guez, Karin E. Santana-Rodrı́guez,
and Heberto Reyes-Barrios
FUNDALAS, Calle Las Flores, C.C. Carabel, PB-L2, MUN 1262, Puerto La Cruz 6023,
Estado Anzoátegui, 6023, Venezuela
luissantanablank@msn.com

This chapter provides an introduction to light–water research with

an emphasis on theories related to laser photobiomodulation (PBM),
also known as low-level laser therapy (LLLT), and their potential
clinical application. Light–water interactions are at the core of
molecular and metabolic mechanisms that coexist with and can
complement and/or substitute for the classic PBM respiratory and
electron transport pathway primarily associated with cytochrome c
oxidase (Cox) (Complex IV). Nevertheless, until now, there had been
a complete dearth of information in PBM textbooks about this topic
and its progress over the last decades.

Handbook of Low-Level Laser Therapy

Edited by Michael R. Hamblin, Marcelo Victor Pires de Sousa, and Tanupriya Agrawal
Copyright c 2017 Pan Stanford Publishing Pte. Ltd.
ISBN 978-981-4669-60-3 (Hardcover), 978-981-4669-61-0 (eBook)
www.panstanford.com
July 6, 2016 17:16 PSP Book - 9in x 6in 08-Hamblin-c08

120 Water as a Photoacceptor, Energy Transducer, and Rechargeable Electrolytic

Since the 1990’s, experimental discoveries have revolutionized

light–water science. The time scale of resonant intermolecular
and intramolecular energy transfer in bulk water, confined spaces,
solvation shells, and aqueous interfaces has been determined,
which further allowed ascertaining molecular relaxation times.
Increasingly small measurements, from nanoseconds down to
femtoseconds, were made possible by major technical advances in
ultra-short pulsed lasers. In parallel, researchers have described the
exclusion zone (EZ): a potential fourth phase of water with broad
applications as a selective, rechargeable, electrolytic bio-battery.
We hope that this text will contribute to a better understanding
of the basis for local and remote clinical applications of light–water
interactions in laser PBM and medicine.

8.1 Introduction

Light and water are essential to life and yet remain mysterious.
Noble laureate A. Zewail once mused that “if one phenomenon
has occupied the thinking of humans throughout history, it is the
phenomenon of light” [1].a But, what is light? According to the
standard model of particle physics, each of the four known forces
is mediated by a fundamental boson, or carrier particle. Photons
are the carrier particles of light and other forms of electromagnetic
force. As bosons, photons obey statistical rules that permit any
number of such particles to occupy the same quantum state. Thus,
they can form energy packets that can effectively interact with
tissues. Streams of photons can also be made into laser beams that
are easy to modulate and have well-defined frequency-dependent
absorption spectra.
On the other hand, von Szent-Györgyi referred to water as “life’s
mater and matrix, mother and medium.” Szent-Györgyi proposed
energy (ion) transport through blood and oxidation–reduction as
part of the circulatory system, where water is critical [2]. He further
introduced “water structures, the electromagnetic field (EMF), and

a Copyright c The Nobel Foundation 2000. We thank the Nobel Foundation, Stockholm,

for granting permission to print this lecture.

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Introduction 121

triplets or some other unusual forms of excitation made possible by

water structures” to our understanding of biological reactions, and
coined the term “bio-energetics,” which is still in use in biology and
medicine. Subsequently, peculiar properties of water in biology have
been discovered [2]. However, until recently, water was presumed to
play only a secondary role in cells and tissues [3].
Such view of water as the ubiquitous neutral solvent has
radically changed. Studies have shown that not only water plays
a primary role in osmosis and diffusion, surface tension, oxygen
exchange, self-assembly, light-induced effects, and vascular effects
[4], but also in the vicinity of common hydrophilic materials
(e.g., biological interfaces), water is extensively ordered [3]. Liquid
water self-organizes and produces extended regions or coherence
domains, which require the intervention of an EMF [5]. Said water
molecules behave in unison with their phase locked with that of
the self-trapped EMF in accordance with the framework of quantum
electrodynamics.
Hence, EMFs may target the collective organization of water to
influence biomolecules. For instance, interfacial water plays a major
part in cellular recognition, especially during first contact events in
which cells decide between survival and apoptosis. The interaction
between light and water may, thus, be capable of steering cells
toward or away from apoptosis-deregulation, which is a hallmark
of cancer, neurodegenerative diseases such as age-related macular
degeneration (AMD), and other complex diseases (CDs) [4, 6–8].
This chapter explores how the modulation of the structure and
function of water by laser (or light) may come to embody a new
mechanistic approach for the treatment of CDs. First, we review
light energy absorption and transport by water. Then, we summarize
the basis for water-mediated, selective, long-range, external radiant
energy supplementation aimed at inducing and modulating phys-
iologically reparative processes, including apoptosis, through the
mechanism termed photo-infrared pulsed biomodulation (PIPBM)
[7]. Next we explore bulk, confined, and interfacial water as
oscillators in near-infrared (NIR) PBM [8]. We complement these
ideas by integrating EZ water and its role as a selective, electrolytic,
rechargeable, bio-battery sensitive to signal parameters relevant to
PBM [4]. Finally, we discuss the implications of Kleiber’s law as a
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122 Water as a Photoacceptor, Energy Transducer, and Rechargeable Electrolytic

Figure 8.1 Schematic representation of main chapter topics.

basis for light energy dose determination [9]. It will be argued that
water offers a potent pathway for external energy supplementation
to re-establish homeostasis/homeokinesis in local and systemic
diseases [10] (Fig. 8.1).

8.2 Absorption and Transport of Light Energy by Water

The Stark–Einstein law states that only absorbed light can trigger
photochemical change. Absorption is directly correlated to the
incident radiation and the absorption coefficient (AC).
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Absorption and Transport of Light Energy by Water 123

Figure 8.2 Black-body curve of water shows absorption increases with

wavelength. Published in Ref. [4] and adapted from Fig. 3 in Ref. [8],
c Mary Ann Liebert, Inc. Publishers 2012 and 2010, respectively.

At wavelengths <800 nm, the AC of water is minimal. At 900

nm, it is still only 0.1 cm−1 and increases to 0.15 cm−1 at 1064 nm.
In materials with a low AC, light propagates with little attenuation.
Hence, pulses as short as 60 fs with a center wavelength of 800 nm
propagate through as much as 6 m of water [11]. Laser bandwidth
to water’s absorption bandwidth is similar at 800 nm (0.36) and
1450 nm (0.34), where there is strong (harmonic/anharmonic)
NIR resonance in water (Fig. 8.2) [11, 12]. These values match a
peak in biologic action spectra found at 800 nm, a region where
absorption by other chromophores, such as Cox, is relatively low
[8]. In the 900–940 nm range, higher AC coincides with another
peak in action spectra [8] and with the black-body radiation of
the human body. As wavelengths increase, absorption by water is
greater. At 3100 nm, there is strong resonance at the level of the
fourth far-infrared (FIR) harmonic/anharmonic, which corresponds
to the fundamental OH stretch [3]. However, at higher wavelengths,
photon energy/penetration and ligand activity decrease.
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124 Water as a Photoacceptor, Energy Transducer, and Rechargeable Electrolytic

Despite its low AC in the 600–1100 nm range, water is a

biologically significant photoacceptor for several reasons. First, the
adult human body is approximately 70% water by weight. In a fetus,
almost 90% by weight is water; whereas in the very old, this can
descend to nearly 50%. In pure numbers, given its small molecular
weight, more than 90% of our molecules are water. It may thus be
said that humans are mostly water. Hence, water represents a major
absorption target, even for wavelengths with small ACs [8]. Second,
high AC frequencies at lower or higher intervals can establish
resonance with water due to the principle of harmonic/anharmonic
resonance, wherein the oscillation may or may not be an exact
integral factor or multiple of the base frequency. For instance,
light bandwidth to water’s absorption bandwidth is similar at 800
nm (0.36) and 1450 nm (0.34). Third, vibrations photo-induced
in water can be seen as Hamiltonian dynamic systems, which are
mathematical functions used to generate equations of motion [13].
Hamiltonian dynamics normally conserve energy, though they can
also exist in dissipative systems where a potential “drift in energy”
plays a significant role in phase transport. When their degrees of
freedom exceed 1, Hamiltonian dynamics are very complex [13].
Water molecules have 3 degrees of freedom in terms of movement,
plus 1/2 degree attributable to time-dependence. Hence, water
vibrations display extremely complex, nonlinear, time-dependent
chaotic behaviors.
Chaos is essential in nature for everything from the stability of
the solar system to human body homeostasis. Remarkably, in chaotic
systems, the interaction among nonlinear molecules with different
degrees of freedom is strongly enhanced despite the smallness of
coupling constants [14]. In addition, for multi-fractal systems such
as biological systems, resonances are not solely on the level set of
the Hamiltonian. On the contrary, such systems can be in resonance
while energy is transferred among different modes or trajectories
[13], magnifying energy absorption and transport through extended
biologic surfaces.
Resonant intermolecular energy transfer in liquid water and
aqueous solutions is much faster than vibrational energy relaxation
[15] and exhibits non-adiabatic relaxation (i.e., with net thermal
change). Vibration-to-electronic energy transfer rates are mode
specific and very fast, with the symmetric stretching mode surviving
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Photo-Infrared Pulsed Biomodulation 125

for less than 10 vibrational periods, or 50 fs (water memory) [16].

Quick energy transfer is possible because protons act as moving
targets, rapidly switching their character from one species to the
next based on the number of water molecules with which they are
associated [17]. At higher photon energies, typically in the visible
to ultraviolet (UV) range, electronic excitation occurs. This involves
promoting electrons to a higher energy set of orbitals.

8.3 Photo-Infrared Pulsed Biomodulation

PIPBM describes a water-based method for selective, noninvasive,

long-range, external light energy supplementation. It stands on
the premise that physiologic processes (oscillations) can often be
activated and synchronized by external stimuli acting through the
body’s preeminent medium, water, helping to restore homeosta-
sis/homeokinesis. While a full elucidation of the PIPBM can be found
elsewhere [7], it is worth reviewing the basis for its selectivity.
As part of PIPBM, multiple modulated laser beams are applied
at right angles to the surface of skin in areas of close proximity
to the biologically closed electric circuits (BCEC) and the vascular
interstitial closed circuit (VICC) [18]. The BCEC and its subset, the
VICC, are conceived to exist with the wall of arteries and veins
functioning as relative insulators around the electrically conducting
medium of blood, the plasma [18]. The vascular system further
satisfies the key geometric constraints of self-similarity and origin
of symmetry that characterize fractals, which behave as frequency-
independent antennas [19].
The multi-scale network of spatiotemporal biological fractals of
which the vascular system is part heightens energy capture and
transport. The efficiency of this fractal network of antennas is
augmented by the structure of the cell’s lipid bilayer, a liquid crystal
(LC) formed through the self-assembly of lipids, water, and other
biomolecules such as proteins and sterols [20]. LCs are sensitive
to pulsed electromagnetic fields (PEMFs), which make membranes
much better detectors of an applied field than isolated molecules in
solution.
Energy is transmitted to target tissues in accordance with the
second law of thermodynamics and its extension, Onsager’s theory
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126 Water as a Photoacceptor, Energy Transducer, and Rechargeable Electrolytic

of non-equilibrium [21]. This is possible because energy prefer-

entially flows to areas with the lowest energy state. Metabolism
and local degrading processes cause electrochemical polarization
leading to redox potential differences between degrading and well-
oxygenated tissues, which favor selective absorption by diseased
tissues.
Energy is transported to macromolecules and biopolymers pri-
marily by water and LCs. As LCs, membranes behave as transistors,
which transmit light energy to molecular bio-motors and ion pumps,
causing a cascade of bio-chemic, metabolic, bio-mechanic, and
hydrodynamic effects. On the other hand, the enhanced structuring
of water in the solvation shell of molecules favors the physiologic
activity of energy-dependent network proteins and signaling nodes
(e.g., P53, PTEN, NF-κB). Photo-induced effects are regulated by
metabolic control levels and are reinforced by the activation of DNA
open-state dynamics, which are consistent with reported reductions
in chromosomal aberrations [22, 23].
The following characteristics of higher-order biological sys-
tems favor the photo-activation and modulation of homeostasis/
homeokinesis via water dynamics in PIPBM:

• Complexity
• Energy dependence
• Electrochemical nature
• Thermodynamic openness (dissipation)
• Preeminence of aqueous media
• Robustness/fragility
• Non-linearity
• Determinism/non-complete determinism

A detailed description of these characteristics and their relationship

with external light energy supplementation can be found in Ref. [10].

8.4 Water Oscillator Paradox

According to the space it occupies, water in biological systems may

be classified as (1) bulk, (2) confined, and (3) interfacial water. The
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Water Oscillator Paradox 127

term “water oscillator paradox” refers to light’s unexpected potential

to induce effects over all three spaces, notwithstanding the relatively
small ACs at wavelengths of interest in PBM [8].

8.4.1 Bulk Water

Water is a polar molecule with more hydrogen bonds (HBs) (3.59
on average) than atoms [24]. A good HB donor and acceptor, water
can saturate polar surface exposed peptide groups nearly as well
as intra-protein HBs [25]. Although HBs are fragile and exist only
fleetingly [26], the average pole movement of condensed-phase
water (e.g., in tissues) can be enhanced 40% by a surrounding
electric field [27], such as one provided by NIR light energy. The
structuring of water by NIR light leads to nanoscale cages via HB
networks, which have deep implications over the modulation of
chemical reactions. This is significant because low-energy electric-
field dynamics and hydrogen bonding play major roles in auto-
ionization, and electric fluctuations in solvation energies can drive
the dissociation of oxygen HBs within tens of femtoseconds [28].
The application of radiant energy transfer via bulk water may be
illustrated by the following examples.

8.4.1.1 Application I: Light energy absorption and enhanced ATP

According to the structural basis of light energy and electron
transfer, NIR energy may undergo excitation energy transfer (very
weak coupling) and/or electron transfer from the excited to the
ground state as indicated by the Boltzmann law. Generally, cofactors,
pigments, and metal ions serve as energy acceptors and redox-
active elements in biologic materials such as proteins, acting as
polydentate ligands ascribed to a “rack mechanism.” The latter
idea is used to explain the unusual reactivity and redox properties
of amino acids and cofactors due to the distortion enforced by
the protein [25, 29]. In this process, enzymes act as intermediary
proteins with catalytic functions. The expected rate constants for
many membrane-associated enzymes lead to the expectation of
optimal coupling in the range from 10−3 to 10−6 V/cm, if a response
is found in a specific band of frequencies (102 –106 Hz, with
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128 Water as a Photoacceptor, Energy Transducer, and Rechargeable Electrolytic

Df > 10 Hz). Enzymes can convert energy from external fields to

drive the reaction that they catalyze. Thus, electric-field modulation
of an enzyme conformational transition can couple the flow of
chemical free energy through the conversion of substrate into
product to the flow of electrical free energy provided by an applied
periodic field [30].
In particular, Cox is essential to the classic photobiology action
mechanism [31]. Cox is an integral transmembrane protein that
contains four redox-active centers: binuclear CuA, CuB, hem a, and
hem a3, all of which absorb red-to-NIR light. Exactly how NIR light
interacts with Cox and increases mitochondrial energy generation
remains unclear [32]. Cox absorbs NIR photons from 700 to 900
nm, but absorption takes place in a part of the enzyme not involved
in nitric oxide (NO) binding, dissociation of which seems to be
a key player in PBM. NIR also seems to affect Cox in conditions
under which NO is unlikely to be present. In addition, as previously
stated, Cox absorbs only 50% of NIR light [8]. Correlated internal
electron- and proton-transfer reactions have been tracked in real
time into the oxidized enzyme (Cox). The resulting time of 3.56
ms [33] is slower by several orders of magnitude than the total
energy transport through water dynamics, from bulk liquid water to
confined spaces. Thus, NIR–water dynamics must precede/coexist
with and complement Cox energy transfer for increased effi-
ciency in ATP production, which can reach a daily turnover of
40 kg [10].

8.4.1.2 Application II: Light-modulated biomolecular motors

and pumps in aqueous media
One of the keys to the functioning of molecular pumps and motors
behind cell membrane activities consists in rectifying directionless
environmental energy to produce non-random effects, an idea
described as the Brownian ratchet principle. Weak PEMFs cause
non-equilibrium fluctuations that can bias Brownian motion [34].
This can induce biologically important phenomena, such as non-
adiabatic mechanisms that provide basis for chemically driven
electron pumping through a molecular wire, such as the D pathway
(for proton water molecules) in Cox.
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Water Oscillator Paradox 129

Experiments on ion pumps show that external electric or light

oscillations and fluctuations can substitute for energy from the
hydrolysis of ATP to power uphill ion transport. ATP synthase can
also synthesize ATP by using energy in the form of protons moving
down an electrochemical gradient [8, 35]. Pulsed light can transfer
energy to a system and, at the same time, rectify and modulate
highly complex vibrational effects induced in aqueous media [7]. For
instance, a frequency of 103 Hz—consistent with the rate of ATP
hydrolysis by many biologic motors—switched on and off can move
a small protein in aqueous media at 106 m/s (equivalent to 100
km/h for a car). Such a ratchet mechanism can overcome a force
of up to 10 pN, or nearly a million times the force of gravity on a
molecule.
Direction, velocity, and force of transport all depend on different
externally controllable parameters such as light phase, frequency,
amplitude, and temperature of the external modulation, as well
as the characteristic internal degrees of freedom of the Brownian
biomotor [36]. Experimentally [34] and clinically [37], weak
pulsed lasers have been found to power and modulate chemical
reaction rates through Brownian motion. Hence, external energy
supplementation may be used to profoundly and positively influence
the evolution of CDs [4, 8]. For instance, energy depletion is
thought to trigger photoreceptor death in macular degeneration and
retinitis pigmentosa [38]. A recent proteomic survey also revealed
altered energetic patterns and metabolic failure in photoreceptor
neurons prior to retinal degeneration [39]. In addition, mitochon-
drial damage has been implicated in the pathogenesis of AMD,
glaucoma, diabetic retinopathy, Parkinson’s disease, Alzheimer’s
disease, and other diseases of old age [8]. Thus, modulation of
Brownian motion through light–water interactions offers significant
therapeutic potential in medicine.

8.4.2 Confined-Space Water

Water confined to nonpolar pores (e.g., aquaporins) and cavities
of nanoscopic dimensions (e.g., proteins) exhibits highly unusual
properties that strongly differ from those of bulk water. Confined
water molecules are strongly cooperative, with the possible coexis-
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130 Water as a Photoacceptor, Energy Transducer, and Rechargeable Electrolytic

tence of filled and empty states, and form tightly hydrogen-bonded

wires or clusters [40]. Energy transfer in confined water has a
1.3–0.2 ps time constant, which is more than 20 times slower than
bulk water [41]. Energy transfer is governed by the rate at which
HBs brake and reform, which matches intramolecular transport
in macromolecules and biopolymers, including proton transport
through pathway D (molecular wire transfer) in Cox [42]. Under
physiologic conditions, weak attractions to the confining wall and
strong interactions between water molecules permit exceptionally
rapid water flow (3 × l09 mol/s) [43]. Proton mobility along ID
water wires exceeds that of the bulk. Proteins exploit this property of
confined water, for instance to ensure fast water flow in aquaporins
or to gate proton flow in proton pumps and enzymes such as
Cox. Conversely, abnormalities in confined water transport play
important roles in the pathophysiology of many CDs, including
neurologic and ophthalmic disorders, as well as cancer [4, 8, 37].
Confined water is sensitive to small perturbations of pore polarity
and solvent conditions, both of which can be manipulated by pulsed
NIR light [43]. Because NIR light can influence the properties
of water in nonpolar confinement, its use may lead to novel
nanofluid technology [44]. The roles of confined water transport
may further be exploited for clinical benefit through NIR modulation
of protein channels, such as those of the aquaporin family, and their
expression/function, for instance as diuretics and in the treatment
of brain swelling, glaucoma, epilepsy, obesity, and several forms of
cancer [8].

8.4.3 Interfacial Water: What is EZ?

The EZ is a quasi-crystalline, solute-free area of interfacial water
that can project from the surface of hydrophilic and hydrophobic
materials such as the cell membrane. The EZ can extend for
hundreds of thousands of layers and remain stable for weeks if
undisturbed. It is robust in physiologic conditions, such as pH values
near 7.35–7.45 and in saline solutions (typically 0.9 g NaCl/L).
It is an electron donor, or reducer, as opposed to non-EZ water,
which is a mild oxidant [3]. EZ’s absorption spectrum overlaps with
wavelengths relevant to PBM. Experimental studies have showed
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Water Oscillator Paradox 131

that all wavelengths can drive EZ expansion, but some wavelengths

were more effective than others. UV (including 270 nm) is the least
effective, visible light is more effective, and IR is the most effective,
particularly at 3000 nm. Ultrasound (US), typically around 7.5 MHz,
initially narrows and then greatly expands the EZ once removed [3],
suggesting a rubber effect and synergism.
Remarkably, the EZ-to-non-EZ interface resembles an electrolytic
pile, with redox potential jumps of fractions of a volt. The EZ can
store charge and return it as current flow with up to 70% of
input energy being readily obtainable [3, 4]. Although the law of
electro-neutrality implies that net charges should not be possible,
this seems violable over restricted volumes of water. Indeed, the
EZ is an unexpectedly effective charge separation, storage, and
release medium. The EZ behaves as a selective, rechargeable,
electrolytic bio-battery, which can act as a dedicated energy reserve.
It may, thus, selectively supplement the cell’s energy demands by
transferring electrons to the intracellular space when intracellular
redox potentials induced by injury or disease differ from those
normally associated with metabolic activity by more than one order
of magnitude. Hence, the EZ can be targeted as an alternative source
and/or reservoir of EM energy to cover energy demands following
injury-induced disturbances [4].
Potentials associated with normal intracellular metabolic activity
(∼−70 to −300 mV) are close to those associated with the EZ
(∼−100 to −200 mV) and are not strong enough to perturb it
[45]. However, injury-induced potentials, which can exceed 1.5 V
[18], can trigger electron transfer and energy supplementation from
the EZ to the injured cell’s intracellular space. Given the complex
electrochemical nature of human eukaryotic cells, electron transfer
from the EZ may activate and modulate not just bio-energetic
function, but also far-reaching signaling, for example via ATP, P1/P2,
and A1/A2/A3 receptors [46], cyclic adenosine monophosphate
(cAMP), and cyclic guanosine monophosphate (cGMP), which are
prominent members of the cell’s energy modulation system.
Experimental and clinical data support both the existence of
EZ and the ability of light to modulate it. EZ measurements
have been conducted using high-resolution (750 MHz) proton
nuclear magnetic resonance (1 H-NMR) spectroscopy in a wide
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132 Water as a Photoacceptor, Energy Transducer, and Rechargeable Electrolytic

Figure 8.3 Left-cheek metastasis of patient with malignant papillary

meningioma (R) post-IPLD treatment. (A) T2 weighted MRI. (B) Raw
microdensitometry: Red tones show high water content caused by photo-
induced cell death. Blue shades correspond to active tumor areas. The wide
divide is consistent with loss of cellular adhesion due to anoikis. Light blue
areas strongly suggest highly ordered water. Bright white zones suggest
fluorescence consistent with the presence of structured matrices associated
with the absorption of 270 nm light, charge separation, and pH differences.
Published in Ref. [49],  c International Journal of Cancer Therapy and
Oncology (IJCTO), 2014.

Figure 8.4 Images showing removal of internal limiting membrane aided

by preservative-free triamcinolone acetonide (TA) during macular hole
surgery. The arrows point to continuous whitish lines indicative of water-
layer ordering at the interface between collagen matrices and TA crystals.
Water ordering is denser and thicker on the image to the right because
of exposure to the surgical microscope’s light in accordance with prior
observations. (Images courtesy of Arturo Santos, MD, and Juan Carlos
Altamirano,MD, Centro de Retina Medica y Quirurgica, Guadalajara, Mexico).
Permission for reproduction granted by Alberto Lifshitz, MD, Editor of
Gaceta Medica de Mexico. Published in Ref. [8] and Ref. [50], 
c Mary Ann
Liebert, Inc. Publishers, 2010 and 2014 respectively.
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Water Oscillator Paradox 133

range of materials such as ion resins, polymers, functionalized

monolayers, and biological tissues [47]. It has been confirmed that
suspended particles (monodisperse colloids) are excluded from
studied surfaces by several hundred micrometers [3]. Moreover, in
prebiotic bacterial cells, it has been shown that the IR region of the
spectrum, of particular importance in LLLT, produces an EZ with
photo-induced charge differentials in the order of −100 to −200 mV
[3].
Using a 90 MHz (1.5 Tesla) 1 H-NMR spectrometer, the authors
showed that an LLLT-relevant 904 nm laser pulsed at 3 MHz (fluence
0.11 J/cm2 , peak power 35 mW) induced significant short-term
changes in T2 and correlation times (τ c) in rat soft tissue [25,
48], indicative of significantly greater structuring of water. An MRI
microdensitometry study of T2-weighted tumor heterogeneities
from a phase I clinical trial study using the same laser device
(fluence 45 J/cm2 ) in patients with advanced neoplasias and an
algorithm for tumor characterization also indicated significantly
increased structuring of water, showing a laser PBM effect over
the EZ associated with histologically confirmed selective photo-
induced tumor cell death [37]. These data support the premise
that laser PBM can increase potential energy in the EZ, which then
acts as an energy repository that can selectively supplement cell
energy demands. It further suggests EZ structuring may be used as a
predicator of anticancer response before measurable tumor volume
reduction (Fig. 8.3) [49].
In addition, a retrospective analysis of published data indicative
of EZ phenomena related to the retina and optic nerve (ON)
was conducted, using surgical microscopy and diffusion-weighted
magnetic resonance imaging, respectively. Pictures of the removal
of the internal limiting membrane aided by preservative-free
triamcinolone acetonide (TA) during macular hole surgery showed
continuous whitish lines, indicative of water-layer ordering at the
interface between collagen matrices and TA crystals (Fig. 8.4).
Apparent diffusion coefficient results further exhibit an axis parallel
to the ON, which may be an ocular expression of the EZ linked
to the steady potential of the eye. While existing results are still
being decoded, they suggest a new understanding of the eye’s
bio-energetic environment, which may have deep implications in
ocular physiology as well as in the pathophysiology, diagnosis, and
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134 Water as a Photoacceptor, Energy Transducer, and Rechargeable Electrolytic

treatment of blinding diseases using light-based therapies such as

PBM. Research is needed to confirm these findings and validate
potential ophthalmic applications [50]. Nevertheless, in view of the
above and given that the eye can be described as an electrochemical
aqueous organ and that water is a key photoacceptor, PBM may
represent a potentially powerful multi-hallmark monotherapy for
AMD and other eye CDs [6].

8.5 Metabolism and Scaling Laws

According to Kleiber’s law, the basal metabolic rate (BMR) of living

organisms is roughly equal to mass (in kg) raised to 3/4 power
(i.e., BMR = M0.75 ) [9]. Namely, as an organism’s size increases,
metabolism must increase less rapidly than volume for heat to be
sufficiently dissipated. Recently, researchers have found that the
existence of this ratio in mammals is made possible by the rate
at which nutrients are carried and heat is removed by the heart,
which allows the positive scaling of the blood velocity (Ml/12).
Kleiber’s law is used to calculate dosage in drug experiments.
We propose it may also be used as a scientific basis to calculate
dosage for human external light energy supplementation. This
implies that PBM doses may be calculated from mass (∼70% water)
and circulatory flow rate, measured by Doppler. Water acts as
a photoacceptor, energy transducer, and rechargeable electrolytic
bio-battery. Thus, calculated metabolism would reflect physiology
or pathophysiology, wherein the relationship between light and
water dynamics has a pivotal role in the (re)establishment of
homeostasis/homeokinesis, expressed as health versus disease. If
confirmed, external energy supplementation would substantiate its
value as a universal approach for the treatment of CDs, alone or in
association with other agents.

8.6 Conclusion

Laser PBM has been proposed as an approach to substitute

and/or complement metabolic energy pathways through oxygen-
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References 135

dependent and/or oxygen-independent mechanisms with critical

signaling pathways in primarily aqueous media. Long considered
an innocuous medium, water has increasingly been found to be
a key player in physiologic mechanisms. Consequently, external
radiant energy may selectively target the organization of water
to steer biologic function. Cellular and molecular bases for water-
mediated, long-range, energy supplementation aimed at inducing
and modulating physiologically reparative processes, including
apoptosis, were presented through PIPBM. Subsequently, the role
of water as an oscillator was discussed, adding to a more coherent
description of the central effects of NIR light over redox centers
and key transmembrane enzymes, such as Cox. Water provides
a pathway for NIR, MIR, and FIR absorption and transportation,
complementing and facilitating Cox energy transfer for increased
efficiency in the production of ATP—a vital molecule required
not only for energy but also as part of the signaling pathways of
growing importance in cancer and other CDs. These ideas were
complemented by integrating the role of the EZ. Data support the
premise that PBM can increase potential energy in the EZ, which
then acts as a rechargeable electrolytic bio-battery for the external
selective supplementation of energy required for cellular work,
signaling pathways, and gene expression in the presence of injury-
induced redox potentials. Lastly, we proposed the use of Kleiber’s
law for external light energy dosage calculation. While not a panacea,
this approach may lead to novel therapies that aim at reestab-
lishing homeostasis/homeokinesis via light–water interaction in
tissues.

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July 6, 2016 17:19 PSP Book - 9in x 6in 09-Hamblin-c09

Chapter 9

Role of Reactive Oxygen Species in

Low-Level Laser Therapy

Vikrant Rai
Center for Clinical and Translational Science, Creighton University,
2500 California Plaza, Omaha, Nebraska 68178, USA
vikrantrai@creighton.edu

Mitochondrial stimulation by low-level laser therapy (LLLT) in-

creases cytochrome c oxidase (Cox) activity, protein and DNA
synthesis, and the production of ATP and reactive oxygen species
(ROS) such as hydrogen peroxide, singlet oxygen, hydroxyl radical,
and superoxide dismutase. Chromophores such as Cox, cytochrome
c, cytochrome b, flavins, porphyrins, hemoglobin, and NADPH
oxidases act as photosensitizers and absorb a specific wavelength
of light, producing ROS. Increased ROS in cell, in turn, causes
oxidative stress, resulting in the activation of various transcription
factors such as NF-κB, AP-1, HSP, and JNK, which increases signaling
pathway and gene expression leading to increased protein synthesis.
This increases cell proliferation, neovascularization, and collagen
synthesis, leading to hastened wound healing and decrease in pain,
swelling, and inflammation.

Handbook of Low-Level Laser Therapy

Edited by Michael R. Hamblin, Marcelo Victor Pires de Sousa, and Tanupriya Agrawal
Copyright c 2017 Pan Stanford Publishing Pte. Ltd.
ISBN 978-981-4669-60-3 (Hardcover), 978-981-4669-61-0 (eBook)
www.panstanford.com
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142 Role of Reactive Oxygen Species in Low-Level Laser Therapy

9.1 Mitochondrial Response to LLLT

Mitochondria are the primary target for red and near-infrared (NIR)
lights (Karu, 1989) and are responsible for cellular response. The
effect of LLLT at cellular level can be attributed to the absorption
of red and NIR lights, by cellular respiratory chain in mitochondria
(Karu, 1989) and Cox (unit IV of mitochondrial respiratory chain)
(Karu and Kolyakov, 2005; Szundi et al., 2001). LLLT-induced
mitochondrial stimulation causes an increased production of ATP
and ROS, influencing redox signaling, affecting intracellular home-
ostasis and proliferation of cells (Tafur and Mills, 2008). Cox in
mitochondria is the main chromophore accepting the laser level
light (Karu and Kolyakov, 2005). Mitochondrial respiration can be
inhibited by competitively displacing oxygen by the nitric oxide
produced in mitochondria and by binding to Cox in a hypoxic or
stressed cell. LLLT can increase nitric oxide concentration in cell
and can also reverse the mitochondrial inhibition due to increased
nitric oxide by photo-dissociating nitric oxide from Cox (Huang
et al., 2009). It has been suggested that after light delivery to
isolated mitochondria, there is an increased activity of Cox after
LLLT (Pastore et al., 2000), increased synthesis of ATP (Karu, 1999),
increased RNA and protein synthesis (Greco et al., 1989), and
increased synthesis of NADH, oxygen consumption, and membrane
potential (Huang et al., 2009). Increased mitochondrial activity,
membrane potential, and ATP synthesis have been seen after the He–
Ne laser illumination of melanoma cell. Increased phosphorylation
of Jun N-terminal kinase (JNK) and activation of AP-1 leading
to cell proliferation along with increased activity of Cox have
been proposed (Hu et al., 2007). Increased electron transport and
increased oxygen consumption by Cox have been shown after the
irradiation of mitochondria after He–Ne laser (Pastore et al., 2000).
To have the maximum effect of LLLT, the set of wavelengths of
laser is specific (Bjordal et al., 2008), and using LLLT below that
range is not effective (Bjordal et al., 2003). A biphasic dose response,
frequently described by Arndt–Schulz curve, shows that low levels
of light have much better effect on cellular level than higher levels
of light. LLLT typically uses light of narrow spectral width in red
or NIR spectrum (600–1000 nm), laser in the range of 1–500 mW
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LLLT-Induced Production of ROS 143

with a power density in the range of 1 mW to 5 W/cm2 (Huang et

al., 2009). It has been proposed that different chromophores act on
different wavelengths. Blue (400–500 nm) and green (510–550 nm)
lights are absorbed by flavins and flavoproteins, yellow (560–590
nm) and red (600–700 nm) lights by porphyrins, and red (600–700
nm) and NIR (710–1100 nm) lights by Cox (Chen et al., 2009).

9.2 LLLT-Induced Production of ROS

Reactive oxygen species are reactive molecules containing oxy-

gen, which are formed as the natural byproduct of the normal
metabolism of oxygen. ROS have important role in homeostasis and
cell signaling. Environmental stress can cause a dramatic increase in
ROS production (Devasagayam et al., 2004). Examples of ROS include
superoxide, hydrogen peroxide, singlet oxygen, and hydroxyl radical.
Photosensitizers such as Cox, flavins, and porphyrins, NADPH
oxidase containing flavoprotein, and cytochrome b and c respond
to LLLT, but Cox is the main chromophore responding to LLLT and
produces ROS (Lubart et al., 2005).

9.2.1 Cytochrome
Cytochromes are inner mitochondrial membrane-bound hemepro-
teins containing heme groups and are found either as monomeric
proteins (cytochrome c) or as subunits of bigger enzymatic
complexes catalyzing redox reactions. Cytochromes are primarily
responsible for the generation of ATP via electron transport chain in
mitochondria. Cox in mitochondria mainly absorbs light in red and
NIR region and produces ROS (Karu, 1989) (Fig. 9.1). Cox (unit IV of
respiratory chain) absorbs red and NIR lights, and the resemblance
of absorption spectra of Cox with the absorption spectra of visible
light for various cellular functions has been proposed (Karu and
Kolyakov, 2005). Several laboratories have reported that LLLT
generates ROS in vitro (Alexandratou et al., 2002; Lubart et al., 2005;
Pal et al., 2007) and that ROS are involved in signaling pathways
initiated after light photons are absorbed by mitochondria (Tafur
and Mills, 2008). Some groups have also reported increase in cellular
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144 Role of Reactive Oxygen Species in Low-Level Laser Therapy

Figure 9.1 Schematic diagram showing the absorption of red and NIR
lights by specific cellular chromophores or photoacceptors localized in the
mitochondrial respiratory chain. Adapted from Huang et al., 2009. Copyright
c 2009 University of Massachusetts.

ROS after LLLT (Callaghan et al., 1996; Grossman et al., 1998; Lavi
et al., 2003; Lubart et al., 2005; Zhang et al., 2008).

9.2.2 Flavins
Flavins are water-soluble molecules present in cells primarily as
FAD and FMN and initiate free radical reaction when excited by
light of wavelength 500 nm. Flavins do not absorb wavelength above
500 nm. Most flavin molecules are found bound to protein in cell,
and this may reduce their activity. Only free or loosely bound flavins
act as photosensitizers. Eichler et al. (2005) have proposed that ROS
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LLLT-Induced Production of ROS 145

are created during low-energy visible light illumination of tissue

by an endogenous photosensitizer, and these ROS are responsible
for reported biostimulative effects. Endogenous photosensitizer
is predominantly found in cytosol and is smaller than 12 kD.
Flavin mononucleotide produces the same signal at concentrations
consistent with reported intracellular free flavin concentrations.
Using cardiac and sperm cells, it has been proposed that flavins are
responsible for the photosensitization of the observed oxyradicals
in cells and these oxyradicals are detected by using electron
paramagnetic resonance (EPR). Oxyradical production has been
determined as a function of illumination wavelength, cell fraction,
and molecular weight. It has been proposed that oxyradicals are
created solely by the 400–500 nm range of visible light (Eichler et al.,
2005).
The study of the relationship between increased intracellular
calcium (Ca2+ ) and ROS production following LEVL illumination of
cardiomyocytes has shown the production of ROS by LEVL. It has
been found that visible light causes the production of singlet oxygen
and H2 O2 and also that exogenously added H2 O2 can imitate the
effect of LEVL (3.6 J/cm2 ) to induce a slow and transient increase
in calcium (Ca2+ ). The elevated level of calcium (Ca2+ ) can be
reduced by verapamil, a calcium channel inhibitor (Lavi et al., 2003).
The dose-dependent nature of morphologic damage following light
or addition of H2 O2 and the kinetics of Ca2+ elevation have also
been suggested. H2 O2 has been measured by luminol, and singlet
oxygen has been measured by the EPR spin-trapping technique
using DEPMPO to show the generation of ROS. The kinetics of the
increase in Ca2+ has been found to reflect an adaptive response of
the cells to oxidative stress (Lavi et al., 2003).

9.2.3 Porphyrins
Porphyrins are organic compounds, and several of them are
naturally occurring. Heme is an example of porphyrin. As mentioned
earlier, porphyrins are photosensitizers having intense absorption
bands (yellow and red) in the visible region and may be deeply
colored. ROS can also be generated in porphyrins after LLLT,
and Kolarova et al. (2008) have proposed that second-generation
July 6, 2016 17:19 PSP Book - 9in x 6in 09-Hamblin-c09

146 Role of Reactive Oxygen Species in Low-Level Laser Therapy

sensitizer porphyrins (TPPS4 , ZnTPPS4 , and PdTPPS4 ) produce ROS

in G361 cell line after photodynamic therapy. It has been proposed
that ZnTPPS4 has the highest efficacy in ROS production and
photodynamic dose depends on sensitizer concentration, oxygen
concentration, and fluence (effective light dose) applied (Kolarova
et al., 2008). ROS production has been measured by dose-dependent
CM-DCF fluorescence, with a light dose of 25 J/cm2 . It has also
been demonstrated that a high dose of irradiation does not have
a significant increase in ROS production and even a higher dose
(more than 25 J/cm2 ) can reduce ROS production. Production of ROS
mainly corresponds to phototoxic effect, and higher ROS generation
can cause larger cell photodamage (Kolarova et al., 2008).
ROS production from porphyrins after photodynamic therapy
can have genotoxic potential in rodents. This photosensitization is
mediated by 5-aminolevulinic acid. Porphyrins and ALA can also
generate ROS in the absence of activating light. Autoxidation of
ALA can produce ROS, and autoxidation of enolic ALA can produce
superoxide anion, hydrogen peroxide, and hydroxyl radical and can
damage cellular structure (Fuchs et al., 2000).
Callaghan et al. (1996) have delivered LLLT to the hemopoietic
cell line U937 using a diode laser (660 nm, 12 mW, 5 kHz).
With fluences of 2.9 and 8.6 J/cm2 , increased superoxide and
hydrogen peroxide production in the differentiated form of the
cells has been found, but decreased DNA synthesis has been found
by the incorporation of tritiated thymidine. Extracellular catalase
addition repudiates the reduction in DNA synthesis caused by LLLT
(Callaghan et al., 1996).
Using a 647 nm laser to illuminate single human fetal foreskin
fibroblast cell on a confocal microscope delivering a small dose of
0.0015 J/cm2 , Alexandratou et al. (2002) have found that DCDHF
oxidation reached a maximum at 8 min post-illumination. An
increase in mitochondrial membrane potential, a periodic oscillation
in cytosolic calcium, and increase in cellular pH have also been
measured, and an increase in ROS production and cell redox
potential has been established (Alexandratou et al., 2002).
Pal et al. (2007) have proposed ROS production from fibroblasts
after LLLT, using fiber-optic-based nanoprobes to deliver LLLT to a
single cell of normal human skin fibroblasts. ROS has been measured
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LLLT-Induced Production of ROS 147

by using DCDHF. A dose-dependent increase in cell proliferation

with a maximum at 16 J/cm2 has been found (Fig. 9.3). The ROS
generation depends on both laser intensity and total laser energy
dose delivered to the single cell. It has also been proposed that there
can be a change in the characteristics of ROS generation, such as
time delay and amount of ROS, by instantaneous irradiation at a
certain threshold energy. It has also been suggested that the dose-
dependent generation of ROS depends on irradiance, total fluence,
and exposure time (Pal et al., 2007).
Lubart et al. (2005) have proposed that the stimulatory effect
of LEVL on different biological systems is by light-induced ROS
formation in living cell. On illuminating both sperm and skin cells
with LEVL, the generation of singlet oxygen has been detected
by using a trapping probe TEMP coupled with EPR spectroscopy.
It has also been shown that together with O2 generation, LEVL
illumination increases the reductive capacity of the cells. The
observed biostimulative effects exerted by LEVL can be explained by
the potential of visible light to change the cellular redox state (Lavi
et al., 2004).
ROS production in mouse embryonic fibroblasts after LLLT
has been shown by using DCDHF and dihydro-rhodamine 123
fluorescent probes. Chen et al. (2011) have used 810 nm and
980 nm lasers and found a very wide dose response with fluences of
0.03 J/cm2 for a positive effect. The maximum effect has been found
between 0.3 J/cm2 and 3 J/cm2 and the reduced effect has been
shown at 30 J/cm2 . The increase in ROS linked with the activation
of NF-κB transcription factor can be prevented by the addition of
antioxidants N-acetylcysteine and vitamin C (Chen et al., 2011).
It has also been proposed that mitochondrial inhibitors increase
ROS and NF-κB activation but not ATP and that LLLT upregulates
ROS production (Chen et al., 2011). An increase in ROS production
and cell redox potential after LLLT has been established by various
laboratories (Alexandratou et al., 2002; Chen et al., 2011; Grossman
et al., 1998; Lavi et al., 2003; Lubart et al., 2005; Pal et al., 2007;
Zhang et al., 2008).
Sunlight can be used as an antidepressant in the treatment of
winter seasonal depression. Oren et al. (2001) have demonstrated
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148 Role of Reactive Oxygen Species in Low-Level Laser Therapy

that the irradiation of normal human NIH/3T3 non-pigmented

fibroblast with visible light source results in the production of ROS
after 10 min of irradiation. The ROS production has been measured
by electron spin resonance (ESR). The production of hydroxyl radical
can be suppressed by the antioxidant vitamin C (Oren et al., 2001).
ROS generation by blue laser has been studied by Kushibiki
et al. (2013) using various cells: mouse preadipocytes (3T3-L1), my-
oblasts (C2C12), prechondrocytes (ATDC5), mesenchymal stromal
cells (KUSA-A1), lung cancer cells (LLC), insulinoma cells (MIN6), fi-
broblasts (NIH-3T3), macrophages differentiated from lymphocytes
(THP-1) after treatment with phorbol ester, rat basophilic leukemia
cells (RBL-2H3), and human cervix adenocarcinoma cells (HeLa).
It has been found that there is an increased level of ROS after
irradiation with blue laser but not after red or NIR. ROS production
has been measured by the intracellular ROS probe CM-H2DCFDA in
a flow cytometer (Kushibiki et al., 2013).

9.3 Role of Reactive Oxygen Species

9.3.1 Oxidative Stress at Cellular Level Due to ROS

Oxidative stress is defined as the imbalance between the production
of ROS and the biological system’s ability to readily detoxify the
reactive species (reduced antioxidant defense) (Huang et al., 2013).
Disturbances in the normal redox state of cells can cause toxic
effects via the production of free radicals and can damage the
cell components by lipid peroxidation, DNA strand break, and
protein fragmentation. Oxidative stress can also cause disruptions
in the normal mechanisms of cellular signaling as some ROS act as
cellular messengers in redox signaling. Moderately increased level
of intracellular ROS causes oxidative stress. ROS-induced oxidative
stress can be harmful even at low concentration (Huang et al.,
2009). ROS has an important role in cell signaling pathways from
mitochondria to nucleus, regulating cell cycle progression, protein
synthesis, nucleic acid synthesis, and enzyme activation. Increased
ROS production can alter the redox activity of cell, and there may be
some transcriptional changes (Huang et al., 2009).
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Role of Reactive Oxygen Species 149

9.3.2 Antioxidant Effect of LLLT

Although LLLT produces ROS resulting in oxidative stress, it can
also be used to decrease oxidative stress. Giuliani et al. (2009) have
proposed that red light can protect the viability of cultured neuronal
cells in the presence of oxidative stress. Red light has also been
found to stimulate neurite outgrowth and to have protective action
on axons (Giuliani et al., 2009).
LLLT causes the production of ROS, but Huang et al. (2014)
have shown the suppression of ROS production after LLLT by using
810 nm laser. It has been found that reduction in the viability
of cortical neurons caused by the oxidative stress of rotenone,
H2 O2 , and CoCl2 can be reversed by LLLT irradiation and that
primary cortical neurons can be rescued from cell death, suggesting
a correlation between LLLT and its antioxidant properties. LLLT can
have beneficial effect on neuronal and non-neuronal pathways pro-
viding improved mitochondrial function, increased cell viability, and
decreased oxidative stress. LLLT-induced increased ROS production
in non-stressed cell and reduced ROS production in stressed cell are
associated with increased MMP activity. In the presence of rotenone,
H2 O2 , and CoCl2 , it has been found that there is a reduction in MMP
activity with increased ROS production, but when LLLT is irradiated
to such cells, there is an increase in MMP activity and decrease
in ROS production from mitochondria (Huang et al., 2014). The
antioxidant effect of LLLT on hypoxic, stressed, or damaged cells is
more than that on normal cell. The correlation between LLLT and its
antioxidant property by the activation of cellular antioxidant system
has also been proposed by Silveira et al. (2007). Lim et al. (2011)
have also proposed the antioxidant property of LLLT by using 635
nm light irradiation on CoCl2 -induced hypoxic human umbilical vein
endothelial cell and have shown that ROS production decreased in
stressed condition, causing increased angiogenesis and cell viability
(Lim et al., 2011).

9.3.3 Cellular Response to Increased ROS (Oxidative Stress)

In response to oxidative stress caused by an increase in superoxide,
hydrogen peroxide, and other ROS, there is an increased gene
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150 Role of Reactive Oxygen Species in Low-Level Laser Therapy

expression or activation of various intracellular signaling pathways

for genes, required for the detoxification of reactive molecules,
for the repair and maintenance of cellular homeostasis. These
induced genes are regulated by transcription factors, and the level of
oxidative stress regulates their subcellular localization, structure, or
affinity for DNA. Activation of these signaling pathways regulates the
transcriptional changes that permit the cell to respond to oxidative
stress (Liu et al., 2005).
These transcription factors can also alter their activity in
accordance with cellular redox conditions along with regulation
achieved by classical cytosolic signaling pathways, such as the
family of mitogen-activated protein kinases, c-Jun N-terminal kinase
(JNK), and p38 kinase family. Protein kinase D, nuclear factor
kappa B, cellular analog of Rous sarcoma virus, glutathione (GSH),
JNK, activator protein-1 (AP1), nuclear factor erythroid 2 related
factor 2 (Nrf2), heat shock factor (HSF-1), and thioredoxin (Trx)
apoptosis signal regulating kinase-1 (ASK1) are some of the signal
transduction intermediates, transcription factors, and ROS sensors,
governing cellular response to oxidative stress. Because of the
ubiquitous nature of oxidative stress and the damaging effects of
ROS, cells respond to these by the modulation of their antioxidant
levels, induction of new gene expression, and protein modification
(Liu et al., 2005) (Fig. 9.2).
The redox status of a cell within a very narrow range is
maintained by homeostatic modulation of oxidant levels. Molecular
sensors within the cells detect ROS by getting oxidized. The new
chemical structures formed initiate signal transduction pathways
and lead to a cascade of reactions. It has been proposed that
some amount of these oxygen radicals are released by different
cell types in response to stimulation with interleukin 1 (IL-1),
tumor necrosis factor (TNF), and phorbol esters and activate
the transcription factor NF-κB by releasing an inhibitory protein
subunit. The activation of transcription factors can be inhibited
by antioxidants or oxygen radicals removing agents resembling
exposure to mild oxidant stress. Oxygen radicals act as second
messengers for the immunomodulatory cytokines TNF and IL-1, in
regulatory pathway activating NF-κB (Schreck and Baeuerle, 1991).
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Role of Reactive Oxygen Species 151

ROS induced oxidave stress

Acvaon of various transcripon

factors such as NF-kB, AP-1, ASK,
NrF-2, HSF-1, PKD, GSH, and JNK

Increased gene expression, protein

synthesis, and cell proliferaon

Figure 9.2 Sensors to oxidative stress and their effect.

9.3.4 Response of Various Transcription Factors to

Oxidative Stress
Activator protein 1 (AP-1) regulates gene expression in response
to a variety of stimuli such as oxidative stress, growth factors,
cytokines, and viral and bacterial infections. AP-1 is a transcription
factor regulating the transcription of various genes. It upregulates
the transcription of 12-O-tetradecanoylphorbol-13-acetate (TPA)-
responsive elements (TREs) in their promoter region of DNA
recognition sequence TGA (C/G) TCA. AP-1 consists of proteins
belonging to Fos and Jun (c-Fos, c-Jun, JunB, JunD), ATF and JDP
protein families. These proteins consist of a “leucine zipper,” which
permits Jun proteins to form homodimers or heterodimers with
Fos proteins or among themselves. Fos proteins cannot dimerize
and bind DNA on their own. AP-1 binds to this DNA sequence via
a leucine zipper. AP-1 also controls some other cellular processes,
including proliferation, differentiation, and apoptosis (Huang et al.,
1997).
Oxidative stress has been involved in the pathogenesis of several
inflammatory processes because of its effect on pro-inflammatory
gene transcription. Oxidative stress activates NF-κB-dependent
transcription of pro-inflammatory mediators. Cysteine residues do
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152 Role of Reactive Oxygen Species in Low-Level Laser Therapy

not form disulfide bonds, unless intracellular redox balance is tilted

toward oxidant stress. The conformation and activity of a number
of enzymes, mainly phosphatases, can be altered by the formation
of disulfide bonds. The activity of protein kinases can be limited
by these enzymes. Prolonged activity for these kinases results
from the inactivation of a specific phosphatase by oxidant stress
(Rajendrasozhan et al., 2008).
Prolonged kinase activity indicates prolonged activation of
intracellular signal cascades. These alterations in the signal cascades
proceeding through successive phosphorylations of kinases con-
clude in the phosphorylation of proteins in many cell compartments
(mitochondria or nucleus). This modification of specific regulatory
proteins may result in some changes, from ionic signals to wide
alterations in patterns of gene expression. As a result, a cell may
die or change its rate of proliferation, depending on the operating
signal network. The maintenance of the rate of cell proliferation has
been linked to the intracellular oscillation of oxidant levels. Irani et
al. (1997) have shown that production of ROS can be suppressed by
flavoprotein inhibitors. The production of ·O2 − involving Rac1 and
a flavoprotein can be caused by H-RasV12 -induced transformation.
The probable mechanism for the effects of antioxidants against
Ras-induced cellular transformation has been suggested by the
insinuation of a ROS as a mediator of Ras-induced cell cycle
progression independent of MAPK and JNK (Irani et al., 1997).
Involvement of ROS in the enhanced proliferation of normal human
keratinocyte by 780 nm laser irradiation has been proposed by
Lubert et al. (Grossman et al., 1998) (Fig. 9.3).
As mentioned earlier, NF-κB is a transcriptional factor regulating
many gene expressions and many cellular functions such as
cellular response to stress, inflammation, and injury. NF-κB activity
is monitored by the level of intracellular ROS. NF-κB plays an
important role in inflammation, oxidative stress, apoptosis, immune
stress, and environmental stress. NF-κB can be activated by LLLT as
well as ROS (Chen et al., 2011).
NF-κB can be activated by the illumination of mouse embryonic
fibroblast with 810 nm or 980 nm light, and its activation can be
measured by luminescence assay. NF-κB activation also depends
on the production of ROS in mitochondria after LLLT. Exposure to
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Role of Reactive Oxygen Species 153

light causes the phosphorylation of NF-κB inhibitor IKKα, and IKKβ

causes the activation of NF-κB. Negative feedback by IKKβ regulates
the activation of NF-κB and ubiquitination, and proteasomal
degradation can also occur, freeing NF-κB and translocating it to
nucleus and initiating transcription (Chen et al., 2011). Exposure
of various cells to H2 O2 can induce the activation of NF-κB, and
this activation is highly cell dependent (Schreck et al., 1991). H2 O2 -
mediated activation of NF-κB may be regulated by the reduced level
of GSH, an intracellular thiol and ROS scavenger, and it may differ
from one cell to another (Meister and Anderson, 1983). NF-κB can
be activated by ROS using 810 nm laser light directly or by the
involvement of tumor necrosis factor alpha (TNFα), interleukin-1
(IL-1), and phorbol ester (Chen et al. 2011). ROS-induced activation
of NF-κB has also been proved by adding antioxidants, which
stopped the activation.
LLLT and mitochondrial inhibitors can produce ROS and ac-
tivate NF-κB, but both have different effect on ATP generation.
Laser increases ATP production, while mitochondrial inhibitors
decrease ATP production (Chen et al., 2011). Activated NF-κB, as
a transcription factor, is responsible for the beneficial effect of
LLLT. Myofibroblast differentiation, proliferation, and migration of
cells regulating proteins, pro-survival and anti-apoptotic proteins all
respond to activated NF-κB (Campbell and Perkins, 2006) (Fig. 9.3).

9.3.5 ROS-Mediated Effect of LLLT on Nervous System

Excessive stimulation of receptors for excitatory neurotransmitter
glutamate in the central nervous system can damage neurons by
excitotoxicity. Exposure of murine primary cultured cortical neuron
to LLLT (810 nm) can increase intracellular calcium level, ATP
synthesis, and mitochondrial membrane potential (MMP). MMP
level increases in both stressed and normal cells. ROS production
reduces in stressed cell but increases in control neurons after LLLT.
Only a significant reduction in ROS can be found in kainate-treated
neurons. Cytoplasmic ROS are measured by CellROX. Excess calcium
opens mitochondrial permeability transition (MPT) pore and ROS
are produced as a result. The organelle’s ability to neutralize ROS
can be decreased by antioxidants such as glutathione exiting from
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154 Role of Reactive Oxygen Species in Low-Level Laser Therapy

Fibroblast
Keratinocyte
Endothelial cell
↑ed cell
proliferation
Lymphocyte
Muscle satellite cells
LLLT induced ↑ROS

Thrombosis and
Platelet activation
hemostasis

Figure 9.3 Cellular effects of reactive oxygen species.

mitochondria owing to opened MPT pore. Two effects of LLLT have

been observed: first, increasing viability, ATP, and MMP in both
normal and excitotoxic neurons; second, raised ROS, NO, and Ca2+
in normal neurons but decreased ROS, NO, and Ca2+ in excitotoxic
neurons. Transient increase in MMP in normal neurons can produce
ROS (Huang et al., 2014).
By illuminating primary cortical neuron culture from embryonic
mice brain with 810 nm laser, Sharma et al. (2011) have proposed
that lower fluences of light cause a significant increase in ATP,
calcium, and MMP and higher fluences decreased their level. Also
there has been an induction of ROS at low fluences, and there has
been a decrease followed by increase in ROS at 30 J/cm2 . Similar
results have been predicted for nitric oxide. It has been suggested
that higher fluences may cause a damaging effect by higher level
of ROS and nitric oxide, while there is a beneficial stimulatory
effect at lower fluences by the induction of cell signaling process
(Sharma et al., 2011). High fluence low-power laser irradiation (HF-
LPLI, 633 nm, 120 J/cm2 ) can increase MPT via the activation of
cytochrome c. An increase in ROS has also been found, and this
increase can cause the onset of MPT. Using human lung carcinoma
cell, Wu et al. (2009) have shown that there is an HF-LPLI-induced
cell apoptosis mediated by MTP, whose activation is ROS dependent.
It has also been suggested that ROS production is endogenous as
well as due to oxidative stress (Wu et al., 2009).
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Role of Reactive Oxygen Species 155

Zhang et al. (2008) have proposed that ROS act as the

second messenger in the biostimulatory effect of low-power laser
irradiation (LPLI) using He–Ne laser on Hela cells. Increase in ROS
generation and Src activation in Hela cells has been seen in a
dose- and time-dependent manner after LPLI. Src tyrosine kinases
act as a target for ROS and oxidative stress. Src activation can be
abolished by antioxidants such as vitamin C, superoxide dismutase,
and catalase but cannot be affected by PKC inhibitor Go6983. Src
tyrosine kinase can also be activated by exogenous H2 O2 in a
concentration-dependent manner. It has been proposed that there
is an ROS-mediated Src activation after LPLI and that low doses
promote and high doses impair the viability of Hela cells (Zhang
et al., 2008). It has also been proposed that activation of protein
kinase Cs (PKCs) by high glucose concentration lead to increased
production of ROS, and this ROS further activate PKCs, providing
signal amplification in cells (Lee et al., 2004) (Fig. 9.3).
Masoumipoor et al. (2014) have suggested the probable use of
LLLT in the treatment of neuropathy using a mouse with sciatic
nerve injury and the chronic constriction injury (CCI) model. ROS
and oxidative stress can induce neuropathic pain. LLLT effect
has been observed on using low-level laser of 660 and 980 nm.
Mechanical and thermal threshold significantly increase after LLLT
but decrease after CCI. A better therapeutic effect is observed
with 660 nm wavelength in comparison to 980 nm wavelength,
and 680 nm can be used in the clinical treatment of neuropathy
(Masoumipoor et al., 2014).
LLLT increases ATP, ROS, and intracellular calcium and can play a
role in the treatment of degenerative or traumatic brain disorders/
injury, stroke, spinal cord injury, peripheral nerve regeneration, and
myocardial infarction mediated by the activation of transcription
factors leading to the expression of many anti-apoptotic, antioxidant,
protective, and pro-proliferation gene products. LLLT can also play a
role in neurorehabilitation (Hashmi et al., 2010).

9.3.6 ROS-Mediated Apoptosis

Photodynamic therapy (PDT) can provoke apoptosis in human
epidermoid carcinoma A431 cells using visible light. Mitochondrial
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156 Role of Reactive Oxygen Species in Low-Level Laser Therapy

involvement in light-induced apoptosis has been studied using

silicon phthalocyanine 4 (Pc 4) localized primarily in cytosolic
membranes but not entirely in mitochondria. ROS are produced
when cells are exposed to Pc 4 and 670–675 nm light. Mitochondrial
inner membrane permeabilization, depolarization and swelling,
cytochrome c release, and apoptotic death have been noted after
the release of ROS. Desferrioxamine can prevent mitochondrial ROS
production. Mitochondrial inner membrane permeabilization and
depolarization can be inhibited by cyclosporin a + trifluoperazine,
but it does not affect mitochondrial ROS generation (Lam et al.,
2001).

9.3.7 ROS Effect on Thrombosis and Hemostasis

Activated platelets play an important role in primary hemostasis
and arterial thrombosis. Activation of platelet is a complex process
involving many signaling pathways. ROS can play an important role
in platelet activation. Reduced nicotinamide adenine dinucleotide
(NADPH) oxidase in phagocytic cells can generate ROS. Intracellular
ROS involving in cellular signaling can also be produced by
nonphagocytic NADPH oxidase isoforms found in endothelial cells,
fibroblasts, vascular smooth muscle cells, and vessel wall. ROS
act as the second messenger in gene expression, apoptosis, and
proliferation. ROS may regulate platelet function by decreasing NO
bioavailability. Platelets also have NADPH oxidase subunits, and
NADPH oxidase activation in platelets produces intracellular ROS,
which are detected by 2 7 -dichlorodihydrofluorescein diacetate
(H2DCF-DA). ROS involvement in the regulation of thrombin-
induced integrin αIIbβ3 activation without affecting granule secre-
tion, NO/cGMP pathway, and platelet shape shows its role in integrin
regulation. ROS can be produced by thrombin receptor activating
peptide 6 (Trap6), collagen-stimulated platelet, stable thromboxane
A2 analog U46619, and thrombin-stimulated platelets but not
with adenosine diphosphate (ADP) stimulation. Intracellular and
extracellular ROS production by neutrophil after phorbol myristate
acetate (PMA) stimulation has also been shown (Begonja et al.,
2005) (Fig. 9.3).
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Role of Reactive Oxygen Species 157

9.3.8 ROS-Mediated Effect of LLLT on Musculoskeletal

System
ROS can have an antibacterial action and can also mediate anti-
inflammatory effect by prostaglandin, cytokine, and interleukin
inhibition. Evidence also suggests a role of ROS in the proliferation
of fibroblast, increased collagen formation reforming connective
tissue, hastened re-epithilization, and increased microcirculation by
increasing the diameter and blood-flow velocity. Possible use of LLLT
as a noninvasive treatment in diabetic foot ulcer has been suggested
by Beckmann et al. It has been found that there is a reduction in the
inflammatory reaction and increased proliferation of myofibroblasts
after LLLT (Beckmann et al., 2014).
ROS-mediated LLLT therapy for bone regeneration has been
studied by Migliario et al. (2014) using 980 nm diode laser on
pre-osteoblast. It has been found that ROS plays an important role
in pre-osteoblast proliferation. ROS mediates cell differentiation,
cell proliferation, and apoptosis by activating signaling pathways.
Irradiation of murine pre-osteoblast MC3T3 cells is done in the
absence and presence of the antioxidant N-acetyl-L-cysteine (NAC)
with varying energy outputs (1–50 J), and it has been found that
there is a fluence-dependent significant increase in ROS generation
in NAC absence, but NAC presence strongly inhibits it. In the absence
of the antioxidant, cell proliferation is found to be significantly
increased both at low and higher energy, with a peak at 10 J, but
there is no cell proliferation by laser irradiation in the presence of
NAC, suggesting the role of ROS in cell proliferation (Migliario et al.,
2014).
Muscle regeneration, regulated by growth factors, involves cell
differentiation, cell proliferation, and migration. LLLT is known to
accelerate tissue repair and promote skeletal muscle regeneration.
Using gastrocnemius muscles of rats postcontusion, Luo et al. (2013)
have proposed that LLLT can promote muscle regeneration along
with reducing scar formation and hemostasis of ROS, and growth
factors IGF-1 and TGF-β1 can be modulated by LLLT (Luo et al.,
2013).
Huang et al. (2009) have proposed the probable use of LLLT
in the ROS-mediated treatment of skin conditions such as vitiligo,
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158 Role of Reactive Oxygen Species in Low-Level Laser Therapy

psoriasis, and acne. Stem cells can also be activated leading to in-
creased tissue repair and healing. Beneficial effects on hypertrophic
scars, wrinkles, acne, and burn scars, and healing of burn have also
been reported (Avci et al., 2013).

9.3.9 ROS-Mediated Effect of LLLT on Oral Cavity

Feuerstein (2012) has suggested the possible use of LLLT as a
complementary antibacterial treatment to eradicate the biofilm
of oral cavity. Light is found to be more effective in biofilm
than suspension, and its effectiveness depends on the type and
other parameters of light. There is an ROS-mediated synergistic
antibacterial effect of blue light (wavelength 400–500 nm) and
H2 O2 , which enhances the phototoxic effect. Increase in the enamel’s
resistance to acid attack can be attributed to noninterference
between biofilm and chemical changes after irradiation (Feuerstein,
2012).
Scwinngel et al. (2012) have proposed the possible ROS-
mediated use of LLLT in the treatment of oral candidiasis in HIV
patients. Oral mucosa has been irradiated with LLLT of wavelength
660 nm, power 30 mW, and fluence 7.5 J/cm2 . Antimicrobial pho-
todynamic therapy (aPDT), a combination of low-power laser and
methylene blue (450 μg/mL), has also been given to patients and
it has been found that LLLT alone is not effective in reducing oral
candidiasis, but there is 100% eradication of candida with aPDT and
no recurrence of candidiasis has been found 30 days post-treatment
(Scwingel et al., 2012).

9.3.10 ROS-Mediated Effect of LLLT on Lungs

LLLT can help in relieving the symptoms of airway and lung
inflammation, and its effect on alveolar macrophage is activated by
oxidative stress and lipopolysaccharides. LLLT irradiation of rat cell
line (AMJ2-C11) cultured with LPS or H2 O2 shows the production
of MIP-2 mRNA and ROS. Increased laser effect is found on
N-acetylcysteine (NAC) pre-treated AMJ2-C11, showing its syner-
gistic effect with NAC. LLLT effect on MIP-2 mRNA expression and
ROS generation can be suppressed by BMS 205820. The synergistic
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References 159

effect of LLLT and NAC results in the reduced expression of NF-κB.

Increased ROS and decreased expression of NF-κB after LLLT and
NAC indicate its involvement in MIP-2 mRNA expression (de Lima
et al., 2010).

References

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Huang, Y. Y., et al. (2009). “Biphasic dose response in low level light therapy.”
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Huang, Y. Y., et al. (2013). “Low-level laser therapy (LLLT) reduces oxidative
stress in primary cortical neurons in vitro.” J Biophotonics 6(10): 829–
838.
Huang, Y. Y., et al. (2014). “Low-level laser therapy (810 nm) protects
primary cortical neurons against excitotoxicity in vitro.” J Biophotonics
7(8): 656–664.
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Irani, K., et al. (1997). “Mitogenic signaling mediated by oxidants in Ras-

transformed fibroblasts.” Science 275(5306): 1649–1652.
Karu, T. (1989). “Laser biostimulation: A photobiological phenomenon.” J
Photochem Photobiol B 3(4): 638–640.
Karu, T. (1989). “Photobiology of low-power laser effects.” Health Phys
56(5): 691–704.
Karu, T. (1999). “Primary and secondary mechanisms of action of visible to
near-IR radiation on cells.” J Photochem Photobiol B 49(1): 1–17.
Karu, T. I., and S. F. Kolyakov (2005). “Exact action spectra for cellular
responses relevant to phototherapy.” Photomed Laser Surg 23(4): 355–
361.
Kolarova, H., et al. (2008). “Production of reactive oxygen species after
photodynamic therapy by porphyrin sensitizers.” Gen Physiol Biophys
27(2): 101–105.
Kushibiki, T., et al. (2013). “Blue laser irradiation generates intracellular
reactive oxygen species in various types of cells.” Photomed Laser Surg
31(3): 95–104.
Lam, M., et al. (2001). “Photodynamic therapy-induced apoptosis in epider-
moid carcinoma cells. Reactive oxygen species and mitochondrial inner
membrane permeabilization.” J Biol Chem 276(50): 47379–47386.
Lavi, R., et al. (2003). “Low energy visible light induces reactive oxygen
species generation and stimulates an increase of intracellular calcium
concentration in cardiac cells.” J Biol Chem 278(42): 40917–40922.
Lavi, R., et al. (2004). “ESR detection of 1O2 reveals enhanced redox activity
in illuminated cell cultures.” Free Radic Res 38(9): 893–902.
Lee, H. B., et al. (2004). “Reactive oxygen species amplify protein kinase
C signaling in high glucose-induced fibronectin expression by human
peritoneal mesothelial cells.” Kidney Int 65(4): 1170–1179.
Lim, W. B., et al. (2011). “Effects of 635 nm light-emitting diode irradiation
on angiogenesis in CoCl(2) -exposed HUVECs.” Lasers Surg Med 43(4):
344–352.
Liu, H., et al. (2005). “Redox-dependent transcriptional regulation.” Circ Res
97(10): 967–974.
Lubart, R., et al. (2005). “Low-energy laser irradiation promotes cellular
redox activity.” Photomed Laser Surg 23(1): 3–9.
Luo, L., et al. (2013). “Effects of low-level laser therapy on ROS homeostasis
and expression of IGF-1 and TGF-beta1 in skeletal muscle during the
repair process.” Lasers Med Sci 28(3): 725–734.
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162 Role of Reactive Oxygen Species in Low-Level Laser Therapy

Masoumipoor, M., et al. (2014). “Effects of 660- and 980-nm low-level laser
therapy on neuropathic pain relief following chronic constriction injury
in rat sciatic nerve.” Lasers Med Sci 29(5): 1593–1598
Meister, A., and M. E. Anderson (1983). “Glutathione.” Annu Rev Biochem 52:
711–760.
Migliario, M., et al. (2014). “Laser-induced osteoblast proliferation
is mediated by ROS production.” Lasers Med Sci 29(4): 1463–
1467.
Oren, D. A., et al. (2001). “Stimulation of reactive oxygen species production
by an antidepressant visible light source.” Biol Psychiatry 49(5): 464–
467.
Pal, G., et al. (2007). “Effect of low intensity laser interaction with
human skin fibroblast cells using fiber-optic nano-probes.” J Photochem
Photobiol B 86(3): 252–261.
Pastore, D., et al. (2000). “Specific helium-neon laser sensitivity of
the purified cytochrome c oxidase.” Int J Radiat Biol 76(6): 863–
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regulation of cigarette smoke-induced lung inflammation: Epigenet-
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811.
Schreck, R., and P. A. Baeuerle (1991). “A role for oxygen radicals as second
messengers.” Trends Cell Biol 1(2–3): 39–42.
Schreck, R., et al. (1991). “Reactive oxygen intermediates as apparently
widely used messengers in the activation of the NF-kappa B transcrip-
tion factor and HIV-1.” Embo J 10(8): 2247–2258.
Scwingel, A. R., et al. (2012). “Antimicrobial photodynamic therapy in the
treatment of oral candidiasis in HIV-infected patients.” Photomed Laser
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Sharma, S. K., et al. (2011). “Dose response effects of 810 nm laser light
on mouse primary cortical neurons.” Lasers Surg Med 43(8): 851–
859.
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role of redox mechanisms.” Photomed Laser Surg 26(4): 323–328.
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Cell Physiol 217(2): 518–528.
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Chapter 10

Molecular Basis for

Photobiomodulation: Light-Induced
Nitric Oxide Synthesis by Cytochrome c
Oxidase in Low-Level Laser Therapy

Robert Oliver Poyton and Marina Hendrickson

Department of Molecular, Cellular, and Developmental Biology, University of Colorado
at Boulder, 347 UCB, Boulder, CO 80309, USA
robert.poyton@colorado.edu

10.1 Introduction

As discussed elsewhere in this book, Low-intensity light in the

visible and near-infrared (NIR) region has beneficial therapeutic
effects. Light in this region of the spectrum lacks the carcinogenic
and mutagenic properties of ultraviolet light and is capable of
penetrating biological tissue and bone. Moreover, wavelengths in the
NIR region are especially effective at penetrating the skull and dense
muscle tissue [27]. This property was recognized in the classical
studies of Jobsis (1977) and subsequently used by Chance et al. [17,
18, 62] in the development of NIR imaging for the noninvasive study
of muscle and brain oxygenation and oxidative metabolism [27, 28].

Handbook of Low-Level Laser Therapy

Edited by Michael R. Hamblin, Marcelo Victor Pires de Sousa, and Tanupriya Agrawal
Copyright c 2017 Pan Stanford Publishing Pte. Ltd.
ISBN 978-981-4669-60-3 (Hardcover), 978-981-4669-61-0 (eBook)
www.panstanford.com
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166 Molecular Basis for Photobiomodulation

Although the molecular mechanisms that underlie the thera-

peutic benefits of low-level light therapy (LLLT) are still poorly
understood, recent research findings have provided useful insights
concerning a photoreceptor for LLLT, potentially important signaling
molecules and target tissues, and the role of mitochondrial energy
metabolism. When considered together, these findings suggest a
working model for LLLT that may help in both optimizing and
monitoring the therapeutic benefits of LLLT. This model and the
evidence upon which it is based are discussed as follows.

10.2 Cytochrome c Oxidase: A Photoreceptor for LLLT

The effective wavelengths for LLLT are broadly distributed between

514 nm and 920 nm [4, 13, 23, 37, 41, 42, 50, 58, 59, 71]. However,
most studies and therapies have used wavelengths between 600 nm
and 830 nm. The major chromophores in mammalian tissues that
absorb light in this NIR range are hemoglobin, myoglobin, melanin,
and mitochondrial cytochrome c oxidase (Cox) [38]. Cox is the
terminal enzyme of the mitochondrial respiratory chain and is the
only chromophore of the four that are present in all mammalian
tissues and cells. Early evidence supporting a role for Cox in LLLT
came from the finding that Cox absorbs most of the infrared light
received by cells [9], and from the determination of the action
spectrum of light on cell attachment and proliferation in vitro [14,
41]. More recent studies with a variety of cells have demonstrated
that Cox inhibitors can mitigate the effects of LLLT, that the toxic
effects of some chemicals on Cox can be reversed by NIR light, and
that wavelengths of light that overlap the absorption spectrum of
oxidized Cox protect neuronal cells from the cytotoxic effects of cell
death by cyanide and azide, both of which are Cox inhibitors (for
review see [53]). When considered together, these findings strongly
suggest that Cox can function as a primary photoreceptor for LLLT.
It has long been known that Cox plays an important role in the
regulation of mitochondrial electron transport and in the regulation
of cellular energy metabolism [40, 51, 57, 68]. More recently,
however, it has been reported that this enzyme can affect intra-
and extracellular signaling pathways and functions in mitochondrial
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Structure/Function of Mitochondrial Cytochrome c Oxidase 167

signaling and adaptation to hypoxia [54, 55], the extension of

lifespan brought about by caloric restriction [43], and possibly
hypoxic vasodilation [3]. Insights from each of these processes can
contribute to the development of a model for the molecular basis
of LLLT. Before considering how Cox may function in LLLT, we will
first briefly review the structure, function, and regulation of this
important respiratory protein.

10.3 Structure/Function of Mitochondrial Cytochrome c

Oxidase

Mitochondrial Cox is a multimeric protein that spans the inner

mitochondrial membrane. It is composed of four redox-active
metal centers and 9–13 subunit polypeptides (Fig. 10.1). Three
of the subunit polypeptides (subunits I, II, and III) are encoded
by the mitochondrial genome, while nuclear genes encode the
rest. The number of nuclear-encoded subunit polypeptides is
species dependent. Some of the nuclear-encoded subunits are
encoded by multigene families, which produce subunit isoforms
that are differentially expressed in response to developmental and
environmental signals and which modulate the enzymatic activities
of the protein [12, 40]. The redox-active metal centers (heme a, heme
a3 , CuA , CuB ) (Fig. 10.1) participate in the electron transfer reactions
within the enzyme. Heme a3 and CuB are bridged in the resting, fully
oxidized form of the enzyme and constitute the binuclear reaction
center. Electrons pass from cytochrome c, via CuA and heme a to
the binuclear reaction center, where they reduce one of two electron
receptors: either oxygen (O2 ) or nitrite (NO2 − ). The reduction of O2
and possibly NO2 − is coupled to the translocation of one proton per
electron across the inner mitochondrial membrane.
The crystal structure of mammalian Cox [66] has shown that
the enzyme is an asymmetric dimer with a transmembrane region
that contains 28 α-helices per monomer (Fig. 10.1), making it one
of the most complex membrane proteins known. All but three of its
subunit polypeptides have at least one hydrophobic transmembrane
helix. Subunit I has 12 transmembrane helices, subunit II has two
transmembrane helices, and subunit III has seven transmembrane
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168 Molecular Basis for Photobiomodulation

Figure 10.1 Crystal structure of mammalian monomeric Cox [65] viewed

within the plane of the inner mitochondrial membrane (indicated by solid
lines). The intermembrane space side and the mitochondrial matrix side of
the membrane are as indicated. The ribbon structures of subunit I (green),
subunit II (blue), subunit III (yellow), and subunit IV (orange) are shown in
color. The remaining nuclear-coded subunits are shown in white [26].

helices. The remaining transmembrane helices lie in the nuclear-

coded subunits. Three of the redox-sensitive metal centers (heme
a, and the binuclear reaction center (heme a3 and CuB )) lie within
subunit I, while CuA lies within subunit II (Fig. 10.2). Subunit III
contains no metal centers but may modulate the proton-pumping
functions of the enzyme, play a role in the assembly or stability of
subunits I or II, or modulate the access of oxygen to the binuclear
reaction center [63]. Together, subunits I–III form the catalytic
core of Cox. This catalytic core is conserved in all members of
the heme/Cu oxidase superfamily [49], making it likely that the
catalytic activities of eukaryotic Cox’s are performed entirely by the
mitochondrially encoded subunits.
Given that the mitochondrially encoded subunits are responsible
for the catalytic activities of Cox, what are the functions of its
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Enzymatic Activities of Cytochrome c Oxidase 169

nuclear-encoded subunits? Genetic studies with yeast Cox suggest

that the nuclear-encoded subunits have at least three functions:
regulation of catalysis, holoenzyme dimerization, and holoenzyme
stability [56]. Yeast V (paralogous to mammalian IV) functions
to regulate the rate of catalysis; another subunit, yeast VIII, is
essential for the dimerization of the holoenzyme, and the other
subunits, yeast IV, VI, VII, and VIIa, are required for the stability
of the catalytic core and/or stable assembly of the holoenzyme. Of
particular interest for the modulation of the catalytic core are the
oxygen-regulated nuclear-encoded yeast subunit V isoforms Va and
Vb (yVa and yVb) and their mammalian paralogs IV-1 and IV-2 (mIV-
1 and mIV-2). These isoforms are encoded by homologous nuclear
genes, which are differentially regulated by oxygen concentration
and have differential effects on the catalytic activities of Cox [12,
35, 36]. The region of mIV that is closest to the binuclear reaction
center is the α helix of its transmembrane domain (Fig. 10.2). This α-
helix is adjacent to transmembrane α-helix XII of subunit I crossing
it near the site at which hemes a and a3 are liganded. Interaction
between the α-helix of mIV and helix XII of subunit I could affect the
relative orientation of hemes a and a3 and, in turn, affect the electron
transfer rates between these two prosthetic groups [12]. The role of
subunit isoforms in the regulation of the catalytic functions of Cox is
discussed as follows.

10.4 Enzymatic Activities of Cytochrome c Oxidase

It has been known for several years that Cox catalyzes the
irreversible reduction of O2 to water (H2 O) (Eq. 10.1).
4H + +4e− + 2O2 → 2H2 O (10.1)
This reaction, designated Cox/H2 O [54], was thought to be the
only reaction catalyzed by Cox. Recently, Cox has been shown to have
another enzymatic activity: the conversion of NO2 − to nitric oxide
(NO), given by Eq. 10.2 [15, 54].
NO2 − + Fe(II) + H+ → NO. + Fe(III) + OH− (10.2)
−
This NO2 -reductase activity of Cox has been called Cox/NO and
has been detected in a variety of eukaryotes [16, 54]. Cox/NO activity
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170 Molecular Basis for Photobiomodulation

Figure 10.2 The spatial relationship between subunits I, III, and IV of

mammalian Cox. The transmembrane α-helix of subunit IV is shown as an
orange ribbon and α-helix XII of subunit I is shown as a green ribbon. The
α-carbon backbones are shown for the rest of subunit I (green), for subunit
IV (orange), and for all of subunit II (blue). Hemes a and a3 are shown in
red, and CuB and CuA clusters are shown in yellow. The location of these
subunits within the plane of the inner mitochondrial membrane is indicated
by the solid lines, with the intermembrane space toward the top and the
mitochondrial matrix toward the bottom [26].

is operative at physiological nitrite concentrations [8, 16], functions

in hypoxic signaling (i.e., hypoxic gene induction) [7, 15, 16], and
may be involved in nitrite recycling [61].
Given that Cox has two distinct activities, Cox/H2 O and Cox/NO,
it is, in essence, a molecular switch. Both activities are regulated
independently, but as discussed in the following sections, only
Cox/NO activity is light regulated.

10.4.1 Regulation of Cox/H2 O Activity

The Cox/H2 O reaction can be modulated in at least three ways:
by the differential expression of oxygen-regulated isoforms [2, 12,
22, 33, 35]; by allosteric regulation via metabolites, especially
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Enzymatic Activities of Cytochrome c Oxidase 171

adenine nucleotides [6]; and by protein phosphorylation [29, 35].

Modulation of Cox/H2 O by its O2 -regulated subunit isoforms, yVa
and yVb or mIV-1 and mIV-2, is well understood [2, 12, 22, 35,
70]. These isoforms are encoded by homologous nuclear genes,
which are differentially regulated by O2 concentration. In yeast,
the aerobic isoform yVa is expressed under normoxic conditions,
while the hypoxic isoform yVb is expressed under hypoxic or anoxic
conditions. These yeast subunit isoforms modulate the turnover
rate of Cox/H2 O, with yVb supporting a rate that is 3–4 times
faster than that supported by yVa [2, 70]. The mammalian O2 -
regulated isoforms have also been reported to modulate the rate of
the Cox/H2 O reaction [22, 35]. Mammalian subunit IV-1 is involved
in the allosteric regulation of Cox/H2 O by ATP [1, 35]. This subunit
contains an adenine nucleotide-binding site, which binds ADP at
low ATP/ADP ratios. At high intramitochondrial ATP/ADP ratios,
ATP binds mIV-1, resulting in the inhibition of Cox/H2 O activity.
Recently, it has been proposed that the allosteric inhibition of
Cox/H2 O by ATP can be prevented by phosphorylation of mIV-
1 [1]. These findings suggest that the aerobic mIV-1 functions to
regulate the Cox/H2 O reaction and under normoxic conditions,
this ATP inhibition effectively regulates the rate of respiration
[39]. Surprisingly, the ATP inhibition of Cox is abolished in cells
expressing the hypoxic mIV-2 [33].

10.4.2 Regulation of Cox/NO Activity

Cox/NO activity is also highly regulated. Oxygen, Cox subunit
isoforms, and adenine nucleotides all modulate its activity. Early
studies demonstrated that Cox/NO activity is inhibited by O2 and
enhanced under hypoxic conditions, and the low cellular pH that
accompanies hypoxia [16]. These studies suggested that Cox/NO
functions best at O2 concentrations below 20 μM. However, recent
studies revealed that both the activity and O2 sensitivity of Cox/NO
are modulated by both the O2 -regulated isoforms yVa and yVb [15]
(Fig. 10.3) and adenine nucleotides (Fig. 10.4). Yeast Cox isozymes
engineered to carry yVb have markedly higher Cox/NO turnover
rates than isozymes with vVa and function at much higher O2
concentrations. The hypoxic isoform yVb supports NO synthesis
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172 Molecular Basis for Photobiomodulation

Figure 10.3 Effects of yV subunit isoforms, yVa and yVb, on Cox/NO activity
and O2 sensitivity.

Figure 10.4 Effects of adenine nucleotides on Cox/NO activity and O2

sensitivity.

at O2 concentrations that are well within the normoxic range for

most tissues [54, 67] and as high as 160 μM O2 [16]. In contrast,
the aerobic isoform yVa supports NO synthesis only under hypoxic
conditions, below 15 μM O2 [16].
Interestingly, ATP and ADP also have differential effects on
Cox/NO activity and its sensitivity to O2 (Fig. 10.4). High ADP/ATP
levels, which accompany exposure to hypoxia and reduced caloric
intake, reduce the sensitivity of Cox/NO to O2 , allowing for NO
synthesis at O2 concentrations that are well within the normoxic
range (30–130 μM O2 ) for most tissues [14]. Together, these findings
indicate that Cox/NO can serve as a source of NO both in normoxia
(130 μM–20 μM O2 ) and hypoxia (2 μM–20 μM O2 ).
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Low-Intensity Light Stimulates Cox/NO but Not Cox/H2 O Activity 173

10.5 Low-Intensity Light Stimulates Cox/NO but Not

Cox/H2 O Activity

To begin to evaluate how Cox functions in LLLT, Ball et al. (2011)

assessed the effect of light on both Cox/NO and Cox/H2 O activities
with a xenon/halogen (Xe–H) lamp. The XE–H lamp used produced
a broad spectrum of visible and NIR light (380–1000 nm) that
included wavelengths previously used for LLLT. The effect of
broadband light from the Xe–H lamp on Cox/NO and Cox/H2 O
activities was assayed by following the production of NO with an NO
electrode and the consumption of oxygen by an oxygen electrode
[8]. This study demonstrated that low-intensity (2–6 mW/cm2 )
broadband light stimulated Cox/NO activity in a dose-dependent
fashion but had no effect on Cox/H2 O activity when either yeast or
mouse brain Cox were assayed. These findings suggested that the
Cox/NO activity of Cox and the NO it produces can serve to mediate
the beneficial effects of LLLT [53].
Further insight concerning how light functions to affect Cox/NO
activity was obtained through the use of selective wavelength
bandpass filters with a Xe–H lamp and wavelength-specific light-
emitting devices (LEDs) [8]. These studies revealed that wave-
lengths between 509 nm and 691 nm all have stimulatory effects
on Cox/NO and that wavelengths above 820 nm are inhibitory. This
stimulatory region (509–691 nm) includes the absorption maxima
of heme a (605 nm), heme a3 /CuB (655 nm), Cox O2 intermediate
P (607 nm), Cox O2 intermediate F (580 nm), the heme a3 −NO
complex (597 nm), as well as wavelengths (630 nm and 670 nm)
that are often used for LLLT. Using LEDs to further narrow down
the wavelength range, Ball et al. (2011) observed that the greatest
simulation of Cox/NO activity was produced by an LED with peak
output at 590 nm and a range between 576 nm and 604 nm.

10.5.1 Possible Mechanism for Light Stimulation of

Cox/NO
The conversion of NO2 − to NO most likely takes place on the
binuclear reaction center (heme a3 -CuB ) of Cox (Fig. 10.2). Evidence
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174 Molecular Basis for Photobiomodulation

for this comes from the findings that a NO2 + -ferric a3 complex
[20, 24, 61, 65] and a heme-nitrosyl complex a23+ -NO [11] are
formed at the binuclear center of Cox in the presence of NO2 − , and,
more recently, that an inorganic reduced heme-copper assembly,
resembling the Cox binuclear reaction center, reduces NO2 − to NO
[30, 31]. Castello et al.[2008] proposed that a one-electron reduction
of NO2 − to NO occurs as part of the catalytic cycle at the binuclear
reaction center of Cox, according to Eqs. 10.3–10.5.

NO2 − + H+ → [Fea3(III) CuB(II) ] − NO2 − (10.3)

[Fea3(III) CuB(II) ] − NO2 − → [Fea3(III) CuB(I) ] − NO+ (10.4)
+ −
[Fea3(III) CuB(I) ] − NO → [Fea3(III) CuB(II) ] + NO + OH (10.5)

It is now clear that these are two distinct pathways in which NO

interacts with Cox: the “nitrite” pathway, in which Cox catalyzes the
conversion of NO2 − to NO, and the inhibitory nitrosylation pathway.
These reactions represent the catalytic intermediates of a reversed
“nitrite” pathway and are not the intermediates of the nitrosylation
pathway long known to inhibit Cox activity [61].
The finding that light has no effect on the Cox/H2 O reaction,
either in the presence or absence of NO2 − [8], suggests that light
does not alter the binding of NO2 − to Cox (Eq. 10.3). This is
consistent with the fact that an NO2 − -ferric a3 complex, like other
ferric hemoprotein adducts, is not photosensitive [61]. In contrast,
the finding that light from an LED with a maximum output at 590 nm
stimulates Cox/NO activity is consistent with the mechanism that
either involves an increase in the rate of formation of an a3 2+ -NO
complex (Eq. 10.4) or the rate of dissociation of NO from an a3 2+ -
NO complex (Eq. 10.5) [8]. This is supported by the observation that
the a3 -NO complex absorbs maximally at 595–597 nm [10] and is
photosensitive when exposed to light [10, 61].

10.6 Cox/NO, NO, and LLLT

Nitric oxide and reactive nitrogen species derived from it are

important signaling molecules that function in several different
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Cox/NO, NO, and LLLT 175

biological pathways and pathologies. These include systemic

blood pressure, hypoxic signaling, stress response pathways, host–
microbe interactions, immune signaling, and apoptosis (for review
see [32]). There is accumulating evidence that NO levels increase
during phototherapy and light-induced cell proliferation [44, 45, 60,
72], that light stimulates vasodilation via NO [5, 19, 47, 48, 52], and
that some of the NO involved in phototherapy and photorelaxation
is generated independently of NOS [25, 44, 72].
Although it has long been known that biological NO is generated
by NO synthases (NOSs), which catalyze the conversion of arginine
to NO, it is now clear that cells are also capable of producing NO
from NO2 − [67, 73]. Interest in this NOS-independent route for
biological NO synthesis is growing because NO2 − is now considered
a circulating reservoir for NO in mammals and because of the
potential therapeutic applications of this alternative route for NO
synthesis [53, 69]. Cox/NO is one of the enzymes that function
to convert NO2 − to NO. Nitric oxide produced from Cox/NO can
be used both inside cells, where it functions in hypoxic signaling
[54] and other intracellular signaling pathways [32], and outside
cells, where it may function in vasodilation and other extracellular
signaling pathways [55]. The discovery that Cox can function as an
NO− 2 -reductase [15, 16] reveals that the interaction between NO and
Cox is more complex than the long held view that NO is merely an
inhibitor of Cox/H2 O activity. Moreover, the finding that Cox/NO can
generate NO under both normoxic and hypoxic conditions indicates
that LLLT can increase NO levels by stimulating new synthesis
[16] and not merely by releasing it from intracellular stores
[46, 64].
As a primary photoreceptor for LLLT, Cox most likely represents
the first step in an intracellular photo-signaling pathway, with
the light-sensitive enzymatic activities of Cox/NO generating the
molecules that serve to initiate this pathway. It has been known for
some time that mitochondria produce low levels of superoxide (O2 − )
under normoxic conditions and that mitochondrial production of NO
and O2 − increases transiently upon exposure to hypoxia [54]. The
recent finding that Cox/NO can function under normoxic (see above)
as well as hypoxic conditions and that Cox/NO is stimulated by light
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176 Molecular Basis for Photobiomodulation

Figure 10.5 A model for light-induced NO synthesis by Cox/NO and its

potential downstream effects in LLLT.

suggests that NO as well as peroxynitrite (ONOO− ), produced from

NO and O2 − , can function as early signaling molecules in an LLLT
photo-signaling pathway. These molecules may initiate signaling via
S-nitrosylation or tyrosine nitration of proteins.
Although Cox/NO-generated NO may have a number of down-
stream targets (Fig. 10.5), one target that is particularly interesting
is HIF-1α, a subunit of HIF-1, which regulates transcription over
many mammalian genes [32]. Short-term exposure to NO can
stabilize HIF-1α under both hypoxic and normoxic conditions. The
HIF-1-NO complex functions in a multitude of cellular processes,
including angiogenesis, apoptosis, autophagy, cell growth and
development, and aging. Recent studies have reported that HIF-1
is involved in LLLT-induced reduction of neuropathic pain [34] and
increased angiogenesis [21]. It will be of interest to determine if
Cox/NO-generated NO is also involved.
July 6, 2016 17:19 PSP Book - 9in x 6in 10-Hamblin-c10

References 177

10.7 Summary

The finding that light stimulates NO synthesis by Cox/NO provides

an important foundation for understanding the molecular basis for
LLLT. However, it is only the first step. Given the multitude of effects
that NO can have on cells and tissues, the challenge for the future
will be to determine how the light-induced production of NO by
Cox/NO functions to mediate the beneficial effects brought about
by LLLT. Given that high levels of NO can also produce nitrosative
stress, it will also be important to more thoroughly evaluate specific
wavelengths and intensities of light for their effects on NO-induced
damage.

Acknowledgments

This work was sponsored by the National Institutes of Health Grant

30228 and a gift from the Leslie F. and Josephine Bailey Trust.

References

1. Acin-Perez, R., Gatti, D., Bai, Y., and Manfredi, G. (2011). Protein
phosphorylation and prevention of cytochrome oxidase inhibition by
ATP: Coupled mechanisms of energy metabolism regulation. Cell Metab.,
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dependence on expression of endothelial nitric oxide synthase. Br. J.

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oxygenation and oxidative metabolism in healthy and diseased hu-

mans. J. Biomed. Opt., 12(6), p. 062105.
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the transcription pattern of subunit isoforms and the kinetics of
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34. Hsieh, Y., Chou, L., Chang, P., Yang, C., Kao, M., and Hong, C. (2012). Low-
level laser therapy alleviates neuropathic pain and promotes function
recovery in rats with chronic constriction injury: Possible involvements
in hypoxia-inducible factor 1α (HIF-1α). J. Comp. Neurol., 520(13), pp.
2903–2916.
35. Hüttemann, M., Lee, I., Liu, J., and Grossman, L. (2007). Transcription of
mammalian cytochrome c oxidase subunit IV-2 is controlled by a novel
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subunit IV isoforms of cytochrome c oxidase. Gene, 267(1), pp. 111–123.
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233–239.
38. Jobsis, F. (1977). Noninvasive, infrared monitoring of cerebral and
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39. Kadenbach, B. (2003). Intrinsic and extrinsic uncoupling of oxidative

phosphorylation. Biochim. Biophys. Acta, 1604(2), pp. 77–94.
40. Kadenbach, B., Hüttemann, M., Arnold, S., Lee, I., and Bender, E.
(2000). Mitochondrial energy metabolism is regulated via nuclear-
coded subunits of cytochrome c oxidase. Free Rad. Biol. Med., 29(3–4),
pp. 211–221.
41. Karu, T., Pyatibrat, L., Kolyakov, S., and Afanasyeva, N. (2005). Ab-
sorption measurements of a cell monolayer relevant to phototherapy:
Reduction of cytochrome c oxidase under near IR radiation. J.
Photochem. Photobiol., 81(2), pp. 98–106.
42. Karu, T. (1999). Primary and secondary mechanisms of action of visible
to near-IR radiation on cells. J. Photochem. Photobiol., 49(1), pp. 1–17.
43. Li, B., Skinner, C., Castello, P., Kato, M., Easlon, E., Xie, L., Li, T., Lu,
S., Wang, C., Tsang, F., Poyton, R., and Lin, S. (2011). Identification of
potential calorie restriction-mimicking yeast mutants with increased
mitochondrial respiratory chain and nitric oxide levels. J. Aging Res.,
2011, pp. 1–16.
44. Lindgård, A., Hultén, L., Svensson, L., and Soussi, B. (2006). Irradiation
at 634 nm releases nitric oxide from human monocytes. Lasers Med. Sci.,
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45. Lipovsky, A., Oron, U., Gedanken, A., and Lubart, R. (2012). Low-level
visible light (LLVL) irradiation promotes proliferation of mesenchymal
stem cells. Lasers Med. Sci., 28(4), pp. 1113–1117.
46. Lohr, N., Keszler, A., Pratt, P., Bienengraber, M., Warltier, D., and Hogg, N.
(2009). Enhancement of nitric oxide release from nitrosyl hemoglobin
and nitrosyl myoglobin by red/near infrared radiation: Potential role in
cardioprotection. J. Mol. Cell. Cardiol., 47(2), pp. 256–263.
47. Matsuo, H., Morimoto, Y., Arai, T., Wada, M., Higo, R., Tabata, S., Nakai, K.,
and Kikuchi, M. (2000). Heat and photolytic nitric oxide are essential
factors for light-induced vascular tension changes. Lasers Med. Sci.,
15(3), pp. 181–187.
48. Mitchell, U., and Mack, G. (2013). Low-level laser treatment with near-
infrared light increases venous nitric oxide levels acutely. Am. J. Phys.
Med. Rehabil., 92(2), pp. 151–156.
49. Moody, A. (1996). ‘As prepared’ forms of fully oxidised haem/Cu
terminal oxidases. Biochim. Biophys. Acta, 1276(1), pp. 6–20.
50. Oron, A., Oron, U., Chen, J., Eilam, A., Zhang, C., Sadeh, M., Lampl, Y.,
Streeter, J., DeTaboada, L., and Chopp, M. (2006). Low-level laser therapy
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applied transcranially to rats after induction of stroke significantly

reduces long-term neurological deficits. Stroke, 37(10), pp. 2620–
2624.
51. Piccoli, C., Scrima, R., Boffoli, D., and Capitanio, N. (2006). Control
by cytochrome c oxidase of the cellular oxidative phosphorylation
system depends on the mitochondrial energy state. Biochem. J., 396(3),
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52. Plass, C., Loew, H., Podesser, B., and Prusa, A. (2012). Light-induced va-
sodilation of coronary arteries and its possible clinical implication. Ann.
Thorac. Surg., 93(4), pp. 1181–1186.
53. Poyton, R., and Ball, K. (2011). Therapeutic photobiomodulation: Nitric
oxide and a novel function of cytochrome c oxidase. Discovery Med., 11,
pp. 154–159.
54. Poyton, R., Ball, K., and Castello, P. (2009). Mitochondrial generation of
free radicals and hypoxic signaling. Trends Endocrinol. Metabol., 20(7),
pp. 332–340.
55. Poyton, R., Castello, P., Ball, K., Woo, D., and Pan, N. (2009). Mitochondria
and hypoxic signaling. Ann. N.Y. Acad. Sci., 1177(1), pp. 48–56.
56. Poyton, R., and McEwen, J. (1996). Crosstalk between nuclear and
mitochondrial genomes. Annu. Rev. Biochem., 65(1), pp. 563–607.
57. Poyton, R., Trueblood, C., Wright, R., and Farrell, L. (1988). Expression
and function of cytochrome c oxidase subunit isologues. Ann. N.Y. Acad.
Sci., 550(1 Cytochrome Ox), pp. 289–307.
58. Rochkind, S., Drory, V., Alon, M., Nissan, M., and Ouaknine, G. (2007).
Laser phototherapy (780 nm), a new modality in treatment of long-
term incomplete peripheral nerve injury: A randomized double-blind
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Stimulatory effect of He-Ne low dose laser on injured sciatic nerves of
rats. Neurosurgery, 20(6), pp. 843–847.
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of nitric oxide in the visible light-induced rapid increase of human
skin microcirculation at the local and systemic levels: II. Healthy
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Cytochrome c oxidase and nitric oxide in action: Molecular mechanisms
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62. Shiga, T., Tanabe, K., Nakase, Y., Shida, T., and Chance, B. (1995).
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63. Shinzawa-Itoh, K., Aoyama, H., Muramoto, K., Terada, H., Kurauchi,
T., Tadehara, Y., Yamasaki, A., Sugimura, T., Kurono, S., Tsujimoto, K.,
Mizushima, T., Yamashita, E., Tsukihara, T., and Yoshikawa, S. (2007).
Structures and physiological roles of 13 integral lipids of bovine heart
cytochrome c oxidase. EMBO J., 26(6), pp. 1713–1725.
64. Shiva, S., and Gladwin, M. (2009). Shining a light on tissue NO stores:
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Cytochrome c oxidase rapidly metabolises nitric oxide to nitrite. FEBS
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The whole structure of the 13-subunit oxidized cytochrome c oxidase at
2.8 A. Science, 272(5265), pp. 1136–1144.
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Shapiro, D., Kozlov, A., Li, H., Lundberg, J., Mason, R., Nohl, H., Rassaf,
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of yeast cytochrome c oxidase subunit V alter the in vivo kinetic
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71. Wu, X., Dmitriev, A., Cardoso, M., Viers-Costello, A., Borke, R., Streeter,
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36–41.
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184 Molecular Basis for Photobiomodulation

light protects cardiomyocytes from hypoxia and reoxygenation injury

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4–14.
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independent formation of nitric oxide in biological tissues. Nat. Med.,
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Chapter 11

Cytoprotective Effect of Low-Level Light

Therapy using LEDs on Neurons

Margaret Wong-Riley and Huan Ling Liang

Department of Cell Biology, Neurobiology, and Anatomy, Medical College of Wisconsin,
8701 Watertown Plank Road, Milwaukee, WI 53226-0509, USA
mwr@mcw.edu

The beneficial effect of low-level light therapy (LLLT) via light-

emitting diodes (LEDs) in the far-red to near-infrared (NIR) range
has been tested and substantiated in cultured primary neurons
exposed to various types of toxins, including the voltage-dependent
sodium channel blocker, tetrodotoxin, and toxins known to inhibit
complex I of the mitochondrial electron transport chain, MPP+
and rotenone. The major biological photoacceptors in the NIR are
hemoglobin, myoglobin, and mitochondrial cytochrome c oxidase
(Cox). Since cultured primary neurons do not contain blood or
muscles, one can study the effects of NIR mainly on Cox in neurons.
This has been verified by the positive correlation between effective
wavelengths for activating Cox and ATP production (i.e., 670, 770,
830, and 880 nm) in neurons exposed to toxins and the absorption
spectrum of Cox, and the lack of an effect at 728 nm, which does
not match the absorption spectrum of Cox. The role of Cox is
further strengthened by the fact that increasing concentrations of

Handbook of Low-Level Laser Therapy

Edited by Michael R. Hamblin, Marcelo Victor Pires de Sousa, and Tanupriya Agrawal
Copyright c 2017 Pan Stanford Publishing Pte. Ltd.
ISBN 978-981-4669-60-3 (Hardcover), 978-981-4669-61-0 (eBook)
www.panstanford.com
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186 Cytoprotective Effect of Low-Level Light Therapy using LEDs on Neurons

KCN, a known irreversible inhibitor of Cox, result in decreasing

effectiveness of NIR. The neuroprotective benefit of LLLT has been
confirmed in vivo when deprived visual cortices of monocularly
enucleated rats up-regulated their Cox activity in response to
NIR treatment. NIR also induces changes in gene expression by
up-regulating neuronal activity-related genes and down-regulating
tumor-associated and stress-induced genes.

11.1 Introduction

Life on our planet, earth, originated in the Archeon Eon aided by light
energy from our sun. Indeed, without sunlight, there will be no life
on earth. Within the wide spectrum of sunlight, however, a narrow
segment from the far-red through the NIR has been found to be
therapeutic. Infected, ischemic, and hypoxic wounds have benefited
from the healing power of low-energy laser irradiation [reviewed
in 5]. The rationale is that NIR stimulates and activates cellular
activity, thereby promoting the healing process [1, 2]. Laser has
and is still being used in such LLLT in a wide variety of clinical
and experimental applications [reviewed in 3, 4, 12, 15, 18, 22], as
illustrated amply in this book.
In recent years, LED arrays developed for the National Aero-
nautics and Space Administration (NASA) manned space flight
experiments have been found to not only help plant growth in space
but aid in human wound healing on earth as well [27]. These arrays
emit light in the far-red to NIR range, produce negligible amount
of heat, have been clinically tested to be safe, and are approved by
FDA for nonsignificant risk status for human trials [27]. LED units
are generally compact, portable, and more affordable than lasers.
Experiments described in this chapter have been done in the first
author’s laboratory using NIR-emitting LEDs.

11.2 Role of Cytochrome c Oxidase in

Photobiomodulation of Cultured Visual Cortical
Neurons

In mammalian tissues, three major photoacceptor molecules are

known to absorb light in the NIR range: hemoglobin in red blood
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Role of Cytochrome c Oxidase in Photobiomodulation of Cultured Visual Cortical Neurons 187

cells, myoglobin in muscle cells, and Cox in the mitochondria. Of

these three, only Cox (EC 1.9.3.1) is part of the energy-generating
machinery in the cell. The specific role of Cox in mediating the
NIR effect on neurons can be investigated in primary neuronal
cultures, in which blood elements and muscle cells are automatically
excluded [29]. When action potentials of these neurons are blocked
by tetrodotoxin (TTX), a voltage-dependent sodium channel blocker,
neuronal Cox level significantly reduces. At the concentration used
(0.4 μM), TTX impedes neuronal activity without causing cell death.
In the presence of TTX, if neurons are exposed to an LED array
(25 cm × 10 cm) at 670 nm (Quantum Devices, Inc., Barnaveld,
WI, USA) for only 80 s a day (equivalent to a power intensity of 50
mW/cm2 and an energy density of 4 J/cm2 ) for the last 5 of the 6
days in culture, their Cox level returns to normal [29]. In the absence
of TTX, normal neurons also increase their Cox levels significantly
above controls after 5 days of LED treatment [29]. These findings
are consistent with the hypothesis that NIR mediates its therapeutic
effect by stimulating cellular activity via increased mitochondrial
Cox activity.
If Cox is directly involved in the NIR-induced photobiomodula-
tion of TTX-inactivated neurons, then the effective action spectrum
of LED should correspond to the NIR absorption spectrum of Cox.
Various wavelengths of LED have been tested to determine if specific
ones are more beneficial. These are 670, 728, 770, 830, and 880 nm,
each administered at a power intensity of 50 mW/cm2 and an energy
density of 4 J/cm2 (equivalent to 80 s exposure) per day for 5 days
on primary neurons bathed in a medium containing 0.4 μM of TTX
for 6 days [31]. Of the five wavelengths tested, 670 and 830 nm
are equally effective in reversing the detrimental effect of TTX on
Cox activity, with 830 nm being the most effective in restoring
enzyme levels to slightly (though not significantly) above that of
controls (111.7%) (Fig. 11.1A). The wavelength of 670 nm is equally
effective in causing a 100% recovery. The wavelength of 880 nm
enables a 98.4% recovery, whereas 770 nm brings about only a
67.2% recovery of enzyme levels (Fig. 11.1A). On the other hand,
728 nm is the least effective and induces only a 27.5% recovery
(Fig. 11.1A).
The ATP content of neurons is down-regulated to 82.5% of
controls by 6 days of TTX exposure. LEDs at 670, 770, 830, and
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188 Cytoprotective Effect of Low-Level Light Therapy using LEDs on Neurons

Figure 11.1 Effects of different wavelengths of LED light (each at 4 J/cm2 ,

80 s per day) on Cox activity (A) and ATP content (B) of cultured primary
neurons exposed to 0.4 μM TTX for 6 days. LED treatment is given for the
last 5 of the 6 days in TTX. Neurons are subdivided into darkly (dark),
moderately (moderate), and lightly (light) reactive for Cox. All P values
indicate comparisons with normal controls. All tested wavelengths are
beneficial with the exception of 728 nm (adapted with permission from Ref.
[31]).

880 nm for the last 5 of the 6 days are all effective in restoring ATP
content to control levels (Fig. 11.1B). On the other hand, 728 nm
is completely ineffective in reversing the detrimental effect of TTX
(Fig. 11.1B).
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Role of Cytochrome c Oxidase in Photobiomodulation of Cultured Visual Cortical Neurons 189

Figure 11.2 Action spectra of Cox and ATP at 670, 725, 770, 830, and
880 nm in primary neurons exposed to TTX for 6 days and LED-treated once
a day (at 4 J/cm2 ) for the last 5 of the 6 days in TTX. Note that the effective
wavelengths (especially 670 and 830 nm) correspond with the reported
absorption spectrum of oxidized Cox [6], whereas the least effective 725 nm
does not (reproduced with permission from Ref. [31]).

When the action spectra of Cox activity and ATP content in

neurons treated with different wavelengths are plotted against
the known absorption spectrum of Cox [6], it is found that the
effective wavelengths for activating Cox and ATP, especially 670 nm
and 830 nm, correlate positively with the absorption spectrum of
oxidized Cox (Fig. 11.2). The least effective wavelength, 728 nm,
does not match with the absorption spectrum of Cox. These results
are comparable to those of Karu, in which the generalized action
spectrum of DNA synthesis in HeLa cells peaks at four wavelengths
in the far-red to NIR range: 620, 680, 760, and 820 nm [17].
Karu suggests that the four peaks correspond to the absorption
spectra of reduced CuA , oxidized CuB , reduced CuB , and oxidized
CuA , respectively, in the redox-active copper centers of Cox. The LED
used for neurons has an effective bandwidth of 25–30 nm, so the
light emitted encompasses the spectra tested in HeLa cells with
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190 Cytoprotective Effect of Low-Level Light Therapy using LEDs on Neurons

monochromatic laser light. The 670 and 830 nm LED treatment falls
within the proposed absorption spectra of oxidized CuB and CuA ,
respectively. The 830 nm wavelength also falls within the maximal
absorption spectrum of oxidized Cox reported in other tissues [11,
28].
Thus, the direct benefit of LED in the NIR range is to increase
Cox activity, so that cells can generate more ATP (Fig. 11.1B) to help
with their healing process and to overcome the detrimental effect of
neurotoxins, such as TTX. Moreover, ATP has recently been found to
serve as a signaling molecule that aids in cell–cell communication
[reviewed in 18].

11.3 Neuroprotective Effect of 670 nm LED on Primary

Neurons Inactivated by Cyanide

If Cox is directly involved in the NIR photobiomodulation reaction,

then a potent and irreversible inhibitor of Cox, such as potassium
cyanide (KCN), should compete with and reduce the beneficial effect
of NIR. When various concentrations of KCN ranging from 10 μM
to 10 mM are administered to primary neurons, they all cause
significant reductions in Cox activity [31]. LED treatment at 670 nm
(with an energy density of 4 J/cm2 , twice a day) can significantly
reverse the detrimental effect on Cox at the lower dosage of KCN
(10–100 μM) [31] (Fig. 11.3A). However, at 300 μM, KCN causes
apoptotic cell death, and at 1 mM or higher concentrations, it
induces necrotic cell death, which is not reversible by NIR [20]. This
is consistent with the hypothesis that NIR activates Cox to mediate
its neuroprotective effect, which is thwarted when Cox itself is
being inactivated by KCN. Similar results are obtained with another
inhibitor of Cox, sodium azide (NaN3 ) [31].
By inactivating Cox, KCN also reduces neuronal ATP content. This
can be partially or completely reversed by 670 nm LED treatment
if the concentration of KCN is kept low (at 10 or 100 μM) [31]
(Fig. 11.3B). At higher concentrations of KCN, NIR is no longer
effective and apoptosis or necrosis will set in [31].
What is the optimal frequency of LED treatment per day for
primary neurons exposed to KCN? Four frequency paradigms have
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Neuroprotective Effect of 670 nm LED on Primary Neurons Inactivated by Cyanide 191

been tested for LED treatment during the first 8 h of 24 h exposure

to 300 μM KCN: (1) once a day immediately after KCN application;
(2) twice a day (immediately and 4 h after KCN); (3) three times
a day (immediately, 2 h 40 min, and 5 h 20 min after KCN); and
(4) four times a day (immediately, 2, 4, and 6 h after KCN). LED
treatment is 670 nm at 50 mW/cm2 and 4 J/cm2 , equivalent to
80 s each time [21]. As shown in Fig. 11.3C, only twice-a-day LED
treatment increases Cox levels significantly above those with KCN
alone (P < 0.05), but it does not reach control values. ATP content
is increased by LED and reaches control levels after two to four

Figure 11.3 (Continued)

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192 Cytoprotective Effect of Low-Level Light Therapy using LEDs on Neurons

Figure 11.3 Neuroprotective effect of LED on primary neurons exposed

to KCN. (A) Various concentrations of KCN are tested with or without LED
treatment (670 nm, 4 J/cm2 for 80 s, twice a day for each of the 5 days
in KCN). Both Cox (A) and ATP content (B) are down-regulated by KCN,
and LED partially reversed the trend only at 10 and 100 μM KCN, but not
at higher concentrations, at which cell death set in. The frequency of LED
treatment has been tested in neurons exposed to 300 μM KCN for 24 h.
Twice-a-day LED within the first 8 h of KCN exposure is the most effective
for increasing Cox activity (C) and ATP content (D), and in decreasing
cell death (E), ROS generation (F), NO production (G), and nitrotyrosine-
positive neurons (H). All *P values are compared with controls. *P <0.05;
**P <0.01, ***P <0.001. All + P values are compared with KCN alone. + P
<0.05; ++ P <0.01, +++ P <0.001. (A, B: adapted with permission from
Ref. [31]. C–F: adapted with permission from Ref. [21], Copyright 2008,
Elsevier).

treatments (Fig. 11.3D). When the KCN and LED experiments are
extended to 3 days, once a day of LED treatment for 3 days is helpful
but does not reach control levels; two to three times per day rescues
neurons’ Cox levels to those of controls; whereas four times a day
is not beneficial. Cellular ATP content returns to control levels with
once-a-day treatment and above control levels with the twice-a-
day paradigm, and increases but does not reach control levels after
three or four times of treatment. Extending the experiment to five
days yields similar results, with increases in Cox activity in all four
paradigms, but only two or three times a day return Cox to control
levels, with twice-a-day treatment giving the best value. ATP content
returns to control levels after once- or twice-a-day LED treatment,
with the latter giving the highest value, whereas three or four times
a day are not helpful [21].
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Neuroprotective Effect of 670 nm LED on Primary Neurons Poisoned by MPP+ 193

KCN of concentration 300 μM induces about 42% of apoptotic

cell death among cultured primary neurons. LED treatment once
a day decreases that percentage by 22.8% (P < 0.05), but twice
a day reduces it by 33.8% (P < 0.001). Three or four times a
day do not rescue neurons from KCN-induced apoptosis [21] (Fig.
11.3E). Generation of reactive oxygen species (ROS) is also induced
by 300 μM KCN, demonstrable with 5-(and -6) chloromethyl-2 ,7-
dichlorodihydrofluorescein diacetate acetyl ester (CM-H2 DCFDA).
Within a 24 h period of exposure to KCN, one LED treatment is
not beneficial, but two to four times do reduce the ROS level,
with two treatments yielding a value closest to that of controls
[21] (Fig. 11.3F). Reactive nitrogen species are also produced
in neurons with KCN exposure [21]. Intracellular nitric oxide
(NO) can be measured with a fluorescent probe DAF-2 DA (4,5-
diaminofluorescein diacetate). The intensity of DAF-2 DA increases
significantly above controls after 24 h of exposure to 300 μM KCN.
This value is significantly reduced below those of KCN alone after
two or three LED treatments, but not with one or four times of
treatment (Fig. 11.3G). Again, two treatments yield the lowest value.
Nitrotyrosine is an indicator of cellular damage and is produced
by the nitration of tyrosine residues in proteins by peroxynitrite, a
product of NO and superoxide. Under normal conditions, the level
of nitrotyrosine is relatively low in primary neurons (∼10.5%) [21].
However, 3 days of exposure to 300 μM KCN significantly increases
the number of nitrotyrosine-positive neurons (P < 0.001). LED
treatment twice a day effectively reduces such numbers (Fig. 11.3H)
[21].
These results consistently indicate that twice-a-day LED treat-
ment is the most beneficial paradigm and that a treatment four times
a day is generally not effective.

11.4 Neuroprotective Effect of 670 nm LED on Primary

Neurons Poisoned by MPP+ and Rotenone:
Implications for Parkinson’s Disease

Parkinson’s disease (PD) is a progressive, debilitating neurodegen-

erative disease characterized by muscle rigidity, tremor at rest,
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194 Cytoprotective Effect of Low-Level Light Therapy using LEDs on Neurons

and bradykinesia caused by the loss of dopaminergic neurons

and accumulation of Lewy bodies in the substantia nigra, pars
compacta of the midbrain [reviewed in 14, 16]. Nigral neuronal
degeneration leads to dopamine deficiency in the striatum, causing
characteristic PD symptoms. In 1983, four individuals used il-
licit drugs containing 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
(MPTP) and developed parkinsonism [19]. The toxicity was caused
by 1-methyl-4-phenylpyridinium (MPP+ ), a metabolite of MPTP
that accumulates in dopaminergic neurons and inhibits complex
I of the mitochondrial electron transport chain [7]. Animals
exposed to MPTP or another complex I inhibitor, rotenone, exhibit
dopaminergic neurodegeneration, and so have been used as animal
models of PD [7, 25].
When primary cultures of striatal neurons are exposed to
100, 250, or 500 μM of MPP+ for 48 h, their ATP content is
significantly reduced (Fig. 11.4A). NIR treatment with 670 nm
LED at 4 J/cm2 twice a day significantly increases the cellular
ATP content at all concentrations of MPP+ tested (P < 0.05–
0.01), reaching control levels in the presence of 100 μM MPP+
(Fig. 11.4A) [21]. Exposure to 50, 100, 200, or 400 nM of rotenone
for 48 h also induces a significant reduction in ATP content of
striatal neurons, and LED treatment twice a day partially reverses
this trend, with the best results achieved at lower concentrations
of rotenone (100 and 200 nm) (Fig. 11.4B). Similar results are
obtained with visual cortical neurons subjected to the same neu-
rotoxins and LED paradigms [21]. LED treatment also partially but
significantly rescues striatal (and cortical) neurons from MPP+ or
rotenone-induced apoptotic cell death (Fig. 11.4C,D), accumulation
of ROS (Fig. 11.4E,F), and excessive NO production (Fig. 11.4G,H)
[21].
Thus, NIR has a promising, therapeutic role in treating Parkin-
son’s disease, and recent findings in animal models are consistent
with that promise [23].
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Neuroprotective Effect of 670 nm LED on Primary Neurons Poisoned by MPP+ 195

Control 250 µM 250 µM MPP+ Control 50 nM 50 nM Rotenone

MPP+ plus LED Rotenone plus LED

Control 250 µM 250 µM MPP+

Control 100 nM 100 nM Rotenone
MPP+ plus LED
Rotenone plus LED

Figure 11.4 Neuroprotective effect of LED on cultured striatal neurons

exposed to various concentrations of MPP+ or rotenone for 48 h. LED
treatment (670 nm, 4 J/cm2 , twice a day) increases the ATP content (A,
B) and decreases the number of dying neurons (C, D), ROS production (E,
F), and NO production in these neurons. All *P values are compared with
controls. *P <0.05; **P <0.01, ***P <0.001. All + P values are compared
with toxins alone. + P <0.05; ++ P <0.01, +++ P <0.001 (adapted with
permission from Ref. [21], Copyright 2008, Elsevier).
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196 Cytoprotective Effect of Low-Level Light Therapy using LEDs on Neurons

11.5 Neuroprotective Effect of Pretreatment with 670

nm LED on Primary Neurons Exposed to KCN,
Rotenone, or MPP+

If LED treatment is beneficial to neurons exposed to toxins, will

there be additional gain in pretreatment prior to toxin exposure?
This question has been tested in primary neurons pretreated with
670 nm LED at an energy density of 30 J/cm2 (power intensity
of 50 mW/cm2 for 10 min) before exposing to various toxins [20,
32]. Pretreatment reduces the number of apoptotic neurons from
24.4% to 12.3% (P < 0.05) when exposed to 28 h of 100 μM
KCN, and from 65.9% to 48.7% with 300 μM KCN (P < 0.001),
an equivalent of 49.6% and 26.1% reduction, respectively (Fig.
11.5A) [20]. The percentage of neurons with single-stranded DNA
(ssDNA), a marker of apoptotic cell death [26], is also reduced
from 36% to 17.9% (P < 0.001) and from 58.9% to 39.6% (P <
0.01) with 100 μM and 300 μM of KCN, respectively, representing
a 50.3% and 32.8% reduction, respectively (Fig. 11.5B) [20]. The
level of active caspase 3, an effector of apoptosis, monitored with
western blotting, is likewise reduced, though not to control levels,
with LED pretreatment (Fig. 11.5C) [20]. The role of caspase in
KCN-induced apoptosis is substantiated when caspase inhibitor I
significantly decreases the number of apoptotic neurons in a dose-
dependent manner (Fig. 11.5D). As programmed cell death is also
regulated by pro- and anti-apoptotic Bcl-2 family of proteins [8],
the levels of pro-apoptotic Bax and anti-apoptotic Bcl-2 become
relevant. LED pretreatment reduces Bax expression up-regulated by
KCN to control levels at both concentrations of KCN (Fig. 11.5E).
At the same time, LED pretreatment increases Bcl-2 levels down-
regulated by KCN back to control values at both concentrations of
KCN (Fig. 11.5F). These findings indicate that KCN at 100–300 μM
range initiates an apoptotic cascade that can be partially rescued by
LED pretreatment.
How effective would LED pretreatment be to neurons poisoned
by rotenone or MPP+ ? This question is addressed in striatal and
cortical neurons in culture [32]. In 29.8% of striatal neurons,
200 nM rotenone for 48 h induces apoptosis. LED treatment (at
4 J/cm2 energy density) twice a day for 2 days during rotenone
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Neuroprotective Effect of Pretreatment with 670 nm LED on Primary Neurons 197

A B
Hoechst Stain 80
ssDNA
Percentage of apoptotic neurons

Percentage of ssDNA-positive
80
70
70
60
60
50 50

neurons
40 40
30 30
20 20
10 10
0 LED+ LED + 0
Control 100 µM 100 µM 300 µM 300 µM Control 100 µM LED+ 300 µM LED +
100 µM 300 µM
KCN Treated
KCN Treated

C Active Caspase 3 D
2.5 Caspase Inhibitor I
90
Percentage of apoptotic
Caspase 3 relative units

2 80
70
neurons
1.5 60
50
1 40
30
0.5 20
0 10
Control 100 µM LED+ 300 µM LED + 0
100 µM 300 µM Control 300 µM 1 µM 3 µM 5 µM 7 µM 10 µM
KCN Caspase inhibitor I pretreated
KCN Treated

E F
Bax 2.5 Bcl-2
2
1.8 2
Bcl-2 relative units
Bax relative units

1.6
1.4 1.5
1.2
1 1
0.8
0.6
0.4 0.5
0.2
0 0
Control 100 µM LED+ 300 µM LED + Control 100 µM LED+ 300 µM LED +
100 µM 300 µM 100 µM 300 µM
KCN Treated KCN Treated

Figure 11.5 Pretreating cultured visual cortical neurons with LED (670 nm,
30 J/cm2 , 10 min) before exposing them to various concentrations of KCN
significantly reduces the number of dying neurons (A), the number of
ssDNA-positive neurons (B), and active caspase 3 levels (C). The percentage
of apoptotic neurons is significantly reduced by increasing concentrations of
caspase inhibitor I pretreatment (D). LED pretreatment also restores BAX
levels up-regulated by KCN (E) and Bcl-2 levels down-regulated by KCN
(F) back to control levels. All *P values (except for D) are compared with
controls. *P <0.05; **P <0.01, ***P <0.001. *P values in D are compared
with 300 μM KCN alone. **P <0.01, ***P <0.001 (adapted with permission
from Ref. [20], Copyright 2006, Elsevier).

exposure reduces the percentage to 19.78%, representing a 33.65%

decrease (P < 0.001) (Fig. 11.6A). Pretreatment with LED (at 4
J/cm2 energy density) twice a day for 2 days before the rotenone
exposure, during which LED is also applied twice a day for 2 days,
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198 Cytoprotective Effect of Low-Level Light Therapy using LEDs on Neurons

A Hoechst Stain
% of apoptopic
neurons

B Hoechst Stain
% of apoptopic
neurons

C ATP Content
% of control

Figure 11.6 Pretreating cultured striatal neurons with LED (670 nm,
4 J/cm2 , twice a day for 2 days) before exposing them to rotenone (A) or
MPP+ (B) for 2 days further reduces the number of dying neurons beyond
that achieved by LED twice a day during toxin exposure. Pretreatment also
increases ATP production beyond that attained by LED treatment during
toxin exposure (C). All *P values are compared with controls. ***P <0.001.
All + P values are compared with toxins alone. ++ P <0.01, +++ P <0.001.
All # P values are compared between LED pretreatment and LED during
toxin exposure. ## P <0.01, ### P <0.001. XXX P (<0.001) in D compared
LED pretreatment plus LED during toxin exposure with pretreatment alone
without LED treatment during toxin exposure (adapted with permission
from Ref. [32], Copyright 2008, Elsevier).
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Neuroprotective Effect of 670 nm LED on Cytochrome c Oxidase 199

reduces the number of apoptotic neurons to 13.07% (P < 0.001),

equivalent to a 56.16% reduction (Fig. 11.6A). Comparable results
are obtained in visual cortical neurons [32]. These experiments have
been repeated in striatal and cortical neurons exposed to 250 μM
MPP+ [32]. Such exposure for 48 h induces apoptosis in 35.1% of
striatal neurons. LED treatment (at 4 J/cm2 energy density) twice
a day for 2 days during MPP+ exposure reduces the percentage to
26.32%, representing a 25.01% decrease (P < 0.001) (Fig. 11.6B).
Pretreatment with LED (at 4 J/cm2 energy density) twice a day for 2
days before the MPP+ exposure, during which LED is also applied
twice a day for 2 days, reduces the number of apoptotic neurons
to 21.95% (P < 0.001), equivalent to a 37.46% reduction (Fig.
11.6B). An additional paradigm is added in the MPP+ experiment:
pretreatment with LED twice a day for 2 days without LED treatment
during KCN exposure. This also reduces the number of apoptotic
neurons to 26.56% (P < 0.001), equivalent to a 24.33 % reduction
from MPP+ values alone, and is comparable to that of LED treatment
during but not before KCN exposure. Similar results are obtained in
cultured visual cortical neurons subjected to the same paradigms of
MPP+ toxins and LED treatments [32].
ATP content in striatal neurons is significantly reduced by 48 h
of exposure to 250 μM MPP+ [32]. Either pretreatment with LED
or LED during MPP+ exposure increases ATP content significantly
above that of MPP+ alone (P < 0.01 for both), and there is no
significant difference between the two treatment paradigms (Fig.
11.6C). Pretreatment plus LED during MPP+ further increase ATP
content above that of MPP+ alone (P < 0.001) (Fig. 11.6C).
Thus, pretreatment with NIR reduces apoptotic response and
increases ATP production in neurons exposed to neurotoxins.
The neuroprotective benefit is even greater when pretreatment is
combined with NIR treatment during toxin exposure.

11.6 Neuroprotective Effect of 670 nm LED on

Cytochrome c Oxidase Activity of Deprived Visual
Cortex of Monocularly Enucleated Rats

If NIR via LED is neuroprotective for cultured neurons, will it

be equally therapeutic in vivo? Monocular enucleation in rats and
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200 Cytoprotective Effect of Low-Level Light Therapy using LEDs on Neurons

A B
0.49 ME 5 Days 0.64 ME 11 Days
Optical density values

Optical density values

0.48 0.63
0.47 0.62

0.46 0.61

0.45 0.6
NDVC DVC NDVC DVC

C D
0.56 ME + LED 5 Days ME + LED 11 Days
Optical density values

0.67

Optical density values

0.55 0.66
0.54 0.65
0.53 0.64
0.52 0.63
NDVC DVC+LED NDVC DVC+LED

E F
0.57 Normal 0.8 Normal + LED 11 Days
Optical density values
Optical density values

0.56 0.75

0.55 0.7

0.54 0.65
0.53 0.5
LVC RVC LVC RVC + LED

G SC: ME 11 Days H
SC: ME + LED 11 Days
Optical density values

0.53 0.54
Optical density values

0.52 0.52
0.51 0.5
0.48
0.5
0.46
0.49 0.44
0.48 0.42
0.47 0.4
NDSC DSC NDSC DSC+LED

Figure 11.7 In vivo rat visual cortex after 5 days (A) and 11 days (B)
of monocular enucleation shows progressive, reduced Cox activity in the
contralateral, deprived visual cortex (DVC) versus the ipsilateral, non-
deprived visual cortex (NDVC). LED treatment (8 J/cm2 for 9 min 33 s
per day) over the cranium of deprived visual cortex (DVC + LED) (C, D)
increases Cox levels to slightly above controls after 11 days of treatment.
LED treatment over normal right visual cortex (RVC) has no effect after 5
days (E), but induces a significant rise in Cox activity after 11 days (F) as
compared to non-treated left visual cortex (LVC). As a control, the deprived
superior colliculus (DSC), which is not in the path of the LED, does not
benefit from either 5 days (G) or 11 days (H) of LED treatment as compared
to the non-deprived superior colliculus (NDSC) (adapted from Ref. [24]).
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cDNA Microarray Analysis of Genes Up- and Down-Regulated by 670 nm 201

primates is known to cause reduced Cox activity in the contralateral,

deprived superior colliculus and visual cortex [13, 33]. Will LED-
mediated NIR applied over the deprived visual cortex bring about
any therapeutic effect? This has been tested in rats monocularly
enucleated and survived for either 5 days or 11 days [24]. Deprived
visual cortex shows a progressive reduction in Cox activity with time
(Fig. 11.7A,B). LED at 670 nm, an aperture of 5 mm, and an energy
density of 8 J/cm2 have been applied for 9 min 33 s over the skull of
the deprived, contralateral visual cortex each day for either 5 days
or 11 days. Results indicate that 5 days of LED treatment increases
slightly Cox activity down-regulated in the deprived visual cortex,
but that 11 days of LED treatment induces a rise in Cox activity
of deprived visual cortex above that of controls (Fig. 11.7C,D).
Surprisingly, LED applied over the visual cortex of normal animals
once a day for 5 days does not have any effect, but an 11-day
treatment results in a dramatic rise in Cox activity well above that
of controls (Fig. 11.7E,F). As an internal control, either 5 days or
11 days of LED treatment has no effect on the deprived superior
colliculus (Fig. 11.7G,H), as this structure is not in the path of the
NIR beam.
Thus far, the neuroprotective effect of NIR (LLLT) has been
repeatedly shown in vivo, both in animals and in humans [3, 9, 10,
15; see also pertinent chapters in this book].

11.7 cDNA Microarray Analysis of Genes Up- and

Down-Regulated by 670 nm LED in Deprived Visual
Cortex of Monocularly Enucleated Rats

What is the molecular mechanism underlying the neuroprotective

effect of NIR via LED treatment? In addition to the direct activation
of Cox and ATP production, NIR is likely to induce a cascade of events
leading to altered gene expression in neurons. To determine if this is
the case, rats have been monocularly enucleated at varying ages (9-,
13-, and 21-day old), and LED at 670 nm, power intensity of 19
mW/cm2 , and energy density of 4 J/cm2 have been administered
over the deprived cortex for 3.5 min each day for 5 of the 6
surviving days. Deprived and non-deprived visual cortices, as well
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202 Cytoprotective Effect of Low-Level Light Therapy using LEDs on Neurons

Figure 11.8 An example of partial microarray data of gene expressions

from visual cortex of rats monocularly enucleated at 9, 13, and 21 days
of age and survived for 6 days. The left three of the six columns denote
comparisons between deprived visual cortex (DVC) versus non-deprived
visual cortex (NDVC) when enucleation is done on days 15, 19, and 27,
respectively. The three right columns compare LED treatment of DVC
(LED) versus non-treated DVC with enucleation on days 15, 19, and 27,
respectively. Green represents down-regulation and red represents up-
regulation. Each row signifies one gene. Many of the same genes are down-
regulated by visual deprivation and up-regulated by LED treatment (see
text for details) (adapted from Ref. [30]). The microarray was done in the
laboratory of Marti Jett at the Walter Reed Army Institute of Research in
Silver Spring, MD. Rina Das, Apsara Dhokalia, and Rasha Hammamieh also
contributed to the processing and analysis of the data.

as deprived cortices treated with LED, have been analyzed with

cDNA microarray (Clontech cDNA expression array Rat 1.2.1 kit)
and Atlas Image 2.01 program as well as Gene Spring program to
determine if any changes occurred in their gene expression [30].
Figure 11.8 illustrates representative examples of the same genes
down-regulated by deprivation but up-regulated by LED, or vice-
versa. As expected, many genes underwent down-regulation with
visual deprivation and up-regulation with LED treatment. Genes that
are up-regulated by LED at all three ages include those that are
July 6, 2016 17:21 PSP Book - 9in x 6in 11-Hamblin-c11

Conclusion 203

involved with neuronal activity, such as glutamate receptors, GABA

receptors, voltage-gated sodium channels, voltage-gated potassium
channels, Ca2+ -ATPase, Na+ /K+ -ATPase, and voltage-dependent
calcium channels [30]. Genes that are down-regulated by LED
include tumor-associated protein 1, Wilms tumor 1, DNA damage-
inducible transcript 1, transforming growth factor beta, RAB11A (a
member of RAS oncogene family), and mineralocorticoid receptors
(aldosterone receptors) [30]. It is striking that many of the same
genes are down-regulated by visual deprivation and up-regulated
by LED treatment (Fig. 11.8). More studies are necessary to
substantiate these findings.

11.8 Conclusion

Light in the far-red to NIR range has known therapeutic benefits.

Primary neurons in culture offer a distinct advantage by enabling
testing of various parameters and toxins under controlled condi-
tions. They reveal the significant role that mitochondrial Cox plays
in the photobiomodulatory process. Activated enzymes increase
energy production and induce a cascade of events leading to changes
in gene expression. A combination of in vitro and in vivo experiments
will lead closer to an optimal paradigm for LLLT.

Acknowledgments

The work done in MWR’s lab cited and illustrated in this chapter
was funded by DARPA Grant N66001-03-1-8906 and NIH/NICAM
Grant R21-AT003002 (grants were shared by Harry Whelan, Janis
Eells, and MWR), NIH Grants EY05439 and EY018441 (to MWR),
and the Department of Molecular Pathology (headed by Marti Jett) at
the Walter Reed Army Institute of Research. MWR thanks Quantum
Devices, Inc. for providing the LED arrays and R. Ignatius for his
ingenious mind. She greatly appreciates helpful discussions with
Drs. H. Whelan, J. Eells, M. Jett, R. Das, A. Dhokalia, R. Hammamieh,
and the late B. Chance during the course of the studies. She also
appreciates the able technical help of M. Scheidt and R. Ying.
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204 Cytoprotective Effect of Low-Level Light Therapy using LEDs on Neurons

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8. Desagher, S., and Martinou, J.C. (2000). Mitochondria as the central
control point of apoptosis. Trends Cell Biol., 10, pp. 369–377.
9. Eells, J.T., Henry, M.M., Summerfelt, P., Wong-Riley, M.T.T., Buchmann,
E.V., Kane, M., Whelan, N.T., and Whelan, H.T. (2003). Therapeutic
photobiomodulation for methanol-induced retinal toxicity. Proc. Natl.
Acad. Sci. USA, 100, pp. 3439–3444.
10. Eells, J.T., Wong-Riley, M.T.T., VerHoeve, J., Henry, M., Buchman, E.V., Kane,
M.P., Gould, L.J., Das, R., Jett, M., Hodgson, B.D., Margolis, D., and Whelan,
H.T. (2004). Mitochondrial signal transduction in accelerated wound
and retinal healing by near-infrared light therapy. Mitochondrion, 4, pp.
559–568.
11. Ferrari, M., Hanley, D.F., Wilson, D.A., and Traystman, R.J. (1990).
Cerebral cytochrome-c-oxidase copper band quantification in perfluo-
rocarbon exchange transfused cats. Adv. Exp. Med. Biol., 277, pp. 85–93.
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References 205

12. Gonzalez-Lima, F., Barkdale, B.R., and Rojas, J.C. (2014). Mitochondrial
respiration as a target for neuroprotection and cognitive enhancement.
Biochem. Pharm., 88, pp. 584–593.
13. Hevner, R.F., and Wong-Riley, M.T.T. (1990). Regulation of cytochrome
oxidase protein levels by functional activity in the macaque monkey
visual system. J. Neurosci., 10, pp. 1331–1340.
14. Hirsch, E.C., Faucheux, B., Damier, P., Mouatt-Prigent, A., and Agid, Y.
(1997). Neuronal vulnerability in Parkinson’s disease. J. Neural. Transm.
Suppl., 50, pp. 79–88.
15. Huang, Y.Y., Gupta, A., Vecchio, D., de Arce, V.J., Huang, S.F., Xuan, W., and
Hamblin, M.R. (2012). Transcranial low level laser (light) therapy for
traumatic brain injury. J. Biophotonics, 5, pp. 827–837.
16. Jankovic, J. (2008). Parkinson’s disease: Clinical features and diagnosis.
J. Neurol. Neurosurg. Psychiat., 79, pp. 368–376.
17. Karu, T. (1999). Primary and secondary mechanisms of action of
visible to near-IR radiation on cells. J. Photochem Photobiol. B, 49,
pp. 1–17.
18. Karu, T. (2010). Multiple roles of cytochrome c oxidase in mammalian
cells under action of red and IR-A radiation IUBMB Life, 62, pp. 607–610
19. Langston, J.W., Ballard, P., Tetrud, J.W. and Irwin, I. (1983). Chronic
Parkinsonism in humans due to a product of meperidine-analog
synthesis. Science, 219, pp. 979–980.
20. Liang, H.L., Whelan, H., Eells, J., Meng, H., Buchmann, E., Lerch-Gaggl,
A. M., and Wong-Riley, M. (2006). Photobiomodulation partially rescues
visual cortical neurons from cyanide-induced apoptosis. Neurosci., 139,
pp. 639–649.
21. Liang, H.L., Whelan, H.T., Eells, J.T., and Wong-Riley, M.T.T. (2008). Near-
infrared light via light-emitting diode treatment is therapeutic against
rotenone- and MPP+ -induced neurotoxicity. Neurosci., 153, pp. 963–
974.
22. Prindeze, N.J., Moffatt, L.T., and Shupp, J.W. (2012). Mechanisms of action
for light therapy: A review of molecular interactions. Exp. Biol. Med.,
237, pp. 1241–1248.
23. Quirk, B.J., DeSmet, K.D., Henry, M., Buchmann, E., Wong-Riley, M.,
Eells, J.T., and Whelan, H.T. (2012). Therapeutic effect of near infrared
(NIR) light on Parkinson’s disease models. Front. Biosci., 4, pp. 818–
823.
24. Scheidt, M., Wong-Riley, M.T.T., Eells, J., and Whelan, H. (2002). Near
infrared LED light treatment of normal and monocularly-deprived rat
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206 Cytoprotective Effect of Low-Level Light Therapy using LEDs on Neurons

visual cortex up-regulates cytochrome oxidase activity in vivo. Soc.

Neurosci. Abstr., #131.21.
25. Sherer, T.B., Betarbet, R., Testa, C.M., Seo, B.B., Richardson, J.R., Kim,
J.H., Miller, G.W., Yagi, T., Matsuno-Yagi, A., and Greenamyre, J.T. (2003).
Mechanism of toxicity in rotenone models of Parkinson’s disease. J.
Neurosci., 23, pp. 10756–10764.
26. Watanabe, I., Toyoda, M., Okuda, J., Tenjo, T., Tanaka, K., Yamamoto,
T., Kawasake, H., Sugiyama, T., Kawarada, Y., Tanigawa, N. (1999).
Detection of apoptotic cells in human colorectal cancer by two different
in situ methods: Antibody against single-stranded DNA and terminal
deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling
(TUNEL) methods. Jpn. J. Cancer Res., 90, pp. 188–193.
27. Whelan, H.T., Buchmann, E.V., Whelan, N.T., Turner, S.G., Cevenini, V.,
Stinson, H., Ignatius, R., Martin, T., Cwiklinski, J., Meyer, G.A., Hodgson, B.,
Gould, L., Kane, M., Chen, G., and Caviness, J. (2001). NASA light-emitting
diode medical applications from deep space to deep sea. Space Tech.
Appl. Int. Forum, 552, pp. 35–45.
28. Wikstöm, M., Krab, K., and Saraste, M. (1981). Cytochrome Oxidase. A
Synthesis. (Academic Press, New York, USA).
29. Wong-Riley, M.T.T., Bai, X., Buchmann, E., and Whelan, H.T. (2001). Light-
emitting diode treatment reverses the effect of TTX on cytochrome
oxidase in neurons. NeuroReport, 12, pp. 3033–3037.
30. Wong-Riley, M.T.T., Whelan, H., Dhokalia, A., Das, R., Hammamieh, R.,
Liang, H.L., Eells, J., and Jett, M. (2002). cDNA microarray analysis of the
visual cortex exposed to light-emitting diode treatment in monocularly
enucleated rats. Soc. Neurosci. Abstr., #131.20.
31. Wong-Riley, M.T.T., Liang, H.L., Eells, J.T., Henry, M.M., Buchmann, E.,
Kane, M., and Whelan, H.T. (2005). Photobiomodulation directly benefits
primary neurons functionally inactivated by toxins: Role of cytochrome
c oxidase. J. Biol. Chem., 280, pp. 4761–4771.
32. Ying, R., Liang, H.L., Whelan, H.T., Eells, J.T., and Wong-Riley, M.T.T.
(2008). Pretreatment with near-infrared light via light-emitting diode
provides added benefit against rotenone- and MPP+ -induced neurotox-
icity. Brain Res., 1243, pp. 167–173.
33. Zhang, C., Granstrom, L., and Wong-Riley, M.T.T. (1996). Deafferentation
leads to a down-regulation of nitric oxide synthase in the rat visual
system. Neurosci. Lett., 211, pp. 61–64.
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Chapter 12

Low-Level Laser and Cultured Neural

Tissue

Patricia J. Armati and Roberta T. Chow

Central Clinical School, Edward Ford Building A27, The University of Sydney,
NSW 2006, Australia
roberta.chow@sydney.edu.au

This chapter provides information on the use of neural tissue

cultures as adjunct models to further define the mechanism by
which low-level laser (LLL) affects either excitatory/stimulating or
inhibitory responses of peripheral nervous system (PNS) sensory
neurons, central nervous system (CNS) neurons, and associated
cells. It also discusses the importance of laser parameters and
conditions.

12.1 Why Use Cell or Tissue Culture Models?

The use of tissue/cell culture models for investigating questions

relating to mechanisms of action of immune-related molecules, drug
effects, and cellular interactions has a long history beginning in 1885
when Willem Roux kept embryonic chick tissue in warmed saline
for some days to observe its development. The simplicity of this
technique contrasts with the complex cell culture techniques of the

Handbook of Low-Level Laser Therapy

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Copyright c 2017 Pan Stanford Publishing Pte. Ltd.
ISBN 978-981-4669-60-3 (Hardcover), 978-981-4669-61-0 (eBook)
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208 Low-Level Laser and Cultured Neural Tissue

present day where organotypic cultures and nonadherent culture

techniques allow three-dimensional organization of individual or
multitypic cell types, which better represent the in vivo arrangement
of the organ or tissue (Kerr et al., 1995). A search of reports of cell
cultures exposed to LLL irradiation found 357 publications from
1982 to the present, showing that there is a relatively long history
of such experiments in a great variety of cell types and cell lines.
This brings up the importance of the types of cells used in culture
experiments, in particular when considering cells of the nervous
system, both peripheral and central.
Reports of experiments with LLL on cultures of cells of the PNS
and CNS predominantly use rodent cells. However, neurons from
human dorsal root ganglia (DRG) and CNS tissue, in general from
fetal tissue, have also been studied. Furthermore, species differences
exist between responses of human cells and those of animals such
as rats, mice, and guinea pigs, to name a few. For example, a study
of glutamate neurocytotoxicity using rat and human cortical tissue
showed a difference in the expression of Ca2+ ATPase isoforms
between rat and human neural tissue (Wanigasekara et al., 2003). An
advantage of using cell culture models to explore cellular responses
(in particular for this chapter) to LLL is that they permit the study
of individual cells or groups of cells that are difficult or not possible
with in vivo or in situ studies. Examples of cell culture models include
explants of mixed cell types, three-dimensional clusters of neurons
in suspension, dissociated cells growing on a defined substratum,
neurospheres derived from brain tissue of the CNS or myelinated
cultures of both peripheral and CNS cells or colonies of a specific
cell type or cell lines that are often transformed and, therefore, ge-
netically abnormal. Cell or tissue cultures further provide a method
that allows rigorous experimental design since each experiment can
have sufficient replicates and each experiment can be replicated so
that data can undergo statistically powerful analyses.

12.2 Cell Lines

Cell lines are also used in many culture models. These cell lines are
generally transformed with specific genetic and cellular responses
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Specific Characteristics of Nervous System Cells 209

that are different from primary cells derived from animal or human
biopsy material. While it is possible to differentiate specific cell
lines into forming neuron-like morphology with multiple neurites
or axonal processes, these are often derived from tumor tissue. For
example, PC-12 cells derived from a human phaeochromocytoma
can be induced by nerve growth factor to form neuron-like
morphology. While cell lines are particularly useful since they are
commercially available, divide rapidly, and have been intensively
characterized, they do not represent primary cell characteristics.
While many culture models employ cell lines, the use of primary
tissue has advantages over cell lines. Of specific importance for
experiments on nervous system cells or tissue is the difference
between neurons and other cell types.

12.3 Specific Characteristics of Nervous System Cells

There are important differences between neurons and other cell

types. Neurons have defined processes and a discrete nerve cell
body. The majority of neurons have an axon and dendrites, each
of which has specific characteristics. Sensory neurons of the PNS,
however, have only a single axon and no dendrites, as discussed
in more detail later. Neuronal morphology and physiology are
substantially different in many aspects such as neurons’ fast
response to stimulation. This is also in contrast to all other cells
of the body and particularly to undifferentiated cell lines. This
difference specifically relates to studies of photobiomodulation-
induced changes in axonal morphology and physiology. Effects
include decreased axonal flow, distribution and abundance of ATP-
bearing mitochondria and other axonal cargo are essential for
nerve conduction. Morphological changes include axonal varicosity
formation (Dekroon and Armati, 2002a). The importance of these
provisos for studies of LLL effects on cultured nervous system cells
is discussed in more detail in the following paragraphs.
The drawback of cell cultures is that they are ex vivo collections
of cells in an artificial environment. They are also often composed of
single cell types without interactions with other cell types associated
with them in vivo. Cell cultures are often an undifferentiated
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210 Low-Level Laser and Cultured Neural Tissue

community of cells. This is particularly relevant to cultures of

neurons where in vivo, all axons including the unmyelinated C
fibres are ensheathed by Schwann cells. In myelinated nerves,
these neuroglia spiral around individual axonal lengths and their
plasma membranes become compacted to form myelin. This results
in saltatory conduction of up to 120 M/s, a function unique to
the nervous system. These considerations of cellular communities,
their development, differentiation and function in normal, damaged
and repair situations are, therefore, particularly important for the
nervous system culture models. While cell culture in itself is an
artificial model, primary neurons can be driven into a mature
myelinated morphology in culture, but in general cell culture models
are of a single cell type.
LLL delivery to cells in culture needs to be correlated with that
in vivo. The discussion and comparison of culture model data and
that from in vivo or in situ data findings are only relevant when
data can be compared. This is particularly important since this
enables correlation of cell responses to LLL and, particularly, laser
parameters. The first step is ensuring that the output power of the
laser device is formally measured at the beginning of a research
project as well as during its conduct and on completion. For example,
in our experiments the same lasers used clinically and in culture
experiments were calibrated and their output measured by the
Australian National Institute of Measurement or at the Department
of Laser Physics, Macquarie University, by Professor Judith Dawes.
This enabled calculations of parameters for irradiation of the
cultured cells for devices that are used clinically, to ensure
equivalence of doses with that delivered transdermally (Chow et al.,
2007). In cultures, cells are directly exposed to incident energy along
its trajectory, whereas in clinical applications, energy is scattered
and attenuated by skin and within deeper tissues. While laser output
should be measured regularly to maintain internal validity within
the experiment, all parameters used must be reported to enable
reproducibility and external validity of experiments. Inconsistent
and incomplete reporting of the complete data set of parameters has
been a feature of many low-level laser therapy (LLLT) studies (Chow
et al., 2011). The critical importance of reporting all parameters has
been discussed by several authors (Calderhead, 1991; Enwemeka,
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LLL, Cell Culture, and Peripheral Nervous System 211

2009; Timberlake and Enwemeka, 2004). The review of Jenkins

and Carroll (2011), in particular, has direct relevance to cell culture
studies of LLL. Not only did they identify standard beam parameters
for clinical studies, including wavelength, output power, duration of
exposure, and beam mode, they also identified the importance of
irradiation covering the whole area of each well in a culture plate
with a uniform power density. Smith (2005) also points out that
insufficient emphasis is given to always providing the wavelengths,
area of irradiation, and dose and the time of irradiation in all
publications. This makes the point that acceptance of LLLT depends
on an understanding of parameters so that such therapy may be
better integrated into clinical practice. The laser parameters of
studies cited in this chapter are given in Table 12.1.
Thermal effects on cultures have not been widely reported,
though change in temperature can be an independent variable
causing changes in cell activity. Cell culture media temperature,
therefore, should be monitored in LLL studies as it is defined as
being a non-thermal modality.

12.4 LLL, Cell Culture, and Peripheral Nervous System

This chapter discusses the effects of LLLT on PNS, but the specific
specialization of the PNS sensory nerves does need to be set
out since the explanation of LLL inhibition in our experiments
relates directly to their organization. Both the PNS and CNS in
vivo are composed of a complex arrangement of neurons and their
associated cells. These include the Schwann cells and fibroblasts of
the PNS and the neuroglial astrocytes and oligodendrocytes of the
CNS as well as the microglia of hemopoietic origin. Most culture
systems, however, are monocultures of neurons or differentiated
neuron-like cell lines such as PC-12 cells, which form PNS-like cells.
The rationale for emphasizing the complexity of cell types is twofold.
First, in the PNS, the sensory nerve cells within the DRG are unique
in their morphology. Sensory neurons have no dendrites and a single
synapse within the dorsal horn (Fig. 12.1).
The neurons are characterized by their defined cell body where
protein and lipid syntheses occur, and mitochondrial charging by
 July 6, 2016 17:22
212 Low-Level Laser and Cultured Neural Tissue
Table 12.1 Studies of laser irradiated cell cultures

Power
Output Duration of ED PD
Reference Cell Type λ (nm) (mW) Exposure Mode (J/cm2 ) (mW/cm2 ) Total J

Shimoyama et al., 1992 Rat cervical 632.5 5.5 180, 300, cw 63 350, 105, 1, 1.5,
sympathetic ganglia 600 210 3,
Jimbo et al., 1998 Murine DRG 830 16.2 120 cw 49 1.9
neurons
Chen et al., 1993 Murine DRG 830 20 3 cw 6
neurons
Miura and Kawatani, Rat nodose 830 2.5–150 1–180 cw 2.5, 5, 0.0025–27

PSP Book - 9in x 6in

1996 ganglion neurons 10, 20,
32.4
Wollman et al., 1996 Rat embryonal 632.8 0.27 480 cw 3.6 0.13
brain cells
Wollman and Rochkind, Rat cortical neurons 632.8 0.27 (i) 480 × 1 cw 3.6 0.13 at each
1998 (ii) 480 × 2 dose
after 24 h
(iii) 480 ×
3@2h
intervals
Wong-Riley et al., 2001 Primary cultured 670 80 cw 4 50
rat visual cortex LED
2x/day
for 5
days

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 July 6, 2016 17:22
Wong-Riley et al., 2005 Primary cultured LED 80 cw 4 50
rat visual cortex 670,
728
770,
830
880 -
pre and
post-Rx
Yoda, 2005 Murine DRG 830 16.2 60 cw 49 ∼1

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Chow et al., 2006 Rat 830 400 15, 30, 60, cw 1.4, 8.3, 300 6, 12,

LLL, Cell Culture, and Peripheral Nervous System

DRG 120 16.7, 24, 48
33.3
Saito et al., 2006 Murine DRG 830 16.2 120 cw 49 1.9
Trimmer et al., 2009 Human trans- 810 50 40 cw 2 50 2å
mitochondrial
hybrid neuronal
cells
Meng et al., 2013 Mouse hippocampal 632.8 10 42, 75, cw 12.74 0.01, 0.75,
neurons 150, 300 1.5,
3

Abbreviations: ED, energy density; PD, power density; cw, continuous wave; LED, light-emitting diode, DRG, dorsal root ganglion

213

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214 Low-Level Laser and Cultured Neural Tissue

oxidative phosphorylation of the mitochondria is driven by ATPase;

then there is the axon hillock—the region between the cell body and,
therefore, the entry point of the high-energy mitochondria, proteins,
and lipids to the axon. The axon is T-shaped with its peripheral
arm ending in the skin or other sensory receptor areas and the
central arm synapsing within the dorsal horn of the spinal cord
(Fig. 12.1). These axons can be up to 1 m in length in contrast

Figure 12.1 Peripheral nervous system neurons (a), central

nervous system interneurons (b), motor neuron and sensory neuron (c).
Note: The nociceptor Aδ fibers are thinly myelinated, and the C-fibers
are unmyelinated, ensheathed by Schwann cells with their endings
unensheathed in the epidermis.
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LLL Irradiation of Cultured Sensory Neurons in Pain-Related Studies 215

to most CNS neurons. This means that intra-axonal transport of

high-energy mitochondria is dependent on the fast axonal transport
system driven by the microtubule motor proteins. The neuronal
organization is, therefore, an important consideration in studies of
cultured DRG neurons especially related to the effects of LLLT. For
example, we have reported that laser at specific parameters causes
depolymerization of axonal microtubules, a significant decrease in
mitochondrial membrane potential and conduction block (Chow
et al., 2007). Jimbo et al. (1998), in an earlier study, have shown the
importance of examining differences in the response of cell body to
LLLT compared to that of axon.

12.5 Delivery of LLL to Neural Tissue in Culture

Culture techniques often involve plastic plates, tubes, or dishes,

and there is little recognition of the knowledge that photons are
transmitted through plastic. Silva et al. (2012) have addressed this
issue. In our experiments, we plated the DRG neurons into alternate
wells of a 24-well cell culture plate and screened each well with a
matt black screen around the sides and beneath each well to avoid
reflected or transmitted photons.

12.6 LLL Irradiation of Cultured Sensory Neurons in

Pain-Related Studies

There is now clear evidence that LLLT is an effective clinical

treatment for many painful conditions (Chow et al., 2006). In
another interesting clinical trial, Nd:YAG laser has been used to effect
dental anesthesia lasting up to 40 min. This is an important finding
for needle-phobic or dento-phobic patients since 80% of people
have fear of dentists (Chan et al., 2012). In a pilot study to dissect
the mechanism by which laser effected dental anesthesia, Chan et al.
(2012) cultured DRG nociceptor neurons using the same Nd:YAG
laser at the same relative dose as used in the clinical trial. Vesicle
formation along the axons was reversed by 24 h (unpublished
data). Control unlasered cultures showed no such changes. This
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216 Low-Level Laser and Cultured Neural Tissue

finding is consistent with our other study discussed below where

the same changes followed laser irradiation (Chow et al., 2007).
By definition LLL is not thermally driven, but its effect is due to
photon absorption. The interesting question regarding such use of
laser is: What is the mechanism of its effectiveness? This is an
important question since there are varying theories currently but
no unified concept. Our group, however, has evidence that LLL at
specific wavelengths, energy densities, and doses affects pain relief
and dental anesthesia by the inhibition of nerve conduction. As the
photoreceptor in eukaryotic cells is mitochondrial cytochrome c
oxidase (Karu, 2010b) and our experiments have shown statistically
significant decrease in mitochondrial membrane potential and a
concomitant decrease in ATP following LLL irradiation less than 8
J/cm2 , this would lead to our reported microtubule destabilization
seen as axonal varicosities, block of fast axonal flow, and conduction
block. The conduction block is reversible, accounting for no adverse
effects of LLL either experimentally or clinically. Further, the
evidence of consistent reporting of laser parameters used in LLLT
is particularly important. This is based on our systematic review
(Chow and Armati, 2010) showing that laser parameters are
variably and often incompletely reported. The concept that neural
inhibition underlies LLLT, however, requires investigation of events
occurring at the molecular and organelle levels in conjunction with
electrophysiological studies in animal models.

12.7 Low-Level Laser: Excitatory or Inhibitory

Further, Karu has shown that the laser effect depends on the
radiation, wavelength, dose, and intensity as well as on other cell
culture conditions (Karu, 2010a, 2014). Therefore, it is possible that
cells in tissue or cell cultures may not respond to LLLT in exactly
the same way so that similar parameters may well have different
effects on different cultured cells. It is clear from the literature
that LLLT can be inhibitory or stimulatory, and to some extent, this
seems contradictory. It is well described in the literature that the
excitatory or stimulating effect of LLL is effective due to the biphasic
dose response, described as the Arndt–Schulz phenomenon. Huang
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Low-Level Laser 217

et al. (2009) have described laser irradiation parameters in medical

terms as being the “medicine” or laser power delivered and the
duration of its delivery or “dose.” They have stressed the importance
of laser parameters in their effectiveness as the ratio between
the two and that this is not necessarily reciprocal (Huang et al.,
2009). In the 1970s, it was first mooted that laser was effective
in wound healing, which stimulated tissue repair by increasing cell
division and macrophage activity in phagocytosing damaged tissue.
From this developed the idea that laser could also play a role in
downregulating pain since with wound healing comes pain relief.
One of the problems in accepting the role of non-ablative laser in
medical conditions was that there was no understanding of the
mechanism by which laser achieved it action. However, considering
the laser parameters for both wound healing and pain relief, it
became clear that the lower end of LLL (0.5 J/cm2 to 3.5 J/cm2 and
4 J/cm2 to 8 J/cm2 ) was stimulatory while a higher LLL irradiation
above 8 J/cm2 was found to be effective for pain relief unlike the
lower doses. We have found that in a cell culture pain model, higher
doses of LLL cause nerve cell changes indicative of neural inhibition.
The findings are consistent with electrophysiological studies on rat
nerve and clinical trials discussed later in the chapter.
The lack of a mechanism for inhibition has been a particular
problem in the acceptance of LLLT for painful conditions. However,
the inhibitor and excitatory effects of LLL on cells in vivo or in
cell culture may well be related to its effect on mitochondria
where the photoacceptors are located. Normally, ATP is released
into the cytosol and the low-energy ADP is then pumped back
into the mitochondrion for recharging. Mitochondria are, therefore,
the starting point of response to LLL—stimulatory or inhibitory—
and related to the laser parameters. ATP, however, is not only an
energy provider but a signaling molecule and neurotransmitter of
significance in the nervous system (Karu, 2010a). In the PNS, it
is an important molecule that signals bidirectionally between the
nerve cell body of the DRG and their surrounding satellite cells. Of
relevance to chronic pain conditions, specific subsets of P2X and
P2Y receptors respond to the pain-signaling ATP released by the
P2X receptor of the satellite cell that signals the nerve cell body.
This upregulates P2Y receptors in sensory neuronal cell bodies
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218 Low-Level Laser and Cultured Neural Tissue

resulting in the downregulation of neuronal P2X receptors. This can

downregulate pain, as shown in elegant studies by Chen et al. (2008),
consistent with inhibiting the development of allodynia.
Another example of LLL modulating important signaling mole-
cules is its effects on nitric oxide (NO), which is formed during
oxidative phosphorylation (Chumak et al., 2000; Karu, 2010b). NO,
a signaling molecule, can cause acute and reversible mitochondrial
inhibition of cytochrome oxidase, which competes with oxygen, or
irreversible inhibition resulting in cell death (Brown and Borutaite,
2002). The effect is concentration dependent. NO can also inhibit
mitochondrial function by a potent reversible interaction with
oxygen or as an irreversible inhibition resulting in cell death (Brown
and Borutaite, 2002). In LLL cell culture studies of PNS neurons, we
discuss studies in which the neurons were reversibly inhibited as
well as those showing increased neurite sprouting.

12.8 Dorsal Root Ganglion Cultures of Nociceptor

Neurons

There are a few reports on the effects of LLL on cultures of

peripheral nerve neurons. One of the few studies investigating the
effect of laser irradiation on neuronal cell cultures is the study by
Chen et al. (1993) showing that LLL causes varicosity formation
in substance P positive mouse DRG neurons and inhibition of
neurite outgrowth. The effect is reversed after 5 h, showing that
such inhibition is neither toxic nor damaging. In another study
examining the effect of LLL on cultured mouse DRG neurons,
Jimbo et al. (1998) have used patch clamping to measure the
neuronal response to an electrical stimulation delivered directly
to the cell body. These cells equate to the nociceptor Aδ and C-
fiber neurons of importance in conducting the painful stimulus to
the CNS dorsal root neurons. The electrical stimulation initiates
an action potential as in vivo. The same culture model, but using
laser irradiation of the cell body, also generates an action potential.
However, when the neurite/axon alone is irradiated with the laser,
there is no measurable action potential at the cell body consistent
with conduction block/inhibition. In contrast, when the algesic
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Dorsal Root Ganglion Cultures of Nociceptor Neurons 219

molecule bradykinin is applied to a neuronal process, it evokes

an action potential, again measured by patch clamping, at the cell
body. When bradykinin is applied followed by laser irradiation,
this does not evoke a measurable action potential at the cell
body. Further, bradykinin stimulation in the absence of further
laser irradiation generates an evoked potential showing return of
function. These early and elegant studies are important as they show
the effectiveness of laser irradiation in causing inhibitory effects and
importantly the reversibility of the laser-induced neural inhibition.
In a study using murine DRG C-fiber nociceptors, Yoda (2005)
has explored the effect of LLL on these cells, again using the
algesic molecule bradykinin to ascertain its effect on intracellular
calcium. The study further dissects the mechanism by which laser
induces changes in cell metabolism as bradykinin is important in
the excitation of cytosolic calcium by elevating its concentration by
calcium flux across the cell membrane and from the mitochondria.
In a parallel experiment when cells are in a Ca2+ -free medium, there
is no such change. LLL at 29 J/cm2 , however, inhibits Ca2+ elevation
by transmembrane flux and Ca2 pools as present in mitochondria
and endoplasmic reticulum. After 30 min, this inhibition is reversed.
This study, like that of Jimbo et al. (1998), is related to the further
understanding of how laser affects pain relief. They postulate that
bradykinin binds normally to its B2 receptor but alters Na+ channel
function and acts via a G-protein-mediated activation, opening
voltage-gated Ca2+ channels. Bradykinin also increases the release
of pooled Ca2+ such as that within the mitochondria. Of further
importance in understanding the mechanism of LLL action on
pain is the finding that LLL does not block the inward Na+ influx
but result in membrane depolarization, a finding supported by
electrophysiological studies, such as ours, showing conduction block
in an in vivo model (Yan et al., 2011). This is also consistent with
our neural inhibition hypothesis. Again a proviso is that the Yoda
(2005) study does not set out the experimental design so that the
number of replicates is not reported, but it does provide a relevant
and important addition to understanding the laser inhibition of
peripheral pain pathways.
A further study by the Yoda group (Saito et al., 2006) has shown
that the laser dose is specifically of relevance in the inhibition or
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220 Low-Level Laser and Cultured Neural Tissue

stimulation of cellular responses. In this study, cell cultures of DRG C-

fiber neurons have been irradiated at a very low dose, which caused
stimulation of action potentials in contrast to higher doses, which
were inhibitory. This study aligns with those of the very early studies
where LLL irradiation was used for wound healing. In these studies,
the irradiation is in the range of 0.5 J/cm2 to 4 J/cm2 (Silveira
et al., 2011). In clinical studies where LLL is delivered directly to
ulcerated skin, the irradiation dose is in the range of 0.5 J/cm2 to
3.9 J/cm2 ; less is not always better, nor is more. Thus, the dose
response is particularly important for pain-related LLLT. Of clinical
relevance is that these studies support such a mechanism and are
consistent with the clinical trial data related to the studies of Chow
et al. (2009) on chronic neck pain, our cell culture studies, and in
vivo electrophysiological experiments on rat sciatic nerve (Yan et al.,
2011). However, a proviso regarding the data of Jimbo et al. (1998)
is that they examined only three neurons in each set of experiments.
But these early cell culture experiments do provide a mechanism
consistent with the hypothesis that laser-induced pain relief has a
neural basis. The reversibility of the LLL effect and the evidence that
it is neither toxic nor damaging are of particular relevance to clinical
studies of laser pain relief where there are no side effects such as
numbness, loss of function, and infection following LLLT since the
therapy is noninvasive.
In earlier in vivo experiments using rats, we have delivered LLL
at a dose equivalent to that delivered in the clinic. We have shown
that LLL irradiation causes conduction block (Yan et al., 2010). To
further dissect the mechanism by which laser affects nociceptive
neurons, we have explored the effect of clinically equivalent laser
irradiation on dissociated rat DRG neurons using our routine culture
technique reported for earlier non-laser studies (Armati et al.,
1990). The suitability of this model for LLL studies is demonstrated
by flow cytometry studies that show that the cells are 95% Aδ or C
nociceptive neurons (Dedov et al., 2001).
Our neuronal studies of dissociated cultures of rat DRG neurons
also address this hypothesis and are related to the mechanism by
which LLL can affect pain relief in which we examined electrophysio-
logical changes following laser irradiation in a rat animal model (Yan
et al., 2010). Our culture model includes satellite cell-ensheathed
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Dorsal Root Ganglion Cultures of Nociceptor Neurons 221

sensory neurons, Schwann cells, and fibroblasts. Although it is

possible to purify and culture each cell types separately, we
considered the interactions between the cell types normally found
within the ganglia provided a more biological system. However,
our method of dissociating the ganglia and the culture medium
is selective for providing cultures, ∼90% small-diameter sensory
neurons, consistent with them being nociceptor C-neurons as shown
by flow cytometry. Although there are some Schwann cells, the
myelin-forming neuroglia of the PNS and some fibroblasts are
present (Mandadi et al., 2004). Aliquots of the single cell suspension
obtained by the same method as that for flow cytometry are plated
onto sets of coverslips coated in Matrigel at a density of ∼10 ×
105 cells/coverslip and maintained in a neurobasal medium, L-
glutamine, penicillin, and streptomycin and a humid atmosphere
with 5% CO2 . Earlier experiments employed Dulbecco’s Modified Ea-
gles Medium supplemented with 10% fetal calf serum with the same
additives as above (Chow et al., 2007; Dekroon and Armati, 2002b).
We then exposed replicate neurons to 830 nm laser at parameters
shown in Table 12.1. The laser was delivered to the cell cultures
from 4.5 cm above the well plate, as was sham laser irradiation using
the protocol of Chow et al. We repeated the same experiment using
808 nm and 650 nm laser (Bokhari, 2012). We found that neuronal
processes are unique to nerve cells that developed varicosities
along their length which are reversed by 24 h. Following that, we
examined the structure of the varicosities, which are assemblies of
depolymerized microtubules, as seen by immunofluorescence, as
well as clusters of mitochondria, as imaged by JC-1 or Mitotracker
Red uptake (Fig. 12.2). JC-1 is a dye that accumulates in mitochon-
dria in a dose-dependent way with higher concentrations having
a different spectral profile indicative of high membrane potential,
i.e., ATP levels and a different profile when the membrane potential
is decreased, indicative of ADP. Mitotracker Red is a vital dye
taken up by mitochondria where the red color is indicative of high
mitochondrial membrane potential and ATP levels. Following 1 h of
laser irradiation and varicosity formation, the mitochondrial mem-
brane potentials significantly decrease as shown by the decrease in
the red signal. These findings are consistent with our hypothesis
and the findings of others that LLL-induced neural changes lead
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222 Low-Level Laser and Cultured Neural Tissue

Figure 12.2 DRG neurons labeled with Mitotracker Red and βIII-tubulin
for 24 h following LLL irradiation. Mitochondrial clusters (white arrows).
Neurons irradiated for 15 s show no varicosity formation or mitochondrial
clusters (b,f). Axons irradiated for 30 s show axonal varicosities containing
mitochondria clusters (c,g, yellow arrows). Neurons irradiated for 60 s have
no mitochondrial clusters (d,h). Nonirradiated neurons have neither vari-
cosities nor mitochondrial clusters (βIII tubulin=green, mitochondria=red,
DAPI=blue, scale bar=10 μm) (Bokhari, 2012).
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Dorsal Root Ganglion Cultures of Nociceptor Neurons 223

to microtubule disorganization, disruption of the fast axonal

flow cargo transport system of organelles, particularly the ATP-
bearing mitochondria being transported from the cell body along
the neuronal processes to provide the ATP requirements of fast
metabolizing neurons at their peripheral endings and CNS synapses.
As sensory neurons are unique in their morphological organization,
disruption of axonal flow and process structure and function and
their reliance on cell body synthesis will compromise the generation
of an action potential.
These culture models, however, do not investigate the effect of
LLL on neurons that are ensheathed by Schwann cells or complex
myelination with many spirals of compacted Schwann cell plasma
membrane. This certainly highlights the importance of comparing
cell culture data with in vivo or clinical studies where the nerve
cells are Schwann cell ensheathed. Importantly, the small-diameter
C-fibers or neurites (axons only in the case of PNS), which are of
great importance in pain conditions, are Schwann cell ensheathed
but not myelinated. Although there are no LLL-treated differentiated
myelinated cultures of rodent or human DRG, it remains a possibility
as the methodology is there (Johnson et al., 2001; Pang et al., 2012).
The high level of lipid in the compact myelin lamellae of myelinated
cultures and the axons may reduce responsiveness to LLL. The main
pain nociceptors or C-neurons, however, are unmyelinated.
In a study using CO2 laser, Lopatina et al. (2012) used organ-
otypic cultures of embryonic chick sensory neurons to examine its
effect on neurite sprouting. The study is related to further dissecting
the mechanism of how LLLT can affect neurite sprouting. They
reported that although at low power there was suppression of
the neurite outgrowth, an increased level of irradiation stimulated
neurite outgrowth. This is, in general, the reverse of most studies
where the lower levels of irradiation are stimulatory and higher
levels inhibitory. Interestingly, they removed the 200 μl of culture
medium prior to irradiating the cells but for the irradiation
time only. Control cells were treated in the same way with no
irradiation. They monitored temperature during the experiment
and reported no increase in temperature, so the response was
due to photomodulation rather than thermomodulation, as in the
experiments of Bec et al. (2012).
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224 Low-Level Laser and Cultured Neural Tissue

Yachnev et al. (2012) have studied the effect of CO2 laser

on Na+ K+ ATPase of rat DRG neurons, specifically nociceptors.
This molecule acts to transduce painful signals received by opioid
receptors of the nociceptors rather than via G protein activation.
Na+ K+ ATPase activates Nav1.8 sodium channels, which are highly
expressed by nociceptor neurons of the DRG, trigeminal and by the
sympathetic ganglionic neurons. This signaling function of Na+ K+
ATPase is particularly important in pain although it does have other
functions in muscle proliferation and apoptosis in malignant cells.
Yachnev et al. (2012) irradiated dissociated cell cultures of rat
DRG neurons, which include the small-diameter nociceptor neurons.
They selected these cells for whole cell patch clamping by their dark
appearance and found that LLL irradiation selectively lowered the
voltage sensitivity of the Nav1.8 channels, which would lower its
resting potential.
The effect of irradiation can be blocked by ouabain, which
blocks Na/K ATPase, demonstrating that the pathway by which
transduction of a painful signal occurs involves Na/K ATP molecule
with an associated conformational change. An interesting aspect of
this study is their confirmation that ATP was the photoacceptor and
LLL irradiation was thermal. Activation by irradiation has direct
relevance to further dissecting how LLLT is an effective clinical
treatment for pain relief. A recent study of NaV1.8 mutations (Han
et al., 2014) reports that mutations in the gene for this channel
contribute to pain pathophysiology.

12.9 LLL Effects on Sympathetic Nervous System Neurons

Shimoyama et al. (1992) used excised rat superior cervical ganglia

to define the effect of He–Ne LLL on the compound action potential
(CAP). These autonomic sympathetic ganglia were maintained in
Krebs-modified solution and stimulation of the ganglia followed He–
Ne irradiation for 3, 5, or 10 min (Shimoyama et al., 1992). There
was a significantly decreased CAP at 3, 5, or 10 min with the greatest
decrease occurring after 10 min. The decreased CAP indicates that
not all the ganglion cells responded. There was no change in latency
in any of the experiments and no change in conduction velocity of the
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Central Nervous System in Culture 225

cells that responded. Non-lasered control ganglia showed no change

from baseline.
For understanding the effects of laser on pain transmission,
Miura and Kawatani (1996) studied sympathetic sensory responses
to LLL using nodose or inferior ganglia of the vagus nerve. To
test if LLL irradiation depolarized the cells, they used a Gal–Al–
As laser, which caused depolarization in a dose-dependent manner
with a slower second depolarization. This indicates an increased
latency in the slower conducting neurons. Further experiments with
tetrodotoxin (TTX), which selectively blocks TTX-sensitive voltage-
gated Na channels highly expressed in nociceptor neurons, also
blocked the inhibiting effect of LLL irradiation, indicating that the
laser acts via these Na channels associated with pain signaling.

12.10 Central Nervous System in Culture

The mammalian CNS includes neurons, astrocytes, oligodendro-

cytes, and monocyte lineage microglia organized in highly specific
spatial arrangements. All these cell types play a pivotal role in
nervous system function in health and disease. The interaction
between the cell types is an active one with bidirectional signaling
occurring constantly, sometimes in response to input from the
PNS, injury, disease, or systemic signaling. Most of the studies
referred to in this chapter are based on neuronal responses to
LLL or light-emitting diode (LED). However, there are some studies
of neural progenitor cells, cybrid cells, or neurospheres. None of
these include myelinated neurons or the full spectrum of cellular
organization so unique and central to CNS function and response
in vivo. They do, however, provide important underpinning for
developing hypotheses related to treatment possibilities, although
not representing the in situ organization, which is a proviso in
interpreting the data.
Although the parameters of LEDs are not directly equated to LLL
as defined earlier, studies with LED irradiation are included in this
chapter as they are increasingly used clinically as phototherapy and
in a number of studies of cultured neurons (Wong-Riley et al., 2001)
and discussed in more detail later in the chapter. As covered earlier
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226 Low-Level Laser and Cultured Neural Tissue

in this book, LED irradiation is not coherent unlike LLL irradiation.

While this primary difference may result in subtle differences in
effect, when photons are delivered by LLL or LED, they reach the
surface of the culture medium and are scattered and reflected and
the LLL coherence is lost. Those photons, regardless of delivery,
should have the same effects on the cells (Desmet et al., 2006).
Although LLL and LED irradiations are generally delivered clinically
by contact of the device with the skin, when irradiation is delivered
with the device above the skin, there is also scattering and reflection
of some photons prior to other photons transmitted transdermally.
In cell cultures, the same effect occurs so that there would not be an
expected difference in effect with either source.
Whereas PNS studies show that LLL irradiation results in sig-
nificantly decreased fast axonal flow and mitochondrial membrane
potentials indicative of conduction block, in contrast, some studies
of CNS neurons or progenitor cells report neuronal stimulation. The
effects include increased neurite formation or neurite sprouting.
To date there are no reports of inhibitory effects of LLLT or LED
on CNS neurons. As stated in Chapter 2, inhibitory effects are
associated with LLLT above 8 mJ/cm2 —a higher dose than those of
the inhibitory studies, which have irradiation doses of 1.5–7.5 J/cm2
(Table 12.1). There are a number of LLL studies in CNS cells, which
range from the retinal cells of the eye to the dopaminergic cells of
the substantia nigra to areas related to stroke.
Bec et al. (2012) used pulsed laser of 1875, 1535, and 1470 nm
(near-infrared) (Table 12.1) to stimulate mouse retinal cells and rat
vestibular ganglion neurons in cell cultures. They developed a novel
simulation program to calculate the accurate level of irradiation
delivered to individual cells for each of the three laser wavelengths.
This assessment as it takes into account “fiber insertion angle,
neuron position in the laser spot, distance between the fiber and
the neurons, and water absorption coefficient for each wavelength.”
They also calculated the energy delivered per unit area for the retinal
neurons with a cell body diameter of ∼20 μm and the vestibular
neurons were 40 μm. Action potentials were measured by whole
cell patch clamping following irradiation at each wavelength. The
response was dependent on the laser parameters and positioning
of the laser tip. Tertrodotoxin which specifically blocks voltage-
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Central Nervous System in Culture 227

gated Na channels inhibited irradiation stimulation in both the

retinal and vestibular neurons. Although this clearly demonstrates
a photothermal effect, the study is included here as it reports a
method of assessing the delivery of irradiation to specific cells at
set parameters. The stimulation they propose is due to the light
absorption by the culture medium and that the heating of the
water stimulates the heat-activated channels such as the transient
receptor potential channels such as TRPV4. However, one caveat
might be the rise in temperature recorded to over 50◦ C, which
they report as non-lethal. The relevance of the experiments is that
such laser stimulation, which is noninvasive, could be a viable
alternative to conventional electrode-delivered stimulation for the
clinical assessment of evoked potentials.
A cell culture study of rat visual cortical cells demonstrated that
LED irradiation could prevent cell death by apoptosis when the cells
were exposed to potassium cyanide (Liang et al., 2006). The study
showed that apoptosis was caspase mediated due to the production
of reactive oxygen species.
In studies using LED, such as those by Wong-Riley et al. (2001,
2005), LED light was found to be effective in wound healing
in humans and animals, which resulted from stimulation of cell
division and downregulation of inhibitory factors. However, the
mechanisms of such LLL stimulatory action on cells have not been
clearly dissected. Wong-Riley hypothesized that light treatment with
an LED array at 670 nm (LED) was therapeutic in stimulating
cellular events involving increase in cytochrome oxidase activity, and
Tina Karu, in an elegant paper in 2012 (Karu, 2014), discussed the
basis of these events.
In a paper by Trimmer et al. (2009), an interesting experi-
ment used transmitotic human cybrid (cytoplasmic hybrid) using
platelets fused with RHoO cells, which lack a mitochondrial genome.
These cells were fused within SH-Sy5Y neuroblastoma cells, which
are adrenergic and express dopamine (http://en.wikipedia.org/
wiki/SH-SY5Y). Some of these were transfected with DNA from
people with Parkinson’s disease (PD), others with DNA from people
with sporadic PD, and a further group from normal volunteers.
The cultures were irradiated with near-infrared laser at 810 nm.
The movement or stationary status of the mitochondria was
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228 Low-Level Laser and Cultured Neural Tissue

calculated from all groups. Trimmer et al. (2009) reported that

the significantly reduced transport of the ATP-bearing mitochondria
via fast axonal flow was restored by LLL. These findings raise the
possibility of using such a technique to improve neuronal function
of the dopamine-deficient neurons although exploration of this as a
treatment remains to be undertaken.
LED irradiation of cultured primary neurons following
tetrodotoxin blocking of the voltage-gated Na channels, cytochrome
oxidase activity returned to normal while LED alone upregulated
cytochrome oxidase also shown in a number of other CNS studies
discussed later. Interestingly these studies are inconsistent with our
studies which show LLL-induced conduction block.
Increased LED also significantly restored neuronal ATP levels.
LED also enhanced the efficacy of LED during exposure to potassium
cyanide (KCN) but did not restore enzyme activity to control levels.
Wong-Riley et al. (2005) explored the hypothesis that 670 nm
LED used in their wound-healing LED therapy acted by activating the
mitochondrial photoacceptor cytochrome c oxidase. In a cell culture
model using postnatal rat cortical cells, Wong-Riley et al. (2005)
examined whether KCN, which completes with cytochrome oxidase
activation, was inhibited by LED. LED treatment partially restored
cytochrome oxidase activity and significantly reduced neuronal cell
death in a dose-dependent way. Increased LED significantly restored
neuronal ATP. Interestingly, pretreatment of the neurons with LED
enhanced its “protective effect” when neurons were given further
LED treatment during exposure to KCN but did not restore enzyme
activity to control levels. In contrast, LED was able to completely
reverse the detrimental effect of the toxin, tetrodotoxin, which only
indirectly downregulated enzyme levels. The tested wavelengths
were from 670 nm to 880 nm. As cytochrome oxidase levels are
controlled by gene expression, the effect of LED appears to be related
to its upregulation of the relevant genes. These results are consistent
with their hypothesis with increased energy metabolism in neurons
functionally inactivated by toxins. This is an interesting study as it
may well have relevance for some neurological conditions.
Wollman et al. (1996, 1998) used fetal aggregate cultures of rat
brain cells and demonstrated that He–Ne LLL at 0.3 mW irradiated
for 8 min promoted neurite spouting from the aggregates and the
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Central Nervous System in Culture 229

(a)

(b) (c)

(d)

Figure 12.3 LLLT rescues dendritic spine loss in neurons derived from
APP/PS1 transgenic mice. (a) Representative photomicrographs of FITC-
phalloidin labeling in hippocampal neurons derived from APP/PS1 mice on
14 DIV under the treatment with or without LLLT. Scale bar, 10 μm. (b)
Quantification of spine density under indicated treatments. For each group,
>25 dendrites from 13 to 15 neurons were measured. (c) Representative
Western blot analysis was performed to detect the levels of PSD-95 in Aβ25–
35 and/or LLLT-treated hippocampal neurons. (d) Quantitative analysis of
the levels of PSD-95 after indicated treatments. All the data in these figures
are presented as mean ± SEM four individual experiments. * p < 0.05
versus control group; # p < 0.05 versus indicated group. Figure reproduced
from Meng et al., Copyright  c 2013, by permission of the Society for
Neuroscience.
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230 Low-Level Laser and Cultured Neural Tissue

migration of neurons to the surface of the aggregates, which were

identified by immunostaining. In nonirradiated cultures, two or
three doses of laser increased the number of neurons that had
migrated from the aggregates. Meng et al. (2013) examined the
effects of laser on an amyloid precursor protein (APP) transfected
human neuroblastoma cell line and mouse hippocampal neurons
exposed to Aβ (β amyloid) in the culture medium. The cells were
irradiated with He–Ne 632.8 nm for 5 min in the dark (Table 12.1).
They hypothesized that if brain-derived growth factor (BDNF)
could be upregulated in these culture models with relevance to
Alzheimer’s disease, it could decrease neuronal death and promote
dendritic growth and synaptic spine formation and density. They
also showed that such irradiation activated transcription factor CRE-
binding protein, which bound to the BDNF promoter and increased
BDNF mRNA and protein expression levels. After 14 days of LLLT,
there was significant increase in dendritic length, branching, and
dendritic spine density in hippocampal cells, which correlates with
synaptic remodeling (Fig. 12.3). Although the authors recognized
that their findings were not a direct correlation between the laser
parameters used in vivo in the mouse model and those used
in the cell culture studies, this finding is of relevance not only
to Alzheimer’s disease but also to other neurological diseases
characterized by a fall-off of synaptic density (Penzes et al., 2009).

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Johnson, M.I., Bunge, R.P., and Wood, P.M. 2001. Primary cell cultures for
the study of myelination. InProtocols for Neural Cell Culture, Fedoroff,
S., and Richardson, A., eds., Humana Press, pp. 95–115.
Karu, T.I. 2010a. Mitochondrial mechanisms of photobiomodulation in
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Karu, T.I. 2010b. Multiple roles of cytochrome c oxidase in mammalian cells

under action of red and IR-A radiation. IUBMB. Life, 62(8), 607–610.
Karu, T.I. 2014. Cellular and molecular mechanisms of photobiomodulation
(low-power laser therapy). IEEE J. Sel. Top. Quantum Electron., 20(2),
143–148.
Kerr, S.J., Armati, P.J., and Brew, B.J. 1995. Neurocytotoxity of quinolinic acid
in human brain cultures. J. Neurovirol., 1(5–6), 375–380.
Liang, H.L., Whelan, H.T., Eells, J.T., Meng, H., Buchmann, E., Lerch-Gaggl,
A., and Wong-Riley, M. 2006. Photobiomodulation partially rescues
visual cortical neurons from cyanide-induced apoptosis. Neuroscience,
139(2), 639–649, available from: http://www.sciencedirect.com/scie-
nce/article/pii/S0306452205014946.
Lopatina, E.V., Yachnev, I.L., Penniyaynen, V.A., Plakhova, V.B., Podzorova,
S.A., Shelykh, T.N., Rogachevsky, I.V., Butkevich, I.P., Mikhailenko, V.A.,
Kipenko, A.V., and Krylov, B.V. 2012. Modulation of signal-transducing
function of neuronal membrane Na+ , K+ -ATPase by endogenous
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Chem., 8(1), 33–39.
Mandadi, S., Numazaki, M., Tominaga, M., Bhat, M.B., Armati, P.J., and
Roufogalis, B.D. 2004. Activation of protein kinase C reverses capsaicin-
induced calcium-dependent desensitization of TRPV1 ion channels. Cell
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dendrite atrophy via upregulating BDNF expression: Implications for
Alzheimer’s disease. J. Neurosci., 33(33), 13505–13517.
Miura, A., and Kawatani, M. 1996. Effect of diode laser irradiation on sensory
ganglion cells from the rat. Pain Res., 11, 175–181.
Pang, Y., Zheng, B., Kimberly, S.L., Cai, Z., Rhodes, P.G., and Lin, R.C.
2012. Neuron-oligodendrocyte myelination co-culture derived from
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Saito, D., Higashi, T., Suzuki, K., and Yoda, K. 2006. Suppressive effects of
low-power laser irradiation on bradykinin-induced action potentials
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234 Low-Level Laser and Cultured Neural Tissue

Shimoyama, M., Fukuda, Y., Shimoyama, M.D., Jima, K., and Mizuguchi, M.D.
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Chapter 13

Shining a Light on Parkinson’s Disease

Daniel McKenzie Johnstone,a Cécile Moro,b Jonathan Stone,a

Alim-Louis Benabid,b and John Mitrofanisc
a Bosch Institute, Department of Physiology F13, University of Sydney,

NSW 2006, Australia

b University Grenoble Alpes, CEA, LETI, CLINATEC, MINATEC Campus,

F38000 Grenoble, France

c Department of Anatomy F13, University of Sydney, NSW 2006, Australia

john.mitrofanis@sydney.edu.au

Parkinson’s disease is a movement disorder with cardinal signs

of resting tremor, akinesia and rigidity. These manifest after a
progressive death of many dopaminergic cells in the midbrain.
Although available therapies can mitigate the signs of the disease,
the progression of this cell death has proved difficult to slow or stop,
and the condition is relentlessly progressive. Hence, there is a real
need to develop treatments that slow the pathology of the disease.
Red to infrared light therapy (λ = 600–1070 nm), particularly light
in the near-infrared (NIR) range, is emerging as an effective therapy
that is capable of stabilizing dying cells. NIR has become a treatment
for tissue stressed by the known causes of age-related diseases:
hypoxia, toxic environments, and mitochondrial dysfunction. Here
we focus on several issues relating to the use of NIR therapy
for Parkinson’s disease. In particular, we consider the evidence

Handbook of Low-Level Laser Therapy

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Copyright c 2017 Pan Stanford Publishing Pte. Ltd.
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238 Shining a Light on Parkinson’s Disease

that NIR mitigates/prevents the degeneration of dopaminergic

cells in the midbrain (the key pathology of the human disease),
the mechanism of this neuroprotection, and finally, the prospect
of using NIR therapy in humans, introducing a novel method of
application. The stage is set for rigorous trial of NIR in Parkinson’s
disease patients, in particular, whether—as in animal models—NIR
provides neuroprotection and slows disease progression, in addition
to mitigating signs.

13.1 Introduction

Several recent studies in animal models of Parkinson’s disease have

reported that treatment with low-level NIR light is neuroprotective,
slowing the underlying death of dopaminergic cells in the midbrain
and reducing the associated motor signs. Current therapies for
Parkinson’s disease do not achieve comparable slowing of degener-
ation and neuroprotection, though they do relieve motor signs. In
this chapter, we consider the evidence for neuroprotection by NIR
in animal models of the disease, how NIR may work to protect cells
against toxic insult, and finally, the prospect of using NIR therapy in
humans.

13.2 Overview of Parkinson’s Disease

Parkinson’s disease is a progressive disorder with distinct car-

dinal signs of resting tremor, lead-pipe rigidity, akinesia, and/or
bradykinesia [1, 2]. These signs manifest after the degeneration
of dopaminergic cells in the substantia nigra pars compacta (SNc)
of the midbrain [1, 3, 4]. The loss of these dopaminergic cells
leads subsequently to a reduction in the levels of dopamine in the
striatum, which in turn manifests as the distinct signs of the disease
[1, 4]. The onset of the disease is insidious: its cause is rarely clear.
There is evidence that it can be caused by exposure to a neurotoxin,
for example paraquat, rotenone, 6OHDA (6-hydroxydopamine), or
MPTP (methyl-4-phenyl-1,2,3,6-tetrahydropyridine) [5]. There is
also evidence, in a small number of cases (10–15%), that mutations
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Evidence for Neuroprotection by NIR Treatment in Parkinson’s Disease 239

in genes (e.g., parkin, PINK1) predispose to the development of

Parkinson’s disease [6]. Further, there are several transgenic animal
models of the disease that, as with the toxin-induced models,
display many Parkinson-like features [7]. Although the mechanisms
underpinning the degeneration of dopaminergic cells in Parkinson’s
disease are not entirely clear, it appears mitochondrial dysfunction
plays a central role [8]. Mitochondria are the “engine rooms” of
cells that produce the energy that fuels cell function (in the form
of adenosine triphosphate, ATP). Under certain conditions, for
example in ageing or after toxic insult/genetic defect, there is a
progressive accumulation of mutations in mitochondrial DNA that
reduces mitochondrial efficacy and ATP yield. This process leads to
an increase in toxic reactive oxygen species, generating oxidative
stress and subsequent cell degeneration, as in Parkinson’s disease
[8].
The current treatment option for most patients with Parkinson’s
disease is dopamine replacement drug therapy, followed by surgery.
The dopamine drug therapy aims to replace the dopamine lost from
the system, while the surgery aims to correct the abnormal function
of the basal ganglia circuitry caused by the loss of the dopamine. The
surgical option is usually recommended to patients after the efficacy
of drug treatment lessens or when the disease has progressed
sufficiently. Both of these treatments provide symptomatic relief
(i.e., treat the signs that characterize the disease) but do not appear
to be neuroprotective (i.e., to slow the underlying degeneration), at
least in humans [9].

13.3 Evidence for Neuroprotection by NIR Treatment in

Parkinson’s Disease

The first studies to report neuroprotection by NIR after parkin-

sonian insult were done in vitro (Table 13.1). NIR treatment
reduces cell death, increases ATP content, and decreases levels of
oxidative stress among rat striatal and cortical cells after exposure
to the parkinsonian toxins rotenone and MPP+ (1-methyl-4-
phenylpyridium) [10, 11]. In cultures of human neuroblastoma cells
engineered to overexpress α-synuclein, NIR increases mitochondrial
 July 6, 2016 17:23
240 Shining a Light on Parkinson’s Disease
Table 13.1 Studies reporting on NIR treatment in Parkinson’s disease

Findings with NIR Study Model Species

Application

↑ cell survival (striatal and Liang et al., 2008 In vitro (rotenone, MPTP) Rat cells
cortical cells) Ying et al., 2008
↑ ATP content
↓ oxidative stress
↑ mitochondrial function Quirk et al., 2012 In vitro (neuroblastoma Human cells
↓ oxidative stress cells overexpressing
α-synuclein)

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↑ mitochondrial movement Trimmer et al., 2009 In vitro (hybrid cells with Human cells
mitochondrial DNA from
Parkinson’s disease
patients)
↑ cell survival (TH+ cells) Shaw et al., 2010 MPTP (acute) Mouse
↑ cell survival (TH+ cells) Peoples et al., 2012 MPTP (chronic)
↑ cell survival (TH+ cells) Moro et al., 2013, 2014 MPTP (acute)
↑ cell survival (TH+ cells) Johnstone et al., 2014 MPTP (acute)
↑ cell survival (TH+ and el Massri et al., 2014 MPTP (acute, sub-chronic)
Nissl-stained cells)
↑ cell survival (TH+ cells) Purushothuman et al., 2013 K369I transgenic (chronic) Mouse
↓ oxidative stress
↓ hyperphosphorylated tau

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 July 6, 2016 17:23
↑ flight Vos et al., 2013 pink1 mutant Flies
↑ complex IV-dependent
respiration
↓ mutant mitochondria

Evidence for Neuroprotection by NIR Treatment in Parkinson’s Disease

defects
↓ abnormal basal ganglia Shaw et al., 2012 MPTP (acute) Mouse
activity (Fos
immunoreactivity)
↑ locomotive behavior Whelan et al., 2008 MPTP (acute) mouse
DeSmet et al., 2009 MPTP (acute)

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Quirk et al., 2012 A53T α-synuclein
Moro et al., 2013 transgenic
MPTP (acute)
↑ cell survival (TH+ and Reinhart et al., 2014 6OHDA hemi-parkinsonian Rat
Nissl-stained cells)
↓ rotational behavior
↓ clinical signs Quietmind Foundation trial Parkinson’s disease patients Human
(http://www.youtube.com/watch?v=9X-
hjgay7pg)
↓ clinical signs Maloney et al., 2010 Parkinson’s disease patients Human
↓ clinical signs Zhao et al., 2003 Parkinson’s disease patients Human
↓ clinical signs Burchman 2011 Parkinson’s disease patients Human

241

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242 Shining a Light on Parkinson’s Disease

function and reduces oxidative stress after MPP+ exposure [12, 13].
Further, mitochondrial movement along axons in hybrid cells, bear-
ing mitochondrial DNA from Parkinson’s disease patients, improves
substantially after NIR treatment, with movement restored to near
control levels [13] (Table 13.1).
The neuroprotective effects of NIR treatment have also been ex-
amined subsequently in vivo in various animal models of Parkinson’s
disease (Table 13.1). In MPTP-treated mice [14–18] and 6OHDA-
lesioned rats [19], NIR treatment saves many dopaminergic cells
from death. In these models, NIR saves not only the expression of
the dopaminergic phenotype—as assessed by tyrosine hydroxylase
(TH) expression (functional neuroprotection)—but also the cells
themselves—as assessed by Nissl staining (true neuroprotection)
[20]. Further, results are similar whether the therapy is applied
at the same time or well after the insult, indicating that NIR both
protects healthy cells against an insult and rescues damaged cells
following an insult [15]. The rescue of cells is particularly relevant to
the clinical reality of the parkinsonian condition, in which individu-
als have, at presentation, already suffered significant degeneration,
so that treatment follows cell loss. In the K369I transgenic mouse
model of frontotemporal dementia, which also shows a chronic
and progressive degeneration of dopaminergic cells in the SNc and
parkinsonian signs [7], NIR treatment decreases oxidative stress and
hyperphosphorylated tau and increases dopaminergic cell survival
in the SNc [21].
Together with preserving dopaminergic cell survival, NIR has
been shown to correct abnormal neuronal activity generated by
the parkinsonian condition [22] (Table 13.1). Using Fos immuno-
histochemistry (a well-established measure of cell activity), the
overactivity of cell firing in the subthalamic nucleus and zona
incerta (two key basal ganglia nuclei), characteristic of parkinsonian
cases, is reduced substantially after NIR therapy. This reduction
does not quite reach control levels, indicating that the restoration
is partial, and is attributed to the surviving SNc dopaminergic
cells being functionally active, continuing to produce and release
dopamine at their terminals in the striatum [22]. This functional
restoration manifests in improved motor behavior. Previous studies
have reported that NIR therapy improves various parameters
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Evidence for Neuroprotection by NIR Treatment in Parkinson’s Disease 243

of locomotive behavior, for example mobility and velocity, after

MPTP treatment [18, 23, 24]. It also delays disease progression
and reduces the severity of the disease phenotype in transgenic
mice expressing the A53T human α-synuclein mutation [12]. In
addition, we have shown recently that NIR treatment reduces
apomorphine-induced rotational behavior in 6OHDA-lesioned rats
[19]. Finally, there is evidence that NIR treatment rescues flight and
mutant mitochondria defects, together with promoting complex-IV-
dependent respiration in pink1 mutant flies [25].
To date, there have been few reports on the effect of NIR treat-
ment on Parkinson’s disease patients. From the Quietmind Foun-
dation trial, there is a linked YouTube video (http://www.youtube.
com/watch?v=9X-hjgay7pg) of a Parkinson’s disease patient dis-
playing improved movement and reduced tremor after extracra-
nially directed application of NIR, but few details are provided. There
is a recent noncontrolled and nonrandomized clinical report indi-
cating improved speech, cognition, freezing episodes, and gait after
extracranial NIR therapy in parkinsonian patients [26]; there are
also some clinical reports suggesting improvements in parkinsonian
“symptoms” in the majority of patients after NIR application through
an intranasal device [27]. Finally, there is a serendipitous finding
in one Parkinson’s disease patient that was treated with NIR for a
dental problem. This patient was reported to display a reduction in
his parkinsonian signs following the NIR treatment [28]. From these
early clinical reports, it remains far from clear whether NIR treat-
ment is neuroprotective (stopping cell death) and/or symptomatic
(treating the motor signs) in humans. If neuroprotective, the saving
of cells would ultimately manifest in symptomatic improvements;
yet it may turn out that NIR treatment is symptomatic only, with no
neuroprotective effects (see Section 13.6).
It should be noted that we use the term NIR to refer specifically
to red to infrared light (λ = 600–1070 nm). Light that includes
wavelengths outside this range may actually be detrimental to cell
survival. A recent study in rats has shown that continuous exposure
to bright light, which includes a peak wavelength of ∼440 nm,
results in a reduction in the number of dopaminergic cells in the SNc
[29]. Indeed, it has been suggested that excess bright light exposure
(i.e., “polluted” light) may be a cause of Parkinson’s disease [29].
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244 Shining a Light on Parkinson’s Disease

In summary, several studies have shown NIR treatment to

be neuroprotective, to restore functional activity, and to improve
movement in various animal models of Parkinson’s disease. The
clinical evidence is far sparser, however, prompting the need for a
systematic, large-scale clinical trial of NIR therapy in patients.

13.4 How Does NIR Work to Neuroprotect: Two

Mechanisms of Action?

The mechanisms that underpin the neuroprotective effects of NIR

are not fully known, but there appears two general modes of action.
The most commonly studied mode of action is that of NIR acting
directly on the cells themselves, repairing mitochondrial function.
The NIR “boosts” mitochondrial function by increasing electron
transfer in the respiratory chain and activating photoacceptors, such
as cytochrome oxidase, resulting in an increase in ATP production
and a reduction in apoptosis [30–33].
A second, emerging hypothesis is that NIR triggers a less direct,
systemic response. Several studies have reported remote, often
bilateral, effects on body tissues after local NIR application to, for
example, skin wounds [34]. Intriguingly, protection of the mouse
brain has been demonstrated after the remote application of NIR to
the dorsum of the animal, with no direct irradiation of the head [16].
While the mechanism remains unknown, it presumably involves the
stimulation of one or more circulating molecules or cell types. One
possibility is the stimulation of immune cells, for example mast cells
and macrophages, which could help neuroprotect cells in the brain
[33, 35, 36]. There may also be effects on inflammatory mediators,
as NIR is associated with downregulation of pro-inflammatory
cytokines and upregulation of anti-inflammatory cytokines [35]. In
addition, bone marrow–derived stem cells may also be involved; a
series of studies have demonstrated that NIR exposure increases
proliferation of c-kit-positive cells in the bone marrow and that,
following myocardial infarction in rats, these cells are mobilized and
recruited specifically to the site of damage where they are associated
with a reduction in myocardial infarct size and ventricular dilatation
[37]. These cells, together with immune cells, may release trophic
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NIR Treatment in Parkinson’s Disease Patients 245

factors such as nerve growth factor and vascular endothelial

growth factor that improves the function of dying (apoptotic)
cells and aids in their survival [38]. Another possibility is for a
signaling system between mitochondria in different body tissues.
Mitochondria in distress in one body tissue have been suggested
to produce an unidentified extracellular signal (mitokine), which
is then transmitted to cells in remote body tissues and as a
consequence induces a mitochondrial stress response [39]. In
relation to NIR and Parkinson’s disease, NIR applied to remote tissue
may prompt a signal system between mitochondria of peripheral
tissues and brain, inducing repair mechanisms in the damaged
cells in the SNc. Taken all together, such indirect mechanisms are
similar to the so-called “abscopal” effect sometimes observed in the
radiation treatment of metastatic cancer, where treatment targeted
at a tumor leads to not only a shrinking of the local tumor but also a
shrinking of tumors far from the treated area [40].
It remains to be determined which of these mechanisms, direct
or indirect, offers the neuroprotection, but we note that they are not
mutually exclusive and that both may contribute to the process [16,
17]. As a working hypothesis, direct stimulation of the mitochondria,
which is supported by the most compelling evidence, is likely to
form the primary mechanism of protection by NIR, while the indirect
stimulation of immune and/or stem cells may form a secondary
and complementary mechanism. Some early results in an animal
model of Parkinson’s disease indicate that, although there was
some neuroprotection of the brain after remote application of NIR
(e.g., to the dorsum of body), the neuroprotection was not as
strong or robust as when NIR was applied directly to the head
[16, 17]. In other words, neuroprotection was achieved with both
direct and indirect irradiation, but the direct irradiation was more
effective.

13.5 NIR Treatment in Parkinson’s Disease Patients: Can

It Work?

A key question is whether NIR can be effective in humans. If

the primary mechanism of action of NIR is direct mitochondrial
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246 Shining a Light on Parkinson’s Disease

stimulation of the damaged or diseased cells (see Section 13.4), then

there may be an issue with its therapeutic potential for Parkinson’s
disease patients. While NIR penetration and dissipation are not a
major consideration when there are few or no tissue barriers, as
in the culture dish [10, 11] or even mouse SNc (4–5 mm inside
the skull) [14, 15], such issues may become prohibitive when NIR
is required to penetrate a thick bony cranium, meninges, skin, and
a large mass of intervening brain parenchyma (80–100 mm inside
skull) in order to reach the human SNc. Indeed, almost all the current
clinical studies reporting the beneficial effects of extracranial NIR
therapy in humans have been in cases where the target region is in
the cortex, lying only 8–10 mm below the cranium [41], whether
in patients suffering trauma [42], stroke [41, 43], or depression
[44]. Structures lying much deeper in the human brain, such as
the SNc, may be beyond the range of the externally applied NIR. It
has been estimated that NIR may traverse a maximum of ∼30 mm
through body tissues [36, 41, 45], with ∼90% attenuation of the
signal [14, 17, 45, 46 47]. Thus, over a distance of 80–100 mm—
the predicted distance between any externally applied NIR and
the SNc in humans—the signal would be extremely weak, perhaps
undetectable [17, 30, 45].
Taken together, these data indicate that extracranially applied
NIR may not reach deeper brain regions in effective doses,
particularly regions deeper than ∼30 mm from cranial surface. This
will likely limit the use of extracranial NIR as a long-term and reliable
neuroprotective treatment in humans, particularly those suffering
from Parkinson’s disease [17, 41]. While there may be some
neuroprotection by NIR therapy via the indirect systemic effect, we
suggest that it may not solely be enough to have a substantial impact
on the SNc. In order for the NIR to have maximum neuroprotective
effect, it may be necessary to place the NIR source within ∼30 mm
of the diseased SNc cells, within the brain itself, to stimulate the
mitochondria directly. This could potentially stimulate an indirect
systemic effect also—for example, by activating circulating immune
cells in nearby blood vessels—providing the secondary, supportive
neuroprotective mechanism of action.
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Developing Methods for Intracranial NIR Delivery 247

13.6 Developing Methods for Intracranial NIR Delivery

Given the limitations of extracranial NIR for treating Parkinson’s

disease, there is a need to develop effective methods of delivering
adequate NIR signal to deeper brainstem structures in humans.
To this end, we are in the process of developing an intracranial
optical fiber device to deliver NIR in regions near the SNc in
order for diseased cells to receive sufficient signal and subsequent
neuroprotection. Our results in mice [17] and rats [19] have shown
that the NIR does not cause toxic damage around the implant
sites in the brain and that the intracranially applied NIR is indeed
neuroprotective to dopaminergic SNc cells against parkinsonian
insult. We have just completed our first optical fiber implants
into a midbrain site close to the SNc (5–10 mm) in monkeys and
our early results are most encouraging. In the two cases to date,
MPTP-treated monkeys irradiated with NIR required less L-dopa for
clinical recovery and had more dopaminergic cells in the SNc and
terminations in the striatum compared to MPTP-treated monkeys
that were not irradiated. We are in the process of analyzing more
cases, and these will hopefully set the platform for future endeavor
in humans.
In summary, although there may be symptomatic treatment,
it appears unlikely that NIR therapy, when applied extracranially,
can be neuroprotective to the human SNc. If applied intracranially,
however, in regions very close to the SNc, the diseased cells may
receive adequate NIR signal and hence be neuroprotected.

13.7 Conclusion

NIR therapy has the potential to develop into a viable treatment

option for patients with Parkinson’s disease. This therapy would
offer patients the advantage of neuroprotection, something that
dopamine replacement drug therapy does not do. In order for the
NIR signal to reach and be neuroprotective to the SNc, located deep
within the brain, it may have to be applied intracranially, using
an optical fiber device. Our early results in rodents and monkeys
indicate that such a device is feasible and can mediate effective
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248 Shining a Light on Parkinson’s Disease

protection of the SNc. We are hence hopeful of success in humans,

where NIR would slow the progression of the disease by rescuing
the critical cells from damage and death. The potential outcomes of
this treatment in Parkinson’s disease are very exciting.

Acknowledgments

We are forever grateful to Tenix Corp, Salteri family, Sir Zelman

Cowen Universities Fund, Fondation Philanthropique Edmond J
Safra, France Parkinson, Michael J Fox Foundation, Fight for Sight
and the International Retinal Research Foundation, and the French
National Research Agency (ANR Carnot Institute) for funding our
work DMJ is an Early Career Research Fellow (NHMRC Australia).

References

1. Bergman H and Deuschl G. Pathophysiology of Parkinson’s disease:

From clinical neurology to basic neuroscience and back. Mov Disord,
2002; 17: S28–40.
2. Jankovic J. Parkinson’s disease: Clinical features and diagnosis. Neurol
Neurosurg Psychiatry, 2008; 79: 368–376.
3. Rinne JO. Nigral degeneration in Parkinson’s disease. Mov Disord, 1993;
8(Suppl1): S31–35
4. Blandini F, Nappi G, Tassorelli C, and Martignoni E. Functional changes of
the basal ganglia circuitry in Parkinson’s disease. Prog Neurobiol, 2000;
62: 63–88.
5. Blesa J, Phani S, Jackson-Lewis V, and Przedborski S. Classic and new
animal models of Parkinson’s disease. J Biomed Biotech, 2012; 845618.
6. Corti O and Brice A. Mitochondrial quality control turns out to be
the principal suspect in parkin and PINK1-related autosomal recessive
Parkinson’s disease. Curr Opin Neurobiol, 2013; 23: 100–108.
7. Ittner LM, Fath T, Ke YD, Bi M, van Eersel J, Li KM, Gunning P, and Götz
J. Parkinsonism and impaired axonal transport in a mouse model of
frontotemporal dementia. Proc Natl Acad Sci USA, 2008; 105: 15997–
16002.
8. Exner N, Lutz AK, Haass C, and Winklhofer KF. Mitochondrial dysfunc-
tion in Parkinson’s disease: Molecular mechanisms and pathophysiolog-
ical consequences. EMBO J, 2012; 31: 3038–3062.
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9. Jankovic P and Poewe W. Therapies in Parkinson’s disease. Curr Opin

Neurol, 2012; 25: 433–447.
10. Liang HL, Whelan HT, Eells JT, and Wong-Riley MT. Near-infrared light
via light-emitting diode treatment is therapeutic against rotenone- and
1-methyl-4-phenylpyridinium ion-induced neurotoxicity. Neuroscience,
2008; 153: 963–974.
11. Ying R, Liang HL, Whelan HT, Eells JT, and Wong-Riley MT. Pretreatment
with near-infrared light via light-emitting diode provides added benefit
against rotenone and MPP+ induced neurotoxicity. Brain Res, 2008;
1243: 167–173.
12. Quirk BJ, DeSmet KD, Henry M, Buchmann K, Wong-Riley M, Eells JT et al.
Therapeutic effect of nearinfrared (NIR) light on Parkinson’s disease
models. Front Biosci, 2012; E4: 818–823.
13. Trimmer PA, Schwartz KM, Borland MK, de Taboada L, Streeter J, and
Oron U. Reduced axonal transport in Parkinson’s disease cybrid neurites
is restored by light therapy. Mol Neurodegen, 2009; 4: 26.
14. Shaw VE, Spana S, Ashkan K, Benabid AL, Stone J, Baker GE et al.
Neuroprotection of midbrain dopaminergic cells in MPTP-treated mice
after near-infrared light treatment. J Comp Neurol, 2010; 1518: 25–
40.
15. Peoples CL, Spana S, Ashkan K, Benabid AL, Stone J, Baker GE et al.
Photobiomodulation enhances nigral dopaminergic cell survival in a
chronic MPTP mouse model of Parkinson’s disease. Parkinsonism Relat
Disord, 2012; 18: 469–476.
16. Johnstone DM, Moro C, el Massri N, Torres N, Jaeger XD, Reinhart F et
al. Indirect application of nearinfrared light induces neuroprotection
in a mouse model of Parkinson’s disease: An abscopal neuroprotective
effect. Neuroscience, 2014 doi: 10.1016/j.neuroscience.2014.05.023.
17. Moro C, el Massri N, Torres N, Ratel D, De Jaeger X, Chabrol C et al.
Photobiomodulation inside the brain: A novel method of intracranial
application of near-infrared light and its impact on dopaminergic
cell survival in MPTP-treated mice. J Neurosurg, 2014; EPub; PMID
24160475.
18. Moro C, Torres N, el Massri N, Ratel D, Johnstone DM, Stone J et al.
Photobiomodulation preserves behaviour and midbrain dopaminergic
cells from MPTP toxicity: Evidence from two mouse strains. BMC
Neurosci, 2013; 14: 40.
19. Reinhart F, el Massri N, Darlot F, Moro C, Costecalde T, Johnstone DM
et al. Evidence for neuroprotection after intracerebral application of
nearinfrared light in a hemi-parkinsonian rat model. In preparation.
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250 Shining a Light on Parkinson’s Disease

20. el Massri N, Johnstone DM, Peoples CL, Moro C, Reinhart F, Torres N et al.
True and functional neuroprotection of midbrain dopaminergic cells in
MPTP-treated mice: The effect of different doses of nearinfrared light on
cell survival and gliosis. ISRN Neuroscience, submitted.
21. Purushothuman S, Nandasena C, Johnstone DM, Stone J, and Mitrofanis
J. The impact of near-infrared light on dopaminergic cell survival in
a transgenic mouse model of parkinsonism. Brain Res, 2013. doi:pii:
S0006-8993(13)01184-0.10.1016.
22. Shaw VE, Peoples CL, Spana S, Ashkan K, Benabid AL, Stone J et al.
Patterns of cell activity in the subthalamic region associated with the
neuroprotective action of near-infrared light treatment in MPTP-treated
mice. Parkinson’s Disease, 2012; 296875.
23. Whelan HT, DeSmet KD, Buchmann E, Henry M, Wong-Riley M,
Eells JT et al. Harnessing the cell’s own ability to repair and
prevent neurodegenerative disease. SPIE Newsroom, 2008; 1–3 doi:
10.1117/2.1200801.1014.
24. DeSmet K, Buchmann E, Henry M, Wong-Riley M, Eells J, VerHoeve J
et al. Near-infrared light as a possible treatment option for Parkinson’s
disease and laser eye injury. Proc SPIE, 2009; 7165; 716503.
25. Vos M, Lovisa B, Geens A, Morais VA, Wagnières G, van den Bergh
H et al. Near-infrared 808 nm light boosts complex IV-dependent
respiration and rescues a Parkinson-related pink1 model. Plos One,
2013; 8: e78562.
26. Maloney R, Shanks S, and Maloney J. The application of low-level laser
therapy for the symptomatic care of late stage Parkinson’s disease:
A non-controlled, non-randomized study. Am Soc Laser Med Surg Abs,
2010; 185.
27. Zhao G, Guo K, and Dan J. 36 case analysis of Parkinson’s disease
treated by endonasal low energy He-Ne laser. Acta Academiae Medicinae
Qingdao Universitatis, 2003; 39: 398 (Chinese).
28. Burchman MA. Using photobiomodulation on a severe Parkinson’s
patient to enable extractions, root canal treatment and partial denture
fabrication. J Laser Dent, 2011; 19: 297–300.
29. Romeo S, Viaggi C, Di Camillo D, Willis AW, Lozzi L, Rocchi C et al. Bright
light exposure reduces TH-positive dopamine neurons: Implications of
light pollution in Parkinson’s disease epidemiology. Sci Rep, 2013; 3:
1395.
30. DeSmet KD, Paz DA, Corry JJ, Eells JT, Wong-Riley MT, Henry MM et al.
Clinical and experimental applications of NIR-LED photobiomodulation.
Photomed Laser Surg, 2006; 24: 121–128.
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31. Hamblin MR and Demidova TN. Mechanisms of lowlevel light therapy.

Proc SPIE, 2006; 6140: 614001.
32. Rojas JC and Gonzalez-Lima F. Low-level light therapy of the eye and
brain. Eye Brain, 2011; 3: 49–67.
33. Chung H, Dai T, Sharma SK, Huang YY, Carroll JD, and Hamblin MR. The
nuts and bolts of low-level laser (light) therapy. Annals Biomed Engin,
2012; 40: 516–533.
34. Braverman B, McCarthy RJ, Ivankovich AD, Forde DE, Overfield M, and
Bapna MS. Effect of helium-neon and infrared laser irradiation on
wound healing in rabbits. Lasers Surg Med, 1989; 9: 50–58.
35. Muili KA, Gopalakrishnan S, Meyer SL, Eells JT, and Lyons JA. Ameliora-
tion of experimental autoimmune encephalomyelitis in C57BL/6 mice
by photobiomodulation induced by 670 nm light. Plos One, 2012; 7:
e30655.
36. Byrnes KR, Waynant RW, Ilev IK, Wu X, Barna L, Smith K et al. Light
promotes regeneration and functional recovery and alters the immune
response after spinal cord injury. Lasers Surg Med, 2005; 36: 171–
185.
37. Tuby H, Maltz L, and Oron U. Induction of autologous mesenchymal
stem cells in the bone marrow by low-level laser therapy has profound
beneficial effects on the infarcted rat heart. Lasers Surg Med, 2011; 43:
401–409.
38. Hou ST, Jiang SX, and Smith RA. Permissive and repulsive cues and
signalling pathways of axonal outgrowth and regeneration. Int Rev Cell
Mol Biol, 2008; 267: 125–181.
39. Durieux J, Wolff S, and Dillin A. The cell-non-autonomous nature of
electron transport chain-mediated longevity. Cell, 2011; 144: 79–91.
40. Postow MA, Callahan MK, Barker CA, Yamada Y, Yuan J, Kitano S
et al. Immunologic correlates of the abscopal effect in a patient with
melanoma. Cancer J, 2012; 18: 153–159.
41. Lapchak PA. Taking a light approach to treating acute ischemic stroke
patients: Transcranial near-infrared laser therapy translational science.
Ann Med, 2010; 42: 576–586.
42. Naeser MA, Saltmarche A, Krengel MH, Hamblin MR, and Knight JA.
Improved cognitive function after transcranial, light-emitting diode
treatments in chronic, traumatic brain injury: Two case reports.
Photomed Laser Surg, 2011; 29: 351–358.
43. Lampl Y, Zivin JA, Fisher M, Lew R, Welin L, Dahlof B et al. Infrared
laser therapy for ischemic stroke: A new treatment strategy: Results of
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252 Shining a Light on Parkinson’s Disease

NeuroThera. Effectiveness and Safety Trial-1 (NEST-1). Stroke, 2007; 38:

1843–1849.
44. Schiffer F, Johnston AL, Ravichandran C, Polcari A, Teicher MH, Webb
RH et al. Psychological benefits 2 and 4 weeks after a single treatment
with nearinfrared light to the forehead: A pilot study of 10 patients with
major depression and anxiety. Behav Brain Functions, 2009; 5: 46.
45. Lapchak PA, Wei J, and Zivin JA. Transcranial infrared laser therapy
improves clinical rating scores after embolic strokes in rabbits. Stroke,
2004; 35: 1985–1988.
46. Zivin JA, Albers GW, Bornstein N, Chippendale T, Dahlofet B, Devlin T
et al. Effectiveness and safety of transcranial laser therapy for acute
ischemic stroke. Stroke, 2009; 40: 1359–1364.
47. Jagdeo JR, Adams LE, Brody NI, and Siegel DM. Transcranial red and
nearinfrared light transmission in a cadaveric model. Plos One, 2012;
7: e47460.
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Chapter 14

Low-Level Laser Therapy and Stem Cells

Qi Zhang, Chang Zhou, and Tingting Dong

Harvard Medical School, Massachusetts General Hospital, 55 Fruit Street,
Boston, MA 02114, USA
qzhang0@mgh.harvard.edu

Adult stem cells are found in many tissues and organs such as
brain, blood, bone, skin, lung, and liver, where they stay in a special
structure called “stem cell niche.” They remain quiescent under
normal conditions and become activated when there is a need to
repair injury or maintain tissue. Stem cells are very promising in
regenerative medicine to treat injury and disease. However, when
isolated and cultured in vitro, stem cells have limited capacity to
divide, which makes it difficult to grow large quantities of stem
cells for medical application. Low-level laser therapy (LLLT) has
been demonstrated to stimulate the proliferation of many kinds
of cultured cells, including stem cells. The augmentation of stem-
cell-based therapies to modulate regenerative process through LLLT
holds great potential. The mechanisms of action, the effects of LLLT
on stem cells, and the potential applications of LLLT on stem-cell-
based treatment are discussed in this chapter.

Handbook of Low-Level Laser Therapy

Edited by Michael R. Hamblin, Marcelo Victor Pires de Sousa, and Tanupriya Agrawal
Copyright c 2017 Pan Stanford Publishing Pte. Ltd.
ISBN 978-981-4669-60-3 (Hardcover), 978-981-4669-61-0 (eBook)
www.panstanford.com
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254 Low-Level Laser Therapy and Stem Cells

14.1 Mechanisms of LLLT Action in Stem Cells

14.1.1 Low-Level Laser Irradiation

LLLT refers to the use of red or near-infrared lights with a
wavelength of 600–1100 nm and an output power of 1–5000 mW,
either a continuous or pulsed wave at a relatively low energy density
of 0.04–50 J/cm2 . The reaction of light energy to modulate biological
function is nonthermal, just like photosynthesis, instead of ablative
or thermal effects. LLLT is also called cold laser, nonthermal laser,
soft laser, biostimulation laser, and photobiomodulation laser and
act as physical therapy in many parts of the world. Unlike solar
energy, which is multidirectional and noncoherent, the light sources
used for LLLT are monochromatic, unidirectional electromagnetic
irradiation that can deliver significant levels of concentrated energy.
LLLT can be performed by lasers or light-emitting diode (LED)
arrays. Lasers can produce 100% of coherent light energy in almost
a single wavelength, whereas LEDs can only produce about 95% of
noncoherent light between a narrow range of wavelengths. Usually,
lasers have high tissue penetration than LEDs. However, LEDs
produce negligible amount of heat and reduce the risk of injury [1].
The most important parameters to describe LLLT are energy (J) or
energy density (J/cm2 ), wavelength (nm), power density (W/cm2 ),
exposure time (s), wave type (continuous or pulsed), fractionation,
contact modality, source type, and physicochemical properties of the
target tissue.

14.1.2 Mechanisms of LLLT

LLLT has been applied to reduce pain and inflammation as well
as to promote wound healing and tissue regeneration. Although
the beneficial effects of LLLT are demonstrated by many pre-
clinical and clinical studies, the underlying mechanisms are not
fully understood, one of which may rely on the ability of LLLT to
directly activate the mitochondrial cytochrome c oxidase, which
is one of the primary photoacceptors operating in LLLT [2]. The
photonic energy is converted into biochemical energy within the
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Mechanisms of LLLT Action in Stem Cells 255

cell, in the form of ATP via mitochondrial respiratory chain [3].

In a good agreement with its effects on the enhancement of
mitochondrial respiration, several investigators, including us, have
shown that LLLT modulates the production of reactive oxygen
species (ROS) and ATP, apoptosis, cellular metabolic process, and
signaling transduction pathways secondarily to more sufficient
function of mitochondria [4–6]. It is wildly considered cytochrome
c oxidase is the primary photoacceptor for red and near-infra
red ranges in mammalian cells since absorption spectra obtained
for cytochrome c oxidase are similar to the action spectra for
biological responses to light [7]. This enzyme is the key molecule
involved in mitochondrial energy metabolism, and any modulation
of cellular metabolism could be anticipated to exert profound effects
in whole-cell physiology. Therefore, during and after LLLT excitation,
numerous intracellular effects have been performed.
When LLLT is applied to cells or tissues, the photons are
absorbed by mitochondria chromophores, especially cytochrome c
oxidase. Absorption of laser energy will trigger the dissociation of
NO from cytochrom c oxidase, leading to a cascade of responses,
including increase in cytochrom c oxidase enzyme activity, electron
transportation, oxidative respiration, mitochondria-related RNA and
protein synthesis, oxygen consumption, membrane potential [8],
and ATP production [9–11]. Meanwhile, the active mitochondria
produce ROS, which serve as a second messenger and activate
various cellular signaling pathways, including regulation nucleic
acid synthesis, protein synthesis, enzyme activation, and cell type
progression [12]. Zhang et al. reported that LLLT could induce
intracellular ROS production in Hela cells, resulting in the activation
of Src tyrosine kinase pathway and enhanced cell viability [13].
Antioxidants, such as vitamin C, could inhibit the effect of LLLT,
indicating that ROS play a critical role during LLLT-induced signaling
pathway activation. Src tyrosine kinase could further activate
downstream pathways, including ERK/MAPK and Akt pathway,
which control cell proliferation and survival. Moreover, Src also
activates transcription factors such as NF-κB and STAT-3 to induce
gene transcription and alter long-term cell behavior.
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256 Low-Level Laser Therapy and Stem Cells

14.1.3 Effects of LLLT

The first demonstration of laser biostimulation was published by
Endre Mester, a few years after the first working laser was invented
[14]. He intended to test if laser would lead to cancer generation
in mice. Surprisingly, the mice did not get cancer but the hair
shaved before experiments grew back quickly by LLLT (694 nm)
illumination than the untreated group. Since then, more than 1000
laboratory studies, supported by approximately 100 clinical trials
with coherent light sources (lasers) or noncoherent light (LEDs),
have been published, symbolizing its booming development. Despite
the lack of consensus on its effectiveness, the use of lasers and LEDs
has shown positive outcomes in relieving short-term osteoarthritis
pain [15], neck pain [16], back pain [17], tendinopathy [18],
wound healing [19], and muscle fatigue [20]. In recent years, the
employment of LLLT has extended beyond wound healing and pain
release. Several studies support its potential benefits in strokes,
neurodegeneration, neuromuscular disorders, memory and mood
disorders, and retinal diseases [21, 22]. Importantly, LLLT given at
energy densities that exert functional effects show no toxic side
effects in both animals and humans [23–25], which highlights the
value of LLLT as a safe and noninvasive countermeasure for clinical
use. There are sufficient evidences for biological effects of LLLT,
but how therapeutic lasers and LEDs work and what the optimal
conditions are still need to be clarified.
Growing evidence supports that LLLT can prevent cell apoptosis
and enhance cell proliferation, migration, and adhesion at low dose
rather than high does. The effect of LLLT on cell proliferation has
been confirmed in several types of cells [26–28], through enhance-
ment of mitochondria function in order to upregulate growth factor
release [29]. In 1987, LLLT first reported its effect on increase
in collagen and basic fibroblast growth factors [30]. Many studies
demonstrate that LLLT promotes wound healing with collagen
synthesis and vascular generation [31]. Recently, the enhancement
of neovascularization, alteration of platelet-derived growth factor
(PDGH) and transforming growth factor α (TGF-α), and inhibition
of cell apoptosis have also been reported [32]. Furthermore, a lot of
studies support the protective role of LLLT on neuronal dysfunction,
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Mechanisms of LLLT Action in Stem Cells 257

Table 14.1 Summary of molecules induced by LLLT

Classification Molecules Biological Effects Benefits

Interleukins IL-1α, IL-6, IL-8, IL-2, Migration, Resolve difficult
IL-4 Immunological infections, Improve
activation inflammatory response
Inflammatory PGE2, COX-2, IL-1β, Inhibition of Reduce pain and
Cytokines TNF-α inflammation swelling, Improve range
of motion, Functional
recovery
Growth Factors BDNF, GDNF, bFGF, Proliferation, Normal tissue
IGF-1, KGF, PDGF, TGF- Differentiation, regeneration, Reduce
β, VEGF Bone nodule scarring, Rapid
formation recovery
Small Molecules ATP, cGMP, ROS, Ca2+ , Cell signaling, Restore normal
NO Angiogenesis vasculature, Resolve
chronic ulcers

Source: Adapted from Ref. [73].

including traumatic brain injury [33], neurodegeneration [34], and

strokes [21]. Since neuronal dysfunctions are often associated with
impaired mitochondrial oxidative metabolism, LLLT might show
beneficial effect of neuroregeneration through improving mitochon-
drial function [35]. LLLT also affects other deeper structures and has
been applied to several internal organs (brain, spinal cord, heart,
liver, and bone marrow) to modulate healing processes after injury
or ischemia [36–39]. Table 14.1 gives a summary of molecules and
effects of LLLT.
One obstacle in the use of LLLT in mainstream medicine is the
lack of a standard (or instructive) laser dose for the treatment
procedure. Different lasers with various parameters, including
wavelength, power density, fluence, application time, and so on,
were used in different studies. Some studies showed positive results
while some negative. It is difficult to tell which parameters are
optimal since the condition in each study is different. However, there
is a basic truth that if the laser energy is too low, there is no effect; if
the energy is too high, the tissue will be damaged. It is reported that
there is a biphasic dose response to LLLT. And for each particular
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258 Low-Level Laser Therapy and Stem Cells

application, there should be optimal doses of LLLT to induce strong

response while not to damage the tissue.

14.2 Effects of LLLT on Stem Cells

14.2.1 Hematopoietic Stem Cells

In the bone marrow, a small fraction of hematopoietic stem
cells (HSCs) could produce heterogeneous populations of actively
dividing hematopoietic progenitors. They give rise to approximately
500 billion blood cells per day, including lymphoid lineage (B-cell,
T-cell, and NK-cell) and myeloid lineage (red blood cell, platelet,
monocyte and macrophage, dendritic cell, neutrophil, eosinophil,
and basophil). During the major phases of blood biogenesis, as il-
lustrated in Fig. 14.1, mitochondrial respiratory chain that produces

Figure 14.1 The hierarchy of human hematopoietic stem cell (HSC), mul-
tipotent progenitor (MPP), common myeloid progenitor (CMP), common
lymphoid progenitor (CLP), megakaryocyte/erythroid progenitor (MEP),
and colony-forming unit (CFU).
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Effects of LLLT on Stem Cells 259

> 90% ATP for a cell plays a vital role. Inadequate mitochondrial
respiration can impair HSC differentiation into myeloid lineage-
committed progenitors, named GEMM, which have the potential
to produce granulocytes (G), erythrocytes (E), monocytes (M), and
megakaryocytes (M) [40, 41]. Furthermore, HSCs reside in the most
hypoxic niche in the bone marrow, and face cell fate decisions
between quiescence, self-renewal, and differentiation [42]. The
hypoxic microenvironment of bone marrow niche highlights the
importance of redox signaling in the maintenance of HSC function.
These reasons reveal the potential effects of LLLT on the modulation
of HSC functions.
LLLT has been used in wound healing for 30 years in clinics
with a long recorded safety and also widely applied to regenerative
medicine and dentistry to enhance healing process. As for its
effects on hematopoiesis, Vacek et al. demonstrated that exposure
of murine bone marrow cells to LLLT increased their differentiation
potential into myeloid progenitors ex vivo [43]. LLLT at 685 nm also
stimulated stem cell proliferation in Dugesia tigrina [44]. Likewise,
the growth and differentiation potential of long-term (3 years)
cryopreserved human peripheral blood progenitors were mostly
restored by LLLT [45]. These studies demonstrated the ability of
LLLT to enhance HSC differentiation in both humans and mice,
provided that a right laser parameter is selected.

14.2.2 Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs), also known as bone marrow
stromal stem cells, are generally found in bone marrow. They have
potential to differentiate into various cell types, including osteoblast,
chondrocytes, and adipocytes, and are used in cell therapy or tissue
engineering. Several studies have shown that LLLT, with different
parameters, can directly increase the proliferation of MSCs. Hou et
al. reported that irradiation at 0.5 J/cm2 with a 635 nm diode laser
significantly stimulated the proliferation of bone-marrow-derived
MSCs from rats. They also observed that the laser at 5 J/cm2
increased the production of vascular endothelial growth factor
(VEGF) and nerve growth factor, and dramatically facilitated the
myogenic differentiation of MSC [46]. Similar results were found in
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260 Low-Level Laser Therapy and Stem Cells

mice: 635 nm laser increased the proliferation of mice MSC [47].

The laser energy density seems to be a critical factor in stimulating
MSCs. Lasers with optimal low energy density could significantly
increase cell proliferation, while high energy density only shows
limited stimulating effects or even inhibiting effects [48]. Using a
660 nm diode laser, Horvát-Karajz et al. found that the proliferation
of MSC was increased by 41% when cells were treated at 1.9 J/cm2
while inhibited by 42% at 11.7 J/cm2 [49]. In another study, it was
found that an 808 nm laser at 4 J/cm2 did not enhance the murine
MSC proliferation [50]. Leonida et al. reported that irradiation with
Nd:Yag laser at 100 mJ or 150 mJ could increase cell proliferation
after 7 days post-irradiation; however, there was no significant
difference after 14 days between the laser group and control group.
Based on previous studies of various laser parameters and cell types,
the energy density ranges from 0.5 to 4.0 J/cm2 , and wavelengths
from 600 to 700 nm of LLLT are effective to enhance the proliferation
of various cell lines [51]. Oron et al. also showed that LLLT can
stimulate bone marrow MSCs and reduce heart scarring after heart
attack [52, 53].
The mechanism by which LLLT stimulate MSC proliferation is not
fully understood. Mechanisms at cellular and molecular levels have
been proposed to explain the phenomenon. In a recent study, Wu
et al. found that LLLT of 0.5 J/cm2 could induce the transcription
and expression of 119 genes, most of which were involved in cell
proliferation and survival [54]. Using microarray analysis, Wang
et al. found that microRNA-193 was significantly upregulated after
LLLT treatment. Blockade of miRNA-193 suppressed the enhancing
effect of LLLT on MSC proliferation. Moreover, the authors reported
that miRNA-193 was involved to regulate the expression of cyclin-
dependent kinase 2 (CDK2) and could be targeted to the inhibitor
of growth family, member 5 (ING5). Inhibition of ING5 was found
to stimulate MSC proliferation [55]. Giannelli et al. reported that
635 nm diode laser increased mouse MSC proliferation, which was
associated with the activation of Notch-1 pathway [47]. The MSC
proliferation and Notch-1 upregulation were closely related to the
increased membrane conductance through voltage-gated channels.
Inhibition of the channels resulted in the attenuation of the laser’s
stimulating effects [56].
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Clinical Applications of LLLT on Stem Cells 261

14.2.3 Adipose-Derived Stem Cells

Recently, adipose-derived stem cells (ADSCs) have become an
appealing source of stem cells due to the ease to obtain these
cells from fat tissues. Low-level laser at a wavelength of 636 nm
and a fluence of 5 J/cm2 was found to increase human ADSC
cellular viability and proliferation in cell cultures [57–59]. Besides
stimulating cell proliferation, Wu et al. reported that LLLT also has
anti-inflammatory effect on ADSCs. The authors found that LLLT at 8
J/cm2 markedly inhibited LPS-induced, pro-inflammatory cytokine
expression in human ADSCs, including Cox-2, IL-1β, IL-6, and IL-
8. The anti-inflammatory effect might be due to the upregulation
of cAMP and downregulation of NF-κB pathway induced by laser
[59]. It is well known that LLLT has anti-inflammatory effect on
many different cells and tissues. Thus, the LLLT may have potential
application for stem cell therapy under inflammatory condition.

14.3 Clinical Applications of LLLT on Stem Cells

14.3.1 LLLT for Stem Cell Transplantation

LLLT has potential applications for stem cell transplantation
treatment. Tuby et al. used LLLT-irradiated MSCs to treat infarcted
hearts in rats. The MSCs were isolated from rat bone marrow,
irradiated with an 810 nm Ga–Al–As laser, and implanted into
infarcted rat hearts. They found that infarcted hearts implanted with
irradiated MSCs showed significant reduction in infarcted size by
53% as compared to hearts implanted with non-irradiated MSCs.
Implantation with LLLT-irradiated MSCs resulted in significantly
increased cell density and proliferation and enhanced angiogenesis
in the infarcted hearts [60]. Consistent with this study, Zhang
et al. found that LLLT pretreatment could remodel the infarcted
myocardium microenvironment to enhance the early survival of
transplanted MSCs and improve therapeutic angiogenesis and heart
function [61]. The authors use a 635 nm, 5 mW diode laser, with
energy density of 0.96 J/cm2 for 150 s and observed increased VEGF,
glucose-regulated protein 78, and superoxide dismutase production
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262 Low-Level Laser Therapy and Stem Cells

as well as decreased malondialdehyde production in the infarcted

myocardium.
LLLT has also been investigated to stimulate liver regeneration
following hepatectomy. Oron et al. applied a diode [Ga–Al–As] laser
with wavelength of 804 nm to irradiate rats after acute hepatectomy
and found that laser significantly enhanced the liver regeneration
by the formation of new hepatocytes and MSCs and angiogenesis
[62].
In a recent study, Choi et al. explored the feasibility of using
ADSCs seeded in acellular dermal matrix along with LLLT to repair
bone defect in athymic nude mice [63]. After application of seeded
ADSCs, the laser group received 632.8 nm laser at 1 J/cm2 daily for
56 days, while the control group received no laser treatment. The
authors reported that bone regeneration was observed in the LLLT
group on day 28, while in the control group on day 56. Moreover,
the bone mineral density in the LLLT group significantly increased
after 7 days, while in the control group after 14 days. The authors
found that LLLT significantly enhanced ADSC proliferation both in
vitro and in vivo, indicating that LLLT combined with ADSCs could
promote bone healing.

14.3.2 LLLT for Wound Healing and Skin Restoring

LLLT is known to stimulate wound healing and skin restoring. Stem
cells in skin can be activated to facilitate wound healing. Kim et al.
investigated the effect of LLLT on ADSC-induced skin healing using
a mouse model. LLLT was found to accelerate the wound closure
and skin regeneration process compared with the non-laser group.
LLLT increased ADSC survival in wound bed by inhibiting apoptosis
of ADSC. In addition, secretion of growth factors was increased in
the LLLT group compared with the non-laser group [64]. Hawkins et
al. reported that LLLT (632.8 nm He–Ne laser) at a dose of 5 J/cm2
significantly stimulated proliferation of wounded skin fibroblasts.
However, high doses of 10 or 16 J/cm2 induced decreased cell
viability and proliferation with damages to cell membrane and DNA
[65].
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Clinical Applications of LLLT on Stem Cells 263

14.3.3 LLLT for Neural Regeneration

Studies have shown supportive evidence that LLLT could accelerate
the regeneration of peripheral nerve tissue. Akgul et al. used a 650
nm diode laser at 25 mW and 10 J/cm2 to treat rats with crushed
sciatic nerve and found that LLLT could accelerate the recovery
of peripheral nerves in this model. Interestingly, they found that
a delayed treatment began 7 days after the injury showed better
effects than immediate treatment [66]. Besides direct effects on
nerve regeneration, LLLT may also be used together with stem
cell transplantation to further stimulate the recovery of injured
nerve tissue. Yang et al. combined LLLT with MSC transplantation
to treat crushed sciatic nerve in rats and found that LLLT plus
MSC transplantation groups showed better recovery than either
LLLT or MSC-treated groups. The MSC–LLLT group also showed
fewer inflammatory cells and less vacuole formation than MSC or
LLLT single treatment groups, suggesting that LLLT combined with
MSC transplantation may achieve better functional recovery than
conventional treatment after nerve crush injury [67].
Unlike the peripheral nervous system, the regeneration of
neurons in the central nervous system (CNS) is not so significant.
It has long been considered that neurons do not regenerate until
recent studies demonstrated that neural stem cells can differentiate
into new neurons under proper circumstances. Neuron regeneration
in CNS would be beneficial for the treatment of neural degenerative
diseases and traumatic brain injury. Using a rat spinal cord injury
(SCI) model, Wu et al. reported that LLLT treatment with a diode
laser of 810 nm, at 150 mW/cm2 , and a daily dose of 1589 J/cm2
significantly promoted the axonal regeneration and induced better
functional recovery after SCI [68]. Xuan et al. found that LLLT of
810 nm, 25 mW/cm2 , and 18 J/cm2 could improve neurological
performance after traumatic brain injury in mice. The authors found
more proliferating cells in the injured sites that were treated by
LLLT than in non-treated ones, suggesting that LLLT may promote
neurogenesis [69].
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264 Low-Level Laser Therapy and Stem Cells

14.3.4 LLLT for Treatment of Hair Loss

The mechanisms through which LLLT promotes hair growth or
regrowth are not fully understood. It has been proposed that LLLT
stimulates mitochondria to produce more ATP and alters the ROS
level, resulting in the induction of downstream signaling pathways,
which further enhance cell proliferation and migration. It is possible
that the epidermal stem cells in hair follicles are stimulated by
LLLT, and the follicles are shifted into anagen phase [70]. A double-
blind randomized controlled trial showed that 665 nm laser or LED
treatment for 16 weeks significantly increased hair counts by 39%
in males with androgenetic alopecia, compared with the placebo
control group [71]. Another recent study found that an FDA-cleared
laser comb was effective for the treatment of male and female
pattern hair loss [72]. A total of 128 male and 141 female subjects
were enrolled in this double-blind randomized controlled clinical
trial. After receiving a 26-week treatment with either a laser comb
or a sham device, there was a significant difference in the increase in
terminal hair density between the laser group and the sham group,
suggesting LLLT may be an effective method to treat the pattern hair
loss in both men and women. More in vitro and in vivo studies are
needed to elucidate detailed mechanisms of LLLT on the treatment
of hair loss.

References

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25. Tuby H, Hertzberg E, Maltz L, and Oron U (2013) Long-term safety

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26. Grossman N, Schneid N, Reuveni H, Halevy S, and Lubart R (1998)
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28. Agaiby AD, Ghali LR, Wilson R, and Dyson M (2000) Laser modulation
of angiogenic factor production by T-lymphocytes. Lasers Surg Med 26:
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29. Bjordal JM, Johnson MI, Lopes-Martins RA, Bogen B, Chow R, and
Ljunggren AE (2007) Short-term efficacy of physical interventions
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30. Abergel RP, Lyons RF, Castel JC, Dwyer RM, and Uitto J (1987)
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31. Corazza AV, Jorge J, Kurachi C, and Bagnato VS (2007) Photobiomodu-
lation on the angiogenesis of skin wounds in rats using different light
sources. Photomed Laser Surg 25: 102–106.
32. Schindl A, Heinze G, Schindl M, Pernerstorfer-Schon H, and Schindl
L (2002) Systemic effects of low-intensity laser irradiation on skin
microcirculation in patients with diabetic microangiopathy. Microvasc
Res 64: 240–246.
33. Oron A, Oron U, Streeter J, de Taboada L, Alexandrovich A, Trembovler
V, and Shohami E (2007) Low-level laser therapy applied transcranially
to mice following traumatic brain injury significantly reduces long-term
neurological deficits. J Neurotrauma 24: 651–656.
34. Shaw VE, Spana S, Ashkan K, Benabid AL, Stone J, Baker GE, and
Mitrofanis J (2010) Neuroprotection of midbrain dopaminergic cells in
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35. Wong-Riley MT, Bai X, Buchmann E, and Whelan HT (2001) Light-

emitting diode treatment reverses the effect of TTX on cytochrome
oxidase in neurons. Neuroreport 12: 3033–3037.
36. Fillipin LI, Mauriz JL, Vedovelli K, Moreira AJ, Zettler CG, Lech O,
Marroni NP, and Gonzalez-Gallego J (2005) Low-level laser therapy
(LLLT) prevents oxidative stress and reduces fibrosis in rat traumatized
Achilles tendon. Lasers Surg Med 37: 293–300.
37. Morrone G, Guzzardella GA, Torricelli P, Rocca M, Tigani D, Brodano GB,
Fini M, and Giardino R (2000) Osteochondral lesion repair of the knee
in the rabbit after low-power diode Ga-Al-As laser biostimulation: An
experimental study. Artif Cells Blood Substit Immobil Biotechnol 28: 321–
336.
38. Weber JB, Pinheiro AL, de Oliveira MG, Oliveira FA, and Ramalho LM
(2006) Laser therapy improves healing of bone defects submitted to
autologous bone graft. Photomed Laser Surg 24: 38–44.
39. Shao XH, Yang YP, Dai J, Wu JF, and Bo AH (2005) Effects of He-Ne laser
irradiation on chronic atrophic gastritis in rats. World J Gastroenterol 11:
3958–3961.
40. Inoue S, Noda S, Kashima K, Nakada K, Hayashi J, and Miyoshi H
(2010) Mitochondrial respiration defects modulate differentiation but
not proliferation of hematopoietic stem and progenitor cells. FEBS Lett
584: 3402–3409.
41. Barbosa CM, Leon CM, Nogueira-Pedro A, Wasinsk F, Araujo RC,
Miranda A, Ferreira AT, and Paredes-Gamero EJ (2011) Differentiation of
hematopoietic stem cell and myeloid populations by ATP is modulated
by cytokines. Cell Death Dis 2: e165.
42. Roy S, Tripathy M, Mathur N, Jain A, and Mukhopadhyay A (2012)
Hypoxia improves expansion potential of human cord blood-derived
hematopoietic stem cells and marrow repopulation efficiency. Eur J
Haematol 88: 396–405.
43. Vacek A, Bartonickova A, and Rotkovska D (1990) Increase in the
capacity of bone marrow exposed to He-Ne laser radiation for growth
of GM-CFC colonies in vitro. Folia Biol (Praha) 36: 65–70.
44. de Souza SC, Munin E, Alves LP, Salgado MA, and Pacheco MT (2005)
Low power laser radiation at 685 nm stimulates stem-cell proliferation
rate in Dugesia tigrina during regeneration. J Photochem Photobiol B 80:
203–207.
45. do Nascimento RX and Callera F (2006) Low-level laser therapy at
different energy densities (0.1–2.0 J/cm2 ) and its effects on the capacity
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of human long-term cryopreserved peripheral blood progenitor cells

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46. Hou JF, Zhang H, Yuan X, Li J, Wei YJ, and Hu SS (2008) In vitro effects
of low-level laser irradiation for bone marrow mesenchymal stem cells:
Proliferation, growth factors secretion and myogenic differentiation.
Lasers Surg Med 40: 726–733.
47. Giannelli M, Chellini F, Sassoli C, Francini F, Pini A, Squecco R, Nosi D,
Bani D, Zecchi-Orlandini S, and Formigli L (2013) Photoactivation of
bone marrow mesenchymal stromal cells with diode laser: Effects and
mechanisms of action. J Cell Physiol 228: 172–181.
48. Huang YY, Chen AC, Carroll JD, and Hamblin MR (2009) Biphasic dose
response in low level light therapy. Dose Response 7: 358–383.
49. Horvat-Karajz K, Balogh Z, Kovacs V, Drrernat AH, Sreter L, and Uher F
(2009) In vitro effect of carboplatin, cytarabine, paclitaxel, vincristine,
and low-power laser irradiation on murine mesenchymal stem cells.
Lasers Surg Med 41: 463–469.
50. Bouvet-Gerbettaz S, Merigo E, Rocca JP, Carle GF, and Rochet N (2009)
Effects of low-level laser therapy on proliferation and differentiation of
murine bone marrow cells into osteoblasts and osteoclasts. Lasers Surg
Med 41: 291–297.
51. AlGhamdi KM, Kumar A, and Moussa NA (2012) Low-level laser therapy:
A useful technique for enhancing the proliferation of various cultured
cells. Lasers Med Sci 27: 237–249.
52. Tuby H, Hertzberg E, Maltz L, and Oron U (2013) Long-term safety
of low-level laser therapy at different power densities and single or
multiple applications to the bone marrow in mice. Photomed Laser Surg
31: 269–273.
53. Tuby H, Maltz L, and Oron U (2011) Induction of autologous mes-
enchymal stem cells in the bone marrow by low-level laser therapy has
profound beneficial effects on the infarcted rat heart. Lasers Surg Med
43: 401–409.
54. Wu YH, Wang J, Gong DX, Gu HY, Hu SS, and Zhang H (2012) Effects
of low-level laser irradiation on mesenchymal stem cell proliferation:
A microarray analysis. Lasers Med Sci 27: 509–519.
55. Wang J, Huang W, Wu Y, Hou J, Nie Y, Gu H, Li J, Hu S, and Zhang H
(2012) MicroRNA-193 pro-proliferation effects for bone mesenchymal
stem cells after low-level laser irradiation treatment through inhibitor
of growth family, member 5. Stem Cells Dev 21: 2508–2519.
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270 Low-Level Laser Therapy and Stem Cells

56. de Villiers JA, Houreld NN, and Abrahamse H (2011) Influence of low
intensity laser irradiation on isolated human adipose derived stem cells
over 72 hours and their differentiation potential into smooth muscle
cells using retinoic acid. Stem Cell Rev 7: 869–882.
57. Mvula B, Mathope T, Moore T, and Abrahamse H (2008) The effect of low
level laser irradiation on adult human adipose derived stem cells. Lasers
Med Sci 23: 277–282.
58. Mvula B, Moore TJ, and Abrahamse H (2010) Effect of low-level laser
irradiation and epidermal growth factor on adult human adipose-
derived stem cells. Lasers Med Sci 25: 33–39.
59. Wu JY, Chen CH, Wang CZ, Ho ML, Yeh ML, and Wang YH (2013) Low-
power laser irradiation suppresses inflammatory response of human
adipose-derived stem cells by modulating intracellular cyclic AMP level
and NF-kappaB activity. PLoS One 8: e54067.
60. Tuby H, Maltz L, and Oron U (2009) Implantation of low-level laser
irradiated mesenchymal stem cells into the infarcted rat heart is
associated with reduction in infarct size and enhanced angiogenesis.
Photomed Laser Surg 27: 227–233.
61. Zhang H, Hou JF, Shen Y, Wang W, Wei YJ, and Hu S (2010) Low level
laser irradiation precondition to create friendly milieu of infarcted
myocardium and enhance early survival of transplanted bone marrow
cells. J Cell Mol Med 14: 1975–1987.
62. Oron U, Maltz L, Tuby H, Sorin V, and Czerniak A (2010) Enhanced liver
regeneration following acute hepatectomy by low-level laser therapy.
Photomed Laser Surg 28: 675–678.
63. Choi K, Kang BJ, Kim H, Lee S, Bae S, Kweon OK, and Kim WH (2013)
Low-level laser therapy promotes the osteogenic potential of adipose-
derived mesenchymal stem cells seeded on an acellular dermal matrix.
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64. Kim H, Choi K, Kweon OK, and Kim WH (2012) Enhanced wound healing
effect of canine adipose-derived mesenchymal stem cells with low-level
laser therapy in athymic mice. J Dermatol Sci 68: 149–156.
65. Hawkins DH and Abrahamse H (2006) The role of laser fluence in cell
viability, proliferation, and membrane integrity of wounded human skin
fibroblasts following helium-neon laser irradiation. Lasers Surg Med 38:
74–83.
66. Akgul T, Gulsoy M, and Gulcur HO (2014) Effects of early and delayed
laser application on nerve regeneration. Lasers Med Sci 29: 351–357.
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67. Yang CC, Wang J, Chen SC, and Hsieh YL (2013) Synergistic effects
of low-level laser and mesenchymal stem cells on functional recovery
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68. Wu X, Dmitriev AE, Cardoso MJ, Viers-Costello AG, Borke RC, Streeter J,
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69. Xuan W, Vatansever F, Huang L, Wu Q, Xuan Y, Dai T, Ando T, Xu T,
Huang YY, and Hamblin MR (2013) Transcranial low-level laser therapy
improves neurological performance in traumatic brain injury in mice:
Effect of treatment repetition regimen. PLoS One 8: e53454.
70. Avci P, Gupta GK, Clark J, Wikonkal N, and Hamblin MR (2014) Low-level
laser (light) therapy (LLLT) for treatment of hair loss. Lasers Surg Med
46: 144–151.
71. Lanzafame RJ, Blanche RR, Bodian AB, Chiacchierini RP, Fernandez-
Obregon A, and Kazmirek ER (2013) The growth of human scalp hair
mediated by visible red light laser and LED sources in males. Lasers Surg
Med 45: 487–495.
72. Jimenez JJ, Wikramanayake TC, Bergfeld W, Hordinsky M, Hickman JG,
Hamblin MR, and Schachner LA (2014) Efficacy and safety of a low-
level laser device in the treatment of male and female pattern hair loss:
A multicenter, randomized, sham device-controlled, double-blind study.
Am J Clin Dermatol 15(2): 115–127.
73. Gao X and Xing D (2009) Molecular mechanisms of cell proliferation
induced by low power laser irradiation. J Biomed Sci 16(1): 4.
July 6, 2016 17:24 PSP Book - 9in x 6in 15-Hamblin-c15

Chapter 15

Antimicrobial Photodynamic Therapy

Vanderlei Salvador Bagnato, Cristina Kurachi,

Kate Cristina Blanco, and Natalia Mayumi Inada
São Carlos Institute of Physics, University of São Paulo, Biophotonics Group,
São Carlos, São Paulo 13566-590, Brazil
vander@ifsc.usp.br

The Biophotonics Group from the São Carlos Institute of Physics

in the University of São Paulo has been focusing on determining
clinical protocols for microbial control using photodynamic therapy
(PDT) as an alternative to conventional treatments. In vitro and
in vivo studies have been performed to establish the best photo-
sensitizers and illumination parameters, as well as to understand
the mechanisms of PDT in microorganisms. Instrumentation has
been developed to perform uniform and controlled illumination
especially designed for the target tissue or organ, and new
photosensitizers have been created for several conditions. The initial
investigation is performed at cell culture assays, and then the
protocol is tested on animal models. Furthermore, many clinical
protocols have been applied. This chapter presents the challenges
faced, developments occurred, and results obtained in the last six
years.
The problem of microbial resistance to antibiotics, as a result
of their inappropriate use, was initially reported in the 1930s

Handbook of Low-Level Laser Therapy

Edited by Michael R. Hamblin, Marcelo Victor Pires de Sousa, and Tanupriya Agrawal
Copyright c 2017 Pan Stanford Publishing Pte. Ltd.
ISBN 978-981-4669-60-3 (Hardcover), 978-981-4669-61-0 (eBook)
www.panstanford.com
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274 Antimicrobial Photodynamic Therapy

and 1940s, with the introduction of the first sulfonamides and

penicillin in infection treatment [1]. Pneumonia is one of the major
causes of morbidity and mortality worldwide, despite advances
in diagnostics and therapeutics in pulmonary infections [2]. This
study aims to find whether lung infection can be reduced through
photosensitizer inhalation and exposure to extracorporeal light.
To evaluate the photodynamic inactivation (PDI) of Streptococcus
pneumoniae, one of the most common microorganisms found
in pulmonary infections, in vitro and in vivo experiments are
ongoing in our Biophotonics Laboratory. Alveolar macrophages
(AM) interaction with photosensitizers were tested, and an animal
model has been used to assess therapeutic effectiveness and safety.
The efficacy of microorganism growth control is under investigation.
This part of our study evaluates the effectiveness of PDT using
indocyanine green (ICG) and infrared light against S. pneumoniae
and the interaction of ICG with AM to determine a safe protocol to
promote the infrared PDI of the microorganism. Initial experiments
aimed to determine this photosensitizer toxicity to AM with different
drug concentrations (ranging from 300 to 9.37 μM) and incubation
times (10, 20, and 30 min). The MTT method, a colorimetric
assay for assessing cell metabolic activity by the dye thiazolyl blue
tetrazolium, was used to obtain the viability of macrophages, and
the results revealed an overall lower viability rates for 150 and 300
μM. Experiments evaluating the PDI of S. pneumoniae with an 850
nm Biotable employed a light dose of 10 J/cm2 and lower ICG
concentrations. Better results were displayed for 10 μM ICG, with
a reduction of 5 log10 colonies forming unities per milliliter for 10
min incubation, and of 4 log10 for 20 and 30 min. Experiments using
a different device (Lasertable emitting at 780 nm) and the bacteria
in co-culture with AM were conducted, verifying the effectiveness
of PDI of the tested drug concentrations and incubation periods
using infrared light. In an animal model for pneumonia, all the PDT
parameters, such as photosensitizer delivery and light distribution,
and the treatment response are under investigation.
Eliminating larvae of mosquito that can spread dengue fever,
chikungunya, zika fever and yellow fever viruses, and other diseases
is a challenge for present scientists. Today, approximately 2.5 billion
people live with the risk of dengue in the world. The World
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Antimicrobial Photodynamic Therapy 275

Health Organization reports 80 million cases per year in 100

countries, affecting every continent except Europe. The problem
has grown dramatically in recent decades because of the rapid
urbanization and the expansion of human mobility. In the Americas,
Aedes aegypti occurs from the United States to Uruguay with
the exception of Canada because of environmental and climate
differences [3, 4]. PDT mechanisms and distinct protocols must
be investigated for effective larvae elimination. In this study, we
test the effectiveness of PDT against different stages of A. aegypti
using Photogem (hematoporphyrin derivative) and curcuminoids,
substances extracted from the roots of turmeric (Curcuma longa), as
photosensitizers. The characterization of photosensitizer distribu-
tion in larvae and the specific PDT response are under investigation
(Fig. 15.1A). Different illumination protocols are tested, and the
population control is monitored. The larvae at 2nd instar were
exposed to different concentrations of photosensitizers, and then
they were illuminated with natural light (sunlight) and artificial
light (fluorescent lamp and light-emitting diodes [LEDs], Fig. 15.1B)
with a specific wavelength for each compound. The photosensitizer
distribution in the larvae was observed by confocal microscopy
(fluorescence images). Preliminary results showed 80%–100% of
larvae mortality after 24 h of PDT in all experimental conditions,
thus indicating a powerful photodynamic effect against A. aegypti
larvae. The best result was obtained by testing exposure to sunlight
during 8 h of consecutive illumination and comparing days of low
and high incidence.
In dentistry, PDT is a promising alternative therapy for com-
bating pathogenic microorganisms. Clinical studies have been
performed to evaluate the reduction in the microorganisms of the
oral cavity using different photosensitizers. Before light irradiation,
each patient performs a mouthwash with a solution containing the
photosensitizer (Fig. 15.2). Saliva is collected at different times, and
the count of colony forming units is recorded. The evaluation of PDT
protocol in a clinical trial is relevant for defining the best parameters
as well as treatment limitations.
PDT also facilitates the treatment of nosocomial and special
patients or patients with oral hygiene difficulties, such as caries,
gingivitis, and periodontal pockets, with higher rates of oral
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276 Antimicrobial Photodynamic Therapy

C
90
15 J/cm²
80 20 J/cm²
25 J/cm²
70
30 J/cm²
60
Mortality (%)

50
40
30
20
10
0
-10
0 24 48 72 96
Time (h)

Figure 15.1 (A) Overlaid fluorescence images highlighting the photosensi-

tizer fluorescence on the larva intestinal tract. (B) Schematics of larvae’s
digestive canal: SG—salivary glands; GC—gastric caeca; MT—malpighi
tubules; and RG—anus. The insect’s digestive apparatus is formed basically
of a tube that runs through their bodies longitudinally from the mouth to
anus, called alimentary canal. (C) Larvae mortality (%) at 2nd instar as the
function of time (hours) after illumination in the presence of 20 μg/mL
Photogem and four different light doses.
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Antimicrobial Photodynamic Therapy 277

Figure 15.2 Homemade handheld prototype based on 450 nm LEDs for

oral decontamination (developed by Technological Support Laboratory, São
Carlos Physics Institute).

infection prevention. This study promotes oral decontamination

through the inactivation of Streptococcus mutans in dental biofilm
formed over a metallic surface using PDT.
Testing the efficiency of PDI in vitro, Dovigo et al. observed the
effect of PDI on different clinical isolates of Candida albicans from
denture stomatitis of 15 patients. They observed that the response
of the strains to PDI was not homogenous within the same species
[5].
Currently, new alternatives for conventional treatments, instru-
mentation development, and definition of new PDT protocols have
become necessary. Our group has been performing clinical PDT
to determine new treatment approaches, especially for cancer and
potentially malignant disorders as well as infectious diseases.
Onychomycosis is characterized by crumbly, thickened or pig-
mented, and dull nails. Conventional treatment involves adminis-
tration of systemic antifungal medication (highly hepatotoxic) and
antibiotics for up to 24 months. The increase in the number of
microbial strains that are resistant to the available drugs and the
high incidence of infection are important aspects to be considered
while developing new technologies and therapeutic options. A PDT
protocol has already been established with satisfactory results
[6], but the association of other techniques and protocols will be
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278 Antimicrobial Photodynamic Therapy

Figure 15.3 Fluorescence of Trichophyton mentagrophytes with

Photogem at 150 μg/mL concentration with different incubation times:
(A) 5 min, (B) 10 min, (C) 15 min and (D) 20 min.

tested to improve the results and reduce treatment time. The in

vitro studies showed the efficacy of PDT on the inactivation of C.
albicans, Trichophyton mentagrophytes, and Trichophyton rubrum
using different curcumins and Photogem as photosensitizers
(Fig. 15.3). The efficiency of curcumin from Sigma Aldrich and
three others variations from a Brazilian company (PDTPharma ,
Cravinhos-SP) was compared. We treated more than 90 patients
using a hematoporphyrin derivative [6], and now we are presenting
the clinical results using and comparing three pharmaceutical
formulations (solution, gel, and emulsion) with the curcumin
produced in Brazil [7]. The light source is a homemade LED device
emitting at 450 nm (Fig. 15.4) and 630 nm for curcumin and
porphyrin activation, respectively. Better results were achieved
using a mixture of curcumin and curcuminoides by PDTPharma ,
with the advantage of lower cost in comparison with Sigma Aldrich
curcumin. The clinical treatment demonstrated an excellent result
with five PDT sessions, confirming the effectiveness of PDT with
PDTPharma curcumin as the photosensitizer for onychomycosis
treatment.
Subcutaneous mycoses are caused by a heterogeneous group
of fungi that infect the skin and, in a few cases, fascia, muscle, or
bone [8]. The most observed in Brazil are paracoccidioidomycosis,
chromoblastomycosis, and lobomycosis (lacaziose). These diseases
are public health problems in some countries of Latin America
and in countries with tropical or subtropical climates. Nodular
lesions and verrucose hyperkeratotic are the most prevalent clinical
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Antimicrobial Photodynamic Therapy 279

Figure 15.4 Equipment for onychomycosis treatment (commercially

available—Gnatus Medical and Odontological Equipment, Ribeirão Preto,
São Paulo, Brazil).

Figure 15.5 Chromoblastomycosis treatment with photodynamic inacti-

vation. (A) Upper limb with a large extension of the disease caused by
Fonsecaea pedrosoi. (B) Illumination of PpIX following our clinical protocol.
(C) Clinical result after five PDT sessions.

presentation of the disease. The current treatment for these is

inefficient and long term. The fungus Fonsecaea pedrosoi is an
etiologic agent of chromoblastomycosis. This study aims to evaluate
the in vitro effect of PDT and determine a clinical protocol to be
tested in patients. It was observed that F. pedrosoi can produce PpIX;
therefore, different protocols of PDT can be applied (Fig. 15.5).
Healing wounds represent a major public health problem, mainly
due to antibiotic resistance. This study aims to evaluate the effects
of PDT associated with artificial skin on disinfection and healing
skin wound in rats contaminated with S. aureus. For performing
PDT, a curcumin concentration of 6 mg/l and a 450 nm LED were
used. The results showed that PDT and artificial skin could promote
disinfection and accelerate healing tissue.
Cervical intraepithelial neoplasia (CIN) I and II are premalignant
lesions with potential indications for PDT. This lesion progression
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280 Antimicrobial Photodynamic Therapy

has a well-established relation to cervical cancer. About 90% of

cervical cancers originate from CIN. Classified on the basis of cyto-
histological characteristics such as low- and high-grade lesions, CIN
is a chronic disease with intraepithelial changes, which turn into
invasive lesions in 5–6 years The major cause of CIN is chronic
infection of the cervix with the sexually transmitted human papillo-
mavirus (HPV), especially the high-risk HPV types 16 or 18. Cervical
cancer is a public health problem, especially in developing countries
such as Brazil. It is still the second leading cancer in women in
Brazil and worldwide [9, 10]. In Brazil, 15,540 new cases of cervical
cancer were estimated in 2014. Conventional treatments show some
limitations, mainly concerning surgical resection. When detected at
initial stages, lesion treatment is simple and has lower morbidity.
Optical diagnostics and PDT may constitute attractive technologies
for cervical lesions. We have a project involving device development,
clinical trial, and evaluation of the safety and efficacy of PDT for the
diagnostics and treatment of CIN I and II [11]. The cream containing
the pro-drug methyl-aminolevulinic acid (MAL) and the device
CerCa 150 System (Fig. 15.6) are made in Brazil (PDTPharma
and MMOptics, respectively), in collaboration with Trubios LLC
(Rockville, MD, USA), and in this sense, this methodology has great
potential to become a standardized technique for the public health
system.

Figure 15.6 CerCa 150 System produced by the Brazilian Company

MMOptics (São Carlos, São Paulo) and Trubios LLC (Rockville, MD, USA).
(A) Front view of the two tips, one for the fluorescence diagnosis of the
cervix, and the other for treatment with PDT. (B) A different view of the same
device.
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Antimicrobial Photodynamic Therapy 281

(A)

(B)
PDT using Curcumin syrup
1.00E+07
Survival fraction

1.00E+06

1.00E+05

1.00E+04
BEFORE PDT
1.00E+03
AFTER PDT
1.00E+02

1.00E+01

1.00E+00
1 2 3 4 5 6 7 8 9 10 11 12 13 14
Patients

Figure 15.7 (A) Treatment of patients using curcumin mixture in drops:

quantification of bacteria (Streptococcus pyogenes) before and after PDT. (B)
Treatment of patients using curcumin mixture in syrup: quantification of
bacteria (S. pyogenes) before and after PDT.

Pharyngitis and tonsillitis are common infections of the upper

respiratory tract commonly caused by bacteria, viruses, and
fungi. The conventional treatment involves oral administration of
antibiotics, which increase the resistant of bacterial strains to
the available drugs and side effects in patients. These treatment
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282 Antimicrobial Photodynamic Therapy

complications, especially in children, prompt development of new

technologies and therapeutic options. In a clinical project started in
early 2015, our group is developing equipment for the diagnosis and
treatment of the disease. The standardized clinical protocol has been
validated with in vitro and clinical studies. In our clinical trial, we are
testing a natural derivative curcumin (PDPharma Ltda., Cravinhos-
SP, Brazil) used in two different pharmaceutical formulations and
activated by a prototype developed at our laboratory, emitting at
450 nm. The main agent of these diseases, S. pyogenes, has been
isolated from 36 patients treated with our standardized protocol
and comparing different synthesis of curcumin (PDTPharma Ltda.,
Cravinhos-SP, Brazil) and two formulations (drops and syrup)
containing curcumin (Fig. 15.7A,B). Adults and children in the
treated groups have acute, recurrent, and chronic infections. The
placebo group and the healthy patients group are under analysis.

References

1. Alanis JA. Resistance to antibiotics: Are we in the post-antibiotic era?

Arch Med Res, 36, 697–705, 2005.
2. Media Centre, 16 June 2006. Produced by Nada Osseiran and
Gregory Hartl. Available in: <http://www.who.int/mediacentre/news/
releases/2006/pr32/en/>. Accessed in: July 18, 2013.
3. Ministry of Health National Dengue Control Program. Epidemiological
Surveillance. Brası́lia (DF), 2002.
4. World Health Organization, “Dengue and dengue haemorrhagic fever,”
Factsheet no. 117, revised March 2009.
5. Dovigo LN, Carmello JC, Carvalho MT, Mima EG, Vergani CE, Bagnato
VS, and Pavarina AC. Photodynamic inactivation of clinical isolates of
Candida using Photodithazine . Biofouling, 29(9), 1057–1067, 2013.
6. Silva AP, Kurachi C, Bagnato VS, and Inada NM. Fast elimina-
tion of onychomycosis by hematoporphyrin derivative-photodynamic
therapy. Photodiagnosis Photodyn Ther, 10(3), 328–330, 2013, doi:
10.1016/j.pdpdt.2013.01.001. [Epub 2013 Feb 12].
7. Silva AP, Carbinatto F, Bagnato VS, and Inada NM. A promising strategy
for the treatment of onychomycosis with curcumin and photodynamic
July 6, 2016 17:24 PSP Book - 9in x 6in 15-Hamblin-c15

References 283

therapy. J Pharm Pharmacol, 3, 434–437, 2015, doi: 10.17265/2328-

2150/2015.09.005.
8. Queiroz-Telles F, Nucci M, Colombo AL, Tobón A, and Restrepo A.
Mycoses of implantation in Latin America: An overview of epidemiology,
clinical manifestations, diagnosis and treatment. Med Mycol, 49(3), 225–
236, 2011.
9. Bosch FX, Lorincz A, Muñoz N, Meijer CJ, and Shah KV. The causal
relation between human papillomavirus and cervical cancer. J Clin
Pathol, 55(4), 244–265, 2002. Review.
10. Cancer incidence in Brazil (2012): In: Instituto Nacional de Câncer
José Alencar Gomes da Silva (INCA), Coordenação Geral de Ações
Estratégicas, Coordenação de Prevenção e Vigilância. Rio de Janeiro.
2011; 118 p.
11. Inada NM, Lombardi W, Leite MFM, Trujillo JR, Kurachi C, and Bagnato
VS. Photodynamic therapy of cervical intraepithelial neoplasia. Proc of
SPIE, 8931, 2014, 89310X-1. doi: 10.1117/12.2040004.
July 6, 2016 17:24 PSP Book - 9in x 6in 16-Hamblin-c16

Chapter 16

Low-Level Laser (Light) Therapy for

Wound Healing in Animal Models

André Luiz Oliveira Ramos,a Felipe Scholz Ramos,b and

Marcelo Victor Pires de Sousaa,c
a Laboratory of Radiation Dosimetry and Medical Physics, Institute of Physics,

Matão Street, Alley R, 187, University of São Paulo, São Paulo,

São Paulo 05508-900, Brazil
b Department of Aesthetic and Cosmetology, Centro Universitário Monte

Serrat—UNIMONTE, Santos, São Paulo 11015-530, Brazil

c Bright Photomedicine Inc., Prof. Linnaeus Prestes Avenue, No. 2242,

Butantã - University City, IPEN - São Paulo, São Paulo 05508-000, Brazil
marcelovictor@usp.br

In this chapter, the fundaments of low-level laser (light) therapy

(LLLT) for wound healing in animal models will be presented.
At the beginning, a brief explanation of the physiology of wound
healing and the corresponding phases (inflammatory, migration,
proliferation, and maturation) will be given. Moreover, the possible
dose-response influence of LLLT on healing phases and wound
depths and the influence of different wavelengths on wound healing
will be discussed. From this point, this chapter will be completed
with low-intensity laser irradiation on infected wounds.

Handbook of Low-Level Laser Therapy

Edited by Michael R. Hamblin, Marcelo Victor Pires de Sousa, and Tanupriya Agrawal
Copyright c 2017 Pan Stanford Publishing Pte. Ltd.
ISBN 978-981-4669-60-3 (Hardcover), 978-981-4669-61-0 (eBook)
www.panstanford.com
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286 Low-Level Laser (Light) Therapy for Wound Healing in Animal Models

16.1 Physiology of Wound Healing

Wound is an opening in the skin, and it can be caused by trauma,

surgery, diabetes, arterial and venous diseases, edema, or pressure.
Generally, wounds heal within a few weeks; however, circulation
problems, diabetes, history of ionizing radiation, collagen, or
autoimmune diseases can delay the healing process. Wound healing
can be enhanced by LLLT; however, researching the healing
therapeutic effects of laser irradiation of blood (LIB) is a hard
task due to the variability of illumination parameters, types of
light delivery (intravenous, transcutaneous, and transmucosa), and
search of local effect due to a systemic treatment. These problems
are enhanced by too many types, sizes, and causes of wounds. So
there are several types of animal models for simulating wounds.
But are they suitable (Karu, 1999)? It is even harder to compare
research on LIB for wound healing in human subjects, as clearly
demonstrated by Lucas et al. (2000). Overall, according to Karu,
there is no evidence that all individuals would respond in the same
way to radiation (Karu et al., 1989).

16.1.1 Mechanisms of Wound Healing with LLLT

Wound healing occurs in four phases (Hopkins et al., 2004):

(1) Coagulation or hemostasis: Immediately after injury, platelets,

endothelial cells, fibrin, and fibronectin act to block or, at least,
decrease the bleeding.
(2) Inflammation: Shortly after the wound, there is an orderly
recruitment of key cells into the wound site.
(3) Migration, proliferation, and angiogenesis: Cells necessary for
wound closure multiply or move to the wound site during a few
days.
(4) Remodeling: Scar formation may last for months.

We found evidences of LIB action during all phases of wound healing.

LLLT decreases platelet aggregation (Brill et al., 2000; Kirichuk et al.,
1999), so it can be a problem during coagulation. So illumination
right before or after a wound formation is controversial. It can be
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Physiology of Wound Healing 287

a contraindication and a possible generator of negative results for

LLLT in wound healing.
On the other hand, reduction in inflammation due to light therapy
is one of the most accepted LLLT effects (Lopes-Martins et al., 2007).
It is evidenced by a decrease in inflammatory mediators such as
prostaglandin E2 (Bjordal et al., 2006; Castano et al., 2007; Pallotta
et al., 2012), leucocytes (Pallotta et al., 2012), and tumor necrosis
factor (TNFα) (Aimbire et al., 2006).
Specifically for wound healing, the most described photobiomod-
ulation effects are connected to cell migration (Haas et al., 1990),
proliferation (Peplow et al., 2010), and angiogenesis (Diegelmann
and Evans, 2004). These processes can be regulated by many
growth factors and are connected with nitric oxide (NO) signaling,
whose release and production can be modulated by LLLT (Chen
et al., 2008; Huang et al., 2011). Many functions in vascular
walls are regulated by NO, including suppression of inflammatory
response, vasodilatation, angiogenesis, inhibition of apoptosis,
and cell migration (Kimura and Esumi, 2003). LLLT promotes
cell proliferation mainly through the activation of mitochondrial
respiratory chain and the initiation of cellular signaling (Gao and
Xing, 2009). Oron et al. showed that laser irradiation caused a 3.1-
fold significant increase in newly formed blood vessels 6 days post
infarction, as compared with nonirradiated rats (Mirsky et al., 2002).
Neovascularization is mediated by matrix metaloproteases (MMP),
which are capable of making the vessels liquid, so that they can
easily enter the interstitial tissue to create new microvascularization
in the healed site. NIR irradiation can upregulate the production
of MMP-2 (gelatinase) and transforming growth factor (TGF)-β1
in cultured human keratinocytes, endothelial cells, and fibroblasts
(Danno et al., 2001).
Near-infrared (NIR) laser irradiation enhances the tensile
strength of cutaneous wounds in a murine diabetic model promoting
an earlier consolidation of scars (Stadler et al., 2001). Wound
illuminated at 830 nm, 20 J/cm2 or 685 nm, 20 J/cm2 showed
better quality of granulation tissue formation (more production and
organization of collagen) than the ones illuminated with higher
fluencies, proving that there is a biphasic dose response (Huang
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288 Low-Level Laser (Light) Therapy for Wound Healing in Animal Models

Figure 16.1 Cellular pathways that can be activated when a light-induced

electric field interacts with ATP, Na+ /K+ ATPase, mitochondria, enzymes,
and membrane phospholipids that regulate Ca2+ signal (legend: ATP*, light-
excited ATP; enzymes*, light-excited enzymes).

et al., 2011) and that enhancement of granulation is possible with

red and NIR light.
The basic biological mechanism behind the effects of LLLT is
thought to be absorption of light energy (red and NIR photons)
by chromophores of the respiratory chain located in mitochondria,
leading to increased enzyme activity and ATP production (Karu et al.,
1995). For visible and NIR wavelengths, chromophores are typically
metals or molecules that contain metallic atoms in their structure
(Silfvast, 2004).
Moreover, low-level light was shown to stimulate the expression
of multiple genes related to cellular migration and proliferation as
well as modulate the production of growth factors and cytokines.
Several mechanisms are investigated and proposed by researchers
to explain the stimulatory effect of LLLT on cells, as shown in
Fig. 16.1.
LLLT can lead to stimulatory or inhibitory effect while cells are
irradiated, according to the light dose absorbed in the tissue. Several
studies suggest that the concept of a biphasic dose response with
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Physiology of Wound Healing 289

Figure 16.2 Clot formation and inflammatory stages in wound healing.

Adapted from Cordeiro et al. (2013).

the total delivered light energy density is responsible for modulating

wound healing.
As we know, healing begins as soon as a wound occurs.
During the healing stages, the optical property of skin is subjected
to constant changes at the wound site in accordance with the
concentration of substances, structural proteins, and the presence
of different types of cells in each phase. After injury, platelet
aggregation is triggered at the wound site, followed by marked
inflammatory phase in the presence of monocytes, macrophages,
and neutrophils, and the formation of new blood vessels (Fig. 16.2).
LLLT can enhance cellular metabolism in order to eliminate
pathogens, clear cell debris, and stimulate neovascularization, which
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290 Low-Level Laser (Light) Therapy for Wound Healing in Animal Models

Figure 16.3 LLLT action on cell migration and proliferation. Adapted from
Cordeiro et al. (2013).

facilitate the migration of immune cells and the transport of oxygen

and nutrients to the wound site.
At the migration and proliferation phase, the granulation tissue
is formed. The tissue optical characteristic is constituted from the
newly formed blood vessels, epithelial cells that proliferate and
migrate into the wound, myofibroblasts and fibroblasts that migrate
from the neighboring tissue, which deposit collagen fibers and other
extracellular matrix proteins (Fig. 16.3).
In this stage (migration and proliferation phase), the adequate
light dose emitted and absorbed by tissue can facilitate the
replacement of endothelial cells, fibroblast proliferation, syntheses
of collagen and contraction wound edges which leads to wound re-
epithelialization.
At the remodeling stage, the immune response and cell migration
and proliferation terminate. LLLT can contribute to the alignment
of collagen fibers, which enhance epidermal formation and the
formation of scar tissue (Fig. 16.4).
LLLT shows good results for in vivo studies on wound healing.
Demidova-Rice et al. (2007) evaluated a single exposure of light
of wavelengths 632.8, 635, 670, 720, and 820 nm and various
fluences for 30 min after wounding a mice. A biphasic dose response
for 635 nm was found, with maximum positive effect at 2 J/cm2 ,
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Physiology of Wound Healing 291

Figure 16.4 LLLT action on remodeling stage on wound healing. Adapted

from Cordeiro et al. (2013).

80–100 mW/cm2 , and a better result was obtained for 820 nm,
where wound contraction occurred 4 days faster than with other
wavelengths (Demidova-Rice et al., 2007).
Fushimi et al. (2011) investigated the effects of different
wavelengths—red (638 nm), blue (456 nm), and green (518 nm)
LEDs—on wound healing in 12 mice sorted out in groups, each with
four mice, and compared with control sham treatment. On the day
after the excision the LEDs have been adjusted at the same power
density 0.25 mW/cm2 for 20 min (0.3 J/cm2 at the wound surfaces)
every other day, and the size of each wound area was evaluated
as relative to the size on day 1. Statistic data are mean SD of four
independent experiments (Figs. 16.5 and 16.6).
The results suggest that red and green lights promote faster
wound healing until day 7, compared with control and blue light.
At day 7, the wound areas irradiated with the green LED were
significantly reduced in size (green: 24.65 16.76%, p < 0.03)
compared with the sham-treated control (57.44 < 13.90%). Total
epithelialization of the wounds irradiated with red or green LEDs
was observed at day 11, while the wounds treated with the blue
LED or the sham-treated control epithelialized at day 13. In vivo
study showed that some cytokines secreted by fibroblasts and
HaCat keratinocytes are significantly induced only by green LED
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292 Low-Level Laser (Light) Therapy for Wound Healing in Animal Models

Figure 16.5 In vivo wound-healing study. Representative wounds at days 1

and 7 are shown. The wound areas on days 1 and 7 are surrounded by solid
lines and by dotted lines, respectively (Fushimi et al., 2011).

Figure 16.6 In vivo wound-healing study. One 8 mm diameter full-thickness

excisional wound was created in the dorsal skin of each animal; animals
were distributed into four groups with four animals in each (Fushimi et al.,
2011).
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Physiology of Wound Healing 293

irradiation, suggesting that the motility of HaCat keratinocytes is

accelerated, although the dose of green LEDs was the lowest among
the three types of LEDs.

16.1.2 Types of Wound Healed by LIB

Low-level LIB was tested in several types of wounds, and positive
effects were claimed for all of them. The most common type of
wound is inflicted by trauma or injury through an incision in skin
with a sharp object, avulsion of body parts, burns, or overexposure
of ionizing radiation. Healing can be enhanced with LIB since many
healing effects are mediated or distributed to the wound site by
blood.
In cases of avulsion, with large loss of blood, He–Ne laser
irradiation (632.8 nm, 1.5–2.0 mW, for 30 min), as part of complex
therapy, promoted increase in plasma albumin transport ability
and the general stimulation of natural detoxification mechanisms
(Kravchenko-Berezhnaia et al., 2002). He–Ne laser irradiation
(632.8 nm, 4.8 mW, for 60 min, daily for 15 days), in addition to
usual treatment, improved hemorheological properties that lead to
improvement in size and quality of survival of avulsed area (31
human patients) compared with 27 patients who received only usual
treatment (Luo et al., 2000).
Hopkins et al. (2004) conducted a randomized, triple-blind,
placebo-controlled experiment with 22 healthy subjects who had
induced in theirs anterior forearms two standardized 1.27 cm2
abrasion wounds. LLLT (820 nm, 8 J/cm2 , for 125 s, pulse rate
of 700 Hz) was applied in only one of the two randomly chosen
wounds. Evidences of a systemic blood (or carried by blood) effect
of laser irradiation were obtained in follow-up testing (in days 6, 8,
and 10) since it revealed that the laser group had smaller wounds
than the sham group for both the treated and untreated wounds
.(Hopkins et al., 2004).
He–Ne laser irradiation (632.8 nm, 6 mW/cm2 , 1 J/cm2 ,
polarized, continuous wave, for 3 min) promoted enhancement of
wound healing due to alterations in blood microcirculation leading
to the acceleration of granulation, tissue formation, and faster re-
epithelization in burned rat skin (da Silva et al., 2006; Ribeiro et al.,
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294 Low-Level Laser (Light) Therapy for Wound Healing in Animal Models

2004). He–Ne laser irradiation (632.8 nm, 1.2 and 2.4 J/cm2 ) caused
significant decrease in the number of macrophage and depth of new
epidermis in deep second-degree burns (Bayat et al., 2005) and a
significant increase in the blood vessel sections and a decrease in the
depth of new epidermis after third-degree burns (Bayat et al., 2006).
There are evidences that laser therapy using different parameters,
such as red (670 nm, 400 mW, 2/week, 8 weeks) (Gaida et al., 2004),
pulsed (585 nm, 5–6 J/cm2 ) (Allison et al., 2003), LED illumination
through hydrogel dressing (Kim and Lee, 2008), and NIR (880 nm,
2.4 and 4 J/cm2 , on two human fibroblast cell lines) (Webb and
Dyson, 2003), can improve the quality of scars from burn wound due
to changes in microvascularization.
Diabetes is known as a factor that delays wound healing. So the
effect of laser (light) illumination of blood on diabetic wounds was
studied. Al-Watban (2009) conducted a huge experiment (with 893
Sprague Dawley rats) to find the optimal light therapy parameters
for healing oval full-thickness wounds and burn wounds in both
diabetic and nondiabetic animals. This series of studies used 532,
633, 810, 980, and 10,600 nm lasers and polychromatic LED clusters
510–872 nm; the highest improvements in wound healing were
obtained using laser at 633 nm, 3/week, 4.71 J/cm2 for diabetic burn
and at 633 nm, 3/week, 2.35 J/cm2 for diabetic wound (Al-Watban,
2009; Al-Watban et al., 2009). Daily LLLT (830 nm, 79 mW/cm2 ,
5.0 J/cm2 /wound) for wound healing in mice with genetic diabetes
showed that the starting time of treatment matters. Dorsal wounds
were irradiated for 1–4 days, or 3–7 days, or nonirradiated, and
higher tensile strength at day 23 occurred in the group irradiated
for 3–7 days (Stadler et al., 2001). It indicates that some negative
results of LLLT for wound healing can be due to early treatment,
which cannot be beneficial to coagulation since it decreases platelet
aggregation.
Post-operative wounds treated with LLLT (904 nm, 5 W,
frequency of 5000 Hz, pulse duration of 180 ns) healed faster, and
the period for patients’ functional recovery (i.e., return to their
ordinary life) decreased (Herascu et al., 2005). LLLT may enhance
epithelization and improve wound healing after gingivectomy
and gingivoplasty operations (Faria Amorim et al., 2006; Ozcelik
et al., 2008). At wound dehiscence post-surgery of saphenectomy
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Thrombosis 295

treatment in diabetics patients, red laser (655 nm, 25 mW, 30

s/point, 4 J/cm2 ) was used. The results obtained were formation
of granulated tissue around the incision, as well as decreased
inflammatory process, reduction of fibrin and wound size. Analgesic
effect was observed since day 1 laser application (Pinto et al., 2008).

16.2 Thrombosis

Thrombosis is the formation of a blood clot (thrombus) inside

a blood vessel, obstructing the flow of blood through the circulatory
system. To prevent blood loss when a blood vessel is injured,
platelets aggregate to form clots. However, clot can sometimes form
without a vessel injury and can break free in the body. Thrombus
can be large enough to reduce blood flow, causing hypoxia or
even anoxia (complete deprivation of oxygen) and infarction (tissue
death). Thrombosis may be triggered by composition of blood
(hypercoagulability), endothelial cell injury, or alterations of the
normal blood flow regimen (stasis or turbulence).
Platelet activation, in vivo and real time, was obtained with
an argon laser (514.5 nm, 20 mW, 300 ms, 120 J/cm2 , 80 μm
of laser beam, focused on a vessel). There are three phases in
platelet activation by laser: recruitment (maximum after 90 ± 20 s),
adhesion (lasts 110 ± 25 s), and detachment (starts 200 ± 20 s
after laser irradiation and completes in less than 120 s). Laser
irradiation with optimal parameters can induce platelet activation
without thrombus formation, since platelets adhere only transiently
(Mordon et al., 2002). Laser irradiation can work in the detachment
of thrombus, decreasing the chances of hypoxia or anoxia by a
thrombus.
Nitric oxide (NO) is a simple biological molecule that inhibits
adhesion and aggregation of platelets (Ramamurthi and Lewis,
1998). It can be another possible mechanism for inhibition of
platelet adhesion or clot formation induced by laser irradiation since
NO release is modulated by LLLT (Huang et al., 2011).
Diabetes may cause angiopathy (with endothelial cell injury),
which can lead to thrombosis and hypoxia. Thirty patients with
diabetic ulcers or gangrenes and elevated levels of glycosylated
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296 Low-Level Laser (Light) Therapy for Wound Healing in Animal Models

hemoglobin received a single low-intensity laser irradiation (non-

thermal, 632.8 nm, 30 mW, 50 min, 30 J/cm2 ) or a sham irradiation
over both forefoot regions in a double-blind placebo-controlled
clinical study. Skin blood circulation, as indicated by temperature
detected by infrared thermograph, increased significantly in the
irradiated forefoot (Schindl et al., 1998). Similar posterior study
by the same group indicated that this effect is systemic and
mediated by blood changes, since there is an increase in temperature
in forefoot contra-lateral to the treated one (Schindl et al.,
2002).
Laser irradiation (0–1.869 J/cm3 , 0–9 min) of blood, in vitro,
changed rheological factors in a dose-dependent way. In addition,
the blood viscosity is closely related to platelet aggregation, clot
formation and coagulation. In this study, the blood viscosity was
significantly altered in 82.5% of cases and was verified alteration
up to 48 h after laser irradiation (Siposan and Lukacs, 2001). These
blood alterations signaling that LLLT can contribute to the treatment
of thrombosis.

16.3 LLLT Influence on Infected Wounds

LLLT has shown many positive results in all phases of wound healing
in animal models, but the treatment of infected wounds is uncertain
and has received significant attention in scientific research in vivo
and in vitro.
Many studies signaling that the same energy of laser beam with
different wavelengths and different radiant intensity may promote
bactericidal effect against Staphylococcus aureus, and the use of
high energy levels (100 ± 5 mW and 50 J/cm2 ) may have more
destructive effects (Dadras et al., 2006; Kaya et al., 2011; Krespi et al.,
2011).
In contrast, Dadras et al. (2006) suggest a proliferative effect
when LLLT is applied to Pseudomonas aeruginosa. Thus, for each
type of bacteria, there are specific parameters for inhibitory action
to occur.
Gonçalves et al. (2013) investigated the action of laser (658 nm,
5 J/cm2 ) irradiated on dorsal infected wounds by S. aureus in Wistar
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References 297

rats (n = 56), by three consecutive days. After that, it was observed

lower bacterial growth at the injured skin.
Escherichia coli concentration has been analyzed in skin wounds
at 3 groups of rats treated with laser (810 nm, 10 W) at different
energy densities: 130, 195, and 260 J/cm2 . After 48 hours it was
observed the absence of E. coli in wounds group irradiated by 260
J/cm2 (Jawhara and Mordon, 2006).
Jawhara and Mordon (2006) assumed that progressive desicca-
tion of the superficial dermis occurs, which dries the wound and
makes the injury site inhospitable for bacteria. However, the exact
physiological mechanism of LLLT and use of other light sources in
infected wounds needs further elucidation.
Another hypothesis of mechanism of LLLT is that the irradiation
of high energy density in infected wounds to promote oxidative
stress generating toxicity for cells and bacteria in the site of the
lesion.
Lipovsky et al. (2008) evaluated the phototoxicity of broadband
white light (400–800 nm) with an intensity of 120 J/cm2 in
pathogenic bacteria such as E. coli 1313, 195 S. aureus, and Serratia
marcescens. The survey results showed a reduction of 62%, 83%,
and 56% in the colony count, respectively.
On the other hand, the action of white light on P. aeruginosa
PA1316 not promote reduction at the viability of this bacteria.
Possibly, this occur because P. aeruginosa is highly resistant to
oxidative stress (Sabra et al., 2002).

References

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Bjordal, J., Lopes-Martins, R., and Iversen, V. 2006. A randomised, placebo-
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Brill, A. G., Shenkman, B., Brill, G. E., Tamarin, I., Dardik, R., Kirichuk, V.
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Castano, A. P., Dai, T., Yaroslavsky, I., Cohen, R., Apruzzese, W. A., Smotrich,
M. H., and Hamblin, M. R. 2007. Low-level laser therapy for zymosan-
induced arthritis in rats: Importance of illumination time. Lasers in
Surgery and Medicine, 39, 543–550.
Chen, C. H., Hung, H. S., and Hsu, S. H. 2008. Low-energy laser irradiation
increases endothelial cell proliferation, migration, and eNOS gene
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Cordeiro, J. V., and Jacinto, A. 2013. The role of transcription-independent
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da Silva, D. D. F. T., de Campos Vidal, B., Zezell, D. M., Zorn, T. M. T., Núñez,
S. C., and Ribeiro, M. S. 2006. Collagen birefringence in skin repair in
response to red polarized-laser therapy. Journal of Biomedical Optics,
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Dadras, S., Mohajerani, E., Eftekhar, F., Hosseini, M. 2006. Different photore-
sponses of 19 Staphylococcus aureus and Pseudomonas aeruginosa to
514, 532, and 633 nm low level lasers in vitro. Current Microbiology, 53,
282–286.
Danno, K., Mori, N., Toda, K. I., Kobayashi, T., and Utani, A. 2001. Near-
infrared irradiation stimulates cutaneous wound repair: Laboratory
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munology and Photomedicine, 17, 261–265.
Demidova-Rice, T. N., Salomatina, E. V., Yaroslavsky, A. N., Herman, I. M.,
and Hamblin, M. R. 2007. Low-level light stimulates excisional wound
healing in mice. Lasers in Surgery and Medicine, 39, 706–715.
Diegelmann, R. F., and Evans, M. C. 2004. Wound healing: An overview of
acute, fibrotic and delayed healing. Frontiers in Bioscience, 9, 283–289.
Faria Amorim, J. C., Sousa, G. R. D., Silveira, L. D. B., Prates, R. A., Pinotti,
M., and Ribeiro, M. S. 2006. Clinical study of the gingiva healing after
gingivectomy and low-level laser therapy. Photomedicine and Laser
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Fushimi, T., Inui, S., Nakajima, T., Ogasawara, M., Hosokawa, K., and
Itami, S. 2012. Green light emitting diodes accelerate wound healing:
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Gaida, K., Koller, R., Isler, C., Aytekin, O., Al-Awami, M., Meissl, G., and Frey,
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Gao, X., and Xing, D. 2009. Molecular mechanisms of cell proliferation
induced by low power laser irradiation. Journal of Biomedical Science,
16, 1–16.
Gonçalves e Silva, D. C. G., Plapler, H., Costa, M. M., Gonçalves e Silva,
R., Aquino de Sá, M. C., Lima e Silva, B. S. 2013. Low level laser
therapy (AlGaInP) applied at 5 J/cm2 reduces the proliferation of
Staphylococcus aureus MRSA in infected wounds and intact skin of rats.
Anais Brasileiros de Dermatologia, 88, 1.
Haas, A. F., Isseroff, R. R., Wheeland, R. G., Rood, P. A., and Graves, P. J.
1990. Low-energy helium-neon laser irradiation increases the motility
of cultured human keratinocytes. Journal of Investigative Dermatology,
94, 822–826.
Herascu, N., Velciu, B., Calin, M., Savastru, D., and Talianu, C. 2005. Low-level
laser therapy (LLLT) efficacy in post-operative wounds. Photomedicine
and Laser Therapy, 23, 70–73.
Hopkins, J. T., Mcloda, T. A., Seegmiller, J. G., and Baxter, G. D. 2004. Low-level
laser therapy facilitates superficial wound healing in humans: A triple-
blind, sham-controlled study. Journal of Athletic Training, 39, 223.
Huang, Y.-Y., Sharma, S. K., Carroll, J., and Hamblin, M. R. 2011. Biphasic dose
response in low level light therapy: An update. Dose-Response, 9, 602–
618.
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Jawhara, S., and Mordon, S. 2006. Monitoring of bactericidal action of laser

by in vivo imaging of 6 bioluminescent E. coli in a cutaneous wound
infection. Lasers in Medical Science, 21, 153–159.
Karu, T., Pyatibrat, L., and Kalendo, G. 1995. Irradiation with He-Ne laser
increases ATP level in cells cultivated in vitro. Journal of Photochemistry
and Photobiology B, 27(3), 219–223.
Karu, T. I. 1999. A suitable model for wound healing: How many times are
we to stumble over the same block? Lasers in Surgery and Medicine, 25,
283–283.
Karu, T., Ryabykh, T., Fedoseyeva, G., and Puchkova, N. 1989. Helium-neon
laser-induced respiratory burst of phagocytic cells. Lasers in Surgery
and Medicine, 9, 585–588.
Kaya, G. Ş., Kaya, M., Gürsan, N., Kireççi, E., Güngörmüş, M., and Balta, H.
2011. The use of 808 nm light therapy to treat experimental chronic
osteomyelitis induced in rats by methicillin-resistant Staphylococcus
aureus. Photomedicine and Laser Surgery, 29, 405–412.
Kim, J. W., and Lee, J. O. 2008. Low level laser therapy and phototherapy
assisted hydrogel dressing in burn wound healing: Light guided
epithelial stem cell biomodulation. In Innovations in Plastic and
Aesthetic Surgery, Eisenmann-Klein, M., and Neuhann-Lorenz, C. (eds),
Springer Berlin Heidelberg, pp. 36–42.
Kimura, H., and Esumi, H. 2003. Reciprocal regulation between nitric oxide
and vascular endothelial growth factor in angiogenesis. Acta Biochimica
Polonica-English Edition, 50, 49–60.
Kirichuk, V., Shenkman, B., Tamarin, I., Dardik, R., Varon, D., and Savion,
N. 1999. Effect of He-Ne laser on platelet activation and aggregation.
Bulletin of Experimental Biology and Medicine, 128, 700–701.
Kravchenko-Berezhnaia, N., Moroz, V., and Kozhura, V. 2002. Laser radiation
to correct disorders of blood albumin transport in severe mechanical
trauma. Anesteziologiia i Reanimatologiia, 22.
Krespi, Y. P., Kizhner, V., Nistico, L., Hall-Stoodley, L., and Stoodley, P.
2011. Laser disruption and killing of methicillin-resistant Staphylo-
coccus aureus biofilms. American Journal of Otolaryngology, 32, 198–
202.
Lipovsky, A., Nitzan, Y., and Lubart, R. 2008. A possible mechanism for visible
light-induced wound healing. Lasers in Surgery and Medicine, 40, 509–
514.
Lopes-Martins, R. A. B., Penna, S. C., Joensen, J., Vereid Iversen, V., and
Magnus Bjordal, J. 2007. Low level laser therapy [LLLT] in inflammatory
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and rheumatic diseases: A review of therapeutic mechanisms. Current

Rheumatology Reviews, 3, 147–154.
Lucas, C., Stanborough, R., Freeman, C., and de Haan, R. 2000. Efficacy of low-
level laser therapy on wound healing in human subjects: A systematic
review. Lasers in Medical Science, 15, 84–93.
Luo, Q., Xiong, M., and Gu, H. 2000. Effect of intravascular low level laser
irradiation used in avulsion injury. Chinese Journal of Reparative and
Reconstructive Surgery, 14, 7–9.
Mirsky, N., Krispel, Y., Shoshany, Y., Maltz, L., and Oron, U. 2002. Promotion
of angiogenesis by low energy laser irradiation. Antioxidants and Redox
Signaling, 4, 785–790.
Mordon, S., Begu, S., Buys, B., Tourne-Peteilh, C., and Devoisselle, J. M. 2002.
Study of platelet behavior in vivo after endothelial stimulation with
laser irradiation using fluorescence intravital videomicroscopy and
PEGylated liposome staining. Microvascular Research, 64, 316–325.
Ozcelik, O., Cenk Haytac, M., Kunin, A., and Seydaoglu, G. 2008. Improved
wound healing by low-level laser irradiation after gingivectomy opera-
tions: A controlled clinical pilot study. Journal of Clinical Periodontology,
35, 250–254.
Pallotta, R. C., Bjordal, J. M., Frigo, L., Junior, E. C. P. L., Teixeira, S., Marcos, R.
L., Ramos, L., de Moura Messias, F., and Lopes-Martins, R. Á. B. 2012.
Infrared (810-nm) low-level laser therapy on rat experimental knee
inflammation. Lasers in Medical Science, 27, 71–78.
Peplow, P. V., Chung, T.-Y., and Baxter, G. D. 2010. Laser photobiomodulation
of proliferation of cells in culture: A review of human and animal
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C. M. R., Zorn, T. M. T., and Zezell, D. M. 2004. Effects of low-intensity
polarized visible laser radiation on skin burns: A light microscopy
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Sabra, W., Kim, E. J., and Zeng, A. P. 2002. Physiological responses
of Pseudomonas aeruginosa PAO1 to oxidative stress in controlled
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302 Low-Level Laser (Light) Therapy for Wound Healing in Animal Models

microaerobic and aerobic cultures. Microbiology, 148(Pt 10), 3195–

3202.
Schindl, A., Heinze, G., Schindl, M., Pernerstorfer-Schön, H., and Schindl, L.
2002. Systemic effects of low-intensity laser irradiation on skin micro-
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Research, 64, 240–246.
Schindl, A., Schindl, M., Schön, H., Knobler, R., Havelec, L., and Schindl,
L. 1998. Low-intensity laser irradiation improves skin circulation in
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Siposan, D. G., and Lukacs, A. 2001. Relative variation to received dose of
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Stadler, I., Lanzafame, R. J., Evans, R., Narayan, V., Dailey, B., Buehner, N.,
and Naim, J. O. 2001. 830-nm irradiation increases the wound tensile
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28, 220–226.
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healing. Journal of Photochemistry and Photobiology B: Biology, 70, 39–
44.
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Chapter 17

Low-Level Laser Therapy for Arthritis in

Animal Models: Beneficial Effect and
Action Mechanism

Flavio Aimbirea and Paulo de Tarso Camilo de Carvalhob

a Science and Technology Department, Federal University of São Paulo,

Avenida Cesare Mansueto Giulio Lattes, 1201, Parque Tecnológico,

São José dos Campos, São Paulo, São Paulo 12247-014, Brazil
b Rehabilitation Science Department, Nove de Julho University, Rua Vergueiro,

235/249, Liberdade, São Paulo 01504-001, Brazil

flavio.aimbire@unifesp.br, paulo.tarso@uninove.br

Applications of low-level laser therapy (LLLT) on muscle skeletal

disorders are probably higher than any other type of laser therapy
applications in health sciences. This is easily visible from the dose
table of the World Association for Laser Therapy (WALT). This
dose table adresses different irradiation protocols according to the
lesion area, which was only described referring to muscle skeletal
disorders, and among them, the rheumatoid arthritis. Although a
reasonable number of studies show the anti-inflammatory effect
of LLLT in a variety of arthritis animal models induced for
different pathways, there is no action mechanism for describing
exactly what cellular signaling is responsible for low-level laser
effects. In fact, diverse authors have found different cellular
mechanisms for describing the beneficial effects of LLLT. This does

Handbook of Low-Level Laser Therapy

Edited by Michael R. Hamblin, Marcelo Victor Pires de Sousa, and Tanupriya Agrawal
Copyright c 2017 Pan Stanford Publishing Pte. Ltd.
ISBN 978-981-4669-60-3 (Hardcover), 978-981-4669-61-0 (eBook)
www.panstanford.com
July 6, 2016 17:25 PSP Book - 9in x 6in 17-Hamblin-c17

304 Low-Level Laser Therapy for Arthritis in Animal Models

not mean that experimental designs from studies with animal

model for arthritis have been poorly delineated; the viewpoint is
that LLLT reveals different signaling pathways according to the
inflammatory environment. For this reason, an increasing number
of authors have reported that instead of LLLT reducing all chemical
mediators of inflammatory process similar to what happens in
treatment with glucocorticoid, laser therapy can modulate the pro-
inflammatory response, increasing both the mRNA expression and
the protein concentration of anti-inflammatory mediators such
as IL-10 (tendinitis model) and heat shock protein-72 (HSP72)
(chondrocytes in rheumatoid arthritis). Thus, these effects suggest
that LLLT, in models of joint inflammation, acts as a homeostasis
regulator to maintain balance between anti- and pro-inflammatory
responses. Naturally, experimental studies reported in this chapter
deserve caution when extrapolated to cellular response in humans.
However, these different signals responsible for laser effect on
rheumatoid arthritis have an important role in the process for
understanding the laser therapy effect in clinical trials as well as the
adjustment of better dosimetry to be used. Another important point
is the resemblance between animal models and clinical symptoms
of rheumatoid arthritis observed in humans. Surely, this gives
reason for trusting experimental results. Moreover, regardless of
the method used to induce rheumatoid arthritis (auto-immune or
septic) in animal models, a majority of studies describe that LLLT
is efficient in attenuating at least one of the symptoms of joint
inflammation without producing any side effect.
Although most arthritis animal models are easily inducible in
rats, mice, and rabbits, if we compare the number of experimental
studies with the clinical trials focused on the study of the beneficial
effects of laser therapy in subjects with arthritis, we find that
the quantity of clinical trials and the constant updating of the
Cochrane Database System Review overcome the investigation in
animal models. It is natural because LLLT for the treatment of
muscle skeletal diseases occupies an important position among anti-
inflammatory therapies which are effective, noninvasive, low cost,
and so far have no side effects in day-to-day clinical practice.
In this chapter, we will discuss the beneficial effect of LLLT on
arthritis in animal models. We begin with describing the findings
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Low-Level Laser Therapy for Arthritis in Animal Models 305

of Lin et al. [12]. They demonstrated that helium–neon (He–Ne)

laser (632 nm, 3.1 mW/cm2 , 15 min) over the knee of arthritic rats
(inducted with papain) markedly increased the density of shock
proteins (anti-inflammatory and anti-oxidant proteins) in cartilage
and was closely related to the repair of arthritic cartilage. They
reinforced that the extragenic production of shock proteins was
well correlated with the therapeutic effect of LLLT in preserving
chondrocytes and the repair of arthritic cartilage in rats. They inves-
tigated the effects of He–Ne laser (632 nm, 3.1 mW/cm2 , irradiation
time 15 min on knee area) on the mucopolysaccharide induction
in experimental osteoarthritic cartilage induced by papain. The
findings demonstrated that He–Ne laser treatment enhances the
biosynthesis of arthritic cartilage resulting in the improvement of
arthritic histopathological changes [13]. Among the animal models
that mimic joint inflammation, Soriano et al. [16] reported the
beneficial effect of LLLT on rat joint subjected to injection of
crystals such as hydroxyapatite, calcium pyrophosphate, and urates.
The authors showed that fibrinogen, prostaglandin E2, and TNF
were markedly increased after the induction of arthropathy; on the
contrary, LLLT reduced the pro-inflammatory markers (fibrinogen,
prostaglandin E2 , and TNF) as well as the anatomopathological
alterations induced by crystal injection.
The illumination time or irradiation time is an important variable
in laser therapy, and thus it should be considered. In some cases,
especially in animal models, the calculation of irradiation time
is in agreement with laser parameters, but the laser effect does
not happen. In this situation, it is important to consider that
the interaction of laser light with arthritic tissue requires more
than a few minutes and daily treatment for stimulating cellular
signaling results in the beneficial effect of LLLT. According to Castano
et al. [7], the use of LLLT (810 nm) in rats subjected to zymosan
injection in their knees presented better efficiency, almost as well
as dexamethasone, with a longer irradiation time (10 or 100 min
compared to 1 min). In addition, laser therapy reduced swelling with
30 J/cm2 , and curiously the serum PGE2 level in arthritic rats was
lower when the animals were irradiated with 3 J/cm2 . Thus, longer
illumination times were more effective than shorter times regardless
of total fluence or irradiance. Still in this context, some authors
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306 Low-Level Laser Therapy for Arthritis in Animal Models

such as Sandoval et al. [15] demonstrated that LLLT (830 nm,

27.5 mW/cm2 , 77 mW), 2 and 5 days after a knee inflammation
induced through an intracapsular injection of terebinthina in rabbit,
produced only a few subtle differences in the inflammatory signals
(body mass, joint perimeter, and temperature) and synovial fluid.
These authors attributed the lack of LLLT effects to the short
irradiation time (0.12 or 0.32 s). Some authors have investigated
the effects of LLLT in the treatment of arthropathies induced by
hydroxyapatite and calcium pyrophosphate (crystals) in rats [17].
In fact, these authors showed that He–Ne laser at 632.8 nm, 5 mW,
8 J/cm2 and 50 s irradiation time for 2 weeks attenuated both the
morphological alterations and the cellular infiltration in the joint
inflamed by crystal injection. In the arthritic joint treated with laser,
the authors found a marked decrease in the percentage of area
with inflammatory infiltrates. The granulomas remained in a less
ostensible form, with adipose cells, fibrosis bands with light residual
inflammation. This result reinforces the importance of dosimetry
and illumination time.
Naturally, the beneficial effect of LLLT on joint inflammation
symptoms in animal models created the mood for some researchers
to investigate the interaction of laser light with the fundamental
cells of joints such as synoviocytes. Some authors demonstrated
that linear polarized near-infrared light irradiation reduced the IL-6
mRNA expression and protein concentration from the supernatant
of fibroblast-like synoviocytes MH7A stimulated with IL-1β [4].
These authors reported that polarized laser light decreased the
joint temperature as well as the IL-6 mRNA expression in the
synovial membrane tissue (immunohistochemical analysis) in rats
with rheumatoid arthritis induced by collagen injection in the joint.
It is important to note that IL-6 plays a key role in the progression
of rheumatoid arthritis. Some researchers used the arthritis animal
model through the intra-articular injection of zymosan for compar-
ing the effects of laser (685 nm and 830 nm) and LED (628 nm)
on edema formation, increase in vascular permeability, and joint
hyperalgesia [9]. In this condition, LLLT reduced the inflammatory
signals more effectively than LED irradiation with similar irradiation
times (100 s), average outputs (20 mW), and energy doses (2 J).
These authors suggested that the anti-inflammatory effect of LLLT
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Low-Level Laser Therapy for Arthritis in Animal Models 307

appeared to be a class effect, which is not wavelength specific

in the red and infrared parts of the optical spectrum. In another
animal model for rheumatoid arthritis induced by collagen injection
in the joint, some authors interested in investigating the action
mechanism evaluated the LLLT (830 nm Ga–Al–As diode; 100–700
mW; 6.4 mW/cm2 , 7.64 J/cm2 ) effect on CCL2 chemokine in synovial
membrane tissue in arthritic rats [18]. The authors demonstrated
that LLLT reduced both CCL2 mRNA expression and production
in the joint of arthritic rats when the animals were irradiated for
20 min, three times a week, for a period of 2 weeks. Considering
that CCL2 is an important chemokine with chemotactic activity
for monocytes and basophils, the reduction in CCL2 in synovial
tissue may be one of the mechanisms by which LLLT relieves the
inflammatory symptoms of arthritis.
Among the different ways to induce arthritis in animal models,
some authors have chosen to inject papain in the knees of rats [8].
These authors analyzed the influence of LLLT at wavelengths of
660 nm (4 J, 100 mW, 3.57 W/cm2 , irradiation time 40 s) and 808 nm
(same parameters as those of 660 nm). They concluded that LLLT
upregulated angiogenesis and downregulated fibrosis formation,
especially at the wavelength of 808 nm. In the animal model
induced by phlogistic agents, Pallotta et al. [14] induced arthritis
by injecting carrageenan in rat knee. These authors reported
that LLLT operating at 810 nm markedly reduced inflammatory
signals such as leukocyte number, myeloperoxidase activity, and
vascular leakage. To understand if LLLT has the ability to act
as glucocorticoids, these authors described that LLLT inhibited
PGE2 production in the knees of arthritic rats, but curiously laser
therapy increased the expression of both COX-1 and COX-2 in
the knee tissue. The augmentation of COX-1 constitutive enzyme
(physiological response) is an interesting mechanism to control
knee inflammation, but it is still not confirmed. Moreover, the rise
of COX-2-inducible enzyme after LLLT is a controversial response
and thereby needs to be evaluated with acuity. A comparative study
of two low-level laser (808 nm, 50 mW, 1.78 mW/cm2 ) doses (2 J,
71.4 J/cm2 , irradiation time 40 s; 4 J, 142.8 J/cm2 , irradiation
time 80 s) on the expression of inflammatory mediators and on
neutrophils and macrophages in acute joint inflammation induced
 July 6, 2016 17:25
308 Low-Level Laser Therapy for Arthritis in Animal Models
Table 17.1 Dosimetry of LLLT in animal model of arthritis

Energy Irradiation Duration of Frequency of

Induction Wavelength density time per treatment treatment Power (mW
Authors Model Animal Type (nm) (J/cm2 ) point (s) (days) (days) or W)

Carlos et al. (2014) Zymosan Rats—Wistar 660 2.5 10 6 h and 12 1 10 mW

h
Kang et al. (2014) Complete Mice—C57BL/6 660 — 150 — — 20 mW
Freund’s
adjuvant
Alves et al. (2013) Papain Rats—Wistar 808 142.4 40 and 80 1 1 irradiation 100 mW

PSP Book - 9in x 6in

(4%) and 50 mW
Alves et al. (2013) Papain Rats—Wistar 808 142.4 40 and 80 21 3× /week 100 mW
(4%) and 50 mW
Dos Santos et al. (2013) Papain Rats—Wistar 808 714 and 40 and 80 1 1 irradiation 50 mW
(4%) 142.8
Alves et al. (2013) Type II Rats—Wistar 780 7.7 65 14 3× /week 22 mW
collagen –
complete
Freund’s
adjuvant
da Rosa et al. (2012) Papain Rats—Wistar 808 and 142 40 21 3× /week 100 mW
(4%) 660
Zhang et al. (2011) Type II Rats—Lewis 830 7.64 20 min 14 3× /week 500 mW
collagen (LEW/CrlCrlj)

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 July 6, 2016 17:25
Pallotta et al. (2011) Kaolin plus Rats—Wistar 810 50, 150, 10, 30, 60, 1 1 irradiation 100 mW
Car- 300, and and 100
rageenan 500
de Morais et al. (2010) Zymosan Rats—Wistar 685 and 2.5 100 1 3× /day 20 mW
830
Sandoval et al. (2009) Commun Rabbit—New 830 3.4 0.12 and 7 Daily 77 mW
Tere- Zealand 0.29
binthina
Castano et al. (2007) Zymosan Rats—Lewis 810 3 and 30 60.6000, and 5 Daily 50 mW

Low-Level Laser Therapy for Arthritis in Animal Models

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6.000
Lin et al. (2006) Papain Rats—Wistar 632 — 900 56 3× /week —
(4%)
Lin et al. (2004) Papain Rats—Wistar 632 — 900 56 3× /week —
(4%)

309

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310 Low-Level Laser Therapy for Arthritis in Animal Models

Table 17.3 Anti-inflammatory effect of LLLT on arthritis animal model

Authors Effect of LLLT

Lin et al. Increases the shock protein arthritic chondrocytes with the preservation of
(2004) chondrocytes and the repair of arthritic cartilage in rats.
Lin et al. He–Ne laser treatment enhances the biosynthesis of arthritic cartilage and
(2006) results in the improvement of arthritic histopathological changes.
Soriano Reduction in fibrinogen prostaglandin E2 and TNF in rats injected with crystal
et al. (2006) in the knee joint. Laser therapy was more effective than diclofenac in patients
with chronic pyrophosphate arthropathy and in patients with chronic apatite
deposition in disease.
Castano LLLT reduced the joint swelling correlated with reduction in the inflammatory
et al. (2007) marker serum prostaglandin E2. Longer illumination times were more effective
than shorter times regardless of the total fluence or irradiance.
Sandoval Laser irradiation with selected parameters produced only a few subtle
et al. (2009) differences in the inflammatory signs and the synovial fluid.
Rubio et al. Laser reduced the percentage of area with inflammatory infiltrates, but it did not
(2010) present histological changes.
de Morais Irradiation with 685 and 830 nm reduced edema formation, vascular permeabil-
et al. (2010) ity, and hyperalgesia. LED treatment with the same fluence of laser had no effect.
Araki et al. Laser reduced IL-6 gene expression in MH7A (synoviocytes cells) and reduced
(2011) inflammation and IL-6 protein expression in knee joint.
Zhang et al. Laser decreased CCL2 expression in arthritic tissue in rat.
(2011)
da Rosa LLLT, especially 808 nm, stimulated angiogenesis and reduced fibrosis formation
et al. (2012) in an experimental model of osteoarthritis.
Pallotta et al. LLLT reduced inflammatory signals but increased both COX-1 and COX-2
(2012) expression in joint inflammation.
dos Santos Both laser doses, especially 2 J decreased histological alterations as well as
et al. (2013) inflammatory mediators and inflammatory cells.
Alves et al. Both 50 and 100 mW repaired joint tissue with reduction in collagen III
(2013) expression and upregulation of collagen I. The dose of 50 mW was more efficient
in decreasing MMP-2 and -9 in arthritic rats.
Alves et al. LLLT reduced mononuclear inflammatory cells exudate of protein medullary
(2013) hemorrhage hyperemia tissue necrosis and improved the distribution of
fibrocartilage chondroblasts and osteoblasts in rats with rheumatoid arthritis.
Alves et al. LLLT, especially 50 mW was efficient in modulating IL-1β and IL-6 as well
(2013) as macrophages and neutrophils migration into inflamed joint tissue which is
correlated with the histology analysis.
Carlos et al. LLLT inhibited leukocytes influx the release of IL-1β and IL-6 and also the
(2014) activity of MMP-2 and -9.
Kang et al. LLLT suppressed paw edema and macrophages population into inflamed joint.
(2014)
Araujo et al. The parameters of LLLT used were not effective in nociception reduction and
(2013) produced no change in the natural history of resolution of the infectious process.
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Low-Level Laser Therapy for Arthritis in Animal Models 311

by papain injection in rat knee has revealed that laser therapy

reduces inflammation signals as well as the mRNA expression of
pro-inflammatory cytokines (TNF, IL-1β, and IL-6) in arthritic rat
knee, but there is no dose-dependent effect [10]. Interestingly, the
study demonstrated that LLLT applied on the lesion area restored IL-
10 expression (an important anti-inflammatory cytokine) to control
values in the joint tissue of arthritic rats. Maybe the absence
of LLLT side effects is justified by laser action on IL-10, which
helps maintain the integrity of joint environment. In another study
on rheumatoid arthritis induced by papain injection in the knee
joint, Alves, Albertini et al. [1] reported the effect of LLLT (808
nm, 50 or 100 mW, 142 J/cm2 , illumination time 40 or 80 s)
on the metaloproteinases-2 and -9 generation and the percentage
of collagen types I and III in the joint tissue of arthritic rats.
These authors showed that LLLT applied at 50 or 100 mW on the
inflamed joint was equally effective in repairing tissue, decreasing
collagen type III, and increasing type I expression in all experimental
periods (7, 14, and 21 days after the initial laser treatment);
however, LLLT at 50 mW reduced metalloproteinase-9 better than
LLLT at 100 mW. This type of laser response where the lower
dose is more beneficial follows the Arndt–Schulz law, which states
that a weak stimulus slightly accelerates vital activity, a stronger
stimulus raises it further, but a peak is reached and an even
stronger stimulus suppresses it, until a negative response is finally
achieved, i.e., LLLT causes inhibition at high doses and stimulation at
low doses.
Alves, Albertini et al. [1] also evaluated the effect of LLLT
in different stages of rheumatoid arthritis in the animal model
through a histological study. In fact, Alves, de Carvalho et al. [2]
irradiated the knee joint with laser (780 nm, 22 mW, 0.10 W/cm2 ,
and 7.7 J/cm2 ) and concluded that LLLT (1.65 J per point and
75 s of irradiation) decreased mononuclear inflammatory cells,
exudate protein, medullary hemorrhage, hyperemia, and necrosis, as
well as improved the distribution of fibrocartilage, chondroblasts,
and osteoblasts. These authors revealed that LLLT was effective
in controlling the inflammatory response both in early and late
stages of rheumatoid progression. In a study on the effect of LLLT
on the expression of inflammatory mediators and on neutrophils
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312 Low-Level Laser Therapy for Arthritis in Animal Models

and macrophages in acute joint inflammation induced by papain

injection in rats, Alves, Vieira et al. [3] reported that LLLT (808
nm, 4 J, and 142.4 J/cm2 ) applied on the inflamed joint at 50
mW (irradiation time 80 s) was more efficient than at 100 mW
(irradiation time 40 s) in reducing TNF, IL-1β, and IL-6 as well as
the number of macrophages and neutrophils. Once again we can
attribute the better laser effect to the Arndt–Schulz law as described
previously. In a model of rheumatoid arthritis induced by zymosan,
Carlos et al. [6] showed that the effect of LLLT (660 nm, 10 mW,
2.5 J/cm2 , and illumination time 10 s) on arthritic rats was driven
by the decrease in both the leukocyte influx and the concentration
of IL-1β and IL-6 as well as the expression of metalloproteinase-
2 and -9 in the inflamed joint cavity. These authors highlighted
that the inhibition of proteases activation after the laser treat-
ment could be one of the mechanisms responsible for the less
degradation of collagen tissue in the animal model of rheumatoid
arthritis.
One of the principal advantages of LLLT is that its use in the
treatment of acute or chronic diseases of the musculoskeletal system
has a positive effect even if applied noninvasively. In fact, in major
cases, laser therapy can be applied transcutaneously to improve
the inflammatory environment of the arthritic joint and reduce
edema and pain. The possibility of irradiating the animal only at the
lesion area can guarantee that a possible effect of LLLT in inducing
a toxic response is cleared. Furthermore, the local effect can be,
at least, one of the reasons that LLLT causes no adverse effects.
Even knowing that in clinical practice there is no possibility of
exactly calculating the energy quantity which interacts with joint
cells; there is no need for the laser light to pass through many
tissue layers in order to reach the inflammation site. Therefore,
most authors prefer using LLLT noninvasively in the treatment
of rheumatoid arthritis, as described in all studies presented in
this chapter. Despite this, some authors reported that a minimally
invasive laser needle system may suppress the acute progression
of rheumatoid arthritis induced by Freund’s adjuvant in mice and
thus it may be used as a potential treatment modality for arthritis
in clinics [11]. These authors evaluated both the edema and the
average of laser speckle contrast images, as well as the macrophage
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Low-Level Laser Therapy for Arthritis in Animal Models 313

population in arthritic mice before and after laser therapy. The laser
therapy (660 nm, 20 mW, 3 J, and illumination time 150 s) reduced
all the measured inflammatory parameters. Naturally, if noninvasive
laser therapy produces beneficial effect, one can imagine that the
minimally invasive technique of irradiation will also have positive
effects on arthritic joint. However, laser applied very close to cells
does not always give satisfactory result. It is reasonable to think
that the closer the site of injury, the better the laser effect, but the
proximity of laser light to the inflamed joint can induce apoptosis
of synoviocytes and consequently hinder remodeling of the arthritic
tissue. It is well known that some laser doses applied at smaller
distances from cells can augment ROS secretion via the activation of
the nuclear transcription factor (NF-κB) in cells in vitro. Therefore,
except for critical situations, the use of LLLT on arthritic joint is
increasingly less invasive with the capacity to produce better results.
Tables 17.1 and 17.3 show some doses and anti-inflammatory effect
of LLLT on arthritis experimental model.
Despite all the results of the effects of LLLT on rheumatoid
arthritis described herein, an animal model has raised the interest
of some researchers because LLLT produced no change in joint
inflammation. This animal model is based on the inoculation of
bacteria in the joint; it is also known as septic arthritis. Clinically,
septic arthritis is the invasion of a joint by an infectious agent that
produces arthritis. Some authors showed that the effect of LLLT (660
nm, 30 mW, and 2 J/cm2 ) on experimental septic arthritis was not
effective in nociception [5]. These same authors also revealed that
the morphological analysis showed a slight cartilage areas recovery
and that the synovial and the inflammatory infiltrate remained high,
even after laser treatment. These results suggest that LLLT in the
animal model of infection presents a singular feature because the
lesion area illuminated by laser also contains Staphylococcus aureus,
an infectious microorganism that also responds to laser light. It
is important to consider that a correct dose of LLLT can control
the inflammation arising from bacterial infection by decreasing the
number of colonies. On the contrary, infectious microorganisms
even not being directly irradiated (in vitro) but irradiated at the
same local (joint cavity) where the bacteria is commonly inoculated
are stimulated by laser therapy, and thereby, a laser low dose
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314 Low-Level Laser Therapy for Arthritis in Animal Models

can potentiate the bacterial action and consequently stimulate the

defense cells attraction into the joint space contributing to the
inflammatory response onset. Currently, no experimental studies
are available using the animal model of infection that show LLLT can
attenuate the symptoms of septic arthritis.
In conclusion, the chapter presents the major studies inves-
tigating the effect of LLLT on arthritis in animal models. We
highlighted that LLLT has a beneficial effect on rheumatoid arthritis
independent of the animal model chosen. It means that laser therapy
can control the secretion of different inflammatory mediators
in any situation in which homeostasis is disrupted. Moreover,
these studies show that LLLT can control both the acute and
chronic inflammation through different pathways, but principally
through downregulating pro-inflammatory cytokines, ROS, and
metalloproteinases. Therefore, the experimental results described
herein are associated to results obtained in clinical practice. This
proves that LLLT can be used for joint inflammation in different
stages of the disease with no side effects. Figure 17.1 illustrates the
action mechanisms of LLLT on experimental arthritis.

Figure 17.1 Scheme illustrating the possible mechanism of action of LLLT

in arthritis.
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References 315

References

1. Alves, A.C., Albertini, R., dos Santos, S.A., Leal-Junior, E.C., Santana, E.,
Serra, A.J., Silva, J.A. Jr., and de Carvalho, P.D. (2013). Effect of low-level
laser therapy on metalloproteinase MMP-2 and MMP-9 production and
percentage of collagen types I and III in a papain cartilage injury model.
Lasers Med. Sci., 29(3), pp. 911–919.
2. Alves, A.C., de Carvalho, P.T., Parente, M., Xavier, M., Frigo, L., Aimbire,
F., Leal-Junior, E.C., and Albertini, R. (2013). Low-level laser therapy in
different stages of rheumatoid arthritis: A histological study. Lasers Med.
Sci., 28(2), pp. 529–536.
3. Alves, A.C., Vieira, R., Leal-Junior, E., dos Santos, S., Ligeiro, A.P., Albertini,
R., Junior, J., and de Carvalho, P. (2013). Effect of low-level laser therapy
on the expression of inflammatory mediators and on neutrophils and
macrophages in acute joint inflammation. Arthritis Res. Ther., 15(5), pp.
116–122.
4. Araki, H., Imaoka, A., Kuboyama, N., and Abiko, Y. (2011). Reduction
of interleukin-6 expression in human synoviocytes and rheumatoid
arthritis rat joints by linear polarized near infrared light (Superlizer)
irradiation. Laser Ther., 20(4), pp. 293–300.
5. Araujo, B.F., Silva, L.I., Meireles, A., Rosa, C.T., Gioppo, N.M., Jorge, A.S.,
Kunz, R.I., Ribeiro, L. de F., Brancalhão, R.M., and Bertolini, G.R. (2013).
Effects of low-level laser therapy, 660nm, in experimental septic arthri-
tis. ISRN Rheumatol. 2013, http://dx.doi.org/10.1155/2013/341832.
6. Carlos, F.P., Silva, M., de Lemos, E., Costa, M.S., and Zamuner, S.R. (2014).
Protective effect of low-level laser therapy (LLLT) on acute zymosan-
induced arthritis. Lasers Med. Sci., 29(2), pp. 757–763.
7. Castano, A.P., Dai, T., Yaroslavsky, I., Cohen, R., Apruzzese, W.A., Smotrich,
M.H., and Hamblin, M.R. (2007). Low-level laser therapy for zymosan-
induced arthritis in rats: Importance of illumination time. Lasers Surg.
Med., 39(6), pp. 543–550.
8. da Rosa, A.S., dos Santos, A.F., da Silva, M.M., Facco, G.G., Perreira, D.M.,
Alves, A.C., Leal-Junior, E.C., de Carvalho, Pde. T. (2012). Effects of low-
level laser therapy at wavelengths of 660 and 808 nm in experimental
model of osteoarthritis. Photochem. Photobiol., 88(1), pp. 161–166.
9. de Morais, N.C., Barbosa, A.M., Vale, M.L., Villaverde, A.B., de Lima,
C.J., Cogo, J.C., and Zamuner, S.R. (2010). Anti-inflammatory effect of
low-level laser and light-emitting diode in zymosan-induced arthritis.
Photomed. Laser Surg., 28(2), pp. 227–232.
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316 Low-Level Laser Therapy for Arthritis in Animal Models

10. dos Santos, S.A., Alves, A.C., Leal-Junior, E.C., Albertini, R., Vieira, R.D.,
Ligeiro, A.P., Junior, J.A., and de Carvalho, P.D. (2013). Comparative
analysis of two low-level laser doses on the expression of inflammatory
mediators and on neutrophils and macrophages in acute joint inflam-
mation. Lasers Med. Sci., 29(3): 1051–1058.
11. Kang, H., Son, T., Lee, A., Youn, I., Seo, D.H., Kim, H.S., and Jung, B. (2014).
The effects of a minimally invasive laser needle system on complete
Freund’s adjuvant-induced arthritis. Lasers Med. Sci., 29(5), 1599–1606.
12. Lin, Y.S., Huang, M.H., Chai, C.Y., and Yang, R.C. (2004). Effects of helium-
neon laser on levels of stress protein and arthritic histopathology in
experimental osteoarthritis. Am. J. Phys. Med. Rehabil., 83(10), pp. 758–
765.
13. Lin, Y.S., Huang, M.H., and Chai, C.Y. (2006). Effects of helium-neon laser
on the mucopolysaccharide induction in experimental osteoarthritic
cartilage. Osteoarthritis Cartilage., 14(4), pp. 377–383.
14. Pallotta, R.C., Bjordal, J.M., Frigo, L., Leal-Junior, E.C., Teixeira, S.,
Marcos, R.L., Ramos, L., Messias, M., and Lopes-Martins, R.A. (2012).
Infrared (810-nm) low-level laser therapy on rat experimental knee
inflammation. Lasers Med. Sci., 27(1), pp. 71–78.
15. Sandoval, M.C., Mattiello-Rosa, S.M., Soares, E.G., and Parizotto, N.A.
(2009). Effects of laser on the synovial fluid in the inflammatory process
of the knee joint of the rabbit. Photomed. Laser Surg., 27(1), pp. 63–69.
16. Soriano, F., Campana, V., Moya, M., Gavotto, A., Simes, J., Soriano, M.,
Soriano, R., Spitale, L., and Palma, J. (2006). Photobiomodulation of pain
and inflammation in microcrystalline arthropathies: Experimental and
clinical results. Photomed Laser. Surg., 24(2), pp. 140–150.
17. Rubio, C.R., Cremonezzi, D., Moya, M., Soriano, F., Palma, J., and Campana,
V. (2010). Helium-neon laser reduces the inflammatory process of
arthritis. Photomed. Laser Surg., 28(1), pp. 125–129.
18. Zhang, L., Zhao, J., Kuboyama, N., and Abiko, Y. (2011). Low-level
laser irradiation treatment reduces CCL2 expression in rat rheumatoid
synovia via a chemokine signaling pathway. Lasers Med. Sci., 26(5), pp.
707–717.
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Chapter 18

Low-Level Laser Therapy for Lung

Diseases: From the Bench to the Bed

Flavio Aimbire
Science and Technology Department, Federal University of São Paulo,
Avenida Cesare Mansueto Giulio Lattes, 1201, Parque Tecnológico,
São José dos Campos, São Paulo, São Paulo 12247-014, Brazil
flavio.aimbire@unifesp.br

18.1 Introduction

Curiously, the research groups that were interested in investigating

the beneficial effects of low-level laser therapy (LLLT) on lung
diseases evaluated both the lung function and the reduction in
clinical symptom without knowing the mechanism of action of LLLT.
Of course, the main target of every treatment used for a disease
is improving the patient’s condition. However, the low-level laser
is an alternative therapy with anti-inflammatory properties which
are known up to the treatment moment. Therefore, there is a need
to characterize that the ideal dosimetry can induce and maintain
an anti-inflammatory response in both airway and lung. In this
context, the investigation of the action mechanism of LLLT was
necessary for understanding if laser light can act like conventional

Handbook of Low-Level Laser Therapy

Edited by Michael R. Hamblin, Marcelo Victor Pires de Sousa, and Tanupriya Agrawal
Copyright c 2017 Pan Stanford Publishing Pte. Ltd.
ISBN 978-981-4669-60-3 (Hardcover), 978-981-4669-61-0 (eBook)
www.panstanford.com
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318 Low-Level Laser Therapy for Lung Diseases

pharmacological therapy for treating lung diseases, and mainly what

immune and inflammatory cells as well as chemical mediators and
transcription factors LLLT can interact with. Another important
view that the investigation could clarify is the mistaken notion that
the same dose used to relieve pain in rheumatoid arthritis can be
used to treat asthma symptoms. Certainly, the wavelength chosen
for treating each disease will be in the “therapeutic window” of
the electromagnetic spectrum, but the wavelength is just a variable
among the physical parameters of lasers that can interfere in
the anti-inflammatory effect of LLLT. A well-established dosimetry
would facilitate the prescription of an appropriate dose that would
be effective in reducing lung inflammation. Furthermore, with a
range of doses already established, it would be possible to evaluate if
LLLT has some interferences with other medicines used for treating
inflammatory lung disorders.
The application of laser light as a non-pharmacological tool
for treating both airway and lung diseases was initiated by three
researchers in the Choithram Hospital and Research Centre in India.
This group has published the first manuscript showing LLLT in
pulmonary tuberculosis [7]. Although controversial, this study has
showed that 90% of patients treated with LLLT presented clinical
improvement. Although this is the first study to recommend LLLT
as an adjuvant to the traditional tuberculosis therapy, the authors
have purposed the LLLT as an invasive intervention, since a needle
carrying the laser was required to reach the patients’ lungs. From
this work, other authors began to investigate the possibility of the
beneficial effect of laser on lung diseases from other etiologies and
noninvasively.
Nowadays, the number of published studies proving the
beneficial effect of LLLT on lung diseases is growing, but it
is still fewer when compared with the use of laser in other
inflammatory diseases. LLLT presents an enormous possibility to
support anti-inflammatory therapy for the treatment of diseases
that compromise airway and lungs. However, some barriers hinder
the widespread use of laser therapy in medical clinic. Among them
is the lack of follow-up of laser-treated patients for a long period
and the absence of investigation of the laser effect on inflammatory
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Asthma 319

Table 18.1 Characteristics of LLLT irradiation on experimental study on

lung inflammation in asthma and chronic bronchitis

Experimental Wavelength Energy density Irradiation Power

Authors study (nm) (J/cm2 ) time (mW or W)

Aimbire et al. (2007) In vivo 650 nm 1.3 42 s 2.5 mW

Wang et al. (2013) In vivo 8
Silva et al. (2014) In vivo 660 nm 3 min 30 mW
Souza et al. (2014) In vitro 660 nm 60s 30 mW

markers responsible for defining lung disease. We also have to

consider the economic impact on public health since LLLT is low
cost, easily manipulated by the patient, and has no side effects.
In this chapter, we will cover the effect and action mechanism
of LLLT in lung diseases where laser has been successful. We will
discuss LLLT in allergic asthma, acute respiratory distress syndrome
(ARDS), chronic obstructive pulmonary disease (COPD), pneumonia,
and tuberculosis.

18.2 Asthma

18.2.1 Clinical Studies

Clinical studies on the effect of laser on asthma are all directed
toward improving the life quality of patients without worrying about
what is really happening between laser light and inflammatory cells
as well as their mediators. Some authors have shown that LLLT
irradiation increased pulmonary compliance and the efficiency of
gas exchange in patients with bronchial asthma [25]. Some other
authors have observed that LLLT can reduce cellular migration
in the lung tissue of patients with chronic lung inflammation
[34]. Although pharmacological therapy is more efficient because
it offers bronchodilator agents, corticoids, and antibiotics, these
authors indicate that LLLT can be effective in conditions of hypoxia,
hipercapnia, and gas exchange dysfunction, which is very exciting
because even though low-level laser is not a pharmacological
therapy, it can attenuate both inflammation and lung mechanical
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320 Low-Level Laser Therapy for Lung Diseases

Table 18.2 Characteristics of LLLT irradiation on experimental study on

lung inflammation in ARDS and ALI

Experimental Wavelength Energy density Irradiation Power

Authors study (nm) (J/cm2 ) time (mW or W)

Aimbire et al. In vivo 685 2.5 and 5 1 min 20 s 12 mW

(2005)
Aimbire et al. In vivo 660 7.5 90 min 30 mW
(2008)
Mafra de In vivo/in vitro 650 1.3 42 s 2.5 mW
Lima et al.
(2010)
de Lima et al. In vivo 685 4.5 252 s 35 mW
(2011)
de Lima et al. In vivo 660 180 s 30 mW
(2011)
Aimbire et al. In vitro 660 7.5 30 mW
(2008)

alterations. The dynamics of gas exchange between capillaries and

blood is an important characteristic of the pulmonary function test
of asthma. Thereby, Polosukhin [35] studied the effect of laser on
gas exchange dynamics between pulmonary capillaries and blood
in asthmatic patients. The author proved that LLLT increased the
metabolic and proliferative activity of endothelial cells as well as the
basal membrane of bronchial mucosa. In addition, LLLT restructured
the bronchial fibrous tissue, permitting the formation of a less dense
bronchial tissue. Still, in endobronchial laser therapy, Prozorova
et al. [36] investigated the efficacy of LLLT in individuals with
chronic bronchitis comparing the efficiency of laser with respect
to anti-oxidants drugs. These authors revealed that LLLT stabilized
the coagulation cascade and increased both humoral and immune
response. In addition, LLLT significantly reduced the concentration
of pro-oxidant compounds and lipidic peroxidation during lung
inflammatory process. On this account, laser therapy has gained sup-
port as a non-pharmacological therapy capable of downregulating
oxidative metabolism in bronchial cellular membranes. It attenuates
pro-oxidant reactions that compromise bronchial reactivity and
structure.
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Asthma 321

Table 18.3 Anti-inflammatory effect of LLLT on experimental study on lung

inflammation in asthma and chronic bronchitis

Authors Effect of LLLT

Aimbire et al. (2007) Attenuation of hemorrhagic lesion induced by immune complex in rat
lungs.
Wang et al. (2013) Adjustment of Th1/Th2 imbalance with anti-inflammatory effect similar
to budenoside.
Silva et al. (2014) Inhibition of bronchoconstriction, Th2 inflammation, and airway
remodeling.
Souza et al. (2014) Suppression of the oxidative stress-induced glucocorticoids resistance
in alveolar macrophages by downregulating cytokine secretion and
histone deacetylase activity.

Within the same context, Rakitina et al. [37] also showed that
LLLT potentiates the antioxidant effect of pharmacological drugs,
decreasing the concentration of free radicals in the blood plasma
of asthmatic individuals. In the early 2000s, Landyshev et al. [20]
reported for the first time that laser light could be irradiated on
skin in the areas of the lungs and great vessels, endobronchially,
in 220 asthmatic patients. The results showed that LLLT reduced
the cytological markers of cell reactions of the bronchopulmonary
system. Moreover, LLLT was cost-effective and safe as well as
had good reproducibility and availability. At this point, the notion
that the beneficial non-pharmacological effects of LLLT could
be used noninvasively for treating asthmatic patients was being
strengthened. Some authors evaluated 50 asthmatic patients who
received laser therapy noninvasively for 10 days [26]. These authors
found that patients with bronchial asthma presented significant
improvement in all estimated lung function parameters just 30 min
after the laser stimulation. Improvements achieved on the third and
the tenth days of treatment were significantly higher in laser-treated
patients. Further investigations confirmed that the improvement in
the measured lung function parameters was significantly higher in
younger patients, patients in whom disease lasted shorter, as well
as women. The authors evidenced that patients with asthma, who
were treated every 3 months for a year, presented significantly lower
frequency and intensity of attacks.
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322 Low-Level Laser Therapy for Lung Diseases

Still in the context of LLLT for the treatment of asthmatic patients,

diverse authors have found similar beneficial effects but with
different methods of irradiation. Among these methods was nonin-
vasive hemolasertherapy in which patients with bronchial asthma
and concomitant rhinosinusitis were irradiated in two different
ways: in tympanic membrane and paranasal sinuses, or exposed
to supravenous laser irradiation [28]. Nikitin and Treshchalina
showed that LLLT associated to conventional medication resulted in
positive changes in clinical and spirometric data [28]. Other authors
used LLLT to treat 228 patients with bronchial asthma [32]. These
authors revealed that patients who were noninvasively irradiated
in the chest area presented a significant reduction in histamine
level in peripheral blood and had an excellent improvement in the
respiratory function. The same authors showed that 466 patients
with bronchial asthma, who were noninvasively irradiated on the
bronchial region, had significant clinical improvement—restoration
of bronchial sensitivity to sympathomimetics and xantine deriva-
tives as well as reduction in glucocorticoid dosage and the duration
of hospital stay and disability [33]. In the same year, Nikitin and
Titova [29] showed that laser infrared radiation of the adrenal
projection region reduced bronchoconstriction attacks, improved
bronchial drainage, and shortened disease duration. Other groups
revealed that intravascular laser irradiation in 17 patients aged 20–
60 years reduced the disease symptoms [17]. Similarly, Sarycheva
et al. [40] showed that intravenous blood laser irradiation in
asthmatic patients improved the morphofuncional parameters of
erythrocytes by restoring their normal forms and increasing their
electrophoretic mobility to normal values. Some authors prefer
to use the acupuncture technique associated to laser therapy in
treating individuals with asthma [16, 18, 30, 38]. For a different
treatment period, varying from three to ten irradiation sessions,
these authors recommend this method (laser plus acupuncture) as a
safe and effective treatment with improvement in forced expiratory
volume, vital capacity, and peak expiratory flow. Moreover, these
studies showed that after LLLT, the corticoid dose was reduced and
the patients stopped with bronchodilators (β2 -agonists) of short
action.
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Asthma 323

Table 18.4 Anti-inflammatory effect of LLLT on experimental study on

lung inflammation in ARDS and ALI

Authors Effect of LLLT

Aimbire et al. (2005) Anti-inflammatory effect on airway hyper-reactivity and
neutrophils influx with the inhibition of COX-2-derived
metabolites (PGE2 and TXA2 ) in BALF.
Aimbire et al. (2008) Reduction of levels of lung neutrophils anti-apoptotic factors by
an NF-κB dependent mechanism.
Aimbire et al. (2008) Reduction of pulmonary microvascular leakage, neutrophil
influx, IL-1β level in the air way and lung after LPS.
Mafra de Lima et al. (2009) Attenuation of cholinergic hyper-reactivity, β2 -adrenergic
hyporesponsiveness and TNF-α mRNA expression in bronchi by
an NF-κB dependent mechanism.
Mafra de Lima et al. (2009) Reduction of lung inflammation and restoration of pulmonary
endothelial cytoskeleton.
de Lima et al. (2010) Associated with N-acetylcysteine lowers MIP-2 mRNA
expression and generation of ROS in alveolar macrophages.
Mafra de Lima et al. (2010) Inhibition of neutrophils recruitment and
chemokines/cytokines levels after LPS.
de Lima et al. (2011) LLLT acts as cAMP-elevating agent to attenuate neutrophil influx
and TNF mRNA expression.
de Lima et al. (2011) Attenuation of intestinal-I/R-induced acute lung inflammation
which favors the IL-10 production and reduces TNF.
de Lima et al. (2013) Suppression of airway reactivity dysfunction and lung
inflammation induced by intestinal-I/R by inhibiting TNF and
iNOS and increasing the IL-10.
de Lima et al. (2013) Attenuation of lung inflammation is driven to increase both the
PPARγ and the HSP70 in lung.

18.2.2 Experimental Studies

In this section, we will discuss the studies focused on understanding
the cellular signaling responsible for the beneficial effects of LLLT
in experimental models of lung inflammation that mimics the
clinical symptoms of allergic asthma. Some studies using the animal
model of asthma describe the action mechanism of laser therapy.
Nonetheless, these studies evaluate important parameters that
define asthma severity such as bronchoconstriction (airway hyper-
reactivity), shift in inflammatory response to Th2 cells (production
of pro-inflammatory cytokines), and airway remodeling (presence
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324 Low-Level Laser Therapy for Lung Diseases

of mucus and collagen in lung tissue). Aimbire et al. [2] conducted

the first experimental study that investigated the effect of LLLT
on the relaxation capacity of airway smooth muscle in an in
vitro model of asthma induced by tumor necrosis factor (TNF). In
fact, many studies show that patients with allergic asthma have
less capacity of relaxing smooth muscle, i.e., these patients are
less responsive to bronchodilators (adrenergic β2 -agonists). These
authors reported beneficial effects of laser therapy (655 nm and 2.6
J/cm2 ) administered perpendicularly to a point in the middle of the
dissected trachea with a partially restored trachea smooth muscle
relaxation response to isoproterenol. In addition, the accumulation
of cAMP (cyclic adenosine monophosphate), a master mediator of
signal transduction for relaxation in airway smooth muscle, was
almost normalized after LLLT. In the asthma model in rats subjected
to the challenge of repeated exposure to ovalbumin, Wang et al.
[46] revealed that laser therapy at 8 J/cm2 once daily for 21 days
decreased the number of eosinophils as well as the levels of IL-4
in bronchoalveolar lavage fluid (BALF) and serum IgE. In addition,
laser therapy increased the interferon-γ (IFN-γ ) levels, showing
that LLLT regulates the Th1/Th2 imbalance of asthmatic rats similar
to budesonide corticoid, which is in accordance with the clinical
studies in which the use of LLLT allows the dose reduction of
glucocorticoids and consequently attenuates some adverse effects of
conventional pharmacological therapy. Some authors have evaluated
the LLLT effect from the point of view of bronchoconstriction
characterized by bronchial hyper-reactivity. We have demonstrated
that LLLT reduces the bronchoconstriction to acetylcholine in mice
challenged with ovalbumin by reducing the RhoA expression in
bronchial muscle [41]. The animals were irradiated on the skin over
the right upper bronchus at four consecutive times—5 min, 1 h,
6 h, and 12 h—after the antigen challenge. Moreover, eosinophil
infiltration and the concentration of interleukins (IL-4, IL-5, IL-
13) and eotaxin were profoundly reduced by LLLT. These authors
have showed that laser therapy attenuated airway remodeling by
reducing both the mucus and the collagen of lungs in mice subjected
to antigenic challenge.
These results demonstrate that LLLT reduced the bronchocon-
striction via RhoA and lessened allergic inflammation via STAT6. Still
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Asthma 325

Table 18.5 Anti-inflammatory effect of LLLT on lung

inflammation in some diseases

Disease Effect of LLLT

COPD There is no report about the LLLT effect on COPD.

Pneumonia There is no report about the LLLT effect on pneumonia.
Tuberculosis There is no report about the LLLT effect on tuberculosis.

in the context of experimental asthma, Souza et al. [42] revealed

that LLLT suppressed the oxidative stress-induced glucocorticoids
resistance in macrophages by decreasing TNF and IL-8 and histone
deacetylase (HDAC) through the activation of protein kinase A (PKA)
via the inhibition of phosphoinositide 3-kinase (PI3K). These results
“open a window” of opportunity in the treatment of patients with
severe asthma in which oxidative stress contributes to the low
response to corticoids through the downregulation of HDAC and
the increase in cytokines. For irradiation, the cover plate of the
culture medium was removed and macrophages were irradiated
once, 1 h after the addition of lipopolysaccharide (LPS). Some
authors demonstrated the effect of LLLT in an experimental model
of immune-complex reaction in which a hemorrhagic lesion is
observed in the lungs of rats subjected to intra-tracheal instillation
of bovine serum albumin [3].
Based on the effects of LLLT on asthmatic individuals as well
as in experimental model, we illustrated in Fig. 18.1a a possible
action mechanism of LLLT in controlling severe asthma by restoring
the capacity of alveolar macrophages to respond to glucocorticoid
treatment in a model of oxidative stress in which HDAC activity is
lesser and pro-inflammatory cytokine is higher. Also in Fig. 18.1b, we
diagramed a putative action mechanism of LLLT on immunological
response in a model of allergic lung inflammation. LLLT presents
a dual effect in this condition by decreasing the Th2 cytokines
(IL-4, IL-5, IL-13) concentration as well as the eotaxin (eosinophil
attractant chemokine) levels and by increasing IFN-γ and IL-
10 (anti-inflammatory cytokines). Besides, LLLT can control the
expression of both mucus and collagen in the lungs of asthmatic
mice. The effect of LLLT on IL-13 is suggested as one of the possible
action mechanisms to relieve bronchoconstriction.
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326 Low-Level Laser Therapy for Lung Diseases

(a) Severe Asthma

Oxidative Stress

α-phosphoinositide-3-kinase (PI3K)

LASER LASER

cytokines secretion HDAC activity

Protein kinase A

cAMP

LASER

LASER
(b) IgG4 mast
cell LASER
Bronchial hyperreactivity,
airway remodelling
Blocking of IgE LASER tissue migration of inflammatory
Cross-Linking Th1 IFN-g cells, IgE production
By IgG4
B cell Eosinophil LASER
SLIT SCIT IL-5 IL-4
IL-10 LASER IL-13
Th2 B cell mast
Tr1 DC subsests cell
IL-9
at the place of IL-10 IL-13
IL-10 administration TGF-β
Mucus production
Treg

IL-10 secreting Tr1 cells or

Adaptative/induced Treg cells LASER

Figure 18.1 Possible action mechanism of LLLT in oxidative stress and

imbalance in Th1/Th2 response in allergic asthma.
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Acute Respiratory Distress Syndrome 327

Figure 18.2 LLLT effect on bronchial hyper-reactivity in asthma as well as

in acute lung inflammation.

It is clear that the effect of LLLT on lung inflammation supports

its effect on bronchial hyper-reactivity in pathological conditions
such as asthma and acute lung inflammation (ALI). However, LLLT
acts directly on the contractile machinery of bronchial smooth
muscle by downregulating both IP3 and RhoA (Fig. 18.2). Tables 18.1
and 18.3 show some doses and effects of LLLT, respectively, on
asthma and chronic bronchitis.

18.3 Acute Respiratory Distress Syndrome

18.3.1 Clinical Studies

Opposite to allergic asthma, the use of LLLT in the treatment of ALI
arising out of ARDS is limited because patients with this pathological
condition have severe lung inflammation in a short time, leading
to a high mortality rate and making long-term treatment difficult.
In most cases, patients with ALI are hospitalized in the intensive
care unit because they need specific drugs that can control the
exacerbation of pulmonary inflammatory response. Of course, LLLT
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328 Low-Level Laser Therapy for Lung Diseases

would be a supporting therapy in these cases, but it would probably

not work because a rapid response to the treatment is required.
Clinical trials that investigate the direct effect of LLLT on ARDS are
not available. Therefore, herein we will present only a clinical study
demonstrating the effect of LLLT on ALI induced by pleurisy. Pleurisy
is an inflammation of the lung pleura (parietal and visceral) with
the augmentation of pleural fluid. It usually occurs when a virus or
bacterium irritates the pleura (double serous membrane that sur-
rounds the lungs), causing inflammation. Some authors conducted
a study on 25 patients with pleurisy and irradiated them with low-
level laser [27]. The results indicated that the laser-treated patients
presented a faster resorption of effusion and remission of subjective
symptoms. Moreover, LLLT increased regenerative mechanisms of
the pleural surface, resulting in better mobility of the diaphragm.

18.3.2 Experimental Studies

Unlike the unique clinical study evaluating the effect of LLLT on
pleurisy in humans, many in vivo and in vitro studies show that
LLLT can control the features of ALI induced by different stimuli.
In this section, we will cover the effect and action mechanism of
LLLT on diverse experimental models of ALI. Curiously, the first
report showing the effect of laser on an acute pathological condition
in the airway and lungs is related to the ability of laser therapy
in inducing apoptosis in human lung adenocarcinoma via the
activation of caspases-3 [45]. Before 2005, no research group was
interested in investigating the anti-inflammatory action mechanism
of LLLT on ALI using experimental models. We demonstrated for
the first time that LLLT reduced the tracheal hyper-reactivity and
the neutrophil influx into BALF and lung tissue in association with
the inhibition of cyclooxygenase-2 (COX-2-derived metabolites) in
rats that were systemically inflamed with LPS from Escherichia coli
[1]. In another study, rats were subjected to LPS administration, and
the authors investigated lung edema, myeloperoxidase activity, and
both the IL-1β mRNA expression and protein concentration. The
findings indicate that LLLT reduced lung permeability (edema) by
a mechanism in which IL-1β seems to have an important role [4]. In
an experimental model in which bronchial smooth muscle segments
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Acute Respiratory Distress Syndrome 329

(BSMS) were bathed with TNF, LLLT reduced the bronchial hyper-
reactivity and the accumulation of inositol triphosphate (IP3) into
BSMS [5]. Moreover, LLLT attenuated cholinergic hyper-reactivity,
β2-adrenergic hyporesponsiveness, and TNF mRNA expression in
rat bronchi segments in E. coli LPS-induced ALI by an NF-κB-
dependent mechanism [23]. The same authors reported that lung
inflammation and endothelial cell damage are decreased after the
laser treatment in a model of ALI induced by E. coli LPS in rats
[24]. Using the same experimental model to induce ALI in rats,
some authors have demonstrated that LLLT reduces the features
of LPS-induced ALI by the inhibition of intercellular adhesion
molecule-1 (ICAM-1) via the downregulation of TNF and IL-1β
[24]. The same authors have found a synergistic action of LLLT
with NAC on MIP-2 mRNA expression from LPS-or H2 O2 -stimulated
AM, and both ROS intracellular generation and NF-κB signaling
seem to be involved [11]. Some authors showed that laser therapy
attenuated the neutrophil influx and TNF in BALF; they also
showed that LLLT increased the cAMP concentration and reduced
the TNF mRNA expression in alveolar macrophages [12]. These
results proved that LLLT indirectly increased cAMP in alveolar
macrophages by a TNF-dependent mechanism. Finally, some authors
have investigated the pro-and anti-inflammatory effects on ALI
induced by intestinal ischemia and reperfusion (i-I/R) in rats. These
authors demonstrated that LLLT attenuated lung inflammation,
which favored IL-10 anti-inflammatory protein production and
reduced TNF pro-inflammatory generation [13]. In the same context
of i-I/R-induced ALI, de Lima et al. [14] reported that LLLT
relieved both the bronchial hyper-reactivity and lung edema by
decreasing ICAM-1, TNF, and inducible nitric oxide synthase (iNOS)
and increasing IL-10 in homogenate lungs. Still in i-I/R-induced
ALI, de Lima et al. [15] showed that LLLT, in order to attenuate
ALI, restored the balance between pro-and antioxidant mediators
rising the peroxisome-activated receptor-γ (PPARγ ) expression and
consequently the heat shock protein (HSP70) production.
In the end, we postulated in Fig. 18.3a,b that independent of
ARDS arising from sepsis or ischemic trauma, the anti-inflammatory
action mechanism of LLLT on ALI is driven to restore the oxidative
stress balance by downregulating ROS secretion via the inhibition
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330 Low-Level Laser Therapy for Lung Diseases

(a)

(b)

Figure 18.3 Possible action mechanism of LLLT on acute lung inflammation

observed in ARDS as well as in mesenteric trauma.

of NF-κB and inversely by upregulating glutathione (antioxidant

enzyme). The effect of LLLT on neutrophilic migration into lungs is
due to both the reduction in pro-inflammatory cytokines and the
increase in IL-10 anti-inflammatory protein. Tables 18.2 and 18.4
show some doses and effects of LLLT, respectively, on ARDS and ALI.
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Chronic Obstructive Pulmonary Disease 331

(a)

(b)

Figure 18.4 Scheme illustrating the putative action mechanism for LLLT in
the treatment of COPD.

18.4 Chronic Obstructive Pulmonary Disease

18.4.1 Clinical Studies

Some studies have evaluated the effect of LLLT on COPD in humans;
however, the laser effect is still controversial. The first study
evaluated the effects of irradiation of blood on the hemodynamic
system in patients with chronic obstructive bronchitis (COB). Fifty
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332 Low-Level Laser Therapy for Lung Diseases

patients with COB exacerbation received LLLT for 20 min for

10 days, and it had a positive effect on the systemic circulation
by changing hyperkinetic hemodynamic into normokinetic one
[8]. Thereby, these authors indicated that it was appropriate to
include laser therapy in COB treatment. While studying blood
cells of patients with COPD, Farkhutdinov and Farkhutdinov [17]
demonstrated that LLLT had a little effect on the chemiluminescence
of blood in patients with low generation of ROS and decreased
functional activity of cells. On the contrary, Kashanskaia and Fedorov
[19] reported that COB patients treated with LLLT presented
reduction in symptoms, increase in the drainage function of bronchi,
normalization of the patient’s immune status, and reduction in lipid
peroxidation processes.

18.4.2 Experimental Studies

No studies on the effect of LLLT on COPD in vivo or in vitro
are available. There are also no published reports on the action
mechanism of LLLT on pulmonar emphysema; however, our group
found that LLLT reduces both the alveolar macrophage activation
and the neutrophilic migration into lungs in mice subjected to
cigarette smoke. LLLT reverted the increase in CD8+ lymphocytes,
KC chemokine, and IL-1β, TNF, IL-6 pro-inflammatory cytokines
through the reduction in NF-κB activation. One of the interesting
effects of LLLT is the increase in both IL-10 anti-inflammatory
cytokine and glutathione antioxidant enzyme level when all phar-
macological and immunological treatments disposable in clinical
practice reduce all chemical mediators that are, in part, responsible
for their adverse effects. Figure 18.4 illustrates a possible action
mechanism of LLLT on COPD.

18.5 Pneumonia

18.5.1 Clinical Studies

A Russian research group [21] conducted the first study describing
the effect of LLLT on pneumonia. The group indicated that LLLT
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Tuberculosis 333

can be used in the routine treatment of patients with pneumonia,

since it improves both the clinical status and the respiratory
function, besides restoring pulmonary blood flow and attenuating
immunological response. Some researchers conducted a clinical
trial with 142 pneumonia patients and found that LLLT controlled
lung edema and improved respiratory function [6]. However, these
authors suggested that LLLT had a better effect in association
with pharmacological drugs. Other groups evaluated 32 pneumonia
patients and demonstrated similar results, but in this study, the
patients received intravenous irradiation [9]. The same authors
investigated 100 pneumonia patients who showed significant
reduction in hemocoagulative disorders after LLLT [10].
There are no experimental studies on the effect of LLLT on
pneumonia in vivo or in vitro. Table 18.5 shows some lung diseases
in which the anti-inflammatory effect of LLLT needs to be more
explored.

18.6 Tuberculosis

18.6.1 Clinical Studies

The application of LLLT in the treatment of tuberculosis is
controversial. Initially, some authors indicated that LLLT applied
directly to lungs through a needle carrying the laser could produce
clinical improvement in 90% of patients [7]. Some authors reported
that LLLT applied in supravenous blood attenuated lung alterations
in 20 patients with tuberculosis [39]. Likewise, Nikolaeva [31]
also reported that tuberculosis patients who were irradiated with
low-level laser in an acupuncture point presented a faster lung
infiltration resorption and resolution of disease symptoms. On the
other hand, some authors have not identified any well-designed
trials using LLLT to treat tuberculosis [43, 44].
There are no experimental studies on the effect of LLLT on
tuberculosis in vivo or in vitro.
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334 Low-Level Laser Therapy for Lung Diseases

References

1. Aimbire, F., Albertine, R., de Magalhães, R.G., Lopes-Martins, R.A., Castro-

Faria-Neto, H.C., Zângaro, R.A., Chavantes, M.C., and Pacheco, M.T.
(2005). Effect of LLLT Ga-Al-As (685 nm) on LPS-induced inflammation
of the airway and lung in the rat. Lasers Med. Sci., 20(1), pp. 11–20.
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Martins, R.A. (2006). Low-level laser therapy partially restores trachea
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Faria-Neto, H.C., Martins, P.S., and Bjordal, J.M. (2007). Effect of low-level
laser therapy on hemorrhagic lesions induced by immune complex in rat
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de Campos, C.B., Lyon, J.P., Silva, J.A. Jr, and Costa, M.S. (2008). Low-
level laser therapy (LLLT) decreases pulmonary microvascular leakage,
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10. Burduli, N.M., and Pilieva, N.G. (2010). Changes in plasma hemostatic
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11. de Lima, F.M., Villaverde, A.B., Albertini, R., de Oliveira, A.P., Faria
Neto, H.C., and Aimbire, F. (2010). Low-level laser therapy associated
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771.
12. de Lima, F.M., Moreira, L.M., Villaverde, A.B., Albertini, R., Castro-Faria-
Neto, H.C., and Aimbire, F. (2011). Low-level laser therapy (LLLT) acts
as cAMP-elevating agent in acute respiratory distress syndrome. Lasers
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13. de Lima, F.M., Villaverde, A.B., Albertini, R., Corrêa, J.C., Carvalho, R.L.,
Munin, E., Araújo, T., Silva, J.A., and Aimbire, F. (2011). Dual effect
of low-level laser therapy (LLLT) on the acute lung inflammation
induced by intestinal ischemia and reperfusion: Action on anti-and pro-
inflammatory cytokines. Lasers Surg. Med., 43(5), pp. 410–420.
14. de Lima, F.M., Vitoretti, L., Coelho, F., Albertini, R., Breithaupt-Faloppa,
A.C., de Lima, W.T., and Aimbire, F. (2013). Suppressive effect of low-level
laser therapy on tracheal hyperresponsiveness and lung inflammation
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L., Castro-Faria-Neto, H.C., França, C., Villaverde, A.B., and Aimbire, F.
(2013). Low-level laser therapy restores the oxidative stress balance in
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16. Elseify, M.Y., Mohammed, N.H., Alsharkawy, A.A., and Elseoudy, M.E.
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17. Farkhutdinov, U.R., and Farkhutdinov, ShU. (2007). Effect of laser
radiation on production of reactive oxygen species in the blood of
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18. Farkhutdinov, U.R. (2007). Intravascular laser irradiation of blood in the
treatment of patients with bronchial asthma. Ter. Arkh., 79(3), pp. 44–
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336 Low-Level Laser Therapy for Lung Diseases

19. Kashanskaia, E.P., and Fedorov, A.A. (2009). Low-intensity laser radi-
ation in the combined treatment of patients with chronic obstructive
bronchitis. Vopr. Kurortol. Fizioter. Lech. Fiz. Kult., (2), pp. 19–22.
20. Landyshev, IuS., Avdeeva, N.V., Goborov, N.D., Krasavina, N.P., Tikhonova,
G.A., and Tkacheva, S.I. (2002). Efficacy of low intensity laser irradiation
and sodium nedocromil in the complex treatment of patients with
bronchial asthma. Ter. Arkh., 74(3), pp. 25–28.
21. Lutai, A.V., Egorova, L.A., and Shutemova, E.A. (2001). Laser therapy of
elderly patients with pneumonia. Vopr Kurortol Fizioter Lech Fiz Kult.,
(3), pp. 15–18.
22. Mafra de Lima, F., Costa, M.S., Albertini, R., Silva, J.A. Jr., and Aimbire,
F. (2009). Low level laser therapy (LLLT): Attenuation of cholinergic
hyperreactivity, beta(2)-adrenergic hyporresponsiveness and TNF-
α mRNA expression in rat bronchi segments in E. coli
lipopolysaccharide-induced airway inflammation by a NF-κB dependent
mechanism. Lasers Surg. Med., 41(1), pp. 68–74.
23. Mafra de Lima, F., Naves, K.T., Machado, A.H., Albertini, R., Villaverde,
A.B., and Aimbire, F. (2009). Lung inflammation and endothelial cell
damage are decreased after treatment with phototherapy (PhT) in a
model of acute lung injury induced by Escherichia coli lipopolysaccha-
ride in the rat. Cell. Biol. Int., 33(12), pp. 1212–1221.
24. Mafra de Lima, F., Villaverde, A.B., Salgado, M.A., Castro-Faria-Neto,
H.C., Munin, E., Albertini, R., and Aimbire, F. (2010). Low intensity
laser therapy (LILT) in vivo acts on the neutrophils recruitment and
chemokines/cytokines levels in a model of acute pulmonary inflamma-
tion induced by aerosol of lipopolysaccharide from Escherichia coli in
rat. J. Photochem. Photobiol. B., 101(3), pp. 271–278.
25. Mikhailov, V.A., Aleksandrova, OIu., and Gol’dina, E.M. (1998). The im-
munomodulating action of low-energy laser radiation in the treatment
of bronchial asthma. Vopr. Kurortol. Fizioter. Lech. Fiz. Kult., (4), pp. 23–
25.
26. Milojević, M., and Kuruc, V. (2003). Low power laser biostimulation in
the treatment of bronchial asthma. Med. Pregl., 56(9–10), pp. 413–418.
27. Milojević, M., and Kuruc, V. (2003). Laser biostimulation in the
treatment of pleurisy. Med. Pregl., 56(11–12), pp. 516–520.
28. Nikitin, A.V., and Treshchalina, IuB. (2004). Efficacy of noninvasive
hemolaserotherapy in patients with bronchial asthma and concomitant
rhinosinusitis. Ter. Arkh., 76(3), pp. 20–23.
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References 337

29. Nikitin, A.V., and Titova, L.A. (2006). Clinical efficacy of target low-
intensity laser radiation on the adrenal projection region in patients
with bronchial asthma. Ter. Arkh., 78(3), pp. 39–40.
30. Nikitin, A.V., Esaulenko, I.E., and Shatalova, O.L. (2008). Effectiveness of
laser puncture in elderly patients with bronchial asthma. Vopr. Kurortol.
Fizioter. Lech. Fiz. Kult., (6), pp. 38–39.
31. Nikolaeva, O.D. (2006). Use of electropuncture diagnostics and lasero-
puncture in patient with pulmonary tuberculosis. Lik. Sprava., (1–2), pp.
31–34.
32. Ostronosova, N.S. (2006). Low-intensity laser radiation in therapy of
bronchial asthma. Vopr. Kurortol. Fizioter. Lech. Fiz. Kult., (2), pp. 8–10.
33. Ostronosova, N.S. (2006). Outpatient use of laser therapy in bronchial
asthma. Ter. Arkh., 78(3), pp. 41–44.
34. Pidaev, A.V. (1997). A mathematical assessment of the efficacy of the
methods for treating patients with chronic nonspecific lung diseases at
a health resort. Lik. Sprava., (6), pp. 168–172.
35. Polosukhin, V.V. (1997). Dynamics of the ultrastructural changes
in blood and lymphatic capillaries of bronchi in inflammation and
following endobronchial laser therapy. Virchows. Arch., 431(4), pp. 283–
290.
36. Prozorova, G.G., Sil’vestrov, V.P., Simvolokov, S.I., and Nikitin, A.V. (1997).
The efficacy of membrane-stabilizing therapy in patients with chronic
obstructive bronchitis. Ter. Arkh., 69(10), pp. 34–36.
37. Rakitina, D.R., Urias’ev, O.M., Garmash, VIa., Ivanova, M.V., Krasnovid,
N.I., and Lebedev, A.V. (1997). Effects of laser therapy on lipids and
antioxidants in blood of patients with bronchial asthma. Ter. Arkh.,
69(12), pp. 49–50.
38. Razumov, A.N., Maliavin, A.G., Ermolaeva, O.A., and Ianushevich, E.A.
(2007). Intra-auricular laser therapy of children suffering
from bronchial asthma. Vopr. Kurortol. Fizioter. Lech. Fiz. Kult., (3),
pp. 5–8.
39. Rusakova, L.I., Dobkin, V.G., and Ovsiankina, E.S. (2002). Efficiency
of supra-venous blood laser radiation used in the treatment of
disseminated pulmonary tuberculosis in adolescents. Probl. Tuberk., (8),
pp. 16–18.
40. Sarycheva, T.G., Tsybzhitova, E.B., Popova, O.V., and Aleksandrov, O.V.
(2009). Morphometry and electrophoretic mobility of red blood cells
from patients with asthma in the intravenous blood laser irradiation.
Klin. Lab. Diagn., (3), pp. 13–14.
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338 Low-Level Laser Therapy for Lung Diseases

41. Silva, V.R., Marcondes, P., Silva, M., Villaverde, A.B., Castro-Faria-Neto,
H.C., Vieira, R.P., Aimbire, F., and de Oliveira, A.P. (2014). Low-level laser
therapy inhibits bronchoconstriction, Th2 inflammation and airway
remodeling in allergic asthma. Respir. Physiol. Neurobiol., 194, pp. 37–
48.
42. Souza, N.H., Marcondes, P.T., Albertini, R., Mesquita-Ferrari, R.A., Fernan-
des, K.P., and Aimbire, F. (2014). Low-level laser therapy suppresses
the oxidative stress-induced glucocorticoids resistance in U937 cells:
Relevance to cytokine secretion and histone deacetylase in alveolar
macrophages. J. Photochem. Photobiol. B., 130, pp. 327–336.
43. Vlassov, V.V., Pechatnikov, L.M., and MacLehose, H.G. (2002). Low-level
laser therapy for treating tuberculosis. Cochrane Database Syst. Rev., (3),
CD003490.
44. Vlassov, V.V., and MacLehose, H.G. (2006). Low-level laser therapy for
treating tuberculosis. Cochrane Database Syst. Rev., (2), CD003490.
45. Wang, F., Chen, T.S., Xing, D., Wang, J.J., and Wu, Y.X. (2005). Measuring
dynamics of caspase-3 activity in living cells using FRET technique
during apoptosis induced by high fluence low-power laser irradiation.
Lasers Surg. Med., 36(1), pp. 2–7.
46. Wang, X.Y., Ma, W.J., Liu, C.S., and Li, Y.X. (2013). Effect of low-level laser
therapy on allergic asthma in rats. Lasers Med. Sci., 2013.
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Chapter 19

Low-Level Laser (Light) Therapy in

Tendon Healing in in Vitro and in Vivo
Models

Lucas F. de Freitasa and Michael R. Hamblinb,c,d

a University of São Paulo, 400 Trabalhador São-Carlense Avenue, São Carlos,

São Paulo 13566-590, Brazil

b Wellman Center for Photomedicine, Massachusetts General Hospital, 50 Blossom

Street, Boston, MA 02114, USA

c Department of Dermatology, Harvard Medical School, Massachusetts General

Hospital, 55 Fruit Street, Boston, MA 02114, USA

d Harvard-Massachusetts Institute of Technology (MIT) Division of Health Sciences and

Technology, 77 Massachusetts Avenue, E25–518, Cambridge, MA 02139, USA

luk freitas@yahoo.com.br

This chapter presents a literature review of the most recent

studies on the application of low-level laser (or light) therapy
(LLLT) in tendon healing. The results published so far point to the
establishment of efficient non-invasive therapeutic approaches to
decrease the time taken by an injured tendon to go back to normality
and regain its full biomechanical properties. The outcomes of
these approaches depend on the laser parameters as well as the
application protocol parameters, which are not always same among
the several authors of the field.

Handbook of Low-Level Laser Therapy

Edited by Michael R. Hamblin, Marcelo Victor Pires de Sousa, and Tanupriya Agrawal
Copyright c 2017 Pan Stanford Publishing Pte. Ltd.
ISBN 978-981-4669-60-3 (Hardcover), 978-981-4669-61-0 (eBook)
www.panstanford.com
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340 Low-Level Laser (Light) Therapy in Tendon Healing in in Vitro and in Vivo Models

19.1 Introduction

Tendons are specialized structures destined to withstand strength

loads, transmitting them from muscles to bone insertions. Their
biomechanical properties can be measured by a parameter called
ultimate tensile strength (UTS), which demonstrates the ability of
tendons to tolerate tensile load and elongation [1]. The staining of
tendons with picrosirius, followed by polarized light microscopic
analysis, allows one to quantify types I and III collagen, while
birefringence measurements are indicated to assess the aggregation
and orientation of collagen fibers [2].
Collagen, the most abundant component of the extracellular
matrix (Fig. 19.1), represents more than 70% of the tendon wet
weight [3], with type I being the collagen primarily incorporated in
the tendon structure. Alongside collagen fibers, proteoglicans play
an important role in tendon composition and tensile strength [4].
Usually, tendons are flexible and extensible, but they are
stronger than muscles, with a tensile strength similar to the
bone (units/area). Even so, repetitive and prolonged low-intensity
activities, as well as an intense activity, lead to a force generation
by the muscles that can cause tendon degeneration and rupture [5].
This condition can take weeks or even months to heal completely
and often require a repair followed by an early immobilization
(Fig. 19.2). During the injury treatment, it is necessary to establish a
permanent repair that allows the withstanding of significant tensile-
strength loads [6, 7].
The tendon gets repaired in three, often overlapping, phases:
inflammatory, proliferative, and remodeling (Fig. 19.3). In general,
the inflammatory phase extends from day 1 to day 7, and it is
characterized by an extrinsic regeneration with the proliferation
of blood capillaries and the migration of elongated fibroblasts
called tenocytes. They are responsible for a significant deposition
of extracellular matrix [8]. This migration is stimulated by TGF-β
expression. The proliferative phase comes next, when an intrinsic
regeneration occurs, mainly because the fibroblasts begin to
synthesize collagen, especially type III. The remodeling phase is the
last one and has its peak around day 21 after injury. The collagen
fibers continue to be produced by the fibroblasts, but the number of
fibroblasts tends to gradually decrease after 14 days [5, 9, 10].
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Introduction 341

Figure 19.1 Typical left-handed helix α-chain representation. (A) The

polypeptide chain composed by the repetition of the triplet Gly-X-Y. X and
Y can be basically any amino acid, but usually X represents prolyne and Y
represents hydroxyproline. (B) The tripeptide composed of three α-chains
wrapped around each other [11].

Athletes tend to develop tendon disorders in the lower extrem-

ities, while manual workers tend to develop injuries in tendons
from the upper extremities, and the excessive loading seems to
be one of the most important risk factors for tendinopathies,
acute or chronic. Age plays an important role as well. Examples of
acute tendinopathies are those that occur after unfamiliar repetitive
movement, friction, and pressure, commonly seen within military
recruits after long marches. The chronic tendon injuries are more
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342 Low-Level Laser (Light) Therapy in Tendon Healing in in Vitro and in Vivo Models

Figure 19.2 Tendon typical structure: collagen fibrils connected to form

a collagen fiber, and the fibers organized in primary, secondary, and
tertiary bundles. The bundles are surrounded by the paratenon. Source:
http://www.pponline.co.uk/.

complicated in terms of etiology and manifestation, as they tend to

develop gradually and may not always be related to excessive tendon
loading [1].
The rehabilitation process after a tendon injury often requires
prolonged immobilization in order to protect the injured tissue
and to prevent other possible rupture during the healing period.
Since immobilization time is long, which leads to damages such as
muscular atrophy, osteoarthritis, tendinocutaneous adhesion, and
infections, strategies to decrease this time have been the focus
of many studies [12]. Although adjuvant treatments have been
developed with this intent, i.e., steroid injections, non-steroidal anti-
inflammatory drugs, ultrasound, hyperbaric oxygen therapy, and
extracorporeal shock wave therapy, a complete functional recovery
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Introduction 343

Figure 19.3 Tendon healing process, and the difference between the repair
with and without immobilization. Source: Lippincott Williams & Wilkins,
2004.

of tendons is hard to achieve [13]. Lasers, however, have shown

promising results in this area.
LASER stands for Light Amplification of Stimulated Emission of
Radiation. It can be divided into two main categories: low-level
lasers (low-power lasers) or high-level lasers (high-power lasers).
While high-level lasers rely on their thermal effect to burn or slice
tissues, low-level lasers interact with biomolecules and activate
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344 Low-Level Laser (Light) Therapy in Tendon Healing in in Vitro and in Vivo Models

signaling cascade, modulating the inflammatory process with no

significant thermal effect [14, 15, 16].
Low-level lasers emit in the range of 600–1000 nm (red and
near-infrared light) and have an output power within 5–500 mW.
These parameters allow light to produce biostimulative effects
on tissues (cell repair, vascular system stimulation) so that low-
level light can be effectively used in wound repair, pain relief,
modulation of inflammation, tendon injuries, and acupuncture [17].
The increased angiogenesis, along with the decreased inflammation
and increased fibroblast activity, leads to an increase in collagen
fibers production and alignment, increase in tensile strength,
decreased pain, oxidative stress prevention, and reduction infibrosis
[18, 19].
The action mechanism by which laser produces biostimulatory
effects involves cellular responses that depend on the combination
of the parameters used (wavelength, energy density, power density,
beam cross-sectional area, application technique, irradiation time,
and treatment intervals) [20]. Although some authors may affirm
that the coherence of light plays an important role in the light
biostimulation of tissues [21], some recent studies using non-
coherent light sources showed similar results when compared to
laser trials, mostly because light coherence is lost in the first layers
of biological tissues [22, 23]. The length of the treatment and the
light dosage seem to be more important than the light source when
it comes to clinical results [24].
Several studies contribute to the understanding of how low-level
light acts to enhance the tendon healing process. The most recent
are discussed in the following sections.

19.2 Low-Level Light Therapy and Inflammation

Inflammation is a physiological process that primarily removes an

offending agent and helps restore the damaged tissue, leading the
injured site back to homeostasis. In summary, after an inflammatory
stimulus, the endothelial cells express adhesion molecules, which
facilitate the migration of inflammatory cells (mainly monocytes
and neutrophils) into the inflamed tissue. The resident cells
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Low-Level Light Therapy and Inflammation 345

release inflammatory mediators (prostaglandin E2, thromboxane

A2, leukotrienes, nitric oxide, tumor necrosis factor, and inter-
leukins, especially IL-1β and IL-6) that cause vasodilation, in-
creasing vascular permeability and, consequently, inflammatory cell
migration [7]. They can also stimulate the synthesis of matrix metal-
loproteinases (MMPs) in several kinds of cells, including tenocytes.
MMPs and their inhibitors are important factors in the maintenance
of tendon matrix morphology, since the former degrade collagen and
proteoglycans in healthy and sickness conditions, and the latter can
control MMPs’ activity [25].
When the inflammation process is impaired for some reason,
it can lead the patient to disabilities. Tendinopathies, for instance,
are tendon damages due to impaired inflammatory response that
can cause tendon rupture, requiring surgical intervention. These
are considered a degenerative disease with a slow healing rate,
and the patients seldom recover the original tendon strength and
elasticity. It is believed that there is a multi-factorial cause for
tendinopathies, although repetitive mechanical stress is the most
significant mechanism involved.
The treatment for tendinopathies often requires the administra-
tion of non-steroidal anti-inflammatory drugs (NSAIDs). However,
the real causes of the disease are not treated, and the side effects
with the long-term use of these drugs can generate secondary
adverse conditions.
In the last years, animal and human studies have demonstrated
that LLLT can modulate the release of inflammatory mediators
[prostaglandin E2 (PGE2), tumor necrosis factor (TNF), IL-1β,
plasminogen activator, cicloxigenase-1 (COX-1), and cicloxigenase-
2 (COX-2)] as well as the leucocyte activity. There is a consequent
decrease in the inflammatory process events, such as edema,
hemorrhagic formation, necrosis and neutrophil migration [25].
There is also evidence that LLLT enhances procollagen synthesis and
activates nuclear factor kappa B (NF-κB), responsible to increase the
expression of inducible nitric oxide synthase (iNOS) [4, 13].
Marcos et al., in 2012, used LLLT after an experimental tendon
injury. The continuous 810 nm light source had an output power
of 100 mW and spot size area of 0.028 cm2 . Laser irradiation was
performed with skin contact at light doses of 1 and 3 J. Even with
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346 Low-Level Laser (Light) Therapy in Tendon Healing in in Vitro and in Vivo Models

a smaller dose, a decrease in COX-2 content to values similar to the

uninjured group was observed. All treatments could decrease levels
of PGE2 and COX-2, as well as decrease the gene expression of TNF
and MMPs, even more than the anti-inflammatory drug diclofenac.
This leads to the conclusion that LLLT applied in the tendon healing
process can prevent the loss of mechanical strength of tendons
under inflammatory conditions [25].
Pires et al. demonstrate that IL-6 produced by T-lymphocytes
aids the activation of other T-lymphocytes, as well as B-cells,
macrophages, neutrophils, and eosinophils. According to the au-
thors, IL-6 enhances fibroblast migration into the inflamed tissue
and induces changes in the extracellular matrix of the tissue, and
those changes may be related to the development of tendinitis [13].
IL-1β takes part in the extracellular matrix degradation and in
the suppression of type I collagen, leading to a reduction in tendon
stiffness and overexpression of elastin, which leads to an increased
elasticity to the tendon. Other mediators such as COX-2, PGE2, and
MMPs also contribute to the degradation of extracellular matrix and
to the loss of the biomechanical strength and durability of tendons
[7].
Another important cytokine involved in tendon healing is IL-
10. This anti-inflammatory cytokine, among others like IL-4 and IL-
13, is responsible for decreasing pro-inflammatory mediators and
suppressing monocytes activation [7, 26]. Reitamo et al. state that
IL-10 can accelerate the healing process due to the reduction in
inflammation and the inhibition of IL-6, IL-8, and IL-12 expression,
helping to maintain the mechanical and histological properties of the
tissue [27].

19.3 Applications of Low-Level Light in Tendon Healing

19.3.1 In Vitro Studies

In vitro studies can be useful in the investigation of the intracellular
mechanisms of LLLT in tendon healing, and the results obtained
so far with in vitro assays are often promising. When 40 mW was
applied in cultured tendon fibroblasts with a pulsed GaAs laser
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Applications of Low-Level Light in Tendon Healing 347

(820 nm) and a GaAsInP laser (635 nm) [4] with a frequency of
50 Hz, good results were obtained. The laser beam was emitted
perpendicularly and evenly to the culture plates, and the energy
densities were 1, 2, or 3 J/cm2 . All the doses were able to enhance
fibroblast proliferation, but 1 and 2 J/cm2 were the most effective
doses, even increasing mRNA expression of type I collagen. It seems
that the light dose can play an important role in the outcome of LLLT
in tendon healing. Using similar light doses, Tsai et al. found that
laser might promote cell proliferation because it induces the passage
of fibroblasts to G1, S, G2, and M phase of cell cycle, due to its early
effect on cell cycle regulatory genes [28].
The tenocytes migration can also be enhanced by low-level light,
as shown by Tsai et al. [29]. The irradiation of 1, 1.5, or 2 J/cm2 with a
660 nm continuous laser (output power of 50 mW) perpendicularly
to culture plates was enough to enhance tenocyte migration in
a dose-dependent manner. The expression of dynamin-2 (large
molecular weight GTPase ubiquitously expressed, which plays an
important role in endocytosis and in cell migration) was upregulated
dose-dependently as well. In a more recent study from the same
authors [28], cell migration was observed dose-dependently again,
and this was associated with the upregulation of the expressions of
proliferating cell nuclear antigen (PCNA) and cyclin E, cyclin A, and
cyclin B-1.

19.3.2 In Vivo Studies

Several studies demonstrate the usefulness of LLLT in tendon
healing. Oliveira et al. induced an experimental lesion by dropping a
186 g weight over the Achilles tendon of rats from a 20 cm height,
and treated them with LLLT [20]. The parameters chosen were
830 nm GaAsAl continuous laser with an output power of 40 mW,
power density of 1.4 W/cm2 , and beam cross-sectional area of 0.028
cm2 . The treatment lasted 3, 5, or 7 days starting on the day of
the lesion, and the total energy applied each day was 0.14 J. The
birefringence analysis showed significant improvement in collagen
fibers realignment. The same power density was used by Salate
et al. [30], and the results showed precocious neovascularization
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348 Low-Level Laser (Light) Therapy in Tendon Healing in in Vitro and in Vivo Models

and an increase in the amount of blood vessels compared to the

other experimental and control groups [30].
When different parameters were used to treat tendon lesion in
humans (810 nm infrared probe, output power of 100 mW, power
density of 100 mW/cm2 , and 3 J per point, with six points of
irradiation in total), three times per week for 4 weeks, there was no
significant difference between the treated and control groups at the
end point. This enforces the idea that the combination of parameters
for the treatment plays an important role in the treatment outcome
[18].
The combination of LLLT with other therapeutic modalities can
be used as well. Wood et al. combined ultrasound pulses of 3 MHz
(20% duty cycle with 2 ms on and 8 ms off, pulse repetition
frequency 100 Hz, spatial average temporal peak intensity of 1.0
W/cm2 , and spatial temporal average intensity of 0.2 W/cm2 ) with
low-power GaAlAs diode laser emitting at 830 nm (output power
of 40 mW, power density of 1.4 W/cm2 , total energy dose of 0.12 J,
and beam cross-sectional area of 0.028 cm2 ). Either the combined
treatment or the individual treatments (ultrasound or LLLT) were
able to increase the percentage and alignment of type I collagen
at the injury site. However, the results do not differ between the
different treatments, which lead to the conclusion that performing
any of the treatments individually would be less expensive and
equally efficient [3].
Aliodoust et al. proved the efficacy of LLLT in Achilles tendon
healing in diabetic and non-diabetic rats. An He–Ne laser (632.8 nm)
with 7.2 mW output power was used, with a surface area of 1.4
W/cm2 and 0.014 J of total energy applied. LLLT was showed to
increase the biomechanical properties of tenotomized Achilles ten-
dons in rats, diabetic or not, through decreasing the inflammatory
process, increasing collagen fiber synthesis and mRNA expression
of TGF-β. However, the results in diabetic rats were some 50%
less intense compared with the non-diabetic ones [10]. Ewakil
et al. used the same laser, but with a light dose of 1 J/cm2 , in
Achilles tendon healing, and the results showed early mobilization
of regenerating tendons, recovery of smooth mobility, and regain of
optimal functional outcome of the neotendons to withstand tensile
strength loads [6].
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Applications of Low-Level Light in Tendon Healing 349

In 2013, Nouruzian et al. used basically the same parameters as

Aliodoust, and the results were similar as well. LLLT improved the
healing process of tenotomized Achilles tendons, as demonstrated
by biomechanical tests, either in diabetic or non-diabetic rats, after
10 days of complete tendon transection. The results were dose
dependent, and a lower dose (2.9 J/cm2 ) has produced a more
desirable outcome, especially in diabetic rats [31].
Pires et al. focused their study on the modulation of the
inflammatory process. Using a 780 nm laser with output power of
22 mW, 7.7 J/cm2 was applied in contact with the tendon of right
calcaneous for 7 days [13]. Albertini et al. [32] had already stated
that LLLT could reduce the expression of COX-2, TNF, IL-1β, and IL-
6, and the results of Pires et al. were similar. The only exception
consisted in the lack of effect over IL-1β expression in the Pires et al.
study. Laraia et al., as well as Albertini, found significant inhibition
of IL-1β at 24 h after the injury and LLLT [7].
Collagenase is an enzyme known for its pro-inflammatory and
degenerative properties, and it was used by Marcos et al. [33] to
induce tendinitis in animal models. In the first days after collagenase
injection, there is an evident inflammatory cell migration and
edema, but these effects were inhibited after LLLT. Marcos et al. used
a single LLLT 1 h after collagenase injection. The laser (810 nm,
near-infrared) emitted a continuous optical output of 100 mW, with
a power density of 3.57 W/cm2 , and three different light doses
were applied: 37.71 J/cm2 (1 J total energy), 107.14 J/cm2 (3 J
total energy), and 214.29 J/cm2 (6 J total energy). Interestingly,
the authors found that COX-1 gene expression was increased in all
LLLT groups, but 3 J of near-infrared light was enough to reduce the
expression of COX-2 gene, as well as to reduce vascular permeability
and edema. The LLLT effects were considered better than the
treatment with the anti-inflammatory diclofenac.
Guerra et al. used near-infrared light as well. Once a day, the
animals received 4 J/cm2 of continuous or pulsed laser irradiation
(GaAlAs laser, 830 nm, output power of 40 mW, and dairy light
dose applied of 4 J/cm2 ). The Western blotting assay for collagen
showed that LLLT increased collagen amount in the injury site
compared to the other groups. Both continuous and pulsed laser
protocols led to a higher type I and type II collagen synthesis, but
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350 Low-Level Laser (Light) Therapy in Tendon Healing in in Vitro and in Vivo Models

the pulsed one was able to elevate in a higher level the type I
collagen content. Eight days after the injury, zymographic analysis
revealed that LLLT stimulated the activation of MMP-2, since a
higher level of intermediate and active isoforms was detected than
the latent isoform. On the other hand, both the latent and the active
isoforms of MMP-9 were detected in higher amounts compared to
the controls. Therefore, LLLT seems to enhance the activity of these
metalloproteases during the first days after the injury [5].
When the animals were euthanized 15 days after the injury,
the analysis of the tendons revealed that LLLT could restore the
collagen content of the tissue to normality, as well as a reduction
in MMPs expression. The authors believe that pulsed LLLT caused a
peak in collagen III, promoting a faster recovery of collagen fibrils
in the injury site. In summary, there was a more marked presence
of active MMP-2 and MMP-9 during the inflammatory phase in
animals treated with pulsed low-level light therapy compared to
the continuous laser, leading to the conclusion that pulsed laser can
be more effective in the process of remodeling the tendon. Altering
the frequency parameters and, consequently, modulating the energy
transmitted to the tissue, different outcomes can be seen [5].
One year later, Guerra et al. published a study in which the
laser (830 nm, output power of 40 mW, and the daily light dose
of 4 J/cm2 ) was enough to increase glycosaminoglycans after 15
days of treatment. The birefringence analysis showed that type
III collagen synthesis was initiated 8 days after injury and LLLT,
but no difference was found regarding type I collagen synthesis
nor collagen organization at this moment. At the end of the data
collection, however, the pulsed LLLT was effective in collagen
organization and in tendon healing [12].
It was mentioned before in this chapter that LLLT can be
used as a complementary therapy, and Barbosa et al. combined
an experimental model of platelet-rich plasma therapy with low-
level light irradiation with two wavelengths—660 nm (InGaAlP
laser) and 830 nm (GaAlAs laser)—both with output power of 100
mW and energy density of 7.0 J/cm2 . PRP treatment alone offered
significant advantages in the tendon healing, as well as LLLT alone,
but the combination of those two procedures at the same time is
significantly more efficient [34].
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Applications of Low-Level Light in Tendon Healing 351

In the same year, Casalechi et al. investigated the effect of

LLLT in IL-10 expression. IL-10 is known as the most important
anti-inflammatory cytokine, and it is capable of blocking the
inflammatory response induced by several stimuli in different
models.
Casalechi et al. used a 780 nm continuous laser (output power
of 22 mW, energy density of 7.5 J/cm2 , and total energy delivered of
1.54 J) for 70 s in contact with the tendon of mice right calcaneous.
At 7 and 14 days after the injury, there was a marked increase in
mRNA expression for IL-10 in treated groups, indicating that the
laser acts also on the early stages of inflammation. Furthermore,
mRNA expression for vascular endothelial growth factor (VEGF) was
increased, and the mRNA expression for MMP-1 was decreased in
the treated animals, contributing to the control of the inflammatory
response [9].
Joensen et al. aimed to investigate the efficacy of LLLT in the
prevention of post-exercise injuries, using a 904 nm pulsed laser
(frequency 700 Hz, output power of 60 mW, and total energy
delivered of 3 J) for 50 s, but there was an increase in edema in
the treated mice 24 h after the injury. The authors discuss that the
optimal light dose for 904 nm lasers in rat muscles seems to be in
the range of 0.1 and 1 J, and higher doses can be ineffective or even
worsen the inflammation. The timing of LLLT irradiation must be
considered as well, although it is seldom discussed in the literature
[1].

19.3.3 LEDs Used in Tendon Healing

The classical idea that the therapeutic effects of low-level light
rely on the coherent properties of the lasers is gradually changing,
because some studies have been demonstrating the efficacy of non-
coherent light sources such as light-emitting diodes (LEDs). In 2009,
Casalechi et al. used an LED (640 ± 20 nm) with an output power
of 100 mW and a 0.5 cm2 area in direct contact with the right
limb of rats. After 120 seconds of irradiation leading to a final dose
of 20 J/cm2 , it was enough to improve the tendon-healing process
when compared to other groups. There was an increase in the
number of fibroblasts and a decrease in inflammation at the injured
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352 Low-Level Laser (Light) Therapy in Tendon Healing in in Vitro and in Vivo Models

site, especially when the rats were treated with LLLT in the first
7 days after injury. There was little effect when the treatment was
performed after the first week [24].
Xavier used a near-infrared LED (880 ± 10 nm) with an output
power of 22 mW, 0.5 cm2 area, and also in direct contact with the
right limb of rats with collagenase-induced Achilles tendinitis. The
applied light dose of 7.5 J/cm2 was able to reduce the number
of inflammatory cells at the site of the lesion. This modulation of
the inflammatory process occurs because of the mRNA expression
for IL-1β, IL-6, and TNF, important mediators in the inflammatory
cascade and in the cell migration process. There was a decrease in
mRNA expression for COX-2, mainly in the initial phase after LED
therapy, confirming that LEDs act via modulation of the chemotaxis
and the inflammatory cascade [35].
More recently, the same author used LED with the same
parameters, but this time to investigate the effect of LLLT in IL-10
expression. The results showed an increase in the mRNA for IL-10
in the initial phase of tendon healing, reinforcing the idea that LEDs
are more useful if used in the initial phase of tendon healing [26].

19.4 Conclusion

Since the 1970s, lasers are being used for medical and biomedical
applications, and new light-based therapeutic protocols for several
pathological conditions are growing in numbers. The use of laser
therapy to heal tendon damage has been investigated with studies in
vitro and in vivo, and the results sometimes are controversial. This
occurs mainly due to the lack of consensus on which parameters
should be ideally used for therapy [16]. Light sources other than
lasers are also to be considered, especially LEDs. Although there are
a few studies using LED, there are data in the literature that support
its efficacy in the acceleration of the tendon-healing process, making
obsolete the affirmation that the effects of low-level light on humans
depend on the coherence of the light.
The results published so far tend to confirm the efficacy of LLLT
in reducing the inflammation due to the decrease in inflammatory
mediators expression, decrease in the capillaries permeability, and
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References 353

the inhibition of inflammatory cell migration, as well as increasing

the expression of anti-inflammatory mediators, especially IL-10.
However, the outcome of the treatment depends on the combination
of laser parameters (i.e., light dose, pulsed or continuous source,
output power, time of irradiation), as well as the protocol of
application (i.e., the frequency of the treatment, the stage of tendon
healing in which light is applied, and the duration of the treatment).
The lack of consensus among the authors in this field about what
parameters should be applied often leads to a skepticism about the
efficacy of LLLT. This situation can be more serious if the studies do
not describe all the relevant data about those procedures. Therefore,
if the researchers of LLLT have a consensus on the parameters to
utilize low-level light, the skepticism over using LLLT in tendon
healing would decrease, and this therapy would be available faster
for clinical purposes.

References

1. Joensen, J., Gjerdet, N. R., Hummelsund, S., Iversen, V., Lopes-Martins, R.

A., and Bjordal, J. M. (2012). An experimental study of low-level laser
therapy in rat Achilles tendon injury, Laser Med Sci, 27, pp. 103–111.
2. Vidal, B. (2003). Image analysis of tendon helical superstructure using
interference and polarized light microscopy, Micron, 34(8), pp. 423–
432.
3. Wood, V. T., Pinfildi, C. E., Neves, M. A. I., Parizoto, N. A., and Hochman, B.
(2010). Collagen changes and realignment induced by low-level laser
therapy and low-intensity ultrasound in the calcaneal tendon, Lasers
Surg Med, 42, pp. 559–565.
4. Chen, C-H., Tsai, Y-H., Lee, C-L., Chen, J-K., and Huang, M-H. (2009). Low-
level laser irradiation promotes cell proliferation and mRNA expression
of type I collagen and decorin in porcine Achilles tendon fibroblasts in
vitro, J Orthop Res, 27, pp. 646–650.
5. Guerra, F. D. R., Vieira, C. P., Almeida, M. S., Oliveira, L. P., Aro, A. A., and
Pimentel, E. R. (2013). LLLT improves tendon healing though increase of
MMP activity and collagen synthesis, Lasers Med Sci, 28, pp. 1281–1288.
6. Ewakil, T. F. (2007). An in-vivo experimental evaluation of He-Ne laser
photostimulation in healing Achilles tendons, Lasers Med Sci, 22, pp.
535–539.
July 6, 2016 17:26 PSP Book - 9in x 6in 19-Hamblin-c19

354 Low-Level Laser (Light) Therapy in Tendon Healing in in Vitro and in Vivo Models

7. Laraia, E. M. S., Silva, I. S., Pereira, D. M., Reis, R. A., Almeida, P., Junior, E.
C. P. L., and Carvalho, P. T. C. (2012). Effect of low-level laser therapy (660
nm) on acute inflammation induced by tenotomy of Achilles tendon in
rats, Photochemn Photobiol, 88, pp. 1546–1550.
8. Tsai, W-C., Cheng, J-W., Chen, J-L., Chen, C-Y., Chang, H-N., Liao, Y-H., Lin,
M-S., and Pang, J-H. S. (2014). Low-level laser irradiation stimulates
tenocyte proliferation in association with increased NO synthesis and
upregulation of PCNA and cyclins, Lasers Med Sci, 29, pp. 1377–1384.
9. Casalechi, H. L., Leal-Junior, E. C., Xavier, M., Silva Jr., J. A., Carvalho, P.
T. C., Aimbire, F., and Albertini, R. (2013). Low-level laser therapy in
experimental model of collagenase-induced tendinitis in rats: Effects in
acute and chronic inflammatory phases, Lasers Med Sci, 28, pp. 989–
995.
10. Aliodoust, M., Bayat, M., Jalili, M. R., Sharifian, Z., Dadpay, M., Akbari, M.,
Bayat, M., Khoshvaghti, A., and Bayat, H. (2014). Evaluating the effect
of low-level laser therapy on healing of tentomized Achilles tendon
in streptozocin-induced diabetic rats by light microscopical and gene
expression examinations, Lasers Med Sci, 29, pp. 1495–1503.
11. Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., and Walker, P.
Molecular Biology of the Cell, 5th Edition (Garland Science, USA).
12. Guerra, F. D. R., Vieira, C. P., Almeida, M. S., Oliveira, L. P., Claro, A. C.
F., Simões, G. F., Oliveira, A. L. R., and Pimentel, E. R. (2014). Pulsed
LLLT improves tendon healing in rats: A biochemical, organizational,
and functional evaluation, Lasers Med Sci, 29, pp. 805–811.
13. Pires, D., Xavier, M., Araújo, T., Silva Jr, J. A., Aimbire, F., and Albertini,
R. (2011). Low-level laser therapy (LLLT; 780 nm) acts differently
on mRNA expression. Of anti- and pro-inflammatory mediators in an
experimental model of collagenase-induced tendinitis in rat, Lasers Med
Sci, 26, pp. 85–94.
14. Baratto, L., Calzà, L., Capra, R., Gallamini, M., Giardino, L., Giuliani, A.,
Lorenzini, L., and Traverso, S. (2011). Ultra-low-level laser therapy,
Lasers Med Sci, 26, pp. 103–112.
15. Round, R., Litscher, G., and Bahr, F. (2013). Auricular acupuncture with
laser, Evid Based Complement Alternat Med, 2013, pp. 1–22.
16. Beckmann, K. H., Meyer-Hamme, G., and Schröder, S. (2014). Low-level
laser therapy for the treatment of diabetic foot ulcers: A critical survey,
Evid Based Complement Alternat Med, 2014, pp. 1–9.
17. Eslamian, F., Shakouri, S. K., Ghojazadeh, M., Nobari, O. E., and
Eftekharsadat, B. (2012). Effects of low-level laser therapy in combina-
July 6, 2016 17:26 PSP Book - 9in x 6in 19-Hamblin-c19

References 355

tion with physiotherapy in the management of rotator cuff tendinitis,

Lasers Med Sci, 27, pp. 951–958.
18. Tumilty, S., McDonough, S., Hurley, D. A., and Baxster, D. (2012). Clinical
effectiveness of low-level laser therapy as an adjunct to eccentric
exercise for the treatment of Achilles’ tendinopathy: A randomized
controlled trial, Arch Phys Med Rehabil, 93, pp. 733–739.
19. Enwemeka, C., Parker, J., Dowdy, D., Harkness, E., Sanford, L., and
Woodruff, L. (2004). The efficacy of low-power lasers in tissue repair
and pain control: A meta-analysis study, Photomed Laser Surg, 31(3),
pp. 283–288.
20. Oliveira, F. S., Pinfildi, C. E., Parizoto, N. A., Liebano, R. E., Bossini, C. E.,
Garcia, E. B., and Ferreira, L. M. (2009). Effect of low level laser therapy
(830 nm) with different therapy regimes on the process of tissue repair
in partial lesion calcaneous tendon, Lasers Sur Med, 41, pp. 271–276.
21. Bouton, M., and Marshall, J. (1986). He-Ne laser stimulation of human
fibroblast proliferation and attachment in vitro, Lasers Life Sci, 1, pp.
125–134.
22. Pontinen, P. (2000). Laser acupuncture. In Simunovie, Z. (ed.), Lasers in
Medicine and Dentistry. Part one: Basic Science, and Up-to-Date Clinical
Application of Low Energy-Level Laser Therapy (LLLT), 1st edn, Vitgraf,
Rijeka, pp. 455–475.
23. Whelan, H. T., Houle, J. M., Hwelean, N. T., Donohoe, D. L., Cwiklinski,
J., Scmitd, M. H., Gould, L., Larson, D. L., Meyer, G. A., Ceverini, V.,
and Stinson, H. (2000). NASA light-emitting diode medical program:
Progress in space flight terrestrial applications, Space Technol Appl Int
Forum, 504, pp. 37–43.
24. Casalechi, H. L., Nicolau, R. A., Casalechi, V. L., Silveira Jr, L., De Paula, A.
M. B., and Pacheco, M. T. T. (2009). The effects of low-level light emitting
diode on the repair process of Achilles tendon therapy in rats, Lasers
Med Sci, 24, pp. 659–665.
25. Marcos, R. L., Leal-Junior, E. C. P., Arnold, G., Magnenet, V., Rahouadj,
R., Wang, X., Demeurie, F., Magdalou, J., Carvalho, M. H. C., and Lopes-
Martins, R. A. B. (2012). Low-level laser therapy in collagenase-induced
Achilles tendinitis in rats: Analyses of biochemical and biomechanical
aspects, J Ortho Res, 30, pp. 1945–1951.
26. Xavier, K., Souza, R. A., Pires, V. A., Santos, A. P., Aimbire, F., Silva Jr, J.
A., Albertini, R., and Villaverde, A. B. (2014). Low-level light-emitting
diode therapy increases mRNA expressions of IL-10 and type I and III
collagens on Achilles tendinitis in rats, Laser Med Sci, 29, pp. 85–90.
July 6, 2016 17:26 PSP Book - 9in x 6in 19-Hamblin-c19

356 Low-Level Laser (Light) Therapy in Tendon Healing in in Vitro and in Vivo Models

27. Reitamo, S., Remitz, A., and Tamai, K. (1994). Interleukin-10 modulates
type I collagen and matrix metalloprotease gene expression in cultured
human skin fibroblasts, J Clin Invest, 94, pp. 2489–2492.
28. Tsai, W-C., Cheng, J-W., Chen, J-L., Chen, C-Y., Chang, H-H., Liao, Y-H, Lin,
M-S., and Pang, J-H. S. (2014). Low-level laser irradiation stimulates
tenocyte proliferation in association with increased NO synthesis and
upregulation of PCNA and cyclins, Lasers Med Sci, 29, 1377–1384.
29. Tsai, W-C., Hsu, C-C., Pang, J-H. S., Lin, M-S., Chen, Y-H., and Liang, F-C.
(2012). Low-level laser irradiation stimulates tenocyte migration with
up-regulation of dynamin II expression, PLoS ONE, 7(5), pp. 1–7.
30. Salate, A. C. B., Barbosa, G., Gaspar, P., Koeke, P. U., Parizoto, N. A.,
Benzem, B. G., and Foschiani, D. (2005). Effect of In-Ga-Al-P diode laser
irradiation on angiogenesis in partial ruptures of Achilles tendon in rats,
Photomomed Laser Surg, 23(5), pp. 470–475.
31. Nouruzian, M., Alidoust, M., Bayat, M., Bayat, M., and Akbari, M. (2013).
Effect of low-level laser therapy on healing of tenotomized Achilles
tendon in streptozotocin-induced diabetic rats, Lasers Med Sci, 28, pp.
399–405.
32. Albertini, R., Villaverde, A. B., Aimbire, F., Bjordal, J., Brugnera, A.,
Mittmann, J., Silva, J. A., and Costa, M. (2008). Cytokine mRNA
expression is decreased in the subplantar muscle of rat paw subjected
to carrageenan-induced inflammation after low-level laser therapy,
Photomed Laser Surg, 26(1), pp. 19–24.
33. Marcos, R. L., Leal-Junior, E. C. P., Messias, F. M., Carvalho, M. H. C.,
Pallotta, R. C., Frigo, L., Santos, R. A., Ramos, L., Teixeira, S., Bjordal, J.
M., and Lopes-Martins, R. A. B. (2011). Infrared (810 nm) Low-level
laser therapy in rat Achilles tendinitis: A consistent alternative to drugs,
Photochem Photobiol, 87, pp. 1447–1452.
34. Barbosa, D., Souza, R. A., Carvalho, W. R. G., Xavier, M., Carvalho, P.
K., Cunha, T. C. R., Arisawa, E. A. L., Silveira Jr, L., and Villaverde, A.
B. (2013). Low-level laser combined with platelet-rich plasma on the
healing calcaneal tendo: A histological study in a rat model, Lasers Med
Sci, 28, pp. 1489–1494.
35. Xavier, M., David, D. R., Souza, R. A., Arrieiro, A. N., Miranda, H., Santana,
E. T., Silva Jr, J.A., Salgado, M. A. C., Aimbire, F., and Albertini, R. (2010).
Anti-inflammatory effects of low-level light emitting diode therapy on
Achilles tendinitis in rats, Lasers Surg Med, 42, pp. 553–558.
July 6, 2016 17:26 PSP Book - 9in x 6in 20-Hamblin-c20

Chapter 20

Bone Repair in Animal Models

Antonio Luiz B. Pinheiro, Luiz G. Pinheiro Soares,

and Aparecida Maria C. Marques
Center of Biophotonics, School of Dentistry, Federal University of Bahia, Salvador,
BA, 40110-150, Brazil
albp@ufba.br

20.1 Introduction

20.1.1 Bone Tissue

Bone is a specialized connective tissue composed of 33% organic
matrix, comprising 28% of type I collagen and the rest of non-
collagenous proteins, including osteocalcin, osteonectin, BMPs,
sialoproteins, and bone proteoglycans, which are arranged to
form bones, rigid and resistant structures that form the skeleton.
Bone tissue is an adaptable structure, which develops according
to the types of mechanical forces received and their metabolic
needs. Bone metabolism also responds to hormonal regulation
and biomechanics, and these two regulatory mechanisms are
antagonists. Nowadays, a major problem in many specialties is the
process of repair of defects caused by trauma, surgical procedures,
or pathologic conditions.

Handbook of Low-Level Laser Therapy

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358 Bone Repair in Animal Models

Despite the seemingly inert aspect, bone grows, remodels, and

remains active throughout the life of the individual. The bone when
injured such as in the case of fractures or after the insertion
of a dental implant, undergoes a series of biological phenomena
leading to its repair. Bone is a highly complex tissue and is
under continuous remodeling, which is primarily mediated by two
cell types: osteoblasts and osteoclasts. Restoring biological and
mechanical properties, unlike other organs and tissues, is then
possible. In response to the trauma of the bone, different and well-
ordered sequences of events occur and result in the repair of the
damaged tissue to a condition very similar to its original structure.
Under physiological conditions, bone tissue has a great regenerative
potential. However, in some situations this regenerative potential
is limited, such as in some systemic diseases that affect bone
metabolism, when a trend to repair occurs through the formation
of fibrous tissue that hinders or even prevents bone repair, in a
phenomenon known as fibrosis.
Several factors can modify, in an important way, the process
of osteogenesis and can cause, for example, delayed consolidation
of fractures and/or pseudoartrosis. In this context, animal studies
have demonstrated the effect of diabetic condition in skeletal tissue
morphology, delayed bone healing, and remodeling of the alveolar
bone. Bone losses are important problems in both medical and
dental fields. They may be caused by several physiologic and/or
pathologic conditions. Physiologic bone losses occur basically
because of ageing.
Bone has a huge repairing capacity and often it can restore
itself to its usual architecture and consequently recovering its
mechanical properties. However, this capacity has limits and its
complete recovery may not be possible in the case of deficient
blood supply, mechanical instability, or competition with highly
proliferating tissues. The loss of bone fragments or the removal
of necrotic or pathologic bone or even some surgical tactics may
result in bone defects as well. These defects may be too large
for spontaneous and physiologic repair. Several methods can be
used to ameliorate bone repair, such as using grafts as well as
phototherapy.
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Introduction 359

20.1.2 Autologous Bone Grafting and Biomaterials

A wide range of biomaterials have been used to obtain an improve-
ment in the repair of bone defects. Both autografts and xenogenous
provide a good structure and stimulating bone formation and the
possibility of association with membranes, a technique known
as guided bone regeneration (GBR). The biomaterials used in
the recovery of bone loss are those obtained from human or
animal tissues and alloplastic. Among those produced synthetically
(alloplastic), the most widely used is hydroxyapatite, followed by
tricalcium phosphate, bioglass, and polymers.
Bone repair is different from the healing of soft tissues because
of both morphology and composition. It is slower and comprehends
consecutive phases, which may differ depending on the type and
intensity of the trauma as well as to the extension of the damage
to the bone. Bone repair, irrespective of the causative agent of
injury, consists of a series of phenomena similar to those occurring
during the healing of soft tissue wounds and include inflammation,
fibroplasias, and remodeling. However, in contrast to the healing
of soft tissue, osteoblasts and osteoclasts are involved in the
remodeling and reconstruction of bone tissue.
Bone grafts can be classified based on their morphological
and/or immunological characteristics. According to morphological
criteria, these are classified into spongy cortical and corticocancel-
lous. The latter is characterized by being faster than the cortical
graft revascularization, showing thin trabeculae separated by wide
spaces filled with bone marrow hematopoietic cells, facilitating cell
migration, but provides a significant mechanical support.
Some procedures can be used to enhance the repair of bone
defects, such as the use of certain types of grafts. These grafts may
be autologous, allogeneic, or alloplastic (biomaterials).
Biomaterials are substances of natural origin or synthetic,
biocompatible, and may temporarily or permanently replace various
tissues, stimulating chemical and biological reactions favorable to
its function. They may act on bone repair by osteoconductive,
osteoinductive, or oseous stimulation. This allows osteoconduction
angiogenesis and growth of bone cells, osteoprogenitor cells from
the bed vessel through the graft surface, giving rise to osteoblasts
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360 Bone Repair in Animal Models

responsible for bone formation at the site of injury. Osteoestimu-

lation promotes bone growth through the transfer of osteoblasts,
together with the graft or stimulation of osteoblasts to the site
receiver. Autologous bone grafts, both intra- and extra-oral, are
examples of such materials. Osteoinduction promotes differenti-
ation of mesenchymal stem cells in perivascular osteoprogenitor
cells under the stimulation of one or more substances released
by inducing bone graft matrix, such as BMPs. Most recently, the
combination of HA +β-calcium triphosphate (β-TCP) graft and
phototherapies has been studied. The use of biomaterials may
modulate the repair of bone defects in a manner similar to that
observed in autologous bone graft, preventing complications and
limitations. However, the use of autologous grafts remains the gold
standard for the treatment of bone defects.

20.1.3 Guided Bone Regeneration

Membranes or biological barriers have become a landmark in the
GBR technique. These structures serve to prevent the growth into
a defect of the tissue or junctional epithelium. There are two
types of membranes: reabsorbable and non-reabsorbable. The main
obstacle for osteogenesis is the faster formation of soft tissues.
In the last decade, several animal and human studies have used
membranes to prevent the invasion of bone defects by soft-tissues.
This principle was developed to regenerate periodontal tissues
lost due to periodontal disease and was called guided periodontal
regeneration, whose effectiveness has been evaluated in animal and
human studies.
Various treatment techniques are used in clinical practice in an
attempt to promote accelerated and/or improved bone repair, and
these include the use of coherent or non-coherent light sources.
Among light sources, laser light is used for laser phototherapy
(LPT). This therapeutic approach has been proved very effective
in biomodulation, the healing of soft tissue, and the bone repair
process. However, many researchers believe that more studies are
needed in this direction because photobiostimulation in humans is
still controversial.
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Light Therapies in the Bone Repair of Animal Models 361

20.1.4 Phototherapy
Over the past 10 years, treatments using light sources have been
used in a number of processes involving the repair of soft tissue
wounds in animals and humans. These treatments are now also
used as adjuvants for the effective repair of mineralized tissues,
as previously suggested by several studies that concluded that
laser/LED phototherapy accelerates the repair of bone directly
affecting osteogenesis.

20.2 Light Therapies in the Bone Repair of Animal Models

Several therapeutic techniques are used in clinical practice to

promote acceleration and/or improve bone repair, including the
use of coherent or non-coherent light sources. Among these light
sources, we will highlight laser light used for LPT. This therapeutic
approach has been very effective in both the biomodulation of soft
tissue wound healing and the bone repair process. However, many
researchers believe that further studies are needed in this direction,
because photobiostimulation in humans is still controversial.
In a comprehensive retrospective study Pinheiro and Gerbi [1]
described the state of the art of the photobioengineering of bone
repair and concluded that the effect of LPT on bone regeneration
depends not only on the total dose of irradiation but also on the time
and mode of irradiation. The threshold energy density and intensity
are biologically independent of one another. This independence
accounts for the success and failure of LPT achieved at low levels
of energy density.
Over the past 10 years, treatments with light sources have been
used in a number of procedures involving the repair of bone in both
animals and humans. These treatments are now used also as aids for
the efficient repair of mineralized tissues.
The first report from our group on the effects of LPT found
better bone healing around dental implants in irradiated animals.
The report observed that after 45 days, it is not possible to detect
differences between irradiated and non-irradiated bone using SEM.
At early stages, differences in bone organization and vascularization
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362 Bone Repair in Animal Models

were detectable [2]. Another study by our team morphometricly

evaluated the amount of newly formed bone after the use of IR laser
light on surgical defects created in the femur of rats. A significant
difference was observed between the areas of mineralized bone
between irradiated and non-irradiated subjects during the early
stages of healing but not after 28 days [3].
Over the last decade, our group also assessed the effects of
the association of light therapies with different biomaterials using
animal models. Since 2003, we carried out several studies using
different protocols in which different light sources were used in
association with biomaterials of different composition, associated or
not to the use of GBR. The results of these studies prove that the use
of adequate protocols improves the repair of different bone defects
[4–20].
Our group has also studied the effect of LPT on the repair of
bone defects associated to autologous bone grafts in animal models.
The results of the previous studies showed that the use of LPT
transoperatively resulted in a positive biomodulative effect on the
healing of bone defects associated to autologous bone grafts [21, 22].
Recently, our team assessed the influence of LPT on bone volume
and bone implant contact interface around implants inserted in
blocks of bovine or autologous bone grafts (autografts), irradiated
or non-irradiated, in rabbit femurs. The results of this investigation
suggest that LPT is effective in enhancing new bone formation with
consequent increase in bone-implant interface in both autologous
grafts and xenografts [23].
Fractures have different etiology and treatment and may be
associated or not to bone losses. Laser light has been shown to
improve bone repair. Our results in animal models indicate that NIR
LPT associated to BMPs and GBR is effective in improving the repair
of fractured bones [24–26].
Tooth movement is closely related to the process of bone
remodeling. The biologic result of application of a force on the
tooth is bone absorption on the pressure side and neoformation
on the traction side of the alveolar bone. Hyalinization, a sterile
necrosis at the pressure zone of the periodontal ligament, observed
during the initial stages of orthodontic movement, and when it
is extensive might cause an important delay in tooth movement.
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Light Therapies in the Bone Repair of Animal Models 363

Changes in alveolar bone during orthodontic movement in rats have

also been studied since LPT affects orthodontic movement. A series
of studies using LPT in rodents in which tooth movement similar to
the one seen during orthodontic treatment showed that LPT causes
increased bone metabolism, blood flow, and lymphatic drainage.
In addition, it was also demonstrated that LPT positively affected
an important aspect of dental movement: hyalinization. Despite
these studies indicated that LPT, with the parameters used, did
not significantly increase the amount of tooth displacement during
induced orthodontic movement in rodents, LPT caused significant
histological changes in the alveolar bone during induced tooth
movement, including alterations in the number of both osteoclasts
and osteoblasts and in collagen deposition in both pressure and
tension areas. Also the use of laser light caused histologic alterations
in hyalinization at early stages and late reduction when compared to
nonirradiated animals [27–29].
In a recent study, we analyzed the effect of laser or light-
emitting diode (LED) phototherapy on the formation of bone at the
midpalatal suture after rapid maxilla expansion by using Raman
spectroscopy. The results of this study using Raman spectral analysis
indicate that laser and LED light irradiation improves deposition of
CHA in the midpalatal suture after orthopedic expansion [30].
Previous studies have shown positive effects of light therapies on
the repair of bone defects, but only a few associate bone healing in
the presence of a metabolic disorder such as diabetes mellitus. We
histologically assessed the effect of LPT on the repair of surgical
defects created in the femur of diabetic and non-diabetic Wistar
Albinus rats. The investigation showed histological evidence of
improved amount of collagen fibers at the early stages of bone
healing and an increased amount of well-organized bone trabeculae
at the end of the experimental period (30 days) in irradiated animals
(diabetic and non-diabetic), compared to the nonirradiated ones.
These results indicate that LPT has a positive biomodulative effect
on the healing process of bone defects even when diabetes mellitus
is present [31].
In order to study the effect of the use of laser or LED light
on the repair of bone tissue grafted or not with hydroxyapatite
(HA) in rodents suffering from iron deficiency anemia (IDA)
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364 Bone Repair in Animal Models

Raman spectroscopy was used as assessment method. These studies

indicated higher HA peaks as well as a decrease in the level
of organic components on healthy animals when graft and LED
phototherapy are associated. On the other hand, IDA interfered
with the incorporation of graft in the bone since LED phototherapy
improved bone repair only when graft was not used. In laser-treated
animals, the results indicate that using HA and laser irradiation
on healthy subjects is favorable to mineral deposition and bone
maturation, which is important for some groups at risk, such as
astronauts. In cases of IDA, the use of graft, associated or not to
laser irradiation, resulted in low collagen and low carbonate and
phosphate HA [32, 33].
Osteoporosis is a disease characterized by the reduction in bone
mineral density. We studied, by Raman spectroscopy, bone defects of
ovariectomized rats treated or not with LED-PT. The results of this
study indicated that infrared LED-PT improved the deposition of HA
on bone defects in ovariectomized rats [34].

20.3 Closing Remarks

Translational research is essential for improving human health due

to a need to transfer new knowledge, mechanisms, and techniques
generated by advances in basic research aimed at offering new
opportunities for the prevention, diagnosis, and treatment of
diseases. It is obvious that both legal and ethical constraints limits
human studies and it is well accepted and strongly recommended
that these studies to be carried out only after in vitro and animal
ones.
Whenever possible, animal models are preferred for research.
Computer models, tissue and cell cultures, and a number of other
non-animal models are used today in biomedical research. However,
animal testing remains a necessity as, for example, blindness cannot
be studied in microorganisms. Also it is not possible to study
the effects of high blood pressure on tissue or cell cultures. The
living system is extremely complex. The nervous system, blood and
brain chemistry, gland and organ secretions, and immunological
responses are all interrelated, making it impossible to explore,
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References 365

explain, or predict the course of diseases or the effects of possible

treatments without observing and testing the entire living system of
an animal. In the meantime, scientists continue to look for ways to
reduce the number of animals needed to obtain valid results, refine
experimental techniques, and replace animals with other research
methods whenever feasible.
Animals make good research subjects for a variety of reasons.
They are biologically similar to humans. In fact, chimpanzees share
more that 99% DNA and mice share more than 98% DNA with
humans; therefore, animals are susceptible to many of the same
health problems as humans. Animals have a shorter life cycle than
humans, and as a result, they can be studied throughout their whole
life span or across several generations. In addition, scientists can
easily control the environment around animals (diet, temperature,
lighting), which would be difficult to do with humans.
Tajali et al. [35] found a statistically significant impact of LPT on
the biomechanical properties of bone, particularly after 14 sessions.
They commented that a large number of negative studies would be
necessary to reject the effect. This suggests that the available animal
research is sufficient to support LPT in bone repair, including in
humans, which corroborates the results of our studies.
Over the past 20 years, our team has been investigating the
effects of laser technology on many medical and dental fields using
animal models. Interesting data were obtained on the mechanisms
of wound healing and repair in both healthy and systemically
compromised animal models. These findings provided new insights
on using light for treating both soft tissue and bone lesions. We could
demonstrate that the use of light effectively improves repair and
healing in different animal models.
Our results indicate that animal models are still needed in many
situations and must follow stricter ethical and legal regulations.
Whenever possible, non-animal models should be preferred.

References

1. Pinheiro, A.L.B., and Gerbi, MEMM. (2006). Photobioenerginnering of

the bone repair process, Photomed. Laser Surg., 24, 169–178.
July 6, 2016 17:26 PSP Book - 9in x 6in 20-Hamblin-c20

366 Bone Repair in Animal Models

2. Pinheiro, A.L.B., Oliveira, M.A.M., and Martins, P.P.M. (2001).

Biomodulação da cicatrização óssea pós-implantar com o uso da lasert-
erapia não-cirúrgica: Estudo por microscopia eletrônica de varredura,
Rev. FOUFBA, 22, 12–19.
3. Pinheiro, A.L.B., Limeira Jr, F.A., Gerbi, MEMM., et al. (2003). Effect of
830 nm laser light on the repair of bone defects grafted with inorganic
bovine bone and decalcified cortical osseus membrane, J. Clin. Laser
Med. Surg., 21, 383–388.
4. Pinheiro, A.L.B., Limeira Jr, F.A., Gerbi, M.E.M.M. et al. (2003). Effect
of lowlevel laser therapy on the repair of bone defects grafted with
inorganic bovine bone, Braz. Dent. J., 14, 177–181.
5. Pinheiro, A.L.B., Limeira Jr, F.A., Gerbi, M.E.M.M., et al. (2003). Effect of
830 nm laser light on the repair of bone defects grafted with inorganic
bovine bone and decalcified cortical osseous membrane, J. Clin. Laser
Med. Surg., 21, 383–388.
6. Gerbi, M., Limeira Júnior, F.A., Pinheiro, A.L.B., et al. (2003). Assessment
of bone repair associated to the use of organic bone graft and membrane
with 830 nm, SPIE Lasers Dentistry, 4950, 137–143.
7. Lopes, C.B., Pinheiro, A.L.B., Sathaiah, S. et al. (2005). Infrared laser light
reduces loading time of dental implants: A Raman spectroscopic study,
Photomed. Laser Surg., 23, 27–31.
8. Gerbi, M.E.M.M., Pinheiro, A.L.B., Marzola, C., et al. (2005). Assessment
of bone repair associated with the use of organic bovine bone and
membrane irradiated at 830 nm, Photomed. Laser Surg., 23, 382–388.
9. Pinheiro, A.L.B., Gerbi, M.E.M.M., Ponzi, EAC., et al. (2008). Infrared
laser light further improves bone healing when associated with bone
morphogenetic proteins and guided bone regeneration: An in vivo study
in a rodent model, Photomed. Laser Surg., 26, 167–174.
10. Pinheiro, A.L.B., Gerbi, M.E.M.M., Limeira, Jr FA., et al. (2009). Bone
repair following bone grafting hydroxyapatite guided bone regeneration
and infra-red laser photobiomodulation: A histological study in a rodent
mode, Lasers Med. Sci., 24, 234–240.
11. Pinheiro, A.L.B., Aciole, G.T.S., Cangussú, M.C.T., et al. (2010). Effects of
laser phototherapy on bone defects grafted with mineral trioxide ag-
gregate, bone morphogenetic proteins, and guided bone regeneration: A
Raman spectroscopic study, J. Biomed. Mat. Res. Part A, 95, 1041–1047.
12. Pinheiro, A.L.B., Soares, L.G.P, Aciole, G.T.S., et al. (2011). Light
microscopic description of the effects of laser phototherapy on bone
defects grafted with mineral trioxide aggregate, bone morphogenetic
July 6, 2016 17:26 PSP Book - 9in x 6in 20-Hamblin-c20

References 367

proteins, and guided bone regeneration in a rodent model, J. Biomed.

Mat. Res. Part A, 98, 212–221.
13. Pinheiro, A.L.B., Soares, L.G.P., Barbosa, A.F.S., et al. (2011). Does
LED phototherapy influence the repair of bone defects grafted with
MTA, bone morphogenetic proteins, and guided bone regeneration?
A description of the repair process on rodents, Lasers Med. Sci., DOI:
10.1007/s10103-011-1033-8.
14. Pinheiro, A.L.B. Soares, L.G.P. Aciole, G.T.S., et al. (2011). Light micro-
scopic description of the effects of laser phototherapy on bone defects
grafted with mineral trioxide aggregate, bone morphogenetic proteins,
and guided bone regeneration in a rodent model, J. Biomed. Mat. Res.
Part A, 98A, 212–221
15. Pinheiro, A.L.B., Soares, L.G.P., Cangussú, MCT., et al. (2012). Effects of
LED phototherapy on bone defects grafted with MTA, bone morpho-
genetic proteins and guided bone regeneration: A Raman spectroscopic
study, Lasers Med. Sci., 27, 903–916.
16. Asouli, G., Amir, A., Rokn, A.R., et al. (2013). Effect of low-level laser
therapy irradiation and Bio-Oss graft material on the osteogenesis
process in rabbit calvarium defects: A double blind experimental study,
Lasers Med. Sci., DOI: 10.1007/s10103-013-1403-5.
17. Soares, L.G.P., Marques, A.M.C., Aciole J.M.S., et al. (2014). Assessment
laser phototherapy on bone defects grafted or not with biphasic
synthetic micro-granular HA +β-tricalcium phosphate: Histological
study in an animal model, Proc. SPIE 8932, Mechanisms for Low-Light
Therapy IX, 893211 DOI: 10.1117/12.2036872.
18. Soares, L.G.P., Marques, A.M.C., Aciole, J.M.S. et al. (2014). Do laser/LED
phototherapies influence the outcome of the repair of surgical bone
defects grafted with biphasic synthetic microgranular HA +β-tricalcium
phosphate? A Raman spectroscopy study, Lasers Med. Sci., DOI:
10.1007/s10103-014-1563-y.
19. Soares, L.G.P., Marques, A.M.C. Guarda, M.G. et al. (2014). Influence of
the λ780 nm laser light on the repair of surgical bone defects grafted
or not with biphasic synthetic micro-granular hydroxylapatite + beta -
calcium triphosphate, J. Photochem. Photobiol. B., Biol, 131, 16–23 DOI:
10.1016/j.jphotobiol.2013.12.015.
20. Pinheiro, A.L.B. Soares, L.G.P., Barbosa, A.F.S. et al. (2012). Does
LED phototherapy influence the repair of bone defects grafted with
MTA, bone morphogenetic proteins, and guided bone regeneration? A
description of the repair process on rodents, Lasers Med. Sci., 27, 1013–
1024 DOI: 10.1007/s10103-011-1033-8.
July 6, 2016 17:26 PSP Book - 9in x 6in 20-Hamblin-c20

368 Bone Repair in Animal Models

21. Weber, J.B.B., Pinheiro, A.L.B., De Oliveira, M.G., et al. (2006). Laser
therapy improves healing of bone defects submitted to autologus bone
graft, Photomed. Laser Surg., 24, 38–44.
22. Torres, C.S., Santos, J.N., Pinheiro, A.L.B., et al. (2008). Does the use of
laser photobiomodulation, bone morphogenetic proteins, and guided
bone regeneration improve the outcome of autologous bone grafts? An
in vivo study in a rodent model, Photomed. Laser Surg., 26, 371–377.
23. Soares, L.G.P., Magalhaes, Jr, E.B., Magalhaes, C.A.B., et al. (2013).
New bone formation around implants inserted on autologous and
xenografts irradiated or not with IR laser light: A histomorphometric
study in rabbits, Braz. Dent. J., 24, 218–223 DOI:10.1590/0103-
6440201302186.
24. Lopes, C.B., Pacheco, M.T.T., Silveira, L., et al. (2010). The effect of
the association of near infrared laser therapy, bone morphogenetic
proteins, and guided bone regeneration on tibial fractures treated with
internal rigid fixation: A Raman spectroscopic study, J. Biomed. Mat. Res.
Part A., 94, 1257–1263.
25. Pinheiro, A.L.B., Lopesm, C.B., Pacheco, M.T.T., et al. (2010). Raman
spectroscopy validation of DIAGNOdent-assisted fluorescence readings
on tibial fractures treated with laser phototherapy, BMPs, guided bone
regeneration, and miniplates, Photomed. Laser Surg., 28, S89–97.
26. Pinheiro, A.L.B., Santos, N.R.S., and Oliveira, P.C. (2013). The efficacy of
the use of IR laser phototherapy associated to biphasic ceramic graft
and guided bone regeneration on surgical fractures treated with wire
osteosynthesis: A comparative laser fluorescence and Raman spectral
study on rabbits, Lasers Med. Sci., 28, 815–822 DOI: 10.1007/s10103-
012-1166-4.
27. Gama, S.K.C., Habib, F.A.L., de Carvalho, J.S.M., et al. (2010). Tooth
movement after infrared laser phototherapy: Clinical study in rodents,
Photomed. Laser Surg., 28, S79–83.
28. Habib, F.A.L., Gama, S.K.C., Ramalho, L.M.P., et al. (2010). Laser-induced
alveolar bone changes during orthodontic movement: A histological
study on rodents, Photomed. Laser Surg., 28, 823–830.
29. Habib, F.A.L., Gama, S.K.C., Ramalho, L.M.P., et al. (2012). Effect of
laser phototherapy on the hyalinization following orthodontic tooth
movement in rats, Photomed. Laser Surg., 30, 179–185.
30. Rosa, C.B., Habib, F.A.L., and Araújo, T.M. (2013). Effect of the laser
and light-emitting diode (LED) phototherapy on midpalatal suture
bone formation after rapid maxilla expansion: A Raman spectroscopy
analysis, Lasers Med. Sci, DOI: 10.1007/s10103-013-1284-7.
July 6, 2016 17:26 PSP Book - 9in x 6in 20-Hamblin-c20

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31. Costa Lino, M.D.M., Carvalho, F.B., Moraes, M.F., et al. (2011). The effects
of photobiomodulation on healing of bone defects in streptozotocin
induced diabetic rats, Proc. SPIE 7887, Mechanisms for Low-Light
Therapy VI, 78870G (2011) DOI: 10.1117/12.875852.
32. Rosa, C.B., Castro, Vieira, I.C., Reis Júnior, JA., et al. (2014). The efficacy
of the use of IR laser phototherapy (LPT) on bone defect grafted
with biphasic ceramic on rats with iron deficiency anemia: Raman
spectroscopy analysis, Lasers Med. Sci., DOI: 10.1007/s10103-013-
1496-x.
33. Castro, I.C.V., Rosa, C.B., Reis Júnior, J.A., et al. (2014). Assessment
of the use of LED phototherapy on bone defects grafted with
hydroxyapatite on rats with iron-deficiency anemia and nonanemic: A
Raman spectroscopy analysis, Lasers Med. Sci., DOI: 10.1007/s10103-
014-1562-z.
34. Aciole, J.M.S., Castro, I.C.V., Soares, L.G.P., et al. (2014). Assessment of the
LED phototherapy on femoral bone defects of ovariectomized rats: A
Raman spectral study, Lasers Med. Sci., DOI: 10.1007/s10103-013-1509-
9.
35. Tajali, S.B., MacDermid, J.C., Houghton, P., et al. (2010). Effects of low
power laser irradiation on bone healing in animals: A meta-analysis, J.
Orthop. Surg. Res., 5, 1.
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Chapter 21

Transcranial Low-Level Laser (Light)

Therapy for Stroke and Traumatic Brain
Injury in Animal Models

Michael R. Hamblin,a,b,c Luis De Taboada,d

and Ying-Ying Huanga,b
a Wellman Center for Photomedicine, Massachusetts General Hospital, 50 Blossom

Street, Boston, MA 02114, USA

b Department of Dermatology, Harvard Medical School, Massachusetts General

Hospital, 55 Fruit Street, Boston, MA 02114, USA

c HarvardMassachusetts Institute of Technology (MIT) Division of Health Sciences and

Technology, 77 Massachusetts Avenue, E25518, Cambridge, MA 02139, USA

d LiteCure LLC, Newark, DE, 19702, USA

hamblin@helix.mgh.harvard.edu

Low-level laser (light) therapy (LLLT) for traumatic events that

cause brain damage is currently an experimental concept. The
broad goals for clinical utilization are the prevention of brain
damage, relief of symptoms, and stimulation of the repair process.
Experimental studies have tested and continue to test these goals
by investigating LLLT in animal models of stroke and traumatic
brain injury (TBI). Animal studies of tLLLT for ischemic stroke
have been summarized in this chapter. Studies by other groups
on LLLT in mouse TBI models have been discussed. Four studies
from our laboratory have been described. The first study looked at

Handbook of Low-Level Laser Therapy

Edited by Michael R. Hamblin, Marcelo Victor Pires de Sousa, and Tanupriya Agrawal
Copyright c 2017 Pan Stanford Publishing Pte. Ltd.
ISBN 978-981-4669-60-3 (Hardcover), 978-981-4669-61-0 (eBook)
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372 Transcranial Low-Level Laser (Light) Therapy for Stroke and Traumatic

pulsed versus continuous wave (CW) laser irradiation in a controlled

cortical impact (CCI)-TBI model and found pulsed 810 nm laser at
10 Hz to be superior. The second study looked at four different
laser wavelengths (660, 730, 810, and 980 nm) in a closed-head
TBI model; only 660 nm and 810 nm were effective. The third study
looked at different treatment repetition regimens (1, 3, and 14 daily
laser treatments). The last study used immunofluorescence tech-
niques to show that neurogenesis, brain-derived neurotrophic factor
(BDNF), and synaptogenesis were upregulated after transcranial
LLLT (tLLLT) for TBI. Limitations in knowledge are still apparent,
such as the optimal wavelength, light source, doses, pulsed or
CW, polarization state, treatment timing, and repetition frequency.
Collaborative efforts between clinicians and basic researchers will
likely increase the usage and understanding of effective laser-based
therapies in the central nervous system (CNS).

21.1 Introduction

Low-level laser (or light) therapy has been clinically applied for
many indications in medicine that require the following processes:
protection from cell and tissue death, stimulation of healing and
repair of injuries, and reduction in pain, swelling, and inflammation.
One area that is attracting growing interest is the use of LLLT to treat
stroke, TBI, neurodegenerative diseases, and spinal cord injuries.
The notable lack of any effective drug-based therapies for most of
these diseases has motivated researchers to consider the use of
light as a real approach to mitigating what is considered to be a
group of serious diseases; the fact that near-infrared (NIR) light
can penetrate into the brain and spinal cord allows noninvasive
treatment to be carried out with a low likelihood of treatment-
related adverse events. Although in the past it was generally
accepted that the CNS could not repair itself, recent discoveries
in the field of neuronal stem cells have brought this dogma into
question. LLLT may have beneficial effects in the acute treatment of
brain damage after stroke or injury, but may also favorably impact
the more chronic degenerative brain diseases.
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Photobiology of Low-Level Laser Therapy 373

21.2 Photobiology of Low-Level Laser Therapy

The first law of photobiology states that for low power light to
have any effect on a living biological system, the photons must
be absorbed by electronic absorption bands belonging to some
molecular chromophore or photoacceptor [1]. One approach to
finding the identity of this chromophore(s) relevant to LLLT is to
carry out action spectra that show which wavelength(s) of light is
most effectively used in a specific chemical reaction. In 1989 Karu
suggested that the mechanism of LLLT at the cellular level was based
on the absorption of monochromatic visible and NIR by components
of the cellular respiratory chain [2].
Perhaps in three main areas of medicine and veterinary practice,
LLLT has a major role to play: (i) wound healing, tissue repair, and
prevention of tissue death; (ii) relief of inflammation in chronic
diseases and injuries with its associated pain and edema; and
(iii) relief of neurogenic pain and some neurological problems.
Figure 21.1 shows the first law of photobiology and the major roles
of LLLT.

Figure 21.1 Schematic representation of the main areas of application of

LLLT.
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374 Transcranial Low-Level Laser (Light) Therapy for Stroke and Traumatic

Biological responses of living cells to photon irradiation are

initiated by photon absorption in intracellular chromophores or
photoacceptors. The absorbed photon’s energy excites the photoac-
ceptor molecule into a more energetic (higher) electronic state,
resulting in a physical and/or chemical molecular change ultimately
leading to the cell’s biological response. Potential intracellular
photoacceptors have been suggested in previous chapters. However,
in all cases the photon/photoacceptor interaction depends on the
absorption spectrum of the photoacceptor and the photon wave-
length; matching the wavelength to the peak in the photoacceptor
absorption spectrum maximizes the probability of absorption and
very likely the cell’s biological response.
The main effects of LLLT on mammalian cells in general and the
molecular mechanisms leading to biological effects are graphically
illustrated in Fig. 21.2. To summarize: there is good evidence that
LLLT increases mitochondrial respiration probably by activating

Figure 21.2 Molecular mechanisms of tLLLT. Light is absorbed by cy-

tochrome c oxidase in the mitochondrial respiratory chain of the cortical
neurons. Cell signaling and messenger molecules are upregulated as a result
of stimulated mitochondrial activity, including reactive oxygen species
(ROS), nitric oxide (NO), and adenosine triphosphate (ATP). These signaling
molecules activate transcription factors, including NF-kB and AP-1, that
enter the nucleus and cause transcription of a range of new gene products.
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LLLT on Neuronal Cells 375

cytochrome c oxidase [3], increases the production of adenosine

triphosphate (ATP) [4], increases the amount of intracellular
reactive oxygen species (ROS) [5]. In many cases, increases in cell
proliferation, migration, and resistance to death have been observed
[6]. Recently, the Hamblin group also found that LLLT using 810 nm
increased activation of the transcription factor NF-kB [7].

21.3 LLLT on Neuronal Cells

The nervous system is divided into two parts: the central nervous
system, which consists of the brain and the spinal cord, and
peripheral nervous system, which consists of cranial and spinal
nerves along with their associated ganglia. The nervous system is,
on a small scale, primarily made up of neurons. Neurons exist in a
number of different shapes and sizes and can be classified by their
morphology and function. The anatomist Camillo Golgi grouped
neurons into two types: type I with long axons used to move signals
over long distances and type II without axons. Type I cells can be
further divided by where the cell body or soma is located. The basic
morphology of type I neurons, represented by spinal motor neurons,
consists of a cell body called the soma and a long thin axon, which is
covered by the myelin sheath. Around the cell body is a branching
dendritic tree that receives signals from other neurons. The end
of the axon has branching terminals (axon terminal) that release
transmitter substances into a gap called the synaptic cleft between
the terminals and the dendrites of the next neuron [8]. Figure 21.3
shows LLLT interacting with neuronal cells.
In cultured human neuronal cells, LLLT resulted in the doubling
of ATP content. LLLT also increased heat shock proteins and
preserved mitochondrial function [3]. Oron et al. showed that
Ga–As laser (808 nm, 50 mW/cm2 , 0.05 J/cm2 ) irradiation can
enhance ATP production in normal human neural progenitor cells
in culture; ATP concentrations were measured 10 min after laser
application [9].
Ignatov et al. [10] used intracellular dialysis and membrane
voltage clamping to show that He–Ne laser irradiation of a pond snail
neuron at a dose of 0.07 mJ increases the amplitude of the potential-
July 6, 2016 17:27 PSP Book - 9in x 6in 21-Hamblin-c21

376 Transcranial Low-Level Laser (Light) Therapy for Stroke and Traumatic

Axon
Dendrites

Cell Body
Nucleus Axon
Schwann’s Terminals
Cells

Figure 21.3 LLLT on neuronal cells. There are reports of LLLT being
effective in many different kinds of neuronal cells.

dependent slow potassium current, while a dose of 7 mJ decreases

this current. Bupivacaine can also suppress the potassium current,
and it was found that combined application of laser irradiation at a
dose of 0.7 mJ increased the blocking effect of 10 μM bupivacaine
on the slow potassium current, while an irradiation dose of 0.07 mJ
weakened the effect of bupivacaine.
Recently, Ying et al. reported that pretreatment with NIR light
via light-emitting diode (LED) significantly suppressed rotenone
or MPP(+)-induced apoptosis in both striatal and visual cortical
neurons from newborn rats, and that pretreatment plus LED
treatment during neurotoxin exposure was significantly better than
LED treatment alone during exposure to neurotoxins [11].
These in vitro findings suggest that LLLT could have beneficial
effects in animal models of neurological disorders and could be a
treatment for stroke.

21.4 Human Skull Transmission Measurements

In 1981 Wan et al. reported the transmittance of nonionizing

radiation in human tissues [12]. Spectral transmittance of 400–
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Human Skull Transmission Measurements 377

Figure 21.4 Ex vivo human skull transmission measurement.

865 nm radiation through various human structures, including

the skull with scalp, the chest wall, abdominal wall and scrotum,
is presented. There is essentially no visible light of wavelengths
shorter than 500 nm transmitted through the chest or the
abdominal wall. Transmittance of all tissues increases progressively
with wavelength from 600 to 814 nm. Tissue thickness, optical
absorption, and scattering are major influencing factors [13].
Lychagov et al. presented the recent results of measurements
of transmittance of high-power laser irradiation through skull
bones and scalp [14]. Figure 21.4 shows the ex vivo human
skull transmission measurement experimental setup. Character of
transmittance was investigated, and characteristics of heterogeneity
of the scattering structure of the skull bones were shown. Besides
that, variation of temperature of skull and scalp surfaces under
exposure of high-power laser irradiation during experiments was
controlled. Experimental results were verified by Monte Carlo
simulations. Figure 21.5 shows the transmission of 808 nm NIR
light through fresh human cadaver brain as measured by an infrared
camera.
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378 Transcranial Low-Level Laser (Light) Therapy for Stroke and Traumatic

Figure 21.5 IR camera photos. Fresh human cadaver brain. 808 nm infrared
wavelength transmits through skin, bone, and dura to reach the cortex.

21.5 Epidemiology of Stroke

Stroke is the third leading cause of death in the United States, after
heart disease and cancer [15]. Each year, approximately 780,000
people experience a new or recurrent stroke. Approximately
600,000 of these are first attacks, and 180,000 are recurrent
attacks [16]. The 3-month mortality rate from ischemic stroke is
approximately 12%. Internationally, millions of people have a new
or recurrent stroke each year, and nearly a quarter of these people
die. Globally, stroke death rates vary widely; the highest rates are in
Portugal, China, Korea, and most of Eastern Europe, and the lowest
rates are in Switzerland, Canada, and the United States.
Strokes can be classified into two major categories: ischemic
and hemorrhagic. Ischemic strokes account for over 80% of all
strokes. The most common cause of ischemic stroke is the blockage
of an artery in the brain by a clot, thrombosis, embolism, or
stenosis. Hemorrhagic strokes are the result of rupture of a cerebral
artery, which can lead to spasm of the artery and various degrees
of bleeding. Until recently, care of subjects with ischemic stroke
was largely supportive, focusing on prevention and treatment
of respiratory and cardiovascular complications. Common acute
complications of stroke include pneumonia, urinary tract infection,
and pulmonary embolism. Long-term morbidity in survivors of
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Mechanisms of Brain Injury after Stroke 379

stroke is common, with ambulation difficulty in 20%, need for

assistance in activities of daily living in 30%, and vocational
disability in 50–70% of patients.

21.6 Mechanisms of Brain Injury after Stroke

Ischemic stroke is a complex entity with multiple etiologies

and variable clinical manifestations. Thrombosis can form in the
extracranial and intracranial arteries when the intima is roughened
and plaque forms along the injured vessel. The endothelial injury
permits platelets to adhere and aggregate; then coagulation is
activated and thrombus develops at the site of plaque. When the
compensatory mechanism of collateral circulation fails, perfusion is
compromised, leading to decreased perfusion and cell death. During
an embolic stroke, a clot travels to brain through an artery. Cells
in the core ischemic zone die within minutes. Ischemia impairs the
patient’s neurologic function. Cells in the penumbra of the injury
remain potentially salvageable for hours [17]. Figure 21.6 shows the
physiology of acute ischemic stroke.

Figure 21.6 Acute ischemic stroke physiology.

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380 Transcranial Low-Level Laser (Light) Therapy for Stroke and Traumatic

When an ischemic stroke occurs, the blood supply to the brain

is interrupted, and brain cells are deprived of the glucose and
oxygen they need to function. The human brain comprises 2%
of body weight but requires 20% of total oxygen consumption
[18]. The brain requires this large amount of oxygen to generate
sufficient ATP by oxidative phosphorylation to maintain and restore
ionic gradients. One estimate suggests that the Na+ /K+ -ATPase
found on the plasma membrane of neurons consumes 70% of
the energy supplied to the brain. This ion pump maintains the
high intracellular K+ concentration and the low intracellular Na+
concentrate ion necessary for the propagation of action potentials.
After global ischemia, mitochondrial inhibition of ATP synthesis
leads to ATP being consumed within 2 min. This causes neuronal
plasma membrane depolarization, release of potassium into the
extracellular space, and entry of sodium into cells [19]. Energy
failure also prevents the plasma membrane Ca2+ ATPase from
maintaining the very low concentrations of calcium that are
normally present within each cell.
Stroke can manifest as focal injuries (contusions, lacerations,
hematomas) or diffuse axonal injury or a combination thereof.
Diffuse axonal injury has been recognized as one of the main con-
sequences of blunt head trauma and blast injuries [20]. Secondary
injuries are attributable to further cellular damage that results from
follow-on effects of the primary injuries and develop over a period
of hours or days following initial traumatic assault. Secondary brain
injury is mediated through excitotoxic cell death in which injured
neurons depolarize and release glutamate [21]. Neighboring cells
are, in turn, depolarized by the excessive glutamate concentrations
and result in a vicious spiral of increasing glutamate concentration.
Depolarized neurons suffer a huge influx of sodium and calcium
but could survive if they had sufficient ATP to power the Na+ /K+ -
ATPase pumps and handle this osmotic load. However, in the setting
of metabolic failure, they exhibit cytotoxic edema, with eventual loss
of viability [22]. An influx of calcium linked to delayed damage,
a decrease in mitochondrial membrane potential, and increased
production of ROS are observed. Other biochemical processes
exacerbating stroke include activation of astrocytes and microglia,
leading to increased cytokines contributing to inflammation and
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Thrombolytic Therapy of Stroke 381

characterized by increased prostaglandin E2 [23]. Excitotoxic cell

death mediated by glutamate also affects glial cells as well as neu-
rons, in particular oligodendrocytes [24]. ROS have been implicated
as key participants of other acute CNS injuries, such as TBI, spinal
cord injury, and ischemia, as well as chronic neurodegenerative
diseases [25]. Stroke causes physical disruption of neuronal tissue,
which sets into motion secondary damage resulting in the death of
additional tissue. Hypoperfusion- and ischemia-induced increases
in the formation of superoxide and nitric oxide (NO) have been
reported after stroke, predicting a role for oxidant stress during
damage [26]. Cellular protection against these ROS involves an
elaborate antioxidant defense system, including that associated with
manganese superoxide dismutase.

21.7 Thrombolytic Therapy of Stroke

Thrombolytic therapy is the only intervention of proven and sub-

stantial benefit for select patients with acute cerebral ischemia [27].
The evidence based for thrombolysis therapy includes 21 completed
randomized controlled clinical trials enrolling 7152 patients, using
various agents, doses, time windows, and intravenous or intra-
arterial modes of administration [28].
The main agent that has been employed is recombinant tissue
plasminogen activator (t-PA) [29]; t-PA is produced endogenously
by endothelial cells and is relatively fibrin specific. T-PA works by
converting the proenzyme plasminogen to the activated enzyme
plasmin. Activated plasmin, in turn, dissolves fibrin clots into low
molecular weight fibrin degradation products. Other thrombolytics
that have been used include streptokinase [30, 31].
Time lost is brain lost in acute cerebral ischemia. In a typical
middle cerebral artery (MCA) ischemic stroke, two million nerve
cells are lost each minute in which reperfusion has not been
achieved [32]. A pooled analysis of all 2775 patients enrolled in
the first six intravenous t-PA trials provided clear and convincing
evidence of a time-dependent benefit of thrombolytic therapy
[33].
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382 Transcranial Low-Level Laser (Light) Therapy for Stroke and Traumatic

21.8 Investigational Neuroprotectants and

Pharmacological Intervention

Neuroprotection is defined as any strategy, or combination of

strategies, that antagonizes, interrupts, or slows the sequence of
injurious biochemical and molecular events that, if left unchecked,
would eventuate in irreversible ischemic injury to the brain. An
enormous variety of agents and strategies have received clinical
scrutiny, each justified by a pathophysiological rationale. In all,
approximately 165 ongoing or completed clinical trials have been
published [34]. The outcome has been an almost universal catalogue
of failure for all of these trials, with exceptions of some small hint of
efficacy in only a few cases.

21.9 Transcranial LLLT for Stroke

The beneficial effects of NIR light on cells and neurons in vitro,

together with the demonstrated ability of NIR light to penetrate
into the brain, strongly suggested that tLLLT should be studied as
a therapy for stroke.
Transcranial LLLT at 808–810 nm can penetrate the brain and
was shown to lead to enhanced production of ATP in the rat cerebral
cortex [3]. Findings of increased neurogenesis in the subventricular
zone (SVZ) were reported in an ischemic stroke animal model
treated with tLLLT [35]. Based on these findings, it is thought
that tLLLT may have multiple mechanisms of action and could be
beneficial in acute ischemic stroke [36].

21.9.1 Transcranial LLLT in Animal Models for Stroke

Although no animal model identically mimics stroke in humans,
the use of animal models is essential for the development of
therapeutic interventions for stroke. Outcome measures for animal
models involve functional measures and infarct size evaluation.
Recommendations from the Stroke Therapy Academic Industry
Roundtable for preclinical animal models are for initial studies to be
conducted in rat followed by a second species, specifically primates.
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Transcranial LLLT for Stroke 383

Figure 21.7 Measurements of 810 nm laser transmission in the skull of

different animal models.

Ischemia is typically induced by occluding the MCA in the animal.

The MCA is most often used to simulate human stroke as most
human strokes are due to occlusion of this vessel or one of its
branches [37]. Animals that have been used in ischemia models
for various specific reasons include rats, mice, gerbils, cats, rabbits,
dogs, pigs, and nonhuman primates. Figure 21.7 shows noninvasive
parameter measurement of animal models’ skull.
The first animal model for tLLLT on brain was reported by Azbel
(1993) in which synaptic conductance in hippocampal neurons in
rat models was demonstrated [38]. In vivo studies have suggested
that infrared laser therapy could be beneficial for the treatment of
acute myocardial infarction as shown by Ad and Oron, who showed
in 2001 that tLLLT reduced the loss of myocardial tissue and the
severity of acute myocardial infarction following chronic ligation of
the left anterior descending coronary artery in laboratory rats [39].
In 2002, Leung et al. used a model of transient cerebral ischemia;
tLLLT inhibited nitric oxide synthase activity and unregulated
expression of TGF beta-1 [40]. In 2004, Lapchak showed that laser
treatment with 7.5 mW/cm2 at 6 h post-stroke onset in a rabbit
small clot embolic stroke model (RSCEM) improved behavioral
performance and produced a durable effect that was measurable
21 days after embolization [41]. Lapchak’s further research showed
tLLLT improved motor function following embolic strokes in rabbits
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384 Transcranial Low-Level Laser (Light) Therapy for Stroke and Traumatic

Figure 21.8 Rat model of stroke being treated with tLLT.

[42, 43]. In 2006, De Taboada et al. [44] showed in two different

animal models a positive impact of infrared laser therapy on the
experimental, ischemic stroke treatment outcomes in New Zealand
rabbits subjected to an RSCEM and also in Sprague Dawley rats
(permanent MCA occlusion). De Taboada et al. have also shown
that laser treatment up to 24 h post-stroke onset in permanent
MCA occlusion showed significant improvement in neurological
deficits, which was evident at 14, 21, and 28 days post-stroke
when compared with the sham control group [44]. Meanwhile, Oron
reported that a noninvasive intervention of tLLLT issued 24 h after
acute stroke may provide a significant functional benefit with an
underlying mechanism possibly being induction of neurogenesis
[35]. Currently, the putative mechanism for infrared laser therapy
in stroke involves the stimulation of mitochondria, which then leads
to preservation of tissue in the ischemic penumbra and enhanced
neuron recovery. The exact mechanistic pathways remain to be
elucidated. Figure 21.8 shows how the treatment is carried out in
a mouse model of stroke.
Table 21.1 summarizes the studies that have looked at tLLLT in
animal models of ischemic stroke.
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Traumatic Brain Injury 385

Table 21.1 Reports of tLLLT used for stroke in animal models

Stroke
Subject model Parameters Effect References
2
Rat MCAO 660 nm; 8.8 mW; 2.64 J/cm ; Suppression of NOS activity and [40]
pulse frequency 10 kHz. Laser upregulation of TGF-β1
was applied at cerebrum at 1 5
and 10 min
Rabbit RSCEM 808 ±5 nm; 7.5 mW/cm2 , 2 min Improvement in behavioral [41]
duration 3 h after stroke and 25 performance and durable effect
mW/cm2 10 min duration 1 or with LLLT within 6 h from
6 h after stroke; stroke onset
Rat MCAO 808 nm; 7.5 mW/cm2 ; 0.9 Administration of LLLT after 24 [35]
J/cm2 ; 3.6 J/cm2 at cortical h after stroke onset induces
surface; CW and 70 Hz, 4 mm functional benefit and
diameter mechanism of neurogenesis
induction
Rat MCAO 808 nm; 0.5 mW/cm2 ; 0.9 LLLT applied at different [44]
J/cm2 on brain 3 mm dorsal to location in the skull improve
the eye and 2 mm anterior to neurological function after
the ear acute stroke
Rabbit RSCEM 808 nm; 7.5 mW/cm2 ; 0.9 LLLT administered 6 h after [42]
J/cm2 ; 3.6 J/cm2 at cortical embolic stroke results in clinical
surface; CW; 300 μs; pulse at improvements in rabbits
1 kHz, 2 ms at 100 Hz

21.10 Traumatic Brain Injury

Traumatic brain injury occurs when an outside force traumatically

injures the brain. TBI includes skull fractures, intracranial hem-
orrhages, elevated intracranial pressure, and cerebral contusions.
Unlike stroke, which is often associated with senior citizens, TBI
affects a predominantly young population. Severe and moderate
TBI, whether accidental or inflicted, is a major health and socioeco-
nomic problem throughout the world. In the United States alone,
approximately 2 million injuries occur each year resulting in 56,000
deaths and 18,000 survivors suffering from permanent neurological
impairment [45–47]. The consequent direct and indirect annual
costs in the United States are estimated at $56 billion [48]. The
World Health Organization has projected that by 2020, road traffic
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386 Transcranial Low-Level Laser (Light) Therapy for Stroke and Traumatic

accidents, a major cause of TBI, will rank third as a cause of the

global burden of disease and disablement, behind only ischemic
heart disease and unipolar major depression [49]. Despite advances
in our understanding of the pathophysiological damage that occurs
following brain injury, current treatments are limited both in their
efficacy and utility [50]. The pathophysiology of TBI is very complex
and still poorly understood. Immediately following the primary
impact, activation of several different pathways begins, resulting
in secondary brain injury. These include inflammation, oxidative
stress, ionic imbalance, increased vascular permeability, mitochon-
drial dysfunction, and excitotoxic damage [51]. These processes
result in brain edema, increased intracranial pressure, and impaired
cerebral perfusion. This combination of cellular and physiologic
disturbances causes increased neuronal cell death, enlargement of
infarct size and neurological, motor, and cognitive impairment. TBI
can result in neurological impairment because of immediate CNS
tissue disruption (primary injury), but, additionally, surviving cells
may be secondarily damaged by complex mechanisms triggered by
the primary event, leading to further damage and long-term disabil-
ity [52]. Efforts to improve the treatment and outcome of TBI must,
therefore, remain the priority for clinicians and researches [53].
Although TBI is a severe concern to global health care, the search
for better therapies in the recent years has not been successful.
This has led to interest in more radical alternatives to existing
procedures, such as tLLLT.

21.10.1 Transcranial LLLT Studies for TBI in Animal Models

The success of tLLLT for stroke encouraged researchers to test
the technique in animal models of TBI. Oron et al. evaluated the
effects of LLLT for TBI in mice. Closed-head injury of mice was
induced by using a weight-drop device. An 808 nm Gs-As diode
laser with two different energy densities (1.2–2.4 J/cm2 over 2
min irradiation with 10 and 20 mW/cm2 ) was delivered to the
brain 4 h after TBI. Neurobehavioral function was assessed by
neurological severity score (NSS). There was no statistical difference
in NSS between the power density of 10 and 20 mW/cm2 . There
was no significant difference between control/non-laser-treated
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Traumatic Brain Injury 387

group and laser-irradiated group at 24 h and 48 h post-CHI. There

was a significant improvement in neurobehavioral function in the
laser-irradiated groups from day 5 up to day 28, where the NSS
was 26–27% lower in the laser-irradiated group. The laser-treated
group showed a lower loss (1.4%) of cortical tissue at the injured
site compared to the sham control group (12.1%) (P < 0.001).
This study suggested that tLLLT significantly reduced long-term
neurological deficits [54].
Moreira et al. reported the effect of low-intensity laser photother-
apy on local and systemic immunomodulation following cryogenic
brain injury in rat. The rats were irradiated with 780 nm and 660 nm
laser on 3 J/cm2 and 5 J/cm2 . This study concluded that LLLT could
modulate TNF-alpha, IL-6, and IL-beta concentrations in the brain
and blood of rats with cryogenic brain injury [55].
Khuman et al. proved that LLLT treatment could improve
cognitive deficits after CCI in mice. CCI was induced by a 3 mm
flat-tipped pneumatic piston at a velocity of 6 m/s and a depth of
0.6 mm, for 100 ms duration. The mice were randomly divided into
opentranscranial group and transcranial group. The former group
underwent 800 nm low-level laser irradiation with different energy
levels (30, 60, 105, 120, 210, and 0 J/cm2 ) for 60–80 min after CCI
and the latter group underwent 60 J/cm2 at different time points
(60–80 mins or 4 h after CCI, or one treatment per day for 7 days).
Cognitive function by Morris water maze (MWM), motor function by
wire-grip test, brain edema, lesion volume, and nitrosative stress by
nitrotyrosine ELISA were assessed. Mice with CCI treated with 60
J/cm2 (500 mW/cm2 × 2 min) had significant improvement in the
latency to the hidden platform and probe trails either via an open
craniotomy or transcranially. An anti-inflammatory effect was noted
via a significant reduction in microgliosis at 48 h with 60 J/cm2 LLLT.
There was no significant difference in motor function (day 1 to 7),
brain edema (24 h), nitrosative stress (24 h), or lesion volume (14
days) between LLLT and control groups [56].
Oron et al. [57] next examined the long-term effect of various
transcranial laser therapy modes (pulsed versus continuous) and
at different treatment time points in mild to moderate closed-head
injury mice (NSS 4–6) induced using a weight-drop device. A Ga–
Al–As 808 nm wavelength laser with an energy level of 1.2 J/cm2
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388 Transcranial Low-Level Laser (Light) Therapy for Stroke and Traumatic

(10 mW/cm2 for 2 min) was delivered 4 h, 6 h, and 8 h post-

injury transcranially. In another experiment, the laser was applied
at a dose of 10 mW/cm2 at 100 Hz, 600 Hz or CW 4 h post-CHI.
The differences in NSS of the laser-treated group 6 h and 8 h post-
injury were 3.4- and 1.8-times that in control non-treated group at
day 56. Compared to the control non-treated group, there was an
approximately 3.5-fold increase in differences in NSS for all three
modes of laser therapy (100 Hz, 600 Hz, and CW). Mice received
tLLLT with PW at 100 Hz 4 h post-injury and had the highest full
recovery (NSS 0) percentage (67%) at day 56. The lesion size was
significant smaller in both CW and PW laser-treated group than the
control group on MRI [57].

21.10.2 Effect of Different Laser Wavelengths in tLLLT in

Closed-Head TBI Model in Mice
The following sections will summarize studies from our laboratory
that have explored the use of tLLLT to treat TBI in animal models.
Wu et al. [58] investigated the effect of different wavelengths of
LLLT for closed-head TBI in mice. Closed-head injury was induced by
using a weight-drop apparatus. Moderate to severe degree TBI mice
(after assessing NSS 6–8, 1 h post-injury) randomly received a single
irradiation with 665, 730, 810, or 980 nm laser with 36 J/cm2 (150
mW/cm2 over 4 min) at 4 h after contusion. The 665 nm and 810
nm groups showed significant improvements in NSS than the sham-
treated control group after day 5 to day 28 as shown in Fig. 21.9
The mean fractional areas of brain determined by morphometry
for 665 nm and 810 nm LLLT groups were significantly decreased
compared to the fractional area for the sham-treated control group
at day 28 [58]. NIR lasers at 730 or 980 nm did not produce the same
beneficial effects on NSS [58]. The principal tissue chromophore
responsible for photobiomodulation effects induced by LLLT is
cytochrome c oxidase (Cox). Cox has distinct absorption bands in
the red (∼665 nm) and NIR (∼810 nm), and there is a minimum
Cox absorption spectrum at 730 nm [59]. Furthermore, the study
mentioned that 980 nm laser did not produce the same positive
effect. However, other previous reports have shown that 980 nm is
an active wavelength in LLLT applications. Wu et al. suggested that
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Traumatic Brain Injury 389

Figure 21.9 Effect of different laser wavelengths in tLLLT in closed-head

TBI in mice. Time course of NSS scores of sham, and laser-treated mice. (A)
Sham-treated control versus 665 nm laser; (B) sham-treated control versus
730 nm laser; (C) sham-treated control versus 810 nm laser; (D) sham-
treated control versus 980 nm laser. Points are means of 8–12 mice and bars
are SD. * p<0.05; ** p,0.01; *** P<0.001 (one-way ANOVA). Reprinted with
permission from Wu et al. [58].

the ineffectiveness of 980 nm in their study might be due to the

different laser dosimetry (irradiance, energy density, etc.) selected
for their study [58].

21.10.3 Effect of Pulsing in LLLT for CCI-TBI in Mice

Although many studies have shown common consensus on the best
wavelengths of laser for treatment of brain disorders and a range
of accepted dosages to be used (irradiance and fluence), there is no
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390 Transcranial Low-Level Laser (Light) Therapy for Stroke and Traumatic

consensus on whether CW or pulse light is best and what factors

govern the pulse parameters to be chosen. Ando et al. [60] compared
the same 810 nm laser parameters delivered in different modes,
pulsed at either 10 Hz or 100 Hz with 50% duty cycle compared with
CW laser in a mouse TBI model. TBI was induced by a controlled
cortical impact device through an open craniotomy. The head was
closed up, and 4 h after injury, LLLT was delivered to the top of
the head using a 1 cm diameter spot. They used an 810 nm Ga–
Al–As diode laser with a single exposure with an average power
density of 50 mW/cm2 for 12 min giving a total fluence of 36 J/cm2 .
At 48 h to 28 days after TBI, NSS showed a significant decrease in
all laser-treated groups. The improvement in the 10 Hz group was
greater than that in the PW 100 Hz and CW groups after day 7,
as shown in Fig. 21.10. The PW 10 Hz group showed a significant
decrease in immobility time in the forced swim test for depression
and anxiety compared to the untreated TBI group at day 28. There

Figure 21.10 Effect of pulsing in tLLLT for CCI-TBI in mice. Time course
of neurological severity score (NSS) of mice with CCI-TBI receiving either
control (no laser treatment) or 810 nm laser (36 J/cm2 delivered at 50
mW/cm2 with a spot size of 0.78 cm2 ) in CW, PW 10 Hz, or PW 100 Hz
modes. Results are expressed as mean ± S.E.M (n = 10). Reprinted with
permission from Ando et al. [60].
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Traumatic Brain Injury 391

was a significant decrease in the immobility periods of the tail

suspension test (another measure of depression and anxiety) in the
PW 10 Hz group compared with the untreated TBI group at day
1 and day 28. These results suggest the antidepressant effect of
LLLT. A significant decrease in lesion size in brain tissue around the
traumatized site was noted in the PW 10 Hz group at day 15 and day
28. It implies that LLLT has a neuroprotective effect at the early stage
of TBI. The therapeutic effects, including the severity of injury, the
antidepressant effects, and the protection of brain tissue of LLLT for
TBI with 810 nm laser were more effective at 10 Hz pulse frequency
than at 100 Hz and CW. Ando et al. also hypothesized that laser
irradiation at 10 Hz pulsed mode was most effective for improving
neurological outcome because the frequency possibly affects the
whole brain. Resonance may occur between the frequency of the
pulsed light and that of the brain waves. Particularly relevant is the
oscillation of theta waves that have a prominent 4–10 Hz rhythm in
the hippocampal region of all mammals [61].

21.10.4 Effects of tLLLT-Repetition Regimen in CCI-TBI in

Mice
The efficacy of LLLT on TBI has been previously investigated to a
limited extent. However, there are still many questions to be solved:
for example, what is the best regimen of treatment repetition?
It has been well established during the 40 years of LLLT studies
that there is a pervasive biphasic dose response relationship that
applies not only in cell culture studies, but also in preclinical animal
studies and even in clinical reports [62]. It has been found that
there is generally an optimum level of energy density (J/cm2 ), power
density (mW/cm2 ), and/or treatment repetition required to give the
best therapeutic effects. A less than optimal choice of parameters
can result in the reduced effectiveness of the treatment, or even a
negative therapeutic outcome [63].
Xuan et al. [64] used a CCI mouse model of severe TBI and
studied the effects of different treatment repetitions of 810 nm LLLT
on neurobehavioral and vestibulomotor functioning, histomorpho-
logical analysis, and histological evidence of neuroprotection and
neurogenesis. The animals of the TBI treatment groups received
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392 Transcranial Low-Level Laser (Light) Therapy for Stroke and Traumatic

Figure 21.11 Effect of treatment repetition of tLLLT for CCI-TBI in mice.

Time course of neurological severity score (NSS) of mice with sham
(craniotomy but no TBI), TBI control receiving no laser treatment, or three
groups of treated mice 810 nm laser (18 J/cm2 delivered at 25 mW/cm2
with a spot size of 0.78 cm2 ) given once at 4 h post-TBI, three times once
each day or 14 times once each day both starting 4 h post-TBI.

tLLLT (CW 810 nm laser, 25 mW/cm2 , 18 J/cm2 ) once at 4 h post-

TBI, three daily treatments, or 14 daily treatments. They found that
LLLT may have beneficial effects in the acute treatment of TBI and
demonstrated that mice with severe TBI treated with once laser
treatment (and to a greater extent three daily laser) had significant
improvements in NSS, and wire-grip and motion test. However, 14
daily laser treatments provided no benefit (Fig. 21.11) as an example
of the biphasic dose response. Furthermore, the study concluded
that LLLT for TBI in mice could significantly improve neural function,
decrease lesion volume, augment cell proliferation, and even protect
the brain against neuronal damage to some degree.
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Traumatic Brain Injury 393

0.07

BrdU/DAPI ration in DG at day 7

0.06

0.05

BrdU/DAPI
0.04

0.03

0.02

0.01

0
sham TBI 1 laser 3 laser

Figure 21.12 Neuroprogenitor cells at the 7 days’ point (BrdU—green and

NeuN—red) in the dentate gyrus; (A) sham, (B) CCI-TBI, (C) 1 × tLLLT, (D) 3
× tLLLT, (E) mean BrdU ratios ± SD (n = 5); normalization of the readings
were done BrdU versus DAPI (blue labeling the nuclei). Scale bar 100 μm.
*** P <0.001 versus sham; ††, †††P<0.01, 0.001 versus TBI.

21.10.5 Transcranial tLLLT in Mice with TBI Stimulates the

Brain to Repair Itself
We used the same model of CCI-TBI in Balb/c mice treated with
either 1 or 3 laser exposures to tLLLT (CW 810 nm laser, 25
mW/cm2 , 18 J/cm2 ) given either once at 4 h post-TBI, or as three
daily treatments [65]. In these studies, we attempted to discover the
cellular mechanisms for the beneficial effect. Immunofluorescence
studies revealed that neuroprogenitor cells (BrdU label retaining
cells that co-stained with the neuronal marker NeuN) were
upregulated in the dentate gyrus of the hippocampus at 7 days (and
less at 28 days) post-TBI in the mice that received one and especially
three tLLLT exposures (Fig. 21.12).
Some evidence was obtained that the newly formed neurons
found in the hippocampus could migrate across the brain to repair
the cortical lesion.
Moreover, BDNF was also upregulated in the dentate gyrus at
7 days in a similar manner to neuroprogenitor cells [66] (see
Fig. 21.13).
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394 Transcranial Low-Level Laser (Light) Therapy for Stroke and Traumatic

Figure 21.13 Brain-derived neurotrophic factor stained cells in the dentate

gyrus at the 7 days’ point. BDNF (red) (A) sham, (B) CCI-TBI, (C) 1 × tLLLT,
(D) 3 × tLLLT, (E) mean BDNF ratios ± SD (n = 5); normalization of the
readings were done BDNF versus DAPI (blue labeling the nuclei). Scale bar
100 μm. *** P <0.001 versus sham; ††, †††P<0.01, 0.001 versus TBI.

Figure 21.14 Synapsin-1 staining in the cortex tissue surrounding the

lesion at the 28 day time point. Synapsin 1 (red). (A) Sham, (B) CCI-TBI,
(C) 1 × tLLLT, (D) 3 × tLLLT, (E) mean BrdU/DAPI ratios ± SD (n = 5);
normalization of the readings were done synapsin-1 versus DAPI (blue
labeling the nuclei). Scale bar 100 μm. *** P <0.001 versus sham; ††,
†††P<0.01, 0.001 versus TBI.
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Conclusion 395

Not only can tLLLT cause the brain to produce new brain cells,
but it can also stimulate the creation of new connections between
existing brain cells, a process known as synaptogenesis or neural
plasticity. This was shown by staining the sections with an antibody
against synapsin-1, a protein that is characteristic of newly formed
synaptic connections, as shown in Fig. 21.14 [66]. However, it was
interesting to note that the highest upregulation of synapsin-1 was
found at 28 days (not at 7 days), and the best region of the brain
was the cortex tissue surrounding the brain lesion, not the dentate
gyrus. This observation implies that the production of BDNF in the
hippocampus can orchestrate brain repair in distant regions of the
brain and that this process takes some time (a few weeks).

21.11 Conclusion

Evidence that tLLLT is a beneficial treatment for acute TBI and acute
stroke is rapidly accumulating. In the past, a number of published
studies that showed that tLLLT was effective for acute stroke
suggested that the same approach would also be effective for acute
TBI, which shares many of the pathophysiological features found in
ischemic stroke. The benefits of tLLLT appear to be based on many
different biological mechanisms. The existence of neuroprotection
or the ability of the laser to prevent the spread of brain cell death
that occurs in the hours and days after a brain lesion is formed is
shown by the smaller size of the lesion area in LLLT-treated animals.
Anti-inflammatory, anti-edema, and pro-angiogenic effects of LLLT
may also have roles to play in the beneficial effects. Perhaps the most
exciting possible beneficial mechanism is that LLLT may stimulate
neurogenesis or increase the ability of the brain to repair itself. Not
only may new brain cells be formed after LLLT, but the existing brain
cells may also be encouraged to form new synaptic connections
in the process known as synaptogenesis or synaptic plasticity. If
these processes can be reliably shown to occur after tLLLT, it
opens the door to the treatment being applied to Alzheimer’s
and Parkinson’s diseases and also for many diverse psychiatric
disorders.
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396 Transcranial Low-Level Laser (Light) Therapy for Stroke and Traumatic

Acknowledgments

Research in the Hamblin laboratory is supported by NIH grant

R01AI050875, Center for Integration of Medicine and Innova-
tive Technology (DAMD17-02-2-0006), CDMRP Program in TBI
(W81XWH-09-1-0514), and Air Force Office of Scientific Research
Military Photomedicine Program (FA9550-11-1-0331).

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24. Matute, C., et al., Excitotoxic damage to white matter. J Anat, 2007.
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26. Cherian, L., and C.S. Robertson, L-arginine and free radical scavengers
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thrombolysis for acute ischaemic stroke. Trials, 2008. 9(1): pp. 37.
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irradiation on the process of bone repair in the rat tibia. Bone, 1995.
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30. Donnan, G.A., et al., Trials of streptokinase in severe acute ischaemic
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2011.
32. Saver, J.L., Time is brain–quantified. Stroke, 2006. 37(1): pp. 263–266.
33. Hacke, W., et al., Association of outcome with early stroke treatment:
Pooled analysis of ATLANTIS, ECASS, and NINDS rt-PA stroke trials.
Lancet, 2004. 363(9411): pp. 768–774.
34. Ginsberg, M.D., Neuroprotection for ischemic stroke: Past, present and
future. Neuropharmacology, 2008. 55(3): pp. 363–389.
35. Oron, A., et al., Low-level laser therapy applied transcranially to rats
after induction of stroke significantly reduces long-term neurological
deficits. Stroke, 2006. 37(10): pp. 2620–2624.
36. Lampl, Y., Laser treatment for stroke. Expert Rev Neurother, 2007. 7(8):
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37. Philip, M., et al., Methodological quality of animal studies of neuro-
protective agents currently in phase II/III acute ischemic stroke trials.
Stroke, 2009. 40(2): pp. 577–581.
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38. Azbel, D.I., et al., The effect of the blood serum from patients subjected to
intravenous laser therapy on the parameters of synaptic transmission.
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39. Ad, N., and U. Oron, Impact of low level laser irradiation on infarct size
in the rat following myocardial infarction. Int J Cardiol, 2001. 80(2–3):
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40. Leung, M.C., et al., Treatment of experimentally induced transient
cerebral ischemia with low energy laser inhibits nitric oxide synthase
activity and up-regulates the expression of transforming growth factor-
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41. Lapchak, P.A., J. Wei, and J.A. Zivin, Transcranial infrared laser therapy
improves clinical rating scores after embolic strokes in rabbits. Stroke,
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42. Lapchak, P.A., et al., Transcranial near-infrared light therapy improves
motor function following embolic strokes in rabbits: An extended
therapeutic window study using continuous and pulse frequency
delivery modes. Neuroscience, 2007. 148(4): pp. 907–914.
43. Lapchak, P.A., and D.M. Araujo, Advances in ischemic stroke treatment:
Neuroprotective and combination therapies. Expert Opin Emerg Drugs,
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44. Detaboada, L., et al., Transcranial application of low-energy laser
irradiation improves neurological deficits in rats following acute stroke.
Lasers Surg Med, 2006. 38(1): pp. 70–73.
45. Sosin, D.M., J.E. Sniezek, and D.J. Thurman, Incidence of mild and
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46. Bruns, J., Jr., and W.A. Hauser, The epidemiology of traumatic brain
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47. Kraus, J.F., and D.L. McArthur, Epidemiologic aspects of brain injury.
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48. Thurman, D.J., et al., Traumatic brain injury in the United States: A public
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50. Vink, R., and A.J. Nimmo, Multifunctional drugs for head injury.
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51. Zink, B.J., J. Szmydynger-Chodobska, and A. Chodobski, Emerging
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52. Teasdale, G.M., and D.I. Graham, Craniocerebral trauma: protection and
retrieval of the neuronal population after injury. Neurosurgery, 1998.
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53. Marklund, N., and L. Hillered, Animal modelling of traumatic brain injury
in preclinical drug development: Where do we go from here? Br J
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54. Oron, A., et al., low-level laser therapy applied transcranially to
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neurological deficits. J Neurotrauma, 2007. 24(4): pp. 651–656.
55. Moreira, M.S., et al., Effect of phototherapy with low intensity laser on
local and systemic immunomodulation following focal brain damage in
rat. J Photochem Photobiol B, 2009. 97(3): pp. 145–151.
56. Khuman, J., et al., Low-level laser light therapy improves cognitive
deficits and inhibits microglial activation after controlled cortical
impact in mice. J Neurotrauma, 2012. 29(2): pp. 408–417.
57. Oron, A., et al., Near infrared transcranial laser therapy applied at
various modes to mice following traumatic brain injury significantly
reduces long-term neurological deficits. J Neurotrauma, 2012. 29(2): pp.
401–407.
58. Wu, Q., et al., Low-level laser therapy for closed-head traumatic brain
injury in mice: Effect of different wavelengths. Lasers Surg Med, 2012.
44(3): pp. 218–226.
59. Karu, T.I., et al., Absorption measurements of a cell monolayer relevant
to phototherapy: Reduction of cytochrome c oxidase under near IR
radiation. J Photochem Photobiol B, 2005. 81(2): pp. 98–106.
60. Ando, T., et al., Comparison of therapeutic effects between pulsed and
continuous wave 810-nm wavelength laser irradiation for traumatic
brain injury in mice. PLoS ONE, 2011. 6(10): pp. e26212.
61. Sushko, B.S., P. Lymans’kyi Iu, and S.O. Huliar, Action of the red
and infrared electromagnetic waves of light-emitting diodes on the
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60.
62. Huang, Y.Y., et al., Biphasic dose response in low level light therapy. Dose
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63. Chung, H., et al., The nuts and bolts of low-level laser (light) therapy. Ann
Biomed Eng, 2012. 40(2): pp. 516–533.
64. Xuan, W., et al., Transcranial low-level laser therapy improves neurolog-
ical performance in traumatic brain injury in mice: Effect of treatment
repetition regimen. PLoS ONE, 2013. 8(1): pp. e53454.
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65. Xuan, W., et al., Transcranial low-level laser therapy enhances learning,
memory, and neuroprogenitor cells after traumatic brain injury in mice.
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66. Xuan, W., et al., Low-level laser therapy for traumatic brain injury
in mice increases brain derived neurotrophic factor (BDNF) and
synaptogenesis. J Biophotonics, 2014. 9999(9999).
July 6, 2016 17:31 PSP Book - 9in x 6in 22-Hamblin-c22

Chapter 22

Phototherapy in Peripheral Nerve Repair

and Muscle Preservation

Shimon Rochkind
Division of Peripheral Nerve Reconstruction, Department of Neurosurgery, Tel Aviv
Sourasky Medical Center, Tel Aviv University, Israel
rochkind@zahav.net.il

Injury of a peripheral nerve frequently results in considerable

disability. In an extremity, such lesions may be associated with loss
of sensory and motor functions, which leads to severe occupational
and social consequences.
Posttraumatic nerve repair and prevention of muscle atrophy
represent a major challenge of restorative medicine. When muscles
are denervated, in cases of complete peripheral nerve injury, they
deteriorate progressively. Surgical repair is the preferred modality
of treatment for the complete or severe peripheral nerve injury.
In most cases, the results can be successful if the surgery is
performed in the first 6 months after injury, in comparison to
long-term cases where surgical management is less successful. The
reason for early surgical intervention has to do with the fact that
between 1 and 3 years post-injury, denervated muscle undergoes
progressive degeneration, which leads to loss of muscle fibers

Handbook of Low-Level Laser Therapy

Edited by Michael R. Hamblin, Marcelo Victor Pires de Sousa, and Tanupriya Agrawal
Copyright c 2017 Pan Stanford Publishing Pte. Ltd.
ISBN 978-981-4669-60-3 (Hardcover), 978-981-4669-61-0 (eBook)
www.panstanford.com
July 6, 2016 17:31 PSP Book - 9in x 6in 22-Hamblin-c22

404 Phototherapy in Peripheral Nerve Repair and Muscle Preservation

and their replacement with fat and fibrous connective tissue. For
most patients who suffer from long-term peripheral nerve injuries,
spontaneous recovery is often unsatisfactory. The usual results after
such an injury are degeneration of the distal axons and retrograde
degeneration of the corresponding neurons of the spinal cord,
followed by a very slow regeneration. Recovery may eventually
occur, but it is slow and frequently incomplete. The secondary effects
of peripheral nerve injury are wasted muscles. Therefore, numerous
attempts have been made to enhance and/or accelerate the recovery
of injured peripheral nerves and decrease or prevent atrophy of the
corresponding muscles.
Among the various proposed methods for enhancing nerve
repair, phototherapy has received increasing attention over the
last two decades. The term phototherapy refers to the use of
light for producing a therapeutic effect on living tissues. Although
a pioneering report on the effects of laser phototherapy on
the regeneration of traumatically injured peripheral nerves was
published in the late 1970s [1], it is only since the late 1980s
that scientific interest was kindled in this therapeutic approach
for neural rehabilitation, leading to the publication of a number
of studies that have shown positive effects of phototherapy on
peripheral nerve regeneration [2, 3].
The possible mechanism of action of phototherapy on the
nervous tissue [4] with respect to peripheral nerve regeneration
has been provided by the in vitro studies which showed that
phototherapy induces massive neurite sprouting and outgrowth in
cultured neuronal as well as Schwann cell proliferation. Also it has
been suggested that phototherapy may enhance the recovery of
neurons from injury by altering mitochondrial oxidative metabolism
and guide neuronal growth cones in vitro, perhaps due to the
interaction with cytoplasmic proteins and, particularly, to the
enhancement of actin polymerization at the leading axon edge.
Phototherapy alters nerve cell activity, including upregulation of
a number of neurotrophic growth factors and extracellular matrix
proteins known to support neurite outgrowth. A possible molecular
explanation was provided by demonstrating an increase in growth-
associated protein-43 (GAP-43) immunoreactivity in early stages
of rat sciatic nerve regeneration after phototherapy. It was shown
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Incomplete Peripheral Nerve Injury 405

Figure 22.1 Histological section of the crushed area of a rat sciatic nerve
showing the response of the nerve to laser phototherapy. (a) Non-irradiated
nerve; note the scar ring of fibrous tissue. (b) Laser-treated nerve shows no
visible scar. Figure reprinted with permission from Ref. [3], Copyright 2009
AANS.

that application of phototherapy upregulates calcitonin gene-related

peptide (CGRP) mRNA expression in facial motor nuclei after
axotomy. By altering the intensity or temporal pattern of injury-
induced CGRP expression, phototherapy may thus optimize the rate
of regeneration and target innervation and neuronal survival of
axotomized neurons. Considerable interest exists in the potential
therapeutic value of laser phototherapy for enhancing regeneration
of severely injured peripheral nerve and restoring or temporary
preventing denervated muscle atrophy.

22.1 Incomplete Peripheral Nerve Injury

Animal studies have shown that in an experimental peripheral

nerve crush injury model (such as traffic accidents, sports-related
injuries, etc.), laser phototherapy decreases scar tissue formation at
the injury site (Fig. 22.1), decreases degeneration in corresponding
motor neurons of the spinal cord (Fig. 22.2), and increases axonal
growth and myelinization (Fig. 22.3). Moreover, direct laser irra-
diation of the spinal cord improves recovery of the corresponding
injured peripheral nerve. These results and others [2, 3] show that
laser phototherapy accelerates and improves the regeneration of
incomplete injured peripheral nerve.
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406 Phototherapy in Peripheral Nerve Repair and Muscle Preservation

Figure 22.2 Paraffin sections from the anterior horn of corresponding

segments of the rat spinal cord 14 days after a crush injury to the sciatic
nerve, showing the spinal cord response to laser treatment of the injured
peripheral nerve. Damaged as well as intact neurons were found in both
laser-treated and control animals. a: Section from a control animal shows
extensive chromatolysis and cytoplasmic atrophy found in 40% of the motor
neurons (arrows). b: Section from a laser-treated animal shows minimal
degenerative changes found in 20% of the motor neurons (arrows). Figure
reprinted with permission from Ref. [3], Copyright 2009 AANS.

Figure 22.3 Photomicrographs showing the axonal response to laser

treatment of the injured (crushed) peripheral nerve in rat. (A) Site of crush
injury in an untreated nerve showing nerve fibers that appear to be smaller
and mostly nonmyelinated. Numerous macrophages and phagocytes are
observed. (B) Site of crush injury in a laser-treated nerve demonstrating
that most axons are ensheathed with myelin and very few infiltrating
macrophages are observed. Figure reprinted with permission from Ref. [3],
Copyright 2009 AANS.
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Complete Peripheral Nerve Injury 407

Figure 22.4 Primary suture after peripheral nerve transection.

22.2 Complete Peripheral Nerve Injury

In acute cases where a peripheral nerve is completely transected

(as in a stab injury, for instance), the treatment of choice is direct
suturing (anastomosis) (Fig. 22.4).
Means of enhancing regeneration are essential, since nerve and
muscle degeneration are always inevitable in severely damaged
peripheral nerves.
The therapeutic effect of 780 nm laser irradiation on peripheral
nerve regeneration after complete transection and direct anastomo-
sis of rat sciatic nerve was evaluated in double-blind randomized
study that showed an increased total number of axons and better
quality of the regeneration process, as evidenced by the increased
number of large-diameter axons compared to the non-irradiated
control group.
In cases where nerve segmental loss exists (for example in cases
of shrapnel or gunshot injury), a double-blind randomized study was
conducted to evaluate the efficacy of laser phototherapy on nerve
growth and regeneration after reconnection using a guiding tube
(Fig. 22.5).
The nerve exhibited good reconnection (growth) through the
tube, which dissolved with time (Fig. 22.6).
Analysis showed growth of myelinated axons, which crossed
through the composite neurotube, and a continuation of axonal
sprouting through the area of the tube to the distal part of the nerve.
July 6, 2016 17:31 PSP Book - 9in x 6in 22-Hamblin-c22

408 Phototherapy in Peripheral Nerve Repair and Muscle Preservation

Figure 22.5 Tube for reconnection of the proximal and distal part of the
injured nerve.

Figure 22.6 Regrowth of the reconstructed nerve through the tube. Four
months after surgery, the tube has completely dissolved.
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Nerve Cells 409

Figure 22.7 Growth of myelinated axons in the distal part of the nerve.

The laser-treated group showed more intensive axonal growth than

the non-irradiated control group (Fig. 22.7).

22.3 Nerve Cells

Neuronal loss and degeneration in the corresponding spinal cord

segments resulting from peripheral nerve injuries led us to explore
the possibility of using laser phototherapy on cells as a way of
preventing or decreasing this phenomenon [5]. In cell cultures, the
laser activates the nerve cells, thereby intensifying axonal sprouting
(Fig. 22.8.)

Figure 22.8 Immunofluorescent staining of controls and laser-irradiated

neuronal cells.
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410 Phototherapy in Peripheral Nerve Repair and Muscle Preservation

Figure 22.9 Graph of the motor function follow-up in injured patients

who underwent either 780 nm laser phototherapy or placebo treatment.
Mean motor function (±SD) of all affected muscles was examined in injured
patients using the Medical Research Council (MRC) Grading System. The
analysis of the results showed that at baseline, the 780 nm laser-treated
and placebo groups were in clinically similar conditions ( p = 0.887). The
analysis of motor function during the 6-month follow-up period compared
with baseline showed statistically significant improvement ( p = 0.0001) in
the laser-treated group compared with the placebo group. Figure reprinted
with permission from Ref. [3], Copyright 2009 AANS.

22.4 Clinical Trial

A pilot clinical double-blind, placebo-controlled randomized study

showed that in patients with incomplete long-term peripheral
nerve injury, 780 nm laser irradiation can progressively improve
peripheral nerve function, which leads to significant functional
recovery (Figs. 22.9 and 22.10).

22.5 Denervated Muscle

Progressive muscle atrophy is common in patients with severe

peripheral nerve injury. Denervated muscles can account for
July 6, 2016 17:31 PSP Book - 9in x 6in 22-Hamblin-c22

Denervated Muscle 411

Figure 22.10 Graph of the motor unit recruitment in injured patients who
underwent either 780 nm laser phototherapy or placebo treatment. Motor
unit recruitment, the mean of all examined muscles (±SD), was monitored
in injured patients. The 780 nm laser-treated and placebo groups were
in similar conditions at baseline ( p = 0.934). In the laser-treated group,
statistically significant improvement ( p = 0.0006) was found in motor
unit recruitment during the 6-month follow-up period, compared with the
placebo group. Figure reprinted with permission from Ref. [3], Copyright
2009 AANS.

significant differences in the extent of acetylcholine receptors

(AChR) and creatine kinase (CK) activity during the denervation
period.
AChR, which play a special role in neuromuscular transmission,
are concentrated at the neuromuscular junction of the adult muscle.
A nerve impulse triggers the release of acetylcholine, producing
a much larger end-plate potential, which excites the muscle
membrane and leads to muscle contraction.
Muscle contraction and relaxation require the action of CK.
Phosphocreatine, formed by the reaction of this enzyme, constitutes
a reservoir of high-energy phosphate, which is available for quick
resynthesis of ATP. This high concentration of ATP is then accessible
for muscle contraction. Following muscle denervation, the level of
CK and muscle weight decreases.
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412 Phototherapy in Peripheral Nerve Repair and Muscle Preservation

This study [6, 7] was designed to assess the status of skele-

tal muscles after laser treatment during long-term denervation
processes, by investigating changes in the level of AChR and CK
activity in the intact and denervated gastrocnemius muscle of rat.
The gastrocnemius muscle was denervated by removing a 10
mm segment of the sciatic nerve. Low-power laser irradiation
was delivered transcutaneously to the right gastrocnemius muscle.
AChR was quantified by the 125I-a-bungarotoxin. CK activity was
measured by a specific spectrophotometric method. Laser treatment
had a significant therapeutic effect on the denervated muscle during
the first 21 days for AChR and the first 30 days for CK activity.
We conclude that in the early stages of muscle atrophy, laser
phototherapy may preserve the denervated muscle by maintaining
CK activity and the amount of AChR.

22.6 Conclusion

Laser phototherapy accelerates and enhances axonal growth and

regeneration after injury or reconstructive peripheral nerve pro-
cedure. Animal and clinical studies show the promoting action
of phototherapy on peripheral nerve regeneration and muscle
preservation, which makes it possible to suggest that the time for
broader clinical trials has come.

References

1. Rochkind S. Stimulation effect of laser energy on the regeneration of

traumatically injured peripheral nerves. Morphogenesis and Regeneration
73: 48–50, 1978.
2. Gigo-Benato D, Geuna S, and Rochkind S. Phototherapy for enhancing
peripheral nerve repair: A review of the literature. Muscle and Nerve 31:
694–701, 2005.
3. Rochkind S. Phototherapy in peripheral nerve regeneration: From basic
science to clinical study. Neurosurgical Focus 26 (2): E8, 2009.
July 6, 2016 17:31 PSP Book - 9in x 6in 22-Hamblin-c22

References 413

4. Rochkind S, Geuna S, and Shainberg A. Phototherapy in peripheral

nerve injury: Effects on muscle preservation and nerve regeneration.
International Review of Neurobiology 87: 445–464, 2009.
5. Rochkind S, El-AniD, Hayun T, Nevo Z, Shahar A. Increase of neuronal
sprouting and migration using 780 nm laser phototherapy as procedure
for cell therapy. Lasers in Surgery and Medicine 41: 277–281, 2009.
6. Rochkind S and Shainberg A. Protective effect of laser phototherapy
on acetylcholine receptors and creatine kinase activity in denervated
muscle. Photomedicine and Laser Surgery 31: 499–504, 2013.
7. Rochkind S, Geuna S, and Shainberg A. Phototherapy and nerve injury:
Focus on muscle response. International Review of Neurobiology 109: 99–
109, 2013.
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Chapter 23

Low-Level Laser Therapy for Spinal Cord

Repair

Takahiro Andoa,b and Michael R. Hamblinc,d,e

a Department of Electronics and Electrical Engineering, Keio University, 3-14-1 Hiyoshi,

Kohoku-ku, Yokohama, Kanagawa 223-8522, Japan

b Hitachi, Ltd., Yokohama Research Laboratory, 292 Yoshida-cho, Totsuka-ku,

Yokohama, Kanagawa, 244-0817, Japan

c Wellman Center for Photomedicine, Massachusetts General Hospital, 50 Blossom

Street, Boston, MA 02114, USA

d Department of Dermatology, Harvard Medical School, Massachusetts General

Hospital, 55 Fruit Street, Boston, MA 02114, USA

e Harvard-Massachusetts Institute of Technology (MIT) Division of Health Sciences and

Technology, 77 Massachusetts Avenue, E25-518, Cambridge, MA 02139, USA

takahiro.ando.dc@hitachi.com

23.1 Introduction

The brain and the spinal cord together form the central nervous
system (CNS). The spinal cord is surrounded and protected by a
vertebral column and has millions of nerve fibers that transmit
electrical impulses between the brain and the rest of the body. The
bundle of nerve cells in the spinal tissue is distally connected to
motor and sensory nerves of the peripheral nervous system (PNS).

Handbook of Low-Level Laser Therapy

Edited by Michael R. Hamblin, Marcelo Victor Pires de Sousa, and Tanupriya Agrawal
Copyright c 2017 Pan Stanford Publishing Pte. Ltd.
ISBN 978-981-4669-60-3 (Hardcover), 978-981-4669-61-0 (eBook)
www.panstanford.com
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416 Low-Level Laser Therapy for Spinal Cord Repair

Spinal cord injury (SCI) occurs when a contusion damages the

cells within the spinal tissue or severs the nerve tracts, leading to
serious residual disabilities such as respiratory difficulty, chronic
pain, urinary problems, and neurologic decline. The worldwide
incidence of traumatic SCI ranges from 15 to 40 cases per million
people each year [1, 2]. There are at least 330,000 patients living
with SCI in the European Union, with about 11,000 new cases every
year [3]. In the United States alone, the number of SCI patients
has been currently estimated to be between 236,000 and 327,000,
with an annual incidence of approximately 12,000 new cases [4].
The leading causes of SCI are motor vehicle accident (43–50%)
followed by falls (19–37%) and violence (18%), especially gunshot
wounds in the United States [5]. Averaged life expectancy after SCI
has been longer nowadays than it was a few decades ago due to
improvements in medical and surgical cures, while SCI alone costs
roughly $40.5 billion per year to the U.S. health care system [6]. Since
patients sustaining traumatic SCI are predominantly young whose
median age is 33 years [5], the impact of the costs for their lifetime
health care on social economy is much greater than that for the
provisional medical cares. Despite such a significant social problem,
no effective and validated interventions for spinal injury have been
established yet. As one of the most promising approaches to treat
SCI, noninvasive low-level laser therapy (LLLT) has received much
attention. In the present chapter, recent advances and developments
in LLLT studies for spinal cord repair, with a focus on treatment of
SCI, are described.

23.2 Therapeutic Strategies for Spinal Injury

SCI consists of a biphasic process involving a primary mechanical

injury followed by inflammatory responses and appearance of
reactive astrocytes [7] (Fig. 23.1). The primary damage is the
result of an initial mechanical insult and is locally restricted to
an area of the vertebral fracture. The pathophysiological process
in the acute phase is characterized by prominent hemorrhage and
ischemia, which is followed by pro-inflammatory and intracellular
events, including free radical production, ionic dysregulation, and
glutamate-mediated excitotoxicity. The secondary injury occurring
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Therapeutic Strategies for Spinal Injury 417

Figure 23.1 Time-dependent changes in the pathogenic state of SCI.

over the time course of days to weeks after trauma is characterized

by further destruction of neuronal and glial cells, leading to an
expansion of the damaged area. The expression of various kinds of
molecules associated with growth-inhibitory properties eventually
results in the formation of glial scar, which is a physical and a
biochemical barrier for axonal regeneration of injured neurons.
Therapeutic strategies after SCI reported so far are generally
categorized into the following three types: (1) neuroprotection,
(2) axon sprouting, and (3) cell/tissue replacement. The concept
of neuroprotection is based on a premise preventing or mini-
mizing pathological consequences after SCI, such as inflammation,
ischemia, excitotoxicity, lipid peroxidation, free radical production,
and apoptosis [8]. For the enhancement of axon sprouting and
plasticity, therapies promote axonal regeneration within the CNS
and, therefore, focus on either augmenting the neuron’s intrinsic
ability to regenerate or attenuating the inhibitory CNS environment
[9]. Cell/tissue replacement aims to substitute lost cells or tissue
for providing growth-promoting factors in a lesioned spinal cord.
In recent reports, stem cells such as embryonic stem and induced
pluripotent stem (iPS) cells have great promise as a source for
introducing new neurons or glial cells to the damaged CNS [10,
11]. The therapeutic strategies for spinal injury in LLLT studies
previously presented are based on either of the above ones.
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418 Low-Level Laser Therapy for Spinal Cord Repair

23.3 LLLT for Spinal Cord Repair

23.3.1 Laser Irradiation in Spinal Cord for Therapy of

Injured Peripheral Nerves
Red or near-infrared (NIR) laser irradiation to the spinal cord was
first carried out aiming at the restoration of traumatized PNS. The
use of helium–neon (He–Ne) laser for the treatment of in vivo
experimental nerve injuries, for the first time, was reported by
Rochkind in 1978 [12]. Although a large number of literatures
published in the 1980s on LLLT for nerve regeneration have dealt
with traumatically injured peripheral nerves alone [13–17], the
systemic effect of LLLT was found in 1989 on the spinal cord
segments corresponding to the crushed sciatic nerves [18].

23.3.1.1 Animal studies

In 1989, Rochkind et al. [18] assessed the systemic effect of He–
Ne laser irradiation on the recovery of the injured peripheral and
central nervous systems in rats, as well as healing of cutaneous
wounds and burns. After crush injuries were inflicted to rat sciatic
nerves by closing hemostatic forceps, a 16 mW He–Ne laser (wave-
length 632.8 nm; continuous wave (CW)) was transcutaneously
irradiated over the right sciatic nerve for 7 min daily (spot size
3 mm2 ; laser fluence 10 J/cm2 ), during 20 days. The laser energy
actually reaching the sciatic nerve had been evaluated to be 2–10%
of the energy delivered to the skin [19]. The bilateral retrograde
degeneration of the motor neurons in the corresponding spinal
segment after the peripheral crush injury was greatly reduced
in the LLLT group. As the systemic effect, the compound action
potential (i.e., electrical activity) in the left non-irradiated leg was
significantly increased. In 1990, Rochkind et al. [20] investigated
the changes in the spinal cord in rats subjected to crush injury
of the sciatic nerve followed by LLLT. The laser parameters were
the same as those in their previous study [18] and the irradiation
duration was 7 min daily for 14 or 21 consecutive days. When the
spinal cord segments corresponding to the crushed sciatic nerve
were histologically analyzed, the laser-treated peripheral nerve was
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LLLT for Spinal Cord Repair 419

found to have mitigated the degenerative changes in neurons in

the spinal tissue and induced the proliferation of neuroglia both in
astrocytes and oligodendrocytes. These results suggest that a higher
metabolism in neurons and a better ability for myelin production
would be driven by He–Ne laser irradiation. More recently, in 2010,
Zhang et al. [21] explored the effects of 660 nm gallium–aluminum–
arsenide (Ga–Al–As) LLLT on neural regeneration after acellular
nerve allograft repair of the sciatic nerve gap in rats. They proved
that LLLT upregulated calcitonin gene-related peptide protein and
the mRNA expression of the spinal cord corresponding to the injury
site and increased the rate of nerve regeneration.
If LLLT is applied to both the crushed nerve and the cor-
responding segments of the spinal cord, the recovery time and
the quality of regeneration of the peripheral nerve have been
revealed to be notably improved [22–24]. In 1988, Rochkind [22]
demonstrated that LLLT applied simultaneously to the injured
sciatic nerve and the corresponding segment of the spinal cord
(L4-S2) accelerated the process of regeneration of the injured
peripheral nerve in rats. A 16 mW He–Ne laser or a 40 mW
CW argon laser or a 20 mW pulsed dye laser was used in the
study, and LLLT was employed daily for 20 consecutive days. The
results showed the regenerative effect diminished with changing
the wavelength from 632 nm to 465 nm. In 2001, Rochkind et
al. [23] similarly examined the recovery of the crushed sciatic
nerve of rats after 632 nm laser application to the corresponding
segments of the spinal cord. After a crush trauma to the sciatic
nerve in rats, each rat was transcutaneously irradiated at four points
between T12-T13-L1 vertebrae along the spinal cord segments
L3-L6, which corresponded to the crushed sciatic nerve (laser
fluence 180 J/cm2 per point). The laser treatment was repeated 30
min daily for 21 consecutive days. The recorded amplitude of an
electrophysiologic activity in the injured nerves was significantly
higher in the laser-treated rats than that of the non-laser-irradiated
group, demonstrating that LLLT administered directly to the spinal
cord improved the recovery of the corresponding insured peripheral
nerve. Also in 2001, Shamir et al. [24] evaluated the therapeutic
effect of 780 nm laser irradiation on peripheral nerve regeneration
after complete transection and direct anastomosis of the rat sciatic
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420 Low-Level Laser Therapy for Spinal Cord Repair

nerve. LLLT was applied transcutaneously for 30 min daily for 21

consecutive days to the corresponding segments of the spinal cord
and to the injured sciatic nerve. Positive somatosensory-evoked
responses were observed in 69% of the irradiated rats and in 18% of
the non-irradiated rats. Immunohistochemical staining in the laser-
treated group showed more intensive axonal growth and better
quality of the regenerative process due to an increased number
of large- and medium-diameter axons. These findings suggest that
LLLT enhances regenerative processes of peripheral nerves after
complete transection and surgical anastomosis.

23.3.1.2 Clinical studies

On the basis of the outcome in animal studies [17, 25], Rochkind et
al. conducted a randomized, double-blind, and placebo-controlled
trial to investigate the effectiveness of 780 nm LLLT for the
treatment of patients suffering from incomplete peripheral nerve
and brachial plexus injuries for 6 months up to several years [26–
28].
In their clinical study [26], 18 patients with a history of
traumatic peripheral nerve/brachial plexus injury with a stable
neurological deficit and a significant weakness were randomly
assigned either placebo (non-active light) or low-power laser
irradiation (wavelength 780 nm; power 250 mW). The placebo
or laser treatment was transcutaneously applied for 3 h to the
injured peripheral nerve (laser fluence 450 J/mm2 ) and for 2 h
to the corresponding segments of the spinal cord (laser fluence
300 J/mm2 ). The irradiation device was placed approximately 40
cm from the skin treatment point (spot size 6 mm2 ), focused
on the injured area of the peripheral nerve or corresponding
level of the spine. Clinical and electrophysiological scores were
evaluated at baseline, at the end of the 21 days of treatment, and
3 and 6 months thereafter. Assessment using Medical Research
Council grading scale for muscle strength showed motor function
of the laser-treated patients was significantly improved during a
6-month follow-up period, compared to that of the placebo group.
Also, electrophysiological analysis revealed a statistically significant
improvement in the recruitment of voluntary muscle activity in the
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LLLT for Spinal Cord Repair 421

laser-irradiated group, compared to the non-treated group. This

pilot study suggests that in patients with long-term peripheral nerve
injury, 780 nm LLLT can progressively improve peripheral nerve
function, leading to significant functional recovery.
Yamany et al. [29] in 2011 applied NIR LLLT to 30 male and
female patients with painful diabetic neuropathy and were able
to modify pain, foot skin microcirculation, and some electrophys-
iological parameters of peripheral nerve function. A commercially
available laser device, which has both a CW He–Ne laser with a
wavelength of 850 nm and a pulsed 905 nm infrared laser, was
utilized in their clinical study. In the laser-treated group of patients,
irradiation was applied (laser fluence 5.7 J/cm2 ) to the lumbosacral
area (one on the L2, one on the S1, and two points laterally to
the spine by about 2 cm) and the plantar surface of the foot
for 15 min each site/session three times per week for 4 weeks.
Pain intensity via visual analogue scale, bilateral peroneal motor
nerves, sural sensory nerves conduction velocity and amplitude was
significantly decreased, and electrophysiological parameters and
foot skin microcirculation were remarkably improved in the LLLT
group, while no significant change was obtained in the control group.

23.3.2 LLLT for Nerve Transplantation of Spinal Injured

Animals
The beneficial effects of LLLT on nerve transplantation after spinal
injury have been reported since the late 1980s. Rochkind [22,
30] proved in 1988 that He–Ne laser irradiation applied to the
spinal cord of dogs underwent transection of the cord at D12-
L1 and implantation of a segment of the autologous sciatic nerve
into the injured area diminished glial scar formation, induced
axonal sprouting in the injured area, and restoration of locomotor
function. In 1992, Rochkind [31] investigated the effect of low-
power laser irradiation on mammalian CNS transplantation. Fetal
brain allografts were transplanted into the brain (fornix region)
of 20 adult rats, and spinal cord allografts were transplanted
into the spinal cord of eight dogs. For 21 days, the closed
operated wounds of 10 rats and 4 dogs were exposed daily to
transcutaneous He–Ne LLLT (wavelength 632.8 nm; CW) with a
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422 Low-Level Laser Therapy for Spinal Cord Repair

spot size of 2 mm2 (laser fluences 30 J/cm2 for rats and 70 J/cm2
for dogs). The results showed that LLLT prevented extensive glial
scar formation between neural transplants and host brain or spinal
cord. Moreover, abundant capillaries were found to be developed
within the transplants in the laser-irradiated animals, indicating the
regeneration potentials in the CNS.
One feasible innovative way of repairing injured mammalian
spinal cord is cell transplantation using a composite implant that
contains cultured cells from autologous or allogeneic source. The
implanted cells grow and serve as a vital bridge to connect
the stumps of the severed spinal cord [32]. A pilot study by
Rochkind et al. [33] in 1997 examined the effects of implants
of cultured neuronal cells and NIR laser irradiation on repairing
injured spinal cords in rats. Embryonal spinal cord nerve cells
which were dissociated from rat fetuses, cultured on biodegradable
microcarriers, and embedded in hyaluronic acid were implanted
in the transected spinal cords (T7-T8) of rats. The whole lesion
area was covered with a thin coagulated fibrin-based membrane.
LLLT was started immediately after surgery and was continued daily
for two weeks (wavelength 780 nm; power 200 mW; irradiation
duration 30 min per day). During the 3–6 months follow up, 14 of
the 15 animals that received laser showed different degrees of active
movements in one or both legs, compared to four of nine animals
that had received cell transplantation without LLLT. In addition, axon
sprouting was observed at 3 days post-surgery in the laser-treated
rats. In 2002, the same research group [34, 35] presented the results
of the effects of cultured embryonal nerve cells and 780 nm LLLT on
the locomotor recovery of the completely transected spinal cords in
24 adult rats. For 14 consecutive post-transfection days, 15 rats were
daily applied to the laser irradiation (power 250 mW; irradiation
duration 30 min). Eleven of the 15 animals (73%) showed different
degrees of active leg movements and gait performance, compared
to four of the nine rats (44%) with implantation alone. In a
control group of seven rats with spinal cord transection and no
transplantation or laser, six (86%) remained completely paralyzed.
Three months after transection, implantation, and laser irradiation,
somatosensory-evoked potentials were significantly elicited in 69%
of rats compared to 38% in the non-irradiated group. These works
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LLLT for Spinal Cord Repair 423

suggest that in vitro composite implants were a regenerative and

reparative source for reconstructing the transected spinal cord and
that post-transplantation LLLT enhanced axonal sprouting, leading
to spinal cord repair.

23.3.3 Effects of NIR Laser Irradiation Alone for SCI Model

Several research groups have shown that transcutaneous LLLT
significantly promotes restoration of locomotor function in SCI
animals. Recently, a clinical trial using red/NIR laser irradiation
treatment has been reported in humans with chronic SCI. Table 23.1
summarizes the most important studies previously published on SCI
animals treated with NIR laser irradiation to the spinal tissue.

23.3.3.1 Experimental SCI model

Animal models of SCI can be mainly classified as contusion and
transection types. The contusion model is the most common one and
widely used in SCI studies because the morphological, biochemical,
and functional changes that occur after trauma are similar to those
in human [36]. Weight-drop tools for the contusion injury such
as the New York University and Multicenter Animal Spinal Cord
Injury Study device are utilized to generate different and consistent
amounts of force. On the other hand, transection injury is not
typical for a clinical situation, but this model is helpful for revealing
the mechanism of axonal regeneration across the transection site.
Following laminectomy, spinal cord transection is performed with
fine scissors. The functional outcomes after making SCI models
and certain treatments are, in general, analyzed by footprint and
locomotor tests [37, 38].

23.3.3.2 Transmittance of transcutaneous NIR laser to spinal

cord
In the NIR wavelength range, light absorption by main chro-
mophores such as water and hemoglobin in tissue is relatively low.
In 2005, Byrnes et al. [39] measured ex vivo spectrophotometric
and light power transmission to assess the extent to which a
transcutaneous laser irradiation penetrates to the injured spinal
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424 Low-Level Laser Therapy for Spinal Cord Repair

Table 23.1 LLLT studies on rat SCI model

Model Irradiation Parameters Effects References

T Transcutaneous, 780 nm; 250 mW; 30 Increased axonal 34
w/cell min daily for 2 weeks sprouting
transplantation
T Transcutaneous 810 nm; 150 mW; 0.3 Increased axonal 39
(6% laser power cm2 ; 1589 J/cm2 ; regeneration and
penetration to the 2997 s daily for functional recovery;
spinal cord depth) 2 weeks suppressed immune
cell activation and
cytokine/chemokine
expression
T, C Transcutaneous 810 nm; 150 mW; 0.3 Higher number of 41
cm2 ; 1589 J/cm2 ; axons;
2997 s daily for significant locomotor
2 weeks recovery
T Transcutaneous; 830 nm; 100 mW; 0.028 Moderately increased 42
applied to two points cm2 ; 250 J/cm2 ; CW; values of inner
of the hindlimb 70 s at three times per diameter and internal
(femur and tibiae) week during 4 weeks and external areas of
tibia diaphysis
T Transcutaneous 808 nm; 35 mW; 0.07 Increased axonal 45
cm2 ; 1500 J/cm2 ; regeneration and
CW or pulsed 100 Hz; functional recovery;
50 min daily for axonal sprouting:
2 weeks CW <pulsed 100 Hz
C Direct, linear 808 nm; 25 mW; 3 cm2 ; Improved locomotor 46
polarization 9.6 J/cm2 ; 20 min function; earlier
(perpendicular or daily for 5 days recovery in the parallel
parallel to the spine) group

Note: Laser parameters are given in the following order: wavelength, power, spot size, energy
density, mode (CW or pulsed), irradiation time, and duration. The parameters are partially
unavailable in several cases. Transection (T) or contusion (C).

cord in rats. Analysis of the spectra revealed that the range of

transmission was the highest through all tissue layers overlying
the spinal cord and through blood between 770 nm and 850 nm
wavelengths. Moreover, analysis of the power penetration showed
that 6% of the power of a 150 mW, 810 nm laser was transmitted
through all the layers of tissue between the dorsal skin surface and
the ventral side of the spinal cord. Another LLLT report [40] using
dogs with acute paraparesis/paraplegia due to acute intervertebral
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LLLT for Spinal Cord Repair 425

disk herniation estimated that transcutaneous 810 nm laser at a

fluence of 1500 J/cm2 applied to the overlying skin penetrated the
spinal cord at an appropriate energy density of 2–8 J/cm2 . Thus, NIR
laser applied transcutaneously transmitted to the depth of the spinal
cord in even comparatively large animals.

23.3.3.3 LLLT for injured spinal cord in rats

Using two different rat models of hemisection and contusion SCI, An-
ders et al. first demonstrated that transcutaneous application of an
810 nm laser promoted axonal regeneration and functional recovery
[39, 41]. In 2005, Byrnes et al. [39] investigated the beneficial effects
of NIR laser irradiation on the treatment of SCI in a rat hemisection
model. Rats that underwent a T9 dorsal hemisection were followed
by a daily treatment with an 810 nm, 150 mW diode laser at the
skin surface overlying the lesion site (spot size 0.3 cm2 ; laser fluence
1589 J/cm2 ; irradiation duration 2997 s). The irradiation was
applied for 14 consecutive days. Axonal regeneration and functional
recovery were assessed by single and double label tract tracing and
a ladder walking and footprint test analysis, respectively. The results
showed increased axonal number and distance of regrowth, as well
as improvement in locomotor functions. In addition, evaluation of
the immune response within the spinal cord revealed that LLLT
significantly decreased immunolabeling for macrophages/activated
microglia, T lymphocytes, and astrocytes, which are involved in
secondary damage of SCI. These findings suggest that 810 nm LLLT
altered the invasion of a number of cell types that play a substantial
role after SCI and led to the recovery of the injured site of the
spinal cord in rats. In 2009, Wu et al. [41] demonstrated that axon
length and number were increased in both dorsal hemisection and
moderate contusion SCI models of rats. They employed 810 nm
laser transcutaneously at the lesion site immediately after injury and
daily for 14 consecutive days with the same irradiation parameters
as their previous study [39]. Functional assessments based on a
footprint test for the hemisection model and an open-field test for
the contusion model showed a statistically significant recovery after
3 weeks post-injury in the LLLT group compared to the untreated
group.
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426 Low-Level Laser Therapy for Spinal Cord Repair

In 2010, Medalha et al. [42] represented a trend toward im-

proved hindlimb diaphysis in NIR laser-treated rats with transected
SCI. The treatment was performed immediately post-surgery, three
times per week for 4 weeks, using a Ga–Al–As laser (wavelength
830 nm; CW; power 100 mW; fluence 250 J/cm2 ). The irradiation
was applied on two points of the hindlimb (femur and tibiae) with
a spot size of 0.028 cm2 for 70 s per point. There is no statically
significant difference in the mechanical strength and mineral density
of bones as well as the locomotor function between the SCI alone
group and the LLLT group. However, values of inner diameter and
internal and external areas of tibia diaphysis increased in the laser-
treated rodents.
In recent studies, the therapeutic efficacy of LLLT for SCI has
been found to depend on irradiation parameters, as reported in
the previous literatures concerning LLLT for other neurological
diseases [43, 44]. The study conducted by Wu et al. in 2012 [45]
compared the effects of CW and pulsed laser on nerve regeneration
in a hemisection model of SCI in rats. An 808 nm diode laser was
utilized with a power density of 0.5 W/cm2 on the skin surface
above the lesion and an energy density of 1500 J/cm2 . Irradiation
was delivered via a 3 mm diameter probe with direct contact to the
skin. The output powers of the CW and 100 Hz pulsed mode were
35 mW and 175 mW, respectively, at a peak power with 20% duty
cycle. Animals were irradiated for 50 min daily for 14 consecutive
days after SCI. The results showed both CW and pulsed 808 nm
LLLT supported axonal regeneration and functional recovery at 3
weeks post-trauma. Furthermore, the regenerative axonal length
distal to the lesion site was significantly greater in the pulsed
LLLT group than in the CW LLLT group. In 2013, Ando et al. [46]
examined the effect of relative orientation of laser polarization
on the efficacy of NIR LLLT for contused spinal cords in rats.
Rat spinal cords were injured with a weight-drop device, and the
lesion sites were “directly” (i.e., not transcutaneously) irradiated
with a linearly polarized 808 nm diode laser positioned either
perpendicular or parallel to the spine immediately after trauma
and daily for 5 consecutive days. The laser power measured at the
injured spinal cord surface was 25 mW, with a spot diameter of
20 mm giving a power density of 8 mW/cm2 (irradiation duration
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LLLT for Spinal Cord Repair 427

20 min; fluence 9.6 J/cm2 per day). Regardless of the polarization

direction, locomotor functional scores of SCI rats treated with the
laser were significantly higher than those of SCI alone group from
day 5 after injury. The locomotive function of SCI rats irradiated
parallel to the spinal column was significantly improved from
day 10 after injury, compared to SCI rats treated with the linear
polarization perpendicular to the spinal column. In these two papers
described earlier, the authors speculated that the higher efficacy
of the recovery with pulsed laser or parallel irradiation could be
attributed to the deeper light penetration into the tissue.

23.3.4 Clinical Study: Intravascular LLLT for Chronic SCI

Patients
In 2012, Huang et al. [47] investigated the clinical effects of
intravascular laser irradiation of blood on oxidative stress and
mitochondrial dysfunction in subjects with chronic SCI resulting
from trauma. Before intravascular laser treatment, the chronic
SCI patients were indisputably in states of mitochondrial damage
and oxidative stress, compared with normal (no SCI) subjects. An
intravascular He–Ne LLLT was conducted using a fiber-optic needle
inserted into the inner cannula of a 24-gauge intravenous catheter
for an elbow venous puncture. The laser irradiation was delivered
for 3600 s per each treatment at a power density of 2.04 W/cm2
(fluence 7347 J/cm2 ; power 4 mW; treatment duration 15 times
over 3 weeks). Significantly higher mitochondrial DNA replication,
adenosine triphosphate synthesis of leucocytes, and total antioxi-
dant capacity were observed in the laser-treated patients compared
to the non-treated group. Low-density lipoprotein in the LLLT group
was significantly reduced at days 10 and 15, with significantly
higher high-density lipoprotein at day 45. These findings suggest the
effectiveness of intravascular laser irradiation to blood in alleviating
oxidative stress and mitochondrial dysfunction in chronic SCI
patients. However, the authors stated that the relationship between
the generation of antioxidant defenses and clinical motor/sensory
improvement was beyond the scope of this study, comprising a
small number of participants undergoing a short-term period of
treatment.
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428 Low-Level Laser Therapy for Spinal Cord Repair

23.4 Mechanism Studies of LLLT for SCI

Although further studies are needed to clarify the therapeutic

mechanisms in LLLT for SCI, as well as the optimum irradiation
conditions, cellular processes such as responses of neuronal growth
and microglial polarization following NIR laser irradiation have been
recently investigated in vitro.
Rochkind et al. [48] examined the effect of 780 nm laser
irradiation on the growth of embryonic rat brain cultures embedded
in NVR-Gel (cross-linked hyaluronic acid with adhesive molecule
laminin and several growth factors). They concluded that LLLT
stimulated migration and fiber sprouting of neuronal cell aggregates,
developed large size neurons with dense-branched interconnected
network of neuronal fibers, and therefore can be considered
potential procedure for cell therapy of neuronal injury or disease.
Another study performed by Leden et al. [49] in 2013 focused on the
effect of 808 nm laser irradiation on microglia, which is the primary
mediator of immune and inflammatory responses in the CNS. To
reveal the microglial polarization followed by NIR LLLT, they utilized
colorimetric assays, immunocytochemistry, proteomic profiling, and
reverse transcriptase-polymerase chain reaction (RT-PCR) after
exposure of microglial cell to laser application with a laser fluence
of 0.2, 4, 10, and 30 J/cm2 (power 50 mW). The results showed
that LLLT had a dose-dependent effect on the spectrum of microglial
for M1 (pro-inflammatory or classically activated) and M2 (anti-
inflammatory or alternatively activated) polarizations. LLLT with
energy densities between 4 J/cm2 and 30 J/cm2 induced expression
of M1 markers in microglia. Markers of the M2 phenotype, including
CD206 and TIMP1, were observed at lower energy densities of
0.2–10 J/cm2 . Additionally, their data obtained from microglia and
neuron co-cultures indicated a dose-dependent effect of LLLT on
microglial-induced neuronal growth and neurite extension. These
findings were of important relevance to not only therapies in the
CNS but also to understanding of LLLT effects on alterations in signal
transduction pathways.
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Conclusion 429

23.5 LLLT for Other Spinal Cord Diseases

Recent reports have dealt with LLLT for the treatment of spinal-
related diseases other than SCI. In 2012, Draper et al. [40] examined
the therapeutic effects of NIR LLLT on intervertebral disk herniation
in dogs. Animals with acute paraparesis/paraplegia due to acute
intervertebral disk herniation were treated post-operatively with
LLLT using a 5 × 200 mW, 810 nm cluster array (power 25 W/cm2 ;
irradiation duration 1 min) daily for 5 days. The irradiation was
applied transcutaneously over the spinal segment associated with
the hemilaminectomy and the two adjacent ones (one cranial and
one caudal). The presented data showed that LLLT in combination
with surgery reduced the time to ambulation in dogs with T3-
L3 myelopathy secondary to intervertebral disk herniation. More
recently, Chen et al. [50] in 2014 observed enhanced neural
regeneration following chronic compression of dorsal root ganglia
(CCD) and improved ambulatory behavior in rats after 808 nm LLLT
at a fluence of 8 J/cm2 for 8 consecutive post-operative days. The
expression levels of the pro-inflammatory cytokines TNF-α and IL-
1β were increased following CCD, while the increases were found to
be reduced by the application of LLLT.

23.6 Conclusion

In the current contribution, we have reviewed the latest efforts of

LLLT for spinal diseases. Evidence has been rapidly accumulating
that red/NIR laser irradiation to the spinal tissue (or peripheral
nerves and blood, as partially reported) is beneficial for spinal cord
repair. Since recent studies have proved that LLLT would upregulate
a number of neurotrophic growth factors and extracellular matrix
proteins for supporting neurite outgrowth, the combination of ap-
plication of drugs, growth factors, procedures of cell transplantation,
and autografts and allografts with LLLT can also be an advantageous
option to treat spinal cord diseases. Moreover, the almost complete
lack of papers on side effects and adverse events associated with
LLLT has promoted acceptation by not only medical profession but
also general public. Meanwhile, possible beneficial mechanisms and
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430 Low-Level Laser Therapy for Spinal Cord Repair

therapeutic processes in LLLT will be deliberately investigated in

further research.

References

1. Ackery, A., Tator, C., and Krassioukov, A. (2004). A global perspective on

spinal cord injury epidemiology, J. Neurotrauma, 21, pp. 1355–1370.
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map for traumatic spinal cord injury epidemiology: Update 2011, global
incidence rate, Spinal Cord, 52, pp. 110–116.
3. Onose, G., Anghelescu, A., Muresanu, D. F., Padure, L., Haras, M. A.,
Chendreanu, C. O., Onose, L. V., Mirea, A., Ciurea, A. V., El Masri, W.
S., and von Wild, K. R. (2009). A review of published reports on
neuroprotection in spinal cord injury, Spinal Cord, 47, pp. 716–726.
4. National Spinal Cord Injury Statistical Center. (2013). Spinal cord injury
facts and figures at a glance, J. Spinal Cord Med., 36, pp. 1–2.
5. Jackson, A. B., Dijkers, M., Devivo, M. J., and Poczatek, R. B. (2004). A
demographic profile of new traumatic spinal cord injuries: Change and
stability over 30 years, Arch. Phys. Med. Rehabil., 85, pp. 1740–1748.
6. The Christopher & Dana Reeve Foundation. One Degree of Separa-
tion: Paralysis and Spinal Cord Injury in the United States, http://
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One Degree of Separation.htm.
7. Renault-Mihara, F., Okada, S., Shibata, S., Nakamura, M., Toyama, Y., and
Okano, H. (2008). Spinal cord injury: Emerging beneficial role of reactive
astrocytes’ migration, Int. J. Biochem. Cell Biol., 40, pp. 1649–1653.
8. Thuret, S., Moon, L. D., and Gage, F. H. (2006). Therapeutic interventions
after spinal cord injury, Nat. Rev. Neurosci., 7, pp. 628–643.
9. Yiu, G., and He, Z. (2006). Glial inhibition of CNS axon regeneration, Nat.
Rev. Neurosci., 7, pp. 617–627.
10. Sahni, V., and Kessler, J. A. (2010). Stem cell therapies for spinal cord
injury, Nat. Rev. Neurol., 6, pp. 363–372.
11. Das, A. K., Gopurappilly, R., and Parhar, I. (2011). Current status and
prospective application of stem cell-based therapies for spinal cord
injury, Curr. Stem Cell Res. Ther., 6, pp. 93–104.
12. Rochkind, S. (1978). Stimulation effect of laser energy on the regenera-
tion of traumatically injured peripheral nerves, Morphogen. Regen., 83,
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13. Rochkind, S., Nissan, M., Razon, N., Schwartz, M., and Bartal, A. (1986).
Electrophysiological effect of HeNe laser on normal and injured sciatic
nerve in the rat, Acta Neurochir., 83, pp. 125–130.
14. Rochkind, S., Barr-Nea, L., Razon, N., Bartal, A., and Schwartz, M. (1987).
Stimulatory effect of He–Ne low dose laser on injured sciatic nerves of
rats, Neurosurgery, 20, pp. 843–847.
15. Rochkind, S., Nissan, M., Barr-Nea, L., Razon, N., Schwartz, M., and Bartal,
A. (1987). Response of peripheral nerve to He–Ne laser: Experimental
studies, Lasers Surg. Med., 7, pp. 441–443.
16. Rochkind, S., Nissan, M., Lubart, R., Avram, J., and Bartal, A. (1988).
The in-vivo-nerve response to direct low-energy-laser irradiation, Acta
Neurochir (Wien), 94, pp. 74–77.
17. Gigo-Benato, D., Geuna, S., and Rochkind, S. (2005). Phototherapy for
enhancing peripheral nerve repair: A review of the literature, Muscle
Nerve, 31, pp. 694–701.
18. Rochkind, S., Rousso, M., Nissan, M., Villarreal, M., Barr-Nea, L., and
Rees, D. G. (1989). Systemic effects of low-power laser irradiation on the
peripheral and central nervous system, cutaneous wounds, and burns,
Lasers Surg. Med., 9, pp. 174–182.
19. Nissan, M., Rochkind, S., Razon, N., and Bartal, A. (1986). HeNe laser
irradiation delivered transcutaneously: Its effect on the sciatic nerve of
rats, Lasers Surg. Med., 6, pp. 435–438.
20. Rochkind, S., Vogler, I., and Barr-Nea, L. (1990). Spinal cord response to
laser treatment of injured peripheral nerve, Spine (Phila Pa 1976), 15,
pp. 6–10.
21. Zhang, L. X., Tong, X. J., Yuan, X. H., Sun, X. H., and Jia, H. (2010).
Effects of 660-nm gallium-aluminum-arsenide low-energy laser on
nerve regeneration after acellular nerve allograft in rats, Synapse, 64,
pp. 152–160.
22. Rochkind, S., Barr-Nea, L., Bartal, A., Nissan, M., Lubart, R., and Razon,
N. (1988). New methods of treatment of severely injured sciatic nerve
and spinal cord. An experimental study, Acta Neurochir. Suppl., 43, pp.
91–93.
23. Rochkind, S., Nissan, M., Alon, M., Shamir, M., and Salame, K. (2001).
Effects of laser irradiation on the spinal cord for the regeneration
of crushed peripheral nerve in rats, Lasers Surg. Med., 28, pp. 216–
219.
24. Shamir, M. H., Rochkind, S., Sandbank, J., and Alon, M. (2001).
Double-blind randomized study evaluating regeneration of the rat
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432 Low-Level Laser Therapy for Spinal Cord Repair

transected sciatic nerve after suturing and postoperative low power

laser treatment, J. Reconstr. Microsurg., 17, pp. 133–137.
25. Rochkind, S., Leider-Trejo, L., Nissan, M., Shamir, M. H., Kharenko, O., and
Alon, M. (2007). Efficacy of 780-nm laser phototherapy on peripheral
nerve regeneration after neurotube reconstruction procedure (double-
blind randomized study), Photomed. Laser Surg., 25, pp. 137–143.
26. Rochkind, S., Drory, V., Alon, M., Nissan, M., and Ouaknine, G. E. (2007).
Laser phototherapy (780 nm), a modality new in treatment of long-
term incomplete peripheral nerve injury: A randomized double-blind
placebo-controlled study, Photomed. Laser Surg., 25, pp. 436–442.
27. Rochkind, S., Geuna, S., and Shainberg, A. (2009). Chapter 25: Photother-
apy in peripheral nerve injury: Effects on muscle preservation and nerve
regeneration, Int. Rev. Neurobiol., 87, pp. 445–464.
28. Rochkind, S. (2009). Phototherapy in peripheral nerve regeneration:
From basic science to clinical study, Neurosurg. Focus, 26, pp. 1–6.
29. Yamany, A. A., and Sayed, H. M. (2012). Effect of low level laser therapy
on neurovascular function of diabetic peripheral neuropathy, J. Adv. Res.,
3, pp. 21–28.
30. Rochkind, S., and Ouaknine, G. E. (1992). New trend in neuroscience:
Low power laser effect on peripheral and central nervous system (basic
science, preclinical and clinical studies), Neurol. Res., 14, pp. 2–11.
31. Rochkind, S. (1992). Central nervous system transplantation benefitted
by low-power laser irradiation, Lasers Med. Sci., 7, pp. 143–151.
32. Rochkind, S., Shahar, A., Fliss, D., El-Ani, D., Astachov, L., Hayon, T.,
Alon, M., Zamostiano, R., Ayalon, O., Biton, I. E., Cohen, Y., Halperin, R.,
Schneider, D., Oron, A., and Nevo, Z. (2006). Development of a tissue-
engineered composite implant for treating traumatic paraplegia in rats,
Eur. Spine J., 15, pp. 234–245.
33. Rochkind, S., Shahar, A., and Nevo, Z. (1997). An innovative approach to
induce regeneration and the repair of spinal cord injury, Laser Ther., 9,
pp. 151–152.
34. Rochkind, S., Shahar, A., Amon, M., and Nevo, Z. (2002). Transplantation
of embryonal spinal cord nerve cells cultured on biodegradable
microcarriers followed by low power laser irradiation for the treatment
of traumatic paraplegia in rats, Neurol. Res., 24, pp. 355–360.
35. Rochkind, S. (2006). Photoengineering of neural tissue repair processes
in peripheral nerves and the spinal cord: Research development with
clinical applications, Photomed. Laser Surg., 24, pp. 151–157.
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36. Metz, G. A., Curt, A., van de Meent, H., Klusman, I., Schwab, M. E., and
Dietz, V. (2000). Validation of the weight-drop contusion model in rats:
A comparative study of human spinal cord injury, J. Neurotrauma, 17,
pp. 1–17.
37. Basso, D. M., Beattie, M. S., and Bresnahan, J. C. (1995). A sensitive
and reliable locomotor rating scale for open field testing in rats, J.
Neurotrauma, 12, pp. 1–21.
38. Metz, G. A., Merkler, D., Dietz, V., Schwab, M. E., and Fouad, K. (2000).
Efficient testing of motor function in spinal cord injured rats, Brain Res.,
883, pp. 165–177.
39. Byrnes, K. R., Waynant, R. W., Ilev, I. K., Wu, X., Barna, L., Smith,
K., Heckert, R., Gerst, H., and Anders, J. J. (2005). Light promotes
regeneration and functional recovery and alters the immune response
after spinal cord injury, Lasers Surg. Med., 36, pp. 171–185.
40. Draper, W. E., Schubert, T. A., Clemmons, R. M., and Miles, S. A. (2012).
Low-level laser therapy reduces time to ambulation in dogs after
hemilaminectomy: A preliminary study, J. Small Anim. Pract., 53, pp.
465–469.
41. Wu, X., Dmitriev, A. E., Cardoso, M. J., Viers-Costello, A. G., Borke, R. C.,
Streeter, J., and Anders, J. J. (2009). 810 nm wavelength light: An effective
therapy for transected or contused rat spinal cord, Lasers Surg. Med., 41,
pp. 36–41.
42. Medalha, C. C., Amorim, B. O., Ferreira, J. M., Oliveira, P., Pereira, R. M.,
Tim, C., Lirani-Galvão, A. P., da Silva, O. L., and Renno, A. C. (2010).
Comparison of the effects of electrical field stimulation and low-level
laser therapy on bone loss in spinal cord-injured rats, Photomed. Laser
Surg., 28, pp. 669–674.
43. Hashmi, J. T., Huang, Y. Y., Osmani, B. Z., Sharma, S. K., Naeser, M.
A., and Hamblin, M. R. (2010). Role of low-level laser therapy in
neurorehabilitation, PM R., 2, pp. S292–305.
44. Chung, H., Dai, T., Sharma, S. K., Huang, Y. Y., Carroll, J. D., and Hamblin,
M. R. (2012). The nuts and bolts of low-level laser (light) therapy, Ann.
Biomed. Eng., 40, pp. 516–533.
45. Wu, X., Moges, H., DeTaboada, L., and Anders, J. (2012). Comparison of
the effects of pulsed and continuous wave light on axonal regeneration
in a rat model of spinal cord injury, Lasers Med. Sci., 27, pp. 525–528.
46. Ando, T., Sato, S., Kobayashi, H., Nawashiro, H., Ashida, H., Hamblin, M.
R., and Obara, M. (2013). Low-level laser therapy for spinal cord injury
in rats: Effects of polarization, J. Biomed. Opt., 18, pp. 098002-1-6.
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47. Huang, S. F., Tsai, Y. A., Wu, S. B., Wei, Y. H., Tsai, P. Y., and Chuang,
T. Y. (2012). Effects of intravascular laser irradiation of blood in
mitochondria dysfunction and oxidative stress in adults with chronic
spinal cord injury, Photomed. Laser Surg., 30, pp. 579–586.
48. Rochkind, S., El-Ani, D., Nevo, Z., and Shahar, A. (2009). Increase of
neuronal sprouting and migration using 780 nm laser phototherapy as
procedure for cell therapy, Lasers Surg. Med., 41, pp. 277–281.
49. von Leden, R. E., Cooney, S. J., Ferrara, T. M., Zhao, Y., Dalgard, C. L.,
Anders, J. J., and Byrnes, K. R. (2013). 808 nm wavelength light induces
a dose-dependent alteration in microglial polarization and resultant
microglial induced neurite growth, Lasers Surg. Med., 45, pp. 253–263.
50. Chen, Y. J., Wang, Y. H., Wang, C. Z., Ho, M. L., Kuo, P. L., Huang, M. H.,
and Chen, C. H. (2014). Effect of low level laser therapy on chronic
compression of the dorsal root ganglion, PLoS One, 9, pp. e89894-1-8.
July 6, 2016 17:31 PSP Book - 9in x 6in 24-Hamblin-c24

Chapter 24

Low-Level Laser (Light) Therapy for the

Treatment of Visual System Injury and
Disease

Janis T. Eells,a Sandeep Gopalakrishnan,b Michele M. Salzman,c

Krisztina Valter,d Jan Provis,d Ricardo Natoli,d John Mitrofanis,e
Jonathan Stone,e and Melinda Fitzgeraldf
a Biomedical Sciences, University of Wisconsin–Milwaukee, Milwaukee, WI 53201, USA
b College of Nursing, University of Wisconsin–Milwaukee, Milwaukee, WI 53201, USA
c Anesthesiology, Vanderbilt University Medical Center, Nashville, TN 37240, USA
d Division of Biomedical Sciences, Research School of Biology, Australian National

University, Acton 0200, Australia

e Bosch Institute, Discipline of Physiology and Save Sight Institute, University of Sydney,

Sydney, NSW 2006, Australia

f Experimental and Regenerative Neurosciences, School of Animal Biology,

The University of Western Australia, Crawley 6009, Australia

jeells@uwm.edu

24.1 Introduction

The retina is one of the highest oxygen-consuming tissues in the

human body. Its inner layers have the high metabolic rate of
all central nervous tissue, and the oxygen consumption rate in

Handbook of Low-Level Laser Therapy

Edited by Michael R. Hamblin, Marcelo Victor Pires de Sousa, and Tanupriya Agrawal
Copyright c 2017 Pan Stanford Publishing Pte. Ltd.
ISBN 978-981-4669-60-3 (Hardcover), 978-981-4669-61-0 (eBook)
www.panstanford.com
July 6, 2016 17:31 PSP Book - 9in x 6in 24-Hamblin-c24

436 Low-Level Laser (Light) Therapy for the Treatment of Visual System Injury and Disease

its photoreceptor layer is, uniquely, several times higher again.

The photoreceptor cells (rods and cones) are correspondingly
rich in mitochondria, which concentrate in their inner segments
and provide the ATP required by the ionic pumps that drive the
“dark current,” whose modulation by light is the beginning of
vision [51]. As a result, photoreceptors consume more oxygen
per gram of tissue weight than any cell in the body [51]. This
intense oxidative phosphorylation in their inner segments, coupled
with high concentrations of polyunsaturated fatty acids in their
outer segments, renders the retina susceptible to oxidative stress
and lipid peroxidation [49]. The retina also contains abundant
photosensitizers and is exposed to visible light [20]. Normally,
oxidative damage is minimized by endogenous antioxidants and
repair systems. With aging and/or retinal disease, there is an
increase in mitochondrial dysfunction and oxidative damage and
corresponding decrements in antioxidants and repair systems,
resulting in retinal dysfunction and retinal cell loss, leading to visual
impairment.
Mitochondrial dysfunction and oxidative damage to the retina
and its components, including photoreceptors, retinal pigment
epithelium (RPE), and retinal ganglion cells (RGC), have been impli-
cated in many forms of retinal injury and degeneration, including
methanol intoxication, light-induced retinal damage, retinopathy
of prematurity, age-related macular degeneration (AMD), retinitis
pigmentosa, and diabetic retinopathy [14]. Mitochondrial repair and
attenuation of oxidative stress are key to the long-term survival of
the retina.
This review concerns the potential, claimed now in many studies,
for red–infrared light to promote the repair of retinal cells in several
of these conditions, and to do so at low energy, and therefore without
side effects. The claim is a large one because the protection and
repair of damaged retinal tissue are not yet concepts in standard
ophthalmological practice; and the application of low-intensity
far-red to near-infrared (FR/NIR) light is minimally invasive and
nontoxic, though dosage is important [8, 10, 21, 35].
We also review mechanisms of action of FR/NIR light, for which
two principal lines of evidence have emerged. Low-intensity FR/NIR
light, when applied directly to damaged tissue, has been shown
July 6, 2016 17:31 PSP Book - 9in x 6in 24-Hamblin-c24

LLLT in Animal Models of Retinal and Optic Nerve Injury 437

to act on mitochondria-mediated signaling pathways to preserve

mitochondrial function, attenuate oxidative stress, stimulate the
production of cytoprotective factors, and prevent neuronal death
in cultured neurons and in animal models of neuronal injury and
disease [6, 17, 23–25, 30, 36]. FR/NIR photons penetrate the
tissue more deeply than shorter or longer wavelengths, and this
treatment, commonly known as low-level laser (or light) therapy
(LLLT) or photobiomodulation (PBM), has documented efficacy in
the prevention and treatment of neurodegenerative diseases in
experimental and clinical studies [28, 29, 35, 50]. Numerous studies
have documented the therapeutic potential and mechanism(s) of
action of LLLT, applied directly to damaged tissue, in the treatment
and pathogenesis of retinal injury and disease [14].

24.2 LLLT in Animal Models of Retinal and Optic

Nerve Injury

24.2.1 Methanol Intoxication

Methanol intoxication produces toxic injury to the retina and optic
nerve, resulting in blindness. Both acute and chronic exposures
to methanol have been shown to produce retinal dysfunction and
optic nerve damage clinically and in experimental animal models
[43]. A toxic acute exposure to methanol results in formic acidemia,
metabolic acidosis, and visual toxicity within 72 h of ingestion
[43]. The toxic metabolite is formic acid, a mitochondrial toxin
known to inhibit the essential mitochondrial enzyme, cytochrome
c oxidase [11, 12, 43]. Eells et al. [11] reported the first direct
link between the actions of far-red to NIR light on mitochondrial
oxidative metabolism in vitro and retinoprotection in vivo in a
well-established rodent model of methanol toxicity [43]. Using
the electroretinogram as a sensitive indicator of retinal function,
these studies demonstrated that three brief 670 nm LED (160 s
at 25 mW/cm2 producing a fluence 4 J/cm2 at the surface of the
eye) (Spectralife, Quantum Devices Inc., Barneveld WI) treatments
delivered at 5, 25, and 50 h of methanol intoxication attenuated
the retinotoxic effects of methanol-derived formate. The authors
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438 Low-Level Laser (Light) Therapy for the Treatment of Visual System Injury and Disease

documented a significant recovery of rod- and cone-mediated

function in LLLT-treated, methanol-intoxicated rats. The 670 nm
LLLT also protected the retina from the histopathologic changes
induced by methanol-derived formate. Not only do these findings
provide a link between the interaction of FR/NIR light with
cytochrome oxidase in vitro and retinoprotection in vivo, they
also suggest that LLLT may enhance recovery from retinal injury
and other ocular diseases in which mitochondrial dysfunction and
oxidative stress are postulated to play a role. More recent studies
have shown that mitochondrial dysfunction and oxidative stress play
key roles in most, if not all, retinal and optic nerve diseases [20].

24.2.2 Light-Induced Retinal Damage

It was first shown in the 1960s that exposure to excessive light
causes photoreceptor damage and death to retinal cells [16].
Oxidative damage produced by photo-oxidation of the photorecep-
tor outer segments is widely accepted as the initiating event in
light-induced retinal damage (LD) [9]. Lesions produced by LD
are characterized by photoreceptor cell death, RPE cell damage,
Müller cell gliosis, and disruption of the outer limiting membrane.
In addition to these structural changes, there is the induction of an
inflammatory state characterized by an invasion of the outer retina
by activated microglia [1–3]. This progressive degeneration has been
used to model many of the factors contributing to the expansion
of the degenerative area, similar to the changes observed in AMD
[40, 41].
Several studies have shown that 670 nm LLLT is protective
against light-induced retinal degeneration [1–3, 34, 38, 41]. The
670 nm LLLT (9 J/cm2 ) [3] administered before, during, or after
exposure to LD protected photoreceptor function as measured by
ERG responses and morphology. This protection involved a reduc-
tion in photoreceptor cell death and inflammatory stress biomarkers
in the retina and reduction in microglial and macrophage invasion
[2, 3]. Pretreatment with LLLT proved to be most effective against
LD compared to treatment during or after LD. However, animals
treated with LLLT post-LD also recovered photoreceptor function by
1 month post-exposure [3].
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LLLT in Animal Models of Retinal and Optic Nerve Injury 439

Microarray analysis showed that gene expression in pathways

involved in inflammation and cell death is downregulated by 670 nm
LLLT in light-damaged retinas [34]. In this study, experimental
groups were protected against light damage (1000 lux for 24 h) by
pretreatment with 670 nm LLLT (9 J/cm2 at the eye, daily for 5 days).
RNA from the retina was hybridized to Affymetrix rat genome ST
arrays. Quantitative real-time PCR analysis of 14 selected genes was
used to validate the microarray results. LD caused the regulation of
175 entities (genes and noncoding RNAs [ncRNAs]) beyond criterion
levels ( p < 0.05 in comparison with controls, fold-change >2).
LLLT pretreatment reduced the expression of 126 of these 175 LD-
regulated entities below criterion. In addition, PBM pretreatment
regulated the expression of 67 entities not regulated by LD. A high
proportion of the entities regulated by LD (>90%) were known
genes. By contrast, ncRNAs were prominent among the entities
regulated by 670 nm LLLT. One of the most highly modulated genes
identified in this study was Ccl2, a potent chemokine involved in
the recruitment of monocytes, T-cells, and dendritic cells to sites of
tissue injury. This chemokine family has also been implicated in the
pathogenesis of AMD. The 670 nm LLLT profoundly downregulated
the expression of Ccl2.
The 670 nm LLLT has been shown to downregulate TNF-alpha,
a key proinflammatory cytokine, in two independent studies in
the eye. Albarracin and Valter [1, 2] showed a reduction in TNF-
alpha expression in LD retina pretreated with 670 nm LLLT using
PCR. Kokkinopoulis et al. [26] showed that 670 nm irradiation
reduced TNF-alpha immunoreactivity in the retinas of aged mice.
They further showed a reduction in the recruitment of IBA-1
positive macrophages to the outer retina and a reduction in C3b and
C3d immunoreactivity in Bruch’s membrane. All these changes are
consistent with the downregulation of inflammatory responses by
LLLT.
Complement activation is associated with the pathogenesis of
AMD and also occurs following LD [3, 41, 42]. The 670 nm LLLT
pretreatment (9 J/cm2 ) reduced the expression of complement
components and receptors in the retina following LD [40]. Moreover,
there was a reduction in the recruitment of C3 expressing
microglia/macrophages in the retina following 670 nm LLLT and
July 6, 2016 17:31 PSP Book - 9in x 6in 24-Hamblin-c24

440 Low-Level Laser (Light) Therapy for the Treatment of Visual System Injury and Disease

a concomitant reduction in the biomarker of oxidative damage, 4-

hydroxynonenal (4-HNE). These findings indicate that the 670 nm
LLLT pretreatment attenuates oxidative damage to photoreceptors
and reduces inflammation, which may reduce the stimulation of the
complement cascade, thus further protecting photoreceptors.

24.2.3 Optic Nerve Injury

Using a rodent model of toxic optic neuropathy induced by the mi-
tochondrial complex I inhibitor, rotenone, Rojas et al. [39] reported
on the neuroprotective actions of 633 nm LLLT. Pigmented rats
received single bilateral intravitreal doses of rotenone, or rotenone
plus different dosage regimens of 633 nm LLLT (3.6 J/cm2 for 3 or
6 days). Treatment effects were evaluated at behavioral, structural,
and neurochemical levels. Rotenone induced a decrease in visual
function, determined by changes in the dark-adapted illuminance
sensitivity threshold, escape latency, and rate of successful trials in
a two-choice visual task, compared with vehicle-treated controls.
Behavioral impairment correlated with a decrease in retinal and
visual pathway metabolic activity, retinal nerve fiber layer thickness,
and ganglion cell layer cell density. These changes were prevented
by LLLT treatments in a dose-dependent manner (the most effective
total dose was 3.6 J/cm2 of 633 nm LLLT delivered once per day
for 6 days following rotenone treatment). Whole-brain cytochrome
oxidase and superoxide dismutase activities were also increased in
LLLT-treated subjects in a dose-dependent manner, suggesting an in
vivo transcranial effect of LLLT. In whole-brain membrane isolates,
LLLT prevented the rotenone-induced decrease in cell respiration.
The results show that LLLT treatment can effectively prevent the
neurotoxic and retinotoxic effects of rotenone and that it might be
used in the treatment of neurodegenerative disorders associated
with mitochondrial dysfunction due to complex I inhibition.
It has been shown that traumatic injury to the central nervous
system (CNS) is accompanied by the spreading damage of secondary
degeneration, resulting in further loss of neurons and function.
Partial transection of the optic nerve has been used as a model of
secondary degeneration. Axons of RGCs in the ventral optic nerve
are spared from initial dorsal injury but are vulnerable to secondary
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LLLT in Animal Models of Retinal and Optic Nerve Disease 441

degeneration mediated by oxidative stress [5]. Using this partial

injury model, Fitzgerald et al. [13, 14] have demonstrated that LLLT
treatment (WARP-10 LED diode array, Quantum Devices Inc. Barn-
eveld, WI, 670 nm, daily 30 min treatments) reduced an indicator
of oxidative stress in areas of optic nerve vulnerable to secondary
degeneration. Importantly, visual function was restored to normal
by LLLT treatment as assessed using optokinetic nystagmus and the
Y-maze pattern discrimination task, providing evidence that 670 nm
LLLT light can reduce oxidative stress and improve function in the
CNS after traumatic injury in vivo.
This team of investigators went on to show that in ventral
optic nerve specifically vulnerable to secondary degeneration,
oligodendrocytes show early signs of oxidative stress, with increases
in advanced glycation end products [46], protein nitration, and
lipid oxidation. Node and paranode structure is also disrupted, with
paranode lengthening and disorganization temporally mirroring
oxidative stress changes in oligodendrocytes [46]. Thirty minute
670 nm LLLT treatments (WARP-75 LED diode array, Quantum
Devices Inc. Barneveld, WI, 28.4 J/cm2 ) increased cytochrome c
oxidase activity and attenuated oxidative stress in oligodendrocytes.
Short-term 670 nm light treatment decreased reactive oxygen
species at the injury site and reduced paranode abnormalities
in optic nerve vulnerable to secondary degeneration. Long-term
treatment preserved RGC vulnerable to secondary degeneration and
maintained behavioral visual function, suggesting that 670 nm LLLT
delivered by LED array may serve as a practical therapeutic strategy
to limit paranode abnormalities and preserve white matter function.

24.3 LLLT in Animal Models of Retinal and Optic Nerve

Disease

24.3.1 Retinopathy of Prematurity

Retinopathy of prematurity (ROP) is a disorder of the developing
retina and one of the leading causes of blindness in developed
countries. Risk factors for ROP include low birth weight and ges-
tational age, supplemental oxygen therapy, and respiratory distress
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442 Low-Level Laser (Light) Therapy for the Treatment of Visual System Injury and Disease

syndrome [4, 33]. ROP is a two-stage disease initiated by delayed

vascular growth following premature birth followed by the release
of hypoxia stimulated factors that stimulate neovascularization.
Current therapeutic interventions include laser photocoagulation or
cryotherapy to target the angiogenic aspect of the disease. These
interventions are invasive, expensive, and only partially effective.
Natoli et al. [33] investigated the therapeutic efficacy of 670 nm
LLLT in two well-established rodent models of oxygen-induced
retinopathy (OIR). Treated animals were exposed to 670 nm LLLT
(WARP-75 LED diode array, Quantum Devices Inc. Barneveld, WI) at
a dose of 9 J/cm2 (50 mW/cm2 × 180 s) once daily during exposure
to hyperoxia. The 670 nm LLLT reduced neovascularization, vaso-
obliteration, and abnormal branching patterns of retinal vessels in
OIR.

24.3.2 Diabetic Retinopathy

Diabetic retinopathy is a common long-term complication of
diabetes. This retinopathy is characterized by damage to the
vasculature and neurons as well as loss of vision itself in severe
cases. Although the pathogenesis of diabetic retinopathy is not
completely understood, a reduction in hyperglycemia has been
shown to exert positive effects on its development and progression.
However, in many patients, maintenance of glycemic control is
difficult; therefore, effective therapies are needed to inhibit the
retinopathy. Tang et al. [47] investigated the therapeutic potential
of LLLT against diabetes-induced damage to the retina. The
effects of transient (240 s, 25 mW/cm2 , 6 J/cm2 ) 670 nm LLLT
(Spectralife, Quantum Devices, Inc., Barneveld, WI) on in vivo
and in vitro pathologic changes relevant to the development of
diabetic retinopathy were studied. The authors found that this
LLLT exerted significant beneficial effects on the retina in diabetes.
In streptozotocin (STZ)-diabetic rats, LLLT attenuated diabetes-
induced abnormalities of retinal function measured by ERGs. At
the cellular level, the 670 nm LLLT decreased RGC death, retinal
superoxide generation, leukostasis, and expression of manganese
superoxide dismutase (MnSOD) and ICAM-1. LLLT also attenuated
oxidative stress and improved cell survival in cultured retinal cells
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LLLT in Animal Models of Retinal and Optic Nerve Disease 443

incubated in diabetic-like concentrations of glucose. The authors

concluded that 670 nm LLLT is a simple adjunct therapy to attenuate
the development of diabetic retinopathy.

24.3.3 Retinitis Pigmentosa

Retinitis pigmentosa is a heterogeneous group of blinding retinal
diseases, with the common feature of photoreceptor degenera-
tion, commonly associated with single-gene mutations. Numerous
investigations have provided considerable information regarding
the genetic basis of these degenerations, and several previous
studies have provided evidence that, in many forms of retinal
dystrophy, oxidative damage to mitochondria is a step in the
death of photoreceptors [44, 45]. Photoreceptors have a dense
concentration of mitochondria in their inner segments to provide
ATP for the energy-intensive processes of outer segment renewal
and maintenance of dark current.
The therapeutic efficacy and mechanism of action of 670 nm
LLLT was investigated in a rodent model of retinitis pigmentosa,
the P23H rat [22]. In this model, the transgene is a rhodopsin
gene engineered to mimic a mutation that causes an autosomal
dominant form of human retinitis pigmentosa common in North
America. P23H rat pups were treated once per day during the
critical period of photoreceptor development with a 670 nm LED
array (180 s treatments at 50 mW/cm2 ; fluence 9 J/cm2 ) (Quantum
Devices Inc., Barneveld WI). Sham-treated rats were restrained but
not exposed to NIR light. In the first series of studies, rats were
treated from postnatal day (p) 16 to p20. The status of the retina
was determined at p22 by the assessment of mitochondrial function,
oxidative stress, and cell death. In a second series of studies, rat pups
were treated from p10 to p25. Retinal status was assessed at p30 by
measuring photoreceptor function by ERG and retinal morphology
by Spectral Domain Optical Coherence Tomography (SD-OCT).
The 670 nm LLLT increased retinal mitochondrial cytochrome
oxidase activity and upregulated the retina’s production of the key
mitochondrial antioxidant enzyme, MnSOD. LLLT also attenuated
photoreceptor cell loss and improved photoreceptor function.
LLLT protects photoreceptors in the developing P23H retina, by
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444 Low-Level Laser (Light) Therapy for the Treatment of Visual System Injury and Disease

augmenting mitochondrial function and stimulating antioxidant

protective pathways. LLLT may have therapeutic potential, where
mitochondrial damage is a step in the death of photoreceptors.

24.3.4 Aging and Age-Related Macular Degeneration

Inflammation is a common feature in the aged retina and in many
retinal diseases, including AMD. In addition, mitochondrial function
has been shown to decline in aging and AMD [20]. Brief exposure
to 670 nm LLLT in the aged retina has been shown to increase
mitochondrial membrane potential and reduce inflammation [26].
Using an aged mouse model of AMD, the complement factor H
knockout (CFH−/− ) in which inflammation is a key feature, Begum
et al. [7] investigated the effects of 670 nm LLLT delivered briefly
in environmental lighting rather than directly focused on the retina.
Mice were exposed to 670 nm for 6 min twice a day for 14
days in the form of supplemented environmental light. Exposed
animals exhibited a significant increase in cytochrome c oxidase.
Complement component C3, an inflammatory marker in the outer
retina, was downregulated, as were vimentin and glial fibrillary
acidic protein expression, which reflect retinal stress in Müller glia.
Hence, 670 nm LLLT is effective in reducing retinal inflammation
likely by cytochrome c oxidase activation in mice with a genotype
similar to that in 50% of AMD patients, even when brief exposures
are delivered via environmental lighting. The efficacy revealed here
supports current early stage clinical trials of 670 nm in AMD
patients.

24.3.5 Parkinson’s Disease

Parkinson disease is mainly characterized by the degeneration
of dopaminergic neurons in the CNS, including the retina [37].
Considerable evidence supports mitochondrial dysfunction and ox-
idative stress as key components in the pathogenesis of Parkinson’s
disease [37]. The Mitrofanis Laboratory [37] tested whether 670 nm
LLLT treatment would enhance the survival of tyrosine hydroxylase
(TH)+ dopaminergic amacrine cells located in the retina using an
acute and a chronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
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LLLT in Clinical Investigations of Retinal Disease 445

(MPTP) mouse model of Parkinson’s disease. For the acute model,

BALB/c mice had MPTP (100 mg/kg) or saline injections over
30 h, followed by a 6-day survival period. For the chronic model,
mice had MPTP (200 mg/kg) or saline injections over five weeks,
followed by a 3-week survival period. The 670 nm LLLT treatment
(90 s at 50 mW/cm2 , 4.5 J/cm2 ) was applied either at the same
time (simultaneous series) or well after (post-treatment series) the
MPTP insult. TH+ cell number immunochemistry was analyzed.
In the MPTP groups, there was significant reduction in TH+ cell
number compared to the saline controls, and the reduction was
greater in the acute (50%) compared to the chronic (30%) model.
In the 670 nm LLLT-treated groups, there were significantly more
TH+ cells than in the MPTP groups of both series (30%). The 670 nm
LLLT treatment was cytoprotective in the simultaneous and rescue
(post-treatment) series of studies.

24.4 LLLT in Clinical Investigations of Retinal Disease

24.4.1 Age-Related Macular Degeneration

Ivandic and Ivandic [19] have shown that 780 nm LLLT with a
laser diode (7.5 mW, 292 Hz, continuous emission for 40 s) aimed
at the macular area (transsclerally) significantly improved visual
acuity in 162/182 (95%) of eyes with cataracts and 142/146 (97%)
of eyes without cataracts in a case series of patients with both
dry and wet AMD. The prevalence of metamorphopsia, scotoma,
and dyschromatopsia was also reduced. In patients with wet AMD,
edema and bleeding improved. The improved vision was maintained
for 3–36 months after treatment. Visual acuity in the control group
remained unchanged. No adverse effects were observed in those
undergoing therapy [27].
To date, only one investigator-initiated human study for AMD has
been conducted with LED treatment in dry AMD [31, 32]. A pilot
study lead by Drs. Devenyi, Merry, and Dotson from The Toronto
and Oak Ridge Study of Photobiomodulation (TORPA) examined
the efficacy of LLLT in the treatment of dry AMD (ARVO Abstract).
This prospective study evaluated validated clinical outcomes in
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446 Low-Level Laser (Light) Therapy for the Treatment of Visual System Injury and Disease

clinically diagnosed dry AMD patients. The subject inclusion criteria

were subjects ≥50 years of age, dry AMD in study eye, and
BCVA between 20/20 and 20/200. The exclusion criteria were
any subjects with visually significant cataract, visually significant
capsular clouding post-cataract/IOL, or any visually significant
disease process. Twenty-two eyes (11 subjects) with dry AMD were
enrolled, and they received 18 treatments over a 6-week period
with two LED devices emitting 590 nm, 670 nm, and 830 nm light.
The three pre-selected wavelengths (590, 670, and 830 nm) were
combined to stimulate mitochondrial cytochrome c oxidase function
and suppress VEGF expression.
Primary outcome measures were change in ETDRS visual acuity,
contrast sensitivity, and fixation stability. Eighteen eyes (nine
subjects) completed the protocol. Four eyes (two subjects) were
excluded from data analysis due to protocol violations. Changes in
visual acuity ( p < 0.0001) and contrast sensitivity ( p < 0.0001
at 3 cycles/degree and p < 0.0032 at 1.5 cycles/degree) were
positive and significant. There were no significant changes in
fixation stability parameters.
The TORPA data for dry AMD show clinically and statistically
significant improvement in visual acuity and contrast sensitivity at
6 weeks and up to 1 year of follow-up, demonstrating the potential
use of LLLT delivered by LED in dry AMD. The study conclusions
were that LED treatment was a noninvasive, easily administered,
and safe treatment with no serious adverse events noted. More
importantly, the studies provide the only 1-year follow-up data of
LED effectiveness in dry AMD. Some loss of clinical benefit was
seen following cessation of treatment, but the vision benefits were
stable in the follow-up to 1 year, suggesting the need to establish
a maintenance dosing schedule and the need to better understand
the underlying cellular benefits. Nevertheless, these findings are the
first to demonstrate a statistically significant clinical benefit [F(4,68)
= 18.86, p < 0.0001] at up to 1 year in dry AMD patients.

24.4.2 Diabetic Retinopathy

The therapeutic efficacy of 670 nm LLLT has been investigated in
a small study involving four patients with noncentered diabetic
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References 447

retinal edema (NCDME) [48]. Although this type of retinal edema

does not impact vision, because the center of the macula is
spared, a substantial percentage of these patients progress to vision
threatening center-involved diabetic macular edema within a year.
Patients were treated twice daily at a fluence of 8 J/cm2 (80 s per
treatment at 50 mW/cm2 ) using a small handheld 670 nm LED array
(WARP10, Quantum Devices, Inc., Barneveld, WI) held 1 inch from
the treated eye. Each patient served as his/her own control, with
one eye treated and the other not treated. Treatment was applied
for a minimum of 2 months with patients having the option to
be treated for 9 months. LLLT treatment resulted in a significant
decrease in NCDME measured by spectral-density optical coherence
tomography (SD-OCT). One patient developed nonarteritic anterior
ischemic optic neuropathy, which the authors speculate was not
related to LLLT treatment. The authors conclude that 670 nm LLLT
has promise as a cost-effective and noninvasive adjunct to inhibit
retinal edema and are planning additional studies.

24.5 Conclusion

Taken as a whole, these studies in experimental models of retinal

and optic nerve injury and disease show that far-red and NIR
LLLT improves mitochondrial function, reduces oxidative stress, and
modulates inflammatory mediators, leading to decreased apoptosis
and retinoprotection. Limited clinical investigations also support the
therapeutic efficacy of FR/NIR LLLT in the treatment of AMD and
diabetic retinopathy. Further studies are necessary to characterize
the effect of LLLT on the human retina and optic nerve and to
define safe protocols for the application of this novel therapy to
mechanistically complex diseases.

References

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H.T. (2004). Mitochondrial signal transduction in accelerated wound
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559–567.
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13. Fitzgerald, M., Bartlett, C.A., Payne, S.C., Hart, N.S., Rodger, J., Harvey, A.R.,
and Dunlop, S.A. (2010). Near infrared light reduces oxidative stress and
preserves function in CNS tissue vulnerable to secondary degeneration
following partial transection of the optic nerve. J. Neurotrauma, 27,
2107–2119.
14. Fitzgerald, M., Hodgetts, S., van den Heuvel, C., Natoli, R., Hart, N., Valter,
K., Harvey, A.R., Vink, R., Provis, J., and Dunlop, S.A. (2013) Red/near-
infrared irradiation therapy for treatment of central nervous system
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16. Hollyfield, J.G., Bonilha, V.L., Rayborn, M.E., Yang, X., Shadrach, K.G., Lu,
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induced inflammation initiates age-related macular degeneration. Nat.
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17. Huang, Y.Y., Sharma, S.K., Carroll, J., and Hamblin, M.R. (2011). Biphasic
dose response in low level light therapy: An update. Dose-Response, 9,
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18. Ishiguro, M., Ikeda, K., and Tomita, K. (2010). Effect of near-infrared
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19. Ivandic, B.T., and Ivandic, T. (2008) Low-level laser therapy improves
vision in patients with age-related macular degeneration. Photomed.
Laser Surg., 26, 241–245.
20. Jarrett, S.G., and Boulton, M.E. (2012). Consequences of oxidative stress
in age-related macular degeneration. Molec Aspects Med., 33, 399–417.
21. Jenkins, P.A., and Carroll, J.D. (2011). How to report low-level laser
therapy (LLLT)/photomedicine dose and beam parameters in clinical
and laboratory studies. Photomed. Laser Surg., 29, 785–787.
22. Kirk, D.K., Gopalakrishnan, S., Schmitt, H., Abroe, B., Stoehr, M., Dubis,
A., Carroll, J., Stone, J., Valter, K., and Eells, J. Photobiomodulation
reduces photoreceptor death and regulates cytoprotection in early
states of P23H retinal dystrophy. Proc. SPIE BIOS, San Francisco, January
2013.
23. Karu, T. (1999). Primary and secondary mechanisms of action of visible
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responses relevant to phototherapy. Photomed. Laser Surg., 2, 355–361.
25. Karu, T.I., Pyatibrat, L.V., Kolyakov, S.F., and Afanasyeva, N.I. (2008).
Absorption measurements of cell monolayers relevant to mechanisms
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of laser phototherapy: Reduction or oxidation of cytochrome c oxidase

under laser radiation at 632.8 nm. Photomed. Laser Surg. 26, 593–599.
26. Kokkinopoulos, I., Colman, A., Hogg, C., Heckenlively, J., and Jeffery, G.
(2012). Age-related retinal inflammation is reduced by 670 nm light
via increased mitochondrial membrane potential. Neurobiol. Aging, 34,
602–609.
27. Kowaltowski, A.J., de Souza-Pinto, N.C., Castilho, R.F., and Vercesi, A.E.
(2009). Mitochondria and reactive oxygen species. Free. Radic. Biol.
Med., 4, 333–343.
28. Liang, H.L., Whelan, H.T., Eells, J.T., Meng, H., Buchmann, E., Lerch-
Gaggl, A., and Wong-Riley, M.W.R. (2006). Photobiomodulation partially
rescues visual cortical neurons from cyanide-induced apoptosis. Neuro-
science, 139, 639–649.
29. Liang, H.L., Whelan, H.T., Eells, J.T., and Wong-Riley, M.T. (2008). Near-
infrared light via light-emitting diode treatment is therapeutic against
rotenone- and 1-methyl-4-phenylpyridinium ion-induced neurotoxicity.
Neuroscience, 153, 963–974.
30. Mason, M.G., Nicholls, P., Wilson, M.T., and Cooper, C.E. (2006). Nitric
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Acad. Sci. USA, 103, 708–713.
31. Merry, G., Dotson, R., Devenyi, R., Markowitz, S., and Reyes, S. (2012)
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degeneration. Results from the Toronto and Oak Ridge photobiomod-
ulation study. ARVO Meeting Abstract, 53, 2049.
32. Merry, G., Devenyi, R., Dotson, R., Markowitz, S., and Reyes, S. (2012)
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ulation WALT Meeting.
33. Natoli, R., Valter, K., Barbosa, M., Dahlstrom, J., Rutar, M., Kent, A., and
Provis, J. (2013). 670 nm photobiomodulation as a novel protection
against retinopathy of prematurity: Evidence from oxygen-induced
retinopathy models. PLoS ONE, 8, e72135.
34. Natoli, R., Zhu, Y., Valter, K., Bisti, S., Eells, J., and Stone, J. (2010). Gene
and noncoding RNA regulation underlying photoreceptor protection:
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tion in rat retina. Mol. Vis., 16, 1801–1822.
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36. Peng, T.I., and Jou, M.J. (2010). Oxidative stress caused by mitochondrial
calcium overload. Ann. NY Acad. Sci., 1201, 183–188.
37. Peoples, C., Shaw, V.E., Stone, J., Jeffery, G., Baker, G.E., and Mitrofanis,
J. (2012). Survival of dopaminergic amacrine cells after near-infrared
light treatment in MPTP-treated mice. ISRN Neurol., 2012, 850150.
38. Qu, C., Cao, W., Fan, Y., and Lin, Y. (2010). Near-infrared light protects the
photoreceptor from light-induced damage in rats. Adv. Exp. Med. Biol.,
664, 365–374.
39. Rojas, J.C., Lee, J., John, J.M., and Gonzalez-Lima, F. (2008). Neuroprotec-
tive effects of near-infrared light in an in vivo model of mitochondrial
optic neuropathy. J. Neurosci., 28, 13511–13521.
40. Rutar, M., Provis, J.M., and Valter, K. (2010). Brief exposure to
damaging light causes focal recruitment of macrophages, and long-term
destabilization of photoreceptors in the albino rat retina. Curr. Eye Res.
35, 631–643.
41. Rutar, M., Natoli, R., Albarracin, R., Valter, K., and Provis, J. (2012). 670
nm light treatment reduces complement propagation following retinal
degeneration. J. Neuroinflamm., 9, 257–263.
42. Rutar, M., Natoli, R., Valter, K., and Provis, J.M. (2011). Early focal
expression of the chemokine Ccl2 by Muller cells during exposure to
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44. Shen, J., Yang, X., Dong, A., Petters, R.M., Peng, Y.W., Wong, F., and
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O’Hare Doig, R.L., Savigni, D.L., Payne, S.C., Harvey, A.R., Dunlop, S.A., and
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Low-intensity far-red light inhibits early lesions that contribute to
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diabetic retinopathy: in vivo and in vitro. Invest. Ophthalmol. Vis. Sci., 54,
3681–3690.
48. Tang, J., Herda, A.A., and Kern, T.S. (2014) Photobiomodulation in
the treatment of patients with non-center involving diabetic macular
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49. Winkler, B.S. (1981). Glycolytic and oxidative metabolism in relation to
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Buchmann, E., Kane, M., and Whelan, H.T. (2005). Photobiomodulation
directly benefits primary neurons functionally inactivated by toxins:
Role of cytochrome c oxidase. J. Biol. Chem., 280, 4761–4771.
51. Yu, D.Y., and Cringle, S.J. (2005). Retinal degeneration and local oxygen
metabolism. Exp. Eye Res., 80, 745–751.
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Chapter 25

Protection from Cardiac Ischemia and

Reperfusion Injury

Agnes Keszler, Svjetlana Dosenovic, and Martin Bienengraeber

Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA
mbieneng@mcw.edu

Photobiomodulation with far-red/near-infrared (NIR) light has

been applied successfully to ameliorate cardiac injury, both in the
acute and in the chronic ischemic heart. This chapter will review the
use of NIR in the protection of the heart from ischemia or ischemia
and reperfusion injury and discuss potentially involved mechanisms.
The prospective for translation into the clinic will also be addressed.

25.1 Introduction

Coronary artery disease continues to represent an important

cause of morbidity and mortality in Western society. Restoration
of blood flow to a region of previously ischemic myocardium
(reperfusion) is a critical life-saving intervention against tissue
necrosis, but reperfusion itself also results in significant damage to
myocardium. In fact, intervention at the time of reperfusion seems
sufficient to mitigate damage, which is of higher clinical impact

Handbook of Low-Level Laser Therapy

Edited by Michael R. Hamblin, Marcelo Victor Pires de Sousa, and Tanupriya Agrawal
Copyright c 2017 Pan Stanford Publishing Pte. Ltd.
ISBN 978-981-4669-60-3 (Hardcover), 978-981-4669-61-0 (eBook)
www.panstanford.com
July 6, 2016 17:32 PSP Book - 9in x 6in 25-Hamblin-c25

454 Protection from Cardiac Ischemia and Reperfusion Injury

compared to treatment before the ischemic event. Mitochondrial

dysfunction, Ca2+ overload, reactive oxygen species (ROS), and
the inflammatory response are some of the main culprits for the
damage of reperfusion injury. Many powerful therapeutic strategies
such as ischemic and pharmacologic pre- and postconditioning
have been developed and are effective in healthy animal models,
but few have translated effectively to patients. A major reason for
the resistance to cardioprotection against infarction by physical or
pharmacological stimuli is the advanced age and/or presence of
comorbidities such as diabetes in patients [1]. Increased oxidative
stress and endothelial dysfunction with disrupted nitric oxide
synthase (NOS) activity appear to contribute to the lack of protection
by ischemic or pharmacologic postconditioning [2, 3]. An acute and
a more chronic phase can be distinguished in the protection of
myocardial damage after an ischemic event. Immediately at the time
of reperfusion, prevention of the large burst in radicals, overload
in cytosolic and mitochondrial Ca2+ , and permeability transition
pore opening have been shown to improve cardiac function and
decrease injury [4]. In the chronically ischemic heart, or after an
extended period of reperfusion, stimulating pro-survival signaling
pathways, extracellular matrix remodeling, and angiogenesis have
been demonstrated to lead to a better outcome [5]. Treatment of
the heart with NIR from low-energy laser or light-emitting diode
(LED) sources has been successfully used for preventing tissue
damage as well as for tissue repair in various animal models. In this
chapter, we will describe various strategies of NIR treatments of the
injured heart, discuss possible underlying mechanisms, and provide
an outlook on remaining open questions and the translatability
into human ischemic heart disease, including acute myocardial
infarction.

25.2 Repair of the Infarcted Heart

In the 1990s in Russia, patients with coronary heart disease with

prior myocardial infarction were exposed to NIR by low-level laser
irradiation (LLLI) applied to the area of the heart on the skin. Lipid
peroxidation was significantly reduced after NIR, but little is known
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Repair of the Infarcted Heart 455

on whether cardiac function improved [6, 7]. Building on previous

experiments with cold or ischemia-injured skeletal muscles of
mammals and amphibians, the effect of NIR by LLLI on the ischemic
heart in various animal models was studied by the group of Uri
Oron [8, 9]. When inducing ischemia by the vasoconstrictor peptide
sarafotoxin-b in mice, the subsequently observed dilation of the
heart was significantly reduced by LLLI through the chest muscle
at 810 nm (5 mW/cm2 at the heart for 2 min) immediately after
peptide injection and 2 days later [10]. In subsequent studies, NIR
was applied after chronic myocardial infarction in rat and dog
models. Infarct was induced by the ligation of the left anterior
descending coronary artery. LLLI (803 nm, 6 mW/cm2 at the surface
of the myocardium for 3 min, at 4–6 different locations) was applied
twice, 15 min and 3 days, after myocardial infarction through the
open chest directly onto the myocardium in dogs, and through the
intercostal muscles in rats. After myocardial infarction, LLLI in dogs
reduced both mortality and infarct size significantly, concomitant
with a decrease in inactive scar tissue [11]. Timing, frequency,
and dosage of LLLI need to be carefully selected. Particularly at
lower power density, early treatment after myocardial infarction and
subsequent repetition is advantageous. On the other hand, repeated
treatment at higher power density (12 mW/cm2 ) is not protective
[11]. Interestingly, similarly to delayed ischemic or pharmacologic
preconditioning, LLLI 7 days prior to induction of myocardial
infarction (5 or 12 mW/cm2 for 2 min) also resulted in a significant
decrease of infarct size [12]. When ischemia in the myocardium
was followed by reperfusion injury, the same treatment regimen
as described above (treatment 15 min and 3 days after myocardial
infarction) reduced dilation of the left ventricle significantly, but
infarct size only tentatively [12]. Overall, ample evidence exists on
the benefits of treatment of the infarcted heart with NIR.

25.2.1 Underlying Mechanisms of Light-Induced Repair

after Myocardial Infarction
Frequently, the beneficial effects of NIR treatment are associated
with the stimulation of mitochondrial metabolism, particularly at
the level of cytochrome c oxidase, complex IV, of the electron
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456 Protection from Cardiac Ischemia and Reperfusion Injury

transport chain [13]. Copper components of cytochrome c oxidase

are believed to be the photoreceptors. Phototherapy thereby en-
hances the mitochondrial ATP production, preserves mitochondrial
function, and, in turn, prevents the degeneration of cardiomyocytes
and other cells in the ischemic heart. In accordance with that,
irradiation of infarcted tissue resulted in preserved ATP content
and mitochondrial ultrastructure [14]. ATP is not only the major
energy currency within cells but is also considered an important
signaling molecule that allows cells and tissues throughout the
body to communicate, either as substrate for signaling kinases
or through ATP-specific receptors that have been associated with
cytoprotection [15].
In addition to altered mitochondrial metabolism upon light
irradiation, several proteins associated with cardioprotection have
been found to be upregulated. Whether this is a consequence of
enhanced metabolism, ATP signaling, or a more direct effect of
NIR on protein expression is currently not known. After myocardial
infarction in rats, NIR caused a significant increase in an inducible
heat shock protein (HSP70), independently of increased tempera-
ture [16]. Higher levels of heat shock proteins have been associated
with myocardial protection from ischemic injury [17]. Further, both
post-infarction irradiated rat hearts and irradiated normoxic hearts
demonstrated a significant increase in inducible NOS (iNOS) and
vascular endothelial growth factor (VEGF) expression compared to
non-irradiated hearts. This was paralleled by a significant elevation
in angiogenesis [12]. The fact that irradiation prior to myocardial
infarction also increased VEGF and iNOS expression may explain the
“preconditioning” effect of light treatment. More recently, increased
angiogenesis and collateralization upon NIR exposure (670 nm, 50
mW/cm2 , 10 min per day for 14 days) with LED have also been
reported in the ischemic hindlimb of mice and rabbits [18]. In
a mouse model for systemic sclerosis, an autoimmune connective
tissue disorder characterized by oxidative stress, impaired vascular
function, and attenuated angiogenesis, NIR stimulated angiogenesis
by increasing angiomotin and decreasing angiostatin expression in
the ischemic hindlimb [19].
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Protection Against Acute Ischemia and Reperfusion Injury 457

25.2.2 Induction of Stem Cells by Phototherapy

In isolated skeletal muscle fibers, LLLI at 632 nm (180 mW/cm2 ,
3 s) stimulated cell cycle entry and the accumulation of myosatel-
lite cells, paralleled with enhanced survival under serum-free
conditions that normally promote apoptosis [20]. Proliferation of
isolated myosatellite cells was enhanced by irradiation concomitant
with the activation of mitogen-activated kinase/extracellular signal-
regulated protein kinase (MAPK/ERK) [21, 22]. In contrast to
the skeletal muscle, the heart possess a rather limited capacity
to regenerate after myocardial infarction. Nevertheless, recent
research suggests a meaningful presence of progenitor cells in the
heart that can be activated after myocardial infarction in mice using
the peptide thymosin β4 [23]. Can stem cells be stimulated by NIR?
Indeed, the proliferation of both mesenchymal stem cells (MSCs) and
cardiac stem cells (CSCs) from rats was stimulated by irradiation
at 803 nm (50 mW/cm2 for 20 s or 60 s). When irradiated MSCs
were implanted into infarcted rat hearts, the hearts exhibited a
higher amount of stem cells, increased angiogenesis, and a reduction
of 53% in infarct size compared to hearts that were implanted
with non-irradiated cells. Thus, irradiation can significantly increase
survival and/or proliferation of MSCs post-implantation into the
ischemic/infarcted heart [24]. Elegantly and less invasively, rats
were treated 4 h after myocardial infarction with LLLI (804 nm, 10
mW/cm2 , 100 s) to the bone marrow of their exposed tibia. Such
treatment decreased infarct size to a larger degree compared to the
direct irradiation of the heart and increased the presence of progeni-
tor cells in the heart [25]. While the origin of those cells is unknown,
it was speculated that the increased number of progenitor cells in
the heart stems from MSCs induced by irradiation to the bone mar-
row, which were then moved by the circulating blood to the heart.

25.3 Protection Against Acute Ischemia and Reperfusion

Injury

Is it possible to not only reverse the extent of myocardial infarction

but prevent it from occurring, or at least limit its severity? This
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458 Protection from Cardiac Ischemia and Reperfusion Injury

was first tested in a cardiomyocyte model of acute hypoxia and

reoxygenation injury. Neonatal cardiomyocytes and HL-1 cells, a
cardiac muscle cell line derived from a mouse atrial myocyte
tumor lineage, were exposed to 6 h hypoxia in the absence of
serum and glucose, followed by 2 h of reoxygenation. At the
time of reoxygenation, cells were treated with NIR from a LED
array (670 nm; 5, 25, or 50 mW/cm2 for 5 min). NIR protected
cardiomyocytes from injury as indicated by decreased lactate
dehydrogenase release, caspase 3 activity, annexin binding, and
the release of cytochrome c from mitochondria into the cytosol,
compared to untreated cells [26]. A significant decrease in cell
damage was only observed at 25 mW/cm2 ; therefore, that dose was
used for subsequent experiments. Importantly, NIR increased NO
levels in cardiomyocytes, and the protective effect of NIR was com-
pletely reversed by the NO scavengers 2-(4-carboxyphenyl)-4,4,5,5-
tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) and oxyhemoglobin,
but only partially blocked by the NOS inhibitor L-NMMA [26]. In
vivo, rabbits or mice subjected to a 30 min left anterior descending
coronary artery occlusion and 2 h reperfusion exhibited a reduced
infarct size when treated with NIR at the onset of reperfusion
(beginning 1 min before and ending 4 min after reperfusion; 660 nm,
150 mW/cm2 at epicardial surface) [27, 42]. When a low-dose
irradiation (3 mW/cm2 ) was combined with low-dose nitrite (0.4
mg/kg), both non-protective alone, a reduction in infarct size was
observed [27]. This suggests a synergism whereby NIR is able to
potentiate the cardioprotective effect of nitrite. Protection again was
abolished in the presence of cPTIO [27].

25.3.1 Alternative Sources of Nitric Oxide in Light-Induced

Cardioprotection
There is large interest in NOS-independent generation of NO in vivo,
particularly under conditions of endothelial dysfunction (decreased
eNOS activity) such as during diabetes, or/and during hypoxia
and ischemia, where low oxygen tensions limit NOS activity [28–
30]. Under hypoxic conditions, heme-containing proteins such as
myoglobin (Mb) and hemoglobin (Hb) exhibit nitrite reductase
activity, which results in an increase in NO liberation (Eq. 25.1) [28,
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Protection Against Acute Ischemia and Reperfusion Injury 459

31]. The NO formed may subsequently react with available deoxyHb

or deoxyMb to yield iron-nitrosylHb (HbNO) or iron-nitrosylMb
(MbNO), respectively (Eq. 25.2):
NO− +
2 + deoxyHb(Fe ) + H → NO + metHb(Fe ) + HO
2+ 3+ −
(25.1)

NO + deoxyHb(Fe2+ ) → Hb(Fe2+ ) − NO (25.2)

Indeed, cardiac Mb has been implicated in the mechanism of
protection of cardiac tissue by nitrite during reperfusion injury [28].
Thus, HbNO and MbNO may represent a significant storage pool
of NO in the heart. Both NO signaling resulting in the activation
of soluble guanylyl cyclase (sGC) and production of cGMP [32–
34] as well as NO-dependent S-nitrosation of a cysteine residue in
complex I [35] have been implicated in protection against cardiac
ischemia and reperfusion injury. Reversible S-nitrosation of complex
I slows the reactivation of mitochondria during the crucial first
minutes of the reperfusion of ischemic tissue, thereby decreasing
ROS production, oxidative damage, and tissue necrosis. Thus,
interventions that can increase NO bioavailability have important
therapeutic potential.
It has been demonstrated both for purified hemoglobin or
myoglobin and for the myocardium that NIR can decay nitrosyl
hemes and release NO. When HbNO was added in a glass vessel
attached to a chemiluminescence-dependent NO analyzer and
irradiated with 670 nm light, a chemiluminescence signal was
detected upon irradiation indicating NO release, and the signal
returned to baseline after switching off the light (Fig. 25.1) [27].
A similar signal was observed for MbNO and also for nitrosylated
cytochrome c oxidase. In addition, when deoxyHb(Fe2+ ) was
exposed to NIR in the presence of sodium nitrite, a significant
amount of NO was released [27]. This strongly suggests that
670 nm light is able to liberate NO from nitrosylated heme
proteins and enhance NO release resulting from nitrite reductase
activity.
These findings were also confirmed in the ischemic rabbit heart
that, after infusion of sodium nitrite, exhibited a large increase
in nitrosyl heme formation as measured by electro-paramagnetic
resonance spectroscopy. When NIR at 670 nm (18 mW/cm2 ; 10 min)
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460 Protection from Cardiac Ischemia and Reperfusion Injury

Figure 25.1 Detection of nitric oxide released from HbNO during exposure
to NIR at 670 nm (20 mW/cm2 ) with ozone-based chemiluminescence.

was applied, the MbNO signal was reduced in the ischemic zone
by ∼60%, suggesting dissociation of the heme–NO bond upon
irradiation [27]. When wavelength dependence of NIR-induced NO
release from HbNO and of NIR-induced protection against ischemia
and reperfusion injury were compared, a good accordance was
found: 670 nm irradiation caused the largest NO release, while
triggered NO release was much less at 740 and particularly at 830
nm. Only irradiation at 670 nm caused a significant decrease in
infarct size in a mouse model of ischemia and reperfusion injury.
Thus, the photodissociation to NO and its synergistic effect with
sodium nitrite may represent a noninvasive and site-specific means
for increasing NO bioavailability (Fig. 25.2).

25.3.2 Cytochrome c Oxidase and NO

Frequently, the beneficial effects of NIR treatment have been
associated with the stimulation of mitochondrial metabolism,
particularly at the level of cytochrome c oxidase, complex IV of
the electron transport chain [13], and concomitant enhancement
of ATP synthesis. NIR may directly affect cytochrome c oxidase
activity through one of its redox active metal centers. In addition,
it has been suggested that the mechanism by which NIR exerts
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Protection Against Acute Ischemia and Reperfusion Injury 461

Figure 25.2 Suggested mechanism for NIR-induced protection from is-

chemia and reperfusion injury. NIR induces NO release from nitrosylated
heme proteins such as myoglobin or hemoglobin both under normal and
diabetic conditions. NO may affect mitochondrial bioenergetics through
reversible inhibition of the electron transport chain, thereby reducing
reactive oxygen species (ROS) generation at the time of reperfusion. Other
effects of NO on guanylate cyclase or permeability transition pore may also
participate in NIR-induced protection.

its action on cytochrome c oxidase is via NO. Nitric oxide that is

bound to the heme a3 CuB center decreases mitochondrial oxygen
consumption [36], which can be reversed by light at 500 nm, as well
as at 670 nm, via dissociation of NO [37]. The activated cytochrome
c oxidase may not only cause changes in electron transport chain
activity, including ROS generation, but released NO is available for
other biological processes such as vasodilation and gene expression.
However, compared to potential NO release from hemoglobin or
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462 Protection from Cardiac Ischemia and Reperfusion Injury

myoglobin, the relative amounts of NO that would be bound to

the enzyme and available for release would be quite limited [38].
Further, it is difficult to perceive how acceleration of cytochrome c
oxidase at the time of reperfusion conveys protection to the heart.
Rather, a mild reversible inhibition of the electron transport chain
has been shown to reduce ROS production during reperfusion and
increase cardiomyocyte survival [39].

25.4 Discussion of Potential Clinical Applications

Due to the reasonably high tissue penetration paralleled by limited

potential of tissue damage, NIR is attractive for the use in ischemic
heart disease. While light dosage needs to be verified for human
cardiac use, by comparing animal studies through various species
and experimental settings, an irradiance of 50–150 mW/cm2 for
2–10 min seems a reasonable starting point to achieve beneficial
effects of NIR. A higher irradiance may be required for acute preven-
tion of ischemia and reperfusion injury at the time of reperfusion.
NIR treatment of the heart may be protective on patients after
acute myocardial infarction or on ischemic heart conditions that are
not accessible to current revascularization procedures. A reduction
in infarct size was achieved when light was applied either at the
time of reperfusion or several hours later, at a reasonable time
after the onset of symptomatic pain of myocardial infarction in
humans.
NIR could be particularly useful in the presence of comorbidities
such as diabetes where many other cardioprotective strategies fail.
Diabetes is an independent predictor of increased cardiovascular
risk, and myocardial infarct size is directly related to increases in
blood glucose concentration in animals with or without diabetes
[40]. The mechanism of light-induced release of NO from heme
protein is likely to be maintained during diabetes, and thus NIR
may be protective from ischemia and reperfusion injury where
other strategies such as ischemic and pharmacologic pre- and
postconditioning fail [3, 41]. Indeed, we found that under acute
hyperglycemia or in a mouse model of type 2 diabetes (db/db
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Discussion of Potential Clinical Applications 463

Figure 25.3 NIR (670 nm, 150 mW/cm2 for 5 min at reperfusion) is
effective in reducing infarct size after ischemia and reoxygenation not only
under euglycemic conditions, but also under acute hyperglycemia (HYPER)
and in a diabetic mouse model (db/db). Adapted from Ref. [42].

mouse), NIR exposure of the mouse heart at the time of reperfusion

reduces infarct size significantly (Fig. 25.3) [42].
While there is significant clinical promise for the use of NIR in
heart disease, several hurdles need to be considered and overcome.
The technical challenge related to the application of NIR to the heart
due to limited penetration through muscle and bone is an important
issue to consider. Obvious scenarios whereby the light can be applied
where needed would be during cardiac surgery such as coronary
artery bypass that carry a significant increased risk of myocardial
infarction, or heart transplantation. It might also be possible to
apply NIR during a balloon angiography, using a catheter bearing
fiber optic through which the light can be delivered to the infarcted
area. In addition, author’s unpublished data on dogs demonstrate
the feasibility of a transesophageal approach to the heart with
a flexible fiber-optic NIR probe. The probe in the esophagus or
stomach (when advanced) is immediately adjacent to the left atrium
and the inferior and posterior walls of the left ventricle. Thus, the
anterior wall that is frequently affected by myocardial infarction
may be as much as 6 cm away from the probe. Still, it may not
be necessary for NIR light to penetrate the area at risk directly.
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464 Protection from Cardiac Ischemia and Reperfusion Injury

A remote effect of NIR, comparable to remote preconditioning,

might still provide protection and lead to a reduction in infarct
size. Signaling factors such as heat shock proteins or NO may
mediate such effect. In fact, for remote ischemic preconditioning,
circulating nitrite derived from shear stress–dependent stimulation
of endothelial NOS at the hindlimbs contributes to cardioprotection
during ischemia and reperfusion [43]. Literature values for NIR
penetration of cardiac tissue are scarce but are usually in the lower
cm range. The advantage of NIR is that absorption is minimal, and
a considerable penetration, depending on the intensity of the light
source, can be achieved. Delivery of NIR through the human chest
wall may require further technology development. An attractive
alternative approach presented in this chapter is the activation of
endogenous stem cells for cell therapy of the infarcted heart. NIR can
be applied non-invasively to the bone marrow of the pelvic girdle,
tibia, or other parts of the skeleton containing bone marrow up to
4 h post-myocardial infarct [25]. Importantly, this would also avoid
the need to isolate stem cells or reprogram other cells, growing them
in vitro and injecting them back into patients.
One may wonder if administering NO donors should not be
equally protective as NIR photobiomodulation if indeed NIR-induced
NO release is part of the protective mechanism. However, systemic
administration of NO donor results in decreases in diastolic arterial
pressure that might significantly reduce coronary blood flow and
hence myocardial oxygen delivery. The use of NIR is advantageous
in the setting of myocardial ischemia and reperfusion injury because
NIR is site specific and produces no significant hemodynamic effects.

25.5 Conclusion

NIR is promising in its ability both to treat the chronically

ischemic heart and to prevent acute ischemia and reperfusion
injury. Two distinct mechanisms are likely in place that converge
on mitochondria but lead to different outcome in bioenergetics:
activation of cytochrome c oxidase versus mild, reversible inhibition
of the electron transport chain. This reversible inhibition is critical
for the prevention of reperfusion injury and is achieved by NO
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References 465

released from heme proteins. Considering the short half-life of NO,

the maximum allowable distance between the site of NIR application
and the infarct location will need to be carefully determined in
future studies. The distance might be unexpectedly high due to NO
oxidation to nitrite and reduction back to nitric oxide at the ischemic
area where required. Ultimately, NIR therapy could alleviate injury
in various organs and could be beneficial during the initiation of
cardiopulmonary resuscitation after cardiac arrest, comparable but
less invasive to other intervention such as short interruptions of
compressions or exposure to volatile anesthetics [44].

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cardiovascular system. Cardiovasc. Res. 69, pp. 309–317.
31. Gladwin, M.T., Raat, N.J., Shiva, S., Dezfulian, C., Hogg, N., Kim-Shapiro,
D.B., et al. (2006). Nitrite as a vascular endocrine nitric oxide reservoir
that contributes to hypoxic signaling, cytoprotection, and vasodilation.
Am. J. Physiol. Heart Circ. Physiol. 291, pp. H2026–H2035.
32. Cuong, D.V., Kim, N., Youm, J.B., Joo, H., Warda, M., Lee, J.W., et al. (2006).
Nitric oxide-cGMP-protein kinase G signaling pathway induces anoxic
preconditioning through activation of ATP-sensitive K+ channels in rat
hearts. Am. J. Physiol. Heart Circ. Physiol. 290, pp. H1808–H1817.
33. Das, A., Xi, L., and Kukreja, R.C. (2008). Protein kinase G-dependent
cardioprotective mechanism of phosphodiesterase-5 inhibition involves
phosphorylation of ERK and GSK3beta. J. Biol. Chem. 283, pp. 29572–
29585.
34. Penna, C., Cappello, S., Mancardi, D., Raimondo, S., Rastaldo, R., Gattullo,
D., et al. (2006). Post-conditioning reduces infarct size in the isolated
rat heart: Role of coronary flow and pressure and the nitric oxide/cGMP
pathway. Basic Res. Cardiol. 101, pp. 168–179.
35. Chouchani, E.T., Methner, C., Nadtochiy, S.M., Logan, A., Pell, V.R., Ding, S.,
et al. (2013). Cardioprotection by S-nitrosation of a cysteine switch on
mitochondrial complex I. Nat. Med. 19, pp. 753–759.
36. Cooper, C.E., Torres, J., Sharpe, M.A., and Wilson, M.T. (1997). Nitric oxide
ejects electrons from the binuclear centre of cytochrome c oxidase by
reacting with oxidised copper: A general mechanism for the interaction
of copper proteins with nitric oxide? FEBS. Lett. 414, pp. 281–284.
37. Sarti, P., Giuffre, A., Forte, E., Mastronicola, D., Barone, M.C., Brunori,
M. (2000). Nitric oxide and cytochrome c oxidase: Mechanisms of
inhibition and NO degradation. Biochem. Biophys. Res. Commun. 274, pp.
183–187.
38. Osipov, A.N., Borisenko, G.G., and Vladimirov, Y.A. (2007). Biological
activity of hemoprotein nitrosyl complexes. Biochemistry (Mosc) 72, pp.
1491–1504.
39. Burwell, L.S., Nadtochiy, S.M., and Brookes, P.S. (2009). Cardioprotection
by metabolic shut-down and gradual wake-up. J. Mol. Cell. Cardiol. 46,
pp. 804–810.
40. van der Horst, I.C., Nijsten, M.W., Vogelzang, M., and Zijlstra, F. (2007).
Persistent hyperglycemia is an independent predictor of outcome in
acute myocardial infarction. Cardiovasc. Diabetol. 6, p. 2.
July 6, 2016 17:32 PSP Book - 9in x 6in 25-Hamblin-c25

References 469

41. Kersten, J.R., Schmeling, T.J., Orth, K.G., Pagel, P.S., and Warltier, D.C.
(1998). Acute hyperglycemia abolishes ischemic preconditioning in
vivo. Am. J. Physiol. 275, pp. H721–725.
42. Keszler, A., Brandal, G., Baumgardt, S., Ge, Z.D., Pratt, P.F., Riess, M.L.,
and Bienengraeber, M. (2014). Far red/near infrared light-induced
protection against cardiac ischemia and reperfusion injury remains
intact under diabetic conditions and is independent of nitric oxide
synthase. Front Physiol. 22, p. 305.
43. Rassaf, T., Totzeck, M., Hendgen-Cotta, U.B., Shiva, S., Heusch, G., and
Kelm, M. (2014). Circulating nitrite contributes to cardioprotection by
remote ischemic preconditioning. Circ. Res. 114, pp. 1601–1610.
44. Riess, M.L., Matsuura, T.R., Bartos, J.A., Bienengraeber, M., Aldakkak,
M., McKnite, S.H., et al. (2014). Anaesthetic postconditioning at the
initiation of CPR improves myocardial and mitochondrial function in a
pig model of prolonged untreated ventricular fibrillation. Resuscitation
85, pp. 1745–1751.
July 6, 2016 17:32 PSP Book - 9in x 6in 26-Hamblin-c26

Chapter 26

Low-Level Laser and Experimental Aortic

Aneurysm: Mechanisms and Therapeutic
Implications

Lilach Gavish and S. David Gertz

Institute for Medical Research–IMRIC, Faculty of Medicine,
The Hebrew University of Jerusalem, PO Box 12272, Jerusalem 91120, Israel
lilachg@ekmd.huji.ac.il

26.1 Introduction

Abdominal aortic aneurysm (AAA) is characterized by a focal

expansion that exceeds the normal diameter by more than 50%. It
is present in approximately 8% of men aged above 65 years and
is the 13th leading cause of death in the United States [1–2]. The
pathology of the aneurysmatic wall includes extensive disruption
and fragmentation of the elastic lamellae associated with marked
inflammatory cell infiltration [3] and progressive decrease in the
number of viable smooth muscle cells (SMCs) [4]. With time and
aggravated by risk factors such as smoking, systemic hypertension,
and hypercholesterolemia, aneurysm growth progresses as a result
of an insidious imbalance between matrix protein degradation and

Handbook of Low-Level Laser Therapy

Edited by Michael R. Hamblin, Marcelo Victor Pires de Sousa, and Tanupriya Agrawal
Copyright c 2017 Pan Stanford Publishing Pte. Ltd.
ISBN 978-981-4669-60-3 (Hardcover), 978-981-4669-61-0 (eBook)
www.panstanford.com
July 6, 2016 17:32 PSP Book - 9in x 6in 26-Hamblin-c26

472 Low-Level Laser and Experimental Aortic Aneurysm

Aneurysm LLLI

Inflammaon

Cell Death

Cell Proliferaon

Matrix Synthesis

Figure 26.1 Low-level laser irradiation versus abdominal aortic aneurysm.

production that increases the risk of rupture of the weakened

wall [5].
Evidence from a variety of experimental systems has shown that
low-level laser irradiation (LLLI) reduces inflammation, prevents
apoptosis, promotes cell proliferation, and augments collagen
synthesis. We, therefore, hypothesized that the seemingly contra-
dictory effects of LLLI—anti-inflammatory and, therefore, presum-
ably anti-atherosclerotic, but pro-wound-healing and, therefore,
pro-arteriosclerotic—might be ideal for retardation of aneurysm
progression where inflammatory processes and weakening of the
arterial wall matrix are major pathologic components (Fig. 26.1).
However, the application of LLLI to the abdominal aorta is techni-
cally problematic since it is a deep structure, lying retroperitoneally
on the posterior abdominal wall, behind the abdominal cavity but
anterior to the vertebral columns.
In this chapter, we will present an integrative review of our
experimental findingsa and comment on the application of this
therapeutic approach to the human clinical setting.

26.1.1 Aortic Elasticity and Resilience

The elasticity of the aorta is necessary to dampen the mechanical
force of the ejected blood and to resist permanent deformation
of the wall, but at the same time assist in forward propulsion.
These properties are derived in large measure from the structural

a In
all our studies, we used 780 nm diode laser coupled to an optic fiber (BWTek,
Newark, Delaware) with an energy density of 1–2 J/cm2 and power density of
2–4 mW/cm2 measured at the experimental plane.
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Introduction 473

organization of the aortic media that is characterized by concentric

layers of elastin that alternate with vascular SMCs. Contributing
to the resilience and integrity of the aorta is the network of
extracellular matrix (ECM) proteins, the largest percentage of which
is collagen fibers that further accommodate the physical forces such
as blood pressure, thereby preventing vessel rupture [6].

26.1.2 Smooth Muscle Cells

Collagen and elastin are synthesized by SMCs and adventitial
fibroblasts. The remodeling and degradation of these proteins, as
well as the other components of the ECM, are regulated by the
matrix metalloproteinases (MMPs) and their respective endogenous
tissue inhibitors of metalloproteinases (TIMPs) that are secreted
by resident cells of vascular wall and by infiltrating inflammatory
cells [7–8]. The vascular SMC is a multipotential cell capable of
migration, proliferation, contraction, and synthesis. In its synthetic
configuration, the SMCs synthesize the bulk of the connective tissue
components of the artery, including collagen (types I and III) and
elastin [9]. The collagen molecules are synthesized initially as
precursor chains that join to form the three-dimensional trihelix
procollagen in the rough endoplasmic reticulum (RER). Procollagen
is transported through the Golgi complex to the exterior of the cell
where assembly of the collagen fibrils is completed [10].

26.1.3 Activated Monocytes/Macrophages

Monocytes are the circulating precursors of tissue macrophages. In
response to injury and other pathological processes within the wall
and the supplied tissues, monocytes cross the endothelial barrier
facilitated by selectins, integrins, and other receptors and associated
adhesion molecules. Once the monocyte leaves the circulation, it
differentiates into macrophages. Activation of macrophages in vitro,
for example by lipopolysaccharide (LPS), a component of the Gram-
negative bacterial wall, elicits an inflammatory-like phenotype
(classical activation) promoting de novo synthesis of a variety of
factors, including chemokines such as macrophage chemotactic
protein (MCP-1), pro-inflammatory cytokines such as interleukin
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474 Low-Level Laser and Experimental Aortic Aneurysm

[IL]-1α, IL-1β, IL-6, and tumor necrosis factor [TNF]-α, anti-

inflammatory cytokines such as IL-10, and a variety of matrix
metalloproteinases. These processes are mediated through well-
established signal transduction pathways [11].

26.2 Effect of LLL on Experimental AAA

In a series of in vitro and in vivo studies, we have shown that LLLI

increases the proliferation of aortic SMCs and elaboration, by these
cells, of collagens I and III [12]. We also found that LLLI reduced
gene expression and secretion of pro-inflammatory chemokines
and cytokines in activated macrophages [13]. Using noninvasive,
high-frequency ultrasonography (HF-u/s), we then showed that
LLLI inhibits the de novo formation of AAA and halts further
progression of pre-induced AAA and the associated deterioration in
the biomechanical integrity of the aortic wall in the angiotensin-II-
infused, apolipoprotein-E-deficient (Apo-E−/− ) mouse model of AAA
[14–16].

26.2.1 LLL Promotes SMC Proliferation and Augments

Collagen Synthesis in Vitro
Because medial SMCs play a pivotal role in AAA [4], we investigated
the effect of LLLI on these cells in vitro with emphasis on
cell proliferation and collagen turnover (i.e., formation, synthesis,
degradation, and differential gene expression of matrix-remodeling
enzymes). For these studies, we used primary cultures of porcine
abdominal aortic SMCs in passage 2–3 in starved (2% serum)
conditions.

26.2.1.1 Proliferation
Using the trypan blue exclusion assay for direct cell count, we found
that LLLI stimulated the proliferation of starved (2% serum) aortic
SMC by up to 32% compared to non-irradiated controls [12].
Starved cells have compromised proliferation capabilities mainly
due to the lack of sufficient energy resources. LLL has been shown
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Effect of LLL on Experimental AAA 475

Figure 26.2 Collagen trihelix formation following LLLI. Note the significant
increase in collagen I immunofluorescent staining over control in the perin-
uclear region reflecting significant increased collagen trihelix formation in
the rough endoplasmic reticulum. Reprinted from Ref. [12] with permission
from John Wiley and Sons.

to upregulate ATP synthesis [17] by the activation of elements of

the mitochondrial respiratory chain [18]. Moreover, we have shown
previously that LLLI has a direct effect on promeylocytic leukemia
protein, a tumor suppressor factor responsible for cell growth arrest
at the G1/S cell cycle phase [18]. Others have shown upregulation
of DNA synthesis following LLLI [19]. It is, therefore, not surprising
that LLLI stimulates the proliferation of a variety of cell types,
including other cells of the vascular wall (not just SMC) such as
fibroblasts from different systems [20, 21] and endothelial cells from
veins [22] as well as arteries [23].

26.2.1.2 Collagens I and III trihelix formation

By immunofluorescent staining, we found that LLLI increased
collagens I and III trihelix formation in aortic SMCs (Fig. 26.2) [12].
Hydroxylation of procollagen chains in the RER prior to the collagen
trihelix formation [10] is catalyzed by prolyl-4-hydroxylase (P4H),
which is upregulated by hypoxia. Since LLLI stimulates collagen
trihelix formation and the formation of reactive oxygen species [24],
which are also characteristic of hypoxia, we hypothesized that LLLI
may directly influence P4H or its downstream targets such as HIF-
1α [12]. Indeed, in a recent study by Cury et al. [25], LLLI was shown
to increase HIF-1α expression in a model of ischemic rat skin flaps.
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476 Low-Level Laser and Experimental Aortic Aneurysm

Figure 26.3 Collagen synthesis by SMCs following LLLI. Left: Monolayer of

fusiform primary porcine aortic SMC grown on culture plate stained with
picrosirius red (PSR). Note the elongated extensions organized in parallel,
secreting PSR-stained collagen at ends to form large PSR-stained bundles
between the cells. Right: Total collagen in medium and on plates of aortic
SMCs quantified by the Sircol assay at 2, 4, and 6 days after (1) LLLI 2 J/cm2 ,
(2) addition of TGF-β (10 ng/ml)— positive control, and (3) non-irradiated
control. Data points are mean ± SE. Note that although collagen secretion
was increased by TGF-β, LLLI had a greater effect (numerical results in
[12]).

26.2.1.3 Collagen secretion

Using the Sircol assay based on the picrosirius red stain, we
found that LLLI increased total collagen synthesis compared to
non-irradiated controls or TGF-β-stimulated positive controls [12]
(Fig. 26.3).

26.2.1.4 MMP activity

By gelatin zymography, we found that the MMP activity in aortic
SMCs was increased following LLLI in a dose-dependent manner as
compared to non-irradiated controls [12].

26.2.2 LLL Attenuates LPS-Induced Secretion of

Inflammatory Factors
LLLI was shown to reduce inflammatory cell infiltrates in other
in vivo experimental systems [26, 27] and to reduce inflammatory
markers in clinical studies [28, 29]. For our in vitro studies, we used
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Effect of LLL on Experimental AAA 477

the murine RAW 264.7 monocyte/macrophage cell line activated by

different concentrations of LPS.

26.2.2.1 Chemokine/cytokine expression

Using semi-quantitative PCR, we found that LLLI reduced the ex-
pression of the chemokine MCP-1, the pro-inflammatory cytokines
IL-1α, IL-1β, and IL-6, as well as the anti-inflammatory cytokine
IL-10, when activated by 1.0 μg/ml LPS [13]. By baseline analysis, we
showed that the effect of LLLI is highly dependent on the extent of
the inflammatory response. At low levels of inflammatory response
(activated by 0.1 μg/ml LPS), LLLI does not appear to inhibit the
inflammatory cytokines [13]. That these effects of laser might be
elicited primarily on inflamed rather than normal tissues would
appear to be of significant clinical importance.

26.2.3 LLLI Prevents de Novo Formation and Halts Further

Progression of Pre-Induced AAA in Vivo
In order to test the hypothesis that LLLI prevents AAA development,
we used the angiotensin-II-infused, C57/Black6, Apo-E−/− mouse
model of AAA developed in the laboratory of Daugherty et al. [30]. In
this model, suprarenal AAA forms with a frequency of up to 80–85%.
High-frequency ultrasonography (0.01 mm resolution) was used to
monitor the aortic expansion over time. The abdominal aorta was
exposed retroperitoneally and was either irradiated with LLL or
sham irradiated.
To determine the effect of LLLI on de novo AAA formation
[14], we irradiated the aorta at the beginning of Ang-II infusion.
However, in a subsequent study to determine if LLLI prevented the
progression [16] of pre-induced aneurysms, we used only those
mice that developed aneurysms more than 35% expansion by
HF-u/s by 2 weeks after the beginning of Ang-II angiotensin infusion.
At this 2-week time point, half of the aneurysms were irradiated
and half were not. At 4 weeks (after the final HF-u/s), the mice
were sacrificed and the aortas perfused, excised, and processed for
histopathologic analysis.
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478 Low-Level Laser and Experimental Aortic Aneurysm

Table 26.1 Effect of LLLI on aneurysmal dilatation at

4 weeks

Group Control LLL p

#of mice with >50% dilatation 7 of 15 0 of 13 0.005*

Diameter by B-mode US [mm] 1.84 ± 0.44a 1.29 ± 0.06 0.0002**
Diameter by histology [mm] 1.99 ± 0.64 1.4 ± 0.16 0.01**

*by Fisher’s Exact test; a Mean ± SD; **by unpaired 2-tailed test

26.2.3.1 De novo aneurysm formation

By B-mode HF-u/s correlated with post-mortem histopathology,
we found that LLLI prevented de novo AAA formation (see
Table 26.1, Fig. 26.4). These results show an overall 94% reduction
in maximum cross-sectional diameter of the suprarenal aneurysm-
prone segments at 4 weeks in LLL-treated mice compared with
baseline [14, 15].

Figure 26.4 LLLI prevents de novo AAA formation. Left: High-frequency

ultrasound measurements of the aortas of control and LLL-irradiated Ang-
II-infused ApoE−/− mice at 4 weeks. Note the prominent dilatation of the
suprarenal aorta region in the control mouse (top), which is not present
in the LLLI mouse (bottom). Arrows indicate medial dissection in the
untreated suprarenal aortic aneurysm. Middle and right: Movat histology of
largest cross section of a control mouse and largest cross section of an LLL-
treated mouse. Arrows indicate the beginning of the missing media. Note
the thick medial layer in the LLLI treated compared to thin medial layer in
the controls. Left: Reprinted in part from Ref. [14] with permission from
Oxford University Press. Middle: Reprinted from Ref. [49], Copyright 2014,
with permission from Elsevier.
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Effect of LLL on Experimental AAA 479

26.2.3.2 Progression of pre-existing aneurysm

Using the pre-induced AAA model, we found that the aortas that
developed AAA at 2 weeks continued to grow as per HF-u/s
at 4 weeks. However, LLL irradiation at 2 weeks halted further
dilatation (Fig. 26.5). Moreover, by correlative histopathology using
the modified Daugherty classification, we found significantly fewer
severe aneurysms at 4 weeks in the LLL-treated animals versus
control [16].

26.2.4 LLL Increases SMC Size and Collagen Deposition

26.2.4.1 Medial SMC size
By measuring the medial area and counting the number of α-actin
positive SMC therein, we found that the mean size of the medial SMC
was significantly greater in the LLL-treated, angiotensin-infused
animals than in either the non-treated, angiotensin-infused mice or
the saline-infused controls [15].
This is consistent with the synthetic rather than contractile SMC
phenotype reflecting upregulated matrix protein synthesis [31].

26.2.4.2 Collagen reinforcement

The area of adventitial collagen measured in picrosirius red-stained
sections, normalized as a percentage of the vessel wall area, was
significantly greater in the LLL-treated mice than in the non-
treated, angiotensin-infused animals, and similar to the saline-
infused controls. In addition, LLLI was found to enhance collagen
reinforcement of transmedial defects at aortic branch orifices that
subtend aneurysm formation in this mouse model (Figs. 26.4 and
26.6) [15].
As indicated earlier, in its synthetic state, the SMC produces the
bulk of the connective tissue components of the artery, including
collagen and elastin, that are responsible for the resilience and
tensile strength of the aortic wall [9]. Studies by others on
skeletal muscles show similar effects. Following LLLI, an increase in
collagens I and III deposition was detected in the tibialis anterior
muscle of rats subjected to cryoinjury [32]. Increases in myofiber
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480 Low-Level Laser and Experimental Aortic Aneurysm

Figure 26.5 High frequency B-mode ultrasonography of the suprarenal

abdominal aorta of Apo-E−/− mice that developed aneurysmal dilatation at
two weeks after infusion of angiotensin-II showing that treatment with LLLI
at 2 weeks inhibited further increase of maximal cross-sectional diameter
(MCD). (A) Ultrasound images are of representative mice from control and
LLI treated groups with the mean MCD values depicted diagrammatically
on the left of A and numerically in B. Colors of captions in A correspond
to colors of profiles in A and bars in B. (B) Bars represent the mean (±SE)
MCD at baseline (0 weeks), 2, and 4 weeks in control (n = 12) and LLL-
treated (n = 14) mice. P-values were calculated by 2-tailed, paired ttest
comparing 2 and 4 weeks for each group separately. (C) Bars represent the
mean (±SE) change in MCD from 2 to 4 weeks in control and LLL- treated
mice. P-value was calculated by 2-tailed, unpaired test. Reprinted from Ref.
[16] with permission from John Wiley and Sons.
July 6, 2016 17:32 PSP Book - 9in x 6in 26-Hamblin-c26

Effect of LLL on Experimental AAA 481

Figure 26.6 LLL-treated, suprarenal AAA of Ang-II-infused Apo-E−/−

mouse. Left: Note marked transmural disruption in the vicinity of the
orifice of the superior mesenteric artery (SMA) with reactive fibromuscular
hyperplasia appearing to wall off the medial defect. Macrophages can be
seen at the site of fragmented elastic lamellae (Movat stain). Right: Note
extensive collagen matrix as viewed with polarized light. Reprinted in part
from Ref. [15] with permission from John Wiley and Sons.

diameter and the number of bromodeoxyuridine (BrdU)-positive

satellite cells were also found after LLLI in deep regions of skeletal
muscles in the atrophied gastrocnemius muscle model [33]. As
mentioned earlier, adventitial fibroblasts also synthesize collagen.
LLLI in both red and near-infrared wavelengths has been shown to
augment collagen production in fibroblasts in a variety of in vitro
and in vivo systems [21, 34–36]. The direct effect of LLLI on aortic
fibroblasts should be investigated.

26.2.5 LLL Attenuates the Number of Macrophages in

Transmedial Aortic Defects
26.2.5.1 Macrophages in area of transmedial defect
By immunohistochemical staining, we found that the number of
mac-2-positive macrophages in the area of the medial defects
was significantly lower in the LLL-treated than the non-treated
mice. As expected, the saline-infused mice had markedly fewer
intramural macrophages in these walling off areas than either of the
angiotensin-infused groups (Fig. 26.7) [15].
The reduced number of macrophages found at 4 weeks after
LLLI may reflect a temporal acceleration of the inflammatory phase
of wound healing as has been described previously in a variety of
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482 Low-Level Laser and Experimental Aortic Aneurysm

Figure 26.7 MAC-2-positive intramural macrophages. Note the signifi-

cantly lower density of macrophages (per 0.01 mm2 ) at the intramural
interface of the disrupted media in the area of attempted repair in the
LLLI-treated, AngII-infused versus the non-treated, AngII-infused controls.
There was little or no effect of LLLI on luminal macrophages. As expected,
the saline- infused mice had markedly fewer intramural macrophages than
either of the AngII-infused groups. Analysis done by Kruskal-Wallis non-
parametric test with Conover-Inman post hoc test. Reprinted from Ref. [15]
with permission from John Wiley and Sons.

studies and discussed by us [15, 37]. Such an accelerated resolution

of the inflammatory phase by LLLI would be expected to modify
the expression and secretion of proteolytic enzymes responsible for
fragmentation and disruption of the lamellar units of the media at
these sites.
In summary, by stimulating SMCs (and possibly fibroblasts) to
produce more collagen, LLL treatment reinforces the media and
increases the resilience of the adventitia. Moreover, by reducing
the number of macrophages at sites of injury, LLLI might attenuate
July 6, 2016 17:32 PSP Book - 9in x 6in 26-Hamblin-c26

Therapeutic Approaches 483

further wall damage thereby reducing the likelihood of further

aneurysmal expansion as seen in this model.

26.3 Therapeutic Approaches

26.3.1 Current Treatments and Early Detection

Pharmacological therapy and risk factor modification have been
only partially successful for the prevention of progression of AAA
[38, 39]. Current forms of treatment focus on open abdomen,
surgical repair, or endovascular exclusion of the diseased segment
of the aorta with large, covered stents (e.g., Gortex covered). These
urgent surgical procedures have a high frequency of major side
effects with life-threatening consequences [40, 41], particularly
in patients of advanced age (the majority of patients) or those
of high risk such as patients with compromised cardiac and/or
pulmonary function [41, 42]. Since the US Preventive Services Task
Force recommendation of 2005 that all males, aged 65–74, whoever
smoked, have a one-time ultrasound screening for AAA, more than
80% of the diagnosed aneurysms are at early stages of the disease
[38, 43], further emphasizing the need for less invasive therapeutic
strategies that target pathogenetic mechanisms of progression and
rupture.

26.3.2 How Can LLL be used for Treatment of AAA?

As indicated, the abdominal aorta is positioned retroperitoneally
on the posterior abdominal wall and anterior to the vertebral
column and adjacent muscles. The depth of penetration of laser light
varies from a few millimeters to several centimeters at the most,
with the light energy getting progressively weaker with increased
depth. Nonetheless, even, and perhaps particularly, after achieving
effective energy levels despite deeper penetration, irradiating the
aorta through other organs and tissues would only be acceptable
after comprehensive and thorough short- and long-term study
of the pathobiological effects of this modality on these adjacent
structures.
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484 Low-Level Laser and Experimental Aortic Aneurysm

26.3.2.1 Noninvasive LLL

LLL therapy was initially used to treat pathologies of superficial
structures such as healing dermal wounds [37]. Subsequently,
structures beneath the skin such as tendons, muscles, and nerves
also became popular targets [28, 44]. More recently, deeper
structures such as brain tissue have also been treated noninvasively
by transcranial irradiation [45].

26.3.2.2 Minimally invasive intravascular LLL

Intravascular LLL has been used for the prevention of restenosis
after coronary angioplasty [46]. Intravascular LLL is one possible
approach to the aorta for the prevention of aneurysm progression.
However, vascular injury and thrombosis from passage of the
associated catheter device and the likely need for multiple treatment
sessions reduce the attractiveness of this treatment approach.

26.3.2.3 Minimally invasive laparoscopic LLL

We have recently suggested an alternative, minimally invasive
laparoscopic approach involving catheter-based irradiation of the
periarterial surface of the aneurysmatic aorta [47]. Assuming the
need for multiple, short course irradiations, as has been used
in a variety of clinical protocols for the use of LLLI in other
pathologies, we have been developing an implantable device for
internal irradiation of body cavities and damaged vessels that could
be attached to the external (periadventitial) surface of the aorta [48].
This implantable device will have an independent energy source
and will be capable of delivering multiple irradiations in a pre-
programmed or externally controlled manner.

26.4 Conclusion

In conclusion, LLLI is a mechanism-based, minimally invasive

therapeutic alternative for the prevention of progression and
rupture of AAA. Its efficacy is easily monitored noninvasively by
ultrasound or CT scan. This novel approach may be applicable to
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References 485

aneurysms in other regions of the body. Retardation of progression

of aneurysmal dilatation in the clinical setting by this novel approach
using LLL- or LED-based technology, delivered intravascularly or la-
paroscopically to small but stubbornly progressing aneurysms, may
prevent the suffering and often serious consequences associated
with the current, urgent surgical procedures, particularly in high-
risk patients [14, 15, 16].

Acknowledgments

These studies were supported in part by The Israel Science

Foundation (grant no. 1298/10), The Rosetrees Trust Fund of the
United Kingdom, and Professor Eliyahu Kelman of Jerusalem. SDG is
The Brandman Foundation Professor of Cardiac and Pulmonary Dis-
eases, Institute for Medical Research—IMRIC, Faculty of Medicine of
The Hebrew University of Jerusalem, Israel.

References

1. Nordon, I.M., et al., Pathophysiology and epidemiology of abdominal

aortic aneurysms. Nat Rev Cardiol, 2011. 8(2): pp. 92–102.
2. Go, A.S., et al., Heart disease and stroke statistics–2014 update: A report
from the American Heart Association. Circulation, 2014. 129(3): pp.
e28–e292.
3. Gertz, S.D., A. Kurgan, and D. Eisenberg, Aneurysm of the rabbit common
carotid artery induced by periarterial application of calcium chloride in
vivo. J Clin Invest, 1988. 81(3): pp. 649–656.
4. Rowe, V.L., et al., Vascular smooth muscle cell apoptosis in aneurysmal,
occlusive, and normal human aortas. J Vasc Surg, 2000. 31(3): pp. 567–
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5. Thompson, R.W., P.J. Geraghty, and J.K. Lee, Abdominal aortic aneurysms:
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39(2): pp. 110–230.
6. Vorp, D.A., Biomechanics of abdominal aortic aneurysm. J Biomech,
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486 Low-Level Laser and Experimental Aortic Aneurysm

7. Sternlicht, M.D., and Z. Werb, How matrix metalloproteinases regulate

cell behavior? Annu Rev Cell Dev Biol, 2001. 17: pp. 463–516.
8. Barnes, M.J., L.F. Morton, and C.I. Levene, Synthesis of collagens types I
and III by pig medial smooth muscle cells in culture. Biochem Biophys
Res Commun, 1976. 70(2): pp. 339–347.
9. Ruckman, J.L., et al., Phenotypic stability and variation in cells of the
porcine aorta: Collagen and elastin production. Matrix Biol, 1994. 14(2):
pp. 135–145.
10. Pacifici, M., and R.V. Iozzo, Remodeling of the rough endoplasmic
reticulum during stimulation of procollagen secretion by ascorbic acid
in cultured chondrocytes. A biochemical and morphological study. J Biol
Chem, 1988. 263(5): pp. 2483–2492.
11. Kopydlowski, K.M., et al., Regulation of macrophage chemokine expres-
sion by lipopolysaccharide in vitro and in vivo. J Immunol, 1999. 163(3):
pp. 1537–1544.
12. Gavish, L., L. Perez, and S.D. Gertz, Low-level laser irradiation modulates
matrix metalloproteinase activity and gene expression in porcine aortic
smooth muscle cells. Lasers Surg Med, 2006. 38(8): pp. 779–786.
13. Gavish, L., et al., Irradiation with 780 nm diode laser attenuates inflam-
matory cytokines but upregulates nitric oxide in lipopolysaccharide-
stimulated macrophages: implications for the prevention of aneurysm
progression. Lasers Surg Med, 2008. 40(5): pp. 371–378.
14. Gavish, L., et al., Low-level laser irradiation inhibits abdominal aortic
aneurysm progression in apolipoprotein E-deficient mice. Cardiovasc
Res, 2009. 83(4): pp. 785–792.
15. Gavish, L., et al., Low level laser arrests abdominal aortic aneurysm by
collagen matrix reinforcement in apolipoprotein e-deficient mice. Lasers
Surg Med, 2012. 44(8): pp. 664–674.
16. Gavish, L., et al., Arrest of progression of pre-induced abdominal
aortic aneurysm in apolipoprotein E-deficient mice by low level laser
phototherapy. Lasers Surg Med, 2014. 46(10): pp. 781–790.
17. Pastore, D., et al., Stimulation of ATP synthesis via oxidative phos-
phorylation in wheat mitochondria irradiated with helium-neon laser.
Biochem Mol Biol Int, 1996. 39(1): pp. 149–157.
18. Gavish, L., et al., Low level laser irradiation stimulates mitochondrial
membrane potential and disperses subnuclear promyelocytic leukemia
protein. Lasers Surg Med, 2004. 35(5): pp. 369–376.
19. Karu, T.I., et al., Absorption measurements of a cell monolayer relevant
to phototherapy: Reduction of cytochrome c oxidase under near IR
radiation. J Photochem Photobiol B, 2005. 81(2): pp. 98–106.
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20. Kreisler, M., et al., Low level 809-nm diode laser-induced in vitro
stimulation of the proliferation of human gingival fibroblasts. Lasers
Surg Med, 2002. 30(5): pp. 365–369.
21. Pereira, A.N., et al., Effect of low-power laser irradiation on cell growth
and procollagen synthesis of cultured fibroblasts. Lasers Surg Med,
2002. 31(4): pp. 263–267.
22. Szymanska, J., et al., Phototherapy with low-level laser influences the
proliferation of endothelial cells and vascular endothelial growth factor
and transforming growth factor-beta secretion. J Physiol Pharmacol,
2013. 64(3): pp. 387–391.
23. Kipshidze, N., et al., Low-power helium: Neon laser irradiation enhances
production of vascular endothelial growth factor and promotes growth
of endothelial cells in vitro. Lasers Surg Med, 2001. 28(4): pp. 355–364.
24. Lubart, R., et al., Low-energy laser irradiation promotes cellular redox
activity. Photomed Laser Surg, 2005. 23(1): pp. 3–9.
25. Cury, V., et al., Low level laser therapy increases angiogenesis in a model
of ischemic skin flap in rats mediated by VEGF, HIF-1α and MMP-2. J
Photochem Photobiol B, 2013. 125: pp. 164–170.
26. Albertini, R., et al., Anti-inflammatory effects of low-level laser therapy
(LLLT) with two different red wavelengths (660 nm and 684 nm) in
carrageenan-induced rat paw edema. J Photochem Photobiol B, 2007.
89(1): pp. 50–55.
27. Lopes, N.N., et al., Effects of low-level laser therapy on collagen
expression and neutrophil infiltrate in 5-fluorouracil-induced oral
mucositis in hamsters. Lasers Surg Med, 2010. 42(6): pp. 546–552.
28. Bjordal, J.M., R.A. Lopes-Martins, and V.V. Iversen, A randomised, placebo
controlled trial of low level laser therapy for activated Achilles tendinitis
with microdialysis measurement of peritendinous prostaglandin E2
concentrations. Br J Sports Med, 2006. 40(1): pp. 76–80; discussion 76–
80.
29. Bjordal, J.M., et al., A systematic review with meta-analysis of the
effect of low-level laser therapy (LLLT) in cancer therapy-induced oral
mucositis. Support Care Cancer, 2011. 19(8): pp. 1069–1077.
30. Daugherty, A., M.W. Manning, and L.A. Cassis, Angiotensin II promotes
atherosclerotic lesions and aneurysms in apolipoprotein E-deficient
mice. J Clin Invest, 2000. 105(11): pp. 1605–1612.
31. Worth, N.F., et al., Vascular smooth muscle cell phenotypic modulation in
culture is associated with reorganisation of contractile and cytoskeletal
proteins. Cell Motil Cytoskeleton, 2001. 49(3): pp. 130–145.
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32. de Souza, T.O., et al., Phototherapy with low-level laser affects the
remodeling of types I and III collagen in skeletal muscle repair. Lasers
Med Sci, 2011. 26(6): pp. 803–814.
33. Nakano, J., et al., Low-level laser irradiation promotes the recovery
of atrophied gastrocnemius skeletal muscle in rats. Exp Physiol, 2009.
94(9): pp. 1005–1015.
34. Abergel, R.P., et al., Biostimulation of wound healing by lasers:
Experimental approaches in animal models and in fibroblast cultures.
J Dermatol Surg Oncol, 1987. 13(2): pp. 127–133.
35. Labbe, R.F., et al., Laser photobioactivation mechanisms: In vitro
studies using ascorbic acid uptake and hydroxyproline formation as
biochemical markers of irradiation response. Lasers Surg Med, 1990.
10(2): pp. 201–207.
36. Ayuk, S.M., N.N. Houreld, and H. Abrahamse, Collagen production
in diabetic wounded fibroblasts in response to low-intensity laser
irradiation at 660 nm. Diabetes Technol Ther, 2012. 14(12): pp. 1110–
1117.
37. Gavish, L., Low level laser therapy for wound healing, in Handbook of
Photomedicine, M.R. Hamblin and Y.-Y. Huang, eds. 2013, Taylor and
Francis: Hoboken. pp. 577–589.
38. Golledge, J., and P.E. Norman, Current status of medical management for
abdominal aortic aneurysm. Atherosclerosis, 2011. 217(1): pp. 57–63.
39. Wemmelund, H., et al., Statin use and rupture of abdominal aortic
aneurysm. Br J Surg, 2014. 101(8): pp. 966–975.
40. Endovascular aneurysm repair versus open repair in patients with
abdominal aortic aneurysm (EVAR trial 1): Randomised controlled trial.
Lancet, 2005. 365(9478): pp. 2179–2186.
41. Drury, D., et al., Systematic review of recent evidence for the safety and
efficacy of elective endovascular repair in the management of infrarenal
abdominal aortic aneurysm. Br J Surg, 2005. 92(8): pp. 937–946.
42. Steinmetz, E.F., C. Buckley, and R.W. Thompson, Prospects for the med-
ical management of abdominal aortic aneurysms. Vasc Endovascular
Surg, 2003. 37(3): pp. 151–163.
43. Screening for abdominal aortic aneurysm: Recommendation statement.
Ann Intern Med, 2005. 142(3): pp. 198–202.
44. Leal Junior, E.C., et al., Effects of low-level laser therapy (LLLT) in the
development of exercise-induced skeletal muscle fatigue and changes
in biochemical markers related to postexercise recovery. J Orthop Sports
Phys Ther, 2010. 40(8): pp. 524–532.
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45. Zivin, J.A., et al., Effectiveness and safety of transcranial laser therapy for
acute ischemic stroke. Stroke, 2009. 40(4): pp. 1359–1364.
46. De Scheerder, I., et al., Long-term follow-up after coronary stenting and
intravascular red laser therapy. Am J Cardiol, 2000. 86(9): pp. 927–930.
47. Gertz, S.D., L. Gavish, and P. Reissman, Device for Irradiating an Internal
Body Surface, United States Patent Application U.S. Patent 8,961,502,
filed June 1, 2009, and issued February 24, 2015; IL Patent 174,858
(Priority Date: April 6, 2006).
48. Gertz, S.D., and L. Gavish, Device for Irradiating an Internal Body Surface,
United States Patent Application US 20120095532 A1: Publication Date:
Apr. 19, 2012 (Priority Date: Nov 3, 2008).
49. Gavish, L., et al., Inadequate reinforcement of transmedial disruptions at
branch points subtends aortic aneurysm formation in apolipoprotein-E-
deficient mice. Cardiovasc Pathol, 2014. 23(3): pp. 152–159.
July 6, 2016 17:32 PSP Book - 9in x 6in 27-Hamblin-c27

Chapter 27

Low-Level Laser Therapy: A Treatment

Modality for Multiple Sclerosis Targeting
Autoimmunity and Oxidative Stress

Zenas George, Miguel A. Tolentino, and Jeri-Anne Lyons

Department of Biomedical Sciences, University of Wisconsin–Milwaukee,
2400 East Hartford Avenue, Milwaukee, WI, 53211 USA
jlyons@uwm.edu

Multiple sclerosis (MS) is the leading cause of neurologic disability

in young adults. The clinical signs and symptoms of MS are caused
by an autoimmune attack on the myelin sheath surrounding the
central nervous system (CNS) neurons, leading to demyelination and
axonal loss. Recent data suggest that permanent disability is due
to oxidative damage to neurons resulting in apoptosis of the cells.
The approved therapeutic agents for MS are costly and only partially
effective because they primarily target the autoimmune aspect of the
disease pathogenesis. Experimental autoimmune encephalomyelitis
(EAE) is the primary animal model of MS. Low-level laser (light)
therapy (LLLT) has shown therapeutic potential in the resolution
of chronic inflammation and protection against neurodegeneration,
both key components in the pathology of MS. Published data in
the EAE model demonstrated the therapeutic potential of 670 nm

Handbook of Low-Level Laser Therapy

Edited by Michael R. Hamblin, Marcelo Victor Pires de Sousa, and Tanupriya Agrawal
Copyright c 2017 Pan Stanford Publishing Pte. Ltd.
ISBN 978-981-4669-60-3 (Hardcover), 978-981-4669-61-0 (eBook)
www.panstanford.com
July 6, 2016 17:32 PSP Book - 9in x 6in 27-Hamblin-c27

492 Low-Level Laser Therapy

light-mediated photobiomodulation, in association with the down-

regulation of pro-inflammatory mediators and the upregulation of
anti-inflammatory proteins. These recent observations indicate that
photobiomodulation is a promising therapeutic approach for the
treatment of MS.

27.1 Introduction

27.1.1 Multiple Sclerosis

Multiple sclerosis is an autoimmune inflammatory disease that
affects the CNS, resulting in demyelination with subsequent neu-
rodegeneration [3] and progressive neurological disability [22]. MS
is diagnosed in young adults between 20 and 40 years of age [22]
and is more prevalent in women than in men, in the ratio of 3:1
[20]. Worldwide, it is estimated that 2.5 million persons are affected
with MS, and disease incidence increases from the equator to the
poles [10], with the highest incidence of disease noted in Northern
Europe, Northern America, New Zealand, and Australia, estimated
to be greater than 100 per 100,000 persons [15]. The cause of MS
is unknown, and a variety of genetic and environmental factors and
infectious agents have been associated with the disease [22]. Recent
genetic analyses indicate an association with immune response
genes, oxidative stress, neurotoxicity, and various other factors in
the susceptibility and progression of disease [2, 7]. Environmental
factors, including pollution, industrialization, lifestyle, diet, low
exposure to sunlight (UV light), and low exposure to infections
during childhood also appear to play a role [22].
The clinical course of MS is variable and unpredictable. The
disease is characterized as relapsing-remitting (RRMS), primary
progressive (PPMS), and secondary progressive (SPMS) [24]. RRMS
is most common and is characterized by repeated attacks of
neurological signs and symptoms, followed by periods of complete
recovery [22]. Within 5–10 years of diagnosis, the majority
of RRMS patients progress to limited mobility and permanent
disability (SPMS). PPMS is characterized by the appearance of
signs and symptoms with progressive loss of neurological function,
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Introduction 493

without relapses or remission. It is important to note that current

therapeutic agents are only effective in the treatment of RRMS [10].
MS histopathology is characterized by the presence of inflam-
matory demyelinating lesions (plaques) in the CNS. These lesions
are dynamic, changing in size and locations over time, leading
to the clinical manifestations associated with the disease [4, 10].
Signs and symptoms associated with the disease are diverse and
unpredictable and include numbness, tingling, weakness, tremor,
nystagmus, paralysis, disorders in speech and vision, vertigo, facial
weakness, trigeminal neuralgia, and hearing loss [22].

27.1.2 Pathogenesis of Multiple Sclerosis

Traditionally, MS is described as an autoimmune, pro-inflammatory
disease of the CNS [22]. Indeed, it is well established that RRMS
is mediated by CD4+ T-cells secreting pro-inflammatory cytokines,
including interferon-γ (IFNγ ), tumor necrosis factor-α (TNFα),
and interleukin (IL)-17. However, recent data indicate that disease
progression and permanent disability are due to the loss of axons
in the brain, mediated by mitochondrial dysfunction and nitroso-
oxidative stress [14]. The recently appreciated shift in pathogenic
mechanism between early and late-stage disease, and between
PPMS and RRMS, explains why the currently approved therapeutic
agents, all of which function to modulate or suppress the immune
response, are only partially effective in RRMS.
While MS pathogenesis is characterized by a pro-inflammatory
immune response and the accumulation of nitroso-oxidative stress
[14, 22], disease remission and successful treatment are charac-
terized by a shift to anti-inflammatory cytokines and anti-oxidant
mechanisms [10]. Production of anti-inflammatory cytokines such
as IL-4 and IL-10 downregulates the pro-inflammatory response
and allows physiologic mechanisms to repair the damage to the
myelin sheath [23]. The current therapeutic agents function by
controlling autoimmunity and slowing disease progression [10].
However, they are ineffective targeting oxidative stress and ongoing
neurodegeneration [10]. Novel approaches targeting this facet of
disease pathology hold the promise of lasting therapeutic benefit,
and perhaps a cure, to MS patients.
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494 Low-Level Laser Therapy

27.1.3 Animal model for Multiple Sclerosis

Experimental autoimmune encephalomyelitis is the primary animal
model for MS [5]. EAE is induced by active immunization with myelin
proteins or passive transfer of activated CD4+ T-cells specific for
these proteins [6]. Different clinical presentations of MS, such as
relapsing-remitting and primary progressive forms, are reproduced
in different animal strains using different myelin antigens [6].
Studies in EAE have been instrumental in defining the pathologic
mechanisms of MS and the development of therapeutic agents for
the treatment of the disease [13].

27.2 LLLT as an Emerging Treatment Modality for

Multiple Sclerosis

LLLT utilizing far-red to near-infrared (FR/NIR; 630–1100 nm)

light has emerged as a simple but powerful means of treating pro-
inflammatory conditions and neurodegenerative diseases. LLLT has
proven effective in the treatment of chronic wounds [25], retinal
disease [1, 8], Parkinson’s disease [11, 21], Alzheimer’s disease [9],
and stroke [12, 18, 26] in human and animal systems. The dual
pathologic mechanisms of a pro-inflammatory immune response
and mitochondrial dysfunction with associated oxidative stress
prompted the investigation of LLLT as a therapeutic strategy for the
treatment of MS.

27.2.1 Efficacy of Phototherapy in Animal Model for

Multiple Sclerosis
Experiments in the EAE model were designed to test the therapeutic
efficacy of LLLT for the treatment of MS [16, 17]. Using the myelin
oligodendrocyte glycoprotein (MOG)-induced model of chronic
disease in C57BL/6 mice, initial studies demonstrated a temporary
effect on disease severity when mice received a 7-day treatment
protocol with 670 nm light (Fig. 27.1A). Treatment (5 J/cm2 ,
2.1 W) was administered to the ventral surface of the animal
using a light-emitting diode (LED) array daily for 7 days, and
July 6, 2016 17:32 PSP Book - 9in x 6in 27-Hamblin-c27

LLLT as an Emerging Treatment Modality for Multiple Sclerosis 495

(A) (B)

Figure 27.1 Amelioration of clinical disease severity in an animal model

of chronic MS. (A) Mice treated for 7 days beginning at the day of onset of
clinical signs; (B) mice treated for 7 days beginning on the day of onset of
clinical signs, followed by 7 days of no light treatment, and a subsequent
7-day treatment period [17].

disease severity was compared with sham-treated (restraint stress,

no light). Treated animals demonstrated less severe disease for
10–14 days following treatment, but severity reached control
levels by 3 weeks post-treatment. Given the hypothesis that the
effects of LLLT are due to the induction of gene expression,
an extended treatment protocol was developed, in which mice
received 7 days of LLLT with 670 nm light, followed by a 7-day
rest period during which no light treatment was given, and a
subsequent 7-day treatment protocol. Using this protocol, a lasting
therapeutic effect was noted in mice receiving LLLT compared
to sham-treated animals (Fig. 27.1B). Preliminary data suggest
that amelioration of the disease is sustained with continued
rest/treatment cycles (data not shown).
A thorough understanding of the mechanism behind the ob-
served therapeutic effect is necessary for the application of LLLT
for the treatment of MS in patients. Subsequent investigation
revealed upregulation of anti-inflammatory cytokines IL-4 and IL-
10 in mice receiving LLLT with 670 nm light compared to sham-
treated animals (Fig. 27.2A,B). Conversely, expression of iNOS, the
enzyme responsible for producing large amounts of nitric oxide
associated with disease pathology, was decreased in treated animals
(Fig. 27.2C). In agreement with less oxidative stress, CNS tissue
sections from LLLT-treated mice also demonstrated less apoptotic
July 6, 2016 17:32 PSP Book - 9in x 6in 27-Hamblin-c27

496 Low-Level Laser Therapy

(A) (B)

(C)

Figure 27.2 Modulation of gene expression in spinal cord tissue isolated

from 670 nm treated and sham mice over the course of clinical EAE. (A) IL-
4, (B) IL-10 [17], (C ) iNOS [16].

cell death than sham-treated animals (Fig. 27.3). These observations

(increased anti-inflammatory markers, decreased pro-inflammatory
markers, decreased apoptotic death) in 670 nm treated mice
support the amelioration of clinical disease noted in treated animals.

27.2.2 LLLT for Treatment of MS

Anecdotal data support the use of LLLT for the treatment of MS.
However, published reports on the application of LLLT for the
treatment of MS patients are limited. A previous report summarizing
three years of data demonstrated a clinical effect of 632 nm light
in MS patients [19]. In this study, patients were treated with
632 nm helium-neon laser at 10 mW output for 10 min 6 days
July 6, 2016 17:32 PSP Book - 9in x 6in 27-Hamblin-c27

Future Directions 497

Figure 27.3 Decreased apoptosis within the central nervous system of

mice receiving 670 nm light treatment compared to sham-treated animals
[16].

a week for 21 days. In patients receiving multiple treatments, a

2-month interval between treatment periods was observed. In an
initial study of 22 patients with PPMS or SPMS, 32% of patients
reported no clinical improvement; 32% of patients reported clinical
improvement, not supported by neurologic tests; and 36% of
patients reported clinical improvement that was supported by
neurologic tests. Over the subsequent 3-year period, the authors
treated 100 MS patients with LLLT, 50 of whom received treatment
for a 3-year period following the above protocol. The authors state
that clinical improvement was generally observed 3–4 weeks after
the completion of the 21-day treatment protocol. Similar to results
in the animal model, if treatments were not continued, the effect of
treatment abated, with patients again deteriorating in neurological
status. Thus, as indicated in the EAE model, LLLT shows promise as
a therapeutic modality for the treatment of MS, although treatments
will likely need to be repeated at regular intervals.

27.3 Future Directions

Studies in the EAE model and limited data in MS patients suggest

that LLLT is safe and effective for the treatment of progressive forms
of MS. Moving forward, it is important to gain a full understanding
of the effects of LLLT on the immune response, the CNS, and the
July 6, 2016 17:32 PSP Book - 9in x 6in 27-Hamblin-c27

498 Low-Level Laser Therapy

neuroinflammation characteristic of MS pathology. Of particular

concern, moving forward, is optimization of treatment protocols
for the use of LLLT in MS patients. Preliminary studies in the
EAE model indicate that treating during the onset of a relapse, or
treating for an extended period of time, may exacerbate disease
(unpublished data). One explanation of this observation is the dual
role for nitric oxide in regulating T-cell activation and in driving
MS pathology. On the one hand, data in the EAE model indicate
that downregulation of oxidative stress protects the CNS from
ongoing neurodegeneration. However, nitric oxide is also important
in limiting T-cell activation and downregulating an active immune
response. Thus, downregulating nitric oxide during the onset of
relapse could result in the over-activation of the autoimmune T-cell
population and exacerbation of the disease process. Our own studies
are progressing to gain a better understanding of the effect of LLLT
on the human immune response. In addition, studies in a relapsing-
remitting EAE model will indicate whether or not LLLT may be safe
in the earlier, immune-mediated stages of disease.

27.4 Conclusion

Our data in the EAE model and limited data obtained from
MS patients indicate that LLLT provides a therapeutic effect in
chronic, progressive stages of the disease. This effect is due to the
downregulation of pro-inflammatory mediators and upregulation of
anti-inflammatory mediators, leading to decreased apoptosis and
neuroprotection within the CNS. Further studies are necessary to
characterize the effect of LLLT on the human immune response and
to define safe protocols for the application of this novel therapy to a
mechanistically complex disease. One important question remaining
is whether LLLT is safe in the earlier, immune-mediated stages
of the disease, or if its application is only useful during chronic
disease when the role of the immune response is limited. The
neuroprotection provided by LLLT could result in lasting clinical
improvement and improved quality of life for MS patients and their
families.
July 6, 2016 17:32 PSP Book - 9in x 6in 27-Hamblin-c27

References 499

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nitrosative stress. PLoS ONE, 8, pp. e67358.
17. Muili, K. A., Gopalakrishnan, S., Meyer, S. L., Eells, J. T., and Lyons, J.-A.
(2012). Amelioration of experimental autoimmune encephalomyelitis
in C57BL/6 mice by photobiomodulation induced by 670 nm light. PLoS
ONE, 7, pp. e30655.
18. Oron, A., Oron, U., Chen, J., Eilam, A., Zhang, C., Sadeh, M. Y. L.,
Streeter, J. L. D., and Chopp, M. (2006). Low-level laser therapy applied
transcranially to rats after induction of stroke significantly reduces long-
term neurological deficits. Stroke, 37, pp. 2620–2624.
19. Peszyñski-Drews, C., Klimek, A., Sopiñski, M., and Obrzejta, D. (2003).
Laser biostimulation of the patients suffering from multiple sclerosis in
respect of biological influence of laser light. Proc. of SPIE, 5229, pp. 97–
103.
20. Sellner, J., Kraus, J., Awad, A., Milo, R., Hemmer, B., and Stuve, O. (2011).
The increasing incidence and prevalence of female multiple sclerosis: A
critical analysis of potential environmental factors. Autoimmun. Rev., 10,
pp. 495–502.
21. Shaw, V. E., Spana, S., Ashkan, K., Benabid, A.-L., Stone, J., Baker, G. E., and
Mitrofanis, J. (2010). Neuroprotection of midbrain dopaminergic cells in
July 6, 2016 17:32 PSP Book - 9in x 6in 27-Hamblin-c27

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MPTP-treated mice after near-infrared light treatment. J. Comp. Neurol.,

518, pp. 25–40.
22. Sospedra, M., and Martin, R. (2005). Immunology of multiple sclerosis.
Ann. Rev. Immunol., 23, pp. 683–747.
23. Steinman, L. (2014). Immunology of relapse and remission in multiple
sclerosis. Ann. Rev. Immunol., 32, pp. 257–281.
24. Trapp, B. D., and Nave, K. A. (2008). Multiple sclerosis: An immune or
neurodegenerative disorder? Annu. Rev. Neurosci., 31, pp. 247–269.
25. Whelan, H. T., Buchmann, E. V., Dhokalia, A., Kane, M. P., Whelan, N. T.,
Wong-Riley, M. T., Eells, J. T., Gould, L. J., Hammamieh, R., Das, R., and Jett,
M. (2003). Effect of NASA light-emitting diode irradiation on molecular
changes for wound healing in diabetic mice. J. Clin. Laser. Med. Surg., 21,
pp. 67–74.
26. Zivin, J. A., Albers, G. W., Bornstein, N., Chippendale, T., Dahlof, B., Devlin,
T., Fisher, M., Hacke, W., Holt, W., Ilic, S., Kasner, S., Lew, R., Nash, M.,
Perez, J., Rymer, M., Schellinger, P., Schneider, D., Schwab, S., Veltkamp,
R., Walker, M., Streeter, J., NeuroThera Effectiveness and Safety Trial-
2 Investigators. (2009). Effectiveness and safety of transcranial laser
therapy for acute ischemic stroke. Stroke, 40, pp. 1359–1364.
July 6, 2016 17:34 PSP Book - 9in x 6in 28-Hamblin-c28

Chapter 28

Low-Level Laser Therapy as an

Alternative Treatment for Snake
Envenomation

Camila Squarzoni Dalea and Stella Regina Zamunerb

a Department of Anatomy, University of São Paulo, Lineu Prestes Avenue, 2415,

São Paulo, São Paulo 05508-900, Brazil

b Universidade Nove de Julho, Rua Vergueiro, 235, São Paulo,

São Paulo 01504-001, Brazil

camila.dale@usp.br

28.1 Introduction

Anti-venom therapy has been ineffective in neutralizing the tissue

damage caused by Bothrops snakebites. Among therapeutic strate-
gies to minimize effects after envenoming, it was hypothesized that a
low-level laser would reduce complications as well as the severity of
local snake venom effects. The purpose of this chapter is to consider
the potential for low-level laser therapy (LLLT) in treating the local
manifestations of Bothrops envenoming by providing background
information on its mechanism of action and delivery parameters.

Handbook of Low-Level Laser Therapy

Edited by Michael R. Hamblin, Marcelo Victor Pires de Sousa, and Tanupriya Agrawal
Copyright c 2017 Pan Stanford Publishing Pte. Ltd.
ISBN 978-981-4669-60-3 (Hardcover), 978-981-4669-61-0 (eBook)
www.panstanford.com
July 6, 2016 17:34 PSP Book - 9in x 6in 28-Hamblin-c28

504 Low-Level Laser Therapy as an Alternative Treatment for Snake Envenomation

28.2 Snake Envenomation of the Brothrops Genus

Ophidic accidents are still an important problem in public health

due to their high frequency and severity, particularly affecting rural
areas of Latin America, Africa, and Asia [9, 22]. In 2009, the World
Health Organization (WHO) incorporated snakebite envenoming
in its list of neglected tropical diseases (www.who.int/neglected
diseases/diseases/en). There are approximately 3000 known snake
species and 20% are venomous [35]. In Latin America, there are
four clinically relevant venomous snake genera: Bothrops, Crotalus,
Lachesis, and Micrurus [35], and the snakes of the Bothrops genus are
responsible for the majority of accidents (Fig. 28.1). In Brazil, 65,911
out of 81,611 snakebite notifications were related to identified
venomous snakes, and 59,619 (73.1%) were caused by Bothrops
genus snakes [28]. This larger prevalence is probably due to the vast
and abundant geographic distribution and the aggressive behavior
of these snakes (Fig. 28.2) [3, 10, 28, 35, 39]. The Bothrops genus
comprises more than 30 species and subspecies spread out from the
South of Mexico to Argentina and some Caribbean Islands. The most
important species are Bothrops asper, Bothrops atrox, and Bothrops

Figure 28.1 Bothrops jararaca. (Image courtesy: Luciano Zandoná).

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Snake Envenomation of the Brothrops Genus 505

Figure 28.2 Distribution of the Bothrops genus snakes in Brazil. Adapted

from Ref. [10].

jararaca. Interestingly, most Bothrops species are found in large

numbers all over Latin and South America, while others are limited
to a specific region, like the Bothrops insularis on Ilha da Queimada
Grande, on the São Paulo state coast [10, 39].
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506 Low-Level Laser Therapy as an Alternative Treatment for Snake Envenomation

Snake venoms are composed of more than 20 different com-

ponents whose functions and interactions have not been entirely
studied. Almost 90–95% of the net weight of a snake venom is
composed of proteins (enzymes, non-enzyme toxins, and non-toxic
proteins). The non-proteic fraction is made up of carbohydrates,
lipids, metals, biogenic amines, free amino acids, and nucleotides
[39]. Consequently, snakebite envenomations are linked with a
remarkable variety of pathophysiological manifestations, which
differ between the various groups of snakes and even within the
same genera and species [19]. But despite venom variability, some
basic actions are universal to all Bothrops species.

28.2.1 Local Manifestations

Local manifestations include local pain, which is induced by
bradykinin, histamine, and venom biogenic amines. Early develop-
ment of edema is frequent. It is usually tense and lilaceous, due to
subcutaneous bleeding caused by small peptides and phospholipase
A2. The venom also causes hemolysis, platelet aggregation, my-
onecrosis, vascular endothelium rupture, and extracellular matrix
component degradation [3, 10, 23, 24, 28, 34, 39]. In up to 24 h,
the whole limb may be impaired due to extracellular leaking.
Tissue necrosis caused by venom proteolytic action associated to
vascular injury is a feared complication, which might be worsened
by tourniquet use and treatment delay. Compartmental syndrome
caused by the inflammatory and hemorrhagic processes in the
injured area is unusual, demands rapid medical intervention when
it occurs, and makes patient management particularly difficult.
Intense pain, paresthesia, cyanoses, and temperature changes may
occur as a result of the nervous-vascular bundle compression by the
edema present in the affected area [3, 10, 28, 39].

28.2.2 Systemic Manifestations

Hemorrhagic manifestations such as gingival bleeding, microscopic
hematuria, purpura, and recent wound bleeding are frequent.
Macroscopic hematuria, hemoptysis, epistaxis, conjunctival bleed-
ing, hematemesis, and central nervous system hemorrhage have
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Snake Envenomation of the Brothrops Genus 507

been reported [3, 10, 34, 35, 39]. Shock and hypotension have been
described following the bite [10, 35, 23, 33, 39]. Kidney injury is
a frequent complication, causing high morbidity and mortality [3,
10, 34–36, 39]. Hemorrhage results from venom action causing
coagulant system activation and intravascular fibrin formation.
Such an action, known as “thrombin-type coagulantaction,” is not
neutralized by heparin. The final result is fibrinogen consumption
and subsequent non-clotting. The venom, while activating factor
X, also consumes the factors V, VII, and platelets causing capillary
microthrombi formation, which contribute to the acute kidney
injury genesis [3, 10, 24, 28, 33, 35, 39].

28.2.3 Anti-Venom Treatment

Treatment of snakebites is still carried out using traditional anti-
venom therapy produced in Brazil by the Instituto Butantan,
Fundação Ezequiel Dias, and the Instituto Vital Brazil. A specific
and early anti-venom application is essential and decisive because
a fast, specific treatment represents an extremely important
positive prognostic factor. The accident classification under mild,
moderate, or severe is based on the patient’s clinical status, which
determines the proper serum therapy. Monitoring of blood pressure,
respiratory condition, cardiac rate, and adequate hydration should
be performed. Attention to bite site injury and systemic bleeding
is of high importance for treatment success [3, 10, 25, 29, 33,
38, 39]. However, this anti-venom is not considered ideal since it
does not prevent local damages caused by the snake venom; it can
induce hypersensitivity reactions and is sometimes difficult to find
in remote areas [4, 11, 22, 23].
Efforts have been made in order to increase the effects of
anti-venom therapy. For example, some authors have investigated
that marsupial serum application against Bothrops erythromelas
neutralizes hemorrhagic, myonecrotic, hyperalgesic, and edemato-
genic effects of the venom [27]. However, the lack of protection
of anti-venom and the severity effect caused by these venoms on
the local site of the bite still encourage the search for alternative
treatments for local reaction induced by snake venom.
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508 Low-Level Laser Therapy as an Alternative Treatment for Snake Envenomation

28.3 Low-Level Laser Therapy for Treatment of Local

Manifestations of Bothrops Envenomation

Low-level light/laser therapy is the direct application of light (usu-

ally delivered via a low-power laser or light-emitting diode, LED) to
stimulate cell responses (photobiomodulation) in order to promote
tissue repair, reduce inflammation, and induce analgesia. There have
been significant studies demonstrating its application and efficacy
at many sites within the body and for the treatment of a range
of musculoskeletal injuries, degenerative diseases and dysfunction.
Envenoming induced by Bothrops snakes is characterized by
drastic local tissue damage involving hemorrhage, myonecrosis, and
prominent inflammatory and hyperalgesic response. Treatments
with low-level laser (LLL) and LED have been demonstrated to
be effective in reducing local effects induced by Bothrops, such as
edema formation, myonecrosis, and hyperalgesia. Experimental data
about the effects of LLL and LED on local manifestations induced by
Bothrops venom are described as follows.

28.3.1 Myonecrosis and LLLT

In survivors of snakebite, the main cause of permanent disability
is the local myonecrosis [45]. Due to the severity of the local
effects, with large areas of skin necrosis, debridement and grafting
of the tissue are common, whereas the destruction of deep tissues
often necessitates amputation [19]. Also, arthrodesis, chronic
ulceration, osteomyelitis, and malignant transformation are long-
term consequences [19]. These alterations are induced by either an
indirect action of venom hemorrhagic toxins or a direct action of
myotoxins upon the plasma membrane of muscle cells [18].
Snake venom hemorrhagic toxins are zinc-dependent metallo-
proteinases, which belong to the family of “metzincins,” together
with astacins, serralysins, matrix metalloproteinases (MMPs), and
ADAMs (enzymes with a disintegrin and metalloproteinase do-
mains) [20]. Snake venom metalloproteinases (SVMPs) comprise
four groups classified according to the domain constitution: (i) P-I,
constituted by enzymes having only the metalloproteinase domain;
(ii) P-II, which includes enzymes presenting the metalloproteinase
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Low-Level Laser Therapy for Treatment of Local Manifestations of Bothrops 509

domain followed by a disintegrin-like (Dis-like) domain; (iii) P-III,

enzymes that have a cysteine-rich (Cys-rich) domain in addition to
metalloproteinase and Dis-like domains; and (iv) P-IV, consisting of
enzymes with two subunits, one constituted by the three domains
characteristic of P-III enzymes and another being a C-type lectin
protein, linked through disulfide bridges to the first one [8].
Myotoxic components, known as “myotoxins,” are molecules that
induce direct cytotoxicity to skeletal muscle cells [19]. The most
important and abundant muscle-damaging components in animal
venoms are phospholipases A2 (PLA2, EC 3.1.1.4), enzymes that
catalyze the hydrolysis of the sn-2 acyl bond of glycerophospho-
lipids, in a calcium-dependent fashion, generating free fatty acids
and lysophospholipids. Venom PLA2s belong to classes I (terrestrial
elapids and sea snake venoms), II (viperid snake venoms), and III
(bee and lizard venoms), among the growing family of secreted
PLA2s [48, 49]. Two different types of myotoxic PLA2s have been
characterized in Bothrops venoms: (i) catalytically active PLA2s,
which have the conserved residues at the catalytic network and
at the calcium-binding loop, including Asp49; and (ii) catalytically
inactive variants having Lys instead of Asp at position 49 [41].
Despite the structural differences in snake venom, PLA2s show a
variety of toxic/pharmacological effects, which include neurotoxic,
myonecrotic, cardiotoxic, hemolytic, hemorrhagic, hypotensive,
anticoagulant, and platelet aggregation inhibition [43, 47].
A number of publications have reported on the benefits of
LLL on local effect induced by Bothrops venom. The ability of
LLLT to decreased myonecrosis induced by Bothrops venom was
first reported by Dourado et al. [14], who evaluated the effects
of a gallium–arsenide (Ga–As) laser (λ 904 nm, energy density of
4 J/cm2 ) on serum creatine kinase (CK) levels and muscle tissue
morphology at different times after the injection of Bothrops moojeni
venom in mice gastrocnemius muscle. They found that LLLT was
able to inhibit the ability of venom to rapidly disrupt the integrity
of the plasma membrane.
The beneficial effect of LLLT on myonecrosis induced by Bothrops
venom was then confirmed using the same Ga–As laser in a different
wavelength (λ, 685 nm; energy density, 4.2 J/cm2 ) [5]. This work
evaluated the effects of LLLT on myonecrosis caused by Bothrops
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510 Low-Level Laser Therapy as an Alternative Treatment for Snake Envenomation

jararacussu venom. As observed for Bothrops moojeni, LLLT signif-

icantly reduced myonecrosis induced by Bothrops jararacussu snake
venom. They also showed that anti-venom treatment combined with
LLLT presented a pattern similar to that exhibited by LLLT alone
[5]. These findings are reinforced by data demonstrating that a
combined treatment of anti-venom with the Ga–As laser application
reduces necrosis in gastrocnemius muscle of mice injected with
Bothrops moojeni venom (λ, 904 nm; energy density, 4 J/cm2 ) [2]. In
another study, irradiation with LLLT (λ, 632.8 nm; energy density,
3.5 J/cm2 ) improved neuromuscular transmission and reduced
the development of myonecrotic changes induced by the Bothrops
jararacussu venom in vitro [14].
As described earlier, myotoxins with PLA2 structure are mainly
responsible for the direct effect of Bothrops venom on plasma
membranes. Therefore, the study of PLA2 myotoxins is of great
relevance. A study with two myotoxins isolated from Bothrops
jararacussu venom—bothropstoxin I (BthTX-I), a basic Lys 49, and
bothropstoxin II (BthTX-II), a basic Asp 49—showed that LLL
treatment significantly reduced the cell damage caused by BthTX-
I or BthTX-II, evidenced by the increase in muscle CK content
and histological observation, which showed a diminished number
of destroyed fibers when compared to muscle injected only with
myotoxins without laser treatment [7].
Local hemorrhage is part of the complex pathological alter-
ations at the bite site characteristic of Bothrops envenoming and
contributes to tissue damage and impaired muscle regeneration
[12]. In this sense, it has been demonstrated that LLLT decreases
the diameter of the hemorrhagic halo caused by Bothrops moojeni
venom, suggesting that LLLT is somehow able to inhibit venom-
induced disruption of microvessels plasma membrane integrity, thus
reducing bleeding [29].
The mechanism by which LLLT improves myonecrosis induced
by Bothrops venoms is still not known. However, recent data
have demonstrated that both He–Ne (λ, 632.8 nm) and Ga–As
(λ, 904 nm) lasers promote vascular endothelial growth factor
receptor-1 expression in endothelial and nonendothelial cells of
mice gastrocnemius exposed to Bothrops moojeni venom [15].
Recent data have also demonstrated that Ga–As (λ, 904 nm; energy
July 6, 2016 17:34 PSP Book - 9in x 6in 28-Hamblin-c28

Low-Level Laser Therapy for Treatment of Local Manifestations of Bothrops 511

density, 3 J/cm2 ) laser irradiation improves myofiber mass recovery

during the regeneration of skeletal muscle of mice previously
damaged by crotoxin, the main component of the South American
rattlesnake Crotalus durissus terrificus venom, demonstrated by an
increase in myogenin gene expression, a marker of muscle differen-
tiation/regeneration, suggesting that LLLT improves skeletal muscle
regeneration by accelerating the recovery of myofiber mass [40].

28.3.2 Local Inflammation and LLLT

A severe inflammatory response characterized by leukocytes influx
and edema formation, with a rapid onset, is a hallmark of
envenomation induced by Bothrops venoms [32]. Local edema
appears very rapidly after the injection of Bothropic venoms in
experimental animals [19, 31], and the pathophysiology involved
is multifactorial. A direct damage to microvessels, i.e., capillaries
and venules, is induced by hemorrhagic toxins, with the consequent
extravasation. Also inflammatory mediators, either released or
synthesized in the course of envenomation, induce increments in the
permeability of microvessels [18, 31].
Most of the studies focusing on the effects of LLLT in the
outcome of the inflammatory reactions induced by Bothrops snake
venoms comprise methods that evaluate the increases in vascular
permeability (mostly paw and muscle edema) and the leukocyte
influx. Leukocytes play an essential role in the host defense
against offending agents and are key mediators in the inflammatory
response [46]. A number of studies have demonstrated that Bothrops
venoms induce a significant leukocyte accumulation at the site of
Bothrops venom injection [5, 16, 17, 46].
The ability of LLLT to decrease inflammatory response induced
by Bothrops venom was first demonstrated in mice injected with
Bothrops jararacussu venom in the gastrocnemius muscle [5]. In
this work, LLLT (λ, 685 nm; energy density, 4.2 J/cm2 ) significantly
reduced the edema formation and leukocyte influx. Moreover,
antiedematogenic action was magnified when laser and anti-venom
therapies were used together [5]. Similar results were obtained in
a mouse footpad model of edema induced by Bothrops jararacussu
venom where LLLT, at the same parameters as described earlier,
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512 Low-Level Laser Therapy as an Alternative Treatment for Snake Envenomation

markedly reduced the edema formation and leukocyte influx to the

peritoneal cavity of mice [42].
More recently it was demonstrated that both LLL (λ, 685 nm;
energy density, 2.2 J/cm2 ) as well as LED device (λ, 635 or
945 nm; energy density, 4 J/cm2 ) reduced paw edema induced by
Bothrops mojenni venom in mice [29]. However, photobiostimulation
associated with anti-venom treatment had the same effect on edema
as LLL or LED irradiation by itself [29]. Similar data were observed
for Bothrops jararacussu venom using LLL at wavelength of 904 nm
and energy density as 4 J/cm2 [42]. Considering isolated myotoxins,
results demonstrate that LLLT (λ, 685 nm; energy density, 4.2
J/cm2 ) reduces edema formation and leukocyte influx after BthTX-
I or BthTX-II [7]. In addition, LLLT acts with the same intensity to
reduce the inflammatory processes for both BthTX-I and BthTX-II,
suggesting that the enzymatic activity of the toxins is not relevant
for LLLT-induced effect [7].

28.3.3 Hyperalgesia and LLLT

Intense pain is also frequently associated with Bothrops en-
venomation. The literature demonstrates that the hyperalgesia
(pain hypersensitivity) induced by Bothrops jararaca venom is
independent from edematogenic response and mediated, at least
partially, by lipid mediators such as prostaglandins, leukotrienes,
platelet activating factor, biogenic amines, and metalloproteases [37,
44]. LLLT is one of the more recent pain treatment modalities.
Literature demonstrates that the analgesic effect of phototherapy
may be mediated by hormonal/opioid mechanisms, the responses of
which depend directly on the dose and wavelength used to irradiate
the tissue [26]. LLLT is also reported to act on peripheral neural
stimulation and the regulation of microcirculation, interrupting pain
mechanisms and promoting analgesia [1]. However, there is only a
little information regarding the effects of LLLT on venom-induced
pain. In this sense, it has been demonstrated that Ga–As LLLT
(λ, 685 nm; energy density, 4.2 J/cm2 ) reduces hyperalgesia induced
by Bothrops jararacussu venom in mice [42]. Similar effects have
been observed for Ga–As (λ, 904 nm; energy density, 4 J/cm2 ),
which have been demonstrated to be effective in reducing pain
July 6, 2016 17:34 PSP Book - 9in x 6in 28-Hamblin-c28

References 513

signs of mice injected with Bothrops moojeni venom [2]. Moreover,

these authors have demonstrated the inhibition of pain signs when
combining LLL and anti-venom treatment, suggesting that the
effects of the combined treatments can help in reducing the pain of
snakebite envenomations [2]. Positive effects on Bothrops moojeni
venom-induced pain have also been described for LED treatment,
where the application of LED at both wavelengths of 635 or 945 nm
(energy density as 4 J/cm2 ) inhibited signs of pain in mice alone or
in combination with anti-venom treatment [30]. More interesting is
the fact that LED treatment is effective in reducing pain even when
the symptoms are already present [30].

28.4 Conclusion

LLL and LED irradiation, applied at the site of the snakebite, are
effective in reducing some of the local effects produced by Bothrops
snakes venom, such as myonecrosis, edema formation, leukocyte in-
flux, local hemorrhage, and hyperalgesia. The mechanisms involved
in these LLL effects are currently not known; however, it is likely that
LLL acts by stimulating phagocytosis, myoblasts proliferation, and
the genes responsible for muscle fibers regeneration. Knowledge of
the mechanisms of action of LLL treatment on Bothrops snakebite
envenomation, in the future, may help to design new alternatives to
treat the local inflammatory reactions characteristic of snakebites,
which are not well neutralized by conventional anti-venoms. Thus,
the effect of phototherapy should be considered a novel therapeutic
tool for the treatment of local symptoms induced by Bothrops snake
envenomation.

References

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pain by physical therapists. Phys. Med. Rehabil. Clin. N. Am., 17, pp. 315–
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Henriques, S. V., dos Santos Picanço, L. C., Lobato, C. P., Ribeiro, J. R.,
July 6, 2016 17:34 PSP Book - 9in x 6in 28-Hamblin-c28

514 Low-Level Laser Therapy as an Alternative Treatment for Snake Envenomation

Pereira, W. L., Carvalho, J. C., and da Silva, J. O. (2013). Effects of a

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therapy decreases local effects induced by myotoxins isolated from
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357.
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nonendothelial cells of mice gastrocnemius exposed to snake venom.
Photochem. Photobiol., 87, pp. 418–426.
16. Farsky, S. H. P., Costa-Cruz, J. W. M., Cury, Y., and Teixeira, C. F. P. (1997).
Leukocyte response induced by Bothrops jararaca crude venom. In vivo
and in vitro studies. Toxicon, 35, pp. 185–193.
17. Flores, C. A., Zappellini, A., and Prado-Franceschi, J. (1993). Lypoxy-
genase-derived mediators may be involved in in vivo neutrophil
migration induced by Bothrops erytromelas and Bothrops alternates
venoms. Toxicon, 31, pp. 1551–1559.
18. Gutiérrez, J. M., and Lomonte, B. (1995). Phospholipase A2 myotoxins
from Bothrops snake venoms. Toxicon, 33, pp. 1405–1424.
19. Gutiérrez, J. M., and Ownby, C. L. (2003). Skeletal muscle degeneration
induced by venom phospholipases A2: Insights into the mechanisms of
local and systemic myotoxicity. Toxicon, 42, pp. 915–931.
20. Gutiérrez, J. M., Rucavado, A., Escalante, T., and Dı́az, C. (2005).
Hemorrhage induced by snake venom metalloproteinases: Biochemical
and biophysical mechanisms involved in microvessel damage. Toxicon,
45, pp. 997–1011.
21. Gutiérrez, J. M., Theakston, R. D. G., and Warrell, D. A. (2006).
Confronting the neglected problem of snake bite envenoming: The need
for a global partnership. PLoS Med., 3, pp. e150.
22. Gutiérrez, J. M., Lomonte, B., León, G., Rucavado, A., Chaves, F., and
Angulo, Y. (2007). Trends in snakebite envenomation therapy: Scientific,
technological and public health considerations. Curr. Pharm. Des., 13, pp.
2935–2950.
23. Gutiérrez, J. M., Escalante, T., and Rucavado, A. (2009). Experimental
pathophysiology of systemic alterations induced by Bothrops asper
snake venom. Toxicon, 54, pp. 976–987.
July 6, 2016 17:34 PSP Book - 9in x 6in 28-Hamblin-c28

516 Low-Level Laser Therapy as an Alternative Treatment for Snake Envenomation

24. Hawgood, B. J., and Rocha e Silva, M. (1997). Snake venom, bradykinin
and the rise of autopharmacology. Toxicon, 35, pp. 1569–1580.
25. Jorge, M. T., and Ribeiro, L. A. (1997). Dose do soro (antiveneno) no
tratamento do envenenamento por serpentes peçonhentas do gênero
Bothrops [Antivenom serum doses in the treatment of poisoning by a
venomous snake of the genus Bothrops]. Rev. Ass. Med. Brasil., 43, pp. 74–
76.
26. Laakso, E. L. (1994). Plasma ACTH and B-endorfhin levels in response to
low level laser therapy (LLLT) for myofascial trigger points. Laser Ther.,
6, pp. 133–142.
27. Martins, A. M. C., Sousa, F. C. M., Barbosa, P. S. F., Toyama, M. H., Toyama,
D. O., Aprı́gio, C. C., Queirós, M. G. R., Guarnieri, M. C., Havt, A., Menezes,
D. B., Fonteneles, M. C., and Monteiro, H. S. A. (2005). Action of anti-
bothropic factor isolated from Didelphis marsupialis on renal effects of
Bothrops erythromelas venom. Toxicon, 46, pp. 595–599.
28. Ministério da Saúde (Brasil). (2001). Manual de Diagnóstico e
Tratamento por Animais Peçonhentos (Fundação Nacional da Saúde,
Brası́lia).
29. Nadur-Andrade, N., Barbosa, A. M., Carlos, F. P., Lima, C. J., Cogo, J. C.,
and Zamuner, S. R. (2012). Effects of photobiostimulation on edema and
hemorrhage induced by Bothrops moojeni venom. Lasers Med. Sci., 27,
pp. 65–70.
30. Nadur-Andrade, N., Zamuner, S. R., Toniolo, E. F., de Lima, C. J., Cogo, J.
C., and Dale, C. S. (2013). Analgesic effect of light-emitting diode (LED)
therapy at wavelengths of 635 and 945 nm on Bothrops moojeni venom-
induced hyperalgesia. Photochem. Photobiol., in press.
31. Nascimento, N. G., Sampaio, M. C., Olivo, R. A., and Teixeira, C. (2010).
Contribution of mast cells to the oedema induced by Bothrops moojeni
snake venom and a pharmacological assessment of the inflammatory
mediators involved. Toxicon, 55, pp. 343–352.
32. Olivo, Rdo. A., Teixeira, C. F. P., Wallace, J. R., Gutiérrez, J. M., and Zamuner,
R. S. (2007). Role of cyclooxygenases in oedemaforming activity of
bothropic venoms. Toxicon, 49, pp. 670–677.
33. Otero-Patiño, R. (2009). Epidemiological, clinical and therapeutic
aspects of Bothrops asper bites. Toxicon, 54, pp. 998–1011.
34. Pinho, F. M. O., and Burdmann, E. A. (2001). Fatal cerebral hemorrhage
and acute renal failure after young Bothrops jararacussu snake bite.
Renal Fail, 23, pp. 269–277.
July 6, 2016 17:34 PSP Book - 9in x 6in 28-Hamblin-c28

References 517

35. Pinho, F. M., Yu, L., and Burdmann, E. A. (2008). Snakebite-induced acute
kidney injury in Latin America. Semin. Nephrol., 28, pp. 354–362.
36. Ribeiro, L. A., and Jorge, M. T. (1997). Acidentes por serpentes do
gênero Bothrops: série de 3.139 casos [Bites by snakes in the genus
Bothrops: A series of 3,139 cases]. Rev. Soc. Bras. Med. Trop., 30, pp. 475–
480.
37. Rocha, S. L., Frutuoso, V. S., Domont, G. B., Martins, M. A., Moussatché, H.,
and Perales, J. (2000). Inhibition of the hyperalgesic activity of Bothrops
jararaca venom by an antibothropic fraction isolated from opossum
(Didelphis marsupialis) serum. Toxicon, 38, pp. 875–880.
38. Saraiva, P., Rojas, E., Escalante, T., Arce, V., Chaves, E., Velásquez, R.,
Lomonte, B., Rojas, G., and Gutiérrez, J. M. (2001). The venom of
Bothrops asper from Guatemala: Toxic activities and neutralization by
antivenoms. Toxicon, 39, pp. 401–405.
39. Sgrignolli, L. R., Mendes, G. E. L., Carlos, C. P., and Burdmann, E. A. (2011).
Acute kidney injury caused by Bothrops snake venom. Nephron Clin.
Pract., 119, pp. c131–136.
40. Silva, L. H., Silva, M. T., Gutierrez, R. M., Conte, T. C., Toledo, C. A., Aoki,
M. S., Liebano, R. E., and Miyabara, E. H. (2012). Ga–As 904-nm laser
irradiation improves myofiber mass recovery during regeneration of
skeletal muscle previously damaged by crotoxin. Lasers Med. Sci., 27, pp.
993–1000.
41. Soares, A. M., Sestito, W. P., Marcussi, S., Stábeli, R. G., Andrião-Escarso,
S. H., Cunha, O. A., Vieira, C. A., and Giglio, J. R. (2004). Alkylation of
myotoxic phospholipases A2 in Bothrops moojeni venom: A promising
approach to an enhanced antivenom production. Int. J. Biochem. Cell
Biol., 36, pp. 258–270.
42. Souza, L. G., Dale, C. S., Nadur-Andrade, N., Barbosa, A. M., Cogo, J. C., and
Zamuner, S. R. (2011). Low-level laser therapy reduces edema, leukocyte
influx and hyperalgesia induced by Bothrops jararacussu snake venom.
Clin. Exp. Med. Lett., 52, pp. 97–102.
43. Stabeli, R. G., Amui, S. F., Sant’ana, C. D., Pires, M. G., Nomizo, A., Monteiro,
M. C., Romao, P. R., Guerra-sa, R., Vieira, C. A., Giglio, J. R., Fontes, M. P.,
and Soares, A. M. (2006). Atividades Biológicas induzidas pela MjTX-
II isolada do veneno de Bothrops moojeni. Comp. Biochem. Physiol. C.
Toxicol. Pharmacol., 42, pp. 371–381.
44. Teixeira, C. F., Cury, Y., Oga, S., and Jancar, S. (1994). Hyperalgesia induced
by Bothrops jararaca venom in rats: Role of eicosanoids and platelet
activating factor (PAF). Toxicon, 32, pp. 419–426.
July 6, 2016 17:34 PSP Book - 9in x 6in 28-Hamblin-c28

518 Low-Level Laser Therapy as an Alternative Treatment for Snake Envenomation

45. Warell, D. A. (2010). Snake bite. Lancet, 375, pp. 77–88.

46. Zamuner, S. R., Gutieérrez, J. M., Muscara, M. N., Teixeira, A. S., and
Teixeira, C. F. P. (2001). Bothrops asper and Bothrops jararaca snake
venoms trigger microbicidal functions of peritoneal leukocytes in vivo.
Toxicon, 39, pp. 1505–1513.
47. Zuliani, J. P., Fernandes, C. M., Zamuner, S. R., Gutierrez, J. M., and Teixeira,
C. F. P. (2005). Inflammatory events induced by Lys-49 and Asp-49
phospholipases A2 isolated from Bothrops asper snake venom: Role of
catalytic activity. Toxicon, 45, pp. 335–346.
48. Six, D. A. and Dennis, E. A. (2000). The expanding superfamily
of phospholipase A(2) enzymes: Classification and characterization.
Biochim. Biophys. Acta., 1488, pp. 1–19.
49. Valentin, E. and Lambeau, G. (2000). Increasing molecular diversity of
secreted phospholipases A(2) and their receptors and binding proteins.
Biochim. Biophys. Acta., 1488(1-2), pp. 59–70.
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Chapter 29

Veterinary Low-Level Laser (Light)

Therapy Applications for Companion
Animals

Richard L. Godine
Ruckersville Animal Hospital and Veterinary Laser Therapy Center,
8301 Seminole Trail, Ruckersville, VA 22968, USA
rlgdvm@gmail.com

29.1 Introduction: Finding Common Ground

As the use of therapeutic lasers and light-emitting devices has

steadily increased in small-animal veterinary medicine over the last
decade, so have the number of manufacturers entering the market.
Little has been taught in veterinary colleges regarding light therapy,
and most veterinarians have gained their knowledge through
the manufacturer’s representatives, literature, and seminars. This
presents a challenge to talk with one another about refining
protocols of light therapy, yet this is what we must do.
The four most common wavelengths found in veterinary lasers
and LEDs are +/− 660 nm, 810 nm, 904 nm, and 980 nm. Powers
range from 5 mW to 12 W. Some units combine more than one
wavelength running simultaneously, while others rotate among four

Handbook of Low-Level Laser Therapy

Edited by Michael R. Hamblin, Marcelo Victor Pires de Sousa, and Tanupriya Agrawal
Copyright c 2017 Pan Stanford Publishing Pte. Ltd.
ISBN 978-981-4669-60-3 (Hardcover), 978-981-4669-61-0 (eBook)
www.panstanford.com
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520 Veterinary Low-Level Laser (Light) Therapy Applications for Companion Animals

wavelengths and alternating frequencies. Almost all of the clinical

trials published in the medical literature have been on humans and
usually using a single wavelength at a time. Very little research has
been done with higher power lasers, yet Class IV lasers represent a
great share of the veterinary market.
To begin our summary of clinical applications, I believe it is
important to state that most veterinary lasers achieve clinical results
when operated per the manufacturer’s instructions. The therapeutic
window is often wide for many conditions. I will be talking about
what I believe to be optimal parameter ranges. This implies that one
is able to measure the parameters and achieve reproducibility.
I use six different laser systems at my hospital and am very
familiar with all the common wavelengths, except 980 nm. In my
opinion, the most efficient and accurate way to deliver therapeutic
doses to your target tissue in fur-covered animals is the use of multi-
probes in the 100–750 mW per diode range. These can be pressed
down toward the target and left in place for as long as it takes to get
your optimal therapeutic response, typically 20–120 s. Techniques
whereby the probe is swept continuously over an area make it more
difficult to approximate your power density and, therefore, energy
density applied to the target tissue in the veterinary patient. This
affects reproducibility and, in certain conditions with a narrower
therapeutic window, can lead to bioinhibition when one was trying
for biostimulation.
As I discuss each clinical application, I will give the anatomical
locations to be treated, the optimal wavelength to use, and the
optimal power density and time to be applied. No matter which
laser one might have, it is hoped that the therapist can get close to
the parameters listed in each protocol. If one desires to apply light
therapy to every condition encountered in a small-animal veterinary
hospital that could benefit from it, then clearly there is not a “one
laser fits all” solution. One of the unique features of veterinary
light therapy is the fact that veterinarians treat every body system
with varying sizes and slight differences in anatomy and physiology.
Treating a Bullmastiff’s hip, a kitten’s corneal ulcer, and everything
in between requires multiple light therapy devices and a sound
knowledge of how parameters affect clinical outcomes in each
situation. The starting place is to know your device well and how
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Treatment Parameters 521

you can manipulate its parameters to achieve the desired clinical

outcome. When this has been accomplished, then one can add
another probe or light therapy unit to supplement the weaknesses
of the first unit.

29.2 Treatment Parameters

As with any medical condition, it is imperative to have an accurate

assessment and diagnosis of the medical condition at hand. What are
the target tissues that need to be treated with light? Do they need to
be bioinhibited for analgesia or biostimulated for healing [1, 2]? Are
there secondary myofascial trigger points that need to be treated?
Are there dermatome spinal roots that need to be stimulated or
dorsal root ganglia that need to be inhibited to attenuate pain
control? How deep is the primary lesion? What reflectors and
absorbers of light lay between your probe and the target tissue? How
thick is the hair coat and how much melanin is in the hair and skin?
Where are the local and regional lymph nodes?
Once a thorough assessment has been made, a protocol can be es-
tablished that identifies various target tissues; optimal wavelength,
power density, and time; and the time for further treatments. In
general, acute injuries will be treated every 12–48 h until resolution,
while chronic injuries will be treated 2–3 times a week for two
weeks, then once a week for two weeks, then every other week, etc.,
until the longest effective interval between treatments is identified.
It should be noted that treating the spinal cord and regional
lymph nodes will often increase and accelerate one’s chances for
success with light therapy. Treating over the spinal roots of a
dermatome can enhance healing of stubborn wounds, while treating
the dorsal root ganglia of a particular area can help downregulate
both acute and chronic pain. The treatment over regional lymph
nodes not only modulates the immune response to an injury but
also helps reduce swelling by opening up lymphatic channels. Many
painful injuries often have myofascial trigger points associated with
them. These foci of oxidative stress can be effectively released with
light therapy.
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522 Veterinary Low-Level Laser (Light) Therapy Applications for Companion Animals

29.3 Musculoskeletal Conditions

Injuries to the musculoskeletal system are the most common

pathologies treated with LLLT in a small-animal veterinary hospital.
Fortunately, they are some of the most rewarding conditions
treated with LLLT. Acute injuries respond rapidly to LLLT, and
chronic injuries treated with LLLT often offer an effective and safer
alternative to pharmaceuticals and surgery. As most patients with
chronic degenerative joint disease (DJD) and osteoarthritis are older,
the worries of side effects of non-steroidal anti-inflammatory drugs
and anesthetic risks might be avoided with LLLT.

29.3.1 Degenerative Joint Disease and Osteoarthritis

This group of diseases is seen daily in the veterinary laser clinic
because of how much longer pets are living as well as the large
number of dysplastic breeds and congenital diseases that exist.
There are three important points to remember when treating this
class of chronic conditions. First, warn the client that their pet may
become sorer after the first treatment and provide temporary oral
pain medication for the first two days. Second, inform the client that
progress will be steady but slow and may take many treatments (6–
10) before significant improvement can be achieved. Finally, be sure
that you are treating all areas of DJD, which are frequently multiple,
and not just the obvious joint (Table 29.1). LLLT failure can often
be attributed to either not going long enough with treatments for
a breakthrough or not treating all the affected joints. Once clinical
relief of symptoms has been achieved, the fluence can be decreased
by a quarter to better promote continuous biostimulation of the
injured and diseased tissues.

29.3.1.1 DJD of the hip

Target tissue (TT): Coxofemoral synovium (2–4 cm deep) (Fig. 29.1)
Wavelength: 810 or 904 superpulsed
Power density: 0.05 to 5 W/cm2
Time: 30–90 s per point depending on power of laser
Frequency: <25 Hz or CW
 July 6, 2016 17:35
Table 29.1 Degenerative joint disease and osteoarthritis

Condition Target Tissue λ Power Density Time Frequency Ideal Energy Application Points Comments
(TT) (nm) (W/cm2 ) (s) (Hz) @TT (J)

Hip DJD Synovium 810 or 0.05–5 30–90 <25 2–4 4–12 lateral and
2–4 cm 904 or 2–4 medial
CW
Inguinal and 660 or <300 20–60 <25 or CW 2–4 Over lymph node
popliteal lymph 810 mW/cm2

PSP Book - 9in x 6in

nodes
DRG 810 1–5 20–60 10 or CW 2–6 Intervertebral Angle probe @ 45◦
(L4-S3) foramen L4-S3
MFTPs 810 1–5 20–40 CW 5–10 Gluteal, iliocostalis, Palpate these
iliopsoas muscles for knots
and pain
DJD stifle Synovium 810 or 0.05–5 20–60 <25 or CW 2–5 3–8 cranial, Move from center to

Musculoskeletal Conditions
1–3 cm 904 4–8 caudal, medial and lateral
2–4 patellar
All other TT Parameters same as
same as hip for hip

(Contd.)

523

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524 Veterinary Low-Level Laser (Light) Therapy Applications for Companion Animals
Table 29.1 (Contd.)

Condition Target Tissue λ Power Density Time Frequency Ideal Energy Application Points Comments
(TT) (nm) (W/cm2 ) (s) (Hz) @TT (J)

DJD elbow Synovium 810 or 0.05–2.5 20–60 <25 or CW 2–5 4–12 around elbow Too high PD and
1–2 cm 904 fluence causes pain

PSP Book - 9in x 6in

Axillary and 660 or <300 mW/cm2 20–60 <25 or CW 2–4 Over lymph node
prescap lymph 810
nodes
DRG 810 1–5 20–60 10 or CW 2–6 Intervertebral
(C6-T1) foramen
C6-T1
MFTPs 810 1–5 20–40 <25 or CW 5–10 Triceps and
infraspinatus

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Musculoskeletal Conditions 525

Figure 29.1 Superpulsed 904 nm, 4 × 60 mW multi-probes treating DJD of

canine hip.

Ideal energy at TT: 2–5 J

Application points: 4–12 points laterally, 2–4 points medi-
ally

Target tissue: Inguinal and popliteal lymph nodes

Wavelength: 810 or 660

Power density: <300 mW/cm2
Time: 20–60 s
Frequency: <25 Hz or CW
Ideal energy at TT: 2–4 J
Application points: Cover directly over all lymph nodes

Target tissue: Dorsal root ganglia (L4-S3)

Wavelength: 810
Power density: 1–5 W/cm2
Time: 20–60 s
Frequency: 10 Hz or CW
Ideal energy at TT: 2–6 J
Application points: Intervertebral foramen (L4-S3)

Target tissue: Myofascial trigger points (MFTPs)3

Wavelength: 810
Power density: 1–5 W/cm2
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526 Veterinary Low-Level Laser (Light) Therapy Applications for Companion Animals

Time: 20–40 s
Frequency: CW
Ideal energy at TT: 5–10 J
Application points: Palpate gluteal, iliocostalis, and iliop-
soas muscles

29.3.1.2 DJD of stifle4

Target tissue: Synovium and cartilage (1–3 cm deep)

Wavelength: 810 or 904 superpulsed

Power density: 0.05–5 W/cm2
Time: 20–60 s per point
Frequency: <25 Hz or CW
Ideal energy at TT: 2–5 J
Application points: 3–8 across cranial stifle, 4–8 across
caudal stifle

All the other target tissues and parameters are the same as for the
hip.

29.3.1.3 DJD of elbow

Target tissue: Synovium (1–2 cm deep)

Wavelength: 810 or 904 superpulsed or 660

Power density: 0.05–2.5 W/cm2
Time: 20–60 s per point
Frequency: <25 Hz or CW
Ideal energy at TT: 2–5 J
Application points: 4–12 points around entire elbow joint
*Higher power densities and fluencies can increase pain

Target tissue: Axillary and prescapular lymph node

Parameters same as previously described for hip

Target tissue: Dorsal root ganglia (C6-T1)

Parameters as previously described for hip dorsal root

ganglia
July 6, 2016 17:35 PSP Book - 9in x 6in 29-Hamblin-c29

Musculoskeletal Conditions 527

Target tissue: Myofascial trigger points (infraspinatus and triceps)

Parameters same as previously described for hip
DJD and OA occur less frequently in other joints such as the shoulder,
vertebral joints, carpus, hock, and phalangeal joints. Treatment
parameters are the same as outlined earlier, taking into account
depth to target tissue and regional lymph nodes and dorsal root
ganglia (DRG) locations.
DJD/osteoarthritis of the shoulder, hock, carpus, tarsus, and
phalangeal joints should also be checked for and treated with
the parameters above, taking into account the depth of the target
tissue. Treatment outcomes often are not optimal because not all
affected joints are treated. The goal is to start 2–3 times a week
and eventually work up to the longest effective interval between
treatments.

29.3.2 Acute Musculoskeletal Injuries5

This group of injuries is quite painful and involves much inflam-
mation and swelling. The goal is to control pain and inflammation
initially and then promote rapid healing of the injured tissue.
Extreme cases may need to be hospitalized and treated with LLLT
every 8 h. Pretreating with ice helps control pain and promotes
the efficacy of the light therapy. Treating the lymph nodes and
venous return tracks from proximal to distal helps reduce swelling
significantly (Table 29.2).

29.3.2.1 Iliopsoas strain

Target tissue: Muscle belly where strained
Wavelength: 810 or 904 superpulsed
Power density: 0.05–5 W/cm2
Time: 40–80 s per point
Frequency: <25 Hz or CW
Ideal energy at TT: 2–6 J
Application points: Cranially: ventral to L1, caudally: the
iliacus and pectineus muscles in the groin.
*Can be chronic strain associated with hip or stifle disease
 July 6, 2016 17:35
528 Veterinary Low-Level Laser (Light) Therapy Applications for Companion Animals
Table 29.2 Acute musculoskeletal injuries

Condition Target Tissue λ Power Density Time Frequency Ideal Energy Application Comments
(TT) (nm) (W/cm2 ) (s) (Hz) @TT (J) Points
Iliopsoas strain Area of strain 810 or 904 0.05–5 40–80 <25 or CW 2–6 Ventral to L1 Can be chronic,
caudally to associated with
groin hip or stifle
disease

PSP Book - 9in x 6in

Biceps and Proximal biceps 810 or 904 0.05–5 60–90 2–1000 or 2–6 Craniomedial PD<100 over
supraspinatus and distal SS CW proximal, longer time
synovitis/strain tendons humerus and work better
distal scapula
DRG (C6-T1) 810 1–5 20–60 10 or CW 2–6 Intervertebral
foramen
C6-T1
Fractures Over fracture 810 or 904 0.05–5 30–90 2–50 or CW 1–4 All around
and soft tissue fracture and
associated soft
tissue

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Musculoskeletal Conditions 529

29.3.2.2 Biceps and supraspinatus tendon strain of the shoulder

Target tissue: Proximal biceps brachii tendon, distal supraspinatus
tendon

Wavelength: 810 or 904 superpulsed

Power density: 0.05–2 W/cm2
Time: 60–90 s
Frequency: 2–1000 Hz or CW
Ideal energy at TT: 2–6 J
Application points: Humeral Intertubercular groove to
supraglenoid for BT; cranial to acromion to greater humeral
tubercle for ST
*Lower power densities (<100 mW/cm2 ) over longer time
period seem to work best
*Often exacerbation of chronic strain. Treat twice a week
until resolved
*Can treat DRG (C6-T1) to help down regulate pain

29.3.2.3 Fractures6
Target tissue: Fracture and associated soft tissue injuries

Wavelength: 810 or 904 superpulsed

Power density: 0.05–5 W/cm2
Time: 30–90 s
Frequency: 2–50 Hz or CW
Ideal energy at TT: 1–4 J
Application points: All around fracture and over soft tissue
injuries
*Without surgery and coaptation alone: Daily for 2–4 days,
then twice weekly
*With surgery: Pre- and post-operation, then daily for 2–4
days, then twice weekly

Treat acute injuries every 12–48 h until pain and inflammation

are under control, then every 2–4 days with 25% less fluence until
healed.
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530 Veterinary Low-Level Laser (Light) Therapy Applications for Companion Animals

29.4 Dermatological Conditions

Skin diseases represent the second most common body system

treated by LLLT in small-animal hospitals. Hot spots, otitis externa,
lacerations, surgical wounds, and insect or snakebites represent
easily treated lesions. Lick granulomas and deep infected wounds
represent more challenging problems; however, LLLT has made
a significant contribution to the treatment of these two difficult
conditions.

29.4.1 Surgical Wounds and Lacerations7–10

Target tissue: Dermis (<1 cm) (Fig. 29.2)

Wavelength: 660 first choice or 810

Power density: <300 mW/cm2
Frequency: 2–50 or CW
Ideal energy at TT: 2–4 J
Application points: Over skin lesion +/− lymph nodes
*Treat pre- and post-surgically, then daily to QOD as needed.

29.4.2 Infected Wounds

Target tissue: Open wound
Treat as surgical wound above. Can protect tip of probe with
cellophane. Can hold probe just above wound. Since there is no
dermal barrier, 1–2 J should be enough energy.
Target tissue: Periphery of wound edges

Wavelength: 810
Power density: 50 mW to 2 W/cm2
Frequency: 2–50 Hz or CW
Ideal energy at TT: 2–4 J
Application points: to skin on the periphery of the wound
*Treat QOD until tissue is able to be surgically closed or
heals by second intention.
*Treat regional lymph nodes as previously described.
*Treat the associated dermatome with 2–4 J at level of DRG.
July 6, 2016 17:35 PSP Book - 9in x 6in 29-Hamblin-c29

Dermatological Conditions 531

Immediate Post-op reduction in

erythema and edema

Figure 29.2 Immediate post-operative reduction in erythema and edema.

Superluminous 660 nm, 4.2 mW × 120 diode arrays treating superficial
dermatitis.

29.4.3 Hot Spots and Otitis Externa

Target tissue: Inflamed superficially infected skin (Fig. 29.2)

Wavelength: 660 followed by 810

Power density: 50 mW to 2 W/cm2
Frequency: 2–50 Hz or CW
Ideal energy at TT: 3–6 J
Application points: Over skin lesions and to regional lymph
nodes
*Treat twice in 24–48 hours

29.4.4 Snake and Insect Bites

Target tissue: Bite wound and surrounding cellulitis

Wavelength: 810 or 904 superpulsed

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532 Veterinary Low-Level Laser (Light) Therapy Applications for Companion Animals
Table 29.3 Dermatological conditions

Condition Target Tissue λ Power Density Time Frequency Ideal Energy Application Comments
(TT) (nm) (W/cm2 ) (s) (Hz) @ TT (J) Points
Lacerations and Dermis 660 #1 or <300 20–120 2–50 or CW 2–4 Over skin lesion Pre- and
surgical <1 cm 810 mW/cm2 post-op ideal
wounds
Infected Open wound 660 #1 or <300 mW/cm2 20–90 2–50 or CW 1–2 Over wound Blue light
wounds 810 (405 nm) is
ideal for
infected tissue

PSP Book - 9in x 6in

Wound edges 810 50 mW to 2 20–60 2–50 Hz or CW 2–4 Periphery of
W/cm2 wound
Regional 660 or <300 mW/cm2 20–60 <25 or CW 2–4 Over lymph
lymph nodes 810 nodes
Dermatome 810 1–5 20–60 10 or CW 2–4 Intervertebral
foramen
Hot spots and Inflamed skin 660 50 mW to 2 30–120 2–50 3–6 Skin lesions Treat again in
otitis externa followed by W/cm2 or CW and regional 24–48 h
810 lymph nodes
Snake and Wound and 810 or 904 0.05–5 W/cm2 30–120 1000 or CW 4–8 Lymph nodes Treat twice in
insect surrounding first, then first 24 h, then
envenomation cellulitis cellulitis daily
proximal to
distal

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Neurological Conditions 533

Power density: 0.05 to 5 W/cm2

Frequency: 1000 Hz or CW
Ideal Energy at TT: 4–8 J
Application points: Lymph nodes first, then cellulitis proxi-
mal to distal.
*Treat twice in first 24 h, then daily until cellulitis resolves.

29.5 Neurological Conditions

LLLT adds an outstanding new treatment modality to several notori-

ously difficult diseases to treat, such as dementia, intervertebral disk
disease, and peripheral neuropathies. Its ability to promote neural
tissue regeneration along with decreasing inflammation, pain, and
oxidative stress make it an ideal therapeutic option in neurological
diseases. It is particularly effective at treating intracranial inflam-
matory (GME) and degenerative diseases (dementia) compared to
conventional management. For disk disease, LLLT can spare many
dogs and cats surgical intervention or high dose of corticosteroids.

29.5.1 Intervertebral Disk Disease11

Target tissue: Spinal cord

Wavelength: 810
Power density: 0.01–5 W/cm2
Frequency: 10 Hz or CW
Ideal energy at TT: 1–4 J
Application points: Directly between dorsal spinous
processes of affected disks, and three spaces cranial and two
spaces caudal.
*Treat daily until improvement (usually 2–3 days), then
QOD until normal
*I frequently give a dexamethasone I.V. injection on day 1.

Target tissue: Paraspinal muscles

Wavelength: 810 or 904 superpulsed

Power density: 0.05–5 W/cm2
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534 Veterinary Low-Level Laser (Light) Therapy Applications for Companion Animals

Figure 29.3 810 nm, 4 × 500 mW multi-probe treating canine dementia.

Frequency: 2–50 Hz or CW
Ideal energy at TT: 3–6 J
Application points: Bilateral paraspinal epaxial muscles
same range as for spinal cord treatment.
*MFTPs can be treated with 6–10 of 810 at TT depth.

29.5.2 Dementia12, 13
Target tissue: Dura and deeper (1–3 cm) (Fig. 29.3)

Wavelength: 810
Frequency: 10 Hz
Ideal energy at TT: 1–3 J
Application Points: Transcranially on poll: 8–16 points on
right and left side of midline with cluster probe
*Treat every 2–3 days until clinically improved, then as
needed.
 July 6, 2016 17:35
Table 29.4 Neurological conditions

Condition Target Tissue λ Power Density Time Frequency Ideal Energy Application Comments
(TT) (nm) (W/cm2 ) (s) (Hz) @TT (J) Points
Intervertebral Spinal cord 810 0.01–5 W/cm2 20–120 10 or CW 1–4 Dorsally over Also treat
disk disease IVD +3 spaces intervertebral
cranially and 2 foramen

PSP Book - 9in x 6in

spaces caudally.
Paraspinal 810 or 904 0.05–5 W/cm2 30–90 2–50 Hz or CW 3–6 Bilateral
muscles paraspinal
muscles same
range as IVDs
Dementia Dura and 810 0.05–2 W/cm2 30–120 10 1–3 Dorsally over Treat every
deeper right and left 2–3 days until
(1–3 cm) hemisphere improved, then

Neurological Conditions
as needed

535

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536 Veterinary Low-Level Laser (Light) Therapy Applications for Companion Animals

29.6 Renal Conditions

The kidneys and urinary bladder are very accessible to light therapy
because of their close proximity to the skin surface, usually only
1–3 cm depending on the size of the animal. Idiopathic feline lower
urinary tract disease is an inflammatory condition affecting the
mucosal wall of the urinary bladder. Sensitization of c-fibers is
believed to play a role in the recurring nature of this common
disease. Treatment with LLLT involves an anti-inflammatory proto-
col for the urinary bladder and a neural blockade at the DRG for
the hypogastraic nerve, pudendal nerve, and pelvic nerve. Renal
failure is the number one chronic organ failure occurring in domestic
dogs and cats. Light therapy offers a palliative treatment that
reduces azotemia, helps combat anemia, and stimulates appetite and
energy levels. Treating the bone marrow of long bones has been
shown to increase circulating mesenchymal stem cells and stimulate
erythropoiesis.

29.6.1 Feline Lower Urinary Tract Disease14

Target Tissue: Urinary bladder wall

Wavelength: 810 or 904 superpulsed

Frequency: 1000 or CW
Ideal energy at TT: 2–6 J
Application points: Caudal ventral abdominal wall, 1–2
points with cluster probe
*Treat 2 days in a row, then every other day until resolved.

Target tissue: DRG for nerves innervating urinary bladder

Wavelength: 810
Frequency: 10 Hz or CW
Ideal energy at TT: 4–8 J
Application points: L2, L3, L4 (hypogastraic n./sympa-
thetic); S1, S2, S3 (Pelvic n. /parasympathetic and Pudendal
n./Somatic)
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Other Internal Organs 537

29.6.2 Chronic Renal Failure15

Target tissue: Kidneys

Wavelength: 810 or 904 superpulsed

Frequency: 2–50 Hz or CW
Ideal energy at TT: 2–4 J
Application points: 2 points with cluster probe on skin over
right and left kidney
*Treat twice a week for 2 weeks, once a week for two weeks,
then every other week.

Target tissue: Bone marrow in long bones16

Wavelength: 810
Frequency: 2–50 Hz or CW
Ideal energy at TT: 1 J
Application points: 2–3 points to center of humerus and
femur
*Treat each time kidneys are treated.

29.7 Other Internal Organs

Other intra-abdominal organs as well as thoracic organs can be

treated successfully with LLLT. Pancreatitis is well suited for light
therapy as the therapeutic goals line up directly with LLLT’s
strengths, i.e., reduce inflammation, reduce oxidative stress, reduce
pain, increase blood flow and angiogenesis, decrease apoptosis, and
speed healing of injured cells.17 In fact, dogs with acute pancreatitis
usually go home eating after the second day of hospitalization
with light therapy as opposed to 4–5 days without LLLT. Prostatitis
and benign prostatic hyperplasia also respond rapidly to LLLT.
Inflammatory bowel disease can be helped greatly by light therapy,
reducing or eliminating the need for corticosteroids. The heart
muscle in end stage heart failure can have its contractility and
oxygenation improved by LLLT, prolonging the quality and length
of these patients that are maxed out on pharmaceutical treatment
options.18, 19 Chronic bronchitis in dogs and asthma in cats will both
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538 Veterinary Low-Level Laser (Light) Therapy Applications for Companion Animals

benefit from light therapy and can have their dose of corticosteroids
reduced or eliminated.20−22

29.8 Other Miscellaneous Applications for Light Therapy

29.8.1 Dental Applications

Red or infrared light can be used to speed up healing following oral
surgery as well as assist in controlling post-operative pain. This
includes teeth extractions, removal of oral tumors, and soft palate
reduction.23 Four joules of 660 nm is typically all that is necessary.
Feline gingivostomatitis is a common and frustrating disease to
treat. It will temporarily have some relief from light therapy, but
rarely can it be controlled without very frequent LLLT treatments.
As one might imagine, this is not an easily accomplished task in the
domestic cat.

29.8.2 Ophthalmic Disorders

Corneal ulcers and anterior uveitis can benefit from LED treatments.
Red or near-infrared wavelengths with power densities less than
150 mW/cm2 and a 2–4 J dose help stubborn corneal ulcers heal
rapidly.24 Doses of 4–6 J to the uvea can reduce the inflammation and
pain associated with anterior uveitis. Treating the sub-mandibular
lymph nodes with 4 J of infrared (810) can augment the direct
treatment.

29.8.3 Neoplasia
Does photobiomodualtion of the immune system help hold neopla-
sia in check or does it fuel an already out of control cellular process?
It appears to do both depending on the tumor type. Certainly LLLT
has a role in palliative veterinary cancer patients where owners
opt not to use chemotherapy, radiation, or surgery. In these cases,
the tumors are frequently reduced in size, progression slowed,
pain controlled, and appetite stimulated. I have treated a number
of dogs with transitional carcinoma of the urinary bladder who
have faired better than with traditional treatment of piroxicam and
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References 539

mitoxantrone. These dogs have all lived longer than a year with no
negative side effects and good quality of life. On the other hand,
I have treated dogs with osteosarcoma that have seemed to get
rapidly worse after treatment. Much work remains to understand
which tumors would be palliated by LLLT and which will be made
worse.25

29.9 Conclusion

With sound knowledge of the principles involved in LLLT, there

are few limitations for which diseases may benefit from this
treatment modality. Veterinarians, by nature of not having as many
legal impediments as human medicine and also being used to a
cash business model, can forge the clinical frontiers of LLLT. In
veterinary medicine, clinical applications often get out in front of the
research and RCTs that are critical for establishing credibility of any
treatment modality. It is imperative that veterinary academia rush
forward in filling this void as more and more veterinarians add light
therapy to their practices.

References

1. Huang YY, Chen AC, Carroll JD, and Hamblin MR. Biphasic dose response
in low level light therapy. Dose-Response, 2009; 7(4): 358–383.
2. Chow R, Armati P, Laasko EL, Bjordal JM, and Baxter GD. Inhibitory
effects of laser irradiation on peripheral mammalian nerves and
relevance to analgesic effects. Photomed Laser Surg, 2011; 29(6): 365–
381.
3. Simunovic Z. Low level laser therapy with trigger point technique: A
clinical study on 243 patients. J Clin Laser Med Surg, 1996;14(4): 163–
167.
4. Alghadir A, Omar MT, Al-Askar AB, and Al-Muteri NK. Effect of low level
laser therapy in patients with chronic knee osteoarthritis: A single-
blinded randomized clinical study. Lasers Med Sci, 2014; 29(2): 749–
755.
5. Alves AC, Vieira R, Leal-Junior E, dosSantos S, Ligeiro AP, Albertini
R, Junior L, and de Carvahlho P. Effect of low level laser therapy on
July 6, 2016 17:35 PSP Book - 9in x 6in 29-Hamblin-c29

540 Veterinary Low-Level Laser (Light) Therapy Applications for Companion Animals

the expression of inflammatory mediators and on neutrophils and

macrophages in acute joint inflammation. Arthritis Res Ther, 2013;
15(5): R16.
6. Son J, Kim YB, Ge Z, Choi SH, and Kim G. Bone healing effects of diode
laser (808 nm) on a rat tibial fracture model. Res Vet Sci, 2010; 88(1):
159–165.
7. Medrado AP, Soares AP, Santos ET, Reis SR, and Andrade ZA. Influence
of laser photbiomodulation upon connective tissue remodeling during
wound healing. J Photochem Photobiol, 2008; 92(3): 144–152.
8. Medrado AP, Pugliese LS, Reis SR, and Andrade ZA. Influence of low
level laser therapy on wound healing and its biological action upon
myofibroblasts. Lasers Surg Med, 2003; 32(3): 239–244.
9. Avci P, Gupta A, Sadasivam M, Vecchio D, Pam Z, and Hamblin MR. Low
level laser (light) therapy (LLLT) in skin: Stimulating, healing, restoring.
Semin Cutan Med Surg, 2013; 32(1): 41–52.
10. Posten W, Wrone DA, Dover JS, Arndt KA, Silapunt S, and Alam M.
Low level laser therapy for wound healing: Mechanism and efficiency.
Dematol Surg, 2005; 31: 334–340.
11. Draper WE, Schubert TA, Clemmons RM, and Miles SA. Low level laser
therapy reduces time to ambulation in dogs after hemilaminectomy: A
preliminary study. J Small Anim Pract, 2012; 53(8): 465–469.
12. Huang YY, Gupta A, Vecchio D, de Arce VJ, Huang SF, Xuan W, and
Hamblin MR. Transcranial low level laser therapy for traumatic brain
injury. J Biophotonics, 2012; 5(11–12): 827–837.
13. Oron A, Oron U, Streeter J, de Taboada L, Alexandrovich A, Trembovler
V, and Shohami E. Low level laser therapy applied transcranially to
mice following traumatic brain injury significantly reduces long-term
neurological deficits. J Neurotrauma, 2007; 24(4): 651–656.
14. Lorenzini L, Giuliani A, Giardino L, and Calza L. Laser acupuncture for
acute inflammatory, visceral and neuropathic pain relief. Res Vet Sci,
2010; 88(1): 159–165.
15. Ucero AC, Sabban B, Benito-Martin A, Carrasco S, Joeken S, and Ortiz A.
Laser therapy in metabolic syndrome-related kidney injury. Photochem
Photobiol, 2013; 89(4): 953–960.
16. Tuby H, Maltz L, and Oron U. Induction of autologous mesenchymal stem
cells in bone marrow by low level laser therapy has profound beneficial
effects in the infarcted rat heart. Lasers Surg Med, 2011; 43(5): 401–409.
17. Burduli NM and Gutnova SK. Types of microcirculation and laser
therapy in chronic pancreatitis. Klin Med (Mosk), 2009; 87(8): 56–61.
July 6, 2016 17:35 PSP Book - 9in x 6in 29-Hamblin-c29

References 541

18. Hentschke VS, Jaenisch RB, Schmeining LA, Cavinato PR, Xavier LL, and
Dal Lago P. Low level laser therapy improves the inflammatory profile of
rats with heart failure. Laser Med Surg, 2013; 28(3): 1007–1016.
19. Vasilev AP, Streltsova NN, and Senatorov IuN. Laser irradiation in the
treatment of ischemic heart disease. Vorp Kurotol Fizoter Lech Fiz Kuly,
2001; 6: 10–13.
20. Wang XY, Ma WJ, Lui CS, and Li YX. Effect of low level laser therapy on
allergic asthma in rats. Lasers Med Sci, 2013; 29(3): 1043–1050.
21. Silva VR, Marcondes P, Silva M, Villaverde AB, Castro-Faria-Neto HC,
Vieira RP, Aimnire F, and de Oliveira AP. Low level laser inhibits
bronchoconstriction, Th2 inflammation and airway remodeling in
allergic asthma. Respir Physiol Neurobiiol, 2014; (194): 37–48.
22. Aimbire F, Bjordal JM, Iversen VV, Albertini R, Frigo L, Pacheco MT,
Castro-Faria-Neto HC, Chavantes MC, Labat RM, and Lopes-Martins RA.
Low level laser therapy partially restores tracheal muscle relaxation in
rats with tumor necrosis factor alpha-mediated smooth airway muscle
dysfunction. Lasers Med Surg, 2006; 38(8): 773–778.
23. Ross G and Ross A. Low level lasers in dentistry. Gen Dent, 2008; 56(7):
629–634.
24. Pellegrino F and Carrasco MA. Argon laser phototherapy in the
treatment of refractory fungal keratitis. Cornea, 2013; 32(1): 95–97.
25. Lui TC-Y, Zhang J, and Li XE. The balance between normal and tumor
tissues in phototherapy of tissue harboring cancer. Photo Med Surg,
31(3): 93–94.
July 6, 2016 17:35 PSP Book - 9in x 6in 30-Hamblin-c30

Chapter 30

Emergence of Low-Level Laser (Light)

Therapy in Clinical Veterinary Practice

Ronald E. Hirschberg
Brockton Animal Hospital, 386 Belmont Street, Brockton, MA 02301, USA
reh1006@gmail.com

Arguably the first recorded use of low-level laser (or light) therapy
(LLLT) in veterinary medicine was in 1967 in Budapest, Hungary,
when Endre Meister irradiated the dermis of mice to assess whether
LLLT would lead to the development of cancer [1]. Though his
attempts were unsuccessful in proving oncogenic capability of LLLT,
he did note that the shaven hair on the dorsum of treated mice grew
back more rapidly than that on the untreated mice. Thus, the clinical
use of LLLT in veterinary medicine was born. Nearly five decades
later, the use of photobiomodulation in clinical veterinary practice is
expanding at a rapid rate.
Though multiple references can be found regarding some early
work in equine medicine in the late 1980s, the primary focus was
equine lameness secondary to injury. In the post-millennium decade,
the use of LLLT in veterinary practice has grown dramatically as
more manufacturers have found a niche in the area of companion
animal practice.

Handbook of Low-Level Laser Therapy

Edited by Michael R. Hamblin, Marcelo Victor Pires de Sousa, and Tanupriya Agrawal
Copyright c 2017 Pan Stanford Publishing Pte. Ltd.
ISBN 978-981-4669-60-3 (Hardcover), 978-981-4669-61-0 (eBook)
www.panstanford.com
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544 Emergence of Low-Level Laser (Light) Therapy in Clinical Veterinary Practice

30.1 Introduction: Factors Influencing Adaptation of LLLT

to Clinical Practice

Multiple factors have led to the recent boom in popularity of pho-

tomedicine in clinical veterinary practice. As technology advances,
the cost and size of the units have declined dramatically. Combined
with increased portability, safety, and ease of operation, LLLT units
have found their place in an increasing number of clinical practices.
In addition to the physical and logistical changes that have led
to this increased usage of light therapy in practice, simplification of
application has enhanced its use. In many practices, the responsi-
bility of providing light therapy has been delegated to a veterinary
technician or other trained staff member. This has occurred since
manufacturers have created “fail safe” protocols in which many
units have preprogrammed exposure parameters that limit, in most
circumstances, the capability of injuring a patient. Although these
protocols may not be the most effective for every range of condition,
they reduce the requirement that a practitioner be well versed in
the calculation of various treatment parameters. Whether this is
positive or negative in the field of photobiomodulation is debatable.
However, the manufacturers of Class IV lasers are well familiar with
the incidents of burning in some patients and have taken steps to
reduce that likelihood. By not having the treatment probe in direct
skin contact or continually scanning it over the treatment area,
one can reduce the injury possibility. However, this also makes the
accurate calculation of the dosage nearly impossible.
LLLT continues to surface in the literature as an effective
veterinary treatment in a multitude of conditions. As reports of
efficacy become more common, the clinical practitioner feels more
comfortable adding this modality to the treatment arsenal. In addi-
tion, peer pressure creates urgency on the part of the veterinarian
to offer at least a similar level of care as his or her colleague. As
many practices have seen a decline in client visits, the practitioner
looks for other new means of income generation within the hospital
setting. LLLT can be a potent driver of practice profits as it offers
a “novel approach” for a variety of conditions that previously have
eluded effective solutions. Though pharmaceutical treatments have
been offered to treat many of these ailments, often they only reduce
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Introduction 545

the symptoms while being accompanied by potentially serious side

effects. Light therapy, however, allows for symptomatic relief while
accelerating healing without fear of side effects. Medical consumers,
who are often aware of pharmaceutical risks, ask for “natural” cures
and treatments in increasing frequency. LLLT provides this solution
while branding a practice as one that continually examines methods
to provide the best alternative for the patient. A practice that offers
LLLT is seen as one that embraces high technology while existing
on the cutting edge of new treatment alternatives. Most veterinary
clients find this a very appealing approach to patient care.

30.1.1 Clinical Applications

As our knowledge has increased regarding the mechanism of LLLT,
likewise clinical applications have expanded. We know that lights in
the red and near-infrared spectrum are effective in exerting action
on the cytochrome c oxidase system. We have also learned that red
light, although very effective in exerting its actions on mitochondria,
tends to be quickly absorbed as it enters the superficial tissue layers
[2]. Near-infrared light with a wavelength in the 810 nm range
strongly affects nitric oxide uncoupling and is capable of deeper
penetration [3]. Interestingly, it also appears that the effectiveness
of light once it enters the mitochondria is independent of whether
the light is coherent (laser) or noncoherent (LED). If we are looking
to use light therapy to reduce inflammation and enhance healing, it
can be LED generated or laser generated depending on the depth
of the target we wish to treat. Laser light is capable of deeper
penetration, while superficial tissues are effectively treated with
LED light. However, when we are seeking pain relief by reducing
nocioceptive input and slowing C-fiber transmission, coherent light
in a continuous wave format is the treatment of choice.

30.1.1.1 Soft tissue, wound healing, and ophthalmological

applications
The initial clinical application of light therapy in veterinary patients
was to relieve inflammation in joints and other associated soft
tissue structures such as tendons and ligaments. In companion
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546 Emergence of Low-Level Laser (Light) Therapy in Clinical Veterinary Practice

animal medicine, LLLT was utilized to treat chronic osteoarthritis

and its associated pain. Wound healing became another favorite
in the light therapy treatment arena as the stimulatory effect of
photons became clearer [4]. The practitioner’s attention next turned
to non-healing wounds and superficial hot spots, skin lesions that
occur from a variety of causes yet persist as the patient tends
toward self-trauma. While these initial applications still comprise
the substantial part of a veterinary laser practitioner’s focus, the
current and future applications grow rapidly. Since most animal
diseases involve inflammation of one or more structures, there
appears to be no limit to the potential use of LLLT as a potent
anti-inflammatory tool. This coupled with the above mentioned
knowledge of LED as an equally effective anti-inflammatory source
allows for novel application to such laser-sensitive structures as the
eye. In ophthalmology, treatment for anterior uveitis and keratitis
has become somewhat common. Perhaps the single most important
ophthalmogical application in our practice is in the area of corneal
ulcers. Although simple corneal ulcers generally are not problematic,
descemetocoeles and indolent or Boxer ulcers present a treatment
challenge. Though not a substitute for currently accepted treatment
for this condition, the biostimulatory effect of LLLT in speeding
up fibroblastic activity results in a far shorter duration. In several
instances, we had patients with indolent ulcers that were previously
treated without light therapy and who subsequently developed a
similar ulcer in the same or contralateral eye. In the initial instance,
the time to cure without light therapy was several weeks, utilizing
appropriate surgical and medical procedures. LLLT shortened the
course of the disease significantly in the recurrent cases from nearly
6 weeks in one particular case to 10 days in the subsequent ulcer.
Though traditional medications were prescribed in all cases, light
therapy clearly is capable of significantly shortening the length of
time required to heal these ulcers.

30.1.1.2 Spinal cord disease

Besides ophthalmological applications, LLLT is emerging as a
favored treatment in a multiple of disease conditions that previously
had minimal options available. Degenerative disc disease with disc
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Introduction 547

herniation currently has few reliable treatments other than surgical

decompression. Medical anti-inflammatory and analgesic therapy
is often used with minimal benefit, yet frequent gastrointestinal
side effects. Light therapy, however, can exert its influence by
reducing inflammation and swelling of the traumatized and inflamed
spinal cord. We can further mitigate the pain with the use of
continuous wave laser application to spinal nerve roots (inhibition)
and use biostimulation to speed up healing of weakened or
damaged connective tissue structures of the vertebral bodies. Light
treatment of degenerative disc disease has become one of the most
common LLLT applications in our practice. These patients can often
achieve a degree of comfort and mobility within a matter of hours
that previously had taken days to accomplish with medications
(Fig. 30.1). In the only published veterinary-specific study to date,
Dr. Tom Shubert of the University of Florida found that LLLT
significantly shortened the interval required for post-laminectomy
canine patients to walk without support. Specifically, dogs that had

Figure 30.1 MRI reveals a severely herniated disc at L3-L4. The patient
(6-year-old Staffordshire terrier) presented with severe back pain and
bilateral hind leg paresis. Improvement was noted within 72 h of two LLLT
treatments, and the dog was clinically normal after three light therapy
treatments over 7 days. Images adapted from patient medical record and
printed with permission of Brockton Animal Hospital LLC.
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548 Emergence of Low-Level Laser (Light) Therapy in Clinical Veterinary Practice

surgery but no post-surgical light treatments required 10–14 days

before being capable of taking a few steps. Patients who received
LLLT post-surgically shortened that interval to 3–4 days [5].
Additionally, in the area of spinal cord and central nervous
system (CNS) disease, several other conditions have shown promise
in their response to light therapy. Brain and spinal trauma, a
common condition in feline medicine, responds well to LLLT
particularly when treatment is instituted in the course of the disease.
Veterinary patients often present with unclear history yet symptoms
that are suggestive of spinal cord disease. The pathology may
have one of many etiologies, yet the effect is usually inflammatory
in nature. Meningitis, syringomyelia, spondylosis, granulomatous
meningoencephalitis (GME), and cervical vertebral instability are a
few of the CNS diseases that have been treated successfully with
light therapy. Unfortunately, as of this writing there have been few
randomly controlled blinded studies. We as practitioners are limited
by the current treatment information available, often anecdotal in
nature. Encouraging further study by veterinary teaching facilities
would greatly enhance the knowledge base in treating many of these
conditions.

30.1.1.3 Orthopedic conditions

Orthopedic applications are one of the most common areas of
utilization of LLLT. Since cranial cruciate ligament rupture is often
preceded by earlier bouts of trauma to this structure, the condition
is thought to be a disease with an insidious course. It is suspected
that the application of light therapy after early strains or partial
tears may promote healing and thereby prevent subsequent total
tear. When the catastrophic event occurs and surgery is required,
recovery (i.e., time to weight bearing) can be reduced dramatically
by pre- and post-surgical application of LLLT. In addition, other
orthopedic conditions such as elbow and hip dysplasia, biceps
brachii tendonitis, soft tissue trauma, and fracture repair have all
shown to benefit from the application of light (Fig. 30.2). The
anti-inflammatory positives of light therapy can be utilized to
decrease discomfort, while the stimulatory effect accelerates bone
healing, allowing for earlier weight bearing. Accelerated migration
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Introduction 549

Figure 30.2 Top left: Original lateral view. Top right: Immediately post-
op. Bottom left: 17 days post-op. Bottom right: Eight weeks post-op; pin is
removed and complete union is noted. Clinically normal. Images adapted
from patient medical record and printed with permission of Brockton
Animal Hospital LLC.

of fibroblasts and osteoblasts, as well as increase in osteoclastic

activity, has been shown to occur after the absorption of light [6].
Callous formation is sped up significantly by LLLT when applied
post-surgically.

30.1.1.4 Dermatology and light therapy

Dermatologic problems are one of the most common reasons that
owners present their pets to veterinarians. Since most of the
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550 Emergence of Low-Level Laser (Light) Therapy in Clinical Veterinary Practice

dermatologic conditions of companion animals are a superficial

inflammatory process, it is easy to deliver light to target tissue.
The discomfort associated with otitis externa is greatly mitigated
by the application of LLLT a few times after diagnosis and medical
treatment of the condition. Hot spot, a self-traumatic lesion that
occurs secondary to a variety of etiologies, is one of the most
frustrating and upsetting conditions that an owner can witness.
Application of light several times during the first few days of medical
treatment will significantly speed up tissue healing (biostimulation)
while reducing inflammation and discomfort. Lick granulomas, a
chronic, relentless disease evidenced by thickened, pigmented, and
proliferative nodules usually on the distal part of the limbs of
canines, is also self-traumatic in nature. Etiology is often behavioral
but once initiated becomes a cyclic habit. Culture/sensitivity
yielding the appropriate antibiotics for the secondary infection is of
some benefit. When LLLT is added to the regimen, resolution occurs
much more quickly.
Allergic skin disease includes inhalant dermatitis (atopy), con-
tact dermatitis, flea bite allergy as well as food allergy. Though
this condition tends to be multifocal with frequently widespread
signs and symptoms, it is primarily immune in nature. Recently,
there have been reports of improvement noted after phototherapy
is administered to multiple regional lymph nodes. Hot spots, as
mentioned above, have shown favorable response to LLLT and are
known in part to be caused by allergic antigenic initiation. With
more knowledge and experience in the light therapy field, the area
of allergic skin disease is surely one that will gain critical attention.
If the early signs of effectiveness are substantiated, this is sure to
be one of the most frequently LLLT-treated diseases in companion
animal practice.

30.1.1.5 LLLT and metabolic disease

Perhaps currently one of the most controversial questions that
needs answering is can we achieve adequate penetration with
appropriate light doses to internal organs? Pancreatitis, cholangio-
hepatitis, inflammatory bowel disease, and chronic kidney disease
are frequently diagnosed in the feline patient. There are LLLT
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Introduction 551

veterinary practitioners who have related anecdotal reports of

seemingly miraculous recovery in patients with these often fatal
diseases. Though no randomly controlled blinded studies have been
initiated as of this date, these claims raise consciousness of light
therapy. Since the basis of most metabolic disease in companion
animals involves primary or secondary inflammation, it encourages
the likelihood of finding benefit from light therapy.
Although obesity has become a major problem in our feline
population, the kidneys remain one of the most superficial and
accessible organs to irradiate. Chronic renal disease is often sequela
to an acute renal event resulting in injury. An inflammatory
process subsequently occurs and promotes further necrosis to
the parenchyma of the renal cortex. As nephrons are destroyed,
kidney function decreases. Kidneys with their tremendous reserve
capacity compensate until nearly 75% of functional tissue has been
destroyed. At this point some degree of renal failure will occur. Logic
dictates that irradiation of the kidneys would reduce inflammation
and prevent further damage. In several cases within our practice,
long-term light therapy (greater than 6 months) appears to have
slowed progression of illness. Patients with chronic renal disease
frequently succumb within a few days to a few months. We have now
managed several light therapy patients that have survived for longer
than a year and continue to have a good quality of life. One particular
patient who receives biweekly LLLT treatments at home has seen a
significant decrease in serum creatinine levels as well as increased
activity and engagement with the family.
Other practitioners have reported similar light therapy benefit in
hepatic, gastrointestinal, and pancreatic disease. Clearly, this is an
area of interest that has tremendous potential. It is incumbent on
the academic veterinary community to recognize the importance of
this science and engage in much needed high-quality randomized
research.

30.1.2 Treatment Parameters

Though there is far from uniform agreement of the precise “best”
treatment protocols for any and all conditions, there are basic
parameters that have shown promise. In laboratory studies as
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552 Emergence of Low-Level Laser (Light) Therapy in Clinical Veterinary Practice

well as blinded trials in human medicine, wavelength, frequency

of pulsation, irradiance, time and treatment frequency, and interval
are all critical in accurately describing the specific dosage of any
application [7]. Though not officially recognized as a treatment
parameter, application pressure is also of essential importance.
Since most veterinary treatments rely primarily on LED therapy, it
is important to apply adequate pressure in order to gain sufficient
doses at the target tissue [8].
Though it is beyond the scope of this chapter to completely
discuss all the aspects of these parameters, there are a few facts
that should be noted. For biostimulation and inhibition, red and
near-infrared light (600–860 nm) is the most effective due to its
penetration and absorption by cells [9]. Though red light is highly
stimulatory, it achieves only minimal penetration due to absorption
by hemoglobin. The near-infrared, however, biostimulates nearly as
well as red but is much less likely to get absorbed until it reaches
some depth. Of course, for superficial dermatologic lesions, red
wavelength is more than adequate as the target is superficial. For
deeper target tissues, near-infrared is more practical, and if these
targets are deeper than 1–1.5 cm, then coherent laser (or light)
needs to be utilized [10].
The discussion of pulsed versus continuous wave is one that
has taken place over a period of several years. Pulsating light
appears more beneficial for stimulation than continuous wave
[11]. Continuous wave appears to be more effective when desiring
inhibition for analgesia [12]. However, one must consider frequency
(speed of pulsation) as well as duty cycle (percentage of time
that light source is on during one pulsation) when discussing the
parameters of light therapy. It is likely that future research will
yield different parameters for certain tissues, such as neural as
compared to connective, muscle, dermal, and osseous targets. As
the use of LLLT expands to include treatment of internal organs,
these parameters will need to be refined to maximize the benefit of
therapy.
Irradiance or power density speaks to the uniqueness of light
therapy and its observed biphasic dose response. The power
density, as expressed in milliwatts per square centimeter, can be
manipulated to stimulate as well as inhibit cellular activity. When
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Introduction 553

applied in the stimulatory range (5–100 mW/cm2 ), we accelerate

healing through a variety of pathways. Usage of a greater irradiance
(300–1730 mW/cm2 ) results in decreasing the rate of transmission
in α, δ, and C-fibers as well as reducing nocioceptive input, hence
promoting analgesia.
The duration or time of each particular probe placement needs
to be sufficient to achieve the energy density required for either
stimulation (healing) or inhibition (analgesia) as prescribed for
the particular LLLT unit. Energy is merely the power density or
irradiance multiplied by the time in seconds. Optimum energy
density is thought to be in the range of 2–3 J/cm2 ) [12]. Though
the above may seem a bit complicated to the new laser therapist,
in many cases these parameters are preset in the treatment unit
and guidelines are provided by the manufacturer. However, a
thorough understanding enables a practitioner to become more
successful by coordinating clinical judgment, response to treatment,
and knowledge of the science of light therapy. Only then can
the veterinarian avail himself or herself of the vast treatment
opportunities that LLLT provides to the patient.

30.1.3 Therapeutic Outline

Treatment protocols vary by time and placement of the probes
depending on the condition, severity, and location of the disease
being treated. In nearly all cases, therapy begins by the treatment
of the regional lymph nodes. Though the pathway is unclear, there
seems to be immune modification by exposure of the lymph nodes
to the correct parameters of light for biostimulation. The treatment
next continues to direct exposure of the target tissue with LED light
if sufficient penetration can be achieved. In the event that the target
is deeper than the LED penetration, then a laser source may be
used to sufficiently treat the target. To briefly recall, it is essential
to understand that penetration is dependent on the wavelength and
the power density of the source, not merely whether the source
is coherent (laser) versus noncoherent (LED) or has a high total
output. Once the target tissue is sufficiently treated, the presence of
edema should be addressed. If, for example, one is treating a limb,
then by continuing irradiation of the venous return pathway of the
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554 Emergence of Low-Level Laser (Light) Therapy in Clinical Veterinary Practice

affected limb, he or she can considerably reduce swelling. Trigger

points may then be treated if desired. Last, a “neural blockade” to
control pain is initiated by utilizing inhibitory levels of light (see
above range) over the spinal segments that innervate the target
area. For example, if one is treating stifle osteoarthritis, a neural
blockade would be effected from the L1-L2 (space between the first
and second lumbar vertebrae), continuing along each space until
reaching L6-L7.
The frequency of treatment is dictated by the severity and the
specifics of the condition. Generally, the “loading dose” for light
therapy requires two to three treatments per week for the first 2
weeks with subsequent sessions being reduced in frequency “to
effect.” For chronic conditions requiring long-term maintenance,
it becomes incumbent on the client to assess the effect of time
as the intervals are prolonged. The treatment schedule becomes
a partnership between the practitioner and the client. Prolonged
intervals stretching to once every 4–6 weeks in frequency are often
enough to achieve ongoing management of a chronic condition.

30.1.4 Safety and Contraindications

When using a laser light source, wearing protective glasses is
imperative for all those who are present in the treatment area. One
should never stare directly into a laser light source. When using a
Class III laser unit, the probe may be held stationery and in contact
with a target tissue without the risk of burning. Occasionally, a
patient with a solid black coat may show some mild discomfort when
treated with a Class III laser source. Wetting the fur with water will
reduce this sensation.
If a Class IV laser unit is utilized for therapy, it is mandatory
to ensure that the light source never directly contacts the tissue
surface. Additionally, the laser probe needs to be continually in
motion to “scan” the target area. Though dose calculation becomes
problematic in this scenario, it is essential to avoid burning the
patient.
LED light requires no special protection for any target tissue
and, as outlined above, may even be utilized to treat certain ocular
conditions.
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Introduction 555

The effect of LLLT on neoplasia is unknown at this point in

time. Understanding that light therapy enhances circulation and
accelerates some growth factors, it is logical to avoid use in any area
where neoplasia is present or suspected.

30.1.5 Clinical and Practical Benefits of LLLT

For the clinical veterinary practice, the benefit of light therapy
extends far beyond its effectiveness in treating a wide variety
of illnesses. It satisfies many other requirements essential to
successfully operate a veterinary facility in the modern world of
the informed medical consumer. At a time when medical treatments
have continually distanced both the practitioner and the client from
the hands on interaction necessary to answer the complex question
“What is best for me and my pet?” LLLT steps up.
First and foremost, light therapy offers a safe and effective
treatment for many of the most common diseases that we see
as veterinarians. Musculoskeletal disorders such as osteoarthritis,
ligament and tendon injuries, degenerative disc disease, and
soft tissue traumas of all types previously have utilized non-
steroidal anti-inflammatory drugs (NSAIDs) as the gold standard of
treatment. Side effects are frequent and, if clients are casual, often
result in serious gastrointestinal side effects. In some instances,
these complications can be life threatening. When NSAIDs are
used chronically, they require periodic blood monitoring and
may, over long term, cause degradation of joint surfaces. In the
current environment, clients are frequently bombarded with media
advertising outlining the effectiveness of certain pharmaceuticals
but then overwhelming us with the narrative of danger. For owners
whose pet companions are a central and critical part of their life,
this pharmaceutical approach is unacceptable. LLLT offers them an
extraordinary alternative.
LLLT is “targeted” modality. By this we mean it is essential
that the practitioner becomes adept at identifying and locating the
specific site of a problem. We cannot use light therapy as a “shotgun”
treatment such as is often done in practice with antibiotics, steroids,
and other pharmaceuticals. Using a targeted therapy such as LLLT,
we increase our understanding of the pathogenesis of the disease.
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556 Emergence of Low-Level Laser (Light) Therapy in Clinical Veterinary Practice

We are, thus, able to relieve symptoms for the patient while

providing a greater comfort for the client as they know we have more
specifically identified the origin of the problem.
Furthermore, this enhanced understanding of the disease
process enables us to involve the client in the wellness of their
pet to a much greater degree. Since the life expectancy of animal
companions is considerably shorter than that of humans, the speed
with which the disease process progresses is greatly accelerated.
This requires the client to be much more observant in partnering
with the veterinary health professional in providing input as to
the status of the patient. Often the client makes the decision as to
how often the pet requires treatment in managing the disease. By
placing a certain responsibility on the client, we are encouraging
“ownership” of the treatment plan and decisions. When the pet
owner is asked to step into this role, treatment compliance increases
dramatically. The client is much more likely to attend to scheduled
appointments as they have an understanding that prolonging
therapy intervals may result in relapse of the patient.
The time spent in the examination room treating the patient
is an invaluable educational opportunity. Whether the veterinarian
or trained veterinary technician attends to the patient during the
treatment, the 10 min or so during therapy invites medical questions
from the client. It also provides time for the practitioner to follow
up on services that may have been suggested in the past yet not
scheduled. This intimate session with client, patient, and doctor
or technician becomes an opportunity to connect with the client,
answer questions, or address other areas of concern. The physically
“hands on” approach to patient care is comforting to the owner in
that they observe the doctor or nurse touching the pet to a much
greater degree than is usually witnessed. The pet is generally very
receptive to the LLLT and in most instances becomes so conditioned
to the sense of comfort and well-being that (suggested to be a
result of endorphin release) they fall asleep. For the client, this
is reassuring seeing their companion so relaxed in the hands of a
practitioner. The whole experience is a great opportunity to build
client and patient rapport and hospital reputation.
Concluding, LLLT provides a highly effective yet minimally
invasive form of treatment for a variety of diseases that, in many
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References 557

instances, have no other alternative therapy. In providing this

modality to the patient, we can rapidly reduce symptoms, modify
the course of disease, and significantly reduce pain and suffering. As
the pet owner is present during the treatment process, light therapy
offers an opportunity to showcase our skill, caring, and scientific
knowledge. We involve the client in decision making, enhancing their
understanding of the disease process. We must not forget that all
of the above can be easily provided with a minimal investment in
technology without the fear of any harmful side effects.

30.1.6 Future of Photobiomodulation in Veterinary

Practice
So where will we go from here in the field of LLLT? As we gain
greater understanding of tissue response to given parameters of
light, it seems as if few conditions will elude exposure to this
therapy. Current work with metabolic and neurological disease has
been most encouraging. As inflammation is often “the root of all
evil” in the disease process, the universal use of light treatment in
nearly every disease can be imagined. Inflammatory liver disease in
felines, seizure disorders in canines, cognitive dysfunction, chronic
renal disease, ophthalmological disorders, traumatic brain injury,
and perhaps every type of orthopedic disorders known to medicine
will likely come under the exposure of light at some time in the near
future. It is now incumbent that our academic institutions answer
the challenge of LLLT. Randomized controlled trials, education of
students as well as continuing education for all veterinarians in the
field of light therapy must be the next logical step in bringing this
science and technology to the position it deserves in the practice of
veterinary medicine.

References

1. Mester, E., Szende, B., and Tota, J.G. (1967). Effect of laser on hair growth
of mice. Kiserl. Orvostud., 19, 628–631.
2. Karu, T.I. (1999). Primary and secondary mechanisms of action of
visible-to-near IR radiation on cells. J. Photochem. Photobiol. B: Biol., 49,
1–17.
July 6, 2016 17:35 PSP Book - 9in x 6in 30-Hamblin-c30

558 Emergence of Low-Level Laser (Light) Therapy in Clinical Veterinary Practice

3. Hudson, D.E., Hudson, D.O., Wininger, J.M., and Richardson, B.D. (2013).
Penetration of laser light at 808 and 980 nm in bovine tissue samples.
Photomed. Laser Surg., 31(4), 163–168.
4. Posten, W., Wrone, D.A., Dover, J.S., Arndt, K.A., Silapunt, S., and Alam,
M. (2005). Low-level laser therapy for wound healing: Mechanism and
efficiency. Dermatol. Surg., 31, 334–340.
5. Draper, W.E., Schubert, T.A., Clemmons, R.M., and Miles, S.A. (2012).
Low-level laser therapy reduces time to ambulation in dogs after
hemilaminectomy: A preliminary study. J. Small. Anim. Pract., 53(8),
465–469.
6. Saygun, I., Nizam, N., Ural, A.U., Serdar, M.A., Avcu, F., and Tözüm, T.F.
(2012). Low-level laser irradiation affects the release of basic fibroblast
growth factor (bFGF), insulin-like growth factor-I (IGF-I), and receptor
of IGF-I (IGFBP3) from osteoblasts. Photomed. Laser Surg., 30(3), 149–
154. doi:10.1089/pho.2011.3079.
7. Jenkins. P.A., and Carroll, J.D. (2011). How to report low-level laser
therapy (LLLT)/photomedicine dose and beam parameters in clinical
and laboratory studies. Photomed. Laser Surg., 29(12), 785–787.
doi:10.1089/pho.2011.9895.
8. Carroll, J.D. (2011). 810 nm low level laser therapy penetration: The
effects of different beam profiles in reaching deep anatomical targets.
2011 Annual ASLMS Pre-Conference.
9. Karu, T.I., and Kolyakov, S.F. (2005). Exact action spectra for cellular
responses relevant to phototherapy. Photomed. Laser Surg., 23, 355–
361.
10. Cheong, W.F., Prahl, S.A., and Welch, A.J. (1990). A review of the optical
properties of biological tissues. IEEE J. Quantum Electron., 26, 2166–
2185.
11. Hashmi, J.T., Huang, Y.Y., Sharma, S.K., Kurup, D.B., De Taboada, L.,
Carroll, J.D., and Hamblin, M.R. (2010). Effect of pulsing in low-level light
therapy. Lasers Surg. Med., 42(6), 450–466.
12. Chow, R., Armati, P., Laakso, E.L., Bjordal, J.M., and Baxter, G.D. (2011).
Inhibitory effects of laser irradiation on peripheral mammalian nerves
and relevance to analgesic effects: A systematic review. Photomed. Laser
Surg., 29(6), 365–381.
July 12, 2016 11:36 PSP Book - 9in x 6in 31-Hamblin-c31

Chapter 31

Photomedicine for Exotic Animals:

A Case-Based Discussion

Narda G. Robinson
Department of Clinical Sciences, Colorado State University College of Veterinary
Medicine and Biomedical Sciences, 300 West Drake Road, Fort Collins, CO 80526, USA
narda.robinson@colostate.edu

31.1 Introduction

Veterinarians are recognizing the need to give more clinical focus on

analgesia and functional restoration in their patients. This extends to
exotic animals, i.e., species not ordinarily seen in day-to-day practice,
though a different set of challenges arise with this patient population
[1]. Nonetheless, many opportunities exist to help this subset of
the veterinary population recover from illness and trauma, reduce
discomfort, and heal from tissue damage [2].

31.2 Hurdles

Impediments to laser therapy include, first, the safe and timely

access to the patient, as in a captive wild animal recovering from

Handbook of Low-Level Laser Therapy

Edited by Michael R. Hamblin, Marcelo Victor Pires de Sousa, and Tanupriya Agrawal
Copyright c 2017 Pan Stanford Publishing Pte. Ltd.
ISBN 978-981-4669-60-3 (Hardcover), 978-981-4669-61-0 (eBook)
www.panstanford.com
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560 Photomedicine for Exotic Animals

injury Second, zoo and wild animals usually experience stress when
encountering humans or being handled by them. In these cases, the
photomedicine practitioner may require extra personnel to restrain
and/or transport the patient. Third, the veterinarian may only have
a presumptive diagnosis due to limited financial resources and/or
proximity to veterinary care. These considerations notwithstanding,
however, wild animals may face euthanasia if they cannot regain
fully functional status in a timely fashion, and domesticated exotics
may also have few other treatment options if other avenues have
already been exhausted and failed.
Another constraint of photomedicine for exotics comes from
the paucity of research in target species, which poses challenges
for the evidence-based photomedicine practitioner [3]. As such,
laser and light-emitting diode (LED) approaches for this population
demand extrapolating results from humans and experimental
animals along with learning how other clinicians have fared with
their treatment protocols. As long as practitioners follow standard
safety guidelines for both patient handling and device usage, the
risks and contraindications for photomedicine approaches resemble
those for domestic animals and humans. The main difference, after
accounting for size, color, and integumental variations, pertains to
eye protection, as standard laser goggles usually will not fit most
birds, dolphins, or elephants.

31.3 Clinical Applications

This section describes several problems typically addressed by

veterinarians who practice photomedicine on exotics, along with the
underlying rationale.

31.3.1 Traumatic Brain Injury

Research on low-level laser (light) therapy (LLLT) for traumatic
brain injury (TBI) has characterized its effects on the brain as
neuroprotective, anti-inflammatory, and reparative [4]. Wild birds
frequently suffer head trauma from collisions with windows and
automobiles (see Fig. 31.1).
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Clinical Applications 561

Figure 31.1 This juvenile Heermann’s gull collided with a motor vehicle on
a busy highway. The bird was picked up from the road, flailing upside down
on the asphalt. Upon inspection, the veterinarian noted blood at the medial
canthus of the eyes and at the nares. The bird was flailing about. Treatment
included corticosteroids and standard neuroprotective measures along with
1 W, 810 nm laser therapy to the skull, as shown, at 2.5 Hz for 1 min per
site. Four hours later, the gull flew away. (Photo and case report courtesy:
Jennifer Conrad, DVM).

Nearly two-thirds of raptors admitted to the Rocky Mountain

Raptor Program in Fort Collins, Colorado, were diagnosed with
physical trauma [5]; about 7% of injured raptors admitted to rescue
facilities have experienced head trauma [6], identified post-mortem
as intracranial hemorrhage [7].
Laser therapy may afford valuable treatment possibilities due
to its safety, tolerability, and noninvasive nature [8]. Ultimately,
more raptors may survive the injury and recover to the point of
being releasable [9]. However, questions remain about the ideal
settings for laser therapy in this population, as well as timing after
trauma; that is, will the risk of a recent intracranial hemorrhage
pose a contraindication for laser therapy to the head immediately
upon admission? Furthermore, what is the safe dose of light
for the avian brain? Should the entire brain receive treatment?
How do parameters concerning wavelength, power density, and
frequency settings shown to be ideal in experimental rodent
research extrapolate to avian species?
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562 Photomedicine for Exotic Animals

31.3.2 Spinal Cord Injury

A common application for laser therapy in small-animal practice is
spinal cord injury (SCI); a common presentation is paraparetic dog
suffering from thoracolumbar intervertebral disk disease (TL IVDD).
As with TBI, animals in the wild may experience traumatic events
that damage their spinal cord, as illustrated by the case in Fig. 31.2.
Compressive SCI causes both primary and secondary tissue
damage. The secondary injury phase occurs 1–2 days after injury
and leads to biochemically mediated neuronal death and spinal
cord inflammation [10]. The best clinical outcomes manifest when
treatments address both the primary, traumatic, and secondary, bio-
chemical, neuronal injury. One measures the degree of improvement
by monitoring improvements in proprioception, voluntary motor
movement, micturition control, and deep pain perception.
In veterinary medicine, slow or disappointing recoveries from
SCI may lead to euthanasia, whereas fuller, quicker neurologic
improvements dissuade clients from opting for euthanasia [11].
Incomplete recovery in dog or cat requires caregivers to manage
an incontinent paraplegic for months to years. This is not usually
an option for a wild, captive, or domesticated exotic animal. This
reinforces the need to integrate a safe and effective therapy such as
photomedicine into the SCI patient’s care.
Laser therapy mitigates neurotrauma through four mechanisms:
(1) prevention of degeneration of motor neurons, (2) higher
metabolism within nerve cells, (3) proliferation of astrocytes
and oligodendrocytes promoting myelinization of nerves, and (4)
increased axonal regeneration [12]. Even patients with long-term
peripheral nerve injury have experienced significant functional
restoration after having laser therapy applied to the damaged
site(s) [13]. Regaining ambulation requires muscle preservation as
well as neural tissue healing; laser biostimulation safely preserves
the physiologic status of denervated muscle tissue while nerve
regeneration is taking place [14].
Furthermore, laser therapy provides analgesia and facilitates
functional neurologic recovery. Low-level laser irradiation alleviates
acute and chronic pain at least in part by causing a reversible
blockade of fast axonal flow and mitochondrial transport along
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Clinical Applications 563

Figure 31.2 The tree squirrel shown here was about 6-month old. It was
seen being picked up by a crow and dropped from about 100 feet. Veterinary
assessment revealed a vertebral fracture at T4. Neurologic examination
revealed loss of deep pain sensation in the pelvic limbs and tail. It received
rehabilitation therapy that included laser treatment with a 1 W, 810 nm
applicator, as shown here, directed at the vertebral fracture for 1 min/cm2
and 2.5 Hz along the thoracic spine. The squirrel received daily treatments
for 7 days, along with acupuncture twice weekly. After this point it received
laser therapy weekly. It regained deep pain in approximately 1 week and
partial motor function in the pelvic limbs by 5 weeks. It continued to
improve and began advancing its limbs in an attempt to ambulate. (Photo
and case report courtesy: Jennifer Conrad, DVM).
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564 Photomedicine for Exotic Animals

nociceptive axons. This leads to a decrease in mitochondrial mem-

brane potential, reduced ATP availability, and blocked conduction
within the A-delta and C nociceptive fibers [15] LLLT induces mRNA
expression of the opioid precursor molecules proopiomelanocortin
and corticotrophin releasing factor within inflammatory tissue,
promoting increased levels of beta-endorphin at the site of damage
[16]. Long-term effects of laser therapy involve neuromodulation
of ascending and descending pain-associated pathways within the
brain and spinal cord.
Photomedicine for SCI in dogs has shown benefits. Studies
suggest that LLLT improves neurologic function following IVDD [17].
Nine to twelve weeks after experimental TL spinal cord transection
and sciatic nerve autograft insertion, dogs that received LLLT were
walking; those without LLLT remained paralyzed. Histologic analysis
of the dogs’ spinal cords in the LLLT group revealed new axons
and blood vessels migrating into the graft as well as absence of
prominent scar tissue, changes that were inapparent in control
animals [18].
A prospective study from the University of Florida evaluated the
time needed to recover ambulation in dogs afflicted with T3-L3
myelopathy. It compared outcomes in dogs with and without LLLT
after hemilaminectomy [19]. LLLT took place daily for 5 days. Dogs
receiving LLLT required 3–5 days to regain ambulation, in contrast
to those in the control group who took a median of 2 weeks. LLLT
consisted of 25 W/cm2 to the skin produced by a head containing
810 nm, 200 mW laser diodes. Researchers estimated an energy
density of 2–8 J/cm2 .
With this information about the effects of laser therapy on the
canine SCI patient, veterinarians treating exotics at least have a place
to start from which to extrapolate treatment parameters to other
animals.

31.3.3 Neuropathic and Orthopedic Pain

Neuropathic pain has been identified in domestic mammals but
has received less attention in birds, except for the result of beak
trimming in commercial poultry where neuroma formation and
chronic pain have been observed [20]. Lack of recognition of the
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Clinical Applications 565

signs of chronic pain thus lead to the undertreatment of neuropathic

and other forms of pain in birds [21].
Self-mutilation in birds that focuses on one localized region can
suggest chronic and/or neuropathic pain, especially if that bird
has a history of injury or trauma. Any type of injury may cause
neuropathic pain, which arises spontaneously as a consequence of
damage to the nervous system. Neuropathic pain can be difficult to
treat, however, and no standard protocol exists for its control or
elimination [22]. Because evidence suggests that nociception and
pain in birds are largely analogous to mammals [23, 24], treatment
strategies that alleviate mammalian pain likely aid in reducing avian
discomfort as well. Figure 31.3 presents a case of treating suspected
neuropathic pain in a prairie falcon. Figures 31.4 and 31.5 illustrate
photomedicine applications in large felidae for pain control.

31.3.4 Wound Healing and Infection

Laser therapy repairs tissue in a number of ways; as a result,
it excels at treating many types of wounds. The biology of
encouraging regrowth after injury involves a complex sequence of
changes, nearly all of which laser supports [25]. From inflammation
and epithelialization to collagen production, wound contraction,
angiogenesis, and more, light therapy creates both local and
systemic responses that foster regrowth and reestablishment of
a healthier cellular milieu. It can also augment the activity of
adipose-derived mesenchymal stem cells, enhance their survival,
and support their secretion of growth factors in the wound bed
[26]. LLLT stimulates fibroblast proliferation, collagen production,
growth factor release, and microvascularization of injured tissue
[27]. It activates local immune cells (macrophages and lymphocytes)
and alters the expression of genes involved in wound healing and
possibly analgesia [27]. Laser fosters resolution of inflammation
by modulating inducible nitric oxide synthase (iNOS) expression,
reducing edema, and speeding normalization of tissue architecture
[28, 29].
Phototherapy boosts activity in the mitochondrial respiratory
chain. It stimulates the enzyme cytochrome c oxidase, thereby
propelling oxidative phosphorylation and augments output of
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566 Photomedicine for Exotic Animals

Figure 31.3 This adult, male prairie falcon exhibited self-inflicted wounds
along the right proximal patagium. The bird started self-traumatizing
approximately 1 month after fracturing the right metacarpus, although the
fracture had stabilized and surface wounds had healed completely. Despite
treatment with a non-steroidal anti-inflammatory drug, the bird continued
to chew along the patagial tendon during this time. Gabapentin, ketamine,
and LLLT were prescribed (dose unavailable). Despite initial improvement,
new self-inflicted wounds appeared along the tendon. Approximately
1.5 months later, a radial and medianoulnar nerve block was performed
with bupivacaine and medetomidine. At this time, the dosing interval of
ketamine was increased to q12h; the dose of gabapentin was increased
7.5-fold. LLLT and ketoprofen remained as previously prescribed. After 2.5
months, the wounds healed completely and no further mutilation took place.
Once deemed ready for release, the falcon was returned to the wild after
181 days in captivity. This is the first reported application of successful
multimodal analgesia in a raptor with uncontrolled neuropathic pain [21].
(Photo and case report courtesy: Narda G. Robinson, DO, DVM, MS, FAAMA).
July 12, 2016 11:36 PSP Book - 9in x 6in 31-Hamblin-c31

Clinical Applications 567

Figure 31.4 This 10-year-old neutered male cougar fell 2 m after a seizure.
He stopped eating for several days after the injury and became extremely
lethargic; this was attributed to pain. He received treatment with an LED
cluster for 1 min at each site, set at 2.5 Hz to the top of the skull at
acupuncture point GV 20 as well as the spine. He also received dry needle
acupuncture (i.e., needling only without electrical stimulation). The cougar
began eating immediately following the treatment. No further treatment
was given afterward because the felid regained his cougar nature. (Photo
and case report courtesy: Jennifer Conrad, DVM).

adenosine triphosphate (ATP). This elevated level of ATP availability

supports healthy cell signaling and the metabolic energy to
encourage tissue regrowth. Laser therapy activates fibroblasts and
keratinocytes and increases DNA replication, angiogenesis, and
analgesia.
For oral surface wounds, laser therapy helps heal stomatitis
as well as mucositis and oropharyngeal ulceration associated with
radiation therapy for cancer of the head and neck [30]. For burns,
laser therapy outperforms ultrasound in the treatment of pain [31].
It reduces the number of inflammatory cells drawn to the region
while it increases formation [32].
Wounds of diabetic patients typically heal more slowly. Whether
in the mouth [33] or elsewhere on the body, photobiomodulation
facilitates wound closure through cytokine and growth factor
release, acceleration of wound resurfacing, and the deposition of
granulation tissue [34].
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568 Photomedicine for Exotic Animals

Figure 31.5 This 21-year-old neutered male bobcat had been declawed
years earlier in the front paws. He developed severe arthritis, altered
conformation, and an abnormal gait as a result of the phalangectomies
(declaw procedure). He received treatment with a device that emitted
640 nm, 870 nm, and 905 nm superpulsed beams, with frequency settings
of 1–250 Hz to the paws and paw pads and 1000–2000 Hz to quell pain
along spinal segments associated with nerves for the forelimb. Laser was
provided daily for between 2 and 5 min per site. Laser application included
a slow scan with the beam over the caudal cervical and cranial thoracic
region at a speed of 1 cm/s. Laser therapy produced mild to moderate
improvement in gait each day. Overall moderate improvement was noted
over time—a reasonable outcome given the chronicity of the problem and
the negative impact to structure and function that may result from the
“declaw” procedure. (Photo and case report courtesy: Jennifer Conrad,
DVM).
July 12, 2016 11:36 PSP Book - 9in x 6in 31-Hamblin-c31

Clinical Applications 569

Considering the plethora of effects of photomedicine on surface

wounds, the potential value of light therapy in exotic animal patients
seems promising. In veterinary patients, laser therapy for wound
healing has been studied in the horse, a species in which non-
or delayed-healing wounds in the equine distal limb have plagued
practitioners for eons. Researcher utilized a line-generated optical
scanner with a dual diode system that emitted a rotating sequence of
frequencies from 4 to 28 Hz. The diodes produced a 635 nm beam by
means of 17 mW diodes. After they excised a 2.5 cm2 , full-thickness
section of skin on the dorsal mid-metacarpal region of each thoracic
limb, one limb received LLLT and the other served as a control [35].
LLLT healed the wounds more quickly; their margins contracted and
epithelialized without forming exuberant granulation tissue. Rates
of healing between LLLT and control limbs differed significantly
after day 17. Considering the safety of LLLT along with its analgesic
and anti-inflammatory advantages, there seems to be little reason
not to offer LLLT as an adjunct for delayed or non-healing wounds.
Exotic animals develop both acute and chronic integumentary
disorders that warrant consideration of phototherapy. The cases in
Figs. 31.6 and 31.7 illustrate different applications as examples of
the versatility this approach offers.

31.3.5 Laser Acupuncture

Acupuncturists first began experimenting with low-power lasers
during the 1970s. A 2008 systematic review of laser acupuncture
research found evidence to support its application for myofascial
pain, postoperative nausea and vomiting, along with the alleviation
of chronic tension headache [36]. Laser acupuncture involves the
process of shining laser light into acupuncture points instead
of inserting needles. While any laser therapy unit may be used
for laser acupuncture, the high-tech acupuncture laboratory at
the Medical University of Graz, Austria, has studied multichannel
laser needle acupuncture, which allows for the stimulation of
several acupuncture points simultaneously by means of multiple
semiconductor injection laser diodes. Flexible optical fibers deliver
the light to the site with minimal loss of intensity. Power density may
reach 20 J/cm2 per acupuncture points, with 30–40 mW per needle,
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570 Photomedicine for Exotic Animals

Figure 31.6 This approximately 45-year-old female Asian elephant was

injured by a landmine and rescued from a logging camp in Thailand. She
developed a grapefruit-sized hard chronic skin abscess in the right caudal
acromion area. She was treated daily with a device that emitted 640 nm,
870 nm, and 905 nm superpulsed beams using a 5 Hz scan over the region
for 5–10 min. The abscess softened daily until it opened on its own in 4 days.
Daily treatments continued during the healing process through a plastic
wrap on laser. (Photo and case report courtesy: Jennifer Conrad, DVM).

and 500 micron diameter spot size [37]. Emission wavelengths vary
but have included 685 nm, 785 nm, and now violet laser (which
appears blue to our eyes) at 405 nm.
In contrast to typical laser therapy treatments that target mainly
injured areas, laser acupuncturists employ an equal or greater
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Clinical Applications 571

Figure 31.7 This dolphin is receiving treatment with 470 nm (blue) light at
50 Hz for 2 min along with the blue light for wounds on the fins, nose, and
a space between the teeth, as well as for preventive medicine in the mouth.
(Photo courtesy: Dolphin Quest Oahu).

number of nonlocal sites. In so doing, they recruit a variety of neural

networks and fascial connections for participation in the healing
process [38–41]. For example, standard laser therapy approaches
to temporomandibular joint (TMJ) disorders typically focus on
the TMJ and/or muscles of mastication [41]. In contrast, laser
July 12, 2016 11:36 PSP Book - 9in x 6in 31-Hamblin-c31

572 Photomedicine for Exotic Animals

acupuncture for TMJ dysfunction may direct light to points on the

forehead, hand, foot, and leg in addition to the TMJ region [42 43].
Although seemingly unrelated and extraneous to a laser therapist,
this type of expanded treatment makes scientific sense to a medical
acupuncturist. What is more, an acupuncture-informed approach
to laser therapy extends its clinical reach to a host of systemic
and autonomic disorders, such as hypertension, depression, and
neuroendocrine dysfunction [44–46].
Laser acupuncture has many advantages over traditional
needling, especially for exotic animal patients. Its noninvasive nature
raises the already high safety profile of acupuncture, aside from the
risk of inadvertent, unprotected ocular exposure. Patients, especially
young ones [47, 48], may more readily accept laser treatment,
especially when approaches aim at sensitive sites on the face, ear,
or perineum. Infrared heating offers an alternative to moxibustion,
i.e., the burning of a herb near acupuncture points [49, 50]. This
eliminates not only the risk of burn but also the heavy smoke and
marijuana-esque odor associated with moxa and the herb Artemisia
vulgaris. For brain research, laser acupuncture circumvents the
hurdles of metal needles in the MRI suite, which may impair image
quality and cause tissue heating [51]. Needle-less acupuncture
reduces risk of infection; certain types of laser light even inactivate
bacteria or diminish their ability to reproduce [52, 53].
On the down side, eliminating the needle may reduce its
effectiveness for trigger points, a staple of acupuncturists’ analgesic
repertoire, meaning that many acupuncturists will want to maintain
the metal instrument for musculoskeletal ailments [54]. While laser
acupuncture is showing effectiveness for a host of problems that
needling traditionally treated, simply shining light cannot supplant
the palpatory feedback delivered by a needle as it encounters the
tone, tension, and texture in muscle tissue.
Laser acupuncturists must guard against excessive heating of
the skin with high power units, especially for neonates [55] and
veterinary patients who cannot clearly communicate about sources
or sites of discomfort, such as anesthetized or neurologically
impaired patients and the veterinary population as a whole.
Nonetheless, research is building insight into ways to optimize
treatment parameters for both laser therapy and acupuncture
July 12, 2016 11:36 PSP Book - 9in x 6in 31-Hamblin-c31

Clinical Applications 573

Figure 31.8 (a) Screech owl presented to the Rocky Mountain Raptor
Program with the inability to fly along with suspected head and right
wing trauma. (b) An example of treatment at an acupuncture point with
an 810 nm, 500 mW, single-beam acupuncture probe. (c) This contrasts
with the relatively oversized four-diode 810 nm, 1 W cluster applied to an
acupuncture point on the right pelvic limb just distal to the stifle joint. Note
how much of the beam courses beyond the bird’s leg and into the holder’s
palm. (Photo and case report courtesy: Narda G. Robinson).
July 12, 2016 11:36 PSP Book - 9in x 6in 31-Hamblin-c31

574 Photomedicine for Exotic Animals

[35, 56–59]. For some problems, two or more wavelengths may

offer roughly equivalent effects, such as red (660 nm) and infrared
(830 nm), to improving muscle performance and reducing fatigue
[59]. On the other hand, 808 nm light reaches more deeply into
tissue and thus may outweigh 980 nm for conditions such as SCI
[60]. Somatosensory-evoked potentials change during high-dose
laser irradiation when treating one locus along an axon as opposed
to several sites along the same axon [61]. This has relevance for
laser acupuncturists who often select one or more points on an
acupuncture channel, and channels commonly follow nerve axons.
Figure 31.8 depicts the type of applicator required for individual
acupuncture point stimulation.

31.4 Conclusion

Laser therapy and LED treatments are gaining a broader foothold

across the board in veterinary medicine, including zoo [62] and wild
animal settings. Exotic animal veterinarians grow accustomed to
improvising treatments for an extensive array of species, some of
which have no reported cases of photomedicine applications in the
literature. This chapter presented examples of the types of patients
that have received laser therapy and LED treatment, along with their
responses.

References

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2011; 14: xiii–xvi.
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140.
3. Godine RL. Low level laser therapy (LLLT) in veterinary medicine.
Photomed Laser Surg. 2014; 23(1): 1–2.
4. Huang YY, Gupta A, Vecchio D, et al. Transcranial low level laser (light)
therapy for traumatic brain injury. J Biophoton. 2012; 5(11–12): 827–
837.
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5. Wendell MD, Sleeman JM, and Kratz G. Retrospective study of morbidity

and mortality of raptors admitted to Colorado State University
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6. Fix AS and Barrows SZ. Raptors rehabilitated in Iowa during 1986 and
1987: A retrospective study. J Wildlife Dis. 1990; 26(1): 18–21.
7. Murphy MD and Robinson NG. A retrospective analysis of head trauma
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Mountain Raptor Program. Honors thesis, Colorado State University,
Jacqueline Murphy, 2013.
8. Zivin JA, Albers GW, Bornstein N. et al. Effectiveness and safety of
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in raptors. J Avian Med Surg. 2015; 29(1): 30–39.
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hemilaminectomy without methylprednisolone sodium succinate for
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Emerg Crit Care. 2007; 17(1): 72–76.
11. Scott HW. Hemilaminectomy for the treatment of thoracolumbar disc
disease in the dog: A follow-up study of 40 cases. J Small Anim Pract.
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12. Rochkind S, Vogler I, and Barr-Nea L. Spinal cord response to laser
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13. Rochkind S, Drory V, Along M, et al. Laser phototherapy (780 nm), a new
modality in treatment of long-term incomplete peripheral nerve injury:
A randomized double-blind placebo-controlled study. Photomed Laser
Surg. 2007; 25(5): 436–422.
14. Rochkind S, Geuna S, and Shainberg A. Chapter 25. Phototherapy in
peripheral nerve injury: Effects on muscle preservation and nerve
regeneration. Int Rev Neurobiol. 2009; 87: 445–464.
15. Chow RT, David MA, and Armati PJ. 830 nm laser irradiation induces
varicosity formation, reduces mitochondrial membrane potential and
blocks fast axonal flow in small and medium diameter rat dorsal root
ganglion neurons: Implications for the analgesic effects of 830 nm laser.
J Peripher Nerv Sys. 2007; 12: 28–39.
16. Hagiwara S, Iwasaka H, Okuda K, et al. CaAlAs (830 nm) low-level laser
enhances peripheral endogenous opioid analgesia in rats. Lasers Surg
Med. 2007; 39: 797–802.
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17. Chyczewski M. Laser biostimulation in neurological diseases of dogs.

Annales Universitatis Mariae Curie-Skodowska. Sectio DD. Medicina
Veterinaria. 2005; 60: 55–58.
18. Rochkind S. The role of laser phototherapy in nerve tissue regeneration
and repair: Research development with perspective for clinical applica-
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Emerging modalities in veterinary rehabilitation. Vet Clin Small Anim.
2005; 35: 1335–1355.
19. Draper WE. Schubert TA, Clemmons RM, et al. Low-level laser
therapy reduces time to ambulation in dogs after hemilaminectomy: A
preliminary study. J Small Anim Pract. 2012; 53: 465–469.
20. Cheng H. Morphopathological changes and pain in beak trimmed laying
hens. Poult Sci 2006; 62: 41–52.
21. Shaver SL, Robinson NG, Wright BD, et al. A multimodal approach to
management of suspected neuropathic pain in a prairie falcon (Falco
mexicanus). J Avian Med Surg. 2009; 23(3): 209–213.
22. Viñuela-Fernández I, Jones E, Welsh EM, et al. Pain mechanisms and
their implication for the management of pain in farm and companion
animals. Vet J 2007; 174: 227–239.
23. Machin KL. Avian analgesia. Sem Avian Exotic Pet Med 2004; 14(4): 236–
242.
24. Garry EM, Jones E, and Fleetwood-Walker SM. Nociception in verte-
brates: Key receptors participating in spinal mechanisms of chronic pain
in animals. Brain Res Rev 2004; 46: 216–224.
25. Prindeze NJ, Moffatt LT, and Shupp JW. Mechanisms of action for light
therapy: A review of molecular interactions. Exp Biol Med (Maywood).
2012; 237: 1241. DOI: 10.1258/ebm.2012.01280.
26. Kim H, Choi K, Kweaon O-K, et al. Enhanced wound healing effect of
canine adipose-derived mesenchymal stem cells with low-level laser
therapy in athymic mice. J Dermatol Sci. 2012; 68: 149–156.
27. Silveira PCL, Streck EL, and Pinho RA. Evaluation of mitochondrial
respiratory chain activity in wound healing by low-level laser therapy.
J Photochem Photobiol B: Biol. 2007; 86: 279–282.
28. Moriyama Y, Moriyama EH, Blackmore K, et al. In vivo study of the
inflammatory modulating effects of low-level laser therapy on the iNOS
expression using bioluminescence imaging. Photochem Photobiol. 2005;
81: 1351–1355.
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29. Chang K-J, Lee D-H, Cho Y-S, et al. Studies on laser therapy for joint
edema in dogs. Korean J Vet Clin Med. 1996; 13(1): 53–56.
30. Verma SK, Maheshwari S, Singh RK, et al. Laser in dentistry: An
innovative tool in modern dental practice. Natil J Maxillofac Surg. 2012;
3(2): 124–132.
31. Sari Z, Polat MG, Ozgul B, et al. A comparison of three different
physiotherapy modalities used in the physiotherapy of burns. J Burn
Care Res. 2013; June 28. [Epub ahead of print].
32. Fiorio FB, Albertini R, Leal-Junior EC, et al. Effect of low-level laser
therapy on types I and III collagen and inflammatory cells in rats with
induced third-degree burns. Lasers Med Sci. 2013; May 16. [Epub ahead
of print].
33. Firat ET, Dag A, Gunay A, et al. The effects of low-level laser therapy
on palatal mucoperiosteal wound healing and oxidative stress status in
experimental diabetic rats. Photomed Laser Surg. 2013; 31(7): 315–321.
34. Dixit S, Maiya A, Umakanth S, et al. Photobiomodulation of surgical
wound dehiscence in a diabetic individual by low-level laser therapy
following median sternotomy. Indian J Palliat Care. 2013; 19(1): 71–75.
35. Jann HW, Bartels K, Ritchey JW, et al. Equine wound healing: Influence of
low level laser therapy on an equine metacarpal wound healing model.
Photon Laser Med. 2012; 1: 117–122.
36. Baxter GD, Bleakley C, and McDonough S. Clinical effectiveness of laser
acupuncture: A systematic review. J Acupunct Meridian Stud. 2008; 1(2):
65–82.
37. Litscher G, Gao X-Y, Wang L, et al. High-tech acupuncture and integrative
laser medicine. Evid Based Complement Alternat Med. 2012; 2012:
363467.
38. Anzinger A, Albrecht J, Kopietz R, et al. Effects of laser needle
acupuncture on olfactory sensitivity of healthy human subjects: A
placebo-controlled, double-blinded, randomized trial. Rhinology. 2009;
47: 153–159.
39. Bjordal JM, Lopes-Martins RAB, Joensen J, et al. A systematic review with
procedural assessments and meta-analysis of low level laser therapy in
lateral elbow tendinopathy (tennis elbow). BMC Musculoskelet Disord.
2008; 9: 75. DOI: 10.1186/1471/2474-9-75.
40. Bjordal JM, Couppe C, Chow RT, et al. A systematic review of low level
laser therapy with location-specific doses for pain from chronic joint
disorders. Aust J Physiother. 2003; 49: 107–116.
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41. Dostalova T, Hlinakova P, Kasparova M, et al. Effectiveness of physiother-

apy and GaAlAs laser in the management of temporomandibular joint
disorders. Photomed Laser Surg. 2012; 30(5): 275–280.
42. Ferreira LA, de Oliveira RG, Guimaraes JP, et al. Laser acupuncture in pa-
tients with temporomandibular dysfunction: A randomized controlled
trial. Lasers Med Sci. 2013; 28(6): 1549–1558. DOI 10.1007/s10103-
013-1273-x.
43. Hotta PT, Hotta TH, Bataglion C, et al. EMG analysis after laser
acupuncture in patients with temporomandibular dysfunction (TMD).
Implications for practice. Complement Ther Clin Pract. 2010; 16: 158–
160.
44. Zhang J, Marquina N, Oxinos G, et al. Effect of laser acupoint treatment
on blood pressure and body weight: A pilot study. J Chiropr Med. 2008;
7(4): 134–139.
45. Quah-Smith I, Smith C, Crawford JD, et al. Laser acupuncture for
depression: A randomised double blind controlled low intensity laser
intervention. J Affect Disorders. 2013; 148: 179–187.
46. Wu J-H, Chen H-Y, Chang Y-J, et al. Study of autonomic nervous activity
of night shift workers treated with laser acupuncture. Photomed Laser
Surg. 2009; 27(2): 273–279.
47. Fontana CR and Bagnato VS. Low-level laser therapy in pediatric Bell’s
palsy: Case report in a three-year-old child. J Alt Complement Med. 2013;
19(4): 376–382.
48. Gottschling S, Meyer S, Gribova I, et al. Laser acupuncture in chil-
dren with headache: A double-blind, randomized, bicenter, placebo-
controlled trial. Pain. 2008; 137: 405–412.
49. Hsiu H, Hsu W-C, Hsu C-L, et al. Microcirculatory changes by laser
doppler after infrared healing over acupuncture points: Relevance to
moxibustion. Photomed Laser Surg. 2009; 27(6): 855–861.
50. Shen X, Zhao L, Ding G, et al. Effect of combined laser acupuncture on
knee osteoarthritis: A pilot study. Lasers Med Sci. 2009; 24: 129–136.
51. Beissner F, Noth U, and Schockert T. The problem of metal needles in
acupuncture-fMRI studies. Evid Based Complement Alternat Med. 2011;
2011: 808203.
52. Dai T, Gupta A, Murray CK, et al. Blue light for infectious diseases:
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53. Fonseca AS, Geller M, Filho MB, et al. Low-level infrared laser effect on
plasmid DNA. Lasers Med Sci. 2012; 27: 121–130.
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54. Robinson NG. Laser vs. acupuncture and massage. Veterinary Practice
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com/vet-dept/small-animal-dept/laser-vs-acupuncture-and-massage.
aspx
55. Raith W, Litscher G, Sapetschnig I, et al. Thermographical measuring
of the skin temperature using laser needle acupuncture in preterm
neonates. Evid Based Complement Alternat Med. 2012; 2012: 614210.
56. Bergamaschi M, Ferrari G, Gallamini M, et al. Laser acupuncture and
auriculotherapy in postural instability: A preliminary report. J Acupunct
Meridian Stud. 2011; 4(1): 69–74.
57. Cunha RG, Rodrigues KC, Salvador M, et al. Effectiveness of laser
treatment at acupuncture sites compared to traditional acupuncture in
the treatment of peripheral artery disease. 32nd Annual International
Conference of the IEEE EMBS. Buenos Aires, Argentina, August 31–
September 4, 2010.
58. He W, Wedig D, Wang L, et al. Violet laser acupuncture – Part 5: An
investigation of different stimulation frequencies on heart rate and
variability. J Acupunct Meridian Stud. 2012; 5(6): 290–294.
59. De Almeida P, Lopes-Martins RAB, De Marchi T, et al. Red (660 nm) and
infrared (830 nm) low-level laser therapy in skeletal muscle fatigue in
humans: What is better? Lasers Med Sci. 2012; 27: 453–458.
60. Hudson DE, Hudson DO, Wininger JM, et al. Penetration of laser light at
808 and 980 nm in bovine tissue samples. Photomed Laser Surg. 2013;
31(4): 163–168.
61. Chow R, Yan W, and Armati P. Electrophysiological effects of single point
transcutaneous 650 and 808 nm laser irradiation of rat sciatic nerve:
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62. Thornton W. Laser therapy helps Birmingham Zoo elephant’s torn
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phan.html on 07-12-14.
July 6, 2016 17:36 PSP Book - 9in x 6in 32-Hamblin-c32

Chapter 32

Recalcitrant Wound: Using Low-Level

Laser (Light) Therapy to Manage
Non-Healing Wounds and Ulcers

Raymond J. Lanzafamea,b and Istvan Stadlerc,d

a Private Practice, 757 Titus Avenue, Rochester, New York 14617, USA
b School of Dental Medicine, State University of New York at Buffalo, Buffalo,

New York 14214, USA

c Laser Surgical Research Laboratory, Rochester General Hospital, Rochester,

New York 14621, USA

d School of Medicine, State University of New York at Buffalo, Buffalo,

New York 14214, USA

raymond.lanzafame@gmail.com

Wound healing is a common and often overlooked process that

restores the host to a functional form following surgery, iatrogenic
injury, or trauma. This process proceeds normally much of the
time. However, several conditions can result in impaired or delayed
healing with resulting increased risks of morbidity and mortality
as a result of complications attributable to chronic wounds. Low-
level laser (light) therapy (LLLT) is being applied both clinically
and experimentally using various light sources to facilitate healing,
particularly in refractory wounds. The most effective wavelengths
appear to be clustered in the red and near-infrared (NIR) portions
of the electromagnetic spectrum [1, 5, 6, 11, 13–16, 20, 27–29, 34,

Handbook of Low-Level Laser Therapy

Edited by Michael R. Hamblin, Marcelo Victor Pires de Sousa, and Tanupriya Agrawal
Copyright c 2017 Pan Stanford Publishing Pte. Ltd.
ISBN 978-981-4669-60-3 (Hardcover), 978-981-4669-61-0 (eBook)
www.panstanford.com
July 6, 2016 17:36 PSP Book - 9in x 6in 32-Hamblin-c32

582 Recalcitrant Wound

35, 38]. This chapter will discuss the applications of LLLT in the
management of poorly healing wounds and ulcers.

32.1 Introduction: An Overview of Normal Wound

Healing

A full treatise on wound healing is beyond the scope of this

chapter. However, it is important for the reader to understand that
wound repair is a highly complex process that involves a series of
overlapping processes that are coordinated to restore functionality.
Five major phases are recognized: wound (injury), hemostasis,
inflammation, proliferation, and remodeling [1, 14, 36].
The initial response to injury is the hemostasis phase, which
results in clot formation due to the action of platelets, fibrin,
complement, and the secretion of kinins and other factors. The clot
serves as a scaffold for subsequent phases, and the inflammation
phase commences as the kinins and other factors attract neutrophils,
macrophages, and lymphocytes. This phase is responsible for re-
moving debris from the wound and managing wound contamination
and controlling infection. Various cellular components secrete a
series of cytokines and produce reactive oxygen species (ROS)
and other mediators such as nitric oxide (NO). This ushers in the
proliferation phase, which is characterized by the production of
proteoglycans and fibroblast-mediated collagen production, as well
as the formation of granulation tissue, which consists of fibroblasts
and endothelium as angiogenesis is stimulated by tumor necrosis
factors-α (TNF-α), β-fibroblast growth factor (β-FGF), and vascular
endothelial growth factor (VEGF), and capillary and blood vessels
proliferate. The epidermal barrier is restored during this phase.
The remodeling phase ensues and persists for weeks or months
thereafter. It is during this phase that collagen crosslinking and
remodeling occurs and scar maturation proceeds. Tissue tensile
strength increases over time, reaching approximately 50% of
uninjured tissue by 6–8 weeks and gradually reaches 80–90% of
normal over the next several months, up to a year after the incident.
Scar tissue differs from normal tissue in that skin is devoid of
appendages and deeper structures are replaced by fibrous tissue.
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Photobiomodulation and Wound Healing 583

As with any complex system, wound healing can be disrupted by

a number of intrinsic and extrinsic forces that can arrest or impair
healing. These include diabetes and other endocrine and metabolic
disorders, cancer, malnutrition, hypoxia, vascular insufficiency and
ischemia, pressure, neuropathy, presence of foreign bodies in the
wound, ionizing radiation, presence of infection, and influence of a
variety of drugs and other substances, and environmental factors,
among other causes.

32.2 Photobiomodulation and Wound Healing

A variety of light sources, including lasers, light-emitting diodes

(LEDs), superluminous diodes (SLDs), and other noncoherent light
sources have been used to treat wounds [1, 3, 5, 6, 11, 13, 15, 16,
20–23, 25–27, 30–36].
Wound treatments are classically delivered on a daily or an
every-other-day basis, most frequently being administered three to
four times per week as a result of the initial observations of Mester
and the success of this empiric regime.
Successful wound management using phototherapy depends on
an understanding of the mechanisms of photobiomodulation and
applying these technologies appropriately.

32.2.1 Photobiomodulation and Its Mechanisms

LLLT, as it is applied to wounds and tissues, uses light of specific
wavelengths to stimulate various cellular processes. Both in vitro
and in vivo studies have demonstrated that LLLT can accelerate
wound healing [2, 4, 6, 8, 10, 13, 14, 19–22, 26, 27, 29–36, 38]. The
photobiomodulation effect occurs as a photochemical rather than
photothermal response to exposure to light.
Photons are absorbed by tissue chromophores, and cellular
metabolism is increased and adenosine triphosphate (ATP) pro-
duction increases. Various secondary messengers are activated, and
cascades of intracellular signals are upregulated [1, 14, 15, 39].
Cytochrome c oxidase has been shown to absorb photon energy
and facilitate the transfer of electrons to molecular oxygen. Increases
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584 Recalcitrant Wound

in this mitochondrial respiratory chain enzyme complex ultimately

result in increased mitochondrial activity and ATP production
through the acceleration of oxidative metabolism [1, 7, 10, 14, 39].
Other mechanisms have also been studied and have been offered
as explanations for the observed photobiomodulation effect. ROS
have been shown to be increased or decreased, and NO production
and intracellular calcium (iCa2+ ) concentrations are increased after
LLLT [10, 12, 14, 16, 34].
LLLT ultimately results in the activation of various transcription
factors, which activate gene transcription. Various growth factors
have been demonstrated to be upregulated, collagen production
increases, and cell migration and proliferation are enhanced [1, 14,
16, 23, 25, 28, 34, 36]. LLLT also reduces inflammation and affects
mast cells, among other cellular responses [14, 25].
The exact mechanisms responsible for the photobiomodulation
effect continue to be debated, and it is likely that the specific
mechanism or mechanisms vary and depend on the cell or tissue
type as well as its location and function. It is important to
understand the fact that biological systems are complex, with
multiple pathways that may work in concert with or in an inhibitory
fashion to one another, depending on the particular needs of
the organism. These pathways often have common denominator
substances that can be influenced by the application of light as well
as other environmental events or mediators.
The resultant of these effects is the acceleration of wound healing
and increased wound tensile strength, which has been demonstrated
in various investigational models, and particularly in diabetic and
ischemic wounds [5, 6, 8, 11, 14, 19, 20–27, 29–38].

32.2.2 Applying Phototherapy to Wounds: Wavelengths

and Energy Density
LLLT has been applied in both experimental models and clinically in
an effort to accelerate or improve wound healing. The majority of
these employ treatment protocols using energy densities between 1
and 10 J/cm2 , although some investigators have used much higher
fluences. Energy densities above 10 J/cm2 tend to be bioinhibitory
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Photobiomodulation and Wound Healing 585

in most models, whereas fluences at or below this level tend to be

biostimulatory [1, 5, 8, 11, 13, 15, 20, 23, 26, 27, 29, 34, 35].
A variety of wavelengths between 390 and 1100 nm have been
investigated and used. However, the most effective wavelengths
appear to be clustered in the red and NIR regions of the spectrum.
Similarly, many clinical protocols employ energy densities of 4–5
J/cm2 and typically apply this to the wound on a daily or more
traditional every-other-day basis [5, 6, 11, 13, 14, 19, 20, 26, 27, 30–
35].

32.2.3 Applying Phototherapy to Wounds: Irradiance,

Exposure Time, and Dose and Treatment Frequency
Conflicting results have been published, demonstrating positive and,
in other cases, negative effects after applying LLLT using empiric
single daily treatments and dosing strategies. It is likely that unique
dose and treatment frequency regimens exist for tissues and cell
types that will result in maximal stimulation and the failure to
determine and utilize them may result in treatment failure [2, 4, 19–
21, 25–27, 33].
Our laboratory has demonstrated these concepts using both
in vitro and in vivo models [2–4, 19]. We demonstrated that cell
proliferation and metabolism can be affected by varying the dose
frequency and treatment interval in vitro [2]. Two treatments per
day were more effective than single daily treatments, and the
individual responses to photoirradiation were different for different
cell types.
Results of a study of the effects of varying irradiance and
exposure times to achieve a daily energy density of 5 J/cm2 at
670 nm in a murine pressure ulcer model again demonstrated that
two treatments per day were more effective in some cases [19].
However, the study also demonstrated that long exposure times
at low intensities (irradiance) were either ineffective or actually
bioinhibitory. There was a specific range of parameters over which
reciprocity of exposure time and irradiance was biostimulatory,
despite the delivery of mathematically equivalent doses to the
tissues.
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586 Recalcitrant Wound

32.2.4 Applying Phototherapy to Wounds: Skin

Pigmentation and Other Considerations
The presence of melanin and other pigments can also influence
treatment efficacy due to the absorption of light by the chro-
mophores. Studies from our laboratory have demonstrated that this
filtering effect is concentration dependent and that these effects
can be mitigated to some degree through the use of pulsing and
coherent light sources [3]. These in vitro experiments demonstrated
that oxidative burst was maximally stimulated at 600 Hz and cell
proliferation was maximally stimulated at 100 Hz in the presence
of a melanin filter, which matched the melanin content of Fitzpatrick
Skin Type V-VI skin.
These results may explain why some investigators have had
variable or poor results when treating wounds in patients with
darker skin. Our results suggest that increasing the delivered light
dose by as much as 25% may be necessary and that using pulsed
noncoherent and/or laser light sources is preferable when treating
this patient population.

32.3 Bacterial Contamination and Wound Infection:

Antimicrobial Effects of LLLT

The presence of an overt wound infection or bacterial contamination

can have serious consequences, resulting in delayed or non-healing
of wounds, in addition to causing significant morbidity or mortality.
Current evidence suggests that 65–80% of wound infections are
caused by bacteria growing in biofilms and that these configurations
are resistant to concentrations of antibiotics or other agents that
would be bactericidal to planktonic cells [24]. Antibiotic resistance
is also becoming increasingly prevalent, which further complicates
patient management [9, 24].
Work from several laboratories has demonstrated that certain
wavelengths of light are bactericidal both in vitro and in vivo, either
alone or when used in combination with other chromophores to
generate ROS, thereby resulting in cell destruction [9, 10, 17, 18,
24, 37]. Blue light between the wavelengths of 405 and 470 nm
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General Considerations for Wound Management 587

has been shown to be particularly beneficial and bactericidal, while

green light at 525 nm is bacteriostatic [9, 17]. Red light had no effect
when used without a photodynamic therapy type agent [17].
Our group demonstrated that using blue light alone at 455
±5 nm when treating Methicillin-resistant Staphylococcus aureus
(MRSA)-infected pressure ulcers was ineffective. However, using
this wavelength in concert with flavins delivered to the wound
in a collagen matrix was bactericidal at specific energy densities
[24]. Others have shown that experimental parameters, bacterial
density, and the mode of treatment significantly affect the outcomes
achieved [9].
This promising area of research suggests that blue light pho-
toirradiation might play a role in routine wound management and
should likely be considered for use in the management of chronic
wounds. As with other applications of LLLT, it is important to be
mindful of treatment parameters and wound conditions.

32.4 General Considerations for Wound Management

Successful management of wounds requires careful attention to

a number of details regardless of whether or not LLLT is used.
Generally accepted clinical management standards should always be
applied and sound clinical judgment should prevail. The clinician
should always be mindful of the fact that unlike experimental
models, patients typically present with differing lesions and lesion
locations, varying comorbidities, a diverse array of wound manage-
ment strategies, and the use of various prescription and over-the-
counter medications. Each of these factors present variables that
must be taken into account when considering and evaluating the
results of LLLT in a particular case. It is also important to recognize
that LLLT is best utilized as an adjunctive therapy.

32.4.1 Initial Evaluation and Management

A thorough history and physical examination should be completed
on each patient, and this information should be updated regularly
in order to provide accurate information about the patient, existing
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588 Recalcitrant Wound

comorbidities as well as medications, allergies, and other environ-

mental factors. Medication reconciliation should be accomplished
at each visit. It is often helpful to ask patients to bring his or her
medications along with them to their initial and periodic follow-up
visits.
Comorbidities such as hypertension, cardiovascular disease,
peripheral vascular insufficiency, diabetes, hypothyroidism, autoim-
mune disorder, malignancy, presence and extent of peripheral
neuropathy, abnormalities of gait and residua of cerebrovascular
events, trauma or other causes should be carefully mapped.
Correction of the underlying conditions, including optimization
of diet and medications, as well as consideration of referral to
physical and/or occupational therapy and prosthetists should be
considered according to the needs of a particular case.

32.4.2 Evaluation of the Wound or Wounds

It is essential to gather as much information as is possible as it
pertains to any wound or wounds for each patient. The history
and physical examination should contain detailed information about
the onset of each lesion, its progress or response to treatments or
care regimes, associated symptoms and any history or presence of
wound-related complications. This information should be updated
regularly in order to provide accurate information about the
patient’s lesions, any existing comorbidities, medications, allergies,
and relevant environmental factors.

32.4.3 Wound Evaluation: Initial Documentation and

Management
The location, nature, and duration of each wound should be noted
and carefully documented. The current care regime should be noted
as well as a history of any prior care.
The wound dimensions and area should be carefully recorded,
and the wound appearance should be documented. This is generally
accomplished by using digital photography and wound perimeter
tracings. Wound status and progress can be verified and tracked by
serial images and tracings. (see Section 32.4.4)
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General Considerations for Wound Management 589

The initial wound status is assessed and documented, and

procedures such as wound cultures, debridement, and biopsy should
be completed after the initial pretreatment documentation has been
accomplished. It is important to biopsy any suspicious lesions in
order to rule out malignancy, as well as to utilize sound surgical
judgment and principles as regards wound care.
It should be remembered that LLLT is an adjunctive therapy
to be used in conjunction with standard management, including
debridement and the use of topical or systemic antibiotics as the
needs of the particular case dictate.

32.4.4 Wound Evaluation: Photodocumentation

Standardized protocols should be developed for the particular prac-
tice as regards wound management and for photodocumentation. It
is important to document the pretreatment status of the wound as
well as the results of any biopsy, debridement, or other management
strategy. In addition, it is necessary to document wound progress
over time and the post-treatment results and outcomes.
Numerous digital cameras and smart phones are available for
image capture. Standardized protocols for wound photodocumenta-
tion facilitate photography and subsequent interpretation of image
data. Figures 32.1 and 32.2 demonstrate the pre- and post-treatment

A B C

Figure 32.1 This series demonstrates the results of LLLT at 50 mW/cm2

× 1.7 min each per wavelegth (4 J/cm2 ) × 4 days per week using 670 nm
and 880 nm LEDs (Spectralife, Quantum Devices, Barneveld, WI) to treat
a diabetic heel ulcer. (A) Pretreatment appearance, (B) wound appearance
after approximately 30 treatments, and (C) the healed ulcer after 60
treatments.
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590 Recalcitrant Wound

A B

Figure 32.2 This series demonstrates use of the THOR-DDII LED Device
(THOR Photomedicine, LTD, Amersham, UK) delivering 660 and 850 nm
at power densities of 50 mW/cm2 and 150 mW/cm2 per wavelength,
respectively, at a total fluence of 4 J/cm2 per 1.7 min treatment, 4 days per
week, to treat a chronic pretibial lesion in an area previously damaged by
radiation therapy. The pretreatment image is shown in (A) and the healed
post-treatment image is shown in (B).

appearance of chronic wounds treated with LLLT in two different

patients. Each image contains a ruler placed in the field, and the
wounds are photographed in the same orientation to the degree
it is possible to do so. These basic features reduce potential
misinterpretation of results due to magnification, image size, or
orientation and do not require any specialized equipment.
Figure 32.3 presents a series of images depicting the clinical
course of an ulcerated lesion of the right wrist imaged using a
proprietary target that provides color, grey, and measurement scales
(Pixcelent, ImArcSys, Rochester, NY). Identifying data are readily
apparent on the target. This system also permits autocorrection
of color and image sizing based on the target if the images are
processed using its accompanying software.
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Clinical Applications and Considerations 591

A B C

Figure 32.3 This series of images illustrates the clinical course of an

ulcerated lesion on the wrist of a 93-year-old female. The lesion proved to
be a basal cell carcinoma (BCC) on biopsy. The initial appearance is shown
in (A). A chronic ulcer persisted after 5-FU ointment application (B). The
healed wound is shown after excision and closure with advancement flaps
(C). This case is shown to demonstrate the use of a multifunction medical
target system for photodocumentation.

32.5 Clinical Applications and Considerations

A number of different light sources and wound management

strategies have been used alone or in concert to manage the
recalcitrant chronic wound. Our center has adopted a strategy of
providing therapy in the office-based setting, treating the wounds
4 days per week while the patients continue their routine wound
care and chronic medications. This strategy assures that the light
therapy is being administered consistently, and any necessary
procedures such as debridement or wound cultures can be attended
to in a timely fashion.
We have successfully used two investigational protocols deliv-
ering red and NIR light to the wounds with equivalent results.
Complete healing occurred in 59.5% of light-treated cases, par-
tial healing occurred in 16.2% of cases, with 10.8% remaining
unchanged and 3.5% were worse after LLLT. The control group
wounds were essentially unchanged, exhibiting on average only a
7% reduction in wound area. None of the sham-treated wounds
healed (P <0.001). The average number of treatments required to
achieve healing or improvement was 67 ± 38 and both regimens
yielded identical results [20, 22]. Clinical results for two wounds and
device parameters are given in Figs. 32.1 and 32.2.
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592 Recalcitrant Wound

32.5.1 Patient Selection

It is important to remember that while LLLT can accelerate healing
and achieve closure of chronic wounds, it is not a panacea.
Phototherapy is most likely to be successfully applied as an early
intervention in high risk and superficial wounds [1, 11–14, 22, 26,
27, 29, 30–33]. Complex and deep wounds, including Stage III and IV
decubitus ulcers, require a multimodal approach and often require
tissue transfer or flap rotation techniques in order to achieve wound
closure [22, 26, 33].

32.5.2 Device Selection and Use

Various devices made by different manufacturers are available for
use in treating wounds. It is important to select the proper device
purchased from a reputable manufacturer, and the device should
provide the required wavelength or wavelengths and be configured
properly for the desired application. The user should be thoroughly
conversant with the device parameters and should verify that the
device is providing the specified output. This is best accomplished
by using a power meter. It should also be noted that it is preferable
to treat large wounds and areas by using multiple “spots” rather
than attempting to cover the area by increasing the spot size, since
reciprocity cannot be assumed [8, 19].
Proper positioning of the light source is also critically important,
particularly when using LED or other noncoherent sources, since
beam divergence from these noncollimated sources will result in
dramatic changes in irradiance intensities as the source is moved
closer or farther from the desired position. The use of a stand
or positioning device is quite helpful in assuring that the proper
distance and exposure are maintained throughout the course of
therapy [8, 19].
As was noted in Section 32.2.3, the use of daily or twice-a-day
treatments may be beneficial in some cases. Such regimes would
present logistical issues in the office or clinic setting. However, in-
patient and home therapy scenarios may be amenable to such a
schedule. Wearable devices and home use technologies can be used
in conjunction with periodic follow-up visits.
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Summary 593

32.6 Summary

Successful use of LLLT is critically dependent on understanding its

mechanisms and strict attention to and use of proper treatment
parameters. This chapter has considered the role of LLLT in the
management of chronic wounds. We have discussed the mechanisms
responsible for the photobiomodulation effect, experimental and
clinical evidence, and wound evaluation and management strategies.
LLLT can serve as a powerful adjunctive therapy and should be part
of the clinical armamentarium of professionals who care for wounds
and injury.

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Photomodulation of oxidative metabolism and electron chain enzymes
in rat liver mitochondria. Photochem. Photobiol., 66(6), pp. 866–871.
July 6, 2016 17:36 PSP Book - 9in x 6in 33-Hamblin-c33

Chapter 33

Clinical Applications with Low-Level

Laser Therapy in Arthritis

Jan M. Bjordal
Physiotherapy Research Group, Department of Global and Public Health,
Kalfarveien 31, University of Bergen Postboks 7804, N-5018, Bergen, Norway
jan.magnus.bjordal@hib.no

33.1 Introduction

There are several categories of arthritis and they differ in etiology

and clinical features. Many rheumatic disorders are characterized
by a type of arthritis arising from systemic inflammation involving
a number of organs and joints. The etiology of diseases like
rheumatoid arthritis is still somewhat unclear, but it is often
described as an autoimmune systemic inflammatory disease. In
this chapter, I will concentrate on osteoarthritis (OA), since the
clinical applications will largely be the same. However, the systemic
inflammatory component seems to be more dominant in rheumatoid
arthritis than in OA [1].
OA is the most common form of arthritis and is broadly
described as a degenerative disease affecting joint cartilage and its

Handbook of Low-Level Laser Therapy

Edited by Michael R. Hamblin, Marcelo Victor Pires de Sousa, and Tanupriya Agrawal
Copyright c 2017 Pan Stanford Publishing Pte. Ltd.
ISBN 978-981-4669-60-3 (Hardcover), 978-981-4669-61-0 (eBook)
www.panstanford.com
July 6, 2016 17:36 PSP Book - 9in x 6in 33-Hamblin-c33

598 Clinical Applications with Low-Level Laser Therapy in Arthritis

surrounding tissues. Because of increasing life expectancy and the

overweight epidemy, OA is becoming an increasing global burden to
modern society [2].
Genetic factors and certain susceptibility genes, asporin (ASPN),
growth differentiation factor 5 (GDF5), and double von Willebrand
factor A domains (DVWA) are among the risk factors and can partly
predict disease progression, especially when they are combined
with clinical findings [3]. The development of our understanding
of risk factors has matured and shown that risk factors can be
subgrouped by partly different risk factors for developing incident
OA and disease progression. For incident OA, age, obesity, and joint
injury are the most prominent risk factors for structural damage,
while inflammatory signs such as synovitis, joint effusion, and
bone marrow edema are the most prominent for progression of
symptoms [4].
Physical activity plays an important role both in the incident
development and modulation of progression of OA. Adherence to
guidelines for recommended physical activity does not negatively
affect the development of incident OA [5], and light physical activity
seems to have a small protective effect against the progression of OA
and disability [6]. However, strenuous weight-bearing activities in
established knee and hip OA have a strong negative effect, especially
when combined with obesity. Recent investigations have suggested
that obesity combined with strenuous activity increases odds ratio
to 11 in need for total knee replacement surgery [7].
Another issue that has achieved increasing focus in recent years
is the different stages of the disease and whether they need stratified
treatment approaches [8]. In the extension of this, the European
League Against Rheumatism targeted a number of priority areas in
OA research. These include predictors of OA progression, if they can
enable stratified interventions; understanding OA pain mechanisms
and tissue communication where multiple tissue pathologies exist.
Furthermore, developing interventions for early OA where both
pain pathophysiology and tissue structural processes may be more
effectively targeted by optimal single or combined therapies [9].
In a trial with a fairly massive exercise intervention program,
small but significant effects were obtained over no exercises [10]. In
non-weight-bearing joints such as wrist, joint protection measures
July 6, 2016 17:36 PSP Book - 9in x 6in 33-Hamblin-c33

Introduction 599

seem to reduce pain and disability slightly [11], whereas a light

home exercise intervention alone or in addition to joint protection
measures seemed to have no significant effects. Other nonsignificant
results of exercise therapy in hip OA have been recently published
[12].
But the overall picture still balances in favor of exercise therapy,
and this is becoming more and more the dominant intervention for
primary prevention and treatment against manifest OA [13]. Several
algorithms for OA management have been provided by different
scientific and professional associations [14–18]. It is a common
feature that none of the current guidelines include low-level laser
therapy (LLLT) as a viable treatment option.
Around the turn of the century such guidelines strongly
advocated the use of pharmacological agents to reduce pain [19].
This was particularly evident when the guidelines were sponsored
by pharmaceutical companies [20]. Later, some associations took
pride in making treatment guidelines evidence based [21]. This
advancement was the first possibility for LLLT to enter the stage
of OA treatment. Before that we performed one systematic review
of LLLT in chronic joint disorders [22], which forms part of the
basis for the dosage recommendations we formed through the World
Association for Laser Therapy (WALT), published on their website in
August 2005 (www.walt.nu) [23]. We then performed another meta-
analysis of anti-inflammatory drugs (NSAID) in the British Medical
Journal in 2004, with considerable international media attention.
Basically, this meta-analysis showed a lack of long-term evidence
and smaller effects than expected. Later in 2007, we investigated
physical therapeutic agents in knee OA in another meta-analysis
[24], where significant effects emerged when trials adhered to
WALT recommendations. These advances led to the inclusion of
our meta-analyses in the literature review, which formed the first
treatment recommendations from Osteoarthritis Research Society
International (OARSI). LLLT was given a 100% score for quality
of study and level 1a score for evidence and the number needed
to treat of 4 [25]. Despite this and due to the mention of LLLT in
only one-sixth of existing guidelines, the opportunity of having LLLT
established was lost. In other words, consensus—not evidence—
killed this opportunity. The positive thing was that the effectiveness
July 6, 2016 17:36 PSP Book - 9in x 6in 33-Hamblin-c33

600 Clinical Applications with Low-Level Laser Therapy in Arthritis

of LLLT was acclaimed in the official statement from OARSI. One

could expect that this positive trend would continue as more positive
LLLT studies were being published. And nine more LLLT studies
with largely positive results in knee OA have been published since
2007. But instead, OARSI updates just mentioned LLLT in the tables,
but omitted LLLT from the recommendations in 2010 [26], and in
the 2014 OARSI update, everything concerning LLLT was completely
omitted [15]. The situation reminds the author of Galileo Galilei and
that a consensus on the world being flat does not mean necessarily
that it is actually flat.
But the important thing is to digest how LLLT works and how
it can be used in different stages of OA. Indeed the possibility of
individually tailoring LLLT to the target pathology and optimizing
combination with other therapy forms is an exciting venue ulti-
mately in line with the current research agenda [9].

33.2 Pathoanatomy and Inflammation in Early-Stage OA

and Avenues for LLLT Irradiation

The dominant feature in early-stage OA is inflammation, with the

classical signs—dolor, calor, and tumor—applying for all affected
anatomical structures. In this stage, structural alterations are small,
but the implications at the tissue level vary by tissue type. The most
common OA joints are in descending order: the knee followed by
lumbar and cervical spine, hand, hip, shoulder, elbow, ankle, wrist,
and foot OA [27].

33.2.1 Synovia
Synovitis in combination with joint effusion is a feature that has
implications for pain, joint nutrition, and progression of disease
[28, 29]. An increasing number of macrophage cells populate the
synovia and partly replace fibroblast cells in symptomatic early OA.
The synovia encapsules the joint on all sides. In some joints such
as the hip joint, synovia is covered by large muscles and thick fat
layers in obese patients, making it difficult to reach synovia with
LLLT. Consequently, LLLT is not a good treatment option for hip OA.
July 6, 2016 17:36 PSP Book - 9in x 6in 33-Hamblin-c33

Pathoanatomy and Inflammation in Early-Stage OA and Avenues for LLLT Irradiation 601

Most other peripheral and spinal joints are more easily accessible
for LLLT and allow irradiation from many angles without overlying
muscle tissue.

33.2.2 Bone
The skeleton has a combination of stiffness and moderate trans-
parency to light [30], which makes LLLT well equipped to deliver
sufficient energy into the bone marrow of most human bones. In
addition, bone marrow edema is a distinct characteristic of OA and
a significant prognostic risk factor for structural deterioration and
disease progression.

33.2.3 Cartilage and Meniscii

The joint surfaces are covered with cartilage cushioning for load and
weight bearing. Cartilage is less transparent to laser light than bone,
but cartilage inflammation can be reduced by LLLT irradiation [31,
32]. It can however, be difficult to irradiate cartilage in the most
central parts of large joints. However, in the knee joint, OA is far more
prevalent in the medial compartment, and consequently fairly easily
accessible from the medial side. Both menisci are also quite suitable
for LLLT as they are situated along the outer edge of the knee joint.
The most posterior parts may be a little more difficult to reach as
they are covered by the m. gastrocnemius and hamstring tendons.

33.2.4 Peripheral Nerves and Pain Receptors

Perpiheral nerves may play a part in sensitization from longstanding
OA pain. The n. peroneus and n. ischiadicus are in some place only
covered by skin and thus easily accessible for LLLT. The distribution
of peripheral nerves and pain receptors also differs between tissue
types. The cartilage and cartilagous meniscii are considered to be
sparsely populated with pain receptors, whereas the bony cortex
and the synovia are more densely populated with pain receptors [1].
Irradiation of nerves in order to inhibit pain from sensitized tissue
has been described in the literature [33, 34], but its clinical relevance
in OA remains to be investigated.
July 6, 2016 17:36 PSP Book - 9in x 6in 33-Hamblin-c33

602 Clinical Applications with Low-Level Laser Therapy in Arthritis

33.3 Complex Relationship between Inflammation,

Tissue Interaction, and Structural Chondral Matrix
Degeneration in OA

Inflammation and degeneration are often interpreted as opponents,

and that they only exist when the other is not present. However,
recent research has changed our understanding in this area, as a
growing amount of evidence suggests that inflammation can be a
catalysator for degenerative processes in cartilage [35, 36]. It has
also become apparent that the interplay between different tissues
is more complex than previously thought [37]. Among other issues,
the important role of subchondral bone and its vascularization has
received more attention due to its contribution to preservation
of cartilage. While the first avenue for LLLT is modulation of
inflammation, the second avenue for LLLT is preservation and
repair of cartilage or meniscii. On the pharmacological side, none
of the pharmaceutical agents offer the same advantages as LLLT.
The drugs typically have one action: either to act as modulators
of inflammation or to act as protectors against cartilage degener-
ation. And drugs have proved less effective in the latter aspects
[38].

33.4 Why LLLT Works in OA?

Whether any of the drugs or LLLT can be characterized as disease-

modifying agents in OA (DMOA) remains an open question in the
absence of clinical evidence. But the theoretical rationale for LLLT
to become a DMOA intervention and the hope for reversing the
disease in early and intermediate stages of OA are still alive. In the
latter stages of OA where structural damage to cartilage matrix and
biomechanical misalignment is prevalent, the hope must be that
LLLT can relieve pain and at best stop the progression of the disease.
The experimental studies of LLLT have been reviewed in detail in
other chapters. A broad overview of the literature is summarized in
Tables 33.1 and 33.2.
July 6, 2016 17:36 PSP Book - 9in x 6in 33-Hamblin-c33

Recommended Doses of LLLT in Arthritis 603

Table 33.1 Summary of an overview of experimental studies of LLLT on

osteoarthritiss

Animal Studies (n) Share (%) Positive Results Human Positive Studies (n =)

Synovia 10 90% 2
Cartilage 19 83%
Meniscus 1 100% 1
Bone 37 67%

Table 33.2 LLLT randomized controlled clinical studies on various arthritic

locations

Number of Studies with

Anatomical Site Studies Positive Results Interpretation
Hand and finger OA 2 0 Negative results explained by
too short irradiation time (3 s)
or too small doses
Knee 14 11 Negative results explained by
(a) too low (810 nm, 2 J) or (b)
too high doses (810 nm, 1250 J),
or (c) only one point irradiated
Hip None
Cervical spine 1 1
Rheumatoid arthritis 9 6 Negative results can be
explained in one study by too
low dose and irradiation time
(785 nm, 0.18 J, 2.5 s)

33.5 Recommended Doses of LLLT in Arthritis

The dosage recommendations of WALT were first published on

their website www.walt.nu in August 2005. Since then the dosage
recommendations have continuously been supervised in view of
emerging literature, but only the dosage recommendations for
lumbar spine with 780–860 nm laser wavelengths have been
revised. The strength of the dosage recommendations has been
proven through two independent systematic reviews, which both
found significantly better effects in trials adhering to the WALT
dosage recommendations [39, 40]. The WALT guidelines for arthritis
are listed in Table 33.3 for the two most common LLLT wavelengths.
July 6, 2016 17:36 PSP Book - 9in x 6in 33-Hamblin-c33

604 Clinical Applications with Low-Level Laser Therapy in Arthritis

Table 33.3 Recommended anti-inflammatory dosage for LLLT

Laser class 3B, 780–860 nm GaAlAs lasersa

Diagnoses Minimum Area Minimum Total Dose
Arthritis Points or cm2 Joules Notes
Finger PIP or MCP 1–2 6
Wrist 2–4 10
Humeroradial joint 1–2 4
Elbow 2–4 10
Glenohumeral joint 2–4 15 Minimum 4 J per point
Acromioclavicular 1–2 4
Temporomandibular 1–2 6
Cervical spine 2–4 15 Minimum 4 J per point
Lumbar spine 2–4 30 Minimum 4 J per point
Hip 2–4 30 Minimum 6 J per point
Knee medial 3–6 20 Minimum 4 J per point
Ankle 2–4 15
Laser class 3B, 904 nm GaAs lasersb
Diagnoses Minimum Area Minimum Total Dose
Arthritis Points or cm2 Joules 904 nm Notes
Finger PIP or MCP 1–2 2
Wrist 2–3 3
Humeroradial joint 1–2 2
Elbow 2–3 3
Glenohumeral joint 2–3 2 Minimum 1 J per point
Acromioclavicular 1–2 2
Temporomandibular 1–2 2
Cervical spine 4 4 Minimum 1 J per point
Lumbar spine 4 4 Minimum 1 J per point
Hip 2 4 Minimum 2 J per point
Knee anteromedial 4–6 4 Minimum 1 J per point
Ankle 2–4 6

Source: Data reprinted with permission from WALT.

Note: Daily treatment for 2 weeks or treatment every other day for 3–4 weeks is recom-
mended. Irradiation should cover most of the pathological tissue in the tendon/synovia.
Therapeutic windows range from typically +/− 50% of given values. Recommended doses
are based on ultrasonographic measurements of depths from skin surface and typical
volume of pathological tissue and estimated optical penetration for the different laser types
in caucasians
a
Continuous or pulse output less than 0.5 W; irradiation time should range between 20 and
300 s; energy dose delivered to the skin over the target tissue.
b
Peak pulse output >1 W, mean output >5 mW, or power density >5 mW/cm2 ; effective
dose range is 0.5–4 J per point depending on depth to target; recommended irradiation
times range between 30 and 600 s depending on output.
Disclaimer: The list may be subject to change at any time when more research trials are
being published. The World Association of Laser Therapy or the authors are not responsible
for the application of laser therapy in patients.
July 6, 2016 17:36 PSP Book - 9in x 6in 33-Hamblin-c33

References 605

References

1. de Lange-Brokaar BJ, Ioan-Facsinay A, van Osch GJ, et al. Synovial

inflammation, immune cells and their cytokines in osteoarthritis: A
review. Osteoarthritis Cartilage. 2012; 20: 1484–1499.
2. Cross M, Smith E, Hoy D, et al. The global burden of hip and knee
osteoarthritis: Estimates from the global burden of disease 2010 study.
Ann Rheum Dis. 2014; 73: 1323–1330.
3. Takahashi H, Nakajima M, Ozaki K, et al. Prediction model for knee
osteoarthritis based on genetic and clinical information. Arthritis Res
Ther. 2010; 12: R187.
4. Felson DT, McLaughlin S, Goggins J, et al. Bone marrow edema and its
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139: 330–336.
5. Barbour KE, Hootman JM, Helmick CG, et al. Meeting physical activity
guidelines and the risk of incident knee osteoarthritis: A population-
based prospective cohort study. Arthritis Care Res (Hoboken). 2014; 66:
139–146.
6. Dunlop DD, Song J, Semanik PA, et al. Relation of physical activity time to
incident disability in community dwelling adults with or at risk of knee
arthritis: Prospective cohort study. BMJ. 2014; 348: g2472.
7. Apold H, Meyer HE, Nordsletten L, et al. Risk factors for knee replace-
ment due to primary osteoarthritis: A population based, prospective
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15: 217.
8. Lane NE, Brandt K, Hawker G, et al. OARSI-FDA initiative: Defining the
disease state of osteoarthritis. Osteoarthritis Cartilage. 2011; 19: 478–
482.
9. Conaghan PG, Kloppenburg M, Schett G, et al. Osteoarthritis research
priorities: A report from a EULAR ad hoc expert committee. Ann Rheum
Dis. 2014; 73: 1442–1445.
10. Hennig T, Haehre L, Hornburg VT, et al. Effect of home-based hand
exercises in women with hand osteoarthritis: A randomised controlled
trial. Ann Rheum Dis. 2014; doi:10.1136/annrheumdis-2013-204808.
11. Dziedzic K, Nicholls E, Hill S, et al. Self-management approaches for
osteoarthritis in the hand: A 2 × 2 factorial randomised trial. Ann Rheum
Dis. 2013; 74(1): 108–118.
12. Bennell KL, Egerton T, Martin J, et al. Effect of physical therapy on pain
and function in patients with hip osteoarthritis: A randomized clinical
trial. JAMA. 2014; 311: 1987–1997.
July 6, 2016 17:36 PSP Book - 9in x 6in 33-Hamblin-c33

606 Clinical Applications with Low-Level Laser Therapy in Arthritis

13. Davis AM and MacKay C. Osteoarthritis year in review: Outcome of

rehabilitation. Osteoarthritis Cartilage. 2013; 21: 1414–1424.
14. Bruyere O, Cooper C, Pelletier JP, et al. An algorithm recommendation for
the management of knee osteoarthritis in Europe and internationally:
A report from a task force of the European Society for Clinical and
Economic Aspects of Osteoporosis and Osteoarthritis (ESCEO). Semin
Arthritis Rheum. 2014; 44(3): 253–263
15. McAlindon TE, Bannuru RR, Sullivan MC, et al. OARSI guidelines for
the non-surgical management of knee osteoarthritis. Osteoarthritis
Cartilage. 2014; 22: 363–388.
16. Hochberg MC, Altman RD, April KT, et al. American College of
Rheumatology 2012 recommendations for the use of nonpharmacologic
and pharmacologic therapies in osteoarthritis of the hand, hip, and
knee. Arthritis Care Res (Hoboken). 2012; 64: 455–474.
17. Smink AJ, van den Ende CH, Vliet Vlieland TP, et al. “Beating
osteoARThritis”: Development of a stepped care strategy to optimize
utilization and timing of non-surgical treatment modalities for patients
with hip or knee osteoarthritis. Clin Rheumatol 2011; 30: 1623–1629.
18. Peter WF, Jansen MJ, Hurkmans EJ, et al. Physiotherapy in hip and knee
osteoarthritis: Development of a practice guideline concerning initial
assessment, treatment and evaluation. Acta Reumatol Port. 2011; 36:
268–281.
19. Hochberg MC, Altman RD, Brandt KD, et al. Guidelines for the medical
management of osteoarthritis. Part II. Osteoarthritis of the knee.
American College of Rheumatology. Arthritis Rheum. 1995; 38: 1541–
1546.
20. Jordan KM, Arden NK, Doherty M, et al. EULAR Recommendations 2003:
An evidence based approach to the management of knee osteoarthritis:
Report of a Task Force of the Standing Committee for International
Clinical Studies Including Therapeutic Trials (ESCISIT). Ann Rheum Dis.
2003; 62: 1145–1155.
21. Roddy E and Doherty M. Guidelines for management of osteoarthritis
published by the American College of Rheumatology and the European
League Against Rheumatism: Why are they so different? Rheum Dis Clin
North Am. 2003; 29: 717–731.
22. Bjordal JM, Couppe C, Chow RT, et al. A systematic review of low level
laser therapy with location-specific doses for pain from chronic joint
disorders. Aust J Physiother. 2003; 49: 107–116.
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23. Bjordal JM. Low Level Laser Therapy (LLLT) and World Association for
Laser Therapy (WALT) Dosage Recommendations. Photomed Laser Surg.
2012; 30: 61–62.
24. Bjordal JM, Johnson MI, Lopes-Martins RA, et al. Short-term efficacy
of physical interventions in osteoarthritic knee pain. A systematic
review and meta-analysis of randomised placebo-controlled trials. BMC
Musculoskelet Disord. 2007; 8: 51.
25. Zhang W, Moskowitz RW, Nuki G, et al. OARSI recommendations for the
management of hip and knee osteoarthritis, part I: Critical appraisal of
existing treatment guidelines and systematic review of current research
evidence. Osteoarthritis Cartilage. 2007; 15: 981–1000.
26. Zhang W, Nuki G, Moskowitz RW, et al. OARSI recommendations for the
management of hip and knee osteoarthritis part III: Changes in evidence
following systematic cumulative update of research published through
January 2009. Osteoarthritis Cartilage. 2010; 18: 476–499.
27. Zhang JF, Song LH, Wei JN, et al. Prevalence of and risk factors for the
occurrence of symptomatic osteoarthritis in rural regions of Shanxi
Province, China. Int J Rheum Dis. 2014; doi: 10.1111/1756-185X.12470.
28. Conaghan PG, D’Agostino MA, Le Bars M, et al. Clinical and ultrasono-
graphic predictors of joint replacement for knee osteoarthritis: Results
from a large, 3-year, prospective EULAR study. Ann Rheum Dis. 2010; 69:
644–647.
29. Hunter DJ, Zhang W, Conaghan PG, et al. Systematic review of
the concurrent and predictive validity of MRI biomarkers in OA.
Osteoarthritis Cartilage. 2011; 19: 557–588.
30. Aziz SM, Khambatta F, Vaithianathan T, et al. A near infrared instrument
to monitor relative hemoglobin concentrations of human bone tissue in
vitro and in vivo. Rev Sci Instrum. 2010; 81: 043111.
31. Carlos FP, de Paula Alves da Silva M, de Lemos Vasconcelos Silva Melo
E, et al. Protective effect of low-level laser therapy (LLLT) on acute
zymosan-induced arthritis. Lasers Med Sci. 2014; 29: 757–763.
32. Kamrava SK, Farhadi M, Rezvan F, et al. The histological and clinical
effects of 630 nanometer and 860 nanometer low-level laser on rabbits’
ear punch holes. Lasers Med Sci. 2009; 24(6): 949–954.
33. Hagiwara S, Iwasaka H, Hasegawa A, et al. Pre-irradiation of blood
by gallium aluminum arsenide (830 nm) low-level laser enhances
peripheral endogenous opioid analgesia in rats. Anesth Analg. 2008;
107: 1058–1063.
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608 Clinical Applications with Low-Level Laser Therapy in Arthritis

34. Chow R, Armati P, Laakso EL, et al. Inhibitory effects of laser irradiation
on peripheral mammalian nerves and relevance to analgesic effects: A
systematic review. Photomed Laser Surg. 2011; 29: 365–381.
35. Tsuzaki M, Guyton G, Garrett W, et al. IL-1 beta induces COX2, MMP-1, -3
and -13, ADAMTS-4, IL-1 beta and IL-6 in human tendon cells. J Orthop
Res. 2003; 21: 256–264.
36. Koshy PJ, Henderson N, Logan C, et al. Interleukin17 induces cartilage
collagen breakdown: Novel synergistic effects in combination with
proinflammatory cytokines. Ann Rheum Dis. 2002; 61: 704–713.
37. Berenbaum F. Osteoarthritis as an inflammatory disease (osteoarthritis
is not osteoarthrosis!) Osteoarthritis Cartilage. 2013; 21: 16–21.
38. Moon SJ, Park JS, Jeong JH, et al. Augmented chondroprotective effect
of coadministration of celecoxib and rebamipide in the monosodium
iodoacetate rat model of osteoarthritis. Arch Pharm Res. 2013; 36: 116–
124.
39. Jang H, Lee H. Meta-analysis of pain relief effects by laser irradiation on
joint areas. Photomed Laser Surg. 2012; 30: 405–417.
40. Tumilty S, Munn J, McDonough S, et al. Low-level laser treatment of
tendinopathy: a systematic review with meta-analysis. Photomed Laser
Surg. 2010; 28(1): 3–16.
July 11, 2016 10:14 PSP Book - 9in x 6in 34-Hamblin-c34

Chapter 34

Use of Low-Level Laser Therapy and

Light-Emitting Diode Therapy to Improve
Muscle Performance and Prevent
Damage: From Animal Models to Clinical
Trials

Cleber Ferraresi,a Nivaldo Parizotto,b Vanderlei Bagnato,c and

Michael R. Hamblina,d,e
a Wellman Center for Photomedicine, Massachusetts General Hospital,

50 Blossom Street, Boston, MA 02114, USA

b Federal University of São Carlos, Rodovia Washington Luı́s, km 235, São Carlos,

São Paulo 13565-905, Brazil

c São Carlos Institute of Physics, University of São Paulo, São Carlos, São Paulo

13566-590, Brazil
d Department of Dermatology, Harvard Medical School, Massachusetts General

Hospital, 55 Fruit Street, Boston, MA 02114, USA

e Harvard-Massachusetts Institute of Technology (MIT) Division of Health Sciences and

Technology, 77 Massachusetts Avenue, E25-518, Cambridge, MA 02139, USA

cleber.ferraresi@gmail.com

34.1 Introduction

Low-level laser therapy (LLLT) and light-emitting diode therapy

(LEDT) have been used as an important therapeutic approach to

Handbook of Low-Level Laser Therapy

Edited by Michael R. Hamblin, Marcelo Victor Pires de Sousa, and Tanupriya Agrawal
Copyright c 2017 Pan Stanford Publishing Pte. Ltd.
ISBN 978-981-4669-60-3 (Hardcover), 978-981-4669-61-0 (eBook)
www.panstanford.com
July 11, 2016 10:14 PSP Book - 9in x 6in 34-Hamblin-c34

610 Use of Low-Level Laser Therapy and Light-Emitting Diode Therapy

manage pain [1, 2], treat tendinopathies [3], or accelerate tissue

repair [2, 4]. Over the last decade, these therapies using red and/or
infrared wavelengths have been applied to skeletal muscles to
prevent damage, mainly analyzed by creatine kinase (CK) levels in
blood, accelerate muscle recovery after intense exercises, and also
increase muscle performance [5–7].
There are literature reports of two main strategies to apply
LLLT or LEDT to increase muscle performance in exercise. The
first strategy is to deliver light before the onset of exercise
(called muscular preconditioning in this chapter). This strategy
has demonstrated huge potential to prevent muscle damage and
increase resistance to muscle fatigue when applied usually about 5
min before a bout of exercise [5–7]. However, recent studies have
suggested that 5 min may not necessarily be the best time [8, 9].
The second strategy is to apply LLLT or LEDT immediately after
the exercise in order to accelerate muscle recovery [5]. Although
there is no consensus about the best moment to use LLLT or LEDT,
recent studies have found that light therapy can achieve better
muscle performance when these therapies are applied after a bout
of exercise [10, 11].
This chapter reviews the literature about the use of LLLT
and LEDT to improve muscle performance and prevent damage
in experimental studies that have provided scientific basis for
the development of clinical trials over the last two decades. All
parameters used in these studies are summarized in Tables 34.1
and 34.2.

34.2 Experimental Models Using LLLT to Enhance Muscle

Performance and Prevent Damage

To our knowledge, the first study using LLLT for muscular

preconditioning, i.e., irradiation before the exercise or induction
of muscle contraction was conducted by Lopes-Martins et al. [12].
The authors induced fatigue in the tibialis anterior muscle of rats
using a model of neuromuscular electrical stimulation. They applied
different doses of light immediately before induction of muscle
fatigue. They found that LLLT (655 nm) with a dose of 0.5 J/cm2
July 11, 2016 10:14 PSP Book - 9in x 6in 34-Hamblin-c34

Experimental Models Using LLLT to Enhance Muscle Performance and Prevent Damage 611

reduced muscle fatigue, and doses of 1.0 or 2.5 J/cm2 also decreased
muscle damage as measured by CK.
These aforementioned results stimulated other researchers to
conduct other experimental studies in order to better identify
LLLT effects on muscle tissue subjected to different protocols of
physical exercises as well as to increase the understanding of the
mechanisms of action involved in light–muscle interaction to reduce
muscle fatigue and damage.
Liu et al. [13] conducted a protocol of training until exhaustion in
rats using a declined treadmill running at 16 m/min. They showed
that LLLT (632.8 nm) applied on one site on the gastrocnemius
muscle in rats after the exercise inhibited inflammation, reduced CK
and malondialdehyde, and also increased oxidative stress defense
through increased activity of superoxide dismutase (SOD).
Using a protocol of swimming exercise to induce fatigue and
muscle damage in rats, Sussai et al. [14] investigated the effects of
LLLT (660 nm) on CK levels in blood and also muscle cell apoptosis.
LLLT was applied during 40 s with one site of irradiation on the
gastrocnemius muscle immediately after the swimming protocol.
Compared to the control group, animals treated with LLLT showed
lower levels of CK and cell apoptosis 24 h and 48 h after muscle
fatigue induction.
Patrocinio et al. [15] investigated the effects of LLLT on muscle
performance using an inclined ladder for training rats. The climbing
exercise on ladder used loads attached to the rat’s tail during a
training program consisting of a strength exercise performed three
times per week during 5 weeks. The initial load was 75% of the rat’s
body weight plus an addition of 30 g per set of exercise with a total of
four sets. After each training session, both tibialis anterior muscles
were irradiated with LLLT (830 nm) at 120 J/cm2 (3.3 J). Although
the biomechanics of the exercise does not require the use of the
tibialis anterior muscle, the results pointed to a reduction in lactate
levels in blood, decreased glycogen depletion, and increased muscle
cross-sectional area in animals treated with LLLT when compared
to control and training group without LLLT, suggesting probably
systemic effects of LLLT.
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612 Use of Low-Level Laser Therapy and Light-Emitting Diode Therapy

The model of electrical stimulation to induce muscle fatigue and

damage reported by Lopes-Martins et al. [12] was replicated in other
studies to investigate additional effects of LLLT on muscle tissue.
As examples of these studies, Leal-Junior et al. [16] investigated the
effects of LLLT (904 nm) on muscle fatigue in rats. LLLT was applied
over one site of the tibialis anterior muscle before the muscle fatigue
protocol (muscular preconditioning). The authors reported that 1 J
and 3 J significantly increased peak force produced by the muscle
compared to control (without LLLT), 0.1 J, and 0.3 J groups. Blood
lactate levels were lower in all groups treated with LLLT. CK levels
had the same response, except for the 3 J group.
Using the same methodology of Leal-Junior et al. [16], de Almeida
et al. [17] showed that LLLT (904 nm, 1 J) significantly decreased
CK levels in blood, reduced gene expression of cyclooxygenase-
2 (COX-2), and increased gene expression of cyclooxygenase-1
(COX-1) when compared to all groups (0.1, 0.3, and 3 J). The
authors concluded that LLLT was effective to prevent muscle damage
and could modulate gene expression related to the inflammatory
process.
Recently, Santos et al. [18] investigated the effects of LLLT when
applied immediately before tetanic contractions induced by neu-
romuscular electrical stimulation during 60 min (one contraction
every 10 min). They compared different wavelengths (660, 830, and
905 nm) and light energies (1, 3, and 10 J). The results showed
a better reduction in fatigue using 660 nm/3 J and 905 nm/1 J.
Regarding muscle damage, only 660 nm/1 J was able to significantly
reduce CK levels in blood. In addition, this study assessed the muscle
morphology that revealed lower muscle damage in all animals
treated with 660 nm/1 J and 3 J; 905 nm/1 J, 3 J, and 10 J.
Albuquerque-Pontes et al. [19] investigated the effects of
different wavelengths of LLLT (660, 830, and 905 nm), light doses
(1, 3, and 10 J), and time response (5, 10, and 30 min; 1, 2, 12, and
24 h) on cytochrome c oxidase activity in muscles of rats without
stress of any kind of exercise. They reported an increased activity of
cytochrome c oxidase with LLLT 660 nm/1 J, 830 nm/3 J, and 905
nm/1 J at all times tested.
July 11, 2016 10:14 PSP Book - 9in x 6in 34-Hamblin-c34

Experimental Models Using LEDT to Enhance Muscle Performance 613

34.3 Experimental Models Using LEDT to Enhance Muscle

Performance and Damage Prevention

Similar to the study reported by Sussai et al. [14], Camargo et al.

[20] used a protocol of swimming exercise to induce muscle fatigue
and damage in rats. This study compared animals subjected to
swimming exercise during 100 min to a control group (without
exercise). In addition, all animals that swam were allocated into
three groups: swim without therapy for muscle recovery after the
exercise; swim and cryotherapy by water immersion at 10◦ C for
10 min; swim plus LEDT (940 nm) applied over both lower limbs.
The authors reported that inflammation and soleus muscle necrosis
decreased significantly when LEDT was used as a therapy for muscle
recovery, compared to other animals that swam and received no
treatment or cryotherapy. In addition, only the group treated with
LEDT significantly decreased CK levels in blood 24 h after the
exercise protocol.
Corazza et al. [21] investigated the effects of LEDT (850 nm) on
muscle volume and inflammatory markers in ovariectomized rats
subjected to a training protocol with progressive loads attached to
the animals (50–80% of the rat’s body weight) during 12 weeks.
The authors found that all groups subjected to exercise (training
alone and training plus LEDT after each training session) and
LEDT (LEDT without training) increased muscle volume and the
concentration of insulin-like growth factor (IGF-1) compared to
the control group (ovariectomized, non-trained and non-irradiated
with LEDT). Furthermore, only the training plus LEDT group
significantly increased interleukin-6 (IL-6, an anti-inflammatory
marker) compared to the training, LEDT and control groups. On the
other hand, the control group had lower pro-inflammatory markers
concentration such as tumor necrosis factor-alpha (TNF-α) and
interleukin-1-beta (IL-1β) compared to all groups.
Another recent study compared different times of LEDT
irradiation (630 nm plus 850 nm simultaneously) over the lower
limbs and lower-back muscles of mice during an acute and
progressive strength training performed on an inclined ladder [11].
The authors assessed muscle performance (maximum load, muscle
power, and work), oxidative stress, energy supply (muscle adenosine
July 11, 2016 10:14 PSP Book - 9in x 6in 34-Hamblin-c34

614 Use of Low-Level Laser Therapy and Light-Emitting Diode Therapy

triphosphate [ATP] and glycogen), muscle cell proliferation, and

mitochondrial metabolism using different regimens of LEDT: LEDT-
Sham, LEDT-Before (5 min, muscular preconditioning), LEDT-
Before-After (5 min, muscular preconditioning and immediately
after each training session), and LEDT-After (immediately after each
training session). The authors found that LEDT-Before and LEDT-
Before-After gave improved muscle performance at the first training
session. However, LEDT-After and LEDT-Before-After were superior
at the end of the training program consisting of six sessions. Both
LEDT-Before-After and LEDT-After groups had higher mitochondrial
metabolism (cytochrome c oxidase activity), energy supply content
(ATP and glycogen), muscle cell proliferation, and oxidative stress
defense. The authors concluded that for a single bout of exercise
about to be undertaken, muscular preconditioning could represent
an important strategy for muscle improvement. However, during
ongoing training programs, the best moment to apply LEDT is after
the exercise or after each training session.
In order to assess the effects of cryotherapy and LEDT on muscle
damage, da Costa Santos et al. [22] evaluated the time to exhaustion,
levels of CK in blood, C-reactive protein, and inflammation in the
gastrocnemius muscle of rats subjected to a swimming protocol.
This exercise was divided into two phases: the first consisted of
45 min of swimming, and during the second phase the animals
swam until exhaustion. During the rest interval between each phase
(25 min), the authors used LEDT (940 nm) or cryotherapy (water
immersion at 10◦ C during 10 min) or passive recovery (without
any therapy) in order to recover muscle performance. Both groups
treated with LEDT or cryotherapy increased the time to achieve
exhaustion at the second phase of the exercise protocol when
compared to passive recovery. However, there was no significant
difference between LEDT and cryotherapy. All groups subjected to
exercise increased the number of neutrophils in the gastrocnemius.
Passive recovery and cryotherapy groups showed leukocytosis when
compared to LEDT and control groups. In addition, the levels of CK in
blood presented no statistical difference among cryotherapy, passive
recovery, and LEDT groups. However, only LEDT group significantly
decreased C-reactive protein compared to the cryotherapy group
24 h after the exercise.
 July 11, 2016 10:14
Table 34.1 Experimental models using LLLT and LEDT to muscle performance/damage prevention

Reference LLLT/LEDT Parameters Muscle/Exercise Irradiation

(12) Diode laser 655 nm Tibialis anterior Contact
Diode area 0.08 cm2 ; 2.5 mW; 31.25 mW/cm2
Groups: 0.5 J/cm2 (32 s)
1 J/cm2 (80 s)
2.5 J/cm2 (160 s)

Experimental Models Using LEDT to Enhance Muscle Performance 615

One site of irradiation Neuromuscular electrical stimulation Muscular preconditioning
(13) Diode laser 632 nm Gastrocnemius Contact
Diode area 0.2 cm2
Groups: 12 J/cm2 ; 4 mW, 20 mW/cm2 , 10 min

PSP Book - 9in x 6in

28 J/cm2 ; 9 mW, 46 mW/cm2 , 10 min
43 J/cm2 ; 14 mW, 71 mW/cm2 , 10 min
One site of irradiation Treadmill After exercise
(16) Diode laser 904 nm Tibialis anterior Contact
Diode area 0.2 cm2 ; 15 mW, 75 mW/cm2
Groups: 0.1 J (7 s)
0.3 J (20 s)
1 J (67 s)
3 J (200 s)
One site of irradiation Neuromuscular electrical stimulation Muscular preconditioning
(14) Diode laser 660 nm Gastrocnemius Contact
Diode area 0.03 cm2 ; 100 mW; 3.3 mW/cm2
4 J por diodo (40 s); 133.3 J/cm2
One site of irradiation Swimming After exercise

(Contd.)

34-Hamblin-c34
 July 11, 2016 10:14
616 Use of Low-Level Laser Therapy and Light-Emitting Diode Therapy
Table 34.1 (Contd.)

Reference LLLT/LEDT Parameters Muscle/Exercise Irradiation

(17) Diode laser 904 nm Tibialis anterior Contact

Diode area 0.2 cm2 ; 15 mW; 75 mW/cm2
Groups: 0.1 J (7 s)
0.3 J (20 s)
1 J (67 s)
3 J (200 s)
One site of irradiation Neuromuscular electrical stimulation Muscular preconditioning
(15) Diode laser 808 nm Tibialis anterior Contact
Diode area 0.028 cm2 ; 100 mW; 3,571 mW/cm2

PSP Book - 9in x 6in

3.3 J por ponto (33 s), 120 J/cm2
One site of irradiation Climbing ladder After exercise
(18) Diode lasers 660 nm, 830 nm, and 905 nm Tibialis anterior Contact
Diode area 0.028 cm2 ; 50 mW; 1,785 mW/cm2
Groups: 660 nm: 1 J (20 s); 3 J (60 s) and 10 J (200 s)
830 nm: 1 J (20 s); 3 J (60 s) and 10 J (200 s)
905 nm: 1 J (20 s); 3 J (60 s) and 10 J (200 s)
One site of irradiation Neuromuscular electrical stimulation Muscular preconditioning
(19) Diode lasers 660 nm, 830 nm, and 905 nm Tibialis anterior Contact
Diode area 0.028 cm2 ; 50 mW; 1,785 mW/cm2
Groups: 660 nm: 1 J (20 s); 3 J (60 s) e 10 J (200 s)
830 nm: 1 J (20 s); 3 J (60 s) e 10 J (200 s)
905 nm: 1 J (20 s); 3 J (60 s) e 10 J (200 s)
One site of irradiation cytochrome c oxidase activity

34-Hamblin-c34
 July 11, 2016 10:14
(20) LED 940 nm Triceps surae Without contact
LED area calculated 17.4 cm2 ; 160 mW; 9.5 mW/cm2
Groups: LED 69.6 J (7 min and 15 s), 4 J/cm2
Cryotherapy 10 min at 10◦ C
One site of irradiation Swimming After exercise
(21) LED 850 nm Greater trochanter and rectus femoris Contact
LED area 0.5 cm2 ; 100 mW, 200 mW/cm2

Experimental Models Using LEDT to Enhance Muscle Performance 617

60 J per site (10 min), 120 J/cm2
Two sites of irradiation: 120 J Jump with load After exercise
(22) LED 940 nm Triceps surae Without contact
LED area calculated 17.4 cm2 ; 160 mW; 9.5 mW/cm2

PSP Book - 9in x 6in

Groups: LED 69.6 J (7 min 15 s), 4 J/cm2
Cryotherapy 10 min at 10◦ C
1 point of irradiation Swimming/recovery and swimming until exhaustion Rest interval (recovery)
(9) Cluster with 20 red LEDs (630 nm) plus 20 infrared LEDs Lower limbs and lower-back muscles Without contact
(850 nm)
LED area cm2 ; 500 mW (red); 1000 mW (infrared);
80 mW/cm2 ; 7.2 J/ cm2 ; 90 s
Groups: LEDT-Sham
LEDT-5min
LEDT-3h
LEDT-6h
LEDT-24h Climbing ladder Muscular preconditioning

(Contd.)

34-Hamblin-c34
 July 11, 2016 10:14
618 Use of Low-Level Laser Therapy and Light-Emitting Diode Therapy
Table 34.1 (Contd.)

Reference LLLT/LEDT Parameters Muscle/Exercise Irradiation

(11) Cluster with 20 red LEDs (630 nm) plus 20 infrared LEDs Lower limbs and lower-back muscles Without contact

PSP Book - 9in x 6in

(850 nm)
LED area cm2 ; 500 mW (red); 1000 mW (infrared);
80 mW/cm2 ; 7.2 J/ cm2 ; 90 s
Groups: LEDT-Sham
LEDT-Before
LEDT-Before-After
LEDT-After Climbing ladder Muscular preconditioning

34-Hamblin-c34
July 11, 2016 10:14 PSP Book - 9in x 6in 34-Hamblin-c34

Clinical Trials Using LLLT to Increase Muscle Performance and Prevent Damage 619

Using red (630 nm) and infrared (850 nm) light simultaneously,
Ferraresi et al. [9] applied LEDT over the lower limbs and lower-
back muscles of mice in a muscular preconditioning protocol.
They identified a time response in skeletal muscles (soleus and
gastrocnemius) for the increased content of ATP. In addition, this
same study identified a very similar time response for resistance
to muscle fatigue tested with a fatigue protocol using an inclined
ladder and a load of 150% of the mouse’s body weight attached to
the mouse tail. The results showed that muscular preconditioning
applied 6 h before exercise (LEDT-6h) sharply increased the ATP
contents compared to all groups: LEDT-Sham, LEDT-5min, LEDT-3h
and LEDT-24h. In addition, LEDT-6h increased the number of climbs
on the ladder that could be accomplished around 600% compared
to LEDT-Sham and LEDT-5min, 200% compared to LEDT-3h, and
300% compared to LEDT-24h. Finally, the authors reported a high
correlation between ATP content and resistance to muscle fatigue.
Table 34.1 presents all the parameters of the LLLT regimens used
in experimental models.

34.4 Clinical Trials Using LLLT to Increase Muscle

Performance and Prevent Damage: Acute Responses

One of the first studies published in this research field was

conducted by Gorgey et al. [23]. They performed a muscular
preconditioning protocol using LLLT (808 nm) for 5 min (low
energy, 3 J) and 10 min (high energy, 7 J) over the quadriceps femoris
muscles before induction of fatigue by neuromuscular electrical
stimulation. Compared to the control group, both groups that
were irradiated with LLLT had less fatigue but without statistical
significance. In addition, there was no statistical difference between
both groups treated with LLLT.
Leal-Junior et al. [24] also performed a muscular preconditioning
protocol applying LLLT (655 nm) on the biceps brachii muscle
before exercise on a Scott bench until exhaustion. This exercise
had a load of 75% of maximum voluntary contraction (MVC), and
LLLT irradiation was applied on four sites of the biceps brachii.
The number of repetitions was increased significantly in all subjects
July 11, 2016 10:14 PSP Book - 9in x 6in 34-Hamblin-c34

620 Use of Low-Level Laser Therapy and Light-Emitting Diode Therapy

that received LLLT compared to the placebo group. However, blood

lactate and time expended to accomplish the exercise were not
significantly altered.
Reproducing the same aforementioned study design, the authors
then investigated the effects of LLLT (830 nm) applied on four sites
of the biceps brachii muscle before MVCs on a Scott bench [25]. The
results were similar to the previous study [24] showing increased
number of repetitions compared to the placebo group. In a similar
study, de Almeida et al. [26] compared red (660 nm) and infrared
(830 nm) wavelengths applied on the biceps brachii muscle before
MVCs on a Scott bench in a muscular preconditioning protocol.
The authors identified significant differences between the placebo
group and both groups irradiated with LLLT. However, there was
no difference between both wavelengths regarding the peak torque
developed by each group irradiated.
Another study reported by Leal-Junior et al. [27] used a cluster
of five laser diodes (810 nm) applied on the biceps brachii muscle
before the same exercise in a Scott bench described in previous
studies. LLLT was applied on two sites of the biceps brachii during
30 s and led to an increased number of repetitions and time of
contraction, decreased lactate levels in blood as well as CK and C-
reactive protein after the exercise.
On the other hand, recently Higashi et al. [28] tried to replicate
the same results reported by Leal-Junior et al. [27]. However,
the LLLT (808 nm) applied on biceps brachii muscle in muscular
preconditioning was not able to increase the number of repetitions
on a Scott bench and was not able to significantly reduce lactate
levels in blood.
Other anatomical muscle groups have been irradiated with LLLT
in order to increase muscle performance in different exercises. As
examples of these studies, Leal-Junior et al. [29] irradiated the rectus
femoris muscle before Wingate tests in a muscular preconditioning
study design. These authors reported no significant effects of the
LLLT (830 nm) on muscle performance, but the levels of CK and
lactate in the blood were decreased when compared to the placebo
group. Baroni et al. [30], also using a muscular preconditioning
with LLLT (cluster of laser diodes, 810 nm), investigated the energy
metabolism, muscle damage, and delayed onset of muscle soreness
July 11, 2016 10:14 PSP Book - 9in x 6in 34-Hamblin-c34

Clinical Trials Using LLLT to Increase Muscle Performance and Prevent Damage 621

after a protocol of eccentric exercise performed using an isokinetic

dynamometer. LLLT was applied on six sites of the quadriceps
femoris muscles and produced increased lactate dehydrogenase
(LDH) activity at 48 h after exercise, decreased CK in blood after 24 h
and 48 h, and improved MVC immediately and 24 h after the exercise
when compared to the placebo group. However, the delayed onset of
muscle soreness was equal for LLLT and placebo groups.
Using surface electromyography to identify muscle fatigue during
60 s of leg extension exercise and measuring the maximal number
of repetitions, Toma et al. [31] applied LLLT (808 nm) on the
quadriceps femoris muscles in a muscular preconditioning regimen.
The authors reported an increased number of repetitions of the
LLLT group compared to placebo, but surface electromyography
showed no statistical difference. In similar studies, another two
clinical trials were conducted in order to identify the effects of
LLLT on the surface electromyography signal. Dos Santos Maciel
et al. [32] investigated the effects of LLLT (780 nm) on muscle
fatigue of the tibialis anterior muscle in a isokinetic dynamometer
associated with surface electromyography. They reported increased
torque exerted by the tibialis anterior muscle with muscular
preconditioning using LLLT, but muscle fatigue analyzed by surface
electromyography was not reduced, and lactate levels in blood
were not significantly different between the groups. On the other
hand, de Brito Vieira et al. [33] applied LLLT (808 nm) on the
quadriceps femoris muscles in subjects doing three sets of 20
maximum voluntary repetitions in an isokinetic dynamometer at
a single training session. Two days after the training session,
all volunteers were evaluated through the number of maximum
repetitions of knee flexion/extension in an isokinetic dynamometer
in conjunction with surface electromyography to identify muscle
fatigue. The authors reported that LLLT increased the number of
repetitions and promoted a lower muscle fatigue compared to the
placebo group.
Other studies have tried to study the acute effects of LLLT on
muscle performance when applied in muscular preconditioning
regimens. De Marchi et al. [34] used a cluster of five laser
diodes (810 nm) applied over the quadriceps femoris muscles,
hamstrings, and triceps surae before a protocol of progressive
July 11, 2016 10:14 PSP Book - 9in x 6in 34-Hamblin-c34

622 Use of Low-Level Laser Therapy and Light-Emitting Diode Therapy

and maximum effort on a treadmill. The authors assessed muscle

fatigue, oxidative stress, and muscle damage. This study showed that
LLLT could increase relative and absolute oxygen uptake and the
duration of exercise (time of running on treadmill) when compared
with placebo group. In addition, LDH, muscle damage (CK), and
lipid peroxidation (TBARS, thiobarbituric acid reactive substances)
were significantly higher in the placebo group. Finally, the SOD
enzyme activity was lower only in the placebo group after the
exercise.
In a similar study, de Brito Vieira et al. [33] and Felismino et al.
[35] applied LLLT (808 nm) on the biceps brachii muscle between
sets of 10 series of 10 repetitions with a load corresponding to
50% of that of the maximum single repetition (1RM) during exercise
consisting of elbow flexion/extension on a Scott bench. The results
of this study pointed to a significant reduction in muscle damage
(CK) 72 h after the exercise test compared to the placebo group.
However, there was no significant difference in muscle performance
(load) between groups.
In an attempt to find the best time to apply LLLT, i.e., before
(muscular preconditioning) or after the exercise, Dos Reis et al. [10]
compared the effectiveness of the LLLT (830 nm) applied on the
quadriceps femoris muscles before or after an exercise protocol of
leg extension with 75% of 1RM. The authors reported that there
was no significant difference regarding the number of maximum
repetitions performed by all groups. However, lactate and CK levels
in blood were lower in the group that received LLLT after the
exercise protocol when compared to placebo and LLLT before the
exercise.
Recently, Antonialli et al. [36] used a diode laser (905 nm)
and LEDs (640 nm and 875 nm) in the same device to stimulate
muscle recovery when applied as muscular preconditioning on
quadriceps femoris muscles before a fatigue protocol with eccentric
contractions and MVCs in an isokinetic dynamometer. Muscular
preconditioning using LLLT and LEDs was performed 3 min before
the exercise protocol. The authors reported that light energies of
10 J and 30 J per site of irradiation increased the peak torque
of MVC immediately and lasting until 96 h after the muscular
July 11, 2016 10:14 PSP Book - 9in x 6in 34-Hamblin-c34

Clinical Trials Using LEDT to Improve Muscle Performance and Prevent Damage 623

preconditioning. Delayed muscle soreness was reduced significantly

using light energies of 30 J and 50 J when compared with the placebo
group. Finally, the authors reported a significant reduction in CK
with light energies of 10, 30, and 50 J.

34.5 Clinical Trials Using LLLT to Enhance Muscle

Performance and Damage Prevention: Chronic
Responses

Ferraresi et al. [37] studied the effects of LLLT (cluster with six
laser diodes, 808 nm) on a strength training program using a load
of 80% of 1RM, 2 days per week during 12 weeks. Immediately
after each training session, the LLLT was applied on seven sites of
the quadriceps femoris muscles. The authors reported an increased
load in the 1RM of the LLLT group compared to control and training
without LLLT groups. In addition, only the LLLT group increased
peak torque assessed by an isokinetic dynamometer.
The second study was conducted by Vieira et al. [38], who
measured the effects of LLLT (cluster with six laser diodes, 808 nm)
on moderate training using a cycle ergometer performed 3 days
per week during 9 weeks. LLLT was applied on five sites of the
quadriceps femoris muscles immediately after each training session.
As results, the authors reported that only the LLLT group showed a
reduced fatigue index of the knee extensor muscles in the isokinetic
dynamometer.

34.6 Clinical Trials Using LEDT to Improve Muscle

Performance and Prevent Damage: Acute Responses

To our knowledge, the first study was conducted by Vinck et al.

[39], who investigated the effects of LEDT (cluster of 32 LEDs,
950 nm) on delayed onset muscle soreness after fatigue induction
of the biceps brachii muscles in an isokinetic dynamometer. The
authors reported no significant difference between LEDT and
July 11, 2016 10:14 PSP Book - 9in x 6in 34-Hamblin-c34

624 Use of Low-Level Laser Therapy and Light-Emitting Diode Therapy

placebo therapies regarding peak torque and pain. However, using

a similar methodology, Douris et al. [40] applied LEDT (cluster
of 36 LEDs, 880 nm and 660 nm) on biceps brachii muscles
after elbow flexion/extension with free weights and reported a
significant decrease in delayed onset muscle soreness after 48 h
when compared to the control group.
Kelencz et al. [41] tested the effectiveness of LEDT (640 nm) to
reduce muscle fatigue in the masseter muscle. The authors tested
three light energies (1.04, 2.08, and 3.13 J) per site of irradiation
and concluded that 2.08 J was the best light energy to increase
the time of exercise (oral occlusion) before appearance of muscle
fatigue.
Recently, Leal-Junior et al. [42] applied LEDT (cluster of 69 LEDs,
850 nm and 660 nm) on one site of irradiation at the belly of the
biceps brachii muscle. They performed a muscular preconditioning
protocol before elbow flexion/extension exercise with a load of 75%
of MVC in a Scott bench. The LEDT group increased the number of
repetitions and the duration of exercise compared to the placebo
group. In addition, LEDT decreased blood levels of lactate, CK, and
C-reactive protein compared to the placebo group. In a similar study,
Baroni et al. [43] also investigated the effects of the same LEDT
cluster (69 LEDs, 850 nm and 660 nm) applied on three sites of the
quadriceps femoris muscles before a fatigue protocol performed in
an isokinetic dynamometer for knee flexion/extension. The authors
reported that LEDT was able to decrease the decay of peak torque
exerted compared to the placebo group.
Investigating the effects of LEDT (640 nm) on muscle recovery af-
ter muscle damage induced by eccentric flexion/extension exercise
for the biceps brachii muscles, Borges et al. [44] reported a delayed
onset of muscle soreness and slower decay in isometric force in the
LEDT group when compared to the placebo group. In addition, the
authors also reported less restriction or a smaller decrease in
the range of motion in the LEDT group 24, 48, 72, and 96 h after
the induction of muscle damage.
Although good results have been reported in previous studies
using LEDT, Denis et al. [45] did not find beneficial results when
LEDT (cluster of 69 LEDs, 950 nm and 660 nm) was applied during
July 11, 2016 10:14 PSP Book - 9in x 6in 34-Hamblin-c34

Clinical Trials Using LEDT to Improve Muscle Performance and Prevent Damage 625

the rest intervals of the Wingate tests. They reported no significant

differences in muscle peak power, fatigue index, and blood lactate
levels.
In order to compare the effects of LLLT (single diode, 810 nm)
and LEDT (cluster of 69 LEDs, 850 nm and 660 nm), Leal-Junior et al.
[46] investigated the muscle performance of athletes subjected to
Wingate tests. LLLT or LEDT was applied on two sites of the rectus
femoris muscle before the exercise (muscular preconditioning).
There was a significant decrease in CK levels in blood of the LEDT
group compared to the placebo and LLLT groups. However, there
was no improvement in the muscle performance or reduction in
lactate levels in the blood of the LEDT group compared to the
placebo and/or LLLT groups.
In a similar study, Leal-Junior et al. [47] also compared the effects
of LEDT (cluster of 69 LEDs, 850 nm and 660 nm) to the use of
cold-water immersion for promoting muscle recovery. Six athletes
performed three Wingate tests on nonconsecutive days and received
either LEDT or cold-water immersion of the lower limbs (5◦ C for
5 min) as therapy. LEDT was applied on two sites of the quadriceps
femoris muscle, two sites of the hamstrings, and two sites of triceps
surae. Comparing both groups, LEDT significantly decreased the
levels of CK and lactate in blood but was not able to increase
muscle work in the Wingate test and did not change C-reactive
protein.
In order to test the effectiveness of LEDT on the prevention
of muscle damage, Ferraresi et al. [8] used an array of 200
LEDs (100 LEDs 850 nm, and 100 LEDs 630 nm) applied on
the quadriceps femoris, hamstrings, and triceps surae muscles of
volleyball players during a national championship. The authors used
LEDT as muscular preconditioning, i.e., LEDT was applied before
each official match to prevent muscle damage (CK). In addition, this
study tested four light energies: 105, 210, and 315 J and placebo.
The authors reported that effective light energies to prevent the
rise in CK were 210 J and 315 J, while 105 J and placebo allowed
significant increase in CK to occur in the blood 24 h after each official
match.
 July 11, 2016 10:14
626 Use of Low-Level Laser Therapy and Light-Emitting Diode Therapy
Table 34.2 Clinical trials using LLLT and LEDT to muscle performance/damage prevention

Reference LLLT/LEDT parameters Músculo/exercı́cio Irradiation

(23) Cluster with four diode lasers 808 nm; Quadriceps femoris Scanning
500 mW; 8.3 mW/cm2
Groups: 7 J (10 min)
3 J (5 min)
One site of irradiation Isokinetic dynamometer Muscular preconditioning
(24) Diode laser 655 nm Biceps brachii Contact
Diode area 0.1 cm2 ; 50 mW; 500 J/cm2 ; 5 W/cm2

PSP Book - 9in x 6in

5 J per diode (100 s)
Four sites of irradiation: 20 J Scott bench Muscular preconditioning
(29) Diode laser 830 nm Rectus femoris Contact
Diode area 0.0028 cm2 ; 100 mW
Groups: Volleyball athletes 4 J (40 s); 1428.57 J/cm2
Soccer athletes 3 J (30 s); 1071.43 J/cm2
Ten sites of irradiation: 40 J (volleyball athletes) Wingate Muscular preconditioning
30 J (soccer athletes)
(25) Diode laser 830 nm Biceps brachii Contact
Diode area 0.0028 cm2 ; 100 mW; 35.7 W/cm2
5 J (50 s), 1785 J/cm2
Four sites of irradiation: 20 J Scott bench Muscular preconditioning

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 July 11, 2016 10:14
(30) Cluster with five diode lasers 810 nm Quadriceps femoris: Contact
Diode area 0.029 cm2 ; 200 mW; 6.89 W/cm2 Two sites on vastus medialis, two sites on vastus
6 J per diode (30 s); 206.89 J/cm2 laterallis, two sites on rectus femoris
30 J per site of irradiation: 5 × 6 J isokinetic dynamometer

Clinical Trials Using LEDT to Improve Muscle Performance and Prevent Damage
Six sites of irradiation: 180 J Muscular preconditioning
(27) Cluster with five diode lasers 810 nm Biceps brachii Contact
Diode area 0.0364 cm2 ; 200 mW; 5.495 W/cm2
6 J per diode (30 segundos), 164.85 J/cm2
30 J per site of irradiation: 5 × 6 J
Two sites of irradiation: 60 J Scott bench Muscular preconditioning
(26) Diode laser 660 nm Biceps brachii Contact
Diode area 0.0028 cm2 ; 50 mW; 17.85 W/cm2

PSP Book - 9in x 6in

5 J (100 s), 1785 J/cm2
Four sites of irradiation: 20 J
versus
Diode laser 830 nm
Diode area 0.0028 cm2 ; 50 mW; 17.85 W/cm2
5 J (100 s), 1785 J/cm2
Four sites of irradiation: 20 J Maximum voluntary contraction (60 s) Muscular preconditioning
(34) Cluster with five diode lasers 810 nm Quadriceps femoris: Contact
Diode area 0.0364 cm2 ; 200 mW; 5.495 W/cm2 Two sites on vastus medialis, two sites on vastus
6 J per diode (30 s), 164.85 J/cm2 laterallis, two sites on rectus femoris
30 J per site of irradiation: 5 × 6 J Hamstrings: four sites
Gastrocnemius: two sites
Twelve sites of irradiation: 360 J Running on treadmill until exhaustion Muscular preconditioning

(Contd.)

627

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628 Use of Low-Level Laser Therapy and Light-Emitting Diode Therapy
Table 34.2 (Contd.)

Reference LLLT/LEDT Parameters Músculo/exercı́cio Irradiation

(28) Diode laser 808 nm Biceps brachii Contact
Diode area 0.0028 cm2 ; 100 mW; 35.7 W/cm2
7 J (70 s), 2500 J/cm2
Eight sites of irradiation: 56 J Elbow flexion Muscular preconditioning
(31) Diode laser 808 nm Biceps brachii Contact
Diode area 0.0078 cm2 ; 100 mW; 12.7 W/cm2
7 J (70 s), 892 J/cm2
Eight sites of irradiation: 56 J Knee extension Muscular preconditioning

PSP Book - 9in x 6in

(32) Diode laser 780 nm Tibialis anterior Contact
Diode area 0.2 cm2 ; 30 mW; 0.15 W/cm2
0.81 J (27 s), 4 J/cm2
Twenty-nine sites of irradiation: 23.49 J Isokinetic dynamometer Muscular preconditioning
(35) Diode laser 808 nm Biceps brachii Contact
Diode area 0.0028 cm2 ; 100 mW; 35.71 W/cm2
1 J (10 s), 357.14 J/cm2
Four sites of irradiation: 4 J Scott bench Between sets of exercise
(10) Cluster with six diode lasers 830 nm Quadriceps femoris: seven sites uniformly Contact
Diode area 0.0028 cm2 ; 60 mW; 21.42 W/cm2 distributed on the quadriceps femoris muscles
0.6 J per diode (10 s), 214.28 J/cm2
3.6 J per site of irradiation: 0.6 J × 6
Seven sites of irradiation of 3.6 J each: 25.2 J Knee extension Muscular preconditioning/after exercise

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(36) Cluster with one diode laser 905 nm, four LEDs 875 nm, Quadriceps femoris: two sites on vastus Contact
and four LEDs 640 nm medialis, two sites on vastus lateralis, and two
Diode area 0.44 cm2 ; 0.3125 mW; 0.07 mW/cm2 sites on rectus femoris

Clinical Trials Using LEDT to Improve Muscle Performance and Prevent Damage
LED area 875 nm: 0.9 cm2 ; 17.5 mW; 19.44 mW/cm2
LED area 640 nm: 0.9 cm2 ; 15 mW; 16.66 mW/cm2
10 J per site of irradiation: 76 s
30 J per site of irradiation: 228 s
50 J per site of irradiation: 381 s
Six sites of irradiation: 60, 180, 300 J Eccentric contractions in isokinetic Muscular preconditioning
dynamometer
(37) Cluster with six diode lasers 808 nm Quadriceps femoris: seven sites uniformly Contact

PSP Book - 9in x 6in

Diode area 0.0028 cm2 ; 60 mW; 21.42 W/cm2 distributed over quadriceps femoris muscles
0.6 J per diode (10 s), 214.28 J/cm2
3.6 J per site of irradiation: 0.6 J × 6
Seven sites of 3.6 J each: 25.2 J Leg press and isokinetic dynamometer After exercise
(38) Cluster with six diode lasers 808 nm Quadriceps femoris: five sites uniformly Contact
Diode area 0.0028 cm2 ; 60 mW; 21.42 W/cm2 distributed over quadriceps femoris muscles
0.6 J per diode (10 s), 214.28 J/cm2
3.6 J per site of irradiation: 0.6 J × 6
Five sites of 3.6 J each: 18 J Cycle ergometer and isokinetic dynamometer After exercise
(39) Cluster with 32 LEDs 950 nm Biceps brachii Contact
Diode area 18 cm2 ; 160 mW
3.2 J/cm2 (360 s)
One site of irradiation Isokinetic dynamometer After exercise

(Contd.)

629

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630 Use of Low-Level Laser Therapy and Light-Emitting Diode Therapy
Table 34.2 (Contd.)

Reference LLLT/LEDT Parameters Músculo/exercı́cio Irradiation

(40) Cluster with 36 LEDs Biceps brachii: one site of irradiation at the Contact
Diode area 5 cm2 ; 100 mW/cm2 ponto muscle-tendon junction and one site of
32 LEDs 880 nm; 80 s, 8 J/cm2 irradiation 5 cm above
Four LEDs 660 nm; 80 s, 8 J/cm2
Two sites of irradiation Eccentric contraction at Scott bench After exercise
(41) LED 640 nm Masseter Contact
LED area 0.522 cm2 ; 116 mW; 222 mW/cm2

PSP Book - 9in x 6in

Groups: 1.04 J (9 s); eight sites of irradiation
(8.35 J); 2 J/cm2
2.08 J (18 s); eight sites of irradiation (16.7 J);
4 J/cm2
3.13 J (27 s); eight sites of irradiation (25 J);
6 J/cm2
Eight sites of irradiation Exercise: oral occlusion Muscular preconditioning
(42) Cluster with 69 LEDs Biceps brachii Contact
LED area 0.2 cm2 mW; 150 mW/cm2
0.3 J LED 660 nm (30 s), 1.5 J/cm2
0.9 J LED 850 nm (30 s), 4.5 J/cm2
41.7 J per site of irradiation (30 s)
One site of irradiation Scott bench Muscular preconditioning

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(43) Cluster with 69 LEDs Quadriceps femoris: Two sites of irradiation on Contact
LED area 0.2 cm2 rectus femoris, two sites on vastus medialis, two
34 LEDs 660 nm; 10 mW; 50 mW/cm2 sites on vastus lateralis
35 LEDs 850 nm; 30 mW; 150 mW/cm2

Clinical Trials Using LEDT to Improve Muscle Performance and Prevent Damage
0.3 J LED 660 nm (30 s), 1.5 J/cm2
0.9 J LED 850 nm (30 s), 4.5 J/cm2
41.7 J per site of irradiation (30 s)
Three sites of irradiation: 125.1 J Isokinetic dynamometer Muscular preconditioning
(44) LED 630 nm Biceps brachii Contact
LED area 1.77 cm2 ; 300 mW; 169.49 mW/cm2
9 J (30 s); 5.1 J/cm2
Four sites of irradiation: 36 J Eccentric contraction Muscular preconditioning

PSP Book - 9in x 6in

(46) Diode laser 810 nm Rectus femoris Contact
Diode area 0.036 cm2 ; 200 mW; 5.5 W/cm2
6 J (30 s), 164.84 J/cm2
Two sites of irradiation: 12 J
versus
Cluster with 69 LEDs
LED area 0.2 cm2
34 LEDs 660 nm; 10 mW; 50 mW/cm2
35 LEDs 850 nm; 30 mW; 150 mW/cm2
0.3 J LED 660 nm (30 s), 1.5 J/cm2
0.9 J LED 850 nm (30 s), 4.5 J/cm2
41.7 J per site of irradiation
Two sites of irradiation: 83.4 J Wingate Muscular preconditioning

(Contd.)

631

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632 Use of Low-Level Laser Therapy and Light-Emitting Diode Therapy
Table 34.2 (Contd.)

Reference LLLT/LEDT Parameters Músculo/exercı́cio Irradiation

(47) Cluster with 69 LEDs Quadriceps femoris: two sites of irradiation Contact
LED area 0.2 cm2 Hamstrings: two sites of irradiation
34 LEDs 660 nm; 10 mW; 50 mW/cm2 Gastrocnemius: one site of irradiation
35 LEDs 850 nm; 30 mW; 150 mW/cm2
0.3 J LED 660 nm (30 s), 1.5 J/cm2
0.9 J LED 850 nm (30 s), 4.5 J/cm2
41.7 J per site of irradiation

PSP Book - 9in x 6in

Five sites of irradiation: 208.5 J
versus
Cryotherapy (water immersion during 5 min at Three Wingates After exercise
5◦ C)
(45) Cluster with 69 LEDs Quadriceps femoris: two sites of irradiation on Contact
Diode area 0.2 cm2 rectus femoris, one point of irradiation on
34 LEDs 660 nm; 10 mW; 50 mW/cm2 vastus lateralis, one point of irradiation on
35 LEDs 950 nm; 15 mW; 75 mW/cm2 vastus medialis
0.30 J LED 660 nm (30 s), 1.5 J/cm2
0.45 J LED 950 nm (30 s), 2.25 J/cm2
25.95 J per site of irradiation
Four sites of irradiation: 103.8 J Wingate Between 2◦ and 3◦ Wingate

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Clinical Trials Using LEDT to Improve Muscle Performance and Prevent Damage
(8) Array with 200 LEDs Whole quadriceps femoris, hamstrings, and Contact
100 LEDs 850 nm (IR) arranged in 25 clusters of triceps surae
four LEDs; 130 mW; 185.74 mW/cm2
100 LEDs 630 nm (red) arranged in 25 clusters
of four LEDs; 80 mW
Groups: 105 J (20 s)
210 J (40 s)

PSP Book - 9in x 6in

315 J (60 s)
Placebo 0 J (30 s) Before official matches of volleyball Muscular preconditioning
(48) Two panels with 2000 LEDs (850 nm) Whole thigh Without contact
Panel area 1110 cm2
2000 LEDs 850 nm; 31 mW/cm2
14.400 J (both lower limbs); 30 min; 55.8 J/cm2 Treadmill During exercise
(49) Two panels with 2000 LEDs (850 nm) Whole thigh Without contact
Panel area 1110 cm2
2000 LEDs 850 nm; 100 mW; 39 mW/cm2 Treadmill During exercise
45 min; 108 J/cm2

633

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634 Use of Low-Level Laser Therapy and Light-Emitting Diode Therapy

34.7 Clinical Trials Using LEDT to Improve Muscle

Performance and Prevent Damage: Chronic
Responses

Paolillo et al. [48] investigated the effects of LEDT combined with

physical training in post-menopausal women during 12 weeks.
Training sessions were performed twice per week with 30 min of
exercise on a treadmill. During each training session, the LEDT group
received LEDT (850 nm) using two panels of LEDs placed 15 cm
away from the women’s thighs. The authors reported increased
muscle power and work of the knee extensor muscles assessed by
an isokinetic dynamometer when compared to the training group
without LED that had a higher fatigue index. In addition, the authors
reported that the same physical training combined with LEDT (850
nm) during 6 months decreased the time for recovery of the heart
rate after exercise [49].
Table 34.2 presents all parameters of the LLLT/LEDT used in
clinical trials.

34.8 Conclusion

LLLT and LEDT are expected to become increasingly widespread

therapies to prevent muscle damage and/or strategies to increase
muscle performance. However, the parameters of wavelength, light
energy, power, timing, and treatment repetition frequency still
remain as the main questions to be ascertained. Further efforts are
needed to elucidate the mechanisms of action that allows the light at
red and near-infrared wavelength to stimulate muscle cells.
Finally, if the use of LLLT and LEDT becomes widespread in
high-performance sports, we believe that the World Anti-Doping
Agency or the International Olympic Committee will need to discuss
these approaches and to take an official position on whether
these therapies and strategies (muscular preconditioning and faster
muscle recovery) will be allowed or not. The lack of any validated
biochemical marker of light exposure of muscles (light-doping) will
hamper these efforts.
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References 635

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and light-emitting diode therapy (LEDT) in short-term skeletal muscle
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640 Use of Low-Level Laser Therapy and Light-Emitting Diode Therapy

recovery after high-intensity exercise in athletes: Preliminary results.

Lasers Med Sci. 2011; 26(4): 493–501. Epub 2010/11/20.
48. Paolillo FR, Milan JC, Aniceto IV, Barreto SG, Rebelatto JR, Borghi-Silva A,
et al. Effects of infrared-LED illumination applied during high-intensity
treadmill training in postmenopausal women. Photomed Laser Surg.
2011; 29(9): 639–645. Epub 2011/07/14.
49. Paolillo FR, Corazza AV, Borghi-Silva A, Parizotto NA, Kurachi C,
and Bagnato VS. Infrared LED irradiation applied during high-
intensity treadmill training improves maximal exercise tolerance in
postmenopausal women: A 6-month longitudinal study. Lasers Med Sci.
2013; 28(2): 415–422. Epub 2012/03/03.
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Chapter 35

Low-Level Laser Therapy of Pain: Clinical

Applications

Roberta T. Chow
Central Clinical School, Edward Ford Building A27, The University of Sydney,
NSW 2006, Australia
roberta.chow@sydney.edu.au

35.1 Background

Low-level laser therapy (LLLT) has been used to treat a wide

spectrum of painful clinical conditions commencing within a decade
of its production by Maiman in 1960 (Maiman, 1960). Initially,
clinical use outstripped an understanding of the mechanisms
for its effects. Further confusion arose with the concurrent use
of laser in acupuncture, a different paradigm. Over subsequent
decades, as research focused on understanding the direct effects
of light on tissue, several mechanisms underlying LLLT analgesia
were proposed (Belkin et al., 1988; Navratil and Dylevsky, 1997;
Schindl et al., 2000). These include anti-inflammatory effects,
neural blockade, stimulation of lymphatic activity, tissue repair,
and reduction of muscle spasm. Each of these mechanisms has

Handbook of Low-Level Laser Therapy

Edited by Michael R. Hamblin, Marcelo Victor Pires de Sousa, and Tanupriya Agrawal
Copyright c 2017 Pan Stanford Publishing Pte. Ltd.
ISBN 978-981-4669-60-3 (Hardcover), 978-981-4669-61-0 (eBook)
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642 Low-Level Laser Therapy of Pain

been studied from subcellular levels through to clinical applications.

Translating and applying knowledge of these multiple mechanisms
from the laboratory to clinical practice are critical to successful
outcomes with LLLT. This chapter will outline the painful conditions
in which LLLT is used and mechanisms for its effects and address
practical considerations.

35.2 What is Pain?

To successfully apply LLLT in this context, one must first understand

the nature of pain. The International Association for the Study of
Pain (IASP) defines it as “an unpleasant sensory and emotional
experience associated with actual or potential tissue damage, or
described in terms of such damage”(IASP Task Force on Taxonomy,
1994). Although this definition encompasses the complexity of
the phenomenon of pain, extending it beyond the activation of
nociceptors into the broader concept of a person’s emotional
response to pain and the social context in which it occurs, it
is in periphery—the immune cells, nociceptors, lymphatics, and
muscles—where LLLT exerts its primary action in modulating pain.

35.3 Types of Pain and Mechanisms

Pain can be divided into three different types based on the underly-
ing mechanism. The first two are nociceptive and neuropathic pain,
both of which can be modulated by LLLT. The third is central pain,
which arises from damage to the central nervous system (CNS), such
as in stroke, and is not currently treated with LLLT.
Nociceptive pain is the most common form of pain and arises
from the activation of nociceptors, the small-diameter thinly
myelinated Aδ and unmyelinated C afferent nerve fibers, which
are found in skin and deeper tissues. When tissue damage occurs,
an inflammatory soup of chemical mediators and proinflammatory
cytokines, such as prostaglandins E2 (PGE2 ) and substance P, is
released by injury or inflammation, which sensitize nociceptors.
Many musculoskeletal conditions are examples of nociceptive pain:
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Types of Pain and Mechanisms 643

osteoarthritis, rheumatoid arthritis, crystallopathies such as gout,

trauma including surgery, whiplash injury, and tendinopathies.
Neuropathic pain, a second form of pain, arises from damage to
nerves, which can occur with infection, for example herpes zoster
causing post-herpetic neuralgia or compression with inflammation,
which occurs with disc prolapse onto nerve roots as in sciatica. Other
examples are trigeminal neuralgia and carpal tunnel syndrome. Both
nociceptive and neuropathic pain can occur concurrently in the
same patient, for example back pain with sciatica. In this and other
clinical scenarios, patients may describe two different types of pain:
the lancinating or burning pain of neuropathic pain and the dull ache
of nociceptive pain.
In recent years, the importance of relieving pain whatever its
origin has been recognized as a clinical imperative, with pain
now being regarded as a disease in its own right (Siddall and
Cousins, 2004). Not only is this important from a patient’s comfort
perspective and is good management, but it is now understood that
reducing the severity of acute pain can prevent pain from becoming
chronic.
The underlying pathophysiology of this is the phenomenon of
“wind up” in the spinal cord leading to long-term potentiation of
pain (Ji et al., 2003). These changes occur initially in the dorsal
horn and result in central sensitization (Arendt-Nielsen and Graven-
Nielsen, 2003), which causes increased sensitivity of nociceptors.
Clinically, this is manifested as hyperalgesia—increased sensitivity
to sensation that is not normally painful—and allodynia—pain with
activity that is not normally painful. Whiplash-associated disorder
(WAD) is a common example in which these symptoms are seen.
These changes occur as a result of neuroplasticity in the dorsal
horn, which is the capacity of neurons in both the peripheral and
central nervous systems, to be modulated by increased or decreased
afferent activity from the somatosensory nerves. It is neuroplasticity,
which underpins the long-term benefits of LLLT.
From a clinical perspective, it is necessary to identify whether the
pathophysiology is neuropathic or nociceptive pain since patients
with neuropathic pain are often more likely to experience a
temporary increase in pain following LLLT.
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644 Low-Level Laser Therapy of Pain

Complicating the picture in many patients are conditions that

appear nociceptive in origin but in which patients experience pain
out of proportion to the pathology. WAD, fibromyalgia, and complex
regional pain syndrome (CRPS) are examples of conditions where
increased sensitization in the “pain matrix” occurs. In these clinical
scenarios, central sensitization reduces pain thresholds and patients
exhibit hyperalgesia and allodynia. These patients have often been
labeled as having psychological problems as a causal factor in
their pain, especially if there are no abnormalities on MRI. It is
now appreciated that changes in the spinal cord underpin these
responses and what are perceived as abnormal psychological factors
are secondary to the pain and limitations it causes. The long-
term benefit of LLLT in such patients is to “re-program” their pain
matrix and cause long-term depression (LTD) of pain (Liu et al.,
1998).

35.4 Mechanisms Underlying Pain Relief

35.4.1 Neural Blockade

Laser can relieve nociceptive and neuropathic pain by partially
inhibiting nerve conduction and reducing afferent stimulation, mim-
icking some functions of local anesthetic injections. Demonstrating
this anesthetic action is a study by Chan et al. in which Nd:YAG
laser (av power: 1.1 mW, 240 s) applied to the cervical surface of
the tooth-induced pulpal anesthesia for dental procedures (Chan
et al., 2012). In other studies, both 650 nm (35 mW, 30 s) and 808
nm (400 mW, 30 s) lasers applied at four points to skin overlying
sciatic nerve reduced action potentials in underlying motor and
sensory components of the sciatic nerve in rats (Yan et al., 2010).
A review of effects of laser on nerves confirmed the “blockade”
effects on nerves in humans as well as animals (Chow et al.,
2011). When laser is applied transdermally, we propose that not
only does it reduce action potentials in underlying nerve trunks, it
also inhibits conduction in the superficial peripheral nerve endings
of nociceptors in skin and deeper tissues. This translates in the
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Mechanisms Underlying Pain Relief 645

clinical situation to patients reporting reduction in tenderness and

pain, sometimes in association with a sensation of “numbness”
during the course of treatment. Importantly, reduction in afferent
stimulation by laser to peripheral nerve endings results in reduced
synaptic activity with second-order dorsal horn neurons, which, in
turn, modulates afferent input to higher centers. Neuroplasticity
underlies this capacity of the nervous system to modulate re-
sponses to laser-induced neural blockade leading to long-term pain
relief.

35.4.2 Reduce Inflammation

Another very important mechanism of nociceptive pain relief with
LLLT is its anti-inflammatory effect. Inflammation is a physiological
response in tissue to injury (for example, trauma or surgery) or
pathological conditions (for example, infection, gout, osteoarthritis,
and rheumatoid arthritis). It is mediated by the release of a
“chemical soup” of proinflammatory peptides released from cells
in the injured area. These molecules sensitize peripheral nerve
endings of nociceptors causing pain as well as acting as chemotactic
stimuli for other cells such as neutrophils to enter the injured
area to initiate healing. Peripheral nerve endings in the skin when
sensitized also contribute to inflammation, so-called neurogenic
inflammation, by releasing substance P and other neuropeptides.
This leads to further activation of cells involved in inflammation,
such as mast cells, and the cascade of pain and inflammation
proceeds. The progress and resolution of inflammation is critical
to normal healing. Specific anti-inflammatory effects of laser have
been demonstrated in several different experimental models. These
include suppression of IL-6 following tendon injury in rats (660
nm, 100 mW, 6 J) (Laraia et al., 2012); decreased mRNA expression
of kinin receptors and of inflammatory cytokines TNFα, IL-1β in
carrageenan-induced inflammation in rat paw (660 nm or 684 nm,
30 mW, 7.5 J/cm2 ) (Bortone et al., 2008); reduced COX-2 expression,
also in a similar model using similar laser parameters (Prianti et al.,
2014). In a case series, human subjects treated with LLLT (830 nm,
1 W, 3 min 10 treatments) for painful conditions, serum PGE2 was
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646 Low-Level Laser Therapy of Pain

significantly lower in patients with successful outcomes than those

who did not get pain relief (Mizutani et al., 2004). In conditions
where inflammation is central to the pathophysiology, LLLT can
effectively reduce pain, which, in some studies, is equivalent to anti-
inflammatory drugs (Albertini et al., 2004).

35.4.3 Reduce Edema

Swelling is a normal physiological response to acute and chronic
inflammation. Extravasation of fluid into the interstitial space occurs
in response to proinflammatory mediators acting on capillaries
and increasing their permeability. Swelling limits movement of an
injured area, splinting a limb and enforcing rest, which is protective
in the initial stages of injury. In acute injuries, reducing edema
will reduce pain and allow early mobilization, which in turn will
activate muscle pump action in limbs. Swelling of limbs also occurs
when there is reduced capacity of the lymphatic system to transport
fluid due either to genetics, known as primary lymphedema or
secondarily as a result of removal, which occurs in the surgical
treatment of cancers. Although edema is a physiological response to
injury, swelling contributes to pain and, when prolonged, results in
pain, pathologically restricts movement, and causes tissue scarring
as fibrin in the interstitial fluid becomes organized. LLLT (650 nm,
energy density: 1 and 2.5 J/cm2 ) reduces edema in experimentally
induced arthritis (Carlos et al., 2014; Pallotta et al., 2011) in
carrageneen-induced inflammation in rat paw (632.8 nm, 2.5 J/cm2 )
(Ferreira et al., 2005) and improves macromolecular clearance via
lymph nodes (CO2 30 s, 0.5–1.5 W) (Shimotoyodome et al., 2001).
Treatment of lymph nodes draining an area of inflammation has
edema-reducing effects, without local laser treatment (830 nm, 35
J/cm2 , 10 s) (Meneguzzo et al., 2013). In some studies, extra oral
LLLT with parameters 808 nm, 100 mW, 120 s (Aras and Gungormus,
2010) and 637 nm, 50 mW, 4 J/cm2 (Markovic and Todorovic,
2007) reduced edema following tooth extraction. LLLT has been
demonstrated to activate the motoricity of lymphatic vessels by
increasing the motility of endothelial cells, which make up the
lymphangion and increase the number of lymphatic channels over
time (Lievens, 1991).
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Mechanisms Underlying Pain Relief 647

35.4.4 Reduce Muscle Spasm

Muscle spasm is a physiological reflex response, which occurs in
response to acute injury or chronic strain. Acute whiplash injury
and the more chronic WAD are examples of such muscle strain
and spasm, which can be prolonged. Chronic postural strain, which
occurs as a result of maintaining non-physiological positions for
long periods of time, for example sitting at a computer or playing a
musical instrument, can also result in muscle spasm, contributing to
the clinical pain picture of many chronic musculoskeletal pain con-
ditions. If the spasm persists over weeks, months, and even years,
“trigger points” form within the muscle (Lavelle et al., 2007), causing
myofascial pain syndrome (MPS) (Gerwin, 2014). Though MPS as a
diagnosis has been controversial, there is now greater understand-
ing of the pathophysiology (Simons, 2008), which is addressed by
the effects of laser on muscles. Once an acute injury resolves or
strain is relieved, these changes revert to normal in most, though not
all, clinical situations, and normal function resumes. In patients with
chronic conditions, LLLT can facilitate more effective rehabilitation,
where they did not respond to physiotherapy prior to LLLT.

35.4.5 Tissue Repair

Trauma and inflammation are associated with tissue damage, which
initiates a cascade of physiological events leading to tissue healing.
The long-term benefits of LLLT result from its capacity to promote
tissue repair, especially in clinical situations where there has been
delayed healing. Much of the research in this area relates to chronic
wound healing, such as chronic venous ulceration, one of the earliest
applications of LLLT (Mester et al., 1972). Other clinical scenarios
where tissue repair is critically important are tendinopathies, e.g.,
Achilles tendinopathy, and enthesopathies, e.g., lateral epicondylitis,
where immediate anti-inflammatory effects of LLLT will relieve pain
but tissue repair is critical for the resumption of normal function.
Tissue repair is a complex process, and LLLT acts in multiple ways.
Laser stimulates migration and activation of cells to the injured area
(Hemvani et al., 1998), decreases inflammatory cytokine production
by neutrophils (Alves et al., 2013), increases phagocytosis of
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648 Low-Level Laser Therapy of Pain

neutrophils, and improves microvasculature and endothelial cell

proliferation (Chen et al., 2008a), increases production of growth
factors (Damante et al., 2009) and procollagen by fibroblasts
(Yamamoto et al., 1996).

35.4.6 Release of Neurotransmitters

The local and systemic modulation of the release of neuropeptide
pain modulators is less studied than many of the above mechanisms.
A number of studies point to significant changes in neuropeptides
in response to LLLT, which are important in endogenous pain
modulation. These include β-endorphins (Hagiwara et al., 2008;
Rico et al., 1994) and their precursors (Laakso et al., 1994) and
serotonin (Ceylan et al., 2004; Walker, 1983), all of which are
molecules which are the pharmacological basis of drugs used in pain
relief. It remains an area for future research.

35.5 Conditions in Which LLLT is Used and Evidence

35.5.1 Reviews of LLLT and Pain

Several reviews have been undertaken into the efficacy of LLLT in
painful conditions. Early reviews stated that there was insufficient
evidence to support LLLT in musculoskeletal pain (de Bie et al.,
1998; Gam et al., 1993) or, at best, were equivocal (Marks
and de Palma, 1999; Schindl et al., 2000). As the number of
randomized controlled trials (RCTs) has increased and there is
greater understanding of the mechanisms of action, more recent
reviews have found positive outcomes in both chronic and acute
pain (Bjordal et al., 2006a; Enwemeka et al., 2004; Jang and Lee,
2012). A common theme among reviewers of LLLT is the need
for complete reporting of parameters used in studies, without
which no comparison of the doses delivered in trials is possible
as the combination and permutation of parameters is almost
infinite (Calderhead, 1991; Jenkins and Carroll, 2011; Tuner and
Hode, 1998). More sophisticated reviews of LLLT emphasize the
importance of finding the appropriate dosage window, which are
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Conditions in Which LLLT is Used and Evidence 649

wavelength specific. The dosage window appropriate for each

condition must be found to achieve the best outcome. Later reviews
of LLLT painstakingly address this in their analyses.

35.5.2 Evidence for Specific Conditions

35.5.2.1 Arthridities
35.5.2.1.1 Knee osteoarthritis
Knee pain, in particular osteoarthritis, is one of the most common
forms of arthritis in people over 65 years of age. Weight loss
and topical anti-inflammatories are now the recommended first
line treatments according to the NICE UK guidelines (National
Institute for Health and Care Excellence, 2014). Even paracetamol,
previously recommended as a first line treatment option, is now
being reconsidered due to long-term toxicity. The serious side
effect profiles of non-steroidal anti-inflammatory drugs (NSAIDs)
now render them second and third line treatments for as short a
period of time as possible. At the end of the treatment spectrum,
arthroscopy of the knee joint results is more harm than benefit in the
long term (Buchbinder and Harris, 2013) and following total knee
replacement, approximately 20% of patients experience ongoing
pain and disability of some degree, particularly women (Beswick
et al., 2012). The case for LLLT as a mainstream therapy is strong
given the limitations of current therapies. Bjordal et al. published
a review of studies of osteoarthritis, which included an analysis of
the doses and application techniques used (Bjordal et al., 2007).
They found that there was good evidence for a clinically relevant
effect provided the appropriate dose was used: for 904 nm, 2–
12 J and for 830 nm, 20–48 J, applied to up to eight points over
the joint capsule. Since that review, a number of other studies,
each using near-infrared (NIR) lasers (810, 830, and 904 nm),
have demonstrated the efficacy of LLLT in knee osteoarthritis
(Alfredo et al., 2012; Gworys et al., 2012; Hegedus et al., 2009)
supported by animal experiments, showing the modulation of
inflammation within the knee joint. In experimentally induced rat
knee inflammation, 810 nm (1, 3, and 6 J) significantly reduced
the number of polymorphonuclear cells at the inflammatory site;
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650 Low-Level Laser Therapy of Pain

COX-1 and COX-2 gene expressions were significantly enhanced by

laser irradiation and PGE2 production was inhibited (Pallotta et al.,
2012). Osteochondral defects were significantly repaired with 890
nm (4.8 J/cm2 ) at 8 weeks (Kamali et al., 2007). A histological study
of laser-irradiated (790 nm, 10 mW, 8 min daily for 6 days) synovial
membrane in patients with rheumatoid arthritis undergoing total
knee replacement demonstrated significantly reduced inflammatory
cell density and reduced synovial villi compared with non-irradiated
areas of the knee joint (Amano et al., 1994).

35.5.2.1.2 Hand osteoarthritis and rheumatoid arthritis

Studies of effects of laser on both forms of arthritis have been
performed. Two reviews (Brosseau et al., 2005; Christie et al., 2007;
Ottawa Panel, 2004) and an overview of the reviews (Christie et al.,
2007) found moderate evidence of efficacy, though with caveats on
the dosages used. The Ottawa Panel, in particular, pointed out the
methodological flaw of using the contralateral hand as a placebo in
LLLT trials because of the systemic effects of laser. One recent study
by Meireles et al. found positive though limited effects and reported
the outcome as negative (Meireles et al., 2011). The dosages used
by Meireles et al. were found to be sub-therapeutic, as shown by
Tuner et al. (Tuner and Hode, 2010), a common finding among
many studies of LLLT (Bjordal and Baxter, 2006; Enwemeka, 2009).
The World Association of Laser Therapy (WALT) recommendation
for Class 3B wavelengths from 780–860 nm for the treatment of
interphalangeal or metacarpophalangeal joints at 1–2 points is 4 J
and for the wrist, 2–4 points with a total of 8 J (World Association of
Laser Therapy, 2010b).

35.5.2.1.3 Gout and other crystallopathies

Crystalline-induced arthropathies include uric acid deposition in
gout, calcium pyrophosphate dihydrate (DCPP) in pseudogout, and
hydroxyapatite in calcific tendinitis. One small clinical trial suggests
the potential of LLLT (904 nm, av power 40 mW) to reduce pain
in acute gouty arthritis (Soriano et al., 2006). A series of animal
experiments provides strong evidence of specific anti-inflammatory
effects of He–Ne laser (632.8 nm, ∼5 mW) induced by urate
(Campana et al., 2004; Soriano et al., 2006), DCPP (Campana et al.,
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Conditions in Which LLLT is Used and Evidence 651

2003; Rubio et al., 2010), and hydroxyapatite crystals (Campana

et al., 1999). In these studies, markers of inflammation such as TNFα,
plasma fibrinogen levels, and PGE2 were reduced to control levels
in laser-treated animals. Levels were also found to be equivalent to
Meloxicam, an anti-inflammatory NSAID used clinically.

35.5.2.2 Neck pain

Neck pain is one of the most common painful conditions encoun-
tered in musculoskeletal medicine, and LLLT is one of the most
evidence-based therapies for neck pain (Chow et al., 2009). The
effectiveness in neck pain may result from the more superficial na-
ture of the anatomical structures, which are within the penetration
depths of most wavelengths of laser. Neural blockade will affect the
medial branch of the dorsal ramus to the facet joints, the target
of radio-frequency ablation or the occipital nerve at the base of
the skull, often the cause of headaches. Reduction in the spasm
of neck muscles, including the trapezius, will improve mobility.
Anti-inflammatory effects will be relevant to enthesitis such as at
the insertion of the levator scapulae and facet joints. Guidelines
suggest that for neck pain, treatment with infrared diode lasers
(780–860 nm) should include up to 12 points overlying lateral
articular pillars of facet joints most affected and muscle bellies with
tender points such as trapezius and sternomastoid muscles, with a
minimum of 4 J per point. Frequency of treatment can vary from
daily to twice a week, depending on the severity of pain, delivered
over 3–4 weeks.

35.5.2.3 Back pain

Back pain is a costly condition both economically and in terms of
poor quality of life in an individual. In the lumbar region, anatomical
structures that are targets of treatment, such as facet joints and
neural foramina, are much deeper than in the neck and, therefore,
dose regimes will be different from that of more superficial
structures. More superficial structures such as entheses around the
sacroiliac joint and iliolumbar and thoracolumbar fascia, which are
implicated in chronic back pain are well within the penetration
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652 Low-Level Laser Therapy of Pain

depths of infrared wavelengths. Spasm and trigger points in paraver-

tebral muscles also a source of myofascial back pain can be relieved
by the application of LLLT (Benjamin et al., 2006). In back pain
associated with failed back surgery (the “failed back pain surgery”
syndrome), the problem can be conceptualized as a “software” ver-
sus “hardware” problem. In this conceptualization of the pathophys-
iology, LLLT cannot alter the bone structures, but reducing inflam-
mation at entheses and reducing muscle spasm can have a profound
effect in pain reduction, even in entrenched painful conditions. The
spectrum ranges from acute first presentations of back pain to the
end stages of failed back surgery syndrome. In acute back pain, two
trials have demonstrated efficacy (Jovicic et al., 2012; Longo et al.,
1991), including with radicular pain (Konstantinovic et al., 2010).
For chronic low back pain, a case series (Ohshiro and Shirono,
1992) and several trials have demonstrated positive outcomes
extending over two decades (Alayat et al., 2013; Basford et al.,
1999; Charlusz et al., 2010; Fiore et al., 2011; Mandic and Rancie,
2011; Snyder-Mackler et al., 1989; Soriano and Rios, 1998; Toya
et al., 1994; Umegaki et al., 1989). The most recent Cochrane
review (Yousefi-Nooraie et al., 2008) was, however, found to be
“inconclusive.” Dr. Jan Bjordal strongly disagreed with the review
conclusions making the following statement: “This is probably the
only Cochrane review where 5 out of 6 RCTs with acceptable
methodological quality and partly or fully positive results, merits a
non-positive review conclusion.” Bjordal et al. have also challenged
the negative findings of an overview of systematic reviews of LLLT
in back pain by Chou and Huffman (Chou and Huffman, 2007). He
identified several methodological shortcomings in their analysis,
showing that there is, indeed, moderate evidence of efficacy of LLLT
in treating chronic nonspecific back pain (Bjordal et al., 2008a). This
is illustrative of the widespread problem of handling technological
data with which reviewers are unfamiliar when evaluating evidence
for non-pharmacological therapies such as LLLT (Bjordal and Greve,
1998; Bjordal et al., 2006b). WALT dosage guidelines for back pain
have been derived from positive studies. For 904 nm laser with
a peak pulse output of 1 W, mean output >5 mW and power
density >5 mW/cm2 a minimum of 4 points with delivery of 4 J
per point is recommended (World Association of Laser Therapy,
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Conditions in Which LLLT is Used and Evidence 653

2010a). For wavelengths of Class 3B lasers from 780–860 nm, the

recommendations are for treatment of eight points, with a minimum
of 4 J per point (World Association of Laser Therapy, 2010b).

35.5.2.4 Shoulder pain

There are fewer studies of LLLT in shoulder conditions, but there is
sufficient evidence to initiate a trial of LLLT especially where other
treatments have failed or patients do not undergo corticosteroid
injection, which has limited evidence of efficacy and greater
evidence of harm (Buchbinder et al., 2003). Conditions for which
there are positive outcomes include shoulder tendinitis (England et
al., 1989), adhesive capsulitis (Green et al., 2003), frozen shoulder
(Favejee et al., 2011), and myofascial pain of the shoulder girdle
(Rayegani et al., 2011). One study with a negative outcome was
found to have administered inadequate dosage due to inadequately
measured device (Bingol et al., 2005; Bjordal and Baxter, 2006).

35.5.2.5 Tendinopathy and enthesitis

A “classical” tendinopathy is Achilles tendinopathy. The pathophys-
iological basis for tendinopathy has swung from being regarded
as inflammatory in origin, then degenerative, and now back again
to having an inflammatory basis (Rees et al., 2013). Bjordal
et al. demonstrated unequivocally that inflammation, at least in an
activated state, has inflammation as a component of the pathophys-
iology (Bjordal et al., 2006c). Evidence on the effectiveness of LLLT
in tendinopathies is equivocal on the basis of a substantial number
of negative trials. Tumilty’s review of the literature demonstrated
that positive outcomes in tendinopathy are critically dependent on
power density (<100 mW/cm2 ), more so than any other parameter
(Tumilty et al., 2010). These findings have now been reflected in
the WALT guidelines. Given the dual pathophysiology, it is likely
that both anti-inflammatory and tissue healing mechanisms are
operational in LLLT in tendinopathy.

35.5.2.6 Lateral epicondylitis

Tennis elbow is an enthesitis, which is archetypal of all enthe-
sopathies in that there is pain arising from the insertion of a muscle
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654 Low-Level Laser Therapy of Pain

to the periosteum at the lateral epicondyle, which is aggravated

by use of the forearm. Both tendons and entheses (and teeth)
attach to the periosteum by a network of Sharpey’s fibers, which
are collagen fibers active in the metabolism of the underlying
bone enabling stress of muscle contraction to be transmitted to
a limb and movement of a joint (Aaron, 2012). Inflammation can
occur as a result of the microtrauma and injury at this junction.
As with tendinopathies, both tissue repair stimulation and anti-
inflammatory activity are important mechanisms of LLLT in these
conditions. A review of the literature shows a moderate benefit
when the appropriate dose guidelines are adhered to (Bjordal et al.,
2008b).

35.5.2.7 Trigger point and myofascial pain

Myofascial pain syndrome and associated trigger points are a
common cause of widespread musculoskeletal pain (Gerwin, 2014),
including headache (Fernandez-de-Las-Penas et al., 2007), shoulder
pain (Alburquerque-Sendin et al., 2013), groin pain (Kim et al.,
2013), and back pain (Chen and Nizar, 2011). LLLT effects on
muscle and trigger points have been demonstrated with both
infrared and visible wavelengths. LLLT (632.5 nm, 0.95 mW)
decreased pain in patients with neck and back trigger points
(Snyder-Mackler et al., 1989); NIR (904 nm) reduced pressure
point tenderness in trapezius muscle trigger points (Airaksinen
et al., 1989); GaAlAs (780 nm, 25 J/cm2 and 60 J/cm2 ) reduced
jaw pain in masseter muscles (Venezian et al., 2010); 670 nm
(cw, 15 mW) improved masseter muscle contraction in patients
with orofacial pain (de Medeiros et al., 2005); 808 nm (100 mW,
energy density: 70 J/cm2 ) increased pressure pain threshold in
masseter muscles and remained for 30 days after treatment (de
Moraes Maia et al., 2014). The effects of laser on muscle and
trigger point activity show several important responses relevant to
clinical response: red laser (660 nm, cw, 9 J/cm2 ) reduced end-plate
noise (EPN) from myofascial trigger points (MTrP) in rabbit skeletal
muscle (Chen et al., 2008b); 660 nm increased muscle cytochrome
c oxidase (Hayworth et al., 2010). Two studies examined effects of
laser of end-plate potentials of neuromuscular junctions of mouse
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Conditions in Which LLLT is Used and Evidence 655

diaphragm, with 655 nm showing no effect (Nicolau et al., 2004b)

and 830 nm (12 J/cm2 ) reduced amplitudes, suggesting decreased
acetylcholine release (Nicolau et al., 2004a).

35.5.2.8 Neuropathic pain

Neuropathic pain occurs as a result of injury to an underlying nerve.
Conditions such as trigeminal neuralgia, post-herpetic neuralgia,
diabetic neuropathy, and carpal tunnel syndrome are examples of
neuropathic pain. Importantly, even in conditions such as back pain,
neuropathic pain is estimated to occur in up to 40% of patients.
Those apparently musculoskeletal conditions where hyperalgesia
and allodynia are present, such as WAD and fibromyalgia, where
central sensitization is present but there is no single identifiable
nerve pathology, have all the characteristics of neuropathic pain and
need to be treated as such. Neuropathic pain conditions are often
very sensitive to laser and can be easily aggravated by too aggressive
treatment.
For post-herpetic neuralgia, there are only two controlled trials:
one evaluating long-term effects (Moore et al., 1988) and the other
the immediate analgesic effect of LLLT on PHN (Kemmotsu et al.,
1991). There are, however, several case series and case reports that
provide a guide to treatment in a condition which is difficult to
control and adjunctive treatment is potentially valuable (Iijima et al.,
1989; Iijima et al., 1991; Knapp, 2013; Mann et al., 1999; McKibbin
and Downie, 1991; Mittal et al., 1996; Namazawa et al., 1996; Otsuka
et al., 1995; Yaksich et al., 1993; Yamada and Ogawa, 1995). Visible
(632.5 nm) and infrared wavelengths (830 nm, 904 nm) are used
in either a scanning or a point-by-point treatment over the affected
dermatome, repeated on several occasions. Occasional worsening of
pain is reported. Age more than 60 years and truncal PHN are poorer
prognostic factors. Treatment must be low dose and short duration
with light or minimal pressure.
Trigeminal neuralgia has also been evaluated in several small
trials (Eckerdal and Bastian, 1996; Shuster et al., 1988; Walker
et al., 1988). Supporting animal experiments show suppression of
neural activity in the trigeminal nucleus with the application of
laser to the face. This suggests the neural blockade effect of laser
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656 Low-Level Laser Therapy of Pain

is operative (Nelson and Friedman, 2001; Wakabayashi et al., 1993).

A small randomized, controlled study of diabetic neuropathy using
905 nm for 5 min approached but did not reach significance for
relief of pain (Zinman et al., 2004). Another study demonstrated
improved conduction velocity in peroneal, sural, and tibial nerves
affected by diabetic neuropathy with patients acting as their own
control (Khamseh et al., 2011). This study used two wavelengths
simultaneously, 808 nm and 904 nm, applied over the nerve root exit
at L4 to S1 and four points along the sciatic nerve. The study did not
assess pain.
Carpal tunnel syndrome is a common neuropathy treated with
LLLT. A review of seven studies suggests that LLLT is a promising,
cost-effective treatment (Naeser, 2006). Since that review there
have been several additional studies that have favorable outcomes
when compared with sham laser (Shooshtari et al., 2008) and
with splinting (Yagci et al., 2009), surgery (Elwakil et al., 2007),
ultrasound and pulsed magnetic field (Dakowicz et al., 2011), and in
patients with rheumatoid arthritis (Ekim et al., 2007). A recent study
did not show any functional difference, but LLLT improved median
nerve conduction (Tascioglu et al., 2012).

35.5.2.9 Lymphedema
Lymphedema is the extravasation of high-protein fluid into tissues,
compared to low protein fluid in acute edema. It can be primary,
which is genetic in origin, or secondary, most commonly following
surgical dissection and removal of lymph nodes. Treatment with
LLLT of upper arm lymphedema after mastectomy for breast cancer
is effective in reducing limb volume, tissue hardness, and pain
(Carati et al., 2003). The US FDA has approved a single laser device
(LTU-904, 904 nm, 5 mW) LLLT for treatment of lymphedema (Piller
and Thelander, 1998). Treatment with this wavelength has been
found to be cost effective (Carati et al., 2003; Pillar and Thelander,
1995) and equivalent to pneumatic compression (Kozanoglu et al.,
2009). A review of five small studies of acceptable methodology has
also demonstrated benefit at a dose of 1–2 J/cm2 per point applied
to several points covering the fibrotic area (Omar et al., 2012). A
single animal experiment demonstrated stimulation of lymphatic
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Pretreatment Pain Relief 657

regeneration with normal structural reorganization occurred in

wounds following combined 632.8 nm and 904 nm LLLT compared
with controls (Lievens, 1991).

35.5.2.10 Post-operative pain

There is a small but seminal study of LLLT modulating post-
operative pain following open cholecystectomy. Moore applied 830
nm, 60 mW, cw laser for 6–8 min immediately following wound
closure, which resulted in significantly decreased post-operative
drug use (Moore et al., 1992). Dental studies have also demonstrated
significant pain relief when administered within 2 h of surgery using
helium–neon laser (∼5 mW, 1.8 J delivered over 3 min) (Clokie and
Al, 1991); 809 nm (7.5 J) for 24 h following endodontic surgery
(Kreisler et al., 2004) and following CO2 laser used for gingivectomy
(Abt, 1992). A recent systematic review found beneficial effects on
trismus but not pain in post-operative dental surgery, so confusion
around optimal dosage for post-operative pain relief remains
(Brignardello-Petersen et al., 2012).

35.6 Pretreatment Pain Relief

Pretreatment with laser for surgery can reduce pain. Several

examples of such preventative effects are found in both animal
and human studies. Pretreatment reduced orofacial pain in rats
following a formalin test (Zeredo et al., 2005) and Er:YAG laser
used before cavity preparation in a pediatric population showed
significant reduction in pain during the procedure in two small
studies (Eren et al., 2013; Tanboga et al., 2011). Painful oral
mucositis associated with chemotherapy can be prevented by
pretreatment with laser (Migliorati et al., 2012).
There are several potential mechanisms underlying effective
pretreatment with laser irradiation to reduce pain or to diminish
effects of chemotherapy on oral mucosa. Laser irradiation inhibits
nociceptive response reducing the release of proinflammatory neu-
ropeptides such as substance P and limits peripheral sensitization,
which modulates the inflammatory cascade, prior to a noxious
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658 Low-Level Laser Therapy of Pain

stimulus. Another mechanism may relate to the stabilization of

mast cells. Mast cell degranulation, which occurs with injury, can
also be dampened down by pretreatment with laser irradiation,
preventing further sensitization of nociceptors (Trelles et al., 1989).
At a systemic level, preirradiation of blood with 830 nm reduced
pain response in rats by enhancing peripheral endogenous opioids
(Hagiwara et al., 2008). Honmura et al. found that inflammatory
edema was reduced with 780 nm (10 mW, power density: 31.8
J/cm2 ) but only when administered before as well as after the
noxious stimulus (Honmura et al., 1992).
The range of conditions discussed above is not exhaustive. Many
conditions are amenable to a trial of LLLT, even in the absence of
high levels of evidence. This is largely because of the safety profile of
LLLT, where no serious events have been reported in any studies.

35.6.1 Unique Effects of LLLT in Pain

What is unique to LLLT is that all the mechanisms described occur
concurrently. Conceptually and practically, this is very different from
pharmacological therapies where a drug has a single action on a
single molecular target, which affects every cell, often the cause
of collateral multiple side effects while having sometimes limited
benefit. Examples are COX inhibitors that have an inflammatory
effect in a target tissue but can equally affect the gut and kidney,
thereby mitigating the beneficial effects. Moreover, addressing a
single pathophysiological component of a painful condition, for
example inflammation in knee osteoarthritis, is often of limited
benefit as pain can arise from muscles, ligaments, and entheses
externally, as well as intra-articular pathology. A modality with
several mechanisms of action has many advantages over single
mechanism drug options.

35.7 Practical Considerations

For successful clinical application of LLLT beyond the confines of

randomized controlled trials, understanding the patient will be a
large factor in determining outcomes of pragmatic treatment. It is
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Practical Considerations 659

important to emphasize that LLLT for pain should be administered

as part of holistic management following a full clinical evaluation,
including differential diagnosis and appropriate investigation. As a
corollary, however, findings on MRI or X-ray are largely irrelevant
to the outcomes of LLLT, apart from excluding potentially serious
disease. “Treat the patient and not the X-ray” is a primary tenant
of clinical LLLT. The ease of application of LLLT may provide a false
sense that everything is amenable to treatment.

35.7.1 Treating Knee Osteoarthritis as an Example

The treatment of knee osteoarthritis provides an example of how
the full repertoire of mechanisms is operationalized in “real life”
management.
In knee pain associated with osteoarthritis, there will be an
inflammatory component that involves the synovium and possibly
the bursa of the pes anserinus; there may be a mechanical
component where both medial and lateral collateral ligaments
are often strained due to abnormal forces on the knee; there
may be muscle spasm in hamstring and quadriceps muscles that
occurs to protect the joint; there may be enthesitis at the muscle
insertions; there may be a joint effusion. LLLT can modify each of
these elements within an osteoarthritic knee. The same principles
can also be applied to all joints and most other nociceptive
conditions. The anti-inflammatory action of laser will be effective
at the synovial surface as well as at entheses at the insertion of
ligaments. The neural blockade effect will occur at all tender points,
causing inhibition in the peripheral endings of nociceptors that
are present in the most superficial layers of skin as well as the
deep nerve tracts. This will further dampen inflammation arising
from nerve endings, so-called “neurogenic inflammation.” Clinically,
the interaction between neural blockade and anti-inflammatory
effects will then downregulate the peripheral sensitization that
occurs in injury and inflammation. Treatment of tender points and
trigger points in muscle bellies will reduce spasm, increasing joint
mobility and improving exercise tolerance. Motoricity of lymphatics
in the local area will also increase so that interstitial edema
and effusions will improve, again improving mobility. This can
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660 Low-Level Laser Therapy of Pain

also be coupled with treating the inguinal lymphatics to increase

lympathatic clearance in the whole limb. Cells involved in tissue
repair, including macrophages, neutrophils, and fibroblasts, will be
activated to initiate tissue repair. Even chondrocyte activity can be
increased so that ultimately some cartilage repair can occur (Holden
et al., 2009; Jia and Guo, 2004).
Repeated treatment is cumulative so that over a number of
treatments, the patient experiences reduction in pain, reduction
in swelling, and improved function. The patient can resume
exercise, lose weight, and reduce medication. If the joint has
advanced disease, a course of treatment may not control symptoms
permanently, so a “maintenance program” that keeps inflammation,
pain, and swelling under control is a reasonable option. Under
these circumstances and in elderly patients in whom surgery is not
indicated and in whom drugs cause serious side effects, the option of
seeing patients once in every 4 to 6 weeks is a safe and cost-effective
option.

35.8 Factors Influencing Outcomes

The deceptively simple application of LLLT belies the complexity

of multiple factors, which influence the clinical outcome. These
factors can be divided into four groups: laser parameters, treatment
protocols, patient and disease factors and are summarized in
Table 35.1.
The interaction of these factors contributes to its complexity and
explains why the outcomes of RCTs can be so variable. Response
to LLLT will depend on the nature of the problem, the site of the
pathology, the wavelength available to use, the sensitivity, tolerance,
and responsiveness of the patient, including the melanin content of
the skin.

35.8.1 Laser Factors

35.8.1.1 Wavelength
Evidence strongly suggests that NIR lasers are the most effective
in musculoskeletal pain. This is likely to relate to the greater
 July 6, 2016 17:37
Table 35.1 Factors affecting outcomes of treatment.

Laser Factors Treatment Patient Factors Disease Factors

Protocols
Wavelength • Red Frequency of Rxs: Skin pigmentation More melanin Duration of • Acute
• NIR • Daily, requires greater condition • Subacute
• Second daily dose • Chronic

PSP Book - 9in x 6in

• Weekly
Output • Very low: Duration of Rx at Obesity More fat = less Nature of the • Nociceptive pain
power <10 mW each point penetration painful condition • Neuropathic pain
• Moderately low: • Seconds
Age • Young, healthy
>10 to <50 mW • Minutes
• Older, frail
• Moderate: >50 Duration of Rx for
Medication • Steroids
to <100 mW whole session

Factors Influencing Outcomes

• Calcium channel
• High: >100 to • Minutes
blockers
<500 mW • Hours

(Contd.)

661

35-Hamblin-c35
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662 Low-Level Laser Therapy of Pain
Table 35.1 (Contd.)

Laser Factors Treatment Patient Factors Disease Factors

Protocols
Parameters Energy density; End point of Nervous System • Facilitated mode Site of pain • Localized
power density; treatment: State (related to (e.g., fibromyalgia) • Diffuse and
total joules; number of points pain) • Normal widespread
area of delivery; treated; • Repressed mode • Deep
• Personality traits • Superficial

PSP Book - 9in x 6in

pulsed versus number of joules Genetics of pain
continuous wave delivered modulation and • Anxiety
Decreased pain; epigenetics of pain • Catastrophising
increased
movement

Mode of • Contact: no Achievement of Duration of the • Acute Pathology • Inflammation

delivery pressure clinical endpoint: clinical condition • Subacute • Degeneration
• Contact: with improved function; • Chronic • Trauma,
pressure decreased pain; Social factors • “Yellow” flags including surgery
• Non-contact, reduced drug intake • Compensation
scanning injuries

35-Hamblin-c35
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Factors Influencing Outcomes 663

penetration depth of these lasers and the deeper structures, which

are the treatment targets in many conditions. Visible lasers with
shallower penetration depths are used for more superficial wound
healing, anti-inflammatory effects, and tissue repair. Transcutaneous
NIR laser as well as visible laser irradiation also inhibits underlying
nerves causing neural blockade, so use of both wavelengths in pain
is supported via that mechanism.

35.8.1.2 What is the correct dose?

What laser therapy does have in common with drug therapies is the
need for the appropriate “dose” to be delivered to achieve optimal
outcomes. The complexity and challenge of the clinical application
of laser therapy, whatever the indication, is to achieve the “correct”
dose in any one patient for the specific condition. In applying laser
clinically, and not within the confines of research, it is the patient’s
response to treatment, which will guide the “dose.” But what is the
dose? Is it the total number of joules? Is it energy density? Is it power
density? These questions have been outlined in several papers
and continue to be the subject of ongoing discussion (Jenkins and
Carroll, 2011). Tendinopathy, for example, appears sensitive to the
power density used as more than 100 mW/cm2 will not be effective.
Wound healing is sensitive to energy density with 1–4 J/cm2 being
most effective and >8 J/cm2 inhibitory. This biphasic response to
LLLT, where small doses of laser are stimulatory and large doses are
inhibitory, manifests at a cellular through to a whole person level
and are relevant across all the clinical applications of LLLT, including
pain (Huang et al., 2009; Sommer et al., 2001). These limits are
very important in research, but a pragmatic approach needs to be
adopted in the clinic as the dosage window can be relatively wide.
WALT has published guidelines for several wavelengths, which are
a good starting point. Ultimately, it will be the patient’s response,
which will be an important guide to dosage.

35.8.1.3 Application technique

Contact pressure is the degree of pressure applied with the probe in
contact with skin. Pressure will temporarily decrease the amount of
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664 Low-Level Laser Therapy of Pain

blood circulating in the target area and reduce the distance between
skin and target tissue, especially in obese patients, allowing more
photons to reach the site of pathology. This will, of course, not be
an issue in superficial conditions. Several considerations dictate the
amount of pressure applied during treatment. If pain is very severe
or a very tender area is identified, then pressure should not be too
great as to cause pain. Not only does this increase the discomfort of
the patient but will stimulate nociceptors, which is the antithesis of
what LLLT aims to achieve, i.e., to silence nociceptors. Depth of the
target tissue is another consideration as sufficient pressure ensures
that enough photons reach the target tissue. Here a compromise
between pain and pressure needs to be reached. Sometimes a tender
area will gradually be less painful during the laser application and
that will be an indicator of adequate treatment.

35.8.2 Treatment Protocol

The frequency at which laser therapy is given and the duration of
each session are dependent on the nature of the condition. This
too, as with dose, is guided to a large extent by patient response.
In general acute conditions can be treated more frequently than
chronic conditions. For acute treatments, a rule of thumb is to deliver
treatment three times in the first week, twice in the second, and once
in the third week and then once a fortnight if necessary. When the
patient starts to experience fewer symptoms, treatment frequency
should decrease. For chronic conditions, treatment can be delivered
more slowly: twice a week for the first two weeks and then once a
week for two to four weeks depending on dose.
As tissue approaches “normality,” it is possible that too frequent
treatments will have less favorable outcomes with increase in pain
after treatment.

35.8.2.1 How long should a course of treatment be?

Patients often want to cease treatment as soon as pain is relieved;
however, reduced pain early in treatment does not indicate tissue
repair, which may take several weeks for full recovery. Many patients
are often very surprised that they can have no or little pain but have
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Factors Influencing Outcomes 665

widespread tenderness. While there is tissue tenderness, patients

may easily reactivate the injury and should take care to avoid
those activities that activate pain. Achieving pain relief with no
tenderness to palpation is the ideal, but it may not be practicable
or indeed possible to treat patients until all tissue tenderness
resolves. Treatment courses can vary; however, six to ten treatments
are suggested as an appropriate course. Followup a month after
completion is often worthwhile to assess the tissue sensitivity.

35.8.3 Patient Factors

Evidence now exists that a number of patient factors will alter
the dose of laser required to achieve a positive outcome. These
include skin pigmentation where the greater the melanin content,
the greater the dose is needed (Liebert et al., 2012; Nussbaum
et al., 2007). Similarly, the greater the amount of adipose tissue
between the skin and target tissue, the greater the dose required to
reach the target. The state of a patient’s “pain” setting, i.e., whether
they exhibit hyperalgesia and allodynia, indicates a lowered pain
threshold and hence the need for a lighter touch and a lower dose
at the initial laser treatments.

35.8.4 Disease Factors

Not all conditions will be responsive to LLLT. Conditions where
there are mechanical factors, such as nerve root compression with
neurological symptoms, are not likely to respond to LLLT. The
presence of hyperalgesia and allodynia is an indicator of central
sensitization and underlying changes in the spinal cord, which
render the need for treatment to be at a lower dose and “light touch”
as even light touch can aggravate the underlying symptoms. One
of the goals of therapy is to reverse these changes by decreasing
nociceptive input over time to induce “long-term depression” (LTD)
of pain. In most conditions, once serious disease is excluded,
a therapeutic trial with a low threshold for cessation can be
considered if there is lack of progress. As a corollary, if a condition is
unresponsive to LLLT, it may be regarded as a “red flag” for further
investigation.
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666 Low-Level Laser Therapy of Pain

35.9 What Are the Goals of Treatment with LLLT?

In “real life” practice where patients present with a variety of

conditions of varying severity in different age groups, a pragmatic
approach must be taken to outcomes that will be as varied as the
patients and conditions. It is important the patient is aware of what
outcomes are likely to be and establishes realistic goals. It is the
outcome that the patient wants that will identify how to manage the
therapeutic process, which should be identified early in the process.
Is the patient an athlete who needs to compete after an injury? Is
it an elderly patient who wants to have less disturbed sleep? Is it
a person who has an injury who needs to return to the workplace
as soon as possible? Each of these individuals will have different
therapeutic goals and LLLT must be tailored to that outcome.

35.9.1 Monotherapy versus Adjunctive Treatment

The pragmatic approach to using LLLT in clinical applications
determines whether it will be a monotherapy or an adjunct to other
treatments. “Cure” is desirable but often unachievable in patients
with chronic pain. In many circumstances, patients will be satisfied
if they can return to a particular activity even if they continue to
experience some pain. Patients may not be able to abandon all
their analgesics, especially opioids or pain modulators but have
them significantly reduced. Using LLLT as a drug-sparing treatment
reduces side effects, costs, and tolerance particularly to opioids and
can mitigate the development of opioid-induced hyperalgesia. Use
of a “home” laser as an interval treatment where patients continue
to use lasers of lower doses to extend the time between treatments
or to reinforce the treatment is also an option for many patients.
For some patients, LLLT can facilitate their moving to a more active
exercise program and be used for flare-ups as they progress through
rehabilitation.

35.9.2 Why Some Patients Do Not Respond to LLLT?

Patient and disease factors are the most likely reason why patients
do not respond to LLLT. This assumes that laser factors have
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Practice Points 667

been evaluated and that basic principles such as checking the

output of the laser device, changing wavelengths, or increasing
(or decreasing) frequency of treatment have been undertaken. The
condition may not respond to treatment, or symptoms are too
widespread to treat cost effectively. Patients’ expectations may
exceed the reality of the treatment, and they may not wish to
continue with treatment if there is not an immediate response.
Drugs may inhibit response to laser. These include corticosteroids,
which have been demonstrated in animal studies to inhibit laser
effects. Calcium channel blockers, for example nifedipine, too have
also been demonstrated to interfere with the effects of laser as
laser modulates calcium channel activity and mediates intracellular
response to photons (Cohen et al., 1998).

35.10 Practice Points

The simplicity of the application of laser is one of the great

advantages of LLLT, but selection of the appropriate treatment sites
is the practical challenge for successful treatment. The aim is to
deliver enough photons to the most clinically relevant sites for the
condition being treated to initiate a tissue response that will lead to
pain relief and healing. In general terms, treatment strategies for all
musculoskeletal conditions can be summarized as follows:

• Treat locally at sites of tenderness. Treat more tender areas

first starting with the most tender points and working
systematically around the area. Treat all aspects of the
joint or pathology. Delivering 1–2 J per point is a safe
and reasonable start. Experimental studies indicate that
the neural blockade effect takes approximately 10 min;
however, for wound healing and tissue repair, delivery of
the total number of joules at an energy density of up to 4
J/cm2 or a power density of up to 100 mW/cm2 is a more
appropriate way to deliver the correct dose.
• Treat tender structures within the dermatome, myotome,
and sclerotome in the nerve root distribution of that of
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668 Low-Level Laser Therapy of Pain

the primary problem, including three levels above and two

below.
• Treat the spine at the level of the nerve root distribution
of the pathology, including the spinous processes where
the dorsal branch of the posterior ramus becomes very
superficial and over the nerve root exits.
• Treat the lymphatics, which drain the primary site, when
there is significant swelling associated with injury or
inflammation or when the patient complains of tightness or
puffiness. Activating the lymphatics will reduce edema.
• Do the “triple test” (T × 3) to assess progress during
treatment where possible. Test a movement or tenderness
to palpation before treatment (T1), treat for an appropriate
time (T2), and then test again (T3). If there is a change
in the “test,” then enough laser has been delivered to that
area. This can be repeated several times during a treatment
session.

35.11 “Tip of the Iceberg” Principle

Patients are often surprised to see how extensive areas of tender-

ness are beyond the primary site of pain, where they have no pain.
The primary site is “the tip of the iceberg,” but a whole limb might
exhibit widespread tenderness on examination. The convergence in
the dorsal horn of sensory afferents from painful sites synapsing
with motor neurons and interneurons results in the enlargement
of the receptive field of an injury so that changes in sensation,
lowered pain thresholds, and muscle spasm occur in uninjured
areas outside the primary site. The therapeutic consequence of this
is the importance of examining the whole dermatome, myotome,
and sclerotome of the affected segment. As an example in knee
osteoarthritis or lateral epicondylitis, all the muscle attachments (at
the periosteum) and the muscle bellies should be examined as well
as the joint or the entheses, and the most tender areas treated even
if the patient is not experiencing pain from these sites.
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Conclusion 669

35.12 Prognostic Factors

Abnormal imaging findings do not preclude good response to laser

therapy unless there are “red flags” that need further investigation.
Equally, the severity of pain or dysfunction does not preclude good
response and a trial of therapy is reasonable. There is no evidence
that older patients respond less well to LLLT, but given that there
are likely to be other co-morbidities with increasing age, there may
be less expectation of complete pain relief. In this scenario, however,
LLLT has a place in ongoing maintenance treatment with the goal of
keeping analgesic drugs to a minimum, and maintaining mobility is
a primary goal. The response to treatment will be the best indicator
of positive outcomes. The more rapid response to treatment is a
positive prognostic indicator of successful outcome.

35.13 Side Effects of Treatment

Side effects of LLLT are relatively infrequent and are minor in

nature. When reported, nausea, fatigue, and temporary exacerbation
of pain for 1–2 days after treatment are the most common. Kert
and Rose have provided a detailed outline of patterns of pain
following treatment, which they suggest reflects differing responses
to treatment depending on the type and chronicity of the pain (Kert
and Rose, 1989).

35.14 Conclusion

LLLT is a modality with a long history and strong evidence to support

its use in pain. It is not a miracle treatment but can achieve good pain
relief in a spectrum of conditions especially where other treatments
have failed. It has few side effects and is tolerated well by older
patients. It can be used as monotherapy or as adjunctive therapy
where it can play a role in drug sparing or facilitating rehabilitation.
The range of conditions able to be treated is wide. It requires some
technical expertise and knowledge, but for most doctors, physio-
therapists, and allied health professionals, it is easy to learn and
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670 Low-Level Laser Therapy of Pain

incorporate in day-to-day practice. Successful outcomes, like all

medical management, depend on good clinical skills linked with
an understanding of the nature of the pain and mechanisms of
laser effects. Laser medicine has the potential to be as popular as
laser surgery and become a stand-alone specialty in mainstream
medicine.

References

Aaron, J. E. 2012. Periosteal Sharpey’s fibers: A novel bone matrix regulatory

system? Front Endocrinol (Lausanne), 3, 98.
Abt, E. 1992. CO2 laser treatment for gingivectomies reduces hemorrhaging,
post-op pain. Clin Laser Mon, 10, 8.
Airaksinen, O., Rantanen, P., Pertti, K., and Pontinen, P. 1989. Effects of
the infrared laser therapy at treated and non-treated trigger points.
Acupunct Electrother Res Int J, 14, 9–14.
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Namazawa, R., Kemmotsu, O., Otsuka, H., Kakehata, J., Hashimoto, T.,
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680 Low-Level Laser Therapy of Pain

National Institute for Health and Care Excellence. 2014. Osteoarthritis: The
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July 6, 2016 17:37 PSP Book - 9in x 6in 35-Hamblin-c35

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power laser irradiation on procollagen synthesis in human fibroblasts.
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Yousefi-Nooraie, R., Schonstein, E., Heidari, K., Rashidian, A., Akbari-

Kamrani, M., Irani, S., Shakiba, B., Mortaz Hejri, S. A., Mortaz Hejri, S. O.,
and Jonaidi, A. 2008. Lowlevel laser therapy for nonspecific low-back
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Zeredo, J. L., Sasaki, K. M., Takeuchi, Y., and Toda, K. 2005. Antinociceptive
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Zinman, L. H., Ngo, M., Ng, E. T., Nwe, K. T., Gogov, S., and Bril, V. 2004. Low-
intensity laser therapy for painful symptoms of diabetic sensorimotor
polyneuropathy: A controlled trial. Diabetes Care, 27, 921–924.
July 6, 2016 17:37 PSP Book - 9in x 6in 36-Hamblin-c36

Chapter 36

Low-Level Laser Therapy and Its

Application in Tinnitus

Alessandra Nara de Souza Rastelli,a

Emanuelle Teixeira Carrera,a Gustavo Nicolodelli,b
and Michael R. Hamblinc,d,e
a Department of Restorative Dentistry, University of São Paulo State-UNESP,

1680 Humaita Street, Araraquara, São Paulo 14801-903, Brazil

b Brazilian Agricultural Research Corporation-Embrapa, CNPDIA,

1452 XV de Novembro Street, São Carlos, São Paulo 13560-970, Brazil

c Wellman Center for Photomedicine, Massachusetts General Hospital,

50 Blossom Street, Boston, MA 02114, USA

d Department of Dermatology, Harvard Medical School, Massachusetts General

Hospital, 55 Fruit Street, Boston, MA 02114, USA

e Harvard-Massachusetts Institute of Technology (MIT) Division of Health Sciences and

Technology, 77 Massachusetts Avenue, E25-518, Cambridge, MA 02139, USA

alrastelli@foar.unesp.br

Several modalities for the treatment of tinnitus have been proposed;

however, an effective standard treatment is still to be confirmed.
Low-level laser therapy (LLLT) has been proposed as a new therapy
for the treatment of tinnitus. However, the application of LLLT on
tinnitus remains obscure in the literature. Then, in the present
chapter, we aimed to show the state of the art of LLLT on tinnitus.

Handbook of Low-Level Laser Therapy

Edited by Michael R. Hamblin, Marcelo Victor Pires de Sousa, and Tanupriya Agrawal
Copyright c 2017 Pan Stanford Publishing Pte. Ltd.
ISBN 978-981-4669-60-3 (Hardcover), 978-981-4669-61-0 (eBook)
www.panstanford.com
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686 Low-Level Laser Therapy and Its Application in Tinnitus

36.1 Introduction

Tinnitus, from the Latin word tinnire, has been defined as a

perception of sound in the ear(s) in which a patient experiences a
ringing, buzzing, or hissing auditory sensation without any external
auditory stimulus [1, 19, 47]. In the general population, almost 15%
has experienced at least one episode of tinnitus. The prevalence
of tinnitus increases by age and reaches 85% in individuals older
than 60 years [42]. This symptom has been described as intolerable
in nearly 20% of the related cases [41]. Reaching as high as 67%,
tinnitus is more prevalent among individuals suffering from hearing
disorders [2]. Additionally, tinnitus is an increasing problem in more
recent birth cohorts [10].
Noise has such deleterious effects on hearing as noise-induced
hearing loss (NIHL) is the second most common form of acquired
hearing loss. It has long been recognized as a problem in noisy-
environment workers [32]. As a possible complication of NIHL,
tinnitus is frequently observed at frequencies equal to or higher
than 3000 Hz, which is one octave band higher than the frequencies
affected in NIHL. Its intensity is usually between 3 and 5 dB (in some
cases up to 15 dB) [13].
Regarding its prevalence and morbidity, tinnitus still remains an
obscure symptom. This is mainly because of the subjective nature
of the disorder and our lack of knowledge of its pathophysiology.
In this way, treatment of tinnitus has been limited, controversial,
and quite often unsuccessful. The treatment of tinnitus using
pharmacological substances has proved to be a difficult task,
although the effect of many substances has been studied and some
used to alleviate the symptoms of tinnitus. However, today there
are no agents specifically recommended for this purpose. Several
therapeutic modalities have been proposed, including medications,
such as sedatives, antiepileptics, antidepressants, antipsychotics,
local anesthetics, antihistamines, and botulinum toxin A [14].
However, these methods have not shown efficient therapeutic effects
[21]. There is no definitive therapy for tinnitus.
Other modalities proposed for the treatment of tinnitus could
be cited and used: antioxidant therapy [48], repetitive transcranial
magnetic stimulation [28], transcutaneous electrical stimulation
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Introduction 687

[26], sound therapy [23], and cognitive behavioral therapy [1]. These
non-pharmacologic and surgical therapeutic approaches have been
used in specific cases. In addition to different standard methods,
transmeatal cochlear low-level laser irradiation has been introduced
as an alternative modality for cochlear dysfunctions such as chronic
cochlear tinnitus [41]. LLLT has been tried with promising results in
patients with subjective tinnitus since 10 years ago [41].
LLLT is a medical treatment that uses low-energy laser to
stimulate or inhibit cellular function [20]. The US Food and Drug
Administration has cleared the use of low-level laser for the
treatment of lymphedema [15] and chronic pain [3, 71]. Clinical
applications of LLLT in otolaryngology include management of
hyperacusis, phonophobia, diplacusis, and sound distortion [69],
and the treatment of tinnitus [54, 55].
Low-level laser has been used for different applications in
medicine, such as wound healing, nerve and tissue repairing, pain
control [56], and treating tinnitus among others [37]. LLLT had
been found useful in treating chronic impaired hearing, sudden
sensorineural hearing impairment [70], and in treating Meniere’s
disease and some other balance disorders [9, 22].
However, the exact mechanism of the effect of LLLT on tinnitus
is not very clear yet, and it has been proposed that it may be
induced by increasing cell proliferation, growth factor secretion,
improvement in inner ear blood flow, and/or activation of the
hair cells mitochondria [41]. The low-level laser procedure has
demonstrated few side effects; only Nakashima et al. reported an
acute onset of hearing deterioration in one patient after the third
bilateral laser irradiation [37].
There is still some degree of controversy concerning the
efficiency of LLLT in tinnitus. Some studies have shown positive
effects [9, 41, 54, 56, 70], but others have found no such effectiveness
[37, 56]. The reason for the discrepancy in different findings is not
well known; however, multiple factors such as differences in study
design, subject characteristics, LLLT methodology, and outcome
measures are used to assess the effects of LLLT.
The purpose of this chapter is to show the more recent state
of the art of LLLT on tinnitus. First, some considerations are
necessary.
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688 Low-Level Laser Therapy and Its Application in Tinnitus

36.2 Symptoms of Tinnitus Ringing in Ears

Many people experience an occasional ringing in the ears or hear

roaring, buzzing, hissing, or whizzing noise. When this constant
internal noise does not diminish, it is referred to as tinnitus. It can
be described as a conscious awareness of a sound in the ears or
head that is not due to an external noise. Individuals have their
own tinnitus tone. It can be a high- or low-frequency sound, and
the volume can vary over time. A significant number of people who
experience tinnitus symptoms become severely distressed by the
sounds. The sounds range from high to low pitch and can be a
single tone, multi-tonal, noise-like, or have no tonal quality. Tinnitus
may be constant, pulsing, or intermittent. It may begin suddenly or
progress gradually [24, 30, 40, 60].

36.3 Types of Tinnitus

Tinnitus can be divided into two different types:

36.3.1 Subjective Tinnitus

Subjective tinnitus can be described as the noises that can be heard
only by the patient. This is the most common type of tinnitus.
Everyone, sitting in a soundproof room, hears noises in their heads.
Usually, these noises are masked in everyday life by all the noise
going on in the world around us. If you cannot hear sounds of
the outside world so well, you tend to notice the natural noises
inside your head much more because they are not masked by
environmental noises [8].
Tinnitus is often linked to hearing loss. If the tiny hair cells
of the cochlea are damaged by certain drugs, noise exposure, or
the aging process, the cochlea becomes less good at differentiating
sounds and your hearing is affected. Tinnitus is also associated
with many different diseases and disorders, and it sometimes
emerges as a side effect of medications used to treat other diseases
[49]. Otologic, neurologic, and infectious causes can be related to
subjective tinnitus. Otologic is the most common cause of this
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Types of Tinnitus 689

kind of tinnitus. This includes NIHL, presbycusis, otosclerosis, otitis,

cerumen impaction, Meniere’s disease, and sudden sensorineural
hearing loss. Neurologic causes include head injury, whiplash, mul-
tiple sclerosis, vestibular schwannoma, and other cerebellopontine-
angle tumors. Tinnitus may arise as a result of different infectious
sources such as otitis media, Lyme disease, meningitis, or syphilis
[8].

36.3.2 Objective Tinnitus

Objective tinnitus can be described as a condition when noises are
heard by somebody examining the patient. This type of tinnitus is
uncommon. Ringing noises in the ears may be caused by spasms of
small muscles in the middle ear (often heard as a clicking sound) or
by abnormalities of the blood vessels in and around the ear. When
tinnitus is related to spasms of the middle ear muscle, namely,
tensor tympani, and stapedius muscles, it is called muscular tinnitus.
Myoclonus of the palatal muscles may result in the clicking noises,
which may indicate an underlying neurologic disorder, such as
multiple sclerosis or neuropathy. Other somatic disorders such as
Eustachian tube dysfunction may be responsible for tinnitus, which
is synchronous with respiratory movements [8, 29].
The turbulent bloodflow is heard directly by the inner ear, usually
when the heart beats (pulsatile tinnitus). Tinnitus can occur because
of an increased bloodflow to the ear, such as during an infection or
inflammation, but also because of anatomical abnormalities of the
blood vessels. Pulsatile tinnitus is frequently linked with different
vascular etiologies, including arterial bruit, dural arteriovenous
shunts, paraganglioma, and venous hum. Arterial bruits may be
present in arteries near the temporal bone, most commonly the
pertrous carotid system [16]. Paraganglioma is a vascular neoplasm
arising from paraganglia cells at the carotid bifurcation in the jugular
bulb or along tympanic arteries. These neoplasms may generate
pulsating sounds that could be transmitted to the cochlea and
produce objective tinnitus. Venous hums may be heard in patients
with a dehiscent jugular bulb, systemic hypertension, or increased
intracranial pressure [8, 16].
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690 Low-Level Laser Therapy and Its Application in Tinnitus

36.3.3 Function and Dysfunction of Inner Ear

The inner ear has two sensory organs: the organ of balance in
the labyrinth and the organ of Corti (hearing) in the cochlea.
The inner ear (internal ear, auris interna) is the innermost part of
the vertebrate ear. In vertebrates, the inner ear is mainly responsible
for sound detection and balance [53]. In mammals, it consists of
the bony labyrinth, a hollow cavity in the temporal bone of the skull
with a system of passages comprising two main functional parts
[68]:

(1) The cochlea, dedicated to hearing; converting sound pressure

patterns from the outer ear into electrochemical impulses,
which are passed on to the brain via the auditory nerve.
(2) The vestibular system, dedicated to balance.

The inner ear is found in all vertebrates, with substantial variations

in form and function. The inner ear is innervated by the eighth cra-
nial nerve in all vertebrates.
The waves of vibration (sound) mechanically transmitted to
the cochlea via the eardrum and the ossicles (middle ear) are first
compressed by a highly complex fluid system and then converted
to electrophysiologic impulses that are transferred to the central
nervous system [63].
This conversion process can be explained in detail with today’s
biological knowledge. It takes place in the auditory cells of the
cochlea and is connected with movements of hair-like receptors on
the cellular membrane and other processes within the cells of the
inner ear. For this to happen properly, the auditory cell requires
energy in the form of ATP produced by the mitochondria (cellular
power plants) inside each cell [63].
If the cochlea is acutely or chronically over-strained, its sensory
(auditory) cells and various cellular organs are affected as well and
they inevitably lose part of their functional capacity. The cells suffer
from a lack of ATP.
A continuous lack of ATP within the inner ear cells of the
cochlea leads to either a gradual or a sudden impairment of the
entire hearing organ. This is experienced by the patient as acute or
insidious symptoms such as pressure in the ear, vertigo, hearing loss,
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Causes of Tinnitus 691

hypersensitivity to sound (hyperacusis), and tinnitus (insidious or

acute) [63].
In the semicircular canals of the organ of balance, any changes
in the position of our head and body are also conveyed via a fluid
system and the movements of hair-like receptors. For this reason,
the ATP-consuming processes within the cells in question are almost
identical to those in the cochlea during the act of hearing.

36.4 Causes of Tinnitus

Although the exact mechanism underlying tinnitus is unknown, it

usually has to do with an abnormality of the hearing or neural
system. Many causes can be linked with tinnitus as follows [31, 72]:

• Disorders in the outer ear, such as ear wax, a hair touching

the eardrum, a foreign body, or a perforated eardrum
• Disorders in the middle ear, such as negative pressure from
Eustachian tube dysfunction, fluid, infection, otosclerosis,
allergies, or benign tumors
• Disorders in the inner ear, such as sensorineural hearing
loss due to noise exposure, aging, inner ear infection, or
Meniere’s disease often accompanied by hearing loss and
dizziness.

Tinnitus can also temporarily result from some medications such as:

• Anti-inflammatories such as aspirin, ibuprofen, non-

steroidal anti-inflammatories, and quinine
• Sedatives
• Antidepressants
• Some antibiotics and chemotherapeutic agents

Other causes of tinnitus can include:

• Systemic disorders such as high or low blood pressure,

anemia, diabetes, thyroid dysfunction, glucose metabolism
abnormalities, vascular disorders, growth on jugular vein,
acoustic tumors, and head or neck aneurysms
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692 Low-Level Laser Therapy and Its Application in Tinnitus

• Nonauditory disorders such as trauma to the head or

neck, temporomandibular (jaw joint) disorders, and neck
misalignment

Although the majority of tinnitus patients have hearing loss, the

presence of tinnitus may not indicate that one is losing hearing.

36.5 Diagnosis of Tinnitus

A complete head and neck examination should be performed, and

otologic and neurotologic evaluations should be conducted [52,
53]. Specific attention should be directed at the auscultation of
periauricular area, observation of palatal movements, and changes
in tinnitus with jaw clenching or eye movements [9].
Sometimes, laboratory and imaging studies are required in order
to obtain the correct diagnosis or to rule out potentially serious
diseases underlying tinnitus. Rapid identification and management
of causal and concurrent conditions in tinnitus may not only reduce
their potentially detrimental consequences, but also resolve or
improve the tinnitus when the conditions are well addressed.
In this way, the diagnosis is made through different techniques.
A complete neurotological examination, including otoscopy and
auscultation of the external ear canal, the periauricular area, and
the neck [52, 53] can be conducted. The medical evaluation will
start with a thorough medical history, followed by a careful physical
examination and diagnostic testing.
Audiological evaluation is an inseparable and important part
of tinnitus assessment. A hearing test is almost always needed to
identify any condition that involves the auditory system. This should
be the first step in an audiological evaluation of tinnitus, because
about 90% of the tinnitus cases occur with hearing loss. In addition
to routine pure tone and speech audiometry, a range of audiological
tests can help assess the health and function of your middle ear,
inner ear, and auditory pathway. They may include tympanometry,
otoacoustic emissions, electrocochleography, auditory brainstem
responses, and vestibular evoked myogenic potentials.
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Mechanisms of LLLT on Tinnitus 693

The audiological evaluation should also include tinnitus test

protocols that can help determine the pitch and loudness of the
tinnitus and how it interacts with an external sound, or what is
termed maskability and residual inhibition [9].
The impact of tinnitus on an individual’s life cannot always
be determined based on simple measures of tinnitus itself. A
comprehensive tinnitus assessment should always include measures
of the impact of tinnitus on your quality of life and ability to function.
A number of tinnitus evaluation questionnaires have been
developed and reported in tinnitus studies. These usually contain
multiple-choice questions covering various aspects of essential
daily activities, sleep, communication, interpersonal interactions,
cognitive functions, and emotional well-being as well as tinnitus
characteristics that can be used to generate an overall index score.
This score reflects how severely tinnitus affects a patient. While all
these questionnaires show certain levels of usefulness to determine
the severity of tinnitus, their differences make comparison among
studies complicated. There is a need to unify the tools used in the
measurement of tinnitus severity [9].
The Tinnitus Handicap Index and the Tinnitus Severity Index
(TSI) are frequently used. The recently published Tinnitus Function
Index has been developed by experts from multiple tinnitus centers
based on existing tinnitus questionnaires [9].
Specialized testing may be necessary in some cases of tinnitus.
For objective tinnitus that is pulsatile, it is prudent to rule out any
life-threatening diseases such as dural arteriovenous malformation,
aneurysm, or skull base tumor. These lesions can be diagnosed with
MR angiography or CT angiography [12]. Skull base tumor such as
paraganglioma can be evaluated by CT temporal bones. MRI can
diagnose central nervous system tumors, cerebellopontine-angle
tumors, increased intracranial pressures, and multiple sclerosis
[12].

36.6 Mechanisms of LLLT on Tinnitus

Although the mechanisms of low-level laser and its effect on

tinnitus are not well understood, some theories have been proposed.
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694 Low-Level Laser Therapy and Its Application in Tinnitus

Increase in cell proliferation, ATP, and collagen production; secretion

of grow factors; improvement in inner ear blood flow; and activation
of the hair cell mitochondria that simulates repair mechanisms have
been cited [39].
Some authors have postulated that LLLT may improve cochlear
function. Animal model studies have found that laser stimulation can
induce anatomic and physiologic changes in the cochlea. According
to Rhee et al. [43], rat hair cells were repaired with LLLT following
noise exposure. Wenzel et al. [62] found that laser stimulation
increased basilar membrane stiffness (and, therefore, resonant
frequency) in guinea pigs. The authors suggested that this could
allow low-frequency regions of the cochlea (where auditory function
is typically less compromised) to respond to high-frequency
sounds.
LLLT also promotes local blood flow in the inner ear and
activates repair mechanisms through photochemical and photo-
physical stimulation of mitochondria in hair cells [61]. Some
patients could show tinnitus as a side effect of Meniere’s disease. An
inner ear disorder, Meniere’s disease is characterized by recurrent
episodic vertigo, fluctuating hearing loss, and tinnitus [17, 22]. The
incidence is estimated to be between 50 and 350 per 100,000
persons per year [62]. The natural history of Meniere’s disease is
typically variable in intensity and frequency. Initial attacks are often
predominantly vestibular, while later attacks are more marked by
hearing loss and tinnitus. The disease is usually unilateral. Raised
endolymphatic pressure (hydrops) is commonly accepted as the
causal condition, although a direct relationship between Meniere’s
and endolymphatic hydrops remains unproven [67].
LLLT can produce a positive biological reaction regardless of the
dysfunction involved. This is due to its elementary and biologically
compelling working mechanism. On account of the basic conception
of our cellular energy-converting system (collector principle), the
electromagnetic energy released by the oxidation of nutrients is
utilized as a source of primary energy for the production of the
cellular fuel ATP. The antennae of the mitochondria can, in addition
to the absorption of the released metabolic energy, utilize both
the photons of the natural solar radiation (apparent biostimulative
effect of sunlight on human cells) and the photons of low/high-level
July 6, 2016 17:37 PSP Book - 9in x 6in 36-Hamblin-c36

LLLT for Tinnitus 695

Figure 36.1 Electromagnetic spectra wavelength.

laser light (clinically proven biostimulative effect of low/high-level

laser light on human cells) as a source of primary energy.

36.7 LLLT for Tinnitus

Low-level lasers are generally used in the visible and near-invisible

range of the electromagnetic spectrum. Figure 36.1 shows the
electromagnetic spectra for different wavelengths.
The most common types of low-level laser are based on
He–Ne (633 nm), InGaAlP (630–685 nm), GaAlAs (780–870 nm),
and GaAs (904 nm). Red laser light (visible) is optimal for superficial
conditions such as mucosa and skin, whereas infrared (invisible)
is better for pain and deeper conditions because of its superior
penetration.
Biological responses of cells to laser irradiation have been
suggested [27] to occur due to physical and/or chemical changes
in photoacceptor molecules and components of the respiratory
chain such as cytochrome c oxidase and NADH-dehydrogenase.
Hypotheses about primary mechanisms at the interface of laser
July 6, 2016 17:37 PSP Book - 9in x 6in 36-Hamblin-c36

696 Low-Level Laser Therapy and Its Application in Tinnitus

Figure 36.2 Different kinds of light tips used for tinnitus application.

irradiation and tissue are redox properties alterations, NO release,

superoxide anion reactions, singlet oxygen production, and local
transient heating of chromophores. Further, secondary processes
are triggered where the mechanisms are performed “in the dark.”
Then, distant effects can be obtained far from the irradiated area.
The redox-regulation mechanism may explain the positive effect of
tissues characterized by acidosis and hypoxia.
Figure 36.2 shows different kinds of light tips that can be used
for tinnitus application.
Although there are few studies on LLLT for tinnitus, it has been
suggested as a new possible therapy modality. We will describe this
therapy modality for tinnitus in the following paragraphs.
Shiomi et al. [51] have investigated the effect of infrared laser
applied to meatus acusticus under 21 J, once a week during 10
weeks. The results of this non-controlled study showed that 26% of
patients reported improved duration, 58% reduced loudness, and
55% just a general reduction in annoyance after LLLT application.
In another study, Shiomi et al. [50] have also verified the effect of
light on the cochlea, using guinea pigs. Direct laser irradiation was
applied to the cochlea through the round window and the amplitude
of compound action potential was reduced to 53–83% immediately
after the onset of irradiation. The amplitude then returned to the
original level. The results of this investigation suggest that LLLT
might lessen tinnitus by suppressing the abnormal excitation of the
eighth nerve or the organ of Corti. In a similar controlled study
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LLLT for Tinnitus 697

conducted by Mirtz et al. [33], a significant effect of LLLT was not

observed.
Some authors [68–70] have applied a different method where the
dose has been increased considerably. The method consists of the
use of one He–Ne low-level laser and three powerful GaAlAs lasers,
covering a large area over and around the ear, using the non-contact
technique. Energy between 3 and 5 J was given at each application
session. Only LLLT was applied. More than 800 patients have been
treated and positive effects have been reported, even for vertigo.
Recent injuries in “the disco generation” are more easily treated than
long-term chronic conditions.
In another study, Wilden et al. [67] reported improvement in the
hearing capacity of these patients, evaluated by audiometry. In order
to evaluate the effect of LLLT on tinnitus, Rogowski et al. [44] divided
32 tinnitus patients into two groups: one group receiving LLLT and
another receiving a placebo procedure. The parameters of low-level
laser were not stated in the available English abstract. The effect was
evaluated using a visual analog scale (VAS). Within the patient group,
transiently evoked otoacoustic emissions (TEOAE) were measured
before, during, and after LLLT. No significant difference between
laser and placebo was found in annoyance or loudness of the tinnitus
and in changes of TEOAE amplitude. These results indicate that there
is no relationship between the effect of low-level laser and changes
in cochlear micromechanics.
In order to evaluate the effectiveness of low-level laser irra-
diation in the treatment of chronic tinnitus, Gungor et al. (2008)
performed a prospective, randomized, double-blind study. For this
investigation 66 ears in 45 patients with chronic unilateral or
bilateral tinnitus were included. A 5 mW laser with a wavelength
of 650 nm or placebo laser was applied transmeatally for 15 min,
once daily for a week. A questionnaire asked patients to score
their symptoms on a five-point scale, before and two weeks after
laser irradiation. A decrease in one scale point, regarding the
loudness, duration and degree of annoyance of tinnitus was accepted
to represent an improvement. According to results obtained, the
loudness, duration and degree of annoyance of tinnitus were
improved, respectively, in up to 48.8, 57.7 and 55.5% of patients
in the laser group. No significant improvement was observed in the
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698 Low-Level Laser Therapy and Its Application in Tinnitus

placebo group. In this way, transmeatal, lowpower laser irradiation

was found to be an effective option for the treatment of chronic
tinnitus [18].
Teggi et al. [56], to determine the efficacy of LLLT for tinnitus,
performed a prospective, randomized double-blind study on 60
patients with tinnitus presenting sensorineural hearing loss in the
affected ear. The patients were randomly divided into two groups.
The first received active laser therapy 20 min a day for 3 months
with a 650 nm, 5 mW soft laser (Group L), and the second received a
therapy using a dummy device that duplicated all aspects of active
laser therapy except for the activation of the laser beam (Group
C). One subject in both groups dropped out due to an increase
in tinnitus loudness. Two more patients in each group ceased to
comply with the protocol due to familiar problems. To evaluate the
patients, the Tinnitus Handicap Inventory (THI) was considered.
Moreover, VAS was established for self-perceived loudness of the
tinnitus and audiometric (frequencies between 125 and 8000 Hz)
threshold in both groups. Psychoacoustic assessment for pitch,
loudness, minimum masking level, and loudness discomfort level
was also investigated. LLLT showed no efficacy as a therapeutic
treatment for tinnitus [56].
More recent studies have shown positive results with the use
of LLLT in tinnitus. Okhovat et al. [41] evaluated the sensation of
tinnitus using a VAS before and two weeks after treatment. It was
a self-controlled clinical trial study on 61 patients with subjective
tinnitus. The patients were irradiated using a 650 nm, 5 mW low-
level laser for 20 days and 20 min per day. Based on the results
obtained in this study, the authors showed that transmeatal low-
level laser irradiation was effective for the treatment of tinnitus and
some variables such as age and job can affect the treatment outcome.
To investigate the effectiveness of LLLT to treat patients who
were suffering from long-term complaints of tinnitus with well-
understood etiology and who were not responding to conventional
therapy in Qatar, Salahaldin et al. [46] included 65 patients aged 15–
76 years with chronic unilateral or bilateral tinnitus with a minimum
duration of illness of one year. The investigation included 101 ears
of 65 patients. A 5 mW laser with a wavelength of 650 nm was
applied transmeatally for 20 min once daily for 3 months. Over half
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LLLT for Tinnitus 699

of the patients (56.9%) had some improvement in their tinnitus

symptoms. Mild improvement was reported in 33.8% of patients,
moderate improvement in 16.9%, and full improvement in 6.15%. Of
the patients who reported dizzy spells as a symptom of their tinnitus
condition, 27.7% reported mild improvement and 16.9% reported
full improvement. Common side effects of LLLT were noted among
20% of patients; however, all of them were mild and disappeared
within a few days. LLLT was found to be useful for treatment of
chronic tinnitus.
Mollasadeghi et al. [35] evaluated patients suffering from tinnitus
accompanied by NIHL. LLLT was applied for 20 sessions, 20 min
each session. Tinnitus was assessed by three different methods (VAS,
THI, and tinnitus loudness) at different times as follow: baseline,
immediately, and 3 months after the intervention. All patients were
male workers in the age group of 30–51 years. The mean tinnitus
duration was 18.5 ± 0.78 years. The three measurement methods
showed improved values after LLLT compared with the placebo,
both immediately and 3 months after treatment. Laser therapy
revealed a U-shaped efficacy throughout the course of follow-up.
Nonresponse rates of the intervention were 57% and 70% in the
two assessment time points, respectively. Based on this study, LLLT
showed to be effective in alleviating tinnitus in patients with NIHL,
although this effect faded after 3 months of follow-up.
Mirvakili et al. used different evaluation criteria to study patients
with tinnitus that was created after sensorineural hearing loss
and reported that low-level laser radiation can improve tinnitus
recovery only for short time and its effect decreases with time. [34].
This was a cross-sectional study on 120 patients with tinnitus and
sensorineural hearing loss. The patients were randomly divided into
two groups; one group received low-level laser and the other group
used the same instrument but it was switched off for 20 sessions of
20 min. THI and VAS were used to evaluate the severity of patients’
symptoms. Severity and frequency of tinnitus were also determined
using audiometric tests. After this study, the authors could show that
the average ages of the 120 patients in the two groups evaluated
were not statistically significant and different. The mean difference
of severity of tinnitus between the two groups was statistically
significant at the end of the study and 3 months after the treatment.
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700 Low-Level Laser Therapy and Its Application in Tinnitus

The VAS and THI mean differences after the treatment were
statistically significant between the two groups but not statistically
significant after 3 months of completion of the study. Thus, it was
possible to show that low-level laser irradiation was effective for
short-term treatment of tinnitus caused by sensorineural hearing
loss and its impact may be reduced over time.
Dehkordi et al. evaluated the effect of LLLT on tinnitus [11]. A
prospective, double-blind, placebo-controlled study was performed
to evaluate the effect of low-dose laser therapy on chronic cochlear
tinnitus. Sixty-six (n = 66) patients were evaluated, where 33
received laser treatment and the remaining 33 received inactive
dummy treatment (control group). Patients from the laser group
received 5 mW under 650 nm for 20 min a day, 5 days a week,
for 4 weeks. The controls followed the same schedule, but they
were “treated” with an inactive device. The degree of tinnitus was
evaluated before and after as follows: (1) TSI, (2) a subjective 10-
point self-assessment scale for tinnitus loudness, and (3) Tinnitus
Evaluation Test. The results showed that LLLT was no better than
placebo for improving overall hearing thresholds or for treating
tinnitus with regard to age, sex, environmental noise level, and
duration [11].
It has been known for decades that patients with temporo-
mandibular disorder (TMD) and craniomandibular disorder may
also have tinnitus problems and that there is a connection between
the two.
In a book, Myrhaug [36] has underlined the fact that two
muscles in the inner ear are innervated by two facial nerves. M.
tensor tympani is innervated by n. trigeminus, and m. stapedius is
innervated by n. facialis. Intensive action in the masticatory muscles
could, therefore, influence these two small muscles and thereby
cause the tinnitus sensation.
Bjorne and Agerberg [5] compared a group of 31 patients
suffering from Meniere’s disease with a control group matched for
sex and age. The patients in the Meniere group showed statistically
significant signs of craniomandibular disorders, such as tenderness
to palpation on the masticatory muscles of the temporomandibular
joint, upper part of the trapezius in the area of the atlas, the axis, and
the third cervical vertebra.
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LLLT for Tinnitus 701

In another study by Bjorne et al. [6], 24 of the 31 patients

from the previous study were compared with 24 control subjects
regarding the frequency of signs and symptoms of cervical spine
disorders. Symptoms of cervical spine disorders, such as head and
neck/shoulder pain, and signs such as limitations in side bending
and rotation movements were more frequent in the patient group
as well as tenderness to palpation of the neck muscles. Thirty-nine
percent of the Meniere patients could influence their tinnitus, both
sound level and pitch, by protrusion or lateral movement of the
mandible or by clenching their teeth. Also 75% of the patients could
trigger their attacks of vertigo by extension, flexion, or side-rotation
of the head and neck.
A correlation between tinnitus and tension of the lateral
pterygoid muscle has been found [4]. Further correlation between
the signs and symptoms of TMD and tinnitus has been indicated in
studies by Rubenstein [45] and Ciancaglini [7].
Wong [69] reports that the styloid process and its attachments
are often the center of TMD problems and that no less than 11
symptoms have been observed in connection with soft tissue lesions
in this region, one of them being tinnitus. The muscular symptoms
are suitable for LLLT, according to the authors.
Tullberg and Ernberg [58] investigated the presence of symp-
toms and signs of TMD in patients with tinnitus. In order to evaluate
the effect of TMD treatment on tinnitus in a long-term perspective
in comparison with a control group of patients on a waiting list,
120 patients with tinnitus were subjected to a clinical examination
of the masticatory system and whether they had coexisting TMD.
Ninety-six patients had TMD, most frequently localized myalgia.
Seventy-three of these completed the treatment and responded to a
questionnaire 2 years later. Fifty patients (n = 50) with tinnitus, who
were on the waiting list, served as the control group. The results
of this study showed that TMD symptoms and signs were frequent
in patients with tinnitus and that TMD treatment had a good effect
on tinnitus in a long-term perspective, especially in patients with
fluctuating tinnitus.
In order to show that tinnitus is one of the otologic symptoms
commonly reported to be associated with TMD, Kanji and Khoza-
Shangase [25] investigated the clinical signs and symptoms of
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702 Low-Level Laser Therapy and Its Application in Tinnitus

tinnitus in a group with TMD (Group A) and compared them

to a group with tinnitus but without TMD (Group B). Twenty
participants were included in the study, 10 in each group. All
participants underwent basic audiological as well as ear, nose,
and throat evaluations to establish Group A and Group B. For
tinnitus assessment, all participants completed a tinnitus survey
questionnaire, and their tinnitus was evaluated using tinnitus
matching procedures. The findings of this study revealed clinically
relevant differences in attributes of tinnitus in patients with and
without TMD. Most of the participants in Group A matched their
tinnitus to a 6000 Hz tone or noise, at lower intensity levels than
participants in Group B, although these results were not statistically
significant. Participants in Group A associated their tinnitus with a
single sound, whereas some participants in Group B associated it
with more than one sound. More participants in Group B reported
the duration of their tinnitus as constant. Tinnitus may occur in
patients with TMD and may be of high frequency. This highlights the
importance of thorough assessment for patients with tinnitus as this
might have implications for diagnosis and management [25].
LLLT is still controversial as a therapeutic modality in cochlear
dysfunctions such as chronic cochlear tinnitus or sensorineural
hearing loss. Low-intensity laser irradiation is a new and effective
method for the treatment of peripheral and central nervous system
injuries and disorders [28]. In addition to different pathophysio-
logical mechanisms of inner ear diseases and diverse theories on
the nature of tinnitus, the methodical differences in study design,
treatment schedules, and irradiation parameters could result in a
wide range of outcomes [54].
As previously described, conflicting results on the effect of LLLT
on hearing have been reported. The current results are further
evidence that LLLT does not have an effect on hearing status. In the
current study, care was taken to blind the subjects, the researchers
administering the treatment, and the researchers administering
auditory testing. It appears that some previous studies were less
careful about controlling placebo effect and potential researcher
bias. Future studies should also implement double blinding, as
well as control and placebo groups. Other factors may also explain
the discrepancy in findings. Some previous studies may have
July 6, 2016 17:37 PSP Book - 9in x 6in 36-Hamblin-c36

References 703

achieved much greater laser penetration by using animal models

[43] and isolated cochleae [62]. The current study, involving external
irradiation of human subjects, likely involved less stimulation of
structures associated with hearing. While a transmeatal approach
to irradiation [24] would have achieved greater penetration, such
an approach represents a less practical delivery method and is
not commonly used by holistic practitioners of LLLT. Differences in
laser wavelength and dosage may also contribute to variable results
across studies.
It is obvious that the available literature on laser therapy
of tinnitus is scarce and ambiguous. Some studies have used a
combination of different techniques, while others used LLLT as
a monotherapy. Differences in wavelengths, pulsing, dosage, and
treatment technique make a firm evaluation impossible. However,
the positive results reported in some studies do merit attention and
further research. Tinnitus is a grave condition, sometimes leading to
suicide. It is also an increasing problem, and the existing treatment
modalities offered to tinnitus patients are not very effective. Young
people suffering from acoustic chocks (concerts, discos) can be more
successfully treated with laser therapy. Understandably enough, a
longstanding condition in elderly persons is a severe condition
taking 10–20 sessions to influence.

36.8 Conclusion

Few have explored the concept of treating tinnitus using low-level

laser irradiation, and there is very little published research on it.
This chapter does not intend to give precise recommendations about
treatment procedures; rather, it wants to put light on the possibility
of improving the quality of life of many tinnitus patients using LLLT.
Of course further research is needed, mainly because the results of
LLLT on tinnitus remain obscure.

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28. Langguth, B., Kleinjung, T., Landgrebe, M., de Ridder, D., and Hajak, G.
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tinnitus, J. Laryngol. Oto., 120, pp. 93–97.
30. Loeb, M., and Smith, R. (1967). Relation of induced tinnitus to physical
characteristics of the inducing stimuli, J. Acoust. Soc. Am., 42, pp. 453–
455.
31. McCormack, A., Edmondson-Jones, M., Fortnum, H., Dawes, P. D,
Middleton, H., Munro, K. J., and Moore, D. R. (2015). Investigating the
association between tinnitus severity and symptoms of depression and
anxiety, while controlling for neuroticism, in a large middle-aged UK
population, Int. J. Audiol., 13, pp. 1–6.
32. Mehrparvar, A. H., Mirmohammadi, S. J., Ghoreyshi, A., Mollasadeghi,
A., and Loukzadeh, Z. (2011). High-frequency audiometry: A means for
early diagnosis of noise-induced hearing loss, Noise Health, 13, pp. 402–
406.
33. Mirtz, F., Zachariae, R., Andersen, S. E., Nielsen, A. G., Johansen, L. V.,
Bjerring, P., and Pedersen, C. B. (1999). The low-power laser in the
treatment of tinnitus. Clin. Otolaryngol. Allied Sci., 24, pp. 346–354.
34. Mirvakili, A., Mehrparvar, A., Mostaghaci, M., Mollasadeghi, A., Mirvakili,
M., Baradaranfar, M., Dadgarnia, M., and Davari, M. (2014). Low-level
laser effect in treatment of patients with intractable tinnitus due to
sensorineural hearing loss, J. Lasers Med. Sci., 5, pp. 71–74.
35. Mollasadeghi, A., Mirmohammadi, S. J., Mehrparvar, A. H., Davari, M. H.,
Shokouh, P., Mostaghaci, M., Baradaranfar, M. H., and Bahaloo, M. (2013).
Efficacy of low-level laser therapy in the management of tinnitus due
to noise-induced hearing loss: A double-blind randomized clinical trial,
Scientific World J., doi: 10.1155/2013/596076.
36. Myrhaug, H. (1981). The theory of otosclerosis and Morbus Ménière
(Labyrinthine vertigo) being caused by the same mechanism: Physical
irritants, an otognathic syndrome. Bergmanns Boktryckeri A/S, Bergen,
Norway.
37. Nakashima, T., Ueda, H., Misawa, H., Suzuki, T., Tominaga, M., Ito, A.,
Numata, S., Kasai, S., Asahi, K., Vernon, J. A., and Meikle, M. B. (2002).
Transmeatal low-power laser irradiation for tinnitus, Otol. Neurotol., 23,
pp. 296–300.
38. NASA. Mission: Science. Introduction to the Electromagnetic Spec-
trum. Available in: http://missionscience.nasa.gov/ems/01 intro.html.
Accessed on 30 March 2015.
July 6, 2016 17:37 PSP Book - 9in x 6in 36-Hamblin-c36

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39. Neri, G., De Stefano, A., Baffa, C., Kulamarva, K., Di Giovanni, P.,
Petrucci, G., Poliandri, A., Dispenza, F., Citraro, L., and Croce, A.
(2009). Treatment of central and sensorineural tinnitus with orally
administered melatonin and sulodexide personal experience from a
randomized controlled study, Acta Otorhinolaryngol. Ital., 29, pp. 86–91.
40. Norena, A., Micheyl, C., Chery-Croze, S., and Collet, L. (2002). Psychoa-
coustic characterization of the tinnitus spectrum: Implications for the
underlying mechanisms of tinnitus, Audiol. Neuro-Otol., 7, pp. 358–369.
41. Okhovat, A., Berjis, N., Okhovat, H., Malekpour, A., and Abtahi, H. (2011).
Low-level laser for treatment of tinnitus: A self-controlled clinical trial,
J. Res. Med. Sci. 16, pp. 33–38.
42. Prochazka, M. (2002). The role of LLLT in treatment of tinnitus,
http://ammhealth.co.za/pdf/info/latest/The%20Role%20of%20
LLLT%20in%20Treatment%20of%20Tinnitus.pdf.
43. Rhee, C. K., Bahk, C. W., Chung, P. S, Jung, J. Y., Suh, M. W., and Kim, S. H.
(2012). Effect of low-level laser treatment on cochlea hair-cell recovery
after acute acoustic trauma, J. Biomed. Optics., 17, pp. 068002.
44. Rogowski, M., Mnich, S., Gindzienska, E., and Lazarczyk, B. (1999). Low-
power laser in the treatment of tinnitus: A placebo-controlled study,
Otolaryngol. Pol. 53, pp. 315–320.
45. Rubenstein, B. (1993). Tinnitus and cranomandibular disorders: Is
there a link? [Thesis]. Swed. Dent. J. Suppl., 95.
46. Salahaldin, A. H., Abdulhadi, K., Nihal, N., and Abdulbari, B. (2012).
Low-level laser therapy in patients with complaints of tinnitus: A clinical
study, Otolaryngology, Article ID 132060.
47. Salvi, R., Lobarinas, E., and Sun, W. (2009). Pharmacological treatments
for tinnitus: New and old, Drugs Future, 34, pp. 381–400.
48. Savastano, M., Brescia, G., and Marioni, G. (2007). Antioxidant therapy in
idiopathic tinnitus: Preliminary outcomes, Arch. Med. Res., 38, pp. 456–
459.
49. Seligmann, H., Podoshin, L., Ben-David, J., Fradis, M., and Goldsher, M.
(1996). Drug-induced tinnitus and other hearing disorders, Drug Saf.,
14, pp. 198–212.
50. Shiomi, Y., Tuji, J., Naito, Y., Shoji, K., Kojima, H., and Honjo, I. (1994).
Effect of low power laser irradiation on inner ear, Pract. Otol. (Kyoto),
87, pp. 1135–1140.
51. Shiomi, Y., Takahashi, H., Honjo, I., Kojima, H., Naito, Y., and Fujiki, N.
(1997). Efficacy of transmeatal low power laser irradiation on tinnitus:
A preliminary report, Auris. Nasus. Larynx., 24, pp. 39–42.
July 6, 2016 17:37 PSP Book - 9in x 6in 36-Hamblin-c36

708 Low-Level Laser Therapy and Its Application in Tinnitus

52. Schoeff, S., Nicholas, B., Mukherjee, S., and Kesser, B. (2014). Imaging
prevalence of sigmoid sinus dehiscence among patients with and
without pulsatile tinnitus, Otolaryngol. Head Neck Surg., 150, pp. 841–
846.
53. Shweel, M., and Hamdy, B. (2013). Diagnostic utility of magnetic reso-
nance imaging and magnetic resonance angiography in the radiological
evaluation of pulsatile tinnitus, Am. J. Otolaryngol. 34, pp. 710–717.
54. Tauber, S., Beyer, W., Schorn, K., and Baumgartner, R. (2003). Trans-
meatal cochlear laser (TCL) treatment of cochlear dysfunction: A
feasibility study for chronic tinnitus, Lasers Med. Sci., 18, pp. 154–161.
55. Teggi, R., Bellini, C., Fabiano, B., and Bussi, M. (2008). Efficacy of low-
level laser therapy in Ménière’s disease: A pilot study of 10 patients,
Photomed. Laser Surg., 26, pp. 349–353.
56. Teggi, R., Bellini, C., Piccioni, L. O., Palonta, F., and Bussi, M. (2009).
Transmeatal low-level laser therapy for chronic tinnitus with cochlear
dysfunction, Audiol. Neurotol., 14, pp. 115–120.
57. Torres, M., and Giráldez, F. (1998). The development of the vertebrate
inner ear. Mechanisms Development, 71, pp. 5–21.
58. Tullberg, M., and Ernberg, M. (2006). Long-term effect on tinnitus by
treatment of temporomandibular disorders: A two-year follow-up by
questionnaire, Acta Odontol. Scand., 64, pp. 89–96.
59. Tyler, R. S., and Baker, L. J. (1983). Difficulties experienced by tinnitus
sufferers, J. Speech Hear. Disord., 48, pp. 150–154.
60. Tyler, R. S. (2000). The psychoacoustical measurement of tinnitus.
In: Tyler, R. S. (Ed.), Tinnitus Handbook (San Diego, USA: Singular
Publishing), pp. 149–179.
61. Vladimirov, I. A., Klebanov, G. I., Borisenko, G. G., and Osipov, A. N. (2004).
Molecular and cellular mechanisms of the low intensity laser radiation
effect, Biofizika, 49, pp. 339–350.
62. Wenzel, G. I., Pikkula, B., Choi, C. H., Anvari, B., and Oghalai, J. S. (2004).
Laser irradiation of the guinea pig basilar membrane, Lasers Surg.
Med., 35, pp. 174–180.
63. Wikipedia, the free encyclopedia. Inner ear. Available in
http://en.wikipedia.org/wiki/Inner ear. Accessed on 30 March 2015.
64. Wilden, L., and Dindinger, D. (1996). Treatment of chronic diseases of
the inner ear with low-level laser therapy (LLLT): Pilot project, Laser
Therapy, 8, pp. 209–212.
July 6, 2016 17:37 PSP Book - 9in x 6in 36-Hamblin-c36

References 709

65. Wilden, L., and Ellerbrock, D. (1999). Verbesserung der Hörkapazität

durch Low-Level-Laser-Licht (LLLL). [Amelioration of the hearing
capacity by low-level-laser-light (LLL)]. Lasermedizin, 14, pp. 129–
138.
66. Wilden, L. (1999). The effect of low-level laser light on inner ear
diseases. In: Tunér, J., and Hode, L. (Eds.), Low Level Laser Therapy:
Clinical Practice and Scientific Background. (Sweden: Prima Books).
ISBN 91-630-7616.
67. Wilden, L. (2009). The different response of the main symptoms of inner
ear exhaustion to a specific high-dosage low-level laser therapy, Med.
Laser Appl., 24, pp. 133.
68. Wolfe, J. M., Kluender, K. R., Levi, D. M., et al. (2009). Sensation and
Perception, 2nd Ed. (Sunderland: Sinauer Associated Inc.)
69. Wong, E., Lee, G., and Mason, T. (1995). Temporal headaches and
associated symptoms relating to the styloid process and its attachments,
Ann. Acad. Med. Singapore, 24, pp. 124–128.
70. Yıldırım, G., Berkiten, G., Ugraş, H., and Saltürk, Z. (2011). Changes in
audiometry results following laser therapy for tinnitus, Eur. J. Gen. Med.,
8, pp. 284–290.
71. Yousefi-Nooraie, R. Schonstein, E. Heidari K. et al. (2008). Low-level
laser therapy for nonspecific low-back pain, Cochrane Database Syst Rev,
Article ID CD005107.
72. Yurgil, K. A., Clifford, R. E., Risbrough, V. B., Geyer, M. A., Huang, M.,
Barkauskas, D. A., Vasterling, J. J., Baker, D. G., and MRS Team. (2015).
Prospective associations between traumatic brain injury and post-
deployment tinnitus in active-duty marines, J. Head Trauma Rehabil.,
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73. Zazzio, M. (2010). Pain threshold improvement for chronic hyperacusis
patients in a prospective clinical study, Photomed. Laser Surg., 28, pp.
371–377.
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Chapter 37

Laser Therapy for the Treatment of

Radiculopathy

Jerome M. Truea,b and Luis C. Verab,c

a Private Practice, 500 SE Dixie Highway, Suite 2, Stuart, FL 34994, USA
b College of Medicine, University of Central Florida, 6850 Lake Nona Boulevard

Orlando, FL 32827,USA
c Palmer College of Chiropractic, 4777 City Center Parkway, Port Orange, FL 32129, USA

jmtrue@gmail.com

37.1 Introduction

Laser phototherapy has the potential to dramatically improve the

clinical outcome in many cases of radiculopathy. The laser therapy
device can be utilized as a singular therapy or in combination with
other therapies and procedures for the treatment of radiculopathy.
Clinicians with proficiency in laser therapy consider it to be an
indispensable modality. Outside of this group of practitioners, many
physicians and healthcare professionals have been slow to recognize
the benefits of laser therapy for the treatment of musculoskeletal
disorders. The scientific literature based on double-blinded studies
utilizing laser therapy for radiculopathy is sparse, yet positive [1–
3]. There are many well-designed animal studies of nerve repair and

Handbook of Low-Level Laser Therapy

Edited by Michael R. Hamblin, Marcelo Victor Pires de Sousa, and Tanupriya Agrawal
Copyright c 2017 Pan Stanford Publishing Pte. Ltd.
ISBN 978-981-4669-60-3 (Hardcover), 978-981-4669-61-0 (eBook)
www.panstanford.com
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712 Laser Therapy for the Treatment of Radiculopathy

regeneration that statistically support the use of therapeutic laser as

a capable technology for promoting neurological recovery [4–7]. The
mechanism of positive therapeutic action from laser irradiation for
the treatment of radiculopathy has not been thoroughly explained
in research to date. Thus far, the mechanisms of action have been
largely extrapolated from cellular and animal studies.
There are many impediments to overcome when treating
radiculopathy. The most confounding dilemma is the lack of clinical
uniformity in the production of patients’ signs and symptoms.
Radiculopathy may result from acute and chronic degenerative
spine disorders, as well as combined predisposing pathologies such
as diabetes and peripheral vascular disease. Radiculopathy most
commonly presents as axial pain in conjunction with peripheral
extremity signs and symptoms in a dermatomal distribution. These
symptoms are radiating pain (dysesthesia), sensory deficit (pares-
thesia), and/or motor deficit (paresis). Entrapment pathologies such
as neurogenic thoracic outlet syndrome in the upper extremity or
piriformis syndrome in the lower extremity may mimic radicu-
lopathy. The diagnosis of radiculopathy is often clinically obvious,
yet its actual etiology may be a singular pathology or combined
factors, including direct nerve root compression, inflammation,
microvascular integrity, metabolic status, and/or other disease
processes. With the exception of radiating extremity pain, little
consensus exists regarding the diagnostic criteria for the diagnosis
of radiculopathy [8–11].
In addition to the lack of clear diagnostic uniformity, another
obvious problem arises when describing treatment protocols for
radiculopathy. There are numerous lasers available with FDA clear-
ance for therapeutic use, each having different technical features,
which influence treatment protocols. These include variations in
power, intensity, wavelength, combined wavelengths, and laser
pulsing, all of which impact the therapeutic effectiveness of the laser
and protocol recommendations.
This chapter covers some of the background research supporting
the use of laser therapy as an adjunctive or individual therapy
for cases of radiculopathy. The treatment protocols discussed
are general anatomically based application guidelines rather than
device, dosage, or wavelength-dependent recommendations. An
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Pathomechanisms of Radiculopathy 713

overview of radiculopathy and its clinical diagnosis is presented

as a basis for patient selection and laser treatment. Patients
with advancing neurological deterioration or “red flags” should be
managed as neurosurgical candidates.
The most common red flag conditions that are a contraindication
to laser therapy are as follows:

• Vertebral fracture with advancing neurological signs

• Cauda equina syndrome
• Spinal infection
• Vertebral malignancy

The current standard in medical practice for the treatment of a

patient with a mild to moderate radiculopathy would be to provide a
trial of medication and follow with a wait-and-see approach prior to
further intervention. In the authors’ experience, this type of stable,
noncritical patient with peripheralized extremity pain frequently
benefits from a timely course of laser therapy.

37.2 Pathomechanisms of Radiculopathy

Radiculopathy is a peripheral neurologic syndrome resulting from

mechanical injury and/or chemical irritation of the spinal nerve
roots. Mechanical injury to the nerve root occurs with compression,
traction, or frictional forces. Neurochemical irritation of the nerve
root occurs as a response to nerve root ischemia, vascular stasis,
or nerve root exposure to inflammatory components released from
herniated disc material and tissue injury [12–14]. When there
is an alteration of the arterial input or venous outflow, the end
result is neuroischemia of the nerve root and generation of nerve
root irritability [15]. The pain associated with radiculopathy is
considered to be neuropathic; however, the exact mechanism is
not clearly understood. The pain component is thought to result
from injury to the endoneurial blood nerve barrier, which increases
permeability to local neuroinflammatory biochemicals, causing
neural membrane instability and ectopic depolarization of nerve
root fibers [16, 17].
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714 Laser Therapy for the Treatment of Radiculopathy

Nerve roots are less protected from biochemical and inflam-

matory insult than peripheral nerves. The nerve root lacks both
the epineurium and the perineurium of the peripheral nerve [18].
This creates an increased permeability within the endoneurial
vasculature of nerve roots and the dorsal root ganglion (DRG)
to plasma proteins compared with the endoneurial vessels of
peripheral nerves [19]. Consequently, the nerve root is more
vulnerable to inflammatory chemicals than the peripheral nerve.
The classic symptoms associated with radiculopathy include
neck or back pain with spasm, shooting pain, mild to moderate
peripheral numbness, or extremity weakness in the distribution of a
nerve root. Radiating extremity pain is the most common complaint.
This may accompany verifiable clinical findings such as dermatomal
sensory deficits, deep tendon reflex changes, and/or motor loss in
muscles supplied by the affected nerve root. The most common
cause of mechanical injury to the nerve root in middle-aged and
young adults is intervertebral disc herniation (Fig. 37.1).
Degenerative disc disease, cervical spondylosis, and osteophytic
spurs causing stenosis of the intervertebral foramen are more
common in patients 55 years of age and older. The stenotic
narrowing of the intervertebral foramen predisposes the nerve

Figure 37.1 Cervical disc herniation with root compression. Copyright

J. True, DC. Used with permission.
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Complex Spinal Pain Patient with Radiculopathy 715

root to direct compression as well as reduced vascular flow. This

creates a state of less-than-adequate perfusion and poor venous
drainage in the nerve root region. As a result, the nerve root may be
vulnerable to inflammation from the accumulation of inflammatory
biochemicals. Additionally, chronic discal disease leads to the
compression of the vertebral venous plexus as it exits the spine
through the lower part of the intervertebral foramen. The impaired
venous outflow creates a vascular stasis leading to perineural
fibrosis and potential radicular compromise [13]. Radiculopathy
may also result from any space-occupying lesion in the lateral recess
or intervertebral foramen, such as synovial cysts or neoplasms that
compress the nerve root and produce symptoms. There are many
metabolic disorders and infections that are capable of predisposing
a patient to radicular compromise or, in the case of infections,
directly inducing a neuropathy that resembles radiculopathy.
Clinically diagnosing a case of radiculopathy, however, is not
always straightforward. Complex injury patterns frequently occur,
giving rise to diagnostically confusing presentations resembling
multi-root injury or signs of plexus involvement.

37.3 Complex Spinal Pain Patient with Radiculopathy

Disc herniation and vertebral degeneration are known causes of

spinal pain, stenosis, and nerve root compression. However, not all
pain referred into the extremities is true radicular or nerve-root-
based pain. The referred pain pattern from pain-sensitive spinal
structures such as the outer annulus or the facet joint may produce
a referred pain pattern that mimics radiculopathy. This type of pain
is referred to as sclerotomal pain. In addition to sclerotomal pain
referral, there is also myotomal pain. In myotomal pain referral,
pain may extend into the hip, buttock, or thigh with injury to the
deep muscles that attach to the spine. It should be noted that in
both sclerotomal and myotomal pain, referral usually does not travel
below the knee in the lower extremity or below the elbow in the
upper extremity [20]. To add to the diagnostic confusion, spinal-
referred pain and radiculopathy may occur simultaneously; thus,
creating an uncertainty for the source of the pain peripheralization.
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716 Laser Therapy for the Treatment of Radiculopathy

37.4 Common Levels of Radiculopathy

This section covers the most common levels of radiculopathy in the

lumbar and cervical spine. The section on thoracic radiculopathy
covers the region in general and discusses information thought to
be most clinically important. Treatment protocols will be limited
to C6 and C7 radiculopathies in the cervical spine and L5 and S1
radiculopathies in the lumbar spine.

37.4.1 Lumbar Radiculopathy

The lower lumbar spine is the most common region of degenerative
disc disease. The vertebral levels of degenerative disc disease most
commonly seen in practice are the L5–S1 level followed by the L4–
L5 level [20]. These intervertebral levels have greater susceptibility
to disc herniation because of the biomechanical stresses from axial
compression and rotational excursion at this region of the spine.
When there is injury to the intervertebral disc or biochemical insult
at the L5–S1 level, the S1 nerve root or the L5 nerve root may be
compromised. The L5 nerve root will be compromised by a central
or paracentral disc herniation, or central canal spondylosis at the
L4–L5 level. The L5 nerve root can also be affected in pathology
where there is an L5/S1 posterolateral and/or lateral recess
intervertebral disc herniation that extends laterally into the neuro-
foramen. This is because the L5 nerve root exits the intervertebral
foramen superior to the L5/S1 intervertebral disc and inferior to the
L5 vertebral pedicle. The S1 nerve root is more likely to be affected in
cases where the L5/S1 disc herniates centrally or slightly paracen-
trally due to its anatomic location in the spinal canal (Table 37.1).

37.4.2 Cervical Radiculopathy

The incidence of cervical radiculopathy is less common than lumbar
radiculopathy. Cervical radicular pain affects 83 per 100,000 adults
annually [21]. The most commonly involved nerve roots producing
upper extremity radiculopathies are the C7 and the C6 nerve
roots. The patient with a C7 radiculopathy will often have a disc
herniation, degeneration, or nerve root inflammation at the C6–C7
 July 6, 2016 17:38
Table 37.1 Common clinical findings in lumbosacral radiculopathy

Vertebral Pain pattern Sensory Motor weakness and Relevant

level Root(s) (Sclerotogenous) paresthesia and deficits atrophy reflex
L1–L2 L1 Anterior hip, lateral Groin and inquinal Slight contribution to Cremasteric
thigh, iliac region region psoas major
L2–L3 L2 Iliac region Proximal anterolateral Quadriceps, adductors Patellar
thigh
L3–L4 L3 Pubic region, lateral Mid-anterolateral thigh Quadriceps, adductors Patellar
thigh

PSP Book - 9in x 6in

L4–L5 L4 Thigh and knee regions Anterior knee, medial Quadriceps, adductors Patellar
foot, medial aspect of
great toe
L5–S1 L5 Hip, femur, and knee Anterior leg distal to Anterolateral compart- Tibialis
regions knee, dorsum of foot ment lower leg, tibialis posterior
anterior, extensor

Common Levels of Radiculopathy

digitorum brevis,
extensor hallucis
longus, gluteal muscles
S1–S2 S1 Ischium, periosteum of Lateral margin of foot Gastrocnemius, soleus, Achilles
lateral foot and little toe flexor hallucis brevis
S2 S2 Lateral ankle Perianal (S2-4) Anal sphincter (S2-4) Anal (S2-4)

Source: Used with permission from Ref. [20].

717

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718 Laser Therapy for the Treatment of Radiculopathy

level due to the anatomical location of the C7 nerve root exiting

the C6/7 intervertebral foramen. C6 radiculopathy results from
compression or irritation of nerve roots at the C5–C6 level [20]
(Table 37.2).

37.4.3 Thoracic Radiculopathy

Thoracic radiculopathy is not as prevalent as cervical or lumbar
radiculopathy. The diagnosis is made through clinical correlation
of magnetic resonance imaging and observed symptoms. Pain and
paresthesia associated with thoracic disc herniation are perceived in
the intercostal space of the affected root. The nerve root divides into
anterior and posterior rami. The anterior rami form the intercostal
nerves. The posterior rami branches carry motor innervation to the
muscles of the back and sensory information from the skin in a
narrow strip surrounding the segmental level of the thoracic spinal
nerve. The T1 nerve root, however, also gives contributions to the
arms and intrinsic muscles of the hand. The T2 root sends a branch
to the brachial plexus to innervate the medial arm and arm pit.
Below the level of T1, the intercostal nerves are usually supplied
by one segmental level. Communicating branches may be present,
which join the intercostal nerve above or below the intercostal
nerve of any given spinal level [22]. The T3–T6 root levels innervate
the thoracic wall, including ribs and facet articulations, intrinsics,
intercostals, serratus posterior superior, and the parietal pleura.
The T7–T11 intercostal nerves are also called the thoracoabdominal
nerves. These nerves innervate the thoracic wall and abdominal
muscles and provide sensation to the pleura, peritoneum, and skin
of lower thorax and abdomen. The 12th ventral ramus is referred to
as the subcostal nerve (Table 37.3). In consideration of differential
diagnosis, the varicella zoster virus, which causes the condition
known as shingles, often affects the intercostal nerves. The neuro-
pathic pain associated with herpetic neuralgia may be mistaken for
radiculopathy although the appearance of a stripe of erupting skin
vesicles confirms the clinical diagnosis. The pain of postherpetic
neuralgia is usually quite severe with any slight movement causing
allodynia. Postherpetic neuralgia and associated allodynia have been
shown to respond favorably to laser therapy [23].
 July 6, 2016 17:38
Table 37.2 Common clinical findings in cervical radiculopathy

Vertebral Pain pattern Sensory Motor weakness and Relevant

level Root (Sclerotogenous) paresthesia and deficits atrophy reflex
C3–C4 C4 Back of neck, top of Supraclavicular region Trapezius None
shoulder
C4–C5 C5 Top of shoulder, Clavicular, region Supraspinatus, deltoid, Biceps
proximal arm anterior aspect of biceps
shoulder

PSP Book - 9in x 6in

C5–C6 C6 Anterior arm, scapula Lateral forearm and Biceps, brachioradialis Biceps, bra-
thumb chioradialis
C6–C7 C7 Posterior arm and wrist Dorsum of arm and Triceps, finger Triceps
forearm, extensors
dorsal and palmar
aspect of second and

Common Levels of Radiculopathy

third digits
C7–T1 C8 Elbow, hand, and wrist Medial arm, Finger flexors, grip Flexor
dorsal and palmar digitorum
aspect of fourth and
fifth digits

Source: Used with permission from Ref. [20].

719

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 July 6, 2016 17:38
720 Laser Therapy for the Treatment of Radiculopathy
Table 37.3 Common clinical findings in thoracic radiculopathy

Vertebral Pain pattern Sensory Motor weakness Relevant

level Root(s) (Sclerotogenous) paresthesia and deficits and atrophy reflex
T1–T2 T1 Corresponding Proximal medial arm Intrinsic hand: None
costovertebral region, interossei, lumbricales,
elbow, wrist, and hand Add, Abd, and opponen

PSP Book - 9in x 6in

pollicis
T3–T6 T3–T6 Corresponding Upper transthoracic Intercostal muscles, None
costovertebral region, region abdominal muscles
chest
T7–T12 T7–T12 Corresponding Lower transthoracic, Intercostal muscles, Abdominal
costovertebral region, abdominal region, abdominal muscles
abdominal region, buttock (T12)
buttock (T12)

Source: Used with permission from Ref. [20]

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Proposed Mechanisms of Laser Therapy on Radiculopathy 721

37.5 Proposed Mechanisms of Laser Therapy on

Radiculopathy

In the clinical setting, the resolution of radiculopathic signs and

symptoms can be remarkable following laser therapy. Whether the
cause of symptoms is a mechanical compression of the nerve roots
or the result of inflammatory chemicals, laser therapy may have
a positive influence on both mechanisms (Fig. 37.2). There are
multiple cellular and biochemical mechanisms in the production of

Figure 37.2 Laser irradiation and the inflammation pathway. Copyright

J. True, DC. Used with permission.
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722 Laser Therapy for the Treatment of Radiculopathy

radiculopathy that are influenced by various wavelengths of light

and laser. Infrared and red wavelengths have been studied to the
greatest extent in laser therapy research.
Chow et al. [24] reviewed the mechanisms of laser-induced
analgesia and the clinical relief of pain in peripheral nerves. This
paper identified experimental studies in humans and animals where
pre- and post-effects of laser irradiation were evaluated. They
compared the effects of laser on the morphological changes seen in
mammalian nerves and neurons before and after laser, the effects
on enzymes and neurotransmitters, and the electrophysiological
changes such as conduction velocity, latency, and evoked potential
responses in whole nerves and selected fiber types.
Laser irradiation of peripheral nerves in animal studies has
been shown to produce slowing of A-delta and C-fiber nerve
conduction with preservation of larger fiber conduction [25–27].
These small-fiber afferents are the nerve fibers responsible for
the transmission of nociception. The mechanism of pain analgesia
and selective reduction in small-fiber nerve conduction may be
the result of a reversible interruption of fast axoplasmic flow,
reduction in mitochondrial membrane potentials, and inhibition
of intraneuronal retrograde transport of adenosine triphosphate
(ATP). This interruption of fast axoplasmic flow was demonstrated
in DRG neurons of the rat. Researchers described multiple small-
fiber axonal varicosities identified as axonal “beading” within the
cultured DRG cells, following irradiation with 830 nm laser [25].
Inflammatory biochemicals generated from tissue injury in the
vicinity of the nerve root are clearly responsible for the initiation
and perpetuation of most cases of radiculopathy. Inflammatory
biochemicals may be generated from facet inflammation, ischemia,
nucleus pulposus material, and disc injury without nuclear ex-
trusion [28–30]. Inflammatory mediators such as tumor necrosis
factor-alpha (TNF-α), interleukin 6 (IL-6), phospholipase A2 (PLA2),
bradykinin, prostaglandin E2 (PGE2), and many others have been
studied for their potential to induce nerve root irritation [14, 31].
Experimental models have consistently demonstrated that nerve
root irritability and radiculopathic signs can be reduced by inacti-
vation of inflammatory biochemicals. Laser therapy has been found
to reduce PGE2, a mediator of inflammatory cytokines [32, 33].
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Clinically Useful Treatment Protocols 723

Laser therapy has also been shown to reduce many inflammatory

cytokines, in particular IL-6, interleukin 1 beta (IL1β), and TNF-α
[34, 35]. Laser irradiation has also been shown to reduce bradykinin,
a neuropeptide associated with nociceptor sensitization [36]. The
positive influence of laser therapy on proinflammatory chemicals
and pain mediators may be a partial explanation for clinical im-
provement following laser treatment. Improved neural regeneration
may result from the rapid rise in nerve growth proteins following
infrared laser exposure. Growth-associated protein-43 (GAP-43) is
a nervous system specific protein, expressed by actively growing
nerve sprouts, which is associated with neural regeneration. Chen
et al. [35] demonstrated in a rat model a greater rise in GAP-43 in the
laser treatment group versus the controls. A painful radiculopathy
was created by experimental compression of DRG. The researchers
concluded that low-level laser was able to enhance neural regenera-
tion following dorsal root compression. The ability of laser therapy
to increase vasodilation through the generation of reactive oxygen
species and nitric oxide has been investigated [37, 38]. Nitric oxide
is a potent vasodilator, and its generation or displacement from the
mitochondria in theory has the potential to reverse ischemia in an
oxygen-depleted nerve root. Cytochrome c oxidase has been widely
accepted as the principle photoacceptor for multiple wavelengths of
light in the mitochondria, including near-infrared [39]. Cytochrome
c oxidase is the fourth protein complex in the mitochondrial electron
transport chain. The end result in this respiratory chain is the
production of ATP. Laser therapy has been shown in cellular studies
to increase ATP levels, thus increasing respiration. Cytochrome c
oxidase levels and respiration decrease in hypoxia. The positive
effects of laser therapy may continue working for hours or days after
treatment because the mitochondrial nitric oxide is displaced, thus
allowing cytochrome c oxidase to function [40].

37.6 Clinically Useful Treatment Protocols

37.6.1 Pulsed or Continuous Laser Therapy

The research community is debating the effects of pulsing or
modulation of the laser output. The optimum pulse repetition
July 6, 2016 17:38 PSP Book - 9in x 6in 37-Hamblin-c37

724 Laser Therapy for the Treatment of Radiculopathy

Figure 37.3 Pulsing versus continuous laser. When a therapy laser is

modulated or pulsed the tissues have time to disperse the heat load. This
allows thermal relaxation or heat quenching. Copyright J. True, DC. Used
with permission.

rates for neurological tissue repair and recovery have not been
reported or confirmed. Gigo-Benato et al. [7] demonstrated a greater
reinnervation and muscle recovery in end-to-side neurorrhaphy
performed on the median nerves of rats using combined dual
wavelengths that delivered continuous wave (808 nm) and pulsed
laser (905 nm) biostimulation. Many studies have demonstrated an
advantage to pulsed laser; however, a few studies found continuous
laser to be equal or better than pulsing for treatment outcome. A
review article by Hashmi et al. [41] discussed the possible benefits
of laser pulsing versus continuous wave (CW) laser therapy. Pulsing
a laser reduces the heat load on tissues, and this “quench period” can
reduce thermal hazards of higher power lasers (Fig. 37.3).
Higher power lasers that are pulsed may have greater effective
depth of penetration with less risk of thermal damage to superficial
tissues. This theory was demonstrated with transcranial laser
exposure of the intact rat brain using an 808 nm laser at various
power densities. Rats were evaluated for neurological function and
histopathological changes following laser exposures up to 750 mW
in CW and pulsed modes. The rat brains exposed to 750 mW
CW mode laser demonstrated neurological deficits and cellular
evidence of thermal damage; however, the rats exposed to 750 mW
July 6, 2016 17:38 PSP Book - 9in x 6in 37-Hamblin-c37

Clinically Useful Treatment Protocols 725

pulsed laser did not show any evidence of neurological damage on

functional assessment or microscopic evaluation [42]. Additionally,
the review article by Hashmi et al. [41] suggested that pulsing may
enhance or entrain normal biological rhythms such as synchronizing
with the normal rhythm of brain waves or influencing voltage-
activated ion channels. Therapeutic laser pulsing may create a
photomechanical effect that influences axonal flow and metabolic
processes in the cell and increases the generation of nitric oxide.
Laser pulsing appears to be more efficacious than CW mode laser,
but the ideal pulse rate frequencies for efficient neural repair are
not scientifically established.

37.6.2 Contact or Coupled Technique

In addition to skin pigment as a barrier to light penetration, the
skin has a degree of reflective property or albedo that causes a
limitation of light penetration. Contacting the skin with the probe
head captures some of the reflected light, which is reflected back into
the patient increasing dosage. With an increasing contact pressure,
the probe head may exsanguinate the superficial capillary bed,
reducing reflection and remittance, which results in deeper light
penetration (Fig. 37.4).
Cleaning the probe between patients with a disinfectant wipe
will prevent cross-contamination. Hygienic protocols should be
maintained with contact technique. Do not contact open wounds
without a transparent wrap barrier. The skin should be intact, clean,
and dry.

37.6.3 Treatment of Associated Guarding Spasm

The spine is an interconnected segmental kinematic chain providing
column support for the head and thorax as well as cantilever
support for the appendages. Spinal pain associated with radicu-
lopathy produces involuntary spasm or splinting that chain up and
down the vertebral column above and below the region of pain
or injury. Recognizing the biomechanical connections associated
with common radicular pain patterns is important for improved
treatment outcomes. Laser treatment of the chain spasm associated
July 6, 2016 17:38 PSP Book - 9in x 6in 37-Hamblin-c37

726 Laser Therapy for the Treatment of Radiculopathy

Figure 37.4 Contact technique increases penetration of the laser beam

and prevents reflectance, which occurs with scanning or non-contact
techniques. Copyright J. True, DC. Used with permission.

with muscle splinting and antalgia is very important for overall

symptom reduction (Fig. 37.5).

37.6.4 Treatment of Segmentally Innervated Musculature

In addition to kinematically connected musculature, the muscles
within the same myotome of the symptomatic spinal level of pain
or radiculopathy should be treated as well. Clinical observation
research from Rochkind et al. [43] demonstrated that peripheral
nerve injuries recover to a greater extent when laser treatment was
directed proximally to the root area as well as to the peripheral
area of direct nerve injury. Ohshiro [44] reported a protocol for
the treatment of spinal pain that was termed Proximal Priority
Laser Therapy. He reported better treatment outcomes when the
contralateral homunculus area of the cortex, the spinal cord, the
lymphatic system, the affected muscles, and vasculature were all
July 6, 2016 17:38 PSP Book - 9in x 6in 37-Hamblin-c37

Clinically Useful Treatment Protocols 727

Figure 37.5 Radiculopathy often creates protective guarding spasm with

secondary areas of pain. These secondary pain sites should be treated along
with the biomechanically connected spasm. Copyright J. True, DC. Used with
permission.

treated in conjunction with dynamic stretching. Although Ohshiro’s

study did not specifically discuss treatment of radiculopathy, the
treatment protocol he utilized increased cerebral blood flow,
reduced back and neck pain, and improved flexibility.
In consideration of the authors’ clinical experiences, successful
treatment of radicular pain requires the following general protocol:

• Treatment of the nerve root area and cord level

• Treatment of the segmentally connected peripheral nerves
of the affected extremity
• Treatment of the biomechanically connected paraspinal
muscles that protectively spasm above and below the level
of radiculopathy
• Treatment of the peripheral muscles innervated from the
involved nerve root
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728 Laser Therapy for the Treatment of Radiculopathy

37.6.5 Treatment of L5 and S1 Radiculopathies

The patient with a lower lumbar disc injury will often exhibit an-
talgia and muscle splinting along with pain and paresthesia into the
anterior shin or the posterior calf depending on the nerve root level.
When the S1 root is compromised, motor weakness may be present
in the gastrocnemius and soleus. With L5 root involvement, the
tibialis anterior and dorsiflexors of the foot may be weak. See Table
37.1 for additional levels and clinically relevant root distributions.
The erector spinae muscles on the same side of spinal pain are
frequently hypertonic as well as the deep intrinsic muscles. This
guarding spasm will extend from the lumbar spine into the thoracic
spine to stabilize the level of injury. This spasm may extend from the
L5–S1 level to the T10 or T8 level and above. In addition, secondary
spasm in the iliocostalis lumborum and quadratus lumborum is
often present to stabilize the lumbar spine. In severe cases of low
back pain, the psoas musculature will need to be treated to relax
spasm and alleviate lumbar pain.
As a general guideline, the laser probe or aperture should be
directed perpendicular to the treatment area and target tissue.
While contacting the skin, the hand piece should follow the contour
of the paravertebral muscles. Contacting a painful trigger point
with the hand piece may elicit a jump response. Quite often, the
immediate level of radicular involvement is too painful to touch with
any pressure. The lateral margin of the quadratus lumborum on both
sides of the spine should be treated (Fig. 37.6).
The hand piece should be pressed into the posterior flank and
the laser beam directed medially along the anterolateral margin of
the muscle. In thin patients, it is easier to treat the lateral aspect of

Figure 37.6 Treating the quadratus lumborum and the hypertonic muscles
of the lumbar spine is beneficial for the reduction of antalgia associated with
lumbar radiculopathy.
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Clinically Useful Treatment Protocols 729

Figure 37.7 The lateral margin of the quadratus lumborum can be treated
with laser by directing the beam with contact and pressure in the posterolat-
eral flank above the iliac crest. Copyright J. True, DC. Used with permission.

the lumbar spine. The beam should be directed along the plane of
the medial and anterior thoracolumbar fascia (Fig. 37.7).
The lumbar nerve roots exit the foramen on the lateral aspect
of the spine. The laser hand piece on some devices may be
too large in circumference to comfortably press into the lateral
flank. Considerable tenderness is often present in the quadratus
lumborum, and deep contact is not possible especially on the
symptomatic side. The posterior primary innervated musculature
and spine should be treated first, followed by the affected extremity.
In the gluteal region and sciatic outlet, the laser should be
pressed firmly along the inferior gluteal fold directing the beam in
a medial and superior direction as well as following the inferior
margin of the piriformis (Fig. 37.8).
Laser treatment should be applied to the sciatic notch, gluteus
medius, hamstrings, and popliteal fossa. The peroneal nerve, the
July 6, 2016 17:38 PSP Book - 9in x 6in 37-Hamblin-c37

730 Laser Therapy for the Treatment of Radiculopathy

Figure 37.8 (A) The anatomical relationship of the piriformis muscle and
sciatic nerve. (B) Example of a treatment pattern for the proximal sciatic
nerve. Copyright J. True, DC. Used with permission.

anterior compartment muscles of the leg, and the top of the foot
should be treated for L5 radiculopathy. The posterior calf muscles,
the lateral side of the foot, and the bottom of the foot should be
treated for S1 radiculopathy.

37.6.6 Treatment of C6 and C7 Radiculopathies

The patient with a cervical radiculopathy will exhibit pain in the
arm, and depending on the vertebral level of involvement, pain
or paresthesia may extend to the fingers. Motor weakness is a
more significant finding and patients with any weakness should be
July 6, 2016 17:38 PSP Book - 9in x 6in 37-Hamblin-c37

Clinically Useful Treatment Protocols 731

carefully monitored for improvement or deterioration. Paresis may

be subtle because of the plurisegmental innervation to muscles in
the upper extremity. A patient with a C6 radiculopathy will report
pain in the anterior forearm, thumb, index finger, and middle finger.
Pain and paresthesia frequently may be felt solely in the hand
and fingers. C6 radiculopathy may be mistaken for carpal tunnel
syndrome when symptoms are only perceived in the hand.
Laser treatment for C6 nerve root complaints should include
the posterior cervical spine at the level of C5–C6, the adjacent
segments above and below C5–C6, the musculature innervated
by the posterior primary rami, the biceps, and the forearm and
thenar muscles. Biomechanically connected muscles may include
the scalenes, levator scapulae, trapezius, splenius capitus, splenius
cervicus, and semispinalis muscles in the upper thoracic spine. To
treat the cervical roots, the laser should be directed posteromedially
and slightly superior in the supraclavicular portion of the posterior
triangle of the neck. The C6 roots join the brachial plexus, and the
plexus passes under the clavicle. The laser should also be directed
at the lateral margin of the pectoralis minor and the proximal
medial arm in order to treat the nerve bundle in the axillary sheath
(Fig. 37.9). The cubital fossa, the forearm, and the volar surface of
the wrist should be treated as well for the distal distribution of the
C6 dermatome and myotome.
C7 radiculopathy is one of the most common radiculopathies.
The patient will complain of pain in the lower cervical spine and
along the midscapular region. A tingling sensation and pain is often
reported in the lateral arm and forearm. Numbness and tingling may
be reported in the index and middle fingers. When paresis is present,
it usually involves the triceps and the wrist and finger extensors.
Paresis of the serratus anterior will sometimes occur causing a
winging of the scapula when the patient pushes away from a wall.
Proximal treatment of a C7 radiculopathy is nearly identical
to a C6 protocol except the treatment is directed to the C6/C7
intervertebral disc. The C7 root is treated in the base of the
supraclavicular region of the posterior triangle in the neck.
Treatment of the brachial plexus, axilla, and proximal medial arm
is identical to a C6 root lesion. The lower digitations of the serratus
anterior should be treated along with the medial arm and posterior
July 6, 2016 17:38 PSP Book - 9in x 6in 37-Hamblin-c37

732 Laser Therapy for the Treatment of Radiculopathy

Figure 37.9 The treatment pattern for a C6 radiculopathy follows the

segmentally innervated muscles in the upper extremity.

arm. The C7 root supplies a large portion of the radial nerve. This
nerve passes into the posterior arm to innervate the triceps and
travels along the posterolateral arm and humerus above the elbow.
The radial nerve crosses the lateral elbow to innervate the muscles
and skin of the posterior forearm. Laser treatment should follow
the course of the radial nerve from the back of the arm to the
lateral cubital fossa, the posterior forearm, and to the back of the
hand. The guarding spasm that stabilizes the lower cervical spine
in a C7 radiculopathy is similar to a C6 pattern of injury. The
muscles usually requiring treatment include the trapezius, levator
scapulae, semispinalis, scalenes, splenius cervicis, splenius capitus,
and occasionally the sternocleidomastoidius.

37.6.7 Treatment of Thoracic Radiculopathies

Thoracic radiculopathy is relatively uncommon as compared to
cervical and lumbar radiculopathies. Radiculopathy involving the
T1 root will cause pain in the medial arm and weakness in the
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Clinically Useful Treatment Protocols 733

hand muscles innervated by the ulnar nerve. A T1 root lesion may

result from an apical lung tumor, and this differential diagnosis
must be excluded before initiation of treatment. Laser treatment
should include the first and second rib, the posterior muscles
of the upper thoracic spine, and the medial arm and hand. The
patient may report chest pain and significant pain on inspiration.
The nerve roots exiting the thoracic spine from T2–T6 may cause
chest pain from the pleural distribution of sensory fibers. Chest
pain may also occur from periosteal irritation as well as from
the rib articulations. Somatic pain is often present in the scapular
region and the intercostal space of the involved thoracic level. The
pattern of pain and paresthesia will frequently extend below the
intercostal space of the involved nerve because of the distribution
of the posterior, lateral, and anterior cutaneous branches. The
distribution of the anterior cutaneous branch may be difficult to
treat in female patients due to the overlying breast. The cutaneous
distribution from the 12th intercostal or subcostal nerve supplies
the skin over the buttock, side of the hip, and lower abdomen.
The pain may be described in abdomen and the top of the hip or
as far down as the posterolateral buttock. The paraspinal muscles
often spasm a few segments above and below the level of injury.
Treatment should include the spinal region, a few segments above
and below the lesion, and the intercostal space of the involved level
(Fig. 37.10).
The intercostal spaces under the shoulder blade are not directly
accessible to direct laser exposure. Typically, the intercostal spaces
underneath the scapula are between ribs 3 and 6. Having the
patient reach the hand to the opposite shoulder will move the
scapula laterally and improve accessibility. The anterior rami of
T11–T12 intercostal nerves provide significant innervation to the
anterolateral abdominal wall. Laser treatment of a lower thoracic
radiculopathy should include the intrinsic muscles of the back that
are innervated by the posterior primary rami. The focus of treatment
should be one or two segments above and below the lesion. Other
targets of treatment in lower thoracic radiculopathies include the
flank, the buttock, the anterior abdominal muscles, and the adjacent
intercostal spaces, in addition to the involved intercostal nerve.
July 6, 2016 17:38 PSP Book - 9in x 6in 37-Hamblin-c37

734 Laser Therapy for the Treatment of Radiculopathy

Figure 37.10 Treatment of a thoracic root lesion. The anterior rami in

the thoracic spine supply cutaneous sensation to the majority of the torso.
Following the intercostal nerve is easy because of the boundary formed by
the superior and inferior rib margin. The cutaneous nerve distribution from
the lower thoracic roots may extend a considerable distance below the rib
interspace. Copyright J. True, DC. Used with permission.
July 6, 2016 17:38 PSP Book - 9in x 6in 37-Hamblin-c37

References 735

Acknowledgments

We are grateful to Anne Canty, Ph.D. for her assistance with reference
organization and manuscript preparation.

References

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738 Laser Therapy for the Treatment of Radiculopathy

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supporting treatment with TNF-α antagonists. Joint Bone Spine, 73(3),
270–277. Epub 2005 Jun 22.
32. Bjordal, J.M., Johnson, M.I., Iversen, V., Aimbire, F., and Lopes-Martins,
R.A. (2006). Low-level laser therapy in acute pain: A systematic review
of possible mechanisms of action and clinical effects in randomized
placebo-controlled trials. Photomed Laser Surg, 24(2), 158–168.
33. Iwatsuki, K., Yoshimine, T., Sasaki, M., Yasuda, K., Akiyama, C., and
Nakahira, R. (2005). The effect of laser irradiation for nucleus pulposus:
An experimental study. Neurol Res, 27(3), 319–323.
34. Alves, A.C., Vieira, R., Leal-Junior, E.C, Dos Santos, S., Ligeiro, A.P.,
Albertini, R., and de Carvalho, P. (2013). Effect of low-level laser therapy
on the expression of inflammatory mediators and on neutrophils and
macrophages in acute joint inflammation. Arthritis Res Ther, 15(5),
R116. doi:10.1186/ar4296.
35. Chen, Y.-J., Wang, Y.-H., Wang, C.-Z., Ho, M.-L., Kuo, P.-L., Huang, M.H.,
and Chen, C.H. (2014). Effect of low-level laser therapy on chronic
compression of the dorsal root ganglion. PLoS ONE, 9(3): e89894.
doi:10.1371/journal.pone.0089894.
36. Jimbo, K., Noda, K., Suzuki, K., and Yoda, K. (1998). Suppressive effects
of low-power laser irradiation on bradykinin evoked action potentials
in cultured murine dorsal root ganglion cells. Neurosci Lett, 240(2), 93–
96.
37. Poyton, R.O., and Ball, K.A. (2011). Therapeutic photobiomodulation:
Nitric oxide and a novel function of mitochondrial cytochrome c oxidase.
Discov Med, 11(57), 154–159.
38. Sharma, S.K., Kharkwal, G.B., Sajo, M., Huang, Y.Y., De Taboada, L.,
McCarthy, T., and Hamblin, M.R. (2011). Dose response effects of 810 nm
laser light on mouse primary cortical neurons. Lasers Surg Med, 43(8),
851–859. doi: 10.1002/lsm.21100.
39. Karu, T.I. (1999). Primary and secondary mechanisms of action of
visible-to-near IR radiation on cells. J Photochem Photobiol B Biol, 49,
1–17.
40. Chen, A.C.H., Huang, Y.Y., Arany, P.R., and Hamblin, M.R. (2009). Role
of reactive oxygen species in low-level light therapy. Mechanisms for
July 6, 2016 17:38 PSP Book - 9in x 6in 37-Hamblin-c37

References 739

low-light therapy IV. Ed. Michael R. Hamblin, Ronald W. Waynant, and

Juanita Anders. San Jose, CA, USA: SPIE, 2009. 716502-11. SPIE—The
International Society for Optical Engineering.
41. Hashmi, J.T., Huang, Y.Y., Sharma, S.K., Kurup, D.B., Taboada, L., Carroll,
J.D., and Hamblin, M.R. (2010). Effect of pulsing in low-level light
therapy. Lasers Surg Med, 42(6), 450–466. doi: 10.1002/lsm.20950.
42. Ilic, S., Leichliter, S., Streeter, J., Oron, A., DeTaboada, L., and Oron, U.
(2006). Effects of power densities, continuous and pulse frequencies,
and number of sessions of low-level laser therapy on intact rat brain.
Photomed Laser Surg, 24(4), 458–466.
43. Rochkind, S., Drory, V., Alon, M., Nissan, M., and Ouaknine, G.E. (2007).
Laser phototherapy (780 nm), a new modality in treatment of long-
term incomplete peripheral nerve injury: A randomized double-blind
placebo-controlled study. Photomed Laser Surg, 25(5), 436–442.
44. Ohshiro, T. (2004). Proximal priority laser therapy: PPLT. Proc SPIE,
5610, Laser Florence 2003: A Window on the Laser Medicine World,
(September 10, 2004), 258–276. doi:10.1117/12.584418.
July 6, 2016 17:38 PSP Book - 9in x 6in 38-Hamblin-c38

Chapter 38

Difficult Path to Treating Acute Ischemic

Stroke Patients with Transcranial
Near-Infrared Laser Therapy

Paul A. Lapchak, Pramod Butte, and Padmesh S. Rajput

Department of Neurology & Neurosurgery, Cedars-Sinai Medical Center,
Advanced Health Sciences Pavilion, Room 8305, 127 South San Vicente Boulevard,
Los Angeles, CA 90048, USA
paul.lapchak@cshs.org

Transcranial near-infrared laser therapy (NILT) or low-level laser

(or light) therapy remains a promising noninvasive neuroprotective
method to treat acute ischemic stroke (AIS), despite the difficulties
observed with translation in clinical trials. Three clinical trials—
NeuroThera Effectiveness and Safety Trial (NEST)-1, NEST-2, and
NEST-3—have evaluated the use of NILT to promote clinical recovery
in patients with AIS; the results have been quite mixed. Can we
recover from this failure reminiscent of the SAINT clinical trial
failure?
This chapter addresses the preclinical studies that supported
the continued development of NILT. The use of NILT was advanced
to clinical trials based on extensive translational research using
multiple species. The results of the three NEST clinical trials will also
be reviewed so that possible new directions to improve the efficacy

Handbook of Low-Level Laser Therapy

Edited by Michael R. Hamblin, Marcelo Victor Pires de Sousa, and Tanupriya Agrawal
Copyright c 2017 Pan Stanford Publishing Pte. Ltd.
ISBN 978-981-4669-60-3 (Hardcover), 978-981-4669-61-0 (eBook)
www.panstanford.com
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742 Difficult Path to Treating Acute Ischemic Stroke Patients with Transcranial

profile of NILT in AIS patients can be considered. Moreover, future

NILT studies should consider administration of a thrombolytic to
enhance cerebral reperfusion alongside NILT neuroprotection.

38.1 Introduction

Acute ischemic stroke is the fourth leading cause of death and the
leading cause of adult disability in the USA with an estimated cost
of $68.9 billion annually [4, 5]. Each year there are approximately
0.8 million new stroke victims in the United States; the majority of
stroke victims suffer from other conditions at the time of the stroke,
primarily diabetes and/or hypertension [4, 5]. The World Health
Organization (WHO) estimates that of the 15 million victims who
suffer strokes annually, more than 5 million die from the brain insult
and approximately 5.5 million are permanently disabled [6].
Currently, the only FDA-approved treatment for stroke is the
thrombolytic, tissue plasminogen activator (rt-PA), a drug that
promotes recanalization [7–10], but it is underutilized as a
treatment (less than 7% of stroke patients are being treated with
rt-PA in the United States [11–13]) despite the fact that rt-PA is
quite useful in up to 50% of patients provided with rt-PA as a
treatment option. rt-PA has shown efficacy up to 4.5 h after a
stroke [14, 15], but it is currently FDA-approved for use within 3 h
of a stroke. Since over 60% of patients do not return to normal
after rt-PA treatment, there is considerable need to develop new
therapies. Furthermore, there are no drugs or devices available to
prevent the nerve cell death associated with ischemic insults. The
failure to develop effective therapies may be the result of inadequate
drug development approaches used by the pharmaceutical and
biotechnology industry.
The potential impact and novelty of NILT for the treatment of
stroke is enormous. However, there is a great challenge to develop
NILT for AIS, since NILT has not been shown to be effective in
all stroke patients [16–18]. Furthermore, there is a paucity of
important information available regarding the best method to apply
NILT to most effectively deliver NILT to cortical and subcortical
brain structures and to improve clinical function. There is also little
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NILT Penetration Profiles in Animals and Humans 743

known regarding the mechanisms of action of NILT even though

we have postulated that NILT may realign homeostatic mechanisms
to promote neuronal survival within the penumbral “at risk” area,
increase the function of neuronal circuits, and then improve clinical
presentation [19–21].
The photobiostimulation, neuroprotective, and clinical improve-
ment effects of NILT are dependent on the quantity of photons that
can cross a barrier, primarily the skull [16, 22–27]. Even though
numerous clinical trials were mounted for stroke, NILT penetration
profiles remain an unknown entity. The first section of this chapter
will deal with this barrier to the continued development of NILT for
neurodegenerative disease.

38.2 NILT Penetration Profiles in Animals and Humans

While it has been demonstrated that irradiation with 808 nm

infrared light does penetrate the scalp, skull and some brain tissues
[3, 25, 26, 28, 29], the level of penetration has not been studied in
detail across species. In a recent study, we used a K-Laser Inc. model
K-1200 dual wavelength (800 nm and 970 nm) Class IV laser with
variable energy output. For this study, we used three different power
densities from 200–700 mW/cm2 to determine NILT penetration
profiles through the skulls/calvaria as a function of power density.
In this study, the laser was used in the 800 nm mode to reproduce
the regimen used in preclinical and clinical studies. The basic study
design is shown in Fig. 38.1. The laser light was coupled into a 3
m, 600 micron fiber (FT600UMT, NA 0.39, Thorlabs), which was
connected to a collimator (F220SMA-780, 780 nm, f = 11.07 mm,
NA = 0.26, Thorlabs). The distance from the lens was adjusted in
order to get a 11.7 mm diameter beam. This gave an area of 107.5
mm2 (1.075 cm2 ), thus in order to achieve power densities of 200–
700 mW/cm2 . The laser energy was detected using an Integrating
Sphere Photodiode Power Sensor (S142C, Thorlabs). To measure the
thickness of animal skulls, we used a Mitutoyo Ratchet Digimatic
Micrometer MDC-1 PJ with 0.00127 mm precision and ±0.00005
error of reproducibility. The thickness of human calvaria was
measured using CT scans at the University of California, San Diego.
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744 Difficult Path to Treating Acute Ischemic Stroke Patients with Transcranial

600 micron
Fiber

Collimating
Lens

Iris

Skull Sample

Integrating Sphere
Detector Laser (800nm)

Figure 38.1 Left: A diagrammatic view of the experimental setup to

measure NILT penetration through skulls and calvaria of various species.
Right: The laser tracking diameter on the surface of a skull from a New
Zealand white rabbit. For the majority of studies, we measured penetration
profiles through the skull at bregma.

Figure 38.2 shows the laser tracking spot on the ventral

surface of the rabbit skull (left) and the diffuse pattern of light
penetration through the skull encompassing the complete skull
(right). Figure 38.3 shows the relationship between the thickness of
skulls from three animal species and human calvaria and the percent
of NILT penetration through the skulls. The data points in the figure
from left to right represent penetration profiles through mouse, rat,
rabbit, and human desiccated dry calvaria.

Figure 38.2 Left: Laser tracking spot on the ventral surface of the
rabbit skull. Right: Diffuse pattern of light penetration through the skull
encompassing the complete skull.
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NILT Penetration Profiles in Animals and Humans 745

Figure 38.3 NILT penetration as a direct function of average skull thickness.

From left to right are mouse, rat, rabbit, and human skulls measured at
bregma in all species. The graph shows a nonlinear correlation between
penetration and thickness.

Additional detailed penetration profiles through human calvaria

measured at bregma (Fig. 38.4) show that there is a correlation
(R 2 = 0.31) for NILT penetration through desiccated dry calvaria
and R 2 = 0.56 for penetration through hydrated degassed calvaria.
That is, NILT penetration decreased as thickness increased.
This type of irradiation results in a variety of photobiostimu-
lation effects, which have been documented for both in vitro and
in vivo conditions [16, 22–27, 30, 31]. The biological effects of
NILT are wavelength specific and are not due to thermal effects
of the laser beam [29, 32, 33]. The original rabbit embolic stroke
study by Lapchak et al. [3] showed that 10 min irradiation of the
rabbit brain at midline using high continuous wave (CW) energy
(25 mW/cm2 , 15 J/cm2 ), through the overlaying shaved skin, which
resulted in significant behavioral improvement, increased surface
skin temperature below the probe by up to 3◦ C; however, the
focal brain temperature directly under the laser probe increased by
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746 Difficult Path to Treating Acute Ischemic Stroke Patients with Transcranial

Figure 38.4 NILT penetration as a direct function of average skull thickness.

Correlation analysis was done for both desiccated dry skull () and
degassed wet calvaria (). There is a weak correlation between NILT
penetration using both conditions.

0.8–1.8◦ C during the NILT treatment. This was considered to be

minimal heating and of no possible deleterious consequence.

38.3 Translational NILT Studies in Stroke Models

In this section, we will present an overview of NILT effects in

preclinical stroke models that supported the NEST series of clinical
trials. In addition, we will discuss the possible future clinical trial
development of NILT.

38.3.1 Is There a Correlation between NILT Power Density

and Improved Behavioral Function in Animal
Models?
As the basis for the NEST trials and as described in detail [2, 3],
the rabbit small clot embolic stroke model (RSCEM) was used to
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Translational NILT Studies in Stroke Models 747

study the effects of NILT on behavior outcome when embolized

rabbits were treated with NILT up to 12 h post-stroke. We should
note that the studies done using rabbits had a relatively small skull
penetration barrier of 2.1 mm, and that using a wide variety of
power densities, we reproducibly measured approximately 12%
mean energy penetration through rabbit skulls at bregma (see Fig.
38.2). It should be noted that the penetration profile through the
dry desiccated rabbit skull may not precisely replicate penetration
through an intact rabbit but is a close approximation that can be
used to species comparison.
In the translational studies, a standardized simple behavioral
outcome assay was used, which used a statistical population analysis
method to determine how a heterogeneous population of embolized
rabbits responded to a treatment [34, 35]. To determine the
therapeutic window for NILT efficacy, we used the RSCEM and
altered the time of NILT administration following embolization,
keeping treatment duration the same [3]. We focused on delayed
treatment initiated 1, 3, 6, 12, or 24 h following embolization to
attempt to define the optimal treatment window for translation into
the stroke patient population.
Figure 38.5 provides information on the therapeutic window in
embolized rabbits. The beneficial effect of NILT over sham-treated
control subjects (* p <0.05) can be measured up to 6 h following
an embolic stroke. NILT administered 12 h following embolization
shifted behavioral scores or clinical function toward a positive
effect, but there was some variability in the response of the group
(p > 0.05). The clinical improvement data from the RSCEM is
important to the development of NILT because the model used has
great diversity, heterogeneity of infarct cores and embolic stroke
in rabbits produces large surface areas of “penumbra” which is
the primary target of neuroprotective treatments, whether they be
small-molecule drugs or devices. When we were able to measure a
beneficial effect of NILT using the RSCEM, the effect was durable and
was maintained up to 21 days after a single NILT application [1, 3].
In addition to the pivotal translational studies done in the
RSCEM, a few other studies with CW NILT have been done using
a rodent stroke model [36] (see Table 38.1). In one experiment
insertion of an intra-luminal filament to occlude the rat middle
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748 Difficult Path to Treating Acute Ischemic Stroke Patients with Transcranial

Figure 38.5 Behavioral improvement in embolized rabbits following

continuous wave (CW) NILT. The beneficial improvement related to NILT
administration is statistically significant (* p <0.05) when applied between
5 min and 6 h following an embolic stroke [1–3], and there was a trend for a
beneficial effect up to 12 h following the stroke.

Table 38.1 Correlation between cortical fluence and behavioral out-

come following stroke in animal models

Cortical Fluence (J/cm2 ) Treatment Regimen Behavioral Outcome* Reference

0.09 CW No effect [2]
0.9 CW Improved up to 3 h [3]
0.9 PW Improved up to 6 h [1]
0.9 CW Improved behavior at 24 h [42]
0.9 CW Improved behavior at 24 h [36]
15 CW Improved up to 6 h [3]

*Treatment time post-stroke; CW: continuous wave; ND: not determined; PW: pulse wave

cerebral artery was used to study NILT effects following ischemia.

In contrast to the rabbit embolic stroke studies (described earlier),
the study showed that NILT was effective when applied 24 h after
ischemia, but not when applied 4 h after the start of ischemia.
During the 28 survival periods used in the study, several behavioral
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NILT Safety Trials 749

tasks were conducted on the rodents. While the effect was small
and only amounted to a 32% improvement in performance in the
NILT-treated group relative, the results were statistically significant.
Interestingly, similar findings using CW NILT (7.5 mW/cm2 ) and the
same filament model were published by de Taboada et al. [25], also
a member of the same research group (formerly Photothera Inc., the
sponsors of the NEST clinical trials).

38.4 NILT Safety Trials

During efficacy trials in the RSCEM, we also turned to a standard

rabbit large clot embolic stroke model (RLCEM) [37–39] to
determine the safety profile of NILT and NILT in combination with
tPA [38], to prepare for possible combination therapy in stroke
patients.
The studies were methodically designed in order to support
the initiation of a four-arm randomized double-blind clinical trial
to include NILT and the combination of NILT plus the current
standard-of-care therapy for stroke, tPA [40]. Based on a wealth
of translational information from the RSCEM, we continued to use
CW NILT at a power density to produce a cortical energy density
of 1.2 J/cm2 and a standard maximally effective (3.3 mg/kg) rabbit
dose of tPA [38, 41]. In this translational study trial design, to
somewhat mimic a clinical trial, NILT was initiated 30 min following
the start of tPA administration, a time when thrombolysis had
occurred.
The study showed that standard tPA dosing increased the
hemorrhage by 160% over baseline, a reproducible finding in this
model [43]. In NILT-treated embolized rabbits, the hemorrhage rate
was 21.7%, whereas sham-treated embolized control rabbits had
a hemorrhage rate of 17.4% ( p > 0.05). Thus, NILT was shown
to be safe and did not significantly affect hemorrhage rate in
embolized rabbits. Moreover, closer group comparison showed that
NILT decreased tPA-induced hemorrhage rate by 30% compared to
the tPA-treated group. Additional analysis showed that there was no
effect of CW NILT on hemorrhage volume measured in tPA-treated
rabbits, and there was no significant effect on overall survival rate
July 6, 2016 17:38 PSP Book - 9in x 6in 38-Hamblin-c38

750 Difficult Path to Treating Acute Ischemic Stroke Patients with Transcranial

[38]. Safety studies in rodents have also documented the safety

of NILT [26]. In a CW dose escalation study, increasing power
density between 7.5 mW/cm2 , 75 mW/cm2 , and 750 mW/cm2 was
measured on both behavioral endpoints as well as histochemical
measures of neuronal damage. The authors found that no significant
tissue damage could be detected using either light or electron
microscopic damage at the two lower doses. However, at the highest
dose, there was significant behavioral and histological damage.
Thus, CW NILT below 75 mW/cm2 appeared to be safe and well
tolerated, at least in animals with skulls of 1 mm thickness

38.5 NILT Stroke Clinical Trial Development

Based on promising efficacy and safety results in two translational

stroke models, Photothera Inc. (now defunct) sponsored three
separate randomized double-blind clinical trials with NILT (10
mW/cm2 , delivering an estimated cortical fluence of 1.2 J/cm2 )
[16–18]. This section will review the NEST clinical trial design and
outcomes (see Table 38.2 for a comparison of all three trials).

38.5.1 NEST-1
The NEST-1 clinical trial results were authored by Lampl et al.
[16]. It was a double-blind, randomized (2:1) study of 120 patients
40–85 years of age, treated within 24 h of ischemic stroke onset
with baseline National Institutes of Health Stroke Scale (NIHSS)
scores of 7–22. The mean enrollment treatment time was 16 h and
20 min. The primary endpoint was the NIHSS scores stratified to
strata 7 to 10, 11 to 15, and 16 to 22, and outcome was collapsed
into a binary National Institutes of Health (NIH) outcome, where
successful treatment could be measured in two ways, as a 90-day
NIHSS score 0 to 1 or as a decrease in score (change) of 9 or
more points from baseline to 90 days. The secondary endpoint was
the modified Rankin scale (mRS), also measured in two ways: The
first used the standard seven-category ordinal form, analyzed across
the whole distribution of scores on the 0–6 mRS scale (full mRS),
whereas the second used a binary mRS with scores of 0,2 considered
 July 6, 2016 17:38
Table 38.2 NEST clinical trial results

Treatment groups: Double-blind and randomized

Criteria NEST-1 [16] NEST-2 [17] NEST-3 [44]
Placebo (41) NILT (79) Placebo (327) NILT (331) 1000 Patients
Age range 40–85 40–85 42–92 19–95 40–80
(mean 68.5) (mean 70.2) (mean 71.5) (mean 61.2)
Sex 26 males 43 males 189 males 183 males Both
15 females 36 females 138 females 148 females
Time to treatment (TT) 4:05–23:22 2:00–23:56 2:30–23:54 2:42–23:54 4:30–24.00
(hours: minutes) (mean 16:20) (mean 16:56) (mean 14:43) (mean 14:38)
Stroke severity

PSP Book - 9in x 6in

• NIHSS (mean 10) (mean 11) (mean 13.2) (mean 13.1) 7–17
Stroke outcome FUTILE
• NIHSS 51% 70% 30.9% 36.3%
◦ stratified (severity) p = 0.048 p = 0.094

NILT Stroke Clinical Trial Development

◦ stratified (TT) p = 0.035 (NIHSS 7–15, p = 0.044)
• mRS
◦ binary outcome p = 0.043
◦ stratified (TT) p = 0.034
◦ full p = 0.026
◦ stratified (TT) p = 0.020
Mortality rate 9.8% 8.9% 17.5% 17.4% FUTILE
SAE 36.6% 25.3% 41.8% 37.8% FUTILE

NIHSS: National Institutes of Health Stroke Scale; mRS: modified Rankin scale; TT: time to treatment; SAE: serious
adverse events

751

38-Hamblin-c38
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752 Difficult Path to Treating Acute Ischemic Stroke Patients with Transcranial

positive or successful and scores of 3,6 considered negative or NILT

treatment failure.
Unlike the rabbit embolic stroke studies where a single applica-
tion was sufficient, for human, because of the size differences, the
probe was applied to 20 points on the skull for 2 min at each spot, for
a power density of 10 mW/cm2 and energy density of 1.2 J/cm2 . This
ensured that NILT could possibly encompass the complete cortex
and allow for limited penetration of laser light energy. There is no
information in the literature to document that the NILT parameters
were optimized prior to the initiation of the NEST clinical trials. It
appears that the power density utilized was based on translational
animal model data.
The result of the NEST-1 study was promising and showed
that more patients in the active treatment group had successful
outcomes than did controls in the sham-treated group, as measured
prospectively on the NIHSS and mRS. As described by Lampl et al.
[16], in NILT-treated patients there was greater improvement from
baseline to 90 days ( p = 0.021) compared to the sham-treated group
(Table 38.2).
Regression analysis of all patients, controlling for parameters
such as age, sex, time to treatment, and baseline score, showed that
38% of all NILT-treated patients realized a final NIHSS score of 0–1
and improved by 9 points or greater. For the binary mRS outcome,
0,2 versus 2,6, 60% of NILT-treated patients had a positive outcome
( p = 0.034–0.043). There were no differences in mortality rate or
serious adverse events between the two groups.

38.5.2 NEST-2
The NEST-1 trial efficacy results served as an impetus for the larger
phase III trial, NEST-2, which was conducted in 660 stroke patients.
The results of clinical trials were published by Zivin et al. [17].
The protocol of the trial was nearly identical to NEST-1, but the
study PI used mRS as the primary endpoints instead of NIHSS;
NIHSS was used for additional analysis (i.e., stratification of results
according to enrollment baseline). Unlike NEST-1, this trial did not
reach statistical significance ( p = 0.094) when data for all enrolled
patients were used. However, post-hoc stratification analysis based
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Need to Optimize NILT in a Standardized Translational Model 753

on enrollment NIHSS scores suggested that moderately affected

patients with NIHSS 7–15 did achieve better performance when
measured at 90 days, but this did not reach statistical significance for
the particular patient group ( p = 0.044). Thus, using pre-specified
subgroup analyses, the study found that a subgroup with NIHSS 7–
15 benefited from the NILT, but patients with high baseline NIHSS
scores were not improved. This may be a technical limitation of the
lack of optimization of NILT and the low CW power density used in
the clinical trials.

38.5.3 NEST-3
What happened? The NEST-3 clinical trial design reported on the
NIH website [44] is similar to the NEST-2 clinical trial (see Table
38.2). The trial results were never reported in the peer-reviewed
literature despite the trial ended in 2012 due to futility [18]. The
trial was originally designed to enroll 1000 patients within 24 h
of a stroke but only 566 patients were enrolled. As per protocol,
patients were enrolled with an NIHSS baseline of 7–17, the range
where beneficial effects of NILT were observed in the NEST-1 and
NEST-2 [16, 17] clinical trials, but more specifically in the NEST-2
trial where NILT was ineffective in patients enrolled with an NIHSS
baseline above 16 points. The trial design did not include standard-
of-care therapy, tPA to be administered within 3–4.5 h of a stroke,
primarily because the time window chosen by the investigators was
well beyond the time approved for standard tPA administration.

38.6 Need to Optimize NILT in a Standardized

Translational Model

Based on experience with the skull penetration characteristics of

four species described earlier in this chapter [46], we demonstrated
that there was optimal NILT penetration of the rabbit skull and our
previous studies showed that this could be achieved to a depth
of 25–30 mm, essentially its complete thickness, when the NILT
therapy was applied at bregma on the midline [3], but this is
related to the thickness of the skull. However, using rats with a skull
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754 Difficult Path to Treating Acute Ischemic Stroke Patients with Transcranial

thickness of 1 mm, it has been suggested that a power density of 10

mW/cm2 (1.2 J/cm2 ) at the level of the rat cortex would be reduced
to 7.5 μW/cm2 (0.9 mJ/cm2 ), 18 mm away from the cortical surface
[42].
According to the NEST-2 trial publication [17], it was estimated
that NILT may only penetrate to a depth of 20 mm [17] using a CW
design with a power density of 10 mW/cm2 (1.2 J/cm2 ). However, if
this is true, then the calvaria information described in this chapter
would suggest that with an average skull thickness of 6–7 mm,
there would only be significant penetration of 13–14 mm below
the skull. With other barrier impediments between the laser probe
and the brain, penetration would be further limited to only a few
millimeters. Using an approximation for the range of thickness of the
human cortex of 1–4.5 mm [45] and because of the complex pattern
of sulci in humans, there would be variable NILT penetration when a
laser probe is placed on the skin surface overlaying the skull. Clearly,
the results of NEST-2 and NEST-3 showed that there was insufficient
NILT coverage of not only cortical structures, but also underlying
structures to produce a “neuroprotective” effect.

38.7 Conclusion

The principal aim of this chapter was to directly evaluate the

literature regarding the development profile of NILT in translational
stroke models and subsequent testing in clinical trials. Clearly, there
is a significant efficacy signal for NILT in translational models,
specifically the rabbit embolic stroke model, which should be
exploited further. The NILT penetration data across species are quite
important if one has to invest in the development of a method to
promote neuroprotection using photons. Since there is differential
photon or energy penetration related to the thickness of skulls,
this should be incorporated into study design when the therapy is
redeveloped. There are a few opportunities to revisit and optimize a
treatment after it has been proved to be less than effective in stroke
patients. The usual path from the bench top in the laboratory to the
clinic is not always a one-way street. We should learn from clinical
July 6, 2016 17:38 PSP Book - 9in x 6in 38-Hamblin-c38

References 755

trial failures and return to the bench top to further optimize NILT so
that the therapy can, once again, be tested in future clinical trials.

Funding: NONE.

Conflict of Interest Statement: NONE.

References

1. Lapchak, P.A., K.F. Salgado, C.H. Chao, and J.A. Zivin, Transcranial near-
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11. Fang, M.C., D.M. Cutler, and A.B. Rosen, Trends in thrombolytic use for
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12. Reeves, M.J., S. Arora, J.P. Broderick, M. Frankel, J.P. Heinrich, S.
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Moomaw, L. Schwamm, and P. Weiss, Acute stroke care in the US: Results
from 4 pilot prototypes of the Paul Coverdell National Acute Stroke
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13. Schwamm, L.H., S.F. Ali, M.J. Reeves, E.E. Smith, J.L. Saver, S. Messe, D.L.
Bhatt, M.V. Grau-Sepulveda, E.D. Peterson, and G.C. Fonarow, Temporal
trends in patient characteristics and treatment with intravenous
thrombolysis among acute ischemic stroke patients at get with the
guidelines-stroke hospitals. Circ Cardiovasc Qual Outcomes, 2013. 6(5):
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14. Hacke, W., M. Kaste, E. Bluhmki, M. Brozman, A. Davalos, D. Guidetti, V.
Larrue, K.R. Lees, Z. Medeghri, T. Machnig, D. Schneider, R. von Kummer,
N. Wahlgren, and D. Toni, Thrombolysis with alteplase 3 to 4.5 hours
after acute ischemic stroke. N Engl J Med, 2008. 359(13): pp. 1317–
1329.
15. Lansberg, M.G., E. Bluhmki, and V.N. Thijs, Efficacy and safety of tissue
plasminogen activator 3 to 4.5 hours after acute ischemic stroke: A
metaanalysis. Stroke, 2009. 40(7): pp. 2438–2441.
16. Lampl, Y., J.A. Zivin, M. Fisher, R. Lew, L. Welin, B. Dahlof, P. Borenstein,
B. Andersson, J. Perez, C. Caparo, S. Ilic, and U. Oron, Infrared laser
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therapy for ischemic stroke: A new treatment strategy: results of the

NeuroThera Effectiveness and Safety Trial-1 (NEST-1). Stroke, 2007.
38(6): pp. 1843–1849.
17. Zivin, J.A., G.W. Albers, N. Bornstein, T. Chippendale, B. Dahlof, T. Devlin,
M. Fisher, W. Hacke, W. Holt, S. Ilic, S. Kasner, R. Lew, M. Nash, J. Perez,
M. Rymer, P. Schellinger, D. Schneider, S. Schwab, R. Veltkamp, M. Walker,
and J. Streeter, Effectiveness and safety of transcranial laser therapy for
acute ischemic stroke. Stroke, 2009. 40(4): pp. 1359–1364.
18. Photothera. PhotoThera NEST-3 Trial Halted Due to Futility. The Stroke
Interventionalist 2012 10/17/2013]; available from: http://www.
tsijournal.com/clinical-trial-updates-body
19. Lapchak, P.A., Taking a light approach to treating acute ischemic stroke
patients: Transcranial near-infrared laser therapy translational science.
Ann Med, 2010. 42(8): pp. 576–586.
20. Lapchak, P.A., Transcranial near-infrared laser therapy applied to
promote clinical recovery in acute and chronic neurodegenerative
diseases. Expert Rev Med Devices, 2012. 9(1): pp. 71–83.
21. Lapchak, P.A. and L. De Taboada, Transcranial nearinfrared laser
treatment (NILT) increases cortical adenosine-5 -triphosphate (ATP)
content following embolic strokes in rabbits. Brain Res, 2010. 1306: pp.
100–105.
22. Desmet, K.D., D.A. Paz, J.J. Corry, J.T. Eells, M.T. Wong-Riley, M.M. Henry,
E.V. Buchmann, M.P. Connelly, J.V. Dovi, H.L. Liang, D.S. Henshel, R.L.
Yeager, D.S. Millsap, J. Lim, L.J. Gould, R. Das, M. Jett, B.D. Hodgson, D.
Margolis, and H.T. Whelan, Clinical and experimental applications of
NIR-LED photobiomodulation. Photomed Laser Surg, 2006. 24(2): pp.
121–128.
23. Byrnes, K.R., R.W. Waynant, I.K. Ilev, X. Wu, L. Barna, K. Smith, R. Heckert,
H. Gerst, and J.J. Anders, Light promotes regeneration and functional
recovery and alters the immune response after spinal cord injury. Lasers
Surg Med, 2005. 36(3): pp. 171–185.
24. Oron, U., T. Yaakobi, A. Oron, D. Mordechovitz, R. Shofti, G. Hayam, U.
Dror, L. Gepstein, T. Wolf, C. Haudenschild, and S.B. Haim, Low-energy
laser irradiation reduces formation of scar tissue after myocardial
infarction in rats and dogs. Circulation, 2001. 103(2): pp. 296–301.
25. Detaboada, L., S. Ilic, S. Leichliter-Martha, U. Oron, A. Oron, and
J. Streeter, Transcranial application of low-energy laser irradiation
improves neurological deficits in rats following acute stroke. Lasers Surg
Med, 2006. 38(1): pp. 70–73.
July 6, 2016 17:38 PSP Book - 9in x 6in 38-Hamblin-c38

758 Difficult Path to Treating Acute Ischemic Stroke Patients with Transcranial

26. Ilic, S., S. Leichliter, J. Streeter, A. Oron, L. DeTaboada, and U. Oron, Effects
of power densities, continuous and pulse frequencies, and number of
sessions of low-level laser therapy on intact rat brain. Photomed Laser
Surg, 2006. 24(4): pp. 458–466.
27. Lapchak, P.A. and D.M. Araujo, Advances in ischemic stroke treatment:
Neuroprotective and combination therapies. Expert Opin Emerg Drugs,
2007. 12(1): pp. 97–112.
28. Zhang, Q., H. Ma, S. Nioka, and B. Chance, Study of nearinfrared
technology for intracranial hematoma detection. J Biomed Opt, 2000.
5(2): pp. 206–213.
29. Mochizuki-Oda, N., Y. Kataoka, Y. Cui, H. Yamada, M. Heya, and K. Awazu,
Effects of near-infra-red laser irradiation on adenosine triphosphate
and adenosine diphosphate contents of rat brain tissue. Neurosci Lett,
2002. 323(3): pp. 207–210.
30. Nissan, M., S. Rochkind, N. Razon, and A. Bartal, HeNe laser irradiation
delivered transcutaneously: Its effect on the sciatic nerve of rats. Lasers
Surg Med, 1986. 6(5): pp. 435–438.
31. Ad, N. and U. Oron, Impact of lowlevel laser irradiation on infarct size in
the rat following myocardial infarction. Int J Cardiol, 2001. 80(2–3): pp.
109–116.
32. Anders, J.J., R.C. Borke, S.K. Woolery, and W.P. Van de Merwe, Low power
laser irradiation alters the rate of regeneration of the rat facial nerve.
Lasers Surg Med, 1993. 13(1): pp. 72–82.
33. Castro-e-Silva, O., Jr., S. Zucoloto, L.G. Marcassa, J. Marcassa, C.
Kurachi, C.A. Melo, F.S. Ramalho, L.N. Ramalho, and V.S. Bagnato,
Spectral response for laser enhancement in hepatic regeneration for
hepatectomized rats. Lasers Surg Med, 2003. 32(1): pp. 50–53.
34. Lapchak, P.A., Translational stroke research using a rabbit embolic
stroke model: A correlative analysis hypothesis for novel therapy
development. Transl Stroke Res, 2010. 1(2): pp. 96–107.
35. Waud, D.R., On biological assays involving quantal responses. J Pharma-
col Exp Ther, 1972. 183: pp. 577–607.
36. Oron, A., U. Oron, J. Chen, A. Eilam, C. Zhang, M. Sadeh, Y. Lampl, J.
Streeter, L. DeTaboada, and M. Chopp, Low-level laser therapy applied
transcranially to rats after induction of stroke significantly reduces long-
term neurological deficits. Stroke, 2006. 37(10): pp. 2620–2624.
37. Lyden, P.D., J.A. Zivin, M. Soll, M. Sitzer, J.F. Rothrock, and J. Alksne,
Intracerebral hemorrhage after experimental embolic infarction. Anti-
coagulation. Arch Neurol, 1987. 44(8): pp. 848–850.
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38. Lapchak, P.A., M.K. Han, K.F. Salgado, J. Streeter, and J.A. Zivin, Safety
profile of transcranial near-infrared laser therapy administered in
combination with thrombolytic therapy to embolized rabbits. Stroke,
2008. 39(11): pp. 3073–3078.
39. Lapchak, P.A., A new embolus injection method to evaluate intracerebral
hemorrhage in New Zealand white rabbits. Brain Res, 2010. 1349C: pp.
129–136.
40. NINDS rt-PA group, Tissue plasminogen activator for acute ischemic
stroke. The National Institute of Neurological Disorders and Stroke rt-
PA Stroke Study Group. N Engl J Med, 1995. 333(24): pp. 1581–1587.
41. Lapchak, P.A., D.M. Araujo, and J.A. Zivin, Comparison of Tenecteplase
with Alteplase on clinical rating scores following small clot embolic
strokes in rabbits. Exp Neurol, 2004. 185(1): pp. 154–159.
42. De taboada, L., S. Ilic, S. Leichliter-Martha, U. Oron, A. Oron, and
J. Streeter, Transcranial application of low-energy laser irradiation
improves neurological deficits in rats following acute stroke. Lasers Surg
Med, 2006. 38(1): pp. 70–73.
43. Lapchak, P.A., D.F. Chapman, and J.A. Zivin, Metalloproteinase inhibition
reduces thrombolytic (tissue plasminogen activator)-induced hemor-
rhage after thromboembolic stroke. Stroke, 2000. 31(12): pp. 3034–
3040.
44. Hacke W., P.D. Schellinger, G.W. Albers, N.M. Bornstein, B.L. Dahlof, R.
Fulton, S.E. Kasner, A. Shuaib, S.P. Richieri, S.G. Dilly, J. Zivin, K.R. Lees,
and NEST 3 Committees and Investigators, Transcranial laser therapy
in acute stroke treatment: results of neurothera effectiveness and safety
trial 3, a phase III clinical end point device trial. Stroke, 2014. 45(11):
3187–3193. doi: 10.1161/STROKEAHA.114.005795.
45. Fischl, B. and A.M. Dale, Measuring the thickness of the human cerebral
cortex from magnetic resonance images. Proc Natl Acad Sci USA, 2000.
97(20): pp. 11050–11055.
46. Lapchak, P.A., P.D. Boitano, P.V. Butte, D.J. Fisher, T. Hoelscher, E.J. Ley,
M. Nuno, A. H. Voie, and P.S. Rajput, Transcranial near-infrared laser
transmission (NILT) profiles (800 nm): Systematic comparison in four
common research species. PLOS ONE, 2015. 10(6): e0127580. doi:
10.1371/journal.pone.0127580.
July 6, 2016 17:38 PSP Book - 9in x 6in 39-Hamblin-c39

Chapter 39

Low-Level Laser (Light) Therapy for

Rehabilitation in Traumatic Brain Injury
and Stroke, including Chronic Aphasia

Margaret A. Naeser,a,b Paula I. Martin,a,b Michael D. Ho,a,b

Maxine H. Krengel,a,b Yelena Bogdanova,a,c Jeffrey A. Knight,a,c,d
Megan K. Yee,a,b Ross Zafonte,e,f,g,h Bang-Bon Koo,i John G.
Roubil,j and Michael R. Hamblinj,k,l
a VA Boston Healthcare System, Jamaica Plain Campus, Boston University Aphasia

Research Center (12-A), 150 South Huntington Avenue, Boston, MA 02130, USA
b Department of Neurology, Boston University School of Medicine, Boston University

Medical Center, 72 East Concord Street, C-3 Boston, MA 02118, USA

c Department of Psychiatry, Boston University School of Medicine, 720 Harrison Avenue,

Doctor’s Office Building, Boston, MA 02118, USA

d Behavioral Sciences Division, National Center for PTSD, VA Boston Healthcare System,

150 South Huntington Avenue Boston, MA 02130, USA

e Department of Physical Medicine and Rehabilitation, Harvard Medical School,

300 1st Avenue, Boston, MA 02129, USA

f Spaulding Rehabilitation Hospital, 300 1st Avenue, Charlestown, MA 02129, USA
g Massachusetts General Hospital, 55 Fruit St, Boston, MA 02114, USA
h Brigham and Women’s Hospital, 75 Francis Street, Boston MA 02115, Boston, USA
i Center for Biomedical Imaging, Boston University School of Medicine, 650 Albany

Street, X-B01, Boston, MA 02118, USA

j Wellman Center for Photomedicine, Massachusetts General Hospital, 50 Blossom

Street, Boston, MA 02114, USA

k Department of Dermatology, Harvard Medical School, Massachusetts General

Hospital, 55 Fruit Street, Boston, MA 02114, USA

l Harvard-Massachusetts Institute of Technology (MIT) Division of Health Sciences and

Technology, 77 Massachusetts Avenue, E25-518, Cambridge, MA 02139, USA

mnaeser@bu.edu

Handbook of Low-Level Laser Therapy

Edited by Michael R. Hamblin, Marcelo Victor Pires de Sousa, and Tanupriya Agrawal
Copyright c 2017 Pan Stanford Publishing Pte. Ltd.
ISBN 978-981-4669-60-3 (Hardcover), 978-981-4669-61-0 (eBook)
www.panstanford.com
July 6, 2016 17:38 PSP Book - 9in x 6in 39-Hamblin-c39

762 Low-Level Laser (Light) Therapy for Rehabilitation in Traumatic Brain Injury

39.1 Introduction

Low-level laser (light) therapy (LLLT) has been suggested as an

effective treatment for a range of brain traumas and diseases [1–
5]. Though the exact mechanism is still being studied, research has
shown that red and near-infrared (NIR) light is able to penetrate
the skull [6, 7] and trigger reactions that elevate mitochondrial
adenosine triphosphate (ATP) production [8]. Transcranial LLLT
(tLLLT) increases local, regional cerebral blood flow in humans with
traumatic brain injury (TBI) [9], and in severe depression cases [10].
Increase in ATP and focal increase in regional cerebral blood flow
are important factors that could contribute to recovery in central
nervous system disorders [11]. In this chapter, we explore a variety
of uses of tLLLT, including improvement in cognition in chronic
TBI and improvement in sequelae of stroke, including improved
language behavior in chronic stroke when aphasia is present (loss
of language abilities, primarily associated with left hemisphere [LH]
stroke).

39.2 Mechanisms of LLLT

While the mechanisms involved are still being elucidated, it

is widely believed that two physiological changes occur when
hypoxic/compromised cells are exposed to red (600 nm) and NIR
(800–900 nm) light via LLLT. The first physiological change is
increased mitochondrial ATP production [12]. The second change
is increased, focal regional cerebral blood flow due to vasodilation.
This occurs for the following reason: In TBI, the mitochondria
become damaged within the cell [13] and there is mitochondrial
dysfunction [14–16]. In hypoxic cells, cytochrome c oxidase (Cox),
a membrane-bound protein that serves as the ultimate electron
acceptor in the cell respiration electron transport chain (among
other functions), becomes inhibited by noncovalent binding of nitric
oxide. When exposed to red or NIR photons, the Cox releases nitric
oxide, which then diffuses outside the cell, increasing local blood
flow and vasodilation [12, 17].
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Traumatic Brain Injury 763

Also following initial exposure to the red/NIR photons, there

is a brief burst of reactive oxygen species (ROS) in the cell and
this activates a number of signaling pathways. The ROS leads to
the activation of redox-sensitive genes and related transcription
factors, including NF-κβ [18, 19]. The LLLT stimulates gene
expression for cellular proliferation, migration, and the production
of anti-inflammatory cytokines and growth factors [3]. Studies
have suggested that the anti-inflammatory, anti-edema, and pro-
angiogenic property of LLLT can also act as an effective treatment
modality in stroke [20].
Results from tLLLT studies with acute, severe TBI in mice suggest
that increased neurogenesis and synaptogenesis are among the most
important mechanisms associated with improved recovery in TBI or
stroke. For example, there was significantly better recovery after 28
days post-TBI in the mice receiving three daily NIR tLLLT treatments
(beginning at 4 h post-TBI), compared to controls [20–22]. The
increases in neurogenesis were located in the dentate gyrus of the
hippocampus, and in the subventricular zone at day 7. Evidence of
synaptogenesis was observed in the perilesional area and in the
subventricular zone at day 28. Thus, results from these studies
suggest that tLLLT can be used to support rehabilitation in patients
suffering from the sequelae of TBI, as well as stroke. The potential
for neurogenesis-targeted clinical therapy in TBI is a high priority
[23].

39.3 Traumatic Brain Injury

39.3.1 Introduction to TBI in Humans

Traumatic brain injury is a major medical problem worldwide,
and in the United States, three TBIs occur every minute [24].
Approximately 1.7 million patients are evaluated annually; over
5 million Americans are living with TBI-related disabilities. The
annual cost is $60–76.5 billion [25]. Closed-head, mild TBI (mTBI)
is the most common (75%), and persistent cognitive dysfunction
occurs in 5–22% of these cases. Mild TBI is associated with the loss
of consciousness (LOC) 30 min or less (including no LOC), and with a
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764 Low-Level Laser (Light) Therapy for Rehabilitation in Traumatic Brain Injury

period of altered mental status, which could include post-traumatic

amnesia—memory gaps—or confusion lasting up to 24 h.
Cognitive dysfunction associated with sports-related mTBI is of
increasing concern, both for males and females (including children)
[26]. Within the past 10 years, the diagnosis of concussion in high
school sports has increased annually, by 16.5% [27]. With each
successive concussion, there is a cumulative effect [28], including a
prolonged period of recovery and a progressively increased risk for
re-injury [29, 30]. Post-season verbal learning scores were observed
to be lower than expected in 24% of college athletes who had
participated in contact sports (versus only 3.6% in noncontact
sports); and the greater the number of head impacts sustained, the
slower the reaction time on ImPACT testing [31]. Among collegiate
football players, those playing in the offensive linemen positions are
at the greatest risk [32].
Closed-head, blast injury is the signature injury of soldiers
returning from Iraq and Afghanistan as part of Operation Enduring
Freedom/Operation Iraqi Freedom (OEF/OIF) [33, 34]. The cogni-
tive sequelae, recovery, and rehabilitation are of increasing concern
[35]. Estimates are as high as 320,000 regarding those who have
returned with TBI [36, 37]. Post-traumatic stress disorder (PTSD) is
also a major concern with OEF/OIF soldiers who have experienced
mTBI [38]. One estimate is that 28% of those diagnosed with mTBI
also report clinical levels of PTSD symptoms. A dose–response
gradient for exposure to blast/combination mTBI on clinical levels
of PTSD symptoms has been observed [39]. The incidence of mTBI
with co-morbidities of PTSD and depression is higher in the military,
than in civilians [40].
In most cases with closed-head mTBI, focal lesions are not
present on CT scan, nor on structural MRI [28, 41–43]. However,
30% of cases without abnormality on CT scan have been observed
to have abnormalities in white matter using diffusion tensor imaging
(DTI) with MRI scans [44].
Diffuse axonal injury (DAI) or traumatic axonal injury has been
recognized as one of the main consequence of closed-head mTBI
[45, 46]. DAI results when shearing, stretching, and/or angular
forces pull on axons and small vessels [14, 47–49]. The frontal
lobes, including medial and lateral prefrontal cortex areas, are
July 6, 2016 17:38 PSP Book - 9in x 6in 39-Hamblin-c39

Traumatic Brain Injury 765

especially vulnerable to damage following mTBI where both linear

and angular, acceleration/deceleration effects occur, e.g., whiplash
in a motor vehicle accident (MVA), concussive blast force, or
even direct impacts to the head [50–52]. This leads to impaired
axonal transport, focal axonal swelling, and may result in axonal
disconnection after several hours [53]. All severities of TBI can
result in a degree of axonal damage [54]. Functionally, the reduction
or loss of interconnectivity produces the cognitive, emotional, and
behavioral problems observed following TBI [55, 56].
White matter degeneration, widespread tau and amyloid-beta
pathology, and inflammation may be present years after only a single
TBI [47, 57]. Increased microglial activity may persist long-term
post-TBI [58, 59]; persistent neuroinflammation may be a mecha-
nistic link between TBI and the development of neurodegenerative
disease, including Alzheimer’s disease [57]. Damaged axons may
serve as a source of the Alzheimer’s disease-associated protein
amyloid-beta [60, 61].
Chronic traumatic encephalopathy (CTE), a progressive tau
protein-linked neurodegenerative disease, is believed to develop
in part, from repeated head trauma [60–63]. Symptoms include
cognitive dysfunction, progressive irritability, suicidal ideation, and
dementia. It may develop years after the head trauma occurred. CTE
has been documented in the US military veterans exposed to blast
injury in Iraq and Afghanistan [50].

39.3.2 Brain Imaging Studies in TBI

Since 1999, mTBI cases have been observed on functional neu-
roimaging studies to have alterations in neural activation during
performance of cognitive tasks [64]. While accuracy during a
working memory task did not differ for the mTBI cases at 1 month
post-mTBI, versus healthy controls, there was more widespread
bifrontal and biparietal activation for the mTBI cases, even in the
initial level of working memory load. This pattern of abnormal
hyperactivation was present even at 1 year post-mTBI [65, 66].
While the mTBI cases showed no significant post-concussive
symptoms at the one-year follow-up, they continued to show mildly
slower reaction speed relative to controls.
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766 Low-Level Laser (Light) Therapy for Rehabilitation in Traumatic Brain Injury

Using a different type of fMRI scan, e.g., resting state, functional

connectivity MRI (rs-fMRI), studies with normals have demon-
strated specific neural networks that function in a widespread
but temporally coordinated manner (with a very low frequency,
0.1 Hz), even while the subject is resting quietly in the MRI
scanner without external, task-related stimuli [67, 68]. One of these
networks, the default mode network (DMN), consists in part of
the ventromedial prefrontal cortex (vmPFC); the precuneus and
posterior cingulate cortex (precun/PCC); postero-lateral parietal
cortices (angular gyri); and medial temporal lobes/hippocampi [67].
The DMN, including the precun/PCC in particular, shows rapid and
highly reactive deactivation in normals during attention-demanding
tasks. Abnormality in the DMN (with failure to deactivate) has been
observed in mTBI cases [67, 69–72].
Abnormalities have also been observed with mTBI cases in
two other rs-fMRI networks, important for normal brain function
during attention-related and cognitive tasks. The first is the salience
network (SN) [73]. The SN controls the DMN, and the SN consists of
the anterior insulae, the pre-supplementary motor areas (preSMAs),
and the dorsal anterior cingulate cortex (dACC) areas. The SN is
critical for normal executive function and inhibition [74–76]. Brain
cortex in the anterior parts of the SN (preSMAs and dACC) promotes
inhibitory control (deactivation) of the posterior parts of the DMN
(precun/PCC), particularly during tasks that require inhibition and
rapid switching for success. The SN is important for signaling
the need to change behavior [77]. The second, additional rs-fMRI
network is the central executive network (CEN). This network
comprises dorsolateral prefrontal cortex areas and the intraparietal
sulci [78, 79]. The CEN is particularly important, as the name
implies, for executive function, including cognitive manipulation of
temporal information, processing speed, and multitasking.

39.3.3 Cognitive Dysfunction in TBI

The most common complaints of cases with mTBI are in the domains
of attention/concentration and working memory, i.e., the ability to
hold information in mind and to manipulate it in light of incoming
material [65, 80, 81]. At 6 months post-injury, indices of executive
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Traumatic Brain Injury 767

function have been found to predict persistence of post-concussive

syndrome in mild and moderate TBI patients [82]. One of the most
debilitating sequelae of mTBI is the failed attempt to re-establish
family and work relationships [83]. Due to the diffuse nature of
damage, however, no single behavioral outcome measure captures
the multidimensional nature of TBI outcome [84].

39.3.4 Poor Sleep in TBI

In addition to the cognitive and psychosocial issues post-TBI, there
are problems with sleep [85–87]. An estimated 53% of individuals
with TBI report sleep disturbances [88]. Sleep problems may exac-
erbate symptoms of TBI [89], increase neuropsychiatric symptoms
(depression, anxiety) post-TBI [90], and interfere with participation
in rehabilitation treatment [91]. Although the etiology of TBI-
related sleep problems is currently under investigation, research
has indicated neurobiological factors, specifically, impairment in the
function of neural circuits involved in sleep/wake regulation [92,
93]. Among many factors, poor sleep would disrupt the normal and
necessary clearing of metabolites, including beta-amyloid and other
potentially neurotoxic waste products that accumulate in the central
nervous system during waking [94].

39.3.5 Pharmacologic Treatments for TBI

Pharmacologic treatments for TBI have been mostly unsuccessful
[84, 95]. Some pharmacologic interventions are available for sys-
temic and intracranial changes associated with moderate and severe
TBI, with a few controlled systematized studies for pharmacologic
treatment of cognitive impairment [96]. There was a large clinical
trial (COBRIT) for moderate, severe, and complicated mTBI, which
utilized the pharmacologic agent, citicoline [97]. Citicoline was
evaluated as a neuroprotective agent and, in TBI, has previously
demonstrated some efficacy in secondary measures for stroke, and
smaller TBI studies [97]. In 2012, the results of the COBRIT study
showed that the use of citicoline compared with placebo for 90 days
did not improve functional and cognitive status [97].
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768 Low-Level Laser (Light) Therapy for Rehabilitation in Traumatic Brain Injury

There are currently no pharmacologic treatments for mTBI

secondary injury or for prevention of cognitive and behavioral
problems associated with mTBI [98, 99]. Further investigation
is warranted to examine the effects of cholinesterase inhibitors,
with preliminary evidence suggesting improvement in attentional
difficulties and mixed results for memory treatment [83]. Regarding
executive dysfunction, no conclusions can be drawn for improve-
ment by pharmacologic intervention post-TBI [83, 96].
McAllister et al. [100] examined the effect of bromocriptine, a
dopamine D2 receptor agonist, on working memory performance
in healthy controls versus mTBI patients. Bromocriptine was
associated with improved working memory performance only in
healthy controls but not in mTBI patients. Imaging showed that
mTBI patients were not able to recruit working memory task-
specific regions of interest. These results suggest that mTBI patients
may have altered response to dopamine. In another study, McAllister
et al. [101] found the opposite effect, using guanfacine, an alpha-
2 adrenergic receptor agonist. Guanfacine was found to selectively
improve working memory performance in mTBI but not in healthy
controls. In the mTBI group, increased activation was observed
within a working memory task-specific region of interest. This
pharmacologic agent may be a promising pharmacologic agent
to test hypotheses about the neural mechanisms of cognitive
dysfunction after mTBI.

39.3.6 Cognitive Rehabilitation Therapies for TBI

Reviews of the effectiveness of current cognitive and behavioral
treatments to improve executive function after TBI have shown
limited evidence for the efficacy of cognitive rehabilitation [35, 102–
104]. Executive dysfunction continues to present a challenge to
the rehabilitation process [105]. Behavioral treatment approaches
for TBI patients attempt to maximize the patients’ behavioral
functioning by working with residual brain-based capacities, but
injured, non-functioning brain cells may limit their potential for
success. Thus, treatments are needed that directly target injured
brain cells to improve the functioning of underlying brain systems
(including functional connectivity in networks such as DMN, SN,
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Traumatic Brain Injury 769

and CEN) that regulate attention, executive function, memory,

emotions, and behavior. Effective treatments to improve cognition
are currently lacking, and these are urgently needed for veterans
as well as non-veterans. Transcranial red/NIR LLLT is a promising
clinical research method to fill these needs.

39.3.7 Transcranial LED Treatments to Improve Cognition

and Sleep in Chronic mTBI
Naeser et al. [106] described two case reports for chronic mTBI
where cognition improved following treatment with red/NIR light-
emitting diodes (LEDs). The LEDs were applied transcranially to
forehead and scalp areas, including midline mid-sagittal areas, as
well as bilateral frontal, temporal, parietal, and occipital areas. Each
red/NIR LED cluster head (MedX Health, Toronto) had a 5.35 cm
diameter and was 500 mW containing 61 diodes (9 × 633 nm, and
52 × 870 nm). The power density was 22.2 mW/cm2 , and 13 J/cm2
(CW) was applied to the scalp from each LED cluster head (estimated
0.4 J/cm2 to surface brain cortex).
Patient 1 (59 years female, professor of web design) began
transcranial LED (tLED) treatments seven years after a closed-
head mTBI from an MVA. Pre-LED, her ability to sustain attention
(computer work) lasted only 20 min. After eight weekly LED
treatments, her sustained attention on the computer increased to 3
h. She reported that if she stopped treating for more than 2 weeks,
she regressed.
Patient 2 (52 years female, high-ranking retired military officer)
had a history of closed-head mTBIs (sports/military, and recent
fall onto concrete from a swing). Her structural MRI brain scan
showed moderate fronto-parietal atrophy, for her age. Pre-tLED, she
was on medical disability for 5 months. After 4 months of nightly
tLED treatments at home, her medical disability was discontinued;
she returned to work full-time as an executive consultant with
an international technology-consulting firm. Neuropsychological
testing after 9 months of tLED indicated significant improvement
(+1SD) in executive function (inhibition, inhibition accuracy) and
(+2SD) in memory, as well as reduction in PTSD. Patient 2 reported
that if she stopped treating for more than 1 week, she regressed.
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770 Low-Level Laser (Light) Therapy for Rehabilitation in Traumatic Brain Injury

Both patients continued home treatments with tLED for at least

5 years (1–3× per week); there have been no adverse events or
negative side effects. Patient 1 is lost to follow-up, and Patient 2
continues with home treatments.
Naeser et al. [5] conducted a pilot, open-protocol study with
a larger number of chronic mTBI patients to examine whether
tLED with the same red/NIR LED cluster heads could improve
cognition when a systematic treatment protocol was used. Eleven
chronic mTBI participants (26–62 years of age, six male) with non-
penetrating brain injury and persistent cognitive dysfunction were
treated for 18 outpatient sessions (Monday, Wednesday, Friday, for
6 weeks), starting at 10 months to 8 years post-mTBI (MVA or
sports-related; and one participant, improvised explosive device
blast injury). Four had a history of multiple concussions. Each LED
cluster head (5.35 cm diameter, 500 mW, 22.2 mW/cm2 ) was applied
for 9 min 45 s to each of 11 scalp placements (13 J/cm2 , CW).
LEDs were placed on the midline from front-to-back hairline, and
bilaterally on frontal, parietal, and temporal areas. See Naeser et al.
[5] for specific LED placements. These placements covered nodes
located on the DMN, SN, and CEN.
Neuropsychological testing was performed pre-LED, and at 1
week and 1 and 2 months after the 18th treatment. A significant
linear trend was observed for the effect of tLED treatment over time
for the Stroop test for executive function, Trial 3 inhibition ( p =
0.004); Stroop, Trial 4 inhibition switching ( p = 0.003); California
Verbal Learning Test (CVLT)-II, Alternating Versions, Total Trials
1–5 ( p = 0.003); and CVLT-II, Long Delay (20 min) Free Recall
( p = 0.006) (see Fig. 39.1). Participants reported improved sleep
and fewer PTSD symptoms, if present (Fig. 39.2). Participants and
family reported better ability to perform social, interpersonal, and
occupational functions.
Bogdonova et al. [107] studied the effects of tLED treatment
on sleep and cognitive function in patients with chronic moderate
TBI at the VA Boston Healthcare System. Two patients (one female)
with moderate TBI and persistent cognitive dysfunction (at least
2 SD below average on one, or 1 SD below average on at least
two neuropsychological tests of executive function and memory)
received 18 sessions of tLED therapy (3× /week for 6 weeks, with
at least 48 h between sessions). Both cases who received tLED
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Traumatic Brain Injury 771

(A) Stroop Test for Executive Function: (B) Stroop Test for Executive Function:
Trial 3, Inhibition Trial 4, Inhibition Switching

1.0 ρ = .004 1.5

ρ = .003
Number of SD Units

1.0

Number of SD Units
0.5
0.5
0.0 0.0
-0.5
-0.5
-1.0
-1.0 -1.5
Pre-Tx 1 Wk. 1 Mo. 2 Mo. Pre-Tx 1 Wk. 1 Mo. 2 Mo.
Post-18 Tx Post-18 Tx Post-18 Tx Post-18 Tx Post-18 Tx Post-18 Tx

(C) California Verbal Learning Test-II: (D) California Verbal Learning Test-II:
Total Trials 1-5 Long Delay Free Recall
1.0 0.5
ρ = .003
ρ = .006
Number of SD Units

Number of SD Units

0.5 0.0

0.0 0.5

0.5 -1.0

-1.0 -1.5
Pre-Tx 1 Wk. 1 Mo. 2 Mo. Pre-Tx 1 Wk. 1 Mo. 2 Mo.
Post-18 Tx Post-18 Tx Post-18 Tx Post-18 Tx Post-18 Tx Post-18 Tx

Figure 39.1 Graphs showing a significant linear trend over time, for the
effect of transcranial LED treatments on specific neuropsychological tests.
(A) Stroop (color-word interference test) for Executive Function: Trial 3,
inhibition (p = 0.004); (B) Stroop, Trial 4 inhibition switching (p = 0.003);
(C) CVLT-II, Alternating Versions, Total Trials 1-5 (p = 0.003); and (D) CVLT-
II, Long Delay (20 minutes) Free Recall (p = 0.006). Data taken from Ref. [5].

showed marked improvement in sleep by increasing an average of

1 h per night (measured with actigraphy), at 1 week after the tLED
treatment series, as compared to pre-tLED. Patient 1 also improved
in executive function, verbal memory, and sleep efficiency, while
Patient 2 improved on measures of PTSD (PCL-M) and depression.
No adverse events were reported.

39.3.8 Intranasal LED Treatments to Improve Cognition

and Sleep in mTBI
A pilot, open-protocol intranasal LED research project with mTBI
participants who have chronic cognitive dysfunction is in progress
at the VA Boston Healthcare System (Naeser lab, personal obser-
vation). Two small diodes (one clipped into each nostril) are used
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772 Low-Level Laser (Light) Therapy for Rehabilitation in Traumatic Brain Injury

(A) D-KEFs Color Word Interference (Stroop) (B) D-KEFs Color Word Interference (Stroop)
for Executive Function: Trial 3, Inhibition for Executive Function:
Trial 4, Inhibition Switching

p = .004 p = .003
1.0 1.5
Number of SD Units

Number of SD Units
1.0
0.5
0.5
0.0 0.0
-0.5
-0.5
-1.0
-1.0 -1.5
Pre-Tx Pre-Tx 1 Wk.
1 Wk. 1 Mo. 1 Mo.
2 Mo. Post-18 Tx Post-18 Tx 2 Mo.
Post-18 Tx Post-18 Tx
Post-18 Tx Post-18 Tx

(C) California Verbal Learning Test-II: (D) California Verbal Learning Test-II:
Total Trials 1-5 Long Delay Free Recall
p = .003 p = .006
1.0 1.5
Number of SD Units

1.0
Number of SD Units

0.5
0.5
0.0 0.0
-0.5
-0.5
-1.0
-1.0 -1.5
Pre-Tx 1 Wk. Pre-Tx
1 Mo. 1 Wk. 1 Mo.
Post-18 Tx 2 Mo. Post-18 Tx Post-18 Tx 2 Mo.
Post-18 Tx
Post-18 Tx Post-18 Tx

Figure 39.2 Four of the eleven mTBI cases treated in the Naeser et al., 2014
study, also had post-traumatic stress disorder (PTSD). All four cases showed
a clinically meaningful or reliable decrease in symptoms of PTSD, after the
transcranial, red/near-infrared LED treatment series [5].

simultaneously for 25 min. The red (633 nm) intranasal diode is 6.5
mW (CW), 7.6 mW/cm2 , with estimated energy delivery to mucosa,
11.4 J/cm2 (Vielight, Toronto). The NIR (810 nm) intranasal diode
has the same parameters, except it is pulsed at 10 Hz (6.5 mW,
net of 50% pulse duty cycle). Both intranasal LEDs are noninvasive,
painless, and nonthermal.
It is hypothesized that some NIR photons can reach the
hippocampal/lateral entorhinal cortex areas via intranasal delivery.
The red photons are hypothesized to improve blood rheology [108]
and to improve sleep by increasing melatonin [109]. Participants are
treated 3×/week for 6 weeks, with 48 h between treatments. The
pre- and post-intranasal LED testing is the same as for mTBI cases
who receive tLED treatments [5, 107].
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Stroke 773

Thus far, one mTBI participant (24 years, female) with a history
of four sports-related concussions (two snowboarding and two
field hockey) has received the intranasal LED treatment series. The
results after the intranasal series were similar to those for our tLED
studies [5, 107]. Significant improvements were observed on tasks
of executive function, verbal memory, attention, and verbal fluency,
at 1, 6, and 12 weeks after the 18th intranasal LED treatment. At
1 week after the 18th intranasal LED treatment, the participant’s
average total sleep time had increased by 61 min per night, and her
sleep efficiency (total sleep time/total time in bed) had increased by
11%. Her sleep efficiency at 12 weeks post-intranasal LED was 5%
above pre-intranasal LED levels, and she reported no longer using
any sleep medication that she had previously been using regularly.
It is possible that her sleep parameters would have continued to
improve even more if she had the opportunity for self-administered
home treatment with intranasal LEDs. There were no negative side
effects or complications.

39.4 Stroke

While stroke trails behind heart disease and cancer as the leading
cause of death in the United States, it is the biggest cause of long-
term disability among American adults [110]. Only 10% of stroke
patients are able to walk away from a stroke with complete or near-
complete recoveries, while 90% continue to suffer from disabilities
that often require them to seek professional care for health and
living support. The economic burdens are also substantial. Between
2012 and 2030, the total direct annual stroke-related medical costs
are expected to increase from $71.55 billion to $183.13 billion [111].

39.4.1 Transcranial LLLT to Treat Acute Stroke

39.4.1.1 Transcranial LLLT studies to treat acute stroke:
Small-animal studies
Multiple studies using tLLLT to treat acute stroke in small animals
were performed prior to the use of tLLLT to treat acute stroke in hu-
mans. A brief review of promising results from the animal studies is
presented here. (See also Chapter 21 in this handbook for additional
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774 Low-Level Laser (Light) Therapy for Rehabilitation in Traumatic Brain Injury

information.) Transcranial LLLT (808 nm) significantly improved

recovery following ischemic stroke in rats, when treated once at
24 h post-stroke [112, 113]. The laser was used transcranially on
the exposed (shaved skin) skull by placing the tip of the fiber optic
(4 mm diameter) onto the skin at two locations on the head (3 mm
dorsal to the eye and 2 mm anterior to the ear) on the contralateral
hemisphere to the stroke [113].
Previous studies with rats had shown that tLLLT of the contralat-
eral, or both hemispheres, demonstrated no difference in functional
outcome [114]. An NIR Ga–As diode laser was used transcranially
to illuminate the hemisphere contralateral to the stroke at a power
density of 7.5 mW/cm2 to brain tissue The neurological deficits at 3
weeks post-stroke were significantly ( p < 0.01) reduced (by 32%)
in the laser-treated rats, relative to controls.
In their study [112, 113], the number of newly formed neuronal
cells assessed by double immunoreactivity to bromodeoxyuridine
and tubulin isotype III, as well as migrating cells (doublecortin
immunoreactivity), was significantly elevated in the subventricular
zone of the hemisphere ipsilateral to the induction of stroke
when treated by tLLLT. There was no significant difference in
the stroke lesion size between control and laser-irradiated rats.
The authors suggested that an underlying mechanism for the
functional benefit post-tLLLT in this study was possible induction of
neurogenesis.
Other studies have also suggested that because improvement
in neurologic outcome may not be evident for 2–4 weeks in the
post-stroke rat model, delayed benefits may be due, in part, to the
induction of neurogenesis and migration of neurons [8, 115]. These
findings in acute stroke studies with rats are similar to those in
severe TBI studies with mice, reviewed above [21, 22], suggesting
a role for neurogenesis to promote improved recovery when tLLLT
is applied in the acute phase. In addition, tLLLT may prevent
apoptosis and improve outcomes by exerting a neuroprotective
effect, although these exact mechanisms are not well understood
[116].
Other studies in rat and rabbit models have also observed
tLLLT to improve functional outcome after stroke [8, 117, 118]. A
rabbit study combined tLLLT with thrombolytic therapy using tissue
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Stroke 775

plasminogen activator, with no increase in bleeding and good safety

[119].
An animal study with rabbits has shown a direct relationship
between the level of cortical fluence delivered (in J/cm2 ) and cortical
ATP content in embolized rabbits [120]. Five minutes following
embolization (right carotid), rabbits were exposed to 2 min of NIR
tLLLT using an 808 nm laser source (CW or pulsed wave, PW,
at 100 Hz, or at 1000 Hz; on skin surface, posterior to bregma
at midline). Three hours after embolization, the cerebral cortex
was excised and processed for the measurement of ATP content.
Embolization decreased cortical ATP content in ischemic cortex by
45% compared to naive rabbits. A linear relationship up to 4.5 J/cm2
in fluence delivered was observed for the relationship between
cortical fluence (in J/cm2 ) versus percent increase in cortical
ATP content (over sham-treated embolized rabbits). This linear
relationship was observed with a power density of 7.5 mW/cm2 CW
(0.9 J/cm2 ), where an increase of 41% in cortical ATP was observed;
and with a power density of 37.5 mW/cm2 PW (100 Hz, 4.5 J/cm2 ),
an increase of 157% in cortical ATP was observed. An increase in
cortical ATP of 221% was observed with a fluence of 31.5 J/cm2 ,
delivered with a power density of 262.5 mW/cm2 PW, 1000 Hz. This
suggests that a near-plateau effect was present regarding the fluence
level delivered above 4.5 J/cm2 . It was surprising, however, that
the increased cortical ATP levels of 157% and 221%, were higher
than those measured in naive rabbits that had never suffered stroke.
Because the authors observed that the PW modes (100 Hz and
1000 Hz) were more effective than the CW mode to increase cortical
ATP, they hypothesized that in future stroke studies in animals and in
humans, even greater improvement in clinical rating scores might be
achieved by optimizing the method of NIR tLLLT delivery, including
the length of treatment and the mode of treatment (PW).

39.4.1.2 Transcranial LLLT to treat acute stroke: Human studies

Transcranial LLLT has been shown to significantly improve outcome
(NIH Stroke Severity Scale) in acute human stroke patients, when
applied at ∼18 h post-stroke, over the entire surface of the head (20
points in the 10/20 EEG system, LH and right hemisphere (RH), but
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776 Low-Level Laser (Light) Therapy for Rehabilitation in Traumatic Brain Injury

not midline, mid-sagittal areas) regardless of stroke location [112].

An NIR (808 nm) laser was used, delivering a fluence of 0.9 J/cm2
over the entire surface (2 min at each of the 20 points; power density
of 7.5 mW/cm2 ).
Only one tLLLT treatment was administered, and 5 days later,
there was significantly greater improvement in the real tLLLT but
not in the sham-treated group ( p < 0.05, NIH Stroke Severity Scale).
This significantly greater improvement was still present at 90 days
post-stroke, where 70% of the patients treated with real tLLLT had
successful outcome, versus only 51% of controls.
In a second, similar study with the same tLLLT protocol, with
an additional 658 acute stroke patients randomized for real or
sham tLLLT, similar significant beneficial results ( p < 0.04) were
observed for moderate and moderate-severe stroke patients (n
= 434) who received the real laser protocol, but not for severe
patients [121]. When all 656 cases were included in the data analysis
(including the severe stroke cases), there was no significant real
versus sham LLLT effect. When data for both stroke studies were
pooled (n = 778; with 120 plus 658) [112, 121], there was a highly
significant beneficial effect for the real tLLLT group ( p = 0.003),
versus those who received the sham laser treatment. Lampl et al.
[112] wrote that “Although the mechanism of action of infrared
laser therapy for stroke is not completely understood. . . infrared
laser therapy is a physical process that can produce biochemical
changes at the tissue level. The putative mechanism. . . involves
stimulation of ATP formation by mitochondria and may also involve
prevention of apoptosis in the ischemic penumbra and enhancement
of neurorecovery mechanisms.” For additional information on tLLLT
to treat acute stroke in humans, see Chapter 38 in this handbook.

39.4.2 Transcranial LLLT to Treat Chronic Stroke

39.4.2.1 Transcranial LLLT to treat chronic stroke: Human
studies
Boonswang et al. [122] described a young stroke patient (29
years, female) with sequelae at 2 years post-stroke that included a
nonfunctional left hand, a very spastic right hand, inability to walk or
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Stroke 777

ambulate, a right sixth cranial nerve palsy producing double-vision,

and severe dizziness. She had received 21 months of rehabilitation
at two different rehabilitation centers prior to LLLT. The patient
then began a photobiomodulation (LLLT) regimen in which she was
treated for 45 min once a week, for 2 months. Her treatment resulted
in “significant improvement in every area of deficit.”
The LLLT included treatment with a hand-held LED cluster
head device with 69 diodes (red, 660 nm and NIR, 850 nm)
that was used at each treatment to deliver 2.95 J/cm2 to each
of 32 locations—8 covering the cerebral cortices, brainstem, and
cervical spine, and 24 locations covering the “core musculature
and lymphatics” for 1 min intervals. Her treatment also included
myofascial release (MR) techniques, physical therapy, cranio-sacral
therapy, and neuromuscular therapy.
After 8 weeks of these treatments (once per week), her
previously nonfunctional left hand could be used to grasp objects,
she regained her ability to move the fingers in her right hand, and
she was able to use a walker. After the first three treatments, she was
no longer dizzy, and she no longer suffered from a right sixth nerve
palsy, “. . . correcting the esotropia and [improving] her diplopia.” She
had previously used an ophthalmologist’s patch and prism therapy
that had not corrected the problem, and surgery was recommended
as the next step. Her results were sustained for another 12 months;
she continued with treatment once a month. It was believed that
more frequent and intensive LLLT treatments (e.g., three treatments
per week) could result in even faster results.
LLLT has been applied to acupuncture points located on the
upper and lower limbs in stroke patients with hemiparesis [123].
The overall results with laser acupuncture were similar to those
following a series of needle acupuncture treatments where an
increase of 11–28% was observed in isolated active range of
motion for knee flexion, knee extension, and/or shoulder abduction
[124]. Significant improvements were also observed in hand paresis
cases, including a reduction in hand spasticity [2] and finger and
hand strength [123, 125]. The Naeser et al. papers are available
at www.bu.edu/naeser/acupuncture (see also Chapter 45 in this
handbook for additional information on laser acupuncture.)
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39.4.3 Transcranial LLLT to Improve Language in Chronic

Aphasia Due to Stroke
39.4.3.1 Aphasia
Aphasia is a language disorder due to acquired brain damage
primarily in the LH (stroke, trauma, tumor, or neurodegenerative
disease). LH damage produces aphasia for the vast majority of cases,
including right-handers and left-handers [125]. In aphasia, the abil-
ity to communicate by oral or written language, or both, is affected
[126]. Aphasia is further defined as “. . . a multi-modality disorder
represented by a variety of impairments in auditory comprehension,
reading, oral-expressive language, and writing. . . but it cannot be
explained by dementia, sensory loss, or motor dysfunction” [127].
When aphasia is caused by stroke, it typically results from
injury to an extended network of cortical and subcortical structures
perfused by the left middle cerebral artery [128], but can also extend
into brain structures perfused by the left anterior or posterior
cerebral arteries. Lesion size is not always a factor in the prognosis
for language recovery [129], but rather lesion site is critically impor-
tant. For example, deep, smaller white matter lesions located near
borders of the left lateral ventricle are often associated with severe
limitation in speech output, and the extent of these white matter
lesions is predictive for the potential of speech recovery (phrase
length, etc.) [130, 131]. Lesion size has been observed, however, to
be associated with the general ability to name pictures in chronic
Wernicke’s aphasia cases [132] and in anomia aphasia cases [133].
In the chronic stage post-stroke onset (greater than 6 months),
approximately 50–60% of aphasia cases have communicative
impairment [134]. In patients with chronic aphasia, improved
language following various therapies has been associated with new
activation in perilesional and remaining LH areas [135–138]. In
fact, Heiss and Thiel [139] have suggested that for better long-term
recovery, RH recruitment may be less efficient than restoring the LH
network.
The RH may play a role in supporting some recovery, however,
when there is a great deal of damage to LH language areas [140].
New RH activation has been observed following speech-language
therapy in some studies [141–143].
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The presence of overactivation in RH inferior frontal and motor/

sensory areas for the mouth in the chronic stage post-stroke has
been associated with poor, hesitant, agrammatic (nonfluent) speech
[144–146]. Our research with repetitive transcranial magnetic brain
stimulation (rTMS) in chronic, nonfluent aphasia cases observed
significant improvement in picture naming and phrase length
following suppression of a portion of the RH Boca’s area (pars
triangularis, in R inferior frontal gyrus) [147]. Functional MRI
studies before and after the rTMS series showed less activation in
the RH frontal areas and increased activation in the LH perilesional
areas post-rTMS [136]. The role of rTMS as another noninvasive
brain stimulation technique to treat stroke sequelae is addressed
further, in the last section of this chapter.
The relative contribution of the LH perilesional areas (versus
the RH homologous language areas) to recovery from aphasia in
stroke has recently been reviewed [128, 148]. Results from recent
studies have continued to support the role of the LH perilesional
areas in better recovery. In the chronic stage post-stroke (1 year or
more), better language recovery was associated with fMRI activation
patterns in the LH areas, including increased activation in the left
frontal and parietal areas, bilateral cerebellum, and decreases in
the RH superior temporal areas. Stroke patients with poor language
recovery had increased activation in the RH [149, 150]. While most
studies have supported a beneficial role for increased LH activation
in better aphasia recovery, some areas within the RH may also
contribute. Less is known, however, regarding the role of the RH, and
additional studies are required [148].
Whether recovery in aphasia is mediated primarily from LH
perilesional areas, or from RH language homologues (or both), all
the aforementioned studies suggest there is potential for brain
reorganization and improved language in chronic, post-stroke
aphasia [151, 152].

39.4.3.2 Importance of specific LED placement areas on the

scalp to treat aphasia in chronic stroke
Naeser et al. [153] carried out a pilot research treatment study
using tLED to treat nonfluent aphasia. Two right-handed, chronic,
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780 Low-Level Laser (Light) Therapy for Rehabilitation in Traumatic Brain Injury

nonfluent aphasia patients with single LH stroke were treated with

tLED (Patient 1: 67 years, male, 18 years post-stroke; Patient 2: 59
years, male, 12 years post-stroke). They were each treated with two
separate LED treatment methods, identical for each case.
Prior to any LED treatments, each patient was tested 3×
(separate visits) for entry/baseline language abilities with the
Boston Diagnostic Aphasia Exam (BDAE) [154] and the Philadelphia
Naming Test (PNT) [155]. Each patient was also tested at 1–2 weeks
after each LED treatment series and again at 1–2 months post-LED.
Significant change was defined as ±2 SD from baseline.
Structural MRI scans showed LH lesion sites compatible with
nonfluent aphasia, even though P2 had primarily subcortical lesion
[130, 131]. Overt naming fMRI scans (3-Tesla Philips Achieva) were
obtained for Patient 1, before and after each LED treatment series,
utilizing the hemodynamic delay method [156]. Patient 2 could not
undergo 3T fMRI due to stents.
The MedX Health (Toronto) LED cluster heads (FDA-cleared,
nonsignificant risk device) were applied to the head. Each LED
cluster head was 5.35 cm diameter (9 red, 633 nm diodes; 52 NIR,
870 nm diodes); 22.48 cm2 in size; 500 mW; 22.2 mW/cm2 power
density; pulsed wave 146 Hz (duty cycle, 80% on). Each aphasia
patient was first treated with bilateral LED placements using the
same tLED placement loci used with chronic TBI cases explained
above [5]. Two months later, each patient was treated with LH-only
tLED placements.

39.4.3.3 Bilateral tLED treatment method

The following midline/mid-sagittal areas, plus L and R cortical areas,
were treated: Frontal poles (LEDs were placed on the forehead,
centered above each eyebrow but below the front hairline); inferior
frontal gyri; and anterior temporal lobes (on temple areas); middle
frontal gyri (MFG) (on the pupil line, immediately posterior to
front hairline); superior temporal gyri (immediately superior to ear
tip); inferior parietal areas (posterior-superior to ear tip); lower
sensori-motor (S-M) cortex, mouth areas (immediately posterior
to LED placement for MFG); and midline (front-hairline to back-
hairline placements), including anterior cingulate cortex and ventral
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Stroke 781

medial prefrontal cortex (at center front hairline); supplementary

motor areas (SMAs) (near vertex); and precuneus areas (superior to
occipital protuberance, halfway toward the vertex), alternating with
inferior to the occipital protuberance, at every other treatment.
Six, 5.35 cm diameter LED cluster heads were applied simulta-
neously (Set A, followed by Set B), 13 J/cm2 (12 min 11 s per set,
and 146 Hz, PW). It was estimated that 0.4 J/cm2 reached the brain
cortex. The LED therapy is noninvasive, painless, and nonthermal.
Patients were treated in a recliner chair. The LED cluster heads were
held in place with a soft nylon elastic cap. Each patient received 18
tLED treatments (M, W, F) for 6 weeks, followed by post-testing.

39.4.3.4 Left-hemisphere-only tLED treatment method

Only some of the same LED placement areas listed above in the
bilateral tLED treatment method were used, and the LED cluster
heads were placed only on LH areas. No midline areas were treated,
including no placement of an LED cluster head on the L&R SMAs
(near vertex). No LEDs were placed on the R side of the head.
The same LED device was used, however, with the application of
an energy density of 39 J/cm2 (36 min 33 s, PW 146 Hz) per LED
cluster head applied to the LH only, placement loci (L side of head).
It was estimated that 1.2 J/cm2 reached brain cortex. Each patient
received 18 LED treatments (M, W, F) for 6 weeks, followed by post-
testing.

Results: Unexpectedly, after the bilateral LED treatment series,

there was a significant decrease in the ability to name pictures
of objects for Patient 1 and no significant change for Patient 2.
Conversely, after the unilateral LH-only LED treatment series, there
was a significant increase in the ability to name pictures for each
stroke patient. On the PNT, Patient 1 improved from 25.33 (SD, 1.53)
at the baseline to 29 at 1 month after the LH-only LED treatment
series. Patient 2 almost doubled his score on the PNT, improving
from 5 (SD, 2) at the baseline to a score of 9 at 1 week after the LH-
only LED treatments.
Changes on the overt naming fMRI scans after each LED
treatment series paralleled changes in the ability to name pictures
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782 Low-Level Laser (Light) Therapy for Rehabilitation in Traumatic Brain Injury

for Patient 1. After the bilateral LED treatment series, there

was increased bilateral activation, including L perilesional S-M,
mouth area; L&R SMAs and R fronto-temporo-parietal areas. High
activation in R frontal and the R SMA has been associated with
poor naming and poor language recovery in aphasia [136, 157–159].
Patient 1 had shown a significant decrease in picture naming at 1
month after the bilateral LED treatment series.
Conversely, on the fMRI scans for Patient 1 after the unilateral
LH-only LED treatment series, there was primarily increased
activation only in the LH. At 2 weeks after the unilateral LH-only
LED series, there was increased activation in the L perilesional, S-
M mouth area; and the L SMA, but not the R SMA. Compared to fMRI
scans after the bilateral LED treatment series, the fMRI scans after
the unilateral LH-only treatment series showed greatly reduced
areas of activation in the RH (only minimal activation was present
in the R frontal and R posterior temporal areas). Activation in the L
SMA (along with the absence of activation in the R SMA) was likely
associated with the significant increase in naming that was present
after the unilateral LH-only LED treatment series for Patient 1 [136,
157–159].
There was significant improvement in sentence-level, auditory
comprehension for each stroke patient after the bilateral LED treat-
ment method. Patient 1 almost doubled his score on comprehension
for commands (BDAE) from 7.67 (SD, 1.53) to 14 at 1 month post-
LED. His score at 2 months after the bilateral LED treatments
(12) remained 2 SD above his baseline level. Patient 2 improved
on auditory comprehension of complex ideational material (BDAE)
from 7.3 (SD, 1.15) at the baseline to 11 at 1 week after the
bilateral LED treatments. The significant improvements in sentence-
level auditory comprehension that were present following the
bilateral LED treatment series remained stable after the LH-only
LED treatment series.
The tLED treatments appeared to target local, cortical neural
networks subjacent to the extra-cranial LED placement loci.
Transcranial LED therapy with appropriate wavelengths, energy
density (J/cm2 ), and power density (mW/cm2 ) has the potential
to modulate brain plasticity and promote recovery in stroke as
well as other central nervous system disorders. The Naeser et al.
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Stroke 783

[153], study was the first report of fMRI documentation for a

focal, hemispheric effect following scalp application of red/NIR LED
therapy to treat stroke.
After the second tLED treatment series (left side of the head to
treat the LH only), there was significant increase in picture naming
for both stroke cases; Patient 1 showed LH perilesional activation.
Thus, these data suggest that in LH stroke patients with aphasia, the
tLED should be applied to the left side of the head only, in order to
have a beneficial effect. A general recommendation in treating acute
or chronic stroke patients would be to only use tLED placements that
are ipsilesional to the side of the brain where the stroke took place,
not contralesional or bilateral.
Perhaps even better results would have been found for the
PhotoThera trials with tLLLT to treat acute stroke patients [112,
121] if, instead of treating both the left and right sides of the
head (irrespective of hemispheric side of the stroke), the tLLLT
placements had been restricted to the ipsilesional side of the head.
Our better language results following LED placements only on the LH
side of the head (ipsilesional to the LH stroke) are supportive of fMRI
research studies reviewed above, where chronic aphasia patients
with increased LH activation (not RH activation) had better language
recovery [149, 150]. Although the PhotoThera studies used a single
measure for overall stroke severity, the NIH Stroke Severity Scale
pre- and post-tLLLT, the same idea would apply to overall stroke
recovery, i.e., the application of the tLLLT may well produce better
results overall if the tLLLT is applied only to the ipsilesional side.
More research studies that examine the short-term and long-term
effects of tLLLT placements are needed.

39.4.3.5 Transcranial LLLT to treat primary progressive aphasia,

neurodegenerative disease
Naeser et al. [153] also reported on the effect of tLED to treat one
aphasia patient with the neurodegenerative disease, primary pro-
gressive aphasia (PPA). Patients with PPA present with symptoms of
aphasia for at least two years before diagnosis (however, no stroke
has taken place); cognitive/memory disturbances are present only
in later stages. Impaired naming is the common deficit among the
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784 Low-Level Laser (Light) Therapy for Rehabilitation in Traumatic Brain Injury

three variants of PPA: nonfluent (nfv), logopenic (lv), and semantic

[160].
The etiology of PPA is often in the clinical spectrum related
to frontotemporal lobar degeneration (FTLD) [161]. Estimates of
the prevalence of FTLD are 2.7–15.0 per 100,000 [161]. FTLD is
the second most common dementia behind Alzheimer’s disease
(below the age of 65) and the fourth most common dementia
in industrialized nations [162]. Twenty to forty percent of FTLD
cases have PPA [161]. The average age of onset is in the late
50s [163] although a wide range is reported (20–82). Survival is
approximately 7 years (range 2–8+ years). The prognosis is highly
variable [161, 162]. There is no gender bias, and there are no known
environmental risk factors [164]. There is no known treatment for
PPA [165, 166].
The three variants of PPA (nfv, semantic, lv) are associated with
cortical thinning in specific language-related regions of the brain,
primarily in the LH, e.g., inferior frontal (nfv), anterior temporal
(semantic), or posterior temporo-parietal (lv) [166, 167]. Data are
inconclusive regarding the histopathological basis for the variants
of PPA [161]. PPA is typically associated with one of three classes
of pathology: tau-positive immunoreactivity, more common in nfv
PPA; frontotemporal lobar dementia—ubiquitin immunoreactivity
(FTLD-U), more common in semantic PPA; amyloid plaques and
neurofibrillary tangles, more common in lv PPA [168–170].
One PPA patient diagnosed with lv PPA (similar to conduction
aphasia) was treated with tLED, using the same LED equipment
described above. She received only the bilateral LED placements (LH
and RH sides, and the midline/mid-sagittal placements). She was
treated 3×/week (M, W, F) for 6 weeks.
After the bilateral LED series, there was significant increase in
sentence-level auditory comprehension (BDAE) at 1 week post-tLED
treatment (but not at 1 and 2 months post-tLED, when picture
naming remained the same or worsened). Her overt naming fMRI
scans showed increased R and L perisylvian activation (but not
SMA), after the tLED treatment series. It was noted that there
was increased L temporo-parietal activation on the fMRI scan at 1
week post-tLED. These are areas that had showed significant cortical
thinning on the structural MRI scan in this case; compatible with
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Stroke 785

her lv (conduction) aphasia. The new L temporo-parietal activation

may have been compatible with her significant improvement in
sentence-level, auditory comprehension at 1 week post-tLED. This
improvement was not present, however, at the 1-month and 2-
month follow-up language testing. Also, the increased L temporo-
parietal activation that was present at 1 week post-tLED on the
overt naming fMRI scan was no longer present on the 1-month
and 2-month follow-up fMRI scans. This rapid deterioration of the
beneficial changes seen on fMRI at 1 week post-tLED, but not at 1
and 2 months post-tLED, suggests that this was likely related to the
progressive nature of the neurodegenerative disease. A case such as
this would require continued tLED treatments, especially to the LH
only.
In summary, there was significant improvement in sentence-
level, auditory comprehension at 1 week post-tLED in this lv PPA
case, along with increased LH activation in LH temporo-parietal
areas where she had significant cortical thinning. These results
suggest that additional tLED studies should be undertaken with PPA
cases; however, the tLED treatments should be placed on the LH
only, and the tLED treatments need to be continued, indefinitely. The
goal would be to improve language and quality of life for as long as
possible, in the PPA cases. These tLED treatments (and intranasal
LED treatments) can be performed in the home [106].

39.4.3.6 Additional tLED treatment studies with chronic aphasia

due to stroke
Four additional chronic aphasia patients with LH stroke have been
treated with tLED (Naeser lab, personal observation). We have
observed that by increasing the energy density to 26 J/cm2 per LED
placement, naming scores were increased even more, than when
13 J/cm2 was used. In addition, we have observed that the best
language results have been observed when midline, mid-sagittal
nodes within the DMN were also treated, simultaneously, along
with language areas in the LH. The DMN nodes include the mesial
prefrontal cortex area (LED cluster head placement at the center
front hairline, halfway on the forehead, halfway behind the hairline),
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786 Low-Level Laser (Light) Therapy for Rehabilitation in Traumatic Brain Injury

and simultaneously, the precun/PCC area (halfway between the

occipital protuberance and the vertex).

39.5 Other Noninvasive Brain Stimulation Therapies to

Treat TBI or Stroke

In addition to the tLLLT therapies reviewed in the earlier sections,

two other noninvasive therapies include transcranial magnetic
brain stimulation (TMS) and transcranial direct current stimulation
(tDCS). Transcranial electrical stimulation of the cortex was first
demonstrated in 1980 [171] but was never used as a medical
therapy because of the undesirable pain it causes. A much more
convenient method was invented by Barker et al. only five years later,
namely TMS [172]. Each of these noninvasive treatment techniques
is reviewed briefly here.

39.5.1 Transcranial Magnetic Brain Stimulation

TMS relies on the induction of small electrical currents in the
brain by a magnetic field, which passes through the skull. Thus,
the application of TMS is painless and, therefore, widely used for
noninvasive stimulation of the human brain. The stimulation works
by passing a large (5 kA or more), brief (<1 ms) current through
the wired and insulated coil placed on the subject’s scalp. The brief
current flowing through the coil generates a magnetic pulse that
penetrates the skull and in turn induces small eddy currents in
electrically conductive regions, i.e., in the underlying brain tissue
[173]. Relatively focal stimulation can be achieved by combining two
circular coils to form a “figure-of-eight” (or butterfly shaped) coil.
The magnetic fields sum up at the point of intersection of both coils.
Using spherical model approximation, it has been estimated that the
spatial resolution of TMS is in the centimeter range (10–20 mm).
Repetitive TMS of appropriate frequency, intensity, and duration
can lead to transient increases or decreases in the excitability of
the targeted cortex that last beyond the duration of the rTMS train
itself [174]. Slow (1 Hz) rTMS has been shown to decrease cortical
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Other Noninvasive Brain Stimulation Therapies to Treat TBI or Stroke 787

excitability in humans [175]. Conversely, fast rTMS (5, 10, or 20 Hz)

can induce a transient increase in cortical excitability [176].
In humans, there is a dominant hemisphere for language, which
in most cases is the LH. After an LH stroke when aphasia is present,
it has been observed that there is much brain reorganization for
language that takes place over the following weeks and 2–3 months.
Indeed, during the first few months, there is increased activation
in the RH. However, after a year or more, in those cases who have
better language recovery, the increased right activation becomes
less, and there is a shift to increased LH activation, mostly in the
left perilesional areas [149, 150]. The increased overactivation that
remains in the RH language homologues is considered by many to
be maladaptive, or only “. . . partially compensatory, and that optimal
neuroplastic changes eventually involve recruitment of perilesional
areas” in the LH [148].
There have been several studies in the past decade that used
slow, 1 Hz, rTMS to inhibit overactivation in the right frontal cortex to
improve language in chronic stroke. With rTMS the motor threshold
is first established for the first dorsal interosseous muscle; and the
rTMS is delivered at 90% of that motor threshold for any given
patient. The optimum area to suppress with rTMS in these chronic
aphasia cases has been the right pars triangularis, located in the
right inferior frontal gyrus (an RH homologue of Broca’s language
area in the LH) [136, 147, 158, 177, 178]. These rTMS treatments
are usually given for 20 min (10 days, 5 days per week). Follow-up
fMRI studies have shown that post-rTMS, there was suppression of
the right frontal areas, and this was associated with improved ability
to name pictures and increased number of words per phrase length
[136], and overall language improvement [158, 178].
The rTMS studies with aphasia patients have been based on
the notion of interhemispheric inhibition, where the overactivation
observed in the RH following LH stroke was associated with less
inhibitory dominance from the injured, dominant LH. Therefore,
parts of the RH became disinhibited and more overactive. Thus,
suppressing a part of the RH was effective to improve language,
beginning with the earliest studies [136, 147]. The role of the RH,
however, is still unclear [128].
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788 Low-Level Laser (Light) Therapy for Rehabilitation in Traumatic Brain Injury

A new type of rTMS, intermittent theta burst (iTBS) with a short,

excitatory stimulation [179] has recently been used to excite the LH
and improve language in chronic aphasia patients who have had
LH stroke [180, 181]. The iTBS treatments consisted of bursts of
three pulses at 50 Hz given every 200 ms in 2 s trains, repeated
every 10 s over 200 s, for a total of 600 pulses. Theta burst was
administered at 80% of motor threshold (10 days, 5 days per week).
An improvement in the white matter integrity (increase in fractional
anisotropy values on DTI, MRI scans) was noted near the stimulation
sites, as well as distal areas [181]. Increased FA [181], corresponded
to perilesional areas that had shown peak activation on semantic
decision fMRI scans [180].
Repetitive TMS has also been used to treat visual neglect and
paralysis following stroke [182, 183]. The role of rTMS and tDCS on
rehabilitation of upper limb and hand paresis has been reviewed
[184], and these authors suggested “a bimodal balance-recovery
model that links interhemispheric balancing and functional recovery
to the structural reserve spared by the lesion.” The noninvasive brain
stimulation would be tailored to the needs of individual patients.

39.5.2 Transcranial Direct Current Stimulation

During the application of tDCS, electrical currents are applied
constantly over a longer period of time than with rTMS, and usually
in the order of minutes, at much lower intensities to achieve
changes in cortical excitability that persist even after stimulation has
ceased. In comparison to TMS, tDCS requires inexpensive hardware
and the procedure is simple. The most important component is
a current generator, which is capable of delivering a constant
electrical current flow of up to 2 mA. The electrical current is
delivered through two sponge electrodes soaked in saline solution.
Typically, these electrodes have a relatively large surface of 20–
35 cm2 that limits the focality of stimulation. However, the large size
keeps current densities low (one of the critical safety parameters).
Nevertheless, subjects may feel a mild tingling or itching sensation
on the scalp beneath the electrodes. Transcranial DCS relies on
the assumption that a weak constant direct current (DC) polarizes
tissue. Stimulation is usually applied for a few minutes (up to
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Other Noninvasive Brain Stimulation Therapies to Treat TBI or Stroke 789

30 min). Depending on the direction of current flow, i.e., polarity,

tDCS can be delivered either as “cathodal, inhibitory” or “anodal,
excitatory.” The use of tDCS for stroke patients has also been
reviewed in the Cochrane Database [185].
Several studies have used tDCS to improve language in chronic
stroke patients with aphasia. Most have applied excitatory anodal
tDCS to the LH [186–188], or inhibitory cathodal tDCS to the RH
[189]. The first tDCS study with chronic aphasia patients, however,
applied cathodal tDCS to the LH and did report improvement in
naming [190].
Some studies have suggested that the relative level of left-
hemispheric dominance for language in an individual person prior
to stroke would also have relevance to variability in recovery
patterns [139]. These authors have suggested that for long-term
recovery, however, RH recruitment may be less efficient than
restoring the LH network. Patients with better recovery have been
observed to have higher activation in specific areas such as the L
superior temporal gyrus and L SMA [149, 157]. It is noteworthy
that in the initial tLED studies in the Naeser lab [153], to treat
chronic stroke patients with LH stroke and aphasia, placing the
LED cluster head at the vertex, stimulating both the R SMA and
the L SMA simultaneously, was not beneficial; in fact, it worsened
naming (this bilateral tLED placement protocol had also placed the
LED cluster heads on the left and the right sides of the head—left
and right perisylvian areas). When the LEDs were placed only on
the left side of the head (left perisylvian areas), improved naming
was observed. Thus, the results from the Naeser et al. [153] study
with tLEDs support the MRI findings that document recovery in
aphasia, i.e., better recovery is associated with increased activation
primarily in the LH perilesional areas only (including the L SMA). In
addition, the results from the Naeser et al. [153] study support the
findings of the rTMS and tDCS studies reviewed earlier with aphasia
patients, suggesting that these other types of noninvasive brain
stimulation are also more effective, when the LH is stimulated with
excitatory stimulation and/or the RH is suppressed with inhibitory
stimulation.
While the noninvasive transcranial treatment methods reviewed
in this chapter (tLLLT, including tLEDs, rTMS, and tDCS; see
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790 Low-Level Laser (Light) Therapy for Rehabilitation in Traumatic Brain Injury

Figure 39.3 Non-invasive brain stimulation modalities. Repetitive transcra-

nial magnetic stimulation (rTMS), transcranial direct current stimulation
(tDCS), and transcranial low-level light therapy (tLLLT) have many features
in common, including the possible stimulation of mitochondria, increased
BDNF, and enhanced neurogenesis and synaptogenesis.

Fig. 39.3) vary in terms of methodology, they all seek to increase

brain function by stimulating neural activity and are thought
to subsequently stimulate neurogenesis (the formation of new
neurons or brain cells) and synaptogenesis (the formation of new
synapses or connections between neurons) in the rehabilitation
of TBI and stroke. Neurogenesis is the generation of neuronal
precursors and birth of new neural cells. Two key sites for adult
human neurogenesis include the subventricular zone of the lateral
ventricles and the subgranular layer of the dentate gyrus in the
hippocampus [191]. “Neurogenesis persists in the adult mammalian
brain, where it can be stimulated by physiological factors, such as
growth factors and environmental enrichment, and by pathological
processes, including ischemia. . . ” [192].
Increased vascular endothelial growth factor (VEGF) has been
associated with enhanced post-ischemic neurogenesis and neuromi-
gration in VEGF-over-expressing transgenic mice, consistent with
a possible role in ischemic brain tissue repair [193]. VEGF could
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Conclusion 791

provide a therapeutic target for more chronic brain repair. NIR

laser therapy has been observed to increase VEGF and angiogenesis
following myocardial infarction in animal studies [194]. Scalp
application of red/NIR LED could stimulate the secretion of VEGF,
thus promoting angiogenesis and neurogenesis.
Brain plasticity has been defined as “the brain’s ability to
undergo functional and structural alterations in response to internal
and external environmental changes” [195]. An example of brain
plasticity in normal adult humans was observed after only five rTMS
sessions to the left superior temporal gyrus, when macroscopic
cortical changes were then observed in Heschl’s gyrus using MRI.
The rTMS was applied at 1 Hz with 2000 pulses per session, at
110% of motor threshold [195]. In another study by this group,
it was observed that after 3 months of learning a new motor task
(juggling), there was transient grey matter increase in extrastriate
motion specific visual area hMT/V5 bilaterally and in the left inferior
parietal sulcus. These changes degraded nearly to baseline 3 months
after the juggling stopped [195–197].
It is believed that increased neuroplasticity is a foundation
for stroke rehabilitation and recovery [184]. Future research
with tLEDs, rTMS, and tDCS should establish the ideal treatment
parameters for any given modality. These modalities would not be
administered at the same time in a stroke patient, but it is possible
that optimal results could be obtained if they were used sequentially,
or in alternate periods of treatment, post-stroke (see Fig. 39.3).

39.6 Conclusion

We believe that we are just at the beginning of the road of discovery

for tLLLT for brain disorders. Clinical testing in patients with chronic
brain damage (TBI and stroke) has just started, but the early results
are highly encouraging. A general recommendation in treating
chronic or acute stroke patients would be to only use LED place-
ments that are ipsilesional to the side of the brain where the stroke
took place, not contralesional or bilateral. When treating TBI cases,
however, bilateral LED placements are highly indicated, because due
to the DAI that occurs with accelerated twisting of the head during
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792 Low-Level Laser (Light) Therapy for Rehabilitation in Traumatic Brain Injury

mTBI, there is bilateral damage. LED treatment protocols for TBI and
stroke cases should include placement over mid-sagittal nodes on
the DMN, such as placement over the center front hairline (to ideally
deliver photons to reach the mesial prefrontal cortex area) and
placement over the mid-sagittal area halfway between the occipital
protuberance and the vertex of the head (to ideally deliver photons
to the precun/PCC portions of the DMN). The large field of neurode-
generative disease is also just beginning to be explored with tLLLT,
including tLED therapies (see also Chapters 13, 27). Considering the
total lack of known side effects, the relatively inexpensive nature of
modern LED light sources, and the ease of home treatment regimens,
we believe that the use of tLLLT will continue to expand [106].

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149. Saur, D., et al., Dynamics of language reorganization after stroke. Brain,
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150. Szaflarski, J.P., et al., Recovered vs. not-recovered from post-stroke
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151. Crosson, B., et al., Regional changes in word-production laterality after
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152. Price, C.J., and J. Crinion, The latest on functional imaging studies of
aphasic stroke. Curr Opin Neurol, 2005. 18(4): pp. 429–434.
153. Naeser, M.A., et al., Improved language after scalp application of
red/near-infrared light-emitting diodes: Pilot study supporting a new,
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804 Low-Level Laser (Light) Therapy for Rehabilitation in Traumatic Brain Injury

noninvasive treatment for chronic aphasia. Procedia Soc Behav Sci,

2012. 61: pp. 138–139.
154. Goodglass, H., and E. Kaplan, Assessment of Aphasia and Related
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155. Roach, A., et al., The Philadelphia Naming Test: Scoring and rationale.
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156. Martin, P.I., et al., Overt naming in aphasia studied with a functional
MRI hemodynamic delay design. Neuroimage, 2005. 28(1): pp. 194–
204.
157. Karbe, H., et al., Brain plasticity in poststroke aphasia: What is the
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215–230.
158. Weiduschat, N., et al., Effects of repetitive transcranial magnetic
stimulation in aphasic stroke: A randomized controlled pilot study.
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159. Winhuisen, L., et al., The right inferior frontal gyrus and poststroke
aphasia: A follow-up investigation. Stroke, 2007. 38(4): pp. 1286–
1292.
160. Kirshner, H.S., Frontotemporal dementia and primary progressive
aphasia: An update. Curr Neurol Neurosci Rep, 2010. 10(6): pp. 504–
511.
161. Grossman, M., Primary progressive aphasia: Clinicopathological corre-
lations. Nat Rev Neurol, 2010. 6(2): pp. 88–97.
162. Reilly, J., et al., Cognition, language, and clinical pathological features
of non-Alzheimer’s dementias: An overview. J Commun Disord, 2010.
43(5): pp. 438–452.
163. Johnson, J.K., et al., Frontotemporal lobar degeneration: demographic
characteristics of 353 patients. Arch Neurol, 2005. 62: pp. 925–930.
164. Rosso, S.M., et al., Frontotemporal dementia in The Netherlands:
Patient characteristics and prevalence estimates from a population-
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165. Mesulam, M.M., Primary progressive aphasia: A 25-year retrospective.
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166. Gorno-Tempini, M.L., et al., Classification of primary progressive
aphasia and its variants. Neurology, 2011. 76(11): pp. 1006–1014.
167. Sapolsky, D., et al., Cortical neuroanatomic correlates of symptom
severity in primary progressive aphasia. Neurology, 2010. 75(4): pp.
358–366.
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168. Forman, M.S., et al., Frontotemporal dementia: Clinicopathological

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169. Lipton, A.M., C.L. White, 3rd, and E.H. Bigio, Frontotemporal lobar de-
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108(5): pp. 379–385.
170. Mann, D.M., et al., Dementia of frontal lobe type: Neuropathology and
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pp. 605–614.
171. Merton, P.A., and H.B. Morton, Stimulation of the cerebral cortex in the
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172. Barker, A.T., R. Jalinous, and I.L. Freeston, Non-invasive magnetic
stimulation of human motor cortex. Lancet, 1985. 1(8437): pp. 1106–
1107.
173. Sparing, R., and F.M. Mottaghy, Noninvasive brain stimulation with
transcranial magnetic or direct current stimulation (TMS/tDCS): From
insights into human memory to therapy of its dysfunction. Methods,
2008. 44(4): pp. 329–337.
174. Pascual-Leone, A., et al., Study and modulation of human cortical
excitability with transcranial magnetic stimulation. J Clin Neurophysiol,
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175. Maeda, F., et al., Modulation of corticospinal excitability by repetitive
transcranial magnetic stimulation. Clin Neurophysiol, 2000. 111(5): pp.
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176. Pascual-Leone, A., et al., Responses to rapid-rate transcranial magnetic
stimulation of the human motor cortex. Brain, 1994. 117(Pt 4): pp.
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177. Hamilton, R.H., et al., Stimulating conversation: Enhancement of
elicited propositional speech in a patient with chronic non-fluent
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113(1): pp. 45–50.
178. Barwood, C.H., et al., Improved language performance subsequent to
low-frequency rTMS in patients with chronic non-fluent aphasia post-
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179. Huang, Y.Z., et al., Theta burst stimulation of the human motor cortex.
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180. Szaflarski, J.P., et al., Excitatory repetitive transcranial magnetic
stimulation induces improvements in chronic post-stroke aphasia.
Med Sci Monit, 2011. 17(3): pp. CR132-9.
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181. Allendorfer, J.B., J.M. Storrs, and J.P. Szaflarski, Changes in white matter
integrity follow excitatory rTMS treatment of post-stroke aphasia.
Restor Neurol Neurosci, 2012. 30(2): pp. 103–113.
182. Lefaucheur, J.P., Stroke recovery can be enhanced by using repetitive
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183. Liew, S.L., et al., Non-invasive brain stimulation in neurorehabilitation:
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184. Di Pino, G., et al., Modulation of brain plasticity in stroke: A novel model
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185. Elsner, B., et al., Transcranial direct current stimulation (tDCS) for
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186. Baker, J.M., C. Rorden, and J. Fridriksson, Using transcranial direct-
current stimulation to treat stroke patients with aphasia. Stroke, 2010.
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187. Fridriksson, J., et al., Transcranial direct current stimulation improves
naming reaction time in fluent aphasia: A double-blind, sham-
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188. Marangolo, P., et al., tDCS over the left inferior frontal cortex improves
speech production in aphasia. Front Hum Neurosci, 2013. 7: pp. 539.
189. Kang, E.K., et al., Improved picture naming in aphasia patients treated
with cathodal tDCS to inhibit the right Broca’s homologue area. Restor
Neurol Neurosci, 2011. 29(3): pp. 141–152.
190. Monti, A., et al., Improved naming after transcranial direct current
stimulation in aphasia. J Neurol Neurosurg Psychiatry, 2008. 79(4): pp.
451–453.
191. Eriksson, P.S., et al., Neurogenesis in the adult human hippocampus.
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192. Jin, K., et al., Evidence for stroke-induced neurogenesis in the human
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193. Wang, Y., et al., VEGF-overexpressing transgenic mice show enhanced
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194. Tuby, H., L. Maltz, and U. Oron, Modulations of VEGF and iNOS in the rat
heart by low level laser therapy are associated with cardioprotection
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688.
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195. May, A., et al., Structural brain alterations following 5 days of

intervention: Dynamic aspects of neuroplasticity. Cereb Cortex, 2007.
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196. Draganski, B., et al., Neuroplasticity: Changes in grey matter induced
by training. Nature, 2004. 427(6972): pp. 311–312.
197. Draganski, B., and A. May, Training-induced structural changes in the
adult human brain. Behav Brain Res, 2008. 192(1): pp. 137–142.
July 6, 2016 17:39 PSP Book - 9in x 6in 40-Hamblin-c40

Chapter 40

Transcranial Near-Infrared Light for

Major Depressive Disorder: Targeting
the Brain Metabolism

Paolo Cassano,a,b Abigail R. Archibald,b and Dan V. Iosifescuc

a Center for Anxiety and Traumatic Stress Disorders, Department of Psychiatry,

Massachusetts General Hospital, 1 Bowdoin Square (6th floor), Boston, MA 02114, USA
b Depression Clinical and Research Program, Massachusetts General Hospital,

1 Bowdoin Square (6th floor), Boston, MA 02114, USA

c Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, PO Box 1230,

NY 10029, USA
cassano@mgh.harvard.edu

40.1 Introduction

In this chapter, we will address the potential use of near-infrared

radiation (NIR) as a future treatment for major depressive disorder
(MDD). The use of near-infrared light as a transcranial treatment
for brain disorders is quite recent, and most of the research in
this arena has been conducted in the past 10 years. There are
currently no psychiatric indications for NIR; at this point, its use
for CNS disorders is strictly experimental. Nonetheless, emerging
preliminary data on the efficacy of NIR in MDD appear to be

Handbook of Low-Level Laser Therapy

Edited by Michael R. Hamblin, Marcelo Victor Pires de Sousa, and Tanupriya Agrawal
Copyright c 2017 Pan Stanford Publishing Pte. Ltd.
ISBN 978-981-4669-60-3 (Hardcover), 978-981-4669-61-0 (eBook)
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July 6, 2016 17:39 PSP Book - 9in x 6in 40-Hamblin-c40

810 Transcranial Near-Infrared Light for Major Depressive Disorder

promising. We will start by describing some of the physical and

biological properties of NIR, with a primary focus on cellular
metabolism (the putative mechanism of its effects in MDD). We will
then review data suggesting MDD may be characterized by abnormal
brain bioenergetic metabolism. Finally, we will examine the clinical
evidence on the effects of NIR on mood and of its efficacy as an
antidepressant. We will first review indirect evidence on the effect
of NIR on mood regulation and then we will discuss studies where
depressed subjects were treated with single or multiple NIR sessions
and with continuous or pulse NIR.

40.2 Transcranial Near-Infrared Light: Biological

Properties and Safety

While light with wavelength lower than the visible spectrum is

potentially harmful due to its high energy (ultraviolet (UV), X-
rays, and γ -rays), electromagnetic waves with higher wavelength
are safely used (micro, radar, TV, radio). Infrared (IR) light is also
ubiquitous on earth. Of the total amount of solar energy reaching
the human skin, 54% is IR and 30% is IR type A, near-infrared
(wavelength range 760–1440 nm) (Kochevar et al., 1999), which
penetrates deeply into the human skin (Schroeder et al., 2010). IR
type A is commonly used to treat a variety of conditions such as
muscle pain, wounds, and migraine (Allais et al., 2003; Chaves et al.,
2014; Ferraresi et al., 2012). IR type A is also used for wellness and
lifestyle purposes such as for cosmetic intervention on peri-orbital
wrinkles (Hersant et al., 2015; Russell et al., 2005). The clinical
use of near-infrared spectroscopy (NIRS) dates from the mid-1980s,
when it was used for monitoring the brain in the neonate and fetus
as well as for monitoring the muscles in adults.
In experimental and animal models, NIR noninvasively delivers
energy to the cytochrome c oxidase; by stimulating the respiratory
chain, NIR leads to increased ATP production (Mochizuki-Oda et al.,
2002; Oron et al., 2007b; Yu et al., 1997). Data suggest that red light
(670 nm) protects the viability of cell culture after oxidative stress,
as indicated by mitochondria membrane potentials (David et al.,
2009). NIR also stimulates neurite outgrowth mediated by nerve
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Transcranial Near-Infrared Light 811

growth factor, and this effect could also have positive implications
for axonal protection (Giuliani et al., 2009). Neuroprotective effects
of red light (670 nm) were documented in in vivo models of
mitochondrial optic neuropathy (Eells et al., 2003; Rojas et al.,
2008). In animal models of traumatic brain injury (TBI), NIR (810
nm) appears to be an effective treatment and improves neurogenesis
(Ando et al., 2011; Oron et al., 2007a; Wu et al., 2012; Xuan et al.,
2013). In addition, NIR improves memory performance in middle-
aged mice (Michalikova et al., 2008). Overall, cellular and animal
studies suggest that NIR increases neurotrophins, neurogenesis,
synaptogenesis, and ATP, while it reduces inflammation, apoptosis,
and oxidative stress (Chung et al., 2012). The aforementioned
biological properties of NIR have led researchers to conduct clinical
experiments using NIR as a potential treatment for specific brain
disorders.
For the transcranial treatment of MDD, researchers have used
both IR light emitting diodes (LEDs) and lasers. A laser (acronym
for light amplification by stimulated emission of radiation) uses
energy (electricity) to excite atoms and then controls the way
these atoms release energy (photons) coherently. Laser light
is monochromatic (a single wavelength), coherent (fixed phase
relationship), directional, and can be visible or “invisible” as in the
case of IR wavelength. Laser devices have been classified in four
classes based on their safety profile: Class I, “fairly safe” if completely
contained (e.g., CD-ROM players/drives); Class II, eye hazard in case
of staring into the beam (e.g., supermarket checkout scanners);
Class III, eye hazard if collected, focused into eye, or the reflected
beam is viewed (e.g., laser pointers and some medical or research
equipment); and Class IV, eye hazard if a direct, reflected, or diffusely
reflected beam is viewed (e.g., research, manufacturing, surgical,
therapeutic use). Laser devices used for the treatment of MDD are
indeed Class IV and have a potential risk for skin and retina hazard
depending on the power level. However, with basic precautions
(such as the use of eye protection with specific lenses and avoiding
reflective surfaces), Class IV lasers are generally safe (Chen et al.,
2013; McCarthy et al., 2010). In three large studies conducted in
1410 stroke patients [NEST-1 (n = 120), NEST-2 (n = 660), NEST-
3 (N = 630)], NIR delivered transcranially with a Class IV laser
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812 Transcranial Near-Infrared Light for Major Depressive Disorder

was safe, with no significant differences in rates of adverse events

between NIR and sham (Hacke et al., 2014; Lampl et al., 2007; Zivin
et al., 2009). To this date, near-infrared transcranial light treatment
has not caused any retinal injury (one of the most likely postulated
adverse events), although care is taken routinely in such studies
to protect the eyes with goggles or eye covers (Kim et al., 2007).
While LEDs are considered safer due to the use of scattered light
and to a more diffuse spreading of their energy in the tissue, their
depth of penetration in the brain tissue has been questioned and
is certainly lower than laser (Tuner and Jenkins, 2012). Moreover,
laser light triggers a potentiating phenomenon, called speckle effect,
when propagating in living tissues, which might further enhance
its biological effects (Tuner and Jenkins, 2012). We will present in
this chapter data on both the use of laser and LED near-infrared
light for the treatment of MDD; due to the paucity of controlled
studies, no comparison between the two modalities of transcranial
treatment in MDD can be made. We will also compare the irradiance
(power density) and the fluence (energy density) of the light used
for transcranial treatment to the natural IR light, to clarify the
magnitude of the exposure in reference to the solar energy. In nearly
all studies and case-reports presented, the NIR irradiance was well
below the safety limit of 320 mW/cm2 set by the American National
Standards Institute (ANSI).

40.3 Depression, Antidepressant Treatment, and Brain

Energy Metabolism

Positron emission tomography (PET) using radioactively labeled

glucose (18F-Fludeoxyglucose: 18F-FDG) allows measurement of
regional brain metabolism. Additionally, magnetic resonance spec-
troscopy (MRS) is an imaging technique that can detect the presence
and abundance of specific molecules within the brain. Multiple PET
and MRS studies have reported regional and global hypometabolism
in MDD, which could be related to the neurobiology of mood
disorders (Iosifescu et al., 2008; Moore et al., 1997; Renshaw et al.,
2001; Volz et al., 1998). FDG-PET studies have shown abnormalities
in glucose use rates and blood flow in several brain regions
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Near-Infrared Light 813

of subjects with major depression (Videbech, 2000). Moreover,

metabolic abnormalities in the anterior cingulate, the amygdala-
hippocampus complex, the dorsolateral prefrontal, and the inferior
parietal cortex seem to improve after antidepressant treatment or
after recovery from depression (Drevets et al., 2002; Kennedy et al.,
2007; Mayberg et al., 2000).
Phosphorus-31 magnetic resonance spectroscopy (31P-MRS)
allows an in vivo measurement of beta-nucleotide triphosphate
(NTP), which largely represents ATP in the brain (Pissarek et al.,
1998), the most important high-energy molecule and a marker of
the cellular energy availability. The brain concentration of ATP is
usually maintained at the expense of phosphocreatine (PCr), due
to the higher phosphate group transfer potential of PCr (Younkin,
1993). In MDD subjects, brain beta-NTP levels were found to
be low (Moore et al., 1997; Renshaw et al., 2001; Volz et al.,
1998). This is also consistent with studies showing a high level
of lactate (a marker of inefficient, anaerobic energy metabolism)
in the brains of MDD subjects (Shungu et al., 2012). Beta-NTP
levels were reported to normalize (i.e., increase) after subjects
responded to antidepressant treatment (Iosifescu et al., 2008).
Iosifescu et al. (2008) demonstrated for the first time with 31P-
MRS a correlation between treatment response to antidepressant
augmentation with triiodothyronine and re-normalization of brain
beta-NTP levels (with compensatory decrease of phosphocreatine).
This study signals a pathway to antidepressant response based on
restoration of a high cellular energy state.

40.4 Near-Infrared Light: Mood Effects in Healthy

Volunteers

In a study from the University of Texas (Austin), Barrett and

Gonzalez-Lima (2013) tested the effect of near-infrared light on
memory, attention, and affect in undergraduates enrolled in an
introductory psychology class. Forty subjects were enrolled in this
double-blind, sham-controlled study. The sham consisted of just
enough light to warm up the skin of the forehead with no significant
irradiation to the brain. The authors delivered one session of near-
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814 Transcranial Near-Infrared Light for Major Depressive Disorder

infrared light to the right forehead, targeting the right frontal pole
(anterior portion of the right prefrontal cortex, Brodmann’s area 9
and 10), using the EEG sites FP2 to center their light source. They
shed low power density light for 4 min at two sites with a Class
IV laser CG-5000 (Cell Gen Therapeutics); the parameters were:
wavelength 1064 nm, irradiance 250 mW/cm2 , fluence 60 J/cm2 ,
time 240 s per site (two sites) (Barrett and Gonzalez-Lima, 2013).
Similar parameters are used clinically with comparable instruments
(by Cell Gen Therapeutics) for the treatment of lower back pain,
sciatica, and migraine headaches. The irradiance (or power density)
was about five times sunlight’s IR irradiance (about 52.7 mW/cm2
at the zenith), and limited to a specific wavelength as opposed
to the sunlight spectrum of IR. The irradiance was roughly twice
the total sun irradiance at ground level (112 mW/cm2 ). It was
estimated that 2% of the near-infrared light passed the cranial
bone and reached the brain. Using a rating scale for positive and
negative affect schedule (PANAS), the authors observed a benefit
lasting 2 weeks in the overall affect of undergraduates treated
with NIR, including improvement in several dimensions related to
depression and anxiety such as being “distressed, irritable, upset,
jittery, nervous, ashamed, afraid and scared.” Undergraduates also
had faster reaction times, measured by the psychomotor vigilance
test (PVT), and improved short-term visual memory 2 weeks after
NIR administration. Reaction time was tested as delay in response to
light signals on the test screen. Short-term visual memory was tested
by the CANTAB Delayed Match to Sample (DMS) test, where the
participant has to recognize a complex visual pattern (the sample),
which they have seen briefly several minutes prior, among four sim-
ilar patterns (Barrett and Gonzalez-Lima, 2013). It is unclear from
the experimental design if the effect on mood was independent from
the cognitive enhancing effect or whether the two were correlated.

40.5 Near-Infrared Light: Effect on Mood in TBI and PTSD

Patients

Two case-reports in patients with TBI [one veteran also affected

by post-traumatic stress disorder (PTSD)] suggested that NIR
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Near-Infrared Light: Effect on Mood in TBI and PTSD Patients 815

(870 nm), administered repeatedly over several months, improved

self-awareness and self-regulation in both social and work envi-
ronments (Naeser et al., 2011). These benefits were attributed to
improved sleep and to reduced impulsivity, irritation, and anger,
consistent with common symptoms of depression and anxiety.
Interestingly, one of the two aforementioned TBI patients treated
with NIR also recovered from a depressive episode complicated
by a suicide attempt. Both patients were treated with a three-
LED-cluster-heads instrument. The NIR parameters used for the
treatment were as follows: wavelengths 870 nm and 633 nm (red
light), irradiance 22.2–25.8 mW/cm2 , fluence 13.3 J/cm2 , and time
10 min per site (Naeser et al., 2011). While the irradiance was
one order of magnitude less compared to the dose used in healthy
volunteers (Barrett and Gonzalez-Lima, 2013), the exposure time
was also about one order of magnitude longer and 3–5 times as
many sites were irradiated. Factoring also the larger surface of
irradiation (greater number of sites), it is likely that the actual
energy delivered to the head of TBI patients by Naeser et al. (2011)
was similar to that delivered to healthy volunteers by Barrett and
Gonzalez-Lima (2013).
The same group recently reported on a cohort of eleven TBI
subjects—eight of whom also had comorbid depression—who were
treated with a very similar NIR protocol (Naeser et al., 2014).
The eight participants were identified as having mild, moderate, or
severe depression based on the Beck Depression Inventory (mean
BDI-II total scores were 22 ± 7.4 SD). All patients were treated with
a three-LED-cluster-heads instrument (MedX Health Model 1100,
Toronto). The light parameters used were as follows: wavelength
870 nm and 633 nm (red light), irradiance 22.2 mW/cm2 , fluence
13 J/cm2 , time 10 min per site. NIR was applied three times per
week for 6 weeks (18 sessions), on 11 sites for 10 min per site (the
total duration of each session was 20 min) (Naeser et al., 2014).
The sites on the skull were chosen on the midline from front to
back, and bilaterally on frontal, parietal, and temporal areas; the
cortical targets included the dorsolateral prefrontal cortex (DLPFC)
and the central executive network. Three subjects (37%) showed
antidepressant response (decrease in BDI-II total score 50% from
baseline) after 6 weeks of NIR treatment (mean BDI-II total scores
July 6, 2016 17:39 PSP Book - 9in x 6in 40-Hamblin-c40

816 Transcranial Near-Infrared Light for Major Depressive Disorder

were 14.8 ± 6.5 SD). Two of them maintained the antidepressant

response after 8 weeks of follow-up (Naeser et al., 2014). The other
five subjects did not respond. Of note, the relatively low response
rate may be related to the comorbid TBI and depression, a condition
where the rates of antidepressant response are generally lower
(Fann et al., 2009).

40.6 Near-Infrared Light For Depression

40.6.1 Near-Infrared Light for Depression and Anxiety:

Single Session
To date, only two preliminary open studies of NIR for the treatment
of MDD have been published. Schiffer et al. (2009) administered
a single NIR session to 10 depressed subjects (mean HAM-D-21
total score 23.9 ± 8.8 SD), 9 with comorbid anxiety disorders
(3 with PTSD). The authors reported good tolerability of NIR and
a significant reduction in anxiety (HAM-A scale) and depression
(HAM-D scale) 2 weeks post-procedure (Schiffer et al., 2009),
which is even more interesting since their subjects were suffering
from resistant depression, where improvement rates have been
traditionally lower (Nierenberg et al., 2007).
All 10 subjects met diagnostic criteria for MDD; comorbid anxiety
disorders were allowed, as well as past history of substance-use
disorder. Resistance to treatment was not exclusionary (4 weeks
of stable therapies were required for inclusion). The treatment
consisted of a single session of NIR at two sites on the forehead,
aiming at the dorsolateral prefrontal cortex (DLPFC on EEG sites
F3 and F4 bilaterally), using an LED instrument (Marubeni America
Corp.) with the following NIR parameters: wavelength 810 nm,
irradiance 250 mW/cm2 , fluence 60 J/cm2 , time 4 min per site
(two sites) (Schiffer et al., 2009). The authors reported that both
depressive and anxiety symptoms (measured with the HAM-D and
HAM-A scales, respectively) were significantly decreased at week
2 and 4 post-treatment. The remission rate of MDD at week 2
(operationalized as HAM-D-21 score <10) was 60%. Four weeks
post-treatment, depressive symptoms were higher than after two
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Near-Infrared Light For Depression 817

weeks, suggesting the duration of the effect of a single treatment

would be less than 4 weeks. These results are very preliminary
given the open and uncontrolled design and the very small sample
size. A placebo-effect could not be assessed and ruled out; however,
placebo-effect tends to be marginal in patients with resistant
depression.

40.6.2 Near-Infrared Light for Depression: Multiple

Sessions
Our group at Massachusetts General Hospital (Boston, MA) has
collected preliminary data on the safety and efficacy of multiple
NIR sessions in MDD patients who exhibited low pretreatment
frontal lobe metabolism, as measured with magnetization trans-
fer phosphorus magnetic resonance spectroscopy (MT-31P-MRS)
(Cassano et al., 2015). We collected data on eight adults with
MDD, with at least moderate depression, and no other comorbid
conditions (except for comorbid anxiety disorders); other inclusion
and exclusion criteria were similar to Schiffer et al. (2009). Data
on only four completers were reported, who completed a 7-week
double-blind treatment, involving 3 weeks of NIR twice a week
followed by cross-over to three more weeks of sham, after a washout
week. At each treatment session, NIR (808 nm) was administered
to the forehead bilaterally at four sites (2 min per site, 12.56 cm2
window each), using a Class IV laser (Photothera Inc.) and the
following treatment parameters: NIR irradiance 700 mW/cm2 and
fluence 84 J/cm2 , for a total NIR energy of 2.40 kJ per session. The
baseline severity of depression was in the moderate range [mean
HAM-D-17 scores = 19.8 ± 4.35 (SD)]. Two of the four subjects
(50%) achieved remission of depressive symptoms, with HAM-D-
17 scores of 4 and 3 at weeks 6 and 7, respectively. At endpoint,
the mean HAM-D-17 total scores was 13 ± 5.35 (SD). Additionally,
suicidal ideation was significantly reduced and remitted in the
aforementioned two subjects (CHRT scale scores decreased from 28
to 5 and from 17 to 2, respectively). The NIR treatment was well
tolerated with only one unrelated adverse event (transient, mild
diarrhea in a subject with irritable bowel syndrome). While our
findings are limited by the very small sample size, they support the
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818 Transcranial Near-Infrared Light for Major Depressive Disorder

hypothesis that NIR could be an effective treatment for MDD and

that its mechanism might be related to correcting brain bioenergetic
deficits (as measured with 31P-MRS).

40.6.3 Near-Infrared Light for Depression: Multiple

Sessions and Pulse Light
While most of the transcranial near-infrared treatment has been
conducted with sources of continuous light, at least one group has
experimented with pulsed light in TBI (Neuro-Laser Foundation in
Denver, CO) (Henderson and Morries, 2015; Morries et al., 2015).
This contribution is potentially relevant to the field, since in animal
models of TBI, it appears that pulse near-infrared light might be
more effective than continuous light (Ando et al., 2011). Morries and
Henderson used a Class IV laser (LiteCure and Diowave) delivering
a dual wave at wavelengths of 980 and 810 nm with 9–13 W of
power. Despite the high power instrument, delivering approximately
double the power used by our group in Boston (MA), Morries
and Henderson did not observe any thermal skin hazard, as the
skin temperature increase was negligible and temperature changes
(checked with a laser thermometer) never exceeded 1◦ C to 3◦ C.
Despite the very high irradiance, nearly two orders of magnitude
above the safety threshold recommended by the ANSI, the reported
fluence (14.8–28.3 J/cm2 ) was comparable with that delivered safely
by the Photothera device for several hundred patients (Lampl et
al., 2007; Zivin et al., 2009, 2012). The high NIR irradiance used by
Morries and Henderson is somewhat mitigated by the pulse source,
which decreases the actual time of exposure to NIR light. The overall
time of exposure of 10 min per hemi-side was twice as long as the
one we used (Cassano et al., 2015) (4 min per hemi-side); however,
Morries and Henderson also used a much larger surface on the
forehead (132 cm2 per hemi-side) while aiming at the frontal cortex,
roughly twice as much as the area of treatment by Cassano et al.
(2015). Morries and Henderson were able to irradiate such a wide
area by using a paced scanning technique (with the light source in
continuous motion over the skin surface), which further explains
why the actual fluence may have reportedly been contained within a
safe range.
July 6, 2016 17:39 PSP Book - 9in x 6in 40-Hamblin-c40

Conclusion 819

All 10 TBI subjects treated in an open pilot study with the

aforementioned methods experienced symptoms of depression
[quick inventory of depressive symptomology (QIDS) mean total
score 12.9 ± 4.6 SD], and six were also diagnosed with MDD. They
underwent 2–3 NIR sessions per week (with at least 1-day interval
between sessions) for a total of 20 sessions over 8 weeks (Morries
et al., 2015). The patients (n = 10) experienced a significant
reduction in depressive symptoms (mean QIDS score 2.2 ± 2.3
SD). Furthermore, all six patients with MDD responded to the
treatment (decrease in QIDS total score ≥50% from baseline) and
five remitted from depression (QIDS total score ≤5). Additionally,
each patient and their partner completed a patient diary and a
spousal-partner diary every week with focus on sleep, self-esteem,
aggression, general health, and anxiety. Patients and their partners
reported resolution of suicidal ideation, irritability, and marital
conflict (Morries et al., 2015). Although they are very preliminary,
these results suggest that repeated administration of Class IV laser
near-infrared light in MDD patients could be safe and effective after
multiple sessions.

40.7 Conclusion

Transcranial NIR treatment appears to be safe and well tolerated.

While no incidents of retinal or skin injury have been documented
in many hundreds of patients treated with transcranial NIR, eye
protections are generally recommended whenever laser devices are
used. The preliminary data reviewed in this chapter suggest that
transcranial NIR could be efficacious for the treatment of depres-
sion, anxiety, and cognitive impairment. The most likely mechanism
by which transcranial NIR might exert an antidepressant effect is
the enhancement of brain bioenergetic metabolism. Controlled trials
testing the antidepressant effect of transcranial NIR in comparison
with sham are necessary to understand the real antidepressant
efficacy and the duration of a possible antidepressant effect.
If transcranial NIR were to be proven safe and effective in large
controlled trials for the treatment of MDD and other psychiatric
disorders, low-cost LED devices (already approved by the FDA for
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820 Transcranial Near-Infrared Light for Major Depressive Disorder

unsupervised use) could be immediately available for clinical off-

label practice and might have significant advantages (cost, side
effects, ability to use at home) over traditional treatments.

References

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Comparison of therapeutic effects between pulsed and continuous
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in mice, Plos One, 6, E26212.
Allais, G., De Lorenzo, C., Quirico, P. E., Lupi, G., Airola, G., Mana, O., et al.
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scutaneous electrical nerve stimulation, laser therapy and acupuncture
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Barrett, D. W., and Gonzalez-Lima, F. 2013. Transcranial infrared laser
stimulation produces beneficial cognitive and emotional effects in
humans, Neuroscience, 230, 13–23.
Cassano, P., Cusin, C., Mischoulon, D., Hamblin, M. R., De Taboada, L.,
Pisoni, A., et al. 2015. Near-infrared transcranial radiation for major
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Chaves, M. E. D. A., Araújo, A. R. D., Piancastelli, A. C. C., and Pinotti, M.
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Chen, Y., De Taboada, L., O’connor, M., Delapp, S., and Zivin, J. A. 2013.
Thermal effects of transcranial near-infrared laser irradiation on rabbit
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Chung, H., Dai, T., Sharma, S. K., Huang, Y. Y., Carroll, J. D., and Hamblin, M. R.
2012. The nuts and bolts of low-level laser (light) therapy, Ann Biomed
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David, D. J., Samuels, B. A., Rainer, Q., Wang, J. W., Marsteller, D., Mendez,
I., et al. 2009. Neurogenesis-dependent and -independent effects of
fluoxetine in an animal model of anxiety/depression, Neuron, 62, 479–
493.
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Kane, M., et al. 2003. Therapeutic photobiomodulation for methanol-
induced retinal toxicity. Proc Natl Acad Sci USA, 100, 3439–3444.
Fann, J. R., Hart, T., and Schomer, K. G. 2009. Treatment for depression after
traumatic brain injury: A systematic review, J Neurotrauma, 26, 2383–
2402.
Ferraresi, C., Hamblin, M. R., and Parizotto, N. A. 2012. Low-level laser (light)
therapy (LLLT) on muscle tissue: Performance, fatigue and repair
benefited by the power of light, Photonics Lasers Med, 1, 267–286.
Giuliani, A., Lorenzini, L., Gallamini, M., Massella, A., Giardino, L., and Calza,
L. 2009. Low infrared laser light irradiation on cultured neural cells:
Effects on mitochondria and cell viability after oxidative stress, Bmc
Complement Altern Med, 9, 8.
Hacke, W., Schellinger, P. D., Albers, G. W., Bornstein, N. M., Dahlof, B.
L., Fulton, R., et al. 2014. Transcranial laser therapy in acute stroke
treatment results of Neurothera Effectiveness and Safety Trial 3: A
Phase III clinical end point device trial, Stroke, 4511, 3187–3193.
Henderson, T. A., and Morries, L. D. 2015. SPECT perfusion imaging
demonstrates improvement of TBI with transcranial near-infrared
laser phototherapy, Adv Mind Body Med, 29, 27–33.
Hersant, B., Sidahmed-Mezi, M., Bosc, R., and Meningaud, J. P. 2015.
Current indications of low-level laser therapy in plastic surgery: A
review, Photomed Laser Surg, 33, 283–297.
Iosifescu, D. V., Bolo, N. R., Nierenberg, A. A., Jensen, J. E., Fava, M., and
Renshaw, P. F. 2008. Brain bioenergetics and response to triiodothy-
ronine augmentation in major depressive disorder, Biol Psychiatry, 63,
1127–1134.
Kennedy, S. H., Konarski, J. Z., Segal, Z. V., Lau, M. A., Bieling, P. J., Mcintyre,
R. S., et al. 2007. Differences in brain glucose metabolism between
responders to CBT and venlafaxine in a 16-week randomized controlled
trial, Am J Psychiatry, 164, 778–788.
Kim, E. A., Kim, B. G., Yi, C. H., Kim, I. G., Chae, C. H., and Kang, S. K. 2007.
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Mayberg, H. S., Brannan, S. K., Tekell, J. L., Silva, J. A., Mahurin, R. K.,
Mcginnis, S. et al. 2000. Regional metabolic effects of fluoxetine in major
depression: Serial changes and relationship to clinical response, Biol
Psychiatry, 48, 830–843.
Mccarthy, T. J., De Taboada, L., Hildebrandt, P. K., Ziemer, E. L., Richieri, S. P.,
and Streeter, J. 2010. Long-term safety of single and multiple infrared
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Emotional responses and memory performance of middle-aged Cd1
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89, 480–488.
Mochizuki-Oda, N., Kataoka, Y., Cui, Y., Yamada, H., Heya, M., and Awazu,
K. 2002. Effects of near-infrared laser irradiation on adenosine
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Lower levels of nucleoside triphosphate in the basal ganglia of de-
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Morries, L. D., Cassano, P., and Henderson, T. A. 2015. Treatments for
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laser phototherapy, Neuropsychiatr Dis Treat, 11, 2159–2175.
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J. A. 2011. Improved cognitive function after transcranial, light-emitting
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Photomed Laser Surg, 29, 351–328.
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M. R., et al. 2014. Significant improvements in cognitive perfor-
mance post-transcranial, red/near-infrared light-emitting diode treat-
ments in chronic, mild traumatic brain injury: Open-protocol study,
J Neurotrauma, 31, 1008–1017.
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Psychiatr Clin North Am, 30, 13–29.
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mice following traumatic brain injury significantly reduces long-term

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July 6, 2016 17:39 PSP Book - 9in x 6in 41-Hamblin-c41

Chapter 41

Low-Level Laser Therapy: A Corner Stone

in the Management of Cancer
Therapy–Induced Mucositis

René-Jean Bensadoun,a Idriss Troussier,b and Raj G. Nairc,d

a Radiation Oncology, Centre de Haute Energie (CHE), 10 Boulevard Pasteur,

06000 Nice, France

b Radiation Oncology Department, CHU de Poitiers, 2 Rue de la Miltrie,

86021 Poitiers, France

c Oral Medicine, Oral Pathology and Human Diseases, School of Dentistry and Oral

Health, Menzies Health Institute, Gold Coast campus, Griffith University,

Queensland 4222, Australia
d Haematology and Oncology, Gold Coast University Hospital, Queensland Health,

Australia
renejean.bensadoun@che-nice.com

41.1 Introduction

Mucositis is recognized as one of the principal dose-limiting factors

during 5FU-based chemotherapy, or treatment with several new
targeted agents. It is also one of the main intensity-limiting acute
toxicities during radiotherapy and radio-chemotherapy for head and
neck cancer, and during hematopoietic cell transplant conditioning.

Handbook of Low-Level Laser Therapy

Edited by Michael R. Hamblin, Marcelo Victor Pires de Sousa, and Tanupriya Agrawal
Copyright c 2017 Pan Stanford Publishing Pte. Ltd.
ISBN 978-981-4669-60-3 (Hardcover), 978-981-4669-61-0 (eBook)
www.panstanford.com
July 6, 2016 17:39 PSP Book - 9in x 6in 41-Hamblin-c41

826 Low-Level Laser Therapy

41.2 What is Mucositis?

Pathologic evaluation of mucositis reveals mucosal thinning leading

to a shallow ulcer thought to be caused by inflammation and
depletion of the epithelial basal layer with subsequent denudation
and bacterial infection. The wound-healing response to this injury is
characterized by inflammatory cell infiltration, interstitial exudate,
fibrin and cell debris producing a “pseudomembrane” analogous to
the eschar of a superficial skin wound [1].

41.3 Low-Level Laser Therapy

“Low” or “low and middle” energy (output power ranged from

5–200 mW) irradiation with He/Ne laser (wavelength 632.8 nm)
or diodes laser (wavelength 630–680 nm, or 780–830 nm) has
been reported to be a simple atraumatic technique (with no
known toxicity in clinical setting), useful in the treatment of
mucositis of various origins. Healing of chronic wounds (skin
or ligaments) and activation of post-surgical healing are some
other potential indications. Irradiation by low-level laser therapy
(LLLT) corresponds to a local application of a high photon density
monochromatic light source. LLLT effects have been confirmed
by numerous in vitro studies; they are influenced by cell type,
laser wavelength, and energy dose. Three main effects are sug-
gested for this type of radiation (with adequate energy rate or
fluence on the target): (1) analgesic effect (λ = 630–650 nm,
λ = 780–900 nm), (2) anti-inflammatory effect (same wavelengths),
and (3) wound-healing effect (proved for He/Ne laser: λ = 632.8 nm;
and suggested for λ = 780–805 nm). All these effects have been
assessed by physical, biological, and experimental studies [1]. The
mechanism of action of the healing effect at molecular and enzy-
matic levels consists mainly of the activation of energy production
in mitochondria (ATP level) or photobiomodulation (PBM).
During oncological treatments, detoxification of free radicals
and/or reduction in free radical formation, induced by chemo- and
radiotherapy are complementary effects (currently being studied
July 6, 2016 17:39 PSP Book - 9in x 6in 41-Hamblin-c41

Clinical Trials 827

by several research teams). The preventive or prophylactic effect of

LLLT raises a lot of interest but needs more experimental data to be
confirmed.

41.4 Clinical Trials

In clinical practice, LLLT (or PBM) with 10–150 mW He/Ne or

diode laser was first reported effective in reducing the severity of
oral mucositis lesions in a non-randomized trial, initiated in Nice
(France) by Ciais et al. in 1985 [2]. Then, Schubert et al. from Seattle
[3] identified a trend toward lower oral mucositis scores, on all
examination days, in a phase I/II study, in which laser application
was performed prophylactically during conditioning before bone
marrow transplant. The efficacy of this method in the prevention
of chemotherapy-induced oral mucositis has been subsequently
confirmed by Cowen et al. [4] in a prospective, double-blind
randomized trial, in patients undergoing bone marrow transplant.
In this study, He/Ne laser was administered to the treatment group
during conditioning (5 days), prior to the day of transplant. It
showed a 33% reduction in grades 3 and 4 mucositis in laser-treated
patients.
High incidence of radiation-induced mucositis prompted a
randomized multicenter trial in France in 1994 to evaluate LLLT for
the prevention of acute radiation-induced oropharyngeal mucosal
lesions [5]. In this phase III randomized study (testing 60 mW, 632.8
nm He/Ne laser), the main objective was to determine whether
preventive He/Ne laser beam applications could reduce or prevent
oropharyngeal mucositis caused by radiotherapy. Daily oral hygiene
(cleaning of the teeth and dental prosthesis) during treatment was
recommended. Patients were assigned to either laser treatment
(L+) or sham-treatment (L−) by computer-blocked randomization.
Analgesics were authorized but not during the 2 days preceding
each week evaluation. Laser was delivered to the tissues by a
straight optical fiber with a 1.2 mm spot size. The nine treatment
areas, each one being a 1 cm2 surface, included posterior third
of buccal mucosa, soft palate, and anterior tonsillar pillars. Laser
July 6, 2016 17:39 PSP Book - 9in x 6in 41-Hamblin-c41

828 Low-Level Laser Therapy

illumination consisted of a continuous beam calibrated at the end

of the optical fiber every day. The treatment time (t) for each
application point was given by the equation t (s) = energy (J/cm2 )
× surface (cm2 )/ power (W). The average energy density delivered
to the treatment areas was 2 J/cm2 . All laser illuminations were
performed by the same individual in each center. This operator
was the only person to know whether or not the patient was
sham-treated, and did not participate in the evaluation and scoring
of mucositis. The whole irradiation field, the oral cavity, and the
visible oropharynx were inspected weekly during 7 weeks by one
specific physician (head and neck surgeon, or radiation oncologist),
blinded to the result of randomization. The evaluation and scoring
of mucositis and pain was performed on the zone of interest
of the study (posterior oropharynx). Criteria for evaluation were
the standard WHO scale for mucositis in the oropharynx, and a
segmented visual analogic scale for pain (patient self-evaluation). In
this “radiotherapy” study that we had the opportunity to coordinate,
laser applications delayed the time of onset, attenuated the peak
severity, and shortened the duration of oral mucositis. Regarding
the degree of objective mucositis, the difference between laser-
treated and nonlaser-treated patients was statistically significant
from week 4 to week 7. Results on decrease in pain intensity
were quite convincing too. Laser applications reduced the incidence
and duration of morphine administration and improved ability to
swallow.
LLLT lead to the optimization of treatment in those different
clinical situations, a tremendous potential for combined treatment
of advanced head and neck cancer with concomitant chemotherapy
and radiotherapy.
Complementary phase III randomized multicenter double-blind
studies, conducted in Europe and America for the same type of
patients, confirmed these results, both for the decrease in mucositis
severity and for pain relief, using 632.8–830 nm laser devices
(He/Ne or diodes) [6–14, 24, 25]. All these studies confirmed the
efficacy of LLLT in the reduction in maximal grade of mucositis,
reduction in the duration of mucositis, oral pain and delay in the
onset of mucositis.
July 6, 2016 17:39 PSP Book - 9in x 6in 41-Hamblin-c41

Conclusion 829

41.5 Recommendations and Future Directions

The Multinational Association of Supportive Care in Cancer (MASCC)

and the International Society of Oral Oncology (ISOO) in 2000
commenced a “Mucositis Consensus Conference,” with complete lit-
erature review to draw guidelines for evidence-based management
of oral mucositis during chemotherapy and radiotherapy. In addition
to basic oral care, bland oral rinses, mucosal coating agents, topical
anesthetics, analgesics, anti-inflammatory agents, antibiotics, antivi-
rals, antifungals, growth factors, cytokines, amifostine, cryotherapy,
enteral nutritional support, and the place of LLLT was addressed,
considering encouraging results [15–23].
In 2014, MASCC–ISOO guidelines was published with a recom-
mendation to use LLLT in preventing oral mucositis in patients
receiving HSCT conditioned with high-dose chemotherapy, with or
without total body irradiation and a ‘suggestion’ for prevention
of oral mucositis in patients undergoing radiotherapy without
concomitant chemotherapy, for head and neck cancer [29]. In 2011,
a new National French cooperative study (12 centers) begun, for
the prevention of mucositis in patients treated with chemora-
diation for head and neck cancer with anticipated confirmatory
results.
A meta-analysis was published by Bjordal et al. in 2012 [26].
This systematic review has revealed evidence for the use of LLLT in
cancer therapy-induced oral mucositis. The evidence behind LLLT
is characterized as promising, but conflicting with large operator
variability. However, this analysis and others [27, 28] showed that
fairly inexpensive lasers (from $2500) with low optical outputs can
be used successfully.

41.6 Conclusion

Finally, LLLT (or PBM) appears to be an efficient and inexpensive

method to control pain and mucositis in head and neck cancer
patients. Moreover, promising results are obtained for other cancer-
therapy side effects, such as cutaneous toxicities.
July 6, 2016 17:39 PSP Book - 9in x 6in 41-Hamblin-c41

830 Low-Level Laser Therapy

References

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Chemotherapy- and radiotherapy-induced mucositis in head and neck
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PM, Soudry M, Franquin JC, and Zattara H. (1992). La laserthérapie dans
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(1994). Effects of low-energy laser on oral mucositis: A phase I/II pilot
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4. Cowen D, Tardieu C, Schubert M, Peterson D, Resbeut M, Faucher C, and
Franquin JC. (1997). Low energy helium-neon laser in the prevention of
oral mucositis in patients undergoing bone marrow transplant: Results
of a double blind randomized trial. Int. J. Radiat Oncol Biol Phys 38(4):
697–703.
5. Bensadoun RJ, Franquin JC, Ciais G, Darcourt V, Schubert MM, Viot M,
Dejou J, Tardieu C, Benezery K, Nguyen TD, Laudoyer Y, Dassonville O,
Poissonnet G, Vallicioni J, Thyss A, Hamdi M, Chauvel P, and Demard F.
(1999). Low-energy He/Ne laser in the prevention of radiation-induced
mucositis. A multicenter phase III randomized study in patients with
head and neck cancer. Support Care Cancer 7: 244–252.
6. Arun Maiya G, Sagar MS, and Fernandes D. (2006). Effect of lowlevel
helium-neon (He-Ne) laser therapy in the prevention and treatment of
radiation induced mucositis in head and neck cancer patients. Indian J
Med Res 124(4): 375–378.
7. Arora H, Pai KM, Maiya A, Vidyasagar MS, and Rajeev A. (2008). Efficacy
of He-Ne laser in the prevention and treatment of radiotherapy induced
oral mucositis in oral cancer patients. Oral Surg Oral Med Oral Pathol
Oral Radiol Endod 105(2): 180–186.
8. Schubert MM, Eduardo FP, Guthrie KA, Franquin JC, Bensadoun RJ,
Migliorati CA, Lloid CM, Eduardo CP, Walter NF, Marques MM, and Hamdi
M. (2007). A phase III randomized double-blind placebo-controlled
clinical trial to determine the efficacy of lowlevel laser therapy for the
prevention of oral mucositis in patients undergoing hematopoietic cell
transplantation. Support Care Cancer 15(10): 1145–1154.
9. Bensadoun RJ, Le Page F, Darcourt V, Bensadoun F, Ciais G, Rostom
YA, Poissonnet G, Dassonville O, Demard F, and MASCC/ISOO Mucositis
July 6, 2016 17:39 PSP Book - 9in x 6in 41-Hamblin-c41

References 831

Group (2006). Radiation-induced mucositis of the aerodigestive tract:

Prevention and treatment. MASCC/ISOO mucositis group’s recommen-
dations. Bull Cancer 93(2): 201–211.
10. Migliorati CA, Massumoto C, Eduardo FP, Muller KP, Carrieri T, Haypek
P, and Eduardo CP. (2001). Low-energy laser therapy in oral mucositis. J
Oral Laser Appl 1: 97–101.
11. Bensadoun RJ and Ciais G. (2002). Radiation and chemotherapy-induced
mucositis in oncology: Results of multicenter phase III studies. J. Oral
Laser App 2: 115–120.
12. Wong SF and Wilder-Smith P. (2002). Pilot study of laser effects on oral
mucositis in patients receiving chemotherapy. Cancer J 8(3): 247–254.
13. Nes AG and Posso MB. (2005). Patients with moderate chemotherapy-
induced mucositis: Pain therapy using low intensity lasers. Int Nurs Rev
52: 68–72.
14. Genot MT and Klastersky J. (2005). Low-level laser for prevention and
therapy of oral mucositis induced by chemotherapy or radiotherapy.
Curr Opinion Oncol 17: 236–240.
15. Braverman B and McCarthy R. (1989). Effect of HeNe and infrared laser
irradiation on wound healing in rabbits. Laser Surg Med 9: 50–58.
16. Boratoletto R, Silva NS, Zangaro RA, Pacheco MT, Da Matta RA, and
Pacheco-Soares C. (2004). Mitochondrial membrane potential after low-
power laser irradiation. Lasers Med Sci 18: 204–206.
17. Sonis ST (2002). The biologic role for nuclear factor-kappaB in disease
and its potential involvement in mucosal injury associated with anti-
neoplastic therapy. Crit Rev Oral Biol Med 13(5): 380–389.
18. Sonis ST, Scherer J, Phelan S, Lucey CA, Barron JE, O’Donnell KE, Brennan
RJ, Pan H, Busse P, and Haley JD. (2002). The gene expression sequence
of radiated mucosa in an animal mucositis model. Cell Prolif 35(1): 93–
102.
19. Pereira AN, Eduardo CP, Matson E, and Marques MM. (2002). Effect of
low-power laser irradiation on cell growth and procollagen synthesis of
cultured fibroblasts. Lasers Surg Med 31: 263–267.
20. Gam AN, Thorsen H, and Lonnberg F. (1993). The effects of lowlevel
laser therapy on musculoskeletal pain: A metaanalysis. Pain 52: 63–66.
21. Whelan HT, Connelly JF, Hodgson BD, Barbeau L, Post AC, Bullard G,
Buchmann EV, Kane M, Whelan NT, Warwick A, and Margolis D. (2002).
NASA light-emitting diodes for the prevention of oral mucositis in
pediatric bone marrow transplant patients. J Clin Laser Med Surg 20:
319–324.
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832 Low-Level Laser Therapy

22. Rubenstein EB, Peterson DE, Schubert M, Keefe D, McGuire D, Epstein

J, Elting LS, Fox PC, Cooksley C, and Sonis ST. (2004). Clinical practice
guidelines for the prevention and treatment of cancer therapy-induced
oral and gastrointestinal mucositis. Cancer 100(supp9): 2026–2046.
23. Sonis ST, Elting LS, Keefe D, Peterson DE, Schubert M, Hauer-
Jensen M, Bekele N, Raber-Durlacher J, Donnellly JP, Rubenstein EB,
Mucositis Study Section of the Multinational Association for Supportive
Care in Cancer, and International Society for Oral Oncology. (2004).
Perspectives on cancer therapy-induced mucosal injury: Pathogenesis,
measurement, epidemiology, and consequences for patients. Cancer
100(supp 9): 1995–2025.
24. Antunes HS, de Azevedo AM, da Silva Bouzas LF, Adão CA, Pinheiro CT,
Mayhe R, Pinheiro LH, Azevedo R, D’Aiuto de Matos V, Rodrigues PC,
Small IA, Zangaro RA, and Ferreira CG. (2007). Low power laser in the
prevention of induced oral mucositis in bone marrow transplantation
patients: A randomized trial. Blood 109(5): 2250–2255.
25. Kuhn A, Antola Porto F, Miraglia P, and Lunardi Brunetto A. (2009). Low-
level infrared laser therapy in chemotherapy-induced oral mucositis. A
randomized placebo-controlled trial in children. J Pediatr Hematol Oncol
31(1): 33–37.
26. Bjordal JM, Bensadoun RJ, Tunèr J, Frigo L, Gjerde K, and Lopes-Martins
RA. (2011). A systematic review with meta-analysis of the effect of low-
level laser therapy (LLLT) in cancer therapy-induced oral mucositis.
Support Care Cancer 19(8): 1069–1077.
27. Epstein JB, Thariat J, Bensadoun RJ, Barasch A, Murphy BA, Kolnick L,
Popplewell L, and Maghami E. (2012). Oral complications of cancer and
cancer therapy: From cancer treatment to survivorship. CA Cancer J Clin
62(6): 400–422.
28. Bensadoun RJ and Nair RG. (2012). Low-level laser therapy in the
prevention and treatment of cancer therapy-induced mucositis: 2012
state of the art based on literature review and meta-analysis. Curr Opin
Oncol 24(4): 363–370.
29. Lalla RV, Bowen J, Barasch A, Elting L, Epstein J, Keefe DM, McGuire DB,
Migliorati C, Nicolatou-Galitis O, Peterson DE, Raber-Durlacher JE, Sonis
ST, Elad S; Mucositis Guidelines Leadership Group of the Multinational
Association of Supportive Care in Cancer and International Society of
Oral Oncology (MASCC/ISOO) (2015). MASCC/ISOO clinical practice
guidelines for the management of mucositis secondary to cancer
therapy. Cancer 120(10): 1453–1461.
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Chapter 42

Photobiomodulation in Dentistry:
Manipulating Biostimulation and
Bioinhibition for Clinical Success

Gerry Rossa and Alana Rossb

a Private Practice, 22 Queen Street North, Tottenham, Ontario L0G 1W0, Canada
b Private Practice, 1440 Don Mills Road, Toronto, Ontario M3B 3P9, Canada

ddsross@rogers.com

Since the conceptualization of using laser sources as a therapeutic

device in the late 1960s, cellular, animal, and human clinical
trials have investigated the mechanisms by which clinical effects
are achieved as well as the ever-expanding clinical applications.
From a clinical perspective, photobiomodulation (PBM) offers
dental practitioners a treatment modality that can be used as an
adjunct to traditional methodologies or as a therapeutic tool on
its own. Applications such as post-extraction healing, reduction
of edema and muscle trismus, dental analgesia, treatment of
dentin hypersensitivity, soft tissue healing, and osseointegration
of implants have been studied in numerous in vivo and in vitro
studies, which have helped identify specific wavelengths and energy
densities for optimal clinical outcomes. PBM is easy to apply by the
dental auxiliary staff (in most jurisdictions) and is well tolerated by
all ages and conditions, making it well accepted by patients. This

Handbook of Low-Level Laser Therapy

Edited by Michael R. Hamblin, Marcelo Victor Pires de Sousa, and Tanupriya Agrawal
Copyright c 2017 Pan Stanford Publishing Pte. Ltd.
ISBN 978-981-4669-60-3 (Hardcover), 978-981-4669-61-0 (eBook)
www.panstanford.com
July 6, 2016 17:39 PSP Book - 9in x 6in 42-Hamblin-c42

834 Photobiomodulation in Dentistry

section will investigate the evidence-based applications of PBM in

a dental clinic, with reference to a number of clinical case studies.

42.1 Introduction

Modern concepts of dentistry today are based on minimally invasive

procedures. Laser therapy using surgical lasers are commonplace
in most dental clinics and allow practitioners to remove soft and
hard tissue with precision and negligible pain. PBM is a subset
of laser therapy that is making its way into dental clinics with
accelerated frequency, supported by extensive cellular and clinical
research. There is virtually no procedure in dentistry that would not
respond positively to PBM; using a drill to prepare a restoration or
an instrument for mechanical debridement starts an inflammatory
response within the tissue and pulp. While important for the release
of growth factors and removing damaged tissues, it also can result
in pain, rubor, and edema. PBM helps modulate the inflammatory
response while reducing pain, making it a tool in every dental
practitioner’s armamentarium that can both ease the stress of the
practitioner and improve the patient experience. PBM is easy to
apply by the dental auxiliary staff (in most jurisdictions) and is well
tolerated by all ages and conditions, making it well accepted by
patients.
From a clinical perspective, PBM offers dental practitioners a
nonthermal and noninvasive treatment modality that can be used
as an adjunct to traditional methodologies or as a therapeutic
tool on its own. Applications such as post-extraction healing,
reduction of edema and muscle trismus, dental analgesia, treatment

Table 42.1 Lasers used in dentistry

Laser Response Absorbing molecule Main application

Hard tissue Photothermal or Water or Removal of tooth

photoaccoustic hydroxyapatite structure and bone
Soft tissue Photothermal Various soft tissues Removal of soft tissue
Low-level laser Photobiomodulation Cytochrome c oxidase Tissue modulation
Photodynamic therapy Photochemical Photosensitizer Bacteria eradication
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Introduction 835

of dentin hypersensitivity, soft tissue healing, and osseointegration

of implants have been studied in numerous in vivo and in vitro
studies, which have helped identify specific wavelengths and energy
densities for optimal clinical outcomes.

Table 42.2 Surgical and photobiomodulatory lasers

Surgical laser Photobiomodulatory laser

Power 1–10 W 10–500 mW

Tip diameter 300–600 μ m 1 cm
Heat generated Yes >0.1◦ C

42.1.1 Keys to Successful Use of PBM in Dentistry

(1) Understand the primary, secondary, and tertiary mechanisms
of PBM. This handbook provides the fundamentals of PBM
that will enable practitioners to improve dental treatments by
understanding the effect of light on tissues, nerve conduction,
inflammatory mediators, and pain pathways.
(2) Understand the effect of light on the lymphatic and circulatory
systems; these pathways make almost every area of the body
accessible to light, directly or indirectly.
(3) Understand the principles of biostimulation and bioinhibition,
or the biphasic response, which allows energy density to be
tailored to the desired physiological response by the cell. The
utilization of the principles of the biphasic response in dental
applications is elucidated in Section 42.2.

42.1.2 Determining the Appropriate Dose

Treatment dose, or energy density, is probably the most important
variable in PBM treatments. Energy density is measured in joules
per square centimeter (J/cm2 ) and is the amount of energy delivered
into the tissue. Clinicians should apply the optimal dose for each
application to maximize the clinical efficacy of PBM. Cellular
responses that lead to wound healing, pain relief, and muscle
relaxation are all sensitive to varying irradiances. Unlike many
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836 Photobiomodulation in Dentistry

Table 42.3 Secondary effects of PBM and dental implications

Effect Relation to dentistry

Increased lymphatic flow Reduces edema and stimulates the immune
response
Production of endogenous opioids Pain relief
(β-endorphins)
Decreased nerve conduction and increased Analgesia
neural latency of C- and Aδ-fibers
Increased collateral circulation and Assists in soft tissue healing and reduces
microcirculation edema
Increased collagen formation and fibroblast Soft tissue healing
stimulation
Stimulation of osteoblasts and odontoblasts Stimulates bone and dentin formation
Reduction in the release of pro-inflammatory Modulates the inflammatory response and
mediators (histamine bradykinins, substance reduces hyperalgesia
P, and acetylcholine)
Increased release of anti-inflammatory Modulates the inflammatory response and
mediators (cytokines) reduces hyperalgesia
Increased migration of leukocytes Metabolizes and necrotizes tissue and
releases opioids

pharmaceuticals, “overdosing” is unlikely to cause any negative

effects but may reduce the efficacy of PBM.
The biomodulatory effects of PBM are governed by the Arndt–
Schultz law, which dictates that cells will respond differently to
varying levels of irradiance (mW/cm2 ) or radiant exposure (J/cm2 ).
Light stimulus will be insufficient to trigger a cellular function if it is
delivered below the recommended dose and will inhibit activation
of these responses if a dose higher than indicated is delivered [1].
For applications in which biostimulation is desired (e.g., stimu-
lation of fibroblasts or osteoblasts), a lower dose is recommended.
Bioinhibitory applications (e.g., reduction of nerve conduction or
inflammatory mediators) require a higher dose.
Irradiances may need to be modified as the treatment progresses
and the clinical outcomes change. An example of this is during im-
plant integration; initially a higher energy density is recommended
to reduce the acute inflammatory response and hyperalgesia. In
the days following, a lower energy density is needed to stimulate
fibroblasts and osteoblasts for improved soft tissue healing and
osseointegration.
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Dental Procedures Using Laser Therapy 837

Research Highlight
Abramese et al. investigated the response of human skin
fibroblasts using a 632 nm He–Ne laser device. Fibroblasts
irradiated with 2.5 J/cm2 or 5.0 J/cm2 responded with stimula-
tory morphological changes, whereas irradiation with 16 J/cm2
inhibited cell migration [2].
A 2006 study by Goulart et al. demonstrated a similar cellular
response on osteoblasts irradiated with 780 nm GaAlAs laser.
PBM accelerated orthodontic movement at a dose of 5.25 J/cm2 ,
whereas a bioinhibitory dosage of 35.0 J/cm2 impeded it [3].

42.2 Dental Procedures Using Laser Therapy

42.2.1 Surgical Extractions

Hyperalgesia (pain) occurs after surgical extractions as a result
of surgical trauma and the release of inflammatory mediators
(bradykinins, substance P, acetylcholine, and prostaglandin). Tra-
ditional pain modulation is achieved through non-steroidal anti-
inflammatory drugs (NSAIDs) and opioids, which are moderately
effective but may in turn result in a number of side effects, including
gastrointestinal irritation, systemic bleeding tendency, decreased
healing, and allergic reactions [4].
While useful after simple extractions, PBM also exhibits signifi-
cant success after difficult extractions and the removal of the lower-
third molars. These procedures are usually followed by hyperalgesia,
swelling, and bruising, which can last 1–5 days after the procedure.

Figure 42.1 Irradiation of a suture line.

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838 Photobiomodulation in Dentistry

Figure 42.2 Application in an extraction socket.

Reduction in pain is the primary indication immediately fol-

lowing dental surgery; therefore, a bioinhibitory dose is used
immediately and preferably directly into the socket. A single
application of laser is customarily all that is necessary and reduces
the need for postoperative pharmaceuticals (Figs. 42.1 and 42.2).
In cases in which a soft tissue flap is involved in the procedure, a
biostimulator is applied in an overlapping method after suturing to
stimulate the fibroblasts and obtain faster wound healing [5].
Post-surgical healing is dramatically improved with PBM by
stimulation of human gingival fibroblasts (HGFs) and osteoblasts.
This intervention improves soft tissue healing and maintains the
integrity of the bone under the extraction site. In addition, an
increase in vascularization results in angiogenesis (blood vessel
formation) and stimulation of endothelial proliferation, both of
which significantly reduce healing time [6, 7].

Research Highlight
A 2009 study by Aras and Gungormus studied the effect of PBM
on trismus and facial swelling following surgical extraction of
the third molar and found that both the swelling and trismus
were significantly less than in the placebo group on both day 2
and 8 [8].
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Dental Procedures Using Laser Therapy 839

42.2.2 Alveolar Osteitis (Dry Socket)

Alveolar osteitis is a painful condition that affects approximately
2% to 5% of people after surgical extractions, primarily in those
who smoke immediately after the procedure [9]. When a tooth is
extracted, a blood clot forms in the socket to protect the bone and
underlying nerves. In some circumstances, the clot can dislodge
or dissolve leaving the bone and nerve exposed to air, food, or
fluid in the oral cavity. Patients with alveolar osteitis present with
excruciating pain and often the early signs of dental infection. The
traditional methodology for treating alveolar osteitis is NSAIDS and
a daily dressing application that helps protect the nerve and bone
while the clot and endothelial lining are repaired.
The prevalence of alveolar osteitis is dramatically reduced
with laser irradiation after surgical extractions as a result of the
stimulation of angiogenesis and HGFs to produce the endothelial
lining [10]. However, in situations of alveolar osteitis, irradiation at
a very high dose into the socket until the patient feels a change in
pain (at times up to 100 J/cm2 ) during the initial visit will decrease
the conduction velocity of Aδ- and C-fibers, which innervate the
pulp as well as stimulate the production of endogenous opioids
(β-endorphins) [11, 12]. When the dressing is changed in the days
following, a biostimulatory dose is applied for stimulation of the
epithelium to cover the bone [9].

42.2.3 Dental Infection

Dental infection primarily occurs as a result of a bacterial infection
from dental decay or a trauma to the tooth. Bacteria will quickly
infect the dental pulp and can spread to the bone supporting the
tooth if not treated. Dental infection results in a collection of pus
and edema in the tissues surrounding the tooth and patients often
present with a dental abscess.
PBM will not have a bacteriocidal effect; however, it can be
useful as an adjunctive treatment modality. Irradiation of the sub-
thoracic lymphatic duct and submandibular lymph nodes with a
biostimulatory dose increases both lymphatic flow and circulation
(Figs. 42.3 and 42.4); this will potentiate the uptake of the antibiotic
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840 Photobiomodulation in Dentistry

Figure 42.3 Irradiation of submandibular lymph nodes.

into the system and stimulate the migration of immune cells to the
site of infection [13].

Research Highlight
A 2003 study by Lopes et al. demonstrated that irradiation
increases lymphatic drainage of the infected area to remove
edema; enhances the proliferation of macrophages, which re-
move necrotized tissue and release growth factors; reduces pro-
inflammatory cells; and stimulates chemotaxis, which brings
immune cells to the site of infection for faster healing [14].
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Dental Procedures Using Laser Therapy 841

Figure 42.4 Submandibular lymph nodes and sub-thoracic lymphatic duct.

42.2.4 Restorations
Restorative dentistry can lead to a lot of stress to both the
patient and the practitioner. Fear of needles, noise, and vibration of
mechanical preparations remain a cause of discomfort in patients
and in many cases contribute to a decrease in dental compliance
[15].
Restorative procedures provoke an inflammatory response
within the pulp that causes the release of pro-inflammatory
peptides, including substance P, bradykinin, histamine, and
prostaglandins [10]. Laser irradiation increases the migration of
leukocytes, which metabolize damaged cells and release endoge-
nous opioids [11, 16]. In addition, PBM induces dentinogenesis
through the biomodulatory activity of pulp cells and stimulation of
odontoblasts [17]. Before placing the restorative materials in deep
preparations, laser irradiation should be directed into the prepared
tooth to decrease pulpal inflammation and stimulate odontoblasts
to create secondary dentin. This, in turn, will reduce the incidence of
pulpal death and negate the need for future endodontic procedures.
A very useful application in laser dentistry is pulpal analgesia,
which allows practitioners to perform primary tooth restorations
and crown cementations comfortably without dental anesthesia.
Local anesthetics serve as a chemical barrier that prevents the
pain impulse from reaching the brain. Conversely, laser analgesia is
the result of endogenous opioids that affect the pain threshold as
opposed to blocking it [18].
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842 Photobiomodulation in Dentistry

Figure 42.5 Application guide for primary teeth.

Laser analgesia is achieved through the irradiation of the pulp

of the tooth with a bioinhibitory dose at the apex application
points noted in Figs. 42.5 and 42.6. This results in the reduction in
the conduction of Aδ- and C-fibers and the release of endogenous
opioids (β-endorphins) [19]. A bioinhibitory dose is then applied
to the cement enamel junction (CEJ) or exposed caries to provide
analgesia lasting 1–2 h [20].
Laser analgesia also decreases the prevalence of soft tissue
trauma, such as lip or cheek biting; a complication often associated
with the long-lasting numbing that accompanies many local anes-
thetics [4]. In addition, if anesthetic is used, a biostimulatory dose at
the lymph nodes and injection site after the procedure will reduce
the duration of anesthesia.
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Dental Procedures Using Laser Therapy 843

Maxillary Jaw

Mandibular Jaw

Figure 42.6 Application guide for permanent teeth.

42.2.4.1 Cementing crowns

Local anesthesia is often applied while cementing crowns, making
the occlusion difficult to adjust. A bioinhibitory dose is first applied
to the apex, according to Fig. 42.6. The temporary crown is then
removed, and a bioinhibitory dose is applied to the buccal, occlusal,
and lingual surfaces. Laser analgesia allows practitioners to cement
the crown comfortably and obtain a precise bite.

42.2.5 Nausea and Gagging

Many patients suffer from nausea and gagging reflexes during dental
treatments or while taking impressions and radiographs. In severe
cases, patients require sedation to perform basic dental procedures,
which in itself carries the potential for serious complications. Laser
irradiation of the pericardium 6 (P6) acupuncture point is effective
for prophylactic treatment of nausea and vomiting. The P6 point
is located three finger lengths midline from the crease in the
wrist [21].
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844 Photobiomodulation in Dentistry

Case Study: Nausea and Gagging

Treating dentist: Martin Kaplan, DDS

Patient: 6-year-old female

Medical history: Her medical history was non-contributory.

There was advanced dental caries of 11 primary teeth. She has
a history of reflux as well as nausea and vomiting from offensive
smells. She was referred to a pedodontist due to several failed
previous visits.

Laser apparatus: 830 nm GaAlAs laser

Treatment: 8 J was applied to the P6 meridian for reduction of

nausea.

Results: PBM reduced nausea enabling the patient to tolerate

X-rays for proper oral diagnosis. A treatment plan for oral con-
scious sedation was proposed. At the oral sedation procedure,
she received 8 J on the P6 point bilaterally and then was offered
the oral medication. While this can be offensive to many, it was
well tolerated by this patient; if the medication had not been
tolerated, she would have required hospitalization. This allowed
for the completion of dental care with the oral sedation of choral
hydrate and vistaril along with nitrous oxide analgesia and local
anesthetic.

Conclusion: Laser irradiation of P6 reduced nausea, eliminated

the risk of aspiration, and behavior tolerance was removed from
clinical hindrance. She safely received care and avoided general
anesthesia.

42.2.6 Dentin Hypersensitivity

Dentinal hypersensitivity is characterized by short, sharp pain
arising in response to a mechanical, chemical, or osmotic stimulation
applied to dentin exposed as a result of a loss of enamel, cementum,
or gingival recession [22]. Pain originates by a fluid movement
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Dental Procedures Using Laser Therapy 845

Figure 42.7 Laser irradiation to P6.

within the tubules in the dentin that activates nociceptive pain.

Conventional therapy includes a chemically induced mechanical ob-
struction of the dental fluid movement; however, this methodology
is limited in its effectiveness and longevity [23].
PBM reduces the conduction velocity of nerve fibers innervating
the dental pulp, which decreases the sensitivity to thermal and
tactile stimuli. In addition, stimulation of odontoblasts produces
secondary dentin, which fills the tubules providing a long-lasting

Figure 42.8 Irradiation of the apex.

July 6, 2016 17:39 PSP Book - 9in x 6in 42-Hamblin-c42

846 Photobiomodulation in Dentistry

Figure 42.9 Irradiation of the CEJ.

solution [24]. Irradiation with a bioinhibitory dose should be

applied first to the apex of the tooth to initiate pulpal analgesia
and then at the CEJ (see Figs. 42.9 and 42.10). One application may
be sufficient; however, depending on the patients’ habits, further
treatments may need to be repeated periodically.

42.2.7 Soft Tissue Lesions

Soft tissue lesions such as herpes lesions, aphthous ulcers, and
denture sores respond well to PBM, due to the stimulation of soft
tissue healing and immune response, as well as the reduction in pain.

Research Highlight
A 2003 study by Marsillo et al. investigated the use of 670
nm GaAlAs laser irradiation on dentin hypersensitivity using
3 J/cm2 and 5 J/cm2 . Irradiation with both energy densities
was effective in 86.53% and 88.88% of the irradiated teeth, an
improvement that was maintained at the 60-day follow-up [25].
Ferreira et al. investigated the effect of 670 nm GaAlAs laser on
reactional dentinogensis and found that the irradiated dental
pulp showed organized odontoblasts, and the pre-dentin layer
depicted a narrow band of matrix and no calcification [16].
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Dental Procedures Using Laser Therapy 847

Endo Diagnosis
Apply laser to apex of sore tooth

Tooth non-vital Hyperemic Tooth

Laser will not have any Pain caused by substance P,

effect and there will be bradykinins, etc.
no pain felt by paent
(nerve is dead) Wait 1 minute for laser to increase
circulaon and lymphac drainage

Apply Laser to Identified Tooth

No Pain Immediate Pain

Indicates tooth can be treated Indicates case of irreversible
with conservave methods pulpis and will require a root canal

Figure 42.10 Diagnosis of pulp hyperemia.

The lymphatic system is treated first with a biostimulatory dose,

followed by the application to the lesion in a noncontact mode.

42.2.7.1 Herpes lesions

Herpes lesions are caused by the human herpes virus type 1, which
present as eruption of lesions with periods of latency. Eruptions
can be triggered by exposure to intense UV irradiation, stress,
and menstruation. Traditional treatments generally utilize topical
creams and are only moderately successful as a result of the
topical application and lack of penetration. Laser irradiation has an
inhibitory effect on the virus and significantly increases the relapse
time [26]. The mechanism by which this happens is not entirely clear
but is thought to be a response of the immune system (decrease
in pro-inflammatory peptides and increase in anti-inflammatory
mediators).
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848 Photobiomodulation in Dentistry

While PBM is effective for significantly speeding the healing of

herpes lesions, the eruption can frequently be prevented if the laser
is applied in the prodromal stage.

Research Highlight
A 2012 study by Sanchez et al. investigated the effect of 670
nm laser irradiation on herpes simplex type 1 and found that
the laser-treated group had a distinct curative effect on the
clinical manifestations and a clear trend toward reduction in the
recurrence frequency [24].

42.2.7.2 Aphthous ulcers

Aphthous ulcers (canker sores) are one of the most common
ulcerative lesions that affect the oral cavity. Patients present
with small round ulcers in the oral epithelium, which expose
nerve endings in the underlying lamina properia, leading to pain
in daily activities such eating, swallowing, and speaking [27].
Common treatment regimens include steroids and mouthwashes,
both of which carry a number of side effects, require patient
compliance, have minimal absorption, and can be quite expensive
[28]. Laser irradiation of the ulcerations decreases the size
and pain intensity, as well as increasing the latency of the
lesions [25].
Aphthous ulcers can be a serious complication in HIV patients.
The lesions are frequently larger and may cause a debilitating pain
that result in malnutrition, weight loss, and compromise their ability

Research Highlight
A 2013 case study investigating the effect of 940 nm laser
irradiation on aphthous ulcers found evidence of a decrease
in healing time, pain intensity, size, and recurrence of lesions
[25].
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Dental Procedures Using Laser Therapy 849

to take medications. PBM is an attractive treatment alternative for

HIV patients due to its nil effect on the immune system and lack of
concern over contraindicating medications [26].

42.2.7.3 Appliance irritation mucosal lesions

Mucosal inflammation and lesions under dentures and orthodontic
appliances cause sores and pain that can sometimes prevent
the denture or appliance from being worn. Laser irradiation of
the mucosal lesions accelerates epithelization, vascularization, and
collagen synthesis of fibroblasts, which significantly increases soft
tissue healing [7].

42.2.8 Oral Mucositis

Oral mucositis is a severe oral lesion resulting from the toxic
effects of radiation and chemotherapy that adversely affects a
patient’s oral function and ultimately quality of life. The condition
occurs in 80–100% of patients undergoing chemotherapy/radiation
and can be so severe that it results in weight loss and reduces
treatment compliance [29]. Patients present with severe pain that
impairs oral functions such as phonation, deglutition, taste, and
dysphagia [30]. Medicine currently offers a few options that can
successfully manage or prevent the condition and are primarily pal-
liative, including topical antimicrobial agents, vitamins, and mouth
washes [31].
PBM can reduce the severity of mucositis by modulating
the inflammatory response and stimulating soft tissue healing.
In addition, the release of endogenous opioids and decreased
conduction of nerve transmission significantly decreases pain.
Numerous studies have also demonstrated the cytoprotective effect
on the mucosa following prophylactic irradiation [32]. The moderate
to strong evidence for the efficacy of PBM in treating and preventing
mucositis suggests that it should be included in the standard of
care for all patients undergoing chemotherapy and/or radiation
[28].
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850 Photobiomodulation in Dentistry

Case Study: Oral Mucositis

Treating dentist: Gerry Ross, DDS

Patient: 61-year-old female

Medical history: Patient undergoing chemotherapy for termi-

nal brain cancer presented with numerous lesions over the
mucosa of the cheeks. The patient could not eat, drink, or
swallow without extreme pain. Treatments (mouth rinses)
assigned by the oncologist were having no effect on the pain or
healing of sores.

Laser apparatus: 660 nm He–Ne laser

Treatment: 2 J/cm2 was applied overlapping throughout the

mouth in noncontact mode for 2 days in a row.

Results: When patient came in on the second day, the pain was
markedly decreased and the sores showed signs of healing. The
patient reported she was able to drink soup and tea. By the
fourth day after the initial treatment, all sores had closed and
patient was able to eat normally.

Conclusion: Laser was successfully applied regularly to pre-

vent the lesion reoccurrence.

Note: Prior to PBM, the oncologist was contacted and ap-

proved laser irradiation as all other methods had failed.

42.2.9 Gingivitis
Gingivitis is an acute inflammatory condition primarily caused by
microorganisms originating from dental plaque [33]. PBM is most
effective in the treatment of acute inflammation; scaling in the
presence of gingival inflammation creates an acute inflammatory
reaction that responds very well to a biostimulatory dose. PBM is
an effective adjunctive treatment that alleviates inflammation in the
periodontal tissue, reduces tissue edema, and stimulates fibroblasts
[34]. In the case that a gingivectomy is performed, PBM utilization
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Dental Procedures Using Laser Therapy 851

will improve soft tissue healing and reduce pain, and reducing the
need for pain medication.

Research Highlight
Healing after a gingivectomy is often a lengthy and painful
process. A 2006 study using a 685 nm laser showed a statisti-
cally significant decrease in pocket depth at 21 and 28 days post-
surgery. Moreover, the laser-treated wounds presented with
factors suggestive of better healing, including color, contour, and
mucosa healing when compared with controls [35].

42.2.10 Periodontitis
Periodontitis is a chronic inflammatory disease resulting from the
accumulation of subgingival bacterial biofilms on tooth surfaces
[36]. Periodontitis presents as tissue recession, profuse bleeding,
and a destruction of the underlying bone [37].
PBM improves the gingival and bleeding index through a
combination of mechanisms:

• Activation of microcirculation in damaged tissue, which de-

creases interstitial and intracellular swelling
• Increase in anti-inflammatory cytokine levels and nonspecific
protection factors (e.g., interferons)
• Decrease in pro-inflammatory mediators (histamine, serotonin,
and PGE2 )
• Increases the formation of connective tissue and alveolar
bone through the stimulation of fibroblasts and osteoblasts,
respectively
• Reduction in pain through the stabilization of nerve cell
membrane and production of β-endorphins

Periodontitis is a great example of how PBM can be used in a

combined care program. Laser irradiation is most efficacious in
treating acute inflammation; mechanical debridement can create a
significant change in the microbiological environment and shift the
pathogenic biofilm from a chronic to acute state. The subsequent
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852 Photobiomodulation in Dentistry

laser irradiation will be much more effective in these conditions

[38].
Periodontitis also requires some manipulation of the energy
density as the treatment progresses. Initially, a bioinhibitory dose
is recommended to modulate the inflammatory response. As the
inflammatory condition settles, a biostimulatory dose is suggested
for healing of the bone and connective tissue.

42.2.11 Endodontics
PBM can assist with endodontic procedures by reducing postoper-
ative pain and edema, as well as lessening the need for analgesics.
Following root canal treatment, a bioinhibitory dose is applied at the
apex of each root (according to Fig. 42.6).
In addition, laser irradiation can assist dentists in identifying
a painful tooth whose cause is not apparent through radiographs
or other diagnostic methods. Irradiation to a hyperemic pulp
stimulates blood and lymphatic flow within the pulp, which is
perceived as pain by the patient. In a healthy tooth or one with a
non-vital pulp, there will be no response.

42.2.11.1 Pulpotomies
In an exposure, the pulp is cauterized followed by the application of
cement to stimulate healing. Laser irradiation with a biostimulatory
dose is applied after cauterization, but before any cement is
placed over the pulp stump, which reduces pain and inflammation,
stimulates secondary dentin, and improves the chances for the tooth
to remain vital.

42.2.12 Nerve Regeneration

Nerve damage within the oral complex, complete or semi-complete,
leads to sensory disabilities. While the peripheral nervous system
(PNS) is capable of regenerating crushed or severed axons in some
situations, it is often incomplete and slow. When a nerve is damaged,
Schwann cells (neuronal macrophages) break down and phagocytize
injured nerve structures. If these cells linger in the area for an
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Dental Procedures Using Laser Therapy 853

extended period of time, they can start to compromise the recovery

of the nerve tissue causing loss of function and pain sensitivity
(neuralgia) [39].
The areas most often damaged are the inferior alveolar nerve
(IAN), which is the sensory nerve of the bottom lip and mandible,
and the lingual nerve, which innervates the anterior two-thirds
of the tongue. These are particularly susceptible to injury during
surgical extractions, lower third molar extractions, sagittal split
osteotomy, implant placement, and nerve blocks. Paresthesia, a
neurosensitive disturbance caused by a lesion in the nerve tissue,
consequently alters the sensorial perception in the affected region,
resulting in a numbing at the affected area [40].
A biostimulatory dose of PBM at the site of injury induces
regenerative effects in the PNS by modulating neurotropic ac-
tors such as nerve growth factor, fibroblast growth factor, and
growth-associated protein, which increases the amount of nerve

Case Study: Nerve Regeneration

Treating dentist: Dr. Gerry Ross, DDS

Patient: 19-year-old male

Medical history: Referred from oral surgeon with paresthesia

of the anterior two-thirds of his tongue and lingual gingiva,
which had not improved in the last 6 months.

Laser apparatus: 830 nm GaAlAs laser

Treatment: 4 J/cm2 was applied at the extraction site three

times per week for 3 weeks.

Results: At the end of the third week, the patient had normal
sensation in the lingual gingiva and tingling in the tongue.
Treatment continued for another 3 weeks, and sensation
returned to both areas.

Conclusion: Laser irradiation successful eliminated paresthe-

sia that had previously not responded to conventional treat-
ments.
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854 Photobiomodulation in Dentistry

Research Highlight
A 2013 study by Gasperinini et al. investigated the effect of
PBM on hypoesthesia of the inferior alveolar nerve (IAN) and
found that 4–6 J/cm2 per treatment along the distribution of
the IAN significantly improved the neurosensory recovery and
mechanoreceptor perception [42].
Akgul et al. demonstrated that 650 nm red light delivered
at an energy density of 10 J/cm2 reduced the migration of
mononuclear cells to damaged nerves, leading to a decrease in
edema and rapid regeneration in an animal model [37].

sprouts [37]. PBM results in a significant acceleration in the time

course and magnitude of neurosensory recovery [41]. Patients may
report a tingling, pricking, crawling, or itching sensations following
PBM, which is a positive sign or neurorehabilitation.

42.2.13 Orthodontics
Orthodontic tooth movement is a process involving both bone
breakdown and bone healing in response to externally applied
forces [43]. Bone is resorbed on the pressure side by osteoclasts and
deposited on the tension side of a tooth by osteoblasts. Following
band placement, an orthodontic force is generated; the teeth are
displaced and an inflammatory response is induced by mechanical
compression of the periodontal ligament, which results in pain [44].
Orthodontic pain is localized, relatively short in duration, and can
be severe depending on the force application applied on the teeth
[45]. Conventional pain management involves the use of NSAIDs,
which are moderately effective for reducing pain but can cause
gastrointestinal upset.
Laser analgesia is the primary indication in orthodontics and
is achieved by delaying the onset and duration of pain. Irradiation
increases nerve latency and stimulates the release of endogenous
opioids, that reduces pain sensations [42, 46]. Bioinhibitory
doses are recommended for 1–2 days after each adjustment
to modulate inflammation and decrease pain. Subsequent visits
using a biostimulatory dose improve healing by stimulating the
July 6, 2016 17:39 PSP Book - 9in x 6in 42-Hamblin-c42

Dental Procedures Using Laser Therapy 855

Figure 42.11 Laser irradiation during orthodontic treatment.

osteoblast/osteoclast turnover. This contributes to a secondary

effect of PBM in orthodontics; increased orthodontic movement,
leading to reduced treatment time.

Research Highlight
Doshi-Mehta et al. investigated the rate of tooth movement on
20 patients irradiated with 810 nm GaAlAs laser and found
that there was an average increase of 30% in the rate of tooth
movement. In addition, the laser-treated group had significantly
less pain than the control group [47].
A 2009 study by Tortamano et al. evaluated the effect of PBM
on reducing pain in orthodontic patients irradiated with 830 nm
GaAlAs laser and found that patients had lower mean scores
for oral pain and intensity of pain on the most painful day. In
addition, the pain ended sooner [43].

42.2.14 Implants
Implantology is an effective prosthetic rehabilitation procedure
for restoring the capacity of mastication, phonation, and esthetics
[48]. A dental implant is an artificial root made of titanium metal,
which is then used to support a prosthetic replacement tooth.
When an implant is placed, approximately 3–4 months are required
for immature bone to become mature, resistant, and capable of
successful loading.
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856 Photobiomodulation in Dentistry

Figure 42.12 Laser irradiation for implant osseointegration.

The use of PBM for bone neoformation and implant osseointe-

gration has been studied extensively due to the positive effect of
infrared light on osteoblastic proliferation, collagen deposition, and
new bone formation [49].

Research Highlight
A 2007 study by Lopes et al. evaluated the effect of 830 nm
laser irradiation on bone tissue around implants. From 0–15
days after irradiation, there was a minimal increase in the
amount of calcium hydroxyapatite (CHA)—a mineral secreted
from mature bone—which was indicative of early stages of
healing when osteoblastic activity is mainly proliferative. From
15–30 days, the distribution of bone tissue was better, the
bone was more organized around the implant, and there was a
significant increase in CHA, indicating bone maturation. At 45
days, there was increased newly formed bone and an absence of
spaces around the irradiated implants, which was a significant
improvement from the control group [47].

Clinically, a bioinhibitory dose is used at the time of implant

placement for pain and inflammation modulation. Irradiation with
biostimulatory doses every 2–3 days for 2 weeks will stimulate
osteoblasts for faster integration of the implant and a more dense
bone surface. Laser irradiation has been demonstrated to reduce
July 6, 2016 17:39 PSP Book - 9in x 6in 42-Hamblin-c42

Dental Procedures Using Laser Therapy 857

the healing time of bone by approximately 30%, allowing for earlier

loading [47]. This same effect applies to bone grafts used around
implants.

42.2.15 Sinusitis
Sinusitis is an inflammatory disease of the nose and paranasal sinus
that presents as nasal congestion, purulent rhinorrhea, facial pain
or pressure, post-nasal drip, headache, anosmia (loss of smell),
and cough. Sinusitis results from the release of local inflammatory
mediators, including bradykinins, prostaglandins, and histamines.
Laser irradiation with a bioinhibitory dose to the frontal or maxillary
sinuses decreases the number of these mediators and significantly
eases congestion by reducing inflammation [50].
Sinusitis can play an important role in dental diagnosis when
patients present with unidentifiable tooth pain. Laser irradiation
along the teeth in question will often not elicit a response from the
patient, and the practitioner is left perplexed as to the cause. In many
cases, sinus congestion in the sinus cavities is the root cause, which
can be dramatically reduced with irradiation of the maxillary and
frontal sinus.

42.2.16 Temporomandibular Joint Pain

Temporomandibular joint disorders (TMDs) are multi-factorial
conditions affecting the muscles of mastication, perauricular area,
and temporomandibular joint [51]. Patients present with headaches,
facial pain, ear ache, dizziness, ringing in the ears, limited movement
or locking of the jaw, grating sounds in the temporomandibular
joint when the mouth is opened and closed, and pain in the
neck, shoulder, and back. Conventional treatments include splints,
occlusal adjustment, and pharmaceuticals, which aim to reduce pain
and improve the function of the masticatory system [52].
PBM improves microcirculation in the muscles of mastication,
which softens and relaxes the affected muscles and normalizes the
intramuscular pressure on sensory nerve endings leading to a reduc-
tion in pain. In addition, increased circulation in irradiated muscles
helps to remove noxious deposits (e.g., lactic acid) associated with
July 6, 2016 17:39 PSP Book - 9in x 6in 42-Hamblin-c42

858 Photobiomodulation in Dentistry

myopathic pain and improves edema through enhanced lymphatic

flow [53]. While PBM is an effective aid in managing patients
with TMD, it is only one tool in a practitioner’s armamentarium.
A combined care program involving splints and other treatment
modalities may still be.
Despite the clinical successes in treating TMD with PBM, there
have been mixed results in the published research studies for a
number of reasons. The main issue is many research studies try
to treat all facial pain conditions as one. TMD is a general term
to describe a number of conditions, including myofascial pain,
disc dislocation, degenerative joint disease, arthritis, neuropathic
pain, referred pain, etc., all of which have different etiologies and
anatomical disturbances. As such, there is no one treatment that will
effectively treat all conditions and should not be studied in this way.
A key to success in treating TMDs is to customize the treatments
on a patient-specific basis with a multidisciplinary team. TMDs
frequently include postural issues, neck pain, psychological factors
as well as many others; patients should be treated with a team of
clinicians able to address these issues to yield the best results.

Figure 42.13 Laser probe treating the temporomandibular joint.

July 6, 2016 17:39 PSP Book - 9in x 6in 42-Hamblin-c42

Dental Procedures Using Laser Therapy 859

Figure 42.14 LED cluster applied over the masseter muscle. (Image
courtesy: James Carroll, President of THOR Laser).

For successful treatment of facial pain, it is essential that clini-

cians understand trigger points and referred pain. The most com-
prehensive text that all clinicians should have in their library is “My-
ofascial Pain and Dysfunction” by Travell and Simons [54]. This text
provides detailed information on trigger points and referred pain,
as well as illustrated diagrams that map the referral pain pattern of
the head and neck muscles. Understanding referred pain is critical
because the source of the pain may be completely different from
the site of pain. Frequently, when patients are not responding to a
treatment protocol, it is a result of referred pain; thus, one must be
sure they are treating the source of the pain, not the site of the pain.
When treating TMDs, you are dealing with both large muscles
(masseter, temporalis, and sternocleidomastoid) and small joint
spaces. If possible, it is advantageous to use clusters of light-emitting
diodes (LEDs) to cover large muscles and the trigger points within
and a laser probe to simulate the lymphatic system and treat joint
spaces.
Acute cases, such as facial pain after long dental appointments,
show the possibility of a quick and lasting response with PBM alone.
Alternatively, chronic conditions require more treatments over an
extended period of time, often in combination with other therapies
(e.g., splints) [55]. The greatest success seems to be treating the
affected joint/area for 2–3 weeks followed by reassessment to
July 6, 2016 17:39 PSP Book - 9in x 6in 42-Hamblin-c42

860 Photobiomodulation in Dentistry

determine the level of improvement. The goal of the break in

treatment is to let the body continue to heal itself.

Research Highlight
A 2007 study by Fikackova et al. investigated the effect of
830 nm GaAlAs laser on a number of different temporo-
mandibular disorders. An improvement after laser therapy was
reported by 82% of patients with myofascial pain, 77% with
temporomandibular joint (TMJ) arthralgia, and 73% with both
myofascial pain and TMJ arthralgia. In addition, there was a
significant improvement in those with chronic pain [56].
Dostalova et al. investigated the effect of PBM on facial pain
using an 830 nm GaAlAs laser. After laser irradiation, pain on the
visual analogue scale reduced from 27.5 to 4.16 and the mouth
opening improved from 34 to 42 mm [57].

42.3 Conclusion

Photobiomodulation continues to be an incredible asset to a dental

practice. It is no longer necessary to fear a visit to the dentist, and
gone are the days when patients leave the office wincing in pain.
Clinical effects such as modulation of inflammations, production of
endogenous opioids, decreased neural latency, increased circulation
and lymphatic flow, and the stimulation of fibroblasts, osteoblasts,
and odontoblasts all contribute to improved dental treatments and
clinical outcomes. With PBM as a prominent tool in a practice,
clinicians are able to provide a higher level of care, with reduced
stress for both the patient and the practitioner.
The ongoing cellular, animal, and clinical researches of PBM
have expanded the applications within a dental clinic and pro-
vided practitioners with precise energy-density recommendations
to achieve optimal clinical outcomes. The ability to manipu-
late the response of the cell with biostimulatory and bioin-
hibitory doses allows practitioners to tackle any application with
confidence.
July 6, 2016 17:39 PSP Book - 9in x 6in 42-Hamblin-c42

References 861

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864 Photobiomodulation in Dentistry

35. Amorim J.C.F., De Sousa G.R., Silveira, L.D.B., Prates R.A., Pinotti M.,
and Ribeiro M.S. (2006). Clinical study of the gingiva healing after
gingivectomy and low-level laser therapy. Photomed Laser Surg, 24(5),
pp. 588–594.
36. Soukos N.S., Mulholland S.E., Socransky S.S., and Doukas A.G. (2003).
Photodestruction of human dental plaque bacteria: Enhancement of the
photodynamic effect by photochemical waves in an oral biofilm model.
Lasers Surg Med, 33, pp. 161–168.
37. Aykol G., Baser U., Maden I., Kazak Z., Onan U., Kucuk S.T., Ademoglu E.,
Issever H., and Yalcin F. (2011). The effect of low-level laser therapy as
an adjunct to non-surgical periodontal treatment. J Periodontol, 82(3),
pp. 481–488.
38. Lui J., Corbe E.F., and Jin L. (2011). Combined photodynamic and low-
level laser therapies as an adjunct to nonsurgical treatment of chronic
periodontitis. J Periodont Res, 43, pp. 89–96.
39. Akgul T., Gulsoy M., and Gulcur H.O. (2014). Effects of early and delay
laser application on nerve regeneration. Lasers Med Sci, 29, pp. 351–
357.
40. Ozen T., Orhan K., Gorur I., and Ozturk A. (2006). Efficacy of low-level
laser therapy on neurosensory recovery after injury to the inferior
alveolar nerve. Head Face Med, 15, pp. 2–3.
41. Miloro M. and Repasky M. (2000). Low-level laser effect on neurosen-
sory recovery after sagittal ramus osteotomy. Oral Surg Oral Med Oral
Pathol Oral Radiol Endod, 89, pp. 12–18.
42. Gasperini G., de Siqueira I.C.R., and Costa L.R. (2014). Lower-level
laser therapy improves neurosensory disorders resulting from bilateral
mandibular sagittal split osteotomy: A randomized crossover clinical
trial. J Craniomaxillofac Surg, 42(5), pp. e130–133.
43. Ekizer A., Uysal T., and Guray E. (2013). Effect of LED-mediated
photobiomodulation therapy on orthodontic tooth movement and root
resorption in rats. Lasers Med Sci, 30(2), pp. 779–785.
44. Bicakci A.A., Kocoglu-Altan B., Toker H., Mutaf I., and Sumer Z. (2012).
Efficiency of low-level laser therapy in reducing pain induced by
orthodontic forces. Photomed Laser Surg, 30(8), pp. 460–465.
45. Tortamano A., Lenzi D.C., Haddad A.C.S.S., Bottino M.C., Dominguez G.C.,
and Vigorito J.W. (2009). Low-level laser therapy for pain caused by
placement of the first orthodontic archwire: A randomized clinical trial.
Am J Orthod Dentofacial Orthop, 136, pp. 662–667.
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46. Ribas M.A., Dominguez J.A., and Pugdollers A. (2012). Analgesic effect
of a low-level laser therapy (830 nm) in early orthodontic treatment.
Lasers Med Sci, 28(1), pp. 335–341.
47. Mehta G.D. and Patil W.A.B. (2012). Efficacy of low-intensity laser
therapy in reducing treatment time and orthodontic pain: A clini-
cal investigation. Am J Ortho Dentofacial Orthop, 141(3), pp. 289–
297.
48. Lopes C.B., Pinheiro A.L.B., Sathaiah S., Duarte J., and Martins
M.C. (2005). Infrared laser light reduces loading time in dental
implants: A Raman spectroscopic study. Photomed Laser Surg, 23(1),
pp. 27–31.
49. Lopes C.B., Pinheiro A.L.B., Sathaiah S., Da Silva N.S., and Salgado M.A.C.
(2007). Infrared laser photobiomodulation (λ 830 nm) on bone tissue
around dental implants: A Raman spectroscopy and scanning electronic
microscopy study in rabbits. Photomed Laser Surg, 25(2), pp. 96–
101.
50. Naghdi S., Ansari N.N., Fathali M., Bartley J., Varedi M., and Honarpishe R.
(2013). A pilot study into the effect of low-level laser therapy in patients
with chronic rhinosinusitis. Physiother Theory Pract, 29(8), pp. 596–
603.
51. Ferreira L.A., de Oliveira R.G., Guimaraes J.P., Carvalho A.C.P., and De
Paula M.V.Q. (2013). Laser acupuncture in patients with temporo-
mandibular dysfunction: A randomized controlled trial. Lasers Med Sci,
28, pp. 1549–1558.
52. Carvalho C.M., de Lacerda J.A., Neto F.D.D.S., Cangussu M.C.T., Marques
A.M.C., and Pinheiro A.L.B. (2010). Wavelength effect in temporo-
mandibular joint pain: A clinical experience. Lasers Med Sci, 25, pp. 229–
232.
53. Öz S., Gökçen-Röhlig B., Saruhanoglu A., and Tuncer E.B. (2010).
Management of myofascial pain: Low-level laser therapy versus occlusal
splints. J Craniofac Surg, 21(6), pp. 722–728.
54. Simons D.G., Travell J.G., and Simons L.S. (1999). Travell & Simons’
Myofascial Pain and Dysfuntion: Upper Half of Body, 2nd Ed. (Lippincott
Williams & Wilkins, USA).
55. Bradley P.F. and Rebliini Z. (1996). Low intensity laser therapy (LILT)
for temporomandibular joint pain: A clinical, electromyographic and
thermographic study. Laser Therapy, pp. 47–48.
56. Fikackova H., Dostalova T., Navratil L., and Klaschka J. (2007). Effective-
ness of low-level laser therapy in temporomandibular joint disorders:
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866 Photobiomodulation in Dentistry

A placebo-controlled study. Photomed Laser Surg, 25(4), pp. 297–

303.
57. Fikackova H., Dostalova T., Vosicka R., Peterova V., Noavratil I., and Lesak
J. (2006). Arthralgia of the temporomandibular joint and low-level laser
therapy. Photomed Laser Surg, 24(4), pp. 522–527.
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Chapter 43

Photobiomodulation for the Clinical

Treatment of Age-Related Macular
Degeneration

Graham Merrya and Robert Dotsonb

a Photospectra Health Sciences Inc., 271 Mutual Street, Toronto, Ontario M4Y1X6,

Canada
b Lumithera Inc., 19332 Powder Hill Place Northeast, Poulsbo, WA 98370, USA

gmerry@photospectra.ca

Age-related macular degeneration (AMD) is the leading cause of

worldwide blindness for the elderly in the western world. It is
a bilateral ocular condition that affects the central area of retina
known as the macula. Although the macula comprises less than 5%
of the retinal area, it is responsible for the central acute vision.
The fovea lies at the center of the macula and is approximately
2 mm in diameter. The fovea contains the highest density of cone
photoreceptor cells and is the only region of the retina where 20/20
vision is attainable. Thus, lesions developing in this region can have
a major impact on visual function.
We will first talk a little about the disease itself, its pathogenesis,
and some of the potential treatments. We will then present evidence
for the use of photobiomodulation (PBM) or low-level laser therapy

Handbook of Low-Level Laser Therapy

Edited by Michael R. Hamblin, Marcelo Victor Pires de Sousa, and Tanupriya Agrawal
Copyright c 2017 Pan Stanford Publishing Pte. Ltd.
ISBN 978-981-4669-60-3 (Hardcover), 978-981-4669-61-0 (eBook)
www.panstanford.com
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868 Photobiomodulation for the Clinical Treatment of Age-Related Macular Degeneration

Figure 43.1 Normal fundus appearance.

(LLLT) as an active treatment for AMD, starting with cellular

responses, small animals, non-human primates, and finally human
Institutional Review Board (IRB)-approved clinical trials.
We want to make it clear at the outset that when we talk about
PBM or LLLT, which are low-level devices, non-thermal and non-
damaging to cells, these should not be confused with “subthreshold”
laser treatment to the retina where powerful lasers are turned down
in intensity to deliver a nonvisible burn to the retina. These devices
are delivering a dose of light that is magnitudes more powerful than
the devices we have used. Figure 43.1 shows a normal retina.
AMD is an enormous burden to the physical and mental health of
affected individuals and their families. AMD usually manifests later
in life at a time when most affected individuals are looking forward
to enjoying retirement activities and maintaining independence.
Instead, millions with AMD suffer bilateral central vision loss
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Photobiomodulation for the Clinical Treatment of Age-Related Macular Degeneration 869

to a degree that causes inability to drive, read a newspaper or

magazine, prepare meals, or enjoy recreational activities. For these
patients, the visual impairment associated with AMD means a loss
of independence, depression, increased financial concerns, and the
need to adapt to vision loss at a time when they may be trying to
cope with other major medical problems.
The cost to society is only now being appreciated, as many as
11 million people in the United States have some form of AMD, and
this number is expected to double to nearly 22 million by 2050 [1].
Age is a prominent risk factor for AMD. Almost 3.8% of Americans in
the age range of 50–59 years have either intermediate or advanced
AMD; by ages 70–79, this increases to 14.4%. The estimated global
cost of visual impairment due to AMD is $343 billion, including $255
billion in direct healthcare costs. The estimated direct healthcare
cost of visual impairment due to AMD in the United States, Canada,
and Cuba is approximately $98 billion [2].
There are two forms of macular degeneration: dry (non-
neovascular) and wet (neovascular). More severe dramatic vision
loss is typically associated with the “wet” form that occurs in about
15% of all patients with AMD, but up to 20% of legal blindness from
AMD is due to the “dry” form [7]. The typical clinical sign of “dry”
AMD is pigment disruption and drusen in the retina. Drusen may
be small “hard” (small with discrete margins) or “soft” (larger with
indistinct edges). They lie between the retinal pigmented epithelium
(RPE) and an adjacent basement membrane complex known as
Bruch’s membrane. Geographic atrophy (GA) and RPE changes are
also observed in dry AMD. Several classification schemes have been
used that subdivide dry AMD into categories based on the number
and size of drusen, amount of GA, and degree of pigmentary changes
in the macula. The initial clinical diagnosis of early AMD is based on
seeing drusen—the hallmark sign of disease (see Fig. 43.2).
A viable and functioning RPE is essential for normal photore-
ceptor cell metabolism and the visual cycle. RPE degeneration
is accompanied by concomitant photoreceptor degeneration. RPE
dysfunction has a profound amplifying impact on the overlying
photoreceptors and central vision. The causes of RPE dysfunction
are yet to be fully elucidated, although a variety of hypotheses
have been advanced, including genetic factors, ischemia, oxidative
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870 Photobiomodulation for the Clinical Treatment of Age-Related Macular Degeneration

Figure 43.2 Fundus photograph showing all stages of dry AMD. There are
soft small and confluent drusen and some hard central drusen within an
area of geographic atrophy.

stress, phagocytic overload, cigarette smoke, lipofuscin toxicity,

inflammation, and apoptosis.
Drusen contain numerous proteins related to the process of
inflammation. Many of these proteins are associated with the
complement cascade and its regulation [3]. Some of the proteina-
ceous components in drusen and the sub-RPE space are activated
complement components and fragments associated with the assem-
bly of the membrane attack complex (MAC) [4–7]. Other drusen
components, including vitronectin, clusterin, complement receptor
1 (CR1), and membrane cofactor protein, are known complement
regulatory proteins. Still others include known activators of the
complement cascade such as C-reactive protein and amyloid b. This
compositional profile forms the basis for the conclusion that drusen
are a manifestation of chronic, local inflammation at the level of
Bruch’s membrane.
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Photobiomodulation for the Clinical Treatment of Age-Related Macular Degeneration 871

Figure 43.3 Fundus autofluorescence photograph. Lipofuscin accumulates

in AMD and auto-fluoresces; the white areas show hyper-fluorescence
and indicate compromised cells. The dark black central area shows no
autofluorescence and corresponds to the area where cell death has occurred
in geographic atrophy.

Genome-wide linkage analyses of extended families with AMD

have identified a number of chromosomal loci that are linked to
AMD [8–16]. The most consistent occurs at chromosome 1q31. In
contrast, the e4 allele of the Apo lipoprotein E (APOE) gene has
consistently been shown to be protective for AMD [17–19], whereas
the e2 allele appears to influence progression and to result in an
earlier age of onset [20].
Common variants [single nucleotide polymorphisms (SNPs)]
in the gene encoding complement factor H (CFH) confer major
susceptibility to, or protection from, AMD. Haplotype analysis
showed that an SNP in the CFH gene could account for up to
50% of AMD cases in the human population, a number close to
50,000,000 globally. Another study showed that allelic variants in
two other complement-related genes, factor B (BF) and complement
component C2, were linked to AMD [20]. Both variants together can
account for nearly 74% of the risk of developing AMD.
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872 Photobiomodulation for the Clinical Treatment of Age-Related Macular Degeneration

In addition to the 1q31 locus, a region on chromosome

10q26 has been identified in several recent studies of AMD [21].
SNPs in chromosomal region 10q26 harboring PLEKHA1, ARMS2,
and HTRA1 showed the strongest association with AMD. Recent
evidence suggests that in patients homozygous for the risk allele,
the lack of synthesis of ARMS2 is causative of AMD. The pivotal
role of the extracellular matrix in the progression of AMD is
underlined by the abnormal deposition of extracellular debris in the
macula, observed frequently in affected individuals. ARMS2 may be
necessary for proper matrix function.
In 1992 the Age-Related Eye Disease Study (AREDS) group
began enrolling subjects in a prospective, multicenter, randomized,
controlled study [22]. Patients enrolled in the AREDS had mild,
moderate, or severe dry AMD in both eyes, or exudative AMD in
one eye only. The study tried to determine whether antioxidant
therapy prevented vision loss, slowed the progression of dry AMD,
or prevented conversion from dry to wet AMD. The study results
demonstrated that daily antioxidant therapy in the form of 15 mg
beta-carotene, 500 mg vitamin C, 50 mg vitamin E, 80 mg zinc and
2 mg copper daily was superior to a placebo in delaying progression
of advanced dry AMD and in slowing conversion from dry to wet
AMD.
In 2006, the National Eye Institute launched AREDS2, a five-
year study designed to test whether the original AREDS formulation
could be improved by adding omega-3 fatty acids, adding lutein
and zeaxanthin, removing beta-carotene, or reducing zinc. The
study also examined how different combinations of the supplements
performed. Omega-3 fatty acids are produced by plants, including
algae, and are present in oily fish such as salmon. Lutein and
zeaxanthin are carotenoids, a class of plant-derived vitamins that
includes beta-carotene; both are present in leafy green vegetables
and, when consumed, they accumulate in the retina. Prior studies
had suggested that diets high in lutein, zeaxanthin, and omega-3
fatty acids protect vision. Removing beta-carotene from the AREDS
formulation did not curb the formulation’s protective effect against
developing advanced AMD, an important finding because several
studies have linked taking high doses of beta-carotene with a higher
risk of lung cancer in smokers. Omega-3 did not show benefit.
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Photobiomodulation for the Clinical Treatment of Age-Related Macular Degeneration 873

Better therapies might not only prevent the progression of

obvious disease that leads to neovascularization, but may also lead
to effective prevention at earlier stages. A preventive treatment that
could delay the onset or progression of AMD by even several years
would amount to essentially a cure in many of these patients.
The ability to arrest the progression of dry AMD, regain normal
macular structure and function, and prevent wet AMD may seem
extremely difficult, but recently many therapeutic targets in the
pathogenesis of dry AMD have been identified and numerous ther-
apies are currently progressing through clinical trials. A multimodal
treatment approach, including antioxidant, anti-inflammatory, and
neuroprotective combination therapy, may eventually yield the
greatest benefit.
Steps in AMD pathogenesis that appear to be good targets
for treatment development include oxidative damage, lipofuscin
accumulation, chronic inflammation, mutations in the complement
pathway and non-complement mutations that influence chronic
inflammation and/or oxidative damage (e.g., mitochondria and the
extracellular matrix structure).
Some traditional pharmaceutical approaches being researched
are mentioned as follows; therapies that have not been shown
to be effective at the time of writing include Anecortave ac-
etate, Eculizumab, POT-4, Tandospirone, the antioxidant OT-551,
Sirolimus, Vitamin E, and Fenretinide.
• Acucela (ACU-4429) (Acucela Inc.) is a non-retinoid visual
cycle modulator that was designed to be selective for rod
photoreceptors.
• ARC-1905 (Ophthotech), an anti-C5 pegylated aptamer, is
administered intravitreally and is currently being investi-
gated in phase 1/2 trials for the treatment of dry and wet
AMD.
• LFG316 (Novartis Pharmaceuticals) is an antibody directed
against C5 and is administered intravitreally. The phase
1 study to assess safety and tolerability of intravitreal
LFG316 in patients with advanced AMD, GA or choroidal
neovascularization has been completed and a phase 2 study
to evaluate the efficacy of six successive monthly doses on
the growth of GA is currently underway.
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874 Photobiomodulation for the Clinical Treatment of Age-Related Macular Degeneration

• FCFD4514S (Genentech/Roche) is a recombinant human-

ized monoclonal antibody fragment directed against com-
plement factor D, which is a rate-limiting enzyme in
the alternative complement pathway. Phase 2 studies are
underway.
• Glatiramer acetate (Copaxone, Reva Pharmaceuticals) is
an immunomodulatory agent approved for the treatment
of multiple sclerosis. It induces suppressor T-cells and
downregulates inflammatory cytokines. A phase 1 study
evaluating the safety and efficacy of weekly treatment over
12 weeks with subcutaneous glatirimer in patients with
drusen has shown that it reduces drusen area. A phase 2/3
study is underway.
• Ciliary neurotrophic factor (CNTF) is a neuroprotective
compound that helps to preserve photoreceptors. CNTF can
be produced and delivered continuously in situ through
encapsulated cell technology. There were no statistically
significant differences observed in the progression of GA
at 12 months for either the high-dose or low-dose groups
compared with the sham group.
• Brimonidine tartrate (Allergan Inc.) is believed to possess
neuroprotective properties. An injectable, sustained release
intravitreal brimonidine polymer is currently being eval-
uated in a randomized, double-blind, phase 2 study with
regard to safety and its effect on the progression of GA.
• RN6G (Pfizer), a humanized monoclonal antibody targeting
amyloid-beta, was found to be safe in patients with dry AMD
and is being studied in a phase 2 clinical trial.
• GSK933776 (GlaxoSmithKline) is another humanized mon-
oclonal antibody directed at amyloid-beta that is admin-
istered intravenously. A phase 2 multicenter, randomized,
double-masked, placebo-controlled study in patients with
GA is underway.

Light or photon therapy can affect several of the pathways

leading to macular damage from AMD. By targeting mitochondrial
respiration by direct absorption at cytochrome c oxidase (Cox),
leading to secondary and tertiary beneficial effects on cellular
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Photobiomodulation for the Clinical Treatment of Age-Related Macular Degeneration 875

metabolism, including anti-inflammatory, antioxidant, and anti-

apoptotic protection and recovery from injury, light appears to have
a very targeted “photoceutical” effect that just cannot be readily
obtained presently by pharmaceutical means.
PBM should therefore be useful as a stand-alone therapy and may
also have a synergistic effect in combination with other treatments
as the multifaceted pathways of AMD are elucidated.
There is a wealth of evidence that LLLT or PBM utilizing lasers
or other light sources (light-emitting diodes [LEDs]) is beneficial in
a host of clinical areas. As clinicians we are ready to ride the wave
of progress that has been provided by the researchers in this field.
Both healthcare providers and patients owe them a large debt of
gratitude.
Very little clinical research in utilizing PBM for ophthalmology
has been undertaken to date; however, there is an urgent need to
properly evaluate this treatment modality given, the large scale and
burden of not just AMD but other ophthalmic diseases that appear
in ever-increasing, almost epidemic proportions.
There is compelling evidence now from cellular, animal and
human studies regarding the benefits of PBM in AMD. David. H.
McDaniel has presented work on cultured RPE cells showing the
revitalization effect of 670 nm LED light at 3.2 J/cm2 continuous
wave after a 425 nm blue light at 30 J/cm2 . There was greater
than 90% cell death with blue light alone but only a 5% death rate
with blue light followed by red light treatment 12 min later. He has
also presented a sevenfold decreased relative vascular endothelial
growth factor (VEGF) expression in 4-week cultured human RPE
cells 24 h after exposure to a 590 nm (yellow) and 870 nm (pulsed
infrared) device at 0.1 J/cm2 .
Janis Eells is one of the foremost researchers of PBM in the
retinal field; her work with her colleagues has been an inspiration
to me and others to move PBM into the clinical sphere. She
has demonstrated that near-infrared (NIR)-LED photo-irradiation
increases the production of cytochrome oxidase in cultured primary
neurons and reverses the reduction of cytochrome oxidase activity
produced by metabolic inhibitors. She has also shown that NIR-LED
treatment prevents the development of oral mucositis in pediatric
bone marrow transplant patients.
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876 Photobiomodulation for the Clinical Treatment of Age-Related Macular Degeneration

Photobiomodulation improves wound healing in genetically

diabetic mice by upregulating genes important in the promotion
of wound healing. More recent studies have provided evidence for
the therapeutic benefit of NIR-LED treatment in the survival and
functional recovery of the retina and optic nerve in vivo after acute
injury by the mitochondrial toxin, formic acid generated in the
course of methanol intoxication. Gene discovery studies conducted
using microarray technology documented a significant upregulation
of gene expression in pathways involved in mitochondrial energy
production and antioxidant cellular protection. These findings pro-
vide a link between the actions of red to NIR light on mitochondrial
oxidative metabolism in vitro and cell injury in vivo. Based on these
findings and the strong evidence that mitochondrial dysfunction
is involved in the pathogenesis of numerous disease processes,
Eells proposed that NIR-LED photobiomodulation represents an
innovative and noninvasive therapeutic approach for the treatment
of tissue injury and disease processes in which mitochondrial dys-
function is postulated to play a role, including diabetic retinopathy,
AMD, Leber’s hereditary optic neuropathy, and Parkinson’s disease
[23].
The effect of PBM on deliberate non-human primate retinal
laser photocoagulation healing could not be better shown than the
photograph in Fig. 43.4.
In each experiment, one monkey was lased without LED
treatment and one lased with LED treatment (670 nm, 4 J/cm2 ).
A laser grid (128 spots delivered to the macula and perimacula)
was created in the central retina of right eye of each animal.
This grid consisted of grade I and II burns, photocoagulating the
photoreceptors and outer nuclear layer of the retina. Assessment of
the severity of the laser burn in LED-treated and untreated animal
demonstrated a greater that 50% improvement in the degree of
retinal healing at 1 month post-laser in the LED-treated monkey
(Fig. 43.3). In addition, the thickness of the retina measured at
the fovea by optical coherence tomography did not differ from
the pre-laser thickness in the LED-treated animal, whereas it was
50% thinner in the untreated animal (Fig. 43.3). Importantly, LED
treatment prevented the loss of cytochrome oxidase staining in the
lateral geniculate nucleus [31], clearly showing that the brain was
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Photobiomodulation for the Clinical Treatment of Age-Related Macular Degeneration 877

Figure 43.4 The effect of PBM on healing of deliberate laser retinal injury—
reported by Janis Eells.

responding to visual input from the “healed” retina in the LED-

treated animal much more effectively than in the untreated animal.
Begum et al. [24] have shown that treatment with 670 nm light
upregulates Cox expression and reduces inflammation in an AMD
mouse model. They asked if 670 nm exposure that can manipulate
the ageing mitochondria had the same ability in an aged mouse
model of AMD, the CFH knockout mouse, where inflammation is
a key feature. Further, they asked whether this occurred when
670 nm was delivered briefly in environmental lighting rather than
directly focused on the retina. They concluded that 670 nm was
effective in reducing inflammation probably via Cox activation in
mice with a genotype similar to that in 50% of AMD patients even
when brief exposures were delivered via environmental lighting.
Further, inflammation can be reduced independent of amyloid b.
The revealed efficacy supports current early stage clinical trials of
670 nm in AMD patients (a reference to the TORPA clinical trial).
Gkotski et al. [25] from the Institute of Ophthalmology, University
College, London have now demonstrated in vivo manipulation of
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878 Photobiomodulation for the Clinical Treatment of Age-Related Macular Degeneration

adenosine triphosphate (ATP) with 670 nm light in mouse retina and

brain.
“Progressive accumulation of age related mitochondrial DNA
mutations reduce ATP production and increase reactive oxygen
species output, leading to oxidative stress, inflammation and
degradation. The pace of this is linked to metabolic demand. The
retina has the greatest metabolic demand and mitochondrial density
in the body and displays progressive age related inflammation and
marked cell loss. Near infrared (670 nm) is thought to be absorbed
by Cox, a key element in mitochondrial respiration and it has been
demonstrated that it improves mitochondrial membrane potentials
in aged eyes. It also significantly reduces the impact of experimental
pathology and ameliorates age related retinal inflammation. We
show ATP decline with ageing in mouse retina and brain. Also, in
these tissues that ATP is significantly increased by 670 nm exposure
in old mice. In the retina this was associated with increased Cox and
reduced acrolein expression. Acrolein, being a free radical marker
of retinal oxidative stress, is up regulated in Alzheimer’s and retinal
degeneration. This is the first demonstration of ATP manipulation in
vivo and may provide a simple non-invasive route to combating age
related tissue decline.”
Natoli et al. [26] looked at the validity of using 670 nm red light
as a preventive treatment for retinopathy of prematurity (ROP) in
two animal models of oxygen-induced retinopathy (OIR). “670 nm
light reduced neovascularisation, vaso-obliteration and abnormal
peripheral branching patterns of retinal vessels in OIR. The neural
retina was also protected against OIR by 670 nm light exposure. OIR-
exposed animals had severe lung pathology, including hemorrhage
and edema, that was significantly reduced in 670 nm + OIR light-
exposed animals. There were no significance differences in the organ
weights of animals in the 670 nm light-exposed animals, and no
adverse effects of exposure to 670 nm light were detected.
Low levels of exposure to 670 nm light protects against OIR
and lung damage associated with exposure to high levels of oxygen,
and may prove to be a non-invasive and inexpensive preventative
treatment for ROP and chronic lung disease associated with
prematurity.”
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Photobiomodulation for the Clinical Treatment of Age-Related Macular Degeneration 879

We now move into the clinical realm of evaluation of PBM in

AMD patients: Boris and Tomislav [27] treated 203 patients with
both dry and wet AMD (with and without cataracts) for four sessions
of 40 s by trans-conjunctival irradiation with a 780 nm laser diode.
This resulted in a total dose of 1.2 J/cm2 . They concluded that LLLT
significantly improved visual acuity and that this was maintained for
3–36 months after treatment. No adverse events were observed.
This study, however, did not have IRB approval, and there
were some questions regarding the methodology and prospective
status of the study in that it did not appear to adhere to rigorous
standardized ophthalmology trial parameters; however, the results
were positive and encouraging.
With the collaboration of clinical researchers at the University
of Toronto, Canada, we decided to undertake an IRB-approved
prospective interventional case series (pilot) study utilizing LEDs.
This was the first study globally to use LED-based PBM in dry AMD—
The Toronto and Oak Ridge study of Photobiomodulation in AMD
(TORPA). The study is summarized as follows:
We designed this study to look at the effect of PBM with low-
powered LED (non-coherent) devices shone through the pupil in
patients with dry AMD. The study was designed as a prospective
non-randomized interventional case series.
Inclusion criteria were documented dry AMD, best corrected
visual acuity (BCVA) of 20/20 to 20/200, and older than 50
years of age. Excluded from the study were subjects with previous
or active wet AMD, with a previous history of epilepsy, with
cognitive impairment, other retinal disease, previous retinal surgery,
significant media opacity or contraindications to dilation drops.
The non-presence of neovascularization was ascertained prior
to enrollment by examination with ocular coherence tomography
(OCT) and intravenous fluorescein angiography (IVFA) and con-
firmed by a retina specialist. All subjects were assessed for visual
acuity with ETDRS charts at 4 m distance (Precision Vision, USA)
recorded in log MAR units, contrast sensitivity at 1.5 and 3 cycles
per degree (Stereo Vision Optec 6500, USA) recorded as log contrast
sensitivity and for fixation stability with the Nidek MP1 micro-
perimeter (Nidek Technologies, Padova, Italy). Accurate estimates of
fixation stability could be obtained from raw data provided by the
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880 Photobiomodulation for the Clinical Treatment of Age-Related Macular Degeneration

instrument by the calculation of a bi-curve ellipse area (BCEA) [28].

Calculations are based on the minor and major axes of an elliptic
area covering fixational eye movements and take into account two
standard deviation measures of each recorded eye movement. The
results are expressed in square degrees. Measurements took place
prior to treatment, immediately following the treatment protocol (6
weeks), 4, 6, and 12 months after.
The intervention consisted of using LLLT in the yellow and red
to NIR range using low-energy delivery with the Warp10 (Quantum
Devices) and the Gentlewaves (Light Bioscience) instruments.
The instruments used are commercially available and have been
approved for use in other conditions by the FDA and Health Canada.
The FDA considers one of the devices a non-significant risk device
for using on the eye.
The treatment parameters followed for the Warp10 delivery
system were 670 nm ± 15 nm at 50–80 mW/cm2 , 4.0–7.68 J/cm2 ,
for 88 ± 8 s. The treatment parameters followed for the Gentlewaves
delivery system were 590 nm ± 8 nm at 4 mW, 790 nm ± 60 nm at
0.6 mW, for 35 s, pulsed at 2.5 Hz (250 ms on, 150 ms off) while
delivering 0.1 J/cm2 /treatment. All subjects were treated with the
two devices used sequentially at each treatment visit for a total of
18 treatments over a 6-week period (thrice per week for 6 weeks).
The primary outcome measures selected for analysis were
visual acuity, contrast sensitivity, and fixation stability estimates.
Data analysis was based on descriptive statistics that include
frequency distributions, a measure of central tendency (mean),
and a measure of dispersion (standard deviation). A statistical
comparison of means between populations was made by t-test
and repeated measures analysis of variance (repeated measures
ANOVA). Differences were considered to be statistically significant
at p values of less than 0.05. The study was performed in adherence
to the guidelines of the Declaration of Helsinki. The study protocol
was approved by an independent Research Ethics Committee (IRB
Services, Aurora, Canada). Informed consent was obtained from all
participants.
Over a span of 12 months 18 AMD study eyes (6 males and 12
females) were recruited and treated, aged 61 to 90 years old (mean
74.3 years/SD 7.7).
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Photobiomodulation for the Clinical Treatment of Age-Related Macular Degeneration 881

0.9 0.9
0.8 0.8
0.7 0.7
0.6 0.6
0.5 0.5

OUTCOME
OUTCOME

0.4 0.4
0.3 0.3
0.2 0.2
0.1 0.1
0.0 0.0
-0.1 -0.1
-0.2 -0.2
-0.3 -0.3
0 1 2 3 4 5 6 7 8 9 10 11 12
TIME

Figure 43.5 log ETDRS VA. There is an immediate post-treatment improve-

ment in visual acuity that declines slightly at 6 months; however it remains
at highly statistically significant levels at 12 months.

Average ETDRS BCVA for the AMD group was measured at 0.25 log
MAR units before the treatment and at 0.13 log MAR units 12 months
after the treatment ( p <0.0001).
Repeated measures ANOVA yielded F(4,68) = 18.86, p less than
0.0001 (Fig. 43.5).

Contrast sensitivity: Repeated measures ANOVA for contrast sensi-

tivity (3 cycles/degree): F(4,68) = 11.44, p less than 0.0001 (Fig.
43.6).

Fixation stability: Repeated measures ANOVA for fixation stability

(BCEA): F(4,68) = 0.90, p less than 0.4661.

Correlation analysis between fixation stability and ETDRS VA: Pear-

son R value of 0.6776, p less than 0.001.
Photobiomodulation, also known as low-level laser therapy, has both
primary and secondary effects on cellular responses to disease,
including anti-inflammatory, antioxidant, and anti-apoptosis effects.
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882 Photobiomodulation for the Clinical Treatment of Age-Related Macular Degeneration

3 3

2 2
OUTCOME

OUTCOME
1 1

0 0
0 1 2 3 4 5 6 7 8 9 10 11 12
TIME

Figure 43.6 log contrast sensitivity at 3 cycles per degree. There is an

immediate improvement in contrast sensitivity following the treatment
with a slight decline at 6 to 7 months, however remaining at highly
statistically significant levels at 12 months.

RPE is the major local source of CFH at the retina/choroid interface.

Mutations or downregulation of CFH may increase the chance of
RPE cells being attacked by activated complement systems. Damage
caused by oxidative stress and inflammation leads to progressive
loss of cell function and thus contributes to the development of
atrophic AMD. Genes in different pathways influence progression to
different stages of AMD. The genes CFH, C3, CFB, and ARMS2/HTRA1
have been associated with progression from intermediate drusen
to large drusen, and from large drusen to GA or NV [29]. By
altering gene expression, PBM can influence factors involved in the
progression of AMD, such as VEGF and inflammatory cytokinins.
Recently, there has been interest in tissue sparing or sub-
threshold laser at 577 nm and 810 nm to produce therapeutic
effects without clinical evidence of intra-retinal damage [30]. It
has been proposed that the benefits might be due to the up- and
downregulation of angiogenic growth factors (e.g., VEGF) [31–34]
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Photobiomodulation for the Clinical Treatment of Age-Related Macular Degeneration 883

mediated by the biological reaction of RPE cells that have been

only sub-lethally injured. We feel that the same benefits to cellular
function can occur with PBM, and that there is no damage to any
cells with the low-powered LED light sources used in our study.
We used fixation stability, a novel testing parameter as one of
our primary outcome measures, and although there were changes
both in the BCEA and PRL location after the treatment, these
were not statistically significant. However, correlation analysis
between visual acuity and fixation stability was improved after the
treatment—further evidence of a treatment effect.
Average ETDRS visual acuity was statistically significantly im-
proved immediately following the treatment, and this improvement
remained at statistically significant levels at 12 months although
some decline in the ETDRS log MAR score is evident after 4 months.
This would suggest that some patients would benefit from re-
treatment somewhere after the 4-month interval. There were no
significant adverse events noted during the course of the study.
One subject with a history of migraine headaches felt that she was
more susceptible to getting a migraine after one treatment session.
However, it did not occur and this sensation lasted less than 1 h.
We believe the results obtained warrant further evaluation of
PBM as an important, safe, and effective treatment in this potentially
devastating disease where there are no proven treatments to date.
The study was presented at ARVO 2012 and also at WALT 2012 [35].
We have been evaluating PBM treatment to patients with dry
macular degeneration in my private practice with a modified
protocol using the same study devices as an “off label” treatment
in Canada. These subjects are not part of an IRB-approved study;
however, treatment and data collection adhere to the same study
standards as TORPA. In addition to ETDRS VA and contrast
sensitivity data collection, he is now collecting high-resolution
spectral domain OCT and fundus autofluorescence scans on all
patients.
The preliminary data of more than 90 subject eyes nicely support
the visual acuity and contrast sensitivity gains seen in the TORPA
study. In addition, there are some cases where anatomical changes
in drusen morphology are seen in conjunction with visual acuity
improvement.
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884 Photobiomodulation for the Clinical Treatment of Age-Related Macular Degeneration

Figure 43.7 (Subject 1) Pretreatment detailed OCT section through a large

central drusen.

In one case (subject 1), there has been a reduction in the height
of a central druse by 11% associated with an increase of 8 letters
of visual acuity, and this was seen immediately after a treatment
protocol of 6 weeks (Figs. 43.7–43.9). In another case, the initial
pretreatment scan a 55-year-old male (subject 2) obtained a 5-letter
gain in visual acuity immediately following the treatment and at the
subsequent scan at 7 months. There was a very significant reduction
in his drusen at the 7-month scan (Figs. 43.1 and 43.11).
Several more cases have been identified where drusen reduction
is seen immediately following a treatment course. This is the first
time anatomical evidence of drusen reduction with serial OCT
scanning has been reported along with benefits to vision and
contrast sensitivity. These and other cases are presently being
collated for publication. A selection of eight cases were presented
at NAALT/WALT 2014 in Washington, D.C.
Lumithera Inc., an ophthalmological medical device company,
is anticipating starting a prospective multicenter clinical trial that
compares PBM treatment arms to placebo or sham groups.
We expect to see that PBM will become an accepted proven
modality for the treatment of dry AMD in the near future,
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Photobiomodulation for the Clinical Treatment of Age-Related Macular Degeneration 885

Figure 43.8 Subject 1 with an approximate 11% reduction in central retinal

thickness immediately following the treatment.

Figure 43.9 Subject 1 with a further reduction in her druse three months
from the initial scan.
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886 Photobiomodulation for the Clinical Treatment of Age-Related Macular Degeneration

Figure 43.10 Subject 2 pretreatment scan.

Figure 43.11 Subject 2 at 7 months after his pretreatment scan.

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References 887

easily administered with no adverse events, providing an active

substantive treatment and hope for the future.

References

1. Rein DB, Wittenborn JS, Zhang X, Honeycutt AA, Lesesne SB, Saaddine
J, and Vision Health Cost-Effectiveness Study Group. Forecasting age-
related macular degeneration through the year 2050: The potential
impact of new treatments. Arch Ophthalmol, 2009; 127(4): 533–540.
2. Access Economics, prepared for AMD Alliance International, The Global
Economic Cost of Visual Impairment, March 2010 (all costs are reported
in 2008 US dollars) March 16, 2010.
3. Anderson D, Ozaki S, Nealon M, Neitz J, Mullins R, Hageman G. et al.
Local cellular sources of apolipoprotein E in the human retina and
retinal pigmented epithelium: Implications for the process of drusen
formation. Am J Ophthalmol, 2001; 131: 767–781.
4. Mullins RF, Johnson LV, Anderson DH, and Hageman GS. Character-
ization of drusen-associated glycoconjugates. Ophthalmology, 1997;
104(2): 288–294.
5. Russell SR, Mullins RF, Schneider BL, and Hageman GS. Location,
substructure, and composition of basal laminar drusen compared with
drusen associated with aging and age-related macular degeneration. Am
J Ophthalmol, 2000; 129(2): 205–214.
6. Mullins RF, Aptsiauri N, and Hageman GS. Structure and composition of
drusen associated with glomerulonephritis: Implications for the role of
complement activation in drusen biogenesis. Eye, 2001; 15(Pt 3): 390–
395.
7. Johnson LV, Leitner WP, Rivest AJ, Staples MK, Radeke MJ, and Anderson
DH. The Alzheimer’s A beta-peptide is deposited at sites of complement
activation in pathologic deposits associated with aging and age-related
macular degeneration. Proc Natl Acad Sci USA, 2002; 99(18): 11830–
11835.
8. Abecasis GR, Yashar BM, Zhao Y, Ghiasvand NM, Zareparsi S, Branham
KE. et al. Age-related macular degeneration: A high-resolution genome
scan for susceptibility loci in a population enriched for late-stage
disease. Am J Hum Genet, 2004; 74(3): 482–494.
9. Iyengar SK, Song D, Klein BE, Klein R, Schick JH, Humphrey J. et al.
Dissection of genome wide scan data in extended families reveals
July 6, 2016 17:40 PSP Book - 9in x 6in 43-Hamblin-c43

888 Photobiomodulation for the Clinical Treatment of Age-Related Macular Degeneration

a major locus and oligogenic susceptibility for age-related macular

degeneration. Am J Hum Genet, 2004; 74(1): 20–39.
10. Klein ML, Schultz DW, Edwards A, Matise TC, Rust K, Berselli CB. et al.
Age-related macular degeneration. Clinical features in a large family
and linkage to chromosome 1q. Arch Ophthalmol, 1998; 116(8): 1082–
1088.
11. Majewski J, Schultz DW, Weleber RG, Schain MB, Edwards AO, Matise
TC. et al. Age-related macular degeneration: A genome scan in extended
families. Am J Hum Genet 2003; 73(3): 540–550.
12. Schick JH, Iyengar SK, Klein BE, Klein R, Reading K, Liptak R. et al. A
whole-genome screen of a quantitative trait of age-related maculopathy
in sibships from the Beaver Dam Eye Study. Am J Hum Genet, 2003;
72(6): 1412–1424.
13. Seddon JM, Santangelo SL, Book K, Chong S, and Cote J. A genome
wide scan for age-related macular degeneration provides evidence for
linkage to several chromosomal regions. Am J Hum Genet, 2003; 73(4):
780–790.
14. Weeks DE, Conley YP, Mah TS, Paul TO, Morse L, Ngo-Chang J. et al. A full
genome scan for age-related maculopathy. Hum Mol Genet, 2000; 9(9):
1329–1349.
15. Weeks DE, Conley YP, Tsai HJ, Mah TS, Rosenfeld PJ, Paul TO. et al. Age-
related maculopathy: An expanded genome-wide scan with evidence of
susceptibility loci within the 1q31 and 17q25 regions. Am J Ophthalmol,
2001; 132(5): 682–692.
16. Weeks DE, Conley YP, Tsai HJ, Mah TS, Schmidt S, Postel EA. et al. Age–
related maculopathy: A genome wide scan with continued evidence of
susceptibility loci within the 1q31, 10q26, and 17q25 regions. Am J Hum
Genet, 2004; 75(2): 174–189.
17. Souied EH, Benlian P, Amouyel P, Feingold J, Lagarde JP, Munnich A. et al.
The epsilon4 allele of the Apolipoprotein E gene as a potential protective
factor for exudative age-related macular degeneration. Am J Ophthalmol,
1998; 125(3): 353–359.
18. Klaver CC, Kliffen M, van Duijn CM, Hofman A, Cruts M, Grobbee DE.
et al. Genetic association of Apolipoprotein E with age-related macular
degeneration. Am J Hum Genet, 1998; 63(1): 200–206.
19. Baird PN, Guida E, Chu DT, Vu HT, and Guymer RH. The epsilon2 and
epsilon4 alleles of the apolipoprotein gene are associated with age-
related macular degeneration. Invest Ophthalmol Vis Sci, 2004; 45(5):
1311–1315.
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20. Gold B, Merriam JE, Zernant J, Hancox LS, Taiber AJ, Gehrs K. et al.
Variation in factor B (BF) and complement component 2 (C2) genes
is associated with age-related macular degeneration. Nat Genet, 2006;
38(4): 458–462.
21. Fisher SA, Abecasis GR, Yashar BM, Zareparsi S, Swaroop A, Iyengar
SK. et al. Meta-analysis of genome scans of age-related macular
degeneration. Hum Mol Genet, 2005; 14(15): 2257–2264.
22. A randomized, placebo-controlled, clinical trial of high-dose supple-
mentation with vitamins C and E, beta carotene, and zinc for age-
related macular degeneration and vision loss: AREDS report no. 8. Arch
Ophthalmol, 2001; 119(10): 1417–1436.
23. Eells JT, Wong-Riley MT, VerHoeve J, Henry M, Buchman EV, Kane
MP, et al. Mitochondrial signal transduction in accelerated wound and
retinal healing by near-infrared light therapy Mitochondrion, 2004; 4(5–
6): 559–567.
24. Begum R, Powner MB, Hudson N, Hogg C, and Jeffery G. Treatment with
670 nm light up regulates cytochrome c oxidase expression and reduces
inflammation in an age-related macular degeneration model. PLoS ONE,
2013; 8(2): e57828. doi:10.1371/journal.pone.0057828
25. Gkotsi D, Begum R, Salt T, Lascaratos G, Hogg C, Chau KY, et al.
Recharging mitochondrial batteries in old eyes. Near infrared increases
ATP. Exp Eye Res, 2014; 122: 50–53 http://dx.doi.org/10.1016/j.exer.
2014.02.023.
26. Natoli R, Valter K, Barbosa M, Dahlstrom J, Rutar M, et al. 670
nm photobiomodulation as a novel protection against retinopathy of
prematurity: Evidence from oxygen induced retinopathy models. PLoS
ONE, 2013; 8(8): e72135. doi:10.1371/journal.pone.0072135.
27. Ivandic BT and Ivandic T. Low-level laser therapy improves vision in
patients with age-related macular degeneration. Photomed Laser Surg,
2008; 26(3): 241–245. doi:10.1089/pho.2007.2132.
28. Tarita-Nistor L, González EG, Markowitz SN, and Steinbach MJ. Fixation
characteristics of patients with macular degeneration recorded with the
MP-1 micro perimeter. Retina, 2008; 28: 125–133.
29. Yu Y, Reynolds R, Rosner B, Daly MJ, and Seddon JM. Prospective
assessment of genetic effects on progression to different stages of age-
related macular degeneration using multi-state Markov models. Invest
Ophthalmol Vis Sci, 2012; 53(3): 1548–1556.
30. Kiire C, Sivaprasad S, and Chong V. Subthreshold micropulse laser
therapy for retinal disorders. Retina Today, 2011.
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890 Photobiomodulation for the Clinical Treatment of Age-Related Macular Degeneration

31. Barnstable CJ and Tombran-Tink J. Neuroprotective and antiangiogenic

actions of PEDF in the eye: Molecular targets and therapeutic potential.
Prog Ret Eye Res, 2004; 23(5): 561–577.
32. Glaser BM, Campochiaro PA, Davis JL, and Jerdan JA. Retinal pigment
epithelial cells release inhibitors of neovascularization. Ophthalmology,
1987; 94: 780–784.
33. Miller H, Miller B, and Ryan SJ. The role of retinal pigment epithelium
in the involution of subretinal neovascularization. Invest Ophthalmol Vis
Sci, 1986; 27: 1644–1652.
34. Ogata N, Tombran-Tink J, Jo N, Mrazek D, and Matsumura M. Upregula-
tion of pigment epithelium-derived factor after laser photocoagulation.
Am J Ophthalmol, 2001; 132(3): 427–429.
35. Merry G, Dotson R, Devenyi R, Markowitz S, and Reyes S. Photobiomod-
ulation as a new treatment for dry age related macular degeneration.
Results from the Toronto and Oak Ridge Photobimodulation Study in
AMD (TORPA). ARVO Meeting Abstracts, 2012; 53: 2049.
July 6, 2016 17:40 PSP Book - 9in x 6in 44-Hamblin-c44

Chapter 44

Laser (Light) Therapy for Postherpetic

Neuralgia

Kevin C. Moorea and R. Glen Calderheadb

a Department of Anaesthesia, Royal Oldham Hospital, Rochdale Road, Oldham,

Manchester OL1 2JH, UK

b Research Division, Clinique L Dermatology, 219 Sowon-ro, Goyang City, Gyeonggi,

South Korea
kevincmoore@outlook.com

44.1 Overview of Postherpetic Neuralgia

44.1.1 Aetiology
Postherpetic neuralgia (PHN) is a neuropathic complication that
may follow an attack of herpes zoster (HZ). The name “herpes
zoster” is derived from the Greek and literally means a creeping
girdle or belt, which aptly describes the appearance in the acute
phase. It is commonly referred to as shingles, a later Latin derivation,
again meaning a belt or girdle, and was popularly called “the ring of
fire,” which is a misnomer because the disease almost always occurs
unilaterally. There was a folk belief that if the rash ever met in the
middle of the chest, the sufferer would die.

Handbook of Low-Level Laser Therapy

Edited by Michael R. Hamblin, Marcelo Victor Pires de Sousa, and Tanupriya Agrawal
Copyright c 2017 Pan Stanford Publishing Pte. Ltd.
ISBN 978-981-4669-60-3 (Hardcover), 978-981-4669-61-0 (eBook)
www.panstanford.com
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892 Laser (Light) Therapy for Postherpetic Neuralgia

The cause of HZ is the varicella zoster virus, which remains

dormant in the cell bodies and occasionally in the ganglia of the
dorsal roots or cranial nerves following a bout of chickenpox. The
virus may be reactivated when the body is stressed due to major
surgery, inter-current infection, or any intervention that impairs the
body’s immune system. As mentioned above, the classic shingles
rash is unilateral and affects one or more dermatomes, usually in the
thoracic or upper lumbar region, but may affect the cephalic area or
even the limbs.

44.1.2 Incidence
More than 70% of cases give a history of varicella in earlier life.
The pain and discomfort of an acute HZ infection usually settle as
the rash and vesicles heal, but if it persists for 3 months or more,
it is then referred to as PHN. While less than 10% of HZ sufferers
under the age of 60 years will develop PHN, the incidence rises
dramatically in old age and approaches 40–50% in the elderly. This
is particularly so in patients who are debilitated or whose immune
systems are compromised. When shingles affects the cephalic
nerve distribution, the possibility of an ensuing attack of PHN is
significantly higher and its treatment a great deal more difficult.

44.1.3 Signs and Symptoms

These are due to neurochemical, physiological, and anatomical
changes in the afferent sensory nerves and posterior root ganglions
in the affected dermatomes and are typified by the experience
of pain, which may be mild, moderate, or severe. Pain may be
described as burning, sharp, stabbing, deep-seated, or a constant
ache. There may be sensitivity to light touch (allodynia) and itching
or numbness. Although PHN may resolve spontaneously, symptoms
can persist for months, years, decades, or even for life. It can be an
extremely debilitating condition, particularly in the elderly, with a
marked effect on patients’ quality of life and, in severe intractable
cases, even inciting suicidal tendencies.
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Laser (Light) Therapy 893

44.1.4 Treatment Options

No single treatment will relieve PHN in all patients. Therefore, the
range of treatment options is wide and varied. Analgesia using
non-opiates may be helpful, but usually more potent opiates
are required. Other pain-modifying medications include tricyclic
antidepressants and anticonvulsants. Topical applications include
lidocaine skin patches, capsaicin cream, and aspirin mixtures.
Nonpharmacological treatments may be trialed, including acupunc-
ture, transcutaneous nerve stimulation, complementary therapy,
cryotherapy, and hypnosis. Occasionally, spinal cord stimulators are
inserted. Antiviral agents used at the onset of the acute attack
may reduce the incidence of PHN. An HZ live vaccine is available
and is recommended for all adults over the age of 50 whose
immune systems are not compromised. This vaccine may reduce the
incidence of HZ in the elderly by about half and thus also reduce the
possible complication of an ensuing attack of PHN.

44.1.5 Prognosis
It follows from the above that frequently a combination of
treatments is required, which can produce unwanted side effects.
Many of the drugs used, especially in the dosage required, are not
well tolerated by the elderly who are frequently taking medication
for coexisting conditions. As a consequence, PHN can be extremely
troublesome for many years, disrupting lifestyles, destroying the
work ethic, and interfering with social interaction. An effective
treatment free of side effects would be a considerable advance in
relieving the symptoms of PHN.

44.2 Laser (Light) Therapy

44.2.1 History
The initial laser system used for medical therapy was the helium–
neon (He–Ne) laser, emitting in the visible red band at 632.8
nm. During the late 1960s and early 1970s, this pioneering work
in phototherapy was spearheaded by Endre Mester in Hungary,
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894 Laser (Light) Therapy for Postherpetic Neuralgia

treating a wide range of body surface lesions and indolent ulcers

[16]. The late 1970s and early 1980s saw the development of
infrared (IR) laser beams, one of the earliest being the gallium–
aluminum–arsenide (GaAlAs) diode-based laser with a continuous
wave output power of 20–1000 mW at the wavelength of 830
nm. With its greater depth of penetration, this system became the
focus for researchers in treating more deep-seated pain conditions.
While the initial stimulus for this work came from Japan by the late
1980s, reports from around the world were raising interest in its
potential for treating chronic pain, and it became known as low-level
laser therapy, with the acronym LLLT, which first appeared in the
literature in 1988 [13, 25, 31].
However, the medico-scientific community remained skeptical,
particularly as many published reports were anecdotal without
accurate treatment regimes and detailed treatment protocols
[4]. Despite these drawbacks, a number of respected scientific
researchers published work that helped to explain the biophysics,
photo-biological tissue effect, and optoelectronic rationale for the
use of this new therapy [10, 18, 22, 32]. As interest in its use for
treating painful conditions spread, the first reports of its clinical
use for PHN began to appear. One of the first English-language
papers described a double-blind crossover trial of patients with
well-established PHN, which had failed to respond to a wide variety
of conventional treatment options [17]. The laser used for the trial
was a GaAlAs diode laser emitting a continuous wave output of 60
mW at 830 nm. The treatment protocol was described in detail, and
pain severity and distribution measurements were taken prior to
each therapy session. Mid- (crossover point) and end-point values of
the trial were subjected to statistical analysis. At the mid-trial point,
the differences in both values were significantly different, but at the
end of the trial, the differences were not significant, although still
in favor of Group A (Fig. 44.1). Ninety-five percent of trial patients
were able to discontinue previous medication and long-term follow-
up showed this improvement to be maintained in 80%. Only 2%
needed to recommence medication. The reduction in pain severity
varied between 40% and 95% (mean 74%) and in pain distribution
by 49–84% (mean 64%). It was concluded that 830 nm LLLT offered
a new potential method of treating PHN.
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Laser (Light) Therapy 895

Figure 44.1 Graphical representation based on the data from Ref. [10]
comparing the response from two groups of PHN patients in this crossover
study. Group A started as the laser diode (LD)-LLLT group with Group B the
unirradiated controls until the crossover point in mid-study, when the roles
were reversed. The figure shows the effect on both pain severity and pain
distribution. At the crossover point, the scores for both pain severity and
pain distribution were statistically significantly better in Group A than in
Group B. Interestingly, after the crossover point, Group A showed a small
but continued improvement even though they were the control group in the
latter half of the study, and their final pain scores were better than those of
Group B, although without significance.

44.2.2 Clinical Research

Within 2 years of the aforementioned report, clinicians from a
number of centers were publishing their own trial results. Most of
these used the same GaAlAs laser and trial protocol as in the above
with only minor differences in the treatment regimes. This meant
that there should be good correlation between the trial results, and
so it proved to be with only minor variations (nonsignificant) in pain
level scores and end trial pain relief achieved [9, 11]. A trial using a
904 nm IR laser produced similar results [15]. New textbooks on
the subject of laser therapy also included a chapter on the treatment
of PHN [23]. What was needed now were greater trial population
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896 Laser (Light) Therapy for Postherpetic Neuralgia

numbers, and so there was a lull in pure PHN papers with their
being included in more widespread pain clinic reports [19, 28].
Conference reports and proceedings, however, continued to record
the wide interest in its potential nowhere more so than in Japan
where Japanese-language papers on the subject were published on a
regular basis. A 1992 paper presented to a Congress in Bologna, Italy,
detailed the cost-effective benefits of using laser therapy in PHN
[20]. The report reviewed the treatment of 100 patients and detailed
the cost over a 1-year period of medical equipment, therapist time,
and prescribed medication. As well as improving the quality of life
in PHN sufferers by 71%, the group treated with laser therapy
demonstrated a total 1 year of treatment cost saving of 28% when
compared with the group treated with conventional means. There
still remained some 15–20% of sufferers who failed to respond
to therapy. Case records evaluation suggested that these patients
fell into one or all of the following groups: patients with severe
debility with an American Society of Anesthesiologists (ASA) Grade
of Physical Fitness of 3 or 4; patients with secondary malignant
disease especially if receiving radiotherapy and/or chemotherapy;
or patients taking polypharmacy, particularly if it included systemic
steroids. Undoubtedly, the suppression of the immune system so
commonly found in all the above played a major role in resistance
to therapy.
By the mid-1990s, the focus of clinical research into the disease
began to change. A 1995 editorial asked the question “Does LLLT
Prevent Postherpetic Neuralgia?” [12] There had already been
anecdotal case reports of the effectiveness of early exposure to
laser therapy in reducing the pain and limiting the duration of the
acute HZ attack, but this editorial positively linked it to a reduced
incidence of subsequent PHN. Certainly, the focus now seemed to
be on prevention and not cure [24]. The other factor that was to
impinge on research was a new generation of laser equipment that
was lighter and more portable with a greater variety of treatment
parameters. Increased beam power and pulsed emission allowed a
wide variety of frequency outputs and new cluster probe heads gave
a much greater power density.
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Laser (Light) Therapy 897

44.2.3 Mechanisms of Action of LLLT

Although all the mechanisms related to the reported efficacy of
LLT in pain attenuation, especially in neuralgias, have not been
fully elucidated, enough has been proved to advance a strong
hypothesis as to the mechanism of action in the control of HZ,
and the attenuation of the pain and neural system hypersensitivity
associated with PHN. In the case of HZ, the proven anti-inflammatory
effect of light in the “phototherapeutic” waveband from 620 nm
to 1000 nm could play a major part in controlling the progress
of the disease and preventing vesicle formation [3]. The control
of inflammatory cytokines and recruitment of anti-inflammatory
agents, release of growth factors from dermal cells and epidermal
keratinocytes, release of nitric oxide, increase in the extracellular
levels of adenosine triphosphate (ATP) are all associated with LLLT
stimulation of tissue. Add to this the potential for LLLT to inhibit
viral replication [6, 21], and there is a strong argument for the
efficacy of LLLT in controlling HZ, and potentially preventing the
eventual onset of PHN, as argued earlier.
Pain control in PHN has been well-demonstrated in the articles
cited earlier. The abnormal antidromic activity in the nerves
associated with the affected dermatome was reportedly well-
controlled with capsaicin [8], so a strong argument could be raised
that the electromagnetic nature of LLLT could have a similar
effect in controlling the abnormal axonal flow, particularly with
the deeper penetration of the near-infrared wavelengths, and a
treatment protocol that involves irradiating dorsal root entry and
exit zones in the intervertebral spaces associated with the affected
dermatomes before treating the area of interest. Random neuronal
firing, consisting of sharp spikes and bursts of rapid firing, has been
associated with neuropathic pain [29]. Ephaptic transmission occurs
between adjacent nerves, so random firing patterns can be induced
in a normal axon, and when this occurs in the chronic situation, the
phenomenon of kindling appears whereby the hyperexcited neural
system enters a vicious circle through which hyperexcited nerves
come to believe that the abnormal activity is normal. LLLT at 830 nm
has been shown to control abnormal firing in clonic spasm activity
[1, 2, 33]; therefore, it is not inconceivable that LLLT is a potent
mediator in the neurogenic imbalance associated with PHN.
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898 Laser (Light) Therapy for Postherpetic Neuralgia

44.3 Enter the Light-Emitting Diode

44.3.1 Background
Clinical reports of trials with LD-LLLT for PHN continued to
appear [27, 36]. The impetus behind the initial enthusiasm seemed,
however, to have waned. It was also suggested that the therapeutic
light source did not have to be coherent and that comparable
results could be gained by using superluminescent diodes (SLDs)
and light-emitting diodes (LEDs) as with laser energy, particularly
if a polarizing filter was used [7].
Up until the late 1990s, the use of LEDs as a therapeutic source
was really clinically not practical, because although old-generation
LEDs were bright, cheap, and cheerful, they had low and unstable
output powers, had an extremely divergent beam, and emitted at
a waveband rather than a wavelength. It was easy to source a red
LED, but almost impossible to source a 633 nm LED. SLDs were
noncoherent but emitted light in a narrow waveband and were,
therefore, quasimonochromatic. They were, however, quite costly,
required a complex driver circuitry, and needed fairly robust cooling
to maintain emission at the rated wavelength. Nevertheless, their
efficacy compared to an LD-based system was very low, as seen in
one article comparing 830 nm LD and SLD energy in flap survival in
an animal model [14].

44.3.2 The “NASA LED”

In 1998, the so-called “NASA LED” was developed by Whelan
et al., working at the NASA Space Medicine Laboratory [34, 35].
These LEDs had an output several orders of magnitude greater
than their predecessors, had a smaller angle of divergence, and
was quasimonochromatic, with over 95% of the emitted photons
at the rated wavelength. Phototherapy had a new clinically useful
light source, low-level laser therapy became low-level light therapy,
though the acronym, LLLT, remained unchanged. Since there was a
wealth of literature on LLLT with laser diodes (LD-LLLT), it made
a lot of sense to source LEDs at the wavelengths proven to elicit
a good response in pain attenuation, wound healing, and so on, so
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Enter the Light-Emitting Diode 899

Figure 44.2 An example of a state-of-the-art LED phototherapy system.

Note the hinged LED panel arrays comprising the treatment head to allow
the head to be adjusted to suit the contour of any part of the body, flat for
the back, rounded for the face, or molded around a limb.

LED arrays emitting at 633 nm and 830 nm were developed and

became commercially available from the early 2000s, which quickly
proved extremely effective in all aspects of pain attenuation and
wound healing [5]. State-of-the-art LED-LLLT systems have planar
arrays that contain more than 1000 LEDs, meaning that the active
area is large and can, therefore, treat a large area of target tissue
in a hands-free manner, allowing the clinician or therapist to do
something else while the treatment is in progress, compared with
the typical point-by-point treatment of probe-mounted LD-LLLT
systems. Figure 44.2 shows an example of a new-generation LED-
LLLT system, with hinged planar arrays to allow even irradiation of
any body contour.
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900 Laser (Light) Therapy for Postherpetic Neuralgia

44.3.3 Efficacy of LED Sources

LEDs have a much lower photon intensity than LDs and are
noncoherent, but they are quasimonochromatic with more than
95% of the photons at the rated wavelength. This means that LED-
LLLT can achieve the same degree of target specificity as an LD-
based system, but over large areas. When LEDs are mounted in
planar arrays, the multiple intersection of the divergent light energy
induces the phenomenon of photon interference, intensifying the
potential effect of the beam through improving directionality of
the photons. Add to this the scattering efficiency of near-infrared
light, and the greatest photon intensity is not at the skin surface,
but beneath the surface, which is where it should be to target
the epidermal and dermal skin cells, vascular plexi, and, most
importantly for HZ and PHN treatment, the cutaneous neural
network. The large active area of an LED-based system can easily
cover multiple intervertebral dorsal root zones, in addition to
treating the entire lesion in a hands-free manner.

44.3.4 Clinical Evidence

The case of acute HZ in a 21-year-old female illustrated in Fig. 44.3
was treated with a continuous wave (CW) 830 nm LED-LLLT system,
with the hinged panel arrays wrapped around the torso, anterior
and posterior, to cover the HZ-affected area. The dose per session
was 60 J/cm2 , three sessions given in one week, two days apart, and
the result was excellent with total resolution by the end of the week.
This was one patient from a series of six HZ subjects, all of whom
experienced resolution of the HZ and associated severe discomfort
in 6–8 days (personal communication and unpublished data, Mario
A Trelles MD PhD, Instituto Médico Vilafortuny, Spain).
Figure 44.4 is based on data from a controlled study on LED-LLLT
following full-face ablative laser resurfacing in 60 patients [30], 30
of whom received the 830 nm LED-LLLT (60 J/cm2 , immediately
after treatment, 24 h and 72 h after treatment, then two more
sessions the following week, 3 days apart). The LED-treated group
healed significantly faster, better than half the time required for
the control group. This was not a study on HZ or PHN, but it
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Enter the Light-Emitting Diode 901

Figure 44.3 Acute-stage HZ in a 21-year-old female treated with 830 nm

LED-LLLT. (a, c) Baseline findings showing the typical hemi-girdle inflamed
and eruptive lesion involving the left chest and back. (b, d) The result
showing total resolution of the lesions 1 week after baseline following three
LED sessions, 2 days apart (Clinical photography courtesy: Mario A Trelles
MD PhD, Cambrils, Spain).

did illustrate the significant LED-LLLT-mediated reduction in pain

and edema, both of which are associated with HZ and PHN. More
relevantly, an article was published recently on 830 nm LED-LLLT
for acute HZ ophthalmicus [26]. The controlled pilot study compared
oral famcyclovir alone with 830 nm LED-LLLT (CW, 60 J/cm2 , four
sessions, 3 days apart) in addition to the oral famcyclovir in 28
patients. The LED-treated group showed resolution of both the
lesions and the pain significantly faster than the control group
( p = 0.006).
One clinic in Seoul, South Korea, has started to use 830 nm
LED-LLLT in a controlled study on PHN, the incidence of which
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902 Laser (Light) Therapy for Postherpetic Neuralgia

Figure 44.4 Results of a controlled study on LED phototherapy after full-

face ablative Er:YAG/CO2 resurfacing, 30 patients per group based on data
from Ref. [36]. Healing was better than one-half of the time compared with
the controls, and pain, edema, and erythema were all significantly reduced
in the group that received 830 nm LED phototherapy.

is increasing rapidly concomitant with the ageing population. The

data collection is at an early stage, but the preliminary results are
extremely promising with a similar efficacy rate to the controlled
studies cited earlier using LD-LLLT (personal communication, Prof
Wonserk Kim, MD, PhD, Sungkyunkwan University, Seoul, South
Korea).

44.4 Conclusion

LD-LLLT is still being used to treat PHN with high degrees of success,
but LED-LLLT is starting to attract attention because of the lower
cost of systems, its ease of use and efficacy, both for the resolution
of the PHN and for the control of the HZ preceding the PHN. As was
stated earlier, a strong case was made that controlling the HZ should
prevent the occurrence of the PHN. Though LLLT for PHN is being
used more and more in pain relief clinics and centers throughout
the world, its use is still not as widespread as it deserves. This
could be due to a variety of reasons, principal among which is the
possible limited interest in the modality at a time when LD-LLLT
was still finding its feet so that the early body of research had
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References 903

little impact on the greater medical and scientific world. The case
for its use in the treatment of PHN remains strong, especially with
its lack of side effects, which is so important in the elderly. The
addition of LED-LLLT to LD-LLLT and the growing body of evidence
for the use of LED phototherapy in all aspects of pain control with
more papers being seen in the peer-reviewed and PubMed-listed
literature should result in the expansion of LLLT in the treatment
of HZ and PHN to the level which it deserves. This will offer benefits
to both clinicians and PHN patients, especially the elderly for whom
this condition can severely curtail their activities of daily living.

References

1. Asagai Y, Ueno R, et al. (1995). Application of low reactive-level laser

therapy (LLLT in patients with cerebral palsy of the adult tension
athetosis type. Laser Ther, 7(3), 113–118.
2. Asagai Y, Watanabe Y, et al. (2005). Suppression of myotonia in cerebral
palsy and adjunctive effect of low-level laser therapy on intensive
functional training. Laser Ther, 14(4), 171–178.
3. Avci P, Gupta A, et al. (2013). Low-level laser (light) therapy (LLLT) in
skin: Stimulating, healing, restoring. Semin Cutan Med Surg, 32(1), 41–
52.
4. Basford JK. (1986). Low energy laser treatment of pain and wounds:
Hype, hope or hokum? Mayo Clin Proc, 61, 671–675.
5. Calderhead RG. (2011). Photoactivation. In: Calderhead RG (Ed.),
Photobiological Basics of Photosurgery and Phototherapy, pp. 69–87.
Hanmi Medical, Seoul, South Korea.
6. Donnarumma G, De Gregorio V, et al. (2010). Inhibition of HSV-1
replication by laser diode-irradiation: Possible mechanism of action. Int
J Immunopathol Pharmacol, 23(4), 1167–1176.
7. Enwemeka CS. (2005). Editorial: Light is light. Photomed Laser Surg,
23(2), 159–160.
8. Fusco BM and Giacovazzo M. (1997). Peppers and pain. The promise of
capsaicin. Drugs, 153(6), 909–914.
9. Hong JN, Kim TH, et al. (1990). Clinical trial of low reactive-level laser
therapy in 20 patients with postherpetic neuralgia. Laser Ther, 2(4),
167–170.
July 6, 2016 17:40 PSP Book - 9in x 6in 44-Hamblin-c44

904 Laser (Light) Therapy for Postherpetic Neuralgia

10. Karu T. (1988). Photobiological fundamentals of low power laser

therapy. J Quantum Electron, 23, 1703–1716.
11. Kemmotsu O, Sato K, et al. (1991). Efficacy of low reactive-level laser
therapy for pain attenuation of postherpetic neuralgia. Laser Ther, 3(2),
71–76.
12. Kemmotsu O. (1995). Editorial: Does LLLT prevent postherpetic
neuralgia? Laser Ther, 7(1), 3–5.
13. King PR. (1989). Low-level laser therapy: A review. Lasers Med Sci, 4,
141.
14. Kubota J and Ohshiro T. (1989). The effects of diode laser low reactive-
level laser therapy (LLLT) on flap survival in a rat model. Laser Ther,
1(3), 127–135.
15. McKibbin LS and Downie R. (1991). Treatment of postherpetic neuralgia
using a 904 nm (infrared) low incident energy laser: A clinical study.
Laser Ther, 3(1), 35–40.
16. Mester E, Ludani G, et al. (1968). The stimulating effects of low power
laser rays on biological systems. Laser Rev, 1, 3.
17. Moore KC, Hira N, et al. (1988). A double-blind crossover trial of low-
level laser therapy in the treatment of postherpetic neuralgia. Laser
Ther, Pilot Issue 7–10.
18. Moore KC and Calderhead RG. (1991). The clinical application of low
incident power density 830 nm Ga Al As diode laser in the therapy of
chronic intractable pain: A historical and optoelectronic rationale and
clinical review. Int J Optoelectron, 6(5), 503–520.
19. Moore KC. (1991). The use of low-level laser biostimulation for the
treatment of pain syndromes. In: Laser Systems for Photobiology and
Photomedicine, pp. 129–135. Plenum Press, New York.
20. Moore KC. (1992). Cost-effective benefits of the use of laser therapy
in the treatment of intractable postherpetic neuralgia. In: Laser
Applications in Medicine and Surgery, pp. 61–63. Monduzzi Editore,
Bologna, Italy.
21. Muñoz Sanchez PJ, Capote Femenı́as JL, et al. (2012). The effect of 670-
nm low laser therapy on herpes simplex type 1. Photomed Laser Surg,
30(1), 37–40.
22. Ohshiro T and Calderhead RG. (1988). Low-Level Laser Therapy: A
Practical Introduction. John Wiley & Sons, Chichester, UK.
23. Ohshiro T. (1991). Low Reactive-Level Laser Therapy: Practical Applica-
tion, pp. 99–103. John Wiley & Sons, Chichester, UK.
July 6, 2016 17:40 PSP Book - 9in x 6in 44-Hamblin-c44

References 905

24. Otsuka H, Numazawa R, et al. (1995). Effects of helium-neon laser

therapy on herpes zoster pain. Laser Ther, 7(1), 27–32.
25. Palmgren N, Jensen GF, et al. (1989). Low power laser therapy in
rheumatoid arthritis. Lasers Med Sci, 4, 193–196.
26. Park KY, Han TY, et al. (2013). The effects of 830 nm light-emitting
diode therapy on acute herpes zoster ophthalmicus: A pilot study. Ann
Dermatol, 25(2), 163–167.
27. Sasaki K, Ohshiro T, et al. (2010). Low reactive level laser therapy in the
treatment of postherpetic neuralgia. Laser Ther, 19(2), 101–105.
28. Shiroto C, Ono K, et al. (1989). Retrospective study of diode laser
therapy for pain attenuation in 3635 patients: Detailed analysis by
questionnaire. Laser Ther, 1(1), 41–48.
29. Study RE, Kral MG. (1996). Spontaneous action potential activity
in isolated dorsal root ganglion neurons from rats with a painful
neuropathy. Pain, 165(2–3), 235–242.
30. Trelles MA, Allones I, and Mayo E. (2006). Light-emitting diode (LED)
phototherapy enhances wound healing after laser ablative resurfacing
of photodamaged facial skin. Med Laser App, 21, 165–175.
31. Walker JB, Akhanajee LK, et al. (1987). Laser therapy for pain of
rheumatoid arthritis. Clin J Pain, 3, 54–59.
32. Walker J. (1988). Low-level laser therapy for pain management: A
review of the literature and underlying mechanisms. In: Ohshiro T and
Calderhead RG (Eds.), Low Level Laser Therapy: A Practical Introduction,
pp. 43–56. John Wiley & Sons, Chichester, UK.
33. Walker JB and Swartzwelder HS. (2011). Suppression of hippocampal
epileptiform activity in vitro after laser exposure. Laser Ther, 14, Pilot
issue, 14–21.
34. Whelan HT, Houle JM, et al. (2000). The NASA light-emitting diode
medical program—progress in space flight and terrestrial applications.
Space Tech App Int’l Forum, 504, 37–43.
35. Whelan HT, Smits RL, et al. (2001). Effect of NASA light-emitting diode
(LED) irradiation on wound healing. J Clin Laser Med Surg, 19(6), 305–
314.
36. Yamada H and Ogawa H. (1995). Comparative study of 60 mW diode
laser therapy and 150 mW diode laser therapy in the treatment of
postherpetic neuralgia. Laser Ther, 7(2), 69–74.
July 6, 2016 17:40 PSP Book - 9in x 6in 45-Hamblin-c45

Chapter 45

Laser Acupuncture

Lucas F. de Freitasa and Michael R. Hamblinb,c,d

a University of São Paulo, 400 Trabalhador São-Carlense Avenue, São Carlos,

São Paulo 13566-590, Brazil

b Wellman Center for Photomedicine, Massachusetts General Hospital, 50 Blossom

Street, Boston, MA 02114, USA

c Department of Dermatology, Harvard Medical School, Massachusetts General

Hospital, 55 Fruit Street, Boston, MA 02114, USA

d Harvard-Massachusetts Institute of Technology (MIT) Division of Health Sciences and

Technology, 77 Massachusetts Avenue, E25-518, Cambridge, MA 02139, USA

hamblin@helix.mgh.harvard.edu

This chapter presents some of the most recent studies about the
stimulation of acupuncture points with low-level light sources. The
so-called laser acupuncture calls the attention of researchers due
to its noninvasiveness and painless application, and the results
published so far corroborate, most of the times, to a future
implementation of this technique as an adjuvant therapy for several
medical applications. Some studies, however, stated otherwise,
leading to the conclusion that the difference between the applied
parameters can lead to different outcomes. This fact leads often to a
skepticism against this technique, and this can only be changed if the
authors standardize the parameters used in their studies, according
to each application.

Handbook of Low-Level Laser Therapy

Edited by Michael R. Hamblin, Marcelo Victor Pires de Sousa, and Tanupriya Agrawal
Copyright c 2017 Pan Stanford Publishing Pte. Ltd.
ISBN 978-981-4669-60-3 (Hardcover), 978-981-4669-61-0 (eBook)
www.panstanford.com
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908 Laser Acupuncture

45.1 Introduction

The possibility of light amplification by stimulated emission of

radiation (laser) was considered for the first time by Albert Einstein
in 1917, but it was only in 1960 that the first laser was constructed
by the physicist Theodor Maiman. His work was based on the
works of Charles H. Townes, who built the first MASER (microwave
amplification by the stimulated emission of radiation) in 1954, and
on the study entitled “Infrared and optical maser,” published by
Townes and Arthur L. Schawlow in 1958. In the early 1960s, there
was already a protocol for photocoagulation of retina with a ruby
laser, but it had to be discontinued due to the damages it caused.
A few years later, a protocol for the treatment of detached retina
was developed using an argon laser, and today, almost 60 years
later, the first laser was built, and its applications have spread to
a great number of fields in medicine other than ophthalmology,
i.e., otolaryngology, dentistry, dermatology, general surgery, and
vascular surgery, as well as in aesthetic procedures [1].
Depending on the desired application, lasers can be of low or high
intensity. While high-intensity lasers rely on their thermal effects to
cauterize, destroy, or cut tissues, low-intensity lasers—also called
low-level lasers, soft lasers, or cold lasers—are believed to interact
with specific biomolecules in the target tissue, activating signaling
cascades without any significant thermal effect. Low-level lasers
usually emit in the wavelengths from 650 to 1000 nm, in the 30–50
mW output power range [1, 2, 3]. These are the most common lasers
used for light stimulation of acupuncture points.
Acupuncture can be described, according to traditional Chinese
medicine (TCM), as a medical treatment using needles to stimulate
energy points through the body. The first archeological findings
about the acupuncture practice could be traced to 3000 years, but
a Stone Age body of a man from 5000 years ago with tattoos of
acupoints was discovered in the Alps, suggesting that this modality
can have had its origin considerably earlier [1, 4, 5].
According to TCM, acupuncture is based on restoring the balance
of the Qi, which is the vital life force of all living beings. Qi
flows through the so-called meridians, the energetic channels that
form a system partially related to the internal organs and their
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Introduction 909

physiological conditions. The acupuncture points are small areas

in this system where the Qi can come to the surface and serve as
an access to the deeper meridian system. Whenever there is an
imbalance of Qi (a deficiency or an excess), it can result in diseases,
and these diseases can be treated by stimulating the acupoints and
restoring the balance of Qi [2, 5].
TCM classification identifies 14 meridians and 361 acupoints,
although there were more points identified over the time. All these
points are related to specific illnesses or problems, but only a few
are used regularly therapeutically. The points to treat are chosen by
the professional through medical examinations and the history of
the patient, and the acupuncture procedure involves the insertion
of needles at the selected points, followed by needle manipulation,
rather a twirling or an up and down motion [5].
On the beginning of the 1970s, laser technology started to be
used as a tool to stimulate acupuncture points throughout the body,
aiming the treatment of hypertension and asthma in the USSR,
although many sources credit the Canadian Friedrich Plog with
the debut of laser acupuncture in 1973–74. Plog’s work enabled
the development of the first commercial laser for the stimulation
of acupoints, named Akuplas. At the end of the 1970s, Zhou,
a Chinese surgeon, started using laser acupuncture to stimulate
acupoints for anesthesia, and in 1984 the first studies about laser
auricular acupuncture were published by Seitz and Kleinkort. The
first laser needles were invented in Germany in 2001, prompting the
stimulation of several acupoints at the same time and minimizing
light scattering that occurs at the skin surface [1, 4, 5].
Low-level light in acupoint stimulation offers some advantages
over classical needle acupuncture, i.e., the fact that it is painless,
infection-free (since there is no insertion in the skin), can be used
in patients that are afraid of needles, and is easier to deliver, which
makes it time efficient and cost effective. Regarding the research
area, it offers another advantage because the lack of sensation
offered by the low-level light makes easy to perform blind-controlled
experiments [6, 7].
The success of laser acupuncture depends on the understanding
of skin properties. The skin represents a barrier to light transmis-
sion, and factors such as skin thickness, age, pigmentation, and
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910 Laser Acupuncture

regional collagen fiber anisotropy alter significantly the effects of

light in the tissue. There are also several biomolecules in the skin
responsible for absorbing and scattering light. Melanin (responsible
for the pigmentation of the skin and located in the dermis layer)
and hemoglobin (present in the blood that irrigates the epidermis)
are the main substances absorbing light, but other compounds such
as carotene, lipids, and filamentous proteins contribute to light
absorption. Keratin and collagen play an important role in light
scattering, although melanosomes, cell nuclei, cell walls, and other
structures can influence light scattering as well [2, 5].
Laser acupuncture’s effects seem to be achieved through a
different mechanism of action than needle acupuncture, although
none of the mechanisms of action are completely elucidated
yet. It seems, for instance, that needle-induced collagen fiber
reorganization is important for needle acupuncture, but laser
acupuncture cannot induce such effect. Mitochondrial cytochromes,
which absorb in the red and near-infrared, have been implicated in
the beneficial effects of laser acupuncture [5]. Cytochrome c oxidase,
for instance, is the terminal enzyme at the respiratory chain and
is an important photoacceptor in these regions of light spectrum.
When this enzyme interacts with red or near-infrared light, there is
an increase in adenosine triphosphate and reactive oxygen species
(ROS) synthesis, the latter ones acting as signaling molecules in
many cell processes [1].
Other biomolecules outside the mitochondria can also act as
photoacceptors, such as NADPH-oxidase (located at the plasma
membrane and produces ROS) and NO-synthases. Superoxide
dismutase and catalase, both important enzymes in redox processes,
absorb red light as well [1].
According to TCM, it is necessary for the patient to have first
a sensory perception named “Deqi sensation” for the acupuncture
treatment to be successful. Some authors say that Deqi sensation
is the central phenomenon of awareness and consciousness, and
it must be taken into account in the research field due to its
importance [8]. The Deqi sensation is described by the patients as
a sensory perception of varying character. It can be a sensation of
heaviness or warmth, an electrical current through the meridians,
a feeling of numbness, prickling, flowing, or heaviness around the
acupuncture point [2].
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Laser Acupuncture in Pain Reduction 911

In needle acupuncture, this sensation can be increased by

rotating or moving the needle up and down. The sensation produced
by the needle is stronger than the one achieved with laser
stimulation, but laser has proven to be effective in Deqi sensation
production, despite all the skepticism caused by the thought that
low-level lasers in the red or near-infrared do not cause any
sensation. Indeed, in the beginning of the treatment, the patients
do not feel any Deqi sensation, but within a few minutes, there
are reports of a warm and pleasant sensation, and sometimes a
feeling of vibration or an ant-bite-like sensation in the treated areas.
These sensations last much longer than the ones produced by needle
stimulation [2, 8].
Stronger Deqi sensations can be achieved with violet light. It
has much less penetration in the skin when compared to red light
(violet can penetrate 1–2 mm, while red light can penetrate 3–4 cm),
because most of it is absorbed in the first layers of skin. While the
red laser usually cannot be felt, the violet laser can evoke sensory
stimulus, inducing a stronger Deqi sensation [4].
The implementation of low-level laser acupuncture encounters
some barriers, which include lack of a systematic approach for
the development of research in this area, as well as lack of a
well-described mechanism of action and some inconsistent reports
of clinical effectiveness in clinical trials. Many studies fail to
give important details about the treatment procedures or were
associated with inappropriate treatment parameters (insufficient
laser power outputs or light doses, for example), mainly those
reporting no significant benefit of laser acupuncture compared to
control or sham conditions. This resulted in a skepticism from some
researchers, which can be eliminated only if the studies take into
account all the important parameters, and if they are performed in
a systematic way for the different therapeutic approaches [9]. In
this chapter, we will go through the most relevant published studies
concerning laser acupuncture as a therapeutic approach.

45.2 Laser Acupuncture in Pain Reduction

Al Rashoud et al. used a combination of low-level light therapy

(LLLT) and quadriceps-strengthening exercises and advice and
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912 Laser Acupuncture

found beneficial effects on pain reduction, as well as a significant

improvement in knee joint function, even 6 months after laser
therapy was discontinued. The authors agree that the positive
outcome can be attributed to the anti-inflammatory effect of LLLT
applied to specific points on the articular capsule and the ability of
LLLT to stimulate reparative properties in human cartilage [10].
Nine studies on laser acupuncture in the treatment of myofascial
pain and musculoskeletal trigger points affecting the neck, shoulder,
thoracic, or lumbar spine were reviewed by Baxter et al. in 2008 [9].
Seven of them reported positive effects in pain reduction after 10 to
12 sessions. The irradiation parameters used in these studies had
a great variability, i.e., the power outputs ranging from 0.95 mW to
25 mW, and light doses from 0.57 J to 5 J per stimulated point, but
this variability was not enough to neutralize the beneficial effects of
low-level light in pain reduction. The only two studies that reported
no significant difference between the experimental and the placebo
groups did not report enough data to determine the actual laser
irradiation parameters used.
In a more recent review on myofascial pain syndrome treated
with laser acupuncture, Uemoto et al. have observed beneficial
effects in the deactivation of myofascial trigger points, and most
of the reviewed studies were successful concerning the recovery of
the normal muscle length and posture, recovery of range of motion
and restoring physiological cell functions, mediating inflammatory
processes, enhancing tissue repair process, and promoting anal-
gesia. Laser application to myofascial trigger points demonstrated
improved local microcirculation, favored oxygen supply to hypoxic
cells, and helped remove waste products of cell metabolism, which
diminishes pain and muscle spasms [11].
Uemoto et al. affirm that the most commonly used laser system
is a gallium–aluminum–arsenide (GaAlAs) laser, and that the most
effective wavelengths in myofascial trigger points deactivation are
in the range of 780–904 nm, due to their deeper penetration into
the tissue. However, positive outcomes were achieved when lower
wavelengths (in the red region) were used, which suggests that
the dosage can play an important role besides the wavelength
selection. The reviewed authors indicate doses from 50 to 100
J/cm2 , but it depends on some aspects, such as pain characteristics
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Laser Acupuncture in Pain Reduction 913

(if chronic, the dose can be smaller and the frequency of applications
is increased, compared to the acute pain), pigmentation of skin
(the more pigmented, the higher light dose applied), and adipose
tissue content (as fat causes light reflection, the light dose must be
increased with higher amounts of adipose tissue) [11].
Baxter et al. also reviewed studies about laser acupuncture in
lateral epicondylitis, but none of them demonstrated any significant
difference between experimental and placebo groups, leading to
the conclusion that low-level light has no beneficial effects in
the treatment of this condition. However, most of the times the
combinations of irradiation parameters used by the reviewed
authors were considered by Baxter et al. to be inadequate to provide
beneficial effects: power outputs within the range of 0.7–12 mW and
light doses below 1 J. One more limiting factor consisted in the use
of a laser not in contact with the skin, which is the desired way to
perform the irradiation, but held 1 mm from the target tissue. This
would limit the penetration of light into the tissue [9].
Summarizing the findings of Baxter et al. in their reviewed
paper, there is moderate evidence to support the use of laser
acupuncture in the treatment of myofacial pain and in chronicle
headaches (based on a study comparing the effectiveness of 10 laser
acupuncture sessions, applied at eight points with a dose of 1.3 J
per point, to placebo in the treatment of 50 patients with chronic
tension headaches; the intensity, duration, and number of headaches
were significantly reduced in the treated patients). There is no
definitive conclusion about the effectiveness of low-level laser (or
light) therapy on acupoints in the treatment of lateral epicondylalgia
because of the lack of appropriated laser irradiation parameters
during the treatments [9].
Glazov et al. performed a double-blind, three-group parallel
randomized controlled trial in 2013, aiming to investigate the effects
of laser acupuncture in chronic low back pain (LBP). It was a
robust randomized controlled trial design that reduced risk of
bias, including concealed allocation procedures and gold standard
randomization. And even with all the controlled parameters, it found
no different outcomes or adverse effects between treated and sham
laser groups at any time/point for laser acupuncture in doses up
to 0. 8 J per point. This trial enforces the lack of biological effect
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914 Laser Acupuncture

from laser acupuncture when treating LBP and supports the idea of
nonspecific effects being responsible for the therapeutic outcomes
in patients that otherwise may have been wrongly attributed to the
laser [13].

45.3 Laser Acupuncture in Wound Healing

Beckmann et al. stated that low-level light has stimulating effects

on wound regeneration, even in chronic ulcers, while high-energy
radiation inhibits this regenerating effect. In fact, near-infrared
light was shown to elevate significantly the levels of TGF-β and
matrix metalloproteinase-2 (MMP-2) in cultured cells. Irradiated
fibroblasts also showed higher amounts of MMP-2 mRNA [3].
A randomized, triple-blind, placebo-controlled trial was per-
formed in healthy volunteers with standardized abrasions on the
anterior forearms, treated with LLLT using light-emitting diodes
(LEDs) (a 46-diode cluster head emitting in 660–820 nm) or a sham
46-diode cluster head. The LLLT enhanced wound healing not only
on the treated wounds, but also the untreated wounds on the same
arm became smaller, in contrast to the sham group, suggesting that
there might be an indirect healing effect after LLLT [14].
Low-intensity laser irradiation of different wavelengths demon-
strated that cellular migration, viability, and proliferation can
be enhanced in diabetic wounded and unwounded human skin
fibroblast cells. The red light (632 nm) was more efficient to elevate
cell haptotaxis and migration than infrared light (830 and 1064 nm)
[15].
A randomized, double-blind, placebo-controlled trial demon-
strated that the combined 660 nm and 890 nm light irradiation
improved the healing effect on 23 diabetic ulcers. Mean ulcer
granulation improved and healing rates were higher in the treated
groups compared to the controls, even after 90 days of treatment
(56% more granulation and 79.2% faster healing than controls at
day 30 and forth). About 58.3% of treated ulcers were fully healed,
and 75% healed at least 90% by day 90 [16].
Blue (470 nm) and red (630 nm) LEDs successfully improved
perfusion in an excision model in nondiabetic rats, through the re-
lease of nitric oxide (NO) from nitrosyl complexes with hemoglobin.
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Laser Acupuncture in Heart Rate and Heart Rate Variation 915

They also enhanced epithelialization and elevated keratin-1-mRNA

levels. NO was released from mitochondrial complexes inhibited by
NO gas, after blue light irradiation. Since NO can induce endothelial
cell migration by activating some growth factors, the treated wound
area became 50% smaller in the blue light group compared to the
control (not illuminated) group, even more than red light [17].

45.4 Laser Acupuncture in Respiratory Diseases

Before 2009 there were insufficient data to support the efficacy of

acupuncture in rhinitis and asthma. Recently, Elseify et al. performed
a study with 50 patients with limited daily activities due to asthma
symptoms. As the laser acupuncture sessions were performed, the
frequency of symptoms was gradually declined, so that at the
10th session, all the daytime symptoms disappeared, and the daily
inhaled corticosteroids dose was reduced at least 50%. At the end
of the study, 48 patients were totally free from asthma symptoms.
The bronchodilator effect of LLLT reflected clinically as a rise in the
forced expired volume in one second (FEV1) as well as in the peak
expiratory flow (PEF). The authors found that the laser acupuncture
can reduce peripheral sputum eosinophil count and leukotrienes
LTC4 and LTD4 production. It also inhibits IgE-mediated allergic
effects (either immediate or delayed), while it elevates plasma cAMP
and cAMP/cGMP [18].
A possible mechanism for the therapeutic outcomes of laser
acupuncture in the treatment of asthma has been speculated as
a bronchodilator action by the activation of the autonomic nerve
afferent pathway from a somatic visceral reflex. The release of
substance P (one of the most important neurogenic inflammatory
mediators) is, therefore, inhibited, and the endogenous opioids that
could influence asthma are released [19].

45.5 Laser Acupuncture in Heart Rate and Heart Rate

Variation

He et al. investigated in 2012 the effects of violet laser (405 nm)

in patient’s heart rate and heart rate variation. The equipment
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916 Laser Acupuncture

Figure 45.1 Anatomical location of some acupoints: (A) Neiguan, (B)

Baihui, (C) Shenmen, and (D) Taichong [13].

used was fixed skin (not inserted) and had an output power of
110 mW, a laser needle spot diameter of 500 μm and frequencies
of 2 Hz or 100 Hz (so that the overall energy doses is half the
one with continuous life mode stimulation). The stimulated points
were Neiguan (PC6) (Fig. 45.1), Shenmen ear acupoint, and Baihui
(GV20). Violet laser did not demonstrate any significant difference
in total heart rate variation, although the heart rate decreased with
the two frequencies, maybe because a pulsed laser was used (the
studies using continuous lasers demonstrated significant alterations
in heart rate variation) or because the smaller tissue penetration of
violet light. The stimulation of Baihui point has shown to increase
brain and peripheral circulation, as well as local temperature [20].
In 2013, the same author investigated the interstitial laser
acupuncture with red laser (658 nm) at the Neiguan acupoint (PC6)
in rats. After 20 min of laser acupuncture stimulation, the heart rate
of all rats decreased significantly, in contrast with the 10 and 30 min
stimulation. Heart rate variation, however, increased during 20 min
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Laser Acupuncture and Brain Activity 917

of laser stimulation, although not significantly, and no alterations

were seen with 10 or 30 min of Neiguan acupoint stimulation [21].
Significant alterations in heart rate and heart rate variation
were found by Litscher et al. after manual acupuncture (using
sterile single-use needles inserted perpendicularly in the skin and
rotated 30 times) and after laser acupuncture (using laser needles
emitting at 685 and 785 nm, 30–40 mW per needle, for 20 min).
Both protocols were used at specific acupoints: Baihui (GV20),
Neiguan (PC6), Shenmen (HT7), and Taichong (LR3). There was no
significant change in heart rate after the first needle acupuncture
session, but in the last session, this parameter was significantly
decreased. On the other hand, heart rate and heart rate variation
were altered already in the first stimulation session using laser
needles. Interestingly, after six acupuncture sessions, the analyzed
parameters were changed more intensely with laser stimulation
than with classic needles [22].
In another study, Litscher et al. stimulated Baihui (GV20) and
Neiguan (PC6) acupoints with violet and red laser as well as
with classic needles. Stimulation of Neiguan did not induce any
changes in heart rate, in contrast with Baihui stimulation, which
diminished the heart rate after red laser and needle stimulation.
Neiguan is a classic acupuncture point known to be effective when
treating cardiovascular disorders, but in this study it demonstrated
inhibitory effects in heart rate variation just with violet laser,
although the heart rate did not show any alterations during the
treatment [4].

45.6 Laser Acupuncture and Brain Activity

Walker and Akhanjee found, already in 1984, that skin irradiation

using a helium–neon laser (632.5 nm) with an output power of
1 mW overlying the median wrist nerve in humans was capable of
producing a somatosensory evoked potential, if the frequency of the
laser was around 3 Hz. This potential was obtained at the Erb’s point
on the shoulder [23].
More recently, in 2012, Litscher investigated the effects of
acupuncture on the brain activity. In the first part of the study,
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918 Laser Acupuncture

Figure 45.2 Anatomical location of some acupoints: (A) Guangming and (B)
Zhiying [12].

the following acupoints were treated with classical acupuncture:

Guangming (GB37) (Fig. 45.2), Taichong (LR3), and Zhiyin (UB67),
the points corresponding for vision, according to TCM. Only the
stimulation of acupoints located on the bladder meridian indicated
the activation of visual, evoked potentials, while the stimulation of
other points on other meridians inhibited those potentials [7].
In the second part of Litscher’s study, Neiguan acupoint was
stimulated with laser needle emitting red light (658 nm), and it
was enough to evoke cortical responses even if the patients did
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Laser Acupuncture and Brain Activity 919

not feel the laser stimulation. Other experiments already stated

that high-heating-rate diode laser pulses can activate selectively the
myelinated Aδ-fiber nociceptors in rats and produce pricking pain
in humans, but low-heating-rate lasers with longer pulses are more
likely to activate unmyelinated C-fibers in rats and to cause burning
pain in humans. Litscher’s results indicate that laser exposure in
order to stimulate acupoints such as Neiguan with a frequency
of 1 Hz can modulate the ascending reticular activating system,
explaining how laser acupuncture can cause Deqi-like sensations
even without any sensory stimulus [8].
Quah-Smith et al. have investigated the difference between
laser and needle acupuncture as an antidepressant treatment,
comparing their brain effects and the impact on the default mode
network (DMN). The needle stimulation of acupoint LR8 (one of
the most important acupuncture points for depression treatment)
demonstrated changes in brain activity, characterized by increased
activation at left right insula and left precentral gyrus, as well as a
deactivation at the left precuneus. If compared, the needle activation
induced greater activation in the left precentral gyrus, while the
laser acupuncture induced greater activation on the left precuneus
[24].
There was no activity changes in the somatosensory cortex
with the laser stimulation of LR8 acupoint; therefore, the afferent
pathways activated by both protocols are likely to be different
with needle acupuncture, due to sensorimotor and somatosensory
inputs, and with laser acupuncture, because of autonomically driven
potentials.
Even though there was no activation of somatosensory cortex,
the significant activation on the left precuneus (which is part of the
posterior DMN) confirms the clinical efficacy of laser acupuncture in
depression [24].
In another study from the same author, the points LR8, LR14
(Fig. 45.3), HT17, and CV14 (Fig. 45.4) were stimulated with
laser at 808 nm (25 mW per 20 s, delivering 0.5 J), and each
acupoint received four laser runs (total 2 J). Both depressed
and nondepressed subjects presented modulation of anterior and
posterior DMNs, but the nondepressed ones had outstanding
modulation of the DMN in the frontal region at medial frontal
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920 Laser Acupuncture

Figure 45.3 Anatomical location of some acupoints: (A) LR8 and (B) LR14
[12].

gyrus. LR8, LR14, and CV14 points induced significant differences

in DMN modulation between depressed and nondepressed subjects,
but there was no difference between groups with HT7 stimulation
[6].
In 2005, Siedentopf et al. selected the left and right acupoints
GB43, which is commonly indicated to deafness, tinnitus, dizziness,
ear diseases, headache, and migraine. After laser stimulation
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Laser Acupuncture and Brain Activity 921

Figure 45.4 Anatomical location of some acupoints. Top scheme: CV14.

Bottom scheme: GB48 [12].

with 670 nm, significant activations in anatomical brain areas

that are known to be related to these problems were identified.
The activations detected were preferentially occurring ipsilaterally,
indicating that peripheral stimulation of acupoints does not enter
the brain by afferent somatosensory pathways, which are counter
lateral. According to TCM, the meridians do not cross to the other
side like somatosensory pathways, so these results support this
traditional hypothesis [25].
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922 Laser Acupuncture

Finally, the HT7 acupoint was stimulated with laser at 405 nm for
10 min in a study carried out by Sutalangka et al. [26]. The results
indicate a cognitive-enhancing effect, as well as a modulation on
oxidative stress and on cholinergic function. LLLT helped to decrease
the oxidative stress in oxidatively stressed neurons and induced
an elevation of acetylcholine (ACh) on the hippocampus. This is
important for memory impairment in Alzheimer’s disease, and the
authors suggest that laser acupuncture at HT17 may help to reduce
the effects of this disease [26].

45.7 Auricular Laser Acupuncture

The acupuncture in the ear was originally performed thinking

about the points and meridians of the auricle as being part of
the whole body system of meridians, but about 60 years ago,
Paul Nogier discovered an inverted fetus-like projection on the
auricle containing acupoints corresponding to the entire body.
Therefore, the ear acupuncture points are more likely to be a
complete acupuncture system for the whole body than a part of
the system itself. This point of view is not based on TCM, but on
the representation of the somatotopic organization of the human
body in the auricle. In this representation, the head with the brain
structures is located in the ear lobe, while the spine runs along the
antihelix. The inner organs can be stimulated with points on the
concha, and the limbs are represented toward the upper rim of the
auricle (Fig. 45.5) [2].
Currently, the applications of auricular acupuncture cover
treatments for acute and chronic sciatalgia, osteoarthritis, headache,
knee arthroscopy, hip fracture, hip arthroplasty, and cancer. The
mechanism of action is believed to occur via activation of the au-
tonomic nervous system, whether sympathetic or parasympathetic
[2].
Heine studied the auricular acupoints and concluded that they
are dot-like structures with 100 μm of diameter at most and consist
of a combination of elastin and collagen fibers pervaded with nerve
endings and small blood vessels [27].
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Other Applications for Laser Acupuncture 923

Musculoskeletal Points
Ankle .C Toss .C Knee .C
Heel .C
Hip .C
Toss .F
Fingers
Knee .F
Hand
Ankle .F Skin
Disorder .C
Heek .F
Wrist
Hip .F Abdomen
Sacral Spine Elbow
Lumbar Spine
Thoracic Spine Arm
External Ear .C
Chest
Muscle Relaxation
Inner Nose .C Shoulder
Inner Ear .F Neck
Cervical Spine Master
Temples Shoulder
Forehead Occiput
Eye Disorder 1 TMJ
Eye Disorder 2
Dental Analgesia Jaw and Teeth
Inner Ear .C
Eye Face

Figure 45.5 Representation of the auricular acupuncture system. Left:

inverted fetus representation; right: musculoskeletal acupoints in the
auricle.

There are few studies in this field yet, and a comparison

between the already published works is difficult because of the
different research subjects and approaches. However, some of the
results indicate that auricular acupuncture can be an adjunctive
treatment during withdrawal therapies for pain relief. Patients
treated with auricular acupuncture usually require less medication,
which reflects in a reduction of side effects and a decreasing of costs
[2].

45.8 Other Applications for Laser Acupuncture

Yang et al. stimulated the acupoints PC-6, CV12 (Fig. 45.6), ST36, and
BL21 (Fig. 45.7) separately, with a laser needle (red light at 658 nm,
output power of 50 mW) inserted 1–3 mm into the skin, as well as
with conventional single-use needles inserted perpendicularly 3–5
mm into the skin of the acupoints [28]. The investigation was based
on previous studies that showed that electroacupuncture at ST36
could cause gastric contractions, and CV12 electrostimulation could
cause gastric relaxations in rats, while conventional acupuncture at
the lower limbs promoted gastric motility enhancement via cholin-
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924 Laser Acupuncture

Figure 45.6 Anatomical location of some acupoints. Top scheme: CV12.

Bottom scheme: ST36 [12].

ergic pathways, and abdomen acupoints stimulation could lead to in-

hibition of gastric motility via sympathetic pathways in rats [28, 29].
The results of Yang’s study in 2013 showed that either
laser acupuncture or conventional acupuncture at PC6 acupoint
increased heart rate, probably via a somatosympathetic reflex
and enhanced gastric motility, probably via sympathetic vagal
regulation. ST36 stimulation by laser and conventional acupuncture
enhanced gastric motility, perhaps via vagal regulation, while CV12
laser and conventional acupuncture stimulation inhibited gastric
July 6, 2016 17:40 PSP Book - 9in x 6in 45-Hamblin-c45

Other Applications for Laser Acupuncture 925

Figure 45.7 Anatomical location of BL21 acupoint [12].

motility via segmental somatosympathetic reflex in the spinal

level. Antinociceptive effects were observed after ST36 stimulation
with laser, and the authors affirm that this effect is mediated by
the activation of serotonergic (5-HT1 and 5-HT2A receptors) and
opioidergic pathways. In summary, Yang et al. found similar results
with laser and manual acupuncture, but the different acupoints lead
to different effects, which indicates that the method of stimulation is
not more important than the choice of acupoints [28].
Ferreira et al. investigated the effects of laser acupuncture in
a child patient who underwent a tumor excision surgery in the
past. The patient showed difficulties in mouth opening, which made
the dental care hard to be performed. The acupoints selected were
divided into three sets: on the face (SI17, SI19, LI19, LI20 (Fig. 45.8),
ST4, ST6, and ST7 (Fig. 45.9)), on the upper extremities (LI4, LI18
(Fig. 45.10) and GV20), and on the lower extremity (LR3), and
they were stimulated in 10 sessions of laser irradiation at 780 nm,
output power 70 mW. The time of irradiation was 60 s at each
point, applying an energy density of 105 J/cm2 per point. Initially,
the mouth-opening length was 33.26 mm, and after 10 sessions, it
reached 53.34 mm, allowing the dental care to be completed. This
mouth-opening length was maintained after 20 months from the
therapy [30].
July 6, 2016 17:40 PSP Book - 9in x 6in 45-Hamblin-c45

926 Laser Acupuncture

Figure 45.8 Anatomical location of some acupoints: (A) SI17, (B) SI19, (C)
LI19, and (D) LI20 [12].

Figure 45.9 Anatomical location of some acupoints: (A) ST4, (B) ST5, (C)
ST6, and (D) ST7 [12].
July 6, 2016 17:40 PSP Book - 9in x 6in 45-Hamblin-c45

Conclusion 927

Figure 45.10 Anatomical location of LI18 acupoint [12].

Finally, Cafaro et al. used a Pointer Pulse laser in the red

region (650 nm), output power 5 mW, and irradiation time of
120 s per acupoint, giving a total dose of 0.6 J over five weekly
sessions, in order to observe the effects on patients with xerostomia.
The acupoints chosen were LI2 (Fig. 45.11), ST5, ST6, ST7, SI19,
and BL13, stimulated bilaterally. A significant increase in saliva
production was observed after the end of the protocol, and the
results are maintained until the third month of follow-up [31].

45.9 Conclusion

The number of emerging technologies with potential applications

in the biomedical field is increasing continuously, but there is an
urgent need to increase the quality of clinical studies in some
areas, especially regarding the use of laser for stimulation of
acupuncture points [3]. After 40 years of investigation, it is still
hard to evaluate the results of low-level light acupuncture because
of many confounding factors [5].
As shown in this chapter, the main trammels for the development
of new therapeutic protocols of laser acupuncture depend on the
quality of published studies, including lack of information about
July 6, 2016 17:40 PSP Book - 9in x 6in 45-Hamblin-c45

928 Laser Acupuncture

Figure 45.11 Anatomical location of acupoints. Top scheme: LI2. Bottom

scheme: LI4 [12].

the procedures and inadequate study parameters. The methods can

vary from combined wavelengths, to a single wavelength, higher
or lower frequencies, and short- or long-time irradiations [3]. Only
a few studies properly described parameters such as irradiance,
wavelength, and light dose [23].
Although there is previous consent that the acupuncture points
are considered to be located at certain depths in the body,
few studies have taken this into account. Even the name “laser
acupuncture” can be questioned, because the coherence of light
is rapidly lost in the skin. Whittaker et al. suggest that “photonic
July 6, 2016 17:40 PSP Book - 9in x 6in 45-Hamblin-c45

References 929

acupuncture” would be a more appropriate term for this therapeutic

approach, based on the fact that non-coherent light sources are
reported to be effective in low-level light therapies.
Even so, a majority of studies show a potential benefit for
the stimulation of acupuncture points by low-level light in many
biomedical applications. In fact, laser acupuncture can be applied
as an adjuvant procedure for many conditions, as long as it is
performed in a controlled and professional manner. It is necessary,
however, that the variation between treatment parameters from one
study to another decreases, so that the conclusions for each desired
application can be reached easily and in a uniform way, and so that a
complete meta-analysis can be performed. The protocol parameters
must be thoroughly described in all the studies, for the same
reasons, and this caveat is directed not only for the acupuncturist,
but for the laser researcher as well. The first must be aware of the
importance of wavelength and beam profile selection, and the latter
must know that the selection of points involves concepts of TCM that
are not readily quantified in western science.

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Lorenzini, L., and Traverso, S. (2011). Ultra-low-level laser therapy.
Lasers Med Sci, 26, pp. 103–112.
2. Round, R., Litscher, G., and Bahr, F. (2013). Auricular acupuncture
with laser. Evid Based Complement Alternat Med, doi: 10.1155/2013/
984763.
3. Beckmann, K. H., Meyer-Hamme, G., and Schröder, S. (2014). Low-
level laser therapy for the treatment of diabetic foot ulcers: A critical
survey. Evid Based Complement Alternat Med, doi: 10.1155/2014/
626127.
4. Litscher, G., Wang, L., Wang, X., and Gaischek, I. (2013). Laser acupunc-
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nm, 405 nm) induce different effects in neurovegetative parameters.
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Lasers Med Sci, 19, pp. 69–80.
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930 Laser Acupuncture

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(2013). Differential brain effects of laser and needle acupuncture at LR8
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controlled trial. Acupunct Med, 32, pp. 116–123.
14. Hopkins, J. T., McLoda, T. A., Seegmiller, J. G., and Baxter, G. D. (2004).
Low-level laser therapy facilitates superficial wound healing in humans:
A triple-blind, sham-controlled study. J Athl Train, 39(4), pp. 223–229.
15. Houreld, N., and Abrahamse, H. (2010). Low-intensity laser irradiation
stimulates wound healing in diabetic wounded fibroblast cells (WS1).
Diab Tech Therap, 12(12), pp. 971–978.
16. Minatel, D. G., Frade, M. A. C., França, S. C., and Enwemeka, C. S. (2009).
Phototherapy promotes healing of chronic diabetic leg ulcers that failed
to respond to other therapies. Lasers Surg Med, 41(6), pp. 433–441.
17. Adamskaya, N., Dungel, P., Mittermayr, R., Hartinger, J., Feichtinger, G.,
Wassermann, K., Redl, H., and van Griensven, M. (2011). Light therapy
by blue LED improves wound healing in an excision model in rats. Injury,
42(9), pp. 917–921.
18. Elseify, M. Y., Mohammed, N. H., Alsharkawy, A. A., and Elseoudy, M. E.
(2013). Laser acupuncture in treatment of childhood bronchial asthma.
Complement Integr Med, 10(1), pp. 199–203.
July 6, 2016 17:40 PSP Book - 9in x 6in 45-Hamblin-c45

References 931

19. Chernyshova, L., Khan, M., Reutova, V., and Semenova, U. (1995). The
effect of low energy laser radiation in the infrared spectrum on
bronchial patency in children with bronchial asthma. Vopr-Kurortol-
Fizioter-Iech-Fiz-Kult, 2, pp. 11–14.
20. He, W., Wedig, D., Wang, L., Gaischek, I., and Litscher, G. (2012). Violet
laser acupuncture — Part 5: An investigation of different stimulation
frequencies on heart rate and variability. J. Acupunct. Meridian Stud,
5(6), pp. 290–294.
21. He, W., Litscher, G., Jing, X-H., Shi, H., Wang, X-Y., Gaischek, I., Su, Y-S.,
Litscher, D., Yang, Z-K., Xin, J-J., and Hu, L. (2013). Effectiveness of
interstitial laser acupuncture depends upon dosage: Experimental
results from electrocardiographic and electrocorticographic recordings.
Evid Based Complement Alternat Med, http://dx.doi.org/10.1155/2013/
934783.
22. Litscher, G., Liu, C-Z., Wang, L., Wang, L-P., Li, Q-Q., Shi, G-X., Gais-
chek, I., Litscher, D., and Wang, X-M. (2013). Improvement of the
dynamic responses of heart rate variability patterns after needle and
laser acupuncture treatment in patients with burnout syndrome: A
transcontinental comparative study. Evid Based Complement Alternat
Med, doi:10.1155/ 2013/128721.
23. Walker, J. B., and Akhanjee, L. K. (1985). Laser-induced somatosensory
evoked potentials: Evidence of photosensitivity in peripheral nerves.
Brain Res, 344, pp. 281–285.
24. Qua-Smith, I., Suo, C., Williams, M. A., and Sachdev, P. S. (2013). The
antidepressant effect of laser acupuncture: A comparison of the resting
brain’s default mode network in healthy and depressed subjects during
functional magnetic resonance imaging. Med Acupunc, 25(2), pp. 124–
133.
25. Siedentopf, C. M., Koppelstaetter, F., Haala, I. A., Haid, V., Rhomberg, P.,
Ischebeck, A., Buchberger, W., Felber, S., Schlager, A., and Golaszewski,
S. M. (2005). Laser acupuncture induced specific cerebral cortical and
subcortical activations in humans. Lasers Med Sci, 20, pp. 68–73.
26. Sutalangka, C., Wattanathorn, J., Supaporn, M., Thukham-mee, W.,
Wannanon, P., and Tong-un, T. (2013). Laser acupuncture improves
memory impairment in an animal model of Alzheimer’s disease. J
Acupunc Meridian Stu, 6(5), pp. 247–251.
27. Heine, H. (1993). Anatomical Correlates of Acupuncture Points, Vol. 7 of
Spektrum der Wissenschaft, Germany.
28. Yang, Z-K., Wu, M-L., Xin, J-J., He, W., Su, Y-S., Shi, H., Wang, X-Y., Hu,
L., Jing, X-H., and Litscher, G. (2013). Manual acupuncture and laser
July 6, 2016 17:40 PSP Book - 9in x 6in 45-Hamblin-c45

932 Laser Acupuncture

acupuncture for autonomic regulations in rats: Observation on heart

rate variability and gastric motility. Evid Based Complement Alternat
Med, http://dx.doi.org/10.1155/2013/276320.
29. Li, Y-Q., Zhu, B., Rong, P.J., Ben, H., and Li, Y-H. (2007). Neural mechanism
of acupuncture-modulated gastric motility. World J Gastroenter, 13(5),
pp. 709–716.
30. Ferreira, D. C. A., De Rossi, A., Torres, C. P., Galo, R., Paula-Silva, F. W.
G., and Queiroz, A. M. (2014). Effect of laser acupuncture and auricular
acupressure in a child with trismus as a sequela of medulloblastoma.
Acupunct Med, 32, pp. 190–193.
31. Cafaro, A., Arduino, P. G., Gambino, A., Romagnoli, E., and Broccoletti, R.
(2015). Effect of laser acupuncture on salivar flow rate in patients with
Sjögren’s syndrome. Lasers Med Sci, 30(6), pp. 1805–1809.
July 6, 2016 17:41 PSP Book - 9in x 6in 46-Hamblin-c46

Chapter 46

Intravascular Laser Irradiation of Blood

Daiane Thais Meneguzzo,a Leila Soares Ferreira,b

Eduardo Machado de Carvalho,c,d
and Cássia Fukuda Nakashimaa
a São Leopoldo Mandic University, Barbosa da Cunha Street 338, Jardim Guanabara,

Campinas, São Paulo 13073-320, Brazil

b School of Dentistry, Ibirapuera University, Interlagos Avenue, 1329 Chácara Flora,

São Paulo 04661-100, Brazil

c APCD SP, Street Volunteers of the Homeland, 547 - Santana, São Paulo 02011-000,

Brazil
d Universidade Cruzeiro do Sul, Avenida Regente Feijo, 1295 - Jardim Analia Franco,

São Paulo 03342-000, Brazil

daitm@uol.com.br

46.1 Introduction

Intravascular laser irradiation of blood (ILIB) is a systemic low-

level laser modality that aims the irradiation of blood. Discovered
by Russian scientists in the 1970s, ILIB consists of laser blood
irradiation through a fiber optic inserted in a vascular channel,
usually a vein in the forearm. ILIB irradiation lasts for about 30
min, which is assumed to be sufficient time for the whole blood to
receive light, enabling systemic effect. Thus, blood components such
as blood lipids, platelets, immune system, and red cells are the main
targets.

Handbook of Low-Level Laser Therapy

Edited by Michael R. Hamblin, Marcelo Victor Pires de Sousa, and Tanupriya Agrawal
Copyright c 2017 Pan Stanford Publishing Pte. Ltd.
ISBN 978-981-4669-60-3 (Hardcover), 978-981-4669-61-0 (eBook)
www.panstanford.com
July 6, 2016 17:41 PSP Book - 9in x 6in 46-Hamblin-c46

934 Intravascular Laser Irradiation of Blood

Although the mechanisms of ILIB are uncertain and need to

be assessed by further randomized controlled trials and clinical
reports, many studies from Russia show favorable effects of the
technique. ILIB has been studied for a wide range of medical
conditions, including diabetes mellitus, asthma, chronic hepatitis,
multiple sclerosis, cardiovascular diseases, hypertension, and au-
toimmune diseases [1–6].
In this chapter, a summary of the main indications and effects of
ILIB will be presented in order to introduce this promising “new”
treatment modality, stimulate new researches on the mechanisms
of action, and propose that further randomized clinical studies are
recommended. Still, new forms of noninvasive systemic irradiation
have already been developed and appear to be promising, such as
modified and intranasal ILIB techniques.

46.2 History of ILIB

In the 1970s, the Russians began research on ILIB with helium–

neon (He–Ne) laser, at 632.8 nm of wavelength, based on the
works of Mester et al. [6]. Originally this method was developed
for the treatment of cardiovascular diseases. Further, improvements
in blood’s rheological properties and microcirculation as well as
reduction in the infarction area have been proved. Basic research
on the mechanisms of action of ILIB has also significantly increased,
showing the antioxidant effect of ILIB therapy by the reactivation of
superoxide dismutase (SOD) enzyme [7]. SOD is a key enzyme that
controls the levels of free radicals and leads to hemostasis and to
prevent many systemic diseases.
The first laser used was the He–Ne laser (632.8 nm), with a
power of 1–3 mW and a period of exposure of 20–60 min. The
technique involves inserting an intravenous catheter in one of the
upper limbs, coupled to an optical fiber that radiates blood with
laser directly and continuously at the application site, distributing
that irradiated blood through the circulation throughout the body
(Fig. 46.1). Usually, the treatments are carried out once or twice a
day up to 10 appointments in all.
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History of ILIB 935

Figure 46.1 Intravenous laser irradiation of blood.

Most of the Russian studies published since the 1970s using the
ILIB technique were little known in the West because of decades of
political separation and were, therefore, regarded with disapproval.
Thus, ILIB remained into discredit for decades due to the lack of
scientific evidence on the mechanisms of action of laser therapy.
At that time, laser therapy was very unclear and empirical, and
only the group of the cell biologist Tina Karu was studying the
mechanisms.
Currently, new studies and clinical findings published in Russian
magazines have contributed to the permanence of ILIB and insti-
gated more scientists to carry out research. Many Western studies
of great scientific importance have brought a better understanding
of the mechanisms of action of laser therapy and elucidated many of
the effects previously reported by the Russians [7, 8].
Among the mechanisms of action known today, we will highlight
light action on cell membrane, a mechanism that may explain the
activation of immune cells, mast cell degranulation, and modulation
of lipids and other blood components; light shutdown of nitric
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936 Intravascular Laser Irradiation of Blood

oxide (NO) from hemoglobin, improving oxygenation of the blood

and vasodilatation; cell biostimulation by light absorption of
mitochondrial enzymes (mainly cytochrome c oxidase), reactivating
the cellular metabolism (ATP, reactive oxygen species [ROS], tumor
necrosis factor-α [TNF-α]) and acting on red and endothelial cells;
the induction of an electric field, polarizing blood substances to
the center of the vessel and temporarily increasing blood flow [9];
and finally, the reactivation of endogenous SOD enzyme [7], an
antioxidant activity that is important in preventing many systemic
diseases. It is believed that the SOD effect is the main mechanism
of action of ILIB; therefore, this technique is also claimed to be an
antioxidant and anti-aging therapy.

46.3 Antioxidant Action of ILIB

In our body, cells produce free radicals during the oxygen

combustion process that converts the nutrients absorbed from
food into energy. External factors may also contribute to the
increased formation of these molecules: environmental pollution; X-
ray and ultraviolet radiation; cigarette; alcohol; pesticide residues;
substances present in foods and beverages (chemical additives,
preservatives, hormones); stress; consumption of saturated fats and
consumption of animal fat.
The interaction of free radicals with the biological system,
regardless of their source, can result in significant health conse-
quences, contributing to the development of certain pathologies
such as aging, Parkinson’s disease, cancer, arthritis, cardiovascular
and cardiorespiratory complications, among others.
To combat the damaging effects of free radicals, the body uses its
defense system. The enzyme system is the main way of combating
free radicals, which prevents their formation or neutralizes them.
Among the enzymes involved in the enzymatic defense system, SOD
plays the primary role. SOD inhibits the superoxide ion, which is
the first free radical formed from molecular oxygen and serves as a
substrate for triggering the formation of more harmful radicals such
as hydroxyl (OH).
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Antioxidant Action of ILIB 937

Table 46.1 General effects of ILIB demonstrated in the literature

ILIB Effects Literature

Regulation of blood lipids Lebed’kov et al. [10]; Poluéktova et al. [11]; Huang
et al. [12]; Babaev et al. [13]
Normalizing action on lipid Azbel et al. [14]; Safafov et al. [15]; Karneev et al.
peroxidation [16]
Inhibition of platelets aggregation Sirenko et al. [17]; Prokopova et al. [18]; Shval’b
(lessening the likelihood of clot et al. [19]; Brill et al. [20]
formation)
Activated platelet aggregation Paleev et al. [21]; Gao et al. [22]
Activation of immune system Dmitriev et al. [23]; Kupnovyts’ka [24]; Palćhun
(dendritic cells, macrophages, and et al. [25]; Koshelev and Chalyk [26]; Hanul et al.
lymphocytes) [27]; Stupak and Rodiukova [28]
Immunomodulation Dedenko [29]; Marchuk and Kuzmich [30]; Alizade
and Karaeva [31]; Sidorov et al. [32]; Tsvettsikh
et al. [33]; Shakhova et al. [5]; Chiran et al. [34]
Interference in the arachidonic acid Tsuman et al. [35]; Sidorov et al. [32]; Polosukhin
cascade/anti-inflammatory effects [36]; Burduli and Krifaridi [37]; Shakhova et al. [5]
Normalize hemostasis system Koshelev and Chalyk [26]
Antioxidant defense Dedenko [29]; Grigor’eva et al. [38]; Koshelev and
Chalyk [26]; Zubkova et al. [39]; Koshelev and
Chalyk [26]; Tsvettsikh et al. [33]; Vladimirov et al.
[7]; Huang et al. [12]; Farkhutdinov [40]; Karneev
et al. [16]; Arnall et al. [41]; Burduli and Krifaridi
[42]; Burduli and Aleksandrova [3]; Shakhova et al.
[5]; Burduli and Balaian [43]
Vasodilatation Sirenko et al. [17]; Alizade and Karaeva [31]; Alizade
and Karaeva [44]
Improves rheological blood Dedenko [29]; Prokopova et al. [18]; Alizade and
characteristics, reduces erythrocyte Karaeva [31]; Paleev et al. [21]; Palćhun et al. [25];
sedimentation and blood viscosity Alizade and Karaeva [44]; Siposan and Lukacs [45];
Mi et al. [46]; Iaitskiı̆ et al. [47]; Sarycheva et al. [4];
Simonenko et al. [48]
Increasing in microcirculation Kirsanova et al. [49]; Iakubenia et al. [50]; Burduli
and Gazdanova [51]; Burduli and Gutnova [52];
Burduli and Tadtaeva [53]; Shakhova et al. [5]

SOD is the fifth most abundant enzyme in the body. But in

some pathological conditions, the enzyme can be inactivated, as in
blood acid conditions, inflammatory situation, and chronic diseases.
Laser radiation of inactive SOD has been proved to reactivate the
July 6, 2016 17:41 PSP Book - 9in x 6in 46-Hamblin-c46

938 Intravascular Laser Irradiation of Blood

enzyme, even at acidic pH conditions in which inactivation occurs

in pathological situations [7]. Thus, ILIB can reverse the oxidative
stress present in various diseases.
Treatment with ILIB is aimed at treating the patient for functional
recovery of the antioxidant enzyme system, balancing the individual
as a whole, to provide a functional optimization of each system.
The effects of ILIB and diseases treated by the ILIB technique are
summarized in Tables 46.1 and 46.2, respectively.

Table 46.2 Clinical effects of ILIB

Diseases Clinical Effects of ILIB Literature

Arthritis • Reduction in the rheumatoid blood factor to 1:20 Tupikin 1985 [54],
titers Sidorov et al. [32],
• No side effects or complications Chiran et al. [34]
• Reduction in the time of patients’ stay at hospital
• Anti-inflammatory and immunomodulating ac-
tion in patients with rheumatoid arthritis
• Normalization of titer of IgA and IgG levels
• Synergistic anti-inflammatory effect of ILBI in
combination with tocilizumab
Asthma • Retained free radical oxidation defects and the Farkhutdinov [40],
disease symptoms Sarycheva et al. [4]
• Better morphofunctional parameters of red blood
cells
Atherosclerosis • Better results in morphofunctional state of ery- Iaitskiı̆ et al. [47]
throcytes and hemorheology
• ILIB therapeutic effect persisted during 3 months
in most patients
Acute period of • Improvement in circulation, gas exchange in lungs, Kirsanova et al. [49]
hemorrhagic and tissue perfusion
shock and after • Activation of regulatory mechanisms of the re-
resuscitation gional blood flow redistribution
Bronchitis • Positive effect on circulatory system by changing Burduli and
hyperkinetic hemodynamics into normokinetic Aksenova [55]
one
Chronic brain • Normalizing action on lipid peroxidation and Karneev et al. [16]
ischemia antioxidant defense
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Antioxidant Action of ILIB 939

Table 46.2 (Contd.)

Diseases Clinical Effects of ILIB Literature

Chronic • Reduction in the functional activity of thrombocytes Spasov et al. [1],

hepatitis and in the presence of fibrinogen and adrenaline hy- Burduli and
reactive drochloride Krifaridi [42],
hepatitis • Improvement in endothelium function Burduli and
• Compensation of NO deficit Krifaridi [37]
• Protection of the organism against free radicals
decreases severity of oxidative stress
• Normalization in the plasma nitric oxide level
• Marked beneficial effect on the cytokine system
Chronic venous • Hypocoagulation, lower hematocrit index Shval’b et al. [19]
insufficiency
Diabetic • Decrease in the activity of free radical oxidation Grigor’eva et al.
angiopathies • Action on antiperoxide protection enzymes [38]
• Improvement on issue microcirculation
Diabetes • Impact on the blood lipid and phospholipid compo- Lebed’kov et al.
nents and erythrocyte membrane [10], Poluéktova
• Favorably affects the blood components et al. [11], Arnall
• Hypolipedemic and hypoglycemic effects et al. [41]
• Decreased concentration of NO in the blood
• Improves peripheral protective sensation in pa-
tients by a mechanism other than an increased NO
production
Coronary heart • Changes in erythrocyte number Simonenko et al.
disease • Effects on the blood antioxidative system [48], Zubkova
• Increase in microcirculation et al. [39], Burduli
• Increase in endothelial oscillations and capillary and Gazdanova
blood flow [51], Azbel et al.
• Increase in endothelial functional activity 1993 [14],
• Decrease in membrane toxicity by 43% Diasamidze 2004
• Decrease in the activity of blood serum components [56], Sirenko et al.
• Significant decrease in the levels of total cholesterol, 1990 [17]
and low-density lipoproteins
• Increase in the content of high-density lipoproteins
• Alleviation of pain syndrome
• Reduction in ventricular arrhythmias frequencies,
heart failures, condition recurrences, and mortality
rate
• Reduction in the hypophyseoadrenocortical and
aldosteron-renin-angiotensin system activities

(Contd.)
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940 Intravascular Laser Irradiation of Blood

Table 46.2 (Contd.)

Diseases Clinical Effects of ILIB Literature

• Reduction in blood levels of dilatants and proaggre-

gants (PGF2 alpha, vasopressin, angiotensin II)
• Increase in vasodilating and antiaggregation hor-
mones (PGE, PGI2) levels
• Improvement in PGI2/TxB2 ratio
Ischemic heart • Increase in the activity of ATP-ase Vasil’ev et al.
disease • Index of erythrocyte deformability [57]
• Improvement in cardiac function
• Optimization of the structural, functional organization
of the cellular membrane
Esophageal • Immunorehabilitation effect Hanul et al. [27]
cancer • Lower postoperative complications rate
• Mortality reduction
• Improvement in three-year survival rate indexes
Gastro- • Increase in PGE2 to the levels typical of those in Burduli and
esophageal healthy individuals Tadtaeva [53],
reflux disease • Significant decrease in all esophageal pH-metry para- Burduli and
meters Tadtaeva [58],
• The DeMeester score achieved normal values Burduli and
• All quality-of-life indicators, except for physical func- Balaian [43]
tion index, significantly improved
• Elevation of pro-inflammatory prostaglandin levels
and the improvement of parameters of microcircula-
tion
• Normalization of plasma levels of stable nitric oxide
metabolites regardless of their initial value, either
high or low
Hypertension • Drop in the arterial blood pressure Alizade and
• Improvement in general condition of patients Karaeva [31],
• Enhancement of the effectiveness of antihypertensive Alizade and
preparations Karaeva [44],
• Favorable shifts in immunological and hemorheologi- Burduli and
cal indices Aleksandrova [3]
• Beneficial clinical effect for up to 4–8 months
• Drop of arterial pressure
• Improvement in viscous and elastic properties of
blood and its hemodynamic index
• Significant decrease in Willebrand factor activity
• Normalization of the NO level regardless of its initial
value
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Antioxidant Action of ILIB 941

Table 46.2 (Contd.)

Diseases Clinical Effects of ILIB Literature

Liver • Alleviation of major clinical symptoms Babaev et al. [13]

problems • Significant positive changes in biochemical parame-
ters (AST, ALT, bilirubin, alkaline phosphatase, lactate
dehydrogenase, and total cholesterol)
Pancreatitis • Recovery of lymphocyte activity Dmitriev et al. [23],
• Increase in the ATP content in erythrocytes Dedenko [29],
• Inhibition of blood proteolytic activity Burduli and
• Enhancement of free radical oxidation, kallikrein– Gutnova [52],
kinin system activity, blood oxygen transport Burduli and
• Correction of endotoxic pancreatogenic syndrome Gutnova [59],
• Positive shifts in the immunological status, morpho- Geı̆nits et al. [60]
functional characteristics of the red blood cells and
hemoglobin, hepatic and renal functions
• Improvement in microcirculation regardless of its
hemodynamic type
• Normalization of ceruloplasmin content in the plasma
• Avoids infection of pancreatic lesions in 67.7%
• Decrease in lethality rate of sterile pancreonecrosis
from 24.4% to 5.1% and of infected pancreonecrosis
from 42.8% to 23.1%
Pneumonia • Positive dynamics in microcirculation in addition to Burduli and Pilieva
conventional medication [61], Paleev et al.
• Pronounced effects on blood rheological character- [21], Prokopova
istics: reduced blood viscosity, improved viscoelastic et al. [18]
properties and osmotic resistance of erythrocytes
• Decrease in blood viscosity
• Hypocoagulative effect of intravascular
• Normalization of clinico-roentgenologic
Psorı́asis • Increased levels of anti-inflammatory cytokines Shakhova et al. [5]
• Decreased levels of pro-inflammatory cytokines
• Immunomodulatory effect
• Stimulates the antioxidative system
• Improves microcirculation
Parkinson’s • Reduction in neurological deficit Komel’kova et al.
disease • Normalization of the activity of MAO B, Cu/Zn-SOD, 2004 [62],
and immune indices Vitreshchak et al.
• Correlation between humoral immunity and activity 2003 [63]
of the antioxidant enzymes (SOD, catalase)
• Interaction between blood cells

(Contd.)
July 6, 2016 17:41 PSP Book - 9in x 6in 46-Hamblin-c46

942 Intravascular Laser Irradiation of Blood

Table 46.2 (Contd.)

Diseases Clinical Effects of ILIB Literature

Pyelonephritis • Stimulates the antioxidant system, in particular, Tsvettsikh et al.

the activity of superoxide dismutase 1999 [33], Safafov
• Immunomodulation effect et al. [15]
• Changes in beta 2-microglobulin, lipid peroxida-
tion, middle-size molecules in blood and urine
• Effective treatment of acute calculous
pyelonephritis
Refractory tach- • Effect on the neurohumoral regulation, normal- Kupnovyts’ka [24]
yarrhythmias ization of barrier, and receptor function of my-
ocardium cell membranes
• Overcomes refractivity of tachyarrhythmias to
antiarrhythmic agents
Septic • Activation of immune system Palchun et al. [25]
complications • Improvement in rheological blood characteristics
in otorhino- • Enhancement of tissue regeneration
laryngology • Immunostimulating effect
Spinal cord • Higher mitochondrial DNA (mtDNA) copy number Stupak and
injury • Higher white blood cell adenosine triphosphate Rodiukova 1999
(WBC ATP) synthesis [28], Huang et al.
• Higher total antioxidant capacity (TAC) with 2012 [12]
significantly reduced malondialdehyde (MDA)
• Reduction in low-density lipoprotein (LDL)
• Significant increase in high-density lipoprotein
(HDL)
• Alleviation of oxidative stress and mitochondrial
dysfunction
Suppurative • More rapid normalization of the indices of mor- Marchuk and
lung diseases phologic composition of gases and acid–base state Kuzmich [30]
of blood
• Immunologic status of the organism, functions of
external respiration
• Incidence of postoperative complications reduced
by 4%
• Duration of hospital stay shortened by 8.1 days
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Modified ILIB Techniques 943

Table 46.2 (Contd.)

Diseases Clinical Effects of ILIB Literature

Suppurative • Stimulates the bone marrow and organs in which Zimon et al. [64]
peritonitis blood elements are stored
• Increase in cell number of intermediate
• Reduction in the number of prehemolytic and
degenerating forms of red cells in various stages
of peritonitis
• Retransformation of stomatocytes into discocytes,
but no marked effect on echinocytic transforma-
tion
Traumatic • Improvement in the results of conventional treat- Koshelev and
abdominal ment and reduction in complications rate Chalyk [26]
organs injuries • Boosts functional activity of leucocytes
• Normalization of hemostasis system
• Antioxidant defense
Tuberculosis • Promotion of pulmonary vascular tone Iakubenia et al.
• Better microcirculatory blood flow [50], Rusakova et al.
• Increased pulse blood filling in the affected [65]
portion of the lung
• Enhancement of the conventional treatment effi-
ciency
• Accelerated positive changes of tuberculosis by
2.5–3.5 months
• Smooth course of tuberculosis developing less
pronounced residual changes in the lung
Infectious, • Reversion of the inflammatory process Polosukhin [36]
destructive • Stabilization of fibroplastic processes
lung diseases

46.4 Modified ILIB Techniques

The original ILIB technique introduced by the Russians presents a

big disadvantage: invasive irradiation. Recently, Chinese literature
and clinical practice in Brazil have suggested other forms of sys-
temic, noninvasive irradiation, such as intranasal laser irradiation
and skin irradiation performed on the wrist. This modification in
the irradiation technique allows more frequent use and expands its
applicability to home care treatment.
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944 Intravascular Laser Irradiation of Blood

46.4.1 Intranasal Irradiation

Since 1989 many Russian groups have studied the therapeutic
effects of intranasal laser photobiomodulation (ILPBM) on the local
inflammation in vasomotor rhinitis [66]. In the mainland of China,
ILPBM has been studied for the treatment of internal diseases. The
special treatment began in 1998 and was called intranasal low-
intensity laser therapy (ILILT).

Figure 46.2 Intranasal laser irradiation.

The technique involves a diode laser device (mainly 650–660

nm) with 10–30 mW of power, attached inside the nose (Fig. 46.2).
The treatment protocol varies from 15 min to 30 min per day for
10 days (consecutively or not). There are four suggested pathways
mediating the ILILT: olfactory nerves, blood cells, meridians in
traditional Chinese medicine, and autonomic nervous system. ILILT
has been applied to treat cerebral injury [67], allergic rhinitis
[68], blood stasis syndrome of coronary heart disease, myocar-
dial infarction, and brain diseases such as insomnia, intractable
headache, Alzheimer’s disease, diabetic peripheral neuropathy, and
acute ischemic cerebrovascular disease [69].
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Modified ILIB Techniques 945

46.4.2 Wrist Skin Irradiation

With the advancement of technology, more powerful diode lasers
appeared on the market. This contributed to the noninvasive idea
(introduced by Brazilian researches a few years ago) of irradiating
the skin on the wrist to reach blood vessels. The idea was supported
by the local industry, which developed a special device for this
modified ILIB (MILIB) technique (Fig. 46.3). This technique and its
benefits became known to health professionals and patients. Among
the advantages of the traditional ILIB technique include improve-
ment in sleep quality and emotional state, increased performance
in sports, and enhanced physical and mental disposition in general.
These effects, however, have not been published yet.

Figure 46.3 Wrist skin laser irradiation (modified ILIB).

The MILIB technique involves a red (660 nm) diode low-level

laser, with a power of 60–100 mW. The laser optical fiber is placed
on the skin of the wrist region (acupuncture point region lung 8),
secured with an elastic band. The irradiation is performed for 30
min continuously, and the dosage is varied from once a week for
10 weeks to daily application for 10 days depending on the type of
disease.
All these new ILIB techniques need to be better studied and
researched. Further investigations on the irradiation time, amount
of light, and rate of light absorption by the irradiated blood vessels
should be done.
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946 Intravascular Laser Irradiation of Blood

46.5 Side Effects and Contraindications of ILIB

There is no information about the side effects of ILIB in the

literature. However, the contraindications of laser therapy should
be considered for ILIB, specially the use of laser without diagnosis,
in blood tumor, on pregnant women, and in the pre-surgery period
(ILIB can increase bleeding).

References

1. Spasov, A.A., Nedogoda, V.V., Konan, K., and Kucheriavenko, A.F. (2000).
Effect of the intravenous laser blood irradiation on efficacy of drug
preparations, Eksp. Klin. Farmakol., 63(5), pp. 65–67.
2. Shval’b, P.G., Zakharchenko, A.Ia., Sigaev, A.A., and Kataev, M.I. (1990).
Intravenous laser irradiation of the blood in occlusive vascular diseases
of the extremities, Sov. Med., 3, pp. 21–23.
3. Burduli, N.M., and Aleksandrova, O.M. (2009). Effect of intravenous
laser blood irradiation on endothelial dysfunction in patients with
hypertensive disease, Klin. Med. (Mosk), 87(6), pp. 22–25.
4. Sarycheva, T.G., Tsybzhitova, E.B., Popova, O.V., and Aleksandrov, O.V.
(2009). Morphometry and electrophoretic mobility of red blood cells
from patients with asthma in the intravenous blood laser irradiation,
Klin. Lab. Diagn., 3, pp. 13–14.
5. Shakhova, A.S., Kulikov, A.G., and Korsunskaia, I.M. (2012). The appli-
cation of intravenous laser irradiation of the blood for the combined
treatment of psoriasis, Vop. Kurortol. Fizioter. Lech. Fiz. Kult., 1, pp. 24–
28.
6. Mester, E., Spiry, T., Szende, B., and Tota, J.G. (1971). Effect of laser
radiation on the wound healing, Z. Exp. Chir., 4(5), pp. 307–312.
7. Vladimirov, Y.A., Osipov, A.N., and Klebanov, G.I. (2004). Photobiological
principles of therapeutic applications of laser radiation, Biochemistry
(Mosc), 69(1), pp. 81–90.
8. Chung, H., Dai, T., Sharma, S.K., Huang, Y.Y., Carroll, J.D., and Hamblin,
M.R. (2012). The nuts and bolts of low-level laser (light) therapy, Ann.
Biomed. Eng., 40(2), pp. 516–533.
9. Amat, A., Rigau, J., Waynant, R.W., Ilev, I.K., and Anders, J.J. (2006).
The electric field induced by light can explain cellular responses to
electromagnetic energy: A hypothesis of mechanism, J. Photochem.
Photobiol. B., 82(2), pp. 152–160.
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References 947

10. Lebed’kov, E.V., Tolstykh, P.I., Marchenko, L.F., Turkina, T.I., and
Krivikhin, V.T. (1998). The effect of the laser irradiation of the blood on
its lipid and phospholipid components in diabetes mellitus, Voen. Med.
Zh., 319(10), pp. 37–38.
11. Poluéktova, M.V., Kharchenko, I.L., Kaplan, M.A., Sokol, N.I., Ershova, L.M.,
Borgul’, O.V., Chirkova, T.V., and Vorob’eva, O.A. (2011). The development
of method of intravenous laser irradiation of blood with green laser in
patients with hyperlipidemia, Klin. Lab. Diagn., 8, pp. 15–17.
12. Huang, S.F., Tsai, Y.A., Wu, S.B., Wei, Y.H., Tsai, P.Y., and Chuang,
T.Y. (2012). Effects of intravascular laser irradiation of blood in
mitochondria dysfunction and oxidative stress in adults with chronic
spinal cord injury, Photomed. Laser. Surg., 30(10), pp. 579–586.
13. Babaev, A.V., Gogolev, D.E., Reiner, O.V., Korochkin, I.M., Fandeev, A.V.,
Pivovarov, V.Y., Fedulaev, Y.N., and Drachan, K.M. (2012). Effect of
intravenous low-intensity laser irradiation of the blood on clinical and
laboratory parameters of hepatocellular insufficiency, Bull. Exp. Biol.
Med., 153(5), pp. 754–757.
14. Azbel’, D.I., Zapara, T.A., Kuznetsova, I.Iu., Ratushniak, A.S., Shergin, S.M.,
Shurgaia, A.M., and Shtark, M.B. (1993). Changes in the neural activity of
the blood serum from patients with ischemic heart disease, Biull. Eksp.
Biol. Med., 116(8), pp. 161–163.
15. Safafov, R.M., Ianenko, E.K., Nikitinskaia, L.P., Golovanov, S.A.,
Drozhzheva, V.V., Kon’kova, T.A., and Danilkov, A.P. (1997). The dynamic
level of beta 2-microglobulin, the basic lipid peroxidation indices and
middle molecules in the blood and urine in patients with acute calculous
pyelonephritis against a background of endovascular helium-neon laser
therapy, Urol. Nefrol. (Mosk), 1, pp. 11–13.
16. Karneev, A.N., Solov’eva, E.Iu., and Fedin, A.I. (2007). Correction of
free-radical processes in patients with chronic brain ischemia with
intravenous laser irradiation of blood, Vopr. Kurortol. Fizioter. Lech. Fiz.
Kult., 3, pp. 22–26.
17. Sirenko, Iu.N., Savitskiı̆, S.Iu., Krasnitskiı̆, S.S., Shabil’ianov, A.V., and
Popova, L.I. (1990). The use of low-intensity laser irradiation of the
blood in myocardial infarction, Sov. Med., 3, pp. 18–21.
18. Prokopova, L.V., Losev, A.A., and Ursol, Iu.I. (1992). Effect of intravascu-
lar laser therapy on the rheologic properties of blood in children with
bilateral destructive pneumonia, Klin. Khir., 6, pp. 7–9.
19. Shval’b, P.G., Kachinskiı̆, A.E., Kolobaev, V.I., and Kataev, M.I. (1992).
Intravenous laser irradiation of the blood in severe forms of chronic
venous insufficiency, Vestn. Khir. Im. I. I. Grek., 149, pp. 78–80.
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948 Intravascular Laser Irradiation of Blood

20. Brill, A.G., Shenkman, B., Brill, G.E., Tamarin, I., Dardik, R., Kirichuk,
V.F., Savion, N., and Varon, D. (2000). Blood irradiation by He–Ne laser
induces a decrease in platelet responses to physiological agonists and
an increase in platelet cyclic GMP, Platelets, 11(2), pp. 87–93.
21. Paleev, N.R., Karandashov, V.I., Voronina, M.A., and Fin’ko, I.A. (1993).
Changes in the blood rheological properties in the transcutaneous
irradiation of the ulnar vascular fascicle with a helium-neon laser, Biull.
Eksp. Biol. Med., 116(10), pp. 428–430.
22. Gao, X., Zhi, P.K., and Wu, X. (2008). Low-energy semiconductor laser
intranasal irradiation of the blood improves blood coagulation status in
normal pregnancy at term, Nan. Fang. Yi Ke Da Xue Xue Bao., 28(8), pp.
1400–1401.
23. Dmitriev, A.E., Iudin, V.A., Arapov, N.A., and Martynov, V.A. (1989).
Effect of intravascular laser irradiation of the blood on blood cells in
pancreatitis, Klin. Med. (Mosk), 67(5), 108–110.
24. Kupnovyts’ka, I.H. (1992). Laser irradiation of the blood in patients with
refractory tachyarrhythmias, Lik. Sprava., 10, pp. 38–41.
25. Palćhun, V.T., Lapchednko, A.S., and Kucherov, A.G. (1995). Intravascular
laser irradiation of the blood in the treatment of suppurative septic
complications in otorhinolaryngology, Vestn. Otorinolaringol., 2, pp. 8–
10.
26. Koshelev, V.N., and Chalyk, Iu.V. (1998). Intravascular laser irradiation of
blood in treatment of traumatic abdominal organs injuries, Khirurgiia
(Mosk), 5, pp. 40–42.
27. Hanul, V.L., Zaı̆tsev, S.L., Kirkilevs’kyı̆, S.I., and Fil’chakov, F.V. (1999).
Intravascular laser irradiation of the blood in complex treatment of the
patient with esophageal cancer, Klin. Khir., 5, pp. 27–29.
28. Stupak, V.V., and Rodiukova, E.N. (1999). The possibility of immuno-
correction in spinal-spinal cord trauma by using intravenous laser
irradiation, Zh. Vopr. Neirokhir. Im. N. N. Burdenko., 1, pp. 20–25.
29. Dedenko, I.K. (1989). Effect of intravenous laser irradiation of blood on
the homeostasis in patients with hemorrhagic pancreatitis, Klin. Med.
(Mosk)., 67(8), pp. 70–73.
30. Marchuk, I.K., and Kuzmich, V.N. (1993). Intravascular laser irradiation
of the blood in the combined treatment of chronic suppurative lung
diseases, Klin. Khir., 6, pp. 9–10.
31. Alizade, I.G., and Karaeva, N.T. (1994). Experience in the use of
autotransfusions of laser-irradiated blood in treating hypertension
patients, Lik. Sprava., 5–6, pp. 29–32.
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References 949

32. Sidorov, V.D., Mamiliaeva, D.R., Gontar’, E.V., and Reformatskaia, S.Iu.
(1999). The interauricular laser therapy of rheumatoid arthritis, Vop.
Kurortol. Fizioter. Lech. Fiz. Kult., 3, pp. 35–43.
33. Tsvettsikh, V.E., Sultanbaev, V.R., Berdichevskiı̆, B.A., Kazeko, N.I., Ovchin-
nikov, A.A., Sultanbaev, R.A., and Murychev, A.V. (1999). Endovascular
helium-neon laser irradiation of the blood in the treatment of chronic
pyelonephritis, Urologiia, 6, pp. 13–15.
34. Chiran, D.A., Weber, M., Ailioaie, L.M., Moraru, E., Ailioaie, C., Litscher,
D., and Litscher, G. (2014). Intravenous laser blood irradiation and
tocilizumab in a patient with juvenile arthritis, Case Rep Med., 2014,
923496.
35. Tsuman, V.G., Mashkov, A.E., Shcherbina, V.I., Surovkina, M.S., and
Sinenkova, N.V. (2005). Intracavitary laser therapy and its effect on the
kallikrein-kinin system of blood in children with pneumothorax, Vestn
Ross Akad. Med. Nauk., 9, pp. 20–26.
36. Polosukhin, V.V. (2000). Ultrastructure of the blood and lymphatic capil-
laries of the respiratory tissue during inflammation and endobronchial
laser therapy, Ultrastruct. Pathol., 24(3), pp. 183–189.
37. Burduli, N.M., and Krifaridi, A.S. (2011). The influence of low-intensity
laser radiation on the vascular endothelium function and the cytokine
system in patients with chronic viral hepatitis, Vopr. Kurortol. Fizioter.
Lech. Fiz. Kult., 2, pp. 30–34.
38. Grigor’eva, I.V., Rakita, D.R, and Garmash, V.Ia. (1991). Effect of
endovascular laser irradiation of the blood of patients with diabetic
angiopathies, Probl. Endokrinol. (Mosk), 37(6), pp. 28–30.
39. Zubkova, S.M., Sorokina, E.I., Kenevich, N.A., Tupitsyna, Iu.Iu., and
Minenkov, A.A. (1993). The blood antioxidant system in patients with
ischemic heart disease undergoing laser therapy, Vopr. Kurortol. Fizioter.
Lech. Fiz. Kult., 6, pp. 4–7.
40. Farkhutdinov, U.R. (2007). Intravascular laser irradiation of blood in the
treatment of patients with bronchial asthma, Ter Arkh., 79(3), pp. 44–48.
41. Arnall, D.A., Nelson, A.G., López, L., Sanz, N., Iversen, L., Sanz, I.,
Stambaugh, L., and Arnall, S.B. (2006). The restorative effects of
pulsed infrared light therapy on significant loss of peripheral protective
sensation in patients with long-term type 1 and type 2 diabetes mellitus,
Acta. Diabetol., 43(1), pp. 26–33.
42. Burduli, N.M., and Krifaridi, A.S. (2009). Effect of low-intensity laser
radiation on the function of vascular endothelium in patients with
chronic viral hepatitis, Klin. Med. (Mosk), 87(1), pp. 49–52.
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950 Intravascular Laser Irradiation of Blood

43. Burduli, N.M., and Balaian, M.M. (2013). The influence of low-intensity
intravenous laser irradiation of the blood on the endothelial function
in the patients presenting with gastroesophageal reflux disease, Vopr.
Kurortol. Fizioter. Lech. Fiz. Kult., 5, pp. 33–35.
44. Alizade, I.G., and Karaeva, N.T. (1997). The effect of plasmapheresis and
laser irradiation of the blood on the hemorheological and hemodynamic
indices in hypertension patients, Lik. Sprava., 3, pp. 110–111.
45. Siposan, D.G., and Lukacs, A. (2000). Effect of low-level laser radiation
on some rheological factors in human blood: An in vitro study, J. Clin.
Laser Med. Surg., 18(4), pp. 185–195.
46. Mi, X.Q., Chen, J.Y., Liang, Z.J., and Zhou, L.W. (2004). In vitro effects
of helium-neon laser irradiation on human blood: Blood viscosity and
deformability of erythrocytes, Photomed. Laser. Surg., 22(6), pp. 477–
482.
47. Iaitskiı̆, N.A., Ageenko, E.M., Davydenko, T.E., Volchkov, V.A., Churzin,
O.A., and Zharskaia, V.D. (2006). Intravascular laser irradiation of
blood in complex treatment of obliterating atherosclerosis of the lower
extremity vessels in elderly and senile patients, Vestn. Khir. Im. I. I. Grek.,
165(4), pp. 34–37.
48. Simonenko, V.B., Siuch, N.I., and Vokuev, I.A. (2002). Diagnostic
implications of changed red cell count in low-intensity laser radiation of
blood in elderly patients with coronary heart disease, Klin. Med. (Mosk),
80(4), pp. 31–33.
49. Kirsanova, A.K., Novoderzhkina, I.S., Kozhura, V.L., Berezina, T.L.,
Dvoretskiı̆, S.V., Talantsev, K.V., and Iakovleva, N.E. (1994). Changes in
the oxygen balance of the body in the acute period of hemorrhagic shock
and after resuscitation under the effect of intravascular helium-neon
laser irradiation of the blood, Anesteziol. Reanimatol., 5, pp. 33–35.
50. Iakubenia, O.N., Tostik, S.I., and Iakubenia, G.I. (1999). Impact of
combined magnetic and laser radiation of regional pulmonary blood
flow in patients with destructive pulmonary tuberculosis, Probl. Tuberk.,
6, pp. 30–32.
51. Burduli, N.M., and Gazdanova, A.A. (2008). Laser Doppler fluometry
in assessment of endothelium state in patients with coronary heart
disease and its correction by intravenous laser irradiation of blood, Klin.
Med. (Mosk), 86(6), pp. 44–47.
52. Burduli, N.M., and Gutnova, S.K. (2009). Types of microcirculation and
laser therapy in chronic pancreatitis, Klin. Med. (Mosk), 87(8), pp. 56–
61.
July 6, 2016 17:41 PSP Book - 9in x 6in 46-Hamblin-c46

References 951

53. Burduli, N.M., and Tadtaeva, D.Ia. (2012). The influence of intravenous
laser therapy on prostaglandin E2 and F2-alpha dynamics and the state
of microcirculation in the patients presenting with gastroesophageal
reflux disease, Vopr. Kurortol. Fizioter. Lech. Fiz. Kult., 6, pp. 17–20.
54. Tupikin, G.V. (1985). Anti-inflammatory and immunosuppressive effects
of laser therapy in patients with rheumatoid arthritis, Ter Arkh., 57(8),
pp. 37–39.
55. Burduli, N.M., and Aksenova, I.Z. (2006). The effects of intravenous
laser irradiation of blood on the system hemodynamics of patients with
chronic obstructive bronchitis exacerbation, Klin. Med. (Mosk), 84(3),
pp. 37–39.
56. Diasamidze, Iu.S. (2004). Impact of photo hemotherapy on blood lipid
composition in coronary heart disease, Klin. Med. (Mosk), 82(7), pp. 34–
36.
57. Vasil’ev, A.P., Strel’tsova, N.N., Shakhov, G.G., and Nikitina, V.I. (1992). The
effect of laser irradiation of the blood on the adenosine triphosphatase
activity of the erythrocyte membranes and on the cardiac activity
indices in patients with ischemic heart disease, Lik. Sprava., 6, pp. 61–
64.
58. Burduli, N.M., and Tadtaeva, D.Ia. (2012). Impact of laser therapy on
PGE2 level, 24-hour pH-metry changes, and quality of life in patients
with gastroesophageal reflux disease, Ter. Arkh., 84(12), pp. 58–61.
59. Burduli, N.M., and Gutnova, S.K. (2010). Effect of intravenous laser
irradiation of the blood on plasma content of ceruloplasmin in patients
with chronic pancreatitis, Bull. Exp. Biol. Med., 149(6), pp. 697–698.
60. Geı̆nits, A.V., Gul’muradova, N.T., and Uspenskaia, T.Z. (2011). The use of
laser beam irradiation by the acute destructive pancreatitis, Khirurgiia
(Mosk), 7, pp. 56–61.
61. Burduli, N.M., and Pilieva, N.G. (2007). The dynamics of microcirculation
parameters in patients with pneumonia receiving intravenous irradia-
tion of blood as a part of complex treatment, Klin. Med. (Mosk), 85(7),
pp. 48–50.
62. Komel’kova, L.V., Vitreshchak, T.V., Zhirnova, I.G., Poleshchuk, V.V.,
Stvolinskiı̆, S.L., Mikhaı̆lov, V.V., Gannushkina, I.V., and Piradov, M.A.
(2004). Biochemical and immunological induces of the blood in
Parkinson’s disease and their correction with the help of laser therapy,
Patol. Fiziol. Eksp. Ter., 1, pp. 15–18.
63. Vitreshchak, T.V., Mikhailov, V.V., Piradov., M.A., Poleshchuk, V.V., Stvolin-
skii, S.L., and Boldyrev, A.A. (2003). Laser modification of the blood in
July 6, 2016 17:41 PSP Book - 9in x 6in 46-Hamblin-c46

952 Intravascular Laser Irradiation of Blood

vitro and in vivo in patients with Parkinson’s disease, Bull. Exp. Biol. Med.,
135(5), pp. 430–432.
64. Zimon, I.N., Agzamov, A.I., Choroshaev, V.A., Kalish. Iu.I., and Dalimov, I.Z.
(1992). Effects of intravascular laser irradiation of blood on erythrocyte
stereo-ultrastructure in the treatment of generalized suppurative
peritonitis, Khirurgiia (Mosk), 9–10, pp. 35–39.
65. Rusakova, L.I., Dobkin, V.G., and Ovsiankina, E.S. (2002). Efficiency
of supra-venous blood laser radiation used in the treatment of
disseminated pulmonary tuberculosis in adolescents, Probl. Tuberk., 8,
pp. 16–18.
66. Tulebaev, R.K., Sadykov, Sh.B., Romanov, V.A., and Khalitova, G.Kh.
(1989). Indicators of the activity of the immune system during laser
therapy of vasomotor rhinitis, Vestn. Otorinolaringol., 1, pp. 46–49.
67. Xiao, X., Guo, Y., Chu, X., Jia, S., Zheng, X., and Zhou, C. (2005). Effects
of low power laser irradiation in nasal cavity on cerebral blood flow
perfusion of patients with brain infarction, Chinese J. Phys. Med. Rehab.,
27(7), pp. 418–450.
68. Yıldırım, Y.S., Apuhan, T., and Koçoğlu, E. (2013). Effects of intranasal
phototherapy on nasal microbial flora in patients with allergic rhinitis,
Iran J. Allergy Asthma Immunol., 12(3), pp. 281–286.
69. Liu, C.Y., and Zhu, P. (Eds.). (2009). Intranasal Low Intensity Laser
Therapy. Beijing: People’s Military Medical Press.
July 6, 2016 17:41 PSP Book - 9in x 6in 47-Hamblin-c47

Chapter 47

Nonsurgical Laser Therapy for Type 1

and Type 2 Diabetes

Leonardo Longo
Institute for Laser Medicine, International Academy Laser Medicine and Surgery,
Borgo Pinti 57, 50121 Firenze, Italy
longo.leonardo@gmail.com

47.1 Epidemiology

Diabetes is a worldwide epidemic pathological condition, and

its frequency is increasing progressively [1]. Diabetes treatment
is complex because diet and physical activity have the same
importance as drug therapy. Often patients do not appreciate this
important principle. There are also nonconventional treatments
such as acupuncture and light-based treatment. The American Heart
Association and the American Diabetes Association have recently
released their statistics for 2012, which are more shocking than
ever [2]:

• Approximately, 86 million Americans less than 20 years old

have diabetes.

Handbook of Low-Level Laser Therapy

Edited by Michael R. Hamblin, Marcelo Victor Pires de Sousa, and Tanupriya Agrawal
Copyright c 2017 Pan Stanford Publishing Pte. Ltd.
ISBN 978-981-4669-60-3 (Hardcover), 978-981-4669-61-0 (eBook)
www.panstanford.com
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954 Nonsurgical Laser Therapy for Type 1 and Type 2 Diabetes

• Each year about 15,000 people less than 20 years old are
diagnosed with Type 1 diabetes.
• In 2012, 29.1 million Americans, or 9.3% of the population,
had diabetes.
• An estimated 8.1 million Americans have undiagnosed
diabetes.
• An estimated 81.5 million Americans have pre-diabetes.
• Every year, 1.6 million new cases of diabetes are diagnosed.

In 2010, diabetes killed 70,553 Americans and was the seventh

cause of deatha Globally, as of 2010, an estimated 285 million
people had diabetes, with Type 2 making up about 90% of the
cases. In 2013, according to the International Diabetes Federation,
about 381 million people had diabetes. Its prevalence is increas-
ing rapidly, and by 2030, this number is estimated to almost
double [1].
Diabetes mellitus occurs throughout the world, but it is more
common in developed countries (especially Type 2). The greatest
increase in prevalence is, however, expected to occur in Asia and
Africa, where most patients will probably be found by 2030. The
increase in developing countries follows the trend of urbanization
and lifestyle changes, perhaps most importantly a “Western-style”
diet. This suggests an environmental (i.e., dietary) effect, but there
is little understanding of the mechanisms at present, though there
is much speculation, some of which have been most compellingly
presented [1].
In conclusion, diabetes mellitus is a widespread disease with
a serious public health problem and a risk factor for many fatal
issues. Its life-long negative influence on the quality of life and its
invalidating consequences are a serious concern for patients and
health authorities [3]. It is understandable that scientists, research
workers, clinicians, health economists, and planners are concerned
with diabetes and try their best to control it through more effective,
easier, and cheaper methods.

a See more at: www.diabetes.org/diabetes-basics/statistics/#sthash.hT1K4hy2.dpuf

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History 955

47.2 History

New therapies based on light energy, electricity and bioresonance

studies have an extensive history as methods for treating diseases.
For 3000 years, it has been known in Chinese medicine that energy
directly tracks organs’ activity and that these activities are subject
to rhythmical vibrations [4]. Around 600 BC, the Greeks found
that when amber is rubbed against fur, it attracted straw particles
[5]. Around 1600 AD, Dr. William Gilbert, who studied magnetism,
was the first to record the word “electric” while investigating the
reactions of amber and magnets. The Mediterranean Torpedo fish
was the earliest reference on the use of electricity in medicine.
There is a bioresonance information laser method of treatment
involving an integrative use of low-level laser irradiation (LLLI),
microwave resonance puncture (MRP), and light-emitting diode
chromatotherapy (LEDCT) (Table 47.1), which would eliminate
insulin by injection or tablets that people with diabetes consume,
thereby allowing them to have a normal life with little diet
restrictions [6]. The beneficial effects of light on biological tissues
have been known since the days of Hippocrates and even earlier.
Approximately 5000 years before Christ, it was known that all

Table 47.1a List of sources already used in

diabetes treatment

• Intense pulse lamps and diodes

• Nonsurgical laser therapy (NSLT) alone
• Laser therapy coupled with electric stimulation
• Laser therapy coupled with photodiodes
• NSLT coupled with magnetic fields

Table 47.1b List of different procedures used

• Regular medicine
• Energetic (quantum) medicine
• Acupuncture
• Intravenous biomodulation
• But for periods, not continued forever
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956 Nonsurgical Laser Therapy for Type 1 and Type 2 Diabetes

Table 47.1c Sources used by us

• Laser near-infrared with magnetic field coupled

• Light red and near-infrared
• But for periods, not continued forever

living matter produces and exchanges different types of energy with

the surrounding environment. Many different names were given to
these phenomena, starting with “prana” for all, including luminous
energies; “chakra” to identify the areas where energy exchange with
the outside is greatest; and finally “aura” for the energy layers
around and immediately interconnected with matter.
J. White, a North American scientist, has studied approximately
100 different ancient civilizations and found 100 different words
used to identify the above concepts [7] (Table 47.2). Up to a few
years ago, the science that studies the human body as if it were
a machine that produces and exchanges energy with the outside
was called “energetic medicine” [8], and today it is known as
“quantum medicine” [9], “prana” as “cosmic energy”, and the chakras
are related to the “coherence domains.” It has been found that
the non-corpuscular electromagnetic radiation produced by the
human body is distributed in areas called “auras” and each aura
has its corresponding wavelength and apparatus. This is where
endogenous energy is immediately exchanged with the outside
exogenous energy. It has also been demonstrated that there is an
interval of approximately 20 days between positive and negative
changes in the energetic aura and body matter, as organs and
apparatus to which it corresponds, and that lasers selectively
influence the auras.
The effects of light on glucose metabolism are not yet clear. We
know, however, that sailors with Type 1 and Type 2 diabetes reduce
the dosage of insulin and anti-diabetic drugs when at high sea. For
over 30 years, evidence-based medicine has showed that lasers have
positive glucose-normalizing effects on Type 1 and Type 2 diabetes
[10].
In the world, there are at least five different types of anti-diabetic
treatments based on light and other energy sources (Table 47.1).
These data encourage us to experiment with the effects of a system
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History 957

Table 47.2 Human energy field

5000 years AC
• Prana, aura, and chakra in India
• Ch’i and Ying-Yang in China
• Ninety seven different names in 97 different countries (J. White, 1977, Future science,
in [8])
Twentieth Century
• Cosmic energy, aura, chakra [8]
• Quantic energy, bioplasma, coherence domain [14]

based on lasers and electromagnetic fields on patients with Type 1

and Type 2 diabetes, following the guidelines of the Declaration
of Helsinki as recently reiterated by the European Union. Our first
aim was to determine whether or not lasers have an effect on
sugar metabolism in diabetic patients. In this case, electromagnetic
fields create a “bioresonance,” which is an increase in intercellular
vibrations so that light can better penetrate intercellular spaces
while maintaining its coherence.
Low-level laser therapy (LLLT) was proposed by Toshio Ohshiro
and Glenn Calderhead in 1978 [11], based on the power of laser
irradiation. Other laser groups are high-level laser therapy and mid-
level laser therapy. Other classifications based on the power of the
tools include “soft,” “mid,” and “hard” laser.
Today we know that apart from power, the physical parameters
responsible for the results are many more and the link between
these parameters has greater importance. For this reason, we prefer
to name LLLT “nonsurgical laser therapy” (NSLT). In NSLT, red and
near-infrared light is directed over tissue for improving healing by
reducing pain and bacterial count. NSLT uses cold (subthermal)
laser light energy to stimulate cellular function. Therapy is accurate,
safe, and can be extremely effective for a wide variety of conditions.
On average, the energy range of low-level laser light lies between
1 mW, and 500 mW, while for surgical lasers, the energy is 3000–
10,000 mW [12].
In Helsinki, Finland, Dr. A. M. Makela performed a study on 24
diabetic patients, seven with Type 1 and 17 with Type 2. A red laser
was used on acupuncture points for 25–30 s on each point, and a
blue cluster was used for 5 min on each area with the dose being
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958 Nonsurgical Laser Therapy for Type 1 and Type 2 Diabetes

increased to 10 min if glucose levels warranted it. These treatments

were performed once a week for 4 weeks, and then once every 2
weeks for 2 months, followed by once a month as needed. Patients
also used the blue cluster treatment at home. Seven Type 1 and 17
Type 2 diabetic patients had treatments for a year or longer. All
patients reported improvement in complications. Of the 17 Type 2
patients, 15 did not need drugs for 6 months. Type 1 patients could
reduce insulin by almost three-fourths, and some of the younger
patients could cut down even more [13].
In Tomar, Portugal [10], I reported the data of an experiment
performed with 15 patients, five of which suffered from diabetes
Type 1 and 10 with diabetes Type 2. Patients were from both
sexes and between 18 and 65 years old. Type 1 patients were
treated once a day for 3 weeks and then once a week until the
glycemia was normal. Type 2 patients were treated once a week
until glycemia was normalized. Both groups decreased anti-diabetic
drugs as they progressed through the treatment. The treatment
included 15 sessions on average. After one month, the follow-up was
excellent, and at six months, it was variable; at the end of 1 year, both
types required another cycle of treatment.
A study with a larger number of patients (and maybe the best
success overall) was performed by Dr. Pretidev Ramdawon from
Mauritius Republic [6]. He had 60 clinical cases, divided into three
age groups—children of age 3.5–15 years, adolescents, and adults—
with the oldest age group of 76. Each control group had three
patients from each age group. The subgroups were those taking
hypoglycemic tablets (no children, two adolescents, and 18 adults)
and those taking insulin injections (five children, six adolescents,
and 29 adults). In this study, a combination of lasers was used,
including helium–neon, light-emitting diode (LED) red, and LED
infrared. These lasers were used intravenously, transcutaneously on
laser-puncture points and coherence domains.
LLLT formed a part of a complex but synergistic method of
treatment, which is actually known as “magneto-infrared laser
therapy” (MILT). This kind of therapy involves the influence of a
constant magnetic field and LED red and infrared light irradiation,
which help the infrared laser irradiation to attain penetration depths
of many centimeters in bio-tissues. This penetrative ability of the
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Background and Objectives 959

LLLT in tissues, through a complex but synergistically acting quanto-

magnetic fields, assures the success of application of the treatment
method for such internal organs as liver and pancreas, which
are directly responsible for the development and manifestation of
diabetes mellitus.
Dr. Ramdawon achieved phenomenal results with his combina-
tion of therapies [6]. By the end of the sixth week, all patients
could stop diabetic medications. Furthermore, all other diabetic
complications, from cardio-myopathy to foot ulcers, were healed
with the exception of three cases, which had undergone plastic
surgery for skin grafting on a foot ulcer. Sixty percent of patients
could return to eating and drinking what they wanted, while the
other 40% had some dietary restrictions. There were also nine
control patients. Nobody had side effects; blood sugar was lowered
(but still in a good range), and their overall general health was
improved. Four of them, who previously had constipation and
flatulence, no longer had gastrointestinal problems. The follow-up
results at the end of a 7-year period were astounding. There was no
recurrence of diabetic symptoms. The results of all test groups have
been positive.

47.3 Background and Objectives

Since human body is composed of 15% matter and 85% water, it

is possible that energy must first get through the water and be
modulated by it before it can be totally absorbed by matter. Some
of light’s biomodulating effects were already known and partially
measured in the 1950s, that is, even before laser was invented.
Inyushin et al. [14] spoke of “bioplasm” to describe the energies
that surround human body and must always be in dynamic
equilibrium with it, in order to avoid organic diseases. According
to Inyushin, light influences and restores bioplasm’s integrity. This
global approach to human body justifies some data of evidence-
based medicine and enormously enlarges our knowledge. Of
course, in medicine and biology, experimentation must follow three
fundamental criteria:
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960 Nonsurgical Laser Therapy for Type 1 and Type 2 Diabetes

(1) Helsinki Declaration rules and EC guidelines.

(2) Virchow’s approach: “At first we study the Facts, then the causes
of Facts.”
(3) WHO approach: “We must study and verify each substance,
energy, tool which modifies a physiological process of the
human body.”

From an exclusively material and Newtonian standpoint, around

1966, Mester et al. [15] were the first to point out that a red light
laser stimulates hair regrowth in the coats of rats and halves the
healing time of experimental ulcers. Many experiments have been
done since then, which incontrovertibly proved that visible and
close to infrared wavelength lasers influence healing time of skin
wounds and ulcers, stimulating or inhibiting the process according
to the radiation dosage and method [16–21]. About 15 years ago,
at the University of Bethesda (Maryland), it was reported that, at
specific doses, some lasers increase cultured fibroblast activity by
98% [22].
Many strictly dose-dependent effects have been demonstrated
in nearly all normal and pathological biological processes: from
cell maturation to reproduction, from inflammation to edema, from
neural irritation to pain inhibition, and also through increased
endogenous endorphin production. The importance of dosage in all
these cases was immediately realized to be fundamental, because
the same type of laser may have opposite effects on the same
biological process and on the same tissues if the irradiation dose is
modified. A good critical review on this topic has been written by
Tuner and Hode [23]. Many other uses of nonsurgical laser were
studied following this concept, as the modulation of metabolisms,
immunological system, nerve cells functions, progenitor cells, and
light energy exchanges [21, 22, 25–29].
In terms of dosage, in order to obtain the desired effects, we
must consider a whole series of physical parameters (wavelength,
emission, fluence, energy density, repetition pulse frequency, spot
size, irradiation time/spot), biological factors (type of tissue,
biological health), and clinical factors (irradiated point, number
and rhythm of sessions, irradiation procedure) [21, 22, 24, 28].
This is what is done in soft tissue regeneration, such as healing of
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Study Design 961

skin wounds and ulcers, in the treatment of bone-muscular-tendon

inflammations and traumas and neuralgias.
A recent review of the available medical literature on NSLT
and diabetes mellitus has produced rather scanty results. Clinical
trials carried out so far are rather few, mainly because of difficulty
in patient recruitment and lack of standardized guidelines for
treatment protocols. It is also possible that some trials carried out
in certain countries are not been given adequate publicity.
In conclusion, treatment of diabetes is very difficult with
insulin, immunosuppressive drugs, diet, and other. These treatments
never follow the need for glucose of patient’s tissues because
it changes continuously, resulting in frequent hypoglycemic and
hyperglycemic crisis, which is very dangerous for patients. In
addition, the continuous introduction of hypoglycemic substances
from the extern completes the total atrophy of the pancreas isles and
cancels the property of pancreas regeneration. The aim of our study
is to verify scientifically if some laser exposure parameters have an
effect on glycaemia and on the management of diabetes, and the
persistence duration of this bio-effect. Laser beam can be used for
obtaining an anti-inflammatory effect on tissues and for increasing
cell functions. We can also suppose a regenerative effect on these
cells.

47.4 Study Design

In this chapter, we would like to unite our previous work with

a proposal for a future research on this topic. For instance, one
of our case reports from Florence, Italy, was a 48-year-old male
who had been taking insulin injections for more than 20 years for
diabetes mellitus Type 1 [30]. He performs sedentary work and
reports hypertension (under treatment with beta-blockers) and has
experienced frequent episodes of hypoglycemia. We performed laser
treatment on his stomach, liver, and spleen regions for 5 min on each
zone, once a day for 3 weeks and then once a week until glycemia
was normalized.
This patient was treated with NSLT for 15 consecutive sessions,
during which he decreased his exogen insulin dose progressively
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962 Nonsurgical Laser Therapy for Type 1 and Type 2 Diabetes

Table 47.3a Inclusion criteria of patients

• Type 1 and Type 2 human diabetes in compensative phase

• Both sexes and middle/old age
• Weight normal or high
• Diabetes started more than 1 year ago

Table 47.3b Exclusion criteria of patients

• History of hypo- and hyperglycemic coma

• History of cardiovascular non-treatable complication
• History of malignancy
• Contemporary participation in a clinical trial for hypoglycemic drug effect control

Table 47.3c Suspension criteria of patients

• Patients with diabetes complication started after laser treatment

• Patients who do not follow the protocol prescribed and the diet (about 60%)
• Patients with adverse effects of laser irradiation (local burns, allergic reaction)
• Patients who present some exclusion criteria that appear after inclusion in the
treatment protocol

and by the end had altogether stopped the insulin injections. He did
a follow-up with the dosage of HbA1c at 3 months and 6 months, at
which times the glycemic values were normal.
NSLT applications with a specific treatment protocol were
carried out between June 2004 and August 2009. We enrolled
patients with diabetes Type 1 and Type 2. Twenty-two Type 1 and
42 Type 2 patients were treated. Five patients per group were given
placebo treatment. Patients were selected on the basis of inclusion,
exclusion, and interruption criteria of treatment (Table 47.3) and
were properly informed about the experimental nature of the study
and the potential benefits and risks connected with it. They gave
their informed consent prior to the start of laser treatment [10].
We used 808 nm diode laser, with a first cycle of 15 sessions on
average of two sessions a day. We repeated one to two applications
a week, then once a month, following glycaemia variations. A total of
74 diabetes mellitus patients were enrolled (Table 47.4)
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Study Design 963

Table 47.4 Features of the series studied

Diabetes Type 1 Diabetes Type 2

Laser treatment Placebo Laser treatment Placebo

Number 22 5 42 5
Sex All All
Age (years) 30 ± 6 45 ± 12
Diabetes duration (years) Minimum 1 year Minimum 1 year
Body mass index NA 32.6 ± 3.7
Median fasting glycemia 20% up 20% up
HbA1c 20% up 20% up

Phase one of clinical experimentation was already done by many

authors [6, 10, 12]. Before treatment, in phase two protocol, patients
underwent a check-up, with control of hematochemical parameters
of blood and urine and function of principal organs such as heart,
liver, kidney, brain, lung, blood, eye, and endocrine system. Blood
sugar was measured before, immediately after, and 10 min after
treatment (Table 47.5). Glycated hemoglobin was measured before
and three months after laser treatment, then every three months. At
the end, patients took their scheduled daily measurements prior to
the treatment, and one extra reading at the same time of day that
the treatment is completed. These measurements were continued
for at least 1 month after concluding the treatment, and then we
recommended taking at least two daily measurements on empty
stomach in the morning and evening, even more than 1 month after
completing the treatment.
Patients fasted for 4 h because previous experiments demon-
strated that laser irradiation (at a dosage level reported to be
effective in the treatment of diabetic patients) could accelerate
gastric emptying. This emptying would result in an increase in the
glycemic level if a patient eats within 5 h before laser irradiation. It
is absolutely necessary that the patient fasts for 4 h prior to the laser
treatment.
Patients in both groups stopped taking insulin 4 h before laser
treatment. They resumed taking exogen insulin only if the glycemic
levels were not reduced by a minimum of 20% immediately after
laser treatment and persisted for 1 day.
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964 Nonsurgical Laser Therapy for Type 1 and Type 2 Diabetes

Table 47.5a Italian experience: Phase two—control parameters

of patients

• Glycated hemoglobin—at first more than 20% up

• Glycemia—at first more than 20% up
• Hematochemical parameters measured once every three months (blood and urine)
• Presence/absence of hypo- and/or hyperglycemic crisis

Table 47.5b Glycemia control

• Regular daily controls

• Immediately before irradiation
• Ten minutes until 1 h after the irradiation
• The day after, at the same hour after irradiation
• Each day, at the same hour after irradiation
• Once a week

Table 47.5c Glycated hemoglobin control

• Before treatment
• Three months after treatment
• Every 3 months

In Type 1 and Type 2 patients, we irradiated the sites of lesions

and the coherence domains of the injured apparatus. Unfortunately,
we could not carry out phase three of clinical experimentation until
today for economic and legal troubles.
However, the same protocol can be used for phase three of
the clinical experimentation, but with more specific tests, such as
dosage of C peptide, serotonin, insulin, and insulin-like substances
measured before and after the conclusion of the first cycle of
treatment, and then twice a year.
Treatment with laser started with the parameters listed in
Table 47.6. Dosage was adjusted following immediate behavior
of glycemia, different for each patient and in the same patient,
depending on a lot of internal and external factors. Five minutes of
irradiation were done on each of the selected areas in succession.
Placebo treatment consisted of only a red laser pointer. Each patient
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Study Design 965

Table 47.6 Dosage parameters

Wavelength 810 nm
Power density (on average) 50 mW/cm2
Time per spot (on average) 5 min
Emission on average PW 50 Hz
Mean power per spot (fluence) Maximum 1 J/cm2
Spot size 1 cm
Irradiated points (zones) Abdomen and coherence domains
Nr and rhythm of sessions Variable

was treated always at the same time of day. The irradiation zones
were different (Table 47.7), depending on the type of diabetes:
coherence domains 2, 3, and 4 for Type 1, region of liver, pancreas,
and spleen, and coherence domain 4 for Type 2. Treatment intervals
varied from 12 h to 7 days (Table 47.8) until blood sugar levels
normalized. Insulin and oral hypoglycemic drugs were gradually
reduced as blood sugar levels decreased under laser irradiation.
We used prototype laser devices made by General Project Srl
from Florence and by Eufoton Srl from Trieste, Italy. This system
comprised an 810 nm laser diode coupled with a low-frequency
magnet and a series of photodiodes (Table 47.6). The duration of
each session was 30 min on average. Sensation during diode laser
irradiation was absent or there was less sense of wind.
Normalization of glycemia was declared by the diabetologist and
the family physician. Patients stopped taking anti-diabetic drugs 4 h
before laser treatment. They could resume taking them only if the
glycemic levels were not reduced by a minimum of 20% immediately
after each laser treatment and persisted for one day. Then insulin
and hypoglycemic drugs were decreased progressively. Diet and

Table 47.7 Irradiation zones

Type 1 diabetes: region of liver, spleen, coherence domains 2, 3, 4 (sovrapubis, gastric,

esophagus-mediastinum regions)
Type 2 diabetes: region of liver, pancreas, and spleen; coherence domain 4 (esophagus-
mediastinum regions)
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966 Nonsurgical Laser Therapy for Type 1 and Type 2 Diabetes

Table 47.8a Diabetes Type 1: Procedure of treatment

• Laser system 810 nm with magnetic field associated

• One or two sessions per day for 15 day
• Then one session per week for 4 week
• Then one session per month, if necessary
• Gradual reduction in exogenous hypoglycemic substances, following the result

Table 47.8b Diabetes Type 2: Procedure of treatment

• One session per week until normalization of glycemia

• Then one session every 2 weeks
• Then one session per month, if necessary

physical activity were never interrupted since they have a primary

role on diabetes management.
The available sample of population obtained during the period
examined was significant for sex and age. All treatment groups
were well balanced with respect to baseline demographics and
concomitant therapies. Pre-existing anti-diabetic therapy included
a wide spectrum of regimens over the whole group. Usually a vast
majority of Type 2 patients were overweight or obese. All statistical
calculations were carried out through EpiInfo.
Withdrawals were noted, but this did not seem to alter the
positive effect of the treatment, which continued up to 12 months
after the end of the trial period. Laser dosages planned for treatment
were well below the dosage necessary to cause local burns. Adverse
effects on patients caused by hypoglycemia induced by laser therapy
could be treated with sugar absorbed under the tongue. Any
potential hyperglycemic complications could be treated with prompt
administration of insulin.
Patients were kept under surveillance by a designated person
for avoiding potential adverse reaction. Potential hypoglycemic
biological effects could be hypoglycemia crisis (cold sweating,
tremor until fainting) never seen in previous personal experiences.
These effects could be immediate, within 8 h before laser treatment
and 1 day after laser treatment. These effects could be discovered by
July 6, 2016 17:41 PSP Book - 9in x 6in 47-Hamblin-c47

Results and Discussion 967

patients themselves or the adequately informed designated person

and could be treated with immediate consumption of sugar.
Potential hyperglycemic effects could give sleepiness until
fainting within 8 h before treatment, immediately, and 1 day after
laser treatment. Also these effects could be discovered directly by
patients (adequately informed) and could be treated with immediate
uptake of prompt insulin. The fact that light irradiation accelerates
gastric emptying, with transitory increase in the glycemic level, as
demonstrated by previous experiments, could be used for increasing
the speed of glucose digestion, avoiding secondary hyperglycemias.
A tool with photodiodes could be given to patients for using on
the gastric region during the period of digestion. Patients would
not be able to change the dosage of photodiodes established by the
physician; they could only switch the button on and off. Physicians
could establish the dosage following the result obtained by the
patients with laser treatment and their speed of digestion.
In total, half of the patients of this project received photodiode
tools for improving the duration of results: 50% of patients with
diabetes Type 1 and 50% with diabetes Type 2, selected randomly.
The others were the control of this experiment. Results were
monitored as usual by diabetologists.

47.5 Results and Discussion

Between, before, and after treatment, diabetes mellitus Type 1

patients showed a 20% mean fall in glycemia (Fig. 47.1) and Type
2 patients showed a 22% mean fall (Fig. 47.2).
From the baseline value, HbA1c fell by 30% in Type 1 and Type
2 after 3 months, by 50% in Type 1 and 35% in Type 2 after 6
months, and by 80% in Type 1 and 60% in Type 2 after 12 months,
on average (Fig. 47.4). Laser alone or along with the pre-existing
treatment with other anti-diabetic drugs produced a significant
glycemic improvement (Table 47.9).
Our data obtained until today show that laser therapy have
a positive role in the treatment of diabetes Type 1 and Type 2.
Although the follow-up is still short, it is positive both in Type 1 and
Type 2 diabetes (Figs. 47.3 and 47.4).
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968 Nonsurgical Laser Therapy for Type 1 and Type 2 Diabetes

Figure 47.1 Results of Type 1 diabetes.

Figure 47.2 Results of Type 2 diabetes.

There were no episodes of biological hypoglycemic effects such

as hypoglycemic crisis induced by laser or due to an excessive
drop in blood sugar on fasting. There were two instances of post-
treatment hypoglycemia in Type 2 diabetic patients, but they were
asymptomatic. There was one case of transitory hyperglycemia in a
patient who had not fasted for 4 h prior to treatment, and it resolved
spontaneously during the course of the day.

Table 47.9 Phase 2 experimentation results

Type 1

• Progressive decrease in glycemia after 15 sessions on average until normalization

• Progressive decrease in exogenous insulin
Type 2

• Progressive decrease in glycemia in different measures until normalization after

15 sessions on average
• Progressive decrease in anti-diabetic drugs and stop exogenous insulin
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Results and Discussion 969

Figure 47.3 Follow-up: 1 year.

Figure 47.4 Follow-up: Glycated hemoglobin.

As previously described, lasers and LED could cause localized

first- and initial second-degree burns, and there was one case
of post-treatment erythema, which cleared up spontaneously.
Allergic (idiosyncratic) reactions to light may trigger an orticarioid
syndrome with itching and erythema. This did not occur in any of
our patients; however, all these adverse reactions could be easily
treated with topical anti-histaminic drugs (Table 47.10).

Table 47.10 Side effects

Potential hypoglycemia
Loss of control of the parameters by patients

There are also some political complications, potentially danger-

ous (Table 47.11).
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970 Nonsurgical Laser Therapy for Type 1 and Type 2 Diabetes

Table 47.11 Complications

• Pharmacological manufactures of insulin and anti-diabetic drugs (potentially lethal for the
MD!)
• Jealousy of classical diabetologists
• Ignorance of many politicians (ethics committees, health offices, and others)
• Economic speculation of some MDs

Insulin replacement therapy and oral anti-diabetic drugs can,

therefore, be gradually suspended to the point of no longer being
needed. The duration of the normal blood sugar level after the
conclusion of laser treatment has not been clearly and uniformly
determined. Further brief cycles of 4–8 sessions of laser therapy
were suggested if basal glycemia peak shows value more than 200
mg%. In addition, it seems that a near-infrared photodiode held
in contact with the skin during the digestion of the main meals,
at individual doses adjusted for each patient, accelerates glucose
metabolism, preventing hyperglycemia secondary to food intake,
and this helps maintain results achieved (Table 47.12 and Figs. 47.5
and 47.6).

Table 47.12 Results of LED therapy

• Twenty patients self-treated with LED did not need further laser irradiation for 2 years
• Twenty patients not treated needed some irradiation after 1 year of treatment, usually for
Christmas and Easter
• Diabetic patients Type 1 and Type 2 had the same results

But this clinic impression must be verified scientifically in

a larger number of patients for a longer time. This is another
experiment. We must also investigate the potential effects of light
and lasers on protein and lipid metabolism as well as on all
neuroendocrine functions of the human body. We will carry out
phase three of the clinical trial as soon as we find an institution to
finance the project.
Regarding the mechanism of action, now we can only present
hypotheses, which have to be verified over time (Table 47.13).
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Results and Discussion 971

(a) (b)

Figure 47.5 LED for hypoglycemic effect (patented).

Figure 47.6 Follow-up: 3 years.

We must remember that in endocrinology each hormonal action

is the result of two opposite groups of substances. For glycaemia,
we know a lot of hyperglycemic hormones and only one principal
hypoglycemic hormone (Table 47.14). Perhaps this is the result of
our ignorance. Of course, more hypoglycemic hormones exist, but we
must discover them. One hypothesis regards insulin-like substances,
such as insulin-like growth factor (IGF), produced in chromaffin cells
of the gastroenteric tube and subcutaneous tissue. Perhaps laser

Table 47.13 Action mechanisms hypothesis

• Anti-inflammatory effects
• Stimulation of beta cells
• Stimulation of chromaffin cells
• Stimulation of microcirculation
• Cleaning of cell walls
• Hypolipidemic effect
• Increase in bioplasma metabolism
• More effects associated
July 6, 2016 17:41 PSP Book - 9in x 6in 47-Hamblin-c47

972 Nonsurgical Laser Therapy for Type 1 and Type 2 Diabetes

Table 47.14 Hyperglycemic hormones

• Cortisol, adrenalin, DEA, ACTH

• GH
• MSH, melatonin
• Androgen, estrogen, progesterone, PRL
• T3, T4, TSH
• Serotonin
• Many local hormones
• Oxytocin, relaxin

beam could increase the formation of these substances, particularly

in diabetes Type 1, where beta cells of the pancreas do not exist.

47.6 Conclusion

Evidence-based medicine indicates that nonsurgical laser therapy

has a hypoglycemic effect on patients with Type 1 and Type 2
diabetes. Further studies will test these evidences and verify if
these treatments could be proposed as an elective treatment for this
pathological condition. The exact dose of laser radiation needed to
produce a hypoglycemic effect is still unknown and is probably not
univocal as it may vary not only from patient to patient but also
in the same patient during the course of treatment. We must test
a range of dosages that surely do not have negative effects on the
irradiated tissue, as reported in the literature.
Previous experiments have demonstrated that laser irradiation
(at a dosage level reported to be effective in the treatment of diabetic
patients) can accelerate gastric emptying. This gastric emptying
would result in an increase in the glycemic level if a patient eats
within 5 h before the laser irradiation. It is absolutely necessary that
the patient fasts some hours prior to the laser treatment.
On the other hand, this effect could be used for avoiding sec-
ondary hyperglycemia post-prandium. Photodiodes used for avoid-
ing post-prandium secondary hyperglycemia could be patented and
given directly to patients for use at home.
One possible adverse reaction of laser irradiation could be local
skin burns. However, laser dosage planned for these studies is
July 6, 2016 17:41 PSP Book - 9in x 6in 47-Hamblin-c47

References 973

well below the dosage necessary to cause local skin burns. Other
reported systemic effects of laser irradiation include dilatation of
microvessels. These systemic effects have not been convincingly
demonstrated.
Possible adverse effects caused by hypoglycemia induced by
laser therapy will be treated with sugar absorbed under the
tongue. No side effects were noted in preliminary experiences.
Any possible hyperglycemic complications will be treated with the
prompt administration of insulin.
Patients must be instructed to measure their blood glucose levels
at the same hour every day. Usually diabetic patients know how
to measure their blood glucose levels. Patients must immediately
contact their physician if they note any change in their health.
Patients not treated with laser (those assigned to the control group)
can be treated at the end of the clinical trial if the treatment is found
to be effective.

References

1. Wild S, Roglic G, Green A, Sicree R, and King H (2004). Global prevalence

of diabetes: Estimates for the year 2000 and projections for 2030.
Diabetes Care, 27(5): 1047–1053.
2. Roger VL, Go AS, Lloyd-Jones DM, Benjamin EJ, Berry JD, Borden WB,
Bravata DM, Dai S, Ford ES, Fox CS, Fullerton HJ, Gillespie C, Hailpern
SM, Heit JA, Howard VJ, Kissela BM, Kittner SJ, Lackland DT, Lichtman
JH, Lisabeth LD, Makuc DM, Marcus GM, Marelli A, Matchar DB, Moy
CS, Mozaffarian D, Mussolino ME, Nichol G, Paynter NP, Soliman EZ,
Sorlie PD, Sotoodehnia N, Turan TN, Virani SS, Wong ND, Woo D, and
Turner MB; on behalf of the American Heart Association Statistics
Committee and Stroke Statistics Subcommittee (2012). Heart disease
and stroke statistics 2012 update: A report from the American Heart
Association. Circulation, published online before print, December 15,
2011, 10.1161/CIR.
3. WHA 42.36. Prevention and control of diabetes mellitus. Hbk Res., Vol. II
(1985), 1.16.17 (Thirteenth Plenary Meeting, 19 May 1989 - Committee
A, Fourth Report).
4. Bioresonance Therapy: History. Bioresonance Therapy. N.p., n.d. Web. 29
Apr. 2012. <http://nano4health.blogspot.com/p/history.html>.
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5. History of Electrotherapy. Bioresonance Diagnostics and Therapy Portal.

N.p., 13 Mar. 2011. Web. 29 Apr. 2012. http://www.bioresonance-
therapy.com/2011/03/history-of-electrotherapy-acute-chronic-pain-
treatment.
6. Ramdawon P (2003). Bioresonance information LASER therapy of
diabetes mellitus. A first clinical experience. Proceedings of LASER
Florence 2001, SPIE Washington, Vol. 4903: 146–153.
7. White J (1979). Kundalini. Evolution and Enlightenment, Anchor Book,
New York.
8. Brennan B (1987). Hands of Light, Bantam Dell Publishing Group, New
York.
9. Spaggiari PG and Tribbia C (2008). Medicina Quantistica, Tecniche
Nuove Publisher, Cremona.
10. Longo L (2008). The role of laser in diabetes management. In Waynant R
and Tata D Eds., Proceedings of Light-Activated Tissue Regeneration and
Therapy Conference, Springer, New York, 215–221.
11. Ohshiro T and Calderhead G (1988). Low Level Laser Therapy: A
Practical Introduction, Wlley & Sons, Chicchester.
12. Kaslow R and Jeremy E (2012). Low Level Lasers (LLLT). Web. 27 Apr.
2012. http://www.drkaslow.com/html/low level lasers -lllt-.html.
13. Makela A (2007). Why the Same Laser Protocols can have Different
Clinical Results in the Treatment of Diabetes: A Review, Laser Florence
2007, Abstracts in Lasers in Medical Science, Springer.
14. Inyushin VM and Chekurov PR (1975). Biostimulation through Laser
Radiation of Bioplasma, Kazakh State University, USSR Hill and Ghosak,
Copenhagen University.
15. Mester E, Ludany G, Selyei M, Szende B, and Tota GJ (1968). The
stimulating effects of lower power laser rays on biological systems.
Laser Rev, 1: 3–9.
16. Baxter GD (1994). Therapeutic Lasers, Churchill Livingstone, UK.
17. Karu T (1998). The Science of Low Power Laser Therapy. Gordon and
Breach Science, Amsterdam, NL.
18. Longo L (1986). Terapia Laser, USES Ed., Firenze, Italy.
19. Marangoni O and Longo L (2006). Lasers in Phlebology, EGT Publisher,
Trieste, Italy.
20. Waynant R and Tata D, eds. (2008). Proceedings of Light-Activated Tissue
Regeneration and Therapy Conference, Springer, New York.
July 6, 2016 17:41 PSP Book - 9in x 6in 47-Hamblin-c47

References 975

21. Longo L. (2014). Laser Manual: Medical Technology, OEO Publisher,

Firenze, Italy.
22. Byrnes KR, Barna L, Chenault VM, Waynant RW, Ilev IK, Longo L, Miracco
C, Johnson B, and Anders JJ (2004). Photobiomodulation improves
cutaneous wound healing in an animal model of Type II diabetes.
Photomed Laser Surg, 22(4): 291–300.
23. Tuner J and Hode L (2003). The Laser Therapy Handbook, Prima Books,
Stockolm, USA.
24. Longo L, Marchi M, and Postiglione M (2001). Comparison between two
different types of lasers for fibromyositis treatment used on different
patients and on the same patients. In Laser Florence 2000: A Window
on the Laser Medicine World, L Longo, A Hofstetter, ML Pascu, and W
Waidelich, Eds., Proceedings of SPIE, Washington, Vol. 4606, 93–103.
25. Rochkind S, Rousso M, Nissan M, Villarreal M, Barr-Nea L, and Rees
DG (1989). Systemic effects of low-power laser irradiation on the
peripheral and central nervous system, cutaneous wounds, and burns.
Lasers Surg Med, 9: 174–182.
26. Rochkind S (2009). Phototherapy in peripheral nerve injury for muscle
preservation and nerve regeneration. Photomed Laser Surg, 27(2): 219–
220.
27. Anders J (2009). The potential of light therapy for central nervous
system injury and disease. Photomed Laser Surg, 27(3): 379–380.
28. Longo L, Lubart R, Friedman H, and Lavie R (2003). A possible
mechanism for visible light-induced skin rejuvenation. In Laser Florence
2003: A Window on the Laser Medicine World, Longo L, Hofstetter A,
Pascu ML, Waidelich W, Eds., Proceedings of SPIE, Washington, Vol. 4606,
93–103.
29. Anders J, Longo L, Waynant R, and Romanczick T (2005). Light as a
Replacement for Mitogenic Factors on Progenitor Cells, Docket No: 9551-
024 Pct U.S. Provisional Application Serial No. 60/666,582 filed March
31, 2005.
30. Longo L, Postiglione MG, Buccioni T, and Longo D (2009). The effects
of Low Level LASER Therapy (LLLT) on blood glucose levels in patients
with Diabetes Mellitus type 1: a case report in Advances in Laserology—
Selected papers of Laser Florence 2008, International Laser Medicine
Congress, Firenze, Italy, 31 October–1 November, 2008, Melville, New
York: American Institute of Physics, 2009, 92–95.
July 6, 2016 17:41 PSP Book - 9in x 6in 48-Hamblin-c48

Chapter 48

Laser Therapy of Traumatic Central

Nervous System Injuries*

Leonardo Longo and Diego Longo

Institute for Laser Medicine, International Academy Laser Medicine and Surgery,
Borgo Pinti 57, 50121 Firenze, Italy
longo.leonardo@gmail.com

48.1 State of the Art and Objectives

Traumatic central nervous system injuries (TCNSIs) are not diseases

but lesions that concern an ever-growing number of people who
are unable to work and have lost their independence. These people
need help to regain at least some of their functional abilities. Causes
of TCNSIs include traffic accidents, diving in shallow water, falls,
gunshots, stab wounds, and natural disasters (earthquakes). The
social aspect is highlighted by the World Health Organization (WHO)
in its definition of health, which is “not the absence of disease

*Lecture presented in Laser Florence 2013, titled Laser and Physical Therapy Applied
to Traumatic Central Nervous System Injuries: Update by L. Longo, MD, F. Giubilo,
MD, C. Romanelli, MD, D. Longo, PT, of Institute of Laser Medicine, Florence, Italy.
Proceedings on-line, reprinted with permission from Medimond Publisher, Italy,
copyright 2013.

Handbook of Low-Level Laser Therapy

Edited by Michael R. Hamblin, Marcelo Victor Pires de Sousa, and Tanupriya Agrawal
Copyright c 2017 Pan Stanford Publishing Pte. Ltd.
ISBN 978-981-4669-60-3 (Hardcover), 978-981-4669-61-0 (eBook)
www.panstanford.com
July 6, 2016 17:41 PSP Book - 9in x 6in 48-Hamblin-c48

978 Laser Therapy of Traumatic Central Nervous System Injuries

or infirmity but a state of complete physical, psychological and

social well being.” Right indications are given in the International
Classification of Functioning, Disability and Health, established by
the WHO in 2001. Trauma epidemiology has significant correlation
with the economic conditions of a community [1]. In the United
States, more than 10% of residents suffered from nonfatal injuries
in 2002. Trauma was the leading cause among those aged 1–4 years.
In Germany, 40% of those injured in 2002 were 20–39 years old (the
greatest incidence was between 20 and 24 years).
The beneficial effects of light on biological tissue have been
known since the times of Hippocrates [2–4]. Around 1966, Mester
et al. [5] were the first to point out that red laser stimulates
hair regrowth in rats and halves the healing time of experimental
ulcers. Many experiments have proved incontrovertibly that visible
and close-to-infrared wavelength lasers influence the healing time
of skin wounds and ulcers, stimulating or inhibiting the process
according to the radiation dosage and method [6–10]. In 1998,
researchers at the Bethesda University Maryland demonstrated that
at specific doses some lasers could increase cultured fibroblast
activity by 98% [11]. Many strictly dose-dependent effects have
been demonstrated in nearly all normal and pathological biological
processes: from cell maturation to reproduction, inflammation
to edema, neural irritation to pain inhibition, and also through
increased endogenous endorphin production. The importance of
dosage in all these cases was immediately realized to be funda-
mental because the same type of laser may have opposite effects
on the same biological process and on the same tissues if the
irradiation dose is modified. Tuner and Hode [12] have written a
critical review on this topic. Many other uses of nonsurgical laser
have been studied following this concept, such as modulation of
metabolism, immunological system, nerve cell functions, progenitor
cells, and light energy exchanges [10, 12, 13, 17, 20].
In terms of dosage, in order to obtain the desired effects, it
is necessary to consider a whole series of physical parameters
(wavelength, emission, fluence, energy density, repetition pulse
frequency, spot size, irradiation time per spot), biological factors
(type of tissue, biological health), and clinical factors (irradiated
point, number and rhythm of sessions, irradiation procedure)
July 6, 2016 17:41 PSP Book - 9in x 6in 48-Hamblin-c48

Study Design and Methodology 979

[7–10, 13, 18]. Therefore it is evident that if the effects of light

are dependent on small quantities of radiations and how they
are administered, we must use lasers that allow us to administer
precise and selective doses to tissue. This is what is done in soft
tissues regeneration such as healing of skin wounds and ulcers in
the treatment of bone–muscle–tendon inflammations, traumas, and
neuralgias [11–18].
TCNSI represents a social problem today, concerning 5% of
population in the developed countries. Scientists, research workers,
clinicians, health economists, and planners are concerned with
TCNSI and are trying their best to control it through more effective,
easier, and cheaper methods. Regarding the effects of lasers on
the central nervous system (CNS), several authors [18–26] have
demonstrated, in vitro, that at given doses, some lasers regenerate
injured neuron cell cultures and cause them to multiply. This
scientific evidence prompted us to verify the effects in vivo.

48.2 Study Design and Methodology

Following this scientific background, since 2004 we have treated

chronic systemic tissue lesions, such as spinal and brain traumatic
injuries, in patients of both genders, aged 16–60 years. The whole
project followed the rule of good clinical practice established by the
European Community, published in the Italian “Gazzetta Ufficiale”
(supplement N◦ 191, 18/8/1997) N◦ 39, 18/6/2001, and by the
Helsinki Declaration.
The patients were selected on the basis of the inclusion–
exclusion criteria and the treatment interruption criteria (Table
48.1). They were informed about the nature, potential benefits, and
risks of the study and were asked to give their informed consent
prior to the laser treatment [20, 21]. The patients admitted to the
study were treated in our consulting room. Their documentation
and treatment schedule were defined. Before the treatment, the
patients were informed about the steps that would be followed.
They were then submitted to the prescribed treatment according
to the protocol and put under medical surveillance. The operator
was attentive to any possible adverse immediate reaction. After
July 6, 2016 17:41 PSP Book - 9in x 6in 48-Hamblin-c48

980 Laser Therapy of Traumatic Central Nervous System Injuries

Table 48.1 Criteria of patient selection

Inclusion criteria
• Both genders, young and middle aged
• Spinal cord injury occurred 1 year ago and more
• NMR: Total and subtotal lesion of SC
• ASIA A classification
• No therapy available
• Progressive withdrawal of any drug treatment
• Rehabilitation fitness continues
Exclusion criteria
• Previous undue surgical intervention (as muscle transposition and tendon severing)
• Patients following other experimental trials
• Drugs not prescribed by us
• Patients stop rehabilitation fitness
Suspension criteria
• No results after the end of the first treatment cycle
• Patients who do not follow the protocol prescribed
• Patients with adverse effects to laser irradiation (local burns, allergic reaction)
• Patients who present some exclusion criteria that appear after the inclusion in our study

the treatment, the operator checked the health of the patients and
kept them under observation for 2 h, before releasing them. The
operator’s comments were noted, and the next appointment was
fixed.
Both sites of the lesions and related coherence domains of the
injured apparatus were irradiated. Laser treatment was carried
out in consulting rooms where instruments and drugs needed
for the treatment of adverse radiation effects were placed. Each
patient was treated at the same place for the entire duration of
the treatment. Before treatment, the patients underwent a check-up
with the control of hemato-chemical parameters of blood and urine
and function of principal organs, such as heart, liver, kidney, brain,
lung, blood, eye, and the endocrine system. The laser treatment was
started with the parameters shown in Table 48.2.
A qualified person controlled the efficiency of lasers and
determined when the lasers were active. The same person was
responsible for the application of safety rules following the
standards of the American National Standard Institute (ANSI) and
the prevention of adverse reactions (WHO and EC publications).
July 6, 2016 17:41 PSP Book - 9in x 6in 48-Hamblin-c48

Study Design and Methodology 981

Table 48.2a Diode laser anti-inflammatory, anti-edema

• Diode laser: 808 nm, 30 W

• Spot size: 5 cm
• Fluence: 12 J/ cm2
• Energy density: 720 J
• PW 3 until 1000 Hz
• Target: column, trigger points of the nerves, related domains
• Four sessions a day
• Cycle of 20 sessions

Table 48.2b CO2 laser anti-inflammatory, anti-edema,

anti-contracture

• CO2 laser: 10,600 nm, 30 W

• Spot size: 5 cm
• Fluence: 12 J/ cm2
• Energy density: 720 J
• CW
• Target: Column
• Four sessions a day
• First cycle of 20 sessions on average

A third person evaluated the rehabilitation results, measured

with the same devices and the same criteria for each patient. An
epidemiologist evaluated the results and their statistical significance
comparing the data obtained for the trials. Energy doses may vary
during the course of the treatment on the basis of the results
obtained from time to time in each patient for each lesion. The
duration of each session was 30 min on average.
No sensation was felt during the diode laser irradiation, except
a slight feeling of wind or heat. The sample of the population
obtained during the period examined was significant for sex and
age. All treatment groups were balanced with respect to baseline
demographics and previous therapies.
All statistical data were calculated with the help of EpiInfo
(statistical analysis software). The effects of laser irradiation are
strictly dose dependent, a rule that applies to all nonsurgical laser
July 6, 2016 17:41 PSP Book - 9in x 6in 48-Hamblin-c48

982 Laser Therapy of Traumatic Central Nervous System Injuries

and light effect on biological tissues. Laser dosages planned for

treatment were below the dosage necessary to cause local burns.
The patients were kept under surveillance in order to avoid po-
tential adverse reactions. Laser could cause minor local burns (first
degree), which are easily treatable with appropriate medication
(antiseptic and laser wound healing stimulation). Allergic reaction
to laser could be orticaria syndrome, itching, and erythema. All these
effects are easily treatable with topical medication (antihistamine
drugs).
Follow-up was carried out each month until 1 year, then every
6 months. We enrolled 216 patients of both genders and age 14–60
years with traumatic spinal cord and brain injuries occurred at least
1 year before laser treatment. Our preliminary experiment started
in January 2004.
Standardization of these patients is difficult because each patient
is totally different in terms of lesion, loss of sensitivity, and
degree of inflammation/degeneration. Since trauma, all patients
treated with CNS injuries had total or subtotal sensory and motor
paralysis under lesion level, clinically classified with the American
Spinal Injuries Association (ASIA) impairment scale, ASCII scale,
EEG, nuclear magnetic resonance (NMR), electromyography (EMG),
evoked somato-sensory potentials (EPSS), evoked somato-motor
potentials (ESMP), and neuro-physiological tests on spinal cord and
brain lesions.
The ASIA impairment scale classifies spinal cord injuries into five
categories depending on the degree of sensory and motor loss of
function, sphincters included. Further evaluations were carried out
using ISCOS datasets.a
An 808 nm diode laser was used with fluence 4–12 J/cm2 on
average, with a first cycle of 20 sessions, four per day. We repeated,
on average, half cycle per month for approximately 12 months. We
do not precisely know how many cycles do these patients need and
what is the end point of their improvement. These aspects will be
the subject of further study.

a www.iscos.org.uk/page.php?content=20, accessed on 19 February 2012.

July 6, 2016 17:41 PSP Book - 9in x 6in 48-Hamblin-c48

Study Design and Methodology 983

Table 48.3 Further monthly cycles

Diode laser (808 nm, 30 W)

• Spot size: 5 cm and 2.5 cm
• Fluence: 12 J /cm2 and 4 J/cm2
• Energy density: variable
• Target: column, trigger points of the nerves, related domains
• Three sessions a day
• Cycle of 10 sessions on average
CO2 laser (10,600 nm, 30 W)
• Spot size: 5 cm
• Fluence: 12 J/ cm2
• Energy density: 720 J
• CW
• Target: column

Further monthly cycles were done, with different dosages

(Table 48.3). This procedure could have anti-inflammatory and
regenerative effects on nervous cells and/or nerve functions.
Patients were selected on the basis of rigid inclusion–exclusion
and suspension criteria, and strict standards were applied for
treatment interruption (Table 48.1). Lasers were used on the lesions
for anti-inflammatory and anti-edema purposes. Lasers used on the
lesions were an 808 nm, 30 W PW laser (3 Hz, spot size 5 cm,
fluence 720 J/cm2 in total, 12 J/cm2 for anti-inflammatory goal)
(Table 48.2a) and a 10,600 nm, 15 W CW laser (spot size 10 cm,
time exposure 20 s for spot), as a skeletal muscle anti-spasm, not
only on the lesion, but also along the nerves in the paralyzed area
(Table 48.2b).
The first cycle of treatment consisted of 20 sessions, with an
average of four sessions per day, each session of 30 min, with a
minimum interval of 2 h between the sessions. It was possible
to computerize laser emissions and to use more than one laser
simultaneously, saving time and discomfort for the patients. All
patients came to us with written opinions from orthopedists,
physiatrists, neurologists, and neurosurgeons who defined the
lesions as complete and incurable and advised against any type of
treatment and/or physiotherapy.
July 6, 2016 17:41 PSP Book - 9in x 6in 48-Hamblin-c48

984 Laser Therapy of Traumatic Central Nervous System Injuries

Table 48.4 Evaluation of the treatment

• Clinic evaluation by specialists: ASCII, ASIA score, Ashworth

Scale modified, others
• Self-evaluation by the patients and their family
• NMR
• EMG
• EPSS, EPMS
• EEG

Table 48.5 Interruption of laser treatment

(49 of the 209 patients treated in total, from
January 2004 to December 2013)

No results after the first cycle 4 patients

Less and slow results 18 patients
Incorrect rhythm of cycles 15 patients
Expensive therapy 9 patients
Temporary results 3 patients

In fact we continued monthly laser therapy only if we noticed

some positive and objective results after the first cycle of treatment.
The results obtained on the patients who underwent laser therapy
were evaluated by physiatrists other than the operator, and after the
first cycle, they did not know if the patients had laser treatment.
For the sensory–motor clinical examination, we used the
standard classifications, such as ASCII, ASIA, Asworth Scales, and
Franklin modified. Before and after the treatment, all the patients
underwent NMR examination and some also had EMG and ESSP
(Tables 48.4 and 48.5).

48.3 Results and Discussion

Results are shown in Table 48.6 and Fig. 48.1.

In patients with spinal lesions at different levels, there was
always a recovery of heat and tactile sensitivity as well as pain- and
proprio-receptivity below the lesion, albeit to different extents and
at different times, but it was always permanent and never transient.
July 19, 2016 10:55 PSP Book - 9in x 6in 48-Hamblin-c48

Results and Discussion 985

Table 48.6 Evaluation of results of 160 patients

Sensory sensibility Minimum increase of two metamers per cycle under the lesion
Involuntary motor Improvement in muscle tone, posture
Voluntary motor Variable; strictly connected with fitness
Anal sphincter Improvement until normalization
Urethral sphincter No for men; normalization in women
Sexual activity Quite normal in 99% of patients
Stand up 135 p, on average, after 100 irradiations
Walking 15 p, on average, after 120 irradiations
ASIA and other classifications Change of one class every six months
NMR Edema and phogosis signs disappeared; lesion of medulla
reduced
EEG Significant improvement, change under stimulation
sEMG Activity at least in two muscles under the level of lesion

C2-4 C5-7 D L Brain

Figure 48.1 Results of 160 patients.

Normal body thermic regulation was always recovered, if inter-

rupted by the lesion, as normal anal sphincter control and sexual
activity in both sexes, including erectile, sensory, and ejaculatory
functions. Bladder control was restored in women, but not in men in
whom there was increased diuresis and urine loss during treatment,
which may be due to the redistribution of bladder tone.
Laser therapy could have results of normalization of muscle tone,
increasing that in floppy paralysis and giving relaxation in spastic
paralysis, changing the dosage of irradiation. The effects are gradual
and progressive, so we can stop progressively the intake of drugs
July 6, 2016 17:41 PSP Book - 9in x 6in 48-Hamblin-c48

986 References

which change the muscle tone, hyper-reflexia, and also fasciculation,

in patients who were restored to almost normal motility.
Previous experiments on spinal cord injury and central nervous
system injury have not shown any significant side effects of
laser irradiation. In any case, all the necessary effective measures
are available and ready in the case of any minor local burn
or microvessel dilatation, in collaboration with the personal
physician.
Appropriate physiotherapy is recommended for voluntary motor
functions. Physiotherapists have noted that patients undergoing
laser therapy have better reaction and strength during exercises and
better muscle tone, and never developed spasms. These results are
transient at the outset during the early treatment cycles, lasting for
about a month, and then become permanent. This may be due to the
accumulation of radiation or the possibility that patients are better
in doing motor exercises, or both. The results were monitored as
usual by neurologists and physical therapists.

48.4 Conclusion

The results obtained demonstrated that traumatic central nervous

system lesions, usually untreatable or less treatable, could be
improved or totally recovered by adequate laser treatment. The
worldwide impact of the study is enormous. Medical, low-density
laser stimulation for nervous tissue regeneration has received
national and international attention and has been widely applied for
the last 30 years. The present research is only a continuation of a
long line of experimentation in this field [27].

References

1. Cinat M.E., Wilson S.E., Lush S., and Atkins C. Significant correlation of
trauma epidemiology with the economic conditions of community. Arch.
Surg., 139: 1350–1355, 2004.
2. White J. Kundalini: Evolution and Enlightenment, Anchor Books, New
York, 1979.
July 6, 2016 17:41 PSP Book - 9in x 6in 48-Hamblin-c48

References 987

3. Brennan B. Hands of Light, Bantam Dell Publishing Group, New York,

1987.
4. Inyushin V.M. and Chekurov P.R. Biostimulation through Laser Radiation
of Bioplasma, Kazakh State University, USSR Hill and Ghosak, Copen-
hagen University, 1975.
5. Mester E., Ludany G., Selyei M., Szende B., and Tota G.J. The stimulating
effects of lower power laser rays on biological systems. Laser Rev., 1: 3–
9, 1968.
6. Baxter G.D. Therapeutic Lasers, Churchill Livingstone, UK, 1994.
7. Karu T. The Science of Low Power Laser Therapy, Gordon and Breach
Science Publishers, Amsterdam, The Netherlands, 1998.
8. Longo L. Terapia Laser, USES ed., Firenze, Italy, 1986.
9. Marangoni O. and Longo L. Lasers in Phlebology, EGT Publisher, Trieste,
Italy, 2006.
10. Waynant R. and Tata D. (Eds.) Proceedings of Light-Activated Tissue
Regeneration and Therapy Conference, Springer, New York, 2008.
11. Byrnes K.R., Barna L., Chenault V.M., Waynant R.W., Ilev I.K., Longo L.,
Miracco C., Johnson B., and Anders J.J. Photobiomodulation improves
cutaneous wound healing in an animal model of Type II diabetes.
Photomed. Laser Surg., 22(4): 291–300, 2004.
12. Tuner J. and Hode L. The Laser Therapy Handbook, Prima Books,
Stockholm, 2003.
13. Longo L., Marchi M., and Postiglione M. Comparison between two
different types of lasers for fibromyositis treatment used on different
patients and on the same patients. In Laser Florence 2000: A Window
on the Laser Medicine World, L. Longo, A. Hofstetter, M.L. Pascu, and W.
Waidelich (Eds.), Proceedings of SPIE, Washington DC, Vol. 4606, 93–
103, 2001.
14. Rochkind S., Rousso M., Nissan M., Villarreal M., Barr-Nea L., and Rees
D.G. Systemic effects of low-power laser irradiation on the peripheral
and central nervous system, cutaneous wounds, and burns. Lasers Surg.
Med., 9: 174–182, 1989.
15. Rochkind S. Phototherapy in peripheral nerve injury for muscle
preservation and nerve regeneration. Photomed. Laser Surg., 27(2):
219–220, 2009.
16. Anders J. The Potential of light therapy for central nervous system injury
and disease. Photomed. Laser Surg., 27(3): 379–380, 2009.
July 6, 2016 17:41 PSP Book - 9in x 6in 48-Hamblin-c48

988 References

17. Longo L., Lubart R., Friedman H., and Lavie R. A possible mechanism
for visible light-induced skin rejuvenation. In Laser Florence 2003: A
Window on the Laser Medicine World, L. Longo, A. Hofstetter, M.L. Pascu,
and W. Waidelich (Eds), Proceedings of SPIE, Washington DC, Vol. 4606,
93–103, 2003.
18. Chow R.T., Johnson M.I., Lopes-Martins R.A., and Bjordal J.M. Efficacy of
low-level laser therapy in the management of neck pain: A systematic
review and meta-analysis of randomised placebo or active-treatment
controlled trials. Lancet, 374(9705): 1897–1908, 2009.
19. Fork R.L. Laser stimulation of nerve cells in aplysia. Science, 171: 907–
908, 1971.
20. Longo L. Non-surgical laser and light in the treatment of chronic
diseases: A review based on personal experiences. Laser Phys. Lett.,
7(11): 771–786, 2010.
21. Anders J.J., Romanczyk T.B., Ilev I.K., Moges H., Longo L., Wu X.,
and Waynant R.W. Light support neurite outgrowth of human neural
progenitor cells in vitro: The role of P2Y receptors. IEEE J. Sel. Top. Quant.
Electron., 14(1): 118–125, 2008.
22. Oron U., Ilic S., De Taboada L., and Streeter J. Ga-As (808 nm) laser
irradiation enhances ATP production in human neuronal cells in culture.
Photomed. Laser Surg., 25(3): 180–182, 2007.
23. Asagai Y. Application of LLLT in patients with cerebral palsy of the adult
tension athetosis type. Nippon Laser Igakkaishi, 28: 74–76, 2007.
24. Oda-Mochizuki N. Neuronal and cellular effects of low level laser: Basic
and clinical research. Nippon Laser Igakkaishi, 28: 57–57, 2007.
25. Rochkind S. Central nervous system transplantation benefitted by low-
power laser irradiation. Lasers Med. Sci., 7: 143–151, 1992.
26. Anders J., Longo L., Waynant R., and Romanczick T. Light as a
Replacement for Mitogenic Factors on Progenitor Cells, Docket No: 9551-
024 Pct U.S. Provisional Application Serial No. 60/666,582 filed March
31, 2005.
27. Longo L. Laser Manual of Medical Technology, OEM Publisher, Firenze,
Italy, 2014.
July 12, 2016 11:37 PSP Book - 9in x 6in 49-Hamblin-c49

Chapter 49

Low-Level Laser (Light) Therapy:

Aesthetic Applications for Hair

Felipe Scholz Ramos,a André Luiz de Oliveira Ramos,b

and Marcelo Victor Pires de Sousab,c
a Department of Aesthetic and Cosmetology, Monte Serrat University Centre -

UNIMONTE, Rangel Pestana Avenue, 99 - Vila Matias, Santos, São Paulo 11015-530,
Brazil
b Laboratory of Radiation Dosimetry and Medical Physics, Institute of Physics, Matão

Street, Alley R, 187, University of São Paulo, São Paulo, São Paulo 05508-900, Brazil
c Bright Photomedicine Inc., Prof. Linnaeus Prestes Avenue, No. 2242, Butantã -

University City, IPEN - São Paulo, São Paulo 05508-000, Brazil

marcelovictor@usp.br

In this chapter, the fundaments of the aesthetic applications of low-

level light therapy (LLLT) for hair growth, hair loss avoidance, and
prevention and treatment of hair diseases will be presented. At the
beginning, the physiology of hair growth and the corresponding
phases (anagen, catagen, and telogen) will be discussed. Moreover,
the main triggers of hair loss (such as aging and chemotherapy)
will be discussed, and a brief explanation of the current treatment
resources, emphasizing LLLT and its main advantages, will be given.
From this point, the historical aspects of LLLT application will be
listed, and in vivo studies and clinical trials will be presented. The
proposed mechanisms of LLLT for hair regrowth and hair loss will

Handbook of Low-Level Laser Therapy

Edited by Michael R. Hamblin, Marcelo Victor Pires de Sousa, and Tanupriya Agrawal
Copyright c 2017 Pan Stanford Publishing Pte. Ltd.
ISBN 978-981-4669-60-3 (Hardcover), 978-981-4669-61-0 (eBook)
www.panstanford.com
July 12, 2016 11:37 PSP Book - 9in x 6in 49-Hamblin-c49

990 Low-Level Laser (Light) Therapy

be presented, along with phototherapy devices for hair loss. The

chapter will be ended with the future perspectives of the aesthetic
applications for hair research.

49.1 Physiology of Hair Growth (Phases)

Human beings have more than 2 million hair follicles, which are
complex and appended skin structures [10]. Despite being small,
they may have major positive and negative influences on the
physical and emotional health of humans. In addition, hair has
many biological functions, including protection and distribution of
sebaceous gland products in scalp [28, 48].
Although hair follicles have similar structure, they range in
size and shape according to their distribution in skin. These
characteristics as well as the density of follicles are genetically
determined during morphogenesis with origin from mesenchymal
cells and across epidermis and deep dermis.
A hair follicle is localized in two compartments: epidermal and
dermal. The follicle has a bulb located slightly above the papillary
dermis. The bulb is formed of germinal matrix cells. The dermal
papilla and sheath are identified as important keys in signaling
hair growth, while the bulb differentiates cells forming the medulla,
cortex, and cuticle of the hair shaft. Dynamic growth occurs by the
differentiation of matrix cells moving up the follicle, and the hair
shape is determined by the dimensions and curvature of the inner-
root sheath, while the size of the hair shaft is defined by the number
of cells from the matrix (Fig. 49.1).
In the bulb, melanocytes synthesize and transfer melanosomes to
the hair shaft, determining the hair color. In the aging melanocytes,
decrease in density and melanin production results in gray hair [28,
48] (Fig. 49.1).
Inside the hair follicle is the arrector pili muscle (APM). Near
the APM is the bulge, an area containing stem cells, which are
important in signaling pathways during the hair growth cycle and
for the regeneration of follicles in the case of epithelium damage
[28]. Hair growth depends on the type and location of the hair follicle
and consists of three phases: anagen, catagen, and telogen. Although
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Physiology of Hair Growth (Phases) 991

Figure 49.1 Structure of hair follicle: (a) E: epidermis; S: sebaceous gland;

APM: arrector pili muscle; B: bulge; HS: hair shaft. (b) ORS: outer-root
sheath; IRS: inner-root sheath; CU: cuticle; CTX: cortex; CTS: connective-
tissue sheath; DP: dermal papilla; M: matrix cells [10].

hair growth occurs in a cyclical manner, each follicular unit acts

independently in a different phase (Fig. 49.2).
The anagen phase corresponds to a period of 3–6 years. This
phase is the growing stage of hair and is the principal determinant
of hair length. Factors such as aging and alopecia may decrease the
duration of this phase. The catagen phase lasts for about 2 weeks.
This is the corresponding transition period when cells of the hair
matrix decrease melanin and other protein synthesis in the hair
shaft structure resulting in club hair. Finally, after the complete
formation of the club hair, the telogen phase begins, which has a
duration of 3–5 months. The hair in this phase is pushed upward
when the anagen phase is initiated again and the cycle is repeated.
Other studies have currently proposed two other phases:
exogenous and kenogen phases. The exogenous phase, subsequent
to the telogen phase, proposes a mechanism for hair detachment,
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992 Low-Level Laser (Light) Therapy

Figure 49.2 Hair growth cycle and selected factors with known hair growth
regulatory roles [39].

suggesting that it occurs at the beginning of a new anagen phase

and involves a proteolytic action on the base shaft cells. The kenogen
phase is the period when the hair follicle remains empty before
beginning a new cycle. In patients with androgenetic alopecia,
kenogen is more frequent and prolonged [12].
In general, around 85% of scalp hair follicles are in the anagen
phase, while 15% are in the telogen phase [28]. The hair growth cy-
cle may vary according to different physiological conditions such as
pregnancy, childbirth, surgery, weight loss, severe stress, drug abuse,
endocrine disorders, anemia, poor diet, and genetic conditions.
Hormones such as estrogen, thyroxine, glucocorticoids, retinoids,
prolactin, and growth hormones can prolong or shorten the stages of
the hair growth cycle. Androgens have a major influence, especially
dihydrotestosterone (DHT), a subproduct of testosterone which acts
through androgen receptors of the, outer root sheath cells, dermal
papilla, pilosebaceous unit and sebaceous gland. This interaction
results androgenetic alopecia (pattern baldness). In order to
have certain plasticity, hair follicles can transform themselves
from vellus to hair terminal and vice versa. Initially androgens
stimulate increase in hair follicle size during adolescence and later
they promote miniaturization of the scalp follicles, resulting in
androgenetic alopecia (AGA) [28].
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Types of Hair Loss and Some Treatments 993

Figure 49.3 Potential triggers for hair disorders. Adapted from Ref. [3].

49.2 Types of Hair Loss and Some Treatments

Hair diseases involve several mechanisms such as hormonal and

stress imbalances due to immunologic responses, inflammation,
genetic mutation, and exposure to toxic substances. Changes
in parameters such as the density and size of hair follicle
and the duration of the hair growth cycle are associated with
hair disorders, diagnosis, and possible causes of hair shedding
(Fig. 49.3).
Hair loss, the gradual and visible decrease in hair (baldness),
is a generic term for alopecia. Some of the most common forms
of hair loss include AGA, alopecia areata, telogen effluvium, and
chemotherapy-induced alopecia [43].

49.2.1 Androgenetic Alopecia

Androgenetic alopecia develops in genetically predisposed individu-
als. AGA follows the pattern of male baldness (Norwood scale) and a
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994 Low-Level Laser (Light) Therapy

pattern of female baldness (Ludwing classification scale). Thus, the

hair follicles located in these specific patterns of male and female
scalp decrease in size promoting a thinning of the hair. In addition,
terminals are miniaturized hair follicles to vellus-like hair follicles
in the same way, the production of hair fibers passes terminals hair
fiber to vellus. This miniaturization seems to occur due to the action
of androgen hormones that reduce the amount of cells in the papilla
dermal.
Testosterone has an important role in the development of AGA
in men. However, the pattern hair loss in women does not have
a consensus whether there is an androgen-dependent relation yet.
In both females and males, alopecia results in a decrease in the
duration of anagen phase and an increase in telogen and exogen
hair follicles. Accordingly, the density of hair shafts in the scalp
also reduces, and the interval of reinitiating hair regrowth increases
(Fig. 49.4). Thus, the hair follicles in the anagen phase change early
to telogen, and it can take months between hair shedding and
regrowth, resulting in baldness [10, 38].

49.2.2 Alopecia Areata

Alopecia areata is the second most common type of hair loss. In most
patients, hair loss occurs at scattered places on the scalp; however,
some individuals lose all scalp and body hair (alopecia totalis and
alopecia universalis, respectively).
The development of alopecia areata is related to the immune
system. It is believed that the self-antigens of hair follicles lead
to an infiltration of inflammatory cells, ceasing the anagen phase
prematurely. Lymphocytes attack the matrix of keratinocytes and
the dermal papillae in alopecia areata. After that, the hair follicles
in the anagen phase enter the dystrophic catagen phase, resulting in
hair fall [10].
The hair loss can be reversed, even after years without hair due
to preserving of the bulge area which is a area rich in stem cell.
However, this possibility of hair regrowth is smaller when the hair
loss is more extensive (alopecia totalis or universalis) [10].
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Types of Hair Loss and Some Treatments 995

Figure 49.4 Changes in hair growth cycle elicit hair disorder [3].

49.2.3 Chemotherapy-Induced Alopecia

Chemotherapy disrupts hair follicles in the anagen phase, forcing
them to enter the dystrophic catagen phase and then interrupt hair
growth and induce fall. This type of alopecia develops rapidly and
extensively, and it affects scalp and body hair. However, after the
chemotherapy, hair usually grows again because the stem cells of the
hair follicle are hardly affected by the treatment.

49.2.4 Telogen Effluvium

Telogen effluvium is one of the most common forms of alopecia,
manifested as accentuated hair loss. There is a synchrony between
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996 Low-Level Laser (Light) Therapy

hair follicles entering the exogenous phase and consequent shed-

ding. Telogen effluvium can be caused by several mechanisms. For
example, hair follicles remain in the anagen phase during pregnancy,
and after the delivery, the hair follicles change for the telogen and
exogen phases (shedding). Some of the causes of telogen effluvium
include surgical trauma, emotional stress, hyper or hypothyroidism,
and consuming food deficient in minerals such as zinc and copper
[38].

49.2.5 Scarring Alopecia

Scarring alopecia, like lichen planus or discoid lupus erythematosus,
occurs as a response to inflammation. Inflammation occurs in the
upper follicle and generally involves the bulge (Fig. 49.4). These
diseases conduce to fibrosis and permanent loss of hair follicle.
The causes of scarring alopecia are unknown but may involve
abnormal functioning of the sebaceous gland, which is necessary
for the normal shedding of the inner-root sheath of the hair
shaft, causing the rupture of the follicle, followed by a healing
process and abnormal fibrotic that leads to the elimination of one
follicle.

49.3 Treatments

Treatments for hair disorders essentially modulate parameters of

size and distribution of hair follicles and promote changes in the
hair growth cycle. Consequently, a priority in the development of
treatments for various alopecia is the development of modulators of
cycle duration that reduce the telogen phase and prolong the anagen
growth.
The anagen phase can be induced and prolonged with pro-
moters of hair growth, including minoxidil, immunophilin ligands
(cyclosporine, tacrolimus), and analogous mediators of hair growth,
such as keratinocyte growth factor, hepatocyte growth factor,
macrophage stimulating protein, Shh, and others [38].
In androgenetic, scarring, and congenital hypotrichosis responsi-
ble for the loss of hair follicle density, the major aim is the formation
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Treatments 997

of new hair follicles. A study revealed that this is possible, and

it is known as hair replication, hair multiplication, or follicular
neogenesis. The implant of dermal papilla cells or dermal sheath
cells cup of bulb region of the hair follicles demonstrated the
developing new hair follicles and thickening of the hair follicles that
already existed [28].
Although the idea of the formation of new follicles was earlier
considered unlikely, today the formation of hair follicles has been
suggested as possible. Recent research has shown that such an event
can occur and is Wnt signaling dependent [15].
In practice, pharmacological treatments modify the size and
growth cycle of hair follicles and induce the formation of new
follicles. Thus, the treatment of AGA is aimed at extending the
anagen stage, converting the telogen hair follicles in anagen,
reversing miniaturization, and possibly generating new hair follicles.
AGA treatment blocks the conversion of testosterone to DHT by the
administration of pharmacological agents, such as finasteride, that
perform some of these targets.
In alopecia areata and inflammatory scarring alopecia (lichen
planus or discoid lupus erythematosus), the treatment target is
to modulate inflammation and protect the hair follicle of the
autoimmune attack mediated by T-cells. T-cells attack the area of
the bulb na alopecia areata and the bulge na alopecia cicatricial
inflammatory [10].

49.3.1 Finasteride
Finasteride prevents the conversion of testosterone to DHT as
byproducts and thus blocks the reverse miniaturization of hair
follicles [34].

49.3.2 Minoxidil
Minoxidil is used to increase the size of miniaturized hair follicles.
It stimulates the growth of anagen hair follicles in the telogen stage
[34].
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998 Low-Level Laser (Light) Therapy

49.4 In Vivo Studies of LLLT

In 1966 Endre Mester, a Hungarian physician, pioneered research

in LLLT by publishing the first studies on the photobiostimulation
effect of low-intensity laser on wound healing in rat skin. One
year later, to investigate if laser radiation might cause cancer in
mice, he carried out a simple experiment: after shaving the hair
off their backs and dividing them into two groups, he applied
low-intensity ruby laser (wavelength 694 nm) to one group and
observed that hair regrew more quickly in the treated group than
in the untreated group. By this work, Mester demonstrated the
biostimulatory effect of low-level laser in hair growth.
Since this in vivo study, many others have shown the efficacy of
LLLT in hair growth. Wikramanayake et al. [45] applied HairMax
LaserComb R
(wavelength 655 nm, 3.7 mW) in six mice with induced
alopecia areata (laser group) for 20 s, three times per week. Hair
regrowth was compared with the control group for 6 weeks. In
the treated group, hair regrowth was first observed 2 weeks after
the initiation of laser treatment. After 6 weeks of treatment, hair
regrowth was observed in all the mice in the laser group (Fig. 49.5),
but none in the control group (sham treated).
On the histological analysis of skin samples collected from
the dorsum of the animals, an increased number of anagen hair
follicles was observed in laser-treated mice in the subcutaneous
layer (Fig. 49.6b). The anagen hair bulbs were larger compared to
the sham-treated mice (control group) in which the majority of hair
follicles were in telogen.
Wikramanayake et al. [46] also reported efficient results of LLLT
applied on neonatal rats subjected to models of chemotherapy-

Figure 49.5 Effects of the HairMax LaserComb R

on hair regrowth in mice
with alopecia areata: (a) before laser, (b) after 2 weeks, and (c) after 6 weeks
of laser treatment.
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In Vivo Studies of LLLT 999

Figure 49.6 Images of hair follicle structures stained with HE. Effects of the
HairMax LaserComb R
on hair regrowth in mice with alopecia areata after 6
weeks of sham treatment (a) and in laser-treated mice (b).

induced alopecia, in which different chemotherapy regimens were

administered: etoposide on days 11, 12, and 13 after birth, or
cyclophosphamide on day 13 after birth, as well as a combination
of cyclophosphamide (35 mg/kg on day 11) and doxorubicin (2.5
mg/kg on days 11, 12, and 13) after birth. They were randomized
into three groups: (i) The control group received chemotherapy
alone (control); (ii) the laser group received chemotherapy and
immediate LLLT (device HairMax LaserComb R
, 1 min, daily for 10
days); and (iii) the sham-treated group received similar treatment
but the laser device was turned off (sham). Each group had
10 rats. During the 15 days post-chemotherapy, all the laser-
treated rats showed hair regrowth, while the other groups did
not show hair regrowth. The non-treated groups did not show
hair regrowth until 5 days later (20 days post-chemotherapy).
Histological analysis showed that the skin was thicker in the
laser group (after cyclophosphamide-induced alopecia) compared
to other groups, with large anagen hair bulbs penetrated deeper into
the subcutaneous tissue.
Light-emitting diodes (LEDs) have been important alternatives
to light therapy for hair regrowth. Fushimi et al. have explored the
effect of a red LED (638 nm with a bandwidth of 3 nm) on hair
growth in mice [50]. The LED was applied at an energy density of
1.0 and 1.5 J/cm2 to in vivo and in vitro studies, respectively.
Six female mice were shaved and after 1 day, they were irradiated
with a red LED light for 20 min at a distance of 5 cm from their
dorsal skins. The mice were irradiated thrice a week. Another six
mice underwent a similar procedure, but the LED was switched off.
Comparing the two groups statistically significant increase in hair
regrowth was detected (indicating anagen induction).
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1000 Low-Level Laser (Light) Therapy

An in vitro study with cultured normal human dermal papilla

cells was carried out with parameters similar to the one described
earlier. The cell dishes were irradiated for the same time with
red LED at a distance of 3 cm from the culture dishes. Eval-
uation of dermal papillae revealed acceleration of hair growth
via human growth factor regulators, such as HGF, leptin, and
vascular endothelial growth factor (VEGF)-A. It was observed that
a decreased number of hair follicles entered the catagen phase with
the induction of the anagen phase and perifollicular angiogenesis,
resulting in an increased hair follicle diameter and accelerated hair
regrowth.

49.5 LLLT for Hair Growth: Clinical Trials

Hair loss is a common disorder that affects men and women, and it
tends to increase with advancing age: In men the rate of hair loss is
50% after 50 years, while in women, the prevalence is about 50%
after 80 years [2].
LLLT showed promising results for in vivo studies on hair
diseases, as demonstrated earlier. Although there are a few clinical
trials of LLLT for hair growth, we can find exciting results from them
(Table 49.1).
Satino et al. [35] evaluated the efficacy of LLLT on hair growth
and tensile strength in 28 male and 7 female AGA patients. They
observed an increase in the tensile strength and stimulation of hair
growth in both sexes in the temporal and vertex regions.
In 2013, Kim et al. [25] reported a randomized, double-blinded,
sham device-controlled trial. The treatment showed an increase in
the density and diameter of hair when compared to the placebo
group, resulting in global improvement. Other studies have been
performed using LLLT, reinforcing AGA in both men and women [24,
26, 27]. In addition, in one multicenter, randomized, sham device-
controlled, double-blinded study with 128 male and 141 female
AGA patients divided into four groups, an increase in the density
of terminal hairs was observed as well as the use of LLLT was
demonstrated to be safe with no serious adverse events. Some minor
effects reported in the study were dry skin (5.1%), pruritus (2.5%),
 July 12, 2016 11:37
Table 49.1 Summary of clinical trials of LLLT for hair loss

Author, Year Groups, Alopecia Type LLLT Parameters, Treatment Regimen Results
TM
Yamazaki et al., Alopecia areata Super Lizer polarized pulsed linear light 1.8 In 47% of the patients, hair growth occurred 1.6
2003 [47] W, 600–1600 nm, 3 min once a week or once months earlier in the irradiated areas than in the
6 male and 9 female patients other week until vellus hair regrowth in at least non-irradiated areas. However, 1 year after irradiation,
50% of the affected area was observed. all the lesions disappeared; hair density, length, and
Carpronium chloride (5%) was applied topically diameter of hair shafts were the same in both
twice daily in combination with oral irradiated and non-irradiated lesions.
antihistamines, cepharanthin, and glycyrrhizin
(extracts of Chinese medicinal herbs).
Kim et al., 2007 Alopecia androgenetic 655 nm red light and 780 nm infrared light Increase in hair density in both the vertex (145.1/cm2

PSP Book - 9in x 6in

[24] Once a day for 10 min for 14 weeks versus 137.3/cm2 pre-treatment, P <0.005) and
24 male patients occiput (163.3/cm2 versus 153.3/cm2 , P <0.005) as
well as the anogen/telogen ratio (vertex: 84.7 versus
79.7 pre-treatment and occiput: 91.9 versus 89.6
pre-treatment) was observed, and 83% of patients
were reported to be satisfied with the treatment.
Satino et al., Alopecia androgenetic HairMax LaserComb 655 nm, 5–10 min every Hair tensile strength revealed greater improvement in
2003 [35] other day for 6 months the vertex area for males and the temporal area for

LLLT for Hair Growth 1001

28 male and 7 female patients females; both sexes significantly benefited in all areas.
Hair count in both sexes and all areas had substantial
improvement (temporal area: 55% in women, 74% in
men; vertex area: 65% in women, 120% in men), with
the vertex area in males having the best outcome.
Contd.

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1002 Low-Level Laser (Light) Therapy
Table 49.1 (Continued)

Author, Year Groups, Alopecia Type LLLT Parameters, Treatment Regimen Results

Leavitt et al., Alopecia androgenetic HairMax LaserComb, thrice a week for 15 min Significant improvements in mean terminal hair density,
2009 [27] 110 male patients for 26 weeks overall hair regrowth, slowing of hair loss, thicker hair,
better scalp health, and hair shine were demonstrated in
terms of patients’ subjective assessment.
Lanzafame et al., Alopecia androgenetic Helmet with 21, 5 mW lasers and 30 LEDs (655 About 35% increase in hair growth was noticed among
2013 [26] 44 male patients ± 5 nm, 67.3 J/cm2 , 25 min, every other day for male AGA patients.
16 weeks LLLT of the scalp at 655 nm significantly improved hair
count in males with androgenetic alopecia.

Kim et al., 2013 Alopecia androgenetic Helmet-type LLLT device, 650 nm laser with Even though mean hair thickness (12.6 ± 9.4 versus 3.9

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[25] 40 male and female patients 630 and 660 nm LEDs, 92.15 mW/cm2 , 47.90 ± 7.3 in control group, P = 0.01) and hair density (17.2
J/cm2 , 18 min for 24 weeks ± 12.1 versus 2.1 ± 18.3 in control group, P = 0.003)
increased significantly in the treatment group, there was
no prominent difference in global appearance between
the two groups.
Jimenez 2014 Alopecia androgenetic Lasercomb (three models: 9-beam with 655 nm In Trial #1, a significant difference in terminal hair
[19] ± 5%, 11 min; 12-beam with 6 beams at 635 nm density change from baseline was observed between the
128 male and 141 female patients ± 5%, 6 beams at 655 nm ± 5%, 8 min; 7-beam 9-beam lasercomb and sham treated [41 of 43 (95%)
divided into four groups: lasercomb (655 nm ± 5%, 15 min) and sham versus 7 of 22 (32%)].
Trial #1: AGA females, 9 beam device for 3 times/week for 26 weeks Trial #2 [37 of 39 (95%) versus 6 of 18 (33%)]
Trial #2: AGA females, 12 beam Trial #3 [20 of 24 (83%) versus 6 of 14 (43%)]
Trials #3 and #4: AGA males, 7- Trial #4 (86% for the 9-beam and 82% for the 12-beam
(#3) or a 9- or 12-beam (#4) models versus 59% sham treated)
Sham device for 7, 9, and 12 beam Lasercomb treatment significantly increases terminal
hair density.

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LLLT for Hair Growth and Hair Loss (Proposed Mechanisms) 1003

scalp tenderness (1.3%), irritation (1.3%), and a warm sensation

at the site (1.3%), although no significant difference was noted
between the active device and the sham device.
LLLT in alopecia areata patients is possibly effective in the
improvement of lesions. When the scalp was irradiated for 3 min,
either once a week or once every other week, along with the use of
supplements and medications, hair regrowth was observed in 47%
of patients. After 1 year of treatment, all lesions disappeared, and
the hair density, length, and diameter in treated and control groups
remained similar [47].

49.6 LLLT for Hair Growth and Hair Loss

(Proposed Mechanisms)

Several in vivo studies suggest that LLLT stimulates telogen hair

follicles to change to the anagen phase and also proliferation of
active hair follicles. Moreover, LLLT prolongs the duration of the
anagen phase with the inhibition of premature catagen development
and a decrease in inflammatory infiltrates compared to the controls.
It is possible to notice that different doses of light irradiated
during in vivo studies and clinical trials have resulted in hair growth.
The adequate light dose for the treatment of hair loss has not been
determined. The knowledge of the light intensity distribution at
skin can help us in knowing the exact mechanism of LLLT in hair
growth because the quantity of photons (light dose) absorbed by
the cell structure of skin can modulate tissue activity to homeostasis.
Although the exact mechanism of LLLT in hair growth is not known,
identifying the laser action in the main cells involved in the hair
growth cycle, it is possible to clearly infer several mechanisms of
LLLT in hair loss diseases.
Studying photobiomodulation in cultured cells, Karu [23] de-
scribed a possible LLLT mechanism at the molecular and cellular
levels. The light absorbed by the mitochondria can increase the
synthesis of adenosine triphosphate (ATP) in cells due to the
absorption of photons by the molecules presents in the respira-
tory chain (flavins, NADH, FAD, cytochromes, and cytochrome c
oxidase) (Fig. 49.7), promoting increased mitochondrial activity.
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1004 Low-Level Laser (Light) Therapy

Figure 49.7 Mechanism of low-level laser on respiratory chain.

This increase, in turn, is characterized by a higher proton gradient

matrix for intermembrane space, which intensifies the proton reen-
try by ATP synthase enzyme promoting oxidative phosphorylation,
more production of ATP [8, 42].
The photons absorbed by the mitochondria can induce modu-
lation of reactive oxygen species (ROS) [2–4, 8]. ROS are involved
in signaling pathways between mitochondria and nuclei, induction
of gene transcription factors such as nuclear factor kappa B and
hypoxia-inducible factor-1, triggering protein synthesis and then
enzymatic reactions that promote modulation of levels of cy-
tokines, growth factors, and inflammatory mediators and increasing
cells proliferation and migration. NO is recognized as an anti-
inflammatory, vasodilatory, angiogenetic, and anti-apoptotic growth
modulator and stem cells stimulator [1, 5–7, 9, 13, 14, 16, 17, 20–
22, 30, 32, 33, 39–41, 44]. Increased blood flow to the skin favors
better oxygenation of hair bulb. These effects promoted by the
action of LLLT on skin cells—fibroblast, keratinocytes, melanocytes,
and stem cells—can contribute to tissue homeostasis and hair
growth.
The anagen phase of hair growth remains active with the aid
of angiogenesis and the regulation of VEGF. The LLLT applied to
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LLLT for Hair Growth and Hair Loss (Proposed Mechanisms) 1005

the scalp releases NO. NO is responsible for vasodilating effect

and a modulating effect of the angiogenic growth. As a result of
increased tissue vascularization and increased synthesis of ATP,
LLLT can promote fibroblast proliferation and increase in collagen
synthesis. Thus the result will be epithelization increased, dermal–
epidermal junction and longer duration of hair follicles in the
anagen phase. The anagen phase can be possibly extended for
the second mechanism of modulation of enzyme that produces
DHT, responsible for altering the expression of VEGF and the
development and aggravation of male pattern baldness. In principle,
the increased blood circulation promoted by the release of NO avoids
the deposition and accumulation of DHT on androgen-dependent
receptors and contributes to the modulation of VEGF.
In addition, processes that induce controlled inflammation in
hair follicles may be beneficial for the development of healthy
follicles due to increased local vascularization [2].
In contrast, cytokines such as IFN-γ , IL-1a and b, TNF-a, MHC,
and Fas-antigen and macrophage migration inhibitory factor are all
involved in the cyclic hair growth and have been shown to play a
role in the pathogenesis of AA. These cytokines are highly disruptive
to hair follicle biology. Wikramanayake et al. [19] supported this
assumption because their study verified a decrease in inflammatory
infiltrates and an increase in anagen hair follicles with LLLT
use. Moreover, the action of LLLT about NO showed be possible
for treat inflammatory processes. Shukla et al. signalize at study
with mice testosterone-treated and untreated that the result of
testosterone-treated group presented better hair growth. Therefore,
it is suggested that cells which grow at a slower rate or cells
under stress conditions respond better the stimulation effects of
LLLT.
The inflammatory process induced and controlled by profes-
sionals has been shown benefits for hair growth and also to
the formation of new hair follicles. It is well known that the
interaction between epithelial cells and underlying cells is essential
to initiate the development of hair follicles. Studies suggest that
new hair follicles may form in response to injury. Studies on
hypertrichosis using lasers and intense pulsed light for hair removal
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1006 Low-Level Laser (Light) Therapy

Anagen

Early Catagen
Anagen

Telogen

Drug Discovery Today: Disease Mechanisms

Figure 49.8 Development of a new hair follicle and activation of hair

growth [28].

have demonstrated an adverse effect on terminal hair growth

known as paradoxical hypertrichosis. This phenomenon occurs
when thermal injury is promoted at low fluences of light inside
biological tissue, as the residual effect of high-power laser at skin
promoting biostimulation [4, 11, 31, 32, 36, 37].
Another important way to explore the mechanisms of LLLT for
hair growth is to understand the relationship between the role that
stem cells develop in hair growth and evidence of the effect of laser
in stem cells. Mesenchymal stem cells have an important role in the
formation of new hair follicles and increase hair growth. These are
located in hair follicles in two distinct reservoirs: (i) bulge area of the
hair follicle and (ii) dermal papilla region (Fig. 49.8). Ito et al. [18]
showed that new follicles are formed from stem cells in epidermal
wounds in rats.
The location and distribution of stem cells tend to vary
throughout the anagen–telogen hair cycle: during the anagen phase,
in the dermal papilla and bulge, and during others, transitions only
in the bulge area.
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LLLT for Hair Growth and Hair Loss (Proposed Mechanisms) 1007

Figure 49.9 High-resolution in vivo multiphoton tomography of living mice.

The locations of hair follicle stem cells are depicted in green.

The action of LLLT on stem cells and the consequent activation

of hair regrowth and the formation new hair follicles can be related
due to stimulating the motility of stem cells (Fig. 49.10). Uchugonova
et al. [49] conducted a study on the application of red and
infrared LLLT in vitro and obtained positive results. When stem cells
were irradiated with laser light, the migration of stem cells were
promoted to the gradient SDF-LCT. High concentrations of SDF-1
also play an important role in the migration of stem cells.
Laser irradiation can activate metabolism of stem cells, increase
ATP production and then migration of stem cells from the bulge
to the dermal papilla, stimulating the anagen phase, cellular
differentiation, and proliferation of hair growth, and from the bulge
to basal layer promoting formation of new hair follicles. More
studies are required to test the influence of laser light on the
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1008 Low-Level Laser (Light) Therapy

Hair fibre

Formation new
follicle hair

Basal layer Epidermis

Sebaceous gland

Outer root sheath The bulge: stem cell niche

Inner root sheath
LLLT
Irradiation

Dermal papilla Active of hair growth

Figure 49.10 Development of a new hair follicle and activation of hair

growth. Adapted from Ref. [15].

mobilization rate of stem cells and to investigate how laser light can
enhance functional abilities of stem cells.

49.7 Phototherapy Devices for Hair Aesthetics

Several light therapy devices are currently available for treating hair
loss. Usually they are for home use and emit in the wavelengths of
630–680 nm and can consist of simple combinations of less than
a dozen lasers or LEDs, or complex arrays with hundreds of light
sources, which may irradiate the whole scalp at the same time.
The handheld home-use devices, such as the Sunetics Laser
Hair Brush (Sunetics International, Las Vegas, NV, USA), X5 Hair
Laser (Spencer Forrest, Inc., Los Angeles, CA, USA), and HairMax
LaserComb (Lexington International, Boca Raton, FL, USA), are
shown in Fig. 49.11. They have red laser modules (less than 5.0 mW)
at different quantities and arrangements.
These devices must be moved and positioned over the affected
area by the patient during the treatment, which may affect
compliance and non-homogeneous dose absorption.
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Phototherapy Devices for Hair Aesthetics 1009

Figure 49.11 Handheld low-level laser devices for home use. Left to right:
Sunetics Laser Hair Brush, X5 Hair Laser, and HairMax LaserComb.

Figure 49.12 In-office low-level laser systems. Left to right Sunetics Clinical
Laser “G”, Super Grow Dome 60 and MEP-90 Hair Growth Stimulation
System.

In-office systems such as the Sunetics Clinical Laser “G”, MEP-

90 Hair Growth Stimulation System (Midwest RF, LLC, Hartland, WI,
USA), and Super Grow Dome 60 (Super Grow Lasers Inc., Houston,
TX, USA) are shown in Fig. 49.12. These devices deliver higher total
doses than the handheld devices.
Home-use systems in helmet-type configuration such as Laser-
Cap (Transdermal Cap, Inc., Cleveland, OH, USA) and iGrowTM Laser
Hair Therapy System TOPHAT655 (Apira Science, Inc., Boca Raton,
FL, USA) are handheld and handsfree (Figs. 49.13 and 49.14),
respectively. The iGrowTM Laser device is fitted with an array of laser
and LED light sources operating at 655 nm. It consists of 21 lasers
diodes (5 mW) and 30 red LEDs.
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1010 Low-Level Laser (Light) Therapy

Figure 49.13 iGrowR Hair Growth System (TOPHAT655): A bicycle helmet–

like hands-free laser therapy device for home use.

Figure 49.14 LaserCap

R
: Handheld laser therapy device for home use.

With more intensity than most in-office systems, LaserCap R

device is a flexible hermetically sealed dome-shaped membrane

with 224 lasers modules (650 nm at 5 mW) that fits in almost any
hat to treat the entire scalp, powered by a rechargeable battery.
Handheld laser devices in helmet-type configuration remain
positioned over the affected area, warranting more homogeneous
light dose absorption in the scalp.
The equipment presented in this chapter are many variations
of phototherapy equipment for hair growth. The knowledge of
the relationship between light dose absorbed at the skin and the
mechanisms of action of this light for hair growth, knowing the
effects of various wavelengths at different doses in the hair cycle,
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Glossary 1011

can promote the development of new phototherapy devices for hair

aesthetic with better and faster clinical results.

49.8 Future Perspectives

The biostimulation effect of laser on hair growth has been known

since 1967 with Mester’s research. Clinical and animal studies
have shown that LLLT stimulates hair growth and effectively
treats alopecia. Despite the effectiveness of LLLT, there are some
controversies around the mechanisms of action in the hair cycle
and the formation of new follicles. The dose dependency of
photobiomodulation for hair growth remains undetermined since
there are many misunderstandings regarding the parameters of the
therapy [29].
New in vivo and clinical studies should be conducted to
determine the effect of LLLT and LEDs on alopecia according to
the intensity of radiated light, wavelength, exposure time, and
interval between applications. It is also important to investigate
the effectiveness of the treatment after staying for a long time
without the application of LLLT. The compatibility of minoxidil and
finasteride with light therapy should be evaluated. This knowledge
may contribute to verify the safety of phototherapy for hair growth
in patients undergoing chemotherapy.
Stem cell research combined with low-level laser irradiation has
opened a new window in photomedicine, looking through which
someone can better understand tissue regeneration assisted by
laser, especially hair growth.

49.9 Glossary

Alopecia: Abnormal hair loss.

Androgenetic alopecia: Loss of hair caused by miniaturization of
genetically predisposed follicles in a male
pattern (frontal recession and thinning
at the vertex) or female pattern (loss of
hair over the crown with sparing of the
frontal hair line).
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1012 Low-Level Laser (Light) Therapy

Alopecia areata: Hair loss in patches caused by an au-

toimmune inflammatory response to the
follicle.
Anagen: Growing stage of the hair follicle cycle.
Bulb: Lowermost portion of the anagen follicle
containing rapidly proliferating cells that
generate the hair.
Bulge: Putative site of epithelial stem cells
within the follicle outer root sheath.
Catagen: Regression stage of the hair follicle cycle.
Club hair: Dead hair possessing thickened base that
anchors it in the follicle during telogen.
Exogen: Stage of the hair follicle cycle when hair
is shed from the follicle.
Telogen: Resting stage of the hair follicle cycle.
Telogen effluvium: Excessive shedding of hair caused by
synchronous entry of many hair follicles
into exogen.

References

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2. Avci P, Gupta GK, Clark J, Wikonkal N, and Hamblin, MR. (2014). Low-
level laser (light) therapy (LLLT) for treatment of hair loss. Lasers Surg
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basic science of hair biology: What are the causal mechanisms for the
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1014 Low-Level Laser (Light) Therapy

18. Ito M, Yang Z, Andl T, Cui C, Kim N, Millar SE, and Cotsarelis G. (2007).
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level laser device in the treatment of male and female pattern hair loss:
A multicenter, randomized, sham device-controlled, double-blind study.
Am J Clin Dermatol, 15, pp. 115–127.
20. Joyner MJ and Dietz NM. (1985). Nitric oxide and vasodilation in human
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11, 5, pp. 1859–1874.
22. Mujoo K, Krumenacker JS, and Murad F. (2011). Nitric oxide-cyclic GMP
signaling in stem cell differentiation. Free Radic Biol Med, 51, 12, pp.
2150–2157.
23. Karu TI. (1989). Photobiology of low power laser therapy. In Laser
Science and Technology: An International Handbook, Vol. 8. London:
Harwood Academy Publishers.
24. Kim SS, Park MW, and Lee CJ. (2007). Phototherapy of androgenetic
alopecia with low level narrow band 655-nm red light and 780-nm
infrared light. J Am Acad Dermatolog, 56, AB112.
25. Kim H, Choi JW, Kim JY, Shin JW, Lee SJ, and Huh CH. (2013). Low-
level light therapy for androgenetic alopecia: A 24-week, randomized,
double-blind, sham device-controlled multicenter trial. Dermatol Surg,
39, 8, pp. 1177–1183.
26. Lanzafame R, Blanche R, Bodian A, Chiacchierini R, Fenandez-Obregon
A, Kazmirek E, and Raymond J. (2013). The growth of human scalp hair
mediated by visible red light laser and LED sources in males. Lasers Surg
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27. Leavitt M, Charles G, Heyman E, and Michaels D. (2009). HairMax
LaserComb laser phototherapy device in the treatment of male andro-
genetic alopecia: A randomized, double-blind, sham device-controlled,
multicentre trial. Clin Drug Investig, 29, 5, pp. 283–292.
28. McElwee KJ and Sinclair R. (2008). Hair physiology and its disorders.
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29. Mester E, Szende B, and Gärtner P. (1968). The effect of laser beams
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626.
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30. Michurina T, Krasnov P, Balazs A, Nakaya N, Vasilieva T, Kuzin B,

Khrushchov N, Mulligan RC, and Enikolopov G. (2004). Nitric oxide is
a regulator of hematopoietic stem cell activity. Mol Ther, 10, 2, pp. 241–
248.
31. Moreno-Arias G, Castelo-Branco C, and Ferrando J. (2002). Paradoxical
effect after IPL photoepilation. Dermatol Surg, 28, 11, pp. 1013–
1016.
32. Mujoo K, Krumenacker JS, and Murad F. (2011). Nitric oxide-cyclic GMP
signaling in stem cell differentiation. Free Radic Biol Med, 51, 12, pp.
2150–2157.
33. Napoli C, Paolisso G, Casamassimi A, Al-Omran M, Barbieri M, Sommese
L, Infante T, and Ignarro LJ. (2013). Effects of nitric oxide on cell
proliferation: Novel insights. J Am Coll Cardiol, 62, 2, pp. 89–95.
34. Rogers NE and Avram MR. (2008). Medical treatments for male and
female pattern hair loss. J Am Acad Dermatol, 59, 4, pp. 547–566.
35. Satino JL and Markou M. (2003). Hair regrowth and increased hair
tensile strength using the HairMax LaserComb for low-level laser
therapy. Int. J. Cosmetic Surg. Aesthetic Dermatol, 5, 2, pp. 113–117.
36. Oshiro T and Calderhead RG. (1991). Progress in Laser Therapy.
Chichester, London: John Wiley.
37. Schneider MR, Schmidt-Ullrich R, and Paus R. (2009). The hair follicle as
a dynamic miniorgan. Curr Biol, 19, 3, pp. R132–R142.
38. Shannon H and Wilma B. (2009). Diffuse hair loss: Its triggers and
management. Cleveland Clin J Med, 76, 6, pp. 361–367.
39. Sciorati C, Galvez BG, Brunelli S, Tagliafico E, Ferrari S, Cossu G,
and Clementi E. (2006). Ex vivo treatment with nitric oxide increases
mesoangioblast therapeutic efficacy in muscular dystrophy. J Cell Sci,
119, Pt 24, pp. 5114–5123.
40. Sciorati C, Buono R, Azzoni E, Casati S, Ciuffreda P, D’Angelo G, Cattaneo
D, Brunelli S, and Clementi E. (2010). Co-administration of ibuprofen
and nitric oxide is an effective experimental therapy for muscular
dystrophy, with immediate applicability to humans. Br J Pharmacol, 160,
6, pp. 1550–1560.
41. Tegenge MA, Rockel TD, Fritsche E, and Bicker G. (2011). Nitric oxide
stimulates human neural progenitor cell migration via cGMP-mediated
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42. Torres BA and Marzzoco A. (1999). Bioquı́mica Básica, Guanabara
Koogan, 2nd edition, 11, pp. 135–158.
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1016 Low-Level Laser (Light) Therapy

43. Weiss R, McDaniel DH, Geronemus RG, and Weiss M. (2005). LED
photomodulation induced hair growth stimulation. Supplement: Amer-
ican Society For Laser Medicine And Surgery Twenty-Eighth Annual
Conference Kissimmee, Florida, 40, 20 June 2008.
44. Weller R, Billiar T, and Vodovotz Y. (2002). Pro- and anti-apoptotic
effects of nitric oxide in irradiated keratinocytes: The role of superoxide.
Skin Pharmacol Appl Skin Physiol, 15, 5, pp. 348–352.
45. Wikramanayake TC, Rodriguez R, Choudhary S, Mauro LM, Nouri
K, Schachner LA, and Jimenez JJ. (2012). Effects of the Lexington
LaserComb on hair regrowth in the C3H/HeJ mouse model of alopecia
areata. Lasers Med Sci, 27, 2, pp. 431–436.
46. Wikramanayake TC, Villasante AC, Mauro LM, Nouri K, Schachner LA,
Perez CI, and Jimenez JJ. (2013). Low-level laser treatment accelerated
hair regrowth in a rat model of chemotherapy-induced alopecia (CIA).
Lasers Med Sci, 28, 3, pp. 701–706.
47. Yamazaki M, Miura Y, Tsuboi R, and Ogawa H. (2003). Linear polarized
infrared irradiation using Super Lizer is an effective treatment for
multiple-type alopecia areata. Int J Dermatol, 42, 9, pp. 738–740.
48. Kolarsick PAJ, Kolarsick MA, and Goodwin C. (2011). Anatomy and
physiology of the skin. J Dermatol Nurses’ Assoc, 3, 4, pp. 203–213.
49. Uchugonova A, Gorjup E, Riemann I, Sauer D, König K. (2008). Two-
photon imaging of stem cells. Proc. of SPIE, 6860 Multiphoton Microscopy
in the Biomedical Sciences VIII, 68601W.
50. Fushimi T, Inui S, Ogasawara M, Nakajima T, Hosokawa K, Itami S.
(2011). Narrow-band red LED light promotes mouse hair growth
through paracrine growth factors from dermal papilla. J Dermatol Sci,
64, 3, pp. 246–248.
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Chapter 50

Low-Level Laser (Light) Therapy for

Cosmetics and Dermatology

Mossum K. Sawhneya and Michael R. Hamblina,b,c

a Wellman Center for Photomedicine, Massachusetts General Hospital, 50 Blossom

Street, Boston, MA 02114, USA

b Department of Dermatology, Harvard Medical School, Massachusetts General

Hospital, 55 Fruit Street, Boston, MA 02114, USA

c Harvard-Massachusetts Institute of Technology (MIT) Division of Health Sciences and

Technology, 77 Massachusetts Avenue, E25 -518, Cambridge, MA 02139, USA

sawhney.mossum@gmail.com

50.1 Introduction

Low-level laser (or light) therapy (LLLT), also called phototherapy or

photobiomodulation, refers to the use of photons to alter biological
activity. Basically, light within a certain range of wavelengths (optical
window) is able to effectively excite tissue chromophores such as
cytochrome c oxidase (possessing high absorption bands in the red
and near-infrared spectral regions where light can penetrate deeply
into tissue), thus bringing about a myriad of cellular events (Ham-
blin and Demidova, 2006). The most widely accepted mechanism
of action of LLLT is through the activation of mitochondria, where
components of the electron transport chain (ETC) or respiratory

Handbook of Low-Level Laser Therapy

Edited by Michael R. Hamblin, Marcelo Victor Pires de Sousa, and Tanupriya Agrawal
Copyright c 2017 Pan Stanford Publishing Pte. Ltd.
ISBN 978-981-4669-60-3 (Hardcover), 978-981-4669-61-0 (eBook)
www.panstanford.com
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1018 Low-Level Laser (Light) Therapy for Cosmetics and Dermatology

chain serve as chromophores and, thus, as targets for the action of

LLLT (Hamblin and Demidova, 2006). Mitochondrial activation, in
turn, stimulates ATP production, causes release of nitric oxide, and
induces reactive oxygen species, which together further result in the
stimulation of redox-sensitive transcription factors and expression
of gene products. Thus, LLLT results in processes such as tissue
repair, wound healing, prevention of cell death, and other long-
term effects (Hamblin and Demidova, 2006). Non-thermal, coherent
light sources (lasers) or non-coherent light sources consisting of
filtered lamps or light-emitting diodes (LEDs) are used in this type
of therapy for reducing pain and inflammation, augmenting tissue
repair and regeneration, deeper tissues and nerves, and preventing
tissue damage (Chung et al., 2012; Gupta et al., 2012). In the last few
decades, non-ablative laser therapies have been used increasingly in
dermatology for the aesthetic treatment of fine wrinkles, photoaged
skin and scars, a process known as photorejuvenation. More recently
they have also been used for treating inflammatory acne (Seaton
et al., 2006). Their potential use for other dermatological conditions
and cosmetic indications such as vitiligo, psoriasis, photoprotection,
hair regrowth, and fat reduction has been shown by several
studies. In this chapter, we will briefly discuss these cosmetic and
dermatological applications of LLLT, starting with its current and
potential use in cosmetic dermatology and various skin conditions,
hair loss treatment, and finally in fat reduction procedures and
cellulite treatment.

50.2 LLLT in Dermatology

50.2.1 LLLT for Skin Rejuvenation

Skin aging is a process that may present with a relatively early
onset, sometimes as early as during the late 20s or early 30s
of an individual’s life. Common signs and symptoms of skin
aging include skin wrinkling, dyspigmentation, telangiectasia, and
reduced elasticity. At the histological and molecular level, common
noticeable features include reduced collagen content, collagen fiber
fragmentation, elastotic degeneration of elastic fibers, presence of
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LLLT in Dermatology 1019

dilated and tortuous dermal vessels, disorientation and atrophy of

the epidermis along with an upregulation of matrix metallopro-
teinases (MMPs), especially MMP-1 and MMP-2 (Kligman, 1989;
Takema et al., 1994). Skin aging is considered a process affected by
both chronological and environmental elements, but the single most
influential factor responsible for accelerated skin aging seems to be
photodamage induced primarily through ultraviolet (UV) radiation
exposure (Takema et al., 1994).
A wide range of modalities have been developed in order to
mitigate the aesthetically undesirable effects of skin aging. Most
of these procedures depend on a controlled form of epidermal
removal and skin wounding to promote new collagen biosynthesis
and dermal matrix remodeling, both serving as a preliminary means
to address the problems associated with photoaging and chrono-
logical aging. Some common methodologies used presently include
application of topical ointments containing vitamin A derivatives
such as retinoic acid, dermabrasion processes, chemical peels,
and ablative laser resurfacing techniques utilizing either a carbon
dioxide (CO2 ) or erbium: yttrium–aluminum–garnet (Er:YAG) lasers
or a combination of both of these (Airan and Hruza, 2005; Branham
and Thomas, 1996; Paasch and Haedersdal, 2011). Nevertheless,
these techniques pose undesirable limitations for the patient such
as intensive post-treatment care, prolonged downtime, and may
also lead to complications such as long-lasting erythema, pain,
infection, bleedings, oozing, burns, hyper- or hypopigmentation, and
scarring (Nanni and Alster, 1998; Sriprachya-Anunt et al., 1997).
These limitations have led to the development of technologies
that transcend such limitations, thus allowing for safer and more
efficient treatment modalities such as non-ablative laser resurfacing
(Hardaway and Ross, 2002; Sachdev et al., 2011; Weiss et al., 2003).
Unlike ablative laser resurfacing, non-ablative laser resurfacing
aims to provide aesthetic improvement of photoaged skin without
destruction of epidermis and thus requires little or no downtime
and offers an attractive alternative for both patients and physicians
(Hardaway and Ross, 2002; Weiss et al., 2003). Intense pulsed light
(IPL) sources, such as 532 nm potassium titanyl phosphate (KTP)
lasers and high-dose 585/595 nm pulsed dye lasers (PDL), primarily
target irregular pigmentation and telangiectasia, and other IPL
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1020 Low-Level Laser (Light) Therapy for Cosmetics and Dermatology

sources, such as low-dose 589/595 nm PDLs, 1064 and 1320 nm

neodymium: yttrium–aluminum–garnet (Nd:YAG) lasers, 1450 nm
diode lasers, and 1540 nm erbium glass lasers, mainly target
skin wrinkle reduction and skin tightening through thermal injury
to the dermis (photothermolysis) (Lee et al., 2007a). Treatments
combining KTP 532 nm lasers with 1064 nm Nd:YAG lasers have
been shown to possess synergistic effects (Lee, 2002). Although
ablative and non-ablative modalities provide means for treatment
of photodamaged skin, they are not without their shortcomings.
Ablative procedures, though effective, have disadvantages such as
risk of dyspigmentation, scarring, postoperative erythema, and
prolonged downtime, while non-ablative technologies are slow
with mild results despite tackling some of the disadvantages of
ablative procedures (Goel et al., 2011). Fractional laser resurfacing
(FSR) or fractional thermolysis is a technology that bridges the
gap between ablative and non-ablative modalities and is gaining
popularity due to its desirable profile of side effects, reduced
recovery period, and significant clinical results (Goel et al., 2011).
The working principle involves using water as a chromophore for
the laser as opposed to melanin, which allows for micro-ablation and
controlled resurfacing of small areas of the skin without damaging
the basement membrane and thus leading to reduced postoperative
problems and progressing toward increased patient comfort (Goel
et al., 2011).
LLLT is a novel treatment option available for non-thermal and
non-ablative skin rejuvenation, which has been shown to be effective
for improving skin conditions such as wrinkles and skin laxity
(Barolet et al., 2009; Bhat et al., 2005; Dierickx and Anderson,
2005; Russell et al., 2005; Weiss et al., 2003; 2004; 2005a). A wide
range of different light sources have been used to deliver light for
these treatments, particularly to the face, and some are shown in
Fig. 50.1. LLLT is a treatment modality that has been shown to
provide increased rates of skin rejuvenation and wound healing
with great efficacy, while also reducing postoperative pain, edema,
and several types of inflammation, making it a highly desirable
modality (Calderhead et al., 2008; Kim and Calderhead, 2011). Early
studies by Abergel et al. and Yu et al. reported an increase in the
production of procollagen, collagen, basic fibroblast growth factors
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LLLT in Dermatology 1021

(bFGF), and proliferation of fibroblasts after exposure to low-energy

laser irradiation in in vitro and in vivo animal models (Abergel et
al., 1987; Yu et al., 1994). Use of light sources with wavelengths of
633 nm/830 nm is most common in cases of clinical applications
involving wound healing and skin rejuvenation. LLLT is now used
for the treatment of even chronic non-healing wounds through
restoration of imbalances in collagenesis/collagenase and allows for
rapid and enhanced wound healing in general (Kim and Calderhead,
2011). Lee et al. conducted a study to investigate the histological and
ultrastructural alterations that followed a series of light treatments
utilizing LEDs with parameters of 830 nm, 55 mW/cm2 , 66 J/ cm2
and 633 nm, 105 mW/cm2 , and 126 J/cm2 . They observed alteration
in the levels of MMPs and tissue inhibitors of metalloproteinases
(TIMPs) (Lee et al., 2007a). The study also showed increased
mRNA levels of interleukin-1 beta (IL-1β), tumor necrosis factor
alpha (TNF-α), intercellular adhesion molecule-1 (ICAM-1), and
connexin 43 (Cx43) following LED phototherapy, whereas IL-
6 levels were decreased (Lee et al., 2007a). Subsequently, the
study also demonstrated a well-marked increase in the amount
of collagen in the post-treatment specimens (Lee et al., 2007a).
In fractional laser resurfacing, it is thought that the deliberate
development of microscopic photothermally mediated wounds is
responsible for the recruitment of pro-inflammatory cytokines IL-
1β and TNF-α in order to cause wound repair. The generation of
such a wound-healing cascade thus contributes to new collagen
synthesis (Lee et al., 2007a). LLLT may also induce this wound-
healing process through athermal and atraumatic induction of
a subclinical “quasi-wound,” even without any actual wounding
created by thermal damage, which can possibly cause complications
as in some other laser treatments (Lee et al., 2007a). MMP activities
are known to be inhibited by TIMPs, suggesting the possibility
of other mechanisms for increased collagen synthesis through the
induction of TIMPs. Collectively viewing these findings, they are
suggestive of the idea that an increased production of IL-1β and
TNF-α might be responsible for the induction of MMP activity
as an early response to light treatment, which might possibly
contribute to the removal of photodamaged collagen fragments in
order to facilitate new collagen biosynthesis. Furthermore, as a
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1022 Low-Level Laser (Light) Therapy for Cosmetics and Dermatology

consequence of the therapy, there may be increased concentrations

of TIMPs that most likely play a role in the protection of the
newly synthesized collagen, from proteolytic degradation by MMPs
(Lee et al., 2007a). Subsequently, heightened expression of Cx43
may possibly enhance cell–cell communication between dermal
components, especially between fibroblasts, allowing for greater
synchrony between cellular responses, following the effects of
photobiostimulation from LLLT in order to promote synthesis of
new collagen in a greater area, including even the regions that did
not receive light irradiation (Lee et al., 2007a).
A clinical study conducted by Weiss et al. demonstrated the
benefits of LLLT over traditional thermal-based rejuvenation modal-
ities. A group of 300 patients received LLLT (590 nm, 0.10 J/cm2 )
alone, and another group of 600 patients received a combination
of LLLT with a thermal-based photorejuvenation procedure. Of
the patients who received just the light treatment, 90% reported
an observed softening of skin textures as well as a reduction in
skin coarseness and fine lines that ranged from small alterations
to significant changes (Weiss et al., 2005c). It was observed that
patients who received a form of LLLT (n = 152) reported a
noticeable reduction in post-treatment erythema and an overall
impression of increased efficacy versus patients that received
treatment through a thermal photorejuvenation laser or light source
lacking any sort of LLLT photomodulation (Kucuk et al., 2010; Weiss
et al., 2005c). Reduction in post-treatment erythema can most likely
be attributed to the anti-inflammatory effects of LLLT (Barolet et al.,
2009). Utilizing different pulse sequence parameters, a multicenter
clinical trial was conducted, wherein 90 patients received eight
LLLT treatments over 4 weeks (Geronemus et al., 2003; McDaniel
et al., 2003; Weiss, 2004; Weiss et al., 2005a). The study found
good overall results with more than 90% of patients improving
by at least one Fitzpatrick photoaging category and 65% of the
patients displaying global improvement in facial texture, fine lines,
background erythema, and pigmentation with results peaking at 4–
6 months following completion of the eight treatments. A noticeable
increase in papillary dermal collagen and reductions in MMP-1 were
generally observed. A study conducted by Barolet et al. also proved
to be consistent with the aforementioned studies. The study used a
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LLLT in Dermatology 1023

Figure 50.1 Examples of LLLT devices used for skin rejuvenation and
treatment of acne.

three-dimensional model of tissue-engineered human reconstructed

skin (HRS) to investigate the potential of LLLT (660 nm, 50
mW/cm, 4 J/cm2 ) in collagen and MMP-1 modulation. The results
showed upregulation of collagen and downregulation MMP-1 in vitro
(Barolet et al., 2009). A split-face, single-blinded clinical study was
then carried out to assess the results of this light treatment on
skin texture and appearance of individuals with aged/photoaged
skin (Barolet et al., 2009). Profilometry quantification (to measure
wrinkles) demonstrated that more than 90% of individuals had a
reduction in rhytid depth and surface roughness, and 87% of the
individuals reported that they have experienced a reduction in the
Fitzpatrick wrinkling severity score following 12 LLLT treatments
(Barolet et al., 2009).

50.2.2 LLLT for Acne

Acne vulgaris is a relatively common skin disorder, with a reported
prevalence of up to 90% among adolescents (Stathakis et al., 1997).
Some studies have reported a comedone prevalence nearing 100%
for both male and females during adolescence (Stathakis et al.,
1997). Although typical acne is neither a serious nor a contagious
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1024 Low-Level Laser (Light) Therapy for Cosmetics and Dermatology

condition, it can greatly impact an individual’s emotional and social

aspects of life. Pathogenesis of acne has not yet been completely
clarified; however, the current consensus is that it involves four main
events: follicular hypercornification, increased sebum secretion,
colonization of Propionibacterium acnes (P. acnes), and inflammation
(Lee et al., 2007b). P. acnes plays a major role in the development of
acne by acting on triglycerides and releasing cytokines, which cause
inflammatory reactions and change infundibular keratinization
(Lee et al., 2007b). Current treatments for acne vulgaris include
topical and oral medications such as topical antibiotics (e.g.,
clindamycin and erythromycin), topical retinoids (e.g., tretinoin
and adapalene), benzoyl peroxide, alpha hydroxy acids, salicylic
acid, or azaleic acid. In severe cases, administration of systemic
antibiotics such as tetracycline and doxycycline, oral retinoids,
and some hormonal treatments is recommended (Aziz-Jalali et al.,
2012). Most medications work by counteracting microcomedone
formation, sebum production, P. acnes, and inflammation (Aziz-Jalali
et al., 2012). Despite the many treatment options currently available,
several patients still have an inadequate response to the treatment,
while others may suffer from actual adverse effects.
In order to mitigate the issues raised by previous treatment op-
tions, phototherapy has been presented as an alternative treatment
modality for acne vulgaris with reduced undesirable side effects
(Rotunda et al., 2004). Sunlight exposure has been reported to have
a significant impact on the treatment of acne with a high efficacy up
to 70%. More recently, techniques applying broad spectrum visible
lights such as LLLT are currently being employed in the treatment
of acne (Cunliffe and Goulden, 2000). One mechanism of action for
phototherapy is through the utilization of porphyrins produced by
P. acnes as part of its normal metabolism. These porphyrins act as
endogenous photosensitizers by absorbing light (specifically blue
light and, to a lesser extent, red light) and bring about photochemical
reactions that produce reactive free radicals and singlet oxygen
species, which prove toxic for P. acnes (Fig. 50.2) (Lee et al., 2007b;
Ross, 2005). Red light has been demonstrated to have a greater
penetration depth when compared to that of blue light (Aziz-Jalali
et al., 2012). Infrared light has been proposed to destroy sebaceous
glands and thereby reduce acne lesions (Lloyd and Mirkov, 2002).
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LLLT in Dermatology 1025

Figure 50.2 Illustration of acne treatment with red and blue light.

Red light has been thought to stimulate cytokine release from

various cells, including macrophages and thus reduce inflammation
(Rotunda et al., 2004; Sadick, 2008).
Many studies have demonstrated the efficacy of red to near-
infrared (NIR) light (spectral range 630 nm to 1000 nm and non-
thermal power less than 200 mW) for the treatment of acne
vulgaris. This red light maybe used alone or in combination with
other modalities (particularly blue light) (Cunliffe and Goulden,
2000; Goldberg and Russell, 2006; Lee et al., 2007b; Posten et al.,
2005; Sadick, 2008). One of these studies demonstrated significant
reduction in active acne lesions after 12 sessions of treatment using
630 nm red spectrum LLLT with fluence of 12 J/cm2 twice a week for
12 sessions in conjunction with 2% topical clindamycin. However,
the same study showed no significant effects when a 890 nm laser
was used (Aziz-Jalali et al., 2012). Other studies have shown that
the use of blue light and red light in combination with each other
resulted in synergistic effects in the treatment of acne (Goldberg and
Russell, 2006; Lee et al., 2007b; Papageorgiou et al., 2000; Sadick,
2008). It was proposed that synergistic effects of mixed light were
due to synergy between the antibacterial and anti-inflammatory
effect of blue and red light, respectively (Fig. 50.2) (Lee et al.,
2007b; Papageorgiou et al., 2000). Notably, in the majority of the
studies, improvements in inflammatory lesions were greater than
the improvements in comedones (Lee et al., 2007b; Papageorgiou
et al., 2000).
Additionally, fractional laser treatment can be used to treat post-
acne scars with the best results obtained in the treatment of macular,
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1026 Low-Level Laser (Light) Therapy for Cosmetics and Dermatology

superficial, and medium-depth scars. Deep scars and ice-pick scars

only show marginal improvement through the use of fractional laser
treatment, while severe scarring can be treated in combination with
other modalities such as chemical peels, surgical dermabrasion,
dermaroller, trichloroacetic acid chemical recreation of skin scars
(CROSS) techniques (Goel et al., 2011). Fractional laser treatment
provides a suitable means for addressing scars in individuals with
darker skin tones and has also shown pore improvement, which
is difficult to treat otherwise (Goel et al., 2011). Like with any
treatment modality, proper counseling and evaluation of effects
should be done to minimize adverse effects.

50.2.3 LLLT for Photoprotection

It is broadly accepted that UV radiation (spectral range <400 nm)
is responsible for most (if not all) of the photodamage inflicted on
skin by chronic exposure to sunlight. Proposed mechanisms for the
action of UV radiation include induction of free radical formation,
formation of DNA cross-links, inhibition of DNA repair, inhibition
and suppression of the immune system, and collagen degradation
(Sinha and Hader, 2002). Presently available treatments involve
reducing UV exposure of the skin by either avoiding sun exposure
or using sunscreens. The former option poses practical difficulties
especially for individuals engaged in outdoor activities, while the
photoprotective efficacy of topical sunscreens leaves a lot to be
desired. The limitations of using topical sunscreens include reduced
efficacy after water exposure or perspiration, spectral limitations,
possible toxicity of nanoparticles present in most sunscreens
(Kimura et al., 2012), compliance, and user allergies.
Recent studies are suggestive of the use of LLLT (specifically red
or NIR radiation) for protection against photodamage induced by UV
radiation. This was based on the fact that earlier on during the day
(morning), red/NIR wavelengths of the solar spectrum predominate
and prepare the skin for the potentially harmful UV radiation that
predominates later on in the day (noon/afternoon) (Barolet and
Boucher, 2008).
In a study conducted by Menezes et al., it was demonstrated
that a form of LLLT utilizing non-coherent NIR radiation (700–2000
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LLLT in Dermatology 1027

nm) was able to generate a strong cellular defense against solar

UV cytotoxicity in the absence of rising skin temperature and it
was assumed to be a long-lasting (at least 24 h) and cumulative
phenomenon (Menezes et al., 1998). Another study conducted
by Frank et al. proposed that NIR irradiation prepares cells to
resist UVB-induced damage by influencing intrinsic (mitochondrial)
apoptotic pathway (Frank et al., 2004). NIR irradiation of human
fibroblasts before subsequent UV exposure was shown to inhibit
UVB-induced activation of caspase-9 and -3, bring about partial
release of cytochrome c and Smac/Diablo, decrease pro-apoptotic
proteins (i.e., Bax), and increase anti-apoptotic proteins (i.e., Bcl-
2 or Bcl-xL) (Frank et al., 2004). The results were suggestive of
UVB-induced apoptosis being inhibited by NIR, which was most
likely brought about by modulating the Bcl2/Bax balance, suggesting
that p53, a sensor of gene integrity involved in cell apoptosis and
repair mechanisms, had an influential role in the process of NIR-
induced photoprotection against UVB radiation. A further study was
conducted, which examined the role of p53 in the cell signaling
pathway in the prevention of UVB toxicity (Frank et al., 2004).
The response to NIR irradiation was shown to be p53 dependent,
which further suggests that NIR irradiation prepares cells to resist
and/or to repair further UVB-induced DNA damage. Finally, the NIR
induction of defense mechanisms was supported by Applegate et al.
(2000), who reported that the protective protein ferritin normally
involved in skin repair (scavenger of ferrous iron otherwise available
for oxidative reactions) was induced by NIR radiation.
In an in vitro study, it has been reported that an increase in
dermal fibroblast procollagen secretion reduces MMP or collagenase
production after non-thermal, non-coherent deep red visible (close
to NIR) light exposures (660 nm, sequential pulsing mode) (Barolet
et al., 2009). These results correlated with significant clinical
improvement in rhytids in vivo (Barolet et al., 2009). In a subsequent
in vivo pilot study, the effect of 660 nm light on protecting the skin
from ultraviolet-light mediated damage was studied in three healthy
subjects using a minimal erythema dose (MED) method (similar
to that used for determination of sunscreen sun protection factor,
SPF) (Barolet and Boucher, 2008). The results displayed that LLLT
had a significant effect in reducing UVB-induced erythema (Barolet
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1028 Low-Level Laser (Light) Therapy for Cosmetics and Dermatology

and Boucher, 2008). Further investigations have been made to

understand the working of this phenomenon in vivo. Effects of non-
thermal, non-coherent 660 nm low-level light pulsed treatments
in enhancing skin resistance prior to subsequent UV damage were
investigated in a group of subjects with normal fair skin and patients
presenting polymorphous light eruption (PLE). Results were sugges-
tive of decreased UVB-induced erythema with the administration of
LLLT prior to UVB exposure. A noticeable reduction in UVB-induced
erythema reaction was observed for 85% of subjects as well as
in patients with PLE. Accompanying the reduced UVB erythema, a
supplemental SPF-15 like effect was observed along with a reduced
post-inflammatory hyperpigmentation response. Yu et al., in an in
vitro study, revealed that LLLT using an He–Ne laser stimulated
an increase in the gene expression of nerve growth factor (NGF),
a major paracrine maintenance factor responsible for melanocyte
survival in skin, as well as its release from cultured keratinocytes
(Yu et al., 2003). NGF has been shown to stimulate the upregulation
of BCL-2 in the cells and thus bring about photoprotection of
melanocytes from UV-induced apoptosis (Zhai et al., 1996). Given
the action of LLLT on NGF regulation, this may serve as an alternative
explanation for the photoprotective abilities of LLLT.

50.2.4 LLLT for Herpes Virus

Herpes simplex virus (HSV) infections are some of the most common
types of infections in the present age and are a bane to patients due
to the lifelong persistence of the virus within the host’s body. Two
types of viral strains are seen to infect humans: HSV-1 and HSV-
2. HSV-1 is primarily responsible for infections of mouth, throat,
face, eye, and central nervous system, while HSV-2 primarily causes
anogenital infections; however, each may cause infections in any
of the previously mentioned systems. Subsequently after initial
infection and resolution of lesions, the virus traverses across nerve
endings and establishes a state of latency in the sensory ganglia,
usually the trigeminal ganglia (de Paula Eduardo et al., 2011).
Several cues can trigger reactivation and migration of the virus to
skin and mucosa through sensory nerves, leading to the reactivation
of the virus especially on basal epithelium of the lips and perioral
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LLLT in Dermatology 1029

areas. These cues may be physical or emotional, such as fever,

exposure to UV light, and immune suppression (de Paula Eduardo
et al., 2011). The expression of the viral infestation can vary from
cold sores in immunocompetent individuals to severe complications
in immunocompromised patients. Up to 60% of all patients,
both immunocompetent and immunocompromised, experience a
prodromal period after which outbreaks develop through the stages
of erythema, papule, vesicle, ulcer, and crust, until finally healing is
achieved. This period is accompanied by pain, burning, itching, or
tingling at the site where blisters form causing great discomfort to
the patients. Immune responses to viral infection encompass action
of macrophages, Langerhans cells, natural killer cells, lymphocyte-
mediated delayed-type hypersensitivity and cytotoxicity (Whitley
et al., 1998).
Although a variety of antiviral drugs are available (e.g., acyclovir
and valacyclovir) for managing outbreaks, they can only operate
within a narrow time window to accomplish any patient benefit
at all, and even then only limited effects are observed in terms of
improving healing times of viral lesions (de Paula Eduardo et al.,
2011). Moreover, drug-resistant strains of the herpes virus pose
an ever-increasing risk especially to immunocompromised patients
(Whitley et al., 1998). Thus, new treatment modalities that can
reduce the frequency of recurrent episodes as well as address the
issue of undesirable effects associated with current treatments are
much needed.
LLLT serves as a suitable alternative to current treatment
technologies, providing for accelerated healing, reduced symptoms,
and affecting the recurrences of outbreaks (Bello-Silva et al., 2010;
de Paula Eduardo et al., 2011; Munoz Sanchez et al., 2012). When
applied to a group of 50 patients suffering from recurrent perioral
HSV infections, during an outbreak-free period, LLLT (wavelength
690 nm, intensity 80 mW/cm2 , and dosage 48 J/cm2 ) showed a
reduction in the recurrence frequency of herpes labialis episodes
(Schindl and Neumann, 1999). In another study with similar
irradiation parameters (intensity 50 mW/cm2 , fluence 4.5 J/cm2 ,
wavelength 647 nm), it was reported that the investigators were
able to significantly prolong intervals of remission from 30 to 73
days in patients with recurrent HSV infections (Landthaler et al.,
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1030 Low-Level Laser (Light) Therapy for Cosmetics and Dermatology

1983). Intriguingly, the treatment proved more effective in patients

suffering from herpes labialis than in those with genital infections.
LLLT treatment, however, did not affect established HSV latency in a
murine model (Perrin et al., 1997).
The mechanism of action of LLLT in inducing antiviral effects
is not known; however, it can be hypothesized that LLLT most
likely acts in an indirect manner, influencing cellular and humoral
components of the immune system as opposed to a mechanism
involving direct viral inactivation (Korner et al., 1989). In one
particular instance, a study conducted by Inoue et al. investigating
suppressed tuberculin reactions in guinea pigs suggested that the
application of LLLT through a low-power laser (fluence of 3.6
J/cm2 ) was responsible for a systemic inhibitory effect on delayed
hypersensitivity (Inoue et al., 1989b). Activation and proliferation of
lymphocytes (Inoue et al., 1989a; Manteifel et al., 1997; Schindl et
al., 1997; Yu et al., 1997) and macrophages (Bolton et al., 1990) as
well as the synthesis and expression of cytokines (Funk et al., 1992;
Yu et al., 1996) accompanying exposure to low intensities of red
and NIR light have been reported on several occasions by numerous
investigators. Whether or not any of these findings are influential
against HSV infection still remains enigmatic.

50.2.5 LLLT for Vitiligo

Vitiligo is an acquired pigmentary disorder characterized by the
depigmentation of skin and hair. The underlying mechanism of
how functional melanocytes are lost from the affected skin is
still under investigation; however, up-to-date findings suggest that
melanocytes, melanoblasts, keratinocytes, and fibroblasts may all be
involved in the repigmentation process of vitiligo (Kitamura et al.,
2004; Lan et al., 2009; Lan et al., 2006; Lee, 2012; Yu et al., 2003;
Yu et al., 2012). Thus, stimulation of such cells may be a possible
treatment method, but due to the obscure pathogenesis of the
condition, treatment outcomes have generally been unsatisfactory.
Topical corticosteroids, phototherapy, and photochemotherapy are
some current modalities that have shown varying degrees of
repigmentation in vitiligo patients (Lan et al., 2006). In 1982, a
group of investigators looked into the effect of LLLT on defective
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LLLT in Dermatology 1031

biosynthesis of catecholamines (involved in melanin biosynthesis)

and discovered that it was able to influence conditions such as
vitiligo and scleroderma (Mandel and Dunaeva, 1982; Mandel
et al., 1997). Later on, one of the investigators from the same
group reported that after 6–8 months of low-energy He–Ne laser
(632 nm, 25 mW/cm2 ) therapy, there was a noticeable degree of
repigmentation in 64% of the patients belonging to a group of
18 individuals, while in another 34% of the patients, follicular
repigmentation was observed (Mandel et al., 1997). Therefore, LLLT
has been suggested as a suitable alternative means for the treatment
of vitiligo (Lan et al., 2006; 2009; Yu et al., 2003).
A certain type of vitiligo (called segmental) is linked to
sympathetic nerve dysfunction in the afflicted areas of skin and has
proved to be resistant to several conventional treatment options (Yu
et al., 2003). Pre-existing studies report that LLLT improves nerve
injury (Anders et al., 1993; Khullar et al., 1996; Rochkind et al., 1989)
and also generates responses to promote repigmentation (Mandel,
1984; Yu, 2000). Thus, previously obtained data suggest that LLLT
may serve as a potential treatment modality for this treatment-
resistant form of vitiligo, i.e., segmental-type vitiligo. Upon local
administration of a low-powered He–Ne laser (3 J/cm2 , 1.0 mW,
632.8 nm), it was observed that 60% of the patients showed notice-
able perilesional and perifollicular repigmentation with successive
treatments. In the same study, upon irradiation of keratinocytes
and fibroblasts with an He–Ne laser (0.5–1.5 J/cm2 ), significant
increases in the amount of NGF released from keratinocytes were
observed. Significant increases in bFGF release from keratinocytes
and fibroblasts have also been reported (Lan et al., 2006). NGF and
bFGF are known to stimulate migration of melanocytes and may
contribute to the development of vitiligo (Peacocke et al., 1988; Wu
et al., 2006; Yu et al., 2012). Furthermore, the medium that had
been irradiated with the He–Ne laser along with keratinocytes led
to the stimulation of deoxythymidine uptake and proliferation of
cultured melanocytes. Finally, enhanced melanocyte migration was
observed, which is thought to have arisen either from the direct
action of the He–Ne laser or indirectly due to some effect induced
by the medium caused by laser exposure. Another study showed
that LLLT could lead to enhanced expression of α2β1 integrins
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1032 Low-Level Laser (Light) Therapy for Cosmetics and Dermatology

and stimulate melanocyte proliferation (Lan et al., 2009). Also LLLT

demonstrated the ability to induce melanocyte growth through
upregulated expression of phosphorylated cyclic-AMP response
element-binding protein (CREB), an important melanocyte growth
regulator (Lan et al., 2009). Components of the ECM also operate as
regulators of physiological functions such as morphology, migration,
tyrosinase activity, and proliferation of pigment cells and thus are
important to the pigmentation process (Hedley et al., 1997; Ma et al.,
2006; Morelli et al., 1993). Type IV collagen is an ECM component
present in the basement membranes of tissues and is known to
have intricate associations with melanocytes in the epidermis such
as promoting melanocyte mobility (Lan et al., 2006). LLLT has
been shown to greatly promote melanocyte attachment to Type
IV collagen and thus also demonstrates the modular capabilities
of LLLT regarding physiological functioning of melanocytes (Lan
et al., 2009). Fibronectin, among other ECM elements, has been
shown to have significant effects on differentiation and migration
of cultured melanoblasts and melanocytes (Ideta et al., 2002;
Takano et al., 2002). In an in vivo study conducted by Gibson
et al. (1983), it was demonstrated that the physical distribution
of fibronectin was closely associated with the migration path
undertaken by melanoblasts during the repigmentation process of
vitiligo. Based on the findings of Lan et al., a significant decrease
in fibronectin binding was displayed by immature melanoblast cell
lines (NCCmelb4), while a more differentiated melanoblast cell line
(NCCmelan5) showed an increase in attachment to fibronectin by
about 20% following LLLT (1 J/cm2 , 10 mW He–Ne laser) (Lan et al.,
2006). Lastly, expression of integrin α5β1 on NCCmelb4 cells was
observed to be enhanced, which is responsible for the regulation of
locomotion of pigmented cells (Lan et al., 2006).

50.2.6 LLLT for Reduction of Pigmented Lesions

Several studies especially for vitiligo show that LLLT exhibits
stimulatory effects on pigmentation. Despite these studies, which
are indicative of the pigmentation-promoting abilities of LLLT, one
of the aforementioned studies showed that the effects of blue light
(415 ± 5 nm, irradiance 40 mW/cm2 , 48 J/cm2 ) and red light (633
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LLLT in Dermatology 1033

± 6 nm, 80 mW/cm2 , 96 J/cm2 ) in combination yielded results

where an overall decrease in melanin was observed (Lee et al.,
2007b). Instrumental measurement results showed that melanin
level increased by 6.7 (the median of differences between before
and after one treatment session) after blue light irradiation without
a statistical significance (P -value > 0.1), whereas it decreased by
15.5 with a statistical significance (P -value < 0.005) after red light
irradiation. This finding could possibly be correlated to the ability
of the laser to brighten the skin tone of the irradiated area, which
was reported by 14 out of 24 subjects after the treatment period.
Up to now, however, no other studies have shown context suggestive
of similar effects of LLLT. Taking into account the differences in
parameters used for vitiligo and acne treatments, different effects
of LLLT on the same tissue could be attributed to the biphasic effects
of LLLT (Huang et al., 2009).

50.2.7 LLLT for Hypertrophic Scars and Keloids

Hypertrophic scars and keloids are benign fibrotic skin tumors
that usually arise following surgery, trauma, or acne and are
difficult to remove. Fibroblastic proliferation and excess collagen
deposition are the main characteristics of these lesions (Uitto
and Kouba, 2000), and imbalances between the rates of collagen
biosynthesis and degradation, superimposed on the individual’s
genetic predisposition, have been implicated in the pathogenesis
of keloids and hypertrophic scars. A broad spectrum of surgical
(e.g., cryotherapy and excision), non-surgical (e.g., pharmacological
administration, mechanical pressure, and silicone gel dressings),
and laser therapies (CO2 , pulsed dye, fractional ablative and non-
ablative lasers) have been tested with variable success; however,
until now, the optimal treatment of these lesions remains undefined
(Bouzari et al., 2007; Louw, 2007; Wolfram et al., 2009). It has been
recently suggested that poor regulation of the transforming growth
factor beta-I (TGF-βI) expression and interleukin 6 (IL-6) signaling
pathways has a significant role in this process, and thus inhibition
of IL-6 pathway and/or TGF-βI expression could potentially serve
as a therapeutic target (Bouzari et al., 2007; Ghazizadeh et al.,
2007; Liu et al., 2004; Uitto, 2007; Wolfram et al., 2009). Reports
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1034 Low-Level Laser (Light) Therapy for Cosmetics and Dermatology

indicating the effects of LLLT on reduction of IL-6 mRNA levels (Lee

et al., 2007a), modulation of platelet-derived growth factor (PDGF),
TGF-β, interleukins such as IL-13 and IL-15, MMPs, all of which
are associated with abnormal wound repair (Barolet and Boucher,
2010; Hamblin and Demidova, 2006), have led to the proposal of
LLLT as an alternative to the currently available therapeutic options.
A new promising treatment modality seems to be the utilization
of PDLs of wavelength 585 nm and is thought to act through the
induction of capillary destruction and alteration of local collagen
formation (Gauglitz, 2013). Moreover, PDL appears to stimulate
the upregulation of MMPs, which also helps to improve keloids
and hypertrophic scars (Gauglitz, 2013). Recommended protocols
call for non-overlapping pulse doses of fluences between 6.0 and
7.5 J/cm2 for 7 mm spots and between 4.5 and 5.5 J/cm2 for 10
mm spot; however, results definitively suggesting the efficacy of
PDL for clinical use are lacking (Gauglitz, 2013). PDL treatments
can present with mild side effects, generally purpura; however,
in certain instances reactivation of keloids has been observed
(Gauglitz, 2013). Also in some cases, prolonged hyperpigmentation
is observed particularly in the case of individuals possessing darker
pigmented skin but can be managed using low fluences. It is worth
mentioning here that the 1064 nm Nd:YAG laser has been suggested
as a means for improving hypertrophic scars and keloids as the
Nd:YAG laser has a greater penetration depth than PDL and would
allow for treatment of thicker scars, but may also be limited by the
decrease in efficacy with increases in depth (Gauglitz, 2013). The
use of LLLT as a prophylactic means in order to avoid and impair
the formation of hypertrophic scars or keloids has been investigated
by Barolet and Boucher. They conducted three different cases,
wherein a single scar was treated by the patient at home on a daily
basis with NIR-LED (805 nm at 30 mW/cm2 , 27 J/cm2 ), following
scar revision by surgery or CO2 laser ablation on bilateral areas
(Barolet and Boucher, 2010). The first patient had pre-auricular
linear bilateral keloids, and post-face lift procedure and surgical
scar revision/excision had been conducted. The second patient
had post-acne bilateral hypertrophic scars on the chest and CO2
laser resurfacing had been performed. For the third patient, CO2
laser resurfacing had also been used for post-excision hypertrophic
July 6, 2016 17:42 PSP Book - 9in x 6in 50-Hamblin-c50

LLLT in Dermatology 1035

bilateral scars on the back (Barolet and Boucher, 2010). Collectively

in these studies, it was observed that the NIR-LED treated scars
showed significant improvement over the control scars in all
measures of efficacy. Additionally, no adverse effects associated with
treatment were reported (Barolet and Boucher, 2010).

50.2.8 LLLT for Healing of Burns

In a clinical study by Weiss et al., 10 patients were treated with LLLT
(590 nm, 0.10 J/cm2 ) for acute sunburn, once or twice a day for 3
days, where only half of the affected area was treated (Weiss et al.,
2005c). Patients reported reduction in symptoms such as redness,
swelling, burning, and peeling. One patient was administered the
treatment twice a day for 3 days on one-half of the affected area
of the patient’s back, while the other half did not receive treatment
(Weiss et al., 2005c). Interestingly enough, the LLLT-treated site
exhibited a reduction in MMP-1, which had been detected with
immunofluorescence staining. Furthermore, the light-treated side
showed decreased MMP-1 gene activity both 4 and 24 h post-UV
injury when analyzed through reverse transcription–polymerase
chain reaction (RT-PCR) when compared to the untreated side.
Four days post-UV exposure, noticeable changes were also observed
associated with inflammation and dermal matrix composition upon
LLLT (Weiss et al., 2005c).
Burns associated with laser therapy pose a major obstacle for
treatment as they cause great discomfort to patients. Studies are
suggestive of the notion that utilizing LLLT as a treatment modality
facilitates faster healing. In one particular case, a group of patients
(n = 9) possessing a range of second-degree burns acquired from
exposure to non-ablative laser devices were administered with
LLLT once daily over the course of a week. According to reports
from patients as well as clinicians, healing was 50% faster (Weiss
et al., 2005c). In a pilot study conducted by the same group of
investigators, a forearm of the patient was intentionally injured on
two sides using a CO2 laser along with a computerized pattern
generator to induce identical effects. Then, both sites were followed
up with daily dressing changes using a nonstick dressing and
Polysporin ointment; however, one of the sites was also exposed
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1036 Low-Level Laser (Light) Therapy for Cosmetics and Dermatology

to LLLT (Weiss et al., 2005c). Results showed that the LLLT-treated

side expressed accelerated re-epithelialization in comparison to the
untreated side (Weiss et al., 2005c).
In a relevant study, Schlager et al. tested the effect of a low-
powered laser (670 nm, 250 mW, 2 J/cm2 ) on the healing of 30
rats that received burns on both their left and right flanks, where
one side received light treatment while the other side was left
untreated (Schlager et al., 2000). Both macroscopic and histological
evaluations of the treated wounds were conducted; however, neither
displayed results that were indicative of accelerated wound healing,
when compared with the control wound sites (Schlager et al., 2000).
In a study conducted by Ezzati et al., a sample size of 74 mice
was taken, where each animal was given two third-degree burns:
one proximal (control) and one distal (experimental). The mice
were divided into four groups. Group 1 mice received sham-LLLT on
distal burn with laser powered-off and thus served as the placebo
group. Groups 2 and 3 were administered a 3000 Hz pulsed infrared
diode laser treatment at the distal burn utilizing fluences of 2.3
J/cm2 and 11.7 J/cm2 , respectively. The final group (Group 4) was
administered with 0.2% nitrofurazone alone (Ezzati et al., 2009).
Microbiological examination of the LLLT treatment groups showed
significant decrease in the incidence of pathogenic organisms such
as Staphylococcus epidermidis, lactobacillus, and diphtheroids when
compared to the baseline (Ezzati et al., 2009). Furthermore, LLLT
showed enhanced tissue-healing ability when compared to the
baseline as well as the nitrofurazone group. The laser with fluence
of 2.3 J/cm2 showed better healing ability but mostly during the
early stages of healing of the burn. The most significant increases
in repair of injury were observed with the group that was treated at
a fluence of 11.7 J/cm2 (Ezzati et al., 2009). Several investigations
have proposed that LLLT serves as an effective treatment modality
for tissue repair through the stimulation of cellular interactions that
bring about repair. LLLT can cause stimulation of macrophages and
mast cells, which are responsible for the release of growth factors
and other mediators. Fibroblast, endothelial cell, and keratinocyte
proliferation maintained during adverse situations can also be
stimulated by means of LLLT (Ezzati et al., 2009).
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LLLT in Dermatology 1037

Burn scars pose a challenge for treatment as they progressively

worsen with hypertrophy and contracture. Thus, due to the lack of
treatment options, LLLT has been tested as a possible treatment
modality. In one particular study, a group of 19 patients possessing
burn scars were treated with a low-powered light source (400 mW,
670 nm, 4 J/cm2 ) twice a week over a course of 8 weeks. Following
treatment it was reported that scars showed increased softness and
pliability (Gaida et al., 2004). Reliefs from pain and pruritus as well
as occasionally improved scar patterns within mesh grafts were also
reported. These effects were sometimes limited, and thus complete
scar disappearances could not be expected. Also it is important
to notice that following treatment, better results were obtained in
scenarios where burn scars were not more than 12 months (Gaida
et al., 2004).

50.2.9 LLLT for Psoriasis

Psoriasis is a chronic, recurrent inflammatory skin disease affecting
about 1–3% of the population (Gelfand et al., 2005; Stern et al.,
2004). Its etiology is not entirely known; however, psoriasis is
known to result from the interactions between systemic, genetic,
immunological, and environmental factors (Zhang, 2012). The
condition presents with well-demarcated plaques formed as a result
of keratinocyte hyperproliferation, mediated by T-lymphocytes that
attack the skin (Griffiths and Barker, 2007).
The regions generally affected in psoriasis include knees, elbows,
scalp, nails, and lower back or sacrum. However, the body in its
entirety could be affected. The severity of the condition is gauged by
measuring the total body area involved or plaque severity. Variants
of psoriasis exist such as chronic plaque psoriasis or psoriasis
vulgaris (Griffiths and Barker, 2007), flexural (inverse) psoriasis
(Laws and Young, 2010; van de Kerkhof et al., 2007), guttate
psoriasis (Krishnamurthy et al., 2010), erythrodermic psoriasis
(Laws and Young, 2010), palmar-plantar psoriasis, facial psoriasis,
scalp psoriasis, and almost all variants result in great morbidity and
diminished quality of life (Finlay et al., 1990). Topical agent use,
systemic drug administration, photodynamic therapy, UV radiation,
and laser therapy are some of the treatment modalities that have
July 6, 2016 17:42 PSP Book - 9in x 6in 50-Hamblin-c50

1038 Low-Level Laser (Light) Therapy for Cosmetics and Dermatology

been evaluated for therapeutic use for psoriasis. Psoralen combined

with UVA and UVB phototherapy provided a revolutionary means
for the treatment of psoriasis when initially introduced. However,
some studies suggested that repeated and excess exposure to UVB
radiation elevated the risk of development of carcinoma of the skin.
Although psoralen + UVA (PUVA) provided a means of treatment
with reduced risk of skin carcinoma induction, it did not completely
eliminate the risk of cancer development. Studies investigating
the use of LLLT as a therapeutic modality, with CO2 ablative
laser (Bekassy and Astedt, 1985), helium–neon laser (Colver et al.,
1984), and red light photodynamic therapy can be dated to the
1980s (Berns et al., 1984). Laser treatment provides a variety of
advantages over conventional treatment modalities; it allows for
selective treatment of the lesion without affecting the surrounding
skin, while having limited or no systemic effects. It can also be
used in combination with other therapeutic options allowing for
more effective treatment of resistant lesions. Several studies were
conducted utilizing a selective excimer laser of 308 nm wavelength
(Asawanonda et al., 2000; Gattu et al., 2009; Trehan and Taylor,
2002). Laser therapy displayed results analogous to those observed
in UVB treatment. The laser treatment was effective in that it
prevented epidermal cell replication while also suppressing the
localized immune responses and thereby reducing the characteristic
inflammation observed in psoriasis (Railan and Alster, 2008).
However, uncertainty exists regarding the carcinogenic ability
of long-term excimer laser exposure. Thus, a PDL possessing a
wavelength of 585 nm was suggested as an alternative. PDLs are
commonly used for the treatment of vascular disorders and thus
have proven to be a legitimate treatment modality for psoriasis, due
to the association of increased vascularity with psoriasis (De Leeuw
et al., 2009; Ilknur et al., 2006). Furthermore, a recent preliminary
study investigated the efficacy of a combination of 830 nm (NIR)
and 630 nm (visible red light) to treat recalcitrant psoriasis using
an LED irradiation and has led to the consideration of LLLT for the
treatment of plaques associated with psoriasis. In the study, patients
that presented with psoriasis, resistant to conventional treatment,
were subjected to sequential treatments with 830 nm and 630 nm
wavelengths for two 20 min sessions, spaced 48 h apart for a total of
July 6, 2016 17:42 PSP Book - 9in x 6in 50-Hamblin-c50

References 1039

4 or 5 weeks. The results from the study did not display any adverse
effects; rather, they showed a resolution of psoriasis (Ablon, 2010).
The study was, however, limited by its small sample size, but the
results provided motivation for future investigations looking at the
use of LLLT as a possible treatment modality.

50.3 Conclusion

LLLT has been investigated as a novel therapeutic modality for the

treatment and management of several dermatological conditions.
The majority of the applications of LLLT has been for some form
of skin rejuvenation (reversal of chronic photodamage for the
most part). Several studies have demonstrated the use of LLLT for
photorejuvenation, treatment of acne, vitiligo, photoprotection, etc.,
and more recent studies demonstrate the potential LLLT possesses
for the treatment of alopecia, fat reduction, and cellulite. Moreover,
LLLT is a modality that is more patient friendly due to its noninvasive
nature with very mild side effects, if any at all. LLLT shows promise
for future applications being a novel treatment modality that also
works with great efficacy in combination with certain existing
options. With growing acceptance and extensive research in the
field of photomedicine, it can be proposed that LLLT among other
phototherapeutic modalities will continue to grow and emerge as a
versatile tool in the field of dermatology.

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and central nervous system, cutaneous wounds, and burns. Lasers Surg
Med, 9: 174–182.
Rotunda AM, Bhupathy AR, and Rohrer TE. 2004. The new age of acne
therapy: Light, lasers, and radiofrequency. J Cosmet Laser Ther, 6: 191–
200.
Russell BA, Kellett N, and Reilly LR. 2005. A study to determine the efficacy
of combination LED light therapy (633 nm and 830 nm) in facial skin
rejuvenation. J Cosmet Laser Ther 7: 196–200.
Sachdev M, Hameed S, and Mysore V. 2011. Nonablative lasers and nonlaser
systems in dermatology: Current status. Indian J Dermatol Venereol
Leprol, 77: 380–388.
Sadick NS. 2008. Handheld LED array device in the treatment of acne
vulgaris. J Drugs Dermatol, 7: 347–350.
Schindl A and Neumann R. 1999. Low-intensity laser therapy is an effective
treatment for recurrent herpes simplex infection. Results from a
randomized double-blind placebo-controlled study. J Invest Dermatol,
113: 221–223.
Schindl L, Schindl M, Polo L, Jori G, Perl S, and Schindl A. 1997. Effects
of low power laser-irradiation on differential blood count and body
temperature in endotoxin-preimmunized rabbits. Life Sci, 60: 1669–
1677.
Schlager A, Oehler K, Huebner KU, Schmuth M, and Spoetl L. 2000. Healing of
burns after treatment with 670-nanometer low-power laser light. Plast
Reconstr Surg, 105: 1635–1639.
Seaton ED, Mouser PE, Charakida A, Alam S, Seldon PM, and Chu AC. 2006.
Investigation of the mechanism of action of nonablative pulsed-dye
laser therapy in photorejuvenation and inflammatory acne vulgaris. Br
J Dermatol, 155: 748–755.
Sinha RP and Hader DP. 2002. UV-induced DNA damage and repair: A review.
Photochem Photobiol Sci, 1: 225–236.
Sriprachya-Anunt S, Fitzpatrick RE, Goldman MP, and Smith SR. 1997.
Infections complicating pulsed carbon dioxide laser resurfacing for
photoaged facial skin. Dermatol Surg, 23: 527–535; discussion 535–
536.
July 6, 2016 17:42 PSP Book - 9in x 6in 50-Hamblin-c50

References 1047

Stathakis V, Kilkenny M, and Marks R. 1997. Descriptive epidemiology of

acne vulgaris in the community. Australas J Dermatol, 38: 115–123.
Stern RS, Nijsten T, Feldman SR, Margolis DJ, and Rolstad T. 2004. Psoriasis
is common, carries a substantial burden even when not extensive,
and is associated with widespread treatment dissatisfaction. J Investig
Dermatol Symp Proc, 9: 136–139.
Takano N, Kawakami T, Kawa Y, Asano M, Watabe H, Ito M, Soma Y, Kubota
Y, and Mizoguchi M. 2002. Fibronectin combined with stem cell factor
plays an important role in melanocyte proliferation, differentiation and
migration in cultured mouse neural crest cells. Pigment Cell Res, 15:
192–200.
Takema Y, Yorimoto Y, Kawai M, and Imokawa G. 1994. Age-related changes
in the elastic properties and thickness of human facial skin. Br J
Dermatol, 131: 641–648.
Trehan M and Taylor CR. 2002. Medium-dose 308-nm excimer laser for the
treatment of psoriasis. J Am Acad Dermatol, 47: 701–708.
Uitto J. 2007. IL-6 signaling pathway in keloids: A target for pharmacologic
intervention? J Invest Dermatol, 127: 6–8.
Uitto J and Kouba D. 2000. Cytokine modulation of extracellular matrix gene
expression: Relevance to fibrotic skin diseases. J Dermatol Sci, 24 Suppl
1: S60–S69.
van de Kerkhof PC, Murphy GM, Austad J, Ljungberg A, Cambazard F, and
Duvold LB. 2007. Psoriasis of the face and flexures. J Dermatolog Treat,
18: 351–360.
Weiss RA, McDaniel DH, and Geronemus RG. 2003. Review of nonablative
photorejuvenation: Reversal of the aging effects of the sun and
environmental damage using laser and light sources. Semin Cutan Med
Surg, 22: 93–106.
Weiss RA, McDaniel DH, Geronemus RG, and Weiss MA. 2005a. Clinical trial
of a novel non-thermal LED array for reversal of photoaging: Clinical,
histologic, and surface profilometric results. Lasers Surg Med, 36: 85–
91.
Weiss RA, McDaniel DH, Geronemus RG, Weiss MA. 2005b. LED photomod-
ulation induced hair growth stimulation. Ann Meet Am Soc Laser Med
Surg, Orlando, Florida.
Weiss RA, McDaniel DH, Geronemus RG, Weiss MA, Beasley KL, Munavalli
GM, and Bellew SG. 2005c. Clinical experience with light-emitting diode
(LED) photomodulation. Dermatol Surg, 31: 1199–1205.
July 6, 2016 17:42 PSP Book - 9in x 6in 50-Hamblin-c50

1048 Low-Level Laser (Light) Therapy for Cosmetics and Dermatology

Weiss RA, McDaniel DH, Geronemus RG, Weiss MA, and Newman J. 2004.
Non-ablative, non-thermal light emitting diode (LED) phototherapy of
photoaged skin. Laser Surg Med, 16: 31.
Weiss RA, Weiss MA, Geronemus RG, and McDaniel DH. 2004. A novel
non-thermal non-ablative full panel LED photomodulation device for
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various skin types. J Drugs Dermatol, 3: 605–610.
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Clin Infect Dis, 26: 541–553; quiz 554–555.
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scars and keloids: A review of their pathophysiology, risk factors, and
therapeutic management. Dermatol Surg, 35: 171–181.
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on melanocytes. Acta Derm Venereol, 86: 498–502.
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Derma 35: 13–18.
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neon laser irradiation stimulates interleukin-1 alpha and interleukin-
8 release from cultured human keratinocytes. J Invest Dermatol, 107:
593–596.
Yu HS, Wu CS, Yu CL, Kao YH, and Chiou MH. 2003. Helium-neon laser
irradiation stimulates migration and proliferation in melanocytes and
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120: 56–64.
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16: 230–244.
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response to sepsis by photobiomodulation. Lasers Surg Med, 21: 262–
268.
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release of bFGF from 3T3 fibroblasts. Photochem Photobiol, 59: 167–
170.
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factor rescues pigment cells from ultraviolet-induced apoptosis by
upregulating BCL-2 levels. Exp Cell Res, 224: 335–343.
Zhang X. 2012. Genome-wide association study of skin complex diseases. J
Dermatol Sci, 66: 89–97.
July 6, 2016 17:42 PSP Book - 9in x 6in 51-Hamblin-c51

Chapter 51

Low-Level Laser Therapy for Body

Contouring and Fat Reduction

Gaurav K. Gupta
Department of Pathology and Laboratory Medicine, Tufts Medical Center,
Biewend Building 3, 800 Washington Street, Boston, MA 02111, USA
ggupta@tuftsmedicalcenter.org

51.1 Background

Low-level laser (or light) therapy (LLLT) is characterized by utiliza-

tion of photons to alter biological activity. Tissue chromophores such
as cytochrome c oxidase are excited by light within a certain range of
wavelength, thus bringing about a series of cellular events. Although
many potential mechanisms of action have been proposed for LLLT,
the largely accepted mechanism of action of LLLT is through the
activation of mitochondria. The components of electron transport
chain in the mitochondria serve as targets for the action of LLLT
(chromophores) and lead to mitochondrial activation and, in turn,
stimulation of ATP production. These events further lead to the
generation of reactive oxygen species (ROS) and release of nitric
oxide, which ultimately result in the stimulation of redox-sensitive
transcription factors and expression of gene products. With the

Handbook of Low-Level Laser Therapy

Edited by Michael R. Hamblin, Marcelo Victor Pires de Sousa, and Tanupriya Agrawal
Copyright c 2017 Pan Stanford Publishing Pte. Ltd.
ISBN 978-981-4669-60-3 (Hardcover), 978-981-4669-61-0 (eBook)
www.panstanford.com
July 6, 2016 17:42 PSP Book - 9in x 6in 51-Hamblin-c51

1050 Low-Level Laser Therapy for Body Contouring and Fat Reduction

cumulative effect of these events, LLLT results in the inhibition of

apoptosis, wound healing, tissue repair, and other long-term effects.
Non-thermal, coherent light sources (lasers) or non-coherent light
sources consisting of filtered lamps or light-emitting diodes (LED)
are used in this type of therapy. LLLT is being increasingly used
for the acceleration of tissue repair and regeneration, reduction of
inflammation and pain, and prevention of tissue damage (Chung et
al., 2012; Gupta et al., 2012). In this chapter, we will briefly discuss
the application of LLLT in fat reduction procedures and cellulite
treatment.

51.2 LLLT in Lipoplasty

The concept of lipoplasty or liposuction was first introduced in the

1920s by Charles Dujarrier, a French surgeon. Dujarrier attempted
to perform body sculpting on the knees of one of his patients,
a ballerina. However, the patient ended up developing gangrene,
leading to amputation of her affected limb, and thus the potential
use of lipoplasty suffered a major setback (Thorek, 1939). In 1974,
Dr. Giorgio Fischer and his son reintroduced liposuction, and they
innovatively utilized oscillating blades within a cannula to chisel
away subcutaneous fat (Fischer, 1990). In 1983, YG Illouz reported
his 5-year experience with a new liposuction technique that could
utilize relatively large cannulas along with suction tubing to securely
remove fat from several regions of the body (Illouz, 1983). This
ushered in the era of modern lipoplasty. Over the following decades,
the concept of tumescent liposuction allowed for better results and
decreased morbidity associated with liposuction. In 2000, Neira
et al. demonstrated the use of low-level laser as new means for
liposuction and successfully utilized it with doses that did not
produce any detectable increases in tissue temperature or cause
any noticeable macroscopic alterations in the tissue structure (Neira
et al., 2002). Prior investigations concerned with the effects of LLLT
on wound healing, pain relief, and edema prevention paved the way
for this therapeutic application (Baxter et al., 1991; King, 1989).
The development of LLLT as a therapeutic modality to augment
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LLLT in Lipoplasty 1051

liposuction while avoiding macroscopic tissue alterations was based

on the determination of optimal parameters such as wavelength
and power output for use (Oschman, 2000). Evidence suggests that
the best wavelengths suitable for biomodulation range between 630
and 640 nm (Al-Watban and Zhang, 1996; Fröhlich, 1968; 1970;
Frohlich, 1975; Sroka et al., 1997; van Breugel and Bar, 1992).
Neira et al. made several intriguing observations regarding the
effects of LLLT on adipocytes. They utilized low-level diode laser
(635 nm) and a maximal power of 10 mW with energy values
ranging from 1.2 to 3.6 J/cm2 (Neira et al., 2002). Using scanning
electron microscopy (SEM) and transmission electron microscopy
(TEM), it was demonstrated that adipocyte plasma membranes
exhibited transitory pore formation as a result of irradiation. It was
formulated that this enabled the release of intracellular lipids from
the adipocytes and thus supplemented the liposuction as it was
expected to reduce the time taken for the procedure, allowed for
extraction of greater volumes of fat, and overall reduced the energy
expenditure of the surgeon.
Although the findings associated with LLLT enjoyed much praise
and enthusiasm, an extensive study by Brown et al. (2004) put
these findings surrounding LLLT into question. In their study,
cultured human pre-adipocytes did not show any differences when
compared to non-irradiated cells after 60 min of irradiation using
an LLLT source (635 nm and fluence 1 J/cm2 ) (Brown et al., 2004).
Furthermore, histological examination of lipoaspirates in a porcine
model exposed to LLLT for 30 min and human lipoaspirates failed
to demonstrate transitory pores when analyzed using SEM (Brown
et al., 2004). Additional data raised questions regarding the ability
of red light (635 nm) to effectively penetrate the skin into the
sub-dermal tissues (Kolari and Airaksinen, 1993). Peter Foddor
supportively stated: “One could postulate that the presence of the
black dots on scanning electron microscopy images on the surface
of fat cells reported by Neira et al. could represent an artifact”
(Brown et al., 2004). Since the data reported by Brown et al. (2004),
there have been several publications reporting the efficacy of
LLLT.
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1052 Low-Level Laser Therapy for Body Contouring and Fat Reduction

51.3 LLLT in Cellulite Treatment

Cellulite is a condition observed in about 85% of post-pubertal

women, posing a major cosmetic concern. Women with the condi-
tion display orange-peel-like dimpling of the skin most commonly
in thighs and buttocks (Gold et al., 2011). The exact underlying
mechanism concerning the pathophysiology of cellulite is still being
looked into, but it is suspected that enlargement of adipocytes,
weakening of connective tissue, and a decrease in microcirculation
are possible triggering factors, helping to initiate the condition
(Gold et al., 2011). Several devices and topical treatment agents are
available for addressing the condition but are limited by their ability
to generate only temporary effects. Given the stimulatory effects
LLLT has been shown to have on circulation, collagen formation, and
fat reduction, it may serve as an alternative to the current treatment
options for cellulite. In a study conducted on 83 subjects possessing
mild to moderate cellulite, administration of a dual wavelength
(650 nm and 915 nm) laser in combination with a massage device
was carried out to test the efficacy of LLLT on cellulite (Gold
et al., 2011). The results demonstrated an improvement in cellulite
appearance as well as a 71% reduction in the circumference of
patient thighs that were treated when compared to a 53% reduction
in the circumference of thighs belonging to the control group
(Gold et al., 2011). Sasaki et al. used a topical phosphotidylcholine-
based anti-cellulite gel along with an LED array (660 nm and
950 nm) to test the effects of LLLT on cellulite reduction (Sasaki
et al., 2007). The results were intriguing as LLLT alone failed to
reduce cellulite, but in combination with a topical anti-cellulite
gel, LLLT was able to bring about cellulite reduction (Sasaki et al.,
2007). Eight out of nine patients were reported to experience
cellulite reduction in thighs when LLLT was used in association with
the anti-cellulite gel. Further clinical examinations, measurements,
and ultrasound evaluations displayed a noticeable reduction in
hypodermal thickness and supported the results already observed
(Sasaki et al., 2007). However, 18 months following treatment, it was
observed that five of the improved thighs reverted to their original
grade of cellulite and only three maintained their improved status.
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LLLT Mechanism of Action 1053

LLLT seems to be a promising alternative to other current

treatment options, especially when used in combination with other
methods. Current literature shows that it has almost no side
effects. However, more studies with a larger number of subjects are
necessary.

51.4 LLLT Mechanism of Action

In the original paper published by Neira et al., the fat-liberating

effects of LLLT on adipocytes were attributed to its ability to
induce transitory micropores, which were visualized with the help
of SEM (Neira et al., 2002). Furthermore, it was postulated that this
stimulated the release of intracellular lipids from the adipocytes.
Based on this, it was formulated that up to 99% of the fat stored
within the adipocytes could be released and subsequently removed
with the help of LLLT (635 nm, 10 mW intensity, 6 min irradiation
time) (Neira et al., 2002). Re-cultured adipocytes exhibited a
tendency to attain their native cellular conformation, which was
further confirmed by Caruso-Davis et al. (2011) utilizing a live–
dead assay to assess the viability of these adipocytes following
irradiation. Increase in ROS following LLLT has been proposed to
bring about lipid peroxidation within the cell membrane, which may
cause damage and may present as the transitory pores. This may
cause temporary damage that presents as transitory micropores.
(Chen et al., 2011; Geiger et al., 1995; Karu, 2008; Tafur and
Mills, 2008). However, when Brown et al. attempted to replicate
the findings of Neira et al. (2002), they failed to visualize any
transitory micropores via SEM (Brown et al., 2004). No further SEM
studies have documented these pores, but many publications have
reported findings that indirectly support the transitory micropore
formation theory. Another proposed mechanism that explains the
release of intracellular lipids from adipocytes suggests the activation
of the compliment cascade, which is responsible for the induction
of adipocyte apoptosis and subsequent release of intracellular lipid
components (Caruso-Davis et al., 2011). To test the feasibility of this
theory, Caruso-Davis et al. exposed differentiated human adipocytes
to plasma and exposed one group of cells to laser, while the control
July 6, 2016 17:42 PSP Book - 9in x 6in 51-Hamblin-c51

1054 Low-Level Laser Therapy for Body Contouring and Fat Reduction

group received no laser intervention (Caruso-Davis et al., 2011).

No differences were noticed with regard to compliment system
components in either group, and it was concluded that LLLT did not
act through the activation of the compliment cascade (Caruso-Davis
et al., 2011).

51.5 Future Directions

LLLT has been investigated as a novel therapeutic adjunct to

lipoplasty for fat reduction. It is a more patient-friendly modality
due to its noninvasive nature with very mild side effects, if any at all.
Additionally, LLLT can be used to facilitate autologous fat transplant.
Application of LLLT to the donor and recipient sites enhances
the viability of adipocytes (fat cells). Another potential application
is improved blood–lipid profiles following LLLT. Therefore, LLLT
shows promise for future applications being a novel treatment
modality that also works with great efficacy in combination with
certain existing options. With growing acceptance and extensive
research in the field of photomedicine, it can be proposed that LLLT,
among other phototherapeutic modalities, will continue to grow and
emerge as a versatile tool in the field of aesthetics.

References

Al-Watban, FAH and Zhang XY. Comparison of the effects of laser therapy
on wound healing using different laser wavelengths. Laser Ther, 1996;
8(2): 127–135.
Brown SA, Rohrich RJ, Kenkel J, Young VL, Hoopman J, Coimbra M. Effect
of low-level laser therapy on abdominal adipocytes before lipoplasty
procedures. Plast Reconstr Surg, 2004; 113(6): 1796–1804; discussion
805–806.
Baxter GD, Bell AJ, Allen JM, and Ravey J. Low level laser therapy: Current
clinical practice in Northern Ireland. Physiotherapy, 1991; 77: 171–178.
Caruso-Davis MK, Guillot TS, Podichetty VK, Mashtalir N, Dhurandhar
NV, Dubuisson O, et al. Efficacy of low-level laser therapy for body
contouring and spot fat reduction. Obes Surg, 2011; 21(6): 722–729.
July 6, 2016 17:42 PSP Book - 9in x 6in 51-Hamblin-c51

References 1055

Chen AC, Arany PR, Huang YY, Tomkinson EM, Sharma SK, Kharkwal GB,
Saleem T, Mooney D, Yull FE, Blackwell TS, and Hamblin MR. Low-level
laser therapy activates NF-kB via generation of reactive oxygen species
in mouse embryonic fibroblasts. PLoS ONE, 2011; 6: e22453.
Chung H, Dai T, Sharma SK, Huang YY, Carroll JD, and Hamblin MR. The nuts
and bolts of low-level laser (light) therapy. Ann Biomed Eng, 2012; 40:
516–533.
Fischer G. Liposculpture: The “correct” history of liposuction. Part I. J
Dermatol Surg Oncol, 1990; 16: 1087–1089.
Fröhlich H. Long-range coherence and energy storage in biological systems.
Int J Quantum Chem, 1968; 2(5): 641–649.
Fröhlich H. Long range coherence and the action of enzymes. Nature, 1970;
228: 1093.
Fukuda TY, Tanji MM, Silva SR, Sato MN, and Plapler H. Infrared low-level
diode laser on inflammatory process modulation in mice: Pro- and anti-
inflammatory cytokines. Lasers Med Sci, 2012 [Epub ahead of print].
Geiger PG, Korytowski W, and Girotti AW. Photodynamically generated 3-
beta-hydroxy-5 alpha-cholest-6-ene-5- hydroperoxide: toxic reactivity
in membranes and susceptibility to enzymatic detoxification. Pho-
tochem Photobiol, 1995; 62(3): 580–587.
Gold MH, Khatri KA, Hails K, Weiss RA, and Fournier N. Reduction in thigh
circumference and improvement in the appearance of cellulite with
dual-wavelength, low-level laser energy and massage. J Cosmet Laser
Ther, 2011; 13(1): 13–20.
Gupta A, Avci P, Sadasivam M, Chandran R, Parizotto N, Vecchio D, de Melo
WC, Dai T, Chiang LY, and Hamblin MR. Shining light on nanotechnology
to help repair and regeneration. Biotechnol Adv, 2012; 31(5): 607–631.
Illouz YG. Body contouring by lipolysis: A 5-year experience with over 3000
cases. Plast Reconstr Surg, 1983; 72: 591–597.
King PR. Low level laser therapy: A review. Laser Med Sci, 1989; 4(3): 141–
150.
Kolari PJ and Airaksinen O. Poor penetration of infra-red and helium neon
low power laser light into the dermal tissue. Acupunct Electrother Res,
1993; 18(1): 17–21.
Neira R, Arroyave J, Ramirez H, Ortiz CL, Solarte E, Sequeda F, and Gutierrez
MI. Fat liquefaction: Effect of low-level laser energy on adipose tissue.
Plast Reconstr Surg, 2002; 110(3): 912–922, discussion 923–925.
Oschman JL. Energy Medicine: The Scientific Basis. 1st ed., 2000. Edinburgh:
Churchill Livingstone.
July 6, 2016 17:42 PSP Book - 9in x 6in 51-Hamblin-c51

1056 Low-Level Laser Therapy for Body Contouring and Fat Reduction

Sasaki GH, Oberg K, Tucker B, and Gaston M. The effectiveness and safety
of topical PhotoActif phosphatidylcholine-based anti-cellulite gel and
LED (red and near-infrared) light on Grade II-III thigh cellulite: a
randomized, double-blinded study. J Cosmet Laser Ther, 2007; 9(2): 87–
96.
Thorek M. Plastic reconstruction of the female breasts and abdomen. Am J
Surg, 1939; 43(2): 268–278.
July 6, 2016 17:42 PSP Book - 9in x 6in 52-Hamblin-c52

Chapter 52

Transcranial Low-Level Laser (Light)

Therapy for Neurocognitive
Enhancement

Julio C. Rojasa and F. Gonzalez-Limab

a Department of Neurology, UCSF Memory and Aging Center, University of California,

1500 Owens Street # 320, San Francisco, California 94158, USA

b Department of Psychology, Institute for Neuroscience, University of Texas at Austin,

108 E. Dean Keeton Stop A8000, Austin, TX 78712-1043, USA

gonzalezlima@utexas.edu

52.1 Introduction

Noninvasive modulation of cognitive brain functions with transcra-

nial low-level laser (or light) therapy (LLLT) is a novel and appealing
neurotherapeutic concept with many potential applications [1]. It
is supported by the groundbreaking discoveries of the beneficial
transcranial effects of infrared laser stimulation on higher-order
cortical functions such as attention, memory, and affective states.
Transcranial LLLT relies on low power density (mW/cm2 ) and
high energy density (J/cm2 ) monochromatic light in the red
to near-infrared wavelengths to modulate brain function in a

Handbook of Low-Level Laser Therapy

Edited by Michael R. Hamblin, Marcelo Victor Pires de Sousa, and Tanupriya Agrawal
Copyright c 2017 Pan Stanford Publishing Pte. Ltd.
ISBN 978-981-4669-60-3 (Hardcover), 978-981-4669-61-0 (eBook)
www.panstanford.com
July 6, 2016 17:42 PSP Book - 9in x 6in 52-Hamblin-c52

1058 Transcranial Low-Level Laser (Light) Therapy for Neurocognitive Enhancement

nondestructive and nonthermal manner. The proposed molecular

mechanism of action of transcranial LLLT is based on its effect on
the predominant endogenous neuronal photoacceptor cytochrome
oxidase, the terminal enzyme in the mitochondrial respiratory chain.
This chapter presents evidence supporting our understanding of
the neurochemical, neurobiological, and brain network mechanisms
mediating observed cognitive effects in preclinical models and
humans. Primary neurochemical effects of transcranial LLLT are
defined as direct enzymatic effects occurring during effective
light stimulation that can be demonstrated in vitro and in vivo.
Secondary neurobiological LLLT effects are long-lasting biological
effects occurring in vivo after light stimulation. Brain network
mechanisms are higher-order neurophysiological effects produced
by the interaction of LLLT primary and secondary effects with
functional neuroanatomical networks that demand more metabolic
energy during a cognitive process. Key studies on LLLT-based
photoneuromodulation of mood and cognition as well as their
implications for future experimental and clinical applications are
also discussed.

52.2 Primary LLLT Neurochemical Effects: Cytochrome

Oxidase Effects on Oxygen and Nitric Oxide

Cytochrome oxidase (also called cytochrome c oxidase, complex IV,

or cytochrome aa3) in the mitochondrial respiratory chain is con-
sidered the main molecular target of transcranial LLLT. Cytochrome
oxidase catalyzes the synthesis of water in mitochondria, the main
oxygen-consuming reaction in neurons, and thus constitutes the
rate-limiting step for cell respiration [2]. The catalytic activity of
cytochrome oxidase is determined by the availability of substrate
(i.e., reduced cytochrome c and oxygen), the availability of the
functional holoenzyme, and the respiratory control exerted by
the transmembrane mitochondrial electrochemical potential, which
couples electron transfer with oxidative phosphorylation. Primary
LLLT effects consist of acceleration of electron transport by directly
modifying the catalytic rate of cytochrome oxidase by increasing the
availability of electrons [3]. The acceleration of electron transport is
July 6, 2016 17:42 PSP Book - 9in x 6in 52-Hamblin-c52

Primary LLLT Neurochemical Effects 1059

expected in systems with suboptimal electron transport, whereas no

effect is expected in conditions with maximal electron transfer rate,
such as those seen in uncoupling. This is supported by observations
that the absorption of red to near-infrared light by cytochrome
oxidase, and any subsequent photobiological effect, is low in the
fully reduced or fully oxidized forms of the enzyme. In contrast,
light absorption by cytochrome oxidase has been demonstrated to
be maximized in the presence of a mixed valence enzyme state
(i.e., partially reduced or oxidized) [4]. The probability of finding a
mixed valence enzyme is higher in highly coupled systems, which
reflect a state of increased energy consumption. This concept has
been tested in whole-brain homogenates that emulate uncoupled
conditions. As expected, the rate of oxygen consumption is not
affected by LLLT in whole-brain homogenates. In contrast, the rate
of oxygen consumption is significantly decreased by the addition of
the mitochondrial complex I inhibitor rotenone to the whole-brain
membrane isolates. It is in these conditions of decreased electron
flow that LLLT is able to accelerate the rate of oxygen consumption
similar to control levels [5]. Evidence of transcranial enhancement
of oxygen consumption by LLLT was provided by measurements of
the in vivo changes in oxygen concentration in the cerebral cortex
of naive rats using fluorescent-quenching oxymetry immediately
following LLLT exposure. The cortical oxygen concentration in
control conditions (i.e., following no light exposure) decreased only
1 ± 0.7%. In contrast, LLLT induced a dose-dependent decrease in
the in situ oxygen concentration of approximately 5 ± 1% after LLLT
1 J/cm2 and 15.8 ± 2% after LLLT 5 J/cm2 . These data support
a clear physiological effect of LLLT on the rate of cortical oxygen
consumption in vivo [6].
In addition, photostimulation of cytochrome oxidase activity not
only increases oxygen consumption in the brain but also leads to
the release of nitric oxide (NO). NO may be produced by LLLT in
two ways related to the photostimulation of cytochrome oxidase
activity: (1) NO may be briefly released from cytochrome oxidase
catalytic sites when the enzyme is first photostimulated; (2) NO may
be produced by cytochrome oxidase activity itself when the oxygen
level falls. Cytochrome oxidase acts to catalyze both the formation of
water at high oxygen levels and the formation of NO from nitrates at
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1060 Transcranial Low-Level Laser (Light) Therapy for Neurocognitive Enhancement

low oxygen levels. This dual enzymatic role of cytochrome oxidase is

based on the biochemical research of Poyton et al. [7]. In both cases,
the primary neurochemical effects of transcranial LLLT depend
on the direct photostimulation of cytochrome oxidase enzymatic
activity. These primary LLLT neurochemical effects can be observed
in vitro, whereas the secondary neurobiological effects occur
in vivo.

52.3 Secondary LLLT Neurobiological Effects: Cytochrome

Oxidase Induction and Cerebral Hemodynamic
Response

The expression of cytochrome oxidase is exquisitely regulated by

the neuronal energetic demands, and it is known that cytochrome
oxidase is more abundant in neurons with high energetic demands.
Neuronal membrane depolarization and glutamatergic receptor ac-
tivation have been shown to induce the transcription of cytochrome
oxidase subunits, an effect that is functionally evident as an increase
in cytochrome oxidase activity [8–11]. Thus, given its central role in
cell respiration, cytochrome oxidase expression is a sensitive marker
of neuronal activity [12, 13]. Photons delivered by LLLT not only
accelerate the catalytic activity of cytochrome oxidase and the rate
of oxygen consumption, but also induce a plethora of secondary
cellular effects. An important effect of LLLT is increasing brain ATP
production, which results from the transduction of photon energy
into ATP-based metabolic energy via the mitochondrial process of
oxidative phosphorylation [14]. Secondary LLLT effects on neural
tissue have been postulated to include long-lasting neurobiological
changes that persist beyond the period of light exposure and
translate into changes in neuronal physiology [2, 3]. These changes
include activation of second messenger cascades and gene expres-
sion that impact, among other things, the metabolic capacity of the
neuron, which is reflected by the expression of cytochrome oxidase
itself. Whereas these secondary effects are remarkably pleiotropic,
they are related to both the parameters of light irradiation ideal
to induce a photoneurobiological effect and the connection of
cytochrome oxidase with numerous cellular pathways beyond cell
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Secondary LLLT Neurobiological Effects 1061

respiration, including gene expression, housekeeping processes and

cell survival [15].
Evidence of secondary mechanisms induced by transcranial LLLT
in vivo has been provided by the quantification of changes in the
cortical cytochrome oxidase activity 24 h after exposure to LLLT
in naive rats. In a regional brain analysis, the mean prefrontal
cortex cytochrome oxidase activity in subjects treated with LLLT
10.9 J/cm2 was 13% higher compared to no-treatment control
subjects. Higher LLLT doses of 21.6 J/cm2 and 32.9 J/cm2 were
less effective at inducing neural cytochrome oxidase activity, which
was consistent with the hormetic dose-response effects typically
associated with LLLT [6]. Dose-response effects have also been
observed in whole-brain levels of cytochrome oxidase activity after
transcranial LLLT irradiation. Compared with untreated subjects,
a dose-dependent increase in whole-brain cytochrome oxidase
activity of 14.8% and 66.6% was observed in subjects treated with
fractionated transcranial LLLT for total doses of 10.8 J/cm2 and 21.6
J/cm2 , respectively [5]. Finally, further evidence of in vivo secondary
transcranial LLLT effects in a dose-response manner was obtained
analyzing the whole-brain cytosolic and mitochondrial superoxide
dismutase (SOD) activity of subjects treated with LLLT. A dose
of 21.6 J/cm2 induced a 51.5% increase in cytosolic SOD activity,
relative to untreated controls [5].
LLLT has also been shown to affect the allosteric interactions
of NO with cytochrome oxidase and hemoglobin [2, 4, 14, 16,
17]. It is hypothesized that LLLT induces dissociation of NO from
these proteins, thus facilitating cell respiration. In line with this
hypothesis, it has been shown that LLLT augments the bioavailability
of NO as quantified by changes in the metabolic byproducts of
NO in venous blood draining from LLLT-treated tissues in humans
[18]. Peak increases in NO occur after 5 min of treatment with a
subsequent decrease. Local increase in NO leads to vasodilation and
a hemodynamic response observed as enhanced cerebral blood flow
[19].
In neurons, the activity-dependent effects of transcranial LLLT
are hypothesized to take place on both presynaptic and postsynaptic
mitochondria. LLLT effects are predicted to preferentially occur
in neuronal terminals within active networks with high energy
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1062 Transcranial Low-Level Laser (Light) Therapy for Neurocognitive Enhancement

Figure 52.1 Activity-dependent primary and secondary neural effects of

LLLT. (A) Light stimulates presynaptic and postsynaptic mitochondria
(Mit), preferentially in active neurons displaying high oxidative metabolic
rates. Whereas the synapsis at the left is at rest and shows no response
to LLLT, the synapsis at the right is active and engages mitochondrial
energy consumption, making it more susceptible to photoneuromodulation.
This activity-dependent enhancing effect is explained by the ability
of cytochrome oxidase in active neurons to act as a more efficient
photoacceptor than in neurons at rest. (B) LLLT directly photostimulates
cytochrome oxidase, thus increasing the rates of oxygen consumption in the
mitochondrial electron transport chain (ETC), and triggering downstream
biochemical pathways that promote synaptic strengthening. Nitric oxide
synthesis (NOS) may also be modulated by LLLT. NOS activation is coupled
to that of excitatory glutamatergic receptors (AMPAR and NMDAR) to
produce NO, which acts as a second messenger and, among other actions
such as vasodilation, regulates cytochrome oxidase activity, thus exerting a
strong influence on respiration. Metabolically active mitochondria induce
upregulation of subunit genes for cytochrome oxidase (COX) nitric oxide
synthase (Nos1), NMDA receptor (Grin 1 and Grin 2b), and AMPA receptor
(Gria2). Energy-consumption-related gene expression is regulated by
the nuclear respiratory transcription factor NRF-1. (C) These secondary
LLLT effects occur in both presynaptic and postsynaptic nerve terminals,
thus supporting Hebbian processes for synaptic strengthening underlying
cognitive enhancement.

demands and active mitochondria. Again, the generalized but, at

the same time, activity-dependent enhancing effect of LLLT is
explained by the excitability of mixed valence cytochrome oxidase,
which is predicted to predominate in neural terminals with high
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Brain Network Mechanisms of LLLT Relevant to Cognitive Function 1063

energy-consumption rates. While LLLT enhances the activity of the

electron transport chain, it also modulates NO synthesis (NOS).
NOS is normally activated by glutamatergic receptors (AMPAR and
NMDAR) to produce NO, which acts as a second messenger and,
among other actions, regulates cytochrome oxidase activity, further
modulating cell respiration. Metabolically active mitochondria com-
municate with the nuclear and mitochondrial genome (red arrows
in Fig. 52.1) to upregulate all cytochrome oxidase (COX) and some
nitric oxide synthase (Nos1), NMDA receptor (Grin 1 and Grin 2b),
and AMPA receptor (Gria2) subunit genes, all under the influence
of the nuclear respiratory transcription factor NRF-1. Thus, gene
expression orchestrated by NRF-1 allows for co-localization of
proteins critical for energy metabolism and synaptic transmission
that aid in synaptic strengthening and improved cognitive function
[8–10].

52.4 Brain Network Mechanisms of LLLT Relevant to

Cognitive Function

Higher-order cortical functions such as attention and memory

are distributed network functions. Functional neuroimaging has
advanced our understanding of brain function and the network
nature of neuroanatomical organization. The functional relevance of
a brain region depends on the functional status of other connected
regions (i.e., the context within which the region is operating) [20]. A
brain region can participate in several behaviors through variations
in its interactions with other areas. Certain regions within a network
are critical because of their role at enabling shifts in functional
connectivity between one set of regions to another. The changes in
metabolic activity in these key brain regions make them important
targets for LLLT-mediated network photoneuromodulation. LLLT
potentially reaches all cortical regions but will selectively enhance
those with higher metabolic demands because of task-dependent
activation. Through the enhancement of regional metabolic capacity,
facilitation of cognitive processes is expected in normal individ-
uals. Furthermore, in pathologic conditions where selective brain
vulnerability impairs normal brain function, LLLT is expected to
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1064 Transcranial Low-Level Laser (Light) Therapy for Neurocognitive Enhancement

Figure 52.2 Theoretical network model of functional neuroanatomical

selectivity of transcranial LLLT. By reaching all the nodes constituting
a neural network, the effects of LLLT are potentially global. Nodes
containing neurons with the highest metabolic rate and thus the highest
energy demand (black nodes) will display the highest cellular benefits
from light energy irradiation. This occurs because cytochrome oxidase
molecules present in neurons within these highly active nodes are highly
effective photoacceptors due to their mixed valence state. Nodes with
intermediate metabolic demand (gray nodes) are less susceptible to
photoneuromodulation, whereas nodes in the resting state (white nodes)
are not altered, as cytochrome oxidase molecules are less frequently
removed from the fully reduced or fully oxidized state. In an activational
neural context A, a given cognitive task has engaged primarily nodes A, B,
and C, which show the highest metabolic rate and also experience the major
benefit from LLLT. In contrast, activational neural context B represents
a different cognitive task and has engaged a different set of nodes: A,
D, and E. This sub-network is now the one that is more amenable to
photoneuromodulation.

modulate the tendencies of subregions to act as either functional

clusters or independent functional nodes. This internal subregional
reconfiguration may allow for the recruitment of additional brain
regions that can compensate for faulty connectivity and mediate an
appropriate behavioral output. These network effects could occur in
addition to direct local neuroprotective effects [21].
Network selectivity represents an important mechanistic insight
that has been tested in vivo. Evidence that the effects of LLLT and
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Dosimetry Parameters Relevant for Transcranial LLLT and Cognitive Enhancement 1065

other metabolic enhancers such as methylene blue (MB) display

brain region activational specificity has been provided by studies of
facilitation of conditioned fear extinction in rats [6, 22]. LLLT given
during the period of memory consolidation induced facilitation of
fear extinction memory, which is known to be mediated by increased
metabolic activity in the prefrontal cortex. When in situ oxygen
consumption and cytochrome oxidase activity were measured in
the prefrontal cortex, subjects treated with LLLT showed increases
in both parameters compared to untreated controls [6]. Similarly,
MB given during the memory consolidation phase of fear extinction
induced selective increases in cytochrome oxidase activity in the
prefrontal cortex. MB has been described as a “magic bullet” as
it concentrates in compartments with high redox activity [22, 23].
Due to its affinity for active oxidoreductases, MB has remarkable
bioavailability to mitochondria with high rates of electron transfer.
Thus, both treatments (LLLT and MB) may reach the totality
of the brain, but only those areas that show higher oxidative
metabolism will maximally benefit from the enhancing effects of
these interventions.

52.5 Dosimetry Parameters Relevant for Transcranial LLLT

and Cognitive Enhancement

The brain is a unique LLLT target, given its intrinsic metabolic

heterogeneity. Thus, the metabolic state of the brain and, more
specifically, the neural context of particular target regions are
expected to impose major restrictions on the responses to LLLT.
Light transmission through tissues is a critical aspect of transcranial
applications of LLLT. The delivery modality of transcranial LLLT with
light-emitting diodes (LEDs) is relevant as contact modalities (i.e.,
applying the light source directly in contact with the skin) will,
in general, offer higher transmittance than non-contact modalities
(i.e., allowing space between the probe and the skin) [24]. Non-
contact delivery modalities, however, allow exposure of extensive
surfaces. LED helmet montages and arrays can be built with
ergonomic considerations for whole-head LLLT. In contrast, laser
sources may be ideal when localized energy delivery is needed. This
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1066 Transcranial Low-Level Laser (Light) Therapy for Neurocognitive Enhancement

Figure 52.3 Transcranial LLLT for cognitive enhancement. Red to near-

infrared light energy offers optimal brain effects due to its higher
transmittance through biological tissues and the affinity of chromophores
in cytochrome oxidase for this specific wavelength range. Low doses in
several fractions are ideal to achieve the desired level of cortical tissue
excitation while minimizing ablative side effects. Cognitive and behavioral
effects have been documented with transcranial forehead contact delivery.
The primary acceptor of light in neural tissue is cytochrome oxidase,
the terminal enzyme of the mitochondrial respiratory chain. Cytochrome
oxidase photostimulation induces acceleration of mitochondrial respiration
(oxygen consumption and nitric oxide release), i.e., primary neurochemical
effects, and a series of secondary neurobiological reactions that lead to
gene expression, blood flow, synaptic strengthening, and improvement in
functional connectivity. Cognitive enhancement is expected when LLLT is
paired with a cognitive task, which allows for a neural context susceptible
to photoneuromodulation.

may be advantageous for boosting cell functions in specific nodes

within dysfunctional neural networks in which connectivity could
be otherwise impaired with broad irradiation.
For transcranial purposes, penetration of light into tissues is also
governed by Beer’s law and is thus contingent upon the properties
of the scalp–bone–dura–brain target sequence and the desired
penetration in the brain [24]. As light travels into tissue, its intensity
decreases because of absorption and scattering. Wavelength is an
important dosimetry factor as red to near-infrared wavelengths
have higher capacity to penetrate tissue. Photons at wavelengths
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Dosimetry Parameters Relevant for Transcranial LLLT and Cognitive Enhancement 1067

between 630 and 800 nm can travel up to 28 mm even in tissue

layers with relatively low transparencies such as skin, connective
tissue, muscle, bone, and spinal cord. The cranium shows absorption
in the red to near-infrared spectrum [25]. Post-mortem human
specimens have helped estimating LLLT transmittance through the
skull. Incident light (I0 ) and transmitted light (I ) are measured with
the tissue directly overlying the aperture of the detector; average
readings are then used to calculate percent transmittance (k) as
100 × I /I0 . Optical density (OD) is calculated as −log (k) [15]. The
cross-sectional width of each set of tissues may be measured with
calipers, and Beer’s law is then applied to calculate the absorption
coefficient (a = OD/width). Using this method, approximately 2% of
1064 nm laser light at 250 mW/cm2 , 60 J/cm2 passes through the
adult human supraorbital frontal bone. This yields an OD of 1.70
and an absorption coefficient of 0.24. This is consistent with the
reported values of transmittance of this wavelength through cranial
human bone of a = 0.22 [26, 27]. Following a similar approach, 5.8%
infrared laser light transmittance through the rat scalp (shaved) plus
skull has been calculated, equivalent to OD = 1.24 and a = 0.35 [6].
In addition, as tissue thickness increases and when bones and an
active vascular supply are present, light penetration decreases but
remains quantifiable when near-infrared light is used [28].
Unique optical properties of the brain are also relevant for
deciding targets for transcranial stimulation. High infrared laser
transmittance has been documented across the entire rat brain
(from dorsal to ventral surface) at 8.5%, corresponding to OD = 1.07
and a = 0.20. Nevertheless, white matter in both the central and
peripheral nervous systems reflects most of the incident power and
shows a low level of absorption and a short penetration depth [29].
In contrast, the transmittance of gray matter is approximately twice
as high as that of white matter. Yet, in gray matter, the optical power
using red to near-infrared light decays up to 80% at 1 mm from
the surface [30]. In solid organs, however, the actual power density
of near-infrared light is estimated to be up to three times higher
than the power at the incident surface because of backscattering and
constructive interference [31].
Besides wavelength, fluency and power density are impor-
tant dosimetry parameters influencing dose-response curves for
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1068 Transcranial Low-Level Laser (Light) Therapy for Neurocognitive Enhancement

transcranial applications. The high number of potential combi-

nations of such parameters may imply that finding the right
combination needed for a particular cognitive effect is a laborious
enterprise. However, achieving a particular fluency in a particular
target should be an operational goal for dose-response studies.
At the end, the desired cognitive effect is the ultimate goal,
but it is known that transcranial LLLT given at different energy
densities still exerts beneficial effects. LLLT has not been associated
with neurohistological or behavioral adverse effects. Preclinical
data using LLLT to stimulate the brain in vivo support that it
is safe. Adverse behavioral effects can be induced with massive
energy densities 100 times higher than those observed to induce
beneficial effects, but even the adverse effects of high doses can be
attenuated if the total energy is delivered using intermittent energy
pulses [32].

52.6 Cognitive Effects of Transcranial LLLT

Among the most fascinating applications of LLLT are those related

to the enhancement of normal brain function and the treatment
of memory loss and mood disorders. This remains, nonetheless,
a largely unexplored research area with enormous clinical po-
tential. Cognitive impairment and neurodegeneration associated
with dementia have been shown to have regional brain metabolic
deficits in early disease stages. For example, early decreases in
brain metabolic activity can be detected in patients at risk of
developing Alzheimer’s disease, especially reductions in cytochrome
oxidase activity [33, 34]. Similarly, the phenotypic expression of
disorders such as major depression and post-traumatic stress
disorder has been associated with decreased metabolic capacity in
prefrontal brain regions [35], and electrical stimulation of prefrontal
cortex has antidepressant effects [36]. LLLT is expected to enhance
metabolic capacity in those regions showing functional deficits, thus
increasing the functional connectivity of the networks involved in
the expression of a particular phenotype. This is in addition to the
neuroprotective effects that LLLT may have in tissue susceptible to
neurodegeneration.
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Cognitive Effects of Transcranial LLLT 1069

Preclinical studies support the potential of transcranial LLLT

for the modulation of cognitive functions. An early demonstration
of improvement in working memory by LLLT in middle-aged mice
tested in an appetitive spatial navigation task [37] occurred in the
absence of nonspecific effects on exploratory activity or anxiety
responses. This study used LLLT at 1072 nm given in 10 fractions,
one fraction per day. Mice treated with LLLT showed higher
latencies to make a choice, but with improved correct choice rates.
Indeed the memory performance of LLLT-treated middle-aged mice
became comparable to that of young mice. These data support that
LLLT could be useful at facilitating the acquisition of a working
memory task in conditions in which information processing speed
is impaired, such as aging.
Transcranial LLLT has also been shown to modulate non-
declarative memory in normal young rats. LLLT improved fear
extinction memory after fractionated LLLT at 660 nm, 9 mW/cm2 ,
5.4 J/cm2 . Fear extinction memory is a form of memory modulated
by prefrontal cortex activation. Extinction is a type of associative
learning in which conditioned behavior extinguishes after repeated
presentations of a conditioned stimulus, and it is clinically relevant
because it constitutes the basis for exposure therapy of phobias
and post-traumatic stress disorder [38]. LLLT-treated rats showed
enhanced extinction memory as compared to controls, and LLLT re-
duced fear renewal by preventing the reemergence of extinguished
conditioned fear responses (reduced freezing behavior in Fig. 52.4)
[6].
Rodent [39] and human [40] studies also support that LLLT may
be effective in the treatment of cognitive and emotional disorders.
A pilot clinical study showed that LLLT applied to the forehead
was able to increase frontal cortex blood flow and induce a 63%
reduction in depression scores in 10 patients with major depression.
Beneficial antidepressant effects were seen 2 and 4 weeks after LLLT
given in one fraction at 810 nm, 250 mW/cm2 [40]. Additionally,
LLLT to the forehead and scalp with an LED cluster at 633 nm and
870 nm improved attention, executive function, and memory in two
patients with chronic traumatic brain injury [41]. LLLT with a power
density of 22.2 mW/cm2 was applied for 10 min per placement
weekly for 2 months or daily for 4 months prior to the testing of
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1070 Transcranial Low-Level Laser (Light) Therapy for Neurocognitive Enhancement

Figure 52.4 LLLT improves memory. Subjects that learned an association

between a tone and shock and exhibited high levels of fear-evoked
behavioral freezing (immobilization) upon tone presentation underwent
four extinction training sessions in a new environment to promote
fear extinction. One day later, subjects were introduced to the original
acquisition environment and underwent tone presentation to induce fear
renewal. Subjects that received LLLT after extinction training showed a
50% reduction in freezing behavior (reduced fear), compared to untreated
controls. This supported the enhancement of fear extinction memory by
LLLT. * = p < .05.

cognitive effects, and these patients have continued daily treatment

at home for up to 5.5 years.
Barrett and Gonzalez-Lima [42] conducted the first controlled
study demonstrating the beneficial effects of transcranial laser
stimulation on cognitive and emotional functions in healthy human
volunteers. Transcranial laser stimulation to the forehead was used
in a placebo-controlled, randomized study to influence cognitive
tasks related to the prefrontal cortex, including a psychomotor
vigilance task and a delayed match-to-sample memory task [42].
LLLT either improved or protected against deterioration, in a
number of behaviors and self-report measures linked to the
functioning of the prefrontal cortex, including reaction time in
a psychomotor vigilance task of sustained attention (Fig. 52.5),
July 6, 2016 17:42 PSP Book - 9in x 6in 52-Hamblin-c52

Conclusion 1071

Figure 52.5 LLLT improves attention. LLLT-treated human subjects dis-

played a decreased reaction time to make a behavioral response compared
to pretreatment conditions in a psychomotor vigilance task. In contrast, a
reduction in reaction time was not observed in placebo-treated controls.
Significant treatment by pre-post score interaction, * = p < .05.

memory retrieval latency, and correct match-to-sample memory

trials and positive emotional states.

52.7 Conclusion

Improvement in brain mitochondrial respiration with transcranial

LLLT constitutes a promising neurotherapeutic principle for brain
enhancement in healthy and diseased individuals. It is also an
important future direction in the research and treatment of condi-
tions associated with cognitive impairment, neurodegeneration, and
psychopathology. Transcranial absorption of photon energy by cy-
tochrome oxidase is proposed as a unique bioenergetics mechanism
underlying LLLT, an intervention with major mechanistic relevance
for the enhancement of cognitive function. Transcranial LLLT
upregulates cortical cytochrome oxidase and enhances oxidative
metabolic capacity in the brain. LLLT improves prefrontal cortex-
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1072 Transcranial Low-Level Laser (Light) Therapy for Neurocognitive Enhancement

related cognitive functions, such as sustained attention, extinction

memory, working memory, and affective state. This fascinating
new intervention may be used to improve neuronal mitochondrial
respiration as a new noninvasive, cognitive-enhancing intervention.
Transcranial LLLT should be able to improve other brain functions
depending on the stimulation parameters used and the functional
state of the stimulated neuroanatomical networks.

Acknowledgements

We gratefully acknowledge the personnel of the Gonzalez-Lima lab,

the lasers supplied by Cell Gen Therapeutics (HD Laser Center,
Dallas, Texas), and the LED arrays built by Frank Lima.

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30. Abdo, A., and Sahin, M. (2007). NIR light penetration depth in the rat
peripheral nerve and brain cortex, Conf Proc IEEE Eng Med Biol Soc,
2007, pp. 1723–1725.
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31. Grimvlatov, V., Rubinshtein, A., and Rubinshtein, M. (2006). Spectral

dosimetry in low light therapy, Proc of SPIE, 6140, pp. S1–S11.
32. Ilic, S., Leichliter, S., Streeter, J., Oron, A., DeTaboada, L., and Oron, U.
(2006). Effects of power densities, continuous and pulse frequencies,
and number of sessions of low-level laser therapy on intact rat brain,
Photomed Laser Surg, 24, pp. 458–466.
33. Gonzalez-Lima, F., Valla, J., and Jorandby, L. (1998). Cytochrome oxidase
inhibition in Alzheimer’s disease. In: Gonzalez-Lima, F. (ed), Cytochrome
Oxidase in Neuronal Metabolism and Alzheimer’s Disease. New York:
Plenum Press, pp. 171–202.
34. Valla, J., Berndt, J.D., and Gonzalez-Lima, F. (2001). Energy hypo-
metabolism in posterior cingulate cortex of Alzheimer’s patients:
Superficial laminar cytochrome oxidase associated with disease dura-
tion. J Neurosci, 21, pp. 4923–4930.
35. Tian, F., Yennu, A., Smith-Osborne, A., Gonzalez-Lima, F., North, C.S., and
Liu, H. (2014). Prefrontal responses to digit span memory phases in
patients with post-traumatic stress disorder (PTSD): A functional near-
infrared spectroscopy study, Neuroimage Clin, 4, pp. 808–819.
36. Hamani, C., Diwan, M., Macedo, C.E., Brandao, M.L., Shumake, J.,
Gonzalez-Lima, F., Raymond, R., Lozano, A.M., Fletcher, P.J., and Nobrega,
J. (2010). Antidepressant-like effects of medial prefrontal cortex deep
brain stimulation in rats, Biol Psychiatry, 67, pp. 117–124.
37. Michalikova, S., Ennaceur, A., van Rensburg, R., and Chazot, P.L. (2008).
Emotional responses and memory performance of middle-aged CD1
mice in a 3D maze: Effects of low infrared light, Neurobiol Learn Mem,
89, pp. 480–488.
38. McLean, C.P., and Foa, E.B. (2011). Prolonged exposure therapy for post-
traumatic stress disorder: A review of evidence and dissemination,
Expert Rev Neurother, 11, pp. 1151–1163.
39. Wu, Q., Xuan, W., Ando, T., Xu, T., Huang, L., Huang, Y.Y., Dai, T., Dhital,
S., Sharma, S.K., Whalen, M.J., and Hamblin, M.R. (2012). Low-level
laser therapy for closed-head traumatic brain injury in mice: Effect of
different wavelengths, Lasers Surg Med, 44, pp. 218–226.
40. Schiffer, F., Johnston, A.L., Ravichandran, C., Polcari, A., Teicher, M.H.,
Webb, R.H., Hamblin, M.R. (2009). Psychological benefits 2 and 4 weeks
after a single treatment with near-infrared light to the forehead: A pilot
study of 10 patients with major depression and anxiety, Behav Brain
Funct, 5, pp. 46.
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1076 Transcranial Low-Level Laser (Light) Therapy for Neurocognitive Enhancement

41. Naeser, M.A., Saltmarche, A., Krengel, M.H., Hamblin, M.R., and Knight,
J.A. (2010). Improved cognitive function after transcranial, light-
emitting diode treatments in chronic, traumatic brain injury: Two case
reports, Photomed Laser Surg, 29, pp. 351–358.
42. Barrett, D.W., and Gonzalez-Lima, F. (2013). Transcranial infrared laser
stimulation produces beneficial cognitive and emotional effects in
humans, Neuroscience, 230, pp. 13–23.
July 12, 2016 11:37 PSP Book - 9in x 6in 53-Hamblin-c53

Chapter 53

Post-Operative Uses of Low-Level Laser

Therapy

Maria Cristina Chavantes,a,b Nathali Cordeiro Pinto,a,c and

Vanessa Milanesi Holandab,d
a Post-Graduate Department in Medical School, University Nove de Julho, Rua

Vergueiro, 235, São Paulo, São Paulo 02111-030, Brazil

b Post-Graduate Department in Biophotonics, University Nove de Julho, Rua Vergueiro,

235, São Paulo, São Paulo 02111-030, Brazil

c Department of Valvar Surgery, Heart Institute, Clinical Hospital, Medical School, 44,

Av. Dr. Eneas de Carvalho Aguiar, 2nd floor, São Paulo, São Paulo 05403-900, Brazil
d Center of Neurology and Neurosurgery Associates (CENNA), Beneficência Portuguesa

of São Paulo Hospital, Maestro Cardim Street, 769 Bela Vista, São Paulo, São Paulo
01323-001, Brazil
mcchavantes@uol.com.br

In our surgical experience, the use of low-level laser therapy

(LLLT) can decrease complications in several major surgeries such
as neurosurgery, major abdominal surgery (bariatric surgery),
breast reconstruction (TRAM flap) surgical procedures, major burn
surgery, and thoracic-cardiovascular surgery. LLLT can accelerate
tissue repair, facilitating cicatrization and providing an analgesic
effect in post-surgical pain. LLLT can also prevent morbidity, avoid-
ing undesirable outcomes that could be evolving to post-operative
death [1]. Although no single tool or technique is yet available to
address the many complex issues surgeons face in their everyday

Handbook of Low-Level Laser Therapy

Edited by Michael R. Hamblin, Marcelo Victor Pires de Sousa, and Tanupriya Agrawal
Copyright c 2017 Pan Stanford Publishing Pte. Ltd.
ISBN 978-981-4669-60-3 (Hardcover), 978-981-4669-61-0 (eBook)
www.panstanford.com
July 12, 2016 11:37 PSP Book - 9in x 6in 53-Hamblin-c53

1078 Post-Operative Uses of Low-Level Laser Therapy

practice, this remarkable device may be highly beneficial for patients

by mitigating their suffering and quite rewarding for doctors.

53.1 LLLT in Post-Cardiovascular Surgery

Approximately 17.3 million people died from cardiovascular dis-

eases (CVDs) in 2008. By 2030, around 23.6 million people would
have died from CVDs [2]. CVD is the leading cause of death in
Brazil (300,000 deaths per year) and worldwide [3]. Cardiovascular
surgery has evolved in the last three decades, particularly in
the treatment of coronary heart disease [4]. In cardiothoracic
surgeries, patients undergo extensive procedures, which may lead
to several types of morbidity, both during surgery and in the post-
operative period. One of the most frequent treatments for ischemic
heart disease is myocardial revascularization, which is generally
performed by applying the saphenous vein as a coronary graft [1].
Complications from cardiac surgery incision are relatively common
in wound dehiscence, along with dreadful morbidities, such as
mediastinitis and/or osteomyelitis, both of which may lead to a
fatal outcome [1]. The incidence of post-sternotomy mediastinitis
following cardiovascular surgery does not decline in the course of
years, and the 47% mortality rate of the condition still poses a
challenge to health care [4].
Pain following cardiothoracic surgery is also a common issue
in the post-operative period. Studies have indicated that 60–70%
of patients undergoing sternotomy and thoracotomy feel intense
pain while making movements. The incidence of pain experienced
by such patients was as high as 30–40% in post-sternotomy and
45–65% in post-thoracotomy, even while in bed rest [5].
Our group at INCOR has been using LLLT after thoracic-
cardiovascular surgeries for over a decade and has been able to
successfully decrease hospital morbidity and mortality rate. Indeed,
it was the first time in the world (1980) that laser was applied in a
cardiac surgery in the Heart Institute (INCOR) of the University of
São Paulo, which was carried out by Prof. Dr. Radi Macruz [6].
In order to evaluate the effects of laser therapy, 80 patients
were divided into four groups, which underwent either sternotomy
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LLLT in Post-Cardiovascular Surgery 1079

or thoracotomy post-cardiovascular surgeries. The groups were

classified as follows: Control Group (G1)—conventional therapy
only; Laser Group (G2)—conventional therapy + red laser irradi-
ation (λ = 655 nm, P = 25 mW, E = 1 J per point) on surgical
incision; Laser Group (G3)—conventional therapy + infrared laser
irradiation (λ = 830 nm, P = 35 mW, E = 1 J per point) on
surgical incision; Placebo Group (G4)—conventional therapy +
“laser pointer” (avoiding psychological factor). A CW diode laser was
applied punctually surrounding the surgical wound perimeter, at 2
cm each, in the following times: in G2 on immediate post-operative
(IPO) period less 12 h, 3rd PO and 6th PO; in G3 on IPO less 6 h,
1st PO and 3rd PO; and in G4 the same parameters as G3, but laser
equipment was off (Fig. 53.1a,b).
All patients were evaluated in the pre-operative period, on IPO,
24 h and 72 h post-surgery, subsequently on the 7th day, for acute
complications prior to discharge, as well as on 15th day, 30th
day, 60th day, and 180th day post-surgical procedure for chronic
morbidity. Surgery incision data were evaluated in the form of first
dehiscence signals in sternotomy/thoracotomy. A visual analogue
scale (VAS) was used to evaluate incision pain and sensibility, and
pain was classified from zero (no pain) to 10 (maximum pain).
Groups were divided into G1 × G2 and G3 × G4, and between
themselves were compared. Patients of G1 and G2 groups were
similar and homogenous, as were the G3 and G4 ones.

(a) (b)

Figure 53.1 LLLT being applied as a preventive tool in surgical incision

(sternotomy) post-cardiovascular surgery (a and b).
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1080 Post-Operative Uses of Low-Level Laser Therapy

(a) (b)

Figure 53.2 (a) Laser group (G2) diabetic patient on 7th PO. (b) Patient
on 30th PO underwent conventional intra-hospital treatment, without laser
application; morbidity occurs (control group G1).

On the 3rd and 7th PO, most G1 and G3 patients showed

unequivocal inflammatory signs around the surgical wounds and
pain when compared to G2 and G4 (Figs. 53.2a and 53.3a). While
patients of G3 did not present any complication, only one patient of
G2 developed a minimal dehiscence, which closed after 4 days. In G1,
five patients presented incisional dehiscence with mediastinitis, as
in hospital for over a year (Fig. 53.2b). In addition, one patient in G4
presented incisional dehiscence and deep infection with secretion,
leading to osteomyelitis; another patient developed mediastinitis
(Fig. 53.3b). Regarding pain intensity mean on the 3rd PO, G1 and
G2 values were 4.5 and 2.8, respectively, as 3 and 4.9 in G3 and
G4, respectively. On 7th PO, G1 and G2 values were 4.1 and 2.4,
respectively, while the values for G3 and G4 were 1.6 and 3.8,
respectively.
Hospitalization time for G1 was 22 days and for G2 was 11 days
(statistically significant, p = 0.015). Hospital stay in G4 was 16 days
and in G3 was 11.8 days (statistically significant, p = 0.001).
Several cardiac studies have shown that patients undergoing
myocardium revascularization or valve repairing developed post-
operative infection and stayed in hospital for a period longer than
18 days, with costs more than $13,000 [7]. Our findings indicate
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LLLT in Post-Cardiovascular Surgery 1081

(a) (b)

Figure 53.3 (a) Laser group (G3) diabetic patient on 7th PO. (b) Patient on
22th PO underwent conventional intra-hospital treatment + “laser pointer”;
morbidity occurs (placebo group G4).

that overall hospitalization time was reduced in both laser groups

(statistically significant), when compared to the control and placebo
groups.
Few papers in the literature have discussed the role of LLLT
in preventing dehiscence. Several preventive measures in both
hospitals and outpatient facilities have also been shown to be
effective in controlling dehiscence. A study by Pinto et al. has
demonstrated that LLLT facilitated the closure of dehiscence post-
saphenectomy in diabetic patients who were not responding to
conventional treatment for over a month post-surgery [8]. Moreover,
LLLT induced analgesia immediately from the first laser therapy
session. Another study by Pinto et al. has demonstrated that LLLT
prevented prodromal signal on saphenectomy complications post-
myocardial revascularization in diabetic patients who, otherwise,
would have most likely developed morbidities [9].

53.1.1 LLLT in Thoracic-Cardiovascular Surgery

Recidivated and severe tracheal stenosis procedure (tracheoplasty)
is done differently from conventional surgical procedure. The
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1082 Post-Operative Uses of Low-Level Laser Therapy

approach is to start from the inside tracheal lumen (endoluminal

tracheobronchoscopic resection by surgical laser), the high power
laser is applied to resect the granulomatous and fibrous tissues,
then LLLT is applied immediately to the wound bed into injured
airway area, avoiding future benign recidivate growth. In such cases,
LLLT successfully contained granuloma formation, thus avoiding
recurrent TS after 3 years of follow-up [10].
LLLT has been an efficient adjuvant tool for the management
of pain in post-surgery procedures and for fast surgical wound
healing. Thus, LLLT seems to be a safe, noninvasive, and cost-
effective treatment, reducing hospital stay and thus benefiting both
patients and health care institutions.

53.2 LLLT in Neurosurgery Procedures

The use of surgical high-power laser (HPL) in neurosurgery has

shown good results since the 1980s [11]. The residual effect of HPL
assists in promoting wound healing and preventing infection and
fistulae formation, similar to the LLLT outcome.

53.2.1 Stroke
Transcranial LLLT has shown considerable effect in acute stroke
patients, for whom a significant improvement in recovery rate was
found, as demonstrated by comparing patients 5 days after LLLT
to control treatment ( p < 0.05), using the National Institutes of
Health Stroke Severity Scale [12]. This difference persisted up to
90 days post-stroke with 70% of LLLT-treated patients having a
successful outcome when compared to 51% of control patients. The
improvement in functional outcome after a stroke due to the use
transcranial LLLT has been well established by several experimental
studies in rat and rabbit models [13, 14].
However, LLLT might have other brain-specific beneficial effects.
Investigations have suggested that such improvements in patient
outcomes are due to the promotion of neurogenesis and migration
of neurons [13, 14]. This assumption is supported by the fact that
the after-stoke LLLT benefits may take 2–4 weeks to manifest,
July 12, 2016 11:37 PSP Book - 9in x 6in 53-Hamblin-c53

LLLT in Neurosurgery Procedures 1083

thus reflecting the time required for new neurons to be formed

and gathered at the damaged site in the brain, although further
multicenter studies should be carried out [15, 16].

53.2.2 Pain
Pain is a major public health problem. In a recent report, the
Institute of Medicine (IOM) has acknowledged pain as a major
challenge in health care. This report stressed that persistent pain
affects more than 100 million Americans, incurring $635 billion in
direct costs and lost wages. Back pain is a leading cause of such
persistent symptoms and is the fifth common reason for visiting
a physician [17]. Back pain also accounted for 50% of all chronic
pain problems in a European survey [18]. According to a US survey,
each year 15% of American adults report frequent low back pain or
pain lasting for more than 2 weeks, with the annual costs exceeding
$100 billion [19]. Several researchers have pointed that LLLT may
successfully treat both acute and chronic pain [20, 21].
In fact, the world population has grown exponentially over the
last centuries, and life expectancy has followed through. As a result,
traumatic lesions and several rheumatic diseases are now more
widespread, particularly among the elderly [22]. Also some patients
cannot cope with the adverse effects of several analgesic drugs, and
the elderly have a heightened risk profile. LLLT would then be a
useful tool, working as an effective side-effect-free painkiller.
Besides being effective for pain management, laser therapy has
been established to improve the cicatrization response and decrease
post-operative morbidity, as seen in wound dehiscence, and the
infection in the post-operative period of neurosurgery [23]. Thus,
LLLT should be listed as a promising tool in all kinds of major
neurosurgical procedures.

53.2.3 Spinal Cord: Trauma and Pain Problems

LLLT has been indicated as a viable treatment for neurological
conditions such as traumatic brain injury (TBI), stroke, spinal cord
injury (SCI), spinal surgery as well as for treating chronic pain
(specially, neuropathic pain) [23, 24].
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1084 Post-Operative Uses of Low-Level Laser Therapy

SCI is a severe central nervous system trauma with no effective

restorative therapies. Rochkind et al. have demonstrated that
LLLT, if applied simultaneously to the injured sciatic nerve and
the corresponding segment of the spinal cord, accelerates the
regeneration process of the injured peripheral nerve [25]. Moreover,
light therapy significantly improves the average length of axonal
regrowth and increases the total axon number for both injury
models. A statistically significant lower angle of rotation of the feet
was observed during a walking test in the hemi-section model. An
overall functional recovery after contusion model was seen in the
LLLT groups [26]. These results suggest that phototherapy could be
a promising therapy for human SCI.
Although TBI is a serious health issue, unfortunately the search
for more effective therapies in recent years has not been successful.
LLLT seems to be a beneficial treatment for TBI. In addition
to increasing mitochondrial activity and activating transcription
factors, as described in a previous chapter, LLLT could help TBI
patients by modulating apoptosis, stimulating angiogenesis, increas-
ing neurogenesis, and lessening cerebral damages, as described in
Chapter 54.

53.2.4 Application of LLLT in Pediatric and Adult

Neurosurgical Procedures
Pinto et al. have observed a significant decline in the dehiscence of
surgical wounds in 13 neonates undergoing LLLT when compared to
23 in newborn control patients (7.69% versus 17.39%) [23]. A CW
diode laser (λ = 685 nm, P = 21 mW, E = 0.19 J) was punctually
applied along the surgical incision perimeter, delivering 4–10 J of
energy per newborn patient. This pioneer work used LLLT in the
post-operative context of a pediatric neurosurgery, thus adding a
preventive role for LLLT, by decreasing the incidence of dehiscence
following surgical repair in myelomeningocele neonates [23].
Trans-operative application of LLLT was described for the first
time by our group to prevent morbidity in failed back surgery
syndrome and evaluate the LLLT effects on spinal surgery. A
prospective randomized, controlled trial was carried out on 48
patients, who underwent laminectomy and were divided into two
July 12, 2016 11:37 PSP Book - 9in x 6in 53-Hamblin-c53

LLLT in Neurosurgery Procedures 1085

Figure 53.4 Transoperative LLLT in three different layers: (a) Over the
duramater. (b) Subcutaneous. (c) Over the skin.

groups: In the LLLT group (G1), CW infrared laser was applied on

25 randomized patients during surgery, on the laminectomy site
(subdural area), on the subcutaneous tissue, and surrounding the
surgical wound site (Fig. 53.4). Parameters used are as follows: λ =
804 ± 2 nm; total exposure time 240 s; energy density 0.62 J/cm2
per minute; power 40 mW; spot area 3.876 cm2 ; fluence 7.44 J/cm2 ;
total time of exposure 4 min, as suggested by the World Association
of Laser Therapy (WALT). WALT recommends a fluence of 4–8 J/cm2
for infrared lasers (780–860 nm), hence contributing to a prompter
recovery of the treated laminectomy area. In the placebo group (G2),
23 patients assumed that they were getting the same treatment as
the G1 group (to avoid psychological influence), although the laser
equipment was not operating.
In the absence of laboratory parameters to assess the surgical
response to phototherapy, our group set out to find a biochemical
marker by using blood tests. Therefore, for both groups, C-reactive
protein (CRP), lactic dehydrogenase (LDH), and creatine kinase
(CK) were assessed in the 2nd and 5th days following surgery.
Digital temperature and VAS were also measured, both pre- and
immediately post-LLLT use. The drainage output were collected
in the first and second days, following neurosurgical procedures
carried out in both groups.
The results showed temperature decrease, pain relief (Fig. 53.5),
and accelerated wound healing in G1 (laser). LLLT may facilitate
lesion healing due to a prompt tissue response while in the acute
inflammation phase. The fastest drop in both LDH and CK occurred
in the next healing phase. Such markers may indicate that these
July 12, 2016 11:37 PSP Book - 9in x 6in 53-Hamblin-c53

1086 Post-Operative Uses of Low-Level Laser Therapy

Pain

1.0

0.9

Placebo
VAS Ratio

0.8 Laser

0.7

0.6

0.5
Post/Prior:24h Post/Prior:72h Period

Figure 53.5 Assessment of pain postoperatively ( p = 0.0001). Kruskal-

Wallis Test.

assessments (G1) may guide LLLT treatment evolution, as it was

suggested by a dramatic CRP drop, in which the proliferation phase
presented statistically significant values ( p < .05), from second to
fifth post-operative day. The decrease in the drain output of the LLLT
group (Fig. 53.6) may be due to a more rapid tissue recovery from
muscles, skin, bone, and vessels (from skin to subdural area). This
statement may be supported by the lower morbidity and infection
risk found, and the decreased dehiscence after spine surgery.
It should be stressed that accurate laser polarization is critical
to LLLT, and in our study LLLT was applied parallel to tension
lines, following Langer lines, anatomical equivalent composed of
preferential parallel orientation and alignment of collagen bundles
[27].
It is then possible to postulate that the polarizing memory is
preserved better in a direction parallel than perpendicular to the
lines of tension, and therefore it could help in improving the healing
of spine surgery. All our applications of LLLT were performed in a
direction parallel to the lines of Langer polarization, further aiding
tissue cicatrization.
July 19, 2016 12:4 PSP Book - 9in x 6in 53-Hamblin-c53

LLLT in Neurosurgery Procedures 1087

Control
Laser

Figure 53.6 Drainage Output (72h p = 0.004). Kruskal-Wallis Test.

In another study by our group, LLLT was applied on the dorsal

ganglion of the second spinal nerve (GDL2, which is a cluster of
neuronal bodies responsible for sensory afferent inputs from more
than 80% of the lumbar region Figs. 53.7 and 53.8) and was indeed
very effective in relieving chronic lumbosacral pain.

53.2.5 LLLT in Neurosurgery Procedures

Only three LLLT sessions in trans-operative and immediately PO
would be adequate to contribute to wound cicatrization, decreased
drainage output, and analgesia in the post-operative follow-up in
pediatric and adult spinal surgery. Given that, LLLT could indeed
be a promising adjuvant therapeutic proposal, accelerating surgical
wound healing, preventing morbidities, reducing hospital stay, and
making it cost effective. Thus, our findings demonstrated that LLLT
could prevent complications in PO for neurological surgery patients.
LLLT is an effective, safe, and noninvasive treatment method, so
it should be regarded as an essential tool in pediatric and adult
neurosurgery procedures.
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1088 Post-Operative Uses of Low-Level Laser Therapy

Figure 53.7 Scottie dog technique to access DGL2 guided by fluoroscopy.

Figure 53.8 Transoperative Diode CW Laser through 600 μm fiber into G14
cannulas guided by fluoroscopy.
July 12, 2016 11:37 PSP Book - 9in x 6in 53-Hamblin-c53

References 1089

53.3 Final Remarks on Post-Operative Surgeries

Several complications after a major abdominal surgery, such as

dehiscence, infection, bleeding, and fistulae, are the main concern
in the surgical field. Major abdominal surgery, such as bariatric
surgery, was carried out in our group study applying LLLT in morbid
obese patients. Laser therapy could avoid formation of seroma and
infection in these patients, while inhibiting dehiscence and assisting
surgical wound cicatrization.
These findings corroborate previous studies on preventive post-
surgical procedures and on strategies aimed at reducing the
morbidity and mortality of high-risk major surgeries. Thus, LLLT
seems to be a safe, efficient, and cost-effective tool in preventing
potential complications following major surgeries.

References

1. Chavantes MC. 2009. Laser em Problemas Cardiorrespiratórios. In:

Laser em Bio-Medicina. Principios e Pratica, Chavantes MC (ed). São
Paulo, Brasil: Atheneu. pp. 149–181.
2. National Center for Health Statistics. Health, United States, 2010: With
Special Feature on Death and Dying. Hyattsville, MD. 2011.
3. Mathers CD and Loncar D. 2006. Projections of global mortality and
burden of disease from 2002 to 2030. PLoS Med, 3(11): e 442.
4. WHO. 2011. Global Atlas on Cardiovascular Disease Prevention and
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5. Pereira MHC. 2009. LLLT e Dor. In: Laser em Bio-Medicina, Chavantes MC
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6. Chavantes MC and Jatene AD. 1990. The use of laser in cardiovascular
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8. Pinto NC, Pereira MHC, Stolf NAG, and Chavantes MC. 2009. Low-level
laser therapy in acute dehiscence saphenectomy: Therapeutic proposal.
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9. Pinto NC, Pereira MHC, Tomimura S, Magalhães AC, Pomerantzeff
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tracheal stenosis using biomodulation effect. In: WALT, 2005, Guaruja,
SP. Photomedicine and Laser Surgery, 1:113.
11. Zamorano L, Chavantes CM, Dujovny M, and Ausman J. 1990. Endoscopic
laser stereotaxis: Indication for cystic and intraventricular lesion. Proc
SPIE 1200, Laser Surgery: Advanced Characterization, Therapeutics, and
Systems II, pp. 253–271.
12. Lampl Y, Zivin JA, Fisher M, Lew R, Welin L, Dahlof B, Borenstein P,
Andersson B, Perez J, Caparo C, Ilic S, and Oron U. 2007. Infrared
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the Neuro-Thera Effectiveness and Safety Trial-1 (NEST-1). Stroke, 38:
1843–1849.
13. Lapchak PA, Salgado KF, Chao CH, and Zivin JA. 2007. Transcranial
near-infrared light therapy improves motor function following embolic
strokes in rabbits: An extended therapeutic window study using
continuous and pulse frequency delivery modes. Neuroscience, 148:
907–914.
14. Oron A, Oron U, Chen J, Eilam A, Zhang C, Sadeh M, Lampl Y, Streeter
J, DeTaboada L, and Chopp M. 2006. Low-level laser therapy applied
transcranially to rats after induction of stroke significantly reduces long-
term neurological deficits. Stroke, 37: 2620–2624.
15. de Taboada L, Ilic S, Leichliter-Martha S, Oron U, Oron A, and Streeter J.
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70–73.
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DC: The National Academies Press.
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18. Breivik H, Collett B, Ventafridda V, Cohen R, and Gallacher D. 2006.

Survey of chronic pain in Europe: Prevalence, impact on daily life, and
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19. Frymoyer JW and Cats-Baril WL. 1991. An overview of the incidences
and costs of low back pain. Orthop Clin North Am, 22(2): 263–271.
20. Chow R, Johnson M, Lopes-Martins R, and Bjordal JM. 2009. Efficacy of
low-level laser therapy in the management of neck pain: A systematic
review and meta-analysis of randomized placebo or active-treatment
controlled trials. Lancet, 374: 1897–1908.
21. Bjordal JM, Couppe C, Chow RT, Tuner J, and Ljunggren EA. 2003. A
systematic review of low-level laser therapy with location-specific doses
for pain from chronic joint disorders. Aust J Physiother, 49: 107–116.
22. CDC. 2010. Prevalence of doctor-diagnosed arthritis and arthritis-
attributable activity limitation: United States, 2007–2009. MMWR Morb
Mortal Wkly Rep, 59(39): 1261–1265.
23. Pinto FC, Chavantes MC, Pinto NC, Alho EJ, Yoshimura EM, Matushita
H, Krebs VL, and Teixeira MJ. 2010. Novel treatment immediately after
myelomeningocele repair applying low-level laser therapy in newborns:
A pilot study. Pediatric Neurosurg, 46: 249–254.
24. Hashimi JT, Huang Y-Y, Osmani BZ, Sharma SK, Naeser MA, and Hamblin
MR. 2010. Role of low-level laser therapy in neurorehabilitation. PM &
R, 2: S292–S305.
25. Rochkind S, Barr-Nea L, Bartal A, Nissan M, Lubart R, and Razon N. 1988.
New methods of treatment of severely injured sciatic nerve and spinal
cord. An experimental study. Acta Neurochir Suppl (Wien), 43: 91–93.
26. Wu Q, Huang YY, Dhital S, Sharma SK, Chen AC, Whalen MJ, and Hamblin
MR. 2010. Low-level laser therapy for traumatic brain injury. Proc SPIE,
7552: 201–206.
27. Silva DFT, Vidal BC, Zezell DM, Zorn TM, Nunez SC, and Ribeiro MS. 2006.
Collagen birefringence in skin repair in response to red polarized-laser
therapy. J Biomed Opt, 11(2): 24002.
July 6, 2016 17:43 PSP Book - 9in x 6in 54-Hamblin-c54

Chapter 54

Bright New World: Future Directions of

Low-Level Laser (Light) Therapy

Marcelo Victor Pires de Sousaa,b and Maria Cristina Chavantesc

a Laboratory of Radiation Dosimetry and Medical Physics, Institute of Physics, Matão

Street, Alley R, 187, University of São Paulo, São Paulo, São Paulo 05508-900, Brazil
b Bright Photomedicine Inc., Prof. Linnaeus Prestes Avenue, No. 2242, Butantã -

University City, IPEN - São Paulo, São Paulo 05508-000, Brazil

c Laser Medical Center of the Heart Institute, General Hospital, Medical School,

University of São Paulo, Av. Dr. Enéas de Carvalho Aguiar, 44 - Pinheiros, São Paulo,
São Paulo 05403-900, Brazil
marcelovictor@usp.br

O wonder!
How many godly creatures are there here!
How beauteous mankind is! O brave new world,
That has such people in’t.
William Shakespeare, The Tempest, Act V, Scene I, II.

Low-level laser (light) therapy (LLLT), as we saw in this handbook,

is a truly multidisciplinary field, providing exciting researching
opportunities for physicians, health professionals, physicists, biol-
ogists, chemists, engineers, and practitioners. Here we have the final
chapter of a vast book, and we will accomplish the hard objective
of making predictions regarding science, which is historically well

Handbook of Low-Level Laser Therapy

Edited by Michael R. Hamblin, Marcelo Victor Pires de Sousa, and Tanupriya Agrawal
Copyright c 2017 Pan Stanford Publishing Pte. Ltd.
ISBN 978-981-4669-60-3 (Hardcover), 978-981-4669-61-0 (eBook)
www.panstanford.com
July 6, 2016 17:43 PSP Book - 9in x 6in 54-Hamblin-c54

1094 Bright New World

known as an ambitious and inglorious task. However, we will be

allowed to open our minds to a broader spectrum of possibilities for
LLLT. Some areas of current activities offering possibilities for future
research are listed here. These topics have been selected based
partly on the author’s personal views and partly on the ongoing
projects in the Wellman Center for Photomedicine (courtesy of
Dr. Michael Hamblin). We will discuss LLLT education and training,
how photomedicine (particularly LLLT) can be positioned as a
branch of medicine, and possible impacts for health and wellness of
people and animals.

54.1 Introduction

The basic principle behind LLLT is that one can increase the
efficiency of adenosine triphosphate (ATP) production in the
interior of a cell. It means that the cells in an organism irradiated
with visible and near-infrared (NIR) light have more energy to
produce any type of endogenous enhancement of function and
endogenous treatment. Photobiomodulation mechanism starts with
light absorption inside mitochondria leading to ATP production.
For this reason, any cell with mitochondria is capable of being
modulated by light. So, that can explain the numerous types of
therapies described in this handbook, and it allows scientists to
imagine even more possible applications.
In this chapter, we will summarize the latest developments and
innovations in LLLT. Therefore, we use this knowledge as a reference
to envision how the next generations of devices, treatments, and
general uses would be to enhance performance even in the case of
non-diseased subjects, such as athletes. To conclude, we will present
a section “A Bright New World for Photobiomodulation,” in which
we will discuss the possible influences of photobiomodulation (in
particular LLLT) for humanity.

54.2 New Clinical Indications for LLLT

The traditional indications for laser therapy are sustained basically

in three pillars: against inflammatory problems, aiding tissue repair-
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New Clinical Indications for LLLT 1095

ing, and analgesic effect. Such roads have been paved throughout
several fields, facilitating the knowledge on laser in the last decades.
Some new avenues opened in the last years, and this will be
discussed in the following paragraphs.

54.2.1 Stem Cells

It was recently observed that low-power NIR laser could direct
differentiation of dental stem cells to a specific mineralized tissue
lineage, dentin [1]. The ability of lasers to induce reparative
dentin had been noted previously although the mechanisms had
remained unknown, hindering the development of effective clinical
protocols. This recent study elucidated the molecular mechanism
of low-power laser treatments that involved activation of a latent
growth factor, TGF-β1. Using chemical biology and transgenic
approaches, this study demonstrated that the laser-generated
reactive oxygen species (ROS)-activated TGF-β1 was critical for
the directed differentiation of endogenous dental stem cells from
both human and rodent (mice and rat) teeth. This is clinically very
relevant as dentin is a key tissue that maintains the vitality of tooth,
and hence the ability to induce dentin prevents the need for root
canal treatments and eventual replacement of non-vital teeth. The
limitations of this study include the lack of morphodifferentiation
of tubular, regenerated dentin. The newly formed dentin was bone
like (osteodentin) and widespread along the tooth pulp chamber
and canals. Better optical focusing techniques and biochemical
strategies to restrict light–tissue interactions could drive to the ideal
regenerative response, instead of the current reparative process
observed. More recently, these observations have been extended to
directing the differentiation of mesenchymal stem cells (MSC) using
a sophisticated, scaffold-based model [2]. This system can generate
exquisitely precise morphogen fields at both temporal and spatial
scales and activate latent TGF-β1 with NIR lasers to direct MSC
differentiation to dentin. Other studies have shown the ability of
LLLT/PBM treatments to mobilize MSC from bone marrow to aid in
healing cardiac, liver, and kidney damage.
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1096 Bright New World

54.2.2 Transcranial LLLT for Brain Disorders

Recently remarkable results have been found in neurology using
transcranial LLLT, a noninvasive treatment for serious metabolic
or degenerative diseases in central nervous system (CNS) or
neurological injuries. Transcranial LLLT improves motor recovery
after strokes in rats [3] and humans [4], reduces significantly
the recovery time in traumatic brain injury (TBI) in mice [5],
and can also be applied in humans to ameliorate chronic TBI
[6]. Encouraging preclinical results have been obtained for some
degenerative CNS diseases such as familial amyotrophic lateral
sclerosis [7], Parkinson’s disease [8], and Alzheimer’s disease [9]
with this technique.
Using a rodent model of toxic optic neuropathy induced by
rotenone, a mitochondrial complex I inhibitor, Rojas et al. [10]
reported the neuroprotective effect of 633 nm LLLT. Whole-brain
cytochrome oxidase and superoxide dismutase activities were
also increased by photoneuromodulation, suggesting an in vivo
transcranial effect of LLLT. It showed that photoneuromodulation
acquired by transcranial LLLT can effectively prevent neurotoxic and
retinotoxic effects of some neurodegenerative disorders associated
with mitochondrial dysfunction because of complex I inhibition.
Parkinson’s disease is mainly characterized by the degeneration
of dopaminergic neurons in the CNS, including the retina [11].
Considerable evidence supports mitochondrial dysfunction and
oxidative stress, as key in the pathogenesis of Parkinson’s disease.
LLLT treatment had cytoprotective effect in many animal research
[12, 13].
The realization that impaired neurogenesis plays an important
role in depression [14] suggested that transcranial LLLT could have
beneficial effects in patients with major depression and anxiety,
and this was confirmed in a pilot clinical trial with 10 subjects
receiving a single transcranial LLLT to forehead [15]. Transcranial
LLLT may be thought to be just in its infancy, but we believe the
stage is set for rapid growth, especially in view of the massive and
continuing failure of clinical trials of pharmaceuticals for many brain
disorders. As the world population continues its senescence and
the epidemic of degenerative diseases related to aging continues
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New Clinical Indications for LLLT 1097

to grow, transcranial LLLT may make a real contribution to these

patients’ health.

54.2.3 Ophthalmology
Retina is one of the highest oxygen-consuming tissues in the human
body because photoreceptor cells (rods and cones) in the retina have
high concentration of mitochondria [16]. Mitochondrial repair and
attenuation of oxidative stress are keys to the long-term survival
of retina. Moreover, retina contains abundant photosensitizers once
it is constantly exposed to visible light. These characteristics make
the retina very susceptible to LLLT effects, and in fact there are
many reports of red and infrared irradiation regenerating damaged
retinal tissue. Eells et al. [17] reported the first direct link between
the actions of far-red to NIR light on mitochondrial oxidative
metabolism in vitro. In order to avoid photodamage, additional care
must be taken during an LLLT procedure in the eye; the use of a
laser could decrease the usability of this procedure. However, the
development of new devices using light-emitting diodes (LEDs) and
standardized dosimetry could lead to an increase in the use of LLLT
in ophthalmology.
Age-related macular degeneration is strongly correlated with
oxidative stress and mitochondria debility. Light therapy can affect
several of the pathways that lead to macular damage. Targeting
mitochondrial respiration by direct absorption at cytochrome c
oxidase leads to secondary and tertiary beneficial effects on cellular
metabolism, including anti-inflammatory, antioxidant, and anti-
apoptotic protection and recovery from injury. LED therapy has been
demonstrated to be a noninvasive, easily administered, and safe
treatment [18]. Nevertheless, there is still an urgent need to properly
evaluate this treatment modality.
Retinopathy of prematurity (ROP) is a disorder of the developing
retina and one of the leading causes of blindness in developed
countries. Risk factors for ROP include low birth weight and
gestational age, supplemental oxygen therapy, and respiratory
distress syndrome. LLLT at 670 nm reduced neovascularization,
vaso-obliteration, and abnormal branching patterns of the retinal
vessels in ROP [19].
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1098 Bright New World

54.2.4 Autoimmune Diseases

Autoimmune diseases arise from an abnormal immune response of
the organism against substances and tissues normally present in the
body, generating lesions or dysfunction of organs or tissues. The
treatment of autoimmune diseases is typically with immunosup-
pression. In both autoimmune and inflammatory diseases, the condi-
tion arises through the exacerbated reactions of the human adaptive
or innate immune systems. Due to this similar mechanism, LLLT,
which is well developed and well recognized in the treatment of in-
flammations, has been hypothesized to have an important role in the
treatment and amendment of symptoms in autoimmune diseases.
Mucous membrane pemphigoid is a heterogeneous group of
autoimmune, subepithelial, blistering diseases. A combination of
topical and systemic steroid treatment is often used when managing
patients with mucous membrane pemphigoid. A patient presenting
mucous membrane pemphigoid was successfully treated with the
application of local corticosteroids and LLLT using an 810 nm diode
laser [20].
Another important perspective for treating autoimmune dis-
eases with LLLT is the treatment of chronic autoimmune thyroiditis.
Hoefling et al. showed that 10 sessions of laser (810 nm)
transcutaneous applications in thyroid were enough to improve
thyroid function in patients with hypothyroidism caused by chronic
autoimmune thyroiditis [21].

54.2.5 Lung Disease and Tracheal Stenosis

Light therapy has gained support as a non-pharmacological therapy
capable of downregulating oxidative metabolism in the respiratory
system [22]. Notwithstanding, the clinical studies on the laser effect
on asthma [23] as well as chronic obstructive pulmonary disease
are usually directed in improving the life of patients without major
concern about mechanisms, dosimetry, or inflammatory cells as well
as their mediators. It is known that LLLT attenuates pro-oxidant
reactions that compromised bronchial reactivity and structure [24].
One of the difficulties in the procedures of LLLT for lungs
and airway is light delivery. It used to be conducted invasively
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New Clinical Indications for LLLT 1099

with optical fibers inside needles, but recent research claims

the transcutaneous efficacy of light delivery to lungs. LLLT has
been proved to treat asthma, tuberculosis, and pleurisy. A future
perspective of treatment could be the prevention of smoking-
induced cancer since it is connected with recurrent inflammatory
processes present in the smoker’s lungs. There is a need to
characterize an internal form of light delivery, as well as ideal
dosimetry is able to induce and maintain an anti-inflammatory
response in both airways and lungs.

54.2.6 Hemodynamic Effect

LLLT can induce a photobiomodulation response inside cells not
only activating ATP production, NO, and ROS, or modulating Na+ –K+
pumps and Ca++ channels. Recent studies indicate that some causal
nexus is involved in inflammation and oxidative stress, leading to
systemic arterial hypertension (SAH).
As LLLT improves lung response as discussed earlier, this treat-
ment acts in the short circulation region (pulmonary circulation)
and then in systemic circulation.
Nowadays, SAH is considered high risk for neuro-cardiovascular
problems. Tomimura et al. indicated that LLLT could diminish
pressure levels in spontaneously hypertensive rats, producing
hemodynamic changes as well as on oxidative stress in blood [33].
Chavantes et al. indicated arterial elasticity and variation in
arterial blood pressure for normotensive and hypertensive patients,
who were submitted to laser therapy. After a while, arterial blood
pressure had dramatic changes due to increase in arterial elasticity
and decrease in vascular resistance [34].
New findings by our study group indicate that pregnant women
with arterial hypertension as well as pre-eclampsia present an
immediate response after LLLT with hemodynamic effect in SAH
[35].

54.2.7 Performance Enhancement

Performance enhancement involves the use of photobiomodulation
in normal healthy subjects to enhance some natural, physiological,
July 6, 2016 17:43 PSP Book - 9in x 6in 54-Hamblin-c54

1100 Bright New World

or cognitive functions. Recently, light therapy using lasers and

LEDs has been used to increase muscle performance in exercises
limited by strength or fatigue. Light therapy may have a role to
play in preparing athletes competing in high performance sports.
Recent reviews have reported positive effects of light therapy on
muscle performance, highlighting protection from exercise-induced
muscle damage; an increased number of repetitions in maximum
exertion tests; increased workload, torque, and muscle fatigue
resistance in training programs; as well as an overview of the
main possible mechanisms of action of light therapy on muscle
tissue [25].
NIR light passes readily through scalp and skull, and a small
percentage of the incident power density can arrive at the cortical
surface in humans [26]. Gonzalez-Lima et al. [27] carried out a
study on healthy college students using LLLT. A single exposure of
the forehead to CW 1064 nm laser (60 J/cm2 at 250 mW/cm2 )
improved the scores in tests that measured attention, memory, and
mood. Reaction time in a sustained-attention psychomotor vigilance
task was significantly improved in the treated (n = 20) versus
placebo-controlled (n = 20) groups, especially in high novelty-
seeking subjects. The LED technology is not expensive. An LLLT
protocol with LEDs has the potential for homecare treatment [28].

54.2.8 Optimizing Treatment

A relevant point to be considered is the dosimetry aspect to get
the best treatment for each health condition. Also for optimizing
the dose, it should be personalized for each patient and animal to
be treated.

54.3 Novel Light Sources for LLLT

Most physicians have never heard of LLLT, and yet more than 1000
devices are already on the world market and many of them have no
proof of efficacy. There are single laser beam devices with power
outputs starting from as little as 1 mW to LED clusters comprising
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Novel Light Sources for LLLT 1101

a few red emitters to arrays with hundreds of LEDs delivering up to

480 W to treat the whole body.
The first LLLT procedures were carried out using lasers, and in
the early days, researchers were not sure if photobiomodulation
processes triggered by light depended on the special properties
of laser light, such as monochromaticity (narrow bandwidth),
coherence, or polarization or if they could be achieved by other
sources of light. In the last decades, evidence have been found that
other light sources can produce photobiomodulation and the major
determining parameter for the effectiveness of LLLT is wavelength,
which must be absorbed by a photoacceptor molecule in the host.
In recent years, many research studies and clinical procedures
have been performed with noncoherent light sources such as LED
or halogen lamps connected to monochromatic filters. Even more
recent are organic LEDs (OLEDs), which extend the possibilities
of LLLT since OLEDs can emit light uniformly from a flexible
surface.

54.3.1 Wearable LLLT Devices: Bandages and Clothing

A variety of technologies are being developed that involve self-
emissive devices rather than discrete emitters embedded in a
substrate. For example, devices have been reported that use OLED,
polymer LED, and thin film flexible electroluminescent sources.
Certainly, light therapy bandages based on these technologies have
several potential advantages, including volume production, readily
customizable temporal and spatial control from the addressing cir-
cuitry, and a very thin form factor, which could help conformability.
Such bandages still have some issues to solve (minimizing toxicity,
handling moisture, and providing sufficient output power), which
will likely delay the appearance of such devices in health-care
markets [29].
A good example of a fabric device that exhibited excellent optical
and thermal properties was introduced by Shen in 2013 [30]. Its
optical power density and operating temperature were stable during
usage for 10 h. A series of tests were conducted for the safety of
the fabric for human skin contact according to ISO 10993-1:2003.
The results showed that there was no potential hazard when the
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1102 Bright New World

luminous fabrics were in direct contact with human skin. Fabrics

made with side-emitting polymer optical fiber (POF) have great
potential to be used for phototherapy.
Combined with deformability, the fabric devices can easily fit
into three-dimensional contours, such as the human body, with
superior permeability to air, light weight, flexibility, and wearing
comfort. A POF is a cylindrical dielectric waveguide usually used to
transport light between the two ends of the fiber by the process
of total internal reflection. By creating certain features in the core
or at the core cladding interface of the fiber, the POF emits light
sideways, which offers possibilities for large-area three-dimensional
illumination.

54.3.2 Implantable LEDs for Brain and Spine

Brain implants, often referred to as neural implants, are technologi-
cal devices that connect directly to the brain (usually placed on the
surface of the brain or attached to the brain cortex). Brain implants
can be used after stroke or other head injuries and diseases.
Neural implants such as deep-brain stimulation and vagus nerve
stimulation are increasingly becoming routine for patients with
Parkinson’s disease and clinical depression, respectively, proving
themselves a boon for people with diseases that were previously
regarded as incurable. Today brain implants work to electrically
stimulate, block, or record signals from single neurons or groups
of neurons in the brain. It is possible to change electrical or
magnetic stimulation to photobiomodulation and doing so increases
the usability of the implants. Implantable LEDs could also be used
in the spine in the case of spinal cord damage. A preliminary animal
study was carried out by Moro et al. [31] who used an implanted
intracranial fiber optic connected to a laser or LED light source to
treat Parkinson’s disease in MPTP-treated mice.

54.3.3 Swallowable LED Source Capsule

The first wireless camera pills created a revolutionary new
perspective for engineers and physicians, demonstrating for the
first time the feasibility of achieving medical objectives deep within
July 6, 2016 17:43 PSP Book - 9in x 6in 54-Hamblin-c54

References 1103

the human body from a swallowable, wireless platform [32]. The

10 years since the first camera pill have been a period of great
innovation in swallowable medical devices. A possible innovation in
the field of photomedicine could be the development of swallowable
LED devices for LLLT since it requires only simple and noninvasive
swallowing of a pill that would be programmed to start emitting red
or NIR light when it reaches the part of the gut affected and could
even be retrieved for reuse.

54.4 A Bright New World with Photobiomodulation

Photobiomodulation (or LLLT) faces several hurdles before becom-

ing mainstream in health care and research; one of these hurdles
is that the field is very broad. A rising percentage of the world’s
population lives in crowded urban areas where air pollution is a key
inducer of lung diseases. LLLT is a promising technique that should
be regarded as a possible treatment in a hospital’s emergency room
in the daily practice.
In fact, LLLT is an important low-cost non-pharmacological
tool, which is safe, noninvasive, and without side effects. Several
specialties in medicine and surgery can find benefits in LLLT to
treat many medical conditions involving inflammation, edema, pain,
tissue damage, and hemodynamic issues.
Imagine a world in which photobiomodulation is mainstream.
In little aches or big surgeries, from little bruises to recovery from
big accidents, LLLT has the potential to be present. LLLT has the
potential to be a disruptive innovation that may lead to a healthier
world.

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