Frost Shaun Michael 2013

Ocular Biomarkers For Early Detection
and Monitoring of Alzheimer’s Disease
By
Shaun M. Frost
B.Sc. (Hons), M.Sc.
Supervisors: Professor Ralph N. Martins1,2
Professor Yogesan Kanagasingam3,4
Dr. Hamid R. Sohrabi1,2
1
School of Medical Sciences, Edith Cowan University
2
School of Psychiatry and Clinical Neurosciences, University of Western Australia
3
Commonwealth Scientific and Industrial Research Organisation (CSIRO), Australia
4
Australian e-Health Research Centre, Perth, Australia
This thesis is presented for the degree of Doctor of Philosophy
The University of Western Australia
School of Psychiatry and Clinical Neurosciences
August 2013
For Michelle and Jake
I
Abstract
Early detection of Alzheimer’s disease (AD) is essential for intervention as subclinical
brain changes occur well before the onset of clinical symptoms. Current imaging of
amyloid plaques in the brain can detect early signs of AD but these tests are expensive
and not practical for population screening. While most AD related pathology occurs in
the brain, the disease has also been reported to affect vision and the eye. The retina, at
the posterior of the eye, is a developmental outgrowth of the brain which is more
accessible for imaging. Retinal degeneration and retinal vascular changes have
previously been reported in AD using specialized techniques. AD has also been reported
to influence the response of the ocular pupil to a bright flash of light, a response
dependent on the cholinergic system which is impaired in AD.
Research for this thesis involved collection and analysis of retinal photographs and
pupil flash response parameters from participants at the McCusker Alzheimer’s Disease
Research Foundation (MARF) in Perth, Australia. Data was collected from AD, mild
cognitively impaired (MCI) and healthy control (HC) participants from the Australian
Imaging, Biomarkers and Lifestyle (AIBL) Flagship Study of Ageing, and from
participants in the Dominantly Inherited Alzheimer Network (DIAN) study.
This is the first study to investigate the retina or pupil flash response with respect to
brain plaque burden, the neuropathological hallmark of AD. This approach has allowed
further illumination of the connection between ocular changes and AD. It is also the
first study to investigate retinal vascular changes in AD using the cost-effective and
widely available retinal photography technique and hence is pertinent to the
development of a population screening test for AD.

II
The results demonstrate relationships between retinal vascular abnormalities,
neocortical plaque burden and AD, and also between pupil flash response parameters,
neocortical plaque burden and AD. Some ocular parameters that are found to be
different in AD are also associated with elevated neocortical plaque burden in pre-
clinical stages of AD, demonstrating potential as biomarkers for early, specific detection
of AD. In addition, some ocular parameters were associated with longitudinal increase
in neocortical plaque burden in healthy controls. These findings have been published or
accepted for publication in a number of peer-reviewed journal articles.
This thesis explores similarities between AD and the retinal diseases glaucoma and age-
related macular degeneration and investigates signs of retinopathy in AD. Advances in
research into these diseases, or pooling of research efforts, may provide novel directions
for therapies and tests, and hence may have broad impact in both ophthalmology and
neurology.
The ocular changes reported in this thesis show potential to contribute toward a non-
invasive, cost-effective screening test for pre-clinical AD and also to monitor response
to treatment. An accurate, early diagnostic test for AD would enable current and future
treatments to be more clinically effective, in addition to accelerating the development of
new treatments.
The findings of this thesis are considered relative to the existing literature.
III
Dedication
This work is dedicated to the research participants who donated their time for this study.
Quotes
A human being is part of a whole, called by us the 'Universe,' a part limited in time and
space. He experiences himself, his thoughts and feelings, as something separated from
the rest - a kind of optical delusion of his consciousness. This delusion is a kind of
prison for us, restricting us to our personal desires and to affection for a few persons
nearest us. Our task must be to free ourselves from this prison by widening our circles
of compassion to embrace all living creatures and the whole of nature in its beauty.
Albert Einstein, physicist born in Germany who formulated the special theory of
relativity and the general theory of relativity; Einstein also proposed that light consists
of discrete quantized bundles of energy (later called photons) (1879-1955).

IV
Declaration
This is to certify that:
(i) This thesis comprises my original work.
(ii) Due acknowledgements are made in the text and the acknowledgement
section to all other materials used and to the assistance of others.
(iii) This thesis has not previously been accepted for any other degree in this
or another institution.
(iv) This thesis has substantially been accomplished during enrolment in the
current degree.
(v) This thesis is less than 100,000 words in length, exclusive of tables,
illustrative matter and appendices.
Shaun M Frost
B.Sc. (Hons), M.Sc.
28th August 2013

V
Publications Arising from this Thesis
Published Papers*
1) Frost S, Martins RN, Kanagasingam Y (2010) Ocular biomarkers for early
detection of Alzheimer's disease. J Alzheimers Dis 22, 1-16.
2) Book Chapter “Retinal Screening for Early Detection of Alzheimer’s Disease”
Frost, Shaun; Martins, Ralph; Kanagasingam, Yogesan. In “Digital Teleretinal
Screening”, Kanagasingam, Yogesan; Goldschmidt, Leonard; Cuadros, Jorge
(Eds.), Springer 2012, ISBN 978-3-642-25809-1
3) Frost S et al. “Retinal Vascular Biomarkers for Early Detection and Monitoring
of Alzheimer’s Disease” Translational Psychiatry 3, e233, February 2013.
4) Frost S et al. “Pupil Flash Response for the Early Detection of Alzheimer’s
Disease”, Current Alzheimer Research, in press, accepted 24th July 2013.
5) Frost S et al. “Pupil flash response biomarkers distinguish amyloid precursor
protein mutation carriers from non-carriers”, Current Alzheimer Research, in
press, accepted 30th April 2013.
*
The first 2 manuscripts form the basis for the introduction Chapter 1. Manuscripts 3, 4
and 5 form the basis of Chapters 3, 4 and 5 respectively. The candidate contributed to
95% of the work in undertaking these projects and preparation of these manuscripts.
VI
Manuscripts Under Preparation
Frost S et al. “Alzheimer’s Disease and Age Related Macular Degeneration”
Frost S et al. “Alzheimer’s Disease and Glaucoma”
Frost S et al. “Retinal Central Reflex and Apolipoprotein E genotype”

VII
Table of Contents
Abstract ..............................................................................................................................I
Dedication ....................................................................................................................... III
Quotes ............................................................................................................................. III
Declaration ......................................................................................................................IV
Publications Arising from this Thesis .............................................................................. V
Table of Contents .......................................................................................................... VII
Acknowledgements .......................................................................................................... X
List of Figures .................................................................................................................XI
List of Tables................................................................................................................ XIII
List of Abbreviations..................................................................................................... XV
Chapter 1: Alzheimer’s Disease and Ocular Biomarkers: A Literature Review ........ 1
1.1) Alzheimer’s Disease.......................................................................................... 1

1.1.1) Treatments for Alzheimer’s Disease ......................................................... 7
1.1.2) Diagnosis for Alzheimer’s Disease ........................................................... 9
1.2) Vision in Alzheimer’s Disease ........................................................................ 14
1.3) Ocular Biomarkers for Early Detection of Alzheimer’s Disease .................... 16
1.3.1) Overview ................................................................................................. 16
1.3.2) Pupil Responses in Alzheimer’s Disease ................................................ 17
1.3.3) The Ocular Lens in Alzheimer’s Disease................................................ 22
1.3.4) The Aqueous and Vitreous Humor in Alzheimer’s Disease ................... 26
1.3.5) The Retina and Optic Disc in Alzheimer’s Disease ................................ 27
1.4) The Scientific and Clinical Significance of the Project .................................. 38
1.5) Hypotheses and Objectives ............................................................................. 40
1.5.1) Retinal Vascular Hypotheses .................................................................. 40
1.5.2) Pupillometric Hypotheses ....................................................................... 41
Chapter 2: Methodological Approaches ................................................................... 42
2.1) Introduction ..................................................................................................... 42

2.2) Participants and Ethical Approvals ................................................................. 43
2.3) Neuroimaging.................................................................................................. 45
2.4) Genetic Analysis ............................................................................................. 46
2.4.1) AIBL participants .................................................................................... 46
2.4.2) DIAN participants ................................................................................... 46
2.5) Retinal Photography and Grading ................................................................... 47
2.5.1) Semi-Automated Analysis of Retinal Topology ..................................... 48
2.5.2) Semi-Automated AMD analysis ............................................................. 52
2.5.3) Clinician Glaucoma Grading .................................................................. 53
2.5.4) Clinician Retinopathy Grading ............................................................... 53
2.5.5) Semi-Automated Analysis of Arteriolar Central Reflex ......................... 54
VIII
2.6) Pupillometry .................................................................................................... 55

2.7) Statistical Analysis .......................................................................................... 58
Chapter 3: Retinal Vasculature in Clinically Diagnosed and Pre-Clinical Sporadic

Alzheimer’s Disease: Published Findings...................................................................... 60
3.1) Introduction ..................................................................................................... 60

3.2) Study 1: Retinal Vasculature in Clinically Diagnosed Sporadic Alzheimer’s
Disease ........................................................................................................................ 61
3.2.1) Methods................................................................................................... 61
3.2.2) Cohort and Demographics ...................................................................... 62
3.2.3) Results ..................................................................................................... 62
3.2.4) Discussion ............................................................................................... 64
3.3) Study 2: Retinal Vasculature and Neocortical Amyloid Plaque burden ......... 67
3.3.1) Methods................................................................................................... 67
3.3.3) Results ..................................................................................................... 69
3.3.4) Discussion ............................................................................................... 71
Chapter 4: Pupil Flash Response in Clinically Diagnosed and Pre-Clinical Sporadic

Alzheimer’s Disease: Published Findings...................................................................... 76
4.1) Introduction ..................................................................................................... 76

4.2) Study 1: PFR in Clinically Diagnosed Sporadic Alzheimer’s Disease ........... 77
4.2.1) Methods................................................................................................... 77
4.2.3) Results ..................................................................................................... 78
4.2.4) Discussion ............................................................................................... 82
4.3) Study 2: PFR and Neocortical Amyloid Plaque burden ................................. 84
4.3.1) Methods................................................................................................... 84
4.3.3) Results ..................................................................................................... 87
4.3.4) Discussion ............................................................................................... 93
Chapter 5: Ocular Biomarkers and Early Onset Familial Alzheimer’s Disease ....... 98
5.1) Introduction ..................................................................................................... 98

5.2) Study Design ................................................................................................. 100
5.2.1) Cohort and Demographics .................................................................... 101
5.2.2) Genetic Analysis ................................................................................... 102
5.3) Results ........................................................................................................... 103
5.4) Discussion ..................................................................................................... 106
Chapter 6: Alzheimer’s Disease, Glaucoma and AMD .......................................... 109
6.1) Introduction ................................................................................................... 109

6.2) Alzheimer’s disease and age-related macular degeneration ......................... 110
6.2.1) Introduction ........................................................................................... 110
6.2.2) Methods................................................................................................. 115
6.2.3) Results ................................................................................................... 115
6.2.4) Discussion ............................................................................................. 118
6.3) Alzheimer’s Disease and Glaucoma ............................................................. 122
IX
6.3.1) Introduction ........................................................................................... 122

6.3.2) Methods ................................................................................................. 126
6.3.3) Results ................................................................................................... 126
6.3.4) Discussion ............................................................................................. 128
Chapter 7: Alzheimer’s Disease and Retinopathy .................................................. 134
7.1) Introduction ................................................................................................... 134

7.2) Clinician-Graded Retinopathy ...................................................................... 139
7.2.1) Methods ................................................................................................. 139
7.2.2) Results ................................................................................................... 140
7.2.3) Discussion ............................................................................................. 142
7.3) Image Analysis of Central Reflex ................................................................. 144
7.3.1) Methods ................................................................................................. 144
7.3.2) Results ................................................................................................... 145
7.3.3) Discussion ............................................................................................. 150
Chapter 8: General discussion and future directions .............................................. 157
8.1) General discussion ........................................................................................ 157

8.1.1) The retina and Alzheimer’s disease ...................................................... 158
8.1.2) The pupil response and Alzheimer’s disease ........................................ 165
8.2) Project Limitations and Suggestions for Future Research ............................ 168
8.3) Concluding remarks ...................................................................................... 173
References ..................................................................................................................... 175
Appendices .................................................................................................................... 206
Appendix 1.1. Consent Forms and Information Sheet .............................................. 206

X
Acknowledgements
First, I acknowledge the support and direction of three outstanding supervisors, Prof.
Ralph Martins, Prof. Yogesan Kanagasingam and Dr. Hamid Sohrabi.
This study was partially supported through the Science and Industry Endowment Fund
and the NIH Grant for the Dominantly Inherited Alzheimer Network Study (Grant
number: U19AG032438) with Principle Investigators John C Morris and Randall J
Bateman.
I also acknowledge the valuable contributions of the research participants who gave
generously of their time, and the researchers at the McCusker Alzheimer Research
Foundation who provided eager support for the inclusion of eye testing into the busy
research schedule. In particular, I am most thankful to Belinda Brown, Samantha
Gardiner and Sabine Matthaes for all their efforts.
I also value the contribution of my wife Michelle in keeping me motivated and moving
forward. Her wisdom, support and guidance over the years have been most important.
XI
List of Figures
Figure 1.1. The microscopic and macroscopic pathology of Alzheimer’s disease. .......... 3
Figure 1.2. Hypothetical model of Alzheimer’s disease pathological progression........... 4
Figure 1.3. Plaque burden in a healthy and an Alzheimer’s diseased (AD) brain. ......... 11
Figure 1.4. Prevalence curves of Alzheimer’s disease (AD) and Aβ plaques. ............... 12
Figure 1.5. Illustration of the visual pathways within the brain...................................... 15
Figure 1.6. Anatomy of the human eye. .......................................................................... 17
Figure 1.7. The ocular pupil and pupil flash response. ................................................... 18
Figure 1.8. Section of the human eye. ............................................................................. 23
Figure 1.9. Cortical cataract in the human intra-ocular lens. .......................................... 23
Figure 1.10. Equatorial supranuclear cataract in an ex-vivo human AD lens................. 24
Figure 1.11. Digital retinal photograph displaying the optic disc in the centre. ............. 28
Figure 1.12. Sectional diagram of the eye with schematic enlargement of the retina. ... 28
Figure 1.13. Anatomy of ocular circulation. ................................................................... 29
Figure 1.14. OCT scan showing the retinal layers around the fovea. ............................. 31
Figure 1.15. OCT scan circling the optic disc. ................................................................ 32
Figure 1.16. Illustration of changes to the optic disc and vision in Glaucoma. .............. 35
Figure 2.1. Retinal photography being carried out for a research participant................. 47
Figure 2.2. Retinal zones utilised for retinal vascular analysis. ...................................... 50
Figure 2.3. Illustration of retinal image analysis............................................................. 52
Figure 2.4. PFR data collection with NeurOptics™ VIP™-200 Pupillometer. ............. 55
Figure 2.5. Illustration of PFR response and parameters measured. ............................... 57
Figure 3.1. Retinal vascular parameters compared across AD and HC groups. ............. 64
Figure 3.2. Asymmetry Factor of the venular network (AFv) compared between groups.
................................................................................................................................. 70
Figure 3.3. Scatter plot for venular length to diameter ratio (LDRv) with SUVR, for
HC- and HC+ groups. ............................................................................................. 71
XII
Figure 3.4. Scatter plot for venular length to diameter ratio (LDRv) with 18-month
change in neocortical plaque burden (SUVR) for the HC group. ........................... 71
Figure 4.1. Mean pupil flash response, relative to resting pupil size, for HC (dashed
line; N=70) and AD (solid line; N=19) groups. ...................................................... 80
Figure 4.2. The PFR differences between healthy controls and Alzheimer’s patients. .. 80
Figure 4.3. Mean pupil flash response relative to resting pupil size. .............................. 88
Figure 4.4. Comparison of Mean Constriction Velocity (VC) between groups. ............ 88
Figure 4.5. PFR parameters and continuous neocortical plaque burden. ........................ 90
Figure 4.6. PFR parameters and longitudinal SUVR. ..................................................... 92
Figure 5.1. Alzheimer Biomarker Pathochronology in Autosomal Dominant AD. ..... 100
Figure 5.2. PFR differences in ADAD mutation carriers. ............................................ 105
Figure 6.1. How people with AMD might see the world. ............................................. 110
Figure 6.2. Retinal changes in AMD. ........................................................................... 112
Figure 6.3. Illustration of changes to the optic disc in Glaucoma. ............................... 123
Figure 6.4. Illustration of changes to the optic disc indicative of atrophy. ................... 123
Figure 7.1. Retinal photograph with clinical signs of retinopathy. ............................... 134
Figure 7.2. Retinal photograph with visible central reflex in both arterioles and venules.
............................................................................................................................... 135
Figure 7.3. Retinal photograph showing mild enhancement of the retinal arteriolar
central reflex (copper wiring). .............................................................................. 136
Figure 7.4. Retinal photograph showing marked enhancement of the retinal arteriolar
central reflex (silver wiring). ................................................................................ 136
Figure 7.5. Grading interface for central reflex quantification. .................................... 145
Figure 7.6. Central reflex ratio results. ......................................................................... 147

XIII
List of Tables
Table 1.1. Reported Ocular Changes in AD. .................................................................. 16
Table 1.2. Summary of reports of PFR changes in AD. ................................................. 21
Table 2.1. Stratification of Perth AIBL cohort. .............................................................. 45
Table 2.2. Description of the 19 retinal vascular parameters.......................................... 49
Table 2.3. PFR parameter descriptions. .......................................................................... 56
Table 3.1. Demographics and descriptive RVP analysis for HC and AD groups. ......... 63
Table 3.2. Demographics of the RVP neuroimaging cohort. ......................................... 69
Table 4.1. Demographics and Pupil Flash Response analysis for Healthy Control and
Alzheimer’s Disease groups.................................................................................... 81
Table 4.2. Demographics of the PFR neuroimaging cohort............................................ 86
Table 4.3. Demographics of the PFR longitudinal neuroimaging cohort. ...................... 87
Table 4.4. Linear associations between PFR parameters and continuous neocortical
plaque burden. ......................................................................................................... 89
Table 4.5. Linear associations between PFR parameters and 18 month change in SUVR.
................................................................................................................................. 91
Table 4.6. ANCOVA associations between PFR parameters and plaque burden increase.
................................................................................................................................. 93
Table 5.1. Demographic characteristics of the mutation carrier and non-carrier groups.
............................................................................................................................... 104
Table 6.1. Demographics and AMD analysis for HC and AD groups. ........................ 116
Table 6.2. Demographics and AMD analysis for HC+ and HC- groups. .................... 118
Table 6.3. Demographics and optic disc analysis for HC and AD groups. ................. 127
Table 6.4. Demographics and optic disc analysis for HC+ and HC- groups. .............. 128
Table 7.1. Demographics and retinopathy analysis for HC and AD groups. ............... 141
Table 7.2. Demographics and retinopathy analysis for HC+ and HC- groups. ........... 142
Table 7.3. Demographics and central reflex analysis for HC and AD groups. ............ 146
Table 7.4. Demographics and central reflex analysis for HC ε4 carriers and non-
carriers. .................................................................................................................. 148
XIV
Table 7.5. Demographics and central reflex analysis for AD ε4 carriers and non-
carriers. .................................................................................................................. 149
Table 7.6. Demographics and CR analysis for HC- and HC+ groups. ........................ 150
XV
List of Abbreviations
AAO Anticipated age at onset

Aβ Amyloid-beta protein
ABI Applied Biosystems ™
ABS Australian Bureau of Statistics
ACh Acetylcholine
AChEI Acetylcholinesterase inhibitors
AD Alzheimer’s disease
ADAD Autosomal dominant Alzheimer’s disease
ADRDA Alzheimer’s Disease & Related Disorders Association
AF Autofluorescence
AIBL Australian Imaging, Biomarkers and Lifestyle study of ageing
AMD Age-related macular degeneration
aMCI Amnesic Mild Cognitive Impairment
ANOVA Analysis of variance
ANCOVA Analysis of covariance
APOE Apolipoprotein E as gene
APOE Apolipoprotein E as a protein
APP Amyloid precursor protein
AUC Area under the curve
AV Arterio-venular
BASE Beta site APP cleaving enzyme
BDNF Brain derived neurotrophic factor
BP Blood pressure
CAA Cerebral amyloid angiopathy
CAMCOG-R Cambridge Cognitive Examination-Revised
CD Cognitive decline
CDR Cup-disc ratio
CSF Cerebrospinal fluid
CI Confidence interval
CNS Central nervous system
CNTF Ciliary neurotrophic factor
CNV Choroidal neovascularisation
CR Central reflex
CVLT® California Verbal Learning Test
DAT Dementia of the Alzheimer's Type
DF Degrees of freedom
DIAN Dominantly inherited Alzheimer’s disease
DLB Dementia with Lewy bodies
DLS Dynamic light scattering
DR Diabetic retinopathy
DSM-III-R Diagnostic & Statistical Manual of Mental Disorders-3rd Edition-
Revised
DSM-IV-TR Diagnostic & Statistical Manual of Mental Disorders, 4th Edition-
Text Revision
DNA Deoxyribonucleic acid
ELISA Enzyme linked immunosorbant assay
EOFAD Early onset familial Alzheimer’s disease
EYO Estimated years to onset
XVI
FDA Food and Drug Administration (USA)

FDG Fluorodeoxyglucose
FDR False discovery rate
FGF Fibroblast growth factor
FTD Frontotemporal dementia
GA Geographic atrophy
GDS Geriatric Depression Scale
HC Healthy control
Hcy Homocysteine
HDL High density lipoprotein
HPH Hollywood Private Hospital
ID Identification
IOP Intra-ocular pressure
IQ Intelligence quotient
LDL Low-density lipoprotein
LOAD Late-onset Alzheimer’s disease
MARF McCusker Alzheimer’s Research Foundation
MC Mutation carrier
MCI Mild cognitive impairment
3MS Modified Mini Mental State Examination
MMSE Mini Mental Status Examination
MRI Magnetic resonance imaging
NC Non (mutation) carrier
NCRAD National Cell Repository for Alzheimer’s Disease
NFTs Neurofibrillary tau tangles
NGF Neurotrophic growth factor
NHMRC National Health & Medical Research Council of Australia
NIA National Institute of Ageing
NIMH National Institute of Mental Health
NINCDS The National Institute of Neurological & Communicative Disorders
& Stroke
NMDA N-methyl-D-Aspartate
NPs Neurotic plaques
NRR Neuro-retinal rim
NS Non-significant (statistical result)
NTs Neuropil threads
OAG Open angle glaucoma
OCT Optical Coherence Tomography
OD Optic disc
OR Odds ratio
PCR Polymerase chain reaction
PERG Pattern electroretinogram
PET Positron emission tomography
PFR Pupil flash response – parameters listed below;
D1 Maximum pupil diameter (mm)
D2 Minimum pupil diameter (mm)
T1 Constriction Latency (sec)
Amp Constriction Amplitude (mm)
%Cons. Constriction Percentage (%)
VC Mean Constriction Velocity (mm/sec)
VCmax Maximum Constriction Velocity (mm/sec)
XVII
ACmax Maximum Constriction Acceleration (mm/sec2)

VD Mean Dilation Velocity (mm/sec)
75%RT 75% Recovery Time (sec)
T2 Latency to Minimum Pupil Size (sec)
PR3.5 Percentage Recovery after 3.5 seconds (%)
PI Pathologically impaired
PiB Pittsburgh Compound-B
PS Presenilin
PS1 Presenilin 1
PS2 Presenilin 2
RFLP Restriction fragment length polymorphism
RGC Retinal ganglion cells
RNFL Retinal Nerve Fiber Layer
ROC Receiver operator characteristic
RPE Retinal pigment epithelium
RVP Retinal Vascular Parameters – listed below;
CRAE Central Retinal Arteriolar Equivalent caliber
CRVE Central Retinal Venular Equivalent caliber
AVR Arteriole - Venular Ratio (CRAE / CRVE)
FDa Fractal Dimension of Arteriolar network
FDv Fractal Dimension of Venular network
BSTDa Zone B Standard Deviation Arteriole
BSTDv Zone B Standard Deviation Venule
TORTa Curvature Tortuosity Arteriole
TORTv Curvature Tortuosity Venule
Num1stBa Number of 1st Branching Arterioles
Num1stBv Number of 1st Branching Venules
BCa Branching Coefficient Arteriole
BCv Branching Coefficient Venule
AFa Asymmetry Factor Arteriole (or Asymmetry Ratio)
AFv Asymmetry Factor Venule (or Asymmetry Ratio)
JEa Junctional Exponent Deviation for Arterioles
JEv Junctional Exponent Deviation for Venules
LDRa Length Diameter Ratio Arteriole
LDRv Length Diameter Ratio Venule
SD Standard deviation
SIVA Singapore I vessel assessment
SLO Scanning laser ophthalmoscopy
SMCs Subjective memory complaints or Subjective memory complainers
SNPs Single nucleotide polymorphisms
SPECT Single photon emission computed tomography
SPs Senile plaques
SPSS Statistical package for social sciences
STR Short tandem repeat
SUVR Standardised uptake value ratio (for PiB-PET)
TGF- β Transforming growth factor- β
USA United States of America
UV Ultraviolet radiation
VD Vascular dementia
1
Chapter 1: Alzheimer’s Disease and Ocular Biomarkers: A
Literature Review
1.1) Alzheimer’s Disease
Alzheimer’s disease (AD) is an age-related neurodegenerative disease. It is the most
common cause of dementia, which describes a serious loss of cognitive ability beyond
that expected from normal ageing. Dementia had a global prevalence of 35.6 million
people in 2010, predicted to reach 65.7 million by 2030 (Alzheimer's Disease
International, 2010). AD is characterised clinically by a progressive decline in memory,
learning and executive function and neuropathologically by the presence of cerebral
amyloid deposits. In addition to the debilitating symptoms endured by AD patients, the
disease imposes a huge social and economic burden on society, with an estimated
worldwide cost of dementia in 2010 of 604 billion, or about 1% of the world’s gross
domestic product (Alzheimer's Disease International, 2010).
Currently, AD is an incurable, progressive and terminal disease usually diagnosed in
people over 65 years of age (Gandy, 2011). It affects 1 in 8 people aged over 65 and
nearly half of those aged over 85 (Alzheimer's Association, 2012). The late-onset form
of AD (LOAD) is the most common form of the disease and in the majority of cases,
where there is no evidence of it being inherited, it is termed “sporadic AD”. A rare form
of AD, termed “early-onset familial AD” (EOFAD), is inherited in an autosomal
dominant manner, and can occur in people as young as 26 years of age (Bertram et al.,
2010, Snider et al., 2005).
Mild Cognitive Impairment (MCI) represents a transitional state between healthy ageing
and dementia. While individuals with MCI demonstrate evidence of cognitive decline,
2
they do not meet established criteria for a diagnosis of dementia. When memory loss is
the primary symptom, amnestic MCI (aMCI) is diagnosed, which is considered to be a
prodromal stage of AD. Reported annual rates of conversion from MCI to AD are about
10-15% compared to 1%-3% for normal individuals (Grundman et al., 2004, Petersen et
al., 1999, Petersen et al., 2001, Rountree et al., 2007, Visser et al., 2006, Wang et al.,
2006). New criteria classify MCI into 3 categories: (1) MCI based on core clinical
criteria, (2) MCI due to Alzheimer's disease (intermediate or high probability), and (3)
MCI unlikely to be due to Alzheimer's disease (Albert et al., 2011, Yaari et al., 2011).
While the presence of core clinical symptoms defines the presence of MCI, further
testing of biomarkers (such as neuroimaging of cerebral amyloid deposits) are required
to gauge the probability that the symptoms are an early manifestation of Alzheimer's
disease.
AD is histopathologically characterised by a substantial loss of neurons in the brain,
atrophy of the brain, as well as the deposition of extracellular amyloid β (Aβ) plaques
and intracellular neurofibrillary tau tangles (NFT) (see Figure 1.1) (Tiraboschi et al.,
2004). The factors that may cause or accelerate the development of AD are not fully
understood, and the exact contribution of plaques and tangles in causing symptoms of
AD also remain to be fully established.

3
A. B.
______________________________________________________________________
Figure 1.1. The microscopic and macroscopic pathology of Alzheimer’s disease.
A. Illustration of an AD neuron with amyloid-beta plaque and tau tangle pathology.
B. Comparison of a healthy brain and a brain showing atrophy due to severe AD (The National Institute
on Aging & The National Institutes of Health, Alzheimer’s disease, Unraveling the mystery, 2008;
p.26,32).
General consensus in the field supports the “Amyloid Cascade Hypothesis”, which
posits that deposition of Aβ in the brain is a crucial step in the process leading to AD
(Hardy and Higgins, 1992, Reitz, 2012, Selkoe, 1991). Many studies suggest that the
effect of Aβ is at least partly mediated by increased tau phosphorylation. A disease
model has emerged beginning with deregulated Aβ proteostasis, leading to changes in
cerebro-spinal fluid (CSF) Aβ concentrations, cerebral Aβ plaques, tau pathology
(tangles), brain atrophy and cognitive decline (see Figure 1.2) (Jack et al., 2013, Reitz,
2012).
4
______________________________________________________________________
Figure 1.2. Hypothetical model of Alzheimer’s disease pathological progression.
(A) Model illustrating the current understanding of the time course of dynamic biomarkers in AD
progression.
CSF Aβ42 / tau = cerebro-spinal fluid Aβ42 / tau concentrations, PET = positron emission tomography
brain imaging scan for Amyloid (Aβ) or FDG (fluorodeoxyglucose: brain glucose metabolism), MRI =
magnetic resonance imaging structural brain scan, Aβ = amyloid β, MCI = mild cognitive impairment.
(B) The vertical black line denotes a given time (T) and the biomarker profile at that time.
Jack et al. Lancet Neurol. 2013 (Jack et al., 2013).
The major protein component of the amyloid plaques is a peptide known as amyloid β
(Aβ). Aβ peptides range from 39 to 43 amino acid residues in length. The longer (Aβ42
or Aβ43) peptides aggregate easily into fibrils, and small soluble oligomers of Aβ are
believed to be a neurotoxic form of Aβ, whereas the large insoluble aggregates and
5
plaques are relatively inert (Shankar et al., 2008). Nevertheless, the Aβ plaques are a
sign of deregulated Aβ proteostasis, and they are in complex equilibrium with other Aβ
species, in particular soluble oligomers, which may attack cell membranes and induce
tau tangle formation, leading to brain atrophy.
The Aβ peptide is proteolytically derived from its parent molecule, the amyloid-β
protein precursor (APP). APP is an integral membrane protein that is metabolized to a
secreted form. A secreted isoform of APP has been implicated in blood clotting (Xu et
al., 2005) and as a regulator of neural plasticity and post-injury repair (Turner et al.,
2003). Aβ peptides are produced as a result of sequential cleavage of APP by enzymes
known as the β-secretase (also known as β-site APP cleavage enzyme or BACE) and γ-
secretase. The most common form is Aβ40, but it is the second most common form,
Aβ42, which is more neurotoxic and is thus associated with disease states (Kang et al.,
1987).
Aβ has been shown to have a constrictive effect on the cerebral vasculature (Suo et al.,
1998), and interestingly, to be neuroprotective at low physiological concentrations
(Chan et al., 1999). For this reason Aβ has been suggested to have a damage response
role in the brain, by sealing the vasculature reducing brain oxygen requirement and
combating oxidative stress (Hardy and Cullen, 2006). A recent study also demonstrated
that Aβ has significant antimicrobial ability against clinically relevant organisms,
introducing an interesting hypothesis that infection might have a role in some forms of
AD (Soscia et al., 2010). Whatever the role of Aβ in the healthy body, a large number
of in vitro and in vivo AD studies, particularly those of EOFAD, have shown that high
levels of Aβ cause oxidative stress in the brain, resulting in synaptic loss, cell
membrane damage, inflammation and ultimately result in neuronal cell death

6
(Butterfield and Lauderback, 2002, Lue et al., 1999). Such studies have shown that
EOFAD-associated mutations in the APP gene or the presenilin 1 and 2 genes
(presenilin being an essential component of the -secretase enzyme) have been found
either to increase total Aβ levels or to increase the production of Aβ42 (Tanzi and
Bertram, 2001, Yin et al., 2007). Such increases in Aβ levels have been implicated in
the pathogenesis of both familial and sporadic AD (Butterfield and Lauderback, 2002,
Lue et al., 1999).
Neurofibrillary tau tangels (NFT) are insoluble, twisted fibers of a protein called tau.
Phosphorylated tau stabilizes the internal structure of healthy neurons, but in AD, tau
proteins are hyperphosphorylated, resulting in the formation of intracellular amyloid
deposits. Again, recent evidence points to soluble tau rather than NFTs as a cause of
neuronal loss in AD (Ramsden et al., 2005, Santacruz et al., 2005, Spires et al., 2006).
When researchers suppressed the mutant tau gene in a mouse model of AD,
neurodegeneration was halted and cognitive performance improved even though NFT
continued to accumulate (Santacruz et al., 2005). While tau hyperphosphorylation is
not specific to AD, there is strong evidence to indicate that it is essential for A-induced
cognitive decline to occur.
The primary neurotransmitter deficit in AD is acetyl-choline (Davies and Maloney,
1976, Herholz et al., 2004, Perry et al., 1981, Tohgi et al., 1994). The cholinergic
hypothesis was the first theory proposed to explain AD (Bartus, 2000, Bartus et al.,
1982), stating that acetyl-choline deficiency is central to AD pathogenesis and cognitive
decline (Contestabile, 2011). In support of this hypothesis, there is substantial evidence
of a bi-directional interaction between cholinergic function and processing of APP
(Pakaski and Kalman, 2008). Additionally, loss of cholinergic fibers has been reported
in various brain areas but found to be most severe in memory-related areas (Dournaud et
7
al., 1995, Geula and Mesulam, 1996), consistent with memory being the primary
cognitive deficit in AD. However, drugs currently administered to enhance cholinergic
function in AD have had only temporary success at improving cognition and do not halt
the progress of the disease (Courtney et al., 2004, Doody et al., 2001, Doody et al.,
2008). Moreover, not all AD patients respond to cholinergic drugs and the differences
between responders and non-responders remain unclear (Connelly et al., 2005, Lemstra
et al., 2007). While the failure of cholinergic drugs to cure AD has been seen by some
researchers as disproving the cholinergic hypothesis, others are continuing to investigate
the role of acetyl-choline in AD, ageing and dementia (Craig et al., 2011).
In recent years, much evidence has been gathered to show that several factors contribute
to the risk of developing AD. These include diabetes, mid-life obesity, and a history of
heart disease or symptoms typically associated with heart disease such as high levels of
low density lipoproteins (LDL) together with low levels of high density lipoproteins (HDL)
(Bates et al., 2009).
1.1.1) Treatments for Alzheimer’s Disease
At present there is no cure or disease modifying drug to effectively treat AD, but there
are drugs that have proven to be beneficial at the level of reducing some symptoms for
up to 18 months. There is an urgent need for effective treatments, with the number of
cases worldwide forecast to exceed 100 million by 2050 (Brookmeyer et al., 2007).
Currently available drugs can delay or alleviate symptoms but do not slow the
progression of the disease. Pharmaceutical drugs currently available to treat the
cognitive manifestations of AD include acetylcholinesterase inhibitors and an N-
methyl-D-aspartate (NMDA) receptor antagonist. To compensate for acetylcholine
deficiency, cholinesterase inhibitors (AChEI; inhibitors of the enzymes that hydrolyze

8
acetylcholine released in the synaptic cleft) were developed. Three such drugs
(donepezil, rivastigmine and galantamine) have been given United States Food and
Drug Administration (FDA) approval and have become widely used in many countries.
Research is continuing into drugs designed to prevent or reduce Aβ production, to break
up Aβ plaques or to prevent metal ion-Aβ interactions which may accelerate Aβ
aggregation (Bush, 2002, Bush, 2003). For example, one suggested avenue of treatment
involves modulating the activity of β and γ-secretases to produce mainly Aβ40 instead
of the more neurotoxic Aβ42 peptide (Weggen et al., 2001).
In other studies, immunizing transgenic AD mouse models with human Aβ42 peptide
was found to prevent the build-up of Aβ plaques (Schenk et al., 1999) and to prevent
memory impairment. However, a successful outcome was not achieved in early human
clinical studies due to serious side-effects. Immunotherapies that either prevent plaque
deposition or enhance removal of plaques are still being actively investigated by
pharmaceutical companies with a number of them in either phase II or III clinical trials.
Anti-aggregation agents with the aim of removing plaques and/or preventing Aβ
fragments from aggregating are also being examined (Parker et al., 2002) and are
expected to enter clinical trials within the next 12 months.
Non-pharmacological therapeutic approaches are also currently being investigated. Of
these, lifestyle modification, particularly physical activity, nutrition and cognitive
training are gaining considerable attention in the field. It has been demonstrated that a
cognitively stimulating environment can reduce Aβ deposition in transgenic mice
(Lazarov et al., 2005), and that increased cognitive activity in humans can reduce the
risk of AD (Wilson et al., 2002). A possible explanation for the mechanism by which
cognitive stimulation might delay onset of AD has been identified (Cirrito et al., 2005).
9
Researchers found that synaptic activity increases Aβ in local interstitial fluid.
Cognitive stimulation transfers electrical activity away from brain regions with high
‘default activity’, hence reducing Aβ levels in these regions – particular regions which
in fact end up with high levels of Aβ deposition in AD (Buckner et al., 2005).
Pharmaceutical treatments can be made available for AD patients only after they have
been clinically diagnosed with the disease. Unfortunately, AD is difficult to diagnose
with certainty. In addition, the cognitive symptoms on which diagnosis is based only
become apparent after irreversible brain damage has already occurred. Hence the search
for better AD treatments needs to be coupled with research into early detection of the
disease (Gandy, 2011).
1.1.2) Diagnosis for Alzheimer’s Disease
There is currently no definitive pre-mortem diagnosis for AD. Conclusive diagnosis of
AD is only achieved following post-mortem examination of the brain for the presence of
plaques and tangles. A pre-mortem diagnosis of “probable AD” is currently made by
clinical observations and testing of cognitive capacity and memory loss, usually using
National Institute of Neurological and Communicative Disorders and Stroke and the
Alzheimer’s disease and Related Disorders Association criteria (NINCDS-ADRDA)
(McKhann et al., 1984). Newer clinical and research guidelines classify AD into 3
categories: (1) probable Alzheimer's disease dementia, (2) possible Alzheimer's disease
dementia, and (3) probable or possible Alzheimer's disease dementia with evidence of
supportive biomarkers (Yaari et al., 2011). Other dementias and conditions such as
depression can have similar symptoms, often confounding diagnosis (Schaffer and
Donlon, 1983). A diagnostic error rate of about 10-15% has been reported for AD
(Galasko et al., 1994, Jellinger, 1996, Thal et al., 2006).

10
A diagnosis of probable or possible AD is only determined when the condition has
progressed significantly and considerable neurological damage has already occurred.
Clinicopathologic studies at autopsy support the hypothesis of a protracted
asymptomatic stage of AD, with brain pathology estimated to begin 10-15 years prior to
cognitive decline (Braak and Braak, 1997, GomezIsla et al., 1996, Hulette et al., 1998,
Markesbery, 2010, Markesbery et al., 2006, Morris and Price, 2001, Price et al., 2001).
The increasing incidence of AD, along with the need to treat the disease before the brain
is irreversibly damaged, calls for a sensitive and specific screening technology to
identify high risk individuals before cognitive decline begins.
Potential biomarker candidates for AD-screening are being sought from many fields
including structural and functional neuroimaging, proteomic analysis of blood and
cerebro-spinal fluid (CSF) and genetic analysis (Bertram and Tanzi, 2008, Corder et al.,
1993, Fagan et al., 2006, Harold et al., 2009, Lambert et al., 2009, Morris et al., 2009,
Rowe et al., 2010, Rowe et al., 2007, Sunderland et al., 2003, Thal et al., 2006). Two
biomarkers are showing particular promise, firstly brain Aβ plaques imaged using
Positron Emission Tomography (PET) with C-11 PiB or F18 ligands (Fagan et al.,
2006, Morris et al., 2009, Rowe et al., 2010, Thal et al., 2006), and secondly CSF
concentrations of beta amyloid (Aβ), total tau and phosphorylated tau peptides
(Blennow et al., 2010, Fagan et al., 2006, Sunderland et al., 2003, Thal et al., 2006).
However, while these are valuable diagnostic and secondary screening biomarkers, they
are not suited to population screening.
Cortical amyloid plaque burden can be evaluated in vivo using PET neuroimaging with
injected ligands such as Pittsburgh Compound-B (PiB), which selectively bind to Aβ
plaques (see Figure 1.3) (Fagan et al., 2006, Morris et al., 2009, Rowe et al., 2010, Thal
et al., 2006). PiB-PET imaging studies have revealed that not only do AD patients
11
exhibit high PiB retention, but also about 30% of cognitively normal elderly individuals
(Fagan et al., 2006, Rowe et al., 2010, Rowe et al., 2007). High PiB retention is
associated with progression to symptomatic AD, hence evidence is building that PiB-
PET imaging provides a test to identify pre-clinical AD (Morris et al., 2009, Okello et
al., 2009, Pike et al., 2007, Rowe et al., 2010, Sperling et al., 2011). Figure 1.4
illustrates the parallel prevalence curves of AD and plaque burden in HC, suggesting
that PET imaging has the potential to detect AD approximately 15 years before
cognitive symptoms arise (Rowe et al., 2010). PET imaging has become highly useful
for AD research purposes, but the expense and invasiveness of the procedure and the
limited availability of PET facilities curtail its usefulness as a primary screening
technology for AD. Similarly, while changes in the concentrations of proteins (Aβ and
tau) in the CSF have been reported in AD, the CSF procedure is invasive and patient
compliance is anticipated to be low.
______________________________________________________________________
Figure 1.3. Plaque burden in a healthy and an Alzheimer’s diseased (AD) brain.
PiB-PET neuroimaging scans of a healthy control and an AD brain, red and yellow coloring represents
high PiB binding (high SUV: standardised uptake value ratio), indicating high plaque load in these
regions. From Klunk et al., Ann Neurol., 2004 (Klunk et al., 2004).
12
Prevalence
of PiB+ve PET
60 in HC
50
Prevalence of plaques
Prevalence (%)
40 in HC
(Davies, 1988, n=110)
(Braak, 1996, n=551)
30 (Sugihara, 1995, n=123)
~15 yrs
20 Prevalence
of AD
(Tobias, 2008)
10
0
30 40 50 60 70 80 90 100
Age (years)
______________________________________________________________________
Figure 1.4. Prevalence curves of Alzheimer’s disease (AD) and Aβ plaques.
Prevalence curve of Alzheimer’s disease (AD) by age, and parallel prevalence curve of amyloid-β (Aβ)
plaques in asymptomatic individuals, by age. PiB+ve PET refers to high plaque burden as measured by
PiB-PET neuroimaging, HC refers to healthy controls (asymptomatic individuals). From Rowe et. al.
(Rowe et al., 2010).
Changes in blood plasma concentration of a number of molecules have been reported to
occur in AD and also prior to subsequent AD diagnosis, including Aβ, tau,
homocysteine and various proteins linked to inflammation or cardiovascular diseases
(Mayeux and Schupf, 2011, Schupf et al., 2008). There have, however, been
inconsistent data across studies. More work is required to elucidate the time-course of
blood biomarker changes as AD progresses, and to establish if blood testing for AD can
be clinically useful.
Genetic association studies have revealed more than 20 genes that have a significant
effect on AD risk (Bertram and Tanzi, 2008, Harold et al., 2009, Lambert et al., 2009).
While these genes may contribute to identifying high risk individuals, they are not
sufficient on their own to make a diagnosis, nor is the major genetic risk factor, the
apolipoprotein E (APOE) 4 allele, which is associated with up to 50% of all AD cases

13
(Corder et al., 1993, Selkoe, 2001). By contrast, the APOE ε2 allele lowers the risk of
AD (Corder et al., 1994). APOE ε4 has been implicated in modulating the metabolism
and aggregation of Aβ (Bu, 2009). Individuals with one copy of the APOE ε4 allele
have a 2-3 fold increased risk of developing AD by age 85 and those with two copies
have a 12 fold increased risk compared to the general population (Corder et al., 1993).
The combined genetic component of sporadic AD results in individuals with a first-
degree relative with dementia having a 10-30% increased risk of AD (van Duijn et al.,
1991). The rarer EOFAD on the other hand is primarily a genetic disorder, with certain
mutations in APP, Presenilin 1 and Presenilin 2 known to cause this disease (Tanzi and
Bertram, 2001, Yin et al., 2007), which affects carriers of specific gene mutations with
penetration approaching 100% (Sherrington et al., 1996).
Neuroimaging studies utilizing magnetic resonance imaging (MRI) have identified that
atrophy of the brain, in particular the hippocampus and entorhinal cortex, is a strong
predictor of AD and cognitive decline (see Figure 1.1) (Apostolova et al., 2006,
Krasuski et al., 1998, Mungas et al., 2005). Functional MRI studies have also found that
AD is associated with reduced brain activity in the parietal and hippocampal regions
(Rombouts et al., 2000). However these structural and functional changes are not
specific to AD, with substantial anatomical overlap between AD and other
neurodegenerative disorders and normal aging. The changes may only be detectable too
late in the disease to be useful for early detection (see Figure 1.2). In addition, MRI
scanning is time-consuming and expensive, and many patients have contraindications
for undergoing MRI scanning, such as claustrophobia, cardiac pacemakers and allergic
reactions to contrast materials.
Despite all these efforts, there is still no screening test or clinically validated biomarker
available for AD. The absence of a suitable screening technology for AD has motivated
14
some researchers to look for biomarkers that might exist elsewhere in the body,
including the eye (Frost et al., 2010). There is hope that the eye might yield biomarkers
that can contribute to an AD-specific screening test, possibly in combination with blood
protein or other markers.
1.2) Vision in Alzheimer’s Disease
Visual disturbance is often an early complaint of AD patients (Katz and Rimmer, 1989,
Sadun et al., 1987) and studies have reported reduced visual performance on tests of
visual field (Trick et al., 1995, Whittaker et al., 2002), color vision (Cogan, 1987,
Cronin-Golomb et al., 1993, Pache et al., 2003), contrast sensitivity (Crow et al., 2003,
Lakshminarayanan et al., 1996, Nissen et al., 1985), backward masking (Mendola et al.,
1995, Schlotterer et al., 1984), visual attention, motion perception, shape-from motion,
visuo-spatial construction, visual memory (Gilmore et al., 1994, Mielke et al., 1995,
Morrison et al., 1991), delayed saccadic initiation and movement and fixation problems
(Fletcher and Sharpe, 1988, Mendez et al., 1990, Sadun and Bassi, 1990, Sadun et al.,
1987). However, none of these deficiencies are specific to AD. The current literature is
controversial and reflects the need for larger and more rigorous studies to be undertaken
before the significance of visual deficits in AD can be conclusively evaluated.
Reported visual deficits in AD have generally been attributed to neuronal damage in the
visual pathways of the brain (Leuba, 1995, McKee et al., 2006) (see Figure 1.5) as well
as deficiency of the neurotransmitter acetylcholine in AD, which is important in visual
processing (Bentley et al., 2008, Herholz et al., 2004, Nobili and Sannita, 1997). Indeed
there is evidence that during the pathogenesis of AD, plaques and tangles occur in
visual processing brain regions prior to their occurrence in the hippocampus (McKee et
15
al., 2006). This supports the hypothesis that visual deficits may be early manifestations
of AD pathology; however visual cortical regions are left mostly spared from
downstream pathology such as NFT and atrophy (Braak and Braak, 1991, Braak and
Braak, 1995, Braak and Braak, 1997, Braak et al., 2006, Thal et al., 2000).
Several studies have demonstrated that visual impairment experienced by some patients
with AD primarily resulted from involvement of primary visual and association cortex,
rather than changes in the retina or optic nerve (Cronin-Golomb et al., 1991, Rizzo et
al., 1992). However, apart from dysfunction in the central visual regions, careful study
of retinal pathology and function in AD has suggested that retinal degeneration also
plays an important role in visual impairment. Research is underway to investigate the
hypothesis that there might be specific pathological changes in the eye that accompany
the disease. If the eye does harbor an endophenotype of AD, this would give hope for an
ocular diagnosis of AD as well as opening up a new avenue for finding other genetic
determinants of the disease. The following section describes reported AD-associated
changes to the eye.
______________________________________________________________________________________________
Figure 1.5. Illustration of the visual pathways within the brain.
Brain regions and pathways involved in visual processing, from the retina, along the optic tracts to the
thalamus and visual cortex (the Department of Neuroscience, Mount Sinai School of Medicine).
16
1.3) Ocular Biomarkers for Early Detection of Alzheimer’s
Disease
1.3.1) Overview
Ocular changes to the pupil, lens, retina and optic disc (optic nerve head) have been
reported to accompany AD (see Figure 1.6 for ocular anatomy). A summary of these
reports is provided in Table 1.1 and discussed in the following sections.
Table 1.1. Reported Ocular Changes in AD.

Part of Reported Ocular Changes in Journal Ref. N (AD,
the eye AD Control)
Pupil Enhanced pupil response to Science (Scinto et al., 1994) 19,32
Cholinergic eye drops Neuroreport (Arai et al., 1996) 25,24
cholinergic drops J Neurol Neurosurg (Idiaquez et al., 1994) 26,23
Neurobiol Aging (Iijima et al., 2003) 14,30
Acta Neurol Scand (Gomez-Tortosa et al., 1996) 24,50
Neuropsychobiol (Grunberger et al., 1999) 29,29
Biol Psychiatry (Kalman et al., 1997) 67,80
Rev Neurol (Robles et al., 1996) 10,20
Nippon Ronen (Kono et al., 1996) 53,29
Altered pupil flash response Aging Clin Exp (Fotiou et al., 2007) 23,23
Int J Psychophysiol (Fotiou et al., 2000) 10,5
Res (Granholm et al., 2003)
Int J Psychophysiol 15,30
J Neurol Neurosurg (Prettyman et al., 1997) 9,9
Lens Aggregation of Aβ, Supra- Lancet (Goldstein et al., 2003) 9,8
nuclear cataract
nuclear cataract (Berisha et al., 2007)
Retina Narrow vein blood column, Invest Ophthal 9,8
decreased venular blood flow
RNFL thinning Invest Ophthal (Berisha et al., 2007) 9,8
Neurosci Lett (Paquet et al., 2007) 26,38
RNFL abnormalities and cell Acta Neurol Scand (Hedges et al., 1996) 26,23
ninning
Loss Arch Ophthal (Tsai, 1991) 26,30
Neurology (Danesh-Meyer et al., 2006) 40,50
Aβ plaques in the retina Neuroimage (Koronyo-Hamaoui et al., 2010) 13,5
Abnormal pattern- Ann Neurol (Katz et al., 1989) 6,6
electroretinogram (PERG) Ann Neurol (Trick et al., 1989) 13,30
Optic Optic disc pallor, pathologic Acta Neurol Scand (Hedges et al., 1996) 26,23
Disc disc cupping, thinning of Arch Ophthal (Tsai, 1991) 30,32
the neuro-retinal rim Neurology (Danesh-Meyer et al., 2006) 40,50
N refers to the number of participants (Alzheimer’s disease and controls indicated separately).
Ref. indicates the bibliographic reference of the publication.
17
______________________________________________________________________
Figure 1.6. Anatomy of the human eye.
1.3.2) Pupil Responses in Alzheimer’s Disease
The pupil is the aperture stop of the eye and controls the retinal illumination (see Figure
1.7). The size of the pupil is regulated by the brain in response to the signals it receives
from the eyes. Pupil size changes with brightness of incident light, emotions (e.g. fear),
pain, cognitive tasks and with use of certain drugs (e.g. alcohol, opioids).
The pupil flash response (PFR) is the response of the pupil to a bright flash of light,
involving rapid contraction followed by re-dilation back to original size (Figure 1.7).
The neurotransmitters responsible for these two processes are acetyl-choline
(constriction) and norepinephrine (dilation). As the primary neurotransmitter deficit in
AD is acetyl-choline (Herholz et al., 2004, Tohgi et al., 1994), pupil responses have
become a research topic for AD diagnosis and monitoring (Ferrario et al., 1998, Fotiou
et al., 2007, Fotiou et al., 2000, Granholm et al., 2003, Prettyman et al., 1997, Tales et
al., 2001). It is possible that AD neurological changes (atrophy, cholinergic depletion)

18
might alter pupil control systems such that PFR parameters could be used for cost-
effective, non-invasive AD screening or monitoring.
A B
______________________________________________________________________
Figure 1.7. The ocular pupil and pupil flash response.
A. Picture of the human eye. The pupil is the central transparent aperture (appearing as black), surrounded
by the coloured iris.
B. An illustrated plot of the pupil flash response, showing the pupil contraction and re-dilation resulting
from a bright flash of white light.
During the 1990’s a number of studies investigated the influence of AD on the effect of
pharmacological drugs delivered for pupil constriction or dilation. A hypersensitive
pupil response to a cholinergic agonist (pilocarpine - constriction) or antagonist
(tropicamide - dilation) was reported for AD patients (Arai et al., 1996, Gomez-Tortosa
et al., 1996, Grunberger et al., 1999, Idiaquez et al., 1994, Iijima et al., 2003, Kalman et
al., 1997, Kono et al., 1996, Pomara and Sitaram, 1995, Robles et al., 1996, Scinto et
al., 1994). A proposed explanation for this hypersensitive response was cholinergic
dysfunction extending to the parasympathetic oculomotor system or iris nerve cells
specifically, although in this case one would expect to find agonist hypersensitivity with
antagonist sub-sensitivity, or vice versa. Alternative explanations more consistent with
the dual hypersensitivity include AD related damage to the locus coeruleus brain region
which is involved in pupillary control (Hou et al., 2006) or increased corneal
penetration of the cholinergic eye-drops. One study using a fluorescent marker to
evaluate corneal penetration of tropicamide found no difference between AD and

19
controls (FitzSimon et al., 1997), but further studies are required to confirm this result.
It should also be noted that not all studies controlled for medications with
anticholinergic effects.
The hypersensitive response was in some cases identified early in the disease progress,
encouraging utilization for AD screening, however other studies have brought into
question both the sensitivity and specificity of the test. Some studies found no
significant hypersensitivity in AD (Caputo et al., 1998, FitzSimon et al., 1997, Fridh et
al., 1996, Granholm et al., 2003, Growdon et al., 1997, Kardon, 1998, Kurz et al., 1997,
Loupe et al., 1996, Marx et al., 1995, Reitner et al., 1997, Treloar et al., 1996), or a
hypersensitive response in APOE ε4 allele carriers rather than AD (Arai et al., 1996,
Higuchi et al., 1997), affecting carriers of this allele who are cognitively normal
(although at increased risk of progressing to AD). A similar hypersensitivity has been
reported in Down’s syndrome subjects – who develop AD as a result of an extra copy of
the amyloid precursor protein (APP) gene (Sacks and Smith, 1989), yet also in healthy
young adults (Marx et al., 1995). Still other studies have demonstrated modulation of
the pupil dilation response by eye color (Sacks and Smith, 1989) or age (Caputo et al.,
1998, Fridh et al., 1996, Higuchi et al., 1997). These results have left the reliability of
the pupil dilation test for AD in question.
As an alternative, PFR then began to be investigated in AD, since it is not reliant on
delivery of drugs to the eye. Changes to a number of PFR parameters have been found
in AD compared to healthy ageing, with a single parameter (reduced “maximum
constriction acceleration”) facilitating perfect classification in one study (Fotiou et al.,
2007). This built upon previous studies which also found significant differences in PFR
between AD and controls (Ferrario et al., 1998, Fotiou et al., 2000, Granholm et al.,
20
2003, Prettyman et al., 1997, Tales et al., 2001). These results are summarized in Table
1.2.
The majority of the results indicate a ‘sluggish’ PFR in AD, with reduced velocities,
accelerations and constriction amplitude, and increased latencies. The result from
Ferrario et al. (Ferrario et al., 1998) reporting increased maximum constriction
acceleration in AD stands in contrast, possibly due to different PFR methodology and
participant selection. Some results indicate a faster recovery after stimulus in AD,
despite slower constriction and dilation velocities, probably due to the reduced
amplitude (Fotiou et al., 2007, Prettyman et al., 1997).
Possible causes of the PFR changes in AD are degeneration in relays in the midbrain, or
central cholinergic depletion (Herholz et al., 2004, Tohgi et al., 1994). The parameters
that demonstrated the most significant differences in AD are calculated from the
constriction phase of the PFR which is primarily a parasympathetic cholinergic response
(Loewenfeld, 1999). Hence these parameters are likely to be the most sensitive markers
of cholinergic deficits in the peripheral parasympathetic pathway. If PFR changes in AD
relate to neurotransmitter status, then PFR testing may be useful as an objective, non-
invasive monitor with which to follow disease progression and treatment efficacy.
21
Table 1.2. Summary of reports of PFR changes in AD.

Reference N (AD, Parameters Change p Medications and
HC) in AD comments
(Prettyman 9,9 Resting pupil diameter - 0.041 All participants
et al., Constriction Amplitude - <0.002 medication-free.
1997) 75% Recovery time - 0.034
Constriction Latency NS
(Ferrario et 20,44 Resting pupil diameter + <0.05 No
al., 1998) Minimum pupil diameter NS anticholinesterase
Constriction percentage NS drugs.
Constriction latency + <0.05
Latency to half contraction NS
Latency to minimum pupil size NS
63% recovery time NS
Maximum constriction velocity NS
Maximum dilation velocity NS
Maximum constriction acceleration + <0.05
(Fotiou et 10,5 Constriction latency NS Medication-free
al., 2000) Constriction amplitude NS participant results
Latency to minimum pupil size - 0.000 reported.
No. of oscillations - 0.000 Additional
donepezil-treated-
AD group
exhibited similar
but non-significant
trends.
(Tales et 12,12 Constriction latency + NS Medication free for
al., 2001) Constriction percentage - <0.05 2 months, diabetes
excluded.
(Granholm 15, 15 Resting pupil diameter NS Trend with
et al., Constriction amplitude - <0.05 donepezil-treated-
2003) Latency to minimum pupil size NS AD having shorter
latency-to-
minimum-pupil
size than AD
without treatment.
Constriction
amplitude was
similar for
treated/untreated
AD.
(Fotiou et 23,23 Resting pupil diameter NS No cardiac
al., 2007) Minimum pupil diameter NS glycosides,
% Recovery 3.5sec after stimulus + <0.001 anticholinergics,
Constriction latency + <0.001 sympathomimetics,
Latency to max velocity + <0.001 beta-blockers.
Latency to minimum pupil size + <0.001
Max constriction velocity - <0.001
Maximum constriction acceleration - <0.001
Constriction amplitude - <0.001
Constriction amplitude percentage + <0.001
N = number of participants in the Alzheimer’s disease (AD) and healthy control (HC) groups. ‘Change in
AD’ indicates parameter increase in AD (+) or decrease (-). P values listed for statistical tests applied in
each study, bold values indicate statistical significance, NS refers to a non-significant statistical test
result.
22
Cholinergic depletion may also occur in other diseases such as Parkinson’s Disease
(Dubois et al., 1990), which has also been reported to influence PFR (Fotiou et al.,
2009, Granholm et al., 2003). AD patients are more likely to have Parkinsonism than
those undergoing healthy ageing (Funkenstein et al., 1993, Merello et al., 1994), and
similarly, individuals with Parkinsonism are more likely to develop AD (Richards et al.,
1993), hence it is possible that PFR changes are related to Parkinsonism or
extrapyramidal signs. For these reasons, the specificity of PFR testing for AD needs
further investigation.
1.3.3) The Ocular Lens in Alzheimer’s Disease
When light enters the eye it passes through the outer ‘corneal’ layer, followed by the
aqueous humor and then the intra-ocular lens (see Figure 1.8). The role of this anterior
region of the eye is to focus an optical image of the outside world onto the retina. One
disorder that can disrupt this role is cataract; an opacification of the lens often due to
protein aggregation (see Figure 1.9). Cataracts are a common problem in the elderly,
with progressive deposition of insoluble protein in the lens and extensive oxidative
damage generally caused by environmental factors such as ultraviolet (UV) radiation
exposure (Hanson et al., 2000, Spector, 1995). According to a World Health
Organization report, cataract is the main cause of blindness in the world, responsible for
51% of blindness registrations, followed by glaucoma (8%) and age-related macular
degeneration (5%) (World Health Organization, 2010).

23
______________________________________________________________________
Figure 1.8. Section of the human eye.
Indicating the fluid compartments (aqueous humor and vitreous humor), the lens, retina and optic disc.
Adapted from Kolb (Kolb, 1995).
A B
______________________________________________________________________
Figure 1.9. Cortical cataract in the human intra-ocular lens.
A,B. Retro-illumination photographs of human intra-ocular lens’, illuminating cortical cataracts (dark
radial lines). The pupils have been dilated with tropicamide eye drops). A different, more difficult to
image type of cataract (equatorial supranuclear cataract) has been linked with Alzheimer’s disease.
24
The ability of the lens to focus light is achieved by its high protein concentration, higher
than any other tissue of the human body. This high protein concentration, along with the
optical accessibility of the lens, makes it ideal for the optical investigation of protein
aggregation in disease, in vivo. The Aβ protein involved in the pathogenesis of AD in
the brain has also been found to exist in the lens (Aβ40 and Aβ42), aqueous humor
(Aβ40) and vitreous humor (Aβ42) of the normal human eye (Goldstein et al., 2003,
Yoneda et al., 2005).
Remarkably, research indicates that a particular type of cataract (equatorial supranuclear
cataract) might be specific to AD sufferers (see Figure 1.10) (Goldstein et al., 2003).
Slit-lamp microscopy of ex-vivo intra-ocular lenses of individuals with AD consistently
revealed equatorial supranuclear cataracts. Subsequent histochemical analysis in the
same study indicated that Aβ aggregates are present in the cytosol of the lens fiber cells
co-localizing with the cataracts. This cataract has also been reported in Down’s
syndrome subjects (Moncaster et al., 2010) – who develop AD as a result of an extra
copy of the APP gene on chromosome 21 (Antonarakis et al., 2004).
______________________________________________________________________
Figure 1.10. Equatorial supranuclear cataract in an ex-vivo human AD lens.
The cataract is the white part of the lens, highlighted by the white arc. It is usually hidden by the iris in-
vivo. Goldstein et. al. (Goldstein et al., 2003).
25
Equatorial supranuclear cataracts (or the preceding Aβ aggregation in the lens) could
thus be a biomarker for AD, although it is unknown at which stage of AD pathogenesis
they occur. The location of these cataracts is the equatorial periphery of the lens
posterior to the iris, which renders them virtually harmless with respect to visual
impairment, as well as difficult to detect on routine physical examination. However,
these AD-linked lesions are readily observed by slit lamp ophthalmological evaluation
in fully dilated subjects. If Aβ is indeed aggregating in the AD lens, leading to these
cataracts, it is possible that the initial molecular changes could be detected non-
invasively as an early screening or diagnostic test. Further research is needed to
establish the specificity of these cataracts and lens Aβ aggregations to AD, since both
APP and Aβ have been shown to increase in concentration in the normal mammalian
lens in response to UV radiation or other oxidative effects (Frederikse et al., 1996).
For early diagnosis of AD, it has been proposed that a suitable eye-drop biomarker may
enable Aβ in the anterior chamber to be stained and quantified (Goldstein et al., 2003).
Alternatively, the size of Aβ aggregates in the eye may facilitate non-invasive detection
with optical scattering (Goldstein et al., 2006), spectral or autofluorescence techniques.
Dynamic light scattering (DLS) uses back-scatter from a low energy laser beam to
determine information about particle size, shape, movement and interactions. The
technique is applicable to all eye tissues and has already shown promise for early
cataract detection (Ansari and Datiles, 1999, Datiles et al., 2002, Datiles et al., 2008). A
decline in the concentration of α–crystallin proteins in the lens, which have the role of
preventing protein aggregation, occurs in the early stages of cataract development, and
can be monitored using DLS.
Raman spectroscopy and autofluorescence techniques both involve illumination of the
sample at a specific wavelength followed by measurement of the in-elastically scattered

26
light at different wavelengths. Raman spectroscopy with principal components analysis
has been used to distinguish AD from control ex vivo post-mortem brain tissues, based
on spectra of protein aggregates (Archer et al., 2007, Hanlon et al., 1999, Hanlon et al.,
2008, Sudworth, 2006). AD brain tissues also exhibit visual and infrared-excited auto-
fluorescence (AF) (Hanlon et al., 1999, Zipfel et al., 2003). These techniques could also
prove useful for non-invasive, early diagnosis of AD using the eye.
1.3.4) The Aqueous and Vitreous Humor in Alzheimer’s Disease
The aqueous humor is the fluid that fills the anterior region of the eye, between the lens
and the cornea (see Figure 1.8). It provides nutrients to the lens and cornea and
maintains the convex curvature of the cornea. The vitreous humor fills the main fluid
chamber of the eye and has functional interactions with the lens and retina.
The vitreous fluid body has been reported to have reduced Aβ42 concentration in AD
patients (Haass and Selkoe, 2007, Tiraboschi et al., 2004). Additionally, a change in
Aβ42 (decrease) and tau protein levels (increase) in the vitreous humor has been linked
to retinal diseases such as diabetic retinopathy and “glaucoma concurrent with other
ocular diseases” (Yoneda et al., 2005). The change in protein levels in the CSF in AD
and these retinal diseases is similar to that observed in the CSF in AD (Blennow et al.,
2010, Fagan et al., 2006, Sunderland et al., 2003, Thal et al., 2006).
Given the retinal degeneration observed in AD (Berisha et al., 2007, Danesh-Meyer et
al., 2006, Hedges et al., 1996, Paquet et al., 2007, Tsai, 1991) and the recently reported
common features between AD and glaucoma (Bayer and Ferrari, 2002, Bayer et al.,
2002, Bayer et al., 2002, Danesh-Meyer et al., 2006, Guo et al., 2007, Hedges et al.,
1996, McKinnon et al., 2002, Nesher and Trick, 1991, Sadun and Bassi, 1990, Tsai,
27
1991, Yin et al., 2008, Yoneda et al., 2005), the vitreous humor is an interesting focus
for future research into ocular protein changes in AD.
1.3.5) The Retina and Optic Disc in Alzheimer’s Disease
While the role of the anterior eye is to focus light onto the retina (see Figure 1.11), the
retina’s task is to convert the light into electrical signals that enter the brain. The retina
is considered to be “nature’s brain slice” due to its organized, laminar structure of
morphologically and functionally distinct cell types, including both neural and
photoreceptor cells (see Figure 1.12). The retina is a developmental outgrowth of the
brain and there is a level of homology between the retinal and cerebral micro-
vasculatures (Patton et al., 2005). The retina is the only place in the body where
vasculature or neural tissue is available for non-invasive optical imaging. It is highly
accessible due to the transparency of the eye, allowing non-invasive imaging of retinal
layers, nerve fibers and vasculature. The optic disc (or optic nerve head) is the interface
between the retina and the optic nerve and is also the location at which retinal blood
vessels and nerve fibers exit the retina.
The transparency of the ocular media combined with the fact that the retina is
developmentally and anatomically an extension of the brain, makes retinal photography
ideal for the detection of systemic diseases that affect the microcirculation. Screening
and monitoring for age-related diseases are growing in importance because of increases
in life expectancy. Retinal photography has become an invaluable tool for prognostic
assessment for systemic diseases such as diabetes and hypertension, and may become
similarly useful for neurodegenerative diseases such as AD. Vascular topography,
including the angles at which blood vessels bifurcate and the relationship between the
widths of parent to daughter blood vessels at vascular junctions is optimized in healthy

28
subjects in order to minimize shear stress across a vascular network (Zamir and Brown,
1982, Zamir and Medeiros, 1982). Variations from the optimal geometrical topography
are known to occur in particular vascular conditions (Chapman et al., 2002, Stanton et
al., 1995). Similar variations may occur in AD due to the disease’s vascular component
and hence are worthy of being explored with retinal photography.
______________________________________________________________________
Figure 1.11. Digital retinal photograph displaying the optic disc in the centre.
Retinal arterioles and venules (darker) are visible, and lightly opaque retinal nerve fibers coursing to the
optic disc.
______________________________________________________________________
Figure 1.12. Sectional diagram of the eye with schematic enlargement of the retina.
(Salk Institute for Biological Studies, 2012).
29
The retinal vasculature has been reported to be altered in a number of disorders that
affect the eye, including hypertension, diabetic retinopathy, glaucoma, age-related
macular degeneration, and AD. The retinal vasculature is divided into two main
supplies; a system of inner-retinal vessels that are anterior to the photoreceptors and are
sparsely distributed to minimize impact on the light path, and a dense choroidal system
supplying the photoreceptors outside the optical path (see Figure 1.13).
Figure 1.13. Anatomy of ocular circulation.

A) Cross-sectional drawing along the superior-inferior axis of the human eye through the optic nerve,
showing the vascular supply to the retina and choroid.
B) Drawing showing vasculature of the retina and choroid.
(a-artery, b-vein, n-nerve)
Drawings by Dave Schumick (Anand-Apte and Hollyfield, 2009).
30
Retinal morphology reported in AD involves changes to the vasculature (Berisha et al.,
2007) and optic disc (Bayer et al., 2002, Danesh-Meyer et al., 2006, Tamura et al.,
2006, Tsai, 1991), retinal cell loss (Blanks et al., 1989, Blanks et al., 1996, Blanks et
al., 1996, Hinton et al., 1986, Sadun and Bassi, 1990, Sadun and Bassi, 1990) and
thinning of the retinal nerve fiber layer (RNFL) (Berisha et al., 2007, Iseri et al., 2006,
Paquet et al., 2007, Parisi et al., 2001). A key study by Berisha et al. (2007) found that
AD participants had a specific pattern of RNFL thinning (measured by optical
coherence tomography, OCT, see Figures 1.14 and 1.15), narrower blood column
diameter in the major superior temporal retinal venule and decreased blood flow in this
venule (both measured by a laser Doppler instrument) (Berisha et al., 2007). A
limitation of the study was the small participant numbers (9 probable AD and 8
controls). Berisha et al. (2007) demonstrated significant thinning of the superior RNFL
in AD; this region corresponds with the inferior visual field and these changes could
explain the vision loss reported in this area in AD (Trick et al., 1995).
Other studies have also reported patterns of RNFL loss in AD (Iseri et al., 2006, Paquet
et al., 2007, Parisi et al., 2001). In a recent RNFL, OCT meta-analysis (He et al., 2012),
there was a significant average RNFL thickness reduction in AD compared with the
control group. The pattern of RNFL loss was not consistent, with significant thinning
reported in many different regions (general, parapapillary, macular, superior, inferior,
nasal and temporal). It has also been demonstrated that persons with MCI already have
reduced RNFL thickness compared to healthy controls, suggesting that retinal
degeneration may occur in the early stages of AD prior to clinical diagnosis (Kesler et
al., 2011, Paquet et al., 2007). Iseri et al. (2006) found macular thinning in AD to be
related to the severity of cognitive impairment (Iseri et al., 2006). Parisi et al. (2001)
found RNFL thinning to be related to retinal dysfunction as revealed by abnormal

31
pattern electroretinogram (PERG) responses (Parisi et al., 2001). Other studies have
also found abnormal pattern electro-retinogram (PERG) responses in AD (Katz et al.,
1989, Trick et al., 1989).
The loss of RNFL thickness in AD is linked to a depletion of retinal ganglion cells
(RGC) and optic nerve axons as identified by histopathological studies (Blanks et al.,
1989, Blanks et al., 1996, Blanks et al., 1996, Hinton et al., 1986, Sadun and Bassi,
1990, Sadun and Bassi, 1990). RGC are the retinal nerve cells that transfer visual
information along their nerve fibers into the brain (see Figure 1.13). A “post-mortem”
study by Blanks et al. (Blanks et al., 1996) demonstrated a 25% decrease in RGC
numbers in the foveal and parafoveal retina.
______________________________________________________________________
Figure 1.14. OCT scan showing the retinal layers around the fovea.
The layer closest to the vitreous humor is the retinal nerve fiber layer (RNFL) which contains fibers
emerging from the retinal ganglion cells below. Also just beneath the RNFL is the retinal vasculature
(evident from the vertical shadows cast in this OCT scan). Beneath the retinal ganglion cells are the
bipolar, amacrine and horizontal cells, followed by a layer of photoreceptor cells. The photoreceptor cells
are nourished by the deeper retinal pigment epithelium (RPE) and a rich posterior vascular layer called
the choroid. (OCT scan courtesy of Chris Barry, Lions Eye Institute, Perth, Australia)
32
______________________________________________________________________
Figure 1.15. OCT scan circling the optic disc.
The image on the right shows the retinal layers detected in an OCT scan traversing a circular path around
the optic disc, as illustrated in the retinal photograph on the left. The RNFL is thickest in the superior and
inferior quadrants. RNFL studies in AD have had varying results, indicating superior, general, macular or
parapapillary thinning. (OCT scan courtesy of Chris Barry, Lions Eye Institute, Perth, Australia)
AD is known to have a vascular component, with small-vessel disease, microinfarction
and cerebral amyloid angiopathy (characterised by Aβ deposition in vessel walls) (de la
Torre, 2009, Ravona-Springer et al., 2003, Ruitenberg et al., 2005, Thal et al., 2003).
Given the homology between the retinal and cerebral microvasculatures (Patton et al.,
2005), it is not unexpected that changes in the retinal vasculature might also occur in
AD. The only study reporting retinal vascular changes in AD was a small study (N=17)
by Berisha et al. (2007) finding that AD participants (N=9) had narrower blood column
diameter in the major superior temporal retinal venule and decreased blood flow in this
venule (Berisha et al., 2007). These findings were made with the use of a laser Doppler
device. No study to date has verified retinal vascular changes in AD using retinal
photography, which is more widely available. Advances in digital retinal imaging have
facilitated accurate and reliable measurements of the optimality of the retinal
vasculature. This includes vascular attenuation, branching geometry and measures of
how effectively the vascular network fills the retinal space. Detection of retinal vascular
33
changes in AD using retinal photography could lead to a more practically applicable AD
screening test.
In addition, no previous studies have investigated retinal vascular changes in AD with
respect to plaque burden and cerebral atrophy, an approach which has the potential to
shed new light on the stage of the disease process at which the retinal changes occur and
hence the suitability of retinal photography for early detection of AD. It should also be
noted that retinal vessel width is influenced by age and race and can be altered in many
disorders (Sun et al., 2009).
The cause of the retinal degeneration in AD is not known. Preliminary evidence is
emerging that Aβ plaques may occur in the human AD retina (Koronyo-Hamaoui et al.,
2010), possibly providing a more accessible location to assess AD-specific neuro-
pathology. The researchers identified retinal Aβ plaques in postmortem eyes from AD
patients (n=8) and suspected early stage cases (n=5), plaques were undetectable in age-
matched non-AD individuals (n=5). The degree of retinal pathology was found to be
consistent with brain pathology. However, further research is needed to determine the
nature of these retinal plaques and their relationship to AD and possible concomitant
ocular disease.
The same researchers demonstrated the ability to non-invasively detect individual
retinal plaques in live AD mice using Curcumin staining with fluorescence imaging,
providing a possible test for early detection of AD in humans. Curcumin, an extract
from the spice Turmeric, is also being tested as a therapeutic for AD due to its ability to
bind to Aβ plaques and affect Aβ proteostasis (Baum et al., 2008, Belkacemi et al.,
2011, Caesar et al., 2012, Garcia-Alloza et al., 2007, Lim et al., 2001).
34
Potential relationships between retinal degeneration reported in AD, retinal Aβ plaques
and retinal vascular changes are intriguing, but require further investigation. If retinal
vascular constriction is associated with AD, it is unclear whether the reduced blood
flow might be responsible for the reported retinal cell death or instead might be a
response to the associated reduction in metabolic demand. Aβ has been reported to
exhibit a constrictive effect on cerebral vessels (Suo et al., 1998) but it is unclear
whether Aβ levels are increased in the AD retina. Research on the retinas of AD
transgenic mice has demonstrated Aβ plaques, hyperphosphorylated tau, increased
microvascular deposition of Aβ and neuroinflammation (Liu et al., 2009, Perez et al.,
2009). Aβ immunotherapy in transgenic mice resulted in the clearance of retinal plaques
but an increase in retinal amyloid angiopathy, identifying non-invasive retinal imaging
as an alternative method for monitoring disease response to immunotherapy in these
mice.
In addition to OCT, retinal photography (see Figure 1.9 and 1.13) has also been used to
identify RNFL abnormalities (nerve fiber loss) in AD (Hedges et al., 1996, Tsai, 1991),
although one study indicated practical difficulties in using this approach for AD
screening. The retinal nerve fibers are thick enough in the inner retina to make them
visible in retinal photographs (see Figures 1.11 and 1.13), hence revealing areas of
RNFL loss.
Retinal photography and Scanning Laser Ophthalmoscopy (SLO) have both been used
to demonstrate optic disc changes in AD, including optic disc pallor, pathologic disc
cupping (hollowing-out), and thinning of the neuro-retinal rim (Danesh-Meyer et al.,
2006, Tsai, 1991). Some of the ocular morphologies found in AD are also found in the
eye disease ‘open-angle glaucoma’, specifically peripapillary RNFL thinning, optic disc
cupping and visual field loss (see Figure 1.16). Glaucoma is characterised by the loss of
35
retinal ganglion cells and their axons, resulting in progressive loss of peripheral vision.
Glaucoma is second only to cataract as a leading cause of blindness worldwide
(Resnikoff et al., 2004) and has ocular hypertension as its largest risk factor. The 5-fold
higher chance of visual field defects and/or optic disc cupping found in AD has been
interpreted as a higher occurrence rate of glaucoma in AD (Bayer et al., 2002).
However no AD participants had a family history of glaucoma, and ocular hypertension
was not found in AD participants but was found in 7.5% of controls, reducing the
likelihood that open-angle glaucoma was the cause. This is still in question though, with
another study supporting the increased incidence of open-angle glaucoma in AD
(Tamura et al., 2006).
(a) (b) (c) (d)
______________________________________________________________________
Figure 1.16. Illustration of changes to the optic disc and vision in Glaucoma.
(a) Healthy optic disc. (b) Optic disc cupping observed in Glaucoma and AD. (c) Healthy visual field. (d)
Peripheral visual field loss resulting from Glaucoma or AD.
(National Eye Institute, National Institutes of Health, 2010)
(http://www.nei.nih.gov/health/glaucoma/glaucoma_facts.asp)
In a retrospective study, a greater than 10% per year decay in visual field and optic disc
cupping were demonstrated in glaucoma patients who were later diagnosed with AD,
whereas an average 3% per year decay was observed in glaucoma patients who did not
develop AD, indicating that AD accelerates the progression of glaucoma symptoms
(Bayer and Ferrari, 2002). Cup-to-disc ratios in AD patients were 39-43% higher than
controls and prevalence of primary open-angle glaucoma was much higher in AD (24%)
36
than controls (10%). However, increased rates of visual field defects and/or optic disc
cupping have also been reported in Parkinson’s disease (Bayer et al., 2002) and since
these changes are observed in glaucoma, they are unlikely to provide a test that has
specificity for AD.
It is possible however, that investigations into these retinal changes in AD and
glaucoma might yield interesting results about the pathogenesis of the diseases as well
as their treatment and monitoring. The similarities between the ocular effects of AD and
glaucoma extend to changes observed in pattern electro-retinogram (PERG) recordings
(Nesher and Trick, 1991), the type of cells lost (large magnocellular RGC (Sadun and
Bassi, 1990)) and possibly to the mechanism of retinal ganglion cell (RGC) death
(apoptosis) (McKinnon et al., 2002, Yin et al., 2008) and retinal vascular changes. In
experimental glaucoma, Aβ co-localizes with RGC apoptosis and induces RGC
apoptosis in vivo (Guo et al., 2007). In addition, targeting the Aβ pathway with a β-
secretase inhibitor, Congo red or Aβ-antibody has been found to be effective in treating
experimental glaucoma (by reducing RGC apoptosis) (Guo et al., 2007).
Several studies have shown decreased blood flow in the optic nerve head, retina and
choroid in Glaucoma patients (Findl et al., 2000, Grunwald et al., 1998, Michelson et
al., 1996, Portmann et al., 2011, Wang et al., 2011), believed to be the result of elevated
intra-ocular pressure (IOP). A decrease in the number of capillaries in the optic nerve
head as well as the atrophy of the peripapillary capillaries supplying the RNFL has also
been reported (Gottanka et al., 2005, Kornzweig et al., 1968). Generalized retinal vessel
thinning has also been reported in Glaucoma (Chang et al., 2011, Jonas and Naumann,
1989, Lee et al., 1998, Mitchell et al., 2005), along with focal (localized) narrowing of
vessels near the optic disc (Papastathopoulos and Jonas, 1995).

37
Chronic ocular hypertension (elevated IOP) can contribute to RGC loss in glaucoma
and has been shown to increase Aβ production in the rat retina (McKinnon et al., 2002).
Interestingly, in addition to its neuroprotective effects, a cholinesterase inhibitor used to
treat AD has demonstrated dual therapeutic potential by reducing IOP in AD patients
(Estermann et al., 2006). Current treatments for glaucoma are directed at reducing IOP,
but evidence indicates that RGC loss still occurs in many glaucoma patients after
successful IOP normalization, indicating that other mechanisms are involved.
In addition to glaucoma, Aβ has also been implicated in other retinal diseases such as
age-related macular degeneration (AMD) (Anderson et al., 2004, Dentchev et al., 2003,
Johnson et al., 2002, Klaver et al., 1999, Luibl et al., 2006, Ohno-Matsui, 2011), which
is the major cause of irreversible blindness in elderly patients worldwide. There are a
number of studies suggesting that AMD is related to dementia, cognitive decline and
AD (Klaver et al., 1999, Ohno-Matsui, 2011). The early stages of AMD are
characterised by drusen, extracellular deposits beneath the photoreceptors. Aβ is present
in drusen and is potentially a key regulator of the progression from drusen to AMD
(Ohno-Matsui, 2011). This suggests that a common pathogenic mechanism might exist
between AMD and AD, hence therapeutic approaches that target Aβ in AD patients may
also be of clinical benefit in AMD. While the choroidal vasculature is altered in late
AMD (Ciulla et al., 2002, Harris et al., 1999, Lutty et al., 1999, Pemp and Schmetterer,
2008) no significant associations between AMD and the inner retinal vasculature have
been reported.
AD has also been associated with retinopathy (Schrijvers et al., 2012), which describes
persistent or acute damage to the retina. Retinopathy is often an ocular manifestation of
systemic disease such as diabetes or hypertension and includes signs such as
generalized and focal arteriolar narrowing, arterio-venous nicking, increased retinal

38
arteriolar light reflex (copper or silver wiring), flame and blot-shaped retinal
hemorrhages, soft and hard exudates, cotton wool spots, macular edema and, in severe
cases, optic disc swelling (Keith et al., 1939, Wong and Mitchell, 2004, Wong and
Mitchell, 2007).
Vascular remodeling in retinopathy occurs over periods of time where the patient may
not be aware of the presence or extent of their disease, until it is too late. The hallmark
of diabetic retinopathy (DR), a microvascular complication of diabetes, include multiple
occlusions due to increased leukocyte entrapment (Ogura, 2000), increased permeability
of the vessel wall with focal leakages (Ben-nun et al., 2004) and, in the proliferative
form, growth of newly-formed vessels (neovascularization). Initially DR is associated
with retinal hypoperfusion, followed by hyperperfusion as diabetic retinopathy
progresses to the neovascularistion stage (Curtis et al., 2009, Pemp and Schmetterer,
2008).
Both diabetes and hypertension are major risk factors for AD and retinopathy. However,
independent of diabetes, hypertension and a number of other covariates, a recent large
population study revealed an association between retinopathy and AD (Schrijvers et al.,
2012).
1.4) The Scientific and Clinical Significance of the Project
The devastating impact of AD, both on those directly affected and on society in general,
creates a pressing need for better treatments. By the time a person is diagnosed with
“probable AD” using current techniques, significant irreversible neuronal degeneration
has already occurred. Therefore, research into better treatments must be paralleled by
39
research into technologies to screen populations for AD, in order to identify cases
before cognitive symptoms arise.
Ocular morphologies reported in AD give hope for a non-invasive, cost-effective
screening test for AD. Evidence is accumulating in support of AD-related changes in the
eye, but finding a sufficiently sensitive and specific ocular biomarker is proving to be a
major challenge. With respect to the retinal microvasculature, new techniques are now
available to measure the health or optimality of the retinal microcirculation, including
vascular topographic parameters such as vascular width indices, branching angles of
blood vessels, retinal vessel tortuosity and fractal dimension. There are no reports of
retinal vascular changes in AD using retinal photography, the least invasive and most
widely available technology for retinal imaging. Hence the first aim of this project is to
determine whether retinal vascular changes in AD can be detected with this technology
and hence become useful for population screening of AD.
Neither retinal vascular changes nor pupil response changes in AD have been previously
investigated with respect to neocortical amyloid plaque burden, an approach that has the
potential to shed light on connections between ocular changes and AD. There remains
scope for ocular changes to be utilised in AD screening or diagnostic purposes with
greater sensitivity and specificity and at an earlier stage in the disease process. An
ocular screening test for AD would benefit AD sufferers and researchers and possibly
provide new insight into the molecular processes and genetic determinants of the
disease. An ocular biomarker or biomarkers could be highly specific for AD, or to be a
useful component in a multidisciplinary approach aimed at producing an earlier and
more accurate diagnosis of AD.

40
1.5) Hypotheses and Objectives
In this thesis the potential application of ocular biomarkers, collected from retinal
photography and pupillometry, in early detection and monitoring of Alzheimer’s disease
will be examined through the hypotheses and objectives detailed below.
1.5.1) Retinal Vascular Hypotheses
1. Retinal abnormalities are associated with AD and can be detected with non-
invasive retinal photography and semi-automated computerized image analysis.
Aim 1.1: To examine the association of abnormal retinal vascular width and
topology with AD diagnosis.
Aim 1.2: To examine the association of signs of retinal disease with AD
diagnosis.
2. Retinal abnormalities occur early in AD pathogenesis and can be detected in
preclinical AD.
topology with neocortical plaque burden in preclinical AD.
Aim 2.2: To examine the association of signs of retinal disease with
neocortical plaque burden in preclinical AD.
topology with EOFAD mutation carrier status and expected years to disease
onset.
41
1.5.2) Pupillometric Hypotheses
3. Pupillometric abnormalities are associated with AD.
Aim 3.1: To examine the association of abnormal PFR with AD diagnosis.
4. Pupillometric abnormalities occur early in AD pathogenesis and can be detected
in preclinical AD.
Aim 4.1: To examine the association of abnormal PFR with neocortical
plaque burden in preclinical AD.
Aim 4.2: To examine the association of abnormal PFR with EOFAD
mutation carrier status and expected years to disease onset.

42
Chapter 2: Methodological Approaches
2.1) Introduction
This Chapter describes the research design, participants, biophysiological measures and
instruments that were used in this project. The methodology was directed toward the
evaluation of retinal photography and pupillometry as primary screening technologies
for AD. Despite a small inherent inaccuracy in clinical diagnosis of AD, a good starting
point to identify ocular biomarkers for AD is to compare a group of clinically diagnosed
AD individuals with a ‘healthy’ control group, demographically similar but evaluated to
be non-AD using the same neuropsychological examination.
In order to further evaluate these biomarkers for the purpose of pre-symptomatic
detection of AD, it would be ideal to follow individuals from health to disease,
exploring temporal changes in these ocular markers on an individual basis. This requires
long-term longitudinal testing and was not possible within the time limitations of this
project. However, this has been possible for cortical amyloid plaque burden in the
Australian Imaging, Biomarkers and Lifestyle (AIBL) and other large studies,
demonstrating that high PiB retention is associated with progression to symptomatic AD
(Morris et al., 2009, Villemagne et al., 2011). Hence, with strong evidence that high
cortical plaque burden, as detected by PiB-PET neuroimaging, identifies the extended
pre-clinical phase of AD (Pike et al., 2007, Rowe et al., 2010, Sperling et al., 2011), the
second component of this study investigated whether ocular AD biomarkers were
associated with plaque burden in asymptomatic individuals.
Ocular biomarkers were further investigated with respect to EOFAD, a rare genetic
form of AD that generally affects persons at a significantly younger age. Despite the
difference in underlying cause and age of onset, EOFAD and the more common
43
sporadic AD have similar neuropathologic hallmarks and clinical features (Bateman et
al., 2011). Hence, new knowledge about the disease process in EOFAD has the
potential to translate into better methods for early detection of sporadic AD.
2.2) Participants and Ethical Approvals
Participants were recruited from the Australian Imaging, Biomarkers and Lifestyle
(AIBL) study (www.aibl.csiro.au) of ageing and the Dominantly Inherited Alzheimer
Network (DIAN) study (www.dian-info.org), at the McCusker Alzheimer’s Research
Foundation (MARF) in Western Australia. Studies into sporadic AD utilised the AIBL
cohort only; the DIAN cohort was utilised separately for the study of EOFAD.
A full description of the AIBL cohort is reported elsewhere (Ellis et al., 2009). Briefly,
participants were excluded if they were less than 55 years old, not fluent in English, had
a Mini-Mental State Examination (MMSE) score lower than 12, or had a history of
brain injury or substance abuse. AD participants met National Institute of Neurological
and Communicative Disorders and Stroke/Alzheimer’s Disease and Related Disorders
Association criteria for probable AD (McKhann et al., 1984). For participants with
MCI, objective impairment was established as at least 1 neuropsychological test score
falling 1.5 SDs or more below relevant normative data (Petersen et al., 1999).
EOFAD participants were recruited from the DIAN study (DIAN, NIA U19 AG032438,
JC Morris, PI, www.dian-info.org, clinicaltrials.gov number NCT00869817). The
DIAN study enrolled offspring of parents carrying a mutation for autosomal dominant
Alzheimer’s disease (ADAD) across 14 international sites (http://www.dian-
info.org/institutions.htm), including the Edith Cowan University, Western Australia.

44
Each participant was a member of a pedigree with a known mutation for ADAD. DIAN
is a longitudinal study involving comprehensive clinical, cognitive, imaging, and
biochemical assessments.
Participants were excluded from the retinal studies if they had history or evidence of
glaucoma, significant cataract or cataract surgery within the last 6 months prior to
screening for this project. Exclusion criteria for the PFR studies were presence of
ophthalmologic pathology (pupillary malformations, severe cataract, glaucoma, retinitis,
iridectomy), receiving anticholinergics, beta-blockers, alpha-agonists or ophthalmic
agents affecting PFR. All participants in the ocular studies were white Caucasians.
To address possible undiagnosed hypertension in this study, the definition of
hypertension was extended to include both physician-diagnosed and identified by
elevated blood pressure (systolic pressure > 140mmHg or diastolic pressure >
90mmHg) on day of retinal imaging.
All participants or legal guardians provided their written informed consent, and all
ocular imaging experiments were approved by the Ethics Committee of the University
of Western Australia (reference number: RA/4/1/2460, 24/08/2009), evaluated
according to the guidelines for human research of the National Health and Medical
Research Council of Australia (NHMRC) and according to the Helsinki Declaration.
The Ethics approval for the parent AIBL study was obtained from the Austin Health
Human Research Ethics Committee (Victoria, Australia) and the Hollywood Private
Hospital (HPH) Ethics Committee in Western Australia. The Ethics approval for the
parent DIAN study was approved by the Washington University Human Research
Protection Office, the Edith Cowan University Ethics Committee and the Hollywood
Private Hospital Ethics Committee, Western Australia.

45
2.3) Neuroimaging
Neuroimaging methodology for the AIBL and DIAN cohorts are reported elsewhere
(Bateman et al., 2012, Bourgeat et al., 2010). Briefly, participants were neuroimaged
for the presence of fibrillar brain amyloid using positron emission tomography (PET)
with Pittsburgh Compound B (PiB) (Klunk et al., 2004, Klunk et al., 2005). Results for
neocortical standardized uptake value ratio (SUVR) were calculated. A bimodal
distribution of PiB-PET SUVR values was observed in the HC group of the AIBL study
(Villemagne et al., 2008). Consequently, hierarchical cluster analysis yielded a cut-off
for neocortical SUVR of 1.5, separating high from low plaque burden (Villemagne et
al., 2008). Subjects were classified as PiB negative (HC-) if their neocortex SUVR was
below 1.5, and PiB positive (HC+) if their neocortex SUVR was above 1.5.
Neuroimaging data was not available for all AIBL study participants, hence clinical
status and neuroimaging studies had different cohorts, as described in the relevant
sections. Table 2.1 presents the clinical and genotypic stratification of the Perth AIBL
cohort at the end of the data collection phase of the ocular biomarkers study (March
2012).
Table 2.1. Stratification of Perth AIBL cohort.
AD MCI HC Total
Full Perth AIBL Cohort 53 57 356 466
APOE ε4 carriers 30 31 93 154
APOE ε4 non-carriers 23 26 263 312
PiB-PET Imaged Cohort 8 13 81 102

APOE ε4 carriers 5 9 45 59
APOE ε4 non-carriers 3 4 36 43
46
2.4) Genetic Analysis
2.4.1) AIBL participants
The APOE genotyping for the AIBL Study follows on the protocol discussed in detail
by Ingelsson et al. (Ingelsson et al., 2001), based on the method described initially by
Hixson and Vernier (Hixson and Vernier, 1990). This protocol has been in use at
Professor Martins’ Laboratory at the Edith Cowan University for routine analysis.
Using one 0.5 ml tube of whole blood, the restriction fragment length polymorphism
(RFLP) was conducted using polymerase chain reaction (PCR) amplification of the
single nucleotide polymorphisms (SNPs) containing DNA region followed by
restriction enzyme cleavage of the PCR product to generate allele-discriminating DNA
fragments.
2.4.2) DIAN participants
Genotyping for familial AD mutations and APOE isoforms was performed by the DIAN
Genetics Core under the direction of Dr. Alison Goate . Ambient blood samples were
shipped from the DIAN performance sites to both the National Cell Repository for
Alzheimer’s Disease (NCRAD) and the DIAN Genetics Core at Washington University.
Deoxyribonucleic acid (DNA) was extracted from blood at both sites using standard
procedures. DNA sequencing of APP, PSEN1 and PSEN2 (the genes that encode for
amyloid precursor, presenilin-1 and presenilin-2 proteins respectively) was performed
by DIAN Genetics Core personnel, using Sanger sequencing methods on an ABI3130xl,
to determine the presence/absence of a disease-causing mutation (Kauwe et al., 2007).
APOE genotyping was performed using an Applied Biosystems ® (ABI) predesigned
real time TaqMan assay “rs7412 & rs429358” according to the manufacturer’s protocol
(ABI, Foster City, California). DNA fingerprinting was performed with the Cell ID kit,
47
using short tandem repeat (STR) analysis of 10 specific loci in the human genome, nine
STR loci and Amelogenin for gender identification (Promega #G9500, Madison,
Wisconsin) in order to confirm that DNA samples obtained by NCRAD and the DIAN
Genetics Core were from the same individual. DNA sequencing, fingerprinting and
genotyping was performed on DNA from NCRAD and the DIAN Genetics Core in
parallel for each individual, and the data were compared for quality control purposes.
All individuals included in this analysis have 100% concordant data for each DNA
sample. Researchers who performed the assessments were not aware of the mutation
status of participants.
2.5) Retinal Photography and Grading
Digital retinal colour photographs (disc centered and macula centred, 45o field) were
collected from both eyes with a Canon CR-1 non-mydriatic camera in a darkened room
(Figure 2.1).
______________________________________________________________________
Figure 2.1. Retinal photography being carried out for a research participant.
The retinal camera and collected retinal image can be seen in this photo.
48
2.5.1) Semi-Automated Analysis of Retinal Topology
Retinal photographs were de-identified and analysed with Singapore I Vessel
Assessment (SIVA) semi-automated software from the Singapore Eye Research
Institute (Cheung et al., 2010, Cheung et al., 2011, Cheung et al., 2011, Cheung et al.,
2010). The analytical principles and reproducibility of measurements using the SIVA
software have been described previously (Cheung et al., 2010). Briefly, the RVPs were
measured from the width and branching geometry of the retinal vessels (see Figure 2.3).
Nineteen RVPs were calculated for each retinal photograph (see Table 2.2 for a
description of RVPs).
The measured retinal zones of interest for the RVPs was 0.5 to 1.0 disc diameters away
from the disc margin (zone B, Figure 2.2) or 0.5 to 2.0 disc diameters away from the
disc margin (zone C, Figure 2.2). Measurement in these zones ensured that the vessels
had attained arteriolar status. The measured zone for each parameter is listed in Table
2.2. A trained grader followed a standardised protocol and performed corrections to
automated procedures as necessary.

49
Table 2.2. Description of the 19 retinal vascular parameters.

Parameter Description Retinal Zone
CRAE Central Retinal Arteriolar Equivalent caliber B
CRVE Central Retinal Venular Equivalent caliber B
AVR Arteriole - Venular Ratio (CRAE / CRVE) B
FDa Fractal Dimension of Arteriolar network C
FDv Fractal Dimension of Venular network C
BSTDa Zone B Standard Deviation Arteriole B
BSTDv Zone B Standard Deviation Venule B
TORTa Curvature Tortuosity Arteriole C
TORTv Curvature Tortuosity Venule C
Num1stBa Number of 1st Branching Arterioles C
Num1stBv Number of 1st Branching Venules C
BCa Branching Coefficient Arteriole C
BCv Branching Coefficient Venule C
AFa Asymmetry Factor Arteriole (or Asymmetry Ratio) C
AFv Asymmetry Factor Venule (or Asymmetry Ratio) C
JEa Junctional Exponent Deviation for Arterioles C
JEv Junctional Exponent Deviation for Venules C
LDRa Length Diameter Ratio Arteriole C
LDRv Length Diameter Ratio Venule C
Description of the 19 retinal vascular parameters (RVPs) measured for each retinal photograph, along
with the retinal zone of interest for calculation of each parameter (see Figure 2.2 for an explanation of the
retinal zones).
50
______________________________________________________________________
Figure 2.2. Retinal zones utilised for retinal vascular analysis.
Zone A is defined as the region from 0 to 0.5 disc diameters away from the disc margin, Zone B is
defined as the region from 0.5 to 1.0 disc diameters away from the disc margin and Zone C (not shown) is
defined as the region from 0.5 to 2.0 disc diameters away from the disc margin. Retinal photograph from
a healthy individual.
Vascular calibers were calculated for the six largest arterioles and six largest venules.
Standard deviation of the width in zone B (BSTD) was calculated for the arteriolar and
venular networks. Summary measures of vascular equivalent caliber were also
calculated (central retinal arterial (CRAE) and venular (CRVE) equivalent caliber),
based on the improved Knudston-Parr-Hubbard formula (Hubbard et al., 1999,
Knudtson et al., 2003). CRAE and CRVE represent the equivalent single-vessel parent
caliber (width) for the six arterioles and venules respectively. From these indices, the
arteriole-to-venule ratio (AVR) was calculated (AVR = CRAE / CRVE).
Natural patterns such as vessel networks often exhibit fractal properties, whereby they
appear the same when viewed over a range of magnifications. The fractal dimension
(FD) describes the range of scales over which this self-similarity is observed. In this
study, the fractal dimension of the retinal vascular network was calculated using the
51
box-counting method (Mainster, 1990). Larger values reflect a more complex branching
pattern.
Retinal vascular tortuosity is defined as the integral of the curvature squared along the
path of the vessel, normalized by the total path length (Hart et al., 1999). All vessels in
the zone of interest with a width larger than 40μm were measured. The estimates were
summarized as the average tortuosity of the measured vessels. A smaller tortuosity
value indicates straighter vessels.
The number of vessels with a first bifurcation (branch) in zone C (Num1stB) was
counted. Average metrics of these branches were then calculated; branching coefficient
(BC), asymmetry factor (AF) and junctional exponent deviation (JE). The branching
coefficient at each vascular bifurcation is defined as BC = (D12+D22)/(D02), where D1
and D2 are the mean vessel widths of each daughter vessel and D0 the mean width of
the parent vessel. The asymmetry factor is defined as AF = (D12)/(D22) (where D1≥D2).
JE expresses the deviation from optimality of the ratio of vessel widths at a bifurcation
(Chapman et al., 2002). It is defined as JE = (D03-(D13+D23))1/3/D0. In terms of
minimising shear stress and work over a bifurcation, the optimum values for BC and JE
are BC=21/3=1.26 and JE=0. All vessels with their first bifurcation within the measured
zone were analysed, with the average value for all vessels reported.
LDR is defined as the vessel length from the midpoint of one vascular bifurcation to the
midpoint of the next bifurcation, expressed as a ratio to the diameter of the parent vessel
at the first bifurcation (King et al., 1996). For all RVP names, a lowercase ‘a’ or ‘v’ at
the end of the name indicates a measurement of the arteriolar or venular network
respectively.
52
A B Adaptive Threshold
C
______________________________________________________________________
Figure 2.3. Illustration of retinal image analysis.
A) Retinal photograph, B) vascular segmentation, C) geometrical analysis.
2.5.2) Semi-Automated AMD analysis
AD and HC participants were stratified by an experienced grader using semi-automated
software to analyse their retinal photographs. Presence of hard drusen (HD, <63 micron
in diameter), soft drusen (SD, either 63-125 micron or >125 micron in diameter),
geographic atrophy (GA, an area of 175 µm of hypopigmentation with a choroidal
vessel in its base) or choroidal neovascularisation (CNV) in either eye was identified.
Participants were identified as having signs of AMD if they had any of the following in
at least one eye:
 soft drusen > 125 micron in diameter, with or without pigmentary abnormalities
 soft drusen 63-125 micron in diameter, with pigmentary abnormalities
 multiple soft drusen 63-125 micron in diameter
Participants were graded as having no signs of AMD only if the images from both eyes
were gradable and the above signs were not present in either eye. Graders were masked
from the disease status of the participants.

53
2.5.3) Clinician Glaucoma and Optic Atrophy Grading
Retinal photographs were examined by an experienced ophthalmologist who determined
the cup-disc-ratio (CDR) of each eye and identified if glaucomatous neuro-retinal rim
(NRR) abnormalities (focal thinning or notching, peripapillary atrophy), peripapillary
drusen or optic disc pallor were present in either eye. Peripapillary drusen and optic disc
pallor are signs of optic atrophy that are non-specific to glaucoma. The mean and largest
CDR value for each pair of eyes was analysed. Graders and clinicians were masked
2.5.4) Clinician Retinopathy Grading
Retinal photographs were examined by an experienced ophthalmologist for the presence
of signs of retinopathy. Only images deemed gradable were considered. The clinician
was masked from the disease status of the participants.
Retinopathy was defined as present if any of the following were detected:
microaneurisms, retinal hemorrhages, soft exudates, hard exudates, macular edema or
optic disc swelling. Arteriovenous nicking and focal arteriolar narrowing were also
reported as present or absent.
Retinal photographs were examined by the ophthalmologist for presence of copper or
silver wire vessels (mild or marked enhancement of the retinal arteriolar central reflex).
This determination is subjective, although clinical guidelines define silver wiring as the
presence of a central reflex with a sharp margin, less than one third of the width of the
arteriolar vessel and consistently present over at least two thirds of the length of the
arteriolar sector (Keith et al., 1939, Svardsudd et al., 1978). Copper wiring is defined as
presence of a central reflex with width greater than one third of the arteriole width,
consistently present over at least two thirds of the length of the arteriolar sector.
54
2.5.5) Semi-Automated Analysis of Arteriolar Central Reflex
A novel quantitative method was utilised for calculating the size of the central reflex
(CR) relative to the size of the retinal vessel, for the largest retinal arteriole in each
retinal photograph. This provides a repeatable, quantitative measure of the central reflex
as a continuous parameter, rather than a coarsely graded or qualitative parameter as has
been used in the past. The CR results are analysed to compare the vessels of AD and
HC participants, and HC- and HC+ participants, as previously described.
Specialized, semi-automated software was developed by the Commonwealth Scientific
and Industrial Research Organisation (CSIRO) to calculate the CR and CR to vessel
width ratio. The method utilises the intensity gradient across the vessel to calculate an
average CR and vessel width in zone B (see Figure 2.2). Data from this study was
utilised to measure the Pearson correlation between the software measured quantitative
CR values and expert graders’ qualitative ranking on CR severity, which was found to
be 0.83 (p<0.01). The inter-grader correlation on CR to arterial calibre ratio was 0.81
and for CR to venular calibre ratio it was 0.85. Graders were masked from participant
characteristics.
Comorbid medical conditions associated with retinal vascular changes are hypertension
and diabetes mellitus, which were therefore treated as confounders in the analysis.
Participant reported smoking history (current or past) was also considered relevant due
to previous reports linking smoking with possible retinal vascular changes (Sun et al.,
2009). Previous cataract surgery was also considered as a confounder, while cataract
surgery within the last 6 months was an exclusion criterion.

55
2.6) Pupillometry
The pupil flash response (PFR) was collected using a NeurOptics™ VIP™-200
Pupillometer (see Figure 2.4). This is a commercial, monocular device providing fully
automated operation and calculation of response parameters. The device produces a
white flash stimulus and then measures the pupil size for 5 seconds using infrared
illumination. The video frame rate is 33Hz, the stimulus/pulse intensity is 180uW and
the stimulus/pulse duration is 31ms. The pupillometer produces diffuse light over the
whole visual field.
The room was darkened for 2 min prior to testing. The test was practiced once before
recording. Occasionally an extra trial was needed to achieve a recording without blinks
or artefacts. Data was rejected if artefacts were present. The right eye was used for all
participants.
A B
______________________________________________________________________
Figure 2.4. PFR data collection with NeurOptics™ VIP™-200 Pupillometer.
A) PFR data collection, B) Video image indicating detected pupil region.
The pupillometer provided automatic calculation of the following eight parameters (see
Table 2.3 and Figure 2.5); resting pupil diameter (mm), minimum pupil diameter (mm),
response latency (msec), mean constriction velocity (mm/sec), maximum constriction
velocity (mm/sec), mean dilation velocity (mm/sec), 75% recovery time (sec) and
56
constriction percentage (%) relative to initial pupil size. A record of the pupil's diameter
as a function of time was exported from the pupillometer. Four extra parameters
(constriction amplitude (mm), maximum constriction acceleration (mm/sec), latency to
maximum constriction (sec) and percentage recovery 3.5 seconds after stimulus (%))
were calculated from the exported PFR data by masked operators using automated
computer algorithms. PFR trials with artefacts or excessive blinking were discarded. A
computer algorithm was used to remove minor blinks.
Table 2.3. PFR parameter descriptions.

PFR Parameter Symbol Description
Maximum pupil diameter (mm) D1 Resting pupil diameter after 2min dark
adaptation
Minimum pupil diameter (mm) D2 Minimum pupil diameter after stimulus
Constriction Latency (sec) T1 Time between stimulus and onset of

constriction
Constriction Amplitude (mm) Amp Difference between initial and minimum

pupil diameters (D1-D2)
Constriction Percentage (%) %Cons. Constriction Amplitude as percentage of

initial resting pupil diameter
Mean Constriction Velocity (mm/sec) VC Mean rate of pupil diameter change

during constriction phase
Maximum Constriction Velocity (mm/sec) VCmax Maximum rate of pupil diameter change
during constriction phase
Maximum Constriction Acceleration (mm/sec2) ACmax Maximum acceleration of pupil

diameter during constriction phase
Mean Dilation Velocity (mm/sec) VD Mean rate of pupil diameter change

during the initial dilation phase
75% Recovery Time (sec) 75%RT Latency between min pupil and 75% re-
dilation
Latency to Minimum Pupil Size (sec) T2 Time from stimulus to reach minimum
pupil size
Percentage Recovery after 3.5 seconds (%) PR3.5 Recovery of pupil after 3.5 seconds,
relative to constriction amplitude
(See Figure 2.5 for illustration of PFR parameters)

57
______________________________________________________________________
Figure 2.5. Illustration of PFR response and parameters measured.
A) Pupil diameter, B) Pupil velocity (rate of change of pupil diameter), C) Pupil acceleration (rate of
change of pupil velocity).
T1: constriction latency, T2: latency to minimum pupil size, T3: time at which 75% recovery attained
(75% recovery time = T3-T2), D1: resting pupil diameter, D2: minimum pupil diameter, VCmax:
maximum constriction velocity (see also Table 2.3).
58
2.7) Statistical Analysis
The data from this study were analysed using a variety of statistical methods that were
deemed appropriate for the hypotheses tested. All statistical analyses were conducted in
XLstat 2011 (Microsoft Excel). Normality of distribution was tested using the Shapiro-
Wilk test and visual inspection of variable histograms (and visual inspection of
residuals from linear regression analyses). Descriptive data analyses were undertaken to
determine the means and standard deviations (SD) for the full cohort and each group.
Demographic comparisons were performed using a χ2 test for categorical variables
(gender, hypertension, diabetes, smoking status and APOE ε4 status), and analysis of
variance (ANOVA) for the continuous age variable (p < 0.05 considered significant).
Across-group ocular measures were compared using analysis of covariance
(ANCOVA), correcting for confounders. Confounders considered for the retinal
measures were age, gender, hypertension, diabetes, smoking status and APOE ε4
status). Confounders considered for the pupil measures were age, gender and APOE ε4
status. The likelihood of false positive results was minimised by adjusting p values
according to the Benjamini and Hochberg false discovery rate (FDR) method
(Benjamini and Hochberg, 1995).
Receiver-operating characteristic (ROC) curve analysis was also performed to further
illustrate the classification accuracy of the ocular measures. The area under the curve
(AUC) of the ROC curves was calculated; an AUC of 1 indicates perfect classification
ability into AD or HC, whereas an AUC near 0.5 indicates poor (random) classification
ability (Swets, 1996). Logistical models combining ocular measures were created to
assess combined classification performance.

59
A bimodal distribution of PiB SUVR was observed in the HC group of the full AIBL
study. Consequently, to identify a PiB cutoff, analysis was performed on all elderly HC
research participants at Austin Health (n=118, age 73.2±7.4 years) using a hierarchical
cluster analysis that yielded a cutoff of 1.5 for neocortical SUVR (Villemagne et al.,
2008). Subjects were classified as PiB negative if their neocortex SUVR was below 1.5,
and PiB positive if their neocortex SUVR was above or equal to 1.5.
All AD and MCI participants in the ocular neuroimaging cohorts tested positive to
elevated plaque burden, hence the groups considered for ANCOVA analysis were HC-,
HC+, MCI+ and AD+, with the sign indicating testing positive or negative for elevated
plaque burden. Unmatched HC-, HC+, MCI+ and AD+ groups were compared using
standard ANCOVA models with Benjamini and Hochberg false discovery rate
adjustment (Benjamini and Hochberg, 1995).
Linear regression, adjusted for the above confounders, was used to model the
relationship between retinal parameters and:
1) SUVR for the HC+ group
2) Change in SUVR over the 18 month period prior to ocular testing for all 30
HC participants
Statistical significance was defined as p < 0.05.

60
Chapter 3: Retinal Vasculature in Clinically Diagnosed and Pre-
Clinical Sporadic Alzheimer’s Disease: Published Findings
3.1) Introduction
While retinal degeneration in Alzheimer’s disease (AD) has been extensively reported,
only one study has reported a retinal vascular abnormality (venular constriction)
(Berisha et al., 2007). This study utilised a laser Doppler device and found both venular
constriction and reduced venular blood flow in AD. Advances in retinal photography
and digital retinal imaging have facilitated a variety of accurate and reliable
measurements of the optimality of the retinal vasculature, using a simpler and more
widely available technology. Therefore, in the present study, retinal images were
collected from AD and healthy control (HC) participants from the AIBL study. Retinal
vascular parameters (RVPs) were calculated and analyses were performed to establish if
retinal vascular abnormalities, that are detectable by retinal photography, occur in AD.
The earliest detectable change in pre-clinical AD is the buildup of amyloid plaque in the
brain. Early detection of AD, prior to irreversible neurological damage, is important for
the efficacy of current interventions as well as for the development of new treatments
and preventative interventions. While PiB-PET imaging and CSF amyloid are the gold-
standards for early AD-diagnosis premortem, these approaches have major practical
limitations for population screening.
In order to investigate whether retinal vascular abnormalities occur early in AD
pathogenesis, before clinical diagnosis is possible, analyses were conducted to
investigate possible associations between the retinal changes and neocortical amyloid
plaque burden.
61
The results, which have been published during the course of my PhD (Frost et al.,
2013), are presented in two sections: i) the association of RVPs and clinical diagnosis of
AD and ii) the association of RVP’s and neocortical amyloid plaque burden.
3.2) Study 1: Retinal Vasculature in Clinically Diagnosed
Sporadic Alzheimer’s Disease
This study addressed whether changes to vascular width or topology are associated with
diagnosis of AD. It also addressed the question of whether these retinal vascular
changes could be detected by non-invasive retinal photography and semi-automated
computerized image analysis.
3.2.1) Methods
Digital retinal colour photographs (disc centered, 45o field) were collected, de-identified
and analysed with semi-automated software. Trained graders followed a standardised
protocol and performed corrections to automated procedures as necessary. Nineteen
retinal vascular parameters (RVPs) were measured from the width and branching
geometry of the retinal vessels (see Table 2.2).
status). The likelihood of false positive results was minimised by adjusting p values
62
3.2.2) Cohort and Demographics
The cohort consisted of 25 probable-AD patients (age 72.4 ± 7.5 yrs, 12 male, 13
female) and 123 healthy control participants (age 71.6 ± 5.6 yrs, 55 male, 68 female).
The demographics of this cohort are presented in Table 3.1. HC and AD groups did not
differ significantly in age, gender, hypertension, diabetes or smoking status. There was
a higher percentage of APOE ε4 carriers in the AD group (p=0.019).
3.2.3) Results
After FDR adjustment, significant differences in 13 of 19 RVPs were found between the
AD and HC groups (Table 3.1 and Figure 3.1). The retinal vascular topographic
differences in AD can be broadly summarized as; 1) vascular attenuation (CRVE,
CRAE, LDRa), 2) increasing standard deviation of vessel width (BSTD), 3) reduced
complexity of the branching pattern (FD, Num1stB), 4) reduced optimality of the
branching geometry (AF, BCv), and 5) less tortuous venules (Tortv).

63
Table 3.1. Demographics and descriptive RVP analysis for HC and AD groups.
Healthy Alzheimer’s Disease P Value FDR adj. P ROC: AUC%
Control (SD%)
Number of Participants [N] 123 25
Age: Years [mean (SD)] 71.6 (5.6) 72.4 (7.5) 0.557‡
Gender; Males: [N (%)] 55 (45) 12 (48) 0.764†
Hypertension: [N (%)] 44 (36) 11 (44) 0.439†
Diabetes: [N(%)] 6 (5) 2 (8) 0.533†
History of Smoking: [N(%)] 5 (4) 2 (8) 0.407†
APOE ε4 Carrier: [N (%)] 38 (31) 14 (56) 0.019†
CRVE [mean (SD)] 182.7 (15.8) 169.7 (15.3) 0.000256§ 0.0049* 0.703 (0.067)
FDv [mean (SD)] 1.210 (0.05) 1.171 (0.048) 0.000350§ 0.0033* 0.716 (0.074)
BSTDa [mean (SD)] 4.101 (0.504) 4.538 (0.984) 0.00135§ 0.0086* 0.595 (0.070)
BSTDv [mean (SD)] 3.983 (0.575) 4.433 (1.333) 0.00188§ 0.0089* 0.541 (0.081)
Num1stBv [mean (SD)] 3.618 (1.052) 2.960 (1.136) 0.00560§ 0.021* 0.660 (0.121)
Num1stBa [mean (SD)] 3.675 (1.075) 3.040 (0.978) 0.00710§ 0.022* 0.675 (0.142)
FDa [mean (SD)] 1.235 (0.052) 1.201 (0.061) 0.00799§ 0.021* 0.644 (0.075)
CRAE [mean (SD)] 129.1 (10.3) 122.9 (12.4) 0.0115§ 0.027* 0.612 (0.082)
AFa [mean (SD)] 0.778 (0.086) 0.824 (0.081) 0.0176§ 0.037* 0.578 (0.081)
BCv [mean (SD)] 1.253 (0.165) 1.347 (0.240) 0.0186§ 0.035* 0.556 (0.084)
Tortv (x10-5) [mean (SD)] 7.660 (1.554) 6.952 (2.601) 0.0244§ 0.042* 0.706 (0.073)
AFv [mean (SD)] 0.701 (0.097) 0.748 (0.095) 0.0301§ 0.047* 0.616 (0.074)
LDRa [mean (SD)] 17.05 (7.87) 21.72 (9.55) 0.0333§ 0.049* 0.651 (0.068)
JEv [mean (SD)] -0.110 (0.378) -0.272 (0.338) 0.0483§ 0.066* 0.539 (0.074)
Only RVPs that were significantly different between groups (p < 0.05) in ANCOVA analysis are shown.
Significant results after FDR adjustment shown in bold type.
‡
Analysis of variance (ANOVA) for the continuous age demographic variable (p < 0.05 considered
significant)
† 2
χ test for categorical demographic variables (gender, hypertension, diabetes, smoking status and APOE
ε4 status) (p < 0.05 considered significant)
§
p value from ANCOVA analysis of differences between groups (including confounders).
*
ANCOVA p values adjusted for false discovery rate (FDR) (Benjamini and Hochberg, 1995) (p < 0.05
considered significant).
Classification accuracy of RVP parameters from ROC analysis, AUC (area under the curve): AUC=0.5
implies random separation of groups, AUC=1.0 implies perfect separation.
Refer to Table 2.2 and Figure 2.2 for a description of the retinal vascular parameters. APOE ε4 Carrier
refers to carrier/non-carrier of an Apolipoprotein E e4 allele.
64
Logistical models combining parameters were created for combined AD classification.
A logistic model combining these 13 RVP’s provided good classification performance
(81.2% sensitivity, 75.7% specificity and 87.7% AUC), compared to the logistic model
including only age and APOE ε4 status (68.0% sensitivity, 61.8% specificity and 63.7%
AUC).
______________________________________________________________________
Figure 3.1. Retinal vascular parameters compared across AD and HC groups.
Boxplot comparison of (A) Central Retinal Venular Equivalent Caliber (CRVE), (B) Fractal Dimension
of the venular network (FDv) across HC (n=123) and AD (n=25) groups. P values from ANCOVA
analysis of differences between groups (including confounders).
3.2.4) Discussion
In this Chapter, the main hypothesis addressed was that retinal vascular abnormalities
are associated with AD. It was found that the AD group differed significantly from the
HC group in 13 of 19 RVPs, after false discovery rate adjustment. The retinal vascular
abnormalities found in AD in the present study can be broadly summarized as; 1)
generalized vascular narrowing (CRVE, CRAE, LDRa), 2) increasing variation in
vessel width (BSTD), 3) reduced complexity of the branching pattern (FD, Num1stB),
65
4) reduced optimality of the branching geometry (AF, BCv) and 5) less tortuous venules
(Tortv).
Among the most significant retinal vascular abnormalities found in AD was generalized
venular narrowing, which opposes the generalized venular widening reported previously
in both vascular dementia (de Jong et al., 2011) and hypertension (Sun et al., 2009).
Since AD and vascular dementia are the most common forms of dementia, these
contrasting results demonstrating lower venular caliber in AD encourage further
research into retinal vascular changes that show potential to discriminate between these
forms of dementia.
It is interesting that while hypertension is a significant risk factor for AD and is known
to cause arteriolar narrowing and venular widening in the retinal circulation (Sun et al.,
2009), these findings indicate an opposing generalized venular narrowing in AD. Some
studies have reported that retinal vascular changes in hypertension may precede clinical
hypertension (Ikram et al., 2006, Kawasaki et al., 2009), a possibility that must be
considered in this study. However, undiagnosed hypertension is unlikely to be the cause
of the observed results in the present study firstly because elevated blood pressure at
time of retinal imaging was adjusted for and secondly because of the opposing changes
to venular width in hypertension and AD.
Another highly significant change observed in this study in AD, reduced fractal
dimension, has also been found to be lower in non-proliferative diabetic retinopathy of
the macular region (Avakian et al., 2002, Daxer, 1993). However, all diabetic
participants in the present study were controlled and did not exhibit diabetic
retinopathy.
66
Participants were excluded from the retinal studies if they had history or evidence of
screening for this project. It is possible that these exclusion criteria may have introduced
some bias in the study, however omitting these criteria would have made the results
more difficult to interpret due to the possible influence of glaucoma on retinal vessels,
discussed further in section 6.3.1, and cataracts or cataract surgery influencing retinal
image quality and retinal vessel properties.
Measurements of ocular refractive error were not available for this study. Dimensional
parameters (CRAE, CRVE, BSTD) were therefore subject to refractive error, unlike the
remaining RVPs which are dimensionless. Bias from magnification differences is not
profound in most eyes within the refractive power range of ±3 Diopter (Wong et al.,
2004) and refractive errors are not likely to be associated with AD and hence are
unlikely to confound the associations assessed. The vessel width reduction observed in
AD, in contrast with the increase in the standard deviation of vessel width, lends
support to vessel width changes in AD that are independent of magnification effects,
since magnification effects alone would be expected to influence both parameters in the
same manner. It is possible that the process of vessel narrowing in AD affects vessels
selectively, hence increasing the standard deviation of vessel width.
These findings add to the growing evidence that retinal changes occur in AD. This is the
first study to go beyond blood column diameters and instead investigated a suite of 19
topographic retinal vascular parameters, finding significant abnormalities in 13 of these
RVPs in AD. The results also demonstrate for the first time that these changes can be
detected using non-invasive, readily available retinal photography. Models combining
RVPs perform well at distinguishing diagnosed AD patients from healthy controls.
However these models are optimised for the present data set and should be tested on
67
other cohorts in future. Retinal photographic analysis shows potential as an adjunct
measure in diagnosis of AD or monitoring of AD-progression or response to treatments.
3.3) Study 2: Retinal Vasculature and Neocortical Amyloid
Plaque burden
This study addressed whether changes to vascular width or topology are associated with
neocortical plaque burden in preclinical AD. Given the results of study 1, showing
retinal vascular changes in diagnosed AD, study 2 investigated whether these changes
occur early enough in disease pathogenesis to be detectable before cognitive symptoms
arise.
3.3.1) Methods
Digital retinal colour photographs (disc centered, 45o field) were collected, de-identified
and analysed with semi-automated software. Trained graders followed a standardised
protocol and performed corrections to automated procedures as necessary. Nineteen
retinal vascular parameters (RVPs) were measured from the width and branching
geometry of the retinal vessels (see Table 2.2).

68
status). The likelihood of false positive results was minimised by adjusting p values
AIBL neuroimaging data was available for 45 HC participants. This neuroimaging
cohort was grouped according to high (SUVR > 1.5) or low (SUVR < 1.5) neocortical
amyloid plaque burden (HC+ and HC- respectively). The demographics of the
neuroimaging cohort are presented in Table 3.2. There were 15 participants in the HC+
group and 30 participants in the HC- group. The HC+ group had a higher percentage of
69
APOE ε4 carriers than the HC- group (p=0.04), there were no significant differences in
the other demographic variables.
Table 3.2. Demographics of the RVP neuroimaging cohort.

HC- HC+ P Value
Number of Participants: [N] 30 15
Age: Years [mean (SD)] 70.4 (5.3) 73.7 (6.3) 0.08‡
Gender; Males: [N (%)] 15 (50) 9 (60) 0.53†
Hypertension: [N (%)] 11 (37) 6 (40) 0.52†
Diabetes: [N (%)] 1 (3) 2 (13) 0.99†
Smokers: [N (%)] 2 (7) 0 (0) 0.99†
APOE ε4 Carrier: [N (%)] 14 (47) 12 (75) 0.04†
HC-: Healthy controls with low plaque burden; HC+: healthy controls with high plaque burden. SD:
standard deviation. No demographic was significantly different between groups. Significant results in
bold type.
‡
significant)
† 2
χ test for categorical demographic variables (gender, hypertension, diabetes, smoking status and APOE
ε4 status) (p < 0.05 considered significant)
3.3.3) Results
ANCOVA analysis revealed larger venular branching asymmetry factor (AFv) and
arteriolar length-to-diameter ratio (LDRa) in the HC+ group (p=0.01 and p=0.02
respectively, after FDR adjustment, see Figure 3.2). These two parameters were also
larger in AD compared to HC; hence these results are consistent with the hypothesis
that RVP changes may precede AD diagnosis.
Combined in a logistic model, AFv and LDRa could identify high plaque burden in the
HC group with 76.9% sensitivity, 69.2% specificity and 74.6% AUC. When combined
70
with age and APOE ε4 status, the classification performance improved to 84.7%
sensitivity, 69.2% specificity and 82.8% AUC (compared to a logistic model with only
age and APOE ε4 status; 66.7% sensitivity, 73.3% specificity and 73.8% AUC).
______________________________________________________________________
Figure 3.2. Asymmetry Factor of the venular network (AFv) compared between
groups.
Boxplot comparison of AFv between (A) HC (n=123) and AD (n=25) groups and, (B) HC- (n=30) and
HC+ (n=15) groups. P values from ANCOVA analysis of differences between groups (including
confounders).
Investigation within the HC+ group, treating neocortical plaque burden (SUVR) as a
continuous parameter (see Figure 3.3), revealed trends for a number of RVPs, the
strongest of which was a negative trend for LDRv (p=0.0067, FDR adj. p=0.13). LDRv
also exhibited a negative trend with longitudinal change in SUVR over 18 months
(p=0.0076, FDR adj. p=0.14), in the HC group (see Figure 3.4). However, none of these
trends were significant after FDR adjustment.

71
35
HC-
Venular Length to Diameter

30
HC+
25
Ratio (LDRv) 20
15
10
0
1 1.5 2 2.5 3
SUVR
______________________________________________________________________
Figure 3.3. Scatter plot for venular length to diameter ratio (LDRv) with SUVR,
for HC- and HC+ groups.
______________________________________________________________________
Figure 3.4. Scatter plot for venular length to diameter ratio (LDRv) with 18-month
change in neocortical plaque burden (SUVR) for the HC group.
3.3.4) Discussion
Many studies have reported retinal degeneration in AD, particularly thinning of the
RNFL and loss of ganglion cells. However, only one previous study has reported retinal
vascular abnormalities in AD, involving thinning of the major superior temporal venule
blood column diameter and reduced blood flow in this vessel, using a laser Doppler
device (Berisha et al., 2007). Retinal photography is a more widely available
technology for investigating the retina, with eye clinics and many optometrists now
72
utilizing the technique to provide regular retinal health checks. In addition, advances in
digital retinal imaging have facilitated accurate and reliable measurements of the
optimality of the retinal vasculature.
The retinal vascular abnormalities found in AD in the present study can be broadly
summarized as; 1) generalized vascular narrowing (CRVE, CRAE, LDRa), 2)
increasing variation of vessel width (BSTD), 3) reduced complexity of the branching
pattern (FD, Num1stB), 4) reduced optimality of the branching geometry (AF, BCv)
and 5) less tortuous venules (Tortv).
An additional question addressed by the present study was whether these changes occur
late in the disease process when AD is clinically diagnosed, or earlier in the disease
process, providing prognostic potential before conventional diagnosis is possible. To
address this question, retinal vascular parameters were compared between healthy
individuals with high (HC+) and low (HC-) neocortical plaque burden, as measured by
PiB-PET imaging. High plaque burden is predictive of progression to AD (Pike et al.,
2007, Rowe et al., 2010, Sperling et al., 2011), so the HC+ group is believed to
represent those participants in the pre-clinical stage of AD.
Two of the RVPs that were found to be elevated in AD, venular branching asymmetry
factor (AFv) and arteriolar length-to-diameter ratio (LDRa), were also higher in the
HC+ group compared to the HC- group. LDRv was not significantly different between
AD and HC groups, but demonstrated a negative trend with continuous SUVR in the
HC+ group, and with rate of change of SUVR in the HC group. These results indicate
that changes to retinal vascular width and branching may be occurring early in AD
pathogenesis, during the asymptomatic plaque deposition stage before subsequent

73
cognitive decline. Hence retinal photography combined with vascular analysis indicates
potential as an adjunct to detect pre-clinical AD.
The findings reported here indicate a relationship between retinal vascular parameters,
neocortical amyloid plaque load, and AD. It is of interest to evaluate the possible
pathophysiological basis of these results. While cerebral amyloid plaques and neuro-
degeneration (particularly hippocampal) are the main hallmarks of AD, cerebral
vascular changes are also known to occur in the disease. In particular, vascular disease
was also evident in the original and disease defining case of Alzheimer (Alzheimer,
1987), and cerebral amyloid angiopathy (CAA), characterised by deposition of amyloid
in vessel walls, has been well documented in AD (Ellis et al., 1996, Jellinger, 2002,
Vinters et al., 1996). Given the homology between the retinal and cerebral
microvasculature (Patton et al., 2005), concomitant amyloid angiopathy in AD might
extend to the retina, with associated destruction of vessel walls resulting in changes to
vascular width and topology.
Since vascular changes and neurodegeneration appear to be occurring in both the brain
and retina in AD, there is some suggestion that AD-specific pathology could also be
occurring in the retina. Fascinatingly, preliminary evidence is emerging that Aβ plaques
may occur in the human AD retina (Koronyo-Hamaoui et al., 2010), possibly providing
a more accessible location to assess AD-specific neuro-pathology. However, further
research is needed to determine the nature of these retinal plaques and their relationship
to AD and possible concomitant ocular disease. In addition, potential relationships
between retinal degeneration reported in AD (Bayer et al., 2002, Berisha et al., 2007,
Blanks et al., 1989, Blanks et al., 1996, Blanks et al., 1996, Danesh-Meyer et al., 2006,
Hinton et al., 1986, Iseri et al., 2006, Paquet et al., 2007, Parisi et al., 2001, Sadun and
Bassi, 1990, Sadun and Bassi, 1990, Tamura et al., 2006, Tsai, 1991), retinal Aβ
74
plaques, and the retinal vascular changes reported in the present study are intriguing, but
require further investigation.
Interestingly, a previous study examining retinal vascular parameters in dementia
reported that wider retinal venules are associated with an increased risk of vascular
dementia (de Jong et al., 2011). Since AD and vascular dementia are the most common
forms of dementia, the contrasting results reported here demonstrating lower venular
caliber in AD encourage further research into retinal vascular changes that show
potential to discriminate between these forms of dementia.
The major limitation of this study is the size of the AD and neuroimaging cohorts.
Future studies with larger cohorts are needed to further examine associations between
RVPs and AD or neocortical plaque burden. The major strength of the study is the well
characterised cohorts and multi-disciplinary investigation, including neuro-imaging data
that enable deeper interrogation of associations between retinal vascular parameters and
AD.
The results of the present study indicate that retinal photography combined with
vascular analysis might provide an adjunct for detecting pre-clinical AD, or for
monitoring disease progression and response to intervention. The study also found
retinal abnormalities in AD that oppose those previously reported in vascular dementia,
suggesting potential for retinal vascular analysis to distinguish between these most
common forms of dementia. Natural variation in RVPs between individuals may limit
the utility of a single retinal photography screening test for AD, hence it is possible that
retinal monitoring, allowing longitudinal analysis of retinal changes, might facilitate
more accurate pre-clinical detection or monitoring of AD. Future longitudinal studies

75
are planned to further explore this possibility and to determine the time-course of retinal
changes in AD.
76
Chapter 4: Pupil Flash Response in Clinically Diagnosed and Pre-
Clinical Sporadic Alzheimer’s Disease: Published Findings
4.1) Introduction
Ocular pathology and changes to vision and ocular function are being investigated for
early detection of AD. Pupil flash response (PFR) parameters are useful clinically in
detecting brain injury and drug use. Previous studies have identified numerous PFR
differences between AD and healthy controls, but a number of studies have also
contradicted these results, possibly due to variation in methodological approaches. It is
also unclear whether these changes are AD-specific or whether they occur early enough
in AD pathogenesis to enable pre-symptomatic screening.
In this study, PFR data were collected from AD, Mild Cognitive Impairment (MCI) and
Healthy Control (HC) participants from the Australian Imaging, Biomarkers and
Lifestyle (AIBL) Flagship Study of Ageing at the McCusker Foundation for
Alzheimer’s Disease Research in Perth, Australia. A commercially available
pupillometer was utilised to establish if PFR abnormalities in AD could be detected
with this instrument. The potential correlations between PFR parameters and brain
amyloid plaque burden (as a specific, pre-clinical feature of AD), were also investigated
in this study.
The results, which have been accepted for publication in the journal Current Alzheimer
Research (Frost et al., in press A), are presented in two sections: i) the association of
PFR parameters and clinical diagnosis of AD, and ii) the association of PFR parameters
and neocortical amyloid plaque burden. To my knowledge, no other study has published
results associating PFR with neocortical amyloid plaque burden.

77
4.2) Study 1: PFR in Clinically Diagnosed Sporadic Alzheimer’s
Disease
This study addressed whether changes to the PFR, as detected by the NeurOptics™
VIP™-200 Pupillometer, are associated with diagnosis of AD.
4.2.1) Methods
After adaptation to a darkened room, the PFR was collected from each participant. The
NeurOptics™ VIP™-200 Pupillometer provided fully automated operation and
calculation of eight response parameters. Graders masked to participant status used
automated software to calculate a further four PFR parameters, providing a total list of
twelve parameters for analysis (see Tables 2.3 and 4.1). The test was practiced once
before recording. Occasionally, an extra trial was needed to achieve a recording without
blinks or artefacts. Data was rejected if artefacts were present. The right eye was used
for all participants. A computer algorithm was used to remove minor blinks.
(ANCOVA), correcting for confounders. Confounders considered for the pupil
measures were age, gender and APOE ε4 status. The likelihood of false positive results
was minimised by adjusting p values according to the Benjamini and Hochberg false
discovery rate (FDR) method (Benjamini and Hochberg, 1995).
78
The cohort consisted of 19 probable-AD patients (age 71.9 ± 7.8 yrs, 10 male, 9 female)
and 70 healthy control participants (age 71.7 ± 5.7 yrs, 34 male, 36 female). The HC
and AD groups did not differ significantly in age, gender composition or APOE ε4
status. The demographics of this cohort are presented in Table 4.1.
4.2.3) Results
Figure 4.2 illustrates the mean PFR profile, relative to resting pupil size, for the AD and
HC groups. After correcting for confounders (age, gender and APOE ε4 status) and
adjusting for FDR, significant differences in 10 of 12 PFR parameters were found
between the AD and HC groups (Table 4.1 and Figure 4.2). The AD group exhibited
reduced resting pupil size (p=0.003), constriction amplitude (p=0.00008) and
constriction percentage (p=0.0003). In terms of velocity and acceleration of the
response, the AD group exhibited a significant reduction in mean constriction velocity
(p=0.0001), maximum constriction velocity (p=0.00009), mean dilation velocity
(p=0.02) and maximum constriction acceleration (p=0.0003). Despite the reduced
velocities, the AD group exhibited quicker recovery, with larger ‘Percentage Recovery
3.5 seconds after stimulus’ (p=0.01) and shorter ‘75% Recovery Time’ (p=0.04). There
was also a slower initiation of response in the AD group, with longer latency to
minimum pupil size (p=0.04) and a non-significant trend for longer constriction latency
79
(p=0.10). All p-values were adjusted for false discovery rate. In summary, these results
indicate a sluggish pupil response in AD.
The ROC curve analysis indicated that mean constriction velocity (VC) provided the
highest classification accuracy with sensitivity 73.7%, specificity 71.4% and AUC
76.1% AUC. The statistically significant PFR parameters were combined in a logistic
model, giving combined classification performance with sensitivity 84.1%, specificity
78.3% and AUC 89.6%. Adding age and APOE ε4 status to the PFR logistic model
improved classification performance (sensitivity 89.2%, specificity 76.8% and AUC
91.8%). A logistic model with only age and APOE ε4 status provided sensitivity 63.1%,
specificity 55.7% and AUC 57.9%, hence inclusion of PFR parameters improved
classification performance markedly.

80
Figure 4.1. Mean pupil flash response, relative to resting pupil size, for HC (dashed
line; N=70) and AD (solid line; N=19) groups.
The AD group exhibited smaller resting and minimum pupil size, constriction amplitude and percentage,
constriction and dilation velocities, constriction acceleration and 75% recovery time. Percentage recovery
3.5 seconds after stimulus was larger in the AD group.
Figure 4.2. The PFR differences between healthy controls and Alzheimer’s
patients.
Boxplot comparisons indicate a significant reduction in constriction velocity and amplitude in AD
patients compared to the control group: A) Mean Constriction Velocity; B) Constriction Amplitude for
Healthy Control (N=70) and Alzheimer’s disease (N=19) groups. P values from ANCOVA analysis of
differences between groups (including confounders).
81
Table 4.1. Demographics and Pupil Flash Response analysis for Healthy Control
and Alzheimer’s Disease groups.
Healthy Alzheimer’s ANCOVA FDR adj. AUC%
Control Disease P Value P Value (SD%)Δ
Age: Years [mean (SD)] 71.7 (5.7) 71.9 (7.8) 0.889‡
Gender; Males: [N (%)] 34 (49) 10 (53) 0.754†
APOE ε4 Carrier#: [N (%)] 30 (43) 11 (58) 0.247†
Mean Constriction Velocity

3.31 (0.46) 2.51 (0.95) 0.000012§ 0.0001* 0.765 (0.073)
[mean (SD)]
Max Constriction Velocity

4.39 (0.59) 3.43 (1.31) 0.000016§ 0.00009* 0.755 (0.079)
[mean (SD)]
Constriction Amplitude [mean

1.55 (0.28) 1.16 (0.50) 0.000020§ 0.00008* 0.772 (0.067)
(SD)]
Max Constriction Acceleration

33.2 (6.3) 25.3 (11.6) 0.000092§ 0.0003* 0.727 (0.079)
[mean (SD)]
Constriction % [mean (SD)] 31.2 (4.4) 26.14 (6.2) 0.00011§ 0.0003* 0.745 (0.054)
Resting Pupil Size [mean (SD)] 5.00 (0.83) 4.26 (1.16) 0.0017§ 0.003* 0.727 (0.072)
% Recovery after 3.5 seconds

70.5 (11.5) 78.8 (12.8) 0.0073§ 0.01* 0.663 (0.074)
[mean (SD)]
Mean Dilation Velocity [mean

0.77 (0.17) 0.65 (0.25) 0.013§ 0.02* 0.667 (0.077)
(SD)]
75% Recovery Time [mean

2.90 (1.06) 2.28 (1.09) 0.029§ 0.04* 0.656 (0.072)
(SD)]
Latency to Min Pupil Size [mean

0.28 (0.05) 0.30 (0.05) 0.036§ 0.04* 0.616 (0.087)
(SD)]
Constriction Latency [mean

227.5 (19.9) 237.2 (29.3) NS§ NS* 0.594 (0.180)
(SD)]
Minimum Pupil Size [mean

3.45 (0.69) 3.10 (0.71) NS§ NS* 0.645 (0.070)
(SD)]
Refer to Table 2.3 and Figure 2.5 for a description of retinal vascular parameters.
‡
significant)
† 2
χ test for categorical demographic variables (gender and APOE ε4 status) (p < 0.05 considered
significant)
§
*
ANCOVA p values adjusted for false discovery rate (FDR) (p < 0.05 considered significant, significant
results after FDR adjustment shown in bold type)
Δ
Classification accuracy of PFR parameters from ROC analysis, AUC: area under the curve, AUC=0.5
implies random separation of groups, AUC=1.0 implies perfect separation).
#
APOE ε4 Carrier refers to carrier/non-carrier of an apolipoprotein E e4 allele.
82
4.2.4) Discussion
When compared to the HC group, the AD patients exhibited smaller resting and
minimum pupil size, reduced constriction amplitude/percentage, reduced constriction
velocity and acceleration and reduced dilation velocity. In summary, this could be
described as a ‘sluggish’ pupil response and is consistent with earlier studies (Fotiou et
al., 2007, Fotiou et al., 2009, Fotiou et al., 2000, Granholm et al., 2003, Prettyman et
al., 1997). Despite the slower velocities, the AD group exhibited a faster overall
recovery to original pupil size, with reduced 75% recovery time and increased recovery
at 3.5 seconds after stimulus. Since both constriction and dilation velocities are slower
in AD, the quicker recovery is likely due to the overwhelmingly smaller amplitude, and
hence reduced dilation required for recovery. A logistic model combining the PFR
parameters provided good classification performance (sensitivity 84.1%, specificity
78.3% and AUC 89.6%).
Possible causes of the PFR changes in AD are degeneration in relays in the midbrain, or
central cholinergic depletion (Herholz et al., 2004, Tohgi et al., 1994). The five PFR
parameters that demonstrated the most significant differences in AD (VC, VCmax,
ACmax, Amp and %Cons.), are calculated from the constriction phase of the PFR
which is primarily a parasympathetic cholinergic response (Loewenfeld, 1999). Hence
these parameters are likely to be the most sensitive markers of cholinergic deficits in the
peripheral parasympathetic pathway. AD patients receiving pharmacological treatment
with anticholinesterase agents (such as Donepezil) have been excluded from this study,
due to the likely effect of these drugs on the PFR. The necessary exclusion of those on
anticholinesterase agents introduces some bias as it is likely that those not treated are
going to be different in some way from the 60-70% of AD subjects who do receive such
therapy.
83
Donepezil has been reported to normalise PFR in some AD patients (Fotiou et al., 2000,
Granholm et al., 2003), supporting the hypothesis that the observed PFR changes in AD
are due to cholinergic depletion. If PFR changes in AD relate to neurotransmitter status,
then PFR testing may be useful as an objective, non-invasive monitor with which to
follow disease progression and treatment efficacy. To investigate this, future
longitudinal studies could follow newly diagnosed AD patients for PFR changes.
Hypotheses that could be investigated include whether PFR testing can predict which
patients will benefit most from anticholinesterase treatment, and whether PFR
normalization during anticholinesterase treatment associates with clinical improvement.
Cholinergic depletion may also occur in other diseases such as Parkinson’s Disease
(Dubois et al., 1990), which has also been reported to influence PFR (Fotiou et al.,
2009, Granholm et al., 2003). AD patients are more likely to have Parkinsonism than
those undergoing healthy ageing (Funkenstein et al., 1993, Galasko et al., 1990, Huff et
al., 1987, Kischka et al., 1993, Merello et al., 1994, Morris et al., 1989), and similarly,
individuals with Parkinsonism are more likely to develop AD (Richards et al., 1993,
Richards et al., 1995), hence it is possible that PFR changes are related to Parkinsonism
or extrapyramidal signs. For these reasons, the specificity of PFR testing for AD needs
further investigation, utilising cohorts of participants with AD, Parkinson’s Disease and
Parkinsonism, as well as healthy controls.

84
4.3) Study 2: PFR and Neocortical Amyloid Plaque burden
This study addressed whether changes to the PFR are associated with neocortical plaque
burden in preclinical AD. Given the results of study 1, showing PFR changes in
diagnosed AD, study 2 investigated whether these changes occur early enough in
disease pathogenesis to be detectable before cognitive symptoms arise. This question
has relevance for evaluating the potential applicability for PFR parameters as
biomarkers for early and specific detection of AD, as part of a non-invasive, cost-
effective screening test for pre-clinical AD.
4.3.1) Methods
A group of participants derived from the AIBL study was recruited for this
investigation. After adaptation to a darkened room, the PFR was collected from each
participant. The NeurOptics™ VIP™-200 Pupillometer provided fully automated
operation and calculation of eight response parameters. Graders masked to participant
status used automated software to calculate a further four PFR parameters, providing a
total list of twelve parameters for analysis (see Table 2.3). The test was practiced once
before recording. Occasionally, an extra trial was needed to achieve a recording without
blinks or artefacts. Data was rejected if artefacts were present. The right eye was used
for all participants. A computer algorithm was used to remove minor blinks.
85
(gender, hypertension, diabetes, smoking status and APOE ε4 status), and one-way
analysis of variance (ANOVA) for the continuous age variable (p < 0.05 considered
significant). Across-group ocular measures were compared using analysis of covariance
(ANCOVA), correcting for confounders. Confounders considered for the pupil
measures were age, gender and APOE ε4 status. The likelihood of false positive results
was minimised by adjusting p values according to the Benjamini and Hochberg FDR
method (Benjamini and Hochberg, 1995), as described in the previous section.
The ROC curve analysis was also performed to further illustrate the classification
accuracy of the ocular measures. The area under the curve (AUC) of the ROC curves
was calculated; an AUC of 1 indicates perfect classification ability into AD or HC,
whereas an AUC near 0.5 indicates poor (random) classification ability (Swets, 1996).
Logistical models combining ocular measures were created to assess combined
classification performance.
AIBL neuroimaging data was available for 43 participants (34 HC, 4 MCI, 5 AD). Each
group was stratified according to high (SUVR > 1.5) or low (SUVR < 1.5) neocortical
amyloid plaque burden (e.g. HC+ and HC- respectively). All AD and MCI participants
had high plaque burden. Due to small numbers in the MCI+ and AD+ groups, they were
combined into a “pathologically impaired with high plaque burden” group (PI+). The
86
demographics of the neuroimaging cohort are presented in Table 4.2. Compared to the
HC- group, there was a higher percentage of APOE ε4 carriers in the HC+ group
(p<0.0001) and the PI+ group (p<0.0001). There were no significant differences in age
or gender. All analyses were adjusted for age, gender and APOE ε4 status.
Table 4.2. Demographics of the PFR neuroimaging cohort.

HC- HC+ MCI+ AD+ PI+
Number of Participants [N] 19 15 4 5 9
Age: Years [mean (SD)] 70.3 (5.8) 74.2 (5.6) 79.0 (5.0) 74.2 (10.0) 76.3 (8.1)
Gender; Males: [N (%)] 10 (53) 9 (60) 2 (50) 4 (80) 6 (67)
APOE ε4 Carrier: [N (%)] 8 (42) 14 (93) 4 (100) 5 (100) 9 (100)
SUVR at eye test: [mean (SD)] 1.30 (0.08) 1.95 (0.33) 2.06 (0.38) 2.27 (0.45) 2.18 (0.41)
Healthy controls with low plaque burden (HC-), healthy controls with high plaque burden (HC+), mild
cognitive impairment with high plaque burden (MCI+), Alzheimer’s disease with high plaque burden
(AD+), pathologically impaired (PI+). The PI+ group is composed of the MCI+ and AD+ groups. SD =
standard deviation.
APOE ε4 Carrier refers to carrier/non-carrier of an apolipoprotein E e4 allele.
SUVR refers to standardised neocortical plaque burden, as measured by PiB-PET neuroimaging.
While PFR data were collected cross-sectionally, longitudinal AIBL neuroimaging data
was available for most of the PFR neuroimaging cohort. Analysis was performed to
compare PFR measures with rate of change of neocortical plaque burden over the 18
month period prior to ocular testing. Groups were stratified as “SUVR increasers” if
their change in SUVR was greater than 0.02, or as non-increasers if their change in
SUVR was less than or equal to 0.02. Six neuroimaging participants did not have prior
(18 month previous) neuroimaging data (2 AD, 2 MCI, 2 HC), hence analysis for 18
month change in SUVR was limited to 37 participants. The demographics of the

87
longitudinal neuroimaging cohort are presented in Table 4.3, there were no significant
demographic differences between the groups. SD = standard deviation.
Table 4.3. Demographics of the PFR longitudinal neuroimaging cohort.

SUVR Increasers Non-Increasers Total
Number of Participants [N] 22 15 37
Age: Years [mean (SD)] 73.5 (6.4) 70.5 (5.8) 72.3 (6.3)
Gender; Males: [N (%)] 14 (64) 8 (53) 22 (59)
APOE ε4 Carrier: [N (%)] 17 (77) 9 (60) 26 (70)
Groups stratified as SUVR increasers (SUVR change > 0.02) and non-increasers (SUVR change ≤ 0.02).
There were no significant demographic differences between the groups. SD = standard deviation.
4.3.3) Results
The neuro-imaging data was used to explore potential PFR differences between the HC-
and HC+ groups, identifying healthy individuals and those at the earliest stages of AD,
respectively. Figure 4.3 illustrates the difference in mean PFR profiles, relative to
resting pupil size, for HC- and HC+ groups. Qualitatively, the difference observed
between the HC- and HC+ groups is similar to the difference between the HC and AD
groups (see Figure 4.1). ANCOVA analysis revealed that mean constriction velocity
was slower (p=0.03 after FDR adjustment) and constriction amplitude was smaller in
HC+ (p<0.05 after FDR adjustment) compared to HC- (see Figure 4.4).
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Figure 4.3. Mean pupil flash response relative to resting pupil size.
Healthy controls with low plaque burden (HC-, dashed line) and with high plaque burden (HC+, solid
line). The HC+ group exhibited reduced amplitude and mean constriction velocity.
Figure 4.4. Comparison of Mean Constriction Velocity (VC) between groups.

Boxplot comparison of VC for Healthy Control groups with low (HC-) and high (HC+) plaque burden. P
values from ANCOVA analysis of differences between groups (including confounders).
Mean constriction velocity alone could detect high plaque burden in the HC group with
66.7% sensitivity, 79.0% specificity and 76.5% AUC. Due to the significantly higher
percentage of APOE ε4 carriers in the HC+ group, a logistic model with only age and
APOE ε4 status provided high classification performance (86.7% sensitivity, 84.2%

89
specificity and 85.6% AUC). Adding mean constriction velocity or constriction
amplitude to this model did not improve the classification performance.
Linear regression was used to test for correlations between PFR parameters and
continuous neocortical plaque burden (SUVR) for the PI+ group, correcting for age and
gender. A significant negative association was found between SUVR and maximum
constriction acceleration (p=0.04 after FDR adjustment). Negative trends were also
found between SUVR and both maximum and mean constriction velocity, and a
positive trend with latency to minimum pupil size, however these trends were not
significant after FDR adjustment (see Table 4.4 and Figure 4.5).
Table 4.4. Linear associations between PFR parameters and continuous

neocortical plaque burden.
PFR Parameter Std. Coeff. Std. Error Model R2 p-value§ FDR adj. p-value*
ACmax -0.844 0.203 0.713 0.004 0.04
VCmax -0.731 0.258 0.535 0.025 0.11
T2 0.728 0.259 0.530 0.026 0.06
VC -0.700 0.270 0.490 0.036 0.11
Results of linear regression for PFR variables with SUVR, in PI+ group.
ACmax = maximum constriction acceleration, VCmax = maximum constriction velocity, T2 = latency to
minimum pupil size, VC = mean constriction velocity.
Std. Coeff. = standardized coefficient, Std. Error = standard error.
§
p value from multivariate linear regression (including confounders).
*
p values adjusted for false discovery rate (FDR) (p < 0.05 considered significant, significant results after
FDR adjustment shown in bold type).
90
50
HC- HC+ MCI+ AD+
45
40
35
ACmax
30
25
20
15
10
1 1.5 2 2.5 3
SUVR
5 6
HC- HC+ MCI+ AD+ HC- HC+ MCI+ AD+
4.5 5.5
4 5
3.5 4.5
VC
VCmax
3 4
2.5 3.5
2 3
1.5 2.5
1 2
1 1.5 2 2.5 3 1 1.5 2 2.5 3
SUVR SUVR
Figure 4.5. PFR parameters and continuous neocortical plaque burden.

ACmax = maximum constriction acceleration, VC = mean constriction velocity, VCmax = maximum
constriction velocity.
While pupil response data were collected at only one time-point for each participant in
this study, prior SUVR data has been collected longitudinally (periodicity 18 months) as
part of the AIBL study. As an alternative perspective on PFR changes during prodromal
stages of AD, the change in neocortical plaque burden (SUVR) over the 18 month
period prior to the retinal imaging was considered (ΔSUVR18month). The full
neuroimaging cohort was considered for this analysis (see Table 4.3). ΔSUVR18month
was negatively associated with maximum constriction acceleration (p=0.03 after FDR
adjustment) and mean dilation velocity (p<0.05 after FDR adjustment). Latency to
minimum pupil size was positively associated with ΔSUVR18month (p=0.02 after FDR
adjustment). Negative trends were also found between ΔSUVR18month and both
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maximum and mean constriction velocity; however these trends were not significant
after FDR adjustment (see Table 4.5 and Figure 4.6).
Table 4.5. Linear associations between PFR parameters and 18 month change in
SUVR.
ACmax -0.466 0.150 0.217 0.004 0.04
T2 0.459 0.150 0.211 0.004 0.02
VD -0.391 0.156 0.153 0.017 <0.05
VC -0.331 0.139 0.370 0.023 0.05
VCmax -0.322 0.144 0.327 0.031 0.06
Results of linear regression for PFR variables with 18 month change in SUVR (full neuroimaging cohort).
ACmax = maximum constriction acceleration, T2 = latency to minimum pupil size, VD = mean dilation
velocity, VC = mean constriction velocity, VCmax = maximum constriction velocity.
§
p value from multivariate linear regression (including confounders).
*
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50 1.2
45
1
40
0.8
35
VD
ACmax
30 0.6
25
0.4
20
0.2
15
10 0
-0.15 -0.1 -0.05 0 0.05 0.1 0.15 -0.15 -0.1 -0.05 0 0.05 0.1 0.15
18M change in SUVR 18M change in SUVR
0.38
0.36
0.34
0.32
LatMinPupil
0.3
0.28
0.26
0.24
0.22
0.2
-0.15 -0.1 -0.05 0 0.05 0.1 0.15
18M change in SUVR
4.5 6
5.5
4
5
3.5 4.5
VC
VCmax
3
3.5
3
2.5
2.5
2 2
-0.15 -0.1 -0.05 0 0.05 0.1 0.15 -0.15 -0.1 -0.05 0 0.05 0.1 0.15
18M change in SUVR 18M change in SUVR
Figure 4.6. PFR parameters and longitudinal SUVR.

ACmax = maximum constriction acceleration, VD = mean dilation velocity, LatMinPupil = latency to
minimum pupil size, VC = mean constriction velocity, VCmax = maximum constriction velocity.
Dividing the neuroimaging cohort into those with increased plaque burden
(ΔSUVR18month > 0.02, n=22) and those that showed no increase (ΔSUVR18month ≤
0.02, n=15), ANCOVA analysis revealed that increased plaque burden was associated
with lower constriction acceleration (p<0.05 after FDR adjustment). Trends were
observed for increased plaque burden and lower mean and maximum constriction
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velocity and mean dilation velocity; however these trends were not significant after
FDR adjustment.
Table 4.6. ANCOVA associations between PFR parameters and plaque burden
increase.
ACmax -0.456 0.150 0.208 0.005 <0.05
VCmax -0.395 0.155 0.156 0.016 0.07
VC -0.387 0.156 0.150 0.018 0.05
VD -0.365 0.157 0.133 0.026 0.06
Results of ANCOVA for PFR variables between SUVR-increasers (ΔSUVR18month > 0.02) and non-
increasers (full neuroimaging cohort).
ACmax = maximum constriction acceleration, VCmax = maximum constriction velocity, VC = mean
constriction velocity, VD = mean dilation velocity.
§
p value from ANCOVA analysis (including confounders).
*
4.3.4) Discussion
Our results demonstrate statistically significant relationships between PFR parameters,
neocortical plaque burden and AD. Many PFR parameters are significantly different
between HC and AD participants and logistic models including these parameters
demonstrate good performance at classifying AD from HC participants. Furthermore,
some of these PFR parameters (mean constriction velocity, constriction amplitude and
maximum constriction acceleration) were also associated with neocortical plaque
burden in pre-clinical AD. In addition, some PFR parameters (maximum constriction
acceleration, latency to minimum pupil size, mean dilation velocity and mean
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constriction velocity) in the HC subjects correlated with rate-of-change in plaque
burden over 18 months prior to pupil testing. The findings indicate potential
applicability for PFR parameters as an adjunct to other biomarkers for early and specific
detection and monitoring of AD. The PFR test shows potential to be included in a non-
invasive, cost-effective screening test for pre-clinical AD.
Consistent with earlier studies (Fotiou et al., 2007, Fotiou et al., 2009, Fotiou et al.,
2000, Granholm et al., 2003, Prettyman et al., 1997), PFR measures were more sluggish
in AD compared to HC, with smaller resting and minimum pupil size, reduced
constriction amplitude/percentage, reduced constriction velocity and acceleration and
reduced dilation velocity. Despite the slower velocities, the AD group exhibited a faster
overall recovery to original pupil size, with reduced 75% recovery time and increased
recovery at 3.5 seconds after stimulus. Since both constriction and dilation velocities are
slower in AD, the quicker recovery is likely due to the overwhelmingly lower
amplitude.
Since the literature had not addressed whether PFR changes occur early enough to
enable pre-symptomatic screening for AD, this study investigated PFR associations with
high neocortical plaque burden (SUVR) in pre-symptomatic individuals. A sluggish
PFR was also observed for the pre-symptomatic group with high plaque burden (HC+)
compared to those with low plaque burden (HC-), exhibiting trends for reduced
constriction amplitude, constriction velocity and constriction acceleration when
compared to the HC- group. However, these trends only reached statistical significance
for mean constriction velocity and constriction amplitude, which were also the
parameters that provided the best AD-classification performance.

95
While a relationship between these PFR parameters and high plaque burden was found,
adding these PFR parameters to a logistic model with age and APOE ε4 status did not
improve classification performance. Due to the high percentage of APOE ε4 carriers in
the HC+ group, it cannot be ruled out that the observed PFR changes in HC+ are in fact
a surrogate for APOE ε4 status. As carriers of the APOE ε4 allele are more likely to
develop AD, it is not unexpected that the HC+ group (considered to be a pre-clinical
AD group) has more carriers, although from a statistical standpoint it would be more
ideal to have included more HC+ non-carriers. Unfortunately these participants were
not available for this study. To reduce the possibility that the PFR differences in the
neuroimaging study were a surrogate for APOE ε4 status, this was included along with
age and gender as confounders in the analyses. APOE ε4 status was not retained in
stepwise ANCOVA models for these PFRs. In addition, for the full PFR cohort of 89
participants, no significant association with APOE ε4 status was found for any PFR
parameter, supporting the hypothesis that the PFR changes are related to high plaque
burden rather than APOE ε4 status.
Linear regression between PFR parameters and continuous SUVR within the
pathologically impaired (PI+) group revealed a significant negative correlation for
ACmax, with negative trends for VCmax and VC and a positive trend for latency to
minimum pupil size. Since longitudinal PiB-PET data preceding the PFR testing was
available for the AIBL cohort, the relationship between PFR variables and the 18-month
change in SUVR was also investigated. ACmax and VD were significantly (negatively)
associated with increasing SUVR. ACmax was also lower in participants who exhibited
an increase in SUVR values, based on ANCOVA analysis. Hence PFR parameters
(particularly ACmax) have the potential to identify individuals with larger or increasing
plaque burden.
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PFR may be influenced by other conditions or factors, such as pseudoexfoliation (a
common cause of secondary glaucoma), past history of trauma or uveitis/iritis, tonic
pupils or even iris colour. Natural variation in pupillary response between individuals
may curtail the utility of PFR testing for detection of high plaque burden in
asymptomatic individuals. However, if longitudinal changes in pupillary response are
related to longitudinal changes in SUVR or conversion to AD, then the value of PFR
testing may be in its use for providing a non-invasive monitor of physiological
abnormality with which to follow disease progression and treatment efficacy.
Relationships demonstrated in this study between PFR parameters and SUVR (or rate of
change of SUVR) suggest a possible role for PFR testing as an adjunct, together with
clinical assessments, for monitoring of disease progress and response to treatment.
However, future longitudinal studies will be needed to further evaluate this possibility.
The major limitation of this study is the size of the AD and neuroimaging cohorts. This
is a preliminary study potentially reporting a finding for others to explore and replicate.
The major strength of the study is the well characterised cohorts and multi-disciplinary
investigation, including neuro-imaging data that enable deeper interrogation of
associations between retinal vascular parameters and AD.
The hypothesis that cholinergic depletion may be behind the observed PFR changes
opens up many opportunities for new studies. Future longitudinal studies could follow
newly diagnosed AD patients for PFR changes and determine whether PFR testing can
predict which patients will benefit most from anticholinesterase treatment, and whether
PFR normalization during anticholinesterase treatment associates with clinical
improvement. As cholinergic depletion and PFR changes may also occur in Parkinson’s
disease (Dubois et al., 1990, Fotiou et al., 2009, Granholm et al., 2003), future studies
should include patients with AD and patients with Parkinson’s disease. Additionally, the
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relationship between PFR and AD could be tested in ‘cleaner’ AD cohorts without co-
morbidity with Parkinson’s disease or Parkinsonism. An example is the Dominantly
Inherited Alzheimer Network (DIAN) study cohort, including autosomal dominant
Alzheimer’s disease mutation carriers who often become symptomatic at a much
younger age, as investigated in the following Chapter.

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Chapter 5: Ocular Biomarkers and Early Onset Familial
Alzheimer’s Disease: Published Findings
5.1) Introduction
A rare form of Alzheimer’s disease (AD), “autosomal dominant AD” (ADAD), affects
carriers of specific gene mutations with penetration approaching 100% (Sherrington et
al., 1996). ADAD represents about 5% of all AD cases and can occur in people as
young as 30 years of age. It is a genetic disorder with specific mutations in primarily
amyloid precursor protein (APP), presenilin 1 and presenilin 2 genes known to cause
the disease (Campion et al., 1995, Goate et al., 1991, Murrell et al., 1991, Sherrington
et al., 1996). It is inherited in an autosomal dominant manner. Children of affected
individuals have fifty-percent risk to develop AD dementia, with a relatively predictable
age at onset within each family.
Despite the difference in underlying cause and age of onset, ADAD and the more
common sporadic AD have similar neuropathologic hallmarks and clinical features
(Bateman et al., 2011). Evidence suggests that AD related pathological changes begin at
least 15 years before the symptomatic stage (Bateman et al., 2012), providing a window
of time in which to detect the disease early and allow emerging therapies the chance to
preserve healthy brain function. Hence, new knowledge about the disease process in
ADAD has the potential to translate into better methods for early detection of sporadic
AD.
Studies of families harboring known autosomal-dominant AD (ADAD) mutations
provide a powerful opportunity to investigate the temporal sequence of ocular and other
AD biomarker changes during disease progression. Studying pre-symptomatic
individuals with ADAD mutations alleviates many problems inherent in studies of pre-
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symptomatic sporadic AD, including uncertainty about age of onset. In addition, the
early age of onset in ADAD means that these studies will be less confounded by age-
related co-morbidities such as hypertension and cardio-vascular disease.
This study investigates ocular biomarkers in ADAD by recruiting West-Australian
participants from the Dominantly Inherited Alzheimer Network (DIAN) study cohort
(Bateman et al., 2011), at the McCusker Alzheimer Research Foundation based in
Perth. The results have been accepted for publication in the journal Current Alzheimer
Research (Frost et al., in press B).
Results from the DIAN study have already established that AD-related CSF pathology
can be detected in mutation carriers more than two decades before their estimated age at
dementia onset (see Figure 5.1) (Bateman et al., 2012). Studying AD biomarkers in
ADAD could prove useful for the development and evaluation of disease-modifying
prevention measures in both EOFAD and sporadic AD populations.
Most DIAN study participants are in the pre-symptomatic stages of the disease and
hence provide a unique means by which to investigate the time-course of biomarker
changes in the lead-up to clinical expression of the disease. Since the parental age of
dementia onset within each family is typically known, the stage of disease progression
for pre-symptomatic family members (e.g. 10 years prior to estimated age of dementia
onset) can be accurately estimated and used as a temporal reference for biomarker
changes. This also enables comparison of biomarker changes across families with
different mutations and typical age of onset.

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CSF Aβ
PiB Binding
CSF Tau
Hippocampal Volume
Brain Metabolism
Episodic Memory
CDR
-25 -20 -15 -10 -5 +5
Years Estimated
Age at Onset
of Symptoms
Figure 5.1. Alzheimer Biomarker Pathochronology in Autosomal Dominant AD.

CSF = cerebro-spinal fluid, Aβ = amyloid-beta, CDR = clinical dementia rating.
The levels of CSF Aβ are initially elevated (~25y before estimated age at onset) but then are marked by
progressive decline (adapted from Morris et al., using data presented in Bateman et al. (Bateman et al.,
2012, Morris et al., 2012)
5.2) Study Design
Alzheimer’s disease (AD) is usually only diagnosed many years after AD pathology
begins. Earlier detection would allow emerging interventions to have a greater chance to
preserve healthy brain function. A rare form of (AD), caused by autosomal-dominant
AD (ADAD) mutations, affects carriers with 100% certainty and at a younger age
specific to their mutation. Studying families with ADAD mutations allows a unique
investigation of the temporal sequence of biomarker changes in AD.

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The objective of this study was to determine whether RVP or PFR measures, previously
reported to be altered in sporadic AD, are different in pre-symptomatic ADAD mutation
carriers. Participants were recruited from the Dominantly Inherited Alzheimer's
Network (DIAN) Study during 2010-2011. Ocular data was collected and both RVP and
PFR measures calculated while masked from participant groupings, then statistical
analysis was performed to compare groups.
Participants were recruited from the DIAN study at the McCusker Foundation for AD
Research, Perth, Western Australia (www.dian-info.org, clinicaltrials.gov number
NCT00869817). The DIAN study methodology has been published elsewhere (Morris
et al., 2012) and were approved by the Washington University Human Research
Protection Office, the Hollywood Private Hospital Ethics Committee and the Edith
Cowan University. All PFR experiments were approved by the Ethics Committee of the
University of Western Australia. All participants provided their written informed
consent for this study.
Participants were excluded from the retinal study if they had history or evidence of
screening for this project. Exclusion criteria for the PFR study were past history of
ocular operations or ophthalmological disease, asymmetrical pupils, receiving
anticholinergics, cardiac glycosides, sympathomimetics or ophthalmic agents affecting
PFR. “Asymmetrical pupils” was defined as an asymmetry of pupil diameter greater
than 2mm.
All participants were white Caucasians from a single family harbouring Dutch cerebral
amyloid angiopathy (hereditary cerebral hemorrhage with amyloidosis-Dutch type,

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HCHWA-D). The APP 693 mutation at position 22 of the Aβ fragment
(APPGlu693Gln), resulting in a glutamine for glutamic acid, was identified in the
proband and his affected sister who died at age 61 and 66 respectively, from recurrent
lobar haemorrhages in the brain. Neuritic amyloid plaques were found but neither
dementia nor cognitive decline have been diagnosed in this family, possibly due to
earlier onset of vascular pathology.
The mean paternal anticipated age at onset (AAO) is 51, all but 2 of the 12 were
younger than the parental AAO at time of PFR, and 8 of the 12 were within 15 years
younger than the parental AAO. All participants performed above the Mini Mental State
Examination (MMSE) cut off score for dementia (>24) (Folstein et al., 1975), indicating
normal cognitive function. The mean MMSE scores were 29.0±0.9 for MC, 27.5±2.1
for NC.
Participants were neuroimaged for the presence of fibrillar brain amyloid using positron
emission tomography (PET) with Pittsburgh Compound B (PiB) (Klunk et al., 2004,
Klunk et al., 2005). Results for neocortical standardized uptake value ratio (SUVR)
were calculated.
5.2.2) Genetic Analysis
Genotyping for familial AD mutations and APOE isoforms was performed by the DIAN
Genetics Core. Ambient blood samples were shipped from the DIAN performance sites
to both the National Cell Repository for Alzheimer’s disease (NCRAD) and the DIAN
Genetics Core at Washington University. DNA was extracted from blood at both sites
using standard procedures. DNA sequencing of APP, PSEN1 and PSEN2 was
performed by DIAN Genetics Core personnel, using Sanger sequencing methods on an
ABI 3130xl, to determine the presence/absence of a disease-causing mutation (Kauwe et

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al., 2007). APOE genotyping was performed using an ABI predesigned real time
TaqMan assay “rs7412 & rs429358” according to the manufacturer’s protocol (ABI,
Foster City, California). DNA fingerprinting was performed with the Cell ID kit, using
short tandem repeat (STR) analysis of 10 specific loci in the human genome, nine STR
loci and Amelogenin for gender identification (Promega #G9500, Madison, Wisconsin)
in order to confirm that DNA samples obtained by NCRAD and the DIAN Genetics
Core were from the same individual. DNA sequencing, fingerprinting and genotyping
was performed on DNA from NCRAD and the DIAN Genetics Core in parallel for each
individual, and the data were compared for quality control purposes. All individuals
included in this analysis have 100% concordant data for each DNA sample.
5.3) Results
The demographic characteristics of the MC and NC groups are described in Table 5.1.
Groups were not significantly different in age, gender, hypertension, APOE ε4 carriers
or MMSE score. Neocortical plaque burden (Standardised Uptake Value Ratio, SUVR)
was higher in the MC group (1.23±0.08) than the NC group (1.08±0.04) (p=0.004).
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Table 5.1. Demographic characteristics of the mutation carrier and non-carrier

groups.
Mutation Carriers Non-Carriers p
Age: Years [mean (SD)] 46.0 ± 5.7 40.0 ± 10.0 0.229
Gender; Males: [N (%)] 5 (83%) 5 (83%) 1.000
Hypertension: [N (%)] 2 (33.3%) 2 (33.3%) 1.000
APOE ε4 Carrier: [N (%)] 2 (33.3%) 1 (16.6%) 0.512
MMSE: [mean ± SD] 29.0 ± 0.9 27.5 ± 2.1 0.135
SUVR: [mean ± SD] 1.23 ± 0.08 1.08 ± 0.04 0.004
Demographic differences assessed using a χ2 test for the categorical variables (gender, hypertension and
APOE ε4 status) and analysis of variance (ANOVA) for the continuous variables (age, MMSE and
SUVR). Significant values in bold font. Groups were not significantly different in age, gender,
hypertension, APOE ε4 carriers or MMSE. SUVR was significantly higher in the mutation carrier group.
SD = standard deviation. MMSE = Mini Mental State Examination (Folstein et al., 1975).
Mutation carrier and non-carrier groups were not significantly different in any retinal
vascular parameter. In terms of pupil flash response parameters, the 75% recovery time
(75%RT) was larger in mutation carriers (p=0.0003, after FDR adjustment) and the
percentage recovery 3.5 seconds after stimulus (PR3.5) was smaller in mutation carriers
(p=0.006, after FDR adjustment) (see Figure 5.2). This slower or ‘sluggish’ PFR trend
was also observed in mutation carriers for mean constriction velocity, max constriction
velocity, mean dilation velocity and max constriction acceleration, but did not reach
statistical significance after FDR adjustment. Confounders (age, gender and APOE ε4
status) were not significant in the ANCOVA models for 75%RT and PR3.5.
Confounders were not significant for other PFR parameters with the exception of
constriction amplitude which was smaller in APOE ε4 carriers (p<0.0001) and in males
(p<0.0001).
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Both the 75%RT and PR3.5 parameters provided perfect classification of mutation
carriers vs non-carriers (sensitivity=100%, specificity=100%) in this cohort. Receiver
operator characteristic (ROC) area under the curve (AUC) was thus 1.0 for both
parameters (75%RT: 95%CI=0.84-1.00, p<0.0001; PR3.5: 95%CI=0.84-1.00,
p<0.0001).
Figure 5.2. PFR differences in ADAD mutation carriers.

Difference in (A) 75% Recovery Time and (B) Percentage recovery 3.5 seconds after stimulus, between 6
mutation carriers and 6 non-carriers from the same family harboring an APP ADAD mutation. Outliers
were not related to age relative to expected onset, hypertension or other co-morbidities. P values from
ANCOVA analysis of differences between groups (including confounders).
The effect of estimated years to onset of symptoms (EYO = AAO-age) on 75%RT and
PR3.5 was then investigated to explore possible changes in these parameters as part of
the temporal sequence of biomarker changes during disease progress. The mean EYO
was 5.0±5.9 for MC and for 11.2±9.5 for NC. No evidence of temporal relationships in
these PFR parameters was found.

106
5.4) Discussion
The results reported in this Chapter indicate that carriers of the APPGlu693Gln
mutation exhibit slower recovery from pupil flash response, with longer 75% recovery
time and smaller percentage recovery 3.5 seconds after stimulus, than non-carriers in the
same family. The parameter ‘75% recovery time’ describes the time taken, after
constriction to minimum pupil size, to recover 75% of the constriction amplitude
through re-dilation. The parameter ‘percentage recovery after 3.5 seconds’ refers to the
percentage of the constriction amplitude recovered by re-dilation at time 3.5 seconds
after stimulus. Both parameters are measures of the recovery time for the pupil after
flash stimulus. Despite the known cerebral vascular effects of the APPGlu693Gln
mutation, no retinal vascular abnormalities were observed in the mutation carriers.
A ‘sluggish’ PFR has previously been reported in sporadic AD (Fotiou et al., 2007,
Fotiou et al., 2000, Granholm et al., 2003, Prettyman et al., 1997), with reduced resting
pupil size, amplitude of constriction and reduced velocity and acceleration of the pupil
size. Despite reduced constriction and dilation velocities, some of these studies reported
a quicker recovery after PFR stimulus in sporadic AD. This is likely due to the
overwhelmingly reduced amplitude of the response. The present study found non-
significant trends for reduced mean constriction velocity, maximum constriction
velocity, mean dilation velocity and maximum constriction acceleration in mutation
carriers. Statistically significant results were found for increased ‘75% recovery time’
and reduced ‘percentage recovery after 3.5 seconds’ in mutation carriers.
APPGlu693Gln mutation carriers appear to exhibit the sporadic-AD trends for reduced
pupil velocity, but not reduced amplitude, resulting in a significantly slower recovery
from stimulus.
107
The study of families with ADAD mutations provides three major benefits over studies
into sporadic AD. Firstly, mutation carriers progress to disease with a very high
certainty and at a well defined age-range. Hence, although all participants in the present
study were pre-symptomatic, genetic testing enabled identification of those destined to
progress to disease. Secondly, a typically younger age of onset (about 51 years for the
present analysis), usually results in fewer confounds due to age-related medical co-
morbidities. However, in the present study, 2 individuals in each group were diagnosed
with hypertension. Thirdly, utilizing within-family comparisons reduces the chance of
other genetic influences.
These PFR results add value to the panel of peripheral markers reported from previous
DIAN studies. Cerebrospinal fluid Aβ42 and tau concentrations, brain amyloid
deposition and glucose hypometabolism, and impaired episodic memory have all been
observed to be altered in MC between 10 to 25 years prior to expected symptom onset
in the DIAN cohort (Bateman et al., 2012). Publications are also in preparation that
report significantly elevated plasma Aβ42 levels, approximately 3 decades prior to
expected symptom onset in MC, and lower platelet APP isoform ratios (APPr) in MC
(unpublished results). Further longitudinal studies will be required to determine if these
findings and biomarkers are relevant to sporadic AD, however such information about
the time course of AD pathology development could prove useful in the design and
development of intervention trials in both ADAD and sporadic AD.
Most families in the DIAN study have genetic mutations that result in AD dementia. Aβ
deposition, as measured by positron-emission tomography with Pittsburgh compound B
(PiB-PET), has been found to be higher in mutation carriers in the DIAN cohort, up to
15 years before expected symptom onset (Bateman et al., 2012). However, the ADAD
mutation investigated in the present study is a mutation of the Amyloid Precursor

108
Protein (APP) at position 22 of the Aβ fragment (APPGlu693Gln), replacing glutamic
acid with glutamine. Mutations in this region of APP are associated with cerebral
amyloid angiopathy (CAA) and ultimately cerebral hemorrhage. Indeed both the
proband and his sister exhibited CAA and recurrent lobar hemorrhages in the brain, but
neuritic amyloid plaques were also found, indicating ADAD pathology may also result
from this mutation. PiB-PET imaging results from the participants in the present PFR
study demonstrated that the mutation carriers had higher SUVR (1.23±0.08) than non-
carriers (1.08±0.04) (p=0.004), although no participants were categorized as high-
SUVR (SUVR>1.5). Neither dementia nor cognitive decline has been observed in this
family, possibly due to earlier onset of CAA and fatal cerebral hemorrhages.
Despite the difference in underlying cause and age at onset, ADAD and sporadic AD
have similar neuropathologic hallmarks and clinical features (Bateman et al., 2011). The
present results indicate that PFR changes may occur in the early pathogenic but pre-
symptomatic stages of CAA or ADAD. The lack of correlation between the PFR
parameters and estimated years to onset (EYO) may indicate that PFR changes occur
early and subsequently stabilize over the EYO range of this cohort, but larger cohorts
will be required to investigate this hypothesis.
PFR changes in AD are considered to be due to either degeneration in pupil-control
relays in the midbrain or cholinergic deficits in the peripheral parasympathetic pathway.
Mechanisms by which CAA could impact on PFR could also include damage to pupil-
control relays in the midbrain. As changes to pupil response have now been reported in
both sporadic AD and APPGlu693Gln mutation carriers, PFR deserves further
investigation as a useful adjunct to assist the diagnosis of early AD. PFR may also be
useful in monitoring CAA or AD for management of risk factors and treatment.

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Chapter 6: Alzheimer’s Disease, Glaucoma and AMD
6.1) Introduction
Two separate retinal diseases have been linked with AD; glaucoma and age-related
macular degeneration (AMD), both leading causes of blindness and vision loss. A
number of similarities have been described between AD pathology in the brain and
retinal pathology in these diseases. All three diseases feature degeneration of central
nervous system (CNS) tissue, deposition of protein aggregates including Aβ plaques
and hyperphosphorylated tau, metabolic and oxidative stress and neuro-inflammation
(McKinnon, 2012, Ohno-Matsui, 2011, Tsolaki et al., 2011).
While most AD-related pathology occurs in the brain, the disease has also been reported
to affect different regions of the retina, including deposits in the macular region,
decreased retinal nerve fiber thickness, loss of retinal ganglion cells, optic disc cupping
and retinal microvascular abnormalities (Bayer et al., 2002, Berisha et al., 2007, Blanks
et al., 1989, Blanks et al., 1996, Blanks et al., 1996, Danesh-Meyer et al., 2006, Hinton
et al., 1986, Iseri et al., 2006, Paquet et al., 2007, Parisi et al., 2001, Sadun and Bassi,
1990, Sadun and Bassi, 1990, Tamura et al., 2006, Tsai, 1991). Some of these retinal
changes are also produced in glaucoma and AMD and may indicate more frequent
comorbidity of these retinal diseases in AD.
For each of these three diseases there is continued debate about which are the damage-
inducing mechanisms. While there are pathological similarities between the three
diseases, there are also important differences, in particular between the retinal
degenerative characteristics of glaucoma and AMD. Retinal changes in AD are being
investigated in the hope of elucidating novel biomarkers for AD. Investigation of the
similarities and differences between the pathogenic mechanisms of AD, AMD and
110
glaucoma may lead to greater understanding of each disease and possible translations of
therapeutic approaches. In this section, signs of glaucoma and AMD and pathology are
investigated in AD and HC participants, including analysis with respect to neocortical
amyloid plaque burden.
6.2) Alzheimer’s disease and age-related macular degeneration
6.2.1) Introduction
Age-related macular degeneration (AMD) describes a group of degenerative retinal eye
diseases that affect the macular area of the retina and hence impair central vision (see
Figure 6.1). AMD is a common age-related eye disease, affecting 15% of people aged
65-74 years, and 25% of those aged 75-84 years (Klein et al., 2004), and accounting for
5% of all blindness registrations (World Health Organization, 2010). It is conservatively
estimated that 50 million people suffer from AMD worldwide, with about one third of
these becoming blind or severely visually impaired due to AMD (Fine et al., 2000,
Gehrs et al., 2006).
Figure 6.1. How people with AMD might see the world.
Central vision is lost while peripheral vision remains intact.
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AMD involves the buildup of drusen; extracellular retinal waste products above the
Bruch’s membrane, between the choroid and retinal pigment epithelial (RPE) cell layer
(see Figure 6.2A) (Sparrow, 2010). The presence of a few small ("hard") drusen is
normal with advancing age, however the presence of larger “soft” and more numerous
drusen in the macula is an early sign of AMD (Buschini et al., 2011). The drusen can
lead to death of RPE cells and subsequent death of the retinal cells that the RPE cells
nourish. This is referred to as “dry” or “atrophic” AMD which causes gradual loss of
central vision. If the RPE layer is severely compromised, it can fail to stop choroidal
neovascularisation (CNV) into the retina, referred to as “wet” or “exudative” AMD (see
Figure 6.2B) (de Jong, 2006). The new blood vessels cause scarring in the retina and
often cause sudden and severe central vision loss.
Comparisons between drusen and AD plaques form the basis for a relationship between
AMD and AD. There are a number of studies suggesting that AMD might be related to
dementia, cognitive decline and AD (Klaver et al., 1999, Ohno-Matsui, 2011). AMD
and AD share several clinical and pathological features, including oxidative stress and
inflammation, which are well-known inducers of protein aggregation (Kaarniranta et al.,
2011). An important characteristic common to both AD and AMD is the presence of Aβ
and tau in the senile plaques of the AD brain and in the drusen of AMD patients
(Anderson et al., 2004, Dentchev et al., 2003, Hoh Kam et al., 2010, Johnson et al.,
2002, Klaver et al., 1999, Luibl et al., 2006, Ohno-Matsui, 2011, Yoshida et al., 2005).
Aβ is potentially a key regulator of the progression from drusen to AMD (Ohno-Matsui,
2011), suggesting that a common pathogenic mechanism might exist between the
diseases.
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Figure 6.2. Retinal changes in AMD.

A) Early stage dry AMD, drusen appear as yellow spots in the retinal image and as pockets of retinal
waste products above the Bruch’s membrane, between the choroid and retinal pigment epithelial cell
layer. B) Wet AMD, choroidal neovacscularisation into retina. From the Macular Degeneration
Foundation (http://www.mdfoundation.com.au/resources/1/factsheets/AMD_Booklet.pdf).
Whatever the initial insult in AD and AMD, a neuro-inflammatory response is observed
in both diseases and may contribute to disease progress. Aβ deposition is inflammatory
(Butterfield et al., 2002, Sutton et al., 1999) and cerebral Aβ deposition in AD is
associated with an inflammatory response, including increased levels of pro-
inflammatory cytokines (e.g. tumor necrosis factor – TNF, and interleukin-1 - IL-1)
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(Akiyama et al., 2000). The IL-1 gene is associated with AD development (Nicoll et al.,
2000) and IL-1 production is known to stimulate neural expression and processing of
APP and hence Aβ (Forloni et al., 1992), possibly providing a feedback loop through
which AD progresses. It is noteworthy that IL-1 also mediates the phosphorylation of
tau protein (Sheng et al., 2001), and the production and activity of acetylcholinesterase
(Li et al., 2000), both of which are altered in AD.
Similarly, with ageing and particularly in AMD, Aβ and other deposits in the Bruch’s
membrane make the interface between the retina and the choroidal blood supply less
permeable and are accompanied by chronic inflammation (Chen et al., 2010, Hoh Kam
et al., 2010). As the Aβ induced inflammatory reaction is mediated by the cytokines
tumor necrosis factor and interleukin-1 (Sutton et al., 1999), a similar feedback loop
could be involved in AMD and hence anti-inflammatory approaches are worthy of
further research in both AD and AMD.
Another aspect to the inflammatory component of AMD is the complement system,
implicated in the formation of drusen, with a key complement gene (complement factor
H gene) providing a genetic marker for AMD (Hageman et al., 2005). Findings relating
to the inflammatory aspects of both diseases provide valuable insight into the disease
process and may lead to advances in treatment.
The apolipoprotein E (APOE) gene, the main genetic risk factor for AD, was also one of
the first genes to be associated with age-related macular degeneration (AMD) (Klaver et
al., 1998, Souied et al., 1998). Carriers of the APOE ε4 allele are at increased risk for
AD but reduced risk for AMD (Baird et al., 2004, Schmidt et al., 2002, Zareparsi et al.,
2004). In contrast, the APOE ε2 allele is protective against AD but might confer an
increased risk of AMD, mainly in men (Schmidt et al., 2002). It should be noted that a
114
large follow-up study found no evidence of an association between APOE genotype and
early signs of AMD (Wong et al., 2006).
A genetic connection between APOE, AD and AMD may be a result of the role of
APOE in Aβ proteolysis and regulation in brain and retina, although the opposing allelic
effects remain unexplained. APOE also has a role in the recycling of cholesterol and
lipids for cell membrane biosynthesis in the retina and it is hypothesized that the
different APOE isoforms influence transport mechanisms through the Bruch’s
membrane and hence may affect macular integrity (Souied et al., 1998). No other AD
related genes have become candidates for AMD pathology, hence it seems that these
diseases have a different genetic background.
Despite the opposed APOE genetic risk factors in AD and AMD, some studies have
indicated increased incidence or prevalence of AMD in AD. Results from the Rotterdam
Study suggested that AMD predicted the 2-years risk of AD in participants >75 years of
age (Klaver et al., 1999). However, another study, the Cardiovascular Health Study with
a large cohort of 2088 participants, found no significant association between AD and
AMD (Baker et al., 2009). A cross-sectional analysis from the Blue Mountains Eye
Study found that persons with late AMD were more likely to have cognitive
impairment, based on the Mini Mental State Examination (MMSE) scores, after
excluding vision related tasks from the examination (Pham et al., 2006). There is a
substantial overlap in the clinical risk factors for AD and AMD. Age, smoking, previous
cataract surgery and family history of AMD all have strong and consistent associations
with AMD, while hypertension has a moderate association (Chakravarthy et al., 2010).
The aim of this study was to investigate early signs of AMD in AD and HC participants,
including analysis with respect to neocortical amyloid plaque burden.

115
6.2.2) Methods
Retinal photographs were collected from AD and HC participants as described in
section 2.5. A trained grader from the Center for Eye Research Australia evaluated
macular-centered images as being gradable or un-gradable for signs of AMD. Only
images deemed gradable were considered.
AD and HC participants were stratified by an experienced grader using semi-automated
software to analyse their retinal photographs. Presence of hard drusen (<63 micron in
diameter), soft drusen (either 63-125 micron or >125 micron in diameter), geographic
atrophy (GA, an area of 175 µm of hypopigmentation with a choroidal vessel in its
base) or choroidal neovascularisation (CNV) in either eye was identified. Participants
were identified as having signs of AMD if they had any of the following in at least one
eye:
 soft drusen > 125 micron in diameter, with or without pigmentary abnormalities
 soft drusen 63-125 micron in diameter, with pigmentary abnormalities
 multiple soft drusen 63-125 micron in diameter
Participants were graded as having no signs of AMD only if the images from both eyes
were gradable and the above signs were not present in either eye. Graders were masked
6.2.3) Results
The cohort consisted of 22 probable-AD patients (age 70.2 ± 9.0 yrs, 13 male, 9 female)
and 101 healthy control participants (age 71.3 ± 6.0 yrs, 40 male, 61 female). The
demographics of this cohort are presented in Table 6.1. The HC and AD groups did not
differ significantly in age, gender, hypertension, diabetes, smoking status or previous

116
cataract surgery. There was a higher percentage of APOE ε4 carriers in the AD group
(p=0.002).
The AD group had a greater percentage of participants with soft drusen (p=0.003, odds
ratio 4.7, 95% CI 1.7-13.1) (see Table 6.1). Only 4 HC participants and no AD
participants had signs of late AMD, all 4 also had early signs of AMD. ANCOVA
analysis revealed that early-AMD did not associate with age, gender, hypertension,
diabetes, smoking, previous cataract surgery or APOE ε4 status, either in the whole
cohort or within each group. An ANCOVA model for soft drusen, adjusting for all
confounders, found a significant association between soft drusen and both age
(p=0.006) and AD (p=0.0007). Hard drusen associated only with age (p=0.043).
Table 6.1. Demographics and AMD analysis for HC and AD groups.

Healthy Control Alzheimer’s Disease P Value
Age: Years [mean (SD)] 71.3 (6.0) 70.2 (9.0) 0.476‡
Gender; Males: [N (%)] 40 (40) 13 (59) 0.099†
Hypertension: [N (%)] 36 (36) 10 (46) 0.391†
Diabetes: [N(%)] 5 (5) 1 (5) 0.936†
Previous Cataract Surgery: [N (%)] 17 (17) 1 (5) 0.171†
APOE ε4 Carrier: [N (%)] 34 (34) 16 (73) 0.002†
Early AMD: [N (%)] 40 (40) 12 (55) 0.202†
Hard Drusen: [N (%)] 38 (38) 11 (50) 0.285†
Soft Drusen: [N (%)] 13 (13) 9 (41) 0.003†
‡
significant)
† 2
χ test for categorical variables (gender, hypertension, diabetes, smoking status, APOE ε4 status and
AMD) (p < 0.05 considered significant)
APOE ε4 Carrier refers to carrier/non-carrier of an apolipoprotein E ε4 allele. SD: standard deviation.
Significant results in bold type.
Hard drusen<63 micron in diameter. Soft drusen >63 micron in diameter.
AIBL neuroimaging data was available for 36 HC participants.
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A subset of healthy control participants were neuroimaged for the presence of fibrillar
brain amyloid using positron emission tomography (PET) with Pittsburgh Compound B
(PiB) (Klunk et al., 2004, Klunk et al., 2005). Subjects were classified as PiB negative
(HC-) if their neocortex SUVR was below 1.5, and PiB positive (HC+) if their
neocortex SUVR was above 1.5. The neuroimaging cohort consisted of 22 HC-
participants (age 69.8 ± 5.5 yrs, 10 male, 12 female) and 14 HC+ participants (age 73.4
± 7.5 yrs, 8 male, 6 female). The demographics of this cohort are presented in Table
6.2. HC- and HC+ groups did not differ significantly in age, gender, hypertension,
diabetes, smoking status, previous cataract surgery, APOE ε4 carriers, early-AMD, hard
drusen or soft drusen.
Within the neuroimaging group, ANCOVA analysis revealed that cataract surgery
associated with early AMD (p=0.018) [hard drusen (p=0.018) and soft drusen
(p=0.006)]. Early-AMD, hard drusen or soft drusen did not associate with age, gender,
hypertension, diabetes, smoking or APOE ε4 status, either in the whole cohort or within
each neuroimaging sub-group.

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Table 6.2. Demographics and AMD analysis for HC+ and HC- groups.
HC- HC+ P Value
Age: Years [mean (SD)] 69.8 (5.5) 73.4 (7.5) 0.106‡
Gender; Males: [N (%)] 10 (46) 8 (57) 0.495†
Hypertension: [N (%)] 9 (41) 6 (43) 0.908†
Diabetes: [N(%)] 1 (5) 0 (0) 1.000†
APOE ε4 Carrier: [N (%)] 11 (50) 11 (79) 0.095†
Early AMD: [N (%)] 8 (36) 4 (29) 0.736†
Hard Drusen: [N (%)] 8 (36) 4 (29) 0.736†
Soft Drusen: [N (%)] 4 (18) 2 (14) 0.832†
‡
significant)
† 2
APOE ε4 Carrier refers to carrier/non-carrier of an Apolipoprotein E ε4 allele. HC-: Healthy controls with
low plaque burden; HC+: healthy controls with high plaque burden. SD: standard deviation. No
demographic was significantly different between groups. Significant results in bold type.
Hard drusen<63 micron in diameter. Soft drusen >63 micron in diameter.
6.2.4) Discussion
AMD and AD are both complex, multifactorial diseases associated with genetic and
environmental factors (Buschini et al., 2011, Chakravarthy et al., 2010, Leveziel et al.,
2011). Aβ deposition and inflammation have been implicated in the pathogenesis of
both diseases and the APOE ε4 allele is a major risk factor for AD but might be
protective against AMD. Despite this, elevated concomitant occurrence of AMD with
AD has been reported. Results from the present study find that AD participants were
significantly more likely to have soft drusen, adding to the evidence that AMD is more
prevalent in AD. While no such association between AD and hard drusen was found,
119
the presence of hard drusen is considered normal with advancing age. However, the
presence of soft drusen in the macula is an early sign of AMD (Buschini et al., 2011).
No association was found between AMD and age or APOE ε4 allele. Other larger
studies have reported such associations and it is possible that the size of this study
affected this result. In addition, no association was found between AMD and high
plaque burden in pre-symptomatic AD, and hence no support was found for AMD as a
predictive factor or risk factor for AD. However, as soft drusen are considered an early
sign of AMD, it is possible that AD increases the risk of developing AMD. Further
longitudinal studies are needed to investigate this possibility. Interesting support for a
link between AMD and dementia comes from studies into cognition and AMD, with a
recent study by Woo et al. finding that persons with AMD are at greater risk for
cognitive decline and MCI (Woo et al., 2012).
In a case-control setting, the concomitant occurrence of AMD with AD may reduce the
sensitivity and specificity of drusen as a biomarker for AD. However, as AMD is not
associated with inner retinal vascular changes or pupil response changes, the ocular
biomarkers discussed in Chapters 2-4 are not likely to be influenced by the relationship
between AD and AMD. In order to assess whether AMD-related retinal changes can
predict AD, future longitudinal studies are needed in which patients free from ocular
disease are included at baseline, and subsequently followed for the occurrence of AD.
It should be noted that Aβ assemblies in drusen may not be equivalent to oligomeric
amyloid assemblies in the aging brain (Anderson et al., 2004). The molecular
composition of Aβ deposits in drusen appear to be more heterogeneous than cerebral
oligomeric Aβ assemblies. It is possible that Aβ deposits in the brain in AD and in the
retina in AMD may be independent pathological processes that are simply correlated
120
through age. However, there is a possibility that both are a consequence of an
underlying deregulated Aβ proteostasis. A decreased capacity to remove damaged
cellular proteins has been strongly implicated in both AD and AMD. Hence therapeutic
approaches that target Aβ production or clearance may be of clinical benefit in both
diseases.
There are also differences in the location of retinal Aβ deposition reported in AMD and
AD. Human and animal model evidence of Aβ deposition in the AD retina suggests that
deposition is within the ganglion cell layer, not confined to the macular region, and
often near retinal vessels (Koronyo-Hamaoui et al., 2010). However, in AMD, Aβ
deposits are locally restricted to the macular area RPE layer, and appear smaller in size
(Anderson et al., 2004). These findings suggest that AMD should be considered a
distinct type of amyloid disease, rather than an ocular form of AD. Based on their
unique distribution, composition and size, retinal Aβ deposits in AD and AMD patients
could potentially be used for differential diagnosis.
Aβ deposition may lead to similar neuro-inflammatory responses that could exacerbate
each condition (Akiyama et al., 2000, Chen et al., 2010, Hoh Kam et al., 2010). So far,
it is still controversial whether non-steroidal anti-inflammatory drugs (NSAIDs) are
beneficial in AD and AMD. Epidemiological studies have found that long-term use of
NSAIDs reduces the risk of AD (Anthony et al., 2000, Breitner et al., 1995, Breitner
and Zandi, 2001, in t' Veld et al., 2001, McGeer and McGeer, 2007, Szekely et al.,
2004), although a clinical trial administrating NSAIDs in AD was disappointing
(Jaturapatporn et al., 2012), indicating that NSAIDs may need to be administered earlier
in the disease process. Studies have also found that NSAIDs reduce the risk of early
AMD progressing to the exudative form (Christen, 2013, Christen et al., 2009, Wilson
et al., 2004).
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The discovery of novel genetic (complement factor H gene) and neuro-inflammatory
markers for AMD have provided crucial insights into the aetiology of this disease
(Hageman et al., 2005). It is possible that important findings such as this in AMD could
also provide novel directions for future research into the pathophysiology of AD. In
addition, there is also hope that AD and AMD could be targeted simultaneously for
treatment and monitoring. However, a caveat for this approach has recently come from
a finding that revealed that inhibition of β-Secretase (BACE1), an important protease
for Aβ production, can cause retinal pathology (Cai et al., 2012). The findings that
BACE1 plays a critical role in retinal homeostasis suggests that the use of BACE
inhibitors for AD should be viewed with caution as they have the potential to lead to
retinal pathology and exacerbate conditions such as AMD. BACE inhibitors need to be
tested for retinal side effects and individuals receiving these drugs should undergo
monitoring of retinal health.
AD and AMD are both neurodegenerative diseases and they share environmental risk
factors and pathological features. The different genetic origin of the diseases challenges
us to further investigate the gene-protein-expression pathways that lead to these
diseases. A greater understanding of the shared features of AD and AMD could lead to
accelerated development of therapies for both diseases. Aβ-targeting therapies or anti-
inflammatory therapies may lead to better outcomes for both blindness and cognitive
decline in an ageing society.

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6.3) Alzheimer’s Disease, Glaucoma and Optic Atrophy
6.3.1) Introduction
Glaucoma represents a group of optic neuropathies that have in common a degeneration
of retinal ganglion cells (RGC) and their axons, resulting in optic atrophy, optic disc
cupping (hollowing-out) and corresponding peripheral visual field defects (Kwon et al.,
2009). Transynaptic degeneration has also been reported in the visual cortex and lateral
geniculate nucleus in glaucoma patients (Gupta et al., 2009, Yucel and Gupta, 2008).
Glaucoma is second only to cataract as a leading cause of blindness worldwide
(Resnikoff et al., 2004), accounting for 8% of blindness registrations (World Health
Organization, 2010).
Open-angle glaucoma (OAG) is the most common type of glaucoma. The largest risk-
factor for glaucoma is age, and the most widely utilised clinical measure is ocular
hypertension (elevated intra-ocular pressure - IOP). Current treatments are directed at
reducing IOP; without treatment glaucoma can cause visual disability and eventually
blindness (Kwon et al., 2009). Chronic ocular hypertension is believed to lead to the
RGC loss in glaucoma, however evidence indicates that RGC loss still occurs in many
glaucoma patients after successful IOP normalization (Flammer and Mozaffarieh, 2007,
Leske et al., 2003), and many OAG patients have normal IOP (Shields, 2008),
indicating that other mechanisms are involved.
Glaucomatous changes can take the form of optic disc cupping, abnormalities of the
neuro-retinal rim (focal thinning or notching, peripapillary atrophy) and in advanced
stages, optic disc pallor (see Figures 6.3 and 6.4). Optic disc cupping refers to the
hollowing out of the disc, as seen in Figure 6.3. Optic disc pallor refers to whitening of
the disc from its normal light-orange colour, due to the loss of axons and the small
123
capillaries surrounding them (see Figure 6.4A). Peripapillary drusen and optic disc
pallor are signs of optic atrophy that are non-specific to glaucoma.
Approximately half of people with Glaucoma don’t know they have the disease, partly
because it is painless and the brain compensates for visual field loss. Additionally,
Glaucoma affects the magnocellular visual processing system which performs visual
functions that are not easily assessed during conventional eye examinations, such as
motion processing and contrast sensitivity (Sadun and Bassi, 1990, Yucel et al., 2000).
This study therefore looks at signs of optic atrophy in the retinal photos of participants,
rather than patient-reported diagnosis of Glaucoma.
A B
______________________________________________________________________
Figure 6.3. Illustration of changes to the optic disc in Glaucoma.
(A) Healthy optic disc. (B) Optic disc cupping observed in Glaucoma and AD.
A B
______________________________________________________________________
Figure 6.4. Illustration of changes to the optic disc indicative of atrophy.
(A) Optic disc with pallor. (B) Optic disc with an abnormal neuro-retinal rim.
124
The two major signs of glaucoma; optic atrophy and peripheral visual field loss, have
also been reported in AD. Optic atrophy, in the form of optic disc pallor, pathologic disc
cupping and thinning of the neuro-retinal rim has been shown to be more common in
AD patients (Danesh-Meyer et al., 2006, Tsai, 1991). In fact, a 5-fold higher chance of
visual field defects and/or optic disc cupping found in AD has been interpreted as a
higher occurrence rate of glaucoma in AD (Bayer et al., 2002). In this study, no AD
participants had a family history of glaucoma, and ocular hypertension was not found in
AD participants but was found in 7.5% of controls, reducing the likelihood that OAG
was the cause. However, other studies have found evidence that AD is associated with
increased prevalence of OAG (Tamura et al., 2006) or glaucoma generally (Chandra et
al., 1986).
Another study found a greater than 10% per year decay in visual field and optic disc
cupping in glaucoma patients who were later diagnosed with AD, compared to an
average 3% per year decay in glaucoma patients who did not develop AD, indicating
that AD accelerates the progression of glaucoma symptoms (Bayer and Ferrari, 2002).
Furthermore, in this study cup-to-disc ratios in patients with AD were increased by 43%
compared to controls, and the proportion of OAG in patients with AD (24%) was much
higher than the control group (10%). However, another study looked retrospectively
over 3 years at a cohort of 62,235 participants in the USA and did not find any
association of incident AD in the baseline OAG patients (Ou et al., 2012).
Preliminary evidence is emerging that AD-pathology, in the form of Aβ plaques, may
occur in the human AD retina (Koronyo-Hamaoui et al., 2010). Several studies have
also explored the possibility that retinal degeneration in glaucoma may be associated
with local AD-like pathology. Evidence for fibrillar tau has been found in the retinas of
glaucoma patients with ocular hypertension (Gupta et al., 2008). It has also been found
125
in aged and glaucoma participants in the optic nerve, peripapillary glia, retina and
vitreous (Leger et al., 2011, Loeffler et al., 1993, Yoneda et al., 2005). Increased Aβ
levels were found in rat models of acute ocular hypertension (Guo et al., 2007,
McKinnon et al., 2002).
Signs of deregulated protein homeostasis in the glaucomatous eye have also come from
studies of the vitreous fluid body. The vitreous has been reported to have reduced Aβ42
and increased tau protein levels in glaucoma patients (Yoneda et al., 2005), and reduced
Aβ42 in AD patients (Haass and Selkoe, 2007, Tiraboschi et al., 2004), consistent with
reduced Aβ42 and increased tau in the CSF of AD patients (Blennow et al., 2010, Fagan
et al., 2006, Sunderland et al., 2003, Thal et al., 2006). Interestingly, some AD patients
exhibit irregularities in intracranial pressure (Wostyn et al., 2010), prompting the
suggestion that some forms of AD could be a cerebral form of glaucoma.
Evidence for a genetic link between AD and glaucoma has come from the implication of
APOE polymorphisms in the pathogenesis of OAG, however conflicting data exists as
to whether they influence the risk of OAG (Copin et al., 2002, Vickers et al., 2002,
Zetterberg et al., 2007). In addition, generalized retinal vessel thinning in AD reported
for the first time in this thesis, has also been reported in glaucoma (Chang et al., 2011,
Jonas and Naumann, 1989, Lee et al., 1998, Mitchell et al., 2005), along with focal
(localized) narrowing of vessels near the optic disc (Papastathopoulos and Jonas, 1995).
Based on the above findings, a relationship between retinal vessel width, OAG and AD
seems to exist. Deregulated Aβ proteostasis could be a common underlying cause of
both diseases. The aim of this study was to use retinal photography to investigate the
optic disc in AD and control participants, and also with respect to neocortical amyloid
plaque burden, the earliest pre-clinical sign of AD.

126
6.3.2) Methods
section 2.5. Retinal photographs were examined by an experienced ophthalmologist
who determined the cup-disc-ratio (CDR) of each eye and identified if glaucomatous
neuro-retinal rim (NRR) abnormalities (focal thinning or notching, peripapillary
atrophy), peripapillary drusen or optic disc pallor were present in either eye. The mean
and largest CDR value for each pair of eyes was analysed. The ophthalmologist was
masked from the disease status of the participants.
6.3.3) Results
Two HC participants and no AD participants had previously been diagnosed with
glaucoma. The demographics of this cohort are presented in Table 6.3. HC and AD
groups did not differ significantly in age, gender, hypertension, diabetes, smoking status
or previous cataract surgery. There was a higher percentage of APOE ε4 carriers in the
AD group (p=0.019).
Neuroretinal rim abnormalities were more prevalent in the AD group (p=0.026, odds
ratio 4.4, 95% CI 1.2-16.5). An ANCOVA model for NRR abnormalities, adjusting for
age, gender, hypertension, diabetes, smoking, previous cataract surgery and APOE ε4
status, revealed a strong association with AD (p=0.006). NRR abnormalities were
weakly associated (p=0.038) with cataracts (cataracts or cataract surgery within the
prior 6 months), but did not associate with any other confounders, including APOE ε4
status. There were also non-significant trends for higher mean and maximum CDR in
the AD group.
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Table 6.3. Demographics and optic disc analysis for HC and AD groups.
Age: Years [mean (SD)] 71.6 (5.6) 72.4 (7.5) 0.557‡
Gender; Males: [N (%)] 55 (45) 12 (48) 0.764†
Hypertension: [N (%)] 44 (36) 11 (44) 0.439†
Diabetes: [N(%)] 6 (5) 2 (8) 0.533†
APOE ε4 Carrier: [N (%)] 38 (31) 14 (56) 0.019†
Mean Cup-Disc-Ratio: [mean (SD)] 0.26 (0.13) 0.29 (0.17) 0.310‡
Maximum Cup-Disc-Ratio: [Mean (SD)] 0.28 (0.13) 0.32 (0.18) 0.235‡
Optic Disc Pallor: [N (%)] 0 (0) 0 (0) 1.000†
NRR Abnormalities: [N (%)] 13 (11) 7 (28) 0.026†
‡
Analysis of variance (ANOVA) for the continuous variables (p < 0.05 considered significant)
†
χ2 test for categorical variables (gender, hypertension, diabetes, smoking status, APOE ε4 status and
APOE ε4 Carrier refers to carrier/non-carrier of an Apolipoprotein E ε4 allele. SD: standard deviation.
AIBL neuroimaging data was available for 45 HC participants. This neuroimaging
cohort was grouped according to high (SUVR > 1.5) or low (SUVR < 1.5) neocortical
amyloid plaque burden (HC+ and HC- respectively). The demographics of the
neuroimaging cohort are presented in Table 6.4. There were 15 participants in the HC+
group and 30 participants in the HC- group. The HC+ group had a higher percentage of
APOE ε4 carriers than the HC- group (p=0.04), there were no significant differences in
the other demographic variables.
There were no significant differences between groups on CDR, pallor or NRR
abnormalities. ANCOVA models for mean and maximum CDR, pallor and NRR
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abnormalities, adjusting for age, gender, hypertension, diabetes, smoking, previous
cataract surgery and APOE ε4 status, did not reveal any significant relationships.
Table 6.4. Demographics and optic disc analysis for HC+ and HC- groups.
HC- HC+ P Value
Age: Years [mean (SD)] 70.4 (5.3) 73.7 (6.3) 0.08‡
Gender; Males: [N (%)] 15 (50) 9 (60) 0.53†
Hypertension: [N (%)] 11 (37) 6 (40) 0.52†
Diabetes: [N (%)] 1 (3) 2 (13) 0.99†
Smokers: [N (%)] 2 (7) 0 (0) 0.99†
APOE ε4 Carrier: [N (%)] 14 (47) 12 (75) 0.04†
Mean Cup-Disc-Ratio: [mean (SD)] 0.25 (0.13) 0.26 (0.13) 0.72‡
Maximum Cup-Disc-Ratio: [Mean (SD)] 0.27 (0.14) 0.28 (0.14) 0.82‡
Optic Disc Pallor: [N (%)] 0 (0) 0 (0) 1.00†
NRR Abnormalities: [N (%)] 3 (10) 1 (7) 0.71†
‡
†
χ2 test for categorical variables (p < 0.05 considered significant)
APOE ε4 Carrier refers to carrier/non-carrier of an Apolipoprotein E ε4 allele.
HC-: Healthy controls with low plaque burden; HC+: healthy controls with high plaque burden. SD:
standard deviation. Significant results in bold type.
6.3.4) Discussion
This study investigated whether there was a relationship between AD and optic atrophy,
a major symptom of glaucoma, in the retinal study cohort. The main finding was an
increased prevalence of NRR abnormalities in AD. While CDR (mean and maximum of
both eyes) was higher in AD, this result was not statistically significant. Optic disc
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pallor was not reported in any of the participants. These results add to the evidence that
optic atrophy occurs in AD.
While there have been conflicting reports in the literature about the influence of APOE
polymorphisms on the risk or pathogenesis of glaucoma (Copin et al., 2002, Vickers et
al., 2002, Zetterberg et al., 2007), in the present study no associations were found
between optic atrophy parameters and APOE genotype. Hence the present study finds
no evidence for a genetic link between AD and glaucoma or optic atrophy.
The cause of glaucoma is not fully understood, although it has long been assumed that
elevated IOP damages RGC’s, causing ocular atrophy and impairing peripheral vision.
However, glaucoma often progresses even after the pressure has been controlled with
drugs. Evidence implicating Aβ in the cell-death process in glaucoma (Guo et al.,
2007), and the present and previously reported findings of optic atrophy in AD, suggest
that Aβ-induced retinal neurodegeneration might be common to these diseases. There is
evidence that the mechanism of RGC loss (apoptosis) is similar in both diseases
(McKinnon et al., 2002, Yin et al., 2008). Recent animal model studies have found that
RGCs produce more Aβ in glaucoma, Aβ co-localizes with RGC apoptosis and induces
RGC apoptosis in vivo, and that drugs directed against Aβ accumulation (Aβ
antibodies), including one currently used to treat AD, might be effective against
glaucoma (Guo et al., 2007).
The possibility that Aβ plaque formation may be a shared pathway that causes AD and
glaucoma was explored using the neuroimaging cohort of the present study. No
significant differences in optic atrophy parameters were found between healthy controls
with high brain plaque burden (HC+) and those with low brain plaque burden (HC-),
suggesting that optic atrophy may occur later in the AD pathogenic process (at the
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symptomatic stage rather than the pre-clinical, cerebral plaque deposition stage). It is,
however, possible that retinal plaques may occur later than cerebral plaques in the AD
disease process, and then go on to produce optic atrophy. While there is preliminary
evidence for the existence of Aβ plaques in the AD retina (Koronyo-Hamaoui et al.,
2010), this has not been confirmed by other studies. Additionally, while the Koronyo-
Hamaoui et al. (2010) study found that in an animal AD model, retinal plaques
preceded cerebral plaques, it has not been established at what stage of the human
disease process retinal plaques may occur.
It is possible that Aβ might be an indirect cause of RGC loss in glaucoma, however
larger studies are required to investigate the link between the mechanisms behind
glaucoma and Alzheimer's disease. Optic atrophy in AD may instead be a consequence
of cerebral neuro-degeneration, through impairment of the visual system or through
reduced CSF pressures altering pressure gradients at the back of the eye (Morgan et al.,
2008, Wostyn et al., 2008).
Generalized retinal vessel thinning in AD, reported for the first time in this thesis, has
also been reported in Glaucoma (Chang et al., 2011, Jonas and Naumann, 1989, Lee et
al., 1998, Mitchell et al., 2005), along with focal (localized) narrowing of vessels near
the optic disc (Papastathopoulos and Jonas, 1995). Several studies have also shown
decreased blood flow in the optic nerve head, retina and choroid in glaucoma patients,
believed to be the result of elevated IOP and the microvasculature being unable to
autoregulate (Findl et al., 2000, Grunwald et al., 1998, Michelson et al., 1996,
Portmann et al., 2011, Wang et al., 2011). A decrease in the number of capillaries in the
optic nerve head as well as atrophy of the peripapillary capillaries supplying the RNFL
has also been reported (Gottanka et al., 2005, Kornzweig et al., 1968).
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Based on the above findings, a relationship between retinal vessel width, OAG and AD
may exist. Retinal vessel signs may reflect vascular dysregulation in the retinal and
cerebral microvasculatures, leading to low perfusion pressure in patients with glaucoma
and AD (de la Torre, 2009, Flammer et al., 2002). The Rotterdam study, a prospective
population-based study of 3469 persons, found that baseline retinal vessel diameters did
not predict incident OAG or optic disc changes, suggesting that retinal vascular changes
in OAG might be a consequence of RGC loss instead of a cause. Further prospective
studies should be conducted to elucidate the role of retinal vessel signs in the
pathophysiology of glaucoma and AD.
The results reported in this Chapter suggest that optic atrophy occurs later in the AD
neurodegenerative process, hence it may not be useful as a screening biomarker for
early AD. However it is possible that investigations into the retinal changes in AD and
glaucoma might yield interesting results about the pathogenesis of these diseases, as
well as their treatment and monitoring. The similarities between the ocular effects of
AD and glaucoma may have therapeutic consequences for both diseases. Currently,
there is a lack of therapies that target the causative cellular processes in glaucoma. AD
drugs directed against Aβ accumulation (Aβ antibodies), show signs that they may be
effective against glaucoma, by reducing RGC apoptosis (Guo et al., 2007). Targeting
Aβ in the retina could provide a more direct therapeutic approach for glaucoma, with
fewer side effects associated with systemic administration of drugs. It may even be
possible to target retinal regions that demonstrate the most degeneration.
Neuroprotection in AD and glaucoma may be achieved through disrupting the role of
Aβ in the signaling pathways that lead to RGC apoptosis.
Another class of AD drugs, acetylcholinesterase (AChE) inhibitors, may also be of
therapeutic benefit in glaucoma. In addition to its neuroprotective effects, one of these

132
drugs (Donepezil) has been shown to reduce IOP in ocular normotensive rabbit eyes,
and could hence potentially be used to treat glaucoma (Estermann et al., 2006). Chronic
ocular hypertension is the most widely utilised clinical measure for glaucoma and has
been shown to increase Aβ production in the rat retina (McKinnon et al., 2002).
Increasing attention is being paid to neuroprotection as a treatment approach for
glaucoma (Whitcup, 2008), and hence advances in neuroprotection research in AD
(Behl and Moosmann, 2002, Streit, 2005, Streit and Xue, 2012) and other
neurodegenerative diseases have potential to flow on to more effective treatments for
glaucoma.
There is controversial evidence that some current treatments for glaucoma, such as the
α2-adrenergic agonist brimonidine, may exhibit neuroprotective effects acting
independently of their IOP reduction effect (Pfeiffer et al., 2013, Saylor et al., 2009).
There is also promising research into neurotrophic molecules for neuroprotection in the
treatment of both AD and Glaucoma (Unsicker, 2013) and therapies targeting the
autocrine and paracrine signaling pathways demonstrating success in animal models of
glaucoma (Weber, 2013). A reduction in the rate of cognitive decline in AD has been
reported after cerebral implantation of neurotrophic growth factor (NGF) (Tuszynski et
al., 2005), and NGF administered to the cornea of animal glaucoma models was shown
to protect RGC’s (Lambiase et al., 2011). Similarly, brain derived neurotrophic factor
(BDNF) has reported therapeutic improvements in animal models of AD (Nagahara et
al., 2009, Nagahara and Tuszynski, 2011) and RGC survival in animal models of
glaucoma (Fu et al., 2009, Ren et al., 2012). There is room for more research on ciliary
neurotrophic factor (CNTF), the transforming growth factor-β family (TGF-β),
fibroblast growth factor (FGF) which show signs of promise in both AD and glaucoma
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(Cui et al., 1999, Dobolyi et al., 2012, Fuchshofer and Tamm, 2012, Garcia et al., 2010,
Joly et al., 2007), as well as combinatorial approaches yet to be explored.
AD, glaucoma and AMD are complex, multifactorial diseases which partially share
genetic and environmental factors (Buschini et al., 2011, Chakravarthy et al., 2010,
Gemenetzi et al., 2012, Kwon et al., 2009, Leveziel et al., 2011), and have some
similarities in pathophysiology. More research is required to establish whether this
substantial overlap is due to the direct contribution of Aβ and tau deposition, or the
secondary involvement of these proteins in other, disease and/or tissue specific insults.
Nevertheless, advances in research into each disease, or pooling of research efforts, may
provide novel directions for therapies and tests, and hence may have broad impact in
both ophthalmology and neurology. However, a challenge to the development of retinal
biomarkers for early detection of AD is the differentiation of AD pathology from AMD
and glaucoma pathology.

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Chapter 7: Alzheimer’s Disease and Retinopathy
7.1) Introduction
Retinopathy refers to persistent or acute damage to the retina (see Figure 7.1). It is often
an ocular manifestation of systemic disease such as diabetes or hypertension. While
different disease processes may produce retinopathy, the clinical signs often overlap and
can include generalized and focal arteriolar narrowing, arterio-venous nicking,
increased retinal arteriolar light reflex (copper or silver wiring), flame and blot-shaped
retinal hemorrhages, soft and hard exudates, cotton wool spots, macular edema and, in
severe cases, optic disc swelling (Keith et al., 1939, Wong and Mitchell, 2004, Wong
and Mitchell, 2007). Vascular remodeling occurs over periods of time where the patient
may not be aware of the presence or extent of their disease, until it is too late.
Flame hemorrhage
Cotton wool spots

Silver wiring
Optic disc Hard exudates
Macula
______________________________________________________________________
Figure 7.1. Retinal photograph with clinical signs of retinopathy.
The hallmarks of diabetic retinopathy, a microvascular complication of diabetes, include
multiple occlusions due to increased leukocyte entrapment (Ogura, 2000), increased

135
permeability of the vessel wall with focal leakages (Ben-nun et al., 2004) and, in the
proliferative form, growth of newly-formed vessels (neovascularization). Initially DR is
associated with retinal hypoperfusion, followed by hyperperfusion as diabetic
retinopathy progresses to the neovascularistion stage (Curtis et al., 2009, Pemp and
Schmetterer, 2008). Hypertensive retinopathy typically involves arteriolar constriction
(resulting in copper/silver wiring and vascular tortuosity), flame and blot-shaped retinal
hemorrhages, cotton wool spots and, in severe cases, optic disc swelling (Keith et al.,
1939, Svardsudd et al., 1978, Wong and Mitchell, 2004, Wong and Mitchell, 2007).
The central light reflex (CR) is the reflection from the interface between the blood
column and the vessel wall (see Figure 7.2). Widening of the retinal arteriolar CR is a
sign of hypertensive retinopathy (Keith et al., 1939, Svardsudd et al., 1978). CR can be
influenced by atherosclerosis (thickening of artery walls – due to deposition of plaques,
cholesterol) and arteriosclerosis (hardening of arteries). The arteriosclerotic changes of
hypertensive retinopathy are caused by chronically elevated blood pressure, defined as
systolic pressure greater than 140 mmHg or diastolic pressure greater than 90 mmHg.
______________________________________________________________________
Figure 7.2. Retinal photograph with visible central reflex in both arterioles and
venules.
The central reflex is the bright central region seen in the arterioles and venules.
136
______________________________________________________________________
Figure 7.3. Retinal photograph showing mild enhancement of the retinal arteriolar
central reflex (copper wiring).
Progression of sclerosis and hyalinization diffuses the CR and causes the arterioles to appear red-brown,
referred to as copper wiring.
______________________________________________________________________
Figure 7.4. Retinal photograph showing marked enhancement of the retinal
arteriolar central reflex (silver wiring).
When the sheathing fully encircles the vessel wall, it is referred to as a silver-wire vessel.
137
The CR can be qualitatively graded as producing copper-wire or silver-wire vessels,
which are two of the signs of hypertensive retinopathy (see Figures 7.3 and 7.4).
Clinical guidelines define silver wiring as the presence of a central reflex with a sharp
margin, less than one third of the width of the arteriolar vessel and consistently present
over at least two thirds of the length of the arteriolar sector (Keith et al., 1939,
Svardsudd et al., 1978). Copper wiring is defined as presence of a central reflex with
width greater than one third of the arteriole width, consistently present over at least two
thirds of the length of the arteriolar sector.
Early atherosclerosis causes the CR to be more diffuse and less bright. Progression of
sclerosis and hyalinization further diffuses the CR and causes the arterioles to appear
red-brown, referred to as copper wiring. Advancing sclerosis increases the optical
density of the retinal blood vessel walls, resulting in a visible sheathing of the vessels.
When the sheathing fully encircles the vessel wall, it is referred to as a silver-wire
vessel.
Increased arteriolar CR width and intensity have previously been linked to systemic
vascular diseases, including hypertension and coronary artery disease (Keith et al.,
1939, Michelson et al., 1979, Svardsudd et al., 1978, Tedeschi-Reiner et al., 2005). For
this reason they have been incorporated into classification schemes for hypertensive
retinopathy (Keith et al., 1939, Leishman, 1957, Scheie, 1953, Wong and Mitchell,
2004). Results from the Blue Mountains Eye Study, a large population-based cohort
study, found that enhanced CR was associated with vascular risk factors and elevated
blood pressure, but not with poor survival (Kaushik et al., 2007).
AD and retinopathy share major risk factors such as diabetes and hypertension.
However, independent of diabetes, hypertension and a number of other covariates, a

138
recent large population study revealed an association between retinopathy and AD
(Schrijvers et al., 2012), suggesting that there is a link between these disorders,
independent of their shared risk factors. However, the same study showed similar
results for vascular dementia, indicating that this association may not be specific to AD.
There is much interest in retinal microvascular signs in AD, in order to investigate the
role of cerebral microvascular disease in the pathogenesis of AD. There is a level of
homology between the retinal and cerebral microvasculatures, in terms of embryology,
anatomy and physiology. The retina is more accessible for imaging than the brain and
hence more useful for early detection and monitoring of disease.
Recent studies are supportive of the hypothesis that abnormal cerebrovascular function
is involved in the progression of AD (de la Torre, 2009, Ruitenberg et al., 2005). The
possibility that abnormal vascular function in AD might extend to the retina is
supported by a hemodynamic study that revealed a significant narrowing of retinal
venular blood column and reduced venous blood flow accompanied by neuronal
damage (Berisha et al., 2007). AD patients have Aβ and collagen fibril deposition in the
walls of cerebral vessels (Kalaria and Pax, 1995, Vinters et al., 1996), hence retinal
vascular changes in AD could be related to a similar process in the retinal vasculature
(Michelson et al., 2007).
Chapter 6 discussed signs of retinal degeneration in AD that are usually observed in the
retinal diseases glaucoma and AMD. In the present Chapter, different retinal changes
are examined in AD; changes that are usually associated with other systemic disease
and referred to as retinopathy. The aim of this study was to use retinal photography to
investigate clinical signs of retinopathy in AD and control participants, and also with
respect to neocortical amyloid plaque burden, the earliest pre-clinical sign of AD.
139
Recent technological developments in retinal photography and image analysis allow
more accurate investigation of retinal microvascular changes such as CR. The aim of the
present study was to use retinal photography to investigate CR both qualitatively
(clinician grading) and quantitatively in AD and control participants, and also with
respect to neocortical amyloid plaque burden, the earliest pre-clinical sign of AD.
7.2) Clinician-Graded Retinopathy
7.2.1) Methods
section 2.5. Retinal photographs were examined by an experienced ophthalmologist for
the presence of signs of retinopathy. Only images deemed gradable were considered.
The clinician was masked from the disease status of the participants.
Retinopathy was defined as present if any of the following were detected:
microaneurisms, retinal hemorrhages, soft exudates, hard exudates, macular edema or
optic disc swelling. Arteriovenous nicking and focal arteriolar narrowing were also
reported as present or absent.
Retinal photographs were examined by the ophthalmologist for presence of copper or
silver wire vessels (mild or marked enhancement of the retinal arteriolar central reflex).
This determination is subjective, although clinical guidelines define silver wiring as the
presence of a CR with a sharp margin, less than one third of the width of the arteriolar
vessel and consistently present over at least two thirds of the length of the arteriolar
sector (Keith et al., 1939, Svardsudd et al., 1978). Copper wiring is defined as presence
140
of a CR with width greater than one third of the arteriole width, consistently present
over at least two thirds of the length of the arteriolar sector.
7.2.2) Results
The demographics of this cohort are presented in Table 7.1. HC and AD groups did not
cataract surgery. There was a higher percentage of APOE ε4 carriers in the AD group
(p=0.019). Silver wiring was not reported in either group but copper wiring was
common in both groups. AD diagnosis was not associated with copper or silver wiring,
retinopathy, arterio-venular (AV) nicking or focal arteriolar narrowing (see Table 7.1).
141
Table 7.1. Demographics and retinopathy analysis for HC and AD groups.

Age: Years [mean (SD)] 71.6 (5.6) 72.4 (7.5) 0.557‡
Gender; Males: [N (%)] 55 (45) 12 (48) 0.764†
Hypertension: [N (%)] 44 (36) 11 (44) 0.439†
Diabetes: [N(%)] 6 (5) 2 (8) 0.533†
APOE ε4 Carrier: [N (%)] 38 (31) 14 (56) 0.019†
Copper Wiring: [N (%)] 110 (89) 24 (96) 0.326†
Silver Wiring: [N (%)] 0 (0) 0 (0) 1.000†
Retinopathy: [N (%)] 1 (1) 0 (0) 1.000†
AV Nicking or Focal Arteriolar

4 (3) 2 (2) 0.288†
Narrowing: [N (%)]
‡
†
χ2 test for categorical variables (gender, hypertension, diabetes, smoking status, APOE ε4 status and
APOE ε4 Carrier refers to carrier/non-carrier of an Apolipoprotein E ε4 allele. SD: standard deviation.
AIBL neuroimaging data was available for 36 HC participants. The neuroimaging
cohort consisted of 22 HC- participants (age 69.8 ± 5.5 yrs, 10 male, 12 female) and 14
HC+ participants (age 73.4 ± 7.5 yrs, 8 male, 6 female). The demographics of this
cohort are presented in Table 7.2. HC- and HC+ groups did not differ significantly in
age, gender, hypertension, diabetes, smoking status, previous cataract surgery or APOE
ε4 carriers. High plaque burden in healthy individuals was not associated with copper or
silver wiring, retinopathy, AV nicking or focal arteriolar narrowing (see Table 7.2).
142
Table 7.2. Demographics and retinopathy analysis for HC+ and HC- groups.
HC- HC+ P Value
Age: Years [mean (SD)] 69.8 (5.5) 73.4 (7.5) 0.106‡
Gender; Males: [N (%)] 10 (46) 8 (57) 0.495†
Hypertension: [N (%)] 9 (41) 6 (43) 0.908†
Diabetes: [N(%)] 1 (5) 0 (0) 1.000†
Previous cataract surgery: [N (%)] 1 (5) 2 (14) 0.327†
APOE ε4 Carrier: [N (%)] 11 (50) 11 (79) 0.095†
Copper Wiring: [N (%)] 19 (86) 13 (93) 0.326†
Silver Wiring: [N (%)] 0 (0) 0 (0) 1.000†
Retinopathy: [N (%)] 1 (1) 0 (0) 1.000†
AV Nicking or Focal Arteriolar Narrowing: [N (%)] 4 (3) 2 (2) 0.288†
‡
significant)
† 2
APOE ε4 Carrier refers to carrier/non-carrier of an Apolipoprotein E ε4 allele. HC-: Healthy controls with
low plaque burden; HC+: healthy controls with high plaque burden. SD: standard deviation. No
demographic was significantly different between groups. Significant results in bold type.
7.2.3) Discussion
This cross-sectional study found no evidence for an association between retinopathy and
AD, or high plaque burden in healthy individuals. It should be noted that this is a small
cohort study, larger studies similar to the Rotterdam study (Schrijvers et al., 2012) are
required to further investigate the possibility of a connection between AD and
retinopathy, particularly longitudinal studies that investigate incidence of AD in
individuals with retinopathy.

143
The Rotterdam study, with a baseline cohort of 6273 participants, found that there was
an association between retinopathy and AD, independent of their common risk factors
(Schrijvers et al., 2012). However, there was a similar relationship for vascular
dementia and retinopathy, and a longitudinal analysis over a mean follow-up of 11.4
years found no association of retinopathy with risk of incident dementia or AD. These
results indicate that retinopathy, rather than a possible early marker for AD, may instead
be a common consequence of dementia.
While the present study did not support the hypothesis of an association between
retinopathy and AD, it was limited by its cross-sectional nature, small cohort size and
the qualitative nature of the clinical retinopathy assessment. Recent technological
developments in retinal photography and image analysis allow for quantitative
investigation of retinal microvascular changes such as central reflex (CR). The next
section will utilise retinal image analysis to calculate quantitative CR values and
investigate this parameter with respect to AD diagnosis and neocortical amyloid plaque
burden, the earliest pre-clinical sign of AD.

144
7.3) Image Analysis of Central Reflex
7.3.1) Methods
Retinal photographs were collected from AD and HC participants in the AIBL study, as
described in sections 2.2 and 2.5. A novel, Commonwealth Scientific and Industrial
Research Organisation (CSIRO) developed, quantitative method was utilised for
calculating the size of the CR relative to the size of the retinal vessel, for the largest
retinal arteriole in each retinal photograph (see Figure 7.5). This provides a repeatable,
quantitative measure of the central reflex as a continuous parameter, rather than a
coarsely graded or qualitative parameter (copper or silver wiring) as has been used in
the past. The CR results were examined to compare the vessels of AD and HC
participants, and HC- and HC+ participants, as previously described.
The specialized, semi-automated software was developed by the CSIRO to calculate the
CR, vessel width and CR to vessel width ratio. It utilises the intensity gradient across
the vessel to calculate an average CR and vessel width in zone B (see Figure 2.2). The
software-measured, quantitative CR values were compared to expert graders’ qualitative
rankings on CR severity, revealing a Pearson correlation of 0.83 (p<0.01). The inter-
grader correlation on CR to arterial calibre ratio was 0.81 and for CR to venular calibre
ratio it was 0.85. Therefore, this semi-automated software provides good agreement
with clinician grading, and through finer quantification has the potential to investigate
CR changes in more detail. All graders were masked from participant characteristics.
145
______________________________________________________________________
Figure 7.5. Grading interface for central reflex quantification.
A quantitative method is utilised for calculating the size of the CR relative to the size of the retinal vessel.
Co-morbid medical conditions associated with retinal vascular changes are hypertension
and diabetes mellitus, which were therefore treated as confounders in the analysis.
Participant reported smoking history (current or past) was also considered relevant due
to previous reports linking smoking with possible retinal vascular changes (Sun et al.,
2009). Previous cataract surgery was also considered as a confounder, while cataract
surgery within the last 6 months was an exclusion criterion.
7.3.2) Results
Of 148 participants, 134 (90.5%) had photographs gradable in at least 1 eye. The cohort
thus consisted of 22 probable-AD patients (age 71.5 ± 7.4 yrs, 10 male, 12 female) and
112 healthy control participants (age 71.5 ± 5.6 yrs, 48 male, 64 female). The
demographics of this cohort are presented in Table 7.3. HC and AD groups did not
differ significantly in age, gender, hypertension, diabetes, smoking status, previous
cataract surgery or APOE ε4 carriers.

146
The central reflex to vessel width ratio (CRR) for the largest arteriole was higher in AD
based on ANOVA analysis (p=0.018, std coef = 0.20, std error = 0.09) (see Figure 7.6).
In an ANCOVA model for CRR including confounders, with stepwise removal for
p>0.1, only disease status (p=0.056, std coef = 0.16, std error = 0.08) and APOE ε4
status (p<0.0001, std coef = 0.34, std error = 0.08) were retained (model R2 = 0.154, DF
= 2, F=11.889, p<0.0001), indicating that CRR is very strongly related to APOE ε4
status. Weak, non-significant trends for narrower arteriole width (consistent with
Chapter 3 results) and wider CR in AD resulted in a significantly higher CRR in AD.
Table 7.3. Demographics and central reflex analysis for HC and AD groups.
Age: Years [mean (SD)] 71.5 (5.6) 71.5 (7.4) 1.000‡
Gender; Males: [N (%)] 48 (43) 10 (46) 0.822†
Hypertension: [N (%)] 41 (37) 9 (41) 0.703†
Diabetes: [N(%)] 6 (5) 2 (9) 0.504†
APOE ε4 Carrier: [N (%)] 36 (32) 11 (50) 0.113†
Central Reflex: [mean (SD)] 22.0 (8.4) 23.0 (3.4) 0.603‡
Arteriole Width: [mean (SD)] 91.9 (11.8) 90.5 (16.9) 0.645‡
Central Reflex Ratio: [mean (SD)] 0.231 (0.039) 0.253 (0.041) 0.018‡
‡
significant)
† 2
χ test for categorical demographic variables (gender, hypertension, diabetes, smoking status, previous
cataract surgery and APOE ε4 status) (p < 0.05 considered significant)
§
APOE ε4 Carrier refers to carrier/non-carrier of an Apolipoprotein E ε4 allele. CRR = ratio CR to vessel
diameter.
147
______________________________________________________________________
Figure 7.6. Central reflex ratio results.
A) Healthy control (HC) and Alzheimer’s disease (AD) participants
B) APOE ε4 non-carriers (ε4-) and carriers (ε4+).
p values from ANCOVA analysis of differences between groups (including confounders).
Within the AD group only, APOE ε4 was associated with larger CRR (p=0.0016, std
coef = 0.660) in an ANCOVA model (model R2 = 0.476, DF = 3, F=5.455, p=0.008).
No confounders were significantly associated with CRR. Within the HC group only,
APOE ε4 was similarly associated with larger CRR (p=0.0017, std coef = 0.291) in an
ANCOVA model (model R2 = 0.114, DF = 2, F=7.036, p=0.001). Again, no
confounders were significantly associated with CRR.

148
Table 7.4. Demographics and central reflex analysis for HC ε4 carriers and non-
carriers.
HC ε4+ HC ε4- P Value
Age: Years [mean (SD)] 71.1 (5.4) 71.7 (5.6) 0.526‡
Gender; Males: [N (%)] 18 (50) 30 (39) 0.294†
Hypertension: [N (%)] 15 (42) 26 (34) 0.445†
Diabetes: [N(%)] 2 (6) 4 (5) 0.949†
Arteriole width: [mean (SD)] 92.3 (9.6) 91.7 (12.7) 0.806‡
‡
significant)
† 2
§
ε4+/ ε4- refers to carrier/non-carrier of an Apolipoprotein E ε4 allele. CRR = ratio CR to vessel diameter.
149
Table 7.5. Demographics and central reflex analysis for AD ε4 carriers and non-
carriers.
AD ε4+ AD ε4- P Value
Age: Years [mean (SD)] 70.3 (8.4) 72.8 (6.5) 0.434‡
Gender; Males: [N (%)] 6 (55) 4 (36) 0.395†
Hypertension: [N (%)] 7 (64) 2 (18) 0.039†
Diabetes: [N(%)] 1 (9) 1 (9) 1.000†
Arteriole Width: [mean (SD)] 86.5 (13.4) 94.6 (19.6) 0.272‡
‡
significant)
† 2
§
ε4+/ ε4- refers to carrier/non-carrier of an Apolipoprotein E ε4 allele. CRR = ratio CR to vessel diameter.
PiB-PET neuroimaging data were available for 41 (37%) of the 112 healthy control
participants. The neuroimaging cohort consisted of 27 healthy individuals with low
plaque burden (HC-, age 70.4 ± 5.5 yrs, 14 male, 13 female) and 14 healthy individuals
with high plaque burden (HC+, age 73.4 ± 6.5 yrs, 8 male, 6 female). The
demographics of this cohort are presented in Table 7.6. HC- and HC+ groups did not
cataract surgery. There were more APOE ε4 carriers in the HC+ group (p=0.019).
The groups also did not differ in CR, CRR or arteriole width. Based on an ANCOVA
model for CRR including confounders, with stepwise removal for p>0.1, only APOE ε4
status (p<0.023, std coef = 0.355) was retained (model R2 = 0.126, DF = 1, F=5.612).
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Table 7.6. Demographics and CR analysis for HC- and HC+ groups.
HC- HC+ P Value
Age: Years [mean (SD)] 70.4 (5.5) 73.4 (6.5) 0.128‡
Gender; Males: [N (%)] 14 (52) 8 (57) 0.747†
Hypertension: [N (%)] 11 (41) 5 (36) 0.755†
Diabetes: [N(%)] 1 (4) 0 (0) 1.000†
APOE ε4 Carrier: [N (%)] 12 (44) 12 (86) 0.019†
Central Reflex: [mean (SD)] 24.5 (15.8) 22.5 (3.4) 0.641
Arteriole Width: [mean (SD)] 89.9 (16.2) 93.3 (10.1) 0.477
Central Reflex Ratio: [mean (SD)] 0.235 (0.052) 0.245 (0.052) 0.528
‡
significant)
† 2
χ test for categorical variables (gender, hypertension, diabetes, smoking status, previous cataract
surgery and APOE ε4 status) (p < 0.05 considered significant)
APOE ε4 Carrier refers to carrier/non-carrier of an Apolipoprotein E ε4 allele.
7.3.3) Discussion
In this study, AD was found to be associated with larger CRR (p=0.018) in an ANOVA
analysis. However, after including confounders in a stepwise ANCOVA analysis for
CRR, disease status (p=0.056, std coef = 0.156) and APOE ε4 status (p<0.0001, std coef
= 0.338) were both retained (model R2 = 0.154, DF = 2, F=11.889), indicating that CRR
is strongly associated with APOE ε4 status and exhibits an independent, non-significant
trend with AD.
In Section 7.2, CR was graded qualitatively by a clinician. No relationship between
CRR and APOE ε4 status was found, but a non-significant trend for greater prevalence
of copper wiring in AD was found. For the present study, a quantitative, semi-
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automated technique was used to find a highly significant association between CRR and
APOE ε4 status, and a positive trend between CRR and AD, independent of APOE ε4
status. The reason that only the automated technique found an association between CRR
and APOE ε4 status might be the more detailed quantification of CRR provided by this
technique.
While the clinician evaluated results found no silver wiring in the cohort, copper wiring
was highly prevalent and exhibited by 89% of the HC group and 96% of the AD group.
Therefore it seems that most individuals in the demographic concerned exhibit some
level of enhanced CR, and that this semi-automated technique shows potential for more
accurate probing of CR changes, through quantification of the CR rather than simply
reporting copper wiring.
The CR is an optical property of retinal vessels that relates to the structure and
composition of the vessel wall. The results presented in Chapter 3 indicate a number of
topographic retinal vascular abnormalities in diagnosed and pre-clinical AD, and it is
reasonable to hypothesise that CR changes might accompany this vascular modulation.
This study investigated whether CR is a marker of AD pathogenesis, however the
results suggest CRR may instead be a surrogate marker for APOE ε4 status.
The APOE ε4 allele has been implicated in modulating the metabolism and aggregation
of Aβ (Bu, 2009), and is the strongest genetic risk factor for AD (Corder et al., 1993).
Individuals with one copy of the APOE ε4 allele have a 2-3 fold increased risk of
developing AD by age 85 and those with two copies have a 12 fold increased risk
compared to the general population (Corder et al., 1993). However, the APOE ε4 status
can already be determined by a simple blood test, hence for CRR to have utility as a
biomarker for AD, a strong association between CRR and AD or neocortical plaque
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burden would have to be demonstrated, independent of the association already provided
by APOE genotype. The lack of CRR difference between HC- and HC+ groups found in
this study, along with the weak association between CRR and AD (after adjustment for
APOE genotype), suggests that CRR may not have utility as a biomarker for early
detection of AD.
The strong association between CRR and APOE ε4 status might be due to
cardiovascular abnormalities and risk factors that are associated with APOE ε4. The
optical reflection that underlies CR depends on the change in refractive index at vessel
surfaces (Fowles, 1989). It is thus possible that changes to the CR in APOE ε4 allele
carriers is related to changes in the vessel wall characteristics that may be associated
with this genotype.
There are no reports directly assessing relationships between APOE genotype and
retinal CR, however an Australian population study investigated risk factors for
enhanced CR and found that the following were positively associated with an increased
likelihood of mildly or markedly enhanced retinal CR; total cholesterol, LDL, body
mass index, mean arterial blood pressure and heavy alcohol consumption (>4 drinks per
day) (Kaushik et al., 2007). None of the participants in the present study consumed
alcohol at >4 drinks per day, however all the other factors above may be associated with
APOE genotype (Davignon et al., 1988, Eichner et al., 1993, Wilson et al., 1994), and
hence are likely to be behind the connection between CR and APOE genotype found in
this study.
APOE ε4 may increase the risk of hypertension which may in turn alter CR through
changes in vessel wall transparency (arteriosclerosis) or changes in blood flow velocity
from microvascular rarefaction (Mayet and Hughes, 2003, Pries and Secomb, 2002),
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which can influence the density of erythrocyte cells lining the vasculature (Brinchmann-
Hansen and Sandvik, 1986, Brinchmann-Hansen and Sandvik, 1986). Increased retinal
CR is a sign of hypertension (Kaushik et al., 2007, Keith et al., 1939, Patel and Kohner,
1994, Svardsudd et al., 1978, Walsh, 1982) and there is some evidence that the APOE
ε4 allele is associated with hypertension (Catalano et al., 1991, Isbir et al., 1997,
Yilmaz et al., 2001).
Yilmaz et al. found the frequency of ε4 allele carriers was higher in hypertensive
individuals (9.95%) than controls (2.38%, p=0.07) and hypertensive individuals positive
for the APOE ε4 allele were at a significantly higher risk of retinopathy (p<0.05)
(Yilmaz et al., 2001). Catalano et al. looked at 50 untreated hypertensive individuals
and 50 controls and found that the APOE genotype was different (P<0.05) (Catalano et
al., 1991). The results suggested that in untreated hypertensive patients, alterations in
the APOE profile are present which, in part, may be responsible for the elevated
incidence of cardiovascular disease, independent of blood pressure.
However, the hypothesis that hypertension is a pathway for APOE ε4 status to affect the
retinal CR was not supported by the results of this study. An ANCOVA model for CRR,
adjusting for hypertension (diagnosed or elevated current blood pressure) revealed no
association between hypertension and CR.
If hypertension-induced arteriosclerosis is not the mechanism through which APOE
genotype may have influenced CR in this study, other cardiovascular factors may be.
The APOE genotype contributes to variation in cholesterol levels, influences the
expression of hyperlipidemia and appears to modulate the susceptibility to
atherosclerosis and ischaemic stroke (Bennet et al., 2007, Davignon et al., 1988, Khan
et al., 2013). Cholesterol levels and atherosclerosis both influence CR (Kaushik et al.,
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2007). There is also evidence that the presence of the APOE ε4 allele predisposes to
coronary artery disease and coronary heart disease (Bennet et al., 2007, Eichner et al.,
1993, Wilson et al., 1994), and increased CR has also previously been linked to
coronary artery disease (Keith et al., 1939, Michelson et al., 1979, Svardsudd et al.,
1978, Tedeschi-Reiner et al., 2005). Findings from some studies suggest that the
stronger the CR abnormality, the greater the severity and extent of coronary vessel
atherosclerosis (Michelson et al., 1979, Tedeschi-Reiner et al., 2005). These studies
incorporated a classification system for CR incorporating “broadening of the light
reflex” together with “copper-wire” and “silver-wire” changes to arterioles, adapted
from the Scheie scheme (Scheie, 1953). Many of these factors also increase risk for AD,
hence the results of this study may be attributed to the influence of APOE genotype on
vascular factors that both increase the risk of AD and result in increased retinal CR.
There is a lack of conclusive or consistent evidence in the literature for an association
between APOE genotype and changes to the vasculature of the retina specifically. Large
population studies have had contrasting results. In the Atherosclerosis Risk in
Communities Study (ARIC), patients with the APOE ε4 allele had a greater risk of non-
diabetic retinopathy than other subjects (Liew et al., 2007), although they did not
consider CR, copper or silver wiring. Other signs were less consistently associated
with APOE polymorphisms. However, in the Cardiovascular Health Study, carriers of
the APOE ε4 and APOE ε2 alleles were more likely to have AV-nicking, and the APOE
ε2 allele was associated with focal arteriolar narrowing, but not other signs of
retinopathy (Sun et al., 2007). Again, CR, copper and silver wiring were not considered.
The clinician-graded study discussed in Section 7.2 did not find any relationship
between AV-nicking and APOE genotype or AD.

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However, APOE gene polymorphisms have been associated with the retinal neuro-
degenerative diseases AMD and glaucoma (Baird et al., 2004, Copin et al., 2002,
Klaver et al., 1998, Schmidt et al., 2002, Souied et al., 1998, Vickers et al., 2002,
Zareparsi et al., 2004, Zetterberg et al., 2007), and the severity of several CNS
degenerative diseases (Bedlack et al., 2000). It is possible that retinal vascular changes
resulting from these diseases might provide a link between APOE and CR, although the
study discussed in Chapter 6 found no evidence for a link between APOE ε4 status and
optic atrophy or the ocular diseases AMD or glaucoma.
Some authors have also suggested that both age and cataract may impair the visibility
and recognition of a mild increase in CR from the retinal photographs (Kaushik et al.,
2007), however no association between CR and cataract or age was found in the present
study. Other retinal vascular signs, AV nicking, and retinopathy in persons without
diabetes were found to be strongly related to increased CR (Kaushik et al., 2007).
Brinchmann-Hansen et al. found a relationship between intensity of the retinal CR and
systolic blood pressure for young individuals, however width of the CR was not found
to be related to age or blood pressure (Brinchmann-Hansen et al., 1987). Width of the
CR is considered a more reliable measure than intensity due to the possible variation of
light intensity associated with camera focus and ocular media transparency.
Previous reports have found that focal arteriolar narrowing is associated with current but
not past blood pressure levels, while AV nicking is related to past but not current levels
(Wong and Mitchell, 2004). Hence the reported association between enhanced light
reflex, AV nicking and retinopathy, rather than focal arteriolar narrowing, suggests that
CR may indicate long-standing hypertension rather than current blood pressure levels,
although as discussed, the present study found no association between hypertension and
CR.
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Retinal microvascular signs have been found to be markers of systemic vascular
diseases, and to predict a number of cardiovascular disorders (Wong and McIntosh,
2005). It is unclear whether these retinal microvascular signs are influenced by genetic
factors. AD and retinopathy share major risk factors such as diabetes and hypertension.
However, independent of diabetes, hypertension and a number of other covariates, a
recent large population study revealed an association between retinopathy and AD
(Schrijvers et al., 2012), suggesting that there is a link between these disorders,
independent of their shared risk factors. Genetic associations such as a possible link
between APOE and CRR, may provide insights into the pathogenesis of ocular and
systemic vascular diseases. The results of the present study suggest a link between the
APOE genotype and changes to the retinal vasculature, likely mediated by systemic
vascular abnormalities which are risk factors for both AD and retinopathy.
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Chapter 8: General discussion and future directions
8.1) General discussion
This Chapter will discuss the findings presented in this thesis, covering retinal changes
and pupil flash response in diagnosed and pre-clinical AD. Possible future directions
will be identified which may enhance our understanding of ocular changes in pre-
clinical AD, AD pathogenesis in general and possibly lead to an ocular screening test
for AD. The limitations of the thesis will be discussed, followed by concluding
comments.
The research for this PhD project has uncovered ocular changes in diagnosed and pre-
clinical AD. The broad aim of this thesis was to contribute toward an ocular screening
test that will facilitate earlier detection of AD throughout the population. The
importance of research into early detection is threefold:
1) Existing interventions (although limited) can be applied at an earlier stage of the
disease, allowing the best chance of a beneficial effect.
2) Emerging preventative interventions can be tested at an earlier stage of the
disease, allowing a better evaluation of their disease modifying effect.
3) Biomarker changes identified in early AD may reveal new insights into the
pathogenesis of the disease and hence inspire new avenues of treatment,
monitoring or diagnosis.
Thus the ocular changes identified for AD will be discussed both for their potential as
screening biomarkers for early detection and monitoring of the disease, as well as for
the possible insights they reveal about AD pathophysiology.

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8.1.1) The retina and Alzheimer’s disease
The findings presented in Chapters 3, 6 and 7 demonstrate retinal morphology in
diagnosed and pre-clinical AD. Individuals with diagnosed AD exhibited extensive and
highly significant topographical alterations to the retinal vasculature, signs of altered
vessel structure affecting vessel reflectance, as well as early signs of AMD (soft drusen)
and glaucoma (neuro-retinal rim abnormalities). Individuals with pre-clinical AD
exhibited some significant topographical changes to the retinal vasculature, consistent
with the changes in diagnosed AD.
The fact that the pre-clinical AD results were fewer and less significant as those for
diagnosed AD might be attributed to the smaller neuroimaging cohort. However, it
cannot be ruled out that pre-clinical retinal changes may occur at a different stage of AD
pathogenesis than cerebral amyloid plaque build-up. Indeed, animal AD model studies
have found that retinal plaques preceded cerebral plaques (Koronyo-Hamaoui et al.,
2010). However, evidence from this thesis in support of retinal changes occurring in a
similar stage of disease progression as cerebral amyloid plaque build-up came from
significant linear relationships between some retinal parameters and continuous
neocortical plaque burden or rate of change of neocortical plaque burden, in healthy
controls. Therefore, further longitudinal, neuroimaging studies with larger cohorts are
required to confirm the temporal relationship between retinal and cerebral changes in
the pathogenesis of AD.
Nevertheless, the fact that retinal vascular changes were detected in diagnosed and pre-
clinical AD in this study is encouraging for the potential of an ocular screening test for
AD. In terms of adding to the understating of AD pathogenesis, this study has expanded
on the Berisha et al. (2007) findings and identified substantial retinal vascular changes
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in AD and pre-clinical AD. This complements another research field that has
demonstrated significant retinal degeneration in AD, and encourages future research
into the causal links between these retinal changes in AD (Bayer et al., 2002, Blanks et
al., 1989, Blanks et al., 1996, Blanks et al., 1996, Danesh-Meyer et al., 2006, Hinton et
al., 1986, Iseri et al., 2006, Paquet et al., 2007, Parisi et al., 2001, Sadun and Bassi,
1990, Sadun and Bassi, 1990, Tamura et al., 2006, Tsai, 1991).
The results on thinning arterioles and venules in AD contrast with another report of
wider retinal venules in vascular dementia (de Jong et al., 2011). Thus, in addition to
potential for early screening for AD, the retinal results presented in this thesis also
suggest that retinal photography might help discriminate between AD and vascular
dementia, the two most common forms of dementia. Venular widening in the retinal
circulation is also associated with hypertension (Sun et al., 2009), which is a major risk
factor for AD. The opposing venular thinning reported in AD in this thesis suggests that
the retinal vascular changes in AD may be independent of the vascular effects of the
risk factor hypertension.
The only prior study reporting retinal vascular changes in AD found reduced blood flow
and reduced blood column width (Berisha et al., 2007). The researchers speculated that
a possible cause could be increased venous wall thickness due to deposition of protein.
Both collagen and Aβ are deposited in cerebral veins in AD (Ellis et al., 1996, Jellinger,
2002, Kalaria and Pax, 1995, van Horssen et al., 2002, Vinters et al., 1996) and hence a
similar process may be occurring in the retina. However, the results from this thesis
indicate a thinning of the full diameter of the retinal vessels, suggesting other
mechanisms are present. Vessel constriction due to Aβ is another possible mechanism,
with animal experiments demonstrating Aβ has a direct and specific constrictive effect
on cerebral vessels, leading to a decreased cerebral blood flow (Suo et al., 1998).
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Addressing the possibility of a causal link between neuro-degeneration and vascular
changes in AD, as discussed in the introduction, there is substantial evidence of both
cerebral and retinal neuro-degeneration in AD. In the pre-clinical AD brain, there is also
strong evidence of vascular changes and neuro-degeneration. While the results of this
thesis suggest retinal vascular changes in pre-clinical AD, no studies have reported on
the possibility that retinal neuro-degeneration might accompany these vascular changes
in pre-clinical AD. Whether cerebral or retinal neurodegeneration precedes vascular
changes and perhaps contributes to these changes through reduced metabolic demand is
an open question. The large population-based prospective Rotterdam Study found that
cerebral hypoperfusion (reduced blood flow) also occurs in pre-clinical AD (Ruitenberg
et al., 2005), however this was related to neuro-degeneration through reduced
hippocampal and amygdala volumes, leaving the causal direction between neuro-
degeneration and vascular effects uncertain in the cerebral system. The Berisha et al.
(2007) study found evidence of retinal hypoperfusion in AD; hence further longitudinal
studies will be required to investigate the link between neuro-degeneration and
vasculature in both brain and retina.
No significant retinal vascular changes were observed in the Dutch APP ADAD
mutation carriers (APPGlu693Gln), indicating a disconnect between retinal vascular
changes and pupil response changes in this group, although small group numbers mean
that similar retinal changes cannot be ruled out in this genetic form of AD. There were
also no signs of glaucoma, AMD or retinopathy in the ADAD cohort, consistent with
the younger age of these participants and the strong age risk factor for these diseases.
The APPGlu693Gln mutation is associated with cerebral amyloid angiopathy (CAA) as
well as cerebral plaques. Neither dementia nor cognitive decline has been observed in
this family, possibly due to earlier onset of CAA and fatal cerebral hemorrhages.
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Despite the known cerebral vascular effects of the APPGlu693Gln mutation, no retinal
vascular abnormalities were observed in the mutation carriers. PiB-PET imaging results
demonstrated that the mutation carriers had higher SUVR than non-carriers, although no
participants had high plaque burden (SUVR > 1.5). Future studies of these rare families
harboring ADAD mutations will be required to investigate if retinal changes do occur
when mutation carriers acquire high plaque burden, consistent with the sporadic AD
results from this thesis. Studies of families harboring known ADAD mutations provide
a powerful opportunity to investigate the temporal sequence of AD biomarker changes,
avoiding the age-related co-morbidities and uncertainty about disease progression in
sporadic AD.
In addition to the retinal vascular changes found in the present study, a greater
prevalence of neuro-retinal rim abnormalities was also found in AD. These
abnormalities are also observed in the retinal disease glaucoma, which has previously
been associated with AD through shared visual deficits and retinal and optic nerve head
pathology. The link between AD and glaucoma, supported by the results of this thesis,
is interesting because the latter is a neuro-degenerative disease of the retina which also
affects the retinal vessels. Several studies have shown decreased blood flow in the optic
nerve head, retina and choroid in glaucoma patients (Findl et al., 2000, Grunwald et al.,
1998, Michelson et al., 1996, Portmann et al., 2011, Wang et al., 2011), believed to be
the result of elevated IOP. A decrease in the number of capillaries in the optic nerve
head as well as the atrophy of the peripapillary capillaries supplying the RNFL has also
been reported (Gottanka et al., 2005, Kornzweig et al., 1968). In addition, generalized
retinal vessel thinning in AD reported for the first time in this thesis, has also been
reported in glaucoma (Chang et al., 2011, Jonas and Naumann, 1989, Lee et al., 1998,
Mitchell et al., 2005), along with focal (localized) narrowing of vessels near the optic
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disc (Papastathopoulos and Jonas, 1995). The large, population-based, prospective
Rotterdam study, found that baseline retinal vessel diameters did not predict incident
glaucoma or optic disc changes, suggesting that retinal vascular changes in glaucoma
are more likely to be a consequence of RGC loss instead of a cause. Further research is
needed to verify this and to investigate if this is also the case for AD.
Investigations into the retinal changes common to AD and glaucoma might yield
interesting results about the pathogenesis of these diseases, and have consequences for
their treatment and monitoring. AD drugs directed against Aβ accumulation may be
effective against retinal degeneration in both glaucoma and AD. Neuroprotection in AD
and glaucoma may be achieved through disrupting the role of Aβ in the signaling
pathways that lead to RGC apoptosis. Acetylcholinesterase inhibitors being tested in
AD may also be of therapeutic benefit in glaucoma, through their ability to reduce IOP.
Neuro-protection and other treatment approaches also have the potential to have
therapeutic benefits in both diseases.
AD has also been previously linked to AMD, which is the major cause of irreversible
blindness in elderly patients worldwide. While the choroidal vasculature is altered in
late AMD and has been implicated as a cause of the disease (Ciulla et al., 2002, Harris
et al., 1999, Lutty et al., 1999, Pemp and Schmetterer, 2008), no associations between
AMD and the inner retinal blood vessels have been reported. In this thesis it was found
that there was an increased prevalence of soft drusen, an early sign of AMD, in AD
patients. However, no association between signs of AMD and pre-clinical AD was
found. More research is required to establish whether the substantial overlap of ocular
AD pathology with AMD and glaucomatous pathology is due to the direct contribution
of Aβ and tau deposition, or whether instead the involvement of these proteins is
secondary to other, disease and/or tissue specific insults. Advances in research into each
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disease, or pooling of research efforts, may provide novel directions for therapies and
tests, and hence may have broad impact in both ophthalmology and neurology. While
the overlap between AD, glaucoma and AMD provides strong research interest, it also
constitutes a challenge to the development of retinal biomarkers for early detection of
AD, which will need to differentiate between AD, AMD and glaucomatous pathology.
There is therefore an opportunity for research into retinal degeneration utilizing both
AD and retinal-disease cohorts (AMD or glaucoma), to further elucidate the connection
between the ocular pathology in each disease and to discover which current or new
biomarkers are useful for disease detection and monitoring.
AD has also previously been associated with retinopathy (Schrijvers et al., 2012),
independent of diabetes, hypertension and a number of other covariates. In this thesis,
signs of retinopathy were investigated, and a quantitative measure of central reflex was
calculated and analysed. The prevalence of clinician-reported retinopathy
(microaneurisms, retinal hemorrhages, focal arteriolar narrowing, arterio-venous
nicking, increased retinal arteriolar light reflex - copper or silver wiring, flame and blot-
shaped retinal hemorrhages, cotton wool spots, macular edema, optic disc swelling) was
not different in diagnosed or pre-clinical AD. However, a semi-automated technique for
quantifying the central reflex of arterioles revealed a larger central reflex to artery width
ratio in AD, but not pre-clinical AD. Closer examination revealed that this ratio was
strongly correlated with APOE ε4 status.
Since the central reflex is a result of the reflectivity of the vessel layers, it is possible
that this change may be related to reduced blood flow and blood column width (Berisha
et al., 2007, Suo et al., 1998) or increased venous wall thickness due to deposition of
protein (Ellis et al., 1996, Jellinger, 2002, Kalaria and Pax, 1995, van Horssen et al.,
2002, Vinters et al., 1996). These results are also consistent with the Chapter 3 results
164
on thinning of retinal vessels in AD; non-significant trends for narrower arteriole width
(consistent with Chapter 3 results) and wider CR in AD resulted in a significantly
higher CRR in AD, hence both vessel thinning and changed vessel structure and blood
flow are likely to be contributing to the reported effect.
In summary, this thesis has addressed the hypotheses detailed in section 1.5.1, finding
several retinal abnormalities that are associated with AD pathogenesis, and
demonstrating that these abnormalities can be detected with non-invasive retinal
photography and semi-automated computerized image analysis. These findings have
provided interesting discussion about the early detection of AD using retinal markers,
and differentiation between AD and vascular dementia. The findings have also been
discussed in relation to the amyloid cascade hypothesis and the possible role of Aβ in
retinal degeneration in AD, as well as vascular changes in the AD brain and retina, and
their relationship to neuro-degeneration.
Ocular changes have been reported in various CNS disorders including AD, Parkinson’s
disease, Multiple Sclerosis and Stroke. Many of these changes will not be specific to a
single disease, but their existence establishes a strong link between the brain and retina.
Additionally, some ocular changes precede the symptomatic hallmarks of these CNS
diseases and hence could be useful for early detection or monitoring. Additionally, the
pathology of some retinal diseases overlaps substantially with both the ocular and
cerebral pathology in AD, prompting pooling of research efforts and providing the
potential of a cascade effect if successful therapeutic approaches can be found.

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8.1.2) The pupil response and Alzheimer’s disease
The findings presented in Chapters 4 and 5 demonstrate pupil flash response changes in
diagnosed and pre-clinical AD, as well as in autosomal-dominant AD (ADAD)
mutation carriers. Individuals with diagnosed AD exhibited extensive and highly
significant changes to the PFR. Individuals with pre-clinical AD exhibited some
significant changes to the PFR, consistent with the changes in diagnosed AD. The fact
that the pre-clinical AD results were not as strong as those for diagnosed AD might be
attributed to the smaller neuroimaging cohort, but it cannot be ruled out that PFR
changes may occur at a different stage of AD pathogenesis than cerebral amyloid plaque
build-up. Exactly how early the PFR changes occur relative to the rise in cortical plaque
deposition remains an open question. Significant linear relationships between PFR
parameters and i) neocortical plaque burden (SUVR) and ii) longitudinal change in
SUVR were found in this project, providing evidence that the PFR changes might occur
early, as plaque burden starts to increase. Further longitudinal, neuroimaging studies
with larger cohorts are required to confirm this.
The fact that PFR changes were detected in pre-clinical AD is encouraging for use in an
early screening test for AD, possibly in conjunction with other biomarkers. Pre-clinical
individuals with known ADAD mutations exhibited highly significant PFR changes,
somewhat related to those in sporadic AD. Recovery from the flash test was slower in
ADAD mutation carriers (MC), indicative of a ‘sluggish’ response, however recovery
was actually faster in sporadic AD despite ‘sluggish’ velocities and accelerations,
because the amplitude of the response was much smaller and hence less re-dilation was
required to attain recovery. The MC participants appeared to exhibit the ‘sluggish’
response without the reduced amplitude. While the pupil velocity and acceleration
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parameters showed trends to be lower in MC participants, only the recovery parameters
were significantly slower, providing perfect separation of the groups.
Studies of families harboring known ADAD mutations provide a powerful opportunity
to investigate the temporal sequence of ocular and other AD biomarker changes during
disease progression. Studying pre-symptomatic individuals with ADAD mutations
alleviates many problems inherent in studies of pre-symptomatic sporadic AD,
including uncertainty about disease progression. The PFR differences in ADAD MC
were thus investigated with respect to their age relative to their expected age of disease
onset. As the age at onset (AAO) due to a familial mutation will be very similar for
individuals with the same mutation, this analysis enabled the PFR biomarker differences
to be evaluated with respect to disease progress. However, no trends were found
between the PFR parameters and age relative to disease onset. This could suggest that
PFR changes occur early and then plateau over the age range of the cohort. Larger,
preferably longitudinal studies will be required to investigate this possibility.
The APPGlu693Gln mutation is associated with cerebral amyloid angiopathy (CAA) as
well as cerebral plaques. Neither dementia nor cognitive decline has been observed in
this family, possibly due to earlier onset of CAA and fatal cerebral hemorrhages. The
PFR changes in MC might be related to early AD pathogenesis, or alternatively CAA
might be impacting on the PFR due to damage to pupil-control relays in the midbrain.
PiB-PET imaging results demonstrated that the MC had higher neocortical plaque
burden (SUVR) than non-carriers, although no participants had high plaque burden
(SUVR > 1.5). Research suggests that neocortical and amygdaloid functional changes of
the cholinergic system are an early and leading event in AD, rather than the
consequence of neurodegeneration (Herholz et al., 2004, Tohgi et al., 1994). Hence the
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higher SUVR in MC could be indicative of cholinergic dysfunction associated with
early AD pathology, resulting in the PFR changes.
Similarly, possible causes of the PFR changes in sporadic AD are degeneration in relays
in the midbrain, or central cholinergic depletion (Herholz et al., 2004, Tohgi et al.,
1994). The five PFR parameters that demonstrated the most significant differences in
sporadic AD (VC, VCmax, ACmax, Amp and %Cons.), are calculated from the
constriction phase of the PFR which is primarily a parasympathetic cholinergic response
(Loewenfeld, 1999). Hence these parameters are likely to be the most sensitive markers
of cholinergic deficits in the peripheral parasympathetic pathway. Reported results from
other studies indicate that Donepezil, which is an anticholinesterase agent used to treat
AD, may normalise PFR in some AD patients (Fotiou et al., 2000, Granholm et al.,
2003). If PFR changes in AD relate to neurotransmitter status, then PFR testing may be
useful as an objective, non-invasive monitor with which to follow disease progression
and treatment efficacy.
In summary, this thesis has addressed the hypotheses detailed in section 1.5.2, finding
several PFR abnormalities that are associated with AD pathogenesis, and demonstrating
that these abnormalities can be detected using a commercial pupillometer. As changes
to pupil response have now been reported in both sporadic AD and APP mutation
carriers, PFR deserves further investigation as a useful adjunct, possibly in conjunction
with blood biomarkers, to assist the diagnosis of early AD. PFR may also be useful in
monitoring CAA or AD for management of risk factors and treatment. Natural variation
in pupillary response between individuals may curtail the utility of PFR testing for
detection of high plaque burden in asymptomatic individuals. However, if longitudinal
changes in pupillary response are related to longitudinal changes in SUVR or
conversion to AD, then PFR testing will be useful as a non-invasive monitor to follow
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disease progression and the response to treatments. Relationships demonstrated in this
study between PFR parameters and SUVR (or rate of change of SUVR) indicate a
possible role for PFR testing as an adjunct, together with clinical assessments, for
monitoring of disease progress and response to treatment. However, future longitudinal
studies will be needed to further evaluate this possibility.
8.2) Project Limitations and Suggestions for Future Research
While this thesis has reported some novel associations between ocular biomarkers and
AD, the findings should be interpreted in the context of certain limitations. Firstly, the
number of AD participants in the studies is quite small (n=20-25), and although
building on previous results with even smaller numbers, e.g. n=9 (Berisha et al., 2007),
n=10 (Fotiou et al., 2000), the results should be verified in larger population based and
preferably longitudinal (prospective or retrospective cohort) studies. This also holds for
the neuroimaging cohorts, which are highly important in terms of identifying early, pre-
symptomatic AD. However the cost and availability of neuroimaging services constitute
a significant barrier to this goal. Interestingly, even such a small number of participants
provided highly significant results indicating that assessing the eye- related pathological
changes may potentially be an accurate and cost-effective way for screening, diagnosis
and participant selection in preventive and ameliorative interventions.
The major strength of the study is the well characterised cohorts and multi-disciplinary
investigation, including neuro-imaging data that enable deeper interrogation of
associations between retinal vascular parameters and AD. Unfortunately, the expense of
PET neuroimaging is high, but a few longitudinal studies worldwide are performing
169
PiB-PET imaging in large cohorts. The findings of this thesis should be further explored
in other studies, to establish if the results apply to other populations.
These findings add to the growing evidence that ocular changes occur in AD. They also
demonstrate that these changes can be detected using non-invasive, readily available
retinal photography and a commercial pupillometer. Models combining ocular
parameters perform well at distinguishing diagnosed AD patients from healthy controls.
However these models are optimised for the present data set and should be tested on
other cohorts in future.
The retinal abnormalities identified in AD in this study, and the overlap between AD
and retinal disease, encourage further research into the possibility that retinal Aβ
plaques occur in these diseases. Some human and animal model evidence for retinal Aβ
plaques in AD has already been uncovered (Koronyo-Hamaoui et al., 2010, Liu et al.,
2009, Perez et al., 2009). If Aβ plaques occur in both the brain and retina in pre-clinical
AD, then the retina could provide a more practical location for screening and
monitoring of the disease. Deposition of Aβ in the ocular lens has also been reported in
AD (Goldstein et al., 2006, Goldstein et al., 2003), and should be considered further in
the investigation of ocular Aβ pathology in AD. Additionally, further investigation of
the role of Aβ in retinal diseases may provide insight into the relationships between
these diseases and AD, and potentially have a cascade effect on therapeutic and
monitoring approaches across the diseases.
Pre-symptomatic individuals with high plaque burden were predominantly carriers of
the APOE ε4 allele, particularly in the case of the pupil markers for sporadic AD study,
where significant PFR differences in pre-clinical AD did not contribute to any
improvement in classification performance above that provided already by age and

170
APOE ε4 status. For the retina study, there were also significantly more APOE ε4
carriers in the HC+ group compared to the HC- group. As carriers of the APOE ε4 allele
are more likely to develop AD, it is not unexpected that the HC+ group has more
carriers, although from a statistical standpoint it would be more ideal to have included
more HC+ non-carriers. While these participants were not available for the present
study, the AIBL study is presently increasing the neuroimaging cohort in Perth to
facilitate larger and better stratified studies in future.
The PFR changes found in AD may relate to cholinergic deficit. Therefore PFR testing
may be useful as an objective, non-invasive monitor with which to follow disease
progression and treatment efficacy. To investigate this, future longitudinal studies could
follow newly diagnosed AD patients for PFR changes. Hypotheses that could be
investigated include whether PFR testing can predict which patients will benefit most
from anticholinesterase treatment, and whether PFR normalization during
anticholinesterase treatment associates with clinical improvement.
Cholinergic depletion, occurring in the AD brain and possibly underlying the PFR
differences in AD, may also occur in other diseases such as Parkinson’s Disease
(Dubois et al., 1990), which has also been reported to influence the PFR (Fotiou et al.,
2009, Granholm et al., 2003). For these reasons, the specificity of PFR testing for AD
needs further investigation, utilising cohorts of participants with AD, Parkinson’s
Disease and Parkinsonism, as well as healthy controls.
Although the APOE ε4 allele is an established risk factor for AD, it was not found to be
associated with any ocular markers, except for central reflex ratio. In addition, for the
full PFR cohort of 89 participants, no significant association with APOE ε4 status was
found for any PFR parameter, supporting the hypothesis that the PFR changes are
171
related to high plaque burden rather than APOE ε4 status. For future studies it may be
important to stratify groups to ensure a sufficient representation of APOE ε4 carriers
and non-carriers, in order to confirm that ocular changes are related to AD rather than a
surrogate for APOE genotype.
Many studies of this nature are limited in their treatment of confounders as they resort
to participant self-reporting of conditions such as cardiovascular problems. The AIBL
study however has collected a vast database including medications, medical history and
test results on related measures such as cholesterol levels and inflammatory markers. In
this study, diagnosed hypertension and high blood pressure were both considered in
confounder adjustments to reduce the likelihood of undiagnosed hypertension impacting
on results. However, it remains possible that undiagnosed, early stage diseases such as
atherosclerosis for example may have been present and may affect the retinal
vasculature.
The EOFAD study described in Chapter 5, while powerful due to the strong genetic
component of the disease and young age of the participants, was limited in participant
numbers due to the rarity of this condition. However the DIAN study has now expanded
its Perth and worldwide cohorts, including many ADAD mutations, providing a basis
for larger future studies into the pupil response changes reported in this thesis.
Natural variation in ocular biomarkers between individuals may limit the utility of an
ocular screening test for AD, hence it is possible that ocular monitoring, allowing
longitudinal analysis of ocular changes, might facilitate more accurate pre-clinical
detection or monitoring of AD. Future longitudinal studies are planned to further
explore this possibility and to determine the time-course of ocular changes in AD.
172
Ocular morphologies reported in AD give hope for a non-invasive, cost-effective
screening test for AD. Evidence is accumulating in support of AD-related changes in the
eye, but finding a sufficiently sensitive and specific ocular biomarker is proving to be a
major challenge. Many reported ocular changes in AD also occur in other disorders.
Optic disc changes, visual field defects and RNFL/retinal cell loss are also observed in
the eye disease glaucoma. Retinal vessel width are influenced by age and race and can
be altered in many disorders. Similarly, pupil responses are influenced by age and eye
color and are altered in many neurological disorders. Also, most studies into ocular
morphology in AD have been limited by small participant numbers, few study groups
and little relevant medical information on participants. Hence larger studies are required
to confirm and investigate further these ocular changes in AD.
It looks likely that multiple biomarkers will have to be combined in a screening and
diagnostic approach for AD. A tiered approach is appropriate, with low-cost, minimally
invasive tests such as retinal photography, pupillometry and blood testing (Watt et al.,
2011) identifying high risk individuals who can then be forwarded for more extensive
testing (PET, MRI, CSF). Multiple modalities are likely to provide higher sensitivity
and specificity for early AD detection, diagnosis and monitoring.

173
8.3) Concluding remarks
The devastating impact of AD, both on those directly affected and on society in general,
creates a pressing need for better treatments. By the time a person is diagnosed with
“probable AD” using current techniques, significant irreversible neuronal degeneration
has already occurred. Therefore, research into better treatments must be paralleled by
research into technologies to screen populations for AD, to identify cases before
cognitive symptoms arise.
Because of the protracted duration of the pre-clinical phase of AD, CSF, blood and
imaging biomarkers associated with AD pathology are being sought in order to develop
a population screening test to identify individuals with pre-clinical AD. A screening test
for AD would allow emerging disease-modifying preventative and interventional
therapies the best chance to preserve normal brain function rather than the current role
of slowing already detectable decline. Thus the AD biomarker field is challenged with
developing prognostic measures that will predict which cognitively normal individuals
will develop AD as well as pathological markers that permit monitoring of disease
progress and response to treatment.
Plaque burden is an indicator of deregulated Aβ proteostasis. The brain amyloid load is
known to increase many years prior to AD diagnosis (Fagan et al., 2006, Rowe et al.,
2010, Rowe et al., 2007). Neocortical Aβ plaques have also been found to produce
aberrant neuronal activity in their vicinity in both animal models (Busche et al., 2008,
Grienberger et al., 2012, Palop et al., 2007) and pre-symptomatic patients (Sperling et
al., 2009). For these reasons, high or increasing SUVR is a strong sign of pre-clinical
AD, and hence the in vivo gold-standard against which this study has evaluated ocular
biomarkers. Therapy aimed at slowing or halting the disease progress must be

174
administered as early as possible, before these discrete alterations in neuronal function
progress to symptomatic AD.
The transparency of the ocular media combined with the fact that the retina is
developmentally and anatomically an extension of the brain, makes retinal photography
ideal for the detection of systemic diseases that affect the microcirculation. Screening
and monitoring for age-related diseases is growing in importance because of increases
in life expectancy. Retinal photography has become an invaluable tool for prognostic
assessment for systemic diseases such as diabetes and hypertension, and may become
similarly useful for neurodegenerative diseases such as AD. Similarly, pupillometry has
the potential to probe a neurotransmitter deficiency that is fundamental to AD, thus
providing a modality for non-invasive, early detection and monitoring of disease
progress and response to treatment.
An ocular screening test for AD would benefit AD sufferers and researchers and
possibly provide new insight into the molecular processes and genetic determinants of
the disease. An ocular biomarker or biomarkers could turn out to be highly specific for
AD, or to be a useful component in a multidisciplinary approach aimed at producing an
earlier and more accurate diagnosis of Alzheimer’s disease.

175
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Appendices
Appendix 1.1. Consent Forms and Information Sheet

207
INFORMATION SHEET FOR PARTICIPANTS
Title of Study: Retinal and Anterior Segment Features as Predictive Markers for
Alzheimer’s Disease and Dementia
Investigators: Professor Ralph Martins and Mr. Shaun Frost
INTRODUCTION
The following information describes a clinical research study and your role in it should
you decide to participate. Please read this carefully and do not hesitate to ask any
questions now or anytime during the study. You may wish to have a family member
read this information sheet with you. Your participation in this study is entirely
voluntary. If you decide to participate in the study you will receive a signed copy of this
Patient Information Sheet and Consent Form for your records.
NATURE AND PURPOSE OF THIS STUDY
This study involves research into changes in the eye that might accompany cognitive
decline or the early stages of dementia. It is hoped that the results might lead to a simple
eye-test that could predict the development of different types of dementia. Participants
with memory problems are being recruited as well as healthy participants. This is a
student PhD project run by Mr. Shaun Frost under the supervision of Professor Ralph
Martins.
208
STUDY PROCEDURES
The study involves a small questionnaire and some simple tests on both eyes of each
participant. Some photographs and scans will be taken of the surface and interior of
each eye, similar to those taken when patients visit an eye specialist. A measurement of
eye-pressure will be made and some vision tests performed. All procedures are non-
invasive (no instrument or fluid will be inserted onto or into your eye). The testing is
expected to take approximately 15 minutes for each participant. The study will involve a
total of approximately 450 participants.
POTENTIAL BENEFITS TO THE COMMUNITY
An eye-test for early detection of Alzheimer’s disease or other dementia’s would allow
intervention before irreversible brain damage has occurred. This could greatly improve
the prognosis of the sufferer. Such an eye-test would also benefit other research into
treatments and cures for dementia.
POTENTIAL BEBEFITS TO THE PARTICIPANT
Retinal and anterior images will be reviewed by an ophthalmologist for signs of ocular
disease and participants will be contacted by mail if further medical investigation is
recommended. However, this study should not replace more thorough clinical eye
testing at a specialist eye centre.
RISKS AND DISCOMFORTS
There may be a slight discomfort experienced from the white flash delivered for each
picture or the air-puff used for measuring eye-pressure.

209
CONFIDENTIALITY AND WITHDRAWAL FROM STUDY
Your records will be kept totally confidential and any publication of results will not
include your name. Your questionnaire answers and your cognitive and medical test
results from the other research study will be used to analyse the results of this eye-study.
Your participation in this study is entirely voluntary and the records can be destroyed at
any time if you change your mind.
MEDICAL CARE / COMPENSATION
Your participation in this study does not prejudice any right to compensation, which
you may have under statute or common law. If any adverse event or injury occurs due to
participation in this study, you will be appropriately compensated under AMACIS’s
Indemnity and Public Liability coverage held by the McCusker Foundation for
Alzheimer’s Disease Research.
INVESTIGATOR CONTACT DETAILS
If you require further information or if you have any problems concerning this project
please contact the principal researcher, Mr. Shaun Frost:
shaun.frost@csiro.au Ph. (08) 9333 6137
ETHICAL APPROVAL
The University of Western Australia Human Research Ethics Committee has given
approval for this study. If you have any concerns about this study, they may be
addressed to the researcher or, alternatively, to the Secretary, Human Research Ethics
Committee, Registrar’s Office, University of Western Australia, 35 Stirling Highway,
Crawley, WA 6009 (telephone number 6488 1610).

210
Consent to Ophthalmological Testing
Study Title: Retinal and Anterior Segment Features as Predictive Markers
for Alzheimer’s Disease and Dementia
INVESTIGATORS: Professor Ralph Martins, PhD
Mr. Shaun Frost, BSc Hons, MSc
Name of the Participant: ____________________________________
I (the participant) have read or have had read to me the information concerning this
study and any questions I have asked have been answered to my satisfaction. I agree to
participate in this activity, realising that I may withdraw at any time without reason and
without prejudice.
I understand that all information provided is treated as strictly confidential and will not
be released by the investigator unless required to by law.
I understand that this research is being conducted to enable screening of populations for
dementia and cognitive decline. I agree to the use of my cognitive and medical test
results for this study. I agree that research data gathered for the study may be published
provided my name or other identifying information is not used.
…………….. ………………………………………. ………………
Date Name of Participant / Legal Guardian Signature
…………….. ………………………………………. ………………
Date Name of Researcher Signature
The Human Research Ethics Committee at the University of Western Australia requires that
all participants are informed that, if they have any complaint regarding the manner in which
a research project is conducted, it may be given to the researcher or, alternatively to the
Secretary, Human Research Ethics Committee, Registrar’s Office, University of Western
Australia, 35 Stirling Highway, Crawley, WA 6009 (telephone number 6488 1610). All
study participants will be provided with a copy of the Information Sheet and Consent Form
for their personal records.
211
Participant Questionnaire
To be completed by the Participant of the study:
Name of the Participant: ____________________________________
Have you ever had any of the following medical conditions?
Diabetes? Yes [ ] No [ ] Unsure [ ]
If yes, please specify type 1/2

_______________
Stroke? Yes [ ] No [ ] Unsure [ ]
High Blood Pressure? Yes [ ] No [ ] Unsure [ ]
Low Blood Pressure? Yes [ ] No [ ] Unsure [ ]
Glaucoma? Yes [ ] No [ ] Unsure [ ]
Cataract? Yes [ ] No [ ] Unsure [ ]
Other Eye Disease? Yes [ ] No [ ] Unsure [ ]
If yes, please specify _________________

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Ocular Biomarkers For Early Detection

and Monitoring of Alzheimer’s Disease

B.Sc. (Hons), M.Sc.

Supervisors: Professor Ralph N. Martins1,2

Professor Yogesan Kanagasingam3,4

Dr. Hamid R. Sohrabi1,2

This thesis is presented for the degree of Doctor of Philosophy

The University of Western Australia

School of Psychiatry and Clinical Neurosciences

Early detection of Alzheimer’s disease (AD) is essential for intervention as subclinical

dependent on the cholinergic system which is impaired in AD.

participants in the Dominantly Inherited Alzheimer Network (DIAN) study.

widely available retinal photography technique and hence is pertinent to the

development of a population screening test for AD.

The results demonstrate relationships between retinal vascular abnormalities,

accepted for publication in a number of peer-reviewed journal articles.

related macular degeneration and investigates signs of retinopathy in AD. Advances in

treatments to be more clinically effective, in addition to accelerating the development of

of discrete quantized bundles of energy (later called photons) (1879-1955).

This is to certify that:

(i) This thesis comprises my original work.

section to all other materials used and to the assistance of others.

illustrative matter and appendices.

B.Sc. (Hons), M.Sc.

28th August 2013

Publications Arising from this Thesis

1) Frost S, Martins RN, Kanagasingam Y (2010) Ocular biomarkers for early

detection of Alzheimer's disease. J Alzheimers Dis 22, 1-16.

2) Book Chapter “Retinal Screening for Early Detection of Alzheimer’s Disease”

Frost, Shaun; Martins, Ralph; Kanagasingam, Yogesan. In “Digital Teleretinal

Screening”, Kanagasingam, Yogesan; Goldschmidt, Leonard; Cuadros, Jorge

(Eds.), Springer 2012, ISBN 978-3-642-25809-1

of Alzheimer’s Disease” Translational Psychiatry 3, e233, February 2013.

Disease”, Current Alzheimer Research, in press, accepted 24th July 2013.

5) Frost S et al. “Pupil flash response biomarkers distinguish amyloid precursor

protein mutation carriers from non-carriers”, Current Alzheimer Research, in

press, accepted 30th April 2013.

Manuscripts Under Preparation

Frost S et al. “Alzheimer’s Disease and Age Related Macular Degeneration”

Frost S et al. “Alzheimer’s Disease and Glaucoma”

Frost S et al. “Retinal Central Reflex and Apolipoprotein E genotype”

Dedication ....................................................................................................................... III

Quotes ............................................................................................................................. III

Publications Arising from this Thesis .............................................................................. V

Table of Contents .......................................................................................................... VII

List of Figures .................................................................................................................XI

List of Tables................................................................................................................ XIII

Chapter 1: Alzheimer’s Disease and Ocular Biomarkers: A Literature Review ........ 1

1.1) Alzheimer’s Disease.......................................................................................... 1

Chapter 2: Methodological Approaches ................................................................... 42

2.1) Introduction ..................................................................................................... 42

2.6) Pupillometry .................................................................................................... 55

Chapter 3: Retinal Vasculature in Clinically Diagnosed and Pre-Clinical Sporadic

3.1) Introduction ..................................................................................................... 60

Chapter 4: Pupil Flash Response in Clinically Diagnosed and Pre-Clinical Sporadic

4.1) Introduction ..................................................................................................... 76

5.1) Introduction ..................................................................................................... 98

Chapter 6: Alzheimer’s Disease, Glaucoma and AMD .......................................... 109

6.1) Introduction ................................................................................................... 109

6.3.1) Introduction ........................................................................................... 122

Chapter 7: Alzheimer’s Disease and Retinopathy .................................................. 134

7.1) Introduction ................................................................................................... 134

Chapter 8: General discussion and future directions .............................................. 157

8.1) General discussion ........................................................................................ 157