Clinical Therapeutics/Volume 43, Number 9, 2021

Review Articles
Novel and Emerging Electrophysiological Biomarkers
of Diabetic Neuropathy and Painful Diabetic
Neuropathy
Anne Marshall, BSc1,2 ; Uazman Alam, PhD2,3 ; Andreas Themistocleous, PhD4,5 ;
Nigel Calcutt, PhD6 ; and Andrew Marshall1,7,8
1
Musculoskeletal Biology, Institute of Life Course and Medical Sciences, University of
Liverpool, Liverpool, United Kingdom; 2 Division of Diabetes, Endocrinology and
Gastroenterology, School of Medical Sciences, Faculty of Biology, Medicine and Health,
University of Manchester, Manchester, United Kingdom; 3 Cardiovascular and Metabolic
Medicine, Institute of Life Course and Medical Sciences, University of Liverpool, Liverpool,
United Kingdom; 4 Nuffield Department of Clinical Neurosciences, University of Oxford,
Oxford, United Kingdom; 5 Brain Function Research Group, School of Physiology, Faculty of
Health Sciences, University of the Witwatersrand, Johannesburg, South Africa; 6 Department
of Pathology, University of California, San Diego, La Jolla, California; 7 Department of
Clinical Neurophysiology, The Walton Centre, Liverpool, United Kingdom; and 8 Division of
Neuroscience and Experimental Psychology, School of Biological Sciences, Faculty of Biology,
Medicine and Health, University of Manchester, Manchester, United Kingdom
ABSTRACT neurophysiological methods with potential to act as
Purpose: Diabetic peripheral neuropathy (DPN) is biomarkers for the diagnosis and monitoring of DPN
the most common complication of diabetes. Small as well as putative future roles as predictors of response
and large peripheral nerve fibers can be involved in to antineuropathic pain medication in pDPN. Nerve
DPN. Large nerve fiber damage causes paresthesia, conduction studies only detect large fiber damage and
sensory loss, and muscle weakness, and small nerve do not capture pathology or dysfunction of small fibers.
fiber damage is associated with pain, anesthesia, foot Because small nerve fiber damage is prominent in DPN,
ulcer, and autonomic symptoms. Treatments for DPN additional biomarkers of small nerve fiber function
and painful DPN (pDPN) pose considerable challenges are needed. Activation of peripheral nociceptor fibers
due to the lack of effective therapies. To meet these using laser, heat, or targeted electrical stimuli can
challenges, there is a major need to develop biomarkers generate pain-related evoked potentials, which are
that can reliably diagnose and monitor progression of an objective neurophysiological measure of damage
nerve damage and, for pDPN, facilitate personalized along the small fiber pathways. Assessment of nerve
treatment based on underlying pain mechanisms. excitability, which provides a surrogate of axonal
Methods: This study involved a comprehensive properties, may detect alterations in function before
literature review, incorporating article searches in abnormalities are detected by nerve conduction studies.
electronic databases (Google Scholar, PubMed, and Microneurography and rate-dependent depression of
OVID) and reference lists of relevant articles with the the Hoffmann-reflex can be used to dissect underlying
authors’ substantial expertise in DPN. This review pain-generating mechanisms arising from the periphery
considered seminal and novel research and summarizes
emerging biomarkers of DPN and pDPN that are based
Accepted for publication March 27, 2021
on neurophysiological methods. https://doi.org/10.1016/j.clinthera.2021.03.020
Findings: From the evidence gathered from 145 0149-2918/$ - see front matter
papers, this submission describes emerging clinical © 2021 Elsevier Inc.

September 2021 1441

 Clinical Therapeutics
and spinal cord, respectively. Their role in informing
mechanistic-based treatment of pDPN as well as
facilitating clinical trials design is discussed.
Implications: The neurophysiological methods dis-
cussed, although currently not practical for use in
busy outpatient settings, detect small fiber and early
large fiber damage in DPN as well as disclosing
dominant pain mechanisms in pDPN. They are suited
as diagnostic and predictive biomarkers as well as end
points in mechanistic clinical trials of DPN and pDPN.
(Clin Ther. 2021;43:1441–1456.) © 2021 Elsevier Inc.
INTRODUCTION
Peripheral neuropathy is the most common compli-
cation of both type 1 (T1DM) and type 2 diabetes
(T2DM), with more than one half of all patients
developing nerve dysfunction in their lifetime.1 Both
chronic and acute diabetic neuropathies are seen, but
distal length-dependent symmetrical polyneuropathy is
the most common and generally referred to as diabetic
peripheral neuropathy (DPN). DPN is the primary
cause of diabetic foot disease, including ulceration
and nontraumatic amputations.2 In addition, up to
one third of patients with diabetes have neuropathic
pain (painful diabetic neuropathy (pDPN),1 ,3–5 which
often leads to sleep disturbance, poor quality of
life, anxiety/depression, and unemployment.6–9 Data
suggest that 10-year mortality is higher in patients with
severe chronic pain.10
Currently, there are no US Food and Drug
Administration–approved disease-modifying therapies
for diabetic neuropathy. Many clinical trials of drugs
targeting presumptive mechanisms of DPN, including
aldose reductase inhibitors, protein kinase C inhibitors,
and benfotiamine, have failed at Phase III of devel-
opment. Potential reasons for these failures include
the use of subjective or insensitive end points such as
physician-based clinical scores and use of biomarkers
that only report function of large myelinated nerve
fibers.11 ,12 Consequently, there is a lack of disease-
modifying medication. Beyond glycemic control and
modulation of cardiometabolic risk factors, the only
option is pain management. Unfortunately, pharmaco-
logic treatments for pDPN are inconsistently effective.
In patients with pDPN treated with monotherapy,
50% pain relief in 50% of cases is considered a
favorable outcome.13 Treatment strategies typically
involve a trial-and-error approach of prescribing anti-
neuropathic pain medications, which have inconsistent
1442
therapeutic benefit.14 There is a major unmet need
for the development of reliable biomarkers that: (1)
capture the onset and progression of DPN; (2) inform
mechanistic-based drug discovery; and (3) facilitate
individualized treatment of neuropathic pain in pDPN.
The purpose of the present narrative review was
to discuss emerging neurophysiological biomarkers
of human DPN and pDPN. Clinical neurophysiology
is a medical specialty in which the recording of
spontaneous or evoked bioelectrical activity is used to
investigate function of the brain, spinal cord, spinal
nerve roots, peripheral nerves, and muscle. Clini-
cal neurophysiology departments may also perform
quantitative sensory testing (QST), such as thermal
threshold analysis, but this review is restricted to
electrophysiological investigations according to the
definition noted (details about QST are given else-
where15–17 ). Investigations of the autonomic nervous
system, including cardiac autonomic testing and sweat
responses, are also not discussed (reviews are presented
elsewhere18–20 ).
MATERIALS AND METHODS
A comprehensive literature review was undertaken, in-
cluding article searches in multiple electronic databases
(Google Scholar, PubMed, and OVID) using key
words (eg, diabetic neuropathy, neuropathic pain, pain
biomarkers) and reference lists of relevant articles
with the authors’ substantial expertise in DPN. This
review considered seminal and novel research and
summarizes emerging biomarkers of DPN and pDPN
that are based on neurophysiological methods. Articles
published from inception of databases to December
2020 were identified. Data from articles that were not
relevant to the aim of the study were excluded from the
review.
RESULTS
In total, 145 papers were cited in the final manuscript.
An evaluation of selected articles was undertaken, and
relevant data were included in the current review.
Authors excluded studies that were not relevant to the
aims and ethos of the manuscript under the guidance
of the senior author (A.M.).
A biomarker is defined by the National Institutes of
Health as “a characteristic that is objectively measured
and evaluated as an indicator of normal biological
processes, pathogenic processes, or pharmacological
responses to a therapeutic intervention.”21 According
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to this definition, biomarkers can have a wide range
of complexity ranging from very simple clinical tests
(eg, deep tendon reflex) or noninvasive diagnostic
investigations (eg, heart rate variability to detect
cardiac autonomic neuropathy) through to complex
laboratory or invasive evaluation (eg, skin biopsy,
microneurography).
The US Food and Drug Administration22 and
European Medicines Agency23 define several categories
of biomarkers, including susceptibility/risk, diagnostic,
prognostic, predictive, enrichment, monitoring, and
surrogate end points. The precise characteristics of
an ideal biomarker depend on its context of use24
but in general terms they include the following: (1)
being scalable and easy to use (eg, be simple, cost-
effective, minimally invasive); (2) have high sensitivity
(ie, high true-positive rate) and specificity (ie, high true-
negative rate); and (3) have a high predictability (ie, a
strong indicator of the condition or of a response to a
particular treatment).
For DPN, the major purposes of biomarkers are to:
(1) act as a diagnostic biomarker to identify individuals
with the biologically defined disorder (eg, large and/or
small fiber DPN); (2) monitor the change in degree of
the disorder over time; and (3) act as an indicator of
drug efficacy in clinical trials. Although detection of
potentially fully reversible changes in nerve function
may theoretically be possible (as discussed in the Nerve
Excitability Testing section), in general, this requires
the detection of nerve pathology or surrogates thereof.
It is therefore important that a diagnostic biomarker
can reliably and reproducibly detect the earliest signs
or the earliest changes of the severity/extent of DPN.
The earliest clinical manifestations of DPN are
typically sensory symptoms or signs in the extremities.
This sensory predominant phenotype is associated with
pathologic alterations in all major classes of sensory
fibers: large myelinated fibers, as well as small thinly
myelinated A-delta and unmyelinated C-fibers.25–29
Therefore, DPN is a mixed large fiber neuropathy
and small fiber neuropathy (SFN). Because small and
large fiber deficits are detected by using different
methodologies, there are distinct biomarkers of small
and of large fiber neuropathy. Biomarkers of somatic
SFN include skin biopsy for quantification of intra-
epidermal nerve fiber density (IENFD) and corneal con-
focal microscopy (CCM) with quantification of corneal
innervation, both of which provide information on
small fiber structure, whereas QST of thermal or pain
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A. Marshall et al.
perception and laser evoked potentials are tests of
small fiber function. The current preferred biomarkers
of large fiber neuropathy are nerve conduction
studies (NCS) and quantitative assessment of vibration
perception.
The current prevailing view is that small fiber
deficits can be detected before objective large fiber
involvement. This is primarily based on cross-sectional
studies of patients with diabetes or prediabetes that
show small fiber abnormalities in a proportion of
patients with normal NCS and in the large majority
of those with abnormal NCS parameters.30–36 Fur-
thermore, normalization of hyperglycemia (diabetes
remission) or intensive treatment of hyperglycemia
with continuous subcutaneous insulin are associated
with early improvements in measures of small, but
not large, fiber neuropathy in severe DPN.37–40 For
example, in severe DPN, although motor nerve
conduction velocity did eventually increase 36 months
after simultaneous pancreas and kidney transplant,
this lagged behind improvements in SFN detected by
using CCM.37 These findings indicate that biomarkers
of SFN are, in general, more sensitive at detecting
early neuropathy or the earliest signs of repair in
DPN. However, in mild to moderate DPN, short-
term modest improvements in glycemic control and
serum triglyceride levels had an independent, additive,
and durable effect on restoration of nerve function
with improvement in lower limb electrophysiological
parameters.41
Relatively large cross-sectional studies of patients
with DPN show that abnormalities of small and large
fibers frequently co-exist.42 ,43 For example, Ziegler
et al,42 in a study of recently diagnosed T2DM found
that although CCM and IENFD detected small nerve
fiber loss, there was a reduction in NCS parameters
in a subpopulation suggestive of large fiber disease.
Furthermore, a recent study of 133 patients with
T2DM and DPN participants were subclassified into
large, small, or mixed neuropathy on the basis of
NCS and IENFD findings.43 The majority (74%) had
evidence of mixed large and small fiber involvement,
although a significant minority had evidence of isolated
large or small fiber DPN. Other studies that stratify
patients based on clinical scales show that NCS and
measures of SFN such as QST, CCM, and IENFD
all progressively decline with increasing severity of
clinical neuropathy.44–46 Although these studies imply
that small and large fiber pathology develop in parallel,
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 Clinical Therapeutics
longitudinal investigations are needed before inferences
can be made regarding the natural history of peripheral
nerve pathology. A recent multicenter longitudinal
study of DPN which assessed large and small fibers
showed that more rapid deterioration in SFN (loss
of ≥6% of nerve fiber length/year detected by using
CCM) is associated with a greater deterioration of
NCS parameters (peroneal motor conduction velocity
and amplitude).47 However, the converse (ie, whether
greater deterioration in NCS was associated with more
rapid small fiber abnormalities) was not addressed.
Therefore, the concomitant use of biomarkers of both
large fiber neuropathy and SFN for DPN represents an
appropriate strategy.
Recent data suggest a greater degree of SFN
(detected by using IENFD and CCM) in patients with
pDPN compared with those with equivalent large fiber
neuropathy but no pain.45 ,48 ,49 However, the findings
in the literature are mixed. For example, other recent
large-scale cross-sectional studies report no difference
in structural or functional small fiber deficits between
painless DPN and pDPN50 or equivalent prevalence
of neuropathic pain in patients with evidence of
small fiber, large fiber, or mixed small and large fiber
polyneuropathy.43 Whether interventions that prevent
or reverse small fiber loss in diabetes can affect the pain
phenotype remains to be determined.
The role of biomarkers of pDPN is somewhat
different from that for DPN. A biomarker of pain per
se is not particularly useful because this information
can be detected, and indeed quantified, by asking
the patient directly. A more useful biomarker would
detect a particular pain phenotype or underlying
pathophysiological mechanism that can be used
to predict an individual’s benefit from particular
treatments, to enrich patient populations for clinical
trials or, potentially, to predict the risk of developing
pain.
NERVE CONDUCTION STUDIES: A GOLD
STANDARD BIOMARKER WITH LIMITATIONS
NCS have long been used as diagnostic, staging, and
prognostic biomarkers for DPN. The investigation
involves supramaximal transcutaneous stimulation of
defined upper and lower limb peripheral nerves.
Recordings are also taken noninvasively with surface
electrodes. Motor and sensory fiber function can
be assessed separately. Measurement of response
amplitude serves as a correlate of axonal loss.
1444
Conduction velocity, which is measured by using
single and 2-site stimulation for sensory and motor
nerves, respectively, is affected by several factors such
as axonal atrophy, internodal distance, and nodal
membrane functions, although it is typically regarded
as a biomarker of demyelination. NCS are routine
procedures and, in skilled hands, provide objective data
that can be compared with local normative ranges.51
Although NCS are widely available, they are most
often used for the assessment of atypical clinical
presentations. A number of point-of-care NCS devices
that may facilitate screening of large fiber neuropathy
in the clinic setting are available. One such device,
DPNCheck (NeuroMetrix, Waltham, Massachusetts),
which measures sural nerve sensory nerve action
potential amplitude and velocity, has been validated
for use in DPN.52 In controlled settings performed
by trained nontechnical individuals, it can be rapidly
performed (<5 minutes) and shows 95% sensitivity
and 71% specificity for DPN detection compared
with conventional NCS53 and 84% sensitivity and
68% specificity compared with composite clinical
scores.53 ,54
NCS have been historically considered a gold
standard for the diagnosis of DPN. Confirmed DPN
is defined as clinical symptom(s) and/or sign(s) of
neuropathy and an abnormal nerve conduction or
adequate small fiber measure if NCS are normal,
as classified by using the updated Toronto consen-
sus guidelines.55 This definition is often used as
an inclusion criterion and/or end point in clinical
trials. NCS findings correlate well with clinical56
and pathologic/morphologic57 measures of severity
of neuropathy. Furthermore, they have prognostic
significance: slowing of motor nerve conduction
velocity is predictive of foot ulceration and death in
DPN.58 However, NCS have significant limitations.
Neurophysiology departments often have normative
reference ranges based on healthy individuals, but
performance standards for NCS are not uniform.
Reference ranges ideally should be standardized
for methodology, age, and for sensory conduction
especially (because of lower signal-to-noise ratio)
and potentially other technical factors such as body
mass index.59 Furthermore, reference ranges based on
populations of healthy individuals can be broad. For
example, a sural sensory amplitude of ≥4 microvolts
might be considered normal by some reference ranges60
but would be highly abnormal for an individual with a
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not uncommonly encountered premorbid value of >20
microvolts. Therefore, detection of early neuropathy
may require serial studies to investigate declining
parameters originally within the “normal” range,
which will limit segregation of patients for clinical
trials based on inclusion criteria requiring “abnormal”
NCS.
The major shortcoming of NCS is that they assess
large myelinated fibers only. It is not possible to
transcutaneously record from thinly myelinated (Aδ)
fibers, which mediate nociception and cold sensation,
or unmyelinated (C) fibers, which mediate warm and
nociceptive sensation. Therefore, an insensitivity to
detecting changes in small nerve fibers, potentially
the most sensitive method for detecting DPN onset
and progression, combined with questionable repro-
ducibility and lack of measurement standardization
across centers,61 limit the use of NCS as a standalone
biomarker of DPN. There is a remarkable paucity
of studies that directly compare the ability of small
and large fiber biomarkers to detect DPN. One
reason for this is that NCS are often used as a
criterion for diagnosis of neuropathy and thus form
the gold standard against which the sensitivity and
specificity of other methods are compared. Studies that
have compared the diagnostic capability of NCS and
SFN biomarkers in DPN used relatively crude and
subjective clinical criteria as a benchmark for neu-
ropathy diagnosis and stratification.44 Nevertheless,
these studies indicate that NCS parameters such as
sural nerve conduction velocity perform with more-or-
less equivalent sensitivity and specificity as structural
and functional measures of SFN. Although NCS are
established as a biomarker of large fiber neuropathy in
DPN, large-scale studies involving detailed neuropathy
phenotyping indicate that they do not differentiate
between pDPN and painless DPN.50 ,62
EMERGING NEUROPHYSIOLOGICAL
BIOMARKERS OF DPN
Pain-related Evoked Potentials
Pain-related evoked potentials involve the recording
of cortical activity through scalp electrodes in response
to selective stimulation of nociceptive skin afferents
(Figure 1). Distinct waveforms can be obtained for
A-delta and C-fiber stimulation, but for the purposes
of clinical evaluation only assessment of A-delta
waveforms is currently believed to be reliable.63 ,64
Nociceptor stimulation is most commonly delivered
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A. Marshall et al.
by using a laser or a brief thermal stimulus, although
stimulus paradigms using pinpricks or specialized
surface electrodes have also been used.
Laser evoked potentials (LEPs) are considered to
represent the most reliable neurophysiological method
to assess nociceptor pathways.63 ,64 The cortical
waveforms generated are termed the N1 and N2/P2
complex. Attenuated, delayed, or absent LEPs to
stimulation of a pathologically involved region such
as the foot in distal symmetrical polyneuropathy can
provide objective evidence of a loss-of-function lesion
affecting the A-delta nociceptive pathways. In a group
of 45 patients with diabetes and varying degrees
of DPN, the most frequent LEP abnormalities were
attenuated or absent cortical waveforms. The degree
of LEP abnormality correlated with the degree of
NCS abnormality, suggesting that large and small
fiber dysfunction occurs in parallel.65 Using carefully
collected age- and site-related normative data, LEPS
showed 78% sensitivity and 81% specificity for
the diagnosis of SFN in patients with DPN and
normal NCS compared with the current gold standard
diagnostic, IENFD.66 Similar sensitivity and specificity
were documented in a large study of 164 patients
with clinical evidence of SFN and normal NCS;
˜20% of the patients had pDPN. LEP abnormalities
had sensitivity and specificity of 66% and 89% for
detecting SFN compared with diagnostic criteria of ≥2
abnormal results from a battery of 6 investigations
(LEP, QST, cardiac autonomic tests, electrodermal
skin conductance, quantitative sweat testing, and
IENFD).67 LEP amplitudes have also been shown to
be attenuated in patients with diabetes of >10 years’
duration and asymptomatic “pure” SFN diagnosed by
using IENFD,68 and they show greater attenuation
in those with neuropathic pain.69 Although this
suggests a greater small fiber burden in painful
neuropathy, it is important to emphasize that LEPs
primarily demonstrate a lesion affecting small (A-
delta) fiber pathways rather than providing evidence
of hyperalgesia or pain mechanisms. However, there
are some data suggesting that differential involvement
of LEPs is associated with that particular symptom of
pDPN. For example, in a recent study of 133 patients
with T2DM and DPN, subclassified into myelinated
versus SFN versus mixed on the basis of NCS and
abnormal IENFD, the presence of burning pain was
associated a greater loss of LEP amplitude, whereas
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 Clinical Therapeutics
Figure 1. Schematic showing established and emerging biomarkers of diabetic peripheral neuropathy (DPN) and
painful diabetic peripheral neuropathy (pDPN). H-Reflex = Hoffmann reflex; RDD = rate-dependent
depression.
allodynia showed a tendency for preservation of LEP
amplitudes.43
LEPs are noninvasive, relatively rapid to perform,
and are an objective neurophysiological biomarker
for SFN. Indeed, in patients with painful sensory
neuropathy, LEP amplitudes positively correlate with
IENFD.70 However, LEPs have several drawbacks as
a biomarker. The technique is expensive, not widely
available, and certain safety measures (eg, wearing of
eye protection) are required during the investigation.
LEPs also habituate, and the waveform amplitudes
are dependent on the participants’ attention.71 Because
LEPs assesses the whole neuraxis through the spinotha-
lamic tracts, the method does not distinguish between
central and peripheral nervous system pathology.
The principles of contact heat evoked potentials
(CHEPs) are similar to those of LEPs. Stimuli are
delivered with a Peltier thermode, with a rapid rate
1446
of rise in temperature to ensure rapid, synchronous
activation of thermal sensitive nociceptors. N1/P1
cortical waveforms are generated that reflect activation
of A-delta nociceptors. Although CHEPs are a potential
biomarker of SFN in DM, this has been addressed in
few studies. Two small-scale investigations involving
˜30 patients with T2DM have shown that CHEP
amplitudes are reduced compared with healthy control
subjects and correlate with other biomarkers of SFN
(QST, autonomic function tests, and IENFD) and
large fiber neuropathy.72 ,73 CHEPs are noninvasive
and do not have safety issues as with LEPs. However,
they require specialized equipment and are subject to
significant habituation effects.71 Both CHEPs and LEPs
are unlikely to be used in a busy clinic setting.
Intra-epidermal electrical evoked potentials (IEEPs)
are performed by using specialized electrodes that
deliver a high current density to epidermal fibers.
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Longer stimulus durations preferentially stimulate
nociceptors. IEEPs offer the advantage of not requiring
a specialized stimulator device such as a laser or
thermode. They are noninvasive and easy to perform.
However, this method is currently experimental, with
few studies that address the use of IEEPs in DM
and none that compare their use with other SFN
biomarkers. Using a concentric planar electrode in
patients with DPN and normal NCS, IEEP cortical
waveforms were shown to be delayed and of lower
amplitude relative to those of healthy volunteers. In
patients with neuropathic symptoms, 95% showed at
least one abnormal finding (greater than the maximum
latency and less than the minimal amplitude of
recordings in healthy volunteers).74
Nerve Excitability Testing
The properties of human peripheral nerve axons can
be assessed noninvasively by using nerve excitability
testing (NET).75 NET is performed in a similar
manner to NCS but uses submaximal as well as
supra-maximal stimulation according to standard-
ized excitability protocols (stimulus–response curve,
strength–duration properties, threshold electrotonus
and the current/threshold relationship, and the recov-
ery cycle) to obtain a reflection of firing threshold
(Figure 1).75–77 The results provide a surrogate of ion
channel and axonal membrane potential properties at
the stimulation site. Although motor axons are more
commonly investigated, because recordings are more
stable and less affected by artifact, sensory nerves can
also be assessed. NET has the advantage in that it
assesses functional measures which may occur before
pathologic axonal or demyelinating features and their
NCS corollary become evident.
In patients with T1DM or T2DM, alterations in
excitability, including features compatible with sodium
channel dysfunction, have been reported in association
with subclinical neuropathy.78–80 These alterations
progress with increasing neuropathy severity and
are seen in patients with normal NCS, raising
the possibility that NET could provide a window
of opportunity to detect abnormalities before they
become irreversible.79 The types of alteration are
different between sensory and motor axons and occur
at an earlier stage in sensory nerves.81
Evidence in individuals with diabetes and no
neuropathy as well as those with “mild to moderate”
neuropathy indicate that glycemic control significantly
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A. Marshall et al.
affects nerve excitability. For example, the alterations
in NET seen in individuals with diabetes and no
neuropathy can be reversed by improvements in
glycemic control,82–85 an effect attributed to reversal of
hyperglycemia-induced suppression of nodal persistent
sodium channels.86 Underlining the potential of NET
to detect changes after intervention, parallel im-
provements in nodal sodium currents and conduction
velocity were shown in a small open clinical trial
with the aldose reductase inhibitor epalrestat in mild
to moderate DPN.87 Evidence of sodium channel
dysfunction detected using NET has also been linked
to painful symptoms in diabetic and other forms of
neuropathy.88 ,89
NET is not widely available and requires specialist
equipment and software. There are no clinically
relevant normative ranges and, although alterations
in NET can be shown despite normal NCS, it has
yet to be validated as a biomarker for either DPN or
pDPN. As with NCS, it does not provide information
about the status of small nerve fibers. However, it
is a useful, noninvasive method to explore disease
pathophysiology and may prove a useful biomarker
when assessing response to treatment for DPN or
pDPN such as in trials of therapeutic agents that act
on voltage-gated ion channels.
Neuropathic Pain in Diabetes
A major unmet need for patients with pDPN is the
ability to predict whether a particular drug is likely to
be efficacious. A more “personalized” and mechanis-
tically based approach to identify the pain generator
or modulatory site(s) would enable greater selectivity
and targeting of drug therapy, which would limit side
effects and improve overall efficacy.90 Neuropathic
pain drugs work in specific locations, as defined by
the receptors that they target. For instance, gabapentin
and pregabalin exert their analgesic effect through
high-affinity binding and modulation of the calcium
channel α 2 -δ proteins in the dorsal root ganglion.91 In
contrast, duloxetine relieves neuropathic pain through
inhibition of serotonin and norepinephrine reuptake,
which enhances descending inhibition of pain.92–94
Consequently, prescribing drugs that target peripheral
nerves when the pain is being generated or modulated
in the central nervous system (CNS) is likely to
fail. The following sections address neurophysio-
logical methods that are potential biomarkers of
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 Clinical Therapeutics
peripheral and centrally generated/modulated pain in
pDPN.
Peripherally Driven Neuropathic Pain and
Nociceptor Hyperexcitability
Small fiber damage95–97 has been proposed to cause
hyperexcitability of nociceptor afferents leading to
abnormal spontaneous activity and sensitization of
peripheral endings. These features could contribute
to spontaneous pain and hyperalgesia or could
drive central sensitization.95 Major determinants of
nociceptor excitability are ion channel and axonal
membrane potential properties.95 ,96 ,98
In support of the nociceptor hyperexcitability
hypothesis, it has been suggested that gain-of-function
variants of genes that encode voltage-gated sodium
channels which cause very rare Mendelian pain disor-
ders may play a role in common acquired neuropathic
pain disorders such as pDPN. For example, rare
SCN9A variants, defined as minor allele frequency
of <1% in the general population, are associated
with pDPN99–101 and idiopathic SFN.102 However, in
a cohort of participants with painful and painless
polyneuropathy, analysis of low-frequency variants in
sodium channels, defined as minor allele frequency
of <5% in the general population, did not associate
with neuropathic pain.103 Therefore, in certain well-
defined clinical contexts, such as pDPN or SFN, rare
sodium channel variants may act as risk factors for the
development of neuropathic pain.
The identification of clinically relevant sodium
channel variants offers the potential for personalized
and more effective treatment.104 ,105 Lacosamide targets
and suppresses activity of voltage-gated sodium
channels, including Nav1.7.106 Lacosamide has limited
efficacy when used in unselected patients with pDPN107
but shows efficacy in alleviating pain in patients
with Nav1.7 mutation–related SFN.104 Although
careful patient selection using molecular genetics can
aid treatment,105 clinically relevant sodium channel
variants in pDPN are currently identified in only a
small minority of patients. With more specific sodium
channel blockers on the horizon, the identification of
pDPN with nociceptor hyperexcitability pDPN is a
priority.
NET has shown evidence of Na+ channel dys-
function that associates with pDPN.88 However, this
method assesses large myelinated fibers and cannot
detect dysfunction in A-delta or C-fiber nociceptors. In-
1448
direct evidence of nociceptor hyperexcitability might be
obtained by using neuropathic pain phenotyping, either
based on detailed symptom-based questionnaires108 or
standardized QST.15 ,50 ,109 However, most patients with
pDPN show a loss of function on QST rather than
evidence of nociceptor hyperexcitability, and there is
no clear correlation between positive symptoms for
hyperexcitability such as allodynia on questionnaires
and QST/clinical findings.50 The only method that
can directly detect nociceptor hyperexcitability is
microneurography.
Microneurography
Microneurography involves insertion of a fine tung-
sten electrode into a peripheral nerve. For evaluation of
peripheral neuropathy, this typically involves recording
from the peroneal or superficial peroneal nerves. The
electrode is micromanipulated to enable recording of
unmyelinated fibers (Figure 1). A typical paradigm
involves stimulation of the cutaneous receptive field(s)
of the unmyelinated fibers by using electrical stimuli.
Using this method, several C-fibers can be recorded
at one time. A raster plot is generated showing
action potentials time-locked to the electrical stimulus
with latencies appropriate for the slow conduction
velocity of C fibers. This enables differentiation of
nociceptor subtypes (eg, polymodal nociceptors vs
silent nociceptors).110 ,111 Normally, there is a stable
baseline latency to low-frequency stimulation of the
receptive field, but when a fiber is spontaneously
active an irregular “saw-tooth” baseline is seen.112–117
Assessment of “abnormal” sensitivity to cutaneous
applied mechanical and thermal stimuli can also be
recorded.112 ,118 The method can therefore provide
evidence of both spontaneous activity and sensitiza-
tion. Patients with painful peripheral neuropathy have
been shown to have abnormal hyperexcitability of
nociceptor fibers.113 ,117 ,119–121
Microneurographic studies in DPN have shown
variable findings. In patients with documented large
fiber neuropathy, one study found an altered dis-
tribution of nociceptor subtypes with a higher
proportion of mechanically insensitive to mechanically
sensitive C-nociceptors, as well as evidence of loss of
mechanical sensitivity in normally sensitive fibers.122
Although a small proportion of nociceptor fibers were
spontaneously active, this did not significantly differ
between pDPN and DPN. Other studies have shown a
higher proportion of hyperexcitable nociceptor fibers
Volume 43 Number 9

in patients with pDPN.117 ,120 For example, Serra
et al117 reported spontaneous activity in 57% of 109
recorded nociceptor fibers in patients with pDPN but
of undefined neuropathy severity. Although this study
did not use a DPN group without pain, the rates of
spontaneous activity (˜5%) were higher than in age-
matched healthy individuals.
Although microneurography is an invaluable tool
for investigating the presence of nociceptor hyper-
excitability, its use as a biomarker is limited. The
technique is invasive, albeit minimally so, and time-
consuming for both the patient and investigator.
Furthermore, it requires specialist equipment and
highly trained operators, and at present is performed
only in a limited number of centers worldwide.
These factors significantly affect the availability and
cost-effectiveness of microneurography. Also, age and
potentially disease-specific definitions of normative and
abnormal data are needed so that its sensitivity and
predictive value can be determined, and it can be
established as a clinical tool. A further drawback is
that each session may yield only small numbers of
nociceptor recordings that are suitable for analysis. For
example, in one study, only 1 to 3 suitable nociceptor
fibers were recorded per session.117 This limits
diagnostic applicability and capacity for monitoring
interventions on an individual level. It is not known
how microneurography compares with the indirect, but
considerably more widely available, QST techniques
in identifying patients with hyperexcitable nociceptors.
Furthermore, the relationship between abnormalities
detected by microneurography and structural changes
of small fibers in the skin is unknown. The method
is potentially of use in clinical trials, especially those
with a focus on underlying pain mechanisms, in which
patients with and without evidence of hyperexcitability
can be segregated.115 ,117
Spinal Inhibitory Dysfunction in pDPN
There is increasing recognition that changes in
the spinal cord123 and brain124–126 may generate and
modulate neuropathic pain in patients with diabetes.
Multiple pathophysiological changes can occur in
the CNS as a result of peripheral nervous system
pathology.127 ,128 These secondary CNS changes can
inappropriately amplify or fail to suppress incoming
signals from the periphery, a mechanism of pain called
disinhibition.
September 2021
A. Marshall et al.
The inhibitory neurotransmitters γ aminobutyric
acid (GABA) and glycine, active through local
inhibitory interneurons, play critical roles in regulating
spinal nociceptive processing. In animal models,
pharmacologic or genetic interventions that enhance
or diminish spinal inhibition result in decreased and
increased behavioral indices of pain, respectively.129–132
In diabetic rodents, spinal inhibitory processes are
dysfunctional. Allodynia in the streptozotocin (STZ)-
rat model of T1DM is driven by spinal disinhi-
bition in which GABA, acting via spinal GABA-A
receptors, is no longer inhibitory and becomes pro-
nociceptive.133 ,134 The mechanism involves downreg-
ulation of the postsynaptic chloride pump KCC2,
which alters the chloride equilibrium potential that
dictates the direction of ion flow through the GABA-
A receptor. Accordingly, spinal GABA-A blockade,
which under normal circumstances is pro-nociceptive,
reverses allodynia in STZ-rats.133
HOFFMANN-REFLEX RATE-DEPENDENT
DEPRESSION: A BIOMARKER OF SPINAL
GABAERGIC INHIBITORY FUNCTION
The Hoffmann-reflex (H-reflex) is regarded as an
electrophysiological corollary of the mechanically
elicited deep tendon reflex.135 The H-reflex arc
comprises Ia afferent fibers, derived from muscle
spindles, forming strong monosynaptic connections
with alpha motor neurons within the spinal cord.
Although originally considered a monosynaptic trans-
spinal reflex,136 it is now accepted that the reflex
mechanism additionally involves both monosynaptic
heteronymous and oligosynaptic connections.137 The
H-reflex is measured in humans using a simple
modification of traditional NCS. The stimulation
protocol evokes 2 waveforms: a direct nerve to muscle
M wave and a longer latency trans-spinally mediated
H wave (Figures 1 and 2). Rate-dependent depression
(RDD) is the measure of the change in amplitude of the
H-wave component over consecutive stimulations. In
normal rats, RDD is driven via activation of inhibitory
spinal GABA-A receptors.133 Loss of RDD occurs
in both humans and animals after disinhibition of
sensory processing caused by spinal cord injury, and in
rats this is linked to reduced spinal KCC2 expression
and subsequent loss of GABAergic inhibition.138–141
STZ-rats exhibiting behavioral indices of neuropathic
pain and reduced spinal KCC2 also exhibit loss
of RDD.133 ,134 Spinal GABA-A blockade, which
1449
 Clinical Therapeutics

Figure 2. Representative electromyogram traces showing M and H waves in response to 3 consecutive

stimulations at 1 Hz frequency in a patient with painless (upper triplicate) and painful (lower triplicate)
diabetic neuropathy. The H-wave amplitude declines in the traces from the patient with painless
neuropathy and illustrates rate-dependent depression. DPN = diabetic peripheral neuropathy.

in control animals impairs RDD,133 paradoxically
restores RDD as well as reverses allodynia.133 Loss
of RDD can be used as a biomarker to separate rats
with behavioral markers of neuropathic pain caused
by spinal disinhibition from rats with similar behav-
ioral manifestations that are of peripheral origin.134
Intriguingly, the selective serotonin and norepinephrine
reuptake inhibitor duloxetine, acting via spinal 5HT2A
receptors, not only alleviates behavioral indices of pain
in STZ-rats142 but also restores RDD via a similar
mechanism.143
There is preclinical evidence that loss of RDD
and indices of neuropathic pain share a common
pathogenic mechanism involving spinal KCC2 deple-
tion and disinhibition caused by inversion of GABA-
A receptor function and exhibit common responses to
spinally acting analgesics outside the root pathogenic
mechanism. This evidence supports the concept that
RDD status may be a viable biomarker for both
identifying the dominant generator site in individual
patients with pDPN and for predicting efficacy of
therapeutic strategies that alleviate spinal disinhibition.
Our research has previously measured the magni-
tude of RDD in patients with T1DM and painful
or painless neuropathy and age-matched healthy
control subjects.143 There was loss of RDD in
patients with pDPN compared with both healthy
control subjects and patients with painless DPN
(Figure 2). These findings were independent of glycemic
control and severity of large fiber neuropathy and
SFN. Loss of RDD correlated with pain sever-
ity, indicating that more severe pain is associated
with the loss of RDD and, by inference, spinal
disinhibition.
Expanding these data from patients with T1DM,
we have recently shown that, similar to findings in
the Zucker Diabetic Fatty rat model of T2DM,143
impaired RDD is also seen in patients with T2DM
and pDPN (unpublished observations, Marshall 2020).
Importantly, not all subjects with pDPN had impaired
RDD (defined as a H3:H1 ratio >2 × SD of
control group mean). This supports the hypothesis that
impaired RDD may serve as a clinical biomarker in a
subset of patients in whom pain arises primarily from
spinal disinhibition. Approximately 60% of patients
with diabetes will develop neuropathy, approximately
one third of those will develop pDPN, and, from
our exploratory studies, 40% of those will exhibit
RDD deficits.143 ,144 This heterogeneity could plausibly
be used to enable definition of abnormal values for
predicting response to spinally acting drugs. It is
important to acknowledge that a proportion of patients
in these studies were taking prescribed antineuropathic
pain medication and that therapeutic responses were
not studied in a systematic manner. Similarly, whether
antineuropathic pain medication affects or normalizes
RDD either in a general or drug-specific manner is
unknown.

1450 Volume 43 Number 9


The measurement of RDD is noninvasive and
potentially as widely available as current NCS. One
drawback is that the H-reflex can be absent or
difficult to elicit with increasing severity of DPN113
or in patients with co-existing S1 radiculopathy.
This may limit the use of RDD as a biomarker in
patients with severe neuropathy. Further larger scale
studies controlling for the use of antineuropathic pain
medication which systematically assess therapeutic
response are also required to determine the sensitivity
and predictive value of RDD, both as a biomarker of
spinal disinhibition and as a predictor of neuropathic
pain medication efficacy in patients with pDPN. It is
not yet known if spinal disinhibition and impaired
RDD in pDPN145 are associated with a distinctive (and
measurable) pain phenotype or whether they relate
to other putative mechanisms that facilitate ascending
nociceptive drive from the spinal cord such as wind-up
or an abnormal descending pain modulatory system146 ;
all of these are potentially easier to measure in an
outpatient setting using targeted QST.
CONCLUSIONS
The early diagnosis of DPN and management of pDPN
continue to pose considerable challenges. NCS are
a long-established biomarker for DPN that predict
clinically relevant outcomes. However, biomarkers
that capture SFN are also required to facilitate the
earliest detection of small, as well as large, fiber DPN.
Noninvasive neurophysiological methods such as pain-
related evoked potentials, which assess the function of
small fiber pathways, and NET, which has the potential
to detect nerve dysfunction before it is irreversible, are
potential biomarkers capable of detecting early DPN.
Although these approaches are currently not practical
for use in busy outpatient settings, they may be suited
as diagnostic biomarkers and end points in mechanistic
clinical trials of DPN.
For pDPN, emerging methods such as H-reflex
RDD and microneurography have the potential to
segregate patients on the basis of pain mechanisms.
They may also allow assessment of the balance
between peripheral versus spinal drive in pDPN. Thus,
they have the potential to function as biomarkers
for trial enrichment and for segregating patients for
mechanistic informed clinical trials in pDPN. Unlike
microneurography, assessment of spinal disinhibition
with RDD is a noninvasive and potentially widely
available method that could be used to inform
September 2021
A. Marshall et al.
physicians about the optimal choice of drug for
individual patients on the basis of pain mechanisms.
CONFLICTS OF INTEREST
The authors have indicated that they have no conflicts
of interest regarding the content of this article.
ACKNOWLEDGEMENTS
This study was supported by American Diabetes As-
sociation Award 1-17-ICTS-062 (Andrew G Marshall
PhD and Nigel Calcutt PhD).
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Address correspondence to: Andrew Marshall, University Hospital Liver-

pool, Institute of Life Course and Medical Sciences, University of Liverpool,
Liverpool, United Kingdom. E-mail: andrew.marshall@liverpool.ac.uk.

1456 Volume 43 Number 9