Diabetes Weblog

61st Scientific Sessions of the American Diabetes
Association
Day 1 - June 22, 2001

Diabetic Neuropathy: A Small-Fiber Disease
Aaron I. Vinik, MD, PhD

Diabetic neuropathy (DN) is the most common and
troublesome complication of diabetes mellitus, leading
to great morbidity and mortality and resulting in a
huge economic burden for diabetes care.[1-3] It is the
most common form of neuropathy in the developed
countries of the world, accounts for more
hospitalizations than all the other diabetic
complications combined, and is responsible for 50% to

75% of nontraumatic amputations.[2,4]
Diabetic neuropathy is a group of clinical syndromes
that alone or in combination affect distinct regions
of the nervous system. It may be silent and go
undetected while exercising its ravages. It is a
heterogeneous disorder that encompasses a wide range
of abnormalities affecting proximal and distal
peripheral sensory and motor nerves as well as the
autonomic nervous systems. The major morbidity
associated with somatic neuropathy is foot ulceration,
the precursor of gangrene and limb loss. Neuropathy
increases the risk of amputation 1.7-fold; 12-fold if
there is deformity (itself a consequence of
neuropathy); and 36-fold if there is a history of
previous ulceration.[5-7] There are 85,000 amputations
in the United States each year, 1 every 2 minutes, and
neuropathy is considered to be the major contributor
in 87% of cases. It is also the most life-spoiling of
the diabetic complications and has tremendous
ramifications for the quality of life in a person with
diabetes. It is now recognized that a major effect of
diabetes is on the small unmyelinated or thinly
myelinated C and A delta nerve fibers that subserve
autonomic function and thermal and pain perception.
Once autonomic neuropathy sets in, life can become
quite dismal and the mortality rate approximates 25%
to 50% within 5-10 years.[5,8-10]

Small-Fiber Dysfunction: The Clinical Syndrome
Patients with small-fiber dysfunction most commonly
present with burning feet. The examination may be
singularly deceptive, with normal reflexes, normal
strength, normal sensory levels, and normal
electrophysiology. It is not surprising that most of
these patients previously have been mislabeled as
neurotic, hysterical, or malingerers. Only with the
advent of new sophisticated methods of detection has
it been possible to establish the real nature of the
disorder. Diabetes remains the most common cause, but
heavy metal poisoning, amyloid, vasculitis, autoimmune
disease, collagen vascular disease, chemotherapeutic
drugs, Chagas’ disease, and idiopathic and familial
causes need to be considered as well. Potentially, an
association also exists between impaired glucose
tolerance (IGT) and neuropathy, especially painful
small-fiber neuropathy. Furthermore, neuropathy may be
the only presenting problem at diagnosis in subjects
with otherwise idiopathic neuropathy.

Autonomic Neuropathy
Autonomic neuropathy can affect virtually every system
in the body. It has major effects on the
cardiovascular system, ranging from sudden death,
silent myocardial infarction, and decreased exercise
tolerance, as well as anorexia, nausea, vomiting,
gastroparesis, constipation sometimes alternating with
diarrhea, genitourinary abnormalities with urinary
retention and erectile dysfunction, and brittle
diabetes due to vagaries of gastric emptying and loss
of the coordinated delivery of fuels from the
gastrointestinal tract and insulin. Autonomic supply
to the skin and subcutaneous tissues is critical for
nutrient delivery, blood flow, and lubrication through
sweating. These can be perturbed in a functional
manner, thus aiding and abetting the loss of sensory
input and collaborating in the process of foot
ulceration, culminating in the process leading to
gangrene and limb loss. Autonomic neuropathy is
therefore a major contributor to the life-spoiling
effects of nerve damage in addition to the reduced
life expectancy. It is important to diagnose
neuropathy before the advent of irreversible changes
occurs.

New Views on the Pathogenesis of Neuropathy
A major emphasis on the role of oxidative stress,
"hot" mitochondria, and the important role of nitric
oxide (NO) in mediating nerve damage was presented at
the 61st Scientific Sessions of the American Diabetes
Association. These views are championed by certain
investigators and hotly challenged by others.
Nonetheless, they set the stage for a new
understanding of pathogenetic mechanisms, which should
help focus the development of therapeutic strategies
on targets other than the traditional ones that have
met with a singular lack of success for more than 3
decades.
James W. Russell, MD, MS,[11] University of Michigan,
presented views on reactive oxygen species (ROS),
mitochondrial dysfunction, and diabetic neuropathy
(Figure 1).

Figure 1.
Recent evidence in both animal models and human sural
nerve biopsies indicates that there is an association
among oxidative stress, mitochondrial (Mt) membrane
depolarization (MMD), and induction of programmed cell
death (PCD), or apoptosis. In streptozotocin
(STZ)-treated diabetic rats, hyperglycemia induces
typical apoptotic changes as well as swelling and
disruption of the Mt cristae in diabetic dorsal root
ganglion (DRG) neurons and Schwann cells, but these
changes are only rarely observed in control neurons.
In human sural nerve biopsies from patients with
diabetic sensory neuropathy, there is
electromicrograph evidence of swelling and disruption
of the Mt and cristae compared with patients without
peripheral neuropathy.

In human SH-SY5Y neurons, rat sensory neurons, and
Schwann cells, in vivo, there is an increase in
reactive oxygen species (ROS) after exposure to 20 mM
added glucose. In parallel, there is an initial Mt
membrane hyperpolarization followed by Mt membrane
depolarization (MMD). In turn, MMD is coupled with
cytochrome-c release and cleavage of caspases.

Oxidative Stress and Cell Death
Various strategies aimed at inhibiting the oxidative
burst, or stabilizing the mitochondrial membrane
potential, block induction of PCD. First, growth
factors such as insulin-like growth factor I (IGF-I)
can block induction of ROS and/or stabilize the
mitochondrial membrane potential. This in turn is
associated with inhibition of PCD. Second, reduction
of ROS generation in neuronal Mt by upregulation of
manganese superoxide dismutase, or downregulation of
neuronal isoform of nitric oxide synthase (nNOS),
prevents neuronal degeneration. Third, upregulation of
uncoupling proteins that stabilize the mitochondrial
membrane potential blocks induction of caspase
cleavage. Collectively, these findings indicate that
hyperglycemic conditions observed in diabetes mellitus
are associated with oxidative stress-induced neuronal
and Schwann cell death, and targeted therapies aimed
at regulating ROS bursts may prove effective in
therapy of diabetic neuropathy.

The Good, the Bad, and the Ugly of Nitric Oxide (NO)
The role NO plays in DN was discussed by both Martin
Stevens, MD,[12] of the University of Michigan, and
Robert Hoeltdke, MD,[13] of the University of West
Virginia. It appears that early during the course of
evolving diabetes, there is an increase in NO and its
derivatives NO2 and NO3, which are collectively known
as Nox (referred to as "noxious"). Under normal
conditions, the synthesis of small amounts of NO are
catalyzed by the constitutive form of nitric oxide
synthase (cNOS), which facilitates vasodilation and
inhibits the aggregatory tendencies of platelets.
Under inflammatory conditions, inducible NOS (iNOS) is
activated and large quantities of NO are formed. In
this scenario NO may be responsible for nerve damage,
neuronal apoptosis, and cell death. Nox may also act
as an oxidative stressor, increasing the production of
ROS, which may also derive from "overheated"
mitochondria, xanthine oxidase, and NADPH among
others. Furthermore, interaction of NO with superoxide
radical (SO2-) results in the formation of
peroxynitrate (ONOO), which nitrotyrosylates proteins,
thereby impairing their function. This postulated
biphasic action of NO is shown in Figure 2. Too much
of a good thing can be harmful!

Figure 2.

Oxidative Stress and Nerve Perfusion
Recently, oxidative stress and nerve perfusion
deficits have emerged as leading candidates in the
pathogenesis of DN. A recently proposed model suggests
that a critical role is played by glucose-induced
oxidative stress and secondary depletion of the
endothelium-derived relaxing factor NO at sites both
within the peripheral nerve trunks and the endoneurial
microvasculature and more proximally in the
sympathetic ganglia. The depletion of NO at these
sites has been suggested to lead both directly and
indirectly to nerve blood flow deficits, nerve
hypoxia, and disruption of nerve mitochondrial
function and energy production. Experimental evidence,
supporting a critical role for disruption of NO in
diabetes at these sites was discussed, together with
data implicating NO repletion as the principal
mediator of many successful therapeutic interventions
in experimental DN in rodent models. The results of
several studies aimed at reducing oxidative stress and
repleting diminished NO production will be available
shortly and will hopefully vindicate these hypotheses.
A caveat that must be considered is the opposing view
that excess NO catalyzed by the enzyme iNOS has also
been shown to cause apoptosis of neurons and other
cells. There are log orders of magnitude differences
in the production of NO catalyzed by endothelial
(e)NOS, nNOS, and iNOS. Whereas diminished nerve blood
flow may derive from decreased endothelial production
of NO and should be the target of future therapeutic
endeavors, the overproduction of NO catalyzed by iNOS
may derive from a host of other cells, including
keratinocytes, macrophages, and leukocytes, which,
among others, may result in apoptosis and dictate an
entirely different approach. Although there is no
doubt that glucose is the major contributor to nerve
damage, the observation that neuropathy is found in
11% of patients who are euglycemic and that apoptosis
of DRG neurons is found in Zucker rats prior to the
development of hyperglycemia suggests that there may
be other factors in the pathogenesis of neuropathy.
The report by Stansberry and Vinik,[14] describing
impaired neurovascular function in first-order,
euglycemic, relatives of patients with diabetes,
emphasizes a need to examine the role that proximal
factors such as hyperinsulinemia, insulin resistance,
and dyslipidemia play in C-fiber dysfunction.

Evaluation of Defects Underlying DN
In parallel with our increased understanding of the
pathogenesis of DN, there must be refinements in our
ability to measure quantitatively the different types
of defects that occur in this disorder, so that
appropriate therapies can be targeted to specific
fiber types. These tests must be validated and
standardized to allow comparison between studies and a
more meaningful interpretation of results. Our ability
to successfully manage the many different
manifestations of DN depends ultimately on our success
in uncovering the pathogenic processes underlying this
disorder.

Peripheral Small-Fiber Function
The development of noninvasive methods for assessing
skin blood flow has enabled clinical measurements of
the effects of diabetes on microvascular
perfusion.[15] Several functional disturbances are
found in the dermal microvasculature in patients with
diabetes. These include decreased microvascular blood
flow, increased vascular resistance, decreased tissue
PO2, and altered vascular permeability
characteristics. Diabetes also disrupts vasomotion,
the rhythmic contraction exhibited by arterioles and
small arteries.[16,17] Unmyelinated C-fibers, which
constitute the central reflex pathway, are assumed to
be damaged in DN, contributing to abnormalities in
cutaneous blood flow. Warm thermal sensation is a
functional measure of C-fiber function in the
periphery, and the impairment of this is paralleled by
a reduction of vasomotion indicating an interaction
between small unmyelinated C-fiber function and
vasomotion.

Loss of Neurogenic Vasodilation in Upper Limb May
Precede Microangiopathy in Lower Limb
In type 2 diabetes, the predominant abnormality in
skin blood flow is the loss of the active neurogenic
vasodilative mechanism in hairy skin.[17] This loss of
neurogenic vasodilation in the upper limb, which is
spared severe neuropathy and ulceration, may precede
other microangiopathic processes that are accelerated
in the lower limb because of increased systemic
pressure. It is feasible that the susceptibility to
foot ulceration is the consequence of impaired
microvascular perfusion of skin and subcutaneous
tissue and a concomitant decrease in vascular supply
to small C-fibers subserving pain and warm thermal
perception, allowing increased heat and tissue injury
in an ischemic limb. These changes are not a
consequence of sclerotic vessels (and therefore are
not permanent).
Two additional indices of peripheral vascular function
have been examined: (1) the hyperemic response to
experimental (occlusive) ischemia and (2) the physical
distensibility of the same vessel beds.[18]
Postischemic hyperemia is thought to be
endothelium-dependent, largely the result of the
release and action of endothelial-derived NO and
prostaglandin. Vessel distension, however, relies
purely on the hydrostatic gradient imposed by
elevating and lowering the upper limb, reflecting the
degree of microvascular distensibility. In these
studies, vessels in the finger pulp responded
similarly in matched groups of patients with and
without diabetes. However, both the upper and lower
limbs showed profoundly impaired heat-stimulated
vasodilation in patients with diabetes.

Both skin sites rely on endothelium for initial
vasodilatation after ischemia or release of
sympathetic output. However, in hairy skin (the hand
dorsum), the active neurogenic vasodilatory mechanism
described above is responsible for most of this
dilation. Thus, these new findings provide evidence of
defective neurogenic vasodilation in hairy skin that
is independent of vascular elasticity (ie, sclerosis
is not the cause of this nondistensibility). These
results suggest that defects, such as the accumulation
of advanced glycation end products (AGE) in vessels
and connective tissues,[19-21] accumulation of
sorbitol in nerves,[22-31] alterations in PKC
metabolism,[20, 28-31] or other metabolic factors that
remain poorly understood, are likely to precede
structural changes in the vessels.

Therefore, blood flow is a sensitive marker of C-fiber
dysfunction, precedes development of diabetes
mellitus, correlates with in vivo indices of the
metabolic syndrome, and may be a "benchmark" for
future studies of agents to improve microvascular
dysfunction in diabetes mellitus.

Evaluating Sensory Nerves in Skin
Skin biopsy is a technique that holds great promise
for the diagnosis, study, and treatment of DN, and may
serve as an endpoint in the investigation of new drugs
in the treatment of small-fiber neuropathies. This
procedure involves taking a minimally invasive 3-mm
punch biopsy from several skin sites over the lower
limb, proximal calf, thigh, and forearm, thus allowing
the evaluation and quantitation of proximal to distal
epidermal nerve fiber (ENF). ENFs are the distal
projections of small DRG neurons that pierce the
dermal-epidermal basement membrane and penetrate the
epidermis, often to the stratum corneum. Here these
thinly myelinated or unmyelinated fibers, which are
not assessed by routine nerve conduction studies, can
be visualized by immunostaining with antibodies to the
protein gene product, PGP 9.5.[32,33] They subserve
heat, pain, and autonomic functions and may be
preferentially affected in diabetes. Reduced numbers
of ENFs are found in diabetic neuropathy[34] and other
small-fiber neuropathies.[31,32]
Periquett and colleagues[37] examined a large cohort
of people with painful feet but normal strength,
minimal neurologic findings, and normal nerve
conduction velocities (NCVs) and found 38% could be
diagnosed by the skin biopsy. Skin biopsy was more
sensitive in a small subset of patients compared with
Quantitative Sensory Tests (QSTs) and Quantitative
Sudomotor Axon Reflexes Testing (QSART). Herrmann and
colleagues[38] compared the results of skin biopsy in
27 patients with different forms of neuropathy and
found 9 patients with decreased skin ENFs who had
normal unmyelinated nerve counts in their sural
nerves. Thus ENF may be the only abnormality in a
significant portion of patients with painful
neuropathy; this has, of course, been demonstrated in
diabetes as well.

The procedures for quantitating ENFs still need some
refining and several techniques are being used.
McArthur[39] reports on routine immunohistochemistry,
and Kennedy and colleagues as well as Periquet and
colleagues[37] use confocal microscopy. The finding of
a large variance in the number of ENFs in normal
subjects indicates that the technique needs to be
refined, possibly by quantifying ENF length and
branching or dendrite length and branching as well as
periodic swellings. Furthermore, the lack of
sensitivity of counts of small unmyelinated fibers in
the nerve biopsies may reflect the fact that these
fibers are destined to supply a variety of organs,
including sweat glands, blood vessels, and arrector
pilorum muscles that may be quantitated in skin
biopsies. The innovation of the skin blister
technique[40] is easily repeated with time, allowing
the measurement of disease progress and reversal, and
is suitable for measuring outcomes.

The Old and the New in Cardiac Autonomic Neuropathy
Cardiovascular autonomic neuropathy (CAN) is a common
complication of diabetes, which results in disabling
clinical manifestations and may predispose individuals
to sudden cardiac death. The onset of loss of heart
rate variability (HRV) is a predictor of premature
death and is associated with a mortality of 25% to 50%
within 5-10 years. This dismal statistic is offset by
the fact that there is evidence that loss in HRV is
reversible with glycemic control,[41] graded
exercise,[42] antioxidants,[43] and a variety of other
agents including beta-blockers,[44]
angiotensin-converting enzyme (ACE) inhibitors,[45]
aldosterone antagonists,[46] and calcium-channel
blockers.[47] The loss in HRV assumes even greater
importance when considered in the context of the
nondiabetic population, in whom loss in HRV is
associated with myocardial infarction and predicts a
poor outcome. Several large trials have shown that
beta-blockers[48] and ACE inhibitors[49] increase
survival. These are, therefore, cogent arguments for
the use of HRV as a standard of evaluating patients
with diabetes. Unfortunately, this has only recently
been brought into the research arena, but with the
standard methodology, testing for CAN may become a
practical reality for practicing clinicians.

Scintigraphic Assessment of Cardiac Sympathetic
Integrity
Recently, direct scintigraphic assessment of cardiac
sympathetic integrity has been made possible by the
introduction of radiolabeled analogues of
norepinephrine which are actively taken up by the
sympathetic nerve terminals of the heart. These
techniques use either [123I] metaiodobenzylguanidine
(MIBG)[50,51] and single photon-emission computed
tomography (SPECT) or [11C] hydroxyephedrine (HED) and
positron-emission tomography (PET).[52] Studies in
diabetic patients have explored the sensitivity of
these techniques to detect CAN, characterize the
effects of glycemic control on the progression of CAN,
and evaluate the effects of CAN on myocardial
electrophysiology, blood flow regulation, and
function.
Deficits of left ventricular (LV) [123I] MIBG and
[11C] HED retention have been identified in diabetic
subjects without abnormalities on cardiovascular
reflex testing consistent with increased sensitivity
to detect CAN. Poor glycemic control results in the
progression of LV tracer deficits, which can be
prevented or reversed by near-euglycemia. Deficits
begin distally in the LV and may extend proximally.
Paradoxically, however, absolute HED retention is
increased in the proximal segments of the severe CAN
subjects consistent with regional "hyperinnervation."
These regions also exhibit abnormal blood flow
regulation. Impaired myocardial MIBG uptake correlates
with altered LV diastolic filling and myocardial
electrophysiologic deficits, and is predictive of
sudden death.[52-55]

CAN Affects Regional Cardiac Perfusion and Electrical
and Chemical Stability
Scintigraphic studies have provided unique insights
into the effects of diabetes on cardiac sympathetic
integrity and the pathophysiologic consequences of LV
sympathetic dysinnervation. It has been proposed that
CAN disrupts myocardial function through effects on
regional perfusion and results in electrical and
chemical stability. Recently, scintigraphic imaging
techniques using radiolabeled neurotransmitter
analogues in animal models and humans have allowed
mechanistic exploration of the associations of CAN
with myocardial instability. These studies have
focused attention on the proximal myocardium as the
critical region of instability. Experimental evidence
supports a role for: (1) myocardial electrical
imbalance, manifest as normal sympathetic tone, but
increased sympathetic innervation in the proximal
myocardium; (2) chemical imbalance with excess
accumulation of plasma norepinephrine in response to
sympathetic stress and increased norepinephrine and
oxidative stress in the proximal myocardium; and (3)
vascular imbalance with impairment of both
endothelium-dependent and endothelium-independent
myocardial blood flow regulation in the proximal
myocardium.
Early recognition of CAN and understanding the causes
and consequences of diabetes complicated by CAN may
lead to the development of effective therapeutic
strategies aimed at preventing sudden cardiac death.
Future studies using complementary neurotransmitter
analogues will allow different aspects of regional
neuronal dysfunction to be characterized with the aim
of developing therapeutic strategies to prevent or
reverse CAN.

Orthostatic Hypotension and Bladder Dysfunction
Therapeutic strategies for the management of
orthostatic hypotension and bladder dysfunction were
also discussed by Roy Freeman, MD.[56] He reviewed the
treatment of neurogenic orthostatic hypotension with
3,4-DL-threo-dihydroxyphenylserine in a randomized,
placebo-controlled crossover trial. Results showed
small but significant effects on blood pressure and
quality of life, but the drug remains in the research
arena.[57] The general approach can be found in a
recent review of the topic.[58]

Summary
Thus, there is now recognition that small nerve fibers
may be targeted in diabetes. These patients
traditionally present with burning feet and a host of
symptoms and physical signs related to autonomic nerve
dysfunction. It was once said, "Know syphilis and you
will know the whole of medicine." Today it can be
said, "Know small-fiber dysfunction and you will know
the whole of medicine." There are new technologies
bringing the clinician closer to making diagnoses at
points of patient contact with reasonable certainty
and sensitivity specificity, accuracy and precision,
allowing him/her to make sound therapeutic decisions
and quantitate the impact and outcome thereof. I do
believe Cinderella has found her slipper; we as
healthcare providers should wear the shoe that fits!

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