Biotechnological Advances for Diagnosis of Peripheral Diabetic
Neuropathy
Received for publication, July 5, 2014
Accepted, August 5, 2014
CONSTANTIN CĂRUNTU1,2, CAROLINA NEGREI3*, DANIEL BODA4, CAROLINA
CONSTANTIN1, ANA CĂRUNTU5, MONICA NEAGU1
1
"Victor Babes” National Institute of Pathology, Immunology Department, Bucharest,
Romania
2
“Carol Davila” University of Medicine and Pharmacy, Department of Physiology,
Bucharest, Romania
3
“Carol Davila” University of Medicine and Pharmacy, Department of Toxicology,
Bucharest, Romania
4
“Carol Davila” University of Medicine and Pharmacy, Dermatology Research Laboratory,
Bucharest, Romania
5
“Dan Theodorescu” Oral and Maxillofacial Surgery Hospital, Bucharest, Romania
*Address correspondence to: Carolina Negrei, Department of Toxicology, “Carol Davila”
University of Medicine and Pharmacy, 6 Traian Vuia Str. 020956, Bucharest, Romania, email: carol_n2002@hotmail.com, tel/fax +40726160275/+40213111152
Abstract
Diabetic neuropathy is a major challenge for the healthcare system, being associated with
multiple local and general complications which involve increased medical and socio-economic costs
and an important reduction of the patient’s quality of life.
In this paper we review the relevant aspects regarding the micro-morphological and
pathophysiological changes in peripheral diabetic neuropathy, we summarize the main techniques used
for diagnosis and staging of diabetic neuropathy and we discuss the biotechnological advances that
were achieved in this field. Thus, recent developments in proteomics and in vivo investigation of
cutaneous nerve structures provides promising data and could lead to the achievement of new
biotechnological diagnostic strategies easy to implement, with a greater accuracy of results, which
allow the diagnosis of early changes in diabetic neuropathy, in a stage in which the preventive and
therapeutical measures have maximum efficiency, thus being able to contribute to the diminishing of
the morbidity associated with the disease.
Keywords: diabetic neuropathy, biotechnology, diagnosis, staging, reflectance confocal microscopy
1.
Introduction
Diabetic neuropathy is a severe chronic complication of diabetes mellitus and is the
most frequent form of neuropathy in the developed countries [1-8]. It affects 40-60% of the
diabetic patients [6, 9], representing a significant cause of morbidity and mortality [2, 5, 7,
10]. Diabetic neuropathy is associated with a whole range of local and general complications
that inflict important health care costs and reduce the patient’s quality of life, with an overall
major socio-economic impact. The local and general complications include the neuropathic
chronic pain, the development of neuro-osteoarthropathy lesions leading to the aspect of
diabetic Charcot foot, or the appearance of recurrent ulcers of the distal extremities, localized
infections that can spread and that can lead to amputation. [11, 12].
The most frequent form of diabetic neuropathy is the distal symmetrical
polineuropathy [2, 7, 10], which is a gradual, diffuse, symmetrical impairment of the
peripheral nerve fibers of the extremities [8, 13]. Sensory, motor but also autonomic nerve
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fibers can be involved [6, 14]. However, the most frequent form of peripheral diabetic
neuropathy is the sensory one [5].
The nerve fibers impairment can appear early in diabetes mellitus [15, 16], some
authors emphasizing as well as an association between the polineuropathic changes and the
impaired glucose tolerance [17]. The disease can be triggered by various pathophysiological
mechanisms, having a heterogeneous symptomatology associated with a variable evolution
[6, 14].
The clinical manifestations of diabetic neuropathy include disorders of the thermoalgesic, tactile, vibratory and pressure sensitivity. Changes of pain sensitivity may range from
hyperalgesia and allodynia to an important decrease of the perception of nociceptive stimuli.
Moreover, the decrease of the sudomotor activity can be accompanied by consecutive
cutaneous xerosis. The symptoms appear and are more severe at the distal regions, having a
“gloves and stockings” distribution, but evolve with a proximal progression [4-6, 18-20].
The distal regions are characterized by a high density of skin nerve fibers and an
increased variety of their types [4]. The diverse symptomatology including changes of tactile,
thermic and pain-perception sensitivity may suggests impairment of the thin myelinated Adelta nerve fibers or unmyelinated C fibers as well as at the level of large diameter
myelinated A-beta nerve fibers; furthermore, the vasoregulatory and sudomotor disorders
indicate the involvement of autonomic innervation [4, 5, 13, 21-26].
The diagnosis of diabetic neuropathy, especially in an early stage where treatment has
maximum efficacy, is a major challenge in clinical practice. The biotechnological advances in
this field, particularly the recent developments in proteomics and in vivo investigation of
cutaneous nerve structures, are very promising and could contribute to the early diagnosis and
the reduction of the morbidity associated with the disease.
2.
Micro-morphological and pathophysiological changes in peripheral diabetic
neuropathy
Previous research has shown that the first changes in peripheral diabetic neuropathy
usually affect the thin myelinated A-delta nerve fibers or unmyelinated type C fibers [22, 27].
Most of the studies concerning the changes of nerve fibers in diabetic neuropathy performed
on human subjects have noted a reduction in the density of cutaneous nerve fibers, in patients
diagnosed with diabetes mellitus type 1 and 2 [23, 28, 29] and in patients with impaired
glucose tolerance [30]; in this cases a significant reduction of the density of intraepidermal
nerve fibers being noticed [31-35]. Previous research [21, 31] has also highlighted a
decreased expression of neuropeptides calcitonin gene-related peptide (CGRP) and substance
P (SP), prevalent in the small diameter sensory nerve fibers, as well as of vasoactive
intestinal peptide (VIP) and neuropeptide Y (NYP), prevalent in the autonomic nerve fibers
[36]. Studies carried out on diabetic patients have emphasised in addition a decreased number
of dermal and epidermal nerve fibers positive for the transient receptor potential cation
channel, subfamily V, member 1 (TRPV1) but also a reduced expression of TRPV1 in the
remaining nerve fibers.
TRPV1 is a ligand-gated, nonselective cation channel that integrates various noxious
stimuli, being involved in the transmission and modulation of pain. It is predominantly
expressed in the unmyelinated type C sensory nerve fibers or the thin myelinated A-delta
nerve fibers [37], and this reduction of the immunoreactivity for TPRV1, possibly triggered
by a decrease of nerve growth factor (NGF), seems to precede the reduction in the density of
these nerve fibers [38]. The diminution of TRPV1 expression can trigger sensitivity disorders
specific for diabetic neuropathy, the TRPV1 receptor being activated by physical triggers like
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high temperatures (>43oC), by the considerable increase in the concentration of H+ ions
(pH<6), as well as by endocannabinoids (anandamide), by capsaicin and other vanilloid
substances [37, 39-41].
Studies carried out on non-human primates have highlighted the association of
diabetes with a significant remodelation of skin innervation of the extremities, involving all
types of nerve fibers [4]. Thus, an accelerated decrease of the intraepidermal thin nerve fibers
density has been noticed, together with the reduction of the expression of neuropeptides like
CGRP and of TRPV1 receptor. Likewise, a hypertrophy and an increased number of
Meissner corpuscles innervated by thick A-beta nerve fibers have been highlighted [4].
Studies performed on murine models of diabetes revealed the early occurrence of
some functional changes including thermal hyperalgesia and mechanical allodynia [15, 4244] caused by the hyperactivity and increased sensitivity of both unmyelinated type C nerve
fibers [15, 45, 46] and myelinated thin A-delta and thick A-beta fibers [42]. The results of
previous studies have shown that the impairment of nociceptive sensitivity in diabetic
neuropathy could be associated with changes in the expression and activity of TRPV1
receptor. Thus, in dorsal root ganglia of diabetic rats it has been shown an increase of TRPV1
expression in the origin neurons of large diameter myelinated A-beta fibers, and a reduction
of TRPV1 expression in the origin neurons of small diameter sensory fibers; however, the
activation of TRPV1 receptor by capsaicin or low pH triggers a higher response, suggesting
the involvement of modulation of expression and activity of the capsaicin-sensitive TRPV1
receptor in phenomena such as hyperalgesia and allodynia in diabetic neuropathy [16]. Other
studies performed on diabetic mice have also emphasized an increase in TRPV1 sensitivity
associated with allodynia and hyperalgesia [45].
Moreover, in diabetic neuropathy, in peripheral nerve fibers subcellular disturbances
have been reported, such as reduction of ATPases activity, mitochondrial dysfunctions and
other metabolic disorders associated with decreased nerve conduction and axo-glial
morphological changes, all these phenomena leading to atrophy and loss of nerve fibers [4749]. The mechanisms that lie at the root of these changes are still incompletely revealed [5],
yet the impairment of the nerve fibers is associated with a precarious control of glycemia,
changes in lipid profile, accumulation of advanced glycation end products and oxidative
stress generated products [6, 50-52]. Microvascular changes and disorders of endothelial
function associated with diabetes could also be involved in the development of neuropathy
[53, 54]. The endoneurial microvessels of diabetic patients show striking changes of the
vessel walls with pericyte degeneration and reduplicated basement membranes [53]. The
damage of vascular endothelium may be induced by the metabolic changes in diabetes with
increased levels of oxygen free radicals. This also may lead to neutralization of nitric oxide
(NO) inducing a reduced vasa nervorum vasodilation response with a consecutive decrease of
nerve perfusion leading to impaired nerve function [54].
3.
Diagnosis and staging of diabetic neuropathy
The diagnosis and staging of diabetic neuropathy can be achieved through various
methods assessing the sensory, motor and autonomic fibers. The assessment also refers to the
changes which appear both in small and large diameter nerve fibers [55].
The clinical examination still remains fundamental in the diagnosis of diabetic
neuropathy [2, 10]. The vibratory, pressure, proprioceptive, tactile and thermo-algesic
perception are investigated and the myotatic reflexes and the muscular force are evaluated.
The presence of clinical signs that are suggestive for neuropathy such as cutaneous xerosis,
infections, ulcers or even deformities of the extremities are also considered [56, 57]. Usually
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a standardized clinical examination which allows the calculation of a neuropathy score is
undertaken; systems that are frequently used being the Michigan Neuropathy Screening
Instrument (MNSI) [56] and the Neuropathy Disability Score (NDS) [57].
The quantitative and semi-quantitative sensory evaluation can be achieved using
various types of devices specific for each type of sensitivity. Thus, in order to test vibratory
sensitivity a tuning fork of 64/128 Hz [56-59] or devices like Neurothesiometer,
Biothesiometer, Vibratron or Vibratron II can be used [60-64]. Tactile sensitivity can be
tested with a 10-g monofilament [63], while the pressure sensitivity can be tested with the
Neuropen [9, 63, 64]. Thermal sensitivity can be investigated using devices equipped with
automatic cooling or heating probes or by devices like Therm Tip [65-67]. The nociceptor
function can be evaluated using a device like Algometer [12, 59, 68, 69] or like Neurotip [9,
63, 64]. Complex, digitally-controlled systems have been developed in order to detect the
sensitivity threshold for various types of sensations [9].
Nevertheless, the clinical evaluation and the quantitative sensory determinations using
various devices are largely biased by subjective reports of the patients and/or even by the
investigators [9, 12, 70, 71].
Nerve conduction studies can emphasize the dysfunctions of diabetic neuropathy [6],
and enable the early diagnosis of neuropathic changes [72, 73]. However, the laborious
character of the investigation makes it difficult to use this technique as a screening test for
diabetic patients [2, 7].
The impairment of the autonomic nerve fibers in distal diabetic neuropathy can be
assessed through different tests of the sudomotor function, as well as through the quantitative
sudomotor axon reflex test, which, however, requires complex equipment and a laborious
methodology [7].
During the last years, investigation of the morphology of cutaneous nerve fibers and
evaluation of their density became more and more important in the early identification of
peripheral neuropathic impairment associated with diabetes mellitus. The histological
evaluation of a skin sample harvested by biopsy allows the investigation of various types of
nerve fibers, ranging from large diameter myelinated fibers to thin unmyelinated fibers and
enabling the investigator with an exact framing of the disease together with the evaluation of
its progress [4, 9, 19, 32, 38]. Immunohistochemstry allows the identification of
intraepidermal nerve fibers, together with the study of dermal peptidergic or non-peptidergic
nerve fibers using antibodies targeted against various parts of neural structures [21, 30, 36,
74-76]. One of the most used markers in immunohistochemistry is PGP 9.5 (protein gene
product 9.5), which allows the labelling of every nervous structure in a certain tissue. Using
antibodies targeted against specific structures, various subpopulations of nerve fibers can be
highlighted. Thus, by means of this technique, especially in the nerve endings of thin sensory
fibers, the presence of a multitude of neuropeptides and neurohormons such as substance P,
CGRP, neurokinin A, galanin or α-MSH (α-melanocyte-stimulating hormone) [77, 78] was
demonstrated. Immunoreactivity for NPY and atrial natriuretic peptide (ANP) was observed
in the autonomic fibers, this labbeling being able to differentiate them from sensory nerve
fibers [36, 79]. Another marker of cutaneous autonomic nerve fibers is tyrosine hydroxylase
[80].
The evaluation of intraepidermal nerve fibers density represents an efficient way to
emphasize the impairment of small-diameter nerve fibers. Also, highlighting of
morphological changes, such as diffuse swellings of intraepidermal nerve fibers may be a
predictive factor for the the progression of neuropathy [81]. Moreover, in the diabetic patients
the histological evaluation of the density of fibers which innervate the sweat glands is
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correlated with the sudomotor function and the neuropathic symptomatology [82]. Thus, the
histological evaluation of cutaneous innervation is an essential element in the diagnostic
strategy of diabetic neuropathy. However, the information provided with regard to the
functionality of nerve fibers is limited, and the invasive character, the technical complexity
and the increased costs of the investigation do not allow the large scale use of this method.
Another way to evaluate the morphological changes of nerve fibers in diabetic
neuropathy is the nerve biopsy, usually performed at the sural nerve, but the technique is
invasive and quite laborious, which significantly limits its applicability in clinical practice [6,
83].
Information regarding the state of the thin nerve fibers can be obtained by
investigating the corneal nerve endings through confocal microscopy [84, 85]. Although the
impairment of the corneal nerve fibers is correlated with the lowering of the density of
intraepidermal nerve fibers [85], their investigation provides only indirect information
concerning the changes of the cutaneous innervation [6].
4.
Biotechnological advances for the assessment of diabetic neuropathy
In spite of the great number of evaluation techniques, in clinical practice diabetic
neuropathy is still underdiagnosed [7, 86]. Developing new methods or protocols useful for
the assessment of diabetic neuropathy represents a major challenge for the scientific research
[7, 8].
4.1.
Early markers - cutaneous nerve fibers evaluation
One of the main topics of interest is the evaluation of the functionality of cutaneous
small diameter nerve fibers as their alteration constitutes an early marker of neuropathy in
diabetes mellitus [9, 85, 87]. At the moment the available techniques have limited
applicability, are biased and involve a great variability [88]. This is why identification of new
methods for evaluation of thin nerve fibers injury is a major challenge.
The vasodilatory response induced by axon reflex expresses directly the activation of
the small diameter nociceptive nerve fibers; previous studies have suggested the possibility of
using the evaluation of this neurovascular response in order to appreciate the functionality of
the nociceptive nerve fibers [89, 90]. Recent research has validated the measurement of
neurovascular cutaneous response as an objective evaluation method of diabetic neuropathy.
The alterations of this vasodilatory response appear in the initial stages of diabetic
neuropathy, when quantitative sensory tests are still unchanged. Thus, the cutaneous
neurovascular response could be an early marker of the thin sensory nerve fibers dysfunction,
even in the subclinical stage of neuropathy [88, 9, 91].
4.2.
Models of cutaneous neurovascular response
In order to trigger the neurovascular response, various methods have been used, such
as the local administration of acetylcholin through iontophoresis [88] or the application of
thermic stimuli [9, 92], the evaluation of the local vasodilatory response being achieved with
laser Doppler flowmetry evaluation.
However, one of the best known and intensely studied models of cutaneous
neurovascular response is the one triggered by the local administration of capsaicin, the hot
ingredient of chilli pepper (Capsicum annuum). Capsaicin directly activates the vanilloid
TPRV1 receptor [37], which, as we have previously mentioned, is predominantly localized in
the thin sensory fibers and plays an important role in the pathogenesis of diabetic neuropathy.
The stimulation of nerve endings by capsaicin activates an orthodromic signal and causes a
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sensation of burning pain. In the mean while, by generating an axon reflex, it triggers the
release of proinflammatory neuropeptides, mainly SP and CGRP [93] and the onset of an
inflammatory process which includes cutaneous vasodilatation, the increase of vascular
permeabillity, plasmatic extravasation and edema [80, 94].
A recent study of our research group [95] has highlighted the possibility of using in
vivo reflectance confocal microscopy (RCM) to assess the cutaneous neurogenic vasodilatory
reaction triggered by capsaicin. This technique allows the investigation of the skin structures
to a depth of around 250 μm, its resolution being comparable with the classical histological
examination. Thus, RCM allows an in vivo study of skin structures and the real time
observation of the manner in which various micro-morphological parameters may vary [9598].
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Figure 1. RCM images of the same dermal papillae (white asterisk) acquired (A) before and (B)
40 min after the application of 1% capsaicin solution, showing an increased diameter of the
capillaries (arrows) after the application of the active substance.
RCM allows the investigation of cutaneous microvascularization both from a
structural and a functional point of view, being considered an excellent assessment method
especially for capillaries localized at the dermo-epidermal junction [95, 99]. Capillary loops
in the dermal papillae appear in transversal section as dark discs representing the vascular
lumina. They are disposed in areas surrounded by bright cells, representing the keratinocytes
and melanocytes from the basal layer of the epidermis which circumscribes the connective
tissue of the papillary dermis (see Figure 1). The real time investigation technique with the
capacity to record video sequences allows the observation and the analysis of blood cells
dynamics through cutaneous vessels. One essential characteristic of this technique is the noninvasive character which offers the possibility of a serial assessment, at various time
intervals, of the same cutaneous region.
In our previously mentioned study the technique of capsaicin administration dissolved
in the immersion oil has enabled a regular distribution of the active substance on the
investigated skin area and also the strict control of the quantity of capsaicin per tegumentary
surface unit. It has also facilitated the investigation of the same cutaneous region at each
experimental stage. These characteristics have laid the foundation for a new test able to
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investigate the functionality of cutaneous small diameter nerve fibers, with obvious clinical
applicability [95].
Moreover, both previous published research [100] and our own preliminary data have
shown that in vivo RCM allows an objective assessment of Meissner corpuscles density and
morphology in glabrous skin, thus suggesting the possibility to estimate the change of this
parameters in diabetic neuropathy (see Figure 2).
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Figure 2 A,B. RCM images showing Meissner corpuscles (arrows) as bright, roundish structures
with a heterogeneous, lobulated internal architecture, located inside the dermal papillae, disposed
in parallel rows on each side of epidermal ridges of the palm skin.
4.3.
Proteomic approaches in diabetic neuropathy
Another promising topic in modern research of diabetic neuropathy is the
investigation of proteomic changes. Published studies do not abound in the description of
proteomic approaches to diabetic neuropathy, but they allow drawing an outline in the field
of proteomics. A recent study searching for serial biomarkers by using mass-spectrometry
technology MALDI-TOF-MS (matrix-assisted-laser-desorbtion/ionisation time of flight mass
spectrometry) and bioinformatics analysis flexAnalysisTM and Clin-ProtTM has identified
peaks relevant to this disease. Among these relevant peaks, a 6631 Da peptide was identified
as a fragment of precursor Apolipoprotein C-I. The identified peptides together with this
precursor can represent good candidates for the study of biomarkers in diabetic neuropathy
[101]. Another research direction was focused on the pathology of sensory neurons in
diabetic neuropathy. In this respect, disorders have been identified in the regulation of
thousands of proteins associated to various cellular pathways, oxidative phosphorylation,
detoxification, mitochondrial dysfunction and so on. Mitochondrial respiration is affected by
hyperglicemia and can lead to remodelations at the level of Schwann cells and thus to
diabetes associated neuropathy [102].
5.
Conclusions
The development of new methods for assessing peripheral diabetic neuropathy is a
major interest topic in research. New advances in proteomics could lead to discoveries in
biomarkers that can indicate the early onset of the disease and/or improve diagnostic and
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evolution of the disease monitoring. In vivo morphological and functional investigation of
cutaneous nerve structures provides promising data and could lead to the development of new
biotechnological tests for the assessment of diabetic peripheral neuropathy, that are easy to
use, minimally invasive, with a higher sensitivity and specificity than the current methods.
Their implementation in clinical practice could enable an early diagnosis of
neurodegenerative changes of the peripheral nervous system, in a stage where treatment has
maximum efficacy, could facilitate the therapeutic monitoring of patients and reduce the risk
for comorbidities.
6.
Acknowledgments
This paper is partly supported by the Sectorial Operational Programme Human
Resources Development (SOPHRD), financed by the European Social Fund and the
Romanian Government under the contract number POSDRU 141531/2014 and by grants PNII-PT-PCCA-2013-4-1386 (NANOPATCH) and PN-II-RU-TE-2011-3-0249 from the
National University Research Council, Romania.
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