Elsevier

Acta Histochemica

Volume 110, Issue 5, 8 September 2008, Pages 351-362
Acta Histochemica

REVIEW
Epidermal nerve fiber quantification in the assessment of diabetic neuropathy

https://doi.org/10.1016/j.acthis.2007.12.004Get rights and content

Summary

Assessment of cutaneous innervation in skin biopsies is emerging as a valuable means of both diagnosing and staging diabetic neuropathy. Immunolabeling, using antibodies to neuronal proteins such as protein gene product 9.5, allows for the visualization and quantification of intraepidermal nerve fibers. Multiple studies have shown reductions in intraepidermal nerve fiber density in skin biopsies from patients with both type 1 and type 2 diabetes. More recent studies have focused on correlating these changes with other measures of diabetic neuropathy. A loss of epidermal innervation similar to that observed in diabetic patients has been observed in rodent models of both type 1 and type 2 diabetes and several therapeutics have been reported to prevent reductions in intraepidermal nerve fiber density in these models. This review discusses the current literature describing diabetes-induced changes in cutaneous innervation in both human and animal models of diabetic neuropathy.

Introduction

Skin biopsies are emerging as a valuable means of diagnosing and staging peripheral nerve disorders. As a minimally invasive technique, skin biopsies allow for assessment of a variety of fiber types, including the small unmyelinated fibers that are difficult to evaluate by other means. There is particular interest in using this technique to provide an assessment of distal symmetrical neuropathies, such as diabetic neuropathy, both to stage and evaluate progression of neuropathy as well as to assess efficacy of potential therapeutics (Kennedy et al., 1996; Lauria et al., 1998; McArthur et al., 1998). Until recently, clinical studies have been restricted to using electrophysiologic and sensory testing as surrogate markers for nerve pathology, or to the evaluation of sural nerve biopsies, which are invasive and not widely approved as a diagnostic tool.

The innervation of the skin consists of low-threshold mechanoreceptors, thermoreceptors and nociceptors, along with their myelinated and unmyelinated axons (Light and Perl, 1993). Epidermal nerve fibers are predominantly capsaicin-sensitive unmyelinated C-fibers involved in detecting thermal nociceptive pain (Nolano et al., 1999; Malmberg et al., 2004). These fibers originate from dorsal root ganglia neurons and form subepidermal bundles in the papillary dermis, immediately subjacent to the stratum basale of the epidermis. Individual fibers lose their Schwann cell ensheathment as axons cross the dermal–epidermal junction and weave through the keratinocytes of the epidermis (Wang et al., 1990; Kennedy and Wendelschafer-Crabb, 1993). These epidermal nerve fibers can be divided into two subsets, peptidergic and non-peptidergic. The peptidergic neurons are nerve growth factor (NGF)-responsive and express calcitonin gene-related peptide (CGRP), substance P and the trkA receptor. The non-peptidergic nerves are glial cell line-derived neurotrophic factor (GDNF)-responsive and express GDNFRα and the P2X3 receptor (Snider and McMahon, 1998). Quantification of epidermal nerve endings may prove to be a valuable diagnostic tool, particularly for the early detection of ‘dying back’ neuropathies.

About half of all diabetic patients will develop neuropathy (Pirart et al., 1978). Distal symmetric neuropathy, which initially affects the hands and feet, is the most common form (Thomas, 1997). The consequences of sensory neuropathy include altered perception of thermal, tactile and vibratory stimuli and can range from hyperalgesia and allodynia to hypoalgesia. Many patients ultimately experience a complete loss of sensation in their hands and feet, which can increase the risk of trauma and lead to infection and amputation. Dysfunction involving the small epidermal C-fibers that respond to thermal stimuli is among the most commonly reported (Polydefkis et al., 2003). Hence, their assessment is particularly valuable, not only for diagnostic and staging purposes, but also for the evaluation of treatments for diabetic neuropathy.

Section snippets

Antibodies

The development of antibodies against a variety of neuronal marker proteins has allowed for the immunohistochemical assessment of intraepidermal nerve fibers (IENFs). The most commonly used antibody is directed against protein gene product 9.5 (PGP9.5), a cytosolic ubiquitin carboxyl-terminal hydroxylase that is found in all neurons and that, in skin biopsies, binds to dermal nerve bundles as well as to both peptidergic and non-peptidergic epidermal nerve profiles (Dalsgaard et al., 1989;

Cutaneous innervation in diabetic humans

Initial studies assessing cutaneous innervation in the skin of diabetic human subjects found reduced immunoreactivity to PGP9.5, along with reduced immunoreactivity to the neuropeptides CGRP, SP, vasoactive intestinal polypeptide (VIP) and neuropeptide Y (NPY) (Levy et al., 1989; Lindberger et al., 1989). Multiple subsequent studies have confirmed the decrease in PGP9.5-immunoreactive IENFs (Levy et al., 1992; Properzi et al., 1993; Kennedy et al., 1996; Lauria et al., 1998; Hirai et al., 2000;

Correlation with other indices of diabetic neuropathy

Issues of assay reproducibility, sensitivity and the rate of disease progression have impeded clinical trials of agents designed to prevent, halt or reverse indices of diabetic neuropathy. The viability of IENF quantification as an alternative or complimentary biomarker will depend on whether the assay correlates with other measures of neuropathy and provides comparable or enhanced sensitivity and reproducibility. A number of studies have begun to address this issue. There is a significant

Animal models

The literature describing cutaneous innervation in non-human primates with diabetes is currently limited to a single paper assessing innervation of glabrous skin from monkeys with naturally occurring type 2 diabetes (Pare et al., 2007). Biopsies from monkeys with short-term hyperglycemia showed a hypertrophy of epidermal nerve fibers. However, in monkeys that were diabetic for a duration of 8 years or longer, a severe reduction in PGP9.5-immunoreactive IENFs was observed. CGRP- and

Functional consequences of epidermal fiber loss in diabetic rodents

While the obvious prediction is that loss of capsaicin-sensitive TRPV-1 bearing epidermal C-fibers should result in thermal hypoalgesia, there have been few attempts to correlate loss of epidermal fibers in rodents with direct functional consequences. In the STZ-diabetic rat, thermal hypoalgesia was present 5 weeks after onset of diabetes, but a reduction in IENF density was not observed until 11 weeks (Bianchi et al., 2004). Similarly, in thy1-YFP mice, cutaneous nerve fiber loss was not

Therapeutic interventions

The effects of therapeutic intervention on cutaneous innervation in animal models of diabetes remain largely unexplored. In the STZ-diabetic rat, there is a report on the neuroprotective properties of erythropoietin (Bianchi et al., 2004). While the density of PGP9.5 immunoreactive profiles in the epidermis was unchanged after 5 weeks of diabetes, a significant reduction was observed after 11 weeks of diabetes and administration of erythropoietin from week 5 onwards prevented the epidermal

Pathogenic mechanisms

In most clinical studies, distinctions have not been made between patients with insulin-dependent type 1 diabetes and patients with insulin-resistant type 2 diabetes. However, whether or not IENF loss is affected by the presence or absence of insulin is an important question. While several studies have reported a reduction in IENF density in rat models of type 1 diabetes (Bianchi et al., 2004; Lauria et al., 2005; Leonelli et al., 2007; Roglio et al., 2007), the effects of hyperglycemia on

Conclusion

Quantification of epidermal innervation is emerging as a reliable and minimally invasive means of diagnosing and staging diabetic neuropathy. Along with the assessment of corneal nerve fiber density using the non-invasive technique of in vivo corneal confocal microscopy (Malik et al., 2003), which has shown a similar progressive reduction in a group of patients with diabetic neuropathy (Quattrini et al., 2007), quantification of IENF density offers the potential for direct assessment of

Acknowledgments

We would like to thank Drs. Itzack Fisher and Corinne Jolivalt for providing the anti-high molecular weight tau antibody and Joshua A. Gregory for providing a micrograph of PGP9.5-immunolabeled rat skin. Supported by NIH grants DK057629 (NAC) and DK078374 (APM) and the Juvenile Diabetes Research Foundation (APM).

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