Measurement of unmyelinated C and A delta nociceptors through punch skin biopsy has been an important development in diabetic peripheral neuropathy over the past decade. The technique provides an objective pathological window into a population of fibers that is invisible to standard electrophysiological techniques and as a result has been difficult to investigate. Clinically, the punch biopsy technique is most often used to define a length-dependent peripheral neuropathy, but can also be used to follow patients longitudinally over time. Epidermal nerve fibers are often lost early in diabetes or impaired glucose tolerance and can be the only objective measure of neuropathy in these patients. The accessibility of cutaneous nerve fibers has also given rise to several nerve injury paradigms from which regeneration can be efficiently measured. The chapter will review the skin biopsy technique and recent findings with respect to diabetes.

Key Words: Epidermal nerve fiber; nociceptor; skin biopsy; regeneration. INTRODUCTION

This chapter describes the skin biopsy and skin blister technique and their role in the evaluation of unmyelinated nerve fibers in skin biopsies from patients with diabetes and impaired glucose tolerance. These techniques are a reliable and reproducible means of assessing C-fiber nociceptors. Historically, pathological examination of these fibers has been limited to nerve biopsies, primarily of the sural nerve. The invasive nature of these biopsies, the insensitivity to detect small degrees of unmyelinated nerve fiber loss, and the wide range of normal values all limited assessment of this population of fibers. Further complicating interpretation of nerve biopsies is the difficulty to discern differences between somatic and autonomic small caliber unmyelinated nerve fibers. Clinically, these fibers are "invisible" because nerve conduction testing assesses only myelinated large sensory and motor fibers. These limitations and the observations that individuals with sensory neuropathies have spontaneous acral neuropathic pain with allodynia led investigators to seek alternative means to assess this population of nerve fibers, and skin biopsy/blister has emerged as a result.

Early studies of epidermal innervation focused on the density and distribution of Meissner's corpuscles (1,2). The availability of antibody to protein gene product (PGP) 9.5, a neuronal ubiquitin hydrolase, rapidly led to sensitive immunohistochemical techniques to visualize nerve fibers in the skin (3). Epidermal innervation has received the most attention although myelinated fibers and autonomic fibers innervating sweat

From: Contemporary Diabetes: Diabetic Neuropathy: Clinical Management, Second Edition Edited by: A. Veves and R. Malik © Humana Press Inc., Totowa, NJ

glands or arrector pili muscles in the deep dermis can also be assessed (4-6). Epidermal fibers represent nociceptors and include both C and AS fibers. Robust normative data have been developed (7,8) and a distal predominant pattern of nerve fiber loss has been demonstrated in several conditions including diabetes, HIV, and idiopathic small fiber neuropathies (9). Less marked reductions in epidermal nerve fiber density as well as morphological changes such as prominent nerve fiber swelling are often present at more proximal and even asymptomatic sites (9).

The association between neuropathic pain and epidermal nerve fiber loss is counterintuitive as nerve fiber loss is generally associated with the absence of sensation. Neuropathic pain has been correlated with epidermal nerve fiber loss in small fiber sensory neuropathy, post herpetic neuralgia (PHN), HIV-SN, and diabetes (9-12). There are several explanations for this apparent paradox. The distal ends of nerve fibers that have withdrawn from the epidermis may act as sensitized nociceptors with reduced thresholds to noxious stimuli (hyperalgesia). Alternatively, spontaneous pain may result from ectopic discharges in peripheral nociceptors. Finally, central changes such as sprouting of injured AS fibers from deep dorsal horn lamina into superficial lamina or changes in descending modulatory pathways may result in innocuous peripheral stimulation being misinterpreted as painful.

Nerves innervating the skin arise within the dorsal root and sympathetic ganglia. As they project toward the skin surface, myelinated fibers branch off to innervate sweat glands, Meisner's corpuscles, and Merkel complexes. Similarly, autonomic fibers envelope sweat glands in a dense matrix of fibers, whereas arrector pili are innervated in a characteristic striated pattern (Fig. 1). Hair follicles are innervated by both myelinated and unmyelinated fibers with specialized nerve endings at the base of the hair shaft. Unmyelinated sensory fibers consist the majority of dermal fibers and project vertically to the subepidermal dermis where they form a horizontally oriented nerve fiber plexus. From this plexus, branches project toward the skin surface penetrating the dermal-epidermal junction. The fibers lose their Schwann cell ensheathment as they enter the epidermis and extend between keratinocytes and Langerhans cells and project toward the stratum corneum as free nerve endings. During embryogenesis, nerve growth factor (NGF) expression within the epidermis is responsible for neuronal survival and targeted growth into the skin (13). Later in development, roughly half of these fibers lose their respon-seiveness to NGF, becoming dependent on glial derived neurotrophic factor (GDNF). NGF-dependent cutaneous nerve fibers respond to noxious stimuli and express neuropep-tides including calcitonin gene related product (CGRP) and SP, whereas GDNF dependent fibers bind the Griffonia lectin IB4 and express thimidine monophosphate and P2X3 but generally not CGRP or SP. Axons from NGF responsive neurons express the high-affinity NGF receptor TrkA as well as the low-affinity receptor p75. GDNF responsive neurons express c-Ret as well as other markers. Abnormalities in both NGF and GDNF have been implicated in the pathogenesis of diabetic neuropathy.

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