We describe methods to quantify epidermal nerve fibers (ENFs) in skin biopsy specimens from diabetic candidates for pancreas transplantation and control subjects. ENFs and the dermal-epidermal basement membrane were stained by immunohistochemical methods, imaged with a confocal microscope, and quantified using a neuron tracing system. The number of ENFs per surface of epidermis was diminished in diabetic subjects. ENF number and summed length of all ENFs per volume of epidermis examined were also decreased. Length and number of branch points of single surviving ENFs were similar in skin of control and diabetic subjects. The methods and results constitute a basis for continued study of the effects of the euglycemia that attends successful pancreas transplantation and the effects of therapy in patients with various types of polyneuropathy.

Capsaicin applied topically to human skin produces itching, pricking and burning sensations due to excitation of nociceptors. With repeated application, these positive sensory responses are followed by a prolonged period of hypalgesia that is usually referred to as desensitization, or nociceptor inactivation. Consequently, capsaicin has been recommended as a treatment for a variety of painful syndromes. The precise mechanisms that account for nociceptor desensitization and hypalgesia are unclear. The present study was performed to determine if morphological changes of intracutaneous nerve fibers contribute to desensitization and hypalgesia. Capsaicin (0.075%) was applied topically to the volar forearm four times daily for 3 weeks. At various time intervals tactile, cold, mechanical and heat pain sensations were assessed in the treated and in contralateral untreated areas. Skin blisters and skin biopsies were collected and immunostained for protein gene product (PGP) 9.5 to assess the morphology of cutaneous nerves and to quantify the number of epidermal nerve fibers (ENFs). Capsaicin resulted in reduced sensitivity to all cutaneous stimuli, particularly to noxious heat and mechanical stimuli. This hypalgesia was accompanied by degeneration of epidermal nerve fibers as evidenced by loss of PGP 9.5 immunoreactivity. As early as 3 days following capsaicin application, there was a 74% decrease in the number of nerve fibers in blister specimens. After 3 weeks of capsaicin treatment, the reduction was 79% in blisters and 82% in biopsies. Discontinuation of capsaicin was followed by reinnervation of the epidermis over a 6-week period with a return of all sensations, except cold, to normal levels. We conclude that degeneration of epidermal nerve fibers contributes to the analgesia accredited to capsaicin. Furthermore, our data demonstrate that ENFs contribute to the painful sensations evoked by noxious thermal and mechanical stimuli.

The ability of capsaicin to excite and subsequently to desensitize a select group of small sensory neurons has made it a useful tool to study their function. For this reason, application of capsaicin to the skin has been used for a variety of painful syndromes. We examined whether intradermal injection of capsaicin produced morphological changes in cutaneous nerve fibers that would account for its analgesic properties by comparing cutaneous innervation in capsaicin-treated skin with psychophysical measures of sensation. At various times after capsaicin injection, nerve fibers were visualized immunohistochemically in skin biopsies and were quantified. In normal skin the epidermis is heavily innervated by nerve fibers immunoreactive for protein gene product (PGP) 9.5, whereas fibers immunoreactive for substance P (SP) and calcitonin gene-related peptide (CGRP) are typically associated with blood vessels. There was nearly complete degeneration of epidermal nerve fibers and the subepidermal neural plexus in capsaicin-treated skin, as indicated by the loss of immunoreactivity for PGP 9.5 and CGRP. The effect of capsaicin on dermal nerve fibers immunoreactive for SP was less obvious. Capsaicin decreased sensitivity to pain produced by sharp mechanical stimuli and nearly eliminated heat-evoked pain within the injected area. Limited reinnervation of the epidermis and partial return of sensation occurred 3 weeks after treatment; reinnervation of the epidermis was ϳ25% of normal, and sensation improved to 50-75% of normal. These data show that sensory dysfunction after capsaicin application to the skin results from rapid degeneration of intracutaneous nerve fibers.

We used immunohistochemistry and confocal microscopy applied to fingertip punch biopsy to study glabrous skin innervation in 14 healthy subjects. In addition to epidermal nerve fibers, we quantified mechanoreceptors and their myelinated afferents. Using digital images and dedicated software, we calculated caliber, internodal and nodal length, and G-ratio of the last four internodes of the myelinated endings. In our skin samples, we found a mean density of 59.0 +/- 29.3 myelinated endings per square millimeter with a mean diameter of 3.3 +/- 0.5 microm and an internodal length of 79.1 +/- 13.8 microm. These findings indicate that Abeta fibers undergo drastic changes in their course from the nerve trunk to the target organ, with repeated branching and consequent tapering and shortening of internodal length. Our work demonstrates that skin biopsy can give information on the status of large myelinated endings as well as unmyelinated sensory and autonomic nerves. Since distal endings are primarily involved in distal axonopathy, skin biopsy can be more suitable than sural nerve biopsy to detect early abnormalities. In addition to diagnostic applications, this technique allows clarification of the mode of termination of Abeta fibers and their relationship with mechanoreceptors, leading to relevant electrophysiological speculations.

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Impaired axonal transport has been postulated to play a role in the pathophysiology of multiple neurodegenerative disorders. In this report, we describe the results of clinical and neuropathological studies in a family with an inherited form of motor neuron disease caused by mutation in the p150 Glued subunit of dynactin, a microtubule motor protein essential for retrograde axonal transport. Affected family members had a distinct clinical phenotype characterized by early bilateral vocal fold paralysis affecting the adductor and abductor laryngeal muscles. They later experienced weakness and atrophy in the face, hands, and distal legs. The extremity involvement was greater in the hands than in the legs, and it had a particular predilection for the thenar muscles. No clinical or electrophysiological sensory abnormality existed; however, skin biopsy results showed morphological abnormalities of epidermal nerve fibers. An autopsy study of one patient showed motor neuron degeneration and axonal loss in the ventral horn of the spinal cord and hypoglossal nucleus of the medulla. Immunohistochemistry showed abnormal inclusions of dynactin and dynein in motor neurons. This mutation of dynactin, a ubiquitously expressed protein, causes a unique pattern of motor neuron degeneration that is associated with the accumulation of dynein and dynactin in neuronal inclusions.

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