Epidermal Innervation In Clinical Trials

Skin biopsy with determination of epidermal nerve fiber density lends itself to application as an outcome measure in regenerative and longitudinal studies. The technique offers the advantage over NCVs that it focuses on a larger subset of nerve fibers than nerve conduction testing and unmyelinated nerve fibers appear to be a more sensitive measure of neuropathy than large myelinated nerve fibers. Several small studies have used serial skin biopsies as an outcome measure. In a trial of recombinant human nerve growth factor (NFG) in HIV-sensory neuropathy, 62 of 270 patients who participated in the trial were included in a substudy examining the density of intraepidermal nerve fibers. Fiber density was inversely correlated with neuropathic pain as measured both by patient and physician global pain assessments (10). The intrasubject reproducibility of the technique, as assessed from correlation between baseline and the week 18 densities was 81% in the distal part of the leg, and 77% in the upper thigh. Although decreased intraepidermal nerve fiber density at the distal leg was associated with lower CD4 counts and higher plasma HIV RNA levels, there was no treatment effect in the relatively short period of 18 weeks (35). The reproducibility of the technique was good in this study and suggests that it could be incorporated in future trials of regenerative agents for sensory neuropathies. An open-label pilot study of acetyl-L-carnitine in patients with antiretroviral toxic neuropathy associated with HIV treatment demonstrated a significant increase in immunoreactivity for PGP9.5 as well as other specific fiber type neurotransmitters. This provocative study used unconventional measures of epidermal innervation and a larger controlled study is currently underway (36). In a small, 18 week open label trial of topiramate in people with moderate diabetic neuropathy, Vinik et al. reported that treatment was associated with improvements in epidermal nerve fiber density and length (37). Enhanced nerve fiber length was observed at all tested sites including the forearm although increases in ENF density were seen only at the proximal leg site. Although further analyses need to be performed, this finding suggests that nerve fiber length may be a useful adjunct measure. There is relatively little longitudinal data using skin biopsy in people with diabetes. Several small series suggest that at the distal thigh, the ENF density declines at a rate of about 1 fiber/mm per year. This combined with the observation that ENF density is correlated with heat pain threshold after adjusting for diabetes duration, age, and gender (27) suggest that a change of 2-3 fibers/mm may represent a clinically meaningful change.

Epidermal nerve fibers offer an additional advantage that they can be easily and repeatedly sampled over time. Their superficial nature also allows them to be injured in a standardized fashion and recovery from such nerve injuries have been used as a measure of nerve fiber regeneration. Several models based on such an approach have been developed to measure collateral (38) and regenerative (18) sprouting in human subjects. Reinnervation of the epidermis following removal of a 3 mm diameter cylindrical tissue plug allows collateral sprouting to be efficiently measured. Following removal of a conventional 3 mm skin biopsy, the site heals by a process of granulation and eventually becomes re-epithelialized. The collagen plug within the dermis of the healing biopsy site acts as a barrier to the distal ends of the transected nerves. Therefore, reinnervation of the epidermis within the original 3 mm biopsy site can only occur through sprouting of uninjured fibers peripheral to the incision line (Figs. 5 and 6). The degree of collateral

Fig. 5. (A) Method to measure collateral sprouting of human epidermal nerve fibers. Following removal of a standard 3 mm biopsy tissue plug (left frame), the site heals by a process of granulation (right frame). Nerve fibers within the dermis are not able to penetrate the collagen scar that forms in the healed biopsy site. The only mechanism for the re-epithe-lialized epidermis to become reinnervated is for fibers to sprout from the epidermis peripheral to the healed 3 mm incision line into denervated central zone. This collateral sprouting can be assessed by taking a larger concentric biopsy centered on the healed, original 3 mm biopsy site (dotted line, right panel). (B) Example of collateral sprouting. The yellow arrow indicated the location of the original 3 mm biopsy incision. Epidermal nerve fibers peripheral to the incision line grow into the central denervated epidermis by a process of collateral sprouting. These sprouts frequently grow along the epidermal side of the dermal-epidermal junction adjacent to the basal keratinocytes which are known to be a source of neurotrophin production. Adapted from ref. 38.

Fig. 5. (A) Method to measure collateral sprouting of human epidermal nerve fibers. Following removal of a standard 3 mm biopsy tissue plug (left frame), the site heals by a process of granulation (right frame). Nerve fibers within the dermis are not able to penetrate the collagen scar that forms in the healed biopsy site. The only mechanism for the re-epithe-lialized epidermis to become reinnervated is for fibers to sprout from the epidermis peripheral to the healed 3 mm incision line into denervated central zone. This collateral sprouting can be assessed by taking a larger concentric biopsy centered on the healed, original 3 mm biopsy site (dotted line, right panel). (B) Example of collateral sprouting. The yellow arrow indicated the location of the original 3 mm biopsy incision. Epidermal nerve fibers peripheral to the incision line grow into the central denervated epidermis by a process of collateral sprouting. These sprouts frequently grow along the epidermal side of the dermal-epidermal junction adjacent to the basal keratinocytes which are known to be a source of neurotrophin production. Adapted from ref. 38.

sprouting can be measured by a concentric 4 or 5 mm biopsy centered on the healed 3 mm biopsy site (Fig. 5). Although detailed time course experiments have not been published in people with diabetes, preliminary evidence suggests that collateral sprouting is reduced in neuropathic states such as diabetes and HIV infection (39).

Regenerative sprouting has been systematically measured following a "chemical axo-tomy" produced by topical capsaicin (18). Application of capsaicin either topically as a cream or injected into the epidermis has been shown to produce a superficial denerva-tion of the skin (40,41). When applied topically in an occlusive bandage, capsaicin produces a standardized injury that is reproducible and well-tolerated (Fig. 6) (18). Studies in healthy control subjects and people with diabetes have demonstrated that functional abnormalities in regenerative sprouting are present among subjects with diabetes with no signs or symptoms of neuropathy (Fig. 7). This observation suggests that nerve is affected early in the course of diabetes and that regenerative abnormalities may be the earliest sign of nerve dysfunction. It could be speculated that an abnormality in nerve

Day 2

Fig. 6. Representative skin biopsy sections demonstrating that topical capsaicin application produces a superficial denervation. The epidermis becomes reinnervated over several months. The day -2 biopsy depicts normal epidermal innervation at baseline. Following capsaicin treatment (day 0), there is complete denervation of the epidermis and subepidermal dermis. Biopsies at 28 and 56 days demonstrate reinnervation of the epidermis. Recovery of ENF density has been correlated with heat pain. From ref. 44.

Fig. 6. Representative skin biopsy sections demonstrating that topical capsaicin application produces a superficial denervation. The epidermis becomes reinnervated over several months. The day -2 biopsy depicts normal epidermal innervation at baseline. Following capsaicin treatment (day 0), there is complete denervation of the epidermis and subepidermal dermis. Biopsies at 28 and 56 days demonstrate reinnervation of the epidermis. Recovery of ENF density has been correlated with heat pain. From ref. 44.

regeneration is followed by development of swellings that are in turn followed by development of neuropathy. Correction of subclinical abnormalities in regeneration as an outcome measure has several attractive features. First, it would allow recruitment of a non-neuropathic population of people with diabetes. This will simplify trial designs and

Fig. 7. For each subject, a regression line from postcapsaicin time-points is generated and the slope of this line is used as the rate of regeneration. The mean line for each group is shown as a thick solid line. The rate of regeneration following denervation is 0.177 ± 0.075 fibers per mm/day for control subjects (red), 0.10 ± 0.07 fibers per mm/day (p = 0.03) for subjects with diabetes but no neuropathy (green), and 0.04 ± 0.03 fibers per mm/day (p = 0.03) for subjects with diabetes and neuropathy (blue).

Fig. 7. For each subject, a regression line from postcapsaicin time-points is generated and the slope of this line is used as the rate of regeneration. The mean line for each group is shown as a thick solid line. The rate of regeneration following denervation is 0.177 ± 0.075 fibers per mm/day for control subjects (red), 0.10 ± 0.07 fibers per mm/day (p = 0.03) for subjects with diabetes but no neuropathy (green), and 0.04 ± 0.03 fibers per mm/day (p = 0.03) for subjects with diabetes and neuropathy (blue).

facilitate the recruitment of homogeneous study subject populations. Second, correction or partial correction of abnormalities in regeneration is a concrete target with a clear clinical interpretation. Furthermore, an improvement in the regeneration rate will precede an improvement in nerve fiber density and is likely to be a more sensitive measure. Finally, such a study designed to detect a 50% normalization of regeneration with 80% power in nonneuropathic subjects with diabetes would require about 65 subjects per treatment arm. A recent trial of Timcodar dimesylate in healthy control subjects used such measures of collateral and regenerative sprouting as outcome measures. Although the compound did not accelerate regeneration by either measure, the trial did demonstrate that such an approach was feasible and the measures were reproducible and robust (42).

In conclusion, skin biopsy with determination of epidermal nerve fiber density is a powerful tool that provides investigators insight into a population of nerve fibers that is prominently affected in diabetes and yet has been relatively under investigated. The superficial nature of epidermal nerve fibers allows repeated sampling of these nerves in a relatively non invasive fashion, and in sites that cannot be assessed through conventional electrodiagnostical techniques. These features have allowed investigators to diagnose neuropathy earlier and to define an association between neuropathy and impaired glucose tolerance. Finally, the ability to injure these fibers in a standardized fashion has led to novel measures of human axonal regeneration that may provide a more sensitive scale by which to assess promising regenerative compounds.

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Peripheral Neuropathy Natural Treatment Options

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