Elsevier

Clinical Neurophysiology

Volume 121, Issue 8, August 2010, Pages 1256-1266
Clinical Neurophysiology

The time course of CO2 laser-evoked responses and of skin nerve fibre markers after topical capsaicin in human volunteers

https://doi.org/10.1016/j.clinph.2010.02.159Get rights and content

Abstract

Objective

To assess the temporal relationship between skin nerve denervation and regeneration (dermal and intra-epidermal fibres, IENF) and functional changes (CO2 laser-evoked potentials, LEPs, and quantitative sensory tests, QST) after topical cutaneous application of capsaicin.

Methods

Capsaicin (0.075%) was applied to the lateral calf for three consecutive days. QST, LEPs and skin biopsies were performed at baseline and time intervals up to 54 days post-capsaicin treatment. Biopsies were immunostained with antibodies for PGP9.5, TRPV1, and GAP-43 (marker of regenerating nerve fibres), and analyzed for IENFs and dermal innervation (for GAP-43).

Results

At 1 day post-capsaicin, cutaneous thermal sensitivity was reduced, as were LEPs. PGP9.5, TRPV1, and GAP-43 immunoreactive-nerve fibres were almost completely absent. By Day 12, LEPs had fully recovered, but PGP9.5 and TRPV1 IENF continued to be significantly decreased 54 days post-capsaicin. In contrast, dermal GAP-43 immunoreactivity closely matched recovery of LEPs.

Conclusions

A good correlation was observed between LEPs and GAP-43 staining, in contrast to PGP9.5 and TRPV1. Laser stimulation is a non-invasive and sensitive method for assessing the initial IENF loss, and regenerating nerve fibres.

Significance

Assessing skin biopsies by PGP9.5 immunostaining alone may miss significant diagnostic and prognostic information regarding regenerating nerve fibres, if other approaches are neglected, e.g. LEPs or GAP-43 immunostaining.

Introduction

Small-fibre neuropathy (SFN) has been described as a subtype of sensory neuropathy with a number of known causes and needing special investigation, since it affects C and Aδ-fibres which cannot be assessed directly with conventional nerve conduction studies. However, a number of investigative tools have been developed over the two last decades to help with the diagnosis of small-fibre neuropathies (Lacomis, 2002). The purpose of the present study was to determine the validity and sensitivity of CO2 laser stimulation for diagnostic and pharmacological investigations in a “volunteer model” of SFN, and its relationship to quantitative sensory tests and skin nerve fibre markers.

Brief (millisecond) laser stimuli selectively activate Aδ and C-nociceptors in the epidermal layer of skin (Bromm et al., 1984), and evoke time-locked brain responses, i.e. laser-evoked brain potentials (LEPs; Carmon et al., 1976, Plaghki and Mouraux, 2005). A validated experimental model of chemical axotomy produces reversible superficial skin denervation by topical capsaicin application in healthy subjects (Nolano et al., 1999, Polydefkis et al., 2004). Repeated topical application of capsaicin has been shown to elevate warm detection and heat pain thresholds (Carpenter and Lynn, 1981, Lynn, 1990, Simone and Ochoa, 1991), and to cause reduction of LEPs (Beydoun et al., 1996), followed by their restoration when topical capsaicin is discontinued.

In this study, two particular aspects were investigated in the cycle of nerve degeneration followed by regeneration following application of topical capsaicin: (1) the temporal relationship between functional and morphological measures, and (2) the sensitivity of these measures to change during the regeneration of the epidermal fibres. The functional measures used were psychophysical (quantitative sensory testing, QST) and electrophysiological (LEPs). Morphological measures were based on the quantification of small nerve fibres in punch biopsies of skin. Intra-epidermal nerve fibre density (IENFd), assessed by PGP9.5 immunohistochemistry, has been validated as a reproducible marker of SFN with high sensitivity and specificity as compared to other diagnostic tools (Lauria et al., 2005, Vlcková-Moravcová et al., 2008, England et al., 2009). Following topical capsaicin application, there is a rapid and profound loss of epidermal nerves. After discontinuation, the regenerative process starts but, in contrast to the functional variables, recovers very slowly over weeks (Nolano et al., 1999) to months, and is usually still incomplete at 6 months (Polydefkis et al., 2004). Although the time lag between functional and morphological recovery after topical capsaicin has been reported by several authors (Simone et al., 1998, Nolano et al., 1999, Khalili et al., 2001, Malmberg et al., 2004), it has, to our knowledge, never been investigated using electrophysiological methods such as LEPs, or histochemically with the nerve markers TRPV1 (heat/capsaicin receptor) and GAP-43 (marker of regenerating nerve fibres).

Section snippets

Subjects

Twelve paid healthy volunteers, with a mean age of 42 years (age range 30–55; 7 men) were included in the study. Ten of these subjects were right-handed with a body mass index (mean ± SD) of 25.3 ± 4.1 kg/m2. The subjects had no history of alcohol or drug abuse, significant illnesses, or clinical findings suggestive of peripheral or central nervous system disorders. All had normal routine lab testing (blood sampling and urine analysis). Written informed consent was obtained from all subjects. The

Nerve fibre densities in skin biopsies

At baseline, skin biopsies taken from the capsaicin-treated leg of the 12 volunteers yielded a significantly higher IENFd using immunofluorescence of PGP9.5 (Mean IF IENF/mm ± SD: 13.8 ± 7.02) as compared to the chromogen assay (Mean IHC IENF/mm ± SD: 6.6 ± 5.04) (z = −0.483; p < 0.013). Furthermore, there was no significant difference observed in baseline IENF/mm between the dominant (control) and non-dominant leg for both staining techniques (z = −0.454; p = 0.65). All subsequent data and analyses of PGP9.5

Discussion

The present study aimed at determining (1) the temporal relationship between functional tests based on CO2 laser technology and morphological measures based on IENFd in skin biopsies and (2) the sensitivity to change of these tests using an experimental model of topical application of capsaicin. The results show that most functional and morphological measures were sensitive to the degenerative and subsequent regenerative processes, and confirm that morphological recovery with conventional

General conclusions

The method consisting of three consecutive days of 0.075% topical capsaicin cream application was successful in mimicking a “reversible small-fibre neuropathy”. Comparative assessment of CO2 laser stimulation and IENFd with the standard PGP9.5 and also TRPV1 immunostaining revealed a mismatch at approximately 12 days post-capsaicin, with a full functional but not morphological recovery – the IENF fibres recovered by only 50% after 54 days. In contrast, the GAP-43 fibres in the sub-epidermis

Conflict of interest

The authors declare that they have no competing interests but M.T., J.S. and T.M. are employees of Johnson and Johnson. M.R. was supported by a grant from Johnson and Johnson.

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