No abstract

Skin biopsies from patients with neuropathic pain often show changes in epidermal innervation, although it remains to be elucidated to what extent such changes can be linked to a particular subgroup of nerve fibers and how these changes are correlated with pain intensity. Here, we investigated to what extent behavioral signs of hyperalgesia are correlated with immunohistochemical changes of peptidergic and non-peptidergic epidermal nerve fibers in a rat model of nerve injury-induced pain. Rats subjected to unilateral partial ligation of the sciatic nerve developed significant mechanical and thermal hyperalgesia as tested by the withdrawal responses of the ipsilateral footpad to von Frey hairs and hotplate stimulation. At day 14, epidermal nerve fiber density and total epidermal nerve fiber length/mm were significantly and consistently reduced compared to the contralateral side, following testing and re-testing by two blinded observers. The expression of calcitonin gene-related peptide, a marker for peptidergic nerve fibers, was not significantly changed on the ipsilateral side. In contrast, the expression of the P2X receptor, a marker for non-peptidergic nerve fibers, was not only significantly reduced but could also be correlated with behavioral hyperalgesia. When labeling both peptidergic and non-peptidergic nerve fibers with the pan-neuronal marker PGP9.5, the expression was significantly reduced, albeit without a significant correlation with behavioral hyperalgesia. In conjunction, our data suggest that the pathology of the P2X epidermal nerve fibers can be selectively linked to neuropathy, highlighting the possibility that it is the degeneration of these fibers that drives hyperalgesia.

Skin innervation is a dynamic process that may lead to changes in nerve fiber density during pathological conditions. We have investigated changes in epidermal nerve fiber density in three different rat models that selectively produce chronic itch (the dry skin model), or itch and inflammation (the dermatitis model), or chronic inflammation without itch (the CFA model). In the epidermis, we identified peptidergic fibers-that is, immunoreactive (IR) for calcitonin gene-related peptide or substance P—and non-peptidergic fibers—that is, IR for P2X3. The overall density of nerve fibers was determined using IR for the protein gene product 9.5. In all three models, the density of epidermal peptidergic nerve fibers increased up to five times when compared with a sham-treated control group. In contrast, the density of epidermal non-peptidergic fibers was not increased, except for a small but significant increase in the dry skin model. Chronic inflammation showed an increased density of peptidergic fibers without itch, indicating that increased nerve fiber density is not invariably associated with itch. The finding that different types of skin pathology induced differential changes in nerve fiber density may be used as a diagnostic tool in humans, through skin biopsies, to identify different types of pathology and to monitor the effect of therapies.

In this study, we investigated whether postburn itch in rats, after a full thickness burn, is correlated to the nervous reinnervation of the burn wound area. For this purpose, we determined scratching duration (expressed as second/hour) at 24 hours, 2, 4, 8, and 12 weeks postburn and combined this with immunohistochemistry for protein gene product 9.5 (PGP9.5) to identify all nerve fibers, calcitonin gene related peptide (CGRP) to identify peptidergic fibers, tyrosine hydroxylase (TH) for sympathetic fibers, and growth-associated protein 43 (GAP-43) for regrowing fibers. We found a modest, but highly significant, increase in scratching duration of all burn wound rats from 3 to 12 weeks postburn (maximally 63 ± 9.5 second/hour compared to sham 3.1 ± 1.4 second/hour at 9 weeks). At 24 hours postburn, all nerve fibers had disappeared from the burn area. Around 4 weeks postburn PGP 9.5- and CGRP-immunoreactive nerve fibers returned to control levels. TH- and GAP-43-IR nerve fibers, which we found to be almost completely colocalized, did not regrow. No correlation was found between scratching duration and nervous reinnervation of the skin. The present results suggest that in rat, like in human, burn wound healing will induce increased scratching, which is not correlated to the appearance of nervous reinnervation.