The reduction of intraepidermal P2X₃ nerve fiber density correlates with behavioral hyperalgesia in a rat model of nerve injury‐induced pain

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.

The mechanisms of protease-activated receptor 2 in mediating pain behaviors through nonselective cation channel signaling

“…In the peripheral terminals of sensory neurons, PLC-PKC cascade can directly activate or sensitize neighboring TRPV1 and TRPA1 channels in DRG [16,28,80,81]. According to the subdivision of DRG neurons, TRPV1-and TRPA1-positive neurons are related to both peptidergic and non-peptidergic nerve fibers, whereas P2 × 3-expressing neurons represent non-peptidergic DRG neurons [82][83][84][85][86][87]. It has been shown that the co-expression of other receptors with P2 × 3 in C-fibers can alter neuronal sensitization induced P2 × 3 activation.…”

Clinical, electrophysiological, and cutaneous innervation changes in patients with bortezomib-induced peripheral neuropathy reveal insight into mechanisms of neuropathic pain

“…The detailed immunohistochemical procedure is described in a recent publication. 36 In short, a two-step immunohistochemistry with Streptavidin-Biotin Complex was used for protein gene product 9.5 (PGP9.5), while additional tyramid signal amplification was applied for calcitonin gene-related peptide (CGRP). Concentrations of primary antibodies were 1:10,000 for rabbit anti-PGP 9.5 (Catalog# ADI-905–520; Enzo Life Sciences, Farmingdale, NY), 1:50,000 for guinea pig anti-CGRP (Catalog # 16013; Progen Biotechnik, Heidelberg, DE), and 1:100,000 for rabbit anti-CGRP (Catalog# PC205L; Millipore, Billerica, MA).…”

“…We also attempted to visualize the nonpeptidergic subclass of nociceptors in the skin, 36 using a histochemical staining method (i.e., acetylcholinesterase) 44 and various immunohistochemical markers (i.e., P2X 3 , IB4, RET, GINIP), 36 , 45 – 47 at varying concentrations and using specific protocols but were unable to obtain reproducible staining patterns allowing for quantification of these fibers.…”

“…Frames were selected so that they comprised the entire epidermis, subdermal layer, and at least 50 µm of upper dermis. The following parameters were manually counted/traced for both PGP9.5 and CGRP, by a single, blinded observer (MB), as previously described 36 : Intraepidermal nerve fiber density (IENFD) of PGP9.5 and GCRP: the number of crossings of the dermal–epidermal junction per millimeter length of the epidermal surface. 34 The length of the epidermal surface was automatically determined by the ImageScope software after tracing.…”

“…Pathological changes in nerve fibers were studied in skin biopsies from the lower leg. 34 , 35 Since we have previously hypothesized that neuropathic pain may be driven by selective degeneration of subsets of unmyelinated nerve fibers in an animal model of nerve-injury induced pain, 36 the second aim of the current study was to use BiPN as a model for nerve-injury induced pain and to study correlations between pathological changes in subsets of unmyelinated nerve fibers in skin biopsies and neuropathic pain descriptors. This way, we aim to test whether the abovementioned hypothesis can be corroborated in humans with neuropathic pain.…”