1090 Analysis of the activation of TRPV1, TRPA1 and PAR-2 receptors with a cytokine profile and electrophysiological response in a re-innervated skin explant model to study pruritus

Abstract

In order to study pruritus in vitro, we used a skin explant model that has been re-innervated by sensory neurons. This model is based on a co-culture between a human skin explant and sensory neurons from dorsal root ganglia of rats. After 5 days of co-culture in transwell, we were able to confirm the presence of nerve fibers in the epidermis. Re-innervation was correlated with decrease of epidermal thickness and apoptosis compared with non-re-innervated skin. We showed by IHC that some of the major actors of non-histaminergic itch (PAR-2, TSLP, TSLP-R, TRPA1, IL31-R, TRPV1) were present in neurons and/or epidermal cells of skin explant. The functionality of the model was assessed by the measurement of TSLP and CGRP release in the supernatant after topically applying a PAR-2 agonist (SLIGKV-NH2, 100μM), TRPV1 agonist (Capsaicin, 10μM) or TRPA1 agonist (Polygodial, 3μM) during 24h on the explant. Interestingly release of TSLP was significantly increased with capsaicin or SLIGKV addition but decreased with polygodial. Release of CGRP was significantly increased by capsaicin and polygodial but decreased with SLIGKV. With qPCR, on skin explant, activation by SLIGK showed a decrease of VEGF expression, polygodial an increase of TSLP, TNF and NGF expression and capsaicin a decrease of sema3 and TNF expression. After topical application of agonists on the explant, patch-clamp analysis on neurons showed activation of 1 fiber for 11 evaluated using SLIGKV, 5/5 with polygodial and 4/6 using capsaicin. In conclusion, this model can be used for studying itch and neurogenic inflammation in vitro. We observed that activation of TRPV1, TRPA1 and PAR-2 lead to different profile of response in skin explant or neurons