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Background: Skin, and epidermis, is innervated by sensory nerve fibres. Interactions between them and signal transduction are only partially elucidated in physiological/pathological conditions, especially in pruritus. Objectives: To study the mechanisms involved in pruritus in vitro, we developed a skin explant model re-innervated by sensory neurons. Methods: This model is based on the co-culture of human skin explants and sensory neurons from dorsal root ganglia of rats. Innervation and the expression of protease activated receptor 2 (PAR2), transient receptor potential vanilloid 1 (TRPV1) and transient receptor potential ankyrin one (TRPA1) was analysed by immunostaining. The response of the model to TRPV1, PAR2 and TRPA1 agonists was analysed by patch-clamp, qPCR and enzyme-linked immunosorbent assay. Results: After 5 days of re-innervating nerve fibres was evidenced in the epidermis. Re-innervation was correlated with decrease of epidermal thickness and the number of apoptotic cells in the tissue. The major actors of non-histaminergic itch (PAR-2, thymic stromal lymphopoietin [TSLP], TSLP-R, TRPA1 and TRPV1) were expressed in neurons and/or epidermal cells of skin explants. After topical exposure of TRPV1-(Capsaicin), TRPA1-(Polygodial) and PAR2-agonist (SLIGKV-NH2) activation of reinnervating neurons could be shown in patch-clamp analysis. The release of TSLP was increased with capsaicin or SLIGKV but decreased with polygodial. Release of CGRP was increased by capsaicin and polygodial but decreased with SLIGKV. Activation by SLIGKV showed a decrease of VEGF; polygodial induced an increase of TSLP, Tumour necrosis factor (TNF) and nerve growth factor and capsaicin lead to a decrease of sema3 and TNF expression. Conclusion: The present model is suitable for studying itch and neurogenic inflammation pathways in vitro. We observed that activation of TRPV1, TRPA1 and PAR-2 leads to different response profiles in re-innervated skin explants.
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BACKGROUND: Close interactions exist between primary sensory neurons of the peripheral nervous system (PNS) and skin cells. The PNS may be implicated in the modulation of different skin functions as wound healing. OBJECTIVE: Study the influence of sensory neurons in human cutaneous wound healing. METHODS: We incubated injured human skin explants either with rat primary sensory neurons from dorsal root ganglia (DRG) or different neuropeptides (vasoactive intestinal peptide or VIP, calcitonin gene-related peptide or CGRP, substance P or SP) at various concentrations. Then we evaluated their effects on the proliferative and extracellular matrix (ECM) remodeling phases, dermal fibroblasts adhesion and differentiation into myofibroblasts. RESULTS: Thus, DRG and all studied neuromediators increased fibroblasts and keratinocytes proliferation and act on the expression ratio between collagen type I and type III in favor of collagen I, particularly between the 3rd and 7th day of culture. Furthermore, the enzymatic activities of matrix metalloprotesases (MMP-2 and MMP-9) were increased in the first days of wound healing process. Finally, the adhesion of human dermal fibroblasts and their differentiation into myofibroblasts were promoted after incubation with neuromediators. Interestingly, the most potent concentrations for each tested molecules, were the lowest concentrations, corresponding to physiological concentrations. CONCLUSION: Sensory neurons and their derived-neuropeptides are able to promote skin wound healing.
Asunto(s)
Neuropéptidos/fisiología , Repitelización , Células Receptoras Sensoriales/fisiología , Piel/citología , Cicatrización de Heridas , Animales , Adhesión Celular , Diferenciación Celular , Proliferación Celular , Técnicas de Cocultivo , Colágeno/metabolismo , Humanos , Técnicas In Vitro , Metaloproteinasas de la Matriz/metabolismo , Miofibroblastos/fisiología , RatasRESUMEN
BACKGROUND: Tacrolimus is an immunosuppressant drug currently used for the treatment of atopic dermatitis and pruritus. This topical therapy is effective and safe, but transient burning, stinging and itch are frequently reported. OBJECTIVES: To understand the mechanisms underlying these burning sensations. METHODS: We examined the impact of tacrolimus on substance P (SP) release in an in vitro model of cutaneous neurogenic inflammation. Because phosphorylation of TRPV1 (transient receptor potential subtype vanilloid 1) plays a role in the induction of pain, we investigated whether tacrolimus regulates the phosphorylation state of TRPV1. Finally, we used a macropatch to evaluate the impact of tacrolimus on voltage-gated calcium currents of sensory neurons. RESULTS: Tacrolimus was able to induce initial SP release by extracellular calcium influx and inhibited SP release induced by capsaicin after 1, 24 and 72 h of pretreatment. Analysis of TRPV1 phosphorylation by Western blot confirmed the capacity of tacrolimus to favour phosphorylation. An electrophysiological study showed inhibitory effects on calcium currents. CONCLUSIONS: The efficacy of tacrolimus in pruritus, as well as the sensory side-effects, could be explained by a direct effect on neurons through an effect on calcineurin, possibly by a desensitization of TRPV1 and calcium currents through the PIP(2) regulation pathway.