THE ROLE OF NERVE GROWTH FACTOR IN EPIDERMAL WOUND REPAIR IN A HUMAN SKIN EX VIVO MODEL

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Abstract

Introduction: One of the main aims of ongoing wound healing research is to identify improved methods for accelerating epidermal repair and/or wound closure. Previously, the impact of nerve growth factor (NGF) on human skin wound healing has been tested in various assays, however the results were inconclusive. Therefore, instead of using in vitro cell culture assays or animals this study opted to assess the effects of NGF in experimentally wounded, organ cultured human skin, since this ex vivo assay mimics clinical wound healing conditions under preclinical conditions more closely than cell culture or 3D skin “equivalent” assays Aims: My main aim was to study epidermal repair in human skin wounds ex vivo in the presence or absence of NGF, NGF-neutralising antibody or the neurotrophin receptor antagonist, K252a. In addition, I aimed to examine the protein expression of endogenous NGF and its cognate receptors; TrkA and p75NTR in healthy human skin and over the course of healing ex vivo, and understand how this might be affected by NGF treatment. Methods: Utilising human skin excised during routine surgery, a partial-thickness 2 mm wound was created within a 6 mm full-thickness skin biopsy (punch-in-a-punch design) and cultured at the air liquid interphase in serum-free Williams E (WE) media for up to six days. Wound healing was assessed by quantitative histomorphometry and en face planimetry. In order to assess epidermal repair by planimetry (longitudinally), organ cultured skin was co-treated with 5-chloromethylfluorescein diacetate (CMFDA) daily and images were taken under a fluorescence upright microscope. The wound bed was treated with the assigned treatments (NGF, K252a and NGF neutralising antibody) on a daily basis. Tissue was processed for immunohistochemistry and immunofluorescence microscopy with antibodies against the proliferative marker Ki-67, or against NGF, TrkA, p75NTR, CD68 (macrophage marker) and CD1a (Langerhans cell marker). Quantification of endogenous NGF in human skin organ culture was performed using enzyme-linked immunosorbent assay (ELISA). Results: Wound healing could be quantified ex vivo via CMFDA fluorescence, which yielded rapid and robust results when compared to histological assessment. CMFDA also successfully traced cells during epidermal repair and indicates that epithelial migration in human skin wounds occurs via a collective migration mechanism. NGF treatment had no beneficial effect on the re-epithelialisation of acute human wounds ex vivo under the current assay conditions. There was little fluctuation in NGF protein expression and of its cognate receptors in wounded human skin in organ culture, suggesting that human skin ex vivo already contains abundant of NGF and its receptors. This was confirmed by ELISA. This study also localised and quantified for the first time the changes of endogenous NGF and its cognate receptor in wounds and confirmed that NGF expression was independently expressed even in denervated human skin ex vivo. However, inhibiting TrkA-mediated signalling with K252a or neutralising endogenous NGF with a specific antibody, revealed significantly impaired epidermal repair. Conclusion: This study confirms that the chosen design for studying experimentally wounded human skin ex vivo is well-suited to interrogate candidate wound healing-promoting agents. Moreover, this thesis project introduces and accurate, yet simple, fluorescent dye-based new method for rapidly assessing human epidermal repair ex vitro. The study also provides evidence that endogenous NGF and TrkA-mediated signalling are required for normal epidermal repair, even in the absence of functional skin innervation, at least under ex vivo conditions. However, the addition of excess exogenous NGF does not accelerate epidermal repair under the examined assay conditions. This raises the possibility that topical NGF administration may promote human skin wound healing only under conditions of relative NGF deficiency. The current thesis project, therefore, suggest to test healing impaired human skin (e.g. leg ulcers) in the future for insufficient NGF skin content, in which case NGF therapy might beneficial.

Keyword(s)

Epidermal repair; Ex vivo; Human skin; Nerve growth factor; Wound healing

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