The effect of topical neuromodulatory medications on orofacial tissue in vitro

Abstract

Systemic neuromodulatory medications (NMs) are the conventional management modality of neuropathic orofacial pain (NOP). Topical administration of NMs such as amitriptyline, carbamazepine and gabapentin have been reported to show promising results when used with NOP, however, the effect of exposure of oral mucosa and skin to topical NMs has not been examined and is therefore unlicensed. The aims of the research contained within this thesis were to: a) investigate the effects of the aforementioned NMs on cell lines and 3D tissue relevant to the orofacial tissue in vitro; b) conduct a preliminary study using RNA extracted from human gingival tissue to investigate whether the potential therapeutic targets for these NMs were expressed. Cellular viability was measured using alamarBlue®, testing the effects of NMs on monolayer cell culture (2D): human skin and oral keratinocytes and mouse fibroblasts. Effects on cell counts were investigated by a CCK-8 assay. Morphological changes and cytokine expression were investigated using scanning electron microscopy and antibody array, respectively. Human gingival tissue biopsy was used to investigate the expression of receptors at the mRNA level using PCR. Amitriptyline exposure was found to decrease cellular viability and count, along with morphological changes as opposed to carbamazepine and gabapentin which had little demonstrable effects. Amitriptyline’s cytotoxic effect was confirmed using 3D oral mucosa models. Amitriptyline’s effect on 3D models was then further examined using immunohistochemistry (H&E, anti-caspase 3 antibodies) and immunofluorescence (anti-amitriptyline antibody). In the 3D model, amitriptyline caused apoptosis after repeated exposure, but was able to traverse tissue barriers, which was also confirmed by High Performance Liquid Chromatography (HPLC). The results of preliminary genetic investigations suggested the expression of the most receptors analysed, this suggests that further studies are required to investigate the efficacy of the topical route as a treatment for NOP. The overall results demonstrate amitriptyline toxicity and avoiding topical amitriptyline is therefore likely to be advisable. Carbamazepine and gabapentin exposure were less harmful and possibly more suitable topical choices, but further studies need to be conducted.