475k A New Gastric-Emptying Mouse Model Based on In Vivo Non-Invasive Bioluminescence Functional Imaging

Abstract

Background & Aims: Immune and non-immune mediators released by gut mucosa play a role in the pathophysiology of irritable bowel syndrome (IBS). In this context, we hypothesize that soluble mediators induce phenotypic changes in gut mucosal innervation in patients with IBS. The aims of the present study were to evaluate in the colonic mucosa of IBS patients compared with healthy controls (HC): 1) Phenotypic changes in the colonic innervation, including the expression of the marker of neuronal sprouting growth-associated protein 43 (GAP-43); 2) source and expression of nerve growth factor (NGF); 3) impact of colonic mediators and NGF on neuronal cell differentiation and sprouting in human cultured neuronal cells. Methods: Mucosal biopsies were obtained from the descending colon of 18 IBS patients and 8 HC. The expression of neuronal specific enolase (NSE), GAP-43 and NGF expression were assessed by quantitative immunohistochemistry. Mucosal mediator release was quantified on colonic biopsies by means of specific immuno-enzymatic assay and/or semi-quantitative western blot. The impact ofmucosal supernatants on nerve sprouting and differentiation was assessed on cultured neuronal cell lines (SH-Sy5Y) by means of morphometric analysis. Results: IBS patients showed a significant increase in the area of lamina propria occupied by NSE+ fibers as compared to HC (2.52% vs 1.48%; P<0.05). A significant increase in GAP-43+ fibers was documented in IBS patients in comparison with HC (1.75% vs 1.05%; P<0.05). NGF immunoreactivity was significantly increased in IBS patients (1.31% vs 0.79%; P<0.05). The majority of NGF+ cells also expressed tryptase immunolabeling, suggesting that mucosal mast cells represent the main source of NGF. NGF mucosal concentration and protein expression was significantly increased in most IBS samples in comparison with HC. Mucosal supernatants obtained from IBS patients induced a 60% neuronal sprouting on cultured neuronal cell lines, which was similar to that obtained with the control stimulus retinoic acid (68%) and significantly higher than that obtained with HC (41%) (P<0.05). Neuronal sprouting was partly NGF-dependent, as it was significantly reduced by NGF neutralization. Conclusions: Nerve fiber density and growth are increased in IBS patients. NGF expression, mainly from mast cell source, is also increased. Mast cellderived NGF participates to neuronal sprouting. Our results provide a new mechanism which may contribute to the pathophysiology of IBS and identify new targets for novel therapies in patients with IBS.