40. Inflammatory Bowel Disease Affects Neuronal Survival in the Dorsal Motor Nucleus of the Vagus

Introduction: Inflammatory bowel disease (IBD) is a common clinical problem. Enteric neurons in the intestinal wall have been found to be reduced in number and functionally altered by intestinal inflammation. However, the effects of IBD on the central neurons projecting to the enteric nervous system are largely unknown. The dorsal motor nucleus of the vagus (DMNV) is located in the brainstem and integrates peripheral and central signals, sending efferent signals to the gastrointestinal system. The purpose of this study was to determine the effects of IBD on neuronal survival of the DMNV in an animal model. Methods: At laparotomy, the fluorescent carbocyanine dye, DiI, was injected into the stomach wall of adult Sprague-Dawley rats in order to retrogradely label neurons of the DMNV. One week later, IBD was induced with enemas containing trinitrobenzene sulphonic acid (TNBS) in 50% ethanol while control animals were given 50% ethanol enemas. After 7 days the rats were sacrificed and tissue obtained for study. Fluorescent microscopy was used to measure DiI staining in the DMNV. In vitro studies were performed using primary culture of DMNV neurons which were isolated from neonatal rat brainstem using microdissection and enzymatic digestion. Cell proliferation was measured by BrdU incorporation assay. Apoptosis was measured by an ELISA assay. Data were analyzed using ANOVA. Results are expressed as mean SEM. Results: Animals treated with TNBS lost weight (8.8 10.5 grams) compared to control animals which gained 44.6 5.5 grams. Food intake for seven days was significantly decreased compared to control animals (66 1.2 vs. 182 2.2 grams, p 0.001). The colon was grossly edematous and inflamed, weighing significantly more in IBD compared to control animals (6.3 0.8 vs. 2.2 0.1 grams, p 0.001). Microscopic analysis demonstrated a 77% decrease of DiI labeling in the DMNV of IBD animals compared to controls (p 0.005). BrdU cell proliferation ELISA assay was performed on cultured DMNV neurons after 24 hours of exposure to cytokine. The cytokines examined were IL-1 , IL-6, or TNF, each at concentrations of 0.1, 1, 10, and 100 ng/ml. Incubation with IL-1 at 0.1, 10, and 100 ng/ml resulted in significantly decreased proliferation (70% 7%, 74% 6%, 65% 8%, and 58% 9% vs. control, respectively; p 0.05), as did incubation with IL-6 at 1, 10 and 100 ng/ml (57% 5%, 58% 9%, and 68% 8% vs. control, respectively; p 0.05) or TNFat 1, 10 and 100 ng/ml (57% 2%, 61% 7%, and 36% 9% vs. control, respectively; p 0.05). Apoptosis was measured after 24 hours incubation with IL-1 , IL-6, or TNFeach at concentrations of 0.1, 1, 10, and 100 ng/ml. Apoptosis of DMNV neurons was increased after exposure to IL-1 at concentrations of 10 and 100 ng/ml (156% 21% and 161% 24% vs. control, respectively; p 0.05), as well as TNFat 10 and 100 ng/ml (160% 25% and 164% 25% vs. control, respectively; p 0.05). Exposure to IL-6 did not significantly change neuronal apoptosis. Conclusions: DMNV neurons projecting to the stomach are reduced in number after induction of colitis in rats. In vitro, pro-inflammatory cytokines diminish DMNV neuronal proliferation and increase apoptosis. These results indicate that IBD results in systemic inflammation which adversely affects neuronal survival in the DMNV.