BACKGROUND: In inflammatory bowel disease, autonomic dysfunction contributes to symptoms, morbidity, and health care resource utilization. Efferent vagal neurons, which provide the primary parasympathetic input to the gastrointestinal tract, are housed in the dorsal motor nucleus of the vagus (DMV) in the brainstem. This study seeks to characterize the effects of IBD on DMV neuronal survival and function. METHODS: TNBS (picrylsulfonic acid) was administered by enema to induce colitis in rats. Brain sections through the DMV were examined for neuronal apoptosis using TUNEL labeling, and for glial cell activation by immunofluorescence. Prothrombin production was evaluated via quantitative RT-PCR from DMV tissue, as well as by double immunofluorescence in DMV sections. To investigate the effects of thrombin in the DMV, thrombin or thrombin and an antagonist to its receptor were administered into the fourth ventricle via a stereotactically placed cannula. DMV sections were then examined for apoptosis by TUNEL assay. To evaluate the effect of thrombin on DMV neuronal function, we examined calcium signaling in primary DMV neuron cultures following exposure to thrombin and other neurotransmitters. RESULTS: TNBS colitis is associated with significantly increased rates of DMV neuronal apoptosis, affecting 12.7 % of DMV neurons in animals with colitis, compared to 3.4 % in controls. There was a corresponding increase in DMV neuron activated caspase-3 immunoreactivity (14.8 vs. 2.6 % of DMV neurons). TNBS-treated animals also demonstrated significantly increased DMV astrocyte and microglial immunoreactivity, indicating glial cell activation. DMV prothrombin production was significantly increased in TNBS colitis, with a close anatomic relationship between prothrombin and microglia. Direct DMV exposure to thrombin replicated the apoptosis and activation of caspase-3 seen in TNBS colitis; these effects were prevented by coadministration of the PAR-1 inhibitor FR171113. Cultured DMV neurons exhibited impaired calcium signaling in response to neurotransmitters following exposure to thrombin. Glutamate-induced calcium transients decreased by 59 %, and those triggered by GABA were reduced by 61 %. PAR-1 antagonism prevented these thrombin-induced changes in calcium signaling. CONCLUSIONS: IBD is associated with DMV microglial activation and production of prothrombin. Thrombin in the DMV causes vagal neuron apoptosis and decreased sensitivity to neurotransmitters.
BACKGROUND: In inflammatory bowel disease, autonomic dysfunction contributes to symptoms, morbidity, and health care resource utilization. Efferent vagal neurons, which provide the primary parasympathetic input to the gastrointestinal tract, are housed in the dorsal motor nucleus of the vagus (DMV) in the brainstem. This study seeks to characterize the effects of IBD on DMV neuronal survival and function. METHODS: TNBS (picrylsulfonic acid) was administered by enema to induce colitis in rats. Brain sections through the DMV were examined for neuronal apoptosis using TUNEL labeling, and for glial cell activation by immunofluorescence. Prothrombin production was evaluated via quantitative RT-PCR from DMV tissue, as well as by double immunofluorescence in DMV sections. To investigate the effects of thrombin in the DMV, thrombin or thrombin and an antagonist to its receptor were administered into the fourth ventricle via a stereotactically placed cannula. DMV sections were then examined for apoptosis by TUNEL assay. To evaluate the effect of thrombin on DMV neuronal function, we examined calcium signaling in primary DMV neuron cultures following exposure to thrombin and other neurotransmitters. RESULTS:TNBS colitis is associated with significantly increased rates of DMV neuronal apoptosis, affecting 12.7 % of DMV neurons in animals with colitis, compared to 3.4 % in controls. There was a corresponding increase in DMV neuron activated caspase-3 immunoreactivity (14.8 vs. 2.6 % of DMV neurons). TNBS-treated animals also demonstrated significantly increased DMV astrocyte and microglial immunoreactivity, indicating glial cell activation. DMV prothrombin production was significantly increased in TNBS colitis, with a close anatomic relationship between prothrombin and microglia. Direct DMV exposure to thrombin replicated the apoptosis and activation of caspase-3 seen in TNBS colitis; these effects were prevented by coadministration of the PAR-1 inhibitor FR171113. Cultured DMV neurons exhibited impaired calcium signaling in response to neurotransmitters following exposure to thrombin. Glutamate-induced calcium transients decreased by 59 %, and those triggered by GABA were reduced by 61 %. PAR-1 antagonism prevented these thrombin-induced changes in calcium signaling. CONCLUSIONS: IBD is associated with DMV microglial activation and production of prothrombin. Thrombin in the DMV causes vagal neuron apoptosis and decreased sensitivity to neurotransmitters.