372 Silencing of Gene Expression for Corticotropin-Releasing Factor (CRF) in the Colon Attenuates Stress-Induced Acceleration of Colonic Transit in Rats

Abstract

We tested a hypothesis that silencing of CRF expression in the enteric nervous system (ENS) attenuates stress-evoked alterations of colonic motor function. Laser capture microdissection (LCM), real time RT-PCR, and immunofluorescence were used to study expression of CRF in the ENS of the rat, mouse and guinea pig colon. Injection of double-stranded RNA (dsRNA) for CRF (dsCRF, 20 μg/rat) into the rat colon was used to achieve RNA interference (RNAi)-mediated inhibition of CRF gene expression. DsRNA for β-globulin was a control (dsControl). Four days after dsRNA administration, rats were exposed to restraint stress for 2 h. Fecal output was monitored at 15 min intervals for 2 h. LCM and RT-PCR revealed CRF mRNA expression in myenteric neurons in rat proximal and distal colon. CRF-like immunoreactivity (IR) was expressed in nerve cell bodies and fibers in the myenteric and submucosal plexuses of the rat, mouse, and guinea pig colon. CRF-IR neurons were 5.4±0.3% of the myenteric population and 21.9±4.5% of the submucosal population in rat colon. In rat colonic myenteric plexus, 45.0±10.5% of CRF-IR neurons expressed choline acetyltransferase-IR and 97.1±1.4% expressed nitric oxide synthase-IR. In the rat colonic submucosal plexus, all CRF-IR neurons expressed vasoactive intestinal peptide-IR. Stress induced widespread immunostaining for c-fos in colonic myenteric ganglia. Double labeling showed that most of the c-fos-positive neurons in stressed animals expressed either CRF-IR or CRF1receptor-IR. Intramural injection of dsRNA for CRF (dsCRF) “knocked-down” basal CRF expression in rat colon, while CRF expression in dsControl animals was unaffected. CRF mRNA was decreased by 60% after four days. CRF peptide-IR was undetectable or very low in myenteric and submucosal neurons of the rat colon. DsControl and dsCRFtreated rats had similar daily food intake, body weight gain, and cumulative fecal output during the 4 days post injection. In dsControl-treated rats, restraint stress induced a significant increase in fecal output when compared to non-stressed controls (stress: 9.7 ± 1.3 vs. nonstress: 2.7 ± 0.8; P 0.05). Stress changes CRF expression in the colonic ENS and is a factor in stress-evoked colon dysmotility. (Support: NIH R01 DK37238 and R01 DK57075 (JD Wood), PhRMA Research Starter Award (S Liu), UNC Center for Functional GI & Motility Disorders seed grant (S Liu), and Hellman Grant (A Bhargava).