Mechanisms of CRF-Induced Intestinal Dysfunction Figure 2. CRF-induced FD4 flux is Prevented with Astressin B. CRF (0.5 mM) induced elevations in the rate of FD4 flux across the porcine ileum mounted on Ussing chambers. Pre-treatment of ileal mucosa with Astressin B (1 mM) 30 minutes prior to CRF exposure prevented CRF-mediated increases in FD4 flux rates. Data for each experimental treatment are expressed as means 6 SE for n = 628 pigs. Symbols (*,{) differ from other treatments by p,0.05; ANOVA. doi:10.1371/journal.pone.0039935.g002 blocked both baseline and CRF mediated mast cell degranulation (Figure 8A) and increases in FD4 flux (Figure 8B) thus indicating an important role of enteric neural signaling in these pathways. Discussion A central pathophysiologic mechanism by which stress induces intestinal diseases, including IBS and IBD, is via disturbances in intestinal epithelial barrier function [5,19,20,21,22,23]. These studies, utilizing an ex vivo Ussing chamber model with porcine ileum, demonstrate that the stress peptide, CRF, triggers disturbances in intestinal epithelial barrier function via a mechanism that involves mast cell dependent TNF-a and protease release and disruption of paracellular tight junctions. Furthermore, we show that the enteric nervous system plays a critical role in CRF-mediated mast cell activation and intestinal barrier injury. The present studies support previous observations in rodent models in that CRF is the key mediator triggering disturbances in intestinal function including motility disorders and intestinal barrier dysfunction [6,11,24,25]. Although the role of CRF in stress-induced intestinal disturbances is well-recognized, the precise mechanism by which CRF signals to trigger epithelial Figure 3. Histological appearance of CRF-treated porcine ileal mucosa on Ussing chambers. Ileal histology (206 magnification) of hematoxylin and eosin-stained porcine ileal sections after 180 minutes on Ussing chamber following treatment with vehicle control (A) or 0.5 mM CRF (B). Images are representative of ileal sections from n = 6 pigs/treatment. doi:10.1371/journal.pone.0039935.g003 Figure 4. Occludin localization in porcine ileum exposed to CRF. In control (vehicle-treated) tissues (A), occludin (green) was localized predominantly to the interepithelial tight junctions as demonstrated by the epithelial membrane staining patterns whereas CRF-treated tissues (B) exhibited a disrupted occluding staining pattern. Images are representative of tissues from n = 3 pigs. doi:10.1371/journal.pone.0039935.g004 PLoS ONE | www.plosone.org 3 June 2012 | Volume 7 | Issue 6 | e39935