investigated the mechanisms of CRF-mediated intestinal epithe- lial barrier dysfunction. Results Influence of CRF on Porcine Ileal Intestinal Barrier Function We employed an ex vivo Ussing chamber system to investigate the role of local CRF signaling on intestinal epithelial barrier function in the porcine ileum. CRF, at concentrations of 0.05 and 0.1 mM, and 0.5 mM induced elevations in FD4 flux across ileal mucosa compared with vehicle-treated controls (Figure 1). In contrast, exposure of ileal mucosa to CRF did not influence TER over the 180 minute time period on the chambers (data not shown). To confirm that CRF was mediating its effects on intestinal permeability via CRF receptors, ileal mucosa was pre- treated with the CRF receptor antagonist, Astressin B (1 mM), prior to exposure of CRF (0.5 mM). Astressin B prevented CRF- induced elevations in FD4 flux (Figure 2). Histological analysis revealed no disruption of intestinal epithelial continuity with CRF (0.5 mM) treatment (Figure 3) indicating that the effects of CRF were due to alterations in the paracellular flux pathways rather than destruction of the epithelium. Immunoflourescence analysis of the tight junction protein occludin revealed marked disruption in occludin staining patterns in ileal tissues exposed to CRF (Figure 4). Mast Cell Degranulation is required for CRF-mediated Increases in Paracellular Permeability in the Porcine Ileum To determine the role of mast cell activation in CRF-mediated intestinal permeability, we evaluated determined if CRF triggered mast cell activation in the porcine ileum and whether blockade of mast cell activity alterned CRF-mediated disturbances in intestinal epithelial permeability. Exposure of porcine ileum to 0.5 mM CRF induced mucosal mast cell degranulation as determined by Mechanisms of CRF-Induced Intestinal Dysfunction histological evidence of mast cell degranulation (Figure 5) and quantified by the increased percentage of degranulated of intestinal mucosal mast cells (Figure 6C). Furthermore, CRF induced the release of mast cell tryptase (Figure 6A) and TNFa (Figure 6B). Pre-treatment of porcine ileal tissues with the mast cell stabilizing agent, sodium cromolyn (1024 M), blocked CRF- mediated mast cell degranulation (Figures 5, 6C) and increases in FD4 flux (Figure 6D). Cromolyn also inhibited elevations in TNFa (Figure 6D) thus indicating that CRF-induced TNFa release was mast cell dependent. To compare the CRF response to a known mast cell degranulation response, tissues were treated with the mast cell degranulating compound c48/80. c48/80 induced marked mast cell degranulation (Figure 6C) and increases in FD4 flux (Figure 6D) that were greater than CRF (p,0.05). Blockade of TNF-a and Protease Activity Prevents CRF- induced Gut Barrier Dysfunction To determine if the mast cell mediators, TNF-a and MC proteases, were responsible for CRF-mediated intestinal barrier dysfunction in the porcine ileum, we treated porcine ileal mucosa on Ussing chambers with a PI cocktail or porcine anti-TNF-a prior to CRF exposure. Protease inhibitors (PI) and anti-TNF-a (0.1 and 10 mg/mL) inhibited CRF-stimulated elevations in FD4 flux (Figure 7A–B). Under baseline condition (no CRF), PI and the higher anti-TNF-a (10 mg/mL) reduced baseline FD4 flux indicating the influence of proteases and TNF-a under basal conditions. Neuronal Activity is required for CRF-induced Gut Barrier Dysfunction To determine whether or not the enteric nervous system played a critical role in CRF-mediated mast cell activation and disturbances in intestinal epithelial permeability, tissues were pretreated on Ussing chambers with the neuronal blocker, tetrodotoxin (TTX) prior to CRF exposure. TTX pretreatment Figure 1. CRF induces elevations in FD4 flux in porcine ileum. Porcine ileum was placed on Ussing Chambers and treated with CRF at increasing concentrations (0.05, 0.1, and 0.5 mM) and the rate of FD4 flux was measured over a 180-minute period. CRF at all concentrations induced elevations in FD4 flux (p,0.01) with the highest FD4 flux rates observed with 0.5 mM CRF. 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.g001 PLoS ONE | www.plosone.org 2 June 2012 | Volume 7 | Issue 6 | e39935