Mechanisms of CRF-Induced Intestinal Dysfunction Figure 7. Neutralization of TNF-a and protease activity inhibits CRF-induced increases in FD4 flux in porcine ileum. Pretreatment of porcine ileum mounted on Ussing chambers with neutralizing anti-TNF-a antibody prevented CRF –induced increased in FD4 flux. Pre-treatment of porcine ileum with a protease inhibitor (PI) cocktail reduced baseline FD4 flux values and CRF-induced increases in FD4 flux. Values represent means SE; n = 628 animals. Symbols (*,{) differ from other treatments by p,0.05. doi:10.1371/journal.pone.0039935.g007 activation indirectly via activation of enteric neurons which release mediators such as substance P and calcitonin gene related peptide that in turn can trigger degranulation of mast cells [43,44]. Alternatively CRF could activate mast cells via direct CRF receptor activation and this pathway could be influence by enteric neural signaling. As stated above, mast cells express CRF receptor Figure 8. The Role of the enteric nervous system in CRF-mediated mast cell activation and FD4 flux. Toluidine blue staining of histological sections (100 X magnification) (A) and quantitation of the % of degranulated tissue mast cells (B) in porcine ileum revealed increased degranulation of mast cells in CRF–treated tissues. Treatment of ileal tissues with tetrodotoxin (TTX) inhibited mast cell degranulation and FD4 flux (C) under basal and CRF-stimulated conditions and inhibited. For histological analysis, values represent means 6 SE; n = 6 animals and are presented as the percentage of total cell count per treatment at 206magnification. FD4 flux values represent means 6 SE; n = 628 animals Symbols (*,{) differs significantly by p,0.05 from vehicle control. doi:10.1371/journal.pone.0039935.g008 PLoS ONE | www.plosone.org 6 June 2012 | Volume 7 | Issue 6 | e39935