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 µM) 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 µM CRF. Data for each experimental treatment are expressed as means ± SE for n = 6−8 pigs. Symbols (*,†) differ from other treatments by p<0.05; ANOVA.
Figure 2.
CRF-induced FD4 flux is Prevented with Astressin B.
CRF (0.5 µM) 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 µM) 30 minutes prior to CRF exposure prevented CRF-mediated increases in FD4 flux rates. Data for each experimental treatment are expressed as means ± SE for n = 6−8 pigs. Symbols (*,†) differ from other treatments by p<0.05; ANOVA.
Figure 3.
Histological appearance of CRF-treated porcine ileal mucosa on Ussing chambers.
Ileal histology (20× magnification) of hematoxylin and eosin-stained porcine ileal sections after 180 minutes on Ussing chamber following treatment with vehicle control (A) or 0.5 µM CRF (B). Images are representative of ileal sections from n = 6 pigs/treatment.
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.
Figure 5.
Histologic analysis of CRF-mediated intestinal mast cell degranulation.
Treatment of porcine ileal tissues mounted on Ussing chambers with CRF caused and increase in mast cell degranulation as determined by toluidine blue staining. Arrows indicate the release of mast cell granules into surrounding tissues. Sodium cromolyn, a mast cell stabilizing agent, prevented CRF-induced mast cell degranulation. Figures are representative of ileal tissue sections from n = 4 pigs.
Figure 6.
The role of mast cell activation in CRF-mediated changes in FD4 flux.
Treatment of porcine ileal tissues mounted on Ussing chambers with CRF increased mast cell degranulation indicated by the increased percentage of degranulated mast cells (C) and the increased release of mast cell tryptase (A) and TNF-α (B). Treatment of porcine ileal tissues with cromolyn sodium 30 minutes prior to exposure to 0.5 µM CRF blocked mast cell degranulation (C) and TNF-α release (B) and prevented CRF-mediated increases in FD4 permeability (D). c48/80 (5 µg/mL) induced marked degranulation of mast cells (C) and increases in FD4 flux (D) that were inhibited with sodium cromolyn. Data for each FD4 flux experiments are expressed as means ± SE for n = 6−8 pigs. Symbols (*,†) differ from other treatments by p<0.05; ANOVA.
Figure 7.
Neutralization of TNF-α 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-α 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 = 6−8 animals. Symbols (*,†) differ from other treatments by p<0.05.
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 ± SE; n = 6 animals and are presented as the percentage of total cell count per treatment at 20× magnification. FD4 flux values represent means ± SE; n = 6−8 animals Symbols (*,†) differs significantly by p<0.05 from vehicle control.