Figure 1.
Characterization of the adult mouse model of cholera using ligated ileal loops inoculated with liveV. cholerae.
(A) Time course of intestinal fluid secretion induced by V. cholerae inoculation. 107 CFU of V. cholerae were inoculated into ileal loops of anesthetized adult mice. At indicated time after bacterial challenge, loops were removed for measurements of loop weight/length ratio. Left, representative images of mouse ileal loops injected with saline or saline containing V. cholerae at specific time points post-inoculation. Right, summary of the data. Data were expressed as mean ± S.E. (n = 4–8 mice per group). *, P<0.05 and ***, P<0.01 compared with 6 h. (B) Survival and diarrhea success rates of the V. cholerae-inoculated mice over the indicated periods of time after inoculation (n = 39 mice).
Figure 2.
Quantitation ofV. cholerae in mouse ileal loops.
Ileal loops were inoculated with 107 CFU of V. cholerae. Amount of V. cholerae recovered from the homogenates of ileal loops was measured at indicated time after V. cholerae inoculation (n = 5 mice per group). Solid lines represent mean values of the number of recovered bacteria. NS, non-statistical difference compared with 6 h.
Figure 3.
Effect of CFTRinh-172 on V. cholerae-induced intestinal fluid secretion in adult mice.
After inoculation of V. cholerae into ileal loops, 20 µg of CFTRinh-172 was administered into mouse peritoneal cavity every 6 h. At 12 h after V. cholerae challenge, ileal loops were removed for measurement of loop weight/length ratio. (A) Representative photographs of ileal loops after indicated treatments. (B) Loop weight/length ratio with indicated treatments. Data were expressed as mean ± S.E. (n = 4). ***, P<0.01 compared with V. cholerae-inoculated control.
Figure 4.
Analysis of intestinal paracellular permeability in the established model.
(A) Transepithelial electrical resistance (TEER) of intestinal tissues. At 12 h after instillation of saline (control), V. cholerae (107 CFU/loop), or cholera toxin (CT, 1 µg/loop) into ileal loops, intestinal tissues were measured for TEER using Ussing chamber systems. Data were expressed as mean ± S.E. (n = 5 mice per group) NS, non-statistical difference compared with control. (B) In vivo measurements of intestinal paracellular permeability using FITC-dextran assays. At 12 h after instillation of saline (control), V. cholerae (107 CFU/loop) or cholera toxin (CT, 1 µg/loop) into ileal loops, intestinal fluid was removed and replaced with FITC-dextran (molecular weight of 4.4 kDa). Thirty min thereafter, blood was taken by cardiac puncture for analysis of serum FITC-dextran level. Data were expressed as mean ± S.E. (n = 4–8 mice per group). NS, non-statistical difference compared with control.
Figure 5.
Histological analysis of intestinal sections.
At 12 h after administration of saline (control) or inoculation with V. cholerae (107 CFU/loop) into ileal loops, ileal loops were removed for histological examination. (A) Representative images of hematoxylin & eosin stained tissues. Arrows indicate edematous submucosa. Arrowheads indicate inflammatory cell infiltration. Photographs were taken at 40× magnification. Scale bar = 500 µm. (n = 5 mice per group) (B) Representative photographs of periodic-acid Schiff (PAS)-stained ileal tissues. Arrows indicate mucin-containing goblet cells, which were positively stained with magenta color. Photographs were taken at 40× magnification. Scale bar = 500 µm. (n = 5 mice per group).