Acute and Chronic Experimental Colitis

Acute and chronic experimental colitis was induced by 3% dextran sulfate sodium (DSS, 36–50 kDa; MP Biomedicals, CA, USA; Cat# 02160110) solution in sterile tap water ad libitum (36). Acute colitis was evaluated on the last day of the experiment (day 8) by using a disease activity index (DAI), colon length and histological scoring system, as described previously (37). DAI was determined as a mean of the following three parameters: weight loss (none 0, 5% 1 point, 5–10% 2 points, 10–15% 3 points, >15% 4 points) stool consistency (solid 0 points, loose stool that do not stick to the anus 2 points, and 4 points for liquid stools that stick to the anus), rectal bleeding (none 0, positive test for occult blood 2 points, and 4 points for gross bleeding). Occult blood in feces was evaluated with Fecal Occult Blood Test (Okult-viditest Rapid; Vidia, Vestec, Czech Republic), which is based on guaiacum reaction. Postmortem, the mesenteric lymph nodes, spleen, and colon were collected from each mouse for further analyses. Colon shortening is an indirect marker of colitis severity, so the entire colon was removed (from caecum to anus) and measured by placing it without tension on a ruler. Next, colon descendens was collected for histological analysis, as described previously. Briefly, tissues were fixed in 4% formalin, dehydrated in ethanol and embedded in paraffin. Four micrometer sections were rehydrated and stained by hematoxylin and eosin. Subsequent microscopic evaluation was made by experienced pathologist in blinded manner.

Chronic DSS colitis was induced by three cycles consisting of 5 days DSS and 9 days tap water. The analyses of colitis severity by DAI, colon shortening, and mucosal damage were performed as described above. The level of acute-phase protein haptoglobin was determined in mouse serum using the mouse haptoglobin ELISA Duoset (Bio-Techne, Minneapolis, MN, USA; Cat# DY4409).