2.3. Treatment of dextran sulfate sodium (DSS)-induced ulcerative colitis mouse model

Forty 8-week old male Swiss Webster mice were randomly assigned into four groups. Group 1 was designed as a healthy control group, in which the animals received sterile water. Groups 2–4 were designed as saline, Tofa and P-Tofa treated group. The experimental groups received 2 cycles of 4% dextran sulfate sodium (DSS, Alfa Aesar, MW 40 kDa) as described previously. [12] Each cycle includes seven-day continuous 4% DSS followed by a seven-day continuous sterile water washout. In Group 2 (Saline), animals received saline injection on day 7 post induction. In Group 3 (Tofa treated), animals received Tofa from day 7 post induction to the end of the study for 21 days. Tofa was suspended in 0.5% methylcellulose and 0.025% Tween 20 solution and was given once daily by oral gavage at a dosing level of 10 mg/kg/day. [13] In Group 4 (P-Tofa treated), animals received a single i.v. injection of P-Tofa on day 7 post induction at a Tofa equivalent dose of 210 mg/kg. The mice’s body weight, stool consistency and presence of hematochezia (tested using ColoScreen™ FOB Test Kit, Helena Laboratories™, Texas, US) were examined and recorded daily. The disease activity index (DAI) was calculated as the sum of the change of body weight, diarrheal score and the bloody stool score (Table 1), according to the method adapted from literature. [14, 15]

The scoring system for the calculation of the disease activity index based on weight loss, stool consistency and the degree of intestinal bleeding [14, 15]

Blood was collected using maxillary sampling method weekly starting a week before colitis induction until necropsy. Animals were sacrificed on day 28 after 24 hours fast. Blood and major organs (heart, liver, spleen, lung, kidney, small intestine, colon, and mesentery lymph nodes) were collected after perfusion. The length of colon was measured. About 1 cm of the distal colon was collected for FACs analyses of cell phenotypes from each animal. The remainder of the colon was assigned to histological evaluation.