Histopathological Evaluation of Colitis

The distal colon was evaluated as this is the most severely affected colonic segment in DSS-induced colitis [6] (link), [9] (link). Severity of colitis was scored on H&E stained colon sections by a blinded observer [10] (link). Sections from segments D1 and D3 were evaluated for each animal and an average of scores in the two segments was used as the score for each individual animal. Segment D2 was scored only on a subset of animals if tissue quality of other segments was poor. Briefly, inflammation severity, inflammation extent and crypt loss were each given a score ranging from 0–4 and each score was multiplied by the extent of the section involved (0 = 0; ≤25% = 1, ≤50% = 2; ≤75% = 3 and ≤100% = 4). Total colitis score is the sum of the scores for inflammation severity, inflammation extent and crypt loss and was calculated as the average of total colitis scores from D1 and D3 in a given animal. Fibrosis was scored by a blinded observer on adjacent sections from segments D1 and D3 stained with Masson’s Trichrome (stains collagen and proteoglycans) or Sirius red (stains fibrillar collagen). The scoring criteria used for fibrosis are shown in Table 1 and fibrosis score for each animal is the average of scores for segments D1 and D3. Histological evaluation and scoring was performed using a Nikon Microphot-FXA microscope equipped with an Optronics DEI 750-3-chip CCD camera for digital imaging.