Fig. 3.

Intestinal pathophysiology. A: histological damage stains. Representative histological stains are from phase-shifted animals with no DSS challenge (left), nonshifted animals challenged with 2% DSS (middle), and phase-shifted 2% DSS-challenged animals (right). There was no significant histology change in the colon of phase-shifted mice (left) compared with that of nonshifted H₂O control mice (stain not shown). The colitis induced by 2% DSS in mice is evidenced by mild mucosal infiltration of inflammatory cells and a reduction of goblet cells (middle). The DSS-induced colitis is exacerbated as evidenced by more extensive destruction of mucosal layer and mucosal ulceration (right). These stains were used for quantitative histological analysis of inflammation (see B). B: histological damage score and myeloperoxidase (MPO) activity. The quantitative measurement for tissue damage was achieved by blindly scoring 3 independent colon sections from each animal (representative stains are shown in A) collected posteuthanasia for visual characterization of destruction, inflammation, and repair using multiple subcategories. The total score (see materials and methods) for each animal was calculated and averaged for the nonshifted and phase-shifted groups. No histological damage could be observed in any of the non-DSS-treated animals, and thus no scores are presented. The DSS challenge led to increased damaged tissue in both groups, but significantly higher levels of tissue damage were observed in the phase-shifted colons (left). MPO activity was determined from 3 colon sections from each animal. Activity levels of both H₂O control groups were indistinguishable and at baseline levels of neutrophil activity (data not shown). Two percent DSS-challenged animals in both groups showed a significant 20- to 40-fold increase in MPO activity in all samples compared with the baseline activity, indicating increased neutrophil infiltration into the colon. The neutrophil infiltration process was significantly increased in the phase-shifted mice (right) compared with nonshifted mice, suggesting higher levels of inflammation in these animals. The standard errors shown indicate the variation between animals in each group, not between individual colon sections (*P < 0.01).