after, during the regenerative phase (day 28), the MUC2 levels in the proximal colon decreased but were still higher than control levels. In the distal colon, however, a trend to decreased MUC2 expression levels was observed during active disease (days 5â€“7), while during the regenerative phase (day 28) MUC2 levels returned to control levels. Similarly to MUC2 expression levels, TFF3 levels seemed to increase in the proximal colon during and after DSS treatment (Fig. 7B). This increase in TFF3 protein levels, however, was not statistically significant. In the distal colon TFF3 expression levels were significantly lower during active disease (days 5â€“7) than in controls. During the regenerative phase (day 28) TFF3 levels had increased again and were comparable to control levels.
Discussion As DSS-induced colitis is commonly used as a model for human inflammatory bowel disease, knowledge is essential to be able to understand the mechanisms underlying the pathology of DSS-induced colitis. Therefore we analyzed the occurrence of clinical symptoms, epithelial gene expression, proliferation, and cell death during DSS-induced colitis. In our study DSS-induced clinical symptoms, such as body weight loss, loose and bloody stools, diarrhea, and gross bleeding, started within several days after the beginning of the DSS treatment, were most severe at the end of DSS treatment, and disappeared only gradually during the recovery period. Previous studies in mice and rats confirm this pattern of occurrence and recovery of clinical symptoms [1, 5, 8, 23]. The pathological features such as crypt loss, ulcerations, and inflammatory infiltrate that appeared in our rat DSS colitis model are similar to those in UC [3, 24] and in DSS-treated mice [3, 5, 24]. Furthermore, DSS-induced damage in rat started and was most severe within the distal colon, and was focal in nature, and was comparable to DSS-induced colitis in mice and UC in human [3, 5, 24]. Our study is the first to describe the response of the colonic epithelium with respect to proliferation and apoptosis during the different phases of DSS-induced disease. DSS administration induced a decrease in proliferative activity and an increase in the number of apoptotic crypt cells, in the proximal colon as well as in the distal colon, during the onset of disease. These data demonstrate that DSS exerts its toxic effects on the epithelium via the relatively undifferentiated crypt cells. This is similar to humans with active UC, in which the numbers of apoptotic cells and proliferating cells in the crypt compartment are also increased [25, 26]. The increased number of apoptotic cells in the surface epithelium suggests that DSS also affects mature surface cells. However, we cannot exclude the possibility that these surface cells were already damaged by DSS when they were relatively immature crypt cells. When DSS feeding was continued, the initial decrease in proliferative activi-
ty was followed by epithelial hyperproliferation in the entire proximal colon and in the proximity of ulcerations in the distal colon. These data clearly demonstrate that the inhibiting effect of DSS on epithelial proliferation can be overruled, leading to hyperproliferation. However, the mechanism(s) and growth factor(s) responsible for this phenomenon remain to be identified. Apart from the DSS-induced alterations in epithelial proliferation and cell death, epithelial cell functions might also be affected. Therefore we examined the effects of DSS on enterocyte specific CA I expression and goblet cell specific MUC2 and TFF3 expression. The effects of DSS feeding on CA I expression levels were most pronounced in the distal colon, where it induced a significant decrease in CA I levels during onset of disease and active disease. This indicates that specific enterocyte functions were down-regulated and thus that colonic functions are impaired during DSS treatment. Immunohistochemical analysis of the distal colon revealed CA I negative surface cells during active disease in areas with apparently normal crypts and surface epithelium. In addition, the majority of flattened surfaces epithelial cells overlying mucosa with crypt distortions were CA I negative, in both the proximal colon and the distal colon. These data suggest that during transition of areas with intact crypts and surface epithelium into areas with crypt loss and flattened surface epithelium, specific enterocyte functions are down-regulated before restitution is initiated. Later in the course of the disease, during the recovery phase, the surface epithelial cells of the distal colon near ulcers were still flattened but were frequently CA I positive. Moreover, during this disease phase CA I protein levels restored to control levels in the distal colon and were even up-regulated in the proximal colon. These data imply that, in addition to epithelial proliferation, epithelial differentiation restored during the recovery phase to accelerate the recovery of colonic functions. Similar results have been reported by Fonti et al.  who observed a reduction in CA I expression in active UC, whereas in humans with UC in remission CA I expression was restored to control levels. Moreover, in rat the down-regulation of CA I expression during DSS treatment was correlated with the occurrence of loose stools and diarrhea, suggesting that CA I plays a role in DSS-induced diarrhea. Quantitative analysis of MUC2 and TFF3 demonstrated a trend toward decreased MUC2 levels and significantly decreased TFF3 levels in the distal colon. As DSS-induced crypt loss and ulcerations were most severe in the distal colon (approx. 50% distally vs. approx. 20% proximally), the decreased MUC2 and TFF3 levels in this segment during active disease are largely due to the dramatic decrease in the overall number of goblet cells. Reduced numbers of goblet cells have also been reported in the colon of humans with active UC . Additionally, MUC2 levels in the sigmoid colon have also been found to be significantly lower in active UC than in controls and UC in remission [18, 19]. Yet, despite the DSS-induced damage
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