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mounted. MUC2, TFF3, and CA I expression was demonstrated according to the above protocol with omission of the HCl incubation, washing with borate buffer, and pepsin treatment. To stain MUC2 and TFF3 sections were boiled in 0.01 M citrate buffer at pH 6.0 for 10 min prior to incubation with a MUC2-specific antibody (WE9, 1:500) [17] or TFF3-specific antibody (1:6000, Prof. Dr. D.K. Podolsky). To detect CA I rabbit anti-CA I (1:16000, Prof. Dr. W.S. Sly) was used. Biotinylated goat anti-rabbit IgG (1:2000, Vector Laboratories) was used as secondary antibody to detect TFF3 and CA I. To study differences in epithelial proliferation between control and DSS-treated animals the number of BrdU-positive cells in six well oriented crypts was counted and expressed per crypt, per intestinal segment, per time point (±SEM). To determine the number of BrdU-positive cells per crypt in each intestinal region sections were judged twice by two independent and blinded observers. Protein dot blotting CA I, TFF3, and MUC2 expression was quantified as described previously [18, 19]. Briefly, segments of proximal and distal colon were homogenized, protein concentration was measured, and 0.30 µg protein of each homogenate was dot-blotted. After blotting the blots were incubated with anti-CA I (1:4000) to detect CA I expression or with a MUC2-specific antibody (WE9, 1:100) to detect MUC2 expression [17, 20]. Thereafter the blots were incubated with 125I-labeled protein A (Amersham, Bucks, UK; specific activity 33.8 mCi/mg). Binding of 125I-labeled protein A to anti-CA I, anti-TFF3 or WE9 was detected by autoradiography using a PhosphorImager and quantified using ImageQuant software (Molecular Dynamics, B&L Systems, Zoetermeer, The Netherlands).

Fig. 1 Effects of DSS treatment on body weight and the occurrence of clinical symptoms. Relative changes in body weight of control (open squares) and DSS-treated (filled triangles) rats. Error bars reflect the standard deviations of the relative changes in body weight on each day. The body weight of each individual animal was set arbitrarily at 1 on day 0 of the experiment. Clinical symptoms, i.e., loose stool, diarrhea, bloody stool, and gross bleeding, are presented as a function of time. Dashed line Days of DSS/water treatment. Note that changes in body weight coincided with changes in clinical symptoms

Statistical analysis

Rats treated with DSS suffered weight loss compared to control animals (Fig. 1). Immediately after the beginning of DSS treatment rats started to lose weight. Weight loss progressed during and shortly after DSS treatment. On day 10, when weight loss of DSS-treated rats was maximal, DSS-treated rats weighed 25% less than control rats. On day 11, 5 days after the end of the DSS administration, rats started to gain weight again. Loose stools, diarrhea, and bloody stools were observed within 3 days after the start of the DSS treatment. Gross bleeding first occurred on day 5, and persisted until day 8. The occurrence of and recovery from the various clinical symptoms over time are presented in Fig. 1.

within the colon, and was divided into three phases: onset of disease, active disease, and regenerative phase. Onset of disease (day 2) was characterized by a slight flattening of the crypt epithelial cells (Fig. 2B), and a slight increase in the number of apoptotic cells in the crypts and surface epithelium in both proximal and distal colon (Fig. 2C). Moreover, in the distal colon focal crypt distortions were observed that varied from atrophy to complete loss (Fig. 2B). During active disease (day 5–7) areas were seen with flattened crypt cells, crypt loss, massive inflammatory infiltrate, flattening of the surface epithelium, focal erosions, and necrotic cells in both colonic segments. The histological damage was focal in nature and most severe in the distal colon on days 5–7 (Fig. 2D). Specifically, approx. 50% of the distal colonic tissue consisted of areas with crypt loss or erosions, while approx. 20% of the proximal colon was severely affected during this phase. During the regenerative phase (day 28) the epithelial morphology of the proximal colon completely recovered. In contrast, in the distal colon branched crypts and pronounced crypt elongations along side erosions, which involved less than 10% of the total surface, were still apparent (Fig. 2E).

Morphology

Proliferation

The colonic tissue showed dramatic morphological changes depending on time of treatment and localization

Epithelial proliferation was studied by immunohistochemical detection and quantification of incorporated

Analysis of variance was performed, followed by an unpaired t test. Differences were considered significant at P<0.05. Data are presented as the mean ±standard error of the mean.

Results Clinical symptoms

Epithelial proliferation, cell death, and gene expression in experimental colitis  

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