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Immunohistochemical analysisImmunostaining of 5-HT and 5-HTR3a was performed using tissues from the small intestine and colon. Six rats each from the restraint and control groups were used for comparison. Since 5-HT is secreted by EC cells, the 5-HT antibody staining of the small intestine and colon tissues was used to stain the EC cells. Positive cells in the control and restraint groups were counted and compared. As a result, EC cell expression tended to increase in the restraint group compared to that in the control group in both the small intestine and colon (Figure 4A, B). The number of positive cells was significantly higher in the restraint group than in the control group in the small intestine instead of the colon (small intestine: p = 0.004; colon: p = 0.3) (Figure 5). When the number of EC cells in the small intestine was compared between the prox-imal and distal small intestine, no significant differ-ence was observed between the restraint and 276Figure 3　A: The expression of 5-Hydroxytryptamine Receptor 3A (5-HTR3a) in the proximal and distal small intestine was compared in the restraint group. B: The expression of 5-HTR3a in the proximal and distal small intestine was compared in the control group. C: The expression of 5-HTR3a was compared between the restraint and control groups only in the distal small intestine.control groups (restraint group: p = 0.95; control group: p = 0.48) (Figure 6A, B). In the distal small intestine, the number of EC cells significantly increased in the restraint group compared to that in the control group (1.33 ± 0.52 vs. 0.53 ± 0.18, respectively; p = 0.012) (Figure 7A). In the prox-imal small intestine, there was no significant differ-ence in the number of EC cells between the restraint and control groups (1.36 ± 0.82 vs. 0.67 ± 0.37, respectively; p = 0.13) (Figure 7B). Moreover, there was no significant difference in 5-HTR3a between the control and restraint groups in both the small intestine and colon (Figure 8).Animal models of bowel motility dysfunction associated with stress induced by cold, acoustics, ether, cold restraint, and wrap restraint have been previously reported. For example, Murakami et al.13) intravenously administered CRH to induce stress Discussion