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Immunohistochemical analysisSmall intestine and colon tissues were dissected from rats and fixed in 4 % paraformaldehyde in 100-mM phosphate buffer at room temperature for 24 hours. Serial sections (4-μm thick) were prepared from formalin-fixed paraffin-embedded tissue sections of the small intestine and colon. For 5-HT detection, cells were incubated with 5HT (1: 10; Thermo Fisher Scientific, Rockford, USA) and then stained using the iVIEW™ DAB Detection Kit (Ventana) and Hematoxylin Counterstain II (Ventana). For 5-HTR3a detection, paraffin sections were heat-treated in Cell Conditioning Solution (Ventana Medical Systems) for 5-HTR3a, incu-bated with normal horse serum (Vector), rabbit anti-rat 5-HTR3a (Abcam), HRP-polymer-conju-gated horse anti-rabbit IgG (Vector), and then stained using ultraView™ Universal DAB Detec-tion Kit (Ventana) and Hematoxylin Counterstain II (Ventana). All the stains mentioned above were used according to the manufacturer’s protocol. An automated immunostainer (BenchMark; Ventana) was used to stain both 5-HT and 5-HTR3a. For each specimen, the number of 5-HT-positive cells was counted in six randomly selected fields per section using a KS400 Image Analyzer System (Zeiss). The data were expressed as the average number of positive cells per 400 µm2 of the mucosa.Similar to real-time PCR, the restraint and control groups were compared for both the small intestine and colon; distal and proximal compari-sons were also made in the small intestine.Statistical methodsAll experimental data are presented as the mean ± standard deviation. All statistical analyses were performed using EZR12) (Saitama Medical Center, Jichi Medical University, Saitama, Japan). More precisely, EZR is a modified version of R commander (version 2.7-0, 2021) designed to add statistical functions frequently used in biostatistics. As appro-priate, the t-test was used for comparisons between the two groups. Differences between means at a level of p < 0.05 were defined as statistically significant.Data Accessibility The data that support the findings of this study are available from Juntendo University, but restric-tions apply to the availability of these data, as they 274were used under license for the current study and are not publicly available. However, data are avail-able from the authors upon reasonable request and with the permission of Juntendo University.Fecal pellet output and water contentWe counted the fecal pellet outputs of 12 restraint rats and 12 control rats. The mean number of fecal pellets was 6.9 ± 1.7 and 0.8 ± 0.8 in the restraint and control groups, respectively, showing that restraint stress significantly increased the number of fecal pellet outputs (p < 0.001, Figure 2A). We also calculated the water content of fecal pellets in six restraint rats and six control rats. The mean water content of the first stool during isolation was 75.5 % ± 7.5 % and 74.7 % ± 5.4 % in the restraint and control groups, respectively, with no significant difference in primary stools between the two groups during isolation (p = 0.80). On the other hand, the mean water content of the fecal pellets after isola-tion was significantly different between the restraint rats and control rats, at 94.8 % ± 4.2 % and 79.1 % ± 6.0 %, respectively (p < 0.001, Figure 2B). This result indicates that restraint stress led to increased water content in the stool and induced diarrhea in the stress models.Small intestinal transitNine restraint rats and 16 control rats were compared for small intestinal motility. The mean length of the small intestine was not significantly different between the restraint and control groups (115.3 ± 3.8 cm vs 113.3 ± 9.2 cm; p = 0.63) (Figure 2C). The mean small intestinal transit rate was 77.9 % ± 7.4 % in the restraint group and 69.5 % ± 8.9 % in the control group, with the restraint group showing a significant increase in the small intestinal transit rate (p < 0.03) (Figure 2D). Real-time PCRSix rats each in the restraint and control groups were compared for the expression of CRH, mast cells, and 5-HTR3a in the small intestine and colon using real-time PCR. The transcripts encoding these were normalized to those encoding GAPDH. We found that the mRNA expression was not significantly different between the restraint and control groups (Table 1). Examining the segment Results