The experiments were performed using adolescent male Sprague-Dawley rats aged 5-6 weeks (to avoid female hormonal effects) weighing 160–250 g (CLEA Japan, Inc., Tokyo), which were housed in cages in a standardized environment with temperature of 24 °C, relative humidity of 55% ± 15%, and a 12-hour/12-hour light-dark cycle for a day. They were allowed to access food and water freely. The animal care and experimental protocols were approved by the Institutional Review Board of Juntendo University.
The animals were randomly assigned to two groups: a restraint group and a control group. The restraint group was placed in isolation among individual compartments of stress cages (Natsume Seisakusho Co. Ltd. Tokyo, Japan; KN-325-C-3) for 1 hour before dissection. Rats in the control group assumed an hour of isolation in the cages without restriction. The rats in both groups did not have free access to food and water during isolation.
All methods were carried out in accordance with relevant guidelines and regulations and this study was carried out in compliance with the ARRIVE guidelines as rats were used in this study.
Fecal pellet output and water contents
The number of fecal pellet outputs during the one-hour isolation was counted. The stool first excreted during isolation was collected, as well as the stool located in the most distal side of their gastrointestinal tract after isolation, for comparison. These fecal pellets were stored at a temperature of -80 °C and were freeze-dried overnight after weighing. The next day, the freeze-dried fecal weight was measured to calculate the water content (%), which was as follows: (fecal weight before drying – fecal weight after drying) / fecal weight before drying × 100%.
Small intestinal transit
We examined the intestinal propulsion of powdered carbon to evaluate small intestinal motility under restraint stress. The rats received 0.5 mL of a powdered carbon suspension in saline per 100 g of their weight (5% W/V) intragastrically through an oral sonde (Primetech Co. Ltd. Tokyo, Japan; FTP-15-78-50). After administration, the restraint group rats were immediately exposed to stress as described above. Rats in both groups underwent an autopsy at 1 hour after intragastric administration of the powdered carbon suspension. The small intestines were collected and their entire lengths were measured. We also measured the length of the small intestine containing the carbon marker. The small intestinal transit rate (%) was calculated as follows: (the length of the small intestine containing the marker / total small intestinal length) × 100%.
The amount of mRNA encoding CRH, mast cells, and 5-HTR3a were quantified using real-time PCR. Full-thickness segments of the small intestine and proximal and distal colonic tissue samples were preserved in RNA stabilization solution and stored at -80 °C until use. Each tissue sample was homogenized in Tri Reagents (Tomy, Japan; MS100) for the extraction of total RNA according to the manufacturer’s instructions (Applied Biosystems). Real-time PCR was performed using the 7500 Fast Real-Time PCR system (Applied Biosystems). The expression of each gene was normalized to the expression of GAPDH using the standard curve method. TaqMan was used for analysis using primers for CRH (Assay number Rn01462137_m1), mast cells (Assay number Rn04342812_g1), and 5-HTR3a (Assay number Rn00667026_m1). The results were compared between the restraint and control groups for both the small intestine and colon. Furthermore, the proximal and distal segments of the small intestine were also compared.
Small 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 (CC1) (Ventana Medical Systems) for 5-HTR3a, incubated with normal horse serum (Vector), rabbit anti-rat 5-HTR3a (Abcam), HRP-polymer-conjugated horse anti-rabbit IgG (Vector), and then stained using ultraView™ Universal DAB Detection Kit (Ventana) and Hematoxylin Counterstain II (Ventana). All the stains below 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, and distal and proximal comparisons were also made in the small intestine.
All experimental data are presented as the mean ± standard deviation. All statistical analyses were performed using EZR  (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 appropriate, 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.
The data that support the findings of this study are available from Juntendo University, but restrictions apply to the availability of these data, as they were used under license for the current study and are not publicly available. However, data are available from the authors upon reasonable request and with the permission of Juntendo University.