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both groups underwent an autopsy 1 hour after intragastric administration of the powdered carbon suspension. The process was shown in the figure (Figure 1). When the abdominal wall was opened during dissection, the intestinal tract was exposed. At that time, the intestines that had been exposed to carbon suspension were stained black and could be observed visually. The small intestines were collected, and their entire lengths placed naturally 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 %.Real-time polymerase chain reaction (PCR)The amount of mRNA encoding CRH, mast cells, and 5-Hydroxytryptamine Receptor 3A (5-HTR3a) was quantified using real-time PCR. Full-thickness segments of the small intestine and proximal and Figure 1　The process from administration of the carbon suspension to dissection was shown. After administration of the carbon suspension, the restraint group was restrained for one hour and the control group was isolated for one hour. After one hour of restraint and isolation, the animals were dissected. In this figure, I presented a picture of a rat in physical restraint. And also presented a picture of the removed small intestine. This picture was taken after the carbon suspension was administered. As shown, the area where the carbon had passed through was stained black, and this length was measured.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) to extract 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 glyceral-dehyde 3-phosphate dehydrogenase (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.273