The Research On The Mechanisms Of Early Dysmotility Following Rat Orthotopic Small Bowel Transplantation

Objectives: To investigate the mechanism of early dysmotility after small bowel transplantation (SBTx) in rats and the role of COX-2 in causing early dysmotility, to address the corresponding alterations of small bowel motility and find the effective therapeutic adjunct for clinical small bowel transplantation.Methods: Through fine microsurgical techniques, a rat orthotopic SBTx model was established. In order to exclude the interferences from rejection and heavy immunosupression required for SBTx, a syngeneic rat(BN - BN)transplantation model was used. Based upon the stable SBTx model, semi-quantitative Reverse transcription-polymerase chain reaction (SQ RT-PCR) method was applied to measure the expression of COX-2 mRNA, which was believed to be a crucial inflammatory regulator, in the intestinal muscularis after each stage of transplantation -before procurement, after preservation, the grafts at 0h,12h,24h and 48h after transplantation. Also LAB-SA immunohistochemistry staining was adopted to detect and localize the expression of COX-2 protein in the whole-mounts of the intestine. And for the first time, transmission electron microscope (TEM) was used to investigate the changes in the intestinal muscularis. Mechanical activity of the normal and 48-hour graft's circular muscular strips were measured in vitro, with intestinal transit and radiology evaluation of the graft motility together to determine the pattern of early dysmotility. At last, through intestinal transit test, the functional motility effect of Sinomenine, a Chinese herbal extraction that was believed to have selective COX-2 inhibition, was determined. Results: 1. Through a stage-oriented learning curve, the techniques for a proficient orthotopicSBTx could be mastered to develop a stable model. In the formal experimental stage, on average, the time for procurement of the entire small intestine was 41.5 +1.5min, for the orthotopic SBTx was 88.2 + 10.5min.The average operating time for the anastomosis of the vein and artery was 22.2+3.8min and 16.0 + 2.8min respectively and 28 + 4.4min for the two end-to-end intestinal anastomoses. Among 60 cases of SBTx in the formal experimental stage, the success rate of 86.7% was achieved. Postoperative bleeding, venous embolization and peritoneal infection were still the major reason of failure. One of the causations of peritoneal infection might be bacterial translocation. The 24-hour transplanted graft looked edematous with a rosy appearance macroscopically and the intestinal diameter augmented. The 48-hour graft appeared similarly but the lesions were more significantly. Routine pathological analysis showed dramatic edema in the 12-hour graft mucosal villi and massive mucosal desquamation was noticed in the 24-hour graft. In the 48-hour graft, the mucosa regenerated markedly and the 96-hour graft showed a essential normal mucosa. In all successful operations, the rats came conscious 3 hours postoperatively, then started to drink within 6 hours approximately and began to eat within 24 hours after the operation. All rats showed an approximate 5% decrease in weight because of the transplantation procedure.2. RT-PCR was performed on the extracts for the presence of COX-2 mRNA after each stage of SBTx. It showed the basal COX-2 expression was very tiny from the normal control group. In contrast, the expression from the donor intestinal muscularis increased swiftly. The expression of COX-2 from the 0-hour graft up-regulated further and peaked from intestinal muscularis of the 24-hour graft, then precipitated. Immunohistochemistry showed that there were resident macrophages within the normal rats' muscularis externa and the expression ofCOX-2 protein was nil or very delicate. Multiple monocytes and phagocytes were stained in their cytoplasm within the 24-hour graft muscularis positively. There was significant expressional difference of COX-2 between the normal control group and 24-hour grafts. For the first time, it was found that COX-2 was expressed in the transplan...