The Experimental Research On Graft Warm Ischemia Promoting Rejection Of Small Bowel Transplantation In Rats

Objective To demonstrate the affect and mechanism of allorejection promoted by graft warm ischemic injury.Methods Isogeneic and Allogeneic orthotopic small bowel transplantation were performed in rats, the survival rates was observed. To evaluate morphological changes and plasma level of D-Lactic and DAO, the recipients were sacrificed at experimental end-point. Iso- and Allotransplantation of hetertopic intestinal were conducted. On the day1 and day2 after surgery, grafts sample were obtained to investigate the protein of TLR-4 and NF-kB by immunohistochemistry, and analyze the expression of TLR-4 mRNA via QRT-PCR. Draining lymph nodes from abdominal of recipients were taken to count the number of double positive cells by CD103 and CD-86 labeled with FACS. On day 3,5 and 7 post-transplantation , graft sample were taken continously for routine pathology test and to measure the CD8~+ cellinfiltration in graft. The expression of IFN-y and IL-4 mRNAs was analyzed via QRT-PCR in graft obtained on day 5 after allo-heretopic small bowel transplantation , at the same time the expression of IgG2a and IgGi were measured by confocal laser scanning microscopy.Results 1. The 14-days survival of small bowel Iso-transplantation and 7-days survival of All-transplantation decreased with increased warm ischemia time. 2, The plasma level of D-Lactic and DAO of Iso-transplantaion on POD 14 increased significantly with prolonged warm ischemia time, in graft, as well as allo-transplantation on POD5. 3. On day 1 postoperatively, the expression of TLR-4 was detected mainly at intestinal epithelium, but its located mostly in monocytes of the lamina propria on day 2. Both increased with warm ischemia time.There was no significant difference in same warm ischemia time groups. 4. The number of TLR-4 labeled monocytes and the proportion of mature DCs increased with warm ischemia time, and correlated positively with the score of small bowel mucosa injury. 5. The significant CD8 positive cells were detected in the allograft of W60 group on day 3 post operatively, that significantly difference with WO group. The number of CD8+ cellsincreased with warm ischemia time. 6. On day 3 and day 5 post operatively, there was no significantly difference between WO and W30 group in score of small bowel rejection, but they differed significantly with W60 group. There were significantly differences among allo-transplantation groups the score of small bowel rejection increased with warm ischemia time on day 5 after transplantation. 7. The expression of IFN-y and IL-4 mRNAs increased with warm ischemia time, and correlated positively with the score of small bowel mucosa rejection. There were significantly differences among allo-transplantation groups. 8. The expression of IgG2a and IgGj increased with warm ischemia time, and correlated positively with the score of small bowel mucosa rejection. There were significantly differences among allo-transplantation groups.Conclusion 1. The graft could tolerance 60min warm ischemia in small bowel Iso-transplantation and Allografts could tolerance 30min warm ischemia without immunosuppressant. 2. Barrier function and permeability of graft was damaged by warm ischemia reperfusion injury. 3. Small bowel mucosa epithelium should be as one of effective celltaken part in TLR-4 pathway, and would take important roles in innate immunity response. 4. Warm ischemia reperfusion injury could induce DCs maturing and active innate immunity response through TLR-4 pathway, then accelerate adoptive immunity response and rejection in allotransplant. 5. Warm ischemia reperfusion injury would up-regulate Th2 immunity response in Allogeneic small bowel transplantation.