The Surgical Experience Of Allogeneic Whole Pancreaticoduodenal Transplantation Model In Rats

Objective To establish the model of allogeneic whole pancreaticoduodenal transplantation(WPDT)in rats.To provide a tool for research grafts rejection and to explore the key points of this operation.Methods Wistar-Furth rats with type 1 diabetes mellitus were induced by intraperitoneal administration of streptozotocin(STZ)at a single dose of 60mg/Kg.On the basis of our primal model, we improved the way of arterial anastomosis.End to side anastomosis was performed for abdominal aorta of donors and recipients.The portal vein of the graft was anastomosed with the recipients left renal vein by cuff technique.And side to side anastomosis was made between the graft duodenum and the host jejunum.According to the technique mentioned above,we successfully established WPDT model between Lewis rats as donors and Wistar rats as recipients.To monitor the levels of blood glucose 3 times weekly and record the survival time(ST)of each rats.And grafts of 3 rats were collected to observe pathohistological changes on day 7 posttransplantation.Results The successful rate of diabetes rats induced by STZ was 85.7%.The incidence rate of diabetic ketoacidosis was 5.6%and the mortality was 7.4%.44 rats were successfully performed WPDT of 50 rats.The mean levels of blood glucose were decreased from(22.83±4.37)mmol/L preoperative to(6.36±2.18)mmol/L postoperative in 44 rats.Among of them,8 rats died in 3 days postoperative,the ST of residual 36 rats was 6-16 days,the mean of ST was(10.45±3.3)d.Three was a progressive increase of blood glucose on day 4,7,10 and 13.The peak of death appeared on day 7-10.The typical acute rejection in pathological changes was observed on day 7.Conclusion The successful rate of diabetes induced by STZ is high.The animal model of diabetes is stable,STZ is a safe drug.Skilled technique and emphasis on details are important to establish WPDT model.The endocrine function of grafts and typical acute rejection can be observed in this model.So,it could be applied to research the theoretical problems involved in grafts rejection. Objective To explore the role and possible mechanisms of spleen cells transfusion via portal vein and anti CD154 monoclonal antibody(mAb)combined with bone marrow transplantation in non-myeloablative preconditioning regimen for induction of immune tolerance in rats with allogeneic pancreaticoduodenal transplantation(PDT).Methods Wistar-Furth(Wistar,Rt1~u)rats with type 1 diabetes mellitus were induced by intraperitoneal administration of streptozotocin at a single dose of 60mg/Kg.We adopt Lewis(Rt1~1)rats as donor,Wistar rats with diabetes as recipient and Sprague-Dawley(SD)rats as the third party donor.Recipient Wistar rats were randomly divided into 3 groups,13 rats in each group.In group transplantation, Wistar rats were performed on PDT alone.In group mAb,Wistar rats were injected 3 mg AH.F5(hamster anti-rat CD154 mAb)via peritonium after PDT.In group tolerance,Wistar rats were performed according to preconditioning regimen.The regimen was portal vein injection of Lewis rats 2×10~8 spleen cells on day 0, intraperitoneal injection 3 mg AH.F5 2 hours later,in combination with intravenous iniection of Lewis rats 1×10~8 bone marrow cells on day 3,PDT on day 20.On day 7 after PDT,the blood glucose of 6 rats from each group were measured.Then enzyme linked immunosorbent assay(ELISA)was applied to determine the concentration of the serum interleukin(IL)-10 and interferon(IFN)-γ.And gafts were collected to observe pathohistological changes and apoptosis in suit with TUNEL and the expression of indoleamine-2,3-dioxygenase(IDO).positive cells by anti-IDO mAb immunochemistry methods.Rt1~1 positive Chimersim in recipient spleen and thymus were followed by flow cytometric(FACS)analysis.Tolerance was assessed by the ratio of stimulation index(SI%)of one-way mixed lymphocyte reaction(MLR).Rat recombined IL-2 reverse assay of one-way MLR and adoptive transfer assay were performed at the same time.Remaining 7 rats of each group were observed the survival time of grafts function.Results The concentration of serum IL-10 and IFN-γwas respectively(188.42±23.15)pg/mL and(202.46±24.07)pg/mL in group tolerance on day 7 posttransplantation,which was significant difference from groups transplantation and mAb.The balance of Th1/Th2 alter to Th2.The pathohistological changes in groups transplantation and mAb were typical acute rejection.The scores of histologic grade were(4.67±0.52)and(4.0±0.63),respectively.There was no significant difference between them(P=0.104).The histologic grade of acute rejection in group tolerance wasⅡ～Ⅲ,score was(2.67±0.82),which was milder than others group(P<0.01).The degree of histologic changes of duodenum was consistent with pancreas.Apoptosis was observed in three groups.But the amount of apoptosis in group tolerance was fewer than others group(P<0.01).The expression of IDO in tissue was no significant difference among groups(P>0.05).The positive staining cells were pancreatic acinal cells and duodenal mucosa endothelial cells and lymphocyte. The positive siteslocated on cytoplasm.The results of chimerism assay revealed chimerism was only detected in group tolerance.The levels were(31.56±6.57)%in spleen and(22.04±8.12)%in thymus.In one-way MLR assay,SI%of group tolerance was(26.41±5.16)%,which was significant lower than other groups (P<0.01).While spleen cells of group mAb could inhibit the stimulus originated by spleen cells of Lewis and SD rats.Exogenous IL-2 could completely or partly reverse immue hyporesponsiveness to allogeneic antibody.In vivo adoptive transfer assay, regulatory and suppressive activity in the spleen cells of rats in group tolerance was observed.The grafts functional survival time of group tolerance was prolonged to (21.83±1.11)days.Conclusion Administration of anti CD 154 mAb alone can not effectively establish chimerism and induce immune tolerance.Combined with transfusion of donor-derived spleen cells and bone marrow cells on the basis of anti CD154 mAb can establish allogeneic mixed chimerism on diabetes rats and successfully induce donor-specific tolerance in PDT which consisted of two immtmogenic organs.Furthermore,the regimen can relieve the degree of rejection and prolong grafts functional survival time.Increased IDO activity in grafts have the effect of anti-injection.However,the preconditioning regimen for induction or maintenance of chimerism and tolerance are not independent of IDO activity.Thus, increasing of IDO activity combined with this regimen have synergistic effect. Multiple mechanisms,including peripheral and central chimersim formation,altering the balance in Th1/Th2,T cells clone deletion by suppression of excessive apoptosis, clonal anergy and regulation/suppression are involved in the tolerance.