Early Kinetic Observation Of Dendritic Cells In The Transplanted Kidney And Graft Draining Lymph Node Of Rats.

Lots of documents indicate that acute and chronic rejections are still the main reasons that lead to the graft function failure.As a basis for the occurrence of cellular immune response,antigen-presenting is one of the major prerequisites of graft rejections.Dendritic cell(DC)is currently recognized as the most powerful antigen presenting cell(APC),and plays an important role in the initiation of immune response.In recent years,studies found that not only can DC leads to graft rejection, but also could DC induce T,B lymphocytes anergy which would result in the graft-specific immune tolerance,the latter has been regarded as the final settlement of long survival of the transplanted kidney.With the DC research in the field of transplantation is gradually warming up,more and more studies focus on the rule of DC changes in vivo after transplantation.By means of immunohistochemical staining, This study was designed to explore the kinetics of DC in the transplanted kidney and the renal hilar lymph node of rats in the early stage after renal transplantation,to explore further the prevention of rejection.This study consists of three parts,as described below.ChapterⅠEstablishment of bilateral renal transplantation in allogeneic rats. Objective:To establish a bilateral renal transplantation model in allogeneic rats.Method:Using SD rats as the donors and Wistar rats as the recipients,we did the bilateral renal transplantation for 30 pairs of rats.The animals were given anesthesia by intraperitoneal injection of 30mg/kg 3%sodium pentobarbital supplemented by inhalation of ether anesthesia.Donor operation was described as follows:Animals were laparotomized with grand cross cut.Firstly the mesenteric artery and celiac artery were freed,ligated,and cut,in order to expose the parts of inferior vena cava (IVC)under the hepatic and the abdominal aorta(AA)above the renal artery.The rectum was Ligated and cut.Then all the gastrointestinal organs were pulled out of abdominal cavity,the bilateral kidneys were thoroughly exposed at the same time. Secondly the kidneys,ureter,and bladder were freed,and then we freed the parts of AA and IVC above the lumbar vein,and cut down the circulation by ligating both of them close to the lumbar vein.We continued free AA up about 1cm long from the point of ligation,and then we inserted the perfusion catheter into the AA's cavity and get it fixed.We blocked the IVC and the AA above the right renal artery,cut open the IVC to establish the outflow tract,we perfuse the kidneys with 4℃Euro-Collins liquid until the kidneys turned to yellow.Then the kidneys,ureters,bladder,together with the AA and IVC nearby the kidneys were en-bloc harvested.The operation of the donor kidneys was described as follows:the connective tissue surrounding the AA was cut open from the back,and the two lumbar artery stumps were found and ligated. Except the 1-1.5cm long AA and IVC adjacent to the renal arteries and veins,other parts of AA and IVC were removed,with the bilateral ureter's openings as the center points,we repaired the bladder wall to oval shape.Recipients operation was described as follows:1 hour before the operation,the rats were intraperitoneally injected with 400U/kg heparin to achieve systemic heparinization.Animals were laparotomized with "+" shape cut,the horizontal incision pointing to the left.We freed the parts of AA and IVC above the lumbar vein up to 1.5cm long,and then we occluded the parts of AA and IVC we freed top and bottom with four vascular clips,1cm long AA and IVC occluded were resected,and the four vascular openings were sheared for the convenience of anastomosis.The donor's AA were anastomosed with AA of the recipient end to end,18 per anastomosis;the same were the anastomosis of IVCs,22 per anastomosis.Removing the clips,the AA pulse recovered and the bilateral kidneys turned to red immediately.The top wall of the bladder was resected and the oval donor bladder flap was then anastomosed with it.Results:The mean operating time was within 180rains and hot ischemic time less than 5s,cold ischemic time less than 90mins,the mean revascularization time was 50rains.The kidney color became a bright pink color immediately after recovery of circulation.Of 30 rats that renal transplants performed,24 survived the procedure and the success rate was 80%.The main failure reasons included:accidents of anesthesia,blooding,bad renal perfusion,and thrombosis of inferior limbs after operation.All of the rats undergoing the operation successfully survived more than one week without using any immunosuppressants.Conclusions:bilateral renal transplantation in allogeneic rats is a feasible animal model for research works in transplant field.Compared with the model of unilateral renal transplantation in rats,this model can decline the demand of vascular anastomosis technique of the operators,and lower the ratio of transplant renal artery stenosis;it also has the advantages of increasing the times of graft harvesting, obtaining paired data and economizing lab animals.ChapterⅡEarly kinetic observation of dendritic cells in the transplanted kidney of rats.Objective:To explore the kinetics of DC in the graft of rats within 72 hours after renal transplantation.Methods:Using SD rats as the donors and Wistar rats as the recipients,we did the renal transplantation for 30 pairs of rats;another 5 donor kidneys were treated as the sham group,which would not undergo transplantation.Respectively,the transplanted kidneys were harvested at 1h,6hs,12hs,24hs,48hs and 72hs averagely after the blood circulation of the grafts were recovered.Samples were paraffin-embedded and sectioned,then were stained by means of H.E.or immunohistochemistry.DC were identified with anti S-100 protein.The pathological changes and the DC density per glomerulus were observed with optical microscope.Results:No signs of acute rejection(AR),such as tubulointerstitial nephritis or artery uveitis,were found in all of the sections.In the section of immunohistochemistry,DC was stained brown,and tubular epithelial cells was stained lightly yellow as a positive control.DC volume in control group:0.50±0.07, 1h after operation:1.06±0.18,6h after operation:2.60±0.31,12h after operation: 3.76±0.11,24h after operation:6.54±0.40,48h after operation:3.92±0.11,72h after operation:2.60±0.26.Obviously there were few DCs in the sham and 1h group,since then DC number gradually increased;and reached the peak in 24h group,then slowly declined.The results of the comparisons between groups:there was no significant difference between control group vs.1h group(P＞0.05),but these two groups is significant different compared with other groups(P(max)=0.042).There were significant differences between 24h and other groups(Pmax=0.038).Conclusions:Within 72 hours after renal transplantation,the number of DC varies following a curve with a single peak.We deduce that DC density increases because DC from the peripheral blood of the recipient migrate into the graft, accordingly,the withdraw of recipient DC results to the density decrease.The revelation of DC changes in graft would be helpful to further improve the remedy aimed to anti- rejection.ChapterⅢEarly kinetic observation of dendritic cells in the graft draining lymph node of rats.Objectives:To locate the position of the left renal hilar lymph node of rats in detail,and to explore the kinetics of DC in the graft draining lymph node of rats within 72 hours after renal transplantation.Methods:5 SD and 5 Wistar rats were anesthetized and Laparotomized, 0.2m1(25%)of methylene blue was injected into the lateral left renal fossa and the cut was closed.6 hours later,the cut was reopened to see if the renal draining lymph node (RDLN)was stained by methylene blue.60 Wistar rats were divided into two groups randomly and averagely.Using SD rats as the donors and Wistar rats as the recipients, we did the renal transplantation in situ for 30 Wistar rats;Respectively,RDLNs were harvested at 1h,6hs,12hs,24hs,48hs and 72hs averagely after the blood circulation of the grafts were recovered.Another 30 Wistar rats underwent the left renal ischemia-reperfusion model,in which we only occluded the left renal artery and vein for 45 min,not remove the kidney,and then the renal circulation was reopened. Similarly as the transplantation group,the renal draining lymph nodes were resected at 1h,6h,12h,48h,and 72h after the renal blood flow was recovered.All of the samples were paraffin-embedded and sectioned,then stained with anti S-100 protein by the method of immunohistochemistry.DC number and morphologic change in the cortex of RDLNs between transplantation and sham groups were observed and compared.Results:6 hours after injection of methylene blue,we found that the lymph nodes we located were stained by the dye in all of the 10 rats.This phenomenon confirmed that the lymph node is the RDLN.Statistical analysis shows that there is a significant difference between the two groups:transplantation and ischemia-reperfusion group(P＜0.01).Speaking quantitatively,in the sham group,the number of DC in the cortex area of RDLN is the least at 1h,and increases to maximum value at 48h,then declines;in transplantation group,the number of DC varies as a rising Linear with time,it is also the least at 1h,and increases steadily, reaches the maximum value at 72h,multiple comparisons show that there is no significant difference in 1h vs.6h(P=0.153),comparisons between other groups are all statistical significantly(24h vs.48h:P=0.011,others:P＜0.01).Speaking morphologically,the DC shape is similar at 1h and 6h in transplantation group,they are both oval and scattered,and we can hardly find any pseudopodia projections on the cellular surface,this is the typical shape of immature DC.But in 12h～72h of transplantation group,DC shape changes totally,a large number of dendritic-like pseudopodium,by which DC embraces lymphocytes,appears on the cellular surface, and DC tends to be distributed densely,this is the typical shape of mature DC.On the contrary,DC in all of the sham groups remains an immature performance steadily.Conclusions:Further clarify the location of the RDLN of rats will be helpful to make it a useful means by which reveal the mechanism of transplantation immunity. The possibility of DC's two stages migration after transplantation(from peripheral blood to graft,graft to RDLN)is very big,there is close relationship between the relocation process of DC and its maturation.Drugs that influence DC migration can also impact the maturation and antigen-presenting process of DC,the research on this aspect has broad prospects.