Impact Of Ischemic Preconditioning On Bacterial Translocation And Rejection In Rats Intestinal Transplantation

Objectives:Previous investigations have proved that ischemic preconditioning (IPC) have significant protective effects on ischemic reperfusion injury (I/R injury) of small intestinal transplantation (SBTx). It is therefore reasonable to expect that IPC will protect the host from acute rejection (ACR) and bacteria translocation (BT) by moderating I/R injury to a certain extent. The objective of the present study is to test the hypothesis that IPC should decrease acute rejection and BT after SBTx in rats. Material and methods:1.AnimalsAndrea Ferencz had proved in his previous study that 2mins ischemia followed by 2 mins reperfusion was long enough to initiate IPC effect. According to this resoult, orthotopic allogenic SBTx was performed from Wistar (weighing 188.4±23.6g) to BN (weighing 198.0±29.2g). Donor-recipient sets were randomly allocated to 4 groups: In group IPC4d (n=7), before the graft was harvested, ischemic preconditioning (IPC) was achieved by a 2min-2min ischemia-reperfusion cycle by clamping the superior mesentery artery (SMA) and subsequent graft reperfusion. After SBTx, the recipients were kept alive for 4 days and then they were sacrificed. In group IPC7d (n=7), 2min-2min IPC was performed and the animal was sacrificed on the 7th day after the SBTx. Two control groups were used: ctrl4d (n=7) and ctrl7d (n=7) with end points on the 4th day and 7th postoperative days respectively.2.Operative ProceduresOrthotopic SBTx was performed as previously described. Animals were operated under isoflurane inhalation anesthesia. Each graft consist the entire jejunum and ilieum from 3-4cm distal Traiz ligment to 3-4cm proximal ileum-cecum valve, of about 80% small bowel of the donor. The vascular pedicle consisted of the superior mesenteric artery with a cuff of aorta and the portal vein. Cold (4°C) Ringer's solution with heparin (10 units/ml) was used to perfuse the vessels and irrigate the intestinal lumen, and the graft was wrapped in moist gauze and stored at 4°C, while recipient operation was prepared. Venous outflow of the graft was reestablished first with end-to-side anastomoses between graft portal vein and recipient vena cava using 9/0 running sutures. Arterial revascularization was achieved by end-to-side arterial anastomoses between the cuff of the donor aorta and the recipient infrarenal aorta with 9/0 monofilament sutures. Gastrointestinal continuity was reestablished by two end-to-end anastomoses between recipient jejunum and ileum and the corresponding segments of the graft. The recipient was kept on warm blanket after the operation until it recovered from anesthesia. Water was given after 2 hours, and the recipient was allowed ordinary chow 24h after the operation.3.SamplingAnimals were sacrificed under deep anesthesia at the end points mentioned above. Under sterile conditions, vena cava blood sample was taken, whole blood was cultured in appropriate media, and serum sample was collected by centrifugating whole blood at 3000rd/min for 5min. Liver, lung, and native and grafted bowel was sampled for histology and biochemical assessment.4.HistologyOrgan weight of the liver and lung were measured before fixation in 10% buffered formalin. Samples of fixed liver, lung and intestine were taken. After dehydrating and embedding, 5μm thick sections were cut and stained with hematoxylin-eosin routinely. Histological grading of acute cellular rejection (ACR) of the grafted intestine was performed according to the grading system established in clinical intestinal transplantation. Villus height and crypt depth were measured at 30 points chosen randomly on the slides of both native and grafted intestine under low power field. Mitotic cells were counted on 5 high power randomly selected fields on each slide. Measurements were blindly repeated by two authors without knowledge of experimental groups.5.ImmunochemistryTNFαand IL-1βlevel were determined by ELISA (eBioscience ref: 88-7340, 88-6010) in serum samples and homogenized intestinal tissue. Myeloperoxidase (MPO) activity was determined by ELISA (Hycult biotechnology ref: HK105) in homogenized intestinal tissue. All procedures were carried out strictly following the instructions offered with the ELISA kit.6.MicrobiologySystemic blood samples (2 ml each) were inoculated into hemoculture bottles for aerobic and anaerobic bacteria (BacT/ALERT? 3D Microbial Detection System Ref: 259791, 259793 Biomerieux Inc.) and incubated in an automatic culture system for 5 days. Cultures were considered positive if more than 100 colony-forming units grew per milliliter culture media. Results1.SurvivalAnimals'diying in the first 3 post-operative days were considered as operative failures. After appropriate trainig, survival rate reached 84.6% (66 out of 78).2.HistopathologyNo distinct of the Organ weight, it was similar in all groups. No abnormalities could be identified upon examination of HE-stained slides of the livers and lungs under microscope. The results of intestinal histopathology grading of damage: IPC and control group samples retrieved on the 4th day received very similar scores. The 2min IPC group of 7th day had higher grades than the ctrl group, but the differences were not significant. The measurements of villus height, crypt depth and proliferation of each group met the same results as histopathology grading, without any significant differences among them.3.Cytokine ImmunochemistryOn the 4th postoperative day, TNFαlevel was significantly higher in the native intestines than in the control ones after 2min IPC. On the 7th day, 2min IPC resulted in a distinct TNFαlevel elevation in the native intestine in comparison with the control group. There were no significant differences in the grafts among the groups. TNFαserum concentration was distinctly elevated in the 2mins IPC group in comparison with the ctrl groups at both endpoints.As regards intestinal tissue cytokines, there were no differences in IL-1βlevel between grafted and native intestine in the 2min IPC group both on the 4th postoperativeday, but it was distinctly raised in the graft on the 7th day. In serum, 2mins IPC group did not show any difference on the 4th postoperative day, but it brought down significantly IL-1βlevel on the 7th postoperative day.4.Myeloperoxidase (MPO) analysis MPO level in the grafts and the native intestine was analysed in order to estimate the infiltration of inflammatory cells of the acutely rejected bowel. At the 4th day after the transplant, MPO level in the grafts and native intestines was very similar in both groups. At the 7th day, MPO level of the grafts of the IPC group was significantly lower.5.MicrobiologyThe results of microbiology cultures: In the early post-operative period, 2min IPC present a protecting effect against bacterial translocation, the positive counts of the bacteria culture is significantly lower than control groups. At the 7th day, the IPC scheme wasn't able to change the result of the culture. DiscussionThe protective effect of IPC on small bowel I/R damage has been observed in some morphological studies but others showed only a slight reduction in lesions that invariably happens after I/R. These studies are heterogeneous in the design of investigating I/R protective effects of IPC. So the adequate period of IPC to confer some ACR protective effect remains unclear.In the present study, Tissue MPO and serum IL-βwere significantly lower, and the degree of rejection tended to be lower in IPC7d compared to Ctrl7d. These differences that were not present at day 4, suggest that IPC 2mins could have a protective effect on acute rejection onset.Proinflammatory cytokines IL-1βand TNFαand MPO levels reflects inflammatory response after IR injury, however their specificity is low and levels would be increased also in case of rejection or other events. Previous studies demonstrated that rejection produces changes in intragraft cytokine level starts from day 5 after SBTx. Regarding native intestine and serum TNFα; levels were significantly higher precisely in IPC group after both 4 and 7 days. This contradictory finding is difficult to explain as native intestine underwent the same procedure in all groups and graft versus host disease is not likely to occur in this model. On the other hand, TNF has sown unexpected results in other IPC models.Regarding blood cultures, BT was improved in IPC4d in comparison to Ctrl4d, this finding suggest that IPC protected the mucosal barriers integrity, and BT as a consequence. After seven days, there were no differences between groups. In fact, BT was commonplace, probably because of the onset of rejection in some degree in almost every animal. The interaction between AGR and BT, and the clinical relevance of BT is not analyzed...