Nitrous Hydrochloride Pretreatment Of Rat Small-size Liver Transplantation Ischemia-reperfusion Injury In Rats And Liver Regeneration

PartⅠEstablishing the Model of 30%Small-for-Size Liver Transplantation in RatsObjective:To establish the model of 30%small-for-size liver transplantation in rats, as a foundation for the forthcoming research on ischemia reperfusion injury(IRI) in small-for-size liver transplantation and liver regeneration post-operatively.Methods:100 pairs of Sprague-Dawley(SD) rats were performed orthotopic liver transplantation(OLT) by using Kamada's 2-cuff method,among which 50 pairs were performed 100%OLT before formal experiment,25 pairs were performed 60%OLT, and 25 pairs were performed 30%OLT.The graft liver was procured by situ rapid perfusion through abdominal aorta.The time for cold ischemia was set as 1 hour. Besides,we randomly selected 10 rats to perform hepatectomy,5 rats by 40%and 5 rats by 70%,and observed the survival rate.Results:For the groups of 60%and 30%partial liver transplantation,the weight ratios of donated liver to receipt liver were 59.70±3.21%and 31.92±2.43%. 92%(23/25) and 72%(18/25) survived for 2 days or above after transplantation. 72%(18/25) and 64%(16/25) were still alive 1 week after transplantation.For the group of hepatectomy,the weight ratios of the excised liver to the theoretical original liver were 42.51±3.62%and 66.83±4.37%,and 100%were alive 1 week after transplantation.Conclusion:Based on past animal experiments,we successfully established the model of 60%and 30%partial liver transplantation in rats with improvement in methods.This model is stable,workable and replicable with high probability of success.The rat model of partial liver transplantation has similar operation procedures and pathological and physiological change as compared with clinical partial liver transplantation,such as living donor liver transplantation;and is an ideal way for research on improving IRI in liver transplantation and promoting liver regeneration. PartⅡExperiment Study on the Role of Diazoxide Preconditioning in Alleviation Ischemia Reperfusion Injury after Rat Small-for-Size Liver Transplantation and the Impact on Liver RegenerationObjective:To investigate the protection mechanism of mitochondrial ATP-sensitive potassium channels(mitoKATP) opening on IRI after rat partial liver transplantation, especially small-for-size liver transplantation,and the impacts on liver regeneration; and offer a better practice to relieve IRI and promote liver regeneration for clinical partial liver transplantation,such as living donor liver transplantation.Methods:The animal model was built as the same way of PartⅠ.300 SD rats with the average weight of 220-300g were randomized into 150 pairs,and divided to 6 groups with 25 pairs in each group.Receipts should be heavier than donators by 10-20g.The 6 groups were:100%orthotopic liver transplantation(Group A),60%partial liver transplantation(Group C),60%partial liver transplantation with diazoxide(Group DA1),60%partial liver transplantation with 5-hydroxidecaprate and diazoxide (Group HD-DA),30%small-for-size liver transplantation(Group S),and 30% small-for-size liver transplantation with diazoxide(Group DA2).For Group DA, receipts received diazoxide(5mg/kg) by intraperitoneal injection 30 min preoperatively.For Group HD-DA,receipts received 5-HD(5mg/kg) 45 min preoperatively,and diazoxide(5mg/kg) by intraperitoneal injection 30 min preoperatively.Besides,50 SD rats with the average weight of 220-300g were randomized into 2 groups with 25 rats in each group.The 2 groups were respectively performed 40%hepatectomy(Group B1) and 70%hepatectomy(Group B2).For each experimental group,5 rats were killed(n=5) respectively at 5 points of time,which were 2 hours,1 day,3 days,5 days,and 7 days postoperatively. Observation and detection includes liver function index(ALT,AST),wet weight of liver tissue postoperatively and ratio of liver regeneration,pathological examination by light microscope,examination of ultra-micro structure of cell by electro-microscope,proliferating cell nuclear antigen(PCNA) index,apoptosis of liver cells by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL),cytokines such as tumor necrosis fator-alpha(TNF-α),interleukin-6(IL-6), and interleukin-1β(IL-1β) in liver tissue and serum by enzyme-linked immunosorbent assay(ELISA),and the activity of NF-κB in liver tissue by ELISA.Results:1.Pathological examination by light microscope:as compared with the control group ~*,the liver cells of Group DA showed less swelling,degeneration and necrosis;the vacuolus showed less regeneration;the structure of hepatic lobule was less disorderly; inflammation was less serious.More cell division was found in Group DA than the control group.2.Ultra-micro structure of cell examined by electro-microscope:the cell structure of Group DA was complete,swelling of mitochondria was less,intercellular space and sinus hepaticus was less distending,Kupffer cell was less active,and phagosome was less observed.However,mitochondrial crista and microvilli of the control group was less observed,endocytoplasmic reticulum was more swelling,Kupffer cell was more active,and some parts were necrosis.3.Index of liver regeneration:as compared with the control group,liver wet weight of Group DA is larger and the liver regeneration ratio increased distinctly.Significant difference was noted 7 days post-operatively(P＜0.05).4.Index of liver function via serum:as compared with the control group,alanine aminotransferase(ALT) and aspartate aminotransferase(AST) of Group DA decreased distinctly,and significant difference was noted 2 hours,1 day,and 3 days post-operatively(P＜0.05).5.Proliferating cell nuclear antigen(PCNA) by immunohistochemistry:as compared with the control group,positive ratio of PCNA of Group DA increased distinctly,and significant difference was noted 3 days and 5 days post-operatively(P＜0.05).6.Apoptosis index(AI) by TUNEL:as compared with the control group,AI of Group DA decreased significantly 2 hours and 1 day post-operatively(P＜0.05).7.Expression of interleukin-1β(IL-1β) in liver tissue:as compared with the control group,IL-1βof Group DA decreased significantly 2 hours and 1 day post-operatively(P＜0.05).8.Expression of tumor necrosis fator-alpha(TNF-α) in serum and liver tissue:TNF-αin serum significant decreased as compared with that in liver tissue.As compared with the control group,TNF-αof Group DA decreased but no significant difference (P＞0.05).For liver tissue,TNF-ot of Group DA decreased significantly as compared with the control group and significant difference was noted 2 hours,1 day and 3 days post-operatively(P＜0.05). 9.Expression of interleukin-6(IL-6) in serum and liver tissue:as compared with the control group,IL-6 in both serum and liver tissue of Group DA increased distinctly and significant difference was noted 2 hours,1 day and 3 days post-operatively (P＜0.05).10.Changes in activity of NF-κB in liver tissue:as compared with the control group, NF-κB of Group DA decreased significantly 2 hours and 1 day post-operatively (P＜0.05).Conclusion:1.DA,which opens the mitoKATP,can relieve IRI after partial liver transplantation, including small-for-size graft,by reducing IL-1βand TNF-αand restraining the activity of NF-κB.2.DA could relieve IRI after transplantation;in the meantime,it could increase the weight,regeneration ratio and PCNA of the transplanted liver,the mechanism of which may be promoting liver regeneration by increasing IL-6 and maintaining essential expression of TNF-αand NF-κB.3.The effect of improving IRI and promoting liver regeneration by using DA could be specially blocked by 5-HD,and it proves that DA produces the effect through mitoKATP and implies that mitoKATP might be a target for improving the treatment effect of partial liver transplantation,including small-for-size graft liver.