Protective Effect Of Somatostatin On The Small-for-Size Graft Injury In Pigs And The Underlying Mechanism

Partial liver transplantation (PLT) is currently gaining wider acceptance, which definitely alleviates donor organ shortage, but also reveals the problem of small-for-size graft syndrome (SFSS). Currently, the precise mechanism behind it remains still unknown. Both clinic and animal studies have shown the portal hyperperfusion, hepatic arterial hypoperfusion, imbalance of hepatic microvesicular regulation, and the abnormal liver regeneration account much for the occurrence and development of SFSS. Rodent models as well as those large animal ones established under the help of veno-venous bypass (VVB) are away from clinical situation. Pigs transplanted with 30% liver grafts without VVB are more suitable for the study of SFSS because of their similarity to clinic procedures in PLT and the physiological status of humans. Somatostatin (SST) has been used for two decades to treat oesophageal variceal haemorrhage for its capacity to decrease portal venous pressure without major side effects. Additionally, SST can induce diverse physiological effects in different tissues and cells, mediated by five different reciptors of SST (SSTR). It was reported recently SST, but other analogues, has intrahepatic effects such as inhibition of ET-1-induced contraction of hepatic stellate cells (HSC), and the transcription and expression of ET-1. It has been indicated low-dose SST-14 can rescue small-for-size (SFS) grafts in rat models and also comfirmed by the individual clinical reports, but further investigations have to be conducted. In this study, we will try to establish a feasible and effective porcine model with 30% liver graft without VVB, and then SST-14 will be applied to the model to assess the effect on the SFS graft injury. Portal pressure gradient (PPG), Changes of microvesicular regulation, apoptosis and the liver regeneration will be addressed to search for the protective mechanism.Part1. Establishing the model of small-for-size graft injury in pigs without veno-venous bypassObjective:To establish a feasible and effective SFS graft injury models in miniature pigs. Materials and Methods:13 pairs of Ba-Ma miniature pigs were randomly assigned into two groups:100% liver graft group (n=5) and 30% liver graft group (n=8), both transplanted with no VVB. The profiles of intra-operative hemodynamics and metabolism were investigated. Animals were followed for 7 days with daily serum for biochemistry exam. The survival rate (non-VVB tolerance rate, operative survival rate, and 7-day accumulative graft survival rate) were respectively calculated as well as the graft weight increase rate at post operative day (POD) 7.Results:All the recipients survived the anhepatic period except one in 30% graft group died of irretrievable acidosis. non-VVB tolerance rate and operative survival rate were all above 75% in both groups.7-day accumulative survival rate in 30% graft group was lower than that in 100% graft group (33.3% vs.100%) but with no significant difference (P=0.0521>0.05). Pigs in 30% graft group showed significantly prolonged prothrombin time, elevated bilirubin and aminotransferase levels during almost the entire follow-up. Serum creatinine concentrations were similar in both groups, with a sharp increase in the first one day, and then settled at values of no significant difference to that before reperfusion. MAP, CVP and BE in both groups, particularly in 30% graft group, decreased significantly at anhepatic phase for the rapid impairment to the effective blood volume (P＜0.01), which inversely evoked a significant elevation of heart rate and K+. After reperfusion, MAP, CVP and K+ restored gradually and got almost complete recovery at the end of operation despite a mild acidosis.Conclusions:Porcine model of 30% liver graft transplantation without VVB can be safely established and used for the study of SFS graft injury as its good clinical simulation on operative procedures and clinicopathological performance.Part2. Protective effect of somatostatin on small-for-size liver grafts in pigs and the underlying mechanismObjective:Investigate the protective effect of SST on the SFS graft injury and explore the preliminary underlying mechanisms.Materials and Methods:12 pairs of Ba-Ma miniature pigs were assigned into two groups: SFS+NS group (n=7), transplanted with 30% liver graft with normal saline as control; SFS+SST group (n=5), transplanted with 30% liver graft with SST administered as following method:Bolus infusion via peripherial veins 3 minutes just before portal reperfusion followed by continous infusion at 5μg/kg/h for 24 hours. The whole liver group (n=4) transplanted with 100% liver graft established in our first part study was used as model control in this research; All animals were followed for 7 days with daily serum for biochemistry exam. The 7-day accumulate graft survival rate and the graft weight increase at POD 7 were respectively calculated.The kinesis of PPG during 7 days follow-up and pathological findings at 2 hours after reperfusion, POD3 and 7 were evaluated. Additional parts of liver tissues were harvested to detect the expression of Ki-67,ET-1 by imnunohistochemistry and the apoptosis by TUNEL. The transcription levels of ET-1 mRNA were examed by Real-Time PCR. ELISA kits were also used to detect the levels of ET-1 and NO in peripheral plasm at 2 hours post reperfusion and on POD1,2,3,5 and 7, and the ratio of ET-1 to NO was further calculated.Results:1. Survival rate:No significant differences were found among the groups in warm ischemia time, cold ischemia time, portal to arterial reperfusion time, infra-hepatic inferior vena cava occlusion time, operating time for recipient, and blood transfusion.7-day accumulative graft survival rate in SFS+SST group was visibly higher than that in SFS+NS group but with no significant difference. (80% vs.42.9%,P=1283>0.05).2. Liver and renal function:Compared to SFS+NS group, the ALT and AST in SFS+SST group on POD1,2 and 3 were significantly decreased as well as the TBIL on POD 1 to 7 and PT on POD 1 to 5 (P<0.05). There were no significant differences of serum creatinine concentrations among three groups.3. Light microscopy examination:Severe sinusoidal congestion and disruption of architecture in the centrilobular region as well as focal endothelial denudation and hemorrhage into the connective tissue at interface zone were observed in SFS+NS group as early as 2 hours after reperfusion, whereas in the whole liver group, these changes were minimal. By POD3, there were more extensive hepatocyte microvesicular steatosis and fibrosis in SFS+NS group and By POD 7, the pathological changes became less severe with evidence of repair of disrupted hepatic architecture. Compared to SFS+NS group, SFS+SST group presented mild injury with almost normal lobules of liver.4. Electronic microscopy examination:In SFS+NS group, the hepatocyte microvesicular steatosis, tremendous mitochondrial swelling, and sinusoidal disruption were observed, whereas in the whole liver group, almost normal hepatocytes with only mild swelling of mitochondria and intact sinusoidal endothelial were observed. The pathological injury in group SFS+SST was less severe with almost integrity of sinusoidal endothelial lining.5. Kinesis of PPG after reperfusion:Over the postoperative period, the values of PPG in SFS+NS group were significantly higher than those in the whole liver group (P＜0.01) with two crest values at 2 hours after reperfusion and on POD 1 whereas in SFS+SST group the values were significantly decreased (P＜0.01) but still higher than the whole liver group with peak at 5 minutes post-reperfusion and thereafter downward to baseline on POD 3.6. Liver regeneration:The Ki-67 proliferation index in SFS+NS group was significantly increased at 2 hours post reperfusion and on POD 3 and 7 (P<0.05). Compared to SFS+NS group, the Ki-67 proliferation index in SFS+SST group was significantly decreased at 2 hours after reperfusion and on POD 3 (P<0.05), but with no significant difference on POD 7. There was also no significant difference of graft weight increase rate on POD 7.7. Apoptosis index:Compared to the whole liver group, the apoptosis index in SFS+NS group was significantly increased at 2 hours post reperfusion and on POD 3 and 7 (P<0.05). Compared to SFS+NS group, the apoptosis index in SFS+SST group was significantly decreased at each time checked (P<0.05).8. Transcription and expression of intrahepatic ET-1:Compared to the whole liver group, the transcription of ET-1 at 2 hours post reperfusion as well as the expression on POD 3 in SFS+NS group was significantly increased (P<0.05), whereas in SFS+SST group they both were significantly decreased when compared to SFS+NS group (P<0.05).9. The ratio of ET-1/NO:Compared to the whole liver group, the ratio of ET-1/NO on POD1,2,3 in SFS+NS group was significantly increased (P＜0.05), whereas in SFS+SST group, it significantly decreased when compared to SFS+NS group (P<0.05).Conclusions:1. Persistent higher PPG and hepatic microcirculation disturbance at the early period of SFS liver transplantation accounts much for the SFS graft injury.2. The over-proliferation of the hepatocytes at the early period of SFS liver transplantation is not necessitous for the size-reduced liver grafts'recovery in volume and function, and instead may even do harm to this process.3. SST-14 can improve the the function and survival of SFS graft in pigs by decreasing the PPG, attenuation of hepatic microcirculation disturbance and reduction of apoptosis.4. Short-term intervention of SST-14 with routine dose decreases the proliferation rate of hepatocytes at the early period of SFS liver transplantation, but has no impairment to the graft recovery in volume and function.