| PART 1 ESTABLISHMENT OF A LIVER TRANSPLANTATION MODEL USING MARGINAL SIZE GRAFT IN RATSObjective: Our aim in this part is to establish an animal model of marginal size graft-orthotopic liver transplantation using reduced size liver transplantation on rats, provides a stable animal model for the investigation of the mechanism of small-for-size syndrome after liver transplantation..Methods: we used male Spraue-Dawley rats, (190~250grams,±20~30 grams), 4 groups designated according to the ratios of donor and recipient liver weight: Group A, whole graft group(n=24); group B, 50% ratio liver transplantation ( the median lobe and right lobe of the liver was selected to be the graft,n=24); group C, 30% ratio liver transplantation (the median lobe of the liver were selected to be the graft, n=24); group D, less than 30% ratio liver transplantation (the median lobe and left lobe of the liver were resected, and remaining lobe was selected to be the graft, n=24). A rat model of nonarterialized orthotopic liver transplantation without veno-venous bypass was used. In the small-for-size graft group, the lobe ligation technique was used to reduce the graft size on the backtable. Operation parameters, portal pressure, liver function indices and survival rate were observed and analyzed for all groups. Liver specimens were morphologically examined under the light microscopy and electron microscopy.Results:1. One week survival rate of the group A, B, C and D was 100%(12/12), 83.3%(10/12), 16.7%(2/12) and 0, respectively. The differences were statistically significant, p<0.001.2. The portal pressure of the whole graft group were relative stable after reperfusion.But there was a significant increase in portal pressure in the reduce size graft group at 5 and 15 minutes after reperfusion compared with that pre-transplantation. This increase in portal pressure reached a peak at 15 minutes (increased by 65%～82%, group C, 16.47cmH20 vs 9.97 cmH20, p<0.05;group D,18.25 cmH20 vs 10.05 cmH20, p<0.05) and gradually subsided after 30 minutes of reperfusion. The relations between the size of graft and the portal pressure at 5,15,30,45,60 minutes after reperfusion are negative, (r=-0.926, r=-0.936, r=-0.904, r=-0.902, r=-0.867, p<0.05).3. In each group ALT and AST levels increased significantly at 24 hours after liver transplantation, the highest in group D , followed by group C, group B, and group A. The differences were statistically significant, p<0.001. The relations between the size of graft and ALT and AST levels are negative, (r=-0.704, r=-0.815, p<0.05).4. H&E staining revealed that the hepatic lobular architecture was well preserved in group A and group B at 24 hours after reperfusion. The hepatocytes and portal tracts showed normal morphological features. On the contrary, Focal necrosis was found mainly around the portal tract in the group C and group D at 24 hours after reperfusion.5. In group C and group D, electron microscopy showed severe mitochondria and endoplasmic reticulum swelling of hepatocytes accompanied by loss of microvilli; while in group A and group B, both the cell nucleus and cellular organelles had no significant breakdown.Conclusions:1. We confirmed again that the rat model of marginal size graft-orthotopic liver transplantation using reduced size liver transplantation was a suitable and stable animal model for the investigation of the mechanism of small- for-size syndrome after liver transplantation.2. We confirmed again that the minimum safely graft volume ratio in rat liver transplantation is 50%, the marginal size graft volume ratio is between 30～35%, and the ratio less than 30% should be considered as extra-small-for-size liver transplantation in the rat.3. To establish an animal model of orthotopic liver transplantation using marginal size graft in rat,we can use rats (190~250grams,±20~30 grams). The median lobe of the liver was selected to be the graft to get the graft ratio in a range between 30～35%.4. In a rat liver transplantation model using marginal size graft, the portal pressure changes in marginal size grafts are transient. Progressive damage of the graft may result from microcirculatory failure due to irreversible endothelial injury after reperfusion. PART 2 PROTECTIVE EFFECT OF IRBESARTAN AND EDARAVONE ON MARGINAL SIZE LIVER TRANSPLANTATION IN RATSObjective: The aim of this study is to investigate the protective effect of IRBESARTAN and EDARAVONE on decreasing portal pressure and reducing ischemia- reperfusion injury in the rat liver transplantation model using marginal size graft and to explore the mechanism of the marginal size graft injury during acute phase of reperfusion.Methods: The previously described rat model of nonarterialized liver transplantation using marginal size graft was used. Survived recipients were divided into five groups: group A, control group; group B, EDARAVONE treatment group; group C, IRBESARTAN treatment group; group D, IRBESARTAN and EDARAVONE treatment group; group E, sham operation group. Six rats in each group were sacrificed randomly at 6 hour and 24 hour after reperfusion respectively. The survival rate of animals and portal pressure were investigated. The rats in every group were sacrificed and its blood sample were collected for liver function measurement. Liver specimens were morphologically examined under light microscopy. The content of SOD and MDA in liver tissues were measured. Fresh liver tissue was used to detect the expression of Egr-1mRNA, ET-1 mRNA and Bax mRNA by RT-PCR. Paraffin-embedded liver specimen were used to histological change(HE), apoptosis (TUNEL) and the protein expression of ET-1,TNF-α, Bax. Six rats at each time pointing in each group were studied.Results:1. One week survival rate of the group A,B,C,D and E were 8.33%(1/12),33.3%(4/12) , 58. 7%(7/12), 83. 3%(10/12),100%(12/12), respectively.The differences were statistically significant, p<0.05.2. The portal pressure in group C and group D were lower than those in group A and group B before transplantation (8.21,8.15 vs. 10.28,10.55,p=0.019) . There was a significant increase of portal pressure in each groups at 5 and 15 minutes after reperfusion compared with the baseline. This increase in portal pressure reached a peak at 15 minutes (increased by 65.5% in group A and group B, 23.1% in group C and group D, 18.11, 18.55 vs. 15.76, 15.80 , p<0.05), gradually subsided after 30 minutes of reperfusion. The portal pressure of group C and group D were relative stable after reperfusion. The portal pressure in group C and group D were lower than those in group A and group B at 5, 15, 30, 45 and 60 minutes after reperfusion. p<0.05.3. Biochemical data showed that the levels of hepatic enzymes(ALT and AST) reached the peak at 6 hours after reperfusion in each group. At 6 and 24 hours after reperfusion, the ALT and AST levels in group A were the highest in four groups, followed by group B, group C, group D and group E. P<0.05.4. H&E staining revealed that the hepatic lobular Architecture was well preserved in group D and group C at 6 hours and 24 hours after reperfusion. The hepatocytes and portal tracts showed normal morphological features. On the contrary,detachment of vascular endothelial cells together with patchy necrosis was present in the group A and group B at 6 hours after reperfusion. Focal necrosis was found mainly around the portal tract in the group A and group B at 24 hours after reperfusion. 5. RT-PCR showed that the intragraft expression of Egr-1mRNA, ET-1 mRNA and Bax mRNA in group E were the lowest at each time point after reperfusion among the five groups, followed by group D, group C, group B and group A. p<0.05.6. The content of MDA after reperfusion was increased by time, inversely; the level of SOD was decreasing by time. The content of MDA in group E were the lowest at each time point after reperfusion among the five groups, followed by group D, group C, group B,and group A. The level of SOD in group E were the highest at each time point after reperfusion among five groups, followed by group D , group C, group B,and group A . p<0.05.7. Immunohistochemical results showed that the intragraft expression of ET-1, TNF-αand BAX in group E were the lowest at each time point after reperfusion among the five groups, followed by group D, group C, group B and group A. p<0.05.8. TUNEL results showed that the apoptotic index of group E was the lowest at each time point after reperfusion among the fiver groups, followed by group D, group C, group B and group A.p<0.05.Conclusion:1. The transient significant increase portal hypertention and ischemia reperfusion injury at the early phase after reperfusion were two significant contributors to the hepatic injury of marginal size liver graft in the rat liver transplantation mole using marginal size graft.2. In the two significant contributors to the hepatic injury of marginal size liver graft in the rat liver transplantation mole using marginal size graft, irreversible endothelial injury by transient significant increase portal hypertention at the early phase was more harmful to ischemia reperfusion injury .3. For two contributors above, IRBESARTAN and EDARAVONE could diminish acute phase injury in marginal size liver graft after reperfusion by anenuating the acute phase shear stress that resulted from transient increase portal hypertention and reduced ischemia reperfusion injury.This two protective methods used together were more effective to one method only. PART 3 EFFECT OF IRBESARTAN AND EDARAVONE ON LIVER REGENERATION ON MARGINAL SIZE LIVER TRANSPLANTATION IN RATSObjective: The aim of this study is to evaluate the effects of IRBESARTAN and EDARAVONE on liver regeneration in marginal size liver transplantation in rat.Methods: The previously described rat model of nonarterialized liver transplantation using marginal size graft was used. Survived recipients were divided into three group: group A, EDARAVONE treatment group; groupB, IRBESARTAN treatment group; group C, IRBESARTAN and EDARAVONE treatment group. Six rats in each group were sacrificed randomly at 2 and 7 days after reperfusion respectively. Each graft was removed and weighed. The regenerative response of liver graft was evaluated by the expression of Ki-67 antigen in hepatocyte that was measured by immunohistochemistry assay.Results:1. On 2 and 7 days after transplantation the wet weight of graft and liver regeneration rate in IRBESARTAN and EDARAVONE treatment group was the highest in three groups, followed by IRBESARTAN treatment group and EDARAVONE treatment group. p<0.05.2. Ki-67 labeling index of the graft reach a peak at 2 days after transplantation in three groups. The Ki-67 labeling index of the graft in IRBESARTAN and EDARAVONE treatment group was the higheset in three groups on 2 days and 7days after transplantation, followed by IRBESARTAN treatment group and EDARAVONE treatment group. p<0.05.Conclusions1. IRBESARTAN and EDARAVONE have no directly effect on hepatocyte regeneration in rat liver transplantation model using marginal size graft. It might protect the marginal size graft by reducing portal pressure and ischemia reperfusion injury. This two protective methods used together were more effective to one method only on hepatocyte regeneration.2. On hepatocyte regeneration in rat liver transplantation model using marginal size graft, reducing portal pressure was more effective to reducing ischemia reperfusion injury.
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