An Experiment About The Protective Effects Of Omega-3Fatty Acids On Rats Subjected To Cryopreserved Liver Transplantation

BackgroudIschemia-reperfusion injury (IRI) caused by graft cryopreservation has triggered a series of problems such as poor early graft function, primary non function and biliary complications. Therefore, IRI has been the hot topics in the study of liver transplantation. Nowdays, the mechanism of IRI is still unclear. The consensus is that the innate immune system will be activated after reperfusion of the graft. And then local proinflammatory response will be appeared. The ischemic damage to the graft mainly show metabolic disorder and parenchyma cell death which lead to ligands activation and mitochondrial release oxygen free radicals. After the reperfusion, the adverse factors above with the involvement of Kupffer cells, NK cells, dendritic cells, T cells, neutrophils and other non parenchymal cells will activate the host’s innate immune system, further aggravate the inflammatory response. The involvement of immune cells in peripheral blood can make the immune activation-inflammatory cascade a continued progress. In short, the IRI is a local proinflammatory response which is mediated by host’s innate immune system and finnaly lead to damage of the liver parenchymal cells.It has been reported that omega-3polyunsaturated fatty acids (n-3PUFA) could improve the prognosis of parenteral nutrition correlation of liver disease, to reduce the production of inflammation medium and adhesion molecules to play a role in anti-inflammatory, to prevent IRI and circulation failure in mice with fatty liver, to alleviate chemically induced acute hepatitis and liver cell damage caused by obstructive jaundice in rats and so on. Recently, it has been proved that n-3PUFA could also attenuate IRI and promote hepatic regeneration following partial liver resection in normal or fatty liver.Although there is report about the liver-protecting effects of n-3PUFA for patients after liver transplantation, the influence of n-3PUFA to the IRI which caused by graft cryopreservation and the exact mechanism is still not very clear. As we know, even with the rapid development of medicine, the IRI of donor liver caused by cryopreservation is still completely unavoidable. Hence, it is clinically meaningful to maximumly reduce IRI and improve liver function for patients after liver transplantation. As mentioned above, the IRI is a local proinflammatory response mediated by host’s innate immune system. In this pathophysiological process, the parenchymal cells release a variety of inflammatory mediators such as TNF alpha, IL-6, IL-1beta and IFN-gamma after the activation of host’s inane immune. This is the key to maintain the inflammatory cascade. It has been proved that omega-3fatty acids could play an anti-inflammatory effect in mouse with chemical-induced hepatitis. In this study, firstly we established an orthotopic liver transplantation model in rats without the reconstruction of hepatic artery. This modal was performed according to Kamada’s two cuff method under the concept of precision surgery. We found the best method for reconstruction of veins and bile duct for ROLT. Secondly, we established an cryopreserved orthotopic liver transplantation model in rats and explored the effect of graft cryopreservation to liver function and microcirculation. In the meantime, we found out the exact length of cropreservation time which was suitable for animal experimental study. At last, we treated the rats wihich subjected to cryopreserved liver transplantation with n-3PUFA. And then the treatment effect of n-3PUFA to the rats was investigated. We try to prove that n-3PUFA could inhibit liver inflammation, improve liver function and further increase the long-term survival of the recipients.Part I Establishment and Evaluation of Liver Transplantation Model in Rats Under the Concept of Precision SurgeryOBJECTIVE:Establishment of rat orthotopic liver transplantation (ROLT) model under the concept of precision surgery. The hepatic artery was not reconstructed. The modal was evaluated for exploring the best microsuture method for reconstruction of suprahepatic vena cava (SHVC), the best cuff and stent for reconstruction of veins and bile ducts.METHODS:Male SD rats weighing250to300g were used as donors and recipients. The weight difference was equal or lesser than20g. According to different microsuture methods for the SHVC and different types of cuffs and stents, three ROLT groups were created to compare the operation times and prognoses. Group1:new microsuture method for SHVC+single-groove cuff+blade-cut stents. Group2:new microsuture method for SHVC+single-groove cuff+scissors-cut stents. Group3: conventional continuous suture method for SHVC+multi-groove cuff+blade-cut stents. Sham operations were performed as controls in group4. The time expenditures with each step were compared among the transplantation groups. The one week-and one month-survival rates of the transplantation groups were recorded and compared. All of the animals that survived one month were sacrificed and the biochemical parameters were tested. The SHVC, infrahepatic vena cava (IHVC), portal vein (PV) and hilar bile duct was detailedly dissected to make sure if there are torsion of the vessels, thrombosis and biliary complications. P＜0.05was considered to indicate statistical significance. All of the analyses were performed using SPSS software, version18(SPSS, Inc., Chicago, IL, USA). RESULTS:Our new microsuture method was faster than the conventional continuous suture method for SHVC anastomosis (P＜0.05). There was no bleeding of the anastomotic stoma of the SHVC. The use of a single-groove cuff for reconstruction of the portal vein and the infrahepatic vena cava shortened the anastomotic time (P＜0.05) and significantly reduce the postoperative thrombosis. The new microsuture method companied with the single-groove cuff shortened the anhepatic time when compared to the conventional continuous suture method with the multi-groove cuff (P＜0.05). The biochemical parameters reached to normal level at one month after the transplantation. The The use of blade-cut stents resulted in fewer biliary complications and better survival over the short and long terms (P＜0.05).CONCLUSIONS:Our new microsuture method and the single-groove cuffs proved to be a precise method for venous reconstruction which shortened the anhepatic time and the anastomotic time significantly. These new methods are more in line with the concept "minimizing surgical invasiveness and maximizing organ protection" of precision surgery. The blade-cut stents apparently decreased the incidence of biliary complications by reducing the injury to the inner wall of the bile duct. In summary, with this precise microsuture method and delicate cuffs and stents, excellent long-term survival can be achieved easily and stably for ROLT. Part II The Effect of Graft Cropreservation to Postoperative Liver Function and Instant Microcirculation For Rats Subjected to Liver TransplantationOBJECTIVE:To establish a mature and stable cryopreserved liver transplantation modal in rats with the reconstruction of hepatic artery. To explore the effect of different cryopreservation time to postoperative liver function and instant microcirculation. To lay the foundation for drug treatment of damage caused by cryopreserved liver transplantation.METHODS:Animals were similar to Part Ⅰ. To establish cryopreserved rat liver transplantation modal with the reconstruction of hepatic artery. The methods mention in Part Ⅰ was applied. According to different cryopreservation time, three ROLT groups were created. In group1, donor liver was perfused and preserved for12h by4℃HTK solution (P12h group) and in group2for24h by4℃HTK solution (P24h group). In group3, sham operations were performed as controls. Anhepatic time, the time used for donor and recipient and general conditions on the7th day after the operation were recorded. For the recipients, the laser speckle perfusion images were performed3,10and20min after the reperfusion of the donor liver. Ten recipients were used for comparison of7day-survival rate. Blood and liver specimens were gained on postoperative day1(24h), day3and day7(POD1,3,7) when the recipients were sacrificed. There were six recipients for each point. The six recipients in the control group were also sacrificed on postoperative day7and blood specimens were collected. Biochemical parameters included alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin (Alb), alkaline phosphatase (ALP) and total bilirubin (TB) were measured for all the blood specimens. Hematoxylin-eosin staining was done for the liver tissue specimens. The liver tissue were semi-quantitatively scored from the following items:portal inflammation, focal necrosis, ductular proliferation, bile duct damage and septal fibrosis. The real-time elastography was performed transabdominally for all the survivors before they were sacrificed. All of the analyses were performed using SPSS software, version18(SPSS, Inc., Chicago, IL, USA). P＜0.05was considered to indicate statistical significance.RESULTS:1. Time expenditure and general conditions. There were no significant difference of the time used in donors and recipients between P12h and P24h groups. The anhepatic time were controlled within14min. The animals in P12h group recovered faster after the operation. There were no icteric sclera, yellow claws and urine for all the survivors in the P12h and P24h groups.2. The microcirculation changes of the graft after the reperfusion. The flux value of the graft liver in the cryopreserved groups were significantly lower than the control. While the flux value in the P12h group was higher than that in the P24h group (P＜0.05).3. The survival rate in the P12h group was significantly higher than that in the P24h group (P＜0.05).4. The biochemical parameters of the serum. The liver function in the cryopreservation groups were heavily damaged when compared with the control group. On POD1,3and7, all the parameters were all worse than that in the control group obviously (P＜0.05). In the cryopreservation groups, ALT and AST decreased over time. While ALP increased at first and then decreased. The Alb and TB value increased over time.5. The semi-quantitative scores of the liver tissue. The liver damage was very obvious even on POD1for the recipients in P12h and P24h groups. Following changes were found in HE staining:Swelling and vacuoles degeneration of the liver cells, hepatic sinusoid congestion, inflammation cells infiltration of the portal tract, bile duct endothelial cells swollen and necrosis. As an extension of time, swelling and vacuoles degeneration of the liver cells and hepatic sinusoid congestion alleviated. Portal inflammation and ductular proliferation were obvious than POD1. Focal necrosis alleviated but bile duct damage aggravated over time. The septal fibrosis was not obvious. At the same points, the P12h group has lower scores than the P24h group with respect to portal inflammation, ductular proliferation, focal necrosis and bile duct damage (P＜0.05). But there were no significant difference of the scores for septal fibrosis (P＞0.05).6. The real-time elastography of recipients’liver. The mean elastic ratio of the liver in the control group was setted as normal value. The elastic ratios in the cryopreservation groups were higher than normal value just on POD1and didn’t change a lot over time. The elastic ratios of liver in the P12h group were similar to that in the P24h group at the same point (P＞0.05). However, the elastic ratios of the cryopreservation groups were always higher than normal value during the observation time (P＜0.05).CONCLUSIONS:1. Cryopreservation of the donor liver caused serious liver function damage. The damage of bile duct cells were heavier than liver cells. The longer the donor liver was cryopreserved, the heavier the liver was impaired.2. Cryopreservation of the donor liver could cause early postoperative damage to hepatic microcirculation. The longer the donor liver was cryopreserved, the heavier the microcirculation was impaired. Laser speckle perfusion imaging can be used as an index for evaluating of early ischemic injury caused by the cryopreservation of the donor liver3. Twelve hours cryopreservation of donor liver was suitable for study of liver impairment after cryopreserved liver transplantation.4. The real-time elastography was a safe and noninvasive method for evaluation of liver function. Part Ⅲ The Effects of Omega-3Fatty Acids to Postoperative Liver Function for Rats Subjected to Cryopreserved Liver TransplantationOBJECTIVE:To investigate the effects of omega-3fatty acids to postoperative liver function for rats subjected to cryopreserved liver transplantation and preliminarily discuss the mechanism.METHODS:According to different time of graft preservation and postoperative administration, rats were randomly divided into three transplantation groups. Our new methods for ROLT which was based on precision liver surgery was used here. In group1, normal liver transplantation was performed. In group2and3, donor liver was perfused and preserved for12h by4℃HTK solution and then transplanted. During postoperative day (POD)1to7, the recipients in group land2received saline solution (12ml/kg/day, via orogastric gavage) while recipients in group3received omega-3fish oil fat emulsion (12ml/kg/day, via orogastric gavage). Ten recipients were used in each group for comparison of four week survival rate. On POD3,7,14,21and28, all survivors in each group were sacrificed, blood and liver specimens were collected. There were six recipients for each point. Biochemical parameters and semi-quantitative score of the hematoxylin-eosin staining of liver tissue were similar to that in Part Ⅰ. At various points after the operation, Ki-67level of the liver tissue was measured by immunohistochemical analysis. Meanwhile, Masson staining was used to describe the degree of fibrosis in liver tissue. The real-time elastography was performed transabdominally for all the survivors just as Part Ⅱ. In addition, on POD3,14and21, the levels of TNF-alpha, IL-6, IFN-gamma and TGF-betal in the serum was measured by ELISA kit. The expression of mRNA of the same cytokines in the live tissue was measured by real time quantitative PCR in the same point. All of the analyses were performed using SPSS software, version18(SPSS, Inc., Chicago, IL, USA). P＜0.05was considered to indicate statistical significance.RESULTS:1. Suvival rate. The normal liver transplantation group was higher than that in the cryopreserved group (P＜0.05). There were no significant difference between group2and3which cryopreserved liver transplantation were performed (P＞0.05).2. The biochemical parameters of the serum. The biochemical parameters except Alb in group2was significantly worse than that in group1and3(P＜0.05). The postoperative liver function in group3was improved but still worse than that in group1(P＜0.05). The biochemical parameters in each group improved during POD14-POD21and flatten out during POD21-POD28. At the end of POD28, the animals in group1and3got nearly normal liver function but the biochemical parameters in group2were still not better than normal value.3. The semi-quantitative scores of the liver tissue. At the same time point, there were no difference for septal fibrosis and focal necrosis among the three groups. For the bile duct damage, group1was the lightest and there were no difference between the other two groups. For portal inflammation, group2was the worst and there were no difference between the other two groups. For ductular proliferation, on POD7and POD14, group1was the lightest and there were no difference between the other two groups. On POD21, group2was the worst and there were no difference between the other two groups.4. The real-time elastography of liver. The elastic ratio of the liver decreased after the operation. There was no difference between group2and3at the same time point(P＞0.05). But the elastic ratio of group2and3were higher than that in group1(P＜0.05).5. The levels of cytokines in the serum. On POD3,7and14, the levels of TNF-alpha, IL-6, IFN-gamma and TGF-betal increased first and then decreased. The levels of cytokines were the highest on POD7. At the same time point, the levels of TNF-alpha, IL-6and IFN-gamma in group2was the highest and there was no difference between the other two groups. The TGF-betal showed no difference among the three groups.6. The expression of mRNA of the cytokines in the live tissue. The changes of the mRNA of the cytokines in liver tissue was similar to that in the serum.The expression of mRNA of the cytokines were also first increased and then decreased. At the same time point, the levels of TNF-alpha, IL-6and IFN-gamma in group2was the highest and there was no difference between the other two groups. The TGF-betal showed no difference among the three groups.7. The proliferation of liver cells. The scores of Ki-67levels in the liver tissue showed that the liver cells proliferated most obviously during one week among the three groups after the operation. Then the proliferatation gradually weakened. There were no difference of the scores of Ki-67between group land3(P＞0.05). During the first two weeks, the scores of Ki-67in group2were higher than that in group1and3(P＜0.05). On POD21, the scores of Ki-67in group2were still higher than that in group3(P＜0.05). And there were no difference among the three groups on POD28.8. Detection of collagen fibers in the liver tissue. The scores of Masson staining showed that the content of collagen fibers in the liver tissue increased first and then decreased. The scores on POD14and POD21were more higher than other time point. But there were no difference between the three groups at the same time points.CONCLUSIONS:1. When compared with normal liver transportation,12hours cryopreservation of the donor liver decreased the four week survival rate of the recipients whether or not usage of omega-3fatty acids.2. Omega-3fatty acids could improve the postoperative liver function in some extent for rats subjected to cryopreservation transplantation. However, it couldn’t change the restore speed of the liver function.3. Omega-3fatty acids probably play aniti-inflammatory role through the inhibition of the synthesis of inflammatory cytokines such as TNF-alpha, IL-6and IFN-gamma in the liver tissue. Then the ischemic reperfusion injury alleviated and the liver function got better. Omega-3fatty acids didn’t increase the risk of fibrosis of the liver. But it couldn’t improve the ductular proliferation and bile duct damage caused by cryopreservation of the donor liver, either.