Clinical And Experimental Study Of Liver Transplantaion Using Marginal Donors

Part I Clinical study of liver transplantaion using marginal donorsOrthotropic liver transplantation has been accepted as an established treatment modality for patients with all forms of end-stage liver diseases. The number of patients on waiting lists has increased dramatically, meanwhile, donations of organs have not increased proportionally, and the discrepancy between available livers and request for transplantation has widened. With innovative surgical techniques and improved immunosuppression, liver transplantation (LT) continues to be the only definitive mode of therapy for patients with end-stage liver diseases. However, the major factor limiting the application of liver transplantation remains the scarcity of donor organs, which presently accounts for a 10 per cent waiting list mortality rate. This has forced many centers to use livers that are considered marginal in terms of performance and survival after implantation. Steatosis is a common feature that identifies marginal liver function, and investigations to evaluate steatosis have reported controversial results. Prolonged cold ischemia time is another feature thatidentifies marginal liver function.The aim of the study here is to present our experience with the use of marginal donor liver, which have resulted in a large increase in the number of liver transplantations.Materials and methodsPatient characteristicsFrom January 2003 to September 2005, 204 adult patients (age > 16 years) receiving cadaveric related liver transplantation at the First Affiliated Hospital, Zhejiang University School of Medicine, China, were enrolled in this retrospective study.During the period of the study, frozen section was not routinely used at the time of procurement for the grading of steatosis. The assessment of donor livers by the donor surgeon was based on visual inspection, palpation, and appearances after flushing with preservation solution. Donor livers that were felt to have moderate steatosis by the donor surgeon were used if there was felt to be a suitable recipient. Postreperfusion liver biopsies were obtained after liver implantation for histologic evaluation of donor liver steatosis. All biopsy specimens were semi-quantitatively graded by the same pathologists after haematoxylin-eosin staining. Fatty infiltration in liver grafts was differentiated in macrovesicular and microvesicular steatosis. Fat droplets displacing the hepatocyte nucleus and occupying the majority of the cytosol were considered macrovesicular steatosis. Data analysisThe following variables were recorded for study population: donor age, donor gender, donor weight, recipient age, cold ischemia time (CIT), recipient gender, recipient weight, etiology of the hepatopathy; pre-transplant liver function [total bilirubin (TB),aminoleucine transferase (ALT), aspartate aminotransferase (AST) and prothrombin time (PT)], pre-transplant kidney function [serum creatinine (SCr) and blood urea nitrogen (BUN)], pre-transplant model for end-stage liver diseases (MELD) score, post-transplant liver function, post-transplant kidney function, immunosuppressive regime (cyclosporine or tacrolimus). The pre-transplant data were collected 24-hour before transplantation. The post-transplant liver and kidney function were observed during the first week of post-transplant. All patients were routinely followed up closely at the outpatient clinic. Patient and graft survival was analyzed at 3 months, 6 months, 1-year or 2-year, using inadequate follow-up as the exclusion criterion.Early allograft dysfunction (EAD) was defined by the presence of at least one of the following characteristics: TB > 10 mg/dl, PT> 17 s, and hepatic encephalopathy from day 2 to day 7 post-transplantation. Early renal dysfunction (ERD) was defined by SCr > 2 mg/dl and/or the need of renal replacement therapy in the first post-transplant week. Statistical analysisThe Kolmogorov—Smirnov test was used to check for normality. Quantitative variables were expressed as mean ± SD with normal distribution and were expressed as median without normal distribution. Categorical variables were presented as values and percentages. The Student's t test or Wilcoxon's rank sum test was used to compare quantitative variables and the Chi-square test was used to compare categorical variables. Kaplan-Meier method and log-rank test were used for survival analysis. SPSS for Windows version 13.0 (SPSS Inc., Chicago, IL) was used to complete all the analyses, and a P value of < 0.05 was considered statistically significant.ResultsThe steatosis scores of the total number of allografts transplanted were as follows: no steatosis and mild steatosis, (n =160; 78.4%); moderate steatosis, (n =44; 21.6%). No donor liver with severe steatosis was used for transplantation. Liver and kidney functionAfter the transplantation, recipient's biochemistry parameters were recorded and the dynamic changes of liver and kidney functions in the first post-transplant week were described. The serum levels of TB, PT, ALT and AST decreased well in the whole week and almost decreased to normal range on the post-transplant day 7. The serum levels of SCr and BUN showed no decline. From the post-transplant day 2 to day 7, the median of serum markers were compared without any significant difference. Patient and graft outcomePNF was really a rare complication in our center (0%). Patients with CIT ≥ 12h presented a significant higher incidence of EAD and lower patient and graft survival than patients with CIT < 12h (P < 0.05). Patients with moderate steatosis presented no significant differences on incidence of EAD, patient and graft survival compared with Patients with no or mild steatosis (P > 0.05). The incidence of EAD was higher in patients with moderate steatosis combined with CIT ≥ 12h than that others (P < 0.05). The 3-month and 6-month patient and graft survival rates were significantly higher in patients with moderate steatosis combined with CIT ≥ 12h than patients with no or mild steatosis combined with CIT < 12h (all P < 0.05).Conclusion1. Moderate steatotic liver can be used to expand the donor pool.2. Allograft with prolonged CIT is associated with poor patient and graft outcome3. Moderate steatotic liver with prolonged CIT is associated with poor patient and graft outcome and should be routinely discarded.Part II Experimental study of liver transplantation using marginal donorsThe discrepancy between available livers and request for transplantation has forced many centers to use livers that are considered marginal donors such as fatty liver. The shortage of organs has led clinicians to continually modify criteria to expand donor pool, particularly those in the so-called expanded or marginal donor pool. As we have mentioned before moderate steatotic liver with prolonged CIT are considered as poor patient and graft outcome should be routinely discarded.Apoptosis or programmed cell death is a genetically controlled response of the cell to commit suicide. Apoptosis is the physiological process for cell deletion in normal, reorganizing, or involuting tissue. A20 was described as a protective gene product induced by tumour necrosis factor a (TNF-α) in human endothelial cells. Both in vivo and in vitro studies have proved its functions in inhibiting apoptosis and inflammatory responses, and its role as a negative regulator of nuclear factor-kappa B (NF-κB) activation has been illustrated recently. NF-κB constitutes a family of more than 150 transcription factors that play a key role in the control of apoptosis and regulation of immune and inflammatory responses. A20 may thus offer itself as a potential therapeutic target for the treatment of various diseases where apoptosis and/or inflammatory responses constitute components of the pathophysiology.The aim of study is to investigate the potential mechanism of the negative effect of steatosis and prolonged cold ischemia time donor on outcome.Materials and methods1. Animal model1.1 Fatty liver rat model :Healthy adult male Lewis rats were fed with a choline-methionine deficient diet for 6 weeks. Five rats were randomly selected and killed for liver biochemical assessment. The pathological change of fatty livers and the activity of liver GSH, MDA, MPO were studied.1.2 In vivo model of rat liver transplantationThe rat liver transplantation was performed according to .Kamada-Calne method with some modification2. Experimental groupsThe present study included male Lewis rats as donor and recipient. Rats were randomly divided into three groups: steatotic donor liver group, steatotic+prolonged cold ischemia time donor liver group, normal donor group. The rates of survive in each group were recorded and rats from each group were killed 24, 48 and 72 hrs after reperfusion. The serum and liver specimens were collected from the killed animals.3. Relevant studies :3.1 Serum biochemistry examination3.2 Liver GSH, MDA, MPO activity assays.3.3 Histology: liver specimens were examined under light microscopy and electron microscopy.3.4 Apoptosis was also quantified by the TUNEL assay.3.5 RT-PCR was used to assess the protective gene A20 mRNA in the liver..4. Statistical analysisData were analyzed using SPSS 13.0 software. Statistical significance was regarded as P value of 0.05 or less.ResultsSurvival study showed the 7-day survival rate of 90 % in N group and 70 % in S group and 40% in SPC group, respectively. The significant difference was investigated between N group and SPC group (P <0.05).Biochemical data showed that the levels of hepatic enzymes and TB reached the peak at 72 hours after reperfusion in each group. At 24 and 48 hours after transplantation, the levels of ALT, AST and TB in SPC group were significantly higher than those in N group (P <0.05). Swelling of hepatocytes, structural derangement, necrosis, regional sinusoidal dilation, congestion and detached epithelial lining were seen in S group and SPC group, especially in the latter. Apoptosis cells in SPC group are much more than those in N group. RT-PCR results showed that the expression of A20 mRNA in SPC group was obviously decreased at each time point after transplantation compared with those in N group.Conclusion1. Moderate steatotic liver can be used to expand the donor pool.2. Moderate steatotic liver with prolonged CIT is associated wth poor patient and graft outcome and should be routinely discarded.3. The negative effect of steatosis combined with prolonged CIT donor might be due to the down-regulated expression of A20, decreased antioxidation ability and apoptosis.