| IntroductionLiver transplantation has become a world-wide accepted alternative treatement for terminal stage of liver disease. Chronic rejection remains an important cause of late liver allograft dysfunction and failure. Chronic rejection is characterized by persistent liver function abnormalities as a consequence of progressive cholestasis which manifest as jaudice, preferential elevation of gamma-glutamyl transpeptidase and alkaline phosphatase. The incidence of CR after transplantation is declining for the past few years from 15% to 20% at the early reports to an incidence of 3% to 5% in current liver allograft recipients, but remains a high level from 8% to 12% after child liver transplantation. The onset of CR is usually from 60 days to 1 year after liver transplantation, except for a small group of early onset occuring within 2-3 weeks. Chronic liver rejection is irreversible, which resulted in graft decompensation, recipient death and retransplantation. The final diagnosis of chronic liver rejection should be based on a combination of the clinical/radiological/laboratory and histopathologic findings. The histopathologic diagnostic criteria for CR are: the presence of bile duct atrophy/pyknosis, affecting a majority of the bile ducts, with or without bile duct loss;(2) convincing foam cell obliterative arteriopathy;or (3) bile duct loss affecting greater than 50% of the portal tracts. The etiology of CR is not yet clear defined. Cytomegalovirus (CMV) infection is a suggested risk factor for CR. Clinical retrospective surveys found that long-playing and subclinical CMV infection increased incidence of chronic rejection in liver transplantation and vanishingbile duct syndrome often occurred in patients with persistent CMV infection. Persistent CMV-DNA was found in hepatocytes, bile duct cells and vascular endothelial cells in those transplant livers with VBDS examined by in situ DNA-hybridization. Other clinical investigations also found a close correlation between CMV infection and VBDS. CMV infection increases incidence of CR in heart, lung and kidney transplantation. Experiment demonstrated in liver allografts undergoing acute rejection, CMV infection significantly increased portal and bile duct inflammation and caused more severe bile duct damage. Immunohistochemistry showed positive CMV antigen express in bile duct cells. In vitro, extensive CMV replication and cytolysis were seen in infected hepatocytes, which mean CMV can cause direct liver parenchyma damage by efficient cytolytic infection of hepatocytes.Human cytomegalovirus (HCMV) infection occurred in 30-65% of liver transplantation recipients, of which 18-40% was symptomatic infection. The role of CMV infection in the pathogenesis of transplanted liver has not been well documented. Whether CMV can directly propagate in bile duct cells and lead to necrosis of biliary epithelium? Or CMV infection triggers or aggravates an immune response by inducing proinflammatory factors (cytokines/ chemotactic factor) which regulate inflammatory reaction and destroy immunological balance? To preliminary study CMV infection on the pathogenesis of chronic liver rejection, we developed a rat model of allograft liver chronic rejection, and evaluated damaging effect of CMV infection on the etiology of chronic liver rejection and effect of CMV infection on fibrosis-inducing factors expressing, which included transforming growth factor P 1 (TGF- P 1), platelet-derived growth factor (PDGF), and basic fibroblast growth factor (bFGF), and effect of CMV infection on Thl/Th2 cytokines (interferon gamma, interleukin-4) expressing in chronic liver rejection.ObjectiveBy developing a rat model of chronic liver rejection, we compare Thl/Th2 cytokines expressing between chronic and acute liver rejection, evaluate damaging effect of CMV infection on the etiology of CR and effect of CMV infection on TGF-P 1, PDGF, bFGF expressing, and preliminary study CMV infection on the pathogenesis of chronic liverrejection.Materials and MethodsInbred male Dark Agouti (DA;RTla) rats served as donors, and inbred male Brown Norway (BN;RTln) rats served as recipients. Animals were assigned to one of four treatment groups at random. Group A: acute rejection group, n=20, no treatment was given after liver transplantation. Group B: low dose cyclosporine A (CsA) treatment group, n=38, animals received low-dose CsA (1 mg/kg/d) from transplantation to day 30 by subcutaneous injection. Group C: CMV infection group, n=32, recipients received CsA lmg/kg/d from transplantation to day 30 and on the transplant day, recipients were infected with HCMV (AD169, 30.0logTCID50, 0.4ml) by peritoneal injection. Group D: syngenetic control group, male BN rats were used as donors and recipients, treatment was given as group B after liver transplantation. All animals received ampicillin (200 mg/kg intramuscularly) on postoperative days 0, 1, and 2. After survival studies (n=8 at each group) were completed, morphometric and histopathologic investigations were performed on the first, 6th, 10th, 20th, 30th, and 60th day after liver transplantation by sacrificing 4-6 rats. Hematoxylin-eosin (H-E) or Masson's trichrome staining were used to study acute or chronic liver rejection and rejection activity index were analyzed. HCMV-IE, HCMV-pp65 antigenemia in peripheral blood mononuclear cell (PBMC) and HCMV-EA, HCMV-LA antigen express in liver tissue were detected by immunohistochemistry. The level and the mRNA express of TGF- P 1> PDGF^ bFGF, INF- y ? IL-4 in peripheral blood serum or liver tissue were detected by transcriptase polymerase chain reaction (RT-PCR) or enzyme-linked immunosorbentassay (ELISA). Liver function test was performed and analyzed.Results1. Survival analysis and general state after liver transplantationThe mean survival time in the group A, B, C, D was 11.88+2.36 days, 58±14.93 days, 41.13+12.45 days, and 169.25+48.71 days (mean+SD) respectively, and the median survival time was 12 days in A group, 54.5 days in B group, 41 days in C group, and 170.5 days in D group. There was no statistic difference between B and C group. The general state of rats in group C were severely bad as compared with other groupswithin 1-3 days after HCMV injection, which manifest as less activity, declined food and drink and lethargy. The symptom was disappeared 3 days after HCMV injection, and no significant difference was seen as compared with other groups.2. Liver function test after rat liver transplantationThe levels of total bilirubin (TBIL) and alanine aminotransferase (ALT) in serum in group A, B, and C increased dramatically on the first day after liver transplantation, and kept at high level till recipient's death, and in group D, recovered to pretransplant level after a transitory increase. The level of TBIL and ALT were no significant differences between group B and C at various time point. However, significant difference existed between group B, C and group D from day 3O.The level of TBIL and ALT significantly increased from day 30 in group B, but increased from day 20 in group C after liver transplantation.3. Histopathologic examination and rejection grade analysis after liver transplantation Acute rejection was discovered in group A by H&.E staining, which showed marked swelling of portal tracts with intense mononuclear cell infiltration and edema;intense cellular infiltration of sinusoids causing considerable separation of liver cell plates;inflammatory cells include macrophages, large pyroinophilic cells, and plasma cells;extensive loss of liver cells with degeneration and necrosis of many of those that survive. Lymphocytes and neutrophilic leukocytes penetrated basement membrane into biliary epithelium and infiltrated surrounding bile duct. Degenerative changes of bile duct in the form of cytoplasmic vacuolation, disordered cell polarity, karyopyknosis and karyokinesis were seen, or moreover, lytic necrosis of bile ductular epithelia and karyolysis may also present. Venular subendothelia! lymphocyte infiltrating was typically. Perivenular inflammatory changes from moderate to severe were showed and extended to liver parenchyma.H&.E staining of rat liver showed chronic rejection from day 30 in B, C group, which manifested as moderate to severe foam cell obliterative arteriopathy, bile duct damage or vanishing, cell infiltration and fibrosis of transplant liver. Portal tracts and sinusoids were swelled or enlarged with intense inflammatory cell infiltration. Medium-sized and small arteries were typically manifest as intimal aggregates of foam cell, fibrosis of arterial wall, which resulted in lumen narrowing or occlusion. Bile ducts showed irregular nuclei, vacuolar degeneration and focal necrosis of biliary epithelium. Some bile ducts were totallydestroyed or vanished due to fibrosis. Portal tract fibrosis plus incomplete or complete septa connecting portal tracts were developed, extensive liver fibrosis and diffuse cirrhosis was presented in more severe cases.Rejection grading analysis showed the overall rejection activity index (RAI) of rat liver in group B was 5.17±1.47, 7.17±1.47 on day 20, day 30 and was 8.0±1.67, 10.83±2.32 (mean±SD) in group C, which existed statistic difference between group B and group C. Semiquantitative histological analysis of transplanted liver found that bile duct damage in group C was significantly high as compared with group D on day 30, though there was not statistic difference between group B and C. Obliterative arteriopathy of rat liver in group C (RAI: 1.33±0.52) was significantly severe than those in group B (RAI: 2.50±0.55) on day 30. Transplant liver fibrosis analysis showed statistic difference from day 20 after liver transplantation between group B and group C, and mean score of liver fibrosis in group B on day 20, day 30 was 1.0±0.63, 1.67±0.52 respectively and 2.67±0.52, 3.17±0.75 in group C.4. HCMV antigen express in PBMC and in liver tissueImmunohistochemistry demonstrated percent of positive HCMV-IE and pp65 express in PBMC was 100%, 75% respectively on day 1 after HCMV injection. The mean number of positive cell per 200,000 leukocytes was 19.0+19^ 7.0+6.83, after that, positive IE express decreased gradually and got to the lowest level on day 20, recovered on day 30. However, positive pp65 express increased to the highest level on day 10;at which mean number of positive cell per 200,000 leukocytes was 12.0± 16.0 and percent of positive express was 80%. The number and percent of positive pp65 express reached to minimal level on day 20, and recovered on day 30.HCMV-EA and LA positive express in liver section was not detected on the first day after HCMV injection. Mild positive EA and LA express was showed from day 6, gentle increased on day 10, and strong positive EA and LA express in transplant liver was found on day 20 and day 30.5. TGF-pi, PDGF, bFGF levels in peripheral blood and mRNA express in transplant liver TGF-31 level in peripheral blood and mRNA express in rat liver in group A was increased by degrees from day 1 to day 10 after liver transplantation and significantly exceeded controlgroup from day 6. TGF-J31 mRNA level of rat liver in group B and C showed strong positive express from day 1 to day 10 after transplantation and was significant high than control group, though there was no statistic difference between group B and C. The TGF-(31 mRNA express was declined little by little from day 20 after liver transplantation. TGF-pi level of rat in peripheral blood transitory increased first, then decreased gradually, which existed statistic difference as compared with group D at 20 day after liver transplantation in group B, and at 6-20 days in group C.PDGF level in peripheral blood and mRNA express in rat liver in group A kept stable after liver transplantation, and was no statistic difference at various time point as compared with control group, which increased continuously in group B and C. The mRNA express of PDGF in group C was significantly high than other groups at day 6, day 10, and than group B, D at day 20. The PDGF level of group C in peripheral blood at day 20 was significantly high than that of group B.The bFGF mRNA level in rat liver of group A and D showed low express, and no statistic difference. The mRNA level of bFGF in rat liver of group B and C increased continuously, which was significantly high than control group from day 10 after liver transplantation. Moreover, the bFGF mRNA express in rat liver of group C significantly exceeded that of group B on day 20 after transplantation. The bFGF level of group B and C in peripheral blood increased slowly, and significantly increased on day 60, day 30 in group B, C respectively. There was no statistic difference at various time point between group B and C, but the level was significantly high than control group on day 60 in group B and day 30 in group C.6. Thl/Th2 cytokine level in peripheral blood and mRNA express in transplant liver Levels of INF-y in peripheral blood and mRNA express in rat liver of group A increased continuously from day 1 to day 10 after liver transplantation. The mRNA express was significantly high than other groups from day 6, and the serum level was exceeded other groups from day 10 after transplantation. Low mRNA express in rat liver of group B, C and D were detected. The serum concentration of INF-y of group C increased slowly, and showed statistic difference as compared with control group at day 20, day 30 after transplantation, though there was no significant differences between group B and C, orgroup B and D.Low IL-4 mRNA express in rat liver of group B, C was detected after liver transplantation. The level of it was significantly increased from day 20 and showed strong positive express. No positive mRNA express was detected in rat liver of group A and D. The levels of IL-4 express in peripheral blood of group B and C were raised from day 20, and showed statistic difference from day 30 as compared with control group. There was no statistic difference at each time point between group B and C, but the serum level was significantly high at day 60 in group B and day 30 in group C. Trivial serum IL-4 express was detected in group A and D.Conclusions1. A reproducible animal model of chronic rejection was developed in liver transplantation in a rat strain combination of DA->BN with low-dose immunosuppression, in which moderate to severe manifestations of foam cell obliterative arteriopathy, bile duct damage or vanishing, cell infiltration and fibrosis of transplant liver was detected from day 30 posttransplantation.2. Fibrosis-inducing factors including TGF-(31, PDGF, bFGF and Th2 cytokine IL-4 express were up-regulated in chronic liver rejection, which may be involved in pathogenesis of chronic rejection. The high IL-4 express was a result of immune activating, and may be a pathway in modulating chronic liver rejection.3. HCMV infection aggravated bile duct damage, foam cell obliterative arteriopathy and liver fibrosis in chronic liver rejection, and accelerated process of chronic rejection.4. The pathogenesis of HCMV infection in chronic liver rejection may be correlated with early high PDGF and bFGF expressing. HCMV infection increased and accelerated fibrosis of chronic rejection was not correlated with level of TGF-(S1 expressing. |
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