Syngeneic Liver Transplantation Research Articles

AAV9-mediated gene delivery to liver grafts during static cold storage in a rat liver transplant model.

Recombinant adeno-associated virus (rAAV) is a novel strategy used clinically for gene delivery, but has not been characterized in the context of organ transplantation. We sought to determine the efficacy of rAAV-mediated gene delivery during static cold storage (SCS) prior to liver transplantation. A triple-plasmid transfection protocol was used to produce rAAV subtype-9 vectors containing firefly luciferase genomes in HEK293 cells. Lewis rat liver grafts were flushed and stored in cold HTK solution. Three experimental groups received rAAV at different doses, administered via the portal vein as a bolus during SCS. A control group did not receive rAAV (N = 2). Recipients then underwent syngeneic liver transplantation. Bioluminescence imaging to quantify in vivo luciferase expression was performed on post-operative days 7, 14, 28, and 56. Control animals demonstrated no bioluminescent activity, while animals receiving rAAV-treated livers had increasing bioluminescence, peaking at four weeks but sustained to the eight-week endpoint. This result was confirmed by experimental endpoint tissue luciferase activity assay. rAAV mediates gene transduction in liver grafts when administered during SCS and has potential for gene therapy applications in solid organ transplantation.

Incompatibility between recipient CD47 and donor SIRPα is not a key risk factor for thrombocytopenia or anemia following rat liver xenotransplantation in mice

Liver xenotransplantation (LXT) is greatly impeded by severe thrombocytopenia, anemia, and coagulopathy. Hepatic phagocytic cells are thought to play an important role in LXT-induced thrombocytopenia and anemia. In this study, we investigated whether the lack of recipient CD47-donor SIRPα interaction, which is known to induce xenograft rejection by macrophages, exacerbates platelet and RBC depletion following LXT. We first addressed this question in the absence of anti-donor immune responses using a syngeneic mouse liver transplantation (LT) model. Neither wild-type (WT) nor CD47KO B6 mice developed thrombocytopenia following LT from WT B6 donors. Although a moderate decline in RBCs was detected following LT, there was no significant difference in RBC counts between WT and CD47KO recipients. Because mouse CD47 is cross-reactive with rat SIRPα, we then compared thrombocytopenia and anemia between WT and CD47KO mice following rat LXT. Unlike syngeneic mouse LT, significant thrombocytopenia and anemia were detected following rat LXT. However, the severities of both platelet and RBC depletions were comparable between WT and CD47KO recipients. Furthermore, WT and CD47KO recipients showed a similar extent of early platelet activation. Our results indicate that CD47-SIRPα signaling does not significantly affect the loss of platelets or RBCs following LXT, suggesting that the limited cross-reactivity between recipient CD47 and donor SIRPα is not a significant risk factor for LXT-induced thrombocytopenia and anemia.

MiR-152 may silence translation of CaMK II and induce spontaneous immune tolerance in mouse liver transplantation.

Spontaneous immune tolerance in mouse liver transplantation has always been a hotspot in transplantation-immune research. Recent studies revealed that regulatory T cells (Tregs), hepatic satellite cells and Kupffer cells play a potential role in spontaneous immune tolerance, however the precise mechanism of spontaneous immune tolerance is still undefined. By using Microarray Chips, we investigated different immune regulatory factors to decipher critical mechanisms of spontaneous tolerance after mouse liver transplantation. Allogeneic (C57BL/6-C3H) and syngeneic (C3H-C3H) liver transplantation were performed by 6-8 weeks old male C57BL/6 and C3H mice. Graft samples (N = 4 each group) were collected from 8 weeks post-operation mice. 11 differentially expressed miRNAs in allogeneic grafts (Allografts) vs. syngeneic grafts (Syngrafts) were identified using Agilent Mouse miRNA Chips. It was revealed that 185 genes were modified by the 11 miRNAs, furthermore, within the 185 target genes, 11 of them were tightly correlated with immune regulation after Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and Genbank data cross-comparison. Verified by real-time PCR and western blot, our results indicated that mRNA expression levels of IL-6 and TAB2 were respectively down regulated following miR-142-3p and miR-155 augment. In addition, increased miR-152 just silenced mRNA of CaMK II and down-regulated translation of CaMK II in tolerated liver grafts, which may play a critical role in immune regulation and spontaneous tolerance induction of mouse liver transplantation.

CD39 Is Critical for Inducing Tolerance in Murine Allogeneic Liver Transplantation.

Background: ATP is abundant in the cell cytosol and important for cell metabolism. Once cells die or are injured, ATP is released into the extracellular space and activates immune cells as a DAMP (damage-associated molecule pattern). CD39 regulates immune activation by hydrolysis of extracellular (e)ATP. We have found that conventional mouse liver myeloid(m) DC express comparatively high levels of surface CD39 and that this contributes to protection of the liver from cold ischemia reperfusion injury after syngeneic liver transplantation (LTx). We hypothesized that CD39 might play an important role in “spontaneous” transplant tolerance after allogeneic mouse LTx. METHODS: 10-12 week-old male B6 WT or CD39 KO (B6 background) livers were transplanted orthotopically into normal 10-12 week-old male C3H mice. Graft rejection was assessed by host survival and histology and also by serum ALT on day 4 after LTx. Incidences of regulatory T cells (Treg) and IFN-γ+ CD8+ T cells in the graft were quantified by flow cytometry at day 4 post LTx. Anti-donor T cell proliferative responses were evaluated by CFSE-MLR. RESULTS: Whereas all the WT B6 liver transplanted C3H mice survived more than 100 days, animals that received CD39KO livers all died within 40 days (MST: WT: >100d vs CD39 KO 8d; p<0.01). Serum ALT at day 4 post LTx was significantly higher in mice given CD39KO livers than in those with WT livers (WT: 480±226 IU/l vs CD39KO 860±484IU/l; p<0.05). The frequency of Treg in liver graft non-parenchymal cells after LTx was significantly lower in CD39KO livers than in WT livers (WT: 3.4±0.5% vs CD39KO 2.6±0.2%; p<0.05). Moreover IFN-γ production by CD8+ liver T cells was significantly higher in CD39KO allografts than in WT allografts (WT: 38.8±1.7% vs CD39KO 46.6±2.0%; p<0.05). Splenic T cells from CD39KO liver transplant recipients exhibited greater proliferation against donor splenocytes than those from WT liver allografted mice (WT: 27.2±5.8% vs CD39KO 36.3±5.7%; p<0.05). CONCLUSION: CD39 deficiency in liver grafts induces stronger anti-donor T cell responses and breaks “spontaneous” tolerance after mouse liver Tx. CD39 may be an important molecule for promoting liver allograft tolerance.

MHC-mismatched mice liver transplantation promotes tumor growth in liver graft

Liver transplantation is a final therapeutic option for treatment of hepatic malignancies, but local recurrence remains high after surgery. However, the underlying mechanisms of local tumor recurrence are still unknown. We speculated that immunological status of transplanted liver may contribute to the progress of tumor development. CT-26 tumor cells are injected into graft after allogeneic or syngeneic liver transplantation. The growth pattern of tumor and the co-relationship of regulatory T cell and effector T cells in liver graft were observed and investigated at 3d, 6d, 9d and 15d post-transplantation. The Hepatic Replacement Area of tumor in allogeneic grafts was significantly larger than that in syngeneic grafts. The activation of tumor growth in allografts was due to the dysfunction of effector T cells mediated by regulatory T cells in liver graft. Using nude mice model, we further confirmed that regulatory T cells from allograft significantly weaken the function of effector T cells in vivo. Our data has showed that MHC-mismatched mice liver transplantation can promote tumor growth in liver graft. For the first time, we demonstrated that susceptibility to tumor development in liver graft is due to the down-regulation of effector T cells’ function mediated by the regulatory T cells.

Pre-retrieval reperfusion decreases cancer recurrence after rat ischemic liver graft transplantation

Liver transplantation from marginal donors is associated with ischemia/reperfusion (I/R) lesions, which may increase the risk of post-transplant hepatocellular carcinoma (HCC) recurrence. Graft reperfusion prior to retrieval (as for extracorporeal membrane oxygenation--ECMO) can prevent I/R lesions. The impact of I/R on the risk of cancer recurrence was assessed on a syngeneic Fischer-rat liver transplantation model. HCC cells were injected into the vena porta of all recipients at the end of an orthotopic liver transplantation (OLT). Control donors were standard heart-beating, ischemic ones (ISC), underwent 10 min or 30 min inflow liver clamping prior to retrieval, and ischemic/reperfused (ISC/R) donors underwent 2h liver reperfusion after the clamping. I/R lesions were confirmed in the ISC group, with the presence of endothelial and hepatocyte injury, and increased liver function tests. These lesions were in part reversed by the 2h reperfusion in the ISC/R group. HCC growth was higher in the 10 min and 30 min ISC recipients (p = 0.018 and 0.004 vs. control, as assessed by MRI difference between weeks one and two), and was prevented in the ISC/Rs (p = 0.04 and 0.01 vs. ISC). These observations were associated with a stronger pro-inflammatory cytokine profile in the ISC recipients only, and the expression of hypoxia and HCC growth-enhancer genes, including Hmox1, Hif1a and Serpine1. This experiment suggests that ischemia/reperfusion lesions lead to an increased risk of post-transplant HCC recurrence and growth. This observation can be reversed by graft reperfusion prior to retrieval.

Hepatic Arterial Perfusion Is Essential for the Spontaneous Recovery From Focal Hepatic Venous Outflow Obstruction in Rats

We previously observed that focal hepatic venous outflow obstruction recovered spontaneously by the formation of sinusoidal canals in a rat model of portal hyperperfusion. We aimed to investigate whether the lack of hepatic arterial perfusion aggravates parenchymal damage, decelerates recovery and influences the formation of sinusoidal canals after focal hepatic venous outflow obstruction. Rats were subjected to arterialized versus nonarterialized syngeneic liver transplantation after ligating the right median hepatic vein in the donor. Hepatic damage, microcirculation, regeneration and vascular remodeling were evaluated. In arterialized-recipients, confluent necrosis interspersed with viable periportal islands of hepatocytes, and vascularized sinusoidal canals with visible blood flow, surrounded by normal sinusoidal structure, were visible on postoperative day (POD) 2. Complete parenchymal recovery was consequently established by resorption of necrosis and hepatocyte proliferation, detected in viable portal islands and border zone. Lack of hepatic arterial perfusion caused complete necrosis in the obstruction zone without viable hepatocytes in the periportal area on POD2. Hepatocyte proliferation was only visible in the border zone. On POD28, perfused vascular structures, without neighboring normal sinusoidal structures, were observed in the scar-like area. Hepatic arterial perfusion determined the extent of hepatic necrosis, the formation of vascularized sinusoidal canals and the parenchymal recovery, after focal hepatic venous outflow obstruction.

Establishment of an Acute Graft-Versus-Host Disease Model Following Liver Transplantation in Donor-Dominant One-Way Major Histocompatibility Complex Matching Rats

Acute graft-versus-host disease is an uncommon but devastating complication following liver transplantation (LTx-aGVHD). We investigated whether a rat model of LTx-aGVHD could be established using Lewis rat donors and (LewisXBN)F1 rats as recipients, which provides favorable conditions for studies of graft-versus-host reaction or disease. Replacement of (LewisXBN)F1 livers by Lewis livers alone was not sufficient to induce aGVHD; all recipients grew in a normal pattern as the syngeneic liver transplantation (LT) from Lewis to Lewis rat. However, when various numbers of donor splenocytes (1 × 10 8, 2 × 10 8, 3 × 10 8, 4 × 10 8) were transferred simultaneously with the LT, the morbidities of lethal aGVHD were 16.7%, 50%, 83.3%, and 100%, respectively. The clinical courses as well as histologic analyses of skin and colon showed typical aGVHD characteristics. However, unlike transfusion-associated aGVHD, the liver graft was not involved. These clinical and histologic characteristics of aGVHD were consistent with those in humans who develop aGVHD after LT. Thus, a reproducible rat model of LTx-aGVHD was developed by performing LT from Lewis to (LewisXBN)F1 rats in combination with donor splenocyte transfusion. This model may be useful for further studies on the mechanisms and effective treatment modalities for LTx-aGVHD.

Regulatory T cells contribute to the immunoregulatory effect on graft versus host reaction after liver transplantation in donor-dominant one-way MHC matching rats

Acute graft-versus-host disease following liver transplantation (LTx-aGVHD) poses a major diagnostic and therapeutic challenge, and the mortality rate is as high as 85%. Even the liver contains large numbers of lymphoid cells in the parenchyma and surgical damage of liver transplantation would induce an immunosuppressive, the incidence of LTx-aGVHD is just approximately 1–2%. CD4 +CD25 +regulatory T (Treg) cells have recently been shown to suppress proliferative responses of CD4 +CD25 −T cells to alloantigenic stimulation in vitro and are required for ex vivo tolerization of donor T cells, which results in their reduced potential to induce aGVHD after bone marrow transplantation. To investigate the role of Treg in LTx-aGVHD, we compared the proportional frequency of Treg in syngeneic liver transplantation recipients (sLT group), semiallogeneic liver transplantation recipients (Semi-LT group), LTx-aGVHD induced recipients (LTx-aGVHD group) and healthy controls. Here we show that replacement of (LewisXBN)F1 liver by Lewis liver alone in Semi-LT group was not sufficient to induce aGVHD, and all recipients grew in a normal pattern as the syngeneic LT from Lewis to Lewis rat. However, when 4 × 10 8 of donor splenocytes were transferred simultaneously with LT, the morbidity of lethal aGVHD were 100%. A relative stable percentage of Treg, defined as CD4, CD25 and Foxp3, was detected in peripheral blood mononuclear cells (PBMCs) of sLT group compared with healthy controls. In early time after transplantation, no significantly change of Treg population was observed in these recipients after semiallogeneic liver transplantation in comparison with healthy controls ( P > 0.05). However, Treg levels showed a relative increase 4 days after transplantation. Especially on 12th and 16th day after transplantation, there was significantly increased proportion of Treg cells compared with healthy controls ( P < 0.05). The present of Treg decreased progressively in LTx-aGVHD group, which was significantly lower than group 1 and group 2 on the 12th and 16th day after liver transplantation ( p < 0.05). In conclusion, recipients in semi-LT group harbor an increased percentage of Treg in peripheral blood compared with controls. Treg have an immunoregulatory effect on graft versus host reaction after liver transplantation. Additional donor splenocyte transplanted with the liver provoke the development of aGVHD after liver transplantation, and reversed the change of Treg in PBMCs as in Semi-LT group, which destroy the balance between Treg and conventional effector T cells which determine the outcome of aGVHD after liver transplantation.

Tezosentan, a novel endothelin receptor antagonist, markedly reduces rat hepatic ischemia and reperfusion injury in three different models

This study investigated the effects of dual endothelin (ET) receptor blockade in rat models of liver ischemia and reperfusion injury (IRI). Three models of IRI were used: (1) in vivo total hepatic warm ischemia with portal shunting for 60 minutes with control (saline) and treatment groups (15 mg/kg tezosentan intravenously prior to reperfusion), (2) ex vivo hepatic perfusion after 24 hours of cold storage in University of Wisconsin solution with control and treatment groups (10 mg/kg tezosentan in the perfusate), and (3) syngeneic liver transplantation (LT) after 24 hours of cold storage in University of Wisconsin solution with control and treatment groups (10 mg/kg tezosentan intravenously prior to reperfusion). Tezosentan treatment significantly improved serum transaminase and histology after IRI in all 3 models. This correlated with reduced vascular resistance, improved bile production, and an improved oxygen extraction ratio. Treatment led to a reduction in neutrophil infiltration and interleukin-1 beta and macrophage inflammatory protein 2 production. A reduction in endothelial cell injury as measured by purine nucleoside phosphorylase was seen. Survival after LT was significantly increased with tezosentan treatment (90% versus 50%). In conclusion, this is the first investigation to examine dual receptor ET blockade in 3 models of hepatic IRI and the first to use the parenterally administered agent tezosentan. The results demonstrate that in both warm and cold IRI tezosentan administration improves sinusoidal hemodynamics and is associated with improved tissue oxygenation and reduced endothelial cell damage. In addition, reduced tissue inflammation, injury, and leukocyte chemotactic signaling were seen. These results provide compelling data for the further investigation of the use of tezosentan in hepatic IRI.

Significance of Portosystemic Shunt on Graft Survival in Liver Transplantation: A Rat Model

Aims Distal splenorenal shunt effectively controls bleeding from esophageal and gastric varices but has a different effect on liver transplantation. This study sought to develop an animal model in rats to mimic the recipient with a portosystemic shunt and to investigate its hemodynamic consequences on liver transplantation. Methods We prepared 5 groups of allogeneic or syngeneic rat liver transplantation models with versus without portosystemic shunt, to investigate its effects on graft survival and portal flow. To explore the effects of excessive portal flow on graft survival in small-for-size liver transplantation, we transplanted partial liver grafts into syngeneic recipients. Results In allogeneic combinations, graft survival among the shunt group was shortened compared with their control counterparts. The graft survival of the large shunt group was significantly lower than that of a small shunt or without shunt group in a syngeneic liver transplantation model. Portal blood pressure of the large shunt group was significantly lower than that of the small shunt group. In contrast, excessive portal flow resulted in dysfunction of liver graft in small-for-size liver transplantation. Conclusions These results suggested that reduction in portal flow by portosystemic shunt lead to an acceleration of acute rejection and subsequent liver graft dysfunction, but it may be applicable to regulate the excessive portal flow in small-for-size transplantations. This study showed a valuable model mimicking the recipient with a portosystemic shunt.

ET-Kyoto Solution plus Dibutyryl Cyclic Adenosine Monophosphate is Superior to University of Wisconsin Solution in Rat Liver Preservation

ET-Kyoto solution (ET-K) is an extracellular-type organ preservation solution containing the cytoprotective disaccharide, trehalose. A previous study reported the supplement of dibutyryl cyclic adenosine monophosphate (db-cAMP) in conventional ET-K to attenuate lung ischemia-reperfusion injury. In this study, the efficacy of this modified ET-K for liver preservation was investigated by comparison with University of Wisconsin solution (UW). ET-K was supplemented with db-cAMP (2 mmol/L). Lewis rats were randomly assigned to two groups, and liver grafts were flushed and stored at 40C for 24 h with ET-K or UW before syngeneic liver transplantation. The graft function and histological changes at 4 h posttransplant as well as 7-day survival were evaluated. Recipient rat survival rate was significantly higher in the ET-K group than in the UW group. Preservation in ET-K resulted in a significant reduction in serum parenchymal transaminase level and promotion of bile production in comparison with UW. The serum hyaluronic acid level, an indicator of sinusoidal endothelial cell injury, was significantly lower after ET-K preservation than that in UW. Histologically, at 4 h after transplantation, the liver grafts preserved in UW solution demonstrated a greater degree of injury than those in ET-K, which appeared to be apoptosis, rather than necrosis. The continuity of the sinusoidal lining was better preserved in ET-K than in UW. In conclusion, ET-K supplemented with db-cAMP is superior to UW in rat liver preservation. This modified ET-K might therefore be a novel candidate for the procurement and preservation of multiple organs.

Protection of transplant-induced hepatic ischemia/reperfusion injury with carbon monoxide via MEK/ERK1/2 pathway downregulation

Carbon monoxide (CO), a product of heme degradation by heme oxygenases (HO), has been shown to provide cytoprotection in various tissue injury models. This study examined the efficacy and molecular mechanisms of exogenously delivered inhaled CO in protecting liver grafts from cold ischemia/reperfusion (I/R) injury associated with liver transplantation. Orthotopic syngenic liver transplantation (OLT) was performed in Lewis rats with 18-h cold preservation in University of Wisconsin solution. Recipients were exposed to air or different concentrations of CO (20-250 ppm) for 1 h before and 24 h after OLT and killed 1-48 h posttransplant. CO inhalation significantly decreased serum alanine transaminase (ALT) levels and suppressed hepatic necrosis and neutrophil accumulation at 24-48 h after OLT in a dose-dependent manner. Reduced hepatic injury with inhaled CO is associated with marked downregulation of early mRNA expression for TNF-alpha and IL-6. Expression in liver grafts of mRNA and protein of the stress-responding enzyme inducible nitric oxide synthase was significantly reduced by CO, while HO-1 was only marginally suppressed. Cold hepatic I/R injury was associated with prompt MAPK phosphorylation in liver grafts at 1 h after OLT, and CO significantly inhibited phosphorylation of ERK1/2 MAPK and its upstream MEK1/2 and downstream transcriptional factor c-Myc. CO also significantly inhibited I/R injury-induced STAT1 and STAT3 activation. In contrast, CO did not inhibit p38 or JNK MAPK pathways during hepatic I/R injury. Results demonstrate that exogenous CO suppresses early proinflammatory and stress-response gene expression and efficiently ameliorates hepatic I/R injury. The possible mechanism may include the downregulation of MEK/ERK1/2 signaling pathway with CO.

Liver I/R injury is improved by the arginase inhibitor, Nω-hydroxy-nor-l-arginine (nor-NOHA)

Liver ischemia-reperfusion (I/R) injury is associated with profound arginine depletion due to arginase release from injured hepatocytes. The purpose of this study was to determine whether arginase inhibition with N(omega)-hydroxy-nor-l-arginine (nor-NOHA) would increase circulating arginine levels and decrease hepatic damage during liver I/R injury. The effects of nor-NOHA were initially tested in normal animals to determine in vivo toxicity. In the second series of experiments, orthotopic syngeneic liver transplantation (OLT) was performed after 18 h of cold ischemia time in Lewis rats. Animals were given nor-NOHA (100 mg/kg) or saline before and after graft reperfusion. In normal animals treated with nor-NOHA, there were no histopathological changes to organs, liver enzymes, serum creatinine, or body weight. In the OLT model, animals treated with saline exhibited markedly elevated serum transaminases and circulating arginase protein levels. Nor-NOHA administration blunted the increase in serum arginase activity by 80% and preserved serum arginine levels at 3 h after OLT. Nor-NOHA treatment reduced post-OLT serum liver enzyme release by 50%. Liver histology (degree of necrosis) in nor-NOHA-treated animals was markedly improved compared with the saline-treated group. Furthermore, use of the arginase inhibitor nor-NOHA did not influence polyamine synthesis owing to the decrease in ornithine levels. Arginase blockade represents a potentially novel strategy to combat hepatic I/R injury associated with liver transplantation.

Hydrodynamic Plasmid DNA Gene Therapy Model in Liver Transplantation

There is great interest in the field of transplantation to genetically modify grafts to decrease preservation injury or allograft rejection. Although adenoviral gene transfer has been effective in experimental liver transplantation, viral toxicity and safety concerns limit potential use in clinical trials. Therefore, the purpose of this study was to develop a model of nonviral gene transfer in the liver transplant setting, allowing for efficient transgene expression. Orthotopic syngeneic rat liver transplantation was performed with 3 h cold ischemia using University of Wisconsin (UW) preservation. A hydrodynamic gene transfer technique was developed where plasmid DNA was delivered to the liver graft by ex vivo rapid infusion of DNA in UW via the IVC with other vessels clamped. Expression plasmids for the marker genes luciferase and secreted human alpha1-antitrypsin (alpha1-AT) were used. Hepatic injury was assessed by graft histology and liver transaminases. Transgene expression was determined by hepatic luciferase relative light units activity (RLU) and serum alpha1-AT protein levels. Variables examined included the effect of (a) volume injected on the intravenous pressure in the liver graft; (b) injury to the liver, as measured by hepatic enzymes and histopathology; (c) variable expression between lobes; (d) volume of UW that the plasmid is administered in; (e) amount of DNA plasmid; (f) type of the promoter used; (g) clamp time; as well as (h) the time course of the marker gene expression. Control rats underwent standard orthotopic syngeneic rat liver transplantation and had no detectable hepatic luciferase activity or serum human alpha1-AT. The optimal DNA plasmid dose was found to be 400 mug/liver graft, as there was no increase in the luciferase expression by increasing the dose. Furthermore, cytomegalovirus promoter yielded greater expression than Rous sarcoma virus. A high injection pressure gradient allowed for more efficient transgene expression, but produced greater liver injury shown by elevated transaminases and centrilobular necrosis. Lowering injection volume from 75 to 50% of graft weight decreased liver injury by 4.5-fold. Although higher UW injection volumes were associated with increased expression, volumes of only 50% led to luciferase expression up to 10,000,000 RLU/mg; this expression was homogeneous between the different liver lobes. Human alpha1-AT was detected in recipient blood as early as 6 h, peaked at 24 h, and remained high for 5 days. We have developed a nonviral gene transfer technique where hydrostatic pressure across the cold-preserved liver vascular bed allows for efficient plasmid DNA delivery. This simple strategy should prove useful to genetically modify liver grafts in the transplantation setting.

Benefit of Kupffer cell modulation with glycine versus Kupffer cell depletion after liver transplantation in the rat: effects on postischemic reperfusion injury, apoptotic cell death graft regeneration and survival*

Inhibition or destruction of Kupffer cells (KC) may protect against ischemia-reperfusion (IR) induced primary graft nonfunction (PNF) in liver transplantation. Besides KC activation, PNF is characterized by microvascular perfusion failure, intrahepatic leukocyte accumulation, cell death and hepatocellular dysfunction. KCs can be inactivated by different agents including gadolinium chloride (GdCl3), methyl palmitate (MP) and glycine. The effects of three KC inactivators on IR-injury after rat liver transplantation were compared in the present study. Lewis liver donors were treated with GdCl3, MP, glycine or saline (control). Liver grafts were transplanted following 24 h storage (UW solution). KC populations and IR damage were assessed by histologic analysis, quantitative real-time polymerase chain reaction (RT-PCR) and intravital microscopy. The number of hepatic ED-1 positive macrophages was diminished after GdCl3 (114.8+/-4.4/mm2 liver tissue) and MP treatment (176.0+/-5.0), versus the glycine (263.9+/-5.5) and control (272.1+/-5.6) groups. All three treatment modalities downregulated phagocytic activity for latex particles, paralleled by reduced microvascular injury (acinar perfusion index, GdCl3: 0.75+/-0.03; MP: 0.83+/-.03; glycine: 0.84+/-0.03; 0.63+/-0.03). Quantitative RT-PCR revealed elevated myeloperoxidase mRNA after glycine versus GdCl3 and MP pretreatment (3.2- and 3.4-fold, P=0.011, respectively), without difference to controls (2.9-fold of glycine). TNFalpha-mRNA was reduced after glycine- (5.2-fold), GdCl3- (19.7-fold), MP-treatment (39.5-fold) compared with controls. However, profound prevention of intrahepatic cell death and liver graft failure was solely achieved with glycine preconditioning. Different than GdCl3 and MP, glycine modulates rather than destroys KCs. Glycine appears to preserve cell viability and to TNFalpha/leukocyte dependent organ regeneration capacity, which is related to increase graft survival following liver transplantation.

Nuclear factor-κB decoy oligodeoxynucleotides attenuates ischemia/reperfusion injury in rat liver graft

To evaluate the protective effect of NF-kappaB decoy oligodeoxynucleotides (ODNs) on ischemia/reperfusion (I/R) injury in rat liver graft. Orthotopic syngeneic rat liver transplantation was performed with 3 h of cold preservation of liver graft in University of Wisconsin solution containing phosphorothioated double-stranded NF-kappaB decoy ODNs or scrambled ODNs. NF-kappaB decoy ODNs or scrambled ODNs were injected intravenously into donor and recipient rats 6 and 1 h before operation, respectively. Recipients were killed 0 to 16 h after liver graft reperfusion. NF-kappaB activity in the liver graft was analyzed by electrophoretic mobility shift assay (EMSA). Hepatic mRNA expression of TNF-alpha, IFN-gamma and intercellular adhesion molecule-1 (ICAM-1) were determined by semiquantitative RT-PCR. Serum levels of TNF-alpha and IFN-gamma were measured by enzyme-linked immunosorbent assays (ELISA). Serum level of alanine transaminase (ALT) was measured using a diagnostic kit. Liver graft myeloperoxidase (MPO) content was assessed. NF-kappaB activation in liver graft was induced in a time-dependent manner, and NF-kappaB remained activated for 16 h after graft reperfusion. NF-kappaB activation in liver graft was significant at 2 to 8 h and slightly decreased at 16 h after graft reperfusion. Administration of NF-kappaB decoy ODNs significantly suppressed NF-kappaB activation as well as mRNA expression of TNF-alpha, IFN-gamma and ICAM-1 in the liver graft. The hepatic NF-kappaB DNA binding activity [presented as integral optical density (IOD) value] in the NF-kappaB decoy ODNs treatment group rat was significantly lower than that of the I/R group rat (2.16+/-0.78 vs 36.78+/-6.35 and 3.06+/-0.84 vs 47.62+/- 8.71 for IOD value after 4 and 8 h of reperfusion, respectively, P<0.001). The hepatic mRNA expression level of TNF-alpha, IFN-gamma and ICAM-1 [presented as percent of beta-actin mRNA (%)] in the NF-kappaB decoy ODNs treatment group rat was significantly lower than that of the I/R group rat (8.31+/-3.48 vs 46.37+/-10.65 and 7.46+/- 3.72 vs 74.82+/-12.25 for hepatic TNF-alpha mRNA, 5.58+/-2.16 vs 50.46+/-9.35 and 6.47+/-2.53 vs 69.72+/-13.41 for hepatic IFN-gamma mRNA, 6.79+/-2.83 vs 46.23+/-8.74 and 5.28+/-2.46 vs 67.44+/-10.12 for hepatic ICAM-1 mRNA expression after 4 and 8 h of reperfusion, respectively, P<0.001). Administration of NF-kappaB decoy ODNs almost completely abolished the increase of serum level of TNF-alpha and IFN-gamma induced by hepatic ischemia/reperfusion, the serum level (pg/mL) of TNF-alpha and IFN-gamma in the NF-kappaB decoy ODNs treatment group rat was significantly lower than that of the I/R group rat (42.7+/-13.6 vs 176.7+/-15.8 and 48.4+/-15.1 vs 216.8+/-17.6 for TNF-alpha level, 31.5+/-12.1 vs 102.1+/-14.5 and 40.2+/-13.5 vs 118.6+/-16.7 for IFN-gamma level after 4 and 8 h of reperfusion, respectively, P<0.001). Liver graft neutrophil recruitment indicated by MPO content and hepatocellular injury indicated by serum ALT level were significantly reduced by NF-kappaB decoy ODNs, the hepatic MPO content (A655) and serum ALT level (IU/L) in the NF-kappaB decoy ODNs treatment group rat was significantly lower than that of the I/R group rat (0.17+/-0.07 vs 1.12+/-0.25 and 0.46+/-0.17 vs 1.46+/-0.32 for hepatic MPO content, 71.7+/-33.2 vs 286.1+/-49.6 and 84.3+/-39.7 vs 467.8+/-62.3 for ALT level after 4 and 8 h of reperfusion, respectively, P<0.001). The data suggest that NF-kappaB decoy ODNs protects against I/R injury in liver graft by suppressing NF-kappaB activation and subsequent expression of proinflammatory mediators.

RESPONSE TO “RECONSTRUCTION OF THE HEPATIC ARTERY IN LIVER TRANSPLANTATION IN MICE: MANDATORY OR USELESS?”

Since Qian et al. (1) first published his results on mouse liver transplantation in 1991, the mouse model has been used for more than 12 years by various groups in a wide range of studies addressing immunological questions, particularly tolerance induction. Initial experiments with this model (2) clearly indicated that in all allogeneic mouse strain combinations, long-term survival could be achieved spontaneously. In some combinations, 10 of 12 recipients survived for more than 100 days (CB57BL/10 to C3H, 83%). In these studies, arterialization of the hepatic artery was not performed and has always been neglected. The aim of our study (3) was to determine whether or not important immunological pathways might be influenced by the missing arterial blood supply. However, no significant differences in graft damage or activation of immune responses were evident after arterialized and non-arterialized syngeneic liver transplantation respectively using CBA/Ca mice. Histological evaluation of non-arterialized syngeneic grafts five days after transplantation also revealed no major signs of bile duct damage, parenchymal necrosis or portal infiltration. In addition, recipients showed constant gain of weight from day 5 on and reached their initial body weight by day 14 (own unpublished data). This is in apparent contrast to the results of Tian et al. (4) who reported a critical phase on days 5–14, resulting in an overall survival rate of only 50% 14 days after non-arterialized mouse liver transplantation. At this point, one may speculate that the use of different mouse strains (BALB/c vs. CBA/Ca in our study) may be responsible for these obvious differences. In very early studies, Qian et al. (1) also observed differences in long term survival rates of B6F1 (74%) and BALB/c (50%) recipients. However, our data using CBA/Ca mice suggest that arterial reconstruction is not essential in mouse liver transplantation and may be neglected when studying mechanisms of tolerance induction. In contrast, our group has previously demonstrated that rearterialization in the rat liver transplantation model is essential in order to achieve physiological conditions resulting in normal liver architecture (5). Ulrich Steger Heinz-Jochen Gassel Karthyn J. Wood

Impact of hepatic rearterialization on reperfusion injury and outcome after mouse liver transplantation.

Nonarterialized mouse liver transplantation is a well-established model for immunologic studies on rejection and tolerance mechanisms. However, the importance of graft arterialization has-in contrast to rat liver transplantation-not been thoroughly examined in the mouse model. The aim of the current study was to investigate the impact of arterial reconstruction on long-term graft survival, histologic alterations, ischemic liver damage, and early immunologic activation pathways. All recipients of arterialized (n=6) and nonarterialized (n=8) syngeneic liver grafts survived indefinitely. There were no differences in their histologic architecture, including no evidence of bile duct proliferation, periductal fibrosis, or alterations in serum transaminases, in long-term survivors from either group. Twenty-four hours after syngeneic liver transplantation, aspartate aminotransferase and alanine aminotransferase levels were increased to an equivalent extent in both groups, in agreement with early reperfusion injury and solitary traumatic injuries as assessed histologically (n=3 per group). Visualized by immunohistochemistry, intercellular adhesion molecule-1 expression was increased on sinusoidal and hepatic vein endothelium at both 1 and 100 days after transplantation, in both arterialized and nonarterialized grafts. Messenger RNA for interleukin-1, interferon-gamma, and tumor necrosis factor-alpha were measured by real-time polymerase chain reaction 24 hr after transplantation. No significant changes in the expression of cytokine mRNA levels were observed. Arterialization of mouse liver grafts does not appear to have a major impact on survival rate or the degree of immunologic activation. Therefore, the value of arterial reconstruction in mouse liver transplantation for experimental investigations is negligible.

Activation of the lipopolysaccharide signaling pathway in hepatic transplantation preservation injury.

Endotoxin or lipopolysaccharide (LPS) initiates a cascade of complications of septic shock and multiple organ failure seen in Gram-negative bacterial infections. The first step of this pathway, which leads to activated nuclear factor (NF)-kappaB, activating protein (AP)-1, and other transcription factors, is the formation of the LPS receptor complex by LPS, LPS-binding protein (LBP), CD14, and toll-like receptor (TLR) 2 or 4. We examined whether the LPS signaling pathway is activated by hepatic ischemia/reperfusion injury in the transplant setting. Orthotopic syngeneic rat liver transplantation was performed with 0 to 18 hr of cold preservation in University of Wisconsin solution. Animals were killed 1 to 48 hr after reperfusion. Northern blot analysis for CD14, LBP, and TLR2 mRNA, immunohistochemistry for LBP, liver enzyme analysis, and gel shift assay for NF-kappaB and AP-1 were performed. LPS levels were elevated early after reperfusion. Aspartate aminotransferase and alanine aminotransferase maximally increased 12 hr after transplantation. LBP mRNA and protein and CD14 mRNA were significantly up-regulated peaking at 6 to 12 hr after reperfusion. TLR2 mRNA was also increased. NF-kappaB activity showed a biphasic peak at 1 to 3 hr and 12 hr after reperfusion, whereas AP-1 activity showed a peak at 3 to 6 hr. The induction of CD14 mRNA correlated with the length of cold ischemia time. These data indicate that multiple components of the LPS signaling pathway are activated during ischemia/reperfusion injury after liver transplantation.