Hepatic Congestion-reperfusion Injury And Its Treatment:Mechanism Study

PartⅠHepatic inflammatory response and microcirculatory disorder of hepatic congestion-reperfusion injury IntroductionLiver transplantation is known to be the most effective way to save end-stage liver disease. The increasing waiting list mortality as a result of ongoing organ shortage has led to the use of living donors with satisfactory results. However, regional congestion of the hepatic lobe can not be avoided in adult to adult living donor liver transplantation (LDLT) using a right lobe. Many institutions procure the right liver without including the middle hepatic vein (MHV) for the sake of donor safety. This method usually leads to congestion in the segment V and VIII of the graft, which occurred in the process of graft harvesting and implantation, due to the deprivation of the regional venous outflow from MHV. Vascular conduits are used for reconstruction of the venous outflow, thus producing a condition:congestion/reperfusion (CR) injury. In general hepatic surgery, we also encounter CR caused by traumatic obstruction of the outflow of partial liver, or iatrogenic ligation of hepatic vein, unexpectedly. Hepatic ischemia/reperfusin (IR) injury has been extensively studied in different animal models. However, few studies have focused on acute CR injury. In this study, we set up a murine model of acute occlusion of venous outflow in the left anterior hepatic lobe. Changes at various time points after reperfusion were observed in order to find out the impairments and response caused by CR. Comparisons of the severity of injury, inflammatory mediators, and the impacts to microcirculation between IR and CR were emphasized.Materials and MethodsAnimalsAdult male C57BL/6mice were assigned to IR, CR or a sham operation (SO) group (n=6for each group). IR Group:The portal vein and hepatic artery of the LAHL were clamped for75min and followed by reperfusion. CR Group:The HV of the LAHL was blocked for75min. Heparin was systemically used by intravenous injection. The animals were sacrificed after reperfusion at three sampling time intervals (2h,6h,24h)(n=6per interval). The blood and liver samples were collected for further analysis and the venous outflow was checked for confirmation of patency in each case.Hepatic function assayBlood samples were obtained from retrobulbar venous plexus to measure ALT and AST by using an Automated Chemical Analyzer. Myeloperoxidase (MPO) assayMPO activity of the liver was analyzed using commercial kits (NJJC Bio Inc., Nanjing, China) according to the manufacturer’s instructions.Histology and TUNEL AssayMorphologic criteria of necrosis such as cell swelling, cell disruption, karyolysis and loss of architecture were evaluated in serial HE-stained sections under light microscopy. Other sections were prepared and stained for apoptotic cells by the TUNEL method using a commercially available kit.The TUNEL assay was performed according to the manufacturer’s protocol. The results were presented as the mean number of TUNEL-positive cells per high power field.Intravital fluorescence microscopyAnalysis of the hepatic microcirculation was performed by means of intravital fluorescence microscopy at2h,6h, and24h after reperfusion. Blood perfusion within individual microvessels was studied after intravenous injection fluorescein-isothiocyanate-labeled dextran (FITC-dextran). In vivo, labeling of leukocytes with intravenous injection rhodamine-6G enabled quantitative analysis of the flow behavior of the leukocytes in both sinusoids and postsinusoidal venules.Real-time PCRTotal hepatic RNA was isolated from small pieces of liver tissue (50to100mg) by using the TRIzol reagent. Reverse transcription was performed on3μg of RNA with an oligo (dT) primer and M-MuL virus reverse transcriptase. SYBR Green was used for the quantification of PCR reactions. The prepared cDNA was subjected to different PCR in the presence of5’and3’murine TNF-α, IL-6, IL-1β,MCP-1primer pairs. Mouse (3-actin was used as an endogenous control to standardize the amount of cDNA added to the reaction. The PCR amplification was placed in an ABI PRISM7500Real-Time PCR System. The PCR reaction consisted of the following steps:1) initial3min denaturation at94℃;2)40cycles of denaturation at94℃for20sec and annealing extension at60℃for45sec.△Ct value was defined as the absolute value of the difference between the Ct value of the target gene and β-actin for each sample.StatisticsAll data are presented as mean values±tandard error of the mean (SEM). Analysis of variance (ANOVA) and T test of independent means were used for statistical analysis. Data were considered significant at a level of P<0.05.ResultsMacroscopic FindingsIn the IR group, the LAHL became pale in color after portal clamping and reperfused after removing the clamp. In the CR group, the LAHL progressively turned dark and swollen within60sec after temporary ligation of the focal hepatic vein.Serum ALT and AST levelIR and CR group led to marked elevation of serum level of ALT and AST at2h and6h after reperfusion, and subsided at24h. In comparison to the IR group, the hepatic enzyme activity of the CR group was evidently higher at2h of reperfusion (ALT:838.5±155.6vs.474.6±123.8, AST:792.5±93.5vs.574.8±188.4, P<0.05) and showed higher values at6h, but did not reach statistical significance (ALT:604.0±187.4vs.498.8±199.9, AST:703.5±162.6vs.584.2±204.9, P>0.05).MPO activityMPO activity increased significantly at2h,6h and24h after reperfusion in both the CR and IR groups. Both the CR and IR groups showed no difference in MPO activity at2h and6h. Interestingly, MPO levels were markedly increased in the CR group at24h compared to the IR group (0.34±0.11U/g vs.0.15±0.04U/g, P<0.01).Histological assessment and TUNEL stainingIn the IR group, significant vacuolization with mild necrosis and neutrophil accumulation was observed at2h after reperfusion. In the CR group, however, significant necrosis with focal sinusoidal congestion and moderate vacuolization of hepatocytes were found. Compared with the few apoptotic cells (0.3±0.2cells/high power field) that were detected in SO group, the apoptotic hepatocytes increased (2.8±0.9cells/high power field) at2h of reperfusion following75min of congestion. The CR and IR groups shared a similar degree of apoptotic changes in hepatocytes using TUNEL staining.Intravital fluorescence microscopySinusoidal non-perfusion rate:Analysis of the hepatic microcirculation by intravital fluorescence microscopy revealed marked perfusion failure after I/R and C/R injury compared with SO group. The non-perfused sinusoid rates increased in the IR and CR groups compared to the SO group (2.5±0.85%) at2h,6h and24h after reperfusion. The non-perfusion rate appeared higher in the CR group than that of the IR group at2h (27.4±1.97%vs.23.8±1.93%, P<0.05). The stagnant flow in the CR group is more notable in the central sinusoids than the peripheral ones.Leukocyte-endothelial cell interactions:Hepatic CR injury provoked activation of leukocytes, as indicated by their enhanced interactions with the endothelia within the hepatic microvasculature. At2h,6h and24h after reperfusion, the numbers of rolling and adherent leukocytes in post-sinusoidal venules were pronouncedly increased in the CR group when compared to the SO group. The adherent leukocytes in sinusoids and the post-sinusoidal leukocytes behaved similarly. Despite these evident alterations of leukocyte-endothelial cell interactions in the liver, CR and IR shared a similar manner of injury and CR displayed no more severity than IR.Analysis of mRNA expression of inflammatory cytokine and chemokineWhen compared with the sham control, the expression of these inflammatory cytokines or chemokines was enhanced between2h to24h after congestion or ischemia. In contrast to the early elevated level of hepatic enzymes, the expression of TNF-a and IL-6showed no apparent enhancement in the CR group in comparison to with those of the IR group. Notably, the mRNA level of IL-1(3at6h and the level of MCP-1at2h or6h were markedly higher in the CR group than in the IR group.ConclusionsIn our murine model, congestion of liver provokes an increased inflammatory response and more severe impairment in hepatic tissue after reperfusion when compared to ischemia alone. Part ⅡIschemic Preconditioning Improves Liver Tolerance to Congestion-Reperfusion Injury in MiceIntroductionIt is common to utilize modified right lobe living donor liver transplantation (LDLT) in many institutions for the sake of donor’s safety. This procedure usually leads to a partial congestion in the anterior hepatic segment, which occurs in the process of graft harvesting and implantation, due to deprivation of the regional venous outflow from the middle hepatic vein. Autogenous or heterogeneous vascular stent are used for reconstruction of the venous outflow, which leads to congestion/reperfusion injury (CRI). Severe CRI results in progressive graft dysfunction and inadequate regeneration of the affected liver segment.Detailed mechanisms and therapies of hepatic ischemia/reperfusion injury (IRI) have been reported in rodent models and clinical practice, but few have focused on CRI and its protective measures. We previously developed a murine hepatic CRI model to examine the difference in severity of injury, inflammatory mediators, and the impacts on microcirculation between CRI and IRI. CRI resulted in more severe hepatic injury due to additional effects of blood stasis, thrombosis, regurgitation in hepatic sinusoids and venous vasculature. However, there hasn’t been adequate treatment options for CRI up to now.Murry et al. disclosed how ischemic preconditioning (IPC) led to an unexpected resistance in the myocardium to a subsequent prolonged ischemia. Clavien et al. described the first clinical beneficial evidence of IPC during major hepatic surgery in patients. Overexpression of HO-1manifested cytoprotection in IR-stressed organ transplants. HO-1was regulated by Nrf2, a master regulator of intracellular redox homeostasis. Nrf2is anchored in the cytoplasm through binding to Keapl, and facilitates ubiquitination/proteolysis of Nrf2. Inactivation of Keapl leads to stabilization of Nrf2, which in turn translocates into the nuclei to activate cytoprotective target genes through binding to ARE. Nrf2/HO-l driven regulation of anti-oxidant and anti-inflammatory functions is important in cytoprotection. This study aimed to prove whether IPC does protect liver tissue from CRI and to study its potential underlying mechanisms through Nrf2/HO-1pathway. Previous murine CRI model was utilized to verify the hypothesis.Materials and MethodsAnimalsAdult male C57BL/6mice assigned to sham operation, CRI, IPC-CRI and congestion precondition (CPC-CRI) group. Mice in the sham group were subjected to laparotomy. In the CRI group, the hepatic vein of the left anterior hepatic lobe (LAHL) was temporarily ligated with10-0suture for75minutes then followed by reperfusion. In the IPC-CRI group, before congestion period (as in CRI group), the portal vein and hepatic artery of the LAHL were previously interrupted by a non-traumatic microvascular clamp for10minutes, followed by10minutes of reperfusion. To imitating IPC in the CPC-CRI group,10minutes of congestion followed by10minutes of reperfusion just before CRI was performed. Heparin (50U/per mouse) was systemically administered by intravenous injection before operation in all groups. The animals were sacrificed after reperfusion at2h,6h,24h,48h and7d sampling time intervals (n=6). The blood and liver samples were collected for hepatic function assay, histology, TUNEL, myeloperoxidase (MPO), and real-time PCR analysis.Hepatic function assayBlood samples were obtained from retrobulbar venous plexus to measure ALT and AST by using an Automated Chemical Analyzer.Myeloperoxidase (MPO) assayMPO activity of the liver was analyzed using commercial kits (NJJC Bio Inc., Nanjing, China) according to the manufacturer’s instructions.Histology and TUNEL AssayMorphologic criteria of necrosis such as cell swelling, cell disruption, karyolysis and loss of architecture were evaluated in serial HE-stained sections under light microscopy. Other sections were prepared and stained for apoptotic cells by the TUNEL method using a commercially available kit.The TUNEL assay was performed according to the manufacturer’s protocol. The results were presented as the mean number of TUNEL-positive cells per high power field. Real-time PCRTotal hepatic RNA was isolated from small pieces of liver tissue (50to100mg) by using the TRIzol reagent. Reverse transcription was performed on3μg of RNA with an oligo (dT) primer and M-MuL virus reverse transcriptase. SYBR Green was used for the quantification of PCR reactions. The prepared cDNA was subjected to different PCR in the presence of5’and3’murine TNF-a, IL-6, IL-1β MCP-1primer pairs. Mouse P-actin was used as an endogenous control to standardize the amount of cDNA added to the reaction. The PCR amplification was placed in an ABI PRISM7500Real-Time PCR System. The PCR reaction consisted of the following steps:1) initial3min denaturation at94℃;2)40cycles of denaturation at94℃for20sec and annealing extension at60℃for45sec.△Ct value was defined as the absolute value of the difference between the Ct value of the target gene and β-actin for each sample.StatisticsAll data are presented as mean values±tandard error of the mean (SEM). Analysis of variance (ANOVA) and T test of independent means were used for statistical analysis. Data were considered significant at a level of P<0.05.RESULTSComparison of serum ALT levelMarked elevation of serum ALT levels in CRI, IPC-CRI and CPC-CRI group were seen at2h and6h after reperfusion, and subsided afterwards. In comparison with the CRI group, the hepatic enzyme activity of the IPC-CRI group was significantly lower at2h (839.2±132.5vs.384.2±94.8, P<0.01) and6h (680±142.4vs.342.3±99.7, P<0.01) of reperfusion but showed no statistical significance at24h,48h, and7d (Fig.2). Nevertheless, compared with the CRI group, the ALT level in CPC-CRI group was notably higher at2h (839.2±132.5vs.1087.5±192.5, P<0.05) and comparable at6h (680±142.4vs.626.7±140.9, P>0.05), which implied that CPC does not work for hepatic congestion by IPC at early stage.Histological assessment and TUNEL stainingHepatic pathological changes were insubstantial in the sham operation group, in the absence of evident vacuolization and necrosis of hepatocytes. Significant necrosis with focal sinusoidal congestion and moderate vacuolization of hepatocytes was seen in the CRI group. Mild vacuolization, necrosis and neutrophil accumulation was observed in IPC-CRI group, compared with the CRI group; its Suzuki score was significantly lower (2h:3.2±0.4vs.1.8±0.4and6h:3.2±0.3vs.1.7±0.5, P<0.05), but showed no statistical significance at24h,48h, and7d. On the other hand, compared with the CRI group, CPC-CRI group demonstrated no beneficial effects but severe necrosis and clotting, whereas its Suzuki score was similar (2h:3.2±0.4vs.3±1.1and6h:3.2±0.3vs.3.5±0.5, P>0.05).In contrast to the multiple apoptotic cells in the CRI group using TUNEL staining, the apoptotic hepatocytes declined dramatically in the IPC-CRI group (2h:22.0±13.8vs.6.1±1.4, P<0.05;6h:10.7±6.0vs.4.5±1.1, P<0.05;24h:12.1±8.5vs.4.3±2.7, P<0.05; positive cells/high power field).MPO activityLiver MPO, a neutrophil marker enzyme, was measured to determine the degree of neutrophil sequestration. CRI caused increment in MPO activity d at2h,6h and24h after reperfusion. Compared with the CRI group, the MPO levels were markedly decreased in the IPC-CRI group at2h and6h (2h:7.1±4.0U/g vs.3.8±1.6U/g, P<0.05; and6h:8.1±1.3U/g vs.5.2±3.0U/g, P<0.05), suggesting less neutrophil infiltration after hepatic congestion by IPC at the early stage. No statistically significant values were observed at24h.Analysis of mRNA expression of inflammatory cytokine and chemokineThe mRNA expression of the inflammatory mediators, i.e. IL-1, IL-6, TNF-α, MCP-1and IFN-γ were analyzed in the reperfused hepatic tissue. In comparison with the sham operation group, the expression of these inflammatory cytokines or chemokines was enhanced between2h to24h after congestion. The expression of IL-1, IL-6, TNF-α and MCP-1in the IPC-CRI group showed apparent decline in comparison with those in the CRI group at24h. Notably, the mRNA level of IL-1and IL-6at2h and6h were also obviously lower in the IPC-CRI group than those in the CRI group. However, there wasn’t any statistical significance in IFN-γ between those two groups.Nrf2/HO-1pathwayCompared with CRI group, the expression of Keapl significantly declined while the expression of Nrf2and HO-1were significantly elevated.ConclusionsThese results provide strong experimental evidence for the use of IPC against hepatic CRI through Nrf2/HO-1pathway. This simple and powerful pretreatment strategy holds great prospect of being translated into clinical use in LDLT. Further research is needed to achieve a better understanding of the underlying protective mechanisms.