Hepatic Reperfusion Injury Research Articles

Pharmacological preconditioning with doxorubicin: Implications of heme oxygenase-1 induction in doxorubicin-induced hepatic injury in rats

Heme oxygenase (HO) is the rate-limiting enzyme in the degradation of heme into biliverdin, carbon monoxide, and iron. HO-1, an inducible form, is thought to contribute to resistance to various types of oxidative stress. Doxorubicin (DOX) produces clinically useful responses in a variety of human cancers. We reported previously that prior administration of DOX ameliorated subsequent hepatic ischemia and reperfusion injury. The aim of this study was to examine whether this pharmacological preconditioning was useful for another type of hepatic injury induced by a non-surgical method. When a high dose of DOX (10 mg/kg body weight) was administered directly to rat liver via the portal vein, serum aspartate transaminase (AST) and alanine transaminase (ALT) levels increased markedly 24 hr after the injection. Under this condition, zinc-protoporphyrin IX, a specific inhibitor of HO-1, caused both serum AST and ALT levels to be elevated further. When a low dose of DOX (5 mg/kg body weight) was administered to rats via the tail vein as pharmacological preconditioning 3 days before the injection of a high dose of DOX via the portal vein, the levels of serum AST and ALT in rats clearly were improved as compared with rats without the preconditioning. Expression of HO-1 in the liver was confirmed 3 days after the administration of a low dose of DOX. In addition, prior administration of zinc-protoporphyrin IX abolished the effect of DOX preconditioning. Immunohistochemical analysis showed that the positive staining of HO-1 protein induced by a low dose of DOX was localized to histiocytes infiltrating periportal areas. These results strongly suggest that pharmacological preconditioning with DOX may generally help to attenuate subsequent oxidant-induced hepatic injury.

Pentoxifylline Contributes to the Hepatic Cytoprotective Process in Rats Undergoing Hepatic Ischemia and Reperfusion Injury

Aim: Considerable efforts have been made to find and/or eliminate the underyling causes of hepatic ischemia-reperfusion injury, but many points are still unclear. Pentoxifylline-related cytoprotection is one of these unclear points. Our study tests the effects of pentoxifylline on the hepatic cytoprotective process in an experimental model. Materials and Methods: The animals were divided into two groups: (1) placebo-pretreated rats and (2) pentoxifylline-pretreated rats. After pretreatment, all rats underwent the hepatic ischemia-reperfusion procedure which was performed by clamping the hepatoduodenal ligament. To evaluate the liver injury, serum levels of alanine transaminase (ALT) and aspartate transaminase (AST), and liver tissue levels of prostaglandin E₂ (PGE₂) were measured before ischemia, immediately after ischemia and immediately after reperfusion. Results: Before ischemia and immediately after ischemia, there were no significant differences between ALT and AST levels of groups 1 and 2 (p >0.05). However, at the end of reperfusion, ALT and AST levels of group 2 were significantly decreased when compared with group 1 (p < 0.05 and p < 0.01, respectively). Additionally, tissue levels of PGE₂ that were obtained before ischemia, immediately after ischemia and immediately after reperfusion in group 2 were significantly higher than those of group 1 (p < 0.001). Conclusion: Pentoxifylline reduces reperfusion injury of the liver through significantly decreased transaminase levels, and contributes to hepatic cytoprotection by increasing tissue levels of PGE₂ significantly. These effects reflect the role of tissue PGE₂ in pentoxifylline-related hepatoprotection against ischemia-reperfusion injury of the liver.

Protective effect of agiotensin II type I receptor antagonist, CV-11974, on ischemia and reperfusion injury of the liver.

Microcirculatory disturbance has been shown to play a critical role in hepatic ischemia and reperfusion (I/R) injury. Angiotensin II (AngII) is one of the most potent endogenous vasoconstrictors. Angiotensin II type I (AT1) receptor antagonist has been reported to have protective effects on I/R injury of the heart and kidney. However, effect on hepatic I/R injury has not been determined. In this study, we investigate our hypothesis that AT1 receptor antagonist, CV-11974, attenuates hepatic I/R injury. Twelve beagle dogs underwent a 2-hr total hepatic vascular exclusion with veno-venous bypass. CV-11974 was given to animals at a dose of 0.002 mg/ kg/min for 5 min followed by 0.001 mg/kg/min for 25 min via portal vein before ischemia (group II, n=6). Nontreated animals were used as the control (group I, n=6). Animal survival, hemodynamics, hepatic tissue blood flow (HTBF), liver function, platelet count, renin activity, and AngII concentration of hepatic vein, energy metabolism, and histopathology were analyzed. Two-week survival was 33% in group I, in contrast, 100% in group II. Mean arterial blood pressure during early reperfusion was maintained, and HTBF after reperfusion was significantly higher in group II. Treatment attenuated liver enzyme release and decrease of platelet count, increased renin and AngII, suppressed ATP degradation during ischemia and enhanced ATP resynthesis after reperfusion. Neutrophil infiltration and histopathological damages were lessened in group II. Our data demonstrated that the local renin-angiotensin system might play a role in hepatic microcirculation. AT1 receptor blockade with CV-11974 attenuated hepatic microcirculatory disturbance and ameliorated I/R injury.

Manipulation of Rac1 and NFBκB activity by adenovirus-mediated gene transfer effectively blocked hepatic reperfusion injury

Manipulation of Rac1 and NFBκB activity by adenovirus-mediated gene transfer effectively blocked hepatic reperfusion injury

Manipulation of Rac1 and NFBκB activity by adenovirus-mediated gene transfer effectively blocked hepatic reperfusion injury

Manipulation of Rac1 and NFBκB activity by adenovirus-mediated gene transfer effectively blocked hepatic reperfusion injury

Attenuation of hepatic ischemia and reperfusion injury by serine protease inhibitor, FUT-175, in dogs

Attenuation of hepatic ischemia and reperfusion injury by serine protease inhibitor, FUT-175, in dogs

Mechanisms of ischemic injury are different in the steatotic and normal rat liver

Hepatic steatosis is associated with significant morbidity and mortality after liver resection and transplantation. Although apoptosis is a key mechanism of reperfusion injury in the normal liver, the pathway leading to cell death in steatotic hepatocytes is unknown. A model of hepatic ischemia and reperfusion injury in fatty and lean Zucker rats was used. Fatty animals had increased aspartate aminotransferase (AST) release and decreased survival after 60 minutes of ischemia compared with lean animals. Apoptosis was the predominant form of cell death in the lean rats (82%), whereas necrosis was minimal. In contrast, fatty animals developed only moderate amounts of apoptosis but showed massive necrosis (73%) after 24 hours of reperfusion. Intracellular mediators of apoptosis, such as caspase 8, caspase 3, and cytochrome c, were significantly lower in the steatotic than in the lean liver indicating dysfunction in activation of the apoptotic pathway. The high percentage of necrosis in the steatotic rats was associated with renal acute tubular necrosis after 24 hours of reperfusion in the fatty, but not in lean rats. Caspase inhibition significantly decreased reperfusion injury in lean animals, but was ineffective in fatty animals. The results indicate that the increased susceptibility of fatty livers to reperfusion injury is associated with a change from an apoptotic form of cell death to necrosis. We conclude that new therapeutic strategies are necessary in the fatty liver. (Hepatology2000;32:1280-1288.)

Topical hepatic hypothermia attenuates pulmonary injury after hepatic ischemia and reperfusion

Background: Prolonged periods of hepatic ischemia are associated with hepatocellular injury and distant organ dysfunction in experimental models. Neutrophils (PMN) and tumor necrosis factor (TNF)-α have been implicated, mostly because of their local deleterious effects on the hepatocyte after hepatic ischemia and reperfusion (I/R) injury. We hypothesize that topical hepatic hypothermia (THH) reduces ischemia and reperfusion-induced hepatic necrosis, PMN infiltration, TNF-α release, and consequent acute pulmonary injury. Study Design: Sprague-Dawley rats (250 to 300 g) were evenly divided into three groups: 90 minutes of normothermic (37°C) partial hepatic ischemia (normothermic I/R), 90 minutes of hypothermic (25°C) partial hepatic ischemia (hypothermic I/R), and sham laparotomy (without ischemia). There were six animals in each experimental group per time point unless otherwise specified. Hepatic necrosis and PMN infiltration were evaluated and scored on hematoxylin and eosin-stained liver specimens 12 hours after reperfusion. Serum TNF-α levels were determined by ELISA at 0 minutes, 15 minutes, 30 minutes, 1 hour, and 12 hours postreperfusion. Pulmonary PMN infiltration and vascular permeability were measured by myeloperoxidase activity and Evans blue dye extravasation, respectively, to quantitate pulmonary injury 12 hours after reperfusion. Results: Normothermic I/R results in a significant increase in TNF-α at 15 and 30 minutes (p < 0.005), PMN infiltration (p < 0.001), and hepatic necrosis (p < 0.001), compared with sham. Institution of THH reduced peak serum TNF-α levels by 54% at 15 minutes (p < 0.005) and by 73% at 30 minutes (p < 0.001) postreperfusion compared with normothermic I/R. Similarly, hepatic PMN infiltration and necrosis at 12 hours were reduced by 60% (p < 0.05) and 47% (p < 0.05), respectively. Myeloperoxidase activity and Evans blue extravasation (measures of acute lung injury) were reduced by 42% and 39%, respectively, with institution of THH compared with animals undergoing normothermic I/R (p < 0.001). Conclusions: These results demonstrate that THH protects the liver from ischemia and reperfusion-induced necrosis and PMN infiltration. In addition, THH reduces the serum levels of TNF-α and associated pulmonary injury. These data suggest that the ischemic liver is a potential source of inflammatory mediators associated with hepatic ischemia and reperfusion-induced pulmonary injury.

Protective effects of herbimycin A on hepatic reperfusion injury

Protective effects of herbimycin A on hepatic reperfusion injury

Role of inducible nitric oxide synthase on hepatic ischemia and reperfusion injury

PROTECTION of hepatocytes from ischemia/reperfusion (I/R) injury is an important issue in the field of hepatic surgery, which includes liver resection and transplantation. Nitric oxide (NO), a well-known endotheliumderived relaxing factor, has been implicated in tissue injury as a result of its capacity to react with superoxides and hydroxyl radical, suggesting that it may influence the pathogenesis of I/R injury. However, little is known about the role of inducible nitric oxide synthase (iNOS) in hepatic I/R injury. In the present study, we investigate the role of iNOS on hepatic I/R injury and study the cellular distribution of endothelial nitric oxide synthase (eNOS) and iNOS by immunohistochemistry, including a double-immunofluorescence technique in combination with confocal laser scanning microscopy. The effect of intraportal administration of aminoguanidine (AG), a selective iNOS inhibitor, is also investigated.

Role of hepatocyte growth factor in hepatic ischemia and reperfusion injury

Role of hepatocyte growth factor in hepatic ischemia and reperfusion injury

Recombinant human hepatocyte growth factor protects the liver against hepatic ischemia and reperfusion injury in rats.

Recent evidence indicates that hepatocyte growth factor (HGF) has a cytoprotective effect against hepatic injury caused by hepatotoxins and inflammatory cytokines. Studies were performed to determine whether HGF influences the survival rate of rats subjected to hepatic warm ischemia/reperfusion (WI/Rp) injury. Male Sprague-Dawley rats were subjected to total or segmental hepatic ischemia by occluding the hepatic artery, portal vein, and bile duct with a microvascular clip. Rats were treated with four intravenous injections of recombinant human HGF (rhHGF 1 mg/kg) or the vehicle 72, 48, 24, and 12 h before surgery. None of the eight animals in the control group were alive 12 h after total hepatic WI/Rp. Seven of eight animals in the rhHGF-treated group were alive more than 2 days after the reperfusion. In the model of segmental hepatic ischemia, rhHGF inhibited the increase in cytokine-induced neutrophil chemoattractant in serum. The number of neutrophils infiltrating the liver was significantly lower in the rhHGF-treated group than in the control group. rhHGF prevented increases in the activity of serum alanine transaminase and in hepatic necrosis. Experiments with proliferating cell nuclear antigen staining demonstrated that hepatocyte proliferation markedly increased in rhHGF-treated rats as compared with controls. These results indicate that HGF facilitates recovery of the liver from hepatic WI/Rp injury, at least in part through the prevention of neutrophil infiltration and the activation of hepatocyte proliferation.

The roles of platelet-activating factor and endothelin-1 in renal damage after total hepatic ischemia and reperfusion.

This study was designed to verify the involvement of platelet-activating factor (PAF) in renal damage associated with hepatic ischemia and reperfusion (HIR) injury through the release of endothelin (ET)-1 and to determine the modulating effect of a specific PAF receptor antagonist on these insults in rats. Male rats pretreated with either normal saline as a vehicle (NS group) or intravenous TCV-309, a PAF receptor antagonist (TCV group), were subjected to 120 min of total hepatic ischemia under an extracorporeal portosystemic shunt. Plasma aspartate transaminase, creatinine, blood urea nitrogen, and ET-1 levels and the relative renal wet weight were determined under nonischemic conditions and at 1, 3, and 6 hr of reperfusion after hepatic ischemia. Changes in mean arterial blood pressure and renal tissue blood flow measurements in the kidney were determined throughout the experiment. Increased plasma aspartate transaminase, creatinine, blood urea nitrogen, and ET-1 levels and the relative renal wet weight after HIR in the NS group were significantly suppressed by TCV-309 pretreatment. Mean arterial blood pressure and renal tissue blood flow after HIR in the TCV group were significantly improved when compared with those in the NS group. These effects resulted in attenuation of structural hepatic and renal damage with the improvement of 7-day survival (62%). The present study demonstrates that renal damage as well as critical liver injury is produced after reperfusion following 120 min of total hepatic ischemia. A PAF receptor antagonist may be therapeutically useful to protect against these types of damage via indirect modulation of plasma ET-1 levels.

Um novo modelo para estudos sobre lesão de isquemia e reperfusão hepáticas em cães

A lesão de isquemia e reperfusão hepáticas que ocorre no transplante de fígado não está completamente elucidada. Vários modelos experimentais com objetivo de estudos fisiopatológico e de modulação farmacológica desta condição têm sido propostos. Um desses modelos, proposto para cães, compreende a isquemia de 30% da massa hepática com descompressão portal translobar através dos restantes 70% de parênquima hepático. No presente trabalho, 10 cães foram submetidos à desvascularização de uma maior massa hepática (70%), seguida de reperfusão, com descompressão venosa esplâncnica através dos lobos caudado e lateral direito (30%) (Grupo Teste), durante o período de isquemia. Dez outros cães foram submetidos à operação simulada (Grupo Controle). Os resultados indicaram com um grau de confiança de 95% que: 1. Durante o período de isquemia verificou-se estabilização da PAM e da PVC, elevação da pressão portal (PP) semelhante à desenvolvida com o uso de "bypass", diminuição significativa da temperatura corporal(TC) sem atingir níveis médios aquém de 36oC, ausência de acidose metabólica ou aumento dos níveis de enzimas (AST, ALT e DL), presença de necrose hepática (NH) e queda do conteúdo de glicogênio hepático (CGH); 2. Após a reperfusão do fígado constatou-se queda da PAM, ausência de diferenças significativas de PVC , permanência de níveis elevados de PP, decréscimo da TC, presença de acidose metabólica (¯ pH, ¯ DB), aumento significativo e progressivo de aminotransferases (AST, ALT), desidrogenase láctica (DL) e necrose hepática (NH), bem como declínio progressivo do conteúdo de glicogênio hepático (CGH). Os resultados sugerem que o modelo proposto pode ser útil para estudos de fisiopatologia e de modulação farmacológica da lesão de isquemia e reperfusão do fígado, utilizando uma maior massa hepática.

Reduction of hepatic reperfusion injury by antithrombin III and aprotinin

Reduction of hepatic reperfusion injury by antithrombin III and aprotinin

Ischemia-reperfusion injury of liver and therapeutic intervention

It is clinically and pathophysiologically important to elucidate the mechanism of cellular injury during hepatic ischemia and subsequent reperfusion. Ischemia-reperfusion injury is complex. Effective prevention strategies will require specific actions during the different phases of ischemia-reperfusion injury, but optimal conditions will only be achieved once the mechanisms of injury at the cellular and molecular levels are understood. It is, therefore, important to study the intracellular signal transduction pathway for its clinical application. This review represents recent advances of the mechanism of hepatic ischemia and reperfusion injury, with special reference to the free radical-mediated injury, the activation of Kupffer cells and neutrophils and the implications of NF-kB activation.

The effect of nitric oxide on ischemia–reperfusion injury in rat liver

A dual role for nitric oxide (NO) in ischemia–reperfusion (I/R) injury is still controversial. This study aims to investigate the role of NO in rat hepatic reperfusion injury. Ischemia was induced by total occlusion of hepatic artery and portal vein for 30 min, then the tissue was reperfused for 30 min. The animals in the l-NAME group ( n=10) received N Gnitro- l-arginine methyl ester ( l-NAME) (15 mg/kg) intraperitoneally 60 min before ischemia. The ischemia group ( n=10) was given an equal volume of saline solution. The control group comprised eight healthy rats which were not exposed to ischemia or reperfusion. An indicator of hepatic injury, plasma alanine amino transferase (ALT) enzyme activities, were increased in the l-NAME group as compared with the ischemia group ( p<0.001). The level of serum nitrite, an index of NO production, and hepatic reduced glutathione (GSH) concentration were lower in the l-NAME group than in the ischemia group ( p<0.001, p<0.01, respectively). Hepatic levels of malondialdehyde (MDA) and conjugated dienes (CD) were significantly increased in the l-NAME group as compared to the ischemia group ( p<0.05, p<0.001, respectively). Our results confirm that l-NAME, an inhibitor of the enzyme NO synthase, increased the lipid peroxidation and possibly tissue injury, due to the inhibition of cytoprotective effects of NO in a rat hepatic I/R model.

Attenuation of Ischemic liver injury in monkeys by anti-LFA1 monoclonal antibody therapy

97 Reperfusion and reoxygenation after a period of ischemia result in tissue injury and are a major cause of graft failure after liver transplantation. The binding of LFA-1 to ICAM family members stabilizes the interaction of T cells and APC's and contributes to T cell stimulation. Odulimomab is a monoclonal IgG1 antibody specific for the CD11a antigen of the leukocyte function associated molecule 1 (LFA-1). In vitro, Odulimomab inhibits cell-cell interaction as well as T-cell adhesion and activation, NK-cell activity, antibody dependent cell mediated cytotoxicity, and T-cell mediated cytotoxicity without activation of complement. Thus, Odulimomab is expected to prevent tissue damage after liver ischemia and reperfusion. Methods: The effect of Odulimomab on liver reperfusion injury was tested in Cynomolgus monkeys: Total hepatic ischemia was achieved in Cynomolgus monkeys by clamping the hepatic artery and portal vein after performing a side-to-side portocaval shunt. Reperfusion of the liver was started after 90 (n=12), 120 (n=12) and 180 minutes (n=5) respectively. Untreated control animals (n=15) were compared with monkeys (n=14) treated with Odulimomab (1.5 mg/kg iv) at the initiation of ischemia and every day (0.75 mg/kg) until sacrifice on day 8. Results: All animals in the 90 and 120 min groups survived until scheduled sacrifice, whereas non of the untreated 180 min animals (n=3) and only one of two treated 180 min animals lived longer then 24 hours. After 90 minutes ischemia, mean LDH levels were lower in the Odulimomab treated group (n=6) than in the control group (n=6) (p=0.048) with a peak on day 1 (1039 ± 101 vs. 1730 ± 353 U/l). Postischemia transaminases were consistently higher in the untreated groups. Due to high interindividual variances, p-values failed to be significant in the first days after the operation and reached significance only when the second peak occurred on day 8 (p=0.047). In the 120 min ischemia group IL-6 production was higher before treatment in the Odulimomab group (183 ± 71 vs. 63 ± 23 U/ml; p=0.1). During antibody treatment this effect was reversed until day 3 (9 ± 4vs. 149 ± 108 pg/ml; p=0.05). Same effect was observed with IL-8 (p=0.05) and TNF-α (p=n.s.). Conclusion: Our results suggest, that Odulimomab therapy of Cynomolgus monkeys attenuates hepatic reperfusion injury after warm ischemia. This research was funded in part by IMTIX - SangStat

Role of P-selectin expression in hepatic ischemia and reperfusion injury.

Researchers have shown that reperfusion of ischemic tissues initiates a complex series of reactions that paradoxically injure tissues. Although several mechanisms have been proposed to explain the pathobiology of ischemic/reperfusion (I/R) injury, much attention has focused on adhesion molecules. Our research is intended to show the kinetics of P-selectin in the liver in response to I/R injury. Left-lobar hepatic ischemia was induced for 30 min in 35 C57BL-6 mice and 20 P-selectin-deficient (K-O) mice. P-selectin expression was measured in these mice at 20 min, 2, 5, 12 and 24 h reperfusion times, as well as in control and sham animals. The animals were injected with radio-labeled P-selectin monoclonal antibody and the organs were harvested for counts/g tissue, expressed as the percentage injected dose. Serum liver enzymes were measured and pathological sections of ischemic and control livers were performed. The unpaired t-test was used for statistical analysis. P-selectin expression showed two peaks in this animal model. The first peak was at 20 min and the second peak at 5 h of reperfusion (p < 0.001). We documented an 8-fold increase in aspartate aminotransferase (AST), alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) levels 10 h following I/R injury. Pathological specimens showed periportal necrosis consistent with an ischemic event. P-selectin K-O mice showed no up-regulation as a separate control group, and the liver enzymes were significantly lower than the wild-type mice at 10 h (p < 0.001). P-selectin has a bimodal expression following hepatic I/R injury. The first peak is attributed to the Weibel-Palade bodies and the second to new translational P-selectin. We noted no difference in the up-regulation of P-selectin in the ischemic and non-ischemic liver lobes in the same animal.

Contribution of endothelin-1 to microcirculatory impairment in total hepatic ischemia and reperfusion injury.

Endothelin (ET)-1 may have a role in hepatic polymorphonuclear leukocyte infiltration as well as microcirculatory disturbance during hepatic ischemia-reperfusion (HIR) injury. This study was conducted to investigate the influence of ET-1 on the hepatic microcirculation after total HIR and to evaluate the effect of a nonselective ET receptor antagonist under these conditions. Male rats pretreated with either normal saline (NS group) or TAK-044, a nonselective ET receptor antagonist (TAK group), were subjected to 120 min of total hepatic ischemia with extracorporeal portosystemic shunting. Plasma ET-1 levels increased significantly from 1 to 6 hr after reperfusion in the NS group when compared with the nonischemic control. In the early phase of reperfusion, the NS group showed significantly narrower sinusoids, lower hepatic tissue blood flow, a lower hepatic tissue oxy-hemoglobin concentration, and more hepatic neutrophil infiltration than the TAK group (P<0.05). Pretreatment with TAK-044 improved hepatic microcirculatory derangement, and resulted in significantly better 7-day survival (61.5%) with more bile production after reperfusion when compared with the NS group (P<0.01). The present study demonstrated that ET-1 is involved in the development of HIR injury by causing deterioration of the hepatic microcirculation. A nonselective ET receptor antagonist successfully ameliorated HIR injury through improvement of hepatic oxygenation and of the microcirculation along with reduced hepatic neutrophil infiltration.