Cyclosporin-A does not prevent cold ischemia/reperfusion injury of rat livers.

Cyclosporin-A (CsA) has been reported to protect livers from warm ischemia/reperfusion (I/R) injury. To study if CsA has also a protective effect on cold I/R injury, two models were used: the isolated perfused rat liver (IPRL) and the orthotopic rat liver transplantation (ORLT). (1) IPRL: Livers were preserved for 24 h (5°C) in University of Wisconsin (UW) solution alone (group 1), with CsA (400 nM) dissolved in dimethylsulfoxide (50 µM) (group 2), and with dimethylsulfoxide (DMSO) alone (group 3). Livers were reperfused for 60 min (37°C) (n = 8/group). Cell necrosis was evaluated by trypan blue uptake and apoptosis by laddering and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay, and by caspase-3 activation. Marked and similar sinusoidal endothelial cell necrosis was found in the three groups while apoptosis was found similarly deceased in groups 2 and 3 compared with group 1. (2) ORLT: Donors received either CsA (5 mg/kg) or corn oil 24 h before transplantation. Recipients were sacrificed after 240 min; cell necrosis and apoptosis were then evaluated. No difference was found between treated and control groups. The current data strongly suggest that CsA has no protective effect on hepatic cold I/R injury. Hepatocyte apoptosis can be reduced by antioxidants, as occurred with DMSO, but introduction of CsA does not provide additional protective effect.

Effect of cold ischemia-warm reperfusion on the cirrhotic rat liver.

Cirrhosis is known to induce capillarization of the sinusoidal endothelial cells (SECs) and collagenization of the space of Disse, resulting in a reduced access of plasma and plasma-dissolved substances to hepatocytes due to their limited diffusion in the extravascular space. The aim of the present study was to use a well known effect of cold ischemia-warm reperfusion (CI-WR) on liver SECs, that is, their retraction and detachment, progressing to a denudation of the SEC lining. The disappearance of the capillarized SEC lining would improve the access of plasma and plasma-dissolved substances to the hepatocytes and consequently might improve the metabolic function of cirrhotic livers. This study was performed using the isolated perfused rat liver model subjected to 24-hour CI followed by a 60-minute WR in thioacetamide-induced cirrhosis. Liver microcirculation was evaluated using the multiple indicator dilution curve (MIDC) technique. Hepatocyte, SEC, and Kupffer cell functions were evaluated using specific elimination processes. As occurs in normal livers, CI-WR induced extensive SEC necrosis with a marked reduction of the hyaluronic acid elimination. By contrast, the hepatic microcirculation was not modified: vascular, extravascular, and the cellular spaces were similar before and following CI-WR. In addition, the hepatic metabolic functions, as evaluated by propranolol and taurocholate hepatic uptake, were neither improved nor decreased, as were Kupffer cell functions. The present data strongly suggest that capillarization of SECs plays a lesser role than collagenization of the space of Disse in the reduced exchange between sinusoids and hepatocytes in thioacetamide-induced cirrhotic rat livers, which appear to be quite resistant to CI-WR.

Matrix metalloproteinase inhibition protects rat livers from prolonged cold ischemia-warm reperfusion injury.

UNLABELLED: Matrix metalloproteinases (MMPs) have been implicated in the hepatic injury induced after cold ischemia-warm reperfusion (CI-WR), by altering the extracellular matrix (ECM), but their precise role remains unknown. The hepatic MMP expression was evaluated after 2 conditions of CI (4 degrees C for 24 and 42 hours: viable and nonviable livers) followed by different periods of WR, using isolated perfused rat livers. CI-WR induced moderate changes in hepatic MMP transcript levels not influenced by CI duration, whereas gelatinase activities accumulated in liver effluents. Therefore, the protective effect of a new phosphinic MMP inhibitor, RXP409, was tested after prolonged CI. RXP409 (10 microM) was added to the University of Wisconsin solution, and livers were preserved for 42 hours (4 degrees C), then reperfused for 1 hour in Krebs solution (37 degrees C), containing 20% erythrocytes. Liver viability parameters were recorded, and the extent of cell necrosis was evaluated on liver biopsies, using trypan blue nuclear uptake. Treatment with RXP409 significantly improved liver function (transaminase release and bile secretion) and liver injury. In particular, the MMP inhibitor significantly modified the extent of cell death from large clusters of necrotic hepatocytes as found in control livers (2%-60% of liver biopsies; mean, 26% +/- 9%) to isolated necrotic hepatocytes as found in treated livers (0.2%-12%; mean, 3% +/- 2%) (P < 0.05). CONCLUSION: These data demonstrate that MMPs, by altering the ECM, play a major role in liver CI-WR injury leading to extensive hepatocyte necrosis and that their inhibition might prove to be a new strategy in improving preservation solutions.

Warm ischemia-reperfusion injury is decreased by tacrolimus in steatotic rat liver.

Ischemia-reperfusion (I-R) injury is poorly tolerated by fatty livers, most probably secondary to reduced cellular adenosine triphosphate (ATP) levels. We investigated the effectiveness of tacrolimus pretreatment on fatty liver I-R injury in obese Zucker rats. Tacrolimus (0.3 mg/kg, intravenously) was injected 24 hours before a 75-minute ischemic period and rats were sacrificed 6 hours later. Tacrolimus modified the response to I-R observed in obese Zucker rats, when compared to nontreated obese rats: a significant reduction in hepatocyte necrosis was associated with a significant increase in hepatocyte apoptosis. In addition, cell necrosis and apoptosis were significantly and inversely correlated in lean nontreated and treated obese Zucker rats following I-R. Tacrolimus also significantly increased the hepatic ATP levels, reduced in nontreated obese rats, toward values found in lean Zucker rat livers. This protective effect of tacrolimus was further confirmed in vivo by a significantly improved survival following pretreatment with tacrolimus, 24 hours prior to ischemia. In conclusion, in obese Zucker rat livers, tacrolimus pretreatment reversed the I-R injury toward the one found in lean Zucker rats. The correlations between ATP levels and the opposite changes in necrosis and apoptotic pathways strongly suggest a cause-effect relationship between tacrolimus and changes in ATP levels.

Hypoxia-reoxygenation-induced chemokine transcription is not prevented by preconditioning or intermittent hypoxia, in mice hepatocytes.

Prolonged ischemia used in liver surgery and/or transplantation causes cellular damage resulting in apoptosis and necrosis. Ischemia-reperfusion (I/R) led Kupffer cells to pro-inflammatory cytokines secretion [tumor necrosis factor (TNF)-alpha, interleukin-1] which involve chemokines secretion by hepatocytes. These chemokines have neutrophil chemotactic properties and neutrophils are involved in the development of I/R-induced necrosis. The aim of this study was to specify the consequence of partial oxygen pressure variation on hepatocyte chemokines synthesis and to verify if intermittent hypoxia and/or preconditioning could decrease it. It was performed on primary cultured mice hepatocytes and Kupffer cells, subjected to continuous, intermittent hypoxia or preconditioning phases, mimicking surgical processes. The chemokine secretion was evaluated by RNase protection assay and enzyme-linked immunosorbent assay method. Only monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-2 (MIP-2) mRNA formation were observed, especially after 1-h hypoxia followed by 10-h (for MCP-1) or 24-h reoxygenation (for MIP-2). In conclusion, TNF-alpha and coculture with Kupffer cells increased hepatocyte chemokines mRNA transcription, whereas surgical split up protocols (intermittent hypoxia and preconditioning) had no significant effect.

Rat liver ischemia-reperfusion-induced apoptosis and necrosis are decreased by FK506 pretreatment.

The aim of this study was to demonstrate that tacrolimus (FK506) has a hepatoprotective effect by reducing ischemia-reperfusion-induced apoptosis and necrosis, both of which lead to post-surgical liver dysfunction. An ischemia-reperfusion model and primary cultured rat hepatocytes subjected to hypoxic and reoxygenation phases, mimicking the surgical process, were used. c-Jun N-terminal kinase 1/stress-activated protein kinase 1 (JNK1/SAPK1) activation leads to caspase 3 activation, a trigger of apoptosis. The activation status of JNK1/SAPK1 was evaluated by immunoprecipitation or Western-blotting experiments. Apoptosis was assessed by measuring caspase activation and by TUNEL (terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate-biotin nick-end labeling) reaction. Necrosis was assessed histologically. Tacrolimus improved the survival rate of rats subjected to ischemia-reperfusion. After FK506 pretreatment, the liver necrosis rate was reduced, and ischemia-reperfusion-induced JNK1/SAPK1 activation and apoptosis were significantly decreased. In hypoxia-reoxygenation-subjected hepatocytes, tacrolimus reduced JNK1/SAPK1 and caspase 3 activation. In the liver, tacrolimus prevented ischemia-reperfusion-induced apoptosis and necrosis.

Diltiazem reduces apoptosis in rat hepatocytes subjected to warm hypoxia-reoxygenation.

Interruption of hepatic blood flow is necessary in surgery, but the liver is sensitive to ischemia and reperfusion. Hypoxia induces an increase in intracellular calcium concentration. In previous studies, we have shown that hypoxia-reoxygenation (H/R) increased calcium influx and induced JNK(1)/SAPK(1) activation which was involved in the triggering of apoptosis. The aim of this study was to demonstrate that diltiazem, a calcium inhibitor, reduced JNK(1)/SAPK(1) activation and consequently could decrease H/R-induced apoptosis. Experiments were performed, in the presence of diltiazem, on primary cultured rat hepatocytes, subjected to warm H/R phases and in a liver ischemia-reperfusion model. The activation status of JNK(1)/SAPK(1) was evaluated by immunoprecipitation and immunohistolocalisation experiments, while apoptosis was assessed by measuring caspase activity and by TUNEL labeling. Diltiazem inhibited H/R-induced JNK(1)/SAPK(1) activation and decreased apoptosis. It could be used to improve postoperative liver function.