Cold Ischemia-warm Reperfusion Research Articles

Hibernation reduces cellular damage caused by warm hepatic ischemia-reperfusion in ground squirrels.

During the hibernation season, livers from 13-lined ground squirrels (Ictidomys tridecemlineatus) are resistant to damage induced by ex vivo, cold ischemia-warm reperfusion (IR) compared with livers from summer squirrels or rats. Here, we tested the hypothesis that hibernation also reduces damage to ground squirrel livers in an in vivo, warm IR model, which more closely resembles complications associated with traumatic injury or surgical interventions. We also examined whether protection is mediated by two metabolites, inosine and biliverdin, that are elevated in ground squirrel liver during interbout arousals. Active squirrels in spring and hibernators during natural arousals to euthermia (body temperature 37 °C) were subject to liver IR or sham treatments. A subset of hibernating squirrels was pre-treated with compounds that inhibit inosine synthesis/signaling or biliverdin production. This model of liver IR successfully induced hepatocellular damage as indicated by increased plasma liver enzymes (ALT, AST) and hepatocyte apoptosis index compared to sham in both seasons, with greater elevations in spring squirrels. In addition, liver congestion increased after IR to a similar degree in spring and hibernating groups. Microvesicular steatosis was not affected by IR within the same season but was greater in sham squirrels in both seasons. Plasma IL-6 increased ~twofold in hibernators pre-treated with a biliverdin synthesis inhibitor (SnPP) prior to IR, but was not altered by IR in untreated squirrels. The results show that hibernation provides protection to ground squirrel livers subject to warm IR. Further research is needed to clarify mechanisms responsible for endogenous protection of liver tissue under ischemic stress.

Participation of autophagy in the initiation of graft dysfunction after rat liver transplantation

Better ways to prevent the cold ischemia-warm reperfusion (CI/WR) injury associated with liver transplantation are needed, and many investigations have focused on the molecular mechanisms of this injury. However, the mechanisms reported to date are controversial and no improvement in therapy has resulted. Here, using prolonged CI and orthotopic transplantation of rat liver grafts, we found that the CI/WR injury was closely associated with autophagy. By 15 min after the start of WR, small masses of hepatocytes that possessed abundant autophagosomes and autolysosomes frequently dissociated from the hepatic cords and obstructed the sinusoid, causing massive necrosis of hepatocytes within 2 hours. The cell masses included TUNEL-positive nuclei without caspase-3 and -7 activation. Autophagy suppression with the phosphatidylinositol 3-kinase (PI3K) inhibitors, wortmannin or LY294002, reduced both liver damage and the mortality rate of recipient rats. To elucidate the downstream mechanisms of this autophagic pathway, liver grafts were treated with aspartic and cysteine proteinase inhibitors, pepstatin and leupeptin. This treatment also significantly improved the survival rate of recipient rats. These data suggest that autophagy-associated hepatocyte death triggers liver graft dysfunction. The protective effects of suppressing autophagy may suggest new ways to prevent CI/WR injury of the liver.

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

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). 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.

Clinical Trial of the Pan-Caspase Inhibitor, IDN-6556, in Human Liver Preservation Injury

Cold ischemia/warm reperfusion (CI/WR) injury remains a problem in liver transplantation. The aim of the current study was to assess the utility of the pan-caspase inhibitor IDN-6556 on CI/WR injury during human liver transplantation. This report is a post hoc analysis of a Phase II, multi-center, randomized, placebo-controlled, double-blinded, parallel group study. Subjects were assigned to four treatment groups: Group 1 (Organ storage/flush: Placebo-Recipient: Placebo); Group 2 (Organ storage/flush: 15 microg/mL-Recipient: Placebo); Group 3 (Organ storage/flush: 5 microg/mL-Recipient: 0.5 mg/kg); and Group 4 (Organ storage/flush: 15 microg/mL-Recipient: 0.5 mg/kg). Liver cell apoptosis was assessed by serum concentrations of the apoptosis-associated CK18Asp396 ('M30') neo-epitope, TUNEL assay and caspase 3/7 immunohistochemistry. Liver injury was assessed by serum AST/ALT determinations. Serum markers of liver cell apoptosis were reduced in all groups receiving drug as compared to placebo. However, TUNEL, caspase 3/7 positive cells and serum AST/ALT levels were only consistently reduced in Group 2 (drug exposed to organ only). This reduction in serum transaminases was significant and observed across the study. In conclusion, IDN-6556 when administered in cold storage and flush solutions during liver transplantation offers local therapeutic protection against CI/WR-mediated apoptosis and injury. However, larger studies are required to confirm these observations.

Implication of mitochondrial dysfunction and cell death in cold preservation – warm reperfusion-induced hepatocyte injury

Cold ischemia--warm reperfusion (CI/WR) injury of liver transplantation involves hepatocyte cell death, the nature and underlying mechanisms of which remain unclear. Isolated hepatocytes and isolated perfused livers were used to determine the prevalence of necrosis and apoptosis as well as mitochondrial dysfunction. In isolated cells, propidium iodide and Hoechst 33342 staining showed a cold-storage, time-dependent increase in necrosis, whereas apoptosis was minimal even after 48 h of hypothermia. Nonetheless, a progressive loss of mitochondrial membrane potential was observed. Translocation of mitochondrial cytochrome c toward microsomes occurred within 24 h of CI/WR, with cytochrome c reaching the cytosol later. Mitochondria isolated from whole livers subjected to CI/WR also display reduced metabolic parameters and increased susceptibility to swelling. These events are associated with increased activity of major initiator (caspase 9) and effector (caspase 3) caspases. The results demonstrate that CI/WR induces mitochondrial dysfunction in isolated cells and in the whole organ; only in the latter is that sufficient to trigger the classical mitochondrial pathway of apoptosis. Our study also provides evidence for the involvement of endoplasmic reticulum stress in CI/WR hepatocyte injury. Combined protection of mitochondria and endoplasmic reticulum may thus represent an innovative therapeutic avenue to enhance liver graft viability and functional integrity.

Natural resistance to liver cold ischemia-reperfusion injury associated with the hibernation phenotype

The success of liver grafts is currently limited by the length of time organs are cold preserved before transplant. Novel insights to improve viability of cold-stored organs may emerge from studies with animals that naturally experience low body temperatures (T(b)) for extended periods. In this study, we tested whether livers from hibernating ground squirrels tolerate cold ischemia-warm reperfusion (cold I/R) for longer times and with better quality than livers from rats or summer squirrels. Hibernators were used when torpid (T(b) < 10 degrees C) or aroused (T(b) = 37 degrees C). Livers were stored at 4 degrees C in University of Wisconsin solution for 0-72 h and then reperfused with 37 degrees C buffer in vitro. Lactate dehydrogenase (LDH) release after 60 min was increased 37-fold in rat livers after 72 h cold I/R but only 10-fold in summer livers and approximately three- to sixfold in torpid and aroused hibernator livers, despite twofold higher total LDH content in livers from hibernators compared with rats or summer squirrels. Reperfusion for up to 240 min had the least effect on LDH release in livers from hibernators and the greatest effect in rats. Compared with rats or summer squirrels, livers from hibernators after 72 h cold I/R showed better maintenance of mitochondrial respiration, bile production, and sinusoidal lining cell viability, as well as lower vascular resistance and Kupffer cell phagocytosis. These results demonstrate that the hibernation phenotype in ground squirrels confers superior resistance to liver cold I/R injury compared with rats and summer squirrels. Because hibernation-induced protection is not dependent on animals being in the torpid state, the mechanisms responsible for this effect may provide new strategies for liver preservation in humans.

Participation of autophagy in the degeneration process of rat hepatocytes after transplantation following prolonged cold preservation

Cold ischemia-warm reperfusion injury of liver grafts has been investigated thoroughly, but its underlying mechanism remains poorly understood. Here we show that autophagy is involved not only during cold preservation but also during warm reperfusion following transplantation. Immunohistochemistry using an antibody against LC3, a microtubule associated protein 1 light chain 3 and a marker of autophagosomes, showed dot-like weak staining in hepatocytes of rat liver grafts during cold preservation. Since University of Wisconsin solution for graft preservation lacks amino acids, the induction of autophagy in hepatocytes was similar to that under starvation conditions. Intense immunopositive punctate structures were detected abundantly in the hepatocytes 30 min after the beginning of reperfusion. LC3-positive granules were often co-localized in ED2-positive Kupffer cells at 60 min of the reperfusion phase. The molecular form of LC3 was mainly LC3-II, a membrane-bound form, during reperfusion, especially at 30 min of the phase. Electron microscopic examination demonstrated numerous vacuolar structures in hepatocytes at 30 min of the reperfusion period, while some hepatocytes with such vacuolar structures were present in the sinusoidal lumen. At the late stage of the reperfusion period, Kupffer cells contained phagocytosed cells that possessed numerous autophagic vacuoles/autolysosomes and nuclei with condensed chromatin. Our results showing the presence of autophagic vacuoles/autolysosomes in hepatocytes of liver grafts after the start of reperfusion suggest that warm reperfusion acted as a stress stimulus to hepatocytes. Moreover, the stress response of hepatocytes may be involved in their degeneration process.

Cathepsin B inactivation attenuates hepatocyte apoptosis and liver damage in steatotic livers after cold ischemia-warm reperfusion injury.

Hepatic steatosis predisposes the liver to cold ischemia-warm reperfusion (CI/WR) injury by unclear mechanisms. Because hepatic steatosis has recently been associated with a lysosomal pathway of apoptosis, our aim was to determine whether this cell-death pathway contributes to CI/WR injury of steatotic livers. Wild-type and cathepsin B-knockout (Ctsb(-/-)) mice were fed the methionine/choline-deficient (MCD) diet for 2 wk to induce hepatic steatosis. Mouse livers were stored in the University of Wisconsin solution for 24 h at 4 degrees C and reperfused for 1 h at 37 degrees C in vitro. Immunofluorescence analysis of the lysosomal enzymes cathepsin B and D showed a punctated intracellular pattern consistent with lysosomal localization in wild-type mice fed a standard diet after CI/WR injury. In contrast, cathepsin B and D fluorescence became diffuse in livers from wild-type mice fed MCD diet after CI/WR, indicating that lysosomal permeabilization had occurred. Hepatocyte apoptosis was rare in both normal and steatotic livers in the absence of CI/WR injury but increased in wild-type mice fed an MCD diet and subjected to CI/WR injury. In contrast, hepatocyte apoptosis and liver damage were reduced in Ctsb(-/-) and cathepsin B inhibitor-treated mice fed the MCD diet following CI/WR injury. In conclusion, these findings support a prominent role for the lysosomal pathway of apoptosis in steatotic livers following CI/WR injury.

THE HEPATIC EXTRACELLULAR MATRIX DURING COLD ISCHEMIA/WARM REPERFUSION OF THE RAT LIVER: ROLE OF MATRIX METALLOPROTEINASES IN ITS ALTERATION.

A310 Background: Poor initial graft function remains a major clinical problem in human liver transplantation. Its incidence (15 to 25% of transplanted patients) appears to depend on the length of cold storage and has thus been ascribed to injury from harvesting, cold storage and warm reperfusion after blood vessel reconnection; i.e. cold ischemia/warm re-perfusion (CI-WR) injury. The underlying mechanism of CI-WR injury has been studied extensively over the last decade, but it remains poorly understood. Recently, it has been demonstrated that different time courses and mechanisms of cell death occur in rat livers after CI-WR, with early sinusoidal endothelial cell necrosis followed by delayed hepatocyte apoptosis. These processes did not appear to be of major importance in the mechanism of graft failure since both were similar under non-lethal and lethal conditions. By contrast, prolonged CI was associated with an alteration of the extracellular matrix (ECM), leading to extensive coagulative necrosis of hepatocytes as found in conditions ending in graft failure. Aims: Since it is known that warm ischemia/WR of livers induces an increased local synthesis of metalloproteinases (MMPs), proteases implicated in the degradation of the hepatic ECM, we looked if the same phenomenon was occurring during prolonged CI/WR of rat livers ensuing in ECM disappearance responsible for the graft malfunction. Methods: Rat livers were preserved for 0, 24 (viable livers) and 42 h (non-viable livers) in the UW solution (4°C), then reperfused for 0, 10, 20, 30, 40, 60, 90, and 120 minutes with a krebs buffer solution (37°C) containing 20% erythrocytes. The first 15 mLs of the liver rinsing solution were taken during early WR and frozen liver biopsies were obtained at the end of each period (n=4 in all groups). Total liver RNAs and proteins were extracted, then MMP and their natural inhibitor (TIMP) expression was analysed by Northern Blot quantitative analysis and MMP activity was nalyzed using a fluorescent substrate (MCA-MAT) or by zymography. Results: although most of these genes are not expressed in the normal rat liver, there was an induction of MMP-13 and 14 expression as well as of TIMP-1 and 2 expression in all livers, independently from CI or WR periods. There was also an increased MMP activity in all livers, as measured ding a in presence or absence of an inhibitor (BB94), whatever the experimental condition. An increased activity of MMPs (particularly gelatinases A and B) was found in the initial flush solution. Comments: These data are quite different from those observed following warm ischemia/WR, particularly when considering that, following CI, the ECM rapidly deteriorates with WR. However since this phenomenon occurs very early during WR, it might be due to the liberation of MMPs already present in sinusoidal endothelial cells and released by their necrosis during prolonged CI, as suggested by our data and others. Conclusion: Induction of hepatic MMP synthesis does not occurs following prolonged CI/WR of rat livers and thus has no major role in the alteration of the hepatic extracellular matrix observed under these conditions. This phenomenon might result from the local passive release of preformed MMPs from dead hepatic cells.

Sinusoidal endothelial cell and hepatocyte death following cold ischemia-warm reperfusion of the rat liver.

Cold ischemia-warm reperfusion (CI-WR) injury of the liver is characterized by marked alterations of sinusoidal endothelial cells (SECs), whereas hepatocytes appear to be relatively unscathed. However, the time course and mechanism of cell death remain controversial: early versus late phenomenon, necrosis versus apoptosis? We describe the occurrence and nature of cell death after different periods of CI with University of Wisconsin (UW) solution and after different periods of WR in the isolated perfused rat liver model. After 24- and 42-hour CI (viable and nonviable livers, respectively), similar patterns of liver cell death were seen: SEC necrosis appeared early after WR (10 minutes) and remained stable for up to 120 minutes. After 30 minutes of WR, apoptosis increased progressively with WR length. Based on morphological criteria, apoptotic cells were mainly hepatocytes within liver plates or extruded in the sinusoidal lumen. In addition, only after 42-hour CI were large clusters of necrotic hepatocytes found in areas of congested sinusoids. In these same livers, the hepatic microcirculation, evaluated by means of the multiple-indicator dilution technique, revealed extracellular matrix disappearance with no-flow areas. In conclusion, different time courses and mechanisms of cell death occur in rat livers after CI-WR, with early SEC necrosis followed by delayed hepatocyte apoptosis. These processes do not appear to be of major importance in the mechanism of graft failure because they are similar under both nonlethal and lethal conditions; this is not the case for the loss of the extracellular matrix found only under lethal conditions and associated with hepatocyte necrosis.

136 Role of mitochondrial permeability transition (MPT) and oxidative stress in cold ischemia/warm reperfusion (CI/WR) injury of rat livers

136 Role of mitochondrial permeability transition (MPT) and oxidative stress in cold ischemia/warm reperfusion (CI/WR) injury of rat livers

Ischemic preconditioning (IPC): Lack of protection against cold-ischemia/warm reperfusion (CI/WR) injury of the rat liver

Ischemic preconditioning (IPC): Lack of protection against cold-ischemia/warm reperfusion (CI/WR) injury of the rat liver

Ischemic preconditioning (IPC): Lack of protection against cold-ischemia/warm reperfusion (CI/WR) injury of the rat liver

Ischemic preconditioning (IPC): Lack of protection against cold-ischemia/warm reperfusion (CI/WR) injury of the rat liver

Apoptosis of sinusoidal endothelial cells occurs during liver preservation injury by a caspase-dependent mechanism.

Cold ischemia/warm reperfusion (CI/WR) liver injury remains a problem in liver transplants. Sinusoidal endothelial cells (SEC) are a target of CI/WR injury, during which they undergo apoptosis. Because caspase proteases have been implicated in apoptosis, our aim was to determine whether liver CI/WR injury induces a caspase-dependent apoptosis of SEC. Rat livers were stored in the University of Wisconsin (UW) solution for 24 hr at 4 degrees C and reperfused for 1 hr at 37 degrees C in vitro. Apoptosis was quantitated using the TUNEL assay, and caspase 3 activation determined by immunohistochemical analysis. Rat liver orthotopic liver transplants (OLT) were also performed using livers stored for 30 hr. Terminal deoxynucleotide transferase-mediated dUTP nick end labeling (TUNEL) positive hepatocytes were rare and did not increase during CI/WR injury. In contrast, TUNEL positive SEC increased 6-fold after reperfusion of livers stored under cold ischemic conditions, compared with controls or livers stored but not reperfused. Immunohistochemical analysis demonstrated active caspase 3 only in endothelial cells after CI/WR injury. When IDN-1965, a caspase inhibitor, was given i.v. to the donor animal and added to UW solution and the reperfusion media, TUNEL positive endothelial cells were reduced 63+/-11% (P<0.05). Similarly, the duration of survival after OLT was significantly increased in the presence of the inhibitor. During liver CI/WR injury: 1) selective apoptosis of endothelial cells occurs; 2) caspase 3 is activated only in endothelial cells; and 3) a caspase inhibitor reduces endothelial cell apoptosis and prolongs animal survival after OLT. The pharmacologic use of caspase inhibitors could prove useful in clinical transplantation.