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In livers excised for transplantation, sinusoidal endothelium appears especially vulnerable to injury during organ preservation in the cold and subsequent reperfusion. The degree of endothelial cell injury correlates with functional impairment of the graft following transplantation. The mechanism of injury remains obscure, but endothelial cell damage has been described as coagulative necrosis secondary to irreversible physico-chemical damage. We investigated whether endothelial cell death is caused by apoptosis rather than by necrosis. Tissue from rat livers stored for varying periods in cold (1ЊC) Euro-Collins solution and then reperfused for 1 hour at 37ЊC were studied for evidence of apoptosis by detection of DNA fragmentation using the in situ terminal deoxynucleotidyl transferase d-uridine triphosphate nick end labeling (TUNEL) assay, DNA gel electrophoresis, and by transmission electron microscopy (EM). DNA fragmentation of the type characteristic of apoptosis was identified in 49.7% ؎ 2.2% of sinusoidal lining cells after 8 hours of ischemia ؉ reperfusion (viable graft) vs. 70.7% ؎ 4.3% after 16 hours ؉ reperfusion (nonviable graft) (P F .001). No such fragmentation was observed after cold preservation without reperfusion or in unpreserved, reperfused livers. EM demonstrated changes characteristic of apoptosis exclusively in endothelial cells. The study suggests that the apoptosis of sinusoidal endothelial cells is a pivotal mechanism of preservation injury in liver transplantation. (HEPATOLOGY 1998;27:1652-1660

Interleukin-6 (IL-6) is an acute reactant cytokine with anti-thought to be the most important risk factor for postoperative inflammatory properties, which has been found to prevent complications in patients undergoing liver resection 1,2 and it injury in a model of acute hepatitis in mice through downregu-is an important cause of primary nonfunction of liver allolation of tumor necrosis factor alpha (TNF-a); to correlate grafts. [3][4][5] The potential mediators involved in WI/Rp injury inversely with markers of hepatocellular injury in patients are numerous, 6-8 and include acute phase reactant cytokines with liver ischemia; and to initiate liver regeneration in mice. and polymorphonuclear neutrophils (PMN). 3,9 Better underIn this study, we investigated the role of IL-6 in rodent models standing of the pathogenesis of WI/Rp injury of the liver and of hepatic warm ischemia/reperfusion (WI/Rp) injury. IL-6-the availability of an agent that could alleviate Rp injury deficient mice (0/0) were subjected to hepatic WI and com-would have important clinical implications. pared with C57BL/6 mice, as well as IL-6 0/0 mice pretreated Interleukin-6 (IL-6) is an acute phase reactant cytokine with recombinant IL-6 (rIL-6). The effects of rIL-6 following with pleiotropic biological effects. This cytokine plays a cenvarious periods of ischemia were further studied in models of tral role in hematopoiesis, host defense, and inflammation. 10 hepatic ischemia in rats. IL-6 0/0 mice had increased reperfu-IL-6 has been found to have potent anti-inflammatory propsion injury as assessed by transaminase levels and a tissue erties, particularly in preventing injuries related to endotoxenecrosis scoring system when compared with controls, an ef-mia. 11,12 In a rat model of endotoxin-induced tracheal injury, fect prevented by pretreatment with rIL-6. Similarly, rats pre-recombinant IL-6 (rIL-6) was found to significantly prevent treated with rIL-6 had reduced reperfusion injury and better neutrophil infiltration and reduce other markers of inflamsurvival than controls in each respective WI group. Tissue mation. 11 TNF-a expression measured by Northern blot analysis andAlthough the liver is an important source of IL-6 and the serum C-reactive protein (CRP) levels, a marker of inflamma-primary site for its clearance, 13 the role of IL-6 in liver disease tion, were significantly reduced in animals pretreated with is unclear. In an acute hepatitis model in mice, rIL-6 was rIL-6. Administration of antibodies to TNF-a reproduced the found to confer a high degree of hepatoprotection when given beneficial effect of rIL-6. Hepatocyte proliferation, as assessed before the induction of the injury. 14 This effect was thought by a scoring method for mitotic index and proliferating nuclear to be related to downregulation of tumor necrosis factor cell antigen staining, was markedly increased in rIL-6-treated alpha (TNF-a) production. In a recent prospective randomrats when compared with controls. In conclusion, this study ized study of hepatic vascular...

storage before transplantation correlates clinically with inThe injury resulting from cold ischemia and warm recreased primary graft nonfunction, graft rejection, and rate perfusion during liver transplantation is a major clinical of re-transplantation. 4,5 problem that limits graft success. Kupffer cell activation Reperfusion injury is difficult to study clinically, but has plays a pivotal role in reperfusion injury, and Kupffer been investigated in animal models, including orthotopic cell products, including free radicals and tumor necrosis liver transplantation in rats. These studies have shown that factor a (TNF-a), are implicated as damaging agents.hepatic reperfusion following ischemia induces Kupffer cell However, the second messengers and signaling pathactivation, superoxide formation, and elevated plasma levels ways that are activated by the stress of hepatic ischemia/ of tumor necrosis factor a (TNF-a). 6,7 Pretreatment with reperfusion remain unknown. The purpose of this study methyl palmitate inhibits Kupffer cell activation and imis to assess the activation of the three known vertebrate mitogen activated protein kinase (MAPKs) and the acti-proves transplant survival threefold, thereby supporting a vating protein 1 (AP-1) transcription factor in response role for Kupffer cells in reperfusion injury. 8 In addition, treatto ischemia and reperfusion in the transplanted rat ment with agents that suppress TNF-a release from activated liver. There was a potent, sustained induction of c-jun Kupffer cells decrease transplant failure.9 Nisoldipine, a Ca 2/ N-terminal kinase (JNK), but not of the related MAPKs channel blocker, reduces plasma levels of TNF-a and inextracellular signal-regulated kinases (ERK) or p38, creases transplant survival time. 10,11 Similarly, pentoxifylupon reperfusion after transplantation. TNF-a messen-line, a methylxanthine which suppresses TNF-a messenger ger RNA (mRNA) levels and transcription factors AP-1 RNA (mRNA) accumulation in response to lipopolysacchaand nuclear factor-kB (NF-kB) were induced in the liver ride, has a protective effect on liver grafts.12 Taken together, after 60 minutes of reperfusion. Finally, there was an these data suggest an important role for TNF-a in mediating elevation of ceramide, but not diacylglycerol or sphingo-reperfusion injury. sine, in the transplanted liver. Ceramide is a second mes-TNF-a is a pleiotropic cytokine that induces cellular effects senger generated by TNF-a treatment and is an activator ranging from proliferation to apoptosis. TNF-a is a potent of JNK. Because JNK activation preceded the elevations activator of activating protein 1 (AP-1) and NF-kB transcripin ceramide and TNF-a mRNA, these results suggest that tion factors and of the c-jun N-terminal kinase (JNK, also increased hepatic TNF-a and ceramide may perpetuate known as stress-activated protein kinase, SAPK).13-15 JNK is JNK induction, but that they are not the initiating sig-a member of the vertebrate mitogen activated protein kinase nals of JNK activation during reperfus...

It is well recognized that consumption of alcohol leads to liver disease in a dose-dependent manner; however, the exact mechanisms remain unclear. Hypoxia subsequent to a hypermetabolic state may be involved; therefore, when it was observed recently that inactivation of Kupffer cells prevented stimulation of hepatic oxygen uptake by alcohol, the idea that Kupffer cells participate in early events that ultimately lead to alcohol-induced liver disease became a real possibility. The purpose of this study was to test that hypothesis. Male Wistar rats were exposed to ethanol continuously by means of intragastric feeding for up to 4 weeks using the model developed by Tsukamoto and French. In this model, ethanol causes fatty liver, necrosis and inflammation--changes characteristic of alcohol-induced liver disease in human beings. Kupffer cells were inactivated by twice weekly treatment with gadolinium chloride (GdCl3), a selective Kupffer cell toxicant. AST levels were elevated to 192 +/- 13 and 244 +/- 56 IU/L in rats exposed to ethanol for 2 and 4 wk, respectively (control value, 88 +/- 7). This injury was prevented almost completely by GdCl3 treatment. Fatty changes, inflammation and necrosis were also all reduced dramatically by GdCl3 treatment. The average hepatic pathological score of rats treated with ethanol for 4 wk was 4.3 +/- 0.6, which was reduced significantly in ethanol- and GdCl3-treated rats to 1.8 +/- 0.5 (p < 0.05). Rates of ethanol elimination were elevated 2- to 3-fold in rats exposed to ethanol for 2 to 4 wk. This elevation was blocked by GdCl3 treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Proteases as well as alterations in intracellular calcium have important roles in hepatic preservationreperfusion injury, and increased calpain activity recently has been demonstrated in liver allografts. Experiments were designed to evaluate (i) hepatic cytosolic calpain activity during different periods of cold ischemia (CI), rewarming, or reperfusion, and (ii) effects of inhibition of calpain on liver graft function using the isolated perfused rat liver and arterialized orthotopic liver transplantation models. Calpain activity was assayed using the f luorogenic substrate Suc-LeuLeu-Val-Tyr-7-amino-4-methyl coumarin (AMC) and expressed as mean ؎ SD pmol AMC released͞min per mg of cytosolic protein. Calpain activity rose significantly after 24 hr of CI in University of Wisconsin solution and further increased with longer preservation. Activity also increased within 30 min of rewarming, peaking at 120 min. Increased durations of CI preceding rewarming resulted in significantly higher activity (P < 0.01). Calpain activity increased rapidly upon reperfusion and was significantly enhanced by previous CI (P < 0.01). Calpain inhibition with Cbz-Val-Phe methyl ester significantly decreased aspartate aminotransferase released in the isolated perfused rat liver perfusate (P < 0.05). Duration of survival after orthotopic liver transplantation using livers cold-preserved for 40 hr was also significantly increased (P < 0.05) with calpain inhibitor. In conclusion, calpain proteases are activated during each phase of transplantation and are likely to play an important role in the mechanisms of preservation-reperfusion injury.

Leukocytes recruited during ischemia-reperfusion to the liver are important mediators of injury. However, the mechanisms of leukocyte adhesion and the role of adhesion receptors in hepatic vasculature remain elusive. L-selectin may critically contribute to injury, priming adhesion for later action of intercellular adhesion molecule-1 (ICAM-1). Paired experiments were performed using mutant mice (L-selectin −/−, ICAM-1 −/−, and L-selectin/ICAM-1 −/−) and wild-type mice (C57BL/6) to investigate leukocyte adhesion in the ischemic liver. Leukocyte adhesion and infiltration were assessed histologically. Aspartate aminotransferase levels were significantly reduced (2- to 3-fold) in mutant vs. wild-type mice in most groups but most significantly after 90 min of partial hepatic ischemia. Leukocyte adhesion was significantly reduced in all mutant mice. Areas of microcirculatory failure, visualized by intravital microscopy, were prevalent in wild-type but virtually absent in L-selectin-deficient mice. After total hepatic ischemia for 75 or 90 min, survival was better in mutant L-selectin and L-selectin/ICAM-1 mice vs. wild-type mice and ICAM-1 mutants. In conclusion, L-selectin is critical in the pathogenesis of hepatic ischemia-reperfusion injury. Poor sinusoidal perfusion due to leukocyte adhesion and clot formation is a factor of injury and appears to involve L-selectin and ICAM-1 receptors.

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