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In liver transplantation, it is currently hypothesized that nonparenchymal cell damage and/or activation is the major cause of preservation-related graft injury. Because parenchymal cells (hepatocytes) appear morphologically well preserved even after extended cold preservation, their injury after warm reperfusion is ascribed to the consequences of nonparenchymal cell damage and/or activation. However, accumulating evidence over the past decade indicated that the current hypothesis cannot fully explain preservation-related liver graft injury. We review data obtained in animal and human liver transplantation and isolated perfused animal livers, as well as isolated cell models to highlight growing evidence of the importance of hepatocyte disturbances in the pathogenesis of normal and fatty graft injury. Particular attention is given to preservation time-dependent decreases in high-energy adenine nucleotide levels in liver cells, a circumstance that (1) sensitizes hepatocytes to various stimuli and insults, (2) correlates well with graft function after liver transplantation, and (3) may also underlie the preservation timedependent increase in endothelial cell damage. We also review damage to bile duct cells, which is increasingly being recognized as important in the long-lasting phase of reperfusion injury. The role of hydrophobic bile salts in that context is particularly assessed. Finally, a number of avenues aimed at preserving hepatocyte and bile duct cell integrity are discussed in the context of liver transplantation therapy as a complement to reducing nonparenchymal cell damage and/or activation. (Liver Transpl 2001;7: 381-400.)A lthough the use of University of Wisconsin (UW) solution has permitted an increase in mean preservation time, the incidence of graft dysfunction still persists, 1,2 contributing significantly to a more than 20% per year mortality rate for patients in liver transplantation centers in the United States. 3 In addition, when storage time exceeds 10 to 12 hours, such late posttransplantation complications as biliary strictures occur in more than 25% of liver transplant recipients. [4][5][6] To prevent or minimize graft dysfunction and posttransplantation complications, it is essential to fully understand the importance of individual liver cell types in graft injury induced by cold storage and reperfusion. Insight into the cellular and molecular basis of these injuries will lead to the development of better intervention strategies, which is very important because graft dysfunction results in increased morbidity and enhances mortality rates. 7,8 Moreover, up to 30% of patients with graft dysfunction need retransplantation as early as within the first 3 months after transplantation, 2,7,8 which increases the demand on an already short pool of donor organs.According to morphological studies, cold preservation leads to injuries of nonparenchymal cells, whereas liver parenchymal cells appear well preserved. It is currently hypothesized that sinusoidal endothelial cell impairment, 9-11 activatio...

With increasing time of cold preservation, levels of highenergy nucleotides in the liver are reducing. The authors hypothesized that cold preservation sensitizes hepatocyte function to ischemic injury occurring during graft rewarming and that the injury can be prevented by short-term reperfusion. Rat livers were cold-preserved in University of Wisconsin solution for 0 to 18 hours and ischemically rewarmed for 0 to 45 minutes to simulate the implantation stage of transplantation. Hepatobiliary function was assessed using a blood-free perfusion model. In comparison with controls, neither 18-hour preservation nor 45-minute ischemic rewarming significantly influenced hepatocyte function. Compared with livers subjected to 45-minute ischemic rewarming, livers subjected to 9-hour preservation and 45-minute rewarming, and livers subjected to 18-hour preservation and 45-minute rewarming exhibited, respectively: 3.8 and 24 times reduced bile production, 4.3-and 116-fold decreased taurocholate excretion, and 3.1 and 42 times depressed bromosulfophthalein excretion. Thirty-minute oxygenated warm reperfusion after 9-and 18-hour preservation nearly completely blunted sensitization of hepatocyte function to rewarming ischemia. The authors found that shortterm oxygenated reperfusion restored adenine nucleotides in liver tissue to the values found before organ preservation and that reperfusion with energy substrate containing solutions increased tissue adenosine triphosphate concentration to a higher level than that found before preservation. In conclusion, sensitization of hepatocyte function to rewarming ischemia increases disproportionally with storage time, suggesting that this phenomenon may play a role in graft dysfunctions with increasing liver preservation time. Shortterm oxygenated reperfusion of the liver may protect hepatocyte functions against warm ischemic insult, even after extended preservation. (HEPATOLOGY 2000;32:289-296.) Preservation injury is a major cause of morbidity and mortality in liver transplant recipients. 1 Because hypothermic preservation causes selective liver injuries to nonparenchymal cells, while hepatocytes appear well-preserved, it is currently hypothesized that sinusoidal endothelial-cell impairment, 2-5 microcirculatory disturbances, 6,7 activation of Kupffer cells, [8][9][10] and sinusoidal accumulation of leukocytes 11,12 are major causes of preservation-related graft failure. This hypothesis is strongly supported by a number of studies showing that sinusoidal endothelial cells lose their function with increasing time of cold storage 13-15 and die during a brief period of reperfusion. 16,17 On the other hand, most parameters of hepatocyte function of livers reperfused after 18-or 24-hour cold preservation were found to be comparable with control livers perfused immediately after hepatectomy. [18][19][20] In clinical transplantation, however, several authors identified that during the implantation stage of transplantation, prolonged warm ischemic time (WIT), preferentially influencing hepat...

The mechanisms of liver injury from cold storage and reperfusion are not completely understood. The aim of the present study was to investigate: 1) whether the inactivation of Kupffer cells (KCs) by gadolinium chloride (GadCl) modulates cold ischemia-reperfusion injury of rat liver; and 2) whether cold storage of rat liver involves injury to biliary epithelial cells (BECs). Hepatobiliary function was assessed using an isolated perfused rat liver model. Compared with control livers, in livers subjected to cold storage at 4 degrees C in Euro-Collins solution (EC) for 18 hours or in University of Wisconsin solution (UW) for 48 hours, portal flow was lower and resistance significantly higher, taurocholate (TC) and bromosulfophthalein (BSP) elimination were markedly impaired, bile flow was reduced, and lactate dehydrogenase (LDH) leakage into the perfusate was increased. Pretreatment of rats with GadCl, a selective KC toxicant, abrogated disturbances of the microcirculation in both models, but it did not influence viability and functional parameters of the liver. Most of the parameters studied in livers stored in UW solution for 18 hours were not significantly different from those found in control livers. As to biliary activity of gamma-glutamyl transferase (GGT), as an index of BEC integrity, it was increased with increasing time of cold storage. The reabsorption of glucose from the bile decreased with longer storage time. The results suggest the following: 1) that cold ischemia-reperfusion injury of rat liver is mediated by KC-dependent (hepatic microcirculation) and -independent (parenchymal cell function) mechanisms; and 2) that cold storage of rat liver induces functional impairment of BECs.

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In vivo effects of N-benzyloxycarbonyl (Cbz)-Leu-Leu-leucinal (MG132) on chymotryptic-like (ChT-L), tryptic-like, and post-glutamyl peptide hydrolytic-like proteasome activities, protein oxidation, lipid peroxidation (LP), glutathione (GSH) level, as well as on the activity of antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and glutathione-reductase) in the rat liver were studied. The possibility of MG132 provoking the formation of free oxygen radicals was also assayed in primary hepatocytes. The following results were obtained: (1) In vivo, MG132 did not change the spontaneous LP, but increased Fe-induced LP and the amount of oxidized proteins; it decreased the GSH level in liver. From the proteasome activities studied in liver cytosol only ChT-L activity was significantly decreased after MG132 administration. Furthermore, MG132 increased antioxidant enzyme activities of SOD, CAT, and GSH-Px. (2) In vitro, MG132 increased free radical oxygen species in hepatocytes; this effect disappeared in the presence of CAT or mannitol. In conclusion, since nowadays proteasome inhibitors are entering into the swing of laboratory and clinical practice, the present data could provide useful information for MG132 action. Consequently, future in vivo experiments with MG132 could highlight the possibility of its use at different pathological conditions.

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