Cold ischemia (CI)-warm reperfusion (WR) liver injury remains a problem in liver transplantation. CI-WR initially causes sinusoidal endothelial cell (SEC) apoptosis through a caspase-dependent mechanism. We previously showed that the caspase inhibitor IDN-1965 prevents CI-WR-induced SEC apoptosis. However, this agent required to be administered to the donor, preservation solution, and recipient for efficacy. Here, we show that a second-generation caspase inhibitor, IDN-6556, effectively prevents CI-WR-induced SEC injury when added only to University of Wisconsin (UW) cold storage media. Rat livers were stored in UW solution for 24 hours at 4°C and reperfused for 1 hour at 37°C. Apoptosis was quantitated using terminal deoxynucleotide transferasemediated deoxyuridine triphosphate nick end labeling (TUNEL) assay and caspase 3 activation determined by biochemical measurement and immunohistochemical analysis. P rimary graft nonfunction caused by preservation injury remains a clinical problem in liver transplantation. 1,2 Moreover, this problem may potentially increase as non-heart-beating donors, marginal donors, and steatotic livers are used more widely in practice. Although preservation injury is multifactorial, ischemia-reperfusion injury of the liver is an important contributing factor. 3 Sinusoidal endothelial cells (SECs) are a cellular target of ischemia-reperfusion injury. 4-6 SEC injury causes microcirculatory disturbances, including leukocyte migration, platelet activation, and ultimately, secondary injury to hepatocytes leading to organ nonfunction and dysfunction. 1,6,7 We and others have shown that SEC injury in this process is caused by cell death by apoptosis. [8][9][10] SECs show the classic morphological feature of apoptosis after ischemia-reperfusion injury. 8,9 Biochemical hallmarks of apoptosis, including DNA fragmentation and caspase 3 activation, also are evident. Finally, protease inhibitors known to block apoptosis also protect against liver ischemia-reperfusion injury. 8,11 Thus, ischemia-reperfusion injurymediated apoptosis of SECs would appear to be a key feature of liver preservation injury.The best-characterized proteases contributing to cell injury by apoptosis are the caspases. 12,13 These proteases are synthesized as proenzymes and activated by proteolytic processing by other caspases. 12 Of caspases cloned and identified to date, caspases 2,3,6,7,8,9, and 10 have been implicated most strongly in apoptosis. 12 All these caspases have a cysteine residue in their active site and recognize aspartate in the p1 position of the substrate. 12 This unique substrate specificity has permitted the development of selective caspase inhibitors. Inhibition of caspases by these protease inhibitors often abrogates apoptosis. We recently showed that IDN-1965, a pan-caspase inhibitor, reduced liver injury in a rodent model of ischemia-reperfusion injury relevant to transplantation. 8 However, for optimal efficacy, the drug had to be included in the University of Wisconsin (UW) storage solution and admin...