Reperfusion injury to endothelial cells following cold ischemic storage of rat livers

Abstract

Storage of donor livers in Euro-Collins solution for human transplantation surgery is limited to about 8 hr. Here, tissue damage to isolated rat livers stored under the same conditions as human livers was characterized following reperfusion. The purpose of this work was to determine the importance of nutritional status on injury due to cold storage and reperfusion, to establish whether lethal injury occurs during cold storage or only after reperfusion, and to identify the cell types most vulnerable to damage. Rat livers were perfused with Krebs-Henseleit-bicarbonate buffer, stored 8 to 48 hr in Euro-Collins solution and reperfused with warm, oxygenated Krebs-Henseleit buffer for 15 min. Nuclear trypan blue uptake and lactate dehydrogenase release were used as indices of cell death. After 8 hr of cold storage and reperfusion, little loss of parenchymal or nonparenchymal viability occurred. After 24 or 48 hr, virtually all parenchymal cells remained viable. However, severe damage to nonparenchymal cells was observed, and about 40% of nonparenchymal cells were trypan blue positive. Nutritional status (fed vs. fasted) did not affect the extent of cell damage. Nonparenchymal cell killing was accompanied by cellular rounding, nuclear pyknosis and protrusion of cells into sinusoidal lumens. Scanning electron micrographs demonstrated denudation of the sinusoidal lining. Rounding and pyknosis were not observed in 24-hr-stored livers which were not reperfused, and trypan blue uptake did not occur in stored livers infused with cold, anoxic Euro-Collins solution. Based on cytochemistry and electron microscopy, lethal cell injury occurred predominantly to endothelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Ischemic preconditioning of rat livers against cold storage-reperfusion injury: Role of nonparenchymal cells and the phenomenon of heterologous preconditioning

“…Conversely, 26% to 27% of nonparenchymal cells were nonviable after storage and reperfusion in control livers not subjected to preconditioning (Table 1), similar to findings in earlier studies. 16,17 When the left half of the liver was preconditioned with 5 minutes of ischemia and 5 minutes of 15 reperfusion, nonparenchymal cell killing in the preconditioned half decreased to 13.4%. In the right half that was not preconditioned, nonparenchymal cell killing also significantly decreased to 17.8%.…”

“…16,17 Briefly, livers were perfused at 30 mL/min for 20 minutes through the portal vein with Krebs-Henseleit bicarbonate buffer (KHB) saturated with 95% oxygen and 5% carbon dioxide at 37°C. No cell killing occurs during this brief perfusion.…”

“…No cell killing occurs during this brief perfusion. 16,17 As the livers were perfused, they were excised and infused at 30 mL/min with ice-cold (0°C to 1°C) University of Wisconsin (UW) solution (Viaspan; Dupont Pharma, Wilmington, DE) for 2 minutes. The livers were then immersed in the same solution inside a plastic container, which was placed in an ice-water bath for 30 hours.…”

“…Under the storage conditions used, cell killing occurs exclusively in sinusoidal endothelial cells, sparing hepatocytes and other types of nonparenchymal cells. 17 Thus, we evaluated sinusoidal endothelial-cell killing as trypan blue-positive nonparenchymal cells observed in eosin-counterstained histological sections. For each liver, 5 periportal areas and 5 pericentral areas were picked at random, and trypan blue-positive nonparenchymal nuclei in eosin-stained sections were counted at high magnification (original magnification ϫ600) without knowledge of the identity of the experimental groups.…”

Addition of simvastatin to cold storage solution prevents endothelial dysfunction in explanted rat livers

“…Liver endothelial cells are particularly vulnerable to I/R injury and develop serious alterations during cold storage, such as retraction, cell body detachment, and apoptosis, which are magnified upon warm reperfusion. 5,6 It is currently accepted that hepatic endothelium damage occurring during cold preservation represents the initial factor leading to hepatic I/R injury, determining poor graft microcirculation, platelet activation, persistent vasoconstriction, up-regulation of adhesion molecules, oxidative stress, Kupffer cell activation, neutrophil infiltration, and hepatocyte death. 7,8 Different mechanisms for endothelial damage during cold storage and/or warm reperfusion have been described.…”