Vol. 290, Issue 1, 235-240, July 1999

Reperfusion Injury in Livers Due To Gentle In Situ Organ Manipulation during Harvest Involves Hypoxia and Free Radicals¹

Peter Schemmer, Henry D. Connor², Gavin E. Arteel³ , James A. Raleigh³, Hartwig Bunzendahl³, Ronald P. Mason² and R. G. Thurman

Laboratory of Hepatobiology, Department of Pharmacology, University of North Carolina, Chapel Hill, North Carolina (P.S., G.E.A., R.G.T.)

Kupffer cell-dependent injury in livers gently manipulated during harvest develops upon transplantation; however, underlying mechanisms remain unknown. Thus, the purpose of this study was to identify factors involved in mechanisms of injury. Livers from female Sprague-Dawley rats (200-230 g) were cold stored for 24 h in University of Wisconsin solution. Subsequently, livers were perfused at 37°C with oxygen-saturated Krebs-Henseleit buffer containing fluorescein-dextran to assess microcirculation. Cell death was assessed by uptake of trypan blue, a vital dye. Minimal dissection during harvest had no effects on sinusoidal lining cells; however, gentle organ manipulation dramatically increased trypan blue uptake about 5-fold (p < .05). In contrast, perfusion with N₂-saturated buffer after cold storage totally prevented cell death due to manipulation. At harvest, portal venous pressure was increased significantly by 70% due to manipulation. Furthermore, vascular space and microcirculation were decreased by more than 50% (p < .05), reflecting the rate of entry and exit of fluorescein-dextran. Pimonidazole, a 2-nitroimidazole marker, was given to rats before harvest to detect hypoxia in liver. Pimonidazole adduct binding was increased significantly about 2-fold by manipulation. To detect free radical adducts by electron spin resonance (ESR) spectroscopy in bile, C-phenyl-N-tert-butylnitrone was given as spin trapping reagent to the donor before operation. Free radical formation was increased about 3-fold by organ manipulation (p < .05). Donors given gadolinium chloride, a selective Kupffer cell toxicant, or dietary glycine, which prevents activation of Kupffer cells, significantly blunted microcirculatory disturbances, hypoxia, and death of endothelial lining cells. These data indicate for the first time that gentle organ manipulation during harvest causes oxygen-dependent reperfusion injury to endothelial lining cells via mechanisms involving hepatic microcirculation, hypoxia, and Kupffer cells.