Abstract
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.
Footnotes
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Send reprint requests to: Dr. Ronald G. Thurman Laboratory of Hepatobiology and Toxicology, Department of Pharmacology, CB# 7365, Mary Ellen Jones Bldg., The University of North Carolina, Chapel Hill, NC 27599-7365. E-mail: thurman{at}med.unc.edu
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↵1 Supported, in part, by Grants AA-09156, National Cancer Institute R42CA68826 and by the Deutsche Forschungsgemeinschaft (Sche 521/1–1, 1–2)
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↵2 Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709.
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↵3 Current addresses: Department of Surgery (H.B.), Department of Radiation Oncology (G.E.A, J.A.R), University of North Carolina, Chapel Hill, NC 27599-7365.
- Abbreviations:
- ELISA
- enzyme-linked immunosorbent assay
- ESR
- electron spin resonance
- GdCl3
- gadolinium chloride
- PBN
- C-phenyl-N-tert-butylnitrone
- UW solution
- University of Wisconsin solution
- Received November 19, 1998.
- Accepted March 22, 1999.
- The American Society for Pharmacology and Experimental Therapeutics
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