Role of anti-tumor necrosis factor-alpha in ischemia/reperfusion injury in isolated rat liver in a blood-free environment.
BACKGROUND
Warm ischemia/reperfusion injury during liver transplantation is the most important cause of primary nonfunction of liver allografts. Tumor-necrosis factor (TNF)-alpha apparently mediates tissue damage by inducing apoptosis and/or necrosis in liver transplants. The aim of the study was to determine, using an isolated rat liver model, if pretreatment with anti-TNF-alpha monoclonal antibodies can attenuate ischemia/reperfusion liver injury. Specifically, its effect on liver cell apoptosis through the modulation of caspase activity was examined in a blood-free environment.


METHODS
Isolated rat livers were perfused with Krebs-Henseleit solution and randomly divided into three groups: (1) continuous perfusion for 165 min (control); (2) perfusion for 90 min, break for 60 min (ischemia), and reperfusion for 15 min; (3) as with group 2, but with administration of monoclonal mouse anti-rat TNF-alpha monoclonal antibodies before induction of ischemia. Caspase-3- and -9-like activity was measured by fluorometric assay, and apoptotic cells were identified by morphological criteria and application of the terminal deoxnucleotidyl transferase-mediated dUTP nick-end-labeling (Tunel) assay.


RESULTS
Portal pressure increased significantly in group 2 (14.8+/-2.3 mm Hg) compared to group 3, which showed no change (P<0.05). Significant amounts of TNF-alpha were detected in the effluent in group 2 at 1 min of reperfusion (147+/-8.9 pg/ml) compared to group 3 (30+/-6.7 pg/ml, P<0.05). Statistically significant reductions in liver enzyme levels were also noted in the animals pretreated with TNF-alpha antibodies (P<0.02). Caspase-3 and -9 activity was significantly decreased (270 and 160%, respectively) in group 3 compared to group 2 (P<0.005 and <0.05, respectively). A significant reduction in postischemic hepatic injury was noted on Tunel assay: many apoptotic hepatocyte cells were detected in group 2 but not in livers pretreated with monoclonal mouse anti-TNF-alpha antibodies (group 3).


CONCLUSIONS
Neutralization with specific monoclonal antibodies against TNF before ischemia induction can attenuate postischemic hepatic injury. Apoptotic injury seems to be ameliorated through modulation of caspase-3- and -9-like activity.
Liver Institute and Department of Medicine D, Rabin Medical Center, Beilinson Campus, Petah Tiqva, Israel.
Ziv
Z
Ben-Ari
Edith
E
Hochhauser
Idan
I
Burstein
Orit
O
Papo
Ella
E
Kaganovsky
Tatyana
T
Krasnov
Alexey
A
Vamichkim
Bernardo A
BA
Vidne
D000818:Animals; D000911:Antibodies, Monoclonal / Q000627:therapeutic use*; D017209:Apoptosis; D020169:Caspases / Q000502:physiology; D007511:Ischemia / Q000628:therapy*; D008099:Liver / Q000098:blood supply* / Q000201:enzymology / Q000473:pathology; D017082:Portal Pressure; D051381:Rats; D017208:Rats, Wistar; D015427:Reperfusion Injury / Q000517:prevention & control*; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors* / Q000378:metabolism / Q000502:physiology
D000911:Antibodies, Monoclonal; D014409:Tumor Necrosis Factor-alpha; D020169:Caspases