reperfusion injury and remote cardiac dysfunction in isolated rat liver

HEPATOLOGY, Vol. 34, No. 4, Suppl. 1, 2003

AASLD ABSTRACTS

IP and IC. Conclusions: Ischemic preconditioning and intermittent clamping are both protective against liver ischemia by preserving hepatic microcirculation and decreasing KC activation. The inverse correlation of TNF-a levels and phagocytic activity is probably caused by an exceeded phagocytic capacity of the KC. Although IC is superior for ischemia of 120 minutes, both procedures similarly reduce I/R injury occurring during the clinically relevant ischemia times below 75 minutes. Disclosures: Pierre-Alain Clavien - No relationships to disclose Roll Graf - No relationships to disclose Stefan Heinrich - No relationships to disclose Yinhua Tian - No relationships to disclose Katharina Vajdova - No relationships to disclose

661

ISCHEMIC PRECONDITIONING PROMOTES LIVER REGENERATION AFTER PARTIAL HEPATECTOMY BY STIMULATION OF ADENOSINE A2 RECEPTOR PATHWAY.

Masahiro Arai, Kazuaki Tejima, Hitoshi Ikeda, Tomoaki Tomiya, Mikio Yanase, Yukiko Inoue, Kayo Nagashima, Masao Omata, University of Tokyo, Tokyo, Japan; Kenji Fujiwara, Saitama Medical School, Saitama, Japan Background A brief period of hepatic ischemia followed by reperfusion reduces sinusoidal endothelial cell killing and Kupffer cell activation and improves recipient survival after liver transplantation, a p h e n o m e n o n called ischemic preconditioning. Such liver graft protection by ischemic preconditioning is produced by stimulation of adenosine receptor pathways (Hepatology 32: 297-302, 2000, Hepatology 32: 249A, 2000). We previously reported that ischemic preconditioning also promotes liver regeneration after partial hepatectomy (Hepatology 36: 210A, 2002). Aim Accordingly, our aim was to investigate the contribution of adenosine receptors to the effect of ischemic preconditioning on liver regeneration. Method Male Sprague-Dawley rats were injected intravenously with 10 mg/kg 8-(p-sulfophenyl)theophilline (SPT; a dual adenosine A1 and A2 receptor antagonist), 0.1 mg/kg 8-cyclopentyl-l,3-dipropylxanthine (DPCPX; an A1 receptor antagonist) or I mg/kg 3,7-dimethyl-l-propargylxanthine (DMPX; an A2 receptor antagonist). After 10 min, hepatic artery and portal vein to the median and left lobes were occluded with a vascular clamp. After another 10 min, the clamp was removed and the liver was then reperfused for 10 min. Subsequently, the median and left lobes, which correspond to 70% of the total liver volume, were resected and the extent of liver regeneration was evaluated with liver regeneration ratio and BrdU labeling index in hepatocytes, a marker of proliferating hepatocytes in S phase. Other rats were injected intravenously with 10/xg/kg CGS-21680 (an A2 receptor agonist) instead of ischemic preconditioning. Subsequently, partial hepatectomy was performed and liver regeneration was evaluated. Liver regeneration ratio was calculated as follows: (A-B)/B, in which A is final liver weight at sacrifice and B is weight of the remnant liver immediately after partial hepatectomy, which is calculated by 0.3/0.7 × weight of the resected liver. Liver regeneration ratio was determined at 4 days after hepatectomy, w h e n increase in regeneration ratio produced by ischemic preconditioning was most prominent in the previous study. For determination of BrdU labeling index in hepatocytes, 50 mg/kg BrdU was injected intraperitoneaily 23 hours after hepatectomy. After 1 hour, the liver was excised and fixed with formalin. Sections of liver tissue were stained immunohistochemically using anti-BrdU antibody as a primary antibody. BrdU labeling index in hepatocytes was expressed as the ratio of BrdU-incorporated hepatocytes to total hepatocytes. Results Ischemic preconditioning increased liver regeneration ratio (1.57 ± 0.03 [Mean ± SEMI vs 1.36 ± 0.04, p < 0.05) and BrdU labeling index (39.8 ± 1.9% vs 31.1 ± 2.2% p < 0.05) compared to control. W h e n both adenosine A1 and A2 receptors were blocked with SPT, increase in regeneration ratio by ischemic preconditioning was reversed (1.49 ± 0.07 [sham] vs 1.38 ± 0.05 [ischemic preconditioning]). DMPX also reversed the effect of ischemic preconditioning on regeneration ratio (1.45 ± 0.04 vs 1.52 ± 0.05) and on BrdU labeling index (31.5 ± 2.5% vs 30.5 ± 2.3%), but DPCPX

479A

did not change the effect of ischemic preconditioning. Adenosine A2 receptor stimulation with CGS-21680 instead of ischemic preconditioning increased regeneration ratio compared to control (1.62 ± 0.05, p < 0.05). Conclusion The effect of ischemic preconditioning on liver regeneration was reversed by adenosine A2 receptor antagonism, whereas the adenosine A2 receptor agonist mimicked ischemic preconditioning. We conclude that promotion of liver regeneration after partial hepatectomy by ischemic preconditioning is mediated by an adenosine A2 receptor pathway. Disclosures: Masahiro Arai - No relationships to disclose Kenji Fujiwara - No relationships to disclose Hitoshi Ikeda - No relationships to disclose Yukiko Inoue - No relationships to disclose Kayo Nagashima - No relationships to disclose Masao Omata - No relationships to disclose Kazuaki Tejima - No relationships to disclose Tomoaki Tomiya - No relationships to disclose Mikio Yanase - No relationships to disclose

662

TPEN ATTENUATES HEPATIC ISCHEMIA/REPERFUSION INJURY AND REMOTE CARDIAC DYSFUNCTION IN ISOLATED RAT LIVER. Ziv Ben-Aft, Rabin Medical Center,

Petah-Tikva, Israel; Bernardo Vidne, Felsenstein Medical Research Center, Petach-Tikva, Israel; Om't Pappo, Rabin Medical Center, Petah-Tikva, Israel; ~ dit Hochhauser, Felsenstein Medical Research Center, Petach-Tikva, Israel Background~Aims: Warm ischemia/reperfusion injury during liver transplantation is the major cause of primary liver graft nonfi~nction. The release of vasoactive substances during ischemia/reperfusion injury generates toxic free radicals that mediate endothelial cell and hepatocyte damage as well as remote cardiac dysfunction. The aim of the study was to determine if pretreatment with TPEN, a potent iron chelator can attenuated these effects. Methods: Isolated rat livers were perfused with Krebs-Henseleit (KH) solution and randomly divided into three groups: 1) continuous perfusion for 165 minutes (control); 2) perfusion for 30 minutes, break for 120 minutes (ischemia), and reperfusion for 15 minutes; 3) similar to group 2, but with administration of TPEN before induction of ischemia. Isolated hearts from other rats were stabilized with KH and reperfused for 15 minutes with the perfusate exiting the livers. Livers were disconnected and the hearts continued to be recirculated with the accumulated liver and heart effluent for an additional 50 minutes. Levels of norepinephrine, epinephrine, dopamine, prostaglandin E2, angiotensin II and thromboxane g 2 were determined. Caspase-3- and -9-1ike activity was measured by fluorometric assay, and apoptotic cells were identified by morphological criteria and the terminal deoxynudeotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay. Left ventricular developed pressure (LVP) and coronary flow were measured during the experimental protocol. Results: At I minute of reperfusion there was a significant increase in iron perfusate levels in groups 2 and 3 compared to group I (p 0.001), and a significant reduction in all vasoac~ve substances in group 3 (TPEN pretreatment) compared to group 2 (ischemia) (p - 0.0001). Caspase-3 but not -9 ac~vity was significantly decreased (2.76-fold) in group 3 compared to group 2 (p - 0.0001). On TUNEL assay, many apoptotic hepatocytes were detected in the ischemic rat livers but not in the livers pretreated with TPEN. When liver effluent following ischemia was directed to the heart, LVP and coronary flow were significantly raised while in the TPEN-treated group LVP and coronary flow increased only mildly. Conclusions: Administration of TPEN before induction of ischemia in isolated rat livers can: 1. attenuate post-ischemic apoptotic hepatic injury by modulating caspase-3-1ike activity and inhibiting the release of vasoactive substance release 2. Decrease remote cardiac dysfunction. Disclosures: Ziv Ben-Ari - No relationships to disclose Edit Hochhauser - No relationships to disclose Orit Pappo - No relationships to disclose Bernardo Vidne - No relationships to disclose