HEPATOLOGY Vol. 22, No. 4, Pt. 2, 1995 AASLD ABSTRACTS 381A

1 0 9 7 PRESENCE OF LIVER NECROSIS IS NEEDED FOR DEVELOPMENT OF INTRACRANIAL HYPERTENSION IN ACUTE LIVER FAILURE. S Chen. FD Watanabe. CC Wane. S Emachi. AA Demetriou. J Rozea. Liver Support Unit Research Laboratory, Cedars-Sinai Medical Cetiter, Los Angeles, CA.

Intracranial hypertension leading to braiastem coning is a major cause of death in fulminant hepatic failure (FHF). In order to study the importance of agents metabolized by the liver and those released by the necrotic liver in the pathogenesis of this complication, we have monitored intracraniai pressure (ICP; subdurai bolt), blood NH3 levels and branched- chain to aromatic amino acid ratios (BCAA/AAA) in pigs with liver necrosis and in pigs rendered anhepatic. We also investigated whether hypothermia can prolong survival and prevent rise in ICP. Methods: Adult swine (40-60kg) were used. In Grp.1 pigs (n=20), liver necrosis was induced by portacaval shunt (PCS) and dearterialization. Grp.2 pigs (n=27) were made anhepatic by PCS, hepatectomy and vena cava grafting (18 mm GoreTex). All pigs were maintained on glucose (5 g/hr i:v.). Eight Grp.l pigs and 20 Grp.2 pigs were kept at 21oc and became hypothermic (25-+0.1°C at 20 hr); 12 Grp.1 pigs and 7 Grp.2 pigs were kept warm and remained normothermic (b.temp.>38°C). Results: In both groups, hypothermic pigs lived longer than normothermic ones. Grp.2 pigs had significantly longer survival than Grp.1 pigs. Significant and persistent intracranial hypertension developed only in normothermic pigs with liver necrosis; only half of the hypothermic ones had a transient rise in ICPat 18-22 hr (33+9mmHg). All pigs had significant hyperammonemia and BCAAJAAA ratios below 1.0

Grp. 1 (Liver Necrosis~ Gro.2 (AnheDatic/ normothermic hvoothermie normothermic h~;Dothermic

Survival (hr) 2 2 + 2 2-9_+1.6" 33 -+ 11t" 42, -+ 4*t NH3 (rag/L) 490 + 163 364 -+ 113 390 -+ 63 356 _+ 83 ICP (mmH~ 29 + 4 10 5: 4* 11 _+ 2? 9 _+ 3 mear~+S.D., *p<5.05 normothermic vs. hypotherrnic, tp<0.05 Grp.1 vs. Grp.2 Conclusions: (1)Hypothermia prolongs survival in anhepatic pigs; (2) Anhepatic pigs survive longer than pigs with liver necrosis. (3) Normal ICP in anhepatic pigs with hyperammonemia, suggests that substances which are metabolized by the liver and accumulate in the blood of the liverless pig, do not cause cerebral edema. (4) Data on pigs with liver necrosis suggest that cerebral edema in FHF is caused by toxemia associated with liver necrosis and that hypothermia has a protective effect.

1 0 9 8 ACTIVATION OF NUCLEAR FACTOR-r.B (NF-r.B) AND EXPRESSION OF I N T E R C E L L ~ ADHESION MOLECULE- 1 (ICAM-1) m R N A DURING HEPATIC ISCHEMIA- REPERFUSION. FP Bell~ AM Manning, and H Jaeschke. Cell Biology and Inflammation Research and Drug Metabolism Research, The Upjohn Company, Kalamazoo, MI.

The neutrophil-dependent injury phase during hepatic ischemia-reperfusion can be attenuated by antibodies against 1~ integrins (CDllb/CD18) on neutrophils (Hepatology 17:915,1993) and its counterreceptor on endothelial cells and hepatocytes, ICAM-1 (J Leukoc Bio157:368, 1995). Translocation of the cytosolic NF-rJ3 to the nucleus is necessary to induce ICAM-1 gene transcription by cytokines in vitro (PNAS 91:11641,1994). To investigate the Potential role of NF-r,B activation in the upregulation of ICAM-1 mRNA in the liver during ischemia-reperfusion in vivo, male Fischer rats were subjected to 30 rain of partial hepatic no-flow ischemia and up to 24h of reperfusion. A minor baseline activation of NF-rJ3 was found in the liver in sham-operated controls and during ischemia, however, at I h and 5 h reperfusion, nuclear NF-r,B DNA-binding activity increased by 270-290% as determined by densitometric analysis of electrophoretic mobility shift assays. At 24 h, NF-~B is still activated by 144% above sham control levels. In the non-ischemic lobes of the liver a similar time course and magnitude of NF-rJ3 activation was observed. Northern blot analysis showed no ICAM-1 mRNA expression in controls, a slight but significant increase during ischemia and a further significant increase during all time points of reperfusion. In contrast, in the non-ischemic lobes ICAM-1 mRNA levels increased only during reperfusion. NF-rJ3 activation and ICAM-1 mRNA expression correlated in all liver samples except during ischemia. We conclude that hepatic ischemia-reperfusion induces ICAM-1 gene transcription during reperfusion by activation of NF-r,B. The fact that ICAM-1 mRNA expression can be induced without change of the activation status of NF-r,B during ischemia suggests that factors other than NF-r,B are also involved in the regulation of ICAM-1 gene transcription in the liver.

1 0 9 9 L-ARG1NINE MINIMIZES REPERFUSION INJURY IN A LOW- FLOW, REFLOW LIVER PERFUSION MODEL. SM Jones and RG Thurman Laboratory of Hepatobiology and Toxicology, Department of Pharmacology, University of North Carolina at Chapel Hill.

The low-flow, reflow model of rat liver perfusion was used to investigate the effects of L-arginine on reperfusion injury in the absence of blood elements. In contrast to in vivo studies, L-arginine cannot minimize hypoxia by improving the microcirculation under these special conditions, but rather can only increase oxygen delivery upon reflow. During reflow, release of lactate dehydrogenase reached a steady-state value of 35 + 3 U/gila in livers perfused in the absence ofL-arginine. L- arginine (0.1 mM) significantly reduced lactate dehydrogenase release to 14 + 1 U/g/h; higher concentrations (1.0 to 3.0 mM) were less effective and the arginine analogue N0~-nitro L-arginine methyl ester (L-NAME, 0.3 mM) reversed the protective effect completely. Malondialdehyde release during reflow averaged 92 + 10 nmol/g/h and was decreased significantly to 47 + 13 nmol/g/h with L-arginine (0.1 raM). Oxygen uptake during reflow was not altered by L-argihine, although the time required to reach steady-state values upon reflow was reduced significantly about two-fold. Trypan blue distribution time, which is used to index the hepatic microcirculation, was decreased significantly from 330 + 17 to 227 + 31 seconds by L-arginine, an effect also blocked by L- NAME. Additionally, L-arginine significantly increased both the rate of entry and exit of fluorescein-dextran, a dye confined to the vascular space, by approximately fifty percent. Collectively, these findings indicate that L-arginine protects against hypoxia/reoxygenation injury in a blood-free perfusion model specifically during the reoxygeaation period. It is likely that L-arginine improves the microcirculation and therefore rapidly removes substrates for free radical generation, since oxygen delivery is increased yet injury is paradoxically reduced.

1 1 0 0 SELECTIVE ZONE 3 INDUCTION OF GLUTATHIONE S-TRANSFER- ASE (GST) YA FOLLOWING ISCHEMIA-REPERFUSION INJURY fiR) OF THE LIVER N Selim. X Liu. R Whalen. GD Branum and TD Booer. Department of Surgery and Medicine, Emory University Medical School, Atlanta, GA Introduction: 1R injury to the liver is common but how hepatocytes respond to IR is poorly understood. Earlier we reported that IR leads to a selective increase in message of a form of GST that detoxifies products of oxidant stress (Ya). Aim: To determine the lobular distribution of Ya induction using in situ hybridi- zation. Methods: Two lobes of rat livers were made ischemic for 30 min and then reperfused, lschemic (I) and non-ischemic (NIl lobes from animals plus livers from sham (S) animals were harvested at 6, 12 and 24 h. Ya message and protein were quantified by Northern and Western analyses using Ya-specific probes; eRNA probes for Ya were used for in situ hybridization. The lobular distribution of Ya transcripts was analyzed by computer image analysis. Results: In Northerns Ya transcript levels increased >2 fold at 12 and 24 h after IR ~n both I and NI lobes relative to S. Ya protein levels also increased signifi- cantly 24 h after IR in I (1.6 fold) and NI (1.4 fold) lobes. Ya transcripts were localized evenly throughout the lobule in S animals but after IR there was an increase in transcript levels in zone 3 in both I and NI lobes (see figure).

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[ ~ Periportal ~ Midzone l Central

12hrl 12hrNI 12hrS 24hrl 24hrNI 24hrS Summary: l ) Ya is normally expressed uniformly in the lobule. 2) IR causes an increase in Ya message and protein levels in I and NI lobes. 3) IR causes a selec- tive increase in Ya message in zone 3 in I (4-8 fold) and NI (2-4 fold) lobes. Conelnsluns: 1) Selective induction ofGST Ya in the region of the lobule most susceptible to IR may be an important defense mechanism against oxidative stress. 2) Ya increase in NI lobe suggests induction is mediated by a circulating factor.