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The effect of augmenter of liver regeneration (ALR) in enhancing survival in experimental acute hepatic failure in rats treated with D-galactosamine
380A
1093
AASLD ABSTRACTS TNFct-INDUCED APOPTOSIS OF HEPATOCYTES PRECEDES MASSIVE HEPATIC NECROSIS DURING THE COURSE OF ACUTE HEPATIC FAILURE Y Takei. S Okumum*.H Na2ai. A Omae. S Kawano.H Fusamoto. T Kamada. Y Makino*. M Kinoshita*. First Dept. of Medidne, Osaka UniversityMedical School, Suita and *RohtoPharm. Co., Osaka, Japan. We have previouslyreported that activated Kupffer cells induce apoptosis in sinusoidal endothelial cells (EC) and parenehymal cells (PC) in a co-culture system. TNFa was suggested to mediate apoptosis by paracrine or juxtaerine. This study was enndueted to determineif TNFa-indueed apoptosisis involved in the pathophysiologyof acute hepatic fedlure in vivo. ~ LPS(0.5p.g/g) and galactosaraine(GalN: ling/g) was administeredsimuhaneenslyvia the tail vein of LPS-sensitive C3H/HeNmice. Livers were harvestedat 0, 1, 2, 4, 6, 8 and 10 h niter LPS/GalN injection. Apoptosisin liver sections was d ~ with TUNEL method, h some experiments, anti-TNF(z mAb (Genzyme, 30F1) was administeredjust beforeLPS/GalN. Results TUNEL-positivePC appeared at 4h after LPS/GalN. Apoptesis increased with time{Table). However,after 10h, liver histologyrevealed a dramatic transitionto massivenecrosiswith hemorrhage. Table !; Time C O ~ of Apgp~sis in Hepatocytes bouts alter iajection 0 I 2 4 6 8 10 LPS/GaIN -+ ++ +++ (necrosis) LPS/GalN+anti-TNFc¢ Serum ALT started to rise at 8 hr. TNF value increased immediately after LPS/GalN injection,reaching a peak (3370+_275 pg/m) at lh. Anti-TNFa mAb prevented completelyapoptosisand ALT elevation (Table 2). .....Tab!e 2: Time C0urse of S e ~ ALT (U/l, *~P<0.05 vs. LPS/GalN) hours 0 1 2 4 6 8
10 LPS/GalN 22+_2 18_+11 10_+4 22+7 23_+8 516+96 5403+179 LPS/GalN+anti-TNFcc 25+7 42+10 32+6 6-+3 53+11 12+1" 32-+6* Conclusion TNFcz was shown to mediate apoptosis in v~vo in this model. Blockade of TNFcc action completely prevented liver damage. Transition from apoptosis to necrosis occurred, which might reflect disruption of hepatic microeirculation due to apoptosisin EC.
1095
A NOVEL EXPERIMENTAL ANIMAL MODEL OF FULMINANT HEPATIC FAILURE. $ Eguchi. A Kamlot. J Liubimova. L Lebow. AA Demetriou. J Roz~a.. Liver Support Unit Research Laboratory, Department of Surgery, Cedars-Sinai Medical Center, Los Angeles, CA. A small animal model of fulminant hepatin failure (FHF) is needed for in vivo studies and assessing liver support systems. We have developed a
new model of FHF in rats by combining resection of the anterior liver lobes (68% of liver mass) with ligation of the right lobe pedicle resulting in necrosis of 24% of the liver mass. The small omental lobes, 8% of the liver mass, remain viable, Post op., glucose is given (5%, 20 ml, s.c.). S-D rats (n---70, -300g) were used to determine survival, changes in blood chemistry and ability of the liver remnant to regenerate [BrdU, proliferation cell nuclear antigen (PCNA), mitotic index], HGF and c-met expression (RTPCR). Results: All rats were comatose by 24 hrs; most rats (90%) died 2448 hrs (mean: 39+11 hrs) post op. Laboratory parameter changes were similar to those seen clinically (meard:S.D.): Base 6h 12h 18h 24h 30h Gluc.(mg/dl) 115+18 251_+97 99-2-_21 86+14 67+36 58_+22 Lact. (mEq/1) 1.0+0.1 3.0+1.7 4.0"2-0.7 4.4-+0.8 5.9_+3.0 5.25+0.9 NH3 (p.gh) 85_+15 424_+147 653_+167 500-+72 724_+119 483_+0.1 ALT(U/Ixl0O) 0.5_+0.2 23_+12 131_+12 95_+7 110-+17 89_+30 LDH(U/IxlOO) 2_+0.5 74_+39 486+151 256_+103 255:L93 88_+66 T.Bilir.(mg/dl) 0.2+0A 0.6 _+0.3 0.7_+0.4 2.0!--0.7 2.5-+1.0 3.5_+1.2 PT (see.) 12-+2 20-+5 26_+2 27_+4 38+_2 25_+2 Of particular interest was that as little as 8% of the liver mass prevented hypoglycemia. HGF and its receptor c-met mRNA were expressed in the regenerating liver remnant but BrdU uptake was minimal and there was no evidence of cell division at 20 hr, 24 hr and 30 hr post op (low PCNA and mitotic indices). In conclusion, we have developed and characterized a novel model of FHF in rats. It has many of the physiologic and biochemical features seen in FHF as well as a severely impaired regenerative response.
1094
HEPATOLOGY October 1995
THE EFFECT OF AUGMENTER OF LIVER REGENERATION (ALR) IN ENHANCING SURVIVAL IN EXPERIMENTAL ACUTE HEPATIC FAILURE IN RATS TREATED WITH D-GALACTOSAMINE A Francavilla. M Ha~,iva. L Polimeno. A Iacobellis, J Prelich. and T Starzl. Dept. of-Surgery, University of Pittsburgh, Dept. of Gastroenterology, University of Bad, Toyobo, Osaka, Japan Galactosamine induces dose-dependent hepatic injury in rats and many other animals. The toxicity of D-Galactosamine appears to be a consequence of the loss of hepatic UTP. We have previously reported that crude ALR and 50,000 and 300,000 Dalton subfractions of ALR are able to prevent, at least in part, the lethal effects of this substance by stimulating hepatic regeneration. Recently we have completed the process of purification of ALR, obtaining a substance with a well defined molecular weight of 28,000 and molecular structure of 198 amino acids (Hepatology 20:747-757, 1994). We reported the ability of ALR to stimulate hepatocyte proliferation when administrated to 40% PH rats and Eck's fistula dogs. This is liver specific and it has the equal potency of insulin, cyclosporine, and FK 506. This report is the application of ALR in successfully reversing the lethality of the Galactosamine that induces hepatic necrosis in rats. D-Galactosamine (2.6 gm per Kg of body weight) was administrated intraperitoneally to 60 male Lewis strain rats. The animals were divided in 2 groups according to the type of treatment: Group 1 (n=30) saline; Group 2 (n=30) ALR. The animals of the control group received I.M. 250 ul of saline every morning for 5 days. The rats of group 2 received 1 ug of ALR in I.M. 250 ul of saline solution. The rats of both groups received 2 ml of nutritional support (5% glucose + vitamin K, 80ug/day/rat) twice a day. Thepercentage of rats surviving in each group was determined daily for 10 days. The results reported demonstrate that ALR significantly improved the survival in intoxicated rats, showing that this growth factor represents a significant advance for the management of clinical fulminant hepatic failure (% of survival: day 1, G1 100%/G2100%; day 2, G1 98%/G2 98%; day 3, G1 52%/G2 76%; day 4, G1 8%/G2 38%*; day 5, G 17%/G2 36%*; day 10, G1 7%/(32 36%*) (*p= <0.05).
1096 DOES CIPROFLOXACIN ALTER SURVIVAL AND HEPATIC REGENERATION IN THE D-GALACTOSAMINE INDUCED FULMINANT HEPATIC FAILURE (FHF) MODEL? KDE Kaita, T Gauthier and GY Minuk. Liver Diseases Unit, Departments of Medicine and Pharmacology, University of Manitoba, Winnipeg, Manitoba. Recently, we reported that ciprofloxacin, an andmicrobial agent with potent GABA^ receptor antagonist properties increaseshepatic regenerative activity in an animal model of alcohol-induced liver disease. In the present study, we documented the effects of ciprofloxacin on survival and hepatic regeneration in a D-galactosamine (D-gal) induced model of FHF in rats. The trial was divided into two parts; a 4 day survival study and a 60 hr hepatic regeneration study. The survival study consisted of 119 adult, male S-D rats divided into six groups (N= 19-20/group); D-gal (2.5 gag 12.) + saline or D-gal + ciprofloxacin (10 mg/kg,.50 mg/kg or 100 mg/kg), norfloxacin (250 mg/kg) or putrescine (300 umole/kg), a potent hepatic growth promoter. The regeneration study consisted of three additional groups (N= 15/group); low dose D-gal ( ! .0 g/kg I.P.) + saline, ciprofloxacin (100 mg/kg) or putrescine (300 umole/kg). Hepatic regeneration was documented by ~H-thymidine incorporation into hepatic DNA. RESULTS: Survival Study: Sur~lwl
D-liai + lallne
D-la~+ clm~l 0
D'I~J + dpro-50
1120(5%)
2/20 (10%)
$/19 (26%)
D-jill + clpro-I00 7/20(35%)
D-pl + norllox
D-I~I + put
J/20 (I 5%)
5/20 (25%)
*p
Regeneration Study: Compared to the D-gal t- saUne-treated group, DNA synthesis rates were significandy increased in the D-gal + ciprofloxacin and D-gal + putrescine groups ( 11.1 vs 18.1 and 18.5 x IO s dpm/mg DNA respectively, p
1093
AASLD ABSTRACTS TNFct-INDUCED APOPTOSIS OF HEPATOCYTES PRECEDES MASSIVE HEPATIC NECROSIS DURING THE COURSE OF ACUTE HEPATIC FAILURE Y Takei. S Okumum*.H Na2ai. A Omae. S Kawano.H Fusamoto. T Kamada. Y Makino*. M Kinoshita*. First Dept. of Medidne, Osaka UniversityMedical School, Suita and *RohtoPharm. Co., Osaka, Japan. We have previouslyreported that activated Kupffer cells induce apoptosis in sinusoidal endothelial cells (EC) and parenehymal cells (PC) in a co-culture system. TNFa was suggested to mediate apoptosis by paracrine or juxtaerine. This study was enndueted to determineif TNFa-indueed apoptosisis involved in the pathophysiologyof acute hepatic fedlure in vivo. ~ LPS(0.5p.g/g) and galactosaraine(GalN: ling/g) was administeredsimuhaneenslyvia the tail vein of LPS-sensitive C3H/HeNmice. Livers were harvestedat 0, 1, 2, 4, 6, 8 and 10 h niter LPS/GalN injection. Apoptosisin liver sections was d ~ with TUNEL method, h some experiments, anti-TNF(z mAb (Genzyme, 30F1) was administeredjust beforeLPS/GalN. Results TUNEL-positivePC appeared at 4h after LPS/GalN. Apoptesis increased with time{Table). However,after 10h, liver histologyrevealed a dramatic transitionto massivenecrosiswith hemorrhage. Table !; Time C O ~ of Apgp~sis in Hepatocytes bouts alter iajection 0 I 2 4 6 8 10 LPS/GaIN -+ ++ +++ (necrosis) LPS/GalN+anti-TNFc¢ Serum ALT started to rise at 8 hr. TNF value increased immediately after LPS/GalN injection,reaching a peak (3370+_275 pg/m) at lh. Anti-TNFa mAb prevented completelyapoptosisand ALT elevation (Table 2). .....Tab!e 2: Time C0urse of S e ~ ALT (U/l, *~P<0.05 vs. LPS/GalN) hours 0 1 2 4 6 8
10 LPS/GalN 22+_2 18_+11 10_+4 22+7 23_+8 516+96 5403+179 LPS/GalN+anti-TNFcc 25+7 42+10 32+6 6-+3 53+11 12+1" 32-+6* Conclusion TNFcz was shown to mediate apoptosis in v~vo in this model. Blockade of TNFcc action completely prevented liver damage. Transition from apoptosis to necrosis occurred, which might reflect disruption of hepatic microeirculation due to apoptosisin EC.
1095
A NOVEL EXPERIMENTAL ANIMAL MODEL OF FULMINANT HEPATIC FAILURE. $ Eguchi. A Kamlot. J Liubimova. L Lebow. AA Demetriou. J Roz~a.. Liver Support Unit Research Laboratory, Department of Surgery, Cedars-Sinai Medical Center, Los Angeles, CA. A small animal model of fulminant hepatin failure (FHF) is needed for in vivo studies and assessing liver support systems. We have developed a
new model of FHF in rats by combining resection of the anterior liver lobes (68% of liver mass) with ligation of the right lobe pedicle resulting in necrosis of 24% of the liver mass. The small omental lobes, 8% of the liver mass, remain viable, Post op., glucose is given (5%, 20 ml, s.c.). S-D rats (n---70, -300g) were used to determine survival, changes in blood chemistry and ability of the liver remnant to regenerate [BrdU, proliferation cell nuclear antigen (PCNA), mitotic index], HGF and c-met expression (RTPCR). Results: All rats were comatose by 24 hrs; most rats (90%) died 2448 hrs (mean: 39+11 hrs) post op. Laboratory parameter changes were similar to those seen clinically (meard:S.D.): Base 6h 12h 18h 24h 30h Gluc.(mg/dl) 115+18 251_+97 99-2-_21 86+14 67+36 58_+22 Lact. (mEq/1) 1.0+0.1 3.0+1.7 4.0"2-0.7 4.4-+0.8 5.9_+3.0 5.25+0.9 NH3 (p.gh) 85_+15 424_+147 653_+167 500-+72 724_+119 483_+0.1 ALT(U/Ixl0O) 0.5_+0.2 23_+12 131_+12 95_+7 110-+17 89_+30 LDH(U/IxlOO) 2_+0.5 74_+39 486+151 256_+103 255:L93 88_+66 T.Bilir.(mg/dl) 0.2+0A 0.6 _+0.3 0.7_+0.4 2.0!--0.7 2.5-+1.0 3.5_+1.2 PT (see.) 12-+2 20-+5 26_+2 27_+4 38+_2 25_+2 Of particular interest was that as little as 8% of the liver mass prevented hypoglycemia. HGF and its receptor c-met mRNA were expressed in the regenerating liver remnant but BrdU uptake was minimal and there was no evidence of cell division at 20 hr, 24 hr and 30 hr post op (low PCNA and mitotic indices). In conclusion, we have developed and characterized a novel model of FHF in rats. It has many of the physiologic and biochemical features seen in FHF as well as a severely impaired regenerative response.
1094
HEPATOLOGY October 1995
THE EFFECT OF AUGMENTER OF LIVER REGENERATION (ALR) IN ENHANCING SURVIVAL IN EXPERIMENTAL ACUTE HEPATIC FAILURE IN RATS TREATED WITH D-GALACTOSAMINE A Francavilla. M Ha~,iva. L Polimeno. A Iacobellis, J Prelich. and T Starzl. Dept. of-Surgery, University of Pittsburgh, Dept. of Gastroenterology, University of Bad, Toyobo, Osaka, Japan Galactosamine induces dose-dependent hepatic injury in rats and many other animals. The toxicity of D-Galactosamine appears to be a consequence of the loss of hepatic UTP. We have previously reported that crude ALR and 50,000 and 300,000 Dalton subfractions of ALR are able to prevent, at least in part, the lethal effects of this substance by stimulating hepatic regeneration. Recently we have completed the process of purification of ALR, obtaining a substance with a well defined molecular weight of 28,000 and molecular structure of 198 amino acids (Hepatology 20:747-757, 1994). We reported the ability of ALR to stimulate hepatocyte proliferation when administrated to 40% PH rats and Eck's fistula dogs. This is liver specific and it has the equal potency of insulin, cyclosporine, and FK 506. This report is the application of ALR in successfully reversing the lethality of the Galactosamine that induces hepatic necrosis in rats. D-Galactosamine (2.6 gm per Kg of body weight) was administrated intraperitoneally to 60 male Lewis strain rats. The animals were divided in 2 groups according to the type of treatment: Group 1 (n=30) saline; Group 2 (n=30) ALR. The animals of the control group received I.M. 250 ul of saline every morning for 5 days. The rats of group 2 received 1 ug of ALR in I.M. 250 ul of saline solution. The rats of both groups received 2 ml of nutritional support (5% glucose + vitamin K, 80ug/day/rat) twice a day. Thepercentage of rats surviving in each group was determined daily for 10 days. The results reported demonstrate that ALR significantly improved the survival in intoxicated rats, showing that this growth factor represents a significant advance for the management of clinical fulminant hepatic failure (% of survival: day 1, G1 100%/G2100%; day 2, G1 98%/G2 98%; day 3, G1 52%/G2 76%; day 4, G1 8%/G2 38%*; day 5, G 17%/G2 36%*; day 10, G1 7%/(32 36%*) (*p= <0.05).
1096 DOES CIPROFLOXACIN ALTER SURVIVAL AND HEPATIC REGENERATION IN THE D-GALACTOSAMINE INDUCED FULMINANT HEPATIC FAILURE (FHF) MODEL? KDE Kaita, T Gauthier and GY Minuk. Liver Diseases Unit, Departments of Medicine and Pharmacology, University of Manitoba, Winnipeg, Manitoba. Recently, we reported that ciprofloxacin, an andmicrobial agent with potent GABA^ receptor antagonist properties increaseshepatic regenerative activity in an animal model of alcohol-induced liver disease. In the present study, we documented the effects of ciprofloxacin on survival and hepatic regeneration in a D-galactosamine (D-gal) induced model of FHF in rats. The trial was divided into two parts; a 4 day survival study and a 60 hr hepatic regeneration study. The survival study consisted of 119 adult, male S-D rats divided into six groups (N= 19-20/group); D-gal (2.5 gag 12.) + saline or D-gal + ciprofloxacin (10 mg/kg,.50 mg/kg or 100 mg/kg), norfloxacin (250 mg/kg) or putrescine (300 umole/kg), a potent hepatic growth promoter. The regeneration study consisted of three additional groups (N= 15/group); low dose D-gal ( ! .0 g/kg I.P.) + saline, ciprofloxacin (100 mg/kg) or putrescine (300 umole/kg). Hepatic regeneration was documented by ~H-thymidine incorporation into hepatic DNA. RESULTS: Survival Study: Sur~lwl
D-liai + lallne
D-la~+ clm~l 0
D'I~J + dpro-50
1120(5%)
2/20 (10%)
$/19 (26%)
D-jill + clpro-I00 7/20(35%)
D-pl + norllox
D-I~I + put
J/20 (I 5%)
5/20 (25%)
*p
Regeneration Study: Compared to the D-gal t- saUne-treated group, DNA synthesis rates were significandy increased in the D-gal + ciprofloxacin and D-gal + putrescine groups ( 11.1 vs 18.1 and 18.5 x IO s dpm/mg DNA respectively, p