Large-animal models of fulminant hepatic failure

 For the development of an artificial liver support system, a clinically relevant large-animal model of fulminant hepatic failure (FHF) is indispensable. Although several large-animal models have been reported so far, they have not been entirely satisfactory. Recently, we have developed a new porcine model of FHF by means of intraportal administration of 0.1 mg/kg of α-amanitin and 1 μg/kg of lipopolysaccharide (LPS). This model has the following superior features: 100% mortality within 5 days along with a marked elevation of aspartate transaminase (AST) levels to around 10 000 IU/l, and severe metabolic disorders such as serum lactate accumulation, hypoglycemia, coagulopathy, plasma amino acid imbalance, and hyperammonemia; an onset of hepatic encephalopathy and a significant increase in intracranial pressure immediately before death; a reversal of FHF by orthotopic liver transplantation, proving that the toxicity is liver-specific and that the graft liver is unaffected; and the capability of the damaged liver to recover and achieve both morphological and functional regeneration in 1 week if supported by an efficient auxiliary graft. Because this porcine model satisfies many of the required criteria of an optimal FHF model, it is expected to provide a useful tool for the study of FHF and the development of new therapies. Received: November 14, 2002 / Accepted: January 23, 2003 Present address: Department of Transplantation and Immunology, Faculty of Medicine, Kyoto University, 54 Kawara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan Tel. +81-75-751-4323; Fax +81-75-751-4877 e-mail: takaday@kuhp.kyoto-u.ac.jp Acknowledgments This work was supported in part by a Grant-in-Aid for Scientific Research of the Ministry of Education, Science, Sports and Culture, Japan; a grant from the Japan Society for the Promotion of Science (JSPS-RFTF96I00202); and a Grant-in-Aid for Research on Human Genome, Tissue Engineering, Food Biotechnology, Health Science Research Grants, Ministry of Health, Labor, and Welfare of Japan. Part of this paper was presented at the 40th Congress of the Japanese Society for Artificial Organs. Correspondence to:Y. Takada     

Extracorporeal treatment in fulminant hepatic failure: pathophysiologic considerations

Fulminant hepatic failure is a life-threatening clinical syndrome following severe hepatic injury leading to cerebral edema and brainstem herniation. Excessive mortality can be currently reduced only by timely orthotopic liver transplantation. Due to the shortage of donor organs, a considerable proportion of patients develop irreversible neurological damage, multiorgan failure or death while waiting for transplantation. Consequently, alternatives to orthotopic liver transplantation and methods of stabilizing patients on the waiting list including extracorporeal detoxification treatment are currently investigated. Recent advances in the pathophysiology of cerebral edema have challenged some of the traditional assumptions on which many blood detoxification systems are based. This article aims to integrate pathophysiology of hepatic encephalopathy and cerebral edema into a proposed future concept of liver support.     

High volume plasma exchange in fulminant hepatic failure.

We investigated the effect of repeated high volume plasma exchange with fresh donor plasma in 11 patients with fulminant hepatic failure, all initially in stage 3 or 4 encephalopathy. A daily exchange of a volume equal to the extracellular volume (20% of body weight) on three consecutive days was intended. We obtained an average of 2.6 exchanges each with a mean volume equal to 16% of the body weight. Five patients (46%, 95% confidence limits 17%-77%) survived, all with acetaminophen induced liver failure. Four of the 6 non-survivors showed a temporary improvement in cerebral function. Two of the patients woke up completely. The 6 non-survivors maintained a stable condition with a systolic blood pressure > 110 mm Hg for a mean of 6.9 days after initiating plasma exchange. Plasma exchange may be considered in acute liver failure in patients with residual liver function before transplantation is finally decided. In addition, plasmapheresis may be used to keep patients with definite liver failure clinically stable until a transplant can be performed.     

Early up-regulation of chemokine expression in fulminant hepatic failure

CC-chemokines recruit and activate macrophages and T lymphocytes, the major components of inflammatory infiltrates in fulminant hepatic failure (FHF). To analyse the role of CC-chemokines in the pathogenesis of FHF, this study examined serum levels and intrahepatic expression of MCP-1, MIP-1alpha, MIP-1beta, and RANTES in the livers and sera of patients with FHF and controls by ELISA, immunohistochemistry, and competitive RT-PCR. Serum levels and intrahepatic expression of all chemokines studied in FHF exceeded the levels in chronic liver diseases and normal controls. Distinct patterns of expression of each chemokine were noted on Kupffer cells, sinusoidal endothelial cells, hepatocytes, lymphocytes, and bile ducts. Intrahepatic chemokine expression correlated closely with the extent of infiltration by macrophages and T lymphocytes (r = 0.65-0.95, p < 0.001). The functional relationship between intrahepatic chemokine release and infiltration was confirmed in chemotaxis assays by inhibiting chemotaxis induced by homogenates of liver tissue obtained from FHF patients with neutralizing MCP-1, MIP-1alpha, MIP-1beta, and RANTES antibodies. The time course of CC-chemokine release was studied in the concanavalin A and the galactosamine/LPS mouse models of FHF. In both models, intrahepatic chemokine up-regulation occurred as an early event prior to hepatic infiltration and liver damage. The data indicate that an abundant intrahepatic release of CC-chemokines is an early and pivotal step in the pathogenesis of FHF.     

Effects of serial resin hemoperfusion in fulminant hepatic failure

Resin hemoperfusion using an albumin coated Amberlite XAD-7 column was performed in 19 patients in coma due to fulminant hepatic failure. The procedure was clinically well tolerated, with good blood compatibility, platelet and white cell levels being 97.3 +/- SE 3.2% and 105 +/- 3.8% of the respective initial values after four hours hemoperfusion. No significant changes were observed in beta-thromboglobulin, screen filtration pressure, plasma electrolytes, calcium, protein or albumin. The total plasma bilirubin fell by a mean of 24 mumol/l, with a reduction in 21 of the 25 perfusions studied of up to 104 mumol/l during perfusion. Mean plasma levels of total bile acids were 137 +/- 19 mumol/l and 115 +/- 16 mumol/l respectively before and after four hours hemoperfusion. The amount of bile acids recovered by elution of the resin column was over three times greater than that apparent from the change in plasma levels. Column chromatography on Sephadex G-25 of material eluted from the resin column showed various peaks to be removed, including substances in the middle molecular weight range (1000-5000 daltons). Of the patients treated, eight (42%) survived to leave hospital.     

肝干细胞移植治疗大鼠暴发性肝功能衰竭的实验研究

目的探索肝干细胞移植对暴发性肝功能衰竭大鼠的治疗效果。方法以二乙酰氨基芴(15mg/kgBW)联合2/3肝切除建立雄性大鼠肝干细胞增殖模型,以改进Seglen胶原酶原位灌注及Percoll密度梯度离心方法分离、纯化肝干细胞,经肝脏注射移植(4×106细胞)治疗由D-氨基半乳糖诱导的暴发性肝功能衰竭雌性大鼠,移植前及移植后24、48、72h及1周取血测定肝功能指标如血氨、ALT、TBiL,移植后1、2、4周取受体肝脏组织应用PCR方法进行性别决定因子检测。结果肝干细胞移植可延长暴发性肝功能衰竭大鼠的中位生存时间(P<0.05),移植后肝干细胞移植组大鼠血清ALT、TBiL及血氨水平明显下降,其测定值各时间点组间比较,治疗组明显低于对照组(P<0.01);肝脏病理损伤减轻,肝干细胞移植2周后雌性受体肝脏组织中性别决定因子呈阳性表达,且随时间延长而表达增强。结论肝干细胞移植可延长暴发性肝功能衰竭大鼠中位生存时间,改善肝功能及病理指标。     

重组腺病毒介导的BCL-Xl基因过表达治疗急性肝衰竭大鼠的研究

目的 明确肝细胞凋亡与急性大鼠肝衰竭模型肝组织损伤程度的关系;明确BCL-Xl过表达对急性肝衰竭大鼠肝脏的治疗保护作用.方法 将60只Wistar大鼠随机分为正常对照组、模型组、处理组三组.正常对照组及模型组给予门静脉注射生理盐水,处理组予门静脉注射重组BCL-Xl腺病毒.预处理7 d后模型组及处理组予D-氨基半乳糖+脂多糖建立肝衰竭模型,观察各组大鼠的BCL-Xl蛋白的表达、ALT、AST水平、肝细胞凋亡率及死亡率.结果 BCL-Xl基因在处理组的表达高于在模型组中的表达;建立大鼠肝衰竭后6 h,处理组的血清ALT、AsT水平低于模型组(P<0.05).处理组的肝细胞凋亡率低于模型组(P<0.05).处理组的大鼠死亡率低于模型组(P<0.05).结论 在急性肝衰竭大鼠中,肝细胞的凋亡率与大鼠的死亡率呈正相关;BCL-Xl在肝组织中的过表达能减少急性肝衰竭模型大鼠的肝细胞凋亡率,降低急性肝衰竭大鼠的死亡率.     

重组腺病毒介导的BCL-Xl基因过表达治疗急性肝衰竭大鼠的研究

目的 明确肝细胞凋亡与急性大鼠肝衰竭模型肝组织损伤程度的关系;明确BCL-Xl过表达对急性肝衰竭大鼠肝脏的治疗保护作用.方法 将60只Wistar大鼠随机分为正常对照组、模型组、处理组三组.正常对照组及模型组给予门静脉注射生理盐水,处理组予门静脉注射重组BCL-Xl腺病毒.预处理7 d后模型组及处理组予D-氨基半乳糖+脂多糖建立肝衰竭模型,观察各组大鼠的BCL-Xl蛋白的表达、ALT、AST水平、肝细胞凋亡率及死亡率.结果 BCL-Xl基因在处理组的表达高于在模型组中的表达;建立大鼠肝衰竭后6 h,处理组的血清ALT、AsT水平低于模型组(P<0.05).处理组的肝细胞凋亡率低于模型组(P<0.05).处理组的大鼠死亡率低于模型组(P<0.05).结论 在急性肝衰竭大鼠中,肝细胞的凋亡率与大鼠的死亡率呈正相关;BCL-Xl在肝组织中的过表达能减少急性肝衰竭模型大鼠的肝细胞凋亡率,降低急性肝衰竭大鼠的死亡率.     

重组腺病毒介导的BCL-Xl基因过表达治疗急性肝衰竭大鼠的研究

目的 明确肝细胞凋亡与急性大鼠肝衰竭模型肝组织损伤程度的关系;明确BCL-Xl过表达对急性肝衰竭大鼠肝脏的治疗保护作用.方法 将60只Wistar大鼠随机分为正常对照组、模型组、处理组三组.正常对照组及模型组给予门静脉注射生理盐水,处理组予门静脉注射重组BCL-Xl腺病毒.预处理7 d后模型组及处理组予D-氨基半乳糖+脂多糖建立肝衰竭模型,观察各组大鼠的BCL-Xl蛋白的表达、ALT、AST水平、肝细胞凋亡率及死亡率.结果 BCL-Xl基因在处理组的表达高于在模型组中的表达;建立大鼠肝衰竭后6 h,处理组的血清ALT、AsT水平低于模型组(P<0.05).处理组的肝细胞凋亡率低于模型组(P<0.05).处理组的大鼠死亡率低于模型组(P<0.05).结论 在急性肝衰竭大鼠中,肝细胞的凋亡率与大鼠的死亡率呈正相关;BCL-Xl在肝组织中的过表达能减少急性肝衰竭模型大鼠的肝细胞凋亡率,降低急性肝衰竭大鼠的死亡率.     

An experimental animal model of fulminant hepatic failure in pigs

The objective of this study was to develop an experimental animal model of fulminant hepatic failure to test the efficacy of the bioartificial liver system. The portal vein and the hepatic artery were clamped intermittently and then the hepatic artery was ligated (ligation group, n=5). Pigs whose hepatic arteries were not ligated after clamping were assigned to the non-ligation group (n=5). The biochemical changes in blood, histologic alterations of the liver and neurologic examination for pigs were checked up. All animals died within 17 hr in the ligation group. On the other hand, all animals survived more than 7 days in the non-ligation group. In the ligation group, the levels of ammonia, lactic acid and creatinine showed a progressively increasing pattern. Prothrombin time was also prolonged gradually. Cytoplasmic condensation and nuclear pyknosis of hepatocytes were detected histologically at autopsy. Neurologic findings such as decreased pain sensation, tachypnea and no light reflex of pupils were observed. The findings shown in the ligation group are similar to the clinical features of fulminant hepatic failure in human and this animal model is reproducible. Therefore, this can be a suitable animal model to evaluate the efficacy of the bioartificial liver system for treating fulminant hepatic failure.     

Acute fulminant hepatic failure in a woman treated with phenytoin and trimethoprim-sulfamethoxazole

Massive hepatic necrosis following exposure to phenytoin and trimethoprim-sulfamethoxazole is a rare occurrence and to the best of our knowledge has not been reported previously. Acute hepatic failure following administration of trimethoprim-sulfamethoxazole has rarely been seen, and only 4 cases have been well documented pathologically. We report a case of acute liver failure in a 60-year-old woman following ingestion of phenytoin and trimethoprim-sulfamethoxazole concomitantly over a 9-day period. Autopsy findings revealed acute fulminant hepatic failure. This case demonstrates the effects of chemical-chemical interactions in the potentiation of hepatotoxicity of single agents and specifically illustrates the need for discontinuing trimethoprim-sulfamethoxazole in the presence of early liver injury.     

Improvement by acetylcysteine of hemodynamics and oxygen transport in fulminant hepatic failure

BACKGROUND. When administered early after an overdose of acetaminophen, intravenous acetylcysteine prevents hepatic necrosis by replenishing reduced stores of glutathione. How acetylcysteine improves the survival of patients with established liver damage induced by acetaminophen, however, is unknown. This study was undertaken to determine whether the beneficial effect of acetylcysteine under such circumstances could be due to enhancement of oxygen delivery and consumption. METHODS. We studied the effect of acetylcysteine on systemic hemodynamics and oxygen transport in 12 patients with acetaminophen-induced fulminant hepatic failure and 8 patients with acute liver failure from other causes. The acetylcysteine was given in a dose of 150 mg per kilogram of body weight in 250 ml of 5 percent dextrose over a period of 15 minutes and then in a dose of 50 mg per kilogram in 500 ml of 5 percent dextrose over a period of 4 hours; measurements were made before treatment began and after 30 minutes of the regimen. RESULTS. In the patients with acetaminophen-induced liver failure, the infusion of acetylcysteine resulted in an increase in mean oxygen delivery from 856 to 975 ml per minute per square meter of body-surface area (P = 0.0036), due to an increase in the cardiac index from 5.6 to 6.7 liters per minute per square meter (P = 0.0021). Mean arterial pressure rose from 88 to 95 mm Hg (P = 0.0054) despite a decrease in systemic vascular resistance from 1296 to 1113 dyn.sec.cm-5 per square meter (P = 0.027). There was an increase in oxygen consumption from 127 to 184 ml per minute per square meter (P = 0.0007) associated with an increase in the oxygen-extraction ratio from 16 to 21 percent (P = 0.022). The effects in the patients with acute liver failure from other causes were similar. CONCLUSIONS. The increase in oxygen delivery and consumption in response to acetylcysteine may account for its beneficial effect on survival in patients with fulminant hepatic failure induced by acetaminophen.     

重组腺病毒介导的BCL-X1基因过表达治疗急性肝衰竭大鼠的研究

目的明确肝细胞凋亡与急性大鼠肝衰竭模型肝组织损伤程度的关系;明确BCL—X1过表达对急性肝衰竭大鼠肝脏的治疗保护作用。方法将60只Wistar大鼠随机分为正常对照组、模型组、处理组三组。正常对照组及模型组给予门静脉注射生理盐水,处理组予门静脉注射重组BCL-Xl腺病毒。预处理7d后模型组及处理组予D-氨基半乳糖＋脂多糖建立肝衰竭模型,观察各组大鼠的BCL-X1蛋白的表达、ALT、AST水平、肝细胞凋亡率及死亡率。结果BCL．XI基因在处理组的表达高于在模型组中的表达;建立大鼠肝衰竭后6h,处理组的血清ALT、AST水平低于模型组（P〈0．05）。处理组的肝细胞凋亡率低于模型组（P〈0．05）。处理组的大鼠死亡率低于模型组（P〈0．05）。结论在急性肝衰竭大鼠中,肝细胞的凋亡率与大鼠的死亡率呈正相关;BCL-XI在肝组织中的过表达能减少急性肝衰竭模型大鼠的肝细胞凋亡率,降低急性肝衰竭大鼠的死亡率。     

The role of hepatic venous congestion and endotoxaemia in the production of fulminant hepatic failure secondary to congestive heart failure

The present study was undertaken to clarify the role of congestion of the liver and endotoxemia in the production of fulminant hepatic failure secondary to congestive heart failure. Induction of congestion of the liver, i.e. an elevation of hepatic venous pressure, in rats was accomplished by partial obstruction of the inferior vena cava. A close relationship was demonstrated between venous pressure and serum levels of transaminases as a measure of hepatic dysfunction. Hepatic dysfunction was mild in rats with venous hypertension alone, and only centrilobular congestion was seen on microscopy. In contrast, severe abnormalities of hepatic function were induced by venous hypertension and endotoxaemia. Histologically, the liver revealed bridging centrilobular necrosis as seen in patients with congestive heart failure who develop fulminant hepatic failure. These data suggest that coexistence of endotoxaemia and congestion of the liver may induce fulminant hepatic failure, and that the latter alone is associated only with slight hepatic dysfunction and liver damage.     

Spectrum of renal tubular damage in renal failure secondary to cirrhosis and fulminant hepatic failure.

Measurements of urinary lysozyme were used to evaluate renal tubular integrity in 34 patients with cirrhosis or fulminant hepatic failure who had developed renal impairment. In 18 of the patients the lysozyme values were normal but in the remaining 16 were increased, supporting previous concepts that renal failure complicating hepatocellular disease may occur both without and with tubular necrosis. The lysozyme values were inversely related to the creatinine clearance, suggesting that the development of tubular necrosis may be determined by the level of renal perfusion. The validity of simpler laboratory tests often used to assess renal tubular integrity--namely, the urine sodium concentration, the urine:plasma osmolality ratio, and casts in the urine sediment--was evaluated by comparison with the lysozyme measurements. The urine sodium concentration was of most value and the findings in the sediment were of no value at all.     

A pilot study of polyunsaturated phosphatidyl choline in fulminant and subacute hepatic failure

Present pilot study was conducted to evaluate efficacy and safety of polyunsaturated phosphatidyl choline (PPC) in a phase III clinical trial in patients of fulminant and subacute hepatic failure over one year period in a prospective randomised blinded controlled design. We found that in patients of fulminant hepatic failure, recovery period from encephalopathy was faster and mortality rate lower in the test group of patients who received PPC in a dose of 350 mg thrice daily for 6 to 8 weeks as compared to the control groups who did not receive it. In the patients of subacute hepatic failure, recovery from encephalopathy was faster, mortality rate lower and regression of ascites was significantly higher (P = 0.0022) in test group of patients who received PPC as compared to the control group. However, as the number of patients in the present pilot study is small, we propose that larger clinical trials are warranted in this direction to prove the efficacy and safety of PPC in fulminant and subacute hepatic failure.     

A dog model of fulminant hepatic failure produced by paracetamol administration.

Oral administration of graded doses of paracetamol to dogs produced hepatic necrosis with some similarities to the clinical syndrome seen in man following a paracetamol overdose. Coma, with raised levels of arterial ammonia, was produced and the aspartate aminotransferase levels became markedly elevated in 2 animals who survived more than 24 h. However, the extent of the hepatic necrosis and the time of survival following paracetamol administration were too variable for this model to be of value for the testing of new methods of temporary liver support. When paracetamol was given by intraperitoneal injection many of the animals died of respiratory distress. Significant methaemoglobinaemia was detected, which was associated with a reduction in the arterial partial pressure of oxygen and was partly reversed by the administration of methylene blue.