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eevaluation of Experimental Model of Hepatic Fibrosis Induced byepatotoxic Drugs: An Easy, Applicable, and Reproducible Model

.H. Jang, K.J. Kang, Y.H. Kim, Y.N. Kang, and I.S. Lee

ABSTRACT

Establishing an easy and reproducible model for hepatic fibrosis is absolutely necessary forresearch on liver reperfusion injury. We compared the characteristics of several hepaticcirrhosis models in terms of the degree of fibrosis, reproducibility, histologic characteris-tics, and success rate to achieve sufficient fibrosis. In mice & rats, we administered threedifferent hepatotoxic drugs (thioacetamide, dimethylnitrosamine, and carbon tetrachloride[CCl4]) through two different routes (oral feeding and intraperitoneal injection). Theanimals fed thioacetamide exhibited little fibrosis; rather, more inflammatory cellsinfiltrated into periportal areas with bile duct proliferation. The livers from hostsadministered dimethylnitrosamine showed greater early injury and severe inflammatoryreactions in the peritoneal cavity. The liver showed a marked degree of piecemeal necrosiswith limited fibrosis. The mice administered a 50% solution of CCl4 (2 mL/kg orally)tolerated the entire induction period of 12 weeks. The degree of fibrosis correlated wellwith the duration of induction. Livers from hosts administered CCl4 orally twice a week for10 weeks was the most effective to achieve sufficient fibrosis and greatest reproducibility

with acceptable animal survival.

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IVER CIRRHOSIS i s a leading cause of death inpatients with chronic liver disease. It causes serious

omplications including portal hypertension, varicealleeding, intractable ascites, and hepatic encephalopa-hy, ultimately resulting in hepatic failure.1 Hepaticbrosis is a basic step in the progression to cirrhosis. Thenimal model of hepatic fibrosis is important for exper-mental research to apply to clinical uses for reperfusionnjury or antifibrosis. The indispensable factors of auitable animal model include easy applicability, ade-uate fibrosis, limited duration of induction, reproduc-

bility, and minimal hazard to personnel. Several animalodels have been introduced to produce hepatic fibrosis

nduced by hepatotoxic agents such as diet, drugs, alco-ol, bile duct ligation, and immunologic activation.2– 4

epatic cirrhosis induced by toxic drugs such as thioac-tamide (TAA), carbon tetrachloride (CCl4 ), and dim-thylnitrosamine (DMN), and bile duct ligation are theost popular experimental models.2,5–7 We examined

xperimental models of hepatic fibrosis induced by hep-totoxic drugs. Our preliminary trials of each model did

ot show good reproducibility in the degree of fibrosis or o

041-1345/08/$–see front matteroi:10.1016/j.transproceed.2008.07.040

700

urvival. Therefore, we compared the characteristics ofeveral popular models in terms of histologic character-stics, degree of fibrosis, reproducibility, and success ratef achieving sufficient fibrosis. In addition, we collectedhe data for survival to reach adequate hepatic fibrosis.

From the Department of Surgery (J.H.J., K.J.K., Y.H.K.), Pa-hology (Y.N.K.), School of Medicine and Institute for Medicalcience, and Center for Traditional Microorganism Resources

I.S.L.), Keimyung University, Daegu, Korea.The present research has been conducted by the Bisa Re-

earch Grant of Keimyung University in 2005 (K.J.K.) and by arant from the Center of Traditional Microorganism Resources

I.S.L.).Address reprint requests to Koo Jeong Kang, MD, Division ofepatobiliary and Pancreatic Surgery, Department of Surgery,eimyung University Dong-San Medical Center, 194 Dongsanong, Jung Gu, Daegu 700-712, Korea. E-mail: kjkang@dsmc.

r.kr

© 2008 by Elsevier Inc. All rights reserved.360 Park Avenue South, New York, NY 10010-1710

Transplantation Proceedings, 40, 2700–2703 (2008)

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DRUG-INDUCED MODEL OF HEPATIC FIBROSIS 2701

ATERIALS AND METHODSxperimental Animal Model of Hepatic Fibrosis

ight-week-old male C57BL/6 mice (20 –25 g) and Sprague-awley rats (150 –200 g) were fed a laboratory diet with water asell as various hepatotoxic agents before excision of the liver

issue. The animals were kept under constant environmentalonditions with 12-hour light-dark cycles. All animals receivedumane care, and all procedures were compliant with ournimal Care Committee guidelines. We administered threeifferent hepatotoxic drugs for development of experimentalbrosis using two different routes: oral feeding and intraperito-eal injection (Table 1). First, the mice and rats were fed a 0.3%r 0.6% solution of TAA in distilled water in their water bottles.he animals were permitted to drink only TAA in a water

olution during the entire experimental period (12 weeks).econd, DMN (10 mg/kg) was injected into the peritoneal cavityf the mice and rats three times a week at 2- to 3-day intervals.hird, CCl4 diluted in corn oil was administered to mice and rats

hrough two routes. The amount of oral feeding was 2 mL/kg50% solution) in mice and 4 mL/kg (50% solution) in rats for 12eeks. Intraperitoneal injection of CCl4 (1 mL/kg) was admin-

stered only in rats, at three concentrations (10%, 30%, and 50%er week) and, in addition, a 50% solution twice a week, for 12eeks.

valuation of Hepatic Fibrosis

o evaluate the degree of hepatic fibrosis, we performed hematox-lin-eosin staining to score the Histologic Activity Index (HAI)8

nd trichrome staining to examine the degree of fibrosis at 6, 8, 10,nd 12 weeks after induction of the hepatic injury. We comparedhe HAI score and the degree of fibrosis according to variousnduction protocols (Fig 1). In addition, we evaluated the survivalate of the animals.

ESULTS

he livers of mice & rats fed TAA exhibited little fibrosis;ather, more inflammatory cells infiltrated to the peri-ortal areas and there was bile duct proliferation. TheAI scores were greater than 12 at six weeks after

dministration of TAA. More than 80% of animals fedAA survived 10 weeks. The livers from hosts adminis-

ered DMN demonstrated greater injury in the earlyeriod and severe inflammatory reactions in the perito-eal cavity. The livers showed marked degrees of piece-eal necrosis with a limited degree of fibrosis. It takesore than 10 weeks to achieve an adequate HAI score.

Table 1. Protocols, Including Animals, Drugs, Route of A

Drugs Animal Co

hioacet-amide (TAA) Mouse 0.3, 0.6% iRat 0.3, 0.6% i

imethyl-nitrosamine (DMN) Mouse 10 mg/kgRat 10 mg/kg

arbon tetrachloride (CCl4) Mouse 2 mL/kg (5Rat 4 mL/kg (5

1 mL/kg (1

Abbreviations: d/w, distilled water; IP, intraperitoneally; PO, orally.

ll of the mice and rats administered DMN were dead n

ithin 5 weeks. Mice and rats administered CCl4 fairlyell tolerated the oral feeding or intraperitoneal injec-

ion of CCl4 to 12 weeks. The animals administered 1L/kg of CCl4 intraperitoneally in three different con-

entrations showed acceptable HAI scores at 8 weeks.he degree of fibrosis at 10 weeks showed marked septalbrosis; the severity of fibrosis correlated with the dura-ion and the total dosage of drug. The mice administered

50% solution of CCl4 (2 mL/kg orally) tolerated thentire induction period of 12 weeks. The degree ofbrosis correlated with the duration of the inductioneriod; the liver showed periportal fibrosis at 8 to 10eeks and septal fibrosis at 12 to 14 weeks (Fig 2). Whene counted the HAI score, the degree of fibrosis corre-

ated with the amount and duration of feeding. The liversrom hosts administered CCl4 intraperitoneally showed aood degree of fibrosis after 10 weeks; however, less than0% of the animals survived for 10 weeks. The survivalate in animals administered the three toxic drugshowed different degrees of liver fibrogenesis. The micend rats treated with TAA had greater than 80% survival.ll of the mice and rats administered DMN were deadithin 5 weeks. Eighty percent of mice given 10% CCl4

olution (2 ml/kg) intraperitoneally survived. However,ess than 60% of the mice given CCl4 intraperitoneallyith 30% or 50% solution survived. The mice adminis-

ered 50% CCl4 solution orally showed sufficient fibrosist 10 weeks, and more than 90% of the mice survived.

ISCUSSION

ur goal in this study was to find an easy reproducibleodel for experimental studies of hepatic fibrogenesis.e compared popular models from several institu-

ions.3,4,7 Our hepatic fibrosis results did not reproduceell the previously reported results in terms of degree ofbrosis, applicability, reproducibility, and survival. Thereas little difference in the degree of fibrosis or HAI scorefter the same induction period among the hepatotoxicgents in experimental models described by a singleesearcher. For feeding agents, the mouse is easier toandle than the rat. For a good experimental model, thenimals must survive until a designated time to obtaindequate hepatic fibrosis. In our study, oral feeding ofAA mixed in the drinking water was easy, without the

istration, and Duration for Induction of Hepatic Fibrosis

ation Route (PO or IP) Duration (weeks)

PO (2 times/week) 1212

IP (3 times/week) 1212

ol.) PO 12ol.) PO 120,50�2%) IP 12

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2702 JANG, KANG, KIM, ET AL

ivers taken at 10 weeks showed an acceptable degree ofbrosis and HAI score but a more toxic inflammatoryeaction and bile duct proliferation. Intraperitoneal in-

ig 1. A–G, At 10 weeks, carbon tetrachloride (CCl4; 1 mg/kgivers. Trichrome-stained livers exhibited moderate to severe fibolution.

ection of DMN yielded a high HAI score within a short t

ime; however, it was too toxic to achieve an adequateegree of hepatic fibrosis before the animals died. Ad-inistration of CCl , orally or by intraperitoneal injec-

arious concentrations was administered intraperitoneally to ratH, Histologic Activity Index scores at 6, 8, and 10 weeks. sol.,

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DRUG-INDUCED MODEL OF HEPATIC FIBROSIS 2703

brogenesis and animal survival; however, feeding ornjection once or twice a week was tedious. Administra-ion of CCl4 or DMN by intraperitoneal injection was lessell tolerated than oral feeding. To obtain an adequateegree of fibrosis earlier required greater amounts ofgents per body weight. However, the animals diedarlier, before reaching an adequate degree of fibrosis.In conclusion, livers from hosts administered CCl4 orally

wice a week for 10 weeks showed the greatest incidencend highest reproducibility of fibrosis, with acceptable

ig 2. Survival rates in animals administered various hepato-oxic agents for 10 weeks to achieve adequate hepatic fibrosisased on the Histologic Activity Index score. In animals given

hioacetamide (TAA) orally (PO), the drug was well tolerated,hereas in animals given carbon tetrachloride (CCl4) or dimeth-lnitrosamine (DMN) by intraperitoneal injection (IP), the drugsere less well tolerated.

nimal survival rates. Although the duration of induction 1

epends on the concentration of the hepatotoxic agent andeekly feeding, 10 weeks was adequate for hepatic fibro-enesis in this experimental model.

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