Reevaluation of Experimental Model of Hepatic Fibrosis Induced by

Hepatotoxic Drugs: An Easy, Applicable, and Reproducible Model

J.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 for
research on liver reperfusion injury. We compared the characteristics of several hepatic
cirrhosis models in terms of the degree of fibrosis, reproducibility, histologic characteristics, and success rate to achieve sufficient fibrosis. In mice & rats, we administered three
different hepatotoxic drugs (thioacetamide, dimethylnitrosamine, and carbon tetrachloride
[CCl4]) through two different routes (oral feeding and intraperitoneal injection). The
animals fed thioacetamide exhibited little fibrosis; rather, more inflammatory cells
infiltrated into periportal areas with bile duct proliferation. The livers from hosts
administered dimethylnitrosamine showed greater early injury and severe inflammatory
reactions in the peritoneal cavity. The liver showed a marked degree of piecemeal necrosis
with 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 well
with the duration of induction. Livers from hosts administered CCl4 orally twice a week for
10 weeks was the most effective to achieve sufficient fibrosis and greatest reproducibility
with acceptable animal survival.

IVER CIRRHOSIS is a leading cause of death in

patients with chronic liver disease. It causes serious
complications including portal hypertension, variceal
bleeding, intractable ascites, and hepatic encephalopathy, ultimately resulting in hepatic failure.1 Hepatic
fibrosis is a basic step in the progression to cirrhosis. The
animal model of hepatic fibrosis is important for experimental research to apply to clinical uses for reperfusion
injury or antifibrosis. The indispensable factors of a
suitable animal model include easy applicability, adequate fibrosis, limited duration of induction, reproducibility, and minimal hazard to personnel. Several animal
models have been introduced to produce hepatic fibrosis
induced by hepatotoxic agents such as diet, drugs, alcohol, bile duct ligation, and immunologic activation.2 4
Hepatic cirrhosis induced by toxic drugs such as thioacetamide (TAA), carbon tetrachloride (CCl4), and dimethylnitrosamine (DMN), and bile duct ligation are the
most popular experimental models.2,57 We examined
experimental models of hepatic fibrosis induced by hepatotoxic drugs. Our preliminary trials of each model did
not show good reproducibility in the degree of fibrosis or

survival. Therefore, we compared the characteristics of

several popular models in terms of histologic characteristics, degree of fibrosis, reproducibility, and success rate
of achieving sufficient fibrosis. In addition, we collected
the data for survival to reach adequate hepatic fibrosis.

From the Department of Surgery (J.H.J., K.J.K., Y.H.K.), Pathology (Y.N.K.), School of Medicine and Institute for Medical
Science, and Center for Traditional Microorganism Resources
(I.S.L.), Keimyung University, Daegu, Korea.
The present research has been conducted by the Bisa Research Grant of Keimyung University in 2005 (K.J.K.) and by a
grant from the Center of Traditional Microorganism Resources
(I.S.L.).
Address reprint requests to Koo Jeong Kang, MD, Division of
Hepatobiliary and Pancreatic Surgery, Department of Surgery,
Keimyung University Dong-San Medical Center, 194 Dongsan
Dong, Jung Gu, Daegu 700-712, Korea. E-mail: kjkang@dsmc.
or.kr

0041-1345/08/$see front matter

doi:10.1016/j.transproceed.2008.07.040

2008 by Elsevier Inc. All rights reserved.

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Transplantation Proceedings, 40, 2700 2703 (2008)

DRUG-INDUCED MODEL OF HEPATIC FIBROSIS

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Table 1. Protocols, Including Animals, Drugs, Route of Administration, and Duration for Induction of Hepatic Fibrosis
Drugs

Thioacet-amide (TAA)
Dimethyl-nitrosamine (DMN)
Carbon tetrachloride (CCl4)

Animal

Mouse
Rat
Mouse
Rat
Mouse
Rat

Concentration

0.3, 0.6% in d/w

0.3, 0.6% in d/w
10 mg/kg
10 mg/kg
2 mL/kg (50% sol.)
4 mL/kg (50% sol.)
1 mL/kg (10,30,50,502%)

Route (PO or IP)

Duration (weeks)

PO (2 times/week)

12
12
12
12
12
12
12

IP (3 times/week)
PO
PO
IP

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

MATERIALS AND METHODS

Experimental Animal Model of Hepatic Fibrosis
Eight-week-old male C57BL/6 mice (20 25 g) and SpragueDawley rats (150 200 g) were fed a laboratory diet with water as
well as various hepatotoxic agents before excision of the liver
tissue. The animals were kept under constant environmental
conditions with 12-hour light-dark cycles. All animals received
humane care, and all procedures were compliant with our
Animal Care Committee guidelines. We administered three
different hepatotoxic drugs for development of experimental
fibrosis using two different routes: oral feeding and intraperitoneal injection (Table 1). First, the mice and rats were fed a 0.3%
or 0.6% solution of TAA in distilled water in their water bottles.
The animals were permitted to drink only TAA in a water
solution during the entire experimental period (12 weeks).
Second, DMN (10 mg/kg) was injected into the peritoneal cavity
of the mice and rats three times a week at 2- to 3-day intervals.
Third, CCl4 diluted in corn oil was administered to mice and rats
through two routes. The amount of oral feeding was 2 mL/kg
(50% solution) in mice and 4 mL/kg (50% solution) in rats for 12
weeks. Intraperitoneal injection of CCl4 (1 mL/kg) was administered only in rats, at three concentrations (10%, 30%, and 50%
per week) and, in addition, a 50% solution twice a week, for 12
weeks.

Evaluation of Hepatic Fibrosis

To evaluate the degree of hepatic fibrosis, we performed hematoxylin-eosin staining to score the Histologic Activity Index (HAI)8
and trichrome staining to examine the degree of fibrosis at 6, 8, 10,
and 12 weeks after induction of the hepatic injury. We compared
the HAI score and the degree of fibrosis according to various
induction protocols (Fig 1). In addition, we evaluated the survival
rate of the animals.

RESULTS

The livers of mice & rats fed TAA exhibited little fibrosis;
rather, more inflammatory cells infiltrated to the periportal areas and there was bile duct proliferation. The
HAI scores were greater than 12 at six weeks after
administration of TAA. More than 80% of animals fed
TAA survived 10 weeks. The livers from hosts administered DMN demonstrated greater injury in the early
period and severe inflammatory reactions in the peritoneal cavity. The livers showed marked degrees of piecemeal necrosis with a limited degree of fibrosis. It takes
more than 10 weeks to achieve an adequate HAI score.
All of the mice and rats administered DMN were dead

within 5 weeks. Mice and rats administered CCl4 fairly

well tolerated the oral feeding or intraperitoneal injection of CCl4 to 12 weeks. The animals administered 1
mL/kg of CCl4 intraperitoneally in three different concentrations showed acceptable HAI scores at 8 weeks.
The degree of fibrosis at 10 weeks showed marked septal
fibrosis; the severity of fibrosis correlated with the duration and the total dosage of drug. 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 with the duration of the induction
period; the liver showed periportal fibrosis at 8 to 10
weeks and septal fibrosis at 12 to 14 weeks (Fig 2). When
we counted the HAI score, the degree of fibrosis correlated with the amount and duration of feeding. The livers
from hosts administered CCl4 intraperitoneally showed a
good degree of fibrosis after 10 weeks; however, less than
50% of the animals survived for 10 weeks. The survival
rate in animals administered the three toxic drugs
showed different degrees of liver fibrogenesis. The mice
and rats treated with TAA had greater than 80% survival.
All of the mice and rats administered DMN were dead
within 5 weeks. Eighty percent of mice given 10% CCl4
solution (2 ml/kg) intraperitoneally survived. However,
less than 60% of the mice given CCl4 intraperitoneally
with 30% or 50% solution survived. The mice administered 50% CCl4 solution orally showed sufficient fibrosis
at 10 weeks, and more than 90% of the mice survived.
DISCUSSION

Our goal in this study was to find an easy reproducible

model for experimental studies of hepatic fibrogenesis.
We compared popular models from several institutions.3,4,7 Our hepatic fibrosis results did not reproduce
well the previously reported results in terms of degree of
fibrosis, applicability, reproducibility, and survival. There
was little difference in the degree of fibrosis or HAI score
after the same induction period among the hepatotoxic
agents in experimental models described by a single
researcher. For feeding agents, the mouse is easier to
handle than the rat. For a good experimental model, the
animals must survive until a designated time to obtain
adequate hepatic fibrosis. In our study, oral feeding of
TAA mixed in the drinking water was easy, without the
nuisance of feeding or injecting drugs every week. The

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JANG, KANG, KIM, ET AL

Fig 1. AG, At 10 weeks, carbon tetrachloride (CCl4; 1 mg/kg) at various concentrations was administered intraperitoneally to rat
livers. Trichrome-stained livers exhibited moderate to severe fibrosis. H, Histologic Activity Index scores at 6, 8, and 10 weeks. sol.,
Solution.

livers taken at 10 weeks showed an acceptable degree of

fibrosis and HAI score but a more toxic inflammatory
reaction and bile duct proliferation. Intraperitoneal injection of DMN yielded a high HAI score within a short

time; however, it was too toxic to achieve an adequate

degree of hepatic fibrosis before the animals died. Administration of CCl4, orally or by intraperitoneal injection was a good model in terms of reproducibility of

DRUG-INDUCED MODEL OF HEPATIC FIBROSIS

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depends on the concentration of the hepatotoxic agent and

weekly feeding, 10 weeks was adequate for hepatic fibrogenesis in this experimental model.
REFERENCES

Fig 2. Survival rates in animals administered various hepatotoxic agents for 10 weeks to achieve adequate hepatic fibrosis
based on the Histologic Activity Index score. In animals given
thioacetamide (TAA) orally (PO), the drug was well tolerated,
whereas in animals given carbon tetrachloride (CCl4) or dimethylnitrosamine (DMN) by intraperitoneal injection (IP), the drugs
were less well tolerated.

fibrogenesis and animal survival; however, feeding or

injection once or twice a week was tedious. Administration of CCl4 or DMN by intraperitoneal injection was less
well tolerated than oral feeding. To obtain an adequate
degree of fibrosis earlier required greater amounts of
agents per body weight. However, the animals died
earlier, before reaching an adequate degree of fibrosis.
In conclusion, livers from hosts administered CCl4 orally
twice a week for 10 weeks showed the greatest incidence
and highest reproducibility of fibrosis, with acceptable
animal survival rates. Although the duration of induction

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