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Abstract

Recently, the need for more standardized operation procedures in experimental liver fibrosis research was suggested due to dramatic changes in European animal welfare rules. Here, we present a short series of standard operation procedures (SOPs) summarizing the most relevant and widely accepted experimental models for the induction of liver injury leading to liver fibrosis. The described procedures are based on the long-term experience of the Collaborative Research Centre ‘Organ Fibrosis: From Mechanisms of Injury to Modulation of Disease’ (http://www.sfbtrr57.rwth-aachen.de/), which is supported by the German Research Foundation (SFB/TRR57). These SOPs will help to improve standardization of fibrosis models and to increase the comparability of data between different laboratories with the aim of reducing animal experimentation according to the principle that was proposed in 1959 by Russell and Burch as an ethical framework for conducting scientific experiments with animals, namely the replacement, refinement and reduction (3R) principle. In the first section we focus on the carbon tetrachloride (CCl₄) model in mice, which is the toxic model of liver fibrosis induction most commonly used worldwide.

Keywords

hepatotoxin cytochrome P450 liver damage fibrosis cirrhosis

Historic background of the model

Experimental liver fibrosis in mice can be induced by surgical intervention (e.g. bile duct ligation), genetic manipulation of fibrosis-related genes (e.g. Mdr2 knockout mice) or application of hepatotoxins.¹ In particular, the single or repeated administration of carbon tetrachloride (CCl₄) has become one of the most commonly used experimental models for inducing toxin-mediated liver fibrosis. However, almost all laboratory use protocols, which might vary with respect to usage of anaesthesia, hepatotoxin preparation, doses, treatment duration, administration routes, or the many other factors that may affect the outcome of experimentation. In the European Union, the laws on the protection of animals used for scientific purposes have recently changed dramatically by the implementation of the new EU Directive 2010/63 that contains 66 restrictive articles.¹ These require that, wherever possible, alternative scientific methods that do not need the use of live animals, should be applied in experimental research. The immediate implementation of the new EU Directive should foster the 3R (replacement, refinement, and reduction) principle that was proposed by William M S Russell and Rex L Burch in 1959 for the more ethical use of animals in testing.² To achieve this challenging objective, it is obvious that researchers in the field that use particular models must ensure that they work according to strict general guidelines and accepted standard operating procedures (SOPs). Therefore, we present here a detailed protocol that induces hepatic fibrosis in a highly reproducible manner. In addition, we discuss the historical background to that model and the pathobiochemical mechanisms that contribute to the formation of hepatic insult.

CCl₄ is widely used as a solvent for dissolving non-polar compounds, such as fats and oils. The acute toxicity of CCl₄ is well established from many animal studies. In particular, studies performed on rats have shown that the lethal dose (LD)₅₀ after acute oral uptake is in the range of 4.7–14.7 mL/kg body weight, depending on nutritional conditions and supplements administered.^3,4 Single oral doses of CCl₄ in corn oil induce increased liver weight, elevated levels of fat, serum urea, liver enzyme activities, and clear histopathological evidence of liver damage with single cell necrosis;⁵ whereas the long-term oral exposure to CCl₄ causes marked hepatotoxicity with resulting fibrosis, bile duct proliferation, cirrhosis and even hepatocellular carcinoma (HCC).⁶ Toxicity of CCl₄ has three or four distinct phases.⁷ The first two or three weeks are mainly characterized by necrosis indicated by rising activities of liver specific enzymes and decreasing values of pseudocholinesterase. During the next two or three weeks massive hepatic fat accumulation occurs and serum levels of triglycerides and aspartate aminotransferase (AST) are significantly increased, while hepatic function is reduced. During the third phase, the increase of AST continues, elevated levels of hydroxyproline and triglycerides are found, and overall liver function further decreases. In the final phase, the values of pseudocholinesterase further decrease, and atrophy of the liver is observed.⁷ This may be combined with a significant decrease of serum albumin and weight loss which indicates progressive loss of hepatic function during prolonged fibrogenesis.

Pathogenic mechanisms of liver damage

CCl₄ is metabolized in the liver by the cytochrome P450 superfamily of monooxygenases (CYP family) to the trichloromethyl radical ( ${CCl}_{3}^{*}$ ). Subsequently, this radical reacts with nucleic acids, proteins, and lipids, thereby impairing key cellular processes resulting in altered lipid metabolism (fatty degeneration and steatosis) and lowered protein quantities (Figure 1). Adduct formation between ${CCl}_{3}^{*}$ and DNA further triggers mutations and the formation of HCC. The formation of trichloromethylperoxy radicals (CCl₃OO*) resulting from oxygenation of ${CCl}_{3}^{*}$ further initiates lipid peroxidation and the destruction of polyunsaturated fatty acids. Consequently, the membrane permeability in all cellular compartments (mitochondria, endoplasmic reticulum, and plasma membrane) is lowered and generalized hepatic damage occurs that is characterized by inflammation, fibrosis, cirrhosis and HCC. A comprehensive summary of the pathogenetic events that occur in the liver during CCl₄-induced damage is given elsewhere.⁸ Besides its strong hepatotoxic effects, minor acute systemic toxicity of CCl₄ has been described decades ago, especially for the peritoneum, mucosa, respiratory tract and central nervous system.⁹

Figure 1.

Pathobiochemical sequence of events during carbon tetrachloride (CCl₄)-induced liver damage. In the liver, CCl₄ is metabolized by cytochrome P450 (CYP) enzymes to a trichloromethyl radical that can be further oxygenated to the trichloromethylperoxy radical. Both radicals are highly reactive and induce complex cellular alterations that result in hepatotoxic damage, inflammation, fibrosis, cirrhosis and hepatocellular carcinoma (HCC).

Experimental procedure

General considerations

Genetic background

It is well accepted that the susceptibility towards CCl₄ in mice is strongly strain-dependent (Table 1). In particular, BALB/c inbred mice are most sensitive to fibrosis induction, whereas FVB/N mice respond poorly to CCl₄. Although C57BL/6 inbred mice develop only intermediate liver fibrosis, this strain is frequently used for fibrosis studies in the CCl₄ model because of the ready availability of genetically-modified mice.

Table 1.

Susceptibility to carbon tetrachloride (CCl₄)-mediated liver fibrosis in different inbreed mouse strains.

Strain	Application route	Susceptibility	References
BALB/c	IP, SC, PO	Susceptible	26 –29
C57BL/6	IP	Intermediate	27,29
DBA/2	IP	Intermediate	27,29
FVB/N	IP	Resistant	27
A	IP	Resistant	27
C3H/He	IP	Resistant, high mortality (>60%)	27
AKR	IP	Resistant, high mortality (>60%)	27

IP: intraperitoneal; SC: subcutaneous; PO: per os (via gavage).

Duration and intervals of treatment

Different responses to CCl₄ due to genetic reasons and severity (strength) of fibrogenesis can basically be modulated by application intervals and duration of treatment. In our experiments, intraperitoneal (IP) application of CCl₄ in C57BL/6 inbred mice twice a week for six weeks, or alternatively application three times a week for four weeks, results in fulminant deposition of intrahepatic collagen matrix and fibrosis resembling human stage 3 according to Desmet-Scheuer scoring.¹⁰ In highly susceptible strains, four weeks of CCl₄ application is usually sufficient for fibrosis induction, while in fibrosis-resistant strains the poor susceptibility can in part be compensated for by prolonged treatment of up to 12 weeks.

Application routes

In principle, CCl₄ can be administered through IP injection, inhalation and gavage. The majority of investigators prefer IP application in mice for reasons of excellent reproducibility, good survival rates,¹¹ ease of performance, and safety. However, for specific aims (such as the induction of portal hypertension) or when the CCl₄ intoxication is established within the laboratory in rats, application via inhalation might be more appropriate, but requires a specific extraction hood in the laboratory and expertise (Figure 2). Only a few authors prefer administration of CCl₄ via gavage; however, in our opinion, this application route is not to be recommended due to frequent early mortality.¹²

Figure 2.

Inhalation exposure apparatus. Before exposing mice or rats to a carbon tetrachloride (CCl₄)-containing atmosphere, the animals are first stimulated with phenobarbital to induce metabolic liver enzymes and to enhance CCl₄-induced liver damage. The animals are then set into an inhalation exposure apparatus and CCl₄-enriched air is conducted into the chamber using exposure cycles and conditions outlined under the ‘Practical implementation’ section of inhalation. To avoid accidental exposure and intoxication of research personnel, the procedure should be carried out under an extraction hood. After exposure, the inhalation chamber should be left in the extraction hood for another 10 min before it is removed.

Biometric calculation of requested cohorts (IP treatment and inhalation)

In a typical CCl₄ experiment one aims to compare fibrosis susceptibility of wild-type mice with a pharmacologically-treated or genetically-modified strain. Suitable readouts for ongoing hepatic fibrogenesis are, for instance, hepatic determination of hydroxyproline, measurement of alpha-smooth muscle actin (α-SMA) or collagen type 1α1 (Col1) gene expression.¹³ Regarding Col1 expression, our data (Figures 3a–3c) typically reveal a variance of approximately 25% in each group.^14–16 In addition, we define a difference of 30% in Col1 mRNA expression to be biologically relevant. From these parameters the calculated effect size d is 1.44, as determined with the G Power V.3.1.5 software (that can be downloaded from the Heinrich Heine University, Düsseldorf, Germany, http://www.gpower.hhu.de/en.html).¹⁷ To achieve a statistical power of 80% and a specific α error probability of 0.05 the minimal required sample size in each group is n = 9 mice (see Table 2 and Figure 3d).

Figure 3.

Hepatic fibrosis after carbon tetrachloride (CCl₄) challenge. Six to eight-week-old male C57BL/6 mice (weighing approximately 18–20 g) received intraperitoneal injections twice weekly of 0.7 mL/kg body weight of CCl₄ in equal volume of mineral oil or mineral oil alone for six weeks. Liver specimen from the control group (a) and CCl₄ challenged animals (b) were stained with Sirius Red. Increased collagen expression and deposition can be further quantified by (c) by quantitative real-time polymerase chain reaction (PCR). Significant differences (*) with P values < 0.05 and in Student's t-test are indicated. More experimental details are given elsewhere¹⁴ (d). The relationship of total sample size (=animals) and statistical power is depicted. Please note that 18 animals (9 animals/group) are needed under the experimental setting described in our experimentation with a statistical power of 80% (marked as a solid blue line). Higher values of statistical power require more animals per group.

Table 2.

Biometric calculation of sample size determined by G Power.

Input:	Tail(s)	= 2
	Effect size d	= 1.44
	α err prob	= 0.05
	Power (1-β err prob)	= 0.8
	Allocation ratio N2/N1	= 1
Output:	Non-centrality parameter δ	= 3.0547013
	Critical t	= 2.1199053
	Df	= 16
	Sample size group 1	= 9
	Sample size group 2	= 9
	Total sample size	= 18
	Actual power	= 0.8178583

Practical implementation

(a) Intraperitoneal

In general, handling mice (i.e. taking them out of the cage, fixation, and exposition to a foreign surrounding) is already stressful for the animals. CCl₄ vapours should be considered to be toxic for humans. Thus prevention of accidental research personnel intoxication is crucial. The level of exposure should be minimized by handling CCl₄ in an appropriate location such as a fume hood. Protective clothing including a long sleeved laboratory coat, rubber gloves, safety goggles and a face mask are minimum standards that should avoid exposure over the recommended threshold limits. After application of CCl₄, the operators should wash their hands thoroughly. In addition, the novel European animal welfare rules require that only trained laboratory personnel should perform the application of CCl₄, ensuring swift and short animal handling times. The actual procedures are as follows:

Mice should be weighed and examined for signs of distress (i.e. changes in respiration, rough hair coat, unusual behaviour, hunched posture). Animals showing these abnormalities should be excluded from the study.

CCl₄ concentrations usually are 0.5 to 0.7 µL/g body weight diluted in corn oil.^18–20 Acute intoxication models with CCl₄ usually use higher doses (0.8–2 µL/g body weight) but mice should be sacrificed after 1 to 3 days.^21,22 Please note that the injection solutions (CCl₄ or CCl₄/corn oil) should be at room temperature or close to body temperature.

The required volume of CCl₄ should be calculated. For practical reasons, we recommend to always inject a constant volume of 50 µL containing a solution of CCl₄ (e.g. Sigma-Aldrich, St Louis, MO, USA) in corn oil (e.g. Sigma-Aldrich). Accordingly, the required volume of CCl₄ (0.5–0.7 µL/g mouse weight is made up to 50 µL of total volume with corn oil. As an example, one would inject 10 µL CCl₄ dissolved in 40 µL of corn oil into a mouse that weighs 20 g to obtain a final dosage of 0.5 µL CCl₄/g body weight.

50 µL of CCl₄/corn oil solution should be injected in mice using disposable, sterile syringes. To this end the mouse should be scuffed behind the neck region between the thumb and forefinger. After turning over the hand the animal should rest on the palm against the base of the thumb using a third finger to stabilize the pelvic region. CCl₄ should be injected into the lower side of the abdomen with the mouse head down to avoid injury to the bladder, diaphragm or intestine using a 27 gauge needle (e.g. 27 G Microlance; Becton Dickinson, Franklin Lakes, NJ, USA). IP injection can lead to local irritation of the skin and CCl₄ can cause mild peritoneal inflammation.

Animals should be re-inspected one hour after injection for abnormalities and every 24 h thereafter. Termination of experiments and thus CCl₄ treatment depends on the specific aim of each study. If immediate proinflammatory alterations are of interest, tissue harvesting after 24 and 48 h is recommended. Established fibrosis can best be investigated after 2–4 weeks of CCl₄ treatment, while severe fibrosis (cirrhosis) can be observed after 6–8 weeks of treatment. At the end of the experimentation animals should be sacrificed by cervical dislocation.

Animal burden/side-effects

Usually mice that receive IP injections of CCl₄ do not develop severe long-term complications. Although CCl₄ intoxication is intended to cause elevated liver enzymes and long-term complications such as portal hypertension, oesophageal bleeding and ascites leading to acute liver injury or liver fibrosis, this condition is usually not associated with pain in humans and thus most likely also not in mice. However, IP CCl₄ application is clearly associated with abdominal adhesions and hepatic inflammation and transient spasms.

(b) Inhalation

Inhalative intoxication with CCl₄ was first used extensively in rats, and was later transferred to mice.²³ Mice in the inhalation group might be treated with protocols that differ in inhalation time or intervals (commonly twice or three times per week) that either lead to significant fibrosis within four weeks¹⁶ or might progress to liver cirrhosis with low serum sodium levels, ascites and portal hypertension after 11–15 weeks of treatment. Under the most damaging conditions, mortality rates can be as high as 70%, while short-cycle thrice-weekly inhalation usually results in significantly lower death rates (0–10%). Studies using inhalative CCl₄ usually aim towards the development of end-stage liver cirrhosis with portal hypertension and are performed for 11–15 weeks. The drawback of this technique is that special equipment and expertise are required to avoid inhalative CCl₄ contamination of research personnel because CCl₄ vapours are considered to be toxic for humans. Thus, prevention of accidental intoxication is crucial (e.g. usage of latex-free gloves is important), and only trained laboratory personnel should perform the experiments. Every contact must occur, and every potentially contaminated subject must be placed under, the extraction hood.

The actual procedures that we favour in our experimentation are as follows:

First the mice should receive phenobarbital (0.3 g/L in drinking water) for one week before starting the CCl₄ inhalation procedure. This treatment should induce liver enzymes and enhance liver damage after CCl₄ inhalation. Those animals that are subjected to CCl₄ inhalation should be kept to a maximum of six animals per cage.

The cage itself and the tube for the gas application should be checked each time before the treatment begins. CCl₄ should be filled in a specific evaporation flask. The tube conducting the air should be placed at least 2–3 cm underneath the CCl₄ fluid surface in the flask. Compressed air with a flow rate of 2 L/min should be pumped into the flasks containing CCl₄ underneath its surface and should bubble out of the fluid CCl₄. The tube feeding the CCl₄-enriched air should be inserted into the cage.

Each treatment should consist of short cycles of CCl₄ exposure three times per week (e.g. Monday, Wednesday, and Friday). The conditions should be set to:

1 min of bubbling and 1 min in the gas atmosphere in the first week

1.5 min of bubbling and 1.5 min in the gas atmosphere in the second week

2 min of bubbling and 2 min in the gas atmosphere throughout all the rest of the experiment

Mice should then be treated with one cycle/treatment in the initial three weeks, then two cycles/treatment in the fourth week, and three cycles/treatment thereafter. Previously treatment consisted of two or more cycles, and these were separated by 2 min of breathing in ambient air not containing CCl₄.

After treatment, the cage should be left in the extraction hood for another 10 min before it is removed.

When a mouse develops ascites, the inhalation should be stopped since ascites is a definitive sign of portal hypertension. The animals should have reached the stage of liver cirrhosis with portal hypertension and at this point the experiments should be conducted (e.g. efficacy analysis of antihypertensive drugs) or the animals should be sacrificed for further analysis (e.g. gene and protein expression in cirrhotic livers).

Animal burden/side-effects

(c) Gavage

Our consortium does not recommend the regular application of CCl₄ by gavage due to high rates of early mortality¹¹ and additional burdens as specified below. Increased mortality would require higher sample numbers, which is not compatible with the 3R principle.² However, if the experimental set-up requires gavage application, investigators should refer to detailed protocols published elsewhere.^24,25

Animal burden/side-effects

CCl₄ oral feeding causes significant distress to the animal and CCl₄ is likely to cause chemically-induced inflammation in the intestinal mucosa. Intubation and application of CCl₄/oil into the trachea needs to be avoided, as accidental CCl₄ application into the lung is usually instantly fatal for the mouse.

Classification of severity of procedure

According to Article 15 of the EU Directive 2010/63 (http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2010:276:0033:0079:en:PDF), the estimated degree of pain, suffering, distress or lasting harm of the animals subjected to CCl₄ application should be classified following a scoring system that contain procedures to be classified as ‘non-recovery’, ‘mild’, ‘moderate’ or ‘severe’.

Details about the classification criteria that underlie this assessment have been established by the Expert Working Group on severity classification of scientific procedures performed on animals. These classification criteria can be found at: http://ec.europa.eu/environment/chemicals/lab_animals/pdf/report_ewg.pdf.

The IP application twice a week of CCl₄ up to four weeks with no major impairment of liver function should be classified as a moderate procedure according to Article 15 of the above-mentioned EU Directive 2010/63.

Prolonged inhalative CCl₄ treatment promotes severe cirrhotic changes in the liver with significant development of ascites²³ and more ascites than IP-treated animals. Therefore, mice subjected to inhalative CCl₄ treatment suffer from the complications of liver cirrhosis, show loss in body weight and less food intake, and are more fatigued. Because of the high mortality rate the procedure is classified as moderate to severe. In line with the 3R principle, the procedure should undergo a refinement, and humane endpoints (such as lethargy, reduced escape reflexes, reduced food intake, extensive body weight loss >20%, continuous spasm or others) must be implemented with frequent observation points to restrict pain, suffering, distress or lasting harm to the animals. Then the procedure can be reclassified as moderate.

Concluding remarks

Application of CCl₄ is a key model in experimental liver research that has been applied worldwide in more than 50,000 studies so far. The model is robust and leads to highly reproducible results. In our opinion, the best treatment route is the application via IP injection. With this SOP we aim to suggest technical and biometric standards facilitating reproducible animal experimentation in line with the new European animal welfare regulations.

Ethical statement

All experiments were approved by the official State animal care and use committee (LANUV, Recklinghausen, Germany).

Footnotes

Acknowledgment

The authors are grateful to Sabine Weiskirchen for preparing .

Funding

This work was supported by grants of the German Research Foundation (SFB/TRR57 P04, P13, P18, and P26).

References

Liedtke

Luedde

Sauerbruch

. Experimental liver fibrosis research: update on animal models, legal issues and translational aspects. Fibrogenesis Tissue Repair 2013; 6: 19.

Russell

WMS

Burch

. The principles of humane experimental technique, London: Methuen, 1959.

McLean

. The effect of diet and 1,1,1-trichloro-2,2-bis-(p-chlorophenyl)ethane (ddt) on microsomal hydroxylating enzymes and on sensitivity of rats to carbon tetrachloride poisoning. Biochem J 1966; 100: 564–571.

Pound

Horn

Lawson

. Decreased toxicity of dimethylnitrosamine in rats after treatment with carbon tetrachloride. Pathology 1973; 5: 233–242.

Korsrud

Grice

McLaughlan

. Sensitivity of several serum enzymes in detecting carbon tetrachloride-induced liver damage in rats. Toxicol Appl Pharmacol 1972; 22: 474–483.

National Cancer Institute, Carcinogenesis, Technical report series. Report on the carcinogenesis bioassay of chloroform (CAS No. 67-66-3). Bethesda, MD: US Department of Health, Education, and Welfare, National Institute of Health, 1 March 1976, http://ntp.Niehs.Nih.Gov/ntp/htdocs/lt_rpts/trchloroform.pdf.

Paquet

Kamphausen

. The carbon-tetrachloride-hepatotoxicity as a model of liver damage. First report: long-time biochemical changes. Acta Hepatogastroenterol 1975; 22: 84–88.

Weber

Boll

Stampfl

. Hepatotoxicity and mechanism of action of haloalkanes: carbon tetrachloride as a toxicological model. Crit Rev Toxicol 2003; 33: 105–136.

Davis

. Carbon tetrachloride as an industrial hazard. JAMA 1934; 103: 962–966.

10.

Desmet

Gerber

Hoofnagle

Manns

Scheuer

. Classification of chronic hepatitis: diagnosis, grading and staging. Hepatology 1994; 19: 1513–1520.

11.

Chang

Yeh

Chang

Chen

. Comparison of murine cirrhosis models induced by hepatotoxin administration and common bile duct ligation. World J Gastroenterol 2005; 11: 4167–4172.

12.

McLean

Sutton

. Instant cirrhosis. An improved method for producing cirrhosis of the liver in rats by simultaneous administration of carbon tetrachloride and phenobarbitone. Br J Exp Pathol 1969; 50: 502–506.

13.

Hillebrandt

Wasmuth

Weiskirchen

. Complement factor 5 is a quantitative trait gene that modifies liver fibrogenesis in mice and humans. Nat Genet 2005; 37: 835–843.

14.

Huss

Stellmacher

Goltz

. Deficiency in four and one half LIM domain protein 2 (FHL2) aggravates liver fibrosis in mice. BMC Gastroenterol 2013; 13: 8.

15.

Nevzorova

Bangen

. Cyclin E1 controls proliferation of hepatic stellate cells and is essential for liver fibrogenesis in mice. Hepatology 2012; 56: 1140–1149.

16.

Granzow

Schierwagen

Klein

. Angiotensin-II type 1 receptor-mediated Janus kinase 2 activation induces liver fibrosis. Hepatology 2014; 60: 334–348.

17.

Faul

Erdfelder

Lang

Buchner

. G*power 3: a flexible statistical power analysis program for the social, behavioral, and biomedical sciences. Behav Res Methods 2007; 39: 175–191.

18.

Liu

Lui

Friedman

. PTK787/ZK22258 attenuates stellate cell activation and hepatic fibrosis in vivo by inhibiting VEGF signaling. Lab Invest 2009; 89: 209–221.

19.

Seki

de Minicis

Inokuchi

. CCR2 promotes hepatic fibrosis in mice. Hepatology 2009; 50: 185–197.

20.

Wasmuth

Lammert

Zaldivar

. Antifibrotic effects of CXCL9 and its receptor CXCR3 in livers of mice and humans. Gastroenterology 2009; 137: 309–319,. 319: e1–3.

21.

Borkham-Kamphorst

van de Leur

Zimmermann

. Protective effects of lipocalin-2 (LCN2) in acute liver injury suggest a novel function in liver homeostasis. Biochim Biophys Acta 2013; 1832: 660–673.

22.

Zaldivar

Pauels

von Hundelshausen

. CXC chemokine ligand 4 (Cxcl4) is a platelet-derived mediator of experimental liver fibrosis. Hepatology 2010; 51: 1345–1353.

23.

Domenicali

Caraceni

Giannone

. A novel model of CCl₄-induced cirrhosis with ascites in the mouse. J Hepatol 2009; 51: 991–999.

24.

Constandinou

Henderson

Iredale

. Modeling liver fibrosis in rodents. Methods Mol Med 2005; 117: 237–250.

25.

Fujii

Fuchs

Yamada

. Mouse model of carbon tetrachloride induced liver fibrosis: histopathological changes and expression of CD133 and epidermal growth factor. BMC Gastroenterol 2010; 10: 79.

26.

Bhathal

Rose

Mackay

Whittingham

. Strain differences in mice in carbon tetrachloride-induced liver injury. Br J Exp Pathol 1983; 64: 524–533.

27.

Hillebrandt

Goos

Matern

Lammert

. Genome-wide analysis of hepatic fibrosis in inbred mice identifies the susceptibility locus Hfib1 on chromosome 15. Gastroenterology 2002; 123: 2041–2051.

28.

Shi

Wakil

Rockey

. Strain-specific differences in mouse hepatic wound healing are mediated by divergent T helper cytokine responses. Proc Natl Acad Sci USA 1997; 94: 10663–10668.

29.

Walkin

Herrick

Summers

. The role of mouse strain differences in the susceptibility to fibrosis: a systematic review. Fibrogenesis Tissue Repair 2013; 6: 18.

The carbon tetrachloride model in mice

Abstract

Keywords

Historic background of the model

Pathogenic mechanisms of liver damage

Experimental procedure

General considerations

Genetic background

Duration and intervals of treatment

Application routes

Biometric calculation of requested cohorts (IP treatment and inhalation)

Practical implementation

(a) Intraperitoneal

Animal burden/side-effects

(b) Inhalation

Animal burden/side-effects

(c) Gavage

Animal burden/side-effects

Classification of severity of procedure

Concluding remarks

Ethical statement

Footnotes

Acknowledgment

Funding

References