PURPOSE: To present a review about a comparative study of bile duct ligation versus carbon tetrachloride Injection for inducing experimental liver cirrhosis. METHODS: This research was made through Medline/PubMed and SciELO web sites looking for papers on the content "induction of liver cirrhosis in rats". We have found 107 articles but only 30 were selected from 2004 to 2011. RESULTS: The most common methods used for inducing liver cirrhosis in the rat were administration of carbon tetrachloride (CCl4) and bile duct ligation (BDL). CCl4 has induced cirrhosis from 36 hours to 18 weeks after injection and BDL from seven days to four weeks after surgery. CONCLUSION: For a safer inducing cirrhosis method BDL is better than CCl4 because of the absence of toxicity for researches and shorter time for achieving it.
Liver Cirrhosis; Drugs; Carbon Tetrachloride; Common Bile Duct; Ratos
OBJETIVO: Apresentar revisão sobre estudo comparativo da indução de cirrose hepática (CH) experimental com a injeção de tetra-cloreto de carbono (CCl4) comparado à ligadura do ducto biliar (BDL). MÉTODOS: A pesquisa foi realizada nas bases de dados do Medline/PubMed e SciELO procurando trabalhos com as palavras indução de CH e ratos. Foram encontrados 107 artigos, mas somente 30 foram selecionados no período de 2004 à 2011. RESULTADOS: Os procedimentos mais comum para indução de CH em ratos foram a injeção de CCl4 e a BDL. O CCl4 induzia CH no período de 36 horas após a injeção e a DBL de sete dias à quatro semanas após a cirurgia. CONCLUSÃO: A BDL é o método mais seguro para indução de CH quando comparado a injeção de CCl4 pela ausência de toxicidade para os pesquisadores e o menor tempo para se obter a lesão hepática.
Cirrose Hepática; Drogas; Tetracloreto de Carbono; Ducto Colédoco; Ratos
13 - REVIEW
Review of experimental models for inducing hepatic cirrhosis by bile duct ligation and carbon tetrachloride injection1 1 Review performed at Department of Gastroenterology, Part of Scientific Initiation Program on Experimental Research Laboratory, LIM-37, School of Medicine, University of Sao Paulo (USP), Brazil.
Revisão de modelos experimentais de cirrose hepática induzida por ligadura do ducto biliar e por injeção de tetracloreto de carbono
Thamirys Guimarães MarquesI; Eleazar ChaibII; Juliana Hamati da FonsecaI; Ana Cecília Rodrigues LourençoI; Felipe Duarte SilvaI; Marcelo Augusto Fontenelle Ribeiro Jr.III; Flavio Henrique Ferreira GalvãoIV; Luiz Augusto Carneiro D'AlbuquerqueV
IGraduate student, School of Medicine, USP, Sao Paulo, Brazil. Acquisition of data, manuscript preparation
IIAssociate Professor of Surgery, Department of Gastroenterology, School of Medicine, USP, Sao Paulo, Brazil. Director of Experimental Research Laboratory, LIM-37,HC/FMUSP. Design of the study, interpretation of data, manuscript preparation
IIIAssistant Professor of Surgery, University of City of Sao Paulo, Brazil. Interpretation of data, manuscript preparation
IVAssistant Professor of Surgery, Department of Gastroenterology, School of Medicine, USP, Sao Paulo, Brazil. Interpretation of data, manuscript preparation
VFull Professor of Surgery, Department of Gastroenterology, School of Medicine, USP, Sao Paulo, Brazil. Interpretation of data, manuscript preparation
PURPOSE: To present a review about a comparative study of bile duct ligation versus carbon tetrachloride Injection for inducing experimental liver cirrhosis.
METHODS: This research was made through Medline/PubMed and SciELO web sites looking for papers on the content "induction of liver cirrhosis in rats". We have found 107 articles but only 30 were selected from 2004 to 2011.
RESULTS: The most common methods used for inducing liver cirrhosis in the rat were administration of carbon tetrachloride (CCl4) and bile duct ligation (BDL). CCl4 has induced cirrhosis from 36 hours to 18 weeks after injection and BDL from seven days to four weeks after surgery.
CONCLUSION: For a safer inducing cirrhosis method BDL is better than CCl4 because of the absence of toxicity for researches and shorter time for achieving it.
Key words: Liver Cirrhosis. Drugs. Carbon Tetrachloride. Common Bile Duct. Ratos.
OBJETIVO: Apresentar revisão sobre estudo comparativo da indução de cirrose hepática (CH) experimental com a injeção de tetra-cloreto de carbono (CCl4) comparado à ligadura do ducto biliar (BDL).
MÉTODOS: A pesquisa foi realizada nas bases de dados do Medline/PubMed e SciELO procurando trabalhos com as palavras indução de CH e ratos. Foram encontrados 107 artigos, mas somente 30 foram selecionados no período de 2004 à 2011.
RESULTADOS: Os procedimentos mais comum para indução de CH em ratos foram a injeção de CCl4 e a BDL. O CCl4 induzia CH no período de 36 horas após a injeção e a DBL de sete dias à quatro semanas após a cirurgia.
CONCLUSÃO: A BDL é o método mais seguro para indução de CH quando comparado a injeção de CCl4 pela ausência de toxicidade para os pesquisadores e o menor tempo para se obter a lesão hepática.
Descritores: Cirrose Hepática. Drogas. Tetracloreto de Carbono. Ducto Colédoco. Ratos.
Liver cirrhosis (LC) is considered a public health, according to World Health Organization, about 800 thousand people die from LC every year. Only in United States LC is responsible for around 27 thousand deaths per year, representing a mortality rate of 9.2 per 100,000, placing it as the 12th overall cause of death1-3.
Nowadays major research centers focus on studying LC, its mechanisms and its behavior, complications and possible treatments. For that reason it has been developed, efficient experimental models of induction of LC in rats. The two most common methods used for experimental LC are the administration of carbon tetrachloride (CCl4) and the bile duct ligation (BDL)7-9. Our aim is to present a comparative study of bile duct ligation versus carbon tetrachloride injection for inducing experimental liver cirrhosis
This research was made through Medline/PubMed and SciELO web sites looking for papers on the content "induction of liver cirrhosis in rats". We found 107 articles but only 30 from 2004 to 2011 were selected. The inclusion criteria were: only rats; bile duct ligation and injection of carbon tetrachloride. Histopathologic examination for confirming cirrhosis; Time of inducing cirrhosis. The exclusion criteria were: large animals cirrhosis; others rodent animals; drug inducing cirrhosis such as dimethylnitrosamine; thiocetamide; butylhydroperoxide. Others drugs association methods: buprenorphine with reduction of portal inflow over a stent inserted in the right renal artery; Others methods: unrestricted flow using an aortic-portal segment; orthotopic liver transplantation with unrestricted portal arterialisation. Thioacetamide associated with partial hepatectomy.
The main methods of induction of LC in rats are the administration of carbon tetrachloride and bile duct ligation. Below are shown induction of cirrhosis by CCl4, dosage of the drug, time for inducing cirrhosis, main test assessment with the method and their results (Table 1) and for bile duct ligation (Table 2).
The main methods of induction of LC in rats are the administration of carbon tetrachloride (CCl4)4 and bile duct ligation (BDL)11. CCl4 is one of the most used methods nowadays however it is considered an extremely toxic method5. Several important basic mechanisms of tissue damages induced by CCl4 have emerged, involving metabolic activation, reactive free radical metabolites, lipid peroxidation, covalent binding and disturbance of calcium homeostasis.
The CCl4 administration results in hepatocyte damage, necrosis, inflammation, and fibrosis, which spreads to link the vascular structures that feed into and drain the hepatic sinusoid (the portal tract and central vein radicle, respectively)3,39. It activates the hepatic stellate cell (HSC) inducing hepatocyte apoptosis and zone III necrosis40. Continuous administration of CCl4 can provide moderate cell necrosis and fatty infiltration in four weeks.
The CCl4 is excreted from the body within the first 24 hour by conjugation reaction mediated by phase. In the literature, three mechanisms have been proposed as the possible explanations for progression of injury: (1) Contribution of inflammatory cells; (2) Production of free radicals; and (3) Leakage of degradative enzymes from the dying and injured cells. Activated resident Kupffer cells and the neutrophils recruited at the site of parenchymal liver injury are considered as the primary culprits in damaging surrounding healthy cells as the result of nonspecific action. However, evidence suggests that the contribution of the inflammatory cells does not or is not sufficient to mediate progression of injury.
The second theory regarding progression of injury is production of free radicals and oxidative stress, and subsequent lipid peroxidation that propagates injury. Though the antioxidants prevent/delay the tissue damage partially, progression of injury still occurs. The inhibition of lipid peroxidation by antioxidants only decreases the initial injury of CCl4, blocking lipid peroxidation fails to prevent progression of injury and subsequent lethality45. By 8 weeks, a micronodular cirrhosis takes place 31. It has been used mainly by intraperitoneal injection12 or oral administration20, the dosage 0,2 to 5ml/kg and LC is achieved between 36 hours to 18 weeks15,20,11,24.
BDL is a safer method comparing to the CCl4. When the BDL is done, it provides an acute obstructive jaundice in two weeks, and progression to cirrhosis in 4 or 6 weeks9,10. BDL stimulates the proliferation of biliary epithelial cells and oval cells (which are hepatocyte progenitors), resulting in proliferating bile ductules with an accompanying portal inflammation and fibrosis43. Cholangiocyte proliferation started after BDL at the edge of the portal tract. During the first week from BDL the hepatic microcirculation did not show any alterations with respect to the normal liver46. Using this method LC is achieved between seven days to four weeks17,26.
Based in our broadly review we concluded that LC can be induced by CCl4 and BDL, however the manipulation of CCl4 can be dangerous for the researcher with risk of inducing liver tumor. BDL is a safer method than CCl4 and cirrhosis could be induced in rats in average mean time of two weeks.
Received: March 23, 2012
Review: May 24, 2012
Accepted: June 21, 2012
Conflict of interest: none
Financial source: none
1Global Status Report on Alcohol 2004. Geneza, 2004. World Health Organization Department of Mental Health and Substance Abuse.
- 2. Schuppan D, AfdhalNH. Liver Cirrhosis. Lancet. 2008; 371:83851.
- 3. Duvoux C, Samuel D. Hepatic transplantation. Gastroenterol Clin Biol. 2009;33:868-81.
- 4. Lee KJ, Kim JY, Jung KS, Choi CY, Chung YC, Kim DH, Jeong HG. Suppressive effects of Platycodongrandiflorum on the progress of carbon tetrachloride-induced hepatic fibrosis. Arch Pharm Res. 2004;27:1238-44.
- 5. Hernandez-Muñoz R, .Balance between oxidative damage and proliferative potential in an experimental rat model of CCl4induced cirrhosis: protective role of adenosine administration. Hepatology. 1997;26:1100-10.
- 6. Assimakopoulos SF, Vagianos CE. Bile duct ligation in rats: a reliable model of hepatorenal syndrome? World J Gastroenterol. 2009;15:121-3.
- 7. Maya-Mendoza A, Hernández-Muñoz R, Gariglio P, Aranda-Anzaldo A. Gene positional changes relative to the nuclear substructure during carbon tetrachloride-induced hepatic fibrosis in rats. J Cell Biochem. 2004;93:1084-98.
- 8. Mikami K, Otaka M, Goto T, Miura K, Ohshima S, Yoneyama K, Lin JG, Watanabe D, Segawa D, Kataoka E, Odashima M, Watanabe S. Induction of a 72-kDa heat shock protein and protection against lipopolysaccharide-induced liver injury in cirrhotic rats. J Gastroenterol Hepatol. 2004;19:884-90.
- 9. Antoine M, Tag CG, Wirz W, Borkham-Kamphorst E, Sawitza I, Gressner AM, Kiefer P. Upregulation of pleiotrophin expression in rat hepatic stellate cells by PDGF and hypoxia: implications for its role in experimental biliary liver fibrogenesis. Biochem Biophys Res Commun. 2005;337:1153-64.
- 10. Sztrymf B, Libert JM, Mougeot C, Lebrec D, Mazmanian M, Humbert M, Herve P. Cirrhotic rats with bacterial translocation have higher incidence and severity of hepatopulmonary syndrome. J Gastroenterol Hepatol. 2005;20:1538-44.
- 11. Peng X, Wang B, Wang T, Zhao Q. Expression of basic fibroblast growth factor in rat liver fibrosis and hepatic stellate cells. J Huazhong Univ Sci Technol Med Sci. 2005;25:166-9.
- 12. Fiorucci S, Rizzo G, Antonelli E, Renga B, Mencarelli A, Riccardi L, Orlandi S, Pruzanski M, Morelli A, Pellicciari R. A farnesoid x receptor-small heterodimer partner regulatory cascade modulates tissue metalloproteinase inhibitor-1 and matrix metalloprotease expression in hepatic stellate cells and promotes resolution of liver fibrosis. J Pharmacol Exp Ther. 2005;314:584-95.
- 13. Kato A, Bamba H, Shinohara M, Yamauchi A, Ota S, Kawamoto C, Yoshida Y. Relationship between expression of cyclin D1 and impaired liver regeneration observed in fibrotic or cirrhotic rats. J Gastroenterol Hepatol. 2005;20:1198-205.
- 14. Lewis MD, Roberts BJ. Role of CYP2E1 activity in endoplasmic reticulum ubiquitination, proteasome association, and the unfolded protein response. Arch Biochem Biophys. 2005;436:237-45.
- 15. Hsu YC, Chiu YT, Lee CY, Wu CF, Huang YT. Anti-fibrotic effects of tetrandrine on bile-duct ligated rats. Can J Physiol Pharmacol. 2006;84:967-76.
- 16. Oria M, Raguer N, Chatauret N, Bartolí R, Odena G, Planas R, Córdoba J. Functional abnormalities of the motor tract in the rat after portocaval anastomosis and after carbon tetrachloride induction of cirrhosis. Metab Brain Dis. 2006;21:297-308.
- 17. Kotsiou A, Tsamouri M, Anagnostopoulou S, Tzivras M, Vairactaris E, Tesseromatis C. H3 Propranolol serum levels following lidocaine administration in rats with CCL4 induced liver damage. Eur J Drug Metab Pharmacokinet. 2006;31:97-101.
- 18. Li G, Xie Q, Shi Y, Li D, Zhang M, Jiang S, Zhou H, Lu H, Jin Y. Inhibition of connective tissue growth factor by siRNA prevents liver fibrosis in rats. J Gene Med. 2006;8:889-900.
- 19. Tsui TY, Lau CK, Ma J, Glockzin G, Obed A, Schlitt HJ, Fan ST. Adeno-associated virus-mediated heme oxygenase-1 gene transfer suppresses the progression of micronodular cirrhosis in rats. World J Gastroenterol. 2006;12:2016-23.
- 20. Cheung PY, Zhang Q, Zhang YO, Bai GR, Lin MC, Chan B, Fong CC, Shi L, Shi YF, Chun J, Kung HF, Yang M. Effect of WeiJia on carbon tetrachloride induced chronic liver injury. World J Gastroenterol. 2006;12:1912-7.
- 21. Peretz G, Link G, Pappo O, Bruck R, Ackerman Z. Effect of hepatic iron concentration reduction on hepatic fibrosis and damage in rats with cholestatic liver disease. World J Gastroenterol. 2006;12:240-5.
- 22. Anan A, Baskin-Bey ES, Bronk SF, Werneburg NW, Shah VH, Gores GJ. Proteasome inhibition induces hepatic stellate cell apoptosis. Hepatology. 2006;43:335-44.
- 23. Liu SQ, Yu JP, Chen HL, Luo HS, Chen SM, Yu HG. Therapeutic effects and molecular mechanisms of Ginkgo biloba extract on liver fibrosis in rats. Am J Chin Med. 2006;34:99-114.
- 24. Mikami K, Otaka M, Watanabe D, Goto T, Endoh A, Miura K, Ohshima S, Yoneyama K, Sato M, Shibuya T, Segawa D, Kataoka E, Yoshino R, Takeuchi S, Sato W, Odashima M, Watanabe S. Zinc L-carnosine protects against mucosal injury in portal hypertensive gastropathy through induction of heat shock protein 72. J Gastroenterol Hepatol. 2006;21:1669-74.
- 25. Xue H, Guo H, Li YC, Hao ZM. Heme oxygenase-1 induction by hemin protects liver cells from ischemia/reperfusion injury in cirrhotic rats. World J Gastroenterol. 2007;13:5384-90.
- 26. Shen H, Fan J, Burczynski F, Minuk GY, Cattini P, Gong Y. Increased Smad1 expression and transcriptional activity enhances trans-differentiation of hepatic stellate cells. J Cell Physiol. 2007;212:764-70.
- 27. Tieppo J, Vercelino R, Dias AS, Silva Vaz MF, Silveira TR, Marroni CA, Marroni NP, Henriques JA, Picada JN. Evaluation of the protective effects of quercetin in the hepatopulmonary syndrome. Food ChemToxicol. 2007;45:1140-6.
- 28. Abe W, Ikejima K, Lang T, Okumura K, Enomoto N, Kitamura T, Takei Y, Sato N. Low molecular weight heparin prevents hepatic fibrogenesis caused by carbon tetrachloride in the rat. J Hepatol. 2007;46:286-94.
- 29. Lee TY, Chang HH, Chen JH, Hsueh ML, Kuo JJ. Herb medicine Yin-Chen-Hao-Tang ameliorates hepatic fibrosis in bile duct ligation rats. J Ethnopharmacol. 2007;109:318-24.
- 30. Thomsen KL, Nielsen SS, Grønbiek H, Flyvbjerg A, Vilstrup H. Effects of lipopolysaccharide endotoxin on the insulin-like growth factor I system in rats with cirrhosis. In Vivo. 2008;22:655-61.
- 31. Yuan LP, Chen FH, Ling L, Bo H, Chen ZW, Li F, Zhong MM, Xia LJ. Protective effects of total flavonoids of Bidensbipinnata L. against carbon tetrachloride-induced liver fibrosis in rats. Pharm Pharmacol. 2008;60:1393-402.
- 32. Borkham-Kamphorst E, Kovalenko E, van Roeyen CR, Gassler N, Bomble M, Ostendorf T, Floege J, Gressner AM, Weiskirchen R. Platelet-derived growth factor isoform expression in carbon tetrachloride-induced chronic liver injury. Lab Invest. 2008;88:1090-100.
- 33. Langer DA, Das A, Semela D, Kang-Decker N, Hendrickson H, Bronk SF, Katusic ZS, Gores GJ, Shah VH. Nitric oxide promotes caspase-independent hepatic stellate cell apoptosis through the generation of reactive oxygen species. Hepatology. 2008;47:1983-93.
- 34. Tiberio GA, Tiberio L, Benetti A, Cervi E, Montani N, Dreano M, Garotta G, Cerea K, Steimberg N, Pandolfo G, Ferrari-Bravo A, Mazzoleni G, Giulini SM, Schiaffonati L. IL-6 Promotes compensatory liver regeneration in cirrhotic rat after partial hepatectomy. Cytokine 2008;42:372-8.
- 35. Tsai JH, Liu JY, Wu TT, Ho PC, Huang CY, Shyu JC, Hsieh YS, Tsai CC, Liu YC. Effects of silymarin on the resolution of liver fibrosis induced by carbon tetrachloride in rats. J Viral Hepat. 2008;15:508-14.
- 36. Vercelino R, Tieppo J, Dias AS, Marroni CA, Garcia E, Meurer L, Picada JN, Marroni NP. N-acetylcysteine effects on genotoxic and oxidative stress parameters in cirrhotic rats with hepatopulmonary syndrome. Basic Clin Pharmacol Toxicol. 2008;102:370-6.
- 37. Hagens WI, Beljaars L, Mann DA, Wright MC, Julien B, Lotersztajn S, Reker-Smit C, Poelstra K. Cellular targeting of the apoptosis-inducing compound gliotoxin to fibrotic rat livers. Pharmacol Exp Ther. 2008;324:902-10.
- 38. Urtasun R, Cubero FJ, Vera M, Nieto N. Reactive nitrogen species switch on early extracellular matrix remodeling via induction of MMP1 and TNFalpha. Gastroenterology. 2009;136:1410-22.
- 39. Alatsakis M, Ballas KD, Pavlidis TE, Psarras K, RafailidisS,Tzioufa-Asimakopoulou V, Marakis GN, Sakantamis AK. Early propranolol administration does not prevent development of esophageal varices in cirrhotic rats. Eur Surg Res. 2009;42:11-6.
- 40. Kim SK, Seo JM, Chae YR, Jung YS, Park JH, Kim YC. Alleviation of dimethylnitrosamine-induced liver injury and fibrosis by betaine supplementation in rats. Chem Biol Interact. 2009;177:204-11.
- 41. Pooranaperundevi M, Sumiyabanu M, Viswanathan P, Sundarapandiyan R, Anuradha CV. Insulin resistance induced by high-fructose diet potentiates carbon tetrachloride hepatotoxicity. Toxicol Ind Health. 2010;26: 89-104.
- 42. Constandinou C, Henderson N, Iredale JP. Modeling liver fibrosis in rodents. Methods Mol Med. 2005;117:237-50.
- 43. Aziz TA, Aziz MA, Fouad HH, Rashed LA, Salama H, Abd-Alla S, Wehab MA, Ahmed T. Interferon-alpha gene therapy prevents aflatoxin and carbon tetrachloride promoted hepatic carcinogenesis in rats. Int J Mol Med. 2005;15:21-6.
- 44. Azmaiparashvili E, Kordzaia D, Dzidziguri D. Biliary hypertension as the cell proliferation trigger in bile duct ligated rats. Georgian Med News. 2009;168:111-6.
- 45. Mehendale HM. Tissue repair: an important determinant of final outcome of toxicant-induced injury. Toxicol Pathol. 2005;33:41-51.
- 46. Priester S, Wise C, Glaser SS. Involvement of cholangiocyte proliferation in biliary fibrosis. World J Gastrointest Pathophysiol. 2010;2:30-7.
Eleazar ChaibFaculdade de Medicina LIM 37Departamento de Gastroenterologia-USPAvenida Dr. Arnaldo 455, 3ºandar/320801296-900 São Paulo SP BrasilTel.: (5511)3061-8322Fax: (5511)3061-7270
Review performed at Department of Gastroenterology, Part of Scientific Initiation Program on Experimental Research Laboratory, LIM-37, School of Medicine, University of Sao Paulo (USP), Brazil.
Publication in this collection
25 July 2012
Date of issue
23 Mar 2012
21 June 2012
24 May 2012