Although there are many causes of chronic liver disease, such as liver cell injury (by alcohol, chemicals, viral hepatitis, etc.), bile duct injury, autoimmune disease/genetic disease, and metabolic dysfunction/disorder SJ, 2009, the consequences are the same. Liver injury causes nodule and scar tissue formation and diffuse fibrosis, characteristics encompassing liver cirrhosis, which leads to reduced liver function and increased risk of cancer SJ, 2009. End-stage liver disease (ESLD) is the final result of acute or chronic liver injury Heidelbaugh JJ, 2006. Moreover, 400 million people are infected with the hepatitis B virus worldwide Kapp, 2009. In Asian countries, the percentage of individuals infected with the hepatitis virus has been reported to be as high as 10% Li et al., 2012. ESLD is classified as the tenth leading cause of death, and 500,000–1,200,000 deaths occur each year because of the progression of viral infection to ESLD Kapp, Truong et al., 2014 2009.

Liver fibrosis is characterized by necrosis and inflammation, which increase numbers of Kupffer cells and activation of hepatic stellate cells, thereby contributing to the degeneration of liver tissues GI-PPEUM LEE, 2005. The liver normally exhibits limited cell turnover; however, as soon as cell loss or damage occurs, a regenerative process is rapidly induced, functioning to recover and maintain organ functions MR Alison,2009. This process then leads to enhancement of extracellular matrix (ECM) production and the proliferation of parenchymal and/or nonparenchymal cells, resulting in fibrogenesis and diffuse hepatic fibrosis. Because cirrhosis is the final stage of fibrosis, further studies are required to determine the molecular mechanisms of cirrhotic changes.

Regardless of the etiology of fibrosis, animal models of liver fibrosis are appropriate for studying hepatic fibrosis and cirrhosis. Although no model that specifically represent the etiologies of human liver cirrhosis have been developed, several animal models of human liver diseases are currently used, including models induced by hepatotoxins, carbon tetrachloride (CCl₄) Domitrovic et al., 2009Ming-Ling Chang, 2005, 3,5- diethoxycarbonyl-1,4-dihydrocollidine (DDC) Ming-Ling Chang, 2005, silica, allyl alcohol (AA), alpha-naphthyl-isothiocyanate (ANIT), and bile duct ligation. These models have provided support for our understanding of the mechanisms of hepatic fibrosis and have enabled us to evaluate the safety and effectiveness of novel potential therapies for liver fibrosis and cirrhosis Ming-Ling Chang, 2005Starkel and Leclercq, 2011Yan Liu, 2013. Among hepatotoxins, CCl₄ is often used to induce hepatic fibrosis and cirrhosis in animals because the underlying biochemical mechanisms and histological characteristics are similar to those observed in human liver cirrhosis Constandinou et al., 2005Fujii et al., 2010GI-PPEUM LEE, 2005Li et al., 2012. CYP2E1, an enzyme expressed in perivenular hepatocytes, metabolizes CCl₄ into the CCl₃+ radical, which causes centrilobular necrosis and alters the permeability of the plasma and mitochondrial membranes of hepatocytes Fujii et al., 2010. This induces inflammation and fibrogenesis and increases the generation of ECM, thereby triggering wound healing. Chronic CCl₄ exposure results in formation of nodules and fibrosis, products of the wound healing process. Many studies have shown that longterm CCl₄administration causes significant changes in histology Ming-Ling Chang, 2005Starkel and Leclercq, 2011. Specifically, CCl₄ treatment has been shown to cause fibrosis after 2–4 weeks, significant bridging fibrosis after 5–7 weeks, cirrhosis after 9–11 weeks, and micronodular cirrhosis after 10–20 weeks Starkel and Leclercq, 2011. However, the dose and duration of CCl₄ treatment required to induce such lesions depend on the species and strain of the animal model used, as well as the method (i.e., intraperitoneal, oral, or inhalation) and frequency of administration Ming-Ling Chang,2005Starkel and Leclercq, 2011. Therefore, we aimed to develop a mouse model of liver cirrhosis using induction by CCl₄. This study mainly concentrated on analyzing the results of oral administration of CCl₄ in Swiss mice, which are popular in Vietnamese laboratories.

Because CCl₄ is toxic and can cause mortality, establishment of a mouse model of liver fibrosis is challenging. According to our current study, gavage is suitable for CCl₄administration as it resulted in low mortality compared to intraperitoneal administration of CCl₄ Ming-Ling Chang, 2005. Moreover, estimating the degree of fibrosis in animal models is critical for determining whether therapeutic treatment could be effective. Traditionally, liver biopsy is considered the ‘gold standard’ for staging liver fibrosis. However, recent data have shown that there is a 30% failure/error rate in diagnoses by liver biopsy Mahato, 2007. Therefore, we used other techniques to accurately determine the liver fibrosis stage Mahato, 2007. To evaluate whether CCl₄ can cause acute liver injury in Swiss mice, we relied on indirect markers, such as serum AST, ALT, total bilirubin, and albumin. Additionally, to confirm liver fibrosis, we evaluated fibrosis biomarkers/direct markers by qRT-PCR and classified liver histology based on the Knodell-Ishak (Ishak Modified HAI) scoring system.

Increases in ALT and AST levels may indicate the occurrence of liver necrosis Field et al., 2008. When liver cells are damaged, intracellular enzymes (including transaminase enzymes) leak into the blood and can be measured as indicators of cell necrosis. In our model, the level of serum ALT increased sharply in the context of hepatitis and acute liver injury. The results of AST and ALT levels in the control group were consistent with other reports Domitrovic et al., 2009. In group I, we observed insignificant changes in serum ALT and AST before and after CCl₄ treatment, suggesting that injured liver cells were likely to be restored because of low CCl₄ concentrations Mederacke, 2013. Moreover, increased AST and ALT levels in groups II and III were equivalent to those reported by Tsai et al (2009) Tsai et al., 2009. These results demonstrated that acute liver injury and inflammation occurred in our mouse model. The increases in AST and ALT levels in liver fibrosis induced by 1.0 mL/kg CCl₄ supported that these mice exhibited substantial hepatic injury. A sharp increase in total bilirubin levels and a drop in albumin levels indicated loss of function in the mouse liver. These results were similar to those reported by Ohashi et al. (2012)Ohashi et al., 2012. Furthermore, our data showed that the ratio of AST/ALT in our mouse model was less than 1, supporting the occurrence of acute hepatitis Thapa and Walia, 2007. In order to confirm the presence of hepatic fibrosis, fibrosis markers and histopathology should also be evaluated.

Fibronectin and other proteins of the ECM create a framework for fibroblast migration. Fibronectin also provides direction and chemical signals for fibroblast proliferation Armbrust et al., 2004. Increased procollagen synthesis contributes to the increase in collagen levels observed in liver cirrhosis, and abnormal mRNA expression of procollagen α1 reflects the level of fiber in liver Du WD, 1999. In this study, we observed increased procollagen expression in CCl₄-treated mice compared to that of normal mice. Because procollagen is a direct marker of fibrosis, abnormally high levels of procollagen indicate that liver tissues have accumulated scar nodules, accompanied by hepatitis and hepatic necrosis. In addition, high levels fibronectin Attallah et al.,2013, TGF-β1 Gressner et al., 2002, and integrin Stickel, 2011 expression may activate cells, such as fibroblasts and hematopoietic stem cells (HSCs). Nt5e has been shown to contribute to increased adenosine levels, which have an important role in hepatic fibrosis Peng et al., 2008. Indeed, synthetic ecto-5'-nucleotidase expression leads to increased levels of extracellular adenosine Peng et al., 2008. The increase in adenosine in injured liver tissue activates the cells through the A2A receptor, enhancing collagen synthesis and expression Che et al., 2007 and eventually leading to liver cirrhosis.

In this study, we applied the Ishak Modified HAI system to classify histological grading and staging of chronic hepatitis and fibrosis in mice. The results showed changes in hepatic histopathology, accumulation of ECM, and formation of nodules and fiber bridges. Our results of liver tissue histopathology were similar to the Massive trichrome staining results reported by Hao et al. (2012.

Based on the results of biochemical tests, anatomical observations, histological staining, and gene expression analysis, we concluded that oral administration of 1.0 mL/kg CCl₄ for 11 weeks (three times per week, or once every two days) was sufficient for high-efficiency induction of liver fibrosis in Swiss mice. This mouse model of fibrosis had meaningfully changes in gene expression, as well as liver structure and function.

AST aminotransferase; ALT Alanine aminotransferase; CCl₄: carbon tetrachloride; CYP2E1: Cytochrome P450 2E1; ESLD: End stage liver disease; ECM: extracellular matrix; H&E: Hematoxylin and Eosin; Nt5e: ecto-5'-nucleotidase; TGFbeta: transforming growth factor beta

Truong Hai Nhung made substantial contributions to conception and design, acquisition of data and analysis and interpretation of data. Being corresponding author, Truong Hai Nhung gave final approval of the manuscript to be submitted and any revised version. Nguyen Hai Nam and Nguyen Thi Kim Nguyen who contributed to conduct some experiments, acquisition of data and participate in drafting the manuscript. Le Minh Huy and Tran Huong Giang made substantial contributions to analyze the histology change by using Knodell-Ishak (Ishak Modified HAI) scoring system. Huynh Nghia and Nguyen Van Thanh are supervisors of this study.