2.3. Diet-based animal models
The animals are fed on specific modified diets for a particular duration which results in cirrhosis (Table 3). The strain of rodents plays a vital role in inducing disease. It has been observed that C57BL/6 mice establish hepatic cirrhosis rapidly with the use of a modified diet (76). These diets are as follows:
2.3.1. Methionine and choline-deficient diet: Methionine is an amino acid that plays important in wound healing and prevents liver damage. Choline is a nutrient essential for metabolism, brain function and helps to treat liver ailments. This diet comprises a sucrose content of up to 40%, fat content of 10%, and is deficient in methionine and choline. After regular administration of this diet for 8-10 weeks, mice and rats develop liver cirrhosis (77, 78). This diet results in increased fatty acid uptake by adipose tissue, which results in increased synthesis of triglycerides. An imbalance between the antioxidant enzymes and free radical production along with the induction of inflammatory response contributes to the development of liver cirrhosis (79).
2.3.2. Liebere De Carli liquid diet: It is also called chronic ethanol feeding and is the first method that involves drinking water incorporated with ethanol. The liquid diet containing ethanol is given as a substitute for water and food. Its constituents include 36% calories of ethanol with a diet concentration of 5% (w/v), carbohydrates 11%, fat 35%, and proteins 18%. The daily ethanol intake by the animal is 14-25g/kg of body weight (80, 81). After taking this diet for 4-12 weeks, mice and rats develop liver cirrhosis. Ethanol oxidizes via the alcohol dehydrogenase pathway, which affects lipid, carbohydrate, protein, and purine metabolism. Cell membrane damage occurs as a result of lipid peroxidation and reactive oxygen species production by the action of cytochrome P-450 (P-4501IEI) causing liver fibrosis (82).
Bin Gao et al developed a slight modification in Liebere De Carli liquid diet containing 5%v/v ethanol continuously for the 10 days, followed by a single dose of ethanol (5mg/kg) on the 11th day and euthanized after nine hours for observation. This experiment can be extended up to 8 weeks for the examination of steatosis, liver cell damage, liver neutrophil migration, and liver function parameters. Thus this model could be used for the evaluation of alcohol-induced liver injury in individuals (83).
2.3.3. High-fat diet: Fats comprise of fatty acid and tri esters of glycerol. These are required for cell growth and provide energy to the body. In this model, the rodents are fed on a high-fat diet. The diet comprises different compositions of nutrients, such as 11% carbohydrates, 71% fat and 18% proteins. The animals fed on this diet develop liver fibrosis within 2-3 weeks in rats and 9 weeks in mice (84, 85). High-fat diet generates aberrant mitochondria and fatty acid influx resulting in the development of insulin resistance and obesity. The beta-oxidation of fatty acids leads to the secretion of very low-density lipoproteins along with free radicals causing liver cirrhosis (86).
The experimental model involves non-alcoholic fatty liver disease (NAFLD) that involves a diet rich in fats without any nutritional deficiency. The diet is composed of 45-74% of fat in total diet intake for the study of hepatic steatosis (87). The C57BL/6 mice are the specialized rodents that are prone to metabolic syndromes i.e. diabetes mellitus. 0.1% cholesterol diet along with fructose-rich diet results in obesity and NAFLD progression in 52 weeks in these animals (88). The physiological, histological, and cellular signaling pathways can be easily understood by this experimental model. There is a limitation in the availability of knockout mice (88).
2.3.4. High fructose diet: In this model, the rodents feed on a high fructose diet leading to development of hepatic fibrosis. At first, 10% fructose is incorporated in the drinking water. The animals are allowed to feed on a high content of fructose (up to 70%) and develop liver cirrhosis within 8 weeks. This method can be used in both mice and rats. Fructose, after metabolism, induces synthesis of fatty acids in the liver. As a result, accumulation of lipids takes place in the liver which leads to cytokine release along with enhanced nodule formation in hepatocytes inducing liver cirrhosis (89-91).
2.3.5. Ethanol Ad libitum feeding: It is one of the oldest methods to establish cirrhosis in rodents. It includes incorporation of alcohol in drinking water but rodents are permitted to feed freely with a chow diet as per standards. The animal fed on ad libitum diet water with 20% (v/v) of ethanol for the duration of 8 weeks in mice, and with 40% (v/v) of ethanol for the duration of up to 29 weeks in rats induces liver cirrhosis.
Experimental alcohol administration in a single bottle, multiple alcohol containing bottles and drinking in dark models were the established models for the evaluation of hepatoprotective drugs. The earliest method of alcohol drinking constituted 5%-20% (v/v) of ethanol supplemented in place of water for 8-10 weeks. In the multiple bottle model, two bottles were incorporated, one of distilled water and the other of 5%-40% ethanol for 29 weeks. Hepatotoxicity is reported when rodents show steatosis, fibrosis and hepatic injury upon acute inflammation and for histological findings include development of focal necrosis and perivenular fibrosis in chronic inflammation (92).
Drinking in the dark model involves 20% alcohol consumption for 2-4 hours in mice quarantined singly. This model is compatible with achieving high blood alcohol concentration i.e. >1.0mg/ml and is used for mimicking prenatal human binge drinking (93). Despite the diversity in alcohol drinking models, several hepatic stressors such as diethyl nitrosamine was used to for progression of hepatic necrosis to hepatocellular carcinoma (94).
Excessive ethanol drinking raises the expression and activity of cytochrome P450 (CYP2E1), which enhances acetaldehyde synthesis by forming reactive oxygen species (ROS). Due to stimulation of interleukins, there is initiation of the inflammatory response and oxidant scavenging capacity is reduced resulting in hepatic fibrosis (95-97).