INTRODUCTION:

Alcoholic liver disease:

Alcohol has been a constituent of man’s society for several millennia, and alcoholism, which is negatively affecting the liver, is the oldest known disease. Alcohol misuse is a major global health issue affecting socioeconomic societies, causing conditions like alcoholic liver disease (ALD).

Preclinical models are used to study human diseases, with a variety of models for Alzheimer's disease (ALD) developed over the past few decades.

These models provide unique insights into the disease's pathophysiology and potential therapeutic approaches. However, not all models accurately replicate the entire range of ALD in humans, and clinically relevant models should exhibit specific features.

This review evaluates the advantages and disadvantages of popular experimental ALD models, discusses the development and application of promising animal models, and highlights common pathogenic events in these models.

Pathophysiology of Alcoholic liver disease:

Alcohol misuse, despite its long history, was not studied until the 20th century. In 1965, Lieber and colleagues discovered the hepatotoxic action of alcohol. Today, Alcohol-Leading Liver Disease (ALD) is a multifaceted illness resulting from alcohol misuse, including simple steatosis and serious damage types like cirrhosis and HCC^1-3

Only 2-10% of ethanol consumed is directly removed by the kidney, lung, and perspiration after absorption in the gastrointestinal tract. Most ethanol is processed in the liver, where hepatocytes convert ethanol into acetaldehyde using alcohol dehydrogenase (ADH). Other paths are; catalase and microsomal ethanol oxidation system (MEOS) and in the latter the cytochrome P450 pathway is used. Acatalyses the removal of hydrogen in acetaldehyde and its conversion to acetate and alterations in the NAD level accelerates hepatic triglyceride and fatty acid synthesis⁴

Figure 1 Pathophysiology of alcoholic liver disease.

The CYP2E1 family regulates ROS production as alcohol metabolism resulting from mitochondria, ER or Kupffer cell. These radicals can worsen hepatocyte oxidative stress. Acetaldehyde, the main ethanol metabolite, is a potent hepatotoxin that causes liver damage through lipid peroxidation, ROS toxicity, and glutathione depletion^5-6

Alcohol metabolism in hepatocytes can lead to biochemical alterations such as cytotoxic metabolites, ROS buildup, and lipid peroxidation, which can trigger pathological reactions resulting in liver destruction, including inflammation, cell death, steatosis, fibrogenesis, and even liver regeneration^7-9. Alcohol-induced liver damage (ALD) leads to fibrosis in 35% of patients. The liver synthesizes cytokines and chemokines and immune system components stimulating the hepatic stellate cells (HSC) and myofibroblasts^13.

This pro-inflammatory condition promotes the synthesis of ECM components leading to fibrogenesis in the liver. HSCs are one of the major focuses in the development of alcoholic liver fibrosis, and the leading producers of ECM components. Thus, acetaldehyde directly interferes with HSC activation and collagen-I genes through the binding of its adducts either to malondialdehyde or 4-hydroxynonenal. In this case, alcohol also supports liver fibrosis by way of endotoxin and immunologic reactions. Ethanol which is recognized to disrupt TJ’s of the intestinal walls enhances permeability of the gut between epithelial cells. High levels of LPS lead to the generate ROS and cytokines by KCs which in turn activate HSC via the TLR4 pathway. Hence, the intense immunological activity which involves different types of liver cells and signal transducers, results in alcohol induced liver fibrosis.^10-20

Preclinical models of ALD:

Lieber – DeCarli

The Lieber-DeCarli liquid diet was described in 1963 particularly for the purpose of using the diet to observe the in vivo effects of alcohol consumption. Rats were given a 15% ethanol solution for 177 days, and those fed ethanol alone showed no liver damage. The researchers hypothesized that starvation caused the liver damage from alcohol use, and alcohol alone was generally considered to pose no hepatotoxic risk^21-22

Already in the 1960s, Lieber et al. synthesized diet with ethanol and other components to investigate the liver pathology. It was discovered that rats are averted from ethanol acceptation and, thus, are unable to imbibe sufficient alcohol to affect their livers. However, in case of a liquid diet formula enriched with ethanol, rats were able to intake 12-18g/kg of ethanol per day, that is, two-three times more compared with the ethanol-only solution. This led him to his last proposition that alcohol is pathogenic and capable of causing liver diseases. This study also established that vitamin A, dietary fat, gender and essential nutrients influenced this process. These results brought a new epoch of the study on liver diseases.^23-29

The Lieber-DeCarli diet is a liquid diet with a constant caloric content of 0.6 to 1.0cal/mL. It consists of 18% casein, 35% fat from olive and corn oils, and 47% energy from maltose mixture and dextrin. 36% of the dextrin and maltose mixture is eliminated from the ethanol-containing formula and replaced with isocalorically measured alcohol. The diet's ethanol content is gradually increased over a primer period of five days, allowing the animal to adjust to the ethanol-contained meal. This brief priming phase ensures the impact of formal feeding on the animal^24-27

The LDE model suggests that rats and mice can be fed for 1 to 9 months, with elevated AST and ALT levels and increased hepatic triglycerides. However, no significant hepatic pathological alterations, especially severe ones like fibrosis, have been linked to the LDE diet, even in rats fed for up to nine months at a time. This is due to the LDE diet's ability to sustain a low BAC in animals^30-38

Again, due to its toxicological effects the LDE diet has been modified to bring about severe hepatic injury in rodents to model ALD in human. Chronic alcohol consumption and the more recent episodes of binge drinking increase the chances of developing ALD. In the chronic-binge ethanol intake preclinical model, the LDE diet is used to give one or more binges of ethanol followed by a chronic phase. The diet is given for 4 weeks, during the chronic eating phase one or more binges are given twice a week. This result in ability to reach BAC’s of 200-500mg/dL in rodent subjects whereby serum transaminases are elevated and steatohepatitis is induced^40-45

Research indicates that binge drinking can introduce additional hepatotoxins, such as APAP, DEN, LPS, and CCL4, during the chronic feeding phase of the liver deprivation diet, worsening liver damage and increasing the application of the LDC diet.

Ethanol ad libitium:

Although there was no observable mortality within the 8-70 weeks of alcohol consumption, the libitum feeding enough to cause liver damage published by this research supported steatosis and more ALT/AST levels though not fibrosis/ cirrhosis lesions in majorities^46-51

Ad libitum feeding is a method for assessing alcohol-related liver disease (ALD) in rodents and mice. Despite its ease of use, rodents have a strong innate dislike for alcohol and have faster metabolism of alcohol compared to humans. This prevents them from reaching high blood alcohol content during long-term ethanol feeding. Early ALD investigations were influenced by low blood alcohol (BAC) levels, with some studies showing a moderate increase in serum ethanol and significant BAC fluctuations. Ad libitum feeding is challenging to assess nutritional status compared to the LDC diet, which ensures equal calories regardless of alcohol consumption ^47,68,52

Subsequently, ethanol ad libitum intake has been accompanied by other stressors to induce inflammation, fibrosis or even HCC in the liver. It is the model of moderate alcoholic liver injury and if desired, can take over from the regular long-term feeding for a certain period because the mortality rate is low. Research has also come up with some secondary indicators to check whether or not particular dietary paradigms worsen chronic alcoholic liver disease. Ethanol ad libitum feeding is a preferred technique for the induction of ALD because it is very acceptable for secondary hits.^52-55

Tuskamoto French intragastric infusion model:

The Tsukamoto-French (TF) model, a new feeding method for mice, was created in 1984 to overcome limitations of oral alcohol administration. The average blood alcohol content in rodents is typically less than 150 mg/dL, and liver steatosis is the predominant pathological alteration in studies without fibrosis or cirrhosis. This approach allows for a more effective and practical m ethod for administering alcohol to mice^56-57

The TF infusion model offers unique benefits over traditional oral feeding strategies by eliminating restrictions on alcohol intake. An early trial used a liquid diet with a 30-day infusion and alcohol, leading to severe liver steatosis and localized necrosis in rats with high blood alcohol content. TF model allows for easy modification of meal content to produce appropriate liver damage models. After 120 days, all animals showed signs of fibrosis, and supplementing the diet with carbonyl iron resulted in fibrosis in most mice, with 2 out of 20 having liver cirrhosis^58-60

The mouse experiment indicated that TF infusion in the stomach will result in high BAC even after 4 weeks with BACs above 400 mg/dL at its heights. Thus, they progress liver injury similar to acute liver disease (ALD) in human beings including cirrhotic changes^61-62

The intra-gastric tube-fasted model in which an intragastric tube is inserted into rats requires technical and surgical skills, the post-operative care needed is stringent and the overall management might prove tedious to sustain for 2 to 3 months. In this case, having open access of the cannula aggravates the danger of infection and irritation, including fatal results. Nevertheless, it is possible to prolong the lifes of rats with daily food infusion and implanted cannula up to six months, so when organized properly; intragastric infusion can become a valid model for studying experimental dietary conditions in ALD.⁶³

The TF model, initially designed to study ethanol intake in mice, has shown severe alcoholic liver impairment compared to other alcohol administration methods. It has also been used to research obesity-related conditions like NAFLD, making it a reliable method⁶⁴

The National institute on Alcohol Abuse and Alcoholism NIAA model:

This acute or chronic alcohol steato/hepatitis model. The mice were alcoholic for 10 days by feeding them with a liquid diet, in which 5% was ethanol, then giving ethanol on the 11th day and euthanizing the mice 9 hours later. It leads to inflammation, generation of liver lesion and increased blood alcohol concentration. The reduction of NIAA, associated with the changes in the paradigm in which alcohol infusions have been done during a single binge or multiple intragastric infusions following chronic feeding with freshly prepared LDE diet enhances neutrophil infiltration in mice.⁵⁹

Non Alcoholic fatty liver disease:

Non-alcoholic fatty liver diseases are hepatic manifestations which are associated with metabolic syndrome. They are characterized by obesity, insulin resistance, fasting hyperglycemia, dyslipidemia, and altered adipokine profiles. As obesity and insulin resistance become more prevalent, their incidence increases, making NAFLD the most common cause of chronic liver disease. The disease progresses from simple liver steatosis to non-alcoholic steatohepatitis (NASH), liver fibrosis, and cirrhosis. NASH in combination with fibrosis or cirrhosis the risk for developing liver cancer (hepatocellular carcinoma) also increases. The histological appearance of the liver changes with the progression of the disease in these individuals^68-74

The inflammatory reaction referred to as NASH is caused by a decrease in the export, or oxidation, of free fatty acids leading to hepatic steatosis. There are still questions about the origin of pathogenicity in NAFLD. In order to study the development of the disease, animal models that express the exact pathology of each stage of NAFLD are employed. This review will cover the various animal models which are commonly used for NAFLD, the identified targets for NAFLD treatment, as well as the increased incidence of NAFLD.

Pathogenesis of NAFLD:

NAFLD is characterized by hepatic steatosis, where triglycerides accumulate in hepatocytes, affecting more than 5% of them. The degree of steatosis can be less (0-33%), medium (33-66%), or high (>66%), and can fill the entire acinus^75-76

NAFLD patients' livers produce triglycerides due to the esterification of glycerol and FFAs. Overproduction of TNF-α triggers the activation of IκB kinase β, leading to insulin resistance (IR). IR raises triglyceride levels in the liver and causes liver steatosis. This occurs due to insulin's failure to inhibit adipose tissue lipolysis, upregulation of transcription factors, and inhibition of β-oxidation of FFAs. These processes increase the buildup of FFA in the liver, leading to triglyceride accumulation and steatosis. Obesity is closely linked to NAFLD, and hyperinsulinemia can inhibit β-oxidation of FFAs.^77-84

Oxidative stress:

The studies show the significant association between the levels of antioxidant activity and the extent of Non-alcoholic steatohepatitis (NASH) in steatohepatitis patients and animals. The combinations of IR and obesity causes elevate level of free fatty acid and this regarded as a primary responsible factor for oxidative stress in NASH. Three subcellular organelles undergo FFA oxidation: It takes place in peroxisomes through the β-oxidation process and in mitochondria through the β-oxidation process and ω-oxidation process that occurs in the endoplasmic reticulum. Thus, when FFA is elevated, MBFA may become overworked and produce more ROS at the same time. Oxidative stress results from a breakdown in the delicate equilibrium that exists between the generation of ROS and the cells’ ability to neutralize these particles and prevent lipid peroxidation and other types of cell damage.^85-88

Proinflammatory cytokines:

Elevated inflammation in the liver is connected to the development of NASH and consequently, deranged cytokines. NASH patients have increased pro-inflammatory cytokines such as TNF-α and IL-6 while adipokine levels could be impacted due to a decrease in adiponectin and an increase in leptin resulting in the enhancement of the obesity related chronic inflammation cycle.^89-90

Figure 2 Pathophysiology of non alcoholic fatty liver disease

Preclinical models of NAFLD:

High-fat diet (HFD):

Development of a high-fat diet (HFD) has occurred because there is an association between obesity and non-alcoholic fatty liver disease (NAFLD). The resulting nutritional composition differed as the control groups of these animal models ate meals with 18% fat, 11% carbohydrate and 71% protein over three weeks. Rats on the HF-diet patterns shown more hepatic steatosis and higher lipid contents than controlAnimals with NAFLD develop insulin resistance, similar to humans suffering from NAFLD. However, the HFD group vs. controls showed no weight gain. This is a typical result in this field because it is inversely related to diabetes and insulin resistance, and supporting information is sparse. ⁹¹

Studies have found male C57BL/6 mice fed a high-fat diet. This is the bad and the good news. The good news is that, up to 16 weeks, in terms of body weight, liver triglycerides, hepatoma, higher fasting serum glucose, and lower adiponectin levels, all changed by diet-induced obesity.” "Changes in the expression of TGF-beta1 and TGF-beta2 were similar in that a high-fat meal (HF-12) with 7 days of 10% fructose was given to male C57BL/6(NTHG) mice to increase the lipid accumulation. Dietary fats produce larger numbers of triglyceride-enriched hepatic cells and therefore more severe liver steatosis and larger body and liver weights⁹²

High-fat diet-fed animals resemble human NAFLD patients due to obesity and insulin resistance, but the severity of hepatic steatosis is influenced by variables like rodent strain, unlike other animal models.

Db/db and ob/ob mice:

For the autosomal recessive Db/Db mouse model are the homozygous status of the targeted gene, which cause inability to signal leptin. This results in normal or even high levels of leptin but does not affect it. These mice are diabetic and obese with hyperphagia, overt hyperglycemia, hyperinsulinaemia, and high serum leptin levels. Still those are good models of NAFLD it may develop methionine and choline-deficient diet. Prolonged intake of calories in amounts above what the body requires might worsen the inflammation of the liver.⁹³

Ob/ob mice: cause autosomal recessive leptin gene mutation; isoform which executes signals contains non-functional, truncated leptin receptors. They are obese, clad in hyperplasia, hyperinsulinaemia, hyperglycemia, insulin resistance, and spontaneous steatosis in the liver. It is provoked by secondary hits such as endotoxin, ethanol, MCD diet, or a high-fat diet. Therefore, hepatic fibrosis could not be induced in Ob/ob mice.⁹⁴

Hence, the ob/ob and db/db mice are useful because they exhibit human metabolic syndrome phenotypes and NAFLD because while on the normal diet the former exhibits hepatic steatosis. However, since leptin resistance is very rare in obese human beings and congenital leptin deficiency, it is impossible to have perfect synchronization between animal models and human IR, hepatic steatosis, and obesity etiology.

Preclinical models of NASH:

MCD dietary model:

A dietary model of NAFLD that is followed is by feeding the mice with a lipogenic MCD diet; methionine and choline deficiency diet, high sugar. Choline is transported and metabolized in liver thus a diet containing low methionine and choline can lead to severe fibrosis and hepatic inflammation. The diet is close to the pathological features of severe clinical NASH and the animals show signs of inflammation and fibrosis, and hepatocyte necrosis. It also enshrines pathways which are associated to the human NASH development. These models experience higher stress in cell organelles, oxidative stress, and autophagocytic stress which make it ideal for modeling histological progressed NASH and research on fibrosis and inflammation in NASH.

This model resembles clinical NASH and is limited by several characteristics that differ from clinical metabolic NASH. The mice on the MCD diet had muscle atrophy, reduced fat mass contributing to weight loss, and low blood insulin concentrations. To induce NASH like condition in mice, different MCD diets are normally provided to db/db or ob/ob mice. Inflammation and fibrosis of the liver can be detected in db/db animal. There are differences regarding the reactions of mouse strains to MCD diet on long-term feeding, which might cause hepatocarcinogenesis in DBA/2 J mice and different levels of liver damage in C57BL/6 mice.^95-96

High-cholesterol diet (HCD):

New studies indicate that dietary cholesterol may play a role in human and animal models’ hepatic inflammation and steatohepatitis. Only a high cholesterol diet elevates the serum insulin concentration, body and liver weight, serum triglyceride, FFA concentration and ALT in mice. The characteristic of NASH becomes further manifested when complemented with high fat or cholate feeding. The mice fed on the diet with high fat content (15%) and high cholesterol content (1%) (HFHC) gained weight faster, had more deposition of lipids in liver, H/E stain showed hepatocytes is 10 folds higher in compared to control, adiponectin levels were reduced, inflammation in white adipose tissue and fibrosis occurred. The experimental rats fed on the diets supplemented with cholesterol (1.25%) and cholate had hepatocellular ballooning and fibrosis, inflammation and steatosis⁹⁷

High-fructose diet:

Consumption of high-fructose foods also increased subjects' likelihood to develop both obesity and non-alcoholic steatosis (NASH). Investigations exhibited a higher hepatic endothelin-1 content and malondialdehyde amount together with increments in hepatic oxidative stress, liver fibrogenesis, and collagen deposition in mice provided NO for 20 weeks. In other words, fructose consumption is required for liver fat deposition, the first step in fibrogenesis. The study, however, found that the CXCR3 receptor might be crucial for the development of NASH in HFHF mouse models, as documented by improved liver histology, decreased lipid peroxidation, and reduced necroinflammation.

Foz/foz mice:

It is compared that an appetitive deficiency was more severe in foz/foz C57BL6/J mice than in foz/foz BALB/c mice, which eliminate strain factor. With regard to NAFLD, foz/foz C57BL6/J mice showed elevated IR, obesity, hyperinsulinemia, and liver fibrosis levels⁹⁸

Db/db mice supplemented with iron:

In db/db mice study excess iron was reported to cause NAFLD, NASH, fibrosis and chow diet containing high iron prompted hepatoma fibrogenesis increased hepatic oxidative stress, inflammation activation and impaired mitochondrial fatty acid β-oxidation.

LIST OF ABBREVIATIONS:

ALD alcoholic liver disease, NAFLD non alcoholic fatty liver disease, ROS reactive oxygen species, NASH non alcoholic steatohepatitis, MCD methionine choline deficiency, HCC hepatocellular carcinoma, NA not available, NF nitrogen free, IR insulin resistance.

CONCLUSION:

ALD and NAFLD are different disease entities of Hepatic diseases. However, they have several similarities in pathogenesis through lipid accumulation, inflammation, and oxidative stress. Despite the continued prominence of preclinical studies in the identification of these diseases and the development of their treatments. Future research directions should shift towards translating these experimental research works into clinical applications that have the potential to better manage ALD and NAFLD patients’ conditions.

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Received on 06.08.2024 Revised on 05.12.2024

Accepted on 11.02.2025 Published on 28.02.2025

Available online from March 03, 2025

Asian J. Pharm. Res. 2025; 15(1):51-59.

DOI: 10.52711/2231-5691.2025.00009

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

Model Name	Species & Age	Animal used	Characteristics	Pros &cons	Mortality
Lieber- Decarli^65-66	C57BL6/J Male 4-12 Weeks	Rat/Mice	Chronic ethanol intake	Easy Procedure Significant ALT elevation Temporary feeding No liver fibrosis	No mortality
	C57BL6/J Male 4-6 Weeks	Rat /Mice	Chronic ethanol + binges	Easy Procedure High ALT with Steatosis Long term feeding No liver fibrosis	High mortality
	Male 4-12 Weeks	Rat/Mice	Ccl4 lipolysaccharide second hit model	Easy Procedure High ALT with Steatosis Fibrosis liver	High mortality
Ethanol ad libitum feeding^65-66	C57BL6/J 1-2 Weeks	Mice	Oal alcohol water	Easy Procedure Minimal ALT rise and mild steatosis	No mortality
Tuskamoto French model ^65-66)	C57BL6/J 2-3 Months	Rat /Mice	Intra gastric infusion	Tough to execute High ALT with steatosis Mild liver fibrosis Cost effective and timely	High mortality
The NIAA mode⁶⁷		Mice	Single ethanol binge + LDE	High alcohol blood levels Fatty liver

Model name	Diet composition	stage	Pros & cons
High fat diet ⁹¹	Fats make 45-75% of the animals total calories. The original said that HFD model initially contained 71% fat, 11% carbohydrate, 18% protein.	Steatosis present Fibrosis present Hepatisis present HCC absent	Weight loss Satiety and reduces hunger
ob/obmice⁹⁴	NA	Steatosis present Fibrosis absent Hepatisis absent HCC absent	Weight gain High blood sugar Increased levels of insulin
Db/dbmice.⁹³	NA	Steatosis present Fibrosis absent Hepatisis absent HCC absent	Weight gain Increased insulin levels
Methonine and choline deficient diet ^(95-96)	The typical diet consists of 40% sugar and 10% fat, although it lacks methonine and chloine.	Steatosis present Fibrosis absent Hepatisis absent HCC absent	Reduces liver steatosis and inflammation.
Highchloestrol Diet⁹⁷	Around 1% of an animal's total calorie intake comes from cholesterol, which is frequently provided in combination with high fat (15%) and high cholesterol (0.5%).	Steatosis present Fibrosis present Hepatisis absent HCC absent	Weight gain High blood sugar
Choline-Deficient L-amino acid-Defined Diet	12.7 kcal/g L-arginine, 15.8 kcal/g L-Aspartic acid, 28.9 kcal/g L-Glutamic acid, and 10.5 kcal/g of Leucine no addition of bitartrate of choline materials available.	Steatosis present Fibrosis present Hepatisis present HCC present	Reduces liver steatosis
High-fat diet+streptozotocin	With a 200 ug injection of streptozotocin, there was 14.4% fat, 46.7% NF extract, and 24.8% protein.	Steatosis present Fibrosis present Hepatisis present HCC present	Weight gain Increased insulin levels
foz/foz mice^.98	NA	Steatosis present Fibrosis present Hepatisis present HCC absent	Significantly reduce adiponectin levels Weight gain