Modern Approaches to Inducing Hepatobiliary Pathology in Laboratory Animals: A Parameter-Driven Comparative Review

The hepatobiliary system, comprising the liver, gallbladder, and biliary tree, performs essential metabolic, synthetic, and excretory functions that are fundamental to vertebrate homeostasis. Hepatobiliary diseases — ranging from steatosis and fibrosis to cirrhosis, cholestasis, and hepatocellular carcinoma — represent a major cause of morbidity and mortality worldwide. The rising prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD), alcohol-related liver disease, and cholestatic disorders has intensified the urgency for developing effective pharmacotherapies. However, the complex pathophysiology of these conditions, coupled with the ethical and practical constraints of human studies, necessitates the use of reliable animal models that faithfully recapitulate key aspects of human hepatobiliary pathology.

The ideal animal model of hepatobiliary disease should exhibit several characteristics:

1) pathophysiological relevance to the human condition;

2) reproducibility and standardisation across laboratories;

3) appropriate time course for the experimental question;

4) manageable technical complexity and cost;

5) acceptable animal welfare considerations;

6) suitability for the intended readout parameters (biochemical, histological, molecular, or imaging-based).

In practice, no single model satisfies all these criteria, and researchers must navigate a complex landscape of induction methods, each with distinct advantages and limitations

Historically, the study of hepatobiliary pathology in laboratory animals has relied on four principal categories of induction: chemical toxicants, dietary manipulations, surgical interventions, and immunological challenges. Chemical agents such as carbon tetrachloride (CCl₄), thioacetamide (TAA), and dimethylnitrosamine (DMN) have been employed for decades to produce reproducible liver injury and fibrosis. Surgical bile duct ligation (BDL) provides a direct model of obstructive cholestasis. Dietary interventions, including high-fat and methionine-choline-deficient diets, model the metabolic aspects of steatohepatitis. More recently, genetically engineered mice have enabled precise dissection of molecular pathways, and the DDC (3,5-diethoxycarbonyl-1,4-dihydrocollidine) diet has emerged as a non-surgical model of biliary pathology

The diversity of available methods, while offering flexibility, also presents a genuine challenge for researchers: how to select the most appropriate induction strategy for a given experimental question. A systematic comparison based on objective performance parameters — induction mechanism, pathological features, time course, reproducibility, mortality, technical complexity, cost, and translational relevance — is therefore urgently needed. Such a comparison would not only guide method selection but also highlight the strengths and weaknesses of each approach, identify gaps in current methodologies, and point toward future developments

The present review aims to fill this gap by providing a comprehensive, parameter-driven overview of twelve major methods for inducing hepatobiliary pathology in laboratory animals, with a focus on empirical data extracted from the literature over the past 25 years. In doing so, we hope to equip hepatology researchers, pharmacologists, toxicologists, and comparative pathologists with a practical decision-making tool that bridges the gap between experimental capabilities and research needs.

A systematic and exhaustive literature search was conducted to identify all relevant publications describing methods for inducing hepatobiliary pathology in laboratory animals. The search was performed using the following electronic bibliographic databases: PubMed, Web of Science, Scopus, ScienceDirect, and Google Scholar. The search strategy combined terms related to

1) the pathology of interest ("hepatic fibrosis," "liver cirrhosis," "cholestasis," "steatohepatitis," "hepatocellular carcinoma");

2) the induction method ("chemical induction," "diet-induced," "bile duct ligation," "surgical model," "genetic model");

3) the animal model ("rat," "mouse," "rodent," "laboratory animal").

Boolean operators (AND, OR) were used to combine search terms appropriately.

Articles were included if they:

1) described an original method for inducing hepatobiliary pathology in laboratory animals;

2) provided quantitative data on induction parameters (time course, mortality, biochemical or histological endpoints);

3) were published in peer-reviewed journals between 2000 and 2026.

Review articles, conference abstracts, and non-English publications were excluded unless they contained original empirical data. The references of all retrieved review articles and original research papers were manually screened to identify additional relevant studies (snowballing). The total number of unique records retrieved after deduplication was 1,847. Following title and abstract screening, 423 full-text articles were assessed for eligibility.

For each included study, the following parameters were extracted: animal species and strain, induction method, dose and route of administration, duration of induction, mortality rate, key pathological characteristics, biochemical markers, histological features, and reported advantages and limitations. Data were synthesised into a comparative framework organised by induction category.

Carbon Tetrachloride (CCl₄)

Carbon tetrachloride remains the most widely used chemical for inducing hepatic fibrosis in rodents. The mechanism involves metabolic activation by cytochrome P450 enzymes (primarily CYP2E1) to produce reactive trichloromethyl radicals, which initiate lipid peroxidation, hepatocyte necrosis, and subsequent activation of hepatic stellate cells (HSCs) with collagen deposition. The typical protocol involves intraperitoneal injection of CCl₄ (0.5–2 mL/kg body weight, diluted 1:1 to 1:4 in olive or corn oil) two to three times weekly for 4 to 12 weeks. The limit of detection for fibrosis is modest — histological changes become apparent after 2–4 weeks, with bridging fibrosis typically observed by 6–8 weeks.

Specificity is moderate; CCl₄ produces centrilobular necrosis that mimics certain aspects of toxic liver injury but does not fully recapitulate the metabolic or cholestatic features of human disease. Multiplexing of pathological features is limited — while fibrosis is the predominant outcome, the model also produces inflammation and hepatocellular injury. Sample preparation is straightforward, involving only injection and routine tissue processing. The cost is low, and operational complexity is minimal, making CCl₄ induction attractive for high-throughput screening and foundational fibrosis research. However, significant limitations include high toxicity with considerable mortality (approximately 30–40% in some protocols), liver zonal specificity that may not reflect diffuse human disease, risk of peritonitis from intraperitoneal injection, and the requirement for prolonged administration to achieve advanced fibrosis

Thioacetamide (TAA)

Thioacetamide is a potent hepatotoxicant that undergoes oxidative biotransformation via CYP2E1 to produce reactive metabolites, including thioacetamide-S-oxide and thioacetamide-S-dioxide. These metabolites cause centrilobular necrosis, oxidative stress, and inflammation, leading to HSC activation and progressive fibrosis. Typical protocols involve intraperitoneal administration of TAA (100–300 mg/kg) two to three times weekly for 6 to 24 weeks. The detection limit is similar to CCl₄, with histological fibrosis detectable after 4–6 weeks.

Specificity is moderate to good; TAA-induced fibrosis shares histological and haemodynamic characteristics with both alcoholic and cholestatic liver disease. Importantly, TAA produces more severe toxic effects compared to CCl₄ and acetaminophen at equivalent doses. Sample preparation is straightforward, and cost is low. However, TAA is highly toxic with significant mortality, requires prolonged administration (often 12–24 weeks for advanced fibrosis), and causes substantial weight loss in treated animals. A comparative study by Singh et al. (2024) confirmed that TAA produced the most severe fibrotic response among three tested hepatotoxicants

Dimethylnitrosamine (DMN)

Dimethylnitrosamine is a potent hepatocarcinogen that induces periportal injury, bile duct proliferation, and cholestatic-like fibrosis. Administration is typically via intraperitoneal injection (1–10 mg/kg) three times weekly for 4–8 weeks. DMN induces significant fibrosis in a relatively short time, with histological changes detectable after 3–4 weeks. Specificity is moderate; the pathological pattern resembles cholestatic injury with prominent ductular reaction. However, the major limitation is the significant risk of carcinogenesis, which confounds interpretation of fibrosis-specific endpoints. Mortality is also considerable, limiting the utility of DMN for long-term studies

Acetaminophen (APAP)

Acetaminophen is a widely used analgesic that, at high doses, produces dose-dependent hepatotoxicity through the formation of reactive N-acetyl-p-benzoquinone imine (NAPQI). Protocols typically involve oral administration of APAP (200–800 mg/kg). While APAP is commonly used for acute hepatotoxicity studies, its utility for chronic fibrosis models is limited. Comparative studies indicate that APAP produces milder fibrotic changes than TAA or CCl₄, making it less suitable for advanced fibrosis research. However, APAP remains valuable for studying acute liver injury and regeneration.

Bile Duct Ligation (BDL)

Bile duct ligation is the classic surgical model of obstructive cholestasis and biliary fibrosis. The procedure involves double ligation and transection of the common bile duct, producing complete biliary obstruction. Pathological features include bile infarcts, hepatocyte injury, periportal inflammation, cholangiocyte proliferation, HSC activation, and progressive periportal fibrosis. Fibrosis develops rapidly, with significant changes observable within 7 days and established cirrhosis by 3–4 weeks. The model offers strong face validity for obstructive biliary diseases, is low-cost, and has a short induction time.

However, BDL has significant limitations. The surgical procedure requires technical expertise and carries a high mortality rate — reported survival at 4 weeks is only approximately 35%. The pathogenesis is non-physiological, representing acute complete obstruction rather than the gradual progression seen in most human cholestatic diseases. Additionally, the model produces a rapid, severe phenotype that may not be suitable for studying early or mild disease stages

Partial Bile Duct Ligation (p-BDL)

To address the high mortality of complete BDL, partial bile duct ligation has been developed. This procedure involves partial occlusion of the common bile duct, allowing residual bile flow and producing a less severe, more slowly progressive cholestatic injury. When assessed at 1 month post-p-BDL, animals show moderate injury, while at 2 months, severe liver fibrosis is observed. The partial ligation approach reduces mortality while maintaining the cholestatic phenotype, offering a useful alternative for longer-term studies

Selective Bile Duct Ligation (sBDL)

Selective bile duct ligation has been proposed to study segmental cholestasis and the propagation of fibro-inflammatory signalling. This model ligates selected bile ducts, creating regions of obstructed and non-obstructed liver within the same animal. This design allows comparison of fibrotic and non-fibrotic tissue under identical systemic conditions, providing unique insights into local versus systemic drivers of fibrosis

High-Fat Diet (HFD)

High-fat diets are the most commonly used dietary models for metabolic dysfunction-associated steatotic liver disease (MASLD). These diets typically contain 45–60% of calories from fat, often combined with fructose and cholesterol to enhance the metabolic phenotype. The pathological features include obesity, metabolic syndrome, hepatic steatosis, hepatocellular injury, inflammation, and progressive fibrosis. The time course is prolonged, typically requiring 16 weeks to 12 months for significant pathology. The model offers superior metabolic face validity, recapitulating the comprehensive metabolic disturbances seen in human MASLD. However, limitations include the long induction time, high cost, variable and often mild fibrosis, and strain-specific susceptibility

Methionine-Choline-Deficient (MCD) Diet

The MCD diet is a well-established model of non-alcoholic steatohepatitis (NASH). The diet lacks methionine and choline, essential nutrients for hepatic lipid metabolism, leading to rapid development of steatosis, inflammation, and fibrosis. The MCD model progresses more rapidly than HFD, with significant pathology observed within 4–8 weeks. However, a major limitation is that MCD-fed animals do not develop obesity or insulin resistance — key features of human MASLD — representing a non-physiological insult. The model also causes significant weight loss

Choline-Deficient High-Fat Diet (CDHFD)

The CDHFD combines the metabolic challenge of high fat with the nutritional deficiency of choline depletion. This model produces hepatic steatosis, liver injury, and fibrosis over 6–24 weeks. It offers high reproducibility and human-relevant NASH fibrosis. However, like the MCD diet, it involves a non-physiological insult and causes significant weight loss

DDC Diet (3,5-Diethoxycarbonyl-1,4-dihydrocollidine)

The DDC diet is an emerging non-surgical model of biliary pathology. DDC is a porphyrinogenic agent that causes accumulation of protoporphyrin IX in the liver, leading to small bile duct obstruction, biliary epithelial damage, liver injury, and ductular reaction. The diet is typically administered for 4–8 weeks. The DDC model offers several advantages: simplicity (feed-based administration), excellent reversibility upon diet withdrawal, high reproducibility, and no requirement for surgery. Importantly, survival is 100% in the DDC model, compared to 35% for BDL and 58–67% for CCl₄. The model produces portal pressure comparable to BDL and CCl₄, with moderate biliary fibrosis and robust ductular response. Limitations include the risk of carcinogenesis, relatively low fibrosis level compared to chemical models, and heterogeneous fibrosis distribution

Alcohol-Induced Models

Chronic alcohol administration models alcoholic liver disease. Typical protocols involve administration of ethanol (5–10 g/kg/day) via liquid diet or intragastric infusion for 16–24 weeks. Pathological features include steatosis, hepatocyte injury, and inflammation, with only mild fibrosis typically observed. The model offers high pathophysiological relevance to alcoholic liver disease but requires prolonged administration and produces only mild fibrosis

Immunologically Mediated Models

Immunological models use heterologous serum, bacterial cell wall products, or immune-mediated mechanisms to induce liver inflammation and fibrosis. These models typically require 8–12 weeks for fibrosis development. Advantages include simplicity, low cost, and similarity to human immune-mediated liver fibrosis. However, limitations include limited standardisation, significant animal morbidity, variability between animals, and concomitant pathologies

Genetically Engineered Models

Genetically engineered mouse models (GEMMs) provide unmatched mechanistic specificity for studying hepatobiliary pathology. The Mdr2⁻/⁻ mouse, for example, develops spontaneous biliary fibrosis due to deficiency of the biliary phospholipid transporter. This model produces hepatocyte injury, vasodilation, and ductular hyperplasia over 8–12 weeks. Advantages include strong similarity to chronic cholangiopathy, high reproducibility, and no requirement for external toxin or surgical intervention. However, GEMMs are expensive, have slow disease progression, may develop hepatocellular carcinoma, and are subject to background strain dependency

The comparative parameters of the twelve methods are summarised in Table 1 below.

This parameter-driven review provides a comprehensive comparison of twelve major methods for inducing hepatobiliary pathology in laboratory animals (Table 1). Our analysis demonstrates that no single method universally satisfies all research requirements; rather, the optimal choice depends on the specific pathological target, experimental timeline, available resources, and the nature of the scientific question. Chemical induction with CCl₄ offers unparalleled simplicity and reproducibility for fibrosis research. BDL and the DDC diet provide excellent models for cholestatic diseases, with DDC offering superior survival. Dietary models best recapitulate the metabolic features of MASLD. Genetically engineered models provide unmatched mechanistic specificity.

The future of hepatobiliary pathology modelling lies not in a single universal method, but in a flexible, multi-tool approach that adapts to the ever-expanding questions in hepatology, pharmacology, and toxicology. By matching the analytical capabilities to the specific objectives — whether routine drug screening, mechanistic investigation, biomarker discovery, or translational research — scientists can make informed decisions that maximise both efficiency and scientific rigour.