Recently, the need for more standardized operation procedures in experimental liver fibrosis research was suggested due to dramatic changes in European animal welfare rules. Here, we present a short series of standard operation procedures (SOPs) summarizing the most relevant and widely accepted experimental models for the induction of liver injury leading to liver fibrosis. The described procedures are based on the long-term experience of the Collaborative Research Centre ‘Organ Fibrosis: From Mechanisms of Injury to Modulation of Disease’ ( http://www.sfbtrr57.rwth-aachen.de/ ), which is supported by the German Research Foundation (SFB/TRR57). These SOPs will help to improve standardization of fibrosis models and to increase the comparability of data between different laboratories with the aim of reducing animal experimentation according to the principle that was proposed in 1959 by Russell and Burch as an ethical framework for conducting scientific experiments with animals, namely the replacement, refinement and reduction (3R) principle. In the first section we focus on the carbon tetrachloride (CCl₄) model in mice, which is the toxic model of liver fibrosis induction most commonly used worldwide.

A major concern in therapy of acute liver failure is protection of hepatocytes to prevent apoptosis and maintain liver function. Small interfering RNA (siRNA) is a powerful tool to silence gene expression in mammalian cells. To evaluate the therapeutic efficacy of siRNA in vivo we used different mouse models of acute liver failure. We directed 21-nt siRNAs against caspase 8, which is a key enzyme in death receptor-mediated apoptosis. Systemic application of caspase 8 siRNA results in inhibition of caspase 8 gene expression in the liver, thereby preventing Fas (CD95)-mediated apoptosis. Protection of hepatocytes by caspase 8 siRNA significantly attenuated acute liver damage induced by agonistic Fas (CD95) antibody (Jo2) or by adenovirus expressing Fas ligand (AdFasL). However, in a clinical situation the siRNAs most likely would be applied after the onset of acute liver failure. Therefore we injected caspase 8 siRNA at a time point during AdFasL- and adenovirus wild type (Adwt)-mediated liver failure with already elevated liver transaminases. Improvement of survival due to RNA interference was significant even when caspase 8 siRNA was applied during ongoing acute liver failure. In addition, it is of particular interest that caspase 8 siRNA treatment was successful not only in acute liver failure mediated by specific Fas agonistic agents (Jo2 and AdFasL) but also in acute liver failure mediated by Adwt, which is an animal model reflecting multiple molecular mechanisms involved in human acute viral hepatitis. Consequently, our data raise hope for future successful application of siRNA in patients with acute liver failure.

Liver fibrosisis defined as excessive extracellular matrix deposition and is based on complex interactions between matrix-producing hepatic stellate cells and an abundance of liver-resident and infiltrating cells. Investigation of these processes requiresin vitroandin vivoexperimental work in animals. However, the use of animals in translational research will be increasingly challenged, at least in countries of the European Union, because of the adoption of new animal welfare rules in 2013. These rules will create an urgent need for optimized standard operating procedures regarding animal experimentation and improved international communication in the liver fibrosis community. This review gives an update on current animal models, techniques and underlying pathomechanisms with the aim of fostering a critical discussion of the limitations and potential of up-to-date animal experimentation. We discuss potential complications in experimental liver fibrosis and provide examples of how the findings of studies in which these models are used can be translated to human disease and therapy. In this review, we want to motivate the international community to design more standardized animal models which might help to address the legally requested replacement, refinement and reduction of animals in fibrosis research.

The toxic properties of various nitrosamines in animals and humans are well established. The parenteral or oral administration of the smallest quantities of diethylnitrosamine (DEN) or dimethylnitrosamine (DMN) results in severe liver damage. Most prominent are intense neutrophilic infiltration, extensive centrilobular haemorrhagic necrosis, bile duct proliferation, fibrosis, and bridging necrosis that ends in hepatocarcinogenesis. Due to the robustness of the induced hepatic alterations, the application of DEN in rodents has become an attractive experimental model for studies aimed at understanding the pathogenetic alterations underlying the formation of liver cancer, which represents one of the most common malignancies in humans worldwide. However, several studies have shown that the hepatocarcinogenic effects of nitrosamines might vary with the genetic background of the animals, their sex, their age, and other factors that might impact the outcome of experimentation. We present general guidelines for working with DEN, and a detailed protocol that allows the establishment of highly reproducible liver cancer in mice. The outcome of liver injury after the application of DEN in mice, as estimated by the formation of cirrhosis and cancer, appears to be a suitable animal model for the analysis of some aspects and processes that promote the pathogenesis of hepatocellular carcinoma in humans.

Idiosyncratic drug-induced liver injury (DILI) is a type of hepatic injury caused by an uncommon drug adverse reaction that can develop to conditions spanning from asymptomatic liver laboratory abnormalities to acute liver failure (ALF) and death. The cellular and molecular mechanisms involved in DILI are poorly understood. Hepatocyte damage can be caused by the metabolic activation of chemically active intermediate metabolites that covalently bind to macromolecules (e.g., proteins, DNA), forming protein adducts—neoantigens—that lead to the generation of oxidative stress, mitochondrial dysfunction, and endoplasmic reticulum (ER) stress, which can eventually lead to cell death. In parallel, damage-associated molecular patterns (DAMPs) stimulate the immune response, whereby inflammasomes play a pivotal role, and neoantigen presentation on specific human leukocyte antigen (HLA) molecules trigger the adaptive immune response. A wide array of antioxidant mechanisms exists to counterbalance the effect of oxidants, including glutathione (GSH), superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPX), which are pivotal in detoxification. These get compromised during DILI, triggering an imbalance between oxidants and antioxidants defense systems, generating oxidative stress. As a result of exacerbated oxidative stress, several danger signals, including mitochondrial damage, cell death, and inflammatory markers, and microRNAs (miRNAs) related to extracellular vesicles (EVs) have already been reported as mechanistic biomarkers. Here, the status quo and the future directions in DILI are thoroughly discussed, with a special focus on the role of oxidative stress and the development of new biomarkers.