**4. Regulatory issues regarding the use of animal models in CLD research**

Biotechnological advances in medicine and pharmaceutical sciences during recent decades have provided us with tools for better diagnosis and treatment of CLD. This has gone hand in hand with the exponential rise in the use of animal models for research. Animal models in preclinical trials have largely served as the scientific basis for the data that has enabled a better understanding of the pathophysiology of human disease. However, researchers around the world are currently expressing concern about the use of animals for scientific experimentation [104].

To make use of animal models in the field of hepatology, as well as in other medical areas, we must essentially consider various aspects regarding the rational use and management of animals for their use in research protocols. Whenever animals are used as research models, we must keep in mind that the main purpose of the study is to obtain feasible, reproducible, and reliable results inasmuch CLD comprises an important health problem that requires prompt study and attention. One way to achieve this is to reduce the stress to which animals are subjected throughout the study. Aforesaid, the

induction of the different stages of CLD requires handling the animals for prolonged periods of time, which can affect and compromise animal welfare. On the other hand, we already know that stress influences affective behavior and stress hormone release may alter the pathophysiology of the CLD [105–108]. For this reason, the scientific community has been made aware of the importance of ensuring the socio-environmental welfare of experimental animals. In Europe, for example, there are regulations regarding the use of experimental animals and a series of standards have been established regarding protection criteria and ethical issues. The scientific community has always been encouraged to implement them insofar as this is possible [109].

Discussions regarding the use of animal models are not recent and have increased over the years, involving the scientific community across different countries [110–112]. Today, there are standards such as Animals in Research: Reporting In Vivo Experiments (ARRIVE), which, since 2010, has become a useful guideline in biomedical science and related areas and is meant to improve the use and management of experimental animals from an ethical, social, and animal welfare-based perspective so as to obtain reliable results [113]. In fact, it has become common practice that, in order to publish scientific research, publishers request letters indicating that the research and ethics committees authorized the use of animals. The legal agreements, regulations, and guidelines for each country, as well as international standards, recommend that each study adheres to the most effective type of test and the most appropriate species for the study in order to reduce the number of animals to the bare minimum. Additionally, the use of more complex animal models, such as animals obtained *via* genetic engineering or humanized animal models, has required the expansion of these regulations.

One of the regulatory standard documents with international recognition is the Guide for the Care and Use of Laboratory Animals (NIH, Guide), which promotes the care and humane use of laboratory animals *via* a comprehensive program of publications, scientific protocols, and opinions based on the scientific experience of researchers using methodologies and practices that enable the desired results [114]. Another body handling international agreements is the Organization for Economic Cooperation and Development (OECD), which is responsible for regulating scientific protocols, tests, and analyses in order to guarantee quality results that adhere to human pathophysiology [115–119].

The implementation, development, and use of more complex animal models requiring special care will necessitate new international standards and agreements to develop better strategies and standardized protocols for scientific research. However, animal replacement is still far from becoming a viable alternative for the comparative study and development of models of human pathophysiology, at least in regard to CLD.

### **5. Conclusion and future outlook**

Clinically speaking, the management of patients with CLD is difficult because, as mentioned above, the disease goes through several stages, each of which requires a different kind of therapeutic intervention. Clinicians and researchers still face many challenges regarding the management and study of CLD. However, there are possible ways of overcoming these challenges. More and more animal models have become available for the study of diseases, parallel to the discovery and development of new drugs. However, it is essential that we continue to improve and validate these models, particularly with regard to the molecular mechanisms that trigger and perpetuate the disease. This will ensure that they truly reflect each of the histopathological stages of

human disease and will increase their predictive validity, as well as their use in the discovery of new therapeutic options.
