Ethics and Science in the Lab. Challenges and Considerations on Animal Experimentation. Interview with Anayátzin Paulina Heredia

UNAM Internacional: We would like to know your opinion about research with animal models. In several articles included in this issue of UNAM International, the authors mention laboratory work with animals and we thought it was important to address this issue, both in the field of neurosciences and in the field of science in general. We would like you to tell us about your approach: what are the problems science faces when experimenting with animals? And, based on this, what care can or should be taken?
Anayatzin Paulina Heredia: The history of the use of animals for scientific research purposes is very long. In fact—and this is something I always mention—the development of medicine has paralleled the use of animals, both in terms of anatomical and physiological knowledge, and what is developed today at the molecular level. Throughout all these years of scientific development, various animal species have been used. Speaking of modern science, as we do it today in the laboratory, that practice is about 100 years old.

In the past, research was done in a less controlled way. Before the 20^th century, there were no research centers or vivariums as we know them today. Each researcher experimented practically at home; there were no spaces designed for research with animals. During the last 100 years, adequate facilities and better care of animals have been addressed. Genetic lines (breeds) are beginning to be developed with—in the case of rats and mice—this particular purpose: to obtain better models, better controlled, from the genetic, the environmental, and the procedural aspects, to obtain better data that can be used and extrapolated to other species.

Criticism of the use of animals for this purposes has been presented for many years. It is said that, as early as the mid-19^th century, Fanny Martin, the wife of Claude Bernard—considered one of the founders of physiology and famous for his use of animals for experimentation—joined the nascent Society for the Protection of Animals, trying to reverse the scientist’s actions. His way of doing research was highly questioned, although his great contribution to the methodology of scientific research is also recognized. He was criticized because at that time vivisection was still done, on fully conscious animals. The technology of anesthetics and drugs that would allow procedures to be carried out on the unconscious animal had not been developed, as they are now. There is a text describing how his wife and daughters were very frightened by the way Bernard did research. So Fanny divorces and creates one of the first animal protection groups, a movement that was then called antivivisectionist, and is the precursor of what we now know as “animalism”, of activists against animal experimentation. These concerns, then, were born in France and spread to other places. Currently we find many groups that openly criticize the way animals are used. The very use of animals for different purposes, including research, is criticized.

So there has always been this social perception, a pressure on the way animals are used. This concern, together with philosophical inquiry, pushed for the development of regulations on the use of animals.

UI: At what point did the international scientific community began to consider that experimentation could be causing pain or suffering in the animals used? When does the concern about what the animal experiences begin?
APH: A starting point is probably recognized in the work of William Russell and Rex Burch, who, in the mid-20th century, proposed the principle of  “the three R’s” [see box]. They used animals, but they criticized the way in which research was carried out with them. They realized that there were procedures that could be ethically questioned, while recognizing the importance of continuing to use animals. They said: there is no alternative; it is necessary to carry out experimental research with animals because of the great benefits we have obtained from it. But the way in which they are used can be improved and that is why they establish this principle, now generally accepted, of the three R’s: replacement, reduction, and refinement. In the 50s, they saw replacement (replacing animals by other means) as very distant, but it is now possible through alternative methods brought about by technological development. For example, in the case of cosmetics, the much-criticized testing of cosmetics in animals already has alternative methods. We should no longer use animals for cosmetics testing.

Norms and regulations change in each country. There are countries that for 20 or 30 years, especially European countries—which have the longest history in this area—have regulations that prohibit testing of cosmetic products on animals. In Mexico, this type of test is still being done. An initiative was presented in 2020 and was accepted; it is expected that laboratories that are still using animals for testing purposes will undertake the transition to alternative methods that rely on cell cultures.

UI: Would these alternative methods be like the organoids that current technology can make—3D structures that function as substitutes for a living organism—or computer simulations?
APH: Precisely, organoids [see box in p.272], cell cultures, software or computer programs that have been very useful in the preclinical stage. For example, the development of a new molecule that could treat a certain type of cancer. Before, it was tested directly on animals; now you have to go through several phases of these methods to evaluate a compound, its chemical characteristics, and so on. One molecule is compared with others and certain mechanisms of action or cellular interaction can be predicted. Out of a 100 products that are sought to have therapeutic potential, very few, less than 10 percent, will move on to an animal model, because they are first tested with these alternative methods.

In the case of teaching, especially for schools such as veterinary medicine, general medicine, clinical medicine, or biology, which considered animal practices in their curricula, there are now alternative methods for students to develop skills or competencies without the need to use animals, at least in the early stages of training. In the last stages it will be necessary to have direct interaction with animals or patients. In veterinary medicine, for example, there has been a lot of commitment to combined education: first they use alternative methods and then they handle the animals directly. Although it has not been replaced absolutely, the use of animals has decreased significantly. That is what replacement refers to.

There are areas in which substitution is going to be harder, such as the development of drugs or vaccines that in some stages do require animal experimentation. Experimental surgery, for example, uses quite a few animal models. But the trend is that they will be progressively replaced. In some areas it is very difficult to justify that animals are still used, as in the case of teaching. Even the use of invertebrates is considering a relative replacement (here regulations are less severe).

In research projects, the first question now is whether an alternative method exists; if not, then the use of animals would be justified, and this is where the other two “R’s” come into play: reduction—using as few animals as possible—in the first term. Based on the variability of what I’m going to measure, I establish the number of animals needed to conduct my research. Not all studies use the same number of animals, sometimes 10 is more than enough and the result is statistically valid. Other times it will be 1000 animals, and then it has to be justified: that is what the “r” for reduction refers to, to consider the minimum number of animals necessary to validate research work.

And finally, the third “r” is refinement, and the greatest development in recent years lies in this area. It addresses the conditions in which the animal will develop from birth to death, going through the experimental procedure to which it will be subjected. In the first definitions of refinement, reference was made only to the moment in which the experiment began: “My experiment will begin when the mice are two months old and weigh 25 grams”, for example. They are subjected to research procedures, such as the administration of some substance, then to a sampling, and finally to the application of an euthanasia method. Today, everything related to the animal’s welfare is considered, from the conditions in which it is born, how it grows, how it is being housed, if it is alone or in a group, what type of interaction it has with its housing mates, the substrate, the type of box, the environment. We are talking about environmental enrichment programs, which include everything that has been developed in terms of well-being. All of this falls under the concept of refinement.

This has been developing along with the consideration we have for other species and the obligation we have in terms of addressing animals’ rights. What is the minimum that an animal requires to be well both physiologically and mentally? Today a lot of importance is given to the mental part: that the animal has many positive stimuli, greater plasticity. In research, retributive justice is being addressed facing the procedures to which I am going to subject the animal to, which can often be annoying or invasive. How am I going to minimize the damage from negative stimuli? How am I going to give the animal positive stimuli so that, somehow, it does not have a bad time?

UI: Do you think that the trend towards humane treatment of research animals has been influenced by critical movements against cultural practices that involve animal abuse, such as bullfighting?
APH: Historically there has been a lot of negative perception in animal research practices. Today there is already a lot of talk about the links we have with other species in general. We have to extend our circle of moral consideration and there is already talk of greater protection for other species, there is already talk of well-being and there is greater concern, at least in big cities. It seems that it is part of a greater general concern about animal welfare.

Some European countries that, as I mentioned, have a longer history of concern for experimental animals, are pioneers in welfare. They have been developing regulations for a longer time and are even beginning to consider invertebrates. I mentioned before that replacement could be based on invertebrates. It was thought that they did not deserve consideration, that is: if I use flies or nematodes, the Caenorhabditis elegans, for example, which is widely used in research, what kind of consideration does it deserve? Now, as the mechanisms of nociception—the physiological process of reaction to damage or pain—are better known and much progress has been made in the field of neurosciences in many species. Well, this is already beginning to be considered for invertebrates; there are already institutions registering projects with invertebrates that include pain assessment.

These changes have been the result of growing knowledge about each species, which invites reflection on how to consider them. At some point this may become a norm and perhaps we will reach the time when invertebrates can also be considered. There is greater empathy; even at a social level there is already talk of “multispecies families”.

UI: Considering that we establish very intense affection relationships with our pets, cats and dogs above all, but sometimes also with some other types of animals, does the researcher in the laboratory, who work in the animal breeding facilities, also establish an affectionate relationship with their animals?
APH: In fact, it is highly recommended that you establish this bond so that the animals get to know you; that is a heavy emotional burden. We don’t want animals to be afraid of us. We don’t want that, the moment they see us putting our hand in the box, they relate it to a negative experience, something that is going to cause them discomfort, displeasure, or stress, so now we talk about conditioning, that is, training them so that it is possible to handle them much more easily.

While this started with non-human primates and vivariums, it has now spread to other species. There are already ways to train, for example, rats to allow themselves to be sampled. Animals learn and there is a lot of interaction. At some point I had a student who even applied reiki with the rats, stretched them and massaged them and those rats looked like stuffed animals, and allowed procedures to be made on them.

So, on the one hand, this has developed quite a bit and has had benefits. On the other hand, for the students this suddenly has an important emotional charge, because you live together, you form this bond throughout the procedure. And, in addition, in the end it is frequently necessary to apply a method of euthanasia, which is complicated. Something that began to be noticed in the medical and nursing area, but that is now also seen a lot in veterinary medicine is compassion fatigue; this emotional exhaustion in the face of certain practices that are done with animals. The vast majority of animals subjected to a procedure eventually have to be killed by a method of euthanasia, which is difficult for some.

UI: Is there any scientific or ethical consensus to define a pain threshold for experiments with animals?
APH: All research projects are now required to set limits. But how do you measure that, how do I set a limit? Well, by knowing various aspects of the animals and weighing them with the objective of the experimental procedure. It is a concept that we call terminal points or humane endpoints, which are indicators of pain or suffering that will allow us to make the best decision about animals.

How do we set the endpoint? Basically, by the behavior of animals in the face of patho-physiological changes or through evaluations of a hormone that is associated with pain, stress, or acute or chronic discomfort. Now researchers are making their pain assessment boards. Not all animals measure pain in the same way. The species are different; some show pain in a very clear way, while in others it is not so obvious. I always give the example of dogs because, in addition, we have a lot of interaction with them. With dogs we can easily identify when something is not right. If I have a dog as a pet, I can identify certain traits: I know then well and I identify changes that catch my attention, that tell me that something is not right: they are not eating as they usually do or they are not as active as normal. Then, given those changes I identify, I can decide if they have something and take them to the vet. The same happens in the lab: you have to observe well, and yes, you have to have that commitment. The researcher should have the commitment to know very well the species he or she is working with.

UI: Does this mean that the researcher who is working in the neurosciences field has to have training in animal behavior? Is this part of the regulations you were talking about?
APH: Maybe not specifically in terms of behavior, but there are training programs on pain management and identification. Although they are not very deep or specialized, they do allow the student of any area to identify signs of pain. And now there are several tools for this that have been developed since the 1980s, such as those that are capable of detecting changes in facial expression, body condition, or weight. There are tools that allow you to measure heart rate, respiratory rate, temperature, which are indicators that something may not be right. I can take a sample and measure cortisol, that is, I can already measure hormones that allow me to make a comprehensive evaluation.

But since we sometimes have very limited vivariums to carry out comprehensive evaluations, what do we have at hand? Always contact with the animal. That’s why many of the tables are based on behavioral changes. Training programs, no matter the area of research in which they are being developed, should have the power to identify pain.

UI: In the case of UNAM, does the research carried out with animals follow a protocol, a regulation? Is it possible to know and consult these regulations?
APH: At the national level we have a standard known as “Standard 062”: the Official Mexican Norm NOM-062-ZOO-1999, Technical specifications for the production, care and use of laboratory animals. In its articles, section 4.2 says that all institutions that carry out research, teaching, technological development, and verification must integrate internal committees for the care and use of laboratory animals. The official standards are mandatory throughout the country, so UNAM must comply with them and form these committees. However, the composition of committees in each entity has not been uniform. Among the first is that of the Faculty of Veterinary Medicine and Zootechnics and institutes such as Biomedical Research. There was an agreement to integrate committees from all over UNAM in 2019, according to which all entities had to integrate committees of different types: biosafety, environmental ethics, academic integrity. This included committees for the care and use of laboratory animals for those entities that used them. Since 2015 there has been greater integration of committees, which are important because they are the institutional mechanisms that ensure that all research projects comply with both the ethical and methodological aspects expected.

UI: One last question: Which animal species are being used the most at UNAM for research? What animals are in our vivariums and how do we take care of them?
APH: Surely the vast majority of vivariums will have rodents, mainly the mouse, which is the animal model par excellence, it is the one that is best known; we have a well-identified genome and that has allowed us to generate genetically modified models. Rats are also used quite a bit. In behavioral studies, in neuroscience studies, the rat is a widely used model. It is mentioned that, for example, for Alzheimer’s disease the best model is the rat, there has been no better animal to induce this behavioral condition, because they are also very intelligent and solve problems quickly.

In recent years, the use of zebra fish has grown a lot; countries like Canada already use zebra fish more than mammals. As a species, it has many advantages. Also the fruit fly, Drosophila melanogaster, or ants are being used as model systems.

There are mammals that are increasingly restricted. The case of dogs: in Mexico City, for example, we have the animal protection law that prohibits the use of dogs and cats for research and teaching purposes.

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