Hardly any other system in the body is as complex as the immune defense system. It is not possible to fully reproduce all its interactions in cell culture, and therefore immunology depends on genetically modified laboratory animals. About six percent of the mice bred and kept at the Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC) also have an immune defect. Because specific immune defense genes have been switched off, certain strains lack T and B lymphocytes – the immune cells that normally fight viruses, bacteria or fungi. Others lack genes to form a receptor for the important signaling molecule interferon gamma, which fights pathogenic germs in the cells and also tumor cells.
“In humans, these immunodeficiencies lead to death within a few months or years if left untreated,” says Dr. Thomas Kammertöns, immunologist at the MDC and Charité. However, the mice live as long as healthy animals if they are protected from all germs, as is common practice. Unlike humans, they are not exposed to pathogens. But does this mean that the mice feel well? This question concerns Thomas Kammertöns, and in various studies since 2012, he has been investigating whether the animals are under any distress. So far, he has only been able to detect insignificant discrepancies in stress levels.
Kammertöns is using the genetically modified mice to develop new immunotherapies against cancer. But also autoimmune diseases and viral infections are being researched with the help of these mouse models. “They are the model for immunological questions,” says Dr. Sarah Jeuthe, veterinarian and animal welfare officer at the MDC. This is why it is so important to systematically study the well-being of immunodeficient mice. Financial support from the Charité 3R center has been provided to enable the research.
Comprehensive picture of the well-being of mice
For the new study, the results of which were recently published in the journal PLOS ONE, scientists, veterinarians and animal keepers worked closely together. “We wanted to know whether we had missed anything,” says Kammertöns, the last author of the paper. “Therefore, we not only looked at physiological parameters, but also systematically observed the behavior of the animals.” According to the EU Directive, laboratory animals are considered to be distressed if they experience pain, suffering, anxiety or permanent damage equal to or exceeding the insertion of a cannula.
The immunologist, lead author Sarah Jeuthe and colleagues from Freie Universität (FU) Berlin and Charité spent nine months studying the behavior and physiology of 90 individual mice. During this time, the team did not find statistically relevant differences in weight or the stress hormone cortisol between the immunocompromised mice and healthy mice in the control group. The cortisol levels did increase over time in animals in all groups, and especially in the female animals. “But this has also been shown in earlier studies,” says Thomas Kammertöns. Depending on their gender, animals become more sensitive as they age.
A standardized protocol for behavioral observation
When the mice built nests, which researchers observed monthly, no abnormalities were detected either. “When an animal is doing well, it also behaves naturally in a cage,” says veterinarian Jeuthe. The mice then shred the hemp pads and puff them up – in the best-case scenario, this turns into a ball into which the mouse completely disappears. During the weekly hour-long observation of facial expressions in accordance with the international standard of the “Mouse Grimace Scale,” just a few mice showed slight signs of discomfort. “Of the five indicators, which include the squinting of eyes or tactile hairs lying flat, only one at most was observed in individual animals and only at a moderate level,” says Jeuthe. Though the presence of humans alone is enough to cause this type of irritation in the animals. “It can already be caused by the smell of disinfectant, which a mouse finds unpleasant.”
In their study, the scientists used a protocol developed by FU Berlin Professor Christa Thöne-Reineke’s team in the Berlin-Brandenburg research platform BB3R as a guide. Similar to the Charité 3R center, the platform aims to support the establishment of alternative and animal-friendly methods in biomedical research. To minimize the “human factor,” the protocol stipulates, among other things, that four people observe the mice independently. None of the participants knows whether the respective mouse has an immunodeficiency and if so, what it is. The task was performed by animal keepers together with scientific employees from the MDC research groups and, in some cases, by the authors of the study themselves. “The animal keepers are used to looking at mice very closely,” says Kammertöns. “They see the animals every day, and this expertise was important to us. This teamwork has further improved the collaboration between research and the animal facilities at the MDC.”
The animals aged normally
Part of the protocol also stipulates that the animals be killed at the end of the assessment period and carefully examined for organ damage. “The mice had aged normally. There was no indication that any organ had developed abnormally,” Kammertöns comments on the report that was prepared by an experienced animal pathologist from the FU Berlin. Only the kidneys were affected by age in nearly half the animals – independent of an immunodeficiency. This is not an unusual finding, since such kidney changes occur regularly in the most frequently used type C57BL/6 laboratory mice, which were also part of this study.
The question of whether immunodeficiency puts a strain on mice bred for animal experiments in germ-free environments is scientifically controversial. Due to the EU Directive 2010/63 and the associated amendment to the German Animal Welfare Act, the breeding of immunodeficient mice is classified as an animal experiment that requires authorization and an application must be sent to the competent authorities. This was confirmed by a recommendation of the National Commission. Previous studies on physiological values sometimes provided evidence of stress. “In contrast to our study,” notes Thomas Kammertöns, “in these studies, the animals did not come from the same litter, so they were not related.” Therefore, it could not be excluded that any observed deviations were due to genetic differences and environmental factors associated with the line of descent. “We have now taken a more comprehensive view of the mice, combining many parameters,” says Kammertöns. “With this in-depth study, we hope to contribute to a fact-based discussion.
Jeuthe, Sarah et al. (2020): „Stress hormones or general well-being are not altered in immune-deficient mice lacking either T- and B- lymphocytes or Interferon gamma signaling if kept under specific pathogen free housing conditions“. PLOS ONE, .