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Different Among Equals – Researchers study deviating behavior of laboratory mice in hopes of developing a skin model to replace animal experimentation

Laboratory mouse. Photo: Karla Fritze.

Laboratory mouse. Photo: Karla Fritze.

Since 2002, animal welfare has been included in the German constitution as an objective of the state. The sheer number of prizes awarded since then for measures that reduce the number of animals in research, or to replace them altogether, speaks to the increasing importance of animal protection. Nevertheless, in 2014 alone, 2.8 million rodents, birds, fish, and others animals were used in scientific research, according to the Federal Ministry of Food and Agriculture. Even though this number dropped for the second straight year, it remains high – and, thus, a challenge. The Berlin-Brandenburg research platform BB3R wants to contribute to solving this problem. Researchers in 12 sub-projects, including a team led by Potsdam nutritional toxicologist Prof. Dr. Burkhard Kleuser, hope to gain important insight into alternative and animal-friendly testing methods. The research association has received almost 4 million euros in funding from the Federal Ministry of Research.

The room in the animal facility of the German Institute of Human Nutrition Potsdam-Rehbrücke (DIfE) is functional, chilly, and disinfected down to the last inch. To the right are cages with small cameras behind them. To the left – also video monitored – is a circular area with a small red hut and some nesting material at the center. A single little mouse is visible on an artificial area one meter in diameter. Doctoral candidate Tina Nitezki knows this mouse – and the 34 others in the test group – quite well by now. The young scientist is in the PhD research group “Innovations in 3R Research – Genetic Engineering, Tissue Engineering and Bioinformatics”, which is affiliated with the BB3R research platform. Her research involving the laboratory mice is part of one of the BB3R research platform sub-projects and aims to better understand the animals’ behavior. The data she collects will contribute to refining animal testing so that it is both less stressful for the animals and more effective for researchers. In particular, she is looking out for stereotypies, i.e. deviant behaviors. She wants to more precisely characterize them to find out why they occur, what keeping conditions may contribute to them, and what they tell us about the animals’ wellbeing. Why under identical living conditions do some mice and not others begin running around in circles after a while? This is just one of the questions researchers still know too little about.

Stereotypies might be a sort of evolutionary dead-end in nature 

In this particular instance, the newly created open field – as the experts call it – is a completely unfamiliar environment for all mice. It confronts them with new odors, lighting, and materials. The first test lasts only five minutes. A second five-minute test follows a week later, followed by a third test that spans 24 hours. Each mouse has three schedules and is video monitored for 30 minutes five times a day. This test phase takes 12 weeks. How will the small mammals move over time? What patterns will they display? In general, the researcher explains, mice are thigmotactic, meaning they avoid moving across open spaces, preferring to stick to walls or natural boundaries. “From an evolutionary biological perspective, this is very reasonable,” says Tina Nitezki. “Their main natural enemies are birds of prey. And a mouse running across an open field would be easy prey.” 

In the very first test, the animals cautiously explore their new environment. Strikingly, the stereotypical mice prefer to stay in the hut and avoid the outer zone. This changes, though, in the second test. “But it was the results of the 24-hour test that impressed us the most,” Tina Nitezki reports weeks later. In the longer-duration test, the stereotypical mice spent more time in the central open space – indicating that stereotypies might be a sort of evolutionary dead-end in nature, yet they slept less than the “normal” FVB/N mice and also moved about more during their active phase. “While the non-stereotypical animals covered 1.77 km, the stereotype ones covered 4.96 km.” And one mouse ran almost 17 km! These distances allow researchers to conclude how much space would be used if it were available. In a normal 18.5x35-cm cage, laboratory mice cover 1.53-3.98 km over the same period.

In the project, Tina Nitezki also studied the animals’ stress levels. One indication of stress is how often they groom themselves. “Here I found no differences between them,” the veterinarian states. Additionally, the level of corticosterone – the most important stress hormone in mice – was established, and the amount of “happiness” hormones – dopamine and serotonin – in their feces was measured. The levels were almost identical. “So stereotypical behavior in FVB/N mice seems not to be stress-dependent.”

Over the course of the study, 19 of the 35 mice developed stereotypies: five started running around in circles, 10 jumped backwards more often, and four latched onto the cage bars with their forelegs and chewed on them. It is not yet clear why siblings kept under identical conditions – FVB/N mice, classically inbred, are almost identical genetically – react so differently. Experts often argue that there is a genetic component. Some studies find proof of “inheritance”. “But in our study, there was no indication of that,” Tina Nitezki underlines. “About 44% of the descendants of stereotypical mothers displayed stereotypical behavior – as did 37% of the offspring of non-stereotypical mothers, too.”

It is, however, well understood why stereotypies develop in the first place. Animals kept under artificial conditions – be it in a zoo, a circus, or for agricultural purposes – cannot exhibit normal behavior and get bored. Wild mice in particular, Tina Nitezki explains, spend a lot of time foraging, whereas laboratory mice are given food and water. In addition, they are put together in same-sex groups to avoid pregnancy, so reproduction rituals cannot be practiced either. Cages are also often small and offer little opportunity to hide, nest, and climb. “However, stereotypies also occur under more stimulating keeping conditions,” Tina Nitezki admits. 

This is a problem in laboratory animal science. Stereotypical mice cannot be used for physiological examinations, since their metabolism differs too much from that of “normal” mice. Tina Nitezki’s project runs through September 2017 and is being supervised by Associate Professor Dr. Stephanie Krämer. She is Veterinary Director of the animal facility of the DIfE and welcomes the research platform’s activities. “The number of experiments conducted on animals needs to be reduced,” she says. Even today, procedures around animal experiments are highly standardized and closely monitored. Nevertheless, more than 1.9 million mice were used for scientific research and usually euthanized at the end of the project in laboratories across Germany in 2014 alone. Prof. Dr. Burkhard Kleuser, deputy spokesperson of the research platform and head of the Potsdam sub-projects, is well aware of this situation. “At the German Institute of Human Nutrition and at the University of Potsdam, a lot is done to minimize animal experimentation in research and replace it with alternative methods whenever possible,” he assures. “But we are not yet able to do all testing in cell cultures.” 

A new skin model could replace animal experimentation

A second Potsdam sub-project of the platform marks another step in the right direction. It addresses immune mechanisms. Burkhard Kleuser’s colleague, Junior Professor Dr. Lukasz Japtok, is researching a new skin model into which immune cells can be integrated. As soon as it reaches the stage of industrial application, it can replace animal experimentation. This would be a major scientific development. Skin models have been available for quite some time but not with inherent immune function. The existing commercial products have been used to establish how certain chemicals damage skin. They are usually marketed to treat burn victims, to molecularly and cytologically clear up dermatological disease, and to test cosmetics and pharmaceuticals. “But none is suited to testing whether substances trigger rashes and allergies,” Lukasz Japtok explains. “This has only been able to be done through animal experimentation.” In the lymph node assay, for instance, certain substances are still brushed onto the ear of a mouse to detect whether there is an immunological reaction.

Lukasz Japtok and his team first grow immune and skin cells from the same person – a test approved by the ethics committee. Volunteers in the department provide hair and blood. From the hair follicles – more precisely, progenitor cells at the hair shaft – the researchers develop the necessary skin cells, while immune cells are isolated from the blood samples. It is very important that both come from the same donor to avoid a defense reaction. This issue is well known in transplantation medicine; immunosuppressants are used by the researchers to block the body’s immune response and prevent transplant rejection.

“Both cell types have been successfully isolated, and we are now about to create co-cultures,” describes Lukasz Japtok the current state of his research. “We are already able to isolate cells from hair follicles and make them develop into keratinocytes, i.e. typical skin cells. And we can generate immune cells from blood cells.” It may sound easy, but it isn’t. Immune cells with different functions need to be created, since the types of immune cells in the skin’s epidermis and dermis differ. In principle, the researchers hope to create two essential antigen presenting cell types: classical dendritic cells and Langerhans cells. To this end, immune cells are isolated from the blood and treated with special cocktails of cytokines – the body’s signaling substances – which trigger a differentiation towards the intended cell types. The aim is to obtain explicitly immature cells. These recognize foreign substances particularly well and incorporate them immediately, thus ensuring that an immune response is initiated.

The team next plans to combine the two cell types to form a co-culture. Lukasz Japtok is aware of the challenges ahead. Whether, how, and under what conditions the two will get along remains to be seen. He is nevertheless very confident in the project’s eventual success. One limitation does remain: The skin model would be just a prototype but one that contains every important cell a mouse has in its skin, as well. If the prototype ends up becoming available, it would be an example of a complete replacement.

BB3R combines the first letters of Berlin and Brandenburg with three Rs: Replacement, Reduction, and Refinement. They delineate what science and research in the field of alternative test methods is all about: to completely replace the use of animals in research, to reduce the number of animal experiments as well as the number of animals used in them, or at least to refine methods to alleviate suffering of the mice, rats, fish, and other animals used.

Animal testing for scientific purposes requires the prior approval by the competent authority. In Brandenburg, this falls under the responsibility of the State Office for Occupational Safety, Consumer Protection and Health, which reports to the Federal Ministry of Food and Agriculture. Test animals are used in biomedical and genetic research and sometimes in university education. The law requires all new drugs to be tested in animal studies before they can be approved for the market.

The Project

Creation of an immunology test platform
Oversight: Prof. Dr. Burkhard Kleuser (University of Potsdam)
Duration: 2014-2017
Funding: Federal Ministry of Education and Research (BMBF)

The Researchers

Prof. Dr. Burkhard Kleuser studied chemistry and food chemistry at the University of Wuppertal. In 1994, he completed his PhD in biochemistry and molecular biology at the University of Hamburg; in 2002 he habilitated in pharmacology and toxicology at Freie Universität (FU) Berlin. Since 2009, Burkhard Kleuser has been Professor of Nutrition Toxicology at the Institute of Nutritional Science at the University of Potsdam, which he has also chaired since 2013.

Universität Potsdam
Institut für Ernährungswissenschaft
Arthur-Scheunert-Allee 114–116, 14558 Nuthetal
Email: kleuser@uni-potsdam.nomorespam.de

Junior Prof. Dr. Lukasz Japtok studied pharmaceutics at Freie Universität (FU) Berlin, where he also earned his PhD in pharmacology and toxicology in 2012. Since 2015, Lukasz Japtok has been Junior Professor of Immune Toxicology at the University of Potsdam.
Email: japtok@uni-potsdam.nomorespam.de

Tina Nitezki studied veterinary medicine at Freie Universität Berlin. Since 2014, she has participated in the PhD research group “Innovations in 3R research – Genetic Engineering, Tissue Engineering and Bioinformatics”.
Email: Tina.Nitezki@dife.nomorespam.de

Associate Prof. Dr. Stephanie Krämer studied veterinary medicine at Freie Universität (FU) Berlin. In 2007, she earned her PhD in veterinary medicine and qualified as an expert veterinarian for laboratory animal science at FU Berlin. In 2014, she habilitated in experimental pharmacology at the University of Potsdam. Since 2009, she has been Veterinary Director of the Max-Rubner-Laboratorium (MRL) of the German Institute of Human Nutrition Potsdam-Rehbrücke. 

Text: Petra Görlich
Translation: Monika Wilke
Published online by: Agnetha Lang
Contact for the online editorial office: onlineredaktion@uni-potsdam.nomorespam.de

Read this and other articles on research at the University of Potsdam in our research magazine Portal Wissen. http://www.uni-potsdam.de/en/explore-the-up/news-and-announcements/university-magazines/archive-portal-wissen.html