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The reduction of suffering, stress and pain in the keeping of laboratory animals and during an animal experiment are central components in the 3R concept.
'Refinement' often takes a back seat to 'Reduce' and 'Replace', but is crucial for the welfare of laboratory animals. With start-up funding of up to 100.000 euros per project, Charité 3R therefore supports research projects with this funding that directly serve the aspect of refinement at Charité.
Quantifying training success with positive reinforcement techniques in laboratory pigs
The aim this project is to minimize the stress of pigs in animal experiments with the help of special training and via the principle of positive reinforcement. The animals are to be accustomed to various examination methods by means of clicker training and in this way lose their fear. In the project "Quantifying training success with positive reinforcement techniques in laboratory pigs", Dr. Dilyana Mangarova and Dr. Julia Brangsch from the Department of Radiology, together with researchers from the Federal Institute for Risk Assessment, want to develop a reproducible training plan for pigs in laboratory animal facilities. In this call, the guidelines of the "Good Evaluation Practice" were implemented by the BIH QUEST Center through the support of the project team Incentivation and Indicators. The quality-oriented evaluation process took place anonymously and in a two-step procedure. In the first step, the project applications were evaluated qualitatively in different categories and the final funding decision was made as a second step in a review meeting.
Pigs are widely used for research and treatment of cardiovascular diseases because they are very similar to humans in terms of organ size and anatomy. They are used, for example, to learn specific human medical surgical techniques before they are applied to humans. In the current project, abdominal aortic aneurysm (AAA), a pathological enlargement of the abdominal aorta, is being researched using imaging techniques on pigs.
However, pigs often need to be restrained for examination, for example for medical procedures such as medications or for clinical examinations. This can lead to stress reactions that affect the health of the animals. In addition, stress can affect research data as well as reduce reproducibility and thus translational potential.
Clicker training is designed to habituate pigs to different research methods by giving the animal a reward each time it performs a desired behavior. In this way, the pigs learn cooperative behavior and the animal's well-being and tolerance to medical interventions are increased. The training success will also be measured and quantified using physiological parameters in a contactless way via different devices (smart ear tags, cameras, biomonitors).
Real-time deep-neural network analysis of head-fixed mice grimace scale for stress prevention
Head-fixing mice is a staple in research for almost 60 years and is used in the most advanced experimental approaches like awake behavior in rodents, multiphoton imaging, and intracellular recording techniques. Maintaining the animal’s head completely stationary during the recording is a prerequisite to a wide range of recording methods. Without it, the experimental methods available to understand brain function in behavior would be substantially more restricted. However, the limitation is, that it can induce stress in animals. In this project, we propose a new method for live stress evaluation in head-fixed mice by utilizing a real-time deep-neural network to analyze the stress level using the grimace scale as reference. This system is simple to implement and only requires a camera and a computer for live analysis of the video feed to inform the researcher if the animal shows any sign of stress to ensure the best possible welfare for head-fixed animals.
Environmental refinement in long-term small animal studies for older age
Due to an aging world population (>2 billion people >60 years of age by 2050) and a resulting focus on the study of age-associated diseases, the need for older laboratory animals will grow. However, behavioral studies suggest that standardized long-term housing, largely shielded from stimuli, may represent an independent stressor for rodents. Therefore, the aim of this study is to develop and document cost-effective and easily implementable solution steps (sensory stimuli, exercise and exploration) for sensory enrichment of the aging process and to investigate their effect on physiological parameters (such as senescence and metabolism). Our project will be supported and extended by the cooperation with the Forschungseinrichtung für experimentelle Medizin (FEM) and other expert scientists.
Development of refinement methods to improve animal welfare in mouse models of liver cancer
Mouse models are still irreplaceable in biomedical research. Many disease models require treatment of animals over long time periods with frequent interventions, such as injections. This results in a high burden on the animals, mainly due to stress. Using two mouse models for hepatocellular carcinoma, we aim to develop a training program that can accustom the mice to the frequent handling and therefore reduce stress. In addition, we aim to develop a method for intraperitoneal injections that is less restraining than currently used methods. These methods are broadly applicable to many mouse models and therefore hold the potential to reduce experimental burden and increase welfare of a large number of animals in biomedical research. This project is a collaboration between scientist from Charité Berlin and the University of Veterinary Medicine Hannover.
Alternative methods for measuring rectal body temperature
Body temperature is an important parameter for analyzing the severity of allergic reactions to different allergens in an animal model. Rectal thermometers are commonly used for this purpose. However, this method requires that animals undergo stressful procedures. The stress, in turn, can affect body temperature and make temperature measurements less reliable. In order to better understand these stress-related effects on temperature measurement and to improve the restrictions with regard to both animal welfare and data accuracy, alternative measurement methods are required. Possibilities are the non-invasive use of infrared thermometers and temperature transponders. Both methods enable non-contact temperature measurements that can contribute to stress reduction in the animal model. So far, there are no reliable studies comparing rectal temperature measurement and the use of infrared thermometers and temperature transponders in the allergy mouse model. Therefore, comparative methodological studies are required to overcome these limitations and improve animal welfare.
In this project, the common rectal measurement method will be compared with the use of non-invasive infrared thermometers and temperature transponders. In this way, animal welfare and data reliability can be massively improved. The results of the study can be transferred easily to other scientific-experimental approaches in which temperature recording are of considerable importance. The gained knowledge will therefore not only be helpful in allergy research, but also in areas such as endocrinology, diabetes and cardiovascular research.
Refinement of a Coxsackievirus B3 acute myocarditis mouse model by analgesia using tramadol.
Myocarditis is an inflammatory heart disease, which may be induced by viruses. Usually, virus-triggered myocardial injury emerges as a second hit after a benign viral affection of the gastrointestinal or respiratory tract. To answer basic research and translational questions, researcher use, inter alia, mouse models. In the mouse model used here mice are infected with a virus (Coxsackievirus B3 (CVB3)) and after a few days, these mice show the features of myocarditis. In addition to heart muscle cells, CVB3 also affects the pancreas, which can cause pain in the mice. The aim of this refinement project is to investigate the effects of the analgesic tramadol. The project will profile if and how tramadol can alleviate pain and, on the other hand, if and how it modulates the immune response and, thereby influences the myocarditis phenotype. For this purpose, a harmonized scoring system documenting standardized parameters is implemented, allowing synergistic data analysis at the different phases after infection.
Alone – but not isolated
Mice are social animals. This is why they are kept in groups. There may be exceptions when individual animals – mostly males – do not integrate into the group or become aggressive against their fellow mice. For the protection of individual health, these animals are kept single in cages and can no longer interact socially.
In this refinement project, the scientists want to test a novel method for the keeping of solitary animals: they are to be integrated as partners. To do this, special cages will be afforded whose individual segments are divided by elements that are open to sight, hearing and smell. In this way, several animals can live in one cage, can sense one another, can have certain interaction with one another – but cannot hurt or disturb one another.
The new system of cages must of course follow the usual regulations: there must be ample room, nesting material, nibbling material and constant access to water and food for the animals. Also, there can be no compromise on hygienic standards. In this project, both male and female mice are to be kept in the new cage system. All indicators for the well-being or stress of the animals will be analyzed. The team is looking to find out whether such separated keeping as partners with the possibility of contact will have a positive influence on the well-being of mice.
An improved examination model for inflammations of the heart muscle
An inflammation of the heart muscle, also known as myocarditis, can be triggered by pathogens such as viruses and bacteria, by toxic effects or by an auto-immune disease attacking the heart proteins. In order to better understand the process of a myocarditis, scientists usually employ a mouse model, where an inflammation of the heart muscle is triggered by an infection with specific viruses. These viruses can also affect other organs such as the pancreas, which leads to additional stress for the test animals.
In this research project, the scientists are looking to improve the animal model in a viral myocarditis, so that the infection remains restricted to the heart muscle and other organs are no longer affected. If successful, the underlying causes of the inflammatory heart muscle disease can be better examined. Stress as well as the absolute number of test animals for the relevant research can be reduced.
Clinical supervision without stress
Test animals must be constantly supervised for scientists to determine whether an animal is appropriately fit for a test – or whether it would inordinately suffer. Most methods for controlling their health status are extremely stressful in themselves for the test animals: mice and rats are lifted from their cages for sight control, which is stressful for the animals. Another method is the supervision of their nesting techniques; if they build their nests neglectfully, this is an indication for their not being well. Especially animals that recently underwent surgery do need a well-built and warm nest with their own smell to recuperate – and no demanding nest building.
It is the goal of this project to facilitate a continuous and stress-free control of the test animals. In order to achieve this, the scientists want to combine various modern techniques: running wheels shall be used that can measure the individual running performance of mice and rats. Another instrument shall determine grip strength to see if animals lose their muscle strength. A location sensor will continuously record the position of the animals in the cage: do they regularly visit the water dispenser and take up sufficient fluids? Do they remain in the nest for too long, which would be indicative of indisposition? A sensor plate in the cage could record relevant patterns of movement.
The scientists intend to develop criteria to infer definite conclusions on the well-being of the animals from the host of data delivered by the combined control systems. This should make it possible that handling stress and pain can be reduced for the data-based completion of the testing before test animals inordinately suffer.
Refinement of animal husbandry in the Department of Experimental Medicine (FEM) at Charité
The Department of Experimental Medicine (FEM) defines the standards for the housing and handling of experimental animals at Charité, considering legal requirements. Quality standards are crucial for the entire biomedical research process, from breeding to experimental husbandry. Furthermore, the FEM trains animal caretakers and qualifies scientific staff to conduct animal experiments. Within this project, the FEM is further developing standard husbandry through innovative refinement measures in breeding and husbandry, further education or training and accompanying ongoing approved projects in terms of innovative animal welfare, beyond established and legal standards. The following focal points are set:
- Digitally assisted monitoring of the animals' well-being
- Improvement of postoperative follow-up
- Improvement of handling techniques
- Multiplication of opportunities for social interaction between animals
Improved examination model for inflammations of the heart muscle (myocarditis)
Inflammations of the heart muscle, also called myocarditis, can be triggerd by viruses and bacteria, but also by toxic effects or by auto-immune diseases damaging the heart proteins. The goal of the research in question has been to profoundly understand the aetiopathology and to develop new therapeutic approaches from there. In order to do this, the scientists employ a mouse model with a myocarditis that is triggered by specific viruses. These viruses can also affect other organs such as the pancreas, which exposes the test animals to additional stress. With this project, the scientists look to optimize the animal model in viral myocarditis, so that the infection remain restricted to the heart muscle and does not affect other organs. If proven successful, the underlying causes of myocarditis can be better examined. The amount of stress as well as the total number of required test animals could be reduced.
Improved pain therapy for laboratory mice in bone research
Disturbances in the healing of bone fractures occur in 5 to 10 percent of all cases. Thus, there is intense research for novel therapeutic strategies – especially through animal models. The use of animals in research is bound to requirements for the careful, gentle and appropriate handling of the animals in question. Pain detection and therapy on laboratory animals, especially laboratory mice, is an elaborate task, and even today only few scientifically evaluated pain treatment protocols for mice are available. Dr. Annemarie Lang and Dr. Anna Rapp are looking to improve the current regime of pain treatment and test a new Buprenorphin-Retard supplement. Buprenorphin is a highly effective pain killer belonging to the opioid segment. Current application calls for an injection every 6 to 8 hours, which is an enormous burden of stress for the mice. A retard supplement by comparison offers a constant supply of the pain-relieving substance over the course of 24 to 48 hours. Presumably, such a supplement would not only replace the repeated daily injections, but would also better shield the animals from pain through the constant level of the pain-relieving agent. In this fashion, the project aims at an improved and stress-relieved pain treatment for laboratory mice. On this project, Dr. Annemarie Lang and Dr. Anna Rapp cooperate with experts from the universities of Zurich and Basel and of the Freie Universität Berlin.
Measuring changes in body temperature without contact
In general therapy as well as in clinical and pre-clinical biomedical research a close supervision of the health status of the patients is indispensable. Body temperature is an extremely important and objectively measurable vital parameter in the treatment of various pathologies. Especially in the case of infections, changes in body temperature, through fever in humans or in the form of hypothermia in the case of small mammals with a highly active metabolism, can be conclusive in the assessment of the actual status of the disease and its course.
In animal research, the methods of measurements currently in use require direct animal contact or the surgical implantation of microchip temperature transponders. These measures not only provoke strain and stress for the animal; they can lead to a change in body temperature by themselves. In this project, a group of scientists are looking to evaluate the implementation of a contact-free method through the use of an infrared camera for the determination of the body temperature of a mouse, while it is free to roam with its group in its accustomed habitat. The objective is to facilitate the valid and stress-free measurement of changes in the body temperature of mice while infected and to significantly reduce the additional strain brought about by conservative temperature measurements according to the goals of refinement. It is further planned to simultaneously record multiple animals of the group via video recordings in order to further reduce the examination time directly at the cage.
Measurement of heart performance – without catheter
Examinations of the heart for the measurement of its performance are elaborate: a catheter must be used quite regularly. In test animals such as mice, the catheter causes damage to the blood vessels, so that the scientists must kill the animal afterwards. It is not only that the mouse is lost – examinations of the cause are also precluded. Meanwhile, there is a new methodology that allows for a non-invasive assessment of the exact heart performance through computing power. Data from blood pressure and from ultrasound examinations play an important role in this. The concentration of certain components in the blood serum (bio markers) also offers important clues. Whereas this methodology is well established with human patients, there is a lack of experience for this with mice.
There is a group of scientists who are looking to change this. Their team has great experience in heart examinations on mice via ultrasound and they can rely on high-end technical equipment to do this. The scientists plan to employ this technique on healthy mice as well as those with a heart disease. The results will then be combined with further data from blood pressure and bio markers in order to assess the pressure-volume-ratio non-invasively. For proof of their computations, the scientists will eventually be forced to examine the animals with catheters and kill them afterwards, but the results could accommodate a future development where animals would have to suffer less in heart examinations and heart catheters would no longer be needed. What’s more, the team is also hoping for a reduction in the total number of test animals: as soon as the non-invasive methodology is established, the animals will stay alive after the examinations in the future.
New ex-vivo-method of “arteries in isolated perfusion” (AIP)
Diseases of the arterial vessels such as arteriosclerosis belong to the class of cardio-vascular diseases and are the most prominent cause of death in industrialized countries. Animal testing is used in this field of research and for the development of novel therapeutic approaches, because the complex properties of arteries and the necessity of blood circulation in vitro –meaning in a test tube or petri dish – cannot be appropriately imitated. An alternative is the examination of arteries that were explanted from a test animal and have been cultivated in a laboratory, but have not been subject to perfusion. This method is called “ex vivo” (Latin for “outside the living”.) Markus Tölle and Mirjam Tölle-Schuchardt have therefore developed an “artery in isolated perfusion” which enables the perfusion of the artery under physiological parameters by use of a puffer over a longer period of time. In one of the funded projects, the two scientists want to optimize and simplify the new ex-vivo-method so that it may be applied more broadly in the research of cardio-vascular diseases. The new methodology is expected to largely replace animal testing with high degrees of stress in the pre-clinical testing phase of medical drugs.
Objective stress tests shall prevent unregistered suffering
In order to explore the immune system, immunologists work with mice who have precisely defined genetic alterations in their defense system. Currently, there are no hints that such animals – when kept pathogen-free – are in any way subject to stress. However, because of the manifold interactions between immune and nerve systems, it cannot be ruled out that the immune defects in such animals may indirectly be a source of stress. It must be noted, though, that such sub-clinical strains which may occur in genetically modified animals are hard to assess, as there are no objectively measureable parameters.
This research project funded by Charité 3R is looking to change this. The assessment of stress shall no longer be done in an analogy between humans and animals, but according to animal-based scientific data.
In order to do so, the team will use a method which was developed by a joint research platform of the federal states Berlin and Brandenburg. This method –originally focusing on animals that were operated on – will be transferred to the assessment of immune-deficient mice. The procedure entails various methods which facilitate animal-based evidence for the well-being of mice: the behavior of the animals will be recorded and the discharge of stress hormones will be measured through fur and scat samples. It will be especially looked at two lines of mice with different immune defects. The results will then be compared to the results won from wild mice. The team is confident to facilitate an objective stress assessment for genetically modified mice under standard parameters – so that their potential suffering does not go unnoticed.
Osteoathrosis: Accurately charting pain – and specifically treating it
Joint pains are the daily fate for many people: worldwide, almost a fifth of all women and a tenth of all men older than 60 years suffer from osteoathrosis. As the current treatment almost exclusively focuses on pain remedy, scientists are intensively looking for new therapies that battle the causes of the disease. They rely on the testing of mice and rats in whom the degeneration of cartilage substance in the joints has been artificially triggered. This leads to a reduction of mobility and the animals develop the pain symptoms typical of osteoathrosis. The pain must be optimally charted and supervised to preclude inordinate suffering of the test animals and to gauge the success of new therapeutic approaches.
Two female scientists of the Charité are looking to more accurately measure the pain of test animals in osteoathrosis research with a novel refinement project. They plan to establish a so-called refinement area in the keeping of test animals. It will be furnished with modern equipment for the detailed behavioral analysis of mice: their ambulation patterns can be determined as well as their behavior in nesting and digging or their mimics. Appliances for the stress-free handling of the animals reduce harmful exterior influences.
In this refinement area, the scientists plan to identify and determine all parameters with whose aid the pain status of the test animals and the success of pain treatment can be best charted specifically in osteoathrosis research. They further plan to share the newly won knowledge with other scientists, so that the suffering of animals and the number of required animals can be reduced.
The isolated heart of large animals in cardio-vascular research
Animal testing and experiments with hearts of pigs especially are of vital importance to cardio-vascular research. In order to reduce both the suffering of animals and the number of experiments, Charité scientists Dr. Marcus Granegger and PD Dr. Simon Sündermann are looking to establish the “isolated large animal heart.” They use the hearts of pigs already available through other lines of research. The hearts will be explanted in general anaesthesia quite similar to a normal organ transplant. The quick removal of the heart effects lesser suffering for the animal than the complex procedure that was used before. The heart will be connected to an artificial blood circuit immediately after the organ explant: the heart begins to pulsate and pumps blood under precisely defined parameters. This methodology allows for the precise quantification of the mechanics of the heart, the oxygen consumption and the blood circulation through the heart chambers. The results are vastly improved over research on living organisms, where natural variances in the circulation of the heart occur. It is thus prevented that experiments are done under non-standardized parameters or that they may prove invalid or in need of repetition through poor data quality. Hence, the number of necessary experiments using animals can be greatly reduced in two ways: for one thing, scientists can perform a number of experiments on an isolated heart, where formerly they would have needed a large number of animals. Secondly, the organs, which would have been discarded before, can now be appropriated to cardio-vascular research and be sensibly re-used.