Evaluation of severity assessment strategies in disease and surgical animal models
Due to the ethical dilemma concerning animal experimentation, special attention is payed to the welfare of laboratory animals. With the EU directive 2010/63/EU more strict regulations for performing animal experiments became effective. Independent review for the necessity of an animal experiment is implemented and the burden for the animals has to be balanced versus the expected benefit of an experiment. To allow careful consideration researchers have to grade the severity of the experimental burden into three categories: mild, moderate and severe. However, this task is hindered by a lack of knowledge about the multiple severity causing conditions like post-operative pain, inflammation, anxiety and other´s. We will investigate well established murineas well as large animal models to develop and validate suitable strategies for severity assessment. Inflammatory bowel disease (IBD) is influenced by environmental and genetic factors. The impact of these factors on severity will be investigated in our murine IBD models under otherwise identical conditions. As an easily gradable model with a different nature of the burden we will investigate mouse stress models. As one of the unique research centre’s breeding and housing germfree mice we plan to create and analyse severity assessment strategies without threating the germfree status. In large animal models we will study severity, especially post-operative pain after surgerieswith a focus on sheep.
In all animal models we plan to perform obligatory clinical investigation as a base. Additionally, we will analyse animal behaviour, physiology and biochemistry to build up a database of manifold different parameter for comparison. Including modern non-invasive imaging technologies will contribute valuable extra readouts to correlate with classical clinical data. Another point that will be addressed is the investigation of the impact arising from the implementation of severity assessment. The main goal is the refinement of existing approaches and the creation of innovative new strategies for assessment of animal burden with defined sound scales.
Severity assessment in animal models of liver resection, liver transplantation in rats and kidney transplantation in pigs
Surgical models that require a laparotomy are commonly used within the field of biomedical research and defined as moderate procedures according to the article 15 of the EU-Directive 2010/63. However, the degree of pain, distress and suffering in different models can be highly variable and raises public concerns and ethical questions.
The aim of the current project is to evaluate severity assessment strategies for animals in surgical procedures by using classical models of (partial) liver resection and transplantation in rats and kidney transplantation in porcine.
Beside clinical observations, we plan to evaluate objective physiological data in combination with inflammatory and stress markers as well as with observational data from scoring systems and behavioural data in order to validate them for the implementation in standard severity assessments. For each of these animal models the heart rate, body temperature and locomotor activity will be evaluated by telemetric measurements also in pigs. Moreover, we will combine the telemetric data with scoring systems (Morton DB 1985) as well as behavioural and locomotion testing to qualify the amount of pain, distress and suffering of the animals during the experimental procedures. This will be accompanied by additional behaviour and locomotion tests e.g. open field (in rodents and pigs), the Rota-rod or Catwalk (rodents only) to find the most suitable test scenario for the experiments. Moreover, we will also analyse different hormone levels e.g. corticosterone and oxytocin and inflammatory parameters in blood and the cortisol metabolites concentration in the faeces of rodents and pigs. We will analyse the liver transplants using transgenic Firefly (Luciferase Tg) rats and in vivo bioluminescence measurement to correlate organ function and injury to the severity of the procedure as well as well-being of the animals via scoring systems.
Goal is to evaluate and to validate objective parameters for the implementation of standard procedures for severity assessment in surgical animal models. With this data generated, we will be able in the future to give information on how severity can be measured and minimized by application of refinement strategies according to the 3R principles.
Physiological and behavioural signs of pain and wellbeing in mice
Estimating severity of experimental procedures is of major importance and stipulated by the EU directive 2010. Severity and suffering in laboratory animals can be affected by pain, a complex, subjective and negative affective state, induced by invasive procedures, diseases and injuries. To provide scientific basis and give recommendations for severity estimation we will evaluate methods of pain and welfare assessment in mice for sensitivity, robustness, reproducibility and transferability. Therefore, we will apply methods ranging from classical scoring of clinical symptoms to recently developed behavioural tests (e.g. burrowing, nest building, mouse grimace scale) as well as new monitoring tools (e.g. Infrared Thermography (IRT)) and recent approaches considering the affective state of the animal (e.g. cognitive bias test).
We will correlate these different methods in our well-established laparotomy model. To prove the transferability of parameters for pain and severity classification in other models we will adapt our tools for models available at University Hospital Zurich like organ transplantation and pancreatitis. To assure reproducibility we will design common score sheets for assessment of pain in surgery and pancreatitis models with collaborators of the DFG research group. We will collaborate within the DFG research group to establish monitoring tools in neurological rodent models as well as surgical, large animal models.
Finally, we will contribute to the 3R principle required by the EU directive by focusing on measures to reduce pain and improve recovery in painful models in mice. Several strategies to refine procedures are available that may improve the animals’ ability to cope with experimental stressors but few have been tested systematically for capability and side effects on experimental read-out. We will analyze improved analgesia protocols, local anesthetics, prolonged warming after surgery and post-procedural housing conditions. We will apply methods with a focus on the affective state of the animal (stress, need for pain relief, positive/negative affective state).
Overall our project will result in easy-to-use, standardized methods to assess and minimize severity in animal experimentation.
Contactless monitoring of vitals, stress and infections
Animal research has played a crucial role in diverse scientific and medical advances. It offered deep insight into various diseases and allows testing of appropriate therapies, treatments and even cures.
However, the use of animals in scientific and medical research is still a controversial topic and is often a subject of heated debate. In general, the bioscience community accepts the use of animals for research, but only within an ethical framework, whose aim is to alleviate and prevent conditions that can harm and kill them.
Therefore, the need for quality in animal-based research and ethical reasons require procedures to minimize or avoid unnecessary discomfort and suffering in animals. In many cases, the level of discomfort or even stress and pain is not quantitatively assessable but is estimated from the clinical point of view and observation.
For that reason, the need for innovative monitoring techniques which do not alter the typical behavior of animals, especially of free-roaming animals, grows continuously. Innovative technologies should be able to remotely monitor not only vital signs (e.g., respiratory rate, heart rate and temperature), pain and stress, but also motion profiles and rumination rate.
Various non-contact measuring techniques have already entered the clinical routine in human medicine. Because of transferability, most of them can be used in animal-based research. Thus, various innovative technologies, such as infrared thermography (IRT), near infrared (NIR), radar and acceleration sensors can be useful in contact-free measuring.
The aim of this project is to further develop camera-based techniques and vital data-derived parameters to estimate severity in various animal models. First, IRT and NIR-based algorithms will be used to assess vital signs from diverse large and small animals. Second, local and systemic inflammation phenomena will be examined by using camera-based methods. Third, assessed vitals and additional parameters will be used to develop aggregated scores classifying severity, distress and pain. Finally, transferability of algorithms developed from sedated animals to free-roaming ones as well as from large animals to small ones will be examined.
Stress assessment in psychiatric rodent models: search for behavioural and biochemical markers, and their potential general application in biomedical animal research
Similar to psychiatric disorders, rodent models for psychiatric diseases are stigmatized and regarded as particularly stressful. However, scientific evaluation of this assumption is scarce.
Specifically, it has not been studied whether models of depression or schizophrenia, to name two most common and severe psychiatric disorders, entail more discomfort than models of oncology and cytostatic treatment, orthopaedics and pain-related disorders. Here, we suggest performing such an evaluation and use simple tests that are sensitive when well-being of rodents is compromised, such as tests for nesting, burrowing, self care and exploring. These tests will be designed in a way which allows implementing them into weekly home cage change routine, to ensure applicability in common animal facilities without disturbing ongoing experimental procedures. In addition, we will study specific biomarkers of stress, e.g. faecal corticosterone levels, at several time points before, during and after paradigms modelling psychiatric disorders.
This will give a time course of stress load. After sacrificing the animals we will investigate several biomarkers known to be altered by chronic stress, such as the inflammatory marker interleukin-6 (IL-6), brain-derived neurotrophic factor (BDNF) and hippocampal neurogenesis, and the microbiome. Optimally, these markers will serve as end point measurements for retrospective severity grading. It will be studied, whether single markers correlate with each other. Moreover, the influence of maintenance conditions in form of housing and enrichment on the behavioural and biochemical/biological markers studied here will be analysed. Last, we plan to apply emerging candidate tests/markers in collaboration with larger animal facilities from the Heidelberg University and the German Cancer Research Center (DKFZ) to a broader spectrum of experimental animal paradigms.
Influence of Imaging on Study Results and Animal Welfare
Non-invasive imaging has been claimed to support the 3R principles (Refinement, Replacement, Reduction) by enabling longitudinal examination of animals and thus studies with lower animal numbers. Many of the non-invasive imaging methods used for rodent imaging are also applied in humans and thus are considered to be well-tolerated. However, there are differences between examinations of humans and rodents, which may be crucial: Animals are imaged under anesthesia, humans not. Since high resolution is often required to scale down to the size of rodents, MRI scans are performed at high field strengths (>4.7T), CT scans with higher doses and ultrasound scans with high frequency. To this add animal preparations like tail vein catheterization and shaving, the latter being necessary for ultrasound, optical and photoacoustic imaging. In addition, subsequent scans are often performed in small time intervals (e.g. every 1 or 2 days).
Surprisingly, there are hardly any data on the impact of non-invasive imaging on rodents’ welfare and on its influence on study results. Therefore, in this project we will study the impact of CT, MRI, ultrasound and optical imaging including the use of common contrast agents on behavioral and physiological characteristics of welfare in mice. We will further investigate in immunocompetent and immunodeficient mice whether the application of imaging has an impact on the growth, spread and vascularization of tumors as well as on their response to therapy. In this context, the impact of the frequency of repetitive imaging examinations, the application of different imaging systems and methods (e.g. MRI at different field strengths) as well as anesthesia methods will be explored.
Since anesthesia leads to a disturbance of the circadian rhythm, it will also be investigated whether imaging experiments are differently tolerated by the animals at day or nighttime.
In summary, with this project we will collect important basic data on the influence of non-invasive imaging on animal welfare and its influence of study results. The obtained results will generate a basis for the refinement of non-invasive imaging applications and thus will significantly support the 3R principles.
Severity assessment from an animal’s point of view
Severity assessment in animal experimentation is a complex biomedical and ethical issue and largely biased by uncertainty. The interpretation of physiological and behavioural measures in relation to animal welfare is difficult and often reflecting an educated gut feeling rather than scientifically sound conclusions. It is thus utmost important to include the perspective of the animals themselves into severity assessment. Choice and preference tests are a straight forward approach in asking the appraisal of different goods. However, preference for one good over another in itself does not necessarily indicate suffering if the access to the preferred good is denied. Nor can the overall severity of an experimental measurement be derived solely from the fact that the animal - if given the choice - would rather not participate in such a procedure. In order to gain a better understanding of the valence of choices made there is a demand for more sophisticated preference tests which allow estimating the strength of the respective preferences. A compelling approach to estimate the value of the choices made is to raise costs and compare the prices the animals are willing to pay. Using choice tests along with consumer demand theory, mice will be "asked" to rate the severity of experimental procedures themselves. Animals usually cannot choose to avoid experimental procedures and such experiences leave traces in internal emotional states. Although emotional states are usually not obvious to others, it has been demonstrated that they can be revealed using tests of cognitive bias. In brief, these tests shed light on the internal emotional state of an animal and ask if future expectations are "optimistic" or "pessimistic".
Advancing these methods on testing cognitive bias in mice will allow a comprehensive severity assessment taking internal emotional states into account. Thus the set of measures proposed here will include the animal's point of view in severity assessment with regard to their preference and valence of future expectations.
Severity assessment in rat and mouse models of focal cerebral ischemia and subarachnoid haemorrhage
Ischemic and hemorrhagic strokes are leading death reasons in humans. However their pathophysiological cascades are not resolved in detail and more preclinical research is needed to improve existing or develop new therapeutic strategies. Severity assessment in existing rodent models of ischemic stroke and subarachnoid hemorrhage (SAH) has to address both the burden induced by the surgical intervention to induce ischemia or bleeding and by the functional deficit caused by the brain lesion itself. We will induce focal ischemia or SAH in rats and mice using the filament models for focal ischemia (MCAo) and SAH, using the advantage that both models need comparable surgical intervention, but are associated with extremely different levels of pain and suffering. In addition we will use the cisterna-magna-blood injection model for SAH that needs small trepanations of the skull, adding to the pain burden. We aim at thoroughly investigating the impact of the surgical intervention and the consequence of the functional impairment on the wellbeing of the animals. Spontaneous behavior (locomotion, nest building, burrowing), motor, sensory and cognitive function will be measured during the course of the experiment and referred to the histological lesion assessed at the end of the experiment. In addition, chemical (corticosterone, prolactin, inflammatory markers) and physiological (heart rate variability, EEG) parameters will be assessed. We aim at figuring out which of them are the most robust tests and parameters and therefore best suited for reliable and applicable assessment in “everyday life” of research. We intend to discriminate the pain- / stress-induced impairment in behavioral tests from the functional deficit induced impairment which itself not necessarily means reduced well-being. Finally we plan to develop recommendations for use of analgesics with the least possible interference with pathophysiological cascades.
The burden of repeated seizures: development of severity assessment schemes in epilepsy models and identification of common distress-associated brain activity patterns
Seizures are a frequent event in experimental animals. They do not only occur in models developed for epilepsy research, but also in various other models of neurological disorders, in genetically modified mice, in preclinical tolerability and safety studies as well as in breeding populations related to inbreeding depression. Taking the high relevance into account, we aim to improve the knowledge about the impact of seizures on the well-being of animals, and to develop suitable severity assessment schemes. Respective information will also render a valuable basis for the development of evidence-based recommendations for refinement measures and animal welfare-based model prioritization. In the first project phase we will focus on three common rat epilepsy models including the kindling model with repeated electrical induction of seizures as well as an electrical and a chemical poststatus epilepticus model with development of spontaneous seizures. Comprehensive and comparative information about the overall pattern of neurobehavioural alterations will be obtained. The respective analysis will consider nest-building activity, burrowing behaviour, social interaction, locomotion, as well as exploratory, anxiety-associated, and anhedonic behaviour. The correlation of behavioural data with chemical (corticosterone, oxytocin, BDNF) and physiological (incl. heart rate variability) parameters will be assessed guiding the identification of the most significant parameters.
In addition, we aim to identify common patterns of brain activity and of molecular alterations in the serotonergic system based on μPET analysis. Relevant patterns will be identified based on correlation with behavioural and biochemical parameters and by comparison of the findings from epilepsy models with those from a stress model and a model of peripheral disease.
Assessment of well-being in rats: intracranial surgery and testing in models for neurological and neuropsychiatric disorders
In the field of neuroscience effective management of pain, discomfort and stress in animal models is crucial not only for ethical and legal considerations but also to achieve high-quality science free from confounding pathophysiological consequences. In our laboratories we investigate pathophysiological mechanisms and innovative neurosurgical treatment options, e.g., the effect of intracranial neuromodulation or ablation on motor function, cognitive and associative-limbic behaviour, as well as on neuronal network activity in rat models for movement disorders, neuropsychiatric conditions and intracranial brain tumors. Most obvious, neurosurgical interventions involve postoperative pain, which is addressed by intra- and postsurgical application of pain-relieving drugs. However, application of chronic electrical stimulation or repeated local microinjection of neuroactive drugs, behavioural testing and electrophysiological recordings in the awake rat may also impose discomfort, stress or even pain, which cannot be easily addressed by analgesic compounds because of possible confounds to the actual experiments. Further, humane endpoints for intracranial tumor development need to be refined.
In this project we will combine traditional clinical scoring with severity assessment tools addressing spontaneous and species specific behaviour in and outside the home cage. These measures will be complemented by more elaborate behavioural paradigms routinely used in our laboratories. Further, biomarkers of stress will be measured. In a second step these data will be used to optimize the postsurgical analgesic protocol, the maintenance conditions, and to refine behavioural testing.
Testing of unwanted side effects of noncoding RNA oligonucleotide therapeutics in small and large animal models
Cardiovascular diseases are the main cause of mortality worldwide and new therapeutic interventions are urgently needed. Cardiac stress such as myocardial infarction leads to cardiac remodelling and heart failure. A new entity of drugs are oligonucleotide therapeutics (ONTs) such as inhibitors of miRNAs or long noncoding RNAs (lncRNAs). We have identified an ONT targeting the lncRNA meg3 effectively, thus reducing cardiac remodeling and fibrosis of cardiac stressed animals. However, during drug development, ONTs are well known to have off-target effects and to induce toxicological effects in many organs. Thus, the aim of this proposal is to thoroughly assess the effects of several different novel anti-fibrotic lncRNA meg3 inhibitors on changes in behaviour, motor, sensory and cognitive function as well as stress markers in the blood that will be measured during the course of the experiment.
In addition, we will develop and validate a novel stress-sensitive marker set of circulating miRNAs through access to various animal disease models available in this consortium.
These approaches and collection of parameters will likely identify the most robust and best suited severity assessment tests especially in the field of oligonucleotide inhibitor development, an increasing research field in universities but also pharmaceutical industry. We anticipate that the broad approach to generate and validate a novel miRNA stress-sensitive test system based on measurements in peripheral blood will add additional knowledge on common markers for distress.
Primate System Neuroscience, Cognitive neuroscience/Sensory selection and Decision making
Invasive basic neuroscience research with rhesus monkeys is a cornerstone of systems and cognitive neuroscience but also belongs to those areas of animal research generating substantial public concern about the animals’ welfare and the severity of the procedures. The goal of this project is to complement and extend behavioural and physiological approaches for welfare assessment in non-human primates (rhesus monkeys) we are developing in our laboratory with choice-based preference testing to rate the relative severity of a variety of different aspects of experimental procedures and the animals’ housing and social conditions. To assess the influence of such experimental procedures and aspects on animal welfare, we are currently developing a welfare scoring system optimized for rhesus monkeys in neuroscience research (see DFG FOR-1847). Yet, such an approach is limited because it focuses on measurable and observable consequences of the animals’ housing and experimental conditions. Furthermore the relative ranking of various observable scoring components might not match the animal’s subjective experience. We thus propose to use preference testing to determine the animals’ individual ranking of various aversive and positive aspects of the experimental, housing and social conditions. Animals will have the possibility to select among multiple two-alternative pairings of various choices. Our approach will build on our extensive experience in training rhesus monkeys in complex and challenging cognitive tasks and will benefit from our recently developed cognitive testing and enrichment kiosk system for rhesus monkeys. As an outcome of the project we expect to have developed a scientific approach that places a multitude of aspects of the animal’s daily experience onto a common ranking scale. This should be a substantial benefit for developing a severity assessment that is free of anthropomorphic biases.
The course of animal distress during gastrointestinal and neurological diseases
It is our goal to evaluate the course of distress for animals during classical gastrointestinal and neurological diseases. The key question is, if distress will increase over time in parallel to the progression of a disease, or if distress will decrease because animals have a coping mechanism for longer lasting stress. If sensitization or desensitization of distress will be measured is of immanent importance for assessing the correct severity of each animal model, especially for animal models which mimic chronic diseases. Thus, this project will provide a solid basis for the refinement of future animal experiments and might also provide data relevant for understanding principles of habituation or lack of habituation to pain and distress in human diseases. Our work will focus on disease models in mouse encompassing three organs: pancreas, liver and brain. We will induce diseases such as chronic pancreatitis, pancreatic carcinoma, liver fibrosis, and Alzheimer disease by chemical induction, surgery or genetic modifications. The progression of each disease will be analyzed by traditional histological, immunohistological and biochemical methods, but also by small animal MRT and PET-CT imaging. The pathophysiological processes of each disease will be correlated with read out parameters quantifying distress. These read out parameters will encompass quantification of allodynia with Von Frey filaments, evaluation of body weight, corticosterone concentrations and the habitus of animals with the help of score sheets as well as burrowing and nesting behavior scores. Moreover, additional methods such as camera based techniques for contactless monitoring, telemetry and cognitive bias tests as well as evaluating miRNAs as biomarkers for distress will be explored. Finally we will also evaluate the influence of chemotherapies and analgetics on animal distress.
Quality Management, Meta-Analyses and Recommendations for Severity Assessment
- Quality Management implementation for the whole research consortium
- To perform Systematic Reviews and Meta-Analyses
- To analyse and to publish recommendations for Severity-Grading
Quality Management: As already outlined in the main proposal, Section 2.7.1 Quality Management and improvement of reproducibility and reliability of biomedical research of biomedical research, we will implement a Quality Management System within this research group. This includes general process instructions, standard operation procedures, as well as central monitoring forms. The issue “Reproducibility” will be addresses by inter-laboratory comparisons as already performed in diagnostic laboratories. Moreover, the project will perform site visits by “Monitors” with auditing of the groups to identify problems or to mentor the groups to work according to SOPs as described in Good Laboratory Practice Guidelines.
Systematic Reviews and Meta-Analyses: In this part the aim of the project is tanalyse specific topics of animal models or methods as well as published evidence of severity in models in a structured way. This will allow the research group to start the project with the highest evidence level possible and to analyse structural problems in the first hand. Furthermore, this is an indispensable step for generating recommendations for severity assessment.
Recommendation for Severity-Grading: As outlined in Section 2.2.1 Recommendations for the grading of models and procedures according to official severity classifications, the aim of this part of the program is to establish recommendations on how defined procedures and animal models, respectively, are to be graded according to EU and national regulations. This will help scientists, authorities and review panels in the evaluation of animal based research. Recommendations will be based on findings of the research unit as well as data gained using Meta-Analyses.
The first goal is to develop SOPs for the use within the FOR-2591 Research Group and to implement this within a QM System for the consortium. The second goal is to perform Meta- Analysis resulting in recommendations for the research community. With this data generated we will be able in the future to give information on how severity can be measured and minimized by application of refinement strategies according to the 3 R principles. The third goal is to establish recommendations on how defined procedures and animal models, respectively, are to be graded for severity assessment according to EU and national regulations.