Evidence-based comparative severity assessment in young and adult mice.

In animal-based research, welfare assessments are essential for ethical and legal reasons. However, accurate assessment of suffering in laboratory animals is often complicated by the multidimensional character of distress and pain and the associated affective states. The present study aimed to design and validate multidimensional composite measure schemes comprising behavioral and biochemical parameters based on a bioinformatics approach. Published data sets from induced and genetic mouse models of neurological and psychiatric disorders were subjected to a bioinformatics workflow for cross-model analyses. ROC analyses pointed to a model-specific discriminatory power of selected behavioral parameters. Principal component analyses confirmed that the composite measure schemes developed for adult or young mice provided relevant information with the level of group separation reflecting the expected severity levels. Finally, the validity of the composite measure schemes developed for adult and young mice was further confirmed by k-means-based clustering as a basis for severity classification. The classification systems allowed the allocation of individual animals to different severity levels and a direct comparison of animal groups and other models. In conclusion, the bioinformatics approach confirmed the suitability of the composite measure schemes for evidence-based comparative severity assessment in adult and young mice. In particular, we demonstrated that the composite measure schemes provide a basis for an individualized severity classification in control and experimental groups allowing direct comparison of severity levels across different induced or genetic models. An online tool (R package) is provided, allowing the application of the bioinformatics approach to severity assessment data sets regardless of the parameters or models used. This tool can also be used to validate refinement measures.

Evidence-based severity assessment of the forced swim test in the rat.

The forced swim test (FST) is a traditional assay, which has been used for more than 40 years to assess antidepressant effects of novel drug candidates. In recent years, a debate about the test has focused on the assumption that the FST is highly aversive and burdening for the animals because of the earlier anthropomorphic interpretation and designation as a "behavioral despair test". The Directive 2010/63/EU and the German Animal Welfare law require a prospective severity classification of the planned experimental procedures. Still, an objective examination of the animals' burden in this test has not been performed yet. To fill this gap, we conducted an evidence-based severity assessment of the forced swim test in rats according to a 'standard protocol' with a water temperature of 25°C. We examined parameters representing the physiological and the affective state, and natural as well as locomotion-associated behaviors in three separate experiments to reflect as many dimensions as possible of the animal's condition in the test. Hypothermia was the only effect observed in all animals exposed to the FST when using this standard protocol. Additional adverse effects on body weight, food consumption, and fecal corticosterone metabolite concentrations occurred in response to administration of the antidepressant imipramine, which is frequently used as positive control when testing for antidepressant effects of new substances. We conclude that this version of the FST itself is less severe for the animals than assumed, and we suggest a severity classification of 'moderate' because of the acute and short-lasting effects of hypothermia. To refine the FST according to the 3Rs, we encourage confirming the predictive validity in warmer water temperatures to allow the rats to maintain physiological body temperature.

Grimace scale assessment during Citrobacter rodentium inflammation and colitis development in laboratory mice.

Introduction: Bacterial infections and chronic intestinal inflammations triggered by genetic susceptibility, environment or an imbalance in the intestinal microbiome are usually long-lasting and painful diseases in which the development and maintenance of these various intestinal inflammations is not yet fully understood, research is still needed. This still requires the use of animal models and is subject to the refinement principle of the 3Rs, to minimize suffering or pain perceived by the animals. With regard to this, the present study aimed at the recognition of pain using the mouse grimace scale (MGS) during chronic intestinal colitis due to dextran sodium sulfate (DSS) treatment or after infection with Citrobacter rodentium. Methods: In this study 56 animals were included which were divided into 2 experimental groups: 1. chronic intestinal inflammation (n = 9) and 2. acute intestinal inflammation (with (n = 23) and without (n = 24) C. rodentium infection). Before the induction of intestinal inflammation in one of the animal models, mice underwent an abdominal surgery and the live MGS from the cage side and a clinical score were assessed before (bsl) and after 2, 4, 6, 8, 24, and 48 hours. Results: The highest clinical score as well as the highest live MGS was detected 2 hours after surgery and almost no sign of pain or severity were detected after 24 and 48 hours. Eight weeks after abdominal surgery B6-Il4/Il10-/- mice were treated with DSS to trigger chronic intestinal colitis. During the acute phase as well as the chronic phase of the experiment, the live MGS and a clinical score were evaluated. The clinical score increased after DSS administration due to weight loss of the animals but no change of the live MGS was observed. In the second C57BL/6J mouse model, after infection with C. rodentium the clinical score increased but again, no increased score values in the live MGS was detectable. Discussion: In conclusion, the live MGS detected post-operative pain, but indicated no pain during DSS-induced colitis or C. rodentium infection. In contrast, clinical scoring and here especially the weight loss revealed a decreased wellbeing due to surgery and intestinal inflammation.

Toward Evidence-Based Severity Assessment in Mouse Models with Repeated Seizures: (II.) Impact of Surgery and Intrahippocampal Kainate.

INTRODUCTION: Chronic epilepsy models require neurosurgical procedures including depth electrode implants. The intrahippocampal kainate model is a frequently used chronic paradigm, which is based on chemoconvulsant administration and status epilepticus induction during the surgical procedure. This experimental approach raises the question of the extent to which this approach affects postsurgical recovery. In addition to the short- and long-term impact of the surgical intervention, a potential impact of highly frequent electrographic seizure events needs to be considered in the context of severity assessment. METHODS: Various behavioral, biochemical, and telemetric parameters were analyzed in four experimental groups of mice: 1st naive, 2nd with transmitter implants, 3rd with transmitter and electrode implants, and 4th with transmitter implants, electrode implants, and kainate-induced status epilepticus. RESULTS: During the early postsurgical phase, transmitter implants caused a transient impact on Mouse Grimace scores and intragroup increase of fecal corticosterone metabolites. Additional craniotomy was associated with an influence on total heart rate variability and fecal corticosterone metabolites. Heart rate and Irwin score increases as well as a prolonged increase in Mouse Grimace scores pointed to an added burden related to the induction of a nonconvulsive status epilepticus. Data from the chronic phase argued against a relevant influence of frequent electrographic seizures on behavioral patterns, fecal corticosterone metabolites, heart rate, and its variability. However, Irwin scores indicated long-term changes in some animals with increased reactivity, body tone, and Straub tail. Interestingly, selected behavioral and telemetric data from the early post-status epilepticus phase correlated with the frequency of electrographic seizure events in the chronic phase. CONCLUSION: In conclusion, our findings argue against the pronounced impact of highly frequent electrographic seizures on the well-being of mice. However, an increased level of nervousness in a subgroup of animals should be considered for handling procedures and refinement measures. In the early postsurgical phase, several parameters indicate an influence of the interventions with evidence that the nonconvulsive status epilepticus can negatively affect the recovery. Thus, the development and validation of refinement efforts should focus on this experimental phase. Finally, the datasets suggest that simple readout parameters may predict the long-term consequences of the epileptogenic insult. Respective biomarker candidates require further validation in the follow-up studies in models with subgroups of animals with or without epilepsy development.

Postoperative Severity Assessment in Sheep.

INTRODUCTION: Sheep are frequently used in translational surgical orthopedic studies. Naturally, a good pain management is mandatory for animal welfare, although it is also important with regard to data quality. However, methods for adequate severity assessment, especially considering pain, are rather rare regarding large animal models. Therefore, in the present study, accompanying a surgical pilot study, telemetry and the Sheep Grimace Scale (SGS) were used in addition to clinical scoring for severity assessment after surgical interventions in sheep. METHODS: Telemetric devices were implanted in a first surgery subcutaneously into four German black-headed mutton ewes (4-5 years, 77-115 kg). After 3-4 weeks of recovery, sheep underwent tendon ablation of the left M. infraspinatus. Clinical scoring and video recordings for SGS analysis were performed after both surgeries, and the heart rate (HR) and general activity were monitored by telemetry. RESULTS: Immediately after surgery, clinical score and HR were slightly increased, and activity was decreased in individual sheep after both surgeries. The SGS mildly elevated directly after transmitter implantation but increased to higher levels after tendon ablation immediately after surgery and on the following day. CONCLUSION: In summary, SGS- and telemetry-derived data were suitable to detect postoperative pain in sheep with the potential to improve individual pain recognition and postoperative management, which consequently contributes to refinement.

Evaluation of score parameters for severity assessment of surgery and liver cirrhosis in rats.

Severity assessment in animals is an ongoing field of research. In particular, the question of objectifiable and meaningful parameters of score-sheets, as well as their best combination, arise. This retrospective analysis investigates the suitability of a score-sheet for assessing severity and seeks to optimise it for predicting survival in 89 male Sprague Dawley rats (Rattus norvegicus), during an experiment evaluating the influence of liver cirrhosis by bile duct ligation (BDL) on vascular healing. The following five parameters were compared for their predictive power: (i) overall score; (ii) relative weight loss; (iii) general condition score; (iv) spontaneous behaviour score; and (v) the observer's assessment whether pain might be present. Suitable cut-off values of these individual parameters and the combination of multiple parameters were investigated. A total of ten rats (11.2%; 10/89) died or had to be sacrificed at an early stage due to pre-defined humane endpoints. Neither the overall score nor any individual parameter yielded satisfactory results for predicting survival. Using retrospectively calculated cut-off values and combining the overall score with the observer's assessment of whether the animal required analgesia (dipyrone) for pain relief resulted in an improved prediction of survival on the second post-operative day. This study demonstrates that combining score parameters was more suitable than using single ones and that experienced human judgement of animals can be useful in addition to objective parameters in the assessment of severity. By optimising the score-sheet and better understanding the burden of the model on rats, this study contributes to animal welfare.

RELSA-A multidimensional procedure for the comparative assessment of well-being and the quantitative determination of severity in experimental procedures.

Good science in translational research requires good animal welfare according to the principles of 3Rs. In many countries, determining animal welfare is a mandatory legal requirement, implying a categorization of animal suffering, traditionally dominated by subjective scorings. However, how such methods can be objectified and refined to compare impairments between animals, subgroups, and animal models remained unclear. Therefore, we developed the RELative Severity Assessment (RELSA) procedure to establish an evidence-based method based on quantitative outcome measures such as body weight, burrowing behavior, heart rate, heart rate variability, temperature, and activity to obtain a relative metric for severity comparisons. The RELSA procedure provided the necessary framework to get severity gradings in TM-implanted mice, yielding four distinct RELSA thresholds L1<0.27, L2<0.59, L3<0.79, and L4<3.45. We show further that severity patterns in the contributing variables are time and model-specific and use this information to obtain contextualized between animal-model and subgroup comparisons with the severity of sepsis > surgery > restraint stress > colitis. The bootstrapped 95% confidence intervals reliably show that RELSA estimates are conditionally invariant against missing information but precise in ranking the quantitative severity information to the moderate context of the transmitter-implantation model. In conclusion, we propose the RELSA as a validated tool for an objective, computational approach to comparative and quantitative severity assessment and grading. The RELSA procedure will fundamentally improve animal welfare, data quality, and reproducibility. It is also the first step toward translational risk assessment in biomedical research.

Grimace scale, burrowing, and nest building for the assessment of post-surgical pain in mice and rats-A systematic review.

Several studies suggested an informative value of behavioral and grimace scale parameters for the detection of pain. However, the robustness and reliability of the parameters as well as the current extent of implementation are still largely unknown. In this study, we aimed to systematically analyze the current evidence-base of grimace scale, burrowing, and nest building for the assessment of post-surgical pain in mice and rats. The following platforms were searched for relevant articles: PubMed, Embase via Ovid, and Web of Science. Only full peer-reviewed studies that describe the grimace scale, burrowing, and/or nest building as pain parameters in the post-surgical phase in mice and/or rats were included. Information about the study design, animal characteristics, intervention characteristics, and outcome measures was extracted from identified publications. In total, 74 papers were included in this review. The majority of studies have been conducted in young adult C57BL/6J mice and Sprague Dawley and Wistar rats. While there is an apparent lack of information about young animals, some studies that analyzed the grimace scale in aged rats were identified. The majority of studies focused on laparotomy-associated pain. Only limited information is available about other types of surgical interventions. While an impact of surgery and an influence of analgesia were rather consistently reported in studies focusing on grimace scales, the number of studies that assessed respective effects was rather low for nest building and burrowing. Moreover, controversial findings were evident for the impact of analgesics on post-surgical nest building activity. Regarding analgesia, a monotherapeutic approach was identified in the vast majority of studies with non-steroidal anti-inflammatory (NSAID) drugs and opioids being most commonly used. In conclusion, most evidence exists for grimace scales, which were more frequently used to assess post-surgical pain in rodents than the other behavioral parameters. However, our findings also point to relevant knowledge gaps concerning the post-surgical application in different strains, age levels, and following different surgical procedures. Future efforts are also necessary to directly compare the sensitivity and robustness of different readout parameters applied for the assessment of nest building and burrowing activities.

Why serology just is not enough: Strategic parvovirus risk assessment using a novel qPCR assay.

Health monitoring of laboratory rodents not only improves animal health but also enhances the validity of animal experiments. In particular, infections of laboratory animals with murine parvoviruses influence biomedical research data. Despite strict barrier housing, prevalence remains high in animal facilities, leading to increased risk of parvovirus introduction after the import of contaminated mice. Unfortunately, hygienic rederivation can be challenging, since gametes often contain residual virus material. Consequently, the process has to be closely monitored with highly sensitive diagnostic methods to verify parvovirus decontamination of the rederived progeny. However, diagnostic sensitivity of traditional methods is often low and requires testing of large animal cohorts. Therefore, we aimed to develop a powerful quantitative real-time polymerase chain reaction (qPCR) assay for the fast and reliable detection of murine parvoviruses in different sample materials. We validated the assay within an infection experiment and systematically analysed various animal-derived and environmental sample materials. We further developed a strategic risk assessment procedure for parvovirus monitoring after embryo transfer. Our novel qPCR assay reliably detected parvovirus DNA in a broad variety of sample materials, with environmental samples dominating in the acute phase of infection, whereas animal-derived samples were more suitable to detect low virus loads in the chronic phase. Here, the assay served as a highly sensitive screening method for parvovirus contamination in mouse colonies, requiring significantly lower sample sizes than traditional methods like conventional PCR and serology. Thus, the use of our novel qPCR assay substantially improves parvovirus diagnostics, enhancing research validity according to the 6Rs.

Health Monitoring of Laboratory Rodent Colonies-Talking about (R)evolution.

The health monitoring of laboratory rodents is essential for ensuring animal health and standardization in biomedical research. Progress in housing, gnotobiotic derivation, and hygienic monitoring programs led to enormous improvement of the microbiological quality of laboratory animals. While traditional health monitoring and pathogen detection methods still serve as powerful tools for the diagnostics of common animal diseases, molecular methods develop rapidly and not only improve test sensitivities but also allow high throughput analyses of various sample types. Concurrently, to the progress in pathogen detection and elimination, the research community becomes increasingly aware of the striking influence of microbiome compositions in laboratory animals, affecting disease phenotypes and the scientific value of research data. As repeated re-derivation cycles and strict barrier husbandry of laboratory rodents resulted in a limited diversity of the animals' gut microbiome, future monitoring approaches will have to reform-aiming at enhancing the validity of animal experiments. This review will recapitulate common health monitoring concepts and, moreover, outline strategies and measures on coping with microbiome variation in order to increase reproducibility, replicability and generalizability.

Toward evidence-based severity assessment in mouse models with repeated seizures: I. Electrical kindling.

OBJECTIVE: Ethical decisions about an allowance for animal experiments need to be based on scientifically sound information about the burden and distress associated with the experimental procedure and models. Thereby, species differences need to be considered for recommendations regarding evidence-based severity assessment and refinement measures. METHODS: A comprehensive analysis of behavioral patterns and corticosterone or its metabolites in serum and feces was completed in kindled mice. The impact of kindling via two different stimulation sites in the amygdala and hippocampus was determined. Data were compared to those from naive and electrode-implanted groups. RESULTS: Amygdala and hippocampus kindled mice exhibited comparable behavioral patterns with increased activity in the open field, reduced anxiety-associated behavior in the elevated-plus maze, and increased anhedonia-associated behavior in the saccharin preference test. In addition, repeated stimulation of the hippocampus caused a reduction in burrowing behavior and an increase in active social interaction. Levels of corticosterone and its metabolites were not altered in serum or feces, respectively. A comparison of mouse data with findings from amygdala kindled rats confirmed pronounced species differences in behavioral patterns associated with the kindling process. SIGNIFICANCE: Taken together the findings suggest a severity classification for the mouse kindling paradigms as moderate regardless of the stimulation site. The outcome of the species comparison provides valuable guidance for species selection for studies exploring behavioral comorbidities. In this context, it is emphasized that the mouse kindling paradigms seem to be well suited for studies exploring the link between ictal events and network alterations on the one hand, and hyperactivity and anhedonia-associated behavior on the other hand. Moreover, the underlying pathophysiological mechanisms and the impact of therapeutic interventions on these behavioral alterations can be studied in these paradigms providing guidance for the clinical management of respective psychiatric comorbidities in patients.

Design of composite measure schemes for comparative severity assessment in animal-based neuroscience research: A case study focussed on rat epilepsy models.

Comparative severity assessment of animal models and experimental interventions is of utmost relevance for harm-benefit analysis during ethical evaluation, an animal welfare-based model prioritization as well as the validation of refinement measures. Unfortunately, there is a lack of evidence-based approaches to grade an animal's burden in a sensitive, robust, precise, and objective manner. Particular challenges need to be considered in the context of animal-based neuroscientific research because models of neurological disorders can be characterized by relevant changes in the affective state of an animal. Here, we report about an approach for parameter selection and development of a composite measure scheme designed for precise analysis of the distress of animals in a specific model category. Data sets from the analysis of several behavioral and biochemical parameters in three different epilepsy models were subjected to a principal component analysis to select the most informative parameters. The top-ranking parameters included burrowing, open field locomotion, social interaction, and saccharin preference. These were combined to create a composite measure scheme (CMS). CMS data were subjected to cluster analysis enabling the allocation of severity levels to individual animals. The results provided information for a direct comparison between models indicating a comparable severity of the electrical and chemical post-status epilepticus models, and a lower severity of the kindling model. The new CMS can be directly applied for comparison of other rat models with seizure activity or for assessment of novel refinement approaches in the respective research field. The respective online tool for direct application of the CMS or for creating a new CMS based on other parameters from different models is available at https://github.com/mytalbot/cms. However, the robustness and generalizability needs to be further assessed in future studies. More importantly, our concept of parameter selection can serve as a practice example providing the basis for comparable approaches applicable to the development and validation of CMS for all kinds of disease models or interventions.

A safe bet? Inter-laboratory variability in behaviour-based severity assessment.

Evidence-based severity assessment is essential as a basis for ethical evaluation in animal experimentation to ensure animal welfare, legal compliance and scientific quality. To fulfil these tasks scientists, animal care and veterinary personnel need assessment tools that provide species-relevant measurements of the animals' physical and affective state. In a three-centre study inter-laboratory robustness of body weight monitoring, mouse grimace scale (MGS) and burrowing test were evaluated. The parameters were assessed in naïve and tramadol treated female C57BL/6J mice. During tramadol treatment a body weight loss followed by an increase, when treatment was terminated, was observed in all laboratories. Tramadol treatment did not affect the MGS or burrowing performance. Results were qualitatively comparable between the laboratories, but quantitatively significantly different (inter-laboratory analysis). Burrowing behaviour seems to be highly sensitive to inter-laboratory differences in testing protocol. All locations obtained comparable information regarding the qualitative effect of tramadol treatment in C57BL/6J mice, however, datasets differed as a result of differences in test and housing conditions. In conclusion, our study confirms that results of behavioural testing can be affected by many factors and may differ between laboratories. Nevertheless, the evaluated parameters appeared relatively robust even when conditions were not harmonized extensively and present useful tools for severity assessment. However, analgesia-related side effects on parameters have to be considered carefully.

Design of a joint research data platform: A use case for severity assessment.

Severity assessment in animal models is a data-driven process. We therefore present a use case for building a repository for interlaboratory collaboration with the potential of uploading specific content, making group announcements and internal prepublication discussions. We clearly show that it is possible to offer such a structure with minimal effort and a basic understanding of web-based services, also taking into account the human factor in individual data collection. The FOR2591 Online Repository serves as a blueprint for other groups, so that one day not only will data sharing among consortium members be improved but the transition from the private to the persistent domain will also be easier.

Where are we heading? Challenges in evidence-based severity assessment.

Evidence-based severity assessment in laboratory animals is, apart from the ethical responsibility, imperative to generate reproducible, standardized and valid data. However, the path towards a valid study design determining the degree of pain, distress and suffering experienced by the animal is lined with pitfalls and obstacles as we will elucidate in this review. Furthermore, we will ponder on the genesis of a holistic concept relying on multifactorial composite scales. These have to combine robust and reliable parameters to measure the multidimensional aspects that define the severity of animal experiments, generating a basis for the substantiation of the refinement principle.

Perspective review of optical imaging in welfare assessment in animal-based research.

To refine animal research, vital signs, activity, stress, and pain must be monitored. In chronic studies, some measures can be assessed using telemetry sensors. Although this methodology provides high-precision data, an initial surgery for device implantation is necessary, potentially leading to stress, wound infections, and restriction of motion. Recently, camera systems have been adapted for animal research. We give an overview of parameters that can be assessed using imaging in the visible, near-infrared, and thermal spectrum of light. It focuses on heart activity, respiration, oxygen saturation, and motion, as well as on wound analysis. For each parameter, we offer recommendations on the minimum technical requirements of appropriate systems, regions of interest, and light conditions, among others. In general, these systems demonstrate great performance. For heart and respiratory rate, the error was <4 beats / min and 5 breaths/min. Furthermore, the systems are capable of tracking animals during different behavioral tasks. Finally, studies indicate that inhomogeneous temperature distribution around wounds might be an indicator of (pending) infections. In sum, camera-based techniques have several applications in animal research. As vital parameters are currently only assessed in sedated animals, the next step should be the integration of these modalities in home-cage monitoring.

Assessment of the Intestinal Barrier with Five Different Permeability Tests in Healthy C57BL/6J and BALB/cJ Mice.

BACKGROUND: Intestinal permeability is thought to be of major relevance for digestive and nutrition-related diseases, and therefore has been studied in numerous mouse models of disease. However, it is unclear which tools are the preferable ones, and how normal values should be defined. AIMS: To compare different in vivo permeability tests in healthy mice of commonly used genetic backgrounds. METHODS: We assessed the intestinal barrier in male and female C57BL/6J and BALB/cJ mice of different ages, using four orally administered permeability markers, FITC-dextran 4000 (FITC-D4000) and ovalbumin (OVA) measured in plasma, and polyethylene glycol (PEG) and lactulose/mannitol (Lac/Man) measured in urine, and by assessing lipopolysaccharide (LPS) in portal vein plasma. RESULTS: After gavage, FITC-D4000, OVA, Lac/Man, and PEG400, but not PEG4000, were detectable in plasma or urine. Female mice tended to have a higher permeability according to the FITC-D4000, OVA, and PEG400 tests, but the Lac/Man ratio was higher in males. No significant differences between the two mouse strains of young and old mice were observed except for mannitol recovery, which was higher in BALB/cJ mice compared to C57BL/6J mice (p < 0.05). Virtually no LPS was detected in healthy mice. For all markers, normal values have been defined based on 5th-95th percentile ranges of our data. CONCLUSION: Selected oral permeability tests, such as FITC-D4000, OVA, PEG400, and Lac/Man, as well as LPS measurements in portal vein plasma, could be suitable for the evaluation of the intestinal barrier in mice, if used in a standardized way.