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

Wheel running behaviour in group-housed female mice indicates disturbed wellbeing due to DSS colitis

Voluntary wheel running (VWR) behaviour is a sensitive indicator of disturbed wellbeing and used for the assessment of individual experimental severity levels in laboratory mice. However, monitoring individual VWR performance usually requires single housing, which itself might have a negative effect on wellbeing. In consideration of the 3Rs principle, VWR behaviour was evaluated under group-housing conditions. To test the applicability for severity assessment, this readout was evaluated in a dextran sodium sulphate (DSS) induced colitis model. For continuous monitoring, an automated system with integrated radio-frequency identification technology was used, enabling detection of individual VWR. After a 14-day adaptation period mice demonstrated a stable running performance. Analysis during DSS treatment in combination with repeated facial vein phlebotomy and faecal sampling procedure resulted in significantly reduced VWR behaviour during the course of colitis and increased VWR during disease recovery. Mice submitted to phlebotomy and faecal sampling but no DSS treatment showed less reduced VWR but a longer-lasting recovery. Application of a cluster model discriminating individual severity levels based on VWR and body weight data revealed the highest severity level in most of the DSS-treated mice on day 7, but a considerable number of control mice also showed elevated severity levels due to sampling procedures alone. In summary, VWR sensitively indicated the course of DSS colitis severity and the impact of sample collection. Therefore, monitoring of VWR is a suitable method for the detection of disturbed wellbeing due to DSS colitis and sampling procedure in group-housed female laboratory mice.Federal Ministry of Economics and Energy, "Zentrales Innovationsprogramm Mittelstand (ZIM)Deutsche Forschungsgemeinschaft https://doi.org/https://dx.doi.org/10.13039/501100001659Peer Reviewe

Running in the wheel: Defining individual severity levels in mice.

The fine-scale grading of the severity experienced by animals used in research constitutes a key element of the 3Rs (replace, reduce, and refine) principles and a legal requirement in the European Union Directive 2010/63/EU. Particularly, the exact assessment of all signs of pain, suffering, and distress experienced by laboratory animals represents a prerequisite to develop refinement strategies. However, minimal and noninvasive methods for an evidence-based severity assessment are scarce. Therefore, we investigated whether voluntary wheel running (VWR) provides an observer-independent behaviour-centred approach to grade severity experienced by C57BL/6J mice undergoing various treatments. In a mouse model of chemically induced acute colitis, VWR behaviour was directly related to colitis severity, whereas clinical scoring did not sensitively reflect severity but rather indicated marginal signs of compromised welfare. Unsupervised k-means algorithm-based cluster analysis of body weight and VWR data enabled the discrimination of cluster borders and distinct levels of severity. The validity of the cluster analysis was affirmed in a mouse model of acute restraint stress. This method was also applicable to uncover and grade the impact of serial blood sampling on the animal's welfare, underlined by increased histological scores in the colitis model. To reflect the entirety of severity in a multidimensional model, the presented approach may have to be calibrated and validated in other animal models requiring the integration of further parameters. In this experimental set up, however, the automated assessment of an emotional/motivational driven behaviour and subsequent integration of the data into a mathematical model enabled unbiased individual severity grading in laboratory mice, thereby providing an essential contribution to the 3Rs principles