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.

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.

Voluntary wheel running behaviour as a tool to assess the severity in a mouse pancreatic cancer model.

Laboratory animals frequently undergo routine experimental procedures such as handling, restraining and injections. However, as a known source of stress, these procedures potentially impact study outcome and data quality. In the present study, we, therefore, performed an evidence-based severity assessment of experimental procedures used in a pancreatic cancer model including surgical tumour induction and subsequent chemotherapeutic treatment via repeated intraperitoneal injections. Cancer cell injection into the pancreas was performed during a laparotomy under general anaesthesia. After a four-day recovery phase, mice received either drug treatment (galloflavin and metformin) or the respective vehicle substances via daily intraperitoneal injections. In addition to clinical scoring, an automated home-cage monitoring system was used to assess voluntary wheel running (VWR) behaviour as an indicator of impaired well-being. After surgery, slightly elevated clinical scores and minimal body weight reductions, but significantly decreased VWR behaviour were observed. During therapy, body weight declined in response to chemotherapy, but not after vehicle substance injection, while VWR activity was decreased in both cases. VWR behaviour differed between treatment groups and revealed altered nightly activity patterns. In summary, by monitoring VWR a high impact of repeated injections on the well-being of mice was revealed and substance effects on well-being were distinguishable. However, no differences in tumour growth between treatment groups were observed. This might be due to the severity of the procedures uncovered in this study, as exaggerated stress responses are potentially confounding factors in preclinical studies. Finally, VWR was a more sensitive indicator of impairment than clinical scoring in this model.

Extracting data from graphs: A case-study on animal research with implications for meta-analyses.

Systematic reviews with meta-analyses are powerful tools that can answer research questions based on data from published studies. Ideally, all relevant data is directly available in the text or tables, but often it is only presented in graphs. In those cases, the data can be extracted from graphs, but this potentially introduces errors. Here, we investigate to what extent the extracted outcome and error values differ from the original data and if these differences could affect the results of a meta-analysis. Six extractors extracted 36 outcome values and corresponding errors from 22 articles. Differences between extractors were compared using overall concordance correlation coefficients (OCCC), differences between the original and extracted data were compared using concordance correlation coefficients (CCC). To test the possible influence on meta-analyses, random-effects meta-analyses on mean difference comparing original and extracted data were performed. The OCCCs and CCCs were high for both outcome values and errors, CCCs were >0.99 for the outcome and >0.92 for errors. The meta-analyses showed that the overall effect on outcome was very small (median: 0.025, interquartile range: 0.016-0.046). Therefore, data extraction from graphs is a good method to harvest data if it is not provided in the text or tables, and the original authors cannot provide the data.

Nest-building performance in rats: impact of vendor, experience, and sex.

Nest building behavior has been intensely applied as a parameter for severity assessment in mice. In contrast, only a limited number of studies have reported nest building data from rats. Here, we assessed nest building in rats in two different facilities addressing the hypotheses that the vendor, previous experience with the nesting material as well as sex of the rats has an impact on the performance. Data from two study sites and three raters were compared to obtain information about the robustness of nest complexity scoring. The findings demonstrate a generally poor nest building performance in rats with a pronounced day-to-day fluctuation, and site-specific differences. Application of a newly developed scoring system resulted in an intermediate inter-rater reliability. Previous experience with the nesting material did not exert a consistent impact on nest complexity scores. Sex differences proved to depend on vendor and animal facility without consistent findings supporting a superior performance in female or male rats. In conclusion, our findings argue against a robust and consistent influence of sex and familiarity with the nesting material. The comparison between facilities suggests that local conditions need to be considered as influencing factors, which should be explored in more detail by future multicenter approaches. Considering the day-to-day fluctuation and the intermediate inter-rater reliability, we highly recommend to base nest complexity evaluation on means from several subsequent days analyzed by a group of experienced raters.

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.

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.

Systematic analysis of severity in a widely used cognitive depression model for mice.

Animal models in psychiatric research are indispensable for insights into mechanisms of behaviour and mental disorders. Distress is an important aetiological factor in psychiatric diseases, especially depression, and is often used to mimic the human condition. Modern bioethics requires balancing scientific progress with animal welfare concerns. Therefore, scientifically based severity assessment of procedures is a prerequisite for choosing the least compromising paradigm according to the 3Rs principle. Evidence-based severity assessment in psychiatric animal models is scarce, particularly in depression research. Here, we assessed severity in a cognitive depression model by analysing indicators of stress and well-being, including physiological (body weight and corticosterone metabolite concentrations) and behavioural (nesting and burrowing behaviour) parameters. Additionally, a novel approach for objective individualised severity grading was employed using clustering of voluntary wheel running (VWR) behaviour. Exposure to the paradigm evoked a transient elevation of corticosterone, but neither affected body weight, nesting or burrowing behaviour. However, the performance in VWR was impaired after recurrent stress exposure, and the individual severity level increased, indicating that this method is more sensitive in detecting compromised welfare. Interestingly, the direct comparison to a somatic, chemically induced colitis model indicates less distress in the depression model. Further objective severity assessment studies are needed to classify the severity of psychiatric animal models in order to balance validity and welfare, reduce the stress load and thus promote refinement.

Semi-automated generation of pictures for the Mouse Grimace Scale: A multi-laboratory analysis (Part 2).

The Mouse Grimace Scale (MGS) is an established method for estimating pain in mice during animal studies. Recently, an improved and standardized MGS set-up and an algorithm for automated and blinded output of images for MGS evaluation were introduced. The present study evaluated the application of this standardized set-up and the robustness of the associated algorithm at four facilities in different locations and as part of varied experimental projects. Experiments using the MGS performed at four facilities (F1-F4) were included in the study; 200 pictures per facility (100 pictures each rated as positive and negative by the algorithm) were evaluated by three raters for image quality and reliability of the algorithm. In three of the four facilities, sufficient image quality and consistency were demonstrated. Intraclass correlation coefficient, calculated to demonstrate the correlation among raters at the three facilities (F1-F3), showed excellent correlation. The specificity and sensitivity of the results obtained by different raters and the algorithm were analysed using Fisher's exact test (p < 0.05). The analysis indicated a sensitivity of 77% and a specificity of 64%. The results of our study showed that the algorithm demonstrated robust performance at facilities in different locations in accordance with the strict application of our MGS setup.