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Translation of animal-based preclinical research is hampered by poor validity and reproducibility issues. Unfortunately, preclinical research has ‘fallen between the stools’ of competing study design traditions. Preclinical studies are often characterised by small sample sizes, large variability, and ‘problem’ data. Although Fisher-type designs with randomisation and blocking are appropriate and have been vigorously promoted, structured statistically-based designs are almost unknown. Traditional analysis methods are commonly misapplied, and basic terminology and principles of inference testing misinterpreted. Problems are compounded by the lack of adequate statistical training for researchers, and failure of statistical educators to account for the unique demands of preclinical research. The solution is a return to the basics: statistical education tailored to non-statistician investigators, with clear communication of statistical concepts, and curricula that address design and data issues specific to preclinical research. Statistics curricula should focus on statistics as process: data sampling and study design before analysis and inference. Properly-designed and analysed experiments are a matter of ethics as much as procedure. Shifting the focus of statistical education from rote hypothesis testing to sound methodology will reduce the numbers of animals wasted in noninformative experiments and increase overall scientific quality and value of published research.

Non-aversive handling is a well-documented refinement measure for improving rodent welfare. Because maternal stress is related to reduced productivity, we hypothesized that welfare benefits associated with non-aversive handling would translate to higher production and fewer litters lost in a laboratory mouse breeding colony. We performed a randomized controlled trial to examine the effects of a standard method of handling (tail-lift with forceps) versus non-aversive handling with transfer tunnels (‘tunnel-handled’) on breeding performance in 59 C57BL/6J mouse pairs. Intervention assignments could not be concealed from technicians, but were concealed from assessors and data analyst. An operationally significant effect of tunnel-handling (large enough differences to warrant programmatic change) was defined before study initiation as a 5% increase in productivity, or one extra pup over the reproductive lifetime of each pair. Pairs were randomly allocated to handling intervention and cage rack location, and monitored over an entire 6-month breeding cycle. For each group, we measured number of pups born and weaned, and number of entire litters lost prior to weaning. Differences between transfer methods were estimated by two-level hierarchical mixed models adjusted for parental effects and parity. Compared to tail-lift mice, tunnel-handled mice averaged one extra pup per pair born (+1.0; 95% CI 0.9, 1.1; P = 0.41) and weaned (+1.1, 95% CI 0.9, 1.2; P = 0.33). More tunnel-handled pairs successfully weaned all litters produced (13/29 pairs, 45% vs 4/30 pairs, 13%; P = 0.015), averaged fewer litter losses prior to weaning (11/29 pairs [38%] vs 26/30 pairs [87%]; P <0.001), and had a 20% lower risk of recurrent litter loss. The increase in numbers of pups produced and weaned with tunnel handling met threshold requirement for operational significance. These data and projected cost savings persuaded management to incorporate tunnel handling as standard of care across the institution. These data also suggest that overlooked husbandry practices such as cage transfer may be major confounders in studies of mouse models.

The laboratory mouse is a key player in preclinical oncology research. However, emphasis of techniques reporting at the expense of critical animal-related detail compromises research integrity, animal welfare, and, ultimately, the translation potential of mouse-based oncology models. To evaluate current reporting practices, we performed a cross-sectional survey of 400 preclinical oncology studies using mouse solid-tumour models. Articles published in 2020 were selected from 20 journals that specifically endorsed the ARRIVE (Animal Research: Reporting of In Vivo Experiments) preclinical reporting guidelines. We assessed reporting compliance for 22 items in five domains: ethical oversight assurance, animal signalment, husbandry, welfare, and euthanasia. Data were analysed using hierarchical generalised random-intercept models, clustered on journal. Overall, reporting of animal-related items was poor. Median compliance over all categories was 23%. There was little or no association between extent of reporting compliance and journal or journal impact factor. Age, sex, and source were reported most frequently, but verifiable strain information was reported for <10% of studies. Animal husbandry, housing environment, and welfare items were reported by <5% of studies. Fewer than one in four studies reported analgesia use, humane endpoints, or an identifiable method of euthanasia. Of concern was the poor documentation of ethical oversight information. Fewer than one in four provided verifiable approval information, and almost one in ten reported no information, or information that was demonstrably false. Mice are the “invisible actors” in preclinical oncology research. In spite of widespread endorsement of reporting guidelines, adherence to reporting guidelines on the part of authors is poor and journals fail to enforce guideline reporting standards. In particular, the inadequate reporting of key animal-related items severely restricts the utility and translation potential of mouse models, and results in research waste. Both investigators and journals have the ethical responsibility to ensure animals are not wasted in uninformative research.

An interactive Sex Inclusive Research Framework (SIRF) supports the evaluation of in vivo and ex vivo research proposals to address the risk of sex bias in preclinical research. The framework delivers a traffic light classification, indicating whether a proposal is appropriate, risky, or insufficient with regard to sex inclusion. This tool is designed for use by researchers, (animal) ethical review boards, and funders to generate a rigorous and reproducible assessment of sex inclusion at the proposal level, thus helping address and resolve the embedded sex bias in preclinical research. In preclinical research, there is a persistent sex bias where research is conducted with a single sex. Here, the authors present a Sex Inclusive Research Framework that provides a traffic light classification of research proposals to facilitate robust assessment and promote equitable sex inclusion

Reproducible science requires transparent reporting. The ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments) were originally developed in 2010 to improve the reporting of animal research. They consist of a checklist of information to include in publications describing in vivo experiments to enable others to scrutinise the work adequately, evaluate its methodological rigour, and reproduce the methods and results. Despite considerable levels of endorsement by funders and journals over the years, adherence to the guidelines has been inconsistent, and the anticipated improvements in the quality of reporting in animal research publications have not been achieved. Here, we introduce ARRIVE 2.0. The guidelines have been updated and information reorganised to facilitate their use in practice. We used a Delphi exercise to prioritise and divide the items of the guidelines into 2 sets, the “ARRIVE Essential 10,” which constitutes the minimum requirement, and the “Recommended Set,” which describes the research context. This division facilitates improved reporting of animal research by supporting a stepwise approach to implementation. This helps journal editors and reviewers verify that the most important items are being reported in manuscripts. We have also developed the accompanying Explanation and Elaboration document, which serves (1) to explain the rationale behind each item in the guidelines, (2) to clarify key concepts, and (3) to provide illustrative examples. We aim, through these changes, to help ensure that researchers, reviewers, and journal editors are better equipped to improve the rigour and transparency of the scientific process and thus reproducibility.

All animal protocols need quantitative justification of animal numbers, but not all will need power calculations.

PurposeVasoplegia is a clinical syndrome marked by severe arteriolar vasodilatation, hypotension, and low systemic vascular resistance refractory to multiple vasopressor treatment. We report our experience with hydroxocobalamin (B12) infusion as a potential rescue adjunct for refractory vasoplegia during cardiopulmonary bypass (CPB).MethodsWe performed a retrospective chart review of 33 patients undergoing cardiac surgery between 1 January 2013 and 31 December 2015, who were given intravenous B12 for refractory hypotension during, or immediately following, CPB. We assessed mean arterial pressure (MAP) responses using semi-parametric group-based models (trajectory analysis). Vasopressor use was evaluated by norepinephrine-equivalent rates calculated five minutes prior, and up to 60 min following, B12 administration.ResultsPatients were mostly male (82%), had a mean (SD) age of 53 (13) yr, and median (IQR) EuroSCORE mortality index of 9 [4-40]. Four patterns of MAP responses to B12 were identified. In Group 1 (“poor responders”) nine of 33 patients (27%) had the highest median [IQR] mortality risk (EuroSCORE 40 [4-52]), lowest mean pre-B12 MAP (50 mmHg), and minimal hemodynamic response in spite of continued vasopressor support. In contrast, Group 2 “responders” (8/33, 24%) showed a brisk MAP response (> 15 mmHg) to B12, sustained for > 60 min post-infusion, with 50% vasopressor reduction. Groups 3 and 4 had the lowest median mortality risk (EuroSCORE 8) and highest pre-B12 MAP (72 mmHg). Although Group 3 patients (“sustainers”; 9/33, 27%) showed a sustained MAP improvement, those in Group 4 (“rebounders”; 7/33, 21%) were characterized by hypertensive overshoot followed by a decrease in MAP.ConclusionThese data indicate considerable heterogeneity in patient response to B12, potentially dependent on both patient preoperative condition and non-standardized time of administration. B12 may provide a useful alternative therapy for refractory hypotension and vasoplegia, but controlled clinical trials to assess efficacy are needed.

The laboratory mouse is a key player in preclinical oncology research. However, emphasis of techniques reporting at the expense of critical animal-related detail compromises research integrity, animal welfare, and, ultimately, the translation potential of mouse-based oncology models. To evaluate current reporting practices, we performed a cross-sectional survey of 400 preclinical oncology studies using mouse solid-tumour models. Articles published in 2020 were selected from 20 journals that specifically endorsed the ARRIVE (Animal Research: Reporting of In Vivo Experiments) preclinical reporting guidelines. We assessed reporting compliance for 22 items in five domains: ethical oversight assurance, animal signalment, husbandry, welfare, and euthanasia. Data were analysed using hierarchical generalised random-intercept models, clustered on journal. Overall, reporting of animal-related items was poor. Median compliance over all categories was 23%. There was little or no association between extent of reporting compliance and journal or journal impact factor. Age, sex, and source were reported most frequently, but verifiable strain information was reported for <10% of studies. Animal husbandry, housing environment, and welfare items were reported by <5% of studies. Fewer than one in four studies reported analgesia use, humane endpoints, or an identifiable method of euthanasia. Of concern was the poor documentation of ethical oversight information. Fewer than one in four provided verifiable approval information, and almost one in ten reported no information, or information that was demonstrably false. Mice are the “invisible actors” in preclinical oncology research. In spite of widespread endorsement of reporting guidelines, adherence to reporting guidelines on the part of authors is poor and journals fail to enforce guideline reporting standards. In particular, the inadequate reporting of key animal-related items severely restricts the utility and translation potential of mouse models, and results in research waste. Both investigators and journals have the ethical responsibility to ensure animals are not wasted in uninformative research.

Refined handling improves laboratory mouse welfare and research outcomes when compared to traditional tail handling, yet implementation does not seem to be widespread. Refined handling includes picking up a mouse using a tunnel or cupped hands. The aim of this study was to determine the current prevalence of and beliefs towards refined handling using the theory of planned behavior. It was predicted that refined handling prevalence is low compared to traditional handling methods, and its implementation is determined by individual and institutional beliefs. Research personnel were recruited via online convenience sampling through email listservs and social media. A total of 261 participants in diverse roles (e.g. veterinarians, managers, caretakers, researchers, etc.) responded primarily from the USA (79%) and academic institutions (61%) Participants were surveyed about their current use, knowledge, and beliefs about refined handling. Quantitative data were analyzed via descriptive statistics and generalised regression. Qualitative data were analyzed by theme. Research personnel reported low levels of refined handling implementation, with only 10% of participants using it exclusively and a median estimate of only 10% of institutional mice being handled with refined methods. Individually, participants had positive attitudes, neutral norms, and positive control beliefs about refined handling. Participants’ intention to provide refined handling in the future was strongly associated with their attitudes, norms, and control beliefs (p<0.01). Participants believed barriers included jumpy mice, perceived incompatibility with restraint, lack of time, and other personnel. However, participants also believed refined handling was advantageous to mouse welfare, handling ease, personnel, and research. Although results from this survey indicate that current refined handling prevalence is low in this sample, personnel believe it has important benefits, and future use is associated with their beliefs about the practice. People who believed refined handling was good, felt pressure to use it, and were confident in their use reported higher implementation. Increased refined handling could be encouraged through education on misconceptions, highlighting advantages, and addressing important barriers.

How to reduce animal numbers and increase the information content of animal-based studies. A ‘well-built’ research question turns the research hypothesis into an actionable, focused, and testable plan of action.