Dissemination of refinements in mouse experimental stroke models to the scientific community.

In the UK, cerebral stroke is the 3rd cause of death, the leading cause of severe adult disability in adults and costs the NHS an estimated £3 billion per year. The generation of novel and rational therapeutics is essential. Thus, every year thousands of animals are used experimentally to model stroke and investigate potential therapies. Mice are commonly used in experimental studies aimed at modelling stroke not least because they are useful for genetic manipulation studies. However, studies using experimental stroke models tend to show a large variability in the data produced. This usually results in researchers using an increased number of animals per study in order to try and detect a significant difference. We proposed that a large portion of this variability may be due to differences in the anatomy of blood vessels in the brain and an alternative approach, which we have previously developed, reduces the variability seen. Our aim here is to disseminate this refined approach within the scientific community so that other labs performing experimental stroke studies will produce data with reduced variability and therefore reduce the number of animals used in such experiments.

Cerebral stroke is the third cause of death and the most common cause of adult disability. However, current treatments are limited both in terms of utility and effectiveness. Efficacy of stroke interventions are tested using rodent middle cerebral artery occlusion (MCAO) models of focal brain ischemia but these have a poor record of translating into clinically effective treatments. The majority of experimental stroke studies use lesion volume as the primary outcome measure. However, variability in MCAO lesion volume is high with significant differences generally tested using parametric analyses which require a normal data distribution. Data from my laboratory over the last 10 years (and confirmed by others) shows that MCAO lesion volume data is not normally distributed and results in a bimodal distribution of small (purely striatal) and large (striato-cortical) lesions. Thus, previous studies using parametric tests, implicitly or explicitly based on an assumption of normal distribution, are likely to have been underpowered and analysed inappropriately thus decreasing their chance of detecting effective drug treatments.
In our previous NC3Rs grant (REF NC/M000117/1 to CG) we have demonstrated that an alternative surgical approach to induce experimental stroke by middle cerebral artery occlusion (MCAO) in mice led to improved welfare and reduced variability in lesion volume. Importantly, such reduced variability should help ensure that lesion volume sizes, within groups, are more consistent and appropriate a priori power calculations are performed. For example, using the data obtained in that study our power analysis shows that fewer animals per group are required to demonstrate a 30% reduction in lesion volume following our new approach compared to the traditional approach.

The overall aim of this project is to produce 3Rs impact within the field of experimental stroke. Through actively engaging with the scientific stroke community we will encourage and promote the adoption of the refinement in technique previously developed in Dr Gibson' lab. By engaging with three key and highly productive preclinical stroke labs worldwide we will speed up the wider adoption of this refinement in technique. We have selected 3 key laboratories in the UK (Professor Stuart Allan, University of Manchester), Europe (Professor Ulrich Dirnagl, Charité University of Berlin, Germany) and USA (Professor Donal Stein, Emory University, Atlanta, GA) who will support us by agreeing to be end users of this new approach (see letters of support). Each lab uses a significant number of mice for experimental stroke studies per year - for example, these key labs typically use 1800-2000 mice per year in experimental stroke studies. Importantly, they also represent, within the field, training hubs for researchers to visit to learn the technique of in vivo stroke induction. Since 2005, approximately 2000 research papers (Web of Science, October 2016) have reported original research findings involving the induction of MCAO in mice. Mice are the most common species exposed to MCAO due to their genetic utility. As a conservative estimate, if each publication contains data from ~30 mice, at least 60,000 mice have undergone MCAO since 2005. This does not take into account unpublished experiments so it is probably a gross underestimation. We have demonstrated, from our previous study, that a surgical refinement to induce MCAO in mice can improve wellbeing and reduce variability. If animal wellbeing is improved then animals undergoing MCAO protocols will recover better from the procedure and the impact upon animal welfare will be improved.