Professor Claire Gibson was awarded funding to improve a mouse model of ischaemic stroke to reduce inter-animal variability.
Principal Investigator: Professor Claire Gibson
Organisation: University of Nottingham
Award type: Pilot study grant
Start date: 2014
Duration: 1 year
Approximately 85% of strokes in humans are ischaemic in nature. This is modelled experimentally, usually in rodents, by temporarily or permanently occluding the middle cerebral artery (MCA). Occlusion can be achieved by various methods including electrocoagulation and pharmacological intervention but mechanical obstruction by intraluminal insertion of a filament is often the method of choice, being used by almost 80% of UK stroke researchers.
The filament is inserted into the intracranial internal carotid artery through an incision usually made in the common carotid artery. The filament prevents blood flow into the MCA which, after a period of ischemia typically up to 90 minutes, is restored by removal of the filament and ligation of the common carotid artery to prevent blood loss through the incision. The surgery is technically demanding and associated with significant animal welfare concerns in the immediate days following the induction of experimental stroke, including death, weight loss, sensorimotor deficits and seizures.
The primary outcome measure used in in vivo stroke research is the infarct or lesion volume. There is, however, considerable interanimal, experiment and laboratory variation in the size of the infarct in the MCA occlusion (MCAO) model even when following the same defined protocol. In some instances, despite the animal undergoing surgery, there is no lesion. Evidence suggests that this variability can be caused by differences in the strain and age of the rodents used, cerebrovascular anatomy, and the size and type of the filament. The variability reduces the statistical power of the experiment necessitating the use of large numbers of animals to power the experiment appropriately. Many studies are underpowered as a result – because of factors such as cost, ethical considerations, and practical issues involved with the complex surgery and subsequent intensive care of the animals required.
3Rs benefits (actual and potential)
During the MCAO procedure, it is typical to permanently ligate the common carotid artery and reperfusion of the MCA occurs via vessels that form the Circle of Willis. This structure is anatomically highly variable, particularly in the commonly used C57Bl/6 mouse strain, which might account for the heterogeneity seen in lesion volume. To address this, Claire investigated whether it was possible to enhance reperfusion by repairing the common carotid artery rather than relying on the Circle of Willis.
Claire used a small tissue pad coated in fibrinogen and thrombin to seal the incision following removal of the filament, rather than permanently ligating the common carotid artery. Using histological assessment and magnetic resonance imaging techniques to monitor cerebral blood flow, Claire demonstrated the repair of the vessel wall was stable and reperfusion was enhanced compared with the standard ligation model. Importantly, repairing the common carotid artery reduces lesion volume variability between animals. Based on this, Claire has shown that a study to determine treatment effect would use 40% fewer mice (35 rather than 58 per group), assuming a power of 0.8, significance level of 0.05 and a predicted 30% reduction in lesion volume between control and test animals.
To make surgical access easier, it is common practice in the MCAO model to temporarily ligate the external carotid artery prior to making an incision in the common carotid artery. However, reducing vascular supply to the external carotid artery, even temporarily, can affect the muscles involved in chewing and swallowing. Claire was also able to demonstrate with the pilot grant that it is possible to conduct the surgery without ligating the external carotid artery with no effect on lesion volume. Preliminary evidence suggests this reduces body weight loss when compared with animals where the external carotid artery is temporarily ligated.
Scientific and technological benefits
The improved surgical methods for the MCAO model were published in Disease Models and Mechanisms in 2017, with the detailed methodology reported in the Journal of Visualized Experiments. Claire was awarded an NC3Rs skills and knowledge transfer grant in 2017 to provide hands-on training to four major stroke research groups in Europe and North America enabling them to adopt the model and subsequently act as training hubs for others. The improved surgical approach may also have applicability in rodent models of embolic stroke.
Claire was awarded NC3Rs funding for a PhD student in 2015. Michaela Bayliss, the PhD student, has investigated further refinements in the MCAO model in rats and mice, including whether improved environmental enrichment benefits animal welfare following stroke surgery and whether it has any impact on ischaemic damage. To date Michaela has one first author paper, published in PLOS ONE, which shows that in normotensive rats (unlike hypertensive rats) a pre-stroke craniotomy does not improve animal welfare in terms of decreased body weight loss, improved survival and fewer neurological deficits.