Bilateral common carotid artery occlusion

Mice were housed in individually-ventilated cages (in groups of up to five mice) under specific pathogen-free conditions and standard 12 hr light/dark cycle with unrestricted access to food and water. All experiments using live animals were conducted under the authority of UK Home Office project and personal licences and adhered to regulations specified in the Animals (Scientific Procedures) Act (1986) and Directive 2010/63/EU and were approved by both The Roslin Institute’s and the University of Edinburgh’s Animal Welfare and Ethics Committees. Experimental design, analysis and reporting followed the ARRIVE guidelines (https://www.nc3rs.org.uk/arrive-guidelines) where possible. The sample size was calculated based on the experimental observations of reporting the effect and standard deviation of BCCAO-induced neuronal loss in both wild-type and Dapk1^–/–(Tu et al., 2010), whose experimental observations using n = 7 per genotype we retrospectively calculated were powered at >99%.

Transient bilateral common carotid artery occlusion (BCCAO) was performed in Dapk1^–/– and wild-type male control mice under isoflurane anaesthesia (with O₂ and N₂O). The operator was unaware of genotype. Core body temperature was maintained at 37 ± 0.5°C throughout the procedure with a feedback controlled heating blanket (Harvard Apparatus, UK). Both common carotid arteries were exposed and dissected from surrounding tissues and occluded by application of an aneurysm clip for 20 min. Clips were removed, the neck wound sutured and topical local anaesthetic (lidocaine/prilocaine) was applied. Mice were recovered on a heated blanket for 4–6 hr and then returned to normal housing. After a 3 day recovery, mice were anaesthetised and perfused transcardially with saline followed by 4% paraformaldehyde. Brains were removed and rostral and caudal blocks prepared using a brain matrix (Harvard Apparatus). Blocks were post-fixed in 4% paraformaldehyde for 24 hr and processed to paraffin blocks. Sections (6 μm) were cut on a microtome (Leica) and stained with haematoxylin and eosin. Ischaemic neuronal death was quantified in the CA1 and CA2 regions of the hippocampus which are the most sensitive regions in this model. Ischaemic (dead) neurons were identified morphologically in two regions of interest (ROIs) in CA1 and the entire CA2 bilaterally. Data are expressed as the number of dead neurons as a % of total neurons in the ROI and show the mean of both hemispheres for each region. All processing and analysis was performed with the operator blind to genotype.