2.4 Infraction volume ratio and brain edema measurement
Excessive administration of pentobarbital sodium was employed for animal euthanasia following ischemic conditions. The brains were sliced into 2mm coronal sections with even thickness. Subsequently, the sections were subjected to staining using a 2% solution of 2,3,5-triphenyl tetrazolium chloride (TTC) (Solarbio, Beijing, China) at a temperature of 37°C for a duration of 20 minutes38. The stained brain sections were fixed in 2% paraformaldehyde and imaged, followed by analysis of the infarct volume (white) percentage using Image J software from the National Institutes of Health, USA. In brief, the infarct volume was calculated by multiplying the total areas of infarction by the section thickness (2mm). The ratio of infarct volume to total brain volume is indicative of cerebral infarction.
Brain edema was evaluated by measuring brain water content using the dry-wet weight method39. In brief, following reperfusion, mice were administered an overdose of anesthesia and their brains were promptly harvested. The brains were then dissected along the midsagittal plane to separate the ischemic hemisphere from the normal hemisphere. Each hemisphere was immediately weighed, and subsequent samples were dried in an oven at 100°C until a constant weight was achieved to determine both wet weight and dry weight. The brain water content was calculated as [(wet weight - dry weight)/wet weight] × 100%.