Microglial morphological analysis
The quantification of microglial morphology performed in this study was based on a protocol previously described and reported by our group . 30μm sagittal brain sections were collected and imaged using Zeiss Observer Z1 widefield microscope with 10X magnification. 3-5 images were taken at the perilesional cortex and at the corresponding location on the contralateral cortex. Images underwent maximum intensity projections before analysis. Images were analysed using a proprietary spanning tree path finding based algorithm with a minimum of 50 microglia per image analysed . Trace images were then analysed on Matlab v2013b using built-in functions. To ensure that only cells with visible major branches were included in the analysis, cells overlapped with each other or had contact with the image border were excluded from analysis.

Data and Statistical Analysis

The data and statistical analysis comply with the recommendations on experimental design and analysis in pharmacology . All statistical analysis was conducted using GraphPad Prism version 8.4.2 for Windows. Data is presented as mean ± standard error of the mean (SEM). Sample size was determined by power analysis. A variance was set at 15% to determine a 25% significant difference will require a group of 8 or more animals for behavioral and infarct size analysis. For comparisons between two treatment groups, a two-tailed student t-test was used. For comparisons between three or more treatment groups, a one-way ANOVA with a Tukey’s post-hoc multiple comparisons test was used. p-values less than 0.05 were deemed statistically significant.