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.