Morphological analysis

Following immunostaining, astrocyte and microglia morphology were evaluated using software (ImageJ; version 2.0.0-rc-69/1.52p; National Institutes of Health). Analysis of dendrite branching features, including number of endpoints, total segment length and maximum branch length, was performed using the plugin Analyze Skeleton (fiji.sc/AnalyzeSkeleton) (39). Briefly, images were converted to 8-bit gray type and filtered with Fast Fourier Transform bandpass filter plugin (imagej.nih.gov/ij/plugins/fft-filter.html). The brightness and contrast were adjusted to show cell morphology and noise was removed. The images were then skeletonized and the number of endpoints, total segment length and maximum branch length were measured automatically using the Analyze Skeleton plugin. The area of IBA1⁺ stained cells and circularity index (CI) were measured using the plugin Shape Descriptors (40). The CI parameter was calculated by the Shape Descriptors plugin as follows: Area=[4p(area)/(perimeter)²] (41). The numbers of vWF⁺, GFAP⁺ and IBA1⁺ cells were counted using Image-Pro-Plus 6.0 software (Media Cybernetics, Inc.).