Quantitative Analysis of Microglial Morphology

For microglial morphology analysis, microglia from cortex and hippocampus were visualized by IBA1 and imaged with a Nikon AIR-MP confocal microscope and then subjected to Sholl analysis as previously described^{41 (link),157 (link)}. In brief, z-stack confocal images of microglia were transformed to 8-bit binary and skeletonized images. Next, skeletonized images were quantified with the Sholl analysis tool of Fiji. Concentric circles were drawn with center on the soma, beginning at 5-μm radius and increasing by 5 μm for each cycle. The number of intersections for each branch and each increasing circle was calculated. About 10–12 microglia were analyzed for each mouse, 100 microglia in total. The microglial senescence index was used to estimate microglial senescence in previous studies^{105 (link)}. This index was calculated as the ratio of the microglial density to the morphological complexity (the area under the curve of Sholl analysis). For immunoreactivity quantification, pixels of target protein or RNA fluorescent signals were normalized and calculated by Fiji^{41 (link),45 (link)}. Statistics and graphics were generated using Prism 8.3.0 and R 3.6.1. The unpaired t-test was used to compare data between two groups. Data are presented as mean ± s.d. unless otherwise stated. Statistical significance was set as P < 0.05.