Figure 4.

The spatial coverage area of microglia is reduced during aging and in a mouse model of Alzheimer’s disease (AD). Brain sections were taken from WT mice aged 3, 9, and 21 months and from two different AD mouse models, namely the APP_Sw,Ind (9 months old) and the amyloid precursor proteins (APP)/PS1 (15 months old) Tg mice, and immunolabeled with anti-IbaI. (A) Confocal z-stack images showing microglia process densities. To classify microglia, gray level, maximum z-projection images were generated and then set to eliminate background based on intensity threshold (upper panels). A heat map visualizing microglia process densities (lower panels) was then generated with the 3D Surface Plot plug-in bundled with FIJI software. Areas highly condensed with microglial processes appear in blue-purple, and areas vacant of microglia processes appear in red-yellow. Bars represent 50 μm. (B–E) Spatial coverage of microglia was performed by grid analysis. Grid analysis was performed by opening a grid of 32*32 squares (each square is 25 μm²) on top of a skeletonized image (see a zoom-in image of the grid in panel B). The total calculated length of processes in each square of the grid is presented as a distribution histogram with binning of 1 μm for 3-, 9-, and 21-month-old mice (C) and for APP_Sw,Ind and, APP/PS1 Tg mice compared with 3-month-old APP_Sw,Ind Tg mice (D). The length of processes per 1 square of a grid was calculated for each mouse (WT, 3 months n = 3; all other groups n = 5) and averaged for each group. Data were then analyzed by a one-way Tukey’s ANOVA. *P < 0.05; **P < 0.01, ***P < 0.001 (E).