Figure 2. Reinjury promotes loss of microglia.

(A) Representative xy (top) and xz (bottom) maximally projected Z stacks (180 μm in depth) show CX3CR1^gfp/+ myeloid cells (green) and vasculature labeled with Evans blue (red) in Cx3cr1^gfp/+ reporter mice without injury, with single injury, or with reinjury. See corresponding Supplemental Video 1. Images are representative of 4 independent mice per group. Scale bar: 20 μm (top, xy) and 20μm (bottom, xz). (B) Concatenated UMAPS generated from high-dimensional flow cytometric analysis (gating shown in Supplemental Figure 1A) of 2 mm cortical punch biopsies from the above groups show 4 different subsets of microglia identified as populations 1 (blue), 2 (green), 3 (pink), and 4 (orange). Dots depict cellularity. (C) Histograms show the relative expression of FSC-area (FSC-A), SSC-A, CD45, CD11b, CX3CR1, F480, P2RY12, CD206, and CD44 on composite, concatenated microglia populations 1 (blue), 2 (green), 3 (pink), and 4 (orange). (D) Quantification of microglia total per experimental group. (E) Quantification of the frequency of each microglia subtype per experimental group. Each symbol in D and E depicts an individual mouse. Bar graphs are representative of 2 independent experiments with 4–5 mice per experimental group. Data are displayed as mean ± SD with *P≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001 using 1-way ANOVA with Fisher’s LSD test (D) and multiple t tests with Holm-Sidak multiple comparisons test (E).