Figure 1.

Autophagy is inhibited in activated microglia & macrophages after TBI. (A-B) Images (20X, scale bar: 100 μm) of microglia/macrophage reporter Cx3cr1-GFP cortical sections from injured mice (3 days after injury) stained with antibodies against macrophage-specific marker ADGRE1/F4/80 (red) and autophagy flux marker SQSTM1 (purple). White boxes in (A) highlight the cells magnified in (B) (scale bar: 20 μm). Macrophages are defined as CX3CR1⁺ADGRE1^high, and microglia as CX3CR1⁺ ADGRE1^low. (C) Corresponding quantification of all CX3CR1⁺ cells (black bars) and CX3CR1⁺ cells with inhibited autophagy (CX3CR1⁺SQSTM1⁺, gray bars) in sham, 1-, 3- and 7 days after injury. 13% of all CX3CR1⁺ cells show inhibition of autophagy at 3 days after injury. (D) Quantification of all macrophages (CX3CR1⁺ ADGRE1^high, black bars) and macrophages with inhibited autophagy (CX3CR1⁺ ADGRE1^high SQSTM1⁺, gray bars). 65% of all CX3CR1⁺ ADGRE1^high cells show inhibition of autophagy at 3 days after injury. Data are mean ± SEM, n = 5 mice/group; *p < 0.05, **p < 0.01 vs. corresponding sham (one-way ANOVA with Dunnet’s post-hoc for multiple comparisons). (E) Images (20X, scale bar: 50 μm) of sham and TBI macrophage reporter Ccr2-RFP mice cortical sections stained with antibodies against autophagy markers LC3 (green) and SQSTM1 (purple). (F) Corresponding quantification of macrophages (CCR2⁺) expressing LC3 (black bars) and LC3 plus SQSTM1 (gray bars). 90% of all CCR2⁺ LC3⁺ cells are SQSTM1⁺ at 3 days post TBI. Data are mean ± SEM, n = 4 mice/group; ***p < 0.001, ****p < 0.0001 vs. corresponding sham (one-way ANOVA with Dunnet’s post-hoc for multiple comparisons). (G-L) Flow cytometry-based assessment of autophagy in microglia and infiltrating monocytes from sham and TBI mouse cortices. (G) Representative dot plot demonstrating strategy for identification of microglia (PTPRC/CD45^int ITGAM/CD11B⁺) and infiltrating myeloid (PTPRC^high ITGAM⁺) populations at 3 days post TBI. (H) Dot plots demonstrating strategy for identifying cells with normal autophagy (LC3⁻) and with inhibited autophagy (LC3⁺) in microglia (MG) and infiltrating myeloid (pMy) cells based on LC3 antibody staining intensity. (I) Comparison of LC3 staining (blue = LC3⁻, red = LC3⁺) with Cyto-ID® autophagy dye and SQSTM1 antibody staining. Cells positive for LC3 and autophagy dye also accumulate higher levels of SQSTM1 indicating inhibition of autophagy flux. (J-L) Quantification of microglia and infiltrating macrophages with inhibited autophagy in sham and TBI mouse cortices based on (J) % of LC3⁺ cells, (K) % of SQSTM1⁺ cells, and (L) % of autophagy dye⁺ cells. Analysis of corresponding blood monocytes is included in (L). Data are mean ± SEM; n = 6–7 mice/group; *p < 0.05, **p < 0.01, ***p < 0.001 vs sham; two-way ANOVA with Dunnet’s post-hoc for multiple comparisons.