Figure 4.
Phagocyte retention in LPC-induced lesions of Myd88−/− mice and impaired phagosome maturation. (A and B) Proportion of the volume occupied by IBA1+ cells in the lesions (devoid of FluoroMyelin) of WT and Myd88−/− mice at 14 dpi. n = 4 or 5 lesions. (C and D) Total accumulation of myelin lipids in IBA1+ cells in mouse spinal cord lesions at 14 dpi, analyzed by the proportion of the volume (V) occupied by both FluoroMyelin (green) and IBA1 (magenta; colocalization, white), shown in A, in the lesions (C) and analyzed per total amount of IBA1 signal (D). n = 4 or 5 lesions. (E and F) Degradation of myelin lipids in cultured microglia. The area of FluoroMyelin in each cell was normalized to the average area of FluoroMyelin per cell at the initial time point (0 h after myelin treatment). n = 3 independent experiments. 6 h after myelin treatment Myd88−/− versus WT: P < 2.2 × 10−16; effect size (Cohen’s d) = 0.542; 1,188 Myd88−/− cells and 1,016 WT cells. 24 h after myelin treatment: Myd88−/− versus WT: P < 2.2 × 10−16; effect size (Cohen’s d) = 0.657; 988 Myd88−/− cells and 947 WT cells. (G–I) Phagosome maturation in myelin-loaded microglia. (G) Myelin+ phagosomes (myelin labeled with PKH67, green) and endolysosomes (LysoTracker Red+, magenta) were detected by live-cell imaging. (H and I) Fusion of phagosomes containing myelin debris with endolysosomes, as analyzed by the area of the overlap between myelin debris and LysoTracker in each cell, 1 h after treatment with myelin debris. n = 3 independent experiments. Myd88−/− versus WT: P = 1.911 × 10−9; effect size (Cohen’s d) = −0.430; 573 Myd88−/− cells and 352 WT cells. (J–L) Oxidative activity in cultured microglia after phagocytosing myelin debris. Representative overview and inset. The organelles labeled with OxyBURST BSA (magenta) identified ROS. Internalized myelin debris was identified by PLP (green). n = 3 independent experiments. Myd88−/− versus WT: P = 4.388 × 10−6; effect size (Cohen’s d) = −0.345; 750 Myd88−/− cells and 378 WT cells. Data are mean ± SEM (error bars) in B, C, and D and 95% CI in F, H, and K. *, P < 0.05; **, P < 0.01; ****, P < 0.0001 by unpaired t test with Welch's correction in B, C, F, H, and K. Scale bars: 100 µm in A; 50 µm in E; and 10 µm in G and J.

Phagocyte retention in LPC-induced lesions of Myd88−/− mice and impaired phagosome maturation. (A and B) Proportion of the volume occupied by IBA1+ cells in the lesions (devoid of FluoroMyelin) of WT and Myd88−/− mice at 14 dpi. n = 4 or 5 lesions. (C and D) Total accumulation of myelin lipids in IBA1+ cells in mouse spinal cord lesions at 14 dpi, analyzed by the proportion of the volume (V) occupied by both FluoroMyelin (green) and IBA1 (magenta; colocalization, white), shown in A, in the lesions (C) and analyzed per total amount of IBA1 signal (D). n = 4 or 5 lesions. (E and F) Degradation of myelin lipids in cultured microglia. The area of FluoroMyelin in each cell was normalized to the average area of FluoroMyelin per cell at the initial time point (0 h after myelin treatment). n = 3 independent experiments. 6 h after myelin treatment Myd88−/− versus WT: P < 2.2 × 10−16; effect size (Cohen’s d) = 0.542; 1,188 Myd88−/− cells and 1,016 WT cells. 24 h after myelin treatment: Myd88−/− versus WT: P < 2.2 × 10−16; effect size (Cohen’s d) = 0.657; 988 Myd88−/− cells and 947 WT cells. (G–I) Phagosome maturation in myelin-loaded microglia. (G) Myelin+ phagosomes (myelin labeled with PKH67, green) and endolysosomes (LysoTracker Red+, magenta) were detected by live-cell imaging. (H and I) Fusion of phagosomes containing myelin debris with endolysosomes, as analyzed by the area of the overlap between myelin debris and LysoTracker in each cell, 1 h after treatment with myelin debris. n = 3 independent experiments. Myd88−/− versus WT: P = 1.911 × 10−9; effect size (Cohen’s d) = −0.430; 573 Myd88−/− cells and 352 WT cells. (J–L) Oxidative activity in cultured microglia after phagocytosing myelin debris. Representative overview and inset. The organelles labeled with OxyBURST BSA (magenta) identified ROS. Internalized myelin debris was identified by PLP (green). n = 3 independent experiments. Myd88−/− versus WT: P = 4.388 × 10−6; effect size (Cohen’s d) = −0.345; 750 Myd88−/− cells and 378 WT cells. Data are mean ± SEM (error bars) in B, C, and D and 95% CI in F, H, and K. *, P < 0.05; **, P < 0.01; ****, P < 0.0001 by unpaired t test with Welch's correction in B, C, F, H, and K. Scale bars: 100 µm in A; 50 µm in E; and 10 µm in G and J.

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