Figure 4.

Mitochondrial alterations occur in responsive UCP2^KO IPL microglia after ONC

(A–J) Comparison of microglial and mitochondrial response in naive UCP2^KO (tan) and 5 days after ONC UCP2^KO (gold). (A–B) Representative IBA1-immunostained microglia (magenta) co-labeled with CD68 (cyan) from naive UCP2^KO (A) or ONC UCP2^KO microglia (B). Next, corresponding 3D-surfaces. Below: Zoom-in of region of interest (dashed line) from image and 3D-surface. Scale bar: 10 μm, zoom-in: 3 μm. (C) Percentage of total CD68 volume per microglia volume. Boxplot minimum and maximum: InterQuartile Range (IQR) around median (center line). Whiskers: 1.5 IQRs. Black diamond points: outliers outside 1.5 IQRs. Wilcoxon rank-sum test: p < 0.0001. (D) CD68 vesicle localization. Scatterplot depicting CD68 vesicle volume vs. distance from the cell soma (0, origin) of the population of vesicles in naive UCP2^KO (left) and ONC UCP2^KO (right, gold) conditions. Point density, pseudo-colored. (E) 3D-filament tracings of microglia from naive UCP2^KO (left) or ONC UCP2^KO (right). Scale bar: 10 μm. Line plot for mean number of Sholl intersections per radial distant from the soma (μm) with 95% confidence interval band. Linear mixed effects model: p < 0.0001. (F–J) Mito-Dendra2 (green) expression in the IBA1-immunostained microglia from (A–B) for naive UCP2^KO microglia (F) or ONC UCP2^KO microglia (G). Next, corresponding 3D-surfaces. Below: Zoom-in of region of interest (dashed line) from image and 3D-surface. Scale bar: 10 μm, zoom-in: 3 μm. (H-J) Mitochondrial parameters. Boxplot minimum and maximum: InterQuartile Range (IQR) around median (center line). Whiskers: 1.5 IQRs. Black diamond: outliers outside of 1.5 IQRs. (H) Mitochondrial network connectivity determined by median mitochondrial volume (left, Wilcoxon rank-sum test: p < 0.0001), number of organelles within a single cell (center, Wilcoxon rank-sum test: p = 0.930881) and mean mitochondrial sphericity (right, Student’s t test: p < 0.0001). (I) Percentage of mitochondrial volume per microglial volume. Wilcoxon rank-sum test: p = 0.0003. (J) Mitochondrial localization. Scatterplot depicting mitochondria volume vs. distance from the cell soma (0, origin) of the population of mitochondria in naive UCP2^KO (left) or ONC UCP2^KO (right) microglia. Point density, pseudo-colored. Percentage of total mitochondrial volume localized within the perinuclear region. Student’s t test: p < 0.0001. ∗∗∗p < 0.001, ^n.s. p > 0.05: not significant. See Table S4 for retina and cell numbers, statistical tests and corresponding data.