Figure 6.
Figure 6. Microglial phagocytosis of apoptotic photoreceptors is decreased with C3 and CR3 deficiency in the rd10 retina. (A–E) Immunohistochemical analysis of microglia translocating into the ONL was performed in flat-mounted retina isolated at P24 from: C3+/+, CR3+/+.rd10, C3−/−.rd10, and CR3−/−.rd10 mice. (A) IBA1+ microglia (green) in the ONL demonstrating a deramified and amoeboid morphology were found in close contact with rhodopsin-expressing rods (red). Many microglia also demonstrated one or more CD68-immunopositive intracellular phagosomes (arrows) that also colocalized with rhodopsin immunopositivity (depicted by white pixels). Scale bars, 10 µm. Densities of ONL microglia were similar between genotypes (B), but microglia from animals with genetic ablation of C3 or CR3 showed significantly smaller mean soma sizes (C) that corresponded to lower mean numbers of intracellular phagosomes per microglia (D) and decreased extents of rhodopsin colocalization within microglia (E), which reflected decreased microglial phagocytosis of rod photoreceptors. (F–H) Comparative analysis of phagocytosis of viable (TUNEL−) and apoptotic (TUNEL+) photoreceptors by ONL microglia was performed for the three genotypes at P24. (F) Phagosomes within IBA1+ microglia were observed to contain both TUNEL+ and TUNEL− nuclei (insets). Scale bar, 50 µm. (G) Decreased phagosomes in C3−/−.rd10 and CR3−/−.rd10 microglia relative to C3+/+, CR3+/+.rd10 microglia was reflected in decreased total phagocytic capacity; these differences were found for the subsets of microglia phagocytosing TUNEL+ photoreceptor nuclei and phagocytosing TUNEL− photoreceptor nuclei. (H) The proportions of TUNEL+ nuclei remaining in the ONL were increased in C3 and in CR3 deficiency, indicating a greater accumulation of uncleared apoptotic photoreceptors in these genotypes. P values indicate comparisons to the C3+/+, CR3+/+.rd10 genotype using a one-way ANOVA with Dunnett’s multiple comparison test; n = 3–4 animals per genotypic group; all data collected from two independent experiments each. All data shown as mean ± SEM.

Microglial phagocytosis of apoptotic photoreceptors is decreased with C3 and CR3 deficiency in the rd10 retina. (A–E) Immunohistochemical analysis of microglia translocating into the ONL was performed in flat-mounted retina isolated at P24 from: C3+/+, CR3+/+.rd10, C3−/−.rd10, and CR3−/−.rd10 mice. (A) IBA1+ microglia (green) in the ONL demonstrating a deramified and amoeboid morphology were found in close contact with rhodopsin-expressing rods (red). Many microglia also demonstrated one or more CD68-immunopositive intracellular phagosomes (arrows) that also colocalized with rhodopsin immunopositivity (depicted by white pixels). Scale bars, 10 µm. Densities of ONL microglia were similar between genotypes (B), but microglia from animals with genetic ablation of C3 or CR3 showed significantly smaller mean soma sizes (C) that corresponded to lower mean numbers of intracellular phagosomes per microglia (D) and decreased extents of rhodopsin colocalization within microglia (E), which reflected decreased microglial phagocytosis of rod photoreceptors. (F–H) Comparative analysis of phagocytosis of viable (TUNEL−) and apoptotic (TUNEL+) photoreceptors by ONL microglia was performed for the three genotypes at P24. (F) Phagosomes within IBA1+ microglia were observed to contain both TUNEL+ and TUNEL− nuclei (insets). Scale bar, 50 µm. (G) Decreased phagosomes in C3−/−.rd10 and CR3−/−.rd10 microglia relative to C3+/+, CR3+/+.rd10 microglia was reflected in decreased total phagocytic capacity; these differences were found for the subsets of microglia phagocytosing TUNEL+ photoreceptor nuclei and phagocytosing TUNEL− photoreceptor nuclei. (H) The proportions of TUNEL+ nuclei remaining in the ONL were increased in C3 and in CR3 deficiency, indicating a greater accumulation of uncleared apoptotic photoreceptors in these genotypes. P values indicate comparisons to the C3+/+, CR3+/+.rd10 genotype using a one-way ANOVA with Dunnett’s multiple comparison test; n = 3–4 animals per genotypic group; all data collected from two independent experiments each. All data shown as mean ± SEM.

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