Figure 3. Infiltrating microglia phagocytose non-apoptotic photoreceptor rods during rod degeneration.

• A–C Phagocytosis of rods by infiltrating microglia. (A) Representative example of a Iba1⁺ microglial process extending into the ONL with a phagosome at its terminal end. Each phagosome contained a photoreceptor nucleus (labeled with DAPI, arrow) that was identified as a rod photoreceptor by rhodopsin immunopositivity (superposition of Iba1⁺ phagosome with rhodopsin⁺ soma in orthogonal views). (B) Example of an amoeboid microglia in the ONL with multiple phagosomes containing both rhodopsin-positive (arrowhead) and rhodopsin-negative (arrow) nuclei. (C) Rhodopsin⁺ nuclei can be localized within CD68-positive phagosomes in infiltrating microglia, indicating phagocytic engulfment of rods. Scale bar, 10 μm.
• D, E The peak of microglial phagocytic activity occurred around P21 and declined subsequently, as measured by the number of rods phagocytosed (D) and the number of photoreceptors phagocytosed per microglial cell (E) (n = 3 animals at each time point).
• F–J Infiltrating microglia phagocytose TUNEL-negative rods in the ONL. (F) Although the peaks of TUNEL positivity and microglial phagocytosis occurred concurrently, the majority of rod photoreceptors undergoing microglial phagocytosis were non-apoptotic (arrows), while many apoptotic photoreceptors were not phagocytosed by microglia (arrowheads); representative example of a P21 retina demonstrates non-overlapping patterns of TUNEL positivity (red) and microglial phagocytosis (Iba1, green). (G) Confocal analysis of a representative amoeboid microglial cell demonstrates that while the photoreceptor nuclei in phagosomes were rhodopsin-positive (white), they were mostly TUNEL-negative. Only a small fraction of microglial phagosomes contained TUNEL⁺, rhodopsin⁺ rods (example shown in H). Scale bars, 20 μm. (I) Quantitative analysis of photoreceptor nuclei within microglial phagosomes according to rhodopsin (black symbols), and TUNEL labeling (red symbols) at different times during rod degeneration. (J) Quantitative analysis of all photoreceptors undergoing either apoptosis or phagocytosis at P21–23 demonstrates that TUNEL⁺ apoptotic photoreceptors and phagocytosed photoreceptors consist of two distinct populations, indicating separate but parallel mechanisms of rod degeneration. Quantitative analyses involved retinal sections from n = 3 animals at each age.

Data information: Data points in (D, E, I) and column heights in (J) indicate mean, error bars indicate ± SEM.