Characterizing the Role of Retinal Microglia in a Mouse Model of Retinopathy

Abstract

Purpose: Retinal microglia have been associated with certain vaso-proliferative retinopathies, although their precise role in these conditions is poorly understood. We hypothesize that resting retinal microglia are neuro-protective and that in response to injury or disease, microglia acquire an intrinsic cytotoxic phenotype perpetuating disease. Methods: Nursing mothers of C57Bl/6 and CX3CR1-EGFP heterozygous mice were placed on a microglia-depleting diet containing PLX5622, an inhibitor of colony stimulating factor 1 receptor (CSF-1R), or on a control diet at the time of litter birth. Retinas from these mouse litters were harvested during early postnatal development [postnatal day (P)0 to P7] as well as during the early (P8) and late (P17) phases in a model of oxygen-induced retinopathy (OIR). Whole retina flatmounts immunostained with lectin to visualize blood vessels and with a novel microglia-specific marker, P2ry12, were imaged via fluorescence and confocal microscopy. Extent of neovascularization (NV) was quantified using the “SWIFT_NV” macro in the software ImageJ. Microglial size and area density were measured using the automated “Analyze Particles” feature in ImageJ. Results: Confocal microscopic images of P2ry12-immunostained retinal flatmounts show increased size and ramification of retinal microglia during normal early postnatal development (P0 – P7). Retinal microglia decrease in size and density during early OIR compared to normoxia (P < 0.001), and co-localize with regressing vessels during phase 1 of OIR. Diet-induced microglial depletion with PLX5622 leads to decreased microglial size (P < 0.01) and area density (P < 0.0001) in P7 mouse retinas compared to those on a control diet. The area density of retinal microglia was greater in the central retina compared to the peripheral retina in both the control diet (P < 0.05) and PLX5622 diet (P < 0.01) groups. Finally, microglial depletion with PLX5622 led to decreased NV in P17 retinas under OIR conditions compared to those on a control diet (P < 0.05). Conclusions: Morphologic changes of retinal microglia as well as microglial co-localization with regressing blood vessels during the early stages of OIR suggest an activated microglial state with phagocytic functions. Thus, depletion or suppression of microglia, which we showed can decrease NV in the late phase of OIR, may be a potential therapeutic target in certain vaso-proliferative retinopathies.