Microglia phagocytosis contributes to clearance of photoreceptor outer segment following retinal detachment

Abstract

Purpose : Retinal detachment (RD) occurs when photoreceptors (PR) are separated from the retinal pigment epithelium (RPE), compromising PR cell survival. PR outer segments (OS) are continuously renewed by a process including daily shedding and phagocytosis by RPE cells, which is interrupted by RD. RD also triggers an innate immune response, with the activation and attraction of retinal microglia to the subretinal space. We hypothesized that subretinal microglia replace the function of RPE by actively phagocytose OS.

Methods : To lineage trace microglia, tamoxifen (Tam)-inducible Cre-driver mice (CX3CR1-CreERT2) were crossed with mice with Cre-dependent expression of tdTomato red-fluorescent protein (RFP). A 4-week period after Tam treatment was used to eliminate RFP-expressing monocytes. The mice then received subretinal injections of 1% hylaluronic acid to cause partial RD. Retinal sections were obtained at 3, 7 and 14 days post-retinal detachment (dprd). Untreated fellow eyes were used as controls. Retinal and subretinal cells were examined by immunofluorescence for expression of RFP and phagocytosis markers. Lysosomal receptor CD68 and phagocytic receptor MerTK were used as phagocytosis markers. Rhodopsin and PNA lectin, were used to identify OS of rods and cones, respectively. Imaris software was used to construct 3D images.

Results : RFP-labeled retinal microglia were observed to migrate to the subretinal space at 3, 7 and 14 dprd. Lineage tracing confirmed that the vast majority of subretinal cells were microglia, averaging 87 ± 2% at 14 dprd. Dramatically increased CD68 and MerTK immunoreactivity were observed in microglia, indicating phagocytic activity. Anti-rhodopsin and PNA-reactive OS were observed anchoring to microglial membranes and drawn within the cell bodies of microglia, indicating phagocytosis of POS. The co-localization of CD68 and rhodopsin implied OS internalization and transfer into phagolysosomes.

Conclusions : The initial innate immune response to RD involves microglia infiltration in the subretinal space. The results reveal microglia OS phagocytosis provides OS clearance in the absence of PR-RPE interaction.

This is a 2021 ARVO Annual Meeting abstract.

 

3D reconstruction of a microglia cell (RFP, red) engulfing PR OS (rhodopsin, green) in the subretinal space. Yellow regions represent co-localization RFP and rhodopsin indicating OS being internalized by the microglia.