Microglial dynamics during photoreceptor death and regeneration in zebrafish

Abstract

Purpose : Microglia are the innate immune cells of the central nervous system and govern injury-induced inflammation by secreting pro- and anti-inflammatory cytokines and chemokines. In mammalian retinas, microglia reside in the retinal parenchyma, nerve fiber/ganglion cell layers and inner and outer plexiform layers. We have identified a novel population of microglia, unique to zebrafish, which reside among the retinal pigment epithelial cells. In this study, we compared the response of subretinal microglia with that of parenchymal microglia following death of photoreceptors. We then assay regeneration of photoreceptors in the csf1ra^j4e1 mutant, where the number of parenchymal microglia, but not subretinal microglia, is diminished.

Methods : Photolytic lesions were used to selectively kill photoreceptors. The transgenic reporter line, Tg(mpeg:eGFP) and 4C4 antibody were used to visualize microglia. PCNA immunocytochemistry and TUNEL staining was used to assay proliferation and cell death, respectively.

Results : At 1 day post lesion (dpl), the number of microglia in the damaged outer retina increases by two-fold, whereas the number of microglia in the inner plexiform layer decreases significantly. This is accompanied by subretinal microglia descending toward the site of photoreceptor death. At 1 dpl, a small subset of microglia is co-labeled with TUNEL and the number of phagocytic microglia increases by 2 dpl. At 2 dpl, more than half of microglia in the subretinal space become PCNA+. By 14 dpl, the subretinal and parenchymal microglia resume their normal distribution, suggesting resolution of inflammation. In the csf1ra^j4e1 mutants, the number of parenchymal microglia was significantly reduced, whereas the number of subretinal microglia matched that of wildtype animals. The diminished number of parenchymal microglia in the mutants did not affect the photoreceptor regeneration.

Conclusions : This study establishes the common and distinct responses of microglia populations that reside in retinal parenchyma and subretinal space in response to the selective death of photoreceptors. Activation, migration, and phagocytosis universally occur among microglia, whereas activated microglia proliferate predominantly in the subretinal space. The normal regeneration of photoreceptors in csf1ra^j4e1 mutants suggests that subretinal microglia are sufficient to trigger the acute inflammation that governs photoreceptor generation.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.