Abstract
Purpose :
Astrocyte-laid honeycomb-shaped template is required for guiding retinal vascular development. Here, we investigated the role of complement in regulating the spatial patterning astrocytic template.
Methods :
C3 expression was examined in C3-tdTomato reporter mice and C3a deposition was determined by immunostaining. Astrocyte spatial distribution, vascular growth, and microglial refinement of astrocytic template were assessed in the WT, C3 KO, and C3aR KO retinal flatmounts by immunostaining. Retinal microglial phagocytosis of astrocyte bodies was analyzed by an in-vitro neutralizing antibody-based assay. Gene expressional changes were determined by RT-PCR and RNA-sequencing.
Results :
Microglia closely interact with the astrocytes and the endothelial tip cells during vascular development in the retina. Prior report and our study show that the depletion of microglia increased astrocyte spatial distribution. Here, we investigated if the complement system facilitates microglial regulation of astrocyte spatial patterning for subsequent vascular growth. Examination of postnatal day (P) 5 retinal flatmounts from C3-tdTomato reporter mice revealed C3-tdTomato expression in microglia around the growing retinal vasculature. Further assessment of P5 retinas displayed C3a (cleaved C3 peptide) deposition on the astrocytic surface in the vascularizing and adjacent avascular zones. Analysis of C3 and C3aR deficient retinas displayed significant reduction in the developmental microglial phagocytic removal of astrocytic bodies when compared to WT retinas. Consequently, retinas lacking C3 and C3aR showed significantly increased astrocyte density. Further examination of C3 and C3aR deficient retinas showed that the increased astrocyte density was associated with elevated proangiogenic extracellular matrix genes expressions resulting in increased vascular density.
Conclusions :
Our findings illustrate that microglia regulate developmental astrocyte and vascular network spatial patterning in the retina via C3a/C3aR complement axis.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.