Microglia appose the intermediate retinal vascular plexus and their ablation induces a decreased physiological and pathological intermediate plexus

Abstract

Purpose: In humans and mice, the neurosensory retina contains three interconnecting vascular plexus layers. In mice these develop in early postnatal stages through mechanisms that are incompletely understood. We reported previously that amacrine cells interact with endothelial cells in the intermediate plexus and genetic ablation of amacrine cells and horizontal cells dramatically interfere with formation of the intraretinal vasculature (Usui Y et al. ARVO 2014). Since neurons and glia are known to function within neurovascular units to control blood supply, in this study we examined the potential contributions of microglia for angiogenesis using pharmacological approaches to deplete host myeloid progenitor cells.

Methods: Transgenic mice expressing Cre recombinase specifically in amacrine and horizontal cells (Ptf1a-Cre mice) were mated with floxed VHL or VEGF with cX3cR1^GFP/+ mice to generate conditional knockouts. The CSF receptor tyrosine kinase inhibitor (Ki20227, 12.5 mg per kg of body weight) was subcutaneously administered daily to ptf1a-Cre; VHL^f/f or VHLl^f/f with cX3cR1^GFP/+ mice. DMSO was used as a vehicle and control. Mice were sacrificed at different time points and branching points and retinal microglia distribution in the intermediate plexus are evaluated by whole mount preparation.<br />

Results: During normal vascular development, retinal microglia are present where the intermediate plexus will form at P12. At P18, concomitant with increases in vascular density, the number of microglia in the intermediate plexus of ptf1a-Cre; VHL^f/f; cx3cr1^GFP/+ mice was significantly increased compared with that in littermates controls, and a statistically significant correlation between the number of branching points of the intermediate plexus and number of microglia was observed in both groups. However, no difference existed between ptf1a-Cre; VEGF^f/f; cx3cr1^GFP/+ and VEGF^f/f; cx3cr1^GFP/+ mice in the number of microglia in the intermediate plexus at P15 and P18. Ki20227 was injected from P11-17, and a pronounced decrease in intermediate vascular density was observed in Ki20227-treated ptf1a-Cre; VHL^f/f; cx3cr1^GFP/+ and VHL^f/f; cx3cr1^GFP/+ mice compared with those of vehicle-treated mice.

Conclusions: These results suggest that amacrine neuron-microglia interactions are fundamental to the proper development of the intermediate retinal vascular plexus.