Characterization of retinal microglia and its pharmacological inhibition in a mouse model for retinopathy of prematurity

Abstract

Retinopathy of prematurity (ROP) is a major cause of childhood blindness. The disease is characterized by the first phase of retinal vessel growth cessation followed by a second phase of abnormal retinal vessel proliferation. Retinal microglia are resident immune cells in the retina, maintaining the homeostasis of the microenvironment. The involvement of microglia in ROP has been noted in human patients and in animal models for ROP, i.e. oxygen-induced retinopathy (OIR). In mouse OIR model, neonatal mouse pups with their nursing mother are placed in an environment containing 75% oxygen for 5 days since postnatal (P) day 7, and returned back to room air (RA) environment on P12, to induce retinopathy. This model mimics the ROP process in human patients. However, the detailed response pattern of retinal microglia in OIR is still lacking, and their general role in OIR is unknown. The present study used mouse OIR model combined with application of liposomes containing clodronate (CLD), which specifically target microglia, to study the role of microglia in the pathogenesis of ROP. Quantification of retinal microglia with their morphology clarified at various time points of OIR helped to elucidate the response pattern of retinal microglia in this model. The number of retinal microglia was increased in the superficial areas of OIR retinae on P12, P17, P21, P25 and P30, and most of them displayed an amoeboid (activated) morphology. The increased retinal microglia were associated with increased superficial retinal vessels in OIR retinae. The amount of retinal microglia in deep retinal areas of OIR retinae also increased from P17 to P30 with a ramified morphology, which were associated with reduced retinal vessels in deep OIR retinae. The activation of retinal microglia displayed both M1 pro-inflammatory and M2 antiinflammatory properties in P17, P21 and P30 OIR retinae tested by real-time PCR. To determine whether retinal microglia are beneficial or detrimental for retinal vasculature during OIR, retinal microglia were transiently inhibited by intravitreal injection of liposomes containing CLD. Intravitreal injection of liposomes-CLD caused significant reduction in retinal microglia amount. Inhibition of retinal microglia before hyperoxia treatment resulted in increased vessel obliteration and subsequent neovascularizations in OIR retinae. Similarly, inhibition of retinal microglia immediately after hyperoxia treatment also led to more neovascularizations in OIR retinae. However, the inhibition of retinal microglia did not interfere with retinal functions and retinal structure, regardless of the time point that microglial inhibition was performed. Taken together, retinal microglia actively participate in the whole process of OIR. Retinal microglia activation is closely associated with vascular abnormalities in the retina. Their activation during OIR process have both pro-inflammatory and antiinflammatory properties. Loss of retinal microglia during either vaso-obliteration stage or proliferation stage leads to exacerbated vascular outcomes, suggesting their overall protective role for retinal vasculature maintenance in OIR. Retinal microglia might play important roles in the pathogenesis of ROP.