Macrophage Depleted Mice Exhibit a Prolonged Neovascular Response in a Mouse Model of Oxygen–Induced Retinopathy

Abstract

Purpose: : Microglial cells (MG) and macrophages (MAC) exhibit both pro– and anti– angiogenic functions. MG/MAC are activated and recruited to areas of retinal neovascularization in the mouse model of oxygen–induced retinopathy (OIR). We previously reported that CCL2 ^–/– mice exhibit a reduction in neovascular tuft–associated MG/MAC in the OIR model. In addition, these mice had a reduction in neovascular tuft apoptotic cells, resulting in a delay in tuft regression (Davies MH, IOVS 2005; ARVO E–Abstract #4185). The present study investigates if retinal neovascularization is altered in C57BL/6 (B6) mice following macrophage and monocyte depletion via systemic treatment with clodronate liposomes.

Methods: : Postnatal day (P)7 B6 mice were exposed to 75% oxygen for 5 days (P12), and then recovered in room air. Intraperitoneal injections of clodronate liposomes or PBS liposome controls were administered on P13 and P15. Macrophage depletion was confirmed in clodronate injected animals by immunolabeling liver sections with an antibody against F4/80 antigen. Eyes from oxygen–exposed (O₂) mice were obtained at various time points for histopathological studies. Retinopathy was qualitatively assessed in FITC–dextran perfused retinal whole mounts via fluorescence microscopy. Retinopathy was quantified by counting preretinal neovascular nuclei in both P17O₂ clodronate (n=8) and PBS (n=8) injected mice as well as P21O₂ clodronate (n=6) and PBS (n=6) injected mice.

Results: : Liver sections from clodronate injected animals had a 60.4% and 88.6% reduction of F4/80 positive cells on P17O₂ and P21O₂, respectively, confirming macrophage depletion. P17O₂ FITC–dextran perfused retinas exhibited prominent neovascular tufts and avascular regions in both clodronate and PBS injected mice. Quantification of preretinal nuclei revealed no significant difference between P17O₂ clodronate (25.59 ± 2.18) and PBS (26.5 ± 2.5) injected mice (P=0.79). However, P21O₂ clodronate (10.83 ± 0.72) retinas exhibited a significantly greater number of preretinal neovascular nuclei compared to PBS controls (6.48 ± 0.83) (P=0.002).

Conclusions: : Our results demonstrate that the depletion of macrophages does not alter the neovascular response on P17O₂. However, on P21O₂, the clodronate injected mice exhibit significantly more preretinal neovascular nuclei, similar to results observed in the CCL2^–/– studies. We speculate that the reduction in tissue macrophages on P21O₂ delays vascular tuft regression.

Keywords: retinal neovascularization