MCP-1 deficiency delays regression of pathologic retinal neovascularization in a model of ischemic retinopathy

Abstract

Purpose: The present study investigates whether retinal neovascularization (NV) and apoptosis are altered in MCP-1-deficient ((-/-)) mice in the OIR model.

Methods: Postnatal day (P) 7 MCP-1(-/-) and C57BL/6 (B6) mice were exposed to 75% oxygen for 5 days and then recovered in room air. Immunostaining was performed to localize macrophages/microglia within retinal whole mounts and cross-sections. Retinopathy was qualitatively assessed in FITC-dextran-perfused retinas, and preretinal NV was quantified on P17, P21, and P24. TUNEL analysis was used to compare apoptosis between B6 and MCP-1(-/-) mice.

Results: MCP-1(-/-) and B6 mice revealed normal vascular development in room air controls and similar vaso-obliteration in oxygen-exposed mice on P12. MCP-1(-/-) mice exhibited significantly reduced vascular tuft-associated F4/80(+) cells compared with B6 mice. FITC-dextran-perfused retinas exhibited prominent neovascular tufts on P17, and quantification of preretinal nuclei revealed no significant differences between MCP-1(-/-) and B6 mice. In contrast, on P21 and P24, MCP-1(-/-) mice exhibited significant increases in preretinal neovascular nuclei compared with B6 controls. These increases in NV in the MCP-1(-/-) mice were associated with a significant reduction in vascular tuft apoptosis.

Conclusions: The results demonstrate that the absence of MCP-1 does not alter normal retinal vascular development. Furthermore, MCP-1(-/-) mice exhibit a similar neovascular response on P17. However, the reduction in tuft-associated macrophages/microglia in the MCP-1(-/-) mice correlates with reduced vascular tuft apoptosis and delayed regression of retinal NV. These findings suggest that macrophages/microglia may contribute to tuft regression through their proapoptotic properties.

Figure 1

Reduced F4/80⁺ macrophages/microglia in MCP-1^−/− mice. F4/80-labeled cells (red) were localized in retinal whole mounts from B6 room air control mice (A, D), along with B6 hyperoxia-exposed mice (B, E), and from MCP-1^−/− mice (C, F). Compared with P17 room air control B6 mice (A, D), an increase in F4/80-positive cells was observed in hyperoxia-exposed B6 mice (B, E). Examination of the hyperoxia-exposed MCP-1^−/− mice (C, F) demonstrated a reduced number of F4/80⁺ cells compared with hyperoxia-exposed B6 mice (B, E) on P17. Immunolabeling of F4/80⁺ macrophages/microglia (green) in retinal cross-sections revealed cells localized in the inner plexiform layer on P17 in B6 room air controls (G, arrows). However, in P17 hyperoxia-exposed retinal cross-sections, F4/80⁺ cells were located within the neovascular tufts in the B6 mice (H, arrow), whereas fewer macrophages/microglia were associated with tufts in the MCP-1^−/− mice (I, arrow). Original magnifications: (A–C) ×25; (D–I) ×400. Quantitative real-time PCR for F4/80 expression normalized to β-actin in B6 OIR time course (J) and in MCP-1^−/− mice compared with B6 mice on P17O₂ (L). Quantification of F4/80⁺ cells (K) in retinal cross-sections. **P < 0.002; *P < 0.05.

Figure 2

Flat-mounted retinas from B6 and MCP-1^−/− mice perfused with FITC-dextran. Central retinal vaso-obliteration (asterisks) was observed in hyperoxia-exposed B6 (A) mice and in hyperoxia-exposed MCP-1^−/− (B) mice on P12. Retinal neovascularization was evident on P17 in B6 (C) and MCP-1^−/− (D) hyperoxia-exposed mice after 5 days of room air recovery. Avascular regions (asterisks) remained present on P17 in both strains of mice. Higher magnification on P17 revealed that, similar to the B6 control mice (E), MCP-1^−/− retinas (F) exhibited a robust neovascular response and had numerous neovascular tufts (arrows). By P21, the neovascular response was attenuated in the B6 mice (G). In contrast, on P21 the MCP-1^−/− hyperoxia-exposed mice exhibited a prolonged neovascular response (H), with several residual tufts remaining (arrows). Original magnifications: (A–D) ×25; (E–H) × 400.

Figure 3

Delayed regression of NV in MCP-1^−/− mice. Preretinal nuclei (arrows) in B6 (A, C, E) and MCP-1^−/− (B, D, F) mice on P17O₂, P21O₂, and P24O₂ were counted after hyperoxia exposure. Quantification of preretinal nuclei revealed a significant difference between B6 and MCP-1^−/− mice on P21O₂ (**P < 0.004) and P24O₂ (*P < 0.02) (G). Original magnification, ×400.

Figure 4

Decreased neovascular tuft apoptosis in MCP-1^−/− mice. Representative TUNEL-stained sections from B6 (A, C) and MCP-1^−/− (B, D) mice on P17O₂ and P21O₂. Apoptotic cells were observed within the neovascular tufts on P17O₂ in B6 mice (A, arrows) and, to a lesser extent, in MCP-1^−/− (B, arrows) mice. On P21O₂, B6 mice (C, arrows) had numbers of apoptotic cells similar to those for MCP-1^−/− mice (D, arrows). TUNEL⁺ cells localized exclusively within the neovascular tufts were counted on P17O₂ and P21O₂ and were reported as a percentage of TUNEL⁺ cells per neovascular nuclei (E). There was a significant difference between B6 and MCP-1^−/− on P17O₂ (*P < 0.0001). Representative cleaved caspase-3–stained sections from B6 (F, arrows) and MCP-1^−/− (G, arrows) mice on P17O₂. Original magnification, ×400.