Purpose: Macrophage recruitment to the choroid has been proposed to contribute to the pathogenesis of choroidal neovascularization (CNV) in AMD. The study was conducted to determine whether treatment with clodronate liposomes (CL(2)MDP-lip), which cause depletion of blood monocytes and lymph node macrophages, diminishes the severity of neovascularization in a mouse model of laser-induced CNV.
Methods: Laser-induced CNV was performed in female 16-month-old C57BL/6 mice. Macrophages were depleted by use of CL(2)MDP-lip intraperitoneally and subcutaneously 72 and 24 hours before and every 2 to 3 days after laser injury. Control mice received injections of either PBS alone or PBS liposomes. Blood monocyte and choroidal macrophage depletion were documented by flow cytometry and choroidal flatmount preparation analysis, respectively. Two weeks after laser injury, mice were injected intravenously with fluoresceinated dextran. The right eyes were removed and prepared for flatmount analysis of CNV surface area (in relative disc areas or DA), vascularity (relative fluorescence), and cellularity (propidium iodide stain). The mice were then perfused with 10% formaldehyde, and the left eyes were removed for histopathology. The means of the various parameters for four CNV lesions per eye were calculated. Fluorescein angiography was also performed.
Results: Flow cytometry of circulating monocytes and immunohistochemical analysis of choroidal macrophage density confirmed the effective depletion of blood monocytes and choroidal macrophages respectively in CL(2)MDP-lip-treated mice. Compared with the control, flatmount analysis of macrophage depleted mice demonstrated a significant reduction in size of the CNV area (2.8 +/- 0.5 DA vs. 1.4 +/- 0.1 DA; P < 0.043). The treated group also revealed less vascularity (1.6 +/- 0.1 units vs. 1.1 +/- 0.0 units; P < 0.0092) and cellularity of CNV lesions (3.3 +/- 0.6 DA vs. 1.7 +/- 0.1 DA, P < 0.04). Histopathology revealed that, in the macrophage-depleted group, CNV was smaller in diameter (1270 +/- 73 pixels vs. 770 +/- 82 pixels, P < 0.0006) and thickness (120 +/- 7 pixels vs. 96 +/- 7 pixels, P < 0.019).
Conclusions: Macrophage depletion using CL(2)MDP-lip reduces size, cellularity, and vascularity of CNV. This observation supports the hypothesis that macrophages contribute to the severity of CNV lesions.