Abstract
Purpose: :
Choroidal neovascularization (CNV) is the main cause of severe vision loss in patients with age-related macular degeneration (AMD). Inflammatory leukocyte infiltration is a critical parameter in pathologic angiogenesis, including CNV. The Rho/ROCK signaling pathway regulates inflammatory leukocyte infiltration. However, involvement of this pathway in CNV is unknown.
Methods: :
C57BL/6 mice were anesthetized and the pupils were dilated. CNV was induced with an argon laser. 4 laser spots (100 mW, 50 µm, 100ms) were placed in each eye using a slit lamp delivery system and a cover glass as a contact lens. Bubble formation at the time of laser injury confirmed the rupture of the Bruch's membrane. The ROCK inhibitor, fasudil (20mg/kg, LC Laboratories) or vehicle were injected intraperitoneally every day. One week after laser injury mice were anesthetized and perfused through the left ventricle with 10 ml PBS followed by 10 ml of 5 mg/ml fluorescein-labeled dextran in 1% gelatin. Micrographs of choroidal flatmounts were taken and the hyperfluorescent areas corresponding to the CNV lesions were measured with Openlab software. The volume of the lesions was quantified, using confocal microscopy. On day 3 after laser injury 10µm frozen sections of the posterior segment were prepared. The sections were incubated with a mouse anti-F4/80 mAb (10µg/ml), and subsequently with the secondary antibody. Photomicrographs of the CNV lesions were taken and the number of F4/80 positive macrophages was counted.
Results: :
7 days after laser injury, the mice treated with fasudil (14000±1292µm², n=16) showed a significant decrease in CNV size, compared with vehicle treated animals (24000± 2864µm², n=16, P=0.005). Fasudil treated animals showed significantly smaller lesion volumes than the vehicle treated group (63856± 5844µm³ vs 25651± 5157µm³, n=16, P=0.00007), determined by confocal microscopy. 3 days after laser injury significantly more macrophages were recruited to the CNV lesion compared to uninjured controls. ROCK inhibition significantly reduced the number of accumulated macrophages in CNV lesions (30± 4vs 9± 3, n=8, P=0.002).
Conclusions: :
ROCK inhibition with fasudil effectively suppresses laser-induced CNV and reduces macrophage recruitment to the lesions. Current results suggest ROCK as an attractive molecular target in the prevention and treatment of AMD.
Keywords: age-related macular degeneration • neovascularization • inflammation