Effect Of Systemic Depletion Of Monocyte-macrophage On Laser-induced Choroidal Neovascularization (CNV) In Mouse

Background Choroidal neovascularization(CNV) affecting the eyes occurs in many diseases such as age-related macular degeneration (AMD), chorioretinitis, ocular histoplasmosis syndrome (OHS), angioid streaks (AS), pathological myopia, and so on. CNV often implicates the macular, leading to exudation or hemorrhage and finally scar forming and thus becomes the most common cause of blindness which makes it a crucial issue to prevent the formation and growth of it. However, The pathogenesis of this angiogenesis process is still uncertain. Many hypothesis have been presented previously. Aging, Hypoxia and many factors which cause abnormal extra cellular matrix (ECM) and rupture of the Bruch membrane were thought to be the key processes. Recently, the importance of imflammation and immunology activiation has been highlighted in the formation and development of CNV,especially the macrophages. Macrophages are one of the common cell constituents that are present in at least 50% of specimens and can be found in the lesions of the laser-induced CNV model. Several imflammatory mediators and angiogenic factors released by macrophages have been demonstrated in the CNV such as tumor necrosis factor-α(TNF-α),vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). The interaction between macrophages and retinal pigmental epithelial (RPE) cells may play a critical role in CNV. Besides,depletion of macrophages could reduce the size and leakage of laser-induced CNV which supports the hypothesis that macrophages contribute to the severity of CNV lesions. Therefore the study of functions of macrophages in CNV can help us better understand the mechanisms of CNV and perhaps helps to find the way to inhibit the development or even formation of CNV.Purpose To investigate the role of macrophages in the development of laser-induced choroidal neovascularization (CNV) by systemic depletion with liposomal clodronate (Cl2MDP-lip).Method To confirm the depletion efficacy of the drugs,4 mice injected Cl2MDP-lip intraperitoneally(Cl2MDP-lip mice, 0.2ml each), 2 mice injected PBS-lip intraperitoneally(PBS-lip mice, 0.2ml) or nothing separately as the control were processd to have the monocytes in the blood counted by flow cytometric and the tissue macrophages in the spleen obseved by immunostaining. The CNV model was estabilished by 532nm laser in adult C57BL/6J mice. One eye each was chosen at random to receive the laser photocoagulation. A total of 70 mice were divided into 3 groups.30 mice assigned to the normal control group received laser treatment without any other intervention; Another two groups involved 20 mice each were administered Cl2MDP-lip (Cl2MDP-lip group) or PBS-lip (PBS-lip group) intraperitoneally at the following time points: 72, 24 hours before and 24, 72 hours after laser injury.Then FFA were performed at day 7and 14 to exam the incidency of the CNV.The poster section of the experimental eyes were processed to undergo the histopathologic examination at 3d, 14d, 28d and56d after laser coagulation in order to assess the activity and severity of CNV. Besides, Macrophages, EC and VEGF proteins were detected by immunofluorescein staining at day3 after laser injury in normal and Cl2MDP-lip mice.Results Compared with the noraml and PBS-lip-treated mice, the amount of blood monocytes decreased and the spleen macrophages were almost abolished 2 days after the administration of Cl2MDP-lip which confirmed its efficacy. The three groups showed similar incidency of CNV by FFA examination at day 7 and 14 after laser treatment. Compared with the other two groups, histopathologic analysis of Cl2MDP-lip mice demonstrated a significant reduction in the cell density of the lesions at day 3 after laser injury; The thichness and area of CNV decreased at day 14 after laser injury(P<0.05); At day 28, the area was still smaller (P<0.05), but the difference of thichness between groups was not significant(P>0.05); At day 56 after the laser injury, both of the differences could not be observed(P<0.05). All the value included the cellularity and the size of CNV showed no difference between the nomal control and PBS-lip groups(P<0.05).Compared with nomal mice, Cl2DMP-lip mice decreased in the number of macrophages invaded in the leision at day 3 (P<0.05). Besides, the number of EC and the expression of VEGF protein also seemed decreased . Conclusion Depletion of macrophages prior to and shortly after laser injury may contribute to reduce the severity of laser-induced CNV but could not stop it from formation. This effect may be attributed to the reduction of the infiltration and proliferation of EC cells and the expression of VEGF protein caused by the depletion of macrophages.