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Choroidal neovascularization (CNV) is one of the primary causes leading to visual damage in many fundus diseases.Many evidences indicate that macrophage activation and monocyte chemoattractant protein-1 (MCP-1) play important roles in CNV.However, the dynamic expression of macrophage and MCP-1 in the initial stage of CNV is not clear.
This study was to investigate the dynamic changes of F4/80 and MCP-1 expressions in retina-choroid tissue with experimental CNV.
Laser-induced CNV models were monocularly established in 105 SPF 8-week-old male wild type C57BL/6 mice.The mice were sacrificed at 6, 12, 24, 48 and 72 hours after photocoagulation, respectively, and the retina-choroid tissue sections and choroidal flatmounts were prepared.The histopathological examination was carried out to observe the changes of morphology and structure as well as inflammatory response in CNV.The expression and distribution of F4/80 and MCP-1 protein in retina-choroid were detected by double immunofluorescence technique.The expression and distribution of F4/80 in choroid were examined by immunofluorescence.The relative expression levels of F4/80 mRNA and the content of MCP-1 protein in RPE-choroid complex were assayed using real-time quantitative PCR and ELISA, respectively.The use and care of the mice complied with the Regulation for the Administration of Affair Concerning Experimental Animals by Ethic Committee of Experimental Animals of Nanjing Medical University.
The rupture of Bruch membrane, RPE, outer nuclear layer and choroid was exhibited under the optical microscope 6 hours after photocoagulation.Infiltration of inflammatory cells and tissue edema were seen as the lapse of photocoagulation time, and proliferation of vascular endothelial cells was found 72 hours after photocoagulation.F4/80 was expressed in photocoagulation area 6 hours later, and MCP-1 was expressed around the area.With the lapse of photocoagulation time, the expression intensity of MCP-1 weakened and that of F4/80 enhanced.The contents of MCP-1 protein in RPE-choroid complex were (31.25±4.73), (276.31±4.20), (331.95±5.86), (221.24±4.42), (179.89±4.10) and (130.80±5.90)pg/mg in the normal control group, photocoagulation 6-, 12-, 24-, 48- and 72-hour groups, respectively, with a significant difference among the groups (F=1 416.46, P<0.01). The contents of MCP-1 protein peaked at 12 hours after photocoagulation and then gradually declined.The expression levels of MCP-1 protein in different time groups were higher than those in the normal control group (all at P<0.01). A significant difference in F4/80 mRNA expression in RPE-choroid complex was also found among the groups (F=762.72, P<0.01, and a gradually raising tendency was seen over time, showing evidently increase in comparison with the normal control group (all at P<0.01).
Inflammatory response occurs in the early stage of experimental CNV.MCP-1 responds to the CNV at early stage, and the accumulation and activation of macrophage play an important role in the development of CNV.