Abstract
Purpose: :
Our previous results demonstrated that complement factor H (CFH) and membrane attack complex (MAC) play an important role in the development of age-related macular degeneration (AMD). The aim of this study was to co-localize CFH and MAC during laser induced CNV in mice and investigate correlation between CFH expression and MAC deposition.
Methods: :
CNV was induced in C57BL/6 mice by photocoagulation using Argon laser and eyes were collected at days 1, 3, 5 and 7 post-laser treatment. Paraffin embedded sections and flat mounted RPE-choroid-sclera were stained for CFH and MAC using immunohistochemistry (IHC). Quantification and correlation analysis of CFH expression and MAC deposition was done by using ImageJ program. RPE and choroidal cells located in area of laser injury and in distant choroid were investigated. IHC and electron microscopy (EM) was used to localize CFH and MAC on cytoplasmic membrane of RPE cells. RNA and protein expression of CFH in RPE-choroid was determined by RT-PCR and Western blotting.
Results: :
CFH was located predominantly in RPE cells of naïve animals. Expression of CFH in RPE cells significantly decreased (38% and 24%, respectively) on day 1 and day 3 post laser compared to naïve. Expression of factor H in RPE cells returned to basal levels (similar to naïve) at day 5 and 7. In naïve mice MAC deposition was found inside choroidal vessels but after laser injury MAC was found also at the apical surface of the RPE cells. Maximum staining for MAC was observed at day 3 and very low at day 7 post-laser. Results of co-localization studies revealed a negative correlation between CFH and MAC expression in the laser spots. These findings were confirmed by electron microscopy (EM).
Conclusions: :
Decrease in CFH production and secretion by RPE cells leads to increased deposition of MAC at the apical surface of RPE cells. Normalization of CFH production will prevent increased MAC formation and CNV progression.
Keywords: age-related macular degeneration • immunohistochemistry • choroid: neovascularization