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Zhuo Shao, Mollie Friedlander, Christian Hurst, Zhenghao Cui, Lucy Evans, Jing Chen, Przemyslaw Sapieha, Sylvain Chemtob, Jean-Sebastien Joyal, Lois Smith; Evaluating Choroidal Microvascular Angiogenesis by Choroid Sprouting Assay. Invest. Ophthalmol. Vis. Sci. 2013;54(15):3049. doi: https://doi.org/.
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Age-related macular degeneration (AMD) is a major cause of blindness. The disease is associated with choroidal vascular dropout (dry AMD) or choroidal neovascularization (wet AMD). The mechanism that causes abnormal choroidal angiogenesis is not completely understood, partially due to the lack of an assay of choroidal vascular growth. Here we characterized and optimized an easily isolated, robust, quantitative and reproducible organotypic microvascular model from the choroid.
Choroid tissue from Sprague Dawley rats, C57BL/6J and 129S6/SvEvTac mice was isolated and incubated in MatrigelTM. After 3-7 days of incubation, the area covered by tube-like sprouts was quantified by ImageJ software and the cell types of the sprouts were analyzed by fluorescence-activated cell sorting (FACS). The normalization and quantification methods for calculating the sprouting area have been standardized, and the impact of retinal pigment epithelial (RPE) cells, age of the animals, the type of media used for incubation and the responses of the assay to pharmacological stimulation has been characterized.
The variation of vascular sprouting area is comparable between choroid samples obtained within or between animals. The sprouting area is highly reproducible between batches when normalized to controls. A semi-automated quantification macro has been developed for efficient quantification. The removal of RPE from the choroid and aging of choroid reduce the sprouting rate. Furthermore, endothelial selective medium CSC and EGM-2 provide a better growth condition for the choroid explants compared to non-selective medium DMEM. Vascular endothelium growth factor (VEGF) promotes choroidal sprouting where 4-HDHA (4-hydroxy-docosahexaenoic acid), an anti-angiogenic metabolite of omega-3 polyunsaturated fatty acid, reduces choroid sprouting dose dependently.
This study defined optimal conditions for a highly reproducible, efficient and quantifiable choroid microvascular ex vivo sprouting assay. This method provides a new experimental tool not only for AMD studies, but also for physiological and pharmacological research related to microvascular diseases in general.
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