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J.B. Kerrison, R.N. Lewis, D.J. Zack; Highthroughput Screening for Molecules That Stimulate Retinal Ganglion Cell Survival and Neurite Outgrowth . Invest. Ophthalmol. Vis. Sci. 2005;46(13):170.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: The objective of this proposal is to develop and implement a high throughput assay for the identification and characterization novel molecules that promote retinal ganglion cell (RGC) survival and neurite outgrowth. Methods: High throughput culture of immunopurified RGCs in 96 well culture plates was performed in order to screen a pharmacologic library for compounds promoting RGC survival and neurite outgrowth. Screening was done in quadruplicate with calculation of the normalized mean and z score. Validation of candidates was performed using a dose response analysis. Results: Purity of RGC culture by DiI retrograde labeling was 94.83 ± 1.32 percent. Morphology in cell culture and biologic response to BDNF and forskolin are typical of RGCs. With optimization of our efficiency and capacity, we are able to screen 160 candidates in quadruplicate per week. Screening of over 1200 pharmacologic compounds of the Chembridge library has identified over 30 candidates. Dose response analysis over a range from 3uM to 120uM has validated one candidate to have a maximal effect comparable to BDNF and forskolin combined. Conclusions: Our system allows us to screen for factors promoting RGC survival and neurite outgrowth in a high throughput manner, adaptable to a variety of libraries. Using this approach we have identified a novel pharmacologic compound that promotes RGC survival and neurite outgrowth in a robust, dose–dependant manner.
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