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V.F. Roche, M. Zhao, C.A. Opere, G.L. Zhan, S.E. Ohia; Effect of Bicyclic Hexahydroaporphines on Apoptosis of Retinal Ganglion Cells (RGC–5) . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2620.
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© ARVO (1962-2015); The Authors (2016-present)
We have previously shown that bicyclic hexahydroaporphines (HHAs), including nor–HHA (1) and its N–methyl congener (2), can attenuate ischemia–induced excitatory neurotransmitter release from isolated bovine retinae. In the present study, we investigated the effect of HHAs on glutamate–induced apoptosis of cultured retinal ganglion cells.
The cell viability assay was carried out using the CellTiter–Blue® Cell Viability Assay (Promega, Madision, WI) following the manufacturer’s instructions. Briefly, utilizing 96 well plates, 1000 RGC–5 cells were plated per well with DMEM medium supplemented with 10% serum. After overnight incubation the medium was replaced with serum–free DMEM and pretreated with HHAs (1 – 100 µM) for one hour before being exposed to glutamate (32 mM) for 24 hrs. After incubation, the cells were treated with 20 µl of CellTiter–Blue cell viability reagent for four hours, followed by 100 µl of stop solution. Fluorescence was measured at 560/590 nm using a Spectra max/Gemini EM fluorescent plate reader (Molecular Device Corp, Sunnyvale, CA). The effect of HHAs on cell viability was reported as percent survival of controls.
Glutamate (8 – 64 mM) induced apoptosis in retinal ganglion cells in a concentration–dependent manner. At 32 mM, glutamate caused a 60% decrease in cell viability, which was attenuated by both 1 and 2 (1 – 100 µM). For instance, 10 µM of 1 or 2 inhibited 32 mM glutamate–induced apoptosis of retinal ganglion cells by 40%. A similar neuroprotective action was elicited by MK–801 (10 µM) on the glutamate–induced response.
We conclude that HHAs can inhibit glutamate–induced apoptosis in retinal ganglion cells. The exact mechanism that underlies HHA’s neuroprotective action on retinal ganglion cells remains to be determined.
The authors gratefully acknowledge Dr. N. Agarwal, University of North Texas Health Sciences Center, for providing RGC–5 cells.
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