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DC Baptiste, C Jollimore, AT E Hartwick, WH Baldridge, BC Chauhan, GM Seigel, ME M Kelly; Effect of Minocycline on the Survival of Cultered Retinal Neurons in Glutamate Excitotoxicity Assays . Invest. Ophthalmol. Vis. Sci. 2002;43(13):747.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Glutamate excitotoxicity may contribute to retinal ganglion cell death in glaucoma and retinal ischemia. We examined whether the tetracycline derivative minocycline, which has been shown to increase neuronal survival in models of neurodegeneration, has the potential to increase the survival of retinal neurons (RNs) in an in vitro cell culture model of glutamate excitotoxicity. Methods: We used the E1A.NR3 rat retinal cell line, which consists of both neuronal and glia cell types. Cells were incubated for 24 hrs with treatment-media containing: i) No added glutamate (TM), ii) 10 or 100 µM glutamate only (TMG) and, iii) 100 µM glutamate plus various doses (0.002-200 µM) of the test drug minocycline (TMG+M). Neuronal survival was quantified using a cell lysis assay. Viable phase-bright nuclei were counted using a haemocytometer. The mRNA for the pro-apoptotic gene caspase-3 was detected using RT-PCR with RNA isolated from glutamate-treated cells and primers specific for rat caspase-3. Band intensities for caspase-3 were normalized to those for a house-keeping gene, cyclophilin. Results: Incubation of retinal cultures for 24 hrs with 10 and 100 µM glutamate produced a 27% and 47% decrease in cell survival compared to control TM groups (p<0.05, n=3). An increase in mRNA expression for caspase-3 was detected in the 100 µM TMG group as early as 2 hrs following glutamate treatment. In cultures treated with 100 µM glutamate plus minocycline (TMG+M), an increase in cell survival was detected at 0.02 µM (23%, n=6), 0.2 µM (18%, n=6), 20 µM (42%, ν=3), and 200 µM (79%, n=3) compared to TMG controls (n=6). Conclusions: The EIA.NR3 retinal cell line is susceptible to cell death with glutamate excitotoxicity via mechanisms that may involve caspase-3. Minocycline was able to increase retinal cell survival following glutamate excitotoxicity and may decrease apoptotic cell death of retinal neurons. Supported by Glaucoma Research Foundation and CIHR.
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