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D.C. Baptiste, C.A. Jollimore, G.M. Seigel, M.E. Kelly; An In Vitro Examination of Minocycline as a Retinal Neuroprotective Agent . Invest. Ophthalmol. Vis. Sci. 2003;44(13):5222.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To study the efficacy of minocycline, a second generation tetracycline, to reduce cell death in primary rat retinal cultures, an immortalized rat retinal cell line (E1A-NR.3) and a rat phenochromocytoma cell line (PC12) following glutamate excitotoxicity or trophic withdrawal. Methods: Cell viability was measured using a cell lysis technique in primary retinal cultures treated for 24-hours with 100 µM glutamate, with or without the inclusion of minocycline (0.002-200 µM). Each treatment group in separate experiments consisted of triplicate wells. For cell lines deprived of serum for 48-hours (E1A-NR.3), or nerve growth factor (NGF) for 12-hours (PC12), in the presence and absence of minocycline, cell viability was measured using a live/dead assay with acridine orange and ethidium bromide. The effects of minocycline on caspase-3 gene expression were studied using semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). Results: Minocycline, but not tetracycline, protected primary retinal cells from excitotoxic cell death. At doses of 2, 20 and 200 µM, minocycline produced an 59%, 82% and 82% increase in cell survival respectively (p<0.05). Minocycline’s protective actions were also confirmed in the E1A-NR.3 cell line, with increases in cell survival of 23% and 25% (p<0.05) at minocycline doses of 0.02 and 0.2 µM following glutamate-induced excitotoxicity, and 14% and 11% (p<0.01 and p<0.05 respectively) following serum withdrawal. Minocycline, at 0.2 and 2 µM also prevented the cell death associated with NGF withdrawal in differentiated PC12 cells (p<0.01). RT-PCR analysis of apoptotic gene expression in retinal E1A-NR.3 cells following 24-hour serum deprivation revealed that 200 nM minocycline decreases caspase-3 mRNA expression. Conclusions: Minocycline increases cell survival and prevents apoptotic cell death of primary retinal cell cultures, the E1A-NR.3 cell line and PC12 cells following glutamate excitotoxicity or trophic withdrawal. The survival-promoting effects of minocycline are not shared by tetracycline. The neuroprotective properties of minocycline may be attributed to a reduction in pro-apoptotic caspase-3 gene expression.
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