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F.B. Kalapesi, M.A. Hill, M.T. Coroneo; Neuroprotective Agent Brimonidine: A Toxicity Study . Invest. Ophthalmol. Vis. Sci. 2003;44(13):5233.
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Glaucoma's pathogenesis has been linked with retinal ganglion cell loss via apoptosis. We have already shown that hydrostatic pressure alone induces significant apoptosis in our in-vitro pressure model using the transformed rat RGC5 cell line. Apoptosis is examined following TUNEL (terminal transferase dUTP nick-end labelling) staining via qualitative analysis by confocal microscopy (LSM) and quantitatively via the laser scanning cytometer (LSC). Recently, alpha-2 receptors have been localised in the inner retina, mainly on ganglion cells. This current study looks at brimonidine, an alpha-2 selective adrenergic agonist which has been shown to be neuroprotective in several animal models. Purpose: To ascertain a concentration range in which brimonidine has no cytotoxic effect on the rgc5 cell line. Methods: Undifferentiated and differentiated RGC5 cells were grown to 70% confluence, split onto 24 wells and grown in varying concentrations of brimonidine varying from 0.2% (2mg/mL) to 2X10-10 %. These cells are then TUNEL stained and analysed by LSM and LSC. Results: Based on morphological analysis, concentrations less that 2X10-5 % do not appear to have a cytotoxic effect on RGC5 cells. Higher concentrations of brimonidine appear to have a cytotoxic effect on the RGC5 cells as evidenced by vacuolation. Conclusions: We have now ascertained an appropriate concentration range of brimonidine to assay in our in-vitro pressure chamber before and after the application of pressure. This will allow us to compare qualitatively (via LSM) and quantitatively (via LSC) the proportion of apoptotic cells with and without brimonidine application. This will show if brimonidine is neuroprotective via reduction of pressure induced apoptosis. This unique in-vitro study and model, the first of its kind, will allow the assessment of multiple potential neuroprotective agents.
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