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P. Balaram, D. J. Calkins; Concentration-Dependent Protection of Retinal Ganglion Cells From Pressure-Induced Apoptosis by Pre-Treatment With a Flavonol Flavonoid. Invest. Ophthalmol. Vis. Sci. 2007;48(13):4944.
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© ARVO (1962-2015); The Authors (2016-present)
Loss of retinal ganglion cells (RGCs) in glaucoma is associated with sensitivity to intraocular pressure. A component of RGC loss involves oxidative stress, so we asked whether a naturally occurring antioxidant, the flavonol flavonoid quercetin, could reduce the susceptibility of RGCs to pressure-induced apoptosis. Here we used the immortalized RGC-5 cell line to test whether a brief pre-treatment with quercetin modulates their survival with elevated hydrostatic pressure in vitro.
Samples of RGC-5 cells were obtained from Dr. N. Agarwal and treated to one of four concentrations of quercetin dihydrate (formula: C15H10O7.2H2O) using DMSO as a solvent: 0, 2.5 uM, 25 uM, and 200 uM. After 30 minutes of treatment, cells were exposed to either ambient pressure or a uniform column of elevated hydrostatic pressure (70 mmHG) for 24 hrs. At the end of this exposure, the RGC-5 cells were fixed, TUNEL-labeled and counter-stained with DAPI. The fraction of TUNEL+ cells was determined by an automated imaging algorithm tuned to apoptotic nuclei. All experiments were completed in triplicate. The statistical difference between samples was determined using ANOVA and Mann-Whitney rank tests, which require no underlying assumption of sample distribution.
Consistent with our previous studies of pressure-induced apoptosis, RGC-5 cells exposed to elevated hydrostatic pressure for 24 hrs demonstrated a significantly higher fraction of TUNEL+ cells (25%) compared to RGC-5 cells at ambient pressure (5%, p<.001) in the absence of quercetin. Pre-treatment with 2.5 uM quercetin reduced pressure-induced apoptosis by a factor of two compared to no treatment (p=.02), a level which was not statistically different from the apoptotic fraction at ambient pressure for the same concentration (4%, p = .48). With 25 uM quercetin, RGC-5 cells at both ambient and elevated pressure experienced an increased rate of apoptosis (7 and 16%, respectively) compared to 2.5 uM. With 200 uM, the fraction of TUNEL+ cells at both ambient and elevated pressure rose significantly to 52% and 62%, respectively (p<.001).
While low concentrations of quercetin do not enhance survival of RGC-5 cells at ambient pressure, for RGC-5 cells challenged by elevated hydrostatic pressure, quercetin at 2.5 uM and 25 uM decreases the fraction of apoptotic cells. Surprisingly, the highest concentration we tested induced a 10-fold increase in apoptosis at ambient pressure and 4-fold increase at elevated pressure (p<<.001), indicating that higher doses of flavonols may actually be harmful.
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