Neuroprotection by Cocktails of Dietary Antioxidants under Conditions of Nerve Growth Factor Deprivation
Effect of antioxidant cocktails on ROS levels and mitochondrial function following NGF deprivation. (a) Quantitation of ROS levels by FACS analysis of DCHF-DA fluorescence. Neuronal PC12 cells were preincubated overnight with Pool-1, Pool-2, or Pool-3 and then exposed to NGF-free medium for 6 hr. Cells were loaded with DCFH-DA (10 μM) during the last 30 min of treatments and flow cytometric measurements (Geo-mean values) were taken on 10,000 cells contained in the gated regions. Data are the mean ± SEM of three experiments in duplicate. versus CTR-NGF (ANOVA and Dunnett’s multiple comparisons test). (b) Overlapping FACS profiles of a representative experiment. (c) Representative merged images of neuronal PC12 cells maintained in the presence of NGF (CTR) or NGF-deprived for 12 h. NGF-differentiated PC12 cells were pretreated overnight with Pool-1, Pool-2, or Pool-3 before NGF withdrawal in the presence of the indicated pools. Cells were stained by MitoTracker Red and Green (50 and 200 nM, resp.) during the last 30 min of treatment and observed with a fluorescence microscope (Nikon) equipped with a CCD camera. Images were captured at 60x magnification. Scale bar = 10 μm. (d) MitoTracker Red fluorescence was quantified by NIH ImageJ, and the MFI was calculated by counting all cells (on average 200 cells) in about 20 random fields for each condition. Data, expressed as MFI, are the mean ± SEM of three experiments in duplicate. versus No-NGF, versus CTR (ANOVA and Dunnett’s multiple comparisons test).