Abstract
Abstract: :
Purpose: After axotomy, retinal ganglion cells (RGC) undergo apoptosis. While this apoptosis is believed to be mediated by mitochondria, events leading up to cell death are still not fully understood. Previous studies in our lab have suggested that reactive oxygen species (ROS) may play a vital role in this signal cascade. In order to study the role of ROS early in the apoptotic cascade, we measured superoxide levels in cultured RGCs after axotomy. Methods: Primary rat mixed retinal cultures of DAPI–labeled axotomized RGCs were treated with Sytox Green and dihydroethidium (HEt). Sytox Green negativity identifies living cells in the culture, and oxidation of HEt to Et is indicative of superoxide production within the cell. The intensity of Et fluorescence in living RGCs was measured and recorded in multiple cells at several time points after axotomy. Results: Superoxide levels in RGCs increase after axotomy, climbing to significantly higher values at 9 (59.2±6.4, p=0.0005), 12 (66.2±7.6, p<0.0002) and 24 hours (70.0±10.4, p<0.002) when compared to 1 hour (30.7±4.3). The presence of polyethylene glycol–conjugated superoxide dismutase (PEG–SOD, 300 U/mL) in the cultures significantly reduced the rise in superoxide levels at 9 and 12 hours (40.4±3.5, p<0.008; 40.9±3.8, p=0.003), but not at 24 hours (70.8±12.6, p=0.5). The combination of several growth factors known to enhance survival after axotomy (BDNF, 50 ng/mL; CNTF, 10 ng/mL; forskolin, 5 µM; insulin, 5 µg/mL) also prevented superoxide levels from rising at the 9 and 12 hour points (43.3±3.9, p=0.02; 51.3±4.2, p<0.05), but not at 24 hours (69.9±6.6, p=0.5). Conclusions: Intracellular superoxide levels rise in RGCs after axotomy, and may be an early event in apoptosis initiation. The presence of growth factors known to promote viability in axotomized RGCs prevents this rise, suggesting that deprivation of neurotrophic factors could be a factor in inducing a superoxide signaling burst.
Keywords: apoptosis/cell death • ganglion cells • imaging/image analysis: non–clinical