The Effects of Oxidative Stress on Mitochondrial Transmembrane Potential in Rat Retinal Ganglion Cells

Abstract

Abstract: : Purpose: To determine the effect of oxidative stress on mitochondrial transmembrane potential (Δ­ψ_m) in retinal ganglion cells (RGCs) in vitro. Methods: Ratiometric imaging of Δ­ψ_m was performed on primary rat retinal cultures containing DAPI-labeled axotomized RGCs using the dual-emission probe JC-1. Hydrogen peroxide was added to a final concentration of 9.47 mM. In conjunction with the hydrogen peroxide, agents known to regulate the mitochondrial permeability transition pore (bongkrekic acid, cyclosporin A), protein synthesis (cycloheximide), and the cellular redox state (TCEP) were added. After acquiring baseline fluorescence levels, the ratio of fluorescence at 580 nm and 535 nm was calculated. The minimum value of this ratio after treatment was chosen as the point of maximal depolarization, and was compared to baseline values for all subsequent analyses. Results: Addition of hydrogen peroxide (9.47 mM) to cultured rat RGCs induced a depolarization event, resulting in a significant decrease in Δ­ψ_m and morphological changes in the mitochondrion associated with pore opening. The fluorescent ratio (F(λ₅₈₀)/F(λ₅₃₅)) decreased dramatically to 14.6 ± 1.3% of the original value after treatment with H₂O₂, compared to 67.7 ± 9.8% for treatment with vehicle alone (p<0.003). Pretreatment with cycloheximide (100 µM) to prevent protein synthesis before addition of H₂O₂ did not block depolarization compared to treatments without (13.5 ± 7.5%, p=0.4). Attempts to block pore opening with bongkrekic acid (1, 10 µM) and cyclosporin A (10 µM) failed to prevent depolarization as well (14.8 ± 4.2%, p=0.5; 13.1 ± 4.0%, p=0.4). Finally, maintaining the redox state to prevent depolarization using TCEP proved unsuccessful (14.9 ± 1.3%, p=0.5). Conclusions: Oxidative stress depolarizes mitochondria in axotomized retinal ganglion cells independent of protein synthesis and inhibitors of transition pore opening. It is possible that mitochondrial depolarization occurs via a non-classical mechanism in these cells. CR: None Support: NIH EY12492

Keywords: apoptosis/cell death • imaging/image analysis: non-clinical • ganglion cells