Abstract
Abstract: :
Purpose: Although in vitro and in vivo models demonstrate caspase activation in retinal ganglion cells (RGCs) undergoing apoptosis, which includes TNF–α–mediated pathway, the caspase–independent component of RGC death in glaucoma is unclear. Here, we aimed to determine whether mitochondrial dysfunction accompanies RGC death induced by TNF–α or ischemia, and whether caspase inhibitor treatment blocks the RGC death. Methods: Primary cultures of rat RGCs were exposed to TNF–α (0.05 ng/ml) or simulated ischemia for up to 48 hours. Temporal relationship of RGC death with the loss of mitochondrial membrane potential and the release of cytochrome c and apoptosis inducing factor (aif) was studied in the absence and presence of caspase inhibitors (Z–IETD–FMK, Z–LEHD–FMK, Z–DEVD–FMK, 50 µM). Cell viability was assessed using calcein assay, and annexin V binding combined with propidium iodide staining was used for the distinction of apoptotic and necrotic cells. Protease activity was measured using a fluorometric assay, and immunocytochemistry was performed using cleavage site–specific antibodies. Time course of mitochondrial alterations in individual cells was assessed using mitotracker orange and subsequent immunocytochemistry. Results:Our findings revealed that the alterations associated with mitochondrial dysfunction (which results in energy depletion and oxidative damage) accompanied the death of RGCs following exposure to TNF–α or ischemia. Caspase inhibitor treatment resulted in an approximately 85% decrease in protease activity. However, despite some early protection, cell viability was less than 70% following 48–hour incubation with death stimuli in the presence of anti–caspase treatment. Both annexin V+ / proprium iodide– apoptotic cells and annexin V+ / proprium iodide+ necrotic cells were detectable in these cultures. In contrast, cell viability was more than 90% in control cultures. Caspase inhibition was not adequate to blockade RGC death if the cell death mediators were released and the mitochondrial membrane potential was lost. More than 70% of RGCs that were mitotracker–positive were rescued with caspase inhibitor treatment. In contrast, approximately 30% of the mitotracker–negative RGCs were capable of rescue with caspase inhibition. Conclusions: These findings imply that the neuroprotective strategies in glaucoma should include tools to prevent initiation of the caspase–independent component of the mitochondrial cell death pathway and/or should improve the intrinsic ability of RGCs to survive the cytotoxic consequences of mitochondrial dysfunction.
Keywords: neuroprotection • ganglion cells • apoptosis/cell death