Activation of Multiple Caspases Involved in the Death of Retinal Ganglion Cells in a Mouse Model of Elevated IOP

Abstract

Abstract: : Purpose: To investigate the activation of multiple caspases involved in the apoptotic cascade leading to the death of retinal ganglion cells (RGCs) in a mouse model of elevated intraocular pressure (IOP). Methods: Argon laser was used to photocoagulate the limbal and episcleral veins of the left eye of wild type C57BL/6J mice. Non–invasive tonometry measured the IOP of both eyes once a week after laser treatment until sacrifice. The mouse RGCs were identified by retrograde labeling with DiI and counted by confocal microscopy. GEArray Q series Mouse Apoptosis Gene Array side–by–side hybridization simultaneously assessed the expression profile of 96 genes involved in apoptosis using RNA samples from the control and treated retinas. Additionally, immunostaining for caspase 8, caspase 9, caspase 3 and cytochrome c was performed in the retina sections from control and elevated IOP eyes. Western blots measured the protein expression of these key factors in control and experimental retinas. Results: The Gene Array showed differences in caspase RNA expression in the control and experimental retinas. Immunostaining demonstrated caspase–3 was activated on day 4 and peaked on day 14. There were more caspase 9 and caspase 8 positive cells in the RGC layer in retina from eyes with elevated IOP compared to control. Cytochrome–c did not co–localize with caspase–9, Western blot results were consistent with the gene array and immunostaining with increased levels of caspases in eyes with elevated IOP. Conclusions:Apoptotic genes are activated in the retinas of mice with elevated IOP. There is also evidence of increased cytochrome–c and caspase activation with a time lag between cytochrome–c release and activation of caspase–9. These findings are consistent with apoptotic RGC death in this mouse model of elevated IOP, allowing greater understanding of the mechanism and regulation of this process.

Keywords: ganglion cells • apoptosis/cell death • intraocular pressure