Abstract
Abstract: :
Purpose: RGC apoptosis in glaucoma has been attributed to excessive activation of N–methyl–D–aspartate (NMDA)–type glutamate receptors. We have recently developed a model of RGC apoptosis using intravitreal staurosporine. Using this model we have investigated the effects of the NMDA receptor antagonists ifenprodil and MK–801 in preventing RGC apoptosis. Methods: 20 DA rats underwent general anaesthetic following which varying intravitreal doses of either ifenprodil or MK–801 (6 µl of 0, 1, 10, 100, 500 µM) were administered either 1 day before or at the same time as intravitreal 0.5µg staurosporine and fluorescent–labeled annexin 5 (2.5 µg). Rat eyes were then imaged using confocal scanning laser ophthalmoscopy (cSLO) with an argon laser (488 nm) for illumination and a wide band–pass filter with short–wavelength cut–off of 521 nm. Videos of scanned retinal areas were recorded and images assessed for fluorescence using a previously established method. A subgroup of rats had RGCs retrogradely labeled by the application of DiAsp to both superior colliculi as previously described 10 days before intravitreal injections. Animals were killed immediately after imaging, and enucleated eyes were fixed in fresh paraformaldehyde for histological analysis. Specimens were viewed by confocal microscopy for retinal ganglion cell apoptosis counts. Results: Staurosporine induced RGC apoptosis as previously demonstrated, with visualized cells fluorescing in the retina with cSLO from 1 hour after treatment. Time–lapse video revealed a random distribution and appearance of fluorescent cells throughout the retina. These were confirmed histologically to be apoptotic retinal ganglion cells by double–labeling. Both ifenprodil and MK–801 were found to significantly reduce RGC apoptosis counts compared to control (p<0.01), although MK–801 was found to be more effective when administered at the same time as staurosporine. Anatomical reconstructions of in vivo imaging and histological results showed a good correlation of methods. Effects of both NMDA receptor agonists were found to be dose–related. Conclusions: Staurosporine is one of the most potent inducers of neuronal apoptosis known and is a protein kinase inhibitor. We have shown both MK801, a general NMDA receptor antagonist and ifenprodil, a selective antagonist to the NMDAR 2B subunit, NR2B, can inhibit staurosporine–induced RGC apoptosis. Both may have potential in preventing RGC apoptosis in glaucoma.
Keywords: apoptosis/cell death • neuroprotection • ganglion cells