Abstract
Abstract: :
Purpose: To define neuronal and glial calcium influx responses to glutamate using cell panning techniques, and to determine the potential protective effect of unoprostone against these glutamate responses. Methods: Retinal ganglion cells (RGCs) were isolated from 1-3 day old Sprague Dawley rat pups, and grown in cell culture using panning techniques. Panning with antibody to Thy 1.1 and the antimitotic cytosine arabinoside were used to isolate retinal ganglion neurons. Cultures without antimitotics were utilized to develop pure retinal glial. Unoprostone (Cayman Chemical) was dissolved in DMSO. Laser scanning confocal microscopy was used to evaluate real-time calcium dynamics. Results: RGCs isolated by panning are extremely sensitive to glutamate, demonstrating sustained calcium influx responses to 100 nM glutamate stimulus. In the presence of glia, RGCs responded to 100 and 500 nM glutamate with transient calcium influx, demonstrating glial buffering effects. Pure retinal glial cultures required 1 mM glutamate stimulus to trigger calcium influx, which was transient and oscillating. In the presence of glia, unoprostone (1 uM), completely blocked neuronal calcium responses to 100 nM glutamate, while DMSO did not. In RGCs isolated by panning, unoprostone, but not DMSO, attenuated calcium responses to 100 nM glutamate. In pure retinal glial cultures, unoprostone (10 uM), inhibited calcium influx stimulated by 5 mM glutamate while DMSO did not. Neither unoprostone nor DMSO inhibited glial calcium influx stimulated by 50 mM KCl. Conclusion: While unoprostone may affect individual retinal ganglion neurons via a receptor-mediated cell signaling event, the presence of retinal glia also plays a key role in RGC survival and unoprostone action. The cellular model described here as well as whole retina preparations are very valuable to further understand the neuroprotective action of unoprostone and its therapeutic usefulness in glaucoma and other retinal diseases. (Support: Marylin Rosenson Fund).
Keywords: 560 retinal culture • 489 neuroprotection • 334 calcium