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A.T. E. Hartwick, W.H. Baldridge; Delayed Calcium Deregulation in Rat Retinal Ganglion Cells Following Prolonged Glutamate Exposure . Invest. Ophthalmol. Vis. Sci. 2005;46(13):4007.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: A sustained increase in intracellular calcium levels [Ca2+]i has been implicated as a key step in the initiation of glutamate–induced neuronal death. In this work, we characterized the calcium dynamics in cultured retinal ganglion cells (RGCs) during prolonged exposure to glutamate, and investigated the relationship between [Ca2+]i and excitotoxic RGC death. Methods: Purified RGC cultures were generated from neonatal (7–8 days old) rats using a 2–step immunopanning procedure. After culture in serum–free Neurobasal–A/B27 medium for 1–3 days, the isolated RGCs were loaded with calcium indicator dye (Fura–2 or Fura 4F) and transferred to Mg2+–free Hank's salt solution for calcium imaging. Changes in [Ca2+]i were monitored before, during, and after a 1 hour exposure to 100 µM glutamate (10 µM glycine present). Following glutamate wash–out, fluorescently–tagged Annexin V and propidium iodide were added to the microscope chamber to compare the calcium imaging data with markers of cell death. Results:Initial calcium influx was mediated primarily through the NMDA receptor, as the NMDA antagonist APV was more effective (66.7 ± 5.5%; n=11) than the non–NMDA glutamate receptor antagonist NBQX (13.4 ± 3.9%; n=18) in blocking the rise in [Ca2+]i induced by 100 µM glutamate. During prolonged (1 hour) glutamate exposure, three distinct patterns of RGC [Ca2+]i dynamics were observed. In one group (67.4% of RGCs imaged), the calcium levels were sustained at a steady level for the full hour and then recovered to baseline following glutamate washout. The second group (28.3% of RGCs) were initially indistinguishable from the first group, but exhibited a large and rapid secondary increase in [Ca2+]i during the 1–hour glutamate exposure. Cells undergoing this delayed calcium deregulation exhibited no recovery following wash and were always Annexin V positive and, in some cells, also stained by propidium iodide. A third small group (4.3%) showed irregular [Ca2+]i dynamics, and underwent cell membrane rupture (disappearance of fura fluorescence and positive staining by Annexin V and propidium iodide). Conclusions: Excitotoxic RGC death is characterized by delayed calcium deregulation, a phenomenon that has been observed in other CNS neurons. Imaging calcium dynamics in purified RGC cultures serves as a useful system for understanding the mechanisms and pathways that underlie this latent loss of calcium homeostasis.
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