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R.G. Corredor, Y. Duan, J.L. Goldberg; Electrical Stimulation Improves Survival of RGCs in vitro and Induces Elevation in Intracellular cAMP and Calcium . Invest. Ophthalmol. Vis. Sci. 2006;47(13):4851.
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Activity works synergistically with other signals to prevent neuronal apoptosis. However, it is not clear what type of activity is optimal for promoting neuronal survival. Particularly, during normal development significant number of RGCs undergo apoptosis and activity deprivation may gates apoptotic pathways in RGCs even during adulthood. Since electrical stimulation has been proposed as a method for inducing activity–dependent neuroprotection, we intend to determine what parameters of electrical stimulation best improves RGCs survival and start elucidating the intracellular molecular pathways involved.
Purified postnatal (P5) RGCs were culture in borosilicate chambers, electrically stimulated using monopolar stainless steel electrodes, and monitored using time lapse microscopy. 24 hours after platting, stimulated and control RGCs were counted in 20 randomly selected microscope fields (n=4, total stimulated cells=924, total control cells=924), and stimulus was applied for two hours. The percentage of surviving cells was quantified at the same spots 24 hour after stimulation. To validate the model intracellular cAMP was measured in RGCs expressing the genetically encoded cAMP sensor EPAC using FRET microscopy, and intracellular calcium was measured by ratiometric fluorescence analysis using the calcium indicator fura–2.
Low frequency electrical stimulation significantly improved RGCs survival in vitro compared with controls (59%, 41.99% respectively) (P<0.0401), RGCs exhibited activity–induced increases in intracellular levels of both cAMP and calcium.
These data demonstrate a significant activity–dependent improvement in survival of purified postnatal RGC after electrical stimulation in vitro. We hypothesize that the better survival is related to activity dependent gene expression synergistically mediated by multiple intracellular molecular pathways, and future work will be focus in identifying those pathways which could be mediated by calcium release from the intracellular calcium storages and could be also mediated by intracellular cAMP elevation.
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