Eye/Vision
Permanent URI for this collectionhttps://hdl.handle.net/20.500.12503/21759
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Browsing Eye/Vision by Subject "glaucoma"
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Item PROLONGED NMDA STIMULATION INDUCES NEUROPROTECTIVE PATHWAYS AND ENHANCES SURVIVABILITY OF PRIMARY RETINAL GANGLION CELLS(2014-03) Mueller, Brett H.; Park, Yong; Ma, Hai-Ying; Yorio, ThomasPurpose (a): Calcium influx through postsynaptic NMDA receptors has been shown to stimulate a number of key pro-survival genes; however, prolonged stimulation has been shown to have excitotoxic effects leading to apoptosis in neurons. Previous studies have shown a rapid dephosphorylation of CREB in primary hippocampal neurons treated for 1-2 h with100µM NMDA . It is hypothesized that the activation of CREB-specific phosphatases is one of the main pathways that cause apoptosis during NMDA excitotoxicity. The current study investigated the role of NMDA stimulation on the phosphorylation of CREB in primary RGCs, and assessed if NMDA overstimulation caused excitotoxic changes similar to those seen in primary hippocampal neurons. In addition, the occurrence of NMDA excitotoxicity in bipolar and photoreceptor cells was also investigated. Methods (b): Purification and culture of RGCs were performed by sequential immunopanning using Thy 1 antibody from P3-P7 Sprague-Dawley rats. Mixed retinal cultures that remained following isolation of RGCs from the retina were plated once the RGCs were separated and purified. Calcium imaging was used to measure the intracellular changes in calcium following treatment of cells with 100µM NMDA. Western blots were performed to determine signaling pathways linked to NMDA induced cell survival or excitotoxicity. Calcein AM and ethidium homodimer were used to quantify cell survival and cell death. Cells were also subjected to a trophic factor deprivation insult for 6 hours and 24 hours. Results (c): Treatment of primary RGCs with NMDA (100 µM) for 6h caused a greater than 2-3 fold induction of the transcription factor pCREB. MK801 (NMDA antagonist) completely abolished endogenous levels of pCREB and blocked NMDA induction of pCREB. NMDA (100 µM) treatment for 6 and 24 hrs under trophic factor deprivation, protected RGCs from trophic factor deprivation induced cellular death. The mixed retinal cultures (retinal cells without RGCs) had an opposite effect, where the levels of pCREB were diminished and the neurons died when treated with 100 µM of NMDA. Conclusions (d): The data suggests that NMDA signaling is essential for RGC survivability and blocking calcium ion influx through this receptor by the NMDA blocker, MK801 can be detrimental to RGC function and survival. These results also demonstrate that primary RGCs behave differently than other neurons in the retina, and are not susceptible to NMDA excitotoxicity.Item SIGMA-1 RECEPTOR STIMULATION PROTECTS PURIFIED RETINAL GANGLION CELLS FROM ISCHEMIC INSULT THROUGH THE PHOSPHORYLATION OF EXTRACELLULAR SIGNAL REGULATED KINASE 1/2(2014-03) Mueller, Brett H.; Park, Yong; Ma, Hai-Ying; Yorio, ThomasPurpose (a): Sigma-1 receptor activation and mitogen-activated protein kinases (MAPKs) have been shown to have neuroprotective roles in protecting retinal ganglion cells (RGCs) from cell death. The purpose of this study was to determine if sigma-1 receptor stimulation with pentazocine could promote neuroprotection under conditions of ischemia through the phosphorylation of extracellular signal regulated kinase (pERK)1/2. Methods (b): Primary RGCs were isolated from P3-P7 Sprague-Dawley rats and purified by sequential immunopanning using a Thy 1.1 antibody. RGCs were cultured for 7 days before subjecting the cells to an ischemic insult (0.5% oxygen in glucose-free medium) for 6 hours. During the ischemic insult, RGCs were treated with pentazocine (sigma-1 receptor agonist) with or without BD1047 (sigma-1 receptor antagonist). In other experiments primary RGCs were treated with pentazocine, in the presence or absence of PD98059 (ERK1/2 inhibitor). Cell survival/death was assessed by staining with the calcein-AM/ethidium homodimer reagent. Levels of pERK1/2, total ERK1/2, and beta tubulin expression were determined with immunoblotting and immunofluorescence. Results (c): RGCs subjected to an ischemic insult demonstrated more than a 40% increase in cell death, compared to untreated controls. RGCs maintained under ischemia also showed a 50% decrease in expression of pERK1/2 (p<0.05). Cell death was attenuated when RGCs were treated with pentazocine under ischemic conditions and levels of pERK1/2 were increased more than 60% (p<0.05), compared to untreated RGCs subjected to ischemia. Treatment with BD1047 abrogated the pentazocine neuroprotection effects, and also attenuated the increase in levels of pERK1/2 (p<0.05). Finally, treatment with PD98059 also reversed the pentazocine mediated neuroprotective effects on RGCs, and abolished the expression of pERK1/2 (p<0.05). Conclusions (d): These results establish a direct relationship between sigma-1 receptor stimulation and neuroprotective effects under ischemia through the involvement of the MAPK/ERK1/2 pathway in purified RGCs. These findings support a role for sigma receptor agonists as potential neuroprotective agents.