Eye/Vision
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Browsing Eye/Vision by Author "Ma, Hai-Ying"
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Item ENDOTHELIN B (ETB) RECEPTORS CONTRIBUTE TO NEURODEGENERATION IN A RODENT MODEL OF GLAUCOMA VIA UPREGULATION OF C-JUN AND BAX(2014-03) Minton, Alena Z.; He, Shaoqing; Ma, Hai-Ying; Krishnamoorthy, Raghu R.Glaucoma is a group of eye conditions that, if left untreated, can result in blindness. It is commonly associated with an increased pressure inside the eye, known as intraocular pressure or simply IOP. As the pressure builds up inside the eye, it causes damage to the optic nerve, which in turn results in the death of retinal ganglion cells (RGCs). Studies from our lab and others have shown that endothelin 1 (ET-1), the potent vasoactive peptide, contributes to glaucoma. Currently, our lab is interested in understanding the role of the endothelin receptor B (ETB) in glaucoma. We are using rats that do not have ETB receptor (ETB KO rats) and those that have the receptor (WT rats). To mimic glaucoma, the high salt solution is injected into the special vein in the eye (episcleral vein). This causes the build up of pressure inside the eye within 7 to 10 days. This model of glaucoma is called the Morrison’s ocular hypertension model. Previously, we found that IOP elevation for 4 weeks in WT rats caused an appreciable loss of RGCs, which was significantly attenuated in ETB KO rats. In addition, pathological changes in the optic nerve were greatly reduced in ETB KO rats, as compared to those in WT rats. To find out the molecular mechanisms responsible for the death of RGCs, we elevated the pressure inside one eye of adult WT and ETB KO rats, while the contralateral eye served as control. After 2 weeks of IOP elevation, retinal sections were obtained and stained with specific antibodies to detect the levels of c-Jun (the member of the activator protein-1 (AP-1) family) and Bax (protein involved in cell death). We found that WT rats have higher levels of c-Jun and Bax in the retina (especially in the ganglion cell layer), as compared to ETB KO rats. Interestingly, using the Promo 3 software, we found 15 binding sites for members of the AP-1 family of proteins on the rat 1.95 kb upstream promoter region of Bax. Therefore, the transcription factor c-Jun may be an upstream regulator of Bax. In conclusion, transcription factor AP-1 could be involved in the elevation of the ETB receptor levels in the Morrison's model of glaucoma. Conversely, deletion of the ETB receptor results in the lower expression of c-Jun. Taken together, there may be a reciprocal relationship between the AP-1 and ETB receptors. Purpose (a): Previously, our lab has demonstrated that increased levels of ETB receptors contribute to the death of retinal ganglion cells (RGCs) and degeneration of optic nerve axons in the Morrison's elevated intraocular pressure (IOP) model of glaucoma in rats. Moreover, these pathological changes were greatly attenuated in ETB receptor-deficient transgenic Wistar Kyoto rats. Interestingly, an increase in ETB receptor levels in RGCs, following 2 weeks of IOP elevation in Brown Norway rats, was shown to be associated with increased expression of c-Jun, a member of the activator protein-1 (AP-1) family. The current study was aimed at investigating whether the increased expression of c-Jun observed in wild type rats is reduced in ETBreceptor-deficient Wistar Kyoto rats subjected to the Morrison’s model of glaucoma. The status of another apoptotic protein, Bax, was also assessed in these rats. Methods (b): IOP was elevated in one eye of adult wild type and ETB receptor-deficient transgenic Wistar Kyoto rats using the Morrison’s method (injection of hypertonic saline through episcleral veins), while the contralateral eye served as control. After IOP was elevated, rats were maintained for 2 weeks and sacrificed. Retinal sections were obtained and stained with specific antibodies to detect the expression of c-Jun and Bax by immunohistochemistry. In addition, retinal sections were immunostained using an antibody to βIII-tubulin, which is selectively expressed by RGCs in the retina. Images were taken using Zeiss LSM-510 confocal microscope with Z-scan. Results (c): Immunohistochemical analysis showed that IOP elevation for 2 weeks caused increased expression of c-Jun and Bax mainly in the ganglion cell layer (GCL) of wild type transgenic Wistar Kyoto rats as compared to ETB receptor-deficient transgenic Wistar Kyoto rats. Interestingly, using the Promo 3 software, we found 15 binding sites for members of the AP-1 family of proteins on the rat 1.95 kb upstream promoter region of Bax. Therefore, the transcription factor c-Jun may be an upstream regulator of Bax (pro-apoptotic factor). Conclusions (d): Transcription factor AP-1 could be involved in the elevation of the ETB receptor levels in the Morrison's model of glaucoma. Conversely, deletion of the ETB receptor results in the downregulation of c-Jun. Taken together, there may be a reciprocal feedback loop between the AP-1 and ETB receptors.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 RETINAL GANGLION CELLS ARE RESISTANT TO AMPA RECEPTOR MEDIATED EXCITOTOXICITY(2014-03) Park, Yong H.; Mueller, Brett H.; McGrady, Nolan; Ma, Hai-Ying; Dibas, Adnan; Yorio, ThomasGlaucoma is an age-related disease that affects nearly 70 million people worldwide. It is characterized by damage to the cells in the back of the eye which eventually die and cause gradual vision loss. The mechanism to how glaucoma occurs is yet unknown but there are many speculations. A protein molecule called the AMPA receptor is speculated to play a role in glaucoma by causing the death of these cells in the back of the eye. In our study, we are isolating the cells from the back of the eye of rats to study the role of the AMPA receptor and how it truly functions. Understanding basic functions of this protein molecule can one day help us develop drugs targeting AMPA receptors and therefore possibly protecting the dying cells in glaucoma. Purpose (a): The ionotropic glutamate receptors (iGluR) have been hypothesized to play a role in glaucoma pathogenesis by mediating excitotoxic death of retinal ganglion cells (RGC). Previous studies on iGluR in the retina have been focused on two broad classes of receptors: NMDA and non-NMDA receptors including the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic receptor (AMPAR) and Kainate receptor. In this study, we examined the specific excitotoxic effects of activation of the AMPAR in RGCs in-vitro. Methods (b): Purified rat RGCs were isolated from P3-P5 Sprague-Dawley rats by a double immunopanning technique using an antibody to Thy 1.1. RGCs were cultured for 7 days before s-AMPA (100μM) treatments. s-AMPA excitotoxicity was determined by Caspase3/7 luciferase activity assay, immunoblot analysis for α-fodrin and Live (calcein AM)/Dead (ethidium homodimer-1) assay. Gap-43 expression was assessed by immunocytochemistry. Results (c): Treatment of cultured RGCs with s-AMPA (100μM) for 24, 48 and 72h, both in the presence and absence of trophic factors (BDNF and CNTF), did not alter caspase 3/7 activity and cleavage of α-fodrin (neuronal apoptosis marker), compared to untreated controls. A significantly higher (p<0.05) cell survival of RGCs (85.3±1.5% alive cells) was observed after a 72h treatment with 100μM s-AMPA compared to control untreated RGCs (74.8±3.1% alive cells). Quantification of s-AMPA (100μM) – mediated excitotoxicity in purified RGCs incubated for 24h in an oxygen/glucose deprived (0.5% oxygen) medium demonstrated no statistically significant differences in cell survival compared to control RGCs maintained under either normoxia or hypoxia. Additionally, immunocytochemical analysis showed increased GAP-43 staining in RGCs after 24h of treatment with s-AMPA (100μM). Conclusions (d): These results indicate that purified RGCs in-vitro are not susceptible to AMPA excitotoxicity as previously hypothesized. Activation of AMPAR increased GAP-43 expression, suggesting AMPAR could possibly increase neurite outgrowth. The ability of AMPA receptors to promote neuroprotection of RGCs remains to be confirmed.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.