Browsing by Subject "Retinal Ganglion Cells"
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Item KNOCKOUT OF CASPASE-7 PROTECTS AGAINST OPTIC NERVE CRUSH-INDUCED RETINAL GANGLION CELL DEATH(2014-03) Choudhury, Shreyasi; Liu, Yang; Clark, Abbot F.; Pang, Iok-HouGlaucoma, a leading cause of blindness worldwide is characterized by injury to the nerve of the eye leading to the death of certain eye cells called retinal ganglion cells (RGCs) and vision loss. Currently available glaucoma therapies only attempt to reduce the eye pressure without addressing the associated RGC death problem. As a result, they do not always sufficiently slow the disease progression in all glaucoma patients. Thus, there is an urgent need to develop strategies for preventing glaucoma associated RGC death. Our preliminary studies have identified a novel protein, caspase-7 as a major player in RGC death pathways. We are studying the role of caspase-7 in RGC death in a mouse glaucoma model and whether it can be targeted for better therapeutic outcome. This project is significant because it will identify a new and potentially critical component of RGC death. This will aid in the design of better therapeutic treatment for glaucoma and other degenerative diseases. Purpose (a): Optic nerve (ON) injury is involved in various ocular diseases, such as glaucoma, which leads to apoptotic death of retinal ganglion cells (RGC) and loss of vision. Caspases have been implicated previously in glaucoma and RGC death. However, the role of caspase-7, a functionally unique caspase, in ON injury and glaucomatous damage has not been studied. Therefore, the purpose of this study is to evaluate the role of caspase-7 in ON injury-induced RGC apoptosis. Methods (b): C57BL/6 (Wt) and caspase-7knockout (casp7KO) mice were used for this study. Optic nerve crush (ONC) was performed on left eyes; right eyes served as control. Western blots of the isolated retinas of Wt mice were used to assess the activation of caspase-7 at 3h, 6h, 12h, 1d, 3d, and 7d after ONC. Immunohistochemistry was performed to detect the localization of caspase-7 in RGC. RGC survival was determined by counting the RBPMS (RGC marker) labeled cells in flat-mounted retinas of Wt and casp7KO mice at 7d, 14d and 28d post injury. Both Wt and casp7KO mice were subjected to spectral-domain optical coherence tomography (SD-OCT) and scotopic threshold response of electroretinography (STR-ERG) to evaluate the retinal structural and RGC functional changes at 7d, 14d, and 28d after ONC. Results (c): Western blot data demonstrated that caspase-7 was activated in Wt retina at 12h, 1d, 3d, and 7d after ONC compared to the uninjured control retinas. The number of surviving RGCs was significantly more (3173±59 cells/mm2, mean±SEM, n=6, p<0.001) in casp7KO retinas compared to Wt retinas (1693±84 cells/mm2) at 28d post ONC. SD-OCT analysis revealed that the thickness of the inner retinal layer (ganglion cell layer, nerve fiber layer, and inner plexiform layer) in casp7KO mice was greater (54±1.1 μm, p<0.05) compared to Wt mice (42.3±1.5 μm). Most importantly, analysis of the STR-ERG response demonstrated a decline in amplitude in Wt ONC eyes (10.5±1.9 μv), whereas the response was significantly higher (20.7±2.3 μv, p<0.05) in casp7KO mice even at 28d post injury. Conclusions (d): The current study indicates that injury to the ON activates caspase-7 and knockout of caspase-7 protects inner retinal layer morphology and RGC function after ONC. Thus, caspase-7 appears to play a critical role in ONC-induced RGC death and inhibition of caspase-7 activity may be a novel therapeutic target for glaucoma and other neurodegenerative diseases of the retina.Item Neuroprotection of Rodent and Human Retinal Ganglion Cells In Vitro/Ex Vivo by the Hybrid Small Molecule SA-2(MDPI, 2022-12-12) Pham, Jennifer H.; Johnson, Gretchen A.; Rangan, Rajiv S.; Amankwa, Charles E.; Acharya, Suchismita; Stankowska, Dorota L.The mechanisms underlying the neuroprotective effects of the hybrid antioxidant-nitric oxide donating compound SA-2 in retinal ganglion cell (RGC) degeneration models were evaluated. The in vitro trophic factor (TF) deprivation model in primary rat RGCs and ex vivo human retinal explants were used to mimic glaucomatous neurodegeneration. Cell survival was assessed after treatment with vehicle or SA-2. In separate experiments, tert-Butyl hydroperoxide (TBHP) and endothelin-3 (ET-3) were used in ex vivo rat retinal explants and primary rat RGCs, respectively, to induce oxidative damage. Mitochondrial and intracellular reactive oxygen species (ROS) were assessed following treatments. In the TF deprivation model, SA-2 treatment produced a significant decrease in apoptotic and dead cell counts in primary RGCs and a significant increase in RGC survival in ex vivo human retinal explants. In the oxidative stress-induced models, a significant decrease in the production of ROS was observed in the SA-2-treated group compared to the vehicle-treated group. Compound SA-2 was neuroprotective against various glaucomatous insults in the rat and human RGCs by reducing apoptosis and decreasing ROS levels. Amelioration of mitochondrial and cellular oxidative stress by SA-2 may be a potential therapeutic strategy for preventing neurodegeneration in glaucomatous RGCs.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 STIMULATION OF THE AMPA RECEPTOR IN RETINAL GANGLION CELLS INCREASES PHOSPHORYLATION OF CREB(2013-04-12) Park, YongPurpose: Activation of the ɑ-amino-3-hydroxy-5-methyl-4-isoxazolepropionic (AMPA) receptor through glutamate or other agonists allows influx of cations including calcium, potassium and sodium. The purpose of this study was to investigate the neuroprotective role of the AMPA receptor in-vitro, in purified retinal ganglion cells (RGCs) and retinal mixed culture lacking RGCs (mixed retinal culture), by assessing the phosphorylation of cAMP response element-binding protein (p-CREB) stimulated by calcium influx. Methods: Purified rat RGCs were isolated from P3-P7 Sprague-Dawley rats and cultured by a double immunopanning technique using an antibody to Thy 1.1. The residual neurons in the retina following RGC isolation (supernates of the panning procedure) were used as the mixed retinal culture (lacking RGCs). Calcium imaging was used to identify the functionality of the AMPA receptors and selectivity of the AMPA agonist. RGCs and mixed retinal neurons were cultured for 7 days before AMPA treatment. Following treatment with AMPA for 6 hours, proteins were extracted and western blot analysis was carried out to determine changes in expression of the p-CREB and p-ERK1/2, which were normalized to total CREB, total ERK1/2, and beta tubulin. Results: AMPA receptors were stimulated through administration of AMPA (100μM), which depolarized the purified RGCs and increased intracellular calcium. The AMPA mediated calcium ion influx was significantly attenuated by approximately 87.8% (p<0.001) following pre-treatment with 20μM of NBQX (AMPA receptor antagonist). Pretreatment with a NMDA receptor (I μM MK801) or Kainate receptor (50μM UBP301) antagonists simultaneous with AMPA administration did not significantly decrease calcium influx. AMPA increased p-CREB by 4.3 ± 1.1 fold in purified RGCs (p=0.004), however, in mixed culture p-CREB did not change appreciably compared to control. ERK1/2 phosphorylation was significantly decreased in mixed retina culture (p<0.05), but not in purified RGC culture treated with AMPA. Conclusions: The data demonstrate distinct differences in the response to AMPA between RGCs and other neuronal populations in the retina. In particular, the lack of significant changes in the phosphorylation of ERK1/2 in purified RGCs following AMPA treatments suggests that an alternative pathway for phosphorylation of CREB maybe more important in RGC cell survival.Item The Neuroprotective Effects of SA-2-NP in a Mouse Model of RGC Injury(2021-05) Ferguson, Jonathan L.; Stankowska, Dorota L.; Millar, J. Cameron; Tovar-Vidales, TaraDetermine if a novel hybrid compound SA-2 can be delivered to the retina in a nanoparticle formulation and have protective effects on retinal ganglion cells (RGCs) following an optic nerve crush (ONC) model of RGC death. Pattern Electroretinography (PERG) was performed on six- to twelve-week-old female (C57BL/6) mice (n = 1-8 mice per group) prior to performing ONC on the left eye to promote RGC death similar to that seen in normotensive glaucoma. Mice were dosed topically for seven or fourteen days either with SA-2 in polylactic glycolic acid (PLGA) nanoparticles, or empty PLGA nanoparticles. Subsequent PERG was performed at seven day following ONC to reassess RGC function after the optic nerve injury and treatments. The mice were subsequently euthanized and both eyes we enucleated and fixed with paraformaldehyde. The retinas were removed, flat mounts were prepared and immunostained with RBPMS antibody to quantify surviving RGCs. Our study demonstrated that SA-2 can be delivered to the retinal tissue with PLGA nanoparticles. However, following optic nerve crush in mice, at the selected doses and delivery regimen of SA-2, neuroprotective effects determined by RGC counts and PERG analysis were not statistically significant. Following ONC in mice, topically delivered SA-2 loaded nanoparticles demonstrated some trend in neuroprotection without statistical significance. Further investigation is required to delineate the efficacious delivery mode and dose.