Browsing by Subject "Neuroprotection"
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Item C1q propagates microglial activation and neurodegeneration in the visual axis following retinal ischemia/reperfusion injury(BioMed Central Ltd., 2016-03-24) Silverman, Sean M.; Kim, Byung-Jin; Howell, Garreth R.; Miller, Joselyn; John, Simon W. M.; Wordinger, Robert J.; Clark, Abbot F.BACKGROUND: C1q represents the initiating protein of the classical complement cascade, however recent findings indicate pathway independent roles such as developmental pruning of retinal ganglion cell (RGC) axons. Furthermore, chronic neuroinflammation, including increased expression of C1q and activation of microglia and astrocytes, appears to be a common finding among many neurodegenerative disease models. Here we compare the effects of a retinal ischemia/reperfusion (I/R) injury on glial activation and neurodegeneration in wild type (WT) and C1qa-deficient mice in the retina and superior colliculus (SC). Retinal I/R was induced in mice through elevation of intraocular pressure to 120 mmHg for 60 min followed by reperfusion. Glial cell activation and population changes were assessed using immunofluorescence. Neuroprotection was determined using histological measurements of retinal layer thickness, RGC counts, and visual function by flash electroretinography (ERG). RESULTS: Retinal I/R injury significantly upregulated C1q expression in the retina as early as 72 h and within 7 days in the superficial SC, and was sustained as long as 28 days. Accompanying increased C1q expression was activation of microglia and astrocytes as well as a significantly increased glial population density observed in the retina and SC. Microglial activation and changes in density were completely ablated in C1qa-deficient mice, interestingly however there was no effect on astrocytes. Furthermore, loss of C1qa significantly rescued I/R-induced loss of RGCs and protected against retinal layer thinning in comparison to WT mice. ERG assessment revealed early preservation of b-wave amplitude deficits from retinal I/R injury due to C1qa-deficiency that was lost by day 28. CONCLUSIONS: Our results for the first time demonstrate the spatiotemporal changes in the neuroinflammatory response following retinal I/R injury at both local and distal sites of injury. In addition, we have shown a role for C1q as a primary mediator of microglial activation and pathological damage. This suggests developmental mechanisms of C1q may be re-engaged during injury response, modulation of which may be beneficial for neuroprotection.Item Endothelin-1 mediated decline in mitochondrial function contributes to neurodegeneration in glaucoma(2020-08) Chaphalkar, Renuka M.; Krishnamoorthy, Raghu R.; Stankowska, Dorota L.; Clark, Abbot F.; Zode, Gulab S.Glaucoma is an optic neuropathy with multifactorial etiologies, commonly associated with elevated intraocular pressure (IOP) and characterized by degeneration of the optic nerve, loss of retinal ganglion cells (RGC), cupping of optic disc and visual field deficits, which could ultimately lead to vision loss. In most cases, glaucoma is a chronic, asymptomatic and gradually progressing neurodegenerative disease, sometimes referred to as the "silent thief of sight," hence, routine eye examinations by an ophthalmologist are critical to determine if there is a likelihood of developing the disease. Elevated IOP is a primary and the only modifiable risk factor in glaucoma. Currently, reducing IOP remains the only proven treatment to delay the progression of RGC death; however, some patients continue to have neurodegenerative effects despite lowering IOP. Therefore, development of novel neuroprotection strategies as an adjunct therapy to IOP-lowering agents will provide a valuable therapeutic strategy in glaucoma. One of the promising targets for neuroprotection is the endothelin system of peptides and their receptors. The endothelin (ET) system comprises of three vasoactive peptides (ET-1, ET-2 and ET-3), which act through two types of G-protein coupled receptors, namely, ETA and ETB receptors. Originally discovered in the cardiovascular system, the diverse expression pattern of endothelin peptides and their receptors implicate their involvement in a variety of physiological processes in the body. A growing body of evidence suggests that endothelins and their receptors are associated with neurodegeneration in glaucoma. Previous studies have demonstrated that ET-1 levels are elevated in aqueous humor (AH) and plasma of glaucoma patients. Our lab previously demonstrated that in an ocular hypertension model in rats, there was an increase in ETB as well as ETA receptor expression primarily in RGCs compared to contralateral eyes. Following IOP elevation, RGC loss was significantly attenuated in the ETB receptor-deficient rats, pointing to a causative role of the ETB receptor in glaucomatous neurodegeneration. However, the precise cellular and molecular mechanisms by which ET-1 promotes neurodegeneration through its actions on the endothelin receptors are not completely understood. Previous studies have shown that ETB receptor stimulation increases the oxidative stress and production of superoxide anions, in sympathetic neurons. Several studies point to the role of mitochondrial dysfunction and oxidative stress as contributors to glaucomatous damage in animal models of glaucoma. To investigate various molecular events contributing to the ET-1 mediated RGC loss in glaucoma, we carried out RNA-seq analysis of the translatome in rat primary RGCs following ET-1 treatment. We identified several key mitochondrial and neurodegenerative gene candidates including Atp5h, Cox17, Foxo1, Moap1 and Map3k11 that were differentially expressed in the translatome by ET-1 treatment in RGCs. Based on our RNA-seq findings, we hypothesized that ET-1 causes an increase in reactive oxygen species (ROS) by acting through the ETB receptor that produces a subsequent decline in mitochondrial function and bioenergetics ultimately predisposing RGCs to cell death. To test this hypothesis, we used an in vitro approach by utilizing rat primary culture of RGCs treated with ET-1 as well as an in vivo approach by intravitreal ET-1 injections in rodents and the Morrison's model of glaucoma in rats. Our data showed that there is a significant decrease in the expression of cytochrome c oxidase 17 copper chaperone (COX17) and ATP synthase, H+ transporting, mitochondrial F0 complex, subunit D (ATP5H), both of which are critical components of the electron transport chain and oxidative phosphorylation pathway. Using a Seahorse mitostress assay, we also found a significant decline of several mitochondrial parameters following ET-1 treatment in primary RGCs, which indicated the possibility of a disruption in the mitochondrial quality control machinery. Hence, we also explored the effect of the ET-1 treatment on the mitophagy pathway, specifically in RGCs. Our findings suggest that there is a decrease in mitophagosome formation in RGCs in the Morrison ocular hypertensive model as well as in GFP-LC3 mice injected with ET-1, indicating an impairment in the mitochondrial quality control mechanism. Our studies reveal several novel candidates that could be targeted for the development of neuroprotective approaches to treat glaucoma.Item EXAMINING PYRUVATE S ANTI-INFLAMMATORY ACTIONS IN BRAIN FOLLOWING CARDIAC ARREST(2013-04-12) Nguyen, AnhPurpose: Approximately 0.5 million Americans suffer cardiac arrest each year. Cardiac arrest is usually lethal, and survivors often face severe neurological disability due to irreversible brain injury inflicted by global ischemia/reperfusion. Brain ischemia and reperfusion activates matrix metalloproteinases (MMPs) that disrupt the blood-brain barrier (BBB), enabling inflammatory cells to invade the brain parenchyma. We have shown that pyruvate, a metabolic intermediate and antioxidant, prevents death of hippocampal and cerebellar neurons and preserves neurobehavioral function in dogs after cardiac arrest. However, pyruvate's neuroprotective mechanism is elusive. We are testing the hypothesis that pyruvate preserves BBB integrity following cardiac arrest by suppressing MMPs and evoking expression of protective proteins hypoxia-inducible factor (HIF)-1ɑ and heat shock protein (Hsp)-70. Methods: Yorkshire swine (30-40 kg) were subjected to cardiac arrest-resuscitation or non-arrest sham protocols. Ventricular fibrillation was induced by a train of electric impulses transmitted to the right ventricle via a pacing wire. Precordial compressions were given at 6-10 min arrest, and then sinus rhythm was restored with defibrillatory transthoracic countershocks. NaCl or Na-pyruvate was infused iv at the rate of 0.1 mmol/kg/min during chest compressions and the first 60 min post-defibrillation. Cerebral cortex was snap-frozen in liquid nitrogen for immunoblot or fixed in 4% paraformaldehyde for immunohistochemistry (IHC) at 4 h after defibrillation. HIF-1ɑ and Hsp-70 contents were assessed by immunoblots and IHC. Four groups were studied: cardiac arrest + NaCl (n = 9) or Na-pyruvate (n = 7), or sham + NaCl (n = 5) or Na-pyruvate (n = 5). Results: Although immunoblots did not reveal appreciable differences in temporal cortex HIF-1ɑ content among the groups, IHC revealed increased HIF-1ɑ content in pyruvate-treated compared to NaCl-treated sham and cardiac arrest groups. Hsp-70 content also was similar among the groups, suggesting that this cytoprotective protein was not augmented within 3 h after 60 min pyruvate treatment. Conclusions: Current studies are examining neurobehavioral function and brain proteins over 7 days post-arrest. MMP activities in brain are being measured by gel zymography. Future experiments will determine plasma concentrations of neurofilament light chain, which, when elevated up to 7 days post-cardiac arrest, is associated with poor neurological outcomes.Item FKBP51 and Methylene Blue as Neuroprotective Targets(2011-05-01) Daudt, Donald R.; Thomas YorioPurpose: Neurodegenerative diseases and neurotraumas typically result in apoptosis of specific neurons leading to the pathology observed during the disease state. Existing treatments target the symptoms instead of preventing the death of these neurons. Although neuroprotective drugs should be useful as a treatment to prevent further loss of neurons, efficacious molecules are lacking. FK506 (tacrolimus), a widely used immunosuppressant drug, has significant neuroprotective and neuroregenerative properties throughout the central nervous system, including the eye. FK506 achieves these properties through interaction with FK506 binding proteins (FKBP), including FK506 binding protein 51 (FKBP51). In this study, we examine the effects of FKBP51 as a neuroprotective agent on a neuronal cell line. Methods: We cultured 661w cell cultures with or without FK506, or stably transfected them with an FKBP51 expression vector. These cells were then exposed to the apoptosis inducing agent staurosporine. Cell viability was determined using a calcein AM/propidium iodide assay. Protein levels and activation of nuclear factor kappa-light chain-enhancer of activated B cells (NF-κB) were determined by western immunoblot analysis. Results: FKBP51 overexpression significantly protected 661w cell cultures from staurosporineinduced apoptosis. FKBP51 overexpression also significantly increased NF-κB p65 protein 35 levels and activated NF-κB p65. FK506 treatment significantly protected 661w neuronal cultures from staurosporine-induced apoptosis. FK506 increased FKBP51, NF-κB p65, and levels of activated NF-κB p65 protein. Conclusions: These results suggest that FKBP51 protects 661w cell cultures from apoptosis induced by staurosporine. Additionally, FK506 protected 661w cell cultures from apoptosis and displayed a mechanism similar to that of FKBP51 overexpression. Both FK506 and FKBP51 appear to act through activation of NF-κB p65 protein, suggesting a common pathway for neuroprotection. These findings suggest that FKBP51 is a compound important to neuronal cell culture survival. FKBP51 may be a potential therapeutic drug target for preventing the neurodegeneration and neurotrauma that occur during neurodegenerative diseases.Item Intermittent Hypoxia Training to Foster Brain Recovery after Ischemic Stroke in rats(2018-05) Ruelas, Steven S.; Mallet, Robert T.; Jung, Marianna E.; Schreihofer, Ann M.; Das, Hriday K.Purpose: Ischemic stroke is the leading cause of disability and #5 cause of death in the US. Annually, nearly 800,000 Americans suffer an ischemic stroke, and 130,000 die. The only FDA approved treatment for stroke is recombinant tissue plasminogen activator, but this thrombolytic agent neither protects the affected tissue, nor mitigates the motor or cognitive impairments resulting from stroke. Intermittent hypoxia training (IHT) has been shown to increase cerebral blood flow, reduce oxidative stress, mobilize cerebroprotective signaling cascades and minimize behavioral deficits in a rat model of Alzheimer's Disease. Moreover, a 20 d IHT program attenuated behavioral deficits and protected neurons in ethanol-withdrawn (EW) rats, even when EW began 35 d after IHT. Therefore, we hypothesize that IHT, initiated in rats after stroke, preserves motor and cognitive function, relative to non-IHT rats. Methods: Ischemic stroke will be produced in rats by 90 min occlusion and abrupt reperfusion of the middle cerebral artery (MCA). Motor function and coordination will be evaluated by the rotarod test before and at 1 week intervals after MCA occlusion (MCAO). Rats must balance on a rotating cylinder that accelerates at a constant speed. High fall latency represents intact motor function. The Morris Water Maze (MWM) assesses spatial learning and memory. Rats are placed in an open, circular pool and must find a sunken platform within 90 s. 24 h after stroke, rats undergoing IHT will breathe moderately hypoxic gas (10% O2) for 5-8 cycles, each lasting 5-10 min, with intervening 4 min room air breathing, for 20 consecutive days. These rats will be compared to an MCAO group continuously exposed to 21% O2. At 21 d post-stroke, the brain will be harvested for analyses of infarct and neuroprotective proteins. Results: In pre-stroke testing, the time taken to solve the MWM fell progressively over 10 days, indicating spatial learning and memory, and fall latency on the rotarod lengthened over 5 days, reflecting improved coordination and possibly a training effect. These studies have established the pre-stroke baselines for assessment of IHT's impact on post-stroke recovery. Conclusions: We expect that IHT given after stroke will minimize motor and cognitive impairment by activating neuroprotective signaling cascades culminating in expression of anti-oxidant and anti-inflammatory proteins.Item Neurodegeneration and Neuroprotection in Transient Cerebral Ischemic Stroke(2004-07-01) Wen, Yi; Simpkins, James W.Yi Wen, Mentor: James W. Simpkins. Neurodegeneration and Neuroprotection in Transient Cerebral Ischemic Stroke. Doctor of Philosophy (Biomedical Science), July 2004, pp287, 2 tables, 44 illustrations, 88 titles. Stroke is the third leading cause of mortality and morbidity in the U.S., with over 750,000 cases per year. Post-stroke symptoms include debilitating chronic neurological complications, which result from neuronal damage. Above 80% of strokes are thrombotic, and drugs like tissue Plasminogen Activator (t-PA) have been clinically applied to dissolve the clot and reestablish blood flow to the affected area. Animal models for stroke have provided much new information on the etiology of the neuronal damage in stroke. Experimental MCA occlusion has been successfully used to produce focal ischemic lesions in rodents. This procedure causes a unilateral ischemic area that typically involves the basal ganglion and frontal, parietal and temporal cortical areas. Interestingly, both early necrotic-type cell death and delayed apoptotic cells death have been described following an ischemic event. In spite of the intensive research in the stroke, effective therapies other than t-PA treatment are not yet available. The current study includes two parts, the first part includes the effects of a sex steroid hormone, estrogen, on several aspects of stroke and other neurotoxicity, including oxidative damage and post-ischemic inflammatory response. Estrogen’s neuroprotective effects have been well demonstrated both in vitro and in vivo. Our studies in this part include the investigation of several potential mechanisms of estrogen’s potent neuroprotective activity that include the activation of neuronal isoform of nitric oxide synthase (nNos), and suppression of NFκB, and its corresponding signal transduction pathways for post-ischemic inflammation. We observed that estrogen induces nNos activation rapidly, NO is highly protective at low concentrations, NOS inhibitors can block the potent neuroprotection of estrogen. At least, part of estrogen’s neuroprotection is mediated by rapid nNOS activation in a way that is independent of estrogen’s traditional transcriptional activity. Estrogen was very effective at suppressing the post-ischemic inflammation, reflected by the suppression of NFκB activity, reduction of IκB phosphorylation and iNOS expression. The other part of the studies includes the research that correlates stroke with neurodegenerative diseases. This part of research includes the investigation of the effects of ischemic stroke on APP processing enzymes, α and β secretases. Ischemic injury induced neurotoxicity caused the hyperphosphorylation of a microtubule protein tau, which is one of the pathological hallmarks of Alzheimer’s Disease. Tau hyperphosphorylation appears to be related with the activation/de-regulation of Cdk5, a cyclin dependent kinase, in post-mitotic neurons. We also observed the induction of aberrant cell cycle reentry, which is also a characteristic feature of many neurodegenerative diseases. This is also related with Cdk5 activation/de-regulation.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 Neuroprotective and neurotoxic outcomes of androgens and estrogens in an oxidative stress environment(BioMed Central Ltd., 2020-03-29) Duong, Phong; Tenkorang, Mavis A. A.; Trieu, Jenny; McCuiston, Clayton; Rybalchenko, Nataliya; Cunningham, Rebecca L.BACKGROUND: The role of sex hormones on cellular function is unclear. Studies show androgens and estrogens are protective in the CNS, whereas other studies found no effects or damaging effects. Furthermore, sex differences have been observed in multiple oxidative stress-associated CNS disorders, such as Alzheimer's disease, depression, and Parkinson's disease. The goal of this study is to examine the relationship between sex hormones (i.e., androgens and estrogens) and oxidative stress on cell viability. METHODS: N27 and PC12 neuronal and C6 glial phenotypic cell lines were used. N27 cells are female rat derived, whereas PC12 cells and C6 cells are male rat derived. These cells express estrogen receptors and the membrane-associated androgen receptor variant, AR45, but not the full-length androgen receptor. N27, PC12, and C6 cells were exposed to sex hormones either before or after an oxidative stressor to examine neuroprotective and neurotoxic properties, respectively. Estrogen receptor and androgen receptor inhibitors were used to determine the mechanisms mediating hormone-oxidative stress interactions on cell viability. Since the presence of AR45 in the human brain tissue was unknown, we examined the postmortem brain tissue from men and women for AR45 protein expression. RESULTS: Neither androgens nor estrogens were protective against subsequent oxidative stress insults in glial cells. However, these hormones exhibited neuroprotective properties in neuronal N27 and PC12 cells via the estrogen receptor. Interestingly, a window of opportunity exists for sex hormone neuroprotection, wherein temporary hormone deprivation blocked neuroprotection by sex hormones. However, if sex hormones are applied following an oxidative stressor, they exacerbated oxidative stress-induced cell loss in neuronal and glial cells. CONCLUSIONS: Sex hormone action on cell viability is dependent on the cellular environment. In healthy neuronal cells, sex hormones are protective against oxidative stress insults via the estrogen receptor, regardless of sex chromosome complement (XX, XY). However, in unhealthy (e.g., high oxidative stress) cells, sex hormones exacerbated oxidative stress-induced cell loss, regardless of cell type or sex chromosome complement. The non-genomic AR45 receptor, which is present in humans, mediated androgen's damaging effects, but it is unknown which receptor mediated estrogen's damaging effects. These differential effects of sex hormones that are dependent on the cellular environment, receptor profile, and cell type may mediate the observed sex differences in oxidative stress-associated CNS disorders.Item Neuroprotective properties of Phytoestrogens(2012-12-01) Brock, Courtney Anne; Singh, MeharvanWomen make up nearly two thirds of total Alzheimer’s cases in the United States. It has been speculated that the loss of endogenous estradiol during menopause is, at least in part, what renders the post-menopausal brain more vulnerable to the effects of aging and Alzheimer’s Disease. While hormone therapy can potentially thwart some of the undesirable consequences and increased risks associated with menopause, women are increasingly rejecting hormone therapy and seeking alternative therapy. There is a strong in interest in phytoestrogens as an alternative to traditional hormone therapy. Phytoestrogens are naturally occurring estrogen like compounds derived from plants which have been shown to have a variety of health benefits. Their effects in the brain however are not fully understood. It was my goal to evaluate the effect of phytoestrogens on brain cells as it relates to neuroprotection. We initially assessed the ability of genistein, the most abundant phytoestrogen found in soy, to protect brain cells against age-associated insults in vitro using the hippocampal cell line (HT22 cells), a cortical cell line (HCN-1A cells), and primary slice cultures of the cerebral cortex. The results of these experiments were such that genistein was protective in the explant model and HCN-1A cells, but not in the HT22 cells suggesting that certain key players must be present for genistein to elicit neuroprotective effects. Based on the known estrogen receptor (ER) profiles for the models used in our study, we hypothesized that ER profiles may dictate the effects of phytoestrogens on brain cells. As such, we evaluated male and female C57/Bl6 mice at 3 different ages for ER expression profile and the effects that a phytoestrogen diet had on BDNF, used in this study as a surrogate marker of neuroprotection. Results showed that phytoestrogens’ effects on the brain differ between the cortex and the hippocampus and are dependent upon the sex of the animal and age at which the diet was initiated. From our results we have proposed a mechanism by which phytoestrogens differentially elicit their effects in the brain. The data presented herein provides valuable insight into phytoestrogens’ effects on the brain.Item NEUROPROTECTIVE PROPERTIES OF SIGMA-1 RECEPTOR IN GLAUCOMA(2014-05-01) Mueller, Brett H.; Thomas YorioGlaucoma is an optic neuropathy commonly associated with elevated intraocular pressure (IOP) that affects over 70 million individuals worldwide. Glaucoma pathology is manifested as cupping of the optic disk, damage to the nerve fiber layer, and visual field deficits. The final pathological step of this disease contributing to visual field loss is the apoptosis of retinal ganglion cells (RGCs). Currently, the only therapeutic agents that are used to treat glaucoma are IOP lowering drugs. However, even when IOP is brought within normal range, a significant number of patients still have progression of visual deficits. Currently, there are no treatment options that have the ability to sustain the viability of RGCs during the disease process of glaucoma. Therefore, neuroprotective drugs that protect RGCs need to be developed as adjunct therapeutic agents to IOP lowering drugs. The sigma-1 receptor (σ-1r) is a non-opioid receptor that has been shown to have the ability to bind to benzomorphans, steroids, and psychotropic drugs. This receptor is ubiquitously expressed throughout the entire body; however, the endogenous ligand and function of σ-1r is not yet known. Several in vitro and in vivo studies have demonstrated the neuroprotective effects of σ-1r stimulation in several models of retinal neurodegenerative diseases including glaucoma and diabetic retinopathy. Numerous studies have linked the neuroprotective effects of σ-1r to its ability to block cytotoxic calcium ion influx through ligand gated and voltage gated ion channels, modulation of ER stress, maintenance of mitochondrial homeostasis, and stimulation of pro-survival intracellular signaling pathways. However in primary RGCs, there have been no studies demonstrating σ-1 receptor mechanism of action. The only proposed neuroprotective mechanism of action of σ-1r that has been performed in retinal flat mounts is blockage of calcium ion influx through activated NMDA receptors. This present research project investigated the mechanism of neuroprotective effects of σ-1rs in primary RGCs, particularly involving L-type voltage gated calcium channels (VGCCs) and activation of extracellular-signal-regulated kinases (ERK 1/2). We demonstrated that VGCCs were activated using KCl (20mM). Pre-treatment with a known L-type VGCC blocker produced a 57% decrease in calcium ion influx through activated VGCCs (following depolarization by KCl). In addition, calcium imaging showed that σ-1r agonists, (+)-N-allylnormetazocine hydrochloride [(+)-SKF10047] and (+)-Pentazocine, inhibited calcium ion influx through activated VGCCs. Treatment with a σ-1r antagonist, BD1047, produced a potentiation of calcium ion influx through activated VGCCs and abolished all inhibitory effects of the σ-1r agonists on VGCCs. This confirms that these ligands were acting through the σ-1r. An L-type VGCC blocker (Verapamil) also inhibited KCl activated VGCCs and when combined with the σ-1r agonists there was not a further decline in calcium entry suggesting similar mechanisms of action of both these agents. Lastly, co-localization studies demonstrated that σ-1rs and L-type VGCCs are co-localized in primary RGCs. Taken together, these results indicated that σ-1r agonists can inhibit KCl induced calcium ion influx through activated L-type VGCCs in primary RGCs. This is the first report of attenuation of L-type VGCC signaling through the activation of σ-1rs in primary RGCs. The ability of σ-1rs to co-localize with L-type VGCCs in primary RGCs implies that these two proteins are in close proximity to each other and that such interactions regulate L-type VGCCs Another signaling pathway that was studied as a potential target of σ-1r mediated neuroprotection was the MAP kinase pathway, in particular, ERK phosphorylation as an index of cell survival. RGCs subjected to oxygen and glucose deprivation (OGD) for 6 hours induced 50% cell death in primary RGCs and inhibited pERK1/2 expression by 65%. Cell death was attenuated when RGCs were treated with pentazocine under OGD and pERK1/2 expression was increased by 1.6 fold compared to OGD treated RGCs without pentazocine treatment. The co-treatment of with an ERK1/2 inhibitor PD098059 with pentazocine significantly abolished the protective effects of pentazocine on the RGCs during this OGD insult. These results established a link between σ-1 receptor stimulation and the neuroprotective effects of the ERK1/2 pathway in purified RGCs subjected to OGD. In conclusion, we have established two novel mechanisms underlying σ-1 receptor mediated neuroprotection in primary RGCs. These findings suggest that activation of the σ-1 receptor in RGCs has a role in calcium regulation and the activation of the ERK1/2 pathway. In addition, this study also demonstrates the robust neuroprotective effects of σ-1 receptor in RGCs when subjected to OGD. These data also provide evidence suggesting that σ-1 receptor may be a therapeutic target to protect RGCs during ocular neurodegenerative diseases like glaucoma.Item PHYTOESTROGENS IN THE BRAIN(2013-04-12) Brock, CourtneyPurpose: Soy is one of the top ten herbal supplements taken in the United States. It is generally marketed as a safe and natural way to improve a diverse array of disease conditions such as osteoporosis and to improve menopausal symptoms. Its efficacy, however, has not been completely validated. Genistein, which is a major constituent of soy, is a phytoestrogen, and is thought to elicit some of soy's beneficial effects through activation of estrogen receptors (ER). Despite its wide use, it is currently unclear how genistein might affect the brain. We hypothesize that like estrogen, genistein can be neuroprotective but its capacity to do so is dependent on the availability of "intact" ER-brain derived neurotrophic factor (BDNF) signaling. We have also tested the hypothesis that because ER expression may change as a function of age and sex, such changes may alter genistein's capacity to influence neuroprotective proteins such as BDNF. Methods: This hypothesis was tested using complementary in vitro and in vivo models. For our in vitro experiments we used mouse cerebral cortical explants. Genistein was applied to the cultures at ER activating concentrations. Glutamate was then applied as an excitotoxic insult to induce cell death. Cell death was quantified by measuring the amount of lactate dehydrogenase (LDH) released by the cells into the media. To corroborate our in vitro data, we assessed whether age related changes in ER expression affect genistein's ability to induce BDNF in male and female mice. Results: Our in vitro data indicate that genistein at 100nM significantly reduces the amount of cell death induced by glutamate in cortical explants which express both ERɑ and ERβ. Our in vivo data indicate that age does affect ER expression but it does so in a sex and brain region specific manner. Genistein's effect on BDNF levels were also age, sex, and brain region specific. Conclusions: Our results indicate that genistein can be protective, but its protective effects may be dependent on the expression of appropriate ERs and their capacity to influence BDNF signaling.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.