Browsing by Subject "Eye Diseases"
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Item A Study of Some Aspects of the Role of Mast Cells in Experimental Autoimmune Uveitis(1994-06-01) Lee, Carol Hamberlin; Edward Orr; Robert Gracy; Laura S. LangLee, Carol Hamberlin, A Study of Some Aspects of the Role of Mast Cells in Experimental Autoimmune Uveitis. Doctor of Philosophy (Biomedical Sciences), June 1994, 141 pp., 6 tables, 29 illustrations, bibliography, 115 titles. Choroidal mast cells have been implicated in experimental autoimmune uveitis (EAU), an ocular inflammatory disease induced by S-antigen (Sag). Activation of ocular mast cells in Lewis rats was evaluated by determining changes in numbers of mast cells, levels of histamine, and wet weights of ocular tissues. A decrease in choroidal mast cells was confirmed statistically, and limbal mast cells were found to be activated earlier than choroidal mast cells. The ocular histamine distribution was altered during EAU, decreasing in the anterior eye, and increasing in the posterior eye. Retinal histamine levels increased when EAU symptoms occurred, but decreased while the disease was still intense. Levels of histamine methyltransferase, which degrades histamine, increased significiantly in retinal tissue when histamine levels fell. Signficant weight increases indicated edema, which can result from mast cell mediator action. Leflunomide, an immunomodulating drug that is known to affect mast cells in vitro, prevented induction of EAU. Leflunomide also suppressed changes in the mast cell-related parameters, histamine levels and wet weights. Mechanisms for activation of ocular mast cells in EAU were investigated. Results suggest that mast cell activation does not occur through mast cell surface IgE-antigen crosslinking. The adjuvant used, complete Freund’s adjuvant, is not conducive to IgE production. Histamine releasing factors, HRFs, are produced by various immune system cellular components. Preliminary efforts did not demonstrate HRF activity. Mast cell numbers, histamine levels, and wet weights were also evaluated in a milder form of EAU induced by M-peptide (Mpep), a peptide fragment of Sag. Mpep/EAU produces few disease symptoms in the anterior eye, but destroys the same retinal area as Sag/EAU—photoreceptor cells and their outer segments. Inflammation is less intense, restricted primarily to the target area. Mast cell numbers did not change, but histamine levels and wet weights changed significantly, suggesting that mast cells are also involved in Mpep/EAU. Overall, the results of this study add to evidence that mast cells are involved in pathogenesis of EAU. The results also point to topics of further investigation into the role of mast cells in EAU and in normal function in ocular tissues.Item Age Related Changes in Rabbit Cornea: Permeability and Membrane Properties(1994-12-01) Tai-Lee, Ke; Clark, Abbot F.; Gracy, Robert W.; McConathy, Walter J.Ke, Tai-Lee, Age Related Changes in Rabbit Cornea: Permeability and Membrane Properties. Doctor of Philosophy (Biochemistry), December, 1994, 139 pp., 26 tables, 13 illustrations, bibliography, 117 titles. This investigation was designed to characterize age-related changes in corneal function and biochemical structure. The specific aims were to: 1) systematically assess changes in permeability to compounds of different molecular weights and lipophilicities, 2) examine differences in tissue binding by utilizing a theoretical transport model, and 3) evaluate the biochemical changes in lipid composition and distribution. Experiments to compare young (six weeks) versus old (three to four years) rabbit corneal permeability were carried out utilizing an in vitro diffusion model. Changes in corneal transmembrane resistance, permeability to various compounds, and metabolic capability were examined by various analytical techniques. In addition, a theoretical penetration model which took into account stromal binding was studied. Corneal lipid composition and distribution were assessed by HPLC and GC. in corneal transmembrane resistance, permeability to various compounds, and metabolic capability were examined by various analytical techniques. In addition, a theoretical penetration model which took into account stromal binding was studied. Corneal lipid composition and distribution were assessed by HPLC and GC. Permeabilities of selected compounds with different physicochemical properties were evaluated in young and old intact and denuded (wounded) rabbit corneas. With age, the membrane permeability significantly decreased in parallel with an increase in transmembrane resistance. Age-related changes in activities of esterase and phosphatase were also found. For some compounds, the aged corneas exhibited longer lag times in penetration studies. This suggested that the binding constant in the cornea from older animals was higher than in young animals. Maximum binding capacity from theoretical model calculations correlated well with experimental results in the young corneal stroma but correlation was less rigorous for old corneal stroma. Age-related changes in lipid composition and distribution in corneas were observed and provide indirect evidence for a decrease in membrane fluidity (decrease in the ratio of phosphatidylcholine/sphingomyelin) in the aged cornea. Results indicate that the aging process in the cornea is associated with changes in biochemical structural matrix including membrane lipid composition and physical properties such as fluidity (microviscosity). Functional correlations include changes in: 1) transmembrane resistance, 2) membrane permeability, 3) enzymatic activities (esterase and phosphatase), and 4) binding properties of the cornea. A possible mechanism for understanding and developing an intervention for age-related changes in the cornea is postulated.Item Characterization of the Bradykinn Receptor in Human Corneal Epithelium(1997-08-01) Wiernas, Terry Kirkham; Michael W. Martin; Glenn Dillon; Michael ForsterWiernas, Terry Kirkham, Characterization of the Bradykinin Receptor in Human Corneal Epithelium. Doctor of Philosophy (Biomedical Sciences), August, 1997, 255 pp., 5 tables, 39 figures, references, 137 titles. Bradykinin (BK) is a well-established mediator of inflammation. High levels of BK in human tears following ocular allergic provocation led to the hypothesis that BK receptors may exist on the corneal epithelium and could play a role in corneal inflammation and/or wound healing, in addition to other functions. To test this hypothesis, human corneal epithelial cells were cultured and used to conduct a series of studies to evaluate and characterize the BK receptor. Due to the limited supply and high cost of primary human corneal epithelial (P-CEPI) cells, in addition to the fact that these cells do not divide and proliferate over more than a few passages, SV40 virus-immortalized human CEPI cells (CEPI-17-CL4) were used as a model system. Extensive studies confirmed that the immortalized cells faithfully represented the primary cells. This study demonstrated the presence of BK receptors on corneal epithelial cells for the first time. The receptors were characterized as the B2 subtype and were found to be represented by an apparent single binding site. Furthermore, stimulation of these receptors was found to elicit concentration-dependent increases in both inositol phosphates, via activation of phospholipase C, and intracellular calcium mobilization. The rank order affinity of BK and its analogs as determined by binding assays was found to correlate well with the rank order potency of BK and its analogs in evoking the latter functional responses, which were blocked by two B2-receptor selective antagonists. A significant, concentration-dependent stimulation of [3H]thymidine uptake in CEPI cell DNA was elicited by BK which suggests a potential mitogenic effect of BK and a role in corneal wound healing. BK did not significantly affect the release of three pro-inflammatory cytokines, prostaglandin E2 or matrix metalloproteinase-1, and seemed to have an inhibitory effect on the release of tumor necrosis factor α. In conclusion, these studies have confirmed that CEPI-17-CL4 cells represent a good in vitro model of human corneal epithelium and have contributed to a better understanding of the ocular effects of BK and characterization of its receptor within the cornea.Item Characterization of the Serotonin Receptors in the Long Posterior Ciliary Artery of the Bovine Eye(2000-08-01) Landry, Theresa A.; Quist, Eugene; Martin, Michael; Pang, Iok-HouLandry, Theresa A., Characterization of the Serotonin Receptors in the Long Posterior Ciliary Artery of the Bovine Eye. Doctor of Philosophy (Biomedical Science), August 2000, 14 pp., 5 tables, 29 illustrations, bibliography, 104 titles. Vascular disease and vasospasm are implicated in the etiology of glaucoma. The long posterior ciliary (LPCA) is the major blood supply for the ciliary body including the ciliary processes that produce aqueous humor. Information about the pharmacological control of this vessel would be helpful in understanding its normal and pathologic function. Serotonin (5-HT) is a neurotransmitter that effectively constricts the LPCA. The objective of this research is to identify the serotonin receptor subtype responsible for the 5-HT induced vasoconstriction of the LPCA and to characterize the cellular mechanisms that mediate that contraction. Ring segments of the LPCA were dissected from bovine eyes and mounted on tungsten triangles attached to a force transducer. Changes in vascular tension were measured and recorded using a physiography recorder. Dose response curves with 5-HT, 5-HT 1-like agonist, 5-CT, and 5-HT2 agonist, α-methyl-5-HT, indicate that the 5-HT 1-like receptor contributed about 15.13% to the contraction and the 5-HT2 receptor contributed to 61.61%. The EC50 for the three agonists were 283 nM (5-HT), 336 nM (5-CT), and 1.7 μM (α-methyl-5-HT). Inhibition curves with selective antagonists indicate that the IC50 is (5-HT 1-like antagonist) and ketanserin (5-HT2 antagonist). Following incubation of the rings with diltiazem 10 μM or nifedipine 10μM, the response to 5-HT was reduced 65.*% and 61.7% respectively. Incubation in calcium free PB produced similar results. Ryanodine inhibited the 5-HT contraction by 58.1% and caffeine inhibited the response 100%. PKC inhibitors bisindolymaleimide II 1 μM, disindolylamalemide II 10 μM, chelerythrine 25 μM and H-7 5 μM decreased the 5-HT response by19.8%, 55.7%, 31.1% and 61.5% respectively. Incubation of the ring segments with one of three PLC antagonists, 2-NCDC 70 μM, U73122 0.5μM, or neomycin 5 mM, prior to the addition of 1 μM serotonin, significantly reduced the contraction of each vessel, p [less than] 0.0001. The 5-HT-induced vasoconstriction of the LPCA of the bovine eye is mediated through activation of both 5-HT2 and 5-HT 1-like receptors. The contraction is dependent on the mobilization of calcium and is mediated in part through PLC activated intracellular calcium release from IP3 sensitive stores.Item Clinical Internship with the Clinical Glaucoma/Viability Group at Alcon Research, Ltd.: The Use of Prostaglandin Analogues in the Treatment of Patients with Open-Angle Glaucoma (OAG) or Ocular Hypertension (OHT)(2003-12-01) Hall, Magali G.; Robert Wordinger; Richard Easom; Victoria RudickHall, Magali. Master of Science, Biomedical Sciences, December 2003. The use of Prostaglandin Analogues (PGAs) in the Treatment of Patients with Open-Angle Glaucoma (OAG) or Ocular Hypertension (OHT). Summary: Glaucoma is an ocular condition that causes damage to the optic nerve leading to a loss of visual function, and permanent blindness if left untreated. It is the leading cause of preventable blindness in the U.S. The main risk factor for glaucomatous optic neuropathy is elevated intraocular pressure (IOP), which can be controlled by pharmaceutical therapy, surgical therapy or both. Topical medication is usually recommended prior to surgical intervention. Objectives: This study had two main objectives. First, to determine the IOP lower safety and efficacy of three concentrations of a new prostaglandin analogues (PGA), and secondly to determine the incidence of ocular hyperemia with once-daily dosing of study medication compared to it’s vehicle and to latanoprost, a marketed PGA. Study Design: This was a Phase II, double-masked, dose-response study with five treatment arms (the three different concentrations of study drug), vehicle, and latanoprost. Study was conducted in fourteen days, with five study visits as follows: Screening and eligibility visit followed by three on-therapy visits scheduled on Day 1, Day 7, and Day 14. The primary efficacy variable was IOP measurements taken at four different time points on study visits. Results: Final data will not available in time to include in this paper.Item Effects of Intravitreal Endothelin-1 on Anterograde Axonal Transport in Rat Optic Nerve: Evaluating a Possible Mechanism for Glaucomatous Optic Neuropathy(2002-05-01) Stokely, Martha Elise Lambert; Thomas Yorio; Scott T. Brady; Glenn DillonStokely, Martha Elise Lambert, Effects of Intravitreal Endothelin-1 on Anterograde Axonal Transport in Rat Optic Nerve: Evaluating a possible mechanism for glaucomatous optic neuropathy. Doctor of Philosophy (Biomedical Sciences and Neuroscience), May 2002; 114 pages; 1 table; 12 figures; bibliography, 274 titles. Glaucoma presents a distinctive dysfunction in anterograde axonal transport that disproportionately affects the delivery of specific types of cargo(s) into the optic nerve. Previous models for pathogenesis of glaucoma have failed to provide an adequate mechanism to explain the characteristic cargo-selectivity. A new theoretical model, the “endothelin receptor-mediated model of neuropathogenesis,” was developed to explain the cargo-selective axonal transport dysfunction seen in glaucomtous optic neuropathy. In addition, a new experimental animal model, the “intravitreal endothlin/axonal transport” model was developed to test hypotheses generated by the new theoretical model. Intravitreal endothelin-1 significantly affected all of the known rate components and subcomponents of anterograde axonal transport in the rat optic nerve. Changes were seen in anterograde axonal transport in the rat optic nerve. Changes were seen in anterograde fast axonal transport for both the fastest moving small tubulovesicles, and slightly slower membrane bound organelles (MBOs), as well as in the slow transport of cytoplasmic matrix and cytoskeletal materials. Endothelin-1’s predominant effect was a severe depression in the mitochondrial subcomponent of fast anterograde axonal transport, which was most pronounced at 28 hours post-treatment. At that time, the effects of endothelin-1 were mimicked by endothelin-3, characteristic of the non-ischemic endothelin-B type of receptor. In addition, analysis of a cohort of 11 distinctive protein bands moving with the mitochondrial subcomponent demonstrated a cargo-selective effect of endothelin-1 and the delayed movement into the optic nerve for a chemically distinct subset of proteins, but not the majority of protein, in transport during this timeframe. These results appear to be consistent with what is known about the pathology of glaucomatous optic neuropathy and the neurochemistry of anterograde axonal transport and suggest that intravitreal may be an excellent model to study the mechanisms of neurodegeneration that occurs in glaucoma.Item Endothelin receptor-mediated neurodegeneration in glaucoma(2017-08-01) McGrady, Nolan; Krishnamoorthy, Raghu R.; Yorio, Thomas; Clark, Abbot F.Primary open-angle glaucoma (POAG) is a complex set of optic neuropathies which are characterized by the degeneration of the optic nerve, cupping of the optic disk and loss of retinal ganglion cells (RGCs). There are approximately 3 million Americans who currently suffer from this disease although this is most likely an underestimation since many individuals with glaucoma are unaware that they have the disease. POAG is an age-related disease progressing slowly over the course of several decades and is most commonly associated with an elevation in intraocular pressure (IOP). Currently available treatments for glaucoma, both surgical and pharmacological, are solely focused on the regulation of IOP; nevertheless, some individuals continue to show progressive damage despite being on available therapies. In recent years, there has been increased momentum towards the development of neuroprotective strategies for POAG, particularly in preclinical models of glaucoma. Despite these efforts, there is still no neuroprotective treatment currently available for glaucoma patients. A potential target for the development of a neuroprotective approach is the endothelin system of peptides and their receptors. The endothelin (ET) system is composed of three vasoactive peptides (ET-1, ET-2 and ET-3) which are comprised of 21-amino acids. The peptides bind to two G-protein coupled receptors (ETA and ETB receptors) leading to activation of numerous signal transduction pathways. Although originally described for its role in the vasculature, all components of the ET system has been shown to be expressed in multiple tissues and cell types and are responsible for diverse cellular effects. Clinical studies have demonstrated an increase in ET-1 concentrations both in the aqueous humor and plasma of glaucoma patients. A previous study by our lab, using a rodent model of ocular hypertension, showed that endothelin B (ETB) receptor expression is increased when compared to control eyes and contributes to neurodegeneration (Minton et al., 2012). Preliminary data in the current study, using Brown Norway rats, demonstrated that ETA expression is also increased in the IOP elevated eyes, suggesting the possibility that the ETA receptor might also have a degenerative role during ocular hypertension. We hypothesize that the ETA expression increases following IOP elevation and contributes to the neurodegeneration of retinal ganglion cells and their axons. To test this hypothesis we employed a well-characterized in vivo model of glaucoma as well as multiple cellular and molecular approaches to understand the role of the ETA receptor in glaucomatous degeneration. Our data suggest that overexpression of the ETA receptor promotes cell death in cultured RGCs. Since both ETA and ETB receptors appear to contribute to neurodegeneration, we tested the ability of an FDA approved medication, macitentan, for neuroprotection in the Morrison model of glaucoma in rats and found it to promote RGC survival. Our studies raise the possibility of testing macitentan as a neuroprotective treatment for glaucoma patients.Item Endothelin-1 Mediated Regulation of Extracellular Matrix Collagens- A Role in Pathology of Primary Open Angle Glaucoma(2007-11-01) Rao, Vidhya Ramachandiran; Thomas Yoroi; Neeraj Agarwal; Raghu KrishnamoorthyEndothelin -1 Mediated Regulation of Extracellular Matrix Collagens –A role in Pathology of Primary Open Angle Glaucoma. Vidhya R. Rao, Doctor of Philosophy. (Pharmacology and Neuroscience), November, 2007, 157 pp., 3 tables, 18 figures. Summary. Primary Open Angle Glaucoma (POAG) is a progressive optic neuropathy characterized by loss of retinal ganglion cells, optic nerve degeneration and characteristic extracellular matrix (ECM) remodeling of the optic nerve head. An increase in collagen type I and VI is observed at the level of lamina cribosa (LC), a distinct connective tissue region of optic nerve in POAG subjects. Extensive ECM remodeling with enhanced collagen deposition observed in POAG is consistent with the pathology of fibrosis. Mechanisms contributing to ECM remodeling in POAG is not known. Endothelin-1(ET-1), a potent vaso-active peptide plays a key role in glaucoma pathology. Intra-vitreal administration of ET-1 in animal models results in optic neuropathy, RGC apoptosis, axonal transport block and ONA activation. An upregulation of ET-1 and ETB receptors is observed in glaucomatous LC and animal models of glaucoma and ET-1 mediated detrimental effects in POAG appears to be mediated by ETB receptors. ET-1 initiatives and maintains enhanced collagen synthesis and deposition in various tissues under pathological conditions and is recognized as a potent profibrotic factor. In the present study we hypothesized that ET-1 increases extracellular matrix collagen deposition in lamina cribrosa and this change in ECM contributes to optic nerve fibrosis. We have demonstrated that cells of lamina cribrose (LC) cells, express functional ETA and ETB receptors. ET-1 increases intracellular calcium mobilization via ETA receptors and increases NO release by mechanisms involving both ETA and ETB receptors. Consistent with POAG pathology we have observed an upregulation ETB receptors in LC cells in response to chronic treatment with ET-1. LC cells also express prepro-ET-1, the primary gene transcript of ET-1. We have demonstrated for the first time that ET-1 exerts its profibrotic effects by enhancing collagen type I and type VI mRNA, protein synthesis, deposition and secretion in LC cells. ET-1 enhanced collagen deposition in LC cells appears to involve both ETA and ETB receptors, as both of the receptor antagonist, individually inhibit ET-1 mediated collagen synthesis. We have demonstrated that ET-1 also exerts its profibrotic effects in vivo by enhancing collagen deposition in rat optic nerve head. We have also observed an apparent decrease in ET-1 mediated collagen VI deposition in optic nerve heads of ETB deficient transgenic rats suggesting that ET-1 mediated collagen VI synthesis involves ETB receptor activation. In conclusion, endothlein-1 stimulates collagen synthesis and deposition both in vitro in LC cells as well as in vivo at the level of rat optic nerve head. ET-1 mediated increase in collage synthesis at the level of optic nerve head could render a fibrotic mechanism that contributes to the progression of POAG.Item Endothelin-1-Induced Proliferation of Human Optic Nerve Head Astrocytes Under Hypoxia(2003-11-01) Desai, Devashish; Thomas Yorio; Ganesh Prasanna; Clark, Abbot F.Desai, Devashish, Endothelin-1-Induced Proliferation of Cultured Human Optic Nerve Head Astrocytes under Hypoxia. Master of Science (Biomedical Sciences). Purpose: Optic nerve head astrocytes (ONAs) normally support and protect the axons of retinal ganglion cells exiting the eye. Along with effects related to elevated intraocular pressure (IOP), proliferation and activation of ONAs, known as ‘astrogliosis’, is also thought to contribute to the pathophysiology of glaucoma by distributing axonal transport and preventing axon regeneration. Concentrations of endothelin-1 (ET-1) are elevated in glaucomatous eyes and in animal models for glaucoma. ET-1 injection into the eye causes reduction of ocular blood flow. ET-1 causes a time-dependent proliferation of human ONAs. Tumor necrosis factor-α (TNF-α), a cytokine, which is also elevated in glaucomatous optic nerve head, promotes ET-1 release from ocular cells and could potentially stimulate ET-1 secretion from the ONAs. Hypoxia resulting from ischemia, which is produced by the elevation of IOP or vasospasm in the retinal vasculature, is considered a significant factor contributing to the stress as the glaucomatous optic nerve head. Methods: Concentrations of ET-1 secreted by hONAs into cell culture media after hypoxia and TNF-α treatment was measured using an enzyme-linked immunosorbent assay (ELISA). Proliferation of hONAs was measured using a proliferation assay (formazan assay), performed at the end of various time periods of incubation with TNPα and ET-1 under normoxia or hypoxia. The involvement of mitogen activated protein kinase (MAPK) in hONA proliferation was examined using MAPK inhibitors and Western blot analyses. Results: Cell culture media collected from hONAs after 24-hour hypoxia with concurrent TNF-α treatment showed a 500% increase in the irET-1. Under normoxia, both TNF-α and ET-1 caused moderate proliferation of hONAs. Under hypoxia, TNF-α-induced proliferation was greatly increased. Conclusion: Hypoxia augments TNF-a and ET-1 growth of optic nerve head astrocytes, by way of increasing ET-1 synthesis and release as well as mitogenesis. Therefore reactive ONAs could be the common denominator underlying optic nerve damage in glaucoma since their localization makes them susceptible to mechanistic and ischemic influences in addition to influences of ET-1 and TNF-α. Keywords: astrocyte; endothelin-1; tumor-necrosis factor-α; hypoxia; proliferation; astrogliosis; glaucoma; optic nerveItem Endothelin-1-Induced Signaling Involved in Extracellular Matrix Remodeling(2006-12-01) He, Shaoqing; Thomas Yorio; Neeraj Agarwal; Peter KoulenET-1-Induced Signaling in ECM Remodeling in Astrocytes. Shaoqing He, Department of Pharmacology & Neuroscience, University of North Texas Health Science Center, Fort Worth, TX 76107. ET-1 levels are elevated under pathophysiological conditions, including glaucoma, however, ET-1’s ocular functions are not fully documented. Therefore, ET-1-induced signaling and ECM remodeling in astrocytes and at the optic nerve head were determined in this study. Three signaling pathways, including ERK1/2, PKC, and P13 kinase, were involved in ET-1-medicated cell proliferation of U373MG astrocytoma cells. Blocking one of these pathways completely abolished cell proliferation. It appeared that ERK1/2 activation was involved, but was independent of PKC and P13 kinase activation by ET-1. It was also determined that the ETB receptor was the dominant receptor involved in ERK1/2 phosphorylation and cell proliferation. In addition, ERK1/2 phosphorylation was not transactivated by the EGF receptor by ET-1. The studies also indicated that there was no activation of c/nPKC, although PKC was involved in cell proliferation. In U373MG astrocytoma cells, MAPK-ERK, PKC and P13K pathways appear to exert their roles in parallel without a direct, apparent “cross-talk”. Based on the signaling pathways obtained from U373MG astrocytoma cells, the regulation of MMPs/TIMPs and fibronectin in ET-1-activated human optic nerve head astroctyes (hONAs) was also determined. ET-1 not only induced rapid phosphorylation of ERK1/2 and PKC βI/ βII/δ but also increased the activity of MMP-2 and the expression of TIMP=1 and 2. The activity of MMP-2 was enhanced in the presence of inhibitors of MAPK or PKC in hONAs, whereas the expression of TIMP-1 and 2 was abolished. ET-1 increased the soluble fibronectin (FN) expression as well as FN matrix formation, however, the expression and deposition of FN were MAPK- and PKC-independent, whereas expression and activity of MMps and TIMPs were MAPK- and PKC-dependent. Therefore, ET-1 shifted the balance of MMPs/TIMPs and substrates that altered the ECM composition and subsequently let to ECM remodeling in activated hONA cells. ET-1’s effects on ECM remodeling at the optic nerve head were also examined following intravitreal administration of ET-1 in rats. The increased expression of MMP-9 and collagen VI was detected in both ETB deficient rats and wildtype Wistar rats post ET-1 intravitreal injection for 2 and 14 days, whereas the deposition of FN and collagen IV was unchanged. There was no significant difference in staining of MMP-9 and collagen VI between ETB deficient rats and wildtype Wistar rats. In this study, ECM remodeling was demonstrated in rats injected with ET-1 into the vitreous. Such changes in the ECM seen in the current study provide additional insight into the mechanisms that might explain the glaucomatous changes observed in ET-1-injection or perfusion models. In summary, ET-1 not only activated several signaling pathways in cell proliferation of astrocytes, but also modulated the expression of ECM molecules in vitro and in vivo, indicating that ET-1 plays a regulatory role in ECM remodeling. These effects coupled with observations that ET-1 levels are elevated in glaucoma patients, suggests that ET-1 may be involved in glaucomatous optic neuropathy.Item Ergonomic Efficiency Field Evaluation of the C-03-35 Intraocular Lens Delivery System(2004-05-01) Kajtoch, Michael; Gwirtz, Patricia A.; Bens, Annita V.; Hileman, KendraObjective: The purpose of this post-market clinical investigation is to validate the ergonomic efficiency of the C-03-35 Intraocular Delivery System following cataract removal by phacoemulsification. The C-O-35 Delivery System is a newly approved device developed by Alcon Laboratories, Inc, which allows the delivery of the ACRYSOF Model SA60AS soft acrylic intraocular lens in a sterile, single-use and disposable unit that combines the hand piece plus the ACRYSOF intraocular lens-contained cartridge in an integrated system. This system is somewhat different from the predecessor MONARCH II Delivery System, which requires the surgeon to correctly insert the ACRYSOF intraocular lens into a cartridge and then assemble the cartridge into a reusable hand piece. The C-03-35 Delivery System eliminates these steps; therefore, it should decrease the risk of damage that may occur to the optic or the haptic as well as reducing surgery time. Materials and Methods: This study is an open label ergonomic assessment of the C-03-35 Delivery System that will be completed after the operative visit (performed on one eye only) of 120 patients by up to twelve investigators. The investigators will enroll patients requiring cataract extraction with intraocular lens implantation into the study that meet predetermined inclusion/exclusion criteria. Data Collection and Analysis: Upon concluding the surgical procedure, the investigator will complete a series of Case Report Forms consisting of questions assessing the ergonomic efficiency of the C-03-35 Delivery System. The Case Report Forms will comprise of questions regarding the optic and haptic placement, ease of use, as well as any adverse events that might have occurred. In addition, the investigator will complete an Exit Case Report Form once the patient concludes the study, is discontinued from the study, or if the patient fails to attend the follow-up visits. The required C-03-35 Case Report Form examination schedule is included in Appendix C. The information obtained from the Case Report Forms will then be entered into a clinical database. The safety information will be analyzed by the Biostatics Department by comparing the safety data obtained from the field evaluation to the Federal Food and Drug Administration’s standards called the FDA Grid of Historical Controls. The FDA Historical Grid provides pharmaceutical companies with performance guidelines by which the investigational test article is measured. In the case of intraocular lenses, the FDA Historical Grid provides standards for overall visual acuity (%20/40), best-case visual acuity (%20/40), and adverse events. Only adverse events will be compared to the FDA Historical Grid and analyzed for this field evaluation. The visual acuity parameters of the intraocular lens will not be analyzed in this study, since the C-03-35 Delivery System uses a FDA approved ACRYSOF Model SA60AS lens with established performance. In addition, the ergonomic efficiency questions such as ease of use will be summarized into a table. Although not currently on the protocol of the study, this information may also be further compared against the MONARCH II Delivery System analysis results, since safety as well as ergonomic efficiency data were also collected during that study. The FDA Grid of Historical Controls is included in Appendix D.Item Exploring Trabecular Meshwork Molecular Pathogenic Mechanisms In Primary Open Angle Glaucoma And Glucocorticoid Induced Glaucoma(2016-08-01) Bermudez, Jaclyn Y.; Clark, Abbot F.; Mao, Weiming; Singh, MeharvanIn a normal functioning eye, the aqueous humor, a fluid secreted by the ciliary body, drains through the trabecular meshwork (TM), a multilayered tissue in the anterior segment of the eye. The TM is the initial site of damage in glaucoma. Damaged TM results in higher aqueous humor outflow resistance and causes elevated IOP, the latter of which leads to optic nerve damage. Numerous clinical studies have shown that lowering IOP can prevent neuronal damage and slow/stop the progression of the disease. In the glaucomatous TM (GTM), there is excessive extracellular matrix protein deposition, cytoskeletal changes and altered cell function. The transforming growth factor β (TGFβ) pathway is activated by TGFβ2 which has been found to be more abundant in the GTM. Additionally, formation of cross-linked actin networks (CLANs) in the GTM is increased compared to non-glaucoma TM. Primary open angle glaucoma (POAG), glucocorticoid-induced glaucoma (GIG) and glucocorticoid-induced ocular hypertension (GCOHT), share similar pathophysiologies. GC-OHT differs from POAG in that about 40% of the population develops GC-OHT after topical treatment with glucocorticoids however, the mechanism that differentiates steroid responders from non-responders is unknown. In our studies we have explored trabecular meshwork molecular pathogenic mechanisms that are responsible for the disease pathology. We have studied epigenetics as a regulatory mechanism for increasing TGFβ2 expression. We have also used proteomics to determine proteins that are associated with CLANs. Lastly, we studied genes that are differentially expressed in glucocorticoid responders versus non-responders in our bovine model of GC-OHT. Overall, our research has enhanced our understanding of the TM and the molecular mechanisms that play a role in glaucoma. We hope to use this information to find new disease modifying therapies.Item Extracellular PACE4 is increased following transient oxygen glucose deprivation in Optic Nerve Astrocytes(2008-05-01) Fuller, John Anthony; Wordinger, Robert J.; Clark, Abbot F.; Krishnamoorthy, Raghu R.Fuller, John Anthony Extracellular PACE4 is increased following transient oxygen glucose deprivation in Optic Nerve Astrocytes. Doctor of Philosophy (Biomedical Sciences), May, 2008, 140 pp., 2 tables, 25 illustrations, bibliography, 218 titles. Primary Open Angle Glaucoma (POAG) is a family of heterogeneous optic neuropathies characterized by progressive retinal ganglion cell (RGC) death that leads to peripheral vision loss and eventually blindness. Various risk factors are associated with glaucoma, however the molecular mechanisms leading to RGC cell death remain unknown. The optic nerve serves as the conduit for the transmission of retinal ganglion action potentials to the brain. The cells that compromise the optic nerve form a scaffold that forms a physical support for the RGC axons. One cell type found throughout the optic nerve and associated with the RGC axon is the optic nerve astrocyte (ONA). Astrocytes are a predominant cell throughout the CNS and are believed to play crucial roles in metabolic, growth factor, and structural support, and respond to protect neurons during injury. The neuronal-glial interface in the optic nerve is poorly understood and believed to plan an important role in POAG pathophysiology, as unmyelenated RGC axons have direct contact with astrocyte processes. IN this study, the subtilisin-like Proprotein Convertases, (SPC) a family of proteases responsible for cleaving a wide variety of protein substrates, were examined in the retina and optic nerve head. PACE4, an SPC found to be secreted and active in the extracellular matrix was found to be highly expressed in the optic nerve, and colocalized to Mϋller cells in the retina and astrocytes in the optic nerve. Exposure of primary optic nerve astrocytes to oxygen-glucose deprivation (OGD) induces an increase in PACE4 mRNA. Furthermore, protein levels of extracellular, processed PACE4 increase following transient ODG, whereas the pro form of the molecule is degraded, and is believed to be chaperoned by the cleaved cysteine rich domain, a product found at high levels in the optic nerve in situ and the ONA in vitro. Due to the extracellular activity of PACE4, we hypothesized that it may regulate the bioactivity of TGF-β2, a growth factor believed to be involved in glaucoma-associated ONH remodeling by inducing the production of extracellular matrix (ECM). When PACE4 is inhibited via siRNA-mediated knockdown, as well as extracellular inactivation, TGF-β2 levels decrease. In addition, fibronectin, a major component of the ECM, is decreased. Furthermore, there is an increase in latent TGF-β2 secreted from the cell. It is therefore possible that PACE4 plays an active role in extracellular growth factor maturation, and may be a central mediator for growth factor bioactivity in the glaucomatous ONA.Item Function of Differentially Expressed Intracellular Calcium Channels in Retinal Neurons(2008-05-01) Nixon, Everett Sheldon; Peter Koulen; Raghu Krishnamoorthy; Rong MaNixon, Everett, Function of differentially expressed intracellular calcium channels in retinal neurons. Doctor of Philosophy (Pharmacology and Neuroscience), May, 2008, pp154, 17 illustrations. The retina, a specialized part of the central nervous system (CNS) is the innermost layer of the eye responsible for capturing light and converting the light response into a signal that can be transmitted through the optic nerve and onto the brain for interpretation. The ability of the retina to perceive light is dependent on its sensory neurons and the neural circuitry present that initiate the primary stage of processing the image being visualized, which then transmits an electrical signal down the optic nerve to the brain for processing and ultimately visual perception. In the vertical pathway of the visual process that involves the photoreceptor cells, bipolar cells and the ganglion cells, glutamate is the main excitatory neurotransmitter. Communication between these cells is dependent upon the release of glutamate into the synaptic region within both the outer plexiform layer and inner plexiform layer, a process that is Ca2+ regulated. In neurons, Ca2+ regulates a plethora of processes such as gene expression, cell death, synaptic plasticity and neurotransmitter release since it serves as a critical intracellular messenger. In view of the involvement of Ca2+ in a variety of physiological processes, it is essential for the intracellular Ca2+ concentration to be tightly regulated within neuronal cell. Regulation of Ca2+ signaling within retinal neurons can occur via inositol 1,4,5-triphosphate (IP3) receptors (IP3Rs) and ryanodine receptors (RyRs). These receptors are involved in the release of Ca2+ from the intracellular stores such as the endoplasmic reticulum (ER) into the cytosol. IP3Rs and RyRs contribute substantially to cytosolic free Ca2+ concentration transients and thereby play an important role in neuronal function. The purpose of the study was to determine the role of mGluRs, IP3Rs and RyRs in increasing intracellular Ca2+ levels in retinal neurons as related to signaling and neurotransmitter release. The present study provides experimental evidence for the following mechanisms: -Activation of mGluR8 in photoreceptor cells reduced cytosolic Ca2+ concentration by inhibition of the voltage gated Ca2+ channels on the plasma membrane. –The distribution of IP3R and RyR isoforms was associated with cytosolic Ca2+ transients and the IP3R induced transients occurs by activation of group I mGluRs. –In rod bipolar cells, the main increase in cytosolic Ca2+ concentrations during depolarization is due to Ca2+ release from internal stores via activation of RyR. The results of the present study contribute to the understanding of intracellular Ca2+ signaling in retinal neurons and Ca2+ signaling mechanisms. This is of relevance for identifying mechanisms controlling neurotransmitter release and possible pharmacological targets in neurodegenerative retinal diseases characterized by Ca2+ dyshomeostasis.Item Histamine Induced Changes in Phospholipase C Activity, Calcium Mobilization, and Contractility in Human Ciliary Muscle Cells(1996-06-01) Markwardt, Kerry L.; Michael W. Martin; Thomas Yorio; Eugene QuistMarkwardt, Kerry L., Histamine induced changes in phospholipase C activity, calcium mobilization, and contractility in human ciliary muscle cells. Doctor of Philosophy (Biomedical Sciences), June, 1996. Histamine has long been known to be an important mediator of inflammation and autocoid throughout the body. It has been shown to cause the contraction of many types of smooth muscle. Due to its known presence in many ocular structures and aqueous humor especially during inflammatory states, it was hypothesized that histamine could have an effect on intraocular pressure (IOP). This could occur if histamine triggered events which ultimately lead to contraction of the ciliary muscle, since it is established that contraction of the ciliary muscle affects aqueous humor outflow. Therefore, it was hypothesized in this study, the histamine causes increases in inositol phosphate production and intracellular calcium in human ciliary muscle cells which ultimately leads to contraction. To test this hypothesis, human ciliary muscle (CM) cells were cultured and used in various experiments to determine the effect of histamine on inositol phosphate production, intracellular calcium mobilization, and contractility. This study, for the first time in CM cells, showed that histamine, via an H1 receptor subtype, caused dose dependent increases in both inositol phosphates and intracellular calcium. Furthermore, it was shown that these histamine-induced events ultimately lead to contraction of the CM cells. Combining the results from all our studies, the data indicate that in human CM cells, histamine via an H1 receptor, activates phospholipase C which generates inositol phosphates such as inositol triphosphate (IP3). IP3 binds to an IP3 sensitive receptor on the endoplasmic reticulum causing the initial release of calcium which is sufficient to cause contraction of the CM cells. The intracellular release of calcium is also involved in activating a calcium channel which allows the influx of extracellular calcium into the cell. The results of these studies suggest that histamine could potentially have an IOP lowering effect in the eye due to contraction of the ciliary muscle. Overall, these studies contribute to a better understanding of the effect of histamine on a key IOP regulating tissue in the eye.Item Impact of Intraocular Pressure Maintenance on Sight Preservation(2002-05-01) Ratliff, Marla D.; Susan Brown; Kristine A. Lykens; Claudia S. CogginRatliff, Marla D., Impact of Intraocular Pressure Maintenance on Sight Preservation. Master of Public Health (Health Services Research), May 2002, 35 pp., 3 tables, 1 figure, 40 titles. Purpose: The objective of the study was to review diurnal IOP control and its impact on sight preservation. Methods: This is a retrospective study used in patients with elevated IOP. Patient IOPs were measured every 4 hours over a 24-hour period. Qualified patients provided open label TRAVATAN to dose once a day for 2 weeks. Following 2 weeks of dosing, IOP measurements were taken every 4 hours for 36 hours. Additional IOP measurements were taken at 60 and 84 hours after the last dose of TRAVATAN. Results: The IOP changes from baseline were statistically significant (p≤0.0001) at all time points out to 36 hours. Even without additional dosing, substantial IOP reductions (6mm Hg) were maintained out to 84 hours. Conclusions: Use of TRAVATAN may have less impact on IOP maintenance due to non-compliance and missed doses. This could help prevent glaucomatous loss and preserve sight.Item In Vitro Effect of CNTF, FGF-9, IL-1α on Human Optic Nerve Head Astrocytes(2004-08-01) Tovar-Vidales, Tara; Wordinger, Robert J.; Alvarez-Gonzales, Rafael; Agarwal, NeerajTovar, Tara., In Vitro Effect of CNTF, FGF-9, and IL-1α on Human Optic Nerve Head Astrocytes. Master of Science (Biomedical Sciences), August 2004, 100 pp., 4 tables, 35 illustrations, bibliography, 163 titles. Glaucoma is a leading cause of blindness worldwide. A major risk factor for glaucoma is increased intraocular pressure that leads to pathological changes in the optic nerve head (ONH). Astrocytes within the ONH become activated in glaucoma and may create an environment detrimental to retinal ganglion cell axons. The factors that cause activation of the ONH astrocytes (ONA) are unknown, although there is evidence that CNTF, FGF-9, and IL-1α activate glial cells within the CNS. The purpose of this research was to determine if exogenous CNTF, FGF-9, and/or IL-1α activate human ONH astrocytes.Item Interleukin-1Alpha-Mediated Signaling Mechanisms in the Human Trabecular Meshwork(2000-12-01) Shade, Debra L.; Pang, Iok-Hou; Yorio, Thomas; Dillon, GlennShade, Debra L., Interleukin-1Alpha-Mediated Signaling Mechanisms in the Human Trabecular Meshwork. Doctor of Philosophy (Biomedical Sciences/Pharmacology), December, 2000, 140 pp., 13 tables, 30 figures, references, 156 titles. This research provides important insights into the means by which interleukin-1alpha (IL-1α) regulates TM cell functions and enhances aqueous outflow, thus lowering IOP. The studies reported herein represent the first known characterization of the central role of the AP-1 transcription factor pathway in IL-1α-mediated production of proMMP-3 by TM cells, as well as the first known evidence that IL-1α can also enhance TM phagocytosis. Using these results as a stepping stone, this research has furthermore led to the identification of “AP-1 activators” as a novel compound class which may be useful in the treatment of glaucoma; it also points to the potential for compounds which regulate MEK, p38, and PKCμ activity as additional means of treatment. Based on these results, it is postulated that such compounds would be expected to lower IOP via upregulations of MMP production, followed by ECM degradation, and potentially, enhanced clearance of degraded ECM via phagocytosis.Item Involvement of Caspase-7 in Photoreceptor and Retinal Ganglion Cell Death(2014-08-01) Choudhury, Shreyasi; Pang, Iok-Hou; Wordinger, Robert J.; Krishnamoorthy, Raghu R.Apoptosis has been implicated in retinal cell death during both retinal differentiation and degeneration. In diseases such as retinitis pigmentosa, glaucoma, age-related macular degeneration, diabetic retinopathy and traumatic optic neuropathy, retinal cell apoptosis plays an important role. Caspases, a family of cysteine proteases, are major players of apoptosis. Thus, one obvious target for modulating apoptosis is the caspase family of proteins. The role of initiator caspases (caspase-1, -2, -8, -9) and effector caspases (caspase-3, -6) in retinal neuronal apoptosis has been studied previously. But the role of a unique effector caspase, caspase-7, has never been studied before. The purpose of this study was to investigate the role of caspase-7 in retinal neuronal cell apoptosis, especially in photoreceptor and retinal ganglion cell (RGC) death. We used the T17M RHO mouse, an animal model for Autosomal Dominant Retinitis Pigmentosa, to study photoreceptor cell apoptosis, and evaluate the role of caspase-7 in a corresponding caspase-7 knockout mouse. Our results show that morphological (evaluated by spectral-domain optical coherence tomography (SD-OCT) and histology) and functional (by electroretinography (ERG)) degenerations in the photo-receptor cells of the T17M RHO mouse are significantly protected by knocking out caspase-7. We further discovered that caspase-7 inhibition reprograms the unfolded-protein response and reduces JNK-induced photoreceptor cell death. To assess the role of caspase-7 in RGC apoptosis, we used the mouse optic nerve crush-induced RGC death as a study model. We found that the insult activates caspase‐7 in RGCs in a time-dependent manner, concomitant with loss of the cells. We also observed the activation of calpain-1, an upstream activator of caspase-7 and the hydrolysis of caspase-7 specific substrates, confirming the involvement of caspase-7. Most importantly, in caspase--‐7 knockout mice, significantly more RGCs survive the optic nerve injury when compared to injured wild type mice as assessed morphologically (immunohistochemistry and SD-OCT) and functionally (ERG) throughout the 28-day post crush study period. Altogether, our findings indicate that caspase-7 appears to play a critical role in photoreceptor and RGC death and inhibition of caspase-7 activity may be a novel therapeutic strategy for retinal degenerative diseases.Item Mechanisms of Photoreceptor Cell Apoptosis(2000-05-01) Crawford, Matthew John; Neeraj Agarwal; Victoria Rudick; Raghu KrishnamoorthyCrawford, Matthew John, Mechanisms of Photoreceptor Cell Apoptosis. Doctor of Philosophy (Biomedical Sciences), May 2000; 168 pp; 3 tables; 23 figures; bibliography, 282 titles. Photoreceptor cell death mediated by programmed cell death pathways is responsible for many disease states of the retina, which result in vision loss. Examples of this include retinal dystrophies and age-related macular degeneration. Correspondingly, the understanding of programmed cell death, or apoptosis, in these cells is important in the formulation of preventative and treatment options. The goals of this dissertation are to characterize a suitable in vitro photoreceptor cell model and explore the molecular mechanisms resulting in apoptotic cell death secondary to oxidative cell death paradigm. Means of interrupting the cell death process were also investigated. An immortalized clonal mouse retinal cell line was shown to express photoreceptor-specific genes and proteins by RT-PCR amplification, Western blot analysis, and immunocytochemical localization. Exposing these cultured cells to visible light resulted in oxidative stress, as exhibited by elevated malonyldialdehyde and reduced gluthathione levels, as well light exposure-dependent apoptosis was shown using multiple techniques which identified fragmentation of chromosomal DNA, a key finding in the apoptotic cell death process. Molecular regulators of apoptotic cell death, including bcl-2 family proto-oncogenes and the nuclear transcription factor NF-kB, were found to be important in oxidative stress-induced pathogenesis of 661 W photoreceptor cells. mRNA and protein levels of the anti-apoptotic proto-oncogene bcl-2 declined following oxidative stress disturbing the balance proto-oncogene regulators and initiating the apoptotic pathway. The nuclear transcription factor NF-kB was found to be constitutionally expressed in the photoreceptor cells with its down-regulation during apoptosis. Permanent transfection of the photoreceptor cells with bcl-2 gene imparted protection from apoptosis and sustained NF-kB levels. The results presented in this dissertation help define the molecular mechanisms which occur during apoptosis of photoreceptor cells. Photo-oxidative stress results in programmed cell death mediated through changes in NF-kB binding activity and bcl-2 family genes. The involvement of caspase-1 in the degradation of NF-kB and the execution of apoptosis is also demonstrated. Over-expression of the proto-oncogene bcl-2 interrupts the apoptotic events, protecting against down-modulation of NF-kb binding activity and cell death. Our proposed mechanism for apoptosis in photoreceptor cells provides several points at which targeted gene expression (bcl-2 or NF-kB), or pharmaceuticals (anti-oxidants, caspase inhibitors, or calcium channel blockers) may prevent apoptotic cell death.