Browsing by Subject "Sense Organs"
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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 and Activity of Endothelin Converting Enzyme-1 in Human Non-Pigmented Ciliary Epithelial Cells(1999-01-01) Finkley, Alvin; Thomas Yorio; S. Dan Dimitrijevich; Victoria J. RudickFinkley, Alvin, Characterization and Activity of Endothelin Converting Enzyme-1 in Human Non-Pigmented Ciliary Epithelial Cells. Master of Science (Biomedical Sciences). Endothelins (ETs) are potent vasoactive peptides, that are present in many ocular tissues including the ciliary epithelium where active ET-1 is produced from the precursor Big ET-1 by a membrane-bound metalloprotease, endothelin-converting enzyme (ECE). Although the role of ocular ET’s are uncertain, ETs have been shown to lower the intraocular pressure. In the current study, ET-1 and Big-ET-1 were detected in SV-40 transformed human ciliary epithelial (HNPE) cells by immunofluorescence suggesting the presence of ECE activity. The presence of ECE was confirmed by Western blotting using polyclonal antibodies against ECE-1 which detected a 124 KDa protein in the membrane fraction and not in the cytosol. Further characterization of the enzymatic activity of ECE (conversion of Big ET-1 to ET-1) was performed using a novel assay involving 121I-Big ET-1 (substrate; 2fmloe) and polyclonal antibodies specific for Big ET-1. Mean ECE-1 activity (expressed as the ratio of 121^1-ET-1 produced to the total 125^I-Big ET-1 incubated X 100) was measured and corresponded to: 26% (0.5 3±0.02 fmole, 1 hr), 63% (1.26±0.07 fmole, 3hr) and 66% (1.33±0.11 fmole, 24 hr) compared to blank controls at 13% (0.25±0.03 fmole). Thiorphan (2mM), an inhibitor of ECE, abolished ECE-1 activity. These results suggest that ECE-1 is localized in HNPE cells and is essential for the production of ET-1. The physiological importance of the proteolytic processing by ECE-1 in ocular tissue may reflect on how ET regulates intraocular pressure. Key Words: endothelin converting enzyme-1; endothelin-1; Big endothelin-1; ciliary epithelium; aqueous humor dynamics; intraocular pressure, Western blotting, ECE-1Item 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 Development of a Subjective Comfort Questionnaire for Hydrogel Contact Lens Wearers(2003-12-01) Hays, Brian Hunter; Sheedlo, Harold; Stein, Jerry; Atiles, LuisThroughout this paper it is written that I would complete this study in its entirety. Due to time constraints and the length of this study, it was planned from the beginning that I would only accomplish the beginning phases, phases one and two. The reason why this paper was written this way, planning the complete research plan, is to aid the individuals that will finish this study in its entirety. I. Purpose. The purpose of this project is to develop a questionnaire that can be used as a tool to measure the subjective symptoms of ocular comfort or discomfort reported by soft contact lens wearers. After a questionnaire has been developed, it will be tested to determine its reliability and validity in capturing the ocular sensations experienced by hydrogel contact lens wearers. II. Overview of the study. The research for the study will be conducted in six phases while pursuing three specific aims. The phases will consist of: A. reviewing literature in the form of reported soft contact lens symptomatology and interviewing skill improvement, compiling and B. examining previously developed questionnaires, developing open-ended interview questions and collecting data from the field. C. developing preliminary questionnaire items based on data gained during the first phase. D. administering the preliminary questionnaire to receive feedback from volunteers with regards to each item’s appropriateness, and tallying the volunteer's responses to graphically analyze each item’s answer distribution. E. refining each item based on the data obtained during the third phase to create a revised draft of the questionnaire. F. determining if the revised draft conveyed and captured the ideas reported by the volunteers by receiving feedback after it is administered. G. demonstrating reliability and validity by psychometrically validating the questionnaire. During each administrational phase of the study (phases three, five and six) two groups of volunteers will be used to gain a broader spectrum of data. Each group will be composed of a sub-set of previously interviewed volunteers and a sub-set of new volunteers. Before any information is obtained, a confidentiality agreement will be discussed with each volunteer. All volunteers will be given a simple, easy to read informed consent form and a randomly assigned number.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 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 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 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 Modulation of Manganese Superoxide Dismutase by 17-Beta Estradiol(2008-05-01) Gottipati, Srinivas; Thomas YorioGottipati, Srinivas. Modulation of manganese superoxide dismutase activity by 17-beta estradiol. Master of Science (Cell Biology and Genetics), May, 2008. We have previously reported that 17β-Estradiol (17β-E2) can protect human lens epithelial cells against oxidative stress by preserving mitochondrial function, acting as a positive regulator of the MAPK signal transduction pathway. While pERK plays a significant role in stabilizing the inner mitochondrial membrane to maintain the mitochondrial membrane potential during oxidative stress, the protective mechanisms activated by 17β-E2 are probably multifactorial acting via both genomic and non genomic pathways. This study examined the effects of 17β-E2 on the expression and activity of MnSOD, which is present exclusively in the mitochondria, as a possible mechanism by which it affords protection against oxidative stress. Our results demonstrate that 17β-E2 rapidly increases the activity of MnSOD in a time dependent manner. This augmentation of activity of MnSOD by 17β-E2 is seen in the absence of a corresponding increase in the mRNA and protein expression, thereby which estrogens protect the cells against oxidative stress will help us in developing estrogens to be useful therapies for the prevention of cataract in postmenopausal women and non feminizing estrogens may provide similar protection in men.Item Molecular Mechanisms of and Potential Therapies for Oxidative Damage to the Retinal Pigment Epithelium(2007-09-01) Wang, Zhaohui; Roque, Rouel S.; Wordinger, Robert J.; Das, HridayWang, Zhaohui, Molecular Mechanisms of and Potential Therapies for Oxidative Damage to the Retinal Pigment Epithelium. Doctor of Philosophy (Biomedical Sciences), September 2007, 161 pages, 34 illustrations, bibliography, 119 titles. Age-related macular degeneration (AMD), the most common cause of irreversible vision loss in the elderly, results mainly from degeneration of the retinal pigment epithelium (RPE) and loss of photoreceptor cells. Oxidative stress has been acknowledged as a leading cause of RPE degeneration and concomitant photoreceptor cell loss, but the exact role of reactive oxygen species (ROS) in RPE cell death remains to be established. Moreover, while mitogen-activated protein kinases (MAPKs) are suggested to be involved in RPE degeneration induced by oxidative stress, the precise functions and molecular mechanisms of MAPKs in RPE degeneration remain elusive. In spite of the numerous therapeutic modalities proposed for AMD, the treatment of AMD remains unsatisfactory. Recent studies suggesting stem cells as a potential source for trophic factors in damaged murine hearts led us to investigate a possible role for stem/progenitor cell-derived factors in protecting RPE cells from oxidative damage. Furthermore, human retinal progenitor cells promote RPE cell survival by regulating p42/p44 MAPK activity. When exposed to oxidative stress produced by glucose oxidase/glucose, human RPE cells exhibited membrane blebbing and cytoskeleton remodeling in the early phase of oxidative stress. Prolonged exposure to oxidative stress induced mitochondrial membrane potential depolarization, cell death and DNA condensation, but not DNA fragmentation. Furthermore, both p38 MAPK and p42/p44 MAPK were activated by oxidative injury. P38 MAPK inhibitor, but not p38 MAPK siRNA, inhibited RPE cell death induced by oxidative stress. Overexpression of constitutively active MEK1 inhibited RPE cell death exposed to oxidative damage. In contrast, interfering p42/p44 MAPK expression accelerated oxidative-stress induced RPE cell death. To investigate the effects of human retinal progenitor cells (hRPC) on RPE cells, we isolated and expanded hRPC in vitro. The hRPCs expressed markers of neuronal and retinal progenitor cells, and were capable of differentiating into neuronal phenotype in defined medium. In the presence of 10% fetal bovine serum, hPRC suppressed RPE cell death induced by oxidative damage. Furthermore, conditioned medium of hRPC induced activation of p42/p44 MAPK, and the protective effect of hRPC and conditioned medium was suppressed by p42/p44 MAPK inhibitor. Our studies increase our understanding of the molecular mechanisms that could be employed to rescue RPE cells from degeneration and support the therapeutic potential of retinal progenitor cells. It will provide further insight into molecular mechanisms of AMD and establish a foundation for the long-term prevention and treatment of AMDItem Mutation in myocilin affect it's processing and secretion in the trabedular meshwork cell(2003-05-01) Jacobson, Nasreen; Robert Wordinger; Richard Easom; Neeraj AgarwalJacobson, Nasreen, Mutations in myocilin affect it secretion and processing in the cell. Doctor of Philosophy (Cell Biology and Ginetics), May 2003, 157 pp., 6 tables, 46 illustrations, 17 movies. Introduction. Myocilin is the protein product of the glaucoma gene MYOC whose function is unknown. Structural predictions of the protein indicate myocilin is secreted. This study uses several techniques to determine whether myocilin is synthesized and processed through the secretory pathway. Methods. Agents known to disrupt the secretory pathway at specific organelles will be used to examine the effect on myocilin secretion. Also, constructs for chimeric myocilin and fluorescent proteins (myoc.504DsRED and myoc.504EGFP) will be used in conjunction with EGFP directed to specific organelles to determine colocalization of myocilin in the cell. The disruption of wild type and disease-causing mutants (myocQ368X.DsRED, myocG364V.504DsRED and myocY437H.504DsRED) of myocilin will be compared. Then in vivo studies will be used to try to determine if myocilin is associated with increased intraocular pressure (IOP). Results. Myocilin appears as a doublet on SDS-PAGE western blots when probed with anti-myocilin antibody (AB129). Treatment of cells with tunicamycin prevents secretion of the upper band of the myocilin doublet, but not secretion of the lower band. Brefelden A prevents secretion of both bands of the myocilin doublet indicating that both bands are processed in the Golgi. Monensin treatment indicates there is no post-Golgi processing of myocilin prior to secretion. Colocalization of fluorescent myocilin with cellular organelles tagged with EGFP indicated that myocilin travels through the ER, Golgi and is secreted from the cell. Disease-causing mutations in myocilin are not secreted. The Q368X associates with wild type myocilin and appears to be degraded. The G364V and Y437H mutants can apparently be retained in the ER and also are closely associated with peroxisomes. Experiments designed to determine if myocilin can be correlated with increased IOP suggest an association of myocilin with increased IOP in an ex vivo human anterior segment perfusion system, but in vivo experiments gave inconclusive results. Conclusions. Myocilin is a secreted glycoprotein in the TM. Glaucomatous mutations in myocilin cause non-secretion. TM cells handle different myocilin mutations differently.Item Myocilin Regulation by Brain-Derived Neurotophic Factor and Transforming Growth Factor-Beta2 in Normal and Glaucomatous Human Trabecular Meshwork Cells(2003-05-01) Liu, Xiaochun; Wordinger, Robert J.; Rudick, Victoria; Clark, Abbot F.Liu, Xiaochun, Myocilin Regulation by Brain-derived Neurotrophic Factor and Transforming Growth Factor beta2 in Normal and Glaucomatous Human Trabecular Meshwork. Doctor of Philosophy (Biomedical Sciences), May 2003, 119 pp., 3 tables, 26 illustrations, bibliography, 188 titles. Glaucoma, of which primary open-angle glaucoma (POAG) is the most common form, is the second leading cause of irreversible blindness in the world. Ocular hypertension is an important risk factor in the development of POAG. The human trabecular meshwork (HTM) is the major regulation site for aqueous humor outflow thus controlling intraocular pressure. In POAG, there are specific morphological and pathological changes in the HTM, including an increase in extracellular matrix components and a decrease in the number of HTM cells. Myocilin (also known as GLC1A or TIGR) is associated with hypertensive POAG by both genetic linkage analysis and glucocorticoid induction studies. Brain-derived neurotrophic factor (BDNF) and transforming growth factor-beta isoforms (TGFβ1-3) have been shown to be present both in normal cultured HTM cells and aqueous humor. In addition, biologically active TGFβ2 levels are increased in the aqueous humor of POAG patients. Mechanical stretch, an important factor in HTM during intraocular hypertension, may up-regulate the expression of BDNF in the HTM cells. Therefore, BDNF and TGFβ2 may be modulators of extracellular proteins in response to the hypertensive glaucomatous injury. However, the regulation of myocilin expression by these growth factors in the HTM has not been studied. Moreover, HTM cells may signal each other via paracrine and autocrine pathways involving BDNF and TGFβ2. In this study, HTM cells were isolated and cultured in vitro. Myocilin gene expression and protein secretion by normal and glaucomatous HTM cells were compared. The regulatory effects of BDNF and/or TGFβ2 or myocilin gene expression and protein secretion by normal and glaucomatous HTM cells were also examined, as well as the reciprocal induction between BDNF and TGFβ2 gene expression and protein secretion. The interdependence between BDNF and TGFβ2 in regulating myocilin expression was determined. The results of the study established the regulatory effects of BDNF and TGFβ2 on myocilin expression as well as on each other. It is possible that both BDNF and TGFβ2 interact with each other in response to an increase of intraocular pressure through paracrine/autocrine mechanisms, resulting in differential gene expression of myocilin.