Browsing by Subject "Vision Science"
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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 Alterations in mRNA Levels of Selected Gene Products During Hypoglycemia, Hypoxia, and Ischemia Induced Apoptosis of Cultured Rat Retinal Ganglion Cells(2001-08-01) Vopat, Kelly S.; Agarwal, Neeraj; Wordinger, Robert J.; Pang, Iok-HouVopat, K., Alterations in mRNA Levels of Selected Gene Products during Hypoglycemia, Hypoxia, and Ischemia Induced Apoptosis of Cultured Rat Retinal Ganglion Cells. Master of Science (Biomedical Science), August 2001. 54 pp., 2 tables, 10 illustrations, bibliography, 105 titles. In order to explore the mechanisms involved in the signal transduction pathways of ischemia-induced apoptosis of RGCs in glaucoma, an in vitro ischmia model of transformed rat retinal ganglion cells (RGC-5) was utilized. RGC-5 cells were exposed to hypoglycemia, hypoxia, and ischemia for six hours. Hypoxia and ischemia resulted in apoptosis of RGC-5 cells as determined by TUNEL assay. The bax mRNA levels increased significantly in cells exposed to hypoxia. The mRNA levels of hemoxygenase, c-fos HSP 70, and BDNF showed a trend of increase in both the hypoxic and ischemic conditions. These results demonstrate that retinal ganglion cells undergo apoptosis in hypoxic conditions likely via an increase in bax/bcl-2. The up-regulation of BDNF and some stress proteins may be part of a cellular rescue effort trying to overcome the damage created by hypoxic and ischemic stresses.Item Cellular Mechanisms in the Ocular Actions of Endothelin(1996-12-01) White, Karen A.; Yorio, Thomas; Pang, Iok-Hou; Dobbs, RichardWhite, Karen A., Cellular Mechanisms in the Ocular Actions of Endothelin. Doctor of Philosophy (Biomedical Sciences/Pharmacology), December, 1996, 151 pp., 25 tables, 23 figures, references, 111 titles. Endothelins are a family of regulatory peptides which could have important implications in this regulation of aqueous humor outflow and intraocular pressure (IOP). The objectives of this dissertation were to investigate the cellular mechanism of endothelin (ET) receptor interactions in ocular tissues focusing on their effect on second messengers such as phospholipase C (PLC) and calcium, and their interactions with phospholipase A2 (PLA2) in ciliary muscle cells. The hypothesis was that in human ciliary muscle (HCM) cells, endothelin-1 (ET-1), via the ETA receptor and a pertussis toxin sensitive G-protein, activates PLC, which in turn stimulates calcium mobilization. Independent of this pathway, ET-1 also activates PLA2 and increases the release of prostaglandins. These two pathways provide a cellular second messenger balance that influences ciliary smooth muscle contraction. The current study demonstrated that ET-1 and endothelin-2 (ET-2) stimulate calcium mobilization in HCM cells via an ETA receptor subtype. It appears that the increase in intracellular calcium ([Ca2+]i) is the result of ET coupled to PLC via a pertussis toxin sensitive G-protein. A biphasic calcium response is elicited with ET stimulation consisting of a transient increase in [Ca2+]I which appears to be primarily due to release of intracellular stores, followed by a lower sustained phase which appears to be dependent on the influx of extracellular calcium. Endothelin-1 also appears to stimulate an increase in prostaglandin E2 (PGE2) formation through activation of PLA2. Furthermore, it appears that the effects of ET-1 on PLC and calcium are independent of the ET-1 effects on PGE2 production, such that the ET-1 induced increase in [Ca2+]I are coupled to the PLC signaling pathway, whereas increase in PGE2 production appears to be the result of an ETA receptor coupled PLA2. Whether there are different subtypes of ETA receptors or the receptor is coupled through different G-proteins is uncertain. Endothelin-1 and Big ET-1 immunoreactivity was also observed in both HCM and human nonpigmented ciliary epithelial (HNPE) cells. This is the first time that ET-1 and Big ET-1 immunoreactivity has been detected in the HCM cells, suggesting that these cells have the capability to synthesize both peptides. Furthermore, the increase in ET-1 and Big ET-1 immunoreactivity upon stimulation with TNF-α suggests that cytokines may be important regulators of ET synthesis and release. The findings of this research aid in the understanding of the mechanism of action whereby ETs regulate aqueous humor dynamics and IOP. Through a better understanding of the cellular actions of ET, insight is gained into the development of new ocular selective agents acting at the ET receptor.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 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 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-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 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 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 Hooper Visual Organization Test (VOT) as a Predictor of Driving Status of Individuals with Dementia(2004-04-01) Budd, Margaret Anne; Doug A. Main; Susan FranksHooper Visual Organization Test (VOT) (Hooper, 1983) items were correlated with driving status of geriatric individuals with dementia to help screen for high-risk drivers. A retrospective review of 87 medical chart on patients, 60-91 years, who underwent a neurocognitive evaluation at the University of North Texas Health Science Center in Fort Worth, Texas, with a complete VOT, driving status, dementia diagnosis, and demographic descriptors (age, gender, marital status) were selected for analysis. Of the 55.2% participants who reported a current driving status, VOT scores ranged: 20.8% normal, 43.8% mildly impaired, 31.3% moderately impaired, and 4.2% severely impaired. An item analysis was followed by direct logistic regression analysis which correctly predicted 85% of the drivers and 74% of the nondrivers with an overall success rate of 80.5% (p=.001). The Wald criterion selected 4 VOT items as reliably predicting driving status: items 6 (hammer), 19 (teapot/pitcher), 22 (mouse), and 25 (block). Models run with gender and/or marital status was not reliably different. These 4 items may add to a brief screening test to identify drivers with dementia potentially at risk. In addition, the large number of current drivers scoring in the impaired range suggests that individuals, their families and others are not intervening with driving behavior, possibly placing the individuals and public at risk.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 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.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 AMD