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Localization and Expression of Et-1 Receptors in the Normal and Glaucomatous Dog Eye


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LOCALIZATION AND EXPR ESSION OF ET-1 RECEPTORS IN THE NORMAL AND GLAUCOMATOUS DOG EYE By MARIA E. KLLBERG A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2003

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Copyright 2003 By Maria E. Kllberg

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Dedicated to my family and my frie nds who made this work come true.

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ACKNOWLEDGMENTS I would like to thank everyone who helped and supported my scientific work and my graduate education, especially those listed below. Dr. Dennis E. Brooks invited me to Florida and made the dream of my life come true. Thanks to Dr Brooks I will be able to dedicate my professional life to ophthalmology as a researcher and clinician. I thank him for his support through the years. The precious friendship we have developed will always help me whatever challenges life has to offer in the future. Dr. William W. Dawson guided me through the world of vision research and electrophysiology, which for me was unknown territory. He has inspired me not only by his knowledge but also by his way of questioning axioms and looking for new truths. I am thankful to Dr Dawson and his wife, Judyth, for their concern and warm generosity. Dr. Adrian M. Timmers meant a great deal to me and my project. He opened his laboratory for me and patiently taught and helped me through every necessary step of the molecular biology part of my project. His positive and supportive attitude was extraordinary. I would like to thank Dr. Kirk N. Gelatt for sharing with tireless enthusiasm his tremendous knowledge in research and clinical ophthalmology. I am also grateful for the many times he helped me in his own positive way whenever I needed good advice. iv

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Dr. Don A. Samuelson broadened my view of comparative ophthalmology. He and his laboratory technicians, Mrs. Patricia Lewis and Ms. Mae Chisholm, were very helpful in accomplishing all the challenges in histologic methods that I encountered during my time as a graduate student. I owe Dr. Adolfo G. Garcia many thanks for his contribution to my project. I am grateful to have gotten not only scientific help but also a friendship for life. My dear friend, Dr. Andrs M. Komromy, has been by my side from the very first page of this new chapter of my life. Andrs made the transition easy by always being there for me whenever I needed help or support. Dr. Franck J. Ollivier, dear friend and colleague, deserves lots of thanks for the wild scientific discussions and many laughs. With all my heart I thank my best friend, Kerstin Bergvall, who helped me through many tough times. Ann Ellis gave me many hours of help teaching me electron microscopy. I would like to thank Dr. Stacy E. Andrew, the ophthalmology residents at the University of Florida, and various ophthalmologists in Florida (especially Dr. Salisbury and Dr. Brogdon) for their contributions. Mr. Harold L. Sapp gave valuable technical assistance. Dr. Carol Detrisac was very helpful in reviewing my histology slides. Dr. George Lambrou, CIBA Vision Ophtha, Novartis, provided very generous financial and technical support. v

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I would like to thank Ms. Marinela Capanu and the Interdisciplinary Center for Biotechnology Research at the University of Florida for their help with statistic analysis of the data. Dr. Nancy Szabo at the Analytical Toxicology Core Laboratory at University of Florida has been extremely helpful with analyzing my samples and sharing her expertise. Dr. Thomas Yorio, Dr. Ganesh Prasanna, Dr. Ragu Krishnamoorthy, Dr. Martha Stokely, Christina Hulet, and Santos Narayan at the Health Science Center, Fort Worth, Texas deserve a special thanks for their tremendous support. I owe many thanks to the Office of Research and Graduate Studies at the College of Veterinary Medicine, University of Florida. Associate Dean Charles Courtney III and Mrs. Sally OConnell made it possible for me to become a graduate student and to successfully finish my dissertation. Sally helped me through several storms. Finally I would like to thank my parents for supporting me the whole way through my training and for helping me to realize my dreams. The sometimes overwhelming distance from home was overridden by the closeness of our relationship. vi

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TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES...............................................................................................................x LIST OF FIGURES...........................................................................................................xi ABSTRACT.....................................................................................................................xiii CHAPTER 1 INTRODUCTION........................................................................................................1 Glaucoma......................................................................................................................1 Role of Endothelin in Glaucoma...........................................................................1 Canine Glaucoma..................................................................................................4 Primary narrow-angle glaucoma in American Cocker Spaniels....................6 Primary open-angle glaucoma in the Beagle................................................10 Ophthalmic Vascular Morphology and Physiology...................................................11 Endothelin and Nitric Oxide.......................................................................................16 Synthesis, Secretion and Clearance of Endothelin..............................................16 Nitric Oxide.........................................................................................................19 Endothelin Receptors...........................................................................................20 Endothelin and the Ophthalmic Circulation........................................................24 Endothelin and Aqueous Humor Production and Outflow.........................................26 Purpose of Study.........................................................................................................27 2 MATERIALS AND METHODS...............................................................................28 Study Design...............................................................................................................28 Clinical Examination of Dogs....................................................................................29 Endothelin-1 and Nitric Oxide Levels in Aqueous Humor and Vitreous...................29 Endothelin-1 Analysis.........................................................................................29 Nitric Oxide Analysis..........................................................................................30 Localization of Endothelin Receptors in the Retina and Choroid..............................30 Light Microscopy................................................................................................31 Transmission Electron Microscopy.....................................................................31 Morphology of Retina and Iridocorneal Angle...................................................33 Endothelin Receptor Protein Expression in the Retina and Choroid..........................33 Protein Extraction................................................................................................34 vii

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Endothelin Receptor and Endothelin-1 mRNA Levels in the Retina.........................36 Isolation of Total RNA........................................................................................36 Reverse Transcriptase Polymerase Chain Reaction............................................37 Real Time Polymerase Chain Reaction...............................................................38 Data Analysis..............................................................................................................40 Morphology.........................................................................................................40 Endothelin-1 and Nitric Oxide Levels in Aqueous Humor and Vitreous...........40 Endothelin Receptor Protein Expression in the Retina and Choroid..................40 Endothelin-1 and ET Receptor mRNA Levels in the Retina..............................41 3 RESULTS...................................................................................................................49 Morphology................................................................................................................49 Iridocorneal Angle...............................................................................................49 Retina...................................................................................................................49 Endothelin Receptors in the Retina and Choroid.......................................................50 Evaluation of Endothelin Antibodies using Light Microscopy...........................50 Evaluation of Endothelin Antibodies using Transmission Electron Microscopy......................................................................................................50 Localization of Endothelin Receptors using Transmission Electron Microscopy......................................................................................................51 ETA receptor................................................................................................51 ETB receptor................................................................................................51 Endothelin-1 Levels in Aqueous Humor, Vitreous and Retina..................................51 Analysis of Aqueous Humor...............................................................................51 Analysis of Vitreous............................................................................................52 Endothelin-1 mRNA Levels in the Retina...........................................................52 Correlation of Retinal Degeneration and ET-1 Levels in Aqueous Humor and Vitreous..................................................................................................................53 Nitric Oxide Levels in Aqueous Humor and Vitreous...............................................53 Analysis of Aqueous Humor...............................................................................53 Analysis of Vitreous............................................................................................54 Endothelin Receptor Protein Expression in the Retina and Choroid..........................54 ETA Receptors in the Retina................................................................................54 ETB Receptors in the Retina................................................................................54 ETA and ETB Receptors in the Choroid...............................................................55 Endothelin Receptor mRNA Levels in the Retina......................................................55 Evaluation of the Real Time PCR Reaction........................................................55 Tissue...................................................................................................................56 ETA Receptor mRNA in the Retina.....................................................................57 ETB Receptor mRNA in the Retina.....................................................................58 Summary of Results....................................................................................................58 4 DISCUSSION.............................................................................................................92 Morphology................................................................................................................92 Endothelin-1 in Aqueous Humor and Vitreous..........................................................93 viii

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Sources of Endothelin-1......................................................................................94 Effect of Steroid Treatment.................................................................................97 Cellular Response to Endothelin-1 Treatment....................................................97 Extravascular Endothelin Receptor Binding Sites..............................................99 Pericytes and Endothelin Receptor Expression.................................................100 Yield of Protein and RNA.................................................................................101 Nitric Oxide in Aqueous Humor and Vitreous.........................................................101 The Cocker Spaniel versus Other Breeds.................................................................103 Implications for the Canine Eye...............................................................................104 LIST OF REFERENCES.................................................................................................108 BIOGRAPHICAL SKETCH...........................................................................................119 ix

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LIST OF TABLES Table page 2-1: Normal dogs in study...............................................................................................42 2-2: Glaucoma dogs in study...........................................................................................43 2-3: Analyses on normal dogs.........................................................................................44 2-4: Analyses on glaucomatous dogs..............................................................................45 2-5: Primers and fluorogenic probes................................................................................46 2-6: Controls for the Real Time PCR..............................................................................47 3-1: Ranking of degree of degeneration in the glaucomatous retinas..............................59 3-2: Endothelin-1 levels in pg/mL for normal dogs........................................................60 3-3: Endothelin-1 levels in pg/mL for glaucomatous dogs.............................................61 3-4: Mean ratios of ET-1/ 18S mRNA in arbitrary units for normal and glaucomatous retinal samples..........................................................................................................62 3-5: Nitrate (NO) levels in M of normal dogs...............................................................63 3-6: Nitrate (NO) levels in M. of glaucomatous dogs...................................................64 3-7: Ratios of ETA receptor/ control protein....................................................................65 3-8: Ratios of ETB receptor/ control protein....................................................................66 3-9: Mean ratios of ETA receptor/ 18S mRNA in arbitrary units for normal and glaucomatous retinal samples...................................................................................67 3-10: Mean ratios of ETB receptor/ 18S mRNA in arbitrary units for normal and glaucomatous retinal samples................................................................................68 3-11: Summary of results................................................................................................69 x

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LIST OF FIGURES Figure page 2-1: Real Time PCR amplification curves of a normal and a glaucomatous retina for the ETA receptor and 18S rRNA...................................................................................48 3-1: Normal iridocorneal angle stained with toluidine blue (Dog M3, original magnification 100X)................................................................................................70 3-2: Iridocorneal angle of a glaucomatous dog (Dog 20, original magnification 40X, PAS stained).............................................................................................................70 3-3: Trabecular meshwork of a glaucomatous dog (Dog 25, original magnification 200X, PAS stained)..................................................................................................71 3-4: Iridocorneal angle of a glaucomatous dog (Dog 19, original magnification 200X, PAS stained).............................................................................................................71 3-5: Iridocorneal angle of a glaucomatous dog (Dog 22, original magnification 100X, PAS stained).............................................................................................................72 3-6: Iridocorneal angle of a glaucomatous dog (Dog 26, original magnification 100X, PAS stained).............................................................................................................72 3-7: Semi-thin (1m) sections of the retina from a normal dog (Dog M4, original magnification 400X) stained with toluidine blue.....................................................73 3-8: Semi-thin (1 m) sections of the retina from a glaucomatous dog (Dog 24, original magnification 400X) stained with toluidine blue.....................................................74 3-9: Semi-thin (1 m) sections of the retina from a glaucomatous dog (Dog 25, original magnification 400X) stained with toluidine blue.....................................................75 3-10: Label of canine scleral vessels and elastic fibers with the ETB antibody..............76 3-11: Arteriole of the choroid of a normal dog...............................................................77 3-12: Endothelin-1 in aqueous humor of normal and glaucomatous eyes......................78 3-13: Endothelin-1 in vitreous of normal and glaucomatous eyes..................................79 xi

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3-14: Scatter plot showing the correlation between the degree of degeneration in central retina and total aqueous humor and vitreal ET-1 levels of glaucomatous dogs........................................................................................................................80 3-15: Nitric oxide in aqueous humor of normal and glaucomatous eyes........................81 3-16: Nitric oxide in vitreous of normal and glaucomatous eyes....................................82 3-17: Endothelin receptor A protein in the retina of normal and glaucomatous eyes.....83 3-18: Endothelin receptor B protein in the retina of normal and glaucomatous eyes.....84 3-19: Endothelin receptor A mRNA in the retina of normal and glaucomatous eyes.....85 3-20: Endothelin receptor B mRNA in the retina of normal and glaucomatous eye......86 3-21: Real Time PCR amplification curves of the cloned ETA receptor DNA sequence run with different mixes of primers and probes....................................................87 3-22: Real Time PCR amplification curves of the cloned ETB receptor DNA sequence run with different mixes of primers and probes.....................................................88 3-23: Real Time PCR run with no template (1) and total RNA (2) run with the primers and probes for the ET receptors and 18S...............................................................89 3-24: RT-PCR of two samples from normal dog retinas.................................................90 3-25: Real Time PCR amplification curves of the ETA receptor for two normal retinal samples stored frozen in RNAlater (sample 1) and frozen dry without preservative (sample 2)..........................................................................................91 xii

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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy LOCALIZATION AND EXPRESSION OF ET-1 RECEPTORS IN THE NORMAL AND GLAUCOMATOUS DOG EYE By Maria E. Kllberg May 2003 Chair: Dennis E. Brooks Major Department: Veterinary Medicine The goal of this study was to document any differences in the levels of the endothelin-1 (ET-1) peptide and nitric oxide (NO) in aqueous humor and vitreous; and in the location and density of the ET-1 receptors, ETA and ETB, in the glaucomatous dog eye as compared to the normal dog eye. Comparisons were made between normal (n=30) and glaucomatous (n=14) samples for the following parameters: Levels of NO and ET-1 in aqueous humor and vitreous, as measured by enzyme immunoassay. Localization of ET receptors in the retina and choroid was done by immunocytochemistry. Expression of receptor protein in the retina and choroid, as measured by Western Blot technique. Retinal mRNA levels of ET-1, and the ET receptors, as measured by Real Time Polymerase Chain Reaction. Retinas were evaluated histologically. xiii

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The numerical data were analyzed using an ANOVA 2*2 factorial analysis. The two factors of interest were the disease, with two levels (glaucomatous and normal); and breed, with two levels (Cocker and non-Cocker). The findings in the glaucomatous eyes were the following: Endothelin-1 increased in aqueous humor and vitreous, with a larger increase in the Cocker group. Nitric oxide increased in aqueous humor and vitreous, with a larger increase in the non-Cocker group. Degree of degeneration of the retina was correlated to ET-1 levels in aqueous humor and vitreous. Distribution of ET receptor labeling was similar in normal and glaucomatous sections for both ET receptors. Endothelin-1 mRNA in the retina increased nonsignificantly in the Cocker group and decreased nonsignificantly in the non-Cocker group. Endothelin receptor expression decreased in both groups with a significant decrease for the ETB receptor in the non-Cocker group. Levels of ET receptor mRNA increased significantly in the Cocker group, while the increase in the non-Cocker group was not significant. The distinct results for the two groups might imply a unique response of ET-1 and its receptors in narrow-angle glaucoma in Cocker Spaniels. xiv

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CHAPTER 1 INTRODUCTION Glaucoma Role of Endothelin in Glaucoma Glaucoma is the final common pathway of a group of diseases with decreased retinal ganglion cell (RGC) sensitivity and function, RGC death and optic nerve head (ONH) cup enlargement, an incremental reduction in visual fields, and blindness. Glaucoma is a neurodegenerative disease as it results in neural cell death [1]. All of these diseases in dogs result in or are associated with increased intraocular pressure (IOP), although the etiology of primary glaucoma is likely to be multifactorial. Mechanical, vascular, and other factors may influence individual susceptibility to optic nerve damage. At every level of IOP there is a risk of glaucomatous damage, although the risk increases with increasing IOP. Damage can occur with extreme rapidity as in angle closure glaucoma, or may progress slowly as in the chronic primary open angle glaucomas (POAGs). Intraocular pressure cannot be used by itself to determine the presence of glaucoma; or to determine whether optic nerve damage will occur or progress. This variation in ONH susceptibility has been suggested to result from varying capacity for circulatory autoregulation to prevent IOP-induced ONH ischemia [1]. Ischemia of the optic nerve and retina may be induced by high IOP, or by vascular dysfunction such as a deficit in autoregulation, hypoperfusion, or vasospasm [2]. Inadequate blood supply of the retina and optic nerve is known to lead to death of ganglion cell neurons partly through the release of glutamate, an excitatory amino acid 1

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2 transmitter [3]. Neurons, which contain ionotropic glutamate receptors [e.g., Nmethyl-D-aspartate (NMDA) receptors], are particularly susceptible to ischemia/reperfusion [4]. The neurons in the retina that express such receptors are the ganglion cells and a subset of amacrine cells. In ischemia/reperfusion, neurotransmitters (e.g., glutamate) are released that overactivate their appropriate receptors. Such overstimulation, particularly of ionotropic glutamate receptors, generally leads to neural cell death [4]. Various ocular blood flow deficits have been seen in patients with POAG or normal tension glaucoma (NTG). Pillunat et al. [5-7] found evidence to suggest defective autoregulation in the ONH in POAG and NTG. Robert et al. [8] showed that the ability of glaucomatous eyes to adjust the blood supply in the optic disc to raised IOP is significantly reduced as compared to that of healthy eyes. Grunwald et al. [9] found a lack of hyperemic response of macular retinal blood flow after increased IOP in humans with POAG suggesting abnormal autoregulation. Patients with POAG with deteriorating visual fields despite an IOP lowered below 21 mm Hg show alterations in ocular blood flow regulation as compared to POAG patients with stable visual fields [10]. Cheng et al. [11] showed that patients with chronic angle-closure glaucoma (ACG) have decreased retrobulbar blood flow velocities and increased vascular resistance in the central retinal artery and temporal posterior ciliary arteries despite well-controlled IOP. Thus, evidence exists that in POAG, ACG and NTG there is defective autoregulation of blood flow to the ONH and retina. Endothelin -1 (ET-1), being a very potent endogenous vasoconstrictor, has been examined as part of the etiology of glaucoma. The presence of endothelin (ET) receptors in the retina and choroid, particularly in retinal blood vessels, suggests that ETs may be

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3 involved in regulating retinal blood flow, and could contribute to retinal ischemia. Chronic ischemia of the anterior optic nerve was induced in animal models [12,13], either by perineural infusion or intravitreal injections of ET-1. These studies have resulted in glaucomatous-like damage to the optic nerve. Henry et al. [14] demonstrated an impairment of ETB receptor-mediated, endothelial cell-dependent, vasodilation in the forearm of human patients with normal-tension glaucoma. This indicates that the eye may be just one manifestation of a more generalized vascular disorder characterized by presumed endothelial cell dysfunction. Such an imbalance in the ET receptors might contribute to ET-induced ischemic damage in glaucoma. Elevations of aqueous ET-1 are found in humans with POAG, and in the plasma and aqueous humor of patients with NTG [15-17]. Endothelin has also been found to induce efflux of glutamate from cultured rat brain astrocytes [18]. This suggests that ET, which is known to be released in ischemia, may exacerbate neurodegeneration by stimulating efflux of glutamate. Temporary retinal ischemia in the piglet leads to an increase in endothelin-mediated vasoconstriction and a loss of tonic nitric oxide-dependent vasodilation remaining several hours after the ischemic insult [19]. Hypoxia-ischemia serves as a stimulus to enhance endothelin production in both brain and heart. Endothelin gene transcription is upregulated and ET release from cultured human vein endothelial cells is increased in response to acute hypoxia. Tissue responsiveness to endothelin, which can be modulated by receptor up and down-regulation, may also increase in response to an ischemic stimulus, but this has not yet been demonstrated in ocular tissues.

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4 Astrocytes are the major glial cell type in the central nervous system and the ONH and are vital for retinal ganglion cell survival [20]. Optic nerve head astrocytes normally maintain the extracellular medium by regulating potassium and glutamate levels and also provide neurotrophic support for nearby neurons [21]. Like brain astrocytes, ONH astrocytes also respond to changes in the physiological state of the neuronal system by becoming metabolically active from a quiescent state and rapidly proliferating to the site of injury (reactive astrogliosis), especially under conditions of injury (ischemia or pressure-related) as seen in glaucoma. Astrogliosis disrupts axonal transport and also inhibits axon regrowth in the glaucomatous ONH [20,22]. Endothelins are implicated in the promotion of astrogliosis in the ONHs of experimental animals [13]. Intravitreal injection of ET-1 resulted in axon loss accompanied by glial proliferation in ONH of rabbits [13]. Prasanna et al. [23] showed that ET-1 induces astroglial proliferation in cultured human ONH astrocytes through ETA and ETB receptor activation suggesting that ET-1 could cause proliferation of ONH astrocytes in glaucoma. Endothelin-1 may also have direct effects on optic nerve function. Intravitreal injections of ET-1 cause alterations in axonal transport in the rat optic nerve [24]. Likewise, continuous administration of ET-1 to the perineural region of the eye in monkeys resulted in altered neuronal activity in the visual cortex [25]. These findings suggest that intravitreal ET-1 can have direct effects on neuronal activity and survival. Canine Glaucoma The dog has the highest frequency of primary glaucomas of all animals, with the narrowor closed-angle type being the most common [26]. The glaucoma classification scheme in dogs based on possible cause includes the primary glaucomas, secondary glaucomas, and congenital glaucomas [27]. In the primary glaucomas, the IOP elevation

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5 develops without concurrent ocular diseases, is inherited in some canine breeds, and has a bilateral potential for development. Primary glaucomas may result from abnormal biochemical metabolism of the trabecular cells of the outflow system [27]. In secondary glaucomas, the increase in IOP is associated with some known antecedent or concurrent ocular disease that physically obstructs the aqueous outflow pathways. In the congenital glaucomas, the increased IOP is associated with an anterior segment anomaly, and the elevation in IOP develops soon after birth. In the dog, the primary glaucomas are divided into open-angle and narrowor closed-angle glaucoma according to the presence of an open or narrow anterior chamber angle and ciliary cleft at gonioscopy. Inherited openand narrow-angle glaucomas occur bilaterally in purebred dogs. The primary glaucomas have been reported in at least 45 breeds and occur most frequently in United States among the American Cocker Spaniel (ACS), English Cocker Spaniel, Smooth and Wire Fox Terriers, Basset Hound, Sealyham Terrier, Miniature and Toy Poodles, and Beagle [27]. Other breeds recently identified with primary glaucomas include the Samoyed, Norwegian Elkhound, Bouvier de Flandres, Siberian Husky, Flat-Coated Retriver, Golden Retriver, Great Dane, Welsh Springer Spaniel, Akita, Chow-Chow, and Shar-Pei [27]. Some breeds appear affected with both the openand narrow closed-angle types, which may suggest these glaucomas are related. Advanced glaucomas in dogs show similar features independent of pathogenesis with an iridocorneal angle (ICA) closure and sclerociliary cleft collapse. The retina is degenerated with attenuated arteries, and the optic disc round, depressed, and atrophied. Intravitreal levels of the neurotransmitter glutamate are increased in dogs with glaucoma,

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6 providing evidence for an ischemic mechanism for RGC death and optic nerve head atrophy in canine glaucoma [28]. In a study by Kallberg et al. [29] aqueous humor ET-1 was 3.5 times normal level in dogs with primary chronic glaucoma. These findings indicate a possible role for ET-1 in the pathophysiology of some types of glaucoma in dogs. Primary narrow-angle glaucoma in American Cocker Spaniels The mechanism of development of narrow and closed ICA in the ACS is not completely clear, but one hypothesis is that tight apposition of the iris slightly increases the pressure within the posterior chamber, which in turn causes forward displacement of the basal iris [27]. Eventually, the basal iris narrows the ICA and opening of the ciliary cleft. Apposition of basal iris across the filtration angle causes a potentially reversible angle closure. With continued apposition, peripheral anterior synechiae (PAS) develop, thereby permanently closing the pathways of aqueous outflow. This mechanism corresponds to the development of angle-closure glaucoma in humans. The most common cause of angle closure in humans is pupillary block, also termed primary angle-closure glaucoma [30]. It impedes the flow of aqueous humor from the posterior to the anterior chamber between the anterior surface of the lens and the posterior surface of the iris. Pupillary block may be absolute, as when the iris is completely bound down to the lens by posterior synechiae, but most often is a functional block, termed relative pupillary block. Angle-closure glaucoma in humans can present with a spectrum of symptoms, from none at all to severe pain, blurred vision, and nausea [31]. Intermittent angle closure defines repeated, brief episodes of angle closure with mild symptoms and elevated IOP. They may continue uneventfully for months or years. Attacks may be accompanied by

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7 progressive PAS formation, leading to chronic angle closure due to a permanent closure of a portion of the angle. The IOP in eyes with chronic angle closure may be normal or elevated. The greatest danger lies in the possibility of sudden conversion to acute angle-closure glaucoma. If the pupillary block becomes absolute, the pressure in the posterior chamber increases and pushes the peripheral iris farther forward to cover the trabecular meshwork and closes the angle with an ensuing rise of IOP. Attacks of acute angle-closure glaucoma are mild at first, but rapidly increase in severity. The symptoms of an acute attack result from the sudden, marked elevation of IOP to as high as 80 mm Hg. Absolute glaucoma refers to an eye with no light perception and a persistently elevated IOP. The time required for a neglected angle-closure attack to cause total blindness in humans is variable and depends on the severity of the acute attack, but appears to be an average of 1 to 2 years [31]. Most affected dogs with angle-closure glaucoma present with either classic clinical signs of unilateral, acute congestive glaucoma of a few days duration, or with chronic, advanced glaucoma with buphthalmia, lens dislocation and cataract, retinal and ONH degeneration, and blindness. Often, the condition becomes bilateral within several months. In a study by Magrane [32] the mean age of affected dogs was 6 years (range, 3-10 years), with the second eye usually affected within 12 months. Both the history and clinical course suggest that this form of glaucoma may be a series of acute IOP attacks, with the subsequent magnitude of the IOP elevation gradually increasing. Tonographic measurements of aqueous humor outflow are usually within normal limits in dogs with narrow ICAs, but they are lower than normal (0.10-0.15

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8 L/min per mm Hg) in dogs with very narrow and closed (due to synechial formation) ICAs and clefts [27]. Tonometry of the acute congestive glaucomas often yields IOPs as great as 50 to 70 mm Hg, and the corneal edema that parallels the elevation in IOP after approximately 40 mm Hg usually prevents gonioscopy. Gonioscopy of the ACS with ocular hypertension usually reveals a narrow to closed ICA and reduced ciliary clefts; as the glaucoma progresses, angle closure and ciliary cleft collapse with peripheral anterior synechial formation commonly occurring. More recently, pectinate ligament dysplasia in the ACS has also been reported, but it does not appear to occur frequently [27]. In a study by Lovekin and Bellhorn [33] three Cocker Spaniels, bred for glaucoma, were followed with repeated clinical examinations, tonometry, aqueous outflow, and water drinking and betamethasone tests. The results were compared with similar test results on 82 controls. Eyes were removed from the three Cocker Spaniels being studied and compared with the eyes of four other Cocker Spaniels that had manifest glaucoma. The studied Cocker Spaniels had reduced IOP regulatory mechanism as compared to the control dogs, and increased IOP was more easily provoked in them than in the control dogs. However, pathologic changes indicating glaucoma were not seen in histologic sections of the ICAs of their eyes. The authors concluded that an abnormal physiology concerned with the regulation of IOP preceded pathologic changes in the angle structures of their eyes. Definite histopathologic changes were shown in the eyes of Cocker Spaniels with manifest glaucoma. The eyes resembled human angle-closure glaucoma or absolute glaucoma with loss of trabecular meshwork, anterior synechia, and attenuation of the intrascleral venous plexus. The histologic ICA structures of an eye of a Cocker Spaniel

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9 with no history of glaucoma showed a lack of trabecular meshwork. This eye, having an IOP of 24 mm Hg, was under prophylactic treatment because of manifest glaucoma in the other eye. Changes of the ocular fundus in the ACS may not correlate with the duration and magnitude of the elevated IOP [27]. It is not unusual for an ACS to present with a high IOP (70-80 mm Hg) and a history of signs of glaucoma being present for less than 1 week, yet after lowering the IOP to less than 20 mm Hg, the dog loses its vision. Ophthalmoscopically, the ocular fundus cannot be visualized until the IOP is lowered and the corneal edema reduced. The optic nerve and retina may initially appear to be normal, with some vascular attenuation detected. With the IOP maintained at 20 mm Hg or lower however, progressive retinal and ONH degeneration eventually become apparent within a few weeks. In some of these dogs, the retinal degeneration may affect only limited areas, appearing as radiating or fan-shaped zones from the ONH that represent areas of retinal and choroidal degeneration caused by ischemia from the occlusion of individual short posterior ciliary arteries. The continued progression of ONH degeneration and deterioration of the animals vision despite lowering of IOP is classic evidence for the role of non-IOP related factors in glaucomatous optic neuropathy. Among animals tested at the University of Florida, intravitreal glutamate levels are much higher in the ACS with glaucoma than in Samoyeds, Shar Peis, and Akitas [28]. In all probability, the initial primary optic nerve injury from the elevated IOP induces RGC degeneration and apoptosis caused by glutamate excitotoxicity, neurotropin deprivation, accumulation of intraneuronal calcium, and formation of nitric oxide (NO), proteases, and oxygen free radicals. The injured,

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10 apoptotic RGC releases more glutamate, predisposing the eye to further, secondary degeneration of adjacent healthy RGC and their axons. This domino effect occurs independent of further IOP-induced primary injury. Primary open-angle glaucoma in the Beagle Primary open-angle glaucoma in the Beagle is the most extensively investigated canine glaucoma. Beagles with spontaneous, hereditary open-angle glaucoma start to develop symptoms between 8 and 16 months of age [26]. The iridocorneal angle and sclerociliary cleft are initially normal. The increase in IOP and decline in the facility of outflow develop slowly. The increased IOP produces slight enlargement of the axial length of the globe, which in turn results in lens subluxation and narrowing of ICA and sclerociliary cleft in dogs between 1 and 4 years of age. Animals 2-4 years of age have IOPs in the range of 25 mm Hg to 40 mm Hg. Eventual ICA and sclerociliary cleft closure result in IOPs of 40 mm Hg to 60 mm Hg in animals 4 to 6 years old. Light microscopic examinations of the aqueous outflow structures indicates no abnormalities in the early affected animals [34,35]. The retinal blood vessels, especially the small peripapillary retinal arterioles and veins, gradually disappear. The optic disc becomes round, depressed and atrophied with the loss of myelin. Optic nerve capillary endothelial cells are ultrastructurally abnormal prior to detectable increases in IOP [36]. Spherical, membrane-bound, electron-dense inclusions resembling Weibel-Palade bodies are found in pericytes and endothelial cells in both preglaucomatous and glaucomatous eyes. These changes are usually associated with microcirculatory abnormalities in humans and diabetic dogs. Abnormal blood flow has been detected in the exterior and internal ophthalmic arteries, the anterior ciliary arteries, and short posterior ciliary arteries, but not the

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11 primary retinal arteries in the glaucomatous Beagle as measured by color Doppler imaging [37]. Furthermore, while treatment with Ca-channel blockers increases blood flow in orbital vessels of normal Beagle dogs, the blood flow of glaucomatous Beagles remains unchanged. This indicates an inability of the vessels to respond to stimuli, which may be due to an altered vascular structure with hypertrophy or dysfunctional regulation by endothelial receptors. Ophthalmic Vascular Morphology and Physiology The eye is one of the most highly perfused organs in the body [38]. In dogs and humans, the eye has two separate systems of blood vessels that differ anatomically and physiologically: the retinal vessels, which supply the inner layers of the retina, and the uveal or ciliary vessels, which supply the rest of the eye. The main supply of blood to the eye and orbit of the dog is via the internal maxillary artery (as a branch of the external carotid artery) that after passing through the alar canal branches to give rise to the external ophthalmic artery (EOA) [39]. The internal ophthalmic artery (IOA) in dogs is a small artery that arises from the anterior cerebral artery at the level of the optic chiasm [40]. It passes through the optic canal on the dorsal surface of the optic nerve in dogs, and runs rostral by the nerve to anastomose with a branch of EOA about midway between the optic canal and the posterior pole of the globe. By comparison, in primates the entire global microcirculation and most of the orbital circulation are supplied via the internal carotid artery, which gives rise to the IOA [39]. Two long posterior ciliary arteries (LPCA), one medial and one lateral, arise from the anastomoses of the external and internal ophthalmic arteries in dogs. Six to ten short posterior ciliary arteries (SPCA) arise, surround the scleral canal of the canine optic nerve and supply the lamina cribrosa, choroid, retina, and ONH circulations in dogs. The SPCA

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12 thus give rise to two very biologically distinct circulations. The peripapillary choroid also derives its blood supply from the SPCA at the ONH margin, although the choroidal and ONH circulations are not continuous. Peripapillary choroidal blood flow is directed anteriorly away from the optic disc as the venous drainage of the peripapillary choroid is anterior to the vortex veins. Choroidal arteries branch into smaller vessels before supplying the choriocapillaris, the innermost layer of choroidal vessels. The retina in dogs and primates has a large vascular network in the major part of the sensory retina. The blood vessels extend from the optic disc to the region of the ora serrata. While the location of the blood vessels within the retina may differ somewhat between species, the general pattern of the major retinal arterioles and venules is generally similar in that they lie superficially in the nerve fiber layer and RGC layer, radiating from the optic disc. The smaller arterioles, venules and capillaries are organized into two dense capillary networks: an inner plexus, being situated at the level of the nerve fiber layer and/or ganglion cell layer; and an outer plexus, which in the dog is situated at the border between the inner nuclear layer and the outer plexiform layer. Primates possess a cone-rich region completely free of rods called fovea. No retina of the domesticated animals has a fovea, but an area of high cone density frequently occurs and is often referred to as the area centralis. This area lies 3to 4-mm dorsolateral to the optic disc in the dog. At the level of the area centralis there are no major retinal vessels, but capillary networks lie in different layers of the retina. The thickness of the innermost vascular plexus varies with the thickness of the nerve fiber layer. In the temporal retina near the raphe where the nerve fiber layer is very thin,

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13 capillaries are almost absent. In the peripapillary retina where the nerve fiber layer is thick, the capillaries form a dense, multilayered plexus. The arrangement (number and location of capillaries) of the superficial capillary network is directly related to the thickness of the nerve fiber layer and the ganglion cell layer, which are thought to be metabolically demanding [41]. The multilayer arrangement leads to a plexus of numerous capillaries fed by a single, relatively large arteriole. Consequently, the retinal microvasculature is subjected to a large pressure head, and changes in its resistance are likely to play a significant role in blood flow regulation. Blood vessels can be identified on the basis of distinct characteristics. Arteries are large, with multiple layers of smooth muscle cells, the muscularis, surrounded by the adventitial layer consisting of circumferentially oriented collagen fibers. An inner elastic lamina separates the muscularis from the endothelium. A basement membrane surrounds the endothelial cells and blends with the internal lamina elastica. Arterioles have smaller caliber, a single layer of smooth muscle cells, and no, or only minimal, internal elastic lamina. The endothelium is continuous and the cells are covered by a basement membrane. Capillaries have a minimal wall thickness consisting of endothelial cells and their basement membrane [42]. The endothelial lining of capillaries is supported by circumferentially oriented pericytes with contractile features [43]. The endothelial cell monolayer lining blood vessels was long thought to simply function as a diffusion barrier. It is now recognized that the endothelium has a crucial role in regulating vascular homeostasis [44]. The location of endothelial cells between the circulating blood and the vascular smooth muscle cells gives them a strategic position to regulate vascular permeability and influence vascular hemostatic metabolic functions

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14 [45]. The vascular endothelium plays an active role in vasomotor f unction of both macroand microvasculatures, including maintenance of vascular tone and regulation of blood flow [46]. Vascular tone depe nds on a balance between the en dothelial vasodilators (e.g., (NO) and vasoconstrictors (e.g., ET) such that reduced formation of vasodilators would result in vasoconstriction and a decrease in vasoconstrictors would result in vasodilation. Therefore, endothelial cells pl ay an important role in modul ating the microvascular tone and autoregulation. Endothelial cells also st rongly affect coagulation, platelet function, and fibrinolysis [45]. Regulation of Blood Flow Autoregulation plays a very important role in the control of bl ood flow in a tissue. The goal of autoregulation in a tissue is to maintain a relatively constant blood flow, capillary pressure, and nutrient supply in spit e of changes in perfusion pressure [46]. This is accomplished through the ability of the cardio vascular system to adjust the resistance of particular vessels by controlli ng the diameter of their lumen. The blood flow in the ONH and intraocular vessels (when pressure in the central retinal vein is normal) is calcula ted by using the following formula: Flow = Perfusion pressure / Resistance to flow Perfusion pressure = Mean blood pressure (BP) intraocular pressure (IOP) Mean blood pressure = Diastolic BP + 1/3 (systolic diastolic BP) From this formula, it emerges that the bl ood flow depends on 1) resistance to blood flow, 2) blood pressure, and 3) IOP. Factors that normally govern microcirculato ry vascular smooth muscle tone involve vasoactive nerves, and circulati ng hormones, as well as endothelial cell derived factors, and myogenic and metabolic factors [47]. A ccording to the metabol ic hypothesis, local arterial smooth muscle tone is regulated by metabolic change in the tissue [46]. Local

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15 concentration of me tabolic products, O2 and CO2, plays a role in maintaining autoregulation. Accumulation of CO2 and a reduction in O2 is due to hypoxia or lack of ability to wash out metabolites, causing vas odilation. The myogenic hypo thesis states that a rise of intravascular pressure causes vaso constriction because stretching of the vessel wall is counteracted by vasoconstriction in the arterioles. Responses to changes in transmural pressure, i.e. stretching are not dependent on endothelium-derived vaso-active substances [48]. The endothelial cytoskeleton together with its extracellular matrix is thought to provide a mechanical linkage betw een the site of the shear stress and the smooth muscle cells. The change in pressure causes activation of pr otein kinase C, an intracellular modulator of Ca 2+ -dependent contractile processes. The role of the endothelium in flow-induced contraction, cau sed by stretching of the vessel wall, may thus be that of a conduit for the mechanical disturbance. This autoregulation of blood flow may also have neurogenic control. Ho wever, there is not much proof of this occurring in the ocular vesse ls because vessels in the re tina and ONH have no autonomic nerve supply [38]. The choroid, by contrast, is richly supplied by the autonomic nerves and yet has no appreciabl e autoregulation [49]. Alterations in blood flow through the microcirculation were long thought to be solely achieved by changes in tone of precapilla ry sphincters. It is now well accepted that retinal pericytes also have th e capacity to act as important regulators of local blood flow. Pericytes are perivascular cells with multifunctional activities. They are contractile cells adjacent to the endothelial lining of capi llaries supporting the microvasculature. The regulatory mechanisms and vessel-wall cr oss-talk between these cells and the microvascular endothelium have been shown to be identical to the those of smooth

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16 muscle cells [43]. These inter actions are very important in the retinal microcirculation where autoregulation is vita l for the maintenance of sm ooth and uninterrupted blood flow. Pericytes are more numerous in the retina than in any other microcirculation in the body [43]. Within the retinal microvasculature there is a rapid transition from vascular smooth muscle cells to pericytes at the interface of the precapi llary and terminal arterioles. Modulation of blood flow by pericy tes in very small ophthalmic arteries and capillaries has been shown in several studi es [50-52]. The capacity of pericytes to regulate the blood flow through capillaries indicate s an important role of the pericytes in the control of microcirculati on of the retina and the ONH. Blood flow in the retina and prelaminar ONH circulations have been shown to possess autoregulatory capacity in severa l species by remaining stable during IOPinduced changes in perfusion pressure, whereas blood flow in the chor oid is sensitive to changes in IOP [53-55]. However, the latter concept remains de batable. In particular, the choroidal tissue just nasal to the optic nerv e may be capable of blood flow autoregulation in instances of increased IO P, as has been shown in the cat [56]. Furthermore, investigations have shown that the choroid is capable of au toregulation in the rabbit [57]. The autoregulatory capacity of the human choroid is unknown [56]. Endothelin and Nitric Oxide Synthesis, Secretion and Clearance of Endothelin In 1988, Yanagisawa et al. [58,59] identif ied endothelin (ET), a very potent endothelium-derived vasoconstrictive factor The substance, a 21-amino acid peptide, was isolated from the endothelial cells of pig aorta and is known today as ET-1. Endothelin-1 has also been found to be expr essed in non-vascular tissues, such as the

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17 brain, kidney, lung and others [ 60]. Subsequent studies have shown that the ET isolated from endothelial cells is one of a family of isopeptides. Three isoforms of the peptide exist: ET-1, ET-2, and ET-3, but only ET-1 can be detected in endothelial cells of vascular tissue. Endothelin2 and ET-3 can be found in the intestine, adrenal gland and br ain. Endothelin-3 is relatively abundant in neuronal tissues [60]. Each ET isoform is a product of separa te genes that code for a precursor protein mRNA that share high sequence homology [ 44]. The endothelins are synthesized from precursors known as preproendothelins (pre proET) comprised of 160 and 238 amino acid residues. Before they are finally processe d into 21 amino acid peptides, these large precursors undergo an intermediate cleavag e by endopeptidases to form the 37-41 amino acid precursors proendothelins (proET), also named Big ET. Big ET is further processed by a specific endothelin converting enzyme (ECE) to form the 21 amino acid active peptide. Smooth muscle cells are targ et cells and so express a nonselective ECE (ECE-1) on their cell surface associated w ith the plasma membrane to convert exogenous Big ET. The pro-ET-1 released from endothelial cells can therefore be converted into ET-1 in the vascular smooth muscle surface [60]. However, the major part of conversion of Big ET1" into ET-1 takes place in the endothelial cells, and it is assumed that the ET-1 is concentrated primarily in these cells, although it is not stored there. The enzyme present in endothelial cells is an EC E (ECE-2) selective for Big ET1" more than Big ET-2" or Big ET-3". The ECE located in vascular smooth muscle acts equally on the different progenitor peptides [60]. In the retinal micr ocirculation pericytes have been shown to respond to ET-1 secreted by the retinal capillary endothelial cells [43,61].

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18 The secretion of ET-1 by endothelial cells is regulated at the level of peptide synthesis, because the endothelial cell cont ains no dense secretory granules in which ET-1 can be stored and then later releas ed. The expression of preproET-1 mRNA and release of the peptide are stimulated by seve ral factors including adrenaline, thrombin, vasopressin, angiotensin II, insulin, cytokines, tr ansforming growth factor (TGF ), arginine, and physical stimuli such as shear stress of the endothelium. Ischemia and hypoxia also result in synthesi s and release of ET-1 [60]. Endothelin-1 synthesis is inhibited via a cGMP-dependent mechanism by NO, prostacyclin, natriuretic peptides and heparin [60]. Hisaki et al. [62] showed that removal of the endothelium does not completely prevent the action of Big ET-1" in the pe rfused rat mesenteric bed, suggesting that smooth muscle cells also can synthesize ET1. Smooth muscle cells in culture express ET-1 mRNA and release ET-1. According to Wa rner [63] within a diseased blood vessel with damaged endothelial cells the vascular smooth muscle may produce ET-1 that is not regulated by endothe lial cell agonists. Most of the ET-1 released by the endoth elial cells (approximately 80% of the synthesized amount) is secreted abluminally acting as a local hormone in a paracrine manner on the surface of the vascular smooth mu scle [60]. The plasma half-life of ET-1 in humans is less than 1.5 min because of its efficient extraction by the splanchnic and renal vascular beds [44]. Endothelin-1 is also reported to be taken up by the lungs but the clearance differs between species [64]. Extraction of ET-1 follows binding to ce ll surface receptors, which are then internalized, allowing degradati on to be carried out within the cell. Endothelin-1 is found

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19 to be internalized by both ETA receptors and ETB receptors [65,66]. Both receptors seem to be agonist-occupied after in ternalization for one to four hours. Endothelin-1 is then dissociated from the receptor and possibly degr aded. It has been shown that internalized ET-1 continues to evoke signaling events in th e cells, which has been suggested to be one of the reasons that ET-1-stimulated effects are long-lasting [65]. A possible candidate for an intracellular degrading enzyme is a soluble protease found in human platelets, vascular smooth muscle and endothelial ce lls [64]. The fate of the inte rnalized receptors will be discussed in the chapter about endothelin receptors. Nitric Oxide Nitric oxide plays an important role in combination with ET-1 to produce a balanced effect on blood vesse l activity. Nitric oxide is an inorganic free radical, produced from L-arginine by the enzyme nitric oxide synthase (NOS ) [63]. Nitric oxide synthase exists in at least three isoforms, but it is only th e calcium-regulated isoform, endothelial NOS (eNOS), th at appears responsible for NO formation within the healthy endothelium. The stimulation of a endothelial cell membrane receptor by an agonist such as acetycholine, histamine, thrombin, ET-1 or bradykinin leads to an increase in intracellular calcium, which in turn activates NOS [49]. Mechanical forces, such as stretch and shear, can also stimulate the production of NO. When NO reaches its target smooth-muscle cell it activates the enzyme guanylate cyclase, responsible for the production of the 3'5'-cyclic guanosine monophosphate (cGMP), a second messenger. The increase in cGMP ultimately leads to smooth-muscle relaxation by reducing the intracellular calc ium content [49]. A basal release of NO maintains ocular vessels in a cons tant state of mid-dilation [67].

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20 Retinal capillary endothelial cells have b een shown to constitutively express the enzyme NOS [68]. The enzyme guanylate cycl ase has been shown to be expressed in retinal pericytes making it possible for the ce lls to synthesize cGMP on exposure to NO. Cultured pericytes relax to sodium nitr oprusside (a NO donor), which stimulates guanylate cyclase and increases cGMP [69]. In addition the pericytes have been shown to respond to prostacyclin [67]. In normal vessels there is a balance be tween the endothelial production of NO and ET-1. The ability of NO to down-regulate ET-1 production and of ET-1 to stimulate NO production by endothelial cells acts to mainta in a matched degree of constrictor-dilator tone. In a number of disease states there is su spected to be a breakdown in the balance between ET-1 and NO [63]. Endothelial ce ll reduction in NO production may be compensated for by the induction of inducable nitric oxide synthase (iNOS) within the vascular smooth muscle. However, iNOS produces NO in an uncontrolled manner. In such disease states, ET-1 production is often increased. This increase in ET-1 production may well underlie deleterious increases in blood vessel reactivity and reductions in tissue perfusion [63]. Endothelin Receptors Endothelin receptors exist both in vascul ar smooth muscle, pericytes, and the endothelium. In 1990, two ET receptors (ETA and ETB) were isolated [44]. Both receptors belong to a family of heptaheli cal G-protein coupled receptors. Although these two receptors have approximately 60% similarity in amino acid sequence, several domains are less homologous to each other and contribute to the functional differences between the ETA and ETB receptors.

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21 The two ET receptor subtypes vary in their affinities to endogenous ligands. Endothelin receptor-A has a high affinity to ET-1 and ET-2, and a low affinity to ET-3. Endothelin receptor-B has an equally potent affinity to all three ligands [70]. Endothelin receptor-A exists on smooth muscle cells and mediates vasoconstriction, while the ETB receptor is located on e ndothelial cells and mediates release of relaxing factors, such as pros tacyclin and nitric oxide. Recent research indicates that ETB also exists on vascular smooth mu scles and mediates vasoconstriction. The relative contributions of ETA and ETB receptors to vasoconstriction is variable, and depends on the species and the vessel type studied [44]. Endothelin causes vasodilation at very low concentrations, and a marked and sustained contraction at high concentrations. The dilator re sponse to endothelin involves activation of the ETB endothelial receptors, linked to NO and/or prostacyclin release by endothelial cells [45]. At higher concentr ations, such that all the endothelial ETB receptors are occupied, ET diffuses through the intima toward the vascular smooth muscle. The constrictive response invol ves the activation by endothelin of ETA and ETB receptors on smooth muscle cells [45]. This vasoconstriction is correlated with a sustained increase of intracellular Ca2+ produced via a two-phase mechanism. The binding of ET-1 to the recep tor activates phosph olipase C (PLC) which hydrolyses phosphatidilinositol-4,5-biphosphate (PIP2) into two products, inositol triphosphate (IP3) and diacylglycerol (DAG). The ini tial increase in intracellular Ca2+ concentration is caused by a rapid mobilization of intracellular calcium stores by IP3. This ET-1 mediated signaling upstream of IP3 interaction with the Ca2+ stores is regulated by tyrosine kinases which also participate in th e contractile response of -adrenoceptor agonists, histamine

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22 and angiotensin II. The maintained increase in intracellular Ca2+ levels is brought about by exterior Ca2+ entry via a calcium channel of the plasma membrane. At least two types of non-se lective cation channels as well as voltage-operated calcium channels are activated by ET-1 [71] One is activated by low and the other by high concentrations of ET-1. The channel ac tivated by low concentrations of ET-1 is inhibited by NO and therefore ET and NO seem to affect the same channel. The increased production of DAG also stimulates the transl ocation of protein kinase C (PKC) from cytoplasm to membrane and its subsequent activation [72]. Activation of PKC leads to phosphorylation of many proteins. Phosphoryl ation of myosin light chains induces smooth muscle contraction. The binding of ET-1 to the ETA receptor gives rise to a long-lasting response while the response of the ETB receptor upon ligand binding is of more transient nature. Moreover, it has been shown that the ETB receptor mediated vasodilation response to repeated injections of ET-1 shows tachyphylaxis whereas the vasoconstrictor response in the same study did not [66]. This implies different pathways for the two receptors following internalization. Bremnes et al. [73] investigated the regulation and intracel lular trafficking pathways of the endothelin receptors. Th eir study demonstrated that both receptor subtypes are rapidly internalized upo n agonist stimulation. Internalized ETA and ETB receptors both enter early endosomes (sorti ng endosomes). However, from this location, the two receptor subtypes are targeted to different intracellular fates. Whereas the ETA receptor is directed to the pericentriola r recycling compartment and subsequently reappears at the plasma membrane, the ligand bound ETB receptor is directed to

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23 lysosomes for degradation. Thus, furthe r clearance of plasma ET-1 by the ETB receptors will be limited by the supply of de novo synt hesized receptor molecules to the cell surface. This mechanism also explains the transient nature of the ETB mediated response and the tendency for development of tac hyphylaxis upon repeated s timulation of the ETB receptor. The conclusion from this group was that the rapid recycling of ETA may provide a basis for the prolonged contractile respons e mediated through this receptor, whereas lysosomal targeting of ETB suggests a role for this recepto r in clearance of ET from the circulation. Chun et al. [74] showed that ET1 remains intact and bound to the ETA receptor for up to two hours after endocytosi s. Their hypothesis for the pr olonged signal response of the ETA receptor was that ligand-occupied ETA receptors may continue to activate the Gprotein after endocytosis. Retinal pericytes express both isoforms of the endothelin receptor, ETA and ETB [75]. The pericyte ETA receptor mediates contraction through the same pathway as the ETA receptor on smooth muscle cells, i.e. th rough the phospholipase C/inositol phosphate signaling pathway [43]. The exact nature of the second messenger signaling downstream to ETB receptor activation in the retinal pericy te remains uncharacterized. However, a study has linked endothelin 3 stimulation of renal mesangial cells (also classified as pericytes) to the production of NO [43]. This mechanism was postulated to buffer the contractile effects of ET-1 esp ecially when local levels are inappropriately high. This is in opposition to the constrictor resp onse evoked by activation of the ETB receptor on smooth muscle cells. Mc Donald et al. [ 76] showed by binding studies of various fragments of ET-1, -2, and -3 using an in vitro model of microvascular pericytes that it is

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24 highly likely that the ETB receptor on retinal pericytes are different from those on the endothelium. There is considerable heterogeneity in pericyte surface endothelin receptor expression with some cell t ypes expressing only the ETA and others ETB [77]. The cell membranes of vascular effector cells may show both receptor types and one may be preferentially expressed. This is certainly the case in retinal pericytes where receptor binding studies have revealed the presence of several hundred thous and high-affinity ETA receptors but only twenty to thirty thousand ETB receptors on a single cell. It is also noteworthy that retinal peri cytes express many more ETA receptors than vascular smooth muscle [75]. At concentrations where ET-1 exerts no dire ct contractile effect, it potentiates the response to other vasoconstric tor hormones such as norepin ephrine and serotonin [45]. The potentiating effects ar e due to an increased Ca2+ sensitivity of smooth muscle cells and can be prevented by pretreatment with calcium antagonists of the dihydropyridine type. Endothelin and the Ophthalmic Circulation The endothelial-dependent re gulatory mechanisms ar e present in the entire cardiovascular system but there is a great he terogeneity of responses from one organ to the other, in arteries and vein s, and in vessels of different diameter. This heterogeneity may be explained by a different smooth musc le content in different vessels, and/or different expression of receptors. Haefliger et al. [78] showed that the effect of both NO and ET-1 increased with decreasing vascular diameter, when compar ing the response to chemical or hormonal agonists of the porcine ophthalmic artery with the response of the ciliary artery.

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25 Endothelin-1 has also been shown to contribute to hyperoxi a-induced retinal vasoconstriction through its ETA receptor in the human retina [79]. Systemic ET-1 administration in healthy subjects reduces pul satile blood flow in the choroid and optic disc at doses which do not affect systemic hemodynamics or flow velocity in the ophthalmic artery [80]. This indicates that ocul ar circulation is par ticularly sensitive to changes in local ET-1 concentration and c onfirms the hypothesis that ET-1 may play a role in ocular vascular diseases. Granstam et al. [81] showed a reduction of retinal blood flow, but no effect on the choroidal blood flow, by intravit real ET-1 injections in cats In contrast, Kiel et al. [82]has shown that the c horoidal circulation in ra bbits is regulated by ETA and ETB receptors mediating opposing effects on choroida l vascular resistance. Their results also suggested that endogenous ET preferentially elicits ETB vasodilation, most likely by stimulating endothelial nitric oxide release. Endothelin-1 is well distributed in ocular tissue and is localized in aqueous humor, iris, ciliary body, retina, and c horoids [83,84]. The ET-1 of retinal origin is mostly vessel oriented but is also found in the retina l ganglion cell layer, the photoreceptor inner segments, and the outer plexiform layer. Naraya n et al. [85] has shown that cultured RPE cells (ARPE-19cells) produce and release ET-1 by stimulation of muscarinic receptors. In the same study mRNA expression for preproET-1, ETA and ETB receptors was detected in the RPE cells. Both ETA and ETB -type receptor binding sites have been detected in the vascular smooth muscle of choroidal and retinal vessels of humans and rats [83]. In the same study extravascular binding sites of the ETB -type were found in the ganglion cell layer. In a

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26 study in humans and rabbits the ETA -like receptor binding sites were localized to the retinal and choroidal blo od vessels, whereas the ETB -like receptor binding sites were localized to the neural and g lial cells of th e retina [86]. Endothelin and Aqueous Humor Production and Outflow In the anterior part of the eye, ETA receptors have been found in human ciliary smooth muscle (HCSM), ciliary non-pigmented epithelial (NPE) cells, and trabecular meshwork (TM) cells [87,88]. Endothelin r eceptor-B receptor expression has been observed in cell cultures in HCSM cells [89]. Sugiyama et al. [90] showed a role for both ETB and ETB receptors in regulating IOP. Endothelin-1 affects the contractility of ciliary smooth muscles (CSM) and TM cells consequently regulating aqueous hum or [91]. Studies ha ve shown that ET-1 decreases aqueous humor formation and incr eases outflow facility in rabbits and monkeys [92-94]. Endothelin-1 has also been shown to decrease the activity of sodium potassium ATPase, a key enzyme involve d in ion transport and aqueous humor production [95]. Since it has been shown that ET-1 decr eases IOP and increases overall outflow facility, the direct effect of ET-1 of the trabecular mesh work cells is functionally antagonistic to the direct effect on ciliary mu scle [96]. Thus intertrabecular spaces could be narrowed by contracting trabecular fibres and widened by ciliary muscle contraction. According to Wiederholt [96] an alternative possibility might exist: in the presence of both tissues, contraction of the trabecular me shwork may increase the rigidity of this tissue allowing the ciliary muscle contracti on to be more effec tive in altering the geometry of the trabecular meshwork. A lthough contraction of the ciliary muscle dominates the overall effect on outflow facility in the human (and most likely in the dog)

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27 eye, the concept of antagonism between ciliar y muscle and trabecular meshwork should be considered in the interpretation of m echanism of action of ET-1 on aqueous outflow and IOP. Purpose of Study The goal of this study was to document any differences in the location and density of endothelin-1 receptors in the glaucoma tous dog eye as compared to the normal dog eye. In addition, the levels of the endothelin -1 peptide and nitric oxide in aqueous humor and vitreous were compared in the normal and glaucomatous dog eye. The hypotheses were the following: The localization of endothelin-1 receptors differs in the re tinal and choroidal vessels in the normal canine eye and the glaucomatous canine eye. The density of endothelin-1 receptors differs in the retina of the normal canine eye and the glaucomatous canine eye. The concentration of endothelin-1 and nitr ic oxide in aqueous humor and vitreous differs between the normal canine eye and the glaucomatous canine eye. The objectives were: To identify the localization of endothelin-1 receptors in the re tinal and choroidal vessels of normal and glaucomatous canine eyes as measured by immunogold labeling. To identify the density of endothelin-1 receptors in the retina of normal and glaucomatous canine eyes as measured by Western Blot and Real Time PCR. To determine the levels of endothelin-1 a nd nitric oxide in the aqueous humor and vitreous of normal and glaucomatous canine eyes as measured by immonoasssay.

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CHAPTER 2 MATERIAL AND METHODS Study Design The normal and glaucomatous canine eyes in this study were from canine clinical cases at the University of Florida and Universidad Nacional Autonoma de Mexico in Mexico City. All dogs went through an ophthalmic examination prior to inclusion in the study (Tables 2-1 and 2-2). Enucleated normal eyes (n=30) of 19 dogs and glaucomatous eyes (n=14) of 14 dogs were collected during one year. Normal and glaucomatous samples were obtained aseptically within 10 minutes after enucleation. The same technique was practiced for all dogs irrespective of the procedure and hospital performing the procedure. Samples of aqueous humor, vitreous, retina, and choroid were stored for future analysis of protein and mRNA content. In addition, samples of the posterior segment of the globe, with retina and choroid attached, and the anterior segment including the iridocorneal angle, were gathered for histological and immunocytochemical studies. The levels of nitric oxide (NO) and endothelin (ET) -1 in aqueous humor and vitreous were measured by enzyme immunoassay. The location of the ETA and ETB receptors were determined in the retina and choroid with immunocytochemistry and the expression of ET receptor protein with Western Blot technique. The mRNA levels of ET-1, and the ETA and ETB receptors, in the retina were assessed with Real Time polymerase chain reaction (PCR). The retinas and iridocorneal angles were evaluated 28

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29 histologically. Comparisons were made between normal and glaucomatous samples for these parameters. A summary of samples and analyses is shown in Tables 2-3 and 2-4. Clinical Examination of Dogs Ophthalmic examinations of the glaucoma dogs in this study included tonometry (Tonopen, Mentor Ophthalmics, MA), slitlamp biomicroscopy (Kowa SL 14,Kowa Optimed, CA), and indirect ophthalmoscopy (Heine Omega 150, Dover, NH). The mean ( SD) intraocular pressure (IOP) of normal dogs, as measured by Tonopen applanation tonometry, is 16.8 4.0 mmHg. The glaucoma dogs in this study were clinically diagnosed with hypertensive glaucoma, i.e. with an IOP exceeding 25 mm Hg. Clinical signs also included episcleral congestion, mydriatic pupils, optic nerve cup enlargement, neuroretinal rim narrowing, optic cup deepening, and retinal degeneration. Slitlamp biomicroscopy, and indirect and direct ophthalmoscopy of each dog were performed by veterinary ophthalmologists with extensive experience. Breeds, ages, and clinical parameters of the normal and glaucomatous dogs are shown in Tables 2-1 and 2-2. Endothelin-1 and Nitric Oxide Levels in Aqueous Humor and Vitreous Endothelin-1 Analysis Samples of aqueous humor and vitreous were gathered from enucleated normal eyes (n=28) of 18 dogs and glaucomatous eyes (n=11) of 11 dogs (Tables 2-3 and 2-4). The samples were stored at -80 C until analysis. Measurements of ET-1 were made by enzyme immunoassay (BBE5 Human Endothelin-1 Parameter ELISA Kit, R&D Systems, MN) by the Analytical Toxicology Core Laboratory at University of Florida. Cross reactivities entail big endothelin, <1%; sarafotoxin, <2%; ET-2, 45%; ET-3, 14%. The fact that canine and human ET-1 have corresponding amino acid sequences validated this

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30 kit for testing canine ET-1. The kit was fi rst evaluated on dog plasma. The extraction method for ET-1 in plasma was then modified and validated for the much smaller sample size and different constituency of the a queous humor samples. Unlike plasma, which requires an extraction prior to assay, aqueous humor samples can be assayed directly. When sufficient volume was available (> 200 L) each sample was run in duplicate. Nitric Oxide Analysis Measurements of total NO were made by enzyme immunoassay (Nitrate/Nitrite Colorimetric Assay Kit; Cayman Chemical Company, Ann Arbor, MI). In this method the two ultimate products of NO activity (nitrite and nitrate) are evaluated together rather than separately to enhance reliability. To achieve this, nitrate in the samples was first converted to nitrite by incubation with nitrate reductase. Griess reagents we re then added to react with nitrite and form a purple azo end-product that absorbs strongly at 540 nm. Although common interfering spec ies include antioxidants (suc h as ascorbic acid, 0.1 mM) and phosphate (>50 mM), the interferan t of greatest concern for this assay is NADPH, an essential cofactor for Nitric Oxide Synthase (NOS) enzyme function. Because this species can inhibi t formation of the azo product, a catalyst is added with the nitrate reductase to prom ote the conversion of NADP+ to NADPH. Aqueous and vitreous humor samples were prepared for the assay by centrifuge filtration through 30 kDa cut-o ff filters (Millipore Corporat ion, Bedford, MA) that had been pre-rinsed with HPLC-grade water. When sufficient volume was available (~100 uL) the samples were analyzed in duplicate. Localization of Endothelin Receptors in the Retina and Choroid To our knowledge, ET receptor antibodies have not been used previously on canine tissue. Therefore it was necessary to determine if the antibodies had an affinity for the ET

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31 receptors in our samples. This was done by immunostaining of scleral vessels of a normal Beagle eye. The staining was evaluated by light microscopy. Following confirmation of the affinity of the antibodies, immunocytochemistry for evaluation of receptor location by transmission electron microscope (TEM) was done on normal and glaucomatous retinas and choroids in the study. Light Microscopy The tissue was immersion fixed in 4% paraformaldehyde 0.1M phosphate buffer solution (PBS), pH 7.4, for 10-12 hours. Following fixation the tissue was washed in 0.1M PBS, pH 7.4, dehydrated in an ascending series of ethanol, and embedded in paraffin. Paraffin sections were cut 6 m thick and mounted on glass slides. After deparaffinization with graded concentrations of xylene and ethanol, slides were immersed in 3% hydrogen peroxide in methanol for 20 minutes at room temperature to block endogenous peroxide activity. The slides were then incubated with 2% non-fat dry milk and 2% cold water fish gelatin in 0.1 M PBS for 30 minutes to reduce non-specific background staining. Primary antibodies, rabbit anti-rat ET receptor type A (1:100) or rabbit anti-rat ET receptor type B (1:100) (Alomone Labs, Jerusalem), were incubated in damp chamber at 4C overnight. The treated slides were then incubated with biotinylated link antibody for 30 minutes at room temperature, then incubated with peroxidase-labeled streptavidin for 30 minutes at room temperature. Peroxidase activity was visualized by the 3-amino-9-ethylcarbazole (AEC) substrate-chromogen system. Transmission Electron Microscopy Normal dog eyes (n=5) from 5 dogs and glaucomatous dog eyes (n=5) from 5 dogs were collected (Tables 2-3 and 2-4).

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32 The tissue was immersion fixed in 4% paraformaldehyde 0.1M PBS, pH 7.4, for 10-12 hours. Following fixation the tissue was washed in 0.1M sodium cacodylate, pH 7.4, with 3.5% sucrose, 0.5% DMSO, and 0.5mM calcium chloride followed by incubation for 1 hour in 1% tannic acid in 0.1M sodium-maleate buffer, pH 6.0. It was then dehydrated in an ascending series of ethanol. At 70% ethanol, 1% p-phenylenediamine (PPD) was added. This was followed by infiltration and embedding in LR White acrylic resin. Blocks were polymerized at 55 C for 12-20 hours. Ultrathin (80-90 nm) sections were collected on formvar carbon filmed, 150 mesh, nickel grids (Electron Microscopy Sciences, Fort Washington, PA). Sections were labeled by placing them on drops of primary antibody, rabbit anti-rat ET receptor type A (1:50) or rabbit anti-rat ET receptor type B (1:200) (Alomone Labs, Jerusalem), in damp chamber at 4C overnight. Goat anti-rabbit IgG labeled with 12 nm collodial gold (Jackson Immuno Research Laboratories, Inc., West Grove, PA) diluted 1:50 in 2% normal goat serum in Tris buffered saline (TBS), pH 7.6, were used as secondary antibodies. All rinses contained 0.05% Tween 20 and 0.2% Triton-X-100. For the negative controls the primary antibodies were excluded and the grids were incubated on PBS followed by incubation on colloidal gold labeled secondary antibody. To assess aspecific binding, the primary antibodies were preabsorbed with the ET antigen. Detection of antibody binding to specimen after pre absorption is considered aspecific and not taken into account in the analysis. Labeling of endothelial cells and smooth muscle cells was used as positive control since label of ETA and ETB receptors in these cell types have been reported extensively in

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33 the literature. Grids were examined and photographed at 75 kV in a Hitachi H-7000 TEM (NSA Hitachi, Palo Alto, CA). Morphology of Retina and Iridocorneal Angle Retinas from 9 glaucomatous eyes and iridocorneal angles (ICAs) from 11 glaucomatous eyes were examined morphologically. Sections for evaluation of morphology were processed in the same manner as described above. Paraffin sections of the anterior segment were stained with Periodic Acid-Schiff (PAS) solution. Staining with toluidine blue was used for sections of both the anterior and posterior segment embedded in LR White resin. The degree of degeneration in the retina of the glaucoma dogs was graded in 3 groups according to severity: 1) No abnormalities in the nuclear layers, minor atrophy in the nerve fiber and ganglion cell layers; 2) Moderate atrophy and /or melding of the inner and outer nuclear layers; 3) Marked atrophy of all retinal layers. The degree of degeneration of the retina was correlated to the ET-1 levels in aqueous humor and vitreous (see Results; Endothelin-1 levels in aqueous humor and vitreous). Endothelin Receptor Protein Expression in the Retina and Choroid Normal dog eyes (n=11) from 11 dogs and glaucomatous dog eyes (n=13) from 13 dogs were used for protein analysis of the retina and choroid (Tables 2-3 and 2-4). The samples were stored in three different ways; 1) snap-freezing of the retina and choroid in liquid nitrogen with no preservative, 2) separation of the retina and choroid prior to freezing with no preservative, 3) separation of the retina and choroid and put in RNaLater solution (Ambion, Austin, TX) prior to freezing. The samples were stored at -80 C until analysis.

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34 Protein Extraction The protein was either extracted form tissue that was frozen without preservative or from tissue where retina and choroid were separated prior to freezing and stored with or without preservative. Protein extraction from snap-frozen retina and choroid with no preservative. Retina and choroid from each eye were separated prior to homogenization. The samples were sonicated (60 Sonic Dismembrator, Fischer, Atlanta, GA) in 400-500 L of Laemmli sample buffer (Bio-Rad Laboratories, Hercules, CA) containing 50 L -mercaptoethanol, 150 L protease inhibitor cocktail [1 tablet Complete Mini EDTA-free (Roche, Chicago IL) dissolved in 1 mL distilled water], 10 L aprotinin (10mg/mL), 10 L leupeptin (10mg/mL), and 10 L phenylmethylsulfonyl fluoride (PMSF) (40mg/mL) per mL of sample buffer. The samples were centrifuged and the supernatant was transferred into fresh tubes for Bradford colorimetric protein assay (Bio-Rad Laboratories, Hercules, CA). Protein isolation from retina and choroid that was separated prior to freezing and stored with or without preservative. The tissue was homogenized in 1 mL of TRIZOL TM Reagent (Total RNA Isolation Reagent, Life Technologies, Gaithersburg, MD) per 50-100 mg of tissue using a power homogenizer (Ultrasonic celldisrupter, Heat Systems, Ultrasonics Inc., Pharmingdale, NY) following the protocol supplied by the manufacturer. Tissue treated with TRIZOL TM Reagent and chloroform for RNA extraction (see Endothelin Receptor mRNA Expression in the Retina) separated into an aqueous phase and an organic phase. After removal of the aqueous phase, containing RNA, the proteins in the organic phase were recovered by precipitation with isopropanol. Protein

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35 content was determined by Bradford colorimetric protein assay (Bio-Rad Laboratories, Hercules, CA). Western Blot The protein samples were loaded on a 4% stacking, 10% resolving Tris-HCl ready gel (Bio-Rad Laboratories, Hercules, CA). Each gel was loaded with the same amount of protein for each sample. Kaleidoscope Prestained Standards (Bio-Rad Laboratories, Hercules, CA) were used as markers, and rat brain extract (SIGMA, Saint Louis, MI) as control on each gel. The electrophoresis was run at 100 V for 1 hour in a running buffer. The proteins in the gel were then electrotransferred on to a nitrocellulose membrane in a transfer buffer at 60 mA overnight. Western-blotting was done using rabbit anti-rat ET receptor type A (1:100) and rabbit anti-rat ET receptor type B (1:200) (Alomone Labs, Jerusalem) as primary antibodies. The receptors were visualized using ECL Western Blotting System by following the manufacturers protocol (Amersham Pharmacia, Piscataway, NJ). Donkey anti-rabbit horse radish peroxidase-linked antibody in Tris buffered saline/Triton X (TBS/T) (1:10,000) was used as secondary antibody. Detection reagents, supplied with the blotting kit, were incubated with the membrane for 1 minute. The membranes were then exposed to an x-ray film (BMX, Light 1; Fisher, Atlanta, GA) for 10, 30, and 120 minutes. The films were scanned with GS-710 Calibrated Imaging Densitometer (Bio-Rad Laboratories, Hercules, CA) and the density of the ET receptor protein bands on each gel was determined using Scion Image for Windows (Scion Corporation, Frederick, MD). This program visualizes the density profile as a curve with optical density on the Y-axis and millimeter (mm) on the X-axis. The density of the bands was expressed as area under

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36 the curve of each peak. The density measurements of the samples were normalized by dividing the value of each band with the value for the control (rat brain extract) of each gel. This resulted in a value expressed in arbitrary units (AU) for each sample. The AU for the ET receptors were compared between normal and glaucomatous retinal and choroidal samples. Endothelin Receptor and Endothelin-1 mRNA Levels in the Retina Isolation of Total RNA Normal dog eyes (n=9) from 9 dogs and glaucomatous dog eyes (n=10) from 10 dogs were used for mRNA analysis (Tables 2-3 and 2-4). Total RNA was isolated from retinas that had been separated prior to freezing and stored dry or in RNALater (Ambion, Austin, TX) using TRIzol Reagent (Life Technologies, Gaithersburg, MD) following the protocol supplied by the manufacturer. The same retinal samples were used for mRNA determination as for Western Blot analysis (Tables 2-3 and 2-4). The samples were homogenized in 1 ml of TRIZOL TM Reagent per 50-100 mg of tissue using a power homogenizer (Ultrasonic celldisrupter, Heat Systems, Ultrasonics Inc., Pharmingdale, NY). The addition of chloroform followed by centrifugation separated the solution into an aqueous and an organic phase. RNA remained in the aqueous phase. After transfer of the aqueous phase, the RNA was recovered by precipitation with isopropyl alcohol. The organic phase was saved for protein isolation (see Endothelin Receptor Protein Expression in the Retina and Choroid). Potential contaminating DNA in the RNA samples was removed using the DNA-free kit from Ambion (Austin, TX). Optical density (OD) measurements were done on the RNA samples at 260nm.

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37 Reverse Transcriptase Polymerase Chain Reaction Primers for the reverse transcriptase polymerase chain reaction (RT-PCR) were designed using the software Vector NTI (Bethesda, MD) (Table 2-5). The primers used for the ETA receptor were designed from the dog ETA nucleotide sequence published by Louden et al. [97]. The ETB primers were designed from a reported canine endothelin receptor B mRNA sequence (Gen Bank Accession number AF034530). The ET-1 primers were designed from canine ET-1 precursor, mRNA (Gen Bank Accession number AF333433). The amplicon sequences produced by the various primers were later used in the design of the primers for the Real Time PCR assay. All primers for the RT-PCR were purchased from Invitrogen Life Technologies (Frederick, MD). Synthesis of cDNA from total RNA was done using the First strand cDNA synthesis kit from Amersham Pharmacia Biotech (Piscataway, NJ); the designed antisense primers were used to prime the cDNA synthesis. PCR was performed in a total volume of 50 L consisting of 6 L cDNA, 5 L 10X Buffer A [(500 mM KCl, 200 mM Tris-HCl,pH 8.4) Invitrogen, Carlsbad, CA], 1 L 50 mM MgCl2, 1 L dNTP, 1 L 5' primer (30 pmole/ L), 1 L 3' primer (30 pmole/ L), and 0.5 L Taq DNA polymerase (5,000 units/mL) (Invitrogen, Carlsbad, CA). All reactions were run in a DNA thermal cycler, Robocycler (Stratagene, La Jolla, CA), using 30 cycles of denaturation at 95 for 0.5 minute, annealing at 53 for 0.5 minute, and extension at 72 for 1.5 minutes. The last cycle contained 10 minutes at 72. The PCR products were run on a 1 % agarose gel alongside a 1000 bp and a 100 bp DNA marker. PCR products were recovered from the gel using the QIAEX II agarose gel extraction kit (Quigen Inc., Valencia, CA). Purified PCR products were ligated into a TA cloning vector, pCR2.1, and transformed into TOP10 cells (Invitrogen, San Diego, CA).

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38 Plasmids from clones containing inserts were isolated using the QIAprep Miniprep (Quigen Inc., Valencia, CA). The inserts were identified by restriction enzyme digestion with EcoR1 (Promega, Madison, WI) and subsequent agarose gel analysis. Plasmid DNA was then used to evaluate the primers for Real Time PCR. Real Time Polymerase Chain Reaction Primers and probes for the Real Time PCR were designed for the amplicons described above using Primer Express for Mac (Perkin-Elmer Applied Biosystems Inc, Foster City, CA) (Table 2-5). The primers and fluorogenic probes for the Real Time PCR were purchased from Perkin-Elmer Applied Biosystems Inc. (Foster City, CA). Random hexadeonucleotides (pd(N)6 primers provided with the Amersham kit were used to prime the cDNA synthesis. The Real Time probes had a reporter dye (6-FAM) conjugated to the 5' end and a quencher dye (TAMRA) conjugated to the 3' end. The principle of the Taqman system is that the fluorescence from the reporter dye is quenched by the quencher dye as long as the dyes are nearby to each other. The specific probe binds to the cDNA being amplified. When the Taqman polymerase replicates the target cDNA it cleaves the probe that is bound to the cDNA. This results in separation of the reporter and quencher dyes allowing the fluorescence of the reporter dye to be measured. The fluorescence increases in each cycle in proportion to the rate of probe cleavage. The Real Time PCR was performed in a total volume of 25 L consisting of 2X Taqman Universal PCR Master Mix, 300nM each of forward and reverse primers, 80 pM of fluorogenic probe, and 3 ng of template. Ribosomal RNA (rRNA) 18S was used as an endogenous control and active reference to normalize quantities of cDNA. In the reaction for the 18S rRNA the primers and probes were replaced with 20X Eukaryotic 18S rRNA

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39 Endogenous Control (Applied Biosystems, Foster City, CA). The Thermal Profile for the Real Time PCR was 1 cycle for 2 min at 50, 1 cycle for 10 min at 95, followed by 40 cycles involving 15 sec at 95 and 1 min at 60 for each cycle. Fluorescence for each cycle was quantitatively analyzed on a Gene Amp 5700 sequence detection system (Perkin-Elmer Applied Biosystems Inc, Foster City, CA). The relative amount of cDNA in the samples was determined by the threshold cycle (CT). The CT is defined as the fractional cycle number at which the level of fluorescence released from the Real Time Probe surpasses the background threshold (Figure 2-1). The higher the starting copy number of the nucleic acid target, the sooner a significant increase in fluorescence is observed. In other words, the higher amount of cDNA in the sample, the lower the CT value. Each sample was run in triplicate to determine reproducibility of the PCR reaction. As a negative control the Real Time PCR was run without any cDNA as template. In addition, the Real Time PCR was run with total RNA, instead of cDNA, to rule out that genomic DNA was present and amplified in the samples. Cloned DNA sequences of both the ETA and the ETB receptor were amplified with primers and fluorogenic probes for the Real Time PCR to evaluate the selectivity and specificity of the primers and the fluorogenic probes. Amplification of both ET receptor DNAs were analyzed with various combinations of primers and probes, as shown in Table 2-6. Additional controls analyzed aspecific amplification. One set of Real Time PCR reaction was carried out using DNAse treated total RNA, and a PCR reaction was also run with omission of cDNA as a negative control. All reactions were run in triplicates.

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40 Data Analysis Morphology The iridocorneal angles were qualitatively interpreted and a comparison was done between ICAs from normal and glaucomatous eyes. The degree of degeneration in the retina of the glaucoma dogs was graded in 3 groups according to severity: 1) No abnormalities in the nuclear layers, minor atrophy in the nerve fiber and ganglion cell layers; 2) Moderate atrophy and /or melding of the inner and outer nuclear layers; 3) Marked atrophy of all retinal layers. Spearmans correlation coefficient was used to determine any correlation between the degree of degeneration and the ET-1 levels in aqueous humor and vitreous. The distribution of labeling of the ETA and ETB receptors in the retina and choroid was qualitatively interpreted in the normal and glaucomatous sections. Endothelin-1 and Nitric Oxide Levels in Aqueous Humor and Vitreous The data were analyzed using an ANOVA 2*2 factorial analysis in which the two factors of interest were the disease, with two levels (glaucomatous and normal), and breed, with two levels (Cocker and non-Cocker). In the presence of interaction the simple effects of each of the two factors were examined separately. The analyses were done on the means of right and left eye from each dog. All the statistical analyses were performed using SAS (version 8). Endothelin Receptor Protein Expression in the Retina and Choroid The expression of the ET receptors in arbitrary units (AU) versus controls were compared between normal and glaucomatous retinal and choroidal samples using the ANOVA 2*2 factorial design described above. Due to lack of tissue no analysis could be done including normal Cocker choroid. All analyses were done ignoring whether the

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41 measurement was taken from left or right eye. In the case of interaction a separate analysis of the simple effects of each of the two factors, disease and breed, was done. Endothelin-1 and ET Receptor mRNA Levels in the Retina The ratios of the mRNA levels versus controls in AU were compared between normal and glaucomatous retinal samples using the ANOVA 2*2 factorial design described above. The variables were first log-transformed to improve the model-fit. All analyses were done ignoring whether the measurement was taken from left or right eye. In the case of interaction a separate analysis of the simple effects of each of the two factors, disease and breed, was done.

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42 Table 2-1: Normal dogs in study. Dog Breed Age Sex University IOP in mm/Hg at enucleation 1 German Shepherd Mix Adult M UF OD: 15.7 OS: 17.7 2 Hound Dog Adult M UF OD: 11.3 OS: 15.7 3 Beagle Adult M UF OD: 21 OS: 10.3 4 Beagle Adult M UF OD: 17 OS: 20.7 5 Beagle Adult F UF OD: 9 OS: 10 6 Beagle Adult F UF OD: 14.7 OS: 15.3 7 Beagle Adult M UF OD: 19 OS: 18 8 Beagle Adult F UF OD: 16 OS: 16 10 Walker Hound 1 y F UF OD: 14 OS: 17.5 11 Greyhound 10 y F UF OS: 9 12 Greyhound 10 y F UF OD: 8 OS: 7 13 Greyhound 1.5 y F UF 15 Walker Hound 1.5 y F UF 18 Hound Dog 2 y F UF M1 Cocker Spaniel 4 y M MX OD: 13 OS: 12 M2 Cocker Spaniel 6 y M MX OD: 9 OS: 11 M3 Cocker Spaniel 4 y M MX OD: 15 OS: 9 M4 Cocker Spaniel 3 y M MX OD: 26 OS: 25 M5 Cocker Spaniel 6 y F MX OD: 18 OS: 21 y, year; M, Male; F, Female; UF, Small Animal Clinical Sciences, University of Florida; MX, Universidad de Mexico, Mexico City; IOP, Intraocular pressure; OD, Right eye; OS, Left eye; ., Missing data point.

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43 Table 2-2: Glaucoma dogs in study Dog Breed Age Sex University IOP at enucleation ICA Duration ofdisease Fundus appearance 14 Cocker Spaniel 10 years SF UF OD: 40 c 1y -c 16 Bouvier deFlandres 3 years CM UF OD: 29 (49) a c 3 mo -c 17 Beagle 12 years M UF OD: 30 (42) a c 10y -c 19 Cocker Spaniel 13 years CM UF OS: 8 b -c > 4 y c 20 Cocker Spaniel 8 years CM UF OS: 30 (48) a c Chronic -c M6 Poodle 4 years F MX OD: 34 Narrow 1 mo Atrophy ONH Vascular attenuation. 21 Samoyed 6 years SF UF OD: 41 c Chronic -c 22 Cocker Spaniel 13 years SF UF OD: 44 c Chronic -c 23 Cocker Spaniel 12 years SF UF OS: 28 (59) a c 3 mo -c 24 Cocker Spaniel 8 years SF UF OD: 50 2 mo Retinal degeneration 25 Cocker Spaniel 15 years SF UF OS: 45 (55) a c 3 mo -c 26 Mix Terrier 8 years SF UF OS: 30 c 6 mo Cupping ONH 27 Cocker Spaniel 6 years SF UF OS: 37 6 mo Cupping ONH 28 Basset 2 years SF UF OS: 38 c 6 mo -c y, year; M, Male; F, Female; UF, Small Animal Clinical Sciences, University of Florida; MX, Universidad de Mexico, Mexico City; IOP, Intraocular pressure; ICA, Iridocorneal angle; OD, Right eye; OS, Left eye; mo, month; ONH, Optic nerve head. a IOP prior to treatment; b No IOP prior to treatment; c Not visible due to corneal edema.

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Table 2-3: Analyses on normal dogs. Dog ELISA ET-1 peptide and NO Aqueous humor/vitreous Immunocytochemistry ET receptors Retina and choroid Western Blot ET receptors Retina and choroid Real Time PCR ET-1 mRNA Retina Real Time PCR ET receptor mRNA Retina 1 OU 2 OU 3 OU 4 OU 5 OU OS 6 OU OD 7 OU OD OD OD 8 OU OD OD OD 10 OU OS OS OS 11 OS OD OD OS 12 OD 13 OS 15 OU 18 OD M1 OS OS OS OS OS M2 OD OD OD OD OD M3 OD OD OD OD OD M4 OS OS OS OS OS M5 OS OS OS OS OS 44 OD, Right eye; OS, Left eye; OU, Both eyes; ET, Endothelin-1; NO, Nitric oxide; -, No analysis.

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Table 2-4: Analyses on glaucomatous dogs. Dog ELISA ET-1 peptide and NO Aqueous humor/vitreous Immunocytochemistry ET receptors Retina and choroid Western Blot ET receptors Retina and choroid Real Time PCR ET-1 mRNA Retina Real Time PCR ET receptor mRNA Retina 14 OD OD OD OD OD 16 OD OD 17 OD OD 19 OS OS OS OS OS 20 OS M6 OD OD OD OD 21 OD 22 OD OD OD OD OD 23 OS OS OS OS OS 24 OD OD OD OD 25 OS OS OS OS 26 OS OS OS 27 OS OS OS OS 28 OS OS OS OS 45 OD, Right eye; OS, Left eye; ET, Endothelin-1; NO, Nitric oxide; -, No analysis.

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46 Table 2-5: Primers and fluorogenic probes. Reagent Primer Sequence Amplicon size Reverse Transcription PCR ETA receptor 380 bp Sense 5'-GTG GCT CTT CGG GTT CTA TT-3' Anti-sense 5'-GGC ATG ACT GGA AAC AAT TT-3' ETB receptor 566 bp Sense 5'-GAA TTA AAG GAA TTG GGG TTC C-3' Anti-sense 5' GCA GTT TTT GAA TCT TTT GCT C3' Real Time PCR ETA receptor 81 bp Sense 5'-CTT GAG AAT TGC CCT CAG TGA A-3' Anti-sense 5'-GCA AAA ATT ACA ACC AAG CAG AAA-3' Fluorogenic Probe 5'-TTT GCC ACT TCT CGA CGC TGC TTA AGA T-3' ETB receptor 76 bp Sense 5'-GGC CGT GGG TTT TGA TAT GA-3' Anti-sense 5'-CTG GGT AGG ATG AAG CAA GCA-3' Fluorogenic Probe 5'-AAC CAT TGA CTA CAA AGG ACG TTA CCT GCG A-3' Endothelin-1 86 bp Sense 5'-TCC TGC TCT TCC CTG ATG GA-3' Anti-sense 5'-GGA ACA ATG TGC TCA GGA GTG TT-3' Fluorogenic Probe 5'-TGT CTA CTT CTG CCA CCT TGA CAT CAT CTG-3'

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47 Table 2-6: The controls for the Real Time PCR were run with the templates listed in the table. The templates were run with various mixes of primers and probes as listed in the table. ETA, Endothelin A; ETB, Endothelin B; 18S, 20X Eukaryotic 18S rRNA Endogenous Control. Template Primer/probe mix ETA receptor sequence A Primer A Probe A Primer B Probe B Primer B Probe B Primer A Probe 18S ETB receptor sequence A Primer A Probe A Primer B Probe B Primer B Probe B Primer A Probe 18S No Template A Primer A Probe B Primer B Probe 18S Total RNA A Primer A Probe B Primer B Probe 18S

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48 Threshold Threshold Figure 2-1: Real Time PCR amplification curves of a normal and a glaucomatous retina for the ETA receptor and 18S rRNA. ETA, Endothelin receptor A; 18S, 20X Eukaryotic 18S rRNA Endogenous Control; CT, threshold cycle.

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CHAPTER 3 RESULTS Morphology Iridocorneal Angle The iridocorneal angles (ICAs) of the clinically normal dogs revealed no abnormalities (Figure 3-1). The outflow apparatus, including trabecular meshwork and scleral venous plexus was distorted in all glaucomatous eyes (Figure 3-2). The anterior corneoscleral trabecular meshwork was completely collapsed in all eyes. The uveoscleral meshwork was completely collapsed in some eyes, while in others it still had openings of varying sizes (Figure 3-3). In most dogs the remaining trabecular cells had a normal, slender appearance, while they were more rounded in other eyes. Melanin-containing cells were clustered in the angle (Figure 3-4). Anterior synechia were present in several globes (Figures 3-4, 3-5 and 3-6). The termination of Descemets membrane was enlarged in four of the six glaucoma Cocker eyes, but had normal thickness in all eyes of the non-Cocker glaucoma group (Figures 3-4 and 3-5). It was retracted in several eyes (Figures 3-4 and 3-6). The ciliary cleft in all eyes, except for the Beagle (dog 17) was covered by light to heavy amount of connective tissue (Figure 3-4). Retina. The clinically normal dogs showed no abnormalities of the retina (Figure 3-7). Retinal changes in the glaucomatous dogs varied from minor thinning of the ganglion cell layer and disorganization of the inner nuclear layer, to atrophy of all retinal layers 49

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50 (Figures 3-8 and 3-9). The peripheral retina was most affected in all eyes with complete atrophy in 5 cases. A thinning of the inner plexiform layer resulting in closer apposition of the nuclear layers, melding, was noticed as an early sign of atrophy, while the nuclear layers still were intact. The photoreceptor nuclei were normal, with some chromatin clumping, in all glaucomatous eyes. The outer segments of the photoreceptors were swollen in several eyes. The degree of degeneration in the central retina of the glaucoma dogs was graded in 3 groups according to severity: 1) No abnormalities in the nuclear layers, minor atrophy in the nerve fiber and ganglion cell layers; 2) Moderate atrophy and /or melding of the inner and outer nuclear layers; 3) Marked atrophy of all retinal layers (Table 3-1). There was a significant correlation at the 0.1 significance level between the total degree of central retinal degeneration, and total aqueous humor and vitreal ET-1 levels of glaucomatous dogs (Spearman correlation coefficient 0.6, p = 0.0901). Endothelin Receptors in the Retina and Choroid Evaluation of Endothelin Antibodies using Light Microscopy Light microscopy showed a weak but distinct label for ET receptors of small scleral vessels of a normal Beagle. No labeling was detected when the primary antibody was omitted. (Figure 3-10). Evaluation of Endothelin Antibodies using Transmission Electron Microscopy There was no labeling when PBS was used instead of the primary antibody. The mixture of ETA antibody with its antigen resulted in very minor labeling. In contrast, the mixture of ETB antibody with its antigen showed only a small decrease in labeling as compared to the primary antibody. Intensity of the specific labeling corresponded to the concentration of the antibody dilution. The most distinct and specific labeling was

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51 achieved with the ETB antibody at a dilution of 1:200. The use of detergents was necessary to minimize extensive nonspecific labeling by the ETB antibody. The difficulties in achieving an optimal protocol with reliable specific label by the ETB antibody excluded any quantitative analysis of the immunocytochemistry. Localization of Endothelin Receptors using Transmission Electron Microscopy The distribution of ET receptor labeling was similar in the normal and glaucomatous sections for both receptors. ETA receptor The ETA receptor was observed in smooth muscle cells and pericytes of vessels in both the retina and the choroid. ETB receptor Localization of the ETB receptor occurred within vascular endothelium, smooth muscle, and pericytes of vessels in both the retina and the choroid (Figure 3-11). Endothelin-1 Levels in Aqueous Humor, Vitreous and Retina The ranges for aqueous humor and vitreal ET-1 levels all normal samples were 0.87 6.3 and 0.23 6.83 pg/mL respectively. The ET-1 aqueous humor and vitreal ranges for all glaucomatous samples were 1.91 14.56 and 1.13 5.96 pg/mL respectively. The ET-1 levels for normal and glaucomatous dogs are shown in Tables 3-2 and 3-3 and the least-squares means from the ANOVA 2*2 factorial analysis are plotted in Figures 3-12 and 3-13. Analysis of Aqueous Humor The mean ( STD) normal and glaucomatous canine ET-1 aqueous levels for all breeds were 2.8 ( 1.52) and 6.86 ( 3.46) pg/mL respectively. There was a significant increase in ET-1 for the glaucomatous eyes (versus the normal eye) (p = 0.0004). The

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52 differences between least-squares means were also analyzed for each level of the two factors due to the presence of interaction. In the Cocker group, there was a significant increase in ET-1 for the glaucomatous eyes (p = 0.0003), while for the non-Cocker group the difference between the normal and glaucomatous eyes was not significant (Figure 3-12). There was no significant difference between the Cocker group and the non-Cocker group nor for the normal or for the glaucomatous eyes. Analysis of Vitreous The mean ( STD) normal and glaucomatous canine ET-1 vitreous levels for all breeds were 1.56 ( 1.86) and 3.47 ( 1.57) pg/mL respectively. The increase in the glaucomatous eyes was considered statistically significant (p = 0.0023). Due to the presence of interaction between the two factors, breed and disease, the differences between least-squares means were analyzed for each level of the two factors. In the Cocker group, there was a significant increase in ET-1 for the glaucomatous eyes (p = 0.0005), while for the non-Cocker group the difference between the normal and glaucomatous eyes was not significant (Figure 3-13). There was a significant difference between the Cocker group and the non-Cocker group (p = 0.0099) for the normal eyes but not for the glaucomatous eyes Endothelin-1 mRNA Levels in the Retina There was insufficient statistical evidence to indicate any significant breed or disease effect on the retinal mRNA levels of ET-1; breed by disease interaction was not found significant either (Table 3-4).

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53 Correlation of Retinal Degeneration and ET-1 Levels in Aqueous Humor and Vitreous The degree of degeneration of the central dorsal retina and central ventral retina was correlated to the ET-1 levels in aqueous humor and vitreous. The grading of the retinal degeneration is described in the Morphology section and the ranking of the glaucomatous central retinas is shown in Table 3-1. The gradual decrease in thickness of the cell layers in the periphery of normal eyes lead to difficulties in comparing and grading any changes in the periphery of the glaucomatous eyes. The peripheral retina was therefore excluded from the correlation analysis. There was a significant correlation at the 0.1 significance level between the total degree of central retinal degeneration, and total aqueous humor and vitreal ET-1 levels of glaucomatous dogs [Spearman correlation coefficient 0.6, p = 0.0901) (Figure 3-14)]. Nitric Oxide Levels in Aqueous Humor and Vitreous The ranges for all normal aqueous humor and vitreal NO levels were 1.41 11.46 and 0.24 16.47 M respectively. The NO aqueous humor and vitreal ranges for all glaucomatous dogs were 2.25 37.94 and 1.07 46.98 M respectively. The NO levels for normal and glaucoma dogs are shown in Tables 3-5 and 3-6 and the least-squares means from the ANOVA 2*2 factorial analysis are plotted in Figures 3-15 and 3-16. Analysis of Aqueous Humor The mean normal and glaucomatous canine NO aqueous levels for all breeds were 3.9 ( 1.93) and 12.48 ( 13.46) M respectively. The increase in the glaucomatous eyes of the non-Cocker group was considered statistically significant (p = 0.0476). There was insufficient evidence to indicate that the two factors, disease and breed, interacted (Figure 3-15).

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54 Analysis of Vitreous The mean normal and glaucomatous canine NO vitreal levels for all breeds were 4.47 ( 2.92) and 15.33 ( 16.22) M respectively. The increase in the glaucomatous eyes was considered statistically significant (p = 0.0085) but due to the presence of interaction, the differences between least-squares means were analyzed for each level of the two factors. In the non-Cocker group, there was a significant increase in NO for the glaucomatous eyes (p = 0.0148), while for the Cockers the difference between the normal and glaucomatous eyes was not significant (Figure 3-16). There was no significant difference between the Cocker group and the non-Cocker group neither for the normal eyes nor for the glaucomatous eyes. Endothelin Receptor Protein Expression in the Retina and Choroid The ratios of the expression levels of ETA versus control, and ETB versus control in the retina and choroid for normal and glaucoma dogs are shown in Tables 3-7 and 3-8 and the least-squares means from the ANOVA 2*2 factorial analysis model are plotted in Figures 3-17 and 3-18. ETA Receptors in the Retina There was a statistically insignificant reduction of the ETA receptors in the retina of the glaucomatous eyes. There was no evidence to indicate that the two factors interacted (Figure 3-17). ETB Receptors in the Retina There was a significant difference in the ETB receptors between the glaucomatous and normal dogs (p = 0.0386); since the interaction between the breed and disease was significant, the simple effects of the two factors were investigated separately. For the non-Cocker group, there was a significant decrease in the ETB receptors between the

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55 glaucomatous and normal dogs (p = 0.0072). Also for the normal eyes, the level of ETB receptors was significantly lower for the Cocker group versus the non-Cocker group. (p = 0.0025) (Figure 3-18). ETA and ETB Receptors in the Choroid Choroidal tissues from normal and glaucomatous dogs in the non-Cocker group were compared. There was a decrease of both receptors in the glaucomatous eyes that was not statistically significant. The Cocker group had a higher expression of the ETA receptor and a lower expression of the ETB receptor than the non-Cocker group, in a comparison of glaucomatous choroidal tissue. Neither difference between the two groups was significant. Endothelin Receptor mRNA Levels in the Retina Parameters for normal and glaucomatous dogs are shown in Tables 3-9 and 3-10 and the least-squares means from the ANOVA 2*2 factorial analysis model are plotted in Figures 3-19 and 3-20. Evaluation of the Real Time PCR Reaction Endothelin A receptor plasmid DNA was amplified with a mix of the ETA receptor primer and ETA receptor fluorogenic probe (A/A), showing an early amplification of the DNA sequence above background in the 13th cycle of the Real Time PCR (Figure 3-21). The mixtures of the ETA receptor primer with ETB receptor fluorogenic probe (A/B), and the ETB receptor primer with ETA receptor fluorogenic probe (B/A), gave only background noise below detectable level. DNA amplification was detected above background when the ETB receptor primer with ETB receptor fluorogenic probe (B/B) was used on ETA DNA. Amplification above background was also seen of 18S rRNA using the 20X Eukaryotic 18S rRNA Endogenous Control on ETA DNA.

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56 Endothelin B receptor plasmid DNA was amplified with the mix of ETB receptor primer with ETB receptor probe (B/B) showing an early amplification of the DNA sequence above background in the 12th cycle of the Real Time PCR (Figure 3-22). The mixtures A/B and B/A give only background noise below detectable level. Amplification was detected above background when the ETA receptor primer with ETA receptor probe (A/A) was used on ETB cDNA. Amplification above background was also seen of 18S rRNA using the 20X Eukaryotic 18S rRNA Endogenous Control on ETB cDNA. Since the amplifications by the mismatched primer/fluorogenic probes were detected after the 29th cycle for both ET receptors, these signals are probably due to PCR artefacts. The difference with the true match of primer and fluorogenic probe is more than 15 cycles, showing that there was no cross-reactivity between different primers and probes. No amplification curves of the ET receptors were seen when the Real Time PCR, with the primers and probes for the ET receptors and 18S, was run with no template. This showed that there was no nonspecific amplification due to contamination of solutions. When using total RNA as template, with the primers and probes for the ET receptors and 18S, no amplification curves were seen for the receptors (Figure 3-23). This proved that there was no genomic DNA in the RNA samples. Traces of 18S rRNA were amplified in both reactions. Tissue The tissue had been stored in two different ways, i.e. frozen dry or frozen in RNase Later preservative. It was therefore necessary to check whether this affected the RNA in the samples. This was done by extracting RNA from two normal dog retinas, one that had been stored frozen in the preservative RNAlater (Retina 1) and the other (Retina 2) stored

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57 frozen dry without any preservative. The yields of total RNA from the samples were 2.1 and 1.9 mg/mL respectively. Both samples were split in half. One half of each sample was treated with DNase to eliminate any residual genomic DNA. RT-PCR was then run on total RNA and DNAse treated RNA for each sample (Figure 3-24). The primers used for the reaction are listed in Table 2-5. The detected bands of the ET receptors and -actin were identical for all samples indicating that the two methods of storage did not alter the size or configuration of the mRNA. The same samples (Retina 1 and 2) were run with the Real Time PCR as for the RT-PCR. Both samples were run in triplicate. The primers used for the reaction are listed in Table 2-5. The mean CT values for the ETA receptor from the two samples were 28.5 (Retina 1) and 28.64 (Retina 2) respectively (Figure 3-25) and for the ETB receptor 26.8 (Retina 1) and 26.6 (Retina 2) respectively. This showed that the two methods of storage had minimal effect on the initial amount of mRNA and amplification of the cDNA of the samples. ETA Receptor mRNA in the Retina There was a significant increase in the mRNA levels (p = 0.0347) in the glaucomatous retinas but it was confounded by interaction. For the Cocker group there was a highly significant increase in mRNA levels (p = 0.0064) between the glaucomatous and normal eyes. There was a small, but statistically insignificant, increase in the non-Cocker group. There was also a significant difference in the glaucomatous eyes with the Cockers having significantly higher levels, at a significance level of 0.1, than the non-Cocker group (p = 0.0917) (Figure 3-19).

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58 ETB Receptor mRNA in the Retina There was a significant increase in the mRNA levels at a significance level of 0.1 (p = 0.0854) in the glaucomatous retinas, but it was confounded by interaction. For the Cocker group the mRNA level was significantly higher for the glaucomatous eyes than for the normal eyes (p = 0.0075). The mRNA level in glaucomatous eyes of the non-Cocker group was slightly lower than for the normal eyes, yet not significant. There was no significant difference in mRNA levels in normal or glaucomatous eyes between the Cocker group and the non-Cocker group (Figure 3-20). Summary of Results Results for all breeds are summarized below. All iridocorneal angles in the glaucomatous eyes were distorted with varying degree of collapse. Retinal changes in the glaucomatous eyes varied from minor thinning of the ganglion cell layer and disorganization of the inner nuclear layer, to complete atrophy of all retinal layers. The peripheral retina was most affected in all eyes. The degree of degeneration of the central retina was positively correlated to the ET-1 levels in aqueous humor and vitreous. The location of ET receptor labeling was similar in the normal and glaucomatous retinal and choroidal sections for both ET receptors. The results for the glaucomatous eyes separating the Cocker and the non-Cocker group are shown in Table 3-11.

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59 Table 3-1: Ranking of degree of degeneration in the glaucomatous retinas. Dog Dorsal Central Ventral Central Total mean 14 2 2 2 16 1 2 1.5 17 1 2 1.5 19 1 1 1 M6 1 2 1.5 22 2 2 2 23 3 3 3 24 2 2 2 25 1 2 1.5

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60 Table 3-2: Endothelin-1 levels in pg/mL for normal dogs. BQL, Below limit of quantification; ., Missing data point; OS, Left eye; OD, Right eye; ET-1, Endothelin-1. Dog Breed ET-1 aqueous ET-1 vitreous 1OS German Shepherd Mix 2.01 1.53 1OD 3.56 0.7 2OS Hound Dog 1.95 0.7 2OD 3.5 1.65 3OS Beagle 2.43 0.64 3OD 2.31 2.49 4OS Beagle 1.42 BQL 4OD 2.07 6.83 5OS Beagle 2.84 1.24 5OD 1.77 3.92 6OS Beagle 2.37 1.53 6OD 1.24 5.64 7OS Beagle 2.49 1.47 7OD 1.65 3.38 8OS Beagle 2.73 2.19 8OD 3.38 0.4 10OS Walker Hound 4.81 4.63 10OD 6.3 3.02 11OS Greyhound 5.29 4.75 12OS Greyhound 5.88 1.06 12OD 5.82 2.84 15OS Walker Hound 2.11 15OD 1.67 M1 OS Cocker 1.7 BQL M2 OD Cocker 0.87 0.32 M3 OD Cocker 0.99 0.23 M4 OS Cocker 2.31 0.48 M5 OS Cocker 3.04 0.29 Non-cocker mean (STD) 3.03 (.54) 2.53 (.82) Cocker mean (STD 1.78 (.91) 0.33(.11) Total mean (STD) 2.80 (.52) 1.56 (.86)

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61 Table 3-3: Endothelin-1 levels in pg/mL for glaucomatous dogs. OD, Right eye; OS, Left eye; ET-1, Endothelin-1. Dog Breed ET-1 aqueous ET-1 vitreous 14 OD Cocker 6.7 5.2 19 OS Cocker 2.52 2.68 22 OD Cocker 4.95 2.09 23 OS Cocker 9.67 4.06 24 OD Cocker 6.8 2.88 25 OS Cocker 8.58 3.16 27 OS Cocker 14.56 5.96 M6 OD Poodle 8.48 1.7 16 OD Bouvier de Flandres 1.91 1.13 17 OD Beagle 5.55 4.05 28 OS Basset 5.6 5.28 Non-cocker mean (STD) 5.38 (.69) 3.04 (.96) Cocker mean (STD) 7.7 (.52) 3.72 (.42) Total mean (STD) 6.86 (.49) 3.47 (.57)

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62 Table 3-4: Mean ratios of ET-1/ 18S mRNA in arbitrary units for normal and glaucomatous retinal samples. ET-1, Endothelin-1; 18S, 20X Eukaryotic 18S rRNA Endogenous Control. Normal Glaucoma Dog Breed ET-1/18S Dog Breed ET-1/18S 7 Beagle 2.05 14 Cocker 2.15 8 Beagle 2.14 19 Cocker 2.12 10 Walker Hound 2.16 22 Cocker 1.94 11 Greyhound 2.19 23 Cocker 2.18 18 Hound Dog 1.95 24 Cocker 2.23 M1 Cocker 2.06 25 Cocker 1.82 M2 Cocker 2.07 26 Mix Terrier 2.09 M3 Cocker 2.12 27 Cocker 2.07 M4 Cocker 2.09 28 Basset 2.18 M5 Cocker 2.0 M6 Poodle 2.21 Non-cocker mean(STD) 2.1 (.1) Non-cocker mean (STD) 2.14 (.07) Cocker mean (STD) 2.07 (.04) Cocker mean (STD) 2.07 (.16) Total mean (STD) 2.08 (.07) Total mean (STD) 2.1 (.13)

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63 Table 3-5: Nitrate (NO) levels in M of normal dogs. BQL, Below limit of quantification; NO, Nitric oxide; ., Missing data point; OS, Left eye; OD, Right eye. Dog Breed NO aqueous NO vitreous 1OS German Shepherd Mix 3.67 2.7 1OD 2.51 3.92 2OS Hound Dog 1.41 10.91 2OD 3.75 4.11 3OS Beagle 1.41 1.19 3OD 7.02 2.32 4OS Beagle 3.26 4 OD 4.3 5 OS Beagle BQL 5 OD 2.23 6OS Beagle 11.46 4.4 6OD 3.84 1 7OD Beagle 2.75 3.73 8OS Beagle 2.58 BQL 8OD 2.58 9.02 10OS Walker Hound 3.73 3.26 10OD 7.6 4.4 11OS Greyhound 8.03 0.24 12OS Greyhound 7.61 2.79 12OD 3.25 16.47 13 OS Greyhound 1.66 15OS Walker Hound BQL 15OD 3.08 M1 OS Cocker 1.58 5.18 M2 OD Cocker 2.75 8.36 M3 OD Cocker 3.42 7.94 M4 OS Cocker 2.5 3.08 M5 OS Cocker 3.17 2.08 Non-cocker mean (STD) 4.51 (.09) 4.12 (.0) Cocker mean (STD) 2.68 (.71) 5.33 (.81) Total mean (STD) 3.9 (.93) 4.47 (.92)

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64 Table 3-6: Nitrate (NO) levels in M. of glaucomatous dogs. BQL, Below limit of quantification; NO, Nitric Oxide; ., Missing data point; OS, Left eye; OD, Right eye. Dog Breed NO aqueous NO vitreous 14 OD Cocker 6.52 9.28 16 OD Bouvier de Flandres 6.10 12.8 17 OD Beagle 8.7 M6 OD Poodle 34.17 33.16 19 OS Cocker 4.34 7.02 22 OD Cocker 8.53 10.46 23 OS Cocker 2.25 1.83 24 OD Cocker 3.75 1.07 25 OS Cocker 37.94 46.98 Non-cocker mean (STD) 16.33 (.51) 22.98 (.4) Cocker mean (STD) 10.56 (.6) 12.77 (.19) Total mean (STD) 12.48 (.46) 15.33 (.22)

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Table 3-7: Ratios of ETA receptor/ control protein in arbitrary units for normal and glaucomatous retinas and choroids. ETA, Endothelin A; ., Missing data point. Normal Glaucoma Dog Breed ETA /control Retina ETA /control Choroid Dog Breed ETA/control Retina ETA/control Choroid 5 Beagle 2.26 1.16 14 Cocker 0.87 5.66 6 Beagle 2.83 2.44 16 Bouvier de Flandres 0.29 7 Beagle 3.69 7.26 17 Beagle 4.04 0.43 8 Beagle 6.55 19 Cocker 0.04 5.10 10 Walker Hound 0.78 7.61 21 Samoyed 1.25 1.95 11 Greyhound 6.08 22 Cocker 0.49 0.36 M1 Cocker 3.69 23 Cocker 1.90 6.06 M2 Cocker 3.29 24 Cocker 0.22 4.25 M3 Cocker 2.36 25 Cocker 2.49 5.27 M4 Cocker 2.49 26 Mix Terrier 1.08 6.7 M5 Cocker 1.11 27 Cocker 6.97 8.69 28 Basset 1.38 8.18 M6 Poodle 0.48 Non-cocker mean (STD) 2.39 (.22) 5.18 (.71) Non-cocker mean (STD) 1.64 (.38) 3.51 (.68) Cocker mean (STD) 2.59 (.99) N/A Cocker mean (STD) 1.86 (.43) 5.06 (.49) Total mean (STD) 2.04 (.87) N/A Total mean (STD) 1.77 (.98) 4.41 (.99) 65

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Table 3-8: Ratios of ETB receptor/ control protein in arbitrary units for normal and glaucomatous retinas and choroids. ETB, Endothelin B; ., Missing data point. Normal Glaucoma Dog Breed ETB /control Retina ETB /control Choroid Dog Breed ETB/control Retina ETB/control Choroid 5 Beagle 5.17 2.18 14 Cocker 0.35 0.06 6 Beagle 0.98 0.95 16 Bouvier de Flandres 0.17 7 Beagle 2.04 1.44 17 Beagle 0.86 0.22 8 Beagle 1.35 0.23 19 Cocker 0.12 0.03 10 Walker Hound 0.84 0.35 21 Samoyed 0.81 1.24 11 Greyhound 0.10 22 Cocker 0.26 0.09 M1 Cocker 0.14 23 Cocker 0.12 0.08 M2 Cocker 0.12 24 Cocker 0.09 0.29 M3 Cocker 0.16 25 Cocker 0.08 0.04 M4 Cocker 0.14 26 Mix Terrier 0.28 0.11 M5 Cocker 0.21 27 Cocker 0.02 0.08 28 Basset 0.07 0.22 M6 Poodle 0.05 Non-cocker mean (STD) 2.08 (.79) 0.88 (.82) Non-cocker mean (STD) 0.41 (.39) 0.39 (.48) Cocker mean (STD) 0.16 (.03) N/A Cocker mean (STD) 0.15 (.11) 0.09 (.09) Total mean (STD) 1.12 (.57) N/A Total mean (STD) 0.26 (.29) 0.22 (.33) 66

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Table 3-9: Mean ratios of ETA receptor/ 18S mRNA in arbitrary units for normal and glaucomatous retinal samples. ETA, Endothelin A; 18S, 20X Eukaryotic 18S rRNA Endogenous Control. Normal Glaucoma Dog Breed ETA /18S Dog Breed ETA /18S 7 Beagle 2.18 14 Cocker 1.80 8 Beagle 2.32 19 Cocker 1.85 10 Walker Hound 2.00 22 Cocker 2.03 11 Greyhound 2.08 23 Cocker 1.87 M1 Cocker 2.19 24 Cocker 1.92 M2 Cocker 2.09 25 Cocker 2.07 M3 Cocker 2.12 26 Mix Terrier 2.15 M4 Cocker 2.21 27 Cocker 2.17 M5 Cocker 2.31 28 Basset 2.01 M6 Poodle 2.14 Non-cocker mean (STD) 2.15 (.14) Non-cocker mean (STD) 2.1(.08) Cocker mean (STD) 2.18 (.09) Cocker mean (STD) 1.96 (.13) Total mean (STD) 2.17 (.11) Total mean (STD) 2.00(.13) 67

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Table 3-10: Mean ratios of ETB receptor/ 18S mRNA in arbitrary units for normal and glaucomatous retinal samples. ETB, Endothelin B; 18S, 20X Eukaryotic 18S rRNA Endogenous Control. Normal Glaucoma Dog Breed ETB /18S Dog Breed ETB /18S 7 Beagle 2.13 14 Cocker 1.9 8 Beagle 2.17 19 Cocker 1.76 10 Walker Hound 2.01 22 Cocker 2.05 11 Greyhound 1.99 23 Cocker 1.98 M1 Cocker 2.21 24 Cocker 1.99 M2 Cocker 2.09 25 Cocker 2.02 M3 Cocker 2.09 26 Mix Terrier 2.13 M4 Cocker 2.17 27 Cocker 2.04 M5 Cocker 2.20 28 Basset 1.89 M6 Poodle 2.22 Non-cocker mean (STD) 2.08 (.09) Non-cocker mean (STD) 2.08 (.17) Cocker mean (STD) 2.15 (.06) Cocker mean (STD) 1.96 (.1) Total mean (STD) 2.12 (.08) Total mean (STD) 2.00 (.13) 68

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69 Table 3-11: Summary of results for the glaucomatous eyes separating the Cocker Spaniel group and the non-Cocker group. Parameter Glaucoma, Cocker Spaniels ET-1 peptide Aqueous humor Vitreous Increase, p = 0.0003 Increase, p = 0.0005 Increase, p = 0.1144 Increase, p = 0.5062 ET-1 mRNA Retina Increase, p = 0.9569 Decrease, p = 0.4273 Nitric Oxide Aqueous humor Vitreous Increase, p = 0.1421 Increase, p = 0.2003 Increase, p = 0.0476 Increase, p = 0.0148 ET receptor protein expression ETA Retina ETB Retina ETA Choroid a, b ETB Choroid a, b Decrease, p = 0.4826 Decrease, p = 0.9916 Higher expression, p = 0.4024 a Lower expression, p = 0.1291 a Decrease, p = 0.5319 Decrease, p = 0.0072 Decrease, p = 0.4066 b Decrease, p = 0.2759 b ET receptor mRNA ETA Retina ETB Retina Increase, p = 0.0064 Increase, p = 0.0075 Increase, p = 0.6248 Decrease, p = 0.9617 Glaucoma, Non-Cockers a No normal choroidal cocker tissue. Choroid from glaucomatous Cockers compared with choroid from glaucomatous non-Cockers. b Normal versus glaucomatous. The level of significance was set at 0.05.

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70 Figure 3-1: Normal iridocorneal angle stained with toluidine blue (Dog M3, original magnification 100X). A) Iris. B) Anterior chamber. C) Pectinate ligament. D) Uveal trabecular meshwork. E) Corneoscleral trabecular meshwork. F) Angular aqueous plexus/sinus. Figure 3-2: Iridocorneal angle of a glaucomatous dog (Dog 20, original magnification 40X, PAS stained). A) Compressed trabecular meshwork. B) Connective tissue at the entrance of the angle.

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71 Figure 3-3: Trabecular meshwork of a glaucomatous dog (Dog 25, original magnification 200X, PAS stained). A) The uveoscleral meshwork has openings of varying sizes. Figure 3-4: Iridocorneal angle of a glaucomatous dog (Dog 19, original magnification 200X, PAS stained). Evidence of previous synechia is present: A) Fibrosis lining the inner surface of Descemets membrane. B) Termination of Descemets membrane is enlarged. The entrance of the angle is covered: C) By connective tissue. D) By melanin-containing cells.

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72 Figure 3-5: Iridocorneal angle of a glaucomatous dog (Dog 22, original magnification 100X, PAS stained). A) Synechia between the cornea and the iris. B) Enlarged termination of Descemets membrane. Figure 3-6: Iridocorneal angle of a glaucomatous dog (Dog 26, original magnification 100X, PAS stained). A) Fibrous tissue covering the endothelium of the cornea.

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73 Figure 3-7: Semi-thin (1m) sections of the retina from a normal dog (Dog M4, original magnification 400X) stained with toluidine blue. The sections are from four different locations: DC) Central retina dorsal of the optic nerve head. VC) Central retina ventral of the optic nerve head. DP) Peripheral retina dorsal of the optic nerve head. VP) Peripheral retina ventral of the optic nerve head. a, nerve fiber layer; b, ganglion cell layer; c, inner nuclear layer; d, outer nuclear layer; e, choroid.

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74 Figure 3-8: Semi-thin (1 m) sections of the retina from a glaucomatous dog (Dog 24, original magnification 400X) stained with toluidine blue. The sections are from four different locations: DC) Central retina dorsal of the optic nerve head. VC) Central retina ventral of the optic nerve head. DP) Peripheral retina dorsal of the optic nerve head. VP) Peripheral retina ventral of the optic nerve head. a, nerve fiber layer; b, ganglion cell layer; c, inner nuclear layer; d, outer nuclear layer; e, choroid; f, ganglion cell; artifact.

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75 Figure 3-9: Semi-thin (1 m) sections of the retina from a glaucomatous dog (Dog 25, original magnification 400X) stained with toluidine blue. The sections are from four different locations: DC) Central retina dorsal of the optic nerve head. VC) Central retina ventral of the optic nerve head. DP) Peripheral retina dorsal of the optic nerve head. VP) Peripheral retina ventral of the optic nerve head. a, nerve fiber layer; b, ganglion cell layer; c, inner nuclear layer; d, outer nuclear layer; e, choroid; complete atrophy.

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76 Figure 3-10: Label of normal canine scleral vessels and elastic fibers with the ETB antibody. A) 1:50 dilution at original magnification of 100X. B) 1:50 dilution at original magnification of 200X. C) Control where the primary antibody is replaced with PBS, original magnification 100X. D) Control where the primary antibody is replaced with PBS, original magnification 200X.

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77 Figure 3-11: Arteriole of the choroid of a normal dog. A) Smooth muscle and endothelial cells labeled with ETB antibody (1:100) at original magnification of 10,000X. B) Smooth muscle and endothelial cells labeled with ETB antibody (1:100) at original magnification of 20,000X.

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78 A ET-1 Aqueous0246810 Normal Glaucoma Cocker Other p = 0.0003pg/ml ET-1 Aqueous0246810 Normal Glaucoma Cocker Other p = 0.0003 ET-1 Aqueous0246810 Normal Glaucoma Cocker Other p = 0.0003pg/ml B ET-1 Aqueous0246810Cocker Other Normal Glaucoma pg/ml ET-1 Aqueous0246810Cocker Other Normal Glaucoma pg/ml Figure 3-12: Endothelin-1 in aqueous humor of normal and glaucomatous eyes (see Tables 3-2 and 3-3). A) In the Cocker group, there is a significant increase in ET-1 for the glaucomatous eyes (p = 0.0003), while for the non-Cocker group the difference between the normal and glaucomatous eyes is not significant. B) There is no significant difference between Cockers and non-Cockers neither for the normal nor for the glaucomatous eyes. ET-1, Endothelin-1

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79 A ET-1 Vitreous01234Normal Glaucoma Cocker Other p = 0.0005pg/ml ET-1 Vitreous01234Normal Glaucoma Cocker Other p = 0.0005 ET-1 Vitreous01234Normal Glaucoma Cocker Other p = 0.0005p = 0.0005pg/ml B ET-1 Vitreous01234Cocker Other Normal Glaucoma p = 0.0099pg/ml ET-1 Vitreous01234Cocker Other Normal Glaucoma p = 0.0099 ET-1 Vitreous01234Cocker Other Normal Glaucoma p = 0.0099pg/ml Figure 3-13: Endothelin-1 in vitreous of normal and glaucomatous eyes (see Tables 3-2 and 3-3). A) In the Cocker group, there is a significant increase in ET-1 for the glaucomatous eyes (p = 0.0005), while for the non-Cocker group the difference between the normal and glaucomatous eyes is not significant. B) There is a significant difference between the Cocker group and the non-Cocker group (p = 0.0099) for the normal eyes but not for the glaucomatous eyes. ET-1, Endothelin-1.

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80 0123 4 246810121416 ET-1 levels aqueous + vitreous, pg/mlMinor degenerationSevere degeneration 0123 4 246810121416 ET-1 levels aqueous + vitreous, pg/mlMinor degenerationSevere degeneration Figure 3-14: Scatter plot showing the correlation between the degree of degeneration in central retina and total aqueous humor and vitreal ET-1 levels of glaucomatous dogs (Spearman correlation coefficient 0.6, p = 0.0901). ET-1, Endothelin-1

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81 A NO Aqueous 05101520Normal Glaucoma Cocker Other p = 0.0476M NO Aqueous 05101520Normal Glaucoma Cocker Other p = 0.0476 NO Aqueous 05101520Normal Glaucoma Cocker Other p = 0.0476M B NO Aqueous05101520Cocker Other Normal Glaucoma M NO Aqueous05101520Cocker Other Normal Glaucoma M Figure 3-15: Nitric oxide in aqueous humor of normal and glaucomatous eyes (see Tables 3-5 and 3-6). A) There is a significant difference between NO levels in normal and glaucomatous eyes for the non-Cocker group (p = 0.0476); but not for the Cocker group. B) There is no significant difference between Cockers and non-Cockers neither for the normal nor for the glaucomatous eyes. NO, Nitric Oxide.

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82 A NO Vitreous0510152025Normal Glaucoma Cocker Other p = 0.0148M NO Vitreous0510152025Normal Glaucoma Cocker Other p = 0.0148 NO Vitreous0510152025Normal Glaucoma Cocker Other p = 0.0148M B NO Vitreous0510152025Cocker Other Normal Glaucoma M NO Vitreous0510152025Cocker Other Normal Glaucoma M Figure 3-16: Nitric oxide in vitreous of normal and glaucomatous eyes (see Tables 3-5 and 3-6). A) In the non-Cocker group, there is a significant increase in NO for the glaucomatous eyes (p = 0.0148), while for the Cockers the difference between the normal and glaucomatous eyes is not significant. B) There is no significant difference between the Cocker group and the non-Cocker group neither for the normal eyes nor for the glaucomatous eyes. NO, Nitric Oxide.

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83 A ETA Receptor Retina00.511.522.53Normal Glaucoma Cocker Other AU ETA Receptor Retina00.511.522.53Normal Glaucoma Cocker Other AU B ETA Receptor Retina00.511.522.53Cocker Other Normal Glaucoma AU ETA Receptor Retina00.511.522.53Cocker Other Normal Glaucoma AU Figure 3-17: Endothelin receptor A protein in the retina of normal and glaucomatous eyes (see Table 3-7). A) There is no significant difference between the normal and glaucomatous eyes. B) There is no interaction with breed. ETA, Endothelin A.

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84 A ETB Receptor Retina00.511.522.5Normal Glaucoma Cocker Other p = 0.0072AU ETB Receptor Retina00.511.522.5Normal Glaucoma Cocker Other p = 0.0072 ETB Receptor Retina00.511.522.5Normal Glaucoma Cocker Other p = 0.0072AU B ETB Receptor Retina00.511.522.5Cocker Other Normal Glaucoma p = 0.0025AU ETB Receptor Retina00.511.522.5Cocker Other Normal Glaucoma p = 0.0025 ETB Receptor Retina00.511.522.5Cocker Other Normal Glaucoma p = 0.0025AU Figure 3-18: Endothelin receptor B protein in the retina of normal and glaucomatous eyes (see Table 3-8). A) The difference between normal and glaucomatous eyes of the non-Cocker group (p = 0.0072) is highly significant while the small decrease of the Cocker group is not significant. B) There is a difference between normal eyes with the non-Cocker group being significantly higher than the Cocker group (p = 0.0025) (B). ETB, Endothelin B.

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85 A ETA mRNA Retina0.260.280.30.320.340.36Normal Glaucoma Cocker Other p = 0.0064log of AU ETA mRNA Retina0.260.280.30.320.340.36Normal Glaucoma Cocker Other p = 0.0064 ETA mRNA Retina0.260.280.30.320.340.36Normal Glaucoma Cocker Other p = 0.0064log of AU B ETA mRNA Retina0.260.280.30.320.340.36Cocker Other Normal Glaucoma p = 0.0917log of AU ETA mRNA Retina0.260.280.30.320.340.36Cocker Other Normal Glaucoma p = 0.0917 ETA mRNA Retina0.260.280.30.320.340.36Cocker Other Normal Glaucoma p = 0.0917log of AU Figure 3-19: Endothelin receptor A mRNA in the retina of normal and glaucomatous eyes (see Table 3-9). A) The increase in the Cocker group is highly significant (p = 0.0064). The small increase in the non-Cocker group is not significant. B) The difference between groups of the glaucomatous eyes is significant at a 0.1 significance level (p = 0.0917). ETA, Endothelin A.

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86 A B ETB mRNA Retina0.26 0.28 0.3 0.32 0.34 Cocker Other Normal Glaucoma log of AU ETB mRNA Retina0.26 0.28 0.3 0.32 0.34 Cocker Other Normal Glaucoma log of AU Figure 3-20: Endothelin receptor B mRNA in the retina of normal and glaucomatous eyes (see Table 3-10). A) The increase in the glaucomatous eyes in the Cocker group is highly significant (p = 0.0075). B) There is no significant difference in normal or glaucomatous eyes between the Cocker group and the non-Cocker group (B). ETB, Endothelin B. ETBmRN A 0.26 0.28 0.3 0.32 0.34 Normal Glaucoma Cocker Other p = 0.0075log of AU ETBmRN A 0.26 0.28 0.3 0.32 0.34 Normal Glaucoma Cocker Other p = 0.0075 ETBmRN A 0.26 0.28 0.3 0.32 0.34 Normal Glaucoma Cocker Other p = 0.0075log of AU ETB mRNA Retina

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87 Figure 3-21: Real Time PCR amplification curves of the cloned ETA receptor DNA sequence run with different mixes of primers and probes (see Table 2-6). All samples are run in triplicate. The (A/A) mix shows an early amplification of the DNA sequence. The mixes A/B and B/A give only background noise below detectable level. Amplification curves are seen late in the reaction with the mix (B/B) and 18S respectively. ETA, Endothelin receptor A; ETB, Endothelin receptor B; A/A, ETA primer/ETA probe; A/B, ETA primer/ETB probe; B/B, ETB primer/ETB probe; 18S, 20X Eukaryotic 18S rRNA Endogenous Control.

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88 Figure 3-22: Real Time PCR amplification curves of the cloned ETB receptor DNA sequence run with different mixes of primers and probes (see Table 2-6). All samples are run in triplicate. The (B/B) mix shows an early amplification of the DNA sequence. The mixes A/B and B/A give only background noise below detectable level. Amplification curves are seen late in the reaction with the (A/A) mix and 18S respectively. ETA, Endothelin receptor A; ETB, Endothelin receptor B; A/A, ETA primer/ETA probe; A/B, ETA primer/ETB probe; B/B, ETB primer/ETB probe; 18S, 20X Eukaryotic 18S rRNA Endogenous Control

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89 Figure 3-23: Real Time PCR run with no template (1) and total RNA (2) run with the primers and probes for the ET receptors and 18S (see Table 2-6). The ET receptors are not amplified in the reaction. Traces of 18S rRNA are amplified in both samples.ET, Endothelin; 18S, 20X Eukaryotic 18S rRNA Endogenous Control

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90 Figure 3-24: RT-PCR of two samples from normal dog retinas. The sample labeled Retina 1 was stored frozen in the preservative RNAlater and the sample labeled Retina 2 stored frozen dry without any preservative. The RT-PCR was run on total RNA and DNAse treated RNA from each sample to visualize any genomic DNA in the samples with total RNA. The primers used for the reaction are listed in Table 2-5.

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91 Figure 3-25: Real Time PCR amplification curves of the ETA receptor for two normal retinal samples stored frozen in RNAlater (sample 1) and frozen dry without preservative (sample 2). Both samples were run in triplicate. The primers used for the reaction are listed in Table 2-5. ETA, Endothelin receptor A; CT, Cycle Threshold

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CHAPTER 4 DISCUSSION Morphology The iridocorneal angle (ICA) is designed specifically for the removal of aqueous humor. Aqueous humor passes between the pectinate ligaments to large extratrabecular spaces of the deep ciliary cleft [uveal trabecular meshwork (Figure 3-1)] [98,99]. It then passes through the more dense meshwork of the corneoscleral trabecular meshwork in the shallow scleral sulcus [98]. The trabecular cells lining the beams of the corneoscleral and uveal meshworks are fibroblast-like cells with slender cell processes [39]. Aqueous humor in the corneoscleral trabecular meshwork drains to a plexus of aqueous humor collector vessels (angular aqueous plexus) where it exits the eye and enters the circulation. A small percentage of aqueous humor (15% in the dog) drains from the eye via unconventional routes (uveoscleral outflow) through the iris stroma and/or between the fibers of the ciliary muscle to the supraciliary and suprachoroidal spaces. The mechanism of development of narrow and closed ICA in the American Cocker Spaniel is not completely clear, but a hypothesis is that tight apposition of the iris slightly increases the pressure within the posterior chamber, which in turn causes forward displacement of the basal iris [27]. Eventually, the basal iris narrows the ICA and opening of the ciliary cleft. Apposition of basal iris across the filtration angle causes a potentially reversible angle closure. With continued apposition, peripheral anterior synechiae (PAS) develop, thereby permanently closing the pathways of aqueous outflow. 92

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93 The ICA progressively narrows, eventually closes, and the ciliary cleft collapses. The intrascleral plexus collapses as a consequence of the enlarged globe [27]. The ICAs of the glaucomatous eyes in our study were partially or completely collapsed, irregardless of the breed and age of the dog, reflecting the fact that the eyes were approaching or at the end-stage of glaucoma (Figure 3-2). The degree of retinal degeneration in our study also did not correlate with the length of disease. This is in accordance with a study by Smedes and Dubielzig [100] where extensive retinal degeneration was found in glaucomatous eyes with a brief clinical history of increased intraocular pressure (IOP) ranging from 1-11 days. There was no difference in degree of retinal degeneration between the Cocker group and the non-Cocker group in our study. Endothelin-1 in Aqueous Humor and Vitreous The mean endothelin (ET)-1 levels in aqueous humor and vitreous in our study were significantly increased in the glaucomatous dog eyes, although the range of ET-1 levels was large (Tables 3-2 and 3-3, and Figures 3-12 and 3-13). Despite this variability, the data indicated a definite increase in ET-1 level in the canine glaucomatous eyes. Elevations of aqueous humor ET-1 are found in human primary open angle glaucoma (POAG) and normal tension glaucoma (NTG) [15-17,101]. The 2.5-fold increase of ET-1 in aqueous humor of the glaucomatous dogs in our study (from 2.8 1.52 to 6.86 3.49 pg/mL) is much higher than the 0.05-fold increase seen in human POAG patients (from 42.17 1.6 to 44.26 2.6 pg/mL) [15]. One explanation for this finding is the much higher IOPs in the canine eyes causing more severe ischemic/reperfusion injury as compared with that of human eyes. The aqueous humor samples in our study were all from advanced cases with uncontrolled IOP and severe

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94 retinal damage. Although no significant correlation was seen between the IOP immediately prior to collection of aqueous humor and the ET-1 levels in the canine glaucoma group, the high IOPs could cause severe ischemic/reperfusion injury, and hence induce ET-1 release at levels far surpassing the ones normally seen in human patients with POAG. Sources of Endothelin-1 The ET-1 in the canine aqueous humor could come from several sources. Cytokines associated with ischemia from elevated IOP might induce increased ET-1 production and release from non-pigmented ciliary epithelial cells or ischemic endothelial cells [102]. Functional endothelin converting enzyme-1 (ECE-1) has been found in human non-pigmented ciliary epithelial cells, which makes these cells a likely source for aqueous humor levels of ET-1 [103]. Transformed human non-pigmented ciliary epithelial cells in culture have also been shown to produce and release ET-1 in response to tumor necrosis factor(TNF-) [102]. According to a study by Smith et al. [104], dogs with glaucoma have a vacuolization, degeneration and atrophy of the ciliary epithelium in late stages of the disease. This could have a negative effect on the production and release of ET-1 from the ciliary epithelium in the chronic stages of the canine disease, making the ciliary epithelium less likely as a source of the elevated ET-1 levels found in dogs with glaucoma. Plasma ET-1 levels in our glaucoma dogs were not determined in this study for technical reasons. Levels of ET-1 have been measured in human aqueous humor and found to be two to three times higher than in plasma both in normals and in patients with POAG [15]. This argues against the influx of ET-1 from plasma unless they were actively secreted into the aqueous. Since systematically administered ET does not cross the blood

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95 brain barrier, it is highly unlikely that it would cross the blood-aqueous barrier under normal conditions [72]. The concentration of ET-1 in plasma in normal dogs varies between 14 and 52 pg/mL and is considerably higher than the levels found in human plasma (12.48 1.3 pg/mL), which may represent a species difference [15,105]. In the event of a disruption of the blood-aqueous barrier, plasma ET-1 might contribute to the increased levels of ET-1 in aqueous humor of glaucomatous dog eyes. This possibility should be investigated in future studies. Vitreal ET-1 levels have to our knowledge not been measured previously in glaucomatous eyes. The levels, mean ( STD) in the normal and glaucomatous eyes were 1.56 ( 1.86) and 3.47 ( 1.57) pg/mL respectively in our study. The vitreal ET-1 levels were lower than the aqueous humor ET-1 levels for both the normal and glaucomatous group. The mRNA levels of ET-1 in the glaucomatous retinas were not significantly changed indicating that the production of ET-1 remains essentially the same in the glaucomatous retinas as in the normal retinas. The increased vitreal ET-1 might emanate from ET-1 in the aqueous humor by reverse diffusion from the anterior chamber or from a decrease in the vitreal clearance of the peptide. Another potential source for ET-1 in the vitreous are the retinal pigment epithelial cells. Narayan et al. [85] showed that human retinal pigment epithelial cells stimulated with carbachol synthesize and release ET-1. The increased levels of ET-1 in aqueous humor and vitreous in our study may be due to reduced removal of the hormone. The ET-1 is normally flushed out with the aqueous humor through the outflow pathways. The function of this mechanism may be decreased in a glaucomatous eye with a collapsed iridocorneal angle. Endothelin-1

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96 inactivating peptidases have been found in human and rat kidney [106,107]. It is not known whether these peptidases exist in the aqueous humor or vitreous. Another mechanism that helps in the clearance of ET-1 is internalization and degradation of the ETB receptor and its associated peptide [44,66,73]. Although the Western Blot technique does not render itself to exact quantification, there were clear trends in our analyses of the receptors. There was a decrease of the protein expression of the ETB receptor in the glaucomatous retinas. (Table 3-8, and Figure 3-18). There was also a decrease of the ETB receptor in the choroid of the non-Cocker group that was not significant. This leads to a decreased clearance of ET-1 by these tissues. Future studies will tell if this trend is true also for the anterior segment, which would in part explain the increase of ET-1 in the aqueous humor. An alternative mechanism explaining the elevated ET-1 levels is the ability of the peptide to upregulate its own gene expression. Saito et al. [108] have demonstrated a dose-dependent increase in prepro-ET-1 levels in cultured rat aortic endothelial cells through activation of the ETB receptor by exogenous ET-1. Primary rat astrocytes were shown to express high-affinity receptors for endothelins [109]. In a study by Ehrenreich et al. [110], rat astrocytes responded to stimulation by ET-1 agonists with an increase in the release of ET-1 through induction of the transcriptional factor AP-1. This group suggested that the astrocytes are capable of a selective, autoregulatory mechanism that maintains high levels of ET-1. The increase of ET-1 mRNA in the glaucomatous retinas of the Cocker group was nonsignificant, while the non-Cocker group showed a nonsignificant decrease, making this mechanism less likely as being responsible for the large increase in ET-1 levels in our study (Table 3-4). We did not determine the content

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97 of ET-1 mRNA in the optic nerve heads. However, an increased release of ET-1 from the astrocytes of the optic nerve head would most likely make only a minor contribution to the ET-1 levels in the vitreous and aqueous humor. The Effect of Steroid Treatment The expression of the ETA receptor was also decreased in the glaucomatous retinal samples compared to normals. One reason for this decrease may have been the fact that a majority of the glaucomatous dogs (7 out of 10 dogs) were treated with corticosteroids due to a concurrent uveitis which could have affected the transcription of the ET receptors and the levels of ET-1. Yorio et al. [111] showed that dexamethasone increased ET-1 levels, down-regulated ETA receptor mRNA and protein levels, while having up-regulated ETB receptor mRNA and protein levels in human non-pigmented ciliary epithelial cells. Decreased amount of high affinity (ETA) binding sites by treatment with dexamethasone has also been shown in cultured rat aortic smooth muscle cells and human cerebromicovascular endothelial cells in culture [112,113]. Endothelin receptor mRNA levels were not measured in these studies. Dexamethasone has also been shown to increase the levels of prepro-ET-1 mRNA and ET-1, and decrease the levels of ETA receptor mRNA in vascular smooth muscle cells [113,114]. Although these results differed somewhat from the results in our study, they clearly showed an impact on ET-1 levels and receptor expression by steroid treatment that might have influenced our data. Cellular Response to Endothelin-1 Treatment The cellular responses to ET-1 treatment vary between various studies and may be an indication of different responses between cell types. Krishnamoorthy et al. [115] showed that ET-1 treatment of human non-pigmented ciliary epithelial cells increased both DNA binding activity of AP-1, a known transcription factor for regulating ET

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98 receptor expression, and the protein expression of ETB receptors. In contrast, studies on human and rat endothelial and smooth muscle cells incubated with ET-1, have shown a decrease in both ETA and ETB receptor binding sites [113,116]. Clozel et al. [117] showed on rat mesangial cells (ETA receptors), and human and rat endothelial cells (ETB receptors), that autocrine production of ET-1 decreases, either by binding or downregulation, the number of binding sites for both receptors. The trend in our study was that the protein expression of the ET receptors was decreased while the mRNA levels were increased in the retina, with exception of the non-Cocker group showing a non-significant decrease of the ETB receptor mRNA levels (Tables 3-7 to 3-10, and Figures 3-17 to 3-20). That would imply that the transcription of the ET receptors is stimulated by the increased ET-1 levels, and therefore the decreased receptor protein expression must be due to either decreased translation or increased destruction. Unfortunately, we were not able to determine the mRNA levels in the choroid with the Real Time PCR, as the choroidal samples gave a very weak and variable fluorescent signal. The weak signal was probably due to the pigment in the choroid. An attempt was made to extract the pigment using the RNAqueous-4 PCR kit (Ambion, Austin, TX). Since most of the pigment remained in the samples we decided not to use the choroidal samples for the Real Time PCR due to unreliable data. The complexity of the response of the eye to glaucomatous damage may be better appreciated by comparing with studies done on neurotrauma models of the brain. Siren et al. [118] characterized the time-course, intensity of expression and cellular origin of components of the ET system in the rat brain after standardized neurotrauma. Their study

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99 showed that cortical injury induced a strong expression of ET-1 in astrocytes while the expression in the basilar artery was not altered. The expression of ETB receptors increased in the astrocytes but decreased significantly in the basilar artery indicating distinct differences in the regulation of the ETB receptor regulation by these cell types. Immunoreactivity of the ETA receptor was only associated to blood vesssels and was not altered by the injury. Neurotrauma failed to induce any alterations in mRNA expression of the ET peptides or the ET receptors in the basilar artery. Extravascular Endothelin Receptor Binding Sites Extravascular ET receptor binding sites might have influenced our results. Mac Cumber et al. [86] described ET receptor binding sites in human and rabbit eyes using radioligand binding that was inferred from pharmacological antagonism. This study showed extravascular ETB -like binding sites of the photoreceptor inner segment layer, the outer nuclear layer, the inner nuclear layer, the ganglion cell layer, and the nerve fiber layer. On the other hand, Stitt et al. [119] using the same technique, found extravascular ETB -like binding sites mainly in the ganglion cell layer in human and rat retinas. In our study we focused on the vascular localization of the ET receptors in the retina and choroid. It is likely though that the canine retina has a similar localization of the ET receptors as that of the species used in previous studies. The Western Blot and Real Time PCR used in our study were done on whole retinas. Consequently, any change in ET receptor levels cannot be attributable to a specific cell type. Thus, the decrease in ETB receptor expression in the glaucomatous eyes seen in our study may, at least in part, be due to atrophy of the nerve fiber layer and the ganglion cell layer. Previous studies have localized the ETA receptor in the retinal and choroidal smooth muscle cells but could not detect any ETA like binding sites in the neural retinal

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100 tissue [86,119]. If this is true also for the canine eye, the decrease in ETA receptor expression in the glaucomatous eyes seen in our study is most probably attributable to ETA receptors located on vascular smooth muscle cells. Future work on cell cultures from normal and glaucomatous eyes is necessary to unravel which cell types are responsible for the changes in ET receptor expression seen in our study. Pericytes and Endothelin Receptor Expression The need for an identification of which cell types change their ET receptor expression in glaucoma is further underlined by the fact that the ET receptors of retinal pericytes function differently from the receptors of smooth muscle cells. The pericyte ETA receptor mediates contraction through the same pathway as the ETA receptor on smooth muscle cells, i.e. through the phospholipase C/inositol phosphate signaling pathway [43]. The exact nature of the second messenger signaling downstream to ETB receptor activation in the retinal pericyte remains uncharacterized. However, a study has linked endothelin-3 stimulation of renal mesangial cells (also classified as pericytes) to the production of NO [43]. This mechanism was postulated to buffer the contractile effects of ET-1, especially when local levels are inappropriately high. This is in opposition to the constrictor response evoked by activation of the ETB receptor on smooth muscle cells. Receptor binding studies have revealed the presence of several hundred thousand high-affinity ETA receptors but only twenty to thirty thousand ETB receptors on a single cell. It is also noteworthy that retinal pericytes have been shown to express many more ETA receptors than vascular smooth muscle [75]. The decreased ET receptor expression in the glaucomatous retinas in our study may reflect changes in the density of ET receptors of the pericytes. Since the ETA receptors far out number the ETB receptors, a decrease in both receptors may have a larger functional effect on the ETB

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101 receptors impairing their buffering effect. This leads to a reduction of blood flow in the retinal microcirculation. Yield of Protein and RNA The changes of ET receptor expression in our study cannot be explained by differences in amount of tissue from the normal and glaucomatous eyes. Although there was a degeneration of the retina in the glaucomatous eyes, the protein yields in both groups were similar. The total protein median levels in the normal and glaucomatous retinas were 250 and 280 g respectively, and in the normal and glaucomatous choroid were 277.5 and 250 g respectively. The yield of total RNA differed between the normal and glaucomatous group. The total RNA median levels for the normal and glaucomatous retinas were 22.5 and 30.14 g respectively. This had no affect on the results of the real time PCR since the quantities of cDNA from each sample were normalized by using the ratio of the sample and the endogenous control rRNA 18S. Nitric Oxide in Aqueous Humor and Vitreous The increase of NO levels in aqueous humor and vitreous in our study is in accordance with a study by Chiou et al. [120] on human patients with acute angle-closure glaucoma (Tables 3-5 and 3-6, and Figures 3-15 and 3-16). In contrast, Galassi et al. [121] found a reduction in cGMP, the intracellular mediator of NO action in the peripheral plasma, in aqueous humor of patients with normal pressure glaucoma. This may imply a difference in NO synthesis in various kinds of glaucoma. All three isoforms of nitric oxide synthase (NOS) are elevated in the optic nerve head of human patients with POAG as shown by Neufeld et al. [122]. This finding is in contrast with a study by Samuelson et al. [25] which showed that the endothelial form (NOS-3) is decreased in the

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102 inner retina of Beagles with POAG. On the other hand, in the same study the inducible form (NOS-2) was localized in the trabecular meshwork of the glaucomatous dogs. Our results are also in contrast with a study by Gunia et al. (personal communication) where NO concentrations were measured in aqueous humor of glaucomatous dogs having transcleral cyclophotocoagulation surgery due to acute onset glaucoma. In their study the mean concentration of NO in the glaucomatous dogs was 9.628 M as compared to 15.17 M in the control group (normal greyhounds). The main difference between the studies is the concentration of NO in the aqueous humor of the control groups. In our study the mean concentration of NO in glaucomatous dogs was 12.48 M as compared to 3.9 M in the control group (Tables 3-5 and 3-6). The lack of agreement between the studies may represent a species difference. Prasanna et al. [123] demonstrated that ET-1 increases NO production, in part via the activation of NOS-2, in cultured human non-pigmented ciliary epithelial cells. In addition, intravitreal injections of ET-1 in rabbits have been shown to increase NO levels [124]. When the synthesis of NOS-2 is induced the enzyme produces massive amounts of NO that is destructive to neighboring cells [122]. Franco-Burland et al. [125] showed an increase in neuronal NOS (NOS-1) in surviving retinal ganglion cells of dogs with glaucoma. The influence of ET-1 on NOS-1 levels have, to our knowledge, not been determined. We did not examine the various isoforms of NOS in our study. However, the increased levels of NO in both aqueous humor and vitreous are likely to influence the mechanisms of both aqueous humor inflow and outflow, and the development of optic nerve degeneration. In addition, if there is a decrease in NOS-3, as shown by Samuelson

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103 et al. [25], the neuroprotective effect caused by circulatory vasodilation is diminished in glaucomatous canine retinas. The Cocker Spaniel versus Other Breeds American Cocker Spaniels with glaucoma can have fan-shaped zones of retinal and choroidal degeneration radiating from the ONH. These areas of retinal ischemia correspond to the areas supplied by individual short posterior ciliary arteries and may be caused by vasospasm of the arteries [27]. The ischemia may be a result of the inability of the vessels to alter their diameter in response to a demand for an increase in blood flow by the tissue due to elevated levels of ET-1. This finding suggests a role for ET-1 in contributing to the glaucomatous damage in American Cocker Spaniels, either by increased release of ET-1 or a nonfunctional circulatory response due to modulation of its receptors. Although several parameters in our study were not significantly different, the Cocker glaucoma group seemed to differ from the non-Cocker glaucoma group. The increase of ET-1 in aqueous humor and vitreous was greater in the Cocker group compared to the non-Cocker group, while the increase in NO was greater in the non-Cocker breed group compared to that of the Cockers (Tables 3-2 to 3-6, and Figures 3-12, 3-13, 3-15, and 3-16). There was a decrease of the ETB receptor protein expression in the glaucomatous retinas which was significant in the non-Cocker group, but not significant in the Cocker group (Table 3-8, and Figure 3-18). For technical reasons we did not have any normal choroid in the Cocker group to compare with the glaucomatous Cockers. A comparison of glaucomatous choroid samples showed that the expression of the ETB receptor in the

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104 choroids of the glaucomatous Cockers was lower, although not significantly, than the non-Cocker breed group (Table 3-8). The expression of the ETA receptor was decreased in both groups in the glaucomatous retinal samples compared to normals (Table 3-7 and Figure 3-17). In contrast, the glaucomatous choroidal samples of the Cocker group had higher expression of the ETA receptor than the non-Cocker group (Table 3-7). None of the differences was significant but did show an interesting trend. The choroid of the glaucomatous Cockers showed a higher expression of the ETA receptor and a lower expression of the ETB receptor compared to the non-Cocker group. This creates an imbalance between the receptors increasing the risk for vasoconstriction by the ETA receptors, which cannot be counter-balanced by the vasodilating effect by the ETB receptors, thus increasing the risk for ischemic ocular damage. The mRNA levels in the retina were significantly increased in the Cocker group for both receptors, while the non-Cocker group showed a non-significant increased level of the ETA receptor and a non-significant decrease of the ETB receptor levels (Tables 3-9 and 3-10, and Figures 3-19 and 3-20). These differences between the groups might imply that the narrow-angle glaucoma in Cocker Spaniels represent a separate type of glaucoma with an unique response to increased IOP. The role of ET-1 in glaucoma in Cocker Spaniels should be further evaluated by characterizing the components of the ET system in the anterior and posterior part of the eye at various stages of the disease. Implications for the Canine Eye Ultrastructural differences in capillaries of the lamina cribrosa, circulatory abnormalities of the mid-peripheral retina, failure of the orbital circulation to respond to

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105 calcium channel blocking drugs, and irregularities in the blood flow of the orbital arteries are present in dogs with hereditary glaucoma prior to elevations in IOP [37,36,126,127]. An ET-1 related ocular micro-circulatory problem may thus be present early in the glaucomatous disease process in dogs. In our study we showed that the aqueous humor and vitreous of dogs with hypertensive glaucoma contains significantly higher levels of ET-1 than normal dogs. There is no indication from our data whether the increase in ET-1 in the aqueous humor of canine glaucomatous eyes is the cause of elevated IOP or just a secondary event. The effects of elevated ET-1 in the anterior chamber must take anatomical differences between species into consideration. The effect on contractility of the trabecular meshwork is functionally antagonistic to the direct effect on ciliary muscle [96]. Since the canine ciliary muscle is poorly developed as compared to primates, the effect of ET-1 might be dominated by the effect on the trabecular meshwork. That could give an increased resistance to aqueous outflow and lead to an increased IOP. The decrease in ET receptors seen in our study is a mass response of the whole retina, including the vessels. As for the vascular component of the ET system, a decrease of the receptors may be a reflection of endothelial cell damage consistent with ischemic events. This would decrease the amount of ETB receptors located on the endothelial cells leading to vasoconstriction. In the case of a concurrent damage to the smooth muscle cells both ET receptors would decrease. Even with a decrease of ETA receptors on the smooth muscle cells, the net effect would probably be an increased vasoconstriction caused by the remaining ETA receptors and ETB receptors located on the smooth muscle

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106 cells. As discussed in the section Pericytes and Receptor Expression, a decrease of both ET receptor types might lead to vasoconstriction. The balance between ET-1 and NO is absent in the canine glaucomatous eyes in our study. The source of the excessive NO production has to be determined. If the increased concentration of NO is produced by glial cells (NOS-1 and NOS-2) it most likely participates in neurodestruction of the retina and ONH. Conversely, NO produced by the vascular endothelial cells (NOS-3) may be neuroprotective by causing vasodilation and increased blood flow in the tissue. The increase of ET-1 in the aqueous humor and vitreous of dogs with glaucoma may contribute not only to the uncontrolled IOP, but also explain the rapid progression to ONH atrophy in canine glaucoma that occurs more slowly in human POAG [128]. Acute increases of IOP induce ischemia of the optic nerve and retina [2]. The ischemia leads to an increase in endothelin-mediated vasoconstriction concomitant with a loss of tonic NO-dependent vasodilation. This effect may persist several hours after the ischemic insult. Ischemia and reperfusion are known to increase both ET-1 and glutamate levels in tissue and plasma [3,129,130]. Elevated intravitreal glutamate levels indicating ischemia are increased during glaucoma in the dog [28]. Defective or inadequate autoregulation and microcirculatory dysfunction of the vessels of the dog eye may result in ischemic injury to the canine retina and optic nerve under normotensive and hypertensive conditions resulting in further release of ET-1. The increased levels of ET may also exacerbate neurodegeneration by stimulating efflux of glutamate from the astrocytes. The rapid degeneration of the optic nerve head found in canine glaucoma despite aggressive IOP reduction therapy may thus be due to the combined, chronic effect of

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107 ET-1 inducing vascular ischemia and associated glutamate release, and a neurotoxic effect of NO [27]. The increased ET-1 levels could also cause astrogliosis disrupting axonal transport and inhibiting axon regrowth in the glaucomatous canine ONH.

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BIOGRAPHICAL SKETCH Maria Elisabeth Kllberg was born on September 26, 1957, in Stockholm, Sweden. She attended elementary and secondary schools in Sweden. Maria attended the College of Veterinary Medicine at the Swedish University of Agricultural Sciences in Uppsala, Sweden, from 1976-1981. After attending veterinary school, Maria worked as a small animal general practitioner for 3 years. In 1985, Maria became co-owner of an animal hospital where she served as chief of staff for the Small Animal Clinic until 1998. During this time Maria took continuous, practical training in eye examination for hereditary diseases in dogs and cats. After passing the exam set by the Swedish Society for Veterinary Ophthalmology, Maria became a member of the Swedish Eye Panel in 1995. In 1998 Maria became a graduate student at the College of Veterinary Medicine, University of Florida in Gainesville, Florida with duties as a teaching and research assistant. In 2002 she started her residency in comparative ophthalmology at the University of Florida, which she will finish in 2005. Her special interests include regulation of blood flow in the healthy and diseased eye. 119


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LOCALIZATION AND EXPRESSION OF ET-1 RECEPTORS IN THE NORMAL
AND GLAUCOMATOUS DOG EYE


















By

MARIA E. KALLBERG


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


2003


































Copyright 2003

By

Maria E. Kallberg


























Dedicated to my family and my friends who made this work come true.















ACKNOWLEDGMENTS

I would like to thank everyone who helped and supported my scientific work and

my graduate education, especially those listed below.

Dr. Dennis E. Brooks invited me to Florida and made the dream of my life come

true. Thanks to Dr Brooks I will be able to dedicate my professional life to

ophthalmology as a researcher and clinician. I thank him for his support through the

years. The precious friendship we have developed will always help me whatever

challenges life has to offer in the future.

Dr. William W. Dawson guided me through the world of vision research and

electrophysiology, which for me was unknown territory. He has inspired me not only by

his knowledge but also by his way of questioning axioms and looking for new truths. I

am thankful to Dr Dawson and his wife, Judyth, for their concern and warm generosity.

Dr. Adrian M. Timmers meant a great deal to me and my project. He opened his

laboratory for me and patiently taught and helped me through every necessary step of the

molecular biology part of my project. His positive and supportive attitude was

extraordinary.

I would like to thank Dr. Kirk N. Gelatt for sharing with tireless enthusiasm his

tremendous knowledge in research and clinical ophthalmology. I am also grateful for the

many times he helped me in his own positive way whenever I needed good advice.









Dr. Don A. Samuelson broadened my view of comparative ophthalmology. He and

his laboratory technicians, Mrs. Patricia Lewis and Ms. Mae Chisholm, were very helpful

in accomplishing all the challenges in histologic methods that I encountered during my

time as a graduate student.

I owe Dr. Adolfo G. Garcia many thanks for his contribution to my project. I am

grateful to have gotten not only scientific help but also a friendship for life.

My dear friend, Dr. Andras M. Komaromy, has been by my side from the very first

page of this new chapter of my life. Andras made the transition easy by always being

there for me whenever I needed help or support.

Dr. Franck J. Ollivier, dear friend and colleague, deserves lots of thanks for the

wild scientific discussions and many laughs.

With all my heart I thank my best friend, Kerstin Bergvall, who helped me through

many tough times.

Ann Ellis gave me many hours of help teaching me electron microscopy.

I would like to thank Dr. Stacy E. Andrew, the ophthalmology residents at the

University of Florida, and various ophthalmologists in Florida (especially Dr. Salisbury

and Dr. Brogdon) for their contributions.

Mr. Harold L. Sapp gave valuable technical assistance.

Dr. Carol Detrisac was very helpful in reviewing my histology slides.

Dr. George Lambrou, CIBA Vision Ophtha, Novartis, provided very generous

financial and technical support.









I would like to thank Ms. Marinela Capanu and the Interdisciplinary Center for

Biotechnology Research at the University of Florida for their help with statistic analysis

of the data.

Dr. Nancy Szabo at the Analytical Toxicology Core Laboratory at University of

Florida has been extremely helpful with analyzing my samples and sharing her expertise.

Dr. Thomas Yorio, Dr. Ganesh Prasanna, Dr. Ragu Krishnamoorthy, Dr. Martha

Stokely, Christina Hulet, and Santos Narayan at the Health Science Center, Fort Worth,

Texas deserve a special thanks for their tremendous support.

I owe many thanks to the Office of Research and Graduate Studies at the College of

Veterinary Medicine, University of Florida. Associate Dean Charles Courtney III and

Mrs. Sally O'Connell made it possible for me to become a graduate student and to

successfully finish my dissertation. Sally helped me through several storms.

Finally I would like to thank my parents for supporting me the whole way through

my training and for helping me to realize my dreams. The sometimes overwhelming

distance from home was overridden by the closeness of our relationship.
















TABLE OF CONTENTS
page

A C K N O W L E D G M E N T S ................................................................................................. iv

L IST O F T A B L E S .............................................................................................. x

LIST OF FIGURES ......... ......................... ...... ........ ............ xi

A B STR A C T ..................... ................................... ........... ................. xiii

CHAPTER

1 IN TR OD U CTION ............................................... .. ......................... ..

G laucom a ................... ..... ....... .... .................................. ............... .
Role of Endothelin in G laucom a ................................ ................... ................
Canine G laucom a ............ .. .... ........................................ .............. .. .4
Primary narrow-angle glaucoma in American Cocker Spaniels ................6
Primary open-angle glaucoma in the Beagle.................................... 10
Ophthalmic Vascular M orphology and Physiology ...................................................11
E ndothelin and N itric O xide.................................................. .................. 16
Synthesis, Secretion and Clearance of Endothelin. .................. ................... 16
N itric O x id e ................................................................................... 19
Endothelin Receptors ..................... .... .......... ...... .. .......... 20
Endothelin and the Ophthalmic Circulation.......... ...................................24
Endothelin and Aqueous Humor Production and Outflow .............. ...............26
Purpose of Study ............... ........ ......... ......... 27

2 M ATERIALS AND M ETHODS ........................................ ......................... 28

S tu d y D e sig n ......................................................................................................... 2 8
Clinical Examination of Dogs ....................... ..... ......................29
Endothelin-1 and Nitric Oxide Levels in Aqueous Humor and Vitreous ...............29
E ndothelin-1 A analysis ............................................... ........ .. ...... ............29
N itric Oxide Analysis ............ ..... .. ....... .. ................... .............. 30
Localization of Endothelin Receptors in the Retina and Choroid ............................30
L ig ht M icro scopy ............................................ ............ .... ....... .... .. 1
Transmission Electron Microscopy ........................ ......... ...............31
Morphology of Retina and Iridocomeal Angle ..............................................33
Endothelin Receptor Protein Expression in the Retina and Choroid..........................33
Protein Extraction .................. ........................... .. ....... ................. 34









Endothelin Receptor and Endothelin-1 mRNA Levels in the Retina.........................36
Isolation of Total RN A .............................................. ... ............... 36
Reverse Transcriptase Polymerase Chain Reaction ........................................37
Real Time Polymerase Chain Reaction...........................................................38
D ata A n aly sis............................. ...................................................... ............... 4 0
M o rp h o lo g y ...................................................................... .. 4 0
Endothelin-1 and Nitric Oxide Levels in Aqueous Humor and Vitreous ...........40
Endothelin Receptor Protein Expression in the Retina and Choroid ..................40
Endothelin-1 and ET Receptor mRNA Levels in the Retina ............................41

3 R E S U L T S .............................................................................4 9

M orp h ology ...............................................................4 9
Iridocorneal A ngle .................. ............................ .. ..... ................. 49
R etina ....................... ........ .............................49
Endothelin Receptors in the Retina and Choroid ....................................... .......... 50
Evaluation of Endothelin Antibodies using Light Microscopy ...........................50
Evaluation of Endothelin Antibodies using Transmission Electron
M icro sco p y ....................... ............................... .. .... .. ............... 5 0
Localization of Endothelin Receptors using Transmission Electron
M icro scopy ...................................................................... 5 1
E T A receptor........................................................................................... 5 1
E T B receptor.......................... ...... .. .......... ..... ............. 51
Endothelin-1 Levels in Aqueous Humor, Vitreous and Retina.............................51
Analysis of Aqueous Hum or. ...........................................................................51
A analysis of V itreous .......... ....................... ................ ................ ... ... .............52
Endothelin-1 mRNA Levels in the Retina................... ......................... ... 52
Correlation of Retinal Degeneration and ET-1 Levels in Aqueous Humor and
V itreous .................. ........... ......... ............. ......... ............ 53
Nitric Oxide Levels in Aqueous Humor and Vitreous ............................................53
A analysis of A queous H um or. ............. ...................................... .....................53
A naly sis of V itreou s .......................................... ..... .....................................54
Endothelin Receptor Protein Expression in the Retina and Choroid.......................... 54
E TA R eceptors in the R etina ........................................... .......... ............... 54
ETB Receptors in the Retina. ........................................ .......................... 54
ETA and ETB Receptors in the Choroid. ............. ........................................55
Endothelin Receptor mRNA Levels in the Retina................... ...............................55
Evaluation of the Real Time PCR Reaction. ...................................................55
T is su e ........................................................................ .. 5 6
ETA Receptor mRNA in the Retina .......................................... ............... 57
ETB Receptor mRNA in the Retina. ........................................ ............... 58
Sum m ary of R esults......... .................................................................. .. .... .. .... .. 58

4 D IS C U S S IO N ...........................................................................................9 2

Morphology .................................. .......................92
Endothelin-1 in Aqueous Humor and Vitreous ................. ................. ..........93









Sources of Endothelin-1 .................................. ...................................... 94
Effect of Steroid Treatment.................................................... 97
Cellular Response to Endothelin-1 Treatment ......................................... 97
Extravascular Endothelin Receptor Binding Sites ............................................99
Pericytes and Endothelin Receptor Expression ....... .................................100
Yield of Protein and RNA ....... ........................................................... 101
Nitric Oxide in Aqueous Humor and Vitreous................................. ... ................ 101
The Cocker Spaniel versus Other Breeds ..... ............................................. ... ...........103
Im plications for the Canine Eye ........................................ .......................... 104

LIST OF REFERENCES ........... ...................................... ...... .... ............... 108

B IO G R A PH ICA L SK ETCH ......... ................. ...................................... .....................119
















LIST OF TABLES


Table page

2-1: N orm al dogs in study ..................................................................... ...................42

2-2: G laucom a dogs in study ................................................ ............................... 43

2-3: A analyses on norm al dogs. ............................................................. .....................44

2-4: Analyses on glaucom atous dogs. ........................................ ......................... 45

2-5: Prim ers and fluorogenic probes........................................ ............................ 46

2-6: Controls for the R eal Tim e PCR ........................................ ......................... 47

3-1: Ranking of degree of degeneration in the glaucomatous retinas............................59

3-2: Endothelin-1 levels in pg/mL for normal dogs ....................................................... 60

3-3: Endothelin-1 levels in pg/mL for glaucomatous dogs. .........................................61

3-4: Mean ratios of ET-1/ 18S mRNA in arbitrary units for normal and glaucomatous
retain al sam p les......................................................................................... .62

3-5: Nitrate (NO) levels in [iM of normal dogs.....................................63

3-6: Nitrate (NO) levels in itM. of glaucomatous dogs .............. ............... 64

3-7: Ratios of ETA receptor/ control protein............... ...................... .............. 65

3-8: Ratios of ETB receptor/ control protein....... ............ .............................. 66

3-9: Mean ratios of ETA receptor/ 18S mRNA in arbitrary units for normal and
glaucom atous retinal sam ples...................................................................... ...... 67

3-10: Mean ratios of ETB receptor/ 18S mRNA in arbitrary units for normal and
glaucom atous retinal sam ples. ........................................ ......................... 68

3-11: Sum m ary of results ................ ........ ....................69















LIST OF FIGURES


Figure pge

2-1: Real Time PCR amplification curves of a normal and a glaucomatous retina for the
ETA receptor and 18S rRN A ................... .................................... .....................48

3-1: Normal iridocorneal angle stained with toluidine blue (Dog M3, original
m agnification 100X).. ................................................ .. ...... .. ........ .... 70

3-2: Iridocomeal angle of a glaucomatous dog (Dog 20, original magnification 40X,
PA S stained) ....................................... ............................. ................. 70

3-3: Trabecular meshwork of a glaucomatous dog (Dog 25, original magnification
200X PA S stained).. .............................................. ... ..... .. ........ .... 71

3-4: Iridocomeal angle of a glaucomatous dog (Dog 19, original magnification 200X,
PA S stained) ....................................... ............................. ................. 71

3-5: Iridocomeal angle of a glaucomatous dog (Dog 22, original magnification 100X,
PA S stained) ....................................... ............................. ................. 72

3-6: Iridocomeal angle of a glaucomatous dog (Dog 26, original magnification 100X,
PA S stained) ....................................... ............................. ................. 72

3-7: Semi-thin (ltm) sections of the retina from a normal dog (Dog M4, original
magnification 400X) stained with toluidine blue................................................73

3-8: Semi-thin (1 [tm) sections of the retina from a glaucomatous dog (Dog 24, original
magnification 400X) stained with toluidine blue................................................74

3-9: Semi-thin (1 [tm) sections of the retina from a glaucomatous dog (Dog 25, original
magnification 400X) stained with toluidine blue................................................75

3-10: Label of canine scleral vessels and elastic fibers with the ETB antibody .............76

3-11: Arteriole of the choroid of a normal dog.. ............... ......................... ........ 77

3-12: Endothelin-1 in aqueous humor of normal and glaucomatous eyes .....................78

3-13: Endothelin-1 in vitreous of normal and glaucomatous eyes..............................79









3-14: Scatter plot showing the correlation between the degree of degeneration in
central retina and total aqueous humor and vitreal ET-1 levels of glaucomatous
dog s. ................................................................................80

3-15: Nitric oxide in aqueous humor of normal and glaucomatous eyes ........................81

3-16: Nitric oxide in vitreous of normal and glaucomatous eyes....................................82

3-17: Endothelin receptor A protein in the retina of normal and glaucomatous eyes.....83

3-18: Endothelin receptor B protein in the retina of normal and glaucomatous eyes.....84

3-19: Endothelin receptor A mRNA in the retina of normal and glaucomatous eyes.....85

3-20: Endothelin receptor B mRNA in the retina of normal and glaucomatous eye ......86

3-21: Real Time PCR amplification curves of the cloned ETA receptor DNA sequence
run with different mixes of primers and probes ................................................87

3-22: Real Time PCR amplification curves of the cloned ETB receptor DNA sequence
run with different mixes of primers and probes..................... ... ...............88

3-23: Real Time PCR run with no template (1) and total RNA (2) run with the primers
and probes for the ET receptors and 18S. .................................... ...............89

3-24: RT-PCR of two samples from normal dog retinas ...............................................90

3-25: Real Time PCR amplification curves of the ETA receptor for two normal retinal
samples stored frozen in RNAlater (sample 1) and frozen dry without
preservative (sam ple 2). ......................... ...... ...... ................ 91















Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

LOCALIZATION AND EXPRESSION OF ET-1 RECEPTORS IN THE NORMAL
AND GLAUCOMATOUS DOG EYE

By

Maria E. Kallberg

May 2003
Chair: Dennis E. Brooks
Major Department: Veterinary Medicine

The goal of this study was to document any differences in the levels of the

endothelin-1 (ET-1) peptide and nitric oxide (NO) in aqueous humor and vitreous; and in

the location and density of the ET-1 receptors, ETA and ETB, in the glaucomatous dog eye

as compared to the normal dog eye. Comparisons were made between normal (n=30) and

glaucomatous (n=14) samples for the following parameters:

* Levels of NO and ET-1 in aqueous humor and vitreous, as measured by enzyme
immunoassay.

* Localization of ET receptors in the retina and choroid was done by
immunocytochemi stry.

* Expression of receptor protein in the retina and choroid, as measured by Western
Blot technique.

* Retinal mRNA levels of ET-1, and the ET receptors, as measured by Real Time
Polymerase Chain Reaction.

* Retinas were evaluated histologically.









The numerical data were analyzed using an ANOVA 2*2 factorial analysis. The

two factors of interest were the disease, with two levels (glaucomatous and normal); and

breed, with two levels (Cocker and non-Cocker).

The findings in the glaucomatous eyes were the following:

* Endothelin-1 increased in aqueous humor and vitreous, with a larger increase in the
Cocker group.

* Nitric oxide increased in aqueous humor and vitreous, with a larger increase in the
non-Cocker group.

* Degree of degeneration of the retina was correlated to ET-1 levels in aqueous
humor and vitreous.

* Distribution of ET receptor labeling was similar in normal and glaucomatous
sections for both ET receptors.

* Endothelin-1 mRNA in the retina increased nonsignificantly in the Cocker group
and decreased nonsignificantly in the non-Cocker group.

* Endothelin receptor expression decreased in both groups with a significant decrease
for the ETB receptor in the non-Cocker group.

* Levels of ET receptor mRNA increased significantly in the Cocker group, while the
increase in the non-Cocker group was not significant.

The distinct results for the two groups might imply a unique response of ET-1 and

its receptors in narrow-angle glaucoma in Cocker Spaniels.














CHAPTER 1
INTRODUCTION

Glaucoma

Role of Endothelin in Glaucoma

Glaucoma is the final common pathway of a group of diseases with decreased

retinal ganglion cell (RGC) sensitivity and function, RGC death and optic nerve head

(ONH) cup enlargement, an incremental reduction in visual fields, and blindness.

Glaucoma is a neurodegenerative disease as it results in neural cell death [1]. All of these

diseases in dogs result in or are associated with increased intraocular pressure (IOP),

although the etiology of primary glaucoma is likely to be multifactorial. Mechanical,

vascular, and other factors may influence individual susceptibility to optic nerve damage.

At every level of IOP there is a risk of glaucomatous damage, although the risk

increases with increasing IOP. Damage can occur with extreme rapidity as in angle

closure glaucoma, or may progress slowly as in the chronic primary open angle

glaucomas (POAGs). Intraocular pressure cannot be used by itself to determine the

presence of glaucoma; or to determine whether optic nerve damage will occur or

progress. This variation in ONH susceptibility has been suggested to result from varying

capacity for circulatory autoregulation to prevent IOP-induced ONH ischemia [1].

Ischemia of the optic nerve and retina may be induced by high IOP, or by vascular

dysfunction such as a deficit in autoregulation, hypoperfusion, or vasospasm [2].

Inadequate blood supply of the retina and optic nerve is known to lead to death of

ganglion cell neurons partly through the release of glutamate, an excitatory amino acid









transmitter [3]. Neurons, which contain ionotropic glutamate receptors [e.g., N-methyl-

D-aspartate (NMDA) receptors], are particularly susceptible to ischemia/reperfusion [4].

The neurons in the retina that express such receptors are the ganglion cells and a subset of

amacrine cells. In ischemia/reperfusion, neurotransmitters (e.g., glutamate) are released

that overactivate their appropriate receptors. Such overstimulation, particularly of

ionotropic glutamate receptors, generally leads to neural cell death [4].

Various ocular blood flow deficits have been seen in patients with POAG or normal

tension glaucoma (NTG). Pillunat et al. [5-7] found evidence to suggest defective

autoregulation in the ONH in POAG and NTG. Robert et al. [8] showed that the ability of

glaucomatous eyes to adjust the blood supply in the optic disc to raised IOP is

significantly reduced as compared to that of healthy eyes. Grunwald et al. [9] found a

lack of hyperemic response of macular retinal blood flow after increased IOP in humans

with POAG suggesting abnormal autoregulation. Patients with POAG with deteriorating

visual fields despite an IOP lowered below 21 mm Hg show alterations in ocular blood

flow regulation as compared to POAG patients with stable visual fields [10]. Cheng et al.

[11] showed that patients with chronic angle-closure glaucoma (ACG) have decreased

retrobulbar blood flow velocities and increased vascular resistance in the central retinal

artery and temporal posterior ciliary arteries despite well-controlled IOP. Thus, evidence

exists that in POAG, ACG and NTG there is defective autoregulation of blood flow to the

ONH and retina.

Endothelin -1 (ET-1), being a very potent endogenous vasoconstrictor, has been

examined as part of the etiology of glaucoma. The presence of endothelin (ET) receptors

in the retina and choroid, particularly in retinal blood vessels, suggests that ETs may be









involved in regulating retinal blood flow, and could contribute to retinal ischemia.

Chronic ischemia of the anterior optic nerve was induced in animal models [12,13], either

by perineural infusion or intravitreal injections of ET-1. These studies have resulted in

glaucomatous-like damage to the optic nerve. Henry et al. [14] demonstrated an

impairment of ETB receptor-mediated, endothelial cell-dependent, vasodilation in the

forearm of human patients with normal-tension glaucoma. This indicates that the eye may

be just one manifestation of a more generalized vascular disorder characterized by

presumed endothelial cell dysfunction. Such an imbalance in the ET receptors might

contribute to ET-induced ischemic damage in glaucoma.

Elevations of aqueous ET-1 are found in humans with POAG, and in the plasma

and aqueous humor of patients with NTG [15-17]. Endothelin has also been found to

induce efflux of glutamate from cultured rat brain astrocytes [18]. This suggests that ET,

which is known to be released in ischemia, may exacerbate neurodegeneration by

stimulating efflux of glutamate.

Temporary retinal ischemia in the piglet leads to an increase in endothelin-

mediated vasoconstriction and a loss of tonic nitric oxide-dependent vasodilation

remaining several hours after the ischemic insult [19]. Hypoxia-ischemia serves as a

stimulus to enhance endothelin production in both brain and heart. Endothelin gene

transcription is upregulated and ET release from cultured human vein endothelial cells is

increased in response to acute hypoxia. Tissue responsiveness to endothelin, which can

be modulated by receptor up and down-regulation, may also increase in response to an

ischemic stimulus, but this has not yet been demonstrated in ocular tissues.









Astrocytes are the major glial cell type in the central nervous system and the ONH

and are vital for retinal ganglion cell survival [20]. Optic nerve head astrocytes normally

maintain the extracellular medium by regulating potassium and glutamate levels and also

provide neurotrophic support for nearby neurons [21]. Like brain astrocytes, ONH

astrocytes also respond to changes in the physiological state of the neuronal system by

becoming metabolically active from a quiescent state and rapidly proliferating to the site

of injury (reactive astrogliosis), especially under conditions of injury (ischemia or

pressure-related) as seen in glaucoma. Astrogliosis disrupts axonal transport and also

inhibits axon regrowth in the glaucomatous ONH [20,22]. Endothelins are implicated in

the promotion of astrogliosis in the ONHs of experimental animals [13]. Intravitreal

injection of ET-1 resulted in axon loss accompanied by glial proliferation in ONH of

rabbits [13]. Prasanna et al. [23] showed that ET-1 induces astroglial proliferation in

cultured human ONH astrocytes through ETA and ETB receptor activation suggesting that

ET-1 could cause proliferation of ONH astrocytes in glaucoma.

Endothelin-1 may also have direct effects on optic nerve function. Intravitreal

injections of ET-1 cause alterations in axonal transport in the rat optic nerve [24].

Likewise, continuous administration of ET-1 to the perineural region of the eye in

monkeys resulted in altered neuronal activity in the visual cortex [25]. These findings

suggest that intravitreal ET-1 can have direct effects on neuronal activity and survival.

Canine Glaucoma

The dog has the highest frequency of primary glaucomas of all animals, with the

narrow- or closed-angle type being the most common [26]. The glaucoma classification

scheme in dogs based on possible cause includes the primary glaucomas, secondary

glaucomas, and congenital glaucomas [27]. In the primary glaucomas, the IOP elevation









develops without concurrent ocular diseases, is inherited in some canine breeds, and has a

bilateral potential for development. Primary glaucomas may result from abnormal

biochemical metabolism of the trabecular cells of the outflow system [27]. In secondary

glaucomas, the increase in IOP is associated with some known antecedent or concurrent

ocular disease that physically obstructs the aqueous outflow pathways. In the congenital

glaucomas, the increased IOP is associated with an anterior segment anomaly, and the

elevation in IOP develops soon after birth.

In the dog, the primary glaucomas are divided into open-angle and narrow- or

closed-angle glaucoma according to the presence of an open or narrow anterior chamber

angle and ciliary cleft at gonioscopy. Inherited open- and narrow-angle glaucomas occur

bilaterally in purebred dogs. The primary glaucomas have been reported in at least 45

breeds and occur most frequently in United States among the American Cocker Spaniel

(ACS), English Cocker Spaniel, Smooth and Wire Fox Terriers, Basset Hound, Sealyham

Terrier, Miniature and Toy Poodles, and Beagle [27]. Other breeds recently identified

with primary glaucomas include the Samoyed, Norwegian Elkhound, Bouvier de

Flandres, Siberian Husky, Flat-Coated Retriver, Golden Retriver, Great Dane, Welsh

Springer Spaniel, Akita, Chow-Chow, and Shar-Pei [27]. Some breeds appear affected

with both the open- and narrow closed-angle types, which may suggest these glaucomas

are related.

Advanced glaucomas in dogs show similar features independent of pathogenesis

with an iridocorneal angle (ICA) closure and sclerociliary cleft collapse. The retina is

degenerated with attenuated arteries, and the optic disc round, depressed, and atrophied.

Intravitreal levels of the neurotransmitter glutamate are increased in dogs with glaucoma,









providing evidence for an ischemic mechanism for RGC death and optic nerve head

atrophy in canine glaucoma [28]. In a study by Kallberg et al. [29] aqueous humor ET-1

was 3.5 times normal level in dogs with primary chronic glaucoma. These findings

indicate a possible role for ET-1 in the pathophysiology of some types of glaucoma in

dogs.

Primary narrow-angle glaucoma in American Cocker Spaniels

The mechanism of development of narrow and closed ICA in the ACS is not

completely clear, but one hypothesis is that tight apposition of the iris slightly increases

the pressure within the posterior chamber, which in turn causes forward displacement of

the basal iris [27]. Eventually, the basal iris narrows the ICA and opening of the ciliary

cleft. Apposition of basal iris across the filtration angle causes a potentially reversible

angle closure. With continued apposition, peripheral anterior synechiae (PAS) develop,

thereby permanently closing the pathways of aqueous outflow.

This mechanism corresponds to the development of angle-closure glaucoma in

humans. The most common cause of angle closure in humans is pupillary block, also

termed primary angle-closure glaucoma [30]. It impedes the flow of aqueous humor from

the posterior to the anterior chamber between the anterior surface of the lens and the

posterior surface of the iris. Pupillary block may be absolute, as when the iris is

completely bound down to the lens by posterior synechiae, but most often is a functional

block, termed relative pupillary block.

Angle-closure glaucoma in humans can present with a spectrum of symptoms, from

none at all to severe pain, blurred vision, and nausea [31]. Intermittent angle closure

defines repeated, brief episodes of angle closure with mild symptoms and elevated IOP.

They may continue uneventfully for months or years. Attacks may be accompanied by









progressive PAS formation, leading to chronic angle closure due to a permanent closure

of a portion of the angle. The IOP in eyes with chronic angle closure may be normal or

elevated.

The greatest danger lies in the possibility of sudden conversion to acute angle-

closure glaucoma. If the pupillary block becomes absolute, the pressure in the posterior

chamber increases and pushes the peripheral iris farther forward to cover the trabecular

meshwork and closes the angle with an ensuing rise of IOP. Attacks of acute angle-

closure glaucoma are mild at first, but rapidly increase in severity. The symptoms of an

acute attack result from the sudden, marked elevation of IOP to as high as 80 mm Hg.

Absolute glaucoma refers to an eye with no light perception and a persistently

elevated IOP. The time required for a neglected angle-closure attack to cause total

blindness in humans is variable and depends on the severity of the acute attack, but

appears to be an average of 1 to 2 years [31].

Most affected dogs with angle-closure glaucoma present with either classic clinical

signs of unilateral, acute congestive glaucoma of a few days duration, or with chronic,

advanced glaucoma with buphthalmia, lens dislocation and cataract, retinal and ONH

degeneration, and blindness. Often, the condition becomes bilateral within several

months. In a study by Magrane [32] the mean age of affected dogs was 6 years (range, 3-

10 years), with the second eye usually affected within 12 months.

Both the history and clinical course suggest that this form of glaucoma may be a

series of acute IOP attacks, with the subsequent magnitude of the IOP elevation gradually

increasing. Tonographic measurements of aqueous humor outflow are usually within

normal limits in dogs with narrow ICAs, but they are lower than normal (0.10-0.15









[L/min per mm Hg) in dogs with very narrow and closed (due to synechial formation)

ICAs and clefts [27]. Tonometry of the acute congestive glaucomas often yields IOPs as

great as 50 to 70 mm Hg, and the corneal edema that parallels the elevation in IOP after

approximately 40 mm Hg usually prevents gonioscopy. Gonioscopy of the ACS with

ocular hypertension usually reveals a narrow to closed ICA and reduced ciliary clefts; as

the glaucoma progresses, angle closure and ciliary cleft collapse with peripheral anterior

synechial formation commonly occurring. More recently, pectinate ligament dysplasia in

the ACS has also been reported, but it does not appear to occur frequently [27].

In a study by Lovekin and Bellhorn [33] three Cocker Spaniels, bred for glaucoma,

were followed with repeated clinical examinations, tonometry, aqueous outflow, and

water drinking and betamethasone tests. The results were compared with similar test

results on 82 controls. Eyes were removed from the three Cocker Spaniels being studied

and compared with the eyes of four other Cocker Spaniels that had manifest glaucoma.

The studied Cocker Spaniels had reduced IOP regulatory mechanism as compared

to the control dogs, and increased IOP was more easily provoked in them than in the

control dogs. However, pathologic changes indicating glaucoma were not seen in

histologic sections of the ICAs of their eyes. The authors concluded that an abnormal

physiology concerned with the regulation of IOP preceded pathologic changes in the

angle structures of their eyes.

Definite histopathologic changes were shown in the eyes of Cocker Spaniels with

manifest glaucoma. The eyes resembled human angle-closure glaucoma or absolute

glaucoma with loss of trabecular meshwork, anterior synechia, and attenuation of the

intrascleral venous plexus. The histologic ICA structures of an eye of a Cocker Spaniel









with no history of glaucoma showed a lack of trabecular meshwork. This eye, having an

IOP of 24 mm Hg, was under prophylactic treatment because of manifest glaucoma in the

other eye.

Changes of the ocular funds in the ACS may not correlate with the duration and

magnitude of the elevated IOP [27]. It is not unusual for an ACS to present with a high

IOP (70-80 mm Hg) and a history of signs of glaucoma being present for less than 1

week, yet after lowering the IOP to less than 20 mm Hg, the dog loses its vision.

Ophthalmoscopically, the ocular funds cannot be visualized until the IOP is lowered and

the corneal edema reduced. The optic nerve and retina may initially appear to be normal,

with some vascular attenuation detected. With the IOP maintained at 20 mm Hg or lower

however, progressive retinal and ONH degeneration eventually become apparent within a

few weeks. In some of these dogs, the retinal degeneration may affect only limited areas,

appearing as radiating or fan-shaped zones from the ONH that represent areas of retinal

and choroidal degeneration caused by ischemia from the occlusion of individual short

posterior ciliary arteries.

The continued progression of ONH degeneration and deterioration of the animal's

vision despite lowering of IOP is classic evidence for the role of non-IOP related factors

in glaucomatous optic neuropathy. Among animals tested at the University of Florida,

intravitreal glutamate levels are much higher in the ACS with glaucoma than in

Samoyeds, Shar Peis, and Akitas [28]. In all probability, the initial primary optic nerve

injury from the elevated IOP induces RGC degeneration and apoptosis caused by

glutamate excitotoxicity, neurotropin deprivation, accumulation of intraneuronal calcium,

and formation of nitric oxide (NO), proteases, and oxygen free radicals. The injured,









apoptotic RGC releases more glutamate, predisposing the eye to further, secondary

degeneration of adjacent healthy RGC and their axons. This domino effect occurs

independent of further IOP-induced primary injury.

Primary open-angle glaucoma in the Beagle

Primary open-angle glaucoma in the Beagle is the most extensively investigated

canine glaucoma. Beagles with spontaneous, hereditary open-angle glaucoma start to

develop symptoms between 8 and 16 months of age [26]. The iridocomeal angle and

sclerociliary cleft are initially normal. The increase in IOP and decline in the facility of

outflow develop slowly. The increased IOP produces slight enlargement of the axial

length of the globe, which in turn results in lens subluxation and narrowing of ICA and

sclerociliary cleft in dogs between 1 and 4 years of age. Animals 2-4 years of age have

IOP's in the range of 25 mm Hg to 40 mm Hg. Eventual ICA and sclerociliary cleft

closure result in lOPs of 40 mm Hg to 60 mm Hg in animals 4 to 6 years old.

Light microscopic examinations of the aqueous outflow structures indicates no

abnormalities in the early affected animals [34,35]. The retinal blood vessels, especially

the small peripapillary retinal arterioles and veins, gradually disappear. The optic disc

becomes round, depressed and atrophied with the loss of myelin.

Optic nerve capillary endothelial cells are ultrastructurally abnormal prior to

detectable increases in IOP [36]. Spherical, membrane-bound, electron-dense inclusions

resembling Weibel-Palade bodies are found in pericytes and endothelial cells in both

preglaucomatous and glaucomatous eyes. These changes are usually associated with

microcirculatory abnormalities in humans and diabetic dogs.

Abnormal blood flow has been detected in the exterior and internal ophthalmic

arteries, the anterior ciliary arteries, and short posterior ciliary arteries, but not the









primary retinal arteries in the glaucomatous Beagle as measured by color Doppler

imaging [37]. Furthermore, while treatment with Ca-channel blockers increases blood

flow in orbital vessels of normal Beagle dogs, the blood flow of glaucomatous Beagles

remains unchanged. This indicates an inability of the vessels to respond to stimuli, which

may be due to an altered vascular structure with hypertrophy or dysfunctional regulation

by endothelial receptors.

Ophthalmic Vascular Morphology and Physiology

The eye is one of the most highly perfused organs in the body [38]. In dogs and

humans, the eye has two separate systems of blood vessels that differ anatomically and

physiologically: the retinal vessels, which supply the inner layers of the retina, and the

uveal or ciliary vessels, which supply the rest of the eye.

The main supply of blood to the eye and orbit of the dog is via the internal

maxillary artery (as a branch of the external carotid artery) that after passing through the

alar canal branches to give rise to the external ophthalmic artery (EOA) [39]. The internal

ophthalmic artery (IOA) in dogs is a small artery that arises from the anterior cerebral

artery at the level of the optic chiasm [40]. It passes through the optic canal on the dorsal

surface of the optic nerve in dogs, and runs rostral by the nerve to anastomose with a

branch of EOA about midway between the optic canal and the posterior pole of the globe.

By comparison, in primates the entire global microcirculation and most of the orbital

circulation are supplied via the internal carotid artery, which gives rise to the IOA [39].

Two long posterior ciliary arteries (LPCA), one medial and one lateral, arise from

the anastomoses of the external and internal ophthalmic arteries in dogs. Six to ten short

posterior ciliary arteries (SPCA) arise, surround the scleral canal of the canine optic nerve

and supply the lamina cribrosa, choroid, retina, and ONH circulations in dogs. The SPCA









thus give rise to two very biologically distinct circulations. The peripapillary choroid also

derives its blood supply from the SPCA at the ONH margin, although the choroidal and

ONH circulations are not continuous. Peripapillary choroidal blood flow is directed

anteriorly away from the optic disc as the venous drainage of the peripapillary choroid is

anterior to the vortex veins. Choroidal arteries branch into smaller vessels before

supplying the choriocapillaris, the innermost layer of choroidal vessels.

The retina in dogs and primates has a large vascular network in the major part of

the sensory retina. The blood vessels extend from the optic disc to the region of the ora

serrata. While the location of the blood vessels within the retina may differ somewhat

between species, the general pattern of the major retinal arterioles and venules is

generally similar in that they lie superficially in the nerve fiber layer and RGC layer,

radiating from the optic disc. The smaller arterioles, venules and capillaries are organized

into two dense capillary networks: an inner plexus, being situated at the level of the nerve

fiber layer and/or ganglion cell layer; and an outer plexus, which in the dog is situated at

the border between the inner nuclear layer and the outer plexiform layer.

Primates possess a cone-rich region completely free of rods called fovea. No retina

of the domesticated animals has a fovea, but an area of high cone density frequently

occurs and is often referred to as the "area centralis". This area lies 3- to 4-mm

dorsolateral to the optic disc in the dog. At the level of the "area centralis" there are no

major retinal vessels, but capillary networks lie in different layers of the retina. The

thickness of the innermost vascular plexus varies with the thickness of the nerve fiber

layer. In the temporal retina near the raphe where the nerve fiber layer is very thin,









capillaries are almost absent. In the peripapillary retina where the nerve fiber layer is

thick, the capillaries form a dense, multilayered plexus.

The arrangement (number and location of capillaries) of the superficial capillary

network is directly related to the thickness of the nerve fiber layer and the ganglion cell

layer, which are thought to be metabolically demanding [41]. The multilayer arrangement

leads to a plexus of numerous capillaries fed by a single, relatively large arteriole.

Consequently, the retinal microvasculature is subjected to a large pressure head, and

changes in its resistance are likely to play a significant role in blood flow regulation.

Blood vessels can be identified on the basis of distinct characteristics. Arteries are

large, with multiple layers of smooth muscle cells, the muscularis, surrounded by the

adventitial layer consisting of circumferentially oriented collagen fibers. An inner elastic

lamina separates the muscularis from the endothelium. A basement membrane surrounds

the endothelial cells and blends with the internal lamina elastica. Arterioles have smaller

caliber, a single layer of smooth muscle cells, and no, or only minimal, internal elastic

lamina. The endothelium is continuous and the cells are covered by a basement

membrane. Capillaries have a minimal wall thickness consisting of endothelial cells and

their basement membrane [42]. The endothelial lining of capillaries is supported by

circumferentially oriented pericytes with contractile features [43].

The endothelial cell monolayer lining blood vessels was long thought to simply

function as a diffusion barrier. It is now recognized that the endothelium has a crucial

role in regulating vascular homeostasis [44]. The location of endothelial cells between the

circulating blood and the vascular smooth muscle cells gives them a strategic position to

regulate vascular permeability and influence vascular hemostatic metabolic functions









[45]. The vascular endothelium plays an active role in vasomotor function of both macro-

and microvasculatures, including maintenance of vascular tone and regulation of blood

flow [46]. Vascular tone depends on a balance between the endothelial vasodilators (e.g.,

(NO) and vasoconstrictors (e.g., ET) such that reduced formation of vasodilators would

result in vasoconstriction and a decrease in vasoconstrictors would result in vasodilation.

Therefore, endothelial cells play an important role in modulating the microvascular tone

and autoregulation. Endothelial cells also strongly affect coagulation, platelet function,

and fibrinolysis [45].

Regulation of Blood Flow

Autoregulation plays a very important role in the control of blood flow in a tissue.

The goal of autoregulation in a tissue is to maintain a relatively constant blood flow,

capillary pressure, and nutrient supply in spite of changes in perfusion pressure [46]. This

is accomplished through the ability of the cardiovascular system to adjust the resistance

of particular vessels by controlling the diameter of their lumen.

The blood flow in the ONH and intraocular vessels (when pressure in the central

retinal vein is normal) is calculated by using the following formula:

Flow = Perfusion pressure / Resistance to flow
Perfusion pressure = Mean blood pressure (BP) intraocular pressure (IOP)
Mean blood pressure = Diastolic BP + 1/3 (systolic diastolic BP)

From this formula, it emerges that the blood flow depends on 1) resistance to blood

flow, 2) blood pressure, and 3) IOP.

Factors that normally govern microcirculatory vascular smooth muscle tone involve

vasoactive nerves, and circulating hormones, as well as endothelial cell derived factors,

and myogenic and metabolic factors [47]. According to the metabolic hypothesis, local

arterial smooth muscle tone is regulated by metabolic change in the tissue [46]. Local









concentration of metabolic products, 02 and CO2, plays a role in maintaining

autoregulation. Accumulation of CO2 and a reduction in 02 is due to hypoxia or lack of

ability to wash out metabolites, causing vasodilation. The myogenic hypothesis states that

a rise of intravascular pressure causes vasoconstriction because stretching of the vessel

wall is counteracted by vasoconstriction in the arterioles. Responses to changes in

transmural pressure, i.e. stretching are not dependent on endothelium-derived vaso-active

substances [48]. The endothelial cytoskeleton together with its extracellular matrix is

thought to provide a mechanical linkage between the site of the shear stress and the

smooth muscle cells. The change in pressure causes activation of protein kinase C, an

intracellular modulator of Ca 2+ -dependent contractile processes. The role of the

endothelium in flow-induced contraction, caused by stretching of the vessel wall, may

thus be that of a conduit for the mechanical disturbance. This autoregulation of blood

flow may also have neurogenic control. However, there is not much proof of this

occurring in the ocular vessels because vessels in the retina and ONH have no autonomic

nerve supply [38]. The choroid, by contrast, is richly supplied by the autonomic nerves

and yet has no appreciable autoregulation [49].

Alterations in blood flow through the microcirculation were long thought to be

solely achieved by changes in tone of precapillary sphincters. It is now well accepted that

retinal pericytes also have the capacity to act as important regulators of local blood flow.

Pericytes are perivascular cells with multifunctional activities. They are contractile cells

adjacent to the endothelial lining of capillaries supporting the microvasculature. The

regulatory mechanisms and vessel-wall cross-talk between these cells and the

microvascular endothelium have been shown to be identical to the those of smooth









muscle cells [43]. These interactions are very important in the retinal microcirculation

where autoregulation is vital for the maintenance of smooth and uninterrupted blood

flow.

Pericytes are more numerous in the retina than in any other microcirculation in the

body [43]. Within the retinal microvasculature there is a rapid transition from vascular

smooth muscle cells to pericytes at the interface of the precapillary and terminal

arterioles. Modulation of blood flow by pericytes in very small ophthalmic arteries and

capillaries has been shown in several studies [50-52]. The capacity of pericytes to

regulate the blood flow through capillaries indicates an important role of the pericytes in

the control of microcirculation of the retina and the ONH.

Blood flow in the retina and prelaminar ONH circulations have been shown to

possess autoregulatory capacity in several species by remaining stable during IOP-

induced changes in perfusion pressure, whereas blood flow in the choroid is sensitive to

changes in IOP [53-55]. However, the latter concept remains debatable. In particular, the

choroidal tissue just nasal to the optic nerve may be capable of blood flow autoregulation

in instances of increased IOP, as has been shown in the cat [56]. Furthermore,

investigations have shown that the choroid is capable of autoregulation in the rabbit [57].

The autoregulatory capacity of the human choroid is unknown [56].

Endothelin and Nitric Oxide

Synthesis, Secretion and Clearance of Endothelin

In 1988, Yanagisawa et al. [58,59] identified endothelin (ET), a very potent

endothelium-derived vasoconstrictive factor. The substance, a 21-amino acid peptide,

was isolated from the endothelial cells of pig aorta and is known today as ET-1.

Endothelin-1 has also been found to be expressed in non-vascular tissues, such as the









brain, kidney, lung and others [60]. Subsequent studies have shown that the ET isolated

from endothelial cells is one of a family of isopeptides.

Three isoforms of the peptide exist: ET-1, ET-2, and ET-3, but only ET-1 can be

detected in endothelial cells of vascular tissue. Endothelin-2 and ET-3 can be found in the

intestine, adrenal gland and brain. Endothelin-3 is relatively abundant in neuronal tissues

[60]. Each ET isoform is a product of separate genes that code for a precursor protein

mRNA that share high sequence homology [44]. The endothelins are synthesized from

precursors known as preproendothelins (preproET) comprised of 160 and 238 amino acid

residues. Before they are finally processed into 21 amino acid peptides, these large

precursors undergo an intermediate cleavage by endopeptidases to form the 37-41 amino

acid precursors proendothelins (proET), also named "Big ET". "Big ET" is further

processed by a specific endothelin converting enzyme (ECE) to form the 21 amino acid

active peptide.

Smooth muscle cells are target cells and so express a nonselective ECE (ECE-1) on

their cell surface associated with the plasma membrane to convert exogenous "Big ET".

The pro-ET-1 released from endothelial cells can therefore be converted into ET-1 in the

vascular smooth muscle surface [60]. However, the major part of conversion of "Big ET-

1" into ET-1 takes place in the endothelial cells, and it is assumed that the ET-1 is

concentrated primarily in these cells, although it is not stored there. The enzyme present

in endothelial cells is an ECE (ECE-2) selective for "Big ET-1" more than "Big ET-2" or

"Big ET-3". The ECE located in vascular smooth muscle acts equally on the different

progenitor peptides [60]. In the retinal microcirculation pericytes have been shown to

respond to ET-1 secreted by the retinal capillary endothelial cells [43,61].









The secretion of ET-1 by endothelial cells is regulated at the level of peptide

synthesis, because the endothelial cell contains no dense secretary granules in which

ET-1 can be stored and then later released. The expression of preproET-1 mRNA and

release of the peptide are stimulated by several factors including adrenaline, thrombin,

vasopressin, angiotensin II, insulin, cytokines, transforming growth factor 0 (TGF 0),

arginine, and physical stimuli such as shear stress of the endothelium. Ischemia and

hypoxia also result in synthesis and release of ET-1 [60]. Endothelin-1 synthesis is

inhibited via a cGMP-dependent mechanism by NO, prostacyclin, natriuretic peptides

and heparin [60].

Hisaki et al. [62] showed that removal of the endothelium does not completely

prevent the action of "Big ET-1" in the perfused rat mesenteric bed, suggesting that

smooth muscle cells also can synthesize ET-1. Smooth muscle cells in culture express

ET-1 mRNA and release ET-1. According to Warner [63] within a diseased blood vessel

with damaged endothelial cells, the vascular smooth muscle may produce ET-1 that is not

regulated by endothelial cell agonists.

Most of the ET-1 released by the endothelial cells (approximately 80% of the

synthesized amount) is secreted abluminally acting as a local hormone in a paracrine

manner on the surface of the vascular smooth muscle [60]. The plasma half-life of ET-1

in humans is less than 1.5 min because of its efficient extraction by the splanchnic and

renal vascular beds [44]. Endothelin-1 is also reported to be taken up by the lungs but the

clearance differs between species [64].

Extraction of ET-1 follows binding to cell surface receptors, which are then

internalized, allowing degradation to be carried out within the cell. Endothelin-1 is found









to be internalized by both ETA receptors and ETB receptors [65,66]. Both receptors seem

to be agonist-occupied after internalization for one to four hours. Endothelin-1 is then

dissociated from the receptor and possibly degraded. It has been shown that internalized

ET-1 continues to evoke signaling events in the cells, which has been suggested to be one

of the reasons that ET-1-stimulated effects are long-lasting [65]. A possible candidate for

an intracellular degrading enzyme is a soluble protease found in human platelets, vascular

smooth muscle and endothelial cells [64]. The fate of the internalized receptors will be

discussed in the chapter about endothelin receptors.

Nitric Oxide

Nitric oxide plays an important role in combination with ET-1 to produce a

balanced effect on blood vessel activity. Nitric oxide is an inorganic free radical,

produced from L-arginine by the enzyme nitric oxide synthase (NOS) [63]. Nitric oxide

synthase exists in at least three isoforms, but it is only the calcium-regulated isoform,

endothelial NOS (eNOS), that appears responsible for NO formation within the

healthy endothelium. The stimulation of a endothelial cell membrane receptor by an

agonist such as acetycholine, histamine, thrombin, ET-1 or bradykinin leads to an

increase in intracellular calcium, which in turn activates NOS [49]. Mechanical forces,

such as stretch and shear, can also stimulate the production of NO.

When NO reaches its target smooth-muscle cell it activates the enzyme guanylate

cyclase, responsible for the production of the 3'5'-cyclic guanosine monophosphate

(cGMP), a second messenger. The increase in cGMP ultimately leads to smooth-muscle

relaxation by reducing the intracellular calcium content [49]. A basal release of NO

maintains ocular vessels in a constant state of mid-dilation [67].









Retinal capillary endothelial cells have been shown to constitutively express the

enzyme NOS [68]. The enzyme guanylate cyclase has been shown to be expressed in

retinal pericytes making it possible for the cells to synthesize cGMP on exposure to NO.

Cultured pericytes relax to sodium nitroprusside (a NO donor), which stimulates

guanylate cyclase and increases cGMP [69]. In addition the pericytes have been shown to

respond to prostacyclin [67].

In normal vessels there is a balance between the endothelial production of NO and

ET-1. The ability of NO to down-regulate ET-1 production and of ET-1 to stimulate NO

production by endothelial cells acts to maintain a matched degree of constrictor-dilator

tone.

In a number of disease states there is suspected to be a breakdown in the balance

between ET-1 and NO [63]. Endothelial cell reduction in NO production may be

compensated for by the induction of inducable nitric oxide synthase (iNOS) within the

vascular smooth muscle. However, iNOS produces NO in an uncontrolled manner. In

such disease states, ET-1 production is often increased. This increase in ET-1 production

may well underlie deleterious increases in blood vessel reactivity and reductions in tissue

perfusion [63].

Endothelin Receptors

Endothelin receptors exist both in vascular smooth muscle, pericytes, and the

endothelium. In 1990, two ET receptors (ETA and ETB) were isolated [44]. Both

receptors belong to a family of heptahelical G-protein coupled receptors. Although these

two receptors have approximately 60% similarity in amino acid sequence, several

domains are less homologous to each other and contribute to the functional differences

between the ETA and ETB receptors.









The two ET receptor subtypes vary in their affinities to endogenous ligands.

Endothelin receptor-A has a high affinity to ET-1 and ET-2, and a low affinity to ET-3.

Endothelin receptor-B has an equally potent affinity to all three ligands [70].

Endothelin receptor-A exists on smooth muscle cells and mediates

vasoconstriction, while the ETB receptor is located on endothelial cells and mediates

release of relaxing factors, such as prostacyclin and nitric oxide. Recent research

indicates that ETB also exists on vascular smooth muscles and mediates vasoconstriction.

The relative contributions of ETA and ETB receptors to vasoconstriction is variable, and

depends on the species and the vessel type studied [44].

Endothelin causes vasodilation at very low concentrations, and a marked and

sustained contraction at high concentrations. The dilator response to endothelin involves

activation of the ETB endothelial receptors, linked to NO and/or prostacyclin release by

endothelial cells [45]. At higher concentrations, such that all the endothelial ETB

receptors are occupied, ET diffuses through the intima toward the vascular smooth

muscle. The constrictive response involves the activation by endothelin of ETA and ETB

receptors on smooth muscle cells [45]. This vasoconstriction is correlated with a

sustained increase of intracellular Ca2+ produced via a two-phase mechanism. The

binding of ET-1 to the receptor activates phospholipase C (PLC) which hydrolyses

phosphatidilinositol-4,5-biphosphate (PIP2) into two products, inositol triphosphate (IP3)

and diacylglycerol (DAG). The initial increase in intracellular Ca2+ concentration is

caused by a rapid mobilization of intracellular calcium stores by IP3. This ET-1 mediated

signaling upstream of IP3 interaction with the Ca2+ stores is regulated by tyrosine kinases

which also participate in the contractile response of a-adrenoceptor agonists, histamine









and angiotensin II. The maintained increase in intracellular Ca2+ levels is brought about

by exterior Ca2+ entry via a calcium channel of the plasma membrane.

At least two types of non-selective cation channels as well as voltage-operated

calcium channels are activated by ET-1 [71]. One is activated by low and the other by

high concentrations of ET-1. The channel activated by low concentrations of ET-1 is

inhibited by NO and therefore ET and NO seem to affect the same channel. The increased

production of DAG also stimulates the translocation of protein kinase C (PKC) from

cytoplasm to membrane and its subsequent activation [72]. Activation of PKC leads to

phosphorylation of many proteins. Phosphorylation of myosin light chains induces

smooth muscle contraction.

The binding of ET-1 to the ETA receptor gives rise to a long-lasting response while

the response of the ETB receptor upon ligand binding is of more transient nature.

Moreover, it has been shown that the ETB receptor mediated vasodilation response to

repeated injections of ET-1 shows tachyphylaxis, whereas the vasoconstrictor response in

the same study did not [66]. This implies different pathways for the two receptors

following internalization.

Bremnes et al. [73] investigated the regulation and intracellular trafficking

pathways of the endothelin receptors. Their study demonstrated that both receptor

subtypes are rapidly internalized upon agonist stimulation. Internalized ETA and ETB

receptors both enter early endosomes (sorting endosomes). However, from this location,

the two receptor subtypes are targeted to different intracellular fates. Whereas the ETA

receptor is directed to the pericentriolar recycling compartment and subsequently

reappears at the plasma membrane, the ligand bound ETB receptor is directed to









lysosomes for degradation. Thus, further clearance of plasma ET-1 by the ETB receptors

will be limited by the supply of de novo synthesized receptor molecules to the cell

surface. This mechanism also explains the transient nature of the ETB mediated response

and the tendency for development of tachyphylaxis upon repeated stimulation of the ETB

receptor. The conclusion from this group was that the rapid recycling of ETA may provide

a basis for the prolonged contractile response mediated through this receptor, whereas

lysosomal targeting of ETB suggests a role for this receptor in clearance of ET from the

circulation.

Chun et al. [74] showed that ET-1 remains intact and bound to the ETA receptor for

up to two hours after endocytosis. Their hypothesis for the prolonged signal response of

the ETA receptor was that ligand-occupied ETA receptors may continue to activate the G-

protein after endocytosis.

Retinal pericytes express both isoforms of the endothelin receptor, ETA and ETB

[75]. The pericyte ETA receptor mediates contraction through the same pathway as the

ETA receptor on smooth muscle cells, i.e. through the phospholipase C/inositol phosphate

signaling pathway [43]. The exact nature of the second messenger signaling downstream

to ETB receptor activation in the retinal pericyte remains uncharacterized. However, a

study has linked endothelin 3 stimulation of renal mesangial cells (also classified as

pericytes) to the production of NO [43]. This mechanism was postulated to buffer the

contractile effects of ET-1 especially when local levels are inappropriately high. This is

in opposition to the constrictor response evoked by activation of the ETB receptor on

smooth muscle cells. Mc Donald et al. [76] showed by binding studies of various

fragments of ET-1, -2, and -3 using an in vitro model of microvascular pericytes that it is









highly likely that the ETB receptor on retinal pericytes are different from those on the

endothelium.

There is considerable heterogeneity in pericyte surface endothelin receptor

expression with some cell types expressing only the ETA and others ETB [77]. The cell

membranes of vascular effector cells may show both receptor types and one may be

preferentially expressed. This is certainly the case in retinal pericytes where receptor

binding studies have revealed the presence of several hundred thousand high-affinity ETA

receptors but only twenty to thirty thousand ETB receptors on a single cell. It is also

noteworthy that retinal pericytes express many more ETA receptors than vascular smooth

muscle [75].

At concentrations where ET-1 exerts no direct contractile effect, it potentiates the

response to other vasoconstrictor hormones such as norepinephrine and serotonin [45].

The potentiating effects are due to an increased Ca2+ sensitivity of smooth muscle cells

and can be prevented by pretreatment with calcium antagonists of the dihydropyridine

type.

Endothelin and the Ophthalmic Circulation

The endothelial-dependent regulatory mechanisms are present in the entire

cardiovascular system but there is a great heterogeneity of responses from one organ to

the other, in arteries and veins, and in vessels of different diameter. This heterogeneity

may be explained by a different smooth muscle content in different vessels, and/or

different expression of receptors.

Haefliger et al. [78] showed that the effect of both NO and ET-1 increased with

decreasing vascular diameter, when comparing the response to chemical or hormonal

agonists of the porcine ophthalmic artery with the response of the ciliary artery.









Endothelin-1 has also been shown to contribute to hyperoxia-induced retinal

vasoconstriction through its ETA receptor in the human retina [79]. Systemic ET-1

administration in healthy subjects reduces pulsatile blood flow in the choroid and optic

disc at doses which do not affect systemic hemodynamics or flow velocity in the

ophthalmic artery [80]. This indicates that ocular circulation is particularly sensitive to

changes in local ET-1 concentration and confirms the hypothesis that ET-1 may play a

role in ocular vascular diseases.

Granstam et al. [81] showed a reduction of retinal blood flow, but no effect on the

choroidal blood flow, by intravitreal ET-1 injections in cats. In contrast, Kiel et al.

[82]has shown that the choroidal circulation in rabbits is regulated by ETA and ETB

receptors mediating opposing effects on choroidal vascular resistance. Their results also

suggested that endogenous ET preferentially elicits ETB vasodilation, most likely by

stimulating endothelial nitric oxide release.

Endothelin-1 is well distributed in ocular tissue and is localized in aqueous humor,

iris, ciliary body, retina, and choroids [83,84]. The ET-1 of retinal origin is mostly vessel

oriented but is also found in the retinal ganglion cell layer, the photoreceptor inner

segments, and the outer plexiform layer. Narayan et al. [85] has shown that cultured RPE

cells (ARPE-19cells) produce and release ET-1 by stimulation of muscarinic receptors. In

the same study mRNA expression for preproET-1, ETA and ETB receptors was detected

in the RPE cells.

Both ETA and ETB -type receptor binding sites have been detected in the vascular

smooth muscle of choroidal and retinal vessels of humans and rats [83]. In the same study

extravascular binding sites of the ETB -type were found in the ganglion cell layer. In a









study in humans and rabbits the ETA -like receptor binding sites were localized to the

retinal and choroidal blood vessels, whereas the ETB -like receptor binding sites were

localized to the neural and glial cells of the retina [86].

Endothelin and Aqueous Humor Production and Outflow

In the anterior part of the eye, ETA receptors have been found in human ciliary

smooth muscle (HCSM), ciliary non-pigmented epithelial (NPE) cells, and trabecular

meshwork (TM) cells [87,88]. Endothelin receptor-B receptor expression has been

observed in cell cultures in HCSM cells [89]. Sugiyama et al. [90] showed a role for both

ETB and ETB receptors in regulating IOP.

Endothelin-1 affects the contractility of ciliary smooth muscles (CSM) and TM

cells consequently regulating aqueous humor [91]. Studies have shown that ET-1

decreases aqueous humor formation and increases outflow facility in rabbits and

monkeys [92-94]. Endothelin-1 has also been shown to decrease the activity of sodium

potassium ATPase, a key enzyme involved in ion transport and aqueous humor

production [95].

Since it has been shown that ET-1 decreases IOP and increases overall outflow

facility, the direct effect of ET-1 of the trabecular meshwork cells is functionally

antagonistic to the direct effect on ciliary muscle [96]. Thus intertrabecular spaces could

be narrowed by contracting trabecular fibres and widened by ciliary muscle contraction.

According to Wiederholt [96] an alternative possibility might exist: in the presence of

both tissues, contraction of the trabecular meshwork may increase the rigidity of this

tissue allowing the ciliary muscle contraction to be more effective in altering the

geometry of the trabecular meshwork. Although contraction of the ciliary muscle

dominates the overall effect on outflow facility in the human (and most likely in the dog)









eye, the concept of antagonism between ciliary muscle and trabecular meshwork should

be considered in the interpretation of mechanism of action of ET-1 on aqueous outflow

and IOP.

Purpose of Study

The goal of this study was to document any differences in the location and density

of endothelin-1 receptors in the glaucomatous dog eye as compared to the normal dog

eye. In addition, the levels of the endothelin-1 peptide and nitric oxide in aqueous humor

and vitreous were compared in the normal and glaucomatous dog eye.

The hypotheses were the following:

* The localization of endothelin-1 receptors differs in the retinal and choroidal
vessels in the normal canine eye and the glaucomatous canine eye.

* The density of endothelin-1 receptors differs in the retina of the normal canine eye
and the glaucomatous canine eye.

* The concentration of endothelin-1 and nitric oxide in aqueous humor and vitreous
differs between the normal canine eye and the glaucomatous canine eye.

The objectives were:

* To identify the localization of endothelin-1 receptors in the retinal and choroidal
vessels of normal and glaucomatous canine eyes as measured by immunogold
labeling.

* To identify the density of endothelin-1 receptors in the retina of normal and
glaucomatous canine eyes as measured by Western Blot and Real Time PCR.

* To determine the levels of endothelin-1 and nitric oxide in the aqueous humor and
vitreous of normal and glaucomatous canine eyes as measured by immonoasssay.














CHAPTER 2
MATERIAL AND METHODS

Study Design

The normal and glaucomatous canine eyes in this study were from canine clinical

cases at the University of Florida and Universidad Nacional Autonoma de Mexico in

Mexico City. All dogs went through an ophthalmic examination prior to inclusion in the

study (Tables 2-1 and 2-2).

Enucleated normal eyes (n=30) of 19 dogs and glaucomatous eyes (n=14) of 14

dogs were collected during one year. Normal and glaucomatous samples were obtained

aseptically within 10 minutes after enucleation. The same technique was practiced for all

dogs irrespective of the procedure and hospital performing the procedure. Samples of

aqueous humor, vitreous, retina, and choroid were stored for future analysis of protein

and mRNA content. In addition, samples of the posterior segment of the globe, with

retina and choroid attached, and the anterior segment including the iridocorneal angle,

were gathered for histological and immunocytochemical studies.

The levels of nitric oxide (NO) and endothelin (ET) -1 in aqueous humor and

vitreous were measured by enzyme immunoassay. The location of the ETA and ETB

receptors were determined in the retina and choroid with immunocytochemistry and the

expression of ET receptor protein with Western Blot technique. The mRNA levels of

ET-1, and the ETA and ETB receptors, in the retina were assessed with Real Time

polymerase chain reaction (PCR). The retinas and iridocomeal angles were evaluated









histologically. Comparisons were made between normal and glaucomatous samples for

these parameters. A summary of samples and analyses is shown in Tables 2-3 and 2-4.

Clinical Examination of Dogs

Ophthalmic examinations of the glaucoma dogs in this study included tonometry

(Tonopen, Mentor Ophthalmics, MA), slitlamp biomicroscopy (Kowa SL 14,Kowa

Optimed, CA), and indirect ophthalmoscopy (Heine Omega 150, Dover, NH). The mean

( SD) intraocular pressure (IOP) of normal dogs, as measured by Tonopen applanation

tonometry, is 16.8 4.0 mmHg. The glaucoma dogs in this study were clinically

diagnosed with hypertensive glaucoma, i.e. with an IOP exceeding 25 mm Hg. Clinical

signs also included episcleral congestion, mydriatic pupils, optic nerve cup enlargement,

neuroretinal rim narrowing, optic cup deepening, and retinal degeneration. Slitlamp

biomicroscopy, and indirect and direct ophthalmoscopy of each dog were performed by

veterinary ophthalmologists with extensive experience.

Breeds, ages, and clinical parameters of the normal and glaucomatous dogs are

shown in Tables 2-1 and 2-2.

Endothelin-1 and Nitric Oxide Levels in Aqueous Humor and Vitreous

Endothelin-1 Analysis

Samples of aqueous humor and vitreous were gathered from enucleated normal

eyes (n=28) of 18 dogs and glaucomatous eyes (n=l 1) of 11 dogs (Tables 2-3 and 2-4).

The samples were stored at -80o C until analysis. Measurements of ET-1 were made by

enzyme immunoassay (BBE5 Human Endothelin-1 Parameter ELISA Kit, R&D Systems,

MN) by the Analytical Toxicology Core Laboratory at University of Florida. Cross

reactivities entail big endothelin, <1%; sarafotoxin, <2%; ET-2, 45%; ET-3, 14%. The

fact that canine and human ET-1 have corresponding amino acid sequences validated this









kit for testing canine ET-1. The kit was first evaluated on dog plasma. The extraction

method for ET-1 in plasma was then modified and validated for the much smaller sample

size and different constituency of the aqueous humor samples. Unlike plasma, which

requires an extraction prior to assay, aqueous humor samples can be assayed directly.

When sufficient volume was available (>200 pL) each sample was run in duplicate.

Nitric Oxide Analysis

Measurements of total NO were made by enzyme immunoassay (Nitrate/Nitrite

Colorimetric Assay Kit; Cayman Chemical Company, Ann Arbor, MI). In this method

the two ultimate products of NO activity (nitrite and nitrate) are evaluated together rather

than separately to enhance reliability. To achieve this, nitrate in the samples was first

converted to nitrite by incubation with nitrate reductase. Griess reagents were then added

to react with nitrite and form a purple azo end-product that absorbs strongly at 540 nm.

Although common interfering species include antioxidants (such as ascorbic acid, 0.1

mM) and phosphate (>50 mM), the interferant of greatest concern for this assay is

NADPH, an essential cofactor for Nitric Oxide Synthase (NOS) enzyme function.

Because this species can inhibit formation of the azo product, a catalyst is added with the

nitrate reductase to promote the conversion of NADP+ to NADPH.

Aqueous and vitreous humor samples were prepared for the assay by centrifuge

filtration through 30 kDa cut-off filters (Millipore Corporation, Bedford, MA) that had

been pre-rinsed with HPLC-grade water. When sufficient volume was available (-100

uL) the samples were analyzed in duplicate.

Localization of Endothelin Receptors in the Retina and Choroid

To our knowledge, ET receptor antibodies have not been used previously on canine

tissue. Therefore it was necessary to determine if the antibodies had an affinity for the ET









receptors in our samples. This was done by immunostaining of scleral vessels of a normal

Beagle eye. The staining was evaluated by light microscopy. Following confirmation of

the affinity of the antibodies, immunocytochemistry for evaluation of receptor location by

transmission electron microscope (TEM) was done on normal and glaucomatous retinas

and choroids in the study.

Light Microscopy

The tissue was immersion fixed in 4% paraformaldehyde 0.1M phosphate buffer

solution (PBS), pH 7.4, for 10-12 hours. Following fixation the tissue was washed in

0.1M PBS, pH 7.4, dehydrated in an ascending series of ethanol, and embedded in

paraffin. Paraffin sections were cut 6 [tm thick and mounted on glass slides. After

deparaffinization with graded concentrations of xylene and ethanol, slides were immersed

in 3% hydrogen peroxide in methanol for 20 minutes at room temperature to block

endogenous peroxide activity. The slides were then incubated with 2% non-fat dry milk

and 2% cold water fish gelatin in 0.1 M PBS for 30 minutes to reduce non-specific

background staining. Primary antibodies, rabbit anti-rat ET receptor type A (1:100) or

rabbit anti-rat ET receptor type B (1:100) (Alomone Labs, Jerusalem), were incubated in

damp chamber at 40C overnight. The treated slides were then incubated with biotinylated

link antibody for 30 minutes at room temperature, then incubated with peroxidase-labeled

streptavidin for 30 minutes at room temperature. Peroxidase activity was visualized by

the 3-amino-9-ethylcarbazole (AEC) substrate-chromogen system.

Transmission Electron Microscopy

Normal dog eyes (n=5) from 5 dogs and glaucomatous dog eyes (n=5) from 5 dogs

were collected (Tables 2-3 and 2-4).









The tissue was immersion fixed in 4% paraformaldehyde 0.1M PBS, pH 7.4, for

10-12 hours. Following fixation the tissue was washed in 0.1M sodium cacodylate, pH

7.4, with 3.5% sucrose, 0.5% DMSO, and 0.5mM calcium chloride followed by

incubation for 1 hour in 1% tannic acid in 0.1M sodium-maleate buffer, pH 6.0. It was

then dehydrated in an ascending series of ethanol. At 70% ethanol, 1%

p-phenylenediamine (PPD) was added. This was followed by infiltration and embedding

in LR White acrylic resin. Blocks were polymerized at 550 C for 12-20 hours. Ultrathin

(80-90 nm) sections were collected on formvar carbon filmed, 150 mesh, nickel grids

(Electron Microscopy Sciences, Fort Washington, PA). Sections were labeled by placing

them on drops of primary antibody, rabbit anti-rat ET receptor type A (1:50) or rabbit

anti-rat ET receptor type B (1:200) (Alomone Labs, Jerusalem), in damp chamber at 4C

overnight. Goat anti-rabbit IgG labeled with 12 nm collodial gold (Jackson Immuno

Research Laboratories, Inc., West Grove, PA) diluted 1:50 in 2% normal goat serum in

Tris buffered saline (TBS), pH 7.6, were used as secondary antibodies. All rinses

contained 0.05% Tween 20 and 0.2% Triton-X-100. For the negative controls the primary

antibodies were excluded and the grids were incubated on PBS followed by incubation on

colloidal gold labeled secondary antibody.

To assess specific binding, the primary antibodies were preabsorbed with the ET

antigen. Detection of antibody binding to specimen after pre absorption is considered

specific and not taken into account in the analysis.

Labeling of endothelial cells and smooth muscle cells was used as positive control

since label of ETA and ETB receptors in these cell types have been reported extensively in









the literature. Grids were examined and photographed at 75 kV in a Hitachi H-7000 TEM

(NSA Hitachi, Palo Alto, CA).

Morphology of Retina and Iridocorneal Angle

Retinas from 9 glaucomatous eyes and iridocorneal angles (ICAs) from 11

glaucomatous eyes were examined morphologically. Sections for evaluation of

morphology were processed in the same manner as described above. Paraffin sections of

the anterior segment were stained with Periodic Acid-Schiff (PAS) solution. Staining

with toluidine blue was used for sections of both the anterior and posterior segment

embedded in LR White resin. The degree of degeneration in the retina of the glaucoma

dogs was graded in 3 groups according to severity: 1) No abnormalities in the nuclear

layers, minor atrophy in the nerve fiber and ganglion cell layers; 2) Moderate atrophy and

/or melding of the inner and outer nuclear layers; 3) Marked atrophy of all retinal layers.

The degree of degeneration of the retina was correlated to the ET-1 levels in aqueous

humor and vitreous (see Results; Endothelin-1 levels in aqueous humor and vitreous).

Endothelin Receptor Protein Expression in the Retina and Choroid

Normal dog eyes (n= 1) from 11 dogs and glaucomatous dog eyes (n=13) from 13

dogs were used for protein analysis of the retina and choroid (Tables 2-3 and 2-4). The

samples were stored in three different ways; 1) snap-freezing of the retina and choroid in

liquid nitrogen with no preservative, 2) separation of the retina and choroid prior to

freezing with no preservative, 3) separation of the retina and choroid and put in RNaLater

solution (Ambion, Austin, TX) prior to freezing. The samples were stored at -80o C until

analysis.









Protein Extraction

The protein was either extracted form tissue that was frozen without preservative or

from tissue where retina and choroid were separated prior to freezing and stored with or

without preservative.

Protein extraction from snap-frozen retina and choroid with no preservative.

Retina and choroid from each eye were separated prior to homogenization. The samples

were sonicated (60 Sonic Dismembrator, Fischer, Atlanta, GA) in 400-500 [tL of

Laemmli sample buffer (Bio-Rad Laboratories, Hercules, CA) containing 50 tL

P-mercaptoethanol, 150 pL protease inhibitor cocktail [1 tablet Complete Mini EDTA-

free (Roche, Chicago IL) dissolved in 1 mL distilled water], 10 tL aprotinin (10mg/mL),

10 tL leupeptin (10mg/mL), and 10 tL phenylmethylsulfonyl fluoride (PMSF)

(40mg/mL) per mL of sample buffer. The samples were centrifuged and the supernatant

was transferred into fresh tubes for Bradford colorimetric protein assay (Bio-Rad

Laboratories, Hercules, CA).

Protein isolation from retina and choroid that was separated prior to freezing

and stored with or without preservative. The tissue was homogenized in 1 mL of

TRIZOL M Reagent (Total RNA Isolation Reagent, Life Technologies, Gaithersburg, MD)

per 50-100 mg of tissue using a power homogenizer (Ultrasonic celldisrupter, Heat

Systems, Ultrasonics Inc., Pharmingdale, NY) following the protocol supplied by the

manufacturer. Tissue treated with TRIZOL TM Reagent and chloroform for RNA extraction

(see Endothelin Receptor mRNA Expression in the Retina) separated into an aqueous

phase and an organic phase. After removal of the aqueous phase, containing RNA, the

proteins in the organic phase were recovered by precipitation with isopropanol. Protein









content was determined by Bradford colorimetric protein assay (Bio-Rad Laboratories,

Hercules, CA).

Western Blot

The protein samples were loaded on a 4% stacking, 10% resolving Tris-HCl ready

gel (Bio-Rad Laboratories, Hercules, CA). Each gel was loaded with the same amount of

protein for each sample. Kaleidoscope Prestained Standards (Bio-Rad Laboratories,

Hercules, CA) were used as markers, and rat brain extract (SIGMA, Saint Louis, MI) as

control on each gel. The electrophoresis was run at 100 V for 1 hour in a running buffer.

The proteins in the gel were then electrotransferred on to a nitrocellulose membrane in a

transfer buffer at 60 mA overnight.

Western-blotting was done using rabbit anti-rat ET receptor type A (1:100) and

rabbit anti-rat ET receptor type B (1:200) (Alomone Labs, Jerusalem) as primary

antibodies. The receptors were visualized using ECL Western Blotting System by

following the manufacturer's protocol (Amersham Pharmacia, Piscataway, NJ). Donkey

anti-rabbit horse radish peroxidase-linked antibody in Tris buffered saline/Triton X

(TBS/T) (1:10,000) was used as secondary antibody. Detection reagents, supplied with

the blotting kit, were incubated with the membrane for 1 minute. The membranes were

then exposed to an x-ray film (BMX, Light 1; Fisher, Atlanta, GA) for 10, 30, and 120

minutes.

The films were scanned with GS-710 Calibrated Imaging Densitometer (Bio-Rad

Laboratories, Hercules, CA) and the density of the ET receptor protein bands on each gel

was determined using Scion Image for Windows (Scion Corporation, Frederick, MD).

This program visualizes the density profile as a curve with optical density on the Y-axis

and millimeter (mm) on the X-axis. The density of the bands was expressed as area under









the curve of each peak. The density measurements of the samples were normalized by

dividing the value of each band with the value for the control (rat brain extract) of each

gel. This resulted in a value expressed in arbitrary units (AU) for each sample. The AU

for the ET receptors were compared between normal and glaucomatous retinal and

choroidal samples.

Endothelin Receptor and Endothelin-1 mRNA Levels in the Retina

Isolation of Total RNA

Normal dog eyes (n=9) from 9 dogs and glaucomatous dog eyes (n=10) from 10

dogs were used for mRNA analysis (Tables 2-3 and 2-4). Total RNA was isolated from

retinas that had been separated prior to freezing and stored dry or in RNALater (Ambion,

Austin, TX) using TRIzol Reagent (Life Technologies, Gaithersburg, MD) following the

protocol supplied by the manufacturer. The same retinal samples were used for mRNA

determination as for Western Blot analysis (Tables 2-3 and 2-4).

The samples were homogenized in 1 ml of TRIzoL TM Reagent per 50-100 mg of

tissue using a power homogenizer (Ultrasonic celldisrupter, Heat Systems, Ultrasonics

Inc., Pharmingdale, NY). The addition of chloroform followed by centrifugation

separated the solution into an aqueous and an organic phase. RNA remained in the

aqueous phase. After transfer of the aqueous phase, the RNA was recovered by

precipitation with isopropyl alcohol. The organic phase was saved for protein isolation

(see Endothelin Receptor Protein Expression in the Retina and Choroid). Potential

contaminating DNA in the RNA samples was removed using the DNA-free kit from

Ambion (Austin, TX). Optical density (OD) measurements were done on the RNA

samples at 260nm.









Reverse Transcriptase Polymerase Chain Reaction

Primers for the reverse transcriptase polymerase chain reaction (RT-PCR) were

designed using the software Vector NTI (Bethesda, MD) (Table 2-5). The primers used

for the ETA receptor were designed from the dog ETA nucleotide sequence published by

Louden et al. [97]. The ETB primers were designed from a reported canine endothelin

receptor B mRNA sequence (Gen Bank Accession number AF034530). The ET-1

primers were designed from canine ET-1 precursor, mRNA (Gen Bank Accession

number AF333433). The amplicon sequences produced by the various primers were later

used in the design of the primers for the Real Time PCR assay. All primers for the RT-

PCR were purchased from Invitrogen Life Technologies (Frederick, MD).

Synthesis of cDNA from total RNA was done using the First strand cDNA

synthesis kit from Amersham Pharmacia Biotech (Piscataway, NJ); the designed

antisense primers were used to prime the cDNA synthesis.

PCR was performed in a total volume of 50 gL consisting of 6 [L cDNA, 5 [L

10X Buffer A [(500 mM KC1, 200 mM Tris-HCl,pH 8.4) Invitrogen, Carlsbad, CA], 1 tL

50 mM MgC12, 1 tL dNTP, 1 tL 5' primer (30 pmole/ tL), 1 tL 3' primer (30 pmole/

tL), and 0.5 tL Taq DNA polymerase (5,000 units/mL) (Invitrogen, Carlsbad, CA). All

reactions were run in a DNA thermal cycler, Robocycler (Stratagene, La Jolla, CA),

using 30 cycles of denaturation at 950 for 0.5 minute, annealing at 530 for 0.5 minute, and

extension at 720 for 1.5 minutes. The last cycle contained 10 minutes at 720. The PCR

products were run on a 1 % agarose gel alongside a 1000 bp and a 100 bp DNA marker.

PCR products were recovered from the gel using the QIAEX II agarose gel

extraction kit (Quigen Inc., Valencia, CA). Purified PCR products were ligated into a TA

cloning vector, pCR2.1, and transformed into TOP10 cells (Invitrogen, San Diego, CA).









Plasmids from clones containing inserts were isolated using the QIAprep Miniprep

(Quigen Inc., Valencia, CA). The inserts were identified by restriction enzyme digestion

with EcoR1 (Promega, Madison, WI) and subsequent agarose gel analysis. Plasmid DNA

was then used to evaluate the primers for Real Time PCR.

Real Time Polymerase Chain Reaction

Primers and probes for the Real Time PCR were designed for the amplicons

described above using Primer Express for Mac (Perkin-Elmer Applied Biosystems Inc,

Foster City, CA) (Table 2-5). The primers and fluorogenic probes for the Real Time PCR

were purchased from Perkin-Elmer Applied Biosystems Inc. (Foster City, CA). Random

hexadeonucleotides (pd(N)6 primers provided with the Amersham kit were used to prime

the cDNA synthesis.

The Real Time probes had a reporter dye (6-FAM) conjugated to the 5' end and a

quencher dye (TAMRA) conjugated to the 3' end. The principle of the Taqman system is

that the fluorescence from the reporter dye is quenched by the quencher dye as long as

the dyes are nearby to each other. The specific probe binds to the cDNA being amplified.

When the Taqman polymerase replicates the target cDNA it cleaves the probe that is

bound to the cDNA. This results in separation of the reporter and quencher dyes allowing

the fluorescence of the reporter dye to be measured. The fluorescence increases in each

cycle in proportion to the rate of probe cleavage.

The Real Time PCR was performed in a total volume of 25 [tL consisting of 2X

Taqman Universal PCR Master Mix, 300nM each of forward and reverse primers, 80 pM

of fluorogenic probe, and 3 ng of template. Ribosomal RNA (rRNA) 18S was used as an

endogenous control and active reference to normalize quantities of cDNA. In the reaction

for the 18S rRNA the primers and probes were replaced with 20X Eukaryotic 18S rRNA









Endogenous Control (Applied Biosystems, Foster City, CA). The Thermal Profile for the

Real Time PCR was 1 cycle for 2 min at 500, 1 cycle for 10 min at 950, followed by 40

cycles involving 15 sec at 950 and 1 min at 600 for each cycle. Fluorescence for each

cycle was quantitatively analyzed on a Gene Amp 5700 sequence detection system

(Perkin-Elmer Applied Biosystems Inc, Foster City, CA). The relative amount of cDNA

in the samples was determined by the threshold cycle (CT). The CT is defined as the

fractional cycle number at which the level of fluorescence released from the Real Time

Probe surpasses the background threshold (Figure 2-1). The higher the starting copy

number of the nucleic acid target, the sooner a significant increase in fluorescence is

observed. In other words, the higher amount of cDNA in the sample, the lower the CT

value.

Each sample was run in triplicate to determine reproducibility of the PCR reaction.

As a negative control the Real Time PCR was run without any cDNA as template. In

addition, the Real Time PCR was run with total RNA, instead of cDNA, to rule out that

genomic DNA was present and amplified in the samples. Cloned DNA sequences of both

the ETA and the ETB receptor were amplified with primers and fluorogenic probes for the

Real Time PCR to evaluate the selectivity and specificity of the primers and the

fluorogenic probes. Amplification of both ET receptor DNAs were analyzed with various

combinations of primers and probes, as shown in Table 2-6.

Additional controls analyzed specific amplification. One set of Real Time PCR

reaction was carried out using DNAse treated total RNA, and a PCR reaction was also

run with omission of cDNA, as a negative control. All reactions were run in triplicates.









Data Analysis

Morphology

The iridocorneal angles were qualitatively interpreted and a comparison was done

between ICAs from normal and glaucomatous eyes. The degree of degeneration in the

retina of the glaucoma dogs was graded in 3 groups according to severity: 1) No

abnormalities in the nuclear layers, minor atrophy in the nerve fiber and ganglion cell

layers; 2) Moderate atrophy and /or melding of the inner and outer nuclear layers; 3)

Marked atrophy of all retinal layers. Spearman's correlation coefficient was used to

determine any correlation between the degree of degeneration and the ET-1 levels in

aqueous humor and vitreous.

The distribution of labeling of the ETA and ETB receptors in the retina and choroid

was qualitatively interpreted in the normal and glaucomatous sections.

Endothelin-1 and Nitric Oxide Levels in Aqueous Humor and Vitreous

The data were analyzed using an ANOVA 2*2 factorial analysis in which the two

factors of interest were the disease, with two levels (glaucomatous and normal), and

breed, with two levels (Cocker and non-Cocker). In the presence of interaction the simple

effects of each of the two factors were examined separately. The analyses were done on

the means of right and left eye from each dog. All the statistical analyses were performed

using SAS (version 8).

Endothelin Receptor Protein Expression in the Retina and Choroid

The expression of the ET receptors in arbitrary units (AU) versus controls were

compared between normal and glaucomatous retinal and choroidal samples using the

ANOVA 2*2 factorial design described above. Due to lack of tissue no analysis could be

done including normal Cocker choroid. All analyses were done ignoring whether the









measurement was taken from left or right eye. In the case of interaction a separate

analysis of the simple effects of each of the two factors, disease and breed, was done.

Endothelin-1 and ET Receptor mRNA Levels in the Retina

The ratios of the mRNA levels versus controls in AU were compared between

normal and glaucomatous retinal samples using the ANOVA 2*2 factorial design

described above. The variables were first log-transformed to improve the model-fit. All

analyses were done ignoring whether the measurement was taken from left or right eye.

In the case of interaction a separate analysis of the simple effects of each of the two

factors, disease and breed, was done.









Table 2-1: Normal dogs in study.
Dog Breed Age Sex University


1 German Shepherd Mix Adult


2 Hound Dog


3 Beagle


4 Beagle

5 Beagle

6 Beagle

7 Beagle

8 Beagle

10 Walker Hound


Greyhound
Greyhound

Greyhound
Walker Hound
Hound Dog
Cocker Spaniel


Adult

Adult

Adult

Adult

Adult

Adult

Adult

ly

10y
10y

1.5 y
1.5 y
2y
4y


M2 Cocker Spaniel

M3 Cocker Spaniel

M4 Cocker Spaniel

M5 Cocker Spaniel


M UF

M UF

M UF

M UF

F UF

F UF

M UF

F UF

F UF


UF
UF

UF
UF
UF
MX


M MX

M MX

M MX

F MX


IOP in mm/Hg at
enucleation
OD: 15.7
OS: 17.7
OD: 11.3
OS: 15.7
OD: 21
OS: 10.3
OD: 17
OS: 20.7
OD: 9
OS: 10
OD: 14.7
OS: 15.3
OD: 19
OS: 18
OD: 16
OS: 16
OD: 14
OS: 17.5
OS: 9
OD: 8
OS: 7


OD: 13
OS: 12
OD: 9
OS: 11
OD: 15
OS: 9
OD: 26
OS: 25
OD: 18
OS: 21


y, year; M, Male; F, Female; UF, Small Animal Clinical Sciences, University of Florida;
MX, Universidad de Mexico, Mexico City; IOP, Intraocular pressure; OD, Right eye;
OS, Left eye;., Missing data point.













Table 2-2: Glaucoma dogs in study
Breed Age Sex University IOP at ICA Duration of Fundus appearance
Dog enucleation disease
14 Cocker Spaniel 10 years SF UF OD: 40 1 y -
16 Bouvier de 3 years CM UF OD: 29 (49) a c 3 mo -


Flandres
Beagle
Cocker Spaniel
Cocker Spaniel
Poodle

Samoyed
Cocker Spaniel
Cocker Spaniel
Cocker Spaniel
Cocker Spaniel
Mix Terrier
Cocker Spaniel
Basset


12 years
13 years
8 years
4 years

6 years
13 years
12 years
8 years
15 years
8 years
6 years
2 years


M
CM
CM
F

SF
SF
SF
SF
SF
SF
SF
SF


UF
UF
UF
MX


OD: 30 (42)a
OS: 8b
OS: 30 (48) a
OD: 34

OD: 41
OD: 44
OS: 28 (59) a
OD: 50
OS: 45 (55) a
OS: 30
OS: 37
OS: 38


c
cNarrow
c
Narrow


10y
>4y
Chronic
1 mo


c Chronic
c Chronic
3 mo
2 mo
3 mo
S 6 mo
6 mo
c 6mo


Atrophy ONH
Vascular attenuation.
c
c
c
Retinal degeneration
c
Cupping ONH
Cupping ONH
c


y, year; M, Male; F, Female; UF, Small Animal Clinical Sciences, University of Florida; MX, Universidad de Mexico, Mexico City;
IOP, Intraocular pressure; ICA, Iridocorneal angle; OD, Right eye; OS, Left eye; mo, month; ONH, Optic nerve head. alOP prior to
treatment;b No IOP prior to treatment; C Not visible due to corneal edema.












Table 2-3: Analyses on normal dogs.
Dog ELISA Immunocytochemistry Western Blot Real Time PCR Real Time PCR
ET-1 peptide and NO ET receptors ET receptors ET-1 mRNA ET receptor mRNA
Aqueous humor/vitreous Retina and choroid Retina and choroid Retina Retina
1 OTT


OU
OU
OU
OU
OU
OU
OU
OU
OS
OD
OS
OU


18
Ml OS
M2 OD
M3 OD
M4 OS
M5 OS
OD, Right eye;


OS
OD
OD
OD
OS
OD


OD
OS OS OS
OD OD OD
OD OD OD
OS OS OS
OS OS OS
OS, Left eye; OU, Both eyes; ET, Endothelin-1; NO, Nitric oxide; -, No analysis.


OD
OD
OS
OS


OS
OD
OD
OS
OS












Table 2-4: Analyses on glaucomatous dogs.
Dog ELISA Immunocytochemistry Western Blot Real Time PCR Real Time PCR
ET-1 peptide and NO ET receptors ET receptors ET-1 mRNA ET receptor mRNA
Aqueous humor/vitreous Retina and choroid Retina and choroid Retina Retina
14 OD OD OD OD OD


OD
OD
OS

OD


OD
OD
OS

OD


21 OD
22 OD OD OD
23 OS OS OS
24 OD OD
25 OS OS
26 OS
27 OS OS
28 OS OS
OD, Right eye; OS, Left eye; ET, Endothelin-1; NO, Nitric oxide; -, No analysis.









Table 2-5: Primers and fluorogenic probes.
Reagent Primer Sequence Amplicon
size
Reverse Transcription PCR


ETA receptor
Sense
Anti-sense
ETB receptor
Sense
Anti-sense


5'-GTG GCT CTT CGG GTT CTA TT-3'
5'-GGC ATG ACT GGA AAC AAT TT-3'

5'-GAA TTA AAG GAA TTG GGG TTC C-3'
5' GCA GTT TTT GAA TCT TTT GCT C- 3'


Real Time PCR


ETA receptor
Sense
Anti-sense

Fluorogenic Probe

ETB receptor
Sense
Anti-sense
Fluorogenic Probe

Endothelin- 1
Sense
Anti-sense
Fluorogenic Probe


5'-CTT GAG AAT TGC CCT CAG TGA A-3'
5'-GCA AAA ATT ACA ACC AAG CAG
AAA-3'
5'-TTT GCC ACT TCT CGA CGC TGC TTA
AGA T-3'

5'-GGC CGT GGG TTT TGA TAT GA-3'
5'-CTG GGT AGG ATG AAG CAA GCA-3'
5'-AAC CAT TGA CTA CAA AGG ACG TTA
CCT GCG A-3'

5'-TCC TGC TCT TCC CTG ATG GA-3'
5'-GGA ACA ATG TGC TCA GGA GTG TT-3'
5'-TGT CTA CTT CTG CCA CCT TGA CAT
CAT CTG-3'


380 bp


566 bp


81 bp


76 bp


86 bp









Table 2-6: The controls for the Real Time PCR were run with the templates listed in the
table. The templates were run with various mixes of primers and probes as
listed in the table. ETA, Endothelin A; ETB, Endothelin B; 18S, 20X
Eukaryotic 18S rRNA Endogenous Control.
Template Primer/probe mix

ETA receptor A Primer A Primer B Primer B Primer 18S
sequence A Probe B Probe B Probe A Probe

ETB receptor A Primer A Primer B Primer B Primer 18S
sequence A Probe B Probe B Probe A Probe
No Template A Primer B Primer 18S
A Probe B Probe
Total RNA A Primer B Primer 18S
A Probe B Probe






48






Thre hold -
S18S ET



Glaucoma/ Nol mal



owlI
I II
4 4 4 P 0 1 1. 2 n r1 3 1 14 'I 17 It 1 X : % '0 Vt 7 iX N' f f4 f % M 40 4

CT CT

Figure 2-1: Real Time PCR amplification curves of a normal and a glaucomatous retina
for the ETA receptor and 18S rRNA. ETA, Endothelin receptor A; 18S, 20X
Eukaryotic 18S rRNA Endogenous Control; CT, threshold cycle.














CHAPTER 3
RESULTS

Morphology

Iridocorneal Angle

The iridocorneal angles (ICAs) of the clinically normal dogs revealed no

abnormalities (Figure 3-1). The outflow apparatus, including trabecular meshwork and

scleral venous plexus was distorted in all glaucomatous eyes (Figure 3-2). The anterior

corneoscleral trabecular meshwork was completely collapsed in all eyes. The uveoscleral

meshwork was completely collapsed in some eyes, while in others it still had openings of

varying sizes (Figure 3-3). In most dogs the remaining trabecular cells had a normal,

slender appearance, while they were more rounded in other eyes. Melanin-containing

cells were clustered in the angle (Figure 3-4). Anterior synechia were present in several

globes (Figures 3-4, 3-5 and 3-6).

The termination of Descemet's membrane was enlarged in four of the six glaucoma

Cocker eyes, but had normal thickness in all eyes of the non-Cocker glaucoma group

(Figures 3-4 and 3-5). It was retracted in several eyes (Figures 3-4 and 3-6). The ciliary

cleft in all eyes, except for the Beagle (dog 17) was covered by light to heavy amount of

connective tissue (Figure 3-4).

Retina.

The clinically normal dogs showed no abnormalities of the retina (Figure 3-7).

Retinal changes in the glaucomatous dogs varied from minor thinning of the ganglion cell

layer and disorganization of the inner nuclear layer, to atrophy of all retinal layers









(Figures 3-8 and 3-9). The peripheral retina was most affected in all eyes with complete

atrophy in 5 cases. A thinning of the inner plexiform layer resulting in closer apposition

of the nuclear layers, melding, was noticed as an early sign of atrophy, while the nuclear

layers still were intact. The photoreceptor nuclei were normal, with some chromatin

clumping, in all glaucomatous eyes. The outer segments of the photoreceptors were

swollen in several eyes.

The degree of degeneration in the central retina of the glaucoma dogs was graded in

3 groups according to severity: 1) No abnormalities in the nuclear layers, minor atrophy

in the nerve fiber and ganglion cell layers; 2) Moderate atrophy and /or melding of the

inner and outer nuclear layers; 3) Marked atrophy of all retinal layers (Table 3-1). There

was a significant correlation at the 0.1 significance level between the total degree of

central retinal degeneration, and total aqueous humor and vitreal ET-1 levels of

glaucomatous dogs (Spearman correlation coefficient 0.6, p = 0.0901).

Endothelin Receptors in the Retina and Choroid

Evaluation of Endothelin Antibodies using Light Microscopy

Light microscopy showed a weak but distinct label for ET receptors of small scleral

vessels of a normal Beagle. No labeling was detected when the primary antibody was

omitted. (Figure 3-10).

Evaluation of Endothelin Antibodies using Transmission Electron Microscopy

There was no labeling when PBS was used instead of the primary antibody. The

mixture of ETA antibody with its antigen resulted in very minor labeling. In contrast, the

mixture of ETB antibody with its antigen showed only a small decrease in labeling as

compared to the primary antibody. Intensity of the specific labeling corresponded to the

concentration of the antibody dilution. The most distinct and specific labeling was









achieved with the ETB antibody at a dilution of 1:200. The use of detergents was

necessary to minimize extensive nonspecific labeling by the ETB antibody. The

difficulties in achieving an optimal protocol with reliable specific label by the ETB

antibody excluded any quantitative analysis of the immunocytochemistry.

Localization of Endothelin Receptors using Transmission Electron Microscopy

The distribution of ET receptor labeling was similar in the normal and

glaucomatous sections for both receptors.

ETA receptor

The ETA receptor was observed in smooth muscle cells and pericytes of vessels in

both the retina and the choroid.

ETB receptor

Localization of the ETB receptor occurred within vascular endothelium, smooth

muscle, and pericytes of vessels in both the retina and the choroid (Figure 3-11).

Endothelin-1 Levels in Aqueous Humor, Vitreous and Retina

The ranges for aqueous humor and vitreal ET-1 levels all normal samples were

0.87 6.3 and 0.23 6.83 pg/mL respectively. The ET-1 aqueous humor and vitreal

ranges for all glaucomatous samples were 1.91 14.56 and 1.13 5.96 pg/mL

respectively. The ET-1 levels for normal and glaucomatous dogs are shown in Tables 3-2

and 3-3 and the least-squares means from the ANOVA 2*2 factorial analysis are plotted

in Figures 3-12 and 3-13.

Analysis of Aqueous Humor

The mean ( STD) normal and glaucomatous canine ET-1 aqueous levels for all

breeds were 2.8 (+ 1.52) and 6.86 ( 3.46) pg/mL respectively. There was a significant

increase in ET-1 for the glaucomatous eyes (versus the normal eye) (p = 0.0004). The









differences between least-squares means were also analyzed for each level of the two

factors due to the presence of interaction. In the Cocker group, there was a significant

increase in ET-1 for the glaucomatous eyes (p = 0.0003), while for the non-Cocker group

the difference between the normal and glaucomatous eyes was not significant (Figure 3-

12). There was no significant difference between the Cocker group and the non-Cocker

group nor for the normal or for the glaucomatous eyes.

Analysis of Vitreous

The mean ( STD) normal and glaucomatous canine ET-1 vitreous levels for all

breeds were 1.56 ( 1.86) and 3.47 ( 1.57) pg/mL respectively. The increase in the

glaucomatous eyes was considered statistically significant (p = 0.0023). Due to the

presence of interaction between the two factors, breed and disease, the differences

between least-squares means were analyzed for each level of the two factors. In the

Cocker group, there was a significant increase in ET-1 for the glaucomatous eyes (p =

0.0005), while for the non-Cocker group the difference between the normal and

glaucomatous eyes was not significant (Figure 3-13). There was a significant difference

between the Cocker group and the non-Cocker group (p = 0.0099) for the normal eyes but

not for the glaucomatous eyes

Endothelin-1 mRNA Levels in the Retina

There was insufficient statistical evidence to indicate any significant breed or

disease effect on the retinal mRNA levels of ET-1; breed by disease interaction was not

found significant either (Table 3-4).









Correlation of Retinal Degeneration and ET-1 Levels in Aqueous Humor and
Vitreous

The degree of degeneration of the central dorsal retina and central ventral retina

was correlated to the ET-1 levels in aqueous humor and vitreous. The grading of the

retinal degeneration is described in the Morphology section and the ranking of the

glaucomatous central retinas is shown in Table 3-1. The gradual decrease in thickness of

the cell layers in the periphery of normal eyes lead to difficulties in comparing and

grading any changes in the periphery of the glaucomatous eyes. The peripheral retina was

therefore excluded from the correlation analysis. There was a significant correlation at

the 0.1 significance level between the total degree of central retinal degeneration, and

total aqueous humor and vitreal ET-1 levels of glaucomatous dogs [Spearman correlation

coefficient 0.6, p = 0.0901) (Figure 3-14)].

Nitric Oxide Levels in Aqueous Humor and Vitreous

The ranges for all normal aqueous humor and vitreal NO levels were 1.41 11.46

and 0.24 16.47 [M respectively. The NO aqueous humor and vitreal ranges for all

glaucomatous dogs were 2.25 37.94 and 1.07 46.98 [M respectively. The NO levels

for normal and glaucoma dogs are shown in Tables 3-5 and 3-6 and the least-squares

means from the ANOVA 2*2 factorial analysis are plotted in Figures 3-15 and 3-16.

Analysis of Aqueous Humor

The mean normal and glaucomatous canine NO aqueous levels for all breeds were

3.9 ( 1.93) and 12.48 (+ 13.46) [iM respectively. The increase in the glaucomatous eyes

of the non-Cocker group was considered statistically significant (p = 0.0476). There was

insufficient evidence to indicate that the two factors, disease and breed, interacted (Figure

3-15).









Analysis of Vitreous

The mean normal and glaucomatous canine NO vitreal levels for all breeds were

4.47 ( 2.92) and 15.33 ( 16.22) iM respectively. The increase in the glaucomatous

eyes was considered statistically significant (p = 0.0085) but due to the presence of

interaction, the differences between least-squares means were analyzed for each level of

the two factors. In the non-Cocker group, there was a significant increase in NO for the

glaucomatous eyes (p = 0.0148), while for the Cockers the difference between the normal

and glaucomatous eyes was not significant (Figure 3-16). There was no significant

difference between the Cocker group and the non-Cocker group neither for the normal

eyes nor for the glaucomatous eyes.

Endothelin Receptor Protein Expression in the Retina and Choroid

The ratios of the expression levels of ETA versus control, and ETB versus control in

the retina and choroid for normal and glaucoma dogs are shown in Tables 3-7 and 3-8

and the least-squares means from the ANOVA 2*2 factorial analysis model are plotted in

Figures 3-17 and 3-18.

ETA Receptors in the Retina

There was a statistically insignificant reduction of the ETA receptors in the retina of

the glaucomatous eyes. There was no evidence to indicate that the two factors interacted

(Figure 3-17).

ETB Receptors in the Retina

There was a significant difference in the ETB receptors between the glaucomatous

and normal dogs (p = 0.0386); since the interaction between the breed and disease was

significant, the simple effects of the two factors were investigated separately. For the

non-Cocker group, there was a significant decrease in the ETB receptors between the









glaucomatous and normal dogs (p = 0.0072). Also for the normal eyes, the level of ETB

receptors was significantly lower for the Cocker group versus the non-Cocker group. (p =

0.0025) (Figure 3-18).

ETA and ETB Receptors in the Choroid

Choroidal tissues from normal and glaucomatous dogs in the non-Cocker group

were compared. There was a decrease of both receptors in the glaucomatous eyes that

was not statistically significant. The Cocker group had a higher expression of the ETA

receptor and a lower expression of the ETB receptor than the non-Cocker group, in a

comparison of glaucomatous choroidal tissue. Neither difference between the two groups

was significant.

Endothelin Receptor mRNA Levels in the Retina

Parameters for normal and glaucomatous dogs are shown in Tables 3-9 and 3-10

and the least-squares means from the ANOVA 2*2 factorial analysis model are plotted in

Figures 3-19 and 3-20.

Evaluation of the Real Time PCR Reaction

Endothelin A receptor plasmid DNA was amplified with a mix of the ETA receptor

primer and ETA receptor fluorogenic probe (A/A), showing an early amplification of the

DNA sequence above background in the 13th cycle of the Real Time PCR (Figure 3-21).

The mixtures of the ETA receptor primer with ETB receptor fluorogenic probe (A/B), and

the ETB receptor primer with ETA receptor fluorogenic probe (B/A), gave only

background noise below detectable level. DNA amplification was detected above

background when the ETB receptor primer with ETB receptor fluorogenic probe (B/B)

was used on ETA DNA. Amplification above background was also seen of 18S rRNA

using the 20X Eukaryotic 18S rRNA Endogenous Control on ETA DNA.









Endothelin B receptor plasmid DNA was amplified with the mix of ETB receptor

primer with ETB receptor probe (B/B) showing an early amplification of the DNA

sequence above background in the 12th cycle of the Real Time PCR (Figure 3-22). The

mixtures A/B and B/A give only background noise below detectable level. Amplification

was detected above background when the ETA receptor primer with ETA receptor probe

(A/A) was used on ETB cDNA. Amplification above background was also seen of 18S

rRNA using the 20X Eukaryotic 18S rRNA Endogenous Control on ETB cDNA.

Since the amplifications by the mismatched primer/fluorogenic probes were

detected after the 29th cycle for both ET receptors, these signals are probably due to PCR

artefacts. The difference with the true match of primer and fluorogenic probe is more

than 15 cycles, showing that there was no cross-reactivity between different primers and

probes.

No amplification curves of the ET receptors were seen when the Real Time PCR,

with the primers and probes for the ET receptors and 18S, was run with no template. This

showed that there was no nonspecific amplification due to contamination of solutions.

When using total RNA as template, with the primers and probes for the ET receptors and

18S, no amplification curves were seen for the receptors (Figure 3-23). This proved that

there was no genomic DNA in the RNA samples. Traces of 18S rRNA were amplified in

both reactions.

Tissue

The tissue had been stored in two different ways, i.e. frozen dry or frozen in RNase

Later preservative. It was therefore necessary to check whether this affected the RNA in

the samples. This was done by extracting RNA from two normal dog retinas, one that had

been stored frozen in the preservative RNAlater (Retina 1) and the other (Retina 2) stored









frozen dry without any preservative. The yields of total RNA from the samples were 2.1

and 1.9 mg/mL respectively. Both samples were split in half. One half of each sample

was treated with DNase to eliminate any residual genomic DNA. RT-PCR was then run

on total RNA and DNAse treated RNA for each sample (Figure 3-24). The primers used

for the reaction are listed in Table 2-5. The detected bands of the ET receptors and P-

actin were identical for all samples indicating that the two methods of storage did not

alter the size or configuration of the mRNA.

The same samples (Retina 1 and 2) were run with the Real Time PCR as for the

RT-PCR. Both samples were run in triplicate. The primers used for the reaction are listed

in Table 2-5. The mean CT values for the ETA receptor from the two samples were 28.5

(Retina 1) and 28.64 (Retina 2) respectively (Figure 3-25) and for the ETB receptor 26.8

(Retina 1) and 26.6 (Retina 2) respectively. This showed that the two methods of storage

had minimal effect on the initial amount of mRNA and amplification of the cDNA of the

samples.

ETA Receptor mRNA in the Retina

There was a significant increase in the mRNA levels (p = 0.0347) in the

glaucomatous retinas but it was confounded by interaction. For the Cocker group there

was a highly significant increase in mRNA levels (p = 0.0064) between the glaucomatous

and normal eyes. There was a small, but statistically insignificant, increase in the non-

Cocker group. There was also a significant difference in the glaucomatous eyes with the

Cockers having significantly higher levels, at a significance level of 0.1, than the non-

Cocker group (p = 0.0917) (Figure 3-19).









ETB Receptor mRNA in the Retina

There was a significant increase in the mRNA levels at a significance level of 0.1

(p = 0.0854) in the glaucomatous retinas, but it was confounded by interaction. For the

Cocker group the mRNA level was significantly higher for the glaucomatous eyes than

for the normal eyes (p = 0.0075). The mRNA level in glaucomatous eyes of the non-

Cocker group was slightly lower than for the normal eyes, yet not significant. There was

no significant difference in mRNA levels in normal or glaucomatous eyes between the

Cocker group and the non-Cocker group (Figure 3-20).

Summary of Results

Results for all breeds are summarized below.

* All iridocorneal angles in the glaucomatous eyes were distorted with varying
degree of collapse.

* Retinal changes in the glaucomatous eyes varied from minor thinning of the
ganglion cell layer and disorganization of the inner nuclear layer, to complete
atrophy of all retinal layers. The peripheral retina was most affected in all eyes. The
degree of degeneration of the central retina was positively correlated to the ET-1
levels in aqueous humor and vitreous.

* The location of ET receptor labeling was similar in the normal and glaucomatous
retinal and choroidal sections for both ET receptors.

* The results for the glaucomatous eyes separating the Cocker and the non-Cocker
group are shown in Table 3-11.






59


Table 3-1: Ranking of degree of degeneration in the glaucomatous retinas.
Dog Dorsal Central Ventral Central Total mean
14 2 2 2
16 1 2 1.5
17 1 2 1.5
19 1 1 1
M6 1 2 1.5
22 2 2 2
23 3 3 3
24 2 2 2
25 1 2 1.5









Table 3-2: Endothelin-1 levels in pg/mL for normal dogs. BQL, Below limit of
quantification;., Missing data point; OS, Left eye; OD, Right eye; ET-1,
Endothelin-1.


Dog Breed
1OS German Shepherd Mix
1OD
20S Hound Dog
20D
30S Beagle
30D
40S Beagle
40D
50S Beagle
5OD
60S Beagle
60D
70S Beagle
70D
80S Beagle
80D
100S Walker Hound
100D
11OS Greyhound
120S Greyhound
120D
150S Walker Hound
150D
Ml OS Cocker
M2 OD Cocker
M3 OD Cocker
M4 OS Cocker
M5 OS Cocker
Non-cocker mean (STD)
Cocker mean (STD
Total mean (STD)


ET-1 aqueous
2.01
3.56
1.95
3.5
2.43
2.31
1.42
2.07
2.84
1.77
2.37
1.24
2.49
1.65
2.73
3.38
4.81
6.3
5.29
5.88
5.82
2.11
1.67
1.7
0.87
0.99
2.31
3.04
3.03 (1.54)
1.78 (0.91)
2.80 (+1.52)


ET-1 vitreous
1.53
0.7
0.7
1.65
0.64
2.49
BQL
6.83
1.24
3.92
1.53
5.64
1.47
3.38
2.19
0.4
4.63
3.02
4.75
1.06
2.84


BQL
0.32
0.23
0.48
0.29
2.53 (1.82)
0.33(0.11)
1.56 (+1.86)









Table 3-3: Endothelin-1 levels in pg/mL for glaucomatous dogs. OD, Right eye; OS, Left
eye; ET-1, Endothelin-1.


Dog Breed
14 OD Cocker
19 OS Cocker
22 OD Cocker
23 OS Cocker
24 OD Cocker
25 OS Cocker
27 OS Cocker
M6 OD Poodle
16 OD Bouvier de Flandres
17 OD Beagle
28 OS Basset
Non-cocker mean (STD)
Cocker mean (STD)
Total mean (STD)


ET-1 aqueous
6.7
2.52
4.95
9.67
6.8
8.58
14.56
8.48
1.91
5.55
5.6
5.38 (+2.69)
7.7 (+2.52)
6.86 (+3.49)


ET-1 vitreous
5.2
2.68
2.09
4.06
2.88
3.16
5.96
1.7
1.13
4.05
5.28
3.04 (+1.96)
3.72 (1.42)
3.47 (+1.57)









Table 3-4: Mean ratios ofET-1/ 18S mRNA in arbitrary units for normal and
glaucomatous retinal samples. ET-1, Endothelin-1; 18S, 20X Eukaryotic 18S
rRNA Endogenous Control.


Normal
Dog Breed
7 Beagle
8 Beagle
10 Walker Hound
11 Greyhound
18 Hound Dog
Ml Cocker
M2 Cocker
M3 Cocker
M4 Cocker
M5 Cocker
Non-cocker mean(+STD)
Cocker mean (STD)
Total mean (STD)


ET-1/18S
2.05
2.14
2.16
2.19
1.95
2.06
2.07
2.12
2.09
2.0
2.1 (+0.1)
2.07 (0.04)
2.08 (0.07)


Glaucoma
Dog Breed
14 Cocker
19 Cocker
22 Cocker
23 Cocker
24 Cocker
25 Cocker
26 Mix Terrier
27 Cocker
28 Basset
M6 Poodle
Non-cocker mean (STD)
Cocker mean (STD)
Total mean (STD)


ET-1/18S
2.15
2.12
1.94
2.18
2.23
1.82
2.09
2.07
2.18
2.21
2.14 (0.07)
2.07 (0.16)
2.1 (0.13)









Table 3-5: Nitrate (NO) levels in [iM of normal dogs. BQL, Below limit of
quantification; NO, Nitric oxide; ., Missing data point; OS, Left eye; OD,
Right eye.


Dog Breed
10S German
1OD
20S Hound
20D
30S Beagle
30D
40S Beagle
40D
5 OS Beagle
5OD
60S Beagle
60D
70D Beagle
80S Beagle
80D
100S Walker
100D
110S Greyhoi
120S Greyhoi
120D
13 OS Greyhoi
150S Walker
150D
Ml OS Cocker
M2 OD Cocker
M3 OD Cocker
M4 OS Cocker
M5 OS Cocker
Non-cocker mean (STD)
Cocker mean (STD)
Total mean (STD)


Shepherd Mix


Dog


Hound

und
und

mnd
Hound


NO aqueous
3.67
2.51
1.41
3.75
1.41
7.02





11.46
3.84
2.75
2.58
2.58
3.73
7.6
8.03
7.61
3.25

BQL
3.08
1.58
2.75
3.42
2.5
3.17
4.51 (+2.09)
2.68 (0.71)
3.9 (1.93)


NO vitreous
2.7
3.92
10.91
4.11
1.19
2.32
3.26
4.3
BQL
2.23
4.4
1
3.73
BQL
9.02
3.26
4.4
0.24
2.79
16.47
1.66


5.18
8.36
7.94
3.08
2.08
4.12 (+3.0)
5.33 (+2.81)
4.47 (2.92)









Table 3-6: Nitrate (NO) levels in riM. of glaucomatous dogs. BQL, Below limit of
quantification; NO, Nitric Oxide; ., Missing data point; OS, Left eye; OD,
Right eye.


Dog Breed
14 OD Cocker
16 OD Bouvier de Flandres
17 OD Beagle
M6 OD Poodle
19 OS Cocker
22 OD Cocker
23 OS Cocker
24 OD Cocker
25 OS Cocker
Non-cocker mean (STD)
Cocker mean (STD)
Total mean (STD)


NO aqueous
6.52
6.10
8.7
34.17
4.34
8.53
2.25
3.75
37.94
16.33 (15.51)
10.56 (13.6)
12.48 (13.46)


NO vitreous
9.28
12.8

33.16
7.02
10.46
1.83
1.07
46.98
22.98 (14.4)
12.77 (17.19)
15.33 (16.22)













Table 3-7: Ratios of ETA receptor/ control protein in arbitrary units for normal and glaucomatous retinas and choroids. ETA,
Endothelin A; ., Missing data point.
Normal Glaucoma


Dog Breed
5 Beagle
6 Beagle
7 Beagle
8 Beagle
10 Walker Hound
11 Greyhound
Ml Cocker
M2 Cocker
M3 Cocker
M4 Cocker
M5 Cocker


Non-cocker mean (STD)
Cocker mean (STD)
Total mean (STD)


ETA /control
Retina
2.26
2.83
3.69

0.78

3.69
3.29
2.36
2.49
1.11


2.39 (+1.22)
2.59 (0.99)
2.04 (1.87)


ETA /control
Choroid
1.16
2.44
7.26
6.55
7.61
6.08








5.18 (2.71)
N/A
N/A


Dog Breed
14 Cocker
16 Bouvier de Flandres
17 Beagle
19 Cocker
21 Samoyed
22 Cocker
23 Cocker
24 Cocker
25 Cocker
26 Mix Terrier
27 Cocker
28 Basset
M6 Poodle
Non-cocker mean (STD)
Cocker mean (STD)
Total mean (STD)


ETA/control
Retina
0.87

4.04
0.04
1.25
0.49
1.90
0.22
2.49
1.08
6.97
1.38
0.48
1.64 (1.38)
1.86 (2.43)
1.77 (1.98)


ETA/control
Choroid
5.66
0.29
0.43
5.10
1.95
0.36
6.06
4.25
5.27
6.7
8.69
8.18

3.51 (3.68)
5.06 (2.49)
4.41 (2.99)













Table 3-8: Ratios of ETB receptor/ control protein in arbitrary units for normal and glaucomatous retinas and choroids. ETB,
Endothelin B; ., Missing data point.
Normal Glaucoma


Dog Breed
5 Beagle
6 Beagle
7 Beagle
8 Beagle
10 Walker Hound
11 Greyhound
Ml Cocker
M2 Cocker
M3 Cocker
M4 Cocker
M5 Cocker


Non-cocker mean (+STD)
Cocker mean (STD)
Total mean (STD)


ETB /control
Retina
5.17
0.98
2.04
1.35
0.84

0.14
0.12
0.16
0.14
0.21


2.08 (1.79)
0.16 (0.03)
1.12 (1.57)


ETB /control
Choroid
2.18
0.95
1.44
0.23
0.35
0.10








0.88 (0.82)
N/A
N/A


Dog
14
16
17
19
21
22
23
24
25
26
27
28
M6


Breed
Cocker
Bouvier de Flandres
Beagle
Cocker
Samoyed
Cocker
Cocker
Cocker
Cocker
Mix Terrier
Cocker
Basset
Poodle


Non-cocker mean (STD)
Cocker mean (STD)
Total mean (+STD)


ETB/control
Retina
0.35

0.86
0.12
0.81
0.26
0.12
0.09
0.08
0.28
0.02
0.07
0.05
0.41 (0.39)
0.15 (0.11)
0.26 (0.29)


ETB/control
Choroid
0.06
0.17
0.22
0.03
1.24
0.09
0.08
0.29
0.04
0.11
0.08
0.22

0.39 (0.48)
0.09 (0.09)
0.22 (0.33)












Table 3-9: Mean ratios of ETA receptor/ 18S mRNA in arbitrary units for normal and glaucomatous retinal samples. ETA, Endothelin A;
18S, 20X Eukaryotic 18S rRNA Endogenous Control.
Normal Glaucoma
Dog Breed ETA/18S Dog Breed ETA/18S
7 Beagle 2.18 14 Cocker 1.80
8 Beagle 2.32 19 Cocker 1.85
10 Walker Hound 2.00 22 Cocker 2.03
11 Greyhound 2.08 23 Cocker 1.87
Ml Cocker 2.19 24 Cocker 1.92
M2 Cocker 2.09 25 Cocker 2.07
M3 Cocker 2.12 26 Mix Terrier 2.15
M4 Cocker 2.21 27 Cocker 2.17
M5 Cocker 2.31 28 Basset 2.01
M6 Poodle 2.14
Non-cocker mean (STD) 2.15 (0.14) Non-cocker mean (STD) 2.1(0.08)
Cocker mean (STD) 2.18 (0.09) Cocker mean (STD) 1.96 (+0.13)
Total mean (STD) 2.17 (+0.11) Total mean (STD) 2.00(0.13)












Table 3-10: Mean ratios of ETB receptor/ 18S mRNA in arbitrary units for normal and glaucomatous retinal samples. ETB, Endothelin
B; 18S, 20X Eukaryotic 18S rRNA Endogenous Control.
Normal Glaucoma
Dog Breed ETB /18S Dog Breed ETB /18S
7 Beagle 2.13 14 Cocker 1.9
8 Beagle 2.17 19 Cocker 1.76
10 Walker Hound 2.01 22 Cocker 2.05
11 Greyhound 1.99 23 Cocker 1.98
Ml Cocker 2.21 24 Cocker 1.99
M2 Cocker 2.09 25 Cocker 2.02
M3 Cocker 2.09 26 Mix Terrier 2.13
M4 Cocker 2.17 27 Cocker 2.04
M5 Cocker 2.20 28 Basset 1.89
M6 Poodle 2.22
Non-cocker mean (STD) 2.08 (0.09) Non-cocker mean (STD) 2.08 (0.17)
Cocker mean (STD) 2.15 (+0.06) Cocker mean (STD) 1.96 (0.1)
Total mean (STD) 2.12 (+0.08) Total mean (STD) 2.00 (0.13)









Table 3-11: Summary of results for the glaucomatous eyes separating the Cocker Spaniel
group and the non-Cocker group.
Parameter Glaucoma, Cocker Spaniels Glaucoma, Non-Cockers
ET-1 peptide
Aqueous humor Increase, p = 0.0003 Increase, p = 0.1144
Vitreous Increase, p = 0.0005 Increase, p = 0.5062
ET-1 mRNA Retina Increase, p = 0.9569 Decrease, p = 0.4273
Nitric Oxide
Aqueous humor Increase, p = 0.1421 Increase, p = 0.0476
Vitreous Increase, p = 0.2003 Increase, p = 0.0148
ET receptor protein
expression
ETA Retina Decrease, p = 0.4826 Decrease, p = 0.5319
ETB Retina Decrease, p = 0.9916 Decrease, p = 0.0072
ETA Choroid a, b Higher expression, p = 0.4024 a Decrease, p = 0.4066 b
ETB Choroid a, b Lower expression, p = 0.1291 a Decrease, p = 0.2759 b
ET receptor mRNA
ETA Retina Increase, p = 0.0064 Increase, p = 0.6248
ETB Retina Increase, p = 0.0075 Decrease, p = 0.9617
aNo normal choroidal cocker tissue. Choroid from glaucomatous Cockers compared with
choroid from glaucomatous non-Cockers. bNormal versus glaucomatous. The level of
significance was set at 0.05.



























Figure 3-1: Normal iridocomeal angle stained with toluidine blue (Dog M3,
original magnification 100X). A) Iris. B) Anterior chamber.
C) Pectinate ligament. D) Uveal trabecular meshwork.
E) Comeoscleral trabecular meshwork. F) Angular aqueous
plexus/sinus.


pa .i. .1


Figure 3-2: Iridocorneal angle of a glaucomatous dog (Dog 20, original magnification
40X, PAS stained). A) Compressed trabecular meshwork. B) Connective
tissue at the entrance of the angle.


























Figure 3-3:


Trabecular meshwork of a glaucomatous dog (Dog 25, original
magnification 200X, PAS stained). A) The uveoscleral meshwork has
openings of varying sizes.


Figure 3-4: Iridocorneal angle of a glaucomatous dog (Dog 19, original magnification
200X, PAS stained). Evidence of previous synechia is present: A) Fibrosis
lining the inner surface of Descemet's membrane. B) Termination of
Descemet's membrane is enlarged. The entrance of the angle is covered: C)
By connective tissue. D) By melanin-containing cells.






















" "In-





Figure 3-5: Iridocorneal angle of a glaucomatous dog (Dog 22, original magnification
100X, PAS stained). A) Synechia between the cornea and the iris. B)
Enlarged termination of Descemet's membrane.


^^a~s


'-U-F;


Figure 3-6: Iridocorneal angle of a glaucomatous dog (Dog 26, original magnification
100X, PAS stained). A) Fibrous tissue covering the endothelium of the
cornea.









DC


a

b
C


d







a/b

C
c
d


e


VC


a
b

C

d





VP
a/b

C
d



e


Figure 3-7: Semi-thin (1l m) sections of the retina from a normal dog (Dog M4, original
magnification 400X) stained with toluidine blue. The sections are from four
different locations: DC) Central retina dorsal of the optic nerve head. VC)
Central retina ventral of the optic nerve head. DP) Peripheral retina dorsal of
the optic nerve head. VP) Peripheral retina ventral of the optic nerve head.
a, nerve fiber layer; b, ganglion cell layer; c, inner nuclear layer; d, outer
nuclear layer; e, choroid.


DP












S alb
*.. ,- .


.. .. ..-




DP
alb A

d


'WI. W


Figure 3-8: Semi-thin (1 im) sections of the retina from a glaucomatous dog (Dog 24,
original magnification 400X) stained with toluidine blue. The sections are
from four different locations: DC) Central retina dorsal of the optic nerve
head. VC) Central retina ventral of the optic nerve head. DP) Peripheral
retina dorsal of the optic nerve head. VP) Peripheral retina ventral of the
optic nerve head. a, nerve fiber layer; b, ganglion cell layer; c, inner nuclear
layer; d, outer nuclear layer; e, choroid; f, ganglion cell; @, artifact.


*4,









VC e


a DC I F .
\"- .l 4-







DP


I n -"'' ,

-ft


-- -,'is

iu, I -o r ';a


Figure 3-9: Semi-thin (1 gm) sections of the retina from a glaucomatous dog (Dog 25,
original magnification 400X) stained with toluidine blue. The sections are
from four different locations: DC) Central retina dorsal of the optic nerve
head. VC) Central retina ventral of the optic nerve head. DP) Peripheral
retina dorsal of the optic nerve head. VP) Peripheral retina ventral of the
optic nerve head. a, nerve fiber layer; b, ganglion cell layer; c, inner nuclear
layer; d, outer nuclear layer; e, choroid; @, complete atrophy.












Kr. -:
? N I:


-nfL7--


-ft.


* -r


7 ."






- .- .
-a: c~~~~~cccc


"-- / -~


. ,. -, .. .

'--: ....' -- l ,f. ,'* -"
-C.. "


--


.-- .
--" --- ,-.--- -""" "" -'

...' .--. : -
-- -

rs 4


Figure 3-10:


--' J .." -" ": f- -- r1
. ...i"q r ". -"-..


^r\ -S p.

---4 --a
.,-. .-- ,.- *,, -. '"" c ". .




i "- C e
.---. 1
. .: _.,.. ,-,


Label of normal canine scleral vessels and elastic fibers with the ETB
antibody. A) 1:50 dilution at original magnification of 100X. B) 1:50
dilution at original magnification of 200X. C) Control where the primary
antibody is replaced with PBS, original magnification 100X. D) Control
where the primary antibody is replaced with PBS, original magnification
200X.


-~I


=-


-1


c-

-,











Smncotn M Jscl


B Label


igure 3-11: Arteriole of the choroid of a normal dog. A) Smooth muscle and
endothelial cells labeled with ETB antibody (1:100) at original
magnification of 10,000X. B) Smooth muscle and endothelial cells labeled
with ETB antibody (1:100) at original magnification of 20,000X.











ET-1 Aqueous


S-*Cocker
-- Other


Normal Glaucoma


ET-1 Aqueous


+- Normal
-i--Glaucoma


Figure 3-12:


Endothelin-1 in aqueous humor of normal and glaucomatous eyes (see
Tables 3-2 and 3-3). A) In the Cocker group, there is a significant increase
in ET-1 for the glaucomatous eyes (p = 0.0003), while for the non-Cocker
group the difference between the normal and glaucomatous eyes is not
significant. B) There is no significant difference between Cockers and
non-Cockers neither for the normal nor for the glaucomatous eyes. ET-1,
Endothelin-1


* p = 0.0003


10
8
6
- 4
2
0


Cocker


Other












ET-1 Vitreous


* D= 0.0005


-- Cocker
-- Other


Normal Glaucoma


ET-1 Vitreous


-- Normal
-I- Glaucoma


Other


Figure 3-13: Endothelin-1 in vitreous of normal and glaucomatous eyes (see Tables 3-2
and 3-3). A) In the Cocker group, there is a significant increase in ET-1 for
the glaucomatous eyes (p = 0.0005), while for the non-Cocker group the
difference between the normal and glaucomatous eyes is not significant. B)
There is a significant difference between the Cocker group and the non-
Cocker group (p = 0.0099) for the normal eyes but not for the glaucomatous
eyes. ET-1, Endothelin-1.


4

3

2.
0
1

0


p= 0.0099


Cocker































0 1 2 3 4


Minor
degeneration


Figure 3-14:


Severe
degeneration


Scatter plot showing the correlation between the degree of degeneration in
central retina and total aqueous humor and vitreal ET-1 levels of
glaucomatous dogs (Spearman correlation coefficient 0.6, p = 0.0901).
ET-1, Endothelin-1












NO Aqueous


Cocker
-- Other


Normal Glaucoma


NO Aqueous


-- Normal
--Glaucoma


Figure 3-15:


Nitric oxide in aqueous humor of normal and glaucomatous eyes (see
Tables 3-5 and 3-6). A) There is a significant difference between NO
levels in normal and glaucomatous eyes for the non-Cocker group (p =
0.0476); but not for the Cocker group. B) There is no significant
difference between Cockers and non-Cockers neither for the normal nor
for the glaucomatous eyes. NO, Nitric Oxide.


20

15

210
5

0


p = 0.0476


20

15

210 -
5

0


Cocker


Other













NO Vitreous


25
20
15
=10
5
0





B





25
20
15

=10

5
0


Cocker


-
-
-

-
-


Other


Figure 3-16:


Nitric oxide in vitreous of normal and glaucomatous eyes (see Tables 3-5
and 3-6). A) In the non-Cocker group, there is a significant increase in NO
for the glaucomatous eyes (p = 0.0148), while for the Cockers the
difference between the normal and glaucomatous eyes is not significant.
B) There is no significant difference between the Cocker group and the
non-Cocker group neither for the normal eyes nor for the glaucomatous
eyes. NO, Nitric Oxide.


, p = 0.0148


Normal Glaucoma






NO Vitreous


-*Cocker

-- Other



















- Normal

--- Glaucoma


-
-

-
-
-


a"












ETA Receptor Retina


+ Cocker
- Other


Figure 3-17:


Endothelin receptor A protein in the retina of normal and glaucomatous
eyes (see Table 3-7). A) There is no significant difference between the
normal and glaucomatous eyes. B) There is no interaction with breed.
ETA, Endothelin A.


3
2.5
2
*1.5
1
0.5
0


Normal


Glaucoma


ETA Receptor Retina


3
2.5
2-
2 Normal
D1.5 -
< 1 --Glaucoma
0.5
0
Cocker Other











ETB Receptor Retina


S-Cocker
--Other


ETB Receptor Retina


- Normal
--Glaucoma


Figure 3-18:


Endothelin receptor B protein in the retina of normal and glaucomatous
eyes (see Table 3-8). A) The difference between normal and glaucomatous
eyes of the non-Cocker group (p = 0.0072) is highly significant while the
small decrease of the Cocker group is not significant. B) There is a
difference between normal eyes with the non-Cocker group being
significantly higher than the Cocker group (p = 0.0025) (B). ETB,
Endothelin B.


2.5
2
,1.5
< 1
0.5
0


p = 0.0072


Normal


Glaucoma


2.5
2
1.5
< 1
0.5
0


Cocker


Other













ETA mRNA Retina


-- Cocker
--Other


ETA mRNA Retina


--Normal
-i- Glaucoma


Figure 3-19:


Endothelin receptor A mRNA in the retina of normal and glaucomatous
eyes (see Table 3-9). A) The increase in the Cocker group is highly
significant (p = 0.0064). The small increase in the non-Cocker group is
not significant. B) The difference between groups of the glaucomatous
eyes is significant at a 0.1 significance level (p = 0.0917). ETA, Endothelin
A.


0.36
0.34
"0.32
0 0.3
S0.28
0.26


p = 0.0064


Normal


Glaucoma


0.36
0.34
00.32
S 0.3 -
- 0.28 -
0.26


--*p = 0.0917
^^^----- ^ *"p = 0.0917


Cocker


Other















ETB mRNA Retina


-- Cocker
Other


ETs mRNA Retina


- Normal
--Glaucoma


Figure 3-20:


Endothelin receptor B mRNA in the retina of normal and glaucomatous
eyes (see Table 3-10). A) The increase in the glaucomatous eyes in the
Cocker group is highly significant (p = 0.0075). B) There is no significant
difference in normal or glaucomatous eyes between the Cocker group and
the non-Cocker group (B). ETB, Endothelin B.


0.34
0.32

< 0.3
0)
.~0.28

0.26


Sp= 0.0075
* p = 0.0075


Normal


Glaucoma


0.34

0.32

o 0.3
0)
0.28
0.26


Cocker


Other