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Matrix Metalloproteinase 2, Matrix Metalloproteinase 9, and Connective Tissue Growth Factor in the Equine Tear Fluid: Po...

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Title: Matrix Metalloproteinase 2, Matrix Metalloproteinase 9, and Connective Tissue Growth Factor in the Equine Tear Fluid: Possible Implications in Corneal Wound Healing
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Copyright Date: 2008

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Source Institution: University of Florida
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Permanent Link: http://ufdc.ufl.edu/UFE0003781/00001

Material Information

Title: Matrix Metalloproteinase 2, Matrix Metalloproteinase 9, and Connective Tissue Growth Factor in the Equine Tear Fluid: Possible Implications in Corneal Wound Healing
Physical Description: Mixed Material
Copyright Date: 2008

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
System ID: UFE0003781:00001


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MATRIX METALLOPROTEINASE 2, MATR IX METALLOPROTEINASE 9, AND CONNECTIVE TISSUE GROWTH FACTOR IN THE EQUINE TEAR FLUID: POSSIBLE IMPLICATIONS IN CORNEAL WOUND HEALING By FRANCK J. OLLIVIER 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 2004

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Copyright 2004 by Franck J. Ollivier

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This work presented in this dissertation shall be a contribution to pres erve sight in horses and it is dedicated to horse owners and veterinary ophthalmologists. This dissertation is also dedi cated to my family and my fr iends wherever they are, for their constant support.

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ACKNOWLEDGMENTS My time as a graduate student ends with this dissertation. The years dedicated to this work represent not only an educational but also a social experience. Many individuals were involved in this endeavor and have been important for my success, especially those listed below. I would like to express my deep gratitude to my mentor and friend, Dr. Dennis Brooks, for his support and understanding throughout these years. I thank him for having given me the great and unique opportunity to come to Florida and pursue my education in veterinary ophthalmology. I would like to sincerely thank Dr. Gregory Schultz who meant a great deal for me, my graduate education, and my project. He opened his laboratory for me and patiently taught and helped me though many steps of my project. His positive and supportive attitude was extraordinary. I am also very grateful to Dr. Kirk Gelatt for sharing, with no limits, his fascinating knowledge and interest in research and clinical veterinary ophthalmology. I am proud to be the last ophthalmology resident he will train before a well-deserved retirement. I am very thankful to Dr. Gysbert van Setten who guided through my project from the other side of the Atlantic Ocean! He not only shared his tremendous knowledge on the tear film and the cornea but also the way of questioning axioms and looking for new ones. His suggestions and our discussions were very valuable. iv

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I owe many thanks to Dr. Stacy Andrew for sharing her indefatigable interest in corneal research and for her precious help in my project at various stages. I would like to thank Dr. James Farese and Dr. Sonal Tuli for being member of my doctoral thesis committee; their advice was important in my success in this dissertation. I owe my dear friend Dr Maria Kllberg many thanks for her scientific but also social support! I will never forget the hilarious moments as well as the tough times we go through together. I would like to sincerely thank Dr. Tim Blalock for his friendship, his patience and his tremendous help in various aspects of my work. I am very thankful to Dr Gary Stevens who helped with statistics. He friendly taught me a lot in this field during our nice and very interesting discussions. I am also grateful to Dr Don Samuelson and his laboratory technicians, Ms. Patricia Lewis, and Ms. Mae Chisholm for their friendly help in the adventure of histology that I went through during my time as a graduate student. I would like to thank Drs. Mary Lassaline, Andrs Komromy, Tim Cutler and Heidi Denis for their contributions in my project. I owe many thanks to Ms. Suzanne Sharra-Maxwell, owner of Pyrite farm in Ocala, Florida, for allowing me to perform parts of my project at her farm and for the laughs shared together. I also 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 helped me through the administrative procedures to successfully finish my dissertation. v

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I would like to extend my thanks to Drs. William Dawson, Michael Goldstein, David Moraga, Colin Serada, Cornelia Gunkel, Hendrik Nollens, as well as Mr. Harold Sapp, Ms. Delena McTeer and Ms. Dottie Holland for all the advice, support and friendship they have shown me throughout my time as a graduate student at the University of Florida. I am grateful to my friends outside of the College of Veterinary Medicine, in Florida, Australia and in France for their understanding, and social support throughout these years. Last but certainly not least, I would like to thank Dr. Alain Regnier for having given me the taste for veterinary ophthalmology and for his friendship, his endless support and his wise advice through these years. Finally, I would like to express my deepest gratitude to my parents Marie-Claire and Gerard Ollivier, my brothers Philippe and Pierre, my sister Claire Ollivier for their love and tremendous support they have offered to me over the years. Merci beaucoup pour votre amour, votre confiance en moi et votre soutien sans mesure et malgr la distance qui nous spare. vi

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TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES.............................................................................................................xi LIST OF FIGURES.........................................................................................................xiii KEY TO ABBREVIATIONS..........................................................................................xvi ABSTRACT.....................................................................................................................xix CHAPTER 1 INTRODUCTION........................................................................................................1 The Cornea and the Precorneal Tear Film (PTF).........................................................1 Structure and Function of the Precorneal Tear Film.............................................1 Structure and Function of the Cornea....................................................................5 Corneal Healing and Scarring.......................................................................................9 Overview on Wound Healing and Corneal Healing..............................................9 Histological Events of Corneal Healing..............................................................11 Epithelial wound healing..............................................................................11 Stromal and endothelial wound healing.......................................................13 Biochemical Mechanisms of Corneal Healing....................................................15 Molecular biology of epithelial wound healing...........................................15 Molecular biology of stromal wound healing..............................................17 Corneal Scarring..................................................................................................19 Proteinases, Proteinase Inhibitors...............................................................................20 Proteinases in The PTF: Types and Origins........................................................20 Serine proteinases.........................................................................................20 Matrix metalloproteinases (MMPs).............................................................21 Origins of the proteinases.............................................................................29 Proteinases, PTF and Corneal Physiopathology..................................................30 Proteinases, Proteinase Inhibitors and the Cornea..............................................33 Growth Factors...........................................................................................................34 Growth Factors, PTF and Corneal Physiopathology...........................................34 Connective Tissue Growth Factor.......................................................................38 Significance to The Horse Racing Industry................................................................42 Purpose of The Study..................................................................................................43 vii

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Hypotheses..........................................................................................................43 Objectives............................................................................................................44 2 DETECTION OF MMP-2 AND MMP-9 IN THE EQUINE TEAR FLUID, CORNEA AND LACRIMAL GLANDS...................................................................45 Introduction.................................................................................................................45 Materials and Methods...............................................................................................47 Materials..............................................................................................................47 MMP-2 and MMP-9 Western Blot......................................................................49 MMP-2 and MMP-9 Immunohistochemistry in Sections of Equine Cornea, Equine Main Lacrimal Gland and Equine Nictitating Membrane Gland........50 Results.........................................................................................................................51 MMP-2 and MMP-9 Western Blot: Detection of MMP-2 and MMP-9 in the Equine Tear Fluid............................................................................................51 Immunohistochemistry: Immunohistochemical Localization of MMP-2 and MMP-9 in the Healthy Cornea and Lacrimal Glands of Horses......................52 Immunohistochemistry: Immunohistochemical Localization of MMP-2 and MMP-9 in Ulcerated Equine Cornea...............................................................55 Discussion...................................................................................................................57 3 EVALUATION OF MMP-2 AND MMP-9 ACTIVITY IN THE EQUINE TEAR FLUID.........................................................................................................................61 Introduction.................................................................................................................61 Materials and Methods...............................................................................................63 Animals................................................................................................................63 Collection of Tear Fluid Samples........................................................................66 Determination of the Tear Fluid Flow (TFF) and Determination of the Release of Proteolytic Activity........................................................................67 MMP Activity Determination by Gelatin Zymography......................................68 Image Analysis....................................................................................................69 Statistical Analysis..............................................................................................71 Results.........................................................................................................................71 Determination of MMP-2 and MMP-9 Activity in the Equine Tear Fluid of Horses with Healthy Eyes................................................................................71 Determination of MMP-2 and MMP-9 Activity in the Equine Tear Fluid of Horses Ulcerative Keratitis..............................................................................74 Determination of TFF in Horses with Healthy Eyes and Horses with Ulcerative Keratitis..........................................................................................76 Determination of The Release of Proteolytic Activity in the Equine Tear Fluid of Horses with Healthy Eyes and Horses with Ulcerative Keratitis.......77 Discussion...................................................................................................................78 viii

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4 MATRIX METALLOPROTEINASE ACTIVITY PROFILES IN THE EQUINE TEAR FILM DURING CORNEAL HEALING IN 10 HORSES WITH ULCERATIVE KERATITIS......................................................................................85 Introduction.................................................................................................................85 Materials and Methods...............................................................................................87 Selection of the Ten Cases..................................................................................87 Collection of Tear Fluid Samples........................................................................88 MMP Activity Determination by Gelatin Zymography......................................89 Image Analysis....................................................................................................90 Statistical Analysis..............................................................................................90 Results.........................................................................................................................92 Discussion...................................................................................................................99 5 IN VITRO INHIBITION OF MATRIX METALLOPROTEINASE ACTIVITY IN THE TEAR FLUID OF HORSES WITH ULCERATIVE KERATITIS.................103 Introduction...............................................................................................................103 Materials and Methods.............................................................................................106 Animals..............................................................................................................106 Collection of Tear Fluid Samples......................................................................106 Determination of MMP Activity and Inhibition Tests by Gelatin Zymography...................................................................................................107 Image Analysis..................................................................................................110 Statistical Analysis............................................................................................111 Results.......................................................................................................................112 Detection and Identification of Proteinases in Pooled Tears by Gelatin Zymography...................................................................................................112 In vitro Inhibition of the Proteinases Present in the Pooled Tears....................113 Inhibition of global proteolytic activity.....................................................113 Inhibition of proteolytic activity for each proteinase.................................115 In vitro Inhibitory Activity and the Duration of Action of Equine Serum Against the Proteinases Present in the Tear Fluid of Horses with Ulcerative Keratitis..........................................................................................................115 Discussion.................................................................................................................116 6 DETECTION OF CTGF IN THE EQUINE TEAR FLUID, CORNEA AND LACRIMAL GLANDS............................................................................................123 Introduction...............................................................................................................123 Materials and Methods.............................................................................................124 Materials............................................................................................................124 CTGF Elisa Assay.............................................................................................125 Dilution Curves.................................................................................................126 CTGF Western Blot...........................................................................................127 CTGF Immunohistochemistry...........................................................................127 Results.......................................................................................................................128 ix

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Detection and Quantification of CTGF in the Horse Tears...............................128 Dilution Curves Bioequivalence.....................................................................130 CTGF Western Blot...........................................................................................131 CTGF Immunohistochemistry...........................................................................131 Discussion.................................................................................................................134 7 CONCLUSIONS......................................................................................................137 LIST OF REFERENCES.................................................................................................143 BIOGRAPHICAL SKETCH...........................................................................................158 x

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LIST OF TABLES Table page 1-1 Proteinases present in the precorneal tear film and involved in corneal wound healing......................................................................................................................25 1-2 Proteinase inhibitors.................................................................................................27 1-3 Growth factors present in the precorneal tear film and involved in corneal wound healing......................................................................................................................40 3-1 Information regarding the horses with healthy eyes involved in the determination of MMP-2 and MMP-9 in the equine tear fluid......................................................65 3-2 Information regarding the horses with ulcerative keratitis involved in the determination of MMP-2 and MMP-9 in the equine tear fluid................................65 3-3 Proteolytic activity (Mean +/SD) in the tear fluid of horses with healthy eyes, in relative standard unit (RSU).....................................................................................72 3-4 Proteolytic activity in the tear fluid of healthy, ulcerated and contralateral normal horse eyes.................................................................................................................74 3-5 TFF in healthy, ulcerated and contralateral normal horse eyes................................76 3-6 Proteolytic activity released in the tear fluid of equine healthy, ulcerated and contralateral normal eyes.........................................................................................78 4-1 Information regarding the ten horses with ulcerative keratitis involved in the determination of the MMP activity during corneal wound healing.........................88 4-2 Level of total MMP activity in relative standard units (RSU) at day 2 and at the day of complete corneal healing in both eyes of 10 horses......................................93 5-1 Percentage of in vitro inhibition of proteolytic activity for the global proteolytic activity, as well as for each proteinase, determined for various proteinase inhibitors by use of pooled tears obtained from ulcerated eyes of horses..............114 6-1 CTGF in tear samples from horses with ulcerated corneas in one eye (12 eyes) and the non diseased contralateral eye...................................................................129 xi

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6-2 CTGF from 9 tear samples from horses with ulcerated corneas in one eye (only tears from this eye were analyzed).........................................................................130 xii

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LIST OF FIGURES Figure page 1-1: Proteinases and corneal wound healing..................................................................18 1-2: Growth factors and corneal wound healing.............................................................18 1-3: Domain Structure of the Matrix Metalloproteinase family......................................29 2-1: Melting ulcer in the right eye of a 12 year old American Quarter horse.................48 2-2: Western Blot of tear fluids from horses with ulcerated eyes (lane 3) and healthy eyes (lane 4).............................................................................................................51 2-3: Western Blot of tear fluids from horses with ulcerated eyes (lane 3) and healthy eyes (lane 4).............................................................................................................52 2-4: Immunolocalization of MMP-2 and MMP-9 in healthy equine cornea...................53 2-5: Immunolocalization of MMP-2 and MMP-9 in equine lacrimal gland...................54 2-6: Immunolocalization of MMP-2 and MMP-9 in equine nictitating membrane gland. 55 2-7: Immunolocalization of MMP-2 and MMP-9 in equine ulcerated cornea. ..............56 3-1: Fungal ulcer in the left eye of a 13 year old American Quarter horse.....................66 3-2: Tear fluid collection in horses by glass capillary tube.............................................67 3-3: Gelatin zymography of equine tear fluid samples....................................................70 3-4: Global MMP activity in the tear fluid of horses with healthy eyes by breed...........72 3-5: Global MMP activity in the tear fluid of horses with healthy eyes by sex..............73 3-6: Global MMP activity in the tear fluid of horses with healthy eyes by age category....................................................................................................................73 3-7: Global MMP activity in the tear fluid of horses with healthy eyes and ulcerative keratitis.....................................................................................................................75 xiii

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3-8: Proteolytic activity in the tear fluid of horses with healthy eyes and ulcerative keratitis by MMP type..............................................................................................75 3-9: TFF and global MMP activity in the tear fluid of horses with healthy eyes and ulcerative keratitis....................................................................................................77 4-1: Gelatin zymogram of tear fluids from case 10.........................................................91 4-2: Case 1 A 6 month Thoroughbred colt presented with a melting corneal ulcer in the left eye (OS).......................................................................................................94 4-3: Case 2 A 17yo Arabian mare presented with a superficial corneal ulcer in the left eye (OS) and a half thickness stromal corneal ulcer in the right eye (OD).......94 4-4: Case 3 A 5yo Thoroughbred mare presented with a fungal keratomalacia in the left eye (OS).............................................................................................................95 4-5: Case 4 A 2yo Thoroughbred filly presented a deep corneal ulcer in the left eye (OS)..........................................................................................................................95 4-6: Case 5 A 2yo Thoroughbred filly presented a deep corneal ulcer in the right eye (OD).........................................................................................................................96 4-7: Case 6 A 7yo Paint gelding presented a corneal ulcer in the right eye (OD). This animal received only medical treatment..........................................................96 4-8: Case 7 A 2 month Thoroughbred colt presented a melting corneal ulcer in the right eye (OD)..........................................................................................................97 4-9: Case 8 A 2 month Thoroughbred colt presented a melting corneal ulcer in the right eye (OD)..........................................................................................................97 4-10: Case 9 A 14yo American Quarter Horse gelding presented a fungal corneal ulcer in the left eye (OS)..........................................................................................98 4-11: Case 10 A 12yo Hanovarian stallion presented a melting corneal ulcer in the left eye (OS).............................................................................................................98 5-1: Image analysis of representative zymogram gels to determine proteinase activity in pooled tears obtained from horses with an active corneal ulcer...........111 5-2: Gelatin zymogram of untreated pooled tears obtained from ulcerated eyes of horses......................................................................................................................112 5-3: In vitro inhibition of global proteolytic activity for various proteinase inhibitors by gelatin zymography...........................................................................................113 xiv

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5-4: Percentage of in vitro inhibition (mean value for triplicate samples) of global proteolytic activity determined by use of gelatin zymography for various proteinase inhibitors in pooled tears obtained from ulcerated eyes of horses........114 5-5: Percentage of in vitro inhibition (mean value for triplicate samples) of global proteolytic activity determined by use of gelatin zymography for equine serum stored in various ways............................................................................................116 6-1: Melting ulcer in the right eye of a 2 month old Thoroughbred horse....................125 6-2: CTGF levels in horse tears.....................................................................................129 6-3: CTGF Dilution curves............................................................................................130 6-4: Western Blot of tear fluids from horses with healthy eyes (lane 3) and ulcerated eyes (lane 4)...........................................................................................................131 6-5: Immunolocalization of CTGF in equine cornea.....................................................132 6-6: Immunolocalization of CTGF in equine lacrimal gland........................................133 6-7: Immunolocalization of CTGF in equine nictitating membrane gland...................133 7-1: The delicate balance between proteinases, proteinases inhibitors, and growth factors.....................................................................................................................139 xv

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KEY TO ABBREVIATIONS ARVO association for research in vision and ophthalmology AUC area under curve CN contralateral normal CTGF connective tissue growth factor DIS diseased ECM extracellular matrix EDTA ethylenediaminetetraacetic acid EGF epithelial growth factor FGF fibroblast growth factor GAG glycosaminoglycan GF growth factor GFR growth factor receptor H&E hematoxilin and eosin HGF hepatocyte growth factor IFNInterferon gamma IL interleukin kDa kilodalton KGF keratocyte growth factor MMP matrix metalloproteinase MT-MMP membrane-type matrix metalloproteinase xvi

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N normal NAC N-acetylcysteine NE neutrophil elastase OD right eye OS left eye PA plasmin PBS phosphate buffer solution PDGF platelet-derived growth factor PMN polymorphonuclear PTF precorneal tear film QH American quarter horse RSU relative standard unit u-PA urokinase-type plasminogen activator t-PA tissue-type plasminogen activator 1-PI 1-proteinase inhibitor TB thoroughbred TBS tris-buffered solution TFF tear fluid flow TGFtransforming growth factor alpha TGFtransforming growth factor beta TIMP tissue inhibitor of matrix metalloproteinase TNFTumor necrosis factor alpha TWH Tennessee walking horse xvii

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VEGF vascular endothelial growth factor xviii

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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 MATRIX METALLOPROTEINASE 2, MATRIX METALLOPROTEINASE 9,AND CONNECTIVE TISSUE GROWTH FACTOR IN THE EQUINE TEAR FLUID: POSSIBLE IMPLICATIONS IN CORNEAL WOUND HEALING By Franck J. Ollivier May 2004 Chair: Dennis E. Brooks Major Department: Veterinary Medicine The goals of this study were to investigate the presence of matrix metalloproteinase 2 (MMP-2), matrix metalloproteinase 9 (MMP-9), and connective tissue growth factor (CTGF) in the equine tear film, cornea, and lacrimal glands; to compare the levels of MMP-2, MMP-9 and CTGF in the tear fluid of horses with healthy eyes and horses with ulcerative keratitis; to document the changes in MMP-2 and MMP-9 levels in horse tear film during corneal healing; and to investigate the in vitro effects of various proteinase inhibitors on the activity of MMP-2 and MMP-9 isolated from the equine tear film. This study demonstrates by immunohistochemistry the expression of MMP-2 protein in the equine lacrimal gland and the gland of the nictitating membrane. The expression of MMP-2 and MMP-9 proteins was also found in the healthy cornea of the horse and was increased in the ulcerated equine cornea. In this study, the level of MMP-2 and MMP-9 activity was determined in a total 330 tear fluid samples by the use of gelatin zymography. MMP proteolytic activity was xix

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detected in all tear samples and was significantly increased in the tear fluid of horses with ulcerative keratitis (1.92 1.06 RSU) compared to normal (0.62 0.47 RSU). Based on the collection and analysis of a total of 124 serial tear fluid samples in 10 horses with ulcerative keratitis, we documented for the first time that total MMP activity decreases in equine tears as corneal epithelial and stromal healing occur. We documented, by gelatin zymography, a high amount of inhibition of equine MMP activity in vitro by the use of potassium edatate diaminetetrataacetatic acid, doxycycline, N-acetylcysteine, equine serum, ilomostat, and -1-proteinase inhibitor. The level of CTGF was determined by enzyme immunoassay in a total 64 tear fluid samples in this study and the molecule was detected in 39 of the samples. These data demonstrate that CTGF was present in the equine tear film. This study also indicates by immunohistochemistry the expression of CTGF protein in the healthy cornea, the lacrimal gland and the gland of the nictitating membrane of the horse (n=10). xx

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CHAPTER 1 INTRODUCTION The Cornea and the Precorneal Tear Film (PTF) The cornea is the gateway of the images into the eye. It is the most powerful refractive ocular structure and must remain transparent. The optical properties of the cornea include clarity, surface smoothness, and refractive index (Nishida, 1997; Samuelson, 1999). The health of the cornea is influenced by the aqueous humor, the intraocular pressure, the eyelids and the precorneal tear film. There are three classifications of tear production: basic, reflex and psychic. Continuous or basal tears are produced at a constant level and permit normal functioning of the precorneal tear film. Additional tear production is stimulated by reflex response to any irritation of the cornea, conjunctiva or nasal mucosa. Human beings are the only species for which psychic tear stimulation has been proven. Structure and Function of the Precorneal Tear Film The precorneal tear film (PTF) is not truly part of the cornea but is anatomically and functionally intimately associated with the cornea. The PTF is usually described as a superimposition of three structurally and functionally unique layers: the outer lipid layer, the intermediate aqueous layer, and the inner mucin layer. However, current belief is that the layers are not so distinct and the PTF is a mucin-dominated gel (Sack et al., 2000). The thickness of the PTF is not known in horses but has been estimated at 3 m in human beings (King-smith et al., 2000). 1

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2 The superficial outer lipid layer of the PTF is relatively thin and composed of oily materials (waxy and cholesterol esters) and phospholipids. This lipid layer retards the evaporation of the underlying aqueous layer between eyelid blinks, and reduces loss of PTF over the eyelids through increases in surface tension between the PTF and the cornea (Chow and Gilbard, 1997; Gum et al., 1999). The lipid phase also promotes a stable and even distribution of tear film over the cornea. The middle layer is the aqueous tear fluid layer. It is the thickest of the three layers and accounts for the majority of the volume. The aqueous layer is a complex mixture of ions, small molecules, glycoproteins, and proteins including enzymes (proteinases), immunoglobulins, cytokines, and growth factors. There is a paucity of information regarding the composition of equine tears. The pH was recently measured in conscious horses with indicator paper and reported to be 8.33 +/0.15 in the range of 8.08.6 (Lowe and Crispin, 2003). In horses, the main source of tears is the orbital lacrimal gland with a relatively minimal contribution from the nictitating membrane gland (Moore, 1990; Williams et al., 1979). Both of these glands are tubuloacinar and histologically similar. The lacrimal gland is located dorsolaterally, in the orbit, against the supraorbital part of the frontal bone, and the nictitating membrane gland is at the base of the T-shaped cartilage (Samuelson, 1999). The aqueous layer flushes foreign material away from the cornea and the conjunctiva, contributing to the mechanical barrier function. Seventy-five percent of the PTF is evacuated through the nasolacrimal drainage system. The PTF enters the puncta by capillary attraction and by eyelid motion which results in lower pressure within the lacrimal sac that acts subsequently as a lacrimal pump (Chow and Gilbard, 1997; Gum et al. 1999; Lemp and Wolfley, 1992). This layer also lubricates the

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3 passage of the eyelids and the nictitating membrane over the cornea. Tear drainage into the nasolacrimal system serves also to remove waste products (dissolved carbon dioxide and lactic acid). The aqueous layer also contains antimicrobial compounds (lysozymes, lactoferrin) and antibodies (IgA, IgG, IgM, IgGT) which chemically protect the cornea (Chow and Gilbard, 1997; Gum et al., 1999; Lemp and Wolfley, 1992; Martin et al., 1997; Marts et al., 1977; Nishida, 1997), and permit transfer of inflammatory cells such as polymorphonuclear cells (PMNs) (Lemp and Wolfley, 1992). The PTF contains soluble proteins that contribute to corneal health, and enhance defense mechanisms (proteinases, immunoglobulins, cytokines, and growth factors). The aqueous layer also delivers nutrients (water, glucose, electrolytes) to the avascular cornea and facilitates transfer of atmospheric oxygen (Gum et al., 1999; Lemp and Wolfley, 1992), Finally, the aqueous layer enhances the optical properties of the cornea by providing an optically smooth surface and by aiding in regulating corneal hydration (Chow and Gilbard, 1997; Gum et al., 1999; Nishida, 1997). The state of relative dehydration, or deturgescence, is dependent upon osmotic forces between the PTF, the aqueous humor, and probably the corneal stroma (Gum et al., 1999). The corneal surface is responsible for most of the light refraction that occurs in the eye, because of the large difference in the indices of refraction between air and cornea, and the angle of incidence of incoming light (Ofri, 1999). The image quality is based upon the regularity of the corneal epithelial surface and quality of the PTF (Nishida, 1997). Furthermore, control of corneal hydration is crucial to maintain the homogenous organization of stromal collagen lamellae and thereby corneal transparency (Gum et al., 1999; Nishida, 1997).

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4 The inner mucin layer consists of mucoproteins derived from goblet cells located in the conjunctiva, predominantly in the fornices (Chow and Gilbard, 1997; Samuelson, 1999). The mucoproteins are bipolar molecules that resemble a gel and bind the PTF aqueous layer (hydrophilic and lipophobic) to the epithelial surface glycocalyx (lipophilic and hydrophobic). This ensures the stability of the PTF and permits redistribution of the PTF after blinking, thus maintaining an optically smooth surface (Chow and Gilbard, 1997; Moore, 1990). The mucin layer also participates in the corneal defense because bacteria and foreign bodies may be entrapped within mucoproteins and the mucin harbors immunoglobulins (IgA) and lyzozyme. The PTF should be perceived as a three-phase system whose components are in dynamic equilibrium (Sack et al., 2000). The PTF serves to lubricate the ocular surface and prevent desiccation. Tear flow, coupled with the cleansing action of the blink, serves as a critical element in an essentially passive barrier defense, and is designed to protect the cornea from effects of trauma, pathogens and noxious agents and to remove waste products. Besides its nutritional value, the PTF also plays an important role in the anti-microbial, anti-inflammatory and proteolytic activities present at the corneal surface. It is important to note that in addition to the production of normal secretory components, the formation of the composite, tristratified tear film depends on eyelid integrity, normal ocular motility, and an intact blink mechanism (Sack et al., 2000). Since the cornea is in close contact with the preocular tear film and the aqueous fluid, the various kinds of proteinases, proteinase inhibitors, growth factors and cytokines in the tear film and aqueous might play an important role in the turnover of the corneal cells and wound healing of the cornea.

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5 Structure and Function of the Cornea The unique organization of the cornea permits clarity, a smooth transparent refractive surface, tectonic strength, differential permeability, and protection (Nishida, 1997; Pepose and Ubels, 1992). The cornea represents the most powerful refractive surface of the eye (Gum et al., 1999; Nishida, 1997). It contributes to 48D of the approximately 60D the refractive power of the human eye (Ofri, 1999; Pepose and Ubels, 1992). This important contribution of the cornea is due to the large difference in refractive indices as the light passes from air into the cornea. The cornea acts like a convex lens as it converges light and its refractive power depends mainly on its curvature (Nishida, 1997; Ofri, 1999). Similarly, in large eyes which are characterized by flat corneas the refractive power of the lens is reduced (48D in humans, 43D in cats, 38-43D in dogs and only 16-20D in horses) (Ofri, 1999). The transmission of the light through the cornea depends on the wavelength of the light and the angle of incidence. Obviously transmission of the light would be reduced by corneal opacities and therefore the cornea should remain transparent (Gum et al., 1999; Nishida, 1997; Ofri, 1999). The transparency is a crucial factor to be maintained in order for the cornea to fulfill its required optical properties, and it is possible because of the following structural features of the cornea: the lack of vessel and blood cells (the normal cornea is avascular (Gum et al.,1999; Nishida, 1997; Pepose and Ubels, 1992), the lack of pigment, the absence of keratinized cells (the corneal epithelium is simple, stratified, squamous and non keratinized), the presence of exquisitely sensitive nerves with free endings. Although the cornea is heavily innervated (Nishida, 1997; Pepose and Ubels, 1992), the specific arrangement of the collagen fibrils in the stroma (Nishida, 1997), the

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6 presence of mechanisms that regulate the hydration of corneal stroma (Gum et al., 1999; Nishida, 1997; Pepose and Ubels, 1992), the anatomic integrity of the epithelium and endothelium that represent physical barriers against the influx of tears and aqueous humor (Gum et al., 1992; Nishida, 1997; Samuelson, 1999), and the role as pumps of the epithelium and endothelium for the maintenance of deturgescence in the cornea (Gum et al., 1999; Pepose and Ubels, 1992; Samuelson, 1999). Finally, the cornea is part of the fibrous coat of the eye and therefore it participates in the maintenance of the eyeball shape and organization (Samuelson, 1999). The mechanical strength of the cornea is provided by its stromal collagen matrix. The equine cornea is 793 to 893 microns thick at the center (Andrew et al., 2001; van der Woerdt et al., 1995) and composed of three distinct layers: the outermost multilayered epithelium and its basement membrane, the middle stroma, the Descemets membrane and the innermost endothelium. The corneal epithelium is arranged in ten to fifteen cell layers: a single layer of mitotically active columnar basal cells, three to six layers of wing cells, and five to ten outer flattened layers of squamous superficial cells (the most differentiated epithelial cells) (Samuelson, 1999; Schultz, 1997). The basal cells of the epithelium are firmly attached to its basal lamina (i.e., its basement membrane) by hemidesmosomes, anchoring collagen fibrils and the glycoprotein laminin. Type IV, VI and VII collagen contribute to the basement membrane (Nishida, 1997) as well as laminin, hyaluronans, fibrin and fibronectin (Friend et al., 1994; Klyce et al., 1998). The hemidesmosomes attach the basal cells to the basement membrane which in turns serve to anchor the epithelium to the stroma. The arrangement of the hemidesmosomes varies among

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7 different species. The epithelial cells have good regenerative powers (basal turnover is approximately 7 days), but after removal of the basal lamina, weeks to months may be necessary for it to completely reestablish and, until the basement membrane is completely reformed, the epithelium can be easily removed from the stroma (Samuelson, 1999). The corneal epithelium is maintained by a constant cycle of shedding of superficial cells, basal cells division, and renewal of basal cells by centripetal migration of new basal cells originating from the limbal stem cells (Neaderland et al., 1987; Nishida, 1997; Pepose and Ubels, 1992) The thickest layer (90% of the corneal thickness), the corneal stroma, is composed of a few keratocytes and fibrocytes, nerve fibers and a large amount of transparent, almost structureless lamellae of fibrous tissue. These lamellae lie in parallel sheets and split easily into surgical planes. Between the lamella are fixed and infrequent wandering cells. The fixed cells are fibrocytes which are called keratocytes and the extension of these cells contributes to formation and maintenance of the stromal lamellae. The keratocytes synthesize collagen molecules (pro-collagen), glycosaminoglycans as well as collagen degradative enzymes such as matrix metalloproteinases (MMPs) (Nishida, 1997). Keratocytes in the normal cornea are quiescent and serve primarily to maintain the slow turnover of extracellular components but they may be activated easily by various types of insults to the corneal stroma. Wandering cells are usually leukocytes that have migrated from the limbus. The lamellae are parallel bundles of collagen fibrils, with each lamella running the entire diameter of the cornea. All the collagen fibrils within a lamella are parallel, but between lamellae, they vary greatly in direction. The precise overall organization of the corneal stroma is the most important factor in maintaining corneal

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8 clarity (Friend et al., 1994; Klyce et al., 1998). The mean diameter of each collagen fiber and the mean distance between these fibers are homogenous and measure less than half of the wavelength of visible light. This anatomic relationship is thought to be responsible for the fact that incident ray scattered by each collagen fiber is cancelled by interference of the other scattered ray, allowing light to pass through the cornea (Gum et al., 1999; Nishida, 1997). If the diameter or the distance between collagen fibers varies (as in fibrosis or edema), the cornea loses its transparency and there is a random scattering of incident rays (Nishida, 1997). The bulk of the corneal stroma is composed of thin uniformly positioned collagen fibrils embedded in glycosaminoglycans that form an extracellular matrix (ECM). The stromal ECM consists of collagen fibrils (native type I, III, V, VI and XII collagens), stromal glycosaminoglycans or GAGs (keratan sulfates, dermatan sulfates, chondroitin sulfates), and glycoproteins. Collagen type I is the most common. Type VI appears to play a role in cell-matrix interactions, which would be especially important during repair. Type III and XII are both believed to be developmental forms. The Descemets membrane is a homogenous, acellular membrane forming an inner protective boundary within the cornea. It is actually an exaggerated basement membrane of the posterior endothelium (20 m in thickness). To some degree, its composition is similar to that of the trabeculum of the iridocorneal angle. This ever-thickening basement membrane contains a number of collagen types I, III, IV, V, and VI, and also type VIII collagen (which is not found elsewhere in the cornea), laminin, fibronectin and heparan sulfates (Friend et al., 1994; Samuelson, 1999).The corneal endothelium rests on the Descemets membrane.

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9 The corneal endothelium produces Descemets membrane and contains an energy dependent pump to maintain corneal deturgescence. The corneal endothelium is only one layer of cells that does not proliferate in humans, monkeys, and cats but does proliferate in rabbits. Endothelial cell count decreases with age and with any trauma, and a critical loss of endothelium may lead to loss of corneal clarity. Corneal Healing and Scarring Horses have large, prominent eyes that are often subject to traumatic injury and resultant corneal infection. Ulcerative keratitis is a common and often vision-threatening condition in horses. Superficial, non-infected ulcers in horses generally heal quickly and without complication, whereas stromal degradation in deep or infected ulcers can rapidly and dramatically progress to corneal perforation in horses in less than 24 hours. When the cornea is injured, multiple systems are activated, which produce a series of complex and coordinated cellular processes that ultimately result in a healed corneal wound. Healing of corneal wounds is an exceptionally complex process involving the integrated actions of multiple proteinases (Table 1-1), growth factors (Table 1-3), and cytokines produced by epithelial cells, stromal keratocytes, inflammatory cells, and lacrimal glands. Multiple autocrine and paracrine interactions occur between epithelial cells and activated stromal fibroblast, and the exocrine actions of factors secreted by lacrimal gland cell into the PTF (Figures 1-1 and 1-2) Overview on Wound Healing and Corneal Healing Wound healing begins at the moment an injury occurs. The sequence of events in the progress of wound healing is as follows: release of soluble chemotactic factors which attract inflammatory cells to the injury site, influx of neutrophils and monocytes to neutralize bacteria and/or fungi in the wound site, the debridement of connective tissue

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10 matrix damage by macrophages, the initiation of neovascularisation, and the stimulation of cell proliferation and connective tissue matrix remodeling. These events occur sequentially until normal tissue architecture is restored. Many times this series of events leads to restoration of normal tissue structure and functions, but sometimes fibrotic disorders occur and scarring results in a loss of function of the particular tissue or organ. When a corneal wound occurs, keratocytes around the wound edge die creating a hypocellular zone (Wachtlin et al., 1999). Chemotactic factors such as PDGF, TGF-, and TGFare released and attract inflammatory cells (Table 1-3), resulting in initiation of reepithelialisation, contraction of connective tissue and stimulation of angiogenesis (Schultz, 1997). The first leukocytes recruited to the site of injury are neutrophils which attack bacteria and /or fungus that may have been introduced into the tissue at the time of injury. Levels of neutrophils begin to decline and macrophages begin to take over as the dominant cell type in the wound. Macrophages function in the degradation and the removal of tissue debris in preparation for reparative phases of wound healing. Activated platelets release several growth factors which result in the recruitment of neutrophils and monocytes. Beyond the site of injury, quiescent keratocytes become activated into fibroblasts and migrate to the site of injury. This migration is followed by cell proliferation, and finally deposition of ECM components. Many growth factors and cytokines have been implicated in stimulating synthesis of ECM components, cell proliferation and migration and angiogenesis (TGF-, TGF-, PDGF, FGF) (Table 1-3). These growth factors have control over the complex processes in wound healing involving migration, mitosis and differentiation of epithelial and stromal cells (Schultz et al., 1992). The fibroblasts deposit additional reparative collagen and eventually

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11 synthesize enough ECM to form a scar, replacing the damage tissue. All these events in the process of inflammation must be reversed for the tissue architecture to return to normal. For this to happen, the removal of the inflammatory mediators which were generated must occur. Infiltration of monocytes and leukocytes must decrease in order for this to occur. Furthermore, removal of extravasated fluid, protein, cellular debris, granulocytes and macrophages occurs as the wound regeneration process continues (Daoud et al., 1985). Histological Events of Corneal Healing Traditionally, corneal wound healing is divided into the healing of those injuries that affect only the epithelium and those resulting in substantial loss of stroma up to the Descemets membrane. Epithelial wound healing Three components are involved in healing of the epithelial surface of the cornea: cell migration to cover the injured area (sliding), mitosis to reconstitute the normal number of epithelial cells and normalization (differentiation) of the corneal epithelial cells (Neaderland et al., 1987; Peiffer et al., 1999; Samuelson, 1999; Schultz, 1997). In the epithelial wound healing, epithelial migration is the initial step for the successful and complete resurfacing of defects (Neaderland et al., 1987; Peiffer et al., 1999; Pepose and Ubels, 1992). After an epithelial injury, signals from disrupted cells or signals generated by exposure of the basement membrane are sent to the surrounding intact epithelial cells. Mitosis ceases (for 96 to 120 hours) and the basal cells at the wound edge retract. Then, there is a phase during which the cell cytoskeleton and intercellular junction of these cells are modified (Cameron, 1997). These events allow the basal cells to begin to migrate by ameboid movement to cover the defect within an hour

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12 after the injury. The edges of the cell membrane ruffle and extend pseudopodia onto the denuded extracellular matrix (ECM), toward the center of the wound (Nishida, 1997; Pepose and Ubels, 1992). Two types of epithelial movements for covering the denuded area in the cornea have been observed: advance groups of sliding monolayered epithelial basal cells and a subsequent landslide-like mass movement of the epithelium (basal and wing cells). The sliding movement precedes the landslide-like mass movement of the epithelium which finally covers the denuded area. During these movements it is important that the cells remain adherent to the neighboring cells or to the ECM. The advance group of the individual cells simply spread over a provisional ECM. Polymorphonuclear neutrophils arrive from the tear film and begin removing remnants of destroyed cells (Schultz, 1997). As mentioned previously, cell migration requires coordinated changes of both the cell cytoskeleton and the cell surface attachments to adjacent cells and substratum. Cell migration is accomplished by rapidly changing the cytoskeleton in order to build the scaffold towards the leading edge of the cell, which forms pseudopods, while disassembling the scaffold at the trailing edge of the cell. At the interface between the dead and dying cells, the intercellular junctions between epithelial cells loosen but do not completely disengage. The direction of the migration are determined by chemical signals in both the fluid environment of the cell (chemotaxis) and in the surface (ECM) to which the cells is attached (haptotaxis).The stimulation factor should exist in a gradient in order to induce a cell movement and then the cell will migrate toward the area of highest concentration (Cameron, 1997). The basal cells migrate until contact inhibition of migration is established by physical contact with adjacent cells (Cameron, 1997).

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13 Once the injured surface has again been covered, mitosis occurs to restore the epithelium to its normal configuration (thickness) (Pepose and Ubels, 1992; Samuelson, 1999). Mitotic replication of the basal cells begins about 24 after epithelial injury: new basal cells are formed to replace those that are migrating forward, and the mitosis occurs in a zone 3 to 5 mm behind the leading edge of the migrating cells (Schultz, 1997). The mitosis continues and may result in a transient corneal epithelial hyperplasia before normalization occurs (Peiffer et al., 1999). Repeated erosions or large defects might overwhelm the replicative capabilities of the transient amplifying population of basal cells adjacent to the site of injury, thus requiring participation of the permanent replicative cells at the limbus (stem cells) (Samuelson, 1999; Peiffer et al., 1999). When normal thickness is reestablished, the highly differentiated cellular characteristics are re-formed (Cameron, 1997). The superficial cells terminally differentiate by synthesizing keratin protein which helps reestablish the barrier properties of the epithelium (Schultz, 1997). Intercellular attachments are also reestablished as well as the basal surface contacts (formation of new hemidesmosomes in the basal cells) (Schultz, 1997). When a corneal abrasion is limited to the epithelium and the basement membrane is not damaged, a normal epithelium with adhesion complexes is formed soon after healing. If the basement membrane is removed or altered, the epithelium must lay down a new basement membrane following healing, and development of normal adhesion complexes is delayed for several months (Pepose and Ubels, 1992). Stromal and endothelial wound healing Deep corneal ulcers heal with a combination of epithelial sliding and replication (previously described), as well as stromal wound healing. Stromal healing involves the re-synthesis and crosslinking of collagen, alterations in proteoglycans synthesis, and

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14 gradual wound remodeling leading to the restoration of tensile strength (Pepose and Ubels, 1992). The earliest event in stromal corneal wound healing appears to be the deposition of fibrin, fibronectin and other elements of the clotting cascade into the wound. Some keratocytes immediately adjacent to the wound margin undergo apoptosis and others begin releasing enzymes involved in the degradation of damaged proteoglycans and collagen lamellae at the wound edge. There is also an influx of PMNs and monocytes in the wound within a few hours that release proteinases to cause the proteolytic debridement of necrotic cellular and extracellular debris. Within a few hours the adjacent keratocytes become activated and begin protein synthesis, and within 3 days the keratocytes are able to secrete collagens and GAGs. There are also proliferation and activation of keratocytes beyond the injury site that migrate then to the site of injury. The proliferation, mitosis, migration and activation of the keratocytes are under influence of many factors such as growth factors and cytokines. Once at the site on injury that has been cleared of debris, keratocytes produce collagens and GAGs. A denervated cornea is at risk for developing epithelial defects. Corneal nerves regenerate after corneal injury, although the process requires months to years. The nerves regenerate from unwounded peripheral nerve trunks. The orientation of the nerve fibers is generally random, and corneal sensitivity is seldom returned to normal (Cameron, 1997). The healing of the corneal stromal wound is slower than in other connective tissues presumably because of the lack of blood vessels (Fagerholm, 2000; Schultz, 1997). Unfortunately, the endothelial cells do not respond to cell loss as quickly as epithelial cells, and endothelial healing varies with both age and species. For this reason when endothelial cells are lost, the defect must be covered by the spreading of cells from

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15 areas adjacent to the wound to cover the wounded area under influence of growth factors such as TGF-, b-FGF, and EGF (Table 1-3) (Friend et al., 1994; Samuelson, 1999). Biochemical Mechanisms of Corneal Healing Molecular biology of epithelial wound healing In the epithelial wound healing, epithelial migration is the initial step for the successful and complete resurfacing of defects. Several biochemical and synthetic events are involved in cell migration. Migration requires energy and one of the earliest changes that occurs during healing is the depletion of glycogen from the cells at the leading edge of migration. Enlargement of the cells is apparently the result of an increase in cell water content, and the actual movement of cells is calcium dependent, as calmodulin inhibition stop migration by preventing microfilament assembly. The processes that initiate migration are mediated by cyclic AMP. Migration of corneal epithelium during wound repair is also accompanied by an increase in protein synthesis. A specific increase in synthesis has been demonstrated for the cytoplasmic protein vinculin (Pepose and Ubels, 1992). The actin component is prominent within the pseudopodia of the basal cells. The actin-containing microfilament system is attached by vinculin to the transmembrane integrin receptors, which selectively bind to ECM proteins such as fibronectin, laminin, and collagens (Schultz, 1997). During the first phase of epithelial wound healing, fibronectin plays an essential role as a provisional, temporal ECM. In the normal unwounded cornea, type IV collagen, laminin, and heparan sulfate proteoglycan are the major components of the basement membrane but not fibronectin (Cameron, 1997; Nishida, 1997; Schultz, 1997). Fibronectin appears shortly after the epithelial injury (produced by adjacent cells or delivered through tears, aqueous humor). Basal cells attach and spread over the fibronectin matrix and fibronectin disappears once the epithelial

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16 wound is healed. Fibronectin stimulates epithelial migration and has chemotactic and haptotactic activities for the corneal epithelial cells. The integrins are the receptors for fibronectin at the surface of corneal cells. Fibronectin provides a suitable ECM for cell attachment and migration of the epithelial cells that become more sensitive to fibronectin through increased expression of integrin. Epithelial growth factor (EGF) and IL-6 regulate integrin expression in corneal epithelial cells (up-regulation), and by this up regulation, they stimulate corneal cell migration. Other components of the ECM (of the basement membrane) such as heparan sulfate and laminin play a role in wound healing by influencing cell adhesion and migration. The motility of the basal cells must involve the simultaneous formation and destruction of attachments between proteins of the plasma membrane of epithelial cells (integrin receptors) and components of the ECM such as fibronectin, and laminin. Therefore proteinases also play a key role in epithelial cell migration by breaking down the attachments: matrix metalloproteinases such as MMP-1, MMP-2 and MMP-9, fibroblast and neutrophil collagenases, stromelysins, as well as serine proteases such as plasminogen activators, plasmin, and neutrophil elastase have been shown to be involved in wound healing (Table 1-1) (Cameron, 1997; Schultz, 1997). Corneal epithelial cells mitosis and sliding are both strongly stimulated by EGF and Keratocyte growth factor (KGF) that are normally present in tears, and also produced by the corneal epithelial cells themselves following injury (Nishida, 1997; Peiffer et al., 1999). On the other end, some member of the TGFfamily inhibits cell proliferation and therefore, counteracts the stimulatory effect of EGF, but most of the TGFfamily does not affect the migration of the epithelial cells (Table 1-3) (Nishida, 1997).

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17 Molecular biology of stromal wound healing The first step in stromal healing is similar to the epithelial healing with the deposition of fibrin, fibronectin and other elements of the clotting cascade into the wound. The debridement of the wound occurs very quickly after the injury. The degradation of the necrotic cellular and extracellular debris (damaged proteoglycans and collagen lamellae) involves many proteinases such as matrix metalloproteinases, collagenases and urokinase-type plasminogen activator (that converts plasminogen to plasmin that causes destruction of fibronectin). These various proteinases are produced and released by keratocytes as well as inflammatory cells (Table 1-1). Once the injury site has been cleared of debris, activated keratocytes produce collagens (type I, type III), and GAGs (keratan sulfate) under the influence of growth factors such as TGF(Table 1-3). To achieve a successful wound healing of the stroma, the fibrous components must be reestablished in a way that follows, at least to some degree, normal development. However, the diameter and other characteristics of the reparative collagen are quite different: the collagen diameter is larger and the individual collagen fibers are more variable in caliber. Similarly the new proteoglycans differ in character and proportion, and the populations of proteoglycans (i.e., GAGs) are selective to keep the collagen fibrils organized and properly sized. Therefore, if the relative concentrations of GAGs are substantially altered during wounding, the possibility of reforming the fibrous architecture for needed transparency is reduced and will remain so until the normal proteoglycan environment is rebuilt. The initial extracellular matrix of the scar produced is the same as the final or resting scar (Schultz, 1997).

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18 Figure 1-1: Proteinases and corneal wound healing. Figure 1-2: Growth factors and corneal wound healing.

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19 Corneal Scarring As we mentioned earlier, corneal transparency is determined in large part by the structure of the stromal ECM. When the precise structure of the stroma is disrupted, it can never be properly restored. Corneal stroma is not regenerated but repaired: it is replaced with unspecialized tissue that creates the scar. The strength of corneal scars and the surrounding tissue never reaches that of the uninjured cornea. It is estimated that the tensile strength returns to 70% of normal native (Cameron, 1997). The repair tissue matrix contains component molecules not usually present in stroma, particularly fibronectin, and the newly synthesized collagen fibrils are thicker than those of the normal stroma and variable in size. Formation of the corneal scar is a dynamic process and clinical changes can be noted up to five years after corneal wound or incision (Eiferman, 1992). Dermatan sulfates and keratin sulfate proteoglycans from the adjacent matrix as well as newly synthesized proteoglycans accumulate with the scar. Collagen type I, III, V, and VI are also quite predominant within the scar (Ljubimov et al., 1998). Some improvement in the transparency of repair tissue can occur over the long term through the progressive remodeling of the repair tissue matrix and the MMPs appear to be involved in this process. Remodeling results in the loss of fibrin and fibronectin and the position of fibrils with a more uniform diameter and a more orderly arrangement. Some of the reparative collagen and proteoglycans are selectively catabolized by specific proteinases (Table 1-1) and new collagen and proteoglycans are then selectively synthesized in a more advantageous orientation, quantity or proportion (Cameron, 1997). In summary, corneal scarring is the result of abundant cell synthesis (collagen, fibronectin, laminin) by fibroblasts which proliferate and differentiate within the

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20 provisional matrix. Stromal scarring can have negative impacts on the refraction or even the light transmission at the level of the cornea. Proteinases, Proteinase Inhibitors Proteinases in The PTF: Types and Origins There are four major classes of proteinases based on the mechanism of catalysis, including the cysteine proteinases (thiols), the aspartate proteinases (acidic), the serine proteinases and the matrix metalloproteinases (MMPs) (Table 1-1) (Birkedal-Hansen et al., 1993). Among these families, two play a major role in corneal physiopathology: the serine proteinases and the MMPs (Fini et al., 1990; Fini, 1998; Matsubara et al., 1991b; Woessner, 1999) (Figure 1-1). Serine proteinases The serine proteinase family includes neutrophil elastase, and the plasmin and the plasminogen activators (Table 1-1). Plasminogen, the inactive form of plasmin, is present in the blood plasma and in interstitial fluids such as tears and the aqueous humor. It accumulates in inflamed tissues. Plasmin and plasminogen are present in the healthy cornea. Conversion to the active form occurs by cleavage at the N terminus mediated by one of the two plasminogen activators (PA): the urokinase type (u-PA) or the tissue type (t-PA) (Stevens et al., 1992). PA can be found at the surface of resident tissue cells and have been reported to be produced by the cornea. However, the tear plasminogen activator seems to be predominantly of the u-PA (Tozser and Berta, 1990). Plasmin has a wide substrate specificity and cleaves a range of extracellular matrix substrates such as fibrin, fibronectin, laminin and it also activates latent collagenases (MMP-1, 3, 9, 14) (Table 1-1) (Berta et al., 1990; Cejkova et al., 1993; Salonen et al., 1987; Stevens et al., 1992). The activity of plasmin is regulated in

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21 the extracellular space by plasma and cellular proteinase inhibitors (1-proteinase inhibitor, 2-anti plasmin, 2-macroglobulin) that are present in the interstitial fluids but also synthesized by the resident tissue cells (Table 1-2). Elevated plasmin activity is considered to be harmful from the point of view of the initiation (Wang et al., 1985) as well as the development of corneal destructive processes. However, it is also suggested that a certain level of serine proteases (plasmin and plasminogen activators) is necessary for the repair process (Hayashi et al., 1991). The levels of plasmin activity depend on the severity of the corneal injury (Cejkova et al., 1993). Neutrophil elastase (NE) is the most abundant serine proteinase in human tears, is found in dog and horse tears (Sathe et al., 1998; Strubbe et al., 2000; Watanabe et al., 1990), and is synthesized by polymorphonuclear leukocytes and macrophages (Sakata et al., 1997). It degrades native III and IV collagen as well as corneal ECM compounds such as laminin, fibronectin (Barletta et al., 1996; Cejkova, 1998; Paterson et al., 1994; Watanabe et al., 1990) (Table 1-1). Matrix metalloproteinases (MMPs) Enzymes of the matrix metalloproteinases (MMP) family are thought to play the major role in ECM remodeling (Birkedal-Hansen et al., 1993; Woessner, 1999). They are now 20 characterized members of this family of zinc containing proteinases (Table 1-1). Substrates include essentially all known matrix molecules, including interstitial collagens as well as the proteinases themselves or their inhibitors. MMPs are synthesized and secreted as inactive pro-enzymes that are activated in the extracellular space by cleavage of a portion at the N terminus (Birkedal-Hansen et al., 1993). The synthesis of the pro-enzymes (i.e. latent forms) has been shown to be under influence of many factors including cytokines and growth factors: IL-1, TNF-, EGF have been shown to induce

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22 the synthesis of MMP-1,-3,-9 whereas IL-4, Il-6, IFN-, TGFinhibit the synthesis of MMP-1, -3 (Twining, 1994) (Tables 1-1 and 1-3). The pro-enzyme activation occurs by means of proteolytic cascades, which probably differs for each enzyme and has been partially characterized (plasmin, membrane -type MMPs) (Table 1-1). Once activated, MMPs require Ca2+ for their stability and Zn2+ as a cofactor (Fini and Girard, 1990; Twining, 1994). MMPs are regulated by non specific inhibitors such as 2-macroglobulin (Table 1-2). They can also be regulated by a specific class of inhibitors, the tissue inhibitors of MMPs (TIMPs), of which there are now 3 types (Table 1-2). Regulatory mechanisms in the extracellular space keep MMP activity under control. MMPs have been identified in the blood plasma and interstitial fluids. They may arrive in tissues this route via blood or tears but most are produced by cells at the remodeling site, either resident tissue cells or infiltrating inflammatory cells. It can be considered a general rule that resident tissue cells will not synthesize MMPs unless there is a demand for tissue remodeling. The process is fine tuned through reciprocal communication between the cells and their extracellular matrix. Synthesis of MMPs has been shown to be stimulated by a number of agents, many of which are products of inflammatory cells or resident tissue cells. Inflammatory cells (PMNs, macrophages) have their own arsenal of matrix degrading proteinases including serine proteinases and MMPs, and they also could be the stimulation of the endogenous proteolytic activity. The latent forms of the MMPs are not biologically active but it is important to consider them as they can be activated by various factors and they can be involved in physiopathological processes (Birkedal-Hansen et al., 1993; Woessner, 1999).

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23 Five sub-families of MMPs exist according to substrate specificity: the stromelysins, the metalloelastase, the membrane-type MMPs (MT-MMPs), the collagenases, and the gelatinases (MMP-2, -9) (Table 1-1). The last two have been shown to play an important role at the level of the cornea. The collagenases (MMP-1, MMP-8, MMP-13, and MMP-18), degrade native type I, II or III collagens. They cleave the collagen molecule into and length fragments and stop, which is a characteristic of mammalian collagenases (Berman et al., 1971; Berman et al., 1973; Kenney et al., 1994). Interstitial collagenase cleaves collagen type I found in the stroma but it can not catalyze degradation of basement membrane collagens which is the controlling step leading to stromal ulceration. For this reason, the relationship between expression of interstitial collagenase and gelatinases is important as they undoubtedly work together in the remodeling process (Fini et al., 1998; Matsubara et al, 1991a). The gelatinases A and B (MMP-2 and MMP-9) (Fini and Girard, 1990; Fini et al., 1992) are of major importance in terms of remodeling and degradation of the corneal stromal collagen. The origin and purpose of MMP-2 and appear to differ at the corneal level. Matrix metalloproteinase-2 is synthesized by corneal keratocytes and performs a surveillance function in the normal cornea, becoming locally activated to degrade collagen molecules that occasionally become damaged as a result of normal wear and tear. (Azar et al., 1998; Matsubara et al., 1991b; Twinning, 1994) Alternatively, MMP-9 is produced by epithelial cells and polymorphonuclear neutrophils (PMNs) following corneal wounding (Fini and Girard, 1990; Matsubara et al., 1991b). It is actually recognized that stromal ulceration does not occur until after the epithelial basement

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24 membrane disappears. It is the controlling step leading to stromal ulceration (Fini and Girard, 1990; Fini et al., 1992; Fini et al., 1996; Mastubara et al., 1991b). Gelatinase B (or MMP-9) plays an important role as it is able to destroys the adhesive structure of the epithelial basement membrane (type VII and IV collagens, laminin, proteoglycans) leading to stromal ulceration, and which delays the re-epithelialization of the injured cornea (Fini et al., 1992; Fini et al., 1996; Kenney et al., 1994). MMP-2 and MMP-9 equally degrade gelatin (denatured collagens), native type IV, V and VII collagens, fibronectin, elastin, and laminin (Table 1-1). This work focused on these last two MMPs.

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Table 1-1: Proteinases present in the precorneal tear film and involved in corneal wound healing. 25 Type Sub-types Proteinase Origin MW (latent/active) Substrate Stimulation Activation MMP-1 (collagenase 1, fibroblast collagenase, interstitial collagenase) Keratocytes Corneal fibroblasts, Macrophages 52/42 kDa Collagen type I,II,III,VII Proteoglycans Pro-MMP 2, 9 Gelatin IL-1 beta Plasmin MMP-3, MMP-10,MMP-7 MMP-8 (collagenase 2, neutrophil collagenase) PMNs Macrophages 85/64 kDa Collagen type I,II,III,VII Proteoglycans Gelatins Neutrophil elastase MMP-13 (collagenase 3) 52/42 kDa Collagen type I,II,III,VII Proteoglycans Gelatins Fibronectin,tenascin TGF alpha TGF beta IL-1 beta bFGF VEGF MMP-3 Interstitial collagenases MMP-18 (collagenase 4) 53/42 kDa Collagen type I MMP-2 (gelatinase A 72-kDa gelatinase) Keratocytes 72/66 kDa Collagen type I,IV,V,VII Proteoglycans Gelatins Elastin, laminin, fibronectin,tenascin Pro-MMP9, 13 TGF alpha TGF beta bFGF Pseudomonas elastase MMP-1,MMP-7,MMP-14, MMP-15, MMP-16, MMP-24 Gelatinases MMP-9 (gelatinase B 92-kDa gelatinase) Corneal epithelial cells, PMNs, 92/84 kDa Collagen type IV,V Proteoglycans Gelatins Elastin, fibronectin TGF alpha TGF beta IL-1 beta, IL-2 bFGF, EGF MMP-1,MMP-2,MMP-3, MMP-7, plasmin MMP-3 (stromelysin 1) Present in the corneal stroma Macrophages 57/45 kDa Collagen type I, II, III, IV,V, IX Proteoglycans Gelatins Fibronectin,laminin,tenascin,elastin Pro-MMP 1, 9 TGF beta, EGF, IL-1, bFGF, PDGF MMP-2 Neutrophil elastase Plasmin MMP-10 (stromelysin 2) 54/44 kDa Collagen typeIII, IV,V Proteoglycans Fibronectin Pro-MMP 1 TGF beta IL-1 beta VEGF, EGF Plasmin Matrix Metallo Proteinases (MMPs) Stromelysins MMP-11 (stromelysin 3) 51/46 kDa Collagen type IV Fibronectin,laminin Serine proteinase inhibitor

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Table 1-1: Continued. 26 Type Sub-types Proteinase Origin MW (latent/active) Substrate Stimulation Activation Matrilysin MMP-7 Macrophages 28/19 kDa Fibronectin,laminin,elastin,tenascin Gelatins Pro-MMP1,2, 9 Neutrophil elastase plasmin MMP-12 (Macrophage elastase) 54/22 kDa Elastin MMP-20 (enamelysin) 54/22 kDa MMP-14 (MT1-MMP) Present in epithelial cells 66/54 kDa Collagen type I, II, III Proteoglycans Fibronectin,laminin,elastin,tenascin Pro-MMP2, 13 Plasmin MMP-15 (MT2-MMP) Present in the corneal stroma 72/60 kDa Pro-MMP-2 TIMP-2 MMP-16 (MT3-MMP) Present in the corneal stroma 64/53 kDa Pro-MMP-2 MMP-17 (MT4-MMP) 57/53 kDa Membrane-type MMPs (MT-MMP) MMP-24 (MT5-MMP) Pro-MMP-2 Matrix Metallo Proteinases (MMPs) Not determined MMP-19, MMP-21, MMP-22, MMP-23 Neutrophil elastase PMNs Macrophages Collagen type III, IV Fibronectin, laminin Heparin sulfate proteoglycans Plasmin Corneal epithelialcells Collagen type III, IV,V Gelatins Fibronectin, fibrin, tenascin, laminin, Proteoglycans Pro MMP1, 3, 9, 14, Latent TGF beta Urokinase type and tissue-type plasminogen activators (u-PA,t-PA) u-PA: Corneal Epithelial cells, keratocytes, PMNs t-PA: lacrimal gland HGF Fibronectin Plasminogen TGF beta, TGF alpha, bFGF, EGF, IL-1, IL-4 Serine proteinases Cathepsin G PMNs Macrophages Fibronectin Aspartate proteinases Cathepsin D, pepsin Proteoglycans Cysteine proteinases Cathepsins B,L and S Proteoglycans Laminin, fibronectin, elastin

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27 Table 1-2:Proteinase inhibitors. Types Proteinases Natural inhibitors Artificial inhibitors Matrix metalloproteinases (MMPs) 2-macroglobulin (Entrapment of the molecule) TIMP-1 (20.6 kDa) TIMP-2 (21.5 kDa, in the cornea, secreted by keratocytes) TIMP-3 (21.6 kDa) TIMP-4 Chelation of the cofactor (Ca2+ and Zn2+): Ascorbic acid, citric acid Disodium ethylene-diaminetetra-acetic acid (EDTA), N-acetylcysteine (NAC) Hydroxamic acid -dipeptides : Ilomostast Thiol peptides Tetracyclines (Doxycycline, oxytetracycline) Urokinase type and tissue-type plasminogen activators (u-PA,t-PA) PA inhibitors Phenylmethylsulfonyl fluoride (PMSF) Aminoethyl-benzne-sulfonyl fluoride (AEBSF) Corticosteroids Neutrophil elastase 1-poteinase inhibitor 2-macroglobulin (Entrapment of the molecule) Phenylmethylsulfonyl fluoride (PMSF) Aminoethyl-benzne-sulfonyl fluoride (AEBSF) Serine proteases Plasmin 1-antitrypsin 2-macroglobulin 2-antiplasmin Aprotinin Phenylmethylsulfonyl fluoride (PMSF) Aminoethyl-benzne-sulfonyl fluoride (AEBSF)

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28 The MMPs form an interesting group of enzymes in that there is a central catalytic domain to which have been added a variety of additional domains or short inserts (Fig 1-3) (Birkedal-Hansen et al., 1993; Woessner, 1999). The typical MMP structure consists of: a signal peptide: stretch of 17-20 residues that is present in all MMPs expect MMP-17 and serves as a signal for secretion into the endoplasmic reticulum and an export from the cell, a propeptide: region of 80 amino-acids with a N-terminal hydrophobic residues and a highly conserved PRCXXPD sequence near the C-terminal that provides the cystein residue (cystein switch) that makes contact with the catalytic zinc atom and maintains the enzyme in its latent form (needs to be cleaved off for the activation of the enzyme). This regulatory region is present in all MMPs even the ones that have the furin-cleavage site for activation (a region of 10-14 residues containing the RXKR sequence, present in MMP-11,-14,-15,-16 and ). a catalytic domain: this domain typically contains about 160-170 residues, including sites for the binding of calcium ions and the structural zinc atom. The 50-54 residues at the C-terminal end of this domain (an highly conserved HEXGH sequence) include the site of biding of the catalytic zinc. The zinc-binding region is independent of the remainder of the catalytic domain because various insertions can occur between these two portions such as fibronectin type II repeats (present in MMP-2 and 9), a hemopexin or vitronectin domain (C terminal domain): this domain of about 200 residues that contains four repeats that resemble to hemopexin and vitronectin with a cystein residue at either end. This domain is present in all MMPs expect MMP-7. This domain does not appear to be essential for catalytic activity by more for the substrate specificity and also contains the binding site for TIMPs. a trans-membrane domain: Membrane-type MMPs (MMP-14,-15,-16 and ) contain this other extension (sequence of 80-100 residues) that governs insertion of these MMPs into the cell membrane (Birkedal-Hansen et al., 1993; Woessner, 1999). The consideration and analysis of the MMP structure allows the understanding of the mode of action of MMPs inhibitors and therefore strategy for the identification of new MMPs inhibitors (Birkedal-Hansen et al., 1993; Woessner, 1999). The new MMPs inhibitors such as (peptide)-based MMP inhibitors not only can effectively interact with

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29 the MMP at subsites proximal to the scissile bond but also functionally bind this critical zinc. The effective zinc chelation in conjunction with the high affinity for the enzymes subsites by functionality provides to the inhibition of the MMPs. The peptide-based MMP inhibitors have been reported to have higher potency and specificity than the previous generation of inhibitors (such as tetracyclines, N-acetyl-cysteine, EDTA). Figure 1-3: Domain Structure of the Matrix Metalloproteinase family. Origins of the proteinases As already mentioned, proteinases can be produced by the corneal cells (MMP-1, MMP-3, MMP-2, MMP-9, as well as t-PA), and the inflammatory cells such as PMNs and macrophages (MMP-7, MMP-8, MMP-9 as well as NE and u-PA). They are therefore called endogenous proteinases (Table 1-1). Infectious organisms also produce

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30 proteinases and these constitute the exogenous proteinases (Hibbetts et al., 1999). For example Pseudomonas aeruginosa has been shown to produces two MMPs (alkaline protease, elastase), and Aspergillus spp and Fusarium spp have been shown to produce serine proteinases (Gopinathan et al., 2001; Hanzi et al., 1993; Kernacki et al., 1996; Kessler et al., 1977; Matsumoto, 2000; Zhu et al., 1990). Extra-cellular enzymes of bacterial or fungal origin contribute to the corneal condition, either directly or indirectly, through the activation of endogenous proteinases (Gopinathan et al., 2000; Twining et al., 1993). Proteinases, PTF and Corneal Physiopathology The maintenance and the repair of the corneal matrix require a tightly coordinated balance of ECM synthesis, degradation and remodeling where proteinases are involved as mentioned earlier (Figure 1-1). Activities of proteolytic enzymes (endogenous proteinases) are normally balanced by natural proteinase inhibitors (including tissue inhibitors of MMPs or TIMPs, 1-proteinase inhibitor, 2-macroglobulin), thus preventing excessive degradation of normal healthy tissue. Proteinases and especially MMPs produce beneficial outcomes for corneal wounds, provided they are produced at the right level, in the right place and at the right time but excessive levels of these proteinases can cause severe complications. Excessive amounts of proteinases can create an imbalance between proteinases and proteinase inhibitors, and increased amounts of proteases can cause pathologic degradation of collagen and proteoglycans in the cornea (Slansky et al., 1969; Twinning, 1994). The tear film proteinases have been previously evaluated in both normal and diseased eyes of animals and human beings (Berman et al., 1971; Berman et al., 1973; Berman et al., 1977; Brooks 1999; Fini and Girard, 1990; Matsubara et al., 1991a;

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31 Matsumoto et al., 1993; Prause, 1983a; Prause, 1983b; Strubbe et al, 2000; Tervo et al., 1988; Tervo et al., 1991). In severely damaged corneas, proteinase activities in the tear film were significantly increased (Berman et al., 1971; Kernacki et al., 1995; Matsubara et al., 1991a; Prause, 1983a; Strubbe et al., 2000; Tervo et al., 1988). If infection is present, the proteinases secreted by infectious organisms are also responsible for the severe corneal damage that is associated with the disease (Kernacki et al., 1995; Twining et al., 1993). In ulcerated horse corneas, tear film levels of MMP-2, MMP-9 and neutrophil elastase are significantly elevated when compared to age-matched normal controls (Strubbe et al., 2000) and are hypothesized to contribute to the breakdown of stromal collagen. Ulcerative keratitis with extensive stromal involvement displays rapid progressive that can lead to corneal perforation in many horse eyes probably due to this upregulated proteolytic activity (Brooks, 1999). In the initial work done by Strubbe et al. (2000), tears were collected using glass capillary tubes. This tear collection method allows the recording of the time needed for collection and therefore the measurement of the tear fluid flow (TFF). The TFF has been measured in various studies performed in human beings (Tervo et al., 1994; van Setten et al., 1989; van Setten et al, 1990; Vesaluoma et al., 1996; Vesaluoma et al., 1997; Vesaluoma and Tervo, 1998) but it has not been measured yet in animals and especially in horses. By taking the TFF changes (the dilution factor) into consideration, it is possible to give a better reflection of the changes in synthesis and release of the proteinases between the different groups of animal studied.

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32 Many studies have been performed on the changes in MMP expression in the skin as well as MMP proteolytic activity in the wound fluids during skin wound healing in animals and human beings (Agren, 1994; Blalock et al., 2001; Ladwig et al., 2002; Lobmann et al., 2002; Herouy, 2001; Parks, 1999; Paul et al., 1997; Wall, 2002). Few studies have reported the proteolytic changes in the tear film during corneal wound healing. Changes in the tear film level of plasmin, a serine protease have been described in animals and human beings (Barlati et al., 1990; Cejkova, 1998; Cejkova et al., 1993; Salonen et al., 1987; Tervo et al. 1998; Tervo et al., 1989a; Tervo et al, 1989b; Tervo et al. 1991; Vesaluoma et al., 1998; van Setten et al., 1989). The changes in level of various MMPs in the tear film (Barro et al., 1998) as well as their expression of MMP in the cornea during wound healing have also been described in rat and rabbit (Fini and Girard, 1992; Fini et al., 1998; Lu et al., 1999; Matsubara et al., 1991a; Ye et al., 1998). To the authors knowledge, there is only one study on the precise profile of the MMP activity in tear film during corneal healing based on the collection and analysis of serial tear fluid samples in human beings (Barro et al., 1998). Few studies have reported the expression of MMPs in healthy and diseased corneas. MMP-7 has been detected in healthy and wounded rat cornea (Lu et al., 1999), MMP-3 in human corneas (Gabison et al., 2003), and MT-MMP-4, and MT-MMP-5 in mouse corneas (Dong et al., 2000). MMP-2 and MMP-9 have been identified by immunohistochemistry in the healthy and ulcerated corneas in humans (Gabison et al., 2003; Kenney et al., 1998), dogs (Chandler et al., 2003), mice (Wall et al., 2002; Yang et al., 2003), and rats (Reviglio et al., 2003; Ye and Azar, 1998) but no similar study has been performed in horses.

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33 An understanding of the physiopathologic processes as well as early diagnosis and aggressive treatment are important to speed healing, reduce scarring, and prevent corneal rupture in case of ulcerative corneal diseases in horses. Despite the abundant literature on ocular proteinases, and despite the prevalence of equine ulcerative keratitis, there is very limited information on tear film proteinases in the horse. MMPs are certainly involved in corneal ulceration and corneal wound healing in horses as Strubbe et al. reported with their preliminary report (Strubbe et al., 2000) and their importance needs to be further assessed. Identification of the proteinases mainly responsible for corneal stromal degradation would be the first logical step to allow specific inhibition Proteinases, Proteinase Inhibitors and the Cornea There are natural proteinase inhibitors that are present in the PTF and in the cornea. 1-proteinase inhibitor, 2-macroglobulin and some TIMPs are found in the blood plasma and interstitial fluids and can also be synthesized by resident tissue cells simultaneously with the MMPs (Table 1-2, Figure 1-1). Disorders occur when there is an imbalance between proteinases and proteinase inhibitors in favor of the proteinases. Proteinase activity in the tear film is believed to speed up degradation of stromal collagen, leading to rapid progression of ulcers. Normalizing proteolytic activity in the tear film is an objective of the treatment of corneal ulcers in horses. Thus, proteinase inhibitors have been recommended for treatment of ulcerative keratitis to reduce the progression of stromal ulcers, speed epithelial healing, and minimize corneal scarring (Berman, 1975; Berman, 1978; Berman et al., 1973; Berman et al., 1975; Brooks, 1999; Tervo et al., 1992; Ward, 1999). However, studies on activity of specific enzymes are limited. Accordingly, recommendations for use of topically administered N-acetylcysteine (NAC), potassium

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34 EDTA, and serum in horses with corneal ulcers are mostly based on anecdotal clinical reports or extrapolations from data obtained from use of these compounds in other species (Brooks, 1999; McLaughlin et al., 1992; Severin, 1976; Strubbe et al., 2000; Tervo et al., 1992; Ward, 1999; Whitley and Gilger, 1999). The availability of newer compounds such as doxycycline, a modified dipeptide that contains hydroxamic acid (i.e., ilomostat), and 1-proteinase inhibitor ( 1-PI), which initially have shown considerable promise as MMP or serine-proteinase inhibitors in other species (Barletta et al., 1996; Dursun et al., 2001; Golub et al., 1984; Perry et al., 1993; Rawal and Rawal, 1984; Schultz et al., 1992) outlines the need for specific studies on possible regulation of enzyme activity in tears in horses with corneal disease (Table 1-2). These new compounds have potential as potent proteinase inhibitors in horses with corneal ulcers. Therefore, there was a need to evaluate the effectiveness of various proteinase inhibitors for reducing the activity of equine tear film MMP-2 and -9 by in vitro testing on samples of tear film obtained from horses with ulcerative keratitis. Growth Factors Growth Factors, PTF and Corneal Physiopathology In the past 15 years, substantial progress has been made in understanding the process of corneal maintenance and corneal wound healing at the molecular level, in order to achieve better outcomes for clinical injuries by rationally design therapeutic strategies that use agents to modulate corneal wound healing. A molecular theory emerged based upon the synthesis and release of several specific growth factors (GFs), at the site of injury which then act through autocrine and paracrine pathways to regulate healing (Pancholi et al., 1998; Schultz, 1994; van Setten et al., 1994). GFs are low molecular weigh polypeptides, are synthesized by a variety of cell

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35 types and act locally on the cell that synthesized the GF or adjacent cells by binding to specific, high-affinity receptors proteins located on the plasma membrane of the target cells and they regulate many physiologic functions of these target cells (Schultz, 1997). They are assumed to control many crucial processes of corneal wound healing including cell migration (chemotactic effects for inflammatory cells including macrophages, neutrophils and also for corneal cells including epithelial and endothelial cells, keratocytes), cell proliferation (mitosis), angiogenesis (neovascularisation includes the proliferation, migration, proteolytic activity, and capillary tube formation of the vascular endothelial cells), and synthesis of ECM components (Casey and Li, 1997; Imanishi et al., 2000; Schultz et al. 1992; Vesaluaoma et al., 1996) (Table 1-3). Cell migration, mitosis, and differentiation as well as reconstruction of adhesion complexes are important features of corneal epithelial wound healing. In addition to regulation of the wound healing process and fibrosis, growth factors are likely to be involved in the maintenance of corneal cellular integrity (Imanishi et al., 2000; Schultz et al., 1992; van Setten et al., 1998; Vesaluoma et al., 1996; Wilson et al., 1990; Wilson, 1998) ( Figure 1-2). The cornea is avascular and the corneal wound healing is controlled in a different manner. Since the cornea is in close contact with the tear film and the aqueous fluid, the various kinds of growth factors and cytokines in the tear film and aqueous play an important role in the turnover of the corneal cells and wound healing of the cornea (Imanishi et al., 2000; Vesaluoma et al., 1996). The tear fluid in particular has certain functions exerted by blood components in other organs. Growth factors, mRNA or protein expression has been shown in the lacrimal gland as well as in all corneal cell

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36 layers. (Table 1-3) About 15 years ago, the first GF was identified in the PTF (van Setten et al., 1989) and the number of GFs discovered in the PTF keeps increasing since then. To date, the following GF have been reported to be present in the PTF of a normal or ulcerated eye of various species: transforming growth factor-alpha (TGF-) (van Setten et al., 1994), fibroblast growth factor (FGF) (van Setten, 1996), epidermal growth factor (EGF), transforming growth factor-beta 1 & 2 (TGF1&2) (Gupta et al., 1996), platelet-derived growth factor (PDGF) (Vesaluoma et al., 1997), vascular endothelial growth factor (VEGF) (van Setten, 1997), hepatocyte growth factor (HGF) (Li et al., 1996), keratocyte growth factor (KGF) (Wilson, 1999), and connective tissue growth factor (CTGF) (van Setten et al., 2003). (Table 1-3). Levels of GFs and their receptors (GFR) in a wound are crucial for its normal healing (Gum et al., 1999; Schultz, 1997). The role of the GF is based on the balance between the presence of the GF in the PTF and the number of GFRs at the surface of the ocular surface. This model has been thoroughly described for EGF (van Setten et al., 1992), and can be extended to other GFs (HGH, PDGF, TGF). A brief description of this model follows hereafter. In case of a wounded cornea, the total amount of GF released in the tear fluid is increased and the increase of the TFF is even greater. Therefore, the concentration of GF is often decreased (Tuominen et al., 2001; van Setten et al., 1992; Vesaluoma et al., 1996; Vesaluoma et al., 1998; Wilson, 1998; Wilson, 1999). The decrease of GF concentration induces an up regulation of the GFR density on the target cells (Steinemann et al., 1990). The increased number of molecules of GF and of GFR leads to an increase number of GF/GFR complexes that exert effect on a

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37 specific aspect of cell metabolism (mitosis, synthesis, migration). The overall balance GF/GFR system is reestablished once complete corneal wound healing is achieved. GFs, Cytokines, proteinases, proteinases inhibitors and endocrine hormones, and the balance between these molecules are crucial for the maintenance of the tissue integrity and its wound healing (Schultz, 1998). The interactions between GFs and proteinases are particularly important in the corneal wound healing process as illustrated by the two examples hereafter: EGF and Plasmin, u-PA, t-PA (van Setten et al., 1992): EGF has been shown to increase the production of u-PA, and t-PA resulting in the increase of plasmin synthesis. On the other end, EGF stimulates the fibronectin synthesis which is a substrate for plasmin. TGF& u-PA, t-PA, MMP-1, MMP-3 (Twining, 1994; Vesaluoma et al., 1998): TGFhas been shown to down-regulate the synthesis of MMP-1 and MMP-3. It has also been reported that TGFinhibits the production of u-PA, and t-PA. Therefore, TGFseems to counteract the stimulatory action of EGF on u-PA, t-PA, and plasmin synthesis. (on another note, TGFalso counteracts the stimulatory action of EGF on corneal epithelial cells proliferation) There are also interactions between the different GFs: EGF and TGF(Nishida, 1997; van Setten et al., 1992), PDGF-BB and HGF (Fagerholm, 2000; Li et al., 1996), TGFand CTGF (Chen et al., 2000; Frazier et al., 1997). Experimental studies show that growth factors influence growth of cultured corneal cells and modulate corneal wound healing in vitro (Blalock et al., 2003; Pancholi et al., 1998; Watanabe et al., 1993) including one using equine corneal cells (Haber et al., 2003). The ability of the GFs to directly regulate key cellular processes such as mitosis, migration, ECM components synthesis suggests that treatment with exogenous GF might enhance healing of corneal wound (Schultz, 1997). Few clinical trials have been done using growth factors. The effects on corneal healing of EGF have been clinically

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38 evaluated in various species including human, primates, dogs, rabbits, and in horses (Burling et al., 2000). There are few reports on growth factors in veterinary ophthalmology as mentioned earlier (Burling et al., 2000; Haber et al., 2003) and there is no information about the presence and the role in equine PTF and equine cornea of a recently discovered growth factor, connective tissue growth factor (CTGF). Connective Tissue Growth Factor Connective tissue growth factor is a secreted, cysteine-rich peptide of about 38 kilodaltons (kDa) that was originally identified as a mitogen for fibroblasts in condition media cultures for human umbilical vein endothelial cells in 1991. This peptide is named CTGF because it has been identified as a major chemotactic and mitogenic factor for the cells of the connective tissue (Table 1-3), and has PDGF related biological and immunological properties. Cuurrently, expression of CTGF is limited to cells derived from the mesenchyme including fibroblasts, vascular endothelial cells, smooth muscle cells, chondrocytes and renal tubules cells (Table 1-3). Leukocytes, lymphocytes and cells derived from the epithelium of ambryos are not known to express CTGF. Transforming growth factor (TGF-) activates fibrocytes to produce CTGF (Igarashi et al., 1996; Wang et al., 2001). CTGF can induce connective tissue cell proliferation and ECM synthesis; it has similar but not identical properties to TGF-, suggesting that it may function as a downstream mediator of some action of TGF-. The overexpression of CTGF has been proposed to play an important role in pathways that lead to fibrosis in various tissues: scleroderma, systemic sclerosis, atherosclerosis, idiopathic pulmonary fibrosis, liver fibrosis, diabetic glomerulosclerosis,

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39 and renal fibrosis (Chen et al., 2000; Gupta et al., 2000; Igarashi et al., 1996; Shi-wen et al., 2000; Takehara, 2001; Wunderlich, 2000). To date, the presence of CTGF has been reported in the human serum (Sato et al., 2000), the anterior chamber fluid (van Setten et al., 2002; van Setten et al., 2003), and recently human tear fluid (van Setten et al., 2003) and rat cornea (Blalock et al., 2003). Limited information is available on the expression of CTGF in ocular tissues but these studies suggest a possible involvement of CTGF in ocular pathology. CTGF is a fibrogenic cytokine that might be involved in corneal wound healing and corneal fibrotic disorders as CTGF enhances fibroblasts to proliferate and produce more collagen (Frazier et al., 1996). Ocular surface diseases such as corneal ulceration and corneal stromal abscessation are common devastating ocular diseases in horses. Healing of corneal ulcers in horses is often associated with profound fibrosis and corneal scar formation that can result in varying degrees of visual impairment (Brooks, 1999). Since no information is available regarding CTGF in horses and since the role of CTGF appears to be so crucial in the regulation of fibrosis in other tissues in other species, its expression and role in the equine PTF and in the normal and fibrotic equine cornea needs to be assessed as CTGF may play an important role in corneal scar formation in horses and might be a target for reducing fibrosis in the future.

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Table 1-3: Growth factors present in the precorneal tear film and involved in corneal wound healing. 40 Factor Origin Structure Main targets Main effects Species EGF Lacrimal gland Corneal epithelial cells Keratocytes Polypeptide 53 AA Molecular weight: 6kDa Corneal epithelial cells Corneal endothelial cells ___ Keratocytes ______________ Vascular endothelial cells Mitogenic *Chemotactic ECM synthesis (fibronectin) Angiogenic Rat Mice Rabbit Human PDGF Platelets Corneal epithelial cells Macrophages Lacrimal gland Dimer of 2 polypeptide chains (A&B) 3 forms: 2 homodimers (AA,BB) and 1 heterodimer (AB) Molecular weight: 30kDa Corneal epithelial cells Keratocytes Neutrophils Angiogenic Mitogenic for epithelial cells and keratocytes Chemotactic for keratocytes and neutrophils ECM component synthesis Rabbit Human TGFLacrimal gland Corneal epithelial cells Keratocytes Macrophages Monomer Molecular weight: 6kDa Corneal epithelial cells Keratocytes Corneal endothelial cells Vascular endothelial cells Mitogenic ECM component synthesis MMP expression ( MMP1) Angiogenic Human Rat TGFMacrophages Platelets Lacrimal gland Corneal epithelial and endothelial cells Keratocytes Various isoforms (1,2&3) Polypeptide (homodimer: 2 identical chains of 112 AA) Molecular weight: 25kDa Corneal epithelial cells Fibroblasts Chemotactic for keratocytes and epithelial cells Anti-proliferative ( mitosis) of epi cells ECM component (collagen, fibronectin) synthesis differentiation MMP expression (uPA, tPA, MMP1, MMP3) and TIMPS induces CTGF synthesis Human VEGF Macrophages Corneal endothelial and epithelial cells Keratocytes Vascular endothelial cells Various isoforms (4) Dimeric heparin binding glycoprotein Molecular weight: 34-42kDa Corneal endothelial and epithelial cells Keratocytes Vascular endothelial cells vasopermeability Angiogenic ( proliferation, migration, proteolytic activity, capillary tube formation of the vascular endothelial cells) Chemotactic for corneal endothelial cells Human

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Table 1-3: Continued. 41 Factor Origin Structure Main targets Main effects Species KGF Lacrimal gland Keratocytes Corneal epithelial cells Mitogenic Rat FGF Lacrimal gland Corneal epithelial and endothelial cells 2 isoforms : acidic and basic (aFGF & bFGF) Corneal epithelial cells Mitogenic for epithelial cells and vascular endothelial cells (bFGF) Angiogenic (bFGF) Rabbit Human HGF Lacrimal gland Keratocytes Heparin biding protein 2 chains (1 60 and 1 30) Molecular weight: 90kDa Corneal epithelial cells Mitogenic Chemotactic Differentiation Human CTGF Corneal epithelium and fibroblasts (TGF beta induced fibroblast cells) Cystein rich peptide Molecular weight: 38kDa Fibroblasts on corneal fibroblasts Chemotactic Mitogenic ECM component (collagen) synthesis Downstream messenger of TGF1 Rat Human EGF= Epidermal growth factor, PDGF= Platelet-derived growth factor, TGF= Transforming growth factor, VEGF= Vascular endothelial growth factor, KGF= Keratocyte growth factor, FGF= Fibroblast growth factor, HGF= Hepatocyte growth factor, CTGF= Connective tissue growth factor

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42 Significance to The Horse Racing Industry Horses have large, prominent eyes that are often subject to traumatic injury and resultant corneal infection. Ulcerative keratitis is a common and often vision-threatening condition in horses. A report from the United States Department of Agriculture mentioned that ocular problems represent the fourth most common cause of operations (7.9%) in horses in the southern part of the United States. This followed trauma (16.2%), leg/hoof problems (15.3%), and colic (13.1%) (NAHMS Equine study, 1998). The Ophthalmology Service of the University of Florida Veterinary Medical Teaching Hospital diagnosed ulcerative keratitis in 527 horses between January 1987 and October 2002. This constituted 35% of all horses evaluated for ophthalmic problems during this period. A simple corneal abrasion can rapidly become vision threatening if it becomes infected with a pathogenic organism, especially in our subtropical environment. Determination of the proteolytic activity in the normal and diseased horse eye as well as during corneal healing and the efficacy of antiproteolytic compounds will allow us to institute appropriate therapy earlier in the course of the disease, decrease the time required for recovery and rehabilitation, and perhaps alleviate the need for surgical therapies. Horses are often laid up in excess of 8 weeks during treatment of corneal diseases. Shortening this time through prompt and adequate treatment would be of a great economic value to the horse racing industry. A pronounced fibrovascular response is also prominent during corneal healing in horses and the growth factors may have an important role in this response. An understanding of the pathophysiologic processes involving the proteinases and the growth factors as well as early diagnosis and aggressive treatment are important for

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43 quick resolution of ulcerative corneal diseases in horses and to speed healing, reduce scarring, and prevent corneal rupture. Purpose of The Study The general goal of this dissertation was to understand the role that MMP-2 and MMP-9 play in corneal ulceration and healing in horses and to assess the efficacy of various agents that inhibit these proteases. An additional goal of this work was to understand the role that CTGF plays in regulating corneal scarring in horses and a long term-goal of this research is to develop agents that reduce corneal scarring, targeting CTGF. Hypotheses The hypotheses were the following: Protease levels in the tear film of horses with normal eyes and horses with corneal ulcers The levels of proteases in tears are increased in horses with an ulcerative keratitis (infectious or sterile) as compared to tears in a normal horse eye. The increased proteolytic activity present in tears from horses with ulcerative keratitis decreases as the corneal ulcer heals. In Vitro effects of various protease inhibitors Protease inhibitors e.g., serum, 2-macroglobulin, ethylene diamine tetraacetate, N-acetyl-L-cysteine, tetracyclines, thiols and hydroxamic acids, 1 proteinase inhibitor are used or represent an interesting alternative in the clinical treatment of corneal ulcers in horses. These protease inhibitors produce a measurable reduction in MMP and NE activity in the tear fluid of horses with ulcerative keratitis that can be measured in vitro. CTFG levels in tear fluid from horses CTGF is present and detectable in the equine PTF.

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44 The level of CTGF in tear fluid is different in an ulcerated eye (infectious or sterile) as compared to a normal eye. Localization of MMP-9, MMP-2, CTGF in the equine cornea and lacrimal glands MMP-9, MMP-2, CTGF are present in the corneal tissue. MMP-9, MMP-2, CTGF are present in the main lacrimal gland as well as then nictitating gland. Objectives The present work had the following goals: To identify the presence of MMP-2 and MMP-9 in the tear fluid, cornea and lacrimal glands of horses. To investigate if ulcerative keratitis has any influence on the activity of MMP-2 and MMP-9 in the equine tear fluid. To follow the changes in the amount of proteolytic activity in horse tear film during corneal healing and stromal remodeling. To investigate what in vitro effects various proteases inhibitors have on the proteolytic activity of MMP-2 and MMP-9 isolated from the equine tear fluid. To investigate the presence and origin of CTGF in the tear fluid, cornea and lacrimal glands of healthy horse eyes. To elucidate what influence ulcerative keratitis has on the presence of CTGF in the equine tear fluid.

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CHAPTER 2 DETECTION OF MMP-2 AND MMP-9 IN THE EQUINE TEAR FLUID, CORNEA AND LACRIMAL GLANDS Introduction The maintenance and repair of the corneal stromal extracellular matrix (ECM) require a tightly coordinated balance of ECM synthesis, degradation and remodeling. Proteolytic enzymes (proteinases) perform important physiological functions in the slow turnover and remodeling of the corneal stroma. The activities of proteolytic enzymes are normally balanced by natural proteinase inhibitors and thus prevent excessive degradation of normal healthy tissue (Hibbetts et al., 1999; Twining et al., 1994). Excessive levels of proteinases can create an imbalance between proteinases and proteinase inhibitor levels, therefore causing pathological degradation of stromal collagen and proteoglycans in the cornea ECM (Geerling et al., 1999; Matsubara et al., 1991a; Slansky et al., 1969; Strubbe et al., 2000; Twining, 1994). Endogenous proteinases are produced by corneal cells and inflammatory cells (Berman et al., 1971; Kernacki et al., 1995; Matsubara et al., 1991a; Prause, 1983a; Strubbe et al., 2000; Tervo et al., 1988). If infection is present, the proteinases secreted by infectious organisms, called exogenous proteinases, are also responsible for corneal damage (Gopinathan et al., 2001; Hanzi et al., 1993; Hibbetts et al., 1999; Kernacki et al., 1995; Matsumoto, 2000; Twining et al., 1993; Zhu et al., 1990). Extra-cellular enzymes of bacterial or fungal origin contribute to the corneal condition, either directly or 45

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46 indirectly, through the activation of endogenous proteinases (Gopinathan et al., 2001; Twining, 1993). Among the various classes of proteinases found in the tear film and cornea of humans and animals, two are thought to play a major role in the corneal metabolism: the matrix metalloproteinases (MMPs) and the serine proteinases (including neutrophil elastase) (Fini et al., 1992; Fini et al., 1998; Matsubara et al., 1991). Two of the 13 known MMPs are of major importance in terms of corneal stromal collagen remodeling and degradation, and are the focus of this study. They include MMP-2 (72-kDa gelatinase A) and MMP-9 (92-kDa gelatinase B) (Fini and Girard, 1990; Fini et al., 1998). Their origin and purpose at the corneal level appear to differ. In ulcerated horse corneas, tear film levels of MMP-2, MMP-9 and neutrophil elastase are significantly elevated when compared to age-matched normal controls (Strubbe et al., 2000), and are hypothesized to contribute to the breakdown of stromal collagen. Ulcerative keratitis with extensive stromal involvement displays rapid progression that can lead to corneal perforation in many horse eyes probably due to this upregulated proteolytic activity (Brooks, 1999). Few studies have reported the expression of MMPs in healthy and diseased corneas. MMP-7 has been detected in healthy and wounded rat cornea (Lu et al., 1999), MMP-3 in human corneas (Gabison et al., 2003), and MT-MMP-4, and MT-MMP-5 in mousecorneas (Dong et al., 2000). MMP-2 and MMP-9 have been identified by immunohistochemistry in the healthy and ulcerated corneas in humans (Kenney et al., 1998; Gabison et al., 2003), dogs (Chandler et al., 2003), mice (Wall et al., 2002; Yang et

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47 al., 2003), and rats (Reviglio et al., 2003; Ye and Azar, 1998) but no similar study has been performed in horses. MMPs are undoubtfully involved in corneal ulceration and corneal wound healing in horses but their importance needs to be assessed. With only one preliminary study published in horses (Strubbe et al., 2000) there is a considerable need for a thorough investigation of the presence of MMP-2 and MMP-9 in tears and their pathophysiological importance. The purpose of this study was to determine the presence of MMP-2 and MMP-9 in the tear fluid of horses by Western Blot analysis, to investigate the localization of MMP-2 and MMP-9 in healthy and pathological equine corneas, and to clarify the possible origins of MMP-2 and MMP-9 by immunohistochemistry. Materials and Methods Materials All procedures were carried out according to the ARVO statement for the Use of Animals in Ophthalmic and Vision research and were approved by the University of Florida Animal Care and Use Committee as well as the UF College of Veterinary Medicine Clinical Research Review Committee. For the detection of MMP-2 and MMP-9 in the equine tear fluid, samples were collected from horses that were either clinical cases (Figure 2-1) from the Large Animal Hospital at the University of Florida College of Veterinary Medicine Veterinary Teaching Hospital or, in the cases of animals without any ocular disease, from a commercial horse farm in Florida. The tear collections were performed after sedation of the animal with an intravenous injection of xylazine hydrochloride (0.5 mg/kg) and before any diagnostic procedure or any treatment. All tear fluid specimens were collected by capillary force from the lower fornix using capillary tubes with an atraumatic tip as

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48 previously described (van Setten et al., 1989). The time of collection of tear fluid samples was not taken in this study. All samples were immediately centrifuged, transferred into Eppendorf tubes (10L aliquots), and stored at -80C until analysis. Two pools of tear fluid (100L) from horses with healthy eyes and horses with ulcerated were then constituted for the identification of MMP-2 and MMP-9 on Western Blot by comparison to human MMP-2 and MMP-9 standards. For immunohistochemistry, five healthy corneas, five main lacrimal glands and five nictitating membrane glands were collected from horses with normal eye following euthanasia due to conditions not related to this project. A 2 year old Thoroughbred mare was presented to the VMTH with corneal perforation and iris prolapse associated with a fungal infection in her right eye. The eye was enucleated, the globe was processed. All samples were fixated in 4% formaldehyde buffer and stored in paraffin. Hematoxylin and Eosin (H&E) staining, and MMP-2 and MMP-9 immunostaining were done on the healthy cornea and lacrimal glands, as well as on the diseased cornea and iris prolapse of this clinical case. Figure 2-1: Melting ulcer in the right eye of a 12 year old American Quarter horse. This case was involved in this study. Note the presence of hyperemic

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49 conjunctiva, and a 8-9 mm melting corneal ulcer surrounded with a corneal edema. MMP-2 and MMP-9 Western Blot Two pools of tear fluid were formed by collection of tears from horses with healthy eyes and horses with ulcerated eyes. The tears were centrifuged at 14,000g for 10 minutes and the supernatant stored at 80 C until their analysis. The samples (pools of tear fluid from healthy and ulcerated horse eyes) as well as a molecular weight markers (MultiMark pre-stained multicolored standards, Invitrogen, Carlsbad, CA) and 0.1 ng of the active and latent forms of the human recombinant MMP-2 and MMP-9 were loaded on a 5% stacking, 12% SDS-PAGE gel. The electrophoresis was run at 125 V for 1 hour in a running buffer. The proteins were electro-transferred onto a PVDF membrane (Immobilon-P, Millipore Corporation, Bedford, MA) in a transfer buffer at 25V at 4 C overnight. The membrane was blocked in Tris-buffered saline (TBS) containing 10% non-fat milk (Blotting grade blocker non-fat dry milk, BioRad Laboratories, Richmond, CA) for one hour and then incubated in the same solution containing a dilution of 1:7000 of the mouse anti-human MMP-2 (1:20 dilution) and MMP-9 (1:25 dilution) (R&D Systems, Mineapolis, MN) for 30 minutes at room temperature. The blot was washed three times with 0.05% Tween-20 TBS and then incubated with rabbit anti-mouse IgG alkaline phosphatase conjugate (Sigma, St Louis, MO) at the dilution of 1:1500 in the blocking solution for 30 minutes at room temperature. The membrane was washed again three times and the BCIP/NBT alkaline phosphatase substrate (Sigma, St Louis, MO) was added and color generated. The reaction was stopped by water washing.

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50 MMP-2 and MMP-9 Immunohistochemistry in Sections of Equine Cornea, Equine Main Lacrimal Gland and Equine Nictitating Membrane Gland Cells expressing MMP-2 and MMP-9 were detected immunohistologically using a standard avidin-biotin amplification method. Briefly, healthy corneas, lacrimal glands, and nictitating membrane glands obtained from five horses, and the globe obtained by enucleation of a clinical case of corneal perforation with iris prolapse were fixed in 4% paraformaldehyde 0.1 M phosphate buffer solution (PBS) overnight at 4 C, dehydrated in an ascending series of ethanol, and embedded in paraffin. Paraffin-embedded sections were prepared and 6 m sections were mounted on microscopic slides (Superfrost/plus; Fischer Scientific, Pittsburgh, PA). Slides were deparaffinized and rehydrated with xylene and a graded series of ethanol. Slides were blocked with 5% horse serum in Tris-buffered saline (TBS) for 30 minutes at room temperature. Slides were then sequentially incubated with mouse anti-human MMP-2 or MMP-9 (R&D Systems, Mineapolis, MN) in TBS and 5% horse serum overnight at 4 C, washed three times with TBS, incubated with biotynilated horse anti-mouse IgG secondary antibody (Vectastain ABC-AP kit,Vector Laboratories, Burlingame, CA) in TBS, washed, and incubated with alkaline phosphatase-conjugated streptavidin in TBS and 5% horse serum (Vectastain ABC-AP kit,Vector Laboratories, Burlingame, CA), washed, and incubated with alkaline phosphatase visualization substrate (Vector Red Alkaline Phosphatase substrate kit I: Vector Laboratories, Burlingame, CA). The reaction was stopped by water washing. The mouse anti-human MMP-2 and MMP-9 antibodies were raised against recombinant human MMP-2 and MMP-9 protein. The sections were photographed with bright-field illumination, and Nomarski phase-contrast microscopy at various magnifications.

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51 Results MMP-2 and MMP-9 Western Blot: Detection of MMP-2 and MMP-9 in the Equine Tear Fluid. Western Blot was used to compare the products detected by the antibody against human MMP-2 and MMP-9 in the two pools of equine tear samples (normal and diseased) to the active and latent forms of the human MMP-2 and MMP-9 standards. A band, similar to those observed with the human latent MMP-2 standard and the human active MMP-2 standard, 66 kDa, was noted in all the equine samples (Figure 2-2). A band, similar to those observed with the human latent MMP-9 standard and the human active MMP-9 standard, 83 kDa, was noted in all the equine samples (Figure 2-3). These results indicate that there is a detectable amount of MMP-2 and MMP-9 in the equine tear fluid. 1 2 3 4 98 kDa 52 kDa 31 kDa Latent Active 17 kDa Figure 2-2: Western Blot of tear fluids from horses with ulcerated eyes (lane 3) and healthy eyes (lane 4). A band similar to those observed with the human latent MMP-2 standard (lane 1) and the human active MMP-2 standard, 66 kDa (lane 2), was noted on all the equine samples (lanes 3 and 4). Molecular Human MMP-2 Equine diseased Equine normal Weight Markers Standard tear fluid tear fluid

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52 1 2 3 4 98 kDa 52 kDa 31 kDa 17 kDa Molecular Human MMP-9 Equine diseased Equine normal Weight Markers Standard tear fluid tear fluid Figure 2-3: Western Blot of tear fluids from horses with ulcerated eyes (lane 3) and healthy eyes (lane 4). A band similar to those observed with the human latent MMP-9 standard (lane 1) and the human active MMP-9 standard, 83 kDa (lane 2), was noted on all the equine samples (lanes 3 and 4). Immunohistochemistry: Immunohistochemical Localization of MMP-2 and MMP-9 in the Healthy Cornea and Lacrimal Glands of Horses Immunoreactivity to MMP-2 specific antibodies was noted in the corneal epithelium, the corneal stroma and in the corneal endothelium (Figure 2-4). In the corneal epithelium, MMP-2 staining was concentrated in the squamous superficial epithelial cells (Figure 2-4). The immunohistochemical staining for MMP-2 in the equine lacrimal glands revealed specific staining for MMP-2 predominantly in the epithelial cells of the intralobular ducts, and the intralobular connective tissue (Figure 2-5). The immunohistochemical staining for MMP-2 in the nictitating membrane glands revealed specific staining for MMP-2 predominantly in the acinar cells and the intralobular connective tissue (Figure 2-6). No immunoreactivity to MMP-9 was found at the dilution used (1:20) in the healthy cornea (Figure 2-4), the equine lacrimal gland (Figure 2-5), or the nictitating membrane glands (Figure 2-6). No immunoreactivity was found in the

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53 tissues on the sections used as controls where the primary antibody was omitted (Figures 2-4, 2-5 and 2-6). 2 1 1 1 2 2 2 3 B C A 3 3 1 E 2 D F 3 1 1 1 2 2 2 3 3 13 G H I Figure 2-4: Immunolocalization of MMP-2 and MMP-9 in healthy equine cornea. H&E staining of the equine cornea (A) where corneal epithelium (1), stroma (2) and Descemets membrane corneal endothelium (3) can be distinguished. MMP-2: Nomarski phase-contrast 1 observation (B) and bright-field observation (C) of the negative control sections showed no staining at all. Immunohistochemical staining for MMP2 in the equine cornea (D, E, and F) showed MMP-2 specific staining in the corneal epithelium, especially in the squamous superficial cell layer (white arrow), a small amount in the stroma underneath the epithelium (black arrow) and the corneal endothelium. MMP-9: Nomarski phase-contrast 1 observation (G) and bright-field observation (H) of the negative control sections showed no staining at all. Immunohistochemical staining for MMP-9 showed no MMP-9 specific staining in the healthy equine cornea (I). Original magnifications were X100 (A, B, C, D, G, H and I) and X200 (E and F)

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54 A B C D E F G I H Figure 2-5: Immunolocalization of MMP-2 and MMP-9 in equine lacrimal gland. H&E staining of the equine lacrimal gland (A) where acini (inset), intralobular ducts, and lymphocytes in the intralobular connective tissue (inset) can be distinguished. MMP-2: Nomarski phase-contrast 1 observation (B) and bright-field observation (C) of the negative control sections showed no staining at all. Immunohistochemical staining for MMP-2 in the equine lacrimal gland (D, E, and F) showed MMP-2 specific staining in the epithelial cells of the intralobular ducts (white arrow), and the intralobular connective tissue (striped arrow). MMP-9: Nomarski phase-contrast 1 observation (G) and bright-field observation (H) of the negative control sections showed no staining at all. Immunohistochemical staining for MMP-9 showed no MMP-9 specific staining in the equine lacrimal gland (I). Original magnifications were X100 (A, B, C, D, G, H and I) and X400 (E), and X1000 (inset in A and F).

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55 2 2 2 1 1 1 B A C 2 1 E F 2 2 2 1 1 1 G H I Figure 2-6: Immunolocalization of MMP-2 and MMP-9 in equine nictitating membrane gland. H&E staining of the equine nictitating membrane (A) where the hyaline cartilage (1) and the nictitating membrane gland (2) composed of acini (inset) can be distinguished. MMP-2: Nomarski phase-contrast 1 observation (B) and bright-field observation (C) of the negative control sections showed no staining at all. Immunohistochemical staining for MMP-2 in the equine nictitating membrane gland (D, E, and F) showed MMP-2 specific staining in the acinar cells (white arrow), and the intralobular connective tissue (striped arrow). MMP-9: Nomarski phase-contrast 1 observation (G) and bright-field observation (H) of the negative control sections showed no staining at all. Immunohistochemical staining for MMP-9 showed no MMP-9 specific staining in the equine lacrimal gland (I). Original magnifications were X100 (A, B, C, D, G, H and I) and X400 (E), and X1000 (inset in A and F). Immunohistochemistry: Immunohistochemical Localization of MMP-2 and MMP-9 in Ulcerated Equine Cornea A 2 year old Thoroughbred mare was presented to the VMTH with corneal perforation and iris prolapse associated with a fungal infection in her right eye. The eye was enucleated and processed. H&E staining, and MMP-2 and MMP-9 immunostaining were done on the whole cornea and iris prolapse.

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56 Immunoreactivity to MMP-2 specific antibodies was noted in the corneal epithelium, the corneal stroma and in the corneal endothelium. In the corneal epithelium, MMP-2 staining was concentrated in the squamous superficial epithelial cells. The immunohistochemical staining for MMP-2 was markedly increased in the epithelium and the stroma of this cornea in comparison to the staining of a healthy cornea (Figure 2-7). Immunoreactivity to MMP-9 specific antibodies was noted in the different layers of the diseased cornea. The MMP-9 staining was predominant in the squamous superficial epithelial cells (Figure 2-7) of this diseased cornea whereas the immunohistochemical staining for MMP-9 was absent in a healthy cornea (Figures 2-4 and 2-7). No immunoreactivity was found in the tissues on the sections used as controls where the primary antibody was omitted (Figure 2-7). 1 1 2 2 1 2 4 4 4 4 A B C D 1 1 1 2 2 2 2 4 4 3 H F 4 E G 1 1 2 1 2 2 2 3 L I J K 3 3 2 1 2 1 1 3 4 2 2 4 4 O Q M Figure 2-7: Immunolocalization of MMP-2 and MMP-9 in equine ulcerated cornea. H&E staining of the ulcerated equine cornea (A) where corneal epithelium (1), stroma (2) and the iris prolapse (4) can be distinguished. Nomarski phasecontrast 1 observation (B) and bright-field observation of the negative control

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57 sections for MMP-2 (C) and MMP-9 (D) showed no staining at all. Immunohistochemical staining of this ulcerated cornea for MMP-2 (and H) and MMP-9 (M, N, O and Q) showed an markedly increased MMP-2and MMP-9 specific staining in the corneal epithelium, especially in the squamous superficial cell layer (white arrow), and the stroma in its wholethickness (black arrow) and the corneal endothelium (striped arrow), in comparison to the immunohistochemical staining of an healthy cornea foMMP-2 (J, K, and L) and for MMP-9 (I). Original magnifications were X5(A, E, M), X100 ( B, C, D, F, I, J, and N) and X200 (G, H, K, L, O and Q) E, F, G r 0 Discussion This study documents the presence of MMP-2 and MMP-9 in the horse tear film by comparison to human MMP Western Blot standards. These results are in accordance with those from Strubbe et al. who first reported the presence of MMP-2 and MMP-9 in equine tears by gelatin zymography (Strubbe et al., 2000). The present study also shows the antigenic similarity between human and equine MMP-2 and MMP-9, and therefore demonstrates the feasibility of localizing MMP-2 and MMP-9 proteins in other horse ocular tissues using human MMP-2 and MMP-9 antibodies. Proteinases can be produced by the corneal cells and the inflammatory cells such as PMNs, macrophages (endogenous proteinases), and infectious organisms (exogenous proteinases) (Hibbetts et al., 1999). Extra-cellular enzymes of bacterial or fungal origin contribute to the corneal condition, either directly or indirectly, through the activation of endogenous proteinases (Gopinathan et al., 2000; Twining et al., 1993). It seems therefore primordial to investigate the possible origins of the proteinases in order to first understand their physiopathological importance in corneal wound and corneal wound healing, and then to intent to regulate the proteolytic activity. In the present study we identified MMP-2 in the lacrimal gland and the glands of the nictitating membrane. Both are probably a source of MMP-2 in equine tear film. The lacrimal acini and cells of the inter-acinar ducts appear involved in the process of MMP-2

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58 release into horse tear fluid. The exact mechanism of this MMP-2 release is yet to be clarified. In this study we were not able to detect the presence of MMP-9 in the lacrimal gland and the glands of the nictitating membrane, suggesting that these glands do not seem to be a source of MMP-9 in horses with healthy eyes (Figures 2-5, 2-6). MMP-2 was identified in the healthy equine cornea at various locations: mainly in the coea reliminary study, only one clinical case of a 2 year old Thoroughbred with iris pr ervations made in this study on the localization of MMP-2 and MMP-9 in healthnd at r et rneal epithelium, but especially in the squamous superficial cell layer; a small amount in the stroma underneath the epithelium (anterior stroma); and some in the corneal endothelium (Figure 2-4). MMP-9 was not found in the healthy equine corn(Figure 2-4). In this p olapse and associated with fungal infection was examined. Immunohistochemical staining of this diseased cornea for MMP-2 and MMP-9 showed an increased MMP-2 and MMP-9 specific staining in the corneal epithelium, especially in the squamous superficial cell layer, the stroma in its whole thickness, and the corneal endothelium(Figure 2-7). The obs y corneas and in one diseased equine cornea correlate with those obtained in other species. In a study on rats with healthy and wounded corneas, MMP-2 was constitutively present in the unwounded corneal epithelium and stroma, and was upregulated after wounding while MMP-9 was found only in the wounded rat cornea. MMP-9 was fouthe level of the basal epithelial cells as well as in the anterior stroma of the wounded corneas (Reviglio et al., 2003; Ye and Azar, 1998). In a study of non-healing corneal ulcer in dogs, MMP-2 and MMP-9 were found at the level of the epithelium (Chandle

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59 al., 2003). In one case report of a man with corneal perforation, MMP-9 was found at the level of the basal epithelial cells as well as in the anterior stroma (Gabison et al., 2003). In another study performed on human corneas, MMP-2 was found in the epithelium of the normal cornea only, upregulated in pathological corneas, but MMP-9 was not foundin normal corneas (Kenney et al., 1997). Yang et al. performed immunohistochemistry inmice corneas and observed the presence of MMP-2 in the corneal epithelium (squamous and basal cells), and the presence of MMP-9 in the corneal epithelium and stroma in the normal corneas (Yang et al., 2003). They also observed that MMP-2 was upregulated andpresent in the corneal epithelium, stroma and endothelium in the pathological corneas. MMP-9 was also upregulated, mainly present in the corneal stroma and epithelium (Yanet al., 2003). MMP-2 a g nd MMP-9 (gelatinases A and B) are of major importance in terms of remodact 1998; utrophils eling and degradation of the corneal stromal collagen in horses as described in other species (Fini and Girard, 1990; Fini and Girard, 1992). This study supports the fthat the origin and purpose of MMP-2 and appear to differ at the corneal level. MMP-2 is synthesized by corneal keratocytes and performs a surveillance function in the normal cornea, becoming locally activated to degrade collagen molecules that occasionally become damaged as a result of normal wear and tear. (Azar et al., Mastubara et al., 1991a; Twining, 1994; Wall et al., 2002; Ye and Azar, 1998). Alternatively, MMP-9 is produced by epithelial cells and polymorphonuclear ne(PMNs) following corneal wounding. (Fini and Girard, 1990; Mastubara et al., 1991a; Ye and Azar, 1998)

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60 It is actually recognized that stromal ulceration does not occur until after the epithelial basement membrane disappears. It is the controlling step leading to stromal ulceration (Fini and Girard, 1990; Fini and Girard, 1992; Mastubara et al., 1991a). Gelatinase B (or MMP-9) plays an important role as it is able to destroys the adhesive structure of the epithelial basement membrane (type VII and IV collagens, laminin, proteoglycans) leading to stromal ulceration, and which delays the re-epithelialization of the injured cornea (Fini and Girard, 1992; Fini, 1998; Kenney et al., 1994; Ye and Azar, 1998). This preliminary study reveals the importance of proteinases in corneal wound and corneal wound healing in horses, and stresses the important need of further investigations in order to understand the unique pathophysiology of the horse cornea.

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CHAPTER 3 EVALUATION OF MMP-2 AND MMP-9 ACTIVITY IN THE EQUINE TEAR FLUID Introduction The Ophthalmology Service of the University of Florida Veterinary Medical Teaching Hospital diagnosed ulcerative keratitis in 527 horses between January 1987 and October 2002. This represented 35% of all horses evaluated for ophthalmic problems during this period. Ulcerative keratitis is a common and often vision-threatening condition in horses. Most superficial, non-infected ulcers in horses heal quickly and without complication. However, stromal degradation in deep or infected ulcers can rapidly and dramatically progress to corneal perforation in horses within 24 hours (Brooks, 1999). The cornea of horses manifests the most severe degree of ulcer-associated stromal collagenolysis seen in animals (Brooks, 1999). This rapid degradation of the corneal stroma in horses with corneal ulcers appears to be caused by various proteolytic enzymes acting on the collagen, proteoglycans, and other components of the stromal ECM. Microorganisms, inflammatory cells, corneal epithelial cells, and fibroblasts all produce and release proteolytic enzymes (Hibbetts et al., 1999; Matsubara et al., 1991a; Matsubara et al., 1991b; Twining et al., 1993). Excessive levels of proteinases can create an imbalance between proteinases and proteinase inhibitor levels, therefore causing pathological degradation of corneal stromal collagen and ECM proteoglycans (Geerling et al., 1999; Matsubara et al., 1991b; Slansky et al., 1969; Strubbe et al., 2000; Twining, 1994). 61

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62 The tear film proteinases have been previously evaluated in both the normal and diseased eyes of animals and humans (Berman et al., 1971; Berman et al., 1973; Berman et al., 1977; Brooks 1999; Fini and Girard, 1990; Matsubara et al., 1991a; Matsumoto et al., 1993; Prause, 1983a; Prause, 1983b; Strubbe et al, 2000; Tervo et al., 1988; Tervo et al., 1991). Proteinase activities in the tear film were significantly increased in severely damaged corneas (Berman et al., 1971; Kernacki et al., 1995; Matsubara et al., 1991a; Prause, 1983a; Strubbe et al., 2000; Tervo et al., 1988). Among the various classes of proteases found in the tear film and cornea of humans and animals, two are thought to play a major role in the corneal metabolism: the matrix metalloproteinases (MMPs) and the serine proteinases (including neutrophil elastase) (Fini and Girard, 1992; Fini et al., 1998; Matsubara et al., 1991a). Two of the 13 known MMPs are of major importance in terms of corneal stromal collagen remodeling and degradation, and are the focus of this study. They include MMP-2 (72-kDa gelatinase A) and MMP-9 (92-kDa gelatinase B) (Fini and Girard, 1990; Fini and Girard, 1992). Tear film levels of MMP-2, MMP-9 and neutrophil elastase are significantly elevated in ulcerated horse corneas, when compared to age-matched normal controls (Strubbe et al., 2000), and are hypothesized to contribute to the breakdown of stromal collagen. In this preliminary work the tears were collected by using glass capillary tubes. This tear collection method allows the recording of the time needed for collection and therefore the measurement of the tear fluid flow (TFF). The TFF has been measured in various studies performed in man (Tervo et al., 1994; van Setten et al., 1989; van Setten et al, 1990; Vesaluoma et al., 1996; Vesaluoma et al., 1997;Vesaluoma and Tervo, 1998) but it has not been measured yet in animals and especially in horses.

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63 An understanding of the pathophysiologic processes as well as early diagnosis and aggressive treatment are important to speed healing, reduce scarring, and prevent corneal rupture in case of ulcerative corneal diseases in horses. Despite the abundant literature on ocular proteinases, and despite the prevalence of equine ulcerative keratitis, there is very limited information on tear film proteinases in the horse. MMPs are certainly involved in corneal ulceration and corneal wound healing in horses as Strubbe et al. reported with their preliminary report (Strubbe et al., 2000) and their importance needs to be further assessed. Identification of the proteinases mainly responsible for corneal stromal degradation would be the first logical step to allow specific inhibition. Hence, with this study, we attempted to more precisely assess the proteolytic activity level due to MMP-2 and MMP-9 in the tear fluid of horses with healthy eyes and in horses with ulcerative keratitis by two means. We first collected and analyzed a large number of equine tear fluid specimens for the measurement of the MMP-2 and MMP-9 levels. We then determined for the first time the tear fluid flow (TFF) in horses by using the same microcapillary tear collection method. We compared the TFF rates between the healthy eyes and the ulcerated eyes in horses, and quantified the excessive tearing that was associated with corneal ulceration in horses. This calculation allowed us to take the tear dilution factor into consideration while measuring the proteolytic activity in the tear fluid of horses with healthy eyes and in horses with ulcerative keratitis. Materials and Methods Animals All procedures were carried out according to the ARVO statement for the Use of Animals in Ophthalmic and Vision research and were approved by the University of

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64 Florida Animal Care and Use Committee as well as the UF College of Veterinary Medicine Clinical Research Review Committee. Tear film MMP activity (for MMP-2 and MMP-9) was measured by quantitative gelatin zymography in horses that were either clinical cases presented to the Ophthalmology Service of the University of Florida Veterinary Medicine Teaching Hospital during 2002 or animals without any ocular disease, from a farm in Florida. A total of 575 samples of tear fluids from about 200 horses were collected between July 2000 and March 2003. Samples were used to develop and standardize the tear fluid collection and tear fluid analysis procedures. A total of 103 horses were specifically involved in this part of the study. Tear fluid was collected from both eyes of these horses (Tables 3-1 and 3-2); 65 horses had two healthy eyes (130 tear samples), and 38 horses presented with unilateral ulcerative keratitis (38 tear samples from ulcerated eyes and 38 tear samples from the contralateral normal eyes). The horses presented with ulcerative keratitis had various diagnoses including bacterial or fungal keratitis, sterile ulcerative keratitis and ulcerative keratitis of unknown etiologic origin, all identified by a fluorescein stain uptake (Figure 3-1).

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65 Table 3-1: Information regarding the horses with healthy eyes involved in the determination of MMP-2 and MMP-9 in the equine tear fluid. Horses with healthy eyes 65 animals Tear fluid samples Timed samples 82 (41 OD 41 OS) Samples not timed 48 (24 OD 24 OS) Total number 130 (65 OD 65 OS) Sex Mare 57 Stallion 3 Gelding 5 Age Mean +/SD 11.6 +/5.2 years 5 yo and younger 8 6 9 yo 19 10 13 yo 16 14 17 yo 12 18 yo and older 10 Breed TB 59 QH 3 Pony 2 TWH 1 OD= right eye, OS= left eye; TB= Thoroughbred, QH= American Quarter Horse, TWH=Tennessee Walking Horse. Table 3-2: Information regarding the horses with ulcerative keratitis involved in the determination of MMP-2 and MMP-9 in the equine tear fluid. Horses with ulcerative keratitis 38 animals Tear fluid samples Timed samples 50 (26 DIS 24 CN) Samples not timed 26 (13 DIS 13 CN) Total 76 (39 DIS 37 CN) Sex Mare 20 Stallion 11 Gelding 9 Age Mean +/SD 8.42 +/8 years Breed TB 24 QH 16 Arab 1 Pinto 1 Microbiology results (bacterial or fungal growth) Negative 24 Positive 14 including 4 bacterial and 10 fungal DIS = ulcerated, diseased eye, CN= contralateral normal eye; TB= Thoroughbred, QH= American Quarter Horse

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66 A B Figure 3-1: Fungal ulcer in the left eye of a 13 year old American Quarter horse. This case was involved in this study. Note the presence of epiphora, hyperemic conjunctiva, and a 6-7 mm fungal corneal ulcer surrounded with a corneal edema and corneal neovascularisation (A). The corneal ulcer is confirmed by a fluorescein stain uptake (B). Collection of Tear Fluid Samples The tear collections were performed after sedation of the animal (xylazine, 0.5 mg/kg IV), akinesia of the upper lid (lidocaine, 2ml SC), and prior to any diagnostic procedure or treatment. Thorough ophthalmic examinations with slit-lamp biomicroscopy, ophthalmoloscopy and photography were performed immediately after the tear collection in order to classify the tear fluid samples in 3 categories: samples from healthy eyes (Normal), samples from ulcerated eyes (Diseased), and samples from contralateral healthy eyes (Contralateral Normal). Eyes with ulcerative keratitis were identified by positive results for retention of fluorescein dye. All tear fluid specimens were collected from the lower fornix by capillary force using 20 L glass capillary tubes with an atraumatic tip as previously described (Figure 32) (van Setten et al., 1989). Briefly, to avoid trauma to the conjunctiva the capillary tip was blunted with a flame and carefully checked. The capillary was held gently in place, touching the tear fluid meniscus, avoiding any contact with the eyelid skin (van Setten et al., 1989). We attempted to collect tears with a minimal component due to the stimulation of the tear

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67 collection. Sample collection is almost not possible without any stimulation but we aimed to reduce the stimulating factors (light, air movement, mechanical irritation, volume collected or simply stress) as much as possible. All samples were immediately centrifuged, transferred into Eppendorf polypropylene microcentrifuge tubes (Brinkmann Instruments Inc., Westbury, NY), and stored at -80C until analysis. Figure 3-2: Tear fluid collection in horses by glass capillary tube. The tear collections were performed after sedation of the animal, akinesia of the upper lid, and prior to any diagnostic procedure or treatment. Tear fluid specimens were collected from the lower fornix by capillary force using capillary tubes with an atraumatic tip as the collection should be rapid and not irritating for the ocular tissues. Determination of the Tear Fluid Flow (TFF) and Determination of the Release of Proteolytic Activity For 41 horses with healthy eyes and for 25 horses with ulcerative keratitis (Tables 3-1 and 3-2), the tear volume and the tear collection time were measured. The volume of the tear fluid sample was divided by the tear collection time to yield the TFF (l/s) in the collection glass capillary tube as described earlier (Tervo et al., 1994; van Setten et al., 1989; van Setten et al, 1990; Vesaluoma et al., 1996; Vesaluoma et al., 1997;Vesaluoma and Tervo, 1998). This parameter was used to compare the tear fluid secretion rates between healthy, diseased and contralateral normal equine eyes.

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68 The release of proteolytic activity was calculated by multiplying the global MMP activity in the tear fluid sample by the TFF in the collection capillary. As corneal ulceration often induced a remarkable hypersecretion of tears, the use of the parameter release (flow corrected proteolytic activity or activity/time) enabled the comparison between healthy, diseased and contralateral normal equine eyes. Even though the microcapillary tear collection method was not quite accurate, it offered a practical means to avoid misinterpretations due to the variable TFF rates, which have caused significant dilution effect on the proteolytic activity in the tear fluid. The superiority of this collection method to others techniques such as Schirmer tests strips and the possible sources of errors of this method have been addressed by other research teams (Berta, 1983; Tervo et al., 1991; Tervo et al., 1994; van Setten et al., 1989; van Setten et al, 1990; Vesaluoma et al., 1996; Vesaluoma et al., 1997;Vesaluoma and Tervo, 1998) and will be discussed later. MMP Activity Determination by Gelatin Zymography Gelatin zymography and measurement of optical density (see Image analysis) were used to evaluate the MMP activity in the tear fluids. Ten L of the tear fluid samples were mixed with an equal volume of Novex Tris-glycine SDS native sample buffer (Invitrogen, Carlsbad, CA). Fifteen L of the mixture were loaded into wells of pre-cast 10% Novex zymogram gelatin gels ( Invitrogen, Carlsbad, CA). Pre-stained molecular weight standards (See Blue pre-stained standards, Invitrogen, Carlsbad, CA) and gelatinases zymography standards for human active and latent forms of MMP-9 and MMP-2 (Oncogen, Boston, MA) were also run on each gel. Gels were electrophoresed at a constant voltage of 125 volts for approximately 2 hours.

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69 Following electrophoresis, the gels were rinsed in distilled water and then gently shaken in a renaturing solution of 2.7% Triton X-100 (Novex zymogram renaturing buffer, Invitrogen, Carlsbad, CA) for 1 hour at 37C to reactivate MMP activities. The gels were then incubated on a rotary shaker in a developing buffer (Novex zymogram developing buffer, Invitrogen, Carlsbad, CA) for 24 hours at 37C to allow the renatured MMPs to digest the gelatin substrate. After the digestion phase, the gels were rinsed and stained by incubation with Coomassie blue Rapid stain (Diversified Biotech, Boston, MA) for 1 hour. Gels were then destained with a 5% acetic acid 7.5 % methanol solution to maximize contrast between lytic areas and non-digested areas. Bands of proteolytic activity appeared uncolored against a dark blue background. The identity of the putative proteases was determined by analysis of the distance that the bands migrated on the gels, compared with the distance for migration of molecular weight and protease standards (Figure 3-3). Image Analysis The levels of proand active-MMP-2, and MMP-9 were measured by optical density scanning of gelatin zymograms of the tears samples. Digital photographs of stained gelatin zymograms were created with a GS-710 imaging densitometer (Bio-Rad, Hercules, CA) and analyzed with the Quantity One quantification software, 4.2.1 beta version (Bio-Rad, Hercules, CA). The four bands present in each lane of the gels were automatically detected by the analyzer after adjusting its sensitivity for each gel. The background was then subtracted for each lane. The image analysis produced an intensity profile curve of each lane with the optical density (OD) function of the distance of migration from the top of the gel (relative front). The higher the optical density, the lower

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70 level of staining, the more gelatin substrate was digested, and the higher the level of proteolytic activity. The area under the curve (AUC) that corresponded to the OD by the width of the band (in mm) was calculated for the four bands of interest, respectively proand active-MMP-2, and MMP-9. The parameters used for comparisons in our study were the AUC which allowed an accurate estimation of proteolytic activity, given in relative standard units (RSU) present in each samples (Figure 3-3, Figure 5-1). Figure 3-3: Gelatin zymography of equine tear fluid samples. Gelatin zymography and measurement of optical density were used to evaluate the MMPs activity in the tear fluids. This image of the gelatin zymogram gel shows tear fluid samples for the ulcerated (DIS, lane 4) and the contralateral (CN, lane 3) eyes of one horse and the tear fluid samples from the two healthy eyes (N OD, lane 5 and N OS, lane 6) of another horse. Human standards of the active forms of MMP-9 (bracket 2, lane 1), MMP-2 (bracket 4, lane 1) and the latent forms of MMP-9 (bracket 1, lane 2), MMP-2 (bracket 3, lane 2) as well as the molecular weight markers (MWM) were loaded on each gel (left hand side of the gel). The red line on the tear fluid samples shows the bracket of the 4 detected bands. According to their location on the gel (based on their molecular weight), the proteases could be identified, compared to the molecular weight markers and the standards. The higher the amount of proteolytic activity, the more gelatin substrate was digested, which was reflected by a concomitant decrease in the amount of 2 4 1 3 1 3 4 2 1 2 3 4 210 M W M 98 64 50 5 6 N OD N OS MMP standard s Active Latent CN DIS

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71 stain. The zymogram gels were then scanned and the levels of each protease (or band) detected on the gel determined by measuring the optical density, transformed to a relative standard unit (RSU).The level of proteolytic activity for each of the protease detected is increased in the tear fluid samples from the diseased and the contralateral eyes in compared to the level in the healthy eyes. Statistical Analysis The ratios of the AUC of the band detected in the sample divided by the AUC of the respective standard loaded on the same gel were calculated for the activity of each individual protease (or band) detected as well as for the global proteolytic activity (sum of all the 4 bands detected in a lane). All analyses were run using the SAS software (SAS Institute Inc., Cary, NC) and the tests were considered statistically significant if the p-value was less than 0.05. The TFF as well as the ratios obtained for healthy right eyes (Normal OD) and healthy left eyes (Normal OS) were compared by T-test analysis (SAS Proc TTEST). Multiple linear regression analysis (SAS Proc GLM) was used to compare ratios obtained for healthy eyes of various breeds, sex categories and age classes. Ratios obtained for healthy eyes (N), healthy contralateral eyes (CN), and ulcerated eyes (DIS) were compared by multiple linear regression analysis. The TFF as well as the release of proteolytic activity were also compared by multiple linear regression analysis (SAS Proc GLM). Results Determination of MMP-2 and MMP-9 Activity in the Equine Tear Fluid of Horses with Healthy Eyes There was no significant difference in the global proteolytic activity (sum of the activity for the active and latent forms of MMP-2 and MMP-9) between the right eye (N

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72 OD) and the left eye (N OS) (n=65, p=0.0706) as well as for each individual protease as illustrated in Table 3-3. Therefore, the values for the right and the left healthy eyes were added to form one group of healthy eyes (Normal, n=130), and that was used in the further analyses. Table 3-3: Proteolytic activity (Mean +/SD) in the tear fluid of horses with healthy eyes, in relative standard unit (RSU) Eye Global MMP activity Latent MMP-9 Active MMP-9 Latent MMP-2 Active MMP-2 Normal OD (n=65) 0.69 .51 2.22 .89 0.47 .53 1.16 .08 0.07 .12 Normal OS (n=65) 0.56 .42 1.83 .50 0.39 .51 1.01 .57 0.05 .07 Normal (n=130) 0.62 .47 2.02 .71 0.43 .52 1.09 .84 0.06 .10 There was no significant difference in the global proteolytic activity between the breeds (p=0.4465) and the sex categories (p=0.1543) of the horses with healthy eyes included in this study as illustrated in Figures 3-4 and 3-5, respectively. It is important to note that the power to detect a difference was limited by the types of breeds and ages of the horses available (Table 3-1, Tennessee Walking Horse n=1; stallion n=3). 00.20.40.60.811.21.4Pony (n = 2)QH (n = 3)TB (n = 59)TW (n = 1)Global MMP activity (RSU ) Figure 3-4: Global MMP activity in the tear fluid of horses with healthy eyes by breed. TB= Thoroughbred, QH= American Quarter Horse, TW=Tennessee Walking Horse

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73 00.20.40.60.811.21.41.6Female (n = 57)Gelding (n = 5)Stallion (n = 3)Global MMP activity (RSU Figure 3-5: Global MMP activity in the tear fluid of horses with healthy eyes by sex. There was no significant difference in the global proteolytic activity between age categories except for the two extreme age categories: the global proteolytic activity was significantly lower in 5 yo or younger horses than in 18 yo or older horses as illustrated in Figure 3-6. However, no correlation between the age of the animal and the proteolytic activity in the tear fluid was found (the Pearson coefficient was 0.074). There was no trend to an increase of activity with age (p=0.069). 00.20.40.60.811.21.41.6 <5yo (n = 8)6-9 yo (n = 19)10-13 yo (n = 16)14-17 yo (n = 12)18 yo < (n = 10)Global MMP activity (RSU ) ** Figure 3-6: Global MMP activity in the tear fluid of horses with healthy eyes by age category. The only significant difference in proteolytic activity between the age groups (P < 0.001).

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74 Determination of MMP-2 and MMP-9 Activity in the Equine Tear Fluid of Horses Ulcerative Keratitis The global proteolytic activity in the tear fluid from diseased eyes was significantly higher than from normal eyes (p<0.0001) and contralateral eyes (p<0.0001). The global proteolytic activity in the tear fluid from the contralateral eyes was higher than from normal eyes but the difference was not significant (p=0.1908) as illustrated in Table 3-4 and Figure 3-7. Similar results were obtained for each individual protease (p<0.0001) (Table 3-4 and Figure 3-8).The proteolytic activity in the equine tear film is mainly due to the latent forms of MMP-9 and MMP-2 (Table 3-4 and Figure 3-8). Table 3-4: Proteolytic activity in the tear fluid of healthy, ulcerated and contralateral normal horse eyes. Eye status Global MMP activity Latent MMP-9 Active MMP-9 Latent MMP-2 Active MMP-2 Normal (n=130) 0.62 .47 2.02 .71 0.43 .52 1.09 1.84 0.06 0.10 CN (n=37) 0.79 .67 2.29 .19 0.51 0.91 1.89 2.48 0.17 0.52 Diseased (n=39) 1.92 .06* 6.30 .49* 1.05 .86* 9.30 .57* 0.71 .97* Proteolytic activity (Mean +/SD) in the tear fluid of healthy (Normal), ulcerated and contralateral normal (CN) eyes, in relative standard unit (RSU). The proteolytic activity in the equine tear film is mainly due to the latent forms of MMP-2 and MMP-9. Proteolytic activity in the tear fluid from diseased eyes differed significantly (P < 0.001) from the proteolytic activity in the tear fluid from normal eyes.

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75 00.511.522.533.5Normal (n = 130)CN (n = 37)Diseased (n = 39)Global MMPs activity (RSU ) Figure 3-7: Global MMP activity in the tear fluid of horses with healthy eyes and ulcerative keratitis. Proteolytic activity (Mean +/SD) in the tear fluid of healthy (Normal), ulcerated (Diseased) and contralateral normal (CN) eyes, in relative standard unit (RSU). Proteolytic activity in the tear fluid from diseased eyes differed significantly (P < 0.001) from the proteolytic activity in the tear fluid from normal eyes. 012345678910Latent MMP-9Active MMP-9Latent MMP-2Active MMP-2MMP activity by type (RSU Normal (n = 130) CN (n = 37) Diseased (n = 39)**** Figure 3-8: Proteolytic activity in the tear fluid of horses with healthy eyes and ulcerative keratitis by MMP type. Proteolytic activity by type of MMP (Mean +/SD) in the tear fluid of healthy

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76 (Normal), ulcerated and contralateral normal (CN) eyes, in relative standard unit (RSU). The proteolytic activity in the equine tear film is mainly due to the latent forms of MMP-2 and MMP-9. *Proteolytic activity for each protease in the tear fluid from diseased eyes differed significantly (P < 0.001) from the proteolytic activity for each protease in the tear fluid from normal eyes. Determination of TFF in Horses with Healthy Eyes and Horses with Ulcerative Keratitis There was no significant difference in the tear fluid flow (TFF) between the right eye (N OD) and the left eye (N OS) (p=0.627) in the 41 healthy horses where the time needed for the tear collection was recorded, and the TFF calculated as illustrated in Table 3-5. Therefore, the TFF values for the right and the left healthy eyes were added to form one group of healthy eyes (Normal, n=82) and that was used in the further analyses. The TFF in diseased eyes was significantly higher than in normal eyes (p<0.0001) and contralateral eyes (p<0.0001) in the 25 horses with ulcerative keratitis where the time needed for the tear collection was recorded, and the TFF calculated as illustrated in Table 3-5. The TFF in the contralateral eyes was higher in normal eyes, but the difference was not significant (p=0.4562) as illustrated in Table 3-5. Table 3-5: TFF in healthy, ulcerated and contralateral normal horse eyes. Eye status TFF (L/s) (Mean +/SD) Normal OD (n = 41) 1.79 +/1.81 Normal OS (n = 41) 1.40 +/0.99 Normal (n = 82) 1.59 +/1.46 CN (n = 24) 2.25 +/2.37 Diseased (n = 26) 5.17 +/4.09 TFF (Mean +/SD) in healthy (Normal), ulcerated (Diseased), and contralateral normal (CN) eyes, in L per second. TFF in diseased eyes differed significantly (P < 0.001) from TFF in normal eyes.

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77 Determination of The Release of Proteolytic Activity in the Equine Tear Fluid of Horses with Healthy Eyes and Horses with Ulcerative Keratitis The figure 3-9 reports the global MMP activity in the tear fluid in parallel to the TFF and this illustrates that the proteolytic activity is maintained high in the tear fluid of the diseased eyes in spite of a high dilution factor due to enhanced TFF. 012345678910Normal (n = 82)CN (n = 24)Diseased (n = 26)TFF (uL/s) or Global proteolytic activity (RSU) Total MMPs activity TFF*** Figure 3-9: TFF and global MMP activity in the tear fluid of horses with healthy eyes and ulcerative keratitis. Global proteolytic activity (Mean +/SD) in the tear fluid of healthy (Normal), ulcerated (Diseased) and contralateral normal (CN) eyes, in relative standard unit (RSU) as well as the TFF in L/s. The proteolytic activity is maintained high in the tear fluid of the diseased eyes in spite of a high dilution factor due to enhanced TFF. Proteolytic activity in the tear fluid from diseased eyes differed significantly (P < 0.001) from the proteolytic activity in the tear fluid from normal eyes. ** TFF in diseased eyes differed significantly (P < 0.001) from TFF in normal eyes. The global proteolytic activity released (global MMP activity multiplied by the TFF) in the tear fluid from diseased eyes was significantly higher than from normal eyes (p<0.0001) and contralateral eyes (p<0.0001). The total proteolytic activity released in the tear fluid from the contralateral eyes was higher than from normal eyes, but the difference was not significant (p=0.6056) as illustrated in Table 3-6.

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78 Table 3-6: Proteolytic activity released in the tear fluid of equine healthy, ulcerated and contralateral normal eyes. Eye status Mean +/SD Normal (n = 82) 0.77 +/0.89 CN (n = 24) 1.39 +/1.73 Diseased (n = 26) 10.52 +/11.85* Global MMP activity released per second (Mean +/SD) in the tear fluid healthy (Normal), ulcerated (Diseased), and contralateral normal (CN) horse eyes, in RSU per second. The activity released in diseased eyes differed significantly (P < 0.001) from the release in normal eyes. Discussion A total of 103 horses were involved in this study: 65 horses had healthy eyes and 38 horses had unilateral ulcerative keratitis. The horses presented with ulcerative keratitis had various diagnoses including bacterial or fungal keratitis, sterile ulcerative keratitis and ulcerative keratitis of unknown etiologic origin, all identified by a fluorescein stain uptake. In this study, we did not take into consideration the etiology in the measurement of the proteolytic activity in the tears; the tear fluid samples were only split into 3 categories: tears from healthy eyes, tears from ulcerated eyes and those from the contralateral healthy eyes. The proteinases present in the tear film can be host-derived and microbial. The importance of the etiology of the ulcerative keratitis on the level of proteolytic activity in the tear film remains to be investigated. Several of these animals had not been treated prior to their first visit at the University of Florida Veterinary Medical Teaching Hospital, but most of them had received ineffective therapy prior to referral to our hospital. Therefore, they did not respond to the treatment and the ulcerative keratitis was considered still active. For this

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79 reason, we did not differentiate those horses that had received medical therapy and those that did not prior to the tear collection. We would like to stress the importance of a proper collection method for investigation of the tear film proteolytic activity in horses. The method of tear collection has been reported to affect the composition of tear fluid obtained (Berta, 1983; Jones et al., 1997; Stuchell et al., 1984; van Haeringen and Glasius, 1976; van Setten et al., 1990). An ideal method of tear collection would permit rapid collection with no ocular irritation, thus permitting differentiation between basal and stimulated reflex tear proteinases level; allow efficient recovery of the tear proteins (and proteinases); and be readily applied in a clinical setting. Conventional methods for tear collection include filter paper or Schirmer test strips, cellulose sponges, porous polyester rods and glass capillary micropipettes (Jones et al., 1997). Tear collection with filter paper or Schirmer test strips is far from ideal as it has been shown that their use leads to activation of proteolytic systems in the tear film (van Haeringen and Glasius, 1976; van Setten et al., 1990). It seems most likely that some micro trauma to the conjunctival and corneal epithelia is caused by the mechanical irritation of the strips that is sufficient to activate or liberate proteinases (Stuchell et al., 1984; van Haeringen and Glasius, 1976). Furthermore, the TFF can be measured by filter paper or Schirmer test strips but this method does not provide an estimation of the actual TFF, since the insertion of the filter paper into the cul-de-sac stimulates reflex lacrimation (Mishima et al., 1966; Stuchell et al., 1984). Similar observations have been reported with the use of cellulose sponges to collect tears (van Agtmaal et al., 1987).

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80 Compared to other methods, the use of blunted capillary tubes to collect tear fluids has been shown to cause less irritation and cellular disruption. Similarly, the use of porous polyester rods has been shown to collect tears atraumatically with minimal ocular irritation (Jones et al., 1997). Even if the use of capillary tubes to collect tears seems a bit awkward in a clinical setting, it ensures a high protein recovery (Jones et al., 1997). The microcapillary tear collection method has been used in various clinical and experimental studies performed in man and animals (Tervo et al., 1991; Tervo et al., 1994; van Setten et al., 1989; van Setten et al., 1990; Vesaluoma et al., 1996; Vesaluoma et al., 1997; Vesaluoma and Tervo, 1998). It seems to be the method of choice to collect tear fluid sample and investigate the tear film proteolytic activity (Tervo et al., 1991; van Haeringen and Glasius, 1976; van Setten et al., 1990). After collecting 575 tear fluid specimens over a 3 year period, we consider that we possess a valid and standardized method to collect tears with 20L blunted glass capillary tubes in horses for the measurement of the equine tear film proteolytic activity. Moreover, this microcapillary tear collection method allows the recording of the time needed for collection and therefore the measurement of the tear fluid flow (TFF) in the capillary. The TFF in the capillary tube has been measured in various studies performed in man and animals (Tervo et al., 1994; van Setten et al., 1990; Vesaluoma et al., 1996; Vesaluoma et al., 1997; Vesaluoma and Tervo, 1998) and served as a relative value of the TFF during the sample collection. For the first time in this study, we reported TFF values for the horse. This study documents that the TFF is increased in the eye of horses with ulcerative keratitis as compared to healthy and contralateral normal eyes. Similar observations have been reported in other species (Tervo et al., 1994; van Setten et

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81 al. 1990; van Setten et al. 1989; Vesaluoma et al., 1996; Vesaluoma et al., 1997; Vesaluoma and Tervo, 1998). Although a rather gross estimate, the capillary tube method seems to be the simplest way to have a reasonable volume correction in determination of the tear fluid proteinases (Tervo et al., 1994; Vesaluoma et al., 1997). This is, to our understanding, necessary in our study because of the clinically evident wide variation in the tear fluid production between healthy and ulcerated eyes. Neglecting the tremendous variations in the TFF rates and giving the proteolytic activity only might be more inaccurate. Since the high TFF caused by reflex tearing in the diseased eye caused a dilution effect, we calculated the release of proteolytic activity by multiplying the global MMP activity by the TFF in the capillary (Tervo et al., 1991; Tervo et al., 1994). The parameter release takes the TFF changes (the dilution factor) into consideration and probably gives a better reflection of the changes in synthesis and release of the proteinases between the different groups studied (healthy, diseased and contralateral normal equine eyes). Despite the inaccuracy of the capillary tube method, we consider it a suitable technique to collect tears for studying the proteolytic activity and the release of proteolytic activity (i.e. activity/time) in the horse tear fluid, once the limitations of the technique are recognized. This study documents the proteolytic activity due to MMP-2 and MMP-9 in the tear film of horses with healthy and ulcerated eyes. We detected some proteolytic activity in the tear fluid of healthy horses and no significant difference in proteolytic activity between right and left was found. These results are in accordance with those from Strubbe et al. who first reported the presence of

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82 MMP-2 and MMP-9 in equine tears by gelatin zymography (Strubbe et al., 2000) by analyzing the tear fluids of 17 healthy horses and 23 horses with unilateral keratitis. It is interesting to note that proteolytic activity was present even in the healthy eyes and similar results have been reported for in humans where some proteases always appear to be present in the tear film (Tervo et al., 1988; van Setten et al., 1990). This support the concept that proteases and in this particular case MMP-2 and MMP-9 are involved in the corneal physiology such as the normal maintenance of the corneal ECM (Fini and Girard, 1992; Twining, 1994). We did not observed any significant difference in the proteolytic activity level of healthy horses between the breeds as well as between the sex categories. The power of the statistical analysis was certainly low as some categories had very low numbers (Table 3-1) but this study was mainly designed to look at the difference between healthy and ulcerated eyes and we were also limited by the distribution of the horse breeds present in Florida and the ones presented at the VMTH. Even if there was a significant difference that we were not able to detect with the statistical analysis because of its low power, the maximal difference in MMP activity in healthy horses observed between breeds and between sex was 0.45 RSU (Figure 3-4) and this difference was much smaller (approximately 3 folds) in comparison with the difference observed (Table 3-4) between healthy and ulcerated eyes (1.30 RSU). For this particular reason, we considered that it was still valid to group all the healthy animals in one category with out taking into account eye side, breed and sex for the comparison horse with ulcerative keratitis. We also noticed a significant difference of activity between the two extreme age classes: the level of proteolytic activity was significantly higher in the healthy old horses (more than 18 years old) than in the healthy young ones (less than 5 years old). However,

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83 we did not find any correlation between age and tear film proteolytic activity. To our knowledge, age-related changes in tear film proteinases have not been evaluated in any species except in horses by Strubbe et al. Strubbe only reported a significantly higher level of MMP-9 in the tear fluid of young horses when compared to horses over 10 years of age (Strubbe et al., 2000). We did not obtain similar results in our study and we unfortunately did not find any studies to explain this phenomenon. In our study, MMP-9 and MMP-2 were present at a significantly higher level in the tear film of horses with ulcerative keratitis versus horses with healthy eyes. These results are similar to findings in other species (Fini and Girard, 1990; Fini et al., 1992; Fini et al., et 1998; Matsubara et al., 1991a), and especially those reported in horses by Strubbe et al. (Strubbe et al., 2000) suggesting that elevation of tear film proteinases is part of a fundamental response of the mammalian eye to corneal injury. We also observed an increased level of proteolytic activity in the contralateral normal eye of affected horses compared to healthy eyes but the difference was not significant in contrast with Strubbes findings. The higher number of animals involved in our study might explain the different results. When the TFF and therefore, the dilution factor, is taken into consideration during the proteolytic activity analysis, we observed the same trends and the differences between the healthy, ulcerated and contralateral normal were even more pronounced. The release of proteolytic activity was significantly higher in the ulcerated eyes compared to healthy, and contralateral normal eyes. The release of proteolytic activity in contralateral normal eyes was also higher than that in the in healthy eyes but not significantly.

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84 This study supports the fact that MMP-2 and MMP-9 (gelatinases A and B) are of major importance in terms of remodeling and degradation of the corneal stromal collagen in horses as described in other species (Fini and Girard, 1990; Fini and Girard, 1992). A particular advantage of the gelatin zymography compared to immunohistochemistry is that both the latent (or proenzyme) and active form of MMPs, which can be distinguished on the basis of the molecular weight, can be detected. This is possible because the proenzymes are activated in situ presumably by the denaturation/renaturation process during the zymography. The latent forms of the MMPs do not have any bioactivity but it is important to quantify them as they can potentially be activated by various factors. Corneal ulcers in horses are associated with initially high levels of tear film proteolytic activity due to MMP-2 and MMP-9 and this study supports the use of anti-proteolytic agents for progressive equine corneal ulcers. Treatment strategies for healing corneal ulcers in horses should be directed towards and reducing tear film concentrations of MMPs. Several anti-proteolytic agents have been proposed as treatments to reduce the activity of tear proteases. Because these compounds use different mechanisms to inhibit various families of proteases in equine tears, a combination of these inhibitors may be indicated for the treatment of severe corneal ulcers in horses (Brooks, 1999; Hibbetts et al., 1999). Objectives for the appropriate use of these compounds early in the course of corneal disease in horses would be to decrease the amount of time required for recovery and rehabilitation, reduce scarring, and potentially alleviate the need for corneal surgical treatment.

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CHAPTER 4 MATRIX METALLOPROTEINASE ACTIVITY PROFILES IN THE EQUINE TEAR FILM DURING CORNEAL HEALING IN 10 HORSES WITH ULCERATIVE KERATITIS Introduction Tissue breakdown occurs with the normal metabolic activity of the cornea. Proteolytic enzymes are important in the slow turnover and remodeling of the normal healthy corneal stroma. The activities of these proteolytic enzymes are normally balanced by inherent proteinase inhibitors in order to prevent excessive degradation of the normal healthy tissue. An imbalance between proteinases and proteinase inhibitor levels due to excessive levels of proteinases can cause pathological degradation of corneal stromal collagen and proteoglycans (Hibbetts et al., 1999; Twining, 1994; Slansky et al., 1969). The tear film proteinases have been previously evaluated in both normal and diseased eyes of animals and man (Berman et al., 1971; Berman et al., 1973; Berman et al., 1977; Brooks 1999; Fini and Girard, 1990; Matsubara et al., 1991a; Matsumoto et al., 1993; Prause, 1983a; Prause, 1983b; Strubbe et al, 2000; Tervo et al., 1988; Tervo et al., 1991). In ulcerated horse corneas, tear film levels of MMP-2, MMP-9 and neutrophil elastase are significantly elevated when compared to age-matched normal controls and are hypothesized to contribute to the breakdown of stromal collagen (Strubbe et al, 2000). Ulcerative keratitis with extensive stromal involvement displays rapid progressive that can lead to corneal perforation in many horse eyes probably due to this upregulated proteolytic activity (Brooks, 1999). 85

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86 Many studies have been performed on the changes in MMP expression in the skin as well as MMP proteolytic activity in the wound fluids during skin wound healing in animals and man (Agren, 1994; Blalock et al., 2001; Ladwig et al., 2002; Lobmann et al., 2002; Herouy, 2001; Parks, 1999; Paul et al., 1997; Wall, 2002). Few studies have reported the proteolytic changes in the tear film during corneal wound healing. Changes in the tear film level of plasmin, a serine proteinase, have been described in animal and man (Barlati et al., 1990; Cejkova, 1998; Cejkova et al., 1993; Salonen et al., 1987; Tervo et al. 1998; Tervo et al., 1989a; Tervo et al, 1989b; Tervo et al. 1991; Vesaluoma et al., 1998; van Setten et al., 1989). The changes in level of various MMPs in the tear film (Barro et al., 1998) as well as their expression of MMP in the cornea during wound healing have also been described in rat and rabbit (Fini and Girard, 1992; Fini et al., 1998; Lu et al., 1999; Matsubara et al., 1991a; Ye et al., 1998). To the authors knowledge, there is only one study on the precise profile of the MMP activity in tear film during corneal healing based on the collection and analysis of serial tear fluid samples in man (Barro et al., 1998). Furthermore, changes in the amount of proteolytic activity in the horse tear films during corneal healing and stromal remodeling have never been reported. Based on the literature and the current knowledge, we hypothesize the tear film MMP activity levels should decrease as the corneal ulcer epithelializes and remodels. In this study, we analyzed tear protease activity during healing of corneal ulcers in horses to test this hypothesis.

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87 Materials and Methods Selection of the Ten Cases All procedures were carried out according to the ARVO statement for the Use of Animals in Ophthalmic and Vision research and were approved by the University of Florida Animal Care and Use Committee as well as the UF College of Veterinary Medicine Clinical Research Review Committee. Tear film MMP activity (for MMP-2 and MMP-9) was followed serially by quantitative gelatin zymography until complete recovery of the ulcerative keratitis in ten horses presented to the Ophthalmology Service of the University of Florida Veterinary Medicine Teaching Hospital during 2002. The horses had various diagnoses including bacterial or fungal keratitis, sterile ulcerative keratitis and ulcerative keratitis of unknown etiologic origin, all identified by a fluorescein stain uptake. Table 4-1 reports the details of the 10 cases involved in this study (Table 4-1). The evolution of the corneal disease in these 10 horses was monitored by ophthalmic examination including slit-lamp biomicroscopy, fluorescein staining and photography. The complete recovery of the ulcerative keratitis was confirmed by negative results for retention of fluorescein dye. Treatment of the ulcerative keratitis in these 10 cases included topical administrations of equine serum, antibiotics, antifungals, atropine, and systemic administration of non-steroidal anti-inflammatory drugs. Surgical treatment was performed on day 2 in addition to medical treatment in 5 cases. The surgical treatments included various techniques such as conjunctival pedicle graft, amniotic membrane transplantation, keratectomy, and penetrating keratoplasty (Table 4-1).

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88 Table 4-1: Information regarding the ten horses with ulcerative keratitis involved in the determination of the MMP activity during corneal wound healing. Case Breed Sex Age (year) Diagnosis Duration of the condition before first visit (days) Microbiology results Medical treatment Surgical treatment Duration of the follow-up (days) 1 TB M 0.5 Melting ulcer OS 3 Negative S AF AB A NS KT + CF 7 2 Arab F 17 Superficial corneal ulcer OS Half thickness stromal corneal ulcer OD 7 Negative S AF AB A NS 9 3 TB F 5 Fungal keratomalacia OS 21 Fungi Pseudomonas S AF AB A NS CS KT + AMT 114 4 TB F 2 Deep corneal ulcer OS 1 Negative S AF AB A NS 22 5 TB F 2 Deep corneal ulcer OD 1 Negative S AF AB A NS 89 6 Paint G 7 Ulcer OD 10 Negative S AF AB A NS 37 7 QH M 5 Melting ulcer OD 1 Negative S AF AB A NS 336 8 TB M 0.1 Melting corneal ulcer OD 2 Negative S AF AB A NS KT + CF 38 9 QH G 14 Fungal corneal ulcer OS 1 Fungi S AF AB A NS PK +CF + AMT 120 10 H M 12 Melting ulcer OS 1 Pseudomonas S AF AB A NS AMT 45 TB= Thoroughbred, QH= American Quarter Horse, H=Hanovarian; M= male, F=female, G=gelding; OD= right eye, OS= left eye; S=serum, AF= antifungal, AB= antibiotic, A=atropine, NS=non steroidal, CS=corticosteroids, CF= conjunctival flap, KT=keratectomy, PK= penetrating keratoplasty, AMT= amniotic membrane transplant Collection of Tear Fluid Samples Samples of tear fluid were obtained from both eyes on the day of admission and at various time points until the complete healing of the cornea. The tear collections were performed after sedation of the animal, akinesia of the upper lid, and prior to any diagnostic procedure or treatment. Thorough ophthalmic examinations with slit lamp biomicroscopy, ophthalmoloscopy and photography were performed immediately after the tear collection. All tear fluid specimens were collected from the lower fornix by capillary force using capillary tubes with an atraumatic tip as previously described (van Setten et al., 1989) The time of collection of each tear fluid sample was not recorded in this study. All samples were immediately centrifuged, transferred into Eppendorf

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89 polypropylene microcentrifuge tubes (Brinkmann Instruments Inc., Westbury, NY), and stored at -80C until analysis. MMP Activity Determination by Gelatin Zymography Gelatin zymography and measurement of optical density (see Image analysis) were used to evaluate the MMP activity in the tear fluids. Ten L of the tear fluid samples were mixed with an equal volume of Novex Tris-glycine SDS native sample buffer (Invitrogen, Carlsbad, CA). Fifteen L of the mixture were loaded into wells of pre-cast 10% Novex zymogram gelatin gels ( Invitrogen, Carlsbad, CA). Pre-stained molecular weight standards (See Blue pre-stained standards, Invitrogen, Carlsbad, CA) and gelatinases zymography standards for human active and latent forms of MMP-9 and MMP-2 (Oncogen, Boston, MA) were also run on each gel. Gels were electrophoresed at a constant voltage of 125 volts for approximately 2 hours. Following electrophoresis, the gels were rinsed in distilled water and then gently shaken in a renaturing solution of 2.7% Triton X-100 (Novex zymogram renaturing buffer, Invitrogen, Carlsbad, CA) for 1 hour at 37C to reactivate MMP activities. The gels were then incubated on a rotary shaker in a developing buffer (Novex zymogram developing buffer, Invitrogen, Carlsbad, CA) for 24 hours at 37C to allow the renatured MMPs to digest the gelatin substrate. After the digestion phase, the gels were rinsed and stained by incubation with Coomassie blue Rapid stain (Diversified Biotech, Boston, MA) for 1 hour. Gels were then destained with a 5% acetic acid 7.5 % methanol solution to maximize contrast between lytic areas and non-digested areas. Bands of proteolytic activity appeared uncolored against a dark blue background. The identity of the putative proteases was determined by analysis of the distance that the

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90 bands migrated on the gels, compared with the distance for migration of molecular weight and protease standards (Figure 4-1). Image Analysis The levels of proand activeMMP-2, and MMP-9 were measured by optical density scanning of gelatin zymograms of the tears samples. Digital photographs of stained gelatin zymograms were created with a GS-710 imaging densitometer (Bio-Rad, Hercules, CA) and analyzed with the Quantity One quantification software, 4.2.1 beta version (Bio-Rad, Hercules, CA). The four bands present in each lane of the gels were automatically detected by the analyzer after adjusting its sensitivity for each gel. The background was then subtracted for each lane triplicate. The image analysis produced an intensity profile curve of each lane with the optical density (OD) function of the distance of migration from the top of the gel (relative front). The higher the optical density, the lower level of staining, the more gelatin substrate was digested, and the higher the level of proteolytic activity. The area under the curve (AUC) that corresponded to the OD by the width of the band (in mm) was calculated for the four bands of interest, respectively proand active-MMP-2, and MMP-9. The parameters used for comparisons in our study were the AUC which allowed an accurate estimation of proteolytic activity, given in relative standard units (RSU) present in each samples (Figure 4-1). The graphs represent the tear film MMP activity profile in RSU in both eyes for each horse function of the time (Figures 4-2 to 4-11). Statistical Analysis The ratios of the AUC of the band detected in the sample divided by the AUC of the respective standard loaded on the same gel were calculated for the activity of each

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91 individual protease (or band) detected as well as for the total proteolytic activity (sum of all the 4 bands detected in a lane). Since the treatments were different between the 10 cases, no statistical comparison was made between the ratios during the corneal wound healing but only for day 2 and the last day when the cornea was completely healed. The ratios obtained for day 2 and for the day of complete healing were compared by T-test analysis. SAS Proc TTEST (SAS Institute Inc., Cary, NC) was used for estimations. In all analyses, the tests were considered statistically significant if the p-value was less than 0.05. Figure 4-1: Gelatin zymogram of tear fluids from case 10. Gelatin zymography and measurement of optical density are used to evaluate the MMP activity in the tear fluids. This image of the gelatin zymogram gel shows tear fluid samples for the ulcerated (DIS) and the contralateral (CN) eyes of the horse #10 collected at different time points from the first day (Day 1, lanes 3 and 4) to the complete corneal healing at Day 45 (lanes 11 and 12). Human standards of the active forms of MMP-9 (bracket 2, lane 1), MMP-2 (bracket 4, lane 1) and the latent forms of MMP-9 (bracket 1, lane 2), MMP-2 (bracket 3, lane 2) as well as the molecular weight markers (MWM) were loaded on each gel (left hand side of the gel). The red line on the tear fluid samples at day 1 (lanes 3 and 4) shows the brackets of the 4 detected bands. According to their location on the gel (based on their molecular weight), the 210 M W M MMP standards Day 38 Day 45 Day 1 Day 9 Day 23 Active Latent 98 64 50 2 4 1 3 1 3 4 2 1 2 3 4 CN DIS DIS CN CN DIS 5 6 7 8 9 10 DIS CN 11 12 CN DIS

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92 proteases could be identified. The zymogram gels are then scanned and the levels of each protease (or band) detected on the gel determined by measuring the optical density, transformed to a relative standard unit (RSU), and the values for each eye and time point reported in figures 2 to 11. Results Total tear film MMP activity decreased as corneal healing progressed compared to levels measured on Day 1 (Figures 4-2 to 4-11). The general trend was a decrease. However, different patterns of tear film MMP activity were observed. There was an increase in proteolytic activity after keratectomy (case 3, Figure 4-4) and penetrating keratoplasty (case 9, Figure 4-10) but not after amniotic membrane transplant or conjunctival graft surgery (cases 1, 8 and 10, Figure 4-2, 4-9 and 4-11 respectively). Table 4-2 reports the level of MMP activity in RSU for both eyes of each animal as well as the mean and standard deviation (SD) for the group of 10 horses at Day 1 and at day of complete corneal healing. The mean of the total MMP activity (SD) measured in the tear fluid of the ulcerated eye (2.44 .44) of the 10 horses was significantly higher than the one in the contralateral eye (0.81 0.68) (p=0.006) on the first day of admission to the VMTH. The mean MMP activity in these ulcerated eyes significantly decreased (-82.4%) between the first of admission and the day when the ulcer was completely healed (p=0.0002). The level activity in the healed eye (0.43 0.17) was not significantly different to the one in the contralateral eye (0.36 0.18) at the day of complete corneal healing (p=0.374). The level of MMP activity in the contralateral eye decreased from 0.81 0.68 to 0.36 0.18, although this 56.0% decrease was not significant (p=0.069) (Table 4-2).

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93 Table 4-2: Level of total MMP activity in relative standard units (RSU) at day 2 and at the day of complete corneal healing in both eyes of 10 horses. Case Eye Eye status Total MMP activity (RSU) at Day 2 Total MMP activity (RSU) at Day of complete healing % change in Total MMP activity 1 OS D 0.83 0.52 37.7 OD CN 0.7 0.43 38.0 2 OS D 0.59 0.12 80.0 OD D 0.47 0.23 51.6 3 OD CN 0.21 0.34 + 63.3 OS D 4.26 0.41 90.3 4 OD CN 0.44 0.68 + 55.2 OS D 2.53 0.33 86.7 5 OS CN 0.49 0.54 + 10.8 OD D 2.75 0.51 81.3 6 OD D 3.27 0.37 88.8 OS CN 0.89 0.13 85.4 7 OD D 2.53 0.67 -73.4 OS CN 1.63 0.36 77.7 8 OD D 1.53 0.66 56.9 OS CN 0.27 0.34 + 27.8 9 OD CN 2.22 0.27 87.6 OS D 3.51 0.54 84.7 10 OD CN 0.47 0.10 -77.6 OS D 4.61 0.37 91.9 Mean SD (D) CI 2.44 .44 1.48 3.41 0.43 0.17 0.32 0.54 82.4 Mean SD(CN) CI 0.81 0.68 0.29 1.33 0.36 0.18 0.22 0.50 56.0 OS= left eye, OD= right eye; D= diseased eye, CN=contralateral normal. CI = 95% confidence interval

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94 Total MMPs activity Case 100.10.20.30.40.50.60.70.80.91237DaysMMPs activity (RSU) OD OS* CF*= ulcerated eye= Fluorescein staining negative a b c d Figure 4-2: Case 1 A 6 month Thoroughbred colt presented with a melting corneal ulcer in the left eye (OS). This animal received a medical treatment, and then a conjunctival graft (CF) on the day after admission. The pictures on the right illustrate the clinical evolution of the ulceration: day 1 (a), day 1 with fluorescein (b), day 2 (c), and day 45 (d). Total MMPs activity Case 200.10.20.30.40.50.60.7159DaysMMPs activity (RSU ) OD* OS* a b *= ulcerated eye= Fluorescein staining negative Figure 4-3: Case 2 A 17yo Arabian mare presented with a superficial corneal ulcer in the left eye (OS) and a half thickness stromal corneal ulcer in the right eye (OD). This animal received only a medical treatment. The pictures on the right illustrate the clinical evolution of the ulceration: day 1 OD (a), day 1 OS (b), day 5 OD (c), and day 5 OS (d). c d

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95 Total MMPs activity Case 300.511.522.533.544.512471112114DaysMMPs activity (RSU) OD OS* KT AMT*= ulcerated eye= Fluorescein staining negative a b Figure 4-4: Case 3 A 5yo Thoroughbred mare presented with a fungal keratomalacia in the left eye (OS). This animal received medical treatment followed by a keratectomy and amniotic membrane transplantation (K+AMT) on the second day. The pictures on the right illustrate the clinical evolution of the ulceration: day 1 (a), day 2 (b), day 7 (c), and day 114 (d). c d Total MMPs activity Case 400.511.522.533.5412346722DaysMMPs activity (RSU) OD OS* a b *= ulcerated eye= Fluorescein staining negative Figure 4-5: Case 4 A 2yo Thoroughbred filly presented a deep corneal ulcer in the left eye (OS). This animal received only medical treatment. The pictures on the right illustrate the clinical evolution of the ulceration: day 1 (a), day 2 (b), day 7 (c), and day 22 (d). c d

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96 Total MMPs activity Case 500.20.40.60.811.21.41.61.821245689DaysMMPs activity (RSU) OD* OS *= ulcerated eye= Fluorescein staining negative Figure 4-6: Case 5 A 2yo Thoroughbred filly presented a deep corneal ulcer in the right eye (OD). This animal received only medical treatment. The pictures on the right illustrate the clinical evolution of the ulceration: day 1 (a), day 2 (b), day 6 (c), and day 89 (d). a b c d a b Total MMPs activity Case 600.511.522.533.51256737DaysMMPs activity (RSU) OD* OS c d *= ulcerated eye= Fluorescein staining negative e f Figure 4-7: Case 6 A 7yo Paint gelding presented a corneal ulcer in the right eye (OD). This animal received only medical treatment. The pictures on the right illustrate the clinical evolution of the ulceration: day 1 (a), day 1 with fluorescein (b), day 2 (c), day 5 (d), day 6 (e), and day 37 (f).

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97 Tobal MMPs activity Case 700.511.522.53123336DaysMMPs activity (RSU) OD* OS *= ulcerated eye= Fluorescein staining negative a b Figure 4-8: Case 7 A 2 month Thoroughbred colt presented a melting corneal ulcer in the right eye (OD). This animal received medical treatment, and a conjunctival graft (CF) on the second day after admission. The pictures on the right illustrate the clinical evolution of the ulceration: day 1 (a), day 1 with fluorescein (b), day 3 (c), and day 38 (d). Tobal MMPs activity Case 800.511.522.53123438DaysMMPs activity (RSU) OD* OS CF*= ulcerated eye= Fluorescein staining negative a b c d Figure 4-9: Case 8 A 2 month Thoroughbred colt presented a melting corneal ulcer in the right eye (OD). This animal received medical treatment, and a conjunctival graft (CF) on the second day after admission. The pictures on the right illustrate the clinical evolution of the ulceration: day 1 (a), day 1 with fluorescein (b), day 3 (c), and day 38 (d).

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98 Tobal MMPs activity Case 900.511.522.5312791418120DaysMMPs activity (RSU) OD OS* PK-CF-AMT*= ulcerated eye= Fluorescein staining negative Figure 4-10: Case 9 A 14yo American Quarter Horse gelding presented a fungal corneal ulcer in the left eye (OS). This animal received medical treatment, and a penetrating keratoplasty with amniotic membrane transplantation and conjunctival graft (PK+ AMT+CF) on the second day after admission. The pictures on the right illustrate the clinical evolution of the ulceration: day 1 (a), day 14 (b), day 18 (c), and day 120 (d). a b c d Tobal MMPs activity Case 1001234516913233845DaysMMPs activity (RSU) OD OS* AMT*= ulcerated eye= Fluorescein staining negative a b Figure 4-11: Case 10 A 12yo Hanovarian stallion presented a melting corneal ulcer in the left eye (OS). This animal received medical treatment and an amniotic membrane transplantation (AMT) on the second day after admission. The pictures on the right illustrate the clinical evolution of the ulceration: day 1 (a), day 1 with fluorescein (b), day 2 (c), and day 45 (d). c d

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99 Discussion Our results of MMP activity in the tear film of horses with ulcerative keratitis prior to initiation of therapy are similar to results reported by Strubbe et al. that MMP-9 and MMP-2 are present at a significantly higher level in the tear film of horses with ulcerative keratitis (Strubbe et al., 2000). The majority of host-derived and microbial tear film enzymes are MMPs. Enzymes of bacterial or fungal origin (i.e. exogenous proteinases) can contribute to ulcerative keratitis, either directly or indirectly, through the activation of corneal proteinases (i.e. endogenous proteinases) (Berman et al., 1971; Berman et al., 1973; Berman et al., 1977; Brooks, 1999; Fini and Girard, 1990; Hibbetts et al., 1999; Matsubara et al., 1991a; Matsumoto et al., 1993; Prause, 1983a; Prause, 1983b; Slansky et al., 1969; Strubbe et al, 2000; Tervo et al., 1988; Tervo et al., 1991). Bacteria were isolated from two cases (cases 3 and 10) and fungi isolated from two cases (cases 3 and 9). Multiple studies have reported elevated levels of proteinases in tears from animals and humans with ulcerative keratitis (Berman et al., 1971; Berman et al., 1973; Berman et al., 1977; Brooks, 1999; Fini and Girard, 1990; Hibbetts et al., 1999; Matsubara et al., 1991a; Matsumoto et al., 1993; Prause, 1983a; Prause, 1983b; Slansky et al., 1969; Strubbe et al, 2000; Tervo et al., 1988) and changes in proteinase activity during the healing process of the ulcerated cornea but precise MMP activity profiles based on the collection and the analysis of many serial tear fluid samples have not been reported in either animals or humans. We document for the first time that total MMP activity decreases in equine tears as the corneal epithelial and stromal healing occur. These results of corneal wound healing are in accordance with those previously reported of skin wound healing (Agren, 1994; Blalock et al., 2001; Herouy, 2001;

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100 Ladwig et al., 2002; Lobmann et al., 2002; Parks, 1999; Paul et al., 1997; Wall, 2002). Agren et al. reported that the total MMP activity was highest in the early phases and then decreased as the skin wound healing proceeded (Agren, 1994). In the same study, the various MMPs (MMP-2 and MMP-9) were distinguished and quantified at each time point and it has been shown that their activity varied depending on the stage of the healing. These results suggest that MMP-2 and MMP-9 have different functions in the skin wound healing process (Parks, 1999; Paul et al., 1997; Agren, 1994). Other studies suggest that the ratio of MMP to tissue inhibitor of MMP (TIMP) in skin wound fluids is inversely correlated with the skin healing process (Herouy, 2001; Ladwig et al., 2002; Lobmann et al., 2002). Therefore, a combination of increased concentrations of MMPs with decreased concentrations of TIMPs is responsible of an increased proteolytic environment that contributes to the failure of skin wound to heal (Blalock et al., 2001; Ladwig et al., 2002; Lobmann et al., 2002). At this point, we were not able to study independently MMP-2 and MMP-9 and we did not look at the TIMPs in the tear film of these 10 horses. The results of our study done in horses are also in agreement with those reported on corneal wound healing. Studies report high level of proteolytic activity in the early stage of the corneal wound healing and lower levels at the corneal ulcer resolution and various roles for the different proteases (Cejkova, 1998; Fini and Girard, 1992; Fini et al., 1998; Tervo et al, 1989b; van Setten et al., 1989). These studies suggest also that persistence of high level of proteolytic in corneal wound is responsible for the failure to heal (Barro et al., 1998; Fini et al., 1998; Tervo et al., 1989). To the authors knowledge, there is only one study that reported the MMP-2 and MMP-9 activity in the tear film during corneal

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101 wound healing based on analysis of serial tear fluid samples (Barro et al., 1998). In this study, Barro et al. showed that enhanced and persistent MMP-2 and MMP-9 activity in tears correlated with corneal damage and graft failure (Barro et al., 1998). It is also interesting to note that the MMP activity decrease during the corneal wound healing is the most pronounced for the three cases with positive cultures (cases 3, 9 and 10). This might underline the participation of the microorganisms in the proteolytic activity detected in the tear film as mentioned earlier. Our data support the use of aggressive therapy to rapidly reduce the activity of tear proteases in case of ulcerative keratitis. Equine serum, which contains strong and broad protease inhibitors such as 2-macroglobulin and 1-proteinase inhibitor, was used topically in all the cases presented here. Various other anti-protease compounds are also available (N-acetylcysteine or NAC, potassium edatate diaminetetrataacetatic acid or EDTA) that utilize different mechanisms to inhibit different families of proteases present in equine tears. A combination of inhibitors may be indicated for severe corneal ulceration in horses (Brooks, 1999; Hibbetts et al., 1999). Medical and surgical treatments of the corneal ulcers in these horses lead to a reduction in tear film proteolytic activity that corresponded with the improvement in the clinical signs of corneal ulceration. Corneal ulcers in horses are associated with initially high levels of tear film proteolytic activity which decrease as the ulcers heal. Measurement of MMP activity in the equine tear film might represent a way to monitor the progression of corneal healing in horses with ulcerative keratitis. Treatment strategies for healing corneal ulcers in horses should be directed towards reducing microbial activity, decreasing uveitis, and reducing tear film concentrations of MMPs as reduced

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102 MMP activity is associated with improvement of the clinical signs of ulcerative keratitis in horses.

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CHAPTER 5 IN VITRO INHIBITION OF MATRIX METALLOPROTEINASE ACTIVITY IN THE TEAR FLUID OF HORSES WITH ULCERATIVE KERATITIS Introduction Ulcerative keratitis is a common and often vision-threatening condition in horses. The Ophthalmology Service of the University of Florida Veterinary Medical Teaching Hospital diagnosed ulcerative keratitis in 527 horses between January 1987 and October 2002. This constituted 35% of all horses evaluated for ophthalmic problems during this period. Superficial, non-infected ulcers in horses generally heal quickly and without complication, whereas stromal degradation in deep or infected ulcers can rapidly and dramatically progress to corneal perforation in horses within 24 hours (Brooks, 1999). A pronounced fibrovascular response is also prominent during corneal healing in horses. An understanding of the pathophysiologic processes as well as early diagnosis and aggressive treatment are important for quick resolution of ulcerative corneal diseases in horses and to speed healing, reduce scarring, and prevent corneal rupture. Proteolytic enzymes perform important physiologic functions in normal tissues, such as turnover and remodeling of the corneal stroma. Activities of proteolytic enzymes are normally balanced by natural proteinase inhibitors, thus preventing excessive degradation of normal healthy tissue. Excessive amounts of proteinases can create an imbalance between proteinases and proteinase inhibitors, and increased amounts of proteases are believed to cause pathologic degradation of collagen and proteoglycans in the cornea (Slansky et al., 1969; Twining, 1994). 103

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104 The cornea of horses manifests the most severe degree of ulcer-associated stromal collagenolysis seen in animals (Brooks, 1999). This rapid degradation of the corneal stroma in horses with corneal ulcers appears to be caused by various proteolytic enzymes acting on the collagen, proteoglycans, and other components of the stromal extracellular matrix. Microorganisms, inflammatory cells, corneal epithelial cells, and fibroblasts all produce and release proteolytic enzymes (Hibbets et al., 1999; Matsubara et al., 1991a; Matsubara et al., 1991b; Twining et al., 1993). Two important families of enzymes that affect the cornea are the matrix metalloproteinases (MMPs) and serine proteinases (including neutrophil elastase) (Fini and Girard, 1990; Fini et al., 1998; Matsubara et al., 1991a). Two MMPs (MMP-2 [72-kd gelatinase A] and MMP-9 [92-kd gelatinase B]) (Fini and Girard, 1990; Fini and Girard, 1992) are of major importance in terms of remodeling and degradation of the corneal stromal collagen. The origin and purpose of MMP-2 and appear to differ at the corneal level. Matrix metalloproteinase-2 is synthesized by corneal keratocytes and performs a surveillance function in the normal cornea, becoming locally activated to degrade collagen molecules that occasionally become damaged as a result of normal wear and tear (Azar et al., 1998; Matsubara et al., 1991b; Twining, 1994). Alternatively, MMP-9 is produced by epithelial cells and polymorphonuclear neutrophils (PMNs) following corneal wounding (Fini and Girard, 1990; Matsubara et al., 1991b). Proteinases have been evaluated in the tear film of normal and diseased eyes of humans and other animals (Berman et al., 1971; Berman et al., 1977; Berman et al., 1973; Fini and Girard, 1990; Matsubara et al., 1991a; Prause, 1983; Strubbe et al, 2000; Tervo et al., 1998). In severely damaged corneas, proteinase activities in the tear film were

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105 significantly increased (Berman et al., 1971; Kernacki et al., 1995; Matsubara et al., 1991a; Prause, 1983a; Tervo et al., 1998). In another study (Strubbe et al., 2000) in horses, higher amounts of MMP-2, MMP-9, and neutrophil elastase were found in the tear film of ulcerated eyes, compared with values for the tear film of age-matched normal control horses. Proteinase activity in the tear film is believed to speed up degradation of stromal collagen, leading to rapid progression of ulcers. Normalizing proteolytic activity in the tear film is an objective of the treatment of corneal ulcers in horses. Thus, proteinase inhibitors have been recommended for treatment of ulcerative keratitis to reduce the progression of stromal ulcers, speed epithelial healing, and minimize corneal scarring (Berman, 1975; Berman, 1978; Berman et al., 1973; Berman et al., 1975; Brooks, 1999; Tervo et al., 1992; Ward, 1999). However, studies on activity of specific enzymes are limited. Accordingly, recommendations for use of topically administered N-acetylcysteine (NAC), potassium EDTA, and serum in horses with corneal ulcers are mostly based on anecdotal clinical reports or extrapolations from data obtained from use of these compounds in other species (Brooks, 1999; McLaughlin et al., 1992; Severin, 1976; Strubbe et al., 2000; Tervo et al., 1992; Ward, 1999; Whitley and Gilger, 1999). The availability of newer compounds such as doxycycline, a modified dipeptide that contains hydroxamic acid (i.e., ilomostat), and 1-proteinase inhibitor (1-PI), which initially have shown considerable promise as MMP or serine-proteinase inhibitors in other species (Barletta et al., 1996; Dursun et al., 2001; Golub et al., 1984; Perry et al., 1993; Rawal and Rawal, 1984; Schultz et al., 1992), outlines the need for specific studies on possible regulation of

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106 enzyme activity in tears in horses with corneal disease. These new compounds have potential as potent protease inhibitors in horses with corneal ulcers. Therefore, in the study reported here, we evaluated the effectiveness of various protease inhibitors for reducing the activity of MMP-2 and -9 in vitro in samples of tear film obtained from horses with ulcerative keratitis. Materials and Methods Animals All procedures were carried out according to the ARVO statement for the Use of Animals in Ophthalmic and Vision research and were approved by the University of Florida Animal Care and Use Committee as well as the UF College of Veterinary Medicine Clinical Research Review Committee. Tear samples were collected from the eyes of 34 horses with ulcerative keratitis. Each horse was evaluated by members of the Ophthalmology Service of the University of Florida Veterinary Medical Teaching Hospital during 2002. The horses had been diagnosed with various conditions, including bacterial or fungal keratitis, sterile ulcerative keratitis, and ulcerative keratitis of unknown etiologic origin. Collection of Tear Fluid Samples Horses were sedated, and akinesia of the upper eyelid was achieved prior to collection of tear samples. Samples were obtained before any diagnostic procedure or application of treatment; however, immediately after collection of tear samples, thorough ophthalmic examinations that included slit-lamp biomicroscopy, tonometry, and ophthalmoscopy were performed. Samples were collected only from eyes with ulcerative keratitis, as identified by positive results for retention of fluorescein dye. All tear samples were collected from the lower fornix by capillary force, using capillary tubes with an

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107 atraumatic tip as described elsewhere (van Setten et al., 1989). Time of collection of each sample was not recorded. All samples were immediately centrifuged, transferred into polypropylene microcentrifuge tubes (Eppendorf tubes, Brinkmann Instruments Inc., Westbury, NY), and stored at oC until analysis. Tear samples collected from the eyes of the 34 horses were pooled to yield a volume of 1,500L. Determination of MMP Activity and Inhibition Tests by Gelatin Zymography Gelatin zymography and measurement of optical density (OD) were used to evaluate MMP activity in untreated pooled tear samples and to conduct inhibition tests of MMP activity in treated tear samples. Aliquots (10 L) of the pooled tears were mixed with an equal volume of SDS-sample buffer (Novex tris-glycine SDS native sample buffer (2X), Invitrogen, Carlsbad, CA). Then, fifteen L of the mixture was loaded into wells of precast 10% zymogram gelatin gels (10% Novex zymogram gelatin gel, Invitrogen, Carlsbad, CA). Untreated samples were constituted on each gel in 3 lanes that remained untreated. Treated samples were constituted in 3 lanes that were treated with a protease-inhibitor compound (triplicate pattern). Prestained molecular-weight standards (See Blue prestained standards, Invitrogen, Carlsbad, CA) and gelatinase-zymography standards for active (Active MMP-2 and active MMP-9 enzymes, Oncogen, Boston, MA) and latent (Proenzyme MMP-2 and MMP-9, Oncogen, Boston, MA) forms of human MMP-2 and -9 were also assayed on each gel. Inhibitory compounds tested in the study included EDTA, doxycycline, NAC, ilomostat, 1-PI, and fresh equine serum. A solution containing 0.2% EDTA was obtained by filling an evacuated, EDTA-containing, 10-mL blood-collection tube (Vacutainer EDTA, Becton-Dickinson, Franklin Lakes, NJ) with 10 mL of developing buffer. Doxycycline 1% (Doxy 100, American Pharmaceutical Partners Inc, Los Angeles,

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108 CA), NAC 20% (Acetylcysteine 20%, Abbott Laboratories, North Chicago, IL), and 1% 1-PI were diluted with developing buffer to obtain concentrations of 0.1% doxycycline, 10% NAC, and 0.1% 1-PI, and 0.5% 1-PI, respectively. Ilomostat (2 mg) was dissolved in 50 L of dimethyl sulfoxide, and then 20 mL of developing buffer was added to achieve a final concentration of 0.1% ilomostat. Ilomostat and 1% 1-PI were provided by 1 of the investigators (GSS). Blood samples were collected from the jugular vein of a horse into dry, sterile, 7-mL blood-collection tubes that did not contain an anticoagulant but did contain gel and clot activator (Vacutainer SST Plus, Becton-Dickinson, Franklin Lakes, NJ). Serum was separated by centrifugation of the tubes at 1000 g for 8 minutes. Serum was harvested and maintained at room temperature (19C); it was used undiluted for in vitro inhibition testing on the day on which it was obtained. Inhibition tests were also conducted with undiluted fresh serum that was stored at cool temperature (4C) during 1 to 8 days, and frozen serum (-20C). Gels were electrophoresed at a constant voltage of 125 volts for approximately 2 hours. After electrophoresis, gels were rinsed in distilled water and then gently shaken in a renaturing solution of 2.7% Triton X-100 (Novex zymogram renaturing buffer (10X), Invitrogen, Carlsbad, CA) for 1 hour at 37oC to reactivate MMP activities. Following the renaturation phase, gels were cut to form 2 sets of triplicate lanes. One set of triplicate lanes was not treated with an inhibitor (untreated samples), whereas the set was treated with one of the inhibitors (treated samples). Gels were then incubated on a rotary shaker in developing buffer (Novex zymogram developing buffer (10X), Invitrogen, Carlsbad, CA) for 24 hours at 37oC to allow the MMPs to digest the gelatin substrate. Inhibitors,

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109 except for the equine serum, were added to the developing buffer during this 24-hour digestion phase to provide inhibitory effects. To test for inhibitory effects of equine serum, gels were incubated in undiluted pure equine serum for 2 hours at 37oC prior to being incubated in the developing buffer for 24 hours at 37oC. After the digestion phase, gels were rinsed and stained by incubation with Coomassie blue (Diversified Biotech, Boston, MA) for 1 hour. Gels were then destained with a solution of 5% acetic acid-7.5 % methanol to maximize contrast between lytic areas and non-digested areas. Bands of proteolytic activity appeared uncolored against a dark-blue background. Inhibition of protease activity was manifested as a lack of band formation. Identity of putative proteases was determined by analysis of the distance that bands migrated on the gels, compared with the distance for migration of molecular weight and protease standards. Investigators were careful to obtain a homogenous pool of tears for use in evaluating the untreated samples and the samples treated with the various inhibitors. An equal volume of the pooled tear sample was loaded into each well; therefore, the same amount of protein and MMPs were contained in the untreated and treated samples. Thus, a difference in MMP activity observed in a gel between an untreated and a treated sample did not result from a difference in the concentration of loaded MMPs; instead it was considered to be the difference in proteolytic activity attributable to the inhibitor. We considered the comparison between the untreated and treated samples to be valid, and we believed that the gels represented the actual in vitro inhibitory effects of the tested compounds.

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110 Image Analysis Digital photographs of stained gelatin zymograms were created by use of an imaging densitometer (GS-710 Calibrated imaging densitometer, Bio-Rad Laboratories, Hercules, CA) and analyzed by use of quantification software (Quantity One quantification software, 4.2.1 beta version, Bio-Rad Laboratories, Hercules, CA). Sensitivity of the analyzer was adjusted for each gel, and bands in each lane of the gels were then automatically detected by the analyzer. Background data was then subtracted for each triplicate set of lanes. Image analysis produced an intensity pattern of each lane by use of the optical density (OD) function for the distance of migration from the top of the gel (relative front). The higher the amount of proteolytic activity, the more gelatin substrate was digested, which was reflected by a concomitant decrease in the amount of stain. Area under the curve (AUC) that corresponded to the OD multiplied by the width of the band (in mm) was calculated for each band. Variables used for comparisons in the study were the AUC, which allowed an accurate estimation of proteolytic activity in each sample (untreated and treated), as well as the inhibition obtained with the various inhibitors. Image analysis provided a report listing all the bands detected on a gel and the AUC for each band (Figure 5-1).

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111 Figure 5-1: Image analysis of representative zymogram gels to determine proteinase activity in pooled tears obtained from horses with an active corneal ulcer. Each gel on the right side of the figure (lane 1, untreated control sample; lane 2, treated with 0.1% 1-proteinase inhibitor [PI]) was scanned by use of an imaging densitometer. Notice that 7 bands were detected on the gels. Intensity patterns generated by the image analysis software for the 2 lanes (lane 1, red tracing; lane 2, green tracing) are indicated on the left side of the figure. The higher the intensity of the band, the higher the optical density (OD), and, therefore, the higher the proteolytic activity. Area under the curve (AUC) that corresponds to the OD multiplied by the width of the band was calculated for each band. Use of the AUC allowed an accurate estimation of proteolytic activity in each pair of samples (untreated and treated), and comparison of the AUCs allowed estimation of the inhibition rate for 0.1% 1-PI in relation to the untreated sample. Relative front = Proportional distance of migration from the top of the gel. Statistical Analysis Ratios of AUC with inhibitor (i.e., treated sample) to AUC without inhibitor (i.e., untreated sample) for global proteolytic activity (i.e., sum of all bands detected in a lane), as well as for activity of each protease (or band) detected, were calculated and compared by use of multiple-regression analysis. A statistical program (Proc GLM, version 8.01, SAS Institute Inc, Cary, NC) was used for estimations. For all analyses, results were considered significant at values of P < 0.05.

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112 Results Detection and Identification of Proteinases in Pooled Tears by Gelatin Zymography All triplicate lanes of untreated pooled tears had 7 bands (Figures 5-1 and 5-2). On the basis of the migration of the bands and standards on the gels, we determined that bands 4, 5, 6, and 7 corresponded to activity of the latent form of MMP-9, active form of MMP-9, latent form of MMP-2, and active form of MMP-2, respectively (Figure 5-2). Thus, pooled tears obtained from ulcerated eyes of horses contained the latent and active forms of MMP-2 and -9. Aggregates of MMP-2 Pro MMP-9 Act MMP-9 Pro MMP-2 Act MMP 2 Figure 5-2: Gelatin zymogram of untreated pooled tears obtained from ulcerated eyes of horses. Pooled tears were loaded on each gel in triplicate and incubated without inhibitor (untreated samples) or with various proteinase inhibitors (treated samples) to assess effects on proteolytic activity of the proteinases in the pooled tears. Molecular weight markers are indicated on the left side of the gel. Each of the 7 bands detected are indicated (red brackets) in the untreated lane on the right side. On the basis of their location on the gel and their molecular weight, proteinases could be identified. MMP = Matrix metalloproteinase. Pro MMP-9 = Latent form of MMP-9. Act MMP-9 = Active form of MMP-9. Pro MMP-2 = Latent from of MMP-2. Act MMP-2 = Active form of MMP-2.

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113 In vitro Inhibition of the Proteinases Present in the Pooled Tears Inhibition of global proteolytic activity When compared with MMP activity in the untreated tear samples, global proteolytic activity observed on the gels was reduced by 99.4% by EDTA, 96.3% by doxycycline, 98.8% by NAC, 98.9% by ilomostat, 52.4% by 0.1% 1-PI, 93.6% by 0.5% 1-PI, and 90.0% by equine serum (Figures 5-3 and 5-4; Table 5-1). We did not detect significant differences in inhibition rates among the various inhibitors tested, except for 0.1% 1-PI, which had a significantly (P < 0.001) lower amount of inhibitory activity than that for any of the other compounds. Band 1 Band 2 Band 3 Band 4 (Pro MMP-9) Band 5 (Act MMP-9) Band 6 (Pro MMP-2) Band 7 (Act MMP-2) Control EDTA Dox NAC PI 0.1 PI 0.5 I ES Figure 5-3: In vitro inhibition of global proteolytic activity for various proteinase inhibitors by gelatin zymography. In vitro inhibition (mean value for triplicate samples) of global proteolytic activity, as well as proteolytic activity for each proteinase, determined for various proteinase inhibitors by use of pooled tears obtained from ulcerated eyes of horses. Samples in each lane were treated as follows: untreated sample (Control), 0.2% EDTA (EDTA), 0.1% doxycycline (Dox), 10% N-acetylcysteine (NAC), 0.1% 1-Pi (Pi 0.1), 0.5% 1-Pi (Pi 0.5); 0.1% solution of a modified dipeptide that contains hydroxamic acid (i.e., ilomostat [I]), and fresh equine serum (ES).

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114 Table 5-1: Percentage of in vitro inhibition of proteolytic activity for the global proteolytic activity, as well as for each proteinase, determined for various proteinase inhibitors by use of pooled tears obtained from ulcerated eyes of horses. Band EDTA 0.2% Doxycycline 0.1% NAC 10% 1 PI 0.1% 1 PI 0.5% Ilomostat 0.1% Fresh serum 1 96.3 86.1 94.3 46.8* 94.5 89.1 90.7 2 99.9 96.3 99.2 22.5* 87.0 95.1 87.4 3 96.6 96.5 97.8 44.6* 96.3 92.1 63.6 4 99.8 98.4 99.8 55.3* 94.9 99.7 91.4 5 98.6 95.9 98.7 46.6* 92.3 99.1 82.4 6 100 95.0 97.0 78.8* 90.5* 98.4 95.9 7 98.5 98.8 96.3 77.8* 70.7* 100 96.6 Total 99.4 96.3 98.8 52.4* 93.6 98.9 90.0 Values reported represent mean value for triplicate samples. Band 4 represented the latent form of matrix metalloproteinase MMP-9. Band 5 represented the active form of MMP-9. Band 5 represented the latent form of MMP-2. Band 7 represented the active form of MMP-2. *Inhibition rate for this compound differed significantly (P < 0.001) from the inhibition rates for the other compounds. EDTA = 0.2% EDTA. Dox = 0.1% Doxycycline. NAC = 10% N-acetylcysteine. 0.1 PI = 0.1% 1-proteinase inhibitor. 0.5 PI = 0.5% 1-proteinase inhibitor. I = 0.1% Solution of a modified dipeptide that contains hydroxamic acid (i.e., ilomostat). ES = Fresh equine serum. 0102030405060708090100Percentage of inhibition EDTA Dox NAC Ilo A1Pi0.1% A1Pi 0.5% Fresh equine serum Figure 5-4: Percentage of in vitro inhibition (mean value for triplicate samples) of global proteolytic activity determined by use of gelatin zymography for various proteinase inhibitors in pooled tears obtained from ulcerated eyes of horses. Inhibitors used were as follows: 0.2% EDTA (white bar), 0.1% doxycycline (black bar), 10% N-acetylcysteine (diagonal lines from upper left to lower right of bar), 0.1% 1-PI (diagonal lines from lower left to upper right of bar),

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115 0.5% 1-PI (dotted bar); 0.1% ilomostat (vertical lines), and fresh equine serum (horizontal lines). *Inhibition rate for this compound differed significantly (P < 0.001) from the inhibition rates for the other compounds. Inhibition of proteolytic activity for each proteinase We also assessed the inhibitory effects of these various compounds detected by gelatin zymography on each individual proteinase present in the tear pool (Figure 5-3; Table 5-1). It is interesting that inhibition was greatest for the latent form of MMP-9 (band 4) and least for the active form of MMP-2 (band 7) for EDTA, NAC, and 0.5% 1-PI, whereas the inhibition was greatest for active form of MMP-2 (band 7) for doxycycline, 0.5% 1-PI, ilomostat, and the fresh serum. There was significant difference in the inhibition rates for each individual band between the 0.1% 1-PI and any other of the tested compounds (P < 0.05). Furthermore, the inhibition of the latent and active MMP2 by 0.5% 1-PI was significantly lower than those for the other tested compounds (p < 0.05). In vitro Inhibitory Activity and the Duration of Action of Equine Serum Against the Proteinases Present in the Tear Fluid of Horses with Ulcerative Keratitis There was no difference in MMP inhibition between fresh serum (-90.0% activity) and frozen serum, i.e. -20C (-90.9% activity). No difference in MMP inhibition between the serum kept at room temperature, i.e. 19C (-92.0 4.9% activity) or in the refrigerator, i.e. 4C (-90.6 2.8% activity) was detected during the eight day trial (Figure 5-5). Inhibitory activity of serum towards tear film MMPs did not significantly change over the eight-day test period.

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116 82848688909294969810012358Time (day)Percentage of inhibition Serum RT Serum Cool Serum frozen Figure 5-5: Percentage of in vitro inhibition (mean value for triplicate samples) of global proteolytic activity determined by use of gelatin zymography for equine serum stored in various ways. Serum RT = serum stored at room temperature (i.e. 19C), Serum Cool = serum stored in the refrigerator (i.e. 4C), Serum frozen = serum stored in the freezer (i.e. -20C). Discussion Analysis of the results we obtained for the untreated (control) samples confirmed those reported elsewhere (Strubbe et al., 2000) in which MMP-2 and -9 are found in substantially high amounts in the tear film of horses with ulcerative keratitis. The majority of host-derived and microbial enzymes in the tear film are MMPs (Barletta et al., 1996; Fini et al., 1992; Fini et al., 1998; Matsumoto, 2000). Enzymes of bacterial or fungal origin (i.e., exogenous proteases) can contribute directly or indirectly to ulcerative keratitis through the activation of corneal proteinases (i.e., endogenous proteinases) (Brooks, 1999; Gopinathan et al., 2001; Matsumoto, 2000; OBrien, 1997; Slansky et al., 1969). In the study reported here, we assessed only the inhibitory effects of the various compounds on the global proteolytic activity that can be detected in ulcerated eyes of horses without distinguishing between proteolytic activity attributable to endogenous

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117 proteinases and activity attributable to exogenous proteinases. Thus, we did not take into account the specific cause of the corneal ulcers. Proteinase inhibitors have been recommended and used in human and veterinary ophthalmology for many years. Their use remains controversial, because the efficacy and local toxic effects of these compounds are frequently questioned. In the study reported here, we documented in vitro a high amount of inhibition of equine MMP activity by the use of EDTA, doxycycline, NAC, equine serum, ilomostat, and 1-PI. For each of these proteinase inhibitors, the proposed mechanisms of inhibition, the current recommendations for use in human and veterinary ophthalmology, and the adverse effects that have been reported with each can be used to justify treatment of horses with ulcerative keratitis. We observed a large reduction in the in vitro activity of equine MMPs with 0.2% EDTA. Doxycycline, EDTA, and NAC are metal-chelating agents, and they inhibit MMPs by chelation of zinc or calcium that MMPs require as a cofactor and stabilizing ion, respectively (Twining, 1994; Ward, 1999). By chelating calcium ions, EDTA interferes with the stability of the MMPs and also decreases the stimulation for the migration of the PMNs to the site of a corneal ulcer. The PMNs release powerful proteinases that are also responsible for breakdown of stromal collagen. EDTA interferes with attachment of opsonized zymosan to the cell membrane of PMNs, leaving the PMNs in a resting, inactivated, granulated state (Pfister et al., 1984). In addition, EDTA has been recommended for the treatment of ulcers associated with corneal collagenolysis (Berman, 1978; Pfister and Pfister, 1997; Ward, 1999); however, corneal tolerance and efficacy of EDTA are subjects of controversy (Corbett et al., 2001; Furrer et al., 1999). In

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118 rabbits, treatment of ulcerated corneas with 0.1% EDTA after keratectomy did not significantly affect the rate of re-epithelialization (Schultz, 1997). Furthermore, EDTA seems to be tolerated well when used at concentrations ranging from 0.05% to 0.2% for treatment of corneal ulcers in horses (Brooks, 1999). However, the long-term use of EDTA may impair formation of tight junction complexes between epithelial cells as a result of the requirement of calcium ions for stable tight junctions. In the study reported here, we documented in vitro inhibition of equine MMPs by 0.1% doxycycline. Tetracyclines inhibit MMP activity independent of their antimicrobial properties (Golub et al., 1984). The proposed mechanism of action of these antimicrobial agents is that tetracyclines bind to zinc and calcium cations that are essential for the MMPs. Tetracyclines form a binding complex with the MMPs, resulting in reduced enzyme activity (Golub et al., 1984; Rawal and Rawal, 1984; Smith et al., 1999). Tetracyclines, and especially doxycycline, inhibit the synthesis of MMPs in human vascular endothelial cells (Rawal and Rawal, 1984). Tetracyclines, such as minocycline and doxycycline, inhibit the breakdown of various connective tissues (i.e., skin, bone, and cornea) mediated by excessive collagenolytic activity (Golub et al., 1984), and they have been used specifically for ophthalmic treatments (Golub et al., 1984; Rawal and Rawal, 1984). Doxycycline promotes healing of persistent ulcers and epithelial defects in humans (Dursun et al., 2001; Perry et al., 1993) and inhibits alkali-induced corneal ulcers in rabbits (Seedor et al., 1987). Tetracyclines are recommended for use in ophthalmic conditions in animals (Ward, 1999; Whitley and Gilger, 1999) but we are not aware of any information on the use of doxycycline in horses.

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119 In our study, 10% NAC proved to be effective for inhibiting in vitro MMP activity in the equine tear film. This supports the fact that NAC is commonly used as an MMP inhibitor to treat humans and other animals with corneal ulcers (Slansky et al., 1972; Ward, 1999). Application of NAC at concentrations of 2% to 10 % every 1 to 4 hours has been recommended for treatment of dogs (Kanao et al., 1993; Ward, 1999; Whitley and Gilger, 1999) and horses (Andrew et al., 1998; Schmidt, 1997; Ward, 1999; Whitley and Gilger, 1999). More specifically, equine serum and 10% NAC has been recommended for the treatment of horses wit severe corneal ulcers (Brooks, 1999). A thiol, NAC contains a sulfhydryl group that binds irreversibly or removes the intrinsic metal cofactor (i.e., zinc) of the MMPs. It may also reduce one or more disulfide bonds of an enzyme (Slansky et al., 1970; Woessner, 1991). Also, NAC inhibits MMP production at the transcriptional level (Galis et al., 1998). In one study (Galis et al., 1998), NAC suppressed MMP-9 synthesis in macrophages. In another study (Thermes et al., 1991), effects of acetylcysteine on rabbit conjunctival and corneal surfaces were examined. Investigators did not detect signs of ocular toxicosis for various concentrations tested, but 20% NAC caused some superficial necrosis and dose-related disruption in the mucus layer of the tear film. Adverse effects were not found for the rate of re-epithelialization of the rabbit corneas in which 10% and 20% NAC were applied as treatment for a superficial epithelial ulcer (Petroutsos et al., 1982). In the study reported here, we detected high amounts of in vitro inhibition of MMP activity attributable to equine serum. The 2-macroglobulin is a nonspecific protease inhibitor that reduces the activity of proteinases of all 4 major proteinase classes (i.e., serine [including neutrophil elastase], aspartic, thiol, and metalloproteinases in human

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120 and rabbit corneas) (Berman, 1975; Hibbetts, 1999; Twining, 1994). The 2-macroglobulin is a tetrameric molecule composed of 2 pairs of identical disulfide-linked subunits. Each subunit contains a region that binds the enzymes and allows the proteinases to cleave peptide bonds. This cleavage leads to a change in conformation of the 2-macroglobulin, resulting in entrapment of the proteinase within the inhibitor (Hibbetts, 1999; Twining, 1984; Woessner, 1991). This particular mechanism tenaciously binds 2 protease molecules/ 2-macroglobulin molecule; thus, 2-macroglobulin is one of the strongest known inhibitors of MMPs. Although 2-macroglobulin mRNA and protein are found in the cornea (Prause, 1983a; Twining, 1994; Twining et al., 1994a), this multifunctional inhibitor is found at much higher quantities in the blood (Prause, 1983a). For this reason, topical application of autologous serum (Bron et al., 2002; Stangogiannis et al., 2002) (1 or 2 drops every 1 to 2 hours) has been recommended for the treatment of corneal ulcers in humans (La Lau, 1979) and other animals (Brooks, 1999; Ward, 1999; Whitley and Gilger, 1999). Blood collected into dry, sterile containers that do not contain anticoagulants will clot rapidly and yield serum that can be separated by centrifugation (Ward, 1999). The serum can be used immediately or stored in a refrigerator or a freezer until needed, and in our experience, its inhibitory effect remains high even after several days of storage (Figure 5-5). However, it is recommended that serum be discarded if not used within 5 days, because it may provide an excellent medium for bacterial growth should it become contaminated (Brooks, 1999; McLaughlin et al., 1992; Ward, 1999). We observed a dose-dependent decrease in proteolytic activity of the MMPs in the pooled tears of horses for 1-PI at concentrations of 0.1 and 0.5%. It is possible that the

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121 dose-related reduction of activity of the MMPs attributable to this serine proteinase inhibitor is not a result of direct inhibition of MMPs by 1-PI; instead, it may result from 1-PI acting as a substrate for the MMPs and competing with the gelatin from the gels. Also called 1-antitryspin, 1-PI is also found in and synthesized by the cornea, (Berman et al., 1973; Twining et al., 1994b) but it is found at a higher concentration in the blood (Berman et al., 1973; Ward, 1999). Furthermore, 1-PI is part of the serpin family of inhibitors that inhibit serine proteinases, such as neutrophil elastase (Berman et al., 1973). The 1-PI exerts its action on proteinases through a mechanism similar to that for 2-macroglobulin. It has the ability to form tight complexes with proteinases and, therefore, reduces proteinase activity (Berman, 1975). It has been documented that 1-PI is a critical substrate for MMP-9 in vivo, and MMP-9 acts upstream to regulate neutrophil elastase activity by inactivating 1-PI (Liu et al., 2000). During the past decade, efforts have been made to design synthetic inhibitors of proteinases (Barletta et al., 1996; Burns et al., 1990a; Burns et al., 1990b; Schultz et al., 1992). Among those that have been reported, ilomostat appears to be promising for the treatment of rapid degradation of the corneal stroma (Schultz et al., 1992). It is more effective in vitro against MMPs in rabbits, compared with the classic chelating agents (Hao et al., 1999a; Hao et al., 1999b). Ilomostat also decreases Pseudomonas alkaline proteinase activity in vitro (Barletta et al., 1996) and is effective in reducing corneal destruction following alkali burns in rabbits (Schultz et al., 1992) or intrastromal injection of pseudomonal culture broth (Barletta et al., 1996). In the study reported here, we reported that use of 0.1% ilomostat caused a high amount of inhibition of in vitro activity of MMPs in the equine tear film. Because the structure of MMPs is highly

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122 conserved among animal species, it is reasonable to expect that the inhibitory effects for this synthetic MMP inhibitor in rabbits would be seen in other domestic animals. Multiple studies have found increased amounts of proteases in tears from humans and other animals with active corneal ulcers. Several agents have been proposed as treatments to reduce the activity of tear proteases, but we are not aware of any reports comparing the relative efficacy of the agents. This places practitioners in a difficult position of not knowing which treatment may be superior. In the study reported here, the relative efficacies of 5 protease inhibitors and equine serum were determined for the inhibition of MMP activity in pooled tears obtained from horses with active corneal ulcers. Analysis of our results indicates that EDTA and ilomostat, followed by NAC and doxycycline, are the most effective inhibitors in vitro and are likely to cause the fewest adverse effects. Because these compounds use different mechanisms to inhibit various families of proteases in equine tears, a combination of these inhibitors may be indicated for the treatment of severe corneal ulcers in horses. Objectives for the appropriate use of these compounds early in the course of corneal disease in horses would be to decrease the amount of time required for recovery and rehabilitation, reduce scarring, and potentially alleviate the need for corneal surgical treatment. Our results that indicate some readily available substances are effective inhibitors of proteases in tears of eyes with active corneal ulcers provide practitioners with a possible valuable adjunctive treatment for vision-threatening diseases of the eyes.

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CHAPTER 6 DETECTION OF CTGF IN THE EQUINE TEAR FLUID, CORNEA AND LACRIMAL GLANDS Introduction Transforming growth factor beta (TGF-) activates fibrocytes to produce connective tissue growth factor (CTGF) (Berman et al., 1973; Igarashi et al., 1996). Overexpression of CTGF apparently plays an important role in fibrosis of various tissues (Chen et al., 2000; Gupta et al., 2000; Shi-wen et al., 2000; Takehara, 2001; Wunderlich, 2000). CTGF may be involved in corneal wound healing via induction of fibroblast proliferation and collagen production (Fraxier et al., 1996). After the report of CTGF presence in human serum (Sato et al., 2000) and its detection in the aqueous humor and tear fluid (van Setten et al., 1992; van Setten et al., 1996), CTGF has been suggested to play a role in ocular wound healing in concert with other proteins, growth factors and enzymes (Schultz et al., 1992; van Setten, 1998). However, one problem in previous studies has been the varying stability of CTGF in the fluids investigated, resulting in difficulties to estimate the correct level of protein concentration. With only one preliminary study so far published there is a considerable need for further thorough investigation on the presence of CTGF in tears and its pathophysiological importance. In melting diseases of the ocular surface such as keratitis and stromal ulceration swift remodeling and reestablishment of tectonic stability is urgently desired. Here CTGF may play an important role. Healing of corneal ulcers in horses, on the other hand, is often associated with profound fibrosis and corneal scar formation that can result in varying 123

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124 degrees of visual impairment (Brooks, 1999). The purpose of this study was hence to determine if CTGF was also present in the tear fluid of horses, to investigate possible alterations of concentration during corneal pathology, and to clarify its possible origin. Materials and Methods Materials All procedures were carried out according to the ARVO statement for the Use of Animals in Ophthalmic and Vision research and were approved by the University of Florida Animal Care and Use Committee as well as the UF College of Veterinary Medicine Clinical Research Review Committee. For the analysis of CTGF concentration in tear fluid samples were collected from 65 eyes of 44 horses that were either clinical cases (Figure 6-1) from the Large Animal Hospital at the University of Florida College of Veterinary Medicine Veterinary Teaching Hospital or, in case of animals without any ocular disease, from a farm in Florida. In total 32 samples were collected from normal eyes; 21 samples from eyes with corneal ulceration, and 12 samples from the unaffected contralateral eyes of horses with ulcers. The tear collections were performed after sedation of the animal with an intravenous injection of xylazine hydrochloride (0.5 mg/kg) and before any diagnostic procedure or any treatment. All tear fluid specimens were collected by capillary force only using capillary tubes with an atraumatic tip as previously described (van Setten et al., 1989) from the lower fornix. The time of collection of all tear fluid sample was not taken in this study. All samples were immediately centrifuged, transferred into Eppendorf tubes (10L aliquots), and stored at -80C until analysis.

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125 For the identification of CTGF in western blot two pools of tear fluid (100L) from horses with healthy eyes and horses with ulcerated eyes were analyzed and compared with human CTGF. For immunohistochemistry, five healthy corneas, five main lacrimal glands and five nictitating membrane glands were collected from horses with normal eye following euthanasia due to condition not related to this project. All samples were fixated in 4% formaldehyde buffer and stored in paraffin. Figure 6-1: Melting ulcer in the right eye of a 2 month old Thoroughbred horse. This case was involved in this study. Note the presence of hyperemic conjunctiva, corneal neovascularization at the periphery, and a 5-6 mm melting corneal ulcer with mild corneal edema and white opacification in the deep stroma in the center of the ulcer. CTGF Elisa Assay CTGF levels were determined using an enzyme immunoassay kit (CTGF Elisa) developed for human tear samples. This assay is a non-commercially available, quantitative "sandwich" enzyme immunoassay technique (Tamatani et al., 1998). Briefly, 96-well microtiter plates were coated with an affinity-purified polyclonal antibody specific for human CTGF at 37 C for one hour (a goat IgG against human CTGF). After washing, standards and samples, rediluted to a volume of 50 l, were added to the

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126 microtiter plates and incubated for 1 hour at room temperature. After another series of washes, a biotin-linked polyclonal antibody specific for human CTGF was added to the wells to bind to the CTGF molecules bound by the first antibody. After an incubation time of 1 hour at room temperature in the dark, alkaline-phosphate conjugated streptavidin was then added and incubated for an additional hour. P-nitrophenyl phosphate, the substrate for alkaline-phosphate, was added to the wells until the color change developed (about 30-45 minutes). Optical densities were obtained by reading the samples at 405 nm with a spectrophotometer Biotek FL340 Microplate reader (Biotek instruments, Winooski, VT). The detection limit of the assay was approximately 0.1 ng/ml CTGF. Dilution Curves A comparison of the human CTGF standard curve with an equine standard curve was made in order to assess the bio-equivalence, and to be able to interpret the results obtained for the equine samples using a human Elisa assay. The human standard curve was obtained by analyzing various samples obtained by serial dilutions of the human CTGF provided with the human Elisa kit. The initial human CTGF standard was at a concentration of 100 ng/ml and corresponded to 100% of the product in the sample tested. It was then diluted to obtain the final concentrations of 50, 10, 1.0, and 0.1 ng/ml. To obtain an equine dilution curve, tear samples from 10 horses (20 eyes) were pooled and an identical serial dilution of the 200 l mixture was made. The diluted samples were analyzed with the human Elisa kit. The equine pool without any dilution corresponds to 100% of product in the samples although its CTGF concentration was not known.

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127 CTGF Western Blot The two pools of tear fluid were formed by collection of tears from horses with healthy eyes and horses with ulcerated eyes. The tears were centrifuged at 14,000g for 10 minutes and the supernatant stored at 80 C until their analysis. The samples (pools of tear fluid from healthy and ulcerated horse eyes) as well as a molecular weight markers (MultiMark pre-stained multicolored standards, Invitrogen, Carlsbad, CA) and 50 ng of human recombinant CTGF were loaded on a 5% stacking, 12% SDS-PAGE gel. The electrophoresis was run at 125 V for 1 hour in a running buffer. The proteins were electrotransferred onto a PVDF membrane (Immobilon-P, Millipore Coporation, Bedford, MA) in a transfer buffer at 25V at 4 C overnight. The membrane was blocked in Tris-buffered saline (TBS) containing 10% non-fat milk (Blotting grade blocker non-fat dry milk, BioRad Laboratories, Richmond, CA) for one hour and then incubated in the same solution containing a dilution of 1:7000 of the goat anti-human CTGF for 30 minutes at room temperature. The blot was washed three times with 0.05% Tween-20 TBS and then incubated with rabbit anti-goat IgG alkaline phosphatase conjugate (Sigma, St Louis, MO) at the dilution of 1:7500 in the blocking solution for 30 minutes at room temperature. The membrane was washed again three times and the BCIP/NBT alkaline phosphatase substrate (Sigma, St Louis, MO) was added and color generated. The reaction was stopped by water washing. CTGF Immunohistochemistry Cells expressing CTGF were detected immunohistologically using a standard avidin-biotin amplification method (Blalock et al., 2003). Briefly, corneas, lacrimal glands, and nictitating membrane glands were obtained from five horses, fixed in 4% paraformaldehyde 0.1 M phosphate buffer solution (PBS) overnight at 4 C, dehydrated

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128 in an ascending series of ethanol, and embedded in paraffin. Paraffin-embedded sections were prepared and 6 m sections were mounted on microscopic slides (Superfrost/plus; Fischer Scientific, Pittsburgh, PA). Slides were deparaffinized and rehydrated with xylene and a graded series of ethanol. Slides were blocked with 10% horse serum in Tris-buffered saline (TBS) for 30 minutes at room temperature. Slides were then sequentially incubated with goat anti-human CTGF in TBS and 10% horse serum overnight at 4 C, washed three times with TBS, incubated with biotinylated horse anti-goat IgG secondary antibody (Vectastain ABC-AP kit,Vector Laboratories, Burlingame, CA) in TBS, washed, and incubated with alkaline phosphatase-conjugated streptavidin in TBS and 10% horse serum (Vectastain ABC-AP kit,Vector Laboratories, Burlingame, CA), washed, and incubated with alkaline phosphatase visualization substrate (Vector Red Alkaline Phosphatase substrate kit I: Vector Laboratories, Burlingame, CA). The reaction was stopped by water washing. The goat anti-human CTGF antibody was raised against recombinant human CTGF protein and purified with a CTGF-affinity column (Frazier et al., 1996). The antibody predominantly recognizes antigenic determinants on the N-terminal sequence of CTGF. The sections were photographed with bright-field illumination, and Nomarski phase-contrast microscopy at various magnifications. Results Detection and Quantification of CTGF in the Horse Tears The human Elisa kit was able to detect the CTGF in the equine sample. CTGF was detected in 23 normal unaffected eyes (72%) and 8 normal contralateral eyes (67%), with the mean CTGF levels ( SEM) being 51.5 19.2 and 13.4 3.9 ng/ml respectively. CTGF was found in 8 of the 21 eyes with corneal ulcers (38%) with the mean CTGF level ( SEM) being 26.3 14.8 ng/ml (Figure 6-2 & Tables 6-1 and 6-2). In contrast to

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129 eyes with corneal ulceration, often showing CTGF levels below detection limit, eyes with corneal laceration (n=3) did always show CTGF in their tears (mean 85.3 ng/ml 38.8 ng/ml). 020406080100120140160180CTGF concentration (ng/ml) Contralateral eyes Ulcerated eyes Normal eyes8/128/2123/32 Figure 6-2: CTGF levels in horse tears. Mean CTGF levels and standard deviations for 12 tear samples from the contralateral eyes of horses with corneal ulceration (CTGF was detected in 8 out of the 12 samples), 21 tear samples from the eyes of horses with corneal ulcers and lacerations (CTGF was detected in 8 out of the 21 samples), and 32 tear samples from the eyes of horses without any corneal condition in both eyes (CTGF was detected in 23 out of the 32 samples). The vertical lines indicate the standard deviation range. Table 6-1: CTGF in tear samples from horses with ulcerated corneas in one eye (12 eyes) and the non diseased contralateral eye Diseased eye Contralateral eye Tear sample # Diagnosis CTGF level (ng/ml) Tear sample # CTGF level (ng/ml) 1 Melting ulcer ND 1 30.40 2 Ulcer 1.07 2 19.73 3 Melting ulcer ND 3 15.73 4 Melting ulcer 23.97 4 15.39 5 Melting ulcer ND 5 0.79 6 Melting ulcer 6.10 6 30.31 7 Laceration 151.88 7 12.36 8 Superficial ulcer ND 8 36.32 9 Ulcer 9.07 9 ND 10 Melting ulcer 283.07 10 ND 11 Melting ulcer ND 11 ND 12 Melting ulcer ND 12 ND

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130 Table 6-2: CTGF from 9 tear samples from horses with ulcerated corneas in one eye (only tears from this eye were analyzed). Tear sample # Diagnosis CTGF level (ng/ml) 1 Corneal laceration 86.95 2 Corneal laceration 17.77 3 Deep stromal ulcer ND 4 Melting ulcer ND 5 Desmetocoele ND 6 Ulcer ND 7 Ulcer ND 8 Superficial ulcer ND 9 Melting ulcer ND Dilution Curves Bioequivalence In figure 6-3 a dilution curve shows the op tical density (values obtained from the microplate reader) represented as a function of the percentage of protein in the sample tested. The human standard curve was very li near with a correlat ion coefficient of 0.9978 whereas the equine dilution curv e was slightly less linear with a correlation coefficient of 0.865. However, the main difference between the human CTGF standard curve and the horse pool dilution curve was regarding th eir slopes: the slope being 0.0076 and 0.0019 in the human and horse re spectively (Figure 6-3). y = 0.0076x + 0.042 R2 = 0.9978 y = 0.0019x + 0.0758 R2 = 0.865 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 020406080100120 Percentage of product in sampleOptical density Figure 6-3: CTGF Dilution curves. A serial dilution of a human CTGF ( ) was made and used as the standard for this Elisa assay (0, 0.1, 1.0, 5.0, 10, 50 & 100 ng/ml). The

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131 concentration of 100 ng/mL of hu man CTGF corresponds to 100% of product in the samples in the graph. An identical serial dilution of a pool of horse tear samples is presented ( ). CTGF Western Blot Western blot showed the two characte ristic CTGF bands on all the equine samples, similar to those observed ar ound 38 kDa with the human CTGF standard (Figure 6-4). These results indicate that th e detectable amount of CTGF in the equine tear fluid is indeed CTGF. Figure 6-4: Western Blot of tear fluids from horses with healthy eyes (lane 3) and ulcerated eyes (lane 4) The two characteristic CTGF bands around 38 kDa were noted on all the equine samples (lanes 3 and 4), similar to those observed with the human CTGF standard (lane 2). CTGF Immunohistochemistry Immunoreactivity to CTGF specific antibodie s was noted in the corneal epithelium and in the corneal endothelium whereas stai ning was absent in the corneal stromal (Figure 6-5). In the corneal epithelium, st aining was concentrated in the squamous superficial epithelial cells (Figure 6-5). The immunohistochemical stai ning for CTGF in the equine lacrimal glands and the nictitating membrane glands revealed specifi c staining for CTGF predominantly in the acinar cells, the epithelial cells of the intr alobular ducts, and the intralobular connective tissue (Figures 6-6 and 6-7). In the acinar cell s, the staining was concentrated close to the 1 2 3 4 52 kDa 31 kDa Molecular Weight Human CTGF Equine normal Equine diseased Markers Standard tear fluid tear fluid

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132 basally located nuclei, opposite to the lume n (Figures 6-6 and 6-7). No immunoreactivity was detected in the control sectio ns (Figures 6-5, 6-6 and 6-7). Figure 6-5: Immunolocalization of CTGF in equi ne cornea. H&E staining of the equine cornea ( A ) where corneal epithelium (1), stroma (2) and Descemets membrane corneal endothelium (3) can be distinguished. Nomarski pha se-contrast 1 observation ( B ) and bright-field observation ( C ) of the negative control sectio ns showed no staining at all. Immunohistochemical staining for CTGF in the equine cornea ( D E F ) showed CTGF specific staining in the co rneal epithelium, especially in the squamous superficial cell layer ( white arrow ) and the corn eal endothelium ( black arrow ). Original magnifications were X100 ( A B C D ) and X200 ( E F ) D A E C B F 1 3 2 1 3 2 1 2 3

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133 A B C D E F Figure 6-6: Immunolocalization of CTGF in equine lacrimal gland. H&E staining of the equine lacrimal gland (A) where acini (inset), intralobular ducts, lymphocytes in the intralobular connective tissue (inset) can be distinguished. Nomarski phase-contrast 1 observation (B) and bright-field observation (C) of the negative control sections showed no staining at all. Immunohistochemical staining for CTGF in the equine lacrimal gland (D, E, F) showed CTGF specific staining in the acinar cells (white arrow), the epithelial cells of the intralobular ducts (black arrow), and the intralobular connective tissue (striped arrow). In the acinar cells, the staining was concentrated close to the basally located nuclei (white arrow), opposite to the lumen. Original magnifications were X100 (A, B, C, D) and X400 (E), and X1000 (inset in A, F). 1 2 1 2 1 2 C A B 1 2 D E F Figure 6-7: Immunolocalization of CTGF in equine nictitating membrane gland. H&E staining of the equine nictitating membrane (A) where the hyaline cartilage (1) and the nictitating membrane gland (2) composed of acini (inset) can be distinguished. Nomarski phase-contrast 1 observation (B) and bright-field observation (C) of the negative control sections showed no staining at all. Immunohistochemical staining for CTGF in the equine nictitating membrane gland (D, E, F) showed CTGF specific staining in the acinar cells (white arrow), the epithelial cells of the intralobular ducts

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134 (black arrow), and the intralobular connective tissue (striped arrow). Original magnifications were X100 (A, B, C, D) and X400 (E), and X1000 (inset in A, F). Discussion This study identifies CTGF in the horse tear film. CTGF was present at detectable levels in approximately 70% of the tears from normal horse eyes and tears from the normal, contralateral eyes of horses with corneal ulcers. The absence of detectable CTGF in the remaining tears fluid samples could indicate the total absence of any CTGF in tears of some horses or a concentration below detection limit. Furthermore, similarly as reported earlier for human tear fluid (van Setten et al., 2003), the processing of the equine tear samples involving centrifugation and storage at -80C until analysis could have affected the data. However, we do believe the comparison between our samples is valid as all samples have been handled and stored in the same manner. The observation that CTGF was found in about 40% of the tears from ulcerated horse eyes at a lower level than in the normal eyes (Figure 6-2) might reflect a dilution effect resulting from increased tear volume, such as shown for EGF (van Setten, 1990). It also could indicate a significantly enhanced receptor binding to the surface at specific stages of the healing period, finally it may be the result of the temporary exhaustion of CTGF reservoir in lacrimal gland cells. The latter may also be the explanation for decreased levels of CTGF found in non-ulcerated sister eyes of horses with corneal ulceration in one eye. The simultaneous presence of enzymes in corneas with corneal ulceration possibly may also contribute to reduced CTGF levels in tears due to proteolysis. The importance of proteolytic enzymes and their regulatory inhibitors in tears has been investigated extensively (Berman et al., 1973; Berman et al., 1988; Kueppers, 1971; Matrisian, 1990; Matsubara et al., 1991b; Tervo et al., 1988; Thorg et al., 1984; Thorg et al., 1983; Tsung

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135 and Holly, 1981; Twining, 1994; Twining et al., 1994a; Twining et al., 1994b; Twining et al., 1994c; van Haeringen and Thorg, 1986). In the present study we identified CTGF in the lacrimal gland and the glands of the nictitating membrane, both probably a major source of CTGF. The localization of the specific staining is very similar to that shown for TGF(Schultz et al., 1992; van Setten et al., 1996). According to our results, both the acini and cells of the inter-acinar ducts are involved in the process of CTGF release into tear fluid. The exact mechanisms of CTGF release has yet to be clarified. CTGF was also identified in the equine healthy cornea which correlates with the findings in previous studies performed other species (Blalock et al., 2003). The present study also shows a strong antigenic similarity between human and equine CTGF, and therefore demonstrates the feasibility of measuring CTGF protein in horse tears using the human CTGF Elisa. There was, however, an incomplete equivalence between the data obtained for human and horse samples as the standard curves were not parallel (Figure 6-3). The values obtained for the horse samples using this human Elisa kit may indicate slightly lower concentrations of CTGF found in the horse tears than a test for horse specific CTGF might do. The occurrence of CTGF specific mRNA in retrocorneal membranes and subepithelial membranes has been recently reported (Wunderlich et al., 2000) and suggests an involvement of CTGF in various wound-healing events in the eye. Considering tear fluid composition as a rough mirror of ocular surface conditions, the current study supports the suggested importance of CTGF in corneal pathology. Its presence in the tears of other horses with ulcers and during healing phases of ulcers

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136 deserves further investigation. The identification of other growth factors in the horse tear fluid that can influence CTGF production or release such as TGFin the human tear fluid (Gupta et al., 2000; Igarashi et al., 1996; Vesaluoma et al., 1997; Wang et al., 2001) would be helpful to understand the mechanism of corneal wound healing and scarring. If not generally, at least in horses with high CTGF levels this growth factor could be of major importance for physiology or wound healing events of the equine cornea. Possibly the CTGF like growth factors are the stromal equivalent to EGF (van Setten et al., 1992) in corneal wound healing.

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CHAPTER 7 CONCLUSIONS When the cornea is injured, multiple systems are activated which produce a series of complex and coordinated cellular processes that ultimately result in a healed corneal wound. Healing of corneal wounds is an exceptionally complex process involving the integrated actions of multiple growth factors, cytokines, and proteinases produced by epithelial cells, stromal keratocytes, inflammatory cells, and lacrimal glands. Multiple autocrine and paracrine interactions occur between epithelial cells and activated stromal fibroblasts, and the exocrine actions of factors secreted by lacrimal gland cell into the PTF. For this reason the tear film can be described as the mirror of the event occurring at the corneal surface. When a corneal wound occurs, keratocytes around the edge die by apoptosis and chemotactic factors including growth factors such as PDGF, TGF-, TGFare released and attract inflammatory cells, resulting in the initiation of re-epithelialization, contraction of connective tissue, and stimulation of angiogenesis (Schultz, 1997). Proteinases are involved in the early phase of corneal wound healing as they play a role in the epithelium cell migration by breaking down the cellular attachments (Cameron, 1997; Schultz et al., 1993). Beyond the site of injury, quiescent keratocytes become activated into fibroblasts and migrate to the site of injury. This migration is followed by cell proliferation, and finally deposition of fibronectin, laminin, heparin sulfate and other ECM components. Many growth factors and cytokines have been implicated in stimulating synthesis of these components, as well as cell proliferation, migration and 137

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138 also differentiation of epithelial and stromal cells and angiogenesis (TGF-, TGF-, PDGF, FGF) (Schultz et al. 1992).The fibroblasts deposit the reparative collagen and eventually synthesize enough ECM to form a scar replacing the damaged tissue. Corneal transparency is determined in large part by the structure of the stromal ECM. In case of corneal injury, corneal stroma is not regenerated but repaired. It is instead replaced with unspecialized tissue that creates the scar (Cameron, 1997). The repair tissue matrix contains component molecules particularly fibronectin, not usually present in stroma, and the newly synthesized collagen fibrils are thicker and more variable in size than those of the normal stroma. All these events in the process of wound healing must be reversed for the tissue architecture to return to normal. The removal of the inflammatory mediators which were generated initially must first occur. Infiltration of monocytes and leukocytes must decline, and then removal of extravasated fluid, protein, cellular debris, granulocytes and macrophages occurs as the wound regeneration process continues. Some improvement in the transparency of repair tissue can occur over the long term through the progressive remodeling of the repair tissue matrix and the MMPs appear to be involved in this process to remove the provisional ECM. A delicate balance between protein synthesis and proteolysis is present in corneal wound healing with many factors including growth factors, proteinases and proteinases inhibitors involved (Figure 7-1). Any imbalance can lead to fibrotic processes (inhibitors > proteinases) or to excessive tissue destruction (proteinases > inhibitors) (Figure 7-1). The major complications of corneal trauma, infection and surgery in horses are corneal liquefaction (i.e., corneal melting) and corneal scarring. A vast amount of research on stromal wound healing and scar formation exists in human and other species

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139 G G r r o o w w t t h h f f a a c c t t o o r r s s p p r r o o t t e e i i n n a a s s e e i i n n h h i i b b i i t t o o r r s s P P r r o o t t e e i i n n a a s s e e s s C C o o r r n n e e a a l l m m e e l l t t i i n n g g F F i i b b r r o o s s i i s s Figure 7-1: The delicate balance between proteinases, proteinases inhibitors, and growth factors. but there is very limited similar information in horses. The major objectives of this research were to begin to understand the events and regulations of stromal wound healing in horses at the molecular level. The long-term goal of this project is to apply this knowledge towards the design of clinical treatments that will regulate certain key molecules in order to reduce the amount corneal liquefaction and scar formation in horses. Prior to this research, very little was known about the involvement of MMPs and growth factors in the tear film and cornea of the horse. These findings support the hypothesis of the involvement of MMP-2 and MMP-9 as well as CTGF in corneal wound healing and scarring in horses. This study demonstrates the expression of MMP-2 protein in the equine lacrimal gland and the gland of the nictitating membrane. The expression of MMP-2 and MMP-9 proteins was also found in the healthy cornea of the horse and was increased in the ulcerated equine cornea. This correlates positively with several experiments showing the same results in other species (Chandler et al., 2003; Gabison et al., 2003; Kenney et al., 1998; Reviglio et al., 2003; Yang et al., 2003; Ye et al., 1998). This is the first example of an analysis of MMP-2 and MMP-9 protein expression in equine healthy corneal tissues

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140 as well as in an injured equine cornea. MMP-2 and MMP-9 are of major importance in terms of remodeling and degradation of the corneal stromal collagen in horses as described in other species (Fini and Girard, 1990; Fini et al., 1992). This study documents the fact that the origin and purpose of MMP-2 and appear to differ at the corneal level. In this study, the level of MMP-2 and MMP-9 activity was determined in a total 330 tear fluid samples by the use of gelatin zymography. Activity was detected in all tear fluids samples, and it was concluded that MMP-2 and MMP-9 are constant components of the equine tear fluid. TFF rate and MMP proteolytic activity were significantly increased in the tear fluid of horses with ulcerative keratitis. Based on the collection and analysis of a total of 124 serial tear fluid samples in 10 horses with ulcerative keratitis, we also documented for the first time that total MMP activity decreases in equine tears as the corneal epithelial and stromal healing occur. Since the long term goal of this project is to develop agents that will reduce corneal melting with minimal side effects, initial experiments using various anti-proteolytic agents were performed. We documented a high amount of inhibition of equine MMP activity in vitro by the use of potassium edatate diaminetetrataacetatic acid (EDTA), doxycycline, N-acetylcysteine (NAC), equine serum (that contains various inhibitors including 2-macroglobulin and 1-proteinase inhibitor), ilomostat, and 1-PI. These anti-protease compounds utilize different mechanisms to inhibit different families of proteases present in equine tears and a combination of inhibitors may be indicated for severe corneal ulceration in horses (Brooks, 1999; Hibbetts et al., 1999). Further animal studies will be performed in the future to confirm that these anti-proteolytic agents have similar effect in vivo. Other studies need to be conducted in order

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141 to investigate the regulation of MMP activity directly by targeting the MMP active site (pro-enzyme activation and active enzyme inhibition), but also indirectly by targeting the MMP synthesis (transcriptional regulation) at the level of the equine cornea and tear film The level of CTGF was determined by Elisa assay in a total 64 tear fluid samples in this study. Since this molecule was detected in 39 of the samples, we concluded that CTGF is a component of the equine tear fluid. This data demonstrates that CTGF was present in the equine tear film. This study also indicates the expression of CTGF protein in the healthy cornea, the lacrimal gland and the gland of the nictitating membrane of the horse. CTGF has been shown to be synthesized and secreted into tears by lacrimal gland cells (van Setten et al., 2002) and has been found in the cornea of other species (Blalock et al., 2003). These findings are important but further studies are needed. The identification of other growth factors in the horse tear fluid that can influence CTGF production or release such as TGFin the human tear fluid (Gupta et al., 2000; Igarashi et al., 1996; Vesaluoma et al., 1997, Wang et al., 2001) would be helpful to understand the mechanism of corneal wound healing and scarring in horses, and to develop anti-fibrotic therapy in horses. It is currently known in other species that the transforming growth factor beta family plays a dominant role in the regulation of stromal fibrosis (Chen et al., 2000). It has recently been shown that CTGF is involved in the process because of its regulation by TGF(Blalock et al., 2003). CTGF induces scar formation by mediating many effects of TGF beta on ECM production and corneal scarring. Future studies should for example investigate the role of TGFin equine corneal scarring and then search for agents inhibiting these molecules in order to reduce the corneal scarring in horses with

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142 minimal side effects. Steroids can reduce the corneal scarring but their use in horse is not without severe risk. Some non specific anti-cancer drugs haven been used to treat corneal scarring such as 5 fluorouracil and mitomycin C, but they cause serious side effect such as epithelial defects and endothelial cell damage (Lee, 1994; Khaw et al., 1993). Recent studies using ribosome therapy targeting CTGF have shown that the ribozyme reduced the expression of CTGF and its biological effects, and might be an interesting alternative to consider in horses. In summary, these experiments have made tremendous progress in developing and evaluating the role of MMP-2, MMP-9, and CTGF in the process of corneal wound healing and scarring in horses. However, the lack of information on the molecular regulation of corneal scarring in horses appear to be almost infinite in extent, promising many exciting years in research on MMPs and growth factors and corneal wound healing and scarring in horses.

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BIOGRAPHICAL SKETCH Franck Jean Ollivier was born on January 6, 1972, in La Tronche, France. He attended elementary and secondary schools in France. Franck attended the National School of Veterinary Medicine in Lyon, France, from 1992-1996. After attending veterinary school, Franck did an ophthalmology internship in the National School of Toulouse under the mentorship of Dr. Alain Regnier. After passing the national exam for veterinary ophthalmology in 1997, he worked as a research project manager in the R&D department for a French veterinary pharmaceutical company, in Sydney, Australia, from 1997 to 1999. Franck returned to Toulouse to finish his thesis titled Experimental Study on the Ocular Tolerance in Rabbit of a Veterinary Cyanoacrylate Tissue Adhesive (Vetbond) and he earned his Dr. Med. Vet. degree in 1999. He worked as a veterinary ophthalmologist in a private referral practice in Grenoble, France, in 1999-2000. In 2000, Franck became a graduate student at the College of Veterinary Medicine, University of Florida, Gainesville, Florida, with duties as a teaching and research assistant under the leadership of Dr. Dennis E. Brooks. In 2003, Franck started his residency in comparative ophthalmology at the College of Veterinary Medicine, University of Florida, Gainesville, Florida, under the mentorship of Dr. Dennis E. Brooks and Dr. Kirk N. Gelatt, which he will finish in 2006. His special interests include the tear film, cornea, anti-proteolytic treatment, and corneal graft. 158