i      ESTABLISHING CANINE TRABECULAR MESHWORK CELL CULTURE  By  Hsiang-Rong Tsai               A THESIS  Submitted to Michigan State University  in partial fulfillment of the requirements  for the degree of   Physiology - Master of Science  2016ii ABSTRACT  ESTABLISHING CANINE TRABECULAR MESHWORK CELL CULTURE  By  Hsiang-Rong Tsai  Glaucoma is a leading cause of incurable blindness. The most common form of glaucoma is primary open-angle glaucoma (POAG) and is associated with pathological alterations of aqueous outflow facilities which lead to increased resistance and elevated intraocular pressure (IOP). Trabecular meshwork (TM) cells residing within the iridocorneal angle are considered key regulators of aqueous humor outflow; their malfunction is considered one of major factors in the pathogenesis of POAG. Glaucoma not only affects humans, but also other species such as dogs.  Dogs are more accessible than other species to study the disease. Canine and human POAG share many features such as plaque formation within the TM with resulting slow progressive elevated IOP, and cupping of the optic nerve head. Because of the similarities between human and canine disease, we posit that the study of canine TM cells will be beneficial for the understanding of disease mechanisms in both dog and human. The purpose of this study was to establish primary canine TM cell cultures. Canine TM explants were carefully isolated and transferred to the cell culture dish. TM cells were identified by their expression of collagen type IV, alpha smooth -SMA) and laminin, but not desmin and keratin. Another key feature of TM cells is their phagocytosis activity.  Finally, the cultured TM cells form typical cross-linked actin networks (CLANs). To the best of our knowledge, this is the first report of primary canine TM (CTM) cultures.     iii ACKNOWLEDGEMENTS     I would like thank all individuals who supported my research and thesis writing, especially my colleagues, staff and professors from both the Department of Physiology and the College of Veterinary Medicine at Michigan State University.    First of all, I would like to appreciate my mentor Dr. András M. Komáromy. Like a lighthouse he provided me guidance for my research and professional life. His dedication for both research and clinical work provide me with an example about the life of a clinical scientist. Under his influence and encouragement, I have chosen to pursuit a degree in veterinary medicine at Michigan State University following my graduate work.  I intend to dedicate my life to both research and clinical work with a hope to have a positive impact in this world. And I would like to thank my committee members Dr. Susanne Mohr and Dr. Arthur Weber who have given me constructive suggestion and guidance for my research. Special thanks to Dr. Susanne Mohr as director of the Physiology Graduate Program who guided both my graduate work and my career decision to pursuit a veterinary degree. I feel fortunate to have joined her program.       I would like to thank Mrs. Christine Herman, research associate, and Ms. Kristin Koehl, veterinary technician, for their full support of my research work. Without their kind support, I would not have been able to complete my research.  I would also like to thank the following past members of the Komáromy Lab for their help and company: Forrest Nussdorfer, Josh Laske, and Gabriel Stewart.    Finally, I would like to thank my family members for their support, especially my parents and brother. Without their support, my pursuit of research would have been much harder. And I iv want to thank the support from Taiwan through their generous scholarship and a grant from Midwest Eye-Banks to assist my study.v TABLE OF CONTENTS  LIST OF TABLES vii  LIST OF FIGURES viii  KEY TO ABBREVIATIONS ix  CHAPTER 1  INTRODUCTION 1   Definition of glaucoma 1    Primary open-angle glaucoma (POAG) 1   Aqueous humor (AH) dynamics 4   Aqueous humor formation 4   Aqueous humor outflow 5   Physiology and pathophysiology of the TM 6   Current treatment options for POAG 8   Animal models of POAG 11   In vitro model systems of POAG 12   Rational for studying canine glaucoma and TM 13  CHAPTER 2  METHODS 15   Isolation of the canine TM (CTM) 15   Primary TM cell culture 17   Fibroblast primary culture 19   Characterization of TM cells  ICC 19   Characterization of TM cells - Phagocytosis assay 22   Dexamethasone (DEX) challenge 22  CHAPTER 3  RESULTS 25   Histological evaluation of isolated CTM explant 25   Characterization of cultured TM cells: Immunocytochemistry 30   Characterization of cultured TM cells: Phagocytosis assay 31   Characterization of cultured TM cells: DEX challenge - CLAN formation 33   Characterization of cultured TM cells: DEX challenge  gene expression 35  CHAPTER 4  DISCUSSION 38   Most TM tissue was excised for cell culture as confirmed by histological examination 38   Multiple TM-specific protein markers are observed at our TM cell strains 38   CLAN formation observed in our non-DEX treated CTM cells 39   Phagocytosis activity of the CTM cells 39   CTM cells didnt have significant DEX response 40   The concern of inadvertently isolating non-TM cells 41   Senescence observed in the CTM cell strains 42  vi CHAPTER 5 - SUMMARY AND FUTURE DIRECTIONS 43   Summary 43   Future direction 43  REFERENCES 47                      vii LIST OF TABLES   Table 1-1: Genes associated with POAG 3 Table 1-2: Current therapies for POAG: Pharmacological therapies 9 Table 1-3: Current therapies for POAG: Surgical therapies 10 Table 1-4: Animal models of POAG & ocular hypertension 11 Table 2-1: Characterization methods previously used for TM cell identification 21 Table 2-2: The list of antibodies used in the experiments 21 Table 3-1: Established primary CTM cell strains  26 Table 3-2: Characterization of canine cell strains 30 Table 3-3: Phagocytosis activity of canine cell strains 33 Table 3-4: Completed q-RTPCR of CTM cell strains and other type of cells 37   viii LIST OF FIGURES   Figure 1-1 AH dynamics in the canine eye 6 Figure 1-2 ECM accumulation in the TM of POAG-affected dogs 13 Figure 2-1 Dissection procedure 16 Figure 2-2 Experimental design 18 Figure 3-1 Cell migration and morphology of canine TM cells and fibroblasts 29 Figure 3-2 The ICC result of CTM 31 Figure 3-3 Phagocytosis activity of cultured CTM cells 32 Figure 3-4 CLAN formation in canine TM cells 33 Figure 3-5 Relative number of CLAN-positive cells in different canine cell strains 35                        Figure 3-6 Relative gene expression of MYOC following 3 days of DEX treatment in                    different canine cell strains 36  ix KEY TO ABBREVIATIONS  AAP angular aqueous plexus AH aqueous humor  -SMA alpha-smooth muscle actin  CE ciliary epithelium CLAN cross-linked actin network CSTM corneoscleral trabecular meshwork CTM canine trabecular meshwork DAPI                                           4', 6-diamidino-2-phenylindole: DEX: Dexamethasone ECM extracellular matrix FBS fetal bovine serum ICA iridocorneal angle ICC immunocytochemistry IOP intraocular pressure iPSCs induced pluripotent stem cells ISVP intrascleral venous plexus  NPE non-pigment epithelium  PACG primary angle-closure glaucoma PBS phosphate-buffered saline PE pigmented epithelium POAG primary open-angle glaucoma q-RTPCR quantitative reverse transcription polymerase chain reaction x rAAV recombinant adeno-associated virus TGF-2 transforming growth factor beta 2  TM trabecular meshwork UTM uveal trabecular meshwork  wt wild type             1 CHAPTER 1  INTRODUCTION  Definition of glaucoma   Glaucoma is a leading cause of incurable blindness. It is defined as a progressive optic neuropathy characterized by the loss of retinal ganglion cells and clinically distinguishable cupping of the optic nerve head 1. There are different forms of glaucoma, both primary and secondary, which all share the same risk factor of elevated intraocular pressure (IOP). This research is focused on primary glaucoma with no clinically observable underlying cause such as inflammation 2, neoplasia 3, or pigmentary dispersion 4.  The two most common forms of primary glaucoma are primary open-angle glaucoma (POAG) and primary angle-closure glaucoma (PACG).  In both of these diseases, alterations along the aqueous humor (AH) outflow pathway result in increased resistance and IOP elevation (Fig. 1-1). While these changes are associated with closure of the iridocorneal angle (ICA) in PACG, the angle appears clinically normal and open in POAG 1. In POAG, it is believed that the pathogenesis of disease is associated with molecular and cellular pathological changes in the trabecular meshwork (TM), a key regulator of AH outflow, resulting in impaired AH drainage from the eye. Those defects within the TM remain largely unknown.   Primary open-angle glaucoma (POAG)   POAG is considered a major type of glaucoma worldwide 5. Elevated IOP is considered a major risk factor of POAG, but it can also develop without obvious IOP change, such as in  50% percent of European- and African- descent patients 6 and 92% for Japanese-descent patients 7,8.  2 In additional to IOP, several other POAG risk factors have been identified 9,10, such as aging, family history, and ethnicity.    Intraocular pressure (IOP): The level of IOP is the only risk factor which can be intervened by medical and surgical treatment 11. In healthy individuals, the range of habitual IOP is 16.3 - 21.3 mmHg. In contrast, the IOPs of hypertensive POAG patients are usually over 22 mmHg 12.  In addition to being elevated, IOPs also fluctuates more in glaucomatous vs. normal eyes 12.    Aging: Both the incidence and prevalence of POAG strongly correlate with aging, especially after 40 years of age, due to gradual increase in AH outflow resistance 13. For example, people of European-descent have a  25% incidence to develop POAG by the age of 64 years and  up to 75% by 81 years 13. A similar increasing age-related POAG incidence has also been observed in people of African-descent. Senescence of TM cells may contribute to this aging-related increase in AH outflow resistance 14. Furthermore, aging seems to have a negative impact on the biomechanical properties of the eye: Elevated scleral stiffness associated with aging can result in increased stress and exacerbated IOP-related damage of the optic nerve head 15. Finally, aging-related decrease of cerebrospinal fluid (CSF) pressure 16 may contribute to optic nerve head damage as well because of greater translaminar gradient 17.     Family history and genetics: Up to 50% of POAG patients have affected family members 18.  In addition, first degree relatives of an affected individual have a 22% risk to develop POAG vs. 2.3% risk in other family members 19. This suggests that genetic predisposition is a critical risk factor of POAG development. The study of POAG-affected families not only identified high risk populations but also POAG candidate genes 20. At least 7 genes have been associated with POAG: MYOC 21,22, CYP1B1 23, WDR36 24, OPTN 25,26, LMX1B 27, NTF4 28 and ASB10 29,30 3 (Table 1-1). Other genes have been associated with POAG risk factors such as optic nerve head morphology, IOP and refractive error 31,32.   Table 1-1: Genes associated with POAG Gene Name Location/Locus Function Reference MYOC Myocilin, TIGR 1q23/ GLC1A Cell migration, mitochondria, ECM turnover and cytoskeleton. 21,22 CYP1B1 Cytochrome P450 family 1 2p21/GLC3A Metabolism of drug and lipid synthesis 23,33 WDR36 WD repeat domain 36 5q22/GLC1G Ribosomal processes and cell growth 24,34,35 OPTN Optineurin 10p14-p15/GLC1E Rab-binding protein, TNF-alpha signaling NFkB pathway, immune response, apoptosis, vesicular transport, mitosis, cellular morphogenesis, oxidative stress protection. 25,36 LMX1B LIM homeobox transcription factor 1-beta 9q34.1/NPS Normal patterning of the dorsoventral axis, establishment of mid brain-hindbrain boundary, development of cerebellum and dopaminergic and serotonergic neurons. 27,37-39 NTF4 Neurotrophin-4 19q13.33/GLC1O Survival and differentiation of mammal neurons, neurotrophin signaling pathway 28,40,41 ASB10 Ankyrin Repeat And SOCS Box Containing 10 7q35-q36/GLC1F IOP regulation 29,30    Ethnicity: The ethnicity was considered another important risk factor for glaucoma. For example, the study on African-derived population in the world-wide glaucoma epidemiology has consistently shown high prevalence of POAG compared with other populations 9,42-45.  4 Aqueous humor (AH) dynamics    The AH is a transparent medium that fills the anterior chamber located between iris and cornea. It does not only provide IOP to support the shape of the eye but also delivers nutrients and removes waste from the eyes 46. In the normal eye, IOP is tightly regulated by well-coordinated production and outflow of AH 47. In POAG-affected eyes, IOP can become elevated due to a dysregulation of the normal AH dynamics, especially an increase of outflow resistance through the TM  this is the main focus of our research. In the following paragraphs we will briefly discuss AH dynamics in the normal eye, before entering the detail of the POAG disease pathogenesis 48.   Aqueous humor formation    The ciliary body produces the AH. It can be subdivided in the pars plicata and pars plana which represent the anterior and posterior portion respectively.  The ciliary epithelium (CE) of the pars plicata is the main location of AH production 49. The CE covers the ciliary processes and is composed of two layers, the superficial non-pigment epithelium (NPE) and deep pigmented epithelium (PE). The basolateral surfaces of NPE cells face the posterior chamber while its apical surface forms gap junction to the counter part of the PE cells (arrow, Figure 1-1) 50. The other side of the PE is in contact with the stroma of the ciliary process where solutes accumulate. In order to avoid blood protein and other inflammatory components to leak uncontrollably into the eye, the NPE cells form tight junction with neighbor cells; this is the blood-aqueous barrier 50.  5   In order to bring the nutrition from the blood to the eye, lipid-soluble and water soluble substances move via diffusion and ultrafiltration from the fenestrated capillaries across the ciliary stroma to the CE.    In the final stage of AH production, the solutes/substances are transported across the CE through diffusion, ultrafiltration and active transport. Among of them, active transport contributes about 80% to 90% of total AH formation 46,51. Active transportation relies on the ion transporters 49. The energy for active transportation is provided by hydrolysis of adenosine triphosphate (ATP) and mediated by Na+-K+-ATPase, which is mostly located in the NPE 50. Besides Na+-K+-ATPase, NPE also requires carbonic anhydrase to secrete bicarbonate to regulate pH and maintain osmotic gradient for continuous AH secretion 52. Carbonic anhydrase is an important target for glaucoma treatment: Inhibitor of carbonic anhydrase reduces AH production and thereby lowers IOP 53.   Aqueous humor outflow   IOP is regulated and maintained mainly by the conventional outflow 54. Once the AH is formed, it flows from posterior chamber via pupil to the anterior chamber then drains through the ICA. AH outflow consists of conventional/trabecular and unconventional/uveosclera outflow. Unconventional outflow occurs via ciliary muscle and sclera, but is only responsible for <15% of AH outflow in the normal human eye 55. In contrast, trabecular outflow provides the major drainage of AH and is regulated by the TM, a loose porous structure within the ICA (Figure 1-1.). The TM is composed by three components through which the AH has to drain: uveal, corneosclera and juxtacanalicular/ cribriform meshworks (Figure 1-1.) 56. The pore size of the TM and AH flow rate are controlled by ciliary muscle tendons that are connected to an elastin 6 net within the juxtacanalicular TM 57. Once it has passed through the juxtacanalicular meshwork,  and through collector channels the episcleral veins and the systemic blood circulation. The circular Schl is a unique structure that can only be found in human, primate and mouse 58-60, while other species such as dog 61, cat 62, pig 63, horse 64, rabbit 65 and zebrafish 66 have an angular aqueous plexus (AAP) instead to drain the AH.          Figure 1-1 AH dynamics in the canine eye. The AH is formed and secreted by the CE covering the ciliary body (arrow). It drains through the TM and angular aqueous plexus within the ICA.   Physiology and pathophysiology of the TM    TM in normal eyes: The TM plays a critical role in regulating AH outflow and maintains the IOP in a normal range. It is derived from neural crest and cranial paraxial mesoderm 67. There is increasing evidence that even once early differentiation is completed, a small population of progenitor cells reside within the TM and serve to replenish the loss of TM cells in the adult eye  68. There is considerable evidence that the major AH outflow resistance is located in the 7 juxtacanalicular TM and the inner wall of  69. This resistance is regulated by cellular and extracellular factors/components. The extra cellular matrix (ECM) production within the TM plays a major role in the resistance regulation 57. Uveal and corneosclera TM form the beams of connective tissue or so called trabecular lamella which contain the core of elastic fibers and collagen surrounded by the sheaths 69. These sheaths are formed by the TM cells, which highly express collagen type IV and laminin, important markers to characterize TM cells, while the core of the beams consist of collagen I and III 70,71. The beams connect to each other in different layers of the TM to form porous filter-like structures. The thickness of beams increases with age, contributing to the increased POAG risk with age 69. In contrast, the juxtacanalicular TM does not form beams of connective tissue but fibril elements of ECM instead. Juxtacanalicular TM cells and ECM fibrils together form an irregular network, the cribriform plexus which is lined against l; this juxtacanalicular TM plays a major role in regulating the resistance of AH outflow 72. Besides ECM turnover, the TM cells also have phagocytosis activity as a self-cleaning mechanism and to reduce/regulate the resistance of AH 73.    TM in POAG eyes: Both cellular and extracellular factors contribute to increased AH outflow resistance in POAG-affected eyes. ECM plaque formation and fibrosis have been found within the juxtacanalicular TM of POAG-affected eyes (Figure 1-2.) 57,74,75. Furthermore, the TM cell structure appears to be altered in these eyes with increased formation of cross-linked actin networks (CLANs) 75. The increased number of CLAN-positive TM cells results in increased cellular stiffness and contractility and higher AH outflow resistance 76. Much effort has been put toward the investigation of the molecular pathways and growth factors involved in the cellular and extracellular changes within the TM 77. Increased activation of transforming growth factor 8 beta 2 (TGF-2) likely plays a major role since elevated concentrations can be found within the AH of POAG patients 78. TGF-2 also plays a major role in wound healing supporting its relevance in tissue fibrosis 79. By increasing TGF-2 in perfused human anterior chambers, outflow resistance increased and related ECM gene expression, such as fibronectin and plasminogen activator inhibitor-1, were also noticeably upregulated 80. In addition, increased MYOC level in human POAG AH is considered another culprit for disease development within the TM 81.    Genetic predisposition and resulting altered gene expression is recognized as a critical factor within the glaucomatous TM (see also Family history and genetics above). Mutations  in genes such as Pax6 82, LTBP2 83, CYP1B1 84, MYOC 85 affect the TM development  and can lead to congenital glaucoma. The MYOC gene is considered a major POAG candidate gene 86. Since MYOC is highly expressed in the TM, the hypothesis was raised that mutant MYOC accumulation within the TM results in TM malfunction and glaucoma 87. Even though MYOC mutations are contributing to the pathogenesis of some forms of POAG and MYOC is highly expressed in glaucomatous TM cells, its major function remains unknown 88.  Current treatment options for POAG   Pharmacological approach: Current pharmacological therapies for POAG aim to either reduce AH formation or increase unconventional outflow (Table 1-2). For example, carbonic anhydrase inhibitors are used to inhibit the production of AH at the CE 89. Prostaglandin analogs increase unconventional outflow 90. Recently, a new class of glaucoma drugs has been developed, Rho-kinase inhibitors, which relax the TM and increase AH outflow 91.  9   Surgical approach: Surgical treatments focus on either reducing AH production or increasing AH outflow (Table 1-3). These include the use of lasers to partially destroy the ciliary processes in order to reduce AH production (cyclophotocoagulation) 92. Trabeculoplasty or trabeculectomy directly target the TM to reduce the outflow resistance. Various types of shunts have been developed to facilitate AH outflow from the anterior chamber.    Table 1-2 and Table 1-3 summarize currently available therapies for POAG. Table 1-2: Current therapies for POAG: Pharmacological therapies Class Mechanism Available Drugs  Reference Prostaglandin analogs Increase unconventional outflow via relaxation of the ciliary muscle and ECM remodeling in ciliary body  Latanoprost, Bimatoprost, Travoprost, Tafluprost, Unoprostone  90,93,94 Beta-blockers Reduce AH production Levobunolol, Carteolol, Metipranolol, Betaxolol, Timolol 94-96 Alpha-agonists Constrict afferent ciliary process vasculature to reduce AH production; increase unconventional outflow Brimonidine Tartrate, Dipivefrin hydrochloride, Apraclonidine hydrochloride 90,96-99 Carbonic anhydrase inhibitors Reduce AH production Brinzolamide, Dorzolamide  Miotics Contracting ciliary muscle and sclera spur to increase conventional outflow Pilocarpine Hydrochloride, Carbachol, Echothiophate 89,90 10 Table 1-2 (contd) Rho kinase inhibitor Inhibit the contraction of ciliary muscle and TM Y-27632 100,101 Hyperosmotics Reduce the volume of aqueous fluid Glycerin, Mannitol 91,102,103 Fix-combination Reduce AH production and elevate draining of AH outflow Timolol maleate and Dorzolamide HCl, Brimonidine Tartrate and Timolol maleate, Brinzolamide and Brimonidine, Latanoprost and timolol, Bimatoprost and timolol, Travoprost and timolol. 104,105  Table 1-3: Current therapies for POAG: Surgery therapies Type of surgery Mechanism Duration Reference Laser cyclophotocoagulation  Destroy ciliary processes to reduce AH production  IOP remains normal after 2 years from surgery 92,111,112 Argon laser trabeculoplasty Targeting pigmented and non-pigmented TM cells but preserve architecture of TM to improve drainage  44% of eyes maintain normal IOP after 2 years from surgery 92,113 Trabeculectomy Excise TM tissue to increase AH outflow 46.9% failure after 5 years from surgery 114,115 Tube shunt Insert the tube through TM to or anterior chamber to increase outflow 29.8% failure after 5 years from surgery 115  11 Animal models of POAG   Glaucoma is not only a human disease but also can be observed in other species. Animals with primary glaucoma provide us with a chance to study disease mechanisms in more detail and may present potential platforms to evaluate novel therapies development. In addition to these spontaneous diseases, experimental and transgenic animal models have also been developed to facilitate POAG study (Table 1-4). Induced ocular hypertension via episcleral vein in experimental animal model was applied to imitate elevated IOP environment of POAG and induce similar clinical phenotypes as POAG 116. However, as previous paragraph, it should be noticed that ocular hypertension is not always associated with pathogenesis of POAG and is not used to determine POAG in clinical diagnosis but considered as major risk factor of POAG 1. The approach of establishing experimental model by using induced ocular hypertension is to overcome the scarcity of spontaneous model and the sample of patients. It is similar but not as equal as the samples from spontaneous model and patients.      Table 1-4 lists some of the established animal models for POAG and ocular hypertension.    Table 1-4: Animal models of POAG & ocular hypertension  Species Type of animal model Mechanism  Clinical phenotype  Reference Monkey Spontaneous Inherited POAG 117  Induced Laser photocoagulation of TM  118  Induced Intracameral injection of latex microspheres  119 Dog Spontaneous  Inherited, ADAMTS10 mutation POAG 120,121 Cat Spontaneous Inherited, LTBP2 mutation POAG 122,123 12 Table 1-4 (contd) Sheep Induced Glucocorticoids induction Ocular hypertension 124 Cow Induced Glucocorticoids induction Ocular hypertension 125 Rabbit Induced Glucocorticoids induction Ocular hypertension 126 Rat Induced Episcleral vein injection with hypertonic saline  POAG 116 Mouse Transgenic MYOC mutation POAG 127 Mouse Transgenic OPTN mutation POAG 128 Mouse Transgenic collagen Type 1 mutation Ocular hypertension 129 Zebrafish Transgenic Lrp2 mutation POAG 130   In vitro model systems of POAG   Although animal models provide us unique opportunities to observe and study disease mechanisms, there are downsides, including the need to sacrifice animals and the high housing expenses. Moreover, species differences may prevent direct translation of findings from animals to humans. In vitro systems that have been developed to study AH outflow pathways include TM cell cultures (Table 2-1) and anterior ocular segment perfusion culture which has been successfully developed on human 131 and bovine 132. These systems allow the use of human surgical TM tissue samples or whole donor eyes.      13 Rational for studying canine glaucoma and TM    Many purebred dogs have a genetic predisposition for primary glaucoma with prevalence comparable or higher than humans; commonly affected dogs breeds include American Cocker Spaniel (5.52%), Basset Hound (5.44%), Chow Chow (4.70%) and Shar-Pei (4.40%) 133. Dogs with primary glaucoma, including POAG, provide us with a unique opportunity to study its pathogenesis. The best established canine glaucoma model is inherited POAG in beagle dogs 134. Its clinical relevant phenotype for human POAG include the slow progressive, sustained IOP elevation 135, response to glucocorticoid challenge 136, cupping of the optic nerve head 134,  and plaque formation within the TM 57,137 (Figure 1-2). Compared with other species, dogs are more accessible and have similar anatomical ocular features as humans, such as similar globe size, TM structure and response to topical IOP-lowering medication 134,136,138-140. Moreover, recent successful developments of ocular recombinant adeno-associated virus (rAAV) mediated gene therapy in dogs were translated into human clinical trials, such as for Leber congenital amaurosis 141 and achromatopsia 142. This explains our interest in canine models of glaucoma, including the study of canine TM cells.    Figure 1-2 ECM accumulation in the TM of POAG-affected dogs.   14   In addition, glaucomatous dogs represent a source of tissue for the study of disease mechanisms. Although primary human TM cell cultures are well established, the size of the trabeculectomy specimens is usually small 74. This increases the difficulty to isolate and culture sufficient TM cells, especially when considering the limited numbers of passages 74. Whole eyes from human donors with well-established history and clinical data are rare and difficult to obtain.    To the best of our knowledge, there are no published reports on the development of primary canine TM cell cultures. Thus, there is a need to develop and characterize primary canine TM cell cultures; this was the purpose of this study.             15 CHAPTER 2  METHODS  Isolation of the canine TM (CTM)    17 canine eyes from 9 adult dogs were collected within 5 minutes of euthanasia. These were either dogs from a local animal shelter or research dogs euthanized for reasons unrelated to this study. The globes were stored on ice until further processing. The 5 eyes from 3 dogs obtained from the shelter were used to refine the steps of tissue dissection and histologic identification of tissue types. The 12 eyes obtained in the laboratory were used to harvest and culture TM cells within 20 hours of enucleation (Table 3-1). The procedures were approved by the Michigan State University IACUC, and were done in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.   The surfaces of the globes were aseptically prepared with 10% povidone iodine and 70% ethanol, then rinsed with 1X working concentration phosphate-buffered saline, pH 7.4 (PBS). The posterior segment was removed from the anterior segment by cutting the globe along the pars plana of the ciliary body with a No. 11 scalpel blade and Westcott tenotomy scissors (Figure 2-1). The lens was then removed from the anterior segment by cutting the lens zonules with Westcott tenotomy scissors (Figure 2-1). The ciliary body and iris were bluntly dissected from the sclera in a posterior to anterior direction (Figure 2-1). The TM tissue could then be recognized as a white band on the ciliary body, delimited anteriorly by the pectinate ligament (Figure 2-1). The identification of the TM was confirmed histologically by processing the dissected tissue routinely for paraffin embedding, sectioning, and staining with hematoxylin and eosin (H&E) (Figure 2-1). Sections were electronically scanned and analyzed by using the Aperior ScanScope slide scanner and software (Leica Microsystems Inc., Buffalo Grove, IL, USA). Only a small trace of juxtacanalicular TM tissue remained connected to the sclera along a 16 pigmented line (Figure 2-1). The TM could be isolated and separated from uveal tract by sharp dissection of the white band (Figure 2-1).   Figure 2-1 Dissection procedure. The globe was first dissected to separate the anterior from the posterior segment (A & B). The ciliary body and iris were separated from the sclera (C). The clear white TM band could be identified on the surface of the ciliary body (C, arrowhead). The white band was carefully dissected from the ciliary body and cut into small pieces for primary culture (D, arrowhead).  Histologic evaluation of the dissected tissue confirmed the successful isolation of the TM: Only traces of the corneoscleral TM (CSTM) and uveal TM (UTM) remained connected to the sclera and ciliary body respectively (E,F, G, narrow arrowhead). And AAP CSTM AAP CSTM ISVP Posterior Chamber UTM TM H E F G D B A C 17 angular aqueous plexus (AAP) can be seen clearly besides remnant CSTM and was connected to the intrascleral venous plexus (ISVP). The histologic appearance of the isolated explants was consisted with TM (H, narrow arrowhead).    Primary TM cell culture    Once the TM was isolated, we followed previously described methods for other species to establish primary cultures 143. TM tissue was carefully cut in approximate 3x3mm square pieces using Westcott tenotomy scissors. Each piece was placed separately in a 15.6-mm diameter well (24-, Corning, Tewksbury, MA) containing 500 l of low glucose Dulbecco's Modified Eagle's Medium (DMEM) (Sigma-Aldrich, St. Louis, MO) with 1% penicillin/streptomycin (Sigma-Aldrich), 10% fetal bovine serum (FBS) (Atlas, Fort Collins, CO) and 1% L-glutamine (Thermo Fisher Scientific, Waltham, MA ) 143.   Within 2 weeks the cells migrated from the tissue onto the bottom surface of the well. The shape of TM cells may vary slightly between species but can generally be described as oval to elongated (see Results) in contrast to spindle-shaped fibroblasts 144-147. Furthermore, TM cells have a unique overlapping-pattern 144-147.    The medium was replaced every 7 days in order to avoid a more frequent interruption of the cell migration process. Once the cells in the well reached confluency within 2-3 weeks, they were detached from the bottom of the well together with the TM tissue remnant with a1x working concentration of trypsin (Thermo Fisher Scientific), re-suspended, and transferred in 5 mL of medium to a T-25 tissue culture flasks (Corning) for passage as previously described 144. The culture medium was then changed every 48 hours until the cells reached confluency, then 18 they were passaged again into three T-75 tissue culture flasks (Corning). After reaching confluency within 7-10 days, the cells were counted and used for either analyses of cell properties (immunocytochemistry (ICC), CLAN quantification, phagocytosis assay and DEX challenge) or long-term storage (Figure 2-2).  Long-term storage was done by cryopreservation: Cells were suspended in cryopreservation medium which contained 10% cell culture quality dimethyl sulfoxide (Sigma-Aldrich) and 90% DMEM (Sigma-Aldrich).The cell numbers were determined with a hemocytometer (Thermo Fisher ScientificA). Then the cell suspension was transferred to 1ml cryovials (USA Scientific, Inc., Ocala, FL) with a concentration of 106 cells /ml.  Cryovials were then transferred into a cell freezing container with 100% isopropyl alcohol (Thermo Fisher Scientific) and kept in a -80 freezer (Thermo Fisher Scientific) overnight.  Then the cryovials were moved into a liquid nitrogen tank (Thermo Fisher Scientific) for long-term storage.     Figure 2-2 Experimental design.   Isolate canine TM explant Primary culture Passage and maintain the CTM strains 1. ICC 2. CLAN  3. Phagocytosis assay 4. DEX challenge  to characterize TM cell properties.  Establish and identify canine TM cells 19 Fibroblast primary culture   Primary fibroblast cell cultures were obtained from wild type (wt) canine skin samples concurrently with eye collection and use as negative controls to compare with primary CTM cultures. In the selected area, the hair was removed with clipper and the skin was aseptically prepared with 70% ethanol and 4% chlorhexidine. Skin samples were then collected with a 4-mm biopsy punch (Acuderm inc., Fort Lauderdale, FL). The procedure of fibroblast isolation was previous described 148,149. Briefly, the skin tissue was aseptically prepared with 70% ethanol and then washed with PBS. Then the tissue was cut into small pieces and each piece was placed separately into the wells of a 6-well receiver plate with poly-D-lysine coating (Millipore, Billerica, MA). DMEM-high glucose medium with 10% FBS and 1% Antibiotic-Antimycotic containing penicillin, streptomycin, and amphotericin B (Thermo Fisher Scientific) was prepared for culture and replaced every 7 days. Once the cells reached confluency, they were treated with trypsin for passage and expansion in T-25 tissue culture flasks (Corning) and subsequent cryopreserved for future experiments. When needed for different experimental purposes, the cells were passaged to T-75 tissue culture flasks (Corning) after reaching confluency in the T-25 tissue culture flasks.    Characterization of TM cells  ICC   Characterizing TM cells was challenging due to lack of a single specific TM cell marker. Rather we had to rely on a combination of different cell markers to identify TM cells; these have been well-established in other species 144-147. TM cells highly express collagen type IV, laminin and alpha-smooth muscle actin (-SMA) 144-147 (Table 2-1). Other markers such as desmin and keratin were also introduced to exclude other types of cells such as corneal epithelium and ciliary 20 smooth muscle cells. Increased expression of myocilin and CLAN formation with DEX stimulation are other TM-specific features.    For ICC, the cells were cultured in the Nunc Lab-Tek II Chamber Slide System (Thermo Fisher Scientific, MA) with seeding density 0.05 x 106 cell number per well until day reached confluency. After approximate 7 days, routine immunostaining and DEX challenge were performed. The cells were fixed in 4% paraformaldehyde for 30 minutes and then washed with 0.25% Triton-X100/1X working concentration PBS, pH 7.4. The cells were then incubated overnight at 4 with primary antibodies (Table 2-2) 144. Secondary antibodies were applied for 1 hour at room temperature (Table 2-2). Finally, Phalloidin/ F-actin AF488 1:100 (Thermo Fisher Scientific) was added and incubated at room temperature for 1 hour to stain the F-actin network in the cells. ProLong Gold Antifade Mountant with 4',6-diamidino-2-phenylindole (DAPI)  (Life Technologies, Carlsbad, CA) was added to the slides before covered with coverslips. The slides were imaged and evaluated with epi-fluorescent microscopy (Eclipse 80i Fluorescent Microscope, Nikon Instruments Inc., NY) and confocal microscopy (FV1000 Laser Scanning Confocal Microscope, Olympus America Inc., PA).                 21 Table 2-1: Characterization methods previously used for TM cell identification. Species  Morphology description  Characterization methods Reference Human Monolayer formation, cobblestone pattern, fast grow after P8 ICC  DEX challenge 150 Human Endothelium-like monolayer ICC,  DEX challenge, phagocytosis assay,  CLAN formation.  145,151-156 Primate Basement membrane, intercellular junction, pinocytotic vesicles, microvillous projections, and branched cell extension and monolayer growth pattern.  Radiolabeling,  DEX challenge, phagocytosis assay   157-159 Porcine Monolayer ICC Southern hybridization Western blot 160 Mouse Similar morphology as HTM cells  ICC DEX challenge CLAN formation  Phagocytosis assay 147,161 Bovine Morphology similar as human TM cells ICC DEX challenge CLAN formation  143,144  Table 2-2: The list of antibodies used in the experiments Antibody  Type Host Species Concentration Manufacturer  -SMA Monoclonal Mouse 1:500 BioGenex, Fremont, CA Laminin Polyclonal Rabbit 1:250 Sigma-Aldrich, St. Louis, MO Collagen type IV Polyclonal Rabbit 1:50 LS Bioscience, Seattle, WA Cytokeratin Monoclonal Mouse 1:50 Invitrogen, Carlsbad, CA Desmin Monoclonal Mouse 1:100 Cell Marque, Rocklin, CA Phalloidin/ F-actin AF488 Dye None  1:100 Thermo Fisher Scientific, Waltham, MA Anti-rabbit IgG conjugated with Texas Red Polyclonal Goat 1:1000 Thermo Fisher Scientific, Waltham, MA Anti-mouse IgG conjugated with Texas Red Polyclonal Goat 1:5000 Thermo Fisher Scientific, Waltham, MA  22 Characterization of TM cells - Phagocytosis assay   The TM cells were first cultured in Nunc Lab-Tek II Chamber Slide System (Thermo Fisher Scientific). Then, E-coli conjugated pHrodo green particles (0.5 mg/well, Invitrogen) were added and incubated for 1 hour at 37 . Those particles remain non-fluorescent outside the cells at neutral pH but emit green fluorescence once engulfed in phagosomes with an acidic pH. Therefore, by observing green fluorescent particles within the cultured cells, we could confirm phagocytosis activity 162. The cells were fixed in 4% paraformaldehyde and co-stained with F-actin to verify the cell structure and location. The slides were coverslipped with ProLong Gold Antifade Mountant with DAPI. Epi-fluorescence microscopy (Eclipse 80i Fluorescent Microscope, Nikon Instruments Inc) was used to observe and co-localize the cells and engulfed particles to verify phagocytosis activity.  Dexamethasone (DEX) challenge   In order to verify that our isolated cell strains consist of TM cells, they were treated with DEX as previous described and briefly outlined below 163. Unique characteristics of TM cells are the up-regulation of myocilin mRNA and protein as well as the increased formation of cross-linked actin networks (CLANs) following treatment with DEX 164. CLANs are defined as geodesic structures composed of at least 3 triangles with 5 hubs 143,163. This arrangement is considered to increase the stiffness of TM cells, resulting in elevated AH outflow resistance in vivo following treatment of POAG-affected eyes with DEX 75.    Induction of myocilin mRNA: Confluent TM cells were cultured in 6-well plates and treated for 3 days with either 100 nM DEX (Sigma-Aldrich, MO) or 0.1% ethanol as vehicle 23 control in DMEM-high glucose medium with 1% penicillin/streptomycin, 10% FBS, and 1% L-glutamine. DEX was first dissolved in ethanol and prepared as 10-3 M stock solution before treatment and therefore ethanol was selected as vehicle control. Approximately 106~107 cells were used for both treatment groups to extract total RNA with RNeasy Mini Kit (Qiagen, Venlo, Netherland) following manufactures instructions. Briefly, cells were harvested as a cell pellet and mixed with 350 l of lysis buffer. Next, 70% ethanol was added to the lysate and filtered through a RNeasy Mini spin column.  Then the column was washed with washing buffer. Finally, RNA was eluted with 30 l RNase-free water and collected for quantification with a Nanodrop ND-1000 Spectrophotometer (Thermo Fisher Scientific). Only the samples with 260nm/280nm ratio of ~ 2.0 or above were considered as pure RNA and accepted for future experiments. The extracted RNA was then treated with DNase (Roche, Basel, Switzerland) to eliminate potential host genome contamination. Reverse transcription of mRNA to cDNA was performed with Superscript II (Invitrogen) with the addition of oligo dT (Life Technologies), 10 mM dNTP (Invitrogen, CA), 0.1M DTT (Invitrogen), and RNase OUT (Invitrogen). The TaqMan probes and primers for canine myocilin (Cat# Cf02627377_m1, Applied Biosystems, Waltham, MA) and the housekeeping gene 18s (Cat# Hs99999901_s1) were used for quantitative-RTPCR (q-RTPCR). mRNA extracted from canine TM tissue was used as positive control for canine myocilin expression 165.    q-RTPCR was performed on a 7500 Fast Real-Time PCR system (Applied Biosystems) using 50 ng cDNA for each sample. cDNA was mixed with 1 l primer /probes and 10 l TaqMan Fast Universal Master Mix 2X (Thermo Fisher Scientific) then added up with DNase-free water to 20 l total volume.  Relative gene expression for each gene was compared with house keeper gene based on the equation 1/[2^(Cttarget gene  Cthousekeeper)] and Ct method was 24 applied for group comparison. Briefly, the Ct value of MYOC mRNA was normalized with 18s to give Ct value for each group. To calculate the difference Ct, the Ct value of ethanol treated group was subtracted from the Ct value of the DEX treated group. Ct was used to calculate relative fold changes of gene expression (2-) 166.    CLAN quantification: Confluent TM cells cultured in Nunc Lab-Tek II Chamber Slide System (Thermo Fisher Scientific) for ICC were treated for 10 days with either 100 nM DEX (Sigma-Aldrich) or 0.1% ethanol as vehicle control in DMEM-high glucose medium with 1% penicillin/streptomycin, 10% FBS, and 1% L-glutamine.  The medium was replaced every other day. At post-treatment day 10, the cells were collected and fixed with 4% paraformaldehyde. Phalloidin/ F-actin AF488 (Thermo Fisher Scientific) was added and incubated at room temperature for 1 hour to stain the cellular F-actin network. Finally, the slides were coverslipped with ProLong Gold Antifade Mountant with DAPI (Life Technologies).  All slides for CLAN formation were evaluated with confocal microscopy (FV1000 Laser Scanning Confocal Microscope, Olympus America Inc., PA) and images were taken at 10x, 20x, 40x, and 60x magnification. For CLAN quantification, five regions in each well of the chamber slides with approximately 50-230 cells per region were imaged with confocal microscopy (FV1000 Laser Scanning Confocal Microscope, Olympus America Inc.) at 20x. The CLAN formation rate was estimated by the ratio of CLAN-positive cells and total number of cells with DAPI staining. The cells were counted in double-blind fashion as previously describe 144. Then the CLANs formation rate was compared between DEX and ethanol treated cells with s-test Only P<0.05 was considered significant.     25 CHAPTER 3  RESULTS  Histological evaluation of isolated CTM explant   Although human and canine ocular anatomy are similar, there are some unique structures within the canine ICA, such as the pectinate ligament and AAP. These slight anatomic differences resulted in the need to develop the methods for TM isolation specifically for the dog. As described under Methods, we performed several dissections of canine cadaver eyes with histologic evaluations to verify isolation of the canine TM (Figure 2-1).    In summary, the CTM was effectively dissected from the uveal tract, and we confirmed histologically that our procedure successfully isolated TM tissue from the sclera and ciliary body. In Table 3-1, we provide the list of established CTM cell strains.26 Table 3-1: Established primary CTM cell strains Canine Participant Eye Gender Age(months) Cell strain ICC Phagocytosis DEX-CLAN DEX-MYOC Passage number BER01 OS F 10.4 BERTM2B Y Y ND ND 2 BER01 OD F 10.4 BERTM7A Y Y Y ND 5 BER01 OS F 10.4 BERTM7B Y Y ND ND 6 BER01 OS F 10.4 BERTM8B Y Y Y Y 6 BER01 OS F 10.4 BERTM5B Y ND ND ND 2 BER01 OD F 10.4    BERTM1A Y ND ND                       ND 2 BER01 OD F 10.4 BERTM3A Y ND ND ND 2 BOST01 OS F 1.1 BOSTM1B Y Y        Y ND 5 BOST01 OD F 1.1 BOSTM2A ND Y ND ND 1 BOST01 OS F 1.1 BOSTM2B ND Y ND ND 2 BOST01 OD F 1.1 BOSTM3A ND Y ND ND 2 BOST01 OS F 1.1 BOSTM3B ND Y ND ND 3 BOST01 OS F 1.1 BOSTM4B ND Y ND ND 2 R01 OD M 2.8 ROYTM3A ND ND Y ND 6 R01 OS M 2.8 ROYTM3B ND ND ND ND 4 R01 OD M 2.8 ROYTM4A ND ND ND ND 4 R01 OS M 2.8 ROYTM4B ND ND ND ND 4 R01 OS M 2.8 ROYSCTM ND ND ND ND 6 R01 OS M 2.8 ROYCRTM ND ND ND ND 4 TEL01 OD M 2.8 TELTM8A ND ND ND ND 4 TEL01 OS M 2.8 TELTM8B ND ND ND ND 4 CUR01 OD F 9.4 CURTM3A ND ND ND ND 11 CUR01 OS F 9.4 CURTM3B ND ND ND ND 4 CUR01 OD F 9.4 CURTM4A ND ND ND ND 4 CUR01 OS F 9.4 CURTM4B ND ND ND ND 4 HOU01 OD M 2.7 HOUTM1A ND ND ND ND 6 HOU01 OS M 2.7 HOUTM1B ND ND ND ND 4 HOU01 OD M 2.7 HOUTM2A ND ND ND ND 4 27 Table 3- HOU01 OS M 2.7 HOUTM2B ND ND ND ND 4          OS: oculus sinister; OD: oculus dexter; F: Female; ND: not done; Y: done28   The migration of TM cells onto the culture plate was noticed within 2 weeks after culturing the explanted tissues (Figure 3-1). These cells continued to expand until they reach confluency within additional 7 to 10 days. The growth pattern of CTM cells was similar as described in other species with features of overlapping cell growth and cobble-stone like monolayer appearance (Figure 3-1) 164,167. The morphology of canine TM cells appeared flat with enlarged intracellular spaces; this has also been observed in the cultured human TM cells 168.     The TM cells that were isolated from adult canine eyes could be successfully passaged at least up to 6 times, although the cells failed to reach confluency and became senescence after passage 5. This phenomenon is consistent with other published results 150,169 and possibly affected by the age of donors. Human TM cells from adult donors have fewer passage numbers and longer population doubling times compared to human fetal TM cells 150,169.     29  Figure 3-1 Cell migration and morphology of canine TM cells and fibroblasts. Cell migration from the TM tissue onto the culture plate was noticed within 2 weeks (1A, arrow head). The newly migrated cells had overlapped pattern and cobble stone-like monolayer (1B, arrow). The small vacuoles around the nucleus suggest phagocytoses activity (1C, arrow). Canine fibroblasts are shown for comparison (1D); compared to TM cells fibroblasts have less intercellular space, and they have a more elongated shape. (calibration bars = 50 m)    30 Characterization of cultured TM cells: Immunocytochemistry    Since there is no specific marker for TM cells, we used combined markers as previously described to identify the cultured canine TM cell strains 167,170. In here, we present the results from 8 different established CTM cell strains and 1 canine fibroblast cell strain which is BERFB01 as negative control.  The confluent canine TM cells express -SMA which can be clearly seen in the cytoplasm with filamentous pattern. This is consistent with the previous results of an IHC study of canine ICA 171. Although the age of the canine can affect -SMA expression in the TM tissue 171, our CTM cell strains strongly express -SMA. Similarly, laminin and collagen type IV stain intracellular vesicles but not the extracellular space of cultured cells (Figure 3-2). No keratin or desmin was observed in the CTM cell strains which indicated there were no cornea epithelium cells and ciliary smooth muscle cells, respectively (Figure 3-2). Table 3-2 summarizes the result of our canine cell strain characterization. Table 3-2: Characterization of canine cell strains  Cell strain Collagen Type IV Laminin -SMA Desmin Keratin BERTM7B  + + + (+) - BERTM8B + + + (+) - BERTM5B  + + + - - BERTM2B + + + - - BERTM7A  + + + - - BERTM3A + + + - - BERTM1A + + + - - ROYTM3A + + + - - BOSTM1B  + + + - - BERFB01 - - - - - +: positive; -: negative; (+): slightly desmin-positive cells observed; BERFB01 is fibroblast  31  Figure 3-2 The ICC result of CTM. ICC staining shows positive expression of collagen IV (A), laminin (C) and -SMA (B) in red, but no expression of desmin (D) and keratin (E). This staining pattern is consistent with TM cells. (Calibration bar = 50 m)  Characterization of cultured TM cells: Phagocytosis assay    In addition to light microscopic observations and ICC, we also performed phagocytosis assays to verify the TM characteristics of our cultured cells. Phagocytosis is considered an important function of TM cells to clear debris within the TM in order to maintain aqueous humor outflow 172-174. Our first observed indication of phagocytosis activity was localization of pigment particles in some of the cells (Figure 3-3). For confirmation, we incubated the cells for 1 hour with acid-sensitive E-coli conjugated pHrodo green particles. When the green particles were engulfed into phagosomes, they become green fluorescent and can be detected by epifluorescence microscopy. We demonstrated that our cultured cells had phagocytosis activity further confirming them to be TM cells (Figure 3-3).  A B C D E 32  Figure 3-3 Phagocytosis activity of cultured CTM cells.  After 1 hour incubation with green fluorescent particles, phagocytosis activity of CTM was observed with particles engulfed clearly in the intracellular space of cells overlapped with F-actin (3A, arrow shows one of many engulfed particles). In comparison, canine fibroblasts did not show any phagocytosis activity (3B). Indications for phagocytosis activity were also observed in unstained, untreated primary TM cell cultures by pigment engulfed in cells. (Arrow, 3C&3D) (calibration bar = 50 m)   33 Table 3-3: Phagocytosis activity of canine cell strains Cell strain Pigment engulfed  Green particle engulfed BERTM7A  - + BERTM7B - + BERTM8B - + BOSTM4B + ND BOSTM3B + ND BOSTM2B + ND BOSTM1B  + + BOSTM3A + ND BOSTM2A + ND BERFB01 - -    -: not observed, +: observed; ND: not done; BERFB01: canine fibroblast  Characterization of cultured TM cells: DEX challenge - CLAN formation    The promotion of CLAN formation after DEX challenge is considered one of critical features of many TM cells 143,145. Such induction was rarely observed in other cell types 175. Therefore, we were interested whether cultured wt CTM cells will increase CLAN formation after DEX treatment 143,147,164.   Figure 3-4 CLAN formation in canine TM cells. CLANs could be observed in some cultured CTM cells with and without DEX treatment (4A, arrow) but not in fibroblasts. (4B, arrow) The 34 structure is similar as previously reported with hub and triangle structure.  (calibration bar = 10 m)   In order to determine DEX-induced upregulation of CLAN formation, we counted CLAN-positive cells of CTM cells following 10 days of DEX treatment and compared them to cells treated with ethanol vehicle control. Ethanol was used as solvent for DEX. Canine fibroblasts were included as negative controls since there are no reports about CLAN formation in this cell type 175. The slides were evaluated with confocal microscopy and 5 cell strains including 3 CTM cell strains (BERTM7A, BERTM8B, BOSTM1B) and 1canine fibroblast strain (BERFB01) were used in this experiment for CLAN evaluation (Figure 3-5). Five regions in each well of the chamber slides with approximately 50-230 cells per region were imaged and counted. However, after 10 days  of CLAN positive cells. Additionally in the CTM cells under normal culture condition. e the result of CLANs number after treatment. There was no significant difference between DEX-treated and ethanol-treated groups (P > 0.05, Figure 3-5). We counted 3±1% CLANs-positive cells (mean ± SD) in BER7ATM, 1±1% in BER8BTM and 1±1% in BOS1BTM following DEX treatment while cells treated with ethanol have 4±3% CLANs-positive cells in BER7ATM, 1±1% in BER8BTM and 1±1% in BOS1BTM. Consistent with previous reports, this suggests that F-actin was altered in our cultured CTM cells 75,143,147, but the effect of DEX challenge was not obvious to be reflected on the number of CLANs.  35  Figure 3-5 Relative number of CLAN-positive cells in different canine cell strains. (mean ± one SD) DEX: DEX treatment, EtOH: Ethanol treatment, FB: fibroblast. P value for BER7ATM is 0.36, for BER8BTM is 0.72, and for BOS1BTM is 0.91. The result was shown there is no significant difference between DEX-treated group and ethanol vehicle control group.   Characterization of cultured TM cells: DEX challenge  gene expression    In order to verify gene expression, we performed q-RTPCR of CTM cells cultured with ethanol vehicle control or DEX treatment (Figure 3-6 & Table 3-6). One of the characteristics of many TM cells strains is the upregulation of MYOC expression with DEX treatment 144.    Our result showed a decreased rather than increased myocilin expression following the DEX treatment. However, the sample size is small and additional experiments needed to be 3% 1% 1% 0% 4% 1% 1% 0% 0.00%1.00%2.00%3.00%4.00%5.00%6.00%7.00%8.00%BER7ATMBER8BTMBOS1BTMFB% of CLAN-postive cells in different cell strain CLAN formation  of CTM  DEXEtOH36 performed. In BER8BTM, the fold expression of MYOC was 0.0002 while in BER7ATM it was 0.76 following DEX treatment (Figure 3-6).    Figure 3-6 Relative gene expression of MYOC following 3 days of DEX treatment in different canine cell strains. The fold changes were variable across CTM cell strains but indicated a decreased MYOC expression. There was 0.0002 fold expression in BER8BTM while BER7ATM had a 0.76 fold change.0.000246 0.760489 0.160428 00.20.40.60.81BER8BTMBER7ATMBERFB01Relative Fold Change MYOC 37 F: Female; M: MYOC; Y: has amplification  Table 3-4:  Completed q-RTPCR of CTM cell strains and other type of cells Canine participant Gender Age (months) Cell strain Treatment Target genes Amplification Ct Housekeeping genes BER01 F 10.4  7ATM DEX/EtOH MYOC Y 0.76 18S   BER01 F 10.4  8BTM DEX/EtOH MYOC Y 0.0002 18S   BER01 F 10.4 FB01 DEX/EtOH MYOC Y 0.16 18S               38 CHAPTER 4  DISCUSSION    To the best of our knowledge this is the first report of the successful isolation and culture of CTM cells. We have identified several well-established TM-cell properties in our primary CTM cell strains, such as phagocytosis activity, expression of -SMA, laminin and collagen type IV, and CLAN formation.    Most TM tissue was excised for cell culture as confirmed by histologic examination     Performing resection of small TM tissue pieces has always been considered a challenge whether for surgery 176,177 or primary culture 74,147,150. Isolating TM tissue without excising other type of tissue is critical to establishing CTM cell strains. Only traces of TM tissue remained attached to the sclera and ciliary body after isolation, according to histologic examination. This suggested the success of TM isolation and reduced the concern of mixing cell types in the cell culture. A few pigmented cells, presumed uveal melanocytes, were observed in the culture dish together with the TM cells. However, from published reports of culturing canine uveal melanocytes, the cell culture medium and supplements for TM cells are not suitable to maintain uveal melanocytes 178. Those pigmented cells lacking the proper culture conditions would not survive after replacing the medium several times.  observe those cells after 1 -2 passages.  Multiple TM-specific protein markers are observed at our TM cell strains   Our CTM cells did express the TM specific proteins mentioned in previously published reports in other animal species 143,147. Because there is no specific marker for TM cells, the 39 expression of a combination of multiple proteins had to be characterized in order to confirm that our cultured cells are indeed TM cells. Following the well-established protocol developed by others, we used the following five antibodies to identify our TM cells by ICC: laminin, -SMA, collagen IV, keratin and desmin 144.  TM cells express laminin, -SMA and collagen IV but not keratin and desmin. Based on this staining pattern, we are confident that we successfully cultured CTM cells.  CLAN formation observed in our non-DEX treated CTM cells   CLAN formation is an important characteristic of TM cells 145. Other ocular cells that have been reported to form CLAN include lamina cribrosa cells 179, human and bovine retinal pigment epithelium, rabbit lens epithelium, bovine corneal endothelium and bovine iris pigment epithelium 175.  But based on the anatomic location and our dissection technique, it is unlikely that we cultured any of these other cell types. Corneal endothelial cells could have been cultured, -SMA stain. This suggests that the CLAN formation we observed occurred in CTM cells.    A low percentage of CLAN-positive cells could be observed under DEX untreated conditions, which has been reported before 143. DEX promotes CLAN formation but CLAN-positive cells also exist under non-DEX treated conditions. Our result has shown ~1 -4% CLAN-positive cells without DEX treatment which is consistent with previous publications 143,163.   Phagocytosis activity of the CTM cells   One of the important phenomena observed during our primary culture of CTM cells was phagocytosis activity. TM cells perform phagocytosis in order to remove debris and reduce the 40 resistance of trabecular AH outflow. Presumed phagocytic vesicles were observed in our cultured cells once they migrated from the tissue explants into the culture plate; this was consistent with previously published reports 180. Moreover, functional phagocytosis activity was confirmed by incubating the cultured cells with bio-particles; these particles could be clearly seen inside the cells, again consistent with previously published reports 162,173.   DEX response    We did observe clear CLAN structures after DEX treatment in our CTM cells. However, this could also be seen in the EtOH-treated control group, and no significant difference in the number of CLANs was observed. Approximately 1-4% of cultured CTM cells were CLAN-positive even after 10 days of DEX treatment. This is in contrast to other species, such as human 145, bovine 143 and mouse 147 where an increase in CLAN positive cells can be observed following DEX treatment. The result of human TM cells has shown that around 18- 55% of CLAN-positive cells appear after 10 days DEX treatment 145, while in bovine an estimated 40% of DEX-treated TM cells were observed with CLAN structure 143. Approximately 30% of cultured murine TM cells were CLAN-positive with DEX treatment 147. We suspect that the induction of CLAN-formation by DEX in cultured TM cells correlates with a particular species IOP-responsiveness to DEX (see explanation below).    Similarly, we did not observe an increase in MYOC mRNA expression following DEX treatment. Our q-RTPCR results indicated a slight difference but not large fold changes as previously published 181-184. However, when evaluating published results more closely, it became clear that not all TM cells respond to DEX treatment, especially cells harvested from normal human eyes 146. Only 38.1% to 52.1% of normal human TM cells responded to DEX treatment 41 with an increased expression of the MYOC protein 146. Moreover, we noticed that MYOC mRNA expression is hard to detect even following DEX treatment. It is possible that our PCR probe will have to be redesigned. The current probe is used effectively to measure MYOC RNA levels in tissue. However, we assume that the quantity of harvested cells for q-RTPCR is much less than in the tissue samples. This is the first time we have applied canine MYOC probes in cell culture. We suspect we will need more sensitive probe or increase the amount of cells used in the experiment. In the future, we will also quantify MYOC protein expression by Western blot as complement to our q-RTPCR experiments.     It is possible that species differences should be considered in order to interpret our data properly. DEX-induced ocular hypertension can been seen in normal cattle 125, sheep 124 and mouse 185. However, based on our clinical experience and previous publications, the majority of wt dogs are non-responders 186. Considering that wt dogs do not respond to topical DEX treatment with an increase in IOP, it is rational that we did not observe a change in CLAN formation and MYOC expression in our wt canine TM cells. It remains to be shown if TM cells from glaucomatous ADAMTS10-mutant dogs respond to DEX since IOP increases in these animals with topical DEX treatment 136.      The concern of inadvertently isolating non-TM cells    We did our best to exclude other, non-TM tissues during the dissection, and we histologically confirmed our isolated TM explants. However, we cannot rule out the  possibility that other cell types were isolated along with the TM tissue, such as fibroblasts 187,188, sclera spur cells 189, and ciliary smooth muscle cells 190. In the future we may use additional antibodies to verify in more detail the presence of other cell types by ICC. 42 Senescence observed in the CTM cell strains   We observed that the time of reaching cell confluency was gradually increased after several passages. Limited passage number of TM cells ranging from 2 to 6 passages has been noticed during cell culture. The phenomenon of TM cell senescence has been reported in several species. Human TM cells have been reported to stop proliferation after 8 to 12 passages 170; and  porcine TM cells have been shown to become flat and senescence at passage number 8 169.  Primary bovine TM cells reached senescence after about 10 passages 144. Similar results have also been recorded for murine TM cells, which can proliferate until passage number 25 before reaching senescence 147. Compared with other species, with the exception of mice, our results on CTM cell passage number do not appear different. Primary TM cells can reach senescence in early passages due to the quality of the cells or the age of the donor.    It has been suggested that senescence of TM cells in vivo may contribute to increase in trabecular outflow resistance in the POAG pathogenesis 191. It remains to be shown if cultured TM cells from canine glaucomatous eyes develop senescence at earlier passage numbers than wt CTM cells. A possible solution to prevent senescence of cultured TM cells is their transfection with mutant defective SV40 virus 170, which can be included in our future investigations.        43 CHAPTER 5 - SUMMARY AND FUTURE DIRECTIONS  Summary   This study documents the first successful isolation and culture of CTM cells. These cells were characterized based on their ability to perform phagocytosis and their expression of -SMA, laminin and collagen type IV, but not keratin and desmin 75,143,144,147.  Moreover, we observed CLAN formation, an alteration of F-actin that is somewhat specific for TM cells, since CLANs are rarely seen in other cells types 175. In summary, all our presented data is consistent with the published results on cultured TM cells from other species.    Our primary cultured wt CTM cell cultures did not respond to DEX treatment. This is consistent with our clinical experience: wt dogs, in contrast to some humans 192 and bovines 125, do not respond to DEX treatment with an increase in IOP. In contrast, ADAMTS10-mutant beagles with POAG have shown such an IOP response to topical DEX treatment 136. Nonetheless, DEX-induced upregulation of MYOC expression 87 and increased CLANs formation 143,145,147 are considered hallmarks of many TM cell strains.   Future directions    The tools developed here can be applied in the detailed study of disease mechanisms involved in canine primary glaucoma, which is a leading cause of incurable blindness. Primary CTM cultures from glaucomatous eyes will provide us with a unique opportunity to study disease mechanisms without the secondary effects of elevated IOP and therapy. We consider this a valuable alternative option to study human TM cells due to the scarcity of glaucomatous human donor eyes. The investigation of CTM from wt and glaucoma-affected eyes will likely assist in providing a better understanding of both canine and human disease.   44    Whether glaucomatous CTM cells can be successful established remains to be shown, one of the initial challenges to isolate CTM from eyes with advanced stages of glaucoma is the scarcity of TM tissue, due to the presumed loss of TM cells affected by chronically elevated IOP 193. This scarcity of glaucomatous TM tissue may increase the difficulty in isolating and maintaining enough number of TM cells for experiments. We have launched initial attempts to isolate TM cells from ADAMTS10-mutant dogs with advanced POAG with promising results.    We performed some additional preliminary gene expression analyses by evaluating ADAMTS10, a gene that has previously been shown to be highly expressed in the TM 120. However, wt CTM cell strains 120. ADAMTS10 is a metalloproteinase responsible for ECM turnover. It may play a major role in the regulation of ECM production within the TM. Mutation of ADAMTS10 can decrease ECM turnover and thus contribute to the elevated resistance of AH outflow as seen in ADAMTS10-mutant humans 194 and dogs with POAG 120. However, the role of ADAMTS10 in the pathogenesis of POAG remains largely unknown, and therefore we would like to further investigate its role in our glaucomatous CTM cells in the future.    The comparison of cultured CTM cells from wt and glaucoma affected dogs will include the evaluation of gene and protein expression patterns in order to determine up- or downregulation epigenetic effects. Moreover, we are interested how the difference in gene expression in the glaucomatous CTM cells affects their biomechanical properties 195.     From our previous research, the alteration of sclera collagen microstructure in the posterior chamber led to the weakness of sclera was noticed in ADAMTS10-mutant dogs with advanced POAG 196. It appears that age-associated weakness of the sclera can increase the severity of the 45 damage to the optic nerve leading to an increase in the progression of the disease. Similar biomechanical changes in the TM tissue has been noticed with DEX treatment 195. This appears that biomechanical alteration in POAG eyes may affect several locations to increase susceptibility to the damage of elevated IOP. However, the mechanisms of biomechanical changes of the tissue under chronically elevated IOP are not clear, nor are the properties of TM cells. Such investigations are currently underway with our collaborators.       Cultured CTM cells provide a useful platform for large scale in vitro compound screening to discover potential therapies for glaucoma and to understand their effects on molecular pathways. For example, a novel Rho kinase inhibitor, K-115, was found to disrupt actin bundles within the TM cells, thereby reducing their stiffness and resulting in decreased trabecular resistance 197. Although most compounds are focused on reducing outflow resistance, measuring outflow resistance can be an issue in vitro. 3D-scaffod with seeded TM cells to simulate TM tissue in vivo are under development developing to overcome this difficulty 198.  This system could be applied to our CTM cells to screen potential drugs and study therapeutic effects. We also intent to perform other compound screenings in collaboration with the MSU Assay Development and Drug Repurposing Core.    Furthermore, CTM cells can be a used for in vitro screening of novel gene therapies. Modification of gene expression in the TM cells by gene therapy is one of the major approaches in the development of future, long-term IOP control 199,200.  For example, self-complementary adeno-associated virus (AAV) vectors containing the MMP1 (matrix metalloproteinase 1) cDNA have been successful delivered to the TM of the in vivo sheep model 201. Non-viral vectors like small interfering RNAs to suppress MYOC expression in TM cells in vitro have been investigated 202. Easy administration and sustained therapeutic effects makes it a potential target 46 for more effective treatment of  glaucoma 203. Moreover, several successful ocular AAV-mediated gene therapies have been or are in the process of being translated from dogs into clinical trials, such as for Leber congenital amaurosis 141 and achromatopsia 142. This has emphasized the important role of canine disease models in gene therapy development. Therefore, we are interested in whether these CTM cells can be transfected by virus vectors, such as AAV, to select the optimal virus vectors for gene therapy in future.     Finally, CTM cells are not only suitable for compound screening and gene therapy development but can also be applied to gene editing with CRISPR-associated protein-9 nuclease (Cas9) 204. Gene-modified TM cells can be transferred back to the TM tissue to replace the malfunctioning TM cells as part of cell-based therapy. Moreover, the possibility of developing cell therapy based on induced pluripotent stem cells (iPSCs) as potential treatment of POAG has been investigated 162. iPSCs-derived TM cells have successfully rescued POAG phenotypes in the MYOC mutant mouse 205. This has been a thriving area in recent years and may be a new approach for treating glaucoma in the future. However, whether such genome editing techniques or iPSCs- based cell therapy can be applied on our CTM cells and dog model remains unknown. We would like to further investigate its therapeutic potential on our CTM cell strains in the future.        47         REFERENCES    48 REFERENCES  1. Kwon YH, Fingert JH, Kuehn MH, Alward WL. Primary open-angle glaucoma. N Engl J Med. 2009;360(11):1113-1124.  2. Moorthy RS, Mermoud A, Baerveldt G, Minckler DS, Lee PP, Rao NA. Glaucoma associated with uveitis. Surv Ophthalmol. 1997;41(5):361-394.  3. Radcliffe NM, Finger PT. Eye cancer related glaucoma: current concepts. 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