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Isolation and Characterization of Normal and Glaucomatous Canine Trabecular Meshwork in Vitro

Permanent Link: http://ufdc.ufl.edu/UFE0022804/00001

Material Information

Title: Isolation and Characterization of Normal and Glaucomatous Canine Trabecular Meshwork in Vitro
Physical Description: 1 online resource (69 p.)
Language: english
Creator: Rinkoski, Tommy
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: beagle, canine, glaucoma, poag
Veterinary Medicine -- Dissertations, Academic -- UF
Genre: Veterinary Medical Sciences thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Over 35 years of research, the unique POAG Beagle model has been a boon to glaucoma researchers. Our study proposes to expand the usefulness of the model by creating protocols for the primary culture of TM cells from normal and glaucomatous canines and confirm the identity of cultured cells, so that TM from the POAG Beagle can be confidently utilized in future research. Primary cultures obtained by enucleation and dissection of normal and glaucomatous canine were cultured in high-glucose DMEM supplemented with 20% FBS initially and 10% FBS after cell establishment. Slices of anterior segment from two eyes were cultured for various times out to one week, fixed and mounted to view expansion histologically. Cell lysates from glaucomatous and normal TM cells incubated with dexamethasone were examined for the presence of myocilin protein. Glaucomatous and normal TM cells were incubated with 0.5 micron latex microbeads to assay phagocytic activity. 79 normal and 16 glaucomatous primary and subsequent cultures were successfully created, as well as cells used for assays and experiments. Passaging, freezing, and thawing of cells was shown to be safe and reliable, leaving 10 aliquots of glaucomatous TM cells for future experimentation. TM cells grew at increasing rates according to percent FBS in their medium, up to a maximum of 20% serum. Histology showed TM cells in culture do not increase in density at their tissue of origin, but migrate along surfaces to which they can attach. Stimulation with dexamethasone increased myocilin expression in both normal and glaucomatous TM cultures. Glaucomatous cell monolayers retained fewer microbeads than normal counterparts, and dexamethasone treatment appeared to impair microbead retention in both normal and glaucomatous cells. Glaucomatous and normal canine TM cells were successfully cultured, safely passaged, and can be frozen for storage and rescued. Cultures behave as predicted for cells of TM origin in myocilin production and phagocytic activity. A population of cultured glaucomatous TM from the POAG Beagle model has been created for future use.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Tommy Rinkoski.
Thesis: Thesis (M.S.)--University of Florida, 2008.
Local: Adviser: Samuelson, Don A.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022804:00001

Permanent Link: http://ufdc.ufl.edu/UFE0022804/00001

Material Information

Title: Isolation and Characterization of Normal and Glaucomatous Canine Trabecular Meshwork in Vitro
Physical Description: 1 online resource (69 p.)
Language: english
Creator: Rinkoski, Tommy
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: beagle, canine, glaucoma, poag
Veterinary Medicine -- Dissertations, Academic -- UF
Genre: Veterinary Medical Sciences thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Over 35 years of research, the unique POAG Beagle model has been a boon to glaucoma researchers. Our study proposes to expand the usefulness of the model by creating protocols for the primary culture of TM cells from normal and glaucomatous canines and confirm the identity of cultured cells, so that TM from the POAG Beagle can be confidently utilized in future research. Primary cultures obtained by enucleation and dissection of normal and glaucomatous canine were cultured in high-glucose DMEM supplemented with 20% FBS initially and 10% FBS after cell establishment. Slices of anterior segment from two eyes were cultured for various times out to one week, fixed and mounted to view expansion histologically. Cell lysates from glaucomatous and normal TM cells incubated with dexamethasone were examined for the presence of myocilin protein. Glaucomatous and normal TM cells were incubated with 0.5 micron latex microbeads to assay phagocytic activity. 79 normal and 16 glaucomatous primary and subsequent cultures were successfully created, as well as cells used for assays and experiments. Passaging, freezing, and thawing of cells was shown to be safe and reliable, leaving 10 aliquots of glaucomatous TM cells for future experimentation. TM cells grew at increasing rates according to percent FBS in their medium, up to a maximum of 20% serum. Histology showed TM cells in culture do not increase in density at their tissue of origin, but migrate along surfaces to which they can attach. Stimulation with dexamethasone increased myocilin expression in both normal and glaucomatous TM cultures. Glaucomatous cell monolayers retained fewer microbeads than normal counterparts, and dexamethasone treatment appeared to impair microbead retention in both normal and glaucomatous cells. Glaucomatous and normal canine TM cells were successfully cultured, safely passaged, and can be frozen for storage and rescued. Cultures behave as predicted for cells of TM origin in myocilin production and phagocytic activity. A population of cultured glaucomatous TM from the POAG Beagle model has been created for future use.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Tommy Rinkoski.
Thesis: Thesis (M.S.)--University of Florida, 2008.
Local: Adviser: Samuelson, Don A.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022804:00001


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ISOLATION AND CHARACTERIZATI ON OF NORMAL AND GLAUCOMATOUS CANINE TRABECULAR MESHWORK IN VITRO By THOMAS A. RINKOSKI A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2008 1

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2008 Thomas A. Rinkoski 2

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To Audrey, Cain, Raechel, and Ethan, and to Woody, with all who came before and after him. 3

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ACKNOWLEDGMENTS Id like to thank everyone I wo rked with at UF. Every one of them contributed to my graduate education in some way. I owe all my prof essors a debt for the doors of research they opened to me, and for showing me some of the people, ideas, and challenges that lay beyond those doors. Dr. Kirk Gelatt was both a friendly, down -to-earth, and helpful mentor and an unbelievably sharp researcher. Its an impressive mind that can recall journal and page number to any glaucoma article I could dig up, but its a ve ry special person who will also tell you how the authors health and marriage are faring. I cant think of a better model for never losing an ounce of humanity to the pressures of research. Dr. Maria Kallberg has been an equally valuable role model. She exemplified a mixture of tenacity, intellect, curios ity, and spirit of collabo ration that ought to exist in every scientist. Thanks go to her and Dr. Caryn Plummer esp ecially for tirelessly playing the roles of veterinarian, professor, or mentor at any point that our research required it. Dr. Don Samuelson was generous with his ti me, expertise, and equipment. Without his support I could never have finished when I did. Special thanks also go to Pat Lewis, who was often the channel through whic h I could gain from the Samuelson lab, not to mention a constantly lovely person to be around Dr. Ed MacKay was showing me the way th rough every step of my time at UF. Professionally and personally, he pl ayed a significant role in my life, and there was no end to the lessons I could learn from him. In particular, I will always be tr y to emulate his ability to find efficiency, to clear away the unnecessary and s ee what is important both in process and product of the scientific effort. 4

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Additional thanks go to Mike Dismuke fo r providing experience and assistance as I embarked into the world of trabecular meshwork culture, and to John and Rachel Kuchtey for their intellectual generosity. Thanks go to Jacki Anderson, without whom I never would have thought to look to veterinary medicine as a field of employment, and would never have found the road I am now travelling. Without my family I would never have had the support to go back to my education. Thanks go to my parents for instilling in me the impor tance of knowledge, and for encouraging all the better sides of my nature. Thanks go to Marie, my big sister for being such a difficult act to follow, and thereby keeping me driven to succeed; and to Brian, my little brother, for his unending stimulus of imagination. Finally and foremost, I give thanks for the love and support of my wife and children, to whom this work is dedicated, and without whom I would be woefully incomplete. 5

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TABLE OF CONTENTS page ACKNOWLEDGMENTS................................................................................................................ .....4 LIST OF TABLES................................................................................................................. ................7 LIST OF FIGURES...............................................................................................................................8 ABSTRACT...........................................................................................................................................9 1 INTRODUCTION................................................................................................................. .......11 Glaucoma......................................................................................................................................11 Trabecular Meshwork Tissue.......................................................................................................13 Trabecular Meshwork Cell Culture..............................................................................................1 5 Characterization of Trabecular Meshwork...................................................................................17 2 METHODS...................................................................................................................... .............21 Primary Cell Culture of Canine TM cells.....................................................................................21 Deviations................................................................................................................................23 Passaging of Canine TM Cells................................................................................................24 Cell Counting of Canine TM Cells..........................................................................................24 Freezing and Thawing of Canine TM Cells............................................................................25 Serum Concentration Assay...................................................................................................... ...26 Histological Time-Course....................................................................................................... .....27 Dexamethasone-Induced Myocilin Expression............................................................................27 Phagocytosis.................................................................................................................................29 3 RESULTS...................................................................................................................... ...............34 Primary Cell Culture........................................................................................................... ..........34 Serum Concentration Assay...................................................................................................... ...36 Histological Time-Course....................................................................................................... .....37 Characterization............................................................................................................... .............38 4 DISCUSSION................................................................................................................... ............54 Cell Culture................................................................................................................... ...............54 Serum Concentration Assay...................................................................................................... ...55 Histological Time-Course....................................................................................................... .....56 Characterization............................................................................................................... .............57 Prostaglandin Pathway.................................................................................................................58 Targeted Genetic Techniques.................................................................................................... ...61 LIST OF REFERENCES.....................................................................................................................63 BIOGRAPHICAL SKETCH............................................................................................................ ...69 6

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LIST OF TABLES Table page 3-1. Master list of cultures................................................................................................... ..........51 7

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LIST OF FIGURES Figure page 1-1. Aqueous humor outflow pathway and relevant anatomy of the drainage angle....................20 2-1. Experimental set-up for serum concentration assay...............................................................31 2-2. Experimental set-up fo r characterization assay......................................................................32 2-3. Lane assignments for electrophoresis.....................................................................................3 3 3-1. Heterogeneous growth in a primary culture plate..................................................................40 3-2. TM cell expansion in culture over time..................................................................................41 3-3. Confluent TM cell growth patterns........................................................................................42 3-4. Debris in primary TM cultures...............................................................................................43 3-5. TM cell senescence over multiple passages...........................................................................44 3-6. Cell growth over time by percent serum in media.................................................................45 3-7. Histological timecourse at the TM........................................................................................ 46 3-8. Expansion of cells of the limbal epithelium by histological time-course...............................47 3-9. Expansion of TM cells away from the TM by histological time-course................................48 3-10. Western blot for myocilin.....................................................................................................49 3-11. Phagocytic activity of canine TM cells................................................................................50 8

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Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science ISOLATION AND CHARACTERIZATI ON OF NORMAL AND GLAUCOMATOUS CANINE TRABECULAR MESHWORK IN VITRO By Thomas A. Rinkoski December 2008 Chair: Don Samuelson Major: Veterinary Medical Sciences Over 35 years of research, the unique POAG B eagle model has been a boon to glaucoma researchers. Our study proposes to expand the us efulness of the model by creating protocols for the primary culture of TM cells from normal and glaucomatous canines and confirm the identity of cultured cells, so that TM from the POAG B eagle can be confidently utilized in future research. Primary cultures obtained by enucleation a nd dissection of normal and glaucomatous canine were cultured in high-glucose DMEM supplemented with 20% FBS initially and 10% FBS after cell establishment. Slices of anterior segment from two eyes we re cultured for various times out to one week, fixed and mounted to vi ew expansion histologic ally. Cell lysates from glaucomatous and normal TM cells incubated with dexamethasone were examined for the presence of myocilin protein. Glaucomatous and normal TM cells were incubated with 0.5 micron latex microbeads to assay phagocytic activity. 79 normal and 16 glaucomatous primary and subsequent cultures were successfully created, as well as cells used for assays and experiments. Passaging, freezing, and thawing of cells was shown to be safe and re liable, leaving 10 aliquots of gl aucomatous TM cells for future experimentation. TM cells grew at increasing rates according to percent FBS in their medium, up 9

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10 to a maximum of 20% serum. Histology showed TM cells in culture do not increase in density at their tissue of origin, but migrate along surfaces to which they can attach. Stimulation with dexamethasone increased myocilin expression in both normal and glaucomatous TM cultures. Glaucomatous cell monolayers retained fewe r microbeads than normal counterparts, and dexamethasone treatment appeared to impair microbead retention in both normal and glaucomatous cells. Glaucomatous and normal canine TM cells were successfully cultured, safely passaged, and can be frozen for storage and rescued. Cultures behave as predicted for cells of TM origin in myocilin production and phagocytic activity. A popul ation of cultured glaucomatous TM from the POAG Beagle model has been created for future use.

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CHAPTER 1 INTRODUCTION Glaucoma The glaucomas are a set of related disorders marked by progressive atrophy of the optic nerve, giving the appearance of an excavated or cupped optic nerve head when viewed by ophthalmoscopy. Untreated or unsuccessfully treate d, the disease can end in blindness in the affected eye(s), and the glaucomas are the s econd leading cause of blindness worldwide. Elevated intraocular pressure (IOP) is a primary ri sk factor shared over mo st forms of glaucoma. The primary glaucomas were estimated to affect 66.8 million people worldwide in the year 2000, with 6.8 million reaching a state of bilateral bl indness. Primary open angle glaucoma (POAG) was estimated to affect 33.2 million, and is the primary form of glaucoma found in those of European, African, and Latin American descent. Primary angle closure glaucoma (PACG) was estimated to affect 33.6 million, and is most prevalen t in those of Asian descent, most notably in Chinese populations (Quigley et al., 1996). In the canine, the primary glaucomas show a similar penetrance in the overall population, which was shown to be 0.5% (Martin, 1977), and furt her described as an in crease from 0.24% to 0.89% in the period from 1964 to 2002 (Gelatt and MacKay, 2004b). As in man, the primary glaucomas in the canine can be classified as POAG or PACG, but there is also the opportunity to organize the many varieties of the disease base d on the breeds in wh ich a given variety is prevalent. For example, the American Cocker Spaniel (ACS) has the highest prevalence of glaucoma of any breed, as high as 5.52%, with a gender-biased distribution. Glaucoma in the ACS is typically narrow or closed angle, often presen ts unilaterally before advancing to the contralateral eye, and seems to generally adva nce in acute IOP elevations that increase in intensity over time (Gelatt et al., 2007). 11

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The POAG Beagle is the disease model which w ill be used to represent the glaucomatous canine eye in our study. This model, when in troduced, was the only heritable POAG disease model available, and continues to be unique, in that other animal models for glaucoma are limited to hypertension or another feature of glau coma without showing the full disease, or are induced glaucomas. In the glaucomatous Beagle, the disease typically shows the first elevations in IOP, often in temporary spikes, between 8 a nd 16 months of age, with continuous elevated IOP and other clinical signs of the disease ap pearing later, between 2 and 5 years of age. Inheritance of the dis ease is autosomal recessive. The spontaneous, inherited POAG of the Beagle has kept the model in active research and publication for more than 35 years (Barrie et al. 1985, Brooks et al. 1995, Gelatt et al. 1976, 1977, 1981, 2007, Gelatt and MacKay 1998, 2001, 2004a, Kllberg et al. 2007, Kuchtey et al. 2008, Samuelson et al. 1989, 2001). The etiology of POAG, whether in man or in the Beagle, is incompletely defined. The progression of the disease seems to depend on the elevation of IOP causing mechanical difficulties, as well as creating a stressful environm ent for various tissues resulting in changes in gene and protein expression and a coincident cha nge in the biochemical environment. A rise in IOP is defined as increasing fluid pressure of the eye, and the fluid in question is aqueous humor, a complex mixture of chemical and biological fact ors that provides circul ation to the avascular tissues of the anterior eye, es pecially the lens and cornea. Aqueous humor is produced by, and enters the eye from, the epithelium of the ciliary processes, posterior to the iris. The primary flow of aqueous humor takes it anteriorly from the posterior chamber where the lens is held, through the iris, and into the anterior chamber wh ere it can come into contact with the cornea. Aqueous then exits the eye primarilythrough th e corneoscleral, or conventional, outflow pathway, i.e. the primary outflow pathway, or th rough a uveoscleral outflow pathway, in which it 12

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either drains sclerally or is reabsorbed into the uvea. The co nventional outflow pathway brings the aqueous humor to the iridocor neal angle, formed by the anteri or iris and the corneoscleral junction. The fluid first flows past the pectinate ligament, a series of fibrous columns anchoring the iris to the corneoscleral wall. Then the a queous encounters and passe s through the trabecular meshwork (TM), the tissue of inte rest for our study, and finally into an angular aqueous sinus, referred to as the Schlemms canal in man, or th e angular aqueous plexus (AAP) in canine, to finally enter the venous system and exit the eye. This pathway and relevant anatomy are shown in Figure 1-1. Trabecular Meshwork Tissue Canine TM consists of a network of filament ous trabeculae coated in fibroblast-like TM cells. The tissue can be subdivided based on the lo cation relative to neighboring tissues as well as the density of the filamentous network and associated cells. The trabeculae filaments, or beams, are composed of a core of collagen and elastin sheathed with granular material and most exteriorly with a basement membrane-like material, on which the cells of the TM attach. The filaments and any incidental extracellular material in the TM are considered extracellular matrix (ECM). The most inner portion of the TM, which is anteriorly attached to the pectinate ligament, is termed the uveal trabecular meshwork (UTM ), and contains the most open area of the TM, with little or no fluid resistan ce. This open network of filame nts in the UTM anchors in the anterior ciliary muscle fibers and at the fibrous tunic at the corneoscleral junction, coincident with the site of the scleral spur in human. Th e portion of TM connecting the UTM to the inner wall of the globes fibrous tunic is the corneoscleral trabecular meshwork (CTM). In the CTM, both the trabeculae and the spaces between them are noticeably reduced, resulting in a tighter network that possibly provides some fluid resistance. Within the scleral wall, at the point of attachment of the CTM, lies the angular aqueous plexus (AAP), the radially aligned drainage 13

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veins through which aqueous humor can exit the a ngle. The AAP veins are internally lined with a monolayer of endothelial cells. The region allowing entry to the AAP from the CTM is often termed the juxtacanalicular region of the TM, wher e fluid must either circumvent cell junctions, or pass through vacuoles and be transported across the endothelial cell to reach the AAP (Samuelson, 2007). Since a great deal of the TM cell culture performed to date has been with human tissue, some anatomical differences from the canine model should be highlighted. The human TM can be divided into sections similar and likely orthologous to those in the canine. The first portion of TM encountered by the exiting a queous flow are beams of ECM co ated with a monolayer of TM cells. Further into the angle is the juxtacanalicular or cribriform region with an increasingly dense mixture of ECM and cells. This differs from the canine in that the juxtacanalicular region seems perhaps a compression of the canine CTM, without separable beams. Schlemms canal, a circumferential drainage vein for exiting aqueous replaces the AAP of the canine eye, but is similarly lined by endothelial cells usually referred to as Schl emms canal endothelial cells (Acott and Kelley, 2008). Glaucomatous TM as differentiated from normal has been documented histologically in the Beagle, showing condensed and less organized trabeculae in glaucomatous TM, as well as a marked increase in extracellular material compared to normal (Samuelson et al., 1989). These findings match similar data found in human nor mal and glaucomatous TM, which show an increase in extracellular material with age for all samples, with a sharper increase in glaucomatous TM (Tripathi, 1972, Lutjen-Drecoll et al., 1981, Rohen, et al., 1981). In canine and human tissue, it has also been noticed that TM cellularity changes in a similar pattern; in both normal and POAG eyes, TM cellularity decrease s with age in a parallel manner, but at any 14

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given age, glaucomatous eyes have fewer cel ls (Alvarado et al., 1984, Samuelson and Gelatt, 1999). These changes are unlikely to account for a restriction of aqueous outflow by themselves, but undoubtedly reflect aspects of the disease not yet fully understood. Trabecular Meshwork Cell Culture The first report of successful growth of a TM culture system (using human TM) was by Polansky et al. in 1979. They described growing explanted TM tissue in Dulbeccos Modified Eagle Medium (DMEM), with 10% human seru m, glutamine, antibiotic and antimicotic supplements. They also experimented with adding fibroblast growth factor (FGF), and found that it modestly accelerated the plating and expansio n of the cultures, slightly decreasing the attachment time from the standard 1-2 weeks. Th ey noted that no proteolytic agents or tissue dissociation techniques were use d, due to concern that cells could be altered in unforeseen ways. On the lookout for potential contaminating cell populations, they cultured corneal endothelium (CE) as well, to compare its growth characterist ics, finding that the CE grew much slower, was morphologically distinct, and could not be sustained in culture beyond 2 passages. Further work in culture, description, and characterization of TM and neighbor ing tissues was described in a follow-up article (Alvarado et al., 1982). The TM cultures from this group were shown to grow into the fourth and fifth passages, although they noted some morphological alterations in later passages, especially when they allowed the cells to remain at confluency for 1-2 weeks. A more recent paper looked further into the senescence of porcine TM cell cultures, showing that cells that had undergone more doublings in culture not only revealed an older, flattened morphology, but had shortened telomeres and upregulat ed expression of a senescence-related -galactosidase protein (Yamazaki et al., 2007). Once a procedure for culture of this tissue, important in the pathology of the glaucomas, was available, it took very little time for others to folow, and 1983, a study using TM cultured 15

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from glaucoma patients was published (Southren, et al.). In this study, TM cells were cultured from small segments of TM removed during tr abeculectomy surgeries rather than dissected whole eyes. Obtaining glaucomatous human eyes for primary culture is an ongoing difficulty, and another reason why an analogous animal model for glaucomatous TM is desirable. Stamer et al. advanced the technique in 1995 by re-evaluating the prot ocol of dissociating the TM cells from the surrounding ECM. These investigators proposed that without some digestion, only the outermost layer of cells were available for culture, an d that by freeing cells from deeper in the tissue, a more representative culture of TM cells could be established, with the added boon of a larger founding population. They used a collagenase digestion procedure to accomplish this, successfully growing cultures and in time showing that the digestion procedure produced better results for the primary culture of glaucomatous human TM (Stamer et al., 2000). This method soon became the preferred met hod for producing monolayer TM cultures. In the aim to recreate more closely a livi ng ocular outflow system, a new TM culture system was developed. After dissecting away the posterior segment, vitreous, and lens, the remaining anterior chamber is clamped into a modified culture dish which allows the segment to be filled with media from the inside and pressuri zed to recreate the IOP of a living eye, allowing outflow to build up outside the eye in a reservoir. This tech nique allows perfusion-based experiments, as well as retaining the cells in thei r natural niche, which can still be sectioned for histological examination after manipulations (Johnson and Tschumper, 1987, 1989). Another technique used to examine perfusion through TM is simply growing a TM monolayer on a filter, which can be placed in a closed system and subj ected to a known pressure (Perkins et al., 1988; Roberts et al., 2007). 16

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With the continuing goal to understand the character and activity of TM cells and how they behave in vivo, some attention has recently been paid to the growth media used for TM or anterior segment organ culture. In vivo, TM grow s in the presence of aqueous humor, and while serum supplementation seems necessary to cause TM cells to replicate and grow in culture, some studies can and have been done noting the differi ng effects of normal growth media and aqueous humor. It was noted that aqueous humor supplemen tation stimulated migration of TM cells in a culture system (Hogg et al., 2000). In more depth, Fautsch et al. showed in 2005 that monolayer cultures grown in 50% aqueous humor differed in morphology and protein expression compared to cells in serum-supplemented media. Aqueous -supplemented cells also dramatically slowed replication, which is certainly more similar to an in vivo situation, where most TM cells also replicate very slowly (Kimpel and Johnson, 1992). A further study showed that TM cells in perfusion culture, treated with recombinant myocilin, showed increased outflow resistance in the presence of aqueous humor, but not in normal media (Fautsch et al., 2006). Characterization of Trabecular Meshwork Simply having successfully isolated a distinct cell line does not always guarantee the accuracy of an investigators expectations about th e identity of the cell line; characterization is a necessary step to lend credence to the isolation. Characterization of many cell types is performed by identifying antigens on the cell surface that distinguish the cell population from neighboring or similar types, but research has so far not turned up any definitive antigens proving the identity of TM tissue in comparison to its neighbors. Fort unately, there are other ch aracteristics that are routinely used: TM cells produce an excess of myocilin protein product when stimulated with certain environmental stresses; and TM cells ar e actively phagocytic, and have been shown to ingest a variety of particles. 17

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The myocilin protein was described in separate publications by Stone et al. and Polansky et al. in 1997 as TIGR, the Trabecular meshwork I nducible Glucocorticoid Response protein. As the name implies, the protein product was iden tified as being produced in the presence of glucocorticoids including corticosteroids, and was isolated from cultures of TM tissue. This finding was supported and clarified in 1999 to show that in vivo and organ-cultured TM produce myocilin constitutively, while TM cell lines prod uce little to undetectable amounts without stimulation. Dexamethasone, TGF or mechanical stress can all induce expression of myocilin as shown by northern blot mRNA analysis (Tamm et al., 1999) Immunoflourescence staining with antibodies against myocilin show that in uninduced TM cell populations, 20% of cells were expressing various levels of m yocilin. Once induced with dexamethasone, 60 to 80% of the cells were showing myocilin expression, and at higher average levels of expression. (SC cells showed no basal staining with 6-15% of cells stai ned when induced) (OBrien et al., 1999). Even before TM cells had been cultured in v itro, they had been characterized as phagocytic (Rohen and van der Zypen, 1968; Grierson and Lee, 1973). It was also made clear that cells in monolayer culture retained phagocytic capacity (T ripathi and Tripathi, 1982), and it was further shown that TM phagocytosis also extended to synthetic particle s, of which latex microbeads have been used most frequently (Grierson et al., 1986). An experime nt using bioparticles (zymosan, 0.3m diameter) in live eyes shed light on the process of TM phagocytosis and indicated that some cells detach ed and migrated after ingesti ng a volume of particles (Sherwood and Richardson, 1988). Perfusion anterior segment culture again confirmed both bioparticle and synthetic particle phagocytosis by TM cells (Buller et al., 1990). TM phagocytosis in the canine has also been confirmed. It was shown that while canine TM cells would engulf latex beads as large as 3 m, 0.5m beads were the most readily ingested. 18

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It was also shown that this phagocytosis was well underway one hour afte r introduction of the particles (Samuelson et al., 1984). Based on this data, our study will use the phagocytosis of 0.5m beads as additional evidence that the cells cultured are indeed of TM origin, and will allow the cells 2 hours of incubation with the beads before analysis. Phagocytosis was demonstrated with cells from POAG human eyes in perfusion organ culture, but there was not a signifi cant increase or decrease in phagocytic activity compared to normal (Matsumoto and Johnson, 1997a). Exposur e to dexamethasone in the same culture system caused a 57% decrease in phagocytic activity by nonglaucomatous TM cells (Matsumoto and Johnson, 1997b). A more recent study showed a significant difference in phagocytic activity between glaucomatous and normal TM cells (Zha ng et al., 2007).While countering the previous finding, it confirmed that in both normal and glaucomatous cells, dexamethasone treatment significantly lowered phagocytic activity. 19

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Figure 1-1. Aqueous humor outflow pathway and relevant anatomy of the drainage angle 20

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CHAPTER 2 METHODS Primary Cell Culture of Canine TM cells Methods and sources for obtaining cadaver ey es for our study were approved by the UF Institutional Animal Care a nd Use Committee (IACUC). Normal eyes were obtained from heartworm positive mixed-breed canines of unknow n age after being euthanized by Alachua County Animal Services and brought to the UF Co llege of Veterinary Medi cine to be used for multiple research and teaching purposes. Glaucomatous eyes were obtained from a single individual upon reaching the endpoint of his research career in the UF POAG Beagle colony. All eyes were enucleated using a subconjunctival method to minimize harvesting of excess tissue. Enucleations were performed 0.5 to 2 hours postmortem for the normal eyes and immediately upon euthanasia for the glaucomatous eyes. All ey es were placed on wet ice for no more than 3 hours, until dissection could be performed. Dissections were performed using aseptic technique with sterile instruments. Excess tissue was removed from the globe using a razor blade, and the globe was then submerged in a 1:1 mixture of phosphate-buffered saline (PBS) (H yclone) and 7.5% Betadyne solution (Webster Veterinary Supply) and rinsed with PBS to remove all traces of Betadyne. The eye was hemisected equatorially using a razor blade a nd the posterior portion wa s removed and placed in formalin (for glaucomatous eyes) or discarded. The lens and vitreous were carefully removed from the remaining anterior segment and discar ded. The iris and ciliary body were then removed in an intact ring, pulling apart points of attachme nt to the sclera. These tissues were placed in formalin if they were from glaucomatous eyes, but were otherwise discarded. The resulting anterior segment was placed in primary cult ure medium (20% FBS, High-glucose DMEM, 100u/mL pen/strep/fungiezone [all Hyclone]) an d incubated briefly at 37C and 5% CO2. 21

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The anterior segment was removed from incubation to a dish or well filled with PBS to cover the segment, as the removal of TM tissue is stabilized by a fluid environment. The anterior segment was quartered, as each quarter eye would provide the starting material for one well of a six-well cell culture plate (Corning costar). On ce quartered, the tissue in its PBS environment was placed under a dissection scope to scrape unwanted tissue away from the TM. Using a scalpel, the corneal en dothelium was scraped away from the anterior TM, and then any excess tissue from the anterior ciliar y body, iris, or pectinate ligament was scraped away from the posterior TM. The PBS environment can be changed at this point to rinse aw ay floating pieces of corneal endothelium, which could otherwise co ntaminate the culture. A curvette was employed to peel up one end of the TM from the sclera, an d then the tissue could be grasped with a forceps and carefully peeled away. The PBS environment assi sted at this step, help ing to keep the fragile strip of TM from breaking as it peeled away from the sclera. The TM strip was removed to a collagenase solution (5mg collagenase A and 5mg human albumin [Roche], in 5mL PBS [Hyclone]), and the TM was removed from the other quarters and each placed in its own aliquot of collagenase. The strips in so lution were agitated and incubated at 37C and 5% CO2 for 30 minutes. The digested TM materi al was pelleted by centrifugation at 2000rpm for 8-10 minutes at room temperature. The collagenase solution was then removed, and the TM material was resuspended in primary cult ure medium. This was plated into either one well of a six-well plate or into a T25 cell culture flask (Corning costar). The culture was incubated at 37C and 5% CO2 for 3-5 days. When phase contrast microscopy showed TM cells adhering to the cu lture surface and migratin g away from the TM tissue, the media was changed to 10% culture media (10% v/v FBS, High-glucose DMEM, 100u/mL pen/strep/fungiezone [all Hyclone]). For cu ltures from glaucomatous eyes, in order to 22

PAGE 23

add to the number of cultures being created, the original media with the TM tissue pieces was moved to another well in the cell culture plat e to begin an additional primary culture, as recommended by Dismuke (personal communication) Once the culture reached confluency in the primary culture well or flask, it was passa ged 1:3 or 1:4 into identical containers. Alternatively, one confluent well of a 6-well plat e could be passaged into a T25 flask, or even 1:2 into T25 flasks; and one confluent T25 flask could be passaged 1:6 or even 1:12 into 6-well plate wells. Deviations The entire TM of eye 05 was placed into a si ngle culture, following the above technique, rather than dividing the quarters into separate cultures. Eyes 03 and 04 were collected as described above, but were sprayed with a 2% solution of Betadyne and rinsed with sterile water to prepare the globe after excess tissue remova l. When quartered, quarters 03C and 03D were dissected differently: after scraping away neighboring tissue, the exterior limbus was also carefully scraped to remove limbal epithelium, and full-thickness cuts were made anterior and posterior to the TM, resulting in a full-thickness explant of TM sitting atop the scleral wall. Quarters 04C and 04D were also dissected di fferently: after scraping away neighboring tissue under the surgery scope, TM was removed as described but without the aid of a PBS bath, and placed directly into culture as an explant rather than subjected to a collagenase digestion. These explants were placed into 10% cu lture media and then treated as described above. Quarters 03A, 03B, 04A, and 04B were dissected as described above, but without the aid of a PBS bath when removing the TM; additionally, while the collagena se solution for these samples was identical, their incubation with the solution deviated fr om above, with nutation for 2 hours at room temperature before centrifugation and plating with 10% culture media. Quarters 03A and 03B 23

PAGE 24

were placed together into collagenase solution an d into culture to compar e use of a half-eye of TM to a quarter. Passaging of Canine TM Cells Passaging was performed when cultures were es timated to be at 90% confluence or higher, and was performed in a laminar flow hood us ing sterile technique. Media was removed by vacuum from the culture containe r. The container was then rinsed with HBSS (HyClone) to wash away remaining serum albumin, and the rinse was removed by vacuum. Tryspin (0.25% Trypsin EDTA, Hyclone) was added to cove r the bottom of the container (1mL was sufficient for either a 6-well plate or a T25 flask), and was left to act on the cells for 3-4 minutes. The trypsinized cells were gently pipetted up and down to assist in loosening cells still attached to the culture surface, and then removed to a prepared conical tube containing the desired cu lture media at a volume that would allow 1mL of media plus cell susp ension to be dispensed to each new culture container (for 6-well plates or T25 flasks). Th e cell suspension was gently pipetted up and down to mix. If a cell count was desired, it could be take n at this stage. The cell suspension was then plated in equal volume to each new culture cont ainer, and additional volume of desired culture medium was added to reach a 5mL total (for 6-well plates or T25 flasks). The new cultures were incubated at 37C and 5% CO2, and occasional observations were made to monitor growth. Media was changed every 4-7 days as require d, and when reaching confluency, cells were passaged again, frozen, or used as desired. Cell Counting of Canine TM Cells Preparation for cell counting was performed in a laminar flow hood using sterile technique, but once cells were placed into the counting cham bers, they would not be re-used for culture, as the hemacytometer is not sterile. Beginning w ith a cell suspension of known volume, a small amount (50ul) was transferred to both counting ch ambers of a freshly cleaned hemacytometer 24

PAGE 25

(with a new or freshly cleaned c over slip). Cells were allowed to settle briefly before counting. The slide was viewed at 100X and cell counts we re performed in 5 squa res (center and four corners) on both counting chambers. The total tall y was kept on a hand counter as each square was sequentially viewed. Cells c overing the border line between two squares were counted if they were on the top or left boundary, but not counted if they were on the bottom or right boundary. If a dilution was thought to be necessary, it would be performed on the cell suspension before removing the aliquot to th e hemacytometer. The average count per square was determined by dividing the total count by 10. The cells/mL va lue was determined by multiplying the average count per square by 104 (the volume correction factor for the hemacytometer), and multiplying by the dilution factor if applicable. Freezing and Thawing of Canine TM Cells Freezing was performed when cultures were esti mated to be at about 90% confluence and still growing. Both freezing and thawing were performed in a laminar flow hood using sterile technique, or with cell suspensions in ster ile containers when not working in the hood (centrifuge, freezer). Media was removed by vacuum from the culture container. The container was then rinsed with HBSS (HyClone) to wash away remaining serum albumin, and the rinse was removed by vacuum. Trypsin was added to cover the bottom of th e container (1mL was sufficient for either a 6-well plate or a T25 flask) and was left to act on the cells for 3-4 minutes. After their short incubation in trypsin, the cells in suspension were gent ly pipetted up and down to assist in loosening cells stil l attached to the culture surface, and then removed to a prepared conical tube containing 2 mL of 4C 10% culture media. The cell suspension was gently pipetted up and down to mix. The resulting suspension wa s centrifuged at 1500rpm for 8-10 minutes, and the supernatant was removed. The pellet was re suspended in 1mL 4C freezing medium (10% v/vFBS, High Glucose DMEM, 100u/mL pen/stre p/fungiezone [all Hyclone], 10% v/v DMSO 25

PAGE 26

[Fisher Bioreagents]). A cell count was performed using this su spension, and the total volume was adjusted to bring the final concentration around 106 cells/mL. The cell suspension was distributed into 2mL cryovials at 1mL/vial (Corning costar) and placed immediately on wet ice for 20-30 minutes, then transferred to a -8 0C freezer for freezing and storage. To thaw frozen aliquots of ce lls, the cryovials were placed into a 37C water bath with constant agitation until thawed (1-2 minutes), after which the contents were transferred to a 15mL conical tube containi ng 2mL 37C primary culture medium. This suspension was centrifuged at 1500 rpm for 8-10 minutes and the supernatant, containing DMSO, was removed. The pellet was resuspended in 1mL 37C culture me dium and plated into either one T25 culture flask or 2 wells of a 6-well culture plate. The new cultures were incubated at 37C and 5% CO2, and occasional observations were made to mon itor growth. Media was changed every 4-7 days as required, and when again reaching confluency cells were passaged or used as desired. Serum Concentration Assay All procedures over the course of this assay were performed in a laminar flow hood using sterile technique, or in sterile containers, with the exception of cell counting, and cell suspension aliquots used for counting were subsequently discarded. The cells used for the assay were growing into their second passage, plated from primary cultures 04A and 03C. Culture 04A was cultured using the collagenase digestion method, while 03C was cultured using a full-thickness explant. Both cultures were passaged into 20 we lls of a 24-well culture plate (Corning costar). Wells derived from 04A were plated at 1.7X104 cells per well, and those derived from 03C were plated at 1.45X104 cells per well. Cells from each source were grown at 5, 10, 15, 20, or 25% FBS-supplemented, high glucose DMEM with 100u/mL pen/strep/fungiezone (all Hyclone). Cells from each source growing in each variety of media were subjected to cell counts at day 2, day 4, day 6 and day 8 from plating. The configurat ion of the experimental plates is shown in 26

PAGE 27

Figure 2-1. Each well was discarded after counti ng rather than re-suspending and counting the same population at every time-point in order to minimize interfer ence with normal growth over time. Histological Time-Course Normal eyes were used to histologically vi sualize migration of TM cells away from an explant in a culture dish. Procedures prior to immobilization were perfor med in a laminar flow hood using sterile technique, or in sterile containers. Four eyes fr om two animals were used for our study. Enucleations were performed as desc ribed above, and dissections were identical through the step removing the vitreous, lens, ciliary body, and iris. After th is point, the anterior segment was cut into 8 slices, and the 6 most re gularly shaped were placed into 10% culture media. On days 0, 1, 2, 3, 4, and 7 one segment from each eye would be immobilized in HistoGel (Richard-Allan Scientif ic) and fixed in 10% buffered formalin for at least 24 hours. The tissue was serially dehydrated and placed into paraffin, then cut into 5m sagittal sections and mounted on glass slides. The slides were reh ydrated for staining with hematoxylin and eosin to show cell nuclei and morphologies and to de fine tissue types, and finally dehydrated and mounted with coverslips for viewing. Dexamethasone-Induced Myocilin Expression All cell culture procedures over the course of this experiment were performed in a laminar flow hood using sterile technique, or in sterile containers, with the exception of cell counting, and cell suspension aliquots used for counting were subsequently discarded. All steps related to cell lysis and protein separati on and detection were performe d using aseptic technique. The glaucomatous cells used for the assay were growing into their second passage, plated from primary culture WOSA. The culture was passa ged into all wells of a 24-well culture plate (Corning costar), plated at 1.04X105 cells per well. Cells were gr own in 10% culture media or 27

PAGE 28

the same media with 100nM dexamethasone (Roc he). Media was changed for all wells (without changing the type of media in each well) on da y 2 to introduce a second dose of dexamethasone to those wells where it was in play. Media was not changed subsequent to day 2 in order that any detectable protein product secreted into the supernatant would be retained. The normal cells used for the assay were grow ing into their third pa ssage, plated from primary cultures 06A-C and 06B-B, combined in order to better approach the plating numbers of the glaucomatous wells. After combining the cells, they were passaged into all wells of a 24-well culture plate as above, plated at 0.42X105 cells per well. Plates were then treated identically to the glaucomatous wells, above. C onfiguration of the experimental plates is shown in Figure 2-2. On day 6, the cellular material of selected wells was subjected to detection for myocilin. The supernatant was removed to collection tubes a nd stored at 4C. The wells were then rinsed with PBS, trypsinized, and the cells in solution placed into 10% culture media in a 1.5mL centrifuge tube to halt the action of the trypsin. The tubes were spun at 2000 rpm for 10 minutes to pellet the cells and supernatants were di scarded. Cells were resuspended in lysis buffer (Fermentas) and set on ice for 10 minutes. An additional centrifugation at 4000 rpm for 10 minutes pelleted the nuclei and fragments of membranous material, leaving the cytoplasmic cellular contents in solu tion. This supernatant was transferred to a collection tube and stored at 4C until Western bl ots were performed, and the pellets were discarded. All samples used for Western blots were combined with SDS reducing buffer (Cell Signaling Technology) and heated at 100C for 5 minutes in a boi ling water bath. Samples were placed on ice for 5 minutes and centrifuged for 5 minutes at 14,000 rpm. Samples were loaded into 15-well 12% Bis-Tris gels in an electrophoresis apparatus. The lane assignments are as shown in Figure 2-3, with normal control TM cells in lanes 1 and 2, normal TM cells stimulated 28

PAGE 29

with dexamethasone in lanes 3 and 4, glaucomat ous TM cells in lanes 5 and 6, glaucomatous TM cells stimulated with dexamethasone in lanes 7 and 8, positive control r ecombinant myocilin in lane 9, and a size ladder in lane 10. The gel was electrophoresed for 1 hour at 200V, then transferred to a 0.2m nitrocellulose membrane at 30V for 1.5 hours. 1% Ponceau S stain was added to visualize proteins, after which the blot was blocked for 30 minutes at room temperature with 1x TBA, 0.5% Tween 20, and 1% BSA. The prim ary antibody, rabbit anti-human myocilin (Santa Cruz Biotechnology) is added at a 1:100 dilution and gen tly rocked overnight at 4C. After washing, the blot was incubated with seco ndary antibody at 1:2000 dilution with gentle agitation for 90 minutes at room temperature. After another wash, Avidin/Biotin conjugate (ABC) solution (Pierce Biotechnology) was added and incubated with gentle agitation for 30 minutes at room temperature. After a final wash, Supersignal West Pico Chemiluminescent Substrate (Pierce Biotechnology) wa s added to cover the membrane in a small dish for 1 minute, after which the blot was sealed in plastic wrap and imaged on a Biorad ChemiDoc XRS. The membrane was exposed to the digital imager fo r 10 minutes and the image used for analysis. Phagocytosis Of the 24 wells of glaucomatous TM cells and 24 wells of non-glaucomatous TM cells, 12 wells from each plate were also used to obser ve the phagocytotic activ ity of the cells. Equal numbers of wells used for this experiment we re growing in dexamethasone-treated media and normal media. Microbeads of 0.5m diameter (Polysciences) were added to each well at a concentration of 109 beads/mL. The beads were allowed to incubate with the cells for 2 hours at 37C and 5% CO2. These wells would all still follo w the procedures for detection of proteins in the supernatant and cell extracts described above, but before the s upernatant was transferred to a sample tube for 29

PAGE 30

storage, a small amount was collected to detect for the concentration of microbeads still present in solution. To assemble a standard curve, known concentrations of beads from 107 beads/mL to 1010 beads/mL were prepared in 10% complete culture media, in a 24-well plate with no cells present. These were allowed to incubate for 2 hours before being collec ted, as above. A drop of solution, whether for the standard curve or from wells containing cells, was placed on a glass slide, coverslipped, and allowed to dry for 24 hours. Sl ides were then observed at 400X magnification and photographed, and the resulting images were overlaid with a grid and microbeads were manually counted. Counts were made over th e entire image, except in the case of 1010 count images, where a tighter grid was used, and representative areas were counted to extrapolate a total count. 30

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Figure 2-1. Experimental set-up for serum concen tration assay. Percent se rum concentration and day of collection a nd count are noted. 31

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Figure 2-2 Experimental set-up fo r characterization assa y. Two plates were prepared as above, one with glaucomatous TM cells, and one w ith normal TM cells. Each well is labeled with the protein to be detected for, the presence of 100nM dexamethasone if applicable, and the addition of 0.5m late x beads to study phagocytic activity, if applicable. 32

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Figure 2-3 Lane assignments for electrophoresis. 33

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CHAPTER 3 RESULTS Primary Cell Culture TM from normal and glaucomatous eyes was successfully grown in culture. Normal TM was successfully grown using three techniques: a full-thickness tissue explant, a TM tissue explant, and a collagenase digestion of TM ti ssue. Glaucomatous TM from POAG Beagle eyes was grown using only the collag enase digestion method, to avoi d the possible contaminations introduced with a full-thickness explant, and base d on the literature indi cation of better success for POAG TM culture (Stamer et al., 2000). Normal TM was grown into the 4th passage to observe morphological signs of senescence, while glaucomatous TM was frozen during the 2nd passage to preserve a maximum number of presenescent cells for future use. Normal TM recovered after freezing and thaw ing, indicating the capability to store canine TM for long periods. All TM cultures created over the course of our study are listed in Table 1, totaling 79 cultures or subcultures derived from normal tissue and 16 cultures or subcultures from glaucomatous tissue. Cultures derived from se gments 03C and 03D were cultured from a fullthickness TM explant; cultures derived from 04C and 04D were cultured from a TM tissue explant without collagenase; all remaining cultu res were produced by collagenase digestion of TM tissue. Typical growth in the primary plate was highly heterogeneous, with growth centered on tissue explants or remains of dige sted tissue, and very little growth in othe r areas of the plate, as seen in Figure 3-1. Growth proceeded at variable rates, but a primary plate was generally confluent and ready to passage at 9-14 days for explant cultures, or 7-11 days for collagenasedigested cultures. There was no significant differen ce detected in establishment and growth rate in culture for normal vs glaucomatous cells. A t ypical course of development in a glaucomatous 34

PAGE 35

primary culture is shown in Figure 3-2. Subcu ltures of passages beyond primary growth did not show the same heterogeneity, and could grow to confluence in slightly less time, dependant on their passage number and the density at which they were passaged. Cells in the second passage generally expanded to confluence in 5-9 days. Whether in primar y culture or subcultures, TM cells isolated in space from other cells would ofte n send out long cellular pr ocesses. As the cells expanded and reached confluence, the elongate spindleor fibroblast-shaped cells would typically be aligned with nei ghboring cells, sometimes linearly, but often creating swirling or fingerprint-like patterns as in Figure 3-3. Cells were frozen to -80 C and recovered successfully. Second-passage cultures 04B-A, 04B-B, 04B-C, 04D-A, 04D-B, and 04D-C were frozen after 5 days of growth. The cells were frozen in 1mL aliquots at approximately 106 cells/mL, producing 6 aliquots of cells. After thawing, all 6 vials produced a vi able culture, and grew to confluence in 10 days, at which point each was passaged 1:3; all subcultures were also viable. Two instances of fungal contamination occurr ed during the study; the first occurrence was isolated to the primary culture 03D and the cult ure was discarded after 6 days of growth. The second occurrence was in isolated wells in the dexamethasone/phagocytosis plates. These wells were not used for analysis and were treated with bleach to prevent the spr ead of the contaminant. At the point of enucleation, it was noted th at eye 05 contained blood in the anterior chamber. Dissection and culture were performed as normal, using TM from the entire eye, in case there was damage to the tissue. The culture, shown in Figure 3-4, contained far more debris associated with the TM than any other culture ove r the course of the study, even when compared to the glaucomatous plates, which themselves contained a high level of debris compared to cultures from non-glaucomatous sources. 35

PAGE 36

As observed above, TM cells in the second passa ge would typically grow to confluence in 5-9 days. This did not seem to vary between normal and glaucomatous cultures, as the second passage glaucoma cultures were near conflu ent when frozen for storage on day 6. Nonglaucomatous cells were observed in their third a nd fourth passages as well. Growth in the third passage cells was slower, taking 12 days to achieve confluence and be ready for further subcultures. The fourth passage was still capabl e of expanding, but showed variable growth, with some areas approaching confluency by day 10 while other areas in the same plate showing only sparse growth at the same time. Day 10 of thes e fourth-passage cultures marked the end of the study, so no further data was co llected, and no further passages were attempted. Cells of advancing passages, particularly the fourth, should marked mor phological difference. Figure 3-5 shows non-glaucomatous cultures during expans ion from primary culture through passage 4. Later passages are characterized by a loss of typical elongate cell shape, with a tendency toward ragged edges, and a lack of typical organization when confluent. Serum Concentration Assay Cultures 04A and 03C were plated into wells of a 24-well culture at 1.7X104 cells per well, and 1.45X104 cells per well, respectively. Results are averaged from cell counts of both cultures, and are summarized in Figure 3-6. Media contai ning 5% serum caused a 2.95-fold increase in cell number in the first 2 days, and a maximum growth of 3.37-fold at day 6. Media containing 10% serum caused a 4.44-fold increase in the fi rst 2 days and a maximum growth of 7.30-fold on day 8. Media containing 15% serum caused a 5.8 4-fold increase on day 2 and a maximum 10.9fold increase on day 8. Media containing 20% se rum caused a 7.97-fold increase on day 2 and a maximum 15.55-fold increase on day 4. Media containing 25% serum caused the most variable results, but averaged 3.59-fold increase in ce lls on day 2, and a maximum of 11.17-fold increase on day 6. 36

PAGE 37

Over the first four days, the growth in media of 5 to 20% showed a clear pattern: higher serum concentration led to increased cell expa nsion. Media containing 25% serum did not follow this pattern, indicating a limit to the growth benefits provi ded by level of serum concentration. Also of note was the duration of sustained gr owth: media with 20% serum caused a population crash after day 4; 15% and 10% serum media continued to gr ow through day 8, though at a reduced rate during the final 4 days; 5% serum media only showed signifi cant growth from day 0 to day 2. Histological Time-Course The methods employed allowed visualization of a process of TM cell outgrowth in culture in the context of a tissue explant. This allowed a rather different perspective than that gleaned with observations of monolayers in culture. It wa s observed that TM cells in their native TM environment expand only minimally, compared to the rapid growth seen in culture plate by phase-contrast microscopy. As shown in Figure 3-7, the cellularity of explanted TM tissue does not change significantly over th e 7 days observed, whereas a 2nd passage population in a culture flask could double many times over in the same peri od. It is not simply th e case that cells do not grow in the context of a media-soaked explant, because other cells in the explant showed proliferation, and there was even spread of TM cells into area s other than their native ECM meshwork. The cell population that was the first to show significant expansion was that of cells along the exterior limbal epithelium. Concern that th ese cells could be a seri ous contaminant is one purpose for the iodine soak of enucleated eyes prior to dissection (see methods). Figure 3-8 shows these cells beginning to sp read off the explant as early as day 1. By day 2 they have travelled along the cornea and around the edges to the interior face, tending to cluster in ragged crevices of the cornea formed as artifacts of the initial dissection. They are distinguishable from 37

PAGE 38

trabecular cells simply by their rounder, thic ker morphology, staining properties, and growth in multiple layers, unlike the cells of the corneal endothelium, whic h they occasionally grew over. They also appear able to migrate away from tissue, as seen in Figure 3-8 A and D. While TM cells showed little proliferati on in their native ECM meshwork, there was noticeable growth into other areas. This usually took the form of TM cells finding open areas in the sclera nearby the ICA and grow ing into them, forming a web-like network of cells, as seen in Figure 3-9 A and B. They would al so occasionally be seen growing along a flat scleral surface, but would still show a tendency towa rd web-like growth, rather than a flat monolayer, as in part C, with shapes somewhat reminiscent of the prot ruding cellular processes s een in culture as cells spread into new areas. Part D shows what appeared to be a more organized, mature outgrowth on day 7, in that the cells furthest from the grow th surface (which in this case was corneal) have connections only to each other and not to the su rface. These cells are growing in an arrangement similar to those in culture, generally aligne d on a shared axis, rather than the web-like morphology that characterized su rface-associated growth. Characterization Western blot analysis of cell lysates showed that TM cells expressed baseline levels of myocilin, and increased expression when stimulat ed by incubation in culture medium containing 100nM dexamethasone. Blots consistently showed smears around bands in both the positive control (recombinant myoc ilin) and dexamethasone-treated cell lysate lanes, but the differences in intensity were clear, as seen in Figure 3-10. This result supports the statement that the cells being grown are of TM origin. The assay for phagocytic activity also indi cated a confirmation of previous findings (Matsumoto and Johnson, 1997a, 1997b; Zhang et al., 2007). A standard curve was created, as shown in Figure 3-11a, and a regr ession equation was fit to the da ta, allowing bead counts from 38

PAGE 39

experimental slides to be converted to concentr ations in beads/mL of supernatant solutions. As each well contained 1mL of media, this concentration is equal to the actual number of beads in the well. The number of beads retained was highest in untr eated normal TM cells at 9.71X108 beads, significantly hi gher than the 9.28X108 beads retained in untreated glaucoma cells. The dexamethasone-treated cells from both nor mal and glaucomatous populations showed a downward trend relative to controls, with 9.47X108 beads retained in treated normals and 9.07X108 beads retained in treated glaucomatous, but comparisons between treated and untreated cells in either population were not significantly different. These results are summarized in Figure 3-11b, and indicate that these cell s are phagocytic, retaining a larg e percentage of the microbeads they were incubated with, and that glaucomat ous canine TM cells are less actively phagocytic than healthy cells. 39

PAGE 40

A B Figure 3-1. Heterogeneous growth in a primary culture plate. A) TM cells expanding from a digested piece of tissue in culture 06A on day 5, B) Id entical culture and day of growth, in a portion of the plate not containing any tissue remnants. 40

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A B C D Figure 3-2. TM cell expansion in culture over time. A) Culture WODA 1 day after plating, B) Culture WODA at 3 days, with long cellular processes, C) Culture WODA at 5 days, D) Culture WODA at 7 days showing come cellular alignment. 41

PAGE 42

A B Figure 3-3. Confluent TM cell growth patterns. A) Culture 04C showing near-confluent cells in a linear arrangement B) Culture 06C show ing near-confluent cells in a swirled arrangement. 42

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A B C Figure 3-4. Debris in primary TM cultures. A) Culture 05A at day 5, much more confluent than other day 5 primary cultures, presumably due to the use of TM from an entire eye rather than a quarter eye. B) Glaucoma tous culture WODA at day 8, also showing some debris. C) Non-glaucomatous culture 06A at day 8. 43

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A B C D Figure 3-5. TM cell senescence over multiple pa ssages. A) Primary culture 06A. B) Second passage culture 06A-C. C) Third passage cu lture 04C-D-C with some loss of typical elongate shape. D) Fourth passage culture 04C-C-A-D showing loss of shape and some development of ragged cell edges. 44

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0 50 100 150 200 250 300Cells/mL, ThousandsDay 5% Serum 1575046500475005300045000 10% serum 157507000084000110000115000 15% Serum 1575092000141000162000172000 20% Serum 15750125500245000183000182000 25% Serum 1575056500138500176000156000 02468 Figure 3-6. Cell growth over time by percent serum in media. 45

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A B C D Figure 3-7. Histological time-course at the TM. AC = anterior chamber, S = sclera. Overall cellularity of the native environment rema ins relatively unchanged over the course of the experiment. Pictures sele cted as representative samples from A) day 0, B) day 2, C) day 4, D) day 7. All images H& E stained and viewed at 250X. 46

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A B C D Figure 3-8. Expansion of cells of the limbal ep ithelium by histological time-course. LS = limbal sclera, EX = extraocular space, C = cornea, AC = anterior chamber, D = Descemets membrane. A) Cells were noted escaping from the exterior surface of the limbus, atop the limbal epithelium at day 1, 100X. B) Ce lls quickly spread around explant onto cut edges of cornea and filled available spaces (cut edges lack corneal epithelium), day 2, 250X. C) Cells appear on the interior surface, growing over the Descemets membrane and corneal endothelium, day 2, 400X. D) Cells expanding away from cut edges of cornea after growing seve ral layers deep by day 3, 100X. 47

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A B C D Figure 3-9. Expansion of TM cells away from th e TM by histological timecourse. AC= anterior chamber, S = sclera. All images 250X. A) TM cells expanding from ECM meshwork into open space, day 2. B) TM cells fully inhabiting a space previously empty, day 3. C) TM cells that have grown away from thei r native environment to inhabit an area of adjacent sclera, forming a web-like networ k as they spread away from their attachments, day 3. D) TM cells that have grown onto adjacent sc lera, spindle shaped with aligned spindle axes, day 7. 48

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Figure 3-10. Western blot for myocilin 49

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50 A y = 5E-05x0.7442R2 = 0.9681 1 10 100 1000 10000 1.E+061.E+071.E+081.E+091.E+10 Beads/ml Bead counts per frame Bead Counts Averages Regression Equation B 8.20E+08 8.40E+08 8.60E+08 8.80E+08 9.00E+08 9.20E+08 9.40E+08 9.60E+08 9.80E+08 1.00E+09Beads retained on cells Control Dexamethasone Control 9.71E+08 9.28E+08 Dexamethasone 9.47E+08 9.07E+08 Normal Glaucomatous Figure 3-11. Phagocytic activity of canine TM cells. A) Bead counts from concentration standards are plotted against known concen trations, and a regression equation was derived, as shown. B) The equation was used to convert bead counts from sample supernatants into concentra tions, which were subtracted from the total concentration to give the concentration retained, equal to the number of beads re tained (in 1 mL of media), which is plotted here by treatment group.

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Table 3-1. Master list of cultures Culture Origin Method Activity Fate 03A Normal alt. Collagenase day 4: media changed day 14: discarded 03C Normal Full-thickness explant day 4: media changed; day 8: explant removed day 10: subcu ltured into serum conc. Assay 03D Normal Full-thickness explant day 4: media change d, explant removed day 6: contaminated, discarded 04A Normal alt. Collagenase day 4: media chan ged day 10: subcultured into serum conc. Assay 04B Normal alt. Collagenase day 4: media changed day 11: passaged 1:3 04B-A Normal subculture day 5: frozen as 04B-X 04B-B Normal subculture day 5: frozen as 04B-X 04B-C Normal subculture day 5: frozen as 04B-X 04B-Xa Normal thawed from 04-B-X day 2: media changed; day 8: media cha nged day 10: passaged 1:3 04B-Xa-A Normal subculture day 6: discarded 04B-Xa-B Normal subculture day 6: discarded 04B-Xa-C Normal subculture day 6: discarded 04B-Xb Normal thawed from 04-B-X day 2: media ch anged; day 8: media cha nged day 10: passaged 1:3 04B-Xb-A Normal subculture day 6: discarded 04B-Xb-B Normal subculture day 6: discarded 04B-Xb-C Normal subculture day 6: discarded 04B-Xc Normal thawed from 04-B-X day 2: media changed; day 8: media cha nged day 10: passaged 1:3 04B-Xc-A Normal subculture day 6: discarded 04B-Xc-B Normal subculture day 6: discarded 04B-Xc-C Normal subculture day 6: discarded 04C Normal TM explant day 4: media changed; day 8: explant remove d day 14: passaged 1:4 04C-A Normal subculture day 9: media changed day 11: discarded 04C-B Normal subculture day 9: media changed day 11: discarded 04C-C Normal subculture day 9: passaged 1:6(wells) 04C-C-A Normal subculture day 8: media changed day 12: passaged 1:4 04C-C-A-A Normal subculture day 4: media changed day 10: discarded 04C-C-A-B Normal subculture day 4: media changed day 10: discarded 04C-C-A-C Normal subculture day 4: media changed day 10: discarded 04C-C-A-D Normal subculture day 4: media changed day 10: discarded 04C-C-B Normal subculture day 8: media changed day 12: passaged 1:4 04C-C-B-A Normal subculture da y 4: media changed day 10: discarded 04C-C-B-B Normal subculture day 4: media changed day 10: discarded 04C-C-B-C Normal subculture day 4: media changed day 10: discarded 04C-C-B-D Normal subculture da y 4: media changed day 10: discarded 04C-C-C Normal subculture day 8: media changed day 12: discarded 04C-C-D Normal subculture day 8: media changed day 12: discarded 04C-C-E Normal subculture day 8: media changed day 12: discarded 04C-C-F Normal subculture day 8: media changed day 12: discarded 51

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Table 3-1. Continued Culture Origin Method Activity Fate 04C-D Normal subculture day 9: passaged 1:6(wells) 04C-D-A Normal subculture day 8: media changed day 12: discarded 04C-D-B Normal subculture day 8: media changed day 12: passaged 1:4 04C-D-B-A Normal subculture day 4: media changed day 10: discarded 04C-D-B-B Normal subculture day 4: media changed day 10: discarded 04C-D-B-C Normal subculture day 4: media changed day 10: discarded 04C-D-B-D Normal subculture day 4: media changed day 10: discarded 04C-D-C Normal subculture day 8: media changed day 12: discarded 04C-D-D Normal subculture day 8: media changed day 12: discarded 04C-D-E Normal subculture day 8: media changed day 12: discarded 04C-D-F Normal subculture day 8: media changed day 12: discarded 04D Normal TM explant day 4: media chan ged, explant removed day 11: passaged 1:3 04D-A Normal subculture day 5: frozen as 04D-X 04D-B Normal subculture day 5: frozen as 04D-X 04D-C Normal subculture day 5: frozen as 04D-X 04D-Xa Normal thawed from 04-D-X day 2: media ch anged; day 8: media cha nged day 10: passaged 1:3 04D-Xa-A Normal subculture day 6: discarded 04D-Xa-B Normal subculture day 6: discarded 04D-Xa-C Normal subculture day 6: discarded 04D-Xb Normal thawed from 04-D-X day 2: media ch anged; day 8: media cha nged day 10: passaged 1:3 04D-Xb-A Normal subculture day 6: discarded 04D-Xb-B Normal subculture day 6: discarded 04D-Xb-C Normal subculture day 6: discarded 04D-Xc Normal thawed from 04-D-X day 2: media ch anged; day 8: media cha nged day 10: passaged 1:3 04D-Xc-A Normal subculture day 6: discarded 04D-Xc-B Normal subculture day 6: discarded 04D-Xc-C Normal subculture day 6: discarded 05A Normal Collagenase day 3: tissue removed to seed a second well (05A-A) day 5: discarded 05A-A Normal seeded from 05A day 2: discarded 06A Normal Collagenase day 8: passaged 1:3 06A-A Normal subculture day 3: media changed day 6: discarded 06A-B Normal subculture day 3: media changed day 6: discarded 06A-C Normal subculture day 3: media changed day 6: discarded 06B Normal Collagenase day 8: passaged 1:3 06B-A Normal subculture day 3: media changed day 6: discarded 06B-B Normal subculture day 3: media changed day 6: discarded 06B-C Normal subculture day 3: media changed day 6: discarded 52

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53 Table 3-1. Continued Culture Origin Method Activity Fate 06C Normal Collagenase day 8: passaged 1:3 06C-A Normal subculture day 3: media changed day 6: discarded 06C-B Normal subculture day 3: media changed day 6: discarded 06C-C Normal subculture day 3: media changed day 6: discarded WOSA Glaucomatous Collagenase day 5: media changed day 7: passaged 1:3 WOSA-A Glaucomatous subculture day 5: media changed day 6: frozen WOSA-B Glaucomatous subculture day 5: media changed day 6: frozen WOSA-C Glaucomatous subculture day 5: media changed day 6: frozen WOSB Glaucomatous Collagenase day 5: media changed da y 7: subcultured into dex&phag exp, media replaced WOSB-A Glaucomatous subculture day 5: media changed day 6: frozen WODA Glaucomatous Collagenase day 5: media changed day 9: passaged 1:4 WODA-A Glaucomatous subculture day 3: media changed day 4: frozen WODA-B Glaucomatous subculture day 3: media changed day 4: frozen WODA-C Glaucomatous subculture day 3: media changed day 4: frozen WODA-D Glaucomatous subculture day 3: media changed day 4: frozen WODB Glaucomatous Collagenase day 5: media changed day 9: passaged 1:4 WODB-A Glaucomatous subculture day 3: media changed day 4: frozen WODB-B Glaucomatous subculture day 3: media changed day 4: frozen WODB-C Glaucomatous subculture day 3: media changed day 4: frozen WODB-D Glaucomatous subculture day 3: media changed day 4: frozen

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CHAPTER 4 DISCUSSION Cell Culture Our study represents a successful step toward broadening the usefulness and scope of research that can be done base d on the POAG Beagle model. This model is already unique in presenting a spontaneous, complete POAG disease in a reliably heritable pattern. The specific genetics behind this trait are being explored, a nd if successfully identified, will only add to the value of an in vitro tissue cultu re representation of the model. Our study has defined a protocol for the successful establishment of TM cells from normal and glaucomatous canines in monolayer culture. Characterization data confir ms that these cells are of TM origin. Time becomes a challenge when working with a canine model, with a reproduction rate far slower than the standard rodents used in research and a disease that progresses over a course of years. The use of monolayer ce ll culture to answer questions about the molecular and cell biology of POAG is a significant advantage. Using the protocols described in our study, half of the tissue from each eye of one animal produced te n aliquots of frozen cells that, when thawed, could each be used for separate experiments that might require multiple eye sacrifices were they to be performed in other ways. From the persp ective of minimizing sacrificed tissue, an even better solution would be to im mortalize lines of normal and glaucomatous canine TM cells, alleviating pressures both to obtai n new eyes and to use cultures at the correct passage to avoid variation do to cell senes cence. Immortalizing primary cells also carries the risk of losing traits specific to the cell type, so it would be advisa ble to use both immortalized and primary cells through a few experiments to show whether their re sponses and properties are similar, or attempt to characterize them with mRNA an d/or protein expr ession profiles. 54

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From an alternate perpective, with the aim to preserve as much similarity to an in vivo situation as possible with an in vitro design, there is th e option of perfusion culture as described by Johnson and Tschumper (1987, 1989). There are ce rtainly advantages to this method, like the ability to measure outflow facility, maintain a ph ysiologic IOP, and have the cells in their own native ECM meshwork. Additionally, after an experiment has been run on a perfusion culture, histology can be performed exactly as it would in a freshly enucleated eye. Unfortunately, this requires full eyes, so the benefits are balanced by the cost in tissue, and this technique is probably a poor solution to appl y to the POAG Beagle model. Serum Concentration Assay Fetal Bovine Serum is used in cell culture fo r the growth factors a nd nutrients it provides. The obtained results support its use within a reasonable range, as increases in the percent composition of serum in the culture media result ed in larger increases in the cell population. Based on the results of this assa y in early cultures, later primar y cultures, including those from glaucomatous sources, were placed in 20% seru m supplemented media to encourage rapid initial growth. With the viability and hardiness of cells from glaucomatous sources in question, this encouraged the best possible expansion. While this was useful for the purposes of establishing a glaucomatous TM culture, it should be remembered that blood serum is not a part of the TM environment in vivo. Previous studies have show n that TM cells grown with aqueous humor as growth media or as a supplement to growth media can alter cell behavior and growth characteristics (Fautsch et al ., 2005, 2006; Hogg et al., 2000). In pa rticular, Fautsch et al. found that myocilin levels were significantly higher in cells grown with aqueous humor-supplemented media. Re-creating experiments with aqueous-su pplemented growth media could be interesting, but aqueous humor is not a readily availabl e resource. The UF CVM Ophthalmology canine aqueous humor library would allow fascinating experiments compar ing the growth of normal and 55

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glaucomatous cells not only in aqueous humor supplements, but even in aqueous supplements from glaucomatous vs. cataract eyes. However, it is possible that growing cells from one animal of origin in aqueous from one or more other animals could provide confounding variables. What might prove an ideal compromise is a culture me dia designed to more closely resemble aqueous humor in types and relative concentrati ons of protein and ot her bio-products. Histological Time-Course An interesting finding was the lack of noticeable increase in cellularity of TM cells in the native ECM meshwork. This obser vation allows for many interpretations, and calls for further experimentation. On one hand, the well-documented success of primary cultu re of TM cells from dissected meshwork seems contradictory to the st atement that TM cells did not expand in their native tissue. However, the observation aligns pe rfectly with the knowledge that TM cells are terminally differentiated and non-mitotic in vivo. A possible line of explanation is that the non-mitotic nature of TM cells is a feature that relies on cues given to the cell from its envi ronment. Perhaps the ECM of the ICA meshwork interacts with the cells in such a way to enc ourage their mitotic quiescence. Aqueous humor may also contain factors suppressing re plication or cell cycle advan cement, a possibility supported by studies of TM cells grown in aqueous-supplemented media (Fautsch et al., 2005). Perhaps more likely than the existence of a single cause is the combination of cellular-ECM signaling and factors in the aqueous humor e nvironment. To set up experiments regarding the degree with which ECM affects cells, it might prove useful to use decellularized ECM meshwork as a substrate for TM cell culture. D ecellularization is a process thr ough which a tissue is exposed to a series of physical and chemical challenges de signed to remove cells and cell debris from a tissue while minimizing disruption to the ECM. Gilbert et al. have a good review of methods that have been used to create decellularized tissue for a variety of applications (2005). Stripping cells 56

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out of an empty canine anterior chamber and th en allowing TM cells that have been passaged through primary culture access to the resulting empty TM as a growing surface could provide interesting results. Modifications to the co mposition of the decellularized ECM could be carefully controlled to note the effect on cells growing to inhabit the tissue. Another line of reasoning that could e xplain the combinati on of expanding and nonexpanding behavior of TM cells is that a sub-pop ulation of TM cells exist that are capable of division and expansion, while the remaining cells are non-replicativ e. Taking this view, a likely candidate is a relatively small population of cells known as the Sc hwalbes line cells. This subpopulation of TM cells was first identified in pr imate and presence in the canine eye was later confirmed (Raviola, 1982; Samuelson et al., 2001) Cells existing at the Schwalbes line have been labeled with markers typically found in st em or progenitor cells, though these methods also mark TM cells generally (Whikehart et al., 2005 ; McGowan et al., 2007). Further, after damage by laser trabeculoplasty, it has been shown that the majority of increase in cell division in the TM localizes to the anterior TM near Schwalbes line (Acott et al., 1989). Taken together, these pieces of evidence point to the anterior, non-filteri ng TM in general, and perhaps Schwalbes line cells in particular, as the origin of new TM cells. In the context of cell culture, it is possible that pre-existing adult TM cells repli cate minimally or not at all, and that cells from this subpopulation are responsible for expansion. A thor ough test for this hypothesis would involve finding a way to selectively label the genome of Schwalbes line cells, grow cells out into culture, and see what proportion of th e culture population becomes labeled. Characterization Both assays used to characterize the cells cult ured confirmed them as being of TM origin. The most widely used method for characteri zation of TM cells, induction of myocilin by dexamethasone treatment, showed positive results by western blots of cell lysates. Assaying for 57

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phagocytic activity, an important and well-documented feature of TM cells, also demonstrated the cultured cells to be in ag reement with the literature. The phagocytic activity of TM cells has been implicated as a possible mechanism for outflow, by transporting fluid and extracellular material across the juxtacanalicular region and into the AAP (or Schlemms canal in humans) by wa y of giant vacuoles. Th is theory, taken with an impairment of phagocytic activity in glaucoma provides an explanation for the findings that glaucomatous TM retains higher le vels of extracellular debris along with the reduced capability for conventional outflow, because both the debris and the fluid would be dependant on a failing mechanism (Tripathi 1972, Lutje n-Drecoll et al., 1981, Rohen et al., 1981). The finding here supports such a model, since glaucomatous TM ce lls were shown to have significantly decreased phagocytic activity compared to normal. While the results obtained by our study were in agreement with previous literature, the technique used in our study to observe phagocytic activity was imperfect in its lack of quantitation of beads that may have been abse nt from the media, yet not engulfed by the TM cells. This population of beads c ould include beads that had settled into inte rcellular spaces, had somehow adhered to cell surfaces, or simply failed to be resuspended into the media. A superior technique has been developed and used in several studies, where the material to be ingested, whether biological or synthetic in nature, is fluorescently labeled. The labeled material is clearly visible, but to further define the picture, a se condary antibody can be added after fixation that will attach only to extracellular particles, such that ingested and uningested particles are labeled differently (Matsumoto and Johnson, 1997a, 1997b; Zhang et al., 2007). Prostaglandin Pathway A significant biochemical pathway in the glauco ma disease process is that employed by the prostaglandin analogue family of IOP-lowering dr ugs. In vivo, prostaglandins are produced from 58

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arachidonic acid in a multi-step synthesis. Cy clooxygenase-1 and -2 (COX-1 and COX-2, also known as prostaglandin H synthase-1 and -2) perform the rate-limiting step in this synthesis, the conversion of arachidonic acid to PGH2 through a PGG2 intermediate. COX-1 is typically considered the constitutive form, and COX-2 the inducible form, and while this doesnt always hold true, it is COX-2 that has be en observed to have correlati ons with aqueous humor flow. A family of synthases creates fina l prostaglandin products from PGG2, including prostaglandin E2 and F2 (Smith et al., 2000) Analogues of these two products have been used as glaucoma drugs for their IOP-lowering ability. COX-2 has been shown to be expressed at higher levels in glaucomatous canine eyes compared to normals. The COX-2 in normal canine eyes was limited to the ciliary epithelium, while glaucomatous eyes showed expression throughout the cornea, iridocorneal angle (including the TM), and ciliary epithelium with increased intensity compared to normal (Marshall et al., 2004). This at first seems counter to a study pr eviously done in human POAG cases, which still found the baseline COX-2 expr ession in the non-pigmented ciliary epithelium of normal controls, but showed no COX-2 expre ssion in glaucomatous tissue (Maihofner et al., 2001). The two studies are actually not in di sagreement, however. The canine study was done only in cases of secondary glaucoma, confirming the human publication which saw expression of COX-2 in the ciliary epithelium of glaucomat ous patients that were not POAG or steroidinduced glaucoma cases. Taken together, this calls for a COX-2 immunolocalization in POAG canine eyes, and concurrently in POAG canine TM cel ls. It would be interesting to note whether there is expression in the ciliary epithelium of the eye, the TM and angle, and whether a monolayer TM culture expresses COX-2 the same or differently than TM in a histological section. It would also be interesting to note whether canine TM cells, both glaucomatous and 59

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normal, produce prostaglandins in the same wa y as seen in human and primate TM cultures (Weinreb et al., 1988; Wein reb and Mitchell, 1989). The prostaglandin products of the aforemen tioned pathway, as well as prostaglandin analogues used in treatment, are recognized by th e prostaglandin FP receptor, which sets events in motion that lead to a decrease in IOP. It has been handily shown that prostaglandin analogues latanoprost, bimatoprost, travopros t, and unoprostone are all ineffective at lowering IOP in mice where the FP receptor has been knocked out (Crowston et al., 2004, 2005; Ota et al., 2005). These receptors have been shown to be expressed in human trabecular meshwork both in vivo and in TM cell culture (Anthony et al., 1998). While it would seem obvious that glaucomatous and normal canines express these receptors, si mply because they show IOP decrease with prostaglandin analogues, it would be revealing to see if there is any upor down-regulation of FP receptors in glaucomatous and normal eyes. TM cell culture would provide a useful screen for the observation of these receptors, as would tissue s ections. It should also be noted that the cells of the smooth muscle of the anterior ciliary body could also be important in their expression of FP receptors, due to the nature of prostaglandin-class drugs to induce increased uveoscleral outflow through this tissue. Once the FP receptors are stimulated by the appropriate prostaglandins, intracellular pathways probably initiate many changes in expression and cellular dynamics in both ciliary smooth muscle and TM cells. It has been shown that prostaglandin F2 stimulates production of matrix metalloproteinases-1, -2, -3, and -9 (MMP -1, -2, -3, and -9) in vitro in ciliary smooth muscle cells (Weinreb et al., 1998). The matrix metalloproteinases are a family of enzymes responsible for digestion of extr acellular gelatin and co llagen fibers, a mechanism that has been implicated in the increased uveoscleral outflow seen with prostaglandin treatment. Glaucomatous 60

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canine (although not necessarily POAG canine) aque ous humor has higher levels of latent MMP2 than normal control, as well as higher levels of both active MMP-2 and latent MMP-9 in ICA tissue (Weinstein, et al., 2007). Ti ssue inhibitors of matrix meta lloproteinases (TIMPs) counter the action of MMPs and have also been studied in the context of aqueous outflow. MMP and TIMP concentrations have been studied both in AH from normal and glaucomatous humans (Maata et al., 2005), and in TM tissue explan ts from normal and glaucomatous humans (Ronkko et al., 2007). The ratios of MMPs to TIMPs have been shown to vary in these tissues, indicating an imbalance of ECM turnover in glaucomatous eyes compared to normal. Performing a study in POAG beagle tissues and fluids comparing the levels and ratios of the MMPs and TIMPs could provide new insight or confirmatory data to all of these ideas. Targeted Genetic Techniques The POAG Beagle model, already a valuable resource in the study of glaucoma disease and treatment, is situated to become an even more exciting model. The autosomal recessive inheritance of the diseas e seen in the Beagle model suggests a single gene may be responsible for their glaucoma. Ongoing research collaborations are underway to use pedigree-based, genomewide single nucleotide polymorphism (SNP) analysis to localize the genetic defect (Kuchtey et al., unpublished data). If this endeavor is fruitful, there could be a target gene or small group of genes of interest for further study. Work in qui ckly-dividing ocular cel l culture could provide valuable information about relationships a target gene and its protein product have with known pathways involved in glaucoma, or elucidation of new pathways. Additionally, simple recessive inheritance with the presence of non-glaucomatous carriers suggests that a single dominant allele is suffi cient to provide healt hy function. This makes a tempting target for gene therapy, because the in troduction of a healthy a llele into cells would theoretically by sufficient to restore function. Ther e already exists a lentiv iral vector that has 61

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been shown to safely and effectively transfect TM in cats and primates (Khare et al., 2008; Barraza et al., unpublished data). Such a vector coul d theoretically be designed to carry a healthy copy of the gene of interest, and permanently a dd it to the genome of transfected TM cells. 62

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LIST OF REFERENCES Acott, T.S., Kelley, M.J., 2008.Extracellular matrix in the trabecular meshwork. Exp Eye Res. 86(4), 543-561. Acott, T.S., Samples, J.R., Bradley, J.M., Bacon, D.R., Bylsma, S.S., Van Buskirk, E.M., 1989. Trabecular repopulation by anterior trabecular meshwork cells after laser trabeculoplasty. Am J Ophthalmol. 107(1), 1-6. Alvarado, J.A., Murphy, C., Juster, R., 1984. Trabecu lar meshwork cellularity in primary openangle glaucoma and nonglaucomatous normals. Ophthalmol. 91(6), 564-579. Alvarado, J.A., Wood, I., Polansky, J.R., 1982. Human trabecular cells II: growth pattern and ultrastructural characteristics. Invest Ophthalmol Vis Sci. 23(4), 464-478. Anthony, T.L., Pierce, K.L., Stamer, W.D., Regan, J.W., 1998. Prostaglandin F2 alpha receptors in the human trabecular meshwork. Inve st Ophthalmol Vis Sci. 39(2), 315-321. Barrie, K.P., Gum, G.G., Samuelson, D.A., Gelatt, K.N., 1985. Quantitation of uveoscleral outflow in normotensive and glaucomatous b eagles by 3H-labeled dextran. Am J Vet Res. 46(1), 84-88. Brooks, D.E., Strubbe, D.T., Kubilis, P.S., MacK ay, E.O., Samuelson, D.A., Gelatt, K.N., 1995. Histomorphometry of the optic nerves of nor mal dogs and dogs with hereditary glacuoma. Exp Eye Res. 60(1), 71-89. Crowston, J.G., Lindsey, J.D., Aihara, M., We inreb R.N., 2004. Effect of latanoprost on intraocular pressure in mice lacking the pros taglandin FP receptor. Invest Ophthalmol Vis Sci. 45(10), 3555-3559. Crowston, J.G., Lindsey, J.D., Morris, C.A., Wh eeler, L., Medeiros F.A., Weinreb R.N., 2005. Effect of bimatoprost on intraocular pressure in prostaglandin FP receptor knockout mice. Invest Ophthalmol Vis Sci. 46(12), 4571-4577. Buller, C., Johnson, D.H., Tschumper, R.C., 1990. Human trabecular meshwork phagocytosis: observations in an organ culture system. I nvest Ophthalmol Vis Sci. 31(10), 2156-2163. Fautsch, M.P., Bahler, C.K., Vrabel, A.M., Howell, K.G., Loewen, N., Teo, W.L., Poeschla, E.M., Johnson D.H., 2006. Perfusion of his-tagg ed eukaryotic myocilin increases outflow resistance in human anterior segments in the presence of aqueous humor. Invest Ophthalmol Vis Sci. 47(1), 213-221. Fautsch, M.P., Howell, K.G., Vrabel, A.M., Charlesworth, C., Muddiman, D.C., Johnson, D.H., 2005. Primary trabecular meshwork cells incuba ted in human aqueous humor differ from cells incubated in serum supplements. I nvest Ophthalmol Vis Sci. 46(8), 2848-2856. Gelatt, K.N., Brooks, D.E., Kllberg, M.E., 2007. The canine glaucomas, in: Gelatt, K.N. (Eds.), Veterinary Ophthalmology. Blackwell Publ ishing Professional, Ames, pp.753-811. 63

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Gelatt, K.N., Gum, G.G., Gwin, R.M., Bromber g, N.M., Meredith, R.E., Samuelson, D.A., 1981. Primary open angle glaucoma: inherited primar y open angle glaucoma in the beagle. Am J Pathol. 102(2), 292-295. Gelatt, K.N., MacKay, E.O., 1998. The ocular hypertensive effects of topical 0.1% dexamethasone in beagles with inherited glaucoma. J Ocul Pharmacol Ther. 14(1), 57-66. Gelatt, K.N., MacKay, E.O., 2001. Effect of different dose schedules of latanoprost on intraocular pressure and pupil size in the glaucomatous beag le. Vet Ophthalmol. 4(4), 283288. Gelatt, K.N., MacKay, E.O., 2004(a). Effect of different dose schedules of travoprost on intraocular pressure and pupil size in the gl aucomatous beagle. Vet Ophthalmol. 7(1), 5357. Gelatt, K.N., MacKay, E.O., 2004(b). Prevalence of the breed-related glaucomas in pure-bred dogs in North America. Vet Ophthalmol. 7, 97-111. Gelatt, K.N., Peiffer, R.L. Jr., Gwin, R.M ., Gum, G.G., Williams, L.W., 1977. Clinical manifestations of inherited glaucoma in th e beagle. Invest Ophthalmol Vis Sci. 16(12), 1135-1142. Gelatt, K.N., Peiffer, R.L. Jr., Gwin, R.M., Sauk, J.J. Jr., 1976. Glaucoma in the beagle. Trans Sect Ophthalmol Am Acad Ophthalmol Otolaryngol. 81(4 Pt 1), OP636-644. Gilbert, T.W., Sellaro, T.L., Badylak, S.F., 2006. Decellularization of tissues and organs. Biomaterials. 27(19), 3675-3683. Grierson, I, Day, J., Unger, W.G., Ahmed, A., 1986. Phagocytosis of latex microspheres by bovine meshwork cells in culture. Graefes Arch Clin Exp Ophthalmol. 224(6), 536-544. Grierson, I, Lee, W.R., 1973. Eryt hrocyte phagocytosis in the huma n trabecular meshwork. Br J Ophthalmol. 57, 400-415. Hogg, P., Calthorpe, M., Batterbury, M., Grie rson, I, 2000. Aqueous humor stimulates the migration of human trabecular meshwork cells in vitro. Invest Ophthalmol Vis Sci. 41, 1091-1098. Johnson, D.H., Tschumper, R.C., 1987. Human tr abecular meshwork organ culture: a new method. Invest Ophthalmol Vis Sci. 28(6), 945-953. Johnson, D.H., Tschumper, R.C., 1989. The effect of organ culture on human trabecular meshwork. Exp Eye Res. 49(1), 113-127. Kllberg, M.E., Brooks, D.E., Gelatt, K.N., Garcia -Sanchez, G.A., Szabo, N.J., Lambrou, G.N., 2007. Endothelin-1, nitric oxide, and glutamate in the normal and glaucomatous dog eye. Vet Ophthalmol. 10(s1), 46-52. 64

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BIOGRAPHICAL SKETCH Thomas Rinkoski, more frequently known as Tommy, was born in 1980 and raised by both his parents in a wide range of United States locales, reaching Green Bay, WI by the time he was completing high school. He was then accepted at Drake University, where he completed his Bachelor of Science degree in Biological Scienc es and his Bachelor of Fine Arts degree in Theatre Performance, along with a minor in cello performance. After graduation, he worked as an informal educator with the Science Center of Iowa while his family grew. Tommy married Audrey Bostrom (now Rinkoski), and had two ch ildren, Cain and Raechel. Having decided to continue his education, Tommy moved the family to Gainesville, Florida and found a job at the University of Florida College of Veterinary Medicine in the Ophtha lmology unit that would allow his to pursue a graduate degree while wo rking as a researcher studying glaucoma in the anterior eye, using the POAG B eagle as a model. Once work on his Master of Science degree was limited to writing and defending the thesis, Tommy applied for and received another glaucoma research position at the Mayo Clinic in Rochester, MN. While in Rochester, Tommy and Audrey added their third child, Ethan, to the family.