Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Prevention of posterior capsule opacification by photodynamic therapy with localized benzoporphyrin derivative… Meadows, Howard Earl 2008

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Notice for Google Chrome users:
If you are having trouble viewing or searching the PDF with Google Chrome, please download it here instead.

Item Metadata


24-ubc_2008_fall_meadows_howard_earl.pdf [ 6.98MB ]
JSON: 24-1.0066942.json
JSON-LD: 24-1.0066942-ld.json
RDF/XML (Pretty): 24-1.0066942-rdf.xml
RDF/JSON: 24-1.0066942-rdf.json
Turtle: 24-1.0066942-turtle.txt
N-Triples: 24-1.0066942-rdf-ntriples.txt
Original Record: 24-1.0066942-source.json
Full Text

Full Text

PREVENTION OF POSTERIOR CAPSULE OPACIFICATION BY PHOTODYNAMIC THERAPY WITH LOCALIZED BENZOPORPHYRIN DERIVATIVE MONOACID RING A (BPD-MA) IN A RABBIT SURGICAL MODEL by Howard Earl Meadows A THESIS SUBMWFED IN PARTIAL FULHLLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in The Faculty of Graduate Studies (Pathology and Laboratory Medicine) THE UNIVERSITY OF BRITISH COLUMBIA August 2008 © Howard Earl Meadows, 2008 ABSTRACT Posterior capsule opacification (PCO) is a major component of secondary cataract, a complication of current cataract surgery practice. This iatrogenic condition occurs in virtually all pediatric cases and to a lesser extent in adults. PCO correlates with the development in the latter half of the 20th Century of extracapsular cataract extraction (ECCE). In these surgeries, the lens capsule is left intact. During ECCE surgery a circular capsulotomy opening is created in the anterior lens capsule, and the cataractous, proteinaceous lens is removed, often via ultrasonic lens liquefaction i.e. phacoemulsification. The posterior, equatorial and remaining anterior portions of the sac-like capsule are left intact, permitting the insertion of an artificial lens into the emptied capsule. However, cells from the monolayer of epithelium on the inner surface of the capsule often begin to proliferate and migrate onto the normally cell-free inner surface of the posterior capsule, and may obscure the central axis of vision. Subsequently, a second surgery is necessary to create a small capsulotomy in the centre of the posterior capsule, usually employing an Nd:YAG laser. However, up to 5% of patients who have capsulotomies may then develop further serious, vision-threatening complications such as macular edema and retinal detachments. This thesis reports the photodynamic therapy (PDT) conditions required to prevent lens epithelial (LE) cell de novo proliferation and migration onto posterior lens capsules in a euthanized rabbit surgical model in order to predict parameters required to prevent PCO in humans. Experiments with primary in vitro cultures of human LE cells have shown rapid delivery of the photosensitizer benzoporphyrin derivative monoacid ring A (BPD-MA) and efficient killing with low light doses of 690 nm red light. Additional studies have shown the efficacy of various viscous agents in protecting the comeal endothelium. During model phacoemulsification ECCE surgeries, the use of hyaluronate viscoelastic carriers addressed the need for containment necessary for localized delivery of photosensitizer in the emptied capsule. Long-term monitoring of PDT-treated rabbit lens capsules in vitro has II demonstrated a phototoxic effect including complete cell kill in this surgical model employing the prophylactic use of PDT. m TABLE OF CONTENTS Abstract ii Table of Contents iv List of Tables vii List of F’igures x List of Abbreviations xii Acnolegeents xii Iedication xiii Chapter 1: Introduction 1 1.1 Core purpose 1 1.2 Anatomy of the eye and lens 1 1.2.1 The eye 1 1.2.2 The mammalian crystalline lens 2 Lens fibre cells 4 Lens capsule and epithelial cells 5 13 Secondary cataract and posterior capsule opacification 7 1.3.1 Cataract surgeries 7 13.2 Vision-impairing complications of the post-surgical capsule 8 1.3.3 Lens cells and the published in vivo rabbit surgical model of PCO 11 1.4 Incidence of posterior capsule opacification (PCO) 12 1.4.1 Overview of secondary cataract incidence 12 1.4.2 A lack of consensus on the incidence of PCO 13 1.43 Meta-analyses of PCO incidence studies (1979 to 1996) 14 1.4.4 Imaging problems and new developments in assessing PCO 16 1.4.5 PCO incidence from N&YAG posterior capsulotomy rates 17 1.5 Secondary cataract prophylaxes 19 1.5.1 Physical approaches 20 Variations in basic primary cataract surgical techniques 20 Ultrasonic, laser and cryogenic surgical adjuncts 21 IOL materials 23 IOL adhesion effects 25 Intraocular inserts 26 IOL designs 26 1.5.2 Physical chemical approaches 27 1.5.3 Pharmacological approaches 28 Antineoplastics 28 Anti-inflammatories and antiallergics 29 1.53.3 Antibodies and cytotoxin delivery systems 32 Intracapsular release of compounds 33 1.53.5 Other compounds 35 Photodynamic therapy 36 1.6 Implications and directions for the current thesis 38 1.7 Primary questions and specific aims 39 iv Chapter 2: Benzoporphyrin Derivative and Viscoelastics 41 2.1 Photodynamic therapy 41 2.1.1 Benzoporphyrin derivative 41 2.1.2 PDT history 43 2.13 Photosensitization mechanisms 48 2.1.4 BPD-MA delivery characteristics 48 2.2 Ocular viscoelastics and BPD-MA mixtures 49 2.3 BPD-MA in sodium hyaluronate 50 Chapter 3: Ocular Anterior Chamber PDT Conditions: Lethal Photosensitizer and Light Titrations for LE cells in vitro 5 1 3.1 Objectives 51 3.2 Materials and methods 52 3.2.1 Comeal endothelium protection from BPD-MA 52 3.2.2 HLE cells in vitro: photosensitizer and light dosimetries with BPD-MA in non-viscous solutions and in diluted ophthalmic viscoelastic 53 In vitro culture of human LE cells 53 BPD-MA in DME /5% FCS and in diluted AMVISC 53 3.2.3 BPD-MA availability from undiluted viscoelastic mixtures 55 3.3 Results 56 3.3.1 Corneal endothelium protection from BPD-MA PDT 56 3.3.2 HLE cells in vitro: photosensitizer and light dosimetries with BPD-MA in non-viscous solutions and in diluted ophthalmic viscoelastic 58 33.2.1 Dark controls: BPD-MA toxicity without light activation 58 BPD-MA in DME /5% FCS and in diluted AMVISC 58 Ten-minute incubations with BPD-MA 60 One-minute incubations with BPD-MA 62 Narrowing the dose range of BPD-MA 63 333 BPD-MA availability from undiluted clinical viscoelastic mixtures 64 3.4 Conclusions 65 Chapter 4: Monitoring Post-surgical Rabbit PDT Test and Control Capsules in vitro 67 4.1 Surgical data: Treated rabbit lens capsules assayed in vitro 100 and 200 J/cm2LED light activation of BPD-MA: Rabbits Ri-Ri 1 67 4.1.1 Rationale 67 4.1.2 Objectives 68 4.13 Materials and methods 68 4.1.4 Phacoemulsification extracapsular lens extraction 69 4.1.5 Pinning and culturing capsules 71 4.1.6 Assessment of cell viability by microscopy 72 4.1.7 Collection of conditioned growth medium 73 4.1.8 Results: 100 and 200 Jfcm2,rabbits Ri-Ru 74 Microscopic observations at 48 h and day 6 74 Calcein and ethidium fluorescence photomicrography 75 Rabbits R2-R5 at 7 weeks 75 Rabbits R6-R11 at 8 days and 3 weeks 79 Rabbits R6-R1 1 at 5 weeks and 7 weeks 86 Ultrasonic energy required for surgeries 87 Volumes of BPD-MA / Ophthalin instilled 88 v 4.1.9 pH of conditioned medium in long-term capsule culture 88 4.1.10 Conclusions: preliminary surgeries Ri-Ru 89 4.2 Surgical data: Rabbit lens capsules: —‘2 J/cm torus laser tip diode laser light activation of BPD-MA compared with 10 JIcm2and 100 JIcm2LED: Rabbits R12-R3 1 90 4.2.1 Rationale: Rabbit pinned capsule model surgeries closely reflecting preferred clinical parameters 90 4.2.2 Objectives 91 4.2.3 Materials 92 Photosensitizer 92 Equipment 92 Rabbits 93 4.2.4 Methods 93 BPD-MA / Ophthalin viscoelastic mixtures 93 Phacoemulsification extracapsular lens extraction 93 Surgical procedures summaries: Sets A, B, and One to Six 93 Detailed surgical descriptions: A, B, and One to Six: Rabbits R12-R31 95 Pinning and culturing capsules 100 Assessment of cell viability by microscopy 100 Collection of conditioned growth medium 101 Cell Kill Index (CKJ) 101 4.2.5 Results: Sets A, B, and One to Six (R12-R3 1) 101 Results summary: Sets A, B, and One to Six (R12-R31) 105 Microscopic observations Days 1, 2 and 7: Sets A, B, and One to Six (R12-R31) 106 Microscopic Observations Day 21 107 Potential ciliary process fibroblast contamination: Sets A, B, and One to Six (R12-R31) 110 Conditionedmedia 121 4.2.6 Conclusions: Surgeries R12-R3 1 124 Chapter 5: Discussion 125 5.1 Chapter outline 126 5.2 The physics of in vitro and pinned rabbit capsule models 127 5.2.1 Drug contact 127 Interface between BSS and Ophthalin/BPD-MA 127 Cell and Ophthalin I BPD-MA interface 127 Lethal BPD-MA (LD100) and PDT Light Doses 128 5.2.2 I/A and phacoemulsification tip microenvironment 132 5.2.3 Lens interactions with phacoemulsification and I/A 133 5.3 The Biology of in vitro and pinned rabbit capsule models 138 5.3.1 Biologicalcommon denominators 139 53.1.1 Post-surgical biological environments 139 The case for preferential photosensitizer uptake 140 5.4 Potential further studies 143 Bibliography 145 Appendices 162 Appendix One: Detailed Materials / Methods for In Vitro Chapter 3 162 vi Appendix Two: Table of Experimental Conditions 100 and 200 3/cm2 (Rabbits: Ri-Ri 1) 164 Appendix Three: Light Doses Delivered by Equatorial Laser 165 Tip #4 (ELT-4) Appendix Four: Animal use 167 Appendix Five: Methods Summary: Human Donor HLE in vitro (BPD) 168 Appendix Six: Flow Chart: Euthanized Rabbit Model Lensectomy Steps 169 Appendix Seven: Results Chart: Euthanized Rabbit Model Lensectomies 170 vii LIST OF TABLES Table 1.1 Exclusion and inclusion criteria for the Schaumberg review 15 Table 4.1 PDT test capsules a 7 weeks (rabbits R2-R1 1) 86 Table 4.2 Rabbit surgeries: Sets A and B (R12-R17); Close-array LED 94 Table 4.3 Rabbit surgeries: Sets One to Six (R19; R21-R3 1); Equatorial Laser Tip-4 94 Table 4.4 PDT capsules at 7 and at 21 days 105 yin LIST OF FIGURES Figure 1.1 Overall topographic anatomy of the eye 2 Figure 1.2 A composite drawing of the lens, cortex, epithelium, capsule and zonular attachments 3 Figure 1.3 A stylized three-piece posterior-chamber intraocular lens (PC IOL) 23 Figure 2.1 Structure of benzoporphyrin derivative (BPD) 43 Figure 2.2 Absorption spectrum of PHOTOFRIN®, oxyhemoglobin andBPD 45 Figure 2.3 Photosensitizers (>650 nm) under clinical and preclinical trials . . . .46 Figure 2.4 The basic chemical structures of porphyrins, chlorins and bacteriochiorins 47 Figure 2.5 Energy states and interactions of photosensitizing agents 47 Figure 3.1 Protection of rabbit corneal endothelium against BPD-MA toxicity by viscoelastic 57 Figure 3.2 Cell survival without photoactivation of human lens epithelial cells in vitro following 10-mm. incubation with liposomal BPD-MA 59 Figure 3.3 Photosensitization with 10 J/cm2LED dose in HLE in vitro following 10-mm. incubation with liposomal BPD-MA 60 Figure 3.4 Comparison of photosensitization with 10 J/cm2LED dose in HLE cells in vitro following 10-mm. incubation with liposomal BPD-MA in either DME /5% FCS or in 25% AMVISC sodium hyaluronate 61 Figure 3.5 Comparison of photosensitization with 10 J/cm2LED dose in FILE cells in vitro following 10-mm. incubation with liposomal BPD-MA in either DME /5% FCS or in 25% AMVISC sodium hyaluronate assayed at 1 day and 20 days post treatment 62 Figure 3.6 Comparison of photosensitization with 10 3/cm2LED dose in HLE cells in vitro following 1-mm. incubation with liposomal BPD-MA 63 Figure 3.7 Comparison of photosensitization with 10 J/cm2LED dose in HLE celisin vitro following 1-mm. incubation with liposomal BPD-MA, treated at 1 or 2 days post trypsinization 64 Figure 3.8 BPD-MA 60 jg/m1 in full-strength AIvIVISC viscoelastic on HLE-19 (48 y female donor) on non-ProNectin-F coverslips for 1.0 mm. then LED69O light dosing 0.5 i/cm2 65 Figure 4.1 Comparison of 100 i/cm2PDT and control capsules at day 6 post surgery (rabbit R3) 74 Figure 4.2 Comparison of PDT test and control capsules at day 6 post surgery in a rabbit RiO treated with 200 i/cm2 75 Figure 4.3 A control capsule at 7 weeks, rabbit R5, 200 i/cm2, showing green calcein fluorescence 76 Figure 4.4 Test capsules at 7 weeks, rabbit R3 at 100 i/cm2and rabbit R5 at 2003/cm 77 Figure 4.5 Test capsules at 7 weeks. Rabbit R4 received 200 J/cm2; assigned a Cell Kill Index = 1. Rabbit R2 received 100 .1/cm2; assigned a CM =0 78 Figure 4.6 Control capsule (rabbit R6) at 8 days after calcein / ethidium treatment, and at 3 weeks with calcein only treatment 79 Figure 4.7 Rabbit R8, 100 J/cm2 control at 8 days; test capsule at 8 days and3 weeks 80 ix Figure 4.8 Rabbit Ri 1, 100 3/cm2 control at 8 days; test capsule at 8 days and3 weeks 81 Figure 4.9 Rabbit R6, 200 J/cm2 control at 8 days; test capsule at 8 days and 3 weeks 82 Figure 4.10 Rabbit RiO, 200 J/cm2 control at 8 days; test capsule at 8 days and 3 weeks 83 Figure 4.11 Rabbit R9, 200 3/cm2control at 8 days; test capsule at 8 days and3 weeks 84 Figure 4.12 Rabbit R7, 2003/cm control at 8 days; test capsule at 8 days and 3 weeks 85 Figure 4.13 Ultrasonic energy times required for phacoemulsification of each BPD-MA / LED-array test capsule (rabbits R2-R1 1) compared with cell kill 87 Figure 4.14 Volume of BPD / Ophthalin used in post phacoemulsification capsules for each BPD / LED-array PDT test capsule (R2-R1 1) compared with cell kill 88 Figure 4.15 Seven-day conditioned DMEM from rabbit test and control capsule cultures 89 Figure 4.16 A control capsule (drug-free Ophthalin, no light) at day 7 shows the typical confluent cell growth on the entire capsule (Set One: R191E2) 107 Figure 4.17 A control capsule (drug-free Ophthalun 1 miii., 2 3/cm)at day 21 shows the typical confluent cell monolayer (Set One; R19/E2); and a typical absolute cell kill PDT capsule (R25/E1, 460 ,ug/ml BPD-MA I Ophthalun, 1 mm., 2 JIcm) 109 Figure 4.18 A confluent test capsule (Ri6/Ei, Set B, 230 g/ml BPD-MA / Ophthalun, 2 mm., 10 J/cm2)at day 21 with reticulated growth.. 110 Figure 4.19 An everted test capsule (R19/E1, 230 ugIm1 BPD-MA / Ophthalin, 2 mm., -.2 JIcm) at days 7 and 21 shows trapped ciliary remnants 112 Figure 4.20 A test capsule (R221E2, 230 ,ug/ml BPD-MA I Ophthalun, 2 mm., -‘2 JIcm)at days 2 and 21 shows trapped ciliary remnants 114 Figure 4.21 A test capsule (R24/E2, 230 jsg/ml BPD-MA / Ophthalin, 1 miii., —‘2 J/cm)at day 21 shows trapped ciliary remnants and the barricade effect 115 Figure 4.22 Suspected ciliary body fibroblast contamination at 4 months 117 Figure 4.23 Wearing away of silver coating in ELT-4 spherical tip 119 Figure 4.24 Modification ofAlcon Terry Squeegee (27G) used for BPD-MA / Ophthalin instillation 120 Figure 4.25 SetA (R12-14) and SetB (R15-17) conditioned DMEM from test and control cultures at 5-6 weeks post-phaco 121 Figure 4.26 pH of nine-day conditioned media from PDT and control pinned-capsule cultures extant 13 months after the initial surgeries 123 x LIST OF ABBREVIATIONS 5-FU 5-fluoruracil AA arachidonic acid A-cells anterior lens capsular epithelial cells BCA bactenochiorinA BLEC bovine lens epithelial cell BPD-MA benzoporphyrin derivative monoacid ring A CCC continuous curvilinear capsulorhexis CCH cortical cleaving hydrodissection COX cyclooxygenase DCM day(s) conditioned medium DHE dihematoporphyrin ethers DM diabetes mellitus DME(M) Dulbecco’s Modified Eagle’s (M)edium E-cells equatorial lens capsular epithelial cells ECCE extracapsular cataract extraction ECLE extracapsular lens extraction ECM extracellularmatrix ELT-4 Equatorial Laser Tip #4 EN/IT epithelial-mesenchymal transition EPCO Evaluation of Posterior Capsule Opacification FCS Fetal Calf Serum, heat-inactivated H&E hematoxylin and eosin HLEC human lens epithelial cell HPD hematoporphyrin derivative ICCE intracapsular cataract extraction xi IOL intraocular lens J joules LD100 lethal dose 100 (percent kill) LE lens epithelium/epithelial EEC lens epithelial cell MDX-RA mouse monoclonal antibody MAb 4197X - ricin A conjugate MI mitotic index MIT (di)Methylthiazol (cli)phenyltetrazolium Nd:YAG neodymium: yttrium-aluminum-garnet laser Ncl:YLF neodymium: yttrium-lithium-fluoride picosecond laser NMR nuclear magnetic resonance NSAIDs non-steroidal anti-inflammatory drugs OPC open posterior capsule P11 Photofrin II PCCC posterior continuous curvilinear capsulorhexis PC IOL posterior chamber intraocular lens PCO posterior capsule opacification PDT photodynamic therapy PLS polylysine-saporin conjugate PMMA polymethylmethacrylate PGs prostaglandins PS photosensitizer Ri rabbit 1 RLEC rabbit lens epithelial cell TUNEL terminal deoxynucleotidyl transferase TdT-mediated dUTP nick end labeling W watts XII ACKNOWLEDGEMENTS A sincere gratitude is extended to Dr. Jack Rootman, UBC Ophthalmology and Visual Sciences for his expert guidance and patience as supervisor for this project, and to the members of the Supervisory Committee, Drs. Wan Lam, Julia Levy, Robert Molday and Martin Trotter. I also thank Drs. David Mallek (MD) and Nick Bussanich (DVM) for their time and care in teaching me the fundamental surgical techniques required for this research. The supply of photosensitizer and the arrangements for the clinical spherical laser tip by QLT Inc. (Vancouver) are also gratefully acknowledged. xm DEDICATION This work is dedicated to the memory of Dr. and Mrs. William A. (Bill) and Rachael Meadows who were inspirational models of the spirit, art and science of the practices of medicine and teaching. xiv Chapter 1: Introduction 1.1 Core purpose The core purpose of this research was to explore the use of an activated photosensitizer to prevent lens epithelial post-surgical de novo proliferation, a principle contributor to the process of posterior capsule opacification (PCO) (Duncan, et a!. 1997; Marcantonio and Vrensen 1999; Spalton 1999) which is in turn a major contributor to the condition called secondary cataract, the second most common cause of visual loss worldwide (Pandey, Apple et a!. 2004). Secondary cataract, a widespread problem following adult cataract surgery (Apple, Solomon et al. 1992), has a near-certain incidence in pediatric cases (Spierer, Desatnik et al. 1992; Knight-Nana, O’Keefe et al. 1996; Hosal and Biglan 2002). The following sections include anatomical and clinical contexts out of which the experiments reported in this current thesis have grown. 1.2 Anatomy of the eye and lens 1.2.1 The eye The human eye consist of two segments, the anterior and the posterior. In turn, the anterior segment includes the anterior and posterior chambers (Fig. 1.1). The anterior chamber is bounded in front by the cornea and in back principally by the iris (Duke-Elder 1961). This chamber contains the majority of the transparent colourless aqueous humour, most often referred to as the aqueous. The much smaller posterior chamber is bounded in front by the iris and in back by the anterior surfaces of the lens, zonules and ciliary processes. 1 Ms Figure 1.1 Overall topographic anatomy of the eye. Jakobiec, F.A., Ocular Anatomy, Embryology, and Teratology, 1982. Harper & Row, Philadelphia. (Reprinted with permission from Lippincott Williams & Wilkins, Philadelphia.) The posterior segment of the eye is bounded posteriorly by the retina, and anteriorly by the area of contact between the exterior of the posterior lens capsule and the anterior region of the vitreous humour. The vitreous is an aggregation of gel-like, transparent, colourless regions responsible in large part for maintaining the shape of the eye, and for providing a tamponade restraint ensuring the regular surface of the retina. 1.2.2 The mammalian crystalline lens The mammalian lens is derived from ectoderm and is made up entirely of epithelial cells and their derivatives (Fig. 1.2). The principle lenticular mass consists of elongated mature central lens fibres and differentiating equatorial lens fibre cells all surrounded by a 2 transparent, tough capsule lined with a monolayer of cuboidal lens epithelium in the Stylized drawing of a human lens in cross-section made from reference slides of hematoxylin and eosin-stained histological sections of human globes. Size of structures are not to relative scale. The anterior epithelial cells (A) appear as a single monolayer on the interior face of that portion of the capsule (C) anterior to the equator (E) of the lens capsule. The inner face of the posterior capsule (PC) is free of epithelial cells. Lens fibre cells (LFC) near the equator appear to still retain nuclei but more central fibres have no visible nuclei. Although the bulk of the lens consists of fibres, only a few representative examples are shown. Zonule insertions into the external equatorial capsule are not shown. anterior and equatorial regions (Duke-Elder 1961; Moore, Persaud et al. 2000). A E Figure 1.2 3 Lens fibre cells Highly-differentiated mature lens fibres make up the bulk of the lens and form a regularly ordered mass interconnected by water channels, ion channels, and gap junctions (Moffat and Pope 2002). Although described by some authors (Moffat and Pope 2002) as a syncytium, the mature lens fibre cells, though packed tightly in regular near-concentric layers, are anucleate when fully mature (McAvoy, Chamberlain et al. 1999), and are therefore not strictly syncytial. However, the abundance of intercellular gap junctions between mature lens fibres would render them an electrical syncytium from a functional perspective. Lens fibre cells are the only transparent cellular entities in the vertebrate body. This transparency is afforded in part by a tight, homogenous packing pattern (Beebe 2003). Image-forming light refraction is also due in part to the lack of all organelles in the mature fibre cells (Lovicu and McAvoy 1989). Immature, peripheral fibre cells have a full complement of organelles (Beebe 2003). This high degree of differentiation of mature fibre cells is likely indicative of an interrupted apoptotic i.e. programmed cell death cascade which halts just before complete cellular destruction. A critical functional aspect of the extreme differentiation of fibre cells is the high content (>30%) of several classes of potentially transparent crystallin proteins, with a minimum of extra-cellular water (Paterson 1970). Also, it is the smallness and uniform regularity of these extracellular spaces along with the structural regularities and lack of membrane-bound organelles of the fibres which afford transparency (Beebe 2003). Mammalian lens fibre cells exist in three principal zones, the most central of which is formed in early embryogenesis (Duke-Elder 1961; Moore and Persaud 1998) and slowly accrues additional anucleate fibre cells to form the lens nucleus. Surrounding are the more recently matured cortical lens fibre cells. Throughout the adult lifespan, new lens fibre cells arise very slowly from lens epithelial cells in the outermost equatorial region (Beebe 2003). Nuclear magnetic resonance (NMR) scans show differences between nuclear and 4 cortical lens fibre cells. Ixperimental measurements of anisotropic water transport i.e. transport in different directions in human lenses aged 13 to 86 years indicate a diffusion barrier which restricts the transport of water between the lens surface and the fibre cells of the lens nucleus (Moffat and Pope 2002). Lens fibre cells have no turnover in the nonpathological state. They exist for the lifespan of the individual, and metabolize very slowly. However there is also evidence of different biochemical activities in the lens nucleus and cortex (Garg 2002). Sweeney and Truscott (Sweeney and Truscott 1998) also showed a restriction of radio-labelled glutathione transport between the lens cortex and nucleus. Although no clear anatomical barrier seems to exist between the cortex and the nucleus, these functional differences coincide with the observation that during experimental lensectomies and in cataract surgeries, hydrodelineation allows the denser nuclear component to be separated from the overlying cortical lens fibres by the forceful injection of balanced salt solution (BSS), partially facilitating removal of the lens nucleus. Lens capsule and epithelial cells Directly adhered to inner capsule surfaces are two main functional populations of cuboidal lens epithelial cells (LECs), the normally quiescent monolayer of the anterior capsule (A cells) and the more regenerative monolayer on the inner capsule equator (E-cells) (Peng, Apple et al. 2000). E-cells are described as having a high lens mitotic index (Ml) (Lang 1999). The anterior face of the posterior capsule has no LECs. Basement membranes co-locate with epithelia and endothelia as boundaries between disparate tissues (Albert, Jakobiec et al. 2000 p.4666). Embryologically, the mammalian lens arises from head surface ectoderm (Moore and Persaud 1998). The capsule, however, does not begin to form until the lens separates, a formation which continues with a slow succession of basal laminae of varying thicknesses laid down at a variable rate in humans for the life of the individual (Seland 1974). This uniquely clear capsule is laid down by the anteriorlens epithelium (Young and Ocumpaugh 1966; Moore and Persaud 1998), areas of 5 which become not only the thickest basement membrane but the thickest membrane in the human body (Seland 1992). The thinnest region happens to be the central posterior of the lens (Ruotsalainen and Tarkkanen 1987), and is that capsule portion through which the central axis of vision passes, and in which posterior capsulotomies are formed in treating debilitating cases of secondary cataract. Rich in type IV collagen, the fibrils of which are arranged to permit transparency (Marcantonio and Vrensen 1999), the lens capsule also contains the proteins laminin, entactin, heparan sulfate proteoglycan and fibronectin, all cross-sectionally co-localized within the anterior lens capsule (Cammarata, Cantu-Crouch et a!. 1986). The capsule is the portion of the lens into which zonules are inserted (Seland 1992), which translate tensile forces from the muscles of the ciliary body to induce accommodative changes of lens thickness and hence focal length. The presence of collagen appears suited to the need for the capsule to withstand these stretching forces (Seland 1992). Although Rafferty etal did not include human lenses in the following study, the cytoskeleton in the lens epithelial cells of rabbits and rats showed a polygonal, mesh-like network at their plasma membrane closest to the outermost fibre cells i.e. the apical LE membrane (Rafferty, Zigman et al. 1993). Lens epithelial cells exhibit a vastly different morphology than lens fibre cells. LECs in situ appear hexagonal, a regularity which is lost in certain lens pathologies (Uusitalo and Kivela 1997) such as anterior subcapsular cataracts (Lovicu, Steven et al. 2004) and posterior capsule opacification (Ibaraki, Ohara et al. 1995; Uusitalo and Kivela 1997). The thin profile of LECs appears consistent with the tamponade forces from the packed, relatively massive underlying lens fibre cells and would appear to minimize any interfering light scatter and refraction, the presence of organelles notwithstanding. Polarity of epithelial cells has been described for many tissue types (Alberts, Bray et a!. 1994; Kendrew 1994). Overt morphological clues and histochemical data in other vertebrate epithelia clearly indicate cellular polarity, a highly-studied example being the 6 mucous-secreting intestinal goblet cells (Alberts, Bray et al. 1994). Although no published studies to date appear to specifically address polarized delivery of plasma membrane proteins in LECs, differences between basal and apical surfaces of LECs are inherent in the morphological disparities between the basal-capsular interface and the apical-lens fibre cell interface. However, there are incidental histochemical indications of LEC polarity. P glycoproteins i.e. multifunctional membrane proteins that act as ATP-dependent efflux pumps (Idriss, Hannun et al. 2000) are observed in the apical cell membrane in LECs in rats (Merrisman-Smith, Young et al. 2002). Polarity of LECs is suggested by the apparent directionality of ion flow in the lens, with strong indications that P-glycoproteins play roles in the circulating flux of Cl- ions within the resting lens, in lens volume regulation and ii the initiation of cortical cataract (Merrisman-Smith, Young et al. 2002). There is also evidence that the metabolically active outer cortex lens fibre cells and epithelial cells supply the inner lens fibre cells and the lens nucleus with water-soluble nutrients (Mathias, Rae et al. 1997) and anti-oxidants such as glutathione (Sweeney and Truscott 1998), functions which would imply directional cellular capabilities. The relationship of LE cells with the overlying capsule and the underlying outermost cortical lens fibre cells has developmental, pathological, and surgical implications. LE cells grown on adhesion-free surfaces differentiate, then mature drastically faster into lens fibre-like cells, complete with gap junctions, expression of gamma-crystallins, and differentiation leading to organelle loss (Arita and Lin 1990), indicating a role of the normal apposition of LECs with capsule in the relative quiescence of the anterior LE cell monolayer in the healthy, undisturbed lens. 1.3 Secondary cataract and posterior capsule opacification 1.3.1 Cataract surgeries In techniques predating current procedures of primary cataract surgery, zonules were interrupted and the entire lens, capsule included, was removed (Shoch 1961). This older 7 technique, called intracapsular cataract extraction (ICCE), had potential for serious complications such as retinal detachment, precipitated by the disruption of the stable interface between the anterior vitreous humour and the apposed external face of the posterior lens capsule. With the entire lens removed, this reduced the stability of the anterior vitreous face thus diminishing the natural tamponade effect of the posterior vitreous against the retina, a force which helps to keep the retina in its natural position. In the majority of current cases in industrialized nations, an anterior capsulectomy is performed which creates a capsulotomy i.e. an opening in the capsule through which a lensectomy is performed, most often via ultrasonic liquefaction i.e. phacoemulsification (Quinlan, Wormstone et al. 1997). The zonules are uninterrupted and all but the central anterior capsule is left in situ, hence the name extracapsular cataract extraction (ECCE). In adults, implantation of an artificial intraocular lens (IOL) usually follows, the majority of which are designed to rest within the capsular bag. An ECCE success rate of 95% is corroborated by various authors (Apple 1984; Apple, Solomon et al. 1992; Kappeihof and Vrensen 1992; Hollick, Spalton et al. 1999; Auffarth and Peng 2000). 1.3.2 Vision-impairing complications of the post-surgical capsule The frequent iatrogenic or physician-induced complication following ECCE is a condition referred to clinically as secondary cataract. The effects include decreased visual acuity, impaired contrast sensitivity and glare disability (Findi, Sycha et al. 2004) and can manifest within weeks in infants (Morgan and Karcioglu 1987) or take several years in older adults to impair vision (Sundelin and Sjostrand 1999). The various peripheral or central components and processes which contribute to this condition can be classified as either cellular or acellular, microscopic, or macroscopic i.e. visible in a low-magnification slit- lamp biomicroscope. They can also be peripheral or central, as well as capsule- or IOL associated. 8 Soemmering’s ring is easily seen via slit lamp. It is the peripheral doughnut-shaped accumulation of residual lens fibre cells, and of swollen and degenerated fibre cells (Kappelhof and Vrensen 1992) in the emptied capsule. The ring may also contain differentiated fibre-like cellular structures plus proliferating lens epithelial cells all of which gradually accrue, causing thickening of the equatorial region in-between the posterior and overlying collapsed anterior aspect of the empty lens capsule (Saika, Miyamoto et al. 1998; Marcantonio and Vrensen 1999). Some of the cells which migrate from Soemmering’s ring onto the posterior capsule attempt maturation to form lentoid bodies known as Elschnig pearls (Kappeihof and Vrensen 1992). These light-scattering entities have been modeled by growing lens epithelial cells on surfaces of varying adhesion, resulting in lentoid bodies exhibiting varying degrees of differentiation and a macroscopic morphology similar to Elschnig pearls (Arita and Lin 1990). A major component of the new activity on posterior capsules occurs when lens epithelial cells in reninant monolayers on the interior face of capsule proliferate de novo and migrate onto the posterior capsule (Saxby, Rosen et al. 1998; Marcantonio and Vrensen 1999; Spalton 1999). Given that the interior face of the posterior capsule is normally devoid of epithelium, the advancing front of new epithelial cells contribute to reduced vision once present in the visual axis. Components of secondary cataract on the periphery of the posterior capsule can also impede clinical examination of the posterior pole and the fundus (Chignell and Wong 1999). Histologically, the material of the posterior capsule proper appears unchanged in PCO (Kappeihof and Vrensen 1992). However, due to the advancing front of epithelial cells and possible presence of Elschnig pearls, the accumulations of these light-scattering microscopic and macroscopic cellular elements renders the surgically exposed surface of the posterior capsule translucent. Although there is never a complete opacity to the light 9 scattered into the posterior segment, the processes described above are nevertheless referred to collectively as posterior capsule opacification or PCO. The presence of the first monolayer of migrating lens epithelial cells onto the posterior capsule as viewed by specular microscopy have been reported as having only a limited effect on vision (Apple, Solomon et al. 1992). However, the transdifferentiation or transformation of these LECs into myofibroblasts (Joo, Lee et al. 1999) or fibroblastic lens cells (Marcantonio and Vrensen 1999) contribute to cell-shape irregularities as well as acellular light-scattering ECM components often observed in PCO development. These cells result from a fundamental epithelial-mesenchymal transition (EMT) (Tanaka, Saika et al. 2004) known also as lens epithelial cell metaplasia (Winther-Nielsen, Johansen et al. 1998). This is corroborated by data which shows that LECs can produce a variety of cytokines (Nishi, Nishi et al. 1996; Caporossi, Casprini et al. 1998). Transformed lens epithelial cells, often spindle-shaped, express the isoform of actin found in smooth muscle (Uusitalo and Kivela 1997). They are commonly found at the edge of the anterior capsulotomy and in the space around the central optic of the IOL (Fig. 1.3). Such cells appear to assume a fibroblastic morphology and secrete fibrin and ECM proteins normally present in neither the lens (Marcantonio and Vrensen 1999) nor in the swollen lens cells, giving rise to globular Elschnig’s pearls (Kato, Kurosaka et al. 1997). The ECM may contribute to light scatter by inducing contracting and wrinkling of the underlying capsule (Obstbaum 1999). Components of this matrix also contribute to plaques containing irregularly amassed collagen fibrils which are therefore translucent, similar to those of another type of primary cataract, the anterior subcapsular cataract (Lovicu, Schulz et al. 1998). On the posterior capsule, the formation of cell aggregates in multilayered islets accompanied by the regression of adjacent cells may also be observed as part of the PCO process (Marcantonio and Vrensen 1999). Other components of secondary cataract include the metaplasia of the anterior capsule LECs plus accompanying fibrosis surrounding the anterior capsulotomy. These 10 changes have been implicated in the purse-string contraction or phimosis of the continuous curvilinear capsulorhexis (CCC) style of anterior capsulotomy (Ursell, Spalton et al. 1997) through which the cataractous lens is removed. When lOLs are present, both macrophages and foreign-body giant cells may also be detected, particularly on the anterior IOL surface (Saika 2004), and although under these circumstances are not by definition part of the process of PCO, can still be considered components of secondary cataract. It is occasional practice to interchange the terms secondary cataract and PCO, but for the purposes of this thesis, PCO will denote the processes described above which contribute to the overall clinical condition, secondary cataract. 1.3.3 Lens cells and the published in vivo rabbit surgical model of PCO The rabbit PCO surgical model (Hansen, Solomon et al. 1988; Lundgren, Jonsson et al. 1992) has been established and used to explore strategies to reduce PCO. The most striking difference between post-surgical human lens capsules and those of rabbits is the very rapid de novo proliferation of LE cells in rabbits. Empty rabbit capsules can completely refill with a massive regeneration of amorphous fibre-like cells (Jones, McLeod et al. 1995), indicative not only of proliferation and migration but also of differentiation and attempted maturation. In humans, any similarity in adult lens capsules has only been reported in cataract patients who have pre-existing proliferative ocular pathologies and have required Nd:YAG posterior capsulotomies to treat secondary cataract. Unusually robust de novo proliferation of LE cells later observed at the margins of this post-ND:YAG posterior capsulotomy is proposed to be influenced by the increased expressions of endogenous growth factors in this sub-population of cataract patients (Jones, McLeod et al. 1995). The usual, relatively slow development of PCO in older humans is exemplified in a study of diabetic and non-diabetic cataract patients, age range 49-89. At one year post surgery, there was no significant difference in PCO gradings between the two groups. It 11 took two years before it could be observed that PCO scores were statistically significantly lower in the patients with diabetes mellitus (Zaczek and Zetterström 1999). In rabbits, post-phacoemulsification refilling of the lens capsule with amorphous lens fibres has been reported as high as 74.5% in control eyes at 3 months and up to 100% at 7 months (Gwon, Gruber et al. 1993). This robustness of actual capsule refilling, as opposed to PCO, is not reported in the human cataract literature. For this thesis the rationale for the use of rabbits, albeit euthanized immediately prior to surgery, was that any PDT-induced prophylaxis of de novo lens cell proliferation which was effective in this robust model (Spierer, Desatnik et al. 1992; Knight-Nana, O’Keefe et al. 1996) could possibly predict an even greater efficacy in adult humans and perhaps a realistic potential for pediatric patients whose LECs exhibit a robust post-surgical phenotype. (The use of euthanized rabbits for the model lensectomies in this thesis is explained in Section 1.6.) 1.4 Incidence of posterior capsule opacilication (PCO) 1.4.1 Overview of secondary cataract incidence Studies regarding the incidence of secondary cataract in adults observed in the 1980s and 1990s reported averaged rates within the range of 15-50% (Apple 1984; Hayashi, Hayashi et al. 1998; Nishi, Nishi et al. 1998; Schaumberg, Dana et a!. 1998; Winther-Nielsen, Johansen et al. 1998; Kim, Lee et al. 1999). This incidence approaches 100% in pediatric cases (Nishi, Nishi et a!. 1998; Winther-Nielsen, Johansen et al. 1998; Nishi 1999). In 2004 it was reported that after primary cataract surgery, the Nd:YAG posterior capsulotomy is the second most common ophthalmic surgery (Findi, Sycha et a!. 2004). However, a downward trend owing to new IOL designs is also discussed in this thesis. As previously indicated in 13.3., the current treatment for secondary cataract in adults and in some pediatric cases is the creation of a permanent posterior capsulotomy via an Nd:YAG laser ablation of a small circular area in the central posterior capsule, a procedure which increases the potential for transient elevated intraocular pressure, retinal 12 detachment, cystoid macular edema, and may also damage the IOL optic and often does not improve the view of the peripheral retina (Javitt, Tielsch et al. 1992). In pediatric cases, a prophylactic posterior capsulotomy is often formed at the time of the primary cataract surgery (Gimbel and DeBroff 1994). Although reasonably safe, Nd:YAG ablation in infants and youth can also lead to similar complications (MacEwen and Baines 1989) for reasons also related to the disruption of the stable interface between the vitreous and capsule. In pediatric cases, advances in the use of both the posterior and anterior continuous curvilinear capsulorhexis, IOL types and their contact with the posterior capsule have been made (Gimbel 1997) and although these advances and ECCE surgery in general have reduced the potential for complications, elimination of secondary cataract would preclude the need for either prophylactic or therapeutic posterior capsulotomies. The following sections relate disparate methods of reporting degrees of PCO and the overall incidence of secondary cataract, pointing to the persistence of this complication and the need to address new prophylaxes. Detailed accounts of disparate imaging techniques used by various research groups to monitor the clinical progress of PCO are presented below, in part to emphasize the appropriateness of being able to detect a single cell in vitro in post-surgical LEC changes, a goal achieved in the experiments reported in the body of this thesis. 1.4.2 A lack of consensus on the incidence of PCO There are numerous reports of the incidence of PCO and the problems inherent in detection and comparison. The first major review (Apple, Solomon et al. 1992) to address the number of adult primary cataract patients who develop this complication noted varying incidence of PCO approaching 50% in 29 cited clinical studies. Apple conservatively assumed that the overall incidence of treated PCO was approximately 30%. Although this major review has been cited in numerous subsequent papers which attempt to address the incidence of PCO in primary cataract patients (Erie, Hardwig et a!. 1998; Hayashi, 13 Hayashi et al. 1998; Michaeli-Cohen, Belkin et a!. 1998; Nishi, Nishi et a!. 1998; Saxby, Rosen et al. 1998; Versura, Torreggiani et al. 1999) a consensus concerning the reliability of the reported PCO rates in adult patients has been problematic. Three main problems have confounded this consensus. Clinical evaluations of PCO have lacked strictly objective grading scales which could be easily reproduced in a wide variety of clinical settings. As well, it has proven difficult to extrapolate statistically- significant data of PCO incidence from the number of reported Nd:YAG capsulotomy treatments to restore the vision of patients presenting with sufficiently severe secondary cataract. As noted by some researchers the Nd:YAG rate, although a clinically relevant parameter, is in fact a surrogate marker of PCO (Spalton 1999). Finally, problems with imaging systems used to measure the presence and extent of PCO have added to the inconsistencies. 1.4.3 Meta-analyses of PCO incidence studies (1979 to 1996) Attempts have been made to address disparate grading protocols and extrapolating PCO incidence from reported Nd:YAG posterior capsulotomy rates. Schaumberg etal (Schaumberg, Dana et al. 1998) have reported a meta-analysis of the statistics on PCO incidence in an extensive body of literature from 1979 to 1996. In this thorough study, 1763 potentially relevant MEDLINE-indexed papers addressing ECCE primary cataract surgery complications were identified. Of these papers, 69 dealt with PCO incidence of which 49 were included in a meta-analysis based the inclusion and exclusion criteria shown in Table 1.1. 14 I. Criteria for inclusion in the Schaumberg 1998 Review: a. study must have been published 1979-1996; b. sample size and postoperative PCO rate reported along with some measure of the length of follow-up for eyes of patients; c. reported in English. II. Criteria for exclusion from the Schaumberg Review: a. animal or post-mortem eyes; b. surgery was not ECCE; c. more than 25% of cases had combined surgical procedures; d. patients had a particular disease such as diabetes; e. no lOLs were used; f. anterior chamber intraocular lenses (AC lOLs) were used; g. outdated (e.g. iris clip) lOLs were used; h. no IOL information was reported; i. N<25 eyes; j. study not related to patient age (i.e. traumatic cataract). Table 1.1 Exclusion and inclusion criteria for the Schaumberg review of posterior capsule opacification incidence (Schaumberg, Dana et al. 1998) Schaumberg (ibid.) concluded that visually significant PCO had developed in more than 25% of adult patients undergoing ECCE, with or without phacoemulsification plus posterior chamber (PC) IOL implantation, monitored 5 years after surgery. Ram and Apple (Ram, Apple et al. 1999) described 3493 eyes with posterior chamber (PC) lOLs (currently the most common type of IOL used, Fig. 1.3) with the two haptics placed behind the iris in sulcus-sulcus, sulcus-bag, or bag-bag position. Included in this report were the generated pooled estimates of eyes developing PCO over three postoperative time points: 1,3 and 5 years. The rate of PCO remained 25% during the five- year postoperative period. As further effort to investigate the confounding lack of strictly objective grading scales, Sundelin and Sjostrand (Sundelin and Sjöstrand 1999) searched for any “hidden” group of patients with PCO five years after cataract surgery. A random sample (n= 164) was selected from 1672 patients who had non-phacoemulsification ECCE cataract surgery with “mostly” polymethylmethacrylate (PMMA), rigid IOL implantation. Patients alive 5 years after cataract removal who had not had Nd:YAG posterior capsulotomies were 15 examined. Follow-up was possible in 51 of 73 non-Nd:YAG patients. Of these, clinically significant PCO was found in 7 cases. The authors calculated an incidence of 43% 5 years after primary cataract surgery. This included the untreated group also exhibiting clinically significant PCO, a factor they assert must be considered in calculating incidence rates. As an illustration of the potential for the disparity of reporting in the literature regarding PCO incidence, a paper published in 1999 (Birinci, Kuruoglu et al. 1999) describes 302 eyes examined retrospectively after various types of lOLs had been implanted within the capsule in non-phacoemulsification ECCE surgeries performed from 1991 to 1996. There were approximately 100 eyes in each group. The mean follow-up time was 27 months, within a range of 12 to 33 months. Posterior capsules were objectively graded on a three-point (1-3) grading scale. However, only those capsules with grade 2 or 3 were deemed to have PCO. Of these, eyes with PMMA lOLs scored 21.7%, heparin-modified PMMA lOLs scored 17.4%, and plate-haptic silicone lOLs scored 7.7%. The average of all groups with a grade 2 or 3 PCO rating was 15.6%. 1.4.4 Imaging problems and new developments in assessing PCO The third consensus-confounding problem in calculating statistically-significant incidences of PCO are poorly-comparable imaging systems. Although the 1998 Schaumberg review (Schaumberg, Dana et al. 1998) addresses the problem of data comparison from disparate studies, the direct comparison between different studies published since then remains difficult, complicated by changes in surgical technique and the non-standardized definitions, measurements and scoring of PCO. In the same year, the Hayashi group indicates that drug effects on PCO remain speculative since the quantification of PCO was yet to be established (Hayashi, Hayashi et al. 1998). In 1999, Coombes and Seward (Coombes and Seward 1999) reasserted that simple slit-lamp retroillumination grading of PCO, and extrapolating rates of PCO incidence from Nd:YAG capsulotomy rates, are both relatively subjective. 16 Earlier, the Tetz group (Tetz, Auffarth et al. 1997) reported a photographic image analysis system of PCO called Morphological PCO Scores in which retroillumination PCO photographs are scanned, and the boundaries of areas of opacification marked. The overall PCO score is calculated by multiplying the density of the opacification (0-4) by the fraction of the capsule area involved behind the IOL central optic. The reliability of the system was tested by having 6 observers examine and score photographs of 5 eyes, presented in different orders, upside-down, and/or reversed to reduce recognition bias. With software, this Evaluation of Posterior Capsule Opacification (EPCO) system evaluates the entire area behind the IOL optic and thus includes a larger area of the posterior capsule than simple visual acuity testing. Revealed was a high reliability and insignificant investigator- dependent variation. However, the extreme equatorial region of the posterior capsule was not visible. EPCO has been used by other authors. Zaczek (Zaczek and Zetterström 1999) prospectively compared patients with heparin-surface-modified PMMA lOLs in 26 diabetes mellitus patients and 26 non-diabetics, all subjected to phacoemulsification surgery. These authors reported, on the basis of the EPCO data of comparative gradings of PCO, that at 2 years the PCO scores were statistically significantly lower in the patients with diabetes mellitus. No percentage of patients exhibiting PCO was directly reported. This result is directly contradictory to a previous study (lonides, Dowler et al. 1994) where a higher PCO incidence was reported in diabetic cataract patients, based however on the Nd:YAG posterior capsulotomy rates. 1.4.5 PCO incidence from Nd:YAG posterior capsulotomy rates There has been a tendency by some authors (Olson and Crandall 1998; Drews 1999; Hollick, Spalton et al. 1999; Kim, Lee et al. 1999) to base the incidence of PCO on reported Nd:YAG posterior capsulotomy rates. These rates have been described (Tetz, Auffarth et al. 1997; Zaczek and Zetterström 1999) as potentially biased for three reasons. 17 The decision to create Nd:YAG posterior capsulotomies may be based solely on changes in visual acuity, and thus not whether PCO was present. Additionally, the Nd:YAG incidence may also be biased because is does not depend on the amount of opacification, but may reflect subjective complaints of poor vision. Finally, economic factors usually not reported may also influence the decision to treat PCO by Nd:YAG, thereby further confounding the correlation between laser treatments and either the degree or incidence of PCO. Although EPCO allows more standardized PCO estimations, other imaging systems have been developed to promote a more precise, reproducible, quantification of PCO, especially with different lens implants. Pande and Ursell (Pande, Ursell et al. 1997) described a high-resolution digital retroillumination of the posterior lens capsule in which images are captured and analyzed for quantitative measures of PCO based on careful boundary assignments of areas of PCO. The resolution permits detection of subtle changes in LEC growth on the posterior capsule. This same research group, in association with Hollick et al measured the percentage of PCO using their digital system in a prospective study to explore the relationship between IOL material and PCO rates (Ursell, Spalton et al. 1998). Involved were 90 eyes in 81 patients 55 years or older. The eyes were randomized to receive PMIVIA, silicone, or polyacrylic AcrySof lOLs. The standardized ECCE surgery did not involve phacoemulsification and no attempts were made to polish epithelial cells off the capsule. Consistent throughout was the formation of a continuous curvilinear capsulorhexis (CCC) as the style of anterior capsulotomy. This technique of tearing the capsule, first described by Gimbel (Gimbel and Neuhann 1990) not only yields a circular opening but also a smooth capsulotomy border which is easily seen. Crucial to the above study (Ursell, Spalton et al. 1998) was the lack of a merely subjective estimation of the PCO incidence based solely on those patients whose vision is sufficiently impaired that a posterior capsulotomy must be performed using an Nd:YAG laser. Instead, the percentage of opacified posterior capsule visible through the area bordered by the CCC opening was measured. At two years (Ursell, Spalton et al. 1998), 18 the opacified area in PMMA IOL eyes ranged from 3.9 % - 67.0%, with a median of 43.7%. In those with silicone, the range was 4.7 % - 75.6%, median 33,5%, whereas the AcrySof range was 2.6 % - 52.0%, median 11.8%. The authors believed this to be the first objective documentation of reduced PCO associated with IOL material, the comparative results deemed statistically significant. Of note, none of the ranges in any IOL test group at any of the follow-up times was zero, indicating that with this image analysis system, at least some measurable PCO was seen in all eyes. In short there was detectable PCO in all 90 of the eyes. 1.5 Secondary cataract prophylaxes Variations of the basic aspects in ECCE cataract surgery such as different diameters of anterior capsule openings, separation of the capsule from the outermost lens cortex, and alternative cleaning or polishing techniques of all the inner capsule surfaces have been tried in order to discover any correlations with changes in the incidence of secondary cataract. Specialized physical treatments of LECs and chemical or biochemical prophylactic approaches have also been widely reported. Finally, intraocular device and IOL designs have been extensively studied, particularly in order to halt PCO progression beneath the artificial lens optic. Although the changes in surgical techniques as well as IOL designs have reduced the degrees of PCO development and the overall incidence of PCO sufficiently severe to require secondary cataract surgeries (Apple, Peng et al. 2000; Peng, Apple et al. 2000; Peng, Visessook et al. 2000), there appear to be no PCO prophylaxes in current clinical practice reported to consistently eliminate all LECs. 19 1.5.1 Physical approaches Variations in basic primary cataract surgical techniques Given that a wider anterior capsulotomy would ostensibly remove more anterior lens epithelial cells and reduce the overall remaining intracapsular load of A-cell LECs, researchers have looked for correlations between capsulotomy size and levels of anterior LEC metaplasia and fibrosis on the inner anterior capsule, anterior capsulotomy contraction (phimosis) as well as overall PCO (Kimura, Yamanishi et al. 1998). Because of the ease of formation and the resulting unique smooth continuous capsulotomy edge (Krumeich and Daniel 1998), considerable attention has been paid to the continuous curvilinear capsulorhexis (CCC) type of capsulotomy (Gimbel and Neuhann 1990), which provides stable IOL fixation in the capsular bag (Nagamoto, Kohzuka et al. 1998). Results of studies of the relationship between CCC size and the diameter of the IOL (Ursell, Spalton et al. 1997) shows some correlation with the degree of LEC metaplasia and resulting capsule CCC phimosis (Joo, Shin et al. 1996). In another study of CCC phimosis, a diabetes mellitus (DM) cohort exhibited statistically-significant greater contraction than the cohort without DM (Hayashi, Hayashi et al. 1998). This indicates the need to statistically correct for, if not at least account for, the possible effects of systemic pathologies on PCO. Other data also indicate that fewer vision-compromising changes and less CCC phimosis in the anterior capsule may be associated with lOLs of greater hydrophilicity (Miyake, Ota et al. 1996). Differences in outcomes of CCC diameter also correlate with different IOL designs (Sickenberg, Gonver et al. 1998). However, there appear to be no studies which correlate CCC size with elimination of the these anterior capsule components of secondary cataract. One study of 302 human eyes with either PMMA or silicone round-edged (biconvex) IOLs shows that the creation of a CCC type of capsulotomy (diameter not reported) was significantly associated (P < .05) with a lower incidence of PCO of 11.5%, 20 versus 24.5% in eyes with an envelope (flap-type) capsulotomy (Birinci, Kuruoglu et a!. 1999). Finally, an apparent contradictory study relating CCC size and PCO (Hollick, Spalton et al. 1999) reports a significantly worse opacification and visual outcome with specific reference to wrinkling of the posterior capsule in human clinical case eyes with large-diameter CCC capsulotomies implanted with PMMA lOLs. The original hypothesis that more PCO-inducing LECs would remain post-surgically in the case of a smaller diameter capsulotomy was not supported by these data, possibly explained in part by the adhesion and possible impedance of the inner anterior capsule LECs now overlapping with and in close contact with the IOL. Attention has also been paid to the prophylactic potentials of other steps which may follow anterior capsulectomy. Cortical cleaving hydrodissection (Faust 1984; Fine 1992) is the separation of the capsule from the outermost lens cortex via careful yet forceful injections, usually of balanced salt solution (BSS, ocular-specific), immediately beneath the capsule under the edge of the capsulotomy. Evidence shows that this step reduces the amount of post-surgical remnant cortical lens cell material (Lehmann 1996). Recent data shows that truly adequate cortical cleanup occurs when relatively copious hydrodissection is employed, and although perhaps counterintuitive, is reported in one paper as resulting in fewer instances of capsule rupture than in those eyes with no or insufficient hydrodissection (Peng, Apple et al. 2000). This procedure usually frees the lens and permits at least partial rotation, facilitating either lens phacoemulsification or lens expulsion into the anterior chamber, depending on the ECCE technique employed. Ultrasonic, laser and cryogenic surgical adjuncts Surgical procedures which attempted physical removal of lens cells have included extensive polishing of the posterior capsule with neodymium: yttrium-lithium-fluoride (Nd:YLF) picosecond lasers (Hanuch, Agrawal et al. 1997), anterior capsule cleaning with an 21 ultrasound inigating scratcher (Meucci, Esente et al. 1991) and cryodisruption (Zaturinsky, Naveh et al. 1990) to rupture lens cells, all techniques having been reviewed (Apple, Solomon et a!. 1992; Liu, Wormstone et al. 1996; Nishi 1999). The approaches had varying degrees of success in removing residual LECs but were time consuming, were not free from inducing their own complications, and as noted (Coombes and Seward 1999), the fact that none of these techniques have been in routine surgical use reflects the difficulty in totally removing all LECs. They further suggest that attempts to remove LECs may simply damage those left in situ, which may then become activated. More recently, a carbon dioxide infrared laser (Michaeli-Cohen, Belkin et a!. 2000) was used to successfully ablate A-cells and E-cells in test halves of two sheep lens capsules, requiring considerable power up to 1.3 W. 22 Figure 1.3 A stylized three-piece posterior-chamber intraocular lens (PC IOL) consisting of the central, circular optic and two thin haptics. The IOL, inserted into the empty lens capsule in the post-surgical aphakic eye, is usually held in place by positioning the distal aspects of the two haptics in the equator of the capsular bag. The optic provides pseudophakic refractive power calculated to restore vision. IOL materials Early developments leading to successful clinical pseudophakia i.e. implantation of an artificial intraocular lens, included models which were anchored within the anterior chamber in front of the iris after ICCE surgery. With the subsequent development of ECCE techniques, the post-surgical lens capsule has by default become an accessible region of the posterior chamber. The posterior chamber intraocular lens (PC IOL), now usually referred to simply as an IOL or in-the-bag IOL, is generally intended for intracapsular fixation and has became the model of choice after ECCE surgery (Fig. 13). These lOLs usually consist of a circular lens, called the optic (biconvex in early models) approximately 6 mm in diameter, most commonly with two thin, arc- or J-shaped anchoring haptics. In some three-piece IOL designs, the haptics are a disparate plastic material and appear inserted into the optic (Linnola and Hoist 1998). Alternatively, haptics and optic of the same material appear as one piece. lOLs are anchored most often with both haptics in the emptied capsular bag (Ram, Apple et al. 1999), although one or both haptics may require anchoring anteriorly within the sulcus of the posterior chamber i.e. the peripheral groove bounded in back by the ciliary processes and in front by the posterior surface of the iris. In such cases the optic is positioned slightly differently relative to the 23 surfaces of the capsule with reported effects on PCO developments (Hansen, Solomon et al. 1988; Tetz, O’Morchoe et al. 1988; Ram, Apple et al. 1999). Three of the most common materials used in lOLs are polymethylmethacrylate (PMMA), silicone, and polyacrylic and have been compared for ocular biocompatiblily (Hollick, Spalton et al. 1998) and relative effect on PCO development with regard to the chemical constituents in the lOLs (Drews 1999; Hollick, Spalton et al. 1999). In 1997 Ursell etal compared foldable silicone and polyacrylic AcrySof lOLs with solid PMMA lOLs, all similar except for material of the optic and angle of haptics. In this study (previously noted in 1.4.5 after its second-year follow up) 90 adult eyes with large (6.5mm) CCCs were then randomized into one of three IOL groups for one-year follow-up (Ursell, Spalton et al. 1997). The polyacrylic lOLs produced significantly less anterior capsule phimosis than PMIvIA or silicone lOLs and were less likely to cause posterior capsule contraction and IOL decentration. Also, polyacrylic lOLs correlated with reduced PCO and also reduced LEC proliferation on the area of contact between the anterior capsule and the IOL optic. After 2 years in the above study (Ursell, Spalton et al. 1998), the percentage of opacification within the prescribed viewing mask area of the digital images showed that all capsules had some degree of PCO. Those with PMMA lOLs scored a median of 43.7%, silicone 33.5% and polyacrylic 11.8%, which at 3 years with 71% of eyes re-examined (Drews 1999), were reported as 56%, 40% and 10% respectively. The corresponding Nd:YAG rates at 3 years were 26% for PMMA, 14% for silicone and 0% for the polyacrylic lOLs, thereby correlating less PCO with polyacrylic material. As previously commented upon in 1.4.5, after 2 years (Ursell, Spalton et al. 1998) the positive percentage values for viewing-mask-area opacification indicate that all capsules exhibited at least partial PCO. This is a strong indication that none of the capsules had all residual LECs removed or severely compromised, other contributors to clinical secondary cataract e.g. macrophages, not withstanding. 24 Similar results have been reported elsewhere (Hayashi, Hayashi et al. 1998) whereby 240 eyes randomized with lOLs made of PMMA (Alcon MZ6OBD), silicone (Allergan SI 3ONB) or foldable acrylic (Alcon AcrySof MA6OBM) showed, at approximately 2 years, Nd:YAG rates of 30.4%, 5.7% and 2.7% respectively. The difference between eyes with PMMA lOLs and the others was statistically significant. Reports have also drawn correlations between IOL material and the types of PCO and rates of formation. In a study of 54 eyes, all of which had Nd:YAG posterior PCO capsulotomies, silicone lOLs (Allergan SI-3ONB) induced PCO faster than PMMA lOLs (Pharmacia 81 1B) (Kim, Lee et al. 1999). In the same report it was noted that fibrosis-like PCO was most common in the silicone group (Kim, Lee et a!. 1999) i.e. a fibrosis from LECs undergoing psuedometaplasia to fibroblast-like cells (Nasisse, Dykstra Ct a!. 1995; Duncan, Wormstone et al. 1997; Saxby, Rosen et al. 1998; Obstbaum 1999; Wormstone, Tamiya et al. 2004). IOL adhesion effects Although PMMA, silicone and soft acrylics have proven to be tolerated within the human anterior chamber, (Hollick, Spalton et a!. 1998), there are differences in adhesion between IOL materials and the lens capsule. AcrySoflOLs are more adhesive than PMTvIA (Nishi and Nishi 1999). It was hypothesized that adhesion may play a role in the behaviour of AcrySof acrylic lOLs in generally diminishing PCO. In 1998, one publication (Oshika, Nagata et a!. 1998) reported significant differences in IOL adhesion to bovine collagen sheets and rabbit posterior capsules, AcrySof the most, PMMA an intermediate adhesion and silicone virtually none. In related bioflim work using radiolabeling, there was significantly greater adsorption of fibronectin, present in normal aqueous humour, to polyacrylic AcySof lOLs than to PMMA (Johnston, Spalton et al. 1999). However, PMMA lenses bound significantly more albumin. Albumin is present in aqueous humour after surgery due ostensibly to the 25 compromise of the ocular blood-aqueous barrier (Tripathi, Li et al. 1998). There is also speculation (Oshika, Nagata et al. 1998) that the increased adsorption of fibronectin to AcrySof lOLs may provide a molecular basis for the increased adhesion to the lens capsule observed in vivo. A shared conjecture (Hollick, Spalton et al. 1998; Oshika, Nagata et al. 1998; Versura, Torreggiani et al. 1999) that the tight adhesion of the IOL optic to the capsular bag, preventing LEC migration in what was earlier proposed as “no space no migration” (Apple, Solomon et al. 1992) may have in turn contributed to the lower clinical PCO rates with AcrySof lOLs in early reports (Mengual, Garcia et aL 1998; Hollick, Spalton et a!. 1999). Intraocular inserts Proposed physical barriers to LEC proliferation include intracapsular tension rings, which are open-ended nearly circular slender polished plastic rings originally designed to be inserted into the equator of an empty capsule as a stabilizer when some of the zonules may have ruptured and to prevent further zonular dehiscence i.e. a bursting of and resulting gap in the attachment of zonules at the periphery of the lens capsule. In human trials, LEC growth was not impeded by the smooth cylindrical ring (Strenn, Menapace et al. 1997). Altered shapes and sharp edges employed in subsequent rabbits studies showed at eight weeks follow-up that the flattened band-shaped circular loop capsule-bending ring caused a marked inhibition of LEC migration attributed to the fact that LECs accumulated outside the ring at the equator (Nishi, Nishi et al. 1998). IOL designs The effects of a squared or sharp edge on the posterior face of lOLs forming a sharp bend in the contact between the IOL and the posterior capsule have been thoroughly described (Peng, Visessook et a!. 2000) as a barrier or contact inhibition to LEC migration on the posterior lens capsule. This barrier effect in rabbits (Nishi, Nishi et al. 1998) prompted 26 later comparisons between AcrySof MA6OBM lOLs with PMMA lOLs which were custom fabricated with matching dimensions and the sharp, rectangular posterior face (Nishi and Nishi 1999). At 3 weeks, rabbit control eyes had typical, abundant PCO but with sharp-edged lOLs, regardless the material, there was minimal migration of LECs under the optic of the IOL. Both IOLs created the same sharp right-angle bend and a complex frill on the posterior capsules of those rabbits implanted, showing that although with robust rabbit LEC proliferation, migration and fibrosis were not eliminated, the cells were instead stopped at the posterior IOL edge. However, evidence indicates that the join of haptic to optic in some 3-part lOLs also serves as a point of entry for LECs under the optic and into the visual axis (Werner, Mamalis et al. 2004). 1.5.2 Physical chemical approaches Two physical chemical approaches used to compromise LECs are reported, water-mediated osmolysis and radiation, both addressing the need to minimize collateral anterior chamber damage particularly to the comeal endothelium after single-dose or slow-release drug application to the capsule. In one study, remnant human post-surgical anterior capsule fragments with the LECs in situ were immersed in distilled deionized water (DDW) then cultured for 1 week (Crowston, Healey et a!. 2004). No viable LECs were observed on capsules exposed for 2 minutes to DDW, and only on 1 of 3 capsules exposed for 1 minute, as determined by photomicrographs of LECs which excluded trypan-blue. In one radiation approach, beta-emitting P-32 was incorporated into (and without physically altering) PMMA intracapsular rings (not specified as sharp-edged) which were inserted into the capsular bag after phacoemulsification ECCE surgery in rabbits (Joussen, Huppertz et al. 2001). At 12 weeks, there was evidence of a dose-dependent decrease in the number of LECs inside the capsules but not a full inhibition of aberrant differentiation of some remaining cells. 27 1.5.3 Pharmacological approaches PCO prophylaxis has been explored with a variety of drugs, including antineoplastics, anti inflammatories, antiallergics, antibodies and antibody-associated toxins, plus chelators, small peptides and local anaesthetics. These have been applied variously as aqueous solutions used for hydrodissection of the capsule before or after anterior capsulectomy, simple introduction into the capsular bag, or incubation within the entire anterior segment during and after ECCE surgery. Some agents have also been used in topical or subconjunctival application, or alternatively via short-term and slow intracapsular release from treated rings and lOLs. Antineoplastics Antimetabolites and hormones used as antineoplastic agents are the two main groups which have been applied to LECs. Three of the principle categories of antimetabolites in the literature are the folic acid analogues, pyrimidine analogues, and antineoplastic antibiotics. Methotrexate, a potent folic acid analogue which inhibits folate-dependent enzymes of de novo purine synthesis (Chabner, Ryan et a!. 2001) has shown effect against LECs but is not used clinically to prevent PCO (man, Ozturk et al. 2001). One halogenated pyrimidine analogue, 5- fluorouracil (5-FU) principally inhibits RNA function once the 5-FU is ribosylated and phosphorylated within the cell to form the nucleotide (Chabner, Ryan et al. 2001). 5-Fl] has a wide use in ophthalmology (Cochener, Bougaran et al. 2003) and has shown antiproliferative activity in epithelial cells of both the pigmented retina (Oshima, Sakamoto et al. 1997) and of the lens (Ruiz, Medrano et al. 1990). Daunomycin (daunorubicin), an anthracycline antibiotic, as well as mitomycin C (MMC), an aziridine-quinone antibiotic, both interfere with DNA synthesis (Chabner, Ryan et a!. 2001) in eukaryotic cells. The former, when simply injected as a solution into 28 the empty capsular bag was reported as an anti-PCO potential with a small, but useable therapeutic window of drug toxicity (Wiedemann 1990). Given the considerable use of mitomycin C in corneal and glaucoma surgeries (Moroi and Lichter 2001), MMC has also received attention and is reported (Shin, Kim et a!. 1998) to inhibit but not prevent PCO. Shin et al (ibid.) treated 81 eyes in patients with combined glaucoma and phacoemulsification ECCE surgery after which a small sponge soaked in 0.5 mg/mI MMC was placed under a subconjunctival flap over a dissected scieral flap and left for 1 or 3 minutes. All eyes had PMMA in-the-bag lOLs. PCO was visually graded (0-4 scale) for up to 2 years, and Nd:YAG posterior capsulectomies were performed when PCO reached grade 3 or 4. Only the three-minute test subgroup showed a statistically- significant effect on PCO. In another study (man, Ozturk et al. 2001), 0.1 ml MMC in solution was used as the hydrodissecting fluid in rabbits. PCO was significantly decreased at 3 months after ECCE surgery. Dexamethasone, a glucocorticoid member of the adrenocorticosteroid hormones has shown effective anti-inflammatory effects as post-surgical drops in cataract patients (Drews 1990), but can also mediate antineoplastic activity via cytoplasmic receptor binding and downstream gene expression leading to apoptosis (Chabner, Ryan et al. 2001). In the man study (man, Ozturk eta!. 2001), 0.1 ml of dexamethasone solution as the hydrodissecting fluid in rabbits resulted in a clearly reduced severity of PCO observed at 3 months, but not prevention of cell proliferation after this single subcapsular dose. Anti-inflammatories and antiallergics In particular, two of the non-steroidal anti-inflammatory drugs (NSAIDs), indomethacin and diclofenac, plus the antimitotic anti-inflammatory drug colchicine are represented in the secondary cataract literature, along with several antihistamines. These drugs have been of interest due to the potential for increased inflammatory cascades initiated by intracapsular cell damage during surgery. This pharmacology is based on the fact that two groups of 29 metabolites of arachidonic acid (AA), the prostaglandins (PGs) and the leukotrienes, collectively identified as eicosanoids, mediate every stage in acute inflammation. Prostaglandins are mediators of inflammatory vasodilation, of increased vascular permeability and of increased chemotactic effects of other inflammatory mediators and cytokines (Cotran, Kumar et a!. 1994). Prostaglandins are released whenever cells are damaged (Roberts and Morrow 2001). In particular, prostaglandin E2 (PGE2) along with cytokines are produced postoperatively by human cataract LECs (Nishi, Nishi et al. 1992). NSAIDs which are potent inhibitors of PGE2 synthesis reduce inflammation in the anterior chamber after cataract surgery (Drews 1990). With regard to PCO research it is worth noting that two enzyme classes yield eicosanoids from AA. The first, lipoxygenases, generate leukotrienes and chemotactic mediators. The second class, comprised of two forms of cyclooxygenase known as COX 1 and COX-2, catalyze AA conversion to prostaglandins (Vane and Botting 1987). NSAIDs such as diclofenac, indomethacin and aspirin inhibit COX-1 and COX-2 i.e. are nonselective COX inhibitors but do not inhibit lipoxygenase pathways. However, the glucocorticoids selectively suppress only COX-2 (Masferrer, Reckly et al. 1994). Indomethacin is a potent COX inhibitor and also inhibits polymorphonuclear leukocyte motility and at very high concentrations it uncouples oxidative phosphorylation and depresses the biosynthesis of mucopolysaccharicles (Roberts and Morrow 2001). It also induces mydriasis, or pupil dilation (Dube and Boisjoly 1990) , is used as a topical anti- inflammatory (Ibaraki, Ohara et al. 1995), and has been applied on its own (see and in drug combinations in PCO studies (below). Diclofenac, a fenamate NSAID derived from N-pheylanthranilic acid (Roberts and Morrow 2001) is also used as a topical ocular anti-inflammatory (Moroi and Lichter 2001), and when applied to human LECs in vitro has been shown to suppress proliferation and fibrous metaplasia (Nishi and Nishi 1991). Given the different mechanisms of actions of anti-inflammatories, combinations of glucocorticoids and NSAIDs have been tested for effects on opacification. In one study 30 with 34 adult ECCE eyes with three-piece PMMA lOLs, a combination therapy of topical 0.1% betamethasone and 0.1% diclofenac in 19 eyes was maintained for 1 month with subsequent tapering. A second group of 15 eyes received 0.1% betamethasone and 0.5% indomethacin(Ibaraki, Ohara et al. 1995). Advancing membranous outgrowth was observed on the IOL surface in 80% of the indomethacin/betamethasone eyes and in 78.9% of the cliclofenac/betamethasone eyes. Specular microscopy showed this outgrowth to be LECs. Colchicine also exhibits anti-inflammatory activity, but via modified cell mobilization. A gout treatment, this antimitotic causes depolymerization of fibrillar microtubules in granulocytes and other motile cells thus inhibiting migration to inflamed areas. The tubulin binding activity of colchicine also disrupts mitotic spindles causing metaphase arrest (Lunn, Baas et al. 1997; Roberts and Morrow 2001). This antimitotic is shown to have reduced the incidence of PCO (Ismail, Alio et al. 1996) but causes significant ocular toxicity (Boulton and Saxby 1998). Three antiallergics, tranilast, ketotifen fumarate and disodium chromoglycate have been applied to bovine LECs in vitro (Suzuki, Iwaki et al. 2001). In post-operative cardiac treatments, tranilast has a potent effect on preventing postangioplasty restenosis (Takahashi, Taniguchi et al. 1999). In the Suzuki study, both tranilast and ketotifen fumarate inhibited LE proliferation and were positive for apoptois TUNEL indicators. These two test culture groups also synthesized less collagen than the control cells but exhibited no changes in transforming growth factor beta-i (TGFI3-1) nor in basic fibroblastic growth factor (b-FGF) expression. No significant changes of any parameters were observed in the chromoglycate cultures. 31 Antibodies and cytotoxin delivery systems Antibodies used in PCO research fall into two groups: those raised against factors considered important in LEC de novo proliferation and transdifferentiation, and toxin- linked antibodies delivered to LECs. One group (Wormstone, Tamiya et al. 2002) has shown that TGFB-2 isoform accelerates LEC transdifferentiation and subsequent contraction in donor human capsular bags from eyes cultured in vitro, resulting in the scattering of light. In this model, the use of the novel human monoclonal anti-TGFI3-2 antibody CAT-152 (lerdelimumab) also negated the TGFI3-2-induced effects, indicating a potential to suppress PCO development. Wormstone etal (Wormstone, Del Rio-Tsonis et al. 2001) also provided evidence that FGF is important for human LEC growth and long-term survival, and proposed that inhibition of FGF receptor-i (FGFR- 1) by synthetics may hold promise in LEC treatment. Antibody-toxin conjugates, or immunotoxins, have also been used to treat LECs. Ricin A, an extremely potent dimerized 3OkD castor bean toxin (O’Neil 2001), has been conjugated with a human LE-specific mouse monoclonal antibody MAb 4197X (MDX-RA; Medarex), (Kelleher, Tarsio et al. 1992; Clark, Emery et al. 1998), the antibody being specific for a glycoprotein epitope on the surface of LECs. In later studies, a specific conjugate ratio of ricin:Mab at 2:1 in an aqueous solution of either 50 or 100 units/mi was used a study of 36 eyes in adult ECCE patients fitted with one-piece PMMA lOLs. One ml of placebo, 50 or 100-unit aqueous solution was injected into the empty capsule, in a protocol stated to displace the residual BSS from the capsule and anterior chamber and the drug then left in the eye (Meacock, Spalton et al. 2000). The percentage of the area of the posterior capsule opacified at one 1 year was reported, the median results being: placebo, 32.0%; MDX-RA 50 units, 3.8%; and MDX-RA 100 units, 7.4% (P=.06) (Meacock, Spalton et al. 2000). All treated patients showed increased postoperative uveitis, but within acceptable limits. Correlations were also observed between treatment groups and the presence of macrophage-derived cells on the anterior IOL surface, a presence also reported 32 by others (Uusitalo and Kivela 1997) in human eyes with secondary cataract which were sectioned post-mortem and screened immunohistochemically. Patients who received low- dose MDX-RA (Meacock, Spalton et al. 2000) showed a greater number of these macrophage-derived cells than placebo and high-dose patients. Despite the extreme toxicity of ricin A, MDX-RA reduced but did not prevent PCO and the authors note that is was clear that MDX-RA did not kill all LE cells and further conjectured whether the remaining cells on the posterior capsule were viable, with the potential to form more PCO. Other cytotoxins in non-antibody conjugates have also been prepared for LEC specific delivery. The cytotoxin saporin does not readily cross mammalian cell membranes, but Bretton etal (Bretton, Swearingen et al. 1999) observed that when conjugated to polylysine, saporin entered and destroyed target bovine LE cells in vitro. The authors observed that polylysine selectively binds to the lens capsule, most likely to the negatively charged glycosaminoglycans e.g. to the abundant heparan sulfate bound to protein as proteoglycans in the lens capsule. Polylysine-saporin conjugate (PLS) in either BSS or 0.5 ml of hydroxyproplymethyl cellulose (Occucoat) viscoelastic was instilled into post-ECCE rabbit capsules for 5 minutes. PMMA lOLs were added and the rabbits monitored for 11 weeks. In 6 test eyes, the time of first cortical regrowth observed was 2 to 3 times longer than in control eyes. One test eye was free of regrowth for 40 weeks with 2 test eyes similar to controls. The results may have been potentially complicated by including antifibrinogenic heparin (in the irrigating BSS) which may have competed with binding sites on the lens capsule for the polylysine in the PLS (Bretton, Swearingen et al. 1999). Intracapsular release of compounds lOLs have been used as a rapid- or slow-release drug delivery system. For example, oil solubility often correlates with adhesion to acrylic IOL optics, leading one group of researchers to coat lOLs with a 1.0% preparation of oil-soluble indomethacin for release in 33 rabbit capsules after ECCE surgeries (Nishi, Nishi et al. 1995). This resulted in reduced PCO and lessened circumoptic regenerated lens masses shown histopathologically at 6 to 12 months. Identically-treated lOLs placed into BSS exhibited a complete release of the indomethacin within 24 hours i.e. a relatively rapid release. Similar studies have been carried out with heparin surface-modified PMMA IOLs in humans, showing a slight PCO reduction. However, the rate of drug release was not reported (Birinci, Kuruoglu et al. 1999). Another hydrophobic toxin, thapsigargin, is an encloplasmic reticulum calcium ATPase inhibitor which induces intracellular Ca2+ release in many types of vertebrate cells (Thastrup, Cullen et al. 1990) and has been used to coat PMMA lOLs, resulting in a 50% release in aqueous solution within 10 hours (Duncan, Wormstone et a!. 1997). ECCE surgeries in human donor eyes were followed by culturing the excised capsules with the IOL in place up to 27 days. Greatly reduced cell growth correlated with 200 nM thapsigargin coatings and total cell death of the residual anterior LECs was observed with lOLs coated in solutions of greater than 2 pM concentrations (Duncan, Wormstone et al. 1997). There was no report of an estimated total amount of drug released. Hydroxy acid polymers have been used as biodegradable surgical materials (Bacakova, Lapcikova et al. 2003). For example, polymers of the dextrorotatory and levorotatory light-rotating isomers of lactic acid (poly-DL-lactid) have been added as carriers in IOL-bound sustained-drug delivery systems (SDDS), slowing drug release into the capsule. Poly-DL-lactid combined with daunorubicin or with indomethacin has been used to pretreat lOLs for rabbit ECCE surgery (Tetz, Ries et a!. 1996). At 8 weeks, measurements of PCO wet mass showed no effect of indometbacin, however daunorubicin still reduced PCO formation by approximately 50%. The authors indicated that it remained to be detennined whether the observed endothelial side effects were specific to the rabbit species or whether the human cornea is as sensitive. 34 A similar material, polylactic-polyglycolic acid has been used as a synthetic absorbable suture material (Vicryl, Polyglactin 910) (Racey, Wallace et al. 1978). Nishi et al(Nishi, Nishi et a!. 1996) fixed a polylactic-polyglycolic acid disc containing 7mg of indomethacin along with an IOL into rabbit capsules after phacoemulsification ECCE surgery. Under these slow-release conditions, indomethacin significantly reduced inflammation (prostaglandin E2 was undetectable) but did not inhibit LEC proliferation, prompting the authors to suggest that exposure to a higher indomethacin concentration, even for a short period, is more effective in inhibiting LEC proliferation than exposure at a lower concentration via sustained release. Other compounds One PCO drug combination of a chelator and tripeptide was based on the observation that ethylene diamine tetraacetate (EDTA) calcium-chelator dissociates epithelial cells from basement membranes in vitro (Nishi, Nishi et al. 1993) by loosening the junctional complexes of LECs. Also, the small ROD tripeptide (arg-gly-aspartic acid) in slow release slightly inhibits PCO in rabbits, putatively via integrin blocking, an inhibition which became significant but not complete when EDTA was included with the RGD (Nishi, Nishi et al. 1997). In the previously-noted man study (man, Ozturk et a!. 2001), the above agents were also tested with 0.1 ml solution of either EDTA or EDTA with ROD as the hydrodissecting fluid in rabbits, both resulting in a significant anti-PCO but non-synergistic effect at three months. An unrelated compound, caffeic acid phenylethyl ester (CAPE) significantly reduced PCO in pigmented rabbits undergoing phacoemulsification surgery with CAPE in the anterior chamber irrigating solution and 3 weeks of post-surgical subeonjunctival injections (Hepsen, Bayramlar et al. 1997). The authors hypothesized that since CAPE selectively inhibits growth of oncogene-transformed fibroblast carcinoma cell lines but not normal 35 cells, the drug would effect LECs as they begin any post-surgical transdifferentiation during fibrous pseudometaplasia. Lidocaine anaesthetic is used during eye surgeries in topical and intracameral administration (Pablo, Ferreras et al. 2004). Preservative-free lidocaine 1% has been applied to rabbit LECs still attached to an anterior capsule fragment or used during cortical cleaving hydrodissection (Vargas, Escobar-Gomez et al. 2003). In the first protocol, enucleated rabbit eyes stripped of cornea and iris permitted a 5.0mm partial capsulectomy yielding a hinged anterior capsule flap which was everted and irrigated with for 1,3 or 5 minutes (n=3 eyes per assigned time). The lidocaine-treated capsules had a range of approximately 18% to 28% mean number of dead cells per square mm compared with an alcohol-treated control capsule, measured by trypan blue dye exclusion. In the second protocol, a complete anterior capsulectomy via CCC was followed by cortical cleaving hydrodissection with 2.0 ml of the same lidocaine preparation, left in situ for the same time periods (n=3 eyes per assigned time). Post-phacoemulsification capsule histology revealed loosening of the cell-capsule attachment in all 3 groups, with nearly-complete dissociation after 3 and 5 minutes, prompting the authors to suggest that this approach might enhance cortical cleanup and potentially decrease PCO via either a direct toxic effect or the additional loosening of the LEC desmosomal intercellular adhesion area. Despite different applications of many agents, none of methotrexate, mitomycin C, daunomycin, 5-fluorouracil or coichicine is routinely used clinically (Inan, Ozturk et al. 2001) and the possible side effects of many other agents including MDX-RA and saporin on intraocular tissues are the major problem in their clinical use (Spalton 1999; man, Ozturket al. 2001). Photodynamic therapy One photosensitizer normally reported in antitumor photodynamic therapy (PDT), Photofrin II, is also reported in the early secondary cataract literature (Lingua, Parel et al. 36 1988; Pare!, Cubeddu et a!. 1990; Lingua, Pare! et al. 1991). Similarly, bacteriochiorin a (BCA), a purple bacterial chlorophyll derivative has also been researched for possible anti PCO potential. In one experiment, when a 10 ,uglml solution of BCA was left for 10 minutes in post-ECCE human donor capsular bags then removed, followed by light exposure of the bag, induced LEC apoptosis and suppression of proliferation was observed 7 days later (van Tenten, Schuitmaker et al. 2001). In subsequent rabbit surgeries by the same group, 1.5 ml of BCA at 10 or 50 pg/mi in a mixture of 30% polyethylene glycol (PEG), with 20% and 50% ethanol was injected into the empty capsule and allowed 10 minutes contact before light irradiation. Examinations at 6 weeks showed variously diminished LEC proliferation and incomplete, thinner Soemmering’s rings. However, corneas of all test rabbits were opaque and swollen and had lost the endothelium (van Tenten, Schuitmaker et al. 2002). PDT with Photofrin II (P11), a mixture of dihematoporphyrin ethers (DHE) made from hematoporphyrin derivative (HPD) has been reported in model rabbit ECCE surgeries (Lingua, Pare! et a!. 1988). Phacoemulsification lensectomy through an extremely small, approximately 1-mm diameter anterior capsulotomy was followed by instillation of 0.75 ml of PIT in saline at 100 pg/rn! into the empty capsule and left for 10 minutes then washed out for 1 minute with BSS. Irradiation in 4 capsule quadrants plus the central anterior capsule via a beveled fibreoptic provided 56 joules (J) of 510 urn light over a period of 10 minutes. At 5 weeks all 4 treated eyes had opacification of the visual axes as well as traction of the capsulotomy. Histology showed healthy fibre and LEC populations plus cell proliferation around the capsulotomy. In one additional eye a combination of 1 part Healon sodium hyaluronate viscoelastic mixed with 1 part P11 stock to give 1.25 mg/ml was introduced through a 1- mm capsulotorny (Lingua, Pare! et a!. 1988). After 20 minutes then BSS flushing for 1 minute, the same five-site fibreoptic light treatment (above) provided approximately 120 J during 20 minutes. At five weeks, clinical opacification of the capsule was not significant, 37 however anterior capsule tears had allowed P11 access into the AC with concomitant endothelial toxicity and corneal edema. Histology showed proliferating fibre cells and LECs in the capsular equator, pericapsular inflammation with ciliary body toxicity and anterior vitritis (Lingua, Pare! et al. 1988). The potential for preferential LEC uptake of P11 was explored by the same research group by determining the ocular distribution of P11 in rabbits. The ECCE protocol described above was employed and 1.0 ml of P11 in saline at 20 or 100 pg/mi was actively rinsed within the empty capsular bag for 3 minutes. The majority of the solution escaped into the anterior chamber. Rabbits were enucleateci at 15 minutes and at various times to 30 hours. Microspectrofluorometric measurements of fluorescence decay curves in various sections of the eyes showed near-equal P11 affinity for LECs, comeal endothelium, iris and ciliary body, with no significant uptake in the retina. Results showed that without PIT intracapsular confinement, there was no preferential LEC accumulation. However, the ciliary body exhibited slightly higher P11 levels (Parel, Cubeddu et al. 1990). 1.6 Implications and directions for the current thesis It has been suggested (Nishi 1999) that the prevention of PCO is more difficult than the treatment of cancer in that any side effects from the inhibition of LEC proliferation are unacceptable. The current thesis reports on the attempt to specifically kill or inhibit LECs based on the idea of localizing the photosensitizer within the post-surgical lens capsule, based on mixing BPD-MA with a gel-like substance (Karamata, Sickenberg et a!. 2000). The BPD MA tested in ECCE lensectomy surgeries in euthanized rabbits, and in relevant preliminary in vitro assays, was applied in biocompatible, ophthalmic surgical-grade viscoelastics, the cohesive gel-like nature of which provided containment and putatively site-specific photosensitizer contact with lens epithelial cells. In addition, the hypothesized photoactivation in only remnant capsular LEA-cells and E-cells is intended by the ring-like 38 illumination provided by the novel laser light dispersion tip (Karamata, Sickenberg et al. 2000) (Surgery Chap. 4, Fig. 4.23, p.1 19) used in the rabbit surgeries. This clinical prototype was designed to distribute a torus-shaped i.e. equatorial red light in order to activate the photosensitizer proposed to be taken up by the A- and E-cells from the visco/BPD-MA mixture laid into the post-lensectomy capsule then removed before irradiation. The back of the spherical tip was silvered in order to minimize exposure of the retina and posterior segment of the eye in the proposed clinical setting. It is important to note that lensectomy surgery-based procedures as a model of human ECCE cataract surgery were carried out for this thesis on freshly-euthanized rabbits for two reasons. First, this was not intended to be a chronic, in vivo animal safety study, which would have required a surgical team for each procedure. Secondly, this study was, in part, to find out whether or not there was proof of principle for the use of light-activated BPD-MA applied to and localized in the emptied “post-surgical” capsule to kill any residual cells. In order to be able to detect even a single surviving intracapsular cell, it was necessary to remove the treated capsule immediately after lensectomy “surgery” for follow- up in incubated cell culture during the course of which living and dead cells in the capsule could be stained and examined microscopically to single-cell resolution. Note: Hereafter, the euthanized-rabbit lensectomy surgical explant model (i.e. follow-up of emptied, treated lens capsules removed to culture) is referred to as the “rabbit surgical model” or “model surgery” and the capsules as “post-surgical capsules” or “pinned capsules”. (See description of in vitro pinning technique in Chap. 4.) 1.7 Primary questions and specific aims Five aims guided the testing of the hypothesis that PDT could effect either the physical ablation, cytocide or permanent compromise of residual LECs and potentially lens fibre cells at the time of primary cataract surgery. 39 I. Initially, the non-irradiated (dark) and light-irradiated effects of benzoporphyrin derivative monoacid ring A (BPD-MA) were tested on human and rabbit lens epithelial cells in vitro to determine photosensitizer and light doses needed to kill residual cells. II. An additional aim was to employ a reproducible, in vitro fluorescence photomicrography assessment technique capable of discriminating single cells in order to monitor epithelial cell growth onto posterior lens capsules after lens removal surgery. III. As well, experiments were performed to determine the bioavailability of BPD MA to lens epithelial cells, and the nature of the photosensitizer interaction with protected and unprotected cornea! endothelium. IV. Based on the successful cell killing observed in the in vitro experiments, drug containment and site-specific laser excitation light delivery strategies were explored in a rabbit surgical model. The short and mid-term effects of PDT in post-surgical capsules cultured in vitro were assessed with a fluorescence assay of the de novo proliferation and migration of cells onto the posterior capsule. V. The final aim was to determine photosensitizer dose, contact time within the empty capsule, and the light dose needed to completely kill photosensitized remnant lens cells after phacoemulsification lens removal, in part, as a model for preventing secondary cataract in humans. 40 Chapter 2: Benzoporphyrin Derivative and Viscoelastics 2.1 Photodynamic therapy Crucial to considerations of a clinical photosensitizer (PS) candidate for photodynamic therapy (PDT) are a low degree of toxicity of the non-irradiated drug (potential “dark effect”), the rate and degree of uptake, any differential uptake by target tissue(s), degree and specificity of light activation, efficiency of generating photocytotoxic products e.g. singlet oxygen (‘02 ) or of direct photoactivated interactions with target cell components. There should also be a safe clearance in the case of systemic application, and a minimum of post-PDT non-specific photosensitivities. 2.1.1 Benzoporphyrin derivative Benzoporphyrin derivative preferred uptake in studies of rapidly-dividing non-adherent cells such as clonogenic chronic myeloid leukemia (CML) cells (Keating, Jamieson et al. 1993; Levy, Dowding et al. 1995), reflect part of the basis of BPD-MA use in both oncologic and non-oncologic clinical applications, differential uptake by various cell types also being a key factor in the application of photosensitizers in solid tumors (Moan, Peng et al. 1987). When first reported, BPD-MA showed 10 times the cytotoxicity when photoactivated in adherent human cell lines compared with hematoporphyrin (HP) and was 10-70 times more cytotoxic to nonadherent cells (Richter, Kelly et al. 1987). The potency of this monoacid form of BPD (Richter, Yip et al. 1996), made it of interest regarding potential prophylaxis of LE cell proliferation. In general, the rapid apoptosis observed in photodynamic therapies reflects cytosolic cytochrome-c release following light-activated photosensitizer damage to the mitochondria of the target cells (Kessel and Castelli 2001). This early release has been observed in the case of BPD-MA (Carthy, Granville et al. 1999) and involves multiple cytochrome-c pathways in various PDT systems (Vantieghem, Xu et al. 2001). 41 Other considerations for the choice of BPD-MA for the research reported in this thesis included PS availability to the lens epithelial cells, and the nature of photoexcitation. BPD-MA has a porphyrin tetrapyrrole ring structure (Fig. 2.1) and has a strong, steep- shouldered well-defined absorption peak at 690 rim (red) light (Fig. 2.2) (Richter, Chowdhary et al. 1993) coincides with commercially available red-light LED panels and diode lasers. In comparison, cytotoxicity studies with an Ml mouse tumor cell line showed that the di-acid BPD derivatives exhibited approximately one fifth the photodynamic cytotoxic effect of BPD-mono-acid (MA) (Richter, Waterfield et al. 1990). Given the poor solubility of native BPD-MA in aqueous solutions (Richter, Waterfield et al. 1990), and its considerable lipophilicity, BPD-MA has been formulated in liposomes for systemic injection (Richter, Waterfield et al. 1993). Liposomes are roughly spherical vesicles, either unilamellar or multilamellar, made in early research from various mixtures of phospholipids, cholesterol, phosphatidyl-choline, -serine, -glycerol andlor palmitic acid, commonly of 200-500 nm in diameter (Szoka and Papahadjopoulos 1978). The experimental results reported in this thesis served to show whether or not BPD-MA in liposomes (QLT, Vancouver, BC) is still available to cells when mixed with ocular viscoelastics in a way to minimally change the original cohesive and elastic properties of the viscoelastic. The euthanized-rabbit lensectomy surgical explant model was designed to determine whether the mixture would provide an easily-manipulated, easily-localized source of photosensitizer in post-surgical lens capsules, allowing PS contact with and uptake by the monolayer of exposed lens epithelial (LE) cells (Chap. 4). Continued development of the rabbit surgical model depended on how rapidly LE cells would take up sufficient photosensitizer during the model cataract surgeries. 42 OMe ‘JUl UM OH Figure 2.1 Structure of benzoporphyrin derivative (BPD) (1) monoacid, ring A analog, (2) monoacid, ring B analog. R = CO2Me. Diacid analogs differ from monoacid analogs only in that they have the ester group replaced with the acid group, therefore, they have two acid groups at C and D rings of the porphyrin. Richter, A. M., E. Waterfield, et a!. (1990). “In vitro Evaluation of Phototoxic Properties of Four Structurally Related Benzoporphyrin Derivatives. Photochemistry and Photobiology 52(3):495-500. (Reprinted with permission from Blackwell Publishing, Oxford, UK) 2.1.2 PDT history Recorded research into photosensitivity appears to have begun in the 19th Century. By 1900, Raab showed that paramecia were killed when exposed to acridine dyes and light (Raab 1900). In 1903, Tappeiner referred to “photodynainic” inactivation to describe observations that eosin in aerobic solutions inactivated enzymes, e.g. invertase and papain, when light-irradiated (von Tappeiner 1903). Other dyes have been investigated: rose bengal, crystal violet, acridine orange, proflavin, methylene blue, and anthraquinonoid dyes as photosensitizers (Garston 1980) metallic salts: zinc oxide, cadmium suffide, zinc sulfide (Spikes 1981); plus transition metal complexes e.g. of osmium and iridium (Demas, Harris et al. 1975). Even relatively recently, reports of PDT mechanisms of membrane changes due to light-irradiated methylene blue with paramecia have been published (Saitow and Nakaoka 1996), a harkening to the influential era of 19th Century German dye research. Light- activated dimethylmethylene blue has also been used in red blood cells (RBC) to elucidate PDT mechanisms which change potassium ion concentration across the membranes of 43 increased permeability and the correlations with damaged RBC band 3 membrane proteins (Trannoy, Brand et al. 2002). The notion that certain physiologic biochemicals or their precursors may act as potential photosensitizers (Straight and Spikes 1985) seemed to have prompted attempts to induce greater fluorescence of endogenous fluorophores to create a clearer outline of tumors (MacDonald and Dougherty 2001). Alternately, other researchers sought to increase the differential tumor fluorescence by supplying exogenous porphyrins to the tumors in humans and mice, reports of which were published mid 20th century (Rasmussen-Taxdal, Ward et al. 1955). It was not until the latter half of the 20th century that three pioneers in the PDT field made significant impacts: R. Lipson (initial tumor localization experiments), S. Schwartz (designed the first clinically useful porphyrin photosensitizer mixture, hematoporphyrin derivative, HpD), and T. Dougherty (clinical development) (Kessel 1998). However, Lipson, finding nonreproducible results when using the parent compound hematoporphyrin as a tumor detector, turned to a hematophorphyrin derivative (HpD) and reported its potential use as a tumor-destroying photosensitizer (Lipson, Baldes et al. 1961). Studies by Weishaupt et at (Weishaupt, Gomer et al. 1976) were turning points in photosensitizer (PS) research wherein singlet oxygen (‘02 ) was identified as the toxic end product of photoactivation in an aerobic environment. Work by the Dougherty group (Dougherty 1974) reinforced the observations that although a fairly simple PS such as fluorescein diacetate is capable of producing 102 , the yield may be low and the penetration of non-red light through chromophore-rich (i.e. hemoglobin) tissues and tumors is poor and prone to tissue scattering (Pandey 2000). This physical limitation implied apparent shortcomings for PDT as a useful clinical and oncological treatment instead of, or as an adjunct to surgical interventions. However, the investigations of the Schwartz synthetic HpD mixture (Dougherty 1996) was an important shift to studying synthetic porphyrins, and other related planar aromatics (Fig. 2.3) and modified derivatives permitting wider ad 44 Cmore systematic research into PS species with more efficient ‘02 yields. Such approaches also aimed at low toxicity of the unexcited ground-state PS (“dark effect”) and more rapid and selective uptake by target tissues. Dougherty etal also isolated the reactive species of photosensitizers in HpD, resulting in Photofrin (Dougherty 1996), and although still a mixture, was the first PDT agent widely used in cancer patients. 4 500 700 900 Wavelength (nm) Figure 2.2 Absorption spectrum of PHOTOFRIN®(---), oxyhemoglobin (- -) and BPD (_). Richter, A. M., R. Chowdhary et al. (1994). “Non-oncologic potentials for photodynamic therapy.” SPIE 2078: 293-304 (Repnnted with permission from SPIE, Bellingham, WA.) The problem of lesser tissue penetration of shorter wavelength light remained. Although more defined than its parent HpD, the mixture of porphyrins in commercial Photofrin (Kessel 1998), as well as the non-aromatic 5-aminolevulinc acid (ALA) share an absorption maximum at 630 nm and have tissue penetration rages of 0.4-3.9 mm (0.4 mm measured in Hb-rich liver tissue), whereas photosensitizers activated with maxima in the 635-700 nm range correlate with nearly double the penetration depth (Pandey 2000). 45 H3CO2 H3CO2 R = CH3, R1 = H and R = H, R1= CR3 (Mixture) Benzoporphin derivative (BPD, 690 nm) (QLT Phototherapeulics, Vancouver, Canada) C020H3 H3C OH3 H3C _1J.\ H5C2 —ICH3 CH3 C2H5 Tin Etiopurpurin (SnEt2 ,650 nm) (PDT Inc. Santa Barbara, USA) R and R1 = Aspartic add (NPe6,665 nm) (Nippon Lab. Japan) m-TetrahydroxyphenylchlQnn (m-THPC, 650 nm) (Scotia Pharmaceuticals, Guildford, U. K.) (660 nm)000H Hexyl ether derivative of Pyropheophorbide a (HPPH or Photoclor, 660 nm) (Roswell Park Cancer Inst., Buffalo, USA) Texaphyrth (730 nm) R = CH2O R1 = O(CH20H Pharmacyctics, California R = n-Hexyl and R = n-Hept1 urpurin-imides, 700 rim) (Roswell Park Cancer Inst., Buffo, USA) Figure 2.3 Photosensitizers (>650 nm) under clinical and preclinical trials. Pandey, R.K. (2000). “Recent advances in photodynaniic therapy.” Journal of Porphynns and Phthalocyanines 4:368-373. (Copyright © 2000 Society of Porphyrins and Phthalocyanines. Reprinted with permission from SPP, Dijon-Cedex, France.) C02R1 ‘xx:: Phthalocyanines (700 rim) (R various groups) -CH2C3 46 As reported in 2000 (Pandey 2000) the majority of photosensitizers being used or investigated at that time were planar aromatics (Fig. 2.3). The potential for many modifications as second- and third-generation derivatives (Sternberg and Dolphin 1993; Boyle and Dolphin 1996) is due first to the variety of residual groups and the relatively plentiful and varied outer excitable pi electrons in these systems (Fig. 2.4). This ensures a more varied potential for electron shifts from normal ground states through photoexcited states and subsequent energy transfer to form reactive oxygen species (Fig. 2.5). The result is a vastly expanded list of PS candidates since the acridine results published by Raab in 1900. NHN NHN NHN NN Porphyrin Chloiixi 22 2t electrons 20 it electrons 18 it electrons Figure 2.4 The basic chemical structures of porphyrins, chiorins and bacteriochiorins. MacDonald, I.J. and T,J. Dougherty (2001). “Basic principles of photodynamic therapy.” Journal of Porphyrins and Phthalocyanines 5: 105-129. (Copyright © 2001 Society of Porphyrins and Phthalocyanines. Reprinted with permission from SPP, Dijon Cedex, France.) Figure 2.5 (Permission was not forthcoming for reproduction of the diagram in Kessel, 1998, showing the absorption of photonic energy by photosensitizers. This diagram shows that a ground-state photosensitizer is raised in its energy level if exposed to a certain wavelength of light. The drawing also shows that at this level of excitation, called a stable excited state, the molecule can either give off photon(s) during a fluorescence event, dictating a return to ground state, or the spin of the excited electron can change via an intersystem crossing giving an intermediate triplet state. This apparently unstable photosensitizer can now transfer its absorbed energy to molecules of oxygen which, in this new, singlet oxygen state, will oxidize any molecule in a biological environment which can be oxidized.) Baterioch1orin 47 2.1.3 Photosensitization mechanisms Porphyrins and their second-generation (Foote 1991) non-metallic chiorin derivatives such as BPD-MA (Richter, Kelly et al. 1987; Richter, Cerruti-Sola et al. 1990; Kessel 1998) make reactive singlet oxygen (‘02 ) efficiently under the right conditions, in part accounting for their initial clinical use in tumor treatments (Stables and Ash 1995). Photodynamic chemical species reactions fall into two general classes, Types I and IL In type I reactions, a photon-excited PS may react, in an anoxic condition, directly with organic substrates or in hypoxic environments, with oxygen to produce superoxide anions or the very reactive hydroxyl radical (Foote 1991). The type II pathways, reported to dominate most PDT reactions, commonly result in 102 production (Foote 1991; Henderson and Dougherty 1992) in a normotensive oxygen environment. The photoelectronic events during photoactivation (and potential photobleaching) of photosensitizers begins with the absorption by an electron of a photon of the appropriate excitation wavelength. This energized electron raises the PS to a stable excited state, from which two outcomes are possible. The PS may fluoresce (Fig. 2.5) and return to ground state, or the spin of the electron can change via an intersystem crossing to a slightly lower energy triplet state. Molecular oxygen in e.g. target tumor tissue is activated by the triplet state energy into ‘02 (Kessel 1998). Singlet oxygen is capable of oxidizing any biochemical molecules prone to oxidation, and is so reactive that in an aqueous environment the lifetime of the’ 02 is 2 js (microseconds) (Foote 1991; Kessel 1998). Because planar aromatics are hydrophobic, the PDT-induced oxidative damage is localized to regions about the same size as the cell membrane thickness (Kessel 1998). 2.1.4 BPD-MA delivery characteristics As BPD-MA is hydrophobic, it is soluble in dimethylsulfoxide (DMSO) (Richter, Jam et al. 1992), but is prepared in lyophilized liposomal formulation for clinical use (Husain, Miller et al. 1996; Kramer, Miller et al. 1996) and reconstituted in sterile water prior to use 48 as was the case for the in vitro and surgical experiments reported in this thesis. Regarding blood plasma interactions, BPD-MA from DMSO stock combines with albumin and low- density lipoprotein (Allison, Waterfield eta!. 1991; Chowdhary, Sharif et a!. 2003) and the distribution of the drug has been studied in many in cancer applications. Where the potential for shutting down the vascular supply to a discrete tumor mass involves the cytotoxic PDT effects on new blood vessels, BPD-MA combines with low-density lipoprotem for delivery to NV tissue and in turn binds with low-density lipoprotein receptors on proliferating endothelium (Ciulla, Danis et al. 1998). In the eye, when BPD MA is pre-complexed with LDL and delivered intravenously to albino rabbits at 2 mglkg, it accumulates in the choroid, RPE, and photoreceptors but is not observed in the lens (Haimovici, Kramer et al. 1997). Given the lack of uptake in the lens in the LDL system, and the proposed localized viscoelastic delivery of the PS to the empty lens capsule in an assumed LDL-poor lens environment, clinically-available liposomal BPD-MA was chosen for this thesis research. 2.2 Ocular viscoelastics and BPD-MA mixtures The notion of mixing BPD-MA with a gel for instillation into the emptied lens capsule, apposed to the monolayer of LE cells (Karamata, Sickenberg et a!. 2000) led to the consideration of viscoelastics currently used in ocular surgery. These are used to protect and maintain the depth of the anterior chamber (AC) when the integrity of the AC is compromised due to incision or accident, and to protect the sensitive corneal endothelium. In the case of the mixture with BPD-MA reported in this thesis, the viscosity factor was to ensure an easily-manipulated, spatially-defined source of photosensitizer in the capsular bag and the cohesiveness to increase the ease with which the visco/BPD-MA mixture might be aspirated after incubation. In one study (van Tenten, Schuitmaker et a!. 2002) (previously noted in Sect. on the effect on light-activated Bacteriochiorin delivered to empty lens capsules in somewhat viscous 30% polyethylene glycol (PEG) with 20% 49 ethanol and 50% water, diminished LE proliferation and Soemmering rings were observed. However, test eyes also had swollen, opaque comeas with damaged or lost endothelium. Three important viscoelastics classes are use in ocular surgery: sodium hyaluronates, hydroxyproplymethylcelluloses and chondroitin sulfates. All are biocompatible, but have different degrees of viscosity, cohesiveness and adhesiveness as well as differing tendencies to produce transient, post-surgical increases in intraocular pressures (McDermott, Hazlett et al. 1998). 2.3 BPD-MA in sodium hyaluronate For this thesis, the decision to use sodium hyaluronate (AMVISC (Bausch & Lomb; Ophthalin (CIBAVision)) in all rabbit surgeries reflects the routine clinical use during primary cataract surgery. (Personal communication, Vancouver General Hospital I Eye Care Centre Surgery and UBC Dept. Ophthalmology). 50 Chapter 3: Ocular Anterior Chamber PDT Conditions: Lethal Photosensitizer and Light Titrations for LE Cells in vitro The preliminary studies reported in this chapter fell into two categories: the availability of liposomal BPD-MA in surgical viscoelastic, and the implications for ocular safety, particularly cornea! endothelium protection. Given that LE cells are a monolayer in the capsule, it was proposed that not only would there be a relatively immediate access to BPD-MA, but also efficient photoactivation given the relative transparency of these cells, Even though there was the potential for a different microenvironment and protein profile on the outer surface of the LE cells in the model surgeries compared with those in culture on plastic or glass, the preliminary experiments with LE cells grown directly from explanted human capsules rather than using transformed or immortalized LE cell lines, were intended to investigate cytotoxic dose ranges to predict those suitable for the rabbit surgical model to follow. Experiments in vitro allowed trials with BPD-MA in non-viscous solution, in diluted and finally in undiluted clinical sodium hyaluronate viscoelastic. 3.1 Objectives A critical objective was to determine whether the corneal endothelium could be adequately protected in a worst-case scenario of direct contact with BPD-MA. The protection of this cell population is vital in any ocular surgery, given that any corneal endothelial cells which are damaged are not regenerated (Doughty and Nguyen 1995; Wang and Hu 1997). A second objective was to determine BPD-MA concentrations, contact time and red (690 nm) light dosimetries necessary to kill LE cells in vitro in non-viscous and semi- viscous drug applications. Thirdly, it was necessary to find out if mixing BPD-MA with full-strength viscoelastics would affect the availability of the drug to LE cells in culture and whether 51 effective activating light doses could be sufficiently low to predict minimal light injury to the adjacent ocular tissues in the clinical setting. 3.2 Materials and methods Generally applicable materials and methods for this chapter are noted in sections 3.2.1- 3.2.3 with additional, detailed information in Appendix One. 3.2.1 Corneal endothelium protection from BPD-MA Corneas were excised from freshly-euthanized New Zealand White rabbits (Staff Veterinarian, Jack Bell Research Centre, Vancouver, BC) and set endothelium side up in a sterile culture dish in Dulbecco’s Modified Eagles Medium (DME) with 5% heat- inactivated fetal calf serum (FCS). Various concentrations of liposomal BPD-MA (QLT, Vancouver, BC) were made in DME, ranging from a potentially clinically-relevant 60 tg/ml to undiluted stock (2.1 mg/mI), a concentration several orders of magnitude stronger than would be encountered clinically. Within 5 minutes, the inverted corneas were coated with a 1-2 mm layer of Ophthalin (10 mg/mI sodium hyaluronate, Ciba Vision) to mimic clinical cornea! endothelium protection. The BPD-MA was gently applied onto the surface of the protective Ophthalin coat, now covering the conical endothelium. Incubations were 1 minute in the case of the ptg/ml range experiments or 10 minutes in the case of the 2.1 mg/mi worst-case scenario. Control corneas received the same amount of photosensitizer free DME onto the Ophthalin layer. All incubations were then tilted and rinsed with DME I 5% FCS to remove the photosensitizer and the Ophthalin, followed immediately by irradiation with 103/cm2LED69O light. Calcein and ethidium homodimer cocktail (Cytoprobe) cytotoxicity fluorescence photomicrography assays were used to determine whether endothelial cells were protected from PDT damage. Compromised cells allow ethidium entry I nuclear (red) fluorescence. Intact cell membranes block ethidium but allow calcein entry and green fluorescence not seen in dead cells. (See Section 3.3 for details.) 52 3.2.2 HLE cells in vitro: photosensitizer and light dosimetries with BPD MA in non-viscous solutions and in diluted ophthalmic viscoelastic In vitro culture of human LE cells (Note: See Appendix Five for general summary of in vitro steps.) Human lens epithelial (HLE) cells were grown from lens capsules harvested from post-mortem (12-48 hours) donor eyes supplied by the Eye Bank of British Columbia. The eyes were received as posterior poles (corneas previously removed) or occasionally as whole eyes. Male and female donors ranged in age from 20 to 72 years. In all cases a 3600 equatorial incision (no more than 0.5 mm anterior to the equator) was made in the lens capsule and the anterior portion lifted away. This explant was placed LE cell-side down onto a 35 mm plastic tissue culture dish containing DME with heat-inactivated 15% FCS, supplemented with penicillin and streptomycin. The capsules were then cut into a series of 1mm-wide parallel strips by using a rocking motion of a rounded scalpel blade. This attached the edges of the capsule strips onto the surface of the dish and increased the amount of explant perimeter exposed to the plastic substrate in an effort to increase the yield of HLE cells. The growth medium on these explants was changed every 2 to 3 days. HLE outgrowth continued until the cell monolayer was nearly confluent then passaged using 0.25% trypsin in EDTA. BPD-MA in UME / 5% FCS and in diluted AMVISC HLE cells, used at either passage 2 or 3, and depending on the number of cells available, were set out in 96-well plates at 17-, 30-, or 42,000 cells / cm2 in DME medium initially with 15% FCS as an enrichment during cell re-stabilization after trypsinization. They were fed fresh medium the following day to remove cell debris and any residual trypsin. The cells were then allowed additional days for re-establishment, that is, to increase the likelihood that no residual trypsin-induced effects on the cell membranes remained before exposure to BPD-MA or use in the appropriate controls. (The difference between the PDT 53 cytotoxic effects on cells allowed 1 day of post-trypsin recovery versus those permitted 2 days was also tested.) Growth medium was then removed and replaced, under subdued light conditions, with BPD-MA at various concentrations (0.010-90.00 pg/mi in DME /5 (five) % FCS or in diluted viscous sodium hyaluronate solution) for 1 or 10-minute incubations at 37°C /5% CO2. Following incubation, the BPD-MA was removed and replaced with BPD-MA-free DME /5% FCS. For PDT test conditions, the cells were then exposed to 10 J/cm2690 nm red LED light. The DME was immediately replaced with DME /5% FCS and the cells returned to 37°C and 5% CO2. For ‘dark’ controls, the growth medium was also changed, under the lowest possible light conditions, to BPD-MA free DME /5% FCS. The LED exposure step was substituted with an equal time at room temperature under the same low-light conditions. After the equivalent time, the BPD-MA free DME /5% FCS on the ‘dark’ control cells was replaced with fresh DME /5% FCS to mimic the PDT test protocol. Both sets of cells were then returned to 37°C and 5% CO2. To explore the availability to cells of BPD-MA in vehicles other that DME /5% FCS, liposomal BPD-MA was mixed with AMVISC clinical viscoelastic. This cohesive and viscous material (12 mg/mi sodium hyaluronate) was first diluted 25:75 (AMVISC: BSS) to facilitate mixing with BPD-MA (0.01 - 0.80 pg/mi) for ease of handling on cells in 96-well plates. The degree of PDT-induced cytotoxicity was measured at either 1 or 20 days via the Ml]’ cytotoxicity assay (Mossman 1983) with the basic premise that cell compromise would occur within 24 hours after BPD-MA-mediated PDT. This also allowed comparisons of cytotoxic effects between short-term and the relatively longer-term i.e. 20 day recovery period, as an in vitro model for any possible recovery of HLE cells in the potential clinical application wherein patients have longer-term post-surgical potential for new proliferation of LE cells. 54 3.2.3 BPD- MA availability from undiluted viscoelastic mixtures To determine the sensitivity of HLE to light-activated BPD-MA mixed with undiluted viscoelastics used during ophthalmic surgery, liposomal BPD-MA 2.1 mg/nfl stock was mixed in undiluted AMVISC, to first give 60 pg/mi BPD-MA. (Using concentrated stock permitted adding only 2.8% v/v to the AMVISC in an attempt to minimize any changes to the cohesiveness.) From the 60 pg/mi mixture, a series of 6-60 pg/mI BPD-MA in AMVISC mixtures was made. HLE (second or third passage) were set out in DMEM / 15% FCS on acid-etched coverslips, some of which had been treated with ProNectin-F delivered as 3 pg/cm2. Most cells were used after an additional 2-3 days or, for comparison, after an additional 2-6 weeks. The cell-covered coverslips were broken into approximately equal wedge-shaped fragments. They were rinsed in clinical BSS at room temperature. The various concentrations of BPD-MA in undiluted AMVISC were layered onto the cells with the same 27G surgical Rycroft cannula used clinically to administer viscoelastics into the eye, allowed precisely 1.0 or 2.0 minutes contact at room temperature then exposed immediately to LED69O light in dose ranges of 0.13 - 2.0 J/cm2.(Note: when the size and number of the broken coverslip fragments allowed, a small area of the cells on the fragment designated for the highest BPD-MA concentration e.g. 60 pg/nil, would be left uncovered as a contact-free control.) The BPD-MA / AMVISC was left on the cells during LED exposure, a condition unique to this set of experiments due to the risk of flushing HLE off the coverslips with a stream of BSS of sufficient force to remove the cohesive mixture. Under very low ambient light conditions, the treated cells on the coverslip fragments were fed an excess of DME / 5% FCS and placed on a slow rocker platform in 37°C /5% CO2 (dark) for an overnight removal of the BPD-MA / AMVISC mixture. The cells were then rinsed in serum-free DME medium and incubated with Cytoprobe for 1 h in 37°C /5% CO9. Excess Cytoprobe was rinsed off in BSS, the cells mounted in DABCO (Appendix One) and examined by fluorescence microscopy and photographed. 55 3.3 Results In the Cytoprobe dual-cocktail cell assay kit, red fluorescence indicates that the outer cell membrane has been compromised and a homodimer form of ethidium, unable to permeate an intact plasma membrane, then enters the nucleus, intercalates and fluoresces red under the microscope FITC-type filter block. Green fluorescence indicates that cells are uncompromised, based on the fact that the poorly-fluorescent calcein protofluorophore has entered through and been contained by an intact cell plasma membrane, cleaved by intracellular catalases, the product of which fluoresce brightly green. 3.3.1 Corneal endothelium protection from BPD-MA PDT In all 5 trials (3 representative results shown) where BPD-MA (in non-viscous solution) was applied to Ophthalin-protected corneal endothelium (CE) in situ on fresh rabbit corneas, the protection was complete, regardless of BPD-MA concentration or incubation time (1 or 10 minutes). The unprotected control cornea (60 ug/ml BPD-MA for a 1-minute incubation followed by 10 i/cm2 red light) showed complete endothelium kill (Fig. 3.1, Upper). (Given that the goal was to test CE protection from an artificially-high concentration e.g. 2.1 mg/nil BPD-MA as a worst-case scenario (below), a dose-response curve was not determined.) In the Ophthalin-only control cornea, an additional internal control was made by touching ultrafine-tip forceps to one spot in order to compromise a few cells before the Ophthalin was applied. This cornea was also subsequently exposed to 10 J/cm2LED69O light. Only the small cluster of cells damaged by the forceps appear dead (Fig. 3.1, Middle). The remainder of the endothelium was not by the handling, Ophthalin coating, rinsing or irradiation. Finally, in the most aggressive test cornea where the extreme high-end concentration of 2.1 mg/nil BPD-MA was incubated on an Ophthalin coated cornea for 10 minutes, and irradiated with 10 J/cm2, there were only 3 serendipitous cells which also appeared red i.e. killed (Fig. 3.1, Lower), the vast majority of the endothelium still viable (green). 56 Figure 3.1 Protection of rabbit cornea! endothelium against BPD-MA toxicity by viscoelastic: Upper: unprotected cornea (60 ug/ml BPD-MA for 1 mm. followed by 10 J/cm2 LED69O light) with 100% kill (ethidium red nuclei) in the absence of Ophthalin coating; Middle: This Ophthalin-only control cornea (no BPD-MA challenge), was also exposed to 10 J/cm2LED69O light. The only dead cells (red nuclei) are those physically compromised with forceps, Ophthalin and light having no toxic effect on the viable cells (calcein green). Lower Ophthalin-coated test endothelium (extreme concentration of 2.1 mg/rn! BP]D-MA for 10 mm., and irradiated as above) shows, with the exception of 3 (red) cells, complete protection from this worst-case adverse collateral PDT damage. 57 3.3.2 IILE cells in vitro: photosensitizer and light dosimetries with BPD MA in non-viscous solutions and in diluted ophthalmic viscoelastic Dark controls: BPD-MA toxicity without light activation In order to determine if cultured HLE cells were killed by liposomal BPD-MA not activated by light (‘dark’ control), the photosensitizer was first applied in a non-viscoelastic DME / 5% FCS solution. Two different HLE cultures (HLE-2: 54 y female, HLE-8: SOy male) in 96-well plates were incubated for 10 minutes with BPD-MA (a maximum of 80 ug/ml with HLE-2 and of 800 ug/ml with HLE-8) in DME with 5% FCS under extremely low- light i.e. ‘dark’ conditions. As determined by MTT assay, there was no significant cell kill. This result was consistent for treated cells whether allowed 1,4 or 5 days of recovery time after contact with the non-illuminated photosensitizer (Fig. 3.2). Standard Deviation error bars in all graphs in Chap. 3 are based on 96-well optical density (OD) readings (Molecular Devices spectrophotometer) for each PBD-MA concentration with n=3 wells of cells tested per concentration represented in each plot point unless otherwise noted. These SD values (n=3 wells) are based on the calculated % cell kill values and are shown as SD value above and’below each plot point, calculated and plotted using Microsoft Excel. 58 A B 160• HLE-2 (1 Day Recovery) 160 HLE-2 (4 Days Recovery) 140 140 .120 120 100 —i.- __ —f-- 100 U) 80 80 c3 60 60 0 C) 40 40 20 20 0 — 0— — 0 5 10 20 40 80 0 5 10 20 40 80 BPD (ug/mi) BPD (ug/mi) C D 160 - HLE-8 (1 Day Recovery) 160 — HLE-8 (5 Days Recovery) 140 I 140 120 120 100 100 ÷ ÷ ÷ 80 80 c60 60 40 ° 40 20 20 0 0 0 50 100 200 400 800 0 50 100 200 400 800 BPD (ug/mi) BPD (ug/mi) Figure 3.2 Cell survival without photoactivation of human lens epithelial cells in vitro following 10-mm. incubation with liposoma] B PD-MA at 0 - 80 jg/ml (A and B) and at 0 - 800 jsg/ml (C and D) in the presence of 5% FCS. Recovery refers to the number of days after BPD-MA exposure the cells were allowed to remain in culture before MTT assay. HLE-2 plated out at 17,000 cells/cm2 ; HLE-8 plated out at 30,000 cells/cm2. SD error bars are based on n=3 wells of cells I optical density readings per BPD concentration. (See Sect. for statistical treatment.) In chart A, there is no error bar for the 10 pg/ml concentration as there was a concurrence of the optical density readings for the triplicate wells. In the experiment reported in chart B, there were only sufficient cells for a single well of cells per drug concentration. Therefore no SD calculation was possible. 59 BPD-MA in DME I 5% FCS and in diluted AMVISC Ten-minute incubations with BPD-MA To determine the baseline sensitivity of HLE to light-activated BPD-MA in non-viscous and semi-viscous carriers, HLE were PDT treated in second and third passage cell culture. Two different cultures (HLE-6: 46y male; HLE-8: 50 y male) were incubated for 10 minutes with BPD-MA in DME /5% FCS and exposed to 10 J/cm2 LED, 690 nm light. As shown in Fig. 3.3, under the above conditions, total cell kill i.e. lethal dose 100% (LD100) was effected in the HLE-8 trials with a BPD-MA concentration of 0.80 jtg/ml (800 nanograms/mi) as determined by MTT assay after a 24-h post-treatment recovery period. A B 100 100 80 80 > 60- 60 1) 40 0400 20- 20 0- I I I 0 0.00 0.01 0.05 0.10 0.20 0.40 0.00 0.05 0.10 0.20 0.40 0.80 BPD (ugIml) BPD (ug/mi) Figure 3.3 Photosensitization with 10 J/cm2 LED dose in FILE in vitro following 10-mm. incubation with liposomal BPD-MA at 0 - 0.40 pg/mi (A) and at 0 - 0.80 pg/mi (B) in the presence of 5% FCS. HLE-6 plated at 30,000 cells/cm2;HLE-8 at 30,000 cells/cm2. Data points are based on n=3 wells of cells. (See Sect. for Standard Deviation error bar statistical treatment.) When BPD-MA was delivered in the diluted AMVISC carrier (diluted to 25% i.e. 1:3 with BSS) (Fig.s 3.4 and 3.5) the LD100 was observed with the -O.8 ug/m1 BPD-MA dose even after 20 days post-PDT recovery time (Fig. 3.5/ B), indicating very little difference in the efficacy of light-activated BPD-MA to kill HLE cells in vitro and that in dilution (25%), sodium hyaluronate neither increased nor significantly decreased the PDT cytotoxic effect. HLE-6 I -- I I 60 100 HLE-9 80 > Z3 CI) a)0 40 20 0 0.00 0.05 0.10 0.02 0.40 0.80 BPD (ug/mi) Figure 3.4 Comparison of cell photosensitization with 10 J/cm2LED dose in HLE in vitro following 10-mm. incubation with liposomal BPD-MA at 0 - 0.80 J1g/ml in either DME / 5% FCS (-•-) or in 25% AMVISC sodium hyaluronate (-0-). HLE-9 plated out at 30,000 cells/cm2. Data points are based on n=3 wells of cells. (See Sect. for Standard Deviation error bars statistical treatment.) To test the potential for long-term recovery of cells treated with BPD-MA and light, an additional HLE cell culture (HLE-10: 58y female) was treated in duplicate with drug in either 5%FCS or 25%AMVISC. One set of cells was assayed with MIT 1 day following PDT treatment while the duplicate cells were allowed 20 days for potential recovery and were fed fresh medium as in the pretreatment schedule (Fig. 3.5). —.—[in 5% FCS] —O--[25% AMVISC] 61 A B 100 100 80 80 > > .5 60 60 Cl) - 040 04Q 20 20 0 0 0.00 0.05 0.10 0.20 0.40 0.80 0.00 0.05 0.10 0.20 0.40 0.80 BPD (ug/mi) BPD (ug/m) Figure 3.5 Comparison of photosensitization with 10 JIcm2 LED dose in HLE cells in vitro following 10-mm. incubation with liposomal BPD-MA at 0 - 0.80 jg/ml in either DME I 5% FCS (-•-) or in 25% AMVISC sodium hyaluronate (-0-) assayed at 1 day post treatment (A) and at 20 days post treatment (B). HLE- 10 plated out at 30,000 cells/cm2. Data points are based on n=3 wells of cells. (See Sect. for Standard Deviation error bars statistical treatment.) One-minute incubations with BPD-MA Further trials with cells in culture (HLE-20; 34y female) were carried out to investigate the dosimetry of BPD-MA with much shorter term drug contact, still using 10 J/cm2 to ensure that the photosensitization was not limited by light-dose (Fig. 3.6). MTT at 1 Day 10: MTT at 20 Days —.—[in 5% FCS] —O—[25% AMVISC] —.—[in 5% FCS] —O.—[25% AMVISC] 62 HLE-20 100 80 Cu > > 60 40 20 0 0.0 1.1 3.3 10.0 30.0 90.0 BPD (ug/mi) Figure 3.6 Comparison of photosensitization with 10 J/cm2LED dose in HLE cells in vitro following 1-mm. incubation with liposomal BPD-MA at 0 - 90.0 J4g/ml in DME / 5% FCS assayed at 1 day post treatment. Cells in plot (i) were exposed to light while in BPD-free DME with 5% FCS while those in plots (ii) and (iii) were exposed while in drug-free and serum-free DME. HLE-20 plated out at 42,000 cells/cm2. Data points are based on n=3 wells of cells. (See Sect. for Standard Deviation error bars statistical treatment. ) Given that some of the cells (above) were treated with BPD-MA and light at 1 day after trypsinization and the remainder after 2 days, the results in Fig. 3.6 also show that no significant effects of trypsinization within the detection limits of the MTT assay on the photosensitivity of cells in vitro were observed. Narrowing the dose range of BPD-MA To explore the BPD-MA short-term dosimetry in the range of 0-30.0 pig/mI BPD-MA, a new culture of cells (HLE- 19: 48y female) was PDT-treated at either 1 or 2 days post trypsinization to determine the LD100 with PD-MA incubations of only 1 minute and ilTadiation of 1OJ/cm2with LED 690 nm light. LD100 was 3.0-3.3 pig/mi BPD-MA in DME /5% FCS (MTT assay at -24 h post-treatment). As shown in Fig. 3.7 there was little difference in LD100 whether the cells were treated at 1 day or 2 days after being trypsinized —O---1 d post trypsiri (I) ——2d post trypsin (ii) —A—2 d post trypsin (iii) 63 and set out in 96-well plates, again indicating the lack of gross interference in BPD-MA uptake of any potential trypsin-induced alterations of the cell membrane. Note: due to the lack of primary cells in culture, the initial cell density of passaged and plated cells was 30,000 cells/cm2. HLE-19BPD 100 • 80 - —.—1 d post trypsin (I) —•—2c1 post trypsin (n) 60 40 20 _ 0 I 4N4 0.0 0.4 1.1 3.3 10.0 30.0 BPD-MA (ugknl) Figure 3.7 Comparison of photosensitization with 10 J/cm2LED dose in FILE ceilsin vitro following 1-mm. incubation with liposomal BPD-MA at 0 - 30.0 J1g/ml in DME I 5% FCS assayed at 1 day post treatment. Cells in plot (i) were treated at 1 day post trypsinization while those in plot (ii) at 2 days. HLE- 19 plated out at 30,000 cells/cm2. Data points are based on n=3 wells of cells. (See Sect. for Standard Deviation error bars statistical treatment.) 3.3.3 BPD-MA availability from undiluted clinical viscoelastic mixtures BPD-MA in full-strength clinical viscoelastics in 6-60 jig/mi concentrations was applied to HLE for precisely 1.0 or 2.0 minutes before immediate light dosing (0.13 - 2.0 J/cm2). These cells showed a definitive 100% lethal dose (LD100) with 1.0 minute exposure to 60 jig/mi followed by 0.5 J/cm2 (e.g. Fig. 3.8; HLE-19: 48y female; on non-ProNectin-F coverslips). Cells exposed to photosensitizer concentrations <60 jig/mi and light doses <0.5 J/cm2 variously exhibited less than LD100 (photographic data not shown). No gross differences in cytotoxicity were observed between those HLE cultures PDT treated 2-3 64 days post-seeding onto coverslips versus those PDT treated 2-6 weeks post-seeding, coverslip treatment being equal (photographic data not shown). No gross differences in cytotoxicity were observed between those trials using HLE seeded onto ProNectin-F treated acid-etched coverslips and those without ProNectin-F (photographic data not shown). BPD-MA 60 ug/ml in full-strength AMVISC viscoelastic layered onto third passage HLE- 19 (48 y female donor) on non-ProNectin-F coverslips for precisely 1.0 mm. before immediate LED69O light dosing (0.5 J/cm2)showed a definitive lethal dose (LD100). Cells are on a single coverslip fragment. Left region: After an overnight gentle rocker incubation to remove the visco/BPD-MA, cells which had been covered with treatment mixture show red ethidium nuclear staining, indicating cell membranes compromised by PDT with cytoplasmic loss and thus minimal green calcein accumulation. Right region: control cells not coated with visco/BPD-MA exhibit calcein green cytoplasmic fluorescence indicating cell viability. 3.4 Conclusions After a one-day minimum recovery in culture, typsinization of FILE cells in 2 or 3 passages from primary culture has little if any gross effect on the susceptibility to killing by light- activated BPD-MA within the photosensitizer and irradiation doses tested. Figure 3.8 65 BPD-MA of concentrations up to and including 8001ug/ml in DME /5% FCS incubated with HLE in vitro for as long as 10 minutes show no significant toxic effect without light activation when assayed up to 5 days post treatment. Cultured NLE cells compromised by light-activated BPD-MA show no significant recovery after 20 days when compared with those assayed only 1 day post exposure. The presence of sodium hyaluronate surgical-grade ophthalmic viscoelastic, either at full or one-fourth clinical concentration, does not overtly impede the cytotoxic effect of light-activated liposomal BPD-MA on primary culture HLE cells. 66 Chapter 4: Monitoring Post-surgical Rabbit PDT Test and Control Capsules in vitro In all rabbits (R1-R3 1) immediate post-mortem lensectomies with and without PDT with subsequent retrieval and culture of all capsules were performed to investigate three principle questions pertaining to RLE cells in the surgical environment rather than in cell culture. First, was liposomal BPD-MA mixed with a cohesive sodium hyaluronate viscoelastic available when applied onto the native RLE on the inner intact capsule in situ rather than in vitro? Second, was there a toxic effect of irradiated BPD-MA and if so at what concentrations and contact incubation times within the emptied capsule? Third, what light dose(s) of 690 nm red light were necessary to activate any cell-associated photosensitizer. 4.1 Surgical data: Treated rabbit lens capsules assayed in vitro at 100 and 200 J/cm2 LED light activation of BPD-MA: Rabbits Ri-Ru 4.1.1 Rationale Using concentrations (230 and 460 pg/mI) of BPD-MA in viscoelastic at least 10 times greater than that used for in vitro cell culture studies decreased the likelihood that concentration was a limiting factor in the initial surgeries. The darker green colour of these higher concentrations of BPD-MA improved the visibility of this mixture of photosensitizer and viscoelastic during instillation into and removal from the empty capsules. In these preliminary rabbit trials (Ri-Ri 1), light doses of 100 and 200 J/cm2,i.e. up to fifty-fold greater than necessary in vitro, were applied in order that light dose would not initially be a limiting variable. Based on the results in Ri-Ri 1, the light doses were subsequently lessened in rabbits R12-R3 1 as reported in section 4.2. Fluorescence photomicroscopy of all post-surgical capsules in culture permitted single-cell resolution e.g. Fig. 4.8. 67 4.1.2 Objectives The objective of the Ri-Ri 1 rabbit surgeries was to compare the effect on RLE cells of BPD-MA subsequently activated with either 100 or 200 J/cm2of LED red light irradiating the entire empty lens capsule in situ. The photosensitizer was delivered as a mixture with Ophthalin into intact post-surgical lens capsules. (Note: see Appendix Six for flow chart.) 4.1.3 Materials and methods (See Appendix Two for Summary of Experimental Conditions.) A 2.0 mg/mi stock of BPD-MA was prepared by adding sterile water (Abbott) to the lyophilized BPD-MA liposomal preparation (QLT, Vancouver, BC). This stock was stored at +4°C and used within 2 to 3 days. A 230 jig/mi mixture of BPD-MA with sterile Ophthalin 10 mg/mi sodium hyaluronate ocular surgical viscoelastic (CibaVision) was prepared from the BPD-MA stock. The relatively high concentration of the stock allowed for a small volume (7% v/v) of liquid added to the viscoelastic, to minimize any potential dilution effects which might alter the cohesive nature of the sodium hyaluronate. This mixture was used as the carrier for delivering BPD-MA in all PDT test capsules. For euthanized rabbits R1-R5, drug-free Ophthalin 10 mg/mI sodium hyaluronate was used in the control capsules and as the corneal endothelial protectant during surgery. For R6-R1 1, AMVISC 12mg/mi sodium hyaluronate (IOLAB) was used likewise. Eleven female NZW rabbits (2.5-3.3 kg, average 2.9 kg) were used in the first surgical group. The PDT test capsule in rabbit number one (Ri) was torn and therefore excluded from the principle data set. All capsules of the remaining rabbits were deemed usable by virtue of post-surgical capsule integrity i.e. were intact. Each animal was euthanized with an intravenous injection of Euthanyl (Staff Veterinarian, Jack Bell Research Centre, Vancouver, BC) and held at room temperature. Note: the first eye (El) in each rabbit was reserved for eventual BPD-MA / LED-array treatment (“PDT test” condition), except rabbit R9. The second eye was assigned for the drug-free viscoelastic 68 control. This approach was adopted to counter potential concern that any killing effect observed in second eyes which may have been used as PDT test eyes could be confounded by potential compromise of the cells due to the wait time post euthanasia. This also addressed any similar concern, although deemed unlikely, of the passage of systemic Euthanyl across the ocular blood barrier then across the capsule. The time for completion of surgery and pinning of the emptied, PDT test capsules in vitro ranged from 1.5 to 3.25 hours post mortem (mean 2.2 hours). For control eyes, completion times were from 2.0 to 4.0 hours (mean 3.3 hours). Lens extraction, BPD-MA / visco instillation into the capsule, contact, subsequent removal, then irradiation were done with the eye in situ in the animal. 4.1.4 Phacoemulsification extracapsular lens extraction With a view to long-term culture of the post-surgical emptied lens capsules, all lensectomies were performed under aseptic and semi-aseptic conditions. Gravity-fed sterile Balanced Salt Solution (BSS, Abbott) with a modified i.v. line was used in conjunction with a Maclntyre anterior eye chamber maintainer gently inserted into the clear cornea immediately anterior to the limbus in order to maintain a constant, dependable chamber depth resulting from the height of the BSS bottle. This height was consistent for all surgeries reported in this thesis. A 0.9 mm wide stab wound was made in the clear cornea and a smooth-edged continuous curvilinear capsulorhexis (CCC) type anterior capsulectomy performed. Because the pupils of the euthamzed rabbits were not dilated, the resulting anterior CCC, formed with a 25G bent-tip needle cystotome, was necessarily small i.e. only 4-5 mm in diameter. The stab wound was then widened to 3.2 mm to accommodate the titanium venturi phacoemulsification ultrasonicating tip (Alcon). In this first set of 11 rabbits, no subcapsular cortical cleaving hydrodissection (SCCH) injection was used to separate the outermost cortical lens fibres from the apical faces of the adjacent lens epithelium, nor hydrodelineation to dissect the lens nuclear region from its adjacent cortical fibres. 69 The clear, proteinaceous lens was phacoemulsified and aspirated (Alcon Series 10,000 clinical surgical unit). The ultrasonic (US) energy level was kept at a consistent 70%, non-pulsed setting and the total US time was noted for each case. In order to diminish the possibility of rupturing the posterior capsule, aspiration vacuum settings of less than the maximum 400 mm Hg were used. Although there was no use of separate, specialty handpieces for irrigationlaspiration (I/A) after the principal lensectomy, nor of specialized cannulae for polishing of the posterior and equatorial capsule to remove potential lens cortex residues, maximal lens material was removed in this first set of surgeries via the basic US phacoemulsification (“US phaco”) handpiece with inherent BSS irrigation and aspiration. The BSS supply to the anterior chamber maintainer was then turned off. Although not necessary, given that no post-treatment corneal endothelial culture was planned for these experiments, drug-free sodium hyaluronate viscoelastic (Ophthalin or AMVISC) was still applied as a protectant to the corneal endothelium, as is done clinically. This was followed by the instillation of either BPD-MA / Ophthalin or drug-free viscoelastic into the empty capsule via a standard, 30-degree angled, smooth, cylindrical 27G Rycroft cannula. The BPD-MA I Ophthalin was laid down first into the inner equatorial region then allowed to fill the remainder of the capsular bag. However, to assure complete filling of the capsule in this initial model surgery and to increase the probability that sufficient BPD-MA had been available to the inner anterior and equatorial lens epithelium monolayer, the BPD-MA / Ophthalin mixture was allowed to overflow into the anterior chamber beyond the smooth, clearly visible circular edge of the CCC capsulotomy. The mixture was left in the capsule for 2.0 minutes from the completion of instillation. BSS flow to the chamber maintainer was then restarted and the BPD-MA / Ophthalin aspirated as a contiguous, cohesive mass from the capsule and anterior chamber using a straight-tip I/A handpiece (Storz) attached to the Alcon 10,000 unit reset into I/A mode. 70 In order to use the close-array LED to irradiate the post-treatment lens capsule, the cornea was removed at the limbus and radial incisions were made in the iris to create 4 equal flaps which were retracted to expose the entire empty capsule, care being taken to expose the entire capsule now lying flat against the anterior vitreous. The exposed capsule was kept moist with a minimum of BSS to reduce the potential flushing of LE cells, then the LED positioned over the eye. The distance between the LED and the empty capsule was uniform and replicable due to the presence of a fixed-length cylinder enclosing the diode array. Irradiation began within 5-8 minutes of removal of either BPD-MA / Ophthalin or drug-free viscoelastic. Light intensity was 237 mW/cm2. For 100 J/cm2, light delivery lasted 7.0 minutes. For 200 J/cm2,the capsule was moistened again with I3SS between 2 successive irradiations of 7 minutes each. (For an example of converting mW/cm2to J/cm2,see Appendix Three.) 4.1.5 Pinning and culturing capsules Lens capsules were immediately removed following LED-array irradiation and placed into in vitro culture. Using a 30G hypodermic needle bent to 90 degrees, the ciliary processes were retracted to allow a clear view of the zonules which were then cut with small iris scissors. A pair of angled capsulorhexis forceps were slid under a capsule which was then folded in half by closing the forceps while tugging the capsule away from the anterior vitreous face. A second pair of fine-tipped forceps positioned behind the posterior capsule engaged the vitreous while the capsule was pulled free. The capsule was transferred immediately to a 35 mm tissue culture dish (Coming) containing DMEM with 15 % beat inactivated FCS, supplemented with penicillin and streptomycin. Using a modification of a mounting technique (Liu, Wormstone et al. 1996) the intact capsules were pinned at the equator directly into the plastic bottom of the culture dish, thus flattened but not overstretched. The capsulotomy was facing upwards and the posterior capsule was in direct contact with the surface of the dish. All post-surgical explant 71 capsules were fed fresh medium every 2-3 days for the first week. After 1 week the medium for capsules from rabbits R2-R5 was supplemented with amphotericin-B antifungal agent (Fungizone, Gibco) as a prophylactic measure. For R6-R1 1, no antifungal agent was used. All test and control capsules were fed identically. 4.1.6 Assessment of cell viability by microscopy (Sectional photomicrographs (non-digital) of each capsule were taken in an ordered sequence to view the entire pinned capsule. The assembled photomosaics were too large to scan for digital quantification of the areas of cell growth.) Light microscopy (Nikon Diaphot) of all capsules permitted determination of the number of clays required for the cell growth in the control capsules to reach confluence on the posterior capsule. Pinned capsules in culture were photographed at day6 with a 4X plan objective via semi-darkfield condenser optics (phase-contrast condenser Ph2 artificially skewed from optical alignment, e.g. Fig. 4.1, to detect refractile, putatively viable cells. At week 7, all test and control capsules from R2-R5 were incubated for 1.5 h with 4 jM calcein-AM and 8 jM ethidium homodimer (Live/Dead Kit, Molecular Probes) in serum-free DMEM and photographed in their entirety (2X plan objective) with both fluorescence and brightfield optics, yielding photomosaics (up to 0.75 square meter) of locations of all cells, residual cortical material plus any outgrowths onto the 35 mm dishes. At day 8, all the capsules from R 6-Ri 1 were treated with calcein-AM and ethidium homodimer as above and photographed (2X plan objective) with both fluorescence and brightfield optics, then rinsed and fed Fungizone-free medium. These same test capsules, and one randomly-chosen control, were then retreated at week 3 with calcein-AM only and photographed again. At week 5, these same capsules (without fluorophore incubation) were photographed again using semidarkfield optics. Finally, at week 7, all test and control capsules were incubated again with calcein-AM and ethidium homodimer. These final fluorescence photomosaics included all areas of cell outgrowth around the capsules. 72 The following Cell Kill Index (CM) was devised to score cell distributions within and around pinned capsules in large fluorescence photomosaics (single-cell resolution): Score 2: posterior capsule entirely clear; either no viable cells or sparse initial cells subsequently diminished in number without short- or long-term (>4 month) recovery; Score 1: subconfluent cells on posterior capsule; few or no cells onto dish via pinholes; Score 0: confluent cells on posterior capsule; cell outgrowth onto dish via pinholes. (Results of surgeries R2-Rl 1 are summarized in Table 4.1 at the end of 4.1.7 Collection of conditioned growth medium DMEM media contains 45 JAM phenol red which turns yellow at acidic pH levels immediately below 7.0 (Wesierska-Gadek, Schreiner et al. 2006). Colour changes of the growth medium of the pinned lens capsules permitted potential correlation with cell growth, given that actively proliferating or quiescent yet metabolizing mammalian cells could be expected to maintain an actively different hydrogen ion exchange with their aqueous environment than would dead cells. At week 14 after the first set of surgeries, all 22 capsules from Ri-Ru were changed to a weekly feeding of 3.0 ml DMEM / 15% FCS. (Fungizone supplementation was maintained for the capsules from Ri-R5.) Although the capsules from Ri had been excluded from the main surgical data, they were maintained on a parallel feeding schedule to serve as a control for observing differences in pH of conditioned medium, At week 15,2.0 ml of the seven-day conditioned medium was collected from each capsule culture dish immediately upon removal from the 5% CO2 / 37°C incubator. The samples were stored in 2.0 ml polyethylene centrifuge tubes (Eppendorf) with an air bubble dead space of less than 5% of the total tube volume, thereby minimizing contact with room air and reducing the potential for phenol red colour change due to possible outgassing of carbon dioxide. All tubes were stored at +4°C overnight and photographed (Fujicolour Daylight film, ASA 400) with indirect sunlight transmitted the full length of the sample tubes. The results of yellow (lowered pH) or pink (raised pH) 73 indicator colour were investigated for possible correlations with cell growth, presence or absence of Fungizone, and with the number of calcein I ethidium treatments. 4.1.8 Results: 100 and 200 J/cm2, rabbits Ri-Ru Microscopic observations at 48 h and day 6 In all 11 control capsules, cells were observed at 48 hours post surgery growing onto some or all of the margin of the central open posterior capsule (OPC) region encompassed by the edge of the anterior capsulotomy. This was not observed in any of the 10 BPD-MA / LED- array treated capsules. By day 5 or 6, all control capsules showed complete cell confluence on the posterior capsule. In contrast, all of the BPD-MA / LED-array treated capsules were free of new cell growth in this same central open region of the posterior capsule. See Fig.s 4.1 and 4.2 for typical examples. (Note: final results in Summary Chart, Appendix Five.) iA Comparison of 100 J/cm2PDT (upper) and control (lower) capsules at day 6 post surgery (rabbit R3). The posterior of the test capsule is essentially clear, having only minor debris which did not result in any cell growth. The control (bottom) shows the typical refractile, confluent cobblestone (single arrow) cell monolayer on the posterior, open sub capsulotomy region. Fine-pattern, stellate wrinkling (double an-ow) of the control capsule was observed only when cells apptoached confluence. Scale bars = 1 mm. Figure 4.1 74 Calcein and ethidium fluorescence photomicrography Rabbits R2-R5 at 7 weeks At 7 weeks, all 4 control capsules in this group exhibited complete epithelial-appearing i.e. cobblestone cell confluence on the posterior capsule plus wide margins of viable cell growth on the culture dish around the margins of the capsule (see Fig. 4.3). The migration path of proliferating epithelial-.appearing cells was observed along the pins and out through the pinholes in the equator of the capsule and extending beyond the capsule periphery to a markedly greater degree than in any of the PDT test capsules. Figure 4.2 Comparison of PDT test (top) and control (bottom) capsules at day 6 post surgery in a rabbit RiO treated with 200 J/cm2. The test capsule appeared clear whereas the control capsule showed, confluent cobblestone growth (single arrow). Scale bars = 1 mm. 75 Figure 4.3 A control capsule at 7 weeks, rabbit RS, 200 J/cm2,showing green calcein fluorescence (right), indicative of viable cells on the interior surfaces of the capsule, on the cell culture dish and on a fallen pin at the 1 o’clock position. These epithelial-appeanng cells have migrated through the pin holes in the periphery of the capsule onto the plastic dish. On the left is the same capsule in the same orientation photographed with phase-contrast optics. Capsule dia. = 10 mm. Of the four test capsules, two (R3, R5) had either low or diminished cell numbers (epithelial-appearing). See Fig. 4.4. 76 Figure 4.4 Test capsules at 7 weeks, rabbit R3 (upper) at 100 3/cm2 and rabbit R5 (lower) at 200 J/cm2. Both (right) show red ethidium fluorescence indicating putative dead cells. The localized green (viable) fluorescence indicates remnants of ciliary bodies mistakenly excised during the removal of the capsule. Very little or no cell outgrowth was observed in these remnants, shown also in corresponding semi-darkfield (upper left) and full darkfield (lower left) capsule views. Capsule dia. = 10 mm. V 77 Of these same four test capsules, the remaining two (R2, R4) showed cell growth (epithelial-appearing) at week 7, only one of which (R2) showed posterior capsule confluence, with significantly less peripheral growth compared with controls (Fig. 4.5). Figure 4.5 Test capsules at 7 weeks. Rabbit R4 (upper) received 200 J/cm2 and was assigned a Cell Kill Index = 1. Rabbit R2 (lower) received 100 J/cm2 and was assigned a CKI 0 on the basis of posterior confluence, although the peripheral growth was significantly limited in comparison with control capsules in this group (e.g. Fig. 4.3). Capsule dia. = 10 mm. 78 Rabbits R6-R11 at 8 days and 3 weeks The BPD-MA / LED-array treated and control capsules were assayed at day 8 with calcein and ethidium. All the test capsules plus a randomly chosen control capsule (from R6) were reassessed at 3 weeks with calcein only. The ongoing vigorous growth in all control capsules (see right side of Fig. 4.6) indicated minimal effect of the initial calcein / ethidium treatment on the gross survival of the cells in the control capsules. Given that the calcein / ethidium was similarly aspirated and rinsed off all test and control capsules immediately after day 8 photography, the reassessments of these capsules at week 3 are deemed comparable. All the control capsules (n=1 1) showed significantly greater degree of migrating cell growth peripheral to the capsule than any of the test capsules. Figure 4.6 Control capsule (rabbit R6) at 8 days (left) after calcein / ethidium treatment, and at 3 weeks (right, same orientation) with calcein only treatment. This week 3 control capsule was typical of all the other control capsules (rabbit R6-R1 1), showing vigorous, well extended growth and a stable confluence (dense green) on the posterior capsule. Capsule dia. = 10 mm. 79 Of these six test capsules (R6-Rl 1), two (R8, Ri 1) showed a decrease from the initial, low number of viable cells in the interior equatorial region of the capsules (Fig.s 4.7 and 4.8), and were, as per the Cell Kill Index definitions, also assigned a CKI value of 2. Figure 4.7 Rabbit R8, 100 J/cm2. Lower right photograph is the control at 8 days. The two upper photographs are of the test capsule at 8 days. Lower left photograph shows the same test capsule (same orientation) at week 3 with fewer cells. Capsule dia. = 10 mm. 80 Figure 4.8 Rabbit Ri 1, 100 J/cm2. Lower right photograph is the control at 8 days. The two upper photographs are of the test capsule at 8 days. Lower left photograph shows the same test capsule (same orientation) at week 3 with fewer cells i.e. individual cells visible in the 4 to 6 o’clock position. Capsule dia. 10 mm. 81 Of the six test capsules (R6-R1 1), two (R6, RiO) showed intermediate growth by week 3, with a preponderance of sparse, rounded cells exhibiting a compromised Figure 4.9 Rabbit R6, 200 J/cm2. Lower right photograph is the control at 8 days. The two upper photographs are of the test capsule at 8 days. Lower left photograph shows the same test capsule (same orientation) at week 3 with sparse, rounded, putatively compromised cells. Capsule dia. = 10 mm. morphological appearance in comparison with the control cells (Fig.s 4.9 and 4.10). 82 Figure 4.10 Rabbit RiO, 200 J/cm2. Lower right photograph is the control at 8 days. The two upper photographs are of the test capsule at 8 days. Lower left photograph shows the same test capsule (same orientation) at week 3 with sparse, putatively compromised cells. Capsule dia.= 10mm. Finally, of the six PDT test capsules from R6-R1 1, two (R9, R7) showed posterior capsule confluence by week 3 (Fig.s 4.11 and 4.12). Of these two, the lens in the eye used for the test capsule of rabbit R9 (Fig. 4.11) was observed at the time of the 83 lensectomy, along with the contralateral eye used as a control, as being resistant to both nuclear and cortical lens removal. Figure 4.11 Rabbit R9, 200 JIcm2. Lower right photograph is the control at 8 days. The two upper photographs are of the test capsule at 8 days. Lower left photograph shows the same test capsule (same orientation) at week 3 with confluent cells. Capsule dia. = 10 mm. 84 Figure 4.12 Rabbit R7, 200 3/cm2. Lower right photograph is the control at 8 days. The two upper photographs are of the test capsule at 8 days. Lower left photograph shows the same test capsule (same orientation) at week 3 with near-confluent cells. Capsule dia. = 10 mm. 85 Rabbits R6-R11 at S weeks and 7 weeks All test and control capsules from rabbits R6-R1 1 were photographed again without treatment at 5 weeks post surgery. The CM values were the same as those at 3 weeks. Repeated treatment with ethidium and calcein at 7 weeks also yielded results consistent with those CM scores obtained at 3 weeks. The fluorescence photomosaics showed viable confluent monolayers of epithelial-appearing cells on the posterior capsule as well as robust cell outgrowth around all the control capsules, indicating that no gross toxic effects resulted from the calcein and ethidium treatment at day 8. A comparison of the control capsule from R6 (the only one of the controls in rabbits R6-R1 1 treated with calcein at week 3) with controls from rabbits R7-R1 1 indicated that no gross toxic effects resulted from treatment by calcein alone. In these 6 test capsules (R6-R1 1) the areas of cell growth correlated with the location of residual lenticular material in the interior (and mostly equatorial) regions of the capsules as shown in Fig.s 4.7 to 4.12. When the Cell Kill Index ratings of all 10 BPD MA I LED-array test capsules from Ri-Ri 1 were compared with light dose (see Table 4.1), the higher dose of 200 J/cm2was not observed to correlate with greater cell kill. Rabbit Number Led Light Dose Poster Capsule Cell Kill Index PDT Test Capsule Joules / cm2 Cell Appearance Arbitrary Units CKI R2 100 Confluent 0 R3 100 t)iminished/unrecovered 2 R8 100 100% initial kill 2 Ru 100 100% initial kill 2 R4 200 Partial confluence 1 R5 200 Diminished/unrecovered 2 R6 200 Partial confluence 1 R7 200 Confluent 0 R9 200 Confluent 0 RiO 200 Partial confluence 1 Table 4.1 PDT test capsules at 7 weeks (rabbits R2-R1 1). Score 2: posterior capsule entirely clear; either no viable cells or sparse initial cells subsequently diminished in number without short- or long-term (>4 month) recovery; Score 1: subconfluent cells on posterior capsule; few or no cells onto dish via pinholes; Score 0: confluent cells on posterior capsule; cell outgrowth onto dish via pinholes. 86 0 6 7 8 Rabbit/Test Capsule Number Figure 4.13 Upper plotline (left axis): ultrasonic energy times required for phacoemulsification of each BPD-MA I LED-array test capsule (rabbits R2-R1 1) compared with cell kill. Lower plotline (right axis): An arbitrary Cell Kill Index score of 2 reflects maximum kill i.e. no growth, or diminished cell numbers, accompanied by a totally clear posterior capsule. Ultrasonic energy required for surgeries Although there were no lensectomies performed by non-ultrasonic means to serve as a control, there was no apparent coffelation between the cell kill in PDT test capsules and the amount of ultrasonic energy delivered to the eye (Fig. 4.13) during lens removal. Similar amounts of US energy were also delivered to control lenses (data not graphed). There were no obvious signs of US-generated heat damage to any of the control capsules at the time of surgery. Full-confluence cell growth was observed in all 11 control capsules during the following observation period. 2.5 2 Cl) 4-. D E E H 0.5 2 0 2 3 4 5 9 10 11 87 Volumes of BPD-MA / Ophthalin instilled A trendline plot (Fig. 4.14) reveals no correlation between the volume of BPD-MA I Ophthalin instilled into (and slightly overflowed out of) the empty test capsules and the CM values for R2-R1 1, despite an overall trend to increased volume in R7-R1 1. 0 3 4 5 6 7 8 9 10 11 Rabbit/Test Capsule Number Upper plotline (left axis): volume of BPD I Ophthalin used in post phacoemulsification capsules for of each BPD / LED-array PDT test capsule (rabbits R2-R1 1) compared with cell kill. Lower plotline (right axis): An arbitrary Cell Kill Index score of 2 reflects maximum kill i.e. no growth, or diminished cell numbers, accompanied by a totally clear posterior capsule. 4.1.9 pH of conditioned medium in long-term capsule culture In the conditioned media samples from all capsule cultures of Ri-Ri 1, the colour of the phenol red in the seven-day conditioned medium from each of the 10 control capsule cultures (week 15 post surgery for rabbits R1-R5; week 10 for rabbits R6-R1 1) was yellow i.e. < pH 7.0 (Fig. 4.15) and correlated with the confluent refractile cell growth. The conditioned medium from Ri/El served as an internal control for pH observations but was otherwise excluded from the CM ratings, as the dish had only a cell-free posterior capsule fragment (due to an error in tearing the capsule free) and exhibited a higher pH similar to those observed in full-capsule PDT test capsules with no viable cells. In the upper row (Fig. 4.15) of conditioned-medium samples from BPD-MA / LED-array test 0.6 0.5 0.4 0.3 0 Figure 4.14 2 2 1 88 capsules, there was a direct correlation between the pH of the conditioned medium and the presence or absence of cells. The yellow appearance (pH < 7.0) of the R2-R5 control conditioned-medium samples was indistinguishable from that of R6-R1 1, with no observable correlation with the use of Fungizone in the R2-R5 cultures. Comparison of the test samples of R2 through Ri 1 (upper row of Fig. 4.15) with the Cell Kill Index results (Table 4.1) shows that all those PDT capsules with a CM rating of 1 or 2 correlate with the basic pH indicated by pink-red phenol red. The 3 test capsules assigned a CM of 0 are the only ones with yellow conditioned medium. These pH results also correlate with the fluorescent photographic observations indicating that the multiple application of calcein and ethidium did not grossly interfere with the ongoing assessment of the differentiation between BPD-MA I LED-array treated and control capsule cell growth in R6-R1 1 capsules. Figure 4.15 Seven-day conditioned DMEM from mbbit test (upper) and control (lower) capsule cultures. Yellow-coloured phenol red indicates a pH < 7.0 associated with viable, metabolically active cells. Pink-red samples (pH> 7.0) correspond to capsules with fewer and/or less metabolically active cells. 4.1.10 Conclusions: preliminary surgeries Ri-Ri 1 Each of the 10 valid test capsules exhibited either a partial or complete cytotoxic effect of light-activated BPD-MA. Stable results at 7 weeks in culture showed that 30% (n=3 cases) BPD-MA/LED CONTROL 89 had confluent de novo epithelial-appearing growth on the posterior capsule but with less overall growth than in the controls. Additionally, 30% (n=3) of the test capsules exhibited subconfluent cell growth, 20% (n=2) showed very few surviving cells which consistently diminished in number over the 7 weeks, and finally 20% (n=2) showed absolute cell kill at every monitoring stage during the 7 weeks. BPD-MA at a concentration of 230 jsg/ml in Ophthalin applied for 2 minutes to a post phacoemulsification capsule in a post-mortem rabbit followed with a 690 nm LED light dose of 100 J/cm2 can effect a complete prevention of growth of lens epithelium onto the central posterior capsule. The consistent survival of cells in all control capsules indicate activated BPD-MA as the sole cause of cell kill. In addition, the lack of correlation between cell kill and light dose indicates that 1003/cm2is sufficient for killing original, quiescent lens epithelial cells in this model and that factors other than light dose are responsible for CKI ratings of less than 2. In PDT-treated test capsules showing growth, the location and degree of cell growth is strongly associated with both the location and amount of residual lenticular material present in the post phacoemulsification capsule, indicating that the contact of the drug with capsule epithelium is critical for maximum cell kill. 4.2 Surgical data: Rabbit lens capsules: —‘2 J/cm torus laser tip diode laser light activation of BPD-MA compared with 10 J/cm2 and 100 J/cm2 LED: Rabbits R12-R31 (Note: see Appendix Six for flow chart.) 4.2.1 Rationale: Rabbit pinned capsule model surgeries closely reflecting preferred clinical parameters Given that no differential killing was observed between 100 and 200 J/cm2activation of BPD-MA in rabbits R2-R1 1, additional sets of rabbit lensectomies were undertaken to determine the effectiveness of photosensitizer activation with lower, more clinically relevant red light doses. Higher concentrations of BPD-MA in Ophthalin also permitted 90 investigation of the potential PDT effect after only a one-minute intracapsular drug incubation, a parameter which would meet the generally accepted surgical dictum that intraoperative clinical procedures should be done with a minimum of time and manipulation. Could this light be delivered effectively via a clinical model fibre optic (see subsequent section, 4.2, rabbits R19-R3 1) with a dispersal tip designed for intracapsular irradiation (Karamata, Sickenberg et al. 2000), exposing the inner equatorial and anterior capsule, but minimizing irradiation of the retina? Also clinically relevant were whether or not the intracapsular instillation, contact time and removal of the BPD-MA I Ophthalin mixture followed by irradiation could be accomplished, in the case of the fibre optic, within a reasonably short time. (This was not applicable in the case of Sets A and B described below in which the close-array LED unit was used to first test light delivery lessened by an order of magnitude i.e. 10 J/cm2 to the test capsules.) Finally, the clinical prototype intraocular laser delivery tip (above) was used to investigate irradiations as low as 2 JIcm in model surgeries where the cornea and iris were left intact, reflecting potential clinical protocol. As previously, lens epithelium kill following light activation was determined by culturing the entire capsules in vitro. 4.2.2 Objectives Although the fundamental objective was to show whether or not in the rabbit model that contact with BPD-MA in Ophthalin followed by red light activation could cause a PDT prophylactic effect of lens epithelial cell de novo proliferation and migration onto the posterior lens capsule, the specific objectives in the following surgeries were to compare BPD-MA activation by LED-array red light (10 or 100 J/cm2 ; sets A, B) with that of laser diode light (—‘2 J/cm ; sets One to Six) of the same wavelength delivered by the clinical prototype Equatorial Laser Tip #4 (ELT-4). Additionally, experiments were carried out to determine if a clinically manageable drug contact time of e.g. 1 minute could effect a significant PDT intracapsular cell kill using relatively low light doses via ELT-4. 91 4.2.3 Materials Photosensitizer Liposomal-formulated BPD-MA was used for all surgeries. Two stocks (2.0 mg/mi for making 230 jg/ml in Ophthalin and 4.0 mg/mi for making 460 J4g/ml in Ophthalin) were prepared by adding sterile water (Abbott) to freeze-dried BPD-MA. For rabbits R12-R17; R21, R22; and R25-R27 the stock was stored at +4°C and used within 1 or 2 days. For rabbits R23 and R24, stock was used at 8 days. For rabbits R28-3 1, stock was used the same day without subsequent refrigeration before mixing with Ophthalin. As with preliminary rabbit surgeries R2-Ri 1, Ophthalin (CibaVision) 10 mg/mi sodium hyaluronate viscoelastic was used as the carrier for BPD-MA delivery in all test capsules. Ophthalin was also used for those control capsules in which drug-free viscoelastic was instilled into the capsule. However, in the following, there was no BPD free viscoelastic instilled into 2 of the 10 control capsules: Rabbit 15, Eye 1 (R15/E1) due to torn posterior capsule; and R271E2 due to scratched cornea which impeded proper viewing of the surgical field. Equipment The phacoemulsification unit and close-anay LED were those used with rabbits R2-R1 1. Laser diode (Applied Optronics) 690 nm red light was delivered via clinical prototype ELT-4 (Fig. 4.23, p.1 19) provided by Karamata, Sickenberg, van den Bergh (Karamata, Sickenberg et al. 2000). A newly refurbished CooperVision surgical microscope with footpedal focus and magnification control was used in surgery sets Five and Six (R28- R30). This footpedal was not available for Ri-Ri 1 surgeries. The same Alcon Series 10,000 phacoemulsification unit and handpieces were used as with Ri-Ri 1. 92 Rabbits Relatively older female NZW rabbits aged 15.5-32.0 weeks (mean 23.7 weeks) weighing 3.4-4.7 kg (mean 4.0 kg compared with mean 2.9 kg for previous surgeries R2-R1 1) were obtained from the UBC Animal Unit, Jack Bell Site, each animal euthanized as with Ri-Ri 1. In each of the surgery sets A, B, and One to Six there was at least one control capsule i.e. photosensitizer-free but light-irradiated, totalling n= 10. Seven of these 10 control capsules were from the second and thus longer-time post-mortem eye of the rabbit in question as previously rationalized. The time for completion of surgery and pinning of the empty capsule was approximately 2-4 hours post-mortem. 4.2.4 Methods BPD-MA / Ophthalin viscoelastic mixtures A 230 jig/mi mixture of BPD-MA with sterile Ophtbalin (460 jig/mi for R25-R27) was prepared from 2.0 mg/mi (or 4.0 mg/mi for R25-R27) BPD-MA stock solutions, thereby maintaining a consistent added volume of 7%v/v for all mixtures, as done in Ri-Ri 1 surgeries. Seven of the 10 control capsules were filled with drug-free Ophthalin. One control capsule (R29/E2) received Ophthalin mixed with the equivalent volume (7%vlv) of sterile water as a control to address the potential for any effect of the slight change in aqueous content i.e. a putative hypotonicity-directed cell-swelling cytotoxic effect on remaining lens epithelium. Phacoemulsification extracapsular lens extraction Surgical procedures summaries: Sets A, B, and One to Six The procedures in common in surgery Sets A, B (R12-R17; Close-array LED irradiation) are outlined in Table 4.2. (See for detailed descriptions) (See Table 4.4, p. 105, for list comparing all sets A, B, and One to Six) 93 1. All had SCCH(2 injections of -4m1 BSS via Rycroft 27G) 2. All had smooth-edged CCC capsule opening via forceps (except R12IE1 which had can-opener capsulotomy) 3. All had ultrasonic phacoemulsification lens removal 4. All had post-phaco. irrigation I aspiration with Storz curved I/A tip 5. None had capsule polishing or scratching 6. All test capsules had 2-mm. contact with 230 g/m1 BPD-MA I Ophthalin 7. All had comeas removed, irises cut into four flaps and retracted 8. All exposed capsules irradiated via 690 nm close-array LED Table 4.2 Rabbit surgeries: Sets A and B (R12-R17); Close-array LED 690 nm photoactivation. Abbreviations: BSS: balanced salt solution; SCCH: subcapsular cortical cleaving hydrodissection; CCC: continuous curvilinear capsulorhexis Specific procedures in surgery Sets A, B (R12-R17) Close-array LED Irradiation were as follows: Set A (4 test eyes): capsules irradiated with either 10 or 100 J/cm2 Set B (5 test eyes): repetition of 10 J/cm2 irradiation The procedures in common in surgery Sets One to Six (R19: P.21-31) Equatorial Laser Tip-4 (ELT-4) Irradiation are outlined in Table 43: 1. All had SCCH (1-3 injections of BSS via Rat-tip and/or J-cannulae) I 2. All had smooth-edged CCC capsule opening via forceps 3. All (except R19, no US energy) had ultrasonic phacoemulsification lens removal 4. All (except P.27/E2) had capsule polishing and scratching 5. Discretionary use of Storz curved I/A tip for irrigation I aspiration 6. Polishing I scratching cannulae used with forced BSS (most capsules) 7. Discretionary use of button iris retractor to locate residual cortex 8. PDT capsules had 2 or 1-mm. contact with BPD-MA I Ophthalin (except R211E2: 3.5 mm. due to technical difficulty) 9. PDT capsules had 230 jg/ml BPD-MA I Ophthalin concentration (except P25-27: 460 jg/ml BPD-MA; 1 mm.) 10. Corneas and irises left intact; wound enlarged to -‘3.5mm for ELT-4 light- ring tip insertion 11. All irradiations with laser diode via ELT-4 clinical prototype 12. All irradiations -2 J/cm2 Table 4.3 Rabbit surgeries: Sets One to Six (R19; R21-R31); Equatorial Laser Tip-4 (ELT-4) 69Onni photoactivation. Abbreviations: BSS: balanced salt solution; SCCH: subcapsular cortical cleaving hydrodissection; CCC: continuous curvilinear capsulorhexis 94 Specific procedures in surgery Sets One to Six (R19; R21-3 1) ELT-4 Irradiation were as follows: Set One (1 test eye): initial trial of ELT-4 -2 J/cm2 (230 jg/m1; two-minute contact) but since no ultrasonic energy used, had to be repeated Set Two (3 test eyes): repetition of set One conditions, all with US phacoemulsification Set Three (2 test eyes): one-minute contact with BPD-MA I Oplithalin instead of two Set Four (4 test eyes): BPD-MA concentration doubled to 460 ptg/ml (one-minute contact) Set Five (2 test eyes): repetition 230 jig/mI BPD-MA (one-minute contact) Set Six (3 test eyes): test of silicone-tipped cannula for BPD-MA / Ophthalin 230 jig/mi delivery (one-minute contact) as close to the empty capsule equator as possible Detailed surgical descriptions: A, B, and One to Six: Rabbits R12-R31 The decision to designate fewer than one third of the total number of capsules as controls (n=i0) arose from the observations previously noted in surgeries Ri-Ri 1 that 100% of control capsules showed consistently robust epithelial cell growth. The majority of the rabbits in these 8 surgical sets were selected because they were older than the previous rabbits Ri-Rh. This decision was based in part on the assumption that the additional steps of subcapsular cortical cleaving hydrodissection (SCCH) and capsule scratching and/or polishing might more closely reflect the clinical case of the older, harder human lens in primary cataract surgery. 95 Aseptic and semi-aseptic conditions, a BSS gravity-fed anterior chamber maintainer and the use of a clear comeal stab wound were employed as previously. For all rabbits R12-R3 1, an opening sufficient for introducing 27G cannulae was formed in the central anterior capsule by creating a small triangular flap via a 25G cystotome. Two basic SCCH regimens were followed. For the rabbits in Sets A and B (LED-array at 10 or 100 J/cm2) an initial injection of BSS (approximately 1 ml) was made just under the capsule via a virgin 27G Rycroft cannula inserted through the small flap and guided 1-2 mm towards the equator of the lens. This point of entry was approximately 30 degrees left of the axis of the stab wound. A second injection (also -.1 ml) was made in the mirror image position to the right. For the rabbits in Sets One to Six (ELT-4 at -2 J/cm2,see Table 4.4) the first SCCH injection was made with a flat-tip hydrodissection carmula (27G) in the same axis as that of the stab wound. The second injection was made with a J-hook cannula (27G) inserted just under the capsule through the triangular flap and pointing back towards the stab wound. For the lenses in Sets One to Six the volumes of BSS were discretionary, based on the appearance of the advancing SCCH ‘wavefront’ visible (at least for the anterior capsule) as the capsule proper separated from the lens. As with Ri-Ri 1, a small anterior capsule opening (4-5 mm diameter) was necessary. With only one exception (Ri3/E1, described below) all capsule openings in Ri2-R3 1 lenses were smooth-edged and circular, via continuous curvilinear capsulorhexis (CCC) made with capsulorhexis forceps (instead of a 25G needle cystotome used in Ri- Ri 1) through an enlarged stab wound, using a needle-formed small central capsular flap as the initial grasping point for the forceps. The exception (Ri2IEi) control eye was notably different in that, as occasionally occurs clinically, a ragged-edged “can-opener” capsulotomy had to be formed in order to stabilize the opening and keep it from running out to the equator and being lost. This was done with a bent-tip 25G needle (cystotome) by making multiple, orderly tears from the margin of the circular capsule opening towards the center of the anterior capsule, leaving many little flaps of anterior capsule, with the 96 possibility of LE cells still attached. This was of serendipitous interest in that these particular LE cells would possibly be less likely to come in contact with the photosensitizer and viscoelastic mixture. Widening of the stab wound to 3.2 mm to accommodate the phacoemulsification tip, as well as the settings for US energy and vacuum were the same as with Ri-Ru. Although as much of the lens material as possible was removed with the phacoemulsification tip, in the following Sets A and B, additional attempts at removing any residual cortical fibres from each capsule were made with a curved-tip I/A handpiece (Storz) with the Alcon 10,000 unit set to I/A mode. However, no separate polishing and/or scratching cannulae were use to further polish the posterior and equatorial capsule to attempt any final removal of potential residual cortical lenticular material. In contrast, the emptied capsules in Sets One to Six (with the notable exception of control capsule R27/E2) were polished. The posterior capsule polishers (cannulae modified in tip shape and texture) included a Jensen olive-tip capsule curved polisher roughened on the bottom; a Knolle capsule scratcher roughened around the circumference of its bent cylindrical tip; a silicone-sleeved bent cylindrical-tip Terry Squeegee (Alcon; Fig. 4.24); plus a smooth J-Hook cannula which was separately used to attempt flushing of resistant cortex observed under the entry wound. With the exception of R19 and R21, the syringes to which the above cannulae were attached were filled with BSS which was flushed through these scratchers and polishers as an adjunct to attempts at direct contact removal of cortex. This BSS stream also prevented roughened tips from catching the posterior capsule, thus eliminating polishing-related tearing. Due to small pupil size, potential residual equatorial cortex was not immediately visible and the use of a button iris retractor was used in attempts to discover lens residue particularly in the area of the equator close to the single, widened main entry wound. As with R2-R1 1, the BSS flow to the anterior chamber maintainer was turned off after the lensectomy i.e. just before instillation of BPD-MA / Ophthalin. All test capsules were overfilled with the photosensitizer mixture to give a consistent baseline drug volume 97 (—0.5 ml) i.e. to ensure contact with BPD-MA / Ophthalin. Only R30-R3 1 capsules received more BPD-MA / Ophthalin (0.7-0.9 ml). In these cases, the capsules appeared just as filled as with 0.5 ml, but with more excess overflow into the anterior chamber of the eye. In contrast to Ri-Ri 1, corneal endothelium viscoelastic protective coating was not applied in Sets A, B, nor in One to Six surgeries in R12-R31. Instillation of BPD-MA / Ophthalin into the empty capsules in all of surgery Sets A and B, and the majority of Sets 1-5 was via 27G Rycroft cannulae. The exceptions in Sets Four and Five were R241E2 and R25/Ei in which silicon-sleeved Terry Squeegees (Fig. 4.24) were used for instillation. The surgeries in Set Six were designed specifically to allow instillation of all test capsules via modified Terry Squeegees in an attempt to distribute BPD-MA I Ophthalin closer to the inner capsule equator while taking advantage of the soft, shielded tips to minimize the risk of capsule puncture. BPD-MA / Ophthalin was left in the capsule for either 2 minutes (Sets A and B; plus Sets One and Two, with the exception of 3.5 minutes for R21/E2) or 1 minute (Sets Three to Six inclusive) timed from the completion of instillation. As previously, the chamber maintainer BSS flow was then restarted and the contiguous, cohesive mass of BPD-MA / Ophthalin was aspirated from the capsule and anterior chamber with a curved-tip Storz I/A handpiece. In Sets A (Ri2-R14) and B (R15-R17) as in R2-R1 1, the comeas were removed at the limbus and radial incisions were made in the iris to create 4 equal flaps which were retracted to expose the entire emptied, to allow access for post-surgical capsule close-array LED irradiation after BPD-MA / Ophthalin had been instilled then removed. The exposed capsule was moistened with a minimum of BSS as with Ri-Ri i, the LED positioned and irradiation began within 5.5-10.5 minutes (mean 6.8, n=9) of removal of either BPD-MA / Ophthalin or control photosensitizer-free viscoelastic as previously. For 100 J/cm2 irradiation, light delivery lasted 7 minutes 41 seconds and for 10 JIcm2, 46 seconds. Light intensity was 217 mW/cm2. 98 For Sets One to Six (irradiation via ELT-4), the cornea and iris were left intact. The single surgical wound was widened further to approximately 3.5 mm for insertion of the 2.0 mm diameter spherical ELT-4 fibreoptic tip (Fig. 4.23, p. 119). Intracapsular irradiation began within 3-5 minutes of completion of BPD-MA / Ophthalin removal. Depth of the anterior chamber (and to some extent the depth of the empty capsule) were maintained with an anterior chamber maintainer BSS drip. The ELT-4 was held steady with the tip positioned in the middle of the capsule opening, pushing slightly against the posterior capsule in aid of keeping the capsule open. Red, 690 nm diode laser light coplanar with the equator was delivered to the capsule interior via the 3600 internal dispersing epoxy ring in the fibreoptic tip. To avoid delays due to calculations during surgeries and so to activate the photosensitizer within a reasonable time, all ELT-4 irradiations (Sets One to Six) were 300 seconds i.e. 5.0 minutes. Test-case irradiations began an average (n=15) 3.3 minutes after BPD-MA removal. (The range was 2.0-5.0 minutes for all, n=15, test cases. With minimum and maximum time extremes eliminated, the range was 2.5-3.5 minutes for n= 11 of the 15 test cases.) This expedited approach was possible given that the laser diode consistently delivered a narrow range of power output (lowest 9.86 mW to highest 13.60 mW, test capsules R19/E1 and R30/E1 respectively). For calculating the light dose spanning the distance to the furthest portion of the capsule i.e. the equator, the small ELT 4, although only 2.0 mm wide, could not simply be considered a theoretical point source in relation to a 10mm average diameter of an empty non-flattened older rabbit capsule. During each surgery, a meter reading was made from the ELT-4 laser-engaged tip inserted into an integrating light meter sphere just before each capsule irradiation (Graseby Optromcs Lightmeter Sphere). From the metering, a range of the amount of light delivered to the equator was based first on the actual tip diameter (potential upper dose due to smaller distance) and secondly, on the tip as a theoretical dimensionless point (potential lower dose due to theoretically greater distance). (See Appendix Three for calculations.) From all 15 99 PDT surgeries (in R19-R3 1), the weakest lower light dose value calculated from 9.86 mW (above) was 1.33 J/cm2. The strongest upper dose value calculated from 13.60mW (above) was 2.86 J/cm2. The range of upper and lower values (nt15 lower; n=15 upper) was 1.59-2.50 JIcm2,with a mean of 2.05 J/cm2,noted hereafter as approximately (‘) 2 J/cm2. Pinning and culturing capsules Immediately following irradiation the capsules were removed from the rabbit eyes and pinned in 35 mm culture dishes as described previously for R2-R1 1. All were fed fresh medium on days 2,5,8, 13 and 16 with minor one-day variations after day 8. All capsules were also rinsed and fed on days 7 and 21, immediately after calcein / ethidium staining and photography (see Fungizone fungistatic agent was not used. PDT and control capsules were fed identically. Assessment of cell viability by microscopy All capsules were examined with a Nikon Diaphot inverted microscope as previously. The posterior capsule region was photographed at day 7 (Sets A and B) or day 2 (Sets One to Six) with either a 2X or 4X plan objective using semi-darkfield condenser optics (i.e. condenser position Ph2 skewed slightly) to detect refractile, viable cells and the location of any residual cortical lens material. At day 21 (Sets A and B) and at day7 and 21 (Sets One to Six), all the PDT and control capsules were incubated for approximately 1.5 h with 4 jM calcein AM and 8 jiM ethidium homodimer (Molecular Probes) in serum-free DMEM and photographed (2X plan objective) with fluorescence optics to give a large photomosaic record of the location of cells including the outer margins of total growth areas around the capsules. 100 Collection of conditioned growth medium Sets A, B, and One to Six: After day 21, all 34 capsules (n=24 test; n=10 control) were changed to a 5-7 day medium replacement schedule and fed exactly the same volume of DMEM I 15% FCS. Control and PDT test capsules, including cell-free test capsules, were maintained on a parallel feeding schedule to permit comparison of differences in pH of conditioned medium. For surgery Sets A and B at 5 to 6 wks, 2.0 ml of the conditioned medium was collected, stored and photographed as previously described. The differential between a yellow (lower pH) and pink (higher pH) colour was correlated with putative cell viability. (Note: At 14 months after the first surgeries of R2-R1 1, conditioned medium from all those capsules still extant from all surgeries R2-R3 1 were sampled, pH metered and the results correlated with the long-term cell presence observed on PDT and control capsules.) Cell Kill Index (CKI) Cell growth on the pinned capsule and on the surrounding culture dish were scored with the same CM used previously (section 4.1.6). 4.2.5 Results: Sets A, B, and One to Six (R12-R31) All 10 photosensitizer-free control capsules exhibited complete posterior capsule epithelial appearing cell growth at 7 days. Cell kill in all PDT capsules is summarized in Table 4.4 (Results summary: Sets A, B, and One to Six, in section Details of microscopic observations of all capsules on Days 1, 2, 7 and 21 follow in sections and The summary discussion of each surgical group (below) highlights the integration of the use of flat-tip hydrodissection cannulae, capsule polishing, BSS flushing, the evolution of the ideas of compromised visco cohesion, the use of the clinical laser tip ELT-4, as well as the use of modified 101 silicone-sleeved cannulae for BPD-MA I Ophthalin instillation. The data in section (conditioned-media pH) provides support for the long-term consistency of the results first shown in Table 4.4 (Results summary: Sets A, B, and One to Six, in section (See also Results Chart: Euthanized Rabbit Model Lensectomies, Appendix Seven.) Set A (n=4 PDT): The purpose was to compare 10 and 100 J/cm2LED doses. Of note, total cell kill was observed only with 10 J/cm2 with only subcapsular cortical cleavage hydrodissection (SCCH) via standard, non-flattened i.e. cylindrical tip 27G Rycroft cannulae, and no capsule polishing, prompting Set B trials of all 10 J/cm2 light doses. Set B (n=5 PDT): The purpose was to repeat 10 J/cm2light LED doses. However, results were confounded by the fact that both PDT lenses R17/E1 and R17/E2 were extremely friable with liquefied cortical lens fibers resulting in considerable residual cortex. Set One (n=1 PDT): The availability of the ELT-4 and a laser diode more suitable for single operator use prompted Set One surgeries. This allowed a more clinically relevant protocol, not requiring cornea removal and iris flap retraction required for LED irradiation. Additionally, SCCH with flat-tip cannulae and polishing were introduced. Surgeries showed that a marked photodynamic cell kill could be effected with as little as 2 J/cm2 via the ELT-4 prototype. Set Two (n=3 PDT): Results confinned that a marked lens epithelial cell kill could be effected with JIcm2 via ELT-4, and the consistent CKI=2 at day 7 for all 3 PDT capsules pointed to improved access of BPD-MA / Ophthalin due perhaps to flat tip SCCH and/or capsule polishing with BSS flushing. As noted previously, the potential for confounding of day 21 results by fibroblasts led to repeating the surgeries. As well, the relatively high kill with two-minute contact prompted trials of one-minute contact. 102 Set Three (w=2 PDT): The purpose was to test a one-minute photosensitizer contact with only-’2 J/cm via ELT-4. One test capsule, R24/E1 was not kept for long-term follow-up due to the puncture of the capsule during instillation of BPD-MA I Ophthalin via a metal 27G Rycroft cannula. This prompted the use of a Terry Squeegee (silicone-sleeved soft-tip cannula, Fig. 4.24) for the instillation of BPD-MA I Ophthalin into R24fE2. The two other PDT capsules in Set Three had CKI scores of 2 at day 7. At day 21 however, reticulate lens cell growth was observed on 80-90% of the posterior capsule of test capsule R23/E1, although it lacked outgrowth on the culture dish when compared with the controls. In contrast, the other PDT capsule (R24’E2) which had also been instilled via a Terry Squeegee had one small locus of cells, not obviously associated with ciliary remnants, which was viable at day 7 but disappeared by day 21, leaving only ciliary remnant- associated cells. This raised the possibility of a need for a critical number of remaining viable lens epithelial cells for long-term survival and reinforced the similar observation in previous rabbit surgeries R2-R1 1. It also provided the first indication that silicone-sleeved extensions should be further tested. Set Four (n=4 PDT): These surgeries explored the effect of doubled BPD-MA concentration (460 jig/mol) on PDT efficacy. Also, a silicone-sleeved Terry Squeegee cannula was used for instillation in one PDT capsule. CKI=0 for 3 of 4 PDT capsules contrast markedly with the CKI=2 score in all PDT capsules in Sets One-Three at day 7. In set Four, the only capsule (R251E1) with CKI=2 was also the only one instilled via the silicone-sleeved cannula. The BPD-MA / Ophthalin for set Four had been mixed with purposely increased vigor to ensure uniform dispersal of the double concentration BPD MA stock. However, the tendency of the extruded mixture to form ropes made instillation into the empty capsules difficult. This change in the BPD-MA / Ophthalin may have been caused by the increased mixing shear forces and/or the increased total lipid from the photosensitizer stock, and prompted Set Five conditions, below. 103 Set Five (n=2 PDT): These surgeries were designed to allow repetition of the use of 230 ugIml BPD-MA I Ophthalin, mixed (without added vigor). The total cell kill in capsule R28/E2 was contrasted by the lesser killing observed in capsule R29/E1. An original note entered at the time of surgery indicated that due to an unusually small capsulorhexis, BPD MA / Ophthalin instillation was difficult in R28/E2. It was also noted during the course of Set Five surgeries that the reflective coating particularly on the retina side of the ELT-4 spherical tip was wearing away (Fig. 4.23). Set Six (n=3 PDT): Given the significant PDT kill in R24/E2 (Set Three) and the total kill in capsule R251E1 (Set Four), both instilled via 27G Rycroft with silicone-sleeved tips (Terry Squeegees) the purpose of Set Six surgeries was to determine if the BPD-MA / Ophthalin could be better delivered to the more potentially proliferative equatorial lens cells by extending the silicone tube at the tip (Fig. 4.24). Although this was hypothesized to minimize potential capsule puncture during instillation and allow closer and/or more complete contact between LE cells and the photosensitizer, only 1 (R3 1/El) of 3 test capsules showed total LE cell kill. Of the 2 remaining PDT capsules, R3 1/E2 was notable in that due to cornea scarring, no polishing or cortical residue checks were undertaken and the poor surgical field of view did not aid in BPD-MA I Ophthalin instillation which could account in part, for the poor kill in this capsule. 104 Results summary: Sets A, B, and One to Six (R12-R31) CKI=2: posterior capsule entirely clear; either no viable cells or sparse initial cells subsequently diminished in number without short- or long-term (>4 month) recovery CKI= 1: subconfluent cells on posterior capsule; few or no cells onto dish via pinholes CKI=0: confluent cells on posterior capsule; cell outgrowth onto dish via pinholes CKI (Cell Kill Index) Note: All 10 control capsules had scores of ‘0’ at both 7 and 21 days Surg. PDT Subcap. Caps. BPD- Contact Light Kill: Kill: Set Capsule Cleavage Polish MA Time Dose 7 21 Conc. days days 1.D. No. jtg/mI (mm.) J/cm2 A R12 / E2 Rycroft - 230 2 10 1 0 R13 I El Rycroft - 230 2 10 2 2 R14 I El Rycroft - 230 2 100 2 2 Rl4 / E2 Rycroft - 230 2 100 1 1 B Rl5 / E2 Rycroft - 230 2 10 1 0 R16 / El Rycroft - 230 2 10 1 0 Rl6 I E2 Rycroft - 230 2 10 1 0 _____ Rl7 / El Rycroft - 230 2 10 1 0 R17 / E2 Rycroft - 230 2 10 1 0 — One R19 I El Flat tip/J-H + 230 2 —2 2 (1) — — Two P.2 1 / E2 Flat tip/i-H + 230 3.5 —2 2 (1) R22 / El Flat tip/J-H + 230 2 —2 2 (1) P.22 / E2 Flat tip/i-H + 230 2 —2 2 (1) . — — Three R23 / El Flat tiplJ-H + 230 1 —2 2 0 R24 / E2 Flat tip/J-H + 230 1 —2 2 (1) — Four R25 / El Flat tip/J-H + 460 1 —2 2 2 P.26 / El Flat tip/i-H + 460 1 —2 0 0 R26 / E2 Flat tip/J-H + 460 1 —2 1 0 R27 / El Flat tip/i-H + 460 1.5 —2 1 0 — — Five P.28 / E2 Flat tip/J.-H + 230 1 —2 2 2 P29 / El Flat tip/J-H + 230 1 —2 1 0 — — Six P.30 / El Flat tip/i-H + 230 1 —2 2 0 R3l/E1 Flat tip/i-H + 230 1 —2 2 2 P.31 / E2 Flat tip/J-H - 230 1 —2 1 0 Table 4.4 PDT capsules at 7 and at 21 days. A score of 2 reflects maximum kill i.e. no growth or diminished number of cells with a totally clear posterior capsule. Irradiations of 10 and 100 3/cm2 were via LED; those of -2 3/cm2 via Equatorial Laser Tip-4. Cell Kill Index 21-day values in parentheses (n=5 of 15, Sets One to Six) are those which, if corrected for putative fibroblast contamination would be CKI2. Flat tip/J-H indicate those lenses hydrodissected with a flat-tipped and/or J-cannula. (El=eye 1; E2=eye 2.) 105 Microscopic observations Days 1, 2 and 7: Sets A, B, and One to Six (R12-R31) Not all controls were observed 24 h post surgery, but those that were (R21/E1; R23/E2 and R301E2) showed a profusion of refractile proliferating cells near the margin of the open posterior capsule (OPC). In all 10 control capsules at 48 hours post surgery, robust cell growth was observed growing onto some or all of the margin of the OPC. This type of growth was not observed in any of the 24 PDT treated capsules in the same time period. However, a few refractile cells were observed on 2 of the 24 PDT capsules at 48 h. One was the PDT capsule R26/E1 of Set Four in which approximately 10 refractile cells were observed on day 2. (Set Four PDT test capsules were the only ones to receive the double concentration 460 ugIml BPD-MA I Ophthalin which proved to be incohesive during instillation.) The second such capsule was R3 1/E2 of Set Six. This capsule had significantly more cortical residue than other capsules and the scattered refractile cells observed at day 2 corresponded with these loci of residue. By days 5-7, and as early as 4.5 days for control R19/E2, all control capsules showed complete posterior capsule confluence, e.g. Fig. 4.16. On day 7, all of the 9 PDT capsules in Sets A and 13 were essentially free of new cell growth in the central OPC, whether irradiated with 10 or 100 J/cm2 (LED). On day7 in Sets One to Six (irradiated with J/cm2via the ELT-4), 14 of 16 test capsules were also essentially free of new cell growth in the central OPC. The two exceptions were the capsules with the rope-like, incohesive and discontiguously instilled BPD-MA I Ophtbalin in Set Four: R261E1 a 100% confluent OPC and R26/E2 with 75%. 106 Figure 4.16 A control capsule (drug-free Ophthalin, no light) at day7 shows the typical confluent cell growth on the entire capsule, including the central open posterior capsule (OPC) region. Viable cells fluoresce green with calcein. Colour shift from green to yellow is a photographic artifact. Set One: R19/E2. Capsule dia. = 10 mm. Microscopic Observations Day 21 In all 10 control capsules at day 21, OPC areas were still 100% confluent with densely packed non-reticulated cell growth. The outgrowth onto the surrounding cell culture dishes was consistently greater than that of any of the PDT capsules in the same surgery set. With the one exception of PDT capsule R26/E1 (poorly cohesive BPD-MA / Ophthalin, Set Four), no PDT capsule showed more outgrowth than any control capsule of any surgery set, regardless of photosensitizer contact time, light dose, light source, cleaving regimen or of polishing regimen. On day 21, 2 of the 9 PDT capsules in the LED Sets A and B showed absolute cell kill, with one capsule at 10 J/cm2 and one at 100 J/cm2.On day 21, 3 of 16 PDT capsules in Sets One to Six irradiated with —P2 J/cm via the ELT-4, showed absolute cell kill (see example in Fig. 4.17, bottom) and another 5 of 16 (or 4 of 15 if the one inside-out PDT capsule R19E/l is disqualified, see below) were also essentially free of 107 new cell growth in the central OPC i.e. subconfluent. The remaining 8 of 16 had confluent OPC areas, but all showed consistently reticulated growth patterns in comparison with the dense confluent cell growth on controls, e.g. Fig. 4.18. 108 Figure 4.17 Upper A control capsule (BPD-free Ophthalin 1 mm., 2 J/cm) at day 21 shows the typical confluent cell monolayer on the open posterior capsule (OPC) region and extensive epithelial-like outgrowth (Set One; R19/E2). Viable cells are green. Lower: A typical absolute cell kill PDT capsule (R25/E1, 460 jsg!ml BPD-MA I Ophthalin, 1 mm., 2 JIcm), the only PDT capsule of 4 in Set Four (poorly cohesive BPD-MA I Ophthalin) with total kill. The nuclei of dead cells stain red with the ethidium from the calcein I ethidium cocktail. Capsule dia. = 10 mm. 109 Figure 4.18 A confluent test capsule (R16/E1, Set B, 230 g/ml BPD-MA I Ophthalin, 2 mm., 10 J/cm2)at day 21 with reticulated growth on the open posterior capsule (OPC) not generally seen in controls (three satellite colonies not shown). Capsule dia. = 10 mm. Potential ciliary process fibroblast contamination: Sets A, B, and One to Six (R12-R31) Noteworthy is that in 5 of 16 test capsules in Sets One to Six which showed significant epithelial cell kill (CKI=1) at day 21, the results were confounded by putative fibroblasts suspected as contaminants from mistakenly excised zonule-associated ciliary processes just outside the outer edge of the capsule equator. These cells e.g. Fig. 4.19, showed distinctly fibroblastic cell morphology of cells corresponding directly with the location of remnants of ciliary processes appearing in close contact with or trapped by the capsule. These 5 test capsules (noted parenthetically as CKI “(1)” at 21 days, in Table 4.4) were R19/E1; R21/E2; R22/E1; R221E2; and R241E2. This potential contamination was observed in the 110 first test capsule irradiated with the ELT-4 fibreoptic (Set One, R19IEI). After phacoemulsification and PDT treatment this capsule was accidentally pulled inside out and could not be reinverted so was pinned as such. At 7 days the ciliary process remnants were clearly trapped inside this everted test capsule (see Fig. 4.19, upper and middle) and were presumed to be in contact with the capsule. The fluorescence photograph of the same region at 21 days (Fig. 4.19, bottom) shows remnant-associated growth with distinctly nonepithelial-like morphology, even when compared with the typical non-confluent advancing cell front of control capsules which present clearly epithelial-like cells which are not yet cobblestoned in appearance. 111 Figure 4.19 Upper: An everted test capsule (R19/E1, 230 ptglml BPD-MA I Ophthalin, 2 mm., 2 J/cm2)at day 7 shows 2 trapped ciliary remnants (transmitted light photomicrograph, 4X dia). Middle: The same view at day7 with combined transmitted light and calcein AM fluorescence shows initial putative fibroblast outgrowth. Lower: The same capsule area at day 21 shows a continued prevalence of fibroblast-like cell morphology (2X dia.). Single and double arrows point to the same two ciliary remnants. Scale bars = 1.0 mm. 112 This scenario of artifactual and non-clinical contamination was also observed in other (i.e. non-everted) test capsules with fibroblast-like cell growth in close proximity to ciliary remnants near or at the outer margins of the capsule. This occurs in normally pinned, non-everted cultured capsules due to the tendency of that portion of equatorial capsule between two pins to roll in on itself in a scroll configuration (see Fig. 4.20). If a ciliary process remnant happens to be in this area, it is occasionally ‘enscrolled’ into this configuration and thus into close contact with the outer surface of the capsule. It is further proposed that contaminating outgrowth occurs onto the outer surface of the capsule with the possibility of migration to the anterior and/or posterior regions. This is further supported by observations of fibroblast-like cell outgrowth from ciliary remnant areas which appears blocked at the lip of the capsulorhexis (Fig. 4.21) indicating exterior cell growth and thus not of interior lens epithelial origin. 113 Figure 4.20 Upper: A test capsule (R221E2, 230 j4g/ml BPD-MA I Ophthalin, 2 mm., 2 JIcm2)at day 2 shows 2 trapped ciliary remnants (transmitted light, 4X dia). Lower: The same capsule area adjacent to the remnants at day 21 shows a prevalence of fibroblast-like cell morphology (4X dia.). The arrow points to cell outgrowth onto the outer surface of the scrolled capsule perimeter, indicating that these cells may be the result of ciliary remnant outgrowth. This is in contrast to the usual pattern of lens epithelial cell growth from the inner equator onto the posterior capsule. Scale bar = 1.0 mm. Capsule pin is uppermost. - 114 Figure 4.21 A test capsule (R241E2, 230 J4g/ml BPD-MA / Ophthalin, I mm., —2 Jfcm) at day 21 shows trapped ciliary remnants. Upper: Single arrows indicate remnants (2X dia.). The barricade effect (double arrow) at the margin of the capsulotomy further supports the notion of cell growth on the exterior of the capsule, and thus not of lens epithelial origin. Lower The same capsule area adjacent to the remnants at day 21 shows a prevalence of fibroblast-like cell morphology (4X dia.). See Fig. 4.19 for exact equivalent 2X and 4X dia. scale bars. 115 If this scenario of ciliary cell outgrowth onto the outer anterior cell capsule has occurred in control capsules, the effect has possibly been masked by the robust lens epithelium growth onto the inner peripheral capsule and OPC. It should also be noted that in some capsules, residual vitreous is still attached to the capsule after zonulectomy and removal of the capsule from the posterior chamber of the eye. This margin of vitreous has been observed to cushion the occasional ciliary fragment and prevent it from contacting capsule or cell culture dish surface. However, in the case of robust outgrowth of epithelial like cells from the pinholes at the equator of capsules onto the dish surface, it is proposed that the cells migrate at least in part down the pins, unobstructed and directly onto the plastic. This is supported by the fact that very robust lens epithelium growth has been observed on several occasions on the surfaces of fallen stainless steel pins in the culture dish. Of course, the possibility of ciliary fibroblast migration along pins and into the interior of the capsule cannot be discounted. The availability of a newly refurbished CooperVision surgical microscope, used in Sets Five and Six (R28.-R30) significantly improved the surgical field of view, enabling far more accurate zonulectomies and the avoidance of accidental excision of ciliary process remnants which preceded empty capsule removal to cell culture. 116 At 4 months, suspected ciliary body fibroblast contamination is observed in phase contrast micrographs (both at 4X dia). Upper: control (R23/E2) shows typical stable cobblestone epithelial cell growth in a capsule with no enscrolled, excised remnant ciliary body. Lower: PDT capsule (R24/E2) shows strong fibroblastic morphology (4 months) similar to that seen in Fig. 4.21, which shows the same capsule at day 21. Scale bar = 1.0 mm. 117 — • . •• ., --. ,. - Figure 4.22 In short, if the 5 previously noted test capsules R19/E1; R21/E2; R22JE1; R22/E2; and R24/E2 which exhibited equatorial and some peripheral capsular subconfluent growth at 21 days are corrected for fibroblast contamination, the effective absolute cell kill ratio would be considered 3+5 i.e. 8 out of 16 in surgery Sets One to Six (or 7 out of 15 if the everted R19/E1 is discounted), all of which were irradiated with —2 J/cm via the clinical ELT-4. All of theseS PDT capsules (R19/E1; R21/E2; R22/E1; R22/E2; and R241E2, above) were treated with 230 ug/ml BPD-MA I Ophthalin but their drug contact times varied: R19/E1 at 2 mm., R21/E2 at 3.5 mm., R221E1 at 2 mm., R22/E2 at 2 mm., and R24/E2 at 1 minute. Each of theseS capsules is assigned a 21 day CM score of ‘(1)’ in Table 4.4 (Results summary: Sets A, B, and One to Six, in section Although R22/E1 showed clear ciliary process fibroblast contamination in day 7 and day 21 photographs, there is evidence of epithelial-like morphology at 4 months. R19IE1 was no longer in long-term culture and therefore not available for photography. 118 Figure 4.23 Wearing away (double arrow) of silver coating in ELT-4 photographed after surgical Set Five. Upper: side view with low laser emission, showing the equatorial dispersing layer in pink (bracket and arrow). Lower: dark-field photo of same orientation indicating light lost from the normally opaque retina side of the spherical tip. Scale bar = 1.0 cm. 119 Figure 4.24 Modification of Alcon Terry Squeegee (27G) used for BPD-MA I Ophthalin instillation in surgical Set Six. Upper: native cannula configuration with extension less than 1 mm. Middle: the sleeve is pulled with gloved fingers to prevent oil or lipid contamination. Lower: finally, tip is trimmed to -‘1.5 mm for instillation use. Rule markings 1.0 mm. Overall, polished capsules in Sets One to Six showed fewer residual cortical and LE cells than unpolished capsules in Sets A and B. In PDT cases showing some growth, it tended to arise from discrete loci rather than from the entire equator (observed previously in certain capsules from R2-R1 1) indicating improved cell accessibility. However, SCCH and polishing have not ensured complete kill in all capsules. However, this study has shown that 100% kill is possible with one-minute BPD-MA I Ophthalin contact and only 2J/cm2 red light delivered via the ELT-4. 120 Conditioned media The long-term differential between control cell survival and PDT capsule results in Sets A and B was confirmed. The colour of the phenol red in the conditioned medium (at 5 to 6 weeks) from each control capsule was distinctly yellow, indicating a lowered pH associated with metabolites from putatively viable cells (Fig. 4.25). CONTROL Jpp, 5] ____ V-MA/LED j )NTROL Figure 4.25 Upper: Set A (R12- 14) and Lower: Set B (R15- 17) conditioned DMEM from test and control cultures at 5-6 wks. Yellow indicates pH <7.0 and metabolically active cells. Pink samples (pH > 7.0) correspond to capsules with no cells or fewer, less metabolically active cells. DCMdays conditioned medium; dpp=days post-phaco Comparison of the R12 through R14 samples in Fig. 4.25 with the CM results given in section shows that those test capsules with a cell kill rating of 2 correlate positively with pink phenol red, pH> 7.0. The control capsules are the only ones with distinctly yellow (i.e. not orange) medium, pH <7.0. Even in R15-R17, where the cell kills were not as effective, the differential between test and control capsules is evident. =37 dpp, 10 DCMJ R12 R13[I3 BPD-MA/LED [5[R16 211 Rh 121 Observations of phenol red colour of Sets One to Six capsules indicated that the differential between test and control capsules was also stable in the same six-week time frame for all capsules. In an attempt to address the long-term stability of the results reported above, conditioned medium from all those capsules from all surgeries R2-R3 1 still extant at 13 months after the first surgeries of R2-R1 1 were sampled and the pH measured by meter. The results, shown in Fig. 4.26 indicate that there was no significant long-term recovery of those capsules exhibiting significant PDT effect as compared with the control capsules from which conditioned medium was sampled. 122 7.70 7.60 7.50 7.40 7.30 7.20 7.10 7.00 6.90 6.80 6.70 6.60 6.50 6.40 6.30 6.20 6.10 D(CD . . I. O>O oe I- i- I-. i-. i- ‘ ‘T ‘ a. a. 6.. a. a. a. a. a. a. a. a. a. a. — a. a. C) C.) 00 00 0C) Category and Culture Age (months post-surgery) Figure 4.26 pH of nine-day conditioned media from PDT and control pinned-capsule cultures extant 13 months after the initial surgeries. Younger cultures are from later surgeries in the sequence of Ri to R3 1. All 7 control capsules, regardless of time in culture, are less than pH 6.90 and correlated with visible cells monolayers in culture. Thirteen of 17 PDT PDT capsules are greater than pH 6.90 and correlated with completely absent or compromised cells. Cultures no longer extant at 13 months had either been accidentally spilled or had mudomly developed fungal contamination. C) C a. a. 123 4.2.6 Conclusions: Surgeries R12-R3 1 All test capsules show a cytotoxic effect of red light-activated BPD-MA and all photoclynamic cytotoxic effects occurred regardless of light device, light dose or contact time. The consistent survival of cells in all control capsules validates the model and indicates activated BPD-MA as the sole cause of cell kill in test capsules. The complete lack of correlation between the light dose and cell kill indicates that the dose of -2 J/cm2delivered by the Equatorial Laser Tip 4 prototype is sufficient for killing lens epithelial cells in this model and that factors other than light dose are responsible for CKI scores less than 2. In test capsules showing new cell growth, the location and degree of growth is strongly associated with both the location and amount of residual lenticular material present, possibly indicating that some LECs are protected from drug uptake by overlying residue(s). 124 Chapter 5: Discussion The use of both in vitro cultured human donor cells followed by model cataract surgery lensectomies in euthanized rabbits eyes allowed a gradual arrival at the potentially clinically- relevant results using an easily seen, easily manipulated BPD-MA / visco mixture and the torus tip for irradiation and activation of intracellular photosensitizer. However, there were several potential anomalies with the in vitro experiments reported in Chapter 3. One, the apparent ‘increase’ in the number of viable cells in only one of the four dark cytoxicity assays of BPD-MA (HLE-8, Fig. 3.2-C) is not likely to be a lone, idiosyncratic case of BPD-MA stimulating cell growth or enzymatic activity (shown as greater than 100% in Fig. 3.2-C) in the two highest concentrations of 400 and 800 jg/ml. As these concentrations are very high and in lipid-based liposomal carriers, a possibly under-rinsed 96-well plate may have had some BPD-MA residue in these wells. Given that the MTT assay uses an OD of 595 urn (Mossman 1983), these readings may reflect some interference from the absorption curve of BPD-MA (peak is 698-690 nm). Although the absorption peaks are somewhat disparate, there may have been overlap in the shoulders of the absorption curves leading to the anomalous viability results greater than 100% for that particular plate. Other potential anomalies may reflect, in part, the use of individual human donor cell cultures rather than pooled cultures. Inconsistent availability of cadaver lenses and the relatively slow growth of HLE from cut strips of human capsules (plus their tendency not to grow beyond early passages in culture) dictated one consistency: there were usually sufficient available cells at passage 2 or3 to do single experiments with BPD in ‘dark’ toxicity or in PDT dosimetries at n=3 wells per drug concentration. Pooling cells from different donors would also have further removed the results from the clinical situation, creating the possibility of unknown interactions between cells from different individuals. In comparing the low ends of BPD-MA concentration ranges, there were examples of donor-to-donor variations. For example, HLE-6 and HLE-8 (Fig. 3.3) show 20% and 100% survival respectively at 0.20 ug/m1 BPD-MA plot points. However, as the dose 125 curve approaches LD100 concentrations, the plots converge i.e. both HLE-6 and HLE-8 fall to approximately 15% survival at 0.40 jig/mi with no statistical difference between the two. This LD100 of 0.40-0.80 jig/mI BPD-MA is also seen in HLE-9 (Fig. 3.4.) and HLE-10 (Fig. 3.5). All of HLE-6, -8 and -9 were plated out at 30,000 cells/cm2,with 10- mill, drug exposure indicating that under similar conditions, cells from different donors behave similarly, specifically with regard to LD100 BPD-MA dosage. This LD100 convergence also appears to apply whether the MTT cytotoxicity assay was done at 1 or 20 days post-treatment (HLE-10, Fig. 3.5). However, in HLE-20 (Fig. 3.6) where cells were at plated at 42,000 cells/cm2and exposed for 1-minute, the LD100 was actually lower, indicating that factors other than donor differences have a greater influence on results. A central goal in all experiments reported in this thesis was to find BPD-MA concentrations, contact times as well as light doses that would ultimately, in the potential clinical situation, kill all intracapsular cells remaining after cataract surgery. Given this and the consistent epithelial morphology of all HLE donor in vitro cultures (and in rabbit pinned capsules not complicated with artifactual ciliary fibroblast-appearing cells), antibody-based identifications of cell types were not done. 5.1 Chapter outline As in any experimental biological system, it is prudent to consider these in vitro and surgical models of PCO in light of physical and biological parameters. The observations in this study prompt discussions based on a clear principle hypothesis, with the fewest assumptions possible and when feasible, the simplest explanations. These include the possible simple barricade of some LB cells by overlying cells, preventing contact with or uptake of effective levels of BPD-MA. 126 There are three principle concerns in this chapter physical and biological explanations for cell survival in test capsules, and potential modifications of the model with additional clinical approaches. 5.2 The physics of in vitro and pinned rabbit capsule models 5.2.1 Drug contact Interface between BSS and Ophthalin/BPD-MA The major assumption made in these studies was that the hypothesized uptake of BPD-MA by target LE cells would require direct contact between the cells and the visco/BPD-MA mixture, providing at least the potential for drug presentation to exposed cells, regardless of the precise release, transfer, and uptake forms and mechanisms. Cell and Ophthalin / BPD-MA interface It was beyond the scope of this thesis to explore the precise biochemical microenvironments at the interface between the advancing front of the viscoelastic photosensitizer mixture, nor to explore the physical stability of the liposome carrier in the viscoelastic mixture. However, the question of liposome stability at the LE surface remains. Although possible effect(s) of the hyaluronate on the stability or longevity of the intact liposomes was not investigated, one could adopt a working hypothesis that they remain intact given that the hyaluronate is essentially an aqueous environment. Also, it seems reasonable to postulate that although the advancing visco-PS front could severely limit the amount of the aqueous BSS leaflet trapped in the interface, there could be a BSS-mediated dilution and lowered viscosity of the containment mixture. This may permit diffusion of BPD-MA liposomes from the viscoelastic margin to the exposed LE cell outer membranes. The question of the stability of liposomes in this now differently-charged ionic interface solution due to the contamination of the hyaluronate with 127 BSS could also be raised. However, the compromise or breakdown of liposomes in the BSS-mixed interface with subsequent cellular uptake of the released PS in this aqueous environment can conceivably be downgraded. This is based on the relative stability of the lyophilized BPD-MA liposomes reconstituted with sterile water as compared with the hydrophobic nature of native, non-liposomal BPD-MA (Richter, Sternberg et al. 1989; Richter, Yip et a!. 1996). Moreover, the noted lipophulicity of BPD (Richter, Waterfield et al. 1993) increases the probability that a diffusion transfer of the drug from the liposome to the cell membrane occurs upon direct contact. Lethal BPD-MA (LD100) and PDT Light Doses As shown in Chap. 3, the investigations of phototoxic concentration ranges of BPD-MA for primary human LE cells in culture on plastic and glass, showed that LD100 values were in the nanogram per ml range of liposomal BPD-MA (with non-limiting light activation). This appeared relatively independent of whether or not the liposomal BPD-MA was presented in aqueous liposomal BPD-MA or of 25% hyaluronate solution, indicating that the presence of hyaluronate was not a hindrance to drug uptake, in one HLE- 10 culture (Chap. 3, Fig. 3.4) indicates that at least with this culture, LE cells appear more susceptible when treated with BPD-MA mixed in the 25% hyaluronate mixture. In the attempt to approximate clinical confinement, BPD-MA was next tested as nanogram and microgram per ml ranges in virtually undiluted hyaluronates such as Ophthalin and AMVISC. The calcein I ethidium homodimer fluorescence photomicrography assays (Chap. 3) showed that LE cells on glass are lethally sensitive to BPD-MA in nanogram and microgram per ml concentration ranges, regardless of their location in relation to each other on the glass coverslip support. As a result, this also supports the assumption that regardless of physical mechanism, contact or at the least, very close proximity between the cells and the advancing visco-drug mixture is necessary for ultimate PDT effect in these assays. By extension, it was hypothesized that native 128 monolayer LE cells which were fully and cleanly exposed by lensectomy surgery would experience the same lethality, a condition subsequently observed in those capsules exhibiting 100% LE cell kill. By extension, this also implies that post-surgical native LE cells still covered by remnant full cortical fibre cells or acellular material such as torn fibre cell membrane fragments in situ would exhibit non-random patterns of cell growth and death, correlated with the location of such detritus, that is, protected from the BPD-MA. Although not of dosimetry interest, it was necessary from the point of view of potential clinical application of possible PCO prophylaxis with PDT to test the efficiency of viscoelastic protection of cornea! endothelia cells against accidental exposure to BPD-MA. Given the protection demonstrated in the exaggerated-case scenario where a non-clinical full-concentration aqueous liposomal BPD-MA stock (2 mg/mi) was placed for a full 10 minutes onto 1-2 mm thick hyaluronate layer over endothelium (Chap. 3), the question is raised that if sodium hyaluronate can effectively prevent BPD-MA exposure, how can BPD-MA be released when mixed in the viscoelastic? The simple explanation is that the protective coat was not only thick, but would be supposed, due to the viscous ttature, to be resistant to the passive diffusion of photosensitizer much deeper than the immediate surface of the hyaluronate. As will be discussed later, the behaviour of liposomal BPD-MA in the immediate surface of viscoelastic, it is the consideration of the immediate surface molecular microenvironment which is important regarding BPD-MA moving out of hyaluronate, as opposed to the macroenvironment of a thick hyaluronate layer protecting corneal endothelium from BPD-MA moving sufficiently into the viscoelastic. The principle hypothesis of this study was that RLE cells in the empty capsule would show a PDT effect up to and including complete kill with BPD-MA delivered in an ocular viscoelastic. The assumption regarding contact led to the secondary hypothesis that removing the central lens nucleus, the inner cortical anucleate lens fibres and the outer cortical fibres would completely expose the LEA-cells and E-cells, permitting contact at the immediate inner (apical) LE cell membrane interface with the visco/BPD-MA. 129 For proving or disproving the principle hypothesis, the simplest objective treatment of the model surgical outcomes is a binary analysis. Uncomplicated treated capsules were either completely devoid of living cells or not. Complete kill was observed: in 4 of 10 non-polished, non-hydrodissected PDT test capsules (R2-R1 1; section 5.1); and in 5 of 24 hydrodissected and/or polished test capsules (Sets A, B, One to Six in section 5.2). Therefore, these observations prove the principle hypothesis of this thesis, a proof which is also supported by observations of reduced LE cell areas of growth and/or cell density in all other test capsules: 6 of 10 in R2-R11; and 13 of 24 in Sets A, B, One to Six. Five more test capsules of 24 in Sets A, B, One to Six in section 5.2 showed no epitbelial-appearing cell outgrowth after PDT. However, these 5 capsules fall outside statistically-acceptable results due to the proliferation of fibroblast-appearing cells clearly growing and extending from accidentally excised distal ciliary processes, enscrolled in the rolled-up outer edges of the capsule. When considered without the putative ciliary fibroblast growth, these 5 capsules lend collateral support for the principle hypothesis. The one exceptional test capsule exhibiting considerable growth (1 of the 24 test capsules in Sets A, B, One to Six) had a lens which was resistant to removal. If one proposes that such a lens had unusual adherence at the cellular level causing cortical remnants to be left behind, there follows an increased likelihood that access to the LE cells of the drug could be restricted and that LE cell kill would be incomplete, as was indeed observed in this test capsule. There may be additional simple physical explanations for the growth in some test capsules. The first of these involves the pinned follow-up niodel itself. Handling the capsule carries its own risks of physical effects on potentially proliferative cells remaining post-treatment in the periphery. After zonulectomy frees the empty, treated capsule, the first disruptive step is handling and folding the capsule with forceps then tugging it away from the vitreous, i.e. breaking the ligamentum hyaloideo capsulare which connects the posterior capsule to the anterior vitreous. This external pressure on the capsule may cause 130 internal rubbing of remaining LE cells, nucleate fibre cells and/or fibres. It could also be argued that these disruptions loosely model the insertion and rotation of an IOL, whereby the haptics rub over any remaining equatorial cortex and lens epithelial equatorial (E-cells), potentially exposing such LE cells originally covered by intact, clandestine outer fibre cells or the detritus of fibre cell or fibre remains. This may permit such LE cells to recover and/or divide once in contact with the rich DMEM / FCS 15% culture medium, as might occur in the clinical setting when the aqueous humour refills the AC bringing with it the normal growth factor components of a recovering eye such as TGF-alpha (Wiskstrom and Madsen 1993) and TGF-beta2 (Wormstone, Tamiya et al. 2002). The subsequent attachment of the capsule to the culture dish by driving pins through the capsule perimeter (Liu, Wormstone et al. 1996) is clearly a potential disrupter for any cells or cortical detritus in the immediate area of the pin tip. These model-specific forces may also render any partially compromised or unaffected remnant LE andlor outer cortical nucleate fibre cells more accessible to cell culture medium. A reasonable explanation for cell growth, protection and death in this surgical model is based on the observations of consistent and robust growth of cells of epithelial morphology in all control capsules. It is important to note that 20 of 22 control capsules underwent the same procedures as the test capsules. In the controls of the latter sets (A, B, and One to Six), this included careful subcapsular cleaving hydrodissection (SCCH), plus capsule polishing in 14 of 15 test capsules in sets One to Six. (In 2 control capsules of Set- Six, the lenses were simply phacoemulsified without the other cleaning steps performed in that set and showed similar growth.) Therefore, the robust growth in every control proves that in this surgical model none of the cleaning or polishing procedures in the author’s hands are capable of completely removing all potential newly proliferative cells. The flourish of newly-dividing cells in confluent week-old control capsules, is clearly contrasted by the consistently meager appearance of non-confluent test capsules with either no growth, discrete growth foci, or attenuated generalized equatorial growth on 131 test capsules. This indicates the presence of remnant cells which did not take up (sufficient) photosensitizer andlor were shielded from or did not receive sufficient 690 inn light. It is unlikely that any remaining intact, newly-differentiating fibre cells (McAvoy, Chamberlain et al. 1999; Lovicu, Steven et al. 2004) would completely occlude light from underlying LE cells which may have theoretically taken up BPD-MA from their surgically- exposed lateral membrane surfaces. However, there appears to be no literature reporting on the potentially lower transparency of these new fibre cells, due to the presence of light- scattering nuclei and organelles yet to be eliminated as the lens fibre cell transforms into an anucleate, organelle-free stable lens fibre. It is also not reported whether or not partially damaged cortical fibre cells and fibres can sufficiently disperse 690 nm red light, preventing sufficient photon absorption by the intracellular BPD-MA for efficient singlet oxygen generation (Foote 1991; Hug, Strand et al. 1997) orcaspase activation (Granville, Carthy et al. 1998; Granville, Jiang et al. 1999; Granville, Shaw et al. 1999) in the vulnerable cells. 5.2.2 I/A and phacoemulsification tip microenvironment During lensectomy, 3 phacoemulsifier settings are used: irrigation only; irrigation and aspiration (I/A); and I/A plus 40,000 Hz (40kHZ) ultrasonic (US) back and forth tip motion. This transduction of piezoelectric signals into US energy breaks up the dense lens fibres in the nucleus (Kelman 1969). Therefore, the physical forces at or near the end of the 1.0mm diameter cylindrical titanium venturi tip include irrigating, aspirating, cutting and punching forces. One could further presume that cutting is due to the 15-degree angled tip end, giving a pointed, can opener-style entry into e.g. a lens fibre. During phacoemulsification, US heat generation along the entire tip is minimized to avoid protein denaturation, reduce edema in the wound margin and to lower the total energy into the anterior chamber of the eye, particularly to the endothelium. These clinical 132 parameters are also applicable to this study such that US energy was engaged only when the tip end was in contact with the nuclear mass or fragments, or resistant cortex. This approach is validated by the lack of correlation between total US time (at the consistent 70% US power setting for all surgeries) and Cell Kill Index (CKI) results plotted on the same graph (Chap. 4, Fig. 4.13) such that any changes in total US time did not correspond with capsules with a maximum CKI=2. BSS irrigation provides cooling and flushing forces approximately 1.5 mm behind the tip end, directed from two 0.75mm irrigation ports 180 degrees apart in a pliable irrigation sleeve surrounding the tip. These two streams of BSS occur in all three control- pedal positions for the phacoemulsification handpiece. The BSS also maintains a positive AC pressure preventing corneal collapse and endothelial damage, and in the case of this study, minimizing the potential for the trituration (rubbing away) of corneal endothelium and iris epithelium, thereby decreasing the possibility that any potentially proliferative dislodged cells will settle into the fully-treated capsule. One confounder in this regard are the moderate changes that were occasionally made to aspiration flow rate throughout the surgeries. However, given the approximately 50-100 ml of BSS used in each eye, it was evident that any such heterologous cells, emulsified nucleus or cortex as well as freed masses of cortex were continuously and copiously flushed and/or aspirated, depending on the pedal position engaged. This flushing effect was also augmented by the consistent use of the gravity-fed BSS AC maintainer, except during either the plain Ophthalin or OphthalinlBPD-MA instillation and incubation in the emptied capsules, arguably times during surgeries when any other dislodged cells would not have opportunity to settle in the inner capsule. 5.2.3 Lens interactions with phacoemulsification and I/A The cohesive nature of lens cortex observed in all 54 lensectomies duplicates pediatric and adult surgeries and supports the proposed covering or protection of remnant LE cells by 133 residual cortical fibre cells or detritus, limiting or preventing PS uptake. For most pediatric cataracts, a non-US I/A handpiece or syringe aspiration can remove the entire lens (Koch and Kohnen 1997). In some adult cases, as also practiced in the study eyes herein, the phacoemulsification handpiece in I/A mode removes the less-dense cortex (Lehmann 1996). This reduces the amount of US energy used and eliminates tip heating, but given the straight shape of the tip, it does not always ensure that all outer cortex is removed, especially that in the inner equator of the capsule directly beneath the access wound. This problem was instrumental in variously subjecting different groups of lenses in section 4.2 to standard Rycroft, flat-tipped and J-cannulae SCCH, and capsule-polishing cannulae. In this study, the phacoemulsification handpiece in I/A pedal position i.e. without US, was used to remove the generally softer fibre cells of the outer cortex and portions of the inner cortex fibres, although US was used in resistant areas. Keeping the tip at a safe distance from the capsule to avoid an aspiration-induced rupture also reflected clinical practice. The consistent pulling away of cortical matter from the capsule perimeter in macroscopic masses and therefore of more than one fibre cell or fibre, points to a comparatively strong intercellular bonds and membrane-membrane attachments in all or some of the following combinations: outermost newly-differentiating cortical fibre cell-to- fibre cell; intermediary fibre cell-to-fibre; innermost mature organelle-free fibre-to-fibre. The formation of macroscopic lens fragment masses during surgical removal suggests a clue that all the cohesions between these various cellular entities are stronger than the attachment between the apical LE membrane and the basal membrane of the immediate, overlying fibre cell. This raises the question of why some residual cortex was still observed in certain post-surgical capsules and not in others, the answer to which would provide clues to an improved technique for ensuring that all LE cells were cleanly and fully exposed. It also appears that the attachment of the basal membrane of LE cells to the capsule is stronger than the attachment of the LE apical membrane to fibre cells. This is supported 134 by the observation of various-sized sheets of dead LE cells fluorescing a consistent and even-appearing ethidium red colour while still attached to the inner surface of test capsules with 100% kill. The fact that in these non-complicated test capsules there were no residual lenticular masses attached to or overlying these sheets of LE cells supports the proposal that as was observed in LE cells grown on coverslips then PDT treated, LE cells on the inner capsule are susceptible to BPD-MA induced photosensitivity when then are unobstructed. Additionally, differences in junctional protein distribution between these two cell types may explain in part this observed weaker interface. As well, recent research shows that more 50% of the protein in fibre cell membranes is major intrinsic protein (MIP) (FitzGerald and Goodenough 1986), an aquaporin involved in the movement of lens water and proposed as a connecting protein between membranes. However, there appear to be no reports that MIP is plentiful in the LE apical cell membrane, reinforcing the notion that the heterologous intermembrane connection between cells of the native LE monolayer and newest fibre cells is comparatively weak, and probably contributes to those inner regions of the capsule where LE cell sheets are rendered cortex free. Given the LE and fibre cell characteristics noted above and the sheets of LE monolayers observed extant on the immediate post-surgical capsules reported in this thesis, how do some areas of the inner capsule, particularly the equator, show greater tendencies to harbour overlying residual cortical remnants? The simplest is a physical explanation. One possible analogy is the removal of an imaginary, very narrow strip of adhesive tape from the inside of a non-spherical, asymmetric curved surface such as the pointed end of an eggshell i.e. similar to the internal capsule equator. When pulling the tape, the force and speed of the rate of change of vector forces will vary depending on the location of the point of detachment. At some shallow angles, the tape may not separate from the surface of the inner shell so that rapid pulling may actually snap the tape apart crosswise, leaving a fragment behind. At other, more acute vector-force angles, the tape may pull cleanly away. Additionally, if a sudden cross 135 vector is applied (i.e. as in the push forward of a phaco tip end currently in lateral cortex), the tape may split lengthwise, leaving a strand in the shell, a possible analogy to damaged fibre cells forming detritus over LE cells. In all surgeries reported here, the portion of the capsules immediately below the tip- entry wound in the cornea was not marked. Such marks would have allowed correlating any cortical amount(s) in this difficult-to-clean clock-hour position of the capsules and any subsequent locations of LE cell outgrowth foci. This relative position in the inner equator is described in the literature as the most difficult to clean (Peng, Apple et al. 2000) and clinically is usually at the patient’s temporal side of the eye. In the lensectomies reported in this thesis there was also a tendency for resistance to pulling away and aspirating outer cortex at the lens equator closest to the entry wound. This increased the probability of leaving either whole fibres, fibre cells or their detritus as potential residue. Although all lens material can be phacoemulsified, the efficiency is in the fragmentation and emulsification of the compressed fibres in the denser nucleus. The 40 kHZ (US) piezoelectric-driven cycle of compression and decompression i.e. punching motion (Kapusta, Chen et al. 1995; Moffat and Pope 2002) also applies a cutting force due to the 15-degree angle at the phaco tip opening. Drawing an analogy with smashing apart a wall the thickness of a single brick, the various forces identified at the tip end would seem likely to disrupt fibres and fibre cells indiscriminately when US power is engaged in the outer cortex. This was often the case in this study. In keeping with the analogy, it would seem possible that some cortical fibres overlying LE cells were damaged and left as detritus of opened and torn-apart outer fibre cells, therefore reducing the intercellular integrity so that cohesive masses of outer fibre cells and fibres can not pull away from the LE membrane. The differences in the apparently stronger attachments between various fibre cell entities within the proteinaceous lens and the weaker attachment between fibre cells and lens epithelial cells also point to a least three possible procedure-induced cellular outcomes 136 related to the I/A and phaco force at the immediate vicinity of the tip of the phacoemulsifier handpiece. First, when the fibre cells are torn away this may physically change the apical LEjunctional, transmembrane and/or intermembrane profile, possibly altering the nature of the interaction with the advancing front of visco/BPD-MA, leaving the apical plasma membrane of the underlying LE cell completely exposed. Second, it may also be due to some remaining outer cortical fibre cells and fibres being ruptured during phacoemulsification in such a way that it greatly reduces the protective blockade effect of these relatively large fibre cells and fibres full of crystallin proteins providing bulk protection against the advancing front of visco/BPD-MA. Third, as seen in the latter sets of surgeries, A, B, and One to Six (Chap. 4, section 4.2) there was a correlation within the test capsules which showed discrete loci of remnant equatorial material and subsequent foci of cell growth, as opposed to the more generalized patterns of growth in the incompletely affected test capsules of the early surgeries (Ri-Ri 1). Although this could result from improved phacoemulsification technique in the author’s hands, there is a strong possibility that the overall attempts in the latter surgeries to make cleaner capsules is due to subcapsular cortical hydrodissection (SCCH) and/or various polishing cannulae, accounting for the discrete growth loci in some of the latter test capsules. Areas of remaining LE cells were seen in all emptied capsules reported here. The same was observed for every cold-storage human donor eye used for explant culture. This is in accord with the published studies of human and rabbit lenses consulted for this thesis (Marcantonio and Vrensen 1999). It is clear that the complete physical ablation or compromise of LE cells during human cataract surgeries and rabbit model lensectomies is not the norm. The absolute cell kill in the statistically significant 40-50% of PDT-test capsules in this thesis shows that at least in principle, all remaining A- and/or E- LE cells can be exposed to an effective uptake of liposomal BPD-MA from sodium hyaluronate, all within one-minute drug exposure and subsequently killed with as little as 2 JIcm. 137 Finally, all of the PDT rabbit surgery capsules with 100% lack of cell growth in pinned culture showed at least some sheets of LE cells, all of which points to a reasonable consistent tendency in all source eyes for the attachment of LE cells to capsule rather that to outermost cortical fibre cells. Regardless of the reason(s) for some remaining viable cells in 6 of 10 and 11 of 24 test capsules (surgery sections 4.1 and 4.2), there is complete and long-lasting (i.e. up to 14 months) absence of post-PDT cells in other PDT capsules. 5.3 The Biology of in vitro and pinned rabbit capsule models In this rabbit model, every extracapsular lens extraction (ECLE) was a unique, multifactorial system, beginning with the potential for different inherent lens conditions e.g. sticky lens, partially liquefied cortex, more or less normal lenses. In those cases with either generalized or multifocal post-surgical residual cortex, no two lenses appeared the same, nor did their posterior capsule cell outgrowth patterns in culture. In adult human lenses, all replications and differentiations occur very slowly (McAvoy, Chamberlain et al. 1999) relative to e.g. skin epithelial cells. However, potential differences exist that may affect the microenvironment encountered by BPD-MA at the molecular level. In short, lens epithelial E-cells replicate slowly, but they are alone in this as A-cells do not appear to divide at all (Peng, Apple et al. 2000). Although outer cortical fibre cell derivatives of E-cells still have nuclei and organelles, they begin to differentiate, destined for massive crystallin(s) synthesis (Reddy, Lin et al. 1988; Wang, Garcia et al. 2004) and the eventual loss of nucleus and organelles to become fibres, slowly pushed more tightly into the inner cortex by yet newer, overlying outer cortical fibre cells. All of these differences contribute to the potential for different cell types and membrane surfaces left in an emptied capsular bag. These differences at the outermost lens layers are present even in the physically and biologically undisturbed symmetric intact lens. It is reasonable to propose that differences would be even greater and more varied in primary cataractous lenses, where for example, 138 there are changes in cytokine signalling (Beebe 1992; Duncan, Wormstone et al. 1997). It follows that that no two lenses would respond the same way to a series of the intracapsular removal forces in the microenvironments of the capsule and basal HLE membrane, nor of the apical HLE membrane and outer fibre cell basal membrane. The results of these responses may be due to the hydrodissection path taken around the entire lens, or by the cell type, cell membrane, or molecular composition of any putative cellular detritus which makes up any possible overlying residue. Therefore, it is likely that no two lenses exhibit the same three dimensional residual cell surface profile encountered by the advancing OphthalinlBPD-MA front. This physically induced multifactorial microenvironmental cellular landscape may also require a multifactorial prophylactic approach to prevent the LE component of PCO. 5.3.1 Biological common denominators Post-surgical biological environments A critical baseline for considering the results of this study stems from fundamental artifacts of the robust rabbit model. The apparent hyperstimulation of post-surgical growth of LE cells in this study is highlighted by a comparison with one study of human donor post- surgical ECCE capsular bags at 4 months to 13 years after IOL implantation (Marcantonio and Vrensen 1999). The A-cells were still attached to the inner anterior capsule and appeared cobblestoned and moderately hexagonal as would native, undisturbed LE cells. This is a strong indicator that in humans, the post-surgical in vivo A-cells, free of the natural tamponade effect of the intact anterior lens, do not all activate or become metaplastic. (If taken to the furthest level, there appears to be is no literature regarding human neoplasias arising from A-cells or even E-cells i.e. the inner lens capsule is a cancer-free region.) The early in vitro results in this study consistently show that A-cells on anterior capsulectomy fragments of donor human and fresh rabbit eyes readily 139 proliferate in the rich 15% FCS-supplemented DMEM medium, not intended to replicate natural aqueous humour, but to encourage growth of even a single residual post-PDT cell. However, contact between A-cells and an IOL changes this. A-cells in contact with the anterior surface of an IOL are modulated via a biocompatibility and foreign-body response (Saika 2004) plus signalling to transdifferentiate into myofibroblasts and contribute to the contraction (phimosis) of the anterior capsulotomy (Findi, Sycha et al. 2004). The lack of lOLs in the rabbit model is therefore a deficit. However, the observation of posterior capsule wrinkling and the apparent change from epithelial to fibroblast-like morphology in 6 of 22 control capsules in this study suggests a metaplasia similar to those reported elsewhere (Saxby, Rosen et al. 1998; Obstbaum 1999) with specific reference to smooth muscle actin expression modulated by TGF-beta (Wormstone, Tamiya et a!. 2002) and fibroblastic growth factors (FGFs) (Lee and Jo0 1999; Wormstone, Del Rio-Tsonis et al. 2001; Tanaka, Saika et al. 2004). Even though in vitro monitoring of post-PDT cell growth onto the native capsule is still a reasonable model, i.e. at least some of the transdifferentiation stages observed in vivo are present in this model, there were no Elschnig Pearls nor Soemmering’s rings seen here nor reported in other applications of this model reviewed in the literature. The case for preferential photosensitizer uptake The theoretical possibility of preferential uptake of BPD-MA by cell types of differing proliferative potential even at the time of cortex disruption, must at least be considered as part of potential artifacts in the rabbit model. Part of the original basis for the clinical use of certain extrinsic photosensitizers (PS) is the preferential drug uptake shown in studies such as those of leukemic cells with BPD-MA (Jainieson, McDonald et al. 1990). Similarly, given that in the rabbit model the LE cells have the potential for rapid growth once the lens is removed, it might be argued that BPD-MA may be preferentially taken up by the more 140 active equatorial LE-cells. To extrapolate, this would mean that if no other factors were involved, then e.g. slower-to-grow human B-cells might not take up BPD-MA as efficiently as those of rabbit capsules and would therefore not be as sensitive to PDT ablation or reduced growth as reported here. If this were true, this could provide a case for the longer-term release of a PS or other preferred agent delivered e.g. from an accommodative life-like lens replacement (Parel, Gelender et a!. 1986; Haefliger, Parel et al. 1987; Nishi, Nishi et al. 1998; Assia, Blumenthal et al. 1999) i.e. pliable and responsive to shape-changing forces delivered from the ciliary muscles. An additional artifact of the rabbit model is the clinical difference between LE cell proliferation and PCO. On close reading of man (man, Ozturk et a!. 2001), it is important to note that in that study, dexamethasone applied in lensectomized rabbits subsequently followed for three months was not antiproliferative but did significantly decreased PCO, highlighting the difference between LE cell proliferation and the complete clinical PCO components of secondary cataract. If 100% non-proliferation of LB cells (and possibly of young, as yet poorly-differentiated nucleate outer cortical fibre cells) were possible in vivo with an IOL inserted, one could consider other established cellular and non-cellular developments of the PCO component of secondary cataract and how they could be eliminated. Alternatively, given that some of these components are systemic i.e. immune cell-mediated, a completely authentic post-euthanasia animal surgical model such as presented in this thesis is not possible. Cellular and one principle acellular (IOL insertion) changes occur in the post operative lens capsule and anterior chamber. Surgical trauma of the lens and LECs and the presence of an IOL induces foreign-body responses and macrophage infiltration (Saika, Miyamoto et al. 1998). We can postulate that this may still occur if all intracapsular nucleate cells have been ablated by PDT, but completely killed LE cells might drastically reduce the cytokine release necessary to maintain the concentration gradient (Gallin and Snyderman 1999) needed for the chemotactic attraction of macrophages. A potentially 141 beneficial collateral scenario in vivo is based on the observation that the reactive oxygen species (ROS) generated by porphyrin photosensitizers in the presence of oxygen, also signal neutrophil infiltration, shown in endothelial cells (Volanti, Matroule et al. 2002) and massively in mouse solid tumors treated with Photofrin II (Cecic, Parkins et al. 2001). The outstanding question is whether or not neutrophils and specifically macrophages would remain in such a theoretical capsule e.g. migrating onto the surfaces of the IOL (Saika 2004) or would autolyze and disintegrate due to the lack of an ongoing, increasing and differentiating cell and matrix wound environment in a typically developing PCO, ACO and secondary cataractous eye. This also poses the question of whether or not any foreign-body giant cells from multiple, fused macrophages (Saika 2004) would also remain or even adhere to newer more biocompatible lenses (Ravalico, Baccara et al. 1997; Hollick, Spalton et al. 1998). Giant cells and macrophages secrete growth factors e.g. TGF-beta has be found in both of the above cells found on lOLs removed from patients (Saika, Miyamoto et al. 2000). Tumor necrosis factor-alpha (TNF-alpha) is also secreted by ocular macrophages and is capable of activating the change of epithelial cells to fibroblastic cells (Bates and Mercurio 2003). As is obvious, unless other triturated non lenticular epithelial cells come to rest in the empty lens capsule, any effect(s) of the neutrophil-derived TGF-beta and TNF-alpha would be negated if the potentially responsive target LE cells had been completely killed via light-activated photosensitizer during lensectomy surgery. Given that Soemmering’s ring is made up of regenerating or swollen lenticular fibres staining exclusively for vimentin (Kivela and Uusitalo 1998), the ring would most likely be absent if putatively proliferative new, nucleate fibre cells or regenerative E-cells (able to produce more lens fibre cells) had been destroyed. As for the elastase-positive differentiating fibroblastic lens cells expressing smooth muscle isoforms of actin with upregulated cytokeratin (Kivela and Uusitalo 1998), the LE cells progenitors would be absent for this scenario. Thus the fibrous component of PCO 142 would likely be absent and the advancing front of newly-proliferating and/or transforming LE/fibroblastic cells would be absent as well. These possible artifacts within the rabbit model as well as the drawback of extrapolating a non-primate model with in vitro follow-up to the clinical setting are potential weaknesses. However, as described earlier the single-cell resolution of this intrinsically and extrinsically robust model capable of follow-up for more that one year, has clearly shown that stopping all cell proliferation via PDT within this system is possible, and seems worth extrapolating to chronic studies. 5.4 Potential Further Studies The complex nature of secondary cataract in vivo, the varied physical responses to phacoemulsification and I/A forces, as well as the potential for a cellular biochemical, cytochemical response to cytotoxicities suggest that a treatment of more than a single modality e.g. PDT with an adjunct compound or compounds deserve investigation. Potential candidates include surfactants in the viscoelastic-photosensitizer mixture to clear away detritus and dissolve membranes. Other possible cytotoxic agents adjunct to PDT, i.e. one of any number reviewed in Chapter 1, could be included in the undiluted viscoelastic carrier introduced into the empty capsular bag. Also, LE cells could be pre-exposed to drugs e.g. photosensitizer, during or immediately after the subcapsular cortical hydrodissection (SCCH) step. Three SCCH deliveries could be explored, the efficiency of which would depend in part on the degree of separation between the LE monolayer and the outermost cortex. First, drug(s) could be mixed with aqueous BSS hydrodissection fluid and delivered to the LE cells during standard SCCH. Second, cortical cleaving could be attempted with injectable viscoelastic drug mixtures of various viscosities, used instead of BSS. This could reduce the exposure to other tissues in the anterior chamber by decreasing the leakage back out through the capsulotomy. Third, a drug-free SCCH procedure could be followed by the injection of a 143 fully-viscous drug mixture by reinjecting along the oiiginal cannula tracks but with a larger gauge cannula. This would possibly allow the mixture to fill the new space between the LE cells and the outermost cortical fibre cells and allow incubation. 144 Albert, D. M., F. A. Jakobiec, et al., Eds. (2000). Principles and Practice of Ophthalmology. Philadelphia, W.B. Saunders. Alberts, B., D. Bray, et al. (1994). Molecular Biology of the Cell. New York, Garland Publishing. Allison, B. A., E. Waterfield, et al. (1991). “The Effects of Plasma Lipoproteins On in vitro Tumor Cell Killing and in vivo Tumor Photosensitization with Benzoporphyrin Derivative.” Photochenaistry and Photobiology 5 4(5): 709-715. Apple, D. J. (1984). “Complications of Intraocular Lenses. A Historical and Histopatbological Review.” Survey of Ophthalmology 2 9(1): 1-54. Apple, D. J., Q. Peng, et al. (2000). “Surgical prevention of posterior capsule opacification Part 1: Progress in eliminating this complication of cataract surgery.” Journal of Cataract and Refractive Surgery 26: 180-187. Apple, D. J., K. D. Solomon, et al. (1992). “Posterior Capsule Opacification.” Survey of Ophthalmology 37(2): 73-116. Arita, T. and L.-R. Lin (1990). “Enhancement of Differentiation of Human Lens Epithelium in Tissue Culture by Changes in Cell-Substrate Adhesion.” Investigative Ophthalmology and Visual Science 31(11): 2395-2404. Assia, E. I., M. Blumenthal, et al. (1999). “Effect of expandable full-size intraocular lenses on lens centration and capsule opacification in rabbits.” Journal of Cataract and Refractive Surgery 2 5(Mar): 347-356. Auffarth, G. U. and Q. Peng (2000). “Posterior Capsule Opacification: Pathology, Clinical Evaluation, and Current Means of Prevention.” Ophthalmic Practice 18(4): 172- 182. Bacakova, L., M. Lapcikova, et a!. (2003). “Adhesion and growth of rat aortic smooth muscle cells on lactide-based polymers.” Advance in Experimental Medicine & Biology 534: 179-89. Bates, R. C. and A. M. Mercurio (2003). “Tumor necrosis factor-alpha stimulates the epithelial-to-mesenchymal transition of human colonic organoids.” Molecular Biology of the Cell 14: 1790-1800. Beebe, D. C. (1992). “The control of lens growth: Relationship to secondary cataract.” ActaOphthalmologica 7 O(Suppl 205): 53-57. Beebe, D. C. (2003). The Lens. Adler’s Physiology of the Eye. P. L. Kaufman and A. Aim. St. Louis, Mosby: 117-158. Birinci, H., S. Kuruoglu, et al. (1999). “Effect of intraocular lens and anterior capsule opening type on posterior capsule opacification.” Journal of Cataract and Refractive Surgery 25(8): 1140-1146. Boulton, M. and L. Saxby (1998). “Adhesion of IOLs to the posterior capsule.” British Journal of Ophthalmology 82(5): 468. 145 Boyle, R. W. and D. Dolphin (1996). “Structure and biodistribution relationships of photoclynamic sensitizers.” Photochemistry and Photobiology 63(3): 469-485. Bretton, R. H., A. Swearingen, et al. (1999). “Use of a polylysine-saporin conjugate to prevent posterior capsule opacification.” Journal of Cataract and Refractive Surgery 25(7): 921-929. Cammarata, P. R., D. Cantu-Crouch, et al. (1986). “Macromolecular organization of Bovine lens capsule.” Tissue Cell 18: 83-97. Caporossi, A., F. Casprini, et al. (1998). “Histology of anterior capsule fibrosis following phacoemulsification.” Journal of Cataract and Refractive Surgery 24: 1343-1346. Carthy, C. M., D. J. Granville, et al. (1999). “Early Release of Mitochondrial Cytochrome c and Expression of Mitochondrial Epitope 7A6 with a Porphyrin-Derived Photosensitizer: Bcl-2 and Bcl-XL Overexpression Do Not Prevent Early Mitochondrial Events but Still Depress Caspase Activity.” Laboratory Investigation 79(8): 953-970. Cecic, I., C. S. Parkins, et al. (2001). “Induction of Systemic Neutrophil Response in Mice by Photodynamic Therapy of Solid Tumors.” Photochemistrv and Photobiologv 74(5): 712-720. Chabner, B. A., D. P. Ryan, et al. (2001). Antineoplastic Agents. Goodman & Gilman’s The Pharmacological Basis ofTherapeutics. 3. G. Hardman, L. E. Limbird and A. G. Gilman. New York, McGraw-Hill: 1389-. Chignell, A. H. and D. Wong (1999). Management of Vitreo-Retinal Disease A Surgical Approach. London, Springer-Verlag. Chowdhary, R. K., I. Sharif, et al. (2003). “Correlation of photosensitizer delivery to lipoproteins and efficacy in tumor and arthritis mouse models; comparison of lipid-based and Pluronic P123 formulations.” Journal of Pharmacy & Pharmaceutical Sciences 6(2): 198-204. Ciulla, T. A., R. P. Danis, eta!. (1998). “Age-Related Macular Degeneration: A Review of Experimental Treatments.” Survey of Ovhthalmologv 43(2): 134— 146. Clark, D. S., J. M. Emery, et al. (1998). “Inhibition of posterior capsule opacification with an immunotoxin specific for lens epithelial cells: 24 month clinical results.” Journal of Cataract and Refractive Surgery 24(12): 1614-1620. Cochener, B., R. Bougaran, et al. (2003). “Non-biodegradable drug-sustained capsular ring for prevention of secondary cataract. Part I: In vitro evaluation.” Journal Francais d’Ophthalamologie 26(3): 223-31. Coombes, A. and H. Seward (1999). “Posterior capsule opacification prevention: IOL design and material.” British Journal of Ophthalmology 83(6): 640-641. Cotran, R. S., V. Kumar, et al. (1994). Inflammation and Repair. Robbins Pathologic Basis of Disease. F. J. Schoen. Philadelphia, W. B. Saunders: 51-92. Crowston, J. G., P. R. Healey, et al. (2004). “Water-mediated lysis of lens epithelial cells attached to lens capsule.” Journal of Cataract and Refractive Surgery 30: 1102-1106. 146 Demas, J. N., E. W. Harris, et al. (1975). “Luminescent osmium (II) and iridium (III) complexes as photosensitizers.” Journal of the American Chemical Society 97: 3838-3839. Dougherty, T. J. (1974). “Activated dyes as antitumor agents.” Journal of the National Cancer Institute 52: 1333-1336. Dougherty, T. J. (1996). “A brief history of clinical photodynamic therapy at Roswell Park Cancer Institute.” Journal of Clinical Laser Medicine and Surgery 14: 219-221. Doughty, M. J. and K. T. Nguyen (1995). “Rapid Morphological Reorganization of the Rabbit Corneal Endothelium after Acute Exposure to Sodium Lactate in vitro.” Ophthalmic Research 27: 80-88. Drews, R. C. (1990). “Management of Postoperative Inflammation: Dexamethasone Versus Flurbiprofen, a Quantitative Study Using the New Flare Cell Meter.” Ophthalmic Surgery 21(8): 560-562. Drews, R. C. (1999). “Discussion of: The Effect of Polymethylmethacrylate, Silicone, and Polyacrylic Intraocular Lenses on Posterior Capsular Opacification 3 Years after Cataract Surgery.” Ophthalmology 106: 54-55. Dube, P. and H. M. Boisjoly (1990). “Comparison of predaisolone acetate and indomethacin for marntaimng mydriasis during cataract surgery.” Canadian Journal of Ophthalmology 25(5): 234-238. Duke-Elder, S., Ed. (1961). The Anatomy of the Visual System. System of Ophthalmology. London, Henry Kimpton. Duncan, G., I. M. Wormstone, et al. (1997). “The aging human lens: structure, growth, and physiological behaviour.” British Journal of Ophthalmology 81: 818-823. Duncan, G., I. M. Wormstone, et al. (1997). “Thapsigargin-coated intraocular lenses inhibit human lens cell growth.” Nature Medicine 3(9): 1026-1028. Erie, J. C., P. W. Harclwig, et al. (1998). “Effect of intraocular lens design on neodymium:YAG laser capsulotomy rates.” Journal of Cataract and Refractive Surgery 2 4(Sept): 1239-1242. Faust, K. J. (1984). “Hydrodissection of soft nuclei.” Journal-AmericanIntra-Ocular Implant Society 10(1): 75-77. Findi, 0., T. Sycha, et al. (2004). “Interventions for preventing posterior capsule opacification.” The Cochrane Database of Systematic Reviews 4. Fine, I. H. (1992). “Cortical cleaving hydrodissection.” Journal ofCataract and Refractive Surgery 18(5): 508-512. FitzGerald, P. G. and D. A. Goodenough (1986). “Rat Lens Culture: MIP Expression and Domains of Intercellular Coupling.” Investigative Ophthalmology and Visual Science 27: 755-771. Foote, C. S. (1991). “Definition of Type I and Type II Photosensitized Oxidation.” Photochemistry and Pbotobiologv 54(5): 659. 147 Gallin, J. I. and R. Snyderman, Eds. (1999). Inflammation Basic Principles and Clinical Correlates. Philadelphia, Lippincott Williams & Wilkins. Garg, A. (2002). Biochemistry of the Lens. Textbook of Ophthalmology. S. Agarwal, A. Agarwal, D. J. Apple et al. New Delhi, Jaypee Brothers Medical Publishers. Vol. 1: 117- 124. Garston, B. (1980). “Evidence for the participation of singlet oxygen in the vat-dye photosensitized degradation of cellulose.” Coloration Technology 96: 535. Gimbel, H. V. (1997). “Posterior continuous curvilinear capsulorhexis and optic capture of the intraocular lens to prevent secondary opacification in pediatric cataract surgery.” Journal of Cataract and Refractive Surgery 23: 652-656. Gimbel, H. V. and B. M. DeBroff (1994). “Posterior capsulorhexis with optic capture: maintaining a clear visual axis after pediatric cataract surgery.” Journal of Cataract and Refractive Surgery 20: 658-664. Gimbel, H. V. and T. Neuhann (1990). “Development, advantages, and methods of continuous circular capsulorhexis technique.” Journal of Cataract and Refractive Surgery 16: 3 1-37. Granville, D. J., C. M. Carthy, et al. (1998). “Apoptosis: Molecular Aspects of Cell Death and Disease.” Laboratory Investigation 78(8): 893-913. Granville, D. J., H. Jiang, et al. (1999). “Bcl-2 Overexpression Blocks Caspase Activation and Downstream Apoptotic Events Instigated by Photodynamic Therapy.” British Journal of Cancer 79(1): 95-100. Granville, D. J., J. R. Shaw, et al. (1999). “Release of Cytochrome c, Bax Migration, Bid Cleavage, and Activation of Caspases 2,3,6,7,9, and 9 during Endothelial Cell Apoptosis.” American Journal of Pathology 155(4): 1021-1025. Gwon, A., L. Gruber, et al. (1993). “Lens Regeneration in New Zealand Albino Rabbits After Endocapsular Cataract Extraction.” Investigative Ophthalmology and Visual Science 34: 2124-2129. Haefliger, E., J.-M. Parel, et al. (1987). “Accommodation of an Endocapsular Silicone Lens (Phaco-Ersatz) in the Nonhuman Primate.” Ophthalmology 94: 471-477. Haimovici, R., M. Kramer, et al. (1997). “Localization of lipoprotein-delivered benzoporphyrin derivative in the rabbit eye.” Current Eye Research 16: 83-90. Hansen, S. 0., K. D. Solomon, et al. (1988). “Posterior capsular opacification and intraocular lens decentration Part I: Comparison of various posterior chamber lens designs implanted in the rabbit model.” Journal of Cataract and Refractive Surgery 14: 605-613. Hanuch, 0., V. B. Agrawal, et al. (1997). “Posterior capsule polishing with the neodymium:YLF picosecond laser: Model eye study.” Journal of Cataract and Refractive Surgery 23(Dec): 1561-1571. Hayashi, H., K. Hayashi, et al. (1998). “Area reduction in the anterior capsule opening in eyes of diabetes mellitus patients.” Journal of Cataract and Refractive Surgery 2 4(Aug): 1105-1110. 148 Hayashi, H., K. Hayashi, et a!. (1998). “Quantitative Comparison of Posterior Capsule Opacification After Polymethylmethacrylate, Silicone, And Soft Acrylic Intraocular Lens Implantation.” Archives of Ophthalmology 1 16(Dec): 1579-1582. Henderson, B. W. and T. J. Dougherty (1992). “How Does Photodynamic Therapy Work?” Photochemistry and Photobiology 55(1): 145-157. Hepsen, I. F., H. Bayramlar, et al. (1997). “Caffeic acid phenethyl ester to irthibit posterior capsule opacification in rabbits.” Journal of Cataract and Refractive Surgery 2 3(Dec): 1572-1576. Hollick, E. J., D. J. Spalton, et al. (1999). “The Effect of Capsulorhexis Size on Posterior Capsular Opacification: One-Year Results of an Randomized Prospective Trial .“ American Journal of Ophthalmology 128: 271-279. Hollick, E. J., D. J. Spalton, et al. (1998). “Biocompatibility of poly(methylmethacrylate), silicone, and AcrySof intraocular lenses: Randomized comparison of the cellular reaction on the anterior lens surface.” Journal of Cataract and Refractive Surgery 2 4(Mar): 361- 366. Hollick, E. J., D. J. Spalton, et al. (1999). “The Effect of Polymethylmethacrylate, Silicone, and Polyacrylic Intraocular Lenses on Posterior Capsular Opacification 3 Years after Cataract Surgery.” Ophthalmology 106: 49-55. Hosal, B. M. and A. W. Biglan (2002). “Risk factors for secondary membrane formation after removal of pediatric cataract.” Journal of Cataract and Refractive Surgery 28:302- 309. Hug, H., S. Strand, et a!. (1997). “Reactive Oxygen intermediates are involved in the Induction of CD95 Ligand mRNA Expression by Cytostatic Drugs in Hepatoma Cells.” Journal of Biological Chemistry 272(45): 28191-28193. Husain, D., J. W. Miller, et al. (1996). “Intravenous infusion of liposomal benzoporphyrin derivative for photodynamic therapy of experimental choroidal neovascularization.” Archives of Ophthalmology 114(14): 978-985. Ibaraki, N., K. Ohara, et al. (1995). “Membranous outgrowth suggesting lens epithelial cell proliferation in pseudophakic eyes.” American Journal of Ophthalmology 119(6): 706- 711. Idriss, H. T., Y. A. Hannun, et a!. (2000). “Regulation of volume-activated chloride channels by P-glycoprotein: phosphorylation has the final say!” Journal of Physiology 524(Pt 3): 629-636. man, U. U., F. Ozturk, et al. (2001). “Prevention of posterior capsule opacification by intraoperative single-dose pharmacologic agents.” Journal of Cataract and Refractive Surgery 27: 1079-1097. lonides, A., J. G. F. Dowler, et a!. (1994). “Posterior Capsule Opacification Following Diabetic Extracapsular Cataract Extraction.” 8: 535-537. Ismail, M. M., J. L. Alio, et al. (1996). “Prevention of secondary cataract by antimitotic drugs: experimental studies.” Ophthalmic Research 28: 64-69. 149 Jamieson, C. H. M., W. N. McDonald, et al. (1990). “Preferential Uptake of Benzoporphyrin Derivative by Leukemic Versus Normal Cells.” LeukemiaResearch 14(3): 209-2 19. Javitt, J. C., J. M. Tielsch, et al. (1992). “National outcomes of cataract extraction. Increased risk of retinal complications associated with Nd:YAG laser capsulotomy. The Cataract Patient Outcomes Research Team.” Ophthalmology 99(10): 1487-1497. Johnston, R. L., D. J. Spalton, et al. (1999). “In vitro protein adsorption to 2 intraocular lens materials.” Journal of Cataract and Refractive Surgery 2 S(Aug): 1109-1115. Jones, N. P., D. McLeod, et al. (1995). “Massive proliferation of lens epithelial remnants after Nd:YAG laser capsulotomy.” British Journal of Ophthalmology 79(3): 26 1-263. Joo, C.-K., E. H. Lee, et al. (1999). “Degeneration and transdifferentiation of human lens epithelial cells in nuclear and anterior polar cataracts.” Journal of Cataract and Refractive Surgery 2 5(May): 652-658. Joo, C. K., J. A. Shin, et al. (1996). “Capsular opening contraction after continuous curvilinear capsulorhexis and intraocular lens implantation.” Journal of Cataract and Refractive Surgery 22: 585-590. Joussen, A. M., B. Huppertz, et al. (2001). “Low-dose-rate ionizing irradiation for inhibition of secondary cataract formation.” International Journal of Radiation Oncology Biology Physics 49(3): 8 17-825. Kappeihof, J. P. and G. F. 3. M. Vrensen (1992). “The pathology of after-cataract: A Minireview.” ActaOphthalmologica 7 0(Suppl 205): 13-24. Kapusta, M. A., J. C. Chen, et al. (1995). “Outcomes of Dropped Nucleus during Phacoemulsification.” Ophthalmology 103: 1184-1187. Karamata, B., M. Sickenberg, et a!. (2000). “A Fibreoptic Light Distributor for the Preventative Photoclynamic Therapy of Secondary Cataract.” Lasers in Medical Science 15: 238-245. Kato, K., D. Kurosaka, et al. (1997). “Elschnig pearl formation along the posterior capsulotomy margin after neodymium:YAG capsulotomy.” Journal of Cataract and Refractive Surgery 2 3(Dec): 1556-1560. Keating, A., C. Jamieson, et al. (1993). “Photodynamic elimination of clonogenic Ph÷ chronic myeloid leukemia cells.” Leukemia&Lvmphoma 1 1(Suppl 1): 265-269. Kelleher, P. J., J. F. Tarsio, et al. (1992). “Cytotoxic effects of a monoclonal antibody ricin a immunotoxin on human ocular cells in culture.” Investigative Ophthalmology and Visual Science 3 3(ARVO abstract 2383): 1168. Kelman, C. D. (1969). “Physics of ultrasound in cataract removal.” International Ophthalmology Clinics 9(3): 739-44. Kendrew, S. J., Ed. (1994). Encyclopedia ofMolecularBiologv, Blackwell Science Ltd. Kessel, D. (1998). Photodynamic Therapy. Science and Medicine. 5: 46-55. 150 Kessel, D. and M. Castelli (2001). “Evidence that bcl-2 is the Target of Three Photosensitizers that Induce a Rapid Apoptotic Response.” Photochemistrv and Photobiologv 74(2): 318-322. Kim, M.-J., H.-Y. Lee, et a!. (1999). “Posterior capsule opacification in eyes with a silicone or poly(methyl methacrylate) intraocular lens.” Journal of Cataract and Refractive Surgery 2 5(Feb): 251-255. Kimura, W., S. Yamanishi, et a!. (1998). “Measuring the anterior capsule opening after cataract surgery to assess capsule shrinkage.” Journal of Cataract and Refractive Surgery 2 4(Sept): 1235-1238. Kivela, T. and M. Uusitalo (1998). “Structure, Development and Function of Cytoskeletal Elements in Non-Neuronal Cells of the Human Eye.” Progress in Retinal and Eye Research 17(3): 385-428. Knight-Nana, D., M. O’Keefe, et al. (1996). “Outcome and complications of intraocular lenses in children with cataract.” Journal of Cataract and Refractive Surgery 22(6): 730- 736. Koch, D. D. and T. Kohnen (1997). “Retrospective comparison of techniques to prevent secondary cataract formation after posterior chamber intraocular lens implantation in infants and children.” Journal of Cataract and Refractive Surgery 2 3(Supplement 1): 657-663. Kramer, M., J. W. Miller, et al. (1996). “Liposomal benzoporphyrin derivative verteporfin photodynamic therapy: selective treatment of choroidal neovascularization in monkeys.” Ophthalmology 103: 427-438. Krumeich, J. H. and J. Daniel (1998). “Blunt, bent needle for continuous curvilinear capsulorhexis.” Journal of Cataract and Refractive Surgery 2 4(Sept): 1180-1183. Lang, R. A. (1999). “Which Factors Stimulate Lens Fiber Cell Differentiation In Vivo?” Investigative Ophthalmology and Visual Science 40(13): 3075-3078. Lee, E. H. and C.-K. Joo (1999). “Role of Transforming Growth Factor-Beta in Transdifferentiation and Fibrosis of Lens Epithelial Cells.” Investigative Ophthalmology and Visual Science 40(9): 2025-2032. Lehmann, R. P. (1996). “Tips on Cortical Cleanup.” Ophthalmic Practice 14(6). Levy, J. G., C. Dowding, et al. (1995). “Selective elimination of malignant stem cells using photosensitizers followed by light treatment.” Stem Cells 13(4): 336-343. Lingua, R., J.-M. Parel, et al. (1988). “Preclinical evaluation of photodynamic therapy to inhibit lens epithelial proliferation.” Lasers and Light in Ophthalmology 2(2): 103-113. Lingua, R. W., J.-M. Parel, et al. (1991). “Photodynamic treatment of lens epithelial cells for cataract surgery.” Proceedings of the Society of Photo-Optical Instrumentation Engineers 1423: 58-61. Linnola, R. J. and A. Holst (1998). “Evaluation of a 3-piece silicone intraocular lens with poly(methyl methacrylate) haptics.” Journal of Cataract and Refractive Surgery 24(1): 1509-1514. 151 Lipson, R. L., E. J. Baldes, et a!. (1961). “The use of a derivative of hematoporphyrin in tumor detection.” Journal of the National Cancer Institute 26: 1-11. Liu, C. S. C., M. I. Wormstone, et a!. (1996). “A Study of Human Lens Cell Growth In Vitro. A Model for Posterior Capsule Opacification.” Investigative Ophthalmology and Visual Science 37(5): 906-914. Lovicu, F. J. and J. W. McAvoy (1989). “Structural analysis of lens epithelial explants induced to differentiate into fibres by fibroblast growth factor (FGF).” Experimental Eye Research 49(3): 479-94. Lovicu, F. 3., M. W. Schulz, et a!. (1998). “TGF-beta-Induced Subcapsular Cataract Involves an Epithelial-Mesenchymal Transition.” Experimental Eye Research 6 7(Suppl 1): S 174. Lovicu, F. J., P. Steven, et a!. (2004). “Aberrant lens fiber differentiation in anterior subcapsular cataract formation: a process dependent on reduced levels of Pax6.” Investigative Ophthalmology and Visual Science 45(6): 1946-1953. Lundgren, B., E. Jonsson, et al. (1992). “Secondary cataract: An in vivo model for studies on secondary cataract in rabbits.” ActaOphthalmologica Suppi. 205: 25-28. Lunn, K. F., P. W. Baas, et al. (1997). “Microtubule Organization and Stability in the Oligodendrocyte.” The Journal of Neuroscience 17(13): 4921-4932. MacDonald, I. J. and T. J. Dougherty (2001). “Basic principles of photodynamic therapy.” Journal of Porphyrins and Phthalocyamnes 5: 105-129. MacEwen, C. J. and P. S. Baines (1989). “Retinal Detachment Following YAG Laser Capsulotomy.” 3: 759-763. Marcantonio, 3. M. and G. F. J. M. Vrensen (1999). “Cell biology of posterior capsular opacification.” 13: 484-488. Masferrer, 3. L., S. T. Reddy, et al. (1994). “In vivo glucocorticoids regulate cylooxygenase-2 but not cyclooxygenase-1 in peritoneal macrophages.” Journal of Pharmacology and Experimental Therapeutics 270: 1340-1344. Mathias, R. T., 3. L. Rae, et al. (1997). “Physiological properties of the normal lens.” Physiological Reviews 77: 21-50. McAvoy, J. W., C. G. Chamberlain, et al. (1999). “Lens Development.” 1 3(pt3b): 425-37. McDermott, M. L., L. D. Hazlett, et a!. (1998). “Viscoelastic adherence to corneal endothelium following phacoemulsification.” Journal of Cataract and Refractive Surgery 2 4(May): 678-683. Meacock, W. R., D. J. Spalton, et a!. (2000). “Double-masked prospective ocular safety study of a lens epithelial cell antibody to prevent posterior capsule opacification.” Journal of Cataract and Refractive Surgery 26: 716-721. 152 Mengual, E., J. Garcia, et al. (1998). “Clinical results of AcrySof intraocular lens implantation.” Journal of Cataract and Refractive Surgery 2 4(Jan): 114—i 17. Merrisman-Smith, B. R., M. A. Young, et al. (2002). “Molecular Identification of P Glycoprotein: A role in Lens Circulation.” Investigative Ophthalmology and Visual Science 43: 3008-3015. Meucci, G., S. Esente, eta!. (1991). “Anterior capsule cleaning with an ultrasound irrigating scratcher.” Journal of Cataract and Refractive Surgery 17: 75-79. Michaeli-Cohen, A., M. Belkin, et al. (2000). “Experimental Intraoperative Use of the Carbon Dioxide Laser for the Prevention of Posterior Capsular Opacification.” Ophthalmic Practice 18(1): 6-8. Michaeli-Cohen, A., M. Belkin, et al. (1998). “Prevention of Posterior Capsule Opacification With the CO2 Laser.” Ophthalmic Surgery and Lasers 29(12): 985-990. Miyake, K., I. Ota, et al. (1996). “Correlation between intraocular lens hydrophilicity and anterior capsule opacification and aqueous flare.” Journal of Cataract & Refractive Surge 22(Suppl 1): 764-9. Moan, J., Q. Peng, et al. (1987). “Photosensitizing Efficiencies, Tumor- and Cellular Uptake of Different Photosensitizing Drugs Relevant for Photodynamic Therapy of Cancer.” Photochemistrv and Photobiology 4 6(5): 713-721. Moffat, B. A. and J. M. Pope (2002). “Anisotropic Water Transport in the Human Eye Lens Studied by Diffusion tensor NMR Micro-imaging.” Experimental Eye Research 74: 677-687. Moore, K. L. and T. V. N. Persaud (1998). The Developing Human. Clinically Oriented Embryology. Philadelphia, W. B. Saunders Company. Moore, K. L., T. V. N. Persaud, et a!. (2000). Color Atlas of Clinical Embryology. Philadelphia, W.B. Saunders. Morgan, K. S. and Z. A. Karcioglu (1987). “Secondary cataracts in infants after lensectomies.” Journal of Pediatric Ophthalmology & Strabismus 2 4(1): 45-48. Moroi, S. E. and P. R. Lichter (2001). Ocular Pharmacology. Goodman & Gilman’s The pharmacological Basis ofTherapeutics. J. G. Hardman, L. E. Limbird and A. G. Gilman. New York, McGraw-Hill: 1819-1849. Mossman, T. (1983). “Rapid Colorimetric Assay for Cellular Growth and Survival: Application to Proliferation and Cytotoxicity Assays.” Journal of Immunological Methods 65: 55-63. Nagamoto, S., T. Kohzuka, et a!. (i998). “Pupillary block after pupillary capture of an AcrySof intraocular lens.” Journal of Cataract and Refractive Surgery 24(9): 1271-1274. Nasisse, M. P., M. J. Dykstra, et a!. (1995). “Lens capsule opacification in aphakic and psuedophakic eyes.” Graefe’s Archive for Clinical and Experimental Ophthalmology 233: 63-70. 153 Nishi, K. and 0. Nishi (1991). “Tissue culture of human lens epithelial cells. Part II: suppressive effect of diclofenac sodium on their proliferation and metaplasia.” Nippon Ganka Gakki Zasshi 1991 95: 581-590. Nishi, 0. (1999). “Posterior capsule opacification Part 1: Experimental investigations.” Journal of Cataract and Refractive Surgery 2 5(Jan): 106-117. Nishi, 0. and K. Nishi (1999). “Preventing posterior capsule opacification by creating a discontinuous sharp bend in the capsule.” Journal of Cataract and Refractive Surgery 2 5(Apr): 52 1-526. Nishi, 0., K. Nishi, et al. (1993). “Removal of lens epithelial cells following loosening of the junctional complex.” Journal of Cataract and Refractive Surgery 19: 56-61. Nishi, 0., K. Nishi, et al. (1992). “Synthesis of interleukin-1 and prostaglandin E2 by lens epithelial cells of human cataracts.” British Journal of Ophthalmology 76: 338-341. Nishi, 0., K. Nishi, et al. (1998). “The Inhibition of Lens Epithelial Cell Migration by a Discontinuous Capsular Bend Created by a Band-Shaped Circular Loop or a Capsule- Bending Ring.” Ophthalmic Surgery and Lasers 29(2): 119-125. Nishi, 0., K. Nishi, et al. (1998). “Lens refilling with injectable silicone in rabbit eyes.” Journal of Cataract and Refractive Surgery 2 4(Jul): 975-982. Nishi, 0., K. Nishi, et al. (1997). “Inhibition of migrating lens epithelial cells by blocking the adhesion molecule integrin: a preliminary report.” Journal of Cataract and Refractive Surgery 23(6): 860-5. Nishi, 0., K. Nishi, et al. (1998). “Capsule-Bending Ring for the Prevention of Capsular Opacifiction: A Preliminary Report.” Ophthalmic Surge and Lasers 29(9): 749-753. Nishi, 0., K. Nishi, et al. (1996). “Effect of intraocular sustained release of indomethacin on postoperative inflammation and posterior capsule opacification.” Journal, of Cataract and Refractive Surgery 22(Suppl 1): 806-10. Nishi, 0., K. Nishi, et al. (1996). “Synthesis of interleukin- 1, interleukin-6, and basic fibroblast growth factor by human cataract lens epithelial cells.” Journal of Cataract and Refractive Surgery 22: 852-858. Nishi, 0., K. Nishi, et al. (1998). “Inhibition of Migrating Lens Epithelial Cells at the Capsular Bend Created by the Rectangular Optic Edge of a Posterior Chamber Intraocular Lens.” Ophthalmic Surgery and Lasers 29(7): 587-594. Nishi, 0., K. Nishi, et a!. (1995). “Effect of indomethacin-coated posterior chamber intraocular lenses on postoperative inflammation and posterior capsule opacification.” Journal of Cataract and Refractive Surgery 21(5): 574-578. O’Neil, M. J., Ed. (2001). The Merck Index: An Encyclopedia of Chemicals. Drugs. and Biologicals. Whitehouse Station, Merck & Co. Obstbaum, S. A. (1999). “Capsule contraction.” Journal of Cataract and Refractive Surgery 25(Oct): 1305. 154 Olson, R. J. and A. S. Crandall (1998). “Silicone Versus Polymethylmethacrylate Intraocular Lenses With Regard to Capsular Opacification.” Ophthalmic Surgery and Lasers 2 9(1): 55-58. Oshika, T., T. Nagata, et al. (1998). “Adhesion of lens capsule to intraocular lenses of polymethylmethacrylate, silicone, and acrylic foldable materials: an experimental study.” British Journal of Ophthalmology 82: 549-553. Oshima, Y., T. Sakamoto, et a!. (1997). “Effect of electric pulses and antiproliferative drugs on cultured bovine retinal pigment epithelial cells.” Current Eye Research 16(1): 64- 70. Pablo, L. E., A. Ferreras, et a!. (2004). “Contact-Topical Plus Intracameral Lidocaine Versus Peribulbar Anesthesia in Combined Surgery: A Randomized Clinical Trial.” Journal of Glaucoma 13: 510-5 15. Pande, M. V., P. G. Ursell, et al. (1997). “High-resolution digital retroillumination imaging of the posterior lens capsule after cataract surgery.” Journal of Cataract and Refractive Surgery 23(Dec): 1521-1527. Pandey, R. K. (2000). “Recent advances in photodynamic therapy.” Journal of Porphyrins and Phthalocyamnes 4: 368-373. Pandey, S. K., D. J. Apple, et al. (2004). “Posterior capsule opacification: a review of the aetiopathogenesis, experimental and clinical studies and factors for prevention.” Indian Journal of Ophthalmology 52(2): 99-112. Parel, J.-M., R. Cubeddu, et al. (1990). “Endocapsular Lavage with Photofrin II as a Photodynamic Therapy for Lens Epithelial Proliferation.” Lasers in Medical Science 5:25- 30. Pare!, J.-M., H. Gelender, et al. (1986). “Phaco-Ersatz: cataract surgery designed to preserve accommodation.” Graef&s Archive for Clinical and Experimental Ophthalmology 224: 165-173. Paterson, C. A. (1970). “Extracellular space of the crystalline lens.” American Journal of Physiology 218(3): 797-802. Peng, Q., D. J. Apple, et al. (2000). “Surgical prevention of posterior capsule opacification Part 2: Enhancement of cortical cleanup by focusing on hydrodissection.” Journal of Cataract and Refractive Surgery 26: 188-197. Peng, Q., N. Visessook, et al. (2000). “Surgical prevention of posterior capsule opacification Part 3: Intraocular lens optic barrier effect as a second line of defense.” Journal of Cataract and Refractive Surgery 26: 198-213. Quinlan, M., I. M. Wormstone, et al. (1997). “Phacoemulsification versus extracapsular cataract extraction: a comparative study of cell survival and growth on the human capsular bag in vitro.” British Journal of Ophthalmology 81: 9O791O Raab, 0. (1900). “Uber die Wirkung fluoreszierencler Stoffe auf Infusorien.” Zeitschrift für Biologie 39: 524-546. 155 Racey, G. L., W. R. Wallace, et al. (1978). “Comparison of a polyglycolic-polylactic acid suture to black silk and plain catgut in human oral tissues.” Journal of Oral Surgery 36(10): 766-70. Rafferty, N. S., S. Zigman, eta!. (1993). “Near-UV Radiation Disrupts Filamentous Actin in Lens Epithelial Cells.” Cell Motility and the Cytoskeleton 26: 4O-4& Ram, J., D. J. Apple, et al. (1999). “Update on Fixation of Rigid and Foldable Posterior Chamber Intraocular Lenses. Part I.” Ophthalmology 106(5): 883-890. Ram, J., D. 3. Apple, et a!. (1999). “Update on Fixation of Rigid and Foldable Posterior Chamber Intraocular Lenses. Part II Elimination of Fixation-induced Decentration to Achieve Precise Optical Correction and Visual Rehabilitation. Choosing the Correct Haptic Fixation and Intraocular Lens Design to Help Eradicate Posterior Capsule Opacification.” Ophthalmology 106(5): 891-900. Rasmussen-Taxdal, D. S., D. E. Ward, et al. (1955). “Fluorescence of human lymphatic and cancer tissue following high doses of intravenous hematoporphyrin.” Surgical Forum 5:610-624. Ravalico, G., F. Baccara, et al. (1997). “Postoperative Cellular Reaction on Various Intraocular Lens Materials.” Onhthalmologv 104: 1084-1091. Reddy, V. N., L.-R. Lin, Ct al. (1988). “Crystallins and their Synthesis in Human Lens Epithelial Cells in Tissue Culture.” Experimental Eye Research 47: 465-478. Richter, A., E. Sternberg, et a!. (1989). “Characterization of benzoporphyrin derivative, a new photosensitizer.” Proceedings of the Society of Photo-Optical Instrumentation Engineers 997: 133-138. Richter, A. M., S. Cerruti-Sola, et al. (1990). “Biodistribution of Tritiated Benzoporphyrin Derivative (3H-BPD-MA), a New Potent Photosensitizer, in Normal and Tumor-Bearing Mice.” Photochemistry and Photobiology 5: 231-244. Richter, A. M., R. Chowdhary, et al. (1994). “Non-oncologic potentials for photodynamic therapy.” Proceedings of the Society of Photo-Optical Instrumentation Engineers 2078: 293-304. Richter, A. M., A. K. Jam, et al. (1992). “Phototsensitizing Efficiency of Two Regioisomers of the Benzoporphyrin Derivative Monoacid Ring A (BPD-MA).” Biochemical Pharmacology 43(11): 2349-2358. Richter, A. M., B. Kelly, et a!. (1987). “Preliminary Studies on a More Effective Phototoxic Agent Than Hematoporphyrin.” Journal of the National Cancer Institute 79(6): 1327-1332. Richter, A. M., E. Waterfield, et al. (1990). “In vitro Evaluation of Phototoxic Properties of Four Structurally Related Benzoporphyrin Derivatives.” Photochemistry and Photobiology 52(3): 495-500. Richter, A. M., E. Waterfield, et al. (1993). “Liposomal Delivery of a Photosensitizer, Benzoporphyrin Derivative Monoacid Ring A (BPD), to Tumor Tissue in a Mouse Tumor Model.” Photochemistrv and Photobiology 57(6): 1000-1006. 156 Richter, A. M., S. Yip, et al. (1996). “Photosensitizing potencies of the structural analogues of benzoporphyrin derivative in different biological test systems.” Journal of Clinical Laser Medicine & Surgery 1 4(November 5): 335-341. Roberts, L. J. and J. D. Morrow (2001). Analgesic-Antipyretic and Antiinflammatory Agents and Drugs Employed in the Treatment of Gout. Goodman & Gilman’s The pharmacological Basis ofTherapeutics. J. G. Hardman, L. E. Limbird and A. G. Gilman. New York, McGraw-Hill: 687-720. Ruiz, J. M., M. Medrano, et al. (1990). “Inhibition of Posterior Capsule Opacification by 5-Fluorouracil in Rabbits.” OphthalmicResearch 22: 201-208. Ruotsalainen, J. and A. Tarkkanen (1987). “Capsule thickness of cataractous lenses with and without exfoliation syndrome.” ActaOphthalmologica 65(4): 444-9. Saika, S. (2004). “Relationship between posterior capsule opacification and intraocular lens biocompatibilty.” Progress in Retinal and Eye Research 23: 283-305. Saika, S., T. Miyamoto, et al. (2000). “Immunolocalization of tgf-B1, -B2, and -B3, and TGF-B receptors in human lens capsules with lens implants.” Graefe’s Archive for Clinical and Experimental Ophthalmology 238(3): 283-293. Saika, S., T. Miyamoto, et al. (1998). “Degenerated lens epithelial cells in rabbit and human eyes after intraocular lens implantation.” Journal of Cataract and Refractive Surgery 2 4(Oct): 1396-1398. Saika, S., T. Miyamoto, et al. (1998). “Immunohistochemical evaluation of cellular deposits on posterior chamber intraocular lenses.” Graefe’s Archive for Clinical and Experimental Ophthalmology 236: 758-765. Saitow, F. and Y. Nakaoka (1996). “Photodynamic Action of Methylene Blue on the Paramecium Membrane.” Photochemistrv and Photobiology 63(6): 868-873. Saxby, L., E. Rosen, et al. (1998). “Lens epithelial cell proliferation, migration, and metaplasia following capsulorhexis.” British Journal of Ophthalmology 82: 945-952. Schaumberg, D. A., M. R. Dana, et al. (1998). “A Systematic Overview of the Incidence of Posterior Capsule Opacification.” Ophthalmology 105: 1213-1221. Seland, H. H. (1992). “The lens capsule and zonulae.” ActaOphthalmologica 70(Suppl 205): 7-12. Seland, J. H. (1974). “Ultrastructural changes in the normal human lens capsule from birth to old age.” ActaOphthalmologica 52(5): 688-706. Shin, D. H., Y. Y. Kim, et al. (1998). “Decrease of Capsular Opacification with Adjunctive Mitomycin C in Combined Glaucoma and Cataract Surgery.” Ophthalmology 105: 1222-1226. Shoch, D. (1961). A Technique of Cataract Extraction. Cataract Surgery and Neuro Ophthalmology. D. T. Vail. Boston, Little, Brown and Co. Vol. 1 No. 3. 157 Sickenberg, M., M. Gonver, et al. (1998). “Change in capsulorhexis size with four foldable loop-haptic lenses over 6 months.” Journal of Cataract and Refractive Surgery 24(7): 925-930. Spalton, D. J. (1999). “Posterior capsular opacification after cataract surgery.” 13: 489-492. Spierer, A., H. Desatnik, et al. (1992). “Secondary cataract in infants after extracapsular cataract extraction and anterior vitrectomy.” Onhthalmic Surgery 23(9): 625-627. Spikes, J. D. (1981). “Selective photooxidation of thiols sensitized by aqueous suspensions of cadmium sulfide.” Photochemistrv and Photobiology 34: 549. Stables, G. I. and D. V. Ash (1995). “Antitumour Treatment Photodynamic therapy.” Cancer Treatment Reviews 2 1: 311-323. Sternberg, E. D. and D. Dolphin (1993). “Second generation photodynamic agents: a review.” Journal of Clinical Laser Medicine and Surgery 1 1(5): 233-241. Straight, R. C. and J. D. Spikes (1985). Photosensitized Oxidation of Biomolecules. Singlet 02”. A. A. Frimer. Boca Raton, CRC Press. IV: 91-143. Strenn, K., R. Menapace, et al. (1997). “Capsular bag shrinkage after implantation of an open-loop silicone lens and a poly(methyl methacrylate) capsule tension ring.” Journal of Cataract and Refractive Surgery 2 3(Dec): 1543-1547. Sundelin, K. and J. Sjöstrand (1999). “Posterior capsule opacification 5 years after extracapsular cataract extraction.” Journal of Cataract and Refractive Surgery 2 S(Feb): 246-250. Suzuki, H., M. Iwaki, et a!. (2001). “Anti-allergic drugs inhibit the proliferation of bovine lens epithelial cells in culture.” Nippon Ganka Gakkai Zasshi - Acta Societatis OphthalmologicaeJaponicae 105(8): 517-523. Sweeney, M. H. and R. J. Truscott (1998). “An impediment to glutathione diffusion in older normal human lenses: a possible precondition for nuclear cataract.” Experimental Eye Research 67: 587-95. Szoka Jr., F. and D. Papahadjopoulos (1998). “Procedure for preparation of liposomes with large internal aqueous space and high capture by reverse-phase evaporation.” Proceedings of the National Academy of Sciences 75(9): 4194-4198. Takahashi, A., T. Taniguchi, et al. (1999). “Tranilast Inhibits Vascular Smooth Muscle Cell Growth and Intimal Hyperplasia by Induction of p21 wqfl/cipl/sdil and p53.” Circulation Research 84: 543-550. Tanaka, T., S. Saika, et al. (2004). “Fibroblast growth factor 2: Roles of regulation of lens cell proliferation and epithelial-mesenchymal transition in response to injury.” MolecularVision 10: 462-467. Tetz, M. R., G. U. Auffarth, et al. (1997). “Photographic image analysis system of posterior capsule opacification.” Journal of Cataract and Refractive Surgery 2 3(Dec): 1515-1520. 158 Tetz, M. R., D. J. C. O’Morchoe, et al. (1988). “Posterior capsular opacification and intraocular lens decentration Part II: Experimental findings on a prototype circular intraocular lens design.” Journal of Cataract and Refractive Surgery 14: 614-623. Tetz, M. R., M. W. Ries, et al. (1996). “Inhibition of posterior capsule opacification by an intraocular-lens-bound sustained drug delivery system: an experimental animal study and literature review.” Journal of Cataract and Refractive Surgery 22(8): 1070-1078. Thastrup, 0., P. J. Cullen, et al. (1990). “Thapsigargin, a tumor promoter, discharges intracellular Ca2+ stores by a specific inhibition of the endoplasmic reticulum Ca2+- ATPase.” Proceedings of the National Academy of Sciences USA 87: 2466-2470. Trannoy, L. L., A. Brand, et al. (2002). “Relation Between K+ Leakage and Damage to Band 3 in Photodynamically Treated Red Cells.” Photochemistrv and Photobiology 75(2): 167-171. Tripathi, R. C., 3. Li, et al. (1998). “Increased Level of Vascular Endothelial Growth Factor in Aqueous Humor of Patients with Neovascular Glaucoma.” Ophthalmology 105(2): 232-7. Ursell, P. G., D. J. Spalton, et al. (1997). “Anterior capsule stability in eyes with intraocular lenses made of poly(methyl methacrylate), silicone, and AcrySof.” Journal of Cataract and Refractive Surgery 2 3(Dec): 1532-1538. Ursell, P. G., D. J. Spalton, et al. (1998). “Relationship between intraocular lens biomaterials and posterior capsule opacification.” Journal of Cataract and Refractive Surgery 2 4(Mar): 352-360. Uusitalo, M. and T. Kivela (1997). “Cell types of secondary cataract: an immunohistochemical analysis with antibodies to cytoskeletal elements and macrophages.” Graefe’s Archive for Clinical and Experimental Ophthalmology 235(8): 506-511. van Tenten, Y., H. J. Schuitmaker, et al. (2002). “A Preliminary Study on the Prevention of Posterior Capsule Opacification by Photodynamic Therapy with Bacterioclilorin A in Rabbits.” Ophthalmic Research 34(3): 113-118. van Tenten, Y., H. J. Schuitmaker, et al. (2001). “The effect of photodynamic therapy with bacteriochlorin a on lens epithelial cells in a capsular bag model.” Experimental Eye Research 7 2(1): 41-8. Vane, J. and R. Botting (1987). “Inflammation and the mechanism of action of antiinflammatory drugs.” Federation ofAmerican Societies for Experimental Biology Journal 1(89-96). Vantieghem, A., Y. Xu, et al. (2001). “Different Pathways Mediate Cytochrome c Release After Photodynamic Therapy with Hypericin.” Photochemistrv and Photobiology 74(2): 133-142. Vargas, L. G., M, Escobar-Gomez, et a!. (2003). “Pharmacologic prevention of posterior capsule opacification: In vitro effects of preservative-free lidocaine 1% on lens epithelial cells.” Journal of Cataract and Refractive Surge 29: 1585-1592. 159 Versura, P., A. Torreggiani, et a!. (1999). “Adhesion mechanisms of human lens epithelial cells on 4 intraocular lens materials.” Journal of Cataract and Refractive Surgery 2 5(Apr): 527-533. Volanti, C., J.-Y. Matroule, et a!. (2002). “Involvement of Oxiclative Stress in NF-kB Activation in Endothelial Cells Treated by Photodynamic Therapy.” Photochemistrv and Photobiology 7 5(1): 36-45. von Tappeiner, H. (1903). “Uber die Wirkung fluoreszierender Substanzen auf Fermente und Toxine.” Berichte der Deutschen Chemischen Gesellschaft 36: 3035. Wang, I.-J. and F.-R. Hu (1997). “Effect of shaking on cornea! endothelial preservation.” CurrentEyeResearch 16(11): 1111-1118. Wang, X., C. M. Garcia, et al. (2004). “Expression and Regulation of alpha-, beta- and gamma-Crystallins in Mammalian Lens Epithelial Cells.” Investigative Ophthalmology and Visual Science 4 5(10): 3608-3619. Weishaupt, K. R., C. J. Gomer, et al. (1976). “Identification of singlet oxygen as the cytotoxic agent in photo-inactivation of a murine tumor.” Cancer Research 36: 2326-23 29. Werner, L., N. Mamalis, et a!. (2004). “Posterior capsule opacification in rabbit eyes implanted with hydrophilic acrylic intraocular lenses with enhanced square edge.” Journal of Cataract and Refractive Surgery 30: 2403-2409. Wesierska-Gadek, J., T. Schreiner, et a!. (2006). “Phenol Red in the Culture Medium Strongly Affects the Susceptibility of Human MCF-7 Cells to Roscovitine.” Cellular& Molecular Biology Letters 12: 280-293. Wiedemann, P. and K. Heimann (1990). “Toxicity of Intraocular Daunomycin.” Lens and Eye Toxicity Research 7(3&4): 305-310. Winther-Nielsen, A., 3. Johansen, et al. (1998). “Posterior capsule opacification and neodymium:YAG capsulotomy with heparin-surface-modified intraocular lenses.” Journal of Cataract and Refractive Surgery 2 4(Jul): 940-944. Wiskstrom, K. and K. Madsen (1993). “The effect of transforming growth factor-alpha (TGFa) on rabbit and primate lens epithelial cells in vitro.” Current Eye Research 12(12): 1123-1128. Wormstone, I. M., K. Del Rio-Tsonis, et a!. (2001). “FGF: An Autocrine Regulator of Human Lens Cell Growth Independent of Added Stimuli.” Investigative Ophthalmology and Visual Science 42(6): 1305-1311. Wormstone, I. M., S. Tamiya, eta!. (2004). “Characterisation of TGF-beta2 signalling and function in a human lens cell line.” Experimental Eye Research 78: 705-714. Wormstone, M., S. Tamiya, et a!. (2002). “TGF-Beta2-Induced Matrix Modification and Cell Transdifferentiation in the Human Lens Capsular Bag.” Investigative Ophthalmology and Visual Science 4 3(7): 2301-2308. 160 Young, R. W. and D. E. Ocumpaugh (1966). “Autoradiographic studies on the growth and development of the lens capsule in rat.” Investigative Ophthalmology and Visual Science 5: 583-593. Zaczek, A. and C. Zetterström (1999). “Posterior capsule opacification after phacoemulsification in patients with diabetes mellitus.” Journal of Cataract and Refractive Surgery 2 5(Feb): 233-237. Zaturinsky, B., N. Naveh, et al. (1990). “Prevention of Posterior Capsular Opacification by Cryolysis and the Use of Heparinized Irrigation Solution During Extracapsular Lens Extraction in Rabbits.” Ophthalmic Surgery 21(6): 431-434. 161 APPENDIX ONE DETAILED MATERIALS / METHODS FOR In Vitro CHAPTER 3 1. Benzoporphyrin derivative monoacid ring A (BPD-MA, QLT, Vancouver, BC) was reconstituted with water as a 2 mg/mi stock from a lyophilized liposomal formulation and stored at 4°C for a maximum of two weeks. 2. Sodium hyaluronate supplied as samples from the Eye Care Centre Day Surgery Unit (AMVISC 10 mg/mi; AMVISC-PLUS 12 mg/mi; Ophthalin 10 mg/mi) 3. ProNectin F Recombinant Attachment Factor (Protein Polymer Technologies Inc., #205001, Stratagene) was used to pretreat acid-etched coverslips onto which cells were passaged for some of the direct calcein / ethidium fluorescence cell membrane compromise assays. 4. Calcein AM! Ethidium homodimer Cytoprobe Kit (PerSeptive Biosciences; Millipore #CPAK CvlOOl) 5. Calcein AM / Ethidium homodimer Live/Dead EukoLight Kit (Molecular Probes; #1.- 3224) 6. Balanced Salt Solution (BSS) (Abbott #3911) 7. Diazobenzocyclo-octane (DABCO) (Aldrich #D2780-2) 2.5% mixed with 10% glycerol in PBS to counteract fluorescence quenching during photographic exposure 8. Dulbecco’s Modified Eagles Medium (DME) 9. Fetal Calf Serum (Sigma), heat inactivated 10. Glass coverslips (Corning 2.5 cm and Goidseal #3246-000-900 1/2”: acid-etched 10 minutes in boiling 0. iN HCI) were used for ProNectin F treatment to enhance cell attachment for direct assay of I3PD-MA in viscous vehicles. 11. Tissue culture plates, 96 well (full area; 0.32 cm2) (Costar) 12. Tissue culture plates, 96 well (one-half area; 0.16 cm2) (Costar #3696) 13. Spectrophotometer plate reader (Molecular Devices Corp.) was used to read MTT cell cytotoxicity assay OD values. 162 14. A microscope (Diaphot Model, Nikon Corp.) with a mercury lamp equipped with filter blocks B-3A and G-2A was used for the fluorescence photomicrography of the cells incubated with calcein and ethidium. 17. LED panel 690 nm ± 12.5 nm at half maximum output (Quantum Devices; power supply: Qbeam200l) were used for exposing BPD-MA-treated cells in multiwell plates or on coverslips. Light intensity at the cell level was approximately 108 mW/cm2as measured by a lightmeter (International Light; Model 1L1350) fitted with a diffuser probe #SEDO38. The diffuser ring (W#5018) and an additional ring (FA#9726) provided a 6 mm distance (measured) from the surface of the LED panel to the internal photocell of the probe. 163 APPENDIX TWO TABLE OF EXPERIMENTAL CONDITIONS 100 and 200 JIcm2 (Rabbits: Ri-Ru) Rabbit Number E BPD or Light Ultrasonic Time from Amount of Control (US) Euthanyl Ophthalin/BPD time to pinning — J/cm2 (mm.) (hours) (ml) Ri 1 Control 100 2.3 1.75 Ri 2 BPD 2.1 2.75 [0.4828] R2 T BPD 100 2.1 1.50 0.28 R2 2 Control “ 1.4 2.50 R3 T BPD 100 1.0 2.00 0.32 R3 2 Control “ 2.1 3.00 R4 T BPD 200 0.9 1.50 0.37 R4 (inside out) 2 Control ‘ 1.4 N/A R5 1 I3PD 200 1.3 2.50 0.45 R5 2 Control “ 0.9 4.00 R6 T BPD 200 1.6 2.50 0.38 R6 2 Control “ 1.0 3.75 R7 T BPD 200 1.1 2.00 0.50 R7 2 Control “ 0.6 3.25 R8 (post. capsule torn) 1 BPD 100 0.9 2.75 0.51 R8 2 Control “ 1.2 4.00 R9 1 Control 200 1.0 2.00 R9 2 BPD 1.2 3.25 0.49 RiO T BPD 200 1.2 1.50 0.51 RiO 2 Control “ 0.9 3.25 Ru V BPD 100 1.2 2.75 0.51 Ru 2 Control “ 1.0 3.75 Averages (n=10): 1.2 mm. 0.43 ml [excluding [excluding Ri] Ri] 164 APPENDIX THREE Light Doses Delivered by Equatorial Laser Tip #4 (ELT-4) (Used in R19-R31: Surgery Sets One to Six) Calculations: capsule irradiation light doses were based on light meter readings taken at the time of surgery. The amount of light reaching the capsule equator was calculated as a range based on the consideration of Equatorial Laser Tip #4 (ELT-4) first as a point source and secondly, as a spherical tip with the actual diameter of 2.0 mm. The most conservative notion was to treat the equatorial capsule as if in a sphere, given that this is the only dimension of the empty ellipsoid-like capsule whose distance from the center of the capsule can be reasonably approximated, and given that the most potentially proliferative cells are at the equator. This also provided a base-line calculation such that in relation to the equator, the anterior of a semi-inflated, empty post-surgical capsule would be closer to the irradiating tip and therefore receive as much or more light than the equator. Given the torus conformation of the light emitted from the tip, 71% of the theoretical sphere was considered as receiving light from a ELT-4 placed centrally in the anterior capsule opening. Semi-inflated, empty post-surgical capsule rabbit capsules were estimated to be 10 mm in diameter, based on pinned and flat capsules capsule diameters measured in vitro and in situ. Given the relatively low power of the laser diode used, it was determined that 2 J/cm2 would be delivered in 5 minutes, manageable in the non-clinical context of these surgeries. ELT-4 considered as point source, functional capsule radius r = 0.5 cm: 1. Torus Irradiated Area of Theoretical Sphere = (4 it r2 ) (0.71) 2. Area= (43t(0.Scm)2)(0.71) 3. Area = 2.2294 cm2 ELT-4 considered actual 2.0 mm dia.., functional capsule radius r = 0.4 cm 4. Torus Irradiated Area of Theoretical Sphere = (4 rt r2 ) (0.71) 5. Area = (4 t (0.4 cm)2 ) (0.71) 6. Area=1.4268cm2 Thus, given these two scenarios, calculated the 3/cm2 range for each capsule: 7. time [in sec] = x [Joules] = x [J/cm 2j y [Watts] y [V//cm 2j 8. thus, mW/cm2calculated from I = mW from light meter area irradiated 165 following example is based on the specific data of surgery: R2 1/El: Example: R21/E1 meter reading = 11.36 mW time of irradiation = 5.0 mm 300 sec 10. From 3, above (point source model; r = 0.5 cm; torus area = 2.2294 cm 2) 11. I = 11.36 mW = 5.0955 mW / cm2 2.2294 cm2 12. From7, above x[J/cm2] 13. x[J/cm2] 114. ELT-4 as ‘point-source’ x 15. From 6, above (2.0 mm dia. model; r = 0.4 cm; torus area = 1.4268 cm 2) 16. I = 11.36mW = 7.9619 mW/cm2 1.4268 cm2 17. From 7, above = y [W/cm2] * time [seconds] 18. = (0.0079619 W/cm2)* (300 see) 119. ELT-4as 2.0 mm dia. x = 2.39 J /cm2 deliveredto capsule Thus, the Equatorial Laser Tip #4 irradiation to the empty capsule in R21/E1 lies within the theoretical range of: 1.53 - 2.39 J / cm2, noted as approximately(-) 2 J/cm 2• The 9. = y [W/cm 2j * time [secondsi = (0.0050955 W/cm2)* (300 sec) = 1.53 J / cm2 delivered to capsule x [J / cm2 x [J / cm2 J 166 APPENDIX FOUR Animal Use Memo: Date: June 29, 2008 Re: Laboratory animal use for the research reported in M.Sc. thesis Thesis Title: “PREVENTION OF POSTERIOR CAPSULE OPACIFICATION BY PHOTODYNAMIC THERAPY WITH LOCALIZED BENZOPORPHYRIN DERIVATIVE MONOACID RING A (BPD-MA) IN A RABBIT SURGICAL MODEL” Copyright: 2008 Program: M.Sc. Pathology and Laboratory Medicine (Experimental Ocular Pathology) Author: Howard Earl Meadows Animals used: New Zealand white rabbits Procedure: post-mortem lensectomy model cataract surgery Guidelines: No rabbits were ordered / housed specifically for this study. Therefore, there is no specific animal certification paperwork. All animals were euthanized by the veterinarian or animal care technician(s) under the supervision of the veterinarian in the Jack Bell Centre for Research (Vancouver), subject to the approved euthanasia protocols in place. All animals were from other certified studies in which the endpoint was post- euthanasia exsanguination for recovery of serum antibodies, free from infectious agents and toxins. The use of the undamaged post-mortem ocular tissues represent a reduction in the need for duplicate animals for the research reported in this study. All model ocular surgeries reported in the above thesis were carried out on post-mortem animals. Howard Meadows Graduate Student Faculty of Medicine University of British Columbia Vancouver, Canada 167 APPENDIX FIVE Methods Summary: Human Donor HEE in vitro with BPD-MA BPD-MA Dark Cytotoxicity: 1. HLE in 96-well plate, including drug-free control wells 2. incubate cells with BPD in dose range 3. remove drug and incubate cells in drug-free medium with FCS for at least one overnight 4. incubate cells with MTT, disrupt cells with acidic alcohol and read optical density 5. calculate % viability for each drug dose Photodynamic Therapy (PDT) Assays: 1. HLE in 96-well plate, including drug-free control wells 2. incubate cells with ranges of BPD-MA concentrations 3. expose cells to a light dose with LED 690 nm panel light source 4. incubate cells in drug-free medium with FCS for at least one overnight 5. determine viability via MIT 6. calculate % kill for each drug concentration 168 APPENDIX SIX Flow Chart: Euthanized Rabbit Model Lensectomy Steps Euthanized NZW rabbit: eye left in place for aseptic lensectomy / treatments Insert gravity-feed BSS anterior chamber maintainer line into anterior chamber (AC) of eye to maintain fluid depth between the inner cornea surface and the lens and iris Make entry wound into AC with keratome Form circular capsulotomy (opening) in anterior lens capsule to allow access to clear lens +1- various lens freeing step(s) e.g. hydrodissection Ultrasonic phacoemulsification removal of clear proteinaceous lens as model of human primary cataract surgery; +1- internal capsule cleaning/polishing Turn off BSS AC maintainer line gravity fluid feed +1- corneal endothelium protective coating with sodium hyaluronate viscoelastic PDT test eyes: instill viscous BPD-MA mixed with clinical sodium hyaluronate viscoelastic to fill the now-emptied lens capsule i.e. to coat any residual lens epithelial target cells remaining on the inner anterior / equatorial capsule surfaces After prescribed BPD-MA contact time, restart BSS AC maintainer Remove (aspirate) contiguous BPD-MA / viscoelastic mixture from the lens capsule (Depending on light source, follow A or B, below.) A. For 200, 100 or 103/cm2LED Or B. For —‘2 J/cm clinical lasertip close-array panel irradiation of irradiation ofBPD-treated capsule: BPD-treated capsule: 1. leave cornea / iris intact 1. remove cornea 2. insert laser tip into AC 2. remove iris 3. place tip in centre of capsulotomy 3. irradiate capsule with 690 nm red light 4. irradiate capsule with 690 nm light 5. remove cornea and iris Common step: cut all zonules around periphery of lens capsule Common step: lift capsule away from anterior face of vitreous Common step: place capsule with capsulotomy facing up in DME/15% FCS in 35mm plastic culture dish Common step: place 8 sterile pins through the outermost edge of the capsule down into the plastic dishjust deep enough to remain upright and keep capsule pinned in place Common step: incubate at 370 C /5% CO2 to monitor for (LE) cell growth onto the posterior capsule surface 169 APPENDIX SEVEN Results Chart: Euthanized Rabbit Model Lensectomies 54 model lensectomy surgeries: 22 control +34 test (PDT) Two Overall Groups: -Non-clinical protocol using LED close-array LED 690 nm panels for capsule light irradiation / excitation of BPD-MA: 200, 100 and 103/cm2 -Clinical protocol using equatorial laser dispersion (torus) tip for intraocular 690 nm light irradiation! excitation of BPD-MA: ‘-2J/cm. (includes R19 / everted PDT capsule: CKI=2 wnen corrected for fibroblasts) Number of PDT capsules showing more outgrowth than control: 1/34 Final full-kill i.e. CKI=2 results of all eutharnzed rabbit lensectomies: Full Kill Corrected for CKI=2 putative fibroblasts lOOvs200J/cm2 4110 4/10 LED panel A 10 vs 100 J/cm2 2/9 2/9 B LEDpanel.) One -Six —‘2JIcm2 3/15 8/15 Clinical laser tip 170


Citation Scheme:


Citations by CSL (citeproc-js)

Usage Statistics



Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            async >
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:


Related Items