ORTHOKERATOLOGY EPITHELIAL CHANGES AND
SUSCEPTIBILITY TO MICROBIAL INFECTION
JENNIFER DENISE CHOO, B.SC., O.D.
A thesis submitted in fulfilment of the requirements for the degree of
Doctor of Philosophy
School of Optometry and Vision Science The University of New South Wales, Sydney, Australia and The Institute for Eye Research Sydney, Australia and The Vision Cooperative Research Centre Sydney, Australia
August 2008
Certificate of Originality
“I hereby declare that this submission is my own work and to the best of my knowledge it contains no materials previously published or written by another person, or substantial proportions of material which have been accepted for the award of any other degree or diploma at UNSW or any other educational institution, except where due acknowledgement is made in the thesis. Any contribution made to the research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis.
I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project's design and conception or in style, presentation and linguistic expression is acknowledged.”
Signed ......
J.D. Choo
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Acknowledgements
“Few things in this world are more powerful than a positive push. - A smile. A word of optimism and hope. A “you can do it” when things are tough.” - Richard M. DeVos
First and foremost, I would like to thank my supervisor Brien Holden for taking a chance on me as a student, colleague and friend these last four years. Words cannot express the gratitude I have for his unconditional support and the tremendous opportunities he has afforded me. It has truly been an honour to work with and learn from such a dynamic and successful mentor. I look forward to working together for years to come.
Thank you also to my co-supervisor Patrick Caroline who introduced me to the world of research and first suggested that I pursue a PhD. This journey would not be possible were it not for the crazy ideas, the brilliant foresight and the tireless support of such a wonderful friend and colleague. I look forward to the other crazy adventures we’ll be embarking upon.
This work was made possible through the incredible support of Denise and Robyn Lawler at the IER Animal Facility and the wonderful administration team of Fabiana, Judith, Maureen, Julie and Vivienne for ensuring that I received all the necessary support when requested. Thank you to Eric Papas for all of his valuable input and for constantly reassuring me that there is a light at the end of the tunnel.
I would like to say a very special thank you to the brilliant Ulli Stahl for all of her help, friendship and optimism; to my buddies, Ajay, Fabian and Tim for their incredible support and willingness to help with everything. A special thank you to my dearest Sunshine who has made this journey fun and supported me unconditionally.
Orthokeratology Epithelial Changes and Susceptibility to Microbial Infection ii
I have made some wonderful friends at the IER who have shared many smiles and laughs with me and have been a constant source of encouragement especially JoJo, Lewigi, Brian S., Martin, Paul H., Paul W., Lynn, Mark, Aldo and Andrea. Thank you to my friends in i-Media, particularly Shane, Baz and Kat, who were always willing to help me produce some new and stunning visual effects to help explain this work to those around the world.
This PhD has provided me with countless opportunities to travel the world and speak internationally. It is a skill that I never knew existed until I began this journey and one that I cannot imagine being without now. My sincerest gratitude goes to all my friends and colleagues from around the world who have embraced this work, supported my journey and made this PhD completely worthwhile. To be involved in such an incredible industry filled with wonderful people is truly a blessing.
Finally, I would like to say a huge Thank You to my wonderful family for their unconditional support and patience as I slowly establish myself in this world.
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Dedicated to my Grandparents, Yun and Sum Tso,
for whom the completion of this work means more
than I can ever understand.
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Orthokeratology Epithelial Changes and Susceptibility to Microbial Infection v
Abstract
Orthokeratology (OK) is a specialty contact lens procedure that involves the overnight wear of lenses to reshape the corneal tissue resulting in clear vision upon lens removal. Currently it is the only way of achieving clear vision without having to wear spectacles or contact lenses during the day or undergoing refractive surgery. This thesis investigated the effects of this procedure on the corneal epithelium and the potential increase in risk of microbial infection in an animal model.
The cat was first established as an appropriate animal model in a pilot study to examine OK epithelial changes. Initial findings of epithelial thickness changes similar to those found with human myopic and hyperopic OK clinical studies led to the further development of this animal model to better mimic human lens wear for the remaining studies undertaken.
Histological studies were used to examine epithelial effects of overnight myopic OK lens wear. Repeatable and differential effects on epithelial thickness and morphology across the cornea were found, including thinning of the central and peripheral epithelium and thickening of the mid-peripheral epithelium. Central thinning was attributed to compression of cells and was less in overnight wear compared to continuous wear. Mid-peripheral thickening was due to increased cell layers and peripheral thinning was attributed to cellular compression. Recovery of epithelial morphologic and thickness changes commenced one day after ceasing lens wear and was complete within one week. Minimal changes to keratocyte populations in regions adjacent to epithelial thickness changes were found.
Microbiological studies investigated the effect of epithelial changes on corneal susceptibility to bacterial infections by exposing OK-treated corneas to large amounts of Pseudomonas aeruginosa. The hypothesis that OK lenses increased susceptibility to infection (within the time tested) was rejected as no infections were produced in any animals (except the positive scratch control). Length of OK treatment, duration and
Orthokeratology Epithelial Changes and Susceptibility to Microbial Infection vi
quantity of bacterial exposure, lens wearing schedule and bacterial strain type did not affect susceptibility to infection.
The epithelium is a major contributor to OK-induced corneal changes. These epithelial changes are reversible and do not appear to predispose to infection provided corneal integrity is maintained.
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Table of Contents
CERTIFICATE OF ORIGINALITY...... I
ACKNOWLEDGEMENTS...... II
ABSTRACT ...... VI
TABLE OF CONTENTS ...... VIII
LIST OF FIGURES ...... XV
LIST OF TABLES ...... XVII
CHAPTER 1: INTRODUCTION...... 1 1.1 Orthokeratology Defined ...... 1 1.2 The Evolution of Orthokeratology ...... 2 1.3 Orthokeratology Lenses...... 4 1.3.1 Orthokeratology Lens Designs ...... 5 1.3.2 Orthokeratology Lens Fitting ...... 7 1.4 Efficacy of Orthokeratology ...... 8 1.4.1 Corneal Topographic Changes...... 12 1.5 Clinical Studies of Orthokeratology Corneal Changes...... 13 1.5.1 Epithelium ...... 13 1.5.2 Stroma ...... 15 1.5.2.1 Edema...... 15 1.5.2.2 Structural effects ...... 16 1.5.2.3 Keratocytes ...... 16 1.5.3 Endothelium...... 16 1.5.4 Posterior Corneal Curvature...... 17 1.6 Animal Studies of Orthokeratology Corneal Changes...... 17 1.7 Orthokeratology and Infections...... 19 1.8 Myopia Control and Orthokeratology...... 23 1.9 Thesis Overview...... 24 1.9.1 Hypotheses...... 24 1.9.2 Aims ...... 25
Orthokeratology Epithelial Changes and Susceptibility to Microbial Infection viii
CHAPTER 2: DEVELOPMENT OF THE CAT MODEL FOR ORTHOKERATOLOGY CORNEAL CHANGES...... 26 2.1 Introduction ...... 26 2.1.1 Aims ...... 27 2.1.2 Hypotheses...... 27 2.2 Materials and Methods ...... 28 2.2.1 Study Design and Sample Size ...... 28 2.2.2 Animals...... 28 2.2.3 Contact Lenses...... 28 2.2.4 Lens Fits...... 29 2.2.5 Lens Wearing Schedules...... 30 2.2.6 Histology ...... 31 2.2.7 Epithelial Thickness Measurements ...... 31 2.3 Results ...... 32 2.3.1 Control Animal with No Lens Wear ...... 32 2.3.2 Four Hour Test Animal ...... 33 2.3.3 Eight Hour Test Animal...... 35 2.3.4 Fourteen Day Test Animal...... 37 2.3.5 Stromal Changes...... 40 2.4 Discussion...... 41 2.4.1 Stromal Changes...... 43 2.4.2 Limitations...... 44 2.4.3 Summary...... 45
CHAPTER 3: EPITHELIAL CHANGES WITH OVERNIGHT ORTHOKERATOLOGY LENS WEAR... 46 3.1 Introduction ...... 46 3.1.1 Aims ...... 46 3.1.2 Hypotheses...... 47 3.2 Materials and Methods ...... 47 3.2.1 Study Design and Sample Size ...... 47 3.2.2 Animal Preparation...... 48 3.2.3 Contact Lenses...... 49 3.2.4 Lens Fits...... 50 3.2.5 Lens Wear...... 51 3.2.6 Corneal Topography ...... 52 3.2.7 Lens Care ...... 53 3.2.8 Lens Wearing Schedule ...... 53
Orthokeratology Epithelial Changes and Susceptibility to Microbial Infection ix
3.2.9 Specimen Preparation for Corneal Histology ...... 54 3.2.10 Epithelial Analysis ...... 54 3.2.10.1 Hematoxylin & Eosin Staining...... 54 3.2.10.2 Epithelial Photography ...... 55 3.2.10.3 Epithelial Thickness Measurements ...... 57 3.2.11 Stromal Keratocyte Analysis...... 58 3.2.11.1 DAPI Staining...... 58 3.2.11.2 Stromal Photography ...... 59 3.2.11.3 Stromal Keratocyte Counts ...... 60 3.2.12 Statistical Analysis...... 61 3.3 Results ...... 61 3.3.1 Topography Results...... 61 3.3.2 Epithelial Thickness of Control Animals...... 62 3.3.3 Epithelial Thickness of Contact Lens Wearing Animals...... 65 3.3.3.1 One Day Animals ...... 70 3.3.3.2 Three Day Animals...... 71 3.3.3.3 One Week Animals...... 72 3.3.3.4 Two Week Animals ...... 73 3.3.3.5 Four Week Animals...... 74 3.3.4 Stromal Keratocyte Counts...... 75 3.3.4.1 Control Animals...... 75 3.3.4.2 Lens Wearing Animals ...... 76 3.4 Discussion...... 78 3.4.1 Epithelium ...... 78 3.4.1.1 Central Epithelial Thickness ...... 78 3.4.1.2 Mid-Peripheral Epithelial Thickness ...... 80 3.4.1.3 Peripheral Epithelial Thickness...... 80 3.4.1.4 Morphological Changes...... 81 3.4.1.5 Temporal Effects...... 82 3.4.2 Stroma ...... 82 3.4.3 Limitations...... 84 3.4.4 Summary...... 86
CHAPTER 4: RECOVERY OF EPITHELIAL CHANGES AFTER CESSATION OF OVERNIGHT ORTHOKERATOLOGY LENS WEAR...... 87 4.1 Introduction ...... 87 4.1.1 Aims ...... 87 4.1.2 Hypotheses...... 88 4.2 Materials and Methods ...... 88
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4.2.1 Study Design and Sample Size ...... 88 4.2.2 Animal Preparation...... 89 4.2.3 Contact Lenses...... 89 4.2.4 Lens Wearing Schedules...... 89 4.2.5 Histology ...... 90 4.2.6 Statistical Analysis...... 90 4.3 Results ...... 90 4.3.1 Epithelial Thickness...... 90 4.3.1.1 One Day Animals ...... 96 4.3.1.2 Three Day Animals...... 97 4.3.1.3 One Week Animals...... 98 4.3.1.4 Four Week Animals...... 99 4.3.2 Stromal Keratocyte Counts...... 100 4.4 Discussion...... 102 4.4.1 Epithelium ...... 102 4.4.1.1 Epithelial Rebound...... 102 4.4.1.2 Variability in Regression Rate ...... 103 4.4.1.3 Speed of Recovery ...... 104 4.4.1.4 Recovery of Morphology ...... 105 4.4.2 Stroma ...... 105 4.4.3 Limitations...... 106 4.4.4 Summary...... 106
CHAPTER 5: ORTHOKERATOLOGY AND CORNEAL SUSCEPTIBILITY TO MICROBIAL KERATITIS ...... 107 5.1 Introduction ...... 107 5.1.1 Aims ...... 108 5.1.2 Hypotheses...... 109 5.2 Materials and Methods ...... 109 5.2.1 Study Design and Sample Size ...... 109 5.2.2 Animal Preparation...... 110 5.2.3 Contact Lenses...... 110 5.2.4 Corneal Topography ...... 110 5.2.5 Lens Wearing Schedule ...... 110 5.2.6 Bacterial Strains and Growth Conditions ...... 111 5.2.7 Lens Bacterial Inoculation ...... 112 5.2.7.1 Test Lenses ...... 112 5.2.7.2 Unworn Control Lenses ...... 112
Orthokeratology Epithelial Changes and Susceptibility to Microbial Infection xi
5.2.8 Enumeration of Bacteria on Lenses ...... 113 5.2.9 Bacterial Adhesion Statistical Analysis...... 113 5.2.10 Validation of Cat Model for Pseudomonas aeruginosa Keratitis: Positive Scratch Control...... 114 5.2.10.1 Corneal Scratch Procedure...... 114 5.2.10.2 Inoculated Contact Lens Wear ...... 115 5.2.10.3 Results of Scratch Control ...... 115 5.2.10.4 Summary of Scratch Control...... 116 5.2.11 Bacterial Exposure Challenges...... 117 5.2.11.1 Challenge I: Two Week Overnight Lens Wear with Bacterial Inoculation on the Last Night of Lens Wear...... 117 5.2.11.2 Challenge II: Six Week Overnight Lens Wear with Bacterial Inoculation on the Last Night of Lens Wear...... 118 5.2.11.3 Challenge III: Two Week Overnight Inoculated Lens Wear with Nightly Instillation of Bacterial Drops...... 118 5.2.11.4 Challenge IV: Two Week Overnight Inoculated Lens Wear with Multiple Daily Bacterial Drops After Lens Removal Each Day...... 118 5.2.11.5 Challenge V: Two Week Continuous Lens Wear followed by a Night of Inoculated Lens Wear ...... 119 5.2.11.6 Challenge VI: Two Week Continuous Inoculated Lens Wear with an Additional Twice Daily Instillation of Bacterial Drops...... 119 5.2.11.7 Challenge VII: Two Week Overnight Lens Wear with Bacterial Inoculation on the Last Night of Lens Wear (using P. aeruginosa 6206)...... 119 5.2.11.8 Clinical Observations and Inflammation Analysis ...... 122 5.3 Results ...... 122 5.3.1 Bacterial Exposure Challenges...... 122 5.3.1.1 Challenge I: Two Week Overnight Lens Wear with Bacterial Inoculation on the Last Night of Lens Wear...... 122 5.3.1.2 Challenge II: Six Week Overnight Lens Wear with Bacterial Inoculation on the Last Night of Lens Wear...... 123 5.3.1.3 Challenge III: Two Week Overnight Inoculated Lens Wear with Nightly Instillation of Bacterial Drops...... 123 5.3.1.4 Challenge IV: Two Week Overnight Inoculated Lens Wear with Multiple Daily Bacterial Drops After Lens Removal Each Day...... 124 5.3.1.5 Challenge V: Two Week Continuous Lens Wear followed by a Night of Inoculated Lens Wear ...... 124 5.3.1.6 Challenge VI: Two Week Continuous Inoculated Lens Wear with an Additional Twice Daily Instillation of Bacterial Drops...... 124 5.3.1.7 Challenge VII: Two Week Overnight Lens Wear with Bacterial Inoculation on the Last Night of Lens Wear (using P. aeruginosa 6206)...... 125 5.3.1.8 Total Adverse Events ...... 126
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5.3.1.9 Detailed Ocular Inflammatory Responses...... 127 5.3.2 Bacterial Adhesion to Lenses...... 131 5.3.2.1 Comparisons Between OK and AF Lenses...... 131 5.3.2.2 Comparisons Between Challenges (Time Points)...... 131 5.4 Discussion...... 132 5.4.1 Bacterial Binding...... 133 5.4.2 Hypoxia ...... 133 5.4.3 Inflammation and Bacteria...... 135 5.4.4 Immunity...... 136 5.4.5 Erosions – Bacteria Driven? ...... 136 5.4.6 Need for Care ...... 137 5.4.7 Bacterial Adhesion to Lenses...... 137 5.4.8 OK Lens Bacteria Retention ...... 139 5.4.9 Compliance...... 140 5.4.10 Bacterial Strain Effect...... 141 5.4.11 Limitations...... 141 5.4.12 Summary...... 142
CHAPTER 6: SUMMARY AND RECOMMENDATIONS...... 143 6.1 Summary...... 143 6.2 Future Directions...... 145 6.3 Conclusions...... 147
REFERENCES ...... 148
APPENDIX 1: SUPPLEMENTAL STATISTICAL ANALYSIS FOR CHAPTERS 3 & 4.... 163 7.1 Supplementary Statistical Analysis for Chapter 3...... 163 7.2 Supplementary Statistical Analysis for Chapter 4...... 168
APPENDIX 2: EPITHELIAL THICKNESS MEASUREMENT SOFTWARE...... 173 8.1 Clamp Function...... 173 8.2 Main Function...... 175
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APPENDIX 3: PUBLICATIONS AND PRESENTATIONS RELATED TO THESIS ...... 183 9.1 Publications ...... 183 9.1.1 Accepted ...... 183 9.1.2 Submitted...... 183 9.2 Oral Presentations ...... 184 9.3 Poster Presentations ...... 186
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List of Figures
Figure 1.1: Cross-sectional drawing of a Paragon CRT lens ...... 7 Figure 1.2: Fluorescein pictures of (a) myopic and (b) hyperopic OK lens fits...... 8 Figure 2.1: Acceptable (a) myopic and (b) hyperopic OK lens fits on the cat eye ...... 30 Figure 2.2: Four day control epithelium...... 32 Figure 2.3: Four hour myopic correction ...... 33 Figure 2.4: Four hour hyperopic correction ...... 34 Figure 2.5: Eight hour myopic correction...... 35 Figure 2.6: Eight hour hyperopic correction ...... 36 Figure 2.7: Fourteen day myopic correction...... 37 Figure 2.8: Fourteen day hyperopic correction ...... 38 Figure 2.9: Central epithelium of myopic (left) and hyperopic (right) corrected eyes of an animal after 14 days of continuous OK lens wear ...... 39 Figure 2.10: Central cat stroma after 14 days of continuous OK lens wear for the correction of 4.00D of myopia (left) and 4.00D of hyperopia (right)...... 40 Figure 3.1: Examples of acceptable (a) AF and (b) OK-fitted lenses ...... 51 Figure 3.2: Set up of animal for topography measurement...... 52 Figure 3.3: Corneal sections at 20X magnification stained with H & E...... 56 Figure 3.4: Example of an epithelial measurement using the customized image analysis software developed for this study...... 58 Figure 3.5: A composite of corneal sections stained with DAPI ...... 60 Figure 3.6: Examples of corneal topographies for (a) AF and (b) OK eyes after 2 weeks of overnight lens wear on the cat eye ...... 62 Figure 3.7: Average non-lens wearing control epithelial thicknesses for each area in right (OD) and left (OS) eyes ...... 63 Figure 3.8: Non-lens wearing control epithelium ...... 64 Figure 3.9: Mean change in epithelial thickness change (µm) across time points in five areas.. 66 Figure 3.10: Profile of average change in epithelial thickness (µm) in five corneal areas over time up to four weeks of lens wear...... 67 Figure 3.11: Mean change in epithelial thickness change (%) across time points in five areas.. 68 Figure 3.12: Mean change in epithelial thickness (%) in OK eyes in five corneal areas up to four weeks of lens wear...... 69 Figure 3.13: One day epithelial changes ...... 70 Figure 3.14: Three day epithelial changes...... 71 Figure 3.15: One week epithelial changes ...... 72 Figure 3.16: Two week epithelial changes ...... 73
Orthokeratology Epithelial Changes and Susceptibility to Microbial Infection xv
Figure 3.17: Four week epithelial changes ...... 74 Figure 3.18: Average keratocyte counts for each corneal area in non-lens wearing control eyes75 Figure 3.19: Mean change in keratocyte counts across time points in five corneal areas...... 77 Figure 4.1: Mean change in epithelial thickness (µm) across time points in five corneal areas after cessation of four weeks of lens wear ...... 91 Figure 4.2: Profile of change in epithelial thickness (µm) in five corneal areas over time after cessation of four weeks of lens wear ...... 92 Figure 4.3: Profile of change in epithelial thickness (%) in five corneal areas after cessation of four weeks of lens wear ...... 94 Figure 4.4: Profile of change in epithelial thickness (%) in five corneal areas after cessation of four weeks of lens wear ...... 95 Figure 4.5: Epithelial changes one day after cessation of lens wear ...... 97 Figure 4.6: Epithelial changes three days after cessation of lens wear...... 98 Figure 4.7: Epithelial changes one week after cessation of lens wear ...... 99 Figure 4.8: Epithelial changes four weeks after cessation of lens wear...... 100 Figure 4.9: Mean change in keratocyte counts in five corneal areas at different time points after cessation of four weeks of lens wear ...... 101 Figure 5.1: Results of a positive corneal scratch control with P. aeruginosa 6294...... 116 Figure 5.2: Illustration of the types of bacterial exposure for different challenges ...... 120 Figure 5.3: Different corneal erosions observed during inoculated continuous wear with the addition of twice daily bacterial drops (Challenge VI) ...... 125 Figure 5.4: Total adverse events versus inoculation score for challenges...... 126 Figure 5.5: Plot of the cumulative inflammation score versus inoculation score for each challenge for AF and OK eyes ...... 127 Figure 5.6: Examples of (a) an eye at baseline and (b) an inflamed eye after bacterial exposure...... 128
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List of Tables
Table 1.1: OK lenses marketed for use in the USA ...... 6 Table 1.2: Summary of studies investigating the efficacy of OK...... 11 Table 1.3: Summary of clinical studies investigating total corneal and epithelial thickness changes in myopic OK ...... 14 Table 1.4: Summary of published cases of microbial keratitis in OK ...... 22 Table 2.1: Average cat epithelial thickness measurements for all eyes ...... 39 Table 3.1: OK lens parameters used in the study...... 49 Table 3.2: Control epithelial thickness comparisons for five corneal areas (SUP, MPS, CEN, MPI, INF) within eyes (ANOVA) and between eyes (t-test)...... 64 Table 3.3: Average change in epithelial thickness (µm) in five areas at different time points..... 65 Table 3.4: Average change in epithelial thickness (%) five areas at different time points ...... 68 Table 3.5: Comparison of average keratocyte counts between areas within each eye (ANOVA) and between eyes (t-test) of control animals...... 76 Table 3.6: Mean change in keratocyte counts in all corneal areas and at all time points ...... 77 Table 4.1: Average change in epithelial thickness (µm) between OK and AF eyes in five corneal areas after cessation of four weeks of lens wear ...... 91 Table 4.2: Mean change in epithelial thickness (%) in five corneal areas after cessation of four weeks of lens wear ...... 94 Table 4.3: Mean change in keratocyte counts in all corneal areas and at all time points after cessation of four weeks of lens wear...... 101 Table 5.1: Summary of bacterial exposure Challenges I to VII ...... 121 Table 5.2: Summary of results for Challenges I to VII...... 126 Table 5.3: Percentage of animals with changes in amount of redness, chemosis, discharge and presence of infiltrates at the end of each Challenge (I-VII) in AF and OK eyes ...... 129 Table 5.4: Cumulative inflammation scores for AF and OK-treated eyes in each challenge ..... 130 Table 5.5: Mean values (cfu/lens x105) for adhered P. aeruginosa strains 6294 and 6206 from AF and OK lenses at different time points...... 131 Table 5.6: Total CFU at different time points ...... 132 Table 7.1: Epithelial thickness of non-lens wearing control animals ...... 163 Table 7.2: Comparisons of change in epithelial thickness (µm) between time points within corneal areas for increasing lens wear ...... 164 Table 7.3: Comparisons of change in epithelial thickness (%) between time points within corneal areas for increasing lens wear ...... 165 Table 7.4: Average keratocyte counts in both eyes of all three control animals...... 166
Orthokeratology Epithelial Changes and Susceptibility to Microbial Infection xvii
Table 7.5: Comparisons of change in keratocyte counts between time points within corneal areas for increasing lens wear...... 167 Table 7.6: Comparisons of change in epithelial thickness (µm) between time points within corneal areas 168 Table 7.7: Comparisons of change in epithelial thickness (µm) between time points within corneal areas 169 Table 7.8: Comparisons of change in epithelial thickness (%) between time points within corneal areas...... 170 Table 7.9: Comparisons of change in epithelial thickness (%) between time points within corneal areas ...... 171 Table 7.10: Comparisons of change in keratocyte counts between time points within corneal areas ...... 172
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Orthokeratology Epithelial Changes and Susceptibility to Microbial Infection xix
Chapter 1: Introduction
CHAPTER 1: Introduction
In the past two decades great strides have been witnessed in all areas of vision correction and today patients are offered a wide range of options that include spectacle lenses, contact lenses and refractive surgery. As will be discussed in this chapter, orthokeratology (OK) contact lens wear is becoming an increasingly popular and preferred option for patients and practitioners in search of a non- surgical modality that can provide stable and reversible vision correction during the day without an appliance or device being worn. This is especially true for younger people who are precluded from surgical correction. Understanding the potential detrimental effects of this procedure on the corneal tissue are therefore highly significant in terms of public health; yet due to the relatively recent revival of the modality, this is an area of research that has yet to be explored. This thesis investigates the effects of OK lenses on corneal epithelial tissue and the implications of these changes with regard to corneal susceptibility to microbial infection.
1.1 Orthokeratology Defined
Orthokeratology (OK), also known as corneal reshaping, corneal refractive therapy
(CRT), and vision shaping technology (VST) is a relatively old contact lens modality.
Used since the late sixties, it was defined as “the reduction, modification, or elimination of refractive anomalies by the programmed application of contact lenses” by Kerns in
1976 (Kerns, 1976a) and it is a definition that still applies today.
Orthokeratology Epithelial Changes and Susceptibility to Microbial Infection 1
Chapter 1: Introduction
Unlike traditional alignment fitted (AF) rigid gas permeable (RGP) lenses that provide optical clarity via the lens optical system worn on eye, OK lenses are deliberately designed to change the contour of the anterior cornea to result in a shape that provides clear vision when the lens is removed. Although OK has been performed for day wear, the current clinical protocol of choice is overnight OK whereby lenses are worn while sleeping and removed upon awakening to afford patients the freedom from glasses and contact lenses during the day. It provides an alternative for patients who cannot or do not want to wear lenses during the day for comfort, environmental, or lifestyle reasons.
Such patients include marginal dry eye sufferers, sports enthusiasts and prior soft and rigid lens wearers. Although permanent changes are frequently desired, OK changes are temporary thus necessitating the wear of lenses on a retainer basis to maintain the visual effects of the procedure.
1.2 The Evolution of Orthokeratology
Reshaping the corneal tissue to improve vision is not a new concept. For centuries people have known that the cornea was the most powerful refractive structure and alterations to its shape could improve visual effects. It is believed that the ancient
Chinese used to sleep with small weights or sand bags on their eyelids to flatten the cornea and reduce myopia (Waring, 1986). Since then, people have invented a variety of unorthodox devices that apply pressure or vacuum to the eye in an attempt to modify corneal shape and reduce refractive errors (Van Vleck, 1999).
Orthokeratology Epithelial Changes and Susceptibility to Microbial Infection 2
Chapter 1: Introduction
Use of contact lenses to reshape the cornea can be traced back to the early pioneers of corneal lenses. In the early 1960s, it was observed that patients fitted with polymethylmethacrylate (PMMA) lenses often reported improved unaided vision following lens removal (Grosvenor, 1963). In 1962, George Jessen introduced his
“Orthofocus” technique, the first deliberate attempt to change the anterior corneal contour using a series of flat-fitting plano-powered PMMA lenses to correct refractive error (Jessen, 1962; Jessen, 1964). Over the next two decades, this basic technique of using flat RGP lenses for corneal reshaping was expanded upon by many others (Grant and May, 1970; Gates, 1971; Fontana, 1972; Ziff, 1976; Carter, 1977). However, several studies (Kerns, 1976b; Binder et al, 1980; Polse et al, 1983; Coon, 1984) found the method to be ineffective and unpredictable, thus traditional OK fell into disfavour for many years.
The practice of OK remained dormant until 1986 when the development of reverse geometry lenses by several individuals began the era of contemporary OK (Wlodyga and Bryla, 1989; Harris and Stoyen, 1992; Wlodyga and Harris, 1993). As will be described later, these new lens designs incorporated a steeper secondary “reverse” curve to help stabilize and centre the flat lens. The subsequent development and revival of
OK was accelerated by the merging of several other technological advancements including: computer-controlled lathing and manufacturing, development of stable high
Dk lens materials, and advancements in computerized corneal mapping techniques
(Lebow, 1996; Dave and Ruston, 1998; Swarbrick, 2004). These advancements helped establish an improved OK procedure that used more sophisticated lens designs, allowed overnight lens wear, yielded more predictable results and provided a more reliable
Orthokeratology Epithelial Changes and Susceptibility to Microbial Infection 3
Chapter 1: Introduction
method for fitting and monitoring corneal changes. OK was now starting to be practiced again within the eye care community.
The modality received an important recognition in the early 2000s when the United
States Food and Drug Administration (FDA) granted approval for overnight wear of several OK lens designs (ParagonVisionSciences, 2002; Bausch&Lomb, 2004). This approval confirmed the efficacy of the modality and subsequently sparked interest in fitting the lenses and research to better understand the procedure.
Today, the popularity of OK is growing. Surveys of contact lens prescribing habits of optometrists in Canada (Woods et al, 2007) and the United Kingdom (Morgan and
Efron, 2008) have found that although use of spherical rigid lenses is on the decline, the use of rigid lenses for specialty fits such as OK is increasing. High levels of patient satisfaction with overnight OK through quality of life surveys have been reported (Rah et al, 2004; Ritchey et al, 2005; Berntsen et al, 2006) and patients have been shown to prefer this technique over conventional contact lens wearing modalities (Lipson et al,
2004; Lipson et al, 2005).
1.3 Orthokeratology Lenses
OK lenses are also referred to as “reverse geometry” lenses, a term coined by Kame
(Mountford, 2004a), to describe the lens design whereby the secondary back curve is steeper than the central back curve. It is this configuration of the posterior lens surface in combination with the tear fluid and lid dynamics that generates the necessary forces
Orthokeratology Epithelial Changes and Susceptibility to Microbial Infection 4
Chapter 1: Introduction
beneath an OK lens to reshape the corneal tissue (Mountford, 2004c). The most common use of OK is for the correction of myopia although designs for hyperopia and astigmatism are currently being developed.
1.3.1 Orthokeratology Lens Designs
The basic design of all myopic OK lenses involve three major components: a central base curve radius that provides a flat lens-to-cornea fitting relationship intended to correct the refractive error, a reverse curve radius to bring the flat central radius back towards the peripheral cornea, and a peripheral curve radius whose main purpose is to facilitate lens movement and tear exchange. Variations from this basic foundation of a reverse geometry OK lens both in design and nomenclature form the basis for different lens designs. Currently a wide range of lenses are marketed for OK in the USA and these are listed in Table 1.1.
A modified Paragon CRT lens for myopic OK will be used in the forthcoming studies thus a brief description of the nomenclature used in this design is warranted. The CRT lens also consists of three primary zones (Figure 1.1). The first zone is the central Back
Optic Zone Radius (BOZR) designed to correct the refractive error. The second zone, the Return Zone Depth (RZD) is a unique sigmoid-shaped curve that controls the amount of lens clearance across the central cornea. The third and final zone, the
Landing Zone Angle (LZA) is a tangent (straight line) angle that provides alignment of the lens across the peripheral cornea.
Orthokeratology Epithelial Changes and Susceptibility to Microbial Infection 5
Chapter 1: Introduction
OK lenses for hyperopia and astigmatism employ the same basic lens design concepts except that the central BOZR is altered to correct the appropriate amount of refractive error. Secondary and peripheral curves are then adjusted to bring proper lens alignment back to the peripheral cornea. These lenses are relatively new and although FDA approval has not been obtained, lens designs for hyperopic correction of up to 4.00 dioptres are available (Table 1.1).
Table 1.1: OK lenses marketed for use in the USA.
FDA Range of Fitting Company Lens Name Material Approval Correction Method Orison for Paragon Up to ABBA Optical Corneal Overnight Topography HDS 100 - 4.00D Reshaping Contex OK E- Boston Up to Empirical Contex Overnight System Equalens II - 5.00D Diagnostic DreimLens Boston Daily and Up to DreamLens Topography International Equalens II overnight - 5.00D Empirical Emerald & Boston Daily and - 1.00D to Euclid Systems Diagnostic Jade Equalens II overnight - 5.00D Topography Eye Care Boston Up to Empirical CKR Overnight Associates Equalens II - 4.00D Topography Wave Contact Up to Wave Boston XO NONE Topography Lens Systems - 4.00D Fargo Corneal Empirical HDS & Daily and Up to GP Specialists Reshaping Diagnostic HDS 100 overnight - 3.00D Therapy Lens Topography Paragon Vision Up to Diagnostic Paragon CRT HDS 100 Overnight Sciences - 6.00D Topography Precision Boston Daily and + 4.00D to BE Retainer Topography Technology Equalens II overnight - 4.00D Daily and Empirical R & R Lens R & R Lens Boston Up to overnight Diagnostic Design Design Equalens II - 4.50D (pending) Topography Advanced SightMove Empirical Boston Daily and Up to Corneal Refractive Lens Diagnostic Equalens II overnight - 5.00D Engineering Series Topography
Orthokeratology Epithelial Changes and Susceptibility to Microbial Infection 6
Chapter 1: Introduction
Back Optic Zone Radius (BOZR) Return Zone Depth Landing Zone Angle (RZD) (LZA)
Figure 1.1: Cross-sectional drawing of a Paragon CRT lens illustrating the three different zones of the lens. Courtesy of Paragon Vision Sciences (Paragon Vision Sciences, Mesa, AZ, USA).
1.3.2 Orthokeratology Lens Fitting
For myopic OK lenses, the resultant lens-to-cornea fitting relationship manifests in a distinctive tear pattern when viewed with sodium fluorescein. A central zone of close apposition of the lens to the cornea (treatment zone) is surrounded by an annular zone of tear pooling corresponding to the mid-peripheral secondary or return zone area (Figure
1.2 (a)). This zone is further surrounded by an annular zone of lens alignment with peripheral lens edge lift. Variations in the size of the treatment zone and the width of the other zones occur with different lens designs.
For hyperopic OK lenses, the tear pattern involves central clearance with tear pooling surrounded by an annular zone of lens apposition to the cornea (Figure 1.2 (b)).
Orthokeratology Epithelial Changes and Susceptibility to Microbial Infection 7
Chapter 1: Introduction
Figure 1.2: Fluorescein pictures of (a) myopic and (b) hyperopic OK lens fits.
Although all OK lenses are similar in design, each design is associated with a particular lens fitting philosophy (Table 1.1). It is the method of lens fitting that is usually the deciding factor for practitioners in determining which lens to prescribe. Practitioners can choose from three different fitting philosophies: empirical, diagnostic/inventory and topography-based (Ruston and Dave, 1997; Caroline, 2001; Mountford, 2004b).
Empirical fitting involves the provision of keratometric and refractive data to the lens manufacturer from which an OK lens is designed and sent back to the practitioner.
Diagnostic and inventory fitting involves a trial lens diagnostic set from which patients are fitted and lenses dispensed the same day. Finally topography-based fitting involves use of corneal topography to provide curvature and sagittal height data to be used in conjunction with custom software to design OK lenses.
1.4 Efficacy of Orthokeratology
Despite the numerous OK lens designs available, it is generally agreed that all designs are equally efficacious with little difference in clinical outcomes (Tahhan et al, 2003).
Considerable evidence from clinical studies has demonstrated the efficacy of the
Orthokeratology Epithelial Changes and Susceptibility to Microbial Infection 8
Chapter 1: Introduction
procedure for mild to moderate amounts of myopia. A summary of refractive outcomes from these studies is listed in Table 1.2. Although the procedure has been FDA approved for treatment of up to 6.00 dioptres with some lenses (Table 1.1), corrections of higher amounts of myopia have been reported (Tung, 2002; Joo, 2008).
The corneal effects of OK lenses are rapid with induction of significant topographic changes within minutes of lens insertion onto the eye (Tahhan et al, 2002; Sridharan and Swarbrick, 2003). The greatest amount of reduction in refractive error usually occurs after the first night of lens wear (Swarbrick et al, 1998; Nichols et al, 2000; Rah et al, 2002; Tahhan et al, 2003; Johnson et al, 2007) with full refractive effects of the procedure achieved after approximately one week (Mountford, 1997a; Nichols et al,
2000; Alharbi and Swarbrick, 2003; Soni et al, 2003; Tahhan et al, 2003; Johnson et al,
2007). Higher refractive error corrections generally take longer to achieve than more modest changes (Fan et al, 1999; Owens et al, 2004).
Because lenses are not worn during the day, the aim of practitioners is to provide visual and corneal changes that remain stable for all waking hours. This is achieved for most patients (Mountford, 1997a; Nichols et al, 2000; Soni et al, 2003) however, slight regression of the effect has been reported to range from 0.25D to 0.75D per day
(Mountford, 1998; Nichols et al, 2000; Rah et al, 2002; Johnson et al, 2007). This regression necessitates lens wear to retain the corneal changes however, variations in this rate can result in variations of lens wear from every night to every second or third night (Mountford, 1997b). Lenses are usually designed to over-correct the cornea by
0.50D to 0.75D to ameliorate daytime regression however the validity of the calculation
Orthokeratology Epithelial Changes and Susceptibility to Microbial Infection 9
Chapter 1: Introduction
to determine the base curve for this has recently been challenged (Chan et al, 2008b).
Longer term OK wear has been found to exhibit higher sustainability of visual acuity, refractive error and apical corneal power over the day than shorter term wear
(Mountford, 1998; Johnson et al, 2007; Kang et al, 2007) suggesting an adaptation to corneal change retention over time.
The procedure was approved for use in children and adults with no age restrictions.
Patient age has been found to influence speed of treatment whereby OK results are slower to take effect in older patients (Jayakumar and Swarbrick, 2005). The effectiveness of OK in children has been specifically investigated in several studies with all results finding comparable safety and efficacy in children as for adults (Fan et al, 1999; Walline et al, 2004; Cho et al, 2005a; Mika et al, 2007;
Lipson, 2008).
Orthokeratology Epithelial Changes and Susceptibility to Microbial Infection 10
Chapter 1: Introduction
Table 1.2: Summary of studies investigating the efficacy of OK.
Study Reduction in # Patients Wear Initial Myopia (Author, year) Age (years) Lens Design Duration Myopia (completed) Schedule (Mean SER) (months) (Mean SER) (Mountford, 1997a) 60 28 ± 12 Contex ON 2 -2.19 ± 0.80 2.19 ± 0.57 (Swarbrick et al, 1998) 6 21 to 27 Contex DW 1 -2.36 ± 0.90 1.71 ± 0.59 (Fan et al, 1999) 54 11 to 15 Orthofocus B, Sightform DW/ON 6 -1.25 to -10.75 Mean 3.00 (Lui and Edwards, 2000) 14 23 ± 3 Contex DW 3 -2.12 ± 0.51 1.50 ± 0.45 (Nichols et al, 2000) 10 (8) 26 ± 4 Contex ON 2 -1.84 ± 0.81 1.83 ± 1.23 (Rah et al, 2002) 60 (31) Did not Fargo 6, Paragon CRT ON 3 R -2.14 ± 0.98 R 2.08 ± 1.11 report L -2.16 ± 1.00 L 2.16 ± 1.05 (Tahhan et al, 2003) 60 (46) 27 ± 4 Rinehart & Reeves, BE, ON 1 R -2.24 ± 0.77 2.00 ± 0.34 Dreimlens, Contex L -2.25 ± 0.81 (Soni et al, 2003) 10 (8) 21 to 43 Contex ON 3 -1.76 ± 0.70 Mean 2.12 (Cho et al, 2003a) 69 5 to 46 Contex ON 1 to 12 -3.93 ± 2.30 90% 6/6 (median 12) (Alharbi and Swarbrick, 18 22 to 29 BE ON 3 -2.63 ± 0.68 2.63 ± 0.57 2003) (Joslin et al, 2003) 9 34 ± 10 Paragon CRT ON 1 -3.33 ± 1.26 3.08 ± 0.93 (Walline et al, 2004) 29 (23) 8 to 11 Paragon CRT ON 6 - 0.75 to - 5.00 To -0.16 ± 0.66 (Owens et al, 2004) 20 17 to 37 BE/ABE ON 1 -2.28 ± 0.84 Mean 2.27 (Hiraoka et al, 2004b) 31 10 to 44 Emerald ON 12 -2.32 ± 1.18 Mean 2.16 (Maldonado-Codina et al, 24 (9) 28 ± 10 BE, No7 ON 7 days BE -2.22 ± 0.63 Mean 2.89 2005) No7 -2.40 ± 0.59 Mean 2.23 (Cho et al, 2005a) 35 7 to 12 Boston XO, Paragon CRT ON 24 -2.27 ± 1.09 2.09 ± 1.34 (Sorbara et al, 2005) 30 (23) 26 ± 7 Paragon CRT ON 1 -3.00 ± 1.03 2.59 ± 0.77 (Berntsen et al, 2005) 20 21 to 37 Paragon CRT ON 1 -3.11 ± 0.96 3.33 ± 0.96 (Hiraoka et al, 2005) 39 15.6 ± 6.2 Emerald ON 3 -2.60 ± 1.13 To -0.17 ± 0.31 (Subramaniam et al, 14 18 to 42 BE ON 1 GP – 2.89 ± 1.18 GP to –0.29 ± 0.55 2007) Non-GP -2.59 ± 1.08 Non-GP to +0.37 ± 0.46 (Mika et al, 2007) 20 (16) 13.0 ± 2.03 Emerald ON 6 -2.06 ± 0.75 -0.16 ± 0.38 (El Hage et al, 2007) 29 (20) 26 ± 3.7 CKR ON 6 R -2.55 ± 0.87 R to 0.45 ± 0.74 L -2.47 ± 0.89 L to -0.17 ± 0.69 (Lipson, 2008) 296 17.7 ± 13.2 Paragon CRT, ON A 20.8 ±14.7 A -3.50 ± 1.50 A 3.30 ± 1.40 custom design B 22.5 ± 21.2 B -3.20 ± 1.50 B 3.10 ± 1.40 *R = right eye, L = left eye; GP = previous gas permeable lens wearers, Non-GP = previous non-gas permeable lens wearer; A = patients 12 yrs old, B = patients .> 12 yrs old
Orthokeratology Epithelial Changes and Susceptibility to Microbial Infection 11
Chapter 1: Introduction
1.4.1 Corneal Topographic Changes
Corneal topography has become an integral part of OK practice as the fitting of
OK lenses requires a higher degree of precision than routine spherical lens fitting.
Practitioners depend on accurate corneal shape data from topographical maps to help design and choose lenses, monitor changes and plan management protocols.
Accurate topographical measurements of corneal shape are thus important to lens design and fitting success. However, repeatability and reproducibility of topographical results have been found to vary between instruments and can effect the OK lens designed (Cho et al, 2002b).
The topographical changes induced by OK mirror the back surface of the lens. For myopic OK this includes a flattening of the apical corneal radius of curvature, referred to as the treatment zone, surrounded by an annulus of mid-peripheral corneal steepening. The central corneal flattening has been reported to correlate with the change in subjective refraction (Mountford, 1997a) although it has been suggested that the mid-peripheral steepening contributes to the overall refractive and visual effects of OK as well (Lu et al, 2004). For hyperopic OK, corneal topography changes are opposite to those of myopic OK with central corneal steepening surrounded by mid-peripheral flattening.
Orthokeratology Epithelial Changes and Susceptibility to Microbial Infection 12
Chapter 1: Introduction
1.5 Clinical Studies of Orthokeratology Corneal Changes
Effects of OK on the cornea have been the focus of much clinical research over the last several years. It is clear from topographic data that the anterior surface of the cornea is modified during the procedure. However, investigations into the relative contributions of the various layers of the cornea to these changes have yielded conflicting results.
1.5.1 Epithelium
The epithelium is the outermost layer of the cornea and due to its proximity to the lens on eye it is susceptible to the pressures of the OK procedure. A summary of clinical studies investigating the effects of OK on total corneal and epithelial thickness is presented in Table 1.3. Coon (Coon, 1984) was the first to report that central corneal thinning and mid-peripheral thickening may be the mechanisms responsible for the optical changes induced by the procedure. Optical pachymetry studies by Swarbrick and colleagues (Swarbrick et al, 1998) confirmed this finding and showed that the majority of changes in corneal thickness were attributed to central epithelial thinning and mid-peripheral stromal thickening. These results suggested that the optical changes could be explained by a compression or redistribution of anterior corneal tissue rather than an overall bending of the cornea. Central epithelial thinning with myopic OK has since been confirmed in other clinical studies (Alharbi and Swarbrick, 2003; Soni et al,
2003; Wang et al, 2003b; Haque et al, 2004; Jayakumar and Swarbrick, 2005; Haque et al, 2007; Lu et al, 2008). However, despite the strong support for central epithelial thinning, some studies did not find any changes to the central epithelial thickness (El
Orthokeratology Epithelial Changes and Susceptibility to Microbial Infection 13
Chapter 1: Introduction
Hage et al, 2007) nor to the central total corneal thickness (Fan et al, 1999; El Hage et al, 2007; Stillitano et al, 2007).
Investigations into the involvement of the mid-peripheral epithelium in the reverse curve area have also yielded conflicting results. Mid-peripheral epithelial thickening has been reported by several groups (Swarbrick et al, 1998; Wang et al, 2003b; Haque et al, 2004; Haque et al, 2007; Lu et al, 2008). However, this has been challenged by studies that have not found any significant changes to mid-peripheral epithelial thickness (Alharbi and Swarbrick, 2003; Jayakumar and Swarbrick, 2005; El Hage et al,
2007). The discrepancies in findings of epithelial thickness both centrally and mid- peripherally may be attributed to differences in the sensitivities of the various instruments used for measurement.
Table 1.3: Summary of clinical studies investigating total corneal and epithelial thickness changes in myopic OK. (-) = decrease, (+) increase, 0 = no significant change, grey boxes = not investigated Centre Mid-Periphery (Author, year) Methods Thickness Thickness Total Epi Total Epi (Coon, 1984) Pachymetry, Ultrasound - + (Swarbrick et al, 1998) Optical Pachymetry - - + + (Fan et al, 1999) A-Scan 0 (Nichols et al, 2000) Orbscan - 0 (Soni et al, 2003) Orbscan + Confocal - - (Alharbi and Swarbrick, 2003) Optical pachymetry - - + 0 (Wang et al, 2003b) OCT - + (Haque et al, 2004) OCT - + (Jayakumar and Swarbrick, 2005) Optical pachymetry - - 0 0 (Haque et al, 2007) OCT Men Z - + OCT EqII - + (El Hage et al, 2007) Orbscan 0 0 0 0 Sonogage 0 0 0 0 Confocal 0 - 0 + (Stillitano et al, 2007) Optical pachymetry 0 + (Lu et al, 2008) OCT - + *OCT = optical coherence tomography, Men Z = Menicon Z lens material, Eq II = Equalens II lens material
Orthokeratology Epithelial Changes and Susceptibility to Microbial Infection 14
Chapter 1: Introduction
Due to the relative novelty of hyperopic OK, only two studies have investigated corneal changes with this procedure (Haque et al, 2008; Lu et al, 2008). Both studies used optical coherence tomography (OCT) to investigate epithelial changes. Haque and colleagues (Haque et al, 2008) found central and mid- peripheral epithelial thickening (more centrally) after one night of lens wear with the degree of central thickening greater for the lens designed to correct a larger degree of hyperopia. Lu and colleagues (Lu et al, 2008) found central thickening and mid-peripheral thinning.
1.5.2 Stroma
1.5.2.1 Edema
As mentioned earlier, OK originally began as a daily wear modality however, with the advent of high oxygen transmissible lens materials, it is now performed as an overnight procedure. Thus the effect of lenses on the overnight corneal edema response is of interest. The corneal swelling patterns induced by OK show greater mid-peripheral compared to central swelling in measurements made immediately after lens removal
(Alharbi and Swarbrick, 2003; Wang et al, 2003b; Haque et al, 2004; Lu et al, 2008).
This pattern of swelling is opposite that induced by conventional RGPs (Wang et al,
2003a; Wang et al, 2003b) and soft lenses (Holden et al, 1985; Fonn et al, 1999; Wang et al, 2003a) whereby the central stroma is found to swell more than the mid-peripheral stroma. The degree of stromal swelling across OK-treated corneas varied between different studies and this was likely due to differences in the instrumentation used for
Orthokeratology Epithelial Changes and Susceptibility to Microbial Infection 15
Chapter 1: Introduction
measurements, timing of measurements, and effects of different OK lens materials on the cornea.
1.5.2.2 Structural effects
Whether the differential change in stromal thickness described above is solely due to alterations in the edema response or whether it is due to changes in the stromal structure is unclear. No clinical studies have investigated the potential structural changes to the stroma with this procedure.
1.5.2.3 Keratocytes
Preliminary investigations into changes in keratocyte density have shown varying results. Wang and colleagues (Wang et al, 2004) found no change to the density in the anterior stroma, a gradual increase in density in the central middle/posterior stroma and a decrease in the peripheral posterior numbers up to six months. Perez-Gomez and colleagues (Perez-Gomez et al, 2003) found an increase in anterior stromal keratocyte density seven days after cessation of one week of OK lens wear however, no pre-lens wear density measurements were made. The effect on and significance of keratocyte density change with OK remain unclear and warrants further investigation.
1.5.3 Endothelium
Only three studies have reported on the endothelial response to OK lenses and all found no significant changes to the endothelium with specular microscopic examination (Fan et al, 1999; Becherer and Kempf, 2004; Hiraoka et al, 2004b).
Orthokeratology Epithelial Changes and Susceptibility to Microbial Infection 16
Chapter 1: Introduction
1.5.4 Posterior Corneal Curvature
Although most research has focussed on anterior corneal changes, modifications to the posterior cornea cannot be dismissed. Investigations involving Purkinje images (Owens et al, 2004) and corneal aberrations after OK (Joslin et al, 2003) suggest that some flattening of the posterior corneal curvature may be occurring with the procedure.
However, recent work using the Pentacam analysis system (Tsukiyama et al, 2008) and
Orbscan IIz corneal tomography (Stillitano et al, 2007) found no changes to the central posterior radius of curvature.
1.6 Animal Studies of Orthokeratology Corneal Changes
The cellular nature of the above corneal changes induced with OK cannot be fully understood and appreciated through clinical studies. It is through the use of histological techniques in animal studies that allow direct visualization and a better understanding of these changes on a cellular level. Only three studies using the rabbit model have been reported for investigation of myopic OK effects and no studies have investigated hyperopic OK changes.
Matsubara and colleagues (Matsubara et al, 2004) were the first to demonstrate OK- induced corneal changes using histology techniques in the rabbit model. Their results found areas of epithelial thinning and mid-peripheral epithelial thickening consistent with the topographical effects observed. Histochemical analysis also demonstrated that there were no marked alterations in epithelial function with the corneal reshaping procedure. However, that study was flawed in methodology as the decentered
Orthokeratology Epithelial Changes and Susceptibility to Microbial Infection 17
Chapter 1: Introduction
histologic effects raised questions about the suitability of fitting human lenses on rabbit eyes given the differences in corneal curvatures and characteristics between species. As described above, the selection of an appropriate OK lens is paramount to achieving the desired corneal changes however, the authors admitted to poor lens fits in the study.
Matsubara and colleagues (Matsubara et al, 2004) also reported an increase in mitosis in the central thinned epithelium of OK-treated rabbit eyes. This finding was contradicted by a study (Shin et al, 2005) that reported a decrease in central epithelial cell proliferation rates in OK-treated rabbit eyes. Again, there were limitations in the OK rabbit model used in the latter study particularly the lack of topographic corneal changes observed has questioned the validity of the results.
Preliminary examination of stromal structure in rabbit eyes with X-ray diffraction analysis of stromal collagen fibrils found no significant difference in collagen fibril diameter between OK-treated and untreated eyes (Owens et al, 2007).
The previous studies were integral to providing insights into the cellular basis for
OK changes but limitations in the animal OK model used raises questions about some of the results. Clearly more studies are needed to support both animal and human clinical findings.
Orthokeratology Epithelial Changes and Susceptibility to Microbial Infection 18
Chapter 1: Introduction
1.7 Orthokeratology and Infections
Although OK is growing in popularity, literature reports of microbial keratitis (MK) in
OK lens wearers have questioned the safety of the modality. MK is the most serious complication of contact lens wear due to its potential to cause permanent vision loss
(Holden et al, 2003). Fortunately, the incidence of MK is very low, particularly with
AF RGP lenses where epidemiological studies have consistently found an incidence of
MK that is the lowest of all the modalities of lens wear (Cheng et al, 1999; Stapleton,
2003; Keay et al, 2007). Although an exact incidence rate of MK with OK is difficult to ascertain due to problems in estimating numbers of OK lens wearers (Walline et al,
2005), this recent increase in reported infections has raised concerns about the safety of the OK modality.
The first 123 cases of OK infections have been summarized by Watt and Swarbrick
(Watt and Swarbrick, 2007b) but since then more have emerged (Chee et al, 2007;
Hsiao et al, 2007). To date, 132 cases of MK in OK have been published and details of these reports are listed in Table 1.4.
An analysis of the distribution of OK infections found that the majority of these MK cases occurred in the same region (Asia = China, Taiwan, Korea, Singapore) and over the same time period (2001) (Watt and Swarbrick, 2007b). This has been attributed to the lack of regulation of the modality in the Asian region at that time which resulted in subsequent government intervention in some countries (DeWoolfson, 2005). OK is slowly being re-introduced into the region but under stricter government regulation
Orthokeratology Epithelial Changes and Susceptibility to Microbial Infection 19
Chapter 1: Introduction
(DeWoolfson, 2005). Further analysis of MK cases showed that a peak year for infections to occur worldwide was in 2001 whereas reporting of the cases occurred several years later (Watt and Swarbrick, 2007b). Additionally, it appears that the number of MK infections being reported has reduced over the last few years.
Most OK infections occurred in children and young adults and this was thought to reflect the demographics of the OK lens-wearing population (Watt and Swarbrick,
2005) rather than a predisposition of children to infections although this still needs to be confirmed.
The most common causative organism in these MK infections was Pseudomonas aeruginosa (P. aeruginosa) (56 cases), not a surprising finding given that it has consistently been the most common organism isolated from other contact lens related infections (Stapleton et al, 1995; Cheng et al, 1999; Lam et al, 2002; Sharma et al,
2003; Mah-Sadorra et al, 2005). P. aeruginosa is an opportunistic pathogen that requires corneal trauma, particularly an epithelial defect, to develop into a corneal infection at least in animal models (Solomon et al, 1994). Studies have shown that contact lens wear by itself can increase the organism’s adhesion to epithelial cells
(Ladage et al, 2001) while extended wear of contact lenses further enhances its adhesion to the corneal epithelium (Fleiszig et al, 1992). It has been reported that OK-treated corneal cells bind more Pseudomonas than their non OK-treated counterparts (Ladage et al, 2004), understanding how and if this may lead to infection in OK needs to be investigated.
Orthokeratology Epithelial Changes and Susceptibility to Microbial Infection 20
Chapter 1: Introduction
A study of the microbial flora of OK patients found no significant differences in levels or types of pathogens with increasing lens wear (Boost and Cho, 2005) suggesting that
MK causing pathogens are usually introduced into the ocular environment. This is supported by the large number of Acanthamoeba OK infections reported.
Acanthamoeba generally occurs in about 3% of infection cases in contact lenses (Cheng et al, 1999; Lam et al, 2002) however it appears to account for about 30% of cases in
OK. As the most common source of Acanthamoeba is exposure to a contaminated water supply (Stehr-Green et al, 1989; Seal et al, 1999; Radford et al, 2002), an official warning to ban the use of tap water in the care regimen of OK lenses has now been encouraged (Walline et al, 2005). There has been very little work conducted to investigate the potential susceptibility of OK-treated corneas to infection after exposure to micro-organisms.
Orthokeratology Epithelial Changes and Susceptibility to Microbial Infection 21
Chapter 1: Introduction
Table 1.4: Summary of published cases of microbial keratitis in OK.
Negative (Author, year) Country Patients Age in yrs (mean) Study Type Pseudomonas Acanthamoeba Unknown Other Details of Other Culture (Lu et al, 2001) China 16 - Case series 7 8 1 Mycotic (Chen et al, 2001) Taiwan 1 9 Case report 1 Serratia marcescens (Hutchinson and Apel, 2002) Australia 2 60, 29 Case report 1 1 (Lau et al, 2003) Taiwan 2 11, 11 Case report 2 (Poole et al, 2003) UK 1 22 Case report 1 (Xuguang et al, 2003) China 4 15 - 19 (17) Case report 4 (Wang and Lim, 2003) Singapore 1 14 Case report 1 (Young et al, 2003) Hong Kong 1 37 Case report 1 (Hsiao et al, 2004) Taiwan 6 9 – 17 (13) Case series 6 (Lang and Rah, 2004) USA 2 12, 29 Case series 1 1 Serratia marcescens (Young et al, 2004) Hong Kong 6 9 – 14 (12.1) Case series 5 1 (Araki-Sasaki et al, 2005) Japan 1 17 Case report 1 20 Retrospective 2 Staphylococcus (Hsiao et al, 2005). Taiwan 9 – 21 (14) 9 1 8 (21 eyes) case series 1 Serratia (Macsai, 2005) USA 2 9, 11 Case report 1 1 Haemophilus influenza 9 Gram negative (Tseng et al, 2005) Taiwan 8 – 17 (12.3) Case series 1 3 1 1 (10 eyes) (Wilhelmus, 2005) USA 1 16 Case report 1 (Yepes et al, 2005) Canada 3 41, 14, 12 Case report 1 1 1 Serratia (Priel et al, 2006) Israel 1 16 Case report 1 2 Fungus 1 Nocardia (Sun et al, 2006) China 28 10 – 21 Case series 8 13 2 1 Providencia stuartii 1 Gram negative rod (Ying-Cheng et al, 2006) Taiwan 1 16 Case report 2 1 Burkholderia cepacia (Voyatzis, 2006) 1 9 Case report 1 (Hsiao et al, 2007) Taiwan 8 (11.2) Case series 4 4 Gram negative (Watt et al, 2007a) Australia 7 12 – 22 (17) Survey 3 1 1 (Robertson et al, 2007) USA 1 19 Case report 1 (Lee et al, 2007) Korea 4 14 - 16 (15) Case series 4 (Chee et al, 2007) Singapore 5 9 – 14 Case series 5 Total 56 38 16 3 19 Grand Total: 132
Orthokeratology Epithelial Changes and Susceptibility to Microbial Infection 22
Chapter 1: Introduction
1.8 Myopia Control and Orthokeratology
As mentioned earlier, OK is growing in popularity. Recent reports of myopia controlling effects of OK lenses have helped fuel interest in the technique. Anecdotal reports of this phenomenon are common amongst patients and practitioners, particularly in Asia where myopia has reached epidemic proportions (Grosvenor, 2003). In a survey of OK lens wearers in Hong Kong, one of the main reasons patients used the modality was for myopia control despite the lack of solid scientific evidence to support this claim
(Chan et al, 2008a). There have been four published articles that have attempted to address this issue (Reim et al, 2003; Cheung et al, 2004; Cho et al, 2005a; Walline,
2007). These were not large randomized clinical studies however all seemed to suggest that OK patients have a slower progression of myopia than their spectacle (Cho et al,
2005a), spherical rigid (Reim et al, 2003; Walline, 2007) and soft lens (Walline, 2007) wearing counterparts.
Recent research suggests that deliberate manipulation of the optics to produce an optical profile whereby the curvature of field moves the peripheral image in front of the retina (Smith et al, 2005; Smith et al, 2007) may slow myopia progression.
A small study looking at peripheral refraction in OK patients found a myopic shift post-lens wear that seems to support this myopia control theory (Charman et al,
2006). More work needs to be done to confirm the effect of OK on the progress of myopia but the mere suggestion of the effect has already stimulated great interest in the modality.
Orthokeratology Epithelial Changes and Susceptibility to Microbial Infection 23
Chapter 1: Introduction
1.9 Thesis Overview
It is evident from the literature that OK has evolved into a form of vision correction that is preferred by many consumers and is growing in acceptance and popularity, particularly in Asia where it is driven by the potential for myopia control. Although our clinical understanding has increased dramatically over the last few years, clarification is still needed to confirm the corneal changes induced by the OK procedure. There is a paucity of animal work conducted to understand the cellular alterations underlying these changes. With the number of MK cases reported in OK wearers and the potential increase in use of the modality if the link to myopia control is confirmed, a better understanding of these corneal changes now becomes paramount to understanding the safety of the procedure. This thesis will investigate the cellular basis for OK corneal changes and determine if they affect susceptibility to infection in an effort to provide insights that may help prevent adverse events in the future.
1.9.1 Hypotheses
Primary Hypotheses:
Orthokeratology involves changes to the cornea that are primarily epithelial in
nature
Orthokeratology induced epithelial changes increase the susceptibility of the
cornea to infection
Orthokeratology Epithelial Changes and Susceptibility to Microbial Infection 24
Chapter 1: Introduction
Secondary Hypotheses:
Cats can be used as a model for OK corneal changes, with epithelial
thickness changes reflecting epithelial changes seen in clinical patients.
Epithelial changes are correlated with OK lens designs whereby flatter lenses
produce flatter epithelium and steeper lenses produce steeper epithelium
Epithelial changes induced by OK are reversible with recovery of thickness
and morphology to pre-lens wear levels after cessation of lens wear
Infection rates in OK corneas will be higher with larger amounts of bacteria
and longer periods of bacterial exposure
1.9.2 Aims
To investigate these hypotheses, the aims of this thesis are:
To establish the cat as a model for OK epithelial changes (Chapter 2)
To obtain a preliminary understanding of the corneal changes with myopic
and hyperopic OK (Chapter 2)
To improve the cat model to better reflect the human lens wearing situation
(Chapter 3)
Having determined if epithelial changes are induced with OK, to gain
further insight into the epithelial changes induced with myopic OK (Chapter
3)
To determine if myopic OK epithelial changes are reversible (Chapter 4)
To determine if the corneal changes induced with myopic OK make the
cornea more susceptible to Pseudomonas aeruginosa infection (Chapter 5)
Orthokeratology Epithelial Changes and Susceptibility to Microbial Infection 25
Chapter 2: Development of the Cat Model for Orthokeratology Corneal Changes
CHAPTER 2: Development of the Cat Model for
Orthokeratology Corneal Changes
2.1 Introduction
Although clinical evidence is mounting in relation to the corneal changes induced with OK lens wear, little is known about the cellular basis for these changes. As mentioned in the last chapter, only one attempt has been made to visualize the OK corneal changes histologically (Matsubara et al, 2004). The rabbit model used in that study was limited in its ability to mimic a typical human overnight OK lens fit and wearing schedule. Thus a better animal model is needed to support and confirm these preliminary findings.
Similarities in the anatomic and physiologic features of the cornea have made the cat a useful model for human ophthalmic research (Bahn et al, 1982; Ren et al,
1994; Habib et al, 1995; Telfair et al, 2000). The cat corneal epithelium shows important similarities to the human epithelium in that it consists of 6-8 cell layers and possesses a Bowman’s membrane (Ehlers, 1970a; Ehlers, 1970b). Previous studies have shown that cats can be used to investigate the corneal epithelial response to contact lens wear (Madigan et al, 1987; Madigan and Holden, 1992).
More specifically, epithelial thinning was found while investigating adherent silicone elastomer lens wear in the cat (Holden et al, 1989). The relative ease with which cats can be fitted with lenses and the previous demonstration of induced epithelial changes with contact lens wear make the cat an ideal model to
Orthokeratology Epithelial Changes and Susceptibility to Microbial Infection 26
Chapter 2: Development of the Cat Model for Orthokeratology Corneal Changes
investigate corneal changes associated with OK. No laboratory studies exist that establish the sequence of morphologic changes induced in the epithelium beneath
OK lenses in the cat.
2.1.1 Aims
This pilot study was undertaken to establish the cat as a new animal model for studying OK epithelial changes and to better understand the nature of these changes as a function of time.
2.1.2 Hypotheses
Primary Hypothesis:
Cats can be used as a model for OK corneal changes, with epithelial
thickness changes reflecting epithelial changes seen in clinical patients
Secondary Hypotheses:
Epithelial changes observed will be dependent upon the OK lens design
used (myopic vs hyperopic)
The magnitude of epithelial changes will increase with increasing duration
of lens wear
Orthokeratology Epithelial Changes and Susceptibility to Microbial Infection 27
Chapter 2: Development of the Cat Model for Orthokeratology Corneal Changes
2.2 Materials and Methods
2.2.1 Study Design and Sample Size
This pilot study involved four animals and employed a contralateral study design.
One cat served as a control with no lens wear and the remaining three animals were each assigned to three different time points. The time points were chosen to represent key milestones in OK lens wear: four hours for early changes, eight hours to represent a typical overnight lens wearing session, and 14 days to represent longer term established changes.
2.2.2 Animals
Four, two-year-old domestic short-haired (DSH) female cats (Liberty Research
Laboratory, Waverly, NY, USA) were used in this study. All the animals in this study were treated according to a protocol approved by the Institutional Animal
Care and Use Committee at Oregon Health Sciences University in Portland, OR,
USA and all experimental procedures conformed to the ARVO statement for the
Use of Animals in Ophthalmic and Vision Research.
2.2.3 Contact Lenses
Contact lenses used in this study were specially designed and manufactured for the cat eye by Paragon Vision Sciences (Mesa, AZ, USA). The design was based on the Paragon CRT lens design using HDS 100 (hyperpurified delivery system) material (nominal Dk of 100 barrers). The right eye of each animal was fitted with
Orthokeratology Epithelial Changes and Susceptibility to Microbial Infection 28
Chapter 2: Development of the Cat Model for Orthokeratology Corneal Changes
a Paragon CRT lens for the correction of myopia and the left eye for the correction of hyperopia. The targets for the refractive changes were – 4.00 dioptres for the myopia correcting lenses and + 4.00 dioptres for the hyperopia correcting lenses.
A custom 12.3 mm diameter CRT diagnostic dispensing set was used throughout the study. Base curves of the myopia and hyperopia correcting lenses were 10.0 mm and 8.0 mm, respectively. The mid-peripheral Return Zone Depths (RZDs) of the lenses ranged from 375 to 475 μm and the peripheral Landing Zone Angles
(LZAs) from 30 to 34 degrees.
2.2.4 Lens Fits
All animals were anesthetized with isoflurane intubation during lens fitting.
Corneal health and clarity were examined with a handheld Burton Lamp (Burton
Manufacturing Co., USA) and each lens was individually fitted and evaluated with sodium fluorescein to establish the optimum lens-to-cornea fitting relationship. An acceptable fitting relationship was based on the same clinical criteria used for human Paragon CRT lens evaluations (Figure 2.1). In the case of the myopic correcting lenses, an acceptable lens fit was one in which there was overall centration of the lens, with 4 to 5 mm of central treatment (fluorescein thinning).
With the hyperopic correcting lenses, an acceptable fit was established when the diagnostic lens centred on the cornea with 3 to 4 mm of central apical clearance.
Orthokeratology Epithelial Changes and Susceptibility to Microbial Infection 29
Chapter 2: Development of the Cat Model for Orthokeratology Corneal Changes
Figure 2.1: Acceptable (a) myopic and (b) hyperopic OK lens fits on the cat eye.
2.2.5 Lens Wearing Schedules
Following insertion of the appropriate fitting lenses, all animals (treated and untreated control), underwent surgical closure of the nictitating membrane. This was accomplished by pulling the membrane over the lens and cornea, and suturing it to the superior-temporal bulbar conjunctiva (Gelatt, 2000). This technique served to preserve the lacrimal gland (Gelatt, 2000) within the nictitans flap and facilitated lens retention. Presence of the sutured nictitating membrane also served to more closely emulate forces present beneath the contact lens in the overnight, closed eye environment.
Each animal was randomly assigned a continuous wearing period of either: four hours, eight hours or 14 days. After the prescribed wearing periods, animals were anesthetized with isoflurane, nictitating membranes were released and lenses were removed. At the end of each wearing period, all four animals were sacrificed with an overdose of pentobarbital sodium (Spectrum Chemicals, Gardena, CA, USA) and eyes collected for histological staining and examination.
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Chapter 2: Development of the Cat Model for Orthokeratology Corneal Changes
2.2.6 Histology
All eyes were enucleated and fixed whole globe in 10% neutral buffered formalin
(NBF) for seven days, after which they were processed on the Sakura VIP tissue processor (Sakura, Japan), dehydrated through graded ethanol (70%, 95%, and absolute alcohol), cleared in Xylene and infiltrated with Paraplast X-Tra tissue embedding medium (Fisher Scientific, USA). Corneas were then sectioned in five
μm intervals with the Leica RM 2155 automatic rotary microtome (Leica,
Germany) and sections were mounted on microscope slides. Slides were incubated overnight in a 37ºC oven and transferred the following day to a 60ºC oven for 30 mins. Sections were hydrated through absolute, 95% and 70% ethanol and washed in running tap water and then stained with routine Hematoxylin and Eosin (H&E).
2.2.7 Epithelial Thickness Measurements
Individual sections were examined under routine light microscopy with a Leica
DM1000 (Leica, Wetzlar, Germany) microscope and the epithelium was photographed with a Leica DCF400 camera at a magnification of 40X. A total of five central and five mid-peripheral epithelial thickness measurements were taken and averaged using the Image-Pro Plus Measurement Software from Media
Cybernetics (Bethesda, MD).
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Chapter 2: Development of the Cat Model for Orthokeratology Corneal Changes
2.3 Results
All epithelial pictures in the following Figures were photographed at 40X magnification.
2.3.1 Control Animal with No Lens Wear
Epithelium of the control eye showed a uniform epithelial thickness (Figure 2.2).
Average central thickness was 37.7 ± 0.8 μm and average mid-peripheral (3mm from centre) epithelial thickness was 37.9 ± 1.5 μm. There were approximately 6 to 8 epithelial cell layers present in all sections of the cornea and morphology of all cell types was consistent with that expected in untreated corneas in both humans (Hogan, 1971) and cats (Ehlers, 1970a; Ehlers, 1970b).
Figure 2.2: Four day control epithelium. Central and mid-peripheral cat corneal epithelium four days post nictitating membrane suturing.
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Chapter 2: Development of the Cat Model for Orthokeratology Corneal Changes
2.3.2 Four Hour Test Animal
Changes in epithelial thickness were observed following four hours of continuous lens wear (Figure 2.3). Average central epithelial thickness in the myopia- corrected right eye was 34.1 ± 0.2 μm and average mid-peripheral thickness was
49.6 ± 1.5 μm. Based on the shape and positioning of the nuclei, central basal cells were compressed (nuclei flatter and closer to stroma) and mid-peripheral basal cells appeared elongated (nuclei vertically longer and further from stroma).
Figure 2.3: Four hour myopic correction. Central and mid-peripheral cat corneal epithelium after four hours of continuous myopic OK lens wear.
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Chapter 2: Development of the Cat Model for Orthokeratology Corneal Changes
With the hyperopia corrected left eye, average central epithelial thickness was 58.0
± 0.9 μm and average mid-peripheral thickness was 42.8 ± 2.2 μm. The central basal cells in this eye were slightly elongated and mid-peripheral cells showed only minimal changes (Figure 2.4). No differences were found in the number of cell layers present in both the centre and the mid-periphery of the epithelium with either the myopic or hyperopic treated eyes.
Figure 2.4: Four hour hyperopic correction. Central and mid-peripheral cat corneal epithelium after four hours of continuous hyperopic OK lens wear.
Orthokeratology Epithelial Changes and Susceptibility to Microbial Infection 34
Chapter 2: Development of the Cat Model for Orthokeratology Corneal Changes
2.3.3 Eight Hour Test Animal
Changes in epithelial thickness were observed following eight hours of continuous lens wear; considered the equivalent of a typical night of OK lens wear (Figure
2.5). The myopia corrected right eye showed an average central epithelial thickness of 35.8 ± 1.1 μm and a mid-peripheral thickness of 73.1 ± 4.7 μm. The central basal cells were compressed and mid-peripheral basal cells appeared elongated. A slight increase in the number of cell layers was seen in the thickened mid-peripheral region whereas no change in cell layers was seen in the central epithelium.
Figure 2.5: Eight hour myopic correction. Central and mid-peripheral cat corneal epithelium after eight hours of continuous myopic OK lens wear.
Orthokeratology Epithelial Changes and Susceptibility to Microbial Infection 35
Chapter 2: Development of the Cat Model for Orthokeratology Corneal Changes
In the hyperopia corrected left eye, average central epithelial thickness was 63.4 ±
1.1 μm and mid-peripheral thickness was 43.7 ± 1.0 μm. The central basal cells appeared slightly elongated whereas the mid-peripheral basal cells showed minimal changes (Figure 2.6). No changes were found in the number of cell layers present in the mid-peripheral epithelium, but a slight increase in cell layers was found in the central epithelium.
Figure 2.6: Eight hour hyperopic correction. Central and mid-peripheral cat corneal epithelium after eight hours of continuous hyperopic OK lens wear.
Orthokeratology Epithelial Changes and Susceptibility to Microbial Infection 36
Chapter 2: Development of the Cat Model for Orthokeratology Corneal Changes
2.3.4 Fourteen Day Test Animal
After 14 days of continuous lens wear, morphologic changes appeared more pronounced (Figure 2.7). Average central epithelial thickness of the myopia corrected right eye was 11.4 ± 0.3 μm and the mid-peripheral thickness was 51.0 ±
5.4 μm. Basal cells in the centre were dramatically compressed whereas those in the mid-periphery appeared to have a shape similar to those of the control epithelium. The dramatic compression and thinning in the central epithelium made evaluation of the exact number of cell layers with light microscopy difficult, however at least 4 cell layers were counted. The mid-peripheral epithelium of the myopic corrected eye showed an increased number of cell layers when compared to epithelium of the control animal.
Figure 2.7: Fourteen day myopic correction. Central and mid-peripheral cat corneal epithelium after 14 days of continuous myopic OK lens wear.
Orthokeratology Epithelial Changes and Susceptibility to Microbial Infection 37
Chapter 2: Development of the Cat Model for Orthokeratology Corneal Changes
The hyperopic corrected left eye showed an opposite epithelial thickness pattern with a central epithelial thickness of 81.9 ± 2.0 μm and a mid-peripheral thickness of 23.6 ± 2.0 μm. The central basal cells had an appearance similar to the control basal cells whereas those in the mid-periphery were compressed (Figure 2.8). The central epithelium showed a large increase in the number of cell layers present however, the mid-peripheral epithelium showed relatively no change in cell layers.
Figure 2.9 shows a comparison of the central epithelium of the myopic and hyperopic eyes of this animal.
Figure 2.8: Fourteen day hyperopic correction. Central and mid-peripheral cat corneal epithelium after 14 days of continuous hyperopic OK lens wear.
Orthokeratology Epithelial Changes and Susceptibility to Microbial Infection 38
Chapter 2: Development of the Cat Model for Orthokeratology Corneal Changes
Figure 2.9: Central epithelium of myopic (left) and hyperopic (right) corrected eyes of an animal after 14 days of continuous OK lens wear.
Table 2.1 is a composite of central and mid-peripheral epithelial thickness measurements for all eyes.
Table 2.1: Average cat epithelial thickness measurements for all eyes.
Epithelial Thickness (µm) 4 Day Central 37.7± 0.8 Mid-Peripheral 37.9±1.5 Control Myopia Hyperopia Hyperopia Myopia Central Mid-peripheral Central Mid-peripheral 4 Hour 34.1± 0.2 49.6± 1.5 58.0± 0.9 42.8± 2.2 8 Hour 35.8± 1.1 73.1± 4.7 63.4± 1.1 43.7± 1.0 14 Days 11.4± 0.3 51.0± 5.4 81.9± 2.0 23.6± 2.0
Orthokeratology Epithelial Changes and Susceptibility to Microbial Infection 39
Chapter 2: Development of the Cat Model for Orthokeratology Corneal Changes
2.3.5 Stromal Changes
Figure 2.10 shows the central stroma of both the right (myopic corrected) and left
(hyperopic corrected) eyes of the 14 day specimen. Histological artefact (white spaces in the stroma) from the corneal sectioning technique precluded accurate measurement of stromal thickness however, gross visual examination of the tissue suggested the central stroma of the myopic eye was thinner relative to the hyperopic corrected eye.
Figure 2.10: Central cat stroma after 14 days of continuous OK lens wear for the correction of 4.00D of myopia (left) and 4.00D of hyperopia (right).
Orthokeratology Epithelial Changes and Susceptibility to Microbial Infection 40
Chapter 2: Development of the Cat Model for Orthokeratology Corneal Changes
2.4 Discussion
Results of this study revealed a lens design (myopia vs. hyperopia corrected) and time-related change in epithelial thickness induced by continuous wearing of OK lenses. As wearing time increased, the myopia-corrected corneas showed increased thinning of the central epithelium and thickening of the mid-peripheral epithelium. These results support clinical evidence suggesting that the epithelium thins in the centre and thickens in the mid-periphery with myopic OK (Swarbrick et al, 1998; Nichols et al, 2000; Alharbi and Swarbrick, 2003; Soni et al, 2003;
Haque et al, 2004; Haque et al, 2007; Lu et al, 2008). These data are also consistent with the histological work conducted in the rabbit model previously described (Matsubara et al, 2004). Since commencing this work, histological analysis of short term (up to 24 hours) OK corneal changes using the primate model has been conducted and is also in agreement with our pattern of epithelial thickness findings across the cornea (Cheah et al, 2008).
The hyperopic lens-corrected corneas showed an opposite epithelial thickness pattern, with thickening of the central epithelium and thinning of the mid- peripheral epithelium with increased lens wear time. These findings are in agreement with the OCT results of Lu and colleagues (Lu et al, 2008) and the central findings are consistent with the OCT findings of Haque and colleagues
(Haque et al, 2008).
Orthokeratology Epithelial Changes and Susceptibility to Microbial Infection 41
Chapter 2: Development of the Cat Model for Orthokeratology Corneal Changes
The epithelial thickness results from the control animal in the present study are in accordance with the published range of epithelial thickness in the cat of 35-50 μm
(Ehlers, 1970a; Ehlers, 1970b). Morphologically, the presence of 6-8 cell layers in the control epithelium is also in agreement with the number of cell layers reported previously (Ehlers, 1970a; Ehlers, 1970b).
Short and Long Term Epithelial Changes
Results of this study suggest various mechanisms may be at work during different stages of the lens wearing cycle. In the early stages of the present study, following four to eight hours of lens wear, epithelial cell compression and deformation appear to be the dominant factors. Compression of cells in the centre and elongation of adjacent cells in the mid-periphery with myopic treatment (and opposite with hyperopic treatment) suggest a transfer of intracellular contents may be taking place between cells (Choo et al, 2004) and this is supported by identical epithelial morphology findings in the monkey OK model (Cheah et al, 2008).
However, with increased wearing time, additional factors such as alterations in cell mitosis (Matsubara et al, 2004; Shin et al, 2005), cell apoptosis, cell exfoliation, and cell redistribution (Greenberg and Hill, 1973; Holden et al, 1989) may contribute to what appears to be a proliferation of cells seen in both the myopic and hyperopic 14 day histologic sections. A decrease in cell exfoliation rate was suggested by Cheah and colleagues (Cheah et al, 2008) based on their finding of an increase in superficial cell size in the mid-peripheral epithelium of the OK-treated eyes in the monkey. Clearly more work is needed to elicit some of these potential physiological changes with OK.
Orthokeratology Epithelial Changes and Susceptibility to Microbial Infection 42
Chapter 2: Development of the Cat Model for Orthokeratology Corneal Changes
The present study showed rather dramatic changes to the corneal epithelium with
14 continuous days of OK lens wear. In current clinical practice, OK patients are instructed to wear lenses overnight (6 to 10 hours) then remove them in the morning to begin an open eye cycle of unaided vision. The exaggerated wearing period of 14 days in the present study provides insight into the long term effects of this procedure on the corneal tissue under extreme conditions. Despite the accelerated wearing period, the myopic eye possessed at least four cell layers in the central thinned epithelium with no obvious histological evidence of epithelial erosion. It is possible that more cell layers may be visible in the centre when viewed using a higher resolution technique such as electron microscopy. Further studies are needed to evaluate tissue response with lens wearing cycles of 6-10 hours a day and at longer time points.
2.4.1 Stromal Changes
Gross examination of the central stroma in the 14 day specimens showed a difference in thickness with the hyperopic eye exhibiting a thicker stroma than the myopic eye. Clinical work (Swarbrick et al, 1998; Alharbi and Swarbrick, 2003;
Haque et al, 2007; Lu et al, 2008) and animal work (Cheah et al, 2008) suggest alterations in stromal thickness do occur with myopic OK. Differential thickening of the mid-peripheral stroma more than the central stroma has been found although the origin of these changes remains unknown. These stromal changes may be physiological (edema), mechanical (lens induced pressure forces) or biochemical
(cell-mediated) in nature. It has been well documented that epithelial cells release
Orthokeratology Epithelial Changes and Susceptibility to Microbial Infection 43
Chapter 2: Development of the Cat Model for Orthokeratology Corneal Changes
a variety of cellular factors, such as matrix metalloproteinases (Birkedal-Hansen,
1995) and cytokines (Wilson et al, 1996a), in response to changes in environment.
Any source of epithelial stress, such as infection, abrasion, refractive surgery or pressure of a contact lens on the ocular surface, can cause release of these factors that can not only modulate the epithelial response, but also affect the stroma in some instances (Wilson et al, 1999).
2.4.2 Limitations
As a pilot study the number of animals was limited to one per time point, thus greater numbers of animals are needed to determine the repeatability of the results and confirm the findings.
Use of both eyes to study myopic and hyperopic OK was useful to gain insights into the ability of the epithelium to change in response to different lens designs.
However, to understand the degree of epithelial thickness change induced by OK, a proper contralateral control is needed for each design.
Suturing of nictitating membranes to facilitate lens retention and to create a closed eye environment may have increased the force of the lens on the cornea and may also have induced a hypoxic response. Although no corneal swelling data was measured, both eyes of each animal were treated similarly to allow direct comparisons of the right and left eyes. If a hypoxic response were induced, the effect would be expected to be more relevant for the longer wearing periods of
Orthokeratology Epithelial Changes and Susceptibility to Microbial Infection 44
Chapter 2: Development of the Cat Model for Orthokeratology Corneal Changes
eight hours and especially 14 days. Studies to better mimic a typical human OK lens wearing schedule and a more natural eyelid dynamic should yield results more representative of those seen clinically.
2.4.3 Summary
Results of this pilot study confirmed the cat was an appropriate model to study
OK-induced epithelial changes. This study suggested that the epithelium plays a major role in changes induced by the OK procedure and the shape and thickness of the epithelium were dramatically influenced by both the wearing time and the posterior lens surface design of the contact lens. Preliminary examination of the stromal structure suggests that the stroma may be influenced by OK lens wear.
Orthokeratology Epithelial Changes and Susceptibility to Microbial Infection 45
Chapter 3: Epithelial Changes with Overnight Orthokeratology Lens Wear
CHAPTER 3: Epithelial Changes with Overnight
Orthokeratology Lens Wear
3.1 Introduction
The previous chapter established that the cat model can be used to investigate OK corneal changes. However, as discussed, the pilot study was limited in many respects. The small number of animals used (only one animal per time point), the continuous wearing schedule (24 hours a day for 14 days) and the presence of nictitating membrane flaps were not a realistic representation of the conditions likely to be experienced by human OK patients. Also, the use of both eyes, one for myopic OK and the other for hyperopic OK, precluded the use of proper contralateral controls. From that pilot study it was clear that a larger study with more animals, a more realistic wearing schedule and the use of better controls was needed to address the repeatability and generalizability of the results with the cat model.
3.1.1 Aims
The purpose of this study was to improve upon the cat OK model from Chapter 2 and to confirm and gain further insight into the epithelial changes induced with increased myopic OK lens wear.
Orthokeratology Epithelial Changes and Susceptibility to Microbial Infection 46
Chapter 3: Epithelial Changes with Overnight Orthokeratology Lens Wear
3.1.2 Hypotheses
Primary Hypothesis:
Overnight myopic OK lens wear induces epithelial thickness changes across
the cornea
Secondary Hypotheses:
Similar patterns of epithelial thickness changes as those seen in myopic
OK-treated eyes of Chapter 2 will be observable
Similar epithelial morphological changes as those seen in myopic OK-
treated eyes of Chapter 2 will be observable
The magnitude of epithelial thickness changes will increase with increasing
duration of lens wear
3.2 Materials and Methods
3.2.1 Study Design and Sample Size
Fifteen, two-year-old cats were used in this contralateral study (Vision CRC & IER
Animal Research Facility, Sydney, Australia). Animals were randomly divided into groups of three for five different time points. The number of animals used was determined based on a significance level of 0.05, power of 0.80 and a difference in central epithelial thickness change between OK-treated and non- treated eyes assumed to be 30 ± 10 microns based on an estimate from the pilot study results reported in Chapter 2. Calculations were made using the PS Power
Orthokeratology Epithelial Changes and Susceptibility to Microbial Infection 47
Chapter 3: Epithelial Changes with Overnight Orthokeratology Lens Wear
and Sample Size Calculations Program (Vanderbilt University Department of
Biostatistics, Nashville, TN, USA).
All the animals in this study were treated according to a protocol approved by the
Vision CRC - Institute for Eye Research Animal Ethics Committee (VIAEC) in
Sydney, Australia. All experimental procedures conformed to the ARVO statement for the Use of Animals in Ophthalmic and Vision Research.
3.2.2 Animal Preparation
To help facilitate lens wear and simulate human lens dynamics, nictitating membranes were surgically removed from both eyes of all animals at approximately six months of age. Animals were fasted overnight and anaesthetised to a depth of Stage II, Plane II using isoflurane vapourized in oxygen via mask.
The analgesic/anti-inflammatory, Rimadyl 4mg/kg SC was administered prior to surgery. The surgical area was then prepared with sterile drapes and eyes were washed with sterile saline and a local anaesthetic oxybuprocaine hydrochloride
0.4%. An ophthalmic antibiotic (Tobramycin 3mg/ml) was applied. Eyelids were retracted with a lid speculum, and the nictitating membrane was clamped for two minutes with mosquito forceps to minimise post-operative bleeding. The membranes were removed with iris scissors at the point of “crushing”. Eyes were rinsed thoroughly with sterile saline and an ophthalmic antibiotic
(Chloramphenical 10mg) was administered immediately and twice daily for seven days following. No further procedures were performed until animals were at least
Orthokeratology Epithelial Changes and Susceptibility to Microbial Infection 48
Chapter 3: Epithelial Changes with Overnight Orthokeratology Lens Wear
one year old in order to allow stabilization of tear film and corneal curvature characteristics.
3.2.3 Contact Lenses
OK and AF lenses were used in this study. Although the main interest was the effect of OK lenses on the epithelium, AF lenses were chosen as a contralateral control to help negate any potential physiologic changes that may be induced by presence of the GP material on the OK eyes. All lenses (both OK and AF) were specially designed for the cat eye with overall lens diameter of 12.3 mm and thickness of 0.16 mm. The same material (Menicon Z, nominal Dk of 160 barrers) and manufacturer (Paragon Vision Sciences, Mesa, AZ, USA) was used throughout the study.
The OK lenses were a modified Paragon CRT design (Paragon Vision Sciences,
Mesa, AZ, USA). The range of lens parameters in the trial lens set is shown in
Table 3.1. A BOZR of 10.0 mm was selected to provide an approximate 4.00D myopic reduction.
Table 3.1: OK lens parameters used in the study. Diameter BOZR RZD LZA 1.025 1.050 34 36 12.3 mm 10.0 mm 1.075 1.100 38 1.125 1.150 *Back Optic Zone Radius (BOZR), Return Zone Depth (RZD) and Landing Zone Angle (LZA).
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Chapter 3: Epithelial Changes with Overnight Orthokeratology Lens Wear
All AF lenses incorporated a posterior optical zone of 10.5 mm and a spherical peripheral design system to create an axial edge lift of approximately 120 microns.
The base curve radii ranged from 8.3 mm to 8.7 mm in 0.1 mm steps. The centre thickness was designed at 0.16 mm to match that of the fellow OK lenses and the powers were plano. Using the OrthoTools 2000 Lens Design Software (EyeDeal
Software and Design, Fresno, California) the AF lens designs resulted in a theoretical apical clearance of 15 microns.
3.2.4 Lens Fits
Three of the 15 cats served as non-contact lens wearing controls and the remaining
12 animals were randomly fitted with an OK lens on one eye and an AF lens on the contralateral eye. Acceptable lens fits were determined based on the same fitting criteria used in humans for alignment and myopic Paragon CRT lenses. For AF lenses, an acceptable fit was one in which the lens exhibited slight apical clearance, mid-peripheral bearing and 360 degrees of peripheral clearance with fluorescein. For the OK lenses, an acceptable lens fit was one in which there was overall centration of the lens, with 4 to 5 mm of central treatment (fluorescein thinning). Figure 3.1 shows examples of acceptable OK and AF lens fits.
Orthokeratology Epithelial Changes and Susceptibility to Microbial Infection 50
Chapter 3: Epithelial Changes with Overnight Orthokeratology Lens Wear
Figure 3.1: Examples of acceptable (a) AF and (b) OK-fitted lenses.
3.2.5 Lens Wear
Both the OK and AF lenses were worn on an overnight basis; they were placed on the eye in the late afternoon and removed in the morning for an average of approximately 16 hours of lens wear. It should be noted that cats are crepuscular animals with extremely unpredictable sleep patterns making it impossible to mimic the overnight wearing schedule of human OK lens wearers (6 to 8 hours of continuous eye closure). Observational and anecdotal reports suggest that cats sleep intermittently with closed eye time totalling more than half the day. Based on this assumption, an overnight wearing period of 16 hours was used to try to ensure half the time (8 hours) of closed eye lens wear was experienced. Although not ideal, this extended open and closed-eye wearing period allowed enhanced corneal changes as daily open-eye wearing of OK lenses results in corneal changes as well (Swarbrick et al, 1998; Fan et al, 1999; Lui and Edwards, 2000; Tahhan et al, 2002; Sridharan and Swarbrick, 2003; Jackson et al, 2004).
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Chapter 3: Epithelial Changes with Overnight Orthokeratology Lens Wear
3.2.6 Corneal Topography
To assess OK treatment centration and help monitor corneal changes, corneal topography maps were taken using the Medmont E300 Corneal Topographer
(Medmont International Pty Ltd, Melbourne, Australia). Animals were placed in cloth restraint bags for the measurement and no sedation was required. For right eye measurements, animals were placed on their right sides such that the horizontal axis of the instrument measurement cone was aligned with the vertical meridian of the right eye; similarly, for left eye measurements, animals were placed on their left sides (Figure 3.2). Topography measurements were taken using the automatic capture option of the instrument for both eyes at baseline, one day, three days, one week, two weeks and four weeks of lens wear. Difference display maps were used to monitor changes in corneal topography relative to baseline at each time point.
Lens parameters and fits were changed as necessary based on these topography plots to achieve a centred treatment pattern in the case of OK lenses and to assure no significant corneal changes in the case of AF lenses.
Figure 3.2: Set up of animal for topography measurement. Example of (a) right eye and (b) left eye measurement using the Medmont E300 corneal topographer.
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Chapter 3: Epithelial Changes with Overnight Orthokeratology Lens Wear
3.2.7 Lens Care
During lens wear, all lenses were removed using a sterilized DMV (Zanesville,
OH, USA) contact lens remover. Lenses were then cleaned, rinsed and stored using Unique pH multipurpose solution (Alcon Laboratories, Fort Worth, TX,
USA) according to the manufacturer’s instructions. Additional cleaning during lens storage in Unique pH was performed by adding one drop of Opti-free
SupraClens solution (Alcon Laboratories, Fort Worth, TX, USA). Prior to being placed on eyes, lenses were rinsed with Unique pH solution.
3.2.8 Lens Wearing Schedule
Five time points were investigated: one day, three days, one week, two weeks and four weeks after commencement of lens wear. The time points were chosen to reflect key milestones in the establishment of corneal changes with OK lenses.
One and three day time points represent early corneal changes while one and two week time points represent times when full corneal changes are generally achieved.
Four weeks was used as a longer term time point to represent established changes.
At the end of each wearing period, one group of three animals was sacrificed with an overdose of pentobarbital sodium (Virbac Australia Pty, Milberra, Australia) and eyes collected for histological staining and examination.
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Chapter 3: Epithelial Changes with Overnight Orthokeratology Lens Wear
3.2.9 Specimen Preparation for Corneal Histology
All eyes were enucleated and fixed (whole globe) in a combination of 2.5% paraformaldehyde and 2% glutaraldehyde in 0.1M phosphate buffer (pH 7.2) fixative for 24 hours, after which the corneas were excised and bisected along the vertical meridian. The left portion of each cornea was then sent to the Histology and Microscopy Unit of the University of New South Wales for histological processing and the right portion was retained for future processing. For histology, the corneas were processed on the Sakura VIP tissue processor (Sakura, Japan) with the following protocol - dehydration through graded ethanol (70%, 80%, 95%, and 3x absolute alcohol), cleared in Xylene and infiltrated with Medite tissue embedding medium (HD Scientific, Sydney Australia). Corneas were then sectioned in four micron intervals with the Leica RM 2165 semi-automated rotary microtome (Leica, Wetzlar, Germany) and sections were mounted on adhesive microscope slides. Slides were dried and placed in a 60ºC oven for 30 minutes.
3.2.10 Epithelial Analysis
3.2.10.1 Hematoxylin & Eosin Staining
Slides were stained with Hematoxylin and Eosin (H&E) to help evaluate epithelial thickness and morphology changes. Briefly, sections were hydrated through absolute, 95% and 70% ethanol, washed in running tap water and then stained with routine H&E.
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3.2.10.2 Epithelial Photography
Slides were viewed on a Leica Leitz DIAPLAN microscope (Leica, Wetzlar,
Germany) with a Leica DFC 480 digital camera attached. Sequential photographs of the cornea were taken at a magnification of 20X then stitched together using
Adobe Photoshop Version 7.0 (Adobe Systems Pty, Australia). Sections of the epithelium corresponding to the five different areas of the OK lens were identified from the stitched picture and photographed at 100X magnification. As the corneas were bisected vertically, corneal areas were labelled per their corneal position: superior periphery (SUP), superior mid-periphery (MPS), centre (CEN), inferior mid-periphery (MPI) and inferior periphery (INF). Figure 3.3 shows an example of the five areas (SUP, MPS, CEN, MPI, INF) examined within OK and AF-treated corneas.
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Figure 3.3: Corneal sections at 20X magnification stained with H & E. Sections stitched together for a panoramic cross section of the whole cornea. Illustrated (a) OK and (b) AF lenses overlayed on top of corneas to demonstrate the areas of interest for epithelial analysis (SUP = superior periphery, MPS = superior mid-periphery, CEN = centre, MPI = inferior mid-periphery, INF = inferior periphery). (c) Examples of magnified epithelium in areas of interest.
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3.2.10.3 Epithelial Thickness Measurements
Epithelial thickness measurements from the photographs (at 100X magnification) were made using customized semi-automated image analysis software developed for exact and repeatable measurements. The software was written in C using the
LabWindows/CVI (National Instruments Corp., Austin, TX, USA) software development environment (Appendix 2). Parts of the image analysis algorithm were performed by functions provided by the IMAQ Vision for LabWindows/CVI library (National Instruments Corp., Austin, TX, USA). This application performs edge detection in a variable number of rectangular search areas. These areas are evenly distributed over a user defined region of interest for the respective image.
Edges are detected along five evenly spaced vertical spoke lines within each measurement rectangle using a user-defined threshold. The first and last edge along each spoke line is displayed to the user for verification. Upon approval, the vertical distance between the two outermost edges is converted into real-world units and saved. For this study, the number of rectangular search areas was set to five, resulting in five thickness values that were averaged for each image. Figure
3.4 shows an example of the measurement software used.
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Figure 3.4: Example of an epithelial measurement using the customized image analysis software developed for this study. (a) User defined region of interest (green box); (b) five vertical measurement spoke lines within original defined box.
3.2.11 Stromal Keratocyte Analysis
As mentioned in Chapter 2, the epithelium and stroma are not independent of each other but are instead in constant communication. Stress exerted on the epithelium can cause the release of various epithelial factors that can affect the stroma, specifically keratocytes (Wilson et al, 1996a). Also, keratocyte density has been found to be affected by contact lens wear in primates (Bergmanson and Chu, 1982) and in humans (Jalbert and Stapleton, 1999; Efron et al, 2002). As a preliminary investigation into potential stromal changes induced by OK lens wear, numbers of keratocytes in regions directly adjacent to areas of epithelial manipulation were enumerated.
3.2.11.1 DAPI Staining
A separate set of slides was stained with 4’,6-diamidino-2-phenylindole (DAPI).
DAPI is a stain that forms a fluorescent complex with natural double-stranded
DNA. It is generally used to label cell nuclei and has been used to identify and
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monitor stromal keratocytes (Kratz-Owens et al, 1992). Sections were hydrated through a series of absolute ethanol, then 70% ethanol and brought to water.
Sections were then stained with DAPI (Molecular Probes, Oregon USA) according to the manufacturer’s instructions. Briefly, slides were incubated with DAPI solution (0.2 g/ml) for two minutes then rinsed in phosphate buffered saline
(PBS; 0.137M NaCl, 2.6 mM KCl, 1.5 mM KH2PO4, 1.66 mM Na2HPO4; pH 7.4) for five minutes before dehydration and cover-slipping.
3.2.11.2 Stromal Photography
Slides were viewed on an Olympus BX51 microscope (Tokyo, Japan) with an
Olympus DP70 digital camera attached. DAPI signal was detected with the U-
MWU2 UV filter cube. Serial photographs of the cornea were taken at magnification of 20X and stitched together using Adobe Photoshop Version 7.0
(Adobe Systems Pty, Australia). The five areas of epithelial thinning and thickening in OK-treated eyes identified with H & E staining (as described above) were identified again in the DAPI stained slides. Rectangular boxes measuring
355 by 565 microns were centred around each of the five areas of interest and overlayed on the composite photograph. Identical boxes were placed on corresponding areas on the composite photograph of the contralateral AF-treated eye of each animal. Figure 3.5 shows an example of the different stromal areas examined within each cornea.
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Figure 3.5: A composite of corneal sections stained with DAPI. Five corneal areas were identified based on previous epithelial examination. Rectangular boxes were overlayed on the cornea and centred on these five areas to delineate boundaries for keratocyte counts.
3.2.11.3 Stromal Keratocyte Counts
All boxes of corneal areas were printed and coded and numbers of keratocytes within each box were enumerated manually by two masked observers and compared to the same area in the contralateral AF eye.
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3.2.12 Statistical Analysis
All statistical analyses in this chapter were performed using SPSS Version 15.0 for
Windows (Chicago, IL, USA). A p-value less than or equal to 5% was considered statistically significant.
3.3 Results
3.3.1 Topography Results
Difference display maps of OK-treated corneas showed topographical changes characteristic of human OK-treated eyes with a large central area of corneal flattening surrounded by a ring of corneal steepening. The average treatment effect obtained at four weeks was 4.75D ± 1.50D of central flattening. Due to the variability in fixation with the animals, topography served as a qualitative analysis to confirm appropriate corneal changes rather than a detailed quantitative analysis.
AF-treated eyes showed minimal corneal changes with slight fluctuations in curvature likely due to tear film irregularities. Figure 3.6 shows examples of difference topography maps for AF and OK-treated eyes of the same animal.
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Figure 3.6: Examples of corneal topographies for (a) AF and (b) OK eyes after 2 weeks of overnight lens wear on the cat eye.
3.3.2 Epithelial Thickness of Control Animals
Mean epithelial thicknesses in the five corneal areas for both eyes of control animals are presented in Figure 3.7 (raw data in Appendix 1 Table 7.1). The average thicknesses across all corneal areas were 39.4 ± 8.4 μm for right eyes and
39.9 ± 6.7 for left eyes. Epithelial thicknesses varied between animals with measurements ranging from 31.6 ± 0.3 to 50.2 ± 0.2 μm.
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OD OS 60 m) 50 40 30 20 10
Epithelial thickness ( 0 SUP MPS CEN MPI INF Corneal Area
Figure 3.7: Average non-lens wearing control epithelial thicknesses for each area in right (OD) and left (OS) eyes. Error bars represent standard deviation of measurements.
To determine uniformity of epithelial thickness within eyes, analysis of variance
(ANOVA) for repeated measures was carried out, using corneal area as within subject factor. No significant differences were found between the five areas within eyes (all p > 0.05; Table 3.2). To determine if epithelial thicknesses differed significantly between eyes of animals, epithelial thicknesses for each area were compared to their counterparts in the other eye with paired t-tests. No significant differences were found between eyes (all p > 0.05; Table 3.2). The uniformity of epithelial thickness within and between eyes of non-lens wearing control animals provides a baseline for future comparisons with epithelial thickness changes in contact lens wearing animals.
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Table 3.2: Control epithelial thickness comparisons for five corneal areas (SUP, MPS, CEN, MPI, INF) within eyes (ANOVA) and between eyes (t-test). Epithelial Thickness (µm) ANOVA Mean (SD) p-value SUP MPS CEN MPI INF OD 39.5 (8.3) 40.1 (8.3) 39.2 (7.7) 39.0 (7.3) 39.3 (8.3) 1.00
OS 39.1 (7.6) 40.9 (6.8) 40.8 (7.8) 40.3 (8.4) 39.4 (9.7) 0.998
t-test 0.825 0.418 0.309 0.298 0.962 p-value
Morphological analysis showed that the epithelium of all three control animals consisted of approximately 7 to 9 cell layers with characteristic appearances of basal, wing and surface cells in both eyes. The uniformity of epithelial morphology of areas within and between eyes of control animals also provides a baseline for future comparisons with lens wearing animals. Figure 3.8 shows an example of the epithelial areas from both eyes of one control animal.
Figure 3.8: Non-lens wearing control epithelium. Representative samples of five corneal areas in right (top) and left (bottom) eyes.
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3.3.3 Epithelial Thickness of Contact Lens Wearing Animals
Having established the uniformity of epithelial thickness within and between eyes in control animals, changes in epithelial thickness for OK lens wearing eyes were investigated at various time points after commencing lens wear. For each time point and corneal area, the difference in epithelial thickness was calculated by subtracting AF thicknesses from contralateral OK thicknesses (OK – AF). For easier terminology, this difference will be called “change in epithelial thickness”.
Changes in epithelial thickness were normally distributed (Shapiro-Wilk, p >
0.108). Mean changes in epithelial thickness (in microns) for the five corneal areas at each time point are shown in Table 3.3 and represented graphically in
Figure 3.9.
Table 3.3: Average change in epithelial thickness (µm) in five corneal areas at different time points. Thickness changes in non-lens wearing control eyes were calculated by subtracting left from right eye values, all other values are expressed as difference from the AF eye. ((-) values represent decreased thickness, (+) values represent increased thickness). Change in Epithelial Thickness (µm) Time N Mean (SD) Point SUP MPS CEN MPI INF
Control 3 + 0.5 (3.2) - 0.8 (1.4) - 1.6 (2.1) - 1.4 (1.7) - 0.1 (1.9) 1 Day 3 - 11.9 (3.6) + 7.9 (7.6) - 12.0 (5.8) + 5.2 (6.3) - 10.5 (3.8) 3 Days 3 - 2.7 (5.2) + 12.6 (10.5) - 9.5 (3.5) + 5.0 (6.5) - 9.5 (5.8) 1 Week 3 - 10.3 (5.7) + 8.9 (3.1) - 16.8 (0.1) + 6.1 (3.9) - 10.5 (7.6) 2 Weeks 3 - 2.8 (1.8) + 7.8 (0.8) - 6.3 (1.5) + 8.2 (3.1) - 9.1 (6.5) 4 Weeks 3 - 9.1 (8.4) + 13.3 (6.9) - 8.4 (5.3) + 8.8 (3.2) - 14.4 (11.2)
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CONTROL 1 DAY 3 DAY 1 WEEK 2 WEEK 4 WEEK
m) 30
20
10
0
-10
-20
-30 Change in Epithelial Thickness ( Thickness Epithelial in Change SUP MPS CEN MPI INF Corneal Area
Figure 3.9: Mean change in epithelial thickness change (µm) across time points in five corneal areas. Error bars represent standard deviation of measurements.
A large change in epithelial thickness in all areas occurred after one night of lens wear with OK eyes showing thinned central (CEN), thickened mid-peripheral
(MPS and MPI) and thinned peripheral (SUP and INF) epithelium. This pattern of epithelial thickness modulation was maintained throughout all time points. To determine if OK thickness changes were significantly different from control animal thicknesses within each area, changes over time points were compared using an
ANOVA test (Appendix 1 Table 7.2). This analysis revealed only the centre area
(CEN) exhibited significant changes in epithelial thickness (ANOVA, p = 0.006).
Post-hoc analysis of this area using Dunnett tests found significance for the one day (p = 0.019) and one week time points (p = 0.001) compared to control animals.
Maximum changes in epithelial thickness were achieved at different time points for each area: SUP at one day (-11.9 ± 3.6), MPS and MPI at four weeks (+13.3 ± 6.9;
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+8.8 ± 3.2), CEN at one week (-16.8 ± 0.1), and INF at four weeks (-14.4 ± 11.2).
To better appreciate the pattern of changes in epithelial thickness within each area over time, a profile of these data is shown as a line graph in Figure 3.10.
SUP MPS CEN MPI INF m)
20 15 10 5 0 -5 -10 -15 -20
Change in Epithelial Thickness ( Thickness Epithelial in Change Control 1 Day 3 Day 1 Week 2 Week 4 Week Time Point
Figure 3.10: Profile of average change in epithelial thickness (µm) in five corneal areas over time up to four weeks of lens wear.
As noted in the previous section with the non-lens wearing control group, epithelial thickness can vary between animals. To determine if inter-animal thickness variations may affect the pattern of thickness changes, changes in epithelial thickness were also expressed as percentage change (from contralateral
AF thickness) in Table 3.4 and Figures 3.11 and 3.12 below.
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Table 3.4: Average change in epithelial thickness (%) five corneal areas at different time points. Control thickness change expressed as change from left eye values, all other percentages expressed as change from AF starting points. ((-) values represent decreased thickness, (+) values represent increased thickness). Change in Epithelial Thickness (%) With Increasing Lens Wear Time N Mean (SD) Point SUP MPS CEN MPI INF Control 3 + 1.3 (9.0) - 2.5 (3.6) - 4.0 (5.6) - 3.1 (4.2) - 0.5 (5.3) 1 Day 3 - 30.7 (10.2) + 22.9 (21.4) - 27.6 (10.0) + 14.1 (15.7) - 30.9 (7.3) 3 Days 3 - 6.6 (12.7) + 33.7 (29.6) - 24.0 (5.7) + 13.2 (16.5) - 22.3 (11.8) 1 Week 3 - 23.5 (11.6) + 22.8 (8.6) - 40.4 (3.5) + 14.1 (8.8) - 25.9 (18.7) 2 Weeks 3 - 7.9 (5.0) + 23.2 (5.1) - 16.7 (5.7) + 21.0 (7.4) - 23.6 (15.8) 4 Weeks 3 - 26.3 (26.4) + 34.8 (19.1) - 21.7 (14.0) + 21.7 (8.7) - 39.0 (31.2)
CONTROL 1 DAY 3 DAY 1 WEEK 2 WEEK 4 WEEK 80 60
40 20 0 -20 -40 -60 -80 Change in Epithelial Thickness (%) Thickness Epithelial in Change SUP MPS CEN MPI INF Corneal Area
Figure 3.11: Mean change in epithelial thickness change (%) across time points in five corneal areas. Error bars represent standard deviation of measurements.
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SUP MPS CEN MPI INF 50 40 30 20 10 0 -10 -20 -30 -40 -50 Change in Epithelial Thickness (%) Thickness Epithelial in Change Control 1 Day 3 Day 1 Week 2 Week 4 Week Time Point
Figure 3.12: Mean change in epithelial thickness (%) in OK eyes in five corneal areas up to four weeks of lens wear.
A comparison of Figures 3.10 and 3.12, found almost identical patterns of thickness change for each corneal area over the different time points suggesting very little inter-animal thickness effects. Statistical analysis of thickness changes using the same tests with percentage values compared to controls was performed.
Again, only the centre area (CEN) showed significant change (ANOVA, p =
0.001) and post-hoc analysis found significance (Dunnett, p < 0.05) for all time points (Appendix 1 Table 7.3).
A detailed qualitative description of morphologic changes at each time point is given in the following sections. All epithelial pictures in the following Figures were photographed at 100X magnification.
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3.3.3.1 One Day Animals
After one night of lens wear, changes in epithelial thickness were observed in OK eyes of all three animals (Figure 3.13). OK eyes showed thinning of the central epithelium (average 28% from Table 3.4), thickening of the mid-peripheral epithelium and thinning of the peripheral epithelium. Based upon the flattened nuclear shape of the basal and lower wing cells central thinning appears to be due to compression of cells. Mid-peripheral thickening appears to be due to a slight increase in numbers of cell layers and a concomitant increase in the height of basal and wing cells. Peripheral thinning appears to be a combination of cellular compression as well as a decrease in the number of cell layers although the magnitude of reduction varied between animals (thickness decrease ranged from 20 to 40%). No changes to the expected epithelial cell morphology were observed in the AF eyes of any animals.
Figure 3.13: One day epithelial changes. Representative sample of five different areas of epithelium after one night of AF (top row) and OK (bottom row) lens wear.
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3.3.3.2 Three Day Animals
After three nights of lens wear, the same pattern of changes in epithelial thickness was seen in the OK and AF-treated eyes as after one day of lens wear (Figure
3.14). The central epithelium thinned by approximately 24% and this also appeared to be due to compression of cells. Mid-peripheral thickening was largely attributed to an increase in the number of cell layers present and peripheral thinning was due to compression of cells with no loss of cell layers observed. AF eyes did not show any appreciable changes to epithelial thickness in all five areas.
Figure 3.14: Three day epithelial changes. Representative sample of five different areas of epithelium after three nights of AF (top row) and OK (bottom row) lens wear.
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3.3.3.3 One Week Animals
After one week of overnight lens wear, epithelial thickness changes were similar to those seen in the previous time points (Figure 3.15). The central epithelium at one week exhibited the largest degree of thinning of all experimental time points
(about 40%) and appears to be due solely to compression of cells as evidenced with the lowered positioning of the basal cell nuclei and flattened shape of the wing cells; the number of cell layers in the centre remained the same as in the contralateral AF eyes. Mid-peripheral epithelium thickened due to an increase in cell number. The peripheral epithelium showed thinning due to compression of cells similar to that observed in the central epithelium.
Figure 3.15: One week epithelial changes. Representative sample of five different areas of epithelium after one week of overnight AF (top row) and OK (bottom row) lens wear.
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3.3.3.4 Two Week Animals
After two weeks of overnight lens wear, epithelial thickness change patterns were maintained (Figure 3.16) however they were not as pronounced as in the one week animals. The least amount of central epithelial thinning was observed with this time point (about 16%) and thinning appeared to be attributed to compression of cells as observed with the general flattening of the nuclei of all cells. Mid- peripheral thickening was due to a slight increase in numbers of cells and peripheral thinning from compression.
Figure 3.16: Two week epithelial changes. Representative sample of five different areas of epithelium after two weeks of overnight AF (top row) and OK (bottom row) lens wear.
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3.3.3.5 Four Week Animals
After four weeks of overnight lens wear the same pattern of epithelial changes as at the other time points were clearly present (Figure 3.17). The central thinning appeared again to be compressive in nature with flattening of basal and wing cell nuclei observed. Mid-peripheral thickening showed a large increase in numbers of cell layers present (the largest increase out of all time points). Peripheral thinning varied in magnitude between animals; for those with large amounts of thinning, cellular compression and a decrease in number of cell layers were observed, for those with less thinning, cellular compression was the main observation.
Figure 3.17: Four week epithelial changes. Representative sample of five different areas of epithelium after four weeks of overnight AF (top row) and OK (bottom row) lens wear.
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3.3.4 Stromal Keratocyte Counts
3.3.4.1 Control Animals
The average numbers of keratocytes in the five corneal areas for all three control animals are presented in Figure 3.18 (raw data in Appendix 1 Table 7.4). The average numbers of keratocytes across all corneal areas were 155 ± 8 for right eyes and 165 ± 6 for left eyes.
OD OS 250
200
150
100
50 Keratocyte Count Keratocyte 0 SUP MPS CEN MPI INF Corneal Area
Figure 3.18: Average keratocyte counts for each corneal area in non-lens wearing control eyes. Error bars represent standard deviation of measurements.
To determine if there was uniformity of keratocyte counts across the five areas within each eye, average keratocyte counts were compared between areas with
ANOVA for repeated measures (Table 3.5). No significant differences were found between the five areas within eyes (all p > 0.05). To determine if keratocyte counts differed significantly between right and left eyes of animals, keratocyte counts were compared using paired t-tests for each corneal area (Table 3.5). Only
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the superior peripheral area (SUP) was found to approach significance between eyes (p = 0.052). The uniformity of keratocyte counts within and between eyes of non-lens wearing control animals provides a baseline for comparisons of keratocyte count changes in contact lens wearing animals.
Table 3.5: Comparison of average keratocyte counts between areas within each eye (ANOVA) and between eyes (t-test) of control animals. Keratocyte Count ANOVA Mean (SD) p-value SUP MPS CEN MPI INF OD 152 (6) 161 (6) 156 (10) 153 (7) 151 (2) 0.399 OS 170 (11) 173 (29) 166 (19) 169 (16) 146 (2) 0.415 t-test 0.052 0.577 0.616 0.253 0.166 p-value
3.3.4.2 Lens Wearing Animals
Having established the uniformity of keratocyte counts within and between eyes in control animals, changes in keratocyte counts for OK lens wearing eyes were investigated at various time points after commencing lens wear. For each time point and corneal area, the difference in keratocyte counts was calculated by subtracting AF counts from contralateral OK counts (OK – AF). For easier terminology, this difference will be called “change in keratocyte counts”. Change in keratocyte counts were normally distributed (Shapiro-Wilk, p > 0.256). Mean changes in keratocyte counts for the five corneal areas at each time point are shown in Table 3.6 and represented graphically in Figure 3.19.
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Table 3.6: Mean change in keratocyte counts in all corneal areas and at all time points. Differences calculated by subtracting contralateral AF from OK counts. Keratocyte count difference Time N Mean (SD) Point SUP MPS CEN MPI INF
Control 3 - 18 (7) - 12 (32) - 10 (29) - 17 (18) + 5 (4) 1 Day 3 - 9 (22) - 14 (11) - 2 (7) - 10 (8) 0 (7) 3 Days 3 - 10 (36) + 14 (22) + 15 (23) + 15 (35) - 12 (13) 1 Week 3 + 4 (18) + 5 (10) + 13 (8) + 7 (9) - 5 (30) 2 Weeks 3 + 5 (8) + 15 (16) + 1 (6) + 4 (13) + 3 (4) 4 Weeks 3 - 5 (24) - 1 (7) - 3 (15) + 15 (16) + 10 (2)
CONTROL 1 DAY 3 DAY 1 WEEK 2 WEEK 4 WEEK 60
40
20
0
-20
-40 Change in Keratocyte Count in Keratocyte Change -60 SUP MPS CEN MPI INF Corneal Area
Figure 3.19: Mean change in keratocyte counts across time points in five corneal areas. Error bars represent standard deviation of measurements.
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To determine if change in keratocyte counts were significantly different from control animal counts within each area, keratocyte changes over time points were compared using an ANOVA test (Appendix 1 Table 7.5). No significant changes in keratocyte counts were found at any time point in any area (all p > 0.05) when compared to non-lens wearing controls.
3.4 Discussion
3.4.1 Epithelium
The epithelium of OK-treated eyes examined in this study confirmed the pattern of thickness changes with central thinning and mid-peripheral thickening as reported in the previous chapter and by others (Matsubara et al, 2004; Cheah et al, 2008).
Additionally, improved corneal sectioning procedures in the current study allowed visualization of the peripheral epithelial thinning occurring beneath the landing zone or fitting curve of the OK lens.
3.4.1.1 Central Epithelial Thickness
Interestingly, the central epithelium did not thin as much as was expected based on the pilot study. It was observed in Chapter 2 that the central epithelium thinned by about 70% (27 μm) with two weeks continuous wear of an OK lens when compared to a separate control animal in that study. In the current study the central epithelium only reached a maximum thinning of approximately 40% (16.8
μm) at the one week time point and despite a longer wearing time only 21% (8.4
μm) thinning after four weeks of lens wear when compared to contralateral AF
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control eyes. This lesser central thinning in the current study compared to the results of the last chapter may be due to a number of factors. Firstly, the different wearing schedules may play a large role in the amount of thinning induced. This study employed an overnight wearing schedule more likely to be experienced in regular human OK lens wear as opposed to the continuous wear schedule used in the pilot study. The period of no lens wear during the day may provide the epithelium with an opportunity to revert to its normal shape and thicken during the day before lenses are placed back on the eye at night. This could potentially prevent a cumulative epithelial thinning effect and therefore illustrates the importance of not over-wearing OK lenses to prevent excessive thinning of the central epithelium.
Secondly, removal of the nictitating membrane in the current study to facilitate lens wear allowed for simulation of normal lid dynamics. This may have eliminated any potential additional pressure exerted on the lenses and subsequent epithelial changes that may have resulted from its presence.
Thirdly, the contralateral design of the current study provided a more reliable comparison of thickness changes as opposed to the inter-animal comparison in the pilot study. As mentioned earlier, the normal epithelial thickness in the cat can vary greatly between animals as was found with a range of 31 μm to 49 μm in the central cornea of AF-fitted eyes. The contralateral control in this study was critical in providing a reliable basis to determine the degree of epithelial thinning or thickening observed. Additionally, use of an AF lens on the control eye made from the same material helped to negate any potential physiological changes induced by lens wear.
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The discrepancy in the magnitude of central thinning between Chapters 2 and 3 may account for the lack of statistical significance seen at some of the CEN time points in the current study. The sample size for this study was calculated based on the assumption that central thickness changes would be similar to those in Chapter
2. As this was not observed, the assumption was incorrect. Thus a larger study involving more animals will be needed to demonstrate statistical significance for epithelial thickness changes at all CEN time points.
3.4.1.2 Mid-Peripheral Epithelial Thickness
The mid-peripheral epithelium thickened in all OK-treated eyes however, the magnitude of thickening at most time points was less than that observed in Chapter
2 (13 μm at 14 days). As mentioned above, the sample size was calculated to detect central epithelial thickness changes thus it was not surprising that the smaller levels of mid-peripheral thickness changes did not achieve statistical significance.
3.4.1.3 Peripheral Epithelial Thickness
Peripheral thinning observed in this study was another interesting and novel finding. The amount of thinning in this area was extremely variable between animals and no discernible pattern in amount of thinning was observed. The most likely cause of these changes was tightness of the peripheral lens fit of some animals due to limitations in the range of parameters for the OK peripheral fitting curve. Questions remain as to the implications for eye health of thinning and constricting this area. Given the natural centripetal pattern of epithelial migration
(Haskjold et al, 1989; Lemp and Mathers, 1989; Lavker et al, 1991), constricting
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this peripheral region may cause a back up in the migration of basal cells that could potentially affect the limbal source stem cells (Lavker et al, 2004; Sun and
Lavker, 2004). One of the benefits of OK is the approximate 16 hours of daytime no lens wear during which the cells have exposure to unlimited oxygen and unrestricted movements. It is believed that during this time cells have an opportunity to revert to their natural state. Further work to investigate OK effects on the migratory pattern of epithelial cells and effects on limbal stem cells would be useful in providing insights on this topic.
3.4.1.4 Morphological Changes
Morphologically, findings from this study are consistent with the shorter term results of the last chapter. Central thinning in the current study was due to compression of the cells particularly in the basal layer with no decrease in the number of cell layers present at all time points. The mid-peripheral thickening appears to be attributed to a combination of cellular elongation and increased cell layers in the first week, then only due to an increase in cell layers for later time points. This latter finding supports the previous suggestion of different mechanisms working to equalize the epithelium over time. More specifically, for time points up to one week, a potential transfer of intracellular fluid from central to mid-peripheral cells plus an alteration in either cell mitosis, apoptosis, exfoliation or migration might be occurring. However, after one week the mid- peripheral basal cells appear to return to baseline shapes and the thickness appears only to be due to an increase in cell numbers.
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3.4.1.5 Temporal Effects
The previous pilot study demonstrated a time-related change in epithelial thickness with a progressive decrease in central thickness and increase in mid-peripheral thickness over the time periods examined. This temporal pattern of change was not observed in the current study. Either the treatment stabilized early or this could just be a reflection of slightly different degrees of treatment.
3.4.2 Stroma
Results from the previous chapter suggested that changes to stromal thickness may be occurring with OK and this has also been reported in several clinical studies
(Swarbrick et al, 1998; Alharbi and Swarbrick, 2003; Haque et al, 2007). Given the constant communication between the epithelium and stroma (Wilson et al,
1999) coupled with the rather dramatic changes to epithelial thickness and morphology observed in this study, it was of interest to investigate any potential effects these epithelial modifications had on the stromal tissue. A measurement of stromal thickness in relation to epithelial thickness change would have yielded the most beneficial results for investigation of this relationship. However, the focus of this study was on the epithelium and as such the tissue processing method was chosen to best maintain the epithelial rather than the stromal integrity. Whilst the processing technique induced artefacts in the stroma that precluded accurate measurement of stromal thickness, it was not anticipated to affect keratocyte numbers and staining, thus allowing analysis of keratocyte populations.
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The results from this study suggest that numbers of keratocytes in areas corresponding to OK epithelial thickness changes did not statistically change during the course of one month of lens wear. These findings contradict preliminary work that has suggested that keratocyte density does change with OK lens wear (Perez-Gomez et al, 2003; Wang et al, 2004). These results are also challenged by studies that have found decreased keratocyte density with contact lens wear in humans (Jalbert and Stapleton, 1999; Efron et al, 2002; Kallinikos and
Efron, 2004) and animals (Bergmanson and Chu, 1982; Yamamoto et al, 2001).
These findings are however, in agreement with studies that have found no effect on keratocyte density in long term contact lens wearers (Patel et al, 2002;
Hollingsworth and Efron, 2004).
Although the keratocyte counts did not significantly change in the current study, the state of these stromal cells is still in question. As mentioned, DAPI is a generic stain for double-stranded DNA regardless of what state of health the cells may be in. Previous studies have found that epithelial injury can mediate keratocyte apoptosis via cytokines such as Interleukin-1 (IL-1) (Wilson et al,
1996a) and soluble Fas ligand (Wilson et al, 1996b). Specific examination of the apoptotic and mitotic states of stromal keratocytes is warranted to help understand keratocyte changes that may be induced with OK lens wear but not necessarily reflected in the keratocyte count in this study due to the non-specificity of the
DAPI stain. More elaborate immuno-histochemical staining of the corneal tissue for cytokine activity could help shed further light on this topic.
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3.4.3 Limitations
The increased number of animals per time point in this study has added more confidence to the repeatability and generalizability of these OK-induced epithelial changes. However, as mentioned above, the statistical analyses suggest that a larger study involving more animals will be needed to achieve statistical significance for epithelial thickness changes for all areas and time points.
Although the OK lenses were designed for a target refractive change of - 4.00D, there was no clear indication as to how much of that refractive change was actually achieved in each animal. Refractive data (auto-refraction or retinoscopy) was not obtained in this study due to logistical reasons. The erratic fixation of animals during topography measurements resulted in less reliable quantitation of corneal shape changes. This precluded a more detailed comparison of epithelial thickness with topographic changes. Improvements in fixation during corneal topography or the addition of a reliable assessment of refraction in the animals could improve interpretation of these epithelial changes in future studies.
This was a relatively short term study with examination of corneal changes only up to four weeks of lens wear. Orthokeratology patients usually wear lenses for several years thus it will be of interest to determine whether longer term wear of the lenses result in different corneal changes. Corneal anomalies such as central corneal epitheliopathy (Ng, 2006), fibrillary lines (Cheung et al, 2006), white lesions (Cheung et al, 2005), corneal iron rings (Cho et al, 2002a; Liang et al,
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2003; Hiraoka et al, 2004a) and pigmented arcs (Cho et al, 2003b; Cho et al,
2005b) have been reported in patients wearing OK lenses for longer periods of time, suggesting that these changes are taking place with repeated lens wear.
Longer term studies are needed to investigate whether OK lens wear imposed over months or years result in different anatomic and physiologic changes to the epithelium and stroma.
As with any histological study, limitations in the preservation of the corneal tissue are always present. It is acknowledged that shrinkage of the tissue may occur with the fixation procedure however, given that the analysis was based on differences between eyes of each animal (intra-specimen), this histological artefact would be minimized as all tissue was treated in the same manner. The tissue processing method was chosen based on its ability to yield a panoramic cross section of the cornea for analysis of changes in epithelial thickness and cellular morphology
(form and shape). However, this method limited the ability to specifically quantify changes in cellular morphometry (height and width) due to limitations in the resolution of the sections. Higher resolution techniques such as transmission electron microscopy would be more useful for an in depth analysis of epithelial changes.
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3.4.4 Summary
This study confirmed the previous findings of central epithelial thinning and mid- peripheral thickening with increasing OK lens wear. It has also demonstrated variability in thinning of the peripheral epithelium likely due to tightness of lens fit. The amount of central thinning seen with a more regular overnight lens wearing schedule was less pronounced than previously reported in the continuous wear pilot study. No time-related patterns for magnitude of thickness changes were observed. The number of stromal keratocytes adjacent to areas of epithelial thickness changes did not change over the course of one month of OK treatment.
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Chapter 4: Recovery of Epithelial Changes After Cessation of Overnight Orthokeratology Lens Wear
CHAPTER 4: Recovery of Epithelial Changes After
Cessation of Overnight Orthokeratology
Lens Wear
4.1 Introduction
The previous chapters have established that substantial changes in epithelial thickness and morphology occur across the cornea with increasing periods of OK lens wear. Questions remain as to the reversibility of these epithelial changes.
One of the claimed major advantages of OK is that it is a reversible refractive procedure whereby the cornea regains its pre-treatment shape shortly after cessation of lens wear. While clinically this has been found to be true from a topographic (Soni et al, 2004), refractive (Barr et al, 2004; Soni et al, 2004) and epithelial thickness (Haque et al, 2004; Soni et al, 2004; Haque et al, 2007) perspective, no studies have investigated recovery of the corneal tissue on a cellular level.
4.1.1 Aims
The purpose of this study was to determine if the epithelial changes induced with myopic OK lens wear are reversible.
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4.1.2 Hypotheses
Primary Hypothesis:
Epithelial changes induced with overnight OK lens wear are reversible.
Secondary hypotheses:
Epithelial thickness is restored after cessation of lens wear
Epithelial cell morphology is restored after cessation of lens wear
4.2 Materials and Methods
4.2.1 Study Design and Sample Size
Twelve, two-year-old cats were used in this study (Vision CRC & IER Animal
Research Facility, Sydney, Australia). Animals were randomly divided into groups of three for four different time points. The number of animals used was determined based on a significance level of 0.05, power of 0.80 and a difference in central epithelial thickness change between OK-treated and non-treated eyes assumed to be 30 ± 10 microns based on an estimate from the pilot study results reported in Chapter 2. Calculations were made using the PS Power and Sample
Size Calculations Program (Vanderbilt University Department of Biostatistics,
Nashville, TN, USA).
All animals in this study were treated according to a protocol approved by the
Vision CRC - Institute for Eye Research Animal Ethics Committee (VIAEC) in
Sydney, Australia. All experimental procedures conformed to the ARVO statement for the Use of Animals in Ophthalmic and Vision Research.
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4.2.2 Animal Preparation
All animals were prepared for this study as described in Chapter 3.
4.2.3 Contact Lenses
OK and AF lenses were used. All contact lenses, lens fits (including topography), lens wear, lens care procedures and descriptions were as described in Chapter 3.
4.2.4 Lens Wearing Schedules
All animals were randomly fitted with an OK lens in one eye and an AF lens on the contralateral eye. All animals wore OK and AF lenses overnight for four weeks after which time lens wear was ceased. Three animals were sacrificed with an overdose of pentobarbital sodium (Virbac Australia Pty, Milberra, Australia) at one day, three days, one week and four weeks following cessation of lens wear and eyes collected for histological staining and examination. Time points were chosen to reflect key milestones in the recovery of corneal changes with OK lenses. One and three day time points represent early corneal regression while one and four week time points represent times when full corneal recovery is thought to be achieved.
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4.2.5 Histology
All histological processing of corneal tissue including slide preparation, H & E staining, DAPI staining, epithelial measurements and keratocyte counts were identical to those described in Chapter 3.
4.2.6 Statistical Analysis
All statistical analyses in this chapter were performed using SPSS Version 15.0 for
Windows (Chicago, IL, USA). A p-value less than or equal to 5% was considered statistically significant.
4.3 Results
4.3.1 Epithelial Thickness
As all animals in this study had worn lenses overnight for four weeks, the assumption that animals attained an epithelial thickness profile similar to the four week time point results from Chapter 3 was made. Thus, four week lens wear thickness data from the previous chapter were included as a comparison to regressive data from this study to measure the magnitude of epithelial recovery after ceasing lens wear. Comparisons to non-lens wearing control thicknesses presented in the last chapter were also made to determine if changes in epithelial thickness regressed to baseline non-lens wearing control levels. For each time point and corneal area, the difference in epithelial thickness was calculated by subtracting AF thicknesses from contralateral OK thicknesses (OK – AF) (again
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Chapter 4: Recovery of Epithelial Changes After Cessation of Overnight Orthokeratology Lens Wear referred to as “change in epithelial thickness”). Changes in epithelial thickness were normally distributed (Shapiro-Wilk, p > 0.144). Mean changes in epithelial thickness (in microns) for the five corneal areas at each time point are shown in
Table 4.1 and represented graphically in Figure 4.1.
Table 4.1: Average change in epithelial thickness (µm) between OK and AF eyes in five corneal areas after cessation of four weeks of lens wear. *Start = 4 week data from Chapter 3. **Control data from Chapter 3. Change in Epithelial Thickness (µm) After Cessation of Lens Wear Time N Mean (SD) Point SUP MPS CEN MPI INF *Start 3 - 9.1 (8.4) + 13.3 (6.9) - 8.4 (5.3) + 8.8 (3.2) - 14.4 (11.2) 1 Day 3 - 3.7 (4.9) + 10.8 (10.6) + 1.4 (10.4) + 9.5 (11.5) - 3.2 (5.9) 3 Days 3 - 2.2 (4.8) + 2.5 (5.1) - 1.2 (5.4) + 3.3 (8.2) - 3.4 (3.9) 1 Week 3 - 1.9 (2.9) - 1.8 (4.1) - 1.7 (4.8) + 1.6 (5.7) - 0.5 (0.5) 4 Weeks 3 - 0.9 (3.3) - 1.8 (2.3) - 0.3 (2.5) + 0.8 (2.4) + 0.4 (2.7) Control** 3 + 0.5 (3.2) - 0.8 (1.4) - 1.6 (2.1) - 1.4 (1.7) - 0.1 (1.9)
START 1 DAY 3 DAY 1 WEEK 4 WEEK CONTROL 30 m) 20
10
0
-10
-20
Change in Epithelial Thickness ( Thickness Epithelial in Change -30 SUP MPS CEN MPI INF Corneal Area
Figure 4.1: Mean change in epithelial thickness (µm) across time points in five corneal areas after cessation of four weeks of lens wear. Error bars represent standard deviation of measurements. *Start = four week data from Chapter 3, Control = non-lens wearing control data from Chapter 3.
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Changes in epithelial thickness commenced recovery towards control thickness values one day after cessation of lens wear and eventually regained control values in all areas within one week. To better appreciate the pattern of changes in epithelial thickness within each area over time, a profile of these data is shown as a line graph in Figure 4.2.
SUP MPS CEN MPI INF m)
20 15 10 5 0 -5 -10 -15 -20
Change in Epithelial Thickness ( Thickness Epithelial in Change *Start 1 Day 3 Day 1 Week 4 Week Control Time Points
Figure 4.2: Profile of change in epithelial thickness (µm) in five corneal areas over time after cessation of four weeks of lens wear. *Start = four week data from Chapter 3
The magnitude of change in epithelial thickness decreased in all areas after one night of ceasing lens wear. This suggests that compared to the starting four week data from Chapter 3, the epithelial thickness in OK eyes thickened centrally
(CEN), thinned mid-peripherally (MPS and MPI) and thickened peripherally (SUP and INF) in this study. This pattern of epithelial thickness recovery continued throughout all time points.
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To determine if changes in epithelial thickness after stopping lens wear were significantly different within each area from the four week starting values of
Chapter 3, an ANOVA was carried out over time points (Appendix 1 Table 7.6).
This analysis revealed only area MPS (p = 0.045) exhibited significant changes in epithelial thickness. Post-hoc analysis using Dunnett tests found this to only approach significance for the one week (p = 0.051) and four week (p = 0.052) time points compared to four week starting values of Chapter 3.
All changes in epithelial thickness decreased towards control values. To determine if epithelial thickness differences regained baseline control levels, an identical statistical analysis was conducted to compare differences between recovery results and non-lens wearing control results from Chapter 3. These results are presented in Appendix 1 Table 7.7. No significant differences from control eyes were found in any of the areas at any time point (ANOVA, all p > 0.05).
As noted in Chapter 3 with the non-lens wearing control group, epithelial thickness can vary between animals. To determine if inter-animal thickness variations may affect the pattern of thickness changes observed, changes in epithelial thickness were also expressed as percentage change from contralateral AF thickness in
(Table 4.2 and Figures 4.3 and 4.4).
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Table 4.2: Mean change in epithelial thickness (%) in five corneal areas after cessation of four weeks of lens wear. ((-) values represent decreased thickness, (+) values represent increased thickness) *Start = four week data from Chapter 3. *Control data from Chapter 3. Change in Epithelial Thickness (%) After Cessation of Lens Wear Time Point Mean (SD) SUP MPS CEN MPI INF *Start - 26.3 (26.4) + 34.8 (19.1) - 21.7 (14.0) + 21.7 (8.7) - 39.0 (31.2)
1 Day - 10.6 (13.7) + 30.6 (30.8) + 5.2 (28.9) + 28.9 (37.0) - 8.5 (19.4)
3 Days - 5.1 (14.0) + 7.2 (13.9) - 2.5 (14.0) + 8.7 (21.3) - 8.5 (9.9)
1 Week - 4.5 (8.0) - 4.3 (10.7) - 4.3 (11.5) + 4.1 (14.7) - 1.4 (1.4)
4 Weeks - 2.5 (9.7) - 4.6 (5.7) - 1.1 (6.8) + 2.0 (6.3) + 0.9 (8.1)
*Control + 1.3 (9.0) - 2.5 (3.6) - 4.0 (5.6) - 3.1 (4.2) - 0.5 (5.3)
START 1 DAY 3 DAY 1 WEEK 4 WEEK CONTROL 80
60
40
20
0
-20
-40
-60
Change in Epithelial Thickness (%) Thickness Epithelial in Change -80 SUP MPS CEN MPI INF Corneal Area
Figure 4.3: Profile of change in epithelial thickness (%) in five corneal areas after cessation of four weeks of lens wear. *Start = four week data from Chapter 3. Control data from Chapter 3.
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SUP MPS CEN MPI INF 50 40 30 20 10 0 -10 -20 -30 -40 -50
Change in Epithelial Thickness (%) Thickness Epithelial in Change *Start 1 Day 3 Day 1 Week 4 Week Control Time Points
Figure 4.4: Profile of change in epithelial thickness (%) in five corneal areas after cessation of four weeks of lens wear. *Start = four week data from Chapter 3. Control data from Chapter 3.
A comparison of Figures 4.2 and 4.4, found almost identical patterns of thickness change for each corneal area over the different time points suggesting very little inter-animal thickness effects. Statistical analysis of thickness changes using the same tests with percentage values compared to four week starting values from
Chapter 3 was performed (Appendix 1 Table 7.8). Again only area MPS (p =
0.031) showed significant change and post-hoc analysis using Dunnett tests found this to be significant for the one week (p = 0.033) and four week (p = 0.033) time points compared to four week starting values.
Statistical analysis of thickness changes using the same tests with percentage values compared to non-lens wearing controls from Chapter 3 was also performed
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(Appendix 1 Table 7.9). No significant differences from control eyes were found in any of the areas at any time point (ANOVA, all p > 0.05).
A detailed qualitative description of morphologic changes is given in the following sections. All epithelial pictures in the following Figures were photographed at
100X magnification.
4.3.1.1 One Day Animals
After one full day of no lens wear, a key finding was that the epithelium thickened centrally in OK eyes compared to starting four week central thickness in Chapter 3.
Interestingly, the central epithelium was found to be thicker than the contralateral
AF eye in two of the three animals in this group. In these two animals, central OK areas showed an increased number of cell layers compared to AF eyes (Figure 4.5).
One animal still showed central epithelium that was thinner than its contralateral
AF eye and the thinning appeared to be due to cellular compression.
Average change in mid-peripheral epithelial thickening in OK eyes varied between areas with MPS decreasing and MPI increasing (Tables 4.1 and 4.2). There were still relatively more cell layers present than in AF eyes in these areas in two of the three animals (Figure 4.5). The large variation in thickness data in this region was attributed to one animal whose mid-peripheral thickness approached that of the contralateral AF eye. This animal showed numbers of cell layers in OK eyes to be the same as AF eyes.
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Compared to four week wear data of Chapter 3, the OK peripheral epithelium appeared to thicken towards AF values but only achieved AF levels in one animal.
No difference in number of cell layers was observed in any animals therefore thinning in this region was attributed to cellular compression as evidenced by the flatter nuclei in OK eyes. As mentioned in the previous chapter, the peripheral area was the most variable and thickness was highly dependent upon individual tightness of lens fit.
Figure 4.5: Epithelial changes one day after cessation of lens wear. Representative sample of five areas of epithelium one day after cessation of four weeks of overnight AF (top row) and OK (bottom row) lens wear.
4.3.1.2 Three Day Animals
At the three day post-cessation time point, the pattern of thickness difference was still present across the cornea though not as pronounced as after one day. Relative to four week wear data from Chapter 3 the central epithelium thickened whereas relative to one day recovery data the central epithelium thinned to achieve control
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AF thickness levels. OK cells appeared to regain their shape as evidenced in the morphologic similarity to epithelial cells of AF eyes (Figure 4.6). Mid-peripheral epithelium thinned considerably from four week wear and one day recovery time points but did not reach AF levels. There were still more cell layers present in this region compared to corresponding areas of AF eyes. Peripheral epithelium thickened to approach AF values and any residual thinning in this region was due to slight compression of cells.
Figure 4.6: Epithelial changes three days after cessation of lens wear. Representative sample of five areas of epithelium three days after cessation of four weeks of overnight AF (top row) and OK (bottom row) lens wear.
4.3.1.3 One Week Animals
Epithelium of all three animals at this time point recovered to thickness values of
AF eyes in all five corneal areas (Figure 4.7). Cellular morphology in OK eyes also regained contralateral AF epithelial shapes. Numbers of cell layers were the same in both eyes for all areas.
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Figure 4.7: Epithelial changes one week after cessation of lens wear. Representative sample of five areas of epithelium one week after cessation of four weeks of overnight AF (top row) and OK (bottom row) lens wear.
4.3.1.4 Four Week Animals
As with the one week results, all three animals at this time point regained epithelial thickness, morphology and cell layer numbers in all five areas of the OK eyes compared to contralateral AF eyes (Figure 4.8).
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Figure 4.8: Epithelial changes four weeks after cessation of lens wear. Representative sample of five areas of epithelium four weeks after cessation of four weeks of overnight AF (top row) and OK (bottom row) lens wear.
4.3.2 Stromal Keratocyte Counts
Having established the uniformity of keratocyte counts within and between eyes in control animals (Chapter 3), changes in keratocyte counts for OK lens wearing eyes were again investigated. For each time point and corneal area, the difference in keratocyte counts was calculated by subtracting AF counts from contralateral
OK counts (OK – AF) (again referred to as “change in keratocyte counts”).
Change in keratocyte counts were normally distributed (Shapiro-Wilk, p > 0.162).
Mean changes in keratocyte counts for the five corneal areas at each time point are shown in Table 4.3 and represented graphically in Figure 4.9. Four week and control animal keratocyte counts from Chapter 3 were included for reference.
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Table 4.3: Mean change in keratocyte counts in all corneal areas and at all time points after cessation of four weeks of lens wear. Differences calculated by subtracting contralateral AF from OK counts. *Start = 4 week data from Chapter 3. *Control data from Chapter 3. Change in keratocyte count Time N Mean (SD) Point SUP MPS CEN MPI INF
*Start 3 - 5 (24) - 1 (7) - 3 (15) + 15 (16) + 10 (2) 1 Day 3 + 5 (18) - 6 (2) + 16 (9) + 6 (8) + 3 (11) 3 Days 3 - 2 (12) + 23 (10) - 14 (11) - 15 (15) + 5 (5) 1 Week 3 0 (8) - 7 (25) - 16 (3) + 9 (7) - 11 (6) 4 Weeks 3 + 22 (1) + 7 (1) - 8 (18) - 7 (24) - 11 (13) *Control 3 - 18 (7) - 12 (32) - 10 (29) - 17 (18) + 5 (4)
START 1 DAY 3 DAY 1 WEEK 4 WEEK CONTROL 40 30 20 10 0 -10 -20 -30 -40 Change in Keratocyte Count -50 SUP MPS CEN MPI INF Corneal Area
Figure 4.9: Mean change in keratocyte counts in five corneal areas at different time points after cessation of four weeks of lens wear. Error bars represent standard deviation of measurements. *Start = 4 week data from Chapter 3. Control data from Chapter 3.
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To determine if change in keratocyte counts were significantly different from control animal counts within each area, keratocyte changes over time points were compared using an ANOVA test (Appendix 1 Table 7.10). Only peripheral area
SUP showed significant changes (ANOVA, p = 0.015) and post-hoc analysis found a significant change for the four week time point (Dunnett, p = 0.004) compared to non-lens wearing controls. This was an unexpected finding and warranted further analysis. Thus keratocyte changes in the four week time point were further compared to the starting values (four week keratocyte changes from Chapter 3).
No significant difference was found (Independent t-test, p = 0.124) however this was likely due to the large variability in the starting four week keratocyte counts from Chapter 3.
4.4 Discussion
4.4.1 Epithelium
Corneal epithelial changes were found to be reversible following cessation of four weeks of overnight myopic OK lens wear. All five areas of OK epithelium examined regained thickness values similar to the contralateral AF eye within one week of cessation of lens wear.
4.4.1.1 Epithelial Rebound
The pattern of early epithelial recovery after one day was interesting. After one full day of no lens wear, the average central epithelium was thicker than the four week lens wear results from Chapter 3. The magnitude of thickening, i.e.
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Chapter 4: Recovery of Epithelial Changes After Cessation of Overnight Orthokeratology Lens Wear exceeding the thickness of contralateral AF eyes in two of the three animals, suggests that in some animals, the epithelium goes through a rather dramatic
“rebound” in the first 24 hours following lens wear. This overshoot of central epithelial thickness has not previously been reported. The presence of a larger number of cell layers in these animals compared to four week lens wear animals suggests either “excessive” cell migration, an increase in cell mitosis or a decrease in cell apoptosis and sloughing occurs. Increased cell mitosis has been reported in imprinted epithelium of corneas fit with steep RGP lenses in the rabbit (Greenberg and Hill, 1973) and thinned central epithelium of OK-treated eyes also in the rabbit
(Matsubara et al, 2004). However, increased mitosis after a period of lens removal has not been investigated.
4.4.1.2 Variability in Regression Rate
Variation in epithelial thickness observed between animals at the one day time point could potentially reflect the variability in regression of OK effects in human patients. In current clinical practice, OK lenses are removed in the morning to begin the cycle of no lens wear during the day. For the majority of patients, the cornea regresses towards its original shape during this time manifesting in a slow degradation of visual acuity throughout the day (Mountford, 1998; Nichols et al,
2000; Rah et al, 2002; Johnson et al, 2007). This regression necessitates nightly wear of lenses to retain corneal changes and subsequent visual effects. However, practitioners have been known to adjust this wearing schedule for individual patients depending on the cornea’s ability to retain the treatment effect. Some patients need only wear lenses every other night, every second night or variations
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Chapter 4: Recovery of Epithelial Changes After Cessation of Overnight Orthokeratology Lens Wear thereof (Mountford, 1997b). The ability of corneas of these patients to retain OK effects longer may be due to variability in speed of epithelial recovery similar to that observed in some animals in this study.
4.4.1.3 Speed of Recovery
These relatively rapid recoveries in epithelial thickness are in agreement with previous clinical findings. Using optical coherence tomography (OCT) Haque and colleagues (Haque et al, 2004) found that after four weeks of lens wear, corneal and epithelial thickness recovered to baseline values after three days. In a more recent study Haque and colleagues (Haque et al, 2007) found that after a single night of lens wear, central epithelial thickness had not completely returned to baseline levels in the subsequent 12 hours whereas paracentral epithelium recovered to baseline thickness only in eyes treated with a higher Dk lens material.
Their results suggest that recovery of epithelial thickness even after just one night of lens wear takes more than 12 hours. Recovery of central thickness findings in the current study are also in agreement to those found by Soni and colleagues (Soni et al, 2004) who observed that central corneal thickness returned to baseline levels within one night after cessation of one month of lens wear. Given the rapidity of the recovery of epithelial changes to original thickness, any residual optical effects experienced by patients beyond three days and especially after one week of cessation of lens wear would likely be due to other longer lasting non-epithelial changes occurring in the stroma.
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4.4.1.4 Recovery of Morphology
The return of epithelial cell morphology in OK corneas to AF shapes after cessation of lens wear demonstrates innate drivers of natural shape and thickness of the epithelial layer. It is unclear whether recovery to baseline is due to cells regaining their original shape or increased epithelial production. The one week timing of full epithelial recovery coincides with the reported epithelial turnover rate of seven days (Hanna et al, 1961) suggesting turnover plays a role. However, for the few animals that recovered within the short three day time period, restoration of cellular morphology is more likely to contribute to recovery. Effects of OK on epithelial homeostasis are still unclear as variant changes to mitotic rates have been observed in two OK rabbit studies (Matsubara et al, 2004; Shin et al,
2005). A more detailed analysis of physiological changes to the epithelium should provide more insight to this question.
4.4.2 Stroma
The significant increase in the keratocyte count in one peripheral area (SUP) at the four week time point compared to controls was an unexpected finding. The reason for such a significant increase is unknown although an increase in anterior stromal keratocyte density has been reported seven days after cessation of one week of OK lens wear (Perez-Gomez et al, 2003). Further work to confirm and understand these changes is warranted.
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4.4.3 Limitations
The present study was relatively short with examination of corneal changes after only four weeks of overnight lens wear. Longer term OK wear has been shown to have higher sustainability of visual acuity (Johnson et al, 2007; Kang et al, 2007) and apical corneal power (Mountford, 1998) throughout the day compared to shorter term wear suggesting that adaptation to retain corneal changes may occur.
It will be of interest to determine whether longer term (years) lens wear results in corneal changes that may alter the reversibility of the epithelium.
Due to similarities in methods employed in this chapter, many of the limitations discussed in Chapter 3 are still applicable to the current study. These include the statistical analysis, topographical analysis and histological processing limitations.
4.4.4 Summary
This study found that short term (up to four weeks) OK is a reversible procedure with epithelial thickness and morphology returning to contralateral control thicknesses and shapes within one week of cessation of lens wear. Additionally, minimal changes to keratocyte numbers were observed after cessation of lens wear.
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CHAPTER 5: Orthokeratology and Corneal
Susceptibility to Microbial Keratitis
5.1 Introduction
The previous chapters have established that OK lenses have the ability to substantially but reversibly alter the shape and thickness of the epithelium.
However, as mentioned in Chapter 1, it has been suggested that manipulation of the corneal tissue with OK may make the cornea more susceptible to infection.
Thus, questions now arise regarding the implications of these epithelial changes.
Are we perhaps predisposing the cornea to microbial infection by manipulating the epithelium this way?
This is a particularly important question in light of numerous reports of microbial keratitis (MK) reported in OK lens wearers and summarized in Chapter 1.
Although an exact incidence rate of MK with OK is difficult to ascertain due to problems in estimating numbers of OK lens wearers, the sudden rise in reported infections has raised concerns about the safety of the OK modality.
Pseudomonas aeruginosa (P. aeruginosa) is the most common bacterial pathogen isolated from contact lens-associated infections (Stapleton et al, 1995; Lam et al,
2002; Sharma et al, 2003) and this trend is no different in the MK cases reported in
OK (Watt and Swarbrick, 2007b). At least two phenotypes of P. aeruginosa
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(cytotoxic and invasive) are involved in causing corneal disease (Cole et al, 1998).
However, in most cases the phenotype of P. aeruginosa causing the infection is not determined. Given the large number of P. aeruginosa cases reported in OK infections, it is of interest to understand whether manipulating the corneal tissue with OK is a compromise sufficient to allow an infection by this opportunistic pathogen.
The epithelium is one of the eye’s most important defences against ocular infections. An intact epithelium is integral to preventing invasion of micro- organisms into the corneal tissue (Fleiszig, 2006; Willcox, 2006). Several animal models have been used successfully to investigate contact lens and non-contact lens induced ocular infections (Davis and Chandler, 1975; Hazlett et al, 1985;
Solomon et al, 1994). These studies suggest that to produce an infection with P. aeruginosa, a compromise or injury that breaches the epithelial barrier is needed to allow for bacterial invasion into the cornea. Given the dramatic fashion in which we are manipulating the epithelium with OK, it is of interest to determine whether these changes represent such a compromise or injury.
5.1.1 Aims
The purpose of this study was to determine if the epithelial changes associated with short term wear of myopic OK lenses render the cornea more susceptible to P. aeruginosa infection.
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5.1.2 Hypotheses
Primary Hypothesis:
OK epithelial changes increase the risk of a corneal infection
Secondary Hypotheses:
Duration of lens wear will increase the risk of infection
Continuous lens wear will increase the risk of infection
Larger amounts of P. aeruginosa will increase the susceptibility to infection
5.2 Materials and Methods
5.2.1 Study Design and Sample Size
This study consisted of a series of seven different bacterial exposure challenges to the eyes of animals after OK and AF lens wear. Twenty-two cats (Vision CRC &
IER Animal Research Facility, Sydney, Australia) were used. The number of animals used in this study was aimed at detecting a difference in infection rate between OK-treated and non-treated eyes initially of 30%, at a significance level of 0.05 based on a best estimate from literature reports (Watt and Swarbrick,
2005).
All animals were treated according to a protocol approved by the Vision
Cooperative Research Centre and Institute for Eye Research Animal Ethics
Committee in Sydney, Australia and all experimental procedures conformed to the
ARVO statement for the Use of Animals in Ophthalmic and Vision Research.
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5.2.2 Animal Preparation
All animals were prepared for this study as described in Chapter 3.
5.2.3 Contact Lenses
OK and AF lenses were used. All contact lenses, lens fits, lens wear, lens care procedures and descriptions are as described in Chapter 3.
5.2.4 Corneal Topography
Corneal changes were monitored using the Medmont corneal topographer
(Medmont International Pty Ltd, Melbourne, Australia) at baseline, one day, three days, one week, two weeks, four weeks and up to six weeks of lens wear. Lens parameters and fits were changed as necessary to achieve a centred treatment pattern in OK eyes and minimal corneal changes in AF eyes.
5.2.5 Lens Wearing Schedule
Two wearing schedules were used in this series of seven challenges. For challenges involving overnight lens wear, lenses were placed on eye in the late afternoon and removed in the morning for an average of 16 hours wear each night.
For challenges involving continuous lens wear, lenses were left on the eye and only taken out for a maximum of 30 minutes to facilitate topographic examination.
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5.2.6 Bacterial Strains and Growth Conditions
Two strains of P. aeruginosa were used (6294 and 6206). Both strains were isolated from human ocular patient specimens and obtained from the School of
Optometry, University of California, Berkeley, USA. Strain 6294 has been shown to have an invasive phenotype whereas strain 6206 has a cytotoxic phenotype
(Fleiszig et al, 1996). Stock cultures of 6294 and 6206 stored in 30% glycerol at -
80 °C were used. Both Pseudomonal strains were grown overnight in 10mL of tryptone soy broth at 37 °C for 18 hours. Bacterial cells were harvested by centrifugation (Eppendorf 5810, Hamburg, Germany) for 10 minutes (2,000g at 18
°C), and washed in sterile phosphate buffered saline (PBS; 0.137M NaCl, 2.6 mM
KCl, 1.5 mM KH2PO4, 1.66 mM Na2HPO4; pH 7.4). Cells were resuspended in
PBS to an optical density of 2.5 measured at 660nm (OD660) in a spectrophotometer (Heliosß, Unicam Instruments, Cambridge, UK). Bacterial concentration was confirmed by plating serially diluted samples (1:10) of bacterial suspension on nutrient agar plates and colonies counted to determine numbers of colony forming units (cfu). Quantification of the OD660 2.5 inoculum found 3.8 x
1010 cfu/mL. One mL of this bacterial suspension was used to soak lenses as described below.
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5.2.7 Lens Bacterial Inoculation
5.2.7.1 Test Lenses
For each challenge, after confirmation of corneal changes through topography, lenses were washed four times by placing in 1 mL of sterile water and shaking for one minute on a plate shaker (175 rpm). Lenses were submerged concave side up for 10 minutes in 1mL of the above bacterial suspension in a 24-well tissue culture plate. Lenses were washed twice in 1mL PBS for one minute on a plate shaker to remove loose bacteria before being placed on animal eyes. Bacterial cells were resuspended to OD660nm = 2.5 in PBS in order to drop bacteria on to the eye either during or after lens wear for Challenges III, IV and VI (described below).
5.2.7.2 Unworn Control Lenses
To estimate numbers of bacteria initially adhering to lenses prior to placement on eye, viable counts were performed on 24 unworn control lenses (12 OK and 12 AF) for each strain of P. aeruginosa. Control lenses were washed four times by placing them in 1mL of sterile water and shaking for one minute on a plate shaker (175 rpm). Lenses were submerged concave side up for 10 minutes in 1mL of bacterial suspension in a 24-well tissue culture plate. Lenses were washed twice in 1mL
PBS for one minute on a plate shaker to remove loose bacteria before undergoing enumeration.
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5.2.8 Enumeration of Bacteria on Lenses
Control lenses and all worn lenses from Challenges I, II, and VII were collected at the end of the study and placed in sterile 5 mL plastic containers filled with 2 mL sterile PBS and vortexed for one minute with a small magnetic stirrer. A 0.1 mL aliquot of the resultant suspension was serially diluted (1:10) in sterile PBS. These samples (20 L) were plated in duplicate on nutrient agar (Oxoid, Sydney,
Australia) plates and incubated for 18 hours at 37 °C. Numbers of colonies per dilution were recorded and used to calculate the number of cfu per contact lens. It should be noted that this study only counted viable bacteria isolated from lenses with this procedure; there was no attempt to identify or quantify dead bacteria or their toxic by-products on the lenses.
5.2.9 Bacterial Adhesion Statistical Analysis
Total counts were summarized using descriptive statistics for each time point (0 = unworn controls, two weeks = Challenges I & VII, six weeks = Challenge II), bacterial strain and lens type. As some of the animals were used more than once in the study, total counts were compared between the two lens types for each bacterial strain and time point using a linear mixed model to adjust for within subject correlation. To test the impact of prior lens wear on adherence, total counts were also compared between time points using linear mixed models for each strain and lens type. For the mixed models analysis, data at the baseline visit (time = 0) were combined effectively to create a single subject (None). However for other visits the animal name was used to identify within subject correlation. Dependent variables
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(Total counts) were log-transformed for the analyses and a p-value less than or equal to 0.05 was considered statistically significant. Post-hoc multiple comparisons were adjusted using Bonferroni correction. All analysis was performed using SPSS for Windows version 15.0 (Chicago, IL).
5.2.10 Validation of Cat Model for Pseudomonas aeruginosa
Keratitis: Positive Scratch Control
To validate the cat infection model and determine if the amount of bacteria adhering to test lenses in our study was enough to cause infection given an effective corneal compromise, a positive scratch control was performed for both strains of P. aeruginosa (6294 and 6206). Two cats (Vision CRC & IER Animal
Research Facility, Sydney, Australia) were used. A single eye of one animal was used for each strain of bacteria. The cat scratch keratitis model was based on similar procedures described by others (Cole et al, 1998; Cowell et al, 1998).
5.2.10.1 Corneal Scratch Procedure
After anaesthetizing animals with isoflurane gas (Veterinary Companies of
Australia Pty, Kings Park, Australia) and a drop of oxybuprocaine hydrochloride
0.4% (Bausch & Lomb, Australia), two horizontal scratches approximately 5 mm long, and 2.5 mm apart were created in the central cornea of one eye with a 25 gauge needle. Effort was made to control the scratches so they were to the full depth of the epithelium but did not penetrate the stroma.
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5.2.10.2 Inoculated Contact Lens Wear
After the corneal scratch procedure, an AF lens (BC = 8.54) inoculated with either
P. aeruginosa 6294 or 6294 as described above was immediately placed on the scratched eye of each animal and left for 16 hours. During this time animals were monitored closely for any signs of discomfort and/or development of infection.
5.2.10.3 Results of Scratch Control
After 16 hours of inoculated lens wear, characteristic signs of an MK were observed in scratched eyes of both positive control animals. Figure 5.1 shows the positive scratch control results for P. aeruginosa 6294. An extensive central area of stromal infiltration was seen, overlying epithelium was ulcerated and stromal destruction with moderate corneal edema was observed. Instilled fluorescein diffused immediately into the stroma, demonstrating a full thickness loss of the epithelium. The extent of epithelial ulceration was considerably more widespread than the initial scratch defect. A moderate to severe anterior chamber reaction, along with severe conjunctival hyperemia was seen. Animals were immediately euthanized with an overdose of pentobarbital sodium (Virbac Australia Pty,
Milperra, Australia).
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Figure 5.1: Results of a positive corneal scratch control with P. aeruginosa 6294. (a) Cat cornea immediately after scratching the epithelium and prior to inoculated lens wear. The same cornea 16 hours after wearing an AF lens soaked with P. aeruginosa 6294 as seen in (b) white light and (c) blue light with fluorescein staining.
.
5.2.10.4 Summary of Scratch Control
This positive scratch control confirmed the cat cornea can become infected with P. aeruginosa strains 6294 and 6206 when the cornea is sufficiently compromised and exposed to bacteria. This study also confirmed the amount of bacteria adherent to lenses with this soaking method is sufficient to cause an infection if an effective corneal compromise is present.
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5.2.11 Bacterial Exposure Challenges
Bacterial challenges of various durations and extents were given in an attempt to elicit a difference in the infection rate between the AF and OK lens wearing eyes.
Animals were re-used for all the challenges if there was no infection, with a washout period of 2.0 ± 0.5 weeks of no lens wear between challenges. All animals were monitored for any adverse effects at baseline, prior to and after inoculated lens wear in each challenge. A description of each bacterial exposure challenge is given below, followed by a summary of the challenges presented in
Table 5.1. Figure 5.2 shows illustrations of the types of bacterial exposures used for the different challenges in this study.
5.2.11.1 Challenge I: Two Week Overnight Lens Wear with Bacterial
Inoculation on the Last Night of Lens Wear
Animals wore daily cleaned and disinfected lenses for 16 hours every night
for two weeks. After confirmation of corneal changes with topography,
lenses were inoculated with strain 6294 as per the protocol described above
and placed on the eye for an additional overnight wearing period of 16 hours
(Figure 5.2(a)). In this group there was one instillation of bacteria. All
lenses were collected after the inoculated wearing period to enumerate
bacteria remaining on the lenses.
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5.2.11.2 Challenge II: Six Week Overnight Lens Wear with Bacterial
Inoculation on the Last Night of Lens Wear
To determine if longer term wear of lenses may predispose to infection, a
study that was similar to Challenge I, but clean lenses were worn for six
weeks prior to inoculation with strain 6294 for the last night of lens wear
(Figure 5.2(a)), i.e. one instillation of bacteria. All lenses were collected
after the inoculated wearing period to enumerate bacteria remaining on the
lenses.
5.2.11.3 Challenge III: Two Week Overnight Inoculated Lens Wear with
Nightly Instillation of Bacterial Drops
Animals wore strain 6294 inoculated lenses for 16 hours every night for two
weeks. Each night, four hours after insertion of inoculated lenses, a 20 μL
drop of the same suspension of strain 6294, was placed onto each eye on top
of the contaminated contact lens (Figure 5.2 (b)), i.e. 28 instillations of
bacteria.
5.2.11.4 Challenge IV: Two Week Overnight Inoculated Lens Wear with
Multiple Daily Bacterial Drops After Lens Removal Each Day
Animals wore strain 6294 inoculated lenses for 16 hours every night for two
weeks. After lens removal each morning, a 20 μL drop of strain 6294 of the
same concentration was placed in each eye directly onto the cornea, two
hours and four hours after lens removal (Figure 5.2 ( c)), i.e. 42 instillations
of bacteria.
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5.2.11.5 Challenge V: Two Week Continuous Lens Wear followed by a
Night of Inoculated Lens Wear
To determine if continuous wear of OK lenses predispose to infection,
animals wore clean lenses continuously for two weeks. After confirmation of
corneal changes with topography at two weeks, lenses were inoculated with
strain 6294 and placed on eye for an additional overnight wearing period of
16 hours (Figure 5.2 (a)), i.e. one instillation of bacteria.
5.2.11.6 Challenge VI: Two Week Continuous Inoculated Lens Wear with an Additional Twice Daily Instillation of Bacterial Drops
To determine if continuous wear of lenses with continuous exposure to
bacteria predisposes to infection, animals wore strain 6294 inoculated lenses
continuously for two weeks; a 20 μL drop of strain 6294 of the same
concentration was placed onto each eye on top of contaminated contact lenses
mid-morning and mid-afternoon (Figure 5.2 (b)), i.e. 29 instillations of
bacteria.
5.2.11.7 Challenge VII: Two Week Overnight Lens Wear with Bacterial
Inoculation on the Last Night of Lens Wear (using P. aeruginosa 6206)
To determine if results of the above challenges were confounded by strain
specificity, animals wore daily cleaned and disinfected lenses as in Challenge
I, but lenses were inoculated with the cytotoxic strain 6206 as per the
protocol described above (Figure 5.2 (a)). In this group there was one
instillation of bacteria. All lenses were collected after the inoculated wearing
period to enumerate planktonic bacteria remaining on the lenses.
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Figure 5.2: Illustration of the types of bacterial exposure for different challenges. (a) lens soaked in bacteria and placed on eye (Challenges I, II, V, VII). (b) lens soaked in bacteria, placed on eye and additional drops of bacteria on top of lens (Challenges III, VI). (c) lens soaked in bacteria and placed on eye at night, bacteria dropped directly onto cornea when lens removed in the morning (Challenge IV).
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Table 5.1: Summary of bacterial exposure Challenges I to VII. Inoculation Score represents the number of instillations of bacteria. Lenses Study Lens Bacterial Strain of Bacterial In/Out Inoculation Challenge Wear Schedule Duration Inoculation Enumeration P. aeruginosa Drops During Score (weeks) Time point Performed Drop I 6294 Overnight 2 End None n/a 1 Yes
II 6294 Overnight 6 End None n/a 1 Yes
III 6294 Overnight 2 Daily Nightly IN 28 No
IV 6294 Overnight 2 Daily Twice Daily OUT 14 No
V 6294 Continuous 2 End None n/a 1 No
VI 6294 Continuous 2 Beginning Twice Daily IN 29 No
VII 6206 Overnight 2 End None n/a 1 Yes
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5.2.11.8 Clinical Observations and Inflammation Analysis
All animals were monitored for any adverse effects at baseline, prior to and after inoculated lens wear in each challenge. The integrity and health of the eye was examined and assessed using a modified CCLRU grading scale. Grades ranged from absent to severe for redness, chemosis, and discharge and presence of infiltrates was noted. Percentages of animals exhibiting different degrees of increases in each category compared to baseline were reported for each challenge.
5.3 Results
5.3.1 Bacterial Exposure Challenges
A description of results obtained in each challenge is presented below and a summary of results is presented in Table 5.2.
5.3.1.1 Challenge I: Two Week Overnight Lens Wear with Bacterial
Inoculation on the Last Night of Lens Wear
Of the original 22 animals, four suffered repeated lens losses and self-induced
scratches to the eyes; these animals were permanently removed from the
study. No infections were observed in the eyes of any of these animals.
Qualitatively, there was a slight increase in the amount of discharge and
redness observed in both eyes of most animals after the inoculated lens
wearing period. No increase in the amount of chemosis or infiltrates was
observed.
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5.3.1.2 Challenge II: Six Week Overnight Lens Wear with Bacterial
Inoculation on the Last Night of Lens Wear
Eighteen animals started and completed this study. No infections were
observed in the eyes of any of the animals. Qualitatively, there was a slight
increase in the amount of discharge and redness observed in both eyes of
most animals after the inoculated lens wearing period. No increase in the
amount of chemosis or infiltrates was observed.
5.3.1.3 Challenge III: Two Week Overnight Inoculated Lens Wear with
Nightly Instillation of Bacterial Drops
Eighteen animals started the study and 14 animals completed the study. Of
the four discontinued animals, three were removed as a result of self-induced
scratches to the eyes; the remaining animal had a broken lens in the eye
which caused a substantial corneal abrasion. No infections were observed.
There was an increase in the amount of redness and discharge in both OK and
AF eyes with no discernible difference between the two. An increase in
chemosis was observed in both eyes of six animals. Peripheral infiltrates
were found in two animals during this challenge. One animal had three large
infiltrates near the temporal limbus in the OK eye and the other had four large
infiltrates near the superior-temporal limbus in the AF eye. Both animals
also showed more redness in the eye with the infiltrates versus the
corresponding eye. These two animals completed the two week study and
infiltrates resolved within a week after cessation of inoculated lens wear.
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5.3.1.4 Challenge IV: Two Week Overnight Inoculated Lens Wear with
Multiple Daily Bacterial Drops After Lens Removal Each Day
Fourteen animals started and completed the study. No infections were
observed, however most animals showed a slight increase in the amount of
redness and discharge in both the OK and AF-treated eyes with no discernible
difference between the two. An increase in chemosis was observed in both
eyes of four animals. One animal developed three large infiltrates in the
peripheral nasal limbal area of the OK eye; this eye also had a more red
appearance. The animal completed the study and infiltrates resolved within
one week of cessation of inoculated lens wear.
5.3.1.5 Challenge V: Two Week Continuous Lens Wear followed by a
Night of Inoculated Lens Wear
Sixteen animals started and completed the study. No infections were
observed. Qualitatively, there was a slight increase in the amount of
discharge (in six animals) and redness (in 13 animals) observed after the
inoculated lens wearing period. Only one animal showed a slight increase in
chemosis in the OK eye. No infiltrates were observed.
5.3.1.6 Challenge VI: Two Week Continuous Inoculated Lens Wear with an Additional Twice Daily Instillation of Bacterial Drops
During this challenge, 15 animals were used. Within one week of study
commencement, three eyes (two OK and one AF) developed erosions during
lens wear whereby patches of epithelium were removed with no stromal
involvement (Figure 5.3). Another three eyes (two AF and one OK)
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developed abrasions from scratches and broken lenses. The intention of this
study was to maintain the integrity of the epithelium after lens wear and
challenge the topographically altered cornea with bacteria to see if there was
any increased predisposition to infection. As an intact epithelium was not
maintained in many of the animals, the decision was made to cease this
particular challenge as it strayed too far from the original study aim.
Figure 5.3: Different corneal erosions observed during inoculated continuous wear with the addition of twice daily bacterial drops (Challenge VI).
5.3.1.7 Challenge VII: Two Week Overnight Lens Wear with Bacterial
Inoculation on the Last Night of Lens Wear (using P. aeruginosa 6206)
Fourteen animals started and completed the study. No infections were
observed. Qualitatively, there was a slight increase in the amount of
discharge and redness observed in both eyes of almost all animals after the
inoculated lens wearing period however, no increase in the amount of
chemosis or infiltrates was observed.
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5.3.1.8 Total Adverse Events
As can be seen from Table 5.2 and Figure 5.4, total adverse events, i.e. inflammatory plus erosions were greatest with the challenges that involved more frequent inoculations.
Table 5.2: Summary of results for Challenges I to VII.
Inoc. Animals Animals Infec- Erosions Abrasions Total Reason for CH Started Completed Adverse Score tions AF OK AF OK Discontinuation Study Study Events 2 not conducive I 1 22 18 0 0 0 1 1 2 to lens wear 2 abrasions II 1 18 18 0 0 0 0 0 0 None 4 abrasions III 28 18 14 0 0 0 1 3 4 (2 due to broken lenses) IV 14 14 14 0 0 0 0 0 0 None V 1 16 16 0 0 0 0 0 0 None Study VI 29 16 N/A 0 1 2 2 1 6 discontinued VII 1 14 14 0 0 0 0 0 0 None
7 6 5 4 3 2 1 Total Adverse Events 0 * 0 5 10 15 20 25 30 35 Inoculation Score
Figure 5.4: Total adverse events versus inoculation score for challenges. *Three data points represented at this position.
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5.3.1.9 Detailed Ocular Inflammatory Responses
The percentage of animals with different degrees of change in redness, chemosis, discharge and presence of infiltrates for AF and OK-treated eyes in each bacterial challenge are presented in Table 5.3. To compare the total level of inflammation between challenges, these percentages were multiplied by a number rating based on the degree of change in each inflammatory factor (i.e. absent = 0, minimal (+) =
1, moderate (++) = 2, severe (+++) = 3) and presented in Table 5.4. A cumulative inflammation score was then calculated by adding all scores within each challenge
(Table 5.4). The cumulative inflammation score for each challenge was then plotted against the total inoculation score which represented the total number of bacterial instillations to the eye with the lenses in place (Figure 5.5).
4.0 3.5 3.0 2.5 OK Eye 2.0 AF Eye 1.5 1.0 0.5 0.0 Cummulative Inflammation Score Inflammation Cummulative 0 5 10 15 20 25 30 Number of Bacterial Instillations
Figure 5.5: Plot of the cumulative inflammation score versus inoculation score for each challenge for AF and OK eyes.
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As can be seen in Figure 5.5, the degree of inflammatory responses observed in this study was associated with the number of bacterial instillations applied. The highest levels of inflammation, indicated by cumulative scores of 3.21 and 3.50 for
AF and OK lenses respectively (Table 5.4), were recorded with the protocol involving the highest number (28) of bacterial instillations to the eye (Challenge
III). The second highest overall levels of inflammation (AF = 2.21 and OK = 2.07) occurred with Challenge IV, which had the second highest number (14) of bacteria instillations with lenses in place. Figure 5.6 shows an example of the appearance of a control versus inflamed eye.
Figure 5.6: Examples of (a) an eye at baseline and (b) an inflamed eye after bacterial exposure.
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Table 5.3: Percentage of animals with changes in amount of redness, chemosis, discharge and presence of infiltrates at the end of each Challenge (I-VII) in AF and OK eyes. Grading was based on a modified CCLRU grading scale. Incomplete challenges are denoted with an (X).
AF EYE OK EYE Challenge Number I II III IV V VI VII I II III IV V VI VII # of Rating Animals 18 18 14 14 16 X 14 18 18 14 14 16 X 14 No Change 0 22% 22% 14% 29% 19% 0% 22% 22% 7% 29% 19% 0% Increase by + 1 78% 78% 64% 64% 81% 100% 78% 78% 57% 71% 75% 100% REDNESS Increase by ++ 2 0% 0% 21% 7% 0% 0% 0% 0% 36% 0% 6% 0% Increase by +++ 3 0% 0% 0% 0% 0% 0% 0% 0% 0% 0% 0% 0% No Change 0 100% 100% 57% 71% 100% 100% 100% 100% 57% 86% 100% 100% Increase by + 1 0% 0% 21% 14% 0% 0% 0% 0% 14% 7% 0% 0% CHEMOSIS Increase by ++ 2 0% 0% 21% 14% 0% 0% 0% 0% 29% 7% 0% 0% Increase by +++ 3 0% 0% 0% 0% 0% 0% 0% 0% 0% 0% 0% 0% No Change 0 22% 28% 0% 14% 63% 14% 28% 22% 0% 21% 69% 14% Increase by + 1 78% 56% 86% 71% 31% 64% 72% 33% 86% 64% 25% 57% DISCHARGE Increase by ++ 2 0% 6% 0% 14% 6% 21% 0% 22% 0% 14% 6% 29% Increase by +++ 3 0% 11% 14% 0% 0% 0% 0% 11% 14% 0% 0% 0% Number of INFILTRATES events 3 0% 0% 7% 0% 0% 0% 0% 0% 7% 7% 0% 0%
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Table 5.4: Cumulative inflammation scores for AF and OK-treated eyes in each challenge calculated from the sum of the percentage of animals exhibiting the different degrees of changes in redness, chemosis and discharge multiplied by the number rating for each change. Inoculation scores were based on number of bacterial instillations into the eye.
Number AF EYE OK EYE Rating I II III IV V VI VII I II III IV V VI VII No Change 0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Increase by + 1 0.78 0.78 0.64 0.64 0.81 1.00 0.78 0.78 0.57 0.71 0.75 1.00 REDNESS Increase by ++ 2 0.00 0.00 0.43 0.14 0.00 0.00 0.00 0.00 0.71 0.00 0.13 0.00 Increase by +++ 3 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 No Change 0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Increase by + 1 0.00 0.00 0.21 0.14 0.00 0.00 0.00 0.00 0.14 0.07 0.00 0.00 CHEMOSIS Increase by ++ 2 0.00 0.00 0.43 0.29 0.00 0.00 0.00 0.00 0.57 0.14 0.00 0.00 Increase by +++ 3 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 No Change 0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Increase by + 1 0.78 0.56 0.86 0.71 0.31 0.64 0.72 0.33 0.86 0.64 0.25 0.57 DISCHARGE Increase by ++ 2 0.00 0.11 0.00 0.29 0.13 0.43 0.00 0.44 0.00 0.29 0.13 0.57 Increase by +++ 3 0.00 0.33 0.43 0.00 0.00 0.00 0.00 0.33 0.43 0.00 0.00 0.00 Number INFILTRATES of events 3 0.00 0.00 0.21 0.00 0.00 0.00 0.00 0.00 0.21 0.21 0.00 0.00 Cumulative Inflammation Score 1.56 1.78 3.21 2.21 1.25 X 2.07 1.50 1.89 3.50 2.07 1.25 X 2.14 Inoculation Score 1 1 28 14 1 29 1 1 1 28 14 1 29 1
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5.3.2 Bacterial Adhesion to Lenses
5.3.2.1 Comparisons Between OK and AF Lenses
Average numbers of cfu adhered to AF and OK lenses for both control and worn lenses (Challenges I, II & VII) and comparisons of bacterial adherence between lenses are presented in Table 5.5. There were no significant differences in adhered bacteria to unworn control OK and AF lenses for both strains of P. aeruginosa (p =
0.352 for strain 6206 and p = 0.980 for strain 6294). However, significantly more bacteria were adherent to the OK lenses than to the AF lenses for both strains of P. aeruginosa in all three challenges.
Table 5.5: Mean values (cfu/lens x105) for adhered P. aeruginosa strains 6294 and 6206 from AF and OK lenses at different time points. p-values show significance levels for comparisons between the two lens designs for each lens wearing time point. Significant values are italicized. Colony Counts (cfu/mL x 105) Time p Strain Challenge Point AF OK Value N Mean (SD) N Mean (SD) Control 0 12 510 (270) 12 480 (160) 0.980
6294 I 2 18 2.4 (4.9) 18 11 (8.8) 0.001
II 6 16 0.1 (0.25) 16 9.2 (7.1) <0.001
Control 0 12 600 (120) 12 650 (130) 0.352 6206 VII 2 9 3.0 (5.8) 9 20 (9.2) 0.014
5.3.2.2 Comparisons Between Time Points (Challenges)
Comparisons of bacterial adherence between time points for each lens type for both Pseudomonal strains are presented in Table 5.6. There was a large decrease in the bacteria adherent to both lens types at two weeks compared to the unworn
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lenses for both bacterial strains (Challenges I and VII). However this only reached statistical significance for the OK lenses (p = 0.001 for strain 6206, p = 0.022 for strain 6294) and not for the AF lenses (p = 0.155 for strain 6206, p = 0.096 for strain 6294). For strain 6294, there were further decreases between two and six weeks in numbers of adherent bacteria with AF lenses (p = 0.012) but not with OK lenses (p = 1.0).
Table 5.6: Total CFU at different time points. Log of mean total colony forming units (cfu) and standard deviations (SD) for adhered P. aeruginosa strains 6206 and 6294 from AF and OK lenses. p-values show significance levels for comparisons between time points (unworn control lenses, 2 weeks and 6 weeks). Significant values are italicized.
Log of Total CFU at different time points
Strain Lens Type Control 2 weeks 6 weeks
Mean (SD) Mean (SD) p* Mean (SD) p* p† AF 17.6 (0.5) 8.1 (5.4) 0.096 3.6 (4.8) 0.031 0.012 6294 OK 17.6 (0.3) 13.1 (1.4) 0.022 13.3 (1.0) 0.019 1.000 AF 17.9 (0.2) 9.0 (5.4) 0.155 6206 OK 18.0 (0.2) 14.3 (0.6) 0.001
*p-values for comparison to unworn lenses † p-values for comparison to 2 week old lenses
5.4 Discussion
This study did not lead to support for the hypothesis that OK-induced epithelial changes predispose to or increase the risk of infection. The epithelial changes induced with OK did not compromise the corneal defensive barrier to infection sufficiently to allow the large amounts of P. aeruginosa instilled to infect the cornea with the length of wear used in this study. There were clearly effects from
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bacterial inoculation resulting in inflammation and surprisingly greater levels of erosions however these were not substantially different between AF and OK lenses and were insufficient to produce infection.
5.4.1 Bacterial Binding
Ladage and colleagues (Ladage et al, 2004) demonstrated that exfoliated OK- treated epithelial cells bound more Pseudomonas than non-OK-treated cells in the rabbit. Bacterial binding to epithelial cells was not specifically investigated in this study, however, given the variety and severity of the challenges presented to the corneas there would have been ample opportunity for the P. aeruginosa to bind to the epithelium. If an increase in P. aeruginosa binding to OK-treated epithelial cells did occur, this study suggests that binding alone may not be enough to cause an infection. The pathogenicity of both strains of P. aeruginosa were confirmed in the positive scratch controls reported as well as the inflammatory responses seen in some of our challenges.
5.4.2 Hypoxia
Previous animal work by Solomon and colleagues demonstrated that moderate to severe hypoxia is a significant risk factor for infectious corneal ulcers in rabbits
(Solomon et al, 1994). The lenses used in this current study were manufactured in the highest oxygen permeable rigid lens material available (Menicon Z, Dk = 160).
The Paragon CRT lens has a parallel surface design resulting in equal thickness
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across the entire lens. Thus the oxygen transmissibility (Dk/t) for the 0.16mm lenses was 100 across the whole lens. This was less than the currently accepted
Dk/t value of 125 for induction of excess contact lens induced overnight corneal swelling in humans (Harvitt and Bonanno, 1999). Assuming that this value holds true in the cat, some corneal swelling was expected. However, no swelling data were obtained as the measurement itself may have disrupted and compromised the epithelial barrier prior to bacterial exposure. In any case, the contralateral design of the experiment involving lenses of the exact same material, thickness and diameter, allowed direct comparisons of the right and left eye of each animal, negating possible confounding effects of lens-induced swelling. Interestingly, the results of a recent epidemiology study suggest that there is a difference in the prevalence of infection rates with high Dk soft lenses (Schein et al, 2005) however the effects of Dk with OK are still unknown. Preliminary work by Matsubara and colleagues (Matsubara et al, 2007) on corneal physiology after OK lens wear showed no difference between an OK lens of Dk = 33 versus Dk = 95. Clinical work has found that higher Dk lens materials yield less stromal swelling in OK
(Haque et al, 2007) and one study has shown increased clinical effects with higher
Dk lenses (Swarbrick and Lum, 2006). As most OK lenses currently are manufactured in materials with less oxygen permeability than those used in this study, it will be beneficial to investigate whether low Dk/t OK lenses affect the susceptibility to infection in an animal model.
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5.4.3 Inflammation and Bacteria
Levels of inflammatory response observed in this study appear to be related to the number of bacterial exposures presented to the eye – more exposure leading to more inflammation. These findings are in agreement with those of Willcox and colleagues (Willcox et al, 2004) who also found that larger numbers of bacteria resulted in more inflammation in the guinea pig model. It is interesting to note that the highest level of inflammation was only achieved with bacterial exposure while lenses were in place as demonstrated by comparing Challenges III and IV.
Both challenges involved two weeks of overnight inoculated lens wear with additional bacterial drops. However Challenge III involved adding one drop on top of inoculated lenses each night whereas Challenge IV involved an additional two drops after lenses were removed each day. Although many more bacteria were added to eyes in Challenge IV, it was the presence of the lens during the addition of the bacterial drop in Challenge III that appeared to be the driving factor behind the increased inflammatory response observed. We speculate that bacteria from the additional drop in Challenge III increased the likelihood of organisms travelling beneath the lens and becoming trapped. This would prolong corneal exposure to the bacteria, allowing more time for pathogenic effects to be exerted and resulting in a more severe inflammatory response. Thus, it appears that it is not simply the presence of bacteria in the eye, but rather the amount of time in direct contact with the cornea that may be important to the induction of inflammation and potential infection.
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5.4.4 Immunity
This study was conducted over the course of nine months and animals were reused for the different challenges. Potential for animals to develop an acquired immunity from repeated exposure to P. aeruginosa during this time was a possibility. To confirm that animals did not develop an immune resistance from repeated exposure to bacteria, a final positive scratch control, performed as described in the Methods, using P. aeruginosa 6294 was conducted on one animal that had undergone all seven challenges. After 16 hours of inoculated lens wear, this animal also developed a microbial keratitis with a large central ulceration similar to the other positive scratch control results reported. This demonstrated that animals were unlikely to develop an acquired immunity that may have affected the results of latter challenges.
5.4.5 Erosions – Bacteria Driven?
The unusually high level of erosions observed in Challenge VI with twice daily inoculation of bacteria in conjunction with extended wear of contaminated lenses is an interesting finding. These observations suggest that bacteria may be able to destabilize the attachment of what are sometimes quite large patches of epithelium.
It has long been a mystery as to what enables MK to be initiated in seemingly successful long and short-term daily and extended contact lens wearers. These observations need to be investigated for if bacteria can produce the erosions seen, particularly in extended wear, this may well be a potential mechanism by which bacteria can gain access to the cornea and induce a microbial keratitis.
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5.4.6 Need for Care
Experimental corneal infection studies such as these cannot be carried out in humans. Use of an in vivo infection model has many advantages as it examines the response of the immune system to bacteria, lid to cornea interactions and simulates a realistic lens wearing situation. This study suggests that an intact cornea, whether OK-treated or not, can resist exposure to pathogenic bacteria in an animal model, and it underscores the importance of good clinical practice to prevent insult to the cornea that may compromise the integrity of the barrier to infection.
Improper lens fits, inappropriate insertion and removal techniques and chipped or damaged lenses from infrequent replacement schedules are some of the preventable sources of potential trauma to the cornea. Proper lens care and hygiene are also important to limit bacterial contamination to prevent infection should trauma to the cornea occur. Boost and Cho (Boost and Cho, 2005) have found that most OK patients had significant contamination of at least one of their lens care items, most frequently the lens case. Elimination of potential sources of pathogenic bacteria is important to help decrease the risk of infection in any modality of contact lens wear.
5.4.7 Bacterial Adhesion to Lenses
Analysis of bacterial adhesion revealed that unworn OK lenses do not adhere more
P. aeruginosa than their AF counterparts when controlled for lens material, diameter and thickness.
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A very large decrease in numbers of adhered bacteria with worn AF and OK lenses compared to unworn lenses was found after each initial contaminated lens wearing period. This result was expected as the eyelids and lacrimal system physically dislodge and remove most of the bacteria from the ocular environment (Milder,
1987; Moses, 1987). This was particularly applicable in the cat as discontinuous closed eye conditions were expected due to their unpredictable sleep patterns. The decrease at the two week time point only reached statistical significance with the
OK lenses (p < 0.05), the lack of significance with the AF lenses (p = 0.155 for strain 6206, p = 0.096 for strain 6294) may be attributed to the larger standard deviation with AF lenses. This increased variability with AF lenses may be due to differences in the tightness of AF lens fits and subsequently the amount of movement on eye that may affect bacteria removal.
The finding that more bacteria are retained on OK compared to AF lenses after the three contaminated lens wearing situations is an interesting finding. There are a number of possible explanations. First, the design of an OK lens includes a reverse curve zone that creates a “reservoir” that may protect bacteria from the shear stress of lens motion. This design may also promote an increase in contact lens deposition in the reverse curve area which, in turn, may help increase bacterial retention. Anecdotal reports from practitioners about accumulated deposits in the reverse curve area of OK lenses and the ability of increased deposits to bind more bacteria (Butrus and Klotz, 1990) support this idea.
A second possibility is related to biochemical factors released into the tears as a result of deliberate compression and expansion of epithelial cells during OK as was
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seen in Chapters 2 and 3. It has been reported that stress exerted on the epithelium, even with soft lenses, can increase bacterial adhesion to the epithelial cell surface (Ladage et al, 2001). Ladage and colleagues found an increase in P. aeruginosa binding to exfoliated OK-treated cells relative to non-treated cells
(Ladage et al, 2004). The current study only looked at bacterial adhesion to contact lenses and not to the epithelium however, both studies found that given bacterial exposure, more P. aeruginosa bound in OK compared with AF situations.
Studies investigating the biochemical changes with OK lenses that may contribute to increased bacterial adhesion to both the cornea and lens are needed to further understand this.
5.4.8 OK Lens Bacteria Retention
Bruinsma and colleagues demonstrated that older, end-stage RGP lenses (ready for replacement) exhibited increased surface roughness with a concurrent increase in bacterial adhesion (Bruinsma et al, 2003). Based on their work, one would expect older worn rigid lenses to adhere more bacteria than less worn lenses. Analysis of the results did not find an increase in adhered bacteria with either lens type in the longer six week compared to the two week time point. This discrepancy is likely due to the relatively short length of lens wear in this study. Interestingly, a further reduction in adhered bacteria was observed in AF lenses at six weeks suggesting either a potential “adaptation” in the factors affecting bacterial adhesion to AF lenses as a mechanism to discourage adhesion or a continued “cleaning” effect.
OK lenses on the other hand, showed no significant further reductions with longer
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lens wear (six weeks). This could constitute a risk factor for infection as almost a million bacteria (of the 5 x 106 initial inoculum) were retained per OK lens compared with only 1 x 103 for the AF lenses (p < 0.001). This suggests that additional vigilance, combined with specific cleaning instructions, are important for OK lens wearers regardless of the length of lens wear.
5.4.9 Compliance
Adhesion results highlight the importance of compliance with an effective lens care regimen for OK lens wearers. Soaking of worn lenses in bacteria in this study was intended to simulate lenses soaking in a contaminated solution. Results suggest that if contaminated lenses are placed on eye, bacteria are more likely to remain adhered to OK lenses than they are to AF lenses thus increasing the risk for an infection. Additionally, current OK practice involves overnight lens wear, a wearing schedule identified as a major risk factor for MK (Stapleton, 2003).
While lens care solutions play an integral role in combating contamination, RGP solutions have been found to have varying anti-microbial efficacy (McLaughlin et al, 1991) and diminished anti-microbial efficacy over time (Boost et al, 2006).
Taken together with the finding that organisms can often survive in ophthalmic solutions (Mayo et al, 1987) and the report that 63% of OK patients had at least one contaminated lens or lens care item (Boost and Cho, 2005), this underscores the need for frequent solution replacement and compliance with lens care instructions.
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5.4.10 Bacterial Strain Effect
Although not a primary goal of the study, a comparison of the two different strains of P. aeruginosa found that they varied in adherence to OK lenses but not to AF lenses. The cytotoxic strain 6206 adhered more than the invasive 6294 strain at the unworn (p = 0.006) and the two week (p = 0.020) time points. This is an intriguing finding as it suggests that lens geometry may play a differential role in determining adhesion between bacterial strains. Further studies to compare adhesion of various micro-organisms to these lenses are warranted.
5.4.11 Limitations
Most research examining the safety and efficacy of OK has taken the form of short term studies i.e. less than six months. Long term effects of OK have yet to be established. Moreover, although there are anecdotal reports of infections with longer term wear, there have been no published epidemiological or prevalence studies of infections with OK lenses. This study investigated the relatively short term corneal changes in relation to susceptibility to bacterial infection; the longest consecutive wearing period was six weeks (Challenge II). Although a relatively short period of time, these findings are still interesting as 20% of the first 50 MK cases reported occurred within three months of commencing lens wear (Watt and
Swarbrick, 2005). Given the relatively recent “rebirth” of the modality, it will be of interest to investigate the physiological changes that accompany the cornea over the long term. Complications such as central corneal epitheliopathy (Ng, 2006), fibrillary lines (Cheung et al, 2006), white lesions (Cheung et al, 2005), corneal
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iron rings (Cho et al, 2002a; Liang et al, 2003; Hiraoka et al, 2004a) and pigmented arcs (Cho et al, 2003b; Cho et al, 2005b) have been reported in patients wearing OK lenses for longer periods, suggesting that these changes are taking place with repeated lens wear. Longer term studies are needed to investigate whether OK corneal changes continually imposed over months or years are more detrimental and predispose further the cornea to infection.
5.4.12 Summary
This study found that epithelial changes associated with myopic OK in the cat model were not sufficient to render the cornea susceptible to infection (in the time used) despite exposure to very high inocula of P. aeruginosa. OK lenses retained more bacteria than AF lenses after one night of bacteria-loaded lens wear and maintained a higher bacterial adherence over time compared with AF lenses, representing a potential higher increased risk for infection. Patient education to maximize proper lens care and minimize potential trauma to the epithelium and exposure to pathogenic bacteria with OK lens wear is very important.
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CHAPTER 6: Summary and Recommendations
6.1 Summary
Orthokeratology is currently the only contact lens modality where lenses do not have to be worn during the day to achieve clear vision during the day. It is growing in popularity around the world however there have been suggestions of a higher rate of infection with this modality. Very few basic science studies have been used to investigate the nature of the corneal changes underlying this procedure. This thesis investigated the effects of OK on epithelial tissue and the implications of these changes with regard to corneal susceptibility to microbial infection.
The cat was first explored as a model to investigate OK-induced corneal changes
(Chapter 2). In a pilot study on four animals, histological evaluation of the corneal tissue after continuous wear of OK lenses found that the procedure induced epithelial thickness and stromal changes that mimicked those seen in clinical practice. These changes included central thinning and mid-peripheral thickening in myopic-treated eyes and the opposite pattern of thickness changes in hyperopic treated eyes. Central epithelial thinning appeared to be attributed to cellular compression and loss of cell layers in longer term lens wear (2 weeks).
The cat OK model was then improved upon by adapting an overnight wearing schedule, corneal monitoring and more animals to better reflect human overnight
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OK lens wearing scenarios (Chapter 3). Histological analysis confirmed the pattern of epithelial thickness and morphologic changes with overnight myopic OK lens wear. Epithelial changes were observed to correspond to the different zones of an OK lens. No changes to keratocyte cell counts in the adjacent stroma were found. The amount of central thinning seen with a more regular overnight lens wearing schedule was less pronounced than previously reported in the extended wear pilot study. Compression of cells was the major factor in central epithelial thinning rather than loss of cell layers previously reported.
The reversibility of epithelial changes after short term (four weeks) lens wear was investigated with similar histological techniques (Chapter 4). Epithelial thickness and morphologic recovery to contralateral control thicknesses and shapes occurred within one week of cessation of lens wear and very minimal changes to keratocyte cell densities were found.
The cat model was then used to investigate OK corneal susceptibility to microbial infection (Chapter 5). Epithelial changes were challenged with very high inocula of P. aeruginosa and found to maintain their ability to resist bacterial infection.
The finding that more bacteria adhered to OK lenses than their AF counterparts suggests a new potential risk factor for infection. Another interesting finding was that instillations of bacteria with continuous wear of inoculated lenses led to development of corneal erosions.
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6.2 Future Directions
The development of the cat as a new model for OK-induced corneal changes provides a useful method to investigate the biological effects of the procedure.
Improvements to the model would include better ways of getting centration with topography so maps can be directly correlated with histological changes. A measure of refraction included or at least attempted either through auto-refraction or retinoscopy would also help correlate amount of refractive change with magnitude of epithelial and corneal change.
Further investigation of the physiological changes underlying these epithelial changes through more advanced immuno-histochemical tissue staining and electron microscopy techniques are warranted. Specifically, a better understanding of mitotic, apoptotic and cell migratory pattern changes would help elucidate the exact mechanisms behind the thickness and morphologic changes observed.
The effects of OK on the stroma are still unclear. Preliminary work from Chapter
2 suggested that stromal thickness may be altered with OK. Improved histological techniques to investigate potential stromal thickness, structural and physiological changes would help in understanding its contribution to the refractive changes induced with the procedure. Although this work suggested that keratocyte populations did not change much with and after four weeks of OK lens wear
(Chapters 3 and 4), no assessment of keratocyte health was performed. Further
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work to investigate potential changes to apoptotic and mitotic rates within the keratocyte population would be useful.
This current body of work was conducted with OK lenses manufactured in the highest Dk rigid lens material (Menicon Z) currently available on the market. The majority of OK lenses sold are manufactured in materials with less oxygen transmissibility. As yet, no cases of MK have been reported in patients wearing
OK lenses made from Menicon Z material. Thus effects of Dk and oxygen availability on epithelial changes and susceptibility to infection would be of great interest and would provide valuable insights into the response of the cornea to different physiologic conditions.
This work investigated short term OK corneal susceptibility to P. aeruginosa infection only and thus precluded the generalization of results to other organisms.
It would be prudent to investigate corneal susceptibility to infection from other organisms particularly Acanthamoeba given the high number of MK cases caused by this pathogen.
The finding of increased bacterial adhesion to OK lenses is significant. This identification of a potential new risk factor for infection in OK warrants further investigation. An analysis of bacterial adhesion to human OK lenses after a period of lens wear would help shed light on this topic and potentially lead to changes in lens care regimens and development of novel lens care products.
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An unexpected finding was the large number of corneal erosions found with the combination of continuous lens wear and exposure of bacteria to the eyes of animals in Challenge VI (Chapter 5). This finding suggests a potential bacterial basis for corneal erosions that may represent an avenue through which bacteria can cause infections. This finding deserves further investigation.
6.3 Conclusions
The epithelium was found to be a major contributor to the corneal changes induced by OK lens wear. These epithelial changes were defined through histological study and the susceptibility of these changes to bacterial infection was tested.
Though longer periods of lens wear need to be investigated, there is no prima facie case for a higher risk of infection with the OK procedure. Nevertheless, given the propensity of OK lenses to trap and collect bacteria, caution needs to be exercised and proper hygiene emphasized to prevent potential adverse events.
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References
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Appendix 1: Supplemental Statistical Analysis for Chapters 3 & 4
APPENDIX 1: SUPPLEMENTAL STATISTICAL
ANALYSIS FOR CHAPTERS 3 & 4
CHAPTER 7:
7.1 Supplementary Statistical Analysis for Chapter 3
Table 7.1: Epithelial thickness of non-lens wearing control animals. Five measurements were taken in each area using customized image analysis software. SUP = superior periphery, MPS = superior mid-periphery, CEN = centre, MPI = inferior mid-periphery, INF = inferior periphery. Epithelial Thickness (µm) Eye Animal Mean (SD) Avg SUP MPS CEN MPI INF (SUP to INF) 1 36.0 (0.8) 35.6 (0.5) 38.5 (0.9) 34.6 (0.4) 33.7 (0.7) 35.7 (1.8)
2 33.6 (0.6) 35.0 (0.8) 31.8 (0.7) 34.8 (0.7) 35.4 (0.6) 33.6 (1.5) OD 49.0 (0.7) 49.6 (0.7) 47.2 (0.3) 47.4 (0.3) 48.8 (0.2) 49.0 (1.1) 3 39.5 (8.3) 40.1 (8.3) 39.2 (7.7) 39.0 (7.3) 39.3 (8.3) 39.4 (8.4) Average 32.9 (0.6) 37.5 (0.4) 37.7 (0.6) 36.8 (0.6) 31.6 (0.3) 35.3 (2.8) 1 36.7 (0.4) 36.5 (0.8) 35.0 (0.2) 34.2 (0.8) 36.3 (0.3) 36.7 (1.1) 2 OS 47.5 (0.2) 48.8 (0.4) 49.7 (0.9) 49.9 (0.2) 50.2 (0.2) 47.5 (1.1) 3 39.1 (7.6) 40.9 (6.8) 40.8 (7.8) 40.3 (8.4) 39.4 (9.7) 39.9 (6.7) Average
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Table 7.2: Comparisons of change in epithelial thickness (µm) between time points within corneal areas for increasing lens wear. Significant values are italicized. 0 = non-lens wearing controls Thickness Post-hoc Time p – value Area Treatment N Change (compared Point (ANOVA) Mean (SD) to Controls) 0 OD - OS 3 + 0.5 (3.2) 1 Day OK - AF 3 - 11.9 (3.6) 3 Day OK - AF 3 - 2.7 (5.2) SUP 0.061 1 Week OK - AF 3 - 10.3 (5.7) 2 Week OK - AF 3 - 2.8 (1.8) 4 Week OK - AF 3 - 9.1 (8.4) 0 OD - OS 3 - 0.8 (1.4) 1 Day OK - AF 3 + 7.9 (6.7) 3 Day OK - AF 3 + 12.6 (10.5) MPS 0.148 1 Week OK - AF 3 + 8.9 (3.1) 2 Week OK - AF 3 + 7.8 (0.8) 4 Week OK - AF 3 + 13.3 (6.9) 0 OD - OS 3 - 1.6 (2.1) 1 Day OK - AF 3 - 12.0 (5.8) 0.019 3 Day OK - AF 3 - 9.5 (3.5) 0.084 CEN 0.006 1 Week OK - AF 3 - 16.8 (0.1) 0.001 2 Week OK - AF 3 - 6.3 (1.5) 0.430 4 Week OK - AF 3 - 8.4 (5.3) 0.156 0 OD - OS 3 - 1.4 (1.7) 1 Day OK - AF 3 + 5.2 (6.3)
Change in Epithelial Thickness 3 Day OK - AF 3 + 5.0 (6.5) MPI 0.155 1 Week OK - AF 3 + 6.1 (3.9) 2 Week OK - AF 3 + 8.2 (3.1) 4 Week OK - AF 3 + 8.8 (3.2) 0 OD - OS 3 - 0.1 (1.9) 1 Day OK - AF 3 - 10.5 (3.8) 3 Day OK - AF 3 - 9.5 (5.8) INF 0.266 1 Week OK - AF 3 - 10.5 (7.6) 2 Week OK - AF 3 - 9.1 (6.5) 4 Week OK - AF 3 - 14.4 (11.2)
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Appendix 1: Supplemental Statistical Analysis for Chapters 3 & 4
Table 7.3: Comparisons of change in epithelial thickness (%) between time points within corneal areas for increasing lens wear. Significant values are italicized. 0 = non-lens wearing controls Thickness Post-hoc Time p – value Area Treatment N Change (compared Point (ANOVA) Mean (SD) to Controls) 0 OD – OS 3 + 1.3 (9.0) 1 Day OK – AF 3 - 30.7 (10.2) 3 Day OK – AF 3 - 6.6 (12.7) SUP 0.111 1 Week OK – AF 3 - 23.5 (11.6) 2 Week OK – AF 3 - 7.9 (5.0) 4 Week OK – AF 3 - 26.3 (26.4) 0 OD – OS 3 - 2.5 (3.6) 1 Day OK – AF 3 + 22.9 (21.4) 3 Day OK – AF 3 + 33.7 (29.6) MPS 0.317 1 Week OK – AF 3 + 22.8 (8.6) 2 Week OK – AF 3 + 23.2 (5.1) 4 Week OK – AF 3 + 34.8 (19.1) 0 OD – OS 3 - 4.0 (5.6) 1 Day OK – AF 3 - 27.6 (10.0) 0.002 3 Day OK – AF 3 - 24.0 (5.7) 0.005 CEN 0.001 1 Week OK – AF 3 - 40.4 (3.5) <0.001 2 Week OK – AF 3 - 16.7 (5.7) 0.033 4 Week OK – AF 3 - 21.7 (14.0) 0.009 0 OD – OS 3 - 3.1 (4.2) 1 Day OK – AF 3 + 14.1 (15.7)
Change in Epithelial Thickness 3 Day OK – AF 3 + 13.2 (16.5) MPI 0.427 1 Week OK – AF 3 + 14.1 (8.8) 2 Week OK – AF 3 + 21.0 (7.4) 4 Week OK – AF 3 + 21.7 (8.7) 0 OD - OS 3 + 0.5 (5.3) 1 Day OK - AF 3 - 30.9 (7.3) 3 Day OK - AF 3 - 22.3 (11.8) INF 0.211 1 Week OK - AF 3 - 25.9 (18.7) 2 Week OK - AF 3 - 23.6 (15.8) 4 Week OK - AF 3 - 39.0 (31.2)
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Table 7.4: Average keratocyte counts in both eyes of all three control animals. Keratocyte Counts Mean (SD) Eye Animal Average SUP MPS CEN MPI INF (SUP to INF) 1 154 (4) 167 (2) 166 (1) 161 (2) 150 (2) 159 (7) 2 145 (2) 161 (3) 156 (4) 147 (2) 150 (3) 145 (7) OD 3 156 (3) 155 (3) 146 (2) 150 (2) 153 (1) 156 (4) Average 152 (6) 161 (6) 156 (10) 153 (7) 151 (2) 155 (8) 1 180 (5) 180 (4) 147 (3) 172 (2) 148 (2) 165 (17) 2 159 (1) 141 (2) 166 (3) 184 (3) 147 (3) 159 (17) OS 3 170 (3) 198 (2) 184 (2) 152 (2) 144 (1) 170 (22) Average 170 (11) 173 (29) 166 (19) 169 (16) 146 (2) 165 (6)
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Appendix 1: Supplemental Statistical Analysis for Chapters 3 & 4
Table 7.5: Comparisons of change in keratocyte counts between time points within corneal areas for increasing lens wear. 0 = non-lens wearing controls Thickness Time p – value Area Treatment N Change Point (ANOVA) Mean (SD) 0 OD – OS 3 - 18 (7) 1 Day OK – AF 3 - 9 (22) 3 Day OK – AF 3 - 10 (36) SUP 0.761 1 Week OK – AF 3 + 4 (18) 2 Week OK – AF 3 + 5 (8) 4 Week OK – AF 3 - 5 (24) 0 OD – OS 3 - 12 (32) 1 Day OK – AF 3 - 14 (11) 3 Day OK – AF 3 + 14 (22) MPS 0.277 1 Week OK – AF 3 + 5 (10) 2 Week OK – AF 3 + 15 (16) 4 Week OK – AF 3 - 1 (7) 0 OD – OS 3 - 10 (29) 1 Day OK – AF 3 - 2 (7) 3 Day OK – AF 3 + 15 (23) CEN 0.466 1 Week OK – AF 3 + 13 (8) 2 Week OK – AF 3 + 1 (6) 4 Week OK – AF 3 - 3 (15) 0 OD – OS 3 - 17 (18) 1 Day OK – AF 3 - 10 (8)
Change in Keratocyte Counts 3 Day OK – AF 3 + 15 (35) MPI 0.278 1 Week OK – AF 3 + 7 (9) 2 Week OK – AF 3 + 4 (13) 4 Week OK – AF 3 + 15 (16) 0 OD – OS 3 + 5 (4) 1 Day OK – AF 3 0 (7) 3 Day OK – AF 3 - 12 (13) INF 0.479 1 Week OK – AF 3 - 5 (30 2 Week OK – AF 3 + 3 (4) 4 Week OK – AF 3 + 10 (2)
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Appendix 1: Supplemental Statistical Analysis for Chapters 3 & 4
7.2 Supplementary Statistical Analysis for Chapter 4
Table 7.6: Comparisons of change in epithelial thickness (µm) between time points within corneal areas. *Start = 4 week lens wear data from Chapter 3. Significant values are italicized. Thickness Post-hoc Time p – value Area Treatment N Change (compared Point (ANOVA) Mean (SD) to *Start) *Start OK - AF 3 - 9.1 (8.4) 1 Day OK - AF 3 - 3.7 (4.9) SUP 3 Day OK - AF 3 - 2.2 (4.8) 0.386 1 Week OK - AF 3 - 1.9 (2.9) 4 Week OK - AF 3 - 0.9 (3.3) *Start OK - AF 3 + 13.3 (6.9) 1 Day OK - AF 3 + 10.8 (10.6) 0.967 MPS 3 Day OK - AF 3 + 2.5 (5.1) 0.045 0.192 1 Week OK - AF 3 - 1.8 (4.1) 0.051 4 Week OK - AF 3 - 1.8 (2.3) 0.052 *Start OK - AF 3 - 8.4 (5.3) 1 Day OK - AF 3 + 1.4 (10.4) CEN 3 Day OK - AF 3 - 1.2 (5.4) 0.420 1 Week OK - AF 3 - 1.7 (4.8) 4 Week OK - AF 3 - 0.3 (2.5) *Start OK - AF 3 + 8.8 (3.2) 1 Day OK - AF 3 + 9.5 (11.5) MPI 3 Day OK - AF 3 + 3.3 (8.2) 0.452 Change in Epithelial Thickness 1 Week OK - AF 3 + 1.6 (5.7) 4 Week OK - AF 3 + 0.8 (2.4) *Start OK - AF 3 - 14.4 (11.2) 1 Day OK - AF 3 - 3.2 (5.9) INF 3 Day OK - AF 3 - 3.4 (3.9) 0.079 1 Week OK - AF 3 - 0.5 (0.5) 4 Week OK - AF 3 + 0.4 (2.7)
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Appendix 1: Supplemental Statistical Analysis for Chapters 3 & 4
Table 7.7: Comparisons of change in epithelial thickness (µm) between time points within corneal areas. *0 = Control data from Chapter 3. Thickness Change p – value Area Time Point Treatment N Mean (SD) (ANOVA) *0 OD – OS 3 + 0.5 (3.2) 1 Day OK – AF 3 - 3.7 (4.9) SUP 3 Day OK – AF 3 - 2.2 (4.8) 0.756 1 Week OK – AF 3 - 1.9 (2.9) 4 Week OK – AF 3 - 0.9 (3.3) *0 OD – OS 3 - 0.8 (1.4) 1 Day OK – AF 3 + 10.8 (10.6) MPS 3 Day OK – AF 3 + 2.5 (5.1) 0.097 1 Week OK – AF 3 - 1.8 (4.1) 4 Week OK – AF 3 - 1.8 (2.3) *0 OD – OS 3 - 1.6 (2.1) 1 Day OK – AF 3 + 1.4 (10.4) CEN 3 Day OK – AF 3 - 1.2 (5.4) 0.959 1 Week OK – AF 3 - 1.7 (4.8) 4 Week OK – AF 3 - 0.3 (2.5) *0 OD – OS 3 - 1.4 (1.7) 1 Day OK – AF 3 + 9.5 (11.5) MPI 3 Day OK – AF 3 + 3.3 (8.2) 0.425 Change in Epithelial Thickness 1 Week OK – AF 3 + 1.6 (5.7) 4 Week OK – AF 3 + 0.8 (2.4) *0 OD – OS 3 - 0.1 (1.9) 1 Day OK – AF 3 - 3.2 (5.9) INF 3 Day OK – AF 3 - 3.4 (3.9) 0.555 1 Week OK – AF 3 - 0.5 (0.5) 4 Week OK – AF 3 + 0.4 (2.7)
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Appendix 1: Supplemental Statistical Analysis for Chapters 3 & 4
Table 7.8: Comparisons of change in epithelial thickness (%) between time points within corneal areas. *Start = 4 week data from Chapter 3. Significant values are italicized. Thickness Post-hoc Time p – value Area Treatment N Change (compared Point (ANOVA) Mean (SD) to *Start) *Start OK – AF 3 - 26.3 (26.4) 1 Day OK – AF 3 - 10.6 (13.7) SUP 3 Day OK – AF 3 - 5.1 (14.0) 0.356 1 Week OK – AF 3 - 4.5 (8.0) 4 Week OK – AF 3 - 2.5 (9.7) *Start OK – AF 3 + 34.8 (19.1) 1 Day OK – AF 3 + 30.6 (30.8) 0.977 MPS 3 Day OK – AF 3 + 7.2 ( 13.9) 0.031 0.157 1 Week OK – AF 3 - 4.3 (10.7) 0.033 4 Week OK – AF 3 - 4.6 (5.7) 0.033 *Start OK – AF 3 - 21.7 (14.0) 1 Day OK – AF 3 + 5.2 (28.9) CEN 3 Day OK – AF 3 - 2.5 (14.0) 0.363 1 Week OK – AF 3 - 4.3 (11.5) 4 Week OK – AF 3 - 1.1 (6.8) *Start OK – AF 3 + 21.7 (8.7) 1 Day OK – AF 3 + 28.9 (37.0) MPI 3 Day OK – AF 3 + 8.7 (21.3) 0.415 Change in Epithelial Thickness 1 Week OK – AF 3 + 4.1 (14.7) 4 Week OK – AF 3 + 2.0 (6.3) *Start OK – AF 3 - 39.0 (31.2) 1 Day OK – AF 3 - 8.5 (19.4) INF 3 Day OK – AF 3 - 8.5 (9.9) 0.054 1 Week OK – AF 3 - 1.4 (1.4) 4 Week OK – AF 3 + 0.9 (8.1)
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Table 7.9: Comparisons of change in epithelial thickness (%) between time points within corneal areas. *0 = Control data from Chapter 3. Thickness Change p – value Area Time Point Treatment N Mean (SD) (ANOVA) *0 OD – OS 3 + 1.3 (9.0) 1 Day OK – AF 3 - 10.6 (13.7) SUP 3 Day OK – AF 3 - 5.1 (14.0) 0.358 1 Week OK – AF 3 - 4.5 (8.0) 4 Week OK – AF 3 - 2.5 (9.7) *0 OD – OS 3 - 2.5 (3.6) 1 Day OK – AF 3 + 30.6 (30.8) MPS 3 Day OK – AF 3 + 7.2 ( 13.9) 0.053 1 Week OK – AF 3 - 4.3 (10.7) 4 Week OK – AF 3 - 4.6 (5.7) *0 OD – OS 3 - 4.0 (5.6) 1 Day OK – AF 3 + 5.2 (28.9) CEN 3 Day OK – AF 3 - 2.5 (14.0) 0.369 1 Week OK – AF 3 - 4.3 (11.5) 4 Week OK – AF 3 - 1.1 (6.8) *0 OD – OS 3 - 3.1 (4.2) 1 Day OK – AF 3 + 28.9 (37.0) MPI 3 Day OK – AF 3 + 8.7 (21.3) 0.441 Change in Epithelial Thickness 1 Week OK – AF 3 + 4.1 (14.7) 4 Week OK – AF 3 + 2.0 (6.3) *0 OD – OS 3 - 0.5 (5.3) 1 Day OK – AF 3 - 8.5 (19.4) INF 3 Day OK – AF 3 - 8.5 (9.9) 0.078 1 Week OK – AF 3 - 1.4 (1.4) 4 Week OK – AF 3 + 0.9 (8.1)
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Table 7.10: Comparisons of change in keratocyte counts between time points within corneal areas. *0 = Control data from Chapter 3. Significant values are italicized. Keratocyte Post-hoc Time p – value Area Treatment N Difference (compared Point (ANOVA) Mean (SD) to *0) *0 OD – OS 3 - 18.0 (6.9) 1 Day OK – AF 3 + 5.0 (18.0) 0.076 SUP 3 Day OK - AF 3 - 2.0 (12.0) 0.015 0.264 1 Week OK - AF 3 + 0.3 (7.5) 0.178 4 Week OK - AF 3 + 21.7 (1.2) 0.004 *0 OD - OS 3 - 12.0 (31.5) 1 Day OK - AF 3 - 6.0 (2.0) MPS 3 Day OK - AF 3 + 22.7 (10.2) 0.217 1 Week OK - AF 3 - 7.0 (24.6) 4 Week OK - AF 3 + 7.3 (0.6) *0 OD - OS 3 - 9.7 (28.5) 1 Day OK - AF 3 + 16.3 (8.5) CEN 3 Day OK - AF 3 - 13.7 (11.0) 0.189 1 Week OK - AF 3 - 15.7 (3.2) 4 Week OK - AF 3 - 7.7 (17.6) *0 OD - OS 3 - 16.7 (18.2) 1 Day OK - AF 3 + 6.0 (7.8) MPI 3 Day OK - AF 3 - 15.3 (14.5) 0.232 Change in Keratocyte Counts Change in 1 Week OK - AF 3 + 8.7 (7.4) 4 Week OK - AF 3 - 7.3 (24.3) *0 OD - OS 3 + 4.7 (3.8) 1 Day OK - AF 3 + 3.3 (10.7) INF 3 Day OK - AF 3 + 5.0 (5.0) 0.072 1 Week OK - AF 3 - 11.3 (5.5) 4 Week OK - AF 3 - 10.7 (13.1)
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Appendix 2: Epithelial Thickness Measurement Software
APPENDIX 2: EPITHELIAL THICKNESS
MEASUREMENT SOFTWARE
CHAPTER 8:
8.1 Clamp Function
////////////////////////////////////////////////////////////////////////////// // Measure Distances ////////////////////////////////////////////////////////////////////////////// ////////////////////////////////////////////////////////////////////////////// // // imaqClampMax // // Description: // Measures a distance from the sides of the search area towards the // center of the search area. // // Parameters: // image - The image that the function uses for distance // measurement. // searchRect - The coordinate location of the rectangular search area // of the distance measurement. // direction - The direction the function search for edges along the // search lines. // distance - Upon return, the distance measured between the two // parallel hit-lines. This parameter is required and // cannot be NULL. // options - Describes how you want the function to detect edges and // what information the function should overlay onto the // image. // transform - An optional specification of the coordinate transform // for searchRect. // firstEdge - Upon return, the coordinate location of the first edge // used to measure the distance. // lastEdge - Upon return, the coordinate location of the last edge // used to measure the distance. // // Return Value: // TRUE if successful, FALSE if there was an error // ////////////////////////////////////////////////////////////////////////////// int __stdcall imaqClampMax(Image* image, RotatedRect searchRect, RakeDirection direction, float* distance, const FindEdgeOptions* options, const CoordinateTransform2* transform, PointFloat* firstEdge, PointFloat* lastEdge) {
ROI* tempROI; ContourID rectID; RakeReport* reportToProcess;
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RakeOptions optionsToUse; LineFloat firstPerpendicularLine, lastPerpendicularLine; PointFloat localFirstEdge, localLastEdge;
// Verify that the distance is not NULL if( distance == NULL ) { imaqPrepareForExit( ERR_NULL_POINTER, imaqClampMaxErrorString, NULL ); return FALSE; }
// Create an ROI, place the rotated rect contour inside it and set the contour color tempROI = imaqCreateROI(); if ( tempROI == NULL ) { imaqPrepareForExit( imaqGetLastError(), imaqClampMaxErrorString, NULL ); return FALSE; } if ( (rectID = imaqAddRotatedRectContour2 (tempROI, searchRect)) == FALSE ) { imaqPrepareForExit( imaqGetLastError(), imaqClampMaxErrorString, tempROI, NULL ); return FALSE; } if ( imaqSetContourColor( tempROI, rectID, &IMAQ_RGB_GREEN ) == FALSE ) { imaqPrepareForExit( imaqGetLastError(), imaqClampMaxErrorString, tempROI, NULL ); return FALSE; }
// If neccesary, transform the rectangle. if ( transform ) { if ( imaqTransformROI2( tempROI, &transform->referenceSystem, &transform- >measurementSystem ) == FALSE ) { imaqPrepareForExit( imaqGetLastError(), imaqClampMaxErrorString, tempROI, NULL ); return FALSE; } }
// Use the supplied rake options (if available) or use default options if( options ) { optionsToUse.threshold = options->threshold; optionsToUse.width = options->width; optionsToUse.steepness = options->steepness; optionsToUse.subsamplingRatio = (int)options->subsamplingRatio; } else { optionsToUse.threshold = DEFAULT_THRESHOLD; optionsToUse.width = DEFAULT_WIDTH; optionsToUse.steepness = DEFAULT_STEEPNESS; optionsToUse.subsamplingRatio = DEFAULT_SUBSAMPLING_RATIO; }
// Set the remaining options optionsToUse.subpixelType = IMAQ_CUBIC_SPLINE; optionsToUse.subpixelDivisions = DEFAULT_SUBPIXEL_DIVISIONS;
// Calculate the edge locations if ( (reportToProcess = imaqRake( image, tempROI, direction, IMAQ_FIRST_AND_LAST, &optionsToUse )) == NULL ) { imaqPrepareForExit( imaqGetLastError(), imaqClampMaxErrorString, tempROI, NULL );
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return FALSE; }
// Use the rake report to calculate the maximum distance if ( imaqMeasureMaxDistance( reportToProcess, &firstPerpendicularLine, &lastPerpendicularLine, distance, &localFirstEdge, &localLastEdge ) == FALSE ) { imaqPrepareForExit( imaqGetLastError(), imaqClampMaxErrorString, tempROI, reportToProcess, NULL ); return FALSE; }
// Display the results if ( imaqOverlayClampResults( image, tempROI, reportToProcess, NULL, &firstPerpendicularLine, &lastPerpendicularLine, localFirstEdge, localLastEdge, TRUE, options ) == FALSE ) { imaqPrepareForExit( imaqGetLastError(), imaqClampMaxErrorString, tempROI, reportToProcess, NULL ); return FALSE; }
// Set the start and end points (if requested) if(firstEdge) *firstEdge = localFirstEdge; if(lastEdge) *lastEdge = localLastEdge;
// Dispose of the rake report if ( imaqDispose(reportToProcess) == FALSE ) { imaqPrepareForExit( imaqGetLastError(), imaqClampMaxErrorString, tempROI, NULL ); return FALSE; }
// Dispose of the ROI if ( imaqDispose(tempROI) == FALSE ) { imaqPrepareForExit( imaqGetLastError(), imaqClampMaxErrorString, NULL ); return FALSE; }
return TRUE;
}
8.2 Main Function
#include "crcertExcel.c" #include "excel8.h" #include
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//======// Defines //======#define DISPLAY_WINDOW 0 #define MAX_BUFFERS 10 #define INIT_POINTS_ARRAY_ELEMENTS 100 #define PX_MM 18.62
#define VisionErrChk(Function) {if (!(Function)) {success = 0; goto Error;}} typedef struct IVA_Data_Struct { Image* buffers[MAX_BUFFERS]; // Vision Assistant Image Buffers PointFloat* pointsResults; // Array of points used for Caliper Measurements int numPoints; // Number of points allocated in the pointsResults array int pointIndex; // Insertion Point } IVA_Data;
static IVA_Data* IVA_InitData(void); static int IVA_DisposeData(IVA_Data* ivaData); static int IVA_CLRExtractGreen(Image* image, Image *plane); void OpenOutput(void); void WriteExcelData(float dataToWrite[], double mean, double stdev, double steps); static int mPnl; char** imagePath; // Image Path int numFiles, current =0, steps; float *distance; double *dist; double mean, stdev, pxPerMicron; ROI *roi; int scaleDown; int main (int argc, char *argv[]) { if (InitCVIRTE (0, argv, 0) == 0) return -1; /* out of memory */ if ((mPnl = LoadPanel (0, "JenCatCorn.uir", MPNL)) < 0) return -1; OpenOutput(); roi = imaqCreateROI (); DisplayPanel (mPnl); RunUserInterface (); DiscardPanel (mPnl); return 0; } int CVICALLBACK OpeneImagesCB (int panel, int control, int event, void *callbackData, int eventData1, int eventData2) { int success, cancelled; switch (event) { case EVENT_COMMIT:
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// Display the Load Image dialog imagePath = imaqLoadImagePopup (NULL, "*.*", NULL, "Open Image", TRUE, IMAQ_BUTTON_LOAD, FALSE, FALSE, TRUE, FALSE, &cancelled,
&numFiles); break; } return 0; } int CVICALLBACK QuitCB (int panel, int control, int event, void *callbackData, int eventData1, int eventData2) { switch (event) { case EVENT_COMMIT: imaqDispose(imagePath); QuitUserInterface (0); break; } return 0; } int CVICALLBACK SameCB (int panel, int control, int event, void *callbackData, int eventData1, int eventData2) { int success, i; double sumofE, meanDiff, acMeanDiff = 0;
switch (event) { case EVENT_COMMIT: GetCtrlVal (mPnl, MPNL_SCALE, &scaleDown); pxPerMicron = PX_MM / scaleDown; success = FindEdges(imagePath[current]);
Sum1D (dist, steps, &sumofE); mean = sumofE / steps; for (i = 0; i< steps; i++){ meanDiff = pow((dist[i] - mean),2); acMeanDiff +=meanDiff; } stdev = sqrt(acMeanDiff / (steps - 1)); mean = mean / pxPerMicron; stdev = stdev / pxPerMicron;
SetCtrlVal (mPnl, MPNL_MEAN, mean); SetCtrlVal (mPnl, MPNL_STDEV, stdev); SetCtrlVal (mPnl, MPNL_N, (double)steps); SetCtrlVal (mPnl, MPNL_DRAW_ROI, 0); break; } return 0; } int CVICALLBACK NextCB (int panel, int control, int event,
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void *callbackData, int eventData1, int eventData2) { int success, i; double sumofE, meanDiff, acMeanDiff = 0;
switch (event) { case EVENT_COMMIT: WriteExcelData(distance, mean, stdev, (double)steps); imaqCloseWindow (IMAQ_ALL_WINDOWS); SetCtrlVal (mPnl, MPNL_DRAW_ROI, 1);
if (current < numFiles-1){ current++; success = FindEdges(imagePath[current]); Sum1D (dist, steps, &sumofE); mean = sumofE / steps; for (i = 0; i< steps; i++){ meanDiff = pow((dist[i] - mean),2); acMeanDiff +=meanDiff; } stdev = sqrt(acMeanDiff / (steps - 1)); mean = mean / pxPerMicron; stdev = stdev / pxPerMicron;
SetCtrlVal (mPnl, MPNL_MEAN, mean); SetCtrlVal (mPnl, MPNL_STDEV, stdev); SetCtrlVal (mPnl, MPNL_N, (double)steps); SetCtrlVal (mPnl, MPNL_DRAW_ROI, 0); } else { MessagePopup ("All Done", "Dear Jennifer, all Images are Analysed"); current = 0; } break; } return 0; }
int FindEdges (char *imagePath) { int success = 1; int err = 0; int cancelled; ImageType imageType; // Image Type Image* image, *scaled; // Image
//get the no of steps (global) from the control GetCtrlVal (mPnl, MPNL_STEPS, &steps); //init distance array distance = malloc (sizeof(float) * steps); dist = malloc (sizeof(double) * steps); // IMAQ Vision creates windows in a separate thread imaqSetWindowThreadPolicy(IMAQ_SEPARATE_THREAD);
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Appendix 2: Epithelial Thickness Measurement Software
// Get the type of the image file to create an image of the right type imaqGetFileInfo(imagePath, NULL, NULL, NULL, NULL, NULL, &imageType); // Create an IMAQ Vision image image = imaqCreateImage(IMAQ_IMAGE_RGB, 7); scaled = imaqCreateImage(IMAQ_IMAGE_RGB, 7); // Read the image from disk imaqReadFile(image, &imagePath[0], NULL, NULL); imaqScale (scaled, image, scaleDown, scaleDown, IMAQ_SCALE_SMALLER, IMAQ_NO_RECT); // Vision Assistant Algorithm success = IVA_ProcessImage(scaled, distance); if (!success) err = imaqGetLastError(); // Dispose resources imaqDispose(image); imaqDispose(scaled); return 0; } int IVA_ProcessImage(Image *image, float distance[]) { int success = 1, okay, i; IVA_Data *ivaData; RotatedRect searchRect; FindEdgeOptions edgeOptions; PointFloat firstEdge; PointFloat lastEdge; Image *plane; Rect boundingRect; int caliperWidth, drawROI;
GetCtrlVal (mPnl, MPNL_DRAW_ROI, &drawROI); GetCtrlVal (mPnl, MPNL_THRESHOLD, &edgeOptions.threshold); edgeOptions.width = 5; edgeOptions.steepness = 6; edgeOptions.showSearchArea = 1; edgeOptions.showSearchLines = 1; edgeOptions.showEdgesFound = 1; edgeOptions.showResult = 1; GetCtrlVal (mPnl, MPNL_SEARCH_RECT_WIDTH, &caliperWidth); edgeOptions.subsamplingRatio = caliperWidth/5; // Creates an 8 bit image that contains the extracted plane. VisionErrChk(plane = imaqCreateImage(IMAQ_IMAGE_U8, 7)); VisionErrChk(imaqExtractColorPlanes(image, IMAQ_RGB, NULL, plane, NULL)); // Initializes internal data (buffers and array of points for caliper measurements) VisionErrChk(ivaData = IVA_InitData()); if (drawROI){ imaqConstructROI2 (plane, roi, IMAQ_RECTANGLE_TOOL, NULL, NULL, &okay); } imaqGetROIBoundingBox (roi, &boundingRect); //t l h w for (i=0; i Orthokeratology Epithelial Changes and Susceptibility to Microbial Infection 179 Appendix 2: Epithelial Thickness Measurement Software else searchRect.left = boundingRect.left + (i*(boundingRect.width / (steps-1))) - (caliperWidth/2); searchRect.height = boundingRect.height; searchRect.width = caliperWidth; searchRect.angle = 0; VisionErrChk(imaqClampMax(plane, searchRect, IMAQ_TOP_TO_BOTTOM, &distance[i], &edgeOptions, NULL, &firstEdge, &lastEdge)); dist[i] = distance[i]; } // Display the image imaqSetWindowTitle (0, imagePath[current]); imaqDisplayImage (plane, 0, TRUE); // Releases the memory allocated in the IVA_Data structure. IVA_DisposeData(ivaData); Error: return success; } //////////////////////////////////////////////////////////////////////////////// // // Function Name: IVA_InitData // // Description : Initializes data for buffer management and caliper points. // // Parameters : None // // Return Value : success // //////////////////////////////////////////////////////////////////////////////// static IVA_Data* IVA_InitData(void) { int success = 1; IVA_Data* ivaData = NULL; int i; // Allocate the data structure. VisionErrChk(ivaData = (IVA_Data*)malloc(sizeof (IVA_Data))); // Initializes the image pointers to NULL. for (i = 0 ; i < MAX_BUFFERS ; i++) ivaData->buffers[i] = NULL; // Initializes the array of points to INIT_POINTS_ARRAY_ELEMENTS elements. ivaData->numPoints = INIT_POINTS_ARRAY_ELEMENTS; ivaData->pointsResults = (PointFloat*)malloc(ivaData->numPoints * sizeof(PointFloat)); ivaData->pointIndex = -1; Error: return ivaData; } Orthokeratology Epithelial Changes and Susceptibility to Microbial Infection 180 Appendix 2: Epithelial Thickness Measurement Software static int IVA_DisposeData(IVA_Data* ivaData) { int i; // Releases the memory allocated for the image buffers. for (i = 0 ; i < MAX_BUFFERS ; i++) imaqDispose(ivaData->buffers[i]); // Releases the memory allocated for the array of points. free(ivaData->pointsResults); ivaData->numPoints = 0; ivaData->pointIndex = -1; free(ivaData); return 1; } //////////////////////////////////Excel Function//////////////////////////////////// /**********************************************************************************/ /* opens Excel and loads the template from the current Eyetracker Output directory*/ /**********************************************************************************/ void OpenOutput (void) { int error; char output_file_name[MAX_PATHNAME_LEN]; error = FileSelectPopup ("", "*.xls", "", "Select an Excel Template for Output", VAL_SELECT_BUTTON, 0, 1, 1, 0, output_file_name); if (error >0){ StartExcel(); error = OpenExcelFile(output_file_name); //opens the template file SelectExcelSheet(1); } } /**********************************************************************************/ /* checks the first sheet for entrys and writes the subject data to the last row***/ /**********************************************************************************/ void WriteExcelData (float toWrite[], double mean, double stdev, double steps) { int sheet_no, row_offset =0, i; char *check; //find the first empty row SelectExcelSheet(1); do Orthokeratology Epithelial Changes and Susceptibility to Microbial Infection 181 Appendix 2: Epithelial Thickness Measurement Software { check = TextFromExcel("A1", 0, row_offset); if (StringLength (check)>0) row_offset++; } while (StringLength (check)>0); TextToExcel ("A1", 0, row_offset, imagePath[current] ); DoubleToExcel ("A1", 1, row_offset, mean); DoubleToExcel ("A1", 2, row_offset, stdev); DoubleToExcel ("A1", 3, row_offset, steps); SelectExcelSheet(2); do { check = TextFromExcel("A1", 0, row_offset); if (StringLength (check)>0) row_offset++; } while (StringLength (check)>0); for (i=0; i< steps; i++){ DoubleToExcel ("A1", i, row_offset, toWrite[i]); } } Orthokeratology Epithelial Changes and Susceptibility to Microbial Infection 182 Appendix 3: Publications and Presentations Related to Thesis APPENDIX 3: PUBLICATIONS AND PRESENTATIONS RELATED TO THESIS CHAPTER 9: 9.1 Publications 9.1.1 Accepted Choo JD, Caroline PJ, Harlin DD (2004). How does the cornea change under corneal reshaping contact lenses? Eye & Contact Lens. 30:211-213. Choo JD, Caroline PJ, Harlin DD, Papas EB, Holden BA (2008). Morphologic changes in cat epithelium following continuous wear of orthokeratology lenses: A pilot study. Contact Lens & Anterior Eye. 31:29-37 Choo JD, Holden BA, Papas EB, Caroline PJ, Willcox MDP (2008). Adhesion of Pseudomonas aeruginosa to orthokeratology and alignment lenses. Optometry & Vision Science. (In press). 9.1.2 Submitted Choo JD, Holden BA, Caroline PJ, Papas EB, Willcox MDP. Orthokeratology and corneal susceptibility to Pseudomonas aeruginosa. Submitted to Cornea (Aug 2008). Choo JD, Holden BA, Caroline PJ. Histological epithelial changes associated with commencement and cessation of one month of orthokeratology lens wear. Submitted to Optometry & Vision Science (Aug 2008). Orthokeratology Epithelial Changes and Susceptibility to Microbial Infection 183 Appendix 3: Publications and Presentations Related to Thesis 9.2 Oral Presentations 1. Hong Kong Optometric Conference, Hong Kong (Nov 2008) Invited Speaker: “Bacterial Adhesion to Contact Lenses” 2. Orthokeratology Society of Oceania, Gold Coast, Australia (Oct 2008) Invited Speaker: “Reversibility of OK” “Bacterial Adhesion to OK Lenses” 3. American Academy of Optometry, Anaheim, USA (Oct 2008) Paper: “Recovery of Orthokeratology Epithelial Changes After Cessation of One Month of Lens Wear: A Histological Study” 4. Association for Italian Orthokeratologists, Bologna, Italy (Oct 2008) Keynote Speaker: “OK Epithelial Changes” “OK and Corneal Susceptibility to Bacterial Infection” 5. Vision 2008 Africa Eyecare Event, Johannesburg, South Africa (July 2008) Keynote Speaker: “Corneal Changes with OK” “OK and Microbial Keratitis” “Bacterial Adhesion to Contact Lenses” 6. Asia Orthokeratology Contact Lens Conference, Seoul, Korea (July 2008) Keynote Speaker: “OK Epithelial Changes” “OK and Microbial Keratitis” 7. 2nd International Symposium on Orthokeratology, Singapore (May 2008) Keynote Speaker: “Orthokeratology Corneal Changes” “OK and Microbial Keratitis” 8. Asia Cornea & Contact Lens Conference, Singapore (May 2008) Invited Speaker: “Bacterial Adhesion to Contact Lenses” 9. Orthokeratology Academy of America, San Diego, USA (Apr 2008) Invited Speaker: “OK Corneal Changes & Microbial Keratitis” 10. American Academy of Optometry, Tampa, USA (Oct 2007) Paper:“Bacterial Adhesion to Orthokeratology vs Alignment RGP Lenses” 11. Contact Lens Association of Ophthalmologists, Las Vegas, USA (Oct 2007) Invited Speaker: “Orthokeratology Corneal Physiology” 12. International Contact Lens Congress, Gold Coast, Australia (Sep 2007) Invited Speaker: “Tales of an OK Cornea” 13. International Society Contact Lens Research, Whistler, Canada (Aug 2007) Paper: “Orthokeratology: Progressive & Regressive Epithelial Changes” 14. Bausch & Lomb Contact Lens Public Forum, Singapore (July 2007) Guest Speaker: “Contact Lenses” Orthokeratology Epithelial Changes and Susceptibility to Microbial Infection 184 Appendix 3: Publications and Presentations Related to Thesis 15. Orthokeratology Academy of America, Houston, USA (Apr 2007) Invited Speaker: “Orthokeratology & Microbial Keratitis” 16. Contact Lens Society of Southern Australia, Adelaide, Australia Invited Speaker: “Orthokeratology Corneal Changes” “Orthokeratology and Microbial Keratitis” “Orthokeratology and Myopia Control” “The Future of Orthokeratology” 17. Pacific University College of Optometry, Portland, USA (Dec 2006) Guest Speaker: “Current Research in Orthokeratology” 18. Contact Lens Society of Australia AGM, Melbourne, Australia (Oct 2006) Invited Speaker: “Orthokeratology & Microbial Keratitis” 19. Orthokeratology Society of Oceania, Gold Coast, Australia (Oct 2006) Invited Speaker: “Myopia Overview” “Orthokeratology & Microbial Keratitis” 20. CIBA Vision Myopia Public Forum, Singapore (Aug 2006) Guest Speaker: “Myopia” 21. CIBA Vision Continuing Education Lecture, Singapore (Aug 2006) Guest Speaker: “Oxygen & Corneal Physiology” 22. Optometric Association of Australia Continuing Education Lecture, Sydney, Australia (July 2006) Invited Speaker: “Corneal Changes and Orthokeratology” 23. University of New South Wales Masters of Optometry Course, Sydney, Australia (July 2006) Guest Speaker: “Orthokeratology: Corneal Changes and Implications” 24. LV Prasad Eye Institute, Hyderabad, India (Nov 2005) Invited Speaker: “Myopia Control and Orthokeratology” 25. Contact Lens Information Session, Delhi, India (Nov 2005) Invited Speaker: “Corneal Changes Associated with Orthokeratology” 26. Orthokeratology Society of Australia, Noosa, Australia (Oct 2005) Invited Speaker: “The Biology of Orthokeratology” “The Optics of Orthokeratology” “Soft Lens Orthokeratology” 27. Contact Lens Society of Australia, Blue Mountains, Australia (Aug 2005) Invited Speaker: “Current Research in Orthokeratology” Orthokeratology Epithelial Changes and Susceptibility to Microbial Infection 185 Appendix 3: Publications and Presentations Related to Thesis 28. 3rd Global Orthokeratology Symposium, Chicago, USA (July 2005) Invited Speaker: “Soft Lens Orthokeratology” “The Optics of Orthokeratology” 29. University of New South Wales Masters of Optometry Course Lecture, Sydney, Australia (May 2005) Guest Speaker: “Review of Histology and Current Research in OK” 30. Society for Orthokeratology in Singapore, Singapore (May 2005) Invited Speaker: “Corneal Changes in Orthokeratology” “Current Research in Orthokeratology” 31. American Academy of Optometry Paper, Tampa, USA (Dec 2004) Paper: “Morphologic Changes in Cat Stroma Following Overnight Lens Wear with the Paragon CRT Lens for Corneal Reshaping” 32. Orthokeratology Society of Australia, Gold Coast, Australia (Oct 2004) Invited Speaker: “Morphologic Changes in Cat Epithelium Following Overnight Lens Wear with the Paragon CRT Lens for Corneal Reshaping” 33. 2nd Global Orthokeratology Symposium, Toronto, Canada (July 2004) Invited Speaker: “Morphologic Changes in Cat Epithelium Following Overnight Lens Wear with the Paragon CRT Lens for Corneal Reshaping” 9.3 Poster Presentations Oct 2008 American Academy of Optometry, Anaheim, USA “Orthokeratology Epithelial Changes with Increasing Lens Wear: A Histological Study” by Jennifer D. Choo, OD, Brien A. Holden, PhD, Patrick J. Caroline, COT Aug 2007 International Society for Contact Lens Research, Whistler, Canada “Overnight Orthokeratology: Progressive and Regressive Epithelial Changes” by Jennifer D. Choo, OD, Patrick J. Caroline, COT, Eric B. Papas, PhD, Mark Willcox, PhD, Brien A. Holden, PhD May 2007 British Contact Lens Association, Manchester, England “Overnight Orthokeratology and Corneal Susceptibility to Infection” by Jennifer D. Choo, OD, Patrick J. Caroline, COT, Eric B. Papas, PhD, Mark Willcox, PhD, Brien A. Holden, PhD Orthokeratology Epithelial Changes and Susceptibility to Microbial Infection 186 Appendix 3: Publications and Presentations Related to Thesis Mar 2007 Asia-ARVO, Singapore “Overnight Orthokeratology and Corneal Susceptibility to a Cytotoxic Strain of Pseudomonas aeruginosa” by Jennifer D. Choo, OD, Patrick J. Caroline, COT, Eric B. Papas, PhD, Mark Willcox, PhD, Brien A. Holden, PhD Dec 2006 American Academy of Optometry, Denver, USA “Overnight Orthokeratology and Corneal Susceptibility to Severe Microbial Challenge” by Jennifer D. Choo, OD, Patrick J. Caroline, COT, Eric B. Papas, PhD, Mark Willcox, PhD, Brien A. Holden, PhD Dec 2006 American Academy of Optometry, Denver, USA “Evaluating the Relative Risk of Microbial Keratitis in Longer Term Overnight Wear of Orthokeratology versus Alignment RGP Lenses” by Jennifer D. Choo, OD, Patrick J. Caroline, COT, Eric B. Papas, PhD, Mark Willcox, PhD, Brien A. Holden, PhD May 2006 British Contact Lens Association, Birmingham, England “Evaluating the Relative Risk of Microbial Keratitis in Orthokeratology versus Alignment Fitting RGP Lenses in an Animal Model” by Jennifer D. Choo OD, Brien A. Holden PhD, Patrick Caroline FAAO, Ajay Kumar, Mark Willcox PhD Aug 2005 International Society for Contact Lens Research, Coolum, Australia “The Ideal Shape of the Cornea After Orthokeratology” by Jennifer D. Choo OD, Brien Holden PhD, Patrick Caroline, Arthur Ho, Simon Evans May 2005 ARVO, Fort Lauderdale, USA “Soft Contact Lenses Can Induce Orthokeratology-like Topographical Changes” by Jennifer D. Choo OD, Patrick Caroline FAAO, Brien Holden PhD, Arthur Ho PhD, Simon Evans, Peter Bergenske OD May 2005 ARVO, Fort Lauderdale, USA “Orthokeratology-like Effects of Everted Soft Contact Lenses: A Mechanical Model” by Simon Evans, Arthur Ho PhD, Jennifer D. Choo OD Orthokeratology Epithelial Changes and Susceptibility to Microbial Infection 187 Appendix 3: Publications and Presentations Related to Thesis Dec 2004 American Academy of Optometry Poster, Tampa, USA “24 Hour OrthoK-induced Epithelial Alterations in Cats” by Blake Hughes, BS, Jennifer D. Choo OD, Patrick Caroline FAAO, Margaret Gondo BS, Jan PG Bergmanson OD PhD FAAO Apr 2004 ARVO, Fort Lauderdale, USA “Morphologic Changes in Cat Epithelium Following Overnight Lens Wear with the Paragon CRT Lens for Corneal Reshaping” by Jennifer D. Choo OD, Patrick Caroline, COT, Dustin Harlin, BS, Bill Meyers, PhD Orthokeratology Epithelial Changes and Susceptibility to Microbial Infection 188