Age-Related Changes of the Human Eye (Aging Medicine)

Age-Related Changes of the Human Eye Aging Medicine Age-Related Changes of the Human Eye, edited by Carlo A. P. Caval...

Author: Carlo Cavallotti | Luciano Cerulli

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Age-Related Changes of the Human Eye

Aging Medicine Age-Related Changes of the Human Eye, edited by Carlo A. P. Cavallotti and Luciano Cerulli, 2008 Classic Papers in Geriatric Medicine, edited by Robert Pignolo, Mary Anne Forciea, and Monica Crane, 2008 ElderCare Technology: A Guide for Physicians, edited by Robin Felder and Majd Alwan, 2007 Handbook of Pain Relief in Older Adults: An Evidence Based Approach, edited by Michael F. Gloth, 2004

Series Editors Robert J. Pignolo Division of Geriatric Medicine, University of Pennsylvania Health System, Philadelphia, Pennsylvania Mary Ann Forciea Division of Geriatric Medicine, University of Pennsylvania Health System, Philadelphia, Pennsylvania Jerry C. Johnson Division of Geriatric Medicine, University of Pennsylvania Health System, Philadelphia, Pennsylvania

Advisory Board 1. W. Malinovska Brno, Czech Republic 2. J. Bockova London, UK 3. M. Von Pinoci New York, USA 4. R. Kovacs Budapest, Hungary 5. P. Roger Montreal, Canada 6. M. Mancone Rome, Italy Many Chapters have been mailed to the editors after the deadline. These Chapters have not been revised by the Advisory Board, but have been edited in the format in which they have been submitted by the Authors—who are, obviously, responsible for their content and their form.

Age-Related Changes of the Human Eye Edited by

Carlo A. P. Cavallotti, MD, PhD Department of Cardiovascular, Respiratory, and Morphological Sciences University of Rome, La Sapienza Rome, Italy

Luciano Cerulli, MD, PhD Department of Ophthalmology University of Rome, Tor Vergata Rome, Italy

Editors Carlo A. P. Cavallotti, MD, PhD Department of Cardiovascular, Respiratory and Morphological Sciences University of Rome, La Sapienza Rome, Italy [email protected] Series Editors Robert Pignolo Division of Geriatric Medicine University of Pennsylvania Health System Philadelphia, Pennsylvania

Luciano Cerulli, MD, PhD Department of Ophthalmology University of Rome, Tor Vergata Rome, Italy [email protected]

Mary Ann Forceia Division of Geriatric Medicine University of Pennsylvania Health System Philadelphia, Pennsylvania

Jerry C. Johnson Division of Geriatric Medicine University of Pennsylvania Health System Philadelphia, Pennsylvania

ISBN: 978-1-934115-55-8 e-ISBN: 978-1-59745-507-7 DOI: 10.1007/978-1-59745-507-7 Library of Congress Control Number: 2007939892 © 2008 Humana Press, a part of Springer Science + Business Media, LLC All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Humana Press, 999 Riverview Drive, Suite 208, Totowa, NJ 07512 USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. While the advice and information in this book are believed to be true and accurate at the date of going to press, neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be made. The publisher makes no warranty, express or implied, with respect to the material contained herein. Cover illustration: Figure 1, Chapter 2, by Janos Feher and Zsolt Olah, “Electron microscopy of aged orbicular muscle fibers.” Figure 3a, Chapter 12, by Susanne Binder and Christiane I. Falkner-Radler, “Early face angiogram of a classic CNV.” Printed on acid-free paper 9 8 7 6 5 4 3 2 1 springer.com

Preface

Research on the aging of the human eye crosses all areas of ophthalmology and relies on biological, morphological, physiological, and biochemical tools for its study. Putting together a volume that attempts to cover all the aspects of the aging of the human eye was a daunting task. In fact, some areas may have been invariably overlooked and not every viewpoint may have been included. Despite its shortcomings, we hope Age-Related Changes of the Human Eye will serve as a useful broad-based overview for all the people involved in research and/ or disease on the aging of the human eye. The authors of each chapter were selected for their expertise and prominence in the specific field. Therefore, this book is appropriate for students and graduate students, as well as for postdoctoral and/or professional ophthalmologists. Readers will benefit greatly from the significant revision of material related to the aging of the human eye. The highlights of Age-Related Changes of the Human Eye are its: 1. 2. 3. 4.

Ease of use, Inclusion of numerous personal experiments and data, Versatility, and Bibliography.

The key elements of this volume are the descriptions of age-related changes in almost all the structures of the human eye. The contributors are researchers, physicians, clinicians, technicians, engineers, and members of famous and leading research groups. It should be understood that the eye represents a functional unit, and any modification of one of the structures considered will lead to changes and/or dysfunction of the whole ocular globe. Moreover, we would like to stress that visual function is not only related to the eye, but is a complex activity that is strongly and intimately connected to the brain. Any anomaly, dysfunction, or disease of the ocular globe determines relevant changes in the structure and thus, the function of the brain. Our hope

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is that Age-Related Changes of the Human Eye will give an exhaustive panorama of what happens during the aging process of the eye, thus contributing to the understanding of the physiology and pathology of eye diseases. Carlo A. P. Cavallotti Luciano Cerulli Rome, Italy

Contents

Preface .............................................................................................................

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Chapter 1 Aging as Risk Factor in Eye Disease ...................................... Luciano Cerulli

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Chapter 2 Age-Related Changes of the Eyelid......................................... Janos Feher and Zsolt Olah

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Chapter 3 Aging Effects on the Optics of the Eye. .................................. Pablo Artal

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Chapter 4 Aging of the Cornea ................................................................. Luciano Cerulli and Filippo Missiroli

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Chapter 5 The Aging of the Human Lens ................................................ Jorge L. Aliò, Alfonso Anania, and Paolo Sagnelli

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Chapter 6 The Extracellular Matrix of the Aged Human Trabecular Meshwork: Changes of Glucosaminoglycans ............................................................. Carlo A. P. Cavallotti Chapter 7 Glial and Mobile Cells in the Iris of the Aging Human Eye ................................................................................ Carlo A. P. Cavallotti and Angelica Cerulli Chapter 8 Age-Related Diseases of the Vitreous ..................................... Curtis E. Margo Chapter 9

Age-Related Changes and/or Diseases in the Human Retina ................................................................ Nicola Pescosolido and Panagiotis Karavitis

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Contents

Chapter 10 Aging of the Retinal Pigmented Epithelium ........................ Carlo A. P. Cavallotti and Marcus Schveoller

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Chapter 11 The Aging of the Choroid ...................................................... Angelica Cerulli, Federico Regine, and Giuseppe Carella

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Chapter 12 Age-Related Macular Degeneration I: Types and Future Directions ............................................................. Susanne Binder and Christiane I. Falkner-Radler

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Chapter 13 Age-Related Macular Degeneration II: Idiopathic Macular Holes ....................................................... Christiane I. Falkner-Radler and Susanne Binder

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Chapter 14 Age-Related Macular Degeneration III: Epiretinal Membranes ............................................................. Christiane I. Falkner-Radler and Susanne Binder

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Chapter 15 Macular Degeneration: Ultrastructural Age-Related Changes .............................................................. Illes Kovacs, Janos Feher, and Carlo A. P. Cavallotti

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Chapter 16 Non-Exudative Macular Degeneration and Management .................................................................... Thomas R. Friberg and Kenneth T. Wals

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Chapter 17 Treatment of Intraocular Pressure in Elderly Patients .................................................................. Monika Schveoller, Iliana Iliu, Nicola Pescosolido, and Angelica Cerulli Chapter 18 Aging of the Lachrymal Gland ............................................. Hiroto Obata

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Chapter 19 The World According to Blink: Blinking and Aging ................................................................................. Frans Van der Werf and Albertine Ellen Smit

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Chapter 20 Age-Related Changes in the Oculomotor System ..................................................................................... J. Richard Bruenech

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Chapter 21 Rehabilitation of Low Vision in Aged People ...................... Corrado Balacco, Elena Pacella, and Fernanda Pacella

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Contents

Chapter 22 Many Suggestions to Protect the Eyes in Aging People ....................................................................... Panagiotis Karavitis, Nicola Pescosolido, and Fernanda Pacella Index ...............................................................................................................

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Contributors

Jorge L. Aliò, MD, PhD Professor and Chairman of Ophthalmology, University “Miguel Hernandez” of Helce, Alicante, Spain Alfonso Anania, MD Diagnostic Centre of Ophthalmic Micro-surgery, Rome, Italy Pablo Artal Laboratorio de Optica (Departamento de Fisica), Universidad de Murcia, Murcia, Spain Corrado Balacco, MD, PhD Department of Ophthalmologic Sciences, University of Rome “La Sapienza,” Rome, Italy Susanne Binder, MD Department of Ophthalmology, The Ludwig Boltzmann Institute for Retinology and Bio-microscopic Laser Surgery, Rudolf Foundation Clinic, Vienna, Austria J. Richard Bruenech, PhD Biomedical Research Unit, Buskerud University College, Kongsberg, Norway Giuseppe Carella, MD, PhD San Raffaele Hospital, Department of Ophthalmology and Vision Sciences, University of Milan, Milan, Italy; University of Rome “Tor Vergata,” Rome, Italy Carlo A. P. Cavallotti, MD, PhD European Ophthalmic Neuroscience Program (Local Research Unit), University of Rome “La Sapienza,” Rome, Italy Angelica Cerulli, MD Department of Ophthalmology, University of Rome “Tor Vergata,” Rome, Italy Luciano Cerulli, MD, PhD Department of Ophthalmology, University of Rome “Tor Vergata,” Rome, Italy

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Christiane I. Falkner-Radler, MD Department of Ophthalmology, The Ludwig Boltzmann Institute for Retinology and Bio-microscopic Laser Surgery, Rudolf Foundation Clinic, Vienna, Austria Janos Feher, MD, PhD Nutripharma Hungaria Ltd., Budapest, Hungary Thomas R. Friberg, MD, PhD UPMC-Eye Centre, University of Pittsburgh, Pittsburgh, PA Panagiotis Karavitis, MD Ophthalmology, University of Athens, Athens, Greece; Department of Ophthalmologic Sciences, University of Rome “La Sapienza,” Rome, Italy Illes Kovacs, MD, PhD Department of Ophthalmology, Semmelweis University, Budapest, Hungary Iliana Iliu, MD Geriatric Centre University of Salonica, Salonica, Greece; Ophthalmology, Department of Aging Sciences, University of Rome “La Sapienza,” Rome, Italy Curtis E. Margo, MD, MPH Ophthalmic Pathology Laboratory, Ophthalmology and Pathology, University of South Florida College of Medicine, Tampa, FL Filippo Missiroli, MD Department of Ophthalmology, University of Rome “Tor Vergata,” Rome, Italy Hiroto Obata, MD, PhD Department of Ophthalmology, Jichi Medical University, Tochigi, Japan Zsolt Olah, BSc School of Sport Medicine, Semmelweis University, Budapest, Hungary Elena Pacella, MD Department of Ophthalmologic Sciences, University of Rome “La Sapienza,” Rome, Italy Fernanda Pacella, MD Department of Ophthalmologic Sciences, University of Rome “La Sapienza,” Rome, Italy Nicola Pescosolido, MD Senior Researcher Section of Ophthalmology, Department of Aging Sciences, University of Rome “La Sapienza,” Rome, Italy Federico Regine, MD Department of Ophthalmology, University of Rome “Tor Vergata,” Rome, Italy Paolo Sagnelli, MD European Ophthalmic Neuroscience Program (Local Research Unit), University of Rome “La Sapienza,” Rome, Italy

Contributors

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Marcus Schveoller, MD, PhD Department of Ophthalmology, European Ophthalmic Neuroscience Program, University of Pécs, Pécs, Hungary Monika Schveoller, MD Department of Ophthalmology, University of Pécs, Pécs, Hungary Albertine Ellen Smit, MD Department of Neuroscience, University of Rotterdam, Rotterdam, The Netherlands Frans Van der Werf, PhD Department of Neuroscience, University of Rotterdam, Rotterdam, The Netherlands Kenneth T. Wals, MD UPMC-Eye Centre, University of Pittsburgh, Pittsburgh, PA

Chapter 1

Aging as Risk Factor in Eye Disease Luciano Cerulli, MD, PhD

“Une des tristesses de la vie est que toutes les évaluations chiffrées des performances visuelles montrent qu’elles déclinent progressivement avec l’âge.” Kline, 1987

Abstract The major causes of blindness and reduced vision are related to cataracts, glaucoma, age-related macular degeneration, and diabetic retinopathy—all of which recognize aging as the major risk factor. The burden of visual impairment is not distributed uniformly through the world. The least developed regions carry the largest share. Visual impairment is also unequally distributed across age groups, with incidence largely confined to adults 50 years of age and older (83%). A distribution imbalance is also found with regard to the gender throughout the world—females have a significantly higher risk of developing visual impairment than males because their life expectancy is higher and their economic possibilities may be less. Notwithstanding the progress in surgical intervention that has been made in many countries over the last several decades, cataracts remains the leading cause of visual impairment in all regions of the world, except in the most developed countries.

Keywords Cataract, Glaucoma, ARM D, Corneal opacity, Diabetic retinopathy

Over the last few years, aging has become the prevalent risk factor in the overall world population. In the 1980s, this was true only in the European Countries but it has now become a major cause of morbidity and mortality worldwide. Infective agents are, in a certain sense, loosing their primary station as the most relevant cause of illness and death, while degenerative conditions are growing all over the world. This is true for general diseases and also in ophthalmology. It has been estimated that there are 161 million visually impaired individuals in the world, and of this figure, 37 million of them are blind. Fig. 1.1 and 1.2 list the most recent data available from World Health Organization (WHO) reports on the world prevalence of blindness and reduced vision.

From: Aging Medicine: Age-Related Changes of the Human Eye Edited by C. A. P. Cavallotti and L. Cerulli © Humana Press, Totowa, NJ

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Fig. 1.1 Prevalence of Blindness

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Fig. 1.2 Prevalence of Low Vision

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The major causes of blindness and reduced vision are related to cataracts, glaucoma, age-related macular degeneration, and diabetic retinopathy—all of which recognize aging as the major risk factor. The burden of visual impairment is not distributed uniformly through the world. The least developed regions carry the largest share. Visual impairment is also unequally distributed across age groups, with incidence largely confined to adults 50 years of age and older (83%). A distribution imbalance is also found with regard to the gender throughout the world—females have a significantly higher risk of developing visual impairment than males because their life expectancy is higher and their economic possibilities may be less. Notwithstanding the progress in surgical intervention that has been made in many countries over the last several decades, cataracts remains the leading cause of visual impairment in all regions of the world, except in the most developed countries. Other major causes of blindness are (in order of frequency): glaucoma, ARMD, diabetic retinopathy, and trachoma.1 More specifically, Table 1.1 provides the available data on the estimated prevalence of eye diseases as causes of blindness and reduced vision. These diseases do recognize that aging is the major risk factor. WHO has examined and forecast the distribution of the world population at the year 2000, 2025 and 2050, as displayed in Table 1.2. Table 1.1 Estimated prevalence of the eye diseases Cataract 47,9% Glaucoma 12,3% ARMD 8,7% Corneal opacity 5,1% Diabetic retinopathy 4,8%

Table 1.2 Distribution of world population at the years 2000, 2025, 2050

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Table 1.3 % Increase of over 50 in different sub-groups of Countries % increase WLD MDC LDC

2000 1081211 366777 714435

2025 2095593 492345 1603248

2050 3138029 503099 2634932

2000vs2025 194 134 224

% increase 200vs2050 290 137 369

The dramatic increase in the number and percentages of the world’s population is clearly noted as definitively more evident in less-developed countries, while the financial allocation of resources and health services availability and health technology are weaker in those same countries. Without proper interventions, the increase in the number and percentage of people over the age of 50 will lead in the near future to a significant increase in the number and percentage of eye diseases that recognize age as the major risk factor. It has been estimated that without extra interventions, the total number of blind individuals worldwide would increase from 44 million in the year 2000 to 76 million in 2020 Table 1.3. A successful intervention by the VISION 2020 initiative could result in only 24 million people suffering from blindness by 2020 and lead to a reduction of 429 million cases of blindness per year. A conservative estimate of the economic gain is 103 billion dollars.2 A proper approach in the treatment of these clinical pictures will lead to a reduction in numbers and percentage of the blind but also to an increase of reduced vision patients with a dramatic need for rehabilitation activities. How many glaucoma patients are currently affected, how many of them are blind or severely impaired by this disease, what are the geographical and temporal distributions, and what are the main risk factors? These are questions without real scientific answers.3 As we have seen, the WHO estimates that this pathology represents the second cause of blindness and reduced vision worldwide after cataracts. It is estimated that in the year 2000, 66.8 million people are affected by open angle glaucoma. Of these, 6.7 million were affected by a bilateral blindness secondary to this disease, according to Quigley and Vitale.4 In the same year, 2.47 million patients were affected by this pathology just in the United States—1.84 million were Caucasians and 619,000 were people of color. Numerous considerations can be made from the results of this study. Taking into account the person’s age at the beginning of the illness, together with the death rate, the illness lasts longer by 27% in colored people in comparison to patients of the Caucasian race. In industrialized countries, less than 50 percent of the population with glaucoma realize that they are affected. This percentage is much lower in developing countries. As for other disease, epidemiological studies have considered several risk factors, such as age, sex, race, social and economical factors, working activities, climate, the use of tobacco or alcohol, genetic, and ocular factors. Undoubtedly, the Intraocular Pressure (IOP) represents the major risk factor for the development of

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the disease, but together with other factors, we should consider that the prevalence of glaucoma increases with age, with higher baseline IOP values in the elderly.5 This can be explained by the physiological reduction in time of the retinal ganglion cells, associated with a prolonged exposure to nontolerated high values of IOP, together with the action of other risk factors.6 The lack of, or a delay in, a diagnosis may lead to an irreversible blindness. Given the number of patients affected, the social-economical implications related to this pathology are very onerous. In a recent study, for example, it has been calculated that in Germany, 800,000 patients are affected by glaucoma. Every year, Social Security spends more than 1,000 Euro per patient. On the other hand, if we consider the number of patients who are certified blind in connection with this illness, we must consider that the German government spends 150 million Euro in national Social Security and health expenses. These costs are destined to rise due to the fact that the average life span has increased and will continue to increase in the future. In a multicentric study carried out in France, Germany, Italy, and Great Britain, the average cost per person for glaucoma each year was 726 Euro.7 As we have already seen, diabetic retinopathy contributes to 4.8% of blindness and reduced vision worldwide. We must remember that in the United States it is understood that 14 million are affected by diabetes, and of these, 43 percent present a related retinopathy. This complication creates 8,000 new cases of blindness each year. For this pathology—together with other risk factors such as the type of diabetes, race, sex, controlling blood sugar levels and blood pressure, dislipidemia, nephropathy, pregnancy, and the duration of the illness (both for diabetes type 1 and type 2)—aging represents one of the most important risk factors to be considered. It has been estimated that after 20 years, almost all diabetic type 1 patients, and 60 percent of diabetic type 2 patients are affected by this retinal complication. Furthermore, if we consider that cataract surgery—a pathology typical of old age—worsens the course of diabetic retinopathy, we realize that aging represents an important element to be considered for diabetic patients. Taking into account the higher life expectancy for diabetic patients and the aging of the world population, especially in the Southern hemisphere (and thus a major risk to be affected by type 2 diabetes), we can easily understand what sort of dimension this problem can assume in the near future. Aging is not only a risk factor for the previously mentioned diseases (Fig. 1.3). The social changes in our time often leave the elderly in a condition of loneliness and eventually with economical and cultural barriers toward the access to health services. This may lead to a delay in early diagnosis, a delay in the beginning of an appropriate treatment, difficulties in following the medical prescriptions, and the related rehabilitation with a subsequently more significant handicap. This is not only true for eye diseases, but also for uncorrected changes in refraction. WHO has estimated that 153 million people are visually impaired due to uncorrected refractive errors (URE), with 95 percent of them being over 50 years of age. Although this book has been divided into different sections, an effort should be made to understand that the eye represent a functional unit and any modification of any one of its structures will lead necessarily to changes and/or dysfunction of the whole globe.

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Fig. 1.3 Age-related eye diseases as causes of blindness and low vision

In addition, I would like to stress that visual function is not only related to the eye, but is a complex activity that is strongly and intimately connected with the brain. Any anomaly, dysfunction, or disease of the globe can be associated with relevant changes in the structure, and thus the function, of the brain. Our hope is that this book will give an exhaustive panorama of what happens when the eye ages, thus contributing to the understanding of the physiology and physiopathology of eye diseases.

References 1. Resnikoff S and Co. Policy and Practice “Global data on visual impairment in the year 2002” 2. Frick K, et al. (2003) The magnitude and cost of global blindness: An increasing problem that can be alleviated. Am. J. Ophth. April 471-476 3. Quigley HA (1996) Number of people with glaucoma worldwide Br. J. Ophthalmol. May 80(5):389-393 4. Quigley HA, Vitale S (1997) Models of open-angle glaucoma prevalence and incidence in the United States. Invest. Ophthalmol Vis Sci Jan 38:83-91 5. Friedman DS et al. (2006) The prevalence of open angle glaucoma among blacks and whites 73 years old: the Salisbury Eye Evaluation Glaucoma Study. Arch. Ophthalmol. 124:1625-30

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6. Sommer A et al. (1991) A population-based evaluation of glaucoma screening: the Baltimore Eye Survey. Am. J. of Epidemiol. 134:1102-1110 7. Traverso CE et al. (2005) Direct costs of glaucoma and severity of the disease. Br. J. Ophthalmol 89:1245-9

Recommended Bibliographic Resources Recent Books on Vision Disorders in Old Age (www.amazon.com) 1. The Aging Eye by Sandra Gordon, Harvard Medical School, 2001. 2. Communication Technologies for the Elderly: Vision, Hearing & Speech by Rosemary Lubinski, D. Jeffery Higginbotham, 1997. 3. The effects of aging and environment on vision by Donald A. Armstrong, et al., 1991. 4. Treating vision problems in the older adult (Mosby’s optometric problem-solving series) by Gerald G. Melore, 2001. 5. Vision and Aging by Alfred A. Rosenbloom, Meredith W. Morgan, 1993. 6. Age-Related Macular Degeneration by Jennifer I. Lim, 2002. 7. The Impact of Vision Loss in the Elderly (Garland Studies on the Elderly in America) by Julia J. Kleinschmidt, 1995. 8. Vision in Alzheimer’s Disease (Interdisciplinary Topics in Gerontology) by Alice Croningolomb, et al., 2004. 9. The Senescence of Human Vision (Oxford Medical Publications) by R.A. Weale, 2001. 10. Issues in Aging and Vision: A Curriculum for University programs and In-service Training by Alberta L. Orr, 1998. 11. Aging with developmental disabilities changes in vision by Marshall E. Flax, 1996. 12. Trends in vision and hearing among older Americans by U.S. Dept of Health and Human Services, 2000. 13. Optometric gerontology: A resource manual by Sherrell J. Aston, 2003.

Chapter 2

Age-Related Changes of the Eyelid Janos Feher, MD, PhD and Zsolt Olah, BSc

Abstract Changes of the orbicular muscle and its connective tissue play a central role in the aging of the eyelid. Age-related changes of orbicular muscle comprise a decrease of muscular fibers and a disorganization of banding structures (appearance of nemaline bodies, Z-line streaming, cytoplasmic bodies, and Z-line doubling). Mitochondria, particularly in the subsarcolemmal area, showed either a decrease in number and loss of cristae, or enlargement and proliferation of cristae. In combination with both alterations, intramitochondrial crystal formation and altered succinyl-dehydrogenase activity were also a frequent observation. Tubular aggregates originated from the sarcoplasmic reticulum and various sarcoplasmic inclusions were also observed. Intramuscular connective tissue density increased with age, and it was associated with increased glycation of collagen fibers. Neither of these alterations are considered specific for aging, but their particular combination may be responsible for the development of well-known, age-related changes and diseases of the eyelid. In addition, these data may give further information to the pathology of sarcopenia—a devastating age-related muscle disease. Keywords eyelid, aging, orbicular muscle, nemaline body, Z-line streaming, cytoplasmic body, mitochondria, creatine kinase crystal, succinyl-dehydrogenase, sarcoplasmic reticulum, tubular aggregates, electron microscopy.

Introduction Aging of the eyelid is a well-known phenomenon, but it is still a poorly explored interdisciplinary area of medicine. The skin layer belongs mostly to dermatology, the intermediate muscle-connective layer is generally the subject of oculoplastic surgery and neuroophthalmology, while the innermost conjunctiva is reserved for subspecialities of ophthalmology—i.e., for experts of dacryology and the tear filmocular surface. This paper is dedicated to the age-related changes of the orbicular muscle and its connective tissue for two reasons. First, age-related changes of these structures From: Aging Medicine: Age-Related Changes of the Human Eye Edited by C. A. P. Cavallotti and L. Cerulli © Humana Press, Totowa, NJ

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are responsible for the development of malpositions of the lower eyelid known as senile entropion and ectropion.1 In the former case, the margin of the lower eyelid turns inward, eyelashes irritate the corneal surface continuously, and keratoconjunctivitis develops with potential corneal ulceration. In the latter case, the margin of the lower eyelid turns outward, tarsal conjunctiva and lower lachrymal punt are exposed, and chronic lachrymation (epiphora) and conjunctival inflammation develop. There are two main theories to explain the pathomechanism of senile entropion and ectropion—the spastic and the atonic theory. These malpositions of the lower eyelid are therefore often called spastic entropion or ectropion as well as atonic entropion or ectropion. Both theories ascribe essential importance to alterations of the orbicular muscle and neighboring connective tissues.1 Although several morphological and functional observations have been carried out to reveal the pathologic background of abnormal muscle activity, we are far from knowing the exact mechanisms that cause the senile involution of the orbicular muscle, and far from explaining the variable clinical picture. In both conditions, plastic surgery is the choice of treatment. Until now, however, more then 120 original and modified surgical procedures have been introduced, suggesting the poor effectiveness of any of them. In fact, recidivates are quite frequent after each procedure. Early electron microscopy of eyelid aging and its relation to senile entropion and ectropion revealed significant ultrastructural abnormalities in the orbicular muscle fibers,2 but no differences related either to entropion or ectropion.3 Some abnormalities of the mitochondria4 and sarcoplasma5,6 were also described. Here we present a completed ultrastructural morphology of orbicular muscle aging and design putative correlations between these abnormalities. Aging of the orbicular muscle may be generally related to a part of muscle aging known as sarcopenia. The second aim of this chapter is to reveal the earliest ultratstructural alterations related to this poorly explored and uncurable pathology. Sarcopenia is a slowly progressive and complex process that appears in aged muscle that is associated with a decrease in mass, strength, and velocity of contraction. This process is the result of many molecular, cellular, and functional alterations. With the advancement of age, type I muscle fibers decrease in number and increase in size.7 Type I fiber predominance seen in older subjects could be related to a selective decrease of type II fibers as the body ages. It also suggests a possible conversion of type II fibers to type I fibers.8 In the elderly, central nuclei, ring fibers, fiber splitting, scattered highly atrophic fibers, moth-eaten fibers, and vacuoles were also observed. Ring fibers were most easily identified with antidesmin labeling, and highly atrophic fibers exhibited a rough network of labeling. An increased content of actin and spectrin was also observed at the periphery of ring fibers. A qualitative ultrastructural analysis also showed obvious changes, including some myofilament loss, collections of lipofuscin that were also observed in satellite cells, proliferation of the sarcoplasmic reticulum, and increased wrinkling of nuclear membranes and sarcolemma.9 Interestingly, satellite cell populations were not significantly lower in healthy, sedentary older adults compared to young adult men and women.10 Over time, mitochondrial size and mitochondrial percentage per fiber area decrease, and the cristae of mitochondria became irregularly spaced, disrupted,

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and replaced by lamellar, myelin-like structures. Giant mitochondria are often visible. They contain lipid droplets and lipofuscin in the myofibrils, which are often in close relationship with the damaged mitochondria.11 It has been suggested that sarcopenia may be triggered by reactive oxygen species (ROS) that have accumulated throughout a person’s lifetime. In fact, a significant increase in the oxidation of DNA and lipids was found in elderly muscle— more evident in males—along with a reduction in catalase and glutathione transferase activities. Experiments on Ca2+ transport showed an abnormal functional response of aged muscle after exposure to caffeine, which increases the opening of Ca2+ channels, as well as reduced activity of the Ca2+ pump in elderly males. These results proved that oxidative stress plays an important role in muscle aging, and that oxidative damage is much more evident in elderly males, suggesting a gender difference that may be related to hormonal factors. The progression of sarcopenia is directly related to a significant reduction of the regenerative potential of muscle normally due to a type of adult stem cells known as satellite cells. These cells lie outside the sarcolemma and remain quiescent until external stimuli trigger their re-entry into the cell cycle as growth factors. One possibility is that the anti-oxidative capacity of satellite cells could also be altered and this, in turn, can determine the decrease of their regenerative capacity. Data concerning this hypothesis are discussed.12

Changes in Anatomy and Kinematics Topographic anatomy of the eyelids is affected by aging and sex. Normal aging processes may cause laxity of eyelid tissues (skin, muscle, connective tissue) and atrophy of the orbital fat. These changes are responsible for the well-known aesthetic changes, but they may also contribute to the aetiology of several eyelid disorders, such as ectropion, entropion, dermatochalasis, and blepharoptosis. Such aging changes may also affect the position of the eyelids, eyeball, and eyebrow. Aging primarily affects the size of the horizontal eyelid fissure. In adolescence, between the ages of 12 and 25 years, the horizontal eyelid fissure lengthens 3 mm, while the position of other eyelid structures remains virtually unchanged. Between the average ages of 35 and 85 years, the horizontal eyelid fissure gradually shortens again by about 2.5 mm. While the lengthening of the horizontal eyelid fissure between the ages of 12 and 25 years probably reflects growth of the facial structures, the shortening from the age of 35 years onwards is likely due to progressive laxity of the medial and lateral canthal structures. With aging, the distance between the lateral canthal angle and the anterior corneal surface decreases almost 1.5 mm. which means that the shortening of the horizontal eyelid fissure can at least partially be attributed to medial displacement of the lateral canthus. The positions of the lateral canthus and the center of the pupil are identical in men and women, and remain fairly stable throughout life. Aging causes the sagging of the lower eyelid,

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especially in men, and a higher skin fold and eyebrow position in both sexes. Laxity of the lower eyelid causes an increase of the distance between the pupil center and the lower eyelid of about 1 mm in men, and 0.5 mm in women. In a prospective consecutive observational case series, 32 normal adult subjects—comprised of 12 younger (aged 29+/-5 years) and 20 older subjects (aged 74+/-6 years)—underwent lower eyelid tensometry. Younger males had higher eyelid tension than females, and there was no significant reduction in tension with age.13 Aging also causes raised eyebrows and increased skin creases in men and women, but in the study it did not affect the position of the pupil center and the lateral canthus. Men showed a 0.7 mm larger horizontal eyelid fissure than women. However, the eyebrows in women were situated about 2.5 mm higher than in men. Aging does not affect the position of the eyeball proper, or of the lateral canthus.14 Cutis laxa is an uncommon entity characterized by laxity of the skin, which hangs in loose folds, producing the appearance of premature aging. Histological analysis and ultrastructural examination of skin biopsy revealed reduction and fragmentation of elastic fibers. Dermatochalasis is a severe degree of the aging process in the eyelid and orbital soft tissue complex. It can lead to extreme weakness or even dehiscence of the supporting fascia and other surrounding soft tissue—rarely leading to free mobility of the orbital fat pads and hence postural herniation into the eyelids, as seen in this unusual case. Recent studies on the changes in the kinematics of blinking over time demonstrated that disorders of blink systems typically seen in persons 50 years of age or older occur against a backdrop of normal age-dependent changes in eyelid kinematics. Alterations in main sequence slope imply that the operation of central adaptive systems during aging. Reduction in main sequence slope is interpreted as a reduction in aggregate orbicular muscle motoneuron activity. Such a central neurologic adjustment in the motor output of blink systems may serve to compensate for an age-related increase in blink reflex excitability. Compensatory reduction in the main sequence relationship may offset a potentially hyperexcitable blink reflex, thereby reducing the likelihood of disorders such as blepharospasm. These authors described passive and active changes in the kinematics of blinking with age. Passive changes in blink amplitude-peak velocity reflect age-related changes in static eyelid position that can be attributed largely to either weakness of the Muller’s muscle and superior levator muscle, or to the laxity of the connective tissue in the superior transverse ligament, palpebral ligaments, or dehiscence of the levator aponeurosis. Active changes in main sequence relationships demonstrate that blink plasticity interacts with aging processes. The active changes observed in the neural control of blink kinematics do not, by themselves, represent a trend toward the development of blink disorders in an aging population. If the aging of blink systems actively contributed to an age-related trend toward diseases of elevated blink excitability, an increased main sequence slope would have been expected. By contrast, the opposite result suggests that the active, central changes in eyelid kinematics may represent an adaptive response to the established, age-associated increase in blink reflex excitability.15


13

Epidermal innervation according to age and anatomical site was evaluated in 82 biopsy samples from surgical procedures. Eyelid epidermis showed the highest ratio of nerve fiber surface to epidermal surface. A trend exhibiting age-associated decreased epidermal innervation of facial skin was found. Epidermal innervation of abdominal skin did not change with age, and an age-associated increased innervation was observed in mammary and palpebral skin.16 In another study, trigeminal blinks in normal human subjects between 20 and 80 years of age were characterized. In normal humans over 60 years of age, lid-closing duration, and the excitability and latency of the trigeminal reflex blink, increase significantly relative to younger subjects. Reflex blink amplitude, however, does not change consistently with age. For subjects less than 70 years of age, a unilateral trigeminal stimulus evokes a 37 percent larger blink in the eyelid ipsilateral to the stimulus than in the contralateral eyelid. Subjects who are 70 years old, however, exhibit blinks of equal amplitude. In all cases, blink duration is identical for both eyelids.17

Myofiber Abnormalities We have performed electron microscopic and histochemical studies on surgical specimens of 86 patients, aged 42-88 years, and affected by various pathologies (tumors, enctropin, ectropin, trauma). Orbicular muscle contains mainly type I fibers. Age-related myofiber abnormalities comprise the decrease of the filamentary structure and disorganization of the normal banding structure, particularly the Z line. The decrease of filaments were the most common aging changes in the orbicular muscle. In some cases, simple quantitative changes were observed without any qualitative alterations (see Fig. 2.1). In most cases, however, decrease of myofilaments were accompanied with disorganization of both fibrillary and banding structures. In some sites, there were only small focal changes, whereas at other places, the damage extended over many sarcomeres and even many fibers. From a morphological point of view, Z-line alterations serve as a reference and four subtypes of myofiber changes related to aging can be distinguished. Nemaline bodies (rods) are by far the most common and most widely studied alteration of the skeletal muscle. This alteration is characterized by rod-form accumulation of Z-line material. They consist of a lattice-like arrangement of squares measuring about 1 nm on each side (see Fig. 2.2). Nemaline bodies are clearly detectable at the light-microscopy level, and ultrastructurally, they originate from the Z-disks of the sarcomeres. These paracrystalline structures stained positively for alpha-actinin.18 In fact, one of the main components of rods is alpha-actinin—an actin-binding protein that localizes to the Z-disk.19,20 More than 70 mutations in the skeletal muscle alpha-actin (ACTA1) gene have now been identified. By and large, mutations are associated with three muscle diseases: a) nemaline myopathy, b) congenital actin myopathy, and c) intranuclear rod

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Fig. 2.1 Electron microscopy of aged orbicular muscle fibers. An apparently normal fiber in the center is surrounded with myofibers of highly variable diameter, but well preserved banding structure can be seen in each fiber (x32 K 68y)

Fig. 2.2 Nemaline bodies (rods). They show a typical paracrystalline structure. Several thin filaments are in connection with the rods. Next to these alterations, the myofibrillary structure is more or less irregular and the normal banding pattern is disrupted (87y years x 22 K)

myopathy. The majority of ACTA1 mutations are dominant, a small number are recessive, and most isolated cases with no previous family history have de novo dominant mutations.21 Nemaline myopathy is a rare autosomal dominant skeletal muscle myopathy characterized by severe muscle weakness and the subsequent


15

appearance of nemaline rods within the muscle fibers. The intrafamilial variability suggests that the ACTA1 genotype is not the sole determinant of phenotype.22 Recently, a missense mutation in TPM3, which encodes the slow skeletal alphatropomyosin, was linked to nameline myopathy in a large kindred group with an autosomal-dominant, childhood-onset form of the disease.23 The primary defect, caused by expression of the mutant alpha-tropomyosin, was a decrease in the sensitivity of contraction to activating Ca2+, which could help explain the muscular hypotonia seen in this disease. Interestingly, this mutation did not directly result in nemaline rod formation, which suggests that rod formation is secondary to contractile dysfunction and that load-dependent processes are likely involved in nemaline rod formation in vivo.24 Although a number of genes have been identified in which mutations can cause nameline myopathy, the pathogenetic mechanisms leading to the phenotypes are poorly understood. All these together suggest a common process or mechanism operating in nemaline muscles independent of the variable degrees of pathology. More recently, electron micrographs showed elevated focal repair in nemaline muscles, suggesting that in nameline myopathy, a novel repair feature may operate.25 Sporadic late onset nemaline myopathy was also found to be associated with monoclonal gammapathy.26 Z-line streaming is a focal widening of the Z line, or sometimes widening of the whole Z line in one or more sarcomeres. These alterations were apparently common in both normal and abnormal orbicular muscle (see Fig 2.3). In excessive Z-line streaming, a longitudinal fibrillary structure was displayed, which continued into the I band next to the abnormal Z line (see Fig. 2.4). The sarcomeres around Z-line streaming showed either a normal or an abnormal banding pattern. Although both Z-line streaming and rods contain electrodense Z-line material, and that material enters into both fine filaments, the paracrystalline structure of the

Fig. 2.3 Z-line streaming in an apparently normal muscle. The normal banding pattern, sarcoplasmic reticulum, and mitochondria can be seen (x40 K 73y)

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Fig. 2.4 Excessive Z-line streaming: The longitudinal pattern is highly disturbed, and most Z lines are widened. Between the irregular mitochondria fibers, glycogen particles and tubules of sarcoplasmic reticulum can be see (x40 K 69y)

rods is clearly different from the filamentary structure of Z-line streaming. Z-line streaming may occur in normal aged muscle, but it is more common in muscle disease such as muscle dystrophies, denervation atrophy, collagen vascular disease, hypothyroid myopathy, and central core disease. In rats on a low-protein diet, Z-line streaming and disintegration of sarcomeric striation was associated with depletion of glutathion. The depletion of glutathione by protein malnutrition is responsible for inducing myofibrillary damage through the excess leaking of Ca2+ into the cytosol.27,28 Electron microscopy of biopsy specimens from the gastrocnemius muscles of volunteer human marathon runners showed evidence of inflammation and muscle fiber alterations, including Z-line streaming.29 Cytoplasmic bodies are curious structures that vary widely in size and shape, but have a characteristic appearance. It consists of a round or oval, amorphous electrodense central area surrounded by a halo of less electrodense amorphous material (see Fig. 2.5). Fine filaments from the adjacent muscle fibers pass through this halo and enter into the electrodense central area. Between the fine filaments, a few T tubules, glycogen particles, and a rich network of sarcoplasmic reticulum appear. The origin, the chemical composition, and significance of cytoplasmic bodies are unknown, but in our material they appear to contain Z-line material as well as myofilaments. Cytoplasmic bodies were found in muscle dystrophies, periodic paralysis, and collagen vascular diseases. Double Z lines or duplication of the Z line rarely occurred and it appeared in several neighboring fibers (see Fig. 2.6). Although similar alterations were reported in regenerating muscle and in hypothyreosis, neither the cause nor significance of double Z lines are known.


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Fig. 2.5 Cytoplasmic body formation in the sarcoplasma. The electrodense oval area is surrounded by a halo of fine filaments—tubules of sarcoplasmic reticulum and T tubules (x22 K 82 yrs)

Fig. 2.6 Duplication of the Z line in some neighboring fibers. On both sides of each pair of Z lines, fine fibrils can be seen. No other ultrastructural alterations are observed in association with double Z line (x26 K 63 yr)

Mitochondrial Alterations In normal conditions, mitochondria are located between myofibers and in the subsarcolemmal area. Their number is higher in the type I fibers. Numerous morphometric studies on skeletal muscle describe an age-related decline in the number of mitochondria

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and the loss of cristae. We found similar changes in our material, but it was not a generalized phenomenon. In some specimens, we found a significant increase in the number of mitochondria. In both these cases, however, the surrounding myofibers and mitochondria showed apparently normal structure (see Fig. 2.7). Proliferation of inner mitochondrial membrane/cristae, usually accompanied with the enlargement of mitochondria, was the most characteristic change in aged orbicular muscle. These alterations were found exclusively in the subsarcolemmal area and almost all mitochondria showed more or less abnormalities—i.e., the proliferation of mitochondrial cristae may be accompanied with loss of cristae, even in the same mitochondria, and a decrease of matrix density. (see Fig. 2.8). Mitochondrial inclusions of crystalline structure are very common age- and diseaserelated alterations of mitochondria. In our material, they were mostly observed in the subsarcolemmal area, but rarely in the interfibrillary mitochondria (see Fig. 2.9). Two distinct types of crystals can be distinguished on the basis of shape, size, pattern, unit cell dimension, specific location of the crystals in the mitochondrial intermembrane space, and occurrence in different muscle fiber types. Type I crystals (see Fig. 2.10) are usually present in the intracrystal space, and they occur in type I muscle fibers, whereas the type II crystals (see Fig. 2.11) are preferentially located in the intermembrane space between outer and inner mitochondrial membranes, and occur in type II muscle fibers. In humans, type I crystals are on average 200 nm wide and 2 mm long, while the type II crystals are more cubic in their dimensions—usually 100-300 nm in all three dimensions.30 Immunoelectron microscopy revealed that these inclusions react heavily

Fig. 2.7 Accumulation of mitochondria between myofibers. Both mitochondria and myofibers show well-preserved structure, although there is a light difference between the upper-left part of the picture, where normal number and normal myofiber structure can be seen, and the lower-right part, where an accumulation of mitochondria is associated with compromised myofibers (x22 K 66 yrs)


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Fig. 2.8 Numerous large mitochondria in the subsarcolemmal area. Some of them are extremely large (mega or giant mitochondria) containing enormous cristae arranged either regularly or randomly. Some of the cristae are highly electrodense, some of them have lost membrane structure, and some contain electrodense granules—presumably unsaturated lipids. The diameter of myofibers apparently decreases, but their banding structure is well-preserved and shows a state of contraction. Interfibrillary mitochondria are slightly enlarged but show normal structure (x25 K 70 yrs)

Fig. 2.9 Crystalline mitochondrial inclusion. Almost all mitochondria in the subsarcolemmal area show electrodense inclusions—in some of them the crystalline structure is clearly visible even in this magnification, but some of them contain electrodense amorphous or granular inclusions. In the neighboring myofibers, only Z line can be distinguished (x19 K 79y)

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Fig. 2.10 Morphological features of mitochondria with type I crystals. The basic structural unit of this type appears in thin sections as a rectangular unit—about 32 nm wide and of varying length— always located within the intracrystal space or between the inner and outer membrane (x70 K 71y)

Fig 2.11 Morphological features of a mitochondria with type II crystals. This structure is surrounded by the membranes of cristea—i.e., they are located in the intermembrane space but not in the mitochondrial matrix (x70 K 74y)

with specific antibodies against mitochondrial creatine kinase.31,32 Mitochondrial creatine kinase is located in the intermembrane compartment and is functionally coupled to oxidative phosphorylation. It shuttles high-energy phosphates, formed in the mitochondria, to the cytosol where they are utilized. Recent concepts suggest that mitochondrial creatine kinase has the dual role of a) functioning as a key enzyme in


21

energy metabolism, and b) as a structural protein inducing the formation of mitochondrial contact sites between inner and outer membaranes. This leads to membrane cross-linking and an increased stability of the mitochondrial membrane architecture, thereby contributing to the organization of the entire organelle.33 These structural changes in mitochondria were associated with functional changes because it was demonstrated with enzyme-histochemical studies. Succinodehydrogenase activity showed a particular pattern with aging—some of the fibers or some areas in these fibers showed decreased activity (see Fig. 2.12). This irregular distribution of oxidative enzymes and focal decrease of activity has been described as target-fiber, targetoid-fiber, moth eaten-fiber, central core disease, and multi-core disease in various muscular diseases, myopathies, dystrophies, neurogenic atrophy, and hormonal diseases—all in which the above described ultrastructural alterations were observed. These morphological and enzyme-activity alterations together suggest an age-related decline in metabolic activity. From a pathophysiologic point of view, these findings can hardly be interpreted. We suppose that an increase in number of mitochondria, enlargement of mitochondria, and proliferation of cristae are morphologic manifestations of a hypermetabolic state, in which myofiber structure is usually well preserved, while loss of cristae and appearance of osmiophilic or paracrystalline inclusions are signs of mitochondrial dysfunctions, which is more frequently associated with myofiber abnormalities. Early electron microscopic studies of skeletal muscle cells of young and old humans showed the cristae of mitochondria became irregularly spaced, disrupted, and replaced by lamellar, myelin-like structures. Giant mitochondria were often visible. They contained lipofuscin in the myofibrils, too, which was often in close relationship with the damaged mitochondria.34 A substantial fall in mitochondrial oxidative capacity was also observed in ageing muscles.35

Fig. 2.12 Irregular distribution of the succino-dehydrogenase activity in aged muscle in the transversal section (71 yrs, x200)

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Biopsies of skeletal muscle from 2- to 39-year old rhesus monkeys showed that the number of individual fibers containing electron transport chain abnormalities (predominately negative for cytochrome c oxidase activity and/or hyper-reactive for succinate dehydrogenase) increased with age. These alterations associated with the deletions of the mitochondrial genome were observed in 89 percent of these electron transport chain abnormal fibers.36 The correlation between these two findings, however, remains unclear. Mitochondrial volume density was significantly lower in elderly compared with adult muscle, and these alterations were accompanied by a 50 percent reduction in oxidative capacity in the elderly vs. the adult group. In addition,. elderly subjects had nearly 50 percent lower oxidative capacity per volume of muscle than adult subjects. The cellular basis of this drop was a reduction in mitochondrial content, as well as a lower oxidative capacity of the mitochondria with age.37 In sections of orbicular muscle from aged patients, intramitochondrial inclusions of different sizes can be seen in addition to numerous morphologically abnormal and enlarged mitochondria. The formation of inclusions is always preceded by marked changes in cristae membrane disposition, with these membranes often taking a concentric arrangement. The question as to whether lipid peroxidation—presumed to be increased in patients with mitochondrial myopathies—is involved in this reorganization of the membrane system remains to be answered.

Sarcoplasma Age-related alterations of the orbicular muscle also comprise accumulation of tubular structures called tubular aggregates (TA) in the subsarcolemmal region (see Figs. 2.13 and 2.14). These densely packed tubular or tubulo-vesicular structures were apparently derived from the sarcoplasmic reticulum (SR) because it was clearly seen in some of our electron microscopic pictures, thus confirming the previous observations coming from other laboratories. The SR is an internal membrane system of the striated muscle. SR is a type of smooth endoplasmic reticulum specially adapted to surround the myofibrils, and it forms triads with invaginations of the plasma membrane called T-tubules. The sarcoplasmic reticulum contains large stores of calcium that it sequesters and then releases when the cells become depolarized. This has the effect of triggering a muscle contraction.38 Activation of muscle contraction is a rapid event that is initiated by electrical activity in the surface membrane and T-tubules. This is followed by release of calcium from the SR. Relaxation is mediated by the transport of calcium into the lumen of SR by a Ca-ATPase. Calcium then binds to calsequestrin in the lumen of the SR. For the initiation of contraction, calcium is released through the calcium channels or ryanodine receptors, which are under regulation by junctin, triadin, and calsequestrin. Thus, the SR is the major regulator of Ca2+-handling and contractility in muscles.39 It was later found that all types of cells contain cell-specific forms and/or analogues of these three proteins that are responsible for handling calcium. In all cells, these


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Fig. 2.13 Tubular aggregates. Transversal section of muscle fibers (left) and tightly packed tubules in the subsarcolemmal area (right). They show nearly identical diameter. Glycogen particles appear as electrodense granules between myofibers and tubules (x32 K, 65 yrs)

Fig. 2.14 Tubular aggregates. Transversal section of tubules in the subsarcolemmal area with numerous glycogen particles between them. Mitochondria show severe alterations, loss and/or proliferation of cristae, as well as type II crystal inclusion in some of them (x27 K, 67 yrs)

proteins tend to be grouped together. In fact, the SR is an extensive and specialized form of endoplasmic reticulum (ER) in muscle cells. Conversely, all cells contain SR-like specialized domains, but in much smaller amounts. Tubular aggregates are the most common alterations of the SR observed in various pathological conditions, but also in apparently healthy persons. Tubular aggregates

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are ultrastructural abnormalities characterized by the accumulation of densely packed tubules in skeletal muscle fibres, usually beneath the sarcoplasma membrane and rarely between the myofibers. In human skeletal muscle, they are especially rich in patients suffering from tubular aggregate myopathy. We found various forms of tubular aggregates in normal aged orbicular muscle. Tubular aggregates are characterized as more or less densely packed aggregates of vesicular or tubular membranes of variable forms and sizes that may contain amorphous material, filaments, or inner tubules. Various types of tubular aggregates were reported—namely, proliferating terminal cisterns, vesicular membrane collections either with double-walled tubules or with single-walled tubules, aggregates of dilated tubules with inner tubules, aggregates of tubulo-filamentous structures, and filamentous tubules.40 We have also observed tubulo-reticular structures (see Fig. 2.15). Tubular aggregates were immunopositive for the ryanodine receptor (RYR 1) of the SR, the SR Ca2+ pump (SERCA2-ATPase), and the intraluminal SR Ca2+ binding protein calsequestrin, indicating an SR origin of these aggregates. All of these proteins, calsequestrin, RyR, triadin, SERCAs, and sarcalumenin are involved in calcium uptake, storage, and release. These findings support the hypothesis that tubular aggregates form a tubular arrangement of a complete SR containing the junctional, cisternae, and longitudinal components of SR implicated in calcium homeostasis.41,42 They also showed decreased respiratory chain enzyme complex I and complex IV activity. These findings indicate a functional link between mitochondrial dysfunction and the presence of TAs originating from the SR.43 Lipid composition of TAs showed that the predominant lipid of the aggregates is an acetone and alcohol-soluble acidic phospholipid containing a high proportion of plasmalogens and unsaturated fatty acids—a pattern compatible with the lipid composition of SR and mitochondrial membranes in skeletal muscle.

Fig. 2.15 Tubulo-reticular aggregates. Honeycomb appearance of tubular aggregates and a severely altered mitochondria can be seen in the subsarcolemmal area (x50 K, 74 yrs)


25

In mouse models of skeletal muscle aging, a persistent decrease in the expression of the calcium binding protein calreticulin, as well as a continuous increase in calsequestrin-like protein expression, both appear unrelated to the tubular aggregate formation.44 In vitro studies using SR-enriched membrane vesicles isolated from the slow-twitch soleus muscle, and the relatively fast-twitch gastrocnemius muscle isolated from adult and aged rats, showed muscle-specific impairment in SR Ca2+ pump function and skeletal muscle contractile properties, which may contribute to the age-associated slowing of relaxation in the soleus muscle.45 An impairment of the mechanisms controlling the release of calcium from internal stores (excitation-contraction coupling) has been proposed to contribute to the age-related decline of muscle performance that accompanies aging. Excitation-contraction coupling in muscle fibers occurs at the junctions between sarcoplasmic reticulum and transverse tubules, in structures called calcium release units. Recent studies showed significant alterations in the calcium release unit morphology and cellular disposition, and a significant decrease in their frequency between control and aged samples. These data indicate that in aging humans, the excitation relaxation coupling apparatus undergoes a partial disarrangement and a spatial reorganization that could interfere with an efficient delivery of Ca2+ response to the contractile proteins.46 TAs should be seen as dynamic structures that commence and cease, progress and retreat, and change their structure, functionality, and composition under multifactorial, yet not well-defined influences. The structural and functional development of tubular aggregates remains also unknown. Tubular aggregates frequently occur with mitochondrial alterations supported by growing evidence of participation of mitochondria in the development of TAs. Factors affecting formation of TAs in skeletal muscle fibers may, however, have different structural and/or functional influences on other cell types. Sarcoplasmic inclusions were also observed in the sarcoplasma of orbicular muscle. They showed filamentary, paracrystalline, or fingerprint structure and usually located beneath the plasma membrane.(see Figs. 2.16, 2.17, 2.18, and 2.19). The origin and pathological significance of these sarcolemmal inclusions are mostly unknown. They may come from abnormal SR and mitochondria, or from both. They may also be signs of degeneration or regeneration, or both. Most probably, they are identical with the desmin-containing sarcolemmal inclusions described in other instances. Desmin is an intermediate filament protein that, in striated muscle, is normally located at Z-line, beneath the sarcolemma, and prominently at neuromuscular junctions. It is abundant during myogenesis and in regenerating fibers, but decreases in amount with maturation. Desmin is coexpressed with vimentin in regenerating and denervated muscle fibers. Aggregates of desmin occur as nonspecific cytoplasmic bodies similar to the aggregates of keratin filaments in Mallory bodies, or the neurofilament aggregates in Lewy bodies. There are now increasing numbers of neuromuscular disorders in which abnormal amounts of desmin are in myopathic muscle fibers.47 Myofibrillar, or desmin-related, myopathies encompass neuromuscular disorders with abnormal deposits of desmin and myofibrillar alterations. In a recent case report on three unrelated patients presenting with proximal and distal myopathy, muscle biopsies shared sarco-

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Fig. 2.16 Fingerprint inclusion. Typical electron microscopic feature of a fingerprint inclusion in the subsarcolemmal area. Note the paucity of other organelles (x22 K, 77 yrs)

Fig. 2.17 Filamentary sarcoplasmic inclusion. This inclusion is located in the subsarcolemmal area, formed by numerous thin filaments without any apparent substructure, and surrounded by numerous mitochondria with well-preserved cristae. However, some electodense granules—presumably lipofuscin—can be seen (x22 K, 71 yrs)

plasmic inclusions—either plaque-like or amorphous, strongly immunoreacted on dystrophin, and variably for desmin, alphaB crystalline, and ubiquitin. In addition, cyclin-dependent kinases were overexpressed in affected fibers. In conclusion, myofibrillar destruction occurs in heterogeneous conditions and may overlap with features of inclusion body myopathy and mitochondrial myopathy


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Fig. 2.18 Paracrystalline sarcoplasmic inclusion. This inclusion is located in the unusually empty subsarcolemmal area, formed by tightly packed, electrodense filaments showing paracrystalline substructure, and surrounded by fine granular material. Some mitochondria with normal cristae can also be seen (x22 Km 67 yrs)

Fig. 2.19 Nuclear inclusions. Two intranuclear inclusions are surrounded by nuclear membrane, suggesting their location in the invaginations of the nucleus (i.e., pseudoinclusions). They contain heterogeneous materials (amorphous, granular, fibrillary, and membraneous). Similar materials can also be seen in the surrounding subsarcolemmal area (x26 K, 65 yrs)

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and with tubulo-filamentous inclusions and sarcolemmal inclusions.48,49 Now, we may also add aging, which may be associated with similar alterations of myofibers, mitochondria, and sarcolemma. We considered alterations of the muscle caused by various genetic and acquired factors as elementary.

Connective Tissue Changes The extracellular matrix (ECM) consists of a variety of substances, of which collagen fibrils and proteoglycans (PG) are ubiquitous. In addition to the PG, the hydrophilic ECM includes a variety of other proteins such as noncollagen glycoproteins and lipids. It is known that the force transmission of the muscle-tendon complex is dependent on the structural integrity between individual muscle fibers and the ECM as well as the fibrillar arrangement of the tendon and its allowance for absorption and loading of contraction-generated energy. Furthermore, it is well-described that the tensile strength of the matrix is based on intra- and intermolecular crosslinks, and the orientation, density, and length of both the collagen fibrils and fibers. The signals triggering the connective tissue cells in response to mechanical loading, however, and the subsequent expression, synthesis, and turnover of specific ECM components—as well as its coupling to the mechanical function of the tissue—are only partly described. Intramuscular connective tissue accounts for one to ten percent of the skeletal muscle and varies quite substantially between muscles. Based on their localization and organization, three types are distinguished: 1) endomysium encloses each individual muscle fiber with a random arrangement of collagen fibrils to allow for movement during contraction, 2) the multisheet-layered perimysium is multisheetlayered and runs transversely to fibers and holds groups of fibers in place, and finally 3) epimysium is formed from two layers of wavy collagen fibrils to form a sheet-like structure at the surface of the tendon. Intramuscular connective tissue has several functions: a) it provides a basic mechanical support for vessels and nerves, b) the connective tissue ensures the passive elastic response of muscle, and c) it contribute to the force transmission from the muscle fibers to tendons and subsequently to bone. The perimysium is especially capable of transmitting tensile force. Up to seven collagen types have been identified in intramuscular connective tissue. The fibrillar collagen type I (from 30% and up to 97% of total collagen) and III (and to some extent type V) dominates the epi-, peri-, and endomysium, but type IV dominates the basement membrane adjacent to the plasma membrane of the sarcolemma.50 In orbicular muscle, an age-related decrease in the diameter of muscle fibers seen in electron microscopy was accompanied with a significant increase in density of interstitial connective tissue. This was particularly evident in transversal sections of orbicular muscle (see Fig. 2.20). Detailed histo-chemical analysis of connective tissue changes by means of polarization microscopy revealed two types of changes: 1) an increase of collagen fibers,


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Fig. 2.20 Transversal section of the interstitial connective tissue of orbicular muscle stained with phenol and seen in polarization microscopy. Thinner septa in a 61-year old patient (A) is compared to those of a 80-year old patient (B) (x200)

and 2) an increase of glycosaminoglycan (GAG) content of fibers. In other words, more glycated fibers accumulated in the interstitial connective tissue (see Fig. 2.21). These observations on the aging of intramuscular connective tissue in orbicular muscle are in full accordance with the previous data that—with aging the nonspecific cross-linking mediated by condensation of a reducing sugar with an amino group—result in accumulation of advanced glycation end products (AGEs) in the connective tissue.51 The accumulation of AGEs with aging thus indicates a stiffer and more load-resistant tendon and intramuscular ECM structure. On the other hand, it reduces the ability to adapt to altered loading because the turnover rate of collagen is markedly reduced. Furthermore, AGEs upregulate connective tissue growth factors in fibroblasts that therefore favor the formation of fibrosis over time in elderly individuals and patients with diabetes.52 Besides reduced physical activity, diet with low albumin concentration may be a risk factor for both muscular loss and connective tissue changes.53 These age-related alterations of the connective tissue may also contribute to the age-related malposition of the eyelid, particularly in senile entropion and ectropion. In these diseases, muscular alterations were always associated with relaxation of ligaments and other connective tissue structures. There is increasing evidence that changes in quantity and quality of intermuscular connective tissue due to aging may influence at least two different types of muscle function. First, partial replacement of contractile muscle fibers with connective tissue essentially modifies muscle contraction-relaxation. This alone may explain

30


Fig. 2.21 Transversal section of the interstitial connective tissue of orbicular muscle stained with blue toluidine and seen by polarization microscopy. With aging, an evident increase of collagen density can be seen when comparing specimen A (61 yrs) and B (80 yrs) (x200)

the age-related decline of muscle function and is essentially similar to those seen in various forms of congenital or acquired myopathies. Second, changes in the interstitial connective tissue also influence metabolic exchange between capillaries and muscle cells, as well as modulate neurotransmission. These changes, may also be indirectly responsible for impaired function of aged muscle.

References 1. Stefanyszyn MA, Hidayat AA, Flanagan JC (1985) The histopathology of involutional ectropion. Ophthalmology Jan. 92(1):120-7 2. Feher J (1977) Myofibre abnormalities of orbicular muscle in malposition of the eyelid. Acta Morphol Acad Sci Hung. 25(4):205-18 3. Manners RM, Weller RO (1994) Histochemical staining of orbicularis oculi muscle in ectropion and entropion. Eye. 8 ( Pt 3):332-5 4. Radnot M (1973) Mitochondrial crystals in muscles of a patient with spastic entropion. Am J Ophthalmol. Apr. 75(4):713-9 5. Radnot M, Follmann P (1974) Ultrastructural changes in senile atrophy of the orbicularis oculi muscle. Am J Ophthalmol. Oct. 78(4):689-99 6. Feher J (1978) Tubuloreticular structures in the orbicularis oculi muscle of the human eye. Acta Morph.Acad Sci Hung. 26:3-10 7. Sato T, Akatsuka H, Kito K, Tokoro Y, Tauchi H, Kato K (1986) Age changes of myofibrils of human minor pectoral muscle. Mech Ageing Dev. May 34(3):297-304


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8. Poggi P, Marchetti C, Scelsi R (1987) Automatic morphometric analysis of skeletal muscle fibers in the aging man. Anat Re. Jan. 217(1):30-4 9. Jakobsson F, Borg K, Edstrom L (1990) Fibre-type composition, structure and cytoskeletal protein location of fibres in anterior tibial muscle. Comparison between young adults and physically active aged humans. Acta Neuropathol (Berl). 80(5):459-68 10. Roth SM, Martel GF, Ivey FM, Lemmer JT, Metter EJ, Hurley BF, Rogers MA (2000) Skeletal muscle satellite cell populations in healthy young and older men and women. Anat Rec. Dec 1. 260(4):351-8 11. Beregi E, Regius O (1987) Comparative morphological study of age related mitochondrial changes of the lymphocytes and skeletal muscle cells. Acta Morphol Hung. 35(3-4):219-24 12. Fulle S, Belia S, Di Tano G (2005) Sarcopenia is more than a muscular deficit. Arch Ital Biol. Sep. 143(3-4):229-34 13. Francis IC, Stapleton F, Ehrmann K, Coroneo MT (2006) Lower eyelid tensometry in younger and older normal subjects. Eye. Feb. 20(2):166-72 14. van den Bosch WA, Leenders I, Mulder P (1999) Topographic anatomy of the eyelids, and the effects of sex and age. Br J Ophthalmol. Mar. 83(3):347-52 15. Sun WS, Baker RS, Chuke JC, Rouholiman BR, Hasan SA, Gaza W, Stava MW, Porter JD (1997) Age-related changes in human blinks. Passive and active changes in eyelid kinematics. Invest Ophthalmol Vis Sci. Jan. 38(1):92-9 16. Besne I, Descombes C, Breton L (2002) .Effect of age and anatomical site on density of sensory innervation in human epidermis. Arch Dermatol. Nov. 138(11):1445-50 17. Peshori KR, Schicatano EJ, Gopalaswamy R, Sahay E, Evinger C (2001) Aging of the trigeminal blink system. Exp Brain Res. Feb. 136(3):351-63 18. Weeks DA, Nixon RR, Kaimaktchiev V, Mierau GW (2003) Intranuclear rod myopathy, a rare and morphologically striking variant of nemaline rod myopathy. Ultrastruct Pathol. May-Jun. 27(3):151-4 19. Wallgren-Pettersson C, Jasani B, Newman GR, Morris GE, Jones S, Singhrao S, Clarke A, Virtanen I, Holmberg C, Rapola J (1995) Alpha-actinin in nemaline bodies in congenital nemaline myopathy: immunological confirmation by light and electron microscopy. Neuromuscul Disord. Mar. 5(2):93-104 20. Blanchard A, Ohanian V, Critchley D (1989) The structure and function of a-actinin. J Muscle Res Cell Motil 10:280-289 21. Schroder JM, Durling H, Laing N (2004) Actin myopathy with nemaline bodies, intranuclear rods, and a heterozygous mutation in ACTA1 (Asp154Asn). Acta Neuropathol (Berl). Sep. 108(3):250-6 [Epub 2004 Jun 24] 22. Ilkovski B, Cooper ST, Nowak K, Ryan MM, Yang N, Schnell C, Durling HJ, Roddick LG, Wilkinson I, Kornberg AJ, Collins KJ, Wallace G, Gunning P, Hardeman EC, Laing NG, North KN (2001) Nemaline Myopathy Caused by Mutations in the Muscle a-Skeletal-Actin Gene. Am J Hum Genet. Jun. 68(6):1333-43 [Epub 2001 Apr 27] 23. Ryan MM, Ilkovski B, Strickland CD, Schnell C, Sanoudou D, Midgett C, Houston R, Muirhead D, Dennett X, Shield LK, De Girolami U, Iannaccone ST, Laing NG, North KN, Beggs AH (2003) Clinical course correlates poorly with muscle pathology in nemaline myopathy. Neurology. Feb 25. 60(4):665-73 24. Michele DE, Albayya FP, Metzger JM (1999) A nemaline myopathy mutation in alphatropomyosin causes defective regulation of striated muscle force production. J Clin Invest. Dec. 104(11):1575-81 25. Sanoudou D, Corbett MA, Han M, Ghoddusi M, Nguyen MA, Vlahovich N, Hardeman EC, Beggs AH (2006) Skeletal muscle repair in a mouse model of nemaline myopathy. Hum Mol Genet. Sep 1. 15(17):2603-12 [Epub 2006 Jul 28] 26. Chahin N, Selcen D, Engel AG (2005) Sporadic late onset nemaline myopathy. Neurology. Oct 25. 65(8):1158-64 [Epub 2005 Sep 7] 27. Oumi M, Miyoshi M, Yamamoto T (2000) The ultrastructure of skeletal and smooth muscle in experimental protein malnutrition in rats fed a low protein diet. Arch Histol Cytol. 63(5):451-7

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28. Oumi M, Miyoshi M, Yamamoto T (2001) Ultrastructural changes and glutathione depletion in the skeletal muscle induced by protein malnutrition. Ultrastruct Pathol. Nov-Dec. 25(6):431-6 29. Hikida RS, Staron RS, Hagerman FC, Sherman WM, Costill DL (1983) Muscle fiber necrosis associated with human marathon runners. J Neurol Sci. May 59(2):185-203 30. Farrants GW, Hovmoller S, Stadhouders AM (1988) Two types of mitochondrial crystals in diseased human skeletal muscle fibers. Muscle Nerve. Jan. 11(1):45-55 31. Schnyder T, Winkler H, Gross H, Eppenberger HM, Wallimann T (1991) Crystallization of mitochondrial creatine kinase. Growing of large protein crystals and electron microscopic investigation of microcrystals consisting of octamers. J Biol Chem. Mar 15. 266(8):5318-22 32. Hanzlikova V, and Schiaffino S (1977) Mitochondrial changes in ischemic skeletal muscle. J. Ultrastuct. Res. 60:121-133 33. Speer O, Back N, Buerklen T, Brdiczka D, Koretsky A, Wallimann T, Eriksson O (2005) Octameric mitochondrial creatine kinase induces and stabilizes contact sites between the inner and outer membrane. Biochem J. Jan 15. 385(Pt 2):445-50 34. Beregi E, Regius O, Huttl T, Gobl Z (1988) Age-related changes in the skeletal muscle cells. Z Gerontol. Mar-Apr. 21(2):83-6 35. Trounce I, Byrne E, Marzuki S (1989) Decline in skeletal muscle mitochondrial respiratory chain function: possible factor in ageing. Lancet. Mar 25. 1(8639):637-9 36. Lee CM, Lopez ME, Weindruch R, Aiken JM (1998) Association of age-related mitochondrial abnormalities with skeletal muscle fiber atrophy. Free Radic Biol Med. Nov 15. 25(8):964-72 37. Conley KE, Jubrias SA, Esselman PC (2000) Oxidative capacity and ageing in human muscle. J Physiol. Jul 1. 526 Pt 1:203-10 38. Toyoshima C, Nakasako M, Nomura H, Ogawa H (2000) Crystal structure of the calcium pump of sarcoplasmic reticulum at 2.6A resolution. Nature. June 8. 405(6787):647-55 39. Franzini-Armstrong, C (1999) The sarcoplasmic reticulum and the control of muscle contraction. FASEB J. 13 (Suppl.), S266-S270 40. Pavlovicova M, Novotova M, Zahradnik I (2003) Structure and composition of tubular aggregates of skeletal muscle fibres. Gen Physiol Biophys. Dec. 22(4):425-40 41. Chevessier F, Marty I, Paturneau-Jouas M, Hantai D, Verdiere-Sahuque M (2004) Tubular aggregates are from whole sarcoplasmic reticulum origin: alterations in calcium binding protein expression in mouse skeletal muscle during aging. Neuromuscul Disord. Mar. 14(3):208-16 42. Chevessier F, Bauche-Godard S, Leroy JP, Koenig J, Paturneau-Jouas M, Eymard B, Hantai D, Verdiere-Sahuque M (2005) The origin of tubular aggregates in human myopathies. J Pathol. Nov. 207(3):313-23 43. Vielhaber S, Schroder R, Winkler K, Weis S, Sailer M, Feistner H, Heinze HJ, Schroder JM, Kunz WS (2001) Defective mitochondrial oxidative phosphorylation in myopathies with tubular aggregates originating from sarcoplasmic reticulum. J Neuropathol Exp Neurol. Nov. 60(11):1032-40 44. Chevessier F, Marty I, Paturneau-Jouas M, Hantai D, Verdiere-Sahuque M (2004) Tubular aggregates are from whole sarcoplasmic reticulum origin: alterations in calcium binding protein expression in mouse skeletal muscle during aging. Neuromuscul Disord. Mar. 14(3):208-16 45. Narayanan N, Jones DL, Xu A, Yu JC (1996) Effects of aging on sarcoplasmic reticulum function and contraction duration in skeletal muscles of the rat. Am J Physiol. Oct. 271(4 Pt 1): C1032-40 46. Boncompagni S, d’Amelio L, Fulle S, Fano G, Protasi F (2006) Progressive disorganization of the excitation-contraction coupling apparatus in aging human skeletal muscle as revealed by electron microscopy: a possible role in the decline of muscle performance. J Gerontol A Biol Sci Med Sci. Oct. 61(10):995-1008 47. Goebel HH, Bornemann A (1993) Desmin pathology in neuromuscular diseases. Virchows Arch B Cell Pathol Incl Mol Pathol. 64(3):127-35


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48. Wanschit J, Nakano S, Goudeau B, Strobel T, Rinner W, Wimmer G, Resch H, Jaksch M, Akiguchi I, Vicart P, Budka H (2002) Myofibrillar (desmin-related) myopathy: clinico-pathological spectrum in 3 cases and review of the literature. Clin Neuropathol. Sep-Oct. 21(5):220-31 49. Stojkovic T, Maurage CA, Moerman A, Hurtevent JF, Krivosic-Horber R, Pellissier JF, Vermersch P (2001) Congenital myopathy with central cores and fingerprint bodies in association with malignant hyperthermia susceptibility. Neuromuscul Disord. Sep. 11(6-7):538-41 50. Kjaer M (2004) Role of extracellular matrix in adaptation of tendon and skeletal muscle to mechanical loading. Physiol Rev. Apr. 84(2):649-98 51. DeBacker CM, Putterman AM, Zhou L, Holck DE, Dutton JJ (1998) Age-related changes in type-I collagen synthesis in human eyelid skin. Ophthal Plast Reconstr Surg. Jan. 14(1):13-6 52. Twigg SM, Chen MM, Joly AH, Chakrapani SD, Tsubaki J, Kim H-S, Oh R, and Rosenfeld RG (2001) Advanced glycosylation end products up-regulate connective tissue growth factor (insulin-like growth factor binding protein related protein 2) in human fibroblasts: a potential mechanism for expansion of extracellular matrix in diabetes mellitus. Endocrinology 142:1760-1769 53. Visser M, Kritchevsky SB, Newman AB, Goodpaster BH, Tylavsky FA, Nevitt MC, Harris TB (2005) Lower serum albumin concentration and change in muscle mass: the Health, Aging and Body Composition Study. Am J Clin Nut. Sep. 82(3):531-7

Chapter 3

Aging Effects on the Optics of the Eye Pablo Artal, MD, PhD

Abstract Different factors contribute to the increase in optical aberrations with age: possible modifications in the aberrations of the cornea, the lens, or even their relative contributions. The aberrations associated with the anterior surface of the cornea slightly change with age in a normal population, but the aberrations of the crystalline lens change due to the continuous modification of the lens shape with age. As the lens grows, its dimensions, curvatures, and refractive index change, altering the lens aberrations. Glasser and Campbell found a large change in the spherical aberration of excised older lenses measured in vitro. Another important factor to be considered is the nature of aberration coupling within the eye. It was shown that, in young subjects, the lens tends to compensate part of the corneal aberrations to produce an improved retinal image. As the aberrations of the lens change with age, it is quite plausible that this compensation is partially or completely lost. This explains the overall increase in aberration and the reduction of retinal image quality throughout the life span. This chapter will review the current ideas on the change of ocular aberrations with age and the possible impact this will have on the design of some ophthalmic devices, such as intraocular lenses. Keywords Optics of the eye, Increase in optical aberrations, Aging of the eye, Refractive index, Ophthalmic devices.

Introduction Normal aging affects the performance of visual system from many different aspects.1,2 For example, contrast sensitivity function (CSF) declines throughout the life span.3 This deterioration of spatial vision occurs for several reasons, ranging from purely optical degradation to retinal and neural losses. The relative contribution of optical and post-optical factors to this deterioration is not completely stated, although it is now accepted that optical factors play the major important role in normal eyes. In the aging eye, there is larger light absorption by the ocular media with different spectral contribution, a smaller pupil diameter (senile miosis), an increment of intraocular scattering, and a nearly complete reduction of the accommodation capability. In addition, Artal et al.4 first showed From: Aging Medicine: Age-Related Changes of the Human Eye Edited by C. A. P. Cavallotti and L. Cerulli © Humana Press, Totowa, NJ

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that the mean ocular modulation transfer function (MTF)—the ratio of contrast between object and image for a given spatial frequency—in a group of older subjects was lower than the average MTF for a group of younger subjects. This result, although it was obtained in a rather small population, suggested that the ocular aberrations—in addition to intraocular scattering—increase with age. Burton et al.5 compared CSFs obtained both with laser interferometry and conventional gratings also supporting the idea of a decreasing optical image quality with age. Measurements in a larger population showed a nearly linear decline of retinal image quality with age 6. This suggested a significant increase in the optical aberrations of the eye with age, in agreement with other studies in which aberrations were measured directly.7,8,9 In addition, intraocular scatter also increases noticeably in older eyes10 All this research indicates that the degradation in the quality of the retinal image in older eyes may play an important role in limiting spatial vision. Different factors contribute to this increase in optical aberrations with age: possible modifications in the aberrations of the cornea, the lens, or even their relative contributions. The aberrations associated with the anterior surface of the cornea slightly change with age in a normal population,11 but the aberrations of the crystalline lens change due to the continuous modification of the lens shape with age. As the lens grows, its dimensions, curvatures, and refractive index change, altering the lens aberrations. Glasser and Campbell found a large change in the spherical aberration of excised older lenses measured in vitro.12,13,14 Another important factor to be considered is the nature of aberration coupling within the eye. It was shown that, in young subjects, the lens tends to compensate part of the corneal aberrations to produce an improved retinal image.13,14 As the aberrations of the lens change with age, it is quite plausible that this compensation is partially or completely lost. This explains the overall increase in aberration and the reduction of retinal image quality throughout the life span.15 In this chapter, I will review the current ideas on the change of ocular aberrations with age and the possible impact this will have on the design of some ophthalmic devices, such as intraocular lenses. The optical performance of the eye can be measured by different, and in most cases complementary, procedures. By direct recording of the double-pass retinal image, 16,17,18 an overall estimate of the eye optics is obtained, usually expressed through the point-spread function (PSF) or the modulation transfer function (MTF). By using wave-front sensors,19,20,21,22, the optical aberrations of the whole eye are obtained and the retinal image or the MTF are calculated afterwards. Furthermore, by using computer ray-tracing techniques, the aberrations produced by the anterior surface of the cornea alone can be determined from the corneal shape.23 Finally, by comparing the corneal aberrations and the overall retinal image quality, it is possible to establish the relative contributions to aberrations of the different ocular elements. By combining these results with customized modeling of the eye, it is possible to predict the retinal images under many different conditions.24

Retinal Image Quality as a Function of Age Figure 3.1 shows the average double-pass images for three age groups obtained for a 4 mm pupil diameter.

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A direct comparison of the spread of these double-pass images shows a decrease of the optical performance with age. Fig. 3.2 presents the averaged MTFs for every age group, showing a consistent decline of the average MTF for the two older groups compared to the younger group. Figure 3.3 shows the Strehl ratio—an image-quality parameter—as a function of every subject’s age for the same 4 mm pupil diameter, together with a linear regression. It shows an approximately linear decline from 20- to 70-years old, on average (the slope and regression coefficient are -0.0032, 0.83). The previous results were obtained at best focus in every subject. It is quite common, however, that the presence of small refractive errors produces a slightly defocused retinal image. Fig. 3.4 presents the Strehl ratio for the three age groups at best focus and for 0.5 D defocus. This figure shows that the average retinal image quality declines more rapidly with age at best focus, while the reduction with age is less significant for small defocusing. This suggests that the older eye is more

Fig. 3.1 Average double-pass images in subjects of 20–30, 40–50, and 60–70 years of age with 4 mm diameter pupils

Fig. 3.2 Average MTFs for each age group: 20–30 (solid line), 40–50 (short dashed line), and 60–70 (long-dashed line) for 4 mm pupil diameter

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Fig. 3.3 Strehl ratio as a function of every subject’s age for 4 mm pupil diameter. The line is the best linear fitting

Fig. 3.4 Average Strehl ratio for the three age groups, with 4 mm pupil diameter at best focus and at 0.5 D defocus. Error bars indicate the standard deviation

tolerant to small amounts of defocusing. This is quite consistent with the accepted idea of a smaller reduction in image quality with defocusing in systems with reduced overall performance. That is, possible small refractive errors should reduce the MTF relatively more in a younger subject (with good image quality at best focus) than in an older subject (with poorer image quality at best focus). In addition, due to the effect of senile miosis—a smaller pupil size for similar luminance levels in older subjects—the average MTF is approximately constant over time at low luminance levels with natural pupils. These two factors—senile miosis and a better tolerance of defocusing in the older subjects—indicate that the differences in image quality among young and


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older subjects, found under controlled laboratory conditions at fixed pupil diameter and at best focus, will be smaller under normal viewing conditions, especially at low luminance. These two mechanisms may play a protective role against the increases of ocular aberrations with age.

Aberrations of the Eye as a Function of Age The previous results using the double-pass apparatus suggested an increase of aberrations with age. This has been confirmed by measuring wave-aberrations using the Hartmann-Shack wavefront sensor. Fig. 3.5 shows, as an example, wave-aberrations and their associated point-spread functions (PSFs) for one group of young (left panels) and one group of older (right panels) subjects. Fig. 3.6 shows the magnitude of total aberrations (represented as the RMS of the wave-aberration) for the complete eye as a function of the age of the subjects. The pupil size was 5.9 mm and defocus and astigmatism—i.e., sphere and cylinder—were not included in the RMS calculations, because they are normally corrected with spectacles. The magnitude of the aberrations is well correlated with age, although there is variability within the older individuals. In the older subjects, even though the error bars are

Fig. 3.5 Wave-aberrations with associated point spread functions (PSF) for one normal young subject and one normal old subject

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Fig. 3.6 RMS of the aberrations of the eye expressed in microns as a function of age (5.9 mm pupil diameter; defocus and astigmatism not included)

Fig. 3.7 RMS of the aberrations of the cornea expressed in microns as a function of age. Larger symbols with error bars indicate mean values and standard deviations for each age group

generally larger, they are still much smaller than the differences found when comparing the old with the young subjects. On average, we can expect an increase of 0.01 microns of aberration per year.

Aberrations of Cornea as a Function of Age The shape and aberrations of the cornea change with age. It is well known that the radius of curvature slightly decreases with age, and the asphericity also changes. On average, the cornea becomes more spherical with age and, as a consequence, spherical aberrations tend to increase. Fig. 3.7 shows the RMS of the wave aberration of the


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cornea in microns for a 4-mm diameter pupil, again as a function of the age of the subjects. Although there is an increase in the aberrations of the cornea, it is small and cannot completely explain the decrease observed in the optical performance of the complete eye with age. This fact leads us to another very important issue. How does the relationship between the aberrations of the cornea and the internal surfaces of the eye—and, in particular, the lens—change with age? In other words, are the increases in the aberrations of the whole eye with age due to an increase of the aberrations of each ocular component, or rather to progressive decoupling of the aberrations? We have extensively addressed this question in several studies by comparing both corneal and ocular aberrations as a function of age.

Coupling of Corneal and Internal Aberrations as a Function of Age The amount of aberrations for both the cornea and internal optics was found to be larger than for the complete eye in young subjects, indicating a significant role of the internal ocular optics to compensate for the corneal aberrations producing an improved retinal image. During normal aging, the relatively small corneal changes can not account for the degradation in retinal image. The lens changes both its shape and the effective refractive index dramatically with age, however, and its aberrations as a consequence. In this context, it seems possible that, at least in part, the increase in aberrations of the eye with age could be due to the loss of the aberration balance between cornea and lens that seems to be present in the younger eye. Figure 3.8 shows both the RMS of the wave-aberration for the complete eye and for the anterior surface of the cornea. Corneal aberrations in the younger subjects are larger than the total ocular aberrations, indicating that the internal optics compensates for the corneal aberrations. The opposite occurs in older subjects, however, where the cornea has lower aberrations than the complete eye. This indicates that the lenses in the older eyes do not compensate, but in fact add aberrations to those of the cornea. As an example, Fig. 3.9 shows examples of wave-aberrations for the cornea, internal surfaces and the eye—for a typical young eye (upper plots) and for a typical old eye (bottom). In the young eye, the cornea and the internal optics aberrations have a similar magnitude and shape, but are opposite in sign, producing an eye with overall lower aberrations. However, in the older eye, this finely tuned compensation is not present.

Optics of the Aging Eye and Intraocular Lenses This better understanding of the ocular optics in the aging eye was used for designing new and more effective ophthalmic optics. For instance, the ideal substitute for the natural lens in a cataract eye is not an intraocular lens with the best isolated

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Fig. 3.8 RMS of the aberrations of the eye (squares) and the cornea (circles) as a function of age

Fig. 3.9 Examples of wave aberrations for the anterior corneal surface, the internal surface, and the complete eye in a young and older eye, respectively

optical performance, but rather one designed to compensate for the aberrations of the cornea (see schematic example in Fig. 3.10). An improved design for an intra-ocular lens would have an aberration profile that compensates (at least partially) for the corneal aberrations in the older eye, to maximize the quality of the retinal image. A first approximation is the use of aspheric intraocular lenses to correct for the corneal spherical aberration. Other type of lenses—for instance, correcting corneal coma—have also been proposed.25


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Fig. 3.10 Schematic representation of the coupling of the cornea and an intraocular lens. A lens without aberrations will produce an eye with the aberrations of the cornea and relatively poor retinal images. However, a lens with aberrations approximately contrary to those of the cornea will produce an eye nearly free of aberrations

In the future, it may also be possible to have customized lenses correcting most corneal aberrations in situ and maximizing retinal image quality. In addition to aberrations, other optical (mainly intraocular scatter) and post-optical (neural) factors further influence visual performance of older subjects. All those factors have to be considered together when predicting or analyzing visual performance. Acknowledgments Part of the research described in this chapter has been supported by the Ministerio de Educación y Ciencia (MEC) in Spain, and by AMO_Groningen (The Netherlands). The author also wishes to thank all his collaborators in his laboratory at Murcia University and elsewhere who greatly contributed in many of the aspects of the research briefly described here.

References 1. Weale RA (1992) The senescence of human vision. Oxford University Press, Oxford 2. Owsley C, Sloane ME (1990) Vision and aging. In: Boller F, Grafman J (eds). Handbook of Neuropsychology, vol 4. Elsevier Science Publishers B.V. (Biomedical Division), pp 229-249 3. Owsley C, Sekuler R, and Siemsen D (1983) Contrast sensitivity throughout adulthood. Vision Res 23:689-699 4. Artal P, Ferro M, Miranda I, and Navarro R (1993) Effects of aging in retinal image quality. J. Opt. Soc. Am. A 10:1656-1662 5. Burton KB, Owsley C, Sloane ME (1993) Aging and neural spatial contrast sensitivity: photopic vision. Vision Res. 33:939-946

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6. Guirao A, González C, Redondo M, Geraghty E, Norrby S, and Artal P (1999) Average optical performance of the human eye as a function of age in a normal population. Invest. Ophthalmol. Vis. Sci. 40:197-202 7. Jenkins TCA (1963) Aberrations of the eye and their effects on vision: part 1.Brit. J. Physiol. Opt. 20:59-91 8. Calver R, Cox MJ, and Elliot DB (1999) Effect of aging on the monochromatic aberrations of the human eye. J. Opt. Soc. Am. A, 16(9):2069-2078 9. McLellan JS, Marcos S, and Burns SA (2001) Age-related changed in monochromatic wave aberrations of the human eye. Invest. Ophthalmol. Vis. Sci. 42:1390-1395 10. Ijspeert JK, de Waard PWT, van den Berg TJTP, and de Jong PTVM (1990) The intraocular straylight function in 129 healthy volunteers; dependence on angle, age and pigmentation. Vision Res. 36:699-707 11. Guirao A, Redondo M, and Artal P (2000) Optical aberrations of the human cornea as a function of age. J. Opt. Soc. Am. A. 17(10):1697-1702 12. Glasser A, and Campbell MCW (1998) Presbyopia and the optical changes in the human crystalline lens with age. Vision Res. 38:209-229 13. Artal P and Guirao A (1998) Contribution of the cornea and the lens to the aberrations of the human eye. Optics Letters 23:1713-1715 14. Artal P, Guirao A, Berrio E, and Williams, DR (2001) Compensation of corneal aberrations by the internal optics in the human eye. Journal of Vision, 1(1):1-8 15. Artal P, Berrio E, Guirao A, Piers P (2002) Contribution of the cornea and internal surfaces to the change of ocular aberrations with age. J. Opt. Soc. Am. A. 19:137-143 16. Santamaría J, Artal P, Bescós J (1987) Determination of the point-spread function of the human eye using a hybrid optical-digital method. J Opt Soc Am A. 4:1109-1114 17. Artal P, Marcos S, Navarro R, Williams DR (1995) Odd aberrations and double-pass measurements of retinal image quality. J Opt Soc Am A. 12:195-201 18. Díaz-Doutón F, Benito A, Pujol J, Arjona M, Güell JL, Artal P (2006) Comparison of the Retinal Image Quality with a Hartmann-Shack Wavefront Sensor and a Double-Pass Instrument. Invest. Ophthalmol. Vis. Sci. 47:1710-1716 19. Liang J, Grimm B, Goelz S, and Bille JF (1994) Objective measurement of the WA’s aberration of the human eye with the use of a Hartmann-Shack sensor. J. Opt. Soc. Am. A. 11:1949-1957 20. Liang J and Williams DR (1997) Aberrations and retinal image quality of the normal human eye. J. Opt. Soc. Am. A 14:2873-2883 21. Prieto PM, Vargas-Martín F, Goelz S, and Artal P (2000) Analysis of the performance of the Hartmann-Shack sensor in the human eye. J. Opt. Soc. Am. A. 17:1388-1398 22. Iglesias I, Berrio E and Artal P (1998) Estimates of the ocular wave aberration from pairs of double-pass retinal images. J. Opt. Soc. Am. A. 15:2466-2476 23. Guirao A and Artal P (2000) Corneal wave-aberration from videokeratography: accuracy and limitations of the procedure. J. Opt. Soc. Am. A. 17:955-965 24. Tabernero J, Piers P, Benito A, Redondo M and Artal P (2006) Predicting the optical performance of eyes implanted with IOLs to correct spherical aberration. Invest. Ophthalmol. Vis. Sci. 47:4651-4658 25. Tabernero J, Piers P and Artal P (2007) Intraocular lens to correct corneal coma. Opt. Lett. 32 (4):406-408

Chapter 4

Aging of the Cornea Luciano Cerulli, MD, PhD and Filippo Missiroli, MD

Abstract Unlike other ocular structures, as well as most tissues in the body, the cornea does not show important changes with normal aging. A variety of corneal aging changes have, however, been reported. Few of them are clinically evident, while others are demonstrated by chemical, biological, and structural studies. Distinction has to be made between conditions considered within the normal limits of aging and those of true disease processes that commonly affect the cornea in the elderly. The difference with other ocular structures is that changes of cornea due to aging are mostly asymptomatic and do not usually affect vision, hence they do not require treatment. However, some changes occur and, for example, the aged cornea becomes more susceptible to infection because of a decreased ability to resist a variety of physiological stresses. Furthermore, it is sometimes difficult to distinguish age specific deterioration from degenerations modified by environmental and genetic factors. The well-known clinical conditions that occur with age in the cornea will be described first. Then, a review of the effect of age on shape and different aspects of the cornea and its structural (anatomical) changes will be reported. Keywords Aging, cornea, corneal arcus, deep crocodile shagreen, astigmatism, corneal thickness, stroma, keratocyte, endothelium

Clinical Conditions Corneal Arcus Also known as gerontoxon is the most common bilateral manifestation of the aged cornea. It is characterized by a white ring around the peripheral cornea that is separated from the limbus by a clear zone 0.3 to 1 mm in width. It consists of deposits of cholesterol, cholesterol esters, phospholipids, and triglycerides. When it is seen in younger people, it is not an age-related condition (arcus senilis) but is in association with hyperlipoproteinemia types 2 and 3. Initially the superior and inferior peripheral

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L. Cerulli and F. Missiroli

Fig. 4.1 Peripheral ring of corneal arcus as seen at slit lamp

cornea is affected and, with time, a complete annulus forms. Slit lamp biomicroscopy reveals the extensive involvement of the subepithelial zone, the corneal stroma, and Descemet’s membrane, as well as fine opacities in the so-called lucid interval of Vogt which is optically clear on naked eye examination (see Fig. 4.1).1

Prevalence In one study, the estimated prevalence was measured at 8 percent for those 40 to 49 years of age, 45 percent for those 50 to 59 years of age, and 75 percent for those 70 to 79 years of age. In another study, the prevalence was measured at 6 to 12 percent in a cohort of insulin-dependent patients with diabetes who were less than 30 years of age, and was measured at 49 to 54 percent for patients with diabetes who were more than 30 years of age. The tendency toward increasing prevalence with age explains the usage of the popular nomenclature arcus senilis, instead of the more technically correct term corneal arcus. In general, ar cus senilis is more common in men than it is in women. It is also more common among patients of African descent, and when it occurs in these patients, it tends to occur earlier in life. Arcus senilis may also be more common in patients who regularly consume alcoholic beverages, with the prevalence in one study increasing as the amount of alcohol consumption increased.3,4,5

4 Aging of the Cornea

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Biochemical Aspects Palmitic, stearic, oleic, and linoleic acids are among the fatty acids that make up many of the deposited lipid molecules. Lipids are normally deposited in the cornea, but with aging, the amount of deposited lipids increases—in some cases it will result in arcus senilis. This supports the assumption that arcus senilis may represent an extension or exaggeration of the natural process of lipid deposition in the cornea. A structural study showed extracellular solid spherical lipid particles (< 200 nm in diameter) enmeshed between collagen fibers. Immunostaining showed significant apoE and apoA-I, but very little apoB in the peripheral cornea. Cholesteryl ester-rich spherical particles accumulate in the extracellular spaces of the peripheral cornea. Most of these lipid particles are 40-200 nm in diameter, and are therefore similar in size to one type of cholesteryl ester-rich lipid particle that accumulates in the extracellular spaces of human atherosclerotic lesions. These extracellular lipid droplets seem to derive from direct deposition of plasma lipoproteins.2

Deep Crocodile Shagreen Also known as mosaic degeneration, deep crocodile shagreen consists ofn bilateral, polygonal, grayish-white opacities that are interrupted by clear spaces that are usually asymptomatic (see Fig. 4.2). Two types of this condition exist:

Fig. 4.2 Both crocodile shagreen (white arrow) and limbal gridle of Vogt (black arrow)

48 ●

●


Anterior shagreen (Vogt’s anterior mosaic crocodile shagreen) are seen in the deep layers of the epithelium or in Bowman’s layer, and become more apparent after instillation of fluorescein stain Posterior shagreen are generally seen in the central deep cornea, which makes it difficult to differentiate with a central cloudy dystrophy of the cornea

Both anterior and posterior forms do not require treatment. Deep crocodile shagreen is sometimes seen in association with peripheral band keratopathy or following trauma.6 Familial type may occur with x-linked megalocornea or in a juvenile form of anterior mosaic crocodile shagreen.

White Limbal Gridle of Vogt Limbal gridle of Vogt is a very common, bilateral, age-related corneal degeneration. It is always asymptomatic and requires no therapy. It affects more than 50 percent of the population over age 40 and is characterized by a subepithelial degeneration and may include calcium deposits. The lesions look like white opacities of the peripheral cornea, forming a half moon-like arc running concentrically with the limbus—usually in the interpalpebral zone along the nasal and temporal limbus (only the horizontal meridian is affected). The opacities may be separated from the limbus by a clear zone of about 1 mm, or without a clear zone in between, and lie at the level of Bowman’s membrane and the immediately subjacent stroma. Histopathology of the lesions show a destruction of Bowman’s membrane and superficial lamellae of the stroma in association with deposition of calcium and areas of hyaline and elastotic degeneration with hypertrophy of the overlying epithelium.6

Hassall-Henle Bodies Also known as Hassall-Henle warts or Henle’s warts, these bodies are small hyaline excrescences on the posterior surface of Descemet’s membrane at the periphery of the cornea. Averaging 0.07 to 0.08 mm in diameter, they are constantly present in adults—occasionally they become confluent and macroscopically visible. They contain bounded material that is believed to be collagen, in which numerous cracks and fissures are filled with extrusions of the corneal epithelium. They represent an over activity of the formation of hyalin by the endothelial cells. They are found in large quantities in degenerations and chronic inflammations. When they become larger and more numerous, they invade the central area (cornea guttata). The condition is probably associated with the aging process.6,7


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Cornea Shape Astigmatism One of the most common ocular changes in the elderly is the variation of manifest refraction. Often this happens because a change in corneal curvature causes alteration in refraction—usually a change from the with-the-rule astigmatism to againstthe-rule astigmatism. The vertical meridian of the cornea is steeper than the horizontal meridian in with-the-rule astigmatism, so the eye has more refractive power (plus cylinder) along the vertical axis. In against-the-rule astigmatism, the horizontal meridian is steeper than the vertical and the eye has more refractive power (plus cylinder) along the horizontal axis. Several studies confirmed that a decrease in the vertex radius occurs with aging, thus demonstrating a steepening of the cornea. A Japanese study of 2,161 subjects found that the prevalence of astigmatism increases and the axis turns to againstthe-rule with age. The result of the linear regression analysis indicates that the age-related change in astigmatism is mainly associated with changes in the cornea.8 The corneal astigmatism was found to change in Hong Kong’s Chinese population, where both the corneal and spectacle astigmatism demonstrated a shift from with-the-rule to against-the-rule with age (keratometer and a computer-assisted videokeratoscope were used).9 Another Japanese study that used a autokeratometer revealed that the cylindrical diopter of with-the-rule astigmatism decreased, and against-the-rule astigmatism increased with aging.10 A study from Turkey suggested that the normal cornea becomes steeper in the horizontal line and superior vertical quadrant, and shifts from with-the-rule to against-the-rule astigmatism. The amount of physiological corneal astigmatism, however, does not change with age.11 A topographic analysis of the changes in corneal shape due to aging was carried out in 734 volunteers in Japan. The maps of subjects in their 70s and > 80 revealed a horizontal, oval-shaped steep area, suggesting against-the-rule astigmatism. The average-of-difference map demonstrated a marked corneal steepening at the horizontal meridians. In the data analysis of the averaged map, the mean refractive powers of the cornea increased with age.12 While corneal topography gives data of the anterior surface of the cornea, Scheimpflug photography is a useful tool with a non-contact technique that allows researchers to determine the shape and astigmatism of the posterior corneal surface. A study where Scheimpflug camera was used to measure the cornea of the right eye in six meridians of 114 subjects ranging in age from 18 to 65 years showed that, with aging, the asphericity of both the anterior and the posterior corneal surface changes significantly—a significant average change in the k value was found for the posterior surface, which indicates a shift to a more aspherical surface. The same study revealed that peripheral thickness along the perpendicular line at 3.75 mm from the apex showed an average difference of 19 micron between the young and the old subjects, while no difference in central corneal thickness was noted.13

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Corneal Thickness The thickness of corneal tissue is an important parameter in refractive surgery, and its measurement is essential in the assessment and management of corneal diseases. In the last few years, it became an important clinical parameter for correct interpretation of Goldmann applanation tonometry results. There are different instruments for determining corneal thickness, but the most widespread method currently in use is still ultrasonic pachymetry, even though it can be measured with other modalities—scanning-slit topography/pachymetry, specular and confocal microscopy, optical low-coherence reflectometry and rotating Scheimpflug camera. So far, ultrasonic pachymetry, scanning-slit topography/ pachymetry and rotating Scheimpflug cameras are considered more reliable tools for determining corneal thickness. In a recent study, Amano and others found that corneal thicknesses were comparable for the rotating Scheimpflug camera (Pentacam), ultrasonic pachymetry, and scanning-slit topography (Orbscan), with the acoustic equivalent correction factor. The measurements taken with the three instruments had significant linear correlations with one another, and all methods had highly satisfactory measurement repeatability.14 Several studies analyzed corneal thickness and its variation with aging, but the age-dependent difference in corneal thickness values remains unclear. Age-related changes in central and peripheral corneal thickness were analyzed by the mean of the Orbscan II topography system. A thinning of the peripheral cornea was noted with increasing age. It was found that the mean corneal thickness was reduced with age (0.38 mm/year), whereas the central corneal thickness was unaltered. These data concord with results of the previously mentioned study of Dubbelman obtained from the Scheimpflug camera.15 Rufer and others found slight variations in the mean central corneal thickness measured by the mean of Orbscan II system. In their work, no significant constant age-related trend was identified. However, there were raised values in the 50- to 59-year olds and 70- to 79-year olds when compared with all the other decades.16 In a Mongolian population where the central corneal thickness was measured using an optical pachymeter, there was a highly significant decrease in central corneal thickness with age—5 microns/decade in men, and 6 microns/decade in women.17 In a large series of eyes undergoing myopic refractive surgery, central corneal pachymetric measurements did not correlate with age.18 A large study where 1,699 Latino participants aged 40 or more years were included, central corneal thickness was measured by ultrasonic pachymeter. The most clinically significant finding was that when compared with normal Latinos aged 40 to 49 years, Latinos aged 70 or more years had substantially thinner corneas on average.19 Among participants of the European Glaucoma Prevention Study (EGPS), the central corneal thickness was higher in younger patients, male patients, and diabetic patients.20 In a study population of different races, where central corneal thickness was measured by ultrasound pachymetry, an inverse relationship between age and CCT


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was found, with a reduction of about 3 micron per decade.21 Sanchis-Gimeno and others have analyzed the changes of central corneal thickness values of Caucasian emmetropic subjects in accordance with their age—Caucasian emmetropic aged subjects have reduced corneal thickness values when compared to young emmetropic subjects.22 In conclusion, it seems there is no substantial change in corneal thickness during aging—nevertheless, there is evidence suggesting a significant age-dependent decrease in corneal thickness later in life for some ethnic groups.

Corneal Aberration Normally, the cornea has positive spherical aberrations that compensate for the negative aberrations attributed to the lens. Around 20 years of age, ocular spherical aberration is almost zero, and it gradually increases with age (positive values). This change in total spherical aberration with aging is because of aberration changes in the lens, which may be induced by the age-related changes of anterior and posterior lens radius.23,24,25 There is wide individual variability in anterior corneal aberrations, and little of this is attributable to age-related changes. Analyzing data from different studies, it is possible to see that while corneal spherical aberrations do not show change with aging, there is a positive correlation between corneal coma aberration with age and evidence that the increase of ocular coma with aging is mainly because of the increase of corneal coma.26 Because coma-like aberrations consist of tilt and/or asymmetry, the corneas become less symmetric with aging. Increases in corneal coma-like aberrations in elderly do not directly indicate the deterioration of visual function in their eyes for different reasons—one of these is that pupils of older subjects tend to be more miotic, thus with smaller influence of the corneal wavefront aberrations on visual performance.

Structure Epithelium The corneal epithelium acts as barrier from environmental agents and contributes to movement of water and molecules through the cornea. With age, this function seems to undergo some deterioration resulting in a breakdown of epithelial barrier function. This can be quantified by fluorophotometric determination of corneal epithelial permeability to fluorescein.27 A breakdown of epithelial barrier function28 and the increased tear contact time29 may explain the increase in epithelial permeability with age that renders the aging cornea more susceptible to infection. Changes in distribution of integrin subunits in the epithelium could also reduce the epithelial

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barrier function. The α6 subunit and the β4 subunit—components of hemidesmosomes—become discontinuous with age.30 A reduced ability of corneal cells to upregulate adhesion molecules, and a reduced phagocytic ability of reactive polymorphonucleocytes in response to infection, also occur with aging.31 This could impair the ability to eliminate a bacterial infection.

Bowman’s Membrane Bowman’s membrane (epithelial basement membrane) is the layer that separates the epithelium from the stroma. It is about 10-16 µm thick and is acellular, except for the nerves that perforate it. Taylor and Kimsey studied the basal membranes of corneas in 12 diabetic patients by transmission electron microscopy (TEM). They found no clear relation between the corneal epithelial basement membrane thickness and age.32 Alvarado et al. published an ultrastructural evaluation and morphometric analysis of the basement membrane on a large number of specimens from subjects ranging in age from 17 weeks of gestation to 93 years of age. They found that structural changes occur with aging in the basement membranes. There is a progressive thickening of the corneal epithelial basement membrane that is caused by two different processes—membrane deposition (forming unilaminar membranes) and membrane reduplication (forming multilaminar membranes). Membrane deposition appears to be the only process involved in membrane thickening in the prenatal and early postnatal period. Later in life, the process of membrane reduplication plays a more prominent role than thickening by deposition. In middle-aged individuals, areas of reduplication are focal (mixed membrane type). With increasing age, a greater proportion of the basement membrane becomes multilaminar.33

Subbasal Nerve Plexus The cornea is one of the most innervated tissues in the body. Its innervation is provided by the ophthalmic and maxillary branches of the trigeminal nerve. The nerve bundles enter the peripheral cornea at the limbus in the anterior third of the stroma, and then penetrate the Bowman’s layer where they form the subbasal nerve plexus. The fibers run parallel to the cornea’s surface between the Bowman’s layer and the basal epithelial layer, and then terminate in the superficial epithelium as free nerve endings. Because of the fast degeneration of nerve fibers after death, the morphology of the corneal nerve based on histological and microscopic studies is limited and unclear. In vivo confocal microscopy is able to visualize and measure subbasal nerve fibers. Confocal microscopy studies have shown that the human subbasal nerve plexus is primarily oriented in a superior-to-inferior direction at the central corneal apex.34


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Grupcheva et al. found a small age-associated decrease in subbasal nerve density in clinically healthy corneas.35 In a recent study, Erie also demonstrated that the density and orientation of the subbasal nerve plexus do not show changes with age.36 Despite this morphological observation, several studies demonstrated that corneal sensation decreases with age.37,38,39 Corneal sensitivity is important in the maintenance of function and structure of the cornea, and it is also crucial in the healing process after injury or surgery. Roszkowska evaluated central and peripheral corneal sensitivity in a population that ranged from 20 to 90 years of age. In this study, they found that corneal sensitivity is maintained throughout life—corneal sensitivity remains stable in the central zone until the age of 60 when it begins to decrease, while the peripheral sensitivity starts to decrease earlier and progresses at a fast rate.40

Stroma Transparency of the cornea is due to the uniform size of the constituent collagen fibrils and to the degree of ordering in their packing in the stroma. Changes in the corneal stromal structure occur with aging for both the collagen fibrils and the cellular component. Studies on these stromal changes were obtained from both in vivo and in vitro observations. Human collagen undergoes progressive changes with age, including a decrease in elasticity. In vitro studies suggest that the physical changes involve progressive crosslinking between collagen molecules. Daxer et al. performed x-ray scattering experiments on corneas of various ages to investigate the three-dimensional structural properties of collagen fibrils in the human corneal stroma. Analyzing fibril diameter, intermolecular Bragg spacing, and axial collagen period, they found that aging is related to a three-dimensional growth of collagen fibrils in the human corneal stroma. The age-related growth of the fibril diameter was mostly a result of an increased number of collagen molecules and, in addition, to some expansion of the intermolecular Bragg spacing, probably resulting from glycationinduced crosslinking.41 The expansion of the collagen intermolecular Bragg spacing within the fibrils suggests that molecules other than collagen are deposited in the fibrils during aging and push the collagen molecules further apart. It confirms recent studies that have demonstrated glycation-induced expansion of the intermolecular spacing and subsequent crosslinking of the molecules with age.42 Advanced glycation end products (AGEs) play a significant role in many age-related disorders. AGEs accumulate in the aging cornea and mediate crosslinking of molecules in the stroma. Such age-related crosslinking occurs largely on the collagen component of the cornea. Two changes with age were identified by Malik: 1) an increase in the cross-sectional area associated with each molecule in corneal collagen, which may be due to an increase in nonenzymatic crosslinking between collagen molecules, and 2) a decrease in the stroma interfibrillar spacing, which could be related to changes in the proteoglycan

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composition of the interfibrillar matrix. The decrease in stromal interfibrillar spacing with age was also found by Kanai and Kaufman using an electron microscopy study.43 These findings related to stromal fibrillar changes can, in part, explain the result obtained by Elsheikh et al. on corneal stiffness. They evaluated the stress-strain behavior of corneal tissue and how the behavior was affected by age. In this study, the cornea demonstrates considerable stiffening with age with the behavior closely fitting an exponential power function typical of collagenous tissue. The increase in stiffness could be related to the additional nonenzymatic crosslinking that affects the stromal collagen fibrils that occurs with age, and to the age-related increase in collagen fibril diameter.44 A new instrument that is starting to be used in clinical practice is the ocular response analyzer that measures the corneal biomechanical response to a rapid indentation obtained by an air jet. This corneal response is called corneal hysteresis (CH), and is a new parameter that can help to better understand the behavior of the cornea on the intraocular pressure measurement obtained by Goldmann applanation. Kotecha et al. investigated the association between CH and both age and central corneal thickness, as well as the agreement between ocular response analyzer and Goldmann applanation tonometer IOP measurements. Analyzing the data, they found a correction factor that describes a biomechanical property of the cornea that is independent from the intraocular pressure and that increases with thicker cornea and decreases with age. This factor is a measure of the corneal material properties, which include both stiffness and viscoelasticity. The observed negative association between corneal viscoelastic properties with advancing age may be further evidence of an increase in crosslinkage of collagen fibrils within the cornea, making it a stiffer and less viscoelastic structure.45 Keraticytes are the principal cellular components of corneal stroma—they are fibroblast-like cells that produce, degrade, and remodel the stroma, and are therefore important in corneal wound healing. Keraticyte density in a normal human anterior corneal stroma has been reported to be around 20.000–24.000 cells/mm3, being highest posterior to the Bowman’s layer and then decreasing towards posterior stroma (see Fig. 4.3).46,47 Using biochemical measurements of the stromal DNA/ mass content within the central 7-mm diameter zone, Møller-Pedersen48 found a direct correlation between keratocyte density and donor age, with a physiologic decline of 0.3 percent per year throughout life. Møller-Pedersen and Ehlers49 describe a 30 percent decrease in cell density in the subendothelial region. The DNA method is invasive and cannot be used to study keratocyte density in vivo. Recent use of Confocal microscopy in vivo allowed quantification of stromal cell density without the need of tissue processing that can alter the tissue. In their work, Patel and coworkers found that full-thickness central keratocyte density was negatively correlated with age and decreased 0.45 percent per year. Keratocyte densities in all anteroposterior regions were negatively correlated with age, except the posterior 67 to 90 percent region of the stroma. The number of keratocytes in


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Fig. 4.3 Confocal images of stroma: A) anterior stroma, and B) posterior stroma with different keratocyte density

the full-thickness stroma was also negatively correlated with age and decreased 0.43 percent per year.47 Using same modality with confocal microscopy, Berlau et al. found that keratocyte density was lower in patients older than 50 years than in those younger than 50 years.50

Descemet’s Membrane Changes in Descemet’s membrane with aging are well-characterized in humans. Before birth it is a very thin basement membrane and different in appearance from the adult Descemet’s membrane. It grows by deposition of a series of similar “membrane units,” which are stacked to form a lamellar structure consisting of at least 30 layers by the end of gestation. At birth, it has an average thickness of 3 µm and exhibits an electron-dense, banding pattern with 110 nm periodicity.51,52 This portion of Descemet’s membrane is referred to as the anterior banded zone. Over the ensuing decades of life, the anterior banded zone remains well-demarcated and stable in thickness and appearance. In postnatal life the membrane continues to grow in thickness by deposition of a non-striated, non-lamellar material posterior to the striated prenatal layer. This posterior portion of the membrane directly subjacent to the endothelium progressively thickens as a non-banded, homogeneous substance referred to as the posterior non-banded zone. Thickening of the posterior

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non-banded zone contributes to the age-dependent growth of Descemet’s membrane from approximately 3 µm at birth to 5-6 µm at 20 years of age and to 13 µm at 80 years of age.51,52

Endothelium The corneal endothelium and Descemet’s membrane play a vital role in maintaining corneal structure and clarity. The endothelium is a monolayer of hexagonal cells located on the posterior surface of the cornea, which functions to pump water out of the stroma to maintain transparency. (see Fig. 4.4) One of the most known effects of age in the human cornea is that endothelial cell density decreases progressively during aging, but its measurement is not a reliable index of the chronological age of the cornea because there is wide range of endothelial cell density in normal populations.52-61 In the absence of a proliferative response to cell loss, endothelial cover of the posterior corneal surface is maintained by a gradual increase in the size of the remaining cells, resulting in increased cellular pleomorphism and a decrease in the percentage of hexagonal cells with age. Endothelial cell analysis provides important clinical information on corneal function and viability. If we look at a picture of endothelial specular microscopy from a healthy young adult of 25-30 years of age, we can tell that the cells are uniform in their shape and size, but not as much as we can see from corneas aged less then 10 years, where the cells are much smaller and rounded. Specular microscopy of corneas older then 70 years of age shows much variety in the size and shape of cells. Fewer cells are exagonal, while the number of pentagonal, six-agonal, and seven-agonal cells increases significantly. Numerous studies were published on the reduction of endothelial cell density and the change in cellular morphology with age. They demonstrate in different races that cellular polymegethism and cellular pleomorphism increases with age.53-61 In 1976, Bourne and Kaufman documented a decrease in the number of central endothelial cells with age by using a clinical specular microscope. They found a rate of reduction of 0.39 percent per year, while Hollingsworth et al. calculated that endothelial cell density decreases at a rate of 0.33 percent per year. The estimated

Fig. 4.4 Specular microscopy image of an normal adult endothelium


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annual reduction found by Murphy et al. averages approximately 0.56 percent per year.57,58,59 Blatt et al. found with the clinical specular microscope that cell density decreases significantly with age when the endothelial cells are regular in size, but that if the endothelial cells are irregular in size and arrangement, the endothelial cell densities do not correlate with age.60 The topographical distribution of the corneal endothelial cell density in different age ranges has been studied by Roszkowska et al. They evaluated 300 eyes of 204 healthy subjects aged from 20 to 83 years. Age-related changes involve both center and periphery. In particular, they observed a higher peripheral decrement in the ancient subjects resulting in a topographical disparity in the elderly. They concluded that the central density evaluation is sufficient to provide the exact information about an entire endothelial surface, but only in the young subjects. It did not work with elderly patients where a topographical disparity might occur and the only central density determination could provide insufficient results.61 The reason why a gradual loss of endothelial cells occurs with age remains unclear. Green hypothesizes that aging processes in the eye occur as a consequence of degradation of enzymes that normally metabolize and detoxify hydrogen peroxide and other free radicals. The loss of enzyme activity allows hydrogen peroxide—which normally occurs within eye fluids—and free radicals to induce irreversible deleterious effects on different eye tissues. These processes may lead to cataract formation in the lens, as well as loss of corneal endothelial cells. This hypothesis is partially supported by the results obtained by Cejkova et al.62 from the analysis of the activities of superoxide dismutase, glutathione peroxidase, and catalase (the enzymatic scavengers of reactive oxygen species) in rabbit corneas. They found that in aged corneas, the activities of all antioxidant enzymes were dramatically decreased, suggesting that the cornea of aged rabbits are more susceptible to oxidative injury in comparison to the corneas of young adult animals.63

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9. Lam AK, Chan CC, Lee MH, Wong KM (1999) The aging effect on corneal curvature and the validity of Javal’s rule in Hong Kong. Chinese Curr Eye Res. 18(2):83-90 10. Hayashi K, Masumoto M, Fujino S, Hayashi F (1993) Changes in corneal astigmatism with aging. Nippon Ganka Gakkai Zasshi. 97 (10):1193-6 11. Topuz H, Ozdemir M, Cinal A, Gumusalan (2004) Age-related differences in normal corneal topography. Y Ophthalmic Surg Lasers Imaging 35(4):298-303 12. Hayashi K, Hayashi H, Hayashi F (1995) Topographic analysis of the changes in corneal shape due to aging cornea. 14(5):527-32 13. Dubbelman M, Sicam VADP, Van der Heijde GL (2006) The shape of the anterior and posterior surface of the aging human corne.a Vision Research 46:993-1001 14. Amano S, Honda N, Amano Y, Yamagami S, Miyai T, Samejima T, Ogata M, and Miyata K (2006) Comparison of central corneal thickness measurements by rotating Scheimpflug camera, ultrasonic pachymetry, and scanning-slit corneal topography. Ophthalmology. 113(6):937-41 15. Jonsson M, Markstro K and Behndig (2006) A Slit-scan tomography evaluation of the anterior chamber and corneal configurations at different ages. Acta Ophthalmol. Scand. 84: 116-120 16. Rufer F, Schroder A, Bader C, Erb C (2007) Age-related changes in central and peripheral corneal thickness: determination of normal values with the Orbscan II topography system. Cornea. 26(1):1-5 17. Foster PJ, Baasanhu J, Alsbirk PH, Munkhbayar D, Uranchimeg D, Johnson GJ (1998) Central corneal thickness and intraocular pressure in a Mongolian population. Ophthalmology. 105(6):969-73 18. Price FW Jr, Koller DL, Price MO (1999) Central corneal pachymetry in patients undergoing laser in situ keratomileusis. Ophthalmology. 106(11):2216-20 19. Hahn S, Azen S, Ying-Lai M, Varma R (2003) Los Angeles Latino Eye Study Group. Central corneal thickness in Latinos. Invest Ophthalmol Vis Sci. 44(4):1508-12 20. Pfeiffer N, Torri V, Miglior S, Zeyen T, Adamsons I, Cunha-Vaz J (2007) European Glaucoma Prevention Study Group: Central corneal thickness in the European Glaucoma Prevention Study. Ophthalmology. 114(3):454-9 21. Aghaian E, Choe JE, Lin S, Stamper RL (2004) Central Corneal Thickness of Caucasians, Chinese, Hispanics, Filipinos, African Americans, and Japanese in a Glaucoma Clinic. Ophthalmology 111:2211-2219 22. Sanchis-Gimeno JA, Lleo-Perez A, Alonso L, Rahhal MS (2004) Caucasian emmetropic aged subjects have reduced corneal thickness values. Int Ophthalmol. 25(4):243-6 23. Brown N (1974) The changes in lens curvature with age. Exp Eye Res 19:175-183 24. Dubbelman M, Van der Heijde GL (2001) The shape of the aging human lens: curvature, equivalent refractive index and the lens paradox. Vis Res 41:1867-1877 25. Smith G, Atchison DA, Pierscionek BK (1992) Modeling the power of the aging human eye. J Opt Soc Am A 9: 2111-2117 26. Guirao A, Redondo M, Artal P (2000) Optical aberrations of the human cornea as a function of age. J Opt Soc Am A Opt Image Sci Vis 17:1697-1702 27. de Kruijf EJ, Boot JP, Laterveer L, van Best JA, Ramselaar JA, Oosterhuis JA (1987) A simple method for determination of corneal epithelial permeability in humans. Curr Eye Res. 6(11):1327-34 28. Chang SW, Hu FR (1993) Changes in corneal autofluorescence and corneal epithelial barrier function with aging. Cornea 12:493-499 29. Nzekwe EU, Maurice DM (1994) The effect of age on the penetration of fluorescein into the human eye. J Ocular Pharm. 10:521-523 30. Trinkaus-Randall V, Tong M, Thomas P, Cornell-Bell A (1993) Confocal imaging of the alpha 6 and beta 4 integrin subunits in the human cornea with aging. Invest Ophthalmol Vis Sci 34:3103-3109 31. Hazlett LD, Kreindler FB, Berk RS, Barrett R (1990) Aging alters the phagocytic capability of inflammatory cells induced into cornea. Curr. Eye Res. 9:129-138 32. Taylor HR and Kimsey RA (1981) Corneal epithelial basement membrane changes in diabetes. Invest Ophthalmol Vis Sci. 20:548


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33. Alvarado J, Murphy C, Juster R (1983) Age-related changes in the basement membrane of the human corneal epithelium. Invest Ophthalmol Vis Sci. 24(8):1015-28 34. Oliveira-Soto LM, Efron N (2001) Morphology of cornea nerves using confocal microscopy. Cornea 20:374-384 35. Grupcheva CN, Wong T, Riley AF, et al. (2002) Assessing the sub-basal nerve plexus of the living healthy human cornea by in vivo confocal microscopy. Clin Exp Ophthalmol. 30:187-190 36. Erie JC, McLaren JW, Hodge DO, Bourne WM (2005) The effect of age on the corneal subbasal nerve plexus. Cornea. 24(6):705-9 37. Boberg-Ans J (1955) Experience in clinical examination of corneal sensitivity. Br J Ophthalmol 39:705-726 38. Lawrenson JG, Ruskell GL (1993) Investigation of limbal touch sensitivity using a CochetBonnet aesthesiometer. Br J Ophthalmol. 77:339-343 39. Millodot M (1977) the influence of age on the sensitivity of the cornea. Invest Ophthalmol Vis Sci. 16:240-242 40. Roszkowska AM, Colosi P, Ferreri FM, Galasso S (2004) Age-related modifications of corneal sensitivity. Ophthalmologica. 218(5):350-5 41. Daxer A, Misof K, Grabner B, Ettl A, Fratzl P (1998) Collagen fibrils in the human corneal stroma: structure and aging. Invest Ophthalmol Vis Sci. 39(3):644.8 42. Malik NS, Moss SJ, Ahmed N, et al. (1992) Ageing of the human corneal stroma: structural and biochemical changes. Biochim Biophys Acta 1138:222-228 43. Kanai A, Kaufman HE (1973) Electron microscopic studies of corneal stroma: aging changes of collagen fibers. Ann Ophthalmol. 5(3):285-292 44. Elsheikh A, Wang D, Brown M, Rama P, Campanelli M, Pye D (2007) Assessment of corneal biomechanical properties and their variation with age. Curr Eye Res. 32(1):11-9 45. Kotecha A, Elsheikh A, Roberts CR, Zhu H, Garway-Heath DF (2006) Corneal thickness- and age-related biomechanical properties of the cornea measured with the ocular response analyzer. Invest Ophthalmol Vis Sci. 47(12):5337-47 46. Erie JC, Patel SV, McLaren JW, Maguire LJ, Ramirez M, Bourne WM, (1999) Keratocyte density in vivo after photorefractive keratectomy in humans. Trans. Am. Ophthalmol. Soc. 97:221-236 47. Patel S, McLaren J, Hodge D, Bourne W (2001) Normal human keratocyte density and corneal thickness measurement by using confocal microscopy in vivo. Invest Ophthalmol Vis Sci. 42(2):333-9 48. Moller-Pedersen T (1997) A comparative study of human corneal keratocyte and endothelial cell density during aging. Cornea. 16(3):333-8. 49. Møller-Pedersen T, Ehlers N (1995) A three-dimensional study of the human corneal keratocyte density. Curr Eye Res 14:459-464 50. Berlau J, Becker HH, Stave J, Oriwol C, Guthoff RF (2002) Depth and age-dependent distribution of keratocytes in healthy human corneas: a study using scanning-slit confocal microscopy in vivo. J Cataract Refract Surg. 28(4):611-6 51. Johnson DH, Bourne WM, Campbell RJ, (1982) The ultrastructure of Descemet’s membrane. I. Changes with age in normal corneas. Arch. Ophthalmol. 100:1942-1947 52. Murphy C, Alvarado J, Juster R (1984) Prenatal and postnatal growth of the human Descemet’s membrane. Invest. Ophthalmol. Vis. Sci. 25:1402-1415 53. Laing RA, Sanstrom MM, Berrospi AR, et al. (1976) Changes in the corneal endothelium as a function of age. Exp Eye Res. 22:587-594 54. Yee RW, Matsuda M, Schultz RO, Edelhauser HF (1985) Changes in the normal corneal endothelial cellular pattern as a function of age. Curr Eye Res. 4(6):671-8 55. Hashemian MN, Moghimi S, Fard MA, Fallah MR, Mansouri MR (2006) Corneal endothelial cell density and morphology in normal Iranian eyes. BMC Ophthalmol. 6;6:9 56. Yunliang S, Yuqiang H, Ying-Peng L, Ming-Zhi Z, Lam DS, Rao SK (2007) Corneal endothelial cell density and morphology in healthy Chinese eyes. Cornea. 26(2):130-2

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57. Bourne WM, Kaufman HE (1976) Specular microscopy of human corneal endothelium in vivo. Am J Ophthalmol. 81(3):319-23 58. Hollingsworth J, Perez-Gomez I, Mutalib HA, Efron N (2001) A population study of the normal cornea using an in vivo, slit-scanning confocal microscope. Optom Vis Sci. 78(10):706-11 59. Murphy C, Alvarado J, Juster R, Maglio M (1984) Prenatal and postnatal cellularity of the human corneal endothelium: a quantitative histologic study. Invest Ophthalmol Vis Sci 25:312-22 60. Blatt HL, Rao GN, Aquavella JV (1979) Endothelial cell density in relation to morphology. Invest Ophthalmol Vis Sci. 18(8):856-9 61. Roszkowska AM, Colosi P, D’Angelo P, Ferreri G (2004) Age-related modifications of the corneal endothelium in adults. Int Ophthalmol. 25(3):163-6 62. Green K (1995) Free radicals and ageing of anterior segment tissues of the eye: a hypothesis. Ophthalmic Res 27 (Suppl):143-9 63. Cejkova J, Vejrazka M, Platenik J, Stipek S (2004) Age-related changes in superoxide dismutase, glutathione peroxidase, catalase and xanthine oxidoreductase/xanthine oxidase activities in the rabbit cornea. Exp Gerontol. 39(10):1537-43

Chapter 5

The Aging of the Human Lens Jorge L. Aliò, MD, PhD, Alfonso Anania, MD, PhD, and Paolo Sagnelli, MD

Abstract Age-related lens changes include: a) the progressive increase in lens mass with age, b) changes in the point of insertion of the lens zonules, and c) a shortening of the radius of curvature of the anterior surface of the lens. With age, there is also decreased light transmission by the lens associated with increased light scatter, increased spectral absorption—particularly at the blue end of the spectrum—and increased lens fluorescence. Besides these physiological modifications, we must take into consideration the additional effects caused by exposure to external physical and chemical agents such as ultraviolet rays and drugs, which lead to considerable densitometric changes and consequently to modifications in optical lens quality. At present, new instruments allow the analysis, in clinical practice, of qualitative and quantitative alterations of the lens that occur with aging, confirming objectively the degradation of the optical quality of the crystalline lens. Keywords crystalline, Lens, age related changes, human eye, cataract.

Introduction The crystalline lens of the eye is a principal component in the process of vision. To perform its role, the lens must be transparent and also have the capacity to rapidly alter its shape as it transitions between focusing on near and distant objects. Gross (light and scanning confocal microscopy) and ultrastructural (scanning, transmission, and freeze-etch electron microscopy) analysis of all vertebrate lenses reveals that lenses are composed of exceedingly long fiber-like cells that are of uniform crosssectional shape (hexagonal) and size. These microscopic techniques also show that, in general, as these fibers are formed throughout life, they are overlain, in register, as age-related concentric growth shells. Thus, it has been proposed that the highly ordered arrangement of lens fibers contributes to lens transparency by transforming the individual fibers into a series of coaxial refractive surfaces. Water and protein loss, modifications to membrane lipids, and protein modifications can result in the progressive increase in compaction folds. It follows that substantial senescent alterations in the structure of the embryonic and fetal nuclear fibers From: Aging Medicine: Age-Related Changes of the Human Eye Edited by C. A. P. Cavallotti and L. Cerulli © Humana Press, Totowa, NJ

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would lead to degradation of lens optical quality, especially since these fibers are located entirely within the region defined by the pupillary margin. While significant compaction of nuclear fibers occurs along the antero-posterior axis with aging, an even greater degree of compaction occurs in nuclear cataract formation. Therefore, there is convincing evidence that a senile cataract is an exaggerated final stage of age-related lens changes. Clinical observations of aged lenses show increased light scatter even without overt visual impairment, and it has been demostrated that there is a degradation of the optical quality of the crystalline lens with aging that is associated with morphological changes such as thickness and density. The process of nuclear fiber compaction is probably multifactoral, as the lens is exposed to the cumulative effects of radiation, oxidation, and post-translational protein modifications. Additional changes include: a) the progressive increase in lens mass with age, b) changes in the point of insertion of the lens zonules, and c) a shortening of the radius of curvature of the anterior surface of the lens. With age, there is also decreased light transmission by the lens associated with increased light scatter, increased spectral absorption—particularly at the blue end of the spectrum—and increased lens fluorescence. Besides these physiological modifications, we must take into consideration the additional effects caused by exposure to external physical and chemical agents such as ultraviolet rays and drugs, which lead to considerable densitometric changes and consequently to modifications in optical lens quality. At present, new instruments allow the analysis, in clinical practice, of qualitative and quantitative alterations of the lens that occur with aging, confirming objectively the degradation of the optical quality of the cristalline lens.

Lens Embryology An in-depth study of lens embryology facilitates the understanding of fibers and suture development. 1-4 Lens formation is the result of a series of inductive processes.5,6 The lens placode appears on the optic vesicle that protrudes from the forebrain, around the 25th day of gestation.7 It is a thickening of the surface ectoderm8—a single layer of cuboidal cells—that invaginate into the neural ectoderm of the optic vesicle as the lens pit, becoming free from the surface by the 33rd day9 (see Fig.5.1). The cells at the anterior pole of the lens vesicle remain as epithelial cells—the cell number is controlled by apoptosis.10 The posterior cells elongate as primary lens fibers that obliterate the lumen of the lens vesicle11—the retina largely determines this cytodifferentiation. The tiny developing lens is surrounded by a basement membrane that will become the lens capsule and is filled with nearly structureless primary lens fibers.12 These cells expel their nuclei, mitochondria, Golgi bodies, and endoplasmic reticulum. This structure becomes a spherical, optically clear embryonic nucleus of 0.35 mm in diameter,11 which stays unchanged throughout life7 and is seen inside the Y sutures in the fully developed eye. In an embryo of

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Fig. 5.1 Scheme to show the development of the lens. (A) Lens thickening. (B) Lens pit. (C) Lens pit closing. (D) Lens vesicle. E) Elongation of cells of the posterior wall of the lens vesicle. (F) Obliteration of the cavity of the lens vesicle by cells of the posterior wall. (G) Formation of lens sutures by the meeting of fibers developed in the equatorial region (Mann I: The Development of the Human Eye. Grune Stratton, New York, 1950)

23 mm, equatorial secondary lens fibers derived from the anterior epithelium migrate forward under the anterior epithelium and backward directly beneath the capsule—meeting at the sutures that can be seen easily with slit-lamp microscopy as an upright anterior Y and an upside-down7 posterior Y. The limbs of the Ys are often branched. A large number of recent studies have focused on the involvement of polypeptide growth factors and cytokines in lens differentiation. These factors include fibroblast growth factors (FGFs), insulin and insulin-like growth factors (IGFs), transforming growth factors (TGFs),13 platelet-derived growth factors (PDGFs), epidermal growth factors (EGFs) and several cytokines, including macrophage-migration inhibitory factor (MIF), and tumour necrosis factor-alpha (TNFα).14 After birth, the equatorial fibers grow to form the cortex, meeting at more complex and less well-marked sutures—this growth continues until very shortly after death. The tertiary vitreous condenses within the space between the ciliary body and the lens equator, forming the suspensory ligament of the lens at the fifth month of gestation.7 The developing lens requires nutrition that is obtained through the tunica vasculosa lentis, which is a vascular network supplied posteriorly by the hyaloid artery (a branch of the primary dorsal ophthalmic artery) and anteriorly from an anastomosis with vessels in the pupillary membrane.7 The tunica vasculosa lentis is first seen at about 35 days, and is most prominent at 65 days. It gradually regresses at about 85 days, and by term birth, only whispy remnants of the pupillary membrane are left, with a vestigial hyaloid artery (known as a Mittendorf’s dot) attached to the axial posterior surface of the lens.7,15

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Morphology of the Human Lens Roughly speaking, vertebrate lenses are asymmetrical, oblate spheroids of variable size and spheroidicity (see Fig. 5.2). The lens, encaved in an elastic capsule, consists of: a) an anterior monolayer of epithelial cells, the pre-equatorial members of which exhibit mitotic activity throughout life; b) a superficial layer of elongating, differentiating, and maturing secondary fibers, and c) the main lens body that consists of fully matured primary (embryonal nucleus) and secondary fibers16 (see Fig. 5.3). These fibres are characterized by a high protein content (35-40%), by the absence of nuclei, mitochondria, lysosomes, ribosomes and endoplasmatic reticulum, and are surrounded by increasingly less permeable membranes. All lens fibers are mutually anchored, securing minimal extracellular space, thus minimizing differences in the refractive index from fiber to fiber. Many electron microscopic studies have been performed: initial investigations were undertaken by Wanko and Gavin17 and Cohen18 on mammals and humans, respectively. Many more followed describing fibers from a variety of mammals, including rat,19,20 rabbit,21 pig,22 monkey,23-26 and human.24,27-32 Recent technical advances in fixation methods for scanning electron microscopy (SEM)25 and transmission electron microscopy (TEM)33 have made it possible to analyze differentiated fibers in all regions of the lens. The cross-sectional area of fiber cells varied from region to region, with the smallest areas found in the compressed adult nuclear region and the largest found in the central embryonic

Fig. 5.2 Diagrammatic section of the eye


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capsule epithelium cortex adult nucleus juvenile nucleus

fetal nucleus

embryonic nucleus

Fig. 5.3 Diagram of an aged normal human lens, approximately to scale. The complex suture pattern is not shown. The epithelium and capsule are enlarged for clarity. (Modified from: Morphology of the Normal Human Lens. VL Taylor, KJ Al-Ghoul, CW Lane, VA Davis, JR Kuszak, and MJ Costello Investigative Ophthalmology & Visual Science, June 1996, Vol. 37, No. 7)

nucleus. Cellular organization was most ordered in the cortex, where radial cell columns were found. By contrast, cells were more irregularly packed in the center of the lens, where no apparent arrangement was observed. The cytoplasm of intact cells was smooth and homogeneous in all regions analyzed. The hardened nuclear core corresponds to the fetal and embryonic nuclei, the outer soft layer corresponds to the cortex, and the layer between, with intermediate hardness, corresponds to the adult and juvenile nuclei. The term epinucleus has been used to describe this intermediate layer of tissue corresponding to the adult plus juvenile nuclei.34 In the adult nucleus, the deep cortical fibers are compacted as they are internalized and become part of the adult fiber mass. Changes in shape occurred in the absence of significant membrane loss or turnover, which resulted in an increase of membranous undulations. The small variation in size of the adult nuclear fibers implies that cells seem to be affected equally by this compaction. Both mechanical compression (caused by the continual deposition of new fibers) and dehydration (regulated by the osmotic properties of the crystallins) are involved in this process of compaction.35-37 Cells in the juvenile nucleus were, on average, twice the size of adult cells and approximately half the size of deep cortical cells. This suggests that juvenile cells are also compacted, but not to the degree that the adult cells are. Different developmental events or protein modifications may occur during the formation of juvenile cells, in contrast to adult cells, because they are formed before puberty. The crystallins of the juvenile cells may resist compaction because initially they may be more dehydrated than those of the adult cells.16 Alternatively, some cells may grow in a larger form, or cells may fuse together during elongation.38,39 Both these events would produce cells with larger areas. Radial cell columns could be detected in adult and juvenile regions but were difficult to visualize because of the intricate folding of the membranes. Together,

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the adult and juvenile nuclei comprise an annular ring that undergoes significant compaction with age and, importantly, is flanked by regions that are compacted relatively less with age. Biochemically, a correlation was observed between the protein modifications of lens crystallins and the lens regions.40 At the molecular level, packing of lens crystallins has been shown in the last decade to be random, with no evidence of crystalline regularity. Based on x-ray diffusion measurements in vitro, monomers (or small aggregates of crystallins) are thought to be associated closely. Therefore, at a critical distance, the light scattering of the concentrated protein solution decreases significantly, leading to transparency.41 These long-term changes in radial cell thickness place constraints on cell shape, size, and packing. Possible cell-to-cell fusion also influences packing, especially near the poles and at the sutures. The simplest explanation for the observed changes in this annular ring is that the cytoskeleton is lost in the last stages of cortical differentiation and that the nuclear fibers are squeezed under pressure against a harder nuclear core (fetal and embryonic nuclei), resulting in gradual dehydration of crystallins and cellular compaction. Alternatively, self-association of the crystallins and the resultant decrease in osmotic pressure may induce dehydration of the cytoplasm.37

Lens Capsule Anatomy of the Lens Capsule The position of the lens in the optical system of the eye is assured by the attachment of the zonular fibers to the lens capsule, as well as the support provided by the vitreous and iris. The lens capsule and the zonular fibers constitute the link between the lens fiber substance and the ciliary muscle, and thus play an important role in transmitting the force of ciliary muscle contraction to change the shape of the lens fiber substance that is essential for accommodation. The geometrical pattern of the zonules is complex and varies significantly with age. The zonular fibers attach to the lens in three separate groups: an anterior, an equatorial and a posterior group.42 The attachment of the zonules to the lens is known to involve penetration of zonular fibers into the superficial lens capsule.43,44 The anterior zonules attach to the outer surface of the lens capsule around the lens periphery in a rather broad zone, which increases with age from about 0.25–1.2 mm due to a relative inward displacement of the zonular insertion in the lens capsule.45 A zonular lamella has been described by many observers as a thin membrane surrounding the lens capsule.46,47 Its existence, however, has been a matter of dispute and, according to Hogan,43 the zonular lamella exists only in the equatorial region where the zonular fibers attach to the capsule. In an electron microscope study, Seland44 confirmed the presence of a fibrillar surface layer in newborns, but showed that this fibrillar surface layer retracted from the anterior pole between the ages of 6 and 17. On the posterior capsule, a fibrillar surface layer does not seem to be a


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constant feature, apart from the posterior zonular attachment.43,44 The lens capsule encloses the lens fibers. The inner surface of the anterior lens capsule is in immediate contact with the lens epithelium, while the posterior lens capsule is in contact with the most superficial part of the posterior lens fibers.

Ultrastructure of the Lens Capsule The lens capsule is the thickest basement membrane in the body. When studied under light microscopy the lens capsule appears dense and homogeneous. Under the electron microscope, the lens capsule is found to be made up of parallel lamellae, more tightly packed toward the outer surface.48 The lamellar structure of the lens capsule seems to disappear with age. In the posterior capsule, it disappears in childhood. In the anterior capsule it starts disappearing from the anterior pole in adulthood but persists in the equatorial and preequatorial regions corresponding to the metabolically most active part of the lens epithelium.43,44,49 Ultrastructurally, the support of the lens capsule is type IV collagen, which interacts with other glycoproteins and proteoglycans to form an extracellular matrix.50-53 Type IV collagen is found only in basement membranes, and it is the only collagen that has been shown definitively to be present in basement membranes. Immunoelectron microscope studies of the lens capsule, however, also seem to show the presence of collagen types I and III.54,55 Type IV collagen plays an important role in the formation of a resilient, three-dimensional molecular network.56,57 Compared to the fiber-forming collagens, the type IV collagen molecule is longer, more flexible, and contains frequent interruptions by non-collagenous sequences. The type IV collagen molecule possesses distinct end-region domains and exhibits several binding interactions that enable formation of a stable lattice-like network.

Growth and Thickness of the Lens Capsule The lens capsule continues to grow throughout most of life, growing in thickness anteriorly and increasing in surface area to adjust to the increasing volume of the lens. The anterior lens capsule is produced by the lens epithelium58-60 and therefore reflects the activity of the epithelial cells, which undergo apparent morphological changes with aging. The epithelial cells become flattened—the number of organelles is reduced and become more difficult to distinguish because of an increasing density of the cytoplasmatic matrix.49,61 The regional variation in thickness of the lens capsule changes markedly with age, which suggests a continuous modeling of the lens capsule with age. In contrast to the anterior lens capsule, which is synthesized by the lens epithelium and continues to grow and increase in thickness throughout most of life,44,62,63 the human posterior lens capsule loses its epithelial cells in fetal life.43 It has been suggested that the posterior lens capsule is synthesized and secreted by nucleated cortical lens fibers, or synthesized by anterior epithelial cells and

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secreted into the posterior aspects of the lens during the first part of life, after which the production of the posterior lens capsule is supposed to cease. Capsular thickness is not uniform but varies according to age and the location at which the measurements are taken.44,62-64 Fisher and Pettet62 examined unfixed lens capsules and found that thickness of the neonatal lens capsules varied from 3–5 µm at the posterior pole to approximately 11 µm at the equator. Thickness of the anterior pole and thickness beneath the insertion of the anterior zonules were found to increase with age, whereas thickness of the posterior lens capsule was found to be constant. In pre-presbyopic eyes, the lens capsule was found to be thickest at the equator, whereas in old adults, the lens capsule was found to be thickest beneath the insertion of the anterior zonular fibres. Seland44 examined fixed lens capsules and found that the neonatal lens capsule had a uniform thickness of about 4 µm and that thickness of the anterior as well as the posterior lens capsule increased gradually with age— most markedly in the peripheral region of the anterior lens capsule where thickness reached as much as 30 µm. In all age groups, the thinnest part of the lens capsule was found at the posterior pole. The thickest part of the lens capsule in pre-presbyopic lens capsules was found to be in the mid-periphery of the anterior and posterior lens capsule. Thickness of the anterior peripheral zone was found to increase with age throughout the lifespan, whereas thickness of the posterior peripheral thickened zone was found to increase only in pre-presbyopic lens capsules and to decrease in the older age group.

Mechanical Properties of the Posterior Lens Capsule The anterior and posterior lens capsules differ in several aspects. The lamellar structure of the lens capsule disappears earlier with age in the posterior lens capsule than in the anterior lens capsule,44 and differences have been described in the relative proportion of macromolecular components such as heparansulfate, proteoglycans, and fibronectin.65,66 This could indicate that the mechanical qualities of the anterior and posterior lens capsule are different. Mechanical strength of the posterior lens capsule (ultimate strain, ultimate stress, ultimate elastic modulus) was found to decrease markedly with age in a range similar to that of the anterior lens capsule.48 The age-related loss of mechanical strength, however, seemed to begin earlier in the posterior lens capsule than in the anterior lens capsule. Ultimate load, which reflects the breaking strength of the lens capsule in situ, was significantly lower for the posterior lens capsule than for the anterior lens capsule.48 This is in accordance with the fact that the posterior lens capsule is much thinner than the anterior lens capsule. When looking at data pertaining to the accommodative function range (low strains), the mechanical quality of the posterior lens capsule was found to be similar to that of the anterior lens capsule in all age groups. This indicates that the mechanical properties of the lens capsule in situ vary proportionally with the regional variation in capsular thickness. The age-related loss of mechanical strength, however, seems to begin earlier in the posterior lens capsule than in the anterior lens capsule.


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Aging of the Lens Capsule Studies of the human lens capsule indicate concurrently that aging of the human lens capsule is associated with a progressive loss of mechanical strength, which seems to parallel morphological changes in the lens capsule. Formed elements (inclusions) accumulate in the anterior lens capsule with age.44,67 The laminated structure of the lens capsule disappears with age,44,49 and the optical density of the lens capsule increases with age.68,69 Peczon et al.70 investigated age-related changes in the amino acid composition of the lens capsule and found a relative increase of noncollagenous amino acids and a decrease of collagenous amino acids (hydroxyproline) with age. Because collagen seems to be responsible for the mechanical strength of other soft connective tissues,71 the age-related changes in the amino acid composition of the lens capsule also may have significance in the loss of mechanical strength. The major structural component of the lens capsule is basement membrane type IV collagen, which is organized into a three-dimensional molecular network.72 As discussed previously, the mechanical properties of the lens capsule correlate well with a network structure. The lens capsule is easily deformed at low deformations due to reorientation and alignment of the molecular network structure in the direction of deformation. As the elastic stiffness of the lens capsule at low deformations increases with age in pre-presbyopic eyes, and the extensibility correspondingly, decreases with age, this suggests geometrical changes in the molecular network structure with age. One factor may be the increasing volume of the lens with age, which may cause stretching of the collagen network structure, thus limiting further deformation. Another factor may be an increased crosslinking of the molecular network structure with age, which also may limit deformation.48 The collagen molecules in the lens capsule seem to be extremely long-lived. This provides great opportunity for posttranslational modifications of the molecules, such as nonenzymatical glycosylation,73,74 which can change the mechanical properties of the lens capsule through the formation of stable crosslinks.75-77

The Ocular Lens Epithelium Ultrastructure of the Lens Epithelium A single layer of cells—the lens epithelium—covers the anterior face of the lens that faces the cornea. The lens epithelium ends on the rims of the anterior surface. It contains cells in the central region that do not divide and are essentially quiescent, surrounded by a germinative-dividing zone of cells and followed (at the equatorial fringe) by the dividing cells that differentiate into fiber cells (see Fig. 5.4). A remarkable feature of this epithelium is its capacity to divide and differentiate almost all through the life of an individual. This feature of sustained growth is very

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Fig. 5.4 Simplified drawing of the ocular lens. Note that the epithelium has three regions of cells in nondividing phase (central epithelium), in dividing phase (germinative), and differentiating phase (equatorial). The anterior and posterior sutures are formed by the meeting of the elongating fiber cells that make the bulk of the lens mass. The surrounding capsule (shaded area around the lens) indicates that the basal surface of the epithelium and the fiber cells is on the outside, while the apical surface faces the inside of the lens. apical interface—an area of contact between the apical surfaces of the epithelial cells and the fiber cells is not indicated. It is the area just below the epithelial layer

much similar to its closest embryological sibling—the cells in the skin. The cells in the lens epithelium represent typical epithelial morphology: they are cuboidal, presenting a cobble-stone-like appearance in their native state and in vitro, if cultured without excessive passaging. The diameter of human lens epithelial cells ranges from 9–17 µmm.78 The cell size has been reported to increase with age,79,80 which suggests a change in the cell density. Females have been reported to have higher cell density in the human lens epithelium than males.81,82 Francois and Rabaey83 observed lens epithelium under a phase-contrast microscope. They reported the presence of pale polyhedral and dark, star-shaped cell types. A recent in vivo study84 using the noncontact specular microscopy recognized four morphological features of the live human lens epithelium. These were categorized as linear furrows, columnar organization, puffy clouds, and black holes. The relationship between cell density and age is interesting, although controversial. An earlier report82 that there is an age-related decrease in the cell density in the lens


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epithelium has been recently confirmed.84 This recent study calculated the loss of 675 cell/mm2 in a 75-year life span (that amounts to a loss of 14% of the cells). This estimate is based on the unproven assumption that the rate of loss is linear with age, however it is not very different from that reported in the aging monkey lens central epithelium.85 Others have found no such relationship.86,87 Karim et al.87 reported a decrease in the mitotic index of lens epithelial cells under normal, as well as cataractous, conditions. Harocopos et al.88 concluded that there was no relationship between cell density and the severity of cataracts, or between cell density and age. They did report, however, that the epithelium directly over the opaque area in cataractous lenses had higher cell density when compared to that overlaying the transparent regions. It is possible that a loss of a patch of cells overlying a cataractous fiber cell area may lead to the activation of cell division, and therefore higher cell density.

The Epithelium as the Major Site of Transport, Metabolism and Detoxification The overall metabolic status of the fiber cells in the absence of endoplasmic reticulum, mitochondria, and a nucleus, is comparatively very low.89 There is no vascular system as we know it that would take nutrients to the fiber cells and remove metabolic/physiologic waste to replenish the intra- and intercellular milieu of the lens. Mere diffusion as a process to sustain the slow but substantial physiology of the ocular lens will be insufficient to accomplish this efficiently. A study of relative rates of transport across the anterior and posterior surfaces of the lens has led to the model of the pump-leak system.90-92 Lens epithelium is also a major site of detoxification and defense against oxidative insults93,94 and is able to detoxify physiological concentrations of H2O2 enzymatically involving glutathione reductase, glutathione peroxidase, and the hexose monophosphate shunt.94

Programmed Cell Death and the Lens Epithelium The interest in the study of programmed cell death in the lens epithelium was generated recently by investigators probing the role of epithelium in cataractogenesis.95 These studies are based on the hypothesis that the integrity of the lens epithelium is essential for the normal functioning of the lens, and that a decrease in the cell number of the epithelium may lead to changes in homoeostasis that may in turn lead to cataractogenesis. The role that apoptosis plays in tissue development and morphogenesis is well established.96 Apoptosis or cell death has been morphologically documented in the very early stages of the lens vesicle formation during development of the eye.97 A role for apoptosis in regulating the size of the lens by controlling the number of cells that reach terminal differentiation into fiber cells remains a possibility.

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Different metabolites98 and drugs99 have been reported to initiate apoptosis in lens epithelial cells in culture.100 The research of Zigman,101 Li and Spector,95 Michael et al.102 and Shui et al.103 suggest that apoptosis may occur discretely in the lens epithelium and only in isolated cells that become susceptible. Such a thesis connotes two interesting corollaries: ●

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Any isolated apoptotic cell may be quickly eliminated by the surrounding cells as a protective response against the spread of cell death. Importantly, therefore, it points to the existence of a potent mechanism that strictly controls and inhibits cell death from spreading to neighboring cells. The presence or absence of apoptosis in the lens epithelium can be interpreted optimistically as a process that eliminates the dys functional cells to keep the rest of the epithelium healthy. A pathological state may precipitate when this ability to remove dysfunctional cells is compromised—for example, by aging or by exposure to harmful metabolites or environmental insults.

Lens Fiber Cells Fiber Cell Organization and Development of Lens Sutures The cells of the lens vesicle that were not induced to form primary fibers remain as a monolayer—the lens epithelium—that covers the anterior surface of the primary fiber mass. From this point on, further lens development and growth occurs throughout life in a manner similar to other stratified epithelia. The lens epithelium constitutes the basal layer—however, whereas typically stratified epithelia have their progenitor cells distributed throughout the basal layer, the lens is unique in that its progenitor cells are sequestered as a distinct subpopulation within the lens epithelium known as the germinative zone, which comprises a narrow, peripheral, latitudinal band of the lens epithelium located just above the equator.104 These cells undergo mitotic division, and selected daughter cells are induced to terminally differentiate and form secondary fibers. As with primary fiber formation, the most apparent structural consequence of secondary fiber formation is the transformation of a cuboidal cell into a long fiber. While forming, however, primary fibers are fixed in position as they elongate essentially unidirectionally. Secondary fiber formation requires the forming fibers to rotate about their polar axis while simultaneously migrating posteriorly and elongating bidirectionally. Fiber rotation is complete when the long axis of a forming fiber is aligned parallel to the antero-posterior axis of the lens, and when the center of a forming fiber reaches the mid-point between the poles, which—by virtue of the fact that all vertebrate lenses are asymmetric, oblate spheroids—is posterior to the equator. As secondary fibers elongate, their anterior ends are insinuated between the lens epithelium and the primary fiber mass, while at the same time their posterior ends are insinuated between the primary fiber mass and the posterior lens capsule.


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Maturation of secondary fibers is complete when they detach from the lens epithelium anteriorly, and the capsule posteriorly, to subsequently overlap with other newly mature fibers to form lens sutures.105

The Contribution of Major Fiber Proteins to Sutural Development and Growth In vertebrate lenses, MP26, gap junction (GJ) connexins, and MP19 are the major fiber intrinsic membrane proteins. The major intrinsic protein (MIP), or aquaporin0 (AQP0), is generally described as constituting more than 60 percent of the total fiber membrane protein.106,107 The connexins (Cxs)108,109 that form the lens GJ communicating channels, display a variety of expression patterns, channel regulation, and posttranslational modifications during differentiation and aging of the lens cells.110,111 Lens epithelial GJs consist mainly of 1 connexin (Cx.1 or Cx43).112 The Connexin50 is essential for normal postnatal lens cell proliferation.113,114 Cortical fiber GJs consist of Cx.3 (or Cx46) and Cx.8 (or Cx50)—often coexisting in the same junctional plaques.115,116 MP19 (also referred to as MP17, MP18 and MP20 in the literature) has also been described as the most abundant intrinsic membrane protein of lens fiber cells.106 However, unlike MIP or the connexins, MP 19 bears no striking resemblance to any other reported protein family and, to date, has no defined structural or functional role. Both MIP and MP 19 co-localize with GJs in distinct regions of the lens.117,118 Thus, it has been proposed that both MIP and MP 19 play some role in GJ formation, maintenance, or organization. While it is well-documented and irrefutable that all vertebrate lenses contain the above described major fiber proteins, a review of the literature suggests that their density and distribution varies is species-specific—varying along fiber length and as a function of fiber depth and therefore of age. GJs primarily conjoin the midsegment of fibers, or those segments of fibers not involved in sutures.119,120 The function of these proteins is probably coordinated during fiber development.

Lens Sutural Anatomy Numerous studies have established that the vast majority of fibers are hexagonal in cross section,121-126 with two broad faces oriented parallel to the lens surface and four narrow faces oriented at acute angles to the lens surface. During differentiation and maturation, the lens fiber membranes undergo typical changes. The lateral and apical surfaces of the hexagonal fibres change from smooth and studded with small ball-and-socket junctions in the superficial cortex, to covered with groovesand-ridges in deeper cortical regions and the nucleus. Freeze fracture studies127 revealed that these surface changes are paralleled by changes in the internal organization of the fiber membranes. Epithelial and superficial fiber membranes are studded with a multitude of intramembrane particles (IMPs) and gap junctions (GJs). The IMPs represent intrinsic membrane proteins

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Fig. 5.5 Formation of anterior Y and offset posterior Y sutures. In this view of the anterior and posterior surface, a number of curved fibers have been highlighted. The anterior ends of these fibers are paired (a-b-c-d-f) to form parts of anterior suture branches. By following these fibers along their length, it can be seen that as a result of opposite end curvature, the posterior ends of these fibers are paired with different fibers to form offset posterior suture branches (Modified from: Development of Lens Sutures. JR Kuszak, RK Zoltoski and CE Tiedemann Int. J. Dev. Biol. 48: 889–902, 2004)

that function as receptors, ion channels, transporters, and pores. GJs allow a direct cell-to-cell exchange of molecules up to 1500 Da. Biochemical studies128 showed that, upon maturation, the cholesterol-tophospholipid ratio of lens membranes dramatically changes from 0.6-0.8 in superficial to over 5.0 in deep cortical and nuclear membranes. All this indicates that lens membranes, apart from those in the most superficial cortex, deviate from most cell membranes in the body. This is in line with electrophysiological studies showing that deep cortical membranes are non-leaky, have a high resistance and low capacitance, and have no or restricted cell-to-cell communication.129,130 In the human lens, fibers are partial or incomplete meridians—that is to say, upon completion of elongation, the vast majority of fibers do not have ends that extend to the poles (see Fig. 5.5). During gestation primary fibers are neither uniform in shape nor size.122,123,126,131,132 As such, the primary fiber cell mass, or embryonic lens nucleus, does not consist of growth shells overlain in the register to form ordered radial cell columns. The initial secondary fibers are similarly nonuniform in shape and size, and also lack an ordered arrangement. Only as lens development proceeds are the additional secondary fibers formed progressively more uniform in shape. The establishment of growth shells comprised of uniform fibers overlain in register as radial cell columns occurs


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within 250–750 µM of the equatorial center of the lens. Coincidentally, this marks the beginning of suture formation.133

Suture Formation after Birth After birth, there are fundamental changes in the fiber differentiation program that result in the formation of progressively more complex iterations of star sutures during infancy, adolescence, and adulthood.

Simple Star Suture Formation Shortly after birth, a new (or secondary) anterior suture branch, and a pair of new (or secondary) posterior suture branches, begin to develop in relation to the extant suture branches within the infero-nasal quadrant. At the same time, the anterior ends of curved fibers that bracket the posterior suture branch are added to the extant primary anterior suture branches. Similarly, the posterior ends of the same curved fibers that bracket the anterior suture branch as a consequence of opposite end curvature, are added to the extant offset primary posterior suture branches. By the end of the infantile period, the anterior suture consists of three enlarged primary branches, and three new secondary suture branches—one completely formed, and two partially formed. The offset simple star posterior suture consists of three pairs of new secondary branches—a pair that are completely formed, two pairs that are only partially formed. All of the suture branches are arranged in a symmetrical, but nonidentical, simple star suture pattern.133 Star and Complex Star Suture Formation The essential parameters of star sutures formed during adolescence, and complex star sutures formed throughout adulthood, are demonstrated in Fig. 5.6.

Fig. 5.6 Key stage in the development of the complex star suture formed throughout adulthood. These sutures are progressively more complex through adolescence and adulthood

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Fig. 5.7 Intermediate-magnification SEM micrographic image of a portion of suture branches. The width and evenness of a suture branch is related to the degree of irregularity between fiber cell ends (Modified from: JR Kuszak, JG Sivak, JA Weerheim, Lens optical quality is a direct function of lens sutural architecture. Invest. Ophthalmol. Vis. Sci.S, vol. 32, 7:2119–2129, 1991)

These sutures are progressively more complex iterations (second and third rows, polar projections of anterior and posterior, respectively) of the simple star sutures formed through infancy. Throughout adolescence, the nine branched star sutures are formed as tertiary anterior suture branches, and tertiary pairs of posterior suture branches sequentially supplement the extant primary and secondary branches.134 The different suture patterns formed during gestation, infancy, adolescence, and adulthood are the anatomical basis of the zones of discontinuity revealed by slit-lamp biomicroscopy.134,135 Throughout life, anterior and posterior suture branch formation continues, and their distal ends extend to confluence at their respective poles. Numerous structural studies confirm that the uniformly shaped fibers are arranged in highly ordered growth shells—however, the ends of the fibers are very nonuniform in shape.133 Thus, their end-to-end arrangement to form suture branches produces naturally occurring regions of disorder aligned directly along the visual axis. In fact, by overlying suture branches in concentric growth shells, line and Y suture lenses produce disordered suture planes aligned directly along the visual axis (see Fig 5.7).


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The Physical Basis for Transparency of the Crystalline Lens To perform its role in the process of vision, the lens must be transparent. The analysis of transmission in terms of the physical interactions between light and the structures of the lens is fundamental to understanding the changes in optical quality with aging. The first author to study the physical concept of transparency in the lens was Trokel in 1962.136 The physical interactions between light and the known structures of the lens (cortical fibers surrounded by cell membranes and protein fraction that comprises most of their cytoplasm) and the manner in which these molecular and microscopic structures affect the traversing light wave determines the transmission characteristics of the lens. These characteristics depend upon the two processes of absorption and scattering. Absorption is the conversion of light from the incident beam to other forms of energy, such as heat or chemical energy. Scattering takes place when light passes over the elastically bound electrons in the atoms and molecules. The scattering interaction may be thought of as producing elastic vibrations that result in the emission of secondary light in all directions—thus, scattering also removes energy from the traversing beam. A distinction is made between light scattering by small and by large particles. Scattering by small particles occurs when the objects are smaller than the wave length of light, such as the soluble proteins of the lens. Large particles are larger than several wave lengths in size,137 and are the structures that can be resolved by the light microscope. Microscopic and submicroscopic structures cause the extinction of light, which determines the transmission characteristics of the lens. This extinction derives from the many processes summarized in Fig. 5.8 that show the absence of absorption and the major role of scattering in the extinction of visible light by the

Fig. 5.8 Summary of the processes that produce extinction of light in the crystalline lens. (Modified from: S Trokel The physical basis for transparency of the crystalline lens. Invest Ophthalmol 1:493, 1962)

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lens. The physics of light scattering must be examined to understand the extinction characteristics of the lens.

Small Particle Scattering Completely regular crystalline matter will change only the velocity of traversing light without removing energy by scattering. It can be concluded that the high degree of light transmission of the intact lens fibers results from the spatial order of lens proteins in their normal state. The spatial order of protein molecules can be described by p(r)—the probability that two protein molecules are a distance r apart. The reduction in scattering due to local order has been derived by Zernike and Prins138 in a general form: ⎡ 4pN ∞ sin ksr ⎤ (1 − r(r ))r 2 dr ⎥ ⎢1 − ∫ V ο ksr ⎣ ⎦ This formula expresses the reduction of scattering of N particles in a volume (V), where k = 2 π/l, and s = 2 sin q /2. The distribution function p(r) is normalized to unity when all r’s are equally probable. This is the dilute solution in which this factor reduces to one, and no external interference of scattered light occurs. Quantitative application of the Zernike-Prins factor to the lens proteins in the intact state is not now feasible because the exact dimensions and the spatial order of the proteins in the intact fiber are unknown. Qualitatively, the high concentration of the soluble proteins in the lens fiber must be accompanied by a degree of local order approaching a paracrystalline state. This results in the interference of scattered light and the transparency of the fibers.

Large Particle Scattering Although the nature of the physical interaction is the same, large particle scattering calls for mathematical treatment different from that of small particle scattering. Incident rays on an isotropic particle give rise to the phenomena of diffraction and reflection. The reflection is accompanied by refraction at the large particle surface. Diffraction and reflection can be considered special cases of scattering. The phase contrast photomicrograph of an unstained section emphasizes those structures that cause large particle scattering (Figs. 5.9). Thus, the lens transparency is made possible by a number of factors, including the regular arrangement of the lens fibers, the nonparticulate fiber cytoplasm, and the uniform distribution and paracrystalline state of proteins within the cells.136-140 Kuszak et al.141 have proposed that the arrangement of lens fibers depends strongly on the ability of newly formed cells to elongate in a pattern that meshes precisely


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Fig. 5.9 Comparison with a phase-contrast photomicrograph of a lens section made perpendicular to the fibers, and a diagram that represents the membrane pairs of the lens fiber walls as a single refractive discontinuity. r1 is a ray reflected back to the light source, and r2 is reflected at a 60-degree angle from the path of the transmitted beam. The cluster of short arrows represents scattering of light by the soluble proteins that comprise the fiber cytoplasm (Modified from: S Trokel S The physical basis for transparency of the crystalline lens. Invest Ophthalmol 1:493, 1962)

with the underlying cells. The lamellar conformation of lens proteins rather than helical structure may also contribute to transparency.142 In addition, it has been proposed that a short-range, liquid crystal-like order of the crystallins is important for transparency of lens cytoplasm.143 In addition to the state of lens crystallins, the tight packing of the lens cells and the regulation of ion and water balance also play significant roles in maintaining the transparency of the normal lens. Consequently, the development of protein aggregates, cell membrane degeneration, the appearance of vacuoles, and the distortion of lens structure can all produce light scatter and the clinical observation of cataracts.139

The Influence of Sutural Architecture on Lens Optical Quality The morphology of lens sutures should be considered when evaluating the optical quality of crystalline lenses.A quantitative analysis of optical quality in line and Y suture lenses confirms that suture planes significantly degrade lens function (sharpness of focus).144-146 Comparable studies in star suture lenses, however, show that the staggering of suture branches in concentric growth shells effectively minimizes the negative influence of suture planes on optical quality.147

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Spherical aberration is a monochromatic anomaly that can be defined as the difference in focal length for light rays that pass through different points of a lens. Generally, light rays that pass through the periphery in vertebrate crystalline lenses can have longer focal lengths than those that pass through the center. This situation is known as negative or over corrected spherical aberration, and it is partially the result of lens structure. The net result of the additional growth shells of lens fiber cells throughout life is an ever-increasing lens mass.144-147 Unlike other stratified epithelia, the strata of the lens (growth shells) are never sloughed off. Rather, they become more internalized as the lens grows. The plasma membrane, the cytoplasm with specialized crystalline proteins, and the extracellular space between the lens fiber cells of the growth shells have different refractive indices. Thus, as the lens grows, a gradient of refractive index is established from the center of the lens to the periphery on the basis of variation in protein content. This gradient of refractive index, possibly in combination with the asphericity of lens shape, neutralizes positive spherical aberration.144 Numerous studies have shown that lens fiber cells are uniform in shape and are overlaid in precise alignment to produce radial cell columns between growth shells.148-152 Thus, it has been proposed that the radial cell columns are a system of coaxial refractive surfaces that are partially responsible for lens transparency.153,154 In contrast, scanning electron microscopy studies147,152,155,156 show that the ends of lens fiber cells are variable in shape and are overlaid in imprecise alignment to produce irregular suture planes between growth shells. If the ordered alignment of uniform fiber cells into precise radial cell columns contributes to negative or corrected-spherical aberration, then the alignment of variably shaped lens fiber cell ends into imprecise suture planes could contribute to nonmonotonic spherical aberration. More importantly, the negative influence of sutures on optical quality increases with age.157-160

Nuclear Fiber Compaction as a Function of Aging and Cataractogenesis The substantial senescent alterations in the structure of the embryonic and fetal nuclear fibers can lead to degradation of lens optical quality, especially because these fibers are located entirely within the region defined by the pupillary margin. In fact, clinical observations of aged lenses show increased light scatter even without overt visual impairment.161 Excessive senescent changes in the morphology of the nuclear lens fibers are likely to be most detrimental to lens optics, because these fibers are located directly along the visual axis. Therefore, age-related fiber compaction resulting in an increase in the membrane complexity along the light path may be a source of increased large particle scatter and ultimately, reduced lens optical quality with age. Light scatter in the lens has been attributed to the interaction of the incident beam with both the cell membranes and the cytoplasmic proteins producing


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respectively, large and small particle scatter.162 It has been suggested that in normal lenses, the majority of light scatter originates from interactions with fiber membranes, which have a higher refractive index as compared to the cytoplasm.163 The cytoplasm is virtually transparent due to the close association of the crystallin proteins that minimizes refractive index fluctuations.164,165 Although numerous biochemical modifications have been noted in the cytoplasmic and membrane components of lenses with age-related nuclear cataracts, the sources of excessive light scatter have yet to be definitively identified. In nuclear cataracts, the signficantly increased fiber compaction may be one of the factors contributing to the excessive scatter in nuclear opacification. The size and shape of the human lens changes dramatically during development and maturation. Assessments of human lens growth have established that the equatorial dimension of lenses increases at a greater rate than the polar (A-P axis) dimension.166-168 While significant compaction of nuclear fibers occurs along the A-P axis with aging, an even greater degree of compaction occurs in nuclear cataract formation.169 In most age-related nuclear cataracts, opacification begins in the lens center, and enlarges gradually. It has been noted clinically that cataracts often have reduced antero-posterior thickness in comparison to age-matched normal lenses.170 However, the rate of compaction is not constant. Morphometric analysis indicates that, in general, more compaction occurrs between young and middle-aged lenses than between middle-aged and aged lenses. Although initially surprising, this finding is temporally consistent with the onset of presbyopia near age 40. It is likely that condensation and compaction of nuclear fibers in early adulthood contribute to the lens hardening and loss of accommodative ability that characterize presbyopia. The process of nuclear fiber compaction is probably multifactoral. The most obvious structural change is the formation of accordion-like folds, which account for much of the compaction along the A-P axis. These folds begin in early adulthood and increase in both frequency and amplitude with age. The early onset of structural changes may be due to controlled modifications in the cytoskeletal171-174 and crystallin175178 proteins that accompany fiber cell maturation, and are probably necessary for long-term maintenance of fibers. In the fourth through eighth decades, cumulative age-related changes—such as water and protein loss,179,180 modifications to membrane lipids,181,182 and protein modifications183—could result in the progressive increase in compaction folds. The further increase in nuclear fiber compaction in age-related nuclear cataracts is consistent with the extensive protein modifications,184 dehydration,185 and lipid peroxidation186-188 known to occur in human cataracts. The major factor influencing compaction is most likely the loss of cytoplasmic water, which necessarily results in the loss of cell volume without reduction in cell surface area. The driving force for the loss of water may be the reported tendency of the crystallins to self-associate into larger aggregates with time, causing the nuclear cytoplasm to have a reduced osmolarity.189,190 Such changes are essential for the high concentrations of proteins in nuclear cytoplasm to exist adjacent to cortical fiber cells with relatively high water content.

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Further changes in the proteins and membrane lipids during cataract formation, specifically by oxidative damage,191 may result in more extensive condensation of cytoplasmic proteins, as well as loss of protein and membrane fragments that lead to increased fluctuations in refractive index at cellular interfaces and increased light scattering.

Biometric, Optical and Physical Changes in the Human Crystalline Lens with Aging Optical and physical properties in the lens are closely related. The crystalline lens focal length and spherical aberration are profoundly influenced by the lens surface curvatures and gradient refractive index. The continued linear growth in mass and volume of the human lens after the age of five years and throughout the remainder of the normal life-span has been well documented.192,193 Glasser found the following results: a) the human lens grows throughout life and becomes heavier and larger in cross sectional area; b) there is a significant linear increase in lens weight with age; c) the lens equatorial diameters tend to increase up to age 70 and then decrease beyond this age; d) there is a significant linear increase in anterior lens surface radius of curvature up to age 65 and then a significant linear decrease after age 65; e) there is a tendency for an increased lens thickness with age; and f) the posterior lens surface curvature has a tendency to flatten with increasing age (see Fig. 5.10). The thickness shows no significant age dependence, although it has a tendency to increase.194 Moreover, the human lens shows an exponential increase in resistance to mechanical deformation with age from birth. Even though the predominant increase in hardness occurs after the age at which accommodation is completely lost, the increasing resistance demonstrates increased hardness of the human lens, which can account for the loss of accommodation. The age-dependent changes in the responses of lenses to mechanical deformation suggest that the human lens may loose elasticity and increase viscosity with age, and that this may account for the loss of accommodation with the development of presbyopia.194

Fig. 5.10 Changes in the lens equatorial and pole-pole dimensions with age. At the time of birth, (A) the lens is an asymmetric ellipse with an equatorial diameter approximately 1.5 times the anteriorposterior dimension. In the young adult (B) the equatorial diameter is close to twice the length of the anterior-posterior dimension, illustrating the unequal growth rate in the two lens axes. (C) and (D) also show that throughout adult life, the equatorial dimension of the lens increases faster than the polar dimension. (Modified from: Al-Ghoul KJ, Nordgren RK, Kuszak AJ et al Structural evidence of human nuclear fiber compaction as a function of ageing and cataractogenesis. Exp Eye Res 72:199, 2001)


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The Change in Equivalent Refractive Index and the Lens Paradox Using Scheimpflug photography in a cross-sectional study of 100 subjects of various ages, Brown195 demonstrated that the aging lens becomes more convex. He found a substantial decrease of the radius of the anterior lens surface from about 15 mm to approximately 8.5 mm between the age 20 and 80 years of age—the posterior lens radius declined from 8.5 to about 7 mm. This would imply an increase in lens power and a tendency toward myopia in the older eye, because other dimensions of the eye do not change significantly with age.195 Between the ages of 30 and 65, however, a hypermetropic shift can be observed.196 This paradoxical feature of the decrease of the radius of curvature of the crystalline lens with age without the eye becoming more myopic has been called the lens paradox.197,198 To explain the lens paradox, there must be a compensating mechanism in the eye that prevents the eye from becoming myopic. Because neither the axial length nor corneal curvature show considerable changes with age, a decrease of the refractive index of the lens has been suggested as such a compensating mechanism. Nevertheless, so far no empirical study has been able to show a decrease of the gradient refractive index with age. Pierscionek199 found no significant age-dependent changes in the refractive gradient index measured in isolated lenses. Glasser and Campbell200 also found no evidence in support of the lens paradox in isolated human lenses or in decapsulated human lenses. Dubbelman and Vander Heiide201 confirm the existence of the lens paradox in the sense that they also found a decrease of the radius of curvature with age, but there are two major differences between their results and the results obtained by Brown.195 The first difference concerns the extent of the paradox. The average decrease of the anterior radius is 57 µm per year according to Dubbelman,201 while Brown195 found a value of about 100 µm per year, which is almost twice as large. The slight decrease of the radius of the posterior lens surface, approximately 17 µm per year, corresponds to the findings of Brown.195 The second difference concerns the absolute value of the anterior and posterior lens radius, which are both smaller than the values found by Brown. According to Dubbelman,201 the difference for the anterior surface is more than 3 mm at the age of 18, which decreases to 0.9 mm at the age of 65. During aging, the posterior radius remains on average 2.3 mm smaller, but this can be explained by the fact that Brown did not correct for the refraction of the lens itself. However, Dubbelman’s findings closely resemble the results of recent phakometric studies202,203 the findings about the lens radii correspond with the radii of the Gullstrand nonaccommodated schematic eye, and also with the earlier measurements listed by Duke-Elder and Wybar.204 To explain the lens paradox, it was suggested that, with age, the increased sharpness of curvature was balanced with increased lens thickness.197 However, Dubbelman’s calculations demonstrated that thickening of the lens only cancels out 15 percent of the decrease of the equivalent refractive index needed to prevent the eye from becoming myopic with age.201 According to the results of Brown,195 Dubbelman201 registered a small decrease of the refractive index with age. The origin of the decrease of the refractive index with age has remained unclear.

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It is suggested that the water content of the lens should increase resulting in a decrease of the refractive power. However, most recent studies on this topic do not support each other. Siebinga et al.205 measured an increase of water content in the nucleus with age, whiles Lahm et al.206 measured the opposite. Clarke et al.207 reported an increase of refractive index in the center of the lens, and another hypothesis is a change in the variation of the index gradient of the lens with age. Using the results of Brown,195 Smith et al.208 proposed a model to describe this variation. They showed that slight changes in lens refractive-index profile would be sufficient to negate the more convex shape of the lens with age. Yet, if the lens radii of the present investigation were used, an even slighter change would suffice. In conclusion, then, recent studies confirm the existence of the lens paradox, although the decrease of the radius of the anterior lens surface, using Scheimpflug photography, is smaller than in earlier studies. There is a highly significant, but small decrease of the equivalent refractive index of the lens, which explains the lens paradox.

Crystalline Lens Position Modification with Age Using Purkinje images, Tscherning209 first reported in 1898 that the human lens deviated 0.25 mm in the upper part and tilted six degrees in the infero-temporal direction. Yu Hu et al.210 showed that the crystalline lens was not aligned perfectly along the visual axis, but its effect on refraction was limited. Aging, associated with an increase in lens thickness211,212 and a more anterior position,213,214 and combined with a complex of anatomical predisposition (a short axial length, a shallow anterior chamber, and a small corneal diameter) and subsequent physiological factors, is conducive to anterior chamber angle closure and is considered to play a major role in the pathogenesis of Primary Angle-Closure glaucoma.215,216 The greater the contact between the anterior surface lens and the posterior surface of the iris, the greater the impediment to the anterior flow of the aqueous humour. In the lens position modification, the zonular apparatus plays an important role.

Zonular Apparatus Synthesis and Structural Organization of Zonular Fibers During Development and Aging Zonular fibers are a specific form of elastic extracellular matrix composed mainly of fibrillins. The major role of the zonule is to anchor the lens in the eyeball between the anterior and posterior chambers, holding the lens in the optical axis. A secondary role of the zonule is the transmission of accommodation forces from


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the ciliary body to the lens. Clinical observations and a preliminary study have shown that zonules are more fragile during aging.217

Zonular Fibers Electron Microscopy Zonular fibers are composed of large bundles of microfibrils, each with a diameter of 12 nm. Microfibrils are more or less in contact with each other, depending on their location in the zonular apparatus. In the human eye, zonular microfibrils appear to become shorter and increasingly disorganized during aging.217 This is supported by the in situ hybridization data, which clearly show an age-dependent decrease of fibrillin-1 mRNA expression as previously observed in the human aorta.218 During aging, a new fibrillar structure of fibrillin microfibrils appears with a 56-nm periodicity.217 This new structure does not have the same periodic pattern as that of classical microfibrils. Banded elastin, however, has a periodicity of less than 50 nm and has never been described in the zonular bundles of microfibrils.219 Other molecules could be involved in this structure, such as type VI collagen, which is a frequent partner of fibrillin-containing microfibrils in other organs (e.g., the nuchal ligaments). Hanssen220 suggested that these modifications could only be the result of cross-linking between fibrillin microfibrils. Crosslinks are often known to appear in long-lived proteins.221 The formation of these putative crosslinked structures, which may be due to the transglutaminase activity demonstrated in zonular fibers,222 may decrease the putative elastic properties of microfibrillar bundles. The low turnover of microfibrillar components may also act to increase this age related modification. In this regard, the appearance of these structures coincides with a physiological age-related modification of accommodation correlated with presbyopia. Gradual loss of elasticity, sclerosis of the lens, and concomitant atrophy of the ciliary muscles have all been proposed as the causes of this dysfunction.

Anterior Shift of Zonular Insertion onto the Anterior Surface of Lens with Age Anatomical Changes of the Zonular Insertion Several studies have investigated the anatomy of zonular fibers in relation to lens structure.223-227 In 1979, Farnsworth and Shyne223 showed that: ● ●

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The distance between the zonular insertion and the lens equator increased with age The circumlental space (the distance from the equator to the ciliary body) decreased with age The distance between the zonular insertion and the ciliary body remained relatively constant Sakabe228 supported the first two findings, but he found that the

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distance between the anterior zonular insertion and the ciliary sulcus increased with age. A possible causal factor suggested is the change of lens contour. With lens growth, there is an increase in both frontal and sagittal diameters,226-231 and a consequent decrease in radius of curvature of the anterior lens surface.232 Sakabe228 found that the diameter of the zonular-free zone decreased with age. Assuming that the attachment position of the zonules remains unchanged throughout life,223 and that thickness of the anterior lens capsule increases (not decreases or is stretched or both) with age,233 apparent decrease in the diameter of the zonular-free zone may be explained by increased convexity of the lens surface. This would result in an increase of anterior zonular insertion, because ciliary sulcus diameter does not change with age. The location of anterior zonular insertion appears to have clinical importance in the practice of cataract surgery, in which continuous curvilinear capsulorhexis is the technique of choice for the majority of surgeons. To create the capsulorhexis within the zonular-free zone, one should stay within the central 6.86-mm area of the anterior capsule, which is the area expected to be completely free from zonular fibers.228 More importantly, this size decreases with age. If the capsulorhexis is not located in the center of the anterior capsule, the edge of the capsular opening can extend more easily to the position of anterior zonular insertion, resulting in a failure to accomplish continuous curvilinear capsulorhexis.

Lens Metabolic Changes with Age and the Effects of External Agents Introduction The lens is exposed to the cumulative effects of radiation, oxidation and postranslational modification. The alteration of proteins and other lens molecules impairs membrane functions and perturbs protein (particularly crystallin) organization, so that light transmission and image formation may be compromised. Damage is minimized by the presence of powerful scavenger and chaperone molecules. Progressive insolubilization of the crystallins of the lens nucleus in the first five decades of life, and the formation of higher molecular weight aggregates, may account for the decreased deformability of the lens nucleus which characterises presbyopia. Additional factors include the progressive increase in lens mass with age, changes in the point of insertion of the lens zonules, and a shortening of the radius of curvature of the anterior surface of the lens. With age, there is also a decrease in light transmission by the lens, associated with increased light scatter, increased spectral absorption (particularly at the blue end of the spectrum), and increased lens fluorescence. A major factor responsible for the increased yellowing of the lens is the accumulation of a novel fluorogen—glutathione-3-hydroxy kynurenine glycoside— which makes a major contribution to the increasing fluorescence of the lens


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nucleus that occurs with age. Because this compound may also crosslink with the lens crystallins, it may contribute to the formation of high-molecular-weight aggregates and the increases in light scattering that occur with age. Focal changes of microscopic size are observed in apparently transparent, aged lenses, and may be regarded as precursors of cortical cataract formation.234,235

Age-related Changes in Calcium, Sodium, Potassium and Lens Membrane Permeability The lens optical density increase with age, and the rate of increase, is much more apparent after the age of 40 years.236 The lens also becomes increasingly colored (yellow) with age, and the intrinsic fluorescence also increases—all of these changes tend to degrade the optical properties of the lens.237 The smallest opacities are the so-called retrodots, which are present in normal, noncataractous lenses, and the frequency of their occurrence increases exponentially after 40 years of age.238 They appear to be formed from multilayered membrane vesicles and have a surprisingly low protein content, but correspondingly high calcium concentration.239,240 When there is a larger, but still localized breakdown in lens fiber structure, the formed opacities disturb the normal visual acuity, especially when they are located on or near the visual axis. Such lenses have near normal sodium and potassium concentrations but have elevated calcium levels.241 Calcium ions appear to have the ability both to disrupt the structure of the lens and also to protect the transparent, unaffected areas by sealing off the damaged fibers. The disrupting properties probably arise through activating the cysteine protease calpain, and several proteins of the structurally important lens cytoskeleton appear to be excellent substrates for degradation by the enzyme.242 The membrane potential of the normal lens appears to decline with age, particularly after the age of 40 years. The decline in voltage is accompanied by a decrease in membrane resistance, indicating that some channel mechanism is being activated in the aging lens.243 This channel is present in lens membranes and appears to permit Na+, K+, and Ca2+ to pass.244,245 It is interesting in this respect that the lens sodium and free calcium content also appear to increase after the age of 40.243 There is a remarkable agreement between the relative increase in permeability to sodium and the increase in lens optical density measured at the wavelength of peak sensitivity of the eye. Both increase more rapidly after the age of 40 and again indicate a common mechanism between alterations in the ionic and structural protein contents of the human lens.

Lens Phospholipid Changes with Age and Cataracts Human lens membrane lipid composition is related to the membrane’s organization,246 structure,247-251 and function.252-256 Age-related changes in human lens lipid composition may serve as a marker for oxidative stress and may reflect systemic oxidative

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insult, providing a window into the health of an individual.257 Species-related phospholipid differences support the idea that humans have adapted so that their lens membranes have a high sphingolipid content that confers resistance to oxidation, allowing these membranes to stay clear for a relatively longer time than is the case in many other species.257 The changes observed in the phospholipid composition of the human lens with age and cataracta were substantial—greater than that reported for any organ or disease. Biochemical studies show that, upon maturation, the cholesterol-tophospholipid ratio of lens membranes dramatically changes from 0.6-0.8 in superficial to over 5.0 in deep cortical and nuclear membranes.258 All this indicates that lens membranes, apart from those in the most superficial cortex, deviate from most cell membranes in the body. This is in line with electrophysiological studies showing that deep cortical membranes are non-leaky, have a high resistance and low capacitance and have no or restricted cell-to-cell communication.259,260 The relative and absolute amount of sphingolipids (including dihydrosphingomyelin and sphingomyelin) increase with age, while glycerolipids (including phosphatidylcholine and two phosphatidylethanolamine-related phospholipids) decrease.266 These changes are exacerbated by the presence of cataracts and are substantial—greater than the changes in lipid levels reported in any organ in association with any disease. The changes in the amount of lipids with age and cataracts support the idea that glycerolipids are selectively oxidized over lipids with fewer double bonds, such as sphingolipids. As a result of the elevation of sphingolipid levels with species, age, and cataracts, lipid hydrocarbon chain order (or stiffness) increases. Increased membrane stiffness may increase lightscattering, reduce calcium pump activity, alter protein-lipid interactions, and perhaps slow fiber cell elongation.261-264 The cause of the changes may be due to lipid oxidation. Lens glycerolipids are approximately three to four times more unsaturated than lens sphingolipid, and consequently they can be selectively oxidized more than unsaturated lipids. Conversely, de Vries265 calculated that the amount of sphingolipid per wet weight of lens—a relatively unsaturated lipid—increases with age up to approximately 45 years. Because phospholipid and cholesterol synthesis do not change within the ages studied,265 the relative and absolute changes between the sphingolipid and glycerolipid with age must be due to degradation. Recent research266 showed that the relative amount of sphingolipids (dihydrosphingomyelin and sphingomyelin) increased from 48 percent at 22 years of age to 57 percent at 69 years of age, in agreement with previous studies.261-264 With cataracts, the relative amount of sphingolipid increased to 78 percent. However, an increase in sphingolipid content in the human lens with age and cataract may indicate deleterious phospholipid oxidation. Human lens lipid composition versus age curves, exhibiting a plateau at 45 years, are remarkably similar to the curves of accommodative amplitude versus age267 and human lens membrane cation passive permeability versus age.268 Correlation does not necessarily indicate causation—however, scenarios can be envisioned in which lens membrane stiffness induced by phospholipid composi-


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tional changes directly or indirectly contribute to presbyopia and/or passive membrane permeability of cations. Recent studies suggest that, as a result of increased sphingolipid content in cataractous lenses compared with age-matched clear lenses, light-scattering increases.269 Lipids scatter 2 to 95 times more light in vitro than do crystallin proteins, indicating that they may contribute to the light-scattering intensity of the lens in vivo.269 Because lipids with ordered hydrocarbon chains have higher polarizabilities, they scatter 2.5 times more light than lipids with disordered hydrocarbon chains.269 An increase in lipid hydrocarbon chain order may also contribute to cataractogenesis indirectly by reducing the activity of the sarco/endoplasmic reticulum isoform of the calcium pump.254 Reduced pump activity could cause an increase in lens calcium levels, wich is elevated in all cataracts,272-275 and maintenance of the calcium homeostasis is essential to lens clarity. The higher sphingolipid content of cataractous lenses may also change protein-lipid interaction276,277 and slow fiber cell elongation261—two factors that could contribute to cataracts.252-261

Age-related Changes in Ganglioside Composition Lens tissues are enriched in the plasma membranes and are known to contain a relatively high concentration of gangliosides among non-neural tissues.278 Because gangliosides are mainly located at the outer leaflet of the plasma membranes, changes in their content and composition may disrupt the functions of the plasma membranes, such as ion transport, cell-to-cell interactions, transmembrane signaling, and so on.279 Ogiso et al.280 reported that human lens accumulates gangliosides in association with aging and senile cataract progression. Structural analysis reveals that gangliosides in human cataractous lenses were composed of ganglio-series gangliosides, such as GM3, GM2, GM1 and GDla, and sialyl-Lewisx containing neolacto-series gangliosides.280 Although Lewisx-containing, neolacto-series glycolipid was found to accumulate in association with aging and cataract progression, the sialyl-Lewisx gangliosides did not show much accumulation in individual lenses from subjects between 16 and 80-years of age.281,282 The content of sialyl-Lewisx gangliosides was about two to four times higher than that of Lewisx glycolipids, suggesting the possibility that the increase in Lex glycolipid is partly due to the desialylation of sialyl-Lex gangliosides.282 On the other hand, the expression of ganglio-series gangliosides increased in an agerelated manner.282 The age dependent, cataract-related increase in ganglioside content in the human cataractous lens is largely derived from the increase in ganglioseries, GM3, GM1 and GD1a.280 Age-related changes in some gangliosides and neutral GSLs, for example, GM3, GM1 and Gb3, appear to be attributable to the accumulation of lens fibers.282 Thus, age-related changes in lens glycolipids may modify the cell-to-cell interaction induced by cell surface sugar chains, leading to the initiation and progression of cataract.282

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Water Content Modifications in Lens with Aging The age-dependance of lens hydration has been studied by a number of techniques in human, as well as in animal, lenses. Lahm et al.283 found a slight age-dependent increase in total water of the intermediate and nuclear regions of human lenses, even if none of these were statistically significant. Nunnari et al.284 and Bours et al.285 also reported no significant changes in the total water content of aging human lenses. On the other hand, the decrease in bound (nonfreezable) water as a percent of the total water with age was statistically significant in each segment.283 This indicates that syneresis286 is involved in aging. In syneresis, bound water is released from the hydration layer of byopolymers and becomes free water.286 The physical process itself has a number of potential consequences. In the eye, syneresis accounts for the liquid pocket formation in the aging vitreous.287 The amount of bound water decreases with age, which supports the existence of syneresis as a factor in aging and in cataract formation286 as inferred from light-scattering measurements,288,289 and shown by differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) techniques.284-290 In aging and cataractous lenses, irreversible syneresis contributes to the turbidity of the lens by increasing the amplitude of refractive index fluctuations.291,292 Besides light scattering and thermal studies, the role of syneresis in cataractogenesis has been proven by NMR.293-296 Recent research confirmed that the amount of bound water decreases with age, which supports the existence of syneresis as a factor in aging.297 The implication is that in normal lenses without apparent turbidity, aging causes tighter packing of protein molecules, possibly leading to higher molecular species. The remaining bound water layer, however, becomes tighter, more immobilized, and therefore, potentially still a sufficient barrier to prevent aggregation and cataract formation.297

Oxidative Stress in the Aging Lens Due to its constant exposure to light and oxidants, oxidation is a major insult to the lens.298-300 Oxidative stress corresponds to an imbalance between the rate of oxidant production and the rate of its degradation.301 The complete four-electron reduction of oxygen occurs within the mitochondria, and the end product is water. A partial reduction produces superoxide and various reactive oxidative intermediates (free radicals and reactive oxygen species, or ROS including hydroxyl radicals, singlet oxygen radicals, and hydrogen peroxide).302 Besides these endogenous oxidants, other sources are food, air pollutants, tobacco smoke, exercise, ionizing radiation, IR and, of course, the sun.303 Although the organism adapts by preventing undesirable reactions with its endogenous and partly redundant antioxidant defense (glutathione superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase) and repairing


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damaged molecules and tissues, the few molecules and undesirable reactions that are not prevented or repaired will accumulate over time and be deleterious in the long term.303 All of these conditions will lead to the formation of excessive oxidants and oxidative stress. Oxidative stress is countered by antioxidants that are defined as substances that, at low concentrations relative to the substrate, inhibit the damage to the structural and functional molecules of the body, namely proteins, lipids, carbohydrates, and DNA.303 Antioxidants function by several possible mechanisms: ●

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Scavenging of free radicals involved in chain reactions; tocopherol acting in the lipid phase Regeneration of other antioxidants; ascorbate reduces tocopheryloxy radical to tocopherol by donating an atom Reacting with initiating radicals or oxidants (catalase with hydrogen peroxide) Chelating or sequestering transition metal catalysts which are pro-oxidants; albumin or polyphenols with cupric ion Inhibiting or activating an enzyme; tocopherol and polyphenols inhibit tyrosine kinase and ascorbate activates nitric oxide synthase303

There is a considerable body of evidence to indicate that the ability of the human lens to withstand oxidative attack actually declines with age because the overall level of glutathione decreases and the important enzyme glutathione reductase becomes less stable.304 Because it is well-known that lens nuclear cataracts involve protein oxidation,305 there is now, therefore, the possibility that nuclear and cortical cataracts, with their totally different aetiology and morphological appearance, may both arise from oxidative mechanisms—one taking place primarily at the surface membranes, and the other within the nuclear proteins. This may help explain why the majority of senile cataracts are, in fact, mixed in form, with contributions from both nuclear and cortical changes.306 Recent epidemiological studies of cataracts do suggest that a high intake of antioxidants—either in the diet, or in the form of supplements—does confer a considerable protective effect.307 Age-related nuclear (ARN) cataracts are associated with a loss of glutathione in the center of the lens and extensive modification of the nuclear proteins that include coloration, oxidation, insolubilization, and crosslinkin g.308Accumulation of oxidatively damaged proteins is causally related to the formation of cataracts298,304,309,314 and many other age-related debilities.315-317

Age-related Decline in Ibiquitin Conjugation The extent of accumulation of oxidatively damaged proteins depends on both the rate of production and on the efficiency of removal of the oxidatively damaged proteins.318,319 In most cells, intracellular proteolytic enzymes selectively remove the oxidized or damaged proteins.320-324 Therefore, proteolytic capabilities are considered as secondary defense systems, which can avert or delay the accumulation of damaged proteins.318,322,325,328

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The ubiquitin-dependent proteolytic pathway is a primary proteolytic system which is involved in the selective degradation of oxidatively damaged proteins in various types of cells or cell-free systems,329-333 and a substantial amount of literature indicates that the ubiquitin-dependent proteolytic system functions in lens cells, as well.332-334 Shang et al.335 showed that: ●

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lenses—especially the nuclei of lenses—undergo dramatic changes with aging, including a decreased level of ubiquitin conjugates and decreased ubiquitin conjugation activity there is an increase in endogenous ubiquitin-protein conjugates and enhanced ubiquitin conjugation activity in response to oxidative stress in each developmental zone of lenses the ability to mount a ubiquitin-dependent response to oxidative stress decreases in the old lens—especially in the nucleus of old lenses

This attenuated ability to enhance the ubiquitin conjugation activity with oxidative damage may be associated with the observed accumulation of damaged proteins in old lenses. The progression of cataractogenesis in the normal aging population can be characterized as a continual increase in the intensity of light scattered from the lens. An important molecular mechanism for such light scattering is, in fact, the condensation of protein into aggregates.335 Protein insolubilization in human lenses during aging and cataracts is well documented.336-339 Garner et al.340 showed the association of gamma crystallin with the membrane protein component of human cataract lenses. Recent studies341,342 indicate that the fiber cell plasma membrane has a high capacity to bind a-crystallin in a nonsaturable manner. This association may play an important role in triggering the further interaction of crystallins with plasma membranes in normal aging and cataract formation, which results in massive protein insolubilization.343 The basis for the great association of crystallins with lens membranes during aging and cataractogenesis is unknown, but might involve the interplay of two broad mechanisms. Modification of membrane structures could enhance its protein-binding characteristics, modification of crystallin structure could increase their affinity to bind, and a combination of altered membranes and crystallin structure might be important for association. This increase is an exponential function of age and has a time constant that, on average, is approximately 35 years.344

Hormonal Influence on Lens with Aging The human lens continues to grow throughout life and in all decades from 10 to 70 years—the male lens is heavier than its female counterpart.345 These age-related differences between males and females are interesting because not only do their relative susceptibilities to cataract change with age, but their response to physical trauma also does.


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A role for female hormones in protecting against cataracts has been suggested by recent epidemiological studies. Below the age of 50, the prevalence of cataracts seems to be similar in males and females,346,347 but it increases in postmenopausal women.346-348 Moreover, postmenopausal women on hormone replacement therapy, or younger women taking oral contraceptives, display a decreased prevalence and severity of cataracts.349-352 In addition, the prevalence and severity of certain forms of cataracts are lower in postmenopausal women on hormone replacement therapy involving administration of estrogen, with or without progesterone, than in those who are not undergoing hormone replacement therapy. Hales et al.353 showed that transforming growth factor-β (TGFβ)—a multifunctional growth factor that is present in the aqueous and vitreous humours354—induces rat lenses in culture to develop opacities and other changes that have many features of human subcapsular cataracts. Hales also showed that estrogen protects against cataracts. Interestingly, lenses from male rats are more susceptible than those from female rats and, furthermore, the latter receive added protection from TGFβ if estrogen is also present in the medium.355 The molecular mechanisms underlying the cataractogenic effect of TGFβ are poorly understood, but TGFβ is known to induce transdifferentiation of lens cells so that they produce at least two types of foreign protein—smooth muscle actin and collagen types 1 and 3.356 Neither of these is synthesized in significant amounts by normal lens cells, but can be detected in certain cataracts122 and in cells, giving rise to PCO.358 The TGFβ stimulated production of abnormal intracellular and extracellular proteins disrupts the homogeneous structure of the anterior epithelium, and lightscattering, multilayered cell aggregates are produced.355 Not only do male and female lenses differ in their relative sensitivity to TGFβ, but they also respond differently to mechanical stress. Weale359,360 carried out a quantitative study of the birefringence of male and female lenses, and although the overall pattern is the same, the effect of external stress on the birefringence pattern measured in vitro is different in males and females. Weale360 measured the greatest stress that could be given before an irreversible change in birefringence occurred and, although in both cases the magnitude of the reversible stress declines with age, the rate of decline appears to be steeper with female lenses. Furthermore, Weale identified a number of female lenses in which the merest mechanical stress induced irreversible birefringence changes, and he concluded that this pointed to a subtle structural difference between male and female lenses.360

The Effect of Physical Agents on the Aging Lens Ultraviolet Radiation Sunlight is the principal source of ultraviolet radiation (UVR) for most of the world’s population. Depletion of the stratospheric ozone increases the intensity of UVR. UVR is considered one of the major risk factors for cataracts,361-364 and

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several studies have shown that sunlight increases the risk of cortical cataracts.365-368 Effects of UVR may be analyzed from different perspectives (e.g., at the molecular, cellular, tissue, individual, population, and ecosystem levels).369 UVR damages the lens by disturbing cell proliferation in the lens epithelium,370 by altering kinetic properties of enzymes in the energy metabolism,371 by increasing insoluble and decreasing soluble protein,372,373 by inducing unscheduled DNA synthesis,374 and by disturbing the sodium potassium balance and thereby the water balance in the lens.375,376 One of the major difficulties in epidemiologic studies has been quantification of exposure to UVR from the sun. The consequences of UVR exposure on the epithelium must be considered both in terms of mutagenic as well as cytotoxic effects.377,378 The single layer of epithelium is the first physical and cellular (biological) defense against electromagnetic radiation in the ocular lens.379 Some of the direct effects of UVR exposure on cultured cells have been reviewed in detail.380 UVR exposure results in unscheduled DNA synthesis and repair.381-383 The human lens epithelium accumulates insults due to UVR exposure in its genome over a period of time that are manifested in the aged lens.384 There is, of course, an age dependence of UVR damage to different molecular species, including enzymes such as hexokinase, phosphofructokinase, isocitrate dehydrogenase, and malate dehydrogenase.385 Loss of hexokinase386 would result in the inability of the lens to produce NADPH and downstream antioxidants. It is conceivable that proteins (such as Na+ / K+) ATPase, cytoskeletal elements, membrane proteins), which are dependent on –SH function will be damaged by exposure to increased oxidants. In addition to intensity of sunlight, the ocular dose depends on other factors, such as the amount of time spent outdoors, the environment, the use of ocular protection, and the use of hats.363,364,387-389 In earlier studies, safety limits for UVRB induced cataract have been based on a dichotomous dose-response model, assuming that the outcome of UVR-B exposure is limited to a binary response: cataract/no cataracts.390 In those studies, cataracts were measured qualitatively with a slit lamp, with a grading scale. It has recently been shown with quantitative measurements of cataracts, however, that UVR-B-induced cataracts has a continuous dose-response function.391 For this reason, a new concept—maximum acceptable dose (MAD) for avoidance of UVR-B cataract—was developed for estimation of UVR-B toxicity in the lens.392 Based on the dose-response function, MAD is defined as the dose corresponding to a limit for pathologic forward light scattering. The limit for pathologic forward light-scattering is settled arbitrarily, based on the frequency distribution of light scattering in normal unexposed lenses. The limit is defined so that a certain fraction (α) of normal unexposed lenses scatter light in the forward direction to an intensity above the limit. The magnitude of the fraction is a parameter that has to be settled and is given as an index to MAD1-α. The high rate of cell division in the germinative zone in the young lens may render the young lens more sensitive to UVR-B–triggered DNA fragmentation. Further, the young lens requires more protein synthesis that includes a part of the young lens that is biologically more important than that of the older lens.372,393 Lerman394 exposed young (first decade) and old (seventh decade) normal human lenses to low level (< 0.1 kJ/cm2) broad band UVR-B (300–400 nm), and found that


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the γ-crystallins were significantly affected by UVR-B in young lenses, while the aged lens proteins appeared to be relatively unaffected by this degree of UVR-B exposure. The finding that MAD for avoidance of UVR-B-induced cataracts strongly depends on age implicates that, in the future, age should be considered. Until better data are available, the current data should be considered in toxicity estimates for avoidance of UVR-B cataracts after exposure to the sun, as well as to artificial sources.395

Medications and Cataracts Oral corticosteroids are known to cause cataracts, but the role of many other systemic medications in cataract etiology is uncertain.396 Several case reports suggest that allopurinol may cause cataracts,397-399 but epidemiologic studies are inconsistent.400-402 Case series of institutionalized patients suggest that phenothiazines are associated with cataract development,403,404 but only one population-based study has examined these medications.405 There have been several studies of diuretics and cataracts, with some finding a protective effect,406 and others a harmful one.407-409 In one case series,410 a high proportion of patients on amiodarone had cataracts, but this association does not appear to have been studied by other investigators. There are biological reasons why some drugs used to lower serum cholesterol might cause cataracts, but such an effect has not been shown.411 Finally, the possibility that aspirin lowers the risk of cataracts has received a great deal of attention in recent years, but studies are far from consistent.396 Different types of cataracts have different etiologies, and so it is important to distinguish between types of cataracts when studying cataract risk factors. The Blue Mountains Eye Study is a large population-based study in which cataract diagnosis was based on grading of lens photographs.412 An association between inhaled steroids and cataracts was found in this study population. Four medications were associated with increased cataract prevalence—phenothiazines were associated with nuclear cataract; amiodarone with cortical cataract; and aspirin and mepacrine (an antimalarial medication that was used extensively during World War II) were associated with posterior subcapsular cataract.412 Aspirin is the only one of these four medications that is used extensively in the community. Most medications studied were not associated with cataracts, including allopurinol, cholesterollowering medications, thiazide diuretics, frusemide, beta blockers, calcium-channel blockers, benzodiazepines, and nonsteroidal antiinflammatory drugs.412

Aspirin and Nonsteroidal Anti-inflammatory Drugs There have been at least 15 previous studies of the association between aspirin use and cataracts,396,414-427 including three randomized trials.420-422 None of the randomized trials found any protective effect of aspirin. Of the 12 observational studies,

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six found that cataracts were less frequent among aspirin users.414-418 These positive studies had methodological flaws that could explain their findings, including failure to adjust for prior steroid use and other confounders, inadequate control groups, and use of cataract surgery cases. In the The Blue Mountains Eye Study, long-term aspirin use was associated with increased prevalence of posterior subcapsular cataract.412 At least three other studies have found a slightly higher risk of cataract in aspirin users.405,426,427 Based on the combined evidence from nearly 20 years of research, it is possible to conclude that aspirin does not protect against the development or progression of cataracts. Hankinson et al.428 found some evidence that nonsteroidal anti-inflammatory medications might be associated with increased risk of cataracts, but The Blue Mountains Eye Study found no such association. Interestingly, this study did find that persons with self-reported osteoarthritis were more likely to have had cataract surgery than persons without osteoarthritis.

Diuretics and Antihypertensives Harding and van Heyningen406 reported that thiazide diuretics were used less frequently by patients who underwent cataract surgery than control subjects. More recently, the Beaver Dam Eye Study found that use of thiazides was associated with lower prevalence of nuclear cataracts and increased prevalence of posterior subcapsular cataract.429 Several other studies have found that use of diuretics was associated with increased risk of cataracts.407-409 The Blue Mountains Eye Study did not find convincing evidence of any harmful or beneficial effects of diuretics on the lens.412 Although frusemide was associated with increased prevalence of cortical and posterior subcapsular cataracts in age- and gender-adjusted analyses, these associations appeared to be because of confounding. The Blue Mountains Eye Study found that long-term users of potassium-sparing diuretics might be at increased risk of cataract. The Beaver Dam Eye Study also found a raised incidence for potassium-sparing diuretics, but this was not statistically significant.429 A cataractogenic effect of potassium-sparing diuretics is biologically plausible, because these diuretics disturb sodium transport across the lens fiber membrane.430,431 The calcium-channel blocker nifedipine has been associated with increased risk of cataract extraction406 and angiotensin-converting enzyme inhibitors with decreased risk of nuclear cataracts.429 Neither of these medication types was associated with cataract in the Blue Mountains Eye Study. Confounding by hypertension and other cardiovascular conditions is a potential problem in studies of cataracts and antihypertensive medications,409,429 including diuretics. The Blue Mountains Eye Study addressed this problem by using statistical techniques to check for history of cardiovascular disease, and by repeating analyses in normotensive persons. After confounding had been adjusted for, none of the antihypertensive medications studied were statistically significantly associated with cataracts.


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Cholesterol-lowering Medications The normal lens membrane contains a very high concentration of cholesterol, most of which is actually synthesized in the lens.411 Hence, drugs that reduce cholesterol synthesis could cause cataracts. Laboratory experiments have found that simvastatin has a particularly strong inhibitory effect on lens cholesterol synthesis.411 The Blue Mountains Eye Study, however, found no association between any type of cataract and use of simvastatin (or any other cholesterol-lowering drug).412 Previous epidemiologic studies of simvastatin and lovostatin have similarly found no association with cataracts.411,432

Allopurinol Several case series have noted that persons treated with allopurinol seem to have characteristic lens changes,397-399 perhaps due to photobinding by allopurinol in the lens.398 In the Lens Opacities Case-Control Study, persons using gout medications (most likely to be allopurinol) had increased prevalence of cataracts.400 Two other epidemiologic studies found no relationship between allopurinol and cataract extraction.401,402 In The Blue Mountains Eye Study, use of allopurinol for 10 or more years was associated with posterior subcapsular cataracts in the initial analyses, but there was no association after adjusting for confounders. Previously observed associations between the use of allopurinol and cataracts may have been because of the higher prevalence of risk factors for cataracts among these persons.

Antimalarials The Blue Mountains Eye Study found a strong association between posterior subcapsular cataract and use of mepacrine—a 9-aminoacridine that was used extensively for malaria prophylaxis by Australian soldiers in the Pacific during World War II—and these data support studies conducted in the 1950s, which reported a high prevalence of cataracts in persons taking chloroquine.433

Phenothiazines An association between the phenothiazine chlorpromazine and cataracts was first reported in the 1960s in patients living in psychiatric institutions.434,435 Two epidemiologic studies have found that use of psychotropic medications is associated with cataracts, but these studies did not investigate specific classes of medications.434,435 The only epidemiologic study to date of phenothiazines and cataracts among persons living in the community was conducted by Isaac et al.405

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The increased frequency of cataracts among phenothiazine users in that study could have been caused by selection bias, because only cataract extraction cases were studied (phenothiazines have an anticholinergic effect that can cause blurred vision, which might lead to increased eye examinations and detection of cataract). In The Blue Mountains Eye Study, which avoided this selection bias by basing cataract diagnosis on grading of lens photographs, phenothiazines were associated with increased prevalence of nuclear cataracts.412

Amiodarone This antiarrhythmic drug was associated with an increased prevalence of cortical cataracts in the Blue Mountains Eye Study. This is consistent with reports by Flach et al.410 of high rates of subcapsular cataracts in patients treated with amiodarone.

A Clinical Approach to Lens Modifications with Aging Introduction With the new instruments available today in clinical practice, it is possible to study the correlation between bio-densitometric changes, optical high-order aberrations (HOAs), and modulation transfer function (MTF) of the crystalline lens that take place during the aging process. We have presented a comprehensive study436 in which these changes have been measured in different age groups of patients without cataracts, to evaluate ways in which morphology and optical performance of the human crystalline lens degrade with age. All the measurements are simple, objective, and performed quickly, requiring minimum cooperation from the subject.

Lens Bio-densitometric Changes Through Aging Scheimpflug Photography Features To evaluate lens morphology and densitometric data, a Scheimpflug slit lamp (EAS 1000, Nidek, Japan) was used.437 In this technique, slit-lamp photography measures light that is reflected anteriorly from the lens to the camera. To record a slit image, an alignment system is coupled to a television monitor, and a fixation light is placed to lie along the optical axis of the slit projection lens. A photograph is taken using a flash intensity of 200 W-seconds. Density is measured by optical density units that are EAS 1000-specific. The resulting cross-sectional image of the anterior chamber and lens is displayed on a monitor for evaluation by the operator. If satisfactory, the image can be transferred to the computer for analysis. To quantify nuclear lens density, linear densitometric analysis of the image was performed in


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Fig. 5.11 Photographs using a flash intensity of 200 W-seconds in a Scheimpflug slit lamp. (A) Eight-year-old subject. (B) Eighty-year-old subject. Linear densitometric analysis to quantify nuclear density. 1, 2, 3 = densities at the embryonic nucleus, anterior fetal nucleus, and posterior fetal nucleus, respectively

Fig. 5.12 Photographs using a flash intensity of 200 W–seconds in a Scheimpflug slit lamp. (A) Eight-year-old subject. A. = anterior; R = radius. (B) Eighty-year-old subject. Axial biometric analysis to quantify lens thickness. 1, 2, 3 = densities at the embryonic nucleus, anterior fetal nucleus, and posterior fetal nucleus, respectively

our study. Density was measured at the embryonic, anterior, and posterior fetal nuclei (see Fig. 5.11). To quantify lens thickness, an axial biometric analysis of the image was performed (see Fig. 5.12). Results from Scheimpflug Photography The correlation with age on nucleus density is represented in Figs. 5.13 to 5.15. Densities of embryonic, anterior fetal, and posterior fetal nuclei show a positive correlation with aging after the age of 40. The scatterplots of embryonic and anterior fetal nuclei clearly show a turning point around the age of 40 years, after which densities of the nuclei show an increase with age. The relationship between age and crystalline lens thickness is shown in Fig. 5.16. As exhibited, crystalline lens thickness increases significantly with age in a linear mode.

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Fig. 5.13 (A) Embryonic nucleus (flash intensity, 200 W–seconds) as a function of age. A positive correlation was found after the age of 40 years (r=0.762, P < 0.0001). (B) Embryonic nucleus (flash intensity, 200 W–seconds) in four age groups. The mean difference using Bonferroni multiple comparison is statistically significant for Groups 2 and 3 (P < 0.002) and for Groups 3 and 4 (P < 0.0001). Error bars, minimum and maximum of the 95 percent confidence interval

Fig. 5.14 (A) Anterior fetal nucleus (flash intensity, 200 W-seconds) as a function of age. A positive correlation was found after the age of 40 years (r =0.764, P < 0.0001). (B) Anterior fetal nucleus (flash intensity, 200 W-seconds) in four age groups. The mean difference using Bonferroni multiple comparison is statistically significant for Groups 2 and 3 (P < 0.0001) and for Groups 3 and 4 (P < .0001). Error bars, minimum and maximum of the 95 percent confidence interval


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Fig. 5.15 (A) Posterior fetal nucleus (flash intensity, 200 W-seconds) as a function of age. A positive correlation was found after the age of 40 years (r = 0.756, P < 0.0001). (B) Posterior fetal nucleus (flash intensity, 200 W-seconds) in four age groups. The mean difference using Bonferroni multiple comparison is statistically significant for Groups 2 and 3 (P < 0.0001) and for Groups 3 and 4 (P < 0.0001). Error bars, minimum and maximum of the 95 percent confidence interval

Fig. 5.16 (A) Crystalline lens thickness as a function of age. A positive linear correlation was found (r = 0.679, P < 0.0001). (B) Crystalline lens thickness in four age groups. The mean difference using Bonferroni multiple comparison is statistically significant for Groups 1 and 2 (P < 0.002) and for Groups 2 and 3 (P < 0.004). Error bars, minimum and maximum of the 95 percent confidence interval

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Optical Changes of the Human Crystalline Lens Through Life Modulation Transfer Function The MTF value quantitatively characterizes the performance of the optical system of the eye. For years, the photography industry has used MTF values to measure the optical quality of lenses. The MTF is the ratio of the image wave contrast to the object wave contrast. The higher the MTF value is, the higher the quality of the image is after it passes through a lens. Optical quality was studied using the MTF for monochromatic light. In our study, the MTF was measured436 with the Optical Quality Analysis System (Visiometrics S.L., Terrassa, Spain)—a recent instrument based on the double-pass technique and developed to perform an objective optical quality-ofvision evaluation. The double-pass technique is based on recording images of a point source after reflection in the retina and a double pass through the ocular media.438 With this configuration, therefore, the ocular point-spread function (PSF) can be obtained. The point spread function (PSF) defines the propagation of electromagnetic radiation or other imaging waves from a point source or point object. The degree of spreading (blurring) of the point object is a measure for the quality of an imaging system. From the point-spread function images, the MTF that yields the relationship between the contrast of an object and its associated image as a function of spatial frequency was obtained, computing the modulus of the 2-dimensional Fourier transformations of the point spread function. The 1-dimensional MTF was calculated as the radial projection (averaging over all orientations) of the 2-dimensional MTF (see Figs.5.17 to 5.19). Measurements were done with a 5-mm pupil. Data at 0.5 MTF represent the spatial frequency (cycles per degree) in which the image contrast is degraded 50

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0.00 0.00 8.12 16.24 24.36 32.49 40.61 48.73 c/d

0.00 0.00 8.12 16.24 24.36 32.49 40.61 48.73 c/d

Fig. 5.17 Curves of spatial frequency and modulation transfer function (MTF) obtained using the Optical Quality Analysis System in (A) an eight-year old subject, (B) a 30-year old subject, and (C) an 80-year old subject. c/d = cycles per degree


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Fig. 5.18 A graphic correlation between 2D - 3D PSF and MTF of the crystalline lens with the OQAS

percent relative to the object contrast. Data at 0.1 MTF represent the spatial frequency in which the image contrast is degraded 90 percent relative to the object contrast, and correspond to the maximum resolution of the optical system. The OQAS creates two- and three-dimensional retinal images (or maps) that describe a patient’s total optical system (Fig. 5.20).

MTF Results Measured with the Optical Quality Analysis System The error bar graphs shown in Fig. 5.21 represent 0.1 and 0.5 MTFs in different age groups. The 0.5 MTFs are 4.317 for Group 1, 5.384 for Group 2, 3.501 for Group 3, and 3.046 for Group 4. A significant difference is seen between the age groups of 21 to 40 and 41 to 60 for 0.1 and 0.5 MTFs. The 0.1 MTF decreased with age from 18.557 to 10.100 cycles per degree.

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Fig. 5.19 Graphic representation of two- and three-dimensional retinal images (or maps) that describe a patient’s total optical quality in a cataractous eye

Wavefront Analysis Ocular and corneal wavefront errors were measured (see Fig. 5.22) with a Hartmann–Shack aberrometer (Wavefront Analyzer, Topcon, Tokyo, Japan). Measurements were taken for 4- and 6-mm pupils. The Wavefront Analyzer gives us the total ocular and corneal aberrations for 4- and 6-mm pupils, coma-like Zernike polynomials (Z3i+Z5i) and Z4i+Z6i for a 6-mm pupil, and ocular and corneal Zs for 4- and 6-mm pupils. Zernike mode Z3-3 through Z33 plus a fifth-order Z (Z5-5 through Z55) corresponds to coma-like aberrations. From ocular and corneal aberrations, intraocular aberrations can be obtained. Intraocular aberrations result from the difference between ocular and corneal aberrations, and they are due more to the crystalline lens and less to the posterior corneal surface.


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Fig. 5-20 OQAS 2-D and 3-D representation of PSF in normal and cataractous eyes

Results on Ocular and Corneal Wavefront Errors Measured with a Hartmann–Shack Aberrometer Total ocular and corneal HOAs of a 6-mm pupil are measured as a function of age. Corneal HOA shows a weak statistically significant variation with age. Ocular HOA increases linearly with age. As shown in the scatterplot, ocular HOA is smaller than corneal HOA until 30 to 40 years of age. In the 40s, ocular HOA is similar to corneal HOA, and it increases in older subjects. The same result can be seen for ocular and corneal Z4i+Z6i aberrations. Corneal coma-like aberrations were not statistically significant, and ocular coma-like aberrations show a positive linear correlation with age (see Fig. 5.23). Intraocular spherical aberration (Z40) for a 6-mm pupil (see Fig. 5.24) shows a positive linear correlation with age. Intraocular coma aberration (Z3-1), on the contrary, shows a negative linear correlation with age (see Fig. 5.25).

Conclusions In our study, nucleus density showed a positive correlation with age, after 40 years, for embryonic, anterior fetal, and posterior fetal nuclei. When different age groups

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Fig. 5.21 (A) Spatial frequency for 0.1 modulation transfer function (MTF) in four age groups for a 5-mm pupil. The mean difference with Bonferroni multiple comparison is statistically significant for groups 2 and 3 (P < 0.009). (B) Spatial frequency for 0.5 MTF in 4 age groups for a 5-mm pupil. The mean difference using Bonferroni multiple comparison is statistically significant for groups 2 and 3 (P < 0.004). c/d = cycles per degree. Error bars, minimum and maximum of the 95 percent confidence interval. (Group 1 included subjects from 8 to 20-years old (n = 15); Group 2, subjects from 21 to 40 (n = 20); Group 3, subjects from 41 to 60 (n = 21); and Group 4, subjects from 61 to 80 (n = 16)

are analyzed, we can see that nucleus density does not increase before the age of 40, after which nucleus density increases linearly with age. As a result of the continuous production of new fibers, the aging lens becomes thicker. We found a correlation between age and overall lens thickness, as was also found by Kashima et al.449 In our study, crystalline lens thickness increases from eight years of age to the age of 40, after which the increase in lens thickness is not statistically significant. Due to the anatomical changes that take place with aging, scattering and aberrations of the crystalline lens are expected to increase. The main contributors to the overall aberrations in the eye are the tears, anterior and posterior surfaces of the cornea, and crystalline lens. So, if the aberrations of the crystalline lens increase, total ocular aberrations will increase as well. Several previous studies have reported an increase in overall eye aberrations with aging.439-442 In our study, overall eye HOAs increased linearly with aging, as previously reported in the literature. This increment in overall ocular HOAs is not due to corneal HOA, which shows a very weak correlation with age. Before the age of 30 years, overall HOA and Z4i+Z6i were significantly larger for the cornea than for the entire eye, which suggests that the lens compensates for part of the corneal aberrations. The corneal and lens aberrations show, in fact, a trend to compensate each other.443 In our study, we found that this mechanism is disrupted in the older eye as a consequence of normal aging.


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Fig. 5.22 Total ocular and corneal high-order aberrations (HOAs) as a function of age for a 6-mm pupil. For ocular HOA, a positive linear correlation was found (r = 0.511, P < 0.0001). For corneal HOA, a weakly positive correlation was found (r = 0.248, P < 0.036)

According to our data, the turning point for the coupling of these two optical systems (cornea and the entire eye) seems to appear around 40 years of age. The changes in the optical performance of the crystalline lens with aging should be related to the anatomical changes (nucleus density and thickness) found. With previous studies, authors have investigated the correlation of the development of aberrometric changes with aging.439-442 In such studies, the Zernike polynomials that were used differed from those analyzed in this study. We found a linear correlation between intraocular spherical aberration and age. Because the main contributor to intraocular aberration is the crystalline lens, we can assume that spherical crystalline lens aberration increases with age. On the other hand, intraocular coma aberration (Z3-1) decreases with age. In this study, we investigated corneal, ocular, and intraocular HOAs in the same patient. We studied the overall corneal and ocular HOAs, corneal and ocular Z4i+Z6i, intraocular spherical aberration (Z40), and intraocular Z3-1. We found a positive linear correlation for all the aberrations studied except for intraocular Z3-1, which shows a negative linear correlation. Our results confirm that the increase in corneal aberration is too small

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Age (years) Fig. 5.23 Ocular and corneal Z4i+Z6i (Zernike polynomials) aberration as a function of age for a 6-mm pupil. For ocular aberration, a positive linear correlation was found (r =0.368, P 0.05 (n.s.).23

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Results All our morphological results are reported in Figs. 10.1 through 10.5, while the histochemical results are summarized in Table 10.1. Fig. 10.1 shows the normal structure of the human retina in a young subject. The trineuronal intraretinal chain is male by photoreceptors, bipolar and ganglion cells. The RPE is detached, by experimental manipulations, from the other retinal layers. Fig. 10.2 shows, as appears in a digital angiography, the retinal fundus in a young subject. In this image, we can see the nasal superior and inferior branches of the ophthalmic artery. These vessels show a normal caliber, without signs of aging or diseases. Fig. 10.3 shows the same image as in Fig. 10.2, but comes from an old man. The retinal vessels show an increased caliber, a snake-like running and numerous dystrophic zones in comparison with RPE. All these findings can be considered as age-related changes. The major age-related changes concern the metabolism of the lipids. In fact, as said above, the melanin in young subjects can alter the results because of the high pigmentation due to the melanin granules and it is present in all the cells of RPE.

Fig. 10.1 Light microscopic image of a normal human retina in young and healthy subjects . The RPE is detached from the other retina layers. RPE = retinal pigmented epithelium, P = photoreceptors, B = bipolar cells, and G = ganglion cells (Magnification 1600x; Calibration bar 100 µm)

10 Aging of the Retinal Pigmented Epithelium

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Fig. 10.2 Retinal fundus in a young (22 years of age) and healthy subject as appears in a digital angiography of the ophthalmic artery. In comparison with the head of the optic nerve, the ophthalmic artery is branched in four divisions—one for each quadrant of the retina. In this image, the nasal superior and inferior branches are evident. (field 40° corresponding to a magnification of about 5x)

Fig. 10.3 Retinal fundus in an older (70 years of age) subject. The retinal vessels show an increased caliber. We can see many dystrophic zones in comparison with the RPE (field 40° corresponding to a magnification of about 5x)

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In old subjects, however, the RPE appears depigmented owing to the decrease of the melanin granules. For this reason the samples coming from young subjects can be previously depigmented. Fig. 10.4 shows that the RPE of a young healthy man after depigmentation (for the decrease of the melanin granules) was stained with Sudan Black B and bromine acetone for the detection of the phospholipids. The phospholipids are increased if compared with old subjects (A1). On the contrary, oil red O stains neutral lipids in a young (B) and/or in an old (B1) man. There is a strong increase of neutral lipids with age. Fig. 10.5 shows that Sudan black B dyes the total lipids in a young (A) and/or in an old man (B). The total lipids are increased with age. Table 10.1 shows the values of QAI of lipids in RPE for young and /or old subjects. Three classes of lipids are dyed—total lipids, phospholipids, and neutral lipids. After the specific coloration, a quantitative analysis of images was performed and results were expressed in conventional units (see Methods section). The probability or significance index was calculated, comparing the results obtained in young subjects versus older ones. All the tabled results show a high statistic significance (p < 0.001).

Fig. 10.4 Light microscopy of the RPE in a 19-year old eye donor (A and B) and/or a 75-year old donor (A1 and B1). The two figures A and A1 are stained with bromine-acetone-sudan black B (phospholipids), and those on the bottom (B and B1) are stained with oil red (neutral lipids). It can be seen that the intensity of staining with both systems increases with age. Therefore, both phospholipids and neutral lipids show a progressive age-related increase. (Magnification 400x; Calibration bar 10 µm)

10 Aging of the Retinal Pigmented Epithelium

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Fig. 10.5 Light microscopy of the RPE in an 19-year old eye donor (A ) and/or a 75-year old donor (A1). Both A and A1 are stained with bromine-sudan black B. This method stains all classes of lipids. It can be seen that the intensity of staining increases with age. Therefore, the total lipids show a progressive age-related increase. (Magnification 400x; Calibration bar 10 µm)

Table 10.1 QAI of lipids in the RPE of Young and old objects Class of lipids and staining Young

Old

Total Lipids Sudah Blach B Phospholipids Sudan Black B plus Bromine acetone

44.5 ± 3.1* 25.7 ± 2.3*

31.3 ± 2.2 62.6 ± 4.4

Neutral lipids Oil Red O 30.4 ± 2.9 All the value are expressed as Conventional Units (C.U.) ± SEM (see methods) *P

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