The Human Eye in Presbyopia: Changes in the Lens and Ciliary Body with Age and Accommodation DISSERTATION Presented in Partial

Changes in the Lens and Ciliary Body with Age and Accommodation

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

Kathryn Richdale

Graduate Program in Vision Science

The Ohio State University

2011

Dissertation Committee:

Professor Karla Zadnik, OD, PhD, Advisor

Professor Donald Mutti, OD, PhD

Associate Professor Petra Schmalbrock, PhD

Kathryn Richdale

2011

Abstract

Despite the ubiquitous nature of presbyopia, numerous questions remain in our understanding of the mechanism of this age-related decline in focusing ability. Previous research was limited by the inability to visualize the internal structures of the human eye in vivo. Studies have been confounded by the use of animal models not entirely similar to humans; pharmacological agents known to affect the normal physiological response; post mortem effects inherent to the use of cadaveric eyes; and ex vivo work using non- physiological forces and discounting the role of the iris, vitreous, and choroid.

The purpose of this dissertation was to objectively quantify changes in the lens and ciliary body of the human eye in vivo in a single cohort of patients, before, during and after onset of presbyopia. This was achieved through the development of optical coherence tomography and magnetic resonance imaging techniques and computer-based image segmentation programs.

Ninety-two patients between the ages of 30 and 50 years were enrolled in the study. This dissertation reports the analysis of a subset of 26 emmetropic subjects.

Regressions of ocular biometric data demonstrate the significant changes in lens shape and size with age. Multivariate mixed model analysis reveals the contributions of both the lens and ciliary body in accommodative function.

Acknowledgments

Through my many academic and personal endeavors, I have been graced with the unwavering support of my sister and brother-in-law, Kristin and Edward Stellingwerf, and my parents, Peter and Joellen Richdale.

I am fortunate to have worked with several exceptional mentors and collaborators at The Ohio State University and beyond, including Donald Mutti, Loraine Sinnott, Mark

Bullimore, Melissa Bailey, Peter Wassenaar, Petra Schmalbrock, Chiu-Yen Kao, Jun Liu,

Samuel Patz, and Adrian Glasser. It is only through their generous support and unique expertise that this project came to fruition.

Finally, I would like to thank my PhD advisor, Karla Zadnik, who was the impetus behind my switch from patient care to patient-based research. For showing me the pleasure and fulfillment of a career in academia, I am eternally grateful.

This work was supported by a National Institutes of Health Mentored Patient-Oriented

Research Career Development award (K23, NEI EY019097), American Optometric

Foundation Ezell Fellowships, and an unrestricted grant from Bausch + Lomb

(Rochester, New York).

iii

Vita

1998...... B.S., University of Notre Dame

2002...... O.D., The Ohio State University

2005...... M.S., The Ohio State University

2005...... Advanced Practice Fellowship in Cornea

and Contact Lenses, The Ohio State

University, The Ohio State University

Publications

Wagner H, Chalmers RL, Mitchell GL, Jansen ME, Kinoshita BT, Lam DY, McMahon

TT, Richdale K, and Sorbara L as the CLAY Study Group. Risk Factors for Interruption to Soft Contact Lens Wear in Children and Young Adults. Optometry and Vision Science

(2011) in press.

Chalmers RL, Wagner H, Mitchell GL, Lam DY, Kinoshita BT, Jansen ME, Richdale K,

Sorbara L, and McMahon TT as the CLAY Study Group. Age and Other Risk Factors for

Corneal Infiltrative and Inflammatory Events in Young Soft Contact Lens Wearers from

iv the Contact Lens Assessment in Youth (CLAY) Study. Investigative Ophthalmology and

Visual Science (2011) in press.

Jansen ME, Chalmers RL, Mitchell GL, Kinoshita BT, Lam DY, McMahon TT, Richdale

K, Sorbara L, and Wagner H as the CLAY Study Group. Characterization of Patients who Report Compliant and Non-Compliant Overnight Wear of Soft Contact Lenses.

Contact Lens and Anterior Eye (2011) in press.

Lam DY, Kinoshita BT, Jansen ME, Mitchell GL, Chalmers RL, McMahon TT, Richdale

K, Sorbara L, and Wagner H as the CLAY Study Group. Contact Lens Assessment in

Youth (CLAY): Methods and Baseline Findings. Optometry and Vision Science (2011)

88(6):708-715.

Kao C-Y, Richdale K, Sinnott L, Ernst L, Bailey MD. Semi-Automatic Extraction

Algorithm for Images of the Ciliary Body. Optometry and Vision Science (2011)

88(2):275-289.

Richdale K, Wassenaar P, Bluestein KT, Abduljalil A, Christoforidis JA, Knopp MV,

Schmalbrock P. 7T MR Imaging of the Human Eye In Vivo. Journal of Magnetic

Resonance Imaging (2009) 30(5):924–932.

Berntsen DA, Merchea M, Richdale K, Mack C, Barr JT. Higher-order Aberrations with

Spherical and Toric Soft Contact Lens Designs. Optometry and Vision Science (2009);

86(2):115-122.

Richdale K, Bullimore MA, Zadnik, K. Lens Thickness with Age and Accommodation by Optical Coherence Tomography. Ophthalmic and Physiological Optics (2008);

28(5):441-447.

Richdale K, Berntsen D, Mack C, Merchea M, Barr J. Visual Acuity with Spherical and

Toric Soft Contact Lenses in Astigmatic Eyes. Optometry and Vision Science (2007);

84(10): 969-975.

Richdale K, Sinnott L, Skadahl E, Nichols J. Frequency of and Factors Associated with

Contact Lens Dissatisfaction and Discontinuation. Cornea (2007); 26(2): 168-174.

Richdale K, Mitchell GL, Zadnik K. Comparison of Multifocal and Monovision Soft

Contact Lens Corrections in Low-Astigmatic Presbyopic Patients. Optometry and Vision

Science (2006); 83(5): 266-273.

Fields of Study

Major Field: Vision Science

Table of Contents

Abstract ...... ii

Acknowledgments...... iii

Vita ...... iv

List of Tables ...... ix

List of Figures ...... x

Chapter 1: Background and Literature Review ...... 1

Accommodation ...... 1

Mechanism...... 1

Measurement ...... 2

Presbyopia ...... 5

Theories ...... 5

Crystalline Lens ...... 7

Ciliary Body ...... 14

Supporting Structures of the Accommodative System ...... 18

Purpose and Aims of Present Study ...... 21

Chapter 2: Methods ...... 24

vii

Study Design ...... 24

Overview ...... 24

Recruitment and Enrollment ...... 24

Data collection ...... 25

Image analysis ...... 32

Statistical Analysis ...... 39

Chapter 3: Results ...... 40

Demographics of the Study Population ...... 40

Accommodative Function ...... 40

Ocular Biometry with Age ...... 43

Change in the Lens and Ciliary Body with Accommodation...... 47

Chapter 4: Discussion ...... 53

Accommodative Function ...... 53

Ocular Biometry and Age ...... 55

Changes in the Lens and Ciliary Body with Accommodation ...... 61

Towards a Better Understanding of Accommodation and Presbyopia ...... 66

Chapter 5: Conclusions ...... 69

References ...... 70

Appendix: Tables and Figures ...... 79

viii

List of Tables

Table 1. Refractive and accommodative variables collected...... 80

Table 2. Biometric variables collected...... 81

Table 3. Demographic information and refractive error ...... 93

Table 4. Accommodative response and amplitude ...... 94

Table 5. Baseline ocular biometry...... 102

Table 6. Ciliary body thickness and ring diameter ...... 115

List of Figures

Figure 1.Visante OCT images...... 82

Figure 2. Ciliary body OCT images ...... 83

Figure 3. Cross sectional slice of a 3D MRI ...... 84

Figure 4. MR imaging set-up...... 85

Figure 5. Ciliary body OCT image analysis...... 86

Figure 6. OCT lens analysis...... 87

Figure 7. MR image analysis pre-processing and region growing...... 88

Figure 8. Extraction of lens and ciliary body ring diameters...... 89

Figure 9. Extraction of lens surface...... 90

Figure 10. Visual inspection of the MRI data...... 91

Figure 11. Intraclass correlations for MRI measures...... 92

Figure 12. Accommodative response with RAF, PowerRefractor and autorefractor...... 95

Figure 13. Accommodative response to 0, 2, 4 and 6 D targets with autorefractor...... 96

Figure 14. Accommodative response to 0, 2, 4 and 6 D targets with PowerRefractor. .... 97

Figure 15. Bland-Altman plots of accommodative response for 0 and 2 D targets...... 98

Figure 16. Bland Altman plots of accommodative response for 4 and 6 D targets...... 99

Figure 17. Bland Altman plots of lens thickness ...... 100

Figure 18. Bland Altman plots of anterior and posterior lens curvature ...... 101

Figure 19. Anterior chamber depth and sagittal lens thickness with age...... 103

Figure 20. Refractive index and anterior lens curvature with age...... 104

Figure 21. Corneal thickness and corneal curvature with age...... 105

Figure 22. Vitreous chamber depth and axial length with age...... 106

Figure 23. Posterior lens curvatureand lens equatorial diameter with age...... 107

Figure 24. Ciliary body ring diameter and thicknesswith age...... 108

Figure 25. Bland Altman plots of lens thickness for 0 and 2D targets...... 109

Figure 26. Bland Altman plots of lens thickness for 4 and 6 D targets...... 110

Figure 27. Change in lens thickness with accommodation ...... 111

Figure 28. Change in anterior and posterior lens curvature with accommodation...... 112

Figure 29. Change in lens equatorial diameter and ciliary body ring diameter with accommodation...... 113

Figure 30. Change in ciliary body thickness with accommodation...... 114

Chapter 1: Background and Literature Review

Accommodation

Mechanism

Helmholtz’s theory of accommodation is generally well accepted and concludes that: contraction of the ciliary muscle leads to a reduction in zonular tension, allowing the crystalline lens to increase in curvature and thickness, thus creating the additional power required for near focus (Atchison 1995; Benjamin and Borish 1998; Glasser 2006).

Although accommodation can be controlled voluntarily, it usually operates under subconscious control by the autonomic nervous system (Marg 1951; Cornsweet and

Crane 1973; Atchison 1995). The ciliary muscle has dual innervation with the parasympathetic system retaining primary control of muscle contraction, and the sympathetic system responsible for a lesser and slow-acting inhibition of accommodation

(Gilmartin 1986; Tamm and Lutjen-Drecoll 1996; Culhane, Winn et al. 1999).

Parasympathetic fibers of the oculomotor nerve originate in the Edinger-Westphal nucleus, synapse in the ciliary ganglion, and continue to the ciliary body via the long and short ciliary nerves, while sympathetic innervation travels through the superior cervical ganglion and along the nasociliary nerves to the long and short ciliary nerves (Atchison

1995; Benjamin and Borish 1998). Unlike most postsynaptic fibers of the autonomic nervous system, many nerve fibers in the ciliary muscle are myelinated, allowing for faster conduction of signals and a swift accommodative response (Atchison 1995).

Accommodation in the human system is stable in less than one second, although there is some decrease in response time with age (Beers and Van Der Heijde 1994;

Kasthurirangan and Glasser 2006).

Measurement

In the late 19th century, F.C. Donders reported the maximum accommodative ability of over 100 subjects and remarked at the almost perfect linear decline with age

(Donders 1864). Donder’s efforts were later expanded by Duane, whose work encompassed over 1,500 patients between the ages of 8 and 72 years (Duane 1908;

Duane 1912; Duane 1915; Duane 1922; Duane 1925; Borsting, Chase et al. 2008).

Recognizing the value of a clinical guideline, Hofstetter used the data points from these two studies to generate formulas to predict the maximum, minimum, and average expected amplitude of accommodation, based solely on a patient’s age (Hofstetter 1944;

Hofstetter 1950). Unfortunately, Hofstetter’s equations were derived from straight lines he constructed “by general inspection to fit the data” and with a desire to “provide constants in the formulas which make calculation easy” (Hofstetter 1950). There was no consideration of repeated measures on the same person or statistical analysis of the data.

The measured amplitude of accommodation can vary widely with the technique, refractive correction, target, lighting, and whether testing is performed monocularly or binocularly (Somers and Ford 1983; Wold, Hu et al. 2003; Ostrin and Glasser 2004;

Sterner, Gellerstedt et al. 2006). Furthermore, the measure is not only of “the subject’s accommodative power, [but] rather his will and ability to exercise it,” which can fluctuate greatly (Duane 1908).

Accommodative amplitude is often measured subjectively using either a “push- up” technique, where a target is moved closer towards the eye, or a “minus lens” test, where increasing negative lens powers are added to stimulate accommodation. Both methods produce a change in retinal image size, the former magnifying the target, the latter minifying it. Although the minus lens test yields lower amplitudes than push-up testing, both subjective measures of accommodation overestimate true accommodative ability (Wold, Hu et al. 2003; Ostrin and Glasser 2004; Win-Hall, Ostrin et al. 2007;

Lopez-Gil, Fernandez-Sanchez et al. 2009). Uncorrected refractive error also leads to variations in amplitude (Dwyer and Wick 1995), as does the type of refractive error and how it is corrected (i.e., spectacle or corneal) (McBrien and Millodot 1986; Rosenfeld

1997; Abraham, Kuriakose et al. 2005).

In general, most patients over-accommodate to distance targets and under accommodate to near targets (Charman 2008) but, differences in target type and size can produce significant changes in accommodative response. More accurate accommodation is observed for targets with good contrast and detail (Toates 1970; Somers and Ford

1983; Rosenfield and Cohen 1995; Rosenfield and Cohen 1996). Light levels affect pupil size and therefore depth of focus and the endpoint of any subjective test. A smaller pupil increases the depth of focus and inflates the subjective accommodative response.

The human convergence and accommodative systems are linked, so, while accommodation is equal between the eyes in normally binocular individuals, binocular measures of accommodation yields higher amplitudes than monocular measures (Duane

1922; Garner and Yap 1997; Rutstein and Daum 1998; Dubbelman, Van der Heijde et al.

2005).

Comparing early measures of accommodation to more recent studies, the reasons for the discrepancies are clear. Donders, instructed subjects to converge their eyes as much as possible to obtain the maximum amount of accommodation, and did not use a cycloplegic agent to determine an accurate refractive error. Duane reported the absolute maximum amplitude achieved with multiple measurements, not an average value. These differences explain why studies controlling for refractive error, target stimuli, lighting, and convergence suggest that the true amplitude of accommodation is lower than those calculated by Hofstetter’s equations (Turner 1958; Wold 1967; Somers and Ford 1983;

Rosenfield and Cohen 1996; Rutstein and Daum 1998; Chen, O'Leary et al. 2000;

Sterner, Gellerstedt et al. 2004; Anderson, Hentz et al. 2008; Scheiman and Wick 2008).

Recently, researchers have suggested alternate formulas for calculating the expected amplitude of accommodation by age. Chen and co-workers formulated an equation based on a linear regression of monocular amplitudes of 405 children and found

values 4 - 6 D lower than Hofstetter’s range (Chen, O'Leary et al. 2000). Unfortunately,

Chen’s equation, with a steeper slope and smaller intercept, suggests that a 32-year-old patient would have zero accommodation. It may be that a single linear equation does not adequately represent the decline in accommodation from birth through presbyopic age.

Indeed, Anderson found that the maximum objectively measured accommodative amplitude of humans through the full life span was best fitted with a sigmoidal function with plateaus in early childhood and beyond mid-life (Anderson, Hentz et al. 2008). Still, between the ages of about 30 and 50 years, the decline remained quite linear.

Presbyopia

Theories

Presbyopia can be defined as the point where accommodative function declines to less than 3 – 4 D, receding the near point beyond arms’ length (Atchison 1995). It is usually manifested in the fourth decade of life. Theories of the mechanism of presbyopia were traditionally categorized as either lenticular or extra-lencticular.

The lenticular theories can be further subdivided into the Hess-Gullstrand and

Duane-Fincham theories (Atchison 1995; Strenk, Strenk et al. 2005). The Hess-

Gullstrand theory suggests that there is a constant force required for each diopter of accommodative response and, therefore, an increasing latent amount of force with age.

The Duane-Fincham theory suggests that each diopter of accommodative response requires additional force such that maximum ciliary muscle contraction occurs at the

maximum accommodative response. Nevertheless, the lenticular theories rest on the fact that the lens is the primary cause of accommodative decline. Traditionally, these theories were based solely on nuclear sclerosis, or “lens hardening,” but more recent studies suggest that overall growth of the lens may contribute to presbyopia.

The extra-lenticular theories of presbyopia attribute the loss of accommodation, at least in part, to aspects of the accommodative system other than the lens. These include either a weakening of the ciliary muscle, a change in the location of the ciliary body, and/or a change in the relationships of the accommodative structures, all of which could limit the ability to effect a change in the crystalline lens. Atchison published a thorough review of these theories (Atchison 1995). In this manuscript, he described extra-lenticular theories proposed by Duane (1922), who suggested that the ciliary muscle weakened with age, and also by Farnsworth and Shyne (1979), and Bito and Miranda (1989), who suggested that presbyopia was a result of a change in elasticity of the zonules or choroid, respectively (Farnsworth and Shyne 1979; Atchison 1995). Koretz and Handelman

(1988) theorized that the increase in lens size, and corresponding shift in the insertion point of the zonules created a change in the direction of force that the ciliary muscle could place on the lens (Koretz and Handelman 1988). Their theory, termed the

Geometric Theory, relied on the premise that the ciliary body remained fixed with age. A

Modified Geometric Theory was later proposed by Strenk, Strenk and Koretz (2005) who, based on MR imaging, showed that the change in geometry occurred from a forward and inward shift of the ciliary body with age (Strenk, Strenk et al. 2005).

Unfortunately, no single theory can fully account for the early-onset, almost perfectly linear decline, and complete loss of accommodative function at such a young age. Thus, as Weale suggested, presbyopia may be the additive result of multiple changes occurring in the human eye with age (Weale 1973; Weale 1995; Weale 1997; Weale

1999). Although great strides have been made over the past decade, the literature remains quite fragmented, with researchers focusing on either lenticular or extra-lenticular factors but failing to provide a comprehensive view of the entire accommodative system. To be certain, there are age-related changes in the entire accommodative system, and only in understanding the full system can one hope to comprehend the mechanism of presbyopia.

Crystalline Lens

Morphology

The crystalline lens develops from surface ectoderm and creates new cells throughout life. The innermost cells of the lens are termed the nuclear region, which is surrounded by a cortex of postnatal fibers and a thin outer capsule (Augusteyn 2007;

Augusteyn 2010).

Numerous studies have shown that the lens increases in sagittal thickness with age and accommodation. Due to normal variations between individuals, and differences in the techniques used to measure the lens, there is a range of reported values, with most studies suggesting a linear increase of about 15 to 30 µm/year and 40 to 80 µm/D of accommodative demand (Koretz, Kaufman et al. 1989; Garner and Yap 1997; Mutti,

Zadnik et al. 1998; Glasser and Campbell 1999; Dubbelman, van der Heijde et al. 2001;

Dubbelman, Van der Heijde et al. 2003; Ostrin, Kasthurirangan et al. 2006; Bolz, Prinz et al. 2007; Bullimore, Mitchell et al. 2007; Jones, Atchison et al. 2007; Atchison, Markwell et al. 2008; Kasthurirangan, Markwell et al. 2008; Sheppard, Evans et al. 2011).

Scheimpflug photography has demonstrated that, with age, the increase in lens thickness occurs in the cortical layers, but, with accommodation, the increased thickness occurs in the nuclear region (Koretz and Cook 2001; Dubbelman, Van der Heijde et al. 2005;

Hermans, Dubbelman et al. 2007).

The first reports of lens equatorial diameter were made ex vivo and suggested an increase in diameter with age (Pierscionek and Weale 1995; Glasser and Campbell 1999;

Rosen, Denham et al. 2006). These measurements were confounded by the fact that a young lens will, by design, take on a more accommodated state and thus measure shorter equatorially, when removed from the eye. In vivo measurements have been conducted using MRI, and while earlier reports confirmed a lack of equatorial growth (Strenk,

Strenk et al. 2004; Jones, Atchison et al. 2007), more recent studies report a slight (less than 10 µm/year) increase with age (Atchison, Markwell et al. 2008; Kasthurirangan,

Markwell et al. 2011). It is generally agreed that equatorial diameter decreases with accommodation. The decrease may be between 15 and 80 µm per D (Storey and Rabie

1985; Strenk, Semmlow et al. 1999; Jones, Atchison et al. 2007; Kasthurirangan,

Markwell et al. 2011; Sheppard, Evans et al. 2011). Another reason for the lack of consensus in the reported values of lens thickness and equatorial diameter is that many

studies compare the increase in lens thickness to the accommodative stimulus rather than the subject’s true accommodative response.

Research has also demonstrated a decrease in the circumlental space with age, but not with accommodation (Strenk, Strenk et al. 2006; Kasthurirangan, Markwell et al.

2011; Sheppard, Evans et al. 2011). Previous authors attributed the decrease in circumlental size to an increase in equatorial diameter of the lens, while MRI studies attribute it to an inward shift of the ciliary body with age (Strenk, Semmlow et al. 1999;

Al-Ghoul, Nordgren et al. 2001; Fea, Annetta et al. 2005; Strenk, Strenk et al. 2006;

Kasthurirangan, Markwell et al. 2011).

The lens is biconvex, hyperbolic and aspheric with the anterior surface flatter than the posterior surface. Both surfaces increase in curvature (decrease in radius) with accommodation (Garner and Yap 1997; Dubbelman and Van der Heijde 2001). As with sagittal lens thickness, the increase in curvature appears linear with increasing accommodative amplitude (Koretz, Cook et al. 1997; Koretz, Cook et al. 2001; Hermans,

Dubbelman et al. 2007). The anterior lens surface steepens by about 20 – 80 μm per year and perhaps 50 – 200 μm per D of accommodation (Garner and Yap 1997; Koretz, Cook et al. 1997; Dubbelman and Van der Heijde 2001; Koretz, Cook et al. 2002; Koretz,

Strenk et al. 2004; Dubbelman, Van der Heijde et al. 2005; Rosales, Dubbelman et al.

2006; Atchison, Markwell et al. 2008). Changes to the posterior lens surface with age are less clear, as optical errors are compounded by photographic methods the deeper the location in the eye. While some authors have suggested that there is no change in

posterior lens curvature with age (Koretz, Strenk et al. 2004; Rosen, Denham et al. 2006;

Atchison, Markwell et al. 2008), others have reported flattening (Glasser and Campbell

1999), and yet others have found steepening (Garner and Yap 1997; Dubbelman and Van der Heijde 2001; Koretz, Cook et al. 2001).

With age and accommodation, the anterior lens surface expands into the anterior chamber and the posterior surface pushes towards the vitreous, although the anterior movement is much greater than the posterior movement, as demonstrated by the nearly equal decrease in anterior chamber depth (Storey and Rabie 1985; Drexler, Baumgartner et al. 1997; Garner and Yap 1997; Dubbelman, Van der Heijde et al. 2005; Strenk, Strenk et al. 2005; Ostrin, Kasthurirangan et al. 2006; Bolz, Prinz et al. 2007).

Not surprisingly, the volume, surface area, and weight of the human lens also increase throughout life (Glasser and Campbell 1999; Koretz, Cook et al. 2001;

Augusteyn 2007). Resolving whether there is a change in lens volume with accommodation has been more contentious, as the lens, being mostly made of water, should be incompressible. Strenk first suggested that there is a change in lens volume with accommodation but did so based only on changes in cross-sectional area (Strenk,

Strenk et al. 2004). Hermans clearly demonstrated that one cannot be surmised from the other (Hermans, Dubbelman et al. 2007; Hermans, Pouwels et al. 2009). The most recent attempt at solving this conundrum came from Sheppard et al. who used a full lens MRI analysis and found an increase in both surface area and volume with accommodation

(Sheppard, Evans et al. 2011). Still, there is some question as to the error induced by their acquisition and image analysis techniques (Levy 2011).

With a continuous increase in thickness and curvature, the lens would be expected to increase in power, leading to a more myopic eye with age. The fact that the human eye loses near focus rather than far has been called “Brown’s lens paradox” (Atchison 1995;

Dubbelman, Van der Heijde et al. 2003). This phenomenon has been attributed to commensurate changes in the refractive index of the lens, with the equivalent refractive index decreasing from about 1.43 at age 20 years to 1.41 at age 80 years (Dubbelman and

Van der Heijde 2001; Atchison, Markwell et al. 2008). Such a change is also supported by a decrease (2 - 3 ms-1/year) in the speed of sound in the lens with age (Koretz,

Kaufman et al. 1989; Augusteyn 2010).

Although often simplified to a single equivalent refractive index, the lens has a gradient refractive index, with the highest index at the center of the lens. The central and peripheral refractive indices do not change with age; rather the area of the central region expands, and the gradient refractive index sharpens in the periphery (Jones, Atchison et al. 2005; Kasthurirangan, Markwell et al. 2008). With accommodation, there is also an increase in equivalent refractive index (0.0013/D), likely due to the increase in thickness of the (higher index) nuclear region (Dubbelman, Van der Heijde et al. 2005).

Mechanical and Functional Properties

Assessment of lenticular elasticity was first undertaken by R.F. Fisher in the

1960s and 70s. Fisher used human cadaver lens and a spinning technique to demonstrate that the lens became less elastic with age, and that the lens cortex was stiffer than the nucleus throughout life (Fisher 1971). Fisher’s work has since been questioned because of the limited sample size, the assumptions used in the calculation of his model, and the inconsistency with clinical findings (Burd, Wilde et al. 2006; Glasser 2006).

Measurement of the elasticity of the lens, often defined as Young’s modulus, does not fully describe the lens, which exhibits both viscous and elastic properties like most biological tissues (Weeber, Eckert et al. 2005; Burd, Wilde et al. 2006). Over the past 30 years, numerous studies have attempted to understand the mechanical properties of the lens through measurements of hardness, penetration ability, stiffness, and resistance, with ex vivo animal or human cadaver tissues or using mathematical modeling.

The excised crystalline lens has been measured using mechanical stretching

(Pierscionek 1993; Pierscionek 1995), mechanical loading (van Alphen and Graebel

1991), compression (Glasser and Campbell 1999), penetration with a dynamometer probe

(Pau and Kranz 1991; Czygan and Hartung 1996), and dynamic mechanical analysis

(Heys, Cram et al. 2004; Weeber, Eckert et al. 2005). These studies have all indicated that there is an increase in stiffness with age; however, the results have ranged from only small increases in lens stiffness before the age where accommodative ability is lost

(Pierscionek 1993; Pierscionek 1995; Glasser and Campbell 1999; Weale 2000) to a

“massive increase” in nuclear stiffness (Heys, Cram et al. 2004; Weeber, Eckert et al.

2005). Recent work has suggested that around the age of 30 years, the stiffness of the nucleus surpasses that of the cortex (Heys, Cram et al. 2004). Examination of the elasticity of the skin in vivo has also shown a marked change at approximately 30 years of age (Alexander and Cook 2006). It has been postulated that the change in the stiffness gradient (cortex versus nucleus) of the lens could account for the entire loss of accommodation with age; unfortunately, this interpretation relies on the compilation of many studies across different populations and assumptions of the dimensions and mechanical properties of the lens, some of which disagree by orders of magnitude

(Hermans, Dubbelman et al. 2006; Weeber, Eckert et al. 2007a; Weeber and van der

Heijde 2007b).

Ex vivo studies of lens stiffness are limited not only by changes in the post- mortem water content of lenses, but also by artifacts from preparation of the lens, and an inability to create an equivalent physiological force on the lens through stretching.

Recognizing the importance of in vivo testing, a method of measuring relative elasticity in the eye was developed by Erpelding and co-workers (Erpelding, Hollman et al. 2007;

Erpelding, Hollman et al. 2007). They used “bubble-based acoustic radiation” to indirectly measure the elastic properties of lenses in cadaveric eyes (Erpelding, Hollman et al. 2007). To date, it has not been performed in humans, and the long-term safety of creating air pockets in the human lens has yet to be established.

Certainly, the lens plays a crucial role in the development of presbyopia; however, the increase in lens stiffness reported by some studies is exponential, contrary to the

linear decline of accommodation; and the major increases in lens stiffness that occur beyond the age of 50 years, long after the majority of accommodative function is lost

(Glasser and Campbell 1999). It seems clear then, that other structures in the eye have a role in accommodative decline.

Ciliary Body

Morphology

The ciliary body is part of the uveal tract of the eye. It can be divided into two sections; the posterior region, called the pars plana, is continuous with the retinal choroid at the ora serrata, and the anterior portion, the pars plicata, is continuous with the iris

(Aiello, Tran et al. 1992). The pars plicata is made up of a series of undulations, known as ciliary processes, which project into the circumlental space (Aiello, Tran et al. 1992;

Tamm and Lutjen-Drecoll 1996).

The ciliary body consists of the ciliary muscle along with connective and vascular tissues. Differentiating the ciliary muscle from the entire ciliary body can only be done decisively by histological study. The ciliary muscle is roughly 4 - 6 mm in length and less than 1 mm in width in adult humans (Tamm and Lutjen-Drecoll 1996; Pardue and Sivak

2000). The muscle has three sections of fibers, primarily differentiated by orientation and location. The longitudinal section is the largest and is located closest to the sclera; radial fibers form a transition to the inner, least abundant, circular fibers (Atchison 1995; Tamm and Lutjen-Drecoll 1996). These regions are not distinct physiologically. The muscle acts

as a single unit and, with both accommodation and age, there is a shift in the relative proportion of the various fibers (Gilmartin 1986; Pardue and Sivak 2000). Importantly, the elastic tendons of the ciliary muscle continue with the elastic fibers of Bruch’s membrane in the choroid (Tamm and Lutjen-Drecoll 1996). It is this elastic connection that is thought to allow so-called “disaccommodation” via a backward tension on the ciliary muscle. During accommodation, contraction of the ciliary muscle causes the circular and radial portions to constrict in a sphincter action (Tamm, Lutjen-Drecoll et al.

1991). As the ciliary body moves inward and the ciliary body ring diameter decreases, the zonular tension is released allowing the lens to “round up.”

The ciliary muscle has many unique characteristics. Unlike most single-unit smooth muscle cells, which contract very slowly, the ciliary muscle is a multi-unit structure and can respond rapidly, more like that of skeletal muscle (Atchison 1995).

Smooth muscle can contract by 50 - 75% of its resting size (Seidel and Weisbrodt 1987;

Atchison 1995). A rough endoplasmic reticulum and well-developed Golgi apparatus typically found in skeletal muscle and responsible for the production of proteins, has been found in the smooth muscle cells of the ciliary body, uterus, and certain blood vessels.

The ciliary muscle also has more mitochondria than any other smooth muscle in the body

(Ishikawa 1962; Tamm and Lutjen-Drecoll 1996).

Histologically, researchers have found that, with age, both the longitudinal and radial portions of the muscle decrease in size while the circular portion increases (Tamm,

Tamm et al. 1992). This may result in a decrease in total length and movement of the

apical edge of the ciliary muscle closer to the lens (Tamm, Lutjen-Drecoll et al. 1991;

Pardue and Sivak 2000). Thus the shape and configuration of the ciliary body in older age may resemble that of the young, accommodated ciliary body. There is an increase in connective tissue beginning in the third decade of life, an increase in the accumulation of lipofuscin, an “aging pigment,” in the fifth decade, and atrophy of the muscle and neurological degeneration after 60 years of age (Tamm, Croft et al. 1992; Atchison 1995;

Tamm and Lutjen-Drecoll 1996; Pardue and Sivak 2000). In other parts of the body where smooth muscle can be studied more readily than the eye, there is a loss of muscle fibers and mitochondria with age (van Alphen and Graebel 1991; Atchison 1995).

Only recently have researchers used imaging techniques to confirm changes in the ciliary body in vivo. These studies have suggested that, with age, the resting ciliary body apex is displaced inward, decreasing the ciliary body ring diameter (Strenk, Semmlow et al. 1999; Stachs, Martin et al. 2002; Fea, Annetta et al. 2005; Strenk, Strenk et al. 2006;

Strenk, Strenk et al. 2010; Kasthurirangan, Markwell et al. 2011; Sheppard and Davies

2011).

Mechanical and Functional Properties

Contraction of the ciliary muscle with age was first examined ex vivo. Fisher alleged that there is an increase in maximum ciliary muscle contraction up to about 45 years of age and a decrease thereafter (Fisher 1977). Other studies suggested that ciliary muscle tissue contracts to pharmacological stimulation well beyond the age of presbyopia

(Poyer, Kaufman et al. 1993; Pardue and Sivak 2000). When studied in sections, Pardue and Sivak reported that the longitudinal portion of the muscle continues to contract, while the circular and radial portions exhibit decreased contractility with age (Pardue and Sivak

2000). Care should be taken when interpreting some of the older ex vivo studies, as researchers clearly demonstrated that dissecting the globe prior to fixation obscures age effects (Lutjen-Drecoll, Tamm et al. 1988; Charman 2008). Furthermore, studying muscle tissue outside the eye ignores the role of the supporting choroidal and zonular system and does not provide information on functional contraction (i.e., the centripetal action required for accommodation).

Studies of sub-human primates have demonstrated a decrease in the ability of the ciliary muscle to move forward and inward with increasing age (Lutjen-Drecoll, Tamm et al. 1988). In monkeys, the ciliary muscle loses its ability to create a usable force on the lens due to a restriction of the elastic attachment at the choroid (Lutjen-Drecoll, Tamm et al. 1988; Tamm, Lutjen-Drecoll et al. 1991; Tamm, Croft et al. 1992). This has not been found in a human model and the use of a primate model to study the mechanism of presbyopia has recently been brought into question (Strenk, Strenk et al. 2006).

Impedance cyclography provided the first in vivo study of ciliary muscle action in humans (Swegmark 1969). Researchers reported ciliary muscle contraction beyond the onset of presbyopia; however, this technique has since been challenged as measuring changes in blood flow, not specific to muscle movement or the development of force

(Swegmark 1969; Saladin, Usui et al. 1974; Atchison 1995). Strenk and colleagues

published a landmark MRI study that provided the first compelling evidence that the ciliary muscle can contract well into the eight decade of life (Strenk, Semmlow et al.

1999; Strenk, Strenk et al. 2006); however, the amount and direction of useable force has yet to be determined.

MRI is the only technique that allows visualization of the entire ciliary body ring, however measurement of fine changes in the ciliary muscle size and function are limited by the resolution of the technique. While MRI can likely not distinguish between ciliary process and muscle, ultrasound biomicroscopy (UBM) and OCT allow cross-sectional imaging of the ciliary body. UBM has recently been used to demonstrate that the ciliary body center of gravity shifts forward with pharmacological stimulation (Stachs, Martin et al. 2002; Park, Yun et al. 2008). This movement decreases with age, but may be restored after cataract extraction. OCT has also been used to measure ciliary body contraction in vivo (Lossing, Richdale et al. 2009; Sheppard and Davies 2010; Sheppard and Davies

2011) Both Lossing and Sheppard found a significant thinning of the ciliary muscle 2 mm posterior to the sclera spur with accommodation.

The ciliary muscle is the “powerhouse” of the accommodative system. And, while it is evident that muscle contraction continues well beyond the development of presbyopia, there seems to be some change in the shape and position of the ciliary body with age. Thus, the force available to control the lens may significantly altered with age.

Supporting Structures of the Accommodative System

In addition to the lens and ciliary body, the zonules, choroid, iris and vitreous are all thought to play a role in the accommodative response.

The anterior zonules connect the ciliary body to the equatorial region of the lens, while the posterior, or vitreous, zonules stretch backwards to the ora serrata (Lutjen-

Drecoll, Tamm et al. 1988; Glasser and Campbell 1999; Bernal, Parel et al. 2006). The zonules are less than 50 µm in diameter and made of protein and glycoprotein, making them very elastic (Davanger 1975; Farnsworth and Shyne 1979; Canals, Costa-Vila et al.

1996). In the unaccommodated eye, the anterior zonules transfer tension to the lens capsule; keeping the lens in a flattened shape. With accommodation, the zonules become slack, and the lens is able to take on a rounded shape.

With age, there is a forward shift in the zonular insertion point on the lens

(Farnsworth and Shyne 1979; Weale 2000; Croft, Glasser et al. 2006). This and the corresponding changes in lens size and ciliary body position mean that the angle, and therefore the amount of force applied to the lens, diminishes with increasing age. Some researchers have stated that zonular structure and elasticity do not change before the age of 45 years (van Alphen and Graebel 1991; Atchison 1995), while others have found that the zonules are thinner, fewer, and more “easily disrupted” with age (Farnsworth and

Shyne 1979; Atchison 1995; Ludwig, Wegscheider et al. 1999). Elasticity declines with age in other parts of the body (Alexander and Cook 2006), thus it seems reasonable that the elasticity of the zonules would also change with age.

The retinal choroid consists of both vascular and elastic tissue. The ciliary muscle is continuous with the elastic tendons of the choroid that is thought to provide the

“restoring force” in disaccommodation (Charman 2008). Studies in animal models suggested that the choroid stiffens with age, restricting the forward and inward movement of the choroid required during accommodation (Glasser and Kaufman 1999; Glasser,

Croft et al. 2001). While posterior restriction occurs in non-human primates, studies in humans demonstrate that the ciliary body may not be restricted in man (Strenk, Semmlow et al. 1999; Strenk, Strenk et al. 2005; Croft, Glasser et al. 2006).

It has been also suggested that the iris aids in accommodation by facilitating the rounding of the anterior lens surface and pulling of the ciliary body forward with accommodative miosis (Crawford, Kaufman et al. 1990; Atchison 1995; Pierscionek and

Weale 1995; Strenk, Strenk et al. 2005). In fact, greater accommodative responses are seen with the use of pilocarpine, a drug that causes both ciliary and iris smooth muscle contraction (Findl, Kiss et al. 2003; Kriechbaum, Findl et al. 2005). The use of strong mydriatics (e.g., 10% phenylephrine) or removal of the iris, marginally decreases the accommodative response (Atchison 1995; Glasser and Kaufman 1999).

Coleman first suggested that the vitreous humor may also play a role in accommodation. He suggested that the differential pressure between the aqueous and vitreous humours promotes forward movement of the lens during accommodation

(Coleman 1970; Coleman and Fish 2001). While accommodation is associated with a

decrease in intraocular pressure, the contribution of the vitreous may be trivial, as accommodation still occurs in patients post-vitrectomy (Win-Hall, Ostrin et al. 2007).

The notion that accommodation occurs solely via the lens and ciliary body is an oversimplification of a very intricate and impressive system. The lens and ciliary muscle are definitely key players, but the supporting roles of the iris, vitreous, zonules, and choroid cannot be ignored and reinforce the importance of studying presbyopia in vivo in the human eye.

Purpose and Aims of Present Study

With age, the size, structure, and/or function of all of the accommodative elements are altered. Still, there remains disagreement as to the relative contribution of the various age-related structural and functional changes. The reason for this is multifold.

For decades, presbyopia research was constrained by the inability to view the internal structures of the human eye. While animal models offer the ability to control accommodation with direct brain stimulation or pharmacological intervention, it has been postulated that there are differences in the basic way in which the human and primate eye change with age and accommodation (Strenk, Strenk et al. 2005). Ex vivo studies are limited by the fact that the largest changes in the dimension, water content, and mechanical or optical properties occur within a few hours post-mortem (Weale 1983).

Studies using in vivo techniques are not without their own limitations. There are distortions inherent to imaging techniques such as Scheimpflug photography. Some of the

errors, including distortion from the camera system and corneal refraction, have been corrected, but others, including refraction by the lens, have not (Atchison 1995;

Dubbelman and Van der Heijde 2001). Studies using pilocarpine and atropine alter the natural accommodative action and create significantly larger responses than possible with normal physiological accommodation (Findl, Kiss et al. 2003; Dubbelman, Van der

Heijde et al. 2005; Koeppl, Findl et al. 2005; Kriechbaum, Findl et al. 2005). Although

MRI has allowed the eye to be visualized without distortion or pharmacological disruption, positioning of the patient in the supine position required for MRI alters the natural position and movement of the lens (Atchison, Claydon et al. 1994;

Kasthurirangan, Markwell et al. 2011).

Finally, there are significant errors induced by the way in which accommodation has been measured and how changes in the lens and ciliary body were calculated. Early studies of presbyopia relied only on subjective measures of accommodation, which are known to overestimate true accommodative function. Other studies assumed an accurate response to a stimulus without measuring the subject’s true accommodation. It is not clear how the accommodative system responds to a target at, or beyond, the maximum residual accommodative ability (e.g. Does the ciliary muscle continue to contract? And, if so, to what extent?).

Thanks to advances in imaging technology, we are now able to image both the overall configuration of the accommodative system, and more detailed structure of the lens and ciliary body. Objective measurements of the accommodative system with age

and step-wise increases in accommodation, will allow a better understanding of the mechanism of accommodation and presbyopia, and hopefully, provide insight into the development of better treatment options.

The aims of this dissertation are to:

1) Develop OCT and MRI techniques for in vivo studies of the human eye;

2) Apply computer-based image processing for objective analysis of these

images; and

3) Quantify age-related and accommodative changes in a single population of

pre-presbyopic and presbyopic adults.

Chapter 2: Methods

Study Design

Overview

In a single cohort of pre-presbyopic and presbyopic subjects, demographic and ocular biometric measurements were recorded to determine which factors were related to age and accommodative function. Accommodative function was measured both subjectively and objectively. Ocular structures were imaged with increasing accommodative demand and with cycloplegia to allow for analysis of age-related changes in both the baseline (cycloplegic) state, and changes in the dimension with accommodative effort. Images were processed with custom segmentation programs.

Linear regression and mixed models were used to analyze the data.

Recruitment and Enrollment

Study volunteers were recruited through flyers, emails, and newspaper advertisements at The Ohio State University. The enrollment criteria are summarized below. The proposed age range is where accommodative decline is theoretically the greatest and begins where previous studies have found significant changes in the lens and ciliary body (Tamm, Tamm et al. 1992; Heys, Cram et al. 2004). Visual acuity of at least

20/25 in each eye and no tropia were required to allow for measurement of

accommodation with magnetic resonance imaging where the contralateral eye was used for fixation and focusing. Refractive error restrictions were based on the limits of the

PowerRefractor. Patients must not have had a cataract greater than Grade I on the Lens

Opacities Classification System III (Chylack, Wolfe et al. 1993) to ensure good acuity and because the presence of a cataract may alter the velocity of sound in the lens (Beers and Van der Heijde 1994). Finally, patients were in good general health and were pre- screened to meet MRI safety criteria.

Enrollment criteria

Between the ages of 30 and 50 years

Not pseudophakic or aphakic

No strabismus, amblyopia or history of vision therapy

Best corrected visual acuity of at least 20/25 in each eye

Emmetropia or contact lens corrected ametropia and < 1.25 D of astigmatism

Less than Grade 1 cataract

Not pregnant or breastfeeding

No systemic disease that would compromise ocular health (e.g., diabetes)

Meet MRI safety restrictions (no metal implants, pacemaker)

Data collection

Subjects were educated on the purpose of the study and informed consent was obtained prior to enrollment. The subject’s self-reported date of birth, gender, race, ethnicity, height, weight, medications, surgeries and ocular and systemic health conditions were recorded. Refractive, accommodative and biometric data were collected on all subjects in the same manner. Details of the testing procedures are provided below and summarized in Tables 1 and 2. All testing was conducted on the right eye. Ametropic subjects wore their habitual contact lens correction. In order to allow adequate pupil dimensions during photorefraction, phenylephrine was used to dilate the pupil. Pupil dilation was defined as one drop of 2.5% phenylephrine, and cycloplegia included the addition of one drop of 1% tropicamide. Testing indicated as under “pupil dilation” or

“cycloplegia” was conducted at least 20 minutes after instillation of the drug.

Accommodative Amplitude

The majority of early research in presbyopia reported only subjective accommodation, despite the fact that it is known to overestimate true accommodative amplitude by up to 4D (Wold, Hu et al. 2003; Ostrin and Glasser 2004; Win-Hall, Ostrin et al. 2007). Nevertheless, to allow comparison of our results with previous and future studies, subjective accommodation was measured. The subject’s left eye was patched, and accommodative amplitude was measured using the push-up to blur technique with the Royal Air Force (RAF) near point rule (Haag-Streit England, Essex, UK), which has been demonstrated to be a repeatable measurement of the subjective amplitude of

accommodation (Rosenfield and Cohen 1996; Antona, Sanchez et al. 2009). If the subject could not clear the card at the most remote position (44 cm), trial lenses were added, starting at +1.00 D and increasing in +0.50 D steps until the subject stated that the image was clear. The target was moved closer until the subject again reported blur. If used, the trial lens power was subtracted from the subject’s accommodative amplitude. Three measurements were made and averaged.

Accommodative Response

Objective measures of monocular accommodative response predict that patients between the ages of 30 and 50 years would have maximum amplitudes between 5.66 D and 0.46 D (Anderson, Hentz et al. 2008). Adults generally over-accommodate to distance targets and under-accommodate to increasing near demands, thus assumptions of a subject’s accommodative response could induce significant calculation errors

(Seidemann and Schaeffel 2003). For the most accurate measurement of true accommodative ability, an objective measurement of accommodation was made using the

Grand Seiko WV 500 Auto-Refractor (Grand Seiko, Ltd., Hiroshima, Japan) and a Badal lens track. Accommodative response was measured at four stimulus levels: 0, 2, 4 and 6

D. The target consisted of four rows of 20/155 Snellen equivalent letters, and subjects were instructed to “pick a letter on the target and carefully focus on it” to provide the most accurate measure of accommodative response for each demand (Stark and Atchison

1994). Five measures were recorded at each level and averaged.

The accommodative response was also recorded continuously using the

PowerRefractor (PlusOptix Inc., FL, USA) during OCT ciliary body imaging. The subject was instructed to focus on a Maltese cross target presented outside the machine.

There is little information regarding changes in the ciliary muscle with accommodation, thus it was important to objectively ascertain accommodative function. The measurement also provided information on the subject’s ability to sustain accommodation, as it required approximately two minutes to acquire the OCT images for each target.

One drop of 2.5% phenylephrine was instilled in the right eye 20 minutes prior to testing, as the PowerRefractor requires at least a 3-4 mm pupil size, and pupil size is known to decrease with both age and accommodation. The PowerRefractor was used in the “monocular mode,” which was shown to agree with both auto-refraction and subjective refraction in adult subjects (Allen, Radhakrishnan et al. 2003). The 95% limits of agreement for repeatability in the monocular mode was +0.26 to -0.32; smaller than that of other modes.

The PowerRefractor provided continuous readings every 0.04 seconds and can include erroneous values during blinks (Harb, Thorn et al. 2006). The data was exported into Microsoft Excel and filtered for blinks by removing changes larger than physiologically possible (>10 D/sec) (Harb, Thorn et al. 2006). As the recordings took approximately 2 minutes per patient, the median accommodative level, standard deviation of the response, and length of recording were collected.

General Ocular Biometry

Measurements of refractive error and eye shape were collected to control for known differences in the lens and ciliary body by eye size or ametropia (Zadnik, Mutti et al. 1995; Oliveira, Tello et al. 2005; Garner, Stewart et al. 2006; Bailey, Sinnott et al.

2008) and to allow for calculations required by other measurements (e.g. phakometry).

Corneal curvature was measured using the Keratron Scout (Optikon, Rome, Italy). Three measurements were captured and averaged. Three measurements of central corneal thickness were recorded and averaged using the Visante Anterior Segment Optical

Coherence Tomography (OCT) System (Software version 3.0, Carl Zeiss Meditec,

Dublin, CA, USA). Axial length was measured using the IOLMaster partial coherence interferometry system (Carl Zeiss Meditec, Dublin, CA, USA). Five measurements of axial length were recorded and averaged. A-scan ultrasound (Allergan-Humphrey Model

820, San Leandro, CA, USA), provided the following dimensions: anterior chamber depth, lens thickness, vitreous chamber depth and axial length. As ultrasound requires corneal contact, a topical anesthetic (0.5% proparacaine) was instilled in the right eye prior to measurement. Five ultrasound readings were made and averaged.

Optical Coherence Tomography of the Accommodative System

The Visante OCT “raw image mode” was used to capture images of the crystalline lens and anterior segment, and “high resolution corneal mode” was used for the ciliary body (Figure 1). To image the crystalline lens, the subject was seated in front

of the machine with the left eye patched. The patient was instructed to fixate on the

Maltese cross target within the machine and the examiner aligned the image until the white fixation line was visible in the center of the lens. The internal lens system was used to add increasing accommodative stimuli of 2, 4, and 6 D. Images of the anterior segment and nasal and temporal equatorial regions of the eye were also captured after cycloplegia.

In each condition, three images were recorded. As the peripheral portion of the lens is not visible behind the pigmented iris (Figure 1, middle), only sagittal lens thickness and anterior and posterior radii of curvature were made.

To image the ciliary body and allow simultaneous photorefraction, the subject was instructed to look outside the instrument at an external Maltese cross target (Figure

2). The distance target was presented on the far wall of the room (> 1 meter), the 2- and

4-D targets were presented on a rod attached to the machine, and the 6-D target was affixed to the mirror. One examiner aligned an image of the ciliary muscle on the Visante while another made continuous measurements of the subject’s accommodative response.

Four images were recorded at each level: 0-, 2-, 4- and 6-D accommodative stimuli, and following cycloplegia. Ciliary body thickness was objectively measured using an image segmentation program written in MATLAB.

Video Phakometry

The anterior and posterior lens radii of curvature and equivalent refractive index were calculated using video phakometry (Mutti, Zadnik et al. 1992). The right eye was

cylcopleged to allow adequate pupil size and a stable crystalline lens surface. The left eye was patched, and the subject fixated a red LED with the right eye. Pairs of Purkinje I, III, and IV images were produced by a dual fiber optic light source at optical infinity and recorded on videotape. The recorded images were digitized, and the separation between the center of each image in the pair was measured using Image Analyst version 8.1

(Acuity Imaging, Nashua, NH USA). The separation yielded an equivalent mirror radius in air, which was then adjusted for refraction through the optical elements preceding the reflecting surface. A spherical fit to the central 2 mm of the anterior and posterior lens surfaces, and an equivalent refractive index was recorded.

Magnetic Resonance (MR) Imaging of the Accommodative System

Three-dimensional (3D) MR images (MRI) were acquired on a separate day from all other measures, but within one month of the primary visit at the College of

Optometry. MRI allowed visualization of the entire crystalline lens and ciliary body ring

(Figure 3).

Subjects were scanned at 7 Tesla (T) (Phillips Achieva, Cleveland, OH, USA) using a volume head coil (Nova Medical, Inc., Wilmington, MA, USA) for transmission, and a custom-built, single loop 2 x 2.3 cm oval radiofrequency coil for reception. The subject was positioned on the MRI bed with his or her head cushioned by foam to limit movement. To decrease motion artifacts common to imaging of the eye, the imaged

(right) eye was taped closed, and the left eye was used to fixate the targets (Figure 4).

The receive coil was fitted within the orbital sulcus and secured with tape. As with OCT testing, a setup using a right-angle mirror and Maltese cross targets at 0, 2, 4, and 6 D stimulus levels were used for MR testing. No dilating drops were used during the MRI due to the cost of scanning time that would have been required by additional scans.

Localizer scans of less than two minutes duration were conducted to plan subsequent scans. Based on pilot work (Richdale, Wassenaar et al. 2009), inversion- recovery turbo field echo (IR-TFE) sequences were acquired with shot and interval times

(TS/TI) of 1800 and 900, repetition and echo times (TR/TE) of 6.8 and 2.3 ms, flip angle

(α) of 8o, TFE-factor of 260, field of view (FOV) of 65 x 65 x 8 mm3, and a scan time of

34 sec. Scans were repeated eight to twelve times at each accommodative level, depending on subject cooperation and quality of the images. Each target took less than 10 minutes of scanning time, and patients were given a alarm button to push if they deemed necessary due to anxiety, a need to move, or to take a break from focusing. Total scanning time was approximately 45 minutes per subject. Images had an acquired voxel volume of 0.25 x 0.25 x 1.0 mm3 and were interpolated for viewing and analysis.

Image analysis

Previous reports of age-related changes in the lens and ciliary body relied on subjective image analysis, which suffer from bias and are limited to the resolution of both the acquisition and measurement technique (e.g. digital calipers). One of the aims of this collaborative research project was to develop computer-based image segmentation

programs to analyze the OCT and MR images and provide objective measurements with sub-pixel accuracy.

OCT Ciliary Body Images

A MATLAB (MathWorks, Natick MA, USA) program written by Chiu-Yen Kao,

PhD from the Department of Mathematics, The Ohio State University, was used to provide ciliary body thickness measurements and is described in detail in Dr. Kao’s manuscript (Kao, Richdale et al. 2011). Briefly, OCT images were exported from the

Visante system as raw image files such that no refractive index was applied. The only input required by the image segmentation algorithm was to manually select the scleral spur (pink asterisk in Figure 5). This was done three times and the average location was used as the starting point of the measurements. The algorithm first segmented the ocular tissue from the background, and then segmented the relatively darker ciliary body from the remaining ocular tissue. Based on values from ex vivo studies, a refractive index of

1.41 was applied to the sclera and 1.38 to the ciliary body, and thickness values were converted from pixels to millimeters (128 pixels/mm for high resolution corneal mode)

(Kao, Richdale et al. 2011).

The images were run with a set criterion for threshold (0.7*mean image level), curvature (deviation from a straight line at mid-point), and cut-point (1 mm anterior to 5 mm posterior to the scleral spur). After processing, all images were visually inspected to ensure that the outline appropriately delineated the ciliary body. Images that failed due to

the curve “running too high” or “cutting across the muscle,” were re-run with the a smaller or larger mid-point value, which allowed the program to “search” either lower or higher to accurately delineate the curve of the ciliary body (Figure 5). Images that failed due to the cut-point (usually from reflections of light from the conjunctiva or dark lines from an eyelash) were progressively cropped. Because the point of cropping can affect the fit of the upper outline of the ciliary body, the first attempt to correct the image was to crop the image 5.5 mm posterior to the scleral spur, followed by 5.0 mm posterior to the sclera spur. Images were never cropped shorter than 5.0 mm. Finally, images that failed to appropriately delineate the ciliary body due to a “noisy” or “indistinct” area at the anterior and/or inner portion, were re-run with progressively lower threshold levels (in

0.1 steps) until the anterior portion of the ciliary body was well defined. This allowed the program to include the noisy area in the ciliary muscle (instead of seeing it as background noise). Finally, the data were analyzed to find subjects who had within-subject standard deviations greater than three standard deviations from the overall sample average. These images were visually inspected once more and re-processed, as needed. Images that did not pass visual inspection and could not be corrected using the methods detailed above were discarded.

OCT Crystalline Lens Images

Images of the crystalline lens were also analyzed using a MATLAB program written by Chiu-Yen Kao, PhD. Due to the limited scan depth of the Visante system, it

was not possible to obtain a picture of the cornea, anterior chamber and lens in one image. OCT is similar to ultrasound, in that it combines multiple line scans to achieve a full image. Thus, while the images are not “refracted” through preceding surfaces, as in

Scheimpflug photography, they are subject to changes in path length due to refractive index. To account for this, the anterior chamber and lens images were stitched together and refractive indices were applied to create “corrected” lens surfaces (Figure 6).

To begin the image analysis program, an examiner selected the midpoint of the images by clicking on the white “fixation line” at the anterior surface of the lens. The fixation line is the reflection normal to the corneal surface. As both the anterior chamber image and lens image contained the anterior lens surface and fixation line reflection, the two images could be overlapped and stitched together.

The images were cropped to the central 8 mm (4 mm to either side of the fixation line) in order to more accurately fit the lens surface. The examiner then selected ten points on the anterior and posterior cornea, and ten points on the anterior and posterior lens surface and a sphere was fitted to each set of points. The same anterior chamber image was used for each of the lens images (0, 2, 4 and 6 D stimulus levels).

The Visante OCT system uses 1310 nm light and refractive index varies with wavelength, so Cauchy’s equation was used to determine adjusted indices of 1.367 for the cornea, 1.328 for the aqueous and 1.408 for the lens (Atchison and Smith 2005). This resulted in a thinner cornea and lens and flatter surfaces than the unadjusted raw image

(Figure 6). Sagittal lens thickness and anterior and posterior curvature measurements

were exported into an Excel spreadsheet and a pixel/mm conversion was applied (64 pixels/mm for raw image mode).

MR Images

To analyze the MR images, an Interactive Data Language (IDL, ITT Visual

Information Solutions, Boulder CO, USA) program was written by Peter Wassenaar, MS,

Department of Radiology, The Ohio State University. The program provided objective measures of lens equatorial diameter, lens sagittal thickness, and ciliary body ring diameter.

To begin, images were interpolated to 0.1 mm isotopic resolution (10 pixels / mm in each direction). The only manual input required by the program was to select a point near the center of the lens in 3D data space. The x, y and z coordinates of this selection were used to begin the iterative 3D image analysis program. From this point, the data were cropped to include 81 pixels (8.1 mm) in the x direction and 116 pixels (11.6 mm) in the y-direction. No cropping was required in the z (slice) direction since the acquisition field was constrained to 80 pixels (8.0 mm).

The image analysis program had three main steps: pre-processing, rotation to an orthogonal plane, and feature extraction. For pre-processing (Figure 7), a 3D bilateral filter was used to reduce noise while maintaining edges (Wassenaar, Richdale et al.

2010). The bilateral filter utilized a Gaussian weighted average of pixels that are neighbors, both in distance and intensity (Tomasi and Manducki 1998). A correction was

applied to compensate for the coil-sensitivity drop-off (lower signal intensity further from the coil). The correction was determined using a linear regression through the signal intensities along each of the three orthogonal axes of the lens, and the inverse of the regressions were multiplied with the original data. This created an image with homogenous brightness throughout the lens.

To correct for rotation, a region growing algorithm was used to determine the location and orientation of the lens (Sonka, Hlavac et al. 1993). A best-fitting plane through the equator of the lens was found using singular value decomposition (Strang

1988). Using the orientation of the best-fitting plane, a transformation matrix was created to re-orient the data to orthogonal planes. The program automatically ran through two iterations of the pre-processing steps and thus did not rely solely on the initial selection point.

For feature extraction, a 2D coronal projection through the lens and ciliary body ring was created (Figure 8). Lens and ciliary body edges were determined by calculating intensity gradients, utilizing both the steepness and the direction of the edges. The maxima and minima in 256 radial directions from the lens center were plotted. The maxima correspond to the edge of the ciliary body and the minima to that of the lens.

Hough transforms were used to fit circles to the plotted maxima and minima points

(Sonka, Hlavac et al. 1993). Constraints were applied such that the lens diameter was limited to between 7 and 11 mm, and the ciliary body diameter to between 0.4 and 4 mm larger than the lens diameter.

Next, sagittal lens thickness was determined. The center of the previously fitted lens circle designated the x and z coordinates, and all points that shared these coordinates, formed a line through the center of the lens. The anterior and posterior limits of the lens were found by calculating another signal intensity gradient along a 3x3 pixel wide channel surrounding the sagittal axis (to reduce signal fluctuations that would have been present had a single line of pixels been used).

Finally, the anterior and posterior lens surfaces were detected (Figure 9). Surface points were initially determined using gradients, as previously described, but this time in three dimensions. Suppression of false edges from noise or artifacts was achieved by creating directional maps (radiating from the lens center) and calculating the inner product of the gradients and the maps. Thinning of the lens surface to a single pixel width was performed using non-maximum suppression and skeleton approaches (Sonka, Hlavac et al. 1993). The lens was assumed to be symmetric about the x (sagittal) axis and the skeletonized lens surface was collapsed, reducing a degree of freedom to more accurately fit the lens shape. The collapsed outline was plotted as the distance from the central lens axis to the surface, along the sagittal axis.

The data were exported into an Excel spreadsheet and sagittal, coronal and skeletonized lens images were saved for visual inspection (Figure 10). Erroneous data from motion, artifacts, or failure of the image analysis program, were deleted (Figure 10).

To determine the ideal number of images required for a reproducible response, intraclass correlation coefficients (ICCs) were calculated for lens thickness, equatorial

diameter and ciliary body ring diameter (Winer 1971). Subjects with at least 6 acceptable images on the 0-D target were used in the ICC analysis (n = 20). As shown in Figure 11, an average of three scans gave ICCs of 0.97 for lens thickness, 0.94 for equatorial diameter and 0.96 for ciliary body ring diameter. Based on this, subjects and targets with at least three useable images were averaged and used for further analysis. If data from a given target did not have at least three acceptable images, data from that target was removed. An entire subject’s MRI data was removed only if all four targets had less than three acceptable images. For purposes of this analysis, two subjects were removed for not having at least three acceptable images at any target, and nine individual targets were deleted from four other subjects. This left 20 subjects with full data sets, and four subjects with partial data sets for analysis.

Statistical Analysis

Descriptive statistics (mean, median, standard deviation and range) were provided for the demographic and biometric and accommodative measures. Bland-Altman methods were used to compare image acquisition and analysis techniques. Linear regressions were used to determine which ocular factors were related to age. Mixed models were used to model the ciliary body and lens changes with accommodative effort and age.

Chapter 3: Results

Demographics of the Study Population

A total of 92 subjects were enrolled in the study to allow for 85 subjects to complete both visits. A minimum of 20 subjects were enrolled in each five-year age bin between 30 and 50 years of age. Their spherical equivalent refractive error ranged from

+1.75 to -10.90 D, and roughly half of the sample wore contact lenses for distance correction (n = 42). Because refractive error is related to biometric and accommodative measurements, a subset of 26 emmetropic subjects (spherical equivalent refractive error between +0.50 and -0.50 D) were analyzed and are reported here. The mean age of the emmetropic subset was 39.5 ± 7.42 years (range: 30.4 to 50.0 years). Fifty-four percent of the subjects (n = 14) were female, and 73% were Caucasian (Caucasian: n = 19, Asian: n = 6, African American: n = 1). The sample is further described in Table 3.

Accommodative Function

Accommodative function was measured both subjectively and objectively (Figure

12). The push-up to blur amplitude (using an RAF rule), which yields only the maximum subjective response, is reported in Table 4. Objective responses to 0-, 2-, 4-, and 6-D stimuli were recorded with both the autorefractor and the PowerRefractor and are also

presented in Table 4. The maximum objectively measured accommodative response to any dioptric target is also reported for comparison, i.e., if one subject’s maximum accommodative response was to the 4-D stimulus and another’s to the 6-D stimulus, those respective maxima are reported. The subjective push-up test (RAF) yielded higher values than either objective test (autorefractor or PowerRefractor). This was not unexpected, as the maximum limit on the RAF rule is roughly 20 D, while the autorefractor and PowerRefractor targets were constrained to a maximum of 6 D.

Despite this, all methods were well correlated (Pearson correlation, r = 0.89 (AR/PR),

0.88 (AR/RAF), 0.87 (PR/RAF), all p < 0.001).

Univariate linear regressions were conducted to determine the relationship between each accommodative measure and age. Regressions of the maximum accommodative response with age (centered at 30 years) yielded the following equations:

AR (D) = 4.75 – 0.21 x (Age – 30), 95% CI = -0.27 to -0.16, p < 0.001

PR (D) = 5.26 – 0.22 x (Age – 30), 95% CI = -0.28 to -0.16, p < 0.001

RAF (D) = 12.43 – 0.54 x (Age – 30), 95% CI = -0.67 to -0.41, p < 0.001

A stepwise regression of age was performed using all three measures of accommodative function as predictors. With backward elimination, the push-up methods was the only predictor retained in the model (backward regression p-values: RAF =

0.045, AR = 0.07, PR = 0.32), likely due to the larger range of amplitudes allowed with the RAF technique.

The subjects’ accommodative response to each stimulus level is plotted in Figures

13 (autorefractor) and 14 (PowerRefractor). In general, accommodative responses lagged behind the accommodative demand (indicated by the dashed line). For most subjects under the age of 40 years (black lines: 30 < 35 years, blue lines: 35 < 40 years), there is a linear increase with increasing demand, while subjects over age 40 (green lines: 40 < 45 years, red lines: 45 – 50 years) show little change in accommodative response to any target. Although the general trends were similar, there was a greater spread with the

PowerRefractor readings than the Autorefractor.

Bland-Altman plots (Bland and Altman 1986) with mean difference (solid line) and 95% limits of agreement (LOA, dashed lines) for the autorefractor and

PowerRefractor responses were constructed for each target (Figures 15 and 16). For the 0

D target, the linear regression of the difference versus the mean was significant (0.38 -

1.39 x Acc Resp, p < 0.001). The mean difference of 0.02 D (95% LOA = 1.30 to -1.26

D) was not statistically different than zero (p = 0.90). Likewise, for the 2-D target, the linear regression was significant (0.39 - 0.54 x Acc Resp, p = 0.001), but the difference of

-0.12 D was not significant (95% LOA = 0.88 to -1.12 D, p = 0.24). For the 4-D target, the linear regression was not significant (0.18 - 0.05 x Acc Resp, p = 0.73). The difference of 0.10 D (95% LOA = 1.48 to -1.28 D) was also not significant (p = 0.50).

For the 6-D target, the regression was not significant (0.21 – 0.10 x Accommodative

Response, p = 0.54), but the difference of -0.50 D was statistically significant (95% LOA

= 1.29 to -2.29 D, p = 0.01). These results indicate that the two methods of measuring accommodative response are not interchangeable. Further discussion of these results are found in Chapter 4, but due to the tight response plots, and the fact that autorefraction is often considered the gold standard, the autorefractor results were used as the indicator of accommodative function for all other analyses.

Ocular Biometry with Age

Both sagittal lens thickness and anterior and posterior lens curvature were measured using more than one technique, thus Bland Altman analysis was used to assess agreement between the methods (Bland and Altman 1986). As discussed in Chapter 2, a refractive index of 1.408 was calculated for adjustment of the lens at 1310 nm. The initial

Bland Altman analysis showed a 0.05 mm bias (p < 0.05), with OCT being thicker than ultrasound. Since ultrasound is considered the gold standard for measurement of lens thickness, it was determined that a refractive index of 1.390 would create zero bias between the OCT and ultrasound lens thickness measurements. Figure 17 shows the

Bland Altman plots of lens thickness with ultrasound compared to MRI and OCT (with a refractive index of 1.39). For OCT, the linear regression of the difference versus the mean was not significant (0.047 – 0.01 x LT, p = 0.56). The mean difference of -0.0005 mm (95% LOA = -0.07 to +0.07 mm) was not statistically different than zero (p = 1.00).

For MRI, the linear regression of the difference versus the mean was also not significant

(-0.124 + 0.023 x LT, p = 0.69). The difference of -0.03 mm (95% LOA = -0.16 to 0.10 mm) was significantly different than zero (p = 0.03). These results indicate that ultrasound and OCT measurements agree, but that the MRI measurements are, on average, 0.03 mm thinner than with ultrasound. These findings also agree with a recent publication by Kasthurirangan and colleagues, who reported lens thickness measurements with 1.5 T MRI to be, on average, 0.05 mm thinner than with ultrasound (Kasthurirangan,

Markwell et al. 2011).

Refractive index adjustments could not be made post-hoc for the OCT lens curvature measurements. Bland Altman plots of anterior and posterior lens curvature with phacometry and OCT are presented in Figure 18. For anterior lens curvature, the linear regression of the difference versus the mean was statistically significant (-4.23 - 0.48 x

ALC, p < 0.001), as was the mean difference of 1.67 mm (95% LOA = -0.66 to 4.00 mm, p < 0.001). For posterior lens curvature, the linear regression of the difference versus the mean was significant (-6.18 + 0.89 x PLC, p < 0.001); as was the mean difference of 3.52 mm (95% LOA = 1.69 to 5.35 mm, p < 0.001). Based on the trend and bias between with

OCT and phakometric curvature measurements, it is likely that there is some error with the OCT lens curvature image analysis methods. Still, the changes in lens curvature

(steepening) with accommodative effort, are statistically significant and physiologically plausible (see next section). Thus, although the OCT lens curvature measurements are not physiologically accurate, the data may still be useful to demonstrate within-subject changes. Further discussion of these findings is presented in Chapter 4.

Baseline ocular biometric results are presented in Table 5. All measurements were conducted under cycloplegic conditions except corneal curvature and corneal thickness

(which are not significantly altered by cycloplegia) and MRI measurements (due to costs that would have been associated with additional scan time). MRI measurements reported are for the distance (0 D) target.

The baseline biometric variables were plotted against age to assess potential relationships (i.e., linear, quadratic). Due to the small sample size and lack of obvious non-linear relationships, linear regressions were conducted.

In this population, age (centered at 30 years) was significantly related to sagittal lens thickness measured by ultrasound (LT_US), OCT (LT_OCT) and MRI (LT_MRI); anterior lens curvature by phakometry (ALC_ph) and OCT (ALC_OCT); anterior chamber depth (ACD); and lens refractive index (RI) (Figures 19 and 20).

LT_US (mm) = 3.67 + 0.031 x (Age – 30), 95% CI = 0.019 to 0.042, p < 0.001

LT_OCT (mm) = 3.65 + 0.031 x (Age – 30), 95% CI = 0.021 to 0.042 p < 0.001

LT_MRI (mm) = 3.65 + 0.031 x (Age – 30), 95% CI = 0.019 to 0.043, p < 0.001

ALC_ph (mm) = 11.82 – 0.11 x (Age – 30), 95% CI = -0.17 to -0.05, p = 0.001

ALC_OCT (mm) = 14.02 – 0.18 x (Age – 30), 95% CI = -0.26 to -0.09, p < 0.001

ACD (mm) = 3.85 – 0.029 x (Age – 30), 95% CI = -0.045 to -0.012, p = 0.001

RI = 1.457 – 0.001 x (Age – 30), 95% CI = -0.001 to 0.000, p = 0.002

Age was not significantly related to: corneal thickness (Pach); corneal curvature

(K); axial length by IOLMaster (AL_IOL) or ultrasound (AL_US); vitreous humor length

(VH); posterior lens curvature by phakometry (PLC_ph) or OCT (PLC_OCT); ciliary body thickness at 1, 2, or 3 mm posterior to the sclera spur (CBT1, CBT2, CBT3); maximum ciliary body thickness (CBTMax); ciliary body ring diameter (CBRD); or lens equatorial diameter (LED) (Figures 21-24).

Pach (microns) = 533.8 + 1.02 x (Age – 30), 95% CI = -0.50 to 2.53, p = 0.18

K (D) = 43.84 - 0.01 x (Age – 30), 95% CI = -0.095 to 0.066, p = 0.73

AL_IOL (mm) = 23.59 + 0.003 x (Age – 30), 95% CI = -0.036 to 0.042, p = 0.88

AL_US (mm) = 23.59 - 0.007 x (Age – 30), 95% CI = -0.044 to 0.032, p = 0.72

VH (mm) = 16.09 - 0.01 x (Age – 30), 95% CI = -0.048 to 0.032, p = 0.66

PLC_ph (mm) = 7.64 - 0.02 x (Age – 30), 95% CI = -0.047 to 0.004, p = 0.09

PLC_OCT (mm) = 10.99 - 0.01 x (Age – 30), 95% CI = -0.066 to 0.040, p = 0.61

CBTMax (mm) = 0.88 – 0.001 x (Age – 30), 95% CI = -0.007 to 0.005, p = 0.74

CBT1 (mm) = 0.81 – 0.001 x (Age – 30), 95% CI = -0.007 to 0.005, p = 0.78

CBT2 (mm) = 0.50 – 0.001 x (Age – 30), 95% CI = -0.008 to 0.005, p = 0.71

CBT3 (mm) = 0.26 – (<0.001) x (Age – 30), 95% CI = -0.004 to 0.005, p = 0.82

LED (mm) = 8.87 - 0.007 x (Age – 30), 95% CI = -0.025 to 0.010, p = 0.40

CBRD (mm) = 11.73 – 0.012 x (Age – 30), 95% CI = -0.034 to 0.009, p = 0.25

A multivariate regression model of age was fitted using all variables significant to the p < 0.10 level in the univariate analyses (accommodative response, anterior chamber depth, lens thickness, refractive index, and anterior and posterior lens curvature). Because there were duplicate measures for lens thickness and curvature, ultrasound and phakometry, considered the gold standard, were used in the multivariate model. Using backwards selection, accommodative response and lens thickness (centered at 3.00 mm) were retained as predictors of age, leaving the final model:

Age (years) = 40.67 - 2.75 x Acc Resp + 6.93 x (LT – 3.00)

Adj R2 = 0.78

95%CI: Acc Resp = -3.86 to -1.62, LT = 0.19 to 13.67

p-value: Acc Resp < 0.001, LT = 0.04

Change in the Lens and Ciliary Body with Accommodation

The lens and ciliary body were also imaged as subjects were presented with stepwise increases in accommodative demand. As described in Chapter 2, OCT and MRI were used to acquire the images, and programs were written to objectively extract measurements of ciliary body thickness, ciliary body ring diameter, lens thickness, and lens equatorial diameter.

Changes in lens thickness with accommodation were available from both OCT and MRI, thus Bland Altman analysis was used to compare the methods (Figures 25 and

26). The OCT-measured lens thickness at each accommodative level was adjusted to a refractive index of 1.39 before comparison to MRI. For the 0 D target, the linear regression of the difference versus the mean was not significant (-0.141 - 0.018 x LT, p =

0.65). The mean difference of -0.07 mm (95% LOA = -0.19 to 0.05 mm) was statistically significant (p < 0.001). Likewise, for the 2-D target, the linear regression was not significant (-2.96 + 0.055 x LT, p = 0.28), but the difference of -0.07 mm was statistically significant (95% LOA = -0.20 to 0.06 mm, p < 0.001). For the 4-D target, the linear regression was significant (-0.643 + 0.14 x LT, p = 0.02). The difference of -0.09 (95%

LOA = -0.22 to -0.04 mm) was also significant (p < 0.001). For the 6-D target, the regression was not significant (-0.573 - 0.116 x LT, p = 0.09), but the difference of -0.09 mm was statistically significant (95% LOA = -0.25 to 0.07 mm, p < 0.001). These results indicate that MRI consistently measured thinner than OCT. This is consistent with our

Bland Altman analysis of MRI with ultrasound, and with previous research

(Kasthurirangan, Markwell et al. 2011). Although the 4-D target showed a significant trend towards MRI measuring even thinner with thinner lenses, the other targets did not.

And, removal of the three subjects that were the furthest outliers made the regression non-significant (-0.532 – 0.106 x LT, p = 0.09). It is plausible that these subjects accommodated during testing on one instrument but “gave up” during the other test.

Mixed model analysis was used to allow inclusion of all four stimulus levels when determining the change in the lens and ciliary body dimensions with accommodation. The subjects’ age, accommodative response, and an interaction term for

accommodative response and age were fixed effects, and the accommodative target was a repeated effect.

For OCT-measured changes in lens thickness and curvature, the interaction between age and accommodative response was not significant (all p > 0.27). With the interaction term removed, both the change in lens thickness and anterior lens curvature were significantly related to accommodative response and age. The change in posterior lens curvature was not significantly related to age or accommodative response. Figures

27 and 28 show the plotted data with lines fitted to the mixed models and age fixed at 30 years:

LT_OCT (mm) = 3.67 + 0.065 x Acc Resp + 0.032 x (Age – 30)

95% CI: Acc Resp = 0.057 to 0.072, Age = 0.020 to 0.043

p-value: Acc Resp < 0.001, Age < 0.001

ALC_OCT (mm) = 13.19 – 0.86 x Acc Resp – 0.16 x (Age – 30)

95% CI: Acc Resp = -1.01 to -0.72, Age = -0.23 to -0.09

p-value: Acc Resp < 0.001, Age < 0.001

PLC_OCT (mm) = 9.99 – 0.12 x Acc Resp + 0.01 x (Age – 30)

95% CI: Acc Resp = -0.29 to 0.04, Age = -0.03 to 0.06

p-value: Acc Resp = 0.15, Age = 0.50

For MRI-measured changes in lens thickness, the interaction between age and accommodative response was also not significant (p = 0.30) and was removed from the model. The change in lens thickness was significantly related to both age and accommodative response (Figure 27).

LT_MRI (mm) = 3.59 + 0.064 x Acc Resp + 0.034 x (Age – 30)

95% CI: Acc Resp = 0.050 to 0.078, Age = 0.021 to 0.047

p-value: Acc resp < 0.001, Age < 0.001

For lens equatorial diameter, neither the interaction term (p = 0.73), nor age (p =

0.41) were statistically significant and both were removed from the model. Likewise, for ciliary body ring diameter, neither the interaction term (p = 0.45), nor age (p = 0.20) were statistically significant and both were removed from the model. Figure 29 shows the data and lines fitted to the models:

LED (mm) = 8.76 - 0.075 x Acc Resp, 95% CI = -0.101 to -0.049, p < 0.001

CBRD (mm) = 11.60 - 0.068 x Acc Resp, 95% CI = -0.044 to -0.092, p < 0.001

For the OCT-measured ciliary body changes, the interaction term and age were not statistically significant for any location (all p > 0.42) and were removed from the

model. Accommodative response was significantly related to the change in thickness at

CBTMax, CBT1, CBT2 and CBT3. Figure 30 shows the plotted data with lines fitted to the mixed model equations:

CBTMax (mm) = 0.832 + 0.026 x Acc Resp, 95%CI = 0.013 to 0.039, p < 0.001

CBT1 (mm) = 0.765 + 0.013 x Acc Resp, 95%CI = 0.004 to 0.022, p = 0.005

CBT2 (mm) = 0.498 – 0.011 x Acc Resp, 95%CI = -0.003 to -0.019, p = 0.005

CBT3 (mm) = 0.282 – 0.015 x Acc Resp, 95%CI = -0.009 to -0.022 p < 0.001

The average (mean ± standard deviation) ciliary body thickness and ring diameter at each accommodative stimulus level are presented in Table 6.

To remove highly correlated variables in the final multivariate model, correlations were conducted using the variables that were significant to the p < 0.10 level in the univariate analyses. If correlations were greater than 0.8, the variables were tested in separate models to determine which the best predictors of accommodative response were.

Examining ciliary body variables, thickness at 1 and 3 mm, and ciliary body ring diameter were the best predictors of accommodative response. For the lens, thickness, and anterior lens curvature were the best predictors of accommodative response. After removal of redundant parameters, the final model included: age, lens thickness, anterior lens curvature, ciliary body thickness at 1 and 3 mm, and ciliary body ring diameter. For the first time in a single population of human adults, we were able to construct a model of

accommodation with both lenticular and extra-lenticular measurements. Using a backwards removal procedure, age, lens thickness, anterior lens curvature, and ciliary body thickness at 3 mm were retained in the final model:

Acc Resp (D) = -11.79 + 7.17 x LT – 4.73 x CBT3 – 0.28 x ALC – 0.30 x Age

95%CI: LT = 5.13 to 9.21, CBT3 = -1.83 to -7.63

ALC = -0.14 to -0.41, Age = -0.19 to -0.41

p-value: LT < 0.001, CBT3 = 0.002, ALC < 0.001, Age < 0.001

Chapter 4: Discussion

Accommodative Function

Over a century has passed since Donders and Duane reported the decline in accommodative ability that accompanies advancement in age. Since then, multiple studies have confirmed their findings, but have also explored the many nuances of the accommodative system. This study of 26 emmetropes, aged 30 to 50 years, further demonstrated that the way in which accommodation is measured has a sizeable effect on the outcome.

Our subjective measure of accommodation, the RAF rule, overestimated the subjects’ true accommodative ability; a finding that agrees with previous research (Ostrin and Glasser 2004; Win-Hall, Ostrin et al. 2007). As subjects approached presbyopia, the subjective and objective results converged. Hofstetter’s equation for average accommodation (18.5 – 0.3 x age) would suggest subjective amplitudes of 3 to 9 D in this population. Our findings of 1.66 to 15.8 D are larger in range; however, some caution should be taken as the data used to determine Hofstetter’s equation was based on the use of a vertical line target, and Hofstetter’s equation was determined by visual inspection of the data, not by statistical methodology (Hofstetter 1950).

As reported previously, subjects tended to over-accommodate to the distance target (0 D), and under-accommodate to the near targets (2, 4, and 6 D) (Charman 2008).

Anderson et al predict a monocular objective accommodative response between 0.46 to

5.66 D for subjects aged 30 to 50 years (Anderson, Hentz et al. 2008). This range agrees with our maximum amplitudes of 0.03 to 5.15 D, as measured with the Grand Seiko

Autorefractor, and 0.17 to 6.67 D with the PowerRefractor.

The reasons for the disparity between the Autorefractor and PowerRefractor results are many. First, the set-up was not identical; subjects viewed a letter target through a Badal lens system on the Autorefractor, and a Maltese cross target on the

PowerRefractor. Target size and magnification has been demonstrated to effect amplitude, although generally by less than 1 D (Somers and Ford 1983; Rosenfield and

Cohen 1995). Research has also demonstrated that patients do not always accommodate to the same level from one test period to another, even with an identical set-up (Harb,

Thorn et al. 2006). Second, the time required to acquire five readings on the

Autorefractor was less than 30 seconds, while the PowerRefractor measurements were continuously recorded over the entire OCT imaging session (approximately two minutes).

Pupil diameter decreases with both age and accommodative effort. In order to maintain a pupil diameter greater than 4 mm, as required for recordings with the PowerRefractor, phenylephrine was used to dilate the pupil of all subjects. Phenylephrine has been shown to reduce the accommodative response, with some researchers purporting that the decrease is due to vasoconstriction of the ciliary body, and others, optical factors

accompanying an increase in pupil size (Atchison 1995; Mordi and Ciuffreda 1998;

Culhane, Winn et al. 1999; Glasser 2006). Although objective measures of accommodation in primates demonstrated no systematic difference in the maximum accommodative response before and after two drops of 10% phenylephrine (Ostrin and

Glasser 2004), and multiple studies have used up to 10% phenylephrine in human studies of accommodation (Dubbelman, van der Heijde et al. 2001; Koretz, Cook et al. 2001;

Koretz, Cook et al. 2002; Dubbelman, Van der Heijde et al. 2005; Hermans, Dubbelman et al. 2007) further research is warranted to better understand the effect of phenylephrine on the accommodative system of adult humans.

Ocular Biometry and Age

In this cross-sectional population, we found no significant age-related differences in corneal thickness, corneal curvature, posterior lens curvature, lens equatorial diameter, ciliary body thickness, ciliary body ring diameter, vitreous humor length or axial length.

Age-related changes were demonstrated for sagittal lens thickness, anterior lens curvature, anterior chamber depth, and lens refractive index.

Previous studies have reported that central corneal curvature remains constant though adulthood, as was confirmed in this study (Koretz, Kaufman et al. 1989;

Dubbelman, Sicam et al. 2006; Atchison, Markwell et al. 2008).

Koretz and coworkers reported a significant increase in corneal thickness with age; however, they attributed the change to a “roughened corneal epithelium” (Koretz,

Kaufman et al. 1989). A meta-analysis by Doughty and Zaman found no significant change in corneal thickness for Caucasian subject, but a slight decrease for other races

(Doughty and Zaman 2000). Recent work by Atchison and colleagues found a decrease of 0.00077 mm/year in a population of 106 emmetropes aged 18 to 69 years (Atchison,

Markwell et al. 2008). Our study population was predominately Caucasian and consisted of only 26 subjects, thus it is not surprising that no statistically significant difference was detected, although our 95% confidence interval does include the results found by

Atchison. Based on the standard deviation and parameter estimate from this study, a sample size of at least 83 subjects would be required to achieve a statistically significant result (www.materresearch.org)

Vitreous humor length does not change with age, as illustrated by this study

(Koretz, Kaufman et al. 1989; Atchison, Markwell et al. 2008). There is also agreement that the anterior chamber depth decreases with age, due to expansion of the lens. Our study found a statistically significant decrease of 0.029 mm/year, with a confidence interval of 0.012 to 0.045, which agrees with previous reports of decrease ranging from

0.011 to 0.022 mm/year (Koretz, Kaufman et al. 1989; Koretz, Strenk et al. 2004;

Atchison, Markwell et al. 2008; Kasthurirangan, Markwell et al. 2011).

There is some question as to whether axial length changes with age, with some studies finding no significant change, others a decrease beyond the sixth decade, and the most recent reports indicating an increase in length during the adult years (Koretz,

Kaufman et al. 1989; Atchison, Markwell et al. 2008; Fotedar, Wang et al. 2010; Nangia,

Jonas et al. 2010). In a population of nearly 5,000 adults aged 30 to 100 years, Nangia and coworkers used ultrasound to calculate an increase of 6 µ/year (Nangia, Jonas et al.

2010). And, with 106 emmetropic adults, Atchison found a similar increase of 11 µ/year using ultrasound; however was not able to confirm this finding with MRI in a subset of

60 subjects (Atchison, Markwell et al. 2008). Koretz and colleagues reported that the velocity of sound in the lens decreases by about 3 (m/sec)/year (Koretz, Kaufman et al.

1989). Assumptions of a constant velocity in ultrasound could lead to errors in axial length with age; however, MR imaging is not subject to optical or acoustic assumptions.

In our study, there was no statistically significant change in axial length with age using either ultrasound or the IOLMaster. Comparing the IOLmaster, ultrasound, and MRI axial length measurements in the full study population could help to better understand the effect of such assumptions.

The anterior lens steepens with age. Our study found a decrease in the radius of curvature of 0.11 mm/year using phakometry, and 0.18 mm/year with OCT. As discussed previously, the OCT lens analysis exhibited steepening of the lens surface with age and accommodation but the absolute values are likely non-physiological resulting from errors in either the refractive index or the algorithm used to analyze the images. The confidence interval of our phakometry data (0.05 to 0.17 mm/year) agree with results from previous research using phakometry, MRI and Scheimpflug photometry which report steepening of between 0.02 and 0.08 mm/year (Dubbelman and Van der Heijde 2001; Koretz, Strenk et al. 2004; Atchison, Markwell et al. 2008).

Earlier studies using Scheimpflug photography suggested an increase in posterior lens curvature with age (Dubbelman and Van der Heijde 2001; Koretz, Cook et al. 2001).

More recent studies, using phakometry and MRI have determined that the posterior lens curvature is not altered with age, a finding that is also supported by this study

(Kirschkamp, Dunne et al. 2004; Koretz, Strenk et al. 2004; Atchison, Markwell et al.

2008; Kasthurirangan, Markwell et al. 2011).

The equivalent refractive index of the lens decreases with age. Our estimate of

0.001 /year is smaller than the 0.004 /year change found by both Atchison and

Dubbelman’s groups (Dubbelman and Van der Heijde 2001; Atchison, Markwell et al.

2008).

Sagittal lens thickness increases throughout life. Despite differences in resolution, and assumptions of index and speed of sound, our ultrasound, OCT and MRI measurements all indicated an increase of about 0.031 mm/year, with a confidence interval of 0.019 to 0.043 mm/year. Although slightly larger, this finding generally agrees with our previous work using OCT, and that of other researchers using ultrasound, MRI and Scheimpflug photography, which reported increases between 0.014 to 0.029 mm/year

(Dubbelman, van der Heijde et al. 2001; Bullimore, Mitchell et al. 2007; Jones, Atchison et al. 2007; Atchison, Markwell et al. 2008; Richdale, Bullimore et al. 2008). It should also be noted that the decrease in anterior chamber depth (0.029 mm/year) is over 90% of the change in lens thickness, suggesting that the posterior lens surface remains relatively stable with age.

Changes in lens equatorial diameter with age are more contentious. Due to the inability to visualize that entire lens in vivo, previous research was conducted in animal models or ex vivo human eyes. As explained previously, removal of the lens from the eye will cause it to take on its most accommodated state, and, therefore, artificially shorten the equatorial diameter in a young eye. To date, the few in vivo studies have been equivocal. The original MRI studies found no change, while more recent work suggested an increase of about 0.01 mm/year (Strenk, Semmlow et al. 1999; Jones, Atchison et al.

2007; Atchison, Markwell et al. 2008; Kasthurirangan, Markwell et al. 2011). In this study, we did not find a statistically significant change in lens equatorial diameter and, while our parameter estimate suggests a decrease in equatorial diameter with age, our confidence interval includes the possibility of an increase of up to 0.01 mm/year. Based on the variation from our study population, and published parameter estimates, a sample size of at least 90 subjects would be required to demonstrate a statistically significant change of 0.01 mm/year. It is also physiologically plausible that lens equatorial diameter decreases with age, due to a displacement of the ciliary body.

Until quite recently, there were no in vivo reports of changes in the human ciliary body with age. Strenk and colleagues first used MRI to measure the ciliary body ring diameter. In her study of 48 subjects aged 22 to 91 years, she reported a decrease of about

0.025 mm/year (Strenk, Semmlow et al. 1999; Strenk, Strenk et al. 2006). A more recent study confirmed Strenk’s results, but suggested the decrease was less, perhaps 0.015 mm/year (Kasthurirangan, Markwell et al. 2011). Although both Strenk and

Kasthurirangan used subjective methods to measure the ciliary body ring diameter, our objective calculation of -0.012 mm/year, with a confidence interval of -0.034 to 0.009, are of the same order and direction as those of Strenk and Kasthurirangan. We were not able to achieve statistical significance in this population of 26 subjects. Assuming our parameter estimate as true, a study of 102 subjects would achieve statistical significance.

Sheppard and Davies recently reported changes in the cross-sectional dimensions of the human ciliary body with age (Sheppard and Davies 2011). While they used a similar technique to acquire the images, they determined ciliary body dimensions based on manual application of straight-line digital calipers, visual selection of a posterior endpoint, and post-hoc application of a refractive index to measurements oblique to the scan line. The potential errors introduced by their methods have been published previously (Bailey 2011). Nonetheless, in a subset of 37 subjects (due to technical failures from a study of 79 subjects) they reported an increase in the maximum ciliary muscle width of about 0.003 mm/year (Sheppard and Davies 2011). In a subset of 45 emmetropic subjects, they found a decrease of 0.002 mm/year at a point 2 mm posterior to the sclera spur; however, this result was not confirmed with their 34 myopic subjects

(Sheppard and Davies 2011). It should also be noted that their “relaxed” ciliary body measurements were made while the subject viewed a 0.2 D stimulus, not under cycloplegia. Our study found no statistically significant changes in the cycloplegic dimensions of the ciliary body with age. Still, it is likely that changes in the ciliary body do occur with age. Ex vivo studies have suggested a change in the relative proportion of

muscle fibers, an increase in connective tissue beginning in the third decade, and atrophy of the muscle beyond the 6th decade (Tamm, Croft et al. 1992; Tamm and Lutjen-Drecoll

1996; Pardue and Sivak 2000). And, although no statistical analysis was presented,

Stachs and coworkers 3D ultrasound biomicroscopy study suggests an alteration in the shape of the ciliary body with age (Stachs, Martin et al. 2006).

This study showed that, as the lens grows in the sagittal direction, its anterior surface becomes more curved, the anterior chamber depth shallows, and the equivalent refractive index of the lens decreases. Using a multivariate model, only accommodative response and lens thickness had significant associations with age. There was a one diopter loss of accommodative response with a 2.75 year increase in age, and a one millimeter increase in lens thickness, with every 7 year increase in age. The need for further analyses is presented at the end of this chapter.

Changes in the Lens and Ciliary Body with Accommodation

In this study, we were able to demonstrate objectively quantified changes in ciliary body thickness and ring diameter, and lens sagittal thickness, equatorial diameter, and anterior curvature, with accommodative effort. We found no statistically significant change in posterior lens curvature with accommodation.

Multiple studies have confirmed an increase in sagittal lens thickness with accommodation. As discussed previously, variations in acquisition technique and assumptions of accommodative response have created a range of values reported in the

literature, the most recent being around 0.04 to 0.08 mm/D (Dubbelman, Van der Heijde et al. 2005; Ostrin, Kasthurirangan et al. 2006; Bolz, Prinz et al. 2007; Jones, Atchison et al. 2007; Richdale, Bullimore et al. 2008; Sheppard, Evans et al. 2011). In this study, we calculated an increase of 0.065 mm/D of accommodative response.

Only recently have MRI studies demonstrated in vivo changes in equatorial lens diameter with accommodation. Our finding of a decrease of 0.075 mm/D agree with the previous reports of 0.06 to 0.09 mm/D (Strenk, Semmlow et al. 1999; Jones, Atchison et al. 2007; Kasthurirangan, Markwell et al. 2011; Sheppard, Evans et al. 2011).

As mentioned earlier, our measurement of lens curvature with OCT is not physiologically correct, due to either errors in the refractive index, or the algorithm; however, we were able to demonstrate statistically significant changes with age and accommodation. For the anterior lens, we found a statistically significant decrease

(steepening) of 0.86 mm/D of accommodative response, with a confidence interval of

0.72 to 1.01. Previous studies, using Scheimpflug photography and MRI, have reported changes of 0.51 to 0.63 mm/D (Garner 1997; Dubbelman, Van der Heijde et al. 2005;

Hermans, Pouwels et al. 2009; Kasthurirangan, Markwell et al. 2011; Sheppard, Evans et al. 2011). We were not able to detect a statistically significant change in posterior lens curvature with accommodation; however, it is likely that the posterior surface steepens by about 0.13 to 0.15 mm/D (Dubbelman, Van der Heijde et al. 2005; Hermans, Pouwels et al. 2009; Sheppard, Evans et al. 2011).

To date, no one has published physiologically accurate measurements of human lens curvature in vivo using the Visante OCT. Using model eyes, Dunne and colleagues pointed out the errors inherent to the built-in software, and proposed further surface corrections (Dunne, Davies et al. 2007). But, as the images used in this study were acquired and exported as raw data files, they were not subject to the fitting algorithms in the system software. There is some question as to the appropriate refractive index at the

1310 nm wavelength. Using the equations provided by Atchison (Atchison and Smith

2005), we calculated refractive indices of 1.367 for the cornea, 1.328 for the aqueous, and

1.408 for the lens. After comparison of our measurements to ultrasound, we determined that a refractive index of 1.390 would eliminate any bias in the lens thickness measurement by ultrasound and OCT. We were not able to correct our lens curvature measurements post-hoc. Still, it is unlikely that such a small adjustment to refractive index would correct the large difference in posterior lens curvature between OCT and phakometry. Furthermore, the anterior lens curvature should only be affected by errors in the cornea and anterior chamber refractive indices, yet significant differences were also found in the anterior lens curvature data. As the Visante is more readily available than

Scheimpflug photographic systems, does not require dilation of the pupil, and is less expensive than MR imaging, it could be a useful technique to measure the lens surface in vivo. Further work should be conducted to determine the true refractive indices at 1310 nm. The image analysis program could also be improved by the introduction of an edge detection algorithm to more precisely and objectively measure lens curvature.

To date, there are only two groups who have published reports of ciliary body ring diameter changes with accommodative effort in the human eye. Strenk and colleagues examined 48 eyes of 40 subjects aged 22 to 91 years and found an average decrease of 0.641 mm with an 8 D accommodative target (Strenk, Strenk et al. 2006).

Unfortunately, there was no measure of the subjects’ true accommodative ability, no intermediate accommodative stimuli, and images were analyzed subjectively.

Kasthurirangan measured the ciliary body ring diameter manually in 15 subjects aged 19 to 29 years, and found a decrease of 0.44 mm with “maximum accommodation,” which ranged from a 4.8 to 6.9 D stimulus (Kasthurirangan, Markwell et al. 2011). Our study imaged the ciliary body ring diameter with incremental accommodative demands, and utilized objective 3D image analysis, to calculate a decrease of 0.068 mm/D of accommodative response, independent of subject age.

Sheppard and Davies studied a population of 50 subjects aged 19 to 34 years, and demonstrated a change in ciliary muscle thickness with accommodation (Sheppard and

Davies 2010). While some of their measurement locations are based on the estimated length of the muscle, the location of their “CM25” would roughly correspond with our

CBT1. They reported a thickening of 7.1 ± 6.4 µ/D of accommodative response, which, while less than our CBT1 increase of 13 µ/D, is within our 95% confidence interval of 4 to 22 µ /D. At CBT2, they found a 2.2 ± 11 µ/D thinning using their 4 D stimulus and measured accommodative response. Our CBT2 result of 11 µ/D (95% CI: 3 to 19 µ/D) is slightly larger. As noted previously, Sheppard and Davies made manual caliper

measurements and adjusted the refractive index of the muscle post-hoc, and oblique to the scan depth. They also did not determine the cycloplegic refractive error of the subject so were not able to calculate the absolute accommodative response. In their most recent publication, which added 29 subjects aged 35 to 70 years, they reported that the change in

CM25 with accommodation was not altered with age, although they did not provide quantitative changes in thickness for this or any other location (Sheppard and Davies

2011). Our study demonstrated statistically significant thickening of the ciliary body in the anterior region (CBT1 and CBTMax), and thinning in the posterior region (CBT2 and

CBT3), that was not related to subject age.

An important caveat in our, and others’ analyses to date, is that it is not possible to differentiate the “will” or “ability” to accommodate. Analysis of changes in structure with accommodation must assume that the subject made an attempt to focus on the target

– whether or not he or she is physically able to do so. But, future analyses should examine the stepwise changes in the ciliary muscle as a subject approaches and exceeds their “optical limit” (as governed by the lack of change in the crystalline lens), to determine if the muscle (and neurological system) behaves in the same linear pattern, or if that pattern is altered with age and the inability to induce the required change in crystalline lens shape.

To date, no study has objectively quantified changes in the lens and ciliary body of a single population with accommodation and age. Our final multivariate analysis demonstrated that both lenticular and ciliary body factors are responsible for the

accommodative response in humans. We reported the following predictions for accommodative response:

With each millimeter increase (thickening) of the lens, the accommodative response increases by about seven diopters;

With each millimeter decrease (thinning) at CBT3, the accommodative response increases by almost five diopters;

With each millimeter decrease (steepening) of the anterior lens radius of curvature, the accommodative response increases by about 0.25 diopters; and

With each year increase in age the accommodative response decreases by a little more than 0.25 diopters.

Towards a Better Understanding of Accommodation and Presbyopia

It is both with age and with accommodation that the lens grows in thickness and the anterior surface steepens. As Brown’s lens paradox suggested, the eye should become more near-sighted with age, but due to the decrease in refractive index, distance focus is preserved (Jones, Atchison et al. 2005; Kasthurirangan, Markwell et al. 2008). The

Geometric Theory of presbyopia, proposed by Koretz and Handelman, suggested that age-related lens growth caused an increased tension in the zonules and altered the directional forces of the ciliary muscle (Koretz and Handelman 1988; Koretz, Kaufman et al. 1989). Based on their MRI work, Strenk, Strenk and Koretz later modified this theory to suggest that lens growth instead forced an anterior and inward displacement of the

ciliary body via the iris, and lead to a slackening of the zonular fibers (Strenk, Strenk et al. 2005). Their Modified Geometric Theory would explain an increase in lens curvature and thickness, and decrease in lens equatorial diameter with age.

The results of this study demonstrate that the lens dimensions are intimately related to both aging and the accommodative response. The ciliary muscle appears to retain its ability to contract, at least through the onset of presbyopia. Still, there is much work left to be done both with this data set, and in future studies. First, further development of the OCT-derived measurements should be undertaken. Ultrasound biomicroscopy can be used to determine the correct refractive index for the cornea, aqueous, lens and ciliary body at 1310 nm. And, the use of model eyes of known parameters can aid in the understanding of distortions or errors in the OCT image acquisition and analysis. Finally, the implementation of computer-based edge detection methods could allow for fast, repeatable and objective measurements of the lens.

There is a wealth of data yet to be uncovered in our MR images. This is the only data set to date with 3D acquisition of the accommodative system in both presbyopic and non-presbyopic subjects, with stepwise increases in accommodative demands. Although beyond the scope of this dissertation, the MR images allow the ability to study changes in anterior chamber depth, circumlental space, whole lens volume and shape, and the geometry of the entire system with age and accommodative effort.

Finally, analysis of the full data set may allow us to determine statistically significant changes in areas where others have reported significant findings, but where,

with a population of only 26, we were unable to do so. Specifically, with the full dataset of 91 subjects, it may be possible to achieve statistically significant regressions of lens equatorial diameter, ciliary body ring diameter, and ciliary body thickness, with age.

Also, as the larger sample size includes 46 myopic and 18 hyperopic subjects, we will be able to study differences in ocular biometry and presbyopia with refractive error.

Chapter 5: Conclusions

In conclusion, we demonstrated the application of MR and OCT techniques to image the human eye in vivo. We incorporated computer-based image analysis programs to objectively measure the lens and ciliary body, and compared the results to established techniques. And, we demonstrated changes in both lenticular and ciliary body dimensions with age and accommodation in a single cohort of emmetropic adults.

The ability to develop new and better treatments for presbyopia depends on an understanding of the entire accommodative system and the significant changes that occur in the human eye with age. It will be imperative to understand the structure and function of the system, before, during, and after the onset of presbyopia, as well as after removal of the aged lens contents and/or capsule. And, it will be necessary to understand the force and direction of ciliary muscle contraction available to harness for accommodating intraocular lenses, lens refilling procedures, or other potential treatments. The use of the image acquisition and analysis techniques presented in this dissertation, as well as the results of this emmetropic dataset and the future analysis of the entire dataset, will hopefully aid in the achievement of these goals.

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Appendix: Tables and Figures

Repeated Variable Instrument Dilation Status Measures Grand Seiko Refractive error 5 cycloplegia autorefractor Accommodative amplitude RAF rule 3 none Grand Seiko 5 at each level none Accommodative response autorefractor (0, 2, 4, and 6 D) PlusOptix continuous pupil dilation PowerRefractor

Table 1. Refractive and accommodative variables collected. (*Pupil dilation: 2.5% phenylephrine; Cycloplegia: 2.5% phenylephrine and 1% tropicamide.)

Repeated Variable Instrument Dilation Status Measures Corneal thickness Visante AS-OCT 3 none Corneal curvature Keratron Scout 3 none ultrasound 5 cycloplegia Anterior chamber depth Visante AS-OCT 3 cycloplegia Vitreous chamber depth ultrasound 5 cycloplegia IOLMaster 5 pupil dilation Axial length ultrasound 5 cycloplegia pupil dilation and Visante AS-OCT 3 at each level cycloplegia Lens sagittal thickness 12 at each (0, 2, 4, and 6 D) 7T MRI none level Ultrasound 5 cycloplegia Lens equatorial thickness 12 at each 7T MRI none (0, 2, 4, and 6 D) level Video Lens refractive index 1 cycloplegia phakometry Video 1 cycloplegia Lens curvature phakometry (0, 2, 4, and 6 D) pupil dilation and Visante AS-OCT 3 at each level cycloplegia Ciliary body thickness pupil dilation and Visante AS-OCT 4 at each level (0, 2, 4, and 6 D) cycloplegia Ciliary body ring diameter 12 at each 7T MRI none (0, 2, 4, and 6 D) level

Table 2. Biometric variables collected. (*Pupil dilation: 2.5% phenylephrine;

Cycloplegia: 2.5% phenylephrine and 1% tropicamide.)

Cornea Fixation Line

Lens

Iris

Lens

Sclera

Cornea

Ciliary body

Figure 1.Visante OCT images of the anterior chamber (top) crystalline lens (middle) and ciliary body (bottom).

Figure 2. Ciliary body OCT images taken with the subject viewing targets through a mirror attached to the machine (top). The PowerRefractor was used to make simultaneous measurements through the viewing mirror (bottom).

Figure 3. Cross sectional slice of a 3D MRI

Figure 4. MR imaging set-up. A receive coil was affixed to the eye (top) and the subject viewed the targets on a rod behind him through a mirror mounted in the transmit head coil

(bottom).

Figure 5. Ciliary body OCT image analysis showing images that would pass visual inspection (top) and fail inspection due to threshold (middle) and upper line cutting too high (bottom).

Figure 6. OCT lens analysis. Points were selected on raw images of the cornea (top left) and lens (bottom left) and fitted with spheres. The images were stitched together at the anterior lens surface (top right) and corrections were made for refractive index.

(Red lines: raw image fitting, cyan line: corrected posterior corneal surface, blue line: corrected anterior lens surface, yellow line: corrected posterior lens surface).

Figure 7. MR image analysis pre-processing and region growing. Left to right: The image is cropped, a 3D bilateral filter is applied, coil sensitivity drop-off correction is applied, and region growing is used to approximate the lens location and orientation.

Figure 8. Extraction of lens and ciliary body ring diameters. Left to right:

Coronal projection through the lens and ciliary body, gradient magnitude, and circles fitted to 256 minima (lens) and maxima (ciliary body) points.

Figure 9. Extraction of lens surface. (2D representation of 3D analysis) Edges were detected using gradients in all three dimensions

(left), and non-maximum suppression (middle) and skeleton (right) methods were applied to thin the lens surface to a single pixel.

Figure 10. Visual inspection of the MRI data (left to right: sagittal, coronal and collapsed images. Top Row: images that would pass visual inspection;

Bottom Row: images that would fail visual inspection.

Figure 11. Intraclass correlations for MRI measures of lens thickness (LT), lens equatorial diameter (LED) and ciliary body ring diameter (CBRD).

Data Mean ± SD Median Range Age (years) 39.48 ± 7.42 38.4 30.4 – 50.0 Height (feet/inches) 5′7″ ± 4″ 5′7″ 5′1″ – 6′3″ Weight (pounds) 167.3 ± 28.0 162.5 126 – 220 Refractive error (D) - 0.03 ± 0.24 - 0.06 - 0.50 – 0.50

Table 3. Demographic information by self-report and cycloplegic spherical equivalent refractive error with the GrandSeiko Autorefractor.

Target Instrument Mean ± SD Median Range Autorefractor 0.28 ± 0.26 0.28 - 0.23 – 0.75 0D target PowerRefractor 0.25 ± 0.58 0.38 - 0.75 – 1.35 Autorefractor 0.90 ± 0.48 0.86 - 0.25 – 1.65 2D target PowerRefractor 1.00 ± 0.77 0.91 - 0.50 – 2.82 Autorefractor 1.93 ± 1.18 2.33 - 0.03 – 3.75 4D target PowerRefractor 1.74 ± 1.23 2.00 - 0.37 – 3.97 Autorefractor 2.77 ± 1.88 3.51 0.03 – 5.15 6D target PowerRefractor 3.10 ± 2.04 3.06 0.04 – 6.67 RAF rule 7.52 ± 4.62 7.52 1.66 – 15.8 Maximum Autorefractor 2.82 ± 1.82 2.82 0.03 – 5.15 response PowerRefractor 3.14 ± 1.98 3.14 0.17 – 6.67

Table 4. Accommodative response and amplitude (D). Positive values indicate greater accommodation.

Figure 12. Accommodative response with RAF rule, PowerRefractor and autorefractor.

Figure 13. Accommodative response to 0, 2, 4 and 6 D targets with the autorefractor. (Black lines: 30 < 35 years, blue: 35 < 40 years, green: 40

< 45 years, and red: 45 - 50 years.)

Figure 14. Accommodative response to 0, 2, 4 and 6 D targets with the

PowerRefractor. (Black lines: 30 < 35 years, blue: 35 < 40 years, green:

40 < 45 years, and red: 45 - 50 years.)

Figure 15. Bland-Altman plots of accommodative response for 0 and 2 D targets. 98

Figure 16. Bland Altman plots of accommodative response for 4 and 6 D targets.

Figure 17. Bland Altman plots of lens thickness by ultrasound with OCT (top) and

MRI (bottom).

100

Figure 18. Bland Altman plots of anterior (top) and posterior (bottom) lens curvature by OCT and phakometry.

101

Structure Dimension Instrument Mean ± SD Median Range average Keratron 43.70 ± 1.42 44.04 41.4 – 46.0 curvature (mm) Cornea thickness OCT 543.0 ± 27.8 545.5 490.3 – 586.7 (microns) Anterior depth (mm) Ultrasound 3.58 ± 0.36 3.54 2.87 – 4.35 chamber Vitreous depth (mm) Ultrasound 16.0 ± 0.7 15.9 15.1 – 17.6 chamber Axial Ultrasound 23.5 ± 0.7 23.4 22.6 –24.9 depth (mm) length IOLMaster 23.6 ± 0.7 23.5 22.7 – 25.2 OCT 3.95 ± 0.30 3.91 3.42 – 4.80 sagittal MRI 3.93 ± 0.30 3.86 3.52 – 4.73 thickness (mm) Ultrasound 3.95 ± 0.30 3.90 3.39 – 4.80 equatorial MRI 8.81 ± 0.29 8.74 8.38 – 9.48 diameter (mm) Lens refractive index Phakometry 1.45 ± 0.01 1.45 1.43 – 1.46 anterior Phakometry 10.75 ± 1.27 10.68 7.70 – 13.52 curvature (mm) OCT 12.36 ± 1.94 12.13 8.80 – 16.54 posterior Phakometry 7.43 ± 0.46 7.44 6.82 – 8.46 curvature (mm) OCT 10.86 ± 0.93 10.60 9.70 – 13.72 thickness OCT 0.80 ± 0.10 0.81 0.61 – 0.98 @ 1mm (mm) thickness OCT 0.49 ± 0.11 0.51 0.19 – 0.72 Ciliary @ 2mm (mm) body thickness OCT 0.27 ± 0.08 0.27 0.08 – 0.40 @ 3 mm (mm) ring diameter MRI 11.62 ± 0.37 11.68 10.99 – 12.34 (mm) Table 5. Baseline ocular biometry.

102

Figure 19. Anterior chamber depth (top) and sagittal lens thickness

(bottom) with age (data collected under cycloplegia except for MRI).

103

Figure 20. Refractive index (top) and anterior lens curvature (bottom) with age (data collected under cycloplegia).

104

Figure 21. Corneal thickness (top) and corneal curvature (bottom) with age.

105

Figure 22. Vitreous chamber depth (top) and axial length (bottom) with age

(data collected under cycloplegia).

106

Figure 23. Posterior lens curvature (top) and lens equatorial diameter (bottom) with age (posterior lens curvature collected under cycloplegia).

107

Figure 24. Ciliary body ring diameter (top) and thickness (bottom) with age (data collected under cycloplegia. 108

Figure 25. Bland Altman plots of lens thickness for 0 and 2D targets.

109

Figure 26. Bland Altman plots of lens thickness for 4 and 6 D targets.

110

Figure 27. Change in lens thickness with accommodation. Lines are fitted using the mixed model equations with age fixed at 30 years.

111

Figure 28. Change in anterior and posterior lens curvature with accommodation. Lines are fitted using the mixed model equations with age fixed at 30 years.

112

Figure 29. Change in lens equatorial diameter (LED) and ciliary body ring diameter

(CBRD) with accommodation (open symbols: subjects aged 40-50 years, closed symbols: subject aged 30-40 years). Lines fitted to mixed models equations.

113

Figure 30. Change in ciliary body thickness at points 1, 2, and 3 mm posterior to the sclera spur (CBT1, CBT2, CBT3) and maximum width (CBTMax) with accommodation. Lines fitted to mixed models equations.

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Ciliary Body 0 D Target 2 D Target 4 D Target 6 D Target Dimension thickness @ 1 0.752 ± 0.085 0.769 ± 0.090 0.779 ± 0.089 0.820 ± 0.118 mm (mm) thickness @ 2 0.493 ± 0.102 0.486 ± 0.106 0.481 ± 0.107 0.469 ± 0.124 mm (mm) thickness @ 3 0.284 ± 0.076 0.270 ± 0.078 0.267 ± 0.074 0.232 ± 0.075 mm (mm) maximum 0.823 ± 0.078 0.846 ± 0.083 0.856 ± 0.075 0.931 ± 0.128 thickness (mm) ring diameter 11.62 ± 0.37 11.55 ± 0.39 11.47 ± 0.37 11.41 ± 0.30 (mm)

Table 6. Ciliary body thickness and ring diameter at distance and with stepwise increases in accommodative demand.

115