Ciliary Muscle Thickness Changes Are Associated with Age THESIS

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Ciliary Muscle Thickness Changes Are Associated With Age

THESIS

Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University

Alyssa Mary Willig, B.S.

Graduate Program in Vision Science

The Ohio State University

2015

Master’s Examination Committee:

Melissa Bailey, OD, PhD, Advisor

Donald Mutti, OD, PhD

Jeffrey Walline, OD, PhD

Alyssa Mary Willig

2015

Abstract

Purpose: To model the cross-sectional relationship of ciliary muscle thickness (CMT) and age throughout the decades of life.

Methods: Subjects (N = 784) were ages 3 to 91 years. Measurements included distance

autorefraction and Zeiss OCT imaging of the ciliary muscle in the right eye. Four ciliary

muscle thickness (CMT) measurements were made at the thickest region (CMTMAX)

and at 1, 2, and 3 mm from the scleral spur (CMT1, CMT2, CMT3). General linear

regression models were used.

Results: Mean ± standard deviation (SD) subject age (years) was 23.4 ± 18.4 (range: 3.4

to 91). Mean ± SD spherical equivalent (D) was −0.53 ± 1.88 (range: −10.87 to +6.00).

The relationship between all CMT measures and age was significant [CMTMAX:

Intercept = 805.75, Age (β = 1.04, p < 0.000001)]; CMT1: Intercept = 746.6, Age (β =

2.3, p = 0.0003), Spherical Equivalent, M (β = −5.8, p = 0.005), Age2 (β = −0.02, p =

0.01), M2 (β = −0.9, p = 0.02); CMT2: Intercept = 483.5, Age (β = 2.4, p = 0.0007), M (β

= −17.7, p < 0.000001), Age2 (β = −0.03, p = 0.001), M2 (β = −1.9, p = 0.00005); CMT3:

Intercept = 254.6, Age (β = 2.8, p < 0.000001), M (β = −13.4, p < 0.000001), Age2 (β =

−0.04, p = 0.00002), M2 (β = −1.2, p = 0.001). The relationship between the location of

ii the point of maximum thickness relative to the scleral spur (SStoMAX) and age was also significant (Intercept = 666.55, Age (β = 4.5, p = 0.08), M (β = −33.75, p < 0.000001),

Age2 (β = −0.07, p = 0.007), M2 (β = −2.2, p = 0.06).

Discussion: These data show that the relationship between cross-sectional ciliary muscle thickness and age is positive in childhood (increasing ciliary muscle thickness with increasing age) but negative in older age (decreasing ciliary muscle thickness with older age). The point of maximum thickness increased across all decades of life and also moves

posteriorly in childhood and then anteriorly in older adults. A similar relationship

showing an increase in thickness in younger ages also has been found in previous studies.

To the best of our knowledge, our study is the first to describe the cross-sectional

relationship between ciliary muscle thickness and age as a quadratic function.

iii

Dedication

This document is dedicated to my family and fiancé, Justin, for their love and support.

Acknowledgments

I would like to acknowledge all those who contributed to the success of this project including Loraine Sinnott, PhD, and Chiu-Yen Kao, PhD, for their advice on statistical and image analysis, and OSU undergraduates who helped me collect my data at the

Center of Science and Industry. I would especially like to thank Melissa Bailey, OD,

PhD, for her constant guidance, enthusiasm, and inspiration as my mentor. This project and its presentation were supported by grants from the National Eye Institute (NEI T35

EY007151) and the Elmer H. Eger Memorial Student Travel Fellowship Grant from The

American Academy of Optometry.

Vita

October 7, 1990………………………………….….Born - Pittsburgh, Pennsylvania

April 2008…………………………………………..Seton LaSalle Catholic High School

April 2012………………………………………….. Bachelor of Science Biological Sciences University of Pittsburgh

December 2015…………………………………….. Master of Science Vision Science The Ohio State University

May 2016…………………………………………....Doctor of Optometry The Ohio State University

Publications

● Lee, Bruce Y., Sarah M. Bartsch, and Alyssa M. Willig. "The Economic Value of a Quadrivalent versus Trivalent Influenza Vaccine." Vaccine 30.52 (2012): 7443- 446 ● Lee, Bruce Y., Kristina M. Bacon, Diana L. Connor, Alyssa M. Willig, and Rachel R. Bailey. "The Potential Economic Value of a Trypanosoma Cruzi (Chagas Disease) Vaccine in Latin America." PLoS Negl Trop Dis PLoS Neglected Tropical Diseases 4.12 (2010)

Fields of Study

Major Field: Vision Science

Table of Contents

Abstract.………...…………………………………………………………………………ii

Dedication……..………………………………………………………………………….iv

Acknowledgments….……………………………………………………………………...v

Vita………………………………………………………………………………………..vi

List of Tables……………………………………………………………………………viii

List of Figures…………………………………………………………………………….ix

Chapter 1: Introduction……………………………………………………………..…….1

Chapter 2: Methods………………………………………………………………………14

Chapter 3: Results……….……………………………………………………………….18

Chapter 4: Discussion…...………………………………………………………….....…20

References………………………………………………………………………………..41

vii

List of Tables

Table 1. Age, Spherical Equivalent Refractive Error, and Ciliary Muscle Thickness….28

Table 2. Regression models of age and ciliary muscle relationships……………………29

Table 3. Regression models of the relationship between age and ciliary muscle dimension for all subjects age 30 years and older…………………………………………………...30

viii

List of Figures

Figure 1. Mean spherical equivalent frequency in the subject population………………31

Figure 2. Example ciliary muscle image…………………………………………..……..32

Figure 3. Ciliary muscle configuration variety across the sample…………………….....33

Figure 4. Ciliary muscle thickness of a 10, 20, and 30 year old example patient based on

model estimates…………………………………………………………………………..34

Figure 5. Ciliary muscle thickness of a 30, 40, 50, 60, and 70 year old example patient based on model estimates………………………………………………………………...35

Figure 6. CMTMAX as a function of mean spherical equivalent refractive error for the

entire sample……………………………………………………………………………..36

Figure 7. CMT1 as a function of mean spherical equivalent refractive error for the entire

sample……………………………………………………………………………………37

Figure 8. CMT2 as a function of mean spherical equivalent refractive error for the entire

sample……………………………………………………………………………………38

Figure 9. CMT3 as a function of mean spherical equivalent refractive error for the entire

sample……………………………………………………………………………………39

Figure 10. Relationship between gender, refractive error, and ciliary muscle dimensions for all subjects age 30 years and older…………………………………………………...40

Chapter 1: Introduction

Eye growth and development have been widely studied areas of vision science.

We know how the majority of ocular structures, such as the crystalline lens, grow,

function, and change with age and time. What we do not know is how the ciliary muscle

is affected by time or the role it plays in eye growth. The ciliary muscle has, up until

recently, only been studied in-depth through histological analyses, as it was difficult to

view in vivo because it was obscured by the iris. Many studies have assessed ciliary

muscles in animal embryos, but human ciliary muscle studies, in general, are lacking.

Because only the histological make-up has been known, the ciliary muscle has largely been ignored in other studies of human eye development and how it changes throughout life. This thesis serves to discover how the ciliary muscle is related to general eye growth

during childhood, refractive error development, and adulthood, including advanced age.

A large portion of what we do know about eye growth after birth comes from the

Orinda Longitudinal Study of Myopia (OLSM) and the Collaborative Longitudinal

Evaluation of Ethnicity and Refractive Error (CLEERE) Study. Longitudinal studies such

as the OLSM extensively studied the eyes of growing children by measuring anterior

chamber depth, lens thickness, vitreous chamber depth, refractive error, corneal power,

axial length, anterior lens radius of curvature, posterior lens radius of curvature, and lens 1 power.1 They discovered that eye growth is coordinated among these structures in all directions and that having myopic parents can impact eye growth and subsequently refractive error in a child somewhat proportionately to whether one or both parents are myopic.1,2

Data from the OLSM suggest that emmetropic subjects exhibited a stable refractive error, whereas the majority of ametropic subjects progressed towards a less positive refractive error between the ages of 6 and 12 years. Crystalline lens thickness declines until the age of 9.5 years, then begins to thicken with time.2 It is thought that although lens fibers are continuously added to the crystalline lens over time, an overall thinning is caused by an equatorial pulling on the lens as the eye grows and anterior and posterior radii of curvature flatten in children.1 As this occurs, increases in anterior chamber are found between the ages of 6-12 years to be 0.22 mm, but these changes differ between refractive error groups and whether or not refractive error is stable or progressive.1,2 Within the same age group, vitreous chamber depth increases by about

0.52 mm and axial length increases by about 0.50 mm.1

Subjects who began the study with higher amounts of hyperopia tended to be arrested in that state while low hyperopes were more likely to reach emmetropia through changes in axial length and anterior chamber depth.2 Corneal power was determined to be the main initial difference between myopic and emmetropic eyes; corneal power remained stable in myopes while gradually decreasing in emmetropes.2 Data from the

CLEERE study showed that the difference in corneal power between emmetropes and

2 those who became myopes was only 0.25D.3 This suggests that there are significant changes being made in emmetropic eyes that allow a balance between eye elongation of the anterior and vitreous chamber depths and changing refractive powers of the cornea and lens to allow clear image focus on the retina.1,2 The majority of changes are found in the crystalline lens, and have been estimated to be around 15D.3 With advancing myopia, an increased growth suggests an uncontrolled developmental process.2 Equatorial restrictions have been proposed where peripheral defocus may increase axial elongation.3

Although a main component of the eye’s equator, the ciliary muscle, however, is missing from this list of growth curve parameters and has long been neglected in all kinds of research, including research related to myopia, because the technology was not available to easily visualize it.

Although little is known of how the ciliary muscle changes with age, its structural basis has been widely studied. Histologically, the ciliary muscle is composed of two main parts: a posterior smooth pars plana which stretches to the vitreous body and is surrounded by predominantly longitudinally projecting zonular fibers and the anterior pars plicata with 70-80 protruding ciliary processes.4 The ciliary process ridges point inwards toward the posterior chamber.4 Concave areas between the processes attach to the zonular fork which is derived from two main zonules connecting to the anterior and posterior lens capsule.4 The pars plicata transitions into the posterior surface of the iris as it moves anteriorly.4

The smooth muscle of the ciliary muscle makes up the majority of the stroma of

the ciliary body.4 The ciliary muscle appears as a right-angled triangle with its outer

surface lying underneath the sclera.4 The supraciliaris, a thin layer of collagen fibers,

fibroblasts, and melanocytes, lies in-between the sclera and ciliary muscle.4 The most

anterior portion of the ciliary muscle is connected to the scleral spur and the trabecular

meshwork.4 No elastic fibers are found in the stroma of the ciliary processes.4 Instead,

connective tissue fuses with the blood vessel layer.4

Three orientations of ciliary muscle fibers are present in the human: radial,

longitudinal, and circular.4 Muscle fiber bundles work together as a syncytium during

accommodation; as the muscle contracts, a longitudinal orientated fibers that were found

in the outer part and fibers become more circular as they move anteriorly and inward.4

This specific organization allows bundles to increase in size as they move inwardly.4 As

the circular fibers thicken, the anterior-inward portion of the muscle becomes more

defined as the muscle contracts during accommodation.4 During this process the zonules

relax and the lens becomes more spherical.4 Muscle sheaths allow coordinating gliding of

muscle bundles, slowly reorganizing fiber placement during muscle contraction.4 It is crucial that a high amount of mitochondria are found within ciliary muscle cells to produce an appropriately high amount of energy for accommodation.4 Each cell also

contains myofilaments in its periphery.4 Among bundles in the extracellular matrix are collagen type I and III.4 The ciliary muscle is not a typical smooth muscle; it contains

dense bands aligned perpendicularly with the cell membrane that continue into the

periphery until reaching myofibrils.4

The unpigmented ciliary epithelium forms the innermost layer of the ciliary

body.5 It consists of a monolayer of cells that project from the base of the iris and

continue along the pars plicata and pars plana until it reaches the retina at the ora serrata.5

The cell type distribution is as follows: cuboidal cells are located at the pars plicata and

tall columnar cells at the pars plana.5 It is believed that the elongated cells anterior to the

ora serrata are shaped according to forces from the vitreous base.5 The basement membrane over the cells in the pars plana and pars plicata thickens with age and serves as the inner limiting membrane of the unpigmented ciliary epithelium.5

The pigmented epithelium is a monolayer of pigmented cells following the same

route as the unpigmented ciliary epithelium but is continuous with the retinal pigmented

epithelium posteriorly.5 Many spherical pigment granules are concentrated anteriorly in

the cytoplasm of the pigmented epithelium.5 Below the epithelium is an inner connective

tissue layer, containing blood vessels and the ciliary muscle.5 This layer is sparse at the

pars plicata and serves as a dividing line between epithelial layers and the ciliary muscle.5

It gradually thickens at the pars plana with the addition of collagen fibers with age.5

There is very little connective tissue in younger eyes.5 The anterior two thirds of the

ciliary body is described as the largest portion as minimal fibers extend posteriorly into

the ora serrata.5 The ciliary body functions include secretion of aqueous humor, strength

for accommodation, supports and creates zonule fibers, and becomes part of the vitreous

base.5

The ciliary body embryonic origins include the neuroectoderm of the optic vesicle

and the neural crest mesenchyme, which become the outer pigmented and inner

unpigmented epithelium and the stroma and ciliary muscle, respectively.5 During the first

year of life, eye growth is accelerated and continues at a fast rate until age 3 years.5

Approximately 76% of ciliary body develops by 24 months and eye growth continues after this time.5 The cornea flattens by about 7 D in the first 6 weeks of life due to

increasing axial length.5 The ciliary body contributes about 20% to the total linear growth

of the eye; It is estimated that out of the 21.7mm net increase in circumferential eye

growth from birth to adulthood, 4.2mm is made up from nasal and temporal ciliary body

lengthening.5

It has been shown through CLEERE that growing eyes become relatively less

oblate or more prolate as they reach myopia.6 A study by Oliveira et. al. concluded that

ciliary bodies tend to be thicker in myopic eyes.6 This may discount the choroid’s role in

eye growth, which suggests that myopic eyes tend to have thinner choroids which tend to change as the eye grows axially.7 Ocular growth in emmetropic eyes exhibit thicker

choroids, implying that this thickening may be a regulating factor to inhibit growth.7 If

the choroid is not part of the solution, more emphasis must be placed on the significance

of equatorial regions.6 In both children and adults, posterior ciliary muscle measurements

increase as refractive error becomes more myopic and eye growth increases.6 It is

6 currently unclear whether or not children that initially have thicker ciliary muscles will become myopic in the future.6 This leads to a question of which came first, the thickened ciliary muscle, or the myopia? Accommodative ability in myopic children should also be considered because it has been speculated that myopes tend to exhibit accommodative lag either before or after refractive error onset.6,8 Retinal defocus, which causes myopic eye growth in animal models, may be a side effect of under accommodating.8 This observation has supported PAL use as a means of myopia control to allow proper focus of near images.8

Our lab has previously looked at ciliary muscle development in children and how the thicknesses differ. It has been speculated that these differences may be due to refractive error. Studies from Pucker et al. (2013) and Kuchem et al. (2013) have independently suggested that there may be multiple causes of myopia development based on varying ciliary muscle thickness values.9,10 One anatomical variant that has been postulated to be related to myopia is the ciliary muscle. Posterior measurements of ciliary muscle thickness (CMT) are typically increased in myopia. Supporting conclusions in a recent study of children have shown that there is significant variability in CMT; posterior

CMT measurements tend to be thicker in myopia whereas anterior CMT measurements are thicker in hyperopia.10 Not every case of myopia, however, shows this relationship.

Buckhurst et. al (2013) did not find a significant correlation between CMT, axial length, and mean spherical equivalent, but instead referenced the choroid’s role in eye growth according to a positive correlation between ocular volume and all CMT measurements.11

Mean spherical equivalent was, however, significantly associated with CMT, with thicker

CMT2 and CMT3 in the myopic eyes.(8) A study of anisometropic patients found no

significant evidence that the longer, more myopic eye had a higher CMT relative to the

fellow eye, suggesting that eye growth can also occur without an increase in CMT.10

These findings led to the follow question: Is the cause of myopia different between

myopes with thin and thick ciliary muscles due to this varying evidence?

We may ultimately learn that the early ciliary muscle appearance can explain how

it contributes to eye growth and refractive error development during childhood and

adolescence, but what happens as the body’s growth curve begins to decline with older

age? This would also be important to know. Due to the wide range in age and number of

sample subjects available at our testing location, we were able to evaluate how ciliary

muscle thickness can change during adulthood and advanced age. Richdale et. al (2013)

and Atchinson et. al. (2008) noted that in middle age groups, age was related to thicker

crystalline lenses, steeper anterior radius of curvature of the lens, and a decreased anterior

chamber depth.12,13 No significant relationship with age was found in the vitreous chamber depth, axial length, posterior radius of curvature of the lens, or any ciliary

12 muscle landmark measurement. To our knowledge, no previous study has evaluated

ciliary muscle changes in a very large cross-sectional sample along the lifetime of a

ciliary muscle. This is of importance for presbyopia studies to determine if the ciliary

muscle can still sustain accommodation after cataract removal and/or replacement with

an accommodating intraocular lens. In an era of increasing visual demands from

electronic devices and a population where the majority of binocular vision disorders are

due to accommodative dysfunction, gaining an in-depth insight into how the focusing

system of the eye works is crucial.14 This information on the ciliary muscle can complete the picture of the ocular anterior segment and may also impact glaucoma or accommodative therapy or treatments.

During accommodation, the ciliary muscle moves forward and inward to release tension on the zonules that connect it to the crystalline lens.15 This method is based on the

Helmholtz theory.15 The anterior ciliary muscle portions (CMTMAX and CMT1) thicken

and also thin posteriorly (CMT2 and CMT3) with accommodation.12,16,17 CMT2 has

been found to be insignificant and variable between subjects when accommodative

14,16 demands change in other studies. Interestingly, the ciliary muscle contracts more

17 temporally than it does nasally. There is also evidence that the center of mass of the

crystalline lens also moves anteriorly as it thickens with age.15 Because about 90% of the

decrease in anterior chamber depth is caused by lens thickening, the posterior lens surface

remains in a fixed position overtime.12 There is also evidence that anterior chamber depth decreases at a rate of 0.011 mm/year and the central crystalline lens thickness increases at

a rate of 0.0235 mm/year.13

Overtime, the ciliary muscle also tends to move forward as well as the crystalline

lens hardens due to sclerosis and develops into a cataract with opacification induced

haze.15 In order to be functionally adequate, the crystalline lens must be clear and

flexible. With age, the lens anterior radius of curvature significantly decreases about

0.0438 mm/year.13 Cataracts are slowly formed over time as alpha, beta, and gamma

crystallin proteins aggregate in staggering amounts within the fiber cells of the lens after an up-regulation of gene transcription.18 As this occurs, lens fiber cells lose crucial

organelles needed for proper function.18 After cataract surgery, the ciliary muscle regains most of its original location as the choroid repositions the ciliary muscle.15 The muscle

may, however, appear to be larger due to accumulation of connective tissue over

time.(12)

Extensive time of the lens being vulnerable to UV light exposure and oxidative stress among other insults also detrimentally affects longevity and configuration of fiber cells.18 Accumulation of these altered proteins causes light scatter as the lens refracts

light, which decreases visual acuity and contrast sensitivity.18 It has been shown that

decrease in normal alpha-crystallin complexes adversely affect chaperone protein activity

and may lead to nuclear sclerosis.18 When these structural changes occur, the lens

undergoes presbyopia, a decreased ability to change lens shape properly to allow

accommodation with ciliary muscle contraction.18 Although presbyopia is a gradual

process, symptoms usually begin in the fourth decade of life.12

When testing accommodative demand in a study by Richdale et. al. (2013), a

linear increase in accommodative response was demonstrated in subjects under the age of

40 years with increasing target demand, while subjects over 40 years held a consistent

accommodative response of high lag while attempting to focus on increasing demands.12

It is unclear, however, if the linear changes are truly linear or how much accommodative

10 lag plays a role in an individual’s accommodative adaptations.16 Lewis et. al (2012)

16 found that a child’s accommodative response is related to ciliary muscle thickness. The loss of accommodative ability was calculated to be approximately 0.2 D each year.12

Sheppard and Davies (2010) estimated accommodative changes on the ciliary muscle based on its length and found that accommodative change per diopter thickened anterior ciliary muscle portions and thinned posterior portions.17 This finding is related to the

Hess-Gullstrand theory of presbyopia; the amount of ciliary muscle contraction per diopter of accommodative response over time is stable and there is an increasing underlying strength of the ciliary muscle over time that does not end in normal accommodation in the presence of an inflexible lens.12 Another non-supported theory of presbyopia is the Schachar Theory which states that equatorial zonules increase tension on the lens when the ciliary muscle contracts.19 As this occurs, anterior and posterior zonules are relaxed.19 The central thickness of the lens then increases its central surfaces steepen and peripheral surfaces flatten.19

When the ciliary muscle contracts as a sphincter by moving forward and centripetally, the lens becomes more spherical in shape.20 This value has been estimated to be around 0.105 mm/D assuming that there is some accommodative lag when viewing

12 targets with a larger demand. Studies have shown that even when the lens hardens, the ciliary muscle still holds onto its ability to contract even with age-related changes, but loses some of its ability to slide forward and centripetally.18,20,21 This functional change may be due to an emphasis on longitudinal muscle fibers and decrease in circular fibers

which exhibit centripetal movement.20 With pilocarpine instillation before and after cataract surgery to determine muscle functionality, it was found that muscle movement increases after cataract surgery compared to pre-surgical movement.21 This suggests that

a hardened lens may arrest the ciliary muscle in a non-accommodative state as it pulls on

zonules.21

Studies have shown that in human eyes, ciliary muscle thickening characteristic of

accommodation was related to accommodative amplitudes and decreased with age as

accommodative abilities also decrease.15 In non-human primate eyes, a decrease in the

22 space between the lens and the ciliary muscle apex with age was found. Also, the apex

appears to increase with age in non-accommodating eyes more than accommodating eyes

because younger ciliary muscles thicken more with pilocarpine instillation.15 This

suggests that apex thickness during contraction does not affect accommodative

amplitude.15

The ciliary muscle intactness and condition after cataract surgery can determine

how effective accommodative training or technologies can be improved, especially

because ciliary muscle changes have been shown to be just as crucial to presbyopia

development as crystalline lens changes.15,21 Because pseudophakic eyes with

accommodative IOLs increase their dioptric power optically by changing location of the

implant independently of the ciliary muscle, advancements in knowledge of the ciliary

muscle can restore an eye’s original and full accommodative mechanism without

compromise such as the need of a low add bifocal or induced haloes or astigmatism from

IOL optics.23 A newer technology called the Synchrony accommodating IOL, made up of a front plus optic and a back minus optic, already takes advantage of ciliary body

contraction to move the a front optic forward as a spring haptic allows the two optics to

come together to focus on near targets.

In summary, the present study was designed to investigate the growth pattern of

the ciliary muscle throughout the various decades of human life. To the best of our

knowledge, our study is the first to describe the cross-sectional relationship between ciliary muscle thickness and age as a quadratic function.

Chapter 2: Methods

Subjects

Subjects were recruited from guests visiting the Center of Science and Industry

(COSI) in Columbus, Ohio, at a laboratory space called the EyePod within the Labs in

Life. All guests over the age of 2 years were eligible. Subjects with a wide range of refractive error were included if best corrected vision was at least 20/32 in both eyes.

Overall, the sample size contained 784 subjects. After potential subjects showed interest in participating, a brief discussion informed them of the benefits, purpose, and procedures of the study. Each adult subject then provided written, informed assent. A parent or legal guardian provided written, parental permission for all subjects under the age of 18. Any child old enough to write his or her name provided written assent and all other children provided verbal assent that was witnessed by an additional staff member. The study procedures and design were approved by the Institutional Review Board at The Ohio

State University.

Testing Procedures

The same testing protocol was followed for each subject, as described below, during a single visit. Subjects who wore contact lenses removed both lenses after best corrected visual acuity was tested.

All measurements, with the exception of visual acuity, were made on the right eye only. Best-corrected visual acuity was tested in both eyes. Subjects were excluded from the study if they could not see at least three letters on the 20/32 line with both eyes. LEA symbol charts were used for subjects under the age of 5 years.

Refractive error was the mean spherical value of five non-cycloplegic measurements taken with a binocular autorefractor/keratometer (WR-5100K; Grand

Seiko Co., Ltd., Hiroshima, Japan). The left eye was occluded as the subject viewed a distant fixation target at 16.54 meters. If the spherical equivalent reading mean was more hyperopic than +0.50 diopters, the autorefraction was repeated while the subject wore

+2.00 spectacles in an attempt to relax accommodation for all subjects who might have hyperopia. The final autorefraction value used for these subjects was the reading that was taken while wearing the +2.00 glasses after adding 2.00 D to each sphere value.

CMT Measurements

Ciliary muscle thickness (CMT) measurements were made with an anterior segment OCT instrument (Visante; Carl Zeiss Meditec). All ciliary muscle images were

15 obtained under non-cycloplegic conditions. Two images were taken of the nasal ciliary muscle for each subject’s right eye as a distant fixation target outside the instrument was viewed. The target was placed approximately 11 meters from the anterior segment OCT.

Ciliary muscle images obtained for each subject were exported as binary files and processed with a semi-automatic segmentation algorithm to obtain thickness measurements at several locations along the length of the ciliary muscle relative to the scleral spur, which is manually marked by an investigator (MDB) prior to processing.

The semi-automatic algorithm has been extensively described in a previous publication.24

The algorithm identifies and outlines only the ciliary muscle, while the ciliary processes are not visible. The algorithm segments the ciliary muscle and sclera from the background of the image. The ciliary muscle is then segmented and thickness measurements at 0.25 mm intervals are made along the length of the muscle. This method of segmenting and measuring ciliary muscle images has been previously shown to be both repeatable and valid.24 It has been used in several previous publications.6,9,10,12,14,16,24

Figure 1 is a labeled representation of analyzed ciliary muscle images. The cross- sectional area of the ciliary muscle is outlined in black. CMTMAX is shown as the point of maximum thickness outlined in dark green. The thickness at 1 mm (CMT1), 2 mm

(CMT2), and 3 mm (CMT3) posterior to the scleral spur are shown in red. The red arrow represents the distance between the scleral spur and the point of maximum thickness

(SStoMAX). The area of the first 3 mm posterior to the scleral spur (CMA3) is highlighted in green.

Statistical Analyses

Stepwise, general linear regression models were used to assess the relationship

between cross-sectional age and ciliary muscle dimensional measurements while

controlling for gender when significant. Because previous reports suggested that the

relationship might not be linear across all of the decades of life, quadratic models were

considered and included when significant. Also, because previous reports from our

laboratory have shown that ciliary muscle thickness can be related to refractive error, and

that this relationship is also non-linear/quadratic,9 the models included spherical

equivalent refractive error as a control variable in linear and or quadratic forms when

significant. Models were completed for the entire sample as well as for only subjects over the age of 30 years (N = 298).

Chapter 3: Results

The study sample included 784 subjects aged 3.4 to 85.1 years (mean + SD = 23.4

+ 18.3 years). Of the total subjects initially enrolled, 11 were disqualified for a history of

LASIK, cataract, or other surgery, 15 subjects left early and were unable to complete

testing, and 99 subjects were unable to participate due to visual acuities worse than 20/32.

Non-cycloplegic refractive errors were measured as the average of five autorefraction

measurements on the right eye. Most subjects were Caucasian. Out of 784 subjects, 43%

were male. Table 1 represents a demographic description of the sample including the

mean, standard deviation, and range for age, CMTMAX, CMT1, CMT2, CMT3, and

spherical equivalent refractive error. Mean thickness values taken at 1 mm, 2 mm, and 3 mm posterior to the scleral spur decrease from anterior to posterior measurements. As shown in Table 1, subjects included in this sample have a wide range of refractive errors.

Table 2 describes values from a regression model analyzing the relationship

between age and each of the ciliary muscle dimensions. All subjects were included in this

analysis. Age was significantly related to all ciliary muscle dimensions. Almost all ciliary

muscle dimensions exhibited a quadratic relationship with cross-sectional age, with an

increase in the dimension across the earlier decades of life and a decrease in the latter

18 decades of life. The exception, CMTMAX, was only related to age in a linear fashion, where ciliary muscle thickness increased with age.

We repeated the analyses shown in Table 2 for all subjects ages 30 years and older (Table 3). In general, the trends were similar to what would be expected from the models shown in Table 2 if one only evaluated the older half of the quadratic relationship between the various ciliary muscle dimensions and age.

Chapter 4: Discussion

Our results showed that the relationship between ciliary muscle thickness and

cross-sectional age was quadratic for all regions, i.e., it increased in thickness across the

first three decades of life and then decreased across the latter decades of life, except for

the point of maximum thickness that increased across all decades of life. The position of the apex also had a quadratic relationship with cross-sectional age. In order to depict this

for the reader, we created figures to show what a subject with values of refractive error

and ciliary muscle thickness would look like at each decade of life for the statistical

models. As shown in Figure 3, across the first three decades of life all muscle dimensions

were larger in older subjects, i.e., closer to 30 years, than younger subjects, i.e., children,

and the point of maximum thickness was progressively more posterior in subjects across

these first three decades of life.

This quadratic relationship was shown as a plotted curve for each decade with

values taken from the linear regression model in Table 2:

2 2 SStoMAX(Age) = Intercept + (Age*SStoMAXAge) + (Age *SStoMAXAge ) +

2 2 (M*SStoMAXM) + (M *SStoMAXM )

An example calculation including average values for a ten year old emmetrope is as

follows:

SStoMAX(10) = 666.55 + (10*4.5) + (102*-0.07) + (0*-33.8) + (02*-2.2)

Across the 4th through the 7th decades of life, however, the pattern of increased

thickness did not continue for all regions. Overall muscle area (CMA3) was less in

elderly subjects than middle-aged subjects. Again, to make it easier to visualize and

interpret the quadratic relationship, Figure 4 demonstrates the statistical model for the

relationship between ciliary muscle dimensions and age for the latter decades of life (ages

30 to 70). While overall area decreased across the latter decades, the muscle tissue

distribution appeared to be different in older age, with older subjects showing thinner

posterior thickness (CMT2, and CMT3), but increasingly thicker maximum thickness

(CMTMAX) measurements. The point of maximum thickness was also shifted more

anteriorly in older subjects. These results were similar to previous studies showing a

forward and inward movement of the ciliary muscle apex with aging.20,25 Our analyses of only subjects ages 30 years and older generally confirmed the quadratic relationship that was found with all subjects.

Interestingly, a study from Pucker et. al. (2013) determined that there is a general increase in ciliary muscle thickness with age in children with a large standard deviation in age groups, even though apical fiber thickness decreases with age.9 Our study found that

the age-related increases in thickness over time for CMTMAX, CMT1, CMT2, and

CMT3 were 5.72 µm, 2.3µm, 2.4 µm and 2.8 µm, respectively. In the study from Pucker et. al. CMTMAX and CMT1 increased by 5.87 µm and 7.32 µm, respectively, in relation to age, which was almost identical to our CMTMAX findings but differed from our

CMT1 measurement.9 The same study by Pucker et. al also found opposite trends for

gender in that females tended to have thicker CMTMAX.6 The interaction between

gender, refractive error, and CMT is shown in Figure 10. Females had thinner CMT

across CMTMAX, CMT2, and CMT3 with myopia, emmetropia, and hyperopia in older

adults. A significant relationship with gender, however, was only noted in the modeling

of older age groups as shown in Table 3.

The frequencies of refractive error in each CMT in each CMT location are

demonstrated in Figures 6-9. Each CMT location contained a wide variety of refractive

errors and also demonstrates overall CMT decrease moving posteriorly to the scleral

spur. A negative association between CMT and refractive error was found at all CMT

locations.

Our study is different from previous studies in many ways. To the best of our

knowledge, our study is the first to describe the cross-sectional relationship between age and ciliary muscle dimensions as quadratic. A study from Richdale et. al (2013) in adults concluded that there was not a significant relationship between age and ciliary muscle ring diameter or changes with each diopter of accommodation.12 Most importantly, our

study had a very large sample size (N=784). Although the majority of subjects were

categorized into early age groups, suggesting that estimates made from data from

younger subjects may be more reliable, we had twice as many subjects between the ages

of 60 and 90 years alone as were in the entire histological sample of a previous study.15,25

Figure 1 represents a histogram of mean spherical equivalent frequency in the subject

population and demonstrates the wide variety of refractive error. Although the vast majority of subjects showed clinical emmetropia, the distribution was skewed to the left, implying that higher refractive errors tended to be myopic.

Although the present study had sound methods and sufficient data, there were some limitations. Subjects were not cyclopleged, leaving the possibility that some accommodation may have been present during ciliary muscle image capture or during auto-refraction. Proximal accommodation due to the position of equipment may also have affected accommodative status during testing. We attempted to limit accommodation by having the subjects look at very distant targets close to optical infinity (approximately

11m or 16m away) and by telling subjects to relax their eyes during ciliary muscle image capture and autorefraction. Given that a previous study from our laboratory by Pucker et. al., (2013) did cycloplege subjects and similarly found an increase in ciliary muscle thickness in children, we do not anticipate that the lack of cycloplegia dramatically affected our results, as they were similar to this previous study.9 In addition, cycloplegia

would not have been necessary in older subjects who already lack accommodative ability,

so our estimates in adult subjects should not have been impacted by the lack of

cycloplegia.

Our study focused on the cross-sectional area of the first 3 mm after the scleral spur. This was chosen because our specific measurement method is unable to measure the length of the ciliary muscle. Supporting evidence has shown that the majority of ciliary muscle changes with age occur in the more anterior aspects, which makes an endpoint less important. It is also histologically debatable that one could determine where the ciliary muscle officially ends in an OCT image. Nonetheless, determining the location of the ciliary muscle endpoint may give future direction to how the choroid contributes to eye growth and age-related changes.

The analyses shown included the mean measurements for all subjects in this large sample; however, obvious inter-individual differences exist. Figure 4 demonstrates how ciliary muscle appearance when at rest can vary drastically between any two individuals.

The top image shows that a prominent apex can be evident in an individual’s ciliary muscle whereas the bottom image does not have a well-defined point of maximum thickness. Our study placed emphasis on discrete thickness points and did not consider overall shape. Considering ciliary muscle shape may give more information on accommodative abilities and prognosis of vision therapy. It may also explain how some young children may have less accommodative abilities than expected based on age and can allude to expectations for near work comfort and academic achievement.

Future studies should consider categorizing subjects based on certain muscle

configurations and model the relationship with age for specific subgroups. A study from

Pucker et. al. also found that the posterior regions of the ciliary muscle (CMT2 and

CMT3) are larger in myopes whereas in hyperopes, the apical regions of the ciliary

muscle are thicker while anterior measurements (CMTMAX and CMT1) were thinner.9

Ciliary muscle shape in general can be drastically different between two individuals of

the same age and more emphasis should be placed on how the shape changes with age

and its consequences for accommodation.

Direction on new presbyopic or glaucoma treatments can also be suggested from

this study. Studies have shown that pharmacologically inducing accommodation on a

post-cataract surgery ciliary muscle does show some functional ability remains even with

redistribution of muscle fibers and changes in shape. A study from Richdale et. al. (2013)

thoroughly studied how accommodative changes occur with age and concluded that there

was not a significant relationship between age and ciliary muscle ring diameter or

changes with each diopter of accommodation.12 They also found that the ciliary muscle

thickens anteriorly and thins posteriorly with accommodation, but did not lose cross-

sectional thickness over time.12 Although Sheppard and Davies found a minimal decrease

in cross-sectional thickness with age, it is questionable whether the present study can be

directly compared to the other studies due to their analyses of muscle length.12,17 Studies have also shown support for a redistribution of muscle fiber arrangements, connective tissue abundance tends to increase in the middle-aged ciliary muscle, and the presence of muscle atrophy in later decades.12

Regardless of thickness changes, it seems that the ciliary muscle is unaffected in its innate or functional thickness.12 These data suggest that the ciliary muscle could

regain the function of its youth with the proper technology implanted and accommodative

therapy. With the anterior and inward movement of the ciliary muscle, the anterior

chamber depth may decrease, increasing the risk of angle closure or glaucoma. This risk

is also compounded with the simultaneous hardening of the crystalline lens in older age

groups. If some muscles are more likely to move anteriorly than others or have an

abundance of muscle fibers due to individual variability, earlier intervention can be

beneficial and may suggest earlier cataract surgery.

Although it is difficult to follow subjects over a human being’s lifetime, a

longitudinal study to follow an individual throughout life would give a perfect picture of

how shape and thickness changes throughout life and could retrospectively look at factors

such as accommodative demands or refractive error that could alter a ciliary muscle’s

physiology. It should be noted that we were only making our measurements in a cross-

sectional design and did not know how an older subject’s ciliary muscle appeared in

childhood nor are we certain how a child’s ciliary muscle can alter over time or with age.

In conclusion, the ciliary muscle does exhibit a meaningful change in thickness with age. Coincidentally, the shapes of the curves describing how the ciliary muscle thickness changes with age look very similar to the changes seen between a young accommodating eye and a non-accommodating eye. The data also demonstrate that it is possible to use our previously described methods for imaging and measuring ciliary muscle dimensions to document the ciliary muscle’s development throughout all the various decades of life.24 There is, however, still much unknown about the ciliary muscle

26 and how it affects and is affected by changes with eye growth both functionally and physically.

Variable Mean SD Range Age (years) 23.4 18.3 3.4 to 91.6 CMTMAX (µm) 829.8 80.6 569.9 to 1079.6 CMT1 (µm) 777.9 75.6 493.4 to 1040.1 CMT2 (µm) 512.6 90.6 235.1 to 821.9 CMT3 (µm) 292.5 73.5 7.9 to 572.7 Spherical Equivalent (D) −0.56 1.88 −10.87 to +6.00

Table 1. Demographic information on study subjects including age and spherical equivalent. Thickness measurements represent the point of maximum thickness (CMTMAX), 1 mm posterior to the scleral spur (CMT1), 2 mm posterior to the scleral spur (CMT2), and 3 mm posterior to the scleral spur (CMT3).

Predictor SStoMAX CMTMAX CMT1 CMT2 CMT3 CMA3 Intercept 666.55 805.75 746.60 483.50 254.60 1.51 4.5(β) 1.0 2.3 2.4 2.8 0.004 Age, years p = 0.08 p < 0.000001 p = 0.0003 p = 0.0007 p < 0.000001 p = 0.002 −0.07 −0.02 −0.03 −0.04 −0.03 Age2 NS p = 0.007 p = 0.01 p = 0.001 p = 0.00002 p = 0.04 −33.8 −5.8 −17.7 −13.4 −0.03 Spherical equivalent (M), D NS p < 0.000001 p = 0.005 p < 0.000001 p < 0.000001 p = 0.04 −2.2 −0.9 −1.9 −1.2 −0.003 Spherical Equivalent2 (M2), D2 NS p = 0.06 p = 0.02 p = 0.00005 p = 0.001 p = 0.003

Table 2. Regression models of the relationship between age and ciliary muscle dimension for all subjects. CMTMAX = maximum ciliary muscle thickness, CMT1, CMT2, CMT3 = ciliary muscle thickness 1, 2, 3 mm posterior to the scleral 29 spur. CMA3 = cross-sectional area of the first 3 mm of the ciliary muscle. SStoMAX = distance between the scleral spur

and the maximum thickness. NS = Not Significant.

Predictor SStoMAX CMTMAX CMT1 CMT2 CMT3 CMA3 Intercept 818.7 821.2 788.5 661.3 432.7 1.8 −2.4(β) 1.3 −3.0 −2.6 −0.002 Age, years NS p = 0.03 p = 0.03 p = 0.003 p = 0.001 p = 0.2 −38.1 −211.5 −167.6 −0.3 Female NS NS p = 0.02 p = 0.0006 p = 0.0006 p = 0.03 −27.6 9.4 −5.4 −12.7 −11.5 −0.02 Spherical equivalent (M), D p = 0.000001 p = 0.1 p = 0.02 p = 0.0002 p = 0.00002 p = 0.02 Female X Spherical Equivalent −12.4 4.0 3.1 0.005 NS NS Interaction p = 0.09 p = 0.001 p = 0.002 p = 0.07

Table 3. Regression models of the relationship between age and ciliary muscle dimension for all subjects age 30 years and 30 older. CMTMAX = maximum ciliary muscle thickness, CMT1, CMT2, CMT3 = ciliary muscle thickness 1, 2, 3 mm posterior to the scleral spur. CMA3 = cross-sectional area of the first 3 mm of the ciliary muscle. SStoMAX = distance between the scleral spur and the maximum thickness. NS = Not Significant.

- 31

Figure 1. Mean spherical equivalent frequency histogram in the total subject population.

Figure 2. Example ciliary muscle (black outline) image. The point of maximum thickness (CMTMAX, dark green), and the thickness at 1 mm (CMT1), 2 mm (CMT2), and 3 mm (CMT3) posterior to the scleral spur (SS) are shown (red). The arrow (red) indicates the distance between the scleral spur and point of maximum thickness (SStoMAX). The area of the first 3 mm (CMA3) posterior to the scleral spur is shaded in light green.

Figure 3. Ciliary muscle thickness of a 10, 20, and 30 year old example patient based on model estimates (Table 2). The dashed lines represent the position of the point of maximum thickness relative to the scleral spur position (x=0, y=0).

Figure 4. Ciliary muscle thickness of a 30, 40, 50, 60, and 70 year old example patient based on model estimates (Table 2). The dashed lines represent the position of the point of maximum thickness relative to the scleral spur position (x=0, y=0).

Figure 5. Example ciliary muscle images showing the variety of configurations of the ciliary muscle across the sample. Note that for some subjects the apex is very prominent (top), and for other subjects it is not prominent (bottom) while the muscle is at rest.

0 0 500 1000 1500 2000 M_MEAN -2 Linear (M_MEAN)

-4

Mean Spherical Equivalent (D) Equivalent Spherical Mean -6

-8

-10 CMT (um)

Figure 6. CMTMAX (um) as a function of mean spherical equivalent refractive error (D) for the entire sample.

0 0 200 400 600 800 1000 1200 M_MEAN -2 Linear (M_MEAN)

-4

Mean Spherical Equivalent (D) Equivalent Spherical Mean -6

-8

-10 CMT (um)

Figure 7. CMT1 (um) as a function of mean spherical equivalent refractive error (D) for the entire sample.

0 0 200 400 600 800 1000 M_MEAN -2 Linear (M_MEAN)

-4 Mean Spherical Equivalent (D) Equivalent Spherical Mean -6

-8

-10 CMT (um)

Figure 8. CMT2 (um) as a function of mean spherical equivalent refractive error (D) for the entire sample.

0 0 100 200 300 400 500 600 700 M_MEAN -2 Linear (M_MEAN)

-4 Mean Spherical Equivalent (D) Equivalent Spherical Mean -6

-8

-10 CMT (um)

Figure 9. CMT3 (um) as a function of mean spherical equivalent refractive error (D) for the entire sample.

1000 900 800 700 Female CMTMAX 600 Male CMTMAX 500 Female CMT2

CMT (um) CMT 400 Male CMT2 300 Female CMT3 200 Male CMT3 100 0 -3 0 3 Mean Spherical Equivalent (D)

Figure 10. Relationship between gender, refractive error, and ciliary muscle dimensions for all subjects age 30 years and older. Mean age chosen was 50 years. CMTMAX is the point of maximum thickness. CMT2, CMT3 = ciliary muscle thickness 2 and 3 mm posterior to the scleral spur. Myopic refractive error is estimated with -3 D and hyperopic refractive error is estimated with +3 D.

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