Isabelle Dortonne
Harvard College 2012

In ophthalmology, accommodation is the act of focusing from distant to near objects. According to the Helmholtz theory of primate accommodation, the ciliary muscle contracts and moves anteriorly inward as the zonules relax. Zonules are fibrous strands that connect the ciliary body with the crystalline lens. Presbyopia, or near vision, is the age-related loss of accommodative ability and is the most common refractive disorder of the elderly. Lens and capsule-based theories of presbyopia assert that the decrease in accommodative amplitude can be attributed to increased hardening of the lens substance and decreased elasticity of the lens capsule with age. One novel technique in the restoration of accommodation is Phaco-Ersatz, or lens refilling. The promise of Phaco-Ersatz as a treatment for near vision can be assessed by characterizing the biomechanical properties of the lens in its natural versus its empty state. Postmortem cynomolgus monkey, rhesus monkey, and human eyes of varying ages were stretched in their natural and empty states in an ex vivo accommodation simulator in eight, 0.25mm steps, which mimics the changes in zonular tension that occur in vivo. Diameter-force relationships were graphed. There was no relationship between the empty-bag diameter slope and age, indicating that the lens capsule’s mechanical properties do not change the setting of accommodation. Moreover, the ratio of the load-diameter slope for empty capsule to natural lens decreased significantly with age, showing that it is the lens material and not the capsule that contributes to presbyopia. The results confirm the postulation that accommodation can be efficiently conducted as long as the lens contents have proper viscoelastic properties. Thus, Phaco-Ersatz is a viable future treatment for presbyopia.

Introduction

Accommodation is the act of focusing from distant to near objects (Bron et al., 1997). According to the Helmholtz theory of primate accommodation, the ciliary muscle contracts and moves anteriorly inward as the zonules relax. This action results in an increase in the anterior and posterior curvatures of the lens. These lens shape changes during accommodation increase lens refractive power, the ability of the lens to focus incoming light rays (Helmholtz, 1855). Among the many contributors to accommodation in the primate lens are the lens material, lens capsule, ciliary muscles, ciliary body, and vitreous (Parel et al., 2006). Particularly, the lens capsule and zonular fibers play a significant role in transmitting the force of ciliary muscle contraction to the lens fibers during accommodation (Krag & Andreassen, 2003).

Presbyopia refers to the age-related loss of accommodative ability and is the most common refractive disorder of the elderly. It usually manifests itself when the patient is about 40 years old. Symptoms become progressively worse, and soon magnifying glasses or bifocals are required to see small objects (Werner et al., 2002). Most likely, presbyopia is initiated by a medley of physical and anatomical changes in the lens and other structures involved in accommodation (Parel et al., 2006). There are three major theories of presbyopia: lens and capsule based theories; extralenticular theories, which focus on the ciliary muscle and choroid; and geometric theories, which examine the changes in the geometry of zonular attachments (Werner et al., 2002). One of the most accepted theories of presbyopia is based on changes that occur in the lens and lens capsule. The lens substance also becomes harder with age. In addition, the lens capsule loses elasticity, which compounds its inability to mold the more rigid lens substance (Fincham, 1937).

The lens capsule is the membrane that surrounds the crystalline lens and serves as an anchorage point for the zonular fibers. The zonules, in conjunction with the lens capsule, mold the crystalline lens contents during the process of accommodation (Courtois, 1987). Fincham stressed the significance of the elastic lens capsule in shaping the lens into its accommodated form. In fact, lesser mammals with a uniform lens capsule thickness have limited accommodative ability (Fincham, 1937).

As humans age, the lens capsule continues to grow anteriorly in thickness and increases in surface area as the lens contents increase in volume. The anterior lens capsule is three to five times thicker than the posterior lens capsule. Although the anterior lens capsule is produced by the lens epithelium and continues to grow throughout life, the posterior lens capsule loses its epithelial cells during the fetal stage. Therefore, the thickness of the posterior lens capsule remains constant throughout life (Krag & Andreassen, 2003).

Over time, the structure of the lens capsule becomes more homogenous (Courtois, 1987). The density of the lens capsule also increases with age, partially due to the increase of non-collagenous amino acids and the decrease of collagenous amino acids (Krag & Andreassen, 2003). For example, in the adult capsule, collagen IV and laminine are abundant, but the glycoprotein fibronectin is scarce (Courtois, 1987). Collagen was recently found to be responsible for the mechanical strength of various soft connective tissues (Krag & Andreassen, 2003). More specifically, collagen IV is a chief structural component of the lens capsule (Krag & Andreassen, 2003).

The lens capsule’s elastic properties have been measured by several researchers. Krag and Andreassen used a uniaxial procedure to produce load-deformation and stress-strain curves for the human lens capsule in relation to age. They found that the lens capsule becomes less elastic and more rigid with age (Krag & Andreassen, 2003). Bowman, a pioneer in the study of lens capsule elastic properties, demonstrated the ability of the lens capsule to return to its original shape after being swollen and punctured (Bowman, 1849). Various studies have reported the elastic modulus under diverse strain levels. It has been found that the lens capsule modulus in the young primate is more than one hundred times that of the lens substance, which contributes to its role as a force transmitter. Although the reported exact values of lens elasticity in the literature vary, it is known that lens capsule extensibility decreases with age. This may be caused by the increasing volume of lens, which stretches the collagen network structure and limits further deformation. Cross-linking of the molecular network structure may also limit further deformation (Krag & Andreassen, 2003).

Currently, there are over 2 billion presbyopes worldwide suffering from the age-related loss of accommodative ability. One novel technique in the restoration of accommodation is Phaco-Ersatz, or lens refilling. The nucleus and cortex are removed from the crystalline lens through a small opening in the capsule known as a mini-capsulorhexis; the capsule, zonules, and ciliary body remain intact. A polymerized gel is then injected into the lens capsule through the mini-capsulorhexis (Parel et al., 2006).

The success of lens refilling relies on the hypothesis that the elasticity of the lens capsule has a negligible effect on its ability to mold the lens material. The postulation is that accommodation can be efficiently conducted as long as the lens contents have proper viscoelastic properties. This is because it is the lens contents and not the lens capsular bag that more greatly maintain accommodation. This hypothesis can be tested by characterizing the biomechanical properties of the lens in its natural state versus its empty state, with the simulation of accommodation.

An ex vivo accommodation simulator can mimic on postmortem lenses the changes in zonular tension that occur during natural (in vivo) accommodation. Thus, power and diameter-force characteristics can be analyzed. If it is found valid that the biomechanical properties of the lens capsular bag do not drastically change with age and have a negligible effect on accommodation, then Phaco-Ersatz, the lens refilling technique, may be an effective treatment of presbyopia (Manns et al., 2007).

Figure 1
Figure 1. Accommodation in the normal eye. Parel, et al.
Figure 2
Figure 2. In a non-presbyopic eye, the image falls directly on the retina (back of the eye). In presbyopia, the image projects behind the retina.
Figure 3
Figure 3. Ex vivo Accommodation Simulator (Manns et al., 2007).

Methods

Preparation of Eyes

Eyes of the cynomolgus monkey (n = 26, 4–10 years, <4 days postmortem), rhesus monkey (n = 10, 1–11 years, <5 days postmortem) and human (n = 29, 8–76 years, <5 days postmortem) were obtained from the University of Miami Division of Veterinary Resources and Florida Lions Eye Bank. All animal experiments abided by the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research; all human eye experiments abided by the Declaration of Helsinki for research involving the use of human tissue. Immediately following euthanasia, the eyes were enucleated, wrapped in gauze, placed in a closed jar, and stored at 4 °C until experimentation.

Dissection

The clean scleral surface of the eye was exposed after removal of the conjuctiva, episcleral tissue, and extraocular muscles. Fluid on the scleral surface was blotted with a surgical sponge and the scleral tissue was bonded, using cyanoacrylate adhesive, to eight custom-made scleral shoes made of plastic (polymethylmetacrylate, PMMA). The resulting arrangement was a ring which covered the circumference of the (eye) globe along the equator. During bonding, the shoes were held in place with fixation pins and a PMMA alignment ring. After the adhesive dried, the posterior portion of the eye was circumferentially dissected and excess vitreous was removed. Extreme caution was taken to ensure that the anterior vitreous, hyaloid membrane, and ciliary body remained intact. Surgical sponges were used to carefully remove vitreous residue on the lens (Figure 4).

The eye was removed from the alignment ring and fixation pins, and transferred to the tissue chamber of the EVAS system, anterior face up. The EVAS contains eight hooks that fit through a hole in each shoe (Figure 4). During transfer, the eye was immersed in DMEM (Dulbeco’s Modified Eagle Medium), a preservation medium, in order to prevent dehydration. Next, the cornea was removed at the limbus with scissors and the iris gently pulled off by its root. A diamond knife was used to make incisions in the sclera between adjacent shoes, producing eight independent segments.

Figure 4a
Figure 4. Dissection and Preparation. a. Eye mounted on alignment ring and fixating pins for dissection.
Figure 4b
Figure 4b. Removal of posterior portion of the eye.
Figure 4c
Figure 4c. Bonding to scleral PMMA shoes.
Figure 4d
Figure 4d. Removal of cornea, iris, and vitreous; incisions made between shoes.
Figure 4e
Figure 4e. Mounting of tissue onto translation stage and shoe attachment to EVAS.

Removal of Lens Contents

By means of hydrodissection, and phacoemulsification in cases of stiff lens contents, the surgeon removed the lens contents through a minicapsulorhexis, a small circular opening in the periphery of the lens capsule (Tahi et al, 1999). Hydrodissection involves the injection of saline (Balanced Salt Solution, BSS), via a blunt cannula, through the minicapsulorhexis between the lens capsule and the cortex. The liquid separates the lens cortex from the surface of the capsular bag so that the lens contents can be removed while leaving the capsule intact. Phacoemulsification uses a piezoelectric motor connected to a titanium tip to ultrasonically break the hard lens nucleus into small fragments, allowing for easier aspiration (Buratto et al., 2003).

EVAS Preparation and Stretching

Each shoe was connected by 6-0 nylon monofilament sutures to a T-shaped bar, which was mounted on a translation stage powered by a stepper motor. Each string also ran through the two sets of pulleys to produce the radial stretching forces, equally distributed among the eight shoes. EVAS was programmed to stretch the lens in eight, 0.25 millimeter steps at a speed of 0.1 mm/s, by the joystick controlled movement of the translation stage. The maximum diameter increase of the outer sclera was 4 millimeters.

A preconditioning stretch cycle was performed on each lens to ensure that there were no problems with the tissues, the suture alignment, or shoe attachment. At the end of each step, the translation stage was stopped for 10 seconds to allow a digital picture recording of the load (force) and lens diameter changes. Each stretching experiment was conducted and recorded a minimum of three times. A trigger pulse sent to the system signaled the start of a new cycle. Stretching was performed on the natural lens and on the empty lens capsule after phacoemulsification or hydrodissection.

Diameter Measurements

Sharp, high-contrast images of the lens were provided by diffuse retroillumination between steps. A digital picture of the top view of the lens was taken at each step during the stretching cycles. The lens and ciliary body diameters were measured along two perpendicular axes, one horizontal and one vertical, in units of pixels, using Canvas 9 imaging software. A calibration factor was determined by measuring a picture of a ruler with 1mm divisions, taken during the stretching experiment, in Canvas 9 software. After recording the length of the lines in Canvas 9, the measurements were divided by the calibration factor and inserted into a Microsoft Excel chart. The average of the horizontal and vertical diameters was used as the measure of the diameter.

The continuous load values were recorded in an Excel chart during stretching and later graphed. The corrected (calibrated) load measures were obtained from the plateaus of the graph, each signifying another EVAS step. This same analysis was used for natural and empty lens capsule experiments.

Lens capsule stretch was defined by the equation:

Various load-diameter relationships were graphed using Origin software. The slopes of the linear regressions of the resulting scatter plots and corresponding ratios were calculated and then analyzed as a function of age.

Results and Discussion

In all species studied, the unstretched empty bag diameter remained constant as a function of age (d = 8.1 ± 0.3 mm cyno, 8.8 ± 0.5 mm rhesus, and 9.8 ± 0.3 mm human). Although the anterior lens capsule thickness increases with age, the surface area of the empty lens capsule of all three primate groups remained constant throughout life (SA = 103.5 ± 6.9 mm2 for cynomolgus, 122.3 ± 13.7 mm2 for rhesus, and 149.6 ± 9.7 mm2 for human). The ratio of empty bag surface area to natural lens surface area decreased with age, although less significantly in the cynomolgus group, which had a smaller age range. These results are plausible because the empty bag surface area stays constant, whereas the lens surface area increases with age. The ratio is constantly less than one, indicating in situ tension even in young animals.

There was no relationship between empty bag load-diameter response and age. Load values ranged from about 5-35 g/mm in all eyes. The absence of an observable relationship indicates that the amount of force required to elicit a diameter change in the empty lens capsule is not dependent on age. Although previous research has demonstrated a decrease in lens capsule elasticity in age, such changes have shown to be insignificant in the setting of accommodation. Thus, loss of elasticity of the lens capsule is not a significant contributing factor to a decrease in accommodative ability. Moreover, it has been shown that the lens capsule of presbyopic humans (>45) is sufficiently elastic to easily deform the novel polymers designed for lens capsule refilling, allowing for restoration of a minimum of 5 diopters of accommodation in patients undergoing the Phaco-Ersatz procedure.

The ratio of the empty capsule load-diameter slope to the natural lens load-diameter slope decreased significantly with age (p< 0.0001). It is more difficult to change the shape of the older natural lens than the younger natural lens, whereas this is not the case with the empty lens capsule. More force is required to get the same results in older primates as when less force is applied to younger primates.

The results support the hypothesis that accommodation can be efficiently conducted as long as the lens contents have proper viscoelastic properties because it is the contents, and not the lens capsular bag, that more greatly maintain accommodation. The findings are contrary to the capsule based theories of presbyopia, which state that changes in the lens capsule’s mechanical properties significantly decrease accommodative amplitude. The findings promote the feasibility of regularly using Phaco-Ersatz, a lens refilling technique, as a treatment of the millions of presbyopes worldwide.

Figure 5
Figure 5. Typical Load-Diameter Response. CY 124-1267. OD Empty Bag. Age: 64 months. PMT: 3hrs.
Figure 6
Figure 6. Mechanical properties of all specimens as functions of age.

Acknowledgements

The full version of this work appears in Investigative Ophthalmology & Visual Science 49:4490-6 (2008). It is authored by Noël M. Ziebarth, David Borja, Esdras Arrieta, Mohamed Aly, Fabrice Manns, Isabelle Dortonne, Derek Nankivil, Rakhi Jain, and Jean-Marie Parel. The work was conducted at the Ophthalmic Biophysics Center, Bascom Palmer Eye Institute of the University of Miami Miller School of Medicine in Miami, Florida.

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