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Prevention of capsule opacification after accommodating lens refilling: Pilot study of strategies evaluated in a monkey model

Koopmans, Steven A. MD, PhD*; Terwee, Thom MSc; Hanssen, Alex MSc; Martin, Heiner Dr-Ing; Langner, Soenke MD; Stachs, Oliver PhD; van Kooten, Theo G. PhD

Author Information
Journal of Cataract & Refractive Surgery: September 2014 - Volume 40 - Issue 9 - p 1521-1535
doi: 10.1016/j.jcrs.2014.02.034
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Abstract

An ultimate goal of modern cataract surgery, beside the restoration of good distance visual acuity, is to restore accommodation1 to enable good intermediate and near visual acuity. Age-related changes that result in hardening of the lens nucleus and cortex are considered by many to be important factors in the development of presbyopia.2–4

Among various surgical procedures to restore accommodation after the onset of presbyopia, replacement of the contents of the capsular bag of the crystalline lens by a flexible polymer was the earliest method proposed. This technique has been given the name lens refilling or phaco-ersatz.5,6 Surgery involves removal of the lens nucleus and cortex through a small capsule opening followed by injection of polymer to refill the capsular bag. The technique has been studied in pig eyes, rabbit eyes, and rhesus monkey eyes.7–9 For in vivo experiments, before human lens-refilling studies, rhesus monkeys were the most suitable animal model because their accommodative mechanism is similar to that of humans10–12 and they develop presbyopia over an age course similar to that of humans.12,13

After the capsular bag is refilled with a synthetic polymer, the development of capsule opacification may cause a decrease in vision. Capsule opacification is caused by a migration, proliferation, and transformation of remaining lens epithelial cells (LECs) that still reside inside the capsular bag after lens surgery.14 Several methods to prevent capsule opacification have been presented in the literature. These include optimizing design parameters of the implanted intraocular lens (IOL),15,16 methods of surgery,17 and the application of chemical agents inhibiting cell adhesion,18 migration, and proliferation, including antiinflammatory and cell death–inducing agents.19–23

For lens refilling, the application of chemical agents could be promising because other methods do not eradicate all LECs. In 2006, Koopmans et al.24 described lens-refilling experiments in young adolescent rhesus monkeys. To prevent the development of capsule opacification, a solution of actinomycin-D and cycloheximide was mixed with sodium hyaluronate to create a chemically active ophthalmic viscosurgical device (OVD). This substance was left in the capsular bag for 5 minutes. After the chemically active sodium hyaluronate was removed, the lens capsule was refilled with a silicone polymer. The choice for a chemically active sodium hyaluronate containing actinomycin-D and cycloheximide was based on cell culture experiments and rabbit implantations.25,26 Despite this treatment of the capsular bag, the initial accommodative amplitude of the monkey eyes decreased during the follow-up, and it was coincident with an increase in capsule opacification. Opacification of the lens capsule is thought to coincide with stiffening of the lens capsule.14 Because lens-refilling surgery is intended to restore long-lasting accommodation, the occurrence of capsule opacification and stiffening should be prevented effectively and permanently.

To improve the efficacy of the prevention of capsule opacification, several strategies are possible. One of them is the use of chemical substances other than actinomycin-D or cycloheximide to induce death of LECs. To predict which substance is most promising for the prevention of capsule opacification, the results of cell-culture experiments may be helpful.26 Another possible strategy is the use of a material other than a silicone polymer for refilling the lens. Physicochemical properties of a material implanted in the capsular bag may directly influence the behavior of the surrounding LECs, and the choice of a more suitable material might reduce the incidence of capsule opacification.27

Earlier experiments with lens refilling using actinomycin-D and cycloheximide showed differences in capsule opacification between rabbits and rhesus monkeys. In a study of rabbit eyes,25 completely clear lens capsules were observed in some animals whereas capsule opacification developed in all rhesus monkeys.24 Given this different outcome in rabbits and rhesus monkeys, we decided that for testing new ways to prevent capsule opacification, a new strategy could best be tested in a pilot study using the rhesus monkey model because this model mostly resembles the human situation.

The present study tested 2 strategies to improve the prevention of capsule opacification after lens-refilling surgery in rhesus monkeys. Due to ethical considerations, treatment variables were studied in individual animals only. Results in our previous study of rhesus monkeys using similar methods24 were included to enable a statistical analysis.

As a first strategy, chemical substances other than an actinomycin-D and cycloheximide combination were used. The substances were selected based on the outcomes of cell-culture experiments. The substances were dissolved in a noncommercial sodium hyaluronate 1.0% solution before gelation and injected in the lens capsular bag; this was followed by refilling the lens capsule with a silicone polymer.

As a second strategy to study the effect of the lens-refilling material on the development of capsule opacification, clinically used sodium hyaluronate (Healon [sodium hyaluronate 1.0%]) was used to fill the lens capsule. Although Healon does not have suitable refractive properties to be used as a lens-replacement material, a study by Gwon and Gruber28 showed that sodium hyaluronate influences the behavior of proliferating LECs to grow in a more regular natural pattern.

After enucleation and before fixation for confocal microscopy, high-resolution magnetic resonance imaging (MRI) images (micro MRI) of the anterior segment were obtained to study the shape and amount of refilling of the lenses.29

In a lens-refilling study in 2006, Koopmans et al.24 did not observe clinically visible damage to structures in the rhesus monkey eye due to treatment of the capsular bag with chemically active sodium hyaluronate; however, detailed microscopic analysis was not performed. Therefore in this study, the corneas of the eyes in which the lens capsular bag had been treated with chemically active substances were studied with confocal microscopy to determine potential corneal endothelial cell damage.

An important variable in conjunction with refill polymer injection is the amount of material injected by the surgeon. Adding material to the capsular bag changes the shape of the lens, increasing the lens power and lens thickness. In a previous study,24 lenses were refilled until the surgeon judged the capsular bag to be filled completely. A-scan ultrasound (US) was used to determine the lens thickness, and a refraction measurement was taken; these values were compared with the preoperative lens thickness and refraction. To obtain more quantitative information about the size and shape of the refilled lenses, high-resolution MRI can be used. In this study, we performed high-resolution MRI of the eyes after they had been enucleated to study the dimensions of the refilled lenses.

Finally, confocal microscopy of the lens capsular bag was performed to study the amount and nature of growth of LECs on the lens capsule.

Materials and methods

Monkeys

One eye of 6 rhesus monkeys (Macaca mulatta) was used for the experiments. Figure 1 shows the data of the monkeys. The animals were approximately 7 years old and weighed between 4.5 kg and 5.5 kg. Four monkeys were females, and 2 were males. The monkeys were treated in accordance with the Association for Vision and Research in Ophthalmology statement for the Use of Animals in Ophthalmic and Vision Research and institutionally approved animal protocols. Four to 15 months before the lens-refilling surgeries, the monkeys had a complete iridectomy in both eyes to prevent miosis and allow unobstructed refraction measurements during pharmacologically induced accommodation. It has been shown that a total iridectomy does not affect the accommodative response in rhesus monkeys.30 Assessment of pharmacologically stimulated accommodation using pilocarpine 4.0% drops was performed once or twice after the iridectomy 2 to 5 months before lens refilling.

Figure 1
Figure 1:
Data listing monkey number and treatments. All animals had a total surgical iridectomy before lens refilling surgeries. The interval between iridectomy and lens refilling is indicated in a time line. In monkeys 2 to 6, after lens removal and before lens refilling, the capsular bag was treated for 5 minutes with chemically active sodium hyaluronate containing the chemicals indicated below the timeline The lens-refilling substance and the duration of its presence are listed above the timeline. Silicone is the silicone polymer that was used before.31 Lens surgeries are indicated by a vertical step in the timeline (Act D = actinomycin-D 10 μg/mL; CAPE = caffeic acid phenethyl ester 3.5 μM; IOL = intraocular lens; MTX = methotrexate 10 μM; PTX = paclitaxel 4 μM; Tween-20 = polysorbate 0.025% [v/v]).

Lens-Refilling Substance

Two materials were used for refilling the lens capsular bag in this study. Figure 1 shows the material used in each monkey. One was a silicone polymer that consists of 2 components that were mixed together and prepared for injection.31 The refractive index of the refilling silicone polymer was 1.42, comparable to the equivalent refractive index of the natural human crystalline lens.

Alternatively, clinically used sodium hyaluronate 1.0% (Healon) was used to fill the capsular bag. Healon has a refractive index of 1.33. In monkey 6, the sodium hyaluronate was left in place during the entire follow-up. In monkey 2, the sodium hyaluronate was surgically replaced by the silicone polymer after 3 months. In monkey 3, the sodium hyaluronate leaked from the capsular bag and the capsular bag was filled with the silicone polymer after 1 month.

Chemically Active Sodium Hyaluronate

Five monkeys received a treatment of the capsular bag with chemically active sodium hyaluronate to prevent capsule opacification. The active chemicals were chosen based on their potential to kill LECs in cell-culture experiments26 and on their potential to prevent capsule opacification in rabbits after lens implantation.25Figure 1 shows the substances used. Actinomycin-D intercalates with DNA resulting in a decreased RNA polymerase (DNA reading) activity, inhibiting RNA synthesis. Methotrexate is a folic acid antagonist and interferes with DNA synthesis. Caffeic acid phenethyl ester (CAPE) interferes with the nuclear transcription factor NF-κB and inhibits proliferation of transformed cells. Paclitaxel interferes with cellular microtubuli and cell division. Polysorbate (Tween-20) lyses biological membranes.

The synthesis of the chemically active sodium hyaluronate was as follows: hyaluronic acid was weighed as a dry fiber-like material. The fibers were then submerged in 70% ethanol in ultrapure water for 5 seconds and then transferred to a sterile petri dish. The now wet fibers were allowed to dry for 2 hours while exposed to the laminar flow of the sterile flow cabinet. The fiber mass was then transferred to a sterile tube. Ultrapure water was added to obtain a final concentration of hyaluronic acid of 1.0%. Directly after the addition of water, a stock solution of the relevant chemical agents was added to reach the final concentrations set for those agents. Stock solutions that were available in dimethylsulfoxide or other organic solvents were diluted at least 1:1000, therewith reaching concentrations that should not affect cells. After the addition of the chemical agents, the solution was allowed to gelate at 4°C overnight to obtain a homogeneous, sterile gel. Figure 1 shows an overview of treatments.

Anesthesia

For anesthesia during surgical procedures and follow-up examinations, the monkeys were given intramuscular ketamine (10 mg/kg) and intubated. Deep anesthesia was achieved by inhalation of isoflurane (Isoflo). After the onset of anesthesia, the monkeys were placed on a vacuum pillow that allowed positioning and fixation of the face upward under an operating microscope (Opmi, Carl Zeiss Meditec GmbH). Additional corneal anesthesia was provided with oxybuprocaine 0.4% eyedrops.

Surgical Technique in General

The periocular region was swabbed with povidone–iodine (10 mg/mL). The head and body of the monkeys were covered with sterile drapes leaving the ocular region exposed. The eyelids were held open with a wire eyelid speculum. A clear corneal tunnel incision was made using a 3.0 mm disposable keratome. The anterior chamber was filled with sodium hyaluronate 1.4% (Healon GV). With a 27-gauge needle, a small peripheral puncture of the anterior lens capsule was made approximately 2.0 to 3.0 mm from the lens equator. With a Utrata forceps, an approximate 1.5 to 2.0 mm diameter curvilinear capsulorhexis was completed in the lens periphery. A clear corneal paracentesis was created using a 15-degree knife, and an anterior chamber maintainer (ACM) connected to an infusion bottle was inserted in the incision. Physiologic saline solution (Endosol) was used in all surgeries.

The lens substance was removed by aspiration. This is possible because the lens substance of a 7-year-old rhesus monkey is softer than that of a cataractous human lens. Aspiration was performed manually using a 20-gauge blunt cannula connected to a 10 mL syringe with a piece of polyethylene tubing. After removal of the lens substance, the anterior chamber was filled with sodium hyaluronate. A purpose-designed silicone membrane32 with a diameter of 2.7 mm and a thickness of 0.2 mm (a capsular plug) was inserted in the capsular bag through the capsulorhexis.

The empty capsular bag was filled with the lens-filling substance through the capsulorhexis beneath the plug by inserting a 25-gauge cannula into the bag and injecting the refill material until the capsular bag was judged by the surgeon (S.A.K. in all cases) to be completely filled. The cannula was retracted and the plug positioned to close the capsulorhexis. The sodium hyaluronate was flushed out of the anterior chamber with the saline solution via the ACM through the corneal incision. Finally, both incisions were sutured with a single 10-0 nylon and the anterior chamber was reinflated with injected saline solution.

At the conclusion of surgery, all monkeys received an approximately 0.2 mL subconjunctival injection of triamcinolone 40 mg/mL and dexamethasone/gentamicin ointment.

Chemical Treatment of Capsular Bag

After removal of the natural lens, the ACM was removed and the anterior chamber was filled with sodium hyaluronate 2.3% (Healon5). The chemically active sodium hyaluronate (Figure 1) was injected in the monkey capsular bag and left in place for 5 minutes in an effort to lyse or kill the remaining LECs and to prevent the early development of postoperative capsule opacification. After this, the ACM was inserted in the corneal incision again and the OVD material was aspirated from the eye, starting with the chemically active sodium hyaluronate solution in the capsular bag. The inner anterior and posterior central capsular bag surfaces were polished with a Kratz capsule polisher (Beaver-Visitec International) as well as possible without risking capsule rupture.

Removal of Lens-Refilling Material

During follow-up, it was decided to remove the silicone refill material in 1 monkey (number 1) and replace it with a regular foldable IOL. To do this, a 3.0 mm clear corneal incision and a paracentesis were made. The anterior chamber was filled with the clinically used sodium hyaluronate, and the mini capsulorhexis was cut with a Vannas scissors to initiate a capsule flap. With a Utrata forceps, the flap was enlarged to a regularly sized capsulorhexis of 5.0 mm. Injecting the sodium hyaluronate between the lens capsule and the refilling material luxated the refilling material in the anterior chamber. With a Utrata forceps, the silicone polymer was pulled from the anterior chamber through the incision where it was grasped with a toothed forceps. As the silicone polymer was continuously pulled on and grasped with a forceps, the material could be removed from the capsular bag in several attempts. After this, the capsular bag was again filled with the clinically used sodium hyaluronate and a regular 30.0 diopter (D) Tecnis Z9000 foldable silicone IOL (Abbott Medical Optics) was implanted in the capsular bag with an IOL implantation forceps. The sodium hyaluronate was irrigated from the anterior chamber, and the incisions were closed with 10-0 nylon. A subconjunctival injection of triamcinolone was given.

Replacement of Sodium Hyaluronate by Silicone Polymer

In 2 monkeys (numbers 2 and 3), the lens capsular bag was first filled with the clinically used sodium hyaluronate. In monkey 3, the sodium hyaluronate had disappeared from the capsular bag after 1 month, possibly due to leakage caused by a larger capsulorhexis and by the plug not covering it completely. It was decided to fill the capsular bag with the silicone polymer. In monkey 2, the capsular bag remained clear after 3 months of follow-up. It was decided to replace the clinically used sodium hyaluronate with the silicone polymer. To do this, an ACM connected to a bottle of saline was inserted through a paracentesis. The capsule plug was depressed with a 20-gauge cannula, and the sodium hyaluronate was aspirated from the capsular bag in a fashion similar to that used for aspiration of the natural lens. Then, the capsular bag was refilled with silicone polymer as described before.

Postoperative Treatment and Examinations

Postoperative examinations were performed every month after surgery in all monkeys using the same anesthesia protocol as for the surgery. At the first postoperative examination, the corneal sutures were removed and a second subconjunctival injection of triamcinolone was given. During the 3 days after the surgery, the monkeys received a daily subcutaneous injection of 4 mg/kg carprofen (Rimadyl) for analgesia.

During the 2 weeks after surgery, the monkeys received a tablet of 5 mg prednisolone daily (hidden in fruit). At each postoperative examination, refraction, A-scan US, and pharmacologically stimulated accommodative responses were measured. Details of these measurements have been describe.24 If refraction measurements were not possible because of media opacities, only A-scan US measurements were performed and follow-up examinations were modified according to clinical judgment. During postoperative slitlamp examinations, photographs were taken through a photographic attachment to the slitlamp and the refractometer.

Enucleation, Magnetic Resonance Imaging, Confocal Microscopy

After a follow-up of 9 to 30 months, monkeys were killed humanely with an overdose of intravenous pentobarbital, 200 mg/kg. Eyes were enucleated and transported in a container on ice to the University of Medicine, Greifswald, Germany, for high-resolution MRI imaging of the anterior segment within 24 hours after enucleation.

Micro-MRI images were acquired using an ultra-high-field magnetic resonance scanner (7.1T Clinscan, Bruker Bioscan). All eyes were imaged using a phased-array surface coil (rat brain) with 2 channels and 2 coil elements for each channel. The eye to be examined was placed in the coil. The examination protocol included T2-weighted spin-echo images in the 3 anatomic planes. A field of view of 40.0 mm × 40.0 mm with a matrix size of 512 pixels × 512 pixels was used. The other imaging parameters were echo time 3300 ms, repetition time 75 ms, and 22 slices with no gap between the slices. The acquisition time was 8 minutes 16 seconds. Lens dimensions were determined from the MRI images as described by Stachs et al.33

After MRI imaging, the eyes were fixed in paraformaldehyde concentrations ranging between 1.0% and 3.7% for staining with fluorescent agents and subsequent microscopy. Corneas and lenses were taken out of the fixed eyes and kept in buffer until further staining. Complete lenses and corneas were permeabilized and stained for nuclei (4′,6-Diamidino-2-phenylindole, dihydrochloride) and cell cytoskeleton (tetramethylrhodamine–phalloidin). Specimens were observed with a confocal laser scanning microscope (TCS SP2, Leica) using a fully water-immersed ×40 objective with high numerical aperture (0.80) and large working distance. Image stacks were made from lenses in situ using confocal microscopy. Projection images were derived from the stacks using Leica software and the maximum projection algorithm. Occasionally, individual sections from image stacks are presented in this paper.

Comparison with an Earlier Study

A 2006 study24 described refraction, accommodative amplitude, lens thickness change, and clarity of the lens capsule after accommodating lens refilling in 9 adolescent rhesus monkeys using a similar surgical procedure with the same silicone polymer used in this study. In 5 of the 9 monkeys, chemically active sodium hyaluronate was used. The results regarding the clarity of the lens capsule of the 2006 study and the present study were combined to enable a statistical comparison between lens refilling with the silicone polymer and lens refilling with the clinically used sodium hyaluronate and whether capsule opacification occurred.

Statistical Analysis

A Fisher exact test in a 2 × 2 contingency table with Bonferroni correction for multiple testing was performed using SPSS software (version 20.0.0.1, International Business Machines Corp.).

Results

Surgeries

Lens removal and refilling of the capsular bag was uneventful in 4 of the 6 monkeys. In 2 monkeys, difficulties occurred during surgery. In monkeys 1 and 3, refilling with the silicone polymer had to be performed 3 times due to leaking of the refill material from the capsular bag because of displacement of the plug during injection. When leaking occurred, the silicone polymer was aspirated from the capsular bag and a new attempt was made to refill the bag. These repeated refilling attempts prolonged the surgery considerably (90 minutes in monkey 1; 105 minutes in monkey 3).

In monkey 1, the anterior lens capsule showed severe amounts of fibrosis at the first postoperative visit, making refraction measurements impossible. To determine the possibility of a backup surgical procedure should lens refilling lead to unacceptable results, removal of the anterior lens capsule and of the silicone material and implantation of the 30.0 D silicone IOL were performed 3 months postoperatively. This procedure was uneventful, and refraction measurements could be performed at each postoperative visit during a follow-up of 12 months.

In monkey 3, the lens capsule was refilled with clinically used sodium hyaluronate after 3 failed attempts to fill the capsular bag with the silicone polymer. One month later, it appeared that the sodium hyaluronate in the capsular bag had disappeared and it was decided to fill the capsular bag with the silicone polymer. This was done uneventfully; however, remains of the silicone polymer adhered to the outside of the anterior lens capsule, making refraction measurements impossible. Accommodation could be established by lens thickness changes measured by A-scan US only.

Preoperative Measurements

The preoperative accommodative amplitude in response to pilocarpine 4.0% eyedrops varied between 3.5 D and 14.8 D. The accommodative lens thickness change in response to pilocarpine 4.0% eyedrops varied between 0.13 mm and 0.85 mm. When the results per eye were summarized, a linear relationship of 0.058 mm lens thickness change per diopter of accommodation was found in the natural lenses.

Postoperative Measurements

The mean postoperative baseline refraction during follow-up of the eyes before stimulation with pilocarpine varied between monkeys. In monkey 1, in which the refilled silicone lens was exchanged with a 30.0 D silicone IOL, the eye became myopic. In the other eyes, the capsular bag was filled with silicone or the clinically used sodium hyaluronate. Monkey 5, in which the lens capsule was filled with silicone, became highly myopic, while monkey 6, in which the lens capsule was filled with the clinically used sodium hyaluronate, became highly hyperopic.

The maximum postoperative accommodative amplitude was 6.25 D in monkey 4 after 13 weeks of follow-up (Table 1). In all monkeys that showed measurable accommodative changes, a decline in accommodative amplitude and/or lens thickness change was seen except in monkey 6, in which the lens capsule was filled with the clinically used sodium hyaluronate. In monkey 2, only 1 data point of accommodative optical power change could be measured due to a decrease in the clarity of the lens capsule. In this monkey, the lens capsule had initially been filled with the clinically used sodium hyaluronate, which was removed after 12 weeks. The capsule was then refilled with silicone polymer. In monkey 3, silicone polymer remaining on the anterior lens capsule prevented refraction measurements.

Table 1
Table 1:
Postoperative measurements.

Slitlamp Evaluation

Slitlamp evaluation of all eyes (operated and unoperated) showed no corneal abnormalities during the follow-up period; this was corroborated by confocal imaging of the corneal endothelium. A gradual increase in capsule opacification of the anterior and posterior lens capsule in monkeys 1, 3, 4, and 5 was noted. These lens capsules had been treated for 5 minutes with a chemically active sodium hyaluronate solution and filled with a silicone IOL (monkey 1) or the silicone refilling polymer (monkeys 3, 4, and 5). The opacification was comparable to the fibrous type of capsule opacification seen after regular IOL implantation and consisted of white strands and patches as well as capsule wrinkles (Figure 2, A). In monkey 2, the lens capsule was filled with chemically active sodium hyaluronate solution for 5 minutes after which this was replaced with the clinically used sodium hyaluronate. After 12 weeks, no capsule opacification was visible. After exchange of the clinically used sodium hyaluronate with silicone polymer, capsule opacification developed and was visible through the slitlamp. In monkey 6, the lens capsule was filled with the clinically used sodium hyaluronate during the entire follow-up of 9 months after a 5-minute treatment of the capsular bag with chemically active sodium hyaluronate. No capsule opacification was visible during the entire follow-up (Figure 2, B).

Figure 2
Figure 2:
Slitlamp image of monkey 4 (refilled with silicone polymer) at the 3-month of follow-up (A) and of monkey 6 (filled with the clinically used sodium hyaluronate) at the 9-month follow-up (B).

In 2006, 9 rhesus monkeys had accommodating lens-refilling surgery with the same silicone polymer used in this study. Five monkeys were treated with a chemically active sodium hyaluronate and 4 were not. The lenses were filled with a silicone polymer. All 9 monkeys developed capsule opacification. Combining the 9 monkey lenses from 2006 with the 5 monkey lenses in this study resulted in a group of 14 monkey lenses refilled with the same silicone polymer. In this study, 2 monkey lenses were filled with the clinically used sodium hyaluronate after treatment of the capsular bag with chemically active sodium hyaluronate. These lenses remained clear. The Fisher exact test (with Bonferroni correction for testing multiple factors) comparing the proportion of no capsule opacification in 2 lenses filled with the clinically used sodium hyaluronate (monkeys 2 and 6) and 14 opacified capsules in 14 silicone-filled lenses results in a P value of less than 0.025, suggesting that lens refilling with the clinically used sodium hyaluronate resulted in a significant difference in capsule opacification, regardless of the other measures taken to prevent capsule opacification.

Magnetic Resonance Imaging

Figure 3 shows the parts of the 7T MRI images of 1 natural rhesus monkey lens and the refilled lenses. Due to loss of intraocular pressure after enucleation, the corneas of the eyes are swollen and have lost their convex shape. The lenses have kept their biconvex shape. Figure 3, A, shows a cross-section of a natural rhesus monkey lens with a thickness of 3.92 mm (indicated in the figure as a rounded number of 0.4 cm). In eyes in which a silicone refill polymer was present (Figure 3, C to F), a chemical shift artifact is visible in the image. Tissue formation can be seen at the inferior equatorial region in Figure 3, D and E, interfering with a complete filling of the capsular bag by the refill material. In Figure 3, C and F, tissue can be seen at the posterior pole. The amount of refilling varies considerably between the lenses in Figure 3, C and F, as reflected by the differences in lens thickness and lens volume (Table 2). Natural lenses also showed variability in lens thickness and volume. Figure 3, G, shows a lens refilled with sodium hyaluronate. The posterior capsule is slightly thickened, representing a thin layer of cellular proliferation. This layer was not visible at the slitlamp (Figure 2, B); thus, it was probably transparent. The lens filled with sodium hyaluronate is thick compared with the natural lenses. This is probably due to the fact that the clinically used sodium hyaluronate binds water molecules and swells in the eye.

Figure 3
Figure 3:
The MRI images of the monkey lenses after enucleation of the eye. A: Natural monkey lens. B: Monkey 1 with a 30.0 D silicone IOL. C to F: Silicone polymer–refilled lenses. G: Lens capsule refilled with sodium hyaluronate (* = chemical shift artifact as reported by Stachs et al.33; ** = regrowth of lens material).
Table 2
Table 2:
Lens volume and thickness determined from MRI images.

Confocal Microscopy

CorneaFigure 4 shows the number of corneal endothelial cells counted in the enucleated eyes and Figure 5 shows a representative confocal microscopy image of the cornea of a treated and untreated eye. In 4 monkeys, the number of endothelial cells was significantly lower in the treated eye than in the untreated eye, although the endothelial cell layer was regular in all eyes.

Figure 4
Figure 4:
Corneal ECD in left eyes and right eyes of the monkeys counted after enucleation. The dashed line represents the mean cell count in the untreated eyes (2970 cells/mm2). In each cornea, 4 fields of view were counted (* = significant decrease in cell density relative to the untreated eye in the same monkey; IOL = intraocular lens).
Figure 5
Figure 5:
Representative confocal projection images of the cornea endothelial cell layer in an untreated eye (A) and treated eye (B) for monkey 3. Images are 375 μm × 375 μm. Nuclei are shown in blue. The actin cytoskeleton is shown in red.

Lens CapsuleFigure 6 shows the confocal microscopy images of the lens capsule of monkeys 2, 3, 4, and 5 and 1 untreated lens. The lenses of monkeys 2, 3, 4, and 5 had been filled with the silicone polymer after treatment with chemically active sodium hyaluronate. In all cases, signs of fibrosis are present in different stages of development and appearance. The cells in Figure 6, A to E, lack the regular lens epithelial–specific regular organization of the untreated lens in Figure 6, F. Figure 7 shows a lens capsule that has been filled with the clinically used sodium hyaluronate after treatment of the capsular bag with chemically active sodium hyaluronate. On the anterior surface, a more regular lens epithelium can be seen, although not perfectly hexagonal in organization, while on the posterior side, lens fibers seem to have formed.

Figure 6
Figure 6:
Confocal projection images of LECs of lenses that were refilled with the silicone polymer in monkey 2 (A), monkey 3 (B), monkey 4 (all anterior) (C), and monkey 5 (D, anterior; E, posterior). An individual section of an images stack from a control (ie, untreated lens) is shown in F. All images are 375 μm × 375 μm. Nuclei are shown in blue. The actin cytoskeleton is shown in red.
Figure 7
Figure 7:
Confocal projection images of LECs of a lens that was refilled with the clinically used sodium hyaluronate (monkey 6). A projection image is shown for anterior (A), whereas for posterior, an individual section is shown (D). Transverse optical slices through the image stacks are shown in B, C, E, and F to indicate the thickness of the cell layers.

Discussion

This study explored methods to improve the efficacy of the prevention of capsule opacification. Due to ethical considerations of a study in monkeys, treatment variables were evaluated in individual animals only. Therefore, each response could be the result of inter-animal variation or of the total surgery, including perisurgical treatment of LECs and IOL implantation. Nevertheless, the clear-cut and well-characterized responses measured with different treatments in the clinical and post-clinical setting led to several observations and statements.

Two strategies were explored to prevent the development of capsule opacification. The first strategy was to treat the capsular bag with chemical agents that had been shown to be effective in the rabbit eyeA other than the combination of actinomycin-D and cycloheximide, which had been used previously.24 The second strategy was to fill the capsular bag with clinically used sodium hyaluronate 1.0% (Healon) instead of the silicone polymer.

The first strategy, using several combinations of chemical agents (methotrexate, CAPE, paclitaxel, and polysorbate in combination with actinomycin-D) to treat the capsular bag, did not prevent the development of capsule opacification. The second strategy (refilling of the capsular bag with the clinically used sodium hyaluronate after a chemical treatment) resulted in an absence of clinically visible capsule opacification in 2 monkeys. Replacement of the sodium hyaluronate by the silicone polymer after 3 months in 1 of those 2 monkeys resulted in a prompt occurrence of capsule opacification that was visible at the slitlamp. In the study published in 2006,24 all 9 monkeys had refilling of the capsular bag with silicone polymer and all 9 monkeys developed capsule opacification. In the present study, all 5 lenses refilled with silicone polymer developed capsule opacification. The absence of any opacification after refilling with the clinically used sodium hyaluronate suggests that some property of the sodium hyaluronate instructs the LECs to refrain from the development of capsule opacification. Because capsule opacification is a universal occurrence after IOL implantation (in human lens surgery and in animal studies), the finding of a clear lens capsule in 2 monkeys after lens removal and refilling is unusual and worth reporting. Refilling the lens capsular bag with the clinically used sodium hyaluronate without a 5-minute pretreatment of chemically active sodium hyaluronate could also have been effective; however, this was not studied in our experiments.

Microscopic examination and MRI imaging of the lens filled with the clinically used sodium hyaluronate in which no clinically visible opacification was present after 9 months of follow-up (monkey 6) showed a thin layer of cells on the anterior and posterior capsule. On the anterior side, a monolayer of epithelial-like cell structures was seen with occasional hexagonal arrangements. On the posterior side, a thick layer of phalloidin-positive material and nuclei were formed, which was assumed to be the onset of lens fiber formation. The absence of fibrosis and the regular organization of the cell layer probably contributed to the fact that the cell layer was not visible at the slitlamp.

Although no fibrotic response could be seen on the lens capsule of monkey 6, the clinically used sodium hyaluronate is not suitable for use as a lens-refilling material. The refractive index is too low (1.33), and the material swells as a result of water uptake,34 making the dimensions of the resulting lens unpredictable. However, the absence of visible capsule opacification after a follow-up of 9 months implies that some properties of the lens-refilling material have a profound effect on the response of the cells residing inside the capsular bag. In our study, the beneficial effect of refilling with the clinically used sodium hyaluronate may have been mediated by the CD44 cell membrane receptor. This receptor is the most important cellular membrane receptor on LECs for hyaluronan.35 Hyaluronan is a component of the extracellular matrix (ECM) in intact lenses. Refilling with the clinically used sodium hyaluronate may have provided the LECs with a more natural environment than refilling with a silicone polymer, resulting in an absence of fibrosis. No loss of lens thickness occurred during 9-month follow-up of the lens filled with the clinically used sodium hyaluronate, meaning that the material is well shielded from enzymes such as hyaluronidase in the anterior chamber when the capsular bag is closed by the silicone plug.

Healon and other sodium hyaluronate formulations have been used in rabbit lens studies by Gwon and Gruber28 and Fernandez et al.36 Gwon and Gruber used crosslinked sodium hyaluronate (Restylane) to act as a scaffold for lens regeneration. In these experiments, the lens nucleus and cortex were removed through a small capsulorhexis and crosslinked sodium hyaluronate was injected in the lens capsule. Optically clear cortical material grew around a central hyaluronate scaffold. Normal lens fiber alignment was seen on histologic examination. Fernandez et al.36 treated the inside of the capsular bag with distilled water and filled the capsular bag with sodium hyaluronate 1.4%. Complete lens regeneration with a regular organization on histology was observed. Our study was different from the study by Gwon and Gruber28 regarding the amount of sodium hyaluronate injected, the closure of the capsule opening by a plug, and the animal model used; however, it was similar in that a clear layer of cortical lens fibers grew around the sodium hyaluronate. More research is needed to understand the mechanisms of interaction between the refilling material and remaining LECs and how to modify the interface between them in such a way that capsule fibrosis and opacification are prevented. Resemblance of the interface between lens implant and LECs to a natural ECM might prevent the fibrotic reaction and lead to a more regular LEC growth pattern. This could be achieved by surrounding the materials of lens implants by appropriate interfaces or coatings.

Our first strategy using several combinations of chemical agents to treat the LECs inside the capsular bag did not result in a permanent absence of capsule opacification. Coincident with the occurrence of capsule opacification, the accommodative amplitude and accommodative lens thickness change decreased with time. Methotrexate, CAPE, paclitaxel, and polysorbate in combination with actinomycin-D were chosen based on positive outcomes in in vivo studies for the prevention of capsule opacification after rabbit lens implantations.26,33 However, fibrosis, in different stages of development and appearance was present in all cases, as visible at the slitlamp, in several MRI images, and on microscopy. The MRI images showed areas of irregular regeneration of lens material at the equator and posterior pole of the lenses refilled with silicone polymer. Microscopically, lens capsules filled with the silicone polymer all showed fibrosis in different stages of appearance or development, despite the perisurgical treatment of the remaining LECs with chemically active sodium hyaluronate. These are representations of the epithelial-to-mesenchymal transformation process.37 This process is thought to start with transforming growth factor-β signaling when the lens epithelium is disturbed and enhanced by the presence of a foreign body such as the silicone polymer. Less capsule opacification in the monkeys had been expected based on the outcome of similar lens-refilling experiments in rabbits. In a study of rabbits,25 a clear anterior capsule and posterior lens capsule were found after lens refilling with a silicone polymer 3 months after treatment of the lens capsule with actinomycin-D and cycloheximide. Also in rabbits, Stachs et al.33 found no capsule opacification after lens refilling with a silicone polymer 40 months after treatment of the capsular bag with methotrexate and actinomycin-D. This indicates that the monkey model for lens refilling and prevention of capsule opacification is different from the rabbit model and that results of 1 animal model cannot be extrapolated to the other. It is still possible that a strategy of chemical ablation of all LECs for long-term prevention of capsule opacification is feasible in monkeys as well. The substances we used were selected based on their efficacy in cell culture experiments.26,A To keep the substances localized to the capsular bag, they were dissolved in a sodium hyaluronate solution. However, their availability and effectivity once they were dissolved in sodium hyaluronate may have been reduced due to binding of molecules to the hyaluronate gel.B More research of the most effective and safe method of applying these chemical substances could improve results.

Preoperative examination of the accommodative amplitude after topical stimulation with pilocarpine 4.0% eyedrops showed variable accommodative amplitudes in the monkeys in this study. This could be caused because topically applied pilocarpine was used to stimulate accommodation,38 resulting in underestimation of the true accommodative amplitude. Because the main interest in this study was the prevention of capsule opacification, no alternative method to stimulate accommodation was chosen.

Postoperative accommodative amplitudes and lens thickness changes decreased and capsule fibrosis developed, similar to the results in our previous study.24 In monkey 6, which did not develop capsule fibrosis, lens thickness changes could be measured during the whole follow-up period, indicating that stiffening of the lens capsule did not develop. In this monkey, accommodating lens power changes could not be measured due to the low refractive index of the clinically used sodium hyaluronate refilling material.

Data on the corneal endothelium indicate the presence of a regular endothelium in all monkey eyes. Endothelial cell counts showed that in 4 out of 6 individual monkeys, a lower endothelial cell density (ECD) was found in the surgical eyes than in the nonsurgical eyes. Although this reduced cell density could have been the result of the implantation procedure, damage from treating the capsular bag with chemical agents cannot be excluded. However, no corneal opacification (as seen in some rabbits after treatment of the capsular bag with chemically active sodium hyaluronate25) was observed. Results in a recent study of capsular permeability of actinomycin-D suggest that it is safe to use within a 5-minute time frame of application.39 Some monkeys (1, 2, and 3) had a second surgical procedure after lens refilling, which may also have caused additional corneal endothelial cell loss. Clinically, no visible signs of corneal endothelial damage (eg, edema) were present. This is confirmed by the observation that endothelial cells were homogeneously distributed. Routine uneventful human cataract surgery may result in an endothelial cell loss of 4.0% 3 months after surgery40 and increase to 28.0% 10 years after surgery,41 depending on the technique used. Optimization of an accommodating lens–refilling procedure may further limit the loss of endothelial cells.

In conclusion, prevention of capsule opacification after lens refilling with a silicone polymer by treating the inside of the capsular bag using several combinations of chemical agents did not result in a permanent absence of capsule opacification. Refilling the capsular bag with Healon after a chemical treatment resulted in an absence of clinically visible capsule opacification in 2 monkeys, also over the long-term. These findings suggest that the physicochemical properties of the refilling material influence the behavior of the remaining LECs at their interface inside the capsular bag, resulting in a clear lens capsule covered with lens epithelium. In the future, it will be important to examine the interaction at the interface between artificial lens materials and LECs. This knowledge may help in the development of a suitable lens-refilling material with a higher refractive index than sodium hyaluronate with similar prevention of the development of capsule opacification.

What Was Known

  • Opacification of the lens capsule reduces the accommodative amplitude after accommodating lens refilling in adolescent rhesus monkeys.
  • A plethora of methods to prevent capsule opacification have been described, including methods to eliminate all LECs from the lens capsule. The choice of IOL material also influences the amount of capsule opacification.

What This Paper Adds

  • The use of sodium hyaluronate as a lens refilling material after a treatment of the capsule with actinomycin-D prevented the development of capsule opacification clinically and microscopically for at least 9 months.
  • It is possible to prevent capsule opacification in a rhesus monkey model when suitable physicochemical characteristics of the IOL material are chosen.

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Other Cited Material

A. Unpublished data on file.
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