Refractive changes induced by electrocautery of the rabbit anterior lens capsule

Refractive changes induced by electrocautery of the rabbit anterior lens capsule Dana A. Jungschaffer, M.D., Essam Saber, M.D., Kerry M. Zimmerman, M.S., Peter J. McDonnell, M.D., Steven E. Feldon, M.D.

ABSTRACT The pathologic basis for presbyopia is classically attributed to lenticular sclerosis or atrophy of the ciliary muscle, but recent work suggests that thickening and loss of elasticity of the anterior lens capsule play an important role. As no practical means for attenuating or reversing the aging process of lens protein has been identified, alteration of the lens capsule eventually might prove to be a desirable alternative to spectacle correction in presbyopic individuals. This paper describes changes in the refractive properties of the lens resulting from alteration of the anterior lens capsule by application of focal cautery, using both an in vitro, in situ and an in vivo rabbit model. In vitro thermal treatment (electrocautery) of the capsule significantly increased the anterior curvature of the lens by an average of +2.95 diopters. Histologic examination of the treated lenses showed thinning of the capsule in the treated areas, as well as focal vacuolar degeneration in the lens substance beneath the lesions. In vivo thermal treatment of eyes induced a significant shift toward myopia, compared with control eyes. The accommodative range increased post-treatment relative to the controls, but the effect diminished over time, stabilizing near baseline at two to three weeks after treatment. Histologic examination showed localized changes but no signs of diffuse cataract formation. We conclude that the anterior capsule may play a significant role in the refractive power and accommodative changes in the crystalline lens. With further study of short-term and long-term effects and with development of noninvasive laser techniques, thermal treatment of the anterior lens capsule might eventually become a practical method of managing hyperopia and loss of accommodation in patients with advanced presbyopia. Key Words: electrocautery, presbyopia, rabbit anterior lens capsule

According to the Helmholtz relaxation theory, the changes that occur during accommodation include contraction of the ciliary muscle and relaxation of zonule fiber tension. I These changes allow the elastic lens capsule to mold the lens into a more curved form, thereby increasing the refractive power. Loss of accommodative amplitude is a ubiquitous and frustrating consequence of aging, and spectacle correction facilitates focus of

objects at only two or three distances, at best. In the United States, the incidence of presbyopia is 31 cases per 100 people, and it is anticipated that this will increase as the average age of the population increases. I The effects of aging on the accommodative mechanism have always been controversial. According to Donders (1864), the ciliary muscle weakens with age, while Helmholtz ( 1855) suggested that the lens becomes less

From the Departments of Ophthalmology (Jungschaffer, Saber, Zimmerman, McDonnell, Feldon) and Neurologic Surgery (Feldon), University of Southern California School of Medicine, Los Angeles, California. Supported in part by a grant from the JosephS. Feldman Foundation to the Doheny Eye Institute; by a Peace Fellow Research grant from the Embassy of the Arab Republic ofEgypt Cultural and Educational Bureau Peace Fellowship Program, Washington, D.C.; an unrestricted grant from Research to Prevent Blindness, Inc., New York, New York; and by a Core Grant for Vision Research (EY 03040) from the National Institutes of Health, Bethesda, Maryland. Argyrios Ziogas helped with statistical analysis; Dr. Narsing Rao, Dr. Jan McDonnell, and Mr. David Stanforth did the histopathology processing, interpretation, and documentation; and Ann Dawson provided editorial assistance for this paper. Reprint requests to Steven E. Feldon, M.D., Doheny Eye Institute, 1450 San Pablo Street, Los Angeles, California 90033. 132

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pliable. 1 More recently, Kaufman 2 proposed that loss of accommodative amplitude with age may result from the collective effects oflenticular growth, changes in internal and external lenticular geometry and optical properties, ciliary muscle mobility, extralenticular elastic tissue, and the vitreous. Studies by Fisher3 and by Strenk and coauthors4 showed that the ciliary muscle does not weaken but may actually undergo compensatory hypertrophy as accommodative amplitude decreases with age. Stiffening of the posterior and outer attachments of the ciliary muscles with age has been shown with histologic5- 7 and videographic8 data in rhesus monkeys. Also, age-dependent changes in lens curvature have been demonstrated in humans9 and rhesus monkeys. 8 •10•11 Nevertheless, if Fisher's observations are correct, complete loss of accommodation by age 61 can be explained by lens changes alone, primarily changes in the lens capsule and lens substance. The capsule loses its elastic properties with age, 12 which may compact the lens fibers, particularly in the nucleus, thereby reducing accommodation. Progressive loss of capsule elasticity may correlate with capsular thickening 13 •14 and may be attributable to an increase in cross-linkage of disulfide bonds within the capsule's beehive-like collagenous structure. 15 If anterior capsule elasticity plays an essential role in accommodation, manipulation of the capsule might alter lens power. For instance, increased capsule tone may be achievable through thermal shrinkage. In the present study, electrocautery disruption was used to alter the anterior capsule in an in vitro, in situ rabbit lens model and an in vivo rabbit model. The changes in lens shape induced by these manipulations were evaluated in terms of changes in curvature of the anterior lens surface and changes in refraction.

MATERIALS AND METHODS Our studies conformed to the Association for Research in Vision and Ophthalmology Resolution on the Use of Animals in Research. Animals were maintained in facilities fully accredited by the American Association of Laboratory Animal Science.

In Vitro Model

The eyes of New Zealand white rabbits were enucleated and placed in normal saline at 4 degrees Celsius for not longer than three days before the experiments. The posterior segment was blotted dry and mounted on a plastic holder, using methyl methacrylate glue (Histoacryl®, B. Braun, Melsungen, Germany). The cornea and the iris were then resected, exposing the entire anterior lens capsule. The capsules (n = 13) were treated with a bipolar coagulator (Storz Endoscopy-America, Culver City, CA) (setting 20). Throughout the experiments the lens capsules were kept moist with frequent applications of artificial tears; six to 12 treatment applications per anterior lens capsule were made. The central optical axis was spared in all cases.

Three sham procedures were performed in which the cornea and iris were resected, but without anterior capsule manipulation. A photocorneascope (Kera Co., Santa Clara, CA), which projects multiple concentric rings oflight (mires), was used to determine the anterior curvature of the anterior lens capsule before and after treatment (Figure 1). 16 For sham procedures, five minutes elapsed between measurements. In addition, the lens surface power of one eye that had a sham procedure was measured ten times in succession. A Kera-scan analyzer was used to measure the mires from the photographic images to determine curvature of the anterior lens surface in air. Average change in anterior curvature was determined for each lens. Statistical differences in anterior curvature before and after treatment were determined by analysis of variance, controlling for variation in ring size. For the one lens that was measured ten times in succession, a coefficient of variation was determined to assess measurement error. Histologic examination was performed on several lenses. These lenses were fixed in half-strength Karnovsky's fixative, dehydrated through graded alcohols, infiltrated and embedded in glycol methacrylate (Historesin, Reichert-Jung [Leica, Inc.], Deerfield, IL), sectioned, and then stained with hematoxylin and eosin.

In Vivo Model

Fourteen pigmented rabbits (Oryctolagus cuniculus) weighing approximately 2.5 kg were anesthetized with an intramuscular injection of ketamine hydrochloride (30 mgjkg) and xylazine hydrochloride (5 mgjkg) (4:1). Refractive measurements of both eyes were taken by standard streak retinoscopy. After these measurements were obtained, tropicamide 1% was instilled into the right (operative) eye four times at five minute intervals to induce mydriasis and cycloplegia, after which refractive measurements were again recorded. The experimental eyes had surgery; the control eyes (left eyes) remained unoperated. A lid speculum was used to hold the eyelids open during surgery. A 2 mm corneal incision was made at the 12 o'clock meridian using a #11 Bard-Parker knife. Hyaluronic acid (Heaton®) was injected into the anterior chamber to maintain its shape. The anterior lens capsule was cauterized with a bipolar surgical diathermy unit (Clinitex) set at the lowest power. We cauterized the lens in eight equally spaced equatorial locations, making two applications to each quadrant. Care was taken to avoid inadvertent rupture of the capsule. The Healon was then washed out of the anterior chamber and replaced with sterile balanced salt solution, after which the corneal wound was closed with 10-0 nylon monofilament. While the rabbit was still on the surgery table, the eyes were again treated with tropicamide 1% and refractive measurements recorded. Topical antibiotics and steroid ointment were applied for three days postoperatively.

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Fig. 1.

(Jungschaffer) Comeascope photographs reflected off the rabbit lens anterior capsule before (left) and after (right) electrocautery.

To induce maximum cycloplegia, drops of tropicamide 1% were instilled into the eyes four times at fiveminute intervals, after which refractive measurements were recorded. To induce maximum accommodation, the eyes were treated with pilocarpine 4% every two minutes for 30 minutes, after which refractive measurements were again recorded. Retinoscopy was easily performed on these rabbits even after treatment with pilocarpine 4% because the pupil aperture remained about 2.5 em. All measurements were taken under general anesthesia. Rabbits were arbitrarily divided into two groups. On a given day, the right eye (experimental) of each rabbit was treated with a single topical medication (antibiotic or steroidal): one group was treated with cycloplegic (mydriatic) drops, the second with cyclospasmic (miotic) drops. Measurements were taken on days 1, 2, 4, 6, 8, 13, 18, and 28 postsurgery. Control measurements (left eye) were recorded after exposure to tropicamide on days 1 and 6 postsurgery. Control measurements for pilocarpine were recorded on the eighth day after surgery. A mortality rate of 30% during the course of the experiment was presumed to be the result of complications of repeated anesthesia induction; all deaths occurred between eight and 13 days after surgery. One-sample t-tests were used to compare the experimental and control groups, under exposure to cyclospasmic and cycloplegic agents separately. Two-sample t-tests were used to compare the ranges of accommodation between the control and experimental groups. Although there were only three control measurements, these values are uniform and are valid for use in statistical analyses; these values should be constant for untreated eyes. Statistical significance wasP< .05. Rabbits were sacrificed by intracardiac administration of a barbiturate overdose in 5% formaldehyde solution. The orbits were then exenterated and placed in 134

5% formaldehyde solution. Lenses were isolated and sections cut perpendicular to the visual axis; alternate sections were stained with hematoxylin and eosin or with periodic acid-Schiff stains.

RESULTS In Vitro Mode/

Histologic examination of the treated lenses (Figure 2) revealed focal thinning of the capsule in the bum areas and vacuolar degeneration in the lens substance directly beneath the treated area. No capsular rupture was evident. The anterior surfaces of 12 of the 13 lenses treated with bipolar coagulation were steepened postopera-

Fig. 2.

(Jungschaffer) Histologic section of an electrocauterized lens shows focal thinning of the capsule in the bum area and underlying vacuolar degeneration of lens substance (hematoxylin and eosin, original magnification X 200).

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76

Postoperative Refractive Power (0)

72

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68

64

60

.

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56 56

.:

/ 60

/ 64

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72

78

Preoperative Refractive Power (0)

Fig. 3.

(Jungschaffer) Postoperative anterior curvature is plotted against preoperative anterior curvature in air for electrocauterized lenses. Each data point represents one lens (n = 13). The line represents identity between the two measurements. The plus (+) and minus (-) symbols indicate an increase or decrease in post-treatment anterior curvature, respectively.

tively, as evidenced by the increase in anterior curvature (Figure 3). Steepening of the lens surface ranged from + 1.79 diopters (D) to +5.40 D. Mean refractive change following electrocautery of the 13lenses was +2.95 D ± 1.50 (± standard error of the mean) (P < .0001 ); mean anterior curvature of the anterior lens surface in air was 62.09 D ± 0.87 and 64.98 D ± 1.13, pretreatment and post-treatment, respectively. The sham procedures resulted in no significant difference between preoperative and postoperative anterior lens curvature (+0.44 D ± 0.34). The coefficient of variation for the lens that was analyzed ten times in succession was small ( 1.18% ).

In Vivo Model

All lenses remained clear centrally, with no evidence of visually significant central cataractous change. No hypopyons or hyphemas were noted in the anterior chamber 24 hours or more after surgery. Lesions found at 28 days after electrocautery demonstrated an intact anterior capsule of normal thickness with underlying amorphous eosinophilic material. Chronic inflammatory cell infiltration and fibroblastic proliferation were present at the periphery of the lesion, demarcating the area of injury from normal lens cortex. There was no evidence of capsule rupture or diffuse cataractous lens change, but some lesions produced small focal changes in the anterior lens cortex (Figure 4). In the control eyes, cycloplegic refraction averaged +5.00 D and the cyclospasmic refraction averaged +2.20 D, for a net pharmacologically induced accommodative amplitude of 2.80 D. Immediately after surgery, cycloplegic-treated experimental eyes demonstrated a shift of approximately 7.00 D toward myopia. This shift regressed gradually but remained statistically

Fig. 4.

(Jungschaffer) Treated lens demonstrating intact capsule and subepithelial scar consisting of amorphous material and chronic inflammatory cell infiltration and fibroblast proliferation. Focal lenticular opacity corresponds to the positive periodic acidSchiff material in the subepithelial cortex (periodic acid-Schiff, original magnification x 100).

significant over the next nine days, stabilizing with a net decrease in hyperopia of about 1.00 D at 28 days after surgery, which was not statistically significant (Figure 5). Refractive changes obtained under induced cyclospasm paralleled those under induced cycloplegia, although individual variation was much greater (Figure 6). Mean experimental accommodative amplitude varied between a minimum of 2.90 D at 13 days and a maximum of 4.20 D at four days (Table 1). The residual effect at 28 days was 3.00 D, for an increase of only 0.20 D over control values. The accommodative amplitude increase related to surgery varied from 0.05 D to 1.40 D. These increases were statistically significant at days four and 18 after surgery (P < .025), but not at days

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Fig. 5.

(Jungschatfer) Refraction (diopters) of eyes after exposure to cycloplegic drops plotted against days after cautery. The values are presented as the mean ± the standard error of the mean. The line with the arrow (--+)represents control values; asterisks(*) represent significant differences from the control value.

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Table l. Differences in range of accommodation in experimental versus control eyes. Accommodative Range Control (C)

Day

Experimental (E)

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1.000 3.729 4.229 3.187

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(Jungschaffer) Refraction (diopters) of eyes after exposure to cyclospasmic drops is plotted against days after cautery. The values are presented as the mean ± the standard error of the mean. The line with the arrow (-)represents control values; asterisks(*) represent significant differences from the control value.

2, 6, 8, or 13. Significance testing could not be performed at 28 days because of the small number of observations.

DISCUSSION Our in vitro and in vivo results demonstrate that the power of the crystalline lens can be increased at least temporarily by focal application of thermal energy to the anterior lens capsule. Even at 28 days, the healing process did not completely reverse the myopic changes. Cataract formation after treatment is a major theoretical concern in the long term, although consistent with our results, manipulation of the capsule per se is not a likely source of cataractogenic change. 17 The strictly focal vacuolar degeneration of lens substance directly beneath the bum area in our in vivo electrocauterized lenses showed no tendency to spread over time. The in vivo model failed to maintain a clinically meaningful increase in accommodative amplitude, the peak effect being noted four days after surgery. Little is 136

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known about the pharmacologic versus physiologic response, although a 2.80 D change for rabbits is reasonable and to be expected in the absence of corroborating studies. The reasons for the transience of the accommodative increase following treatment require further investigation. The rabbit eye is known to have limited accommodative capability, 18 and the ciliary muscle may be incapable of sustaining the increased amplitude of response to repeated pharmacologic stimulation. Alternatively, the rabbit eye may become refractory to pharmacologic manipulation because of depletion or alteration of receptor sites, or the healing process itself may reverse the mechanical effects. These possibilities are subject to experimental testing by using a species with a greater range of accommodation and known presbyopic changes with age, such as the monkey. 18 •19 Also, monkeys can be trained to accommodate to visual stimuli, 20•21 thereby obviating the dependency on pharmacologic induction of accommodative spasm and cycloplegia. More intense application of electrocautery and use of anti-inflammatory or immune suppressive agents (e.g., corticosteroids) may lead to a more permanent effect. In addition, our future experiments will include Scheimpflug videography to examine lenticular changes, particularly cornea-to-lens distance and corneal curvature, to determine how much of the refractive changes might have resulted from alteration of the lenticular curvature. We have shown that the anterior curvature of the lens-capsule complex is consistently increased by thermal applications to the anterior lens capsule in vitro and in vivo. How this treatment also increases accommodative amplitude is unknown and requires further investigation. The resultant increase in lens power and accommodation might reflect an increase in anterior capsule elasticity. Alternatively, the anterior lens capsule may change shape, increasing tone without a corresponding change in elasticity. Deformation of the lens might also be facilitated by a change in the relative position of the zonule attachments to the lens. 10 Future

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studies to determine Young's modulus for the treated anterior lens capsule are contemplated to resolve this issue. The thermally induced changes in the lens capsule may prove analogous to those noted in the cornea following thermal keratoplasty. 22 •23 Our results are consistent with the reports of others suggesting that the anterior capsule is an important component of the refractive power of the crystalline lens, both in the unaccommodated and accommodated state. Potentially, thermal manipulation of the lens capsule could eventually prove useful for refractive surgery in a clinical setting. However, basic questions such as stability and predictability of the procedure have to be addressed using animal models. Even then, widespread therapeutic application would await development of a precise noninvasive system for safe delivery of infrared energy. Availability of such a system would minimize risks such as infection, capsular rupture, and inadvertent damage to anterior chamber structures.

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8. Neider MW, Crawford K, Kaufman PL, Bito LZ. In vivo videography of the rhesus monkey accommodative apparatus: age-related loss of ciliary muscle response to central stimulation. Arch Ophthalmol 1990; 108:69-74 9. Koretz JF, Handelman GH, Brown NP. Analysis of human crystalline lens curvature as a function of accommodative state and age. Vision Res 1984; 24: 1141-1151 10. Koretz JF, Neider MW, Kaufman PL, et al. Slit-lamp studies of the rhesus monkey eye: I. Survey of the anterior segment. Exp Eye Res 1987; 44:307-318 11. Koretz JF, Bertasso AM, Neider MW, et al. Slit-lamp studies of the rhesus monkey eye: II. Changes in crystalline lens shape, thickness and position during accommodation and aging. Exp Eye Res 1987; 45:317-326 12. Fisher RF. The significance of the shape of the lens and capsular energy changes in accommodation. J Physiol 1969; 201:21-47 13. Last RJ. Eugene Woltrs Anatomy of the Eye and Orbit, 6th ed. Philadelphia, WB Saunders Co, 1968; 383-396 14. Niesel P. Visible changes of the lens with age. Trans Ophthalmol Soc UK 1982; 102:327-330 15. Courtois Y. The capsule of the crystalline lens. In: Stark L, Obrecht G, eds, Presbyopia. New York, Professional Press Books, 1987; 45-53 16. Rowsey JJ, Reynolds AE, Brown R. Corneal topography: corneascope. Arch Ophthalmol 1981; 99: 1093-1100 17. Pau A, Novotny GEK, Kern W. The lenticular capsule and cellular migration in anterior capsular cataract. Graefes Arch Clin Exp Ophthalmol 1986; 224: 118-121 18. Bito LZ, Kaufman PL, DeRousseau CJ, Koretz J. Presbyopia: an animal model and experimental approaches for the study of the mechanism of accommodation and ocular ageing. Eye 1987; 1:222-230 19. Bito LZ, DeRousseau CJ, Kaufman PL, Bito JW. Agedependent loss of accommodative amplitude in rhesus monkeys: an animal model for presbyopia. Invest Ophthalmol Vis Sci 1982; 23:23-31 20. Wurtz RH. Visual receptive fields of striate cortex neurons in awake monkeys. J Neurophysiol 1969; 32:975-986 21. Schwarz U, Miles FA. Ocular responses to translation and their dependence on viewing distance. I. Motion of the observer. J Neurophysiol1991; 66:851-864 22. Neumann AC, Fyodorov S, Sanders DR. Radial thermokeratoplasty for the correction of hyperopia. Refract Corneal Surg 1990; 6:404-412 23. Seiler T, Matallana M, Bende T. Laser thermokeratoplasty by means of a pulsed holmium:YAG laser for hyperopic correction. Refract Corneal Surg 1990; 6:335-339

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