Lens Regeneration in New Zealand Albino Rabbits After Endocapsular Cataract Extraction

Transcription

1 Lens Regeneration in New Zealand Albino Rabbits After Endocapsular Cataract Extraction Arlene Gwon,* Lawrence Gruber, Christine Mantras, and Crystal Cunanan Purpose. To evaluate the regenerative capacity of the adult rabbit lens after removal of a Concanavalin A-induced posterior subcapsular cataract. Methods. Cataractogenesis was induced by intravitreal injection of Concanavalin A in adult New Zealand albino rabbits. At 7 mo postinjection, the cataracts were removed. Endocapsular lens extraction was performed by phacoemulsification and irrigation/aspiration with Balanced Salt Solution. Results. Postoperative!)', lens regeneration wasfirstnoted in the Balanced Salt Solution normal lens group at 3 weeks and the Concanavalin A cataract group at 6 weeks. By the 3-mo postoperative examination, lens regrowth, measured by digital image analysis, filled 74.5% of the capsule bag in the Balanced Salt Solution normal lens group and 46.6% in the Concanavalin A cataract group. In the latter group, less lens material was regenerated and at a slower rate than in eyes with extraction of a normal lens. Conclusion. This experimental model is the first to show that lens regeneration can occur after removal of cataracts secondary to inflammation. Invest Ophthalmol Vis Sci 1993;34: Hixtracapsular cataract extraction with intraocular lens (IOL) implantation is currently the procedure of choice for the treatment of adult cataracts. Although it has been highly successful in adults, it is associated with numerous complications in children. As many as 68% of cases may require additional surgery for secondary membranes, dislocated lenses, intraocular lens removal, and glaucoma. 1 The greater tendency for lens epithelial proliferation, which complicates intraocular lens surgery in the pediatric group, may be advantageous if surgery could be modified to allow the lens to regenerate after cataract removal. Since 1827, lens regeneration has been known to occur after extracapsular or endocapsular extraction of a normal lens, which leaves the anterior and posterior lens capsule relatively intact in rabbits. 2 " 10 Milliot also showed it to occur in other mammals including dogs, cats, sheep, and pigs. 4 The only reported attempt to regenerate a lens after extraction of a cataractous lens was by Stenhouse Stewart in I In a previous unpublished study, cataractogenesis was induced in New Zealand albino rabbits by intravitreal injection of Concanavalin A (Con A). Injections of this immunogenic agent produced an anterior and posterior uveitis that persisted for approximately 2 mo and resulted in the development of a posterior subcapsular cataract by 3 mo. The present study was designed to evaluate the regenerative capacity of the adult rabbit lens after removal of this Con A-induced posterior subcapsular cataract. From Allergan Pharmaceuticals and the. *University of California at /mine, Irvine, California. Submitted far publication December IS, 1991; accepted August 7, Proprietary interest category: E. Reprint requests: Arlene Ciuon, Allergan Pharmaceuticals, 2525 Dupont Drive, P.O. Hox 19534, Irvine, CA MATERIALS AND METHODS Concanavalin A Intravitreal Injection Four adult female New Zealand Albino rabbits weighing approximately 4-5 kg were anesthetized with Investigative Ophthalmology & Visual Science, May 1993, Vol. 34, No. 6 Copyright Association for Research in Vision and Ophthalmology

2 Lens Regeneration After Cataract Extraction 2125 cc intramuscular injection of mixture of 5 mg/kg xylazine base (Haver, Shawnee, KS) and 50 mg/kg ketamine HC1 (Aveco, Fort Dodge, IA) combined with sterile water. A wire lid speculum was inserted in the interpalpebral fissure and intravitreal injections were placed approximately 2 mm posterior to the corneoscleral limbus in the superotemporal quadrant, using a 30 gauge needle. One eye of each rabbit received a JU.1 injection of Con A (Sigma Chemical Co., St. Louis, MO; ICN Biochemicals, Cleveland, OH) and the fellow eyes received an equal volume of balanced salt solution (BSS, Allergan Medical Optics, Irvine, CA). Animals were examined with slit lamp biomicroscopy at days 2 and 8, and months 1, 5, and 7. Slit lamp photography was performed at months 1, 5, and 7. Endocapsular Phacoemulsification Bilateral lens extraction was performed by endocapsular phacoemulsification on the four adult female New Zealand albino rabbits that had developed cataracts in the Con A-treated eye and had a normal lens in the BSS-treated eye Rabbits were anesthetized with approximately 5 mg/kg xylazine base and 50 mg/kg ketamine HC1, intramuscularly. The surgical eye was dilated with 1% cyclopentolate (Alcon, Fort Worth, TX) and 10% phenylephrine (Winthrop, New York, NY); eyelashes were trimmed; and the ocular area was disinfected with povidone iodine (Professional Disposables, Inc., Orangeburg, NY). A wire lid speculum was inserted to retract the lids, and a limbal incision was made at the 11:00 position with a 2.85 mm keratome. A 21-gauge phacoemulsification tip was inserted through the corneal wound and used to perform the anterior capsulotomy and removal of the lens nucleus with BSS as the irrigant. Care was taken to remove all lens cortical material by irrigation and aspiration. At the completion of the procedure, the corneal incision was closed with three interrupted 10-0 nylon sutures. At the end of surgery, 0.25 ml (20 mg) of gentamicin (Solo Pak, Franklin Park, IL) was injected subconjunctivally, and polymyxin B bacitracin-neomycin ointment (Pharmaderm, Melville, NY) was applied topically. Postoperatively, all surgical eyes were treated topically with 1% tropicamide (Alcon, Humacao, Puerto Rico) and 0.3% gentamicin (Allergan, Irvine, CA), four times each day for 7 days. Animals were treated in accordance with USDA guidelines and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Slit Lamp Biomicroscopy and Photography The animals were evaluated postoperatively with slit lamp biomicroscopy and photography at regular intervals: weeks 3, 4, and 6 and at months 3, 4, 5, 6, and 7. Each time the lens regeneration that filled the capsular bag was photographed and measured with the pupil maximally dilated. Lens regeneration proceeds from the periphery of the capsular bag to the center and full growth (100%) was noted when the capsular bag appeared full when viewed frontally in the maximally dilated eye. This was established by observing animals at the slit lamp and later by evaluating slit lamp photographs at the end of the study period. Photographs were taken with a Nikon camera (Nikon, Tokyo, Japan) attached to a Nikon slit lamp using Kodak Ektachrome 100 film rated at ISO 100/21 (Eastman Kodak, Rochester, NY). Digital Image Analysis The slit lamp photographs were evaluated with the aid of a Macintosh II CX computer with 32-bit Quickdraw (Apple Computer, Cupertino, CA). A charge-coupled device camera (Sony XC-77) with a 55-mm Micro-Nikkor lens (f 2.8) was used to create the photographic slides. The images were captured digitally and stored on the computer. All image analysis was conducted with two programs (Enhance version and Image version 1.22y Micro Frontier Inc., Des Moines, IA) as previously described. 13 Briefly, the earliest image in the series for each animal was designated as the baseline image. All other images of the eye for that animal were aligned geometrically with the baseline image by the computer. Geometric alignment involved image rotation, size scaling, and translational (lateral) movements. The region of lens regrowth in each image was traced with a program drawing tool and the computer calculated the area within the boundaries of the tracing. The computer measurements were given in arbitrary units. The units from each measurement from the different images in a sequence were followed and recorded over time. Measurement results in units were then calculated into percents using zero as the baseline. For each rabbit, the largest or oldest growth time measurements were scaled to 100% or maximal fill (subtracting area of capsulotomy adhesion and not necessarily equivalent to 100% filling of the capsular bag). All results were graphed individually and displayed as a mean value with standard error bars. These results then determined the amount of lens regeneration in the capsule bag. Lens Weights After the rabbits were lethally injected with Eutha-6 (Western Medical Supply Company, Inc., Arcadia, CA), eyes were enucleated and the lenses removed using a posterior instrument approach. Careful dissection was used to remove all vitreous and fibrin. The lenses were then weighed. Two lenses, one from the Con A test group and one from the control group, were not weighed. These lenses were reserved for future in vitro studies.

3 2126 Investigative Ophthalmology & Visual Science, May 1993, Vol. 34, No. 6 FIGURE 1. Localized posterior subcapsular opacity due to needle perforation at 5 mo after intravitreal BSS injection. FIGURE 3. Nuclear and cortical cataract at 7 mo after intravitreal Con A injection. FIGURE 2. Granular/vacular posterior subcapsular opacity at 5 mo after intravitreal Con A injection. FIGURE 4. Translucent lens capsule with a few areas of adhesion of anterior and posterior capsule at 3 weeks after endocapsular cataract extraction. RESULTS subcapsular opacity, which progressed to a dense opacity at 5 mo (Figure 2). At 7 mo, one eye had progressed to full cortical and nuclear cataract (Figure 3). The other three eyes had dense granular/ vacuolar posterior subcapsular cataracts. Cataract Development All eyes that received BSS injection were normal on slit lamp biomicroscopy without evidence of inflammation throughout the 7-mo observation period. One eye showed a localized posterior subcapsular lens opacity at the site of needle perforation during the intravitreal injection. This lens opacity was first noted 2 days postoperatively and remained localized through the 7-mo examination (Figure 1). This eye was excluded from subsequent lens regeneration analysis. All eyes that received Con A intravitreally showed moderate cells and fibrin in the anterior chamber and on the anterior lens capsule at 2 days postoperatively. By day 8, cells were still present in the anterior chamber and there was a grainy/lacy pattern of cells and fibrin on the posterior surface of the posterior lens capsule. At 1 mo all eyes had a grainy posterior Lens Regeneration During the initial postoperative period (days 1-7), all surgical eyes had mild to moderate fibrin in the anterior chamber. By 3 weeks after surgery, all eyes were without evidence of inflammation and the lens capsular bag was relatively translucent with a few folds (Figure 4). Lens regeneration was first noted in the BSS normal lens group at 3 weeks and the Con A cataract group at 6 weeks. One eye from the Con A group exhibited posterior synechiae and pupil constriction

4 2127 Lens Regeneration After Cataract Extraction FIGURE 5. SevenLy-five percent lens regrowth in BSS group 3 mo postoperatively. FIGURE 7. Full lens regrowth in frontal plane in BSS group 7 mo postoperative!)'. FIGURE 6. Forty-seven percent lens regrowih in Con A group 3 mo postoperatively. FIGURE 8. Full lens regrowth in frontal plane in Con A group at 7 mo postoperatively. for the duration of the study, making it difficult to evaluate lens regrowth. By the 3-month postoperative examination, lens regrowth filled 74.5% of the capsule bag in the BSS normal lens group (Figure 5) and 46.6% in the Con A cataract group (Figure 6). At 6-7 mo, maximal fill of the lens capsule bag in the frontal plane was noted (Figures 7, 8, 9). Continued lens regrowth appeared in an anteroposterior direction, which was not measured in the present study. No difference in the biomicroscopic appearance of the regenerated lenses after normal vs Con A cataract removal was noted. All eyes had anteroposterior capsule adhesions, most often at the capsulotomy site. The regenerated lenses of both groups were donut shaped caused by the adhesions, and had variable translucency caused by irregular fiber alignment and presence of vacuoles. Lens Regrowth (Digital Image Analysis) FIGURE 9. Lens regrowth measured by digital image analysis. Lens regeneration in Con A cataract group was slower than in BSS control group.

5 2128 Investigative Ophthalmology & Visual Science, May 1993, Vol. 34, No. 6 After the 7-mo postoperative examination, the rabbits were killed. Gross dissection showed the lenses to be relatively intact, fairly translucent, and donutshaped. Lens regrowth had filled the lens capsule bag except at the anterior capsulotomy site adhesion to the posterior capsule. The Con A eye that had shown posterior synechiae for the duration of the study exhibited inflammation in the posterior chamber. This eye had fibrin attached to the posterior lens surface, and the capsular bag contained approximately 30% lens regrowth. The Con A eye that displayed a vitreous opacity at 6 mo also displayed fibrin in the vitreous upon dissection. The fibrin peeled easily from the back of the capsule. The lenses were dissected, free of vitreous and fibrin attachments, and then weighed. The mean lens weight in the BSS normal regenerated group (0.24 ± 0.02 g) was greater than the Con A cataract group (0.14 ± 0.09). It was noted that the two lenses in the Con A group that had fibrin in the vitreous (0.06 and 0.07g) weighed significantly less than the one remaining lens (0.27g). DISCUSSION The lens, like all ectodermal tissue, continues to grow throughout life and after injury the lenticular epithelium naturally tends to regenerate itself. Thus, after extracapsular cataract extraction with removal of the anterior capsule the residual lens epithelium proliferates across the posterior capsule contributing to posterior capsule opacification. 14 If the anterior capsule is left relatively intact, as in endocapsular cataract extraction, limited lens regeneration may occur. 15 Most of the investigative work to date has entailed the removal of normal lenses in the study of lens regeneration. The only report of lens regeneration after cataract removal was the work of Stenhouse Stewart in a radiation cataract model. 7 He irradiated one eye of mature rabbits with 14 Gy and 6 mo later noted the formation of multiple small vacuoles in a radial pattern in the posterior cortex, which barely interfered with lens transparency. Seven mo after irradiation he removed the lens capsule contents and noted poor transparency in the regenerated lenses. No further information is given on the nature of these regenerated lenses. In the present study, the regenerated lenses of the cataract group were similar to the control normal lens group in transparency. The regenerated lenses are fairly translucent but because of abnormalities in the rate of regrowth in different parts of the capsule bag these lenses are not optically clear and irregularities in structure exist. In addition, the lenses have varying degrees of vacuolization and some areas of opacification. In this small sample no differences in structure and translucency of the regenerated lenses in the normal versus cataract lens group was visible by slit lamp biomicroscopy. However, the regenerated lenses were noted to be smaller in the Con A cataract eyes with more extensive capsular adhesions. More extensive studies may be helpful in understanding any qualitative differences in the regenerative capacity of the lens in normal versus cataract removal. Previous studies on lens regeneration in New Zealand albino rabbits have shown that lens regrowth begins as early as 2 weeks and fills the capsular bag at approximately three months after extraction of normal lenses in juvenile rabbits. In the present study of adult rabbits, lens regrowth was first noted at 3 weeks in the normal lens group and 6 weeks in the Con A cataract group. In the latter group, less lens material was regenerated and at a slower rate than in eyes with extraction of a normal lens. These eyes appeared to have more adhesion of the anterior and posterior capsules which may be related to the induced inflammation. In summary, lens regeneration was shown to occur after endocapsular extraction of posterior subcapsular cataracts induced by intravitreal injection of Concanavalin A. The regenerated lenses weighed less than lenses regenerated after normal lens extraction. Further studies of lens regeneration using other cataract models may be of value in understanding its potential in the treatment of pediatric cataract. Key Words lens regeneration, conconavalin A, posterior subcapsular cataract, uveitic cataract, endocapsular cataract extraction Acknowledgments The results of this paper were presented at ARVO in 1990 in a poster entitled "Lens Regeneration in NZA Rabbits Following Endocapsular Phacoemulsification to Remove Cataracts Induced by Intravitreal Injection with Concanavalin A." ( ) Digital image analysis data on adult normal lens group was also reported in prior publication: Gwon AE, Jones RL, Gruber LJ, Mantras C: Lens Regeneration in Rabbits Measured by Image Analysis. Invest Ophthalmol Vis Sci 33: , June References 1. Johnson DA, Parks MM. Cataracts in childhood: prognosis and complications. Seminars in Ophthalmology. 1991;6: Cocteau LL. Reproduction du crystallin. Journal de Physiologie Experimental et Pathologique. 1827; 1: Mayer. Ueber die reproduction der krystallinse./oiwnal der Chirurgie und Augenheilkunde (Berlin, von Graefe und Walther) 1832;17: Milliot B. De la regeneration du crystallin chez quel-

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