Concanavalin A-Induced Posterior Subcapsular Cataract: A New Model of Cataractogenesis

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1 Articles Concanavalin A-Induced Posterior Subcapsular Cataract: A New Model of Cataractogenesis Arlene Gwon,*~f Christine Mantras,* Lawrence Gruber* and Crystal Cunanan* Purpose. To evaluate the effect of Concanavalin A (Con A) on cataract formation in New Zealand Albino rabbits. Uveitis is a chronic inflammatory condition of the eye involving the anterior and/or posterior segments. It may be acute or chronic and is associated with the development of posterior subscapular cataract over time. Con A is a nonspecific inflammatory agent and mitogen for T cells and some B cells. Used extensively in immunogenic studies Con A has been shown to induce uveitis after intravitreal injection in New Zealand Albino rabbits. Methods. In two separate studies, Con A was injected intracamerally or intravitreally into one eye of 12 New Zealand Albino rabbits and an equal volume of balanced salt solution was injected into the opposite eye as a control. In a third study, the effect of topical steroids after intravitreal injection of Con A was evaluated. In all studies, anterior and posterior inflammation and the development of cataract was monitored by slit lamp biomicroscopy and photography. Cataract formation was also studied histopathologically. Results. Initially, all eyes treated with Con A demonstrated moderate anterior chamber inflammation while eyes treated with balanced salt solution showed no inflammation. Three months after treatment, posterior subcapsular cataracts were present in all rabbit eyes treated with intravitreal Con A. In the third study, topical steroid treatment of Con A-induced inflammation significantly reduced anterior chamber inflammation but had no effect on vitreous humor and posterior subcapsular cataract formation. Conclusion. This experimental model was the first to demonstrate the development of posterior subcapsular cataracts after Con-A induced inflammation. The cataract was clinically and histologically similar to human posterior subscapular cataracts. Invest Ophthalmol Vis Sci. 1993; 34: IL osterior subcapsular (PSC) cataract is a type of presenile and senile opacification of the human lens occurring in 6% of adults between the ages of 43 and 84 years in the Beaver Dam Eye Study. 1 It may be caused by a variety of conditions and toxins and has been referred to as "cataracta complicata." It is known to occur in inflammation, widespread degenerative states, and when ocular circulation is gravely impaired. Such cataracts are presumably due to abnormal lens metabolism and associated with diffusion into the lens From *Allergan Pharmaceuticals and the ^University of California at Irvine, Irvine, California. The results of this paper were presented in part at ARVO, 1992 in a poster titled "Concanavalin A-Induced Posterior Subcapsular Cataract: A New Model of Cataractogenesis" ( ). Submitted for publication: December 11, 1992; accepted May 27, Proprietary interest category: E. Reprint requests: Arlene Gwon, Allergan Inc., 2525 Dupont Drive, P.O. Box 19534, Irvine, CA of toxins from an inflammatory focus, exogenous drugs or from products of degeneration caused by disease. The earliest clinical changes are typically seen in the central or axial region of the posterior lens and thus decreases vision early in its course. 23 The lectin Concanavalin A (Con A) is a nonspecific inflammatory agent and mitogen for T cells and some B cells. It has been used extensively in immunogenic studies and shown to induce uveitis after intravitreal injection in New Zealand albino rabbits. 4 " 6 Because of the prolonged nature of the inflammatory response with Con A seen in animal studies, it was a good candidate for study of the development of complications, such as cataract formation in uveitis. In the current study, we investigated the role of Con A-induced inflammation in the formation of PSC cataracts. Intravitreal injection of Con A was associated with anterior and posterior uveitis and cataract formation, whereas intracameral injection was asso- Investigative Ophthalmology & Visual Science, December 1993, Vol. 34, No. 13 Copyright Association for Research in Vision and Ophthalmology 3483

2 3484 Investigative Ophthalmology & Visual Science, December 1993, Vol. 34, No. 13 ciated with acute mild anterior uveitis and no cataract development. We also evaluated the role of topical corticosteroids in decreasing the Con A-induced inflammation and subsequent PSC cataract formation. Whereas anterior uveitis was significantly less in the steroid treated group, no difference was noted in the severity of posterior uveitis and the" development of PSC cataract. MATERIALS AND METHODS Intracameral Injections Topical anesthesia with.5% proparacaine solution (Allergan, Irvine, CA) was applied to both eyes of six juvenile female New Zealand Albino rabbits weighing 2.5 to 3 kg. Approximately 1 ml Con A at 1 mg/ml (total dose of 1 ug) was injected into the anterior chamber of one eye of each rabbit and the contralateral eye received an equal volume of balanced salt solution (without prior paracentesis). After the injections, the animals received one drop each of 1 % tropicamide (Alcon, Humacao, Puerto Rico) and.3% gentamicin (Solo Pak, Franklin Park, IL), four times daily for 7 days. Intravitreal Injections Eighteen juvenile female New Zealand albino rabbits weighing approximately 2.5 to 3 kg were anesthetized with a 2 to 3 ml intramuscular injection of a 1:5 mixture of 1 mg/ml xylazine base (Haver, Shawnee, KS) and 5 mg/ml ketamine HC1 (Aveco, Fort Dodge, IA) combined with sterile water. The eyelashes were trimmed and the fur surrounding the eye was prepped with povidone iodine (Professional Disposables, Inc., Orangeburg, NY). A lid speculum was inserted and intravitreal injections were placed at approximately 2 to 3 mm posterior to the corneoscleral limbus in the superotemporal quadrant, using a 3-gauge needle. Group 1. Six rabbits received a 1 ml injection of Con A at 1 mg/ml (Sigma Chemical Co., St. Louis, MO and ICN Biochemicals, Cleveland, Ohio), yielding a total dose of 1 /tg in one eye. The fellow eye received an injection of equal volume of balanced salt solution (Allergan Medical Optics, Irvine, CA). Group 2. Twelve rabbits received intravitreal Con A. Postoperatively, six of these rabbits received 1% Pred Forte (Allergan, Irvine, CA) four times daily in the test eye for 3 weeks. Postoperatively, all test eyes received 1% tropicamide (Alcon, Humacao, Puerto Rico) and 1% phenylepherine (Winthrop, New York, NY) four times daily to each eye for 2 weeks to maintain dilation. Slit Lamp Biomicroscopy/Photography All eyes were examined with slit lamp biomicroscopy at least biweekly for 1 month, weekly for 2 months, and monthly thereafter. Slit lamp photography was performed at months 2, 3, and 4. Biomicroscopy findings were graded on a scale from to 4 with = none, 1 = trace, 2 = mild, 3 = moderate, and 4 = severe (Table 1). Intergroup comparisons used the exact P value derived from the Wilcoxon Rank Test. Histopathology Rabbit eyes were fixed in 1% neutral buffered formalin. After washing the eyes in tap water, the globe was sectioned from the central cornea through the pupil to the optic nerve with the lens in situ. Tissue was processed in an automatic tissue processor overnight. The tissue was then dehydrated in reagent grade alcohol, cleared with xylene and infiltrated with paraffin. Paraffin-embedded tissues were sectioned at 5 nm and stained with hematoxylin and eosin. All animals were handled in accordance with USDA guidelines and the ARVO Resolution on the Use of Animals in Research. RESULTS Biomicroscopy Intracameral Injections. Four eyes that received balanced salt solution intracamerally had no evidence of irritation or inflammation at any time during the 6- week observation period. Two eyes had minor irritation, which resolved by day 4 and remained clear through the 6-week observation period: one eye showed mild anterior chamber cells, which resolved by TABLE l. Anterior Chamber Inflammation Grading Scale Cells None Trace Mild Moderate Severe Flare None Trace Mild Moderate Severe No cells seen per high power field 1-9 cells seen per high power field 1-25 cells seen per high power field 26-5 cells seen per high power field Too many cells to count per high power field No Tyndall effect Tyndall beam in the anterior chamber has mild intensity Tyndall beam in the anterior chamber has strong intensity Tyndall beam is very intense, aqueous has white, milky appearance Tyndall beam has marked intensity, fibrin fills anterior chamber and obscures view of the pupil

3 Posterior Subcapsular Cataract Model 3485 day 4; one eye that had a slight iris nick during injection had some fibrin on the iris at 24 hours, which resolved by day 4. All eyes receiving intracameral injection of Con A had slight irritation and pupil miosis immediately after injection. At 24 hours, there was mild to moderate cells and fibrin in the anterior chamber. Two eyes also had moderate corneal edema and haze. These eyes were treated with 1% prednisolone acetate (Allergan, Irvine, CA), one drop four times a day for 5 days. By 1 week postinjection, all inflammatory signs had resolved and all eyes remained normal until the animals were killed at 6 weeks. Intravitreal Injections. Inflammation. At day one, all eyes receiving balanced salt solution were normal without evidence of inflammation in the anterior or posterior segment and remained normal throughout the 6-month observation period. All eyes that received Con A intravitreally had fibrin in the vitreous humor and a few had a preretinal hemorrhage on indirect ophthalmoscopy at day 1. The anterior segments of these eyes were normal (Table 2). By day 3, there was evidence of inflammation in all Con A-treated eyes with moderate cells and fibrin in the anterior chamber, on the anterior lens capsule and in the vitreous humor. Anterior segment inflammation gradually subsided by 4 to 7 days while posterior segment inflammation increased with moderate cells and fibrin noted in the vitreous and on the posterior surface of the posterior lens capsule. Inflammation persisted through day 9 in the steroid group and day 16 in the nonsteroid group. Anterior chamber cells were significantly less in the steroidtreated group at all times except days 3, 7, and 9 (Table 2). Anterior chamber flare and fibrin was minimal in both groups throughout the evaluation period, and significantly less in the steroid group at day 9 only (Table 3). Vitreous cells were moderate in both groups TABLE 2. Anterior Days Intravitreal Concanavalin A: Chamber Cells Steroids (n = 6).16 ±.15.8 ± ±.19.3 ± ±.3 No Steroids (n = 12) 1.41 ± ± ± ± ± ± ±.14 P Value Values are mean ± SE. Biomicroscopy findings are graded on a scale from to 4; = none, 1 = trace, 2 = mild, 3 = moderate, and 4 = severe. TABLE 3. Intravitreal Concanavalin A: Anterior Chamber Flare/Fibrin Days Steroids (n = 6).5 ±.2.16 ±.15 No Steroids (n = 12).16 ± ± ± ± ±.3.16 ±.11 P Value Values are mean ± SE. Biomicroscopy findings are graded on a scale from to 4; = none, 1 = trace, 2 = mild, 3 = moderate, and 4 = severe. and not significantly different (Table 4). After the seventh week it was difficult to evaluate vitreous inflammation because of the development of cataracts. Cataract development. The lenses of steroid and nonsteroid Con A groups remained clear until 2 weeks when a grainy, lacy pattern of cells and fibrin were noted on the posterior capsule surface (Fig. 1). As early as 5 weeks, vacuoles were noted in the posterior subcapsular lens area in 3 of 6 steroid treated eyes and 11 of 12 nontreated eyes. By 7 weeks, a posterior subcapsular cataract was noted in 3 of 6 steroid eyes and all of the nontreated eyes. By 3 months postintravitreal Con A, PSC cataracts were present in all eyes and were granular/vacuolar in appearance (Fig. 2). In the six steroid-treated eyes, the PSC opacities were localized in the central optical axis TABLE 4. Intravitreal Concanavalin A: Vitreous Cells Days 1 2 Q O W5 W6 W7 Steroids (n = 6) ± ± ± ± ± ± ± ± ± ±.24 No Steroids (n = 12).42 ±.14 o F.Q + n 14 U.JO T" U.I i.83 ± ±.3 3. ± ± ± ± ± ± ± ±.25 P Value > > >.999 Values are mean ± SE. Biomicroscopy findings are graded on a scale from to 4: = none, 1 = trace, 2 = mild, 3 = moderate, and 4 = severe.

4 3486 Investigative Ophthalmology 8c Visual Science, December 1993, Vol. 34, No. 13 FIGURE 1. Slit lamp photograph. A lacy pattern of cells and fibrin is noted on the posterior surface of the lens in intravitreal Con A-treated eye at 2 weeks. or diffuse involving 2% to 4% (n = 3) or 8% to 1% (n = 3) of the posterior circumference, respectively. In the 12 nonsteroid eyes, PSC opacity was localized in 5, diffuse in 3, and mixed with anterior and posterior cortical opacity in 4. By 16 weeks, four eyes, two in each group, had developed mature cortical and nuclear cataracts. In the remaining eyes, the PSC cataracts appeared stable and nonprogressive. No cataracts were noted in any of the balanced salt solution-treated eyes at any time. Histopathology At 2 weeks, the lens capsule and anterior epithelium appeared intact. Small vacuoles were seen in the epithelial layer that were probably related to a fixation artifact. Lens fibers appeared normal for the most 2. Slit lamp photograph. A globular, edematous posterior subcapsular opacity is noted in intravitreal Con Atreated eye at 11 weeks. FIGURE FIGURE 3. Photomicrograph of intravitreal Con A-treated eye at 14 months. A monolayer of epithelial cells lines the anterior capsule. Incomplete cell differentiation is noted in the equator with nuclei displaced toward the posterior lens. (Bar = 5 //.) part, staining more deeply in the nuclear region and paler in the cortical area. There was an occasional separation of the cortical fibers from the posterior capsule. Adjacent cortical fibers appeared foamy or swollen and separation between fibers was somewhat prominent. Occasional vacuoles were seen. The lens nucleus appeared normal and cell differentiation in the equatorial region appeared unremarkable. The vitreous humor contained numerous inflammatory cells and fibrin strands. At 3 months, the lens capsule appeared intact. A semicontinuous monolayer of epithelium extended FIGURE 4. Photomicrograph of intravitreal Con A-treated eye at 3 months. A multilayer of large, rounded bladder-type cells of Wedl is seen on the posterior capsule. Adjacent corticalfibersare swollen or globular and there is loss of normal architecture in the posterior cortex. Multiple inflammatory cells and afibrovascularmembrane are noted in the vitreous humor. (Bar = 5 M-)

5 Posterior Subcapsular Cataract Model 3487 along both the anterior and posterior capsule with loss of the equatorial bow region. Lens epithelial cell differentiation was incomplete with nuclei displaced toward the posterior lens (Fig. 3). The anterior epithelial layer contained vacuoles and there were some areas of cell loss. Cells along the posterior capsule were larger and more rounded in appearance, the so-called "bladder" cells of Wedl. Adjacent cortical fibers were swollen or globular (Fig. 4). In other areas, there was loss of architecture and the cortex was amorphous with an occasional cell nucleus noted in the more central cortical regions. The vitreous humor contained numerous inflammatory cells and afibrovascular membrane. Similar changes were noted in the micrographs examined at 4, 5, and 14 months. DISCUSSION Posterior subcapsular cataracts can occur spontaneously in the aging Wistar rat, 7 can occur after the intravitreal injection of docosahexenoic acid 8 and bacterial endotoxin 9 and can occur after microwave and ionizing radiation exposure. 911 In the current studies, we describe the occurrence of PSC cataracts after Con A-induced inflammation. We have shown that intravitreal injection of the lectin Con A will induce chronic inflammation followed by development of posterior subcapsular cataract in New Zealand Albino rabbits. The prolonged Con A-induced inflammation does not occur when the compound is injected intracamerally. Although slight inflammation did occur for a few days after intracameral injection, chronic inflammation and cataract formation was not noted. It is possible that the injected Con A was washed out through the trabecular meshwork too rapidly to induce a chronic inflammation and cataract. In contrast, intravitreal injection of Con A induced a chronic anterior and posterior uveitis with exacerbations and remissions for up to 2 months. The inflammation is similar to some types of human uveitis in its clinical course of exacerbations and remissions, inflammatory signs of keratoprecipitates, anterior chamber cells, posterior synechiae formation, vitreous cells, and cataract formation. Treatment with topical corticosteroids resulted in less severe anterior uveitis but had minimal effect on the posterior uveitis. PSC cataract formation appeared to be less severe in the steroid group but differences were small and further studies are needed for verification. In humans, both uveitis and steroid therapy are known to induce PSC cataract independently, so it would be of interest to learn if higher doses of steroid had an inhibitory effect in this model. The clinical and morphologic changes in PSC cataract in humans and radiation-induced PSC in mice, rats, and the bullfrog have been well described."" 17 Clinically, the posterior subcapsular cataract seen at 3 months after intravitreal injection was similar in clinical appearance to the vacuolar or lacy type of cataract described by Eshagian. 2 These cataracts are granular and appear to be made of multiple watery cysts. They are seen in senile or age-related, diabetic, retinitis pigmentosa, and corticosteroid cataracts. 2 The Con A-inflammatory PSC cataract is histologically similar to that reported in humans 212 " 14 and in radiation-induced PSC cataract in animals " 17 Cell differentiation in the equatorial region is incomplete with displacement of the lens bow nuclei toward the posterior lens pole. Cortical fibers appear irregular in size and shape. There is migration of cells along the posterior capsule. These posterior cells are swollen, "bladder" type cells of Wedl and there is swelling in the adjacent cortical fibers, with areas of liquefaction and loss of fiber architecture. It is also noteworthy the Con A-induced inflammation remained relatively inactive after 2 months and the posterior subcapsular cataract progressed very little from 3 to 14 months in most cases. These results can be interpreted several ways: the cataract progression may be dependent on an active inflammatory stimulus; cataract progression may require the continued presence of Con A; or the Con A is somehow removed or inactivated over time. In addition to being a nonspecific inflammatory agent and a mitogen for T cells, Con A is widely used as a probe for studying cell-surface oligosaccharides Suzuki et al demonstrated the binding of Con A to the lens epithelial cells and lens fibers with nuclei. It is possible that one cause of the cataract formation in this model is disruption of cell-cell contacts by the binding of the Con A to the N-acetyl-glucosamine residues in the cell membranes. This aberrant binding could lead to cataract formation early on, but with time the cells may internalize the Con A, effectively returning the cell surface to its original state, and thus halting the progression of the cataract. Whether Con A is binding to the N-acetyl-glucosamine residues in this model has not been determined. Nor does there exist any literature describing the use of lectins such as Con A to alter cell-cell contacts in any animal model. Finally, there have been no studies of cell membrane cycling of bound lectin molecules, although such a precedence exists for growth factor receptors. Further studies are needed to discern among these possible mechanisms of action in the formation of Con A-induced cataractogenesis. In summary, we have developed a model of Con A-induced inflammatory posterior subcapsular cataract that has many characteristics desirable in a model, including a cataract development rate that would al-

6 3488 Investigative Ophthalmology & Visual Science, December 1993, Vol. 34, No. 13 low this to be a good assay system for drugs and other factors affecting cataractogenesis. Key Words lens opacity/cataract, Concanavalin A, posterior subcapsular cataract, steroids Acknowledgments The authors thank John Conlon, PhD, for statistical assistance. References 1. Klein BEK, Klein R, Linton K. Prevalence of age-related lens opacities in a population: the Beaver Darn Eye Study. Ophthalmology 1992;99: Eshagian J. Human Posterior Subcapsular Cataracts. Trans Ophthal Soc UK. 1982; 12: Luntz MH. Clinical types of cataract. In: Datiles MB, Kinoshita JH. Duane's Clinical Ophthalmology. JB Lippincott: Philadelphia; 1992: Pirie A, Van Heyningen R. Biochemistry of the Eye. Blackwell Scientific: Oxford; 1956: Shier WT, Trotter JT, Reading CL. Inflammation induced by Concanavalin A and other lectins. Proc Soc ExpBiolMed. 1974;146: Hall JM, PribnowJF. Effect of Concanavalin A on ocular immune responses. Journal of the Reticuloendothelial Society. 1977;21: Gorthy WC. Cataracts in the aging rat lens. Ophthal Res. 1977;9: Goosey JD, Tuan WM, Garcia CA. A lipid peroxidative mechanism for posterior subcapsular cataract formation in the rabbit: a possible model for cataract formation in tapetoretinal diseases. Invest Ophthalmol Vis Sci 1984;25: Worgul BV, Merriam GR. The role of inflammation in radiation cataractogenesis. Exp Eye Res 1981;33: Lipman RM, Tripathi BJ, Tripathi RC. Cataracts induced by microwave and ionizing radiation. Surv Ophthalmol 1988;33: Holsclaw DS, Merriam GR, Medvedovsky C, Rothstein H, Worgul BV. Stationary radiation cataracts: an animal model. Exp Eye Res 1989;48: Eshaghian J, Streeten B. Human posterior subcapsular cataract. Arch Ophthal. 198;98: Nagata M, Matsuura H, Yutaka F: Ultrastructure of posterior subcapsular cataract in human lens. Ophthalmic Res. 1986;18: Greiner JV, Chylack LT. Posterior subcapsular cataracts. Arch Ophthal. 1979;97: Yang VC, Ainsworth EJ. A histological study on the cataractogenic effects of heavy charged particles. Proc Natl Sci Counc B Roc. 1987; 11: Palva M, Palkama A. Ultrastructural lens changes in x-ray induced cataract of the rat. Ada Ophthalmologica. 1978;56: Zintz C, Beebe DC: Morphological and cell volume changes in the rat lens during formation of radiation cataracts. Exp Eye Res 1986;42: Suzuki T. Lectin binding pattern of the normal rat lens. Ada Soc Ophthalmol Jpn. 199;93: Panjawani N, Moulton P, AlroyJ, Baum J: Localization of lectin binding sites in human, cat and rabbit cornea. Invest Ophthalmol Vis Sci. 1986;27: