A C T A O P H T H A L M O L O G I C A VOL. 5 7 1 9 7 9

Department of Ophthalmology (Head: David Miller), and Department of Neurolom (Head: Norman Getrhwind),

Beth Irrael Hotpatal and Hanrard Medical Srhool, Botton, Mast, USA

IN VlTRO PRODUCTION OF STEROID CATARACT IN BOVINE LENS

Part I: Measurement of optical changes

BY

DAVID MILLER, M. L. TlJERlNA and CHAIM MAYMAN

Fresh calf lenses were incubated in nutritive media, to which was added ouabain and dexamethasone phosphate at concentrations of 1 x 10-4 M. At the end of a three day incubation period, cortical opacification developed in the control series of lenses as well as those lenses incubated in both ouabain and dexamethasone phosphate. Using a light transmission device which quantitates lens opacification, it was noted that dexamethasone produced a level of cortical opacification significantly greater than that of the control series. Ouabain produced a level of cortical opacification statistically identical to that produced by the dexamethasone. It is suggested that the aforementioned light trans- mission device is an accurate and reproducible method of quantitating cataractous opacification.

Kqv word\: steroid cataract - in i~itro cataract -cataract quantification - ouabain cataract -cataract turbiditv.

Research on cataracts has been conducted by scientists representing medicine, biochemistry and physics. Cdllecting and comparing the results of research from these disciplines is important if cross fertilization of ideas is to take place. To this end, we feel that standardizing the measurement of the severity of the cataractous process is an important first step in encouraging communicaton between re- searchers.

In this paper, we intend to (a) describe a simple and precise method for measuring experimental cataracts and (b) use the technique in assessing the severity of cataracts produced by ouabain (Bonting et al. 1963; Bonting 1965; Palva 8c

Received March 27. 1979.

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Palkama 1974) and dexamethasone (Wood et al. 1967; Bettman et al. 968; Cremer-Bartels et al. 1968; Ono et al. 1974; Friedburg et al. 1974; VanVenrooij et al. 1974; Mayman et al. 1979).

Most cataracts interfere with light transmission by an increase in both forward and backward light scattering. The forward light scattering tends to wash out the contrast of the retinal image. As the cataract progresses, the lens becomes white or more opaque, which implies an increase in backscatter, and a consequent decrease in light transmission (Miller & Benedek 1973).

The method to be described takes advantage of the fact that as the cataract progresses, backscattering and forward scattering progress, and thus, a smaller percentage of a narrow beam of transmitted light arrives at an equally small sensor.

Methods

Calf eyes were placed on ice at a local abattoir, and were delivered to the laboratory within one to two hours of the death of the animal. The lenses were removed atraumatically from the enucleated globe, and incubated in 20 ml of incubation media in a 60 X 15 mm plastic Petri dish (Falcon Plastics, Oxnard, Calif.) in a water jacketed incubator (National Appliance Comp., Model 3321, Portland, Ore.), containing an atmosphere of 5% C042, 95% air and 100% humidity. The incubation medium was based on a medium devised by Kinoshita & Obazawa (unpublished) and contained (per 100 ml): TC-199 (without glucose or phenol red, Grand Island Biological Company, Grand Island, N.Y.): 64.0 ml; bicorbonate buffer, 25.6 ml (containing in grams per liter NaHC03, 7.650; KHC03, 0.368; NaCI, 0.7431; KCI, 0.08533); distilled water, 10.4 ml; glucose, 216.4 mg (12 mM); fructose, 540.48 mg (30 mM); glutamine, 3.652 mg (0.25 Mm); CaCIz. 2Hz0.24.54 mg (10 mg/100 ml Ca++); fetal calf serum, (GIBCO No. 614), 5.0 ml; penicillin G (50000 U per ml) 0.02 ml; streptomycin (5.0 Gm per 9.0 ml), 0.025 ml. The medium was then gassed with 5 per cent COz -95 per cent air for 30 min at 27°C. The final osmolality of the medium was 290 f 2mOsm per liter.

In this medium, which will be called the control medium, were placed 28 lenses. A second group of 27 lenses was placed in the control medium, to which was added the disodium salt of dexamethasone-2 I-P, in a final concentration of 1 X lO-4M. All lenses were incubated for 72 h a t 37.0"C.

At the end of 72 h, the lenses were removed from the incubator and the amouqt of light transmitted through each lens was determined.

The device for light transmission measurement is shown in Fig. 1. In essence, white light from a fiber optic bundle (3) (Applied Fiberoptics, Southbridge, Ma) is transmitted through an aluminum chamber containing the calf lens (2), and the light is measured via the fiberoptic probe of a photomultiplier (1). (Gamma Scientific, San Diego, Cat.). To insure rapid and predictable lens placement in the chamber, each lens was centered in the Petri dish (6) using a teflon doughnut (2). and the ensemble was placed in a drawer (5). The drawer was pushed into the housing, automatically insuring a precise relationship between light source, lens and photomultiplier probe.

The light transmission for each lens was recorded on the photomultiplier in arbitrary units. During each series of experiments, light transmission of a freshly dissected calf lens was also

Number I.ens type

Fig. I J

Mean light transmission (arb. units)

Standard deviation

U

Diagrammatic representation of the devicde used for measuring light transmission through a cataractous lens (1) probe from photomultiplier (2) teflon doughnut which centers lens (3) fiber optic light source (4) sleeve to hold fiber optic light source ( 5 ) drawer which houses lens

in Petri dish -doughnut ensemble (6) Peteri dish (7) legs of entire ensemble.

measured. Preliminary studies showed that light incident to the lens was scattered by all portions of the lens. Thus, the light received by the photomultiplier probe gathered a representative sample from all portions of the experimental lenses.

Results

Table I presents the light transmission data in arbitrary units for each group of differently treated lenses, along with a comment on the statistical difference between each series and the control.

Table I reveals that both dexamethasone and ouabain induce opacities o f the same degree.

Fig. 2 demonstrates the appearance of typical lenses in the control (C), ouabain (OUA), dexamethasone (DEX), and fresh (F) groups.

Both the ouabain and dexamethasone induced opacities were significantly greater than the control lens opacity.

Fig. 2. The appearance of calf lenses after 3-day incubation. Top left: fresh control lens. Top right: incubated control lens (3 days). Lower left: lens incubated in 1 x 10-4 M dexamethasone. Lower right: lens incubated in 1 X M ouabain. Whiteness signifies transparency, and

blackness signifies opacity.

In Vitro Steroid Cntrimrt I

In each case, the opacity was always located in the lens cortex, leaving the lens nucleus clear. Using the dissecting microscope set at 30 x magnification, one could see that the individual lens fibers of the cortex were opaque, as was the layer of epithelial cells just under the lens capsule. If the fresh lens were left in incubation media, plus dexamethasone, for two hours, small vacuoles were noted to form along the boundary between cortex and nucleus.

Thus, from a macroscopic point of view, the experiment seems to demonstrate the following series of events in a lens incubated in culture medium: First, there is an accumulation of vacuoles, which ultimately coalesce at the border between the cortex and nucleus. Then an opacification of the cells and fibers of the cortex develops. Finally, a peeling off of the opacified cortex takes place, leaving a clear nucleus.

Discussion

The present study follows the cortical opacification of fresh calf lenses incubated for three days in nutritive media. The process appears to be accelerated by a factor of 7 if either ouabain or dexamethasone, at a concentration of 1 x 10-4 M is added to the medium.

Since the degree of lens opacification produced by dexamethasone was identical to that produced by ouabain in our series of experiments, and since ouabain has been shown to inhibit Na+-K+ ATP’ase in the lens, the data suggest that both may act via the same mechanism, that of inhibition of Na+-K+ ATP’ase in the lens.

We also noted that use of the light transmission apparatus described in the study gave precision to the measurement of cataract opacification far beyond the 1 to 4+ system conventionally used.

Acknowledgments

T h e authors would like to thank Dr. Leo T. Chylack for his advice in organizing the study, and his constant counsel throughout the work. T h e authors would also like to thank Professor George Benedek and Dr. Jin Kinoshita for their comments and suggestions during the study.

References

Bettman J. W., Jung W. E. & Noyes P. 0. (1964) Potentiating action of prednisone on galactose cataracts in rats. Inuest. Ophthal. 3 , p. 628.

Bonting S. L. ( 1965) Na-K activated adenosine-triphosphatase and active cation transport in the lens. In: Symposium un the Lens. Harris J. E., ed. p. 347. C.V. Mosby Comp., St. I.ouis.

Bonting S. L., Caravaggio L. L. & Hawkings N. M. (1963) Studies on sodium-potassium activated adenosine-triphosphatase. VI. Its role in cation transport in the lens of cat, calf and rabbit. Arrh. Biochem. 101, p.47.

D. Millrr. M . L. Tijrr inn nnd C . Mnyninn

Kinoshita J. (1974) Mechanism initiating cataract formation. Inorrt. Ophthul. 13, p. 7 13. Kinoshita J. & Obazawa H. (unpublished data). Mayman C. I. (1972) Inhibitory effect of dexamethasone on sodium-potassium activated

adenosine triphosphatase of choroid plexus in cat and rabbit. F d . Proc. 31, p. 591 abs. Mayman C. I. (1974) Developmantal aspects of sodium-potassium activated adenosine

triphosphatase (Na+-K+ ATP'ase) in choroid plexus. Neurology (Minneap.) 24, 370-37 1. Mayman C. I., Tijerina M. L. (1976) Effect of dexamethasone on sodium-potassium activated

adenosine triphosphatase of choroid p1exus.J. Neurochem. (submitted for publication). Mayman C. I., Miller D. & Tijerina M. L. (1979) In vitro production of steroid cataract in

bovine lens. Part I1 - Measurement of sodium-potassium adenosine triphosphatase activity. Acta ophthal. (Kbh.) 57, 1107- 1 1 16.

Miller D., Benedek G. (1973) Intraocular Light Smttering. C . C . Thomas, Springfield, Ill. Palva M., Palkama A. (1974) Histochemically demonstrable sodium-potassium activated

adenosine triphosphatase (Na-K ATP'ase) activity in the rat lens. Exp. Eyr Rer. 19, p. 1 17. Tarkkanen A., Esila R. & Liesmaa M. (1966) Experimantal cataracts following long-term

administration of corticosteroids. Actn nphthal. (Kbh.) 44,665-668. Wood D. C., Contaxis I., Sweet D., Smith J. C., Jr. & VanDolah J. (1967) Response of rabbits

to corticosteroids. I. Influence on growth, intraocular pressure and lens transparency. Amer. J. Ophthal. 63,849-856.

Wood D. C., Sweet D., Contaxis L. & VanDolah J. (1967) Response of rabbits to cortico- steroids. 11. Influence of topical therapy on lens, aqueous humor, serum and urine composition. Amer. J. Ophthal. 63, 849-856.

Author'.$ address: David Miller, M. D., Department of Ophthalmology, Beth Israel Hospital, 330 Brookline Avenue, Boston, MA 02215, USA.

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