Galactose Cataract Prevention with Sorbinil, an Aldose Reductase Inhibitor: a Light Microscopic Study

Galactose cataract prevention with sorbinil, an aldose reductase inhibitor: a light microscopic study

M. Datiles, H. Fukui, T. Kuwabara, andj. H. Kinoshita

Cataract formation in galactosemic rats was studied by ophthalmoscopy, slit-lamp biomicros- copy, and by light microscopy using plastic embedding with methacrylate. Untreated rats developed nuclear cataracts by 14 days and mature cataracts by 21 days. However, rats treated with the aldose reductase inhibitor sorbinil did not develop any cataractous change for up to 8 months of 50% galactose feeding and could not be distinguished from normal controls. This strongly suggests that aldose reductase is the common factor involved in the formation of sugar cataracts. (INVEST OPHTHALMOL VIS SCI 22:174-179, 1982.)

Key words: galactosemic cataract, aldose reductase inhibitor, methacrylate plastic embedding

Aildos, e reductase (AR) has been implicated Methods as the common factor which initiates the cata- Male Sprague-Dawley albino rats weighing 50 ractous process in both diabetic and galac- gin were fed 50% galactose feed. Rats were treated tosemic rats.1 A variety of AR inhibitors with with sorbinil given by oral intubation at a dose of diverse chemical structures has been shown 60 mg/day/kg body weight. Subsequent experi- to effectively delay the onset of cataracts in ments showed that the same results were obtained diabetic and galactosemic animals.2"6 More- when the sorbinil was mixed with rat feed (425 mg/kg of food). A total of 250 rats in each of the over, the inhibitor sorbinil (d-6-fluorospiro- untreated and sorbinil-treated groups were used. chroman 4,4'imidazolidine 2',5'dione) has The eyes were examined daily with pupils di- recently been reported to essentially prevent lated by a cycloplegic-mydriatic (0.5% to 1.0% cy- cataractous changes from occurring in dia- clopentolate (Cyclogyl); Alcon Laboratories, Inc., 6 betic rats. To complete documentation on Ft. Worth, Texas), and the lenses were examined the treatment of sugar cataracts with AR in- with an American Optical direct ophthalmoscope hibitors, we present a light microscopic study and an American Optical slit-lamp Biomicroscope. of the lenses of galactosemic rats treated with For histology, samples were taken at 3, 7, 10, 14 sorbinil. and 21 days. The rats were sacrificed with an overdose of sodium pentobarbital; the eyes were carefully enucleated and then fixed overnight in 4% glutar- aldehyde in 0.15M sodium-potassium phosphate buffer, pH 7.2. The eyes were slit open at the From the Laboratory of Vision Research, National Eye limbus 10 min after start of fixation to allow the Institute, National Institutes of Health, Bethesda, Md. rapid entry of the fixative. The eyes were trans- Submitted for publication Jan. 30, 1981. ferred to 10% buffered formaldehyde the next day Reprint requests: Jin H. Kinoshita, Laboratory of Vision and further fixed for several days. They were then Research, Bldg. 6, Room 222, National Eye Institute, dehydrated for 2 hr each in a series of ethyl al- 9000 Rockville Pike, Bethesda, Md. 20205. cohols (50%, 70%, 80%, 90%, and 95%) and

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infiltrated with three changes of plastic (methacrylate) embedding mixture, solution A with catalyst (JB-4 embedding kit; Polysciences Inc.) at 2 hr intervals. A final embedding mixture, 20 to 25:1 ratios of solution A with catalyst and solution B, was poured into plastic molds. The well-infiltrated whole eyes were then dropped into the molds and positioned, and the medium allowed to harden. Within 30 to 45 min, the medium had poly- merized sufficiently hard to allow an oily residue to be washed off the surface with soapy water. The molds were then left overnight to harden, and the block was cut the next day on a Sorvall JB-4A mi- crotome. Sections 1 to 2.5 fim thick were cut with a Vi-inch thick glass knife. The sections were floated on water, mounted on a glass slide, dried on a hot plate, and stained (hematoxylin/eosin, periodic acid-Schiff stain (PAS), or toluidine blue). They were then covered with a cover slip, Fig. 1. Gross photograph of mature galactosemic examined, and photographed with a Carl Zeiss mi- cataract in the rat. croscope. Samples were also processed by Ameri- can Histolabs (Rockville, Md.). In the histological studies we found that the equator at the third day on the galactose diet. plastic-embedding method, a procedure not widely All rats developed a nuclear opacity that was used for the lens, yielded results far superior to grossly visible by 2 weeks and had mature that of the paraffin-embedding technique. The cataracts by day 21 (Fig. 1). In striking con- whole eye was routinely embedded as in the paraf- trast, all rats treated with sorbinil showed no fin procedure. This avoids introducing artifacts lens changes when examined by the oph- caused in dissecting out the fresh lens. Moreover, with the plastic method it is possible to section the thalmoscope or slit lamp Biomicroscope al- eye so as to obtain sections as thin as 1 /xm. The though they had been maintained on the 50% water-iniscible plastic rapidly infiltrates and is not galactose diet for up to 8 months. The affected by high humidity. Furthermore, the sorbinil-treated rats were examined daily for simple procedure does not require ultraviolet the first month on the galactose diet and irradiation. Routine staining methods used for weekly thereafter. These lenses could not be paraffin are also applicable to the plastic fixed ma- distinguished from the normal control rat terial. lenses. Sorbinil effectively blocked the syn- The simple method allows for a magnification of thesis of galactitol, so that the level of polyol up to 1000X with light microscopy, gives excellent accumulation in the lenses were less than definition of cataractous changes, and is relatively 10% of those in the untreated galactosemic free of artifacts. When many cataracts are being rats. examined, the use of the relatively thin section of plastic-embedded specimens is the procedure of Histological changes of the lenses choice. When greater definitions of a particular Untreated, galactose-fed rats area of the cataract is required, electron micros- INITIAL VACUOLAR STAGE. Three days after copy can be employed. initiation of the 50% galactose feed, the lenses of all rats showed pre-equatorial and Results equatorial vacuoles {Fig. 2). These were de- Ophthalmoscopic and slit lamp observa- scribed previously as located between fibers tions. Cataracts in the galactose-fed rats and were called intercellular cysts.7 The lens progressed almost at the same rate in all ani- fibers were swollen, and those in the bow mals, and the dense nuclear opacity formed area appeared particularly vulnerable. usually 14 days after the start of the diet. The INTERMEDIATE VACUOLAR STAGE. Seven days first obvious change were vacuoles at the after the start of the galactose diet, the swell-

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Figs. 2 to 6. For legends see facing page.

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ing of the lens fibers and formation of vacu- cells continued to proliferate and form multi- oles were prominent in the anterior and layers of cells. The nucleus was displaced. posterior cortical area. In some areas, there The deep lens fibers were disintegrating. was already liquefaction of cortical lens fi- Sorbinil-treated galactose-fed rats. Close bers. Abnormal local proliferation of anterior histological examinations of the lenses of the lens epithelial cells was also observed (Fig. sorbinil-treated rats fed galactose for 3, 7, 10, 3). These abnormally proliferating cells con- 14, and 21 days revealed no change, and such tinued to produce basement membrane in a lenses could not be distinguished from nor- disorderly fashion. mal control rat lenses. Changes seen in the LATE VACUOLAR STAGE. There was further untreated group could not be detected in the liquefaction of the cortical lens fibers as well treated group. as proliferation of anterior epithelial cells at Similarly, histological examination of the 10 days. The anterior and posterior Y sutures lenses of rats fed galactose and treated with were seen with gaping spaces, and the gaps sorbinil for 1, 3, 6, and 8 months revealed no were filled with what appeared to be lique- cataractous changes, and such lenses could fied cortical material. The supranuclear lens not be distinguished from normal control rat fibers were swollen, and the ends of the lenses (Fig. 6). fibers appeared to have swollen many times the normal size (Fig. 4). The cells in the bow Discussion area were also edematous. Cataracts that can be induced in galac- NUCLEAR CATARACT STAGE. At 14 days, the tosemic rats occur much more readily than in cortex was mainly liquefied. The bow area diabetic rats. Early lens changes detectable still showed actual formation of new lens fiber in lenses of rats fed galactose for only 3 days cells. Extensive liquefaction occurred in the require 2 to 3 weeks to occur in diabetic rats. area of the Y sutures. The lens epithelial cells A dense nuclear opacity in galactose-fed rats showed foci of proliferation and evidence of occurs in 2 weeks, whereas in diabetic rats it multilayering of cells. The lens nucleus was appears in 9 to 12 weeks.8"12 noted to be floating in the fluid cortex. Sugar level obviously is an important fac- MATURE CATARACT STAGE. At 21 days, the cor- tor. However, the difference in the rate of tex, as already seen on day 14, was liquefied cataract formation between the galactose and (Fig. 5). The bow area still showed formation diabetic rats is not related to the sugar levels. of new lens fiber cells, although the new fiber In fact, the blood galactose level in the galac- cells were markedly swollen. The epithelial tosemic rats is usually one half or less of the

Fig. 2. Initial vacuolar stage. Bow area of the lens shows vacuoles forming in the pre-equatorial and equatorial cortex. (Methacrylate, toluidine blue; X200.) Fig. 3. Intermediate vacuolar stage. Anterior subcapsular area shows abnormal proliferation of cells with formation of basement membrane in a disorderly fashion (arrows). Area of the anterior suture is filled with liquified cortical material (asterisk). Note the swollen ends of the cortical lens fibers. (Methacrylate, toluidine blue; X200.) Fig. 4. Late vacuolar stage. Cross section of the lens fibers in the posterior supranuclear area shows swollen fibers many times the normal size. (Methacrylate, toluidine blue; X200.) Fig. 5. Mature cataract stage. Bow area of the lens still shows formation of new lens fibers despite the complete liquefaction of the cortex. The newly formed fibers are edematous. (Methacrylate, toluidine blue; X200.) Fig. 6. Sorbinil-treated galactosemic rat, after 8 months on 50% galactose diet. Section of bow area shows none of the changes seen in the untreated rats and cannot be distinguished from the normal controls. (Methacrylate, toluidine blue; X200.)

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Table I. Galactose cataracts in rats

Effect tose in Route of on polyol diet AR inhibitor administration level (%) End point of cataract Cataract development

50 Alrestatin Oral 50 Nuclear opacity Delay, 3 weeks 30 Alrestatin Oral 50 Nuclear opacity Delay 50 AY 20,263 Vitreous injection 50 Vacuoles and nuclear opacity Delay, 2 weeks 50 Quercitrin Topical 50 Nuclear opacity Delay, 1 week 30 Sorbinil Oral 96 Vacuoles Marked delay (10 mg/kg/day) 35 R.S. 7535 Topical and oral — Nuclear opacity Delay 50 Quercitin Topical Lens fiber swelling 50 Gossypin Oral 50 Nuclear opacity Delay 50 Sorbinil Oral 90 Vacuoles and nuclear opacity No lens changes in 8 mo (60 mg/kg/day)

Table II. Diabetic cataracts

Route of Effect AR adminis- on polyol End point Cataract Animal inhibitor tration level of cataract development Investigator Ref No. Year

Degu Quercitrin Oral 50% Vacuoles and Delay Varma et al. 3 1977 nuclear opacity Degu Quercitrin Oral 50% Vacuoles and Delay 2 weeks Varma 16 1978 nuclear opacity Rat Sorbinil Oral 95% Vacuoles and No lens changes Fukushi et al. 6 1980 nuclear opacity in 6 mo Degu Sorbinil Oral 95% Vacuoles and No lens changes Datiles and Unpub 1980 nuclear opacity in 6 mo Fukui data

Animals were made diabetic with streptozotocin injections.

glucose level in diabetic rats. The difference the striking effectiveness of sorbinil (Tables I is explained by the fact that galactose is a and II). The sorbinil dose of 60 mg/kg/day better substrate for AR than glucose. Second, used in the present study was much lower sorbitol is further metabolized but galactitol than that of AR inhibitors in other studies. is not. As a result of these two factors, the For example, alrestatin was used at 700 to osmotically active products of the sorbitol 1100 mg/kg/day and quercitrin at 3000 mg/ pathway never accumulate as highly in the kg/day. In all likelihood a lower level of sor- lens of a diabetic rat as in the galactosemic binil may be effective, since the 60 mg dose rat. was the only level used. However, Peterson In sugar cataract formation, age is another et al.,4 who first introduced sorbinil, re- factor affecting the rate of opacification with ported that a 10 mg/kg/day dose was not effec- cataract more easily induced in the younger tive in completely preventing lens changes. animal. It is therefore most impressive that The most severe test of any AR inhibitor is to sorbinil is so effective in preventing cataract- block cataracts when a 50% galactose diet is ous changes from occurring in young wean- fed to young rats. In some studies in Table I a ling rats fed a 50% galactose diet. The his- lower cataractogenic concentration of galac- tological studies revealed that there was no tose was used to demonstrate the effective- evidence of precataractous changes during ness of the AR inhibitor. Thus far, sorbinil at the 8 months of observation. a sufficient dose is the only inhibitor to suc- A summary of the various studies of AR cessfully prevent cataracts under these con- inhibitors on sugar cataract formation reveal ditions.

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betic and galactosemic rats. Metabolism 28(Suppl. 1):456, 1979. 5. Beyer-Mears A and Farnsworth P: Diminished sugar cataractogenesis by quercitrin. Exp Eye Res 28:709, Investigator Ref. No. Year 1979. 6. Fukushi S, Merola L, and Kinoshita JH: Altering Gabbay and Kinoshita 17 1972 Dvornik et al. 2 1973 cataract in diabetic rats. INVEST OPHTHALMOL VIS Kinoshita 1 1974 SCI 19:313, 1980. Varma and Kinoshita 15 1976 7. Kuwabara T, Kinoshita JH, and Cogan D: Electron Peterson et al. 4 1979 microscopic study of galactose-induced cataract. IN- VEST OPHTHALMOL 8:133, 1969. Waterbury et al. 13 1979 8. Gifford S and Bellows J: Histologic changes in the Beyer-Mears and Farnsworth 5 1979 lens produced by galactose. Arch Ophthalmol 21: Parmar and Ghosh 14 1979 Kinoshita et al. 1980 346, 1939. (present 9. Friedenwald J and Rytel D: Contributions to the study) histopathology of cataract. Arch Ophthalmol 53:825, 1955. 10. Sippel TO: Changes in the water, protein and glutathione contents of the lens in the course of ga- The fact that so many structurally different lactose cataract development in rats. INVEST OPH- AR inhibitors are effective is strong support THALMOL 5:568, 1966. for the concept that AR is involved in the 11. Sakuragawa M, Kuwabara T, Kinoshita JH, and formation of sugar cataracts. The fact that Fukui H: Swelling of lens fibers. Exp Eye Res 21:381, 1975. these inhibitors are consistent in delaying the 12. Cottlier E: Hypophysectomy effect in lens epithe- formation of cataracts in both diabetic and lium mitosis and galactose cataract development in galactosemic rats is further proof that AR is rats. Arch Ophthalmol 67:476, 1962. the common factor involved in the initiation 13. Waterbury LD, Mahoney JM, and Pfister JR: Inhi- of these cataracts. bition of lens aldose reductase (A.R.) by 7-Dimeth- ylsulfamoylxanthone-2-carboxylic acid (RS 7535). Fed Proc 38:254, 1979. REFERENCES 14. Parmar NS and Ghosh MN: Effect of Gossypin, a 1. Kinoshita JH: Mechanisms initiating cataract forma- flavonoid, on the formation of galactose-induced tion. INVEST OPHTIIALMOL 13:713, 1974. cataracts in rats. Exp Eye Res 29:229, 1979. 2. Dvornik D, Simard-Duquesne N, Krami M, Sestanj 15. Varma SD and Kinoshita JH: Topical treatment of K, Gabbay KH, Kinoshita JH, Varma SD, and galactose cataract. Doc Ophthalmol Proc Ser 8:305, Merola LO: Polyol accumulation in galactosemic 1976. and diabetic rats: control by an aldose reductase in- 16. Varma SD: Delaying the formation of cataracts in hibitor. Science 182:1146, 1973. diabetes. In Proceedings of the 3rd International 3. Varma SD, Mizuno A, and Kinoshita JH: Diabetic- Congress on Eye Research, Osaka, Japan, May cataracts and flavonoids. Science 195:205, 1977. 1978, p. 113. 4. Peterson MJ, Sarges R, Aldingen C, and Mac- 17. Gabbay KH and Kinoshita JH: Mechanism of devel- Donald DP: CP-45, 634: a novel aldose reductase opment and prevention of cataracts. Isr J Med Sci inhibitor that inhibits polyol pathway activity in dia- 8:1557, 1972.

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