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 174 Downloaded from iovs.arvojournals.org on 10/01/2021 Volume 22 Number 2 Galactose cataract prevention with sorbinil 175 infiltrated with three changes of plastic (methacry- late) 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- Downloaded from iovs.arvojournals.org on 10/01/2021 176 Datiles et al. Invest. Ophthalmol. Vis. Sci. February 1982 Figs. 2 to 6. For legends see facing page. Downloaded from iovs.arvojournals.org on 10/01/2021 Volume 22 Number 2 Galactose cataract prevention with sorbinil 111 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.