Vitamin E deficiency and the retina: photoreceptor and pigment epithelial changes. W. Gerald Robison, Jr., Toichiro Kuwabara, and John G.

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1 Vitamin E deficiency and the retina: photoreceptor and pigment epithelial changes W. Gerald Robison, Jr., Toichiro Kuwabara, and John G. Bieri* To investigate the role of normal vitamin E levels and the interrelationships between vitamins E and A in maintaining the visual cells of the retina, weanling female Sprague-Dawley rats were fed vitamin E-free diets differing tenfold in their vitamin A content (O.S and.0 mg of retinol per kilogram of diet). Rats on vitamin E-free diets with the higher vitamin A level exhibited, marked disruption of photoreceptor outer segment membranes and. a fivefold increase in the number of lip of us cin granules in the pigment epithelial cells which ingest these membranes. Rats on vitamin E-free diets with the lower vitamin A level showed the same retinal damages plus significant loss of photoreceptor cells compared to age-matched rats on control diets. Rods and cones were involved, equally, and. their pattern of loss was not like that found in vitamin A deficiency. Normal levels of vitamin E probably protect photoreceptor membranes from oxidative damage and retard the accumulation of their remnants and other products of lipid breakdown in the pigment epithelium. The vitamin A status of rats has a significant influence on the extent of damage induced by vitamin E deficiency. Key words: vitamin E, vitamin A, lipofuscin, retina, photoreceptor membranes, retinal pigment epithelium, lipid peroxidation outer segment membranes of photoreceptor cells contain extraordinarily high proportions of polyunsaturated fatty acids,' and therefore may be unusually susceptible to lipid peroxidation. 2 Vitamin E (a-tocopherol) is an antioxidant shown to be effective in protecting such membranes 2-4 and in retarding the intracellular accumulation of the lipomscin pigments which contain end products of lipid peroxidation. 31 ~ 7 From the Laboratory of Vision Research, National Eye Institute, and *Laboratory of Nutrition and Endocrinology, National Institute of Arthritis, Metabolism and Digestive Diseases, National Institutes of Health, U.S. Department of Health, Education and Welfare, Bethesda, Md. Submitted for publication Oct. 1, 197. Reprint requests: W. Gerald Robison, Jr., Ph.D., Building 6, Room 201, National Institutes of Health, Bethesda, Md Vitamin E normally occurs in high concentrations in bovine rod outer segments. When it is lowered by seasonal dietary changes, the maintenance of intact rod outer segment membranes for in vitro studies becomes more difficult unless vitamin E is added to the preparations and other precautions are taken against oxidation. 2 So far, most of the evidence regarding an in vivo role of vitamin E in maintenance of the retina comes from studies by Hayes, 9 who reported photoreceptor outer segment degeneration limited to the macula of monkeys deficient in vitamin E for 2 years, and from the recent work by Katz et al., 10 who found massive accumulations of autofluorescent pigment in the pigment epithelium but no apparent damage to the neural retina in rats on diets lacking various antioxidants, including a-tocopherol. 63

2 64 Robison, Kuwabara, and Bieri Invest. Ophthalmol. Visual Sri. July 1979 The purpose of the present study was to elucidate the contribution of vitamin E in protecting rod and cone membranes. Previous work was extended by quantitating the increase in lipofuscin of the pigment epithelium, by examining ultrastructural changes in photoreceptor outer segment membranes which were not noted before, and by evaluating irreversible damage to the retina with the use of counts of surviving rod and cone nuclei in vitamin E-deficient rats. Also, the interrelationships between vitamin E and vitamin A in maintaining the retina were investigated by vitamin E deprivation of rats with controlled vitamin A status. Materials and methods Female Sprague-Dawley rats were housed individually in suspended, stainless steel wire cages and maintained with food and water ad libitum at room temperature under 12 hr cyclic illumination of 4 foot-candles at cage level. Half the vitamin E-deprived rats were given adequate vitamin A to compensate for an anticipated loss of this vitamin by increased peroxidation in tissues lacking vitamin E." The others were maintained on a marginal level of vitamin A, receiving enough to prevent vitamin A deficiency but not enough to permit significant tissue storage of this vitamin All rats were fed from weaning a basal diet without vitamins A and E 13 consisting of 20% vitamin-free casein, 3.% mineral mix, 0.1% trace mineral mix, 10% corn oil stripped of vitamin E but preserved with 0.02% 2,6-ditertiary butyl-p-cresol (BHT),* 4% cellulose, 2% vitamin mix (no A or E), and 60.4% sucrose. After 1 week of adjustment to this purified basal diet, the animals were divided into four groups. Two were designated the "adequate" vitamin A series and received.0 mg of retinol per kilogram of diet, and two groups were designated "marginal" vitamin A series and received 0. mg of retinol per kilogram of diet. The retinol was in the form of stabilized, microencapsulated retinyl pahhitate (Nopco Chemical Co., Louisville, Ky.). Within each series, one group received no vitamin E and the other received a supplement of dl-atocopheryl acetate (General Biochemicals, Cleveland, Ohio), 20 mg/kg of diet. Groups of rats were sacrificed with vapors of ether after 2,, and months on the diets. Retinol This small amount of BHT prevents rancidity in the diet but has no vitamin E-like activity in vivo. levels were determined for the plasma 14 and liver. 1 Portions of extraocular muscle, uterus, liver, heart, and brain were taken for studies to be reported elsewhere. The left eye was removed and slit at the limbus, fixed for 30 min at room temperature in a solution of 2.% glutaraldehyde and 6% sucrose buffered to ph 7.2 with 0 mm sodium cacodylate, fixed for another 30 min in 10% phosphate-buffered formalin, then bisected horizontally. Half the eye was processed for histological sectioning and staining, including the periodic acid-schiff (PAS) reaction, by embedding in either paraffin or in glycol methacrylate, and the other half was frozen on Dry Ice for fluorescence microscopy. Frozen sections 10 jam thick were prepared and mounted on slides in glycerol. They were examined for autofluorescence under a Leitz Orthoplan microscope equipped with a mercury vapor lamp and a GB 3 heat filter. A 2 mm UGI excitation filter (peak transmission near 360 nm) and a 430, 460, 470, or 490 barrier filter were used to localize and characterize the autofluorescence typical of lipofuscin. Then, owing to its greater transmittance, a KP 490 excitation filter combined with a 10 or 30 barrier filter was used to photograph this fluorescence with a Leitz Orthomat camera loaded with Ektachrome Daylight, ASA 200, 3 mm film. The right eye was fixed at room temperature for 6 to 9 hr in the 2.% glutaraldehyde solution mentioned. Portions of the retina within 2 mm of the optic disc were taken from the superior temporal and inferior nasal quadrants and processed independently for electron microscopy by dehydration in ethanol and embedding in Epon. Sections 1 ju.m thick were stained with toluidine blue and analyzed for alterations in structure and number of retinal elements by means of photomicrographs printed to obtain a total magnification of 1000 times the specimen size. Counts were taken from at least 10 separate locations in the superior temporal and 10 locations in the inferior nasal quadrants of two rats for each diet and age group. Since the data varied more between animals than between quadrants in the same eye, all the data from each animal were pooled. Cone nuclei were distinguished from rod nuclei morphologically following the descriptions by Carter-Dawson et al. 16 The numbers of cone nuclei, rod nuclei, and lipofuscin-like granules were determined for 200 jum lengths of retina. Ultrathin sections were taken of representative regions and were examined with a JEM 100B electron microscope following staining with uranyl acetate and lead citrate.

3 Volume 1 Number 7 Vitamin E deficiency and retina 6 Results There were no differences in growth rates between groups of rats receiving adequate or marginal vitamin A, but the groups fed vitamin E grew slightly faster than did those on E-free diets. After weeks, the E-adequate, A-marginal group and the E-adequate, A- adequate group weighed 263 ± and 29 ± gm, respectively, whereas the E-free, A- marginal and E-free, A-adequate groups weighed 232 ± 3 and 223 ±9 gm, respectively (means ± S.E.M. for five rats). These differences were maintained through the eighth month. By months, rats fed the vitamin E-free diets had barely detectable plasma levels of a-tocopherol (<100 /Ag/dl), whereas rats receiving vitamin E-adequate diets had plasma levels greater than 1000 /xg/dl. At all times, the eyes and hair coat of all rats except one were normal. One rat fed E-adequate, A-marginal diet began secreting porphyrin from the Harderian glands after 7 weeks even though growth was normal. This rat was omitted from the study. As expected, ll tissue vitamin A levels were markedly affected by the vitamin E status of the animals (Table I). At months, the rats fed the E-free, A-marginal diet had one-tenth the liver concentration of retinol that the E-adequate, A-marginal group had, and between the E-free, A-adequate and E-adequate, A-adequate groups the difference was twofold. At months, the E-free groups showed slight decreases from the -month values, but the E-adequate, A-adequate group doubled its liver retinol in this time. All plasma concentrations were normal except for those in the E-adequate, A-adequate group at months, which were higher than usual but within the range expected for unfasted rats receiving these dietary levels of retinol. No toxic effects were noted. Histology. At 2 months the retinas of rats on the E-free, A-adequate and the E-free, A-marginal diets appeared similar and varied only slightly from retinas of the E-adequate, A-adequate controls. Their retinal pigment epithelium exhibited a greenish-yellow autofluorescence (presumed to originate mainly from vitamin A derivatives) that faded within Fig. 1. Fluorescence micrographs of the neural retina, pigment epithelium, choroid, and solera of rats fed an E-adequate, A-adequate diet (left) or an E-free, A-adequate diet (right) for months. The E-deprived rat exhibited unusually bright yellow fluorescence in the pigment epithelium, in the veins (v) but not in the arteries (a) of the sclera, and in various regions of the choroid (arrow). The plasma retinol levels at time of sacrifice were 132 and 34 /-tg/dl, respectively. (Calibration bar: 100 /am.) Table I. Plasma and liver vitamin A* Diet group E-free, A-marginal E-free, A-adequate E-adequate, A-marginal E-adequate, A-adequate Time (months) Plasma (figlrll) 32 ± 1 23 ± ± 6 34 ± ± 4 40 ± ± 100 Liver (faglgn 3 ± 13 1 ± 612 ± ± ± 23 1,307 ± 2, * Values tire means ± S. E. for 3 or 4 ruts. Blanks indicate that no rats were taken at that time. a few minutes and did not appear to be significantly brighter or longer-lived than the autofluorescence in controls. On the other hand, the number of PAS-staining (PASpositive), lipofuscin-like granules in the pigment epithelium was slightly greater in E-free than in E-adequate rats. After months the retinas of rats in the various diet groups were clearly different. The eyes of all rats on the vitamin E-free diets exhibited an intense yellow autofluorescence typical of lipofuscin throughout the pigment epithelium, in many parts of the choriocapillaris, and in veins of the sclera. All

4 66 Robison, Kuwabara, and Bieri Invest. Ophthalmol. Visual Set. July 1979 Fig. 2. Outer retinal regions from rats fed an E-free, A-marginal diet for months (left), an E-adequate, A-marginal diet for months (middle), or an E-free, A-adequate diet for months (right). E-deprived rats exhibited massive accumulations of granules in the pigment epithelium (pe), disorganization and swelling (circle) of photo receptor outer segments (OS), and loss of photoreceptor nuclei from the outer nuclear layer (ONL). More nuclei were lost after months on E-free diets (right micrograph shows 46 remaining) than after months (66 remaining). Cone nuclei (arrows) were lost proportionately. (Epon sections, 1 jltm thick, toluidine blue stain; calibration bar: 10 /im.) Table II. Retinal changes in Vitamin E-deficient rats Diet group Time (months) Cone nuclei* (per 200 fjun) Rod nuclei* (per 200 pm) RPE height* 6.9 ± 1 7. ± O.lt 6.7 ± 0.2t 7.3 ± ± 0.4 RPE granules* (per 200 pm) E-free, A-m;irgin;i1 E-free, A-adequate E-adeqiiate, A-marginal E-udequate, A-adequate «Q 4.9 ± ± O.lf 3.7 ± 0.6t 4.0 ± 0..1 ± 0.3. ± ± ± 20f 1 ± 30t 339 ± ± ± 7 37 ± 294 ± 22.1 ± 0..2 ± ± 20t t 292 ± 4t 406 ± 7t ± 13 4 ± * Values are means ± S.E. for two rats. Blanks indicate that no rats were taken at that time. t Significantly different from control (E-adequate, A-adequate group) at less than the 0.0 level of significance as determined by Student's t test and a single-tail t distribution. the fluorescent cells showed an obvious increase in PAS-positive cytoplasmic inclusions of granular structure that were interpreted to be lipofuscin granules. The autofluorescence of the retinal pigment epithelium had two components, a greenish-yellow component which faded too rapidly to permit satisfactory photography and a bright yellow component which remained with very little fading. The E-adequate rats showed more of the fast-fading component (probably originating from vita-

5 Volume 1 Ntimber 7 Vitamin E deficiency and retina 67 Fig. 3. Retinal pigment epithelium and portions of outer segments (OS) from rats fed an E-free, A-marginal diet for months (left), an E-adequate, A-adequate diet for S months (middle), or an E-free, A-adequate diet for months (right). E-deprived rats accumulated unusual quantities of lipofuscin pigment granules (G) and secondary lysosomes (Ly) in their swollen retinal pigment epithelium. They showed some mitochondria] changes (arrow) of undetermined origin and consequence. (Calibration bar: 1.0 /ini.) min A derivatives), whereas the E-free rats showed markedly more of the stable, bright yellow fluorescence typical of lipofuscin (excitation ca. 360 nm, maximum emission ca. 430 nra). Fig. 1 shows the striking differences in the amount of stable fluorescence observed after months on the diets. Photomicrographs of 1 fim sections (Fig. 2) permitted quantitation of changes in numbers of lipofuscin granules and measurements of other retinal changes for each diet group. At months (Fig. 2, Table II), the distal portions of rod outer segments in rats fed vitamin E-free diets were disorganized and swollen, with maximum widths averaging 2. jam and representing a 44% increase over controls. The retinal pigment epithelial cells were 30% to 0% taller (ca. 6.9 /im), and the numbers of lipofuscin granules in their cytoplasm were increased three to five times the normal number regardless of whether the groups compared received marginal or adequate levels of vitamin A. Rod nuclei were decreased by 16% in the E-free, A-niarginal group compared to the E-adequate, A-adequate control group. At months (Fig. 2, Table II) there was somewhat more swelling (up to 4 ixm in width) and more extensive disorganization of rod outer segments in the vitamin E-free rats concomitant with a slight decrease in outer segment length. Again the retinal pigment epithelium was taller and contained up to five times the control levels of lipofuscin pigment. Histiocytes were found relatively infrequently in the subretinal space. The 40% increase in lipofuscin pigment that occurred in the E-adequate, A-adequate con-

6 6 Robison, Kuwabara, and Bieri Invest. Ophthalmol. Visual Sri. July 1979 trol group between and months (Table II) probably represented normal aging changes. Another probable indication of aging or simply an increase in eye size was the 1% decrease in the number of rod nuclei from 37 ± to 294 ± 22 per 200 fxm of retina between and months in these controls. This probable aging change was closely mimicked in the E-free, A-adequate group, where a 17% decrease in rod nuclei was found between and months on the diet. Neither of these changes approached the 4% decrease in rod nuclei between the and month groups on the E-free, A-marginal diet which left those rats with 46% fewer rod nuclei than rats fed the E-adequate, A-adequate diet for months (Table II, Fig. 2). The fact that the E-free, A-marginal diet led to a significant loss of rod nuclei, whereas the E-free, A-adequate diet did not, suggests that the levels of vitamin A in vitamin E- deficient rats may be critical for rod preservation. The numbers of cone nuclei were decreased significantly in the E-free, A-marginal rats compared to the E-adequate, A- adequate controls, and the proportions of cone to rod nuclei remained essentially normal in all diet and time groups. Also, the proportion of photoreceptor nuclei in the central and peripheral retina remained essentially unchanged between and months in all rats. Vltrastructure. Electron microscopy revealed changes in the rod outer segments as well as in the retinal pigment epithelium of vitamin E-deprived rats. The membrane disks in the distal regions of rod outer segments were swollen and disorganized, sometimes exhibiting expansion of intradisk spaces and other times expansion of interdisk spaces (Fig. 3). The more proximal regions of rod outer segments remained quite normal in structure. However, the proportion of rod outer segments remaining organized was less after months than it was after only months, and it was less in the A-marginal rats than in the A-adequate rats. The granules which accumulated in the cytoplasm of retinal pigment epithelial cells of rats on E-free diets varied in size, shape, and electron lucidity. They were located more centrally than melanin pigment granules and seldom did they invade apical microvilli. They were heterogeneous in substructure, having regions of variable densities, regions with lamellar inclusions, and regions with fine granular structure. Their infrastructure was consistent with that reported for lipofuscin granules, as were their autofluorescence in frozen sections and their PAS-positive staining following paraffin embedding.' These lipofuscin granules were increased in numbers and maximum size in rats deprived of vitamin E for months compared to those in rats deprived for months. However, there were no consistent differences in the ultrastructure of the granules with different times on the E-free diets or with different levels of vitamin A. The lipofuscin granules in control rats were similar in infrastructure but were smaller in average size and were found much less frequently. More secondary lysosomes were found in E-deprived rats, and several mitochondria exhibited vesicular inclusions. Discussion The marked disruption of photoreceptor outer segments and massive accumulations of lipofuscin in the pigment epithelium following vitamin E deficiency (E-free, A-adequate diet) probably resulted from a chain of oxidative events beginning with the highly reactive polyunsaturated fatty acids of outer segment membranes Being deprived of the antioxidant environment normally provided by tissue vitamin E, these photorecepor membranes exhibited an instability in vivo, previously reported for in vitro preparations of rod outer segment membranes depleted of vitamin E. 2 The fivefold acceleration of lipofuscin accumulation in the pigment epithelium occurring simultaneously with loss of structural integrity of outer segments suggests that breakdown products of these photoreceptor membranes do, in fact, contribute to the contents of the lipofuscin granules. The role of the retinal pigment epithelium in regularly phagocytizing, concentrating, and degrading numerous packets of outer segment mem-

7 Volume 1 Number 1 Vitamin E deficiency and retina 69 brane disks is well established, 17 as is its involvement in accumulating lipofuscin, thought to represent the end products of I9 lipid peroxidation. The photoreceptor membrane remnants in granules of the pigment epithelium might simply represent end products of lipid peroxidation, 3 " 7 or they might include other materials such as the proposed retinoyl complexes which were extracted from lipofuscin granules of brain tissue. 20 Increased levels of dietary retinol, within the range used, did not increase the autofluorescence or the numbers of granules accumulated (compare granules in rats fed A-adequate and A-marginal diets). However, what relation the levels of retinol might have to the possible formation of retinoyl complexes through conversion to retinoic acid remains to be elucidated. The vitamin A status had striking effects on the eyes of vitamin E-deprived rats, which probably represented additive rather than independent effects of vitamin A deficiency. Lowering the dietary vitamin A (E-free, A-marginal diet) increased the damage resulting from vitamin E deficiency. In addition to outer segment disorganization and lipofuscin accumulation, there was a significant loss of photoreceptor nuclei in rats fed the E-free, A-marginal diet, which was not seen in the E-free, A-adequate rats. Although the rats studied were not deficient in plasma retinol (Table I), localized tissue deficiencies in vitamin A may have arisen by peroxidative loss of this vitamin" in rats fed the E-free, A-marginal diet. The vitamin A levels stored in the pigment epithelium 21 or present in the microenvironment surrounding the rods and cones may have dropped below a critical level for the retina in the present experiment. However, although the retinal changes mimicked, at first, the damage reported in vitamin A deficiency, 22 ' 23 later the changes differed significantly. At first, the distal portions of the outer segments became swollen, with their disk membranes disorganized and vesiculated. However, later there was no extensive formation of small vesicles, the loss of rod nuclei was not greater in the central than in the peripheral retina, and cone nuclei were not spared preferentially as with vitamin A deficiency. Such additive effects of vitamin A status may be critical in influencing the final outcome in vitamin E-deprived animals. The E-free diet fed to monkeys for 2 years by Hayes 9 resulted in outer segment disruption but no reported loss of photoreceptor nuclei. None of the antioxidant-deficient diets utilized by Katz et al. I0 resulted in either photoreceptor damage or drop out. Probably their diets provided adequate levels of vitamin A. Studies on the interrelationships of vitamin E and vitamin A in maintaining retinal structure and function as well as general health should be fruitful. 24 Since photoreceptor loss was equivalent in all regions of the retina and involved cones and rods equally, why the vitamin E-deprived monkeys reported by Hayes 9 exhibited preferential damage in the cone-rich macula remains unknown. Further investigations are needed to determine how the effects of vitamin E and vitamin A deficiencies differ between diurnal and nocturnal animals. The outer segments of photoreceptor cells are surrounded by a well-oxygenated environment, yet their component membranes are unusually susceptible to oxidation. Loss of membrane integrity, accumulation of lipofuscin, and cell death were observed in retinas following vitamin E deprivation. This study suggests that a normal vitamin E status of an animal is important for protecting the retina from such changes, all of which probably result from a series of oxidative events. Also, vitamin E may protect the retina from light damage, if lipid peroxidation is involved as has been proposed. 2-2C> Singlet oxygen which is released upon illumination of retinal may trigger a series of reactions resulting in peroxidation of lipids and membrane instability. If so, a scavenger of free radicals such as a-tocopherol should provide some protection from light damage. Presently, albino rats on various vitamin E/vitamin A diets are being exposed to several light intensities and cycles.

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