Reduced and Oxidized Ascorbates in Guinea Pig Retina under Normal and Light-exposed Conditions

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1 Reduced and Oxidized Ascorbates in Guinea Pig Retina under Normal and Light-exposed Conditions Barbara J. Woodford,* Mark O. M. Tso,* and Kwok-Wai Lamf Both reduced and oxidized ascorbates were measured in aqueous, neural retina, and pigment epithelium-choroid complex (PE-C) of pigmented guinea pigs. Normal values for total ascorbate of 16 mg/ dl in aqueous, 22 mg/dl in neural retina, and 7 mg/dl in PE-C were found. After mild photic damage caused by varying lengths of exposure of 10,000 to 20,000 lux of fluorescent lighting, reduced ascorbate concentrations generally decreased in the neural retina, while oxidized ascorbate generally increased in PE-C. In both normal and light-exposed retinas, reduced ascorbate was predominant in the neural retina, and oxidized ascorbate was predominant in the PE-C. Histochemical localization of reduced ascorbate occurred in the Miiller cell fibers and at the apices of the retinal pigment epithelium. Invest Ophthalmol Vis Sci 24: , 1983 Low-level illumination has produced structural and functional damage to the retina in a variety of experimental conditions in rats, rabbits, and monkeys (for a review, see Noell 1 ). In such photic injury of the retina, a mechanism has been proposed that involves the production of singlet oxygen or superoxide radical and subsequent lipid peroxidation of disc membranes of photoreceptor outer segments. The rod outer segments are particularly susceptible to such peroxidation because they are rich in polyunsaturated fatty acids. A number of agents have been suggested as natural protectants against peroxidation damage. Superoxide dismutase, catalase, peroxidase, a-tocopherol, glutathione, and ascorbic acid have been thought to play such a role in the eye. 2 Ascorbate is found in high concentrations in the aqueous humor (3-28 mg/dl) and the lens ( mg/dl) of many species, although plasma levels in most are less than 1 mg/dl. 3 Ascorbate levels are also high in the retinas of the few species studied. The concentration of ascorbate in the retina of rats is 28 mg/dl, although aqueous and lens levels are 0-3 mg/ dl. 4 Heath et al 5 reported that the concentration of From the Georgians Theobald Pathology Laboratory, Department of Ophthalmology, University of Illinois Eye and Ear Infirmary, Chicago,* and the University of Texas Health Service Center, San Antonio, f Supported in part by Public Health Service grants EY1903 and Core grant EY from the National Institute of Health. Presented in part at the annual meeting of the Association for Research in Vision and Ophthalmology, Sarasota, Florida, Submitted for publication August 23, Reprint requests: Mark O. M. Tso, MD, Georgiana Theobald Pathology Laboratory, Department of Ophthalmology, University of Illinois Eye and Ear Infirmary, Chicago, IL ascorbate is 20 mg/dl in the retinas of rabbits, 22 mg/ dl in cows, and 28 mg/dl in guinea pigs. Several functions of the high level of ascorbate in the eye have been proposed. Friedenwald et al 6 considered ascorbate vital in aqueous secretion; Cristiansson 7 believed ascorbate to be critical in the polymerization of vitreal components; Varma et al 8 implicated it in the protection of lens against oxidation; and recently, Nirankari et al 9 established the importance of ascorbate in corneal lesion healing. Because of the strong evidence that ascorbate acts as an antioxidant in other systems, eg, brain, 10 mast cells, 11 and liver, 12 we investigated its possible role as protectant against light damage in the retina of guinea pigs. The guinea pig was chosen because it is one of the few species other than primates, in which the scorbutic state may be produced by dietary control. We determined the level of reduced ascorbic acid (AA) and the oxidized form, dehydroascorbate (DHA), of the normal neural retina and pigment epitheliumchoroid complex (PE-C) of the guinea pig, and localized histochemically the reduced ascorbate in the normal and scorbutic retina. We then established a light level that produced mild damage to the outer retina and retinal pigment epithelium (RPE) of the guinea pig, and compared the normal ascorbate values with those of the light-exposed neural retina and PE-C. Materials and Methods Twenty pigmented (Peruvian) and two albino guinea pigs ( g) were housed under normal conditions of cyclic lighting (10 hours dark, 14 light) and fed a vitamin-c-enriched guinea pig diet /83/0700/862/$ 1.10 Association for Research in Vision and Ophthalmology 862

No. 7 ASCOftBATE IN GUINEA PIG RETINA / Woodford er ol. 863 (Purina ). Two albino guinea pigs were made scorbutic with a vitamin-c-deficient diet (Purina ), supplemented with dry oatmeal.

2 No. 7 ASCOftBATE IN GUINEA PIG RETINA / Woodford er ol. 863 (Purina ). Two albino guinea pigs were made scorbutic with a vitamin-c-deficient diet (Purina ), supplemented with dry oatmeal. These animals were kept on the diet for at least two weeks and were judged scorbutic by lack of weight gain, weakness, and/or skin lesions. Subsequent confirmation of the scorbutic state was obtained by an analysis of aqueous, which contained less than 1 mg/dl ascorbate. The normal room lighting consisted of a luminance at the floor of the cage of 3.2 to 15.8 Ft-L as measured with a Spectra Spotmeter photometer. The illumination then was calculated to be about 10 Ft-C or 115 lux. Constant environmental lighting was obtained by placing Plexiglas cages on a shelf with two 40-watt cool-whitefluorescentbulbs, 8-9 inches from each side. White cardboards were placed under each cage and on the roof of the enclosure, 7 inches above the cages. The system provided a luminance at the floor of the cage of 10,000 to 20,000 lux or 800 to 1600 Ft-C. Sixteen guinea pigs were exposed for 6 (2 animals), 12 (2), 24 (2), or 48 (10) hours. The temperature of the enclosure was kept below 30 C which maintained the normal guinea pig body temperature of 37.8 C. Two animals were kept in each cage; throughout light exposure they were fed the same diet as when in normal cyclic lighting conditions. Eyes were enucleated immediately after exposure or, in five animals, after 7 days of normal cyclic lighting following 48 hours of light exposure. Normal and light-exposed eyes were processed for electron microscopy to determine morphologic damage caused by light exposure. Guinea pigs were anesthetized with 25 mg/kg nembutol intraperitoneally. A drop of aqueous was aspirated from the anterior chamber with a No. 30 needle and frozen directly in a syringe covered with parafilm. The eyes were then rapidly removed, and the anterior portions of the eyes were cut away. The remaining eye cyps were cleansed of vitreous, the neural retina was detached, and PE-C was dissected from sclera. Each sample was placed in a pre-weighed, prelabeled capsule, which was again weighed and kept at 70 C until sent on dry ice for analysis. Extracts of retinal tissues were obtained by homogenizing each tissue in 4% perchloric acid (one ml per 200 mg of wet tissue) and centrifuging. Aqueous was untreated. The ascorbate concentrations in aqueous humor and eye tissue extract were analyzed by high pressure liquid chromatography (HPLC), according to the method of Fox et al. 13 Aqueous fluid or tissue extract was injected directly into the column, and the AA was measured by absorbance at 254 nm. Other aliquots of tissue extract were treated with dithioerythritol to reduce DHA. The total ascorbate concentration (AA and DHA) was then measured by HPLC. Aqueous was not treated further, since all ascorbate in aqueous was in the reduced form Histochemical localization of AA was accomplished in the whole retinas of two normal and two scorbutic albino guinea pigs by the silver method of Chinoy. 14 Eye cups were fixed overnight at 3-5 C in a solution of 5 g AgNO 3, 34 ml H 2 O, 66 ml absolute ethanol, and 5 ml glacial acetic acid, final ph 2.0 to 2.5, in lightproof containers. Eye cups were then washed two to three times in 70% alcoholic ammonia for minutes, and then dehydrated and embedded in paraffin. Paraffin sections were counterstained with cresyl violet. A brown precipitate of silver grains indicated sites of AA. Using the above conditions of incubation insured that other reducing agents such as glutathione, cystein, or reducing sugars were not involved and that oxidation of ascorbic acid did not occur. 14 Albinos were used to decrease any reducing effect or masking of reaction product by melanin granules. Results The levels of AA and DHA of aqueous, neural retina, and PE-C of normal pigmented guinea pigs are shown in Fig. 2A. The form of ascorbate was predominantly AA in the neural retina and DHA in the PE-C. Guinea pig retinas exposed to 10,000-20,000 lux for 48 hours showed evidence of morphologic damage, exhibiting vacuolation of the RPE and disruption, disorganization, and vacuolation of the outer and inner segments in focal areas of the retina (cf, Figs. 1A and B). The levels of AA and DHA of aqueous, neural retina, and PE-C of pigmented guinea pigs, light-exposed for 6, 12, 24, and 48 hours are shown in Figs. 2B-E. After 6 hours of exposure (Fig. 2B), ascorbate levels of neural retina lowered because of a decrease in AA that was statistically significant when compared with the normal (P < 0.02). Total ascorbate in PE-C was also less than that in the normal. When animals were exposed to successively longer periods (Figs. 2C-E) total retinal ascorbate levels gradually rose but still were below those of the normal retina. Total ascorbate of PE-C rose to that of the normal, but DHA was higher than in the normal, and AA remained low. Aqueous ascorbate after light exposure was slightly higher than that of the normal. Ascorbate levels in eyes of animals allowed to recover for seven days after 48 hours of continuous fluorescent light are shown in Fig. 2F. In this recovery phase, there was a decrease of total ascorbate in the neural retina when compared with normal animals and with animals immediately after 48 hours of light

3 864 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / July 1960 Vol. 24 Fig. 1A. Retina of a control pigraented guinea pig showing outer (OS) and inner segments (IS) and pigment epithelium (RPE) (X7,500). Inset: All layers of normal retina of guinea pig (Mallory's blue; X480). B. Retina of pigmented guinea pig after 48 hours of continuous fluorescent light exposure, showing swollen and vacuolated RPE (arrows) and distorted and vacuolated OS (arrowheads) (X9,000). Inset: Outer layers of light exposed retina are affected with vacuolated RPE and OS (arrows) (Mallory's blue; X480). RPE * K exposure. However, in recovery, the percentage of AA in the neural retina remained about the same as in the normal or immediately after exposure, but in the PE-C the percentage of AA decreased further. When the histochemical localization of reduced ascorbate was performed in the normal retina, silver grains were seen in Miiller cellfibers,especially prominent in the inner plexiform layer, in focal areas of the outer plexiform layer, and around nuclei of the inner and outer nuclear layers. Silver grains also were

4 No. 7 ASCOftBATE IN GUINEA PIG RETINA / Woodford er ol. 865 Fig. 2. Reduced (shaded area) and oxidized (clear area) aqueous (), neural retina (R), and retinal pigment epithelium-choroid complex (PE-C). Values are expressed as mg/dl of aqueous fluid or mg/dl of wet tissue weight. The mean values of n eyes are represented along with the standard errors of the mean (I). The percent of total ascorbate which is in the reduced form (shaded area) is indicated above the bar. A. Normal light-adapted pigmented guinea pig. B. Immediately after 6 hours of fluorescent light exposure of 10,000-20,000 lux. C. Immediately after 12 hours of light exposure. D. Immediately after 24 hours of light exposure. E. Immediately after 48 hours of light exposure. F. One week after 48 hours of light exposure. UJ 5 CD zo 8» CD O O * n=8 97% 32% NORMAL A n = 4 100% 71% I IT 1 8% 6 Hrs B n = 4 100% rh 100% 22% 12 Hrs C n=io 100% rf"i 93% 17 /, Hrs D 48 Hrs E 1 WK POST F noted at the apical surface of the RPE and in the vitreous (Figs. 3A and B). Electron microscopy of companion eyes of the normal albino guinea pig indicated that some structures that could be interpreted as premelanosomes were present. However, these were relatively few in number and could not account for the amount of silver reduction in this area of the RPE. Specificity of the stain is demonstrated by the comparative lack of silver granules in the retina from scorbutic albino guinea pigs (Figs. 3C and D). The contralateral retinas of the scorbutic eyes used for histochemistry were analyzed and found to contain 0-1 mg/dl ascorbate. Discussion In this study we have established concentrations of reduced and oxidized ascorbates in the neural retina and in the PE-C in the normal guinea pig. In a previous study, Heath et al 5 measured total ascorbate without differentiating reduced from the oxidized form in retinas of guinea pigs. Furthermore, it is not clear whether their reported value of 28.2 mg/dl included the RPE. Likewise, when Heath et al 4 reported on both reduced and oxidized forms of ascorbate in retinas of rats (total:24.5 mg/dl; 70-75% AA), it again was not clear whether their values included the RPE. In the normal guinea pigs in the present study, the predominance of AA in the neural retina and of DHA in the PE-C was evident. Because blood ascorbate is in the reduced form, it is reasonable to assume that the DHA in PE-C exists in the RPE and not in the stromal vessels. Varma 8 showed that the cation pump (as demonstrated by uptake of Rb+) of the in vitro lens was damaged by photochemical production of superoxide radicals. This damage was prevented by the addition of ascorbate to the medium. In more recent work, 16 Varma has shown that lipid peroxidation, as measured by the formation of malonaldehyde, is caused by fluorescent light in in vitro rat lens. Such increases in malonaldehyde were prevented by addition of ascorbic acid. Hence, Varma suggested that the high level of reduced ascorbate in the aqueous and lens probably acts as an antioxidant to protect the lens from light damage. The presence of ascorbate in a similarly reduced form in the retina of the guinea pig and rat suggests that the ascorbate in the retina may likewise act as an antioxidant. The decrease of reduced ascorbate in the neural retina after light exposure further supports this hypothesis. The presence of DHA as the predominant form of

5 866 INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / July 1983 Vol. 24 Figs. 3A and 3B. Sections of the retina of a normal guinea pig. C. and D. Sections of the retina of a scorbutic guinea pig (silver stain for reduced ascorbate, and cresyl violet; X600). A and C are bright-field micrographs, while B and D are taken with dark-field illumination. The presence of reduced ascorbate is demonstrated in the inner plexiform layer (IPL) of the normal retinal as dark silver grains in A and bright spots in B in a pattern corresponding to Miiller cell fibers. There are also silver grains at the apices of the RPE (arrowheads). In the scorbutic guinea pig, Miiller cell fibers, although visible in the bright field micrograph (arrows), are not stained with silver. Only the vitreous attached to the inner limiting membrane (arrowheads) continues to demonstrate the presence of reduced ascorbate. ascorbate in the PE may indicate the mechanism of transport of ascorbate into the retina. Like the aqueous, high concentrations of reduced ascorbate in the retina may indicate a process of active transport from the blood. Friedenwald6 reported that ascorbate was mainly in the oxidized form in the ciliary epithelium, although it was in the reduced form in blood, ciliary stroma, and aqueous. He suggested that the oxidation-reduction potential across the stromal-epithelial barrier is the driving force of aqueous secretion and of the active transport of ascorbate into the aqueous. After being secreted into the aqueous in the oxidized form, it is quickly reduced by an unknown mechanism.3 In a like manner, ascorbate as DHA might be actively transported from the blood and choroidal stroma through the RPE to the retina, where it exists in large concentrations. This theory is supported further by our histochemical localization of reduced ascorbate in the apices of the RPE cells. In addition, the elevated levels of DHA in PE-C immediately after light exposure might indicate increased transport of ascorbate by the RPE to replenish decreased AA levels in the neural retina caused by photochemical oxidation.

No. 7 ASCORBATE IN GUINEA PIG RETINA / Woodford er ol. 867 Key words: retina ascorbate, guinea pig, aqueous, retinal pigment epithelium Acknowledgment The authors wish to thank Dr.

6 No. 7 ASCORBATE IN GUINEA PIG RETINA / Woodford er ol. 867 Key words: retina ascorbate, guinea pig, aqueous, retinal pigment epithelium Acknowledgment The authors wish to thank Dr. Rockefeller Young and Dr. Stuart Starr for their measurements of illumination in these experimental conditions. References 1. Noell WK: Possible mechanisms of photoreceptor damage by light in mammalian eyes. Vision Res 20:1163, Feeney-Burns L, Berman ER,and Rothman H: Lipofuscin of human retinal pigment epithelium. Am J Ophthalmol 90:783, Kuck JFR Jr: Chemical constituents of the lens. In Biochemistry of the Eye, Graymore CN, editor. London, Academic Press, 1970, pp Heath H, Rutter AC, and Beck TC: Changes in the ascorbic acid and glutathione content of the retinae and adrenals from alloxan-diabetic rats. Vision Res 2:431, Heath H, Beck TC, and Rutter AC: Biochemical changes in aphakia. Vision Res 1:274, Friedenwald JS, Buschke W, and Michel HO: Role of ascorbic acid (vitamin C) in secretion of intraocular fluid. Arch Ophthalmol 29:535, Cristiansson J: Changes in the vitreous body in scurvy; studies on guinea pigs in vivo. I. The biopolymers of the vitreous body. Acta Ophthalmol 35:336, Varma SD, Kumar S, and Richards RD: Light-induced damage to ocular lens cation pump: prevention by vitamin C. Proc Natl Acad Sci USA 76:3504, Nirankari VS, Varma SD, Lakhanpal V, and Richards RD: Superoxide radical scavenging agents in treatment of alkali burns; an experimental study. Arch Ophthalmol 99:886, Seregi A, Schaefer A, and Komlos M: Protective role of brain ascorbic acid content against lipid peroxidation. Experientia 34:1056, Ortner MJ: The oxidation of endogenous ascorbic acid during histamine secretion by rat peritoneal mast cells. Exp Cell Res 129:485, Kato N, Kawai K, and Yoshida A: Effect of dietary level of ascorbic acid on the growth, hepatic lipid peroxidation and serum lipids in guinea pigs fed polychlorinated bephenyls. J Nutr 111:1727, Fox RR, Lam KW, Lewen R, and Lee PF: Ascorbate concentration in tissues from normal and buphthalmic rabbits. J Hered 73:109, Chinoy NJ and Sanjeevan AG: On the specificity of alcoholic acidic silver nitrate reagent for the histochemical localization of ascorbic acid; a reappraisal. Histochemistry 56:275, Heath H. The distribution and possible functions of ascorbic acid in the eye. Exp Eye Res 1:362, Varma SD, Srivastava VK, and Richards RD: Photoperoxidation in lens and cataract formation: preventive role of superoxide dismutase, catalase and vitamin C. Ophthalmic Res 14:167, 1982.

Histology of the Eye

Histology of the Eye Objectives By the end of this lecture, the student should be able to describe: The general structure of the eye. The microscopic structure of:»cornea.»retina. EYE BULB Three coats