The Protective Effect of Ascorbic Acid in Retinal Light Damage of Rats Exposed to Intermittent Light

Transcription

1 July 1990 Vol. 31/7 Investigative Ophthalmology G Visual Science Articles The Protective Effect of Ascorbic Acid in Retinal Light Damage of Rats Exposed to Intermittent Light Daniel T. Organisciak, Yih-ling Jiang, Hih-min Wang, and Ina Bicknell Retinal light damage in dark-reared rats supplemented with ascorbic acid and exposed to multiple doses of intermittent light was studied and compared with damage in unsupplemented dark-reared and cyclic-light-reared rats. The extent of photoreceptor cell loss from intense light exposure was determined by whole-eye rhodopsin levels and retinal DNA measurements two weeks after light treatment. Two weeks after 3 or 8 hr of intermittent light, ascorbate-supplemented animals had rhodopsin and retinal DNA levels that were two to three times higher than in unsupplemented dark-reared rats. In both types of rats rhodopsin levels were influenced by the number of light doses, the duration of light exposure, and to a lesser extent, by the length of the dark period between exposures. Rhodopsin levels in the dark-reared ascorbate-supplemented rats were significantly higher than in unsupplemented dark-reared rats, and were similar to the levels in unsupplemented cyclic-light-reared animals. Ascorbate treatment had no effect on the rate of rhodopsin bleaching. However, regeneration was greater in supplemented rats after multiple 1-hr light exposures. Intermittent light also resulted in lower ascorbate levels in the retinas of supplemented and unsupplemented rats, with dramatic losses from the retinal pigment epithelium (RPE)-choroid in both types of animals. We conclude that ascorbic acid protects the eye by reducing the irreversible Type I form of light damage in dark-reared rats. Ascorbate appears to shift light damage to the Type II form typical of cyclic-light-reared animals. Invest Ophthalmol Vis Sci 31: ,1990 A long-term light-rearing environment affects both the rhodopsin level in the rat eye and the overall metabolic state of the retinal photoreceptor cell. 1 ' 6 Prior light history is also a major determinant of the extent and reversibility of retinal photoreceptor cell damage in rats exposed to intense visible light. 7 " 9 Under identical exposure conditions, rats reared in darkness exhibit extensive loss of both photoreceptors and the adjacent retinal pigment epithelium (RPE), whereas visual cell loss without major RPE involvement predominates in rats previously main- From the Department of Biochemistry, School of Medicine, Wright State University, Dayton, Ohio. Supported by grant EY from the National Institutes of Health and the Petticrew research fund at Wright State University (DTO). Presented by Y-L. Jiang in partial fulfillment of the requirements for the Masters Degree in Science, Wright State University. Submitted for publication: October 5, 1989; accepted December 6, Reprint requests: D.T. Organisciak, Department of Biochemistry, Wright State University, Dayton, OH tained in a weak cyclic-light environment. 7 These forms of retinal light damage have been classified by Noell 7 as Type I and Type II. The manner or schedule by which light is administered also can influence the extent of damage. Multiple intermittent light exposures cause more retinal damage than continuous light of the same duration Histologic findings show that RPE damage is greater in rats treated with intermittent light than in animals exposed to continuous illumination." In both cyclic-light- and dark-reared rats, supplementation with ascorbic acid has been found to reduce visual cell loss from damaging light. 912 " 14 Recent electrophysiologic, histologic, and biochemical evidence obtained from dark-reared rats exposed to intense light demonstrates that ascorbate supplementation dramatically protects the RPE. 15 ' 16 In the current paper we describe biochemical measures of retinal cell damage due to intermittent light in rats reared in darkness or in weak cyclic light. Our evidence supports the notion that ascorbic acid reduces retinal damage from intermittent light exposure by interfering with the progression of Type I light damage. 1195

2 1196 INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / July 1990 Vol. 31 Materials and Methods Animal Maintenance, Light Exposure, and Ascorbate Treatment Weanling male albino Sprague-Dawley rats were obtained from Harlan (Indianapolis, IN) and maintained in darkness or in a weak cyclic-light environment (20-40 lux, 12 hr/day) until the age of 60 days. The dark environment was interrupted for less than 0.5 hr/day for routine animal maintenance. During these periods, dim red light was used. All animals were fed Purina rat chow, ad libitum, and had free access to water. Prior to light exposure, all rats were dark adapted for hr. Twenty-four hours before and just prior to the first light exposure, one half of the dark-reared rats were injected intraperitoneally (IP) with L-ascorbic acid (Sigma, St. Louis, MO) at a dose of 0.5 mg/g body wt. 13 Ascorbate-supplemented and unsupplemented rats then were simultaneously exposed to intense green light ( nm) of approximately 2000 lux illumination in green Plexiglas chambers. All chambers were equipped with programmable timers set for multiple light-dark periods and with small electric fans which maintained the environmental temperature at C." The normal experimental paradigm consisted of 1-hr light exposures followed by 2-hr dark periods, with light treatment beginning at 09:00. In some experiments, the light exposure varied from 5-60 min. In other experiments, 1-hr light exposures were used with intervening dark periods of hr. After light treatment the rats were either sacrificed immediately or returned to the dark environment and maintained there for various periods before use. All animals were sacrificed in halothane-saturated chambers. The use of animals in this investigation conformed to the ARVO resolution on the Use of Animals in Research. Measurements of Retinal Light Damage, Rhodopsin Bleaching, and Regeneration Rhodopsin and retinal DNA levels in the eyes of experimental animals maintained in darkness for 2 weeks after intense light treatment were measured to determine the extent of retinal photoreceptor cell loss. Unexposed animals maintained for 2 weeks under the same dark conditions were used as controls. Techniques for the measurement of rhodopsin and retinal DNA have been described. 9 " 15 Bleaching of rhodopsin was determined in ascorbate-supplemented and unsupplemented rats by exposing animals to light for up to 1 hr and then immediately measuring rhodopsin. Rhodopsin regeneration in vivo was measured in other rats exposed to light for 1 hr followed by darkness for as long as 4 hr. In some experiments, three intermittent 1-hr exposures were used with dark periods of hr. Rhodopsin was measured at the end of the third dark period. To determine rhodopsin regeneration in vitro, a crude rod outer segment (ROS) fraction from intermittent light-exposed rats was mixed with a 2- fold molar excess of 9-ds-retinal (Sigma) and incubated in the dark for 1 hr. The ROS were precipitated and isorhodopsin extracted with 1% /3-octyl glucoside (R. K. Crouch, personal communication). Control ROS from unexposed rats were treated with green light for 5 min in vitro before regeneration with 9-cw-retinal. The extent of regeneration was then determined from the AA 480 nm (measured before and after bleaching of the extracts) of experimental and control samples. All detergent extracts were mixed with 10/nl 1 Mhydroxylamine, previously adjusted to ph 7.0. Measurement of Tissue Ascorbate Levels After Light Exposure Ascorbic acid was measured in isolated retinas and in the remaining RPE-choroid-scleral complex by a high-performance liquid chromatography (HPLC) procedure essentially as described. 12 Tissues were excised immediately after light exposure, homogenized in chloroform:methanol (2:1), and mixed with 0.2 volume water. The upper aqueous phase obtained after centrifugation at 6000 g was dried and resolubilized with water. HPLC was performed on a 4.5 X 250 mm octadecyl (C 18) column (5-/xm particle size) at 25 C, with a mobile phase consisting of 3% 0.1N citric acid:97% water, ph 3.5, at a flow rate of 0.5 ml/min. Ascorbate (reduced) was detected at 245 nm and quantitated by comparison with standards of known concentration. As determined by the recovery of L-[l- 14 C]-ascorbic acid (New England Nuclear, Boston, MA), extraction efficiency for retinal and eyecup ascorbate was 93% and 87%, respectively. In some experiments, blood in the ocular tissues of control and ascorbate-supplemented rats was removed by transcardiac perfusion with normal saline. Ether-anesthetized animals were perfused with 200 ml saline immediately before tissue excision. The retina was removed, and the remaining eyecup was everted over a small plastic tip which was placed into the chloroform:methanol solution. A brief (20-sec) sonication was used to separate the RPE-choroid from the sclera; tissues were then homogenized and extracted separately. Blood obtained by cardiac puncture before perfusion was mixed with 2 volumes of ice cold 4% per-

3 i-oi 4 i l-o-l 1- No. 7 INTERMITTENT LIGHT DAMAGE AND ASCORDATE / Orgonisciok er ol 1197 Table 1. Rhodopsin and visual cell DNA in rats exposed to intermittent light Rhodopsin* (nmol/eye) DNA (9, o of control) Exposure time (hr) Unsupplemented Unsupplemented Ascorbatesupplemented Ascorbatesupplemented ±0.1 (26) 0.8 ±0.2(10) 0.3 ±0.1 (20) 0.1 ±0.1 (18) 2.2 ±0.2 (10) 1.7 ±0.2 (12) 1.0 ±0.1 (14) 0.1 ±0.1(4) 40 ± 9(13) 18± 8(8) 8± 7(8) loof 83 ± 7(4) 46 ± 14(6) 6± 4(6) * Rhodopsin measured in unexposed rats without 2-week dark maintenance was 2.0 ± 0.2, in both the unsupplemented and ascorbate-supplemented animals. Results are the mean ± SD for the number of eyes shown in parentheses. Ascorbate, 0.5 mg/g 2 X IP, was given 24 hr before and just prior to light exposure (1 hr light 2 hr dark periods). f Retinal DNA in the unexposed dark-reared rats maintained for 2 weeks in darkness. chloric acid. After centrifugation at g for 15 min, aliquots of the supernatant were injected directly onto the same HPLC column as used for retinal analysis. Results Rhodopsin and Retinal DNA as Indicators of Light Damage The protective effect of ascorbic acid was determined in dark-reared rats by measuring rhodopsin levels and retinal DNA 2 weeks after intermittent light exposures. Table 1 shows that for 3- and 8-hr exposures, rhodopsin and visual cell DNA were higher in the ascorbate-supplemented rats. For 3 hr of intermittent light, rhodopsin in the ascorbate-treated rats was 77% of the level in dark-maintained controls (2.2 nmol/eye). Unsupplemented rats had only 35% of the control rhodopsin level. After 8 hr of intermittent light the difference between the ascorbate-supplemented and unsupplemented rats was about 3- fold 45% compared to 13% of the control rhodopsin level. However, after 24 1-hr doses of light, rhodopsin was only 4-5% in both types of rats. As shown by the 0-hr rhodopsin values, ascorbate treatment had no effect on the level of rhodopsin in unexposed control rats. Visual cell DNA, determined in other rats, exhibited the same dramatic differences as seen for rhodopsin. DNA levels in ascorbate-supplemented and unsupplemented animals were 83% and 40% 2 wks after 3 hr of intermittent light; the levels were 46% and 18% after eight 1 -hr light exposures. In both types of rats, DNA was low (6-8%) after 24 1-hr light doses. The similar recoveries of visual cell DNA and rhodopsin show that either measurement is a good indicator of light-induced photoreceptor cell damage. Retinal Damage as a Function of Light or Dark Duration Figure 1 shows that, in dark-reared rats, the length of the light period had a greater effect on retinal dam- Fig. 1. Rhodopsin recovery after intermittent exposure using three doses of light of increasing duration (A) or three 1-hr light doses with increasing dark duration between light exposures (B). Dark-reared ascorbatetreated rats (triangles) (0.5 mg/gm 2 X IP) and unsupplemented rats (rilled circles) were exposed simultaneously and maintained subsequently in total darkness for 2 weeks. Cycliclight-reared rats (open circles), were similarly treated, but without ascorbate treatment. In (A), 0 min refers to (/) Q. o o o i -1 - t 1 N r " ~~ i > i l i.. I pre Un- 0 exposure exposed Control Light duration (min) A - I r^ i ^ i i i Dark duration (hrs) no light exposure. Preexposure rhodopsin levels were nmol/eye; 2 weeks later, rhodopsin in the unexposed control rats had increased to nmol. Values are given as the mean ± SD (n = 8-10). I z B 1 -*

4 1198 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / July 1990 Vol. 31 age than did the length of the dark period. Rats were exposed to three doses of light of 5-60 min, each followed by a 2-hr dark period (Fig. 1A), or to three 1-hr light periods with intervening and equivalent dark periods of 30 min to 8 hr (Fig. IB). For all light exposures, ascorbate-treated animals recovered significantly more rhodopsin than did their untreated counterparts (Fig. 1A). After three 10-min exposures, the unsupplemented rats had 1.7 nmol rhodopsin/eye compared to 2.2 nmol in the ascorbate-supplemented animals. With 30-min light exposures, the unsupplemented rats had about 0.9 nmol rhodopsin/eye; ascorbate-treated animals recovered more than twice as much rhodopsin (1.9 nmol). The unsupplemented dark-reared rats had about 0.8 nmol of rhodopsin 2 weeks after three 1-hr light exposures, and the ascorbate-supplemented rats had 1.7 nmol/eye. Regardless of the length of the dark period, the unsupplemented rats had rhodopsin losses of about 65% 2 weeks after three intermittent 1-hr exposures (Fig. 1B). Light damage in the ascorbate-treated rats exhibited a similar lack of dependence on dark periods of up to 2 hr (20-25% rhodopsin loss), but rhodopsin was lower when the dark period was extended beyond 2 hr. In all cases, however, rhodopsin levels were significantly higher than in unsupplemented dark-reared rats, and close to the levels found in cyclic-light-reared rats similarly exposed. To compare further the light damage in ascorbatesupplemented dark-reared rats with damage in cyclic-light-reared rats, rhodopsin was measured in animals exposed to light for 3 hr, but exposed in a variable number of doses. Figure 2 shows that light damage in all rats increased as a function of the number of light exposures. In the unsupplemented darkreared rats, 180 min of light given as one 3-hr continuous light exposure resulted in a rhodopsin level of about 1.5 nmol/eye. The same light given in nine 20-min doses (alternating with 2-hr dark periods), resulted in only 0.35 nmol rhodopsin/eye. Ascorbatesupplemented dark-reared rats also exhibited a dependence on the number of light doses, but the effect was much less dramatic. Two weeks after one or nine consecutive light exposures, rhodopsin was 1.95 and 1.55 nmol/eye, respectively. Light damage in the cyclic-light-reared rats was less. In these animals, rhodopsin levels were 2.1 and 1.8 nmol/eye, respectively, after one or nine light doses. In these experiments preexposure rhodopsin levels were nmol/eye for dark-reared rats and 1.75 nmol/eye for the cycliclight-reared animals. In all unexposed control rats (zero doses of light), rhodopsin levels were 2.25 nmol/eye after 2 weeks in darkness. pre- 0 exposure 3 6 Number of light doses Fig. 2. Rhodopsin levels 2 weeks after multiple light exposures. Dark-reared rats treated with ascorbate (triangles) or untreated (filled circles), and cyclic-light-reared rats (open circles) without ascorbate, were exposed to light for a total of 3 hr. The number of light exposures was varied from one (continuous) to nine (intermittent) doses. Two-hour dark periods were used for intermittent light treatment. Values are the mean ± SD for eight to ten eyes (four to five rats). Rhodopsin Levels During and After Light Exposure Ascorbic acid treatment had no effect on the rate of rhodopsin bleaching (Figure 3). In both ascorbatesupplemented and unsupplemented dark-reared rats, about 90% of the rhodopsin was bleached after 10 min of light and nearly 95% bleached after a 30-min exposure (Fig. 3A). Rhodopsin regeneration, however, was influenced by ascorbic acid supplementation. If light was given in a single 1-hr dose, the rates of regeneration were the same in both types of rats (Fig. 3B). If light was given intermittently with dark periods of various durations, the ascorbate-treated rats had higher rhodopsin levels than the unsupplemented animals (Fig. 3C). Thirty minutes after three light exposures with 30- min dark periods, 42% more rhodopsin was found in the ascorbate-treated rats than in the unsupplemented animals (1.0 vs 0.7 nmol/eye). The ascorbate-treated rats had 33% more rhodopsin with 2-hr dark periods, and 14% more rhodopsin with 4-hr dark periods between exposures. These differences do not appear to have resulted from a direct (damaging) effect of light on rhodopsin's ability to regenerate. Both ROS from unsupplemented light-exposed rats (three 1-hr light periods) and ROS from unexposed controls formed % isorhodopsin when incubated in vitro with 9-ds-retinal (data not shown).

5 No. 7 INTERMITTENT LIGHT DAMAGE AND ASCORBATE / Organisciak er al 1199 three 1 - hr light doses C Light exposure (min) Dark (hrs) Dark (hrs) Fig. 3. Rhodopsin bleaching and dark regeneration as a function of light or dark duration. Ascorbate-treated (triangles) and untreated (filled circles) dark-reared rats were exposed simultaneously to light for up to 1 hr, and rhodopsin was measured immediately (A) or after 1 hr of light exposure followed by various periods in darkness (B). (C) Data for ascorbate-treated (triangles) or untreated (filled circles) rats exposed to 3 1-hr light doses with intervening dark periods of from 30 min to 4 hr. Values are given as the average of four to six determinations (A), or the mean ± SD (n = 6-8) (B, C). Tissue Ascorbate Distribution in Rats Before or After Light Exposure Two methods were used to obtain tissues for the determination of ascorbate distribution. In method A, tissues were excised from rats after removal of blood by transcardiac perfusion with normal saline. In method B, tissues were excised from animals without perfusion. Method A: Prior to light exposure, the average ascorbic acid level in the retinas of unsupplemented rats was 12.8 nmol; retinal ascorbate was 36% higher in the supplemented animals (Table 2, row 1). In the intact RPE-choroid-scleral complex (whole eyecup) of unsupplemented rats, 9.8 nmol of ascorbate was found; it was distributed 14%/86% between the RPE-choroid and the sclera. The ascorbic acid level in the whole eyecup of supplemented rats was 2.5 times higher with a distribution of 28% in the RPEchoroid and 72% in the sclera. In the supplemented rats the RPE-choroid contained almost six times more ascorbic acid than in unsupplemented animals; the corresponding scleral tissue was 2.5-fold higher in ascorbate. No differences were apparent between the cyclic-light and dark-reared rats used in these experiments. A comparison of ascorbate values in rats exposed to three doses of light (Table 2, row 2) with those of rats before light exposure shows a decrease of 7-20% from the retina (unsupplemented and supple- Table 2. Tissue ascorbate distribution in perfused rats (cyclic-light- and dark-reared) (nmol/tissue ± SD) Unsupplemented Ascorbate-supplemented* Retina^ Whole eyecup RPEchoroid Sclera Bloody Retina Whole eyecup RPEchoroid Sclera Bloody Unexposed (n = 5-6) 12.8 ± ± ± ± ± ± ± ± ± ±470 Exposed, 3 1-hr light periods (n = 5-7) 11.9 ± ± ± ± ± ± ± ± ± ±4.5 * Ascorbated-supplemented rats were given 0.5 mg/g body weight 2 X IP 24 hr before and min prior to tissue excision or perfusion. t Retinal values are the average of the left and right retinas from a single animal. Whole eyecup values (intact RPE-choroid-sclera) were obtained from the left eyecup and the RPE-choroid/sclera distribution from the corresponding right eyecup. % Blood values are in nanomoles per milliliter.

6 1200 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / July 1990 Vol. 31 mented). Ascorbate was similar in the eyecups of unsupplemented rats, whereas in the supplemented animals 50% of the eyecup ascorbate was lost. Although there was no loss of ascorbate from the intact eyecup of unsupplemented rats, approximately 40% of the ascorbate contained in the RPE-choroid fraction was lost after three 1-hr exposures. In the RPE-choroid of ascorbate-treated rats, an 83% reduction in ascorbate occurred. About 50% of the ascorbate was also lost from the sclera of ascorbate-treated rats, whereas scleral ascorbate was actually higher in the unsupplemented animals. Despite these differences, only 8% of the eyecup ascorbate in unsupplemented rats and 12% in supplemented animals was contained in RPE-choroid after light, compared to 14% and 28% before light exposure. After light exposure, blood ascorbate was lower in both types of rats, but the decrease in the supplemented animals was large. In these rats, blood ascorbate after three light doses (7 hr after injection) was the same as in the unsupplemented control rats. It is unlikely, therefore, that ascorbate in the choroidal circulation has a major effect on the eyecup levels of unperfused rats after three or more light exposures (see below). Method B: Ascorbic acid in the retinas of unsupplemented and ascorbate-treated rats decreased by similar amounts during intermittent light exposure (Table 3). Compared to the retinal values for unexposed dark-reared animals (Table 3, row 1), the unsupplemented rats lost an average of 13% retinal ascorbate after three 1-hr light exposures and 34% after 8 hr of light. Retinal ascorbate in the supplemented rats was 6% lower after three light doses and 30% lower after eight doses of light. After 24 1-hr exposures (72 hr overall), retinal ascorbate levels were the same in both the supplemented and unsupplemented rats. These values were higher than those of rats exposed to light for only 8 hr. Light exposure had little or no effect on ascorbate levels in the intact RPE-choroid-scleral complex of the unsupplemented dark-reared rats. Values of nmol were measured after light exposure; nearly the same as found in the 0-hr control rats. In the supplemented rats, ascorbate was 10.5 nmol/eyecup after 3 hr of intermittent light, and nmol after the longer light exposures. Discussion The high damaging potency of an intermittent light schedule was originally described by Noell et al in Using rats maintained under hyperthermic conditions, which potentiates damage by continuous light, Noell found irreversible electroretinogram (ERG) changes after administering only three or four 5-min doses of light. 10 Similarly, O'Steen et al 17 found dramatic histologic damage as a function of age in hyperthermic rats exposed to intermittent light for 17.5 hr using 5 equivalent doses. Sperling 18 studied cone damage in monkeys exposed to blue light in a continuous or intermittent fashion and concluded that irreversible blue cone damage involved repeated partial bleaching and recovery of the cone pigment. Recently, we described retinal damage from intermittent light in rats maintained at normal body temperature." In agreement with earlier studies, we concluded that multiple intermittent exposures led to increased retinal damage compared with continuous light of the same duration." In the current study, we found a remarkable protective effect by ascorbic acid in dark-reared rats treated with intermittent light. As measured by the levels of rhodopsin or visual cell DNA, 3 or 8 hr of light resulted in significantly less damage in ascorbate-treated rats than in unsupplemented animals (Table 1). In all rats, retinal damage was found to depend on the number of light doses (Table 1, Fig. 2) and on the duration of exposure (Fig. 1A). A protective effect by ascorbate in dark-reared rats exposed to continuous light has recently been reported by Noell et al. 15 In that study, electrophysiologic measures of the intactness of the RPE layer, the azide and thiocyanate response, were dramatically higher in the ascorbate-treated animals. 15 As deter- Table 3. Tissue ascorbate levels in light-exposed unperfused Unsupplemented rats (nmol/tissue)* Ascorbate-supplemented Exposure time (hr) Retina (10) Whole eyecup (10) Retina (5) Whole eyecup (5) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 1.5 * Results are the mean ± SD for n retinas or whole eyecups (RPE-choroid-scleral complex). Tissues were isolated immediately after the last light period. Ascorbate-supplemented rats were given 0.5 mg/g body weight 2 X IP, 24 hr before and just prior to light exposure. One-hr light, 2-hr dark periods used throughout.

7 No. 7 INTERMITTENT LIGHT DAMAGE AND ASCORDATE / Orgonisciok er ol 1201 mined at both the light and electron microscopic levels, 16 ascorbate-treated rats exhibit less RPE damage than do unsupplemented animals. The ascorbate-treated rats also had lower levels of ROS phagosomes, 16 an indication of reduced photoreceptor damage. In the current study, similar rhodopsin levels were found in ascorbate-treated dark-reared rats and in unsupplemented cyclic-light-reared animals, indicating that ascorbic acid prevents the dramatic RPE and photoreceptor cell loss typically associated with Type I light damage. The protective effect of ascorbate, therefore, appears to be related to its ability to shift the form of light damage from Type I, to the Type II form usually found in cyclic-lightreared rats. 7 Earlier studies 1213 and the current work show that ascorbic acid decreases in the rat retina in response to light. For both supplemented and unsupplemented rats, similar decreases were found in the retinas after 3 or 8 hr of intermittent light (Table 2, 3). In addition, after 3 hr of light, there was a dramatic loss of ascorbate from the RPE-choroid in both types of rats and a change in its distribution between RPE-choroid and sclera. Whether the loss of ocular ascorbate during light occurs by leakage from the RPE cell layer or by oxidation to dehydroascorbate is currently unknown. In other animal species, 19 " 21 it is known that the RPE contains both ascorbate and dehydroascorbate and that their levels are altered by light exposure Ascorbate supplementation resulted in an almost 6-fold increase in ascorbic acid levels in the RPEchoroid of unexposed rats (Table 2). Based on kinetic considerations for RPE cell uptake of ascorbate 22 and the blood levels in supplemented rats after injection, a rise in RPE levels would be expected. For the supplemented rats, blood ascorbate was the same as control after three 1- hr light doses, and whole eyecup values were nearly the same as in the unsupplemented rats (Tables 2, 3). The determining factor for elevating RPE ascorbate, therefore, appears to be its concentration in blood and the length of time that that level remains above normal. In the rats treated with 24 1-hr light doses, retinal ascorbate also was elevated (Table 3), but light damage was extensive in both types of animals (Table 1). This suggests that ascorbate may be concentrated in the surviving inner retinal layers of these rats, independent of the normal RPE transport mechanism. 22 Thus, ascorbate supplementation effectively reduces or delays retinal damage for up to 24 hr after injection (eight 1-hr light doses), but its protective effect can be overcome with longer periods of exposure. Our study also shows that the damaging effects of intermittent light in the eye are not entirely dependent on the rhodopsin concentration at the time of exposure. Although light damage increased with the number and duration of bleaching light doses, in the unsupplemented rats, retinal damage was nearly the same regardless of the length of the dark period between light exposures (Fig. IB). In the ascorbate-supplemented rats, rhodopsin losses were only 20-25% for multiple exposures with dark periods of up to 2 hr in length. These rats also regenerated higher levels of rhodopsin between exposures (Fig. 3C), yet had significantly less retinal damage than the unsupplemented animals. Likewise, the cyclic-light-reared rats demonstrated less retinal damage from intermittent light, even though rhodopsin regeneration between exposures is known to be greater than in comparable dark-reared animals.'' Clearly, factors in addition to the bleaching of rhodopsin must influence the extent of retinal damage by light. In both the ascorbate-treated and unsupplemented rats, slightly lower rhodopsin levels were found with 1-hr dark periods in comparison to 30-min or 2-hr dark periods (Fig. IB). Although the individual changes were not significant, they do suggest complex interactions between the generation of a toxic substance^) and the damage and repair mechanisms that may occur during the light or dark periods. Previous work suggests that ascorbic acid may act by interrupting the formation, or action, of a light-generated toxic substance in the eye. 15 This remains to be proven. An increasing body of evidence points to an effect of long-term light rearing environment on the metabolic state of visual cells. 1 " 3 ' 6 ' 8 Adaptive changes in the levels of ROS docosahexaenoic acid (ROS 22:6) of rats born and reared under different light intensities have been reported. 6 It is also known that ROS 22:6 is lost from the ROS of rats exposed to intense visible light 13 ' 23, and that intermittent light increases this loss.'' In the current study, we found that ascorbate treatment reduced the loss of ROS 22:6 during light exposure. After eight 1-hr exposures, ROS 22:6 was 47 mol % compared to 39 mol % in the ROS of unsupplemented rats (data not shown). It is not yet clear if the loss of ROS 22:6 during light exposure is a causative factor in light damage or is a result of the damage process. Environmental light-dependent changes in the levels of retinal S-antigen (S-ag) (48K protein, arrestin) and the alpha subunit of transducin (T«) (G-protein) have recently been reported. 24 Several other studies indicate that S-ag migrates from the photoreceptor cell body into the ROS during light 25 " 28 ; there, it may be involved in the inactivation of photolyzed rhodopsin. 29 More importantly, however, S-ag and Ta compete for binding to the phosphorylated C-ter-

8 1202 INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / July 1990 Vol. 31 minal region of rhodopsin during illumination. 29 Thus, adaptive changes in the levels of transduction proteins in the photoreceptors and their movement into or out of the ROS may modulate light damage. How proteins normally involved in the transduction of light influence retinal damage during prolonged exposure remains to be determined. Further studies of these proteins may provide important clues to understanding the mechanisms involved in the Type I and Type II forms of retinal light damage. Key words: light damage, rats, ascorbic acid, intermittent exposure References 1. Noell WK, Delmelle MS, and Albrecht R: Vitamin A deficiency effect on retina: Dependence on light. Science 172:72, Noell WK and Albrecht R: Irreversible effects of light on the retina: Role of vitamin A. Science 172:76, Organisciak DT and Noell WK: The rod outer segment phospholipid/opsin ratio of rats maintained in darkness or cyclic light. 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