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(Investigative Ophthalmology and Visual Science. 2000;41:3694-3701.)
© 2000 by The Association for Research in Vision and Ophthalmology, Inc.

Circadian-Dependent Retinal Light Damage in Rats

Daniel T. Organisciak1, Ruth M. Darrow1, Linda Barsalou1, R. Krishnan Kutty2 and Barbara Wiggert2

1 From the Petticrew Research Laboratory and the Departments of Biochemistry/Molecular Biology and Ophthalmology, Wright State University School of Medicine, Dayton, Ohio; and the 2 Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, National Institutes of Health, Bethesda, Maryland.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. To determine the relative susceptibility of rats to retinal light damage at different times of the day or night.

METHODS. Rats maintained in a dim cyclic light or dark environment were exposed to a single dose of intense green light beginning at various times. Normally, light exposures were for 8 or 3 hours, respectively, although longer and shorter periods were also used. Some animals were treated with the synthetic antioxidant dimethylthiourea (DMTU) before or after the onset of light. The extent of visual cell loss was estimated from measurements of rhodopsin and retinal DNA levels 2 weeks after light treatment. The time course of retinal DNA fragmentation, and the expression profiles of heme oxygenase-1 (HO-1) and interphotoreceptor retinol binding protein (IRBP) were determined 1 to 2 days after exposure.

RESULTS. When dark-adapted, cyclic light–reared or dark-reared rats were exposed to intense light during normal nighttime hours (2000–0800) the loss of rhodopsin or photoreceptor cell DNA was approximately twofold greater than that found in rats exposed to light during the day (0800–2000). The relative degree of light damage susceptibility persisted in cyclic light–reared rats after dark adaptation for up to 3 additional days. For rats reared in a reversed light cycle, the light-induced loss of rhodopsin was also reversed. Longer duration light treatments revealed that dim cyclic light–reared rats were three- to fourfold more susceptible to light damage at 0100 than at 1700 and that dark-reared animals were approximately twofold more susceptible. Intense light exposure at 0100 resulted in greater retinal DNA fragmentation and the earlier appearance of apoptotic DNA ladders than at 1700. The extent of retinal DNA damage also correlated with an induction of retinal HO-1 mRNA and with a reduction in IRBP transcription. Antioxidant treatment with DMTU was effective in preventing retinal light damage when given before but not after the onset of light.

CONCLUSIONS. These results confirm earlier work showing greater retinal light damage in rats exposed at night rather than during the day and extend those findings by demonstrating that a single, relatively short, intense light exposure causes a circadian-dependent, oxidatively induced loss of photoreceptor cells. The light-induced loss of photoreceptor cells is preceded by DNA fragmentation and by alterations in the normal transcriptional events in the retina and within the photoreceptors. The expression profile of an intrinsic retinal factor(s) at the onset of light exposure appears to be important in determining light damage susceptibility.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Retinal photoreceptor cells are paradoxically efficient in their ability to capture photons for the initiation of visual transduction while being vulnerable to cellular damage from excess light. Because rhodopsin bleaching serves as the trigger for light’s physiological or pathologic1 effects in the retina, the initial site of photon absorption is at the level of the rod outer segments (ROS). Under prolonged or intense light conditions this initial response leads to cellular damage, resulting in the death and disappearance of photoreceptors. Visual cell death occurs by an apoptotic process involving nuclear chromatin condensation and DNA fragmentation,2 3 4 which appears to require the proapoptotic gene c-fos,5 as well as an alteration in calcium homeostasis.6 Intense light exposure also triggers an oxidative process in the retina as natural or synthetic antioxidants reduce photoreceptor cell death, DNA fragmentation,7 8 damage to the opsin gene,9 the expression of heme oxygenase-1 (HO-1), a 32-kDa antioxidative stress protein,10 and the loss of polyunsaturated fatty acids from ROS membranes.11

The extent of retinal light damage is modulated by light intensity and the duration of exposure,1 the wavelengths of light used,1 12 13 diet, age, and genetic factors (reviewed in Refs. 14 15 ). Long-term adaptive processes regulated by different light-rearing intensities16 17 or by dark rearing18 19 are additional extrinsic factors that alter the susceptibility of visual cells to light-mediated damage. Circadian-dependent alterations in opsin gene expression20 and in the expression of other intrinsic proteins involved in visual transduction have been reported.21 22 23 24 Circadian rhythmicity and/or diurnal light cycles are also known to affect ROS disc shedding25 26 27 and retinal melatonin and dopamine levels28 29 30 31 (reviewed in Refs. 32 33 ). Melatonin injections affect disc shedding in rats34 and increase light damage susceptibility.35 36 37 The administration of luzindole, a melatonin receptor antagonist, reduces the extent of retinal light damage38 as does pinealectomy,39 although pinealectomy may not affect retinal melatonin levels or its circadian rhythm.40

Because earlier studies describing the diurnal dependence of retinal light damage used multiple light exposures over several days41 42 or multiple melatonin injections,35 36 37 which could change circadian rhythms and light detection thresholds,43 we exposed rats to a single dose of intense visible light at various times of the day or night. Our study confirms that the circadian-dependent loss of visual cells is greatest when intense light exposure begins during the normal nighttime phase of the diurnal cycle. We also show that retinal light damage is almost completely prevented when light treatment begins during the day or when an antioxidant is given before exposure, irrespective of the prior long-term rearing conditions of the animals. This study suggests that an intrinsic factor(s) expressed in the retina in a circadian manner influences the extent of intense light-induced photoreceptor cell death.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animal-Rearing Conditions
Weanling male albino Sprague–Dawley rats from Harlan Inc. (Indianapolis, IN) were maintained under dim cyclic light conditions or in darkness for a period of 40 days before use. The cyclic light environment consisted of 12 hours white incandescent light (20–30 lux) per day, with lights normally on at 0800 and off at 2000. Other weanling rats were maintained for 40 days in a reversed light cycle: lights on 2000 and off 0800. The dark-rearing environment was interrupted for less than one-half hour each day for routine animal maintenance, under dim red illumination >600 nm. These maintenance times were within the same 2-hour period (1000–1200) for both rearing conditions. Animal activity profiles were monitored by using small microphones and strip chart recorders, placed near the cage racks in each room. Cyclic-reared rats were normally dark adapted for 16 hours before light treatment, but in one experiment the animals were dark adapted for up to 3 additional days. The use of rats in this investigation conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

Intense Light Exposure
Rats were exposed to 490- to 580-nm light in green Plexiglas chambers1 (no. 2092; Dayton Plastics, Dayton, OH) beginning at various times of the day or night. The animals were unanesthetized and unrestrained during light exposure. Light intensity during exposure was 1200 to 1400 lux, approximately 200 µW/cm2 corneal irradiance (light meter 1 L 1400A; International Light, Newburyport, MA). Rats from the dim cyclic light environment were normally treated with intense light for 8 hours. Because of their greater light damage susceptibility,7 dark-reared rats were exposed to light for 3 hours. These exposure times resulted in approximately 50% visual cell loss for rats treated with light beginning at 0100 and were selected as the criterion for relative light damage susceptibility in this study (cf. Figs. 1 and 4 ) and as a means to demonstrate the efficacy of antioxidant treatments in previous work.8 9 10 11 In some experiments longer or shorter light treatments were used. Some rats were given the synthetic antioxidant dimethylthiourea (DMTU) at a dose of 500 mg/kg IP, one or two times, before or during light treatment.7 11 After treatment the rats were either killed immediately in a CO2-saturated chamber or were maintained in darkness for 2 additional weeks before use.



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  		Figure 1. Rhodopsin recovery after light exposure starting at different times. Results are presented as the mean ± SD for n = 4–20 pairs of eyes from dark-adapted rats exposed to intense light. ({blacktriangleup}), Rats treated with DMTU before light exposure10 at 0100; ({blacksquare}), unexposed control rhodopsin values are from 5 to 10 animals in darkness for the same 2 week period as the light-exposed rats and represent 100% recovery of visual pigment. The bar at the bottom represents periods of animal activity, measured with microphones, in the animal rearing facility. *Rhodopsin values significantly lower than for the average of 0800–2000 (P < 0.001).

 


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  		Figure 4. Effects of intense light duration on rhodopsin recovery in rats exposed at different times. Rhodopsin for cyclic light–reared rats (•) treated with light at 0100 (n = 4–18); 0900 (n = 8–12) or 1700 (n = 8–12), mean ± SD. Unexposed control value 2.26 ± 0.11 nmol/eye (n = 11). For dark-reared rats at 0100 (•) rhodopsin from (n = 4–12) animals; for 1700 ({blacktriangleup}) (n = 4–16) rats. Unexposed control rhodopsin value 2.19 ± 0.14 nmol/eye (n = 12). *For cyclic light– and dark-reared rats rhodopsin values at 0100 were significantly lower than for 0900 or 1700 (P < 0.001).

 
Rhodopsin and Retinal DNA
End point determinations of photoreceptor cell survival were made by measuring the whole eye rhodopsin content44 and retinal DNA levels7 in the same rats 2 weeks after intense light exposure. In some experiments both eyes were used for rhodopsin, and the average was taken as a single value. Unexposed control animals, moved to darkness at the same time as experimental rats, were used to estimate 100% recovery of rhodopsin or retinal DNA. Although rhodopsin levels are maximized by dark maintenance, DNA levels are unaffected by dark rearing,7 and a linear correlation has been found between the two measurements in light-damaged rats.14

The DNA content of rat photoreceptors was calculated by subtracting the DNA remaining in the inner retinal layers of 6-month-old RCS dystrophic rats from total retinal DNA in control and experimental animals.7 14 The DNA values for intact retinas from normal rats and those from 6-month-old RCS rats were found to be 263 and 75 µg, respectively.

Because rhodopsin gene expression increases during the night20 and phagocytosis of the shed tips of ROS is greatest after the onset of light in the morning,25 26 we also determined its content in whole eye preparations, excised retinas, and the remaining eye cups of rats. Dark-adapted, cyclic light– and dark-reared rats were killed at 0100, 0900, or 1700, and the tissues were dissected as described.44 The fraction of rhodopsin in the eye cups was used to estimate the relative degree of phagocytosis by retinal pigment epithelium (RPE), but also includes rhodopsin from mechanically dissociated ROS.44 Rhodopsin was extracted with 1.5% Emulphogene BC-720 detergent (Sigma, St. Louis, MO) and measured as previously described.7

DNA Extraction and Gel Electrophoresis
Individual retinas were digested and DNA extracted as previously described.7 The DNA extracts were electrophoresed on 1.5% neutral agarose gels for 1.5 hours in 40 mM Tris-acetate containing 1 mM EDTA, gels stained with ethidium bromide, and photographed under UV light (302 nm) using a Fotodyne/Analyst camera with computer-controlled display (Hartland, WI). Each lane in the gels was loaded with 2 µg DNA representing the combined DNA from four retinas of four separate rats. A 100-bp marker (Gibco BRL, Gaithersburg, MD) was used as a DNA size standard.

Northern Blot Analysis
The fellow eyes from rats used for DNA gel electrophoresis were extracted with RNAzol B (Tel-Test, Friendswood, TX). Total RNA was then ethanol precipitated and dissolved in diethylpyrocarbonate-treated water and electrophoresed on agarose gels in the presence of formaldehyde. After electrophoresis, RNA was capillary blotted onto a Nytran membrane (Schleicher & Schuell, Inc., Keene, NH), UV cross-linked, and then hybridized at 68°C with a rat HO-1 probe labeled with 32P.10 The membranes were washed under stringent conditions and then exposed to x-ray film (X-OmatAR; Kodak, Rochester, NY). Blots were then stripped with a solution of 0.1x SSC and 0.1% SDS heated to boiling, rehybridized with a 32P probe for human interphotoreceptor retinol-binding protein (IRBP),45 washed, and exposed to x-ray film as above. The HO-1 probe was generated by RT-PCR from spleen poly(A)+ RNA preparations as described.10 The IRBP probe was a gift from Federico Gonzalez-Fernandez. Both probes were labeled with 32P by random priming.46

Statistical Evaluation
The data were analyzed by one- or two-way analysis of variance (ANOVA) with post hoc Tukey’s multiple comparison procedure or by using Student’s 2-sample, 2-tailed t-test. P values < 0.05 were considered to represent significant differences.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Retinal Light Damage Varies with Circadian Time
To determine relative light damage susceptibility, we used intense visible light exposures beginning at various times of the day or night. As shown in Figure 1 , both the dim light–reared rats and those reared in darkness recovered less rhodopsin when light treatment was initiated at night (2000–0800). For dim light–reared rats exposed for 8 hours to intense light at 2300 or 0100, the recovery of rhodopsin was only 54% and 42% of control, respectively (P < 0.001). For light treatment at 0700 the recovery of rhodopsin was 79% of the unexposed control value (2.24 ± 0.11 nmol/eye; n = 10). When intense light exposure was initiated during the day (0800–2000), the average recovery of rhodopsin was twofold higher than that found in rats exposed at 0100 (P < 0.001).

A similar profile of retinal light damage was found in the more sensitive dark-reared rats.7 For example, 3-hour light exposures beginning at 2300 and 0100 resulted in nearly the same rhodopsin recoveries as for dim cyclic light–reared rats. Light treatment during the middle to late morning hours (0900–1100) resulted in a greater rhodopsin recovery than at night. Remarkably, light exposure of dark-reared rats beginning in midafternoon to early evening (1500–1900) caused little retinal damage. These animals averaged 86% rhodopsin recovery compared with unexposed controls (2.20 ± 0.09 nmol/eye; n = 5). By ANOVA there was a significant correlation between light exposure start time and rhodopsin recovery in both cyclic light–reared (F = 26.12; P < 0.001) and dark-reared rats (F = 15.00; P < 0.001). When dark- or cyclic light–reared rats were pretreated with the antioxidant DMTU and then exposed to light at 0100, retinal light damage was completely prevented. For rats not given DMTU, reduced light damage susceptibility correlated with the periods of decreased activity in their rearing environments.

In separate experiments we measured rhodopsin in one eye and retinal DNA in the fellow eyes of rats exposed to light beginning at 0100, 0900, or 1700. By either measurement, light exposure starting at 0100 resulted in greater visual cell loss than exposure at the other times (Table 1) . In both cyclic light– and dark-reared rats, intense light treatment at 0100 led to rhodopsin and visual cell DNA levels that were significantly lower than those in unexposed controls (P < 0.001). Light exposure at 1700 resulted in substantially greater recoveries of rhodopsin and DNA, with no significant differences from control animals.


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  		Table 1. Rhodopsin and Photoreceptor Cell DNA in Rats Exposed to Light

 
Light Damage in Rats from a Reversed Light Cycle
To further characterize the circadian dependence of retinal light damage, rats from the normal dim light cycle and others maintained for 40 days in a reversed light cycle were treated for 8 hours with intense light. Figure 2 shows that rhodopsin levels for rats exposed to damaging light during their respective daytime periods were greater than those for rats exposed during their respective night. For normal cycle rats treated with light at 0100, rhodopsin recovery was 37% of control (2.20 ± 0.10 nmol/eye; n = 8) and 74% for rats from the reverse cycle. Conversely, reversed cycle rats exposed to light at 1700 recovered only 41% rhodopsin, compared with 82% for rats from the normal rearing environment. Reversed cycle rats exposed to light 1 hour into their dark period (0900) also recovered less rhodopsin than normal cycle rats exposed 1 hour into their respective day. By ANOVA there were significant differences among the three light exposure start times for both the normal and reversed light cycle rats (P < 0.001; P < 0.05, respectively). In both groups, rhodopsin values for rats exposed to light at 0100 were significantly different from those of rats exposed at 1700.



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  		Figure 2. Rhodopsin levels in rats reared in a normal or a 12 hour reversed light cycle. Results are the average rhodopsin levels ± SD for n = 4 for rats exposed to intense light for 8 hours beginning at 0100, 0900, or 1700. All rats were dark-adapted for 16 hours before exposure beginning at the same time. The shaded areas show the normal and reversed light/dark-rearing conditions. *Rhodopsin values at 0100 significantly different from for values at 1700 (P < 0.05).

 
Persistence of Light Damage in Dark-Adapted Rats
Because circadian rhythms are retained when animals from a diurnal light cycle are kept in darkness for several days,25 26 27 we extended the normal 16-hour, dark adaptation period for up to 3 days before intense light treatment. As determined by rhodopsin or DNA measurements, extended dark adaptation did not substantially alter the profile of retinal light damage (Fig. 3) . For rats exposed to intense light at 0100, rhodopsin was 42% of the unexposed control after 16 hours of dark adaptation and approximately 30% of control (2.20 ± 0.10 nmol/eye; n = 11) for the longer dark periods. Photoreceptor cell DNA was between 38% and 57% of control (185 ± 15 µg/eye; n = 11). Light exposure initiated at 0900 or 1700 resulted in about twofold greater rhodopsin recovery and DNA levels during the normal or extended dark periods. Pairwise comparisons for rhodopsin and DNA levels at 0100 revealed that the values for 16 hours of darkness were significantly higher than those for 40 hours but no different from for the 88-hour dark period.



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  		Figure 3. Rhodopsin and photoreceptor cell DNA in light-exposed rats after long-term dark adaptation. Dim cyclic light–reared rats were dark-adapted for the normal 16-hour period, for the normal period plus 1 day (40 hours), or for 3 additional days (88 hours) and then exposed to intense light for 8 hours at 0100, 0900, or 1700. After 2 weeks in darkness rhodopsin was measured in one eye and retinal DNA in the fellow eye. (•), Results are the mean ± SD for 7 to 8 individual rats exposed to light or ({blacktriangleup}) 11 unexposed control animals. Unexposed control rhodopsin value 2.20 ± 0.10 nmol/eye; DNA 185 ± 15 µg/retina. *Values significantly higher at 16 hours than for 40 hours of dark adaptation (P < 0.05). Values for 0900 and 1700 were not significantly different from each other.

 
Light Duration and Relative Light Damage Susceptibility
Using 50% rhodopsin loss as the criterion, we compared light damage susceptibility at various times by extending the duration of light treatment. For cyclic light–reared rats, light exposure starting at 0900 or 1700 resulted in the same relative degree of photoreceptor cell loss (Fig. 4) . In both cases, rhodopsin was 50% of control when the duration of light exposure was 24 hours. For rats treated with light beginning at 0100, the same 50% rhodopsin loss required only 6 to 8 hours, one-third to one-quarter of the time required for animals treated with light at 0900 or 1700. Two-way ANOVA revealed significant effects associated with both the start time and duration of light exposure (P < 0.001) but that each is an independent factor (P < 0.60). Tukey’s test indicates that rhodopsin values for both the 0900 and 1700 start times were significantly higher than those for 0100 but not significantly different from each other. In dark-reared rats treated with light at 0100, rhodopsin recovery was 50% after approximately 2.5 hours, whereas 4.5 hours of exposure was required at 1700. Dark-reared rats, therefore, are almost twofold more susceptible to retinal light damage at 0100 than at 1700 (P < 0.001).

Rhodopsin at Different Times of the Day or Night
Because rhodopsin synthesis and the phagocytosis of ROS tips by the RPE are circadian dependent25 26 27 and the bleaching of rhodopsin triggers retinal light damage,1 12 we measured its baseline levels at various times. Whole eye rhodopsin levels and its content in excised retinas and in the remaining eye cups (RPE fraction) were determined for dark-adapted, cyclic light– and dark-reared rats. As shown in Table 2 , whole eye rhodopsin levels measured at 0100 were 5% to 10% higher than at 0900 or 1700. The distribution of rhodopsin between retina and RPE was also different. Approximately 97% of the rhodopsin was contained in the retinal fractions at 0100, whereas slightly less was present at 0900 or 1700. All dark-reared rats had significantly higher rhodopsin levels than those in cyclic light–reared rats, but only the rhodopsin value at 0100 in cyclic light–reared animals was significantly higher than that at 1700.


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  		Table 2. Ocular Tissue Levels of Rhodopsin at Different Times

 
Changes in Retinal DNA and in RNA after Light Exposure
To confirm that retinal damage is enhanced by intense light exposure at 0100, we studied DNA fragmentation patterns by neutral gel electrophoresis. We also determined the expression profiles of retinal HO-1 and IRBP mRNAs as markers of oxidative stress and photoreceptor cell transcription, respectively. The bottom panels of Figure 5 show that DNA ladders were present 1 to 2 days after intense light treatment. For rats reared in dim cyclic light, a prominent pattern of DNA ladders appeared 1 day after light exposure at 0100. Higher-molecular-weight DNA fragments, seen as a smear near the top of the gel, were present when light exposure was initiated at 0900, whereas little DNA fragmentation occurred at 1700. For dark-reared rats exposed to light at 0100 or 0900, a smear of high-molecular-weight DNA fragments and well-defined DNA ladders were present 1 day later. Considerably less DNA fragmentation was present from light exposure at 1700. In both types of rats, little DNA damage was present immediately after intense light treatment. These patterns were similar to those for rats unexposed to intense light (not shown).



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  		Figure 5. Changes in retinal DNA and mRNA after intense light exposure. Rats were treated with light beginning at 0100, 0900, or 17001 9 17 for 8 hours (cyclic-reared) or 3 hours (dark-reared) and then killed immediately after (0 hours) or 1 or 2 days later. Four retinas from 4 separate rats were used for DNA extraction with RNA extracted from the fellow eyes. Aliquots from each extract were combined and loaded on gels for DNA (2 µg) or RNA (10 µg) analysis. Blots of RNA gels were probed for HO-1 and then stripped and reprobed for IRPB. 18S rRNA is shown for comparison of RNA loading differences. Tissues from rats unexposed to light were extracted at the same times.

 
As shown by the expression profile for HO-1 mRNA, oxidative stress was present in retinas from cyclic light–reared rats immediately after light exposure at 0100, whereas less HO-1 induction occurred at 0900 or 1700. The relative expression of HO-1 mRNA was greatest 1 day after light treatment and less thereafter. Similar patterns of HO-1 expression were found in dark-reared rats 1 to 2 days after intense light treatment. As shown by the relative lack of signal for HO-1 in unexposed controls, this mRNA is not constitutively expressed in the retina.

In contrast to HO-1 in unexposed rat retinas, IRBP is constitutively expressed to approximately the same degree during the day or night. However, after intense light exposure IRBP mRNA levels were reduced. The relative reductions in IRBP expression correlated with higher levels of HO-1 expression and DNA damage, for example, 0100. Because IRBP is synthesized within photoreceptors,47 this indicates that intense light exposure also affects the normal transcriptional events in these cells.

Light Exposure and Antioxidative Treatment in Rats
Because intense light induces an oxidative insult in the rat retina, we used DMTU as a probe to study the early time course of light damage. The animals were given a single dose of DMTU before or during intense light treatment at 0100, and rhodopsin subsequently was measured as an index of protection by the antioxidant. When DMTU was given before light exposure, the relative degree of protection was greater than that in rats given the antioxidant at later times (Fig. 6) . For cyclic light–reared rats rhodopsin recovery was 93% of control (2.20 nmol/eye) when DMTU was given 30 minutes before an 8-hour exposure. Visual pigment levels were significantly lower in rats treated with DMTU at the start of light exposure or at times up to 60 minutes after the start of light (P < 0.005). In dark-reared rats treated with DMTU 30 minutes before a 3-hour light exposure, rhodopsin recovery was 88% of the unexposed control (2.15 nmol/eye). It was significantly lower in rats injected with DMTU at time 0 (P < 0.025) or 15 to 60 minutes after the onset of light (P < 0.010). In both groups of animals, saline-treated rats recovered about the same level of rhodopsin as found in those given DMTU after the start of light treatment.



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  		Figure 6. Average rhodopsin levels in rats treated with DMTU before or after the start of light exposure. Dark-adapted cyclic light–reared ({circ}) or dark-reared (•) rats were treated with DMTU (1X, IP) at 500 mg/kg before or after the start of light exposure beginning at 0100. Other rats were given the saline vehicle 30 minutes before exposure. Results are the average for 4 to 5 pairs of eyes from rats exposed to light for 8 hours (cyclic) or 3 hours (dark) or 5 saline injected ({triangleup}, {blacktriangleup}) animals. Unexposed control rhodopsin values ({square}, {blacksquare}) were from 4 rats not given DMTU or saline. Standard deviations were 10% to 25% of the mean. *Rhodopsin recoveries in rats given DMTU 30 minutes before exposure were significantly higher than for rats given DMTU at later times.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study shows that intense visible light exposure during the nighttime phase of the diurnal cycle results in extensive visual cell loss in rats, whereas comparable light treatment during the day caused considerably less damage (Fig. 1 , Table 1 ). We also found that reversing the normal 12-hour light–dark cycle31 of rats reversed their susceptibility to light damage (Fig. 2) and that light damage profiles were retained after dark adaptation for several days (Fig. 3) . Our results confirm earlier studies demonstrating enhanced retinal light damage of rats at night41 42 and extend those findings by showing that a single, relatively short, light treatment is capable of initiating the damage. On the basis of results with longer duration light exposures, we estimate that cyclic light–reared rats are three- to fourfold more sensitive to light damage at 0100 than at 1700 and that dark-reared rats are approximately twofold more susceptible (Fig. 4) . In both types of rats the circulating levels of ACTH and corticosterone were similar and varied in the same circadian manner (data not shown). Accordingly, it appears that our dark-reared rats have sufficient alternative environmental cues, such as noise, so that their circadian rhythms are not entirely free running.

An important finding from this work is that the same types of animals that exhibit enhanced retinal light damage at 0100 are practically resistant to damage when light exposure was initiated during the period 1500 to 1700. This result is not due to partial bleaching of rhodopsin during the dim light phase of the light cycle, because the animals were either dark adapted for 16 hours before light exposure or previously reared in darkness. This suggests that the retina expresses an endogenous factor(s) that enhances or retards the process of cell death from intense light. Opsin and other visual transduction proteins are expressed in a circadian fashion,20 21 22 23 24 and retinal light damage can be modulated by long-term, environmental light–regulated changes in some of these.16 17 18 19 Although we found higher levels of rhodopsin in unexposed rats at 0100 than at other times and significantly more rhodopsin in dark-reared rats (Table 2) , the prebleach differences do not appear to be sufficient to account for the differences in light damage susceptibility. During prolonged light exposure, however, there could be a significant effect on the amplification of visual transduction by continuous rhodopsin bleaching. Other evidence points to the relative levels of retinal dopamine and melatonin as effectors of light damage susceptibility.34 35 36 37 Wiechmann and O’Steen37 reported that melatonin injections of rats enhances visual cell loss from light, irrespective of the time of day. Bush et al.38 showed that luzindole, which blocks melatonin receptors, reduces the extent of retinal light damage. This suggests that a receptor-mediated process is involved with light damage susceptibility and that, whether circulating or endogenous, melatonin is not simply a photosensitizer in the retina.

The mechanism of retinal light damage remains elusive, but photoreceptor cell status at the start of light exposure appears to be a key factor in determining the outcome of light’s pathologic effect. By using relatively brief light treatments during the sensitive nighttime period or the resistant daytime period, we found remarkable differences in the extent of damage. This was also true for longer duration exposures, in that light treatment starting at 1700 always resulted in the recovery of more rhodopsin than for rats exposed at 0100 (Fig. 4) . Because a 24-hour light treatment of rats beginning at 1700 would progress through the more sensitive nighttime period, we conclude that the critical factor is the protein expression profile of the retina upon the initiation of light exposure. Although systemic factors, such as circulating hormones, can influence the effects of light in the retina,35 36 37 39 in adrenalectomized rats we found a similar profile of retinal light damage (data not shown). More work will be required to understand the role of systemic factors on protein expression and light damage in the retina.

Intense light exposure affected the transcription of retinal genes, as shown by reduced levels of IRBP mRNA and by the enhanced expression of HO-1 mRNA. IRBP is constitutively expressed in retinal photoreceptor cells,47 whereas HO-1 is an antioxidative protein that serves as a useful marker of oxidative stress in the retina.7 10 Light treatment also resulted in the appearance of apoptotic DNA ladders, which correlated with elevated HO-1 mRNA levels and reduced IRBP expression. Thus, the mechanism by which light induces changes in the normal transcriptional events within photoreceptors and induces a stress response in retina is temporally related to a program(s) involving DNA damage and cellular death.

Retinal light damage involves an oxidative process that, once initiated, is not easily reversed. Previous work has shown that synthetic or natural antioxidants reduce or eliminate retinal light damage,11 14 DNA damage,7 8 and damage to the opsin gene9 when given to rats before light exposure. We also found that prior administration of DMTU prevented rhodopsin loss (Figs. 1 6) but that it was ineffective when given 15 to 60 minutes after the onset of light. Light-induced apoptosis is widely believed to be a primary cause of visual cell death,2 3 4 5 although oxidative damage occurs simultaneously.2 8 9 Because rhodopsin photobleaching is the trigger for retinal light damage,1 12 13 this indicates that damage is initiated within ROS and that it spreads to the entire visual cell. Our results also indicate that the period normally associated with the initial bleach of rhodopsin coincides with the onset of an oxidative process in the retina. The extent to which reactive oxygen(s) is directly involved in light-induced damage to visual cells or to which it affects proapoptotic genes such as c-fos5 remains to be determined. It is entirely possible that the balance between pro- and antiapoptotic gene expression in the retina is determined by the balance between oxidative forces and antioxidative processes during light exposure.

It is tempting to speculate that the timing of intense light exposure of patients undergoing ocular surgery could affect the extent of retinal damage.48 Although we recognize that rats are nocturnal and primates are diurnal, each has well-established circadian rhythms that affect hormone secretion and cellular metabolism. Additional work will be required to determine whether intense exposure affects patients in the same ways as it affects experimental animal models of retinal degeneration.


   Acknowledgements
 
The authors thank Federico Gonzalez-Fernandez (University of Virginia Health Sciences Center, Charlottesville, VA) for providing us with a cDNA clone for rat IRBP.


   Footnotes
 
Supported in part by Grant EY-01959 from the National Institutes of Health, Bethesda, Maryland; by M. Petticrew, Dayton, Ohio; and by the Ohio Lions Research Foundation, Dayton, Ohio (DTO).

Submitted for publication March 6, 2000; revised June 26, 2000; accepted July 21, 2000.

Commercial relationships policy: N.

Presented in part at the annual meeting of the Association for Research in Vision and Ophthalmology, Fort Lauderdale, Florida, April 1997.

Corresponding author: Daniel T. Organisciak, Department of Biochemistry and Molecular Biology, Wright State University School of Medicine, 3640 Colonel Glenn Highway, Dayton, OH 45435. dto{at}wright.edu


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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