September 2012
Volume 53, Issue 10
Free
Retina  |   September 2012
Functional and Morphologic Consequences of Light Exposure in Primate Eyes
Author Affiliations & Notes
  • Ryo Mukai
    From the Department of Ophthalmology and Visual Sciences, Gunma University Graduate School of Medicine, Gunma, Japan; and the
  • Hideo Akiyama
    From the Department of Ophthalmology and Visual Sciences, Gunma University Graduate School of Medicine, Gunma, Japan; and the
  • Yuki Tajika
    Department of Anatomy, Gunma University Graduate School of Medicine, Gunma, Japan.
  • Yukitoshi Shimoda
    From the Department of Ophthalmology and Visual Sciences, Gunma University Graduate School of Medicine, Gunma, Japan; and the
  • Hiroshi Yorifuji
    Department of Anatomy, Gunma University Graduate School of Medicine, Gunma, Japan.
  • Shoji Kishi
    From the Department of Ophthalmology and Visual Sciences, Gunma University Graduate School of Medicine, Gunma, Japan; and the
  • Corresponding author: Ryo Mukai, Department of Ophthalmology and Visual Sciences, Gunma University Graduate School of Medicine, 3-39-15 Showa-machi, Maebashi-shi, Gunma 371-8511, Japan; rmukai@showa.gunma-u.ac.jp
Investigative Ophthalmology & Visual Science September 2012, Vol.53, 6035-6044. doi:10.1167/iovs.12-9608
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      Ryo Mukai, Hideo Akiyama, Yuki Tajika, Yukitoshi Shimoda, Hiroshi Yorifuji, Shoji Kishi; Functional and Morphologic Consequences of Light Exposure in Primate Eyes. Invest. Ophthalmol. Vis. Sci. 2012;53(10):6035-6044. doi: 10.1167/iovs.12-9608.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: To evaluate the morphologic and functional changes of the primate retina after light exposure using spectral-domain optical coherence tomography (SD-OCT) and electroretinography (ERG).

Methods.: Seven monkey eyes with light-emitting diode (LED) contact lenses were exposed to light for 8 hours. SD-OCT and ERG were performed bilaterally before, after the light exposure, and on days 1 and 14 in three monkeys. The tests were repeated after 4 months, followed by enucleation 12 hours later. Six eyes of three other animals were enucleated 12 hours after the first light exposure, and two eyes of one monkey were enucleated after 14 days, followed by microscopy.

Results.: Immediately after light exposure, SD-OCT showed increased reflectivity of the outer segment (OS). Electron microscopy showed intracellular vacuolated and irregular lamellar structures at the proximal OS, while the distal end of the OS was unfolded at the RPE interface. At 14 days, the histologic changes and the OCT images returned to normal. ERG showed decreased cone and rod responses immediately after light exposure and decreased cone responses on day 1. Normalization occurred on day 14.

Conclusions.: Light exposure caused increased reflectivity of the photoreceptor OS, which corresponded to intracellular vacuolization and irregularity of the lamellar structure of the OS. OCT images returned to normal along with the histologic restoration. Rod and cone responses decreased transiently immediately after light exposure, which might be attributed to incomplete recovery from retinal bleaching.

Introduction
Solar retinopathy, a retinal injury caused by gazing directly at the sun during a solar eclipse, was first commonly reported in the 17th century. 1 In 1968, it also was reported that histologic changes occur in the photoreceptor outer segment after exposure to fluorescent lamps in a murine model. 2 In 1972, indirect ophthalmoscopy determined there was loss of regularity and pyknosis in the photoreceptors along with disruption of the RPE in rhesus monkeys after prolonged light exposure. 3 Sun et al. recently reported that intense light exposure damaged the rod outer segment of the photoreceptors, which ultimately resulted in loss of the visual cells in rats. 4 A previously reported rat model mimics human retinal diseases and has long been used for the studies that examine light-induced photoreceptor degeneration. Rod outer segment losses after light exposure also have been shown to occur in the rhodopsin mutant dog model of human retinitis pigmentosa. 5  
Using electroretinography (ERG), Noell et al. in 1966 reported finding retinal functional changes in rats after prolonged light exposure. 6 Subsequently, it was reported that after continuously exposing mice to light intensities of 1700 lux for 12 to 72 hours, the b-wave amplitude of the combined rod-cone response in ERG decreased more than 40% compared with pre-exposure values. 7,8  
Recent advances in spectral-domain optical coherence tomography (SD-OCT) make it possible to observe specific details of the retinal structures, such as the inner and outer plexiform layers, external limiting membranes (ELMs), and the photoreceptor inner and outer segments (IS/OS). The reflective line at the junction between the photoreceptor IS/OS is now considered to be the hallmark for evaluating the integrity of the photoreceptor outer segment. 9,10 SD-OCT studies also have found defects in the IS/OS line in a case of solar retinopathy, 11 and transient disruption of the IS/OS line in subjects after gazing at a computer display. 12  
The goals of the current study were to define the morphologic and functional consequences of light-emitting diode (LED) exposure in the primate retina using both SD-OCT and ERG in an in vivo monkey model. We also attempted to determine the correlation between the SD-OCT findings and the light and electron micrographs in primate retinas after light exposure. 
Methods
The current study included two experiments. In the first, we used SD-OCT and ERG to examine the time course of the acute in vivo changes that occurred after LED exposure. In the second experiment, we determined whether the changes were reproducible and if there were any histologic correlations. 
Animals
Seven adult monkeys ( Macaca fuscata ; weight, 5–10 kg, supplied by Amami Wild Animal Research Center, Kagoshima, Japan) were treated according to the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. The experimental protocol was approved by the Experimental Animal Committee of the Gunma University Graduate School of Medicine. Before each procedure, all animals were anesthetized with intramuscular ketamine (35 mg/kg) and xylazine (5 mg/kg). The room temperature was maintained between 24° and 30°C, while respiration rates were monitored through visual management. Topical oxybuprocaine (0.5%) was instilled to induce anesthesia followed by dilation of the pupils using 0.5% phenylephrine and 0.5% tropicamide. All eyes were examined with a slit lamp and indirect ophthalmoscopy to confirm that there was no corneal or lens opacity or any retinal damage at the beginning of the procedure. 
Light Exposure
The right eye of each monkey was exposed to light for 8 hours via the use of a white LED contact lens-type electrode (LS-W; Mayo, Nagoya, Japan). All left eyes served as the controls. During the 8-hour LED exposures, the left eyes were covered by a patch. The LED intensity was 8912 candelas (cd/m2), and each LED emitted a broad-spectrum light, with a wavelength ranging from 400 to 750 nm (Fig. 1). Using a light meter (LX-105; Reed Instruments, Wilmington, NC), the LED intensity was converted to an illuminance of approximately 7000 lux. The illuminance was measured by the light meter, which contained a sensor that was in contact with the LED of the contact lens. 
Figure 1. 
 
Spectral distribution of the white LED.
Figure 1. 
 
Spectral distribution of the white LED.
Before adopting this intensity and duration, we tested various conditions to determine the optimal settings. When the preliminary tests were started, we gradually increased the light intensity from 400 to 8912 cd/m2 over 2 to 10 hours (400 cd/m2 for 2 hours; 800 cd/m2 for 2 hours; 800 cd/m2 for 4 hours; 4000 cd/m2 for 2 hours; 6000 cd/m2 for 4 hours; 8912 cd/m2 for 3 hours; 8912 cd/m2 for 8 hours; and 8912 cd/m2 for 10 hours). The first changes observed for the SD-OCT images occurred after 8 hours at an LED intensity of 8912 cd/m2
SD-OCT and full-field ERG were performed before, immediately after light exposure, and on days 1 and 14 in both eyes of three monkeys. To overcome any potential adverse effects in the monkeys due to the general anesthesia that was required during the long experiment, we did not reexamine the animals until 4 months after their first experiment. During those 4 months, we performed the same experiment in another two monkeys. At that time, we confirmed normalization of the findings on the OCT scans and the electrical responses. Once normalized, the animals underwent the same experiment a second time, after which the eyes were enucleated 12 hours after the LED light exposure. In addition, another six eyes of three animals were enucleated 12 hours after the first unilateral light exposure, while two eyes of one monkey were enucleated at 14 days. The enucleated eyes were submitted for light and transmission electron microscopy (LM, TEM). All eyes were enucleated with animals under deep pentobarbital anesthesia.  
SD-OCT
We performed SD-OCT (Carl Zeiss Meditec Inc., Dublin, CA) to examine the morphologic retinal changes. All monkeys underwent a 6.0-mm line scan before, immediately after the LED exposure, and on postexposure days 1 and 14. All OCT images were compared with those of the fellow eyes. 
ERG Analysis
ERGs, which were recorded before and immediately after LED exposure and on postexposure days 1 and 14, were performed using a synchronized trigger and summing amplifier (Primus; LACE Elettronica S.R.L., Pisa, Italy) with a stimulation device (Mayo). ERGs were recorded via the use of a white LED contact lens-type electrode (Mayo) instead of a Ganzfeld stimulator. The LED was used as a corneal bipolar electrode, with the negative and ground electrodes placed in the subcutaneous skin of the forehead and earlobe, respectively. After the monkeys were anesthetized, the pupils were dilated with 1% tropicamide and 2.5% phenylephrine hydrochloride. After 8 hours of light exposure, the monkeys were dark adapted for at least 40 minutes before any recordings. The stimulus and recording systems were used according to the protocol of the International Society for Clinical Electrophysiology of Vision. 13  
Statistical Analysis
A t-test was used to assess whether the amplitude and peak implicit ratio of the LED-exposed eye to the normal control eye changed after LED exposure. P < 0.05 was considered significant. The sequential changes in the relative ratio of the maximal flash responses for the rods and the cones, compared with normal control eyes, were analyzed by a repeated measured analysis of variance immediately after LED exposure and on postexposure days 1 and 14. 
Preparation of Retinal Tissue Sections for LM and TEM
Eyes were enucleated at 12 hours and 14 days after light exposure. After the animals were euthanized by an intravenous injection of pentobarbital, they were perfused through the bilateral carotid artery with freshly prepared 3% paraformaldehyde containing 2.5% glutaraldehyde in PBS (pH, 7.4). The eyes were enucleated, fixed overnight in 2.5% glutaraldehyde and 3% paraformaldehyde in 0.1 M PBS, and then cryoprotected with sucrose in 0.1 M PBS, with adjustments in the sucrose concentration as follows: 10% sucrose (1 hour); 20% sucrose (1 hour); and 30% sucrose (overnight). The eyecups were embedded in OCT compound and frozen with liquid nitrogen. The embedded eyecups were sliced into 10-μm sections with a cryostat (Leica Microsystems, Wetzlar, Germany). Serial horizontal sections were made from the superior to inferior portions of the eyecups. When the serial sections approached the fovea, the foveal depression appeared. We obtained approximately 20 frozen sections with a foveal depression. Two 10-μm–thick sections were obtained from the central fovea and the adjacent area and then submitted for the TEM study. The remaining sections were examined by LM using hematoxylin and eosin (H-E stain) or terminal deoxynucleotidyl transferase-mediated, dUTP-biotin catalyzed DNA nick-end labeling (TUNEL) staining. 
TEM
After light exposure of the retina, TEM was performed at the Electron Microscopy Facility (Filgen Inc., Nagoya, Japan). Briefly, specimens were prefixed in 3% paraformaldehyde containing 2.5% glutaraldehyde in PBS, postfixed with 2% osmium tetroxide, and dehydrated for 10 minutes each in sequential baths of 30%, 50%, 70%, 90%, and 100% ethanol. The specimens were put into propylene oxide for 30 minutes followed by a mixture of propylene oxide and epoxy resin for 1 hour, with the samples then embedded into a gelatin capsule with epoxy resin that was maintained at 60°C for 1 day. Subsequently, 80- to 90-nm ultrathin sections were obtained using an ultramicrotome. The ultrathin sections were then stained with 2% tannic acid in distilled water for 5 minutes followed by 2% uranyl acetate in DW for 15 minutes and then by lead staining solution for 5 minutes. In the final step, sections were coated with thin carbon film and placed in a vacuum. The specimens were examined by TEM using a high-resolution instrument at 80 kV. 
TUNEL Staining
The TUNEL method was used to detect apoptotic cells (In Situ Cell Death Detection Kit, POD; Roche, Basel, Switzerland). The TUNEL enzyme (1 hour at 37°C) and peroxidase converter (30 min at 37°C) were applied to the 10-μm–thick sections after incubation in a permeabilizing solution of 0.1% Triton-X in 0.1% sodium citrate for 2 minutes. An enhancement reagent diaminobenzidine (DAB; Dako, Glostrup, Denmark) was used as the chromogen. After staining, image analysis was used to count the TUNEL-positive cells in the outer nuclear layer (ONL), with the number of TUNEL-stained nuclei quantified in four random slides per macula. 
Reproducibility
To confirm the reproducibility, the right eyes were treated using the same procedure 4 months after the first experiment. OCT and ERG then were performed immediately after 8 hours of light exposure. 
Results
OCT Findings
We used OCT to obtain 6-mm vertical and horizontal scans through the fovea at each time point. Before light exposure, SD-OCT showed that the retinas had a normal layered structure, similar to that of human retinas. In addition, the fovea had a normal depression. The minimally reflective line of the ELM was anterior to the IS/OS line (Figs. 2A, 3A). In the outer retina, the junction between the photoreceptor IS/OS was a highly reflective line, which was separated from the highly reflective line of the RPE by a dark zone between the two lines (Fig. 2A). Immediately after 8 hours of light exposure, there was an increase in the reflectivity of the dark zone between the IS/OS and RPE, which made it difficult to differentiate the IS/OS and RPE as distinct lines (Figs. 2B, 3B). Although the dark zone remained highly reflective 24 hours after light exposure, a dark zone between the IS/OS and RPE appeared at the fovea (Fig. 2C). At 2 weeks, the reflectivity of the space between the IS/OS and RPE was low and comparable with that observed before light exposure. The IS/OS and RPE appeared as two independent highly reflective lines with a dark zone between them (Fig. 2D). There were no changes observed for the ELM and inner retina throughout the experiment. 
Figure 2. 
 
SD-OCT before and after light exposure. (A) Before light exposure, the IS/OS and RPE are seen as two reflective lines. (B) Immediately after light exposure, the reflectivity of the dark layer between IS/OS and RPE increases, making it difficult to differentiate the IS/OS from the RPE (arrows). (C) Although the dark zone is still highly reflective 24 hours after light exposure, restoration of the dark zone between the IS/OS and RPE is seen at the fovea (arrows). (D) At 2 weeks, the reflectivity of the layer between the IS/OS and RPE is low, with the IS/OS and RPE is seen as two distinct highly reflective lines. The ELM and inner retina do not change throughout the experiment.
Figure 2. 
 
SD-OCT before and after light exposure. (A) Before light exposure, the IS/OS and RPE are seen as two reflective lines. (B) Immediately after light exposure, the reflectivity of the dark layer between IS/OS and RPE increases, making it difficult to differentiate the IS/OS from the RPE (arrows). (C) Although the dark zone is still highly reflective 24 hours after light exposure, restoration of the dark zone between the IS/OS and RPE is seen at the fovea (arrows). (D) At 2 weeks, the reflectivity of the layer between the IS/OS and RPE is low, with the IS/OS and RPE is seen as two distinct highly reflective lines. The ELM and inner retina do not change throughout the experiment.
Figure 3. 
 
High-magnification SD-OCT images. (A) Before light exposure. (B) Immediately after light exposure. An increase in the reflectivity of the dark layer between the IS/OS and RPE is seen immediately after light exposure. The length between the IS/OS and RPE (arrows) remains unchanged.
Figure 3. 
 
High-magnification SD-OCT images. (A) Before light exposure. (B) Immediately after light exposure. An increase in the reflectivity of the dark layer between the IS/OS and RPE is seen immediately after light exposure. The length between the IS/OS and RPE (arrows) remains unchanged.
ERG Analysis
We showed the representative waveforms of case 1 before and after light exposure (Fig. 4). Immediately after light exposure, significant decreases (P < 0.01) were found in the exposed eyes for the amplitude of the b-wave in the rod response, the a-wave trough to the b-wave peak in the cone, and the combined rod-cone responses compared with the fellow eyes. In addition, while there were slight decreases of the a-wave of the combined rod-cone response immediately, 24 hours, and 14 days after the light exposure, these decreases were not statistically significant compared with the fellow eyes (Fig. 5A). All eyes returned to normal 14 days after light exposure (Figs. 5B, 5C, 5D). Although there was a significant decrease (P < 0.05) in the cone response at 24 hours, it recovered 14 days after exposure (Fig. 5C). 
Figure 4. 
 
ERG before and after light exposure in case 1. Top: For the rod response, a reduction of the b-wave is seen immediately after exposure compared with the fellow eye (control). The response recovers at 24 hours and 2 weeks. Middle: A decrease in the amplitude from the trough of the a-wave to the peak of the b-wave is seen immediately after exposure and at 24 hours compared with the fellow eye. At 2 weeks, there is no difference between the eyes. Bottom: Reductions of the combined rod-cone response for the a- and b-wave are observed immediately after and at 24 hours, with all responses recover at 2 weeks.
Figure 4. 
 
ERG before and after light exposure in case 1. Top: For the rod response, a reduction of the b-wave is seen immediately after exposure compared with the fellow eye (control). The response recovers at 24 hours and 2 weeks. Middle: A decrease in the amplitude from the trough of the a-wave to the peak of the b-wave is seen immediately after exposure and at 24 hours compared with the fellow eye. At 2 weeks, there is no difference between the eyes. Bottom: Reductions of the combined rod-cone response for the a- and b-wave are observed immediately after and at 24 hours, with all responses recover at 2 weeks.
Figure 5. 
 
The mean amplitudes of the electrical responses in six eyes of three monkeys (n = 3 light-exposed eyes; n = 3 control eyes). (A) The a-wave of the combined rod-cone response decreases slightly immediately, 24 hours, and 14 days after light exposure. No significant differences are seen compared with the fellow eyes. (B) Although a significant decrease from normal (P < 0.01) is seen for the b-wave of the combined rod-cone response, the response returns to normal 24 hours after the light exposure. (C) A significantly lower cone response (P < 0.05) is seen immediately and 24 hours after light exposure, with recovery at 14 days. (D) A significantly lower rod response (P < 0.05) is seen immediately after exposure, with a return to normal at 24 hours.
Figure 5. 
 
The mean amplitudes of the electrical responses in six eyes of three monkeys (n = 3 light-exposed eyes; n = 3 control eyes). (A) The a-wave of the combined rod-cone response decreases slightly immediately, 24 hours, and 14 days after light exposure. No significant differences are seen compared with the fellow eyes. (B) Although a significant decrease from normal (P < 0.01) is seen for the b-wave of the combined rod-cone response, the response returns to normal 24 hours after the light exposure. (C) A significantly lower cone response (P < 0.05) is seen immediately and 24 hours after light exposure, with recovery at 14 days. (D) A significantly lower rod response (P < 0.05) is seen immediately after exposure, with a return to normal at 24 hours.
LM and TEM
Normal retinal structures were observed in all three fellow control eyes when examined by LM with toluidine blue stain (Figs. 6A, 6C) and by TEM. In addition, LM showed that the fovea had a normal depression, and the ELM was clearly visible. Although the photoreceptor inner segments were oriented perpendicularly. 
Figure 6. 
 
LM of the retina in the control and exposed eyes 12 hours after LED exposure with 8912 cd/m2. Although the photoreceptor outer segments are obliquely sectioned in the exposed eye, no abnormality is seen. (A, B) Toluidine blue staining, ×20 magnification. Higher magnifications (×40) of A and B are shown in (C) and (D). No abnormality is seen in the photoreceptors in the exposed eyes compared with the fellow eyes. The ONL is slightly pyknotic. Microvesicles in the exposed eyes are seen in Henle's fiber.
Figure 6. 
 
LM of the retina in the control and exposed eyes 12 hours after LED exposure with 8912 cd/m2. Although the photoreceptor outer segments are obliquely sectioned in the exposed eye, no abnormality is seen. (A, B) Toluidine blue staining, ×20 magnification. Higher magnifications (×40) of A and B are shown in (C) and (D). No abnormality is seen in the photoreceptors in the exposed eyes compared with the fellow eyes. The ONL is slightly pyknotic. Microvesicles in the exposed eyes are seen in Henle's fiber.
Twelve hours after light exposure, LM showed there were both perpendicularly arrayed inner segments along with a regular arrangement of dots in the outer segment layer, which suggested a cross-section (Figs. 6B, 6D). The ONL was slightly pyknotic (Fig. 6D). Microvesicles also were seen in Henle's fiber layer in the exposed eyes. The ganglion cell layer showed cellular vacuolization. The RPE appeared normal in the exposed and fellow eyes. 
TEM showed a perpendicularly arrayed inner segment along with regular ovoid structures that corresponded to the cross-section of the outer segments at 12 hours after light exposure (Fig. 7A). The ovoid structures consisted of a fingerprint pattern of intracellular lamellae and scant cytoplasm with an intact plasma membrane. The outer segments were obliquely oriented or cross-sectioned from the proximal portion, but they were perpendicularly sectioned at the distal end where the intracellular lamellar structure was observed. A higher magnification further showed intracellular microvesicles with fragments of the lamellar structure at the proximal portion of the outer segment that was adjacent to the inner segment (Figs. 7B, 7C, 7D). At the distal end of the outer segments in the control eyes, the cylindrical outer segments were enveloped by RPE microvilli, while the outer segments were plump and folded with their tips buried in the RPE (Figs. 7E, 7F). 
Figure 7. 
 
TEM of the retina in the control and exposed eye 12 hours after light exposure. (A) TEM shows a perpendicularly arrayed inner segment and regular arranged ovoid structures of the outer segments (magnification, ×600). The junction between the IS/OS in the control (B) and the exposed eyes (C) (magnification, ×6000). Although the intracellular lamellar structure is normal in the control eye, the exposed eye had intracellular microvesicles with fragments of lamellar structure at the proximal portion of the outer segment that is adjacent to the inner segment. (D) shows structural changes similar to those in (C) but in a different area. TEM ×6000. (E, F) At the distal end of the outer segments, the cylindrical outer segments are enveloped by the RPE microvilli in the control eye (E), while the outer segments are plump and folded, with their tips buried in the RPE in the exposed eye (F).
Figure 7. 
 
TEM of the retina in the control and exposed eye 12 hours after light exposure. (A) TEM shows a perpendicularly arrayed inner segment and regular arranged ovoid structures of the outer segments (magnification, ×600). The junction between the IS/OS in the control (B) and the exposed eyes (C) (magnification, ×6000). Although the intracellular lamellar structure is normal in the control eye, the exposed eye had intracellular microvesicles with fragments of lamellar structure at the proximal portion of the outer segment that is adjacent to the inner segment. (D) shows structural changes similar to those in (C) but in a different area. TEM ×6000. (E, F) At the distal end of the outer segments, the cylindrical outer segments are enveloped by the RPE microvilli in the control eye (E), while the outer segments are plump and folded, with their tips buried in the RPE in the exposed eye (F).
Changes 14 Days after Light Exposure
LM showed no obvious differences in the retinal structures between light-exposed and fellow eyes (Figs. 8A, 8B). TEM showed a perpendicularly arranged inner segment and a regular ovoid structure of the outer segment, which suggested a cross-section (Figs. 9A, 9B). The intracellular degeneration of the outer segment at the proximal portion was restored within a normal lamellar structure (Fig. 9C). At the distal end of the outer segment, no unfolding was seen at the apical plane of the RPE (Fig. 9D). The appearance of the outer tip of the outer segment was normal, with the area enveloped by the RPE microvilli. 
Figure 8. 
 
LM of the retina in the control and the exposed eyes 14 days after the light exposure. H-E staining ×20 (A, B). Normal alignment and length of the photoreceptor inner and outer segments are seen in control and exposed eyes.
Figure 8. 
 
LM of the retina in the control and the exposed eyes 14 days after the light exposure. H-E staining ×20 (A, B). Normal alignment and length of the photoreceptor inner and outer segments are seen in control and exposed eyes.
Figure 9. 
 
TEM of the retina in the control and exposed eye 14 days after light exposure. TEM of the photoreceptors. (A) Control eye 14 days after the light exposure. (B) Exposed eye 14 days after the light exposure. TEM (×1000; A, B) shows a perpendicularly arranged inner segment and a normal ovoid structure of the outer segment, which suggests a cross-section. (C) Higher magnification (×3000) shows that the intracellular degeneration of the outer segment at the proximal portion is restored in the normal lamellar structure. (D) TEM of the exposed eye (×3000). No unfolding at the apical plane of the RPE is seen at the distal end of the outer segment. The outer tip of the outer segment is normally enveloped by the RPE microvilli.
Figure 9. 
 
TEM of the retina in the control and exposed eye 14 days after light exposure. TEM of the photoreceptors. (A) Control eye 14 days after the light exposure. (B) Exposed eye 14 days after the light exposure. TEM (×1000; A, B) shows a perpendicularly arranged inner segment and a normal ovoid structure of the outer segment, which suggests a cross-section. (C) Higher magnification (×3000) shows that the intracellular degeneration of the outer segment at the proximal portion is restored in the normal lamellar structure. (D) TEM of the exposed eye (×3000). No unfolding at the apical plane of the RPE is seen at the distal end of the outer segment. The outer tip of the outer segment is normally enveloped by the RPE microvilli.
TUNEL Assay
A TUNEL assay of the retina 12 hours and 14 days after light exposure showed no TUNEL-positive cells in any of the macular sections or the peripheral retina in the exposed or fellow eyes (Figs. 10A–D). 
Figure 10. 
 
TUNEL assay of the retina in the control and exposed eye 12 hours and 14 days after LED exposure. No TUNEL-positive cells in the photoreceptors are seen in the macula in the normal controls or the exposed retinas (AD).
Figure 10. 
 
TUNEL assay of the retina in the control and exposed eye 12 hours and 14 days after LED exposure. No TUNEL-positive cells in the photoreceptors are seen in the macula in the normal controls or the exposed retinas (AD).
Discussion
In the current study, SD-OCT showed increased reflectivity of the layer between the IS/OS and RPE immediately after 8 hours of light exposure, which led to one highly reflective band in the outer segment. Twenty-four hours after light exposure, a separation of the IS/OS and RPE was observed at the fovea. Complete separation of the two reflective lines and a return to normal occurred 14 days after light exposure. Because the low reflective zone between the IS/OS and RPE corresponds to the photoreceptor outer segment layer, this increased reflectivity suggests that morphologic changes occurred in the outer segment. 
In the TEM study, the photoreceptor outer segment exhibited intracellular vacuolization and irregular lamellar structures at the proximal portion immediately after 8 hours of light exposure. In addition, the distal end of the outer segments was unfolded at the interface of the RPE. Fourteen days after light exposure, the structural changes of the outer segments at the proximal and distal portions returned to normal and there was normalization of the IS/OS and RPE reflective lines. This suggested that both the intracellular vacuolization and irregularity of the lamellar structure of the outer segment at the proximal portion, in conjunction with the unfolded distal end, were responsible for the increased reflectivity of the layer between the IS/OS and RPE immediately after 8 hours of light exposure. The normal physiologic renewal of the outer segment that occurs within the eye was most likely the reason behind the restoration of the light exposure–induced morphologic damage after 14 days. It also has been reported that while the physiologic renewal of the rods requires approximately 10 days, the cones require additional time. 14,15  
Immediately after 8 hours of light exposure to 8912 cd/m2, we performed full-field ERG after a 40-minute dark adaptation period. The results showed that the electrical responses in all exposed eyes decreased compared with the fellow eyes. It is well known that a 40-minute dark adaptation period is sufficiently long for rhodopsin to regenerate in a normal rod. However, because our animals had increased reflectivity of the outer segment in SD-OCT immediately after 8 hours of light exposure, the poor recovery from the bleaching might be attributed to the decreased ERG response immediately after 8 hours of light exposure. Cameron et al. previously reported 16 that longer dark adaptation times are needed for the retina after exposure to strong light intensities. In Oguchi disease with a golden-yellow fundus reflex, the rod response was absent after a 40-minute dark adaptation and OCT showed increased reflectivity of outer segment. 17,18 However after 2 hours of dark adaptation, the golden-yellow reflex disappeared (Mizuo-Nakamura phenomenon) and the IS/OS line became normal. Miyake et al. reported that the rod response was fully recovered after 3 hours of dark adaptation. 19 The 40-minute dark adaptation might not have been sufficiently long for recovery from retinal bleaching in our animals. However, the data represent the electrical responses of the retina with increased reflectivity of outer segment. At 24 hours after light exposure, the rod response returned to normal, but the cone response decreased along with mostly normalized reflectivity of the outer segment on OCT. Complete recovery from bleaching of rhodopsin in the rods may be responsible for recovery of the rod response 24 hours after light exposure, despite the intracellular vacuolization and irregularity of the lamellar structure of the outer segment. 
The LM and TEM findings showed that the pattern of the photoreceptor outer segments varied depending on the perpendicular or oblique section. The outer segment appeared to have a cylindrical array in the perpendicular section, while the oblique section had a dotted or ovoid pattern. However, although we observed a dotted pattern in the outer segment in four eyes immediately after light exposure for 8 hours, we also observed this pattern in four eyes that were not exposed to light. Thus, the dot pattern in the outer segment could have been a tissue-processing artifact that was not induced by light exposure. 
The cone morphology is specific at the fovea and differs from the other parts of the retina. The cone inner segment is plumper than the rod segment, while the cone outer segment is cone-shaped, with its length approximately half that observed for the rod segment in mammals. 20 The short cone outer segment is enveloped by elongated RPE microvilli and is referred to as the cone sheath. 21 At the fovea, however, the cone features are different, with the cones more cylindrical in shape, appearing very rod-like. In fact, the lengths of the cones within the fovea are approximately the same as the rods. The distal end of the foveal cone attaches to the RPE and is enveloped by RPE microvilli. Thus, since our specimens were obtained from the fovea, the photoreceptors consisted of rod-like cones. We did not observe any short cones with elongated RPE microvilli. 
A previous SD-OCT study reported a third reflective line between the IS/OS and RPE, which was referred to as the cone outer segment tips (COST). 22 Although the origin of the COST remains controversial, it is believed to represent the distal end of the cone. Because the length of the cone outer segment is half that of the rod, this means the COST will end right in the middle between the IS/OS and RPE. The SD-OCT results for the monkey eyes examined in the current study did not show a COST line, even in the normal eyes. In addition, while the current LM and TEM histologic sections did not show a short outer segment forming a cone sheath, the current findings did indicate there were cylindrical cones that were similar to the rods. If indeed the cone outer segment can be definitively shown to be morphologically similar between monkeys and humans, then the interpretation of COST will need to be revised. 
In an electron microscopy study in 1970, Kuwabara reported that the retinas of albino rats exposed to a hot lamp for 24 hours recovered. 23 A few weeks after the initial exposure, vacuolated and swollen photoreceptor outer segments along with irregular lamellar membranes in the photoreceptor outer segment were observed. However, after a few months, the vacuolated outer segments disappeared and new lamellar membranes were seen in the retinal sections. 
The light sources used in that study were either a cool fluorescent lamp or a hot incandescent lamp, with intensities that ranged from 750 to 1000 foot candles (approximately 7500–10,000 lux). The animals were forced to gaze at these lights, with exposure times varying from 1 hour to 2 weeks. In our current experiment, although the cornea was exposed to LED light, the light intensities were approximately 700 to 1000 lux, which was one-tenth of that used by Kuwabara. 23 Thus, the weaker light intensity used in the current study might have led to localized damage of the photoreceptor outer segment. 
In 1981, Sykes et al. 24 performed an experiment that examined monkeys exposed to a fluorescent lamp for 12 hours while under general anesthesia. The peak wavelength of the fluorescent lamp used was almost 400 to 700 nm, with the intensities ranging from 5900 to 24,700 lux. After light exposure, the investigators reported changes at the proximal and distal portions of the outer segment, similar to the results of the current study. 
Recent SD-OCT studies have shown an increased reflectivity between the IS/OS and RPE under various conditions, with all of these previous OCT findings similar to those observed in the monkeys exposed to light in the current study. 22,23 We also previously reported finding focal increased reflectivity between the IS/OS and RPE at the fovea after prolonged gazing at displays and recovery. 12 While similar OCT findings have been reported for Oguchi disease, differentiation between the IS/OS and RPE is possible in these patients, provided there is a prolonged dark adaptation period. 17,18 While similar OCT images also have been found for commotio retinae, the IS/OS and RPE appear to be separate structures when the shiny retina finally returns to normal. 25 Thus, the lesions in all of these diseases appear to have outer segment abnormalities. 
In conclusion, the increased reflectivity of the outer segment in monkey eyes exposed to light corresponded to intracellular vacuolization and irregularity of the lamellar structure of the outer segment at the proximal portion and unfolded distal ends. After histologic recovery, all OCT images of the IS/OS and RPE returned to normal. The rod and cone responses transiently decreased immediately after light exposure, which might be attributed to incomplete recovery from retinal bleaching. 
References
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Footnotes
 Disclosure: R. Mukai, None; H. Akiyama, None; Y. Tajika, None; Y. Shimoda, None; H. Yorifuji, None; S. Kishi, None
Figure 1. 
 
Spectral distribution of the white LED.
Figure 1. 
 
Spectral distribution of the white LED.
Figure 2. 
 
SD-OCT before and after light exposure. (A) Before light exposure, the IS/OS and RPE are seen as two reflective lines. (B) Immediately after light exposure, the reflectivity of the dark layer between IS/OS and RPE increases, making it difficult to differentiate the IS/OS from the RPE (arrows). (C) Although the dark zone is still highly reflective 24 hours after light exposure, restoration of the dark zone between the IS/OS and RPE is seen at the fovea (arrows). (D) At 2 weeks, the reflectivity of the layer between the IS/OS and RPE is low, with the IS/OS and RPE is seen as two distinct highly reflective lines. The ELM and inner retina do not change throughout the experiment.
Figure 2. 
 
SD-OCT before and after light exposure. (A) Before light exposure, the IS/OS and RPE are seen as two reflective lines. (B) Immediately after light exposure, the reflectivity of the dark layer between IS/OS and RPE increases, making it difficult to differentiate the IS/OS from the RPE (arrows). (C) Although the dark zone is still highly reflective 24 hours after light exposure, restoration of the dark zone between the IS/OS and RPE is seen at the fovea (arrows). (D) At 2 weeks, the reflectivity of the layer between the IS/OS and RPE is low, with the IS/OS and RPE is seen as two distinct highly reflective lines. The ELM and inner retina do not change throughout the experiment.
Figure 3. 
 
High-magnification SD-OCT images. (A) Before light exposure. (B) Immediately after light exposure. An increase in the reflectivity of the dark layer between the IS/OS and RPE is seen immediately after light exposure. The length between the IS/OS and RPE (arrows) remains unchanged.
Figure 3. 
 
High-magnification SD-OCT images. (A) Before light exposure. (B) Immediately after light exposure. An increase in the reflectivity of the dark layer between the IS/OS and RPE is seen immediately after light exposure. The length between the IS/OS and RPE (arrows) remains unchanged.
Figure 4. 
 
ERG before and after light exposure in case 1. Top: For the rod response, a reduction of the b-wave is seen immediately after exposure compared with the fellow eye (control). The response recovers at 24 hours and 2 weeks. Middle: A decrease in the amplitude from the trough of the a-wave to the peak of the b-wave is seen immediately after exposure and at 24 hours compared with the fellow eye. At 2 weeks, there is no difference between the eyes. Bottom: Reductions of the combined rod-cone response for the a- and b-wave are observed immediately after and at 24 hours, with all responses recover at 2 weeks.
Figure 4. 
 
ERG before and after light exposure in case 1. Top: For the rod response, a reduction of the b-wave is seen immediately after exposure compared with the fellow eye (control). The response recovers at 24 hours and 2 weeks. Middle: A decrease in the amplitude from the trough of the a-wave to the peak of the b-wave is seen immediately after exposure and at 24 hours compared with the fellow eye. At 2 weeks, there is no difference between the eyes. Bottom: Reductions of the combined rod-cone response for the a- and b-wave are observed immediately after and at 24 hours, with all responses recover at 2 weeks.
Figure 5. 
 
The mean amplitudes of the electrical responses in six eyes of three monkeys (n = 3 light-exposed eyes; n = 3 control eyes). (A) The a-wave of the combined rod-cone response decreases slightly immediately, 24 hours, and 14 days after light exposure. No significant differences are seen compared with the fellow eyes. (B) Although a significant decrease from normal (P < 0.01) is seen for the b-wave of the combined rod-cone response, the response returns to normal 24 hours after the light exposure. (C) A significantly lower cone response (P < 0.05) is seen immediately and 24 hours after light exposure, with recovery at 14 days. (D) A significantly lower rod response (P < 0.05) is seen immediately after exposure, with a return to normal at 24 hours.
Figure 5. 
 
The mean amplitudes of the electrical responses in six eyes of three monkeys (n = 3 light-exposed eyes; n = 3 control eyes). (A) The a-wave of the combined rod-cone response decreases slightly immediately, 24 hours, and 14 days after light exposure. No significant differences are seen compared with the fellow eyes. (B) Although a significant decrease from normal (P < 0.01) is seen for the b-wave of the combined rod-cone response, the response returns to normal 24 hours after the light exposure. (C) A significantly lower cone response (P < 0.05) is seen immediately and 24 hours after light exposure, with recovery at 14 days. (D) A significantly lower rod response (P < 0.05) is seen immediately after exposure, with a return to normal at 24 hours.
Figure 6. 
 
LM of the retina in the control and exposed eyes 12 hours after LED exposure with 8912 cd/m2. Although the photoreceptor outer segments are obliquely sectioned in the exposed eye, no abnormality is seen. (A, B) Toluidine blue staining, ×20 magnification. Higher magnifications (×40) of A and B are shown in (C) and (D). No abnormality is seen in the photoreceptors in the exposed eyes compared with the fellow eyes. The ONL is slightly pyknotic. Microvesicles in the exposed eyes are seen in Henle's fiber.
Figure 6. 
 
LM of the retina in the control and exposed eyes 12 hours after LED exposure with 8912 cd/m2. Although the photoreceptor outer segments are obliquely sectioned in the exposed eye, no abnormality is seen. (A, B) Toluidine blue staining, ×20 magnification. Higher magnifications (×40) of A and B are shown in (C) and (D). No abnormality is seen in the photoreceptors in the exposed eyes compared with the fellow eyes. The ONL is slightly pyknotic. Microvesicles in the exposed eyes are seen in Henle's fiber.
Figure 7. 
 
TEM of the retina in the control and exposed eye 12 hours after light exposure. (A) TEM shows a perpendicularly arrayed inner segment and regular arranged ovoid structures of the outer segments (magnification, ×600). The junction between the IS/OS in the control (B) and the exposed eyes (C) (magnification, ×6000). Although the intracellular lamellar structure is normal in the control eye, the exposed eye had intracellular microvesicles with fragments of lamellar structure at the proximal portion of the outer segment that is adjacent to the inner segment. (D) shows structural changes similar to those in (C) but in a different area. TEM ×6000. (E, F) At the distal end of the outer segments, the cylindrical outer segments are enveloped by the RPE microvilli in the control eye (E), while the outer segments are plump and folded, with their tips buried in the RPE in the exposed eye (F).
Figure 7. 
 
TEM of the retina in the control and exposed eye 12 hours after light exposure. (A) TEM shows a perpendicularly arrayed inner segment and regular arranged ovoid structures of the outer segments (magnification, ×600). The junction between the IS/OS in the control (B) and the exposed eyes (C) (magnification, ×6000). Although the intracellular lamellar structure is normal in the control eye, the exposed eye had intracellular microvesicles with fragments of lamellar structure at the proximal portion of the outer segment that is adjacent to the inner segment. (D) shows structural changes similar to those in (C) but in a different area. TEM ×6000. (E, F) At the distal end of the outer segments, the cylindrical outer segments are enveloped by the RPE microvilli in the control eye (E), while the outer segments are plump and folded, with their tips buried in the RPE in the exposed eye (F).
Figure 8. 
 
LM of the retina in the control and the exposed eyes 14 days after the light exposure. H-E staining ×20 (A, B). Normal alignment and length of the photoreceptor inner and outer segments are seen in control and exposed eyes.
Figure 8. 
 
LM of the retina in the control and the exposed eyes 14 days after the light exposure. H-E staining ×20 (A, B). Normal alignment and length of the photoreceptor inner and outer segments are seen in control and exposed eyes.
Figure 9. 
 
TEM of the retina in the control and exposed eye 14 days after light exposure. TEM of the photoreceptors. (A) Control eye 14 days after the light exposure. (B) Exposed eye 14 days after the light exposure. TEM (×1000; A, B) shows a perpendicularly arranged inner segment and a normal ovoid structure of the outer segment, which suggests a cross-section. (C) Higher magnification (×3000) shows that the intracellular degeneration of the outer segment at the proximal portion is restored in the normal lamellar structure. (D) TEM of the exposed eye (×3000). No unfolding at the apical plane of the RPE is seen at the distal end of the outer segment. The outer tip of the outer segment is normally enveloped by the RPE microvilli.
Figure 9. 
 
TEM of the retina in the control and exposed eye 14 days after light exposure. TEM of the photoreceptors. (A) Control eye 14 days after the light exposure. (B) Exposed eye 14 days after the light exposure. TEM (×1000; A, B) shows a perpendicularly arranged inner segment and a normal ovoid structure of the outer segment, which suggests a cross-section. (C) Higher magnification (×3000) shows that the intracellular degeneration of the outer segment at the proximal portion is restored in the normal lamellar structure. (D) TEM of the exposed eye (×3000). No unfolding at the apical plane of the RPE is seen at the distal end of the outer segment. The outer tip of the outer segment is normally enveloped by the RPE microvilli.
Figure 10. 
 
TUNEL assay of the retina in the control and exposed eye 12 hours and 14 days after LED exposure. No TUNEL-positive cells in the photoreceptors are seen in the macula in the normal controls or the exposed retinas (AD).
Figure 10. 
 
TUNEL assay of the retina in the control and exposed eye 12 hours and 14 days after LED exposure. No TUNEL-positive cells in the photoreceptors are seen in the macula in the normal controls or the exposed retinas (AD).
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