Prolonged Rhodopsin Phosphorylation in Light-Induced Retinal Degeneration in Rat Models

Abstract

purpose. The effects of various light-induced stresses on the retina were examined in the retinal degenerative rat model.

methods. Retinal morphology and electroretinograms (ERGs) were analyzed after application of light-induced stress of several intensities (650, 1300, 2500, or 5000 lux). For evaluation of rhodopsin (Rho) function, the kinetics of Rho regeneration and dephosphorylation were studied by spectrophotometric analysis and immunofluorescence labeling with antibodies specifically directed toward the phosphorylated residues ³³⁴Ser and ³³⁸Ser in the C terminus of Rho. Retinal cGMP concentration was determined by ELISA. Expression levels of neurotrophic factors (FGF2, brain-derived neurotrophic factor [BDNF], platelet-derived growth factor [PDGF], and ciliary neurotrophic factor [CNTF]) were evaluated quantitatively by RT-PCR.

results. Light intensity–dependent deterioration of ERG responses and thinning of the retinal outer nuclear layer were observed in wild-type and Royal College of Surgeons (RCS) rat retinas. Under dark adaptation after light-induced stress, the kinetics of Rho regeneration were not different between wild-type and RCS rat retinas. Rho dephosphorylation at ³³⁴Ser and ³³⁸Ser was extremely delayed in RCS rat retinas compared with wild-type without light-induced stress, but Rho dephosphorylation at those sites became slower in both RCS and wild-type rat retinas. In terms of expression of neurotrophic factors, almost no significant changes were observed between the animals after light-induced stress.

conclusions. The present study indicates that light-induced stress causes intensity-dependent deterioration in retinal function and morphology in wild-type and RCS rat retinas. Disruption of the phototransduction cascade resulting from slower kinetics of Rho dephosphorylation appears to be involved in retinal degeneration.

Retinitis pigmentosa (RP) is a heterogeneous disease group of progressive hereditary retinal degeneration clinically characterized by night blindness, visual field defects, and abnormal electroretinogram (ERG) responses due to photoreceptor cell death. ¹ Mutations of retinal genes, ¹ light damage, ² and autoimmune responses ³ are all involved in photoreceptor cell death. Several animal models with gene deficits and in vivo retinal cell death induced by intense light–induced stress ² or intravitreous administration of antibodies ³ have been developed recently. Among these, the Royal College of Surgeons (RCS) rat, in which the retinal pigment epithelium (RPE) cell is affected by the gene encoding the receptor tyrosine kinase Mertk has been the most widely used in the study of RP. In terms of the cause of retinal degeneration in this rat, it has been suggested that an inability of the shed tips to perform phagocytosis of rod outer segment (ROS) debris by RCS RPE is primarily involved. ^{4 5 6} Nir et al. ^{7 8} described an interesting observation that light-induced stress promotes photoreceptor cell survival and function in the RCS rat, whereas light-induced stress causes apoptotic cell death in wild-type and other types of animal models with retinal degeneration. As a possible mechanism of the light-induced protective mechanism in the RCS rat retina, they suggested that light-induced stress may enhance the levels of expression of FGF2, which has been shown to be a possible survival factor of RCS retinal degeneration. ^{7 8} This beneficial effect toward RCS retinal degeneration suggests that some modulation in functions of the photoreceptor outer segment (OS) by light exposure must be related, because it is the only receptor for light within retinal neurons, and light triggers the visual transduction cascade reactions within OS. Therefore, to elucidate what kinds of mechanisms are involved on light-induced stress, systematic studies of photoreceptor cell functions including each step of the visual transduction processes, photoexcitation, quenching, and adaptation are essentially required. 

To gain new insights into the mechanism of light-induced stress on RCS retinal degeneration, we exposed RCS and wild-type rats to various illumination conditions, and the resultant retinal function was evaluated by ERG, rhodopsin (Rho) regeneration, Rho phosphorylation, cGMP concentration, and morphologic analysis. In addition, mRNA expression levels of several neurotrophic factors were investigated. 

Materials and Methods

All experimental procedures were designed to conform to the ARVO Statement for Use of Animals in Ophthalmic and Vision Research and our own institution’s guidelines. Unless otherwise stated, all procedures were performed at 4°C or on ice in ice-cold solutions. 

Light-Induced Stress Conditions to Wild-Type (SD, BN) Rats and Retinal Degeneration (RCS) Rats

In the present study, 3- to 5-week-old Sprague-Dawley (SD) rats, Brown Norway (BN) rats (Crea, Tokyo, Japan) and 3- to 5-week-old inbred RCS (rdy ^−/−) rats (albino type; Crea) reared in cyclic light conditions (650 lux, 12 hours on-off) were used. SD, BN, or RCS rats were exposed to 650, 1300, 2500, and 5000 lux white light for 24 hours in a light-induced stress box equipped with white fluorescent lamps and covered inside by mirrors (Fig. 1) . The rates were kept under usual cyclic light conditions or dark adaptation for ERG measurement and histologic examination. ERG measurements were performed at 1, 7, and 30 days in SD and BN rats, and 1 and 7 days in RCS rats after light-induced stress. Histologic examination was performed at 7 and 60 days in SD and BN rats and at 7 days in RCS rats after the light-induced stress. 

ERG Measurement

Details of the preparation, recording technique, and measurements of ERG have been described elsewhere. ⁹ Before ERG measurement, rats were subjected to 24 hours of dark adaptation. Under anesthesia, the each rat was laid on its side with its head fixed in place with surgical tape in an electrically shielded room for overnight dark adaptation. The pupils were dilated with drops of 0.5% tropicamide. ERGs were recorded with a contact electrode equipped with a suction apparatus to fit on the cornea (Kyoto Contact Lens Co., Kyoto, Japan). A ground electrode was placed on the ear. Responses evoked by white flashes (3.5 × 10² lux, 200-ms duration) using a Ganzfeld dome (SG-2002; Meiyo Co., Tokyo, Japan) were recorded and studied with ERG-analysis software (PowerLab Scope version 3.7; ADInstruments Ltd., Castle Hill, NSW, Australia). The a-wave amplitude was determined from the baseline to the bottom of the a-wave. The b-wave amplitude was determined from the bottom of the a-wave to the top of the b-wave. 

Preparation of Specific Antibodies toward Phosphorylated Rho at 334Ser or 338Ser

Specific antisera toward phosphorylated Rho at ³³⁴Ser or ³³⁸Ser were obtained by immunization of phosphorylated authentic peptides P-Rho334 peptide (DDEApSATASK) or P-Rho338 peptide (CEASATApSKT) chemically conjugated with bovine thyroglobulin, and antisera were each further purified into IgG by protein G Sepharose column chromatography as described recently. ¹⁰ Each purified antibody (0.1 mg IgG) was then incubated with urea-washed rat ROS (20 mg) at room temperature for 2 hours, and then the mixture was ultracentrifuged at 100,000g for 1 hour. The resultant supernatant was used as a specific antibody toward phosphorylated Rho at ³³⁴Ser or ³³⁸Ser. Antibody titers and their specificities were determined by ELISA and Western blot, respectively, as described in our recent report. ¹⁰  

Light and Immunofluorescence Microscopy

Enucleated eyes were fixed with methacarn (60% methanol, 30% chloroform, 10% glacial acetic acid) overnight, dehydrated, and embedded in paraffin. Posterior segments cut from the enucleated eyes were embedded in paraffin. Retinal sections were cut vertically through the optic disc at 2-μm thickness, mounted on subbed slides, and dried. The sections were processed with hematoxylin-eosin staining after deparaffinization with graded ethanol and xylene solutions. For evaluation of photoreceptor cell survival, the sections including the full length of the retina from the optic nerve head through the ora serrata were photographed, and the rows of cell nuclei in the photoreceptor outer nuclear layer (ONL) were counted at 200-μm intervals along the whole horizontal retinal axis. 

For immunofluorescence labeling, after the deparaffinization, sections were blocked with phosphate-buffered saline (PBS) containing 5% goat serum and 3% bovine serum albumin (BSA) for an hour and then incubated overnight with anti-P-Rho334 peptide antibody or anti-P-Rho338 peptide antibody (1:500) at 4°C. Sections were washed and incubated with fluorescein-isothiocyanate (FITC)-conjugated antibodies to rabbit IgG (Cappel, Durham, NC) for an hour at room temperature. Specificity controls were obtained by omitting the primary antibodies. Sections were observed, and pictures were taken with a fluorescence microscope (model BH-2; Olympus, Tokyo, Japan) using a blue filter. 

Quantitative RT-PCR Analysis

Total RNA from retinas (four eyes, two rats were used in each experimental condition) was isolated (Isogen Reagent) according to the procedure recommended by the manufacturer (Nippon gene, Tokyo, Japan.). The cDNAs were generated from 2 μg retinal RNA in a 12-μL reaction with 1 μL oligo(dT) primer (0.5 mg/mL; Invitrogen-Gibco, Rockville, MD). The reaction mix was denatured at 70°C for 10 minutes. Four microliters of first-strand buffer (250 mM Tris-HCl, 375 mM KCl, 15 mM MgCl₂; Superscript; Invitrogen-Gibco), 2 μL dithiothreitol (0.1 M; DTT; Invitrogen-Gibco), 1 μL deoxyribonucleoside triphosphate (10 mM; dNTP; Invitrogen-Gibco), 1 μL RNase inhibitor (40 U/μL; RNase inhibitor; Invitrogen-Gibco), and 1 μL reverse transcriptase (200 U/μL; Superscript II; Invitrogen-Gibco) were added to the mixture. The incubation was performed at 42°C for 50 minutes and at 70°C for 15 minutes. The PCR amplifications were performed using 4 μL from the RT reaction, 5 μL 10 × PCR buffer (200 mM Tris-HCl, 500 mM KCl), 2 μL MgCl₂ (50 mM), 1 μL dNTP, 5 μL sense and antisense primers (10 pM/μL), and 0.5 μL Taq polymerase (5 U/μL; Invitrogen-Gibco). The PCR mixture was denatured at 94°C for 4 minutes and then run for 30 cycles of 94°C for 1 minute, 55°C for 1 minute, and 72°C for 2 minutes. 

For PCR analysis (Taqman; Applied Biosystems, Inc. [ABI], Foster City, CA), the primers and probes were designed on computer (Primer-Express software; ABI) as follows: 

FGF2 (Gene ID 54250) forward, 5′-28CGCACCCTATCCCTTCACA-3′, reverse, 5′-128TCCACCCAAAGCAGTAGAAGGA-3′, detection probe, 5′-73TCCAAAACCTGACCCGATCCCTCC-3′. 

CNTF (Gene ID 25707) forward, 5′-78ATGGCTTTCGCAGAGCAAA-3′, reverse, 5′-262TCAGTGCTTGCCACTGGTACA-3′, detection probe, 5′-102CTGACCCTTCACCGCCGGGA-3′. 

BDNF (Gene ID 24225) forward, 5′-254CAAGCCACCATCAAAAGACTGA-3′, reverse, 5′-333GCTTGCCGGTTCCTCTCTCT-3′, detection probe, 5′-292ACAAGCGGCGGCACTTCCTCG-3′. 

PDGF (Gene ID 25266) forward, 5′-307TGACAGCCTCCCTGACT-3′, reverse, 5′-376CACCTGATTGAACTTGCAC-3′, detection probe, 5′-329AGCCTCGCTTCCACCTCCACACAA-3′. 

Rodent GAPDH as an internal control was amplified by using a commercially available kit (ABI) at the same time. The PCR mix contained 1 μL cDNA template; 1 × Taqman buffer A 8% glycerol; 5 mM MgCl₂; 200 μM each of dATP, dCTP, and dGTP; and 400 μM dUTP; 1.25 units DNA polymerase (AmpliTaq Gold; ABI), 0.25 units uracil-N-glycosylase (AmpErase; ABI), 300 nM each of the primers in total 50 μL. Standard reactions were performed with a sequence detection system (Prism; ABI). All experiments were performed in triplicate. 

Other Analytical Methods

Rho regeneration was determined by spectrophotometric analysis, as described previously. ¹¹ Briefly, rats were exposed to the different illumination intensities as described earlier and then subjected to dark adaptation. At different times during dark adaptation, under dim red light, rats were euthanatized, and eyes were enucleated and halved into the anterior and posterior segments. The posterior segments were then homogenized with 10 mM HEPES buffer (pH 7.5), containing 10 mM dodecyl β-maltoside and 20 mM hydroxylamine by a glass–glass homogenizer. The sample was centrifuged at 20,000g, and the spectra were recorded before and after complete bleaching. Rho concentrations were determined from the light-sensitive OD at 498 nm, assuming a molar extinction coefficient of 40,600 at 498 nm. Retinal cytosolic cGMP concentrations were determined by ELISA kit (Assay Designs, Inc., Ann Arbor, MI), with retinal extracts used according to the manufacturer’s protocol. 

Results

An intensity-dependent deterioration of the ERG response was observed the day after light-induced stress in SD and BN rats, when the wild-type rat species were exposed to several intensities of light (650, 1300, 2500, or 5000 lux for 24 hours; Fig. 2A ). Such ERG impairment was not significantly changed during the following 7 days or 30 days (Fig. 2A) . Although no significant changes in retinal morphology were observed until a few days after the illumination, intensity-dependent thinning of the ONL was recognized as early as 1 week after light-induced stress (Fig. 2B) . Further deterioration in the retinal morphology was not observed until 2 months after exposure to light (Fig. 2B) . Similarly, retinal ONL thinning and lowering of ERG responses were detected in the RCS rat, and thereafter the ERG responses continued to decrease (Fig. 3) . However, the rates of change after exposure to 650 and 1300 lux were almost identical with those of control conditions. These data indicate that intensity-dependent bright-light–induced ERG impairment was simply added to the RCS-dependent ERG deterioration. Light-induced retinal rescue effects proposed by Nir et al. ^{7 8} and other additional effects were not detected. 

Next, to get insight into which steps in the phototransduction cascade were affected by such light-induced stress within photoreceptor cells, Rho regeneration and Rho dephosphorylation and cytosolic cGMP concentrations were evaluated. These factors are known to be involved in the following mechanisms: (1) initial photoreception by Rho, (2) a critical step of quenching the photoexcitation, and (3) resultant metabolite of the final steps of the vertebrate phototransduction cascade. As shown in Figure 4 , rates of Rho regeneration during dark-adaptation from different light-induced bleaching conditions (1300 or 2500 lux) were almost identical in 3-week-old RCS and SD rats, suggesting that light-induced stress does not affect Rho regeneration in wild-type and RCS rats. Next, the effects of light-induced stress on Rho phosphorylation and dephosphorylation were studied by means of a recently developed immunohistochemical method involving a specific antibody against phosphorylated ³³⁴Ser or ³³⁸Ser, both of which have been identified as major sites of phosphorylation in Rho in vivo. ¹⁴ In SD rat retina, ³³⁸Ser antibody specifically recognized ROS of light-adapted but not of dark-adapted retina. The immunopositivity then gradually diminished from base to tip of the ROS after dark adaptation (Fig. 5A) . The ³³⁴Ser antibody showed identical immunolabeling properties, except that its immunoreactivity took a longer time to be diminished. Estimation of the kinetics of dephosphorylation of phosphorylated ³³⁴Ser and ³³⁸Ser sites of BN and SD were determined by measuring the vertical lengths of immunofluorescence-labeled ROS during the dark adaptation before and after the light-induced stress. Dephosphorylation of ³³⁴Ser and ³³⁸Ser sites went to completion within 1 or 2 hours and 3 or 4 hours in BN and SD rats, respectively, without light-induced stress, and dephosphorylation of these sites was significantly prolonged by light-induced stress (1300 lux; Fig. 5B ). To evaluate the influence of light-induced stress on Rho dephosphorylation kinetics, the times necessary to reach 50% dephosphorylation at these phosphorylation sites during dark adaptation were determined under several conditions, as indicated in Table 1 . Kinetics of Rho dephosphorylation were significantly delayed in RCS rats compared with wild-type rats, as described previously, ¹⁰ and those in wild-type and RCS rats were markedly delayed in a light-intensity–dependent manner. 

Levels of retinal cytosolic cGMP in the SD rat were significantly decreased by light exposure compared with that in dark adaptation. In contrast, light-dependent reduction in cytosolic cGMP was less in the RCS and SD rats pretreated with exposure to bright light (1300 lux) for 24 hours (Fig. 6) . 

Several neurotrophic factors, including FGF2, platelet derived growth factor (PDGF), brain-derived neurotrophic factor (BDNF), and ciliary neurotrophic factor (CNTF) and other factors are known to protect both the animal model of inherited retinal degeneration and light-induced retinal damage. Nir et al. ^{7 8} recently reported that some neurotrophic factors may be involved in light-induced rescue from RCS retinal degeneration. To test whether these phenomena take place, quantitative RT-PCR was performed. However, statistically significant changes were not observed in FGF2, PDGF, or CNTF on light-induced stresses with several light intensities between RCS and SD rats during the 7 days after light-induced stress. 

Discussion

Light-induced stress on the retina causes a series of reactions that lead to apoptotic cell death of photoreceptors. ² Its severity depends on the light’s intensity, duration of exposure, and wavelength, ^{12 13 14} as well as the animal species, such as albino and pigmented. ¹⁵ Alternatively, it has also been reported that light exposure of the retina in some conditions causes protective effects against retinal photoreceptor apoptosis. ^{7 8} This beneficial effect by light exposure may be the result of light-induced stimulation for the expression of some trophic factor such as FGF2. However, the molecular mechanisms causing this response to exposure to light, which includes both destructive and beneficial effects, are controversial and have not been fully clarified. Therefore, to elucidate what kinds of mechanisms are involved in this phenomenon, we systematically studied the effects of light-induced stress of several intensities on wild-type (SD and BN) and retinal degenerative (RCS) rat retinas and made the following observations: (1) On light-induced stress, intensity-dependent deterioration in retinal function analyzed by ERG and thickness of ONL occurred in wild-type rats, whereas levels of such light-induced deterioration in retinal function and morphology were different between SD (albino) and BN (pigmented) species, as described by Iseli et al. ¹⁵ (2) Similarly, deterioration in ERG response and retinal ONL thickness were observed in the RCS rat, but no beneficial effects on light-induced stress were observed. (3) Kinetics of Rho regeneration after light-induced stress was completed within approximately 2 hours of dark-adaptation in both wild-type and RCS rats. (4) The kinetics of Rho dephosphorylation was commonly delayed in wild-type and RCS rat retina by light-induced stress. However, these changes in kinetics exclusively depended on the rat species: albino, pigmented, and retinal degeneration. (5) Cytosolic cGMP concentrations were modulated by light-induced stress. (6) There were no significant changes in expression of the mRNA of several neurotrophic factors, including FGF2, PDGF, and CNTF in the light-induced retinal degeneration model. 

In terms of the abnormalities of photoreceptor cells in RCS rats, several changes were noted in the RCS ROS including opsin, ¹⁶ arrestin, ^{17 18} and ROS protein phosphorylation levels, ¹⁹ which may affect quenching of the phototransduction pathway. In our previous study in RCS rats compared with SD rats, ²⁰ we performed proteome analysis and found significantly lower levels of expression of the mRNA of α-A crystalline and rhodopsin kinase (RK), which are thought to be involved in post-Golgi processing of opsin and Rho phosphorylation, respectively. In contrast, expression levels of other major proteins of ROS were almost comparable to those in 3-week-old SD rats according to SDS- PAGE analysis. ²⁰ Therefore, we suggested that the kinetics of Rho phosphorylation and dephosphorylation are specifically affected in RCS rats. Thus, in 3-week-old RCS rats, it is reasonable to think that only the kinetics of Rho dephosphorylation is impaired, whereas the kinetics of Rho regeneration is comparable to wild-type rats in the present study. Our previous study involving the in vitro biochemical assay revealed that levels of Rho phosphorylation in 3-week-old RCS rats were slightly lower than those in 3-week-old SD rats, but, in contrast, dephosphorylation of phosphorylated Rho showed much slower kinetics in RCS than in SD rat ROS (3 weeks old). Furthermore, with our recent method of using specific antibodies to Rho phosphorylated at the ³³⁴Ser or ³³⁸Ser sites, which are known to be major phosphorylation sites in Rho in vivo, ^{11 21} we were able to evaluate the kinetics of dephosphorylation of phosphorylated photolyzed Rho in RCS and wild-type rats and SD and BN rats in vivo. This method applied during dark adaptation showed that dephosphorylation of ³³⁸Ser and ³³⁴Ser sites was completed within several hours (0.2–2 hours) in SD and BN rat retinas. However, those antibodies directed toward phosphorylated-³³⁸Ser and -³³⁴Ser sites were diminished within 4 to 7 days in RCS rat retinas. Therefore, we hypothesized that extremely prolonged survival of phosphorylated forms of Rho may contribute to persistent misregulation of phototransduction processes in retinal degeneration in RCS rat. ^{10 22} Our present results suggest that light-induced retinal degeneration may be caused by the same mechanism of RCS rat retinal degeneration as just described. since we found that light-induced stress also caused significant delay in the kinetics of Rho dephosphorylation. Furthermore, the retinal photoreceptor degenerative model of cancer-associated retinopathy (CAR), which is produced by intravitreous administration of anti-recoverin antibody to rats, showed significant high levels of Rho phosphorylation. ^{23 24} In addition, Rho mutants within the C terminus, in which ³⁴⁵Val and ³⁴⁷Pro are the most common sites of mutations causing autosomal dominant retinitis pigmentosa (adRP), ^{25 26} were also phosphorylated at significantly higher levels than in wild-type. ²⁷ Therefore, prolonged survival of phosphorylated Rho by lower phosphatase activities or enhanced Rho kinase activities may be one of the common mechanisms responsible for most of the retinal photoreceptor degeneration. Although slow kinetics in Rho dephosphorylation was commonly observed in these retinal degenerations, the degree of kinetic change did not correspond to the severity of retinal degeneration. Additional unknown mechanisms must therefore be present to account for retinal degeneration. 

As possible events occurred after the prolonged survival of the phosphorylated form of Rho in the light-damaged model, we can suggest the following mechanisms: (1) Phosphorylated Rho continuously suppresses light-dependent transducin activation causing cGMP accumulation within the cytosol; (2) cGMP-gated channels on plasma membranes are continuously open; (3) there is an increase in intracellular Ca²⁺ levels; and (4) a Ca²⁺-dependent apoptotic pathway is activated. In our present study, retinal cytosolic cGMP levels were significantly high in the RCS rat retina and light-induced, stress-treated wild-type retina under light adaptation to 650 lux for 1 hours (P < 0.05; Fig. 6 ). Because it is well known that cytosolic cGMP concentrations were strictly regulated by PDE and guanylate cyclase, ²⁸ despite a relatively small difference as shown in Figure 6 , this may induce misregulation of the phototransduction pathway by changing states of cGMP gated channels. This speculated mechanism is almost identical with the molecular pathology of CAR. ³ In the CAR rat model obtained by intravitreal administration of anti-recoverin antibody, the following set of events have been experimentally been determined: (1) anti-recoverin antibody penetrating the photoreceptor cells, ^{23 29} (2) binding of the anti-recoverin antibody with recoverin, (3) blocking of recoverin function and inhibition of RK in a calcium-dependent manner causing enhancement of Rho phosphorylation, ²⁴ (4) marked suppression of light-dependent transducin activation, (5) continued opening of cGMP gated channels on plasma membranes resulting in an increase of intracellular Ca²⁺ levels, ³⁰ and (6) activation of the caspase-dependent apoptotic pathway. ^{23 31} Therefore, an increase of intracellular Ca²⁺ levels that activate apoptosis is the key mechanism causing both CAR and light-induced retinal degeneration. In fact, our previous study demonstrated that normalization of elevated intracellular Ca²⁺ levels by nilvadipine, a Ca²⁺ channel blocker that preferably transfers to central nervous system (CNS), significantly protected against retinal degeneration in light-induced retinal degeneration, RCS, ³² and CAR model rats. ³³  

Protective effects toward retinal disease in RCS rats and other retinal degenerations have been demonstrated with mechanical damage, ³⁴ laser burns, ³⁵ and light-induced stress. ² Regarding the possible mechanisms of their retinal protective effects, they commonly induced enhancement of intrinsic neurotrophic factors, such as FGF2. In fact, intravitreous administration of several neurotrophic factors, including FGF2, ^{36 37 38} lens epithelium–derived growth factor (LEDGF), ³⁹ and hepatocyte growth factor (HGF) ⁴⁰ have shown significant protective effects. This mechanism is still the most likely because our previous study demonstrated significant upregulation of FGF2 and Arc during nilvadipine-induced retinal protection of RCS rat retina by DNA microarray analysis. ⁴¹ However, in contrast, there were not any significant changes in expression of neurotrophic factors in our present study, including FGF2, PDGF, and CNTF on light-induced stresses to wild-type and RCS rat retinas. It may be speculated that upregulation of neurotrophic factors by the retina within various species could require certain specific conditions of light-induced stress. Evidently, Nir et al. ^{7 8} described variations over time for the beneficial effects of light-induced stress to RCS rats. They found that the most notable beneficial effects in 23-day-old RCS rats, whereas only a slight effect was detected in 18-day-old rats. In our experiment, 18-day-old RCS rats were exposed to various intensities of light, and retinal morphology was almost normal in light microscopy examination, with slight deterioration of the ERG amplitude. Nevertheless, a light-induced effect on 23-day-old RCS rats could not suitably be evaluated because ERG responses were diminished and retinal morphology had severely deteriorated within 30 days in our RCS rat strain. Another possible reason for the difference in the light-induced effects observed by Nir et al. ^{7 8} and that in our present study is that the genetic background of the RCS rats may be somewhat different. In addition, in the experimental methods of Nir et al., rats were exposed to bright white light of 110 to 130 ft-c before intense light-induced stress, as a preconditioning protocol. In contrast, in our light-induced stress experiments, no such preconditioning was used. This difference in methods may be an alternative reason for the discrepancy in terms of the neurotrophic factor expression on light-induced stress. Nevertheless, Nir et al. reported that light-induced retinal rescue was found only in the RCS rat but not in the P23H rat, another model of RP. 

In conclusion, light-induced stress caused significant delay in the kinetics of Rho dephosphorylation resulting in misregulation of the visual transduction cascade in both normal and RCS retinal degeneration, and light-induced retinal rescue may occur in some specific animal species under certain special conditions. 

 Supported by grants from the Japanese Ministry of Health and Welfare and by a Grant-in-Aid for Scientific Research from the Japanese Ministry of Education, Sports and Culture.

 Submitted for publication August 28, 2005; revised January 24, March 25, and July 4, 2006; accepted October 23, 2006.

 Disclosure: F. Ishikawa, None; H. Ohguro, None; I. Ohguro, None; H. Yamazaki, None; K. Mamiya, None; T. Metoki, None; T. Ito, None; Y. Yokoi, None; M. Nakazawa, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Figure 1.

 

Light-induced stress for normal and RCS rats. Light-induced stress was administrated to wild-type and RCS rats using a light-induced stress box. Inner walls of the light-induced stress box are all covered by mirrors, and white fluorescence lights are attached under the top cover by which the light intensity can be controlled (top). Six rats could be housed in a cage which was divided into six rooms by acryl boards to keep the rats separate (bottom). The luminance levels differed less than 3% among each of the boxes within the cages.

Figure 2.

 

Effects of light-induced stress with several light intensities toward ERG responses (A) and retinal morphology (B) in BN and SD rats. Three-week-old BN or SD rats were exposed to light-induced stress with several light intensities (650, 1300, 2500, or 5000 lux) for 24 hours. Fifty rats were used in each experimental condition. After the light-induced stress, 10 rats were immediately subjected to 24 hours of dark adaptation, followed by ERG measurement. Another 10 rats were raised in regular cyclic light conditions (12 hours in 650 lux and 12 hours in the dark) for 6 or 29 days and then kept in the dark for 24 hours, followed by ERG measurements. The remaining 10 rats were kept in regular cyclic light conditions for 7 or 60 days and then euthanatized. The eyes (n = 10, from 10 rats) were enucleated, the retinas were processed for hematoxylin-eosin [HE] staining of paraffin sections, and the ONL nuclei were counted. Rats raised in cyclic light conditions without light-induced stress were used as control subjects. Mean ± SD of ERG amplitudes (b-wave) and rows of ONL nuclei are plotted in (A) and (B), respectively. (C) Representative ERGs obtained from rats exposed to light-induced stress with several light intensities at 1 day after exposure. *P < 0.01, **P < 0.001 (Mann-Whitney test).

Figure 3.

 

Effects of light-induced stress with several light intensities toward ERG responses (A) and retinal morphology (B) in RCS rats. Three-week-old RCS rats were exposed to light-induced stress with several light intensities (650, 1300, 2500, or 5000 lux) for 24 hours. Thirty rats were used in each experimental condition. After the light-induced stress, 10 rats were immediately subjected to 24 hours of dark adaptation, followed by ERG measurements. Another 10 rats were raised in regular cyclic light (12 hours in 650 lux and 12 hours in the dark) for 6 days and then kept in the dark for 24 hours followed by ERG measurements. The remaining 10 rats were kept in regular cyclic light conditions for 7 days and then euthanatized. The eyes (n = 10, from 10 rats) were enucleated, the retinas were processed for HE staining of paraffin-embedded sections, and the ONL nuclei were counted. Rats raised in cyclic light conditions without light-induced stress were used as control subjects. (A–C) ERG data are as described in Figure 2 . *P < 0.001 (Mann-Whitney test).

Figure 4.

 

Time course of Rho regeneration after stress induced by light several intensities. Three-week-old SD or RCS rats treated with 1300 or 2500 lux for 24 hours were then kept in the dark. In our RCS rat strain, retinal morphology and Rho concentrations were almost comparable, although ERG responses had already deteriorated within 3 weeks after birth. At several time points—0, 1, 2, 4, and 6 hours—rats were euthanatized, and enucleated eyes were processed to direct Rho concentration analysis. For each analysis, both eyes were used from one rat. Experiments were performed in triplicate using fresh preparations. No difference was observed between RCS and SD rats (P > 0.05, Mann-Whitney test). Data are expressed as the mean ± SD.

Figure 5.

 

Kinetics of dephosphorylation in phosphorylated 334^Ser and 338^Ser sites in BN and SD rats pretreated by light-induced stress. Three-week-old BN or SD rats were exposed to light-induced stress (1300 lux) for 24 hours and then were maintained in the dark. At different time points (BN rats at 0, 0.1, 0.2, 0.5, 1, 2, 4, and 6 hours; SD rats at 0, 1, 2, 3, 4, 6, and 12 hours), three rats (three eyeballs) in each category were subjected to immunofluorescence labeling by anti-P-Rho antibodies. Photographs of the sections were taken. Representative photographs of SD rat retinas under light (650 lux) or after different intervals of dark adaptation were treated by anti-P-Rho 338 antibody (A). Vertical length of photoreceptor outer segment layers and that of fluorescence labeling were measured at temporal points 1.0 mm apart from optic disc from six different points from three different eyeballs and their ratios were plotted in (B). Data are expressed as the mean ± SD. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; OS, outer segment. Scale bar, 25 μm. *P < 0.01, Mann-Whitney test.

Table 1.

 

Time Required for 50% Dephosphorylation of Rho at Specific Sites during Dark Adaptation from Different Bleach Conditions

	50% Dephosphorylation of Rho (h)
		No Light-Induced Stress	650 lux, 24 h	1300 lux, 24 h	2500 lux, 24 h	5000 lux, 24 h
BN 334Ser	0.15 ± 0.02	0.15 ± 0.02	1.8 ± 0.2	2.5 ± 0.5	3 ± 0.5
BN 338Ser	0.1 ± 0.05	0.1 ± 0.05	0.2 ± 0.04	0.5 ± 0.1	1 ± 0.2
SD 334Ser	2 ± 0.2	2.5 ± 0.3	3.5 ± 0.5	5 ± 0.5	6 ± 0.8
SD 338Ser	0.7 ± 0.1	1.5 ± 0.2	3 ± 0.6	4 ± 0.8	5 ± 0.5
RCS 334Ser	84 ± 12	120 ± 10	168 ± 15	>200	>200
RCS 338Ser	48 ± 10	60 ± 8	72 ± 10	120 ± 15	150 ± 12

 BN, SD, or RCS rats treated by light-induced stress with 650, 1300, 2500, or 5000 lux for 24 hours were then kept in the dark. At several time points (up to 400 h), rats were euthanized, and enucleated eyes were processed with immunofluorescence labeling by anti-P-Rho334 or anti-P-Rho338 antibody Rates of dephosphorylation in phosphorylated ³³⁴Ser and ³³⁸Ser sites were estimated by plotting of vertical length of the immunofluorescence labeled ROS as described in Figure 5 , and time required for 50% dephosphorylation of Rho at specific sites was calculated. For one analysis, three eyes from three rats were used. Experiments were performed on fresh preparations. Data are expressed as the mean ± SD.

Figure 6.

 

Retinal cytosolic cGMP concentrations in RCS rats and light-induced stress-treated SD rats. Retinal cytosolic cGMP concentrations were determined by ELISA. Analysis was performed using SD or RCS rats kept under dark for 6 hours and 650 lux light adaptation for 1 hour. In light-induced stress treated SD rat, rats kept at 650 lux light adaptation after light-induced stress (1300 lux) for 24 hours were analyzed. For one analysis, two eyes from one rat were used. Data are expressed as the mean ± SD. Experiments were performed in triplicate using fresh preparations. *P < 0.05, **P < 0.01 (Mann-Whitney test).

The authors thank Tadao and Akiko Maeda for excellent editing of the manuscript.