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(Investigative Ophthalmology and Visual Science. 2002;43:2834-2840.)
© 2002 by The Association for Research in Vision and Ophthalmology, Inc.

NF-{kappa}B Activation in Light-Induced Retinal Degeneration in a Mouse Model

Tinghuai Wu, Yueguo Chen, Samuel K. S. Chiang and Mark O. M. Tso

From the Wilmer Eye Institute, Johns Hopkins School of Medicine, Baltimore, Maryland.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. To investigate the modulation of nuclear factor (NF)-{kappa}B in light-induced photoreceptor degeneration in a mouse model.

METHODS. Mice were exposed to intense green light. Light-induced activation of NF-{kappa}B and its nuclear localization were studied by immunohistochemistry. The NF-{kappa}B DNA–binding activity in the retinas after exposure to light was measured by electrophoretic mobility shift assay (EMSA). Nuclear transactivation of NF-{kappa}B in the photoreceptor cells was determined by quantitative real-time (qRT)-PCR. The amount of NF-{kappa}B p65 in the photoreceptor cells after exposure to light was assessed by Western blot analysis. To obtain more photoreceptor-specific information, microdissected photoreceptor cells were used in some studies.

RESULTS. By an immunohistochemical method, the perinuclear region of the photoreceptor cells was heavily labeled with an antibody to activated NF-{kappa}B after a 1-hour exposure to light. Nuclear localization of NF-{kappa}B in the photoreceptor nucleus was seen at 12 hours. In the experiments involving 3 hours of exposure to light followed by recovery in the dark, nuclear localization of NF-{kappa}B was also noted after 12 hours’ recovery in the dark. During continuous exposure to light, the NF-{kappa}B DNA–binding activity gradually increased and reached its maximum at 12 hours. There was an increase of NF-{kappa}B p65 protein at 3 hours. The mRNA levels of I{kappa}B{alpha} were upregulated after 6 hours’ exposure to light.

CONCLUSIONS. Intense light activated NF-{kappa}B in the photoreceptor cells in vivo, increased the NF-{kappa}B DNA–binding activity, and increased the expression of mRNA of I{kappa}B{alpha}, a target gene of NF-{kappa}B.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The primary function of the photoreceptor cells is perception of light, but exposure of the rodent retina to intense light leads to photoreceptor degeneration. Although the molecular mechanisms underlying this pathogenetic process are not clearly understood, there is evidence that the process is influenced by a variety of factors. Noell1 has demonstrated that this process is mediated by rhodopsin. The formation of free radicals is another important pathogenetic factor in light-induced retinal degeneration.2 3 4 5 Wenzel et al.6 and Hafezi et al.7 have reported that transcription factor activator protein-1 also plays an essential role in the regulation of light-induced apoptosis in photoreceptors at the execution stage.6 7 Calcium overload blockers, which inhibit inositol 1,4,5-triphosphate-induced release of intracellular stores of calcium, ameliorate light-induced retinal degeneration.8 Survival factors, such as brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), basic fibroblast growth factor (bFGF), nerve growth factor, and insulin-like growth factor-II, are thought to promote survival of the photoreceptor cells damaged by intense light.9 Recently, Wong et al.10 have noted the expression of MRG1 in the photoreceptor cells, a primary response gene that functions as a non–DNA-binding transactivator during light-induced retinal degeneration in rats.

The transcription factor nuclear factor (NF)-{kappa}B acts as a master regulator of stress responses in a variety of experimental conditions by exerting a strong modulatory effect on apoptosis. NF-{kappa}B is composed of homodimers and heterodimers, including p50, p52, p65 (RelA), c-Rel, and RelB. The dominant form of the dimer is p50/p65. In most cells, NF-{kappa}B is present in an inactive form in the cytoplasm of the cells, where it is bound to inhibitory I{kappa}B proteins. All I{kappa}B proteins—I{kappa}B{alpha}, I{kappa}Bß, I{kappa}B{gamma}, I{kappa}B{delta}, I{kappa}B{epsilon}, and Bcl-3—inhibit NF-{kappa}B’s activity by masking a nuclear localization signal (NLS) at the C terminus of the NF-{kappa}B subunits.11 12

NF-{kappa}B is a key regulator of apoptosis in a variety of experimental conditions. The analysis of NF-{kappa}B–deficient mice and cells provides distinct support of an antiapoptotic role for NF-{kappa}B.13 14 15 16 For example, NF-{kappa}B p65-/- mice die during embryonic development as a result of massive apoptosis in hepatocytes. Recent evidence suggests that NF-{kappa}B also contributes to neuroprotection in the central nervous system.17 18 Krishnamoorthy et al.19 and Crawford et al.20 have studied the role of NF-{kappa}B in mouse 661W photoreceptor cells. When 661W cells are exposed to light, NF-{kappa}B’s activity is decreased, and photoreceptor apoptosis ensues. Transfection of these cells with dominant–negative I{kappa}B{alpha} greatly increases the kinetics of downregulation of NF-{kappa}B, resulting in faster apoptosis.19 These observations suggest that NF-{kappa}B may play an important antiapoptotic role in 661W cells during light stress. However, the modulation of NF-{kappa}B in photoreceptor degeneration in vivo has not yet been examined.

In this study, we reproduced mouse model of photic injury, originally produced in rats.1 Modulation of light-induced NF-{kappa}B in the photoreceptor cells in the mouse model was investigated by immunohistochemistry, electrophoretic mobility shift assay (EMSA), quantitative real-time (qRT)-PCR, and Western blot analysis. Microdissected photoreceptor cells from mouse retinas were used for qRT-PCR and Western blot analysis to obtain more photoreceptor-specific information.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Male albino BALB/cJ mice (Jackson Laboratories, Bar Harbor, ME), aged 4 to 5 weeks, were used in the study. All protocols adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Animal Care and Use Committee of Johns Hopkins University. The mice were fed standard laboratory chow (Prolab RMH 1000; Quality Laboratory Products, Inc., Elkridge, MD) and allowed free access to water in an air-conditioned room with a 12-hour light–dark cycle at a light intensity of 20 to 40 lux.

Exposure to Light
The mouse model of photic injury was used in this study. Exposure to lights was performed in a chamber of green plexiglas (model 2092; Dayton Plastics, Dayton, OH), which transmits green light at an illuminance of 3.1 to 3.5 Klux. The illumination inside the chamber was equidirectional, and the chamber temperature during exposure to light was 25 ± 1.5°C.

After 14 days of cyclic light followed by 48 hours of dark adaptation, mice were exposed to intense light. Two schedules of exposure were used: First, 18 mice in groups of 3 were exposed to intense light and killed immediately after 1, 3, 6, 9, 12, or 24 hours of exposure. Second, 12 mice in groups of 3 were exposed to intense light for 3 hours, placed in a dark room for recovery, and killed after 0, 3, 6, or 12 hours of recovery. Three mice maintained in the dark for 48 hours without exposure to light served as the control. Both sets of experiments were repeated three times.

Tissue Preparation
The mouse eyes were enucleated, embedded in optimal cutting temperature compound, fresh-frozen on dry ice, and stored at -80°C. Blocks were cut vertically at 9 µm through the optic nerve head and ora serrata with a cryostat.

Morphometric and TUNEL Studies
The effects of intense light on the mouse retinas were examined in hematoxylin and eosin–stained sections by light microscopy. Nine sections from three mice were used for morphometric measurements and TUNEL. The thicknesses of the outer nuclear layer (ONL) of the entire retina was evaluated quantitatively on computer with image analysis software (MicroPlan II; Donsanto, Natick, MA), as previously described.22 The morphometric measurements were recorded in retinal sections obtained along the posterior-anterior axis of the globe. Photoreceptor apoptosis was determined by TUNEL, as previously described.23 The number of TUNEL-positive photoreceptor cells was expressed as a percentage of the total number of photoreceptor cells per section in the retinas. Results are expressed as the mean ± SD.

Immunohistochemistry
NF-{kappa}B p65 immunolabeling was performed with a kit (MOM; Vector Laboratories, Burlingame, CA) according to the manufacturer’s instructions. In brief, frozen sections were fixed in acetone, quenched in 0.3% H2O2 in methanol, and incubated in a mouse IgG blocking solution for 1 hour. A monoclonal antibody that recognizes the active form of NF-{kappa}B p65 (12H11; Roche Diagnostics, Indianapolis, IN)24 25 26 was applied at a 1:200 dilution, and sections were incubated at 4°C overnight. NF-{kappa}B p65 immunoreactivity was detected by a biotinylated secondary antibody, and 3,3'-diaminobenzidine was used as the chromogen. An isotype control antibody, a mouse anti-Salmonella poona monoclonal antibody (IgG3; MAB 750; Chemicon International, Inc., Temecula, CA), was used at the same concentration as the anti-NF-{kappa}B p65 monoclonal antibody.

Electrophoretic Mobility Shift Assay
The preparation of retinal nuclear extracts and determination of the NF-{kappa}B DNA–binding activity were performed with a nuclear and cytoplasmic reagent kit (NE-PER; Pierce, Rockford, IL) and an electrophoretic mobility shift assay (EMSA) chemiluminescence kit (LightShift; Pierce), respectively, according to the manufacturer’s protocols. A double-stranded oligonucleotide containing an NF-{kappa}B DNA–binding consensus sequence, 5'-AGT TGA GGG GAC TTT CCC AGG C-3' (Santa Cruz Biotechnology, Santa Cruz, CA), and a mutant double-stranded oligonucleotide, 5'-AGT TGA GGC GAC TTT CCC AGG C-3', were used to study NF-{kappa}B DNA–binding activity, as previously described.27 Briefly, 2 µg of nuclear extracts from the whole retina was preincubated in a reaction mixture for 20 minutes, and biotin end-labeled, double-stranded oligonucleotide containing the {kappa}B consensus sequence was added. Five microliters of loading buffer was added to each sample. A 20-µL aliquot of the samples was electrophoresed through a 6% nondenaturing polyacrylamide gel.

Microdissection of the ONL
Frozen sections were fixed in 70% ethanol for 30 seconds. Sections were washed in dH2O for 10 seconds and 70% ethanol for 10 seconds; stained with eosin-Y for 30 seconds; and dehydrated in 95% ethanol for 10 seconds, 100% ethanol for 10 seconds, and 100% xylene for 30 seconds. Sections were then dried. Laser capture microdissection (LCM) was performed on a commercially available apparatus (PixCell II with a beam diameter of 7.5 µm; Arcturus, Mountain View, CA). The photoreceptor cells were placed on the transfer film on the undersurface of a tube cap. The caps were then placed in matching tubes and stored at -80°C for qRT-PCR analysis.

For Western blot analysis, manual dissection of the ONL was performed as previously described.28

Western Blot Analysis
Microdissected photoreceptor cells were homogenized with a tissue grinder in 50 µL of cold suspension buffer (20 mM HEPES-KOH [pH 7.5], with 250 mM sucrose, 10 mM KCl, 1 mM MgCl2, 1 mM EDTA, 1 mM EGTA, and 1 mM DTT) containing proteinase inhibitor cocktail (Roche Diagnostics). The homogenates were centrifuged at 750g at 4°C. Samples (5 µg) were separated on a 12% SDS-PAGE gel, and Western blot analysis was then performed as previously described.29 The polyclonal antibody (sc-372; Santa Cruz Biotechnology), which reacts with an epitope consisting of 20 amino acids at the C terminus of NF-{kappa}B p65, was used for immunodetection.

To evaluate the specificity of antibody binding, the immunoblot was stripped and reprobed as follows. The blot was incubated for 30 minutes in 62.5 mM Tris buffer (pH 6.7), containing 100 mM ß-mercaptoethanol and 2% SDS at 50°C. The stripped blots were then reprobed with the antibody, which was preincubated with a fivefold excess of the blocking peptide (sc-372P; Santa Cruz Biotechnology).

RNA Extraction and qRT-PCR
Fifty microliters guanidinium isothiocyanate lysis solution was added to tubes containing the ONL of the retina obtained by LCM. The tubes were inverted and agitated with an extraction solution until all the fluid was in contact with the surface of the cap containing the LCM-prepared samples. RNA recovery from the caps was performed as previously described.30 RNA was amplified using an RNA PCR kit (GeneAmp; PE-Applied Biosystems, Foster City, CA) according to the manufacturer’s protocol. The mRNA levels of I{kappa}B{alpha} were determined by qRT-PCR with Smart Cycler (Cepheid, Sunnyvale, CA) using a fluorescent dye specific for DNA-double strands (LightCycler-FastStart DNA Master SYBR Green1 kit; Roche Diagnostics). qRT-PCR was performed in 25-µL reactions (2–5 µL of RT reaction product, 2.8 µL fluorescent dye, and 200 nM of each I{kappa}B{alpha} primer). The following primer pairs were used: I{kappa}B{alpha}, 5'-ATGAAGGACGAGGAGTACGAGCAA-3' and 5'-TCTCTTCGTGGATGATTGCCAA-3; 18S rRNA, 5'-CTTGACCATGTTCCCAGAGTCG-3' and 5'-AGCACCTCGACGCTTACA-AGA-3'. The melting temperatures for amplicons ranged from 79°C to 80°C. Control experiments established that the signal for each amplicon was derived from cDNA and not from primer dimers.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Morphologic, Morphometric, and TUNEL Studies
Morphologic study of the retinas after exposure to light (<6 hours) without dark recovery showed a few scattered, densified retinal RPE cells, but the number of densified photoreceptor nuclei increased substantially between 6 and 24 hours’ exposure to light. No subretinal macrophages were seen, and no morphologic changes were observed in the inner nuclear layer (INL) of the retinas. The thickness of the ONL of the retinas remained at 42–51 µm in retinal sections (Fig. 1A) .



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  		Figure 1. (A) Thickness of the ONL of retinal sections in mouse eyes subjected to continuous exposure to light. (B) Number of TUNEL-positive photoreceptor cells in the ONL of the retinas of mice exposed to light for various times. Note the significant increase in the number of TUNEL-positive photoreceptor cells after 6 hours of exposure.

 
In TUNEL analysis, a small number of apoptotic photoreceptor cells were found to be scattered throughout the ONL of the retina after 3 hours’ exposure to light. The number of positive nuclei was markedly increased at 6 hours (Fig. 2) . The percentage of TUNEL-positive photoreceptor cells at 9 hours was about three times that at 6 hours (Fig. 1B) . After 24 hours of exposure to light, many TUNEL-positive photoreceptor cells had coalesced, and accurate counting of the individual TUNEL-positive cells was not feasible. Therefore, the data at that time point are not included.



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  		Figure 2. TUNEL analysis of retinal sections in mouse eyes subjected to continuous exposure to light. (A) A negative (dark-adapted) control retina. (B–F) TUNEL-positive photoreceptor cells of the retinas at 1, 3, 6, 12, or 24 hours of exposure to light, respectively. (G–I) Hematoxylin and eosin staining of the superior retina from a negative (dark-adapted) control eye and from eyes exposed to light for 6 or 24 hours, respectively. Original magnification, x400.

 
Light-Induced Activation of NF-{kappa}B in Photoreceptor Cells
The light-induced activation of NF-{kappa}B in the photoreceptor cells was investigated by immunohistochemical labeling using a monoclonal antibody specific for NLS of NF-{kappa}B. The NLS epitope is not accessible in the inactive form of NF-{kappa}B in the cytoplasm of the cells, where NF-{kappa}B is masked by its inhibitor I{kappa}B{alpha}. Our immunohistochemical study indicated that the active NF-{kappa}B p65 immunoreactivity in the perinuclear region of photoreceptor cells was markedly increased over control levels after 1 to 6 hours of exposure to light. The definitive nuclear localization was observed at 12 hours (Figs. 3E 3F) . In addition, a low level of immunoreactivity to the anti-NF-{kappa}B p65 antibody was noted in the photoreceptor cells in dark-adapted control samples.



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  		Figure 3. Immunochemical localization of NF-{kappa}B p65 in the mouse retina after continuous exposure to light. (A) After 48 hours of dark adaptation, weak NF-{kappa}B p65 immunoreactivity was present in scattered photoreceptor nuclei (arrows). (B–D) After 1, 3, or 6 hours of exposure to light, respectively, NF-{kappa}B p65 immunoreactivity in the perinuclear region of the photoreceptor cells increased in the ONL. (E, F) After 12 or 24 hours of exposure, respectively, nuclear localization of NF-{kappa}B was noted in the photoreceptor cells (arrows). Original magnification, x1000.

 
To assess timing of the nuclear translocation of activated NF-{kappa}B in the cytoplasm of the photoreceptor cells, mice were exposed to light for 3 hours; allowed to recover in the dark for 0, 3, 6, or 12 hours; and killed immediately at those time points. Nuclear localization of NF-{kappa}B in the photoreceptor cells was prominently evident after 12 hours of dark recovery (Fig. 4E) . In contrast, an isotype control at that time point was negative (Fig. 4F) .



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  		Figure 4. Immunochemical localization of NF-{kappa}B p65 in the mouse retina after 3 hours’ exposure to light, followed by recovery in the dark. (A) After 48 hours of dark adaptation without exposure to light. (B–E) Recovery for 0, 3, 6, or 12 hours, respectively. (F) Sections from eyes exposed to intense light for 12 hours that were immunolabeled with an isotype control antibody (anti-Salmonella). OS, outer segment. Original magnification x 1000.

 
We performed EMSAs to determine the levels of nuclear NF-{kappa}B DNA–binding activity in the retina after exposure to light. In the EMSA blot, there were two prominent bands (Fig. 5) . The intensity of the upper band was increased after 3 hours of exposure to light, reached its maximum at 12 hours, and decreased at 24 hours. The specificity of the upper band was determined by the application of excess cold (unlabeled) and mutant {kappa}B double oligonucleotides in the assay.



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  		Figure 5. EMSA blot showing effects of exposure to light on the NF-{kappa}B DNA–binding activity in mouse retinas. Lane 1: NF-{kappa}B was constitutively active in the photoreceptor cells. Lanes 2 to 6: NF-{kappa}B DNA–binding activity in the nuclei of retinal cells after 3, 6, 9, 12, and 24 hours of exposure to light, respectively. Lanes 7 and 8: NF-{kappa}B DNA–binding activity by competition EMSA, with 100-fold molar excess of cold and mutant NF-{kappa}B oligonucleotides, respectively. LS, lower-shifted band.

 
We performed Western blot analysis to assess the protein levels of NF-{kappa}B in the ONL of the retinas after exposure to light. We found that the amount of NF-{kappa}B p65 in the ONL was notably increased at 3 hours (Fig. 6A) . The blot was stripped and then reprobed with the antibody preincubated with its blocking peptide, to verify the specificity of the 65-kDa bands. The 65-kDa band was not visible after reprobing (Fig. 6B) . In addition, to exclude the possibility that the disappearance of the 65-kDa bands was not the result of a loss of protein after stripping, the immunoblot was again stripped and reprobed with the antibody. The reappearance of the 65-kDa band confirmed that NF-{kappa}B p65 protein was not significantly lost after stripping; thus, the 65-kDa band specifically corresponded to NF-{kappa}B p65 subunits (Fig. 6C) .



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  		Figure 6. Western blot of NF-{kappa}B p65 protein level in photoreceptor cells after exposure to light. (A) ONL proteins (5 µg) obtained from retinas of mice exposed to intense light for 1, 3, or 24 hours and from dark-adapted control mice were electrophoresed on a 12% SDS-PAGE gel and immunolabeled with a polyclonal antibody against NF-{kappa}B p65. One microgram of NIH/3T3 whole-cell extract was used as a positive control. NS, nonspecific. Solid arrow: localization of the specific NF-{kappa}B p65 band. (B) The membrane was stripped and reprobed with antibody to NF-{kappa}B p65 that was preincubated with its blocking peptide. Open arrow: disappearance of the specific NF-{kappa}B p65 band. (C) The membrane was restripped and reprobed with an antibody to activated NF-{kappa}B p65. Solid arrow: localization of the specific NF-{kappa}B p65 band. (D) Retinal section after manual microdissection of the ONL of the retina.

 
Light-Induced Upregulation of I{kappa}B{alpha} Expression in Photoreceptor Cells
To monitor the nuclear transactivation of NF-{kappa}B in the photoreceptor cells after exposure to light, we determined the mRNA levels of I{kappa}B{alpha}, a target gene of NF-{kappa}B, by qRT-PCR with total RNA isolated from LCM-obtained ONL (Fig. 7A) . The levels of I{kappa}B{alpha} mRNA were upregulated after 6 hours of exposure to light and increased by a factor of 9 at 12 hours, compared with that of the dark-adapted control.



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  		Figure 7. Light-induced expression of I{kappa}B{alpha} mRNA in the photoreceptor cells. (A) Relative expression levels of I{kappa}B{alpha} and 18S rRNAs in the photoreceptor cells at various exposure times were determined by qRT-PCR. 18S rRNA was used as an internal control. (B) The residual retinal section after LCM. OS, outer segment; IS, inner segment; OPL, outer plexiform layer.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
To study light-induced photoreceptor degeneration, we reproduced in a mouse model of photic injury, originally produced in rats.1 The alterations in the mouse photoreceptor and RPE cells were morphologically comparable to those in the light injury rat model.1 The reasons for using a mouse model for this study are the ready availability of transgenic mice and the possible generation of transgenic mice for future study.

In this study, light-induced activation of NF-{kappa}B in the photoreceptor cells in vivo was demonstrated by immunohistochemistry, EMSA, and qRT-PCR. The subcellular localization of the activated NF-{kappa}B after exposure to light was investigated by immunohistochemistry. The activated NF-{kappa}B was first noted in the perinuclear region and later in the nucleus of the photoreceptor cells 12 hours after continuous exposure (Fig. 3F) . In a further evaluation the nuclear translocation of the activated NF-{kappa}B, mice were exposed to intense light for 3 hours and allowed to recover in the dark. Photoreceptor nuclei immunolabeled with an antibody specific for NF-{kappa}B p65 subunits were seen after 12 hours of recovery (Fig. 4E) , indicating that 3 hours’ exposure to light was sufficient to induce the nuclear translocation of NF-{kappa}B in the photoreceptor cells.

During continuous exposure to light, the NF-{kappa}B DNA–binding activity in retinal nuclear extracts progressively increased until 12 hours and then decreased at 24 hours (Fig. 5) . The banding pattern of the EMSA blot was similar to that reported in hippocampal neurons secondary to excitatory injury.31 The upper band corresponded to NF-{kappa}B and the lower band to the novel neuronal {kappa}B-binding protein described by Moerman et al.32 The physiological roles of the novel protein remain to be determined. Our immunohistochemical study showed that the major contribution of the NF-{kappa}B DNA–binding activity in the retinal nuclear extracts came from the photoreceptor cells. The decreased level of the NF-{kappa}B DNA–binding activity noted after 24 hours of exposure to light may be a consequence of extensive photoreceptor cell death, because apoptotic photoreceptor cells were markedly increased at that time point. Another possibility is that the decreased NF-{kappa}B DNA–binding activity at 24 hours reflects a negative-feedback regulation of NF-{kappa}B modulation. The relationship between decreased NF-{kappa}B–binding activity and photoreceptor apoptosis requires further investigation. It is attractive to speculate that the decreased NF-{kappa}B DNA–binding activity may play a role in light-induced photoreceptor degeneration.

Expression of I{kappa}B{alpha} is tightly regulated by NF-{kappa}B through an autoregulatory mechanism.33 When the activated NF-{kappa}B is bound to {kappa}B sites in the I{kappa}B{alpha} promoter region, the mRNA level of I{kappa}B{alpha} increases.12 To monitor nuclear transactivation of NF-{kappa}B in the photoreceptor cells, the levels of I{kappa}B{alpha} mRNA were determined by qRT-PCR. After 6 and 12 hours of exposure to light, the mRNA levels of I{kappa}B{alpha} were dramatically increased (Fig. 7A) . The upregulation of I{kappa}B{alpha} mRNA was an indicator of light-induced nuclear transactivation of NF-{kappa}B in the photoreceptor cells.

In short, the increase in nuclear NF-{kappa}B DNA–binding activity was noted after 3 hours of exposure to light, and the level of I{kappa}B{alpha} mRNA was upregulated after 6 hours, whereas photoreceptor nuclei immunolabeled with anti-NF-{kappa}B p65 antibody were not visible until 12 hours of continuous exposure to light. Thus, light-induced nuclear transactivation of NF-{kappa}B in photoreceptor cells detected by the EMSA and qRT-PCR analysis was much earlier than the nuclear localization of NF-{kappa}B observed by immunohistochemistry. These observations indicate that a small amount of the activated NF-{kappa}B translocated into the nuclei of the photoreceptor cells before 12 hours.

In the immunohistochemical and EMSA studies, low levels of NF-{kappa}B p65 immunoreactivity and NF-{kappa}B DNA–binding activity were observed in the dark-adapted photoreceptor cells. These results indicate that NF-{kappa}B was constitutively active, which is consistent with observations in a study of 661W cells.19 It has been reported that electrical activity within neurons and synaptic transmission between neurons are potent stimuli for activation of NF-{kappa}B,34 and such neuronal activity in the dark-adapted retinas may account for the constitutive activation of NF-{kappa}B in the photoreceptor cells.

For our Western blot analysis, we used microdissected ONL of the retinas, which consisted of the photoreceptor cells as well as the processes of Müller cells. Increased NF-{kappa}B p65 protein was observed after 3 hours of exposure to light (Fig. 6A) . It was interpreted as contributions from both the active and inactive forms of NF-{kappa}B p65 in the ONL of the retina, because the antibody did not differentiate the active NF-{kappa}B from the inactive one in Western blot analysis. The increased NF-{kappa}B p65 may be due to an increase in NF-{kappa}B translation and/or transportation of NF-{kappa}B from other parts of the cells to the ONL in response to exposure to light.

In contrast to light-induced activation of NF-{kappa}B in photoreceptor cells in vivo, NF-{kappa}B activity in the 661W cells is downregulated on exposure to light.19 The difference in the modulation of NF-{kappa}B in response to light in vivo and in vitro may reflect the effects of different cellular context and environment. The photoreceptor cells in vivo are surrounded by Müller cells, which not only provide structural support for the photoreceptors, but also perform important physiologic functions, such as the facilitation of the transmission of nerve impulses, the removal of excess neurotransmitters, and the production and transportation of growth factors.35 Recently, Harada et al.36 have noted that light stress upregulates both P75ntr and the high-affinity neurotrophin receptor TrkC in different parts of the fibers of Müller cells. The blockade of P75ntr prevents reduction of bFGF and results in both structural and functional photoreceptor survival in vivo. The absence of P75ntr significantly prevents light-induced photoreceptor apoptosis. Because cultured photoreceptor cells do not have the support from Müller cells and interactions with other neuronal cells in the retina, the progressively decreased NF-{kappa}B activity without an initial increase of its activity after exposure to light is not surprising. It is possible that light-induced activation of NF-{kappa}B in the photoreceptor cells requires cellular factors such as growth factors from other retinal cells. Walsh et al.37 have shown that the levels of bFGF and CNTF in the retinas are upregulated after exposure to light. LaVail et al.9 have demonstrated that growth factors, particularly bFGF and CNTF, protect the rat photoreceptor cells from light damage. It has been reported that bFGF enhances NF-{kappa}B’s activity in vascular smooth muscle cells in a dose-dependent manner.38

In addition, cultured photoreceptor cells can proliferate, whereas the photoreceptor cells in vivo are terminally differentiated without proliferative ability. This may also affect light-induced modulation of NF-{kappa}B in the photoreceptor cells. Accordingly, in vivo studies are crucial to elucidate the mechanisms of light-induced photoreceptor degeneration.

Although in vivo study may better reflect the actual scenario of the photoreceptor cells’ response to light stress, a major challenge in such studies is the complexity of the retinal tissue, which makes obtaining photoreceptor-specific information difficult. To overcome this obstacle, relatively pure photoreceptor cells were dissected from the ONL of the retina for some of our studies.

In conclusion, we have demonstrated that intense light activates NF-{kappa}B in photoreceptor cells in vivo. The specific role of NF-{kappa}B in light-induced photoreceptor degeneration is under investigation.


   Footnotes
 
Supported by a Research to Prevent Blindness senior scientific investigator award (MOMT), Oliver Birckhead Fund, and the Michael B. Panitch Research Fund.

Submitted for publication March 19, 2002; revised April 26, 2002; accepted May 16, 2002.

Commercial relationships policy: N.

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.

Corresponding author: Mark O. M. Tso, Wilmer Eye Institute, Johns Hopkins Hospital, Room 457, Woods Building, 600 N. Wolfe Street, Baltimore, MD 21287-9238; mtso{at}jhmi.edu.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Noell, W. (1980) Possible mechanisms of photoreceptor damage by light in mammalian eyes Vision Res 20,1163-1171[Medline][Order article via Infotrieve]
  2. Li, Z, Tso, M, Wang, H, Organisciak, D. (1985) Amelioration of photic injury in rat retina by ascorbic acid: a histopathologic study Invest Ophthalmol Vis Sci 26,1589-1598[Abstract/Free Full Text]
  3. Tso, M. (1989) Experiments on visual cells by nature and man: in search of treatment for photoreceptor degeneration (Friedenwald Award and Lecture) Invest Ophthalmol Vis Sci 30,2421-2454
  4. Tso, MO. (1987) Retinal photic injury in normal and scorbutic monkeys Trans Am Ophthalmol Soc 85,498-556[Medline][Order article via Infotrieve]
  5. Organisciak, D, Wang, H, Li, Z, Tso, M. (1985) The protective effect of ascorbate in retinal light damage of rats Invest Ophthalmol Vis Sci 26,1580-1588[Abstract/Free Full Text]
  6. Wenzel, A, Grimm, C, Marti, A, et al (2000) c-Fos controls the "private pathway" of light-induced apoptosis of retinal photoreceptors J Neurosci 20,81-88[Abstract/Free Full Text]
  7. Hafezi, F, Marti, A, Grimm, C, et al (1999) Differential DNA binding activities of the transcription factors AP-1 and Oct-1 during light-induced apoptosis of photoreceptors Vision Res 39,2511-2518[Medline][Order article via Infotrieve]
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