Mechanism of Protection from Light-Induced Retinal Degeneration by the Synthetic Antioxidant Phenyl-N-tert-Butylnitrone

Abstract

purpose. A prior study demonstrated that pretreatment with phenyl-N-tert-butylnitrone (PBN), a synthetic antioxidant and free radical trapping agent, protects rats from light-induced photoreceptor cell death. The objective of the present study was to elucidate the molecular mechanism of PBN neuroprotection.

methods. Sprague-Dawley rats (5–6 weeks old) raised in dim (5 lux) cyclic light (12 hours ON/OFF) from birth were injected intraperitoneally with PBN or water 30 minutes before exposure to three columns of fluorescent light (∼2700 lux intensity) for 0, 3, 6, 12, or 24 hours. mRNA levels were measured by RNase protection assay and DNA fragmentation by TUNEL assay. Activator protein (AP)-1 complex was determined by electrophoretic mobility shift assay. Immunocytochemistry and Western blots were used to measure changes in c-fos levels.

results. Typical apoptotic features (TUNEL staining and DNA laddering) were seen in rat retinas after 24 hours of continuous exposure to light, but not in PBN-injected rats. FasL, Bax, and caspase-3 were upregulated in a time-dependent manner. PBN treatment markedly inhibited caspase-3 gene expression, but neither PBN nor bright light exposure had any effect on caspase-3 activity. AP-1 activation by exposure to light was inhibited by PBN. Western blot analysis showed that the c-fos protein level increased in the nuclear fraction after a 6-hour exposure to light, but was decreased in PBN-treated rats.

conclusions. Inhibition of c-fos activation by PBN may be the key event in protection. The involvement of oxygen free radicals has been suggested in c-fos activation and the action of PBN could be through its antioxidant activity.

It is well known that excessive exposure to light induces photoreceptor degeneration in albino rats ^{1 2 3} and mice, ^{4 5 6 7} and these animals have been used for investigations on the mechanism of photoreceptor cell death. In addition, several animal models of inherited retinal degeneration have been developed and shown to have increased susceptibility to light damage. ^{6 7 8} Cell death in inherited photoreceptor degeneration and light-induced retinal damage has been shown to occur by apoptosis as the final common pathway. ^{3 9 10} Mechanistic studies of light-induced photoreceptor degeneration have demonstrated that the activation of the transcription factor activator protein (AP)-1 is an important step in the apoptotic pathway. ^{11 12} Furthermore, c-fos-deficient mice demonstrate decreased susceptibility to retinal light damage, ¹³ supporting the essential role of c-fos in this model. 

Our laboratory reported that pretreatment with the synthetic antioxidant phenyl-N-tert-butylnitrone (PBN) protects albino rats from light-induced photoreceptor cell depletion. ^{14 15} PBN is a free radical scavenger that was originally developed as a spin trapping agent to detect free radicals in chemical and biological systems. ^{16 17} PBN has demonstrated a variety of pharmacologic activities in animal models of inflammatory disease in which PBN′s free-radical-trapping action is considered to play an essential role. ^{18 19} In the central nervous system (CNS), PBN has been shown to increase the lifespan of mice ²⁰ and to provide neuroprotection from ischemia-reperfusion and oxidative injuries. ^{21 22 23 24} Other effects of PBN include reduction in the mortality associated with endotoxin shock ²⁵ and traumatic shock. ²⁶ In a rat endotoxin-shock model, PBN treatment inhibited LPS-mediated activation of AP-1 and NF-κB. ²⁷ Although PBN was effective in protecting rats from retinal light damage, no studies have been performed to elucidate the mechanism. In this study, we investigated the molecular mechanism of PBN′s protective effect against light-induced photoreceptor degeneration in albino rats and obtained evidence that it acts as an inhibitor of c-fos activation and apoptosis gene expression. 

Materials and Methods

Reagents

We synthesized and purified PBN in our laboratory to assure its purity. ²⁸ c-Fos and full-length human caspase-3 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and EMD Bioscience (San Diego, CA). The rat Apo-1 probe set (multiple apoptosis-related genes) for the RNase protection assay (RPA; RiboQuant) was purchased from BD BioScience (San Jose, CA). The biotin-labeled and unlabeled AP-1 double-stranded DNA was purchased from Panomics Inc. (Redwood City, CA). A caspase-3 colorimetric assay kit was purchased from Sigma-Aldrich (St. Louis, MO). 

Animals

Male and female Sprague-Dawley rats (6–7 weeks old) were kept in dim (5–10 lux) cyclic light (12 hours on/off, 6 AM–6 PM) after birth and fed laboratory chow ad libitum with free access to water. The animal care strictly conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and the University of Oklahoma Health Sciences Center (OUHSC) Guidelines for Animals in Research. All protocols were reviewed and approved by the Institutional Animal Care and Use Committees of the OUHSC and the Dean A. McGee Eye Institute. 

Light-Exposure and PBN Treatment Regimens

At 6 AM on the day of an experiment, rats that had been dark adapted overnight were exposed to a 2700-lux intensity of fluorescent light for various lengths of time. In PBN-treated groups, rats were injected intraperitoneally (50 mg/kg of an aqueous solution of 20 mg PBN/mL) 30 minutes before exposure to light. Rats were killed immediately after exposure to light and eyes processed for TUNEL assay or retinas removed for RNA preparation or gel analysis. In some instances, nuclei were prepared by centrifugation. 

Preparation of Retinal Sections

Rat eyes were fixed overnight at 4°C in 4% paraformaldehyde in phosphate-buffered saline (PBS; pH 7.4). The tissues were rinsed with PBS and immersed in 10%, 20%, and 30% sucrose in PBS. Samples were embedded in optimal cutting temperature (OCT) compound (Sakura, Tokyo, Japan) under liquid nitrogen and stored at −80°C. Cryosections (12 μm) of tissue were mounted on slides and air dried. 

TUNEL Staining

TUNEL staining was performed with an in situ cell death detection kit (Roche Applied Science, Indianapolis, IN) according to the manufacturer’s protocol. 

DNA Fragmentation

Retinas were gently homogenized in 100 mM Tris-HCl (pH 8.0) containing 20 mM EDTA. Proteinase K and SDS were added to the homogenized solution at a final concentration of 20 μg/mL and 0.8%, respectively, and the mixture incubated for 18 hours at 50°C. The mixture was extracted twice with phenol-chloroform and treated with RNase (20 μg/mL) at 37°C for 2 hours. DNA was precipitated with 3 M sodium acetate (pH 5.2) and ethanol, solubilized in TE buffer, and electrophoresed on a 1.5% agarose gel. 

Electrophoretic Mobility Shift Assay

AP-1’s binding activity to a consensus DNA sequence was determined with an electrophoretic mobility shift assay (EMSA). Retinal nuclear proteins were extracted using nuclear and cytoplasmic reagents (NE-PER; Pierce, Rockford, IL). Light-shift chemiluminescent EMSA kit (Pierce) was used according to the manufacturer’s protocol. 

Western Blot of c-Fos

For Western blots of c-fos in the nucleus, nuclear extracts (10 μg protein) were electrophoresed on 4% to 20% Tris-HEPES-SDS polyacrylamide gels (Pierce) and transferred onto a polyvinylidene difluoride (PVDF) membrane. The membranes were bathed with the c-fos antibody and then washed three times with TBST (10 mM Tris-HCl; [pH 8.0], 150 mM NaCl, and 0.1% Tween 20). Peroxidase-conjugated donkey anti-rabbit IgG (Amersham Biosciences, Amersham, UK) was used as secondary antibody. Protein bands were developed by enhanced chemiluminescence (ECL) according to the manufacturer’s instructions. 

Immunohistochemistry

Retina sections were washed with PBS containing 0.25% Triton X-100 (PBS-Tx) and treated for 10 minutes with PBS containing 3% Triton X-100. Sections were blocked with 3% normal goat serum in PBS for 30 minutes at room temperature and incubated with rabbit anti-c-fos antibody (1 μg/mL) overnight in a moist chamber at 4°C. After the slides were washed, they were incubated for 1 hour with AlexaFluor 488-conjugated goat anti-rabbit IgG at room temperature. Nuclear staining was performed with a 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI). The sections were observed under a fluorescence microscope (Eclipse E8000; Nikon, Tokyo, Japan). 

RNase Protection Assay

To determine the RNA levels of apoptosis-related genes, an RPA was used. Retinas were collected at indicated time points after exposure to light and frozen in liquid nitrogen. Total RNA extraction was performed (TRIzol Reagent; Sigma-Aldrich). RNA was hybridized with a probe set, digested with RNase, and electrophoresed as previously described. ²⁷  

Caspase-3 Western Blot Analysis and Activity

Retinas were gently homogenized in CHAPS (3-([3-cholamidopropyl]dimethylammonio-2-hydroxy-1-propanesulfonate) buffer (50 mM HEPES; [pH 7.4], 5 mM CHAPS, and 5 mM dithiothreitol [DTT]) and incubated for 20 minutes at 4°C. Retinal lysates were centrifuged at 16,000g for 15 minutes at 4°C and subjected to Western blot (100 μg protein) and a caspase-3 activity assay (50 μg protein) with a caspase-3 colorimetric assay kit (Sigma-Aldrich), used according to the manufacturer’s protocol. Results were calculated with a p-nitroaniline (pNA) calibration curve and are reported as nanomoles of pNA released per milligram protein per minute. 

Results

PBN Protection of Albino Rat Retina from Light-Induced Apoptosis

Dark-adapted rats were exposed to 2700-lux light for 0, 3, 12, or 24 hours, and retinas were processed for TUNEL or DNA fragmentation analysis. TUNEL-positive cells were not seen in the dark-adapted retina or after 3 hours of 2700-lux exposure (Figs. 1A 1B) , but appeared in the outer nuclear layer of the retinas of rats exposed to light for 12 or 24 hours (Figs. 1C 1D) . In rats treated with water, DNA laddering analysis indicated that the retina exposed to light for 24 hours showed characteristic DNA fragmentation on agarose gel electrophoresis (Fig. 2C)and on TUNEL staining (Fig. 2A) . One injection of PBN given 30 minutes before exposure to light protected the retinas against light-induced apoptosis. No TUNEL-positive cells were visible (Fig. 2B) , and DNA electrophoresis showed no DNA laddering (Fig. 2C)after 24 hours of exposure. Occasionally, a TUNEL-positive reaction occurred in other retinal layers (Fig. 2A) , but to a much less extent than in the ONL. These studies confirm and expand our previous reports of retinal protection by systemic administration of PBN before acute exposure to bright light. ^{14 15}  

PBN Effects on Light-Induced Upregulation of c-Fos

EMSA of retinas exposed to 2700-lux light for 0, 3, 6, 12, or 24 hours indicated that there was an increase in AP-1 binding activity for the first 12 hours, with maximum binding at 6 hours (Fig. 3A) . Administration of PBN 30 minutes before exposure to light prevented much of the increase in AP-1 binding (Fig. 3B) . Densitometry of the AP-1 band showed significant reduction in the retinas of PBN-injected animals (Fig. 3C) . 

Western blot analysis of retinal nuclear proteins showed that c-fos was increased after exposure to light (Fig. 4A) . Six hours of 2700-lux light markedly increased the c-fos level in the water-injected rats, but not in the retinas of PBN-injected animals (Fig. 4B) . 

Immunoreactivity of c-fos was present in all retinal layers in the dark-adapted retina (Fig. 5B) . In the retina of rats exposed to light for 6 hours, c-fos immunofluorescence increased in the outer plexiform layer and the outer nuclear layer (Fig. 5C) . PBN reduced the increase of c-fos in both layers (Fig. 5D) . 

Effects of PBN on Light-Induced Expression of Apoptosis-Related Genes

The effect of PBN on the expression of apoptosis-related genes was investigated by RPA. RNA was prepared from retinas of rats exposed to 0, 3, 6, 12, 24, or 72 hours of 2700-lux light and probed with rat Apo-1. Various levels of mRNAs of FasL, Bcl-xl, Bax, caspase-2, and caspase-3 were detected in the RPA (Fig. 6A) . Caspase-3, caspase-2, and Bax were upregulated in rats after exposure to light, whereas Bcl-xl and FasL were downregulated. Densitometric analysis indicated that retinal caspase-3 expression in animals exposed to 2700-lux light for 12 to 24 hours was ∼20 times higher than in dark-adapted rats and that this increase was reduced in PBN-treated rats (Fig. 6B) . The expression of FasL, caspase-2, Bcl-xl, and Bax was not affected by PBN (Fig. 6C) . Caspase-1 was present in the Apo-1 probe, but was not detected in the RPA. 

The full-length human caspase-3 antibody we used has immunoreactivity to both procaspase-3 and cleaved caspase-3. A small procaspase-3 band was found in all samples (Fig. 7A)and the level of pro-caspase-3 activity in the retina from PBN-injected animals was same as that of retinas from dark-adapted or water-injected animals after 12 or 24 hours of exposure to 2700-lux light. Caspase-3 activity in dark-adapted retina was 0.025 ± 0.001 nanomoles pNA released per milligram protein per minute and in retinas of water- or PBN-injected animals exposed to light for 12 hours was 0.029 ± 0.005 and 0.027 ± 0.006 nmol/mg, respectively (Fig. 7B) . In retinas exposed to light for 24 hours, the caspase activity of water- and PBN-injected animals was 0.026 ± 0.006 and 0.024 ± 0.003 nmol/mg, respectively, indicating that caspase-3 activity was not affected by exposure to light and/or PBN treatment (Fig. 7C) . 

Discussion

Apoptotic cell death occurs in both light-induced ^{10 29 30} and inherited retinal degeneration. ^{9 31 32 33 34} Previous studies have demonstrated the presence of two distinct pathways in light-induced apoptosis in mice. ³⁵ Bright light is thought to induce apoptosis that is independent of transducin, which is accompanied by induction of transcriptional factor AP-1. By contrast, low-light-induced apoptosis has been shown to require transduction. In the present study, AP-1 activation was clearly seen in bright-light-induced retinal degeneration, but was inhibited by pretreatment with PBN. In lipopolysaccharide (LPS)-treated rats and in LPS-stimulated cells, preadministration of PBN has been shown to inhibit significantly the activation of LPS-mediated NF-κB and AP-1, ^{27 36} both of which are considered to be involved in the LPS effect. 

A single administration of PBN before exposure to light was protective, suggesting that PBN is prophylactic rather than therapeutic in this model. Therefore, it is speculated that for PBN-mediated protection, its presence is necessary during the early signaling events that occur immediately after exposure to light. To explore early events in PBN protection from light damage, we investigated changes in AP-1, which mainly consists of c-fos/junD heterodimer. ¹¹ AP-1 is often involved in cellular responses caused by various external stimuli and regulates various gene expressions. In retinal light damage models, AP-1 DNA binding activity has been shown to increase during exposure to light. ⁴ DNA binding of c-fos is essential for a specific apoptotic pathway induced by light, but not for the execution of apoptosis induced by other stimuli such as N-methyl-N-nitrosourea. ¹¹ In the present study, even in retinas exposed to light for 6 hours, AP-1 activation was detected despite the absence of TUNEL staining and histologic changes, which confirms that AP-1 activation is an early event for light-induced photoreceptor degeneration. To our knowledge, this is the first report of AP-1 activation induced by light in rat photoreceptors. Western blot analysis showed a significant increase in c-fos level in retinal nuclear proteins obtained from light-exposed rats, which is consistent with DNA AP-1 binding activity. PBN inhibited the increase of both c-fos protein level and AP-1 activation. The presence of PBN in the EMSA gel did not affect AP-1 DNA binding activity (data not shown). In addition to the fact that PBN inhibits LPS-induced cytokine gene upregulation and AP-1 activation in rats, ²⁷ Ahmed et al. ³⁷ have shown that PBN suppresses IL-1-stimulated expression of matrix metalloproteinase-13 in human osteoarthritis chondrocytes, through the inhibition of JNK and AP-1. Inhibition of AP-1 activation has also been demonstrated in the protection from retinal light damage mediated by dexamethasone-induced glucocorticoid receptor activation. ³⁸ Thus, AP-1 inhibition may be a critical target in drug-mediated protection of retina from damaging light. 

Caspase-dependent apoptotic pathways are suggested to play a major role in photoreceptor degenerations. ^{39 40 41 42} In the present experiments, there was upregulation of caspase-3 gene expression after exposure to light, and this upregulation was significantly inhibited by preadministration of PBN. Other genes that were upregulated by exposure to light were unaffected by PBN. However, there was no significant change in caspase-3 amounts or catalytic activities in any animal (Fig. 7) , suggesting that there were discrepancies between mRNA and protein levels. Others have noted increased transcription in the absence of an increase in translation. ^{43 44 45 46 47 48} Under oxidative stress conditions, it has been reported that reactive oxygen inhibits the synthesis of proteins. ⁴⁹ Wu et al. ⁵⁰ exposed rats to 6 hours of intense blue light and then dark adapted them for various times up to 24 hours and measured caspase-3 activity in retinal homogenates. No enzyme activity was seen immediately after the animals were removed from the exposure to light, but significant activity was found after 8 and 16 hours of dark adaptation. Their Western blots showed an increase in a 32-kDa protein at 8 and 16 hours, which was not found in our Western blots performed on retinas removed immediately after exposure to bright light. Taken together, these results suggest that activation of caspase-3 occurs in the dark and may not occur as long as the animals remain in the light. Because we found apoptotic nuclei in the absence of increased caspase-3 activity, we suggest that this enzyme is not involved in apoptotic cell death in light-induced retinal degeneration. 

Caspase-1 has been shown to be a major player in light-induced photoreceptor degeneration in mice. ^{39 41 51 52} In our present study, probes in an RPA include Fas, Bcl-xl/S, FasL, caspase-1, caspase-2, caspase-3, Bax, Bcl-2, L32, and GAPDH. We did not detect any caspase-1 mRNA expression. The RPA is less sensitive than RT-PCR methods, and so we attempted to maximize its sensitivity by using radiolabeled probes that can detect mRNA of 5 × 10⁵ molecules. We cannot exclude the possibility that the RPA’s sensitivity was too low to allow detection of caspase-1 in our animals. The other possibility is that there are differences in the apoptotic process of light-induced retinal degeneration between rats and mice. Further studies are needed to clarify this point. 

Because photoreceptor cell death in light damage and hereditary retinal degenerations is by apoptosis, the light damage model has been used extensively ^{14 38 53 54 55} to screen putative neuroprotective drugs that ultimately were tested in mutant models of human retinal disease. Although some cytokines ^{47 56 57 58 59 60 61 62 63} and other drugs ⁸ were effective in both models, some were not. ^{15 64} We found that PBN protected against light-induced apoptosis in transgenic rats with a P23H or S334ter rhodopsin mutation, but not against photoreceptor degeneration due to expression of the mutant proteins, ¹⁵ suggesting that different apoptotic pathways may be involved in photoreceptor degeneration mediated by light damage and heredity. This lack of effect in the mutant rats is understandable in light of our current finding that PBN reduced c-fos expression in light-stressed rats. Hafezi et al. ¹³ showed that mice lacking c-fos were not damaged by light, but that reducing c-fos in mice with inherited retinal degeneration (c-fos ^−/− Rpe65L) did not prevent the loss of photoreceptor cells. ⁶⁵ Thus, although the final common pathway in light-induced and hereditary retinal degenerations may be apoptosis, there are fundamental differences in the upstream events that ultimately lead to cell death. 

 Supported by National Eye Institute Grants EY04149, EY00871, and EY12190; National Center of Research Resources Grant RR17703; OCAST-OARS; Research to Prevent Blindness, Inc.; and the Foundation Fighting Blindness.

 Submitted for publication August 4, 2004; revised October 10, 2004; accepted October 25, 2004.

 Disclosure: H. Tomita, None; Y. Kotake, None; R.E. Anderson, 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 micrograph of retinal sections from rats after dark adaptation (A) and immediately after exposure to 2700 lux for 3 (B), 12 (C), or 24 (D) hours. TUNEL-positive cells are visible in the outer nuclear layer (ONL) of the retina exposed for 12 or 24 (C, D) hours. Red: TUNEL-positive staining. RPE, retinal pigment epithelium; OS, outer segment; IS, inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Bar, 50 μm.

Figure 2.

 

Effects of PBN on light-induced photoreceptor cell death. Sterile water or PBN was intraperitoneally injected into rats 30 minutes before exposure to light (24 hours at 2700 lux). TUNEL-positive cells are evident in the water-injected animals (A), but not in the retinas of PBN-injected animals (B). Red: TUNEL-positive staining. Agarose gel electrophoresis of retinal DNA obtained from 12 hours of dark-adapted, water- or PBN-injected rats exposed to light for 24 hours (C). Lane 1: retina from dark-adapted rats; lane 2: retina from PBN-injected rats; lane 3: retina from sterile water-injected rats; and lane 4: 100-bp ladder marker.

Figure 3.

 

EMSA for AP-1 in the retina. Nuclear fractions were isolated from retinas of rats exposed to 2700-lux light for various times (A). The band disappeared when unlabeled AP-1 consensus double-stranded DNA was added to the reaction mixture as a competitor. (B) Effects of PBN on the activity of AP1 in the retina exposed to 2700 lux for 6 hours. The activity of AP-1 was significantly decreased by PBN injection (C). Results are shown as the mean ± SD, n = 3, *P < 0.05, unpaired t-test.

Figure 4.

 

Western blot analysis of c-fos in the retinal nuclear fraction. The c-fos content was increased by exposure to light (A) and was inhibited by PBN injection (B).

Figure 5.

 

Immunohistochemistry of c-fos in the retina exposed to 2700-lux light. c-Fos immunoreactivity in the dark-adapted retina were observed in all retinal layers (B), but were absent when the antibody was preincubated with the blocking peptide (A). In the retina of rats exposed to light for 6 hours and preinjected with water, c-fos immunofluorescence increased in the outer plexiform layer and to a lesser extent in the ONL (C). Preinjection with PBN inhibited the increase of c-fos (D). Bar, 20 μm.

Figure 6.

 

RPA for apoptosis-related genes. (A) Rat retinas were removed immediately after various times of exposure to 2700-lux light, and RNA was prepared for RPA. (B) RPA was performed on retinas from water- or PBN-injected animals exposed to 2700-lux light for 12 hours. (C) Densitometric analysis revealed that PBN inhibited only caspase-3 upregulation (n = 3).

Figure 7.

 

Caspase-3 Western blot analysis and activity in the retina exposed to light. Pro- and cleaved caspase-3 were detected by Western blot analysis (A). No differences in cleavage of caspase-3 were detected in water- or PBN-injected retinas after exposure to light. The activities of caspase-3 did not change in 12 (B) or 24 (C) hours of 2700-lux exposure to light.

The authors thank Mark Dittmar of the Dean McGee Eye Institute for maintaining the animal colonies used in this study and Charles Stewart of the Oklahoma Medical Research Foundation for help in developing the RPA.