December 2007
Volume 48, Issue 12
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Retina  |   December 2007
Photoreceptor Protection against Light Damage by AAV-Mediated Overexpression of Heme Oxygenase-1
Author Affiliations
  • Ming-Hui Sun
    From the Department of Ophthalmology and the
    Graduate Institute of Clinical Medical Sciences, Chang Gung University, Taoyuan, Taiwan; the
  • Jong-Hwei Su Pang
    Graduate Institute of Clinical Medical Sciences, Chang Gung University, Taoyuan, Taiwan; the
  • Show-Li Chen
    Graduate Institute of Microbiology, College of Medicine, National Taiwan University, Taipei, Taiwan; the
  • Ping-Chang Kuo
    Graduate Institute of Microbiology, College of Medicine, National Taiwan University, Taipei, Taiwan; the
  • Kuan-Jen Chen
    From the Department of Ophthalmology and the
  • Ling-Yuh Kao
    From the Department of Ophthalmology and the
  • Ju-Yun Wu
    Departments of Medical Research and
  • Ken-Kuo Lin
    From the Department of Ophthalmology and the
  • Yeou-Ping Tsao
    Departments of Medical Research and
    Ophthalmology, Mackay Memorial Hospital, Taipei, Taiwan; and the
    Department of Microbiology and Immunology, National Defense Medical Center, Taipei, Taiwan.
Investigative Ophthalmology & Visual Science December 2007, Vol.48, 5699-5707. doi:10.1167/iovs.07-0340
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      Ming-Hui Sun, Jong-Hwei Su Pang, Show-Li Chen, Ping-Chang Kuo, Kuan-Jen Chen, Ling-Yuh Kao, Ju-Yun Wu, Ken-Kuo Lin, Yeou-Ping Tsao; Photoreceptor Protection against Light Damage by AAV-Mediated Overexpression of Heme Oxygenase-1. Invest. Ophthalmol. Vis. Sci. 2007;48(12):5699-5707. doi: 10.1167/iovs.07-0340.

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

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Abstract

purpose. To investigate whether overexpression of the cytoprotective gene heme oxygenase-1 (HO-1) in photoreceptors by gene delivery attenuates cellular injury caused by intense light damage and to document the possible mechanisms of protection.

methods. Recombinant adeno-associated virus type 5 (rAAV5) expressing the mouse HO-1 gene (mHO-1) was delivered to cyclic-light reared Sprague-Dawley (SD) rats by subretinal injection. Three weeks after transfer of HO-1 gene, animals were subjected to 2-hour intense light exposure then were returned to darkness. Expression of HO-1, p53, p38, and cellular FLICE inhibitory protein (c-FLIP) at different times after intense light damage was evaluated by Western blot analysis. HO-1 transgene expression, along with expression of c-fos and bcl-2, was analyzed by immunohistochemistry. In addition, the protective effects of HO-1 were evaluated by determining the morphology of the retina. Finally, apoptosis in photoreceptors was measured using TdT-dUTP terminal nick-end labeling (TUNEL) 24 hours after photic injury.

results. Exogenous administration of HO-1 by gene transfer led to HO-1 transgene expression in photoreceptors. Protection of retina by HO-1 overexpression is evident from the partially preserved retina structure and attenuated apoptosis in photoreceptors after photic injury. Concurrently, overexpression of HO-1 was associated with a decrease in the expression of c-fos and p53, an increase in the activation of p38 and bcl-2, and preserved the expression of c-FLIP.

conclusions. Overexpression of HO-1 in photoreceptors protected themselves from subsequent cellular damage caused by intense light exposure. The anti-apoptotic mechanisms of HO-1 may be related to the induction of p38, bcl-2, and c-FLIP and to the suppression of c-fos and p53.

Retinal photoreceptor degeneration is the main result of several vision-threatening retinal diseases, such as retinitis pigmentosa, age-related macular degeneration, and retinal dystrophies. Intense light exposure induces damage to rod outer segments (ROS) of photoreceptors and ultimately results in loss of visual cells in rats. 1 This model mimics these human retinal diseases and has long been used for the study of light-induced photoreceptor degeneration. Normally, the initial visual cascade is triggered by rhodopsin bleaching in ROS. However, in this model, intense light exposure also damages ROS first, which is then followed by photoreceptor cell apoptosis. Because the retina is an oxygen-rich environment and the ROS membranes are full of polyunsaturated fatty acids, it has been speculated that oxygen-free radicals are responsible for this light-induced retinal damage through lipid peroxidation. 2  
Expression of heme oxygenase (HO), a heat shock and acute stress protein, is induced in response to various stimuli 3 4 and primarily consists of three isoenzymes—HO-1, HO-2, and HO-3. 5 6 HO-1 is found to overexpress in the rat retina in response to intense light exposure 7 8 9 ; therefore, HO-1 is considered an effective marker to evaluate the effect of intense light damage to the retina. Furthermore, induction of HO-1 may be a cellular response to oxidative stress and could be a part of antioxidant defense system. Antioxidants such as 1,3-dimethylthiourea, a flavonoid, protect retinal photoreceptor cells and retinal pigment epithelial cells against intense light damage by reducing oxidative stress-induced apoptosis through either reducing or increasing HO-1 expression levels. 7 8 10 However, it is not clear whether HO-1 induction in retina in response to intense light damage is beneficial or detrimental to photoreceptors. 
Recombinant viral vectors are powerful tools for delivering genes to target tissue in vivo. In this study, we transduced photoreceptors by double-strain adeno-associated virus serotype 5 (AAV 5) encoding the HO-1 gene (AAV5-HO-1) to investigate whether the overexpression of HO-1 in retina could protect retina photoreceptors from light damage. It was also of interest to document the possible mechanisms through which HO-1 protects photoreceptors from light damage-induced apoptosis. 
Materials and Methods
Animals
Four- to five-week-old male Sprague-Dawley rats, each weighing 150 to 250 g, were housed in a temperature-controlled room. The animals were kept on a 12-hour light/12-hour dark schedule and had free access to food and water. All animals were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
Generation of rAAV-HO-1
The mouse HO-1 gene was amplified by primers 5′-ATGGAGCGTCCACAGCCCGAC-3′ and 5′-TTACATGGCATAAATTCCCA-3′ from the mouse cDNA library. The amplified products of polymerase chain reaction (PCR) were verified for their DNA sequences and cloned into the BamHI/ NotI site of a double-strain AAV vector plasmid. For generation of rAAV, the constructed AAV vector plasmids, AAV packaging plasmids (pXR5), and adenovirus helper plasmids (pXX6) were cotransfected into T3 cells. The rAAV5-HO-1 (expressing HO-1 gene) and rAAV5-LacZ (expressing Escherichia coli LacZ gene) virus particles were produced by freeze/thaw cycles and cesium chloride density gradient purification, as previously described. 11 Titers of rAAV-HO-1 and rAAV-LacZ were determined by dot blot hybridization using HO-1 and LacZ DNA as probes, respectively. 
Transduction of HO-1
Rats were anesthetized with intramuscular injections of 0.8 mL/kg of an equal-volume mixture of 50 mg/mL ketamine (Ketalar; Parke-Davis, Morris Plains, NJ) and 2% xylazine (Rompun; Bayer AG, Leverkusen, Germany). Because the superior hemisphere is more susceptible to light damage than the inferior hemisphere in rodent animals, 1 we chose to inject the viral particles into the superior quadrant of the retina to evaluate the protective effect of HO-1 on photoreceptor. Therefore, a sclerotomy was made 1 mm behind the superior limbus with the tip of a 30-gauge needle. A 33-gauge blunt-tip needle (Hamilton, Reno, NV) was inserted tangentially toward the superior and posterior pole of the eye, and 3 μL viral suspension containing 1.1 × 1010 viral particles (approximately 1.1 × 108 infectious units or 1.1 × 107 transduction units) was injected. 12 Successful injection was confirmed by identifying a retinal detachment roughly one fourth the size of the retina. Eyes with massive subretinal hemorrhage, lens trauma, or other complication were excluded. The contralateral eye of each rat was left without injection or eyes were injected with rAAV-LacZ as control. 
Light Damage
All rats were reared in a cyclic light environment for 12 hours/day. Before intense light treatment, rats were dark adapted for 24 hours. After pupil dilation, one dark-adapted rat was placed in each chamber (consisting of five circular acrylic resin [Plexiglas; Altuglas International, Philadelphia, PA] tubes) and was exposed to intense light (13,000 Lux) for as long as 2 hours, beginning at 9 AM. Each rat was housed separately in individual chambers to prevent them from shielding each other from the light. During light exposure, the room temperature was kept at 24°C and the animals were unrestrained; food and water were also available. After light exposure, all rats were returned to darkness. 
Semiquantitative RT-PCR
Total RNA was extracted from retinas with reagent (TRIzol; Invitrogen, Carlsbad, CA). Synthesis of cDNA was performed with 1 μg total RNA at 50°C for 50 minutes, using oligo (dT) primers and reverse transcriptase (Superscript III; Invitrogen). The amplification mixture (final volume, 20 μL) contained 1 × Taq polymerase buffer, 0.2 mM dNTPs, 1.5 mM MgCl2, 1 μM primer pair, and 0.5 U Taq DNA polymerase (Life Technologies). cDNA was equalized in an 18- to 22-cycle amplification reaction with HO-1 primers (forward, 5′-GTG-GAG-ACG-CTT-TAC-GTA-GTG-C-3′; reverse, 5′-CTT-TCA-GAA-GGG-TCA-GGT-GTC-C-3′), yielding a 250-bp product. The number of cycles for the HO-1 primer set (denaturation, 30 seconds, 94°C; annealing, 40 seconds, 57°C; and polymerization, 45 seconds, 72°C) was chosen to be in the linear range of amplification. 
Western Blot Analysis
At intervals of 0, 6, 12, 24 hours, and 1 week after 2-hour intense light exposure, rats were killed in a CO2-saturated chamber, anterior segments were removed, and retina wholemounts were isolated and shock frozen at −80°C within 2 minutes of enucleation. Retinas were later ultrasonically homogenized into 300 μL Ripa buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 10 mM EDTA, 0.1% SDS, 1% NP-40, 0.5% sodium deoxycholate, 1 mM Na3VO4, 1 mM NaF, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, and proteinase inhibitor at 4°C. Protein extracts (20 μg protein in each lane) were separated by 12% SDS-PAGE, and the proteins were transferred to a nitrocellulose membrane. The membranes were then blocked and probed with rabbit polyclonal anti-HO-1 (Abcam, Cambridge, UK), anti-p38 (Promega, Madison, WI), anti-p53 (Chemicon, Temecula, CA), anti-cellular FLICE inhibitory protein (c-FLIP; Abcam), and anti-actin (Sigma, St. Louis, MO) antibody at different dilution. A peroxidase-conjugated anti-rabbit secondary antibody (PerkinElmer, Norwalk, CT) was used at a dilution of 1:15,000. Immunoblots were visualized by chemiluminescence (ECL Plus; Amersham Pharmacia Biotech UK, Little Chalfont, Buckinghamshire, UK). 
Immunohistochemistry
Immunohistochemistry was performed to examine the localization of the endogenous and exogenous HO-1, c-fos, and bcl-2 after intense light exposure and to determine the difference between retina transduced by rAAV-HO-1 and rAAV-LacZ. Rats were killed 0 hour and 6, 12, and 24 hours after light damage. Eyeballs were fixed in 2% paraformaldehyde for overnight storage and then were embedded in paraffin. After dewaxing and rehydration of the paraffin sections, the tissue specimens were incubated with one of the following primary antibodies: rabbit polyclonal antibody against rat HO-1, c-fos, or bcl-2 (all Abcam). Immunoreactivity was detected by a fluorescein isothiocyanate (FITC)-labeled or a Cy3-labeled anti-rabbit antibody (Abcam), and cell nuclei were counterstained with 4′-6-diamidino-2-phenylindole (DAPI). 
In Situ TUNEL Labeling
Eyeballs were harvested 24 hours after 2 hours of intense light exposure and were sectioned along the vertical meridian to include a full length of retina passing through the optic nerve head and the superior and inferior regions of the eye. For each rat, two 3-μm thick retinal sections that included the ora serrata and the optic nerve were stained by TdT-mediated dUTP nick-end labeling (TUNEL)-based kit (TdT FragEL; Oncogene, Darmstadt, Germany). The number of TUNEL-positive cells for each retinal section was counted in two selected superior retinal areas, each 0.66 mm in length. The first segment chosen was located 0.66 mm superior to the optic nerve head, and the second segment was 0.66 mm superior to the first segment The total number of TUNEL-positive cells of these four retina areas was averaged as a representative of TUNEL-positive cells per one eye sample (n = 6 eye samples in each group). 
Morphology and Transmission Electron Microscopy
Seven days after intense light exposure, rats were killed in a CO2-saturated chamber. Before enucleation, the superior conjunctiva was sutured with 8.0 vicryl as a reference that provided easy orientation. Eyecups were fixed in 2.5% glutaraldehyde for transmission electronic microscopy preparation. The superior hemisphere along the vertical meridian, including the optic nerve, was chosen to evaluate the photoreceptor damage. The retina from the optic nerve head to peripheral area was divided equally into six points, and the outer nuclear layer (ONL) thickness and the combined thickness of ONL, rod outer segment (ROS), and rod inner segment (RIS) of the retina were measured using a standard microscope (original magnification, ×400; n = 7 in each group). 
Results
Expression of Exogenous HO-1 by Gene Transfer in Retina and Expression of Endogenous HO-1 in Retina
Western blot analysis of homogenized retina tissue without light exposure revealed that the expression of HO-1 was undetectable in normal retina and rAAV-LacZ-treated retina. However, in retina transduced with AAV-HO-1 for 3 weeks, high-level synthesis of HO-1 was detected, and the amount of HO-1 production was 5.3-fold greater than that in normal retina and in retina treated with AAV-LacZ (Fig. 1A)
As shown in Figures 1B and 1C , the expression of endogenous HO-1 protein and mRNA in retina was detectable at 6 hours after intense light damage, peaked at 12 hours, decreased at 24 hours, and gradually returned to basal level at 1 week. On the other hand, after intense light exposure, total amounts of HO-1 mRNA and protein in rAAV-HO-1-treated retina also peaked at 12 hours, decreased at 24 hours, and were still constantly expressed at 1 week. 
Immunohistochemistry was performed to study the localization of HO-1 expression. Immunohistochemical analysis showed prominent HO-1 immunoactivity in the ONL of the treated retina (Fig. 2B)compared with untreated retina (Fig. 2A) . This finding indicated that AAV-mediated delivery of the HO-1 gene successfully contributed to exogenous HO-1 protein expression in photoreceptors. Twenty-four hours after light exposure, immunoactivity of endogenous HO-1 was observed mainly in internal limiting membrane (ILM), basal Müller cell processes, and external limiting membrane (ELM) (Fig. 2C) . In contrast, a strong HO-1 signal remained detectable in ONL in rAAV-HO-1-transduced retina, but less HO-1 signal was detected in ILM (Fig. 2D)
rAAV-HO-1-Mediated Anti-apoptotic Effect
Fewer TUNEL-positive cells were present in the ONL in rAAV-HO-1-treated retina than in the rAAV-LacZ-treated retina or the untreated retina 24 hours after intense light injury (Fig. 3A) . Quantitative analysis of the number of TUNEL-positive cells in the ONL showed statistically fewer apoptotic cells in rAAV-HO-1-treated retinas (33.2 ± 17.2 cells) than in rAAV-LacZ-treated retinas (226.4 ± 79.35 cells) or untreated retinas (269.58 ± 95.3 cells) (P < 0.05; n = 6; Fig. 3B ). 
rAAV-HO-1-Mediated Preservation of Retina Structure
In untreated retina or rAAV-LacZ-treated retina, significant reduction of RIS and ROS thickness, near disappearance and shrinkage of photoreceptors, and complete disruption of the external limiting membrane (ELM) was found 7 days after intense light damage (Figs. 4C 4D) . However, ELM, ONL, RIS, and ROS were partially preserved in rAAV-HO-1-treated retina (Fig. 4B) . Quantitative analysis for retina thickness revealed that reduction in the thickness of the ONL or in the combined thickness of ONL, RIS, and ROS was significantly ameliorated in rAAV-HO-1-treated retina than in rAAV-LacZ-treated and untreated retina (Figs. 4E 4F)
rAAV-HO-1-Mediated Suppression of c-fos and p53 and Activation of bcl-2 and p38
Immunohistochemical analysis of retinal tissue revealed that the proto-oncogene c-fos protein was induced 6 hours after intense light injury in rAAV-LacZ-treated retina (Figs. 5D 5E 5F)and that activation of c-fos protein was suppressed in rAAV-HO-1-treated retina (Figs. 5A 5B 5C) . The level of p53 expression was significantly upregulated at 12 hours and 24 hours after intense light damage in rAAV-LacZ-treated retina compared with rAAV-HO-1-treated retina (Fig. 6 ; 6.71 ± 0.55-fold-vs. 2.8 ± 0.83-fold and 6.93 ± 0.77-fold vs. 5.65 ± 0.7-fold, respectively; P < 0.05). In contrast, expression of p53 was not significantly detected in rAAV-HO-1-treated retina until 24 hours after intense light damage (Fig. 6) , indicating that rAAV-HO-1 treatment delayed the expression of p53. Expression of p38 was induced in rAAV-HO-1-treated retina at 6 hours and was induced in rAAV-LacZ-treated retina 12 hours after intense light damage (Fig. 6) . The level of p38 was significantly higher in rAAV-HO-1-treated retina at 6, 12, and 24 hours after intense light damage than in rAAV-LacZ-treated retina (Fig. 6 ; 2.73 ± 0.66-fold vs. 1.12-fold ± 0.11-fold, 4.91-fold ± 0.26-fold vs. 2.07-fold ± 0.24-fold, and 5.92-fold ± 1.1-fold vs. 5.22-fold ± 1.05-fold, respectively; P < 0.05). The increase in p38 expression appeared in a time-dependent fashion. These findings indicate that rAAV-HO-1 transduction activates the expression of p38. 
We also documented the effect of HO-1 on the expression of survival-promoting bcl-2 protein after photic injury. Higher immune reactivity of bcl-2 in ONL was found in rAAV-HO-1-treated retina 24 hours after photic injury (Figs. 7A 7B 7C)than in rAAV-LacZ-treated retina. Throughout our experiments, however, the levels of other apoptosis-related proteins (bax, caspase 3) were not changed by HO-1 gene delivery when assayed by Western blot assay or immunohistochemistry (data not shown). 
rAAV-HO-1-Mediated Preservation of c-FLIP
The long variant of cellular FLICE inhibitory protein (c-FLIPL) was found to exist in control retina immediately after intense light damage and was significantly upregulated 6 hours after intense light damage, then was nearly undetectable at 12 and 24 hours after intense light damage (Fig. 8) . Similarly, the short variant of cellular FLICE inhibitory protein (c-FLIPS) was decreased gradually in control retina after intense light damage. However, these decreases in c-FLIPL and c-FLIPS levels were significantly attenuated in rAAV-HO-1-treated retinas at 12 hours and 24 hours after intense light damage compared with rAAV-LacZ-treated retinas, indicating that HO-1 gene delivery preserved the level of c-FLIP (Fig. 8 ; c-FLIPL: 3.27 ± 1.08-fold vs. 0.97 ± 0.76-fold at 12 hours, 3.01 ± 0.63-fold vs. 0.82 ± 0.68-fold at 24 hours; c-FLIPS: 1.38 ± 1.06-fold vs. 1.0 ± 0.05-fold at 12 hours, 1.18 ± 0.11-fold vs. 1.0 ± 0.04-fold at 24 hours; P < 0.05). 
Discussion
Overexpression of HO-1 by gene transfer has been shown to inhibit apoptosis and to protect cells from oxidative damage in microvessel endothelial cells, rat cardiac myocytes, and hyperoxia-induced lung injury. 13 14 15 16 In this study, the administration of exogenous HO-1 by gene transfer led to the overexpression of HO-1 protein in photoreceptor cells (Fig. 2) , attenuated apoptosis in photoreceptors 24 hours after intense light exposure (Fig. 3) , and ameliorated the reduction of ONL thickness and combined ONL+RIS+ROS thickness caused by light damage (Fig. 4) , suggesting that overexpression of HO-1 in photoreceptors themselves could further provide protection against light-induced damage. 
Our study revealed the AAV-HO-1 treatment could partially inhibit intense light-induced apoptosis in retinas (Fig. 3) . Interestingly, treatment also induced p38 and bcl-2 overexpression in retina (Figs. 6 7) . This correlation led us to suggest that the protection effect of HO-1 may occur in part through the induction of p38 and bcl-2. On the other hand, intense light induced c-fos and p53 in retina, which were significantly attenuated by AAV-HO-1 treatment (Figs. 5 6) . Because it has been found that c-fos and p53 are involved in triggering apoptosis in several types of tissue, 17 18 our observations further support the hypothesis that HO-1 overexpression can provide protection signaling against apoptosis. 
An earlier study shows that 12- to 24-hour intense light exposure causes endogenous HO-1 expression in the retina at 24 hours to be higher than that at 12 hours. 7 Our finding revealed that endogenous HO-1 levels peaked at 12 hours after light exposure but decreased at 24 hours (Figs. 1B 1C) . We propose that this difference may result from different light exposure times. The rats were exposed to intense light for 12 to 24 hours in Kutty et al. 7 but to only 2 hours in our study. This 2-hour light exposure might have failed to sustain the levels of endogenous HO-1 protein that resulted from the longer 12-hour light exposure. 
HO-1 is a rate-limiting enzyme in heme degradation and has been shown to be involved in cellular responses to oxidative stress. 19 HO-1 is also induced in retina after light damage. 7 8 9 Although most studies speculated that this might be a protective response, 19 20 the exact role of HO-1 in the retina in response to light damage has not been clarified. In the present study, our results demonstrated for the first time that endogenous expression of HO-1 was induced in ILM, basal Müller cell process, and ELM in vivo after intense light exposure (Fig. 2) , which was comparable to previous findings in a rat ischemia/reperfusion injury model 21 and in retinal organ culture. 20 This may be an intrinsic protective mechanism, but it is not enough to inhibit intense light-induced damage. In this regard, transduction of the HO-1 gene in retina by rAAV was mainly confined to photoreceptors (Fig. 2)that provided a significantly protection from intense light-induced apoptosis (Fig. 3) , reflecting that the specific induction of HO-1 in ONL was more essential than in other type cells in retina. This finding can provide the very important explanation for the observed protection of photoreceptors in rAAV-HO-1-treated retinas after light exposure. In addition, we observed that at 1 week after intense light exposure, the total amounts of HO-1 mRNA and protein in the rAAV-HO-1-treated retina remained a level as high as before light exposure, which suggested that maintaining an adequate level of HO-1 expression is beneficial to support the survival of photoreceptors. 
The protective effect of HO-1 is thought to come from the byproducts of heme catabolism, such as ferritin, bilirubin, and carbon monoxide (CO). Ferritin, induced by free iron release activity of HO-1, serves as a free radical scavenger and a cytoprotective antioxidant. 22 23 Bilirubin acts as an antioxidant and attenuates lipid peroxidation. 24 CO, a gaseous molecule with biological activity similar to that of nitric oxide, 25 is an important anti-apoptotic agent 26 and is associated with the prevention of ischemia/reperfusion-induced retinal damage. 27 Evidence indicates that the anti-apoptotic effect of CO is mediated by the activation of p38 mitogen-activated protein kinase (MAPK). 26 28 Our observation that HO-1 transgene expression in photoreceptors led to the activation of p38 MAPK (Fig. 6)suggests that p38 MAPK upregulation may mediate the protection offered by HO-1 transgene expression in photoreceptors against light-induced apoptosis. It is still unclear to us whether CO also mediates the protection effect of HO-1 in light-injured retina. 
Accumulating reports have revealed the mechanism of apoptosis in photoreceptors in response to intense light damage. 1 8 29 Activation of c-fos is also essential for light-induced apoptosis of photoreceptor cells in the adult mouse retina. 17 Overexpression of bcl-2 in transgenic mice protects photoreceptors from apoptosis caused by constant light damage. 30 Caspase-1 and caspase-3 have also been demonstrated to participate in light-induced apoptosis in photoreceptors. 31 32 Recently, light-induced photoreceptor apoptosis was further shown to be mediated by the activation of neuronal nitric oxide synthase (nNOS) and guanylate cyclase, which is caspase-3 independent. 33 Our study demonstrated for the first time that HO-1 overexpression in photoreceptors by gene transfer rescued retina and attenuated apoptosis in photoreceptors, concomitant with a decrease in the activation of proto-oncogene c-fos induced by intense light exposure and an increase in activation of survival-promoting gene bcl-2. Our observation provided in vivo evidence supporting the notion that the modulation of c-fos and bcl-2 gene expression may mediate the protection of light-damaged photoreceptors by HO-1. 
The proapoptotic effect and mechanism of p53 are well documented. Many types of stress response activate p53, including DNA damage response. 34 35 p53 responds to DNA damage by arresting the cell cycle for DNA repair. 36 In addition, p53 upregulates the proapoptotic gene Bax but downregulates the anti-apoptotic gene bcl-2, 37 thus contributing to mitochondria-dependent apoptosis. p53 also results in apoptosis through transcriptional induction of oxidation-related genes, such as p53-induced genes (PIG3, PIG6, PIG7), and the generation of reactive oxygen species. 38 Interestingly, HO-1-mediated neuroprotection in a mouse model of ischemic stroke is correlated with altered p53 protein expression and upregulated bcl-2 levels. 18 In our study, the protein level of p53 was increased in the retina after intense light exposure, and HO-1 gene transfer delayed the increase in protein level of p53 (Fig. 6) . This result along, with the findings of significant reductions in light-induced photoreceptor apoptosis and the activation of bcl-2 achieved by HO-1 gene delivery, suggested that prevention of the activation of p53 protein in retina in response to light damage might play an important role in the rescue of photoreceptor apoptosis by HO-1. 
The mechanism of HO-1-mediated p53 protein suppression is unclear. p53 expression requires the activation of c-fos, 39 and our study demonstrated that activation of c-fos was suppressed in the HO-1-expressing eye (Fig. 5) . Therefore, it is possible that HO-1 inhibited p53 expression through the suppression of c-fos. However, the mechanisms involved in preventing the activation of p53 may be more complex. For instance, overexpression of HO-1 can protect against DNA damage 40 and, hence, prevent the activation of the DNA damage response. In addition, HO-1-derived CO inhibits smooth muscle cell apoptosis by blocking p53 activation. 41 These other mechanisms involved in the suppression of p53 expression by HO-1 await further investigation. Furthermore, in addition to the suppression of p53 we observed, other mechanisms of HO-1 may arrest apoptosis. For instance, p21, which can be induced by HO-1, confers resistance to apoptosis in renal tubular epithelial cell line and in human gastric cancer cells. 42 43 In addition, p21 overexpression protects human melanoma cells against apoptosis. 44 The involvement of p21 and other HO-1-inducible genes in mediating the photoreceptor protection offered by HO-1 remains to be determined. 
The autoproteolytic generation of caspase-8 from procaspase-8 is the initial event that induces downstream caspase directly or that leads to the activation of caspase-9 through the mitochondrial pathway to result in apoptosis. 45 c-FLIP, with its long (c-FLIPL) and short (c-FLIPS) variants, competitively inhibits the autoproteolytic activation of procaspase-8 within death-inducing signal complexes by its death effector domains, which display homology to the N-terminal region of procaspase-8 but lack catalytic activity. 46 47 The caspase-8 homologue c-FLIP is considered a key regulator for survival. 48 In addition, the expression of c-FLIP protects neural stem cells from lipopolysaccharide-induced apoptosis 49 and prevents TNF-α-mediated death in cultured retinal ganglion cells. 50 In our study, c-FLIPL was upregulated in control retina 6 hours after intense light damage, but its level was nearly undetectable after 12 hours. Similarly, the amount of c-FLIPS initially present decreased gradually in control retina after intense light damage (Fig. 8) . It is possible that the presence of c-FLIP might be part of a cellular defense mechanism against this form of oxidative stress, but this effect could not be maintained in control retina. In contrast, c-FLIPL and c-FLIPS expression in rAAV-HO-1-treated retina persisted (Fig. 8) . This indicated that HO-1 overexpression rescued photoreceptors from light-induced apoptosis by preserving the presence of c-FLIPL and c-FLIPS.  
In summary, the present study demonstrated that rAAV-mediated HO-1 gene delivery resulted in transgene expression in photoreceptors and consequently attenuated light-induced apoptosis in photoreceptors and preserved the retina structure, as evidenced by the partial preservation of thickness of ONL, RIS, and ROS. HO-1 may protect photoreceptors through regulating the expression of c-fos, p53, and bcl-2, the activation of p38, and the preservation of c-FLIPL and c-FLIPS
 
Figure 1.
 
(A) Western blot analysis for HO-1 transgene expression. Three weeks after subretinal injection of either rAAV-HO-1 or rAAV-LacZ, retina tissues were harvested and subjected to Western blot analysis to detect HO-1 transgene expression. Retina tissues with no treatment were also analyzed by Western immunoblotting. The results are presented as expression fold of HO-1 activation, normalized against values of actin controls to adjust for protein loading. Groups marked with an asterisk were significantly different from rAAV-HO-1-treated retinas (Mann-Whitney U test, P < 0.05, N = 5). (B) Western blot analysis for HO-1 expression after light exposure. Animals with transduction by rAAV-HO-1 in the right eye for 3 weeks and rAAV-LacZ in the fellow eye were subjected to 2-hour intense light exposure. At 0 hour, 6, 12, and 24 hours, and 1 week after 2-hour intense light exposure, retina tissues were harvested and homogenized ultrasonically in RIPA buffer and then analyzed by immunoblotting to detect expression of endogenous and exogenous HO-1. Data are expressed as mean ± SD of independent experiments (n = 8 experiments) and are presented as expression fold of HO-1 activation normalized against values of actin controls to adjust for protein loading. (C) RT-PCR analysis of HO-1 mRNA at 0 hour, 6, 12, and 24 hours, and 1 week after light exposure. Data are expressed as mean ± SD (n = 5 experiments). Glyceraldehyde-3-phosphate dehydrogenase (G3PDH) expression was examined for normalization purposes.
Figure 1.
 
(A) Western blot analysis for HO-1 transgene expression. Three weeks after subretinal injection of either rAAV-HO-1 or rAAV-LacZ, retina tissues were harvested and subjected to Western blot analysis to detect HO-1 transgene expression. Retina tissues with no treatment were also analyzed by Western immunoblotting. The results are presented as expression fold of HO-1 activation, normalized against values of actin controls to adjust for protein loading. Groups marked with an asterisk were significantly different from rAAV-HO-1-treated retinas (Mann-Whitney U test, P < 0.05, N = 5). (B) Western blot analysis for HO-1 expression after light exposure. Animals with transduction by rAAV-HO-1 in the right eye for 3 weeks and rAAV-LacZ in the fellow eye were subjected to 2-hour intense light exposure. At 0 hour, 6, 12, and 24 hours, and 1 week after 2-hour intense light exposure, retina tissues were harvested and homogenized ultrasonically in RIPA buffer and then analyzed by immunoblotting to detect expression of endogenous and exogenous HO-1. Data are expressed as mean ± SD of independent experiments (n = 8 experiments) and are presented as expression fold of HO-1 activation normalized against values of actin controls to adjust for protein loading. (C) RT-PCR analysis of HO-1 mRNA at 0 hour, 6, 12, and 24 hours, and 1 week after light exposure. Data are expressed as mean ± SD (n = 5 experiments). Glyceraldehyde-3-phosphate dehydrogenase (G3PDH) expression was examined for normalization purposes.
Figure 2.
 
Immunohistochemical localization of HO-1 protein before and after intense light exposure. Expression of HO-1 in retina was identified by rabbit polyclonal anti-rat HO-1 antibody. HO-1 immunoactivity (red) was detected by Cy3-labeled secondary antibody. Cell nuclei (blue) were counterstained with DAPI. (A) Untreated retina without light exposure. (B) rAAV-HO-1-treated retina without light exposure. Exogenous HO-1 immunoactivity was detected in photoreceptors. (C) Untreated retina 24 hours after light exposure. Endogenous HO-1 immunoactivity was mainly confined to ILM (arrowheads), basal Müller cell processes (short arrows), and ELM (long arrows). (D) rAAV-HO-1-treated retina 24 hours after light exposure. Scale bar, 40 μm. INL, inner nuclear layer.
Figure 2.
 
Immunohistochemical localization of HO-1 protein before and after intense light exposure. Expression of HO-1 in retina was identified by rabbit polyclonal anti-rat HO-1 antibody. HO-1 immunoactivity (red) was detected by Cy3-labeled secondary antibody. Cell nuclei (blue) were counterstained with DAPI. (A) Untreated retina without light exposure. (B) rAAV-HO-1-treated retina without light exposure. Exogenous HO-1 immunoactivity was detected in photoreceptors. (C) Untreated retina 24 hours after light exposure. Endogenous HO-1 immunoactivity was mainly confined to ILM (arrowheads), basal Müller cell processes (short arrows), and ELM (long arrows). (D) rAAV-HO-1-treated retina 24 hours after light exposure. Scale bar, 40 μm. INL, inner nuclear layer.
Figure 3.
 
Anti-apoptotic effect of HO-1. (A) Apoptosis in photoreceptor cells in each group was detected by TUNEL analysis 24 hours after intense light exposure. Original magnification, ×400. (B) Quantitative analysis for number of TUNEL-positive cells. Data were expressed as mean ± SD. Groups significantly different from the rAAV-HO-1-treated group by *Mann-Whitney U test (P < 0.05) and by **Wilcoxon signed-rank test (P < 0.05); n = 6 in each group.
Figure 3.
 
Anti-apoptotic effect of HO-1. (A) Apoptosis in photoreceptor cells in each group was detected by TUNEL analysis 24 hours after intense light exposure. Original magnification, ×400. (B) Quantitative analysis for number of TUNEL-positive cells. Data were expressed as mean ± SD. Groups significantly different from the rAAV-HO-1-treated group by *Mann-Whitney U test (P < 0.05) and by **Wilcoxon signed-rank test (P < 0.05); n = 6 in each group.
Figure 4.
 
Effect of HO-1 on preservation of retinal structure 7 days after intense light exposure. Upper row: toluidine blue staining. Lower row: ultrastructural examination of the same retinas as upper row. (A) Normal eye. (B) rAAV-HO-1-treated retina. (C) rAAV-LacZ-treated retina. (D) Untreated retina. White arrows: densified photoreceptors with condensed chromatins. Short black arrows: apoptotic bodies of photoreceptors. Long black arrows: external limiting membrane. Transmission electron microscopy; original magnification, ×2500. INL, inner nuclear layer. (E, F) Quantitative analysis of HO-1 on preservation of thickness of ONL, ROS, and RIS 7 days after intense light damage. The superior hemisphere retina from optic nerve head to peripheral area was divided equally into six segments (S1–S6). Thickness of each divided segment was counted and compared among each group. Data were expressed as mean ± SD. Groups significantly different from the rAAV-HO-1-treated group by *Mann-Whitney U test (P < 0.05) and by **Wilcoxon signed-rank test (P < 0.05); n = 7 in each group.
Figure 4.
 
Effect of HO-1 on preservation of retinal structure 7 days after intense light exposure. Upper row: toluidine blue staining. Lower row: ultrastructural examination of the same retinas as upper row. (A) Normal eye. (B) rAAV-HO-1-treated retina. (C) rAAV-LacZ-treated retina. (D) Untreated retina. White arrows: densified photoreceptors with condensed chromatins. Short black arrows: apoptotic bodies of photoreceptors. Long black arrows: external limiting membrane. Transmission electron microscopy; original magnification, ×2500. INL, inner nuclear layer. (E, F) Quantitative analysis of HO-1 on preservation of thickness of ONL, ROS, and RIS 7 days after intense light damage. The superior hemisphere retina from optic nerve head to peripheral area was divided equally into six segments (S1–S6). Thickness of each divided segment was counted and compared among each group. Data were expressed as mean ± SD. Groups significantly different from the rAAV-HO-1-treated group by *Mann-Whitney U test (P < 0.05) and by **Wilcoxon signed-rank test (P < 0.05); n = 7 in each group.
Figure 5.
 
Immunohistochemical analysis for expression of c-fos protein 6 hours after intense light damage. Animals with transduction by rAAV-HO-1 in right eye for 3 weeks and rAAV-LacZ in fellow eye were subjected to 2-hour intense light exposure. Six hours after intense light exposure, expression of c-fos protein in retina tissue was detected by anti-c-fos primary antibody. Immunoactivity of c-fos (green) was identified by fluorescein isothiocyanate (FITC)-labeled secondary antibody. Cell nuclei (blue) were counterstained with DAPI. (AC) rAAV-HO-1-treated retina. (DF) rAAV-LacZ-treated retina. (GI) rAAV-LacZ-treated retina without the anti-c-fos antibody (negative control). The background level in specimens without the anti-c-fos primary antibody displayed barely detectable signal. (C, F, I) Merged images.
Figure 5.
 
Immunohistochemical analysis for expression of c-fos protein 6 hours after intense light damage. Animals with transduction by rAAV-HO-1 in right eye for 3 weeks and rAAV-LacZ in fellow eye were subjected to 2-hour intense light exposure. Six hours after intense light exposure, expression of c-fos protein in retina tissue was detected by anti-c-fos primary antibody. Immunoactivity of c-fos (green) was identified by fluorescein isothiocyanate (FITC)-labeled secondary antibody. Cell nuclei (blue) were counterstained with DAPI. (AC) rAAV-HO-1-treated retina. (DF) rAAV-LacZ-treated retina. (GI) rAAV-LacZ-treated retina without the anti-c-fos antibody (negative control). The background level in specimens without the anti-c-fos primary antibody displayed barely detectable signal. (C, F, I) Merged images.
Figure 6.
 
Western blot analysis for expression of p53 and p38 protein after intense light damage. Animals with transduction by rAAV-HO-1 in right eye for 3 weeks and rAAV-LacZ in fellow eye were subjected to 2-hour intense light exposure. At 0 hour and 6, 12, and 24 hours after 2-hour intense light exposure, retina tissues were analyzed by Western immunoblotting to detect p53 and p38 protein. Results are presented as expression fold of p53 and p38 activation, normalized against values of actin controls to adjust for protein loading. Group significantly different from the rAAV-LacZ-treated group by *Wilcoxon signed-rank test (P < 0.05); n = 5.
Figure 6.
 
Western blot analysis for expression of p53 and p38 protein after intense light damage. Animals with transduction by rAAV-HO-1 in right eye for 3 weeks and rAAV-LacZ in fellow eye were subjected to 2-hour intense light exposure. At 0 hour and 6, 12, and 24 hours after 2-hour intense light exposure, retina tissues were analyzed by Western immunoblotting to detect p53 and p38 protein. Results are presented as expression fold of p53 and p38 activation, normalized against values of actin controls to adjust for protein loading. Group significantly different from the rAAV-LacZ-treated group by *Wilcoxon signed-rank test (P < 0.05); n = 5.
Figure 7.
 
Immunohistochemical analysis for expression of bcl-2 protein 24 hours after intense light damage. Animals with transduction by rAAV-HO-1 in right eye for 3 weeks and rAAV-LacZ in fellow eye were subjected to 2-hour intense light exposure. At 24 hours after intense light exposure, expression of bcl-2 protein in retina tissues was detected by anti-bcl-2 primary antibody. Immunoactivity of bcl-2 (green) was identified by FITC-labeled secondary antibody. Cell nuclei (blue) were counterstained with DAPI. (AC) rAAV-HO-1-treated retina. (DF) rAAV-LacZ-treated retina. (GI) rAAV-HO-1-treated retina without the anti-bcl-2 antibody (negative control). The background level in specimens without the anti-bcl-2 primary antibody displayed barely detectable signal. (C, F, I) Higher magnification.
Figure 7.
 
Immunohistochemical analysis for expression of bcl-2 protein 24 hours after intense light damage. Animals with transduction by rAAV-HO-1 in right eye for 3 weeks and rAAV-LacZ in fellow eye were subjected to 2-hour intense light exposure. At 24 hours after intense light exposure, expression of bcl-2 protein in retina tissues was detected by anti-bcl-2 primary antibody. Immunoactivity of bcl-2 (green) was identified by FITC-labeled secondary antibody. Cell nuclei (blue) were counterstained with DAPI. (AC) rAAV-HO-1-treated retina. (DF) rAAV-LacZ-treated retina. (GI) rAAV-HO-1-treated retina without the anti-bcl-2 antibody (negative control). The background level in specimens without the anti-bcl-2 primary antibody displayed barely detectable signal. (C, F, I) Higher magnification.
Figure 8.
 
Western blot analysis for expression of c-FLIP after intense light damage. Animals with transduction by rAAV-HO-1 in right eye for 3 weeks and rAAV-LacZ in fellow eye were subjected to 2-hour intense light exposure. At 0 hour and 6, 12, and 24 hours after 2-hour intense light exposure, retina tissues were analyzed by Western immunoblotting to detect long and short variants of cellular FLICE inhibitory protein (c-FLIPL and c-FLIPS). Results are presented as expression fold of c-FLIPL and c-FLIPS activation, normalized against values of actin controls to adjust for protein loading. Group significantly different from the rAAV-LacZ-treated group by *Wilcoxon signed-ranks test (P < 0.05); n = 5.
Figure 8.
 
Western blot analysis for expression of c-FLIP after intense light damage. Animals with transduction by rAAV-HO-1 in right eye for 3 weeks and rAAV-LacZ in fellow eye were subjected to 2-hour intense light exposure. At 0 hour and 6, 12, and 24 hours after 2-hour intense light exposure, retina tissues were analyzed by Western immunoblotting to detect long and short variants of cellular FLICE inhibitory protein (c-FLIPL and c-FLIPS). Results are presented as expression fold of c-FLIPL and c-FLIPS activation, normalized against values of actin controls to adjust for protein loading. Group significantly different from the rAAV-LacZ-treated group by *Wilcoxon signed-ranks test (P < 0.05); n = 5.
The authors thank Hong-Kong Chen, An-Ling Cheng, and I-Pin Choung for excellent technical support. 
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Figure 1.
 
(A) Western blot analysis for HO-1 transgene expression. Three weeks after subretinal injection of either rAAV-HO-1 or rAAV-LacZ, retina tissues were harvested and subjected to Western blot analysis to detect HO-1 transgene expression. Retina tissues with no treatment were also analyzed by Western immunoblotting. The results are presented as expression fold of HO-1 activation, normalized against values of actin controls to adjust for protein loading. Groups marked with an asterisk were significantly different from rAAV-HO-1-treated retinas (Mann-Whitney U test, P < 0.05, N = 5). (B) Western blot analysis for HO-1 expression after light exposure. Animals with transduction by rAAV-HO-1 in the right eye for 3 weeks and rAAV-LacZ in the fellow eye were subjected to 2-hour intense light exposure. At 0 hour, 6, 12, and 24 hours, and 1 week after 2-hour intense light exposure, retina tissues were harvested and homogenized ultrasonically in RIPA buffer and then analyzed by immunoblotting to detect expression of endogenous and exogenous HO-1. Data are expressed as mean ± SD of independent experiments (n = 8 experiments) and are presented as expression fold of HO-1 activation normalized against values of actin controls to adjust for protein loading. (C) RT-PCR analysis of HO-1 mRNA at 0 hour, 6, 12, and 24 hours, and 1 week after light exposure. Data are expressed as mean ± SD (n = 5 experiments). Glyceraldehyde-3-phosphate dehydrogenase (G3PDH) expression was examined for normalization purposes.
Figure 1.
 
(A) Western blot analysis for HO-1 transgene expression. Three weeks after subretinal injection of either rAAV-HO-1 or rAAV-LacZ, retina tissues were harvested and subjected to Western blot analysis to detect HO-1 transgene expression. Retina tissues with no treatment were also analyzed by Western immunoblotting. The results are presented as expression fold of HO-1 activation, normalized against values of actin controls to adjust for protein loading. Groups marked with an asterisk were significantly different from rAAV-HO-1-treated retinas (Mann-Whitney U test, P < 0.05, N = 5). (B) Western blot analysis for HO-1 expression after light exposure. Animals with transduction by rAAV-HO-1 in the right eye for 3 weeks and rAAV-LacZ in the fellow eye were subjected to 2-hour intense light exposure. At 0 hour, 6, 12, and 24 hours, and 1 week after 2-hour intense light exposure, retina tissues were harvested and homogenized ultrasonically in RIPA buffer and then analyzed by immunoblotting to detect expression of endogenous and exogenous HO-1. Data are expressed as mean ± SD of independent experiments (n = 8 experiments) and are presented as expression fold of HO-1 activation normalized against values of actin controls to adjust for protein loading. (C) RT-PCR analysis of HO-1 mRNA at 0 hour, 6, 12, and 24 hours, and 1 week after light exposure. Data are expressed as mean ± SD (n = 5 experiments). Glyceraldehyde-3-phosphate dehydrogenase (G3PDH) expression was examined for normalization purposes.
Figure 2.
 
Immunohistochemical localization of HO-1 protein before and after intense light exposure. Expression of HO-1 in retina was identified by rabbit polyclonal anti-rat HO-1 antibody. HO-1 immunoactivity (red) was detected by Cy3-labeled secondary antibody. Cell nuclei (blue) were counterstained with DAPI. (A) Untreated retina without light exposure. (B) rAAV-HO-1-treated retina without light exposure. Exogenous HO-1 immunoactivity was detected in photoreceptors. (C) Untreated retina 24 hours after light exposure. Endogenous HO-1 immunoactivity was mainly confined to ILM (arrowheads), basal Müller cell processes (short arrows), and ELM (long arrows). (D) rAAV-HO-1-treated retina 24 hours after light exposure. Scale bar, 40 μm. INL, inner nuclear layer.
Figure 2.
 
Immunohistochemical localization of HO-1 protein before and after intense light exposure. Expression of HO-1 in retina was identified by rabbit polyclonal anti-rat HO-1 antibody. HO-1 immunoactivity (red) was detected by Cy3-labeled secondary antibody. Cell nuclei (blue) were counterstained with DAPI. (A) Untreated retina without light exposure. (B) rAAV-HO-1-treated retina without light exposure. Exogenous HO-1 immunoactivity was detected in photoreceptors. (C) Untreated retina 24 hours after light exposure. Endogenous HO-1 immunoactivity was mainly confined to ILM (arrowheads), basal Müller cell processes (short arrows), and ELM (long arrows). (D) rAAV-HO-1-treated retina 24 hours after light exposure. Scale bar, 40 μm. INL, inner nuclear layer.
Figure 3.
 
Anti-apoptotic effect of HO-1. (A) Apoptosis in photoreceptor cells in each group was detected by TUNEL analysis 24 hours after intense light exposure. Original magnification, ×400. (B) Quantitative analysis for number of TUNEL-positive cells. Data were expressed as mean ± SD. Groups significantly different from the rAAV-HO-1-treated group by *Mann-Whitney U test (P < 0.05) and by **Wilcoxon signed-rank test (P < 0.05); n = 6 in each group.
Figure 3.
 
Anti-apoptotic effect of HO-1. (A) Apoptosis in photoreceptor cells in each group was detected by TUNEL analysis 24 hours after intense light exposure. Original magnification, ×400. (B) Quantitative analysis for number of TUNEL-positive cells. Data were expressed as mean ± SD. Groups significantly different from the rAAV-HO-1-treated group by *Mann-Whitney U test (P < 0.05) and by **Wilcoxon signed-rank test (P < 0.05); n = 6 in each group.
Figure 4.
 
Effect of HO-1 on preservation of retinal structure 7 days after intense light exposure. Upper row: toluidine blue staining. Lower row: ultrastructural examination of the same retinas as upper row. (A) Normal eye. (B) rAAV-HO-1-treated retina. (C) rAAV-LacZ-treated retina. (D) Untreated retina. White arrows: densified photoreceptors with condensed chromatins. Short black arrows: apoptotic bodies of photoreceptors. Long black arrows: external limiting membrane. Transmission electron microscopy; original magnification, ×2500. INL, inner nuclear layer. (E, F) Quantitative analysis of HO-1 on preservation of thickness of ONL, ROS, and RIS 7 days after intense light damage. The superior hemisphere retina from optic nerve head to peripheral area was divided equally into six segments (S1–S6). Thickness of each divided segment was counted and compared among each group. Data were expressed as mean ± SD. Groups significantly different from the rAAV-HO-1-treated group by *Mann-Whitney U test (P < 0.05) and by **Wilcoxon signed-rank test (P < 0.05); n = 7 in each group.
Figure 4.
 
Effect of HO-1 on preservation of retinal structure 7 days after intense light exposure. Upper row: toluidine blue staining. Lower row: ultrastructural examination of the same retinas as upper row. (A) Normal eye. (B) rAAV-HO-1-treated retina. (C) rAAV-LacZ-treated retina. (D) Untreated retina. White arrows: densified photoreceptors with condensed chromatins. Short black arrows: apoptotic bodies of photoreceptors. Long black arrows: external limiting membrane. Transmission electron microscopy; original magnification, ×2500. INL, inner nuclear layer. (E, F) Quantitative analysis of HO-1 on preservation of thickness of ONL, ROS, and RIS 7 days after intense light damage. The superior hemisphere retina from optic nerve head to peripheral area was divided equally into six segments (S1–S6). Thickness of each divided segment was counted and compared among each group. Data were expressed as mean ± SD. Groups significantly different from the rAAV-HO-1-treated group by *Mann-Whitney U test (P < 0.05) and by **Wilcoxon signed-rank test (P < 0.05); n = 7 in each group.
Figure 5.
 
Immunohistochemical analysis for expression of c-fos protein 6 hours after intense light damage. Animals with transduction by rAAV-HO-1 in right eye for 3 weeks and rAAV-LacZ in fellow eye were subjected to 2-hour intense light exposure. Six hours after intense light exposure, expression of c-fos protein in retina tissue was detected by anti-c-fos primary antibody. Immunoactivity of c-fos (green) was identified by fluorescein isothiocyanate (FITC)-labeled secondary antibody. Cell nuclei (blue) were counterstained with DAPI. (AC) rAAV-HO-1-treated retina. (DF) rAAV-LacZ-treated retina. (GI) rAAV-LacZ-treated retina without the anti-c-fos antibody (negative control). The background level in specimens without the anti-c-fos primary antibody displayed barely detectable signal. (C, F, I) Merged images.
Figure 5.
 
Immunohistochemical analysis for expression of c-fos protein 6 hours after intense light damage. Animals with transduction by rAAV-HO-1 in right eye for 3 weeks and rAAV-LacZ in fellow eye were subjected to 2-hour intense light exposure. Six hours after intense light exposure, expression of c-fos protein in retina tissue was detected by anti-c-fos primary antibody. Immunoactivity of c-fos (green) was identified by fluorescein isothiocyanate (FITC)-labeled secondary antibody. Cell nuclei (blue) were counterstained with DAPI. (AC) rAAV-HO-1-treated retina. (DF) rAAV-LacZ-treated retina. (GI) rAAV-LacZ-treated retina without the anti-c-fos antibody (negative control). The background level in specimens without the anti-c-fos primary antibody displayed barely detectable signal. (C, F, I) Merged images.
Figure 6.
 
Western blot analysis for expression of p53 and p38 protein after intense light damage. Animals with transduction by rAAV-HO-1 in right eye for 3 weeks and rAAV-LacZ in fellow eye were subjected to 2-hour intense light exposure. At 0 hour and 6, 12, and 24 hours after 2-hour intense light exposure, retina tissues were analyzed by Western immunoblotting to detect p53 and p38 protein. Results are presented as expression fold of p53 and p38 activation, normalized against values of actin controls to adjust for protein loading. Group significantly different from the rAAV-LacZ-treated group by *Wilcoxon signed-rank test (P < 0.05); n = 5.
Figure 6.
 
Western blot analysis for expression of p53 and p38 protein after intense light damage. Animals with transduction by rAAV-HO-1 in right eye for 3 weeks and rAAV-LacZ in fellow eye were subjected to 2-hour intense light exposure. At 0 hour and 6, 12, and 24 hours after 2-hour intense light exposure, retina tissues were analyzed by Western immunoblotting to detect p53 and p38 protein. Results are presented as expression fold of p53 and p38 activation, normalized against values of actin controls to adjust for protein loading. Group significantly different from the rAAV-LacZ-treated group by *Wilcoxon signed-rank test (P < 0.05); n = 5.
Figure 7.
 
Immunohistochemical analysis for expression of bcl-2 protein 24 hours after intense light damage. Animals with transduction by rAAV-HO-1 in right eye for 3 weeks and rAAV-LacZ in fellow eye were subjected to 2-hour intense light exposure. At 24 hours after intense light exposure, expression of bcl-2 protein in retina tissues was detected by anti-bcl-2 primary antibody. Immunoactivity of bcl-2 (green) was identified by FITC-labeled secondary antibody. Cell nuclei (blue) were counterstained with DAPI. (AC) rAAV-HO-1-treated retina. (DF) rAAV-LacZ-treated retina. (GI) rAAV-HO-1-treated retina without the anti-bcl-2 antibody (negative control). The background level in specimens without the anti-bcl-2 primary antibody displayed barely detectable signal. (C, F, I) Higher magnification.
Figure 7.
 
Immunohistochemical analysis for expression of bcl-2 protein 24 hours after intense light damage. Animals with transduction by rAAV-HO-1 in right eye for 3 weeks and rAAV-LacZ in fellow eye were subjected to 2-hour intense light exposure. At 24 hours after intense light exposure, expression of bcl-2 protein in retina tissues was detected by anti-bcl-2 primary antibody. Immunoactivity of bcl-2 (green) was identified by FITC-labeled secondary antibody. Cell nuclei (blue) were counterstained with DAPI. (AC) rAAV-HO-1-treated retina. (DF) rAAV-LacZ-treated retina. (GI) rAAV-HO-1-treated retina without the anti-bcl-2 antibody (negative control). The background level in specimens without the anti-bcl-2 primary antibody displayed barely detectable signal. (C, F, I) Higher magnification.
Figure 8.
 
Western blot analysis for expression of c-FLIP after intense light damage. Animals with transduction by rAAV-HO-1 in right eye for 3 weeks and rAAV-LacZ in fellow eye were subjected to 2-hour intense light exposure. At 0 hour and 6, 12, and 24 hours after 2-hour intense light exposure, retina tissues were analyzed by Western immunoblotting to detect long and short variants of cellular FLICE inhibitory protein (c-FLIPL and c-FLIPS). Results are presented as expression fold of c-FLIPL and c-FLIPS activation, normalized against values of actin controls to adjust for protein loading. Group significantly different from the rAAV-LacZ-treated group by *Wilcoxon signed-ranks test (P < 0.05); n = 5.
Figure 8.
 
Western blot analysis for expression of c-FLIP after intense light damage. Animals with transduction by rAAV-HO-1 in right eye for 3 weeks and rAAV-LacZ in fellow eye were subjected to 2-hour intense light exposure. At 0 hour and 6, 12, and 24 hours after 2-hour intense light exposure, retina tissues were analyzed by Western immunoblotting to detect long and short variants of cellular FLICE inhibitory protein (c-FLIPL and c-FLIPS). Results are presented as expression fold of c-FLIPL and c-FLIPS activation, normalized against values of actin controls to adjust for protein loading. Group significantly different from the rAAV-LacZ-treated group by *Wilcoxon signed-ranks test (P < 0.05); n = 5.
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