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Retinal Cell Biology  |   September 2014
NF-κB–Mediated Nitric Oxide Production and Activation of Caspase-3 Cause Retinal Ganglion Cell Death in the Hypoxic Neonatal Retina
Author Affiliations & Notes
  • Gurugirijha Rathnasamy
    Department of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
  • Viswanathan Sivakumar
    Department of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
  • Parakalan Rangarajan
    Department of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
  • Wallace S. Foulds
    Singapore Eye Research Institute, Singapore National Eye Centre, Singapore
    Emeritus Professor, University of Glasgow, Glasgow, Scotland
  • Eng Ang Ling
    Department of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
  • Charanjit Kaur
    Department of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
    Singapore Eye Research Institute, Singapore National Eye Centre, Singapore
  • Correspondence: Charanjit Kaur, Department of Anatomy, MD10, 4 Medical Drive, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117594; antkaurc@nus.edu.sg
Investigative Ophthalmology & Visual Science September 2014, Vol.55, 5878-5889. doi:10.1167/iovs.13-13718
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      Gurugirijha Rathnasamy, Viswanathan Sivakumar, Parakalan Rangarajan, Wallace S. Foulds, Eng Ang Ling, Charanjit Kaur; NF-κB–Mediated Nitric Oxide Production and Activation of Caspase-3 Cause Retinal Ganglion Cell Death in the Hypoxic Neonatal Retina. Invest. Ophthalmol. Vis. Sci. 2014;55(9):5878-5889. doi: 10.1167/iovs.13-13718.

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

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Abstract

Purpose.: Hypoxic insult to the developing retina results in apoptosis of retinal ganglion cells (RGCs) through production of inflammatory mediators, nitric oxide (NO), and free radicals. The present study was aimed at elucidating the pathway through which hypoxia results in overproduction of NO in the immature retina, and its role in causing apoptosis of RGCs.

Methods.: Wistar rats (1 day old) were exposed to hypoxia and their retinas were studied at 3 hours to 14 days after exposure. The protein expression of nuclear factor-κB (NF-κB) and neuronal nitric oxide synthase (nNOS) in the retina and primary cultures of RGCs was analyzed using Western blotting and double-immunofluorescence, whereas the concentration of NO was determined calorimetrically. In cultured RGCs, hypoxia-induced apoptosis was evaluated by caspase-3 immunolabeling.

Results.: Following hypoxic exposure, NF-κB–mediated expression of nNOS, which was localized to the RGCs, and subsequent NO production was significantly increased in the developing retina. In primary cultures of RGCs subjected to hypoxia, the upregulation of nNOS and NO was significantly suppressed when treated with 7-nitroindazole (7-NINA), an nNOS inhibitor or BAY, an NF-κB inhibitor. Hypoxia-induced apoptosis of RGCs, which was evident with caspase-3 labeling, also was suppressed when these cells were treated with 7-NINA or BAY.

Conclusions.: Our results suggest that in RGCs, hypoxic induction of nNOS is mediated by NF-κB and the resulting increased release of NO by RGCs causes their apoptosis through caspase-3 activation. It is speculated that targeting nNOS could be a potential neuroprotective strategy against hypoxia-induced RGCs death in the developing retina.

Introduction
Hypoxic insult to the immature retina results in death of retinal ganglion cells (RGCs) 1,2 and leads to visual impairments in the neonate. Growing evidence suggests that several factors, such as apnea, placental insufficiency, pulmonary dysfunction, respiratory distress, and cyanotic heart disease, all of which can result in hypoxia, are important etiological factors in the development of retinal damage in the immature eye. 36  
We have shown recently that hypoxic damage in the developing retina, through enhanced production of free radicals, nitric oxide (NO), and inflammatory mediators, results in the death of RGCs. 7,8 Nitric oxide, synthesized from L-arginine by nitric oxide synthase (NOS), is known to mediate a wide range of physiological processes, such as vasodilation, neurotransmission, and host cell defense. 911 In response to various stimuli, all three isoforms of NOS (endothelial NOS [eNOS], neuronal NOS [nNOS], and inducible NOS [iNOS]), have been demonstrated in the developing retina. 1,1216 Among these isoforms, nNOS has been reported to be expressed in RGCs. 13,14,17 We have reported previously an increased expression of nNOS in the RGCs of retinas of hypoxic neonatal rats 1 and it has been proposed that nNOS contributes significantly to the death of RGCs. 17  
Although NO regulates many physiological and cellular processes, a high concentration of NO has been reported potentially to be cytotoxic 1820 and has been postulated as a key factor mediating various forms of retinopathy. 21 Koeberle and Ball 22 previously demonstrated the death of RGCs in adult retina following the intraocular administration of an NO donor. Also, NO has been demonstrated to have toxic effects on RGCs in vitro under hypoxic conditions, 23 and suppression of NOS under such conditions has been reported to protect RGCs. Although a toxic role for NO has been proposed, its role in mediating apoptosis of RGCs in the hypoxic developing retina has not been elucidated to our knowledge. 
The present study was aimed at demonstrating the pathway involved in hypoxia-mediated upregulation of nNOS in RGCs and the subsequent production of NO, which may result in apoptosis of RGCs. Previous studies have suggested that nuclear factor κB (NF-κB) might be involved in the upregulation of nNOS 2426 and hypoxia has been reported to enhance the nuclear translocation of NF-κB in neural tissues. 27,28 The present study evaluated the role of NF-κB, in inducing nNOS, by treating hypoxic RGCs with the NF-κB–specific inhibitor, BAY. In addition, the nNOS-specific inhibitor, 7-nitroindazole (7-NINA), was used to evaluate the role of NO in causing the death of RGCs. 
Materials and Methods
Animals
A total of 62 Wistar rats (1 day old) was exposed to hypoxia by placing them for two hours in a multigas chamber (Model MCO 18M; Sanyo Biomedical Electrical Co., Ltd., Tokyo, Japan), filled with a gas mixture of 5% oxygen and 95% nitrogen. The rats then were allowed to recover under normoxic conditions for 3 or 24 hours, or 3, 7, or 14 days before euthanasia. Another group of 56 rats kept outside the chamber was used as age-matched controls. A total of 40 (6–8 days old) rats was used for the preparation of primary cultures of RGCs. The study was approved by the Institutional Animal Care and Use Committee of National University of Singapore and was conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
Primary Cultures of RGCs
Preparation of Retinal Suspensions.
Retinas were dissociated enzymatically to make a suspension of single cells, essentially as described previously. 29,30 Briefly, the retinas derived from 6- to 8-day-old Wistar rats were incubated at 37°C for 30 minutes in Eagle's balanced salt solution containing papain (15 U/mL), collagenase (70 U/mL), bovine serum albumin (BSA, 0.2 mg/mL; Sigma-Aldrich Corp., St. Louis, MO, USA) and DL-cysteine (0.2 mg/mL). To yield a suspension of single cells, the tissue then was triturated sequentially through a narrow-bore Pasteur pipette in a solution containing 2 ng/mL ovomucoid, 0.004% DNase I, and 1 mg/mL BSA. After centrifugation at 100g for 5 minutes, the cells were rewashed in a solution containing ovomucoid and BSA (10 mg/mL each). The cells then were resuspended in 0.1% BSA in PBS. 
Preparation of Panning Tubes.
Culture flasks (25 cm2) were incubated with OX-42 antibody (1:50; Harlan Sera-Lab, Ltd., Edinburgh, UK) diluted in 2.5 mL of PBS at 4°C overnight. Corning polypropylene tubes (50 mL) were incubated with Thy-1 antibody diluted in 3 mL of PBS (1:200; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). The flasks and tubes were washed twice with 3 mL of PBS. To prevent nonspecific binding of cells to the panning flasks and tubes, 3 to 4 mL of 0.1% BSA were placed on the coated area. 
Panning Procedure.
The retinal cell suspension was incubated in the OX-42–coated flasks at room temperature for 30 minutes. The suspension was shaken gently every 10 minutes to ensure access of all cells to the surface of the coating area. Nonadherent cells were removed and placed in the Thy-1–coated tubes. The cells were incubated for 30 minutes and the tubes then were washed gently five times with 3 mL of PBS. Finally adherent cells on Thy-1–coated tubes were washed with culture medium (described below) and after centrifugation at 100g for 5 minutes, the supernatant was discarded carefully and the cells were seeded onto 12-mm glass cover slips that had been coated with poly-L-lysine (50 μg/mL; Sigma-Aldrich Corp.). The purity of the RGCs in cultures was determined by staining with the antibody Thy-1, a specific RGC marker. 31 The percentage of RGCs in the cultures was approximately 90%. 
Culture of Purified RGCs.
Purified RGCs were plated at a low density of approximately 500 to 2000 cells/well and were cultured in 400 μL of a serum-free Neurobasal medium containing glutamine (1 mM; Sigma-Aldrich Corp.), gentamicin (10 μL/mL; Invitrogen Life Technologies, Carlsbad, CA, USA), B27 supplement (1:50; Invitrogen Life Technologies), brain-derived neurotrophic factor (50 ng/mL; Sigma-Aldrich Corp.), ciliary neurotrophic factor (50 ng/mL; Sigma-Aldrich Corp.), and forskolin (10 μM; Sigma-Aldrich Corp.). The cultures were maintained at 37°C in a humidified atmosphere containing 5% CO2 and 95% air. 
Treatment of RGCs.
To study the effects of hypoxia on the expression of nNOS and NF-κB in RGCs, the cells were exposed to hypoxia in a chamber (Model MCO 18M, Multi-gas incubator; Sanyo Company Pte., Ltd., Moriguchi, Japan) for 4 hours at 37°C in a 3% oxygen, 5% CO2, and 92% nitrogen mixture. In all the experiments, RGCs in matching controls were incubated in an incubator at 37°C with 95% air and 5% CO2. In addition, serum-free medium containing 10 μM of 7-NINA 32 (Tocrisis Bioscience, Ellisville, MO, USA) or BAY (NF-κB inhibitor 33 ; Sigma-Aldrich Corp.) was added to each well for 3 hours immediately after hypoxic exposure. In various groups, the concentration of NO was measured, and cell death was investigated by caspase-3 labeling. 
Western Blotting.
Retinas (2 retinas from each rat) were removed from rats exposed to hypoxia (n = 5 at each time point) and their corresponding controls (n = 5 at each time point). Protein was extracted from the retinas using tissue protein extraction reagent and from cultured RGCs using mammalian protein extraction reagent (Thermo Fisher Scientific, Waltham, MA, USA) containing protease inhibitors. All procedures were done at 4°C. Homogenates were centrifuged at 15,000g for 10 minutes and the supernatant was collected. Cytoplasmic and nuclear extracts of cultured RGCs were isolated using an NE-PER (Thermo Fisher Scientific) kit following the manufacturer's instructions. Protein concentrations were determined by the Bradford method 34 using BSA (Sigma-Aldrich Corp.) as a standard. Samples of supernatants containing 20 μg protein were heated to 95°C for 5 minutes and were separated by SDS-PAGE in 10% SDS gels, in a Mini-Protean 3 apparatus (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Protein bands were electroblotted onto 0.45-μm polyvinylindene difluoride membranes (Bio-Rad Laboratories, Inc.) and then blocked with 5% nonfat milk for 1 hour at room temperature. The membranes were washed and subsequently incubated with anti-nNOS (1:1000; BD Biosciences, San Jose, CA, USA) antibody diluted in blocking solution (5% nonfat milk), overnight at 4°C. The membranes then were washed and incubated with secondary antibody; anti-rabbit IgG, 1:5000 (Thermo Fisher Scientific) conjugated with horseradish peroxidase. Specific binding was revealed by an enhanced-chemiluminescence kit (Thermo Fisher Scientific) following the manufacturer's instructions. For loading control, after intensive washing, the membranes were incubated with monoclonal mouse anti-β-actin (1:5000; Sigma-Aldrich Corp.). X-ray films (Thermo Fisher Scientific) were scanned with a computer-assisted G-710 densitometer (Bio-Rad Laboratories, Inc.) to quantify band optical density using Quantity One software (Bio-Rad Laboratories, Inc.). 
NO Colorimetric Assay.
The total amount of NO in the retinal tissue supernatant from the control and hypoxic rats (n = 5 at each time point) was determined by the Griess reaction using a colorimetric assay kit (US Biological, Swampscott, MA, USA) according to the manufacturer's instructions. The optical density was measured at 520 nm with a precision microplate reader (Molecular Devices Corporation, Sunnyvale, CA, USA) that detects nitrite (NO2-), a stable reaction product of NO. 
The concentration of NO in the culture medium from control and hypoxic RGCs and those treated with 7-NINA/BAY was measured using the above colorimetric assay kit. 
NF-κB Assay.
The level of NF-κB in the cytosolic/nuclear fractions of cultured RGCs from the control, hypoxia, hypoxia + BAY, and hypoxia + 7-NINA groups was determined with NF-κB p65 (pSer536) phosphotracer ELISA kit (Abcam, Cambridge, UK) according to the manufacturer's instructions. The relative fluorescence of the samples was measured at 530 nm excitation/590 nm emission using a SpectraMaxM5 microplate reader (Molecular Devices Corporation). 
Double Immunofluorescence.
Rats at 3 days after hypoxic exposure and their corresponding controls (n = 3 in each group) were used for double immunofluorescence studies. Following deep anesthesia with 6% pentobarbital, the rats were killed by perfusion with 2% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. Frozen coronal sections of the retina with a thickness of 40 μm were cut with a cryostat (Model 3050; Leica Instruments GmbH, Nubloch, Germany) and rinsed in PBS. Endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide in methanol for 30 minutes and the sections subsequently were washed with PBS. Sections then were incubated at room temperature with a cocktail mix of two primary antibodies: nNOS (1:500; BD Biosciences)/NF-κB (1:100; Santa Cruz Biotechnology, Inc.) and NeuN (1:200; Millipore, Billerica, MA, USA) the latter being a specific marker for RGCs. 35 Subsequent antibody detection was done with a cocktail mix of two secondary antibodies: Cy3-conjugated goat anti-rabbit IgG and FITC-conjugated sheep anti-mouse IgG (1:100; Sigma-Aldrich Corp.). After three washes with PBS, the sections were mounted with a fluorescent mounting medium (DakoCytomation, Glostrup, Denmark). Colocalization of nNOS/NF-κB with NeuN was observed under a confocal microscope (Olympus, FV 1000; Olympus Optical Co., Ltd., Tokyo, Japan). The isotypic control confirmed the specificity of all primary antibodies used (data not shown). 
Purified RGCs were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4 for 20 minutes, and blocked with 3% normal goat serum and 1% BSA for 30 minutes. The cells then were incubated overnight at 4°C with a mixture of two primary antibodies against NF-κB/nNOS and Thy1.1; Thy1.1 is a specific marker for RGCs. Subsequent antibody detection was done with the mixture of secondary antibodies; Cy3-conjugated goat anti-rabbit IgG and FITC-conjugated sheep anti-mouse IgG (1:100; Sigma-Aldrich Corp.) and processed as described above. 
Caspase-3 Labeling in Retinal Flat Mounts and in Cultured RGCS.
Retinal flat mounts were prepared from retinas collected from rats at 3 days after hypoxia along with their age-matched controls and hypoxic rats treated with BAY (20 mg/kg 36 )/7-NINA (10 mg/kg, 37 n = 3 in each group), following the instruction as described previously. 38 For the detection of apoptosis, the flat mounts were washed with PBS and the endogenous peroxide activity was blocked with 0.3% hydrogen peroxide for 30 minutes. The flat mounts then were incubated overnight at room temperature with a cocktail of two primary antibodies: anti-caspase-3 (1:200; Cell Signaling Technology, Inc., Beverly, MA, USA) and anti-Thy1.1 antibodies. Subsequently the flat mounts were washed in PBS and incubated with a cocktail mix of secondary antibodies and were mounted with a fluorescent mounting medium (DakoCytomation) following the steps detailed above. For apoptosis detection in primary cultures, RGCs were incubated at 4°C overnight with a cocktail mix of anti-caspase-3 and anti-Thy1.1 antibodies, and processed as described above. The number of caspase-3–positive RGCs was obtained by counting cells in six randomly selected microscopic fields obtained from each slide at ×40 magnification. The percentage of caspase-3–positive RGCs against the total number of RGCs was calculated and averaged. 
Statistical Analysis
The data are presented as mean ± SD. A 1-way ANOVA followed by post hoc analysis using Dunnett's test (GraphPad Software, San Diego, CA, USA) was used to determine the statistical significance of differences between normal versus hypoxic and between hypoxic versus hypoxic + 7-NINA/BAY groups. A value of P < 0.05 (*) was considered statistically significant. 
Results
nNOS Protein Expression by Western Blotting
Protein expression of nNOS showed a significant difference between the control and hypoxic groups. An immunoreactive band for nNOS was detected at 155 kDa (Fig. 1A) and was increased significantly at 24 hours and 3 days after the hypoxic exposure (Fig. 1B), but decreased below control levels at 7 and 14 days. 
Figure 1
 
Western blot analysis showing the protein expression of nNOS in the retina of postnatal rats at 3 and 24 hours, and 3, 7, and 14 days after hypoxic exposure and their corresponding controls. (A) Shows the immunoreactive bands of nNOS (155 kDa) and β-actin (43 kDa). (B) Bar graph showing significant changes in the optical density following hypoxic exposure. Each bar represents the mean ± SD. The experiment was repeated five times and a representative blot is shown here. Significant differences in protein level between hypoxic and control groups are expressed as *P < 0.05. (C) Bar graph showing fold change in NO content in the retina of postnatal rats at 3 and 24 hours, and 3, 7, and 14 days after hypoxic exposure and their corresponding controls. Significant differences in NO level between hypoxic and control groups are expressed as *P < 0.05.
Figure 1
 
Western blot analysis showing the protein expression of nNOS in the retina of postnatal rats at 3 and 24 hours, and 3, 7, and 14 days after hypoxic exposure and their corresponding controls. (A) Shows the immunoreactive bands of nNOS (155 kDa) and β-actin (43 kDa). (B) Bar graph showing significant changes in the optical density following hypoxic exposure. Each bar represents the mean ± SD. The experiment was repeated five times and a representative blot is shown here. Significant differences in protein level between hypoxic and control groups are expressed as *P < 0.05. (C) Bar graph showing fold change in NO content in the retina of postnatal rats at 3 and 24 hours, and 3, 7, and 14 days after hypoxic exposure and their corresponding controls. Significant differences in NO level between hypoxic and control groups are expressed as *P < 0.05.
NO Assay
The concentration of NO in the retina was significantly increased at 24 hours and 3 days after hypoxic exposure when compared to controls (Fig. 1C). However, the changes in NO levels observed at 3 hours, 7 and 14 days were not significant. 
Cellular Localization of nNOS and NF-κB
Expression of nNOS was localized in NeuN–labeled cells in the ganglion cell layer (GCL) of the retina that were identified as RGCs. A weak immunoexpression of nNOS was observed in the GCL (Figs. 2A–C) of control rat retinas. The expression of nNOS in the GCL (Figs. 2D–F) was enhanced at 3 days following hypoxic exposure when compared to the controls. At 3 days after hypoxic exposure, parallel to nNOS the expression of NF-κB also was increased in NeuN-labeled RGCs in the hypoxic retina when compared to controls (Figs. 2G–L) and there was increased nuclear translocation of NF-κB into the nucleus of RGCs in the hypoxic retina (Figs. 2K, 2L). 
Figure 2
 
Confocal images showing the distribution of NeuN (A, D, G, J; green), nNOS (B, E; red), and NF-κB (H, K; red) in the RGCs (arrows) in the GCL in the retinas of rats at 3 days after hypoxic exposure (DF, JL) and their corresponding controls (AC, GI). Colocalized labeling of NeuN with nNOS/NF-κB in RGCs is seen in (C, F, I, L). Note the increased expression of nNOS/NF-κB in RGCs in hypoxic rats (DF, JL) when compared to control rats (AC, GI) and the increased nuclear translocation of NF-κB in the RGCs in hypoxic retina (JL). Scale bars: 20 μm.
Figure 2
 
Confocal images showing the distribution of NeuN (A, D, G, J; green), nNOS (B, E; red), and NF-κB (H, K; red) in the RGCs (arrows) in the GCL in the retinas of rats at 3 days after hypoxic exposure (DF, JL) and their corresponding controls (AC, GI). Colocalized labeling of NeuN with nNOS/NF-κB in RGCs is seen in (C, F, I, L). Note the increased expression of nNOS/NF-κB in RGCs in hypoxic rats (DF, JL) when compared to control rats (AC, GI) and the increased nuclear translocation of NF-κB in the RGCs in hypoxic retina (JL). Scale bars: 20 μm.
Hypoxia-Induced Nuclear Translocation of NF-κB
An ELISA analysis indicated a significant increase in nuclear and cytoplasmic levels of NF-κB in RGCs exposed to hypoxia when compared to controls (Fig. 3A). However, this increase was significantly suppressed when hypoxic RGCs were treated with NF-κB inhibitor BAY. In hypoxic RGCs treated with 7-NINA, there was no significant change in NF-κB levels when compared to hypoxic RGCs. 
Figure 3
 
(A) An ELISA analysis showing cytoplasmic and nuclear NF-κB levels in primary cultured retinal RGCs in control, hypoxia, hypoxia + 10 μM BAY (Hyp+BAY) and hypoxia + 10 μM 7-NINA (Hyp+7-NINA) groups. Data represent mean ± SD of the fold change in the fluorescent intensity of p-P65 subunit of NF-κB in cultured RGCs. Significant differences in NF-κB levels compared to control group is indicated by *P < 0.05, **P < 0.01, and with respect to hypoxia as #P < 0.05, ##P < 0.01. (BG) Confocal images showing the localization of NF-κB (C, F; red) in Thy1 (green)–labeled control (BD) and hypoxic (EG) cultured RGCs (arrows). Nuclear translocation of NF-κB is evident following hypoxia (F, G). Scale bars: 20 μm.
Figure 3
 
(A) An ELISA analysis showing cytoplasmic and nuclear NF-κB levels in primary cultured retinal RGCs in control, hypoxia, hypoxia + 10 μM BAY (Hyp+BAY) and hypoxia + 10 μM 7-NINA (Hyp+7-NINA) groups. Data represent mean ± SD of the fold change in the fluorescent intensity of p-P65 subunit of NF-κB in cultured RGCs. Significant differences in NF-κB levels compared to control group is indicated by *P < 0.05, **P < 0.01, and with respect to hypoxia as #P < 0.05, ##P < 0.01. (BG) Confocal images showing the localization of NF-κB (C, F; red) in Thy1 (green)–labeled control (BD) and hypoxic (EG) cultured RGCs (arrows). Nuclear translocation of NF-κB is evident following hypoxia (F, G). Scale bars: 20 μm.
Double immunofluorescence showed the cytoplasmic localization of NF-κB in the control group of cultured RGCs (Figs. 3B–D) and the nuclear translocation in cells subjected to hypoxia (Figs. 3E–G). 
NF-κB–Mediated the Expression of nNOS and NO Production in Hypoxic RGCS
Western blot analysis showed that the protein expression of nNOS was increased in primary cultures of RGCs subjected to 4 hours of hypoxia when compared to that of controls (Figs. 4A, 4B), and was suppressed by 7-NINA and BAY. The NO levels in RGCs culture media as determined by the colorimetric assay were significantly increased after 4 hours of hypoxic exposure when compared to control cell culture medium (Fig. 4C). The NO levels were reduced in the RGCs culture medium from hypoxia + 7-NINA and hypoxia + BAY groups. 
Figure 4
 
(A) Western blot analysis showing the protein expression of nNOS in RGCs of control, hypoxia, hypoxia + 10 μM 7-NINA (Hyp+7-NINA), and hypoxia + 10 μM BAY (Hyp+BAY) groups. The upper panel shows the immunoreactive band of nNOS and its corresponding β-actin band. Bar graph in (B) shows the significant differences in optical density between control and treatment groups. The bar graph in (C) shows a significant difference in the concentration of NO released into the culture medium by RGCs in various groups. Significant changes are indicated by *P < 0.05 with respect to control and #P < 0.05 with respect to hypoxia.
Figure 4
 
(A) Western blot analysis showing the protein expression of nNOS in RGCs of control, hypoxia, hypoxia + 10 μM 7-NINA (Hyp+7-NINA), and hypoxia + 10 μM BAY (Hyp+BAY) groups. The upper panel shows the immunoreactive band of nNOS and its corresponding β-actin band. Bar graph in (B) shows the significant differences in optical density between control and treatment groups. The bar graph in (C) shows a significant difference in the concentration of NO released into the culture medium by RGCs in various groups. Significant changes are indicated by *P < 0.05 with respect to control and #P < 0.05 with respect to hypoxia.
Double immunofluorescence showed that the nNOS protein expression was enhanced in RGCs subjected to 4 hours of hypoxia (Figs. 5D–F) compared to that of control cells (Figs. 5A–C). Hypoxia-induced nNOS expression was diminished by 7-NINA (Figs. 5G–I) and by BAY (Figs. 5J–L), confirming the results obtained from Western blot and colorimetric analyses. 
Figure 5
 
Confocal images showing the distribution of Thy-1 (A, D, G, J) and nNOS (B, E, H, K) in primary cultures of RGCs in control, hypoxia, hypoxia + 10 μM 7-NINA (Hyp+7-NINA), and hypoxia + 10 μM BAY (Hyp+BAY) groups. The colocalized expression of nNOS with Thy-1 immunoreactive cells (arrows) can be seen in (C, F, I, L). Following hypoxia nNOS expression is upregulated (E, F), which is prevented in hypoxia + 7-NINA (H, I) and hypoxia + BAY (K, L) groups. Scale bars: 20 μm.
Figure 5
 
Confocal images showing the distribution of Thy-1 (A, D, G, J) and nNOS (B, E, H, K) in primary cultures of RGCs in control, hypoxia, hypoxia + 10 μM 7-NINA (Hyp+7-NINA), and hypoxia + 10 μM BAY (Hyp+BAY) groups. The colocalized expression of nNOS with Thy-1 immunoreactive cells (arrows) can be seen in (C, F, I, L). Following hypoxia nNOS expression is upregulated (E, F), which is prevented in hypoxia + 7-NINA (H, I) and hypoxia + BAY (K, L) groups. Scale bars: 20 μm.
Caspase-3 Activation in Retinal Flat Mounts and in Cultured RGCS
In retinal flat mounts (Fig. 6A) obtained from control rats, only a few Thy-1 immunoreactive RGCs were positive for caspase-3 (Figs. 6B–D). There was a significant increase in caspase-3–positive RGCs in retinal flat mounts obtained from hypoxic rats (Figs. 7E–G). However, there was reduced caspase-3 labeling in retinal flat mounts from hypoxic rats treated with either BAY (Figs. 6H–J) or 7-NINA (Figs. 6K–M). The percentage of caspase-3–positive RGCs was significantly increased in the hypoxic group when compared to the control group. This was, however; significantly reversed when hypoxic RGCs were treated with 7-NINA or BAY (Fig. 6N). Similar results were obtained in RGC cultures in control, hypoxia, hypoxia + BAY, and hypoxia + 7-NINA groups (Figs. 7A–M). 
Figure 6
 
(A) The confocal image of retinal flat mount prepared from a 4-day-old control rat. (BM) Confocal images showing the apoptosis of Thy-1 (B, E, H, K)–positive RGCs (arrows), as marked by caspase-3 labeling (C, F, I, L) on retinal flat mounts prepared from control, hypoxia, hypoxia + 20 mg/kg BAY (Hyp+BAY), and hypoxia + 10 mg/kg 7-NINA (Hyp+7-NINA) groups of rats at 3 days following hypoxic exposure. The colocalized expression of caspase-3–positive cells and Thy-1 can be seen in (D, G, J, M). Bar graph in (N) represents the significant differences in the percentage of caspase-3–positive RGCs in various groups. Significant differences with respect to control are indicated by *P < 0.05, **P < 0.01, and with respect to hypoxia as #P < 0.05, ##P < 0.01. Scale bars: 500 μm (A), 50 μm (BM).
Figure 6
 
(A) The confocal image of retinal flat mount prepared from a 4-day-old control rat. (BM) Confocal images showing the apoptosis of Thy-1 (B, E, H, K)–positive RGCs (arrows), as marked by caspase-3 labeling (C, F, I, L) on retinal flat mounts prepared from control, hypoxia, hypoxia + 20 mg/kg BAY (Hyp+BAY), and hypoxia + 10 mg/kg 7-NINA (Hyp+7-NINA) groups of rats at 3 days following hypoxic exposure. The colocalized expression of caspase-3–positive cells and Thy-1 can be seen in (D, G, J, M). Bar graph in (N) represents the significant differences in the percentage of caspase-3–positive RGCs in various groups. Significant differences with respect to control are indicated by *P < 0.05, **P < 0.01, and with respect to hypoxia as #P < 0.05, ##P < 0.01. Scale bars: 500 μm (A), 50 μm (BM).
Figure 7
 
Confocal images showing apoptotic cells labeled with Thy-1 (A, D, G, J) and caspase-3 (B, E, H, K) in primary cultured RGCs in control, hypoxia, hypoxic + 10 μM 7-NINA (Hyp+7-NINA), and hypoxic + 10 μM BAY (Hyp+BAY) groups. The colocalized expression of caspase-3–positive cells and Thy-1 can be seen in (C, F, I, L). (M) Bar graph represents the significant differences in the mean percentage of caspase-3–positive RGCs. When hypoxic RGCs were treated with 7-NINA and BAY, the incidence of caspase-3–positive cells is significantly decreased as indicated by *P < 0.05 with respect to control and #P < 0.05 with respect to hypoxia. Scale bars: 20 μm.
Figure 7
 
Confocal images showing apoptotic cells labeled with Thy-1 (A, D, G, J) and caspase-3 (B, E, H, K) in primary cultured RGCs in control, hypoxia, hypoxic + 10 μM 7-NINA (Hyp+7-NINA), and hypoxic + 10 μM BAY (Hyp+BAY) groups. The colocalized expression of caspase-3–positive cells and Thy-1 can be seen in (C, F, I, L). (M) Bar graph represents the significant differences in the mean percentage of caspase-3–positive RGCs. When hypoxic RGCs were treated with 7-NINA and BAY, the incidence of caspase-3–positive cells is significantly decreased as indicated by *P < 0.05 with respect to control and #P < 0.05 with respect to hypoxia. Scale bars: 20 μm.
Discussion
In this study, we have shown that in neonatal retina, apoptosis of RGCs following a hypoxic exposure is associated with hypoxia-mediated nuclear translocation of NF-κB and increased expression of nNOS. It appears that the nuclear translocation of NF-κB had a role in the increased expression of nNOS in hypoxic RGCs. This notion lends its support from the fact that nNOS expression in cultured RGCs exposed to hypoxia was significantly reduced by a NF-κB–specific inhibitor, BAY. Our study further indicated that an increased production of NO through the enhanced nNOS expression in hypoxic RGCs causes the death of the RGCs by activated caspase-3–mediated apoptosis. 
In the developing retina, NOS and NO are required for the timely maturation of the inner plexiform layer 12 and for early retinal differentiation, 15 but an excessive induction of NOS isoforms has been implicated in damage to the retina. 17,39 In response to hypoxic insult, in the developing retina, although excessive production of inflammatory mediators 7 and destructive effects of free radicals 8 are implicated in death of RGCs, it appears that increased nNOS expression in RGCs in the hypoxic retina and the subsequent production of NO also may result in their apoptosis via caspase-3 activation. Hypoxia-mediated nNOS expression is well documented 1,39,40 and NO produced from nNOS has been demonstrated as being highly detrimental to RGCs in adult retina, 17,22 yet the mechanisms involved remain to be fully characterized. 
It has been speculated that hypoxia-mediated activation of transcription factor, NF-κB, 27,28 may have a critical role in the regulation and activation of genes involved in inflammation, oxidative stress, and apoptosis. 27,4143 Under physiological conditions, NF-κB is localized in the cytoplasm and its activation is inhibited by IκB. 44 Hypoxic exposure causes degradation of IκB and results in activation and translocation of NF-κB 28,45 to the nucleus, where it regulates the expression of target genes. In the present study, following hypoxic exposure, the expression of NF-κB was increased in the RGCs of developing retina. This was supported further by the finding from cultured RGCs, wherein the concentration of NF-κB was upregulated in the cytoplasmic and nuclear fractions of hypoxic RGCs. However, this increase was abolished when hypoxic cultured RGCs were treated with BAY. Hypoxia-induced NF-κB activation has been reported previously in human retinal progenitor cells, 46 and in RGC-5, an RGC cell line 47,48 and the activation of NF-κB in RGCs has been implicated in the apoptosis of these cells. 4850 In light of the above and from our results, it appears that the nuclear translocation of NF-κB in RGCs, in response to hypoxia, could lead to the transcription of genes that might result in the death of RGCs. 
Additional support for the role of NF-κB in the hypoxia-induced expression of nNOS comes from a previous study, which reported the presence of NF-κB binding site in the promoter of the nNOS gene. 25 The suppression of nNOS expression and NO production in hypoxic RGCs treated with BAY or 7-NINA also supports the view that hypoxia-mediated nuclear translocation of NF-κB is essential for the induction of nNOS and the subsequent production of NO in hypoxic RGCs. 
A number of reports claim that an excess production of NO through nNOS expression could mediate RGC death. These include the ability of NO to induce apoptosis in cultured retinal neurons when treated with advanced glycation end products 51 /S-nitroso-N-acetyl-penicillamine (SNAP), a NO donor, 23 and the increased survival of cultured RGCs against NO-mediated neurotoxicity, by the addition of NOS inhibitors, such as L-NAME, to the culture medium. 52 Previously, NO was shown to induce the proapoptotic cascade, in hypoxic neural tissues, by phosphorylating Bcl-2. 53 Once phosphorylated, Bcl-2 loses its antiapoptotic potential and its ability to heterodimerize with the proapoptotic protein Bax, resulting in Bax-mediated activation of caspases and initiation of apoptosis. 5456 The NO-mediated injury to the RGCs is believed to occur via a caspase-dependent pathway. The addition of caspase inhibitor, Z-VAD-FMK, to SNAP-treated hypoxic RGC-5 cells resulted in partial protection. 23 In the present study, following hypoxia, parallel to the increased NO production there was increased expression of caspase-3 in RGCs in the developing retina. Our in vitro study also depicted the same, wherein there was increased caspase-3 labeling in hypoxic cultured RGCs. This increase in caspase-3-positive RGCs, however, was reduced when treated with 7-NINA or BAY, in vivo and in vitro. The results supported the view that excess NO produced by nNOS in hypoxic RGCs leads to their apoptosis through activation of caspase cascade. 
Conclusions
Taken together, our results indicated that in hypoxic immature retina, activation of NF-κB in the RGCs results in the increased expression of nNOS, which subsequently leads to increased production of NO. This enhanced production of NO in turn causes the death of RGCs through caspase-3 activation. Inhibitors of nNOS and NF-κB, such as 7-NINA and BAY, significantly reduced hypoxia-induced nNOS expression and NO production, and decreased the death of RGCs following hypoxia, suggesting that they could be potential therapeutic agents against hypoxia-associated damage in the developing retina. 
Acknowledgments
Supported by Research Grant R-181-000-120-213 from National Medical Research Council (NMRC) of Singapore and R-181-000-148-750 from National University Health System (NUHS), Singapore. The authors alone are responsible for the content and writing of the paper. 
Disclosure: G. Rathnasamy, None; V. Sivakumar, None; P. Rangarajan, None; W.S. Foulds, None; E.A. Ling, None; C. Kaur, None 
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Figure 1
 
Western blot analysis showing the protein expression of nNOS in the retina of postnatal rats at 3 and 24 hours, and 3, 7, and 14 days after hypoxic exposure and their corresponding controls. (A) Shows the immunoreactive bands of nNOS (155 kDa) and β-actin (43 kDa). (B) Bar graph showing significant changes in the optical density following hypoxic exposure. Each bar represents the mean ± SD. The experiment was repeated five times and a representative blot is shown here. Significant differences in protein level between hypoxic and control groups are expressed as *P < 0.05. (C) Bar graph showing fold change in NO content in the retina of postnatal rats at 3 and 24 hours, and 3, 7, and 14 days after hypoxic exposure and their corresponding controls. Significant differences in NO level between hypoxic and control groups are expressed as *P < 0.05.
Figure 1
 
Western blot analysis showing the protein expression of nNOS in the retina of postnatal rats at 3 and 24 hours, and 3, 7, and 14 days after hypoxic exposure and their corresponding controls. (A) Shows the immunoreactive bands of nNOS (155 kDa) and β-actin (43 kDa). (B) Bar graph showing significant changes in the optical density following hypoxic exposure. Each bar represents the mean ± SD. The experiment was repeated five times and a representative blot is shown here. Significant differences in protein level between hypoxic and control groups are expressed as *P < 0.05. (C) Bar graph showing fold change in NO content in the retina of postnatal rats at 3 and 24 hours, and 3, 7, and 14 days after hypoxic exposure and their corresponding controls. Significant differences in NO level between hypoxic and control groups are expressed as *P < 0.05.
Figure 2
 
Confocal images showing the distribution of NeuN (A, D, G, J; green), nNOS (B, E; red), and NF-κB (H, K; red) in the RGCs (arrows) in the GCL in the retinas of rats at 3 days after hypoxic exposure (DF, JL) and their corresponding controls (AC, GI). Colocalized labeling of NeuN with nNOS/NF-κB in RGCs is seen in (C, F, I, L). Note the increased expression of nNOS/NF-κB in RGCs in hypoxic rats (DF, JL) when compared to control rats (AC, GI) and the increased nuclear translocation of NF-κB in the RGCs in hypoxic retina (JL). Scale bars: 20 μm.
Figure 2
 
Confocal images showing the distribution of NeuN (A, D, G, J; green), nNOS (B, E; red), and NF-κB (H, K; red) in the RGCs (arrows) in the GCL in the retinas of rats at 3 days after hypoxic exposure (DF, JL) and their corresponding controls (AC, GI). Colocalized labeling of NeuN with nNOS/NF-κB in RGCs is seen in (C, F, I, L). Note the increased expression of nNOS/NF-κB in RGCs in hypoxic rats (DF, JL) when compared to control rats (AC, GI) and the increased nuclear translocation of NF-κB in the RGCs in hypoxic retina (JL). Scale bars: 20 μm.
Figure 3
 
(A) An ELISA analysis showing cytoplasmic and nuclear NF-κB levels in primary cultured retinal RGCs in control, hypoxia, hypoxia + 10 μM BAY (Hyp+BAY) and hypoxia + 10 μM 7-NINA (Hyp+7-NINA) groups. Data represent mean ± SD of the fold change in the fluorescent intensity of p-P65 subunit of NF-κB in cultured RGCs. Significant differences in NF-κB levels compared to control group is indicated by *P < 0.05, **P < 0.01, and with respect to hypoxia as #P < 0.05, ##P < 0.01. (BG) Confocal images showing the localization of NF-κB (C, F; red) in Thy1 (green)–labeled control (BD) and hypoxic (EG) cultured RGCs (arrows). Nuclear translocation of NF-κB is evident following hypoxia (F, G). Scale bars: 20 μm.
Figure 3
 
(A) An ELISA analysis showing cytoplasmic and nuclear NF-κB levels in primary cultured retinal RGCs in control, hypoxia, hypoxia + 10 μM BAY (Hyp+BAY) and hypoxia + 10 μM 7-NINA (Hyp+7-NINA) groups. Data represent mean ± SD of the fold change in the fluorescent intensity of p-P65 subunit of NF-κB in cultured RGCs. Significant differences in NF-κB levels compared to control group is indicated by *P < 0.05, **P < 0.01, and with respect to hypoxia as #P < 0.05, ##P < 0.01. (BG) Confocal images showing the localization of NF-κB (C, F; red) in Thy1 (green)–labeled control (BD) and hypoxic (EG) cultured RGCs (arrows). Nuclear translocation of NF-κB is evident following hypoxia (F, G). Scale bars: 20 μm.
Figure 4
 
(A) Western blot analysis showing the protein expression of nNOS in RGCs of control, hypoxia, hypoxia + 10 μM 7-NINA (Hyp+7-NINA), and hypoxia + 10 μM BAY (Hyp+BAY) groups. The upper panel shows the immunoreactive band of nNOS and its corresponding β-actin band. Bar graph in (B) shows the significant differences in optical density between control and treatment groups. The bar graph in (C) shows a significant difference in the concentration of NO released into the culture medium by RGCs in various groups. Significant changes are indicated by *P < 0.05 with respect to control and #P < 0.05 with respect to hypoxia.
Figure 4
 
(A) Western blot analysis showing the protein expression of nNOS in RGCs of control, hypoxia, hypoxia + 10 μM 7-NINA (Hyp+7-NINA), and hypoxia + 10 μM BAY (Hyp+BAY) groups. The upper panel shows the immunoreactive band of nNOS and its corresponding β-actin band. Bar graph in (B) shows the significant differences in optical density between control and treatment groups. The bar graph in (C) shows a significant difference in the concentration of NO released into the culture medium by RGCs in various groups. Significant changes are indicated by *P < 0.05 with respect to control and #P < 0.05 with respect to hypoxia.
Figure 5
 
Confocal images showing the distribution of Thy-1 (A, D, G, J) and nNOS (B, E, H, K) in primary cultures of RGCs in control, hypoxia, hypoxia + 10 μM 7-NINA (Hyp+7-NINA), and hypoxia + 10 μM BAY (Hyp+BAY) groups. The colocalized expression of nNOS with Thy-1 immunoreactive cells (arrows) can be seen in (C, F, I, L). Following hypoxia nNOS expression is upregulated (E, F), which is prevented in hypoxia + 7-NINA (H, I) and hypoxia + BAY (K, L) groups. Scale bars: 20 μm.
Figure 5
 
Confocal images showing the distribution of Thy-1 (A, D, G, J) and nNOS (B, E, H, K) in primary cultures of RGCs in control, hypoxia, hypoxia + 10 μM 7-NINA (Hyp+7-NINA), and hypoxia + 10 μM BAY (Hyp+BAY) groups. The colocalized expression of nNOS with Thy-1 immunoreactive cells (arrows) can be seen in (C, F, I, L). Following hypoxia nNOS expression is upregulated (E, F), which is prevented in hypoxia + 7-NINA (H, I) and hypoxia + BAY (K, L) groups. Scale bars: 20 μm.
Figure 6
 
(A) The confocal image of retinal flat mount prepared from a 4-day-old control rat. (BM) Confocal images showing the apoptosis of Thy-1 (B, E, H, K)–positive RGCs (arrows), as marked by caspase-3 labeling (C, F, I, L) on retinal flat mounts prepared from control, hypoxia, hypoxia + 20 mg/kg BAY (Hyp+BAY), and hypoxia + 10 mg/kg 7-NINA (Hyp+7-NINA) groups of rats at 3 days following hypoxic exposure. The colocalized expression of caspase-3–positive cells and Thy-1 can be seen in (D, G, J, M). Bar graph in (N) represents the significant differences in the percentage of caspase-3–positive RGCs in various groups. Significant differences with respect to control are indicated by *P < 0.05, **P < 0.01, and with respect to hypoxia as #P < 0.05, ##P < 0.01. Scale bars: 500 μm (A), 50 μm (BM).
Figure 6
 
(A) The confocal image of retinal flat mount prepared from a 4-day-old control rat. (BM) Confocal images showing the apoptosis of Thy-1 (B, E, H, K)–positive RGCs (arrows), as marked by caspase-3 labeling (C, F, I, L) on retinal flat mounts prepared from control, hypoxia, hypoxia + 20 mg/kg BAY (Hyp+BAY), and hypoxia + 10 mg/kg 7-NINA (Hyp+7-NINA) groups of rats at 3 days following hypoxic exposure. The colocalized expression of caspase-3–positive cells and Thy-1 can be seen in (D, G, J, M). Bar graph in (N) represents the significant differences in the percentage of caspase-3–positive RGCs in various groups. Significant differences with respect to control are indicated by *P < 0.05, **P < 0.01, and with respect to hypoxia as #P < 0.05, ##P < 0.01. Scale bars: 500 μm (A), 50 μm (BM).
Figure 7
 
Confocal images showing apoptotic cells labeled with Thy-1 (A, D, G, J) and caspase-3 (B, E, H, K) in primary cultured RGCs in control, hypoxia, hypoxic + 10 μM 7-NINA (Hyp+7-NINA), and hypoxic + 10 μM BAY (Hyp+BAY) groups. The colocalized expression of caspase-3–positive cells and Thy-1 can be seen in (C, F, I, L). (M) Bar graph represents the significant differences in the mean percentage of caspase-3–positive RGCs. When hypoxic RGCs were treated with 7-NINA and BAY, the incidence of caspase-3–positive cells is significantly decreased as indicated by *P < 0.05 with respect to control and #P < 0.05 with respect to hypoxia. Scale bars: 20 μm.
Figure 7
 
Confocal images showing apoptotic cells labeled with Thy-1 (A, D, G, J) and caspase-3 (B, E, H, K) in primary cultured RGCs in control, hypoxia, hypoxic + 10 μM 7-NINA (Hyp+7-NINA), and hypoxic + 10 μM BAY (Hyp+BAY) groups. The colocalized expression of caspase-3–positive cells and Thy-1 can be seen in (C, F, I, L). (M) Bar graph represents the significant differences in the mean percentage of caspase-3–positive RGCs. When hypoxic RGCs were treated with 7-NINA and BAY, the incidence of caspase-3–positive cells is significantly decreased as indicated by *P < 0.05 with respect to control and #P < 0.05 with respect to hypoxia. Scale bars: 20 μm.
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