1
NF- B mediated nitric oxide production and activation of caspase-3 cause retinal ganglion cell death in the hypoxic neonatal retina
Gurugirijha Rathnasamy1, Viswanathan Sivakumar1, Parakalan Rangarajan1, Wallace
S Foulds, 2, 3 , Eng Ang Ling1, Charanjit Kaur 1,2*
1. Department of Anatomy, Yong Loo Lin School of Medicine, Blk MD10, 4 Medical
Drive, National University of Singapore, Singapore 117597
2. Singapore Eye Research Institute c/o Singapore National Eye Centre, 11 Third
Hospital Avenue, Singapore 168751
3. Emeritus Professor, University of Glasgow, Glasgow, Scotland G12 8QQ
*Corresponding author
E-mail: [email protected]
Fax: 65-67787643
Phone: 65-65163209
Running title: nNOS mediates apoptosis of RGCs in hypoxic retina
IOVS Papers in Press. Published on August 19, 2014 as Manuscript iovs.13-13718
Copyright 2014 by The Association for Research in Vision and Ophthalmology, Inc.
2
Abstract 1
Purpose: Hypoxic insult to the developing retina results in apoptosis of retinal ganglion cells 2
(RGCs) through production of inflammatory mediators, nitric oxide (NO) and free radicals. 3
The present study was aimed at elucidating the pathway through which hypoxia results in 4
overproduction of NO in the immature retina, and its role in causing apoptosis of RGCs. 5
6
Methods: One-day-old Wistar rats were exposed to hypoxia and their retinas were studied at 7
3h to 14 days after exposure. The protein expression of nuclear factor- B (NF- B) and 8
neuronal nitric oxide synthase (nNOS) in the retina and primary cultures of RGCs was 9
analysed using western blotting and double-immunofluorescence; whereas, the concentration 10
of NO was determined calorimetrically. In cultured RGCs, hypoxia-induced apoptosis was 11
evaluated by caspase-3 immunolabelling. 12
13
Results: Following hypoxic exposure, NF- B mediated expression of nNOS, which was 14
localized to the RGCs, and subsequent NO production was significantly increased in the 15
developing retina. In primary cultures of RGCs subjected to hypoxia, the up-regulation of 16
nNOS and NO was significantly suppressed when treated with 7-nitroindazole (7-NINA), a 17
nNOS inhibitor or BAY, a NF- B inhibitor. Hypoxia-induced apoptosis of RGCs, which was 18
evident with caspase-3 labelling, was also suppressed when these cells were treated with 7-19
NINA or BAY. 20
21
Conclusion: Our results suggest that in RGCs, hypoxic induction of nNOS is mediated by 22
NF- B and the resulting increased release of NO by RGCs, results in their apoptosis through 23
caspase-3 activation. It is speculated that targeting nNOS could be a potential neuroprotective 24
strategy against hypoxia-induced RGCs death in the developing retina. 25
3
26
Key words: hypoxia; nitric oxide; neuronal nitric oxide synthase; 7-nitroindazole; NF- B; 27
retinal ganglion cells; caspase-3 28
29
Introduction 30
Hypoxic insult to the immature retina results in death of retinal ganglion cells (RGCs) 1, 2 and 31
leads to visual impairments in the neonate. Growing evidence suggests that several factors 32
such as apnoea, placental insufficiency, pulmonary dysfunction and respiratory distress and 33
cyanotic heart disease, all of which can result in hypoxia, are important etiological factors in 34
the development of retinal damage in the immature eye 3-6 35
We have shown recently that hypoxic damage in the developing retina, through 36
enhanced production of free radicals, nitric oxide (NO) and inflammatory mediators results in 37
the death of RGCs 7, 8. Nitric oxide (NO), synthesized from L-arginine by nitric oxide 38
synthase (NOS), is known to mediate a wide range of physiological processes such as 39
vasodilation, neurotransmission and host cell defense 9-11. In response to various stimuli, all 40
three isoforms of NOS (endothelial NOS (eNOS), neuronal NOS (nNOS) and inducible NOS 41
(iNOS)), have been demonstrated in the developing retina 1, 12-16. Among these isoforms, 42
nNOS has been reported to be expressed in RGCs 13, 14, 17. We have earlier reported an 43
increased expression of nNOS in the RGCs of retinas of hypoxic neonatal rats 1 and it has 44
been proposed that nNOS contributes significantly to the death of RGCs 17. 45
Although NO regulates many physiological and cellular processes, a high concentration of 46
NO has been reported to be potentially cytotoxic 18-20 and has been postulated as a key factor 47
mediating various forms of retinopathy 21. Koeberle and Ball 22 have previously demonstrated 48
the death of RGCs in adult retina following the intraocular administration of an NO donor. 49
NO has been demonstrated to have toxic effects on RGCs in vitro under hypoxic conditions 50
4
23, and suppression of NOS under such conditions has been reported to protect RGCs. 51
Although a toxic role for NO has been proposed, its role in mediating apoptosis of RGCs, in 52
the hypoxic developing retina, has not been elucidated. 53
The present study was aimed at demonstrating the pathway involved in hypoxia-54
mediated up-regulation of nNOS in RGCs and the subsequent production of NO, which may 55
result in apoptosis of RGCs. Previous studies have suggested that nuclear factor kappa B 56
(NF-kB) might be involved in the up-regulation of nNOS 24-26 and hypoxia has been reported 57
to enhance the nuclear translocation of NF-kB in neural tissues 27, 28. The present study 58
evaluated the role of NF- B, in inducing nNOS, by treating hypoxic RGCs with NF- B 59
specific inhibitor, BAY. In addition, the nNOS specific inhibitor, 7-nitroindazole (7-NINA), 60
was used to evaluate the role of NO in causing the death of RGCs. 61
Materials and Methods 62
Animals 63
Forty-seven 1-day-old Wistar rats were exposed to hypoxia by placing them for two hours 64
in a multi-gas chamber (Model: MCO 18M; Sanyo Biomedical Electrical Co, Ltd, Tokyo, 65
Japan), filled with a gas mixture of 5% oxygen and 95% nitrogen. The rats were then allowed 66
to recover under normoxic conditions for 3h, 24h, 3d, 7d or 14d before sacrifice. Another 67
group of 41 rats kept outside the chamber was used as age matched controls. A total of 40 (6-68
8 days old) rats were used for the preparation of primary cultures of RGCs. The study was 69
approved by the Institutional Animal Care and Use Committee of National University of 70
Singapore and was conducted in accordance with the ARVO Statement for the Use of 71
Animals in Ophthalmic and Vision Research. 72
73
74
5
Primary cultures of retinal ganglion cells 75
Preparation of retinal suspensions 76
Retinas were dissociated enzymatically to make a suspension of single cells, essentially 77
as described previously29, 30. Briefly, the retinas derived from 6 to 8 day-old Wistar rats were 78
incubated at 37°C for 30 min in Eagle’s balanced salt solution containing papain (15U/ml), 79
collagenase (70U/ml), bovine serum albumin (BSA; 0.2mg/ml; Sigma-Aldrich, MO, USA) 80
and DL-cysteine (0.2mg/ml). To yield a suspension of single cells, the tissue was then 81
triturated sequentially through a narrow-bore Pasteur pipette in a solution containing 2ng/ml 82
ovomucoid, 0.004% DNase I, and 1mg/ml BSA. After centrifugation at 800 rpm for 5 min, 83
the cells were rewashed in a solution containing ovomucoid and BSA (10mg/ml of each). The 84
cells were then re-suspended in 0.1% BSA in phosphate buffered saline (PBS). 85
Preparation of panning tubes 86
Culture flasks (25 cm2) were incubated with OX-42 antibody (1:50; Harlan Sera-Lab Ltd, 87
Edinburgh, UK) diluted in 2.5 ml of PBS at 4°C overnight. Corning polypropylene tubes 88
(50ml) were incubated with Thy-1 antibody diluted in 3 ml of PBS (1:200; Santacruz 89
Biotechnology Inc., Santacruz, USA). The flasks and tubes were washed twice with 3 ml of 90
PBS. To prevent non-specific binding of cells to the panning flasks and tubes, 3-4 ml of 0.1% 91
BSA was placed on the coated area. 92
Panning procedure 93
The retinal cell suspension was incubated in the OX-42-coated flasks at room temperature 94
for 30 min. The suspension was gently shaken every 10 min to ensure access of all cells to 95
the surface of the coating area. Non-adherent cells were removed and placed in the Thy-1 96
coated tubes. The cells were incubated for 30 min and the tubes were then washed gently five 97
times with 3 ml of PBS. Finally adherent cells on Thy-1 coated tubes were washed with 98
culture medium (described below) and after centrifugation at 800 rpm for 5 min, the 99
6
supernatant was carefully discarded and the cells were seeded on to 12-mm glass cover slips 100
that had been coated with poly-L-lysine (50μg/ml; Sigma-Aldrich). The purity of the RGCs 101
in cultures was determined by staining with the antibody Thy-1, a specific RGC marker 31. 102
The percentage of RGCs in the cultures was about 90%. 103
Culture of purified retinal ganglion cells 104
Purified RGCs were plated at a low density of approximately 500-2000 cells/well and 105
were cultured in 400μl of a serum-free Neurobasal medium containing with glutamine (1mM; 106
Sigma-Aldrich), gentamicin (10μl/ml; Invitrogen Life Technologies, CA, USA), B27 107
supplement (1:50; Invitrogen Life Technologies), brain-derived neurotrophic factor (50ng/ml; 108
Sigma-Aldrich), ciliary neurotrophic factor (50ng/ml; Sigma-Aldrich), and forskolin (10μM; 109
Sigma-Aldrich). The cultures were maintained at 37°C in a humidified atmosphere 110
containing 5% CO2 and 95% air. 111
Treatment of retinal ganglion cells 112
To study the effects of hypoxia on the expression of nNOS and NF- B in RGCs, the cells 113
were exposed to hypoxia in a chamber (Model MCO 18M; Multi-gas incubator, Sanyo 114
Company Pte Ltd, Japan) for 4h at 37°C in a 3% oxygen, 5% CO2 and 92% nitrogen mixture. 115
In all the experiments, RGCs in matching controls were incubated in an incubator at 37°C 116
with 95% air and 5% CO2. In addition, serum-free medium containing 10μM of 7-117
nitroindazole (7-NINA; 32 Tocrisis Bioscience, MO, USA) or BAY (NF- B inhibitor; 33 118
Sigma-Aldrich, MO, USA) was added to each well for 3h immediately after hypoxic 119
exposure. In various groups, the concentration of NO was measured, and cell death was 120
investigated by caspase-3 labeling. 121
Western blotting 122
Retinas (2 retinas from each rat) were removed from rats exposed to hypoxia (n=5 at each 123
time point) and their corresponding controls (n=5 at each time point). Protein was extracted 124
7
from the retinas using tissue protein extraction reagent and from cultured RGCs using 125
mammalian protein extraction reagent (Thermo Scientific, MA, USA) containing protease 126
inhibitors. All procedures were carried out at 4°C. Homogenates were centrifuged at 15000 g 127
for 10 min and the supernatant was collected. Cytoplasmic and nuclear extracts of cultured 128
RGCs were isolated using a NE-PER© (Thermo Scientific, MA, USA) kit following the 129
manufacturer's instructions. Protein concentrations were determined by the Bradford method 130
34 using BSA (Sigma-Aldrich) as a standard. Samples of supernatants containing 20μg 131
protein were heated to 95°C for 5 min and were separated by SDS-PAGE in 10% SDS gels, 132
in a Mini-Protean 3 apparatus (Bio-Rad, CA, USA). Protein bands were electro-blotted onto 133
0.45μm polyvinylindene difluoride membranes (Bio-Rad) and then blocked with 5% non-fat 134
milk for one hour at room temperature. The membranes were washed and subsequently 135
incubated with anti-nNOS (1:1000; BD Biosciences, USA) antibodie diluted in blocking 136
solution (5% non-fat milk), overnight at 4°C. The membranes were then washed and were 137
incubated with secondary antibody; anti-rabbit IgG, 1:5000 (Thermo Scientific, MA, USA) 138
conjugated with horseradish peroxidase. Specific binding was revealed by an enhanced-139
chemiluminescence kit (Thermo Scientific, MA, USA) following the manufacturer’s 140
instructions. For loading control, after intensive washing, the membranes were incubated with 141
monoclonal mouse anti- -actin (1:5000; Sigma-Aldrich). X-ray films (Thermo Scientific, 142
MA, USA) were scanned with a computer-assisted G-710 densitometer (Bio-Rad) to quantify 143
band optical density using Quantity One software (Bio-Rad). 144
Nitric oxide colorimetric assay 145
The total amount of NO in the retinal tissue supernatant from the control and hypoxic rats 146
(n=5 at each time point) was determined by the Griess reaction using a colorimetric assay kit 147
(US Biological, Swampscott, MA, USA) according to the manufacturer’s instructions. The 148
8
optical density was measured at 520nm with a precision microplate reader (Molecular 149
Devices Corporation, CA, USA) that detects nitrite (NO2-), a stable reaction product of NO. 150
The concentration of NO in the culture medium from RGCs subjected to hypoxia and 151
those subjected to hypoxia and of 7-NINA/BAY was measured using the above colorimetric 152
assay kit. 153
NF- B assay 154
The level of NF- B in the cytosolic/nuclear fractions of cultured RGCs from the control, 155
hypoxia, hypoxia + BAY and Hypoxia + 7-NINA groups was determined with NF- B p65 156
(pSer536) phosphotracer ELISA kit (Abcam) according to the manufacturer's instructions. 157
The relative fluorescence of the samples was measured at 530nm excitation/590nm emission 158
using a SpectraMaxM5 microplate reader (Molecular Devices Corporation, CA, USA). 159
Double immunofluorescence 160
Rats at 3d after hypoxic exposure and their corresponding controls (n=3 in each group) 161
were used for double immunofluorescence studies. Following deep anesthesia with 6% 162
pentobarbital, the rats were sacrificed by perfusion with 2% paraformaldehyde in 0.1M 163
phosphate buffer, pH 7.4. Frozen coronal sections of the retina with a thickness of 40μm 164
were cut with a cryostat (Model 3050; Leica Instruments GmbH, NUBLOCH, Germany) and 165
rinsed in PBS. Endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide in 166
methanol for 30 min and the sections were subsequently washed with PBS. Sections were 167
then incubated at room temperature with a cocktail mix of two primary antibodies: nNOS 168
(1:500; BD Biosciences, USA)/NF- B (1:100; Santacruz Biotechnology Inc, USA) and NeuN 169
(1:200; Millipore, MA, USA) the latter being a specific marker for RGCs 35. Subsequent 170
antibody detection was carried out with a cocktail mix of two secondary antibodies: Cy3-171
conjugated goat anti-rabbit IgG and FITC-conjugated sheep anti-mouse IgG (1:100; Sigma-172
Aldrich). After three washes with PBS, the sections were mounted with a fluorescent 173
9
mounting medium (DakoCytomation, Glostrup, Denmark). Co-localization of nNOS/NF- B 174
with NeuN was observed under a confocal microscope (Olympus, FV 1000 Olympus Optical 175
Co. Ltd, Tokyo, Japan). The isotypic control confirmed the specificity of all primary 176
antibodies used (data not shown). 177
Purified RGCs were fixed in 4% paraformaldehyde in 0.1M phosphate buffer, pH 7.4 for 178
20 min, and blocked with 3% normal goat serum and 1% BSA for 30 min. The cells were 179
then incubated overnight at 4°C with a mixture of two primary antibodies against NF- B / 180
nNOS and Thy1.1 separately. Thy1.1 is a specific marker for RGCs. Subsequent antibody 181
detection was carried out with the mixture of secondary antibodies; Cy3-conjugated goat anti-182
rabbit IgG and FITC-conjugated sheep anti-mouse IgG (1:100; Sigma-Aldrich) and processed 183
as described above. 184
Caspase-3 labelling in Retinal Flatmounts and in cultured RGCs 185
Retinal flatmounts were prepared from retinas collected from rats at 3d after hypoxia 186
along with their age matched controls and hypoxic rats treated with BAY (20mg/kg36)/ 7-187
NINA (10mg/kg37) (n=3 in each group), following the instruction as described previously 38. 188
For the detection of apoptosis, the flatmounts were washed with PBS and the endogenous 189
peroxide activity was blocked with 0.3% hydrogen peroxide for 30 minutes. The flatmounts 190
were then incubated overnight at room temperature with a cocktail of two primary antibodies: 191
anti-caspase-3 (1:200; Cell Signaling Technology, Inc., MA, USA) and anti-Thy1.1 192
antibodies. Subsequently the flatmounts were washed in PBS and incubated with a cocktail 193
mix of secondary antibodies and were mounted with a fluorescent mounting medium (Dako 194
Cytomation) following the steps detailed above. For apoptosis detection in primary cultures, 195
RGCs were incubated at 4°C overnight with a cocktail mix of anti-caspase-3 and anti-Thy1.1 196
antibodies and processed as described above. The number of caspase-3 positive RGCs was 197
obtained by counting cells in six randomly selected microscopic fields obtained from each 198
10
slide at X40 magnification. The percentage of caspase-3 positive RGCs against the total 199
number of RGCs was calculated and averaged. 200
Statistical analysis 201
The data are presented as mean ± SD. One-way ANOVA followed by post-hoc analysis 202
using Dunnett’s test (GraphPad Software, San Diego, CA, USA) was used to determine the 203
statistical significance of differences between normal vs hypoxic and between hypoxic vs 204
hypoxic+7-NINA/BAY groups. A value of P<0.05 (*) was considered statistically 205
significant. 206
Results 207
nNOS protein expression by western blotting 208
Protein expression of nNOS showed a significant difference between the control and 209
hypoxic groups. An immunoreactive band for nNOS was detected at 155 kDa (Fig.1A) and 210
was significantly increased at 24h and 3d after the hypoxic exposure (Fig. 1B) but decreased 211
below control levels at 7 and 14d. 212
Nitric Oxide assay 213
The concentration of NO in the retina was significantly increased at 24h and 3d after hypoxic 214
exposure when compared with controls (Fig. 1C). However, the changes in NO levels 215
observed at 3h, 7d and 14d were not significant. 216
Cellular localization of nNOS and NF- B 217
Expression of nNOS was localised in NeuN labelled cells in the ganglion cell layer (GCL) 218
of the retina that were identified as RGCs. A weak immunoexpression of nNOS was observed 219
in the GCL (Fig. 2A-C) of control rat retinas. The expression of nNOS in the GCL (Fig, 2D-220
F) was enhanced at 3d following hypoxic exposure when compared to the controls. At 3d 221
after hypoxic exposure, parallel to nNOS the expression of NF- B was also increased in 222
NeuN labelled RGCs in the hypoxic retina when compared to controls (Fig 2G-L) and there 223
11
was increased nuclear translocation of NF- B into the nucleus of RGCs in the hypoxic retina 224
(Fig 2 K, L). 225
Hypoxia-induced nuclear translocation of NF- B 226
ELISA analysis indicated a significant increase in both nuclear and cytoplasmic NF- B in 227
RGCs exposed to hypoxia when compared to controls (Fig. 3A). However, this increase was 228
significantly suppressed when hypoxic RGCs were treated with NF- B inhibitor BAY. In 229
hypoxic RGCs treated with 7-NINA there was no significant change in NF- B levels when 230
compared to hypoxic RGCs. 231
Double immunofluorescence showed the cytoplasmic localization of NF- B in the control 232
group of cells (Fig. 3B-D) and the nuclear translocation in cells subjected to hypoxia (Fig. 233
3E-G). 234
NF- B mediated the expression of nNOS and NO production in hypoxic RGCs 235
Western blot analysis showed that the protein expression of nNOS was increased in 236
primary cultures of RGCs subjected to 4h of hypoxia when compared to that of controls (Fig. 237
4A, B) and was suppressed by both 7-NINA and BAY. NO levels in RGCs culture media as 238
determined by the colorimetric assay were significantly increased after 4h of hypoxic 239
exposure when compared to control cell culture medium (Fig. 4C). NO levels were reduced 240
in the RGCs culture medium from hypoxic+7-NINA and hypoxic+BAY groups. 241
Double immunofluorescence showed that the nNOS protein expression was enhanced in 242
RGCs subjected to 4h of hypoxia (Fig. 5 D-F) compared to that of control cells (Fig. 5A-C). 243
Hypoxia-induced nNOS expression was diminished by 7-NINA (Fig. 5G-I) and by BAY 244
(Fig. 5J-L), confirming the results obtained from western blot and colorimetric analyses. 245
Caspase-3 activation in retinal flatmounts and in cultured RGCs 246
In retinal flatmounts (Fig. 6A) obtained from control rats, only a few Thy-1 247
immunoreactive RGCs were positive for caspase-3 (Fig. 6B-D). There was a significant 248
12
increase in caspase-3 positive RGCs in retinal flatmounts obtained from hypoxic rats (Fig. 249
7E-G). However, there was reduced caspase-3 labelling in retinal flatmounts from hypoxic 250
rats treated with either BAY (Fig. 6H-J) or 7-NINA (Fig. 6K-M). The percentage of caspase-251
3 positive RGCs was significantly increased in hypoxic group when compared to the control 252
group. This was however; significantly reversed when hypoxic RGCs were treated with 7-253
NINA or BAY (Fig. 6N). Similar results were obtained in RGC cultures in control, hypoxia, 254
hypoxia+BAY and hypoxia+7-NINA groups (Fig. 7A-M). 255
Discussion 256
In this study, we have shown that in neonatal retina, apoptosis of RGC following a hypoxic 257
exposure is associated with hypoxia-mediated nuclear translocation of NF- B and increased 258
expression of nNOS. It appears that the nuclear translocation of NF- B played a role in the 259
increased expression of nNOS in hypoxic RGCs. This notion lends its support from the fact 260
that nNOS expression in cultured RGC exposed to hypoxia was significantly reduced by a 261
NF- B specific inhibitor, BAY. Our study further indicates that an increased production of 262
NO through the enhanced nNOS expression in hypoxic RGCs causes the death of the RGCs 263
by activated caspase-3 mediated apoptosis. 264
In the developing retina, NOS and NO are required for the timely maturation of the inner 265
plexiform layer 12 and for early retinal differentiation 15 but an excessive induction of NOS 266
isoforms has been implicated in damage to the retina 17, 39. In response to hypoxic insult, in 267
the developing retina, although excessive production of inflammatory mediators 7 and 268
destructive effects of free radicals 8 are implicated in death of RGCs, it appears that increased 269
nNOS expression in RGCs in the hypoxic retina and the subsequent production of NO may 270
also result in their apoptosis via caspase-3 activation. Hypoxia-mediated nNOS expression is 271
well documented 1, 39, 40 and NO produced from nNOS has been demonstrated as being 272
13
highly detrimental to RGCs in adult retina 17, 22, yet the mechanisms involved remain to be 273
fully characterized. 274
It has been speculated that hypoxia-mediated activation of transcription factor, NF- B 27, 28, 275
may play a critical role in the regulation and activation of genes involved in inflammation, 276
oxidative stress and apoptosis 27, 41-43. Under physiological conditions, NF-κB is localized in 277
the cytoplasm and its activation is inhibited by IκB 44. Hypoxic exposure causes degradation 278
of IκB and results in activation and translocation of NF-κB 28, 45 to the nucleus, where it 279
regulates the expression of target genes. In the present study, following hypoxic exposure, the 280
expression of NF- B was increased in the RGCs of developing retina. This was further 281
supported by the finding from cultured RGCs, wherein the concentration of NF- B was up-282
regulated in both the cytoplasmic and nuclear fractions of cultured hypoxic RGCs. However, 283
this increase was abolished when hypoxic cultured RGCs were treated with BAY. Hypoxia-284
induced NF- B activation has been reported previously in human retinal progenitor cells 46 285
and in RGC-5, a RGC cell line 47, 48 and the activation of NF- B in RGCs has been implicated 286
in the apoptosis of these cells 49 50 48. In light of the above and from our results, it appears that 287
the nuclear translocation of NF- B in RGCs, in response to hypoxia, could lead to the 288
transcription of genes that might result in the death of RGCs. 289
Additional support for the role of NF- B in the hypoxia induced expression of nNOS 290
comes from a previous study, which reported the presence of NF- B binding site in the 291
promoter of the nNOS gene 25. The suppression of nNOS expression and NO production in 292
hypoxic RGCs treated with BAY or 7-NINA also supports the view that hypoxia-mediated 293
nuclear translocation of NF- B is essential for the induction of nNOS and the subsequent 294
production of NO in hypoxic RGCs. 295
14
A number of reports claim that an excess production of NO through nNOS expression could 296
mediate RGC death. These include the ability of NO to induce apoptosis in cultured retinal 297
neurons when treated with advanced glycation end products 51/ S-nitroso-N-acetyl-298
penicillamine (SNAP), a NO donor 23 and the increased survival of cultured RGCs against 299
NO mediated neurotoxicity, by the addition of NOS inhibitors such as L-NAME to the 300
culture medium 52. Previously, NO was shown to induce the pro-apoptotic cascade, in 301
hypoxic neural tissues, by phosphorylating Bcl-2 53. Once phosphorylated, Bcl-2 loses its 302
anti-apoptotic potential and its ability to heterodimerize with the pro-apoptotic protein Bax, 303
resulting in Bax-mediated activation of caspases and initiation of apoptosis 54-56. NO 304
mediated injury to the RGCs is believed to occur via a caspase dependent pathway. The 305
addition of caspase inhibitor, Z-VAD-FMK, to SNAP treated hypoxic RGC-5 cells resulted 306
in partial protection 23. In the present study, following hypoxia, parallel to the increased NO 307
production there was increased expression of caspase-3 in RGCs in the developing retina. 308
Our in vitro study also depicted the same; wherein there was increased caspase-3 labelling in 309
hypoxic cultured RGCs. This increase in caspase-3 positive RGCs however, was reduced 310
when treated with 7-NINA or BAY, both in vivo and in vitro. The results support the view 311
that excess NO produced by nNOS in hypoxic RGCs leads to their apoptosis through 312
activation of caspase cascade. 313
Conclusion 314
Taken together, our results indicate that in hypoxic immature retina, activation of NF- B 315
in the RGCs results in the increased expression of nNOS, which subsequently leads to 316
increased production of NO. This enhanced production of NO in turn causes the death of 317
RGCs through caspase-3 activation. Inhibitors of nNOS and NF-κB, such as 7-NINA and 318
BAY significantly reduced hypoxia-induced nNOS expression and NO production and 319
15
decreased the death of RGCs following hypoxia, suggesting that they could be potential 320
therapeutic agents against hypoxia associated damage in the developing retina. 321
Acknowledgements 322
This study was supported by a research grant R-181-000-120-213 from National Medical 323
Research Council (NMRC) of Singapore and R-181-000-148-750 from National University 324
Health system (NUHS), Singapore. The authors do not have any conflict of interest. 325
326
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481
Figure Legends 482
Figure. 1 Western blot analysis showing the protein expression of neuronal nitric oxide 483
synthase (nNOS) in the retina of postnatal rats at 3, 24h, 3, 7 and 14d after hypoxic exposure 484
and their corresponding controls. The upper panel A shows the immunoreactive bands of 485
nNOS (155kDa) and -actin (43kDa). (B) Bar graph showing significant changes in the 486
optical density following hypoxic exposure. Each bar represents the mean ± SD. The 487
experiment was repeated three times and a representative blot is shown here. Significant 488
differences in protein level between hypoxic and control groups are expressed as *P<0.01. 489
(C) Nitric oxide (NO) content in the retina of postnatal rats at 3, 24h, 3, 7 and 14d after 490
hypoxic exposure and their corresponding controls. Significant differences in NO level 491
between hypoxic and control groups are expressed as *P<0.01. 492
Figure. 2 Confocal images showing the distribution of NeuN (A, D; G, J: green), neuronal 493
nitric oxide synthase (nNOS; B, E; red) and nuclear factor- B (NF- B: H, K) in the retinal 494
20
ganglion cells (RGCs; arrows) in the ganglion cell layer (GCL) in the retina at 3d after 495
hypoxic exposure. Co-localized labeling of NeuN with nNOS/NF- B in RGCs is detected in 496
C, F, I, L. Note the increased expression of nNOS/NF- B in RGCs in hypoxic rats (D-F; J-L) 497
when compared to control rats (A-C; G-I) and the increased nuclear translocation of NF- B 498
in the RGCs in hypoxic retina (J-L). Scale bar, A-F = 20 m. 499
Figure. 3 Panel A represents an Enzyme-linked immunosorbent assay analysis showing 500
cytoplasmic and nuclear NF-κB levels in primary cultured retinal ganglion cells (RGCs) in 501
control, hypoxia, hypoxia + 10 M BAY (Hyp+BAY) and hypoxia + 10 M 7-NINA (Hyp+7-502
NINA) groups. Data represents mean ± SD of the fluorescent intensity of p-P65 subunit of 503
NF-κB in cultured RGCs. Significant differences in NF-κB levels in comparison to control 504
group is indicated by *P<0.05, **P<0.01 and with respect to hypoxia as #P<0.05, ##P<0.01. 505
B-G - Confocal images showing the localisation of NF-κB (C, F; red) in Thy1 (green) 506
labelled control (B-D) and hypoxic (E-G) cultured retinal ganglion cells (arrows). Nuclear 507
translocation of NF-κB is evident following hypoxia (F, G). Scale bar, B-G = 20 m. 508
Figure. 4 A. Western blot analysis showing the protein expression of neuronal nitric oxide 509
synthase (nNOS) in retinal ganglion cells (RGCs) of control, hypoxia, hypoxia+10 M 7-510
NINA (Hyp+7-NINA) and hypoxia+10 M BAY (Hyp+BAY) groups. The upper panel shows 511
the immunoreactive bands of nNOS and its corresponding -actin band. Bar graph in panel B 512
shows the significant difference in optical density between control and treatment groups and 513
are indicated by *P<0.01 and #P<0.01. 514
Figure. 5 Confocal images showing the distribution of Thy-1 (A, D, G, J) and neuronal nitric 515
oxide synthase (nNOS; B, E, H, K) in primary cultures of retinal ganglion cells (RGCs) in 516
control, hypoxia, hypoxia +10 M 7-NINA (Hyp+7-NINA) and hypoxia + 10 M BAY 517
(Hyp+BAY) groups. The co-localized expression of nNOS with Thy-1 immunoreactive cells 518
(arrows) can be seen in C, F, I and L. Following hypoxia nNOS expression is upregulated (E, 519
21
F) which is prevented in hypoxic+7-NINA (H, I) and hypoxic+BAY (K, L) groups. Scale bar 520
(A-F): 20 m. 521
Figure. 6 Panel A shows the confocal image of retinal flatmount prepared from a 4d old 522
control rat. B-M- Confocal images showing the apoptosis of Thy-1 (B, E, H, K) positive 523
RGCs (arrows), as marked by caspase-3 labelling (C, F, I, L), on retinal flatmounts prepared 524
from control, hypoxia, hypoxia+20mg/kg BAY (Hyp+BAY) and hypoxia+10mg/kg 7-NINA 525
(Hyp+7-NINA) groups of rats at 3d following hypoxic exposure. The co-localized expression 526
of caspase-3 positive cells and Thy-1 can be seen in D, G, J, M. Bar graph in N represents the 527
significant differences in the percentage of caspase-3 positive RGCs in various groups. 528
Significant differences with respect to control is indicated by *P<0.05, **P<0.01 and with 529
respect to hypoxia as #P<0.05, ##P<0.01. Scale bar, A= 500 m; B-M = 50 m. 530
Figure. 7 Confocal images showing apoptotic cells labeled with Thy-1 (A, D, G, J) and 531
caspase-3 (B, E, H, K) in primary cultured retinal ganglion cells (RGCs) in control, hypoxia, 532
hypoxic+10 M 7-NINA (Hyp+7-NINA) and hypoxic+10 M BAY (Hyp+BAY) groups. The 533
co-localized expression of caspase-3 positive cells and Thy-1 can be seen in C, F, I and L. 534
Panel M, bar graph represents the significant differences in the mean percentage of caspase-3 535
positive RGCs. When hypoxic RGCs were treated with 7-NINA and BAY, the incidence of 536
caspase-3 positive cells is significantly decreased as indicated by *P<0.01; #P<0.01. Scale 537
bar, A-L = 20 m. 538
539
Abbreviations 540
541
BSA : bovine serum albumin 542
Caspase-3 : cas-3 543
GCL : ganglion cell layer 544
22
HIF-1 : hypoxia inducible factor-1 545
NF- B : nuclear factor-kappaB 546
NO : nitric oxide 547
nNOS : neuronal nitric oxide synthase 548
iNOS : inducible nitric oxide synthase 549
eNOS : endothelial nitric oxide synthase 550
7-NINA : 7-nitroindazole 551
PBS : phosphate buffered saline 552
RGCs : retinal ganglion cells 553
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