Increased NADPH oxidase-derived superoxide is involved in the neuronal cell death induced by hypoxia-ischemia in neonatal hippocampal slice cultures

Abstract

Neonatal brain hypoxia-ischemia (HI) results in neuronal cell death. Previous studies indicate that reactive oxygen species, such as superoxide, play a key role in this process. However, the cellular sources have not been established. In this study we examine the role of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex in neonatal HI brain injury and elucidate its mechanism of activation. Rat hippocampal slices were exposed to oxygen glucose deprivation (OGD) to mimic the conditions seen in HI. Initial studies confirmed an important role for NADPH oxidase-derived superoxide in the oxidative stress associated with OGD. Further, the OGD-mediated increase in apoptotic cell death was inhibited by the NADPH oxidase inhibitor apocynin. The activation of NADPH oxidase was found to be dependent on the p38 mitogen-activated protein kinase-mediated phosphorylation and activation of the p47(phox) subunit. Using an adeno-associated virus antisense construct to selectively decrease p47(phox) expression in neurons showed that this led to inhibition of both the increase in superoxide and the neuronal cell death associated with OGD. We also found that NADPH oxidase inhibition in a neonatal rat model of HI or scavenging hydrogen peroxide reduced brain injury. Thus, we conclude that activation of the NADPH oxidase complex contributes to the oxidative stress during HI and that therapies targeted against this complex could provide neuroprotection against the brain injury associated with neonatal HI.

Figure 1. Oxygen glucose deprivation increases p47^phox protein levels in rat hippocampal slice cultures

Rat hippocampal slice cultures were exposed to OGD in the presence or absence of the NADPH oxidase inhibitor, apocynin (0–250μM, 2h prior to OGD). Slices were harvested 8h after OGD and subjected to Western blot analysis to determine effects on total p47^phox protein levels. A representative blot is shown (A). Protein loading was normalized by reprobing with β-actin and the data plotted as the ratio of p47^phox:β-actin. In addition the affect of OGD on membrane (B) and cytosolic (C) localized p47^phox was determined. Again representative images are shown. Loading of the membrane fraction was normalized by reprobing with NaK ATPase and the data plotted as the ratio of p47^phox: NaK ATPase normalized to 1.0 for the control. Similarly, loading of the cytosolic fraction was normalized by reprobing with GAPDH and the data plotted as the ratio of p47^phox: GAPDH normalized to 1.0 for the control. Data are presented as mean ± S.E from 4 independent experiments using 24 pooled slices per experiment. * P<0.05 vs. control, † P<0.05 vs. OGD alone.

Figure 1. Oxygen glucose deprivation increases p47^phox protein levels in rat hippocampal slice cultures

Rat hippocampal slice cultures were exposed to OGD in the presence or absence of the NADPH oxidase inhibitor, apocynin (0–250μM, 2h prior to OGD). Slices were harvested 8h after OGD and subjected to Western blot analysis to determine effects on total p47^phox protein levels. A representative blot is shown (A). Protein loading was normalized by reprobing with β-actin and the data plotted as the ratio of p47^phox:β-actin. In addition the affect of OGD on membrane (B) and cytosolic (C) localized p47^phox was determined. Again representative images are shown. Loading of the membrane fraction was normalized by reprobing with NaK ATPase and the data plotted as the ratio of p47^phox: NaK ATPase normalized to 1.0 for the control. Similarly, loading of the cytosolic fraction was normalized by reprobing with GAPDH and the data plotted as the ratio of p47^phox: GAPDH normalized to 1.0 for the control. Data are presented as mean ± S.E from 4 independent experiments using 24 pooled slices per experiment. * P<0.05 vs. control, † P<0.05 vs. OGD alone.

Figure 2. NADPH oxidase inhibition attenuates superoxide generation in rat hippocampal slice cultures exposed to oxygen glucose deprivation

Rat hippocampal slice cultures were exposed to OGD in the presence or absence of the NADPH oxidase inhibitor, apocynin (100μM, 2h prior to OGD) then harvested at 8h then subjected to electron paramagnetic resonance (EPR) using the spin-trap compound 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine HCl (CMH) to determine superoxide levels. Representative EPR waveforms are shown (A). Absolute levels of superoxide generation were then determined as nmols superoxide generated/min/mg protein (B). Apocynin attenuates the OGD-mediated increase in superoxide levels. Values are presented as mean ± S.E from 4 independent experiments using 24 pooled slices per experiment. * P<0.05 vs. no OGD, † P<0.05 vs. OGD alone.

Figure. 3. Apocynin attenuates oxygen glucose deprivation mediated regional cell injury in rat hippocampal slice cultures

Rat hippocampal slice cultures were exposed to OGD in the presence or absence of the NADPH oxidase inhibitor, apocynin (100μM, 2h prior to OGD). The effect on cell injury was then quantified by measuring changes in PI uptake fluorescence either in the whole slice, CA1, CA3, or DG subregions 8h after OGD. Representative images are shown for the PI uptake in the entire slice (A). PI uptake in the whole slice exhibited a time-dependent increase and apocynin pretreatment significantly decreased PI uptake (B). Similar effects were found in the CA1 (B) and DG sub-region (B). However, in the CA3 area apocynin pretreatment did not show a significant decrease in PI uptake (B). In addition, LDH release from the slice cultures was also determined (C). The LDH absorbance at 490nm was divided by protein content. Data are presented as mean ± S.E. from 4 independent experiments. * P<0.05 vs. no OGD, † P<0.05 vs. OGD alone.

Figure 4. NADPH oxidase inhibition attenuates apoptosis in rat hippocampal slice cultures exposed to oxygen glucose deprivation

Rat hippocampal slice cultures were exposed to OGD in the presence or absence of the NADPH oxidase inhibitor, apocynin (0–100μM, 2h prior to OGD). Slices were harvested 8h after OGD and subjected to Western blot analysis to determine effects on cleaved caspase-3 (A). A representative blot is shown (A). OGD increases cleaved caspase-3 levels and this is attenuated by apocynin pretreatment at 50- and 100-μM (A). Slices were also subjected to TUNEL analysis. Representative images are shown demonstrating the TUNEL staining of apoptotic cells (green) co-localized with PI staining of all the nuclei (red) resulted in more yellow (merged) nuclei than in apocynin pretreated slices (B). The magnification used was 10×. Quantification of the percentage of apoptotic nuclei to total nuclei was also carried out indicating that apocynin decreased the level of apoptotic nuclei in response to OGD (C). Data are presented as mean ± S.E from 4 independent experiments using 24 pooled slices per experiment. * P<0.05 vs. no OGD, † P<0.05 vs.

Figure 4. NADPH oxidase inhibition attenuates apoptosis in rat hippocampal slice cultures exposed to oxygen glucose deprivation

Rat hippocampal slice cultures were exposed to OGD in the presence or absence of the NADPH oxidase inhibitor, apocynin (0–100μM, 2h prior to OGD). Slices were harvested 8h after OGD and subjected to Western blot analysis to determine effects on cleaved caspase-3 (A). A representative blot is shown (A). OGD increases cleaved caspase-3 levels and this is attenuated by apocynin pretreatment at 50- and 100-μM (A). Slices were also subjected to TUNEL analysis. Representative images are shown demonstrating the TUNEL staining of apoptotic cells (green) co-localized with PI staining of all the nuclei (red) resulted in more yellow (merged) nuclei than in apocynin pretreated slices (B). The magnification used was 10×. Quantification of the percentage of apoptotic nuclei to total nuclei was also carried out indicating that apocynin decreased the level of apoptotic nuclei in response to OGD (C). Data are presented as mean ± S.E from 4 independent experiments using 24 pooled slices per experiment. * P<0.05 vs. no OGD, † P<0.05 vs.

Figure 5. Targeted decreases in p47^phox expression in neuronal cells attenuates superoxide generation in rat hippocampal slice cultures

Rat hippocampal slice cultures were transduced with the AAV-SYN-1-p47^phoxAS, an AAV-SYN-1null construct, or were untransduced. After 7 days slices were exposed or not to OGD, harvested 8h later then subjected to Western blot analysis to determine effects on p47^phox protein levels. A representative image is shown (A). The AAV-SYN-1-p47^phoxAS construct significantly decreases p47^phox levels in control slices (B) and prevents the increase in p47^phox protein levels in response to OGD (B). Slices were also subjected to electron paramagnetic resonance (EPR) using the spin-trap compound 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine HCl (CMH) to determine superoxide levels. Representative EPR waveforms are shown (C). Absolute levels of superoxide generation were then determined as nmols superoxide generated/min/mg protein (D). Reducing p47^phox protein levels significantly reduces the OGD-mediated increase in superoxide. Values are presented as mean ± S.E from 4 independent experiments using 24 pooled slices per experiment. *p<0.05 vs. uninfected no OGD, †p<0.05 vs. OGD exposed untransduced slices.

Figure 6. Targeted decreases in p47^phox expression in neuronal cells attenuates neuronal cell death in rat hippocampal slice cultures

Rat hippocampal slice cultures were transduced with the AAV-SYN-1-p47^phoxAS, an AAV-SYN-1null construct, or were untransduced. After 7 days slices were exposed to OGD and the effect on PI uptake (A & B) and LDH release (C) was determined. Representative images are shown. Decreasing p47^phox expression reduces PI uptake in the whole slice as well as in the CA1 and DG regions but not in the CA3 (B). Similarly, Decreasing p47^phox expression reduces the OGD-mediated increases in LDH release (C). Values are presented as mean ± S.E from 4 independent experiments using 24 pooled slices per experiment. *p<0.05 vs. uninfected no OGD, †p<0.05 vs. OGD exposed untransduced slices.

Figure 7. Targeted decreases in p47^phox expression in neuronal cells attenuates apoptotic cell death in rat hippocampal slice cultures

Rat hippocampal slice cultures were transduced with the AAV-SYN-1-p47^phoxAS, an AAV-SYN-1null construct, or were untransduced. After 7 days slices were exposed to OGD and the effect on cleaved caspase-3 (A & B), and TUNEL positive nuclei (C & D) determined. Representative images are shown. Decreasing p47^phox expression reduces the OGD-mediated increases in cleaved caspase-3 (B) and the increase in TUNEL positive nuclei (D). Values are presented as mean ± S.E from 4 independent experiments using 24 pooled slices per experiment. *p<0.05 vs. uninfected no OGD, †p<0.05 vs. OGD exposed untransduced slices.

Figure 8. Oxygen glucose deprivation increases the p38MAP kinase mediated phosphorylation of p47^phox

Rat hippocampal slice cultures were exposed to OGD then harvested 4h later and subjected to immunoprecipitation/Western blotting to determine the effect on the interaction of p38MAPK with p47^phox (A & B). The effect of the p38MAPK inhibitor, SB203580 (50μM, 2h prior to OGD) on the OGD-mediated increase in plasma membrane translocation of p47^phox 4h after OGD was also determined (C & D) as well as the effect on phospho-serine p47^phox (E & F). Representative images are shown (A, C, E). OGD increases the interaction of p38MAPK with p47^phox while both the OGD-mediated increases in p47^phox membrane translocation and phospho-serine levels are attenuated by p38MAPK inhibition. Values are presented as mean ± S.E from 4 independent experiments using 24 pooled slices per experiment. *p<0.05 vs. no OGD, †p<0.05 vs. OGD alone.

Figure 8. Oxygen glucose deprivation increases the p38MAP kinase mediated phosphorylation of p47^phox

Rat hippocampal slice cultures were exposed to OGD then harvested 4h later and subjected to immunoprecipitation/Western blotting to determine the effect on the interaction of p38MAPK with p47^phox (A & B). The effect of the p38MAPK inhibitor, SB203580 (50μM, 2h prior to OGD) on the OGD-mediated increase in plasma membrane translocation of p47^phox 4h after OGD was also determined (C & D) as well as the effect on phospho-serine p47^phox (E & F). Representative images are shown (A, C, E). OGD increases the interaction of p38MAPK with p47^phox while both the OGD-mediated increases in p47^phox membrane translocation and phospho-serine levels are attenuated by p38MAPK inhibition. Values are presented as mean ± S.E from 4 independent experiments using 24 pooled slices per experiment. *p<0.05 vs. no OGD, †p<0.05 vs. OGD alone.

Figure 9. gp91ds-tat treatment attenuates NADPH oxidase activity and superoxide generation in the neonatal rat brain exposed to hypoxia-ischemia

P7 neonatal rats were pre-treated with gp91ds-tat, the scrambled control peptide, or vehicle then exposed to HI. Two hours after HI, NADPH oxidase activity (A) was determined in the left and right hemispheres of the brain. Superoxide levels were also determined using both EPR (B) and DHE oxidation (C & D). There is a significant increase in both NADPH oxidase activity and superoxide levels in the right hemisphere of the neonatal brain that is attenuated by gp91ds-tat, but not the scrambled peptide. Values are presented as mean ± S.D. from 5–6 animals per group. *p<0.05 vs. left hemisphere, †p<0.05 vs. HI + vehicle.

Figure 10. gp91ds-tat treatment attenuates neuronal injury in the neonatal rat brain exposed to hypoxia-ischemia

P7 neonatal rats were pre-treated with gp91dstat, the scrambled control peptide, or vehicle then exposed to HI. Twenty-four hours after HI the brains were removed, sectioned and subjected to TTC staining to determine the infarct volume in the right hemisphere. Representative TTC stained sections are shown (A). The HI-mediated increase in infract volume is attenuated by gp91ds-tat but not the scrambled peptide (B). TUNEL staining was also carried out 24h post-HI to determine the affect on apoptosis in the neonatal brain. Sections were counterstained with PI (red) and representative images are shown (C). Quantitation of TUNEL positive cells shows that gp91ds-tat, but not the scrambled peptide, attenuates the increase in apoptosis in the right hemisphere by HI (D). Values are presented as mean ± S.D. from 6 animals per group. *p<0.05 vs. left hemisphere, †p<0.05 vs. HI + vehicle.

Figure 10. gp91ds-tat treatment attenuates neuronal injury in the neonatal rat brain exposed to hypoxia-ischemia

P7 neonatal rats were pre-treated with gp91dstat, the scrambled control peptide, or vehicle then exposed to HI. Twenty-four hours after HI the brains were removed, sectioned and subjected to TTC staining to determine the infarct volume in the right hemisphere. Representative TTC stained sections are shown (A). The HI-mediated increase in infract volume is attenuated by gp91ds-tat but not the scrambled peptide (B). TUNEL staining was also carried out 24h post-HI to determine the affect on apoptosis in the neonatal brain. Sections were counterstained with PI (red) and representative images are shown (C). Quantitation of TUNEL positive cells shows that gp91ds-tat, but not the scrambled peptide, attenuates the increase in apoptosis in the right hemisphere by HI (D). Values are presented as mean ± S.D. from 6 animals per group. *p<0.05 vs. left hemisphere, †p<0.05 vs. HI + vehicle.

Figure 11. Scavenging H₂O₂ attenuates neuronal injury in the neonatal rat brain exposed to hypoxia-ischemia

Amplex Red assays demonstrated that H₂O₂ levels were significantly increased in hippocampal slice cultures exposed to OGD (A) and in the hippocampi of neonatal rats exposed to HI at P8 (B). The delivery of AAV GPx-1 significantly increased GPx-1 protein (C) and activity (D) in the hippocampus of the neonatal rat and significantly reduced H₂O₂ levels after exposure to HI (B). Twenty-four hours after HI the brains were removed, sectioned and subjected to TTC staining to determine the infarct volume in the right hemisphere. Representative TTC stained sections are shown (E). The HI-mediated increase in infract volume in the right hemisphere is significantly attenuated by the over-expression of GPx-1 (F). Values are presented as mean ± S.E. from 4 independent experiments using 24 pooled slices per experiment and as mean ± S.D. for 6 animals per group. In the slice cultures *p<0.05 vs. no OGD; in the rat brain *p<0.05 vs. sham, †p<0.05 vs. HI + artificial (a) CSF.