Hypoglycemic neuronal death is triggered by glucose reperfusion and activation of neuronal NADPH oxidase (original) (raw)
Superoxide is produced at the time of GR after HG. To characterize the source of superoxide production in hypoglycemic neuronal injury, we used a rat model of insulin-induced HG and evaluated the production of ROS with dihydroethidium (28). Dihydroethidium is oxidized by superoxide and superoxide reaction products to form fluorescent ethidium (Et) species (29), which are then trapped within cells by DNA binding. Rats subjected to 60 minutes of profound HG (producing an isoelectric EEG) showed only a modest increase in neuronal Et fluorescence, but rats undergoing only 30 minutes of HG followed by a subsequent 30-minute interval of GR showed a several-fold increase in neuronal Et fluorescence despite the shorter hypoglycemic interval (Figure 1A), suggesting that ROS are produced primarily during GR. The Et formation induced by HG/GR was blocked by the superoxide dismutase (SOD) mimic tempol (4-hydroxy-2,2,6,6-tetramethylpiperidinyloxy) (50 mg/kg) and was also blocked in transgenic rats that overexpress SOD-1 (30), confirming that the Et fluorescence signal results primarily from the production of superoxide (Figure 1B). Tempol and SOD-1 overexpression also reduced neuronal death (Figure 1, C and D), thus linking superoxide production to HG-induced neuronal death. Blood glucose concentrations were not significantly different among the 3 treatment groups (Table 1).
GR induces neuronal superoxide production after HG. (A) Neuronal superoxide production as imaged by Et fluorescence in rat brain hippocampal sections after sham HG (Sham), 60 minutes of HG without GR (HG only), or 30 minutes of HG plus 30 minutes of GR (HG/GR). Et signal intensity is expressed as the ratio of the mean fluorescence in neuronal perikarya (example outlined in white dashed line) to background (stratum radiatum; white square). Scale bar: 100 μm. Data are mean + SEM; n = 4; *P < 0.05; **P < 0.01. (B) Et fluorescence induced by HG/GR was blocked by tempol and by overexpression of SOD-1 (SOD-1 Tg). Data are mean + SEM; n = 3–5; *P < 0.05. (C) Tempol and SOD-1 overexpression also reduced neuronal death after HG/GR. H&E-stained sections of the hippocampal CA1 cell layer prepared 7 days after HG/GR show degenerating neurons with pyknotic, eosinophilic changes (arrows). Scale bar: 100 μm. (D) The number of degenerating neurons was quantified in 4 brain regions: hippocampal CA1, subiculum (Sub), dentate gyrus (DG), and perirhinal cortex (Ctx). Data are mean + SEM; n = 7–10; *P < 0.05 versus HG/GR alone in each region.
Blood glucose concentration during HG and GR
Superoxide is produced primarily by NADPH oxidase. To identify the site and mechanism of HG-induced superoxide production, we subjected cortical cell cultures, containing neurons and glia, to glucose deprivation (GD) followed by GR. As observed with HG in vivo, GD alone caused only a negligible increase in the Et signal, but GR produced a rapid increase in neuronal Et fluorescence (Figure 2A). Control cultures, in which 10 mM glucose was added back after less than 5 minutes of GD, exhibited a negligible Et signal (Figure 2B). GR with 2 mM glucose produced a slightly smaller signal than GR with 10 mM glucose (Figure 2B), and all subsequent studies were performed using 10 mM glucose replacement. Detection of the lipid peroxidation product 4-hydroxynonenal in neurons after GD/GR, but not GD alone, provided additional evidence that GR triggers superoxide formation (Figure 2C).
Neuronal superoxide production after GD/GR is due to NADPH oxidase activation. (A) Et fluorescence in cultured mouse neurons at time points after GD and GR. Top row shows neurons subjected to 2 hours of GD followed by GR; bottom row shows neurons subjected to GD during the entire 3-hour interval. Line graphs show the change in Et fluorescence over time in each of the labeled neurons, with values normalized to the background signal. Scale bar: 30 μm. (B) Quantification of Et-positive neurons in cultures treated with 3 hours GD alone or with 2 hours GD followed by 1 hour GR at the designated glucose concentration. Controls (Cont) received 10 mM glucose after less than 5 minutes of GD. Data are mean + SEM; n = 7; *P < 0.01. (C) Immunostaining for 4-hydroxynonenal in cultured neurons. Representative of n = 3. Scale bar: 100 μm. (D) Neuronal superoxide production induced by GD/GR was not blocked by inhibitors of mitochondrial superoxide production. ROT, rotenone; TTFA, 2-thenoyltrifluoroacetone; FCCP, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; OHCA, α-cyano-4-hydroxycinnamate. n = 4. (E) Superoxide production was also unaffected by the glycolytic inhibitor iodoacetate (Iodo) but was blocked by 6-aminonicotinamide (6AN) and by apocynin (Apo). This pattern was also observed in neurons cultured in the absence of glia (bottom panel). Data are mean + SEM; n = 4; **P < 0.05. (F) Immunostaining for the p47phox (left panels) or p67phox (right panels) subunits of NADPH oxidase showed their migration to the neuronal plasma membrane (arrows) after GD/GR but not after GD alone. MAP2 immunostaining demarcates the neuronal cytoplasmic space. Scale bar: 10 μm.
Mitochondria have been identified as a source of neuronal superoxide production in glutamate excitotoxicity (31), and glutamate excitotoxicity contributes to hypoglycemic neuronal death (7, 10, 12). We therefore evaluated superoxide production in the presence of the complex I inhibitor rotenone (5 μM), the complex II inhibitor 2-thenoyltrifluoroacetone (10 μM), and the mitochondrial uncoupler carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (0.5 μM), each of which prevents superoxide production by the mitochondrial electron transport chain (32). None of these agents prevented neuronal superoxide production during GD/GR (Figure 2D). Superoxide can also be generated from dehydrogenase complexes of the mitochondrial tricarboxylic acid cycle (33); however, neuronal superoxide production after GD/GR was similarly unaffected by inhibiting glucose-derived substrate delivery to mitochondria with either 500 μM iodoacetate to inhibit glycolysis or with 250 μM α-cyano-4-hydroxycinnamate (OHCA) to block mitochondrial pyruvate uptake (Figure 2, C and D).
A third potential source of superoxide generation is NADPH oxidase, which requires glucose for regeneration of NADPH by the hexose monophosphate shunt (22). Incubation with either 500 μM 6-aminonicotinamide, an inhibitor of the hexose monophosphate shunt (34), or with 500 μM apocynin (4-hydroxy-3-methoxyacetophenone), an inhibitor of NADPH oxidase activity (35), blocked the superoxide production induced by GD/GR in cortical cell cultures (Figure 2E). Since cortical cultures contain astrocytes and microglia in addition to neurons, these studies were also performed in cultures containing neurons alone to determine which cell type was the source of superoxide production. Cultures containing neurons in the absence of glia produced the same pattern of results obtained with the neuron/glia cocultures, although a shorter interval of GD was required to induce comparable superoxide production (Figure 2E).
NADPH oxidase is composed of several subunits, including the p47phox and p67phox subunits, which coalesce at the plasma membrane to form the active enzyme complex (22, 36). Immunostaining confirmed the presence of these subunits in neurons and showed their translocation to the neuronal plasma membrane after GD/GR but not after GD alone (Figure 2F).
Inhibition of NADPH oxidase prevents superoxide production and subsequent neuronal death. To confirm further that NADPH oxidase is the source of superoxide production after GD/GR, we prepared cortical cocultures consisting of neurons from WT mice or from p47phox-deficient mice, with the neurons plated onto WT glia. Cells from p47phox-deficient mice are unable to assemble an active NADPH oxidase complex (37, 38). The p47phox-deficient neurons generated much less superoxide after GD/GR and showed less neuronal death than WT neurons (Figure 3A). This effect was comparable to that obtained in WT neurons treated with 500 μM apocynin or 500 μM 6-aminonicotinamide (Figure 3B). We then used the p47phox-deficient mice to evaluate superoxide production and neuronal survival after HG in vivo. Mice lacking p47phox showed much less neuronal death than WT mice of the same C57/B6 background strain (Figure 3C), and this reduction in cell death was accompanied by reduced neuronal Et fluorescence at the time of reperfusion (Figure 3D). A comparable reduction in cell death was observed in WT rats treated with 15 mg/kg apocynin to inhibit NADPH oxidase previous to GR (Figure 3E).
Inhibition of NADPH oxidase prevents HG-induced neuronal death. (A) GD/GR-induced superoxide production in cortical neurons is attenuated by p47phox deficiency. Data are mean + SEM; n = 3; *P < 0.05 versus WT. (B) GD/GR-induced neuronal death is blocked by p47phox gene deletion and by pharmacological inhibitors of NADPH oxidase activity. Photomicrographs show propidium iodide (PI) staining of dead neurons 22 hours after GD in WT neurons, p47phox-deficient neurons, or WT neurons treated with 6-aminonicotinamide or apocynin. Scale bar: 100 μm. Data are mean + SEM; n = 4–6; *P < 0.05. (C) Photomicrographs show dead neurons stained green by Fluoro-Jade B in the hippocampal CA1 region of WT and p47phox-deficient mice 7 days after HG/GR. Graph quantifies hypoglycemic neuronal death in 4 vulnerable brain regions. Scale bar: 100 μm; data are mean + SEM; n = 3; *P < 0.05. (D) HG/GR-induced superoxide production, as evidence by Et fluorescence, was attenuated in neurons of p47phox-deficient mice. Scale bar: 100 μm; data are mean + SEM; n = 3–4; *P < 0.05. (E) Apocynin reduced neuronal death in WT rats evaluated 7 days after HG/GR. Data are mean + SEM; n = 5; *P < 0.05.
Zinc chelation prevents superoxide production during GR. The mechanism by which NADPH oxidase is activated in nonphagocytic cells such as neurons is not well understood, but zinc has been identified as both an inducer of neuronal NADPH oxidase activity (39) and a contributor to hypoglycemic neuronal death (15). We examined intracellular free zinc concentrations in cultured neurons using the fluorescent dye FluoZin-3 (40). FluoZin-3 fluorescence intensity was increased by 52% ± 11% in neurons subjected to 3 hours of GD and by 203% ± 19% in neurons treated with only 2 hours of GD followed by 1 hour of glucose replacement (n = 4; P < 0.01). Cultures treated with 50 μM of the extracellular zinc chelator calcium disodium EDTA (CaEDTA) (41) showed a significantly reduced FluoZin-3 signal after GD/GR (Figure 4A). Specificity of the FluoZin-3 signal was confirmed by near-complete suppression of the fluorescent signal by 100 μM of the cell-permeant heavy metal chelator TPEN [N,N,N',N'-tetrakis(2-pyridyl-methyl)ethylenediamine] (40). The translocation of p47phox and p61phox to the neuronal cell membrane in cortical cultures treated with GD/GR was similarly blocked by CaEDTA but not zinc EDTA (ZnEDTA), which lacks zinc chelating capacity (Figure 4B).
Zinc chelation prevents activation of neuronal NADPH oxidase. (A) FluoZin-3 images of intracellular free zinc levels in cultured neurons after 3 hours of GD or 2 hours of GD and 1 hour of glucose replacement (GD/GR). Fluorescence induced by GR was attenuated in cultures treated with CaEDTA during the GD/GR incubations and eliminated by 10-minute incubation with N,N,N',N'-tetrakis(2-pyridyl-methyl)ethylenediamine (TPEN). n = 4. (B) Immunostaining for the p47phox and p67phox subunits of NADPH oxidase in cultured neurons. GD/GR-induced migration of these subunits to the neuronal plasma membrane was blocked by the zinc chelator CaEDTA but not by ZnEDTA used as a control. Scale bar: 10 μm. Representative of 3 cultures under each condition. (C) Vesicular zinc in the rat hippocampal hilus is imaged with TSQ fluorescence (white). The TSQ signal loss was greater after 30 minutes HG and 30 minutes GR (HG/GR) than after 60 minutes HG without GR. Fluorescence intensity is expressed as arbitrary units, with subtraction of background measured in the lateral ventricle (white square). Scale bar: 200 μm. Data are mean + SEM; n = 10; *P < 0.05; **P < 0.01. (D) TSQ fluorescence was increased in the postsynaptic pyramidal cells after HG/GR, and this increase was blocked by CaEDTA (n = 3). Scale bar: 100 μm. (E) CaEDTA reduces GR-induced superoxide production in the CA1 neurons. ZnEDTA was the control. Graph shows quantified CA1 neuronal Et fluorescence intensity in rats treated with intracerebroventricular saline, CaEDTA, or ZnEDTA. Scale bar: 100 μm. Data are mean + SEM; n = 3–5; *P < 0.05.
Extracellular zinc in brain is derived primarily from release of synaptic vesicles (42). We used the fluorescent dye TSQ [(6 methoxy 8 quinolyl) para toluenesulfonamide], which binds chelatable zinc (43), to determine whether vesicular zinc release is associated with NADPH oxidase activation after induction HG and GR (Figure 4C). Vesicular zinc in the hippocampal hilus region stains brightly with TSQ under basal conditions. The vesicular zinc signal was partially depleted by 60 minutes of HG alone but was almost completely depleted by 30 minutes of HG followed by 30 minutes of GR. Conversely, TSQ staining for zinc in the postsynaptic pyramidal cell bodies was absent under control conditions or HG alone but increased after HG/GR (Figure 4D), suggesting that zinc released from synaptic vesicles is translocated into the postsynaptic cells under these conditions. This increase was blocked in rats given intracerebroventricular injection of the zinc chelator CaEDTA. Rats treated with CaEDTA also showed reduced neuronal superoxide formation, suggesting that vesicular zinc release is upstream of NADPH oxidase activation. ZnEDTA, used as a control, had no effect (Figure 4E).
Post-HG glucose concentrations influence neuronal superoxide production. Results of the cell culture studies presented here are consistent with prior reports that glucose flux through the hexose monophosphate shunt is required for regeneration of the NADPH substrate used by NADPH oxidase (34, 44). A previous study also suggested that high blood glucose concentrations following HG can exacerbate brain injury (45). We therefore assessed the effects of differing blood glucose concentrations during the immediate reperfusion period on neuronal superoxide production and survival. Rats were given glucose at rates that maintained blood glucose concentration in the range of 1–2 mM, 5–10 mM, or 10–15 mM following HG (Table 1). Anesthesia was discontinued after 2 hours, at which time the rats were allowed to feed ad libitum. Rats in the 1–2 mM glucose group exhibited reduced neuronal death (evaluated at 7 days) relative to the 5–10 mM group (Figure 5A). Rats in the 1–2 mM glucose group also showed reduced neuronal superoxide production during GR (Figure 5B). Rats in the 10–15 mM glucose group did not exhibit any further increase in neuron death relative to the 5–10 mM group. There was negligible Et signal in the “sham HG” rats given glucose (in addition to insulin) to maintain blood glucose in the 5–10 mM range.
Effects of reperfusion glucose concentrations on neuronal superoxide production and survival. (A) Effects of reperfusion blood glucose concentrations on neuronal survival after HG/GR. Data are mean + SEM; n = 7–10; *P < 0.01 versus the 5–10 mM GR group in each region. (B) Neuronal superoxide production as imaged by Et fluorescence in rat brain hippocampal sections after HG and GR at the indicated blood glucose concentrations. Sham HG rats received glucose infusions immediately after insulin to prevent HG. Scale bar: 100 μm. (C) Et signal intensity was quantified for the CA1 region as described for Figure 1A. Data are mean + SEM; n = 4; **P < 0.05.