Hypoglycemic neuronal death is triggered by glucose reperfusion and activation of neuronal NADPH oxidase
J. Clin. Invest. Sang Won Suh, et al. 117:910 doi:10.1172/JCI30077 [
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Figure 2Neuronal 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 p47
phox (left panels) or p67
phox (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.
