Article Figures & Data
Figures
- Figure 1.
Primary neuronal cell cultures were exposed to key oxidants in TBI: ONOO−, NO, and O2−. NO reacts with O2− to form ONOO−. ONOO− causes protein nitration, lipid peroxidation, DNA damage, and inhibition of mitochondrial electron transport, leading to necrotic cell death. O2− is removed by MnSOD and ONOO− is removed by PRDX5.
- Figure 2.
Genetic background regulated antioxidative enzyme MnSOD after oxidative stress. ONOO− caused higher MnSOD synthesis in DA compared to PVG. Baseline levels were higher in PVG compared to DA. No increase was seen at 2 h, confirming a de novo protein synthesis. Dotted lines mark baselines. Pictures are constructed from different parts of gels and marked as such. Densiometric quantification was made as a mean of three consecutive gels which were normalized to both α-tubulin and a specific control identical for all gels; *p < 0.05.
- Figure 3.
Genetic background did not regulate antioxidative enzyme PRDX5 after oxidative stress. PRDX5 was decreased by ONOO−, increased by NO and O2−, and unchanged by 4-HNE. Baseline expression did not differ between strains. Dotted lines mark baselines. Pictures are constructed from different parts of gels and marked as such. Densiometric quantification was made as a mean of three consecutive gels, which were normalized to both α-tubulin and a specific control identical for all gels.
- Figure 4.
Genetic background effected 4-HNE formation after oxidative stress. PVG had higher levels of 4-HNE after oxidative stress by ONOO−, NO, and O2− compared to DA at 24 h. A discrete increase was seen at 2 h. ONOO− caused a 35× increase of 4-HNE in PVG, compared to 7× by NO and 12× by O2−, why ONOO− was confirmed as the most powerful oxidant. Baseline levels did not differ between strains. Neuronal iNOS was not induced by any of the oxidants ONOO−, NO, O2−, or 4-HNE. Dotted lines mark baselines. Pictures are constructed from different parts of gels and marked as such. Densiometric quantification was made as a mean of three consecutive gels which were normalized to both α-tubulin and a specific control identical for all gels; *p < 0.05 and **p < 0.01.
- Figure 5.
Genetic background effected 3-NT formation after oxidative stress. PVG had higher levels of 3-NT after oxidative stress by ONOO− and NO. ONOO−, NO, and O2− consistently caused a 10–15× increase of 3-NT, why nitrosylation occurred indiscriminate of oxidant. A discrete increase was seen at 2 h. Baseline levels did not differ between strains. Dotted lines mark baselines. Pictures are constructed from different parts of gels and marked as such. Densiometric quantification was made as a mean of three consecutive gels which were normalized to both α-tubulin and a specific control identical for all gels; *p < 0.05.
- Figure 6.
Genetic background effected the acute oxidative stress at 2 h. Oxidative stress levels measured by CellROX fluorescent marker were consistently higher in DA after NO, O2−, and ONOO−, compared to PVG. ONOO− caused a 10× increase of oxidative stress compared to a 2× increase for NO and 3× for O2− why ONOO− was confirmed as the most powerful oxidant. 4-HNE did not cause oxidative stress in the neurons. Dotted lines mark baselines; *p < 0.05.
- Figure 7.
Genetic background effected neuronal death, measured by LDH, after oxidative stress. PVG had increased neuronal death after oxidative stress consistently by ONOO−, NO, and O2− compared to DA at 24 h. Baseline levels did not differ between strains. Dotted lines mark baselines; *p < 0.05, ***p < 0.005, and ****p < 0.001.
- Figure 8.
Photomicrographs by Cell-IQ of neuronal cell cultures. Morphologic changes were detected after ONOO− (SIN-1), NO (DETA NO), O2− (DMNQ), and 4-HNE. Control cultures did not exhibit signs of cell death. Cell death was extensive after ONOO− compared to NO, O2−, and 4-HNE. Morphologic signs of cell death were reversed in the 4-HNE groups by the addition of antioxidants.
- Figure 9.
Graphic presentation of the phenotype regulated neuronal antioxidative response.
Tables
Antibody/assay Specificity Species Source Product number Dilution MnSOD SOD2 Rb Abcam ab13533 1:5000 PRDX5 Peroxyredoxin V Rb Abcam ab180587 1:1000 4-HNE 4 Hydroxynonenal Rb Abcam ab46545 1:1000 3-NT 3-Nitrotyrosine [39B6] Mo Abcam ab61392 1:500 iNOS Inducible NO synthase Rb Abcam ab15323 1:250 α-Tubulin Loading control Mo Abcam ab7291 1:10,000 α-Tubulin Loading control Rb Abcam ab176560 1:1000 NeuN Neuron-specific nuclear protein Mo Millipore MAB377 1:500 GFAP Glial fibrillary acidic protein Rb Abcam ab33922 1:1000 Alexa Fluor 680 Secondary 680 nm GoαRb Molecular Probes A21076 1:15,000 800CW Secondary 800 nm DoαMo Li-Cor 926-32212 1:15,000 800CW Secondary 800 nm GoαRb Li-Cor 926-32211 1:15,000 LDH assay Lactate dehydrogenase release N/A Abcam ab102526 N/A
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