Figure 5

Inhibition of catalase activity attenuates pioglitazone-induced protection from H ₂ O ₂ in cortical neuronal cultures. (a) The effect of hydrogen peroxide exposure (MIN H₂O₂; 250 μM) on cortical neuronal viability in vitro compared to serum free minimal media (MIN) (**P < 0.01 as compared with MIN); the effect of pioglitazone (1 μM) and the specific catalase inhibitor, 3-aminotriazole (3-AT) (10 mM) (**P < 0.01 MIN H₂O₂ compared to MIN H₂O₂ Pio 1 μM; and **P < 0.01 MIN H₂O₂ Pio 1 μM compared to MIN H₂O₂ Pio 1 μM 3-AT 10 mM) and the effect of catalase exposure (MIN H₂O₂ CAT; 100 U/ml) (**P < 0.01 MIN H₂O₂ compared to MIN H₂O₂ CAT) on cortical neuronal viability. Cultures were treated with pioglitazone for 1 hour prior to incubation with 3-AT for 1 hour followed by exposure to hydrogen peroxide. Cultures were treated with catalase for 1 hour prior to exposure to hydrogen peroxide. Cell viability was assessed by MTT assay. Data are expressed as percentage of cells grown in MIN medium. Statistical significance was obtained by one-way ANOVA followed by Bonferroni post-hoc test. (b) Effect of hydrogen peroxide exposure (250 μM H₂O₂) on cortical neuronal cell survival (number of βIII-tubulin cells per field; **P < 0.01 as compared with MIN, Student's t-test); the effect of pioglitazone (1 μM) on cortical neuronal viability exposed to H₂O₂ (*P < 0.05 compared with MIN H₂O₂); the effect of catalase exposure (MIN H₂O₂ CAT; 100 U/ml) (**P < 0.01 MIN H₂O₂ compared to MIN H₂O₂ CAT) and the effect of the specific catalase inhibitor, 3-AT (10 mM) on pioglitazone-induced neuroprotection from H₂O₂ (**P < 0.01 comparing MIN H₂O₂ Pio 1 μM with MIN H₂O₂ Pio 1 μM 3-AT 10 mM). (c) Effect of hydrogen peroxide exposure (250 μM H₂O₂) on axon length within neuronal cultures (determined by SMI312 staining; **P < 0.01 as compared with MIN, Student's t-test); the effect of pioglitazone (1 μM) on axon length in neurons exposed to H₂O₂ (*P < 0.05 compared with MIN H₂O₂); the effect of catalase exposure (MIN H₂O₂ CAT; 100 U/ml) (**P < 0.01 MIN H₂O₂ compared to MIN H₂O₂ CAT) and the effect of the specific catalase inhibitor, 3-AT, (10 mM) on pioglitazone-induced axon protection from H₂O₂ (**P < 0.01 comparing MIN H₂O₂ Pio 1 μM with MIN H₂O₂ Pio 1 μM 3-AT 10 mM). Values represent the mean ± SEM from at least three separate experiments. ANOVA, analysis of variance; MTT, 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide; SEM, standard error of the mean.