Figure 4

Effect of pioglitazone on hydrogen peroxide-mediated reductions in neuronal survival in cortical neuronal culture. (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 compared with MIN); the effect of pioglitazone (0.1 μM to 10 μM; MIN H₂O₂ PIO) on cortical neuronal viability exposed to H₂O₂ (*P < 0.05 and **P < 0.01 as compared with MIN H₂O₂); and the effect of the PPAR-γ antagonist, GW9662 (1 μM) on pioglitazone-induced neuroprotection from H₂O₂ (**P < 0.01 comparing MIN H₂O₂ Pio 1 μM GW9662 1 μM with MIN H₂O₂ Pio 1 μM). Cultures were treated with GW9662 for 1 hour prior to incubation with pioglitazone for 1 hour followed by 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; MIN H₂O₂) on cortical neuronal cell survival (number of βIII-tubulin cells per field; ***P < 0.001 as compared with MIN, Student's t-test); the effect of pioglitazone (1 μM) on cortical neuronal survival exposed to H₂O₂ (***P < 0.001 compared with MIN H₂O₂); and the effect of the PPAR-γ antagonist, GW9662 (1 μM) on pioglitazone-induced neuroprotection from H₂O₂ (**P < 0.01 comparing MIN H₂O₂ Pio 1 μM GW9662 1 μM with MIN H₂O₂ Pio 1 μM). (c) Effect of hydrogen peroxide exposure (250 μM H₂O₂) on axon length within neuronal cultures (determined by SMI312 staining; ***P < 0.001 as compared with MIN, Student's t-test); the effect of pioglitazone (1 μM) on axon length in neurons exposed to H₂O₂ (**P < 0.01 compared with MIN H₂O₂); and the effect of the PPAR-γ antagonist, GW9662 (1 μM) on pioglitazone-induced axon protection from H₂O₂ (***P < 0.001 comparing MIN H₂O₂ Pio 1 μM GW9662 1 μM with MIN H₂O₂ Pio 1 μM). 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; PPAR-γ, peroxisomal proliferator activated receptor γ; SEM, standard error of the mean. To further examine the neuroprotective properties of pioglitazone, neurons were exposed to identical test conditions and cultures were then fixed and stained for βIII tubulin, SMI312 and the nuclear marker DAPI. The numbers of viable neurons (determined by nuclear appearance) expressing βIII tubulin were counted for each condition. As expected, neuronal survival was significantly increased following exposure to pioglitazone (1 μM) (Figure 4b). In addition, we examined the influence of pioglitazone on axonal morphology in these cultures. Neurofilament phosphorylation, as identified by the antibody SMI312, was examined as a marker of axonal integrity and length. Pre-treatment with pioglitazone (1 μM) caused a significant increase in total axon survival (Figure 4c). Pre-treatment with GW9662 led to a significant reduction in the neuroprotective (Figure 4b) and axonoprotective (Figure 4c) effects of pioglitazone. To determine further the role of catalase in pioglitazone-induced neuroprotection, we pre-incubated the cultures with the catalase inhibitor 3-amino triazole (10 mM) (3-AT) for 1 hour, prior to incubation with H₂O₂ (250 μM). The neuroprotective and axonoprotective effects of pioglitazone were attenuated by the addition of 3-AT (10 mM) (Figure 5a-5c). Furthermore, addition of exogenous catalase (100 U/ml) to cultures exposed to H₂O₂ improved neuronal survival (measured by MTT assay (Figure 5a) and neuronal morphology (Figure 5b) and axonal length within cultures (Figure 5c).