Figure 2

Effect of pioglitazone on microglial-induced neuronal and axonal loss in transwell co-culture. (a) Effect of pioglitazone (Pio; 1 and 10 μM) on nitrite production by microglial cultures either in the absence (MIN) or presence of LPS and IFN-γ (MIN LPS IFN). Cultures were exposed to IFN and LPS for 48 hours and nitrite levels measured in the culture supernatant (μM nitrite, **P < 0.01 compared to MIN; statistical significance was obtained by one-way ANOVA followed by Bonferroni post-hoc test. (b) Effect of pioglitazone (Pio; 1 and 10 μM) on nitrite production by neuron-microglia transwell co-cultures either in the absence (MIN) or presence of LPS and IFN-γ (MIN LPS IFN). Cultures were exposed to IFN and LPS for 48 hours and nitrite levels measured in the culture supernatant (μM nitrite, **P < 0.01 compared to MIN; statistical significance was obtained by one-way ANOVA followed by Bonferroni post-hoc test). (c) Effect of pre-treatment with pioglitazone (1 and 10 μM) on cortical neuronal survival in the presence of LPS and IFN-γ activated microglia in transwell co-culture (number of β-III tubulin cells per field) (**P < 0.01 compared to MIN LPS IFN, Student's t-test). (d) Effect of pre-treatment with pioglitazone (1 and 10 μM) on total axon length (**P < 0.01 compared to MIN LPS IFN, Student's t-test) in the presence of LPS and IFN-γ activated microglia in transwell co-culture (length of SMI-312 axons per field). Photomicrographs showing immunoreactivity for β-III tubulin (red) and DAPI (blue) in neuron-microglia transwell co-cultures in the presence of MIN LPS and IFN-γ (MIN LPS IFN) (e) and (f) MIN LPS IFN-γ and pioglitazone (10 μM) (MIN LPS IFN Pio 10 μM). ANOVA, analysis of variance; DAPI, 4',6-diamidino-2-phenylindole; IFN, interferon; LPS, Lipopolysaccharide. Microglial derived-nitric oxide has been shown to mediate significant neuronal and axonal loss in vitro. We examined the influence of pioglitazone pre-treatment on neuronal survival and axonal morphology in neuronal-microglial transwell co-cultures. Co-cultures were fixed and stained for βIII tubulin, SMI312 and the nuclear marker DAPI. Pre-treatment with pioglitazone (10 μM) conferred a neuroprotective effect with a significant increase in neuronal (Figure 2c) and total axon survival (Figure 2d) in transwell co-cultures of neurons and activated microglia. These data suggest that pioglitazone provides protection for cortical neurons in neuron-microglia co-cultures through mechanisms which may be independent of its effects on nitrite reduction and that cell-to-cell contact is not required for this to occur.