Figure 1.
Effects of DMSO on survival and viability of mouse cortical astrocytes in culture.
(A) General growth profile of incubated with various concentrations of DMSO for 24 h. Astrocytes were grown well and closely adhered to each other on the surface of coverslips under normal circumstances and exposed to 1% DMSO. Many astrocytes were detached from the coverslips in 5% DMSO culture medium. (B–C) Quantitative analysis of the cell density (B) and cell viability (C) of astrocytes incubated with different concentrations of DMSO for 24 h. Data are shown as a mean ± SEM of five independent experiments performed in triplicate. *P<0.05 and **P <0.01 versus control group.
Figure 2.
Effects of DMSO on substructures of cultured mouse cortical astrocytes.
(A) Representative transmission electron micrographs showing substructural morphology of astrocytes after treatment with different concentrations of DMSO for 24 h. The disruption of mitochondria integrity becomes more severe with increased DMSO concentrations. Fragmentation of the nucleus, with condensation and margination of nuclear chromatin, was frequently observed in astrocytes exposed to 5% DMSO. (B) Quantitation of mitochondrial cross-sectional area. The results confirmed DMSO-induced mitochondrial swelling, with a significant rightward shift in the mitochondrial area cumulative frequency curve, relative to untreated control. (C) The quantitative analysis showed increases in the percentage of mitochondrial vacuolization in astrocytes treated with DMSO in a dose-dependent manner. Data are shown as a mean ± SEM of five independent experiments. *P<0.05 and **P<0.01 versus control group.
Figure 3.
Effects of DMSO on ΔΨm of cultured mouse cortical astrocytes.
(A) Representative micrographs showing ΔΨm, revealed by TMRE fluorescent staining, in astrocytes treated with various concentrations of DMSO for 24 h. The left first micrograph is a positive control for depolarized mitochondria by incubated with 20 µM FCCP, an uncoupler of electron transport and oxidative phosphorylation, for 10 minutes prior to staining with TMRE. (B) The quantitative analysis revealed that TMRE fluorescence intensity was decreased in astrocytes treated with DMSO in a dose-dependent manner. Data are shown as a mean ± SEM of five independent experiments performed in triplicate. *P<0.05 and **P<0.01 versus control group.
Figure 4.
Effects of DMSO on and the release of mitochondrial Cyt c and intracellular ROS generation in cultured mouse cortical astrocytes.
(A) Representative flow cytometry data showing mitochondrial Cyt c-FITC fluorescence within the cytoplasm of astrocytes treated with various concentrations of DMSO for 24 h. (B) Quantitative analysis of Cyt c-FITC fluorescence intensity. (C) Representative western blot bands showing expression levels of Cyt c in cytosolic and mitochondrial fractions of astrocytes treated with various concentrations of DMSO for 24 h. β-tubulin and COX IV, which were exclusively expressed within the cytosol and mitochondria, respectively, were used as loading controls. (D) The quantitative analysis of the relative optical density of cytosol and mitochondrial Cyt c showing that DMSO caused translocation of Cyt c from the mitochondria into the cytoplasm of astrocytes. Data are shown as a mean ± SEM of five (for flow cytometry) or four (for Western blot) independent experiments performed in triplicate. *P<0.05 and **P<0.01 versus control group.
Figure 5.
Effects of DMSO on mitochondrial and intracellular ROS generation in cultured mouse cortical astrocytes.
(A) Representative flow cytometry data showing Mito-SOX fluorescence, a highly selective indicator of superoxide in live cell mitochondria, in astrocytes treated with various concentrations of DMSO for 24 h. (B) Quantitative analysis revealed that the Mito-SOX fluorescence intensity increased in astrocytes treated with DMSO with a dose-dependent manner. (C) Representative flow cytometry data showing DCF fluorescence in astrocytes treated with various concentrations of DMSO for 24 h. (D) The quantitative analysis showed that ROS levels were increased in astrocytes treated with DMSO at 5% concentration but not at 1%, by detecting the fluorescence intensity of DCF. (E) Data are shown as a mean ± SEM of five independent experiments performed in triplicate. *P<0.05, **P<0.01 versus control group. #P<0.05, ## P<0.01 versus 5% DMSO treated group.
Figure 6.
Effects of DMSO on apoptosis of cultured mouse cortical astrocytes.
Astrocytes were incubated with various concentrations of DMSO for 24 h. (A) Representative micrographs showing TUNEL positive apoptotic astrocytes (cyan-blue). Cell nuclei counterstained with Hoechst 33342 (blue). (B) Quantitative analysis of astrocyte apoptosis. (C) Representative western blot bands showing expression levels of procaspase-3, cleaved caspase-3 and Bcl-2 in astrocytes. (D) The quantitative analysis showed that Bcl-2 and procaspase-3 expression levels were decreased, but cleaved caspase-3 expression level was increased in astrocytes treated with 1% or 5% DMSO. Data are shown as a mean ± SEM of five (for TUNEL) or four (for Western blot) independent experiments performed in triplicate. *P<0.05 and **P<0.01 versus control group.
Figure 7.
Effects of DMSO on expression of GLT-1 and GLAST in cultured mouse cortical astrocytes.
(A) Representative micrographs showing immunoreactivity of GLT-1 and GLAST in astrocytes treated with various concentrations of DMSO for 24 h. (B) Mean integrated optical density (MIOD) of immunostaining for GLT-1 and GLAST. (C) Representative western blot bands showing expression levels of GLT-1 and GLAST in astrocytes. (D) Quantitative analysis revealed that GLT-1 and GLAST protein levels were decreased in astrocytes treated with DMSO in a dose-dependent manner. Data are shown as a mean ± SEM of five (for immunostaining) or four (for Western blot) independent experiments performed in triplicate. *P<0.05 and **P<0.01 versus control group.