Fig 1.
Methylene blue protects astrocytes against OGD-reoxygenation induced cell death.
Quantitative analysis of astrocyte viability by Calcein AM assay A. Exposure of primary astrocytes to OGD-reoxygenation condition caused significant loss in viability and MB administration during OGD increased cell death. B. Administration of MB (0.1 μM and 1 μM) during reoxygenation significantly protected astrocytes from OGD-reoxygenation induced cell death. C. Representative images of Calcein AM staining at 24 hours after reoxygenation with or without MB treatment. *** p < 0.0001 Vs. CTL. # p < 0.05, ## and ### p < 0.0001 Vs. OGD-reoxygenation control /0 μM MB.
Fig 2.
Methylene blue increases oxygen consumption rate (OCR) in primary astrocytes.
A. Depictions are the changes in OCR in astrocytes following vehicle or MB (10 μM) treatment. MB increased OCR in astrocytes at all-time points. B. Quantitative analysis of OCR in control and MB (10 μM) treated astrocytes before oligomycin treatment. MB (10 μM) significantly increased OCR in astrocytes compared to control. *** p < 0.0001 Vs. Control.
Fig 3.
Methylene blue increases intracellular oxygen flux in astrocytes.
A. Representative image of Ruthenium (II) fluorescence lifetime obtained using FLTIM. All the groups had similar fluorescence before treatment. Ruthenium (II) fluorescence lifetime was shorter in MB (10 μM) treated astrocytes as compared with control. As a positive control, Glucose oxidase (GO) increased Ruthenium (II) fluorescence lifetime in astrocytes. B. Quantitative analysis demonstrated that MB treatment significantly decreased Ruthenium (II) fluorescence lifetime in astrocytes. **, *** p < 0.0001 Vs. control.
Fig 4.
Methylene blue does not increase superoxide production.
A. Representative image depicting MitoSOX fluorescence in Control, Antimycin-A (AA) and different concentrations of Methylene blue treatment. B. Quantitative analysis of MitoSOX assay results. Antimycin-A (50 μM) significantly increased superoxide production in astrocytes compared to control and MB treatment. MB did not increase superoxide production in astrocytes. **, *** p < 0.0001.
Fig 5.
Methylene blue increases glucose uptake and hexokinase activity in astrocytes.
A. Representative image of 2-NBDG fluorescence in control and MB treated astrocytes under normoxic condition. B. Quantitative analysis of 2-NBDG uptake demonstrated that MB concentration-dependently increased 2-NBDG uptake in astrocytes under normoxic condition. C. Quantitative analysis of hexokinase activity in astrocytes. Methylene blue (1 μM) significantly restored OGD induced loss of hexokinase activity. **, *** p < 0.0001 Vs. Control. ### p < 0.0001 Vs. OGD-reoxygenation control / 0 μM MB.
Fig 6.
Methylene blue increases ATP production and attenuate phosphorylation of glycogen synthase in astrocytes.
A. Quantitative analysis of ATP production in MB treated astrocytes at 60 min after reoxygenation. MB significantly increased ATP production in primary astrocytes after transient OGD. B. Representative Western blots of glycogen synthase (GS), Phospho glycogen synthase (PGS) and actin in astrocytes 24 hours after OGD-reoxygenation. C. Quantitative analysis of Western blots demonstrated that ratio P-GS/GS was significantly increased in astrocytes following OGD-reoxygenation which was significantly reduced in MB (1 μM) treated astrocytes. D. Quantitative analysis of glycogen content in astrocytes. At 24 hours following OGD-reoxygenation, astrocytes had less glycogen content compared to normoxia control. MB (1 μM) treated astrocytes had significantly higher glycogen content compared to non-MB treated cells following OGD-reoxygenation. * p < 0.05; *** p < 0.0001 Vs. Control. ## p < 0.001 Vs. OGD-reoxygenation control / 0 μM MB.