Figure 1.
Schematic representation of the experimental design and most important results.
Figure illustrates crucial steps in investigation of Δψm changes during simulated reperfusion within one hour after extended exposure of astrocytes to GD or OGD. The culturing media are designated as: high glucose (hG), low glucose (lG) or no glucose (nG). During the experiments cells were cultured either in normoxic (normO2) or hypoxic (hypoO2) conditions. Ellipsoid shapes show conditions the cells were subjected to. Text on connector lines shows whether we found significant effect of given experimental condition(s). Octagons indicate conclusions we made.
Figure 2.
OGD in lG partly preserves Δψm (i.e. delays depolarization) caused by subsequent NaN3 treatment.
(A) Sodium azide (NaN3) causes concentration-dependent decline of Δψm in both experimental conditions. However, OGD in lG medium significantly delayed the decrease in JC-1 fluorescence ratio caused by treatment with 5 mM NaN3 during simulated reperfusion. (B) Expectedly, both NaN3 and H2O2 show concentration-dependent effect by lowering the JC-1 red/green fluorescence ratio. Astrocytes were stained with JC-1 either before or during the treatment with NaN3 (marked as pre-dye loading and dye loading under ETC inhibition, respectively). NaN3 has not affected the cell membrane organization allowing at the same time JC-1 to enter the cytoplasm and mitochondria within. (C) Representative fluorescent micrographs of astrocytes labeled with JC-1 Original micrographs were converted to tritanope color palette (ImageJ 1.48a). Depolarization is visible in the normoxic group (b, c) (seen as concentration-dependent decrease in magenta and increase in blue color), but it is more pronounced than in OGD group after treatment with NaN3 (e, f). Some mitochondria remained partly depolarized. Data are expressed as a percentage normalized to the red/green fluorescence ratio values of untreated control (the first bar from left). Significant differences are indicated by **p<0.01 with respect to untreated control, ##p<0.01 between treatment and its respective control, ΔΔp<0.01 between different inhibitor concentrations.
Figure 3.
Glucose deprivation leads to hyperpolarization of Δψm after both normoxic and OGD conditions.
(A) Glucose deprivation for 8 h increases the ratio of JC-1 fluorescence. This effect was not detected when combining GD and OD, but level of significance was noticable low (p = 0.059). (B) Preconditioning for 12 h with media containing reduced or no glucose promotes hyperpolarization after OGD during simulated reperfusion. After preconditioning, astrocytes were subjected to hypoxic conditions for additional 6 h (grey bars) or they were maintained in normoxic conditions (white bars). (C) Lowering glucose in the incubation medium leads to an increased ratio of JC-1 fluorescence during simulated reperfusion after normoxic and OGD conditions. There was significant interaction between effect of glucose in the culturing media and the effect of OGD. Astrocytes were incubated for two days either in hG or lG medium. Subsequently, OGD was conducted for 6 h (grey bars). (D) Schematic representation of the experimental design of C. The duration of each step is shown in brackets. (E) Representative red signal from fluorescent micrographs of astrocytes labeled with JC-1. Original micrographs were converted to rainbow pseudocolor pallete using LUTs (ImageJ 1.48a). Increase in red fluorescence is observed when astrocytes are incubated in OGD in nG medium as compared to normoxic conditions in lG medium (Fig. 2B). The scale bar represents 100 µm. Data are expressed as a percentage normalized to the JC-1 red/green fluorescence ratio values of untreated control astrocytes (first bar on the left). Significant differences are indicated by **p<0.01 with respect to control (normoxia in hG for Fig. 3A and 3C, normoxia in lG for Fig. 3B), ##p<0.01 between normoxia and OGD (in lG or nG), ΔΔp<0.01 between two OGD treatments.