Astrocytic Mitochondrial Membrane Hyperpolarization following Extended Oxygen and Glucose Deprivation
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.