Figure 1.
(A, B) Under normal conditions, primary adult astrocytes present a polygonal to fusiform and flat morphology, as shown by phase contrast microscopy. We did not observe morphological alterations during the culture period. The arrowhead indicates a polygonal cell, and the arrow indicates a fusiform cell. Scale bars: (A) = 100 µm, (B) = 50 µm.
Figure 2.
Adult astrocyte cultures present classical astroglial markers.
(A) Astrocytes show intense immunolabeling for GFAP. (B) The cells were counterstained with DAPI (blue). Shown are the merged images only. The data are from 5 independent experiments. Scale bars: (A) = 50 µm, (B) = 100 µm. Representative immunoblot band of rat cortex homogenate and adult astrocytes for (C) GFAP, (D) vimentin, (E) GAPDH. N = 5 for all immunoblot experiments.
Figure 3.
Adult astrocytes cultures express GS, ALDH1L1 and S100B proteins.
(A) Immunocytochemistry of adult astrocyte cultures for GS with a merge for DAPI staining. Representative immunoblot band for (B) GS, (C) ALDH1L1 and (D) S100B. The left band represents rat cortex homogenate and the right band represents adult astrocytes. N = 5 for all immunoblot experiments.
Table 1.
Analysis of the main glial parameters in adult astrocytes under basal conditions.
Figure 4.
Adult astrocyte cultures express the main glutamate transporters.
Representative immunoblot band for (A) GLAST and (B) GLT1. The left band represents rat cortex homogenate and the right band represents adult astrocytes. N = 5 for all immunoblot experiments.
Figure 5.
Effects of H2O2 on adult astrocyte cell viability.
Membrane integrity (PI incorporation) and metabolic activity (MTT reduction) were measured as described in the Materials and Methods section. The medium was removed, followed by the addition of DMEM/F12 supplemented with 5% FBS, and the cells were maintained at 5% CO2 (37°C) in the presence or absence of H2O2 for 3 h. Data are expressed as the ratio of PI incorporation to MTT reduction and represent the mean ± S.E. from 4 to 6 experimental determinations performed in triplicate. * P<0.05 and ** P<0.01 indicate significant differences from control conditions.
Figure 6.
Adult astrocytes take up glutamate (A) and glucose (B).
To determine the glutamate uptake, the culture medium was removed followed by the addition of DMEM/F12 supplemented with 5% FBS in the presence or absence 50 µM H2O2 for 3 h. To determine the glucose uptake, the medium was then replaced by DMEM/F12 supplemented with 1% FBS containing 1 µCi/ml [3H]2DG, in the presence or absence of 500 µM glutamate for 20 min. In all procedures, the cells were maintained at 5% CO2/37°C. The assays were performed as described in the Materials and Methods section. Absolute values obtained under control conditions were 0.8±0.08 nmol/mg protein/min for glutamate uptake and 172±20 fmol/mg protein for glucose uptake. The data represent the mean ± S.E. from 4 to 6 experimental determinations performed in triplicate. * P<0.05 indicates significant differences from the control values.
Figure 7.
The effect of resveratrol on (A) intracellular ROS production, (B) GS activity and (C) intracellular GSH content during H2O2-induced oxidative insult.
The culture medium was removed followed by the addition of DMEM/F12 supplemented with 5% FBS, and the cells were pre-incubated for 1 h with 100 µM resveratrol followed by the addition of 50 µM H2O2 for 3 h. Absolute values obtained under control conditions were 2.2±0.6 µmol/mg protein/min for GS activity and 14±1.1 nmol/mg protein for GSH content. The data represent the mean ± S.E. from 4 to 6 experimental determinations performed in triplicate. ** P<0.01 indicates significant differences from the control values.
Figure 8.
Astrocyte responses to inflammatory stimuli.
The culture medium was removed, followed by the addition of DMEM/F12 supplemented with 5% FBS, and the cells were pre-treated with resveratrol (50 µM) for 1 h. After the pre-treatment, H2O2 (50 µM) was added for 3 h in the presence or absence of resveratrol. Cells were also treated with LPS (10 µg/ml) for 3 h. The data represent the mean ± S.E. from 3 experimental determinations performed in triplicate. ** P<0.01 indicates significant differences from the control value.
Figure 9.
Effects of H2O2 and Zn2+ on actin reorganization in adult astrocyte cultures.
The culture medium was removed, followed by the addition of DMEM/F12 supplemented with 5% FBS, and the cells were maintained at 5% CO2 (37°C) in the presence or absence of H2O2 or Zn2+ for 3 h. Representative images of the rhodamine-phalloidin fluorescence signals of cells exposed to (A, D) basal conditions, (B) 50 µM H2O2, (C) H2O2 + SB203580 (5 µM), (E) 50 µM Zn2+ and (F) Zn2+ + LPA (2 µM). All images are representative fields from at least 3 experiments performed in duplicate. Scale bars: (A, B, D, E) = 50 µm, (C, F) = 100 µm.