As described above, intracellular Ca2+ can also be increased by blocking intracellular storage calcium pumps with thapsigargin. Therefore, the effect of thapsigargin on activation was compared for astrocytes in DMEM and EDTA-supplemented calcium-free DMEM (Fig. 3.6). 1μM thapsigargin induced significant activation of astrocytes in DMEM (60.85±2.77%; p < 0.001; Fig. 3.6B, F) that was not significantly different from the activation induced by PMA/ionophore (p > 0.05; Fig. 3.6D, F). However, in calcium- free medium, thapsigargin only increased activation (6.48±1.04%; p < 0.05; Fig. 3.6C, F) to the basal levels of untreated astrocytes in DMEM. In the calcium-free conditions, PMA/ionophore activated significantly more astrocytes (14.1±1.87%) than thapsigargin (p < 0.05) and combining the treatments induced a further significant additive effect (22.66±2.11%; p < 0.05; Fig. 3.6E, F), however the activation in both cases remained significantly less than for similarly treated astrocytes in DMEM (p < 0.001; Fig. 3.6F). The elongated bipolar astrocyte morphology with sparse GFAP fibres induced by
Figure 3.5 Calcium-dependence of NF-κB mediated astrocyte activation. A: Activation was compared for astrocytes treated for 1 hour with 20ng/mL PMA/500nM ionophore in DMEM, calcium-free DMEM or in calcium-free DMEM supplemented with 200μM EDTA after 4 hours pre-incubation in this medium, with untreated astrocytes 1 hour after a change of DMEM or calcium-free DMEM with EDTA or from DMEM to calcium-free DMEM for comparison controls. B-G: Immunostaining for GFAP (green) was used to identify astrocytes and NF-κB (red) to identify activated astrocytes. Nuclei were counterstained with Hoechst blue (HB). A change from DMEM (B) to calcium-free medium induced astrocyte activation (D) similarly to treatment with PMA/ionophore (C, E). Incubation of astrocytes in calcium-free medium supplemented with the chelating agent EDTA resulted in more elongated astrocyte morphologies with only sparse GFAP fibres (F, G). In these conditions astrocytes were not activated by medium change (F) and only a low percentage were activated by PMA/ionophore (G). Scale bar is 50 μm. H: Quantitative analysis of astrocyte activation. Incubation in calcium-free DMEM induced a significant increase in astrocyte activation (p < 0.001) that was not significantly different (p > 0.05) from the increase induced by PMA/ionophore for astrocytes in DMEM. However, compared to untreated astrocytes in DMEM, there were significantly less activated astrocytes 1 hour after a change to fresh calcium-free DMEM supplemented with EDTA following 4 hours pre-incubation in the same medium conditions (p < 0.05). Although PMA/ionophore induced a significant increase in activation for the pre-incubated astrocytes in EDTA-supplemented calcium-free DMEM (p < 0.01), the increase was significantly less than for astrocytes in DMEM (p < 0.001). In contrast there were significantly more activated astrocytes after PMA/ionophore treatment in calcium-free DMEM without pre-incubation than for astrocytes in DMEM (p < 0.05). Data were analysed by 2-way ANOVA and post-hoc Tukey’s multiple comparison test. Results are means ± SEM, n = 8. Asterisks denote significant differences: * p < 0.05, ** p < 0.01, *** p < 0.001.
Figure 3.6 Thapsigargin activates astrocytes. A: Activation was compared for astrocytes treated for 1 hour with PMA/ionophore and/or the specific internal store Ca2+-ATPase blocker, thapsigargin (1μM) in DMEM or calcium-free DMEM supplemented with 200μM EDTA (after 4 hours pre-incubation in this medium) with untreated astrocytes in the same media as comparison controls. B-E: Immunostaining for GFAP (green) was used to identify astrocytes and NF-κB (red) to identify activated astrocytes. Nuclei were counterstained with Hoechst blue (HB). Thapsigargin activated astrocytes in DMEM (B) to a similar extent as PMA/ionophore (D) but not when astrocytes were incubated in calcium-free conditions (C). Calcium-free medium induced an elongated astrocyte morphology with sparse GFAP fibres (Figs. 3.4F, 3.5A) that was not altered by thapsigargin treatment (C). However when thapsigargin was combined with PMA/ionophore, astrocytes adopted a spindly morphology with compressed GFAP fibres and some were activated (E). Scale bar is 50 μm. F: Quantitative analysis of astrocyte activation. Thapsigargin induced significant activation of astrocytes in DMEM (p < 0.001) that was not significantly different from the activation induced by PMA/ionophore (p > 0.05). However, for astrocytes in calcium-free medium, thapsigargin only increased activation (p < 0.05) to the basal levels of untreated astrocytes in DMEM. Although PMA/ionophore activated significantly more astrocytes than thapsigargin (p < 0.05) and combining the treatments induced a further significant additive effect (p < 0.05) in the calcium-free conditions, the activation remained significantly less than for similarly treated astrocytes in DMEM (p < 0.001). Data were analysed by 2-way ANOVA and post-hoc Tukey’s multiple comparison test. Results are means ± SEM, n = 8. Asterisks denote significant differences: * p < 0.05, ** p < 0.01, *** p < 0.001.
Figure 3.7 Calcium-dependent effects of PMA/ionophore on astrocyte morphology and GFAP expression. A: PMA induced some stellation of astrocytes but did not alter GFAP immunoreactivity. B: Ionophore induced some apoptosis of astrocytes that was associated with decreased intensity of GFAP immunoreactivity in relatively intact cells and more intense immunoreactivity in detached cells displaying GFAP depolymerisation (arrows) and cytoplasmic condensation (asterisks). C: The apoptosis and depolymerisation were accentuated by combined PMA/ionophore treatment. Scale bar is 50μm. D: GFAP expression was analysed by Western blot for untreated astrocytes (lane 1) and astrocytes treated for 48 hours with 20ng/mL PMA (lane 2), 500nM ionophore (lane 3) and combined PMA/ionophore (lane 4). Protein bands were obtained at about 55kDa with the anti-GFAP primary antibody. GFAP levels appeared to be highest for untreated astrocytes with slightly lower expression levels following PMA treatment. GFAP levels decreased a little further in ionophore-treated astrocytes and were noticeably much lower following combined PMA/ionophore treatment. Equal quantities of total protein were loaded for each lane as confirmed by similar intensity immunoreactivity in each lane for the β actin (~ 42kDa) loading control. E: Calcium and ionophore dose dependence of GFAP expression. GFAP expression was analysed by Western blot for untreated astrocytes in calcium-free DMEM (lane 1) or calcium-free DMEM with a 5mM addition of calcium chloride (lane 5), astrocytes treated with 250nM ionophore in calcium-free DMEM (lane 2) or calcium-free DMEM with 5mM calcium chloride (lane 6), astrocytes treated with 500nM ionophore in calcium-free DMEM (lane 3) or calcium-free DMEM with 5mM calcium chloride (lane 7) and astrocytes treated with 1μM ionophore in calcium-free DMEM (lane 4) or calcium-free DMEM with 5mM calcium chloride (lane 8). All treatments were for 48 hours. The blot image is representative of 4 blots performed on separate samples. There was little difference in GFAP expression for untreated astrocytes in DMEM with 5mM calcium chloride (lane 5) and astrocytes treated with 250nM or 500nM ionophore in either medium condition (lanes 2-3 and 6-7). There was possibly slightly lower GFAP expression for untreated astrocytes (lane 1) and 1μM ionophore treated astrocytes (lane 4) in calcium-free DMEM and noticeably much less GFAP expression for astrocytes treated with 1μM ionophore in calcium-free DMEM with 5mM calcium chloride (lane 8). All differences between lanes seen for the GFAP bands corresponded with similar differences for the bands for β actin and GAPDH loading controls.
calcium-free medium conditions (Figs. 3.5) was not altered by thapsigargin treatment (Fig. 3.6C), however combining thapsigargin with PMA/ionophore in calcium-free medium induced an even more elongated, spindly astrocyte morphology with compression of the sparse GFAP fibres (Fig. 3.6E).