Fig. 6
Astrocytes were stimulated, in a fluorescence-based assay for detecting changes in intracellular Ca2+ over time, with the following: 5-HT(10−5 M), glutamate (10−3 M), or ATP (10−4 M). AUC and peak values of Ca2+ transients are shown. The cells were cultivated in 5.5 mM glucose the whole cultivation period. Ca2+ responses were measured after the cells were incubated with LPS (10 ng/ml) for 24 h, when incubated with LPS for 24 h followed by LPS, 25 mM glucose, a combination of naloxone (Nal) (10−12 M), endomorphin-1 (EM-1) (10−6 M), and levetiracetam (Lev) (10−4 M), or a combination of sildenafil (Sild) (1 μM) and vitamin D3 (D3) (100 nM) or combination of all substances for another 24 h. Unstimulated cells were used as controls. The area under the Ca2+ peak (AUC) was calculated for each Ca2+ transient, and the amplitude (peak) was expressed as the maximum increase. The cells were obtained from four experiments with quadruple wells in each. The level of significance was calculated against LPS (5.5) and analyzed using one-way ANOVA followed by Dunnett’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001. Additional data, small histograms, for LPS + glucose, LPS + glucose + sildenafil, LPS + glucose + vitamin D3, and LPS + glucose + sildenafil + vitamin D3. * P < 0.05