Figure 1

Effect of citicoline, hypoxia, and the Ca ionophore A23187 on cell death in hCMEC/D3. A) Shows control hCMEC/D3 without citicoline or staurosporin treatment (i), cells treated with staurosporin (ii; 10 μM), and cells pre-incubated with citicoline for 4 h before apoptosis was induced with 10 μM staurosporin (24 h; iii). The presence of citicoline (10 μM) was sufficient to significantly inhibit cell apoptosis (iii) B) Shows control hCMEC/D3 without citicoline or hypoxia treatment (i), cells treated after exposure to hypoxia (ii; 12 h; 1% O₂) and cells pre-incubated with citicoline for 4 h prior to subjecting to hypoxia (iii). The data shows that citicoline (10 μM) was able to significantly reduce the number of cells undergoing apoptosis (iii). C) Shows that citicoline protected the cells against apoptosis induced by calcium ionophore. (i) Shows control cells, (ii) the effect of 10 μM/24 h treatment with Ca ionophore, and (iii), the effect of 4 h pre-treatment with citicoline before addition of 10 μM Ca ionophore. Citicoline significantly protected against apoptosis induced by the ionophore. The bar graphs shows data from one representative experiment carried out in triplicate wells. All experiments were performed three times. Cells were considered apoptotic when cell nuclei demonstrated positive PI/Hoechst staining and apoptotic morphology (Data not shown). For quantification of PI/Hoechst -positive cells, four fields per section were examined at 200-fold magnification. The apoptotic index was calculated using the formula: Apoptotic index = 100 * (number of PI/Hoescht + cell nuclei per field/total number of cell nuclei per field).