Figure 4.

CDP-choline attenuates oxidative stress-induced postreperfusion apoptosis. Cardiac myocyte cultures were subjected to coverslip hypoxia/reperfusion. To induce oxidative stress, 5 mM H₂O₂ was added immediately after reperfusion (Postreperfusion oxidative stress). Preconditioning was performed by incubating the cells with CDP-choline (50 mg/mL) prior to reperfusion (Postreperfusion oxidative stress CDP-choline preconditioning). Cells were further incubated for 6 hrs. A group of untreated cells was included as control (negative control). To assess changes in cell nucleus morphology, the cells were fixed and stained with Hoechst 33342 fluorescent dye (upper panel). Changes in nuclear morphology associated with apoptosis, such as nuclear condensation and fragmentation, were detected by fluorescence microscopy (63X). Apoptosis and necrosis were evaluated by flow cytometry analysis of cells labeled with Annexin-V-FLUOS (Annexin V) and propidium iodide (PI) (lower panel). The proportion of labeled cells is depicted in the lower panel. The proportion of viable cells, with low Annexin and low PI staining, is shown in the left, lower quadrant of each dot plot. Apoptotic cells, with high Annexin and low PI staining, are depicted in the right, lower quadrant. Necrotic cells, with high Annexin and high PI staining, are showed in the right, upper quadrant. Results shown are representative figures from three independent assays.