Fig. 7. Neuroprotection of pyruvate and succinate against apoptosis. Serum withdrawal from the culture medium resulted in 50-60% cell death in 30 hours, measured by LDH release and Trypan Blue extrusion. (A) Bright phase photos of Trypan-Blue-treated pure-neuronal cultures before and after serum deprivation. Normal cells do not show positive staining with Trypan Blue (dark color); after a 30-hour incubation in the serum-free medium many Trypan-Blue-positive cells represent the injured or dead cells lack of ability to extrude the dye from the intracellular space. Addition of 5 mM pyruvate in the serum-free medium attenuated Trypan Blue staining and cell death. Bar, 50 µm. (B) Pyruvate attenuated serum deprivation-induced cell death in a concentration-dependent manner; the neuroprotection was mostly reversed by co-applied 4-CIN (2 mM). Neuroprotective effects were achieved even when pyruvate was given up to 4 hours after the onset of serum deprivation. (C) Succinate at 5 mM showed little neuroprotective effect, increasing its concentration to 20 mM reduced serum deprivation-induced apoptosis, consistent with its effect on I_pump and ROS production at this concentration. The succinate downstream metabolite oxaloacetate (5 mM) also showed comparable neuroprotection against serum deprivation-induced apoptosis. (D) Co-applied pyruvate (5 mM) showed no attenuating effect on the neuronal death induced by C₂-ceramide (25 µM, 48-hour exposure). In fact, pyruvate appeared to increase the C₂-ceramide toxicity, which is consistent with a lower pump current in the presence of pyruvate and C₂-ceramide (see Fig. 2C). n=8-31 cultures. *P<0.05 compared with serum deprivation alone; #P<0.05 compared with serum deprivation plus pyruvate.