Apoptotic mechanisms triggered by in vitro ischemia

Cultured mouse cortical neurons undergo apoptosis when exposed to staurosporine. The cell-permeable caspase inhibitor Z-VAD.FMK attenuated this death as well as apoptosis induced by serum deprivation. However, Z-VAD.FMK did not attenuate the excitotoxic necrosis induced by NMDA, kainate, or 90 min exposure to oxygen-glucose deprivation (OGD). We have previously shown that blockade of the excitotoxic component of oxygen-glucose deprivation-induced neuronal death with glutamate antagonists unmasks an apoptosis. Treatment with Z-VAD.FMK attenuated this OGD-induced neuronal apoptosis. These data support the idea that brain caspases mediate the apoptotic component of OGD-induced neuronal death.;The BCL-2 family member BAX, promotes apoptosis. Cultured neurons from Bax-deficient mice exhibit reduced vulnerability to staurosporine-induced or serum deprivation-induced apoptosis, but little or no change in vulnerability to excitotoxic necrosis induced by exposure to excitotoxins or OGD. However, if the excitotoxic component of OGD-induced death was blocked by the addition of glutamate antagonists then wild type but not Bax-deficient neurons underwent apoptosis. Consistent with the idea that Bax-deficiency attenuates ischemic neuronal apoptosis, mice deficient in Bax also had smaller infarcts following transient middle cerebral artery occlusion than wild type littermates. These data implicate BAX in the pathogenesis of ischemic central neuronal apoptosis.;In cultured cortical neurons [Ca2+]i decreased during staurosporine induced-apoptosis. The voltage gated Ca2+ channel agonist (--)Bay K 8466 (BayK)restored [Ca2+] i and attenuated cell death in a dose dependent manner. Similarly, kainate restored [Ca2+]i and attenuated cell death. It has previously been shown that OGD triggers a rise in [Ca2+] i and excitotoxic necrosis. Glutamate antagonists present during the deprivation prevent neuronal excitotoxic necrosis; however following long periods of deprivation neurons will undergo apoptosis. Concomitant with this apoptosis we found a decrease in [Ca2+]i. Adding BayK to the medium following OGD termination ameliorated both the decrease in [Ca2+]i and neuronal death. Low concentrations of kainate (1--10 m M) also attenuated this cell death. Interestingly, the ability of kainate to rescue neurons from this death depended on when it was added to the medium, with three hours after OGD termination being the most efficacious. These data suggest Ca2+ may play a more complicated role in ischemia than previously thought.