The effect of three metabolic inhibitors—iodoacetate, potassium cyanide, and potassium arsenate—on neuronal viability was studied in primary rat cortical and hippocampal CA1 neuronal cultures. Iodoacetate (0.1 mM) applied for 5 min to 8‐day‐old cultures resulted in delayed neuronal death within 3–24 h in cortical and hippocampal CA1 neurons. Neuronal degeneration was preceded by transient inhibition of energy metabolism to ∼40% and a permanent inhibition of protein synthesis to ∼50%. The inhibition of protein synthesis and the neuronal death were prevented by the free radical scavenger vitamin E but not by the glutamate antagonist MK‐801. Removal of calcium during iodoacetate exposure could not protect against toxicity, and there was no increase of intracellular calcium concentration during and shortly after iodoacetate treatment. Cyanide and arsenate produced only partial neuronal degeneration, even at a dose of 10 mM. These observations demonstrate that brief exposure of neurons to low concentrations of iodoacetate produces a delayed type of neuronal death that is not mediated by either calcium or glutamate. The therapeutic effect of vitamin E points to a free‐radical mediated injury and suggests that this type of pathology may also be involved in delayed neuronal death after transient energy depletion in vivo.

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Neurons from cerebral cortex and hippocampal CA1 sector exhibit a striking difference in vulnerability to transient ischemia. To establish whether this difference is due to the inherent (pathoclitic) properties of these neurons, the ischemic susceptibility was studied in primary cortical and hippocampal cultures by using a new model of argon‐induced in vitro ischemia. Neuronal cultures were exposed at 37°C for 10–30 min to argon‐equilibrated glucose‐free medium. During argon equilibration, Po2 declined to <2.5 torr within 1 min and stabilized shortly later at ∼1.3 torr. After 30 min of in vitro ischemia, total adenylate was <45% and ATP content <15% of control in both types of culture. Cytosolic calcium activity increased from 15 to 50 nM. Reoxygenation of cultures after in vitro ischemia led to delayed neuronal death, the severity of which depended on the duration of in vitro ischemia but not on the type of neuronal cultures. Energy charge of adenylate transiently returned to ∼90% of control after 3 h, but ATP content recovered only to 40% and protein synthesis to <35%. Cytosolic calcium activity continued to rise after ischemia and reached values of ∼500 nM after 3 h. The new argon‐induced in vitro ischemia model offers major advantages over previous methods, but despite this improvement it was not possible to replicate the differences in cortical and hippocampal vulnerability observed in vivo. Our study does not support the hypothesis that selective vulnerability is due to an inherent pathoclitic hypersensitivity.

The effect of trophic factors on neuronal survival after 30 min oxygen and glucose deprivation (in vitro ischemia) was studied in primary hippocampal and cortical neuronal cultures of rat. In vitro ischemia was produced at 37 degrees C by placing cultures in glucose-free medium, the oxygen content of which was removed by gassing with pure argon. After in vitro ischemia neurons were allowed to recover either in serum-free minimal essential medium (MEM) or in MEM containing 5% native horse serum, 100 ng/ml basic fibroblast growth factor (bFGF) or 10 ng/ml transforming growth factor-beta 1 (TGF-beta 1), respectively. Cultures that recovered in serum-free medium suffered a progressive type of neuronal injury: survival of either cortical or hippocampal neurons declined from about 60% after 1 h to 50% after 3 h, 40% after 6 h and less than 20% after 24 h. Addition of serum proteins to the incubation medium did not influence early survival (up to 3-6 h) but significantly improved survival after 24 h (more than 40% in both hippocampal and cortical cultures). Addition of TGF-beta 1 and bFGF had only minor effects. These data show that serum reduces delayed ischemic cell death by a mechanism which is different from that of TGF-beta 1 or bFGF protection.

Primary cortical and hippocampal neuronal cultures submitted to brief histotoxic hypoxia suffer delayed neuronal death after 24 h [Uto et al. (1995) J Neurochem 64: 2185-2192]. In this study the ultrastructural changes were monitored during the first 6 h following 5-min histotoxic hypoxia induced by exposure to 100 microM iodoacetate. In both cortical and hippocampal CA1 neurons, disaggregation of ribosomes was the earliest sign of histotoxic pathology. Vacuolizations of mitochondria, endoplasmic reticulum and Golgi apparatus, as well as fragmentation and disintegration of neurofilaments followed later. Signs of apoptotic nuclear degeneration were absent. Our observations demonstrate that, similar to that seen in ischemia, disaggregation of ribosomes after brief histotoxic hypoxia is one of the first pathological alterations heralding delayed neuronal death.