Removal of extracellular sodium prevents anoxia-induced injury in freshly dissociated rat CA1 hippocampal neurons

Anoxia is believed to cause nerve injury and death in part, by inducing sustained, elevated levels of intracellular Ca²⁺. The increased concentration of intracellular Ca²⁺ is capable, by itself, of inducing nerve injury and death, even without the added stress of anoxia. However, we have recently shown that an increased level of intracellular Ca²⁺ is not necessary for anoxia-induce CA1 nerve injury. Since we have observed that extracellular Na⁺ decreases during anoxia, we studied the role of extracellular Na⁺ in anoxia-induced nerve injury. Removal of extracellular Na⁺ and its replacement with the impermeant cation N-methyl-d-glucamine (NMDG⁺) completely protected freshly dissociated CA1 neurons during and after severe anoxia, for up to 90 min. Intracellular Ca²⁺ decreased during anoxia, recovering during reoxygenation. Propidium iodide was excluded from the neurons for as long as Na⁺ was absent. Addition of Na⁺ (by replacing NMDG⁺) following anoxia resulted in rapid bleb formation, swelling and intracellular Ca²⁺ rise. Removal of Na⁺ before the rupture of blebs caused either shrinkage or pinching off of blebs so that the neuron apparently returned to its previous undisturbed state. We conclude that: (1) replacement of Na_o⁺ with NMDG⁺ totally prevents anoxia-induced nerve injury as manifested by morphological changes, e.g., swelling, bleb formation and membrane injury; (2) upon re-exposure to Na_o⁺ following anoxia in the absence of Na_o⁺, neurons swell and bleb; (3) the appearance of blebs following anoxic exposure in the absence of Na_o⁺ is reversible and Na_o⁺ dependent. (4) Bleb formation itself is not necessarily immediately lethal to the neuron.