Importance of Sodium Homeostasis during Hypoxia-Induced Apoptosis

Abstract 2000 Perinatal Brain Injury: Patterns and Mechanisms Platform, Tuesday, 5/4

Previous observations from our laboratory have shown that, under hypoxic conditions, an increase in intracellular sodium (Na_i⁺) is critical in inducing neuronal death. However, the nature of cell death associated with increased Na_i⁺ during hypoxia is unknown. We hypothesized that an increase in Na_i⁺ is a critical event in hypoxia-induced neuronal apoptosis. Methods. Cultured neocortical neurons (age 7-14 days) were exposed to an environment of 1% O₂ or maintained in 21% O₂ for 72 hours. Since voltage-gated Na⁺ channels and non-NMDA receptors are major routes of sodium entry in neurons, we chose to treat neurons in both the hypoxic and control groups with tetrodotoxin (TTX; 1 µM) and NBQX (10 µM) at the time of hypoxic exposure to block Na⁺ entry through their respective routes. Untreated neurons maintained in 21% O₂ or exposed to 1% O₂ served as controls. The presence of apoptosis was determined by: change in cell morphology, in situ DNA fragmentation (TUNEL method), annexin V staining, and electron microscopy. The proportion of apoptotic cells was assessed by annexin V staining and neuronal viability was assessed by exclusion of propidium iodide. Results. After 72 hours of exposure to 1% O₂, untreated neurons showed evidence of apoptosis. Cell death in untreated neurons exposed to hypoxia was ∼2 times that of untreated neurons maintained in 21% O₂ with 75% of cell death being attributable to apoptosis. Treatment of neurons with TTX and NBQX at the time of hypoxic exposure reduced the proportion of apoptotic neurons by ∼40% as compared to untreated neurons (p< 0.05). There was no noticeable effect of TTX and NBQX on neuronal survival or apoptosis when compared to untreated neurons at 21% O₂. Conclusions. Blocking Na⁺ entry during hypoxia attenuates neuronal death by apoptotic mechanisms. We speculate that these results provide a link between changes in Na⁺ homeostasis and gene activation that occurs during apoptotic cell death.

The work described above is funded in part by the following grants: UCP, Gaylord Rehabilitation Research Institute, and YCHRC