Effects of ion channel blockade on the distribution of Na, K, Ca and other elements in oxygen-glucose deprived CA1 hippocampal neurons

The pathophysiology of brain ischemia and reperfusion injury involves perturbation of intraneuronal ion homeostasis. To identify relevant routes of ion flux, rat hippocampal slices were perfused with selective voltage- or ligand-gated ion channel blockers during experimental oxygen-glucose deprivation and subsequent reperfusion. Electron probe X-ray microanalysis was used to quantitate water content and concentrations of Na, K, Ca and other elements in morphological compartments (cytoplasm, mitochondria and nuclei) of individual CA1 pyramidal cell bodies. Blockade of voltage-gated channel-mediated Na⁺ entry with tetrodotoxin (1 μM) or lidocaine (200 μM) significantly reduced excess intraneuronal Na and Ca accumulation in all compartments and decreased respective K loss. Voltage-gated Ca²⁺ channel blockade with the l-type antagonist nitrendipine (10 μM) decreased Ca entry and modestly preserved CA1 cell elemental composition and water content. However, a lower concentration of nitrendipine (1 μM) and the N-, P-subtype Ca²⁺ channel blocker ω-conotoxin MVIIC (3 μM) were ineffective. Glutamate receptor blockade with the N-methyl-d-aspartate (NMDA) receptor-subtype antagonist 3-(2-carboxypiperazin-4-yl) propyl-1-phosphonic acid (CPP; 100 μM) or the alpha-amino-3-hydroxy-5-methyl-4-isoazole propionic acid (AMPA) receptor subtype blocker 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 μM/100 μM glycine) completely prevented Na and Ca accumulation and partially preserved intraneuronal K concentrations. Finally, the increase in neuronal water content normally associated with oxygen-glucose deprivation/reperfusion was prevented by Na⁺ channel or glutamate receptor blockade. Results of the present study demonstrate that antagonism of either postsynaptic NMDA or AMPA glutaminergic receptor subtypes provided nearly complete protection against ion and water deregulation in nerve cells subjected to experimental ischemia followed by reperfusion. This suggests activation of ionophoric glutaminergic receptors is involved in loss of neuronal osmoregulation and ion homeostasis. Na⁺ channel blockade also effectively diminished neuronal ion and water derangement during oxygen-glucose deprivation and reperfusion. Prevention of elevated Na_i⁺ levels is likely to provide neuroprotection by decreasing presynaptic glutamate release and by improving cellular osmoregulation, adenosine triphosphate utilization and Ca²⁺ clearance. Thus, we suggest that voltage-gated tetrodotoxin-sensitive Na⁺ channels and glutamate-gated ionotropic NMDA or AMPA receptors are important routes of ion flux during nerve cell injury induced by oxygen-glucose deprivation/reperfusion.