Magnesium and Cobalt, not Nimodipine, Protect Neurons Against Anoxic Damage in the Rat Hippocampal Slice

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Abstract
Brain tissue, maintained in vitro, was used to determine whether agents that block calcium entry into neurons can improve the recovery of evoked responses after anoxia. The hippocampus was dissected from a rat brain and sliced perpendicular to its long axis such that its main neuronal circuits remain functional. A pathway in the slice was stimulated electrically, and an extracellular potential, the evoked population spike, recorded from the neurons postsynaptic to that pathway. A bipolar stimulating electrode was placed in either the perforant path or the Schaeffer collaterals and a monopolar metal microelectrode placed, respectively in either the dentate granule cell layer or the CA1 pyramidal cell layer. The slices were maintained in vitro by superfusing them with oxygenated (95% O2, 5% CO2) artificial cerebrospinal fluid (aCSF). In order to generate anoxia, the tissue was superfused with aCSF bubbled with 95% N2, 5% CO2 for either 5 or 10 min. All drugs examined were present in the aCSF before, during, and immediately after the anoxic period. Percentage recovery was expressed as the amplitude of the evoked population spike 60 min after anoxia divided by its preanoxic amplitude. Protection in this model is defined as a significant (P < 0.05) improvement in percentage recovery compared with the recovery of untreated slices. There was no recovery of the response recorded from untreated dentate granule cells aftr 10 min of anoxia (0 .+-. 0%, n = 5; mean .+-. SE), whereas 5 min of anoxia was sufficient to cause damage to the untreated CA1 pyramidal cells (4 .+-. 3%, n = 6). When nimodipine (10-7 M) was present, there was no significant improvement in the recovery of the evoked population spike from either the dentate granule cells (11 .+-. 11%, n = 5) or the CA1 pyramidal cells (5 .+-. 5%, n = 5). Cobalt (2 mM), which had improved the recovery of dentate granule cells, protected the CA1 pyramidal cells from anoxic damage (64 .+-. 12%, n = 5). Magnesium (10 mM) significantly improved recovery of both the dentate granule cells (76 .+-. 5%, n = 5) and the CA1 pyramidal cells (35 .+-. 10%, n = 8) after anoxia in this in vitro model. ATP levels during anoxia were measured in order to determine how magnesium might protect against the anoxic damage. ATP was maintained at a significantly higher level during anoxia when 10 mM magnesium was present in the bathing medium (1.7 .+-. 0.2 vs. 1.1 .+-. 0.15 nM/mg dry weight). Nimodipine did not maintain ATP levels during anoxia. The authors conclude that magnesium and cobalt, but not nimodipine, protect against anoxic damage to the hippocampus in this in vitro model. Any potential clinical benefit of magnesium (cobalt is highly toxic) would have to be tested in an in vivo model, and serious problems such as the limited permeability of the blood-brain barrier to magnesium would have to be overcome. Their results support the importance of calcium influx as one trigger for anoxic damage.