Hypothermia and rewarming injury in hippocampal neurons involve intracellular Ca2+ and glutamate excitotoxicity

This study examines the causes of hypothermia and rewarming injury in CA1, CA3, and dentate neurons in rat hippocampal slice cultures. Neuronal death, assessed with propidium iodide or Sytox® fluorescence, Fluoro-Jade labeling, and Cresyl Violet staining, depended on the severity and duration of hypothermia. More than 6 h at temperatures less than 12 °C followed by rewarming to 37 °C (profound hypothermia and rewarming, PH/RW) caused swelling and death in large number of neurons in CA1, CA3, and dentate. During PH, [ATP] decreased and [Ca2+]i and extracellular [glutamate] increased, with neuron rupture and nuclear condensation following RW. The data support the hypothesis that neuronal death from PH/RW is excitotoxic, due to ATP loss, glutamate receptor activation and Ca2+ influx. We found that antagonism of N-methyl-D-aspartate (NMDA) receptors, but not 2-amino-3-(5-methyl-3-oxo-1,2- oxazol-4-yl) propanoic acid or metabotropic glutamate receptors, decreased neuron death and prevented increases in [Ca2+]I caused by PH/RW. Chelating extracellular Ca2+ decreased PH/RW injury, but inhibiting L- and T-type voltage-gated Ca2+ channels, K+ channels, Ca2+ release from the endoplasmic reticulum, and reverse Na+/Ca2+ exchange did not affect the Ca2+ changes or cell death. We conclude that the mechanism of PH/RW neuronal injury in hippocampal slices primarily involves intracellular Ca2+ accumulation mediated by NMDA receptors that activates necrotic, but not apoptotic processes.

▶Despite its therapeutic utility, hypothermia can be injurious to brain. ▶Profound hypothermia/rewarming injury was studied in hippocampal slice cultures. ▶The injury is accompanied by increased [Ca2+]i and extracellular [glutamate]. ▶Inhibitors of NMDA, but not AMPA or metabotropic receptors rescued the cultures. ▶NMDA receptor-mediated glutamatergic excitotoxicity causes cold injury.