Anesthetic protection of neurons injured by hypothermia and rewarming: roles of intracellular Ca2+ and excitotoxicity.

BACKGROUND: Mild hypothermia is neuroprotective after cerebral ischemia but surgery involving profound hypothermia (PH, temperature less than 18°C) is associated with neurologic complications. Rewarming (RW) from PH injures hippocampal neurons by glutamate excitotoxicity, N-methyl-D-aspartate receptors, and intracellular calcium. Because neurons are protected from hypoxia-ischemia by anesthetic agents that inhibit N-methyl-D-aspartic acid receptors, we tested whether anesthetics protect neurons from damage caused by PH/RW. METHODS: Organotypic cultures of rat hippocampus were used to model PH/RW injury, with hypothermia at 4°C followed by RW to 37°C and assessment of cell death 1 or 24 h later. Cell death and intracellular Ca were assessed with fluorescent dye imaging and histology. Anesthetic agents were present in the culture media during PH and RW or only RW. RESULTS: Injury to hippocampal CA1, CA3, and dentate neurons after PH and RW involved cell swelling, cell rupture, and adenosine triphosphate (ATP) loss; this injury was similar for 4 through 10 h of PH. Isoflurane (1% and 2%), sevoflurane (3%) and xenon (60%) reduced cell loss but propofol (3 µM) and pentobarbital (100 µM) did not. Isoflurane protection involved reduction in N-methyl-D-aspartate receptor-mediated Ca influx during RW but did not involve γ-amino butyric acid receptors or KATP channels. However, cell death increased over the next day. CONCLUSION: Anesthetic protection of neurons rewarmed from 4°C involves suppression of N-methyl-D-aspartate receptor-mediated Ca overload in neurons undergoing ATP loss and excitotoxicity. Unlike during hypoxia/ischemia, anesthetic agents acting predominantly on γ-aminobutyric acid receptors do not protect against PH/RW. The durability of anesthetic protection against cold injury may be limited.

Reduction in N-methyl-D-aspartate Receptor-mediated Cell Death in Hippocampal Neurons by Glucose Reduction Preconditioning.

BACKGROUND: Repeated episodes of reduced glucose availability can precondition the brain against damage caused by severe hypoglycemia. Because N-methyl-D-aspartate (NMDA) receptor activation may contribute to neuronal loss in the hippocampus following glucose deprivation, we tested the hypothesis that preconditioning with reduced glucose decreased NMDA receptor-mediated cell death in hippocampal neurons. METHODS: Hippocampal slice cultures from 7-day old rats were used to study glucose reduction preconditioning and N-methyl-D-aspartate receptor (NMDAR)-mediated cell death. Preconditioning involved reductions in glucose to the following levels: 0.1 mM, 0.5, or 1.0 mM for 30 minutes, 60 minutes, or 90 minutes on 3 consecutive days. Cell death following 1-hour total glucose deprivation was measured with a vital dye technique (SYTOX fluorescence). As an index of NMDAR activity, cell death following application of 1 mM NMDA, was also measured. RESULTS: A preconditioning protocol of 30 minutes of 0.1 mM glucose per day for 3 days reduced cell death following 1-hour total glucose by 65% to 70%, depending on cellular region. No reduction in NMDAR-mediated cell death was seen following any of the preconditioning treatments. However, when NMDAR-mediated cell death was assessed following preconditioning combined with subsequent total glucose deprivation, cell death was reduced in the cultures that had been preconditioned with 0.1 mM glucose for 30 minutes×3 days. CONCLUSIONS: We found that that glucose reduction preconditioning protects hippocampal neurons against severe glucose deprivation-induced neuronal damage. This preconditioning was not associated with reductions in NMDAR-mediated cell death except when the preconditioning was combined with an additional exposure to a period of total glucose deprivation.

Limitations of Mild, Moderate, and Profound Hypothermia in Protecting Developing Hippocampal Neurons After Simulated Ischemia.

Mild hypothermia (33°C-34°C) after cerebral ischemia in intact animals or ischemia-like conditions in vitro reduces neuron death. However, it is now clear that more profound hypothermia or delayed hypothermia may not provide significant protection. To further define the limitations of hypothermia after cerebral ischemia, we used hippocampal slice cultures to examine the effects of various degrees, durations, and delays of hypothermia on neuron death after an ischemia-like insult. Organotypic cultures of the hippocampus from 7- to 8 day-old rat pups were cooled to 32°C, 23°C, 17°C, or 4°C immediately or after a 2-4 hour delay from an injurious insult of oxygen and glucose deprivation (OGD). Cell death in CA1, CA3 and dentate regions of the cultures was assessed 24 hours later with SYTOX® or propidium iodide, both of which are fluorescent markers labeling damaged cells. OGD caused extensive cell death in CA1, CA3, and dentate regions of the hippocampal cultures. Hypothermia (32°C, 23°C and 17°C) for 4-6 hours immediately after OGD was protective at 24 hours, but when hypothermia was applied for longer periods or delayed after OGD, no protection or increased death was seen. Ultra-profound hypothermia (4°C) increased cell death in all cell areas of the hippocampus even when after a milder insult of only hypoxia. In an in vitro model of recovery after an ischemia-like insult, mild to profound hypothermia is protective only when applied without delay and for limited periods of time (6-8 hours). Longer durations of hypothermia, or delayed application of the hypothermia can increase neuron death. These findings may have implications for clinical uses of therapeutic hypothermia after hypoxic or ischemic insults, and suggest that further work is needed to elucidate the limitations of hypothermia as a protective treatment after ischemic stress.