Effect of mild and moderate hypothermia on hypoxic injury in nearly pure neuronal culture.
OBJECTIVE
The effects of mild and moderate hypothermic therapy on cerebral injury are still controversial. Our hypothesis is that mild and moderate hypothermia should have some effects on neurons themselves if they really have protective effects. By using a nearly pure neuronal culture, we evaluated the effects and mechanism of hypothermia against hypoxic insult.


METHODS
A nearly pure neuronal culture from cortices of 18-day-old Wister rats was used. The neurons were exposed to below 1% oxygen at 3 different temperatures (30, 33 and 37°C). First, cell viability was measured by assessing viable neurons with trypan blue. Second, to evaluate the mechanism, the extracellular glutamate concentration was measured by high-performance liquid chromatography after hypoxia; cell viability after exposure to extrinsic glutamate was also evaluated. Next, mitochondrial membrane potential was estimated, by monitoring aggregation of MitoCapture™, and the percentage of apoptotic cells was evaluated by staining with Hoechst 33342 and propidium iodide.


RESULTS
After 24-h hypoxic insult, cell viability at 30 and 33°C was significantly higher than at 37°C. There was no significant difference between extracellular concentrations of glutamate after hypoxia or cell viability after glutamate exposure among the 3 temperature groups. In moderate hypothermia, the number of neurons with mitochondrial injury and the percentage of apoptotic cells were significantly reduced.


CONCLUSIONS
Mild and moderate hypothermia inhibited hypoxic neuronal cell death. The mechanism of this effect may be related to protection of mitochondrial function, presumably followed by inhibition of apoptosis, at least in moderate hypothermia.
Hua
Yu
Y
Department of Anesthesiology and Critical Care Medicine, Hokkaido University Graduate School of Medicine, N15 W7, Sapporo 0608638, Japan.
Hisano
Kenjiro
K
Morimoto
Yuji
Y
D000818:Animals; D017209:Apoptosis / Q000502:physiology; D002118:Calcium / Q000378:metabolism; D015687:Cell Hypoxia / Q000502:physiology*; D002470:Cell Survival / Q000502:physiology; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D004563:Electrochemistry; D005260:Female; D018698:Glutamic Acid / Q000378:metabolism; D007036:Hypothermia, Induced; D008928:Mitochondria / Q000502:physiology; D009336:Necrosis; D009474:Neurons / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D011247:Pregnancy; D051381:Rats; D017208:Rats, Wistar
D018698:Glutamic Acid; D002118:Calcium