Department of Anesthesiology and Critical Care Medicine, Hokkaido University Graduate School of Medicine, N15 W7, Sapporo 0608638, Japan.
J Anesth. 2010 Oct;24(5):726-32. doi: 10.1007/s00540-010-0999-x. Epub 2010 Aug 5.
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.
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.
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.
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.
亚低温和中度低温治疗对脑损伤的影响仍存在争议。我们的假设是，如果亚低温和中度低温真有保护作用，它们应该对神经元本身有一定的影响。我们通过使用近乎纯的神经元培养物，评估了低温对缺氧损伤的作用及其机制。
我们使用了来自 18 天大的 Wister 大鼠皮质的近乎纯神经元培养物。神经元在 3 种不同温度（30、33 和 37°C）下暴露于低于 1%的氧气中。首先，通过用台盼蓝评估存活神经元来测量细胞活力。其次，为了评估机制，在缺氧后通过高效液相色谱法测量细胞外谷氨酸浓度；还评估了暴露于外源性谷氨酸后细胞活力。接下来，通过监测 MitoCapture™的聚集来估计线粒体膜电位，并通过用 Hoechst 33342 和碘化丙啶染色来评估凋亡细胞的百分比。
在 24 小时缺氧损伤后，30 和 33°C 时的细胞活力明显高于 37°C。在 3 个温度组中，缺氧后细胞外谷氨酸浓度或谷氨酸暴露后细胞活力均无显著差异。在中度低温下，具有线粒体损伤的神经元数量和凋亡细胞的百分比显著减少。
亚低温和中度低温抑制了缺氧神经元的死亡。这种作用的机制可能与保护线粒体功能有关，至少在中度低温下可能会抑制细胞凋亡。
