Kharlamov E, Cagnoli C M, Atabay C, Ikonomović S, Grayson D R, Manev H
Allegheny-Singer Research Institute, Medical College of Pennsylvania, Pittsburgh, USA.
J Neurochem. 1995 Sep;65(3):1395-8. doi: 10.1046/j.1471-4159.1995.65031395.x.
Typically, primary cultures of rat cerebellar granule neurons are grown in the presence of 25 mM KCl and are considered to mature by approximately 7 days in vitro. Potassium deficiency was created by growing the neurons from days 1 to 4 in the presence of 12.5 mM KCl (immature cultures) or by switching the mature neurons grown with 25 mM KCl to 12.5 mM KCl. In both conditions we observed neuronal death that bears the signs of apoptosis, i.e., DNA fragmentation determined qualitatively by agarose gel electrophoresis of DNA and quantitatively by in situ terminal deoxynucleotidyl transferase assay. The protein synthesis inhibitors cycloheximide and anisomycin provided neuroprotection in the mature cultures but potentiated the toxic effect of KCl deprivation in the immature neurons. The results suggest that a prudent use of protein synthesis inhibitors is critical in experiments with primary neuronal cultures.
通常，大鼠小脑颗粒神经元的原代培养物在含有25 mM氯化钾的条件下生长，并被认为在体外培养约7天时成熟。通过在第1天至第4天期间将神经元培养于含有12.5 mM氯化钾的环境中（未成熟培养物），或通过将在25 mM氯化钾中生长的成熟神经元转换至12.5 mM氯化钾中，来制造钾缺乏状态。在这两种情况下，我们都观察到神经元死亡，并伴有凋亡迹象，即通过DNA琼脂糖凝胶电泳定性测定以及原位末端脱氧核苷酸转移酶测定法定量测定的DNA片段化。蛋白质合成抑制剂环己酰亚胺和茴香霉素在成熟培养物中提供神经保护作用，但在未成熟神经元中增强了氯化钾剥夺的毒性作用。结果表明，在原代神经元培养实验中谨慎使用蛋白质合成抑制剂至关重要。
