Viel J J, McManus D Q, Cady C, Evans M S, Brewer G J
Department of Medical Microbiology and Immunology, Southern Illinois University School of Medicine, Springfield, Illinois 62794-9626, USA.
J Neurosci Res. 2001 May 15;64(4):311-21. doi: 10.1002/jnr.1081.
For a model of neurological disease and ischemia, we extended recent work to culture adult postmortem rat brain neurons. Frontal cortex sections were removed from adult rats immediately following sacrifice and at different postmortem intervals and with the brain at either 22 degrees C or 4 degrees C. Brain could be stored four times longer at 4 degrees C between sacrifice and neuronal disaggregation to achieve the same 20% recovery of live cells from those plated compared to 22 degrees C. Each milligram of rat frontal cortex was estimated by the optical disector method to contain 160,000 neurons. When cells were isolated as rapidly as possible, 9% of the neurons originally present in the brain were viable. Various postmortem intervals from 2 to 24 hr resulted in a reduction from 6% to 3% of the cells originally present. After 5 days in culture, viable neurons were 23-42% of those isolated. Neuron-like cells that survived represented 40-75% of the viable cells, or 0.5-2.75% of those originally estimated to be present in the brain. Electrophysiology experiments show that cells isolated 0 and 24 hr postmortem had neuronal electrical properties, including an average resting membrane potential of -48 mV, voltage-sensitive currents, and action potentials. Neuron-like cells were immunoreactive for neuron-specific enolase, neurofilament 200, glutamate, MAP2, and tau after 2 weeks in culture. These experiments show that neuron-like cells can be reliably cultured from adult rat cortex up to 6 hr postmortem when stored at 22 degrees C and up to 24 hr postmortem when stored at 4 degrees C. These findings should encourage donation of human postmortem brain neurons for studies on ischemia, adult pharmacology, and neurological disease.
对于神经疾病和缺血模型，我们拓展了近期的工作，培养成年大鼠死后的脑神经元。在成年大鼠处死后，立即从其大脑中取出额叶皮质切片，并在不同的死后间隔时间，且大脑分别处于22℃或4℃的条件下进行处理。与22℃相比，在处死后至神经元解离之间，大脑在4℃下可保存的时间延长四倍，以实现与接种的活细胞相同的20%回收率。通过光学分割法估计，每毫克大鼠额叶皮质含有160,000个神经元。当尽可能迅速地分离细胞时，大脑中最初存在的神经元中有9%是存活的。2至24小时的各种死后间隔导致最初存在的细胞从6%减少到3%。培养5天后，存活的神经元占分离出的神经元的23 - 42%。存活的神经元样细胞占活细胞的40 - 75%，即最初估计存在于大脑中的细胞的0.5 - 2.75%。电生理学实验表明，死后0小时和24小时分离的细胞具有神经元电特性，包括平均静息膜电位为 -48 mV、电压敏感电流和动作电位。培养2周后，神经元样细胞对神经元特异性烯醇化酶、神经丝200、谷氨酸、微管相关蛋白2和tau具有免疫反应性。这些实验表明，当在22℃下保存时，可从成年大鼠皮质可靠地培养死后长达6小时的神经元样细胞；当在4℃下保存时，可培养死后长达24小时的神经元样细胞。这些发现应会鼓励捐赠人类死后的脑神经元用于缺血、成人药理学和神经疾病的研究。
