跳跃探针式离子电导显微镜动态观察大鼠海马神经元坏死★◆

摘要

背景：目前关于神经元坏死的分子机制研究已取得一定进展，然而由于技术水平的限制，对神经元坏死过程细胞的动态变化研究较少。应用跳跃探针式离子电导显微镜可对体外培养神经元进行实时、非接触式的连续观察。　　目的：采用跳跃探针式离子电导显微镜动态观察大鼠海马神经元坏死过程中细胞表面形态随时间的动态变化。　　方法：体外原代培养大鼠海马神经元，用1 mmol/L过氧化氢溶液处理30 min致神经元坏死。采用跳跃探针式离子电导显微镜连续扫描，观察神经元坏死过程中其胞体与突起的形态变化，并测量其细胞高度与体积变化。　　结果与结论：跳跃探针式离子电导显微镜连续扫描9 h结果显示，过氧化氢组大鼠海马神经元由正常的三角形或梭形逐渐肿胀变成球形，轴突与树突出现串珠样改变，最终神经突断裂并溶解消失。同时测量细胞高度与体积后结果显示，过氧化氢组神经元细胞高度比及体积比于扫描后2-7 h之间持续增加(P<0.05)，扫描后7-9 h之间维持于相对稳定水平，最终高度与体积值约为初始值的2倍。线性回归分析结果示在扫描后1-7 h之间过氧化氢组神经元高度与体积增加量与时间呈线性相关(b=0.15，P <0.05；b=0.17，P<0.05)。结果证实，利用跳跃式离子电导显微镜技术观察大鼠海马神经元坏死，可以得到了高质量的细胞图像，更准确的细胞高度与体积的信息。%BACKGROUND:The molecular mechanism underlying neuronal cel death has been made some progress in the past years. However, the cel morphology dynamics after neuron death is stil missing because of technical shortcomings. Even the time required for the execution of the death program is stil unknown. However, neurons cultured in vitro can be observed continual y in a non-contact manner by a hopping probe ion conductance microscopy, therefore the morphology changes of a death process can be obtained. OBJECTIVE:To dynamical y observe morphological changes of rat hippocampus neurons during a cel necrotic process by the hopping probe ion conductance microscopy. METHODS:Primary Wistar rat hippocampal neurons were used for the experiment, and cel necrosis was induced by 1 mmol/L hydrogen peroxide solution for 30 minutes. Neurons without hydrogen peroxide-treatment were used as control. The morphology of neuronal soma and neurites were continual y imaged by the hopping probe ion conductance microscopy after hydrogen peroxide treatment, and the height and volume of a neuron were simultaneously measured. RESULTS AND CONCLUSION:Continuous imaging for 9 hours after hydrogen peroxide treatment under the hopping probe ion conductance microscopy showed that:Hippocampal neurons gradual y transformed from a normal triangular or fusiform shape to a more spherical shape due to cel swel ing. Axon beading and dendritic beading were gradual y formed after scanning, and final y the neurites were ruptured and dissolved. Measurement of the cel height and volume showed that:The height and volume of hydrogen peroxide-treated neurons elevated after 1 hour of scanning, and continued increasing up to 7 hours after scanning (P<0.05). The final height and volume were approximately twice as beginning. The increase of height and volume manifested as linear correlation with time within 1 to 7 hours by linear-regression analysis (cel height b=0.15, P<0.005;cel volume b=0.17, P<0.05). Meanwhile, no significant morphological change was observed during the time of scanning a normal neuron. It is successful to observe necrotic process of hippocampus neurons by the hopping probe ion conductance microscopy. Higher-quality cel image and more accurate information about cel height and volume can be obtained.