CXCL12对原代培养大鼠交感神经元新生轴突形态的影响

CXCL12 AFFECTS THE AXON MORPHOGENESIS OF PRIMARY CULTURED RAT SYMPATHETIC NEURONS

目的:观察CXCL12与其受体CXCR4对原代培养的大鼠交感神经元轴突生长的影响,并探讨其与神经病理性疼痛交感神经轴突可塑性的相关性及可能机制。方法:极低密度原代培养SD大鼠交感神经元,免疫荧光染色法检测CXCR4受体与酪氨酸羟化酶(tyrosine hydroxylase,TH)表达情况,应用激光共聚焦显微镜和荧光显微镜进行共定位或计数。采用200 ng/ml浓度CXCL12干预极低密度培养的交感神经元,观察对其轴突生长的影响,对照组加入等量PBS,两组培养12小时后对全部神经元进行活细胞荧光染色,并对神经元逐一拍照处理,测量每细胞的轴突总长度和轴突分支点数量,并对神经元进行同心圆法(Sholl Analysis)分析。结果:CXCR4主要定位于原代培养的交感神经元胞体部位,少量表达于轴突与生长锥,细胞计数共定位率为99.7%。使用外源性CXCL12干预会使培养的交感神经元的轴突复杂度增加(P<0.001)和分支点数量增加(P<0.05),但对轴突总长度无明显影响。只对靠近胞体的第一级轴突进行Sholl分析,第一级轴突复杂度较对照组明显下降(P<0.0001)。结论:CXCL12干预可以增加原代培养大鼠交感神经元的轴突复杂度。

Objective： The roles of CXCL12 and its major receptor CXCR4 in the axon morphogenesis of primary cultured sympathetic neurons were explored in our present study, we aim to discuss the relationship between the plasticity of sympathetic axon terminals and the development and maintenance of chronic neuropathic pain. Methods： For immunocytochemistry and co-localization of tyrosine hydroxylase（TH） and CXCR4, primary sympathetic neurons were cultured at a very low density. The images were acquired by confocal or fluorescence microscope. Sympathetic neurons were cultured at a very low density for axonal morphological analysis. Neurons treated with CXCL12 at 200 ng/ml or the vehicle（PBS as control group）, were fluorescently labeled with vital dye. All neuronal images were acquired and analyzed to obtain total neurite length, number of branch point and profiles of Sholl analysis. Results： CXCR4 was co-expressed with 99.7% of TH positive neurons. Axons and growth cones exhibited lower fluorescent intensity than the somas. Neurons treated with CXCL12 showed more complex axonal Sholl morphology（P〈0.001） and more branch points（P〈0.05）, but no significant changes of total cable length of axons（P〉0.05）. However, when the root（first process） of the exposed axons was solely analyzed, the neurons of CXCL12 treated group showed less complexity by Sholl plot compared with the control group（P〈0.0001）. Conclusion： Primary cultured rat sympathetic neurons treated with CXCL12 show more complex axon morphogenesis.