Core Unit Chip Application (CUCA), Institute of Human Genetics and Anthropology, University Hospital Jena, Jena, Germany.
J Neurosci Methods. 2009 Dec 15;185(1):23-8. doi: 10.1016/j.jneumeth.2009.09.003. Epub 2009 Sep 11.
Recent studies using molecular and genetic techniques just have started to elucidate the complex process that drives successful peripheral nerve regeneration. Introducing proteomics to this field, we unilaterally performed a facial nerve axotomy in 13 adult Wistar rats. Seven days later, a total of 40 20-microm coronary cryostat sections of the operated and contralateral unoperated nucleus facialis were microdissected. On the one hand, microdissected areas were pooled for each side, lysed and applied to ProteinChip Arrays. On the other hand, one microdissected area from the right and left facial nucleus each was directly placed on the affinity chromatographic array. Facial motoneurons were lysed in situ and released their proteins to spatially defined points. 215 laser addressable distinct positions across the surface of the spot enabled a high spatial resolution of measured protein profiles for the analysed tissue area. Protein profiles of the single positions were plotted over the used tissue section to visualize their distribution. The comparative analysis of the protein lysates from operated and normal nuclei facialis revealed, for both approaches used, differentially expressed proteins. Although by direct application of one cryostat section only a few hundred motoneurons were analysed, results comparable to these using lysates were obtained. Additionally, the applied technique revealed differences in the intensity distribution of several proteins of unknown function in the lesioned in comparison to the contralateral normal facial nucleus. This proteomic analysis with ultra high sensitivity paired with potential for a spatial resolution is a promising methodology for peripheral nerve regeneration studies.
最近使用分子和遗传技术的研究刚刚开始阐明驱动成功外周神经再生的复杂过程。我们将蛋白质组学引入该领域，在 13 只成年 Wistar 大鼠中进行了面神经轴突切断术。7 天后，对手术侧和对侧未手术的面神经核总共进行了 40 个 20 微米冠状冷冻切片的显微解剖。一方面，将每个侧的显微解剖区域混合，裂解并应用于 ProteinChip 阵列。另一方面，将右侧和左侧面神经核的一个显微解剖区域直接放置在亲和色谱阵列上。面神经运动神经元原位裂解，将其蛋白质释放到空间定义的点。在斑点表面跨越的 215 个激光可寻址的不同位置实现了对分析组织区域的高空间分辨率的测量蛋白质图谱。将单个位置的蛋白质图谱绘制在使用的组织切片上，以可视化其分布。对手术和正常面神经核的蛋白质裂解物进行的比较分析显示，两种方法均显示出差异表达的蛋白质。尽管直接应用一个冷冻切片仅分析了几百个运动神经元，但仍获得了与使用裂解物相当的结果。此外，所应用的技术还揭示了损伤侧与对侧正常面神经核相比，几种未知功能蛋白的强度分布存在差异。这种具有超高灵敏度并具有潜在空间分辨率的蛋白质组学分析是外周神经再生研究的一种很有前途的方法。
