Lewis Gwendolyn M, Kucenas Sarah
Department of Biology, University of Virginia, VA, USA.
Department of Biology, University of Virginia, VA, USA.;
J Vis Exp. 2013 Jun 20(76):50621. doi: 10.3791/50621.
The nervous system is often described as a hard-wired component of the body even though it is a considerably fluid organ system that reacts to external stimuli in a consistent, stereotyped manner, while maintaining incredible flexibility and plasticity. Unlike the central nervous system (CNS), the peripheral nervous system (PNS) is capable of significant repair, but we have only just begun to understand the cellular and molecular mechanisms that govern this phenomenon. Using zebrafish as a model system, we have the unprecedented opportunity to couple regenerative studies with in vivo imaging and genetic manipulation. Peripheral nerves are composed of axons surrounded by layers of glia and connective tissue. Axons are ensheathed by myelinating or non-myelinating Schwann cells, which are in turn wrapped into a fascicle by a cellular sheath called the perineurium. Following an injury, adult peripheral nerves have the remarkable capacity to remove damaged axonal debris and re-innervate targets. To investigate the roles of all peripheral glia in PNS regeneration, we describe here an axon transection assay that uses a commercially available nitrogen-pumped dye laser to axotomize motor nerves in live transgenic zebrafish. We further describe the methods to couple these experiments to time-lapse imaging of injured and control nerves. This experimental paradigm can be used to not only assess the role that glia play in nerve regeneration, but can also be the platform for elucidating the molecular mechanisms that govern nervous system repair.
神经系统常被描述为身体的一个硬连接组件，尽管它是一个相当灵活的器官系统，能以一致、刻板的方式对外界刺激做出反应，同时保持令人难以置信的灵活性和可塑性。与中枢神经系统（CNS）不同，外周神经系统（PNS）具有显著的修复能力，但我们才刚刚开始了解控制这一现象的细胞和分子机制。以斑马鱼作为模型系统，我们有前所未有的机会将再生研究与体内成像及基因操作相结合。外周神经由被胶质细胞层和结缔组织包围的轴突组成。轴突被有髓鞘或无髓鞘的施万细胞包裹，这些施万细胞又被称为神经束膜的细胞鞘包裹成束。受伤后，成年外周神经具有清除受损轴突碎片并重新支配靶标的显著能力。为了研究所有外周胶质细胞在PNS再生中的作用，我们在此描述一种轴突横断试验，该试验使用市售的氮泵染料激光对活的转基因斑马鱼的运动神经进行轴突切断。我们还进一步描述了将这些实验与受伤神经和对照神经的延时成像相结合的方法。这种实验范式不仅可用于评估胶质细胞在神经再生中的作用，还可作为阐明控制神经系统修复的分子机制的平台。
