Kretz Alexandra, Marticke Julia K, Happold Caroline J, Schmeer Christian, Isenmann Stefan
Neuroregeneration Laboratory, Department of Neurology, University of Jena Medical School, Erlanger Allee 101, D-07747 Jena, Germany.
Nat Protoc. 2007;2(1):131-40. doi: 10.1038/nprot.2007.12.
This protocol details a tissue culture technique that allows for quantified regeneration studies on adult retinal ganglion cells (RGCs), that is, CNS neurons. The method may also allow for elucidation of molecular cues, for example of signals relevant in neuronal survival and axon regeneration. The procedure relies on fractioned stripe culture of previously injured retina in defined culture media. Naive dendritic cell contacts of RGCs are preserved, and the system is independent of growth factors. In contrast to other techniques, the protocol is based on tissue grown from adult animals; it dispenses immature co-cultures and evaluates the outgrowth of unmyelinated neurites in a milieu lacking CNS myelin. The technique is suitable for rodent retina from mouse or rat. A growth-conditioning injury of the optic nerve is set 10 days before retinal explantation. Explants are cultured for 5-7 days. Mere preparation of a single retina should be completed within 20 min.
本方案详细介绍了一种组织培养技术，该技术可用于对成年视网膜神经节细胞（RGCs），即中枢神经系统神经元进行定量再生研究。该方法还可能有助于阐明分子线索，例如与神经元存活和轴突再生相关的信号。该程序依赖于在特定培养基中对先前受损视网膜进行分段条纹培养。RGCs的天然树突状细胞接触得以保留，并且该系统不依赖生长因子。与其他技术相比，该方案基于成年动物的组织生长；它省去了不成熟的共培养，并在缺乏中枢神经系统髓磷脂的环境中评估无髓神经突的生长。该技术适用于小鼠或大鼠的啮齿动物视网膜。在视网膜外植前10天对视神经进行生长调节损伤。外植体培养5 - 7天。仅制备单个视网膜应在20分钟内完成。
