Mitchell P J, Hanson J C, Quets-Nguyen A T, Bergeron M, Smith R C
Biotechnology Discovery Research, Lilly Research Labs, DC 0344, Lilly Corporate Center, Indianapolis, IN 46285, United States.
J Neurosci Methods. 2007 Aug 30;164(2):350-62. doi: 10.1016/j.jneumeth.2007.04.021. Epub 2007 May 6.
The adult mammalian CNS is extremely limited in its ability to regenerate axons following injury. Glial scar, neuroinflammatory processes and molecules released from myelin impair axonal regrowth and contribute to the lack of neural regeneration. An in vitro assay that quantitates neurite outgrowth from cultured neurons as a model of neuronal regenerative potential is described. Specifically, the neurite outgrowth from primary neurons (rat cerebellar granule neurons; CGNs) and a neuronal cell line (NG108-15) were quantitatively measured after optimization of culture conditions. After cultures were fixed and immunostained to label neurons and nuclei, microscope images were captured and an image analysis algorithm was developed using Image-Pro Plus software to allow quantitative analysis. The algorithm allowed the determination of total neurite length, number of neurons, and number of neurons without neurites. The algorithm also allows for end-user control of thresholds for staining intensity and cell/nuclei size. This assay represents a useful tool for quantification of neurite outgrowth from a variety of neuronal sources with applications that include: (1) assessment of neurite outgrowth potential; (2) identification of molecules that can block or stimulate neurite outgrowth in conventional culture media; and (3) identification of agents that can overcome neurite outgrowth inhibition by inhibitory substrates.
成年哺乳动物的中枢神经系统在损伤后轴突再生能力极为有限。胶质瘢痕、神经炎症过程以及髓磷脂释放的分子会损害轴突再生，并导致神经再生的缺乏。本文描述了一种体外试验，该试验将培养神经元的神经突生长定量作为神经元再生潜力的模型。具体而言，在优化培养条件后，对原代神经元（大鼠小脑颗粒神经元；CGNs）和一种神经元细胞系（NG108-15）的神经突生长进行了定量测量。在培养物固定并进行免疫染色以标记神经元和细胞核后，拍摄显微镜图像，并使用Image-Pro Plus软件开发了一种图像分析算法以进行定量分析。该算法可确定总神经突长度、神经元数量以及无神经突的神经元数量。该算法还允许最终用户控制染色强度和细胞/细胞核大小的阈值。此试验是一种用于定量多种神经元来源神经突生长的有用工具，其应用包括：（1）评估神经突生长潜力；（2）鉴定在传统培养基中可阻断或刺激神经突生长的分子；（3）鉴定可克服抑制性底物对神经突生长抑制作用的药物。
