Lariosa-Willingham Karen, Leonoudakis Dmitri
Teva Pharmaceuticals Biologics Discovery, Redwood City, CA, USA.
Methods Mol Biol. 2018;1791:179-192. doi: 10.1007/978-1-4939-7862-5_14.
The formation of new myelin in persistent multiple sclerosis (MS) lesions is compromised, leading to a reduction in neuron function and subject to degeneration and death. Current MS therapies can control autoimmune-mediated demyelination, but none directly promote the regeneration of myelin in the central nervous system (CNS). To identify new drugs that stimulate remyelination, we established a high-throughput cell-based assay to identify compounds that promote myelination. Methods were developed for initiating myelination in vitro using a preparation of primary embryonic rat cortical cells. We developed an immunofluorescent phenotypic image analysis method to quantify the morphological alignment of myelin characteristic of the initiation of myelination. The assay scalability and consistency was validated by screening the NIH clinical collection library of 727 compounds and identified ten compounds that promote myelination (Lariosa-Willingham et al., BMC Neurosci 17:16, 2016). Here, we present the detailed methods for a high capacity in vitro assay that assesses myelination of live axons.
在持续性多发性硬化症（MS）病灶中，新髓鞘的形成受到损害，导致神经元功能下降，并易发生退化和死亡。目前的MS疗法可以控制自身免疫介导的脱髓鞘，但没有一种疗法能直接促进中枢神经系统（CNS）中髓鞘的再生。为了鉴定刺激髓鞘再生的新药，我们建立了一种基于细胞的高通量检测方法，以鉴定促进髓鞘形成的化合物。我们开发了利用原代胚胎大鼠皮质细胞制剂在体外启动髓鞘形成的方法。我们开发了一种免疫荧光表型图像分析方法，以量化髓鞘形成起始时髓鞘特征的形态排列。通过筛选美国国立卫生研究院（NIH）的727种化合物临床收藏库，验证了该检测方法的可扩展性和一致性，并鉴定出10种促进髓鞘形成的化合物（拉里奥萨-威林厄姆等人，《BMC神经科学》17:16，2016年）。在此，我们展示了一种评估活轴突髓鞘形成的高通量体外检测方法的详细方法。
