Use of high content image analyses to detect chemical-mediated effects on neurite sub-populations in primary rat cortical neurons
Use of high content image analyses to detect chemical-mediated effects on neurite sub-populations in primary rat cortical neurons
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DOI：
10.1016/j.neuro.2012.10.013
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发表时间：
2013-01-01
期刊：
影响因子：
3.4
通讯作者：
Mundy, William R.
中科院分区：
文献类型：
--
作者：
Harrill, Joshua A.;Robinette, Brian L.;Mundy, William R.
传统的发育神经毒性试验在体内进行，成本高，耗时长，并使用大量的动物。为了解决这些效率低下的问题，在发育神经毒性危害识别的第一层筛选方法中使用了神经元发育的体外模型。一种常用的体外评估发育神经毒性的终点是测量神经突生长。该生物过程适合于使用基于高含量成像（HCI）的方法的高通量测量。迄今为止，大多数神经突生长的HCI研究都集中在总神经突生长的测量上，而没有检查定型的神经元生长模式是否被破坏或特定的神经突亚群（即轴突或树突）是否被选择性地影响。本研究描述了两个HCI为基础的分析方法的开发和实施，用于评估神经元成熟的化学效应。这些方法利用培养物中原代大鼠皮质神经元的常规生长模式（即分期方法）以及β（III）-微管蛋白和MAP 2的差异细胞质分布（即减法）来定量响应于化学暴露的神经突起始、轴突生长和次级神经突（或树突）生长的抑制。结果表明，这些不同的成熟过程受到不同的药理学化合物（K252 a，Na 3VO 4，Bis-1）已知抑制神经突生长的影响。此外，一组已知的发育神经毒物也差异影响原代皮层培养的轴突和次生神经突的生长。这项工作改进了以前的HCI方法，提供了一种手段，在其中快速和具体量化的轴突和树突在体外的生长的化学作用。(c)2012 Elsevier Inc. All rights reserved.
Traditional developmental neurotoxicity tests performed in vivo are costly, time-consuming and utilize a large number of animals. In order to address these inefficiencies, in vitro models of neuronal development have been used in a first tier screening approach for developmental neurotoxicity hazard identification. One commonly used endpoint for assessing developmental neurotoxicity in vitro is measurement of neurite outgrowth. This biological process is amenable to high-throughput measurement using high content imaging (HCI) based methodologies. To date, a majority of HCI studies of neurite outgrowth have focused on measurements of total neurite outgrowth without examining whether stereotypic neuronal growth patterns are disrupted or whether specific sub-populations of neurites (i.e. axons or dendrites) are selectively affected. The present study describes the development and implementation of two HCI based analysis methods for assessing chemical effects on neuronal maturation. These methods utilize the stereotypical growth pattern of primary rat cortical neurons in culture (i.e. the Staging Method), as well as the differential cytoplasmic distribution of beta(III)-tubulin and MAP2 (i.e. the Subtraction Method), to quantify inhibition of neurite initiation, axon outgrowth and secondary neurite (or dendrite) outgrowth in response to chemical exposure. Results demonstrate that these distinct maturational processes are differentially affected by pharmacological compounds (K252a, Na3VO4, Bis-1) known to inhibit neurite outgrowth. Furthermore, a group of known developmental neurotoxicants also differentially affected the growth of axons and secondary neurites in primary cortical culture. This work improves upon previous HCI methods by providing a means in which to rapidly and specifically quantify chemical effects on the growth of axons and dendrites in vitro. (c) 2012 Elsevier Inc. All rights reserved.