Multiparametric Image Analysis of Rat Dorsal Root Ganglion Cultures to Evaluate Peripheral Neuropathy-Inducing Chemotherapeutics
Multiparametric Image Analysis of Rat Dorsal Root Ganglion Cultures to Evaluate Peripheral Neuropathy-Inducing Chemotherapeutics
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DOI：
10.1093/toxsci/kfw254
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发表时间：
2017-03-01
影响因子：
3.8
通讯作者：
Davis, Myrtle
中科院分区：
文献类型：
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作者：
Guo, Liang;Hamre, John, III;Davis, Myrtle
化疗引起的周围神经病变（CIPN）是癌症患者经历的一种主要的、剂量限制性的不良反应。CIPN的基于机制的风险缓解和有效治疗的进展可以通过合适的体外测定来辅助。为此，我们开发了一个多参数的形态学为中心的大鼠背根神经节（DRG）测定。神经元和非神经元的亚细胞结构的形态学变化进行了分析与自动显微镜系统。NeuN（神经元特异性核蛋白）和Tuj-1（β-III微管蛋白）的染色分别用于鉴定神经元细胞核和神经元细胞体/神经突。波形蛋白染色（雪旺细胞中间丝的组分）用于标记非神经元支持细胞。用DAPI染色但缺乏NeuN的细胞核代表非神经元细胞。在连续暴露于CIPN诱导剂24小时后和药物去除后72小时分析图像，以提供从初始药物作用恢复的动态测量。硼替佐米、顺铂、艾日布林、紫杉醇或长春新碱的治疗诱导了剂量依赖性的神经突/突起区域的损失，模拟了轴突的“死回”变性，这是体内临床CIPN的组织病理学标志。对于所有五种CIPN诱导药物，神经突损失的IC 50在最大临床暴露（Cmax）的3倍内，但是对于2种非CIPN诱导剂，IC 50为Cmax的> 4倍或>= 28倍。还观察到化合物特异性效应，例如顺铂或硼替佐米导致的神经突断裂和紫杉醇导致的神经元细胞体增大。总的来说，这些结果支持使用定量、形态学评价和DRG细胞培养模型来告知CIPN的风险和检查CIPN的机制。
Chemotherapy-induced peripheral neuropathy (CIPN) is a major, dose-limiting adverse effect experienced by cancer patients. Advancements in mechanism-based risk mitigation and effective treatments for CIPN can be aided by suitable in vitro assays. To this end, we developed a multiparametric morphology-centered rat dorsal root ganglion (DRG) assay. Morphologic alterations in subcellular structures of neurons and non-neurons were analyzed with an automated microscopy system. Stains for NeuN (a neuron-specific nuclear protein) and Tuj-1 (beta-III tubulin) were used to identify neuronal cell nuclei and neuronal cell bodies/neurites, respectively. Vimentin staining (a component of Schwann cell intermediate filaments) was used to label non-neuronal supporting cells. Nuclei that stained with DAPI, but lacked NeuN represented non-neuronal cells. Images were analyzed following 24h of continuous exposure to CIPN-inducing agents and 72h after drug removal to provide a dynamic measure of recovery from initial drug effects. Treatment with bortezomib, cisplatin, eribulin, paclitaxel or vincristine induced a dose-dependent loss of neurite/process areas, mimicking the ` dying back' degeneration of axons, a histopathological hallmark of clinical CIPN in vivo. The IC50 for neurite loss was within 3-fold of the maximal clinical exposure (Cmax) for all five CIPN-inducing drugs, but was > 4-or >= 28-fold of the Cmax for 2 nonCIPN- inducing agents. Compound-specific effects, eg, neurite fragmentation by cisplatin or bortezomib and enlarged neuronal cell bodies by paclitaxel, were also observed. Collectively, these results support the use of a quantitative, morphologic evaluation and a DRG cell culture model to inform risk and examine mechanisms of CIPN.