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Organotypic Brain Slice Culture Microglia Exhibit Molecular Similarity to Acutely-Isolated Adult Microglia and Provide a Platform to Study Neuroinflammation
Frontiers in Cellular Neuroscience ( IF 5.3 ) Pub Date : 2020-11-24 , DOI: 10.3389/fncel.2020.592005
Alex R. D. Delbridge , Dann Huh , Margot Brickelmaier , Jeremy C. Burns , Chris Roberts , Ravi Challa , Naideline Raymond , Patrick Cullen , Thomas M. Carlile , Katelin A. Ennis , Mei Liu , Chao Sun , Normand E. Allaire , Marianna Foos , Hui-Hsin Tsai , Nathalie Franchimont , Richard M. Ransohoff , Cherie Butts , Michael Mingueneau

Microglia are central nervous system (CNS) resident immune cells that have been implicated in neuroinflammatory pathogenesis of a variety of neurological conditions. Their manifold context-dependent contributions to neuroinflammation are only beginning to be elucidated, which can be attributed in part to the challenges of studying microglia in vivo and the lack of tractable in vitro systems to study microglia function. Organotypic brain slice cultures offer a tissue-relevant context that enables the study of CNS resident cells and the analysis of brain slice microglial phenotypes has provided important insights, in particular into neuroprotective functions. Here we use RNA sequencing, direct digital quantification of gene expression with nCounter® technology and targeted analysis of individual microglial signature genes, to characterize brain slice microglia relative to acutely-isolated counterparts and 2-dimensional (2D) primary microglia cultures, a widely used in vitro surrogate. Analysis using single cell and population-based methods found brain slice microglia exhibited better preservation of canonical microglia markers and overall gene expression with stronger fidelity to acutely-isolated adult microglia, relative to in vitro cells. We characterized the dynamic phenotypic changes of brain slice microglia over time, after plating in culture. Mechanical damage associated with slice preparation prompted an initial period of inflammation, which resolved over time. Based on flow cytometry and gene expression profiling we identified the 2-week timepoint as optimal for investigation of microglia responses to exogenously-applied stimuli as exemplified by treatment-induced neuroinflammatory changes observed in microglia following LPS, TNF and GM-CSF addition to the culture medium. Altogether these findings indicate that brain slice cultures provide an experimental system superior to in vitro culture of microglia as a surrogate to investigate microglia functions, and the impact of soluble factors and cellular context on their physiology.



中文翻译:

器官型脑切片培养小胶质细胞与急性分离的成年小胶质细胞表现出分子相似性,并提供了研究神经炎症的平台

小胶质细胞是中枢神经系统(CNS)驻留的免疫细胞,已与多种神经系统疾病的神经炎性发病机制有关。它们对神经炎症的多种环境依赖性贡献才刚刚被阐明,这部分可以归因于研究小胶质细胞的挑战体内 而且缺乏治疗 体外研究小胶质细胞功能的系统。器官型脑片培养物提供了与组织相关的环境,使中枢神经系统驻留细胞的研究成为可能,而脑片小胶质细胞表型的分析提供了重要的见解,尤其是在神经保护功能方面。在这里,我们使用RNA测序,使用nCounter®技术进行基因表达的直接数字量化以及对单个小胶质细胞特征基因的靶向分析,以相对于急性分离的对应物和二维(2D)初级小胶质细胞培养物(相对广泛使用)表征脑切片小胶质细胞体外替代。使用单细胞和基于人群的方法进行的分析发现,相对于急性分离的成年小胶质细胞,脑切片小胶质细胞显示出更好的保存规范性小胶质细胞标记物和整体基因表达,对急性分离的成年小胶质细胞具有更高的保真度。体外细胞。我们表征了在培养中电镀后随时间变化的脑切片小胶质细胞的动态表型变化。与切片制备相关的机械损伤促使炎症开始,随着时间的流逝逐渐消退。基于流式细胞仪和基因表达谱,我们确定了两周的时间点是研究小胶质细胞对外源性刺激反应的最佳选择,例如LPS,TNF和GM-CSF加入培养物中后,在小胶质细胞中观察到了治疗引起的神经炎症改变。中。总而言之,这些发现表明脑片培养提供了优于体外 小胶质细胞培养作为替代指标,以研究小胶质细胞的功能以及可溶性因子和细胞环境对其生理的影响。

更新日期:2020-12-21
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