Delbridge Alex R D, Huh Dann, Brickelmaier Margot, Burns Jeremy C, Roberts Chris, Challa Ravi, Raymond Naideline, Cullen Patrick, Carlile Thomas M, Ennis Katelin A, Liu Mei, Sun Chao, Allaire Normand E, Foos Marianna, Tsai Hui-Hsin, Franchimont Nathalie, Ransohoff Richard M, Butts Cherie, Mingueneau Michael
Multiple Sclerosis and Neuroimmunology Research Unit, Biogen, Cambridge, MA, United States.
Biogen Postdoctoral Scientist Program, Biogen, Cambridge, MA, United States.
Front Cell Neurosci. 2020 Dec 21;14:592005. doi: 10.3389/fncel.2020.592005. eCollection 2020.
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 and the lack of tractable 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 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 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 culture of microglia as a surrogate to investigate microglia functions, and the impact of soluble factors and cellular context on their physiology.
小胶质细胞是中枢神经系统（CNS）中的常驻免疫细胞，与多种神经系统疾病的神经炎症发病机制有关。它们对神经炎症的多种背景依赖性贡献才刚刚开始被阐明，这在一定程度上可归因于研究小胶质细胞的挑战以及缺乏易于处理的系统来研究小胶质细胞功能。器官型脑片培养提供了一个与组织相关的环境，能够研究中枢神经系统常驻细胞，并且对脑片小胶质细胞表型的分析提供了重要的见解，特别是在神经保护功能方面。在这里，我们使用RNA测序、用nCounter®技术对基因表达进行直接数字定量以及对单个小胶质细胞特征基因进行靶向分析，以相对于急性分离的对应物和二维（2D）原代小胶质细胞培养物（一种广泛使用的替代物）来表征脑片小胶质细胞。使用单细胞和基于群体的方法进行分析发现，相对于细胞，脑片小胶质细胞表现出对经典小胶质细胞标志物和整体基因表达的更好保留，对急性分离的成年小胶质细胞具有更高的保真度。我们表征了脑片小胶质细胞在培养接种后随时间的动态表型变化。与切片制备相关的机械损伤引发了炎症的初始阶段，该阶段会随着时间的推移而消退。基于流式细胞术和基因表达谱分析，我们确定2周时间点是研究小胶质细胞对外源施加刺激的反应的最佳时间点，如在向培养基中添加脂多糖（LPS）、肿瘤坏死因子（TNF）和粒细胞 - 巨噬细胞集落刺激因子（GM - CSF）后在小胶质细胞中观察到的治疗诱导的神经炎症变化所例证。总之，这些发现表明，脑片培养提供了一个优于小胶质细胞培养的实验系统，作为研究小胶质细胞功能以及可溶性因子和细胞环境对其生理学影响的替代方法。
