Adams Alexandra C, Kyle Michele, Beaman-Hall Carol M, Monaco Edward A, Cullen Matthew, Vallano Mary Lou
Department of Neuroscience & Physiology, SUNY Upstate Medical University, Syracuse, NY, 13210, USA.
Department of Pulmonary and Critical Care, Mount Sinai Beth Israel Medical Center, New York, NY, 10003, USA.
Cell Mol Neurobiol. 2015 Oct;35(7):961-75. doi: 10.1007/s10571-015-0191-9. Epub 2015 Apr 18.
A simple method to co-culture granule neurons and glia from a single brain region is described, and microglia activation profiles are assessed in response to naturally occurring neuronal apoptosis, excitotoxin-induced neuronal death, and lipopolysaccharide (LPS) addition. Using neonatal rat cerebellar cortex as a tissue source, glial proliferation is regulated by omission or addition of the mitotic inhibitor cytosine arabinoside (AraC). After 7-8 days in vitro, microglia in AraC(-) cultures are abundant and activated based on their amoeboid morphology, expressions of ED1 and Iba1, and ability to phagocytose polystyrene beads and the majority of neurons undergoing spontaneous apoptosis. Microglia and phagocytic activities are sparse in AraC(+) cultures. Following exposure to excitotoxic kainate concentrations, microglia in AraC(-) cultures phagocytose most dead neurons within 24 h without exacerbating neuronal loss or mounting a strong or sustained inflammatory response. LPS addition induces a robust inflammatory response, based on microglial expressions of TNF-α, COX-2 and iNOS proteins, and mRNAs, whereas these markers are essentially undetectable in control cultures. Thus, the functional effector state of microglia is primed for phagocytosis but not inflammation or cytotoxicity even after kainate exposure that triggers death in the majority of neurons. This model should prove useful in studying the progressive activation states of microglia and factors that promote their conversion to inflammatory and cytotoxic phenotypes.
本文描述了一种从单一脑区共培养颗粒神经元和神经胶质细胞的简单方法，并评估了小胶质细胞对自然发生的神经元凋亡、兴奋性毒素诱导的神经元死亡以及添加脂多糖（LPS）的激活情况。以新生大鼠小脑皮质作为组织来源，通过省略或添加有丝分裂抑制剂阿糖胞苷（AraC）来调节神经胶质细胞的增殖。在体外培养7 - 8天后，基于其阿米巴样形态、ED1和Iba1的表达以及吞噬聚苯乙烯珠的能力和大多数经历自发凋亡的神经元，AraC（-）培养物中的小胶质细胞丰富且被激活。AraC（+）培养物中的小胶质细胞和吞噬活性稀少。暴露于兴奋性毒性浓度的红藻氨酸后，AraC（-）培养物中的小胶质细胞在24小时内吞噬了大多数死亡神经元，而不会加剧神经元损失或引发强烈或持续的炎症反应。添加LPS会基于小胶质细胞中TNF-α、COX-2和iNOS蛋白及mRNA的表达诱导强烈的炎症反应，而在对照培养物中这些标志物基本无法检测到。因此，即使在触发大多数神经元死亡的红藻氨酸暴露后，小胶质细胞的功能效应状态也倾向于吞噬作用，而非炎症或细胞毒性。该模型在研究小胶质细胞的渐进激活状态以及促进其转变为炎症和细胞毒性表型的因素方面应会证明有用。
