Microglial Cell Migration Stimulated by ATP and C5a Involve Distinct Molecular Mechanisms: Quantification of Migration by a Novel Near-Infrared Method
Microglial Cell Migration Stimulated by ATP and C5a Involve Distinct Molecular Mechanisms: Quantification of Migration by a Novel Near-Infrared Method
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DOI：
10.1002/glia.20813
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发表时间：
2009-06-01
期刊：
影响因子：
6.2
通讯作者：
Stella, Nephi
中科院分区：
文献类型：
--
作者：
Miller, Aaron M.;Stella, Nephi
小胶质细胞，即大脑的巨噬细胞，在神经炎症的传播中发挥重要作用。增加小胶质细胞迁移响应特定的化学引诱剂已被记录，但鲜为人知的是，这些刺激和介导其影响的信号转导通路之间的差异。目前测量细胞迁移的方法通常是劳动密集型的，并且依赖于手动计数细胞数量，因此需要更有效和客观的方法。在这里，我们提出了一种改进的和更高通量的Boyden室技术，通过使用DRAQ 5，一种在近红外发射的核染料来测量小胶质细胞迁移。在测试的一组化学引诱物中，我们发现ATP和C5 a有效地刺激小鼠原代小胶质细胞的迁移。ATP和C5 a的刺激作用显示出显著的加和性，表明每个化学引诱物通过独立的分子机制刺激迁移。因此，我们发现这些反应的关键差异：ATP刺激了趋化作用和趋化性的组合，这种反应是由ROCK信号通路介导的;而C5 a只刺激趋化性，这种反应是由Rac 1信号通路介导的。最后，我们发现，功能性PI 3-激酶只需要随机基底小胶质细胞迁移。因此，我们的研究结果表明，不同的非重叠的信号转导通路控制不同模式的小胶质细胞迁移，并建议这些不同的分子机制的靶向调节神经炎症传播的不同方面。(C)2008 Wiley-Liss，Inc.
Microglial cells, the macrophages of the brain, play an essential role in the propagation of neuroinflammation. Increased microglial cell migration in response to specific chemoattractants has been documented, but less is known about the differences between these stimuli and the signal transduction pathways that mediate their effects. Current methods to measure cell migration are often labor-intensive and rely on the manual counting of cell number, so more efficient and objective methods are needed. Here we present an improved and higher-throughput Boyden chamber technique that measures microglial cell migration by using DRAQ5, a nuclear dye that emits in the near-infrared. Out of a panel of chemoattractants tested, we found that ATP and C5a potently stimulate the migration of mouse primary microglial cells. The stimulatory effects of ATP and C5a displayed significant additivity, suggesting that each chemoattractant stimulated migration through independent molecular mechanisms. Accordingly, we found key differences in these responses: ATP stimulated a combination of both chemokinesis and chemotaxis, and this response was mediated by the ROCK signaling pathway; whereas C5a stimulated only chemotaxis and this response was mediated by the Rac1 signaling pathway. Finally, we found that functional PI3-kinase is only required for random basal microglial cell migration. Thus, our results show that distinct nonoverlapping signal transduction pathways control different modes of microglial cell migration and suggest that the targeting of these distinct molecular mechanisms should modulate different aspects of neuroinflammation propagation. (C) 2008 Wiley-Liss, Inc.