Stence N, Waite M, Dailey M E
Department of Biological Sciences, 355 Biology Building, University of Iowa, Iowa City, IA 52242, USA.
Glia. 2001 Mar 1;33(3):256-66.
The dynamics of microglial cell activation was studied in freshly prepared rat brain tissue slices. Microglia became activated in the tissue slices, as evidenced by their conversion from a ramified to amoeboid form within several hours in vitro. To define better the cytoarchitectural dynamics underlying microglial activation, we performed direct three-dimensional time-lapse confocal imaging of microglial cells in live brain slices. Microglia in tissue slices were stained with a fluorescent lectin conjugate, FITC-IB(4), and stacks of confocal optical sections through the tissue were collected repeatedly at intervals of 2-5 min for several hours at a time. Morphometric analysis of cells from time-lapse sequences revealed that ramified microglia progress to amoeboid macrophages through a stereotypical sequence of steps. First, in the withdrawal stage, the existing ramified branches of activating microglia do not actively extend or engulf other cells, but instead retract back (mean rate, 0.5-1.5 microm/min) and are completely resorbed into the cell body. Second, in the motility stage, a new set of dynamic protrusions, which can exhibit cycles of rapid extension and retraction (both up to 4 microm/min), abruptly emerges. Sometimes new processes begin to emerge even before the old branches are completely withdrawn. Third, in the locomotory stage, microglia begin translocating within the tissue (up to 118 microm/h) only after the new protrusions emerge. We conclude that the rapid conversion of resting ramified microglia to active amoeboid macrophages is accomplished not by converting quiescent branches to dynamic ones, but rather by replacing existing branches with an entirely new set of highly motile protrusions. This suggests that the ramified branches of resting microglia are normally incapable of rapid morphological dynamics necessary for activated microglial function. More generally, our time-lapse observations identify changes in the dynamic behavior of activating microglia and thereby help define distinct temporal and functional stages of activation for further investigation.
在新鲜制备的大鼠脑组织切片中研究了小胶质细胞活化的动力学。小胶质细胞在组织切片中被激活，体外数小时内它们从分支状转变为阿米巴样形态即可证明这一点。为了更好地定义小胶质细胞活化背后的细胞结构动力学，我们对活脑切片中的小胶质细胞进行了直接三维延时共聚焦成像。组织切片中的小胶质细胞用荧光凝集素偶联物FITC-IB(4)染色，每次以2 - 5分钟的间隔重复收集穿过组织的共聚焦光学切片堆栈，持续数小时。对延时序列中的细胞进行形态计量分析表明，分支状小胶质细胞通过一系列刻板的步骤转变为阿米巴样巨噬细胞。首先，在退缩阶段，活化小胶质细胞现有的分支状突起不再积极延伸或吞噬其他细胞，而是回缩（平均速率为0.5 - 1.5微米/分钟）并完全被吸收回细胞体。其次，在运动阶段，一组新的动态突起突然出现，这些突起可表现出快速延伸和回缩的循环（两者速度均可达4微米/分钟）。有时甚至在旧分支完全退缩之前新的突起就开始出现。第三，在移动阶段，小胶质细胞仅在新突起出现后才开始在组织内移位（速度可达118微米/小时）。我们得出结论，静息的分支状小胶质细胞快速转变为活跃的阿米巴样巨噬细胞不是通过将静止的分支转变为动态的分支，而是通过用一整套全新的高运动性突起取代现有的分支来实现的。这表明静息小胶质细胞的分支状突起通常不具备活化小胶质细胞功能所需的快速形态动力学。更普遍地说，我们的延时观察确定了活化小胶质细胞动态行为的变化，从而有助于定义不同的激活时间和功能阶段以供进一步研究。
