The significance of activated microglia around motoneurons axotomized after nerve injuries has been intensely debated. In particular, whether microglia become phagocytic is controversial. To resolve these issues we directly observed microglia behaviors with two-photon microscopy in ex vivo spinal cord slices from CX3CR1-GFP mice complemented with confocal analyses of CD68 protein. Axotomized motoneurons were retrogradely-labeled from muscle before nerve injuries. Microglia behaviors close to axotomized motoneurons greatly differ from those within uninjured motor pools. They develop a phagocytic phenotype as early as 3 days after injury, characterized by frequent phagocytic cups, high phagosome content and CD68 upregulation. Interactions between microglia and motoneurons changed with time after axotomy. Microglia first extend processes that end in phagocytic cups at the motoneuron surface, then they closely attach to the motoneuron while extending filopodia over the cell body. Confocal 3D analyses revealed increased microglia coverage of the motoneuron cell body surface with time after injury and the presence of CD68 granules in microglia surfaces opposed to motoneurons. Some microglia formed macroclusters associated with dying motoneurons. Microglia in these clusters display the highest CD68 expression and associate with cytotoxic T-cells. These observations are discussed in relation to current theories on microglia function around axotomized motoneurons.

神经损伤后轴突切除的运动神经元周围激活的小胶质细胞的意义一直存在激烈争论。特别是，小胶质细胞是否具有吞噬能力存在争议。为了解决这些问题，我们用双光子显微镜直接观察 CX3CR1-GFP 小鼠离体脊髓切片中的小胶质细胞行为，并辅以 CD68 蛋白的共聚焦分析。轴突运动神经元在神经损伤前从肌肉中逆行标记。接近轴突运动神经元的小胶质细胞行为与未受伤运动神经元内的小胶质细胞行为有很大不同。它们早在损伤后 3 天就形成了吞噬细胞表型，其特征是频繁的吞噬杯、高吞噬体含量和 CD68 上调。轴突切除后小胶质细胞和运动神经元之间的相互作用随着时间而变化。小胶质细胞首先延伸终止于运动神经元表面吞噬杯的过程，然后它们紧密附着在运动神经元上，同时在细胞体上延伸丝状伪足。共聚焦 3D 分析显示，随着损伤后时间的推移，小胶质细胞对运动神经元细胞体表面的覆盖范围增加，并且与运动神经元相反的小胶质细胞表面存在 CD68 颗粒。一些小胶质细胞形成与垂死的运动神经元相关的大簇。这些簇中的小胶质细胞表现出最高的 CD68 表达并与细胞毒性 T 细胞相关。这些观察结果与当前有关轴突运动神经元周围小胶质细胞功能的理论进行了讨论。