Abstract

We used two-channel three-dimensional time-lapse fluorescence confocal imaging in live rat hippocampal slice cultures (1-7 days in vitro) to determine the motility behaviors of activated microglia as they engage dead and dying cells following traumatic brain tissue injury. Live microglia were labeled with a fluorescently conjugated lectin (IB(4)), and dead neurons were labeled with a membrane-impermeant fluorescent DNA-binding dye (Sytox Orange or To-Pro-3). Tissue injury during the slicing procedure induced neuronal death and microglial activation, but the density of dead cells diminished approximately 10-fold by 7 days in vitro as resident microglia cleared dead cells. In time-lapse movies (4-20 h long), activated microglia exhibited varying levels of motile and locomotory activity. The motility of microglia could change abruptly following contact by other microglia or death of nearby cells. When neighboring cells died, some microglia rapidly moved toward or extended a process to engulf the dead cell, consistent with a chemotactic signaling response. Dead cell nuclei usually were engulfed and carried along by highly motile and locomoting microglia. The mean time to engulfment was approximately 5 times faster for newly deceased cells (33 min) than for extant dead cells (160 min), suggesting that the efficacy of microglial phagocytosis in situ might vary with time after cell death or mode of cell death. These observations demonstrate that activated microglia are heterogeneous with respect to motile activity following traumatic tissue injury and further indicate that cell motility in situ is temporally regulated at the single cell level, possibly by direct cell-cell contact and by diffusible substances emanating from nearby dead cells.