Microglial motility in the rat facial nucleus following peripheral axotomy

Microglial motility was studied in living mammalian brain tissue using infr ared gradient contrast microscopy in combination with video contrast enhanc ement and time lapse video recording. The infrared gradient contrast allows the visualization of living cells up to a depth of 60 mu m in brain slices , in regions where cell bodies remain largely uninjured by the tissue prepa ration and are visible in their natural environment. In contrast to other t echniques, including confocal microscopy, this procedure does not require a ny staining or labeling of cell membranes and thus guarantees the investiga tion of tissue which has not been altered, apart from during preparation. M icroglial cells are activated and increase in number in the facial nucleus following peripheral axotomy. Thus we established the preparation of longit udinal rat brainstem slices containing the axotomized facial nucleus as a s ource of activated microglial cells. During prolonged video time lapse reco rdings, two different types of microglial cell motility could be observed. Microglial cells which had accumulated at the surface of the slice remained stationary but showed activity of the cell soma, developing pseudopods of different shape and size which undulated and which were used for phagocytos is of cell debris. Microglial phagocytosis of bacteria could be documented for the first time in situ. In contrast, ameboid microglia which did not di splay pseudopods but showed migratory capacity, could be observed exclusive ly in the depth of the tissue. Some of these cells maintained a close conta ct to neurons and appeared to move along their dendrites, a finding that ma y be relevant to the role of microglia in "synaptic stripping", the displac ement of synapses following axotomy. This approach provides a valuable oppo rtunity to investigate the interactions between activated microglial cells and the surrounding cellular and extracellular structures in the absence of staining or labeling, thus opening a wide field for the analysis of the ce llular mechanisms involved in numerous pathologies of the CNS.