MICROGLIAL ACTIVATION IN BRAIN SLICES: TIME-COURSE OF NUCLEAR TRANSLOCATION OF NF-κB AND CHANGES IN SURFACE EXPRESSION OF CELL ADHESION MOLECULES.

M.E. Dailey, L. Fuller, A. Hoffman, and D. Kurpius.

Dept. of Biological Sciences, University of Iowa, Iowa City, IA  52242

Microglia activate and mobilize in response to traumatic brain injury, but the intracellular signaling events underlying activation remain obscure.  We used an in vitro hippocampal tissue slice model to study the time course of molecular changes in activating microglia following acute tissue injury (tissue slicing).  A quantitative, two-channel fluorescence image analysis protocol was developed to define spatio-temporal changes in immunohistochemical staining for signaling and adhesion molecules in identified microglia.  Rat or mouse tissue slices were harvested and fixed at various times (2.5min-48hr) following tissue slicing, then tissues were double-labeled with fluorescent probes to visualize microglia (IB4 lectin) and one of several antigens.  We found that immunolabeling for the transcription factor, NF-κB, rapidly increased about 3-fold in nuclei of activating microglia, peaking at 60-75min following tissue injury and returning to baseline within 3hr.  Many microglia showed a 1.5 to 4 fold increase in cell surface expression of Leukocyte Function Antigen (LFA-1), an αLβ2 integrin, but this was slower, peaking at ~18 hr after tissue injury.  Not all microglia showed comparable changes in NF-κB and LFA-1 immunostaining.  Moreover, time-lapse confocal imaging showed differences in microglial cell motility dynamics, suggesting heterogeneity in the microglial activation response.  To determine whether motility behaviors correlate with differences in LFA-1 immuno-staining, activating microglia were imaged for several hours in live tissue slices, then after fixation the same cells were assessed by quantitative immunohistochemistry.  Preliminary results show a wide range of LFA-1 immunostaining intensities even among highly motile and locomotory microglia, suggesting that LFA-1 expression alone does not autonomously specify the motility behavior of activating microglia in situ.

Supported by NIH (NS43468).