Three-dimensional quantification of microglial ramification in the mouse brain after chronic restraint stress

Abstract

Microglia play a pivotal role in the brain development and plasticity through synaptic pruning. Morphological change of microglia reflects a key feature in this process and has been used to define microglial activation. Most studies have applied two-dimensional (2D) imaging method to evaluate microglial morphology so far. We have established a 3D analytical approach in a mouse chronic restrain-stress (CRS, 2 h/day × 10 days) model with C57BL/6J male mice. Eighty microglia (20 microglia/brain area/treatment) in the infralimbic and M2 areas of the medial prefrontal cortex were imaged and reconstruct into 3D digital images, from which a semi-automatic method was used to track down microglial branches and several parameters were calculated by using the 3D analysis tools in ImageJ. In Ctrl animals, microglia in the infralimbic area had significantly lower average branch number and longer average branch length than in the M2 area (both p 0.05). After CRS, significant increase in the average branch number of microglia was only observed in the infralimbic area (p 0.05). However, the average branch length was decreased in the infralimbic microglia after CRS (p 0.05), whereas the M2 microglia showed increased maximal branch length (p 0.05). These results suggests that activated microglia increase their structural complexity in an area-specific manner, but it is hard to simply use over- or de-ramification to describe their activation status.