Morphological analysis of microglia was performed on 3-dimensional fluorescence images using Imaris × 64 version 9.6–9.9 (Bitplane, Zurich, Switzerland) algorithms. Microglial cells in which the nucleus was at least 15 µm away from the image border were selected for analysis. The modules "Filament tracer" and "Surface" were used for microglia reconstruction. A total of 50 cells from 3 different mice were analyzed for each group. The background was minimized with an appropriate filter width (20–40 µm) and the region of the analyzed cell was selected manually. The parameters Filament Length, Filament No, Dendrite Branch Pts, Filament No, Sholl Intersections, and Soma Volume were obtained from the specific values calculated by Imaris. Although tracing was performed automatically by the algorithm, we individually verified that processes originated from one defined cell. False connections were removed manually which were commonly less than 1%. The number of Sholl intersections was defined as the number of process intersecting concentric spheres, defining the spatial distribution of segments as a function of distance from the soma (Sholl analysis). All spheres have their center at the soma (beginning point) with a 5 µm step resolution for the spheres.
Free full text: Click here
