Microglial Cell Morphological Analysis

Using the custom-designed script cited above, the following morphological criteria could be extracted for each microglial cell (Fig. 1b, c): a set of measured criteria as cell body and cytoplasm area, defined as the cell body area associated with the cytoplasmic area of the primary ramifications, expressed in μm²; branching characteristics such as the total number and length (μm) of ramifications and the number of primary, secondary and tertiary ramifications; and roundness (ratio between surface and perimeter squared of the cell body) and GFP intensity by whole cell.
A second set of calculated criteria extrapolated from the previous ones yielded the complexity index (CI) and the covered environment area (CEA). First, we defined the CI using two different criteria extracted from the Acapella™ script: the number of segments of each cell, a segment being defined as the length of process between two nodes, and the number of its primary ramifications. By dividing these two criteria, we obtain also a mean complexity by primary ramification for each microglial cell (Additional file 2). On the other hand, CEA represents the 2D total surface covered by its ramifications and defined as the area of the polygon formed by linking the extremities of its processes, expressed in μm². The areal density of microglial cells by region or by brain was calculated by dividing the number of microglial cells selected by the scanned tissue area.
The CI revealed a completely distinct microglial phenotype, the amoeboid cells. The amoeboid or rod cells are characterized by a CI = 1 (no nodes) and are characteristic for activated cells, displaying engulfing, phagocytic properties [11 (link)]. Because of their particular role, we distinguish them from the other microglial cells.