Figure 4

Overview of vascular network and neurovascular inflammation processing and analysis pipelines. (a) Three-dimensional 90-µm sub-stack of light sheet fluorescence microscopy of glucose transporter 1 (GLUT1). (b) Schematic representation of vascular network analysis parameters included. Vascular network length is depicted as dotted line following a vascular tree since it corresponds to the total vascular network course. Junctions are points where the vascular network bifurcates. (c) Maximum projection obtained from the sub-stack shown in (a). Images were later (d) binarized and (e) dilatated. The dilatation step prevents very small branches (yellow arrowhead) visible on the binary image as adjacent from beginning to end to be misclassified as round junctional sections. (f) Skeletonized and (g) tagged topological vascular network (branches are shown in red, junctions in purple). The visible junctions correspond to both two-dimensional and three-dimensional junction sections. (h) Three-dimensional 30-µm sub-stack of light sheet fluorescence microscopy of ionized calcium-binding adaptor molecule 1 (IBA1). (i) Maximum projection obtained from the sub-stack shown in (h). (j) Microglia refined segmentation output by cell size [9–900 µm²] and circularity [0.2–1.0]. (k) Three-dimensional exemplification of vascular inflammation (yellow; white arrowheads) of three-dimensional light sheet fluorescence microscopy of GLUT1 (red) together with IBA1 (green). Scale bars: 100 µm (a,c–k).