Figure 2. Cluster formation and progression over the course of EAE.

(a) 3D reconstruction of a representative spinal cord volume imaged in vivo from the pial surface to 75 μm deep shows a pial vessel (red) in longitudinal orientation that turns perpendicularly and penetrates the spinal cord parenchyma (left). Overlay of the green channel shows the spatial relationship between GFP-positive cells and the vessel at the peak of EAE (middle). A full rotation showing the same volume of tissue from bottom to top shows increased accumulation of perivascular microglia around the parenchymal segment of the blood vessel (dotted line, right). The deepest point of the penetrating vessel is marked with white stars. Grid scale, 14 μm. See corresponding Supplementary Movie 2. (b) Time-lapse in vivo imaging showing pial macrophages and microglia rapidly associating with the vasculature at the peak of EAE. (Left) The leading edge of individual cell bodies or processes closest to the vasculature was tracked over a period of 60 min. Blue tracks identify cells that maintained their proximity to the vasculature and magenta tracks identify cells that either moved closer to the vasculature or extended processes toward it. (Middle and right) Yellow arrowheads show a cell approaching the blood vessel and associating with pre-existing perivascular cells over time. Blue arrowheads show a cell with processes already in contact with the blood vessel that migrates towards the vessel and eventually extends a new process toward it. Red star identifies a cell that maintains its point of contact with the vessel wall throughout the 60-min time course (white arrowheads), while it extends a new process toward the same blood vessel (magenta arrowheads), and retracts one of its processes that was directed away from the vessel (red arrowheads). Scale bar, 10 μm. See corresponding Supplementary Movie 3. (c) Repetitive in vivo imaging in the same spinal cord area 21 and 32 days after immunization shows a newly formed perivascular cluster (solid oval) on one side of a blood vessel and a resolving cluster (dotted oval) on the opposite side of the same vessel. Scale bar, 10 μm.