Figure 3. Microglial clusters form in areas of BBB disruption and perivascular fibrin deposition.

(a) In vivo time-lapse imaging in the spinal cord of Cx3cr1^GFP/+ EAE mice at the onset of clinical symptoms (clinical score 1) identified sites of active BBB disruption correlating with perivascular clusters. Imaging started immediately after a bolus i.v. injection of a 70 kDa rhodamine dextran solution (red). As time progressed, dye continuously leaked in the perivascular tissue that was occupied by an extensive microglial cluster (green). Scale bar, 20 μm. See corresponding Supplementary Movie 4. (b) In vivo imaging of microglial clusters (green) in the spinal cord of Cx3cr1^GFP/+ EAE-challenged mice after 3–7 daily i.v. injections of Alexa594-fibrinogen (red). Cluster formation overlaps with areas of perivascular fibrin deposition at the peak of EAE. The 91.8% calculated proportion of extracellular fibrin that colocalizes with microglia is significantly higher than the 30.6% predicted by Fisher’s combined probability test if the correlation were random. Bar graph represents mean±s.e.m. from 20 clusters from n=8 mice injected with Alexa594-fibrinogen (****P<0.0001, paired t-test). Scale bar, 10 μm. (c) Double immunofluorescence of laminin (basal membranes, green) and fibrin (red) shows fibrin deposition in the Virchow Robins space (arrows) as well as parenchymally (arrowheads, boxed area). Higher magnification images of the boxed area show parenchymal fibrin deposition correlating with perivascular inflammation shown with DAPI staining (blue). Scale bars, left: 30 μm; right: 10 μm. (d) Left: Correlated EM within the perivascular space of an inflamed spinal cord vessel shows abundant deposition of densely packed fibrillar material, consistent in ultrastructure with precipitated fibrin. The spinal cord parenchyma shows demyelinated fibers and dystrophic axons. Right: The perivascular space of a larger vessel contains abundant extracellular fibrillar material and macrophage/microglial cells with intracytoplasmatic lipid degradation products. (E, endothelial cell; F, fibrin; M, macrophage/microglia; GL, astrocytes of the glia limitans; BV, blood vessel; L, leukocyte, arrows: demyelinated/partly dystrophic axons). Scale bars, 2 μm.