Figure 5: Perivascular cluster formation requires fibrin deposition and precedes demyelination.

Representative in vivo images (a) and correlated histology (b,c) from the same mice are shown at pre-onset EAE, peak EAE, and peak EAE after hirudin administration. (a) In vivo imaging of microglial clusters (green) in relation to the vasculature (red) in the mouse EAE spinal cord. Scale bar, 10 μm. (b) Fibrinogen immunoreactivity. Scale bar, 10 μm. (c) Myelin staining with LFB (blue) counterstained with PAS (phagocytes, purple). Scale bar, 10 μm. (d) Number of clusters, fibrinogen immunoreactivity and demyelination in the spinal cord of healthy, EAE, and hirudin-treated mice. In healthy controls (n=5), microglial clusters, fibrin and demyelination were undetectable (UN). In pre-onset EAE (n=5), microglial clusters and fibrin were present, but there was no demyelination. At the peak of EAE, there was an increase in microglial clusters (n=9), fibrin deposition (n=7) and demyelination (n=8). At the peak of EAE in hirudin-treated mice (n=9), microglial clusters, fibrin deposition and demyelination were significantly reduced. Values are mean±s.e.m. **P<0.01, ***P<0.001 (one-way analysis of variance (ANOVA)), *P<0.05 (Mann–Whitney test). (e) Correlation analysis of the number of clusters with fibrin deposition (n= 15, R²=0.5054, P<0.01, F-test) and demyelination (n=16, R²=0.5692, P<0.001, F-test).