Studies on Vascular Changes and Immune Cell Role in the Ischemic Brain By In Vivo Two-Photon Microscopy

Fumagalli, Stefano (2013). Studies on Vascular Changes and Immune Cell Role in the Ischemic Brain By In Vivo Two-Photon Microscopy. PhD thesis The Open University.



Mechanisms contributing to the inflammatory cascade, including vascular modifications and immune cell recruitment/activation, act with high dynamism within a three-dimensional space, thus being ideally investigated by in vivo two-photon microscopy (2-PM). In this thesis I applied in vivo 2-PM and quantitative bright field and confocal microscopy to explore cerebrovascular remodelling, immune cell dynamism and phenotype in two models of ischemia in mice achieved by transient or permanent middle cerebral artery occlusion. To specifically visualize microglia and T-cells, I used cx3crl eGFP and hCD2_eGFP mice, respectively.

I imaged animals before and at different time points after ischemia, and quantified blood flow velocity and extravasation in individual vessels. After reperfusion, blood flow exhibited >80% drop in most vessels and extravasation established as early as 20min after ischemia onset. In this ischemic territory, I analyzed motility and morphology of GFP+ microglia. Microglia were stationary and became ameboid at 24h after injury. The absence of fractalkine receptor (CX3CR1) prevented the ameboid switch and favoured a protective M2 microglia polarization, characterized by decreased CD68 and iNOS expression and increased Yml expression. To assess the temporal evolution of microglia/macrophage polarization in the ischemic brain, I investigated the expression and coexpression pattern of CD11b, CD45, CD68, Yml, CD206 and iNOS after ischemia by conventional immunohistochemistry. Microglia/macrophages showed multiple polarization states, with a specific pattern of distribution and association with globular or ramified CD11b morphology. M2 microglia/macrophage peaked at 24h after injury, whereas Ml cells peaked at 48h. The phagocytic activity (CD68), mainly confined at the lesion borders at 6h-48h, dramatically increased and occupied all the ischemic territory at 7d.

I finally investigated T-cells dynamism within the ischemic territory by in vivo 2-PM. I described two populations characterized by different track velocities and found that motile cells preferentially moved along the perivascular space, where they contacted astrocytes and perivascular macrophages.

These data provide novel information on the inflammatory response after stroke and pave the way for developing strategies resulting in promotion of a protective inflammatory phenotype.

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