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-P:M). 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 I 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 offractalkine receptor (CX3CRl) 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 CDllb, CD4S, 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 CDII b morphology. M2 microglia/macrophage peaked at 24h after injury, whereas MI 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.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:594845 |
| Date | January 2012 |
| Creators | Fumagalli, Stefano |
| Publisher | Open University |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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