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Background: Circumventricular organs (CVO) are cerebral areas with incomplete endothelial blood-brain barrier (BBB) and therefore regarded as "gates to the brain". During inflammation, they may exert an active role in determining immune cell recruitment into the brain. Methods: In a longitudinal study we investigated in vivo alterations of CVO during neuroinflammation, applying Gadofluorine M- (Gf) enhanced magnetic resonance imaging (MRI) in experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis. SJL/J mice were monitored by Gadopentate dimeglumine- (Gd-DTPA) and Gf-enhanced MRI after adoptive transfer of proteolipid-protein-specific T cells. Mean Gf intensity ratios were calculated individually for different CVO and correlated to the clinical disease course. Subsequently, the tissue distribution of fluorescence-labeled Gf as well as the extent of cellular inflammation was assessed in corresponding histological slices. Results: We could show that the Gf signal intensity of the choroid plexus, the subfornicular organ and the area postrema increased significantly during experimental autoimmune encephalomyelitis, correlating with (1) disease severity and (2) the delay of disease onset after immunization. For the choroid plexus, the extent of Gf enhancement served as a diagnostic criterion to distinguish between diseased and healthy control mice with a sensitivity of 89% and a specificity of 80%. Furthermore, Gf improved the detection of lesions, being particularly sensitive to optic neuritis. In correlated histological slices, Gf initially accumulated in the extracellular matrix surrounding inflammatory foci and was subsequently incorporated by macrophages/microglia. Conclusion: Gf-enhanced MRI provides a novel highly sensitive technique to study cerebral BBB alterations. We demonstrate for the first time in vivo the involvement of CVO during the development of neuroinflammation. [ABSTRACT FROM AUTHOR]

Background: Two-photon laser scanning microscopy (TPLSM) has become a powerful tool in the visualization of immune cell dynamics and cellular communication within the complex biological networks of the inflamed central nervous system (CNS). Whereas many previous studies mainly focused on the role of effector or effector memory T cells, the role of naïve T cells as possible key players in immune regulation directly in the CNS is still highly debated.Methods: We applied ex vivo and intravital TPLSM to investigate migratory pathways of naïve T cells in the inflamed and non-inflamed CNS. MACS-sorted naïve CD4+ T cells were either applied on healthy CNS slices or intravenously injected into RAG1 -/- mice, which were affected by experimental autoimmune encephalomyelitis (EAE). We further checked for the generation of second harmonic generation (SHG) signals produced by extracellular matrix (ECM) structures.Results: By applying TPLSM on living brain slices we could show that the migratory capacity of activated CD4+ T cells is not strongly influenced by antigen specificity and is independent of regulatory or effector T cell phenotype. Naïve T cells, however, cannot find sufficient migratory signals in healthy, non-inflamed CNS parenchyma since they only showed stationary behaviour in this context. This is in contrast to the high motility of naïve CD4+ T cells in lymphoid organs. We observed a highly motile migration pattern for naïve T cells as compared to effector CD4+ T cells in inflamed brain tissue of living EAE-affected mice. Interestingly, in the inflamed CNS we could detect reticular structures by their SHG signal which partially co-localises with naïve CD4+ T cell tracks.Conclusions: The activation status rather than antigen specificity or regulatory phenotype is the central requirement for CD4+ T cell migration within healthy CNS tissue. However, under inflammatory conditions naïve CD4+ T cells can get access to CNS parenchyma and partially migrate along inflammation-induced extracellular SHG structures, which are similar to those seen in lymphoid organs. These SHG structures apparently provide essential migratory signals for naïve CD4+ T cells within the diseased CNS. [ABSTRACT FROM AUTHOR]