Neuronal damage in autoimmune neuroinflammation is the correlate for long-term disability in multiple sclerosis (MS) patients. Here, we investigated the role of immune cells in neuronal damage processes in animal models of MS by monitoring experimental autoimmune encephalomyelitis (EAE) by using twophoton microscopy of living anaesthetized mice. In the brainstem, we detected sustained interaction between immune and neuronal cells, particularly during disease peak. Direct interaction of myelin oligodendrocyte glycoprotein (MOG)-specific Th17 and neuronal cells in demyelinating lesions was associated with extensive axonal damage. By combining confocal, electron, and intravital microscopy, we showed that these contacts remarkably resembled immune synapses or kinapses, albeit with the absence of potential T cell receptor engagement. Th17 cells induced severe, localized, and partially reversible fluctuation in neuronal intracellular Ca 2+ concentration as an early sign of neuronal damage. These results highlight the central role of the Th17 cell effector phenotype for neuronal dysfunction in chronic neuroinflammation.

Chronic inflammation in various organs, such as the brain, implies that different subpopulations of immune cells interact with the cells of the target organ. To monitor this cellular communication both morphologically and functionally, the ability to visualize more than two colors in deep tissue is indispensable. Here, we demonstrate the pronounced power of optical parametric oscillator (OPO)-based two-photon laser scanning microscopy for dynamic intravital imaging in hardly accessible organs of the central nervous and of the immune system, with particular relevance for long-term investigations of pathological mechanisms (e.g., chronic neuroinflammation) necessitating the use of fluorescent proteins. Expanding the wavelength excitation farther to the infrared overcomes the current limitations of standard Titanium:Sapphire laser excitation, leading to 1), simultaneous imaging of fluorophores with largely different excitation and emission spectra (e.g., GFP-derivatives and RFP-derivatives); and 2), higher penetration depths in tissue (up to 80%) at higher resolution and with reduced photobleaching and phototoxicity. This tool opens up new opportunities for deep-tissue imaging and will have a tremendous impact on the choice of protein fluorophores for intravital applications in bioscience and biomedicine, as we demonstrate in this work.

In the course of autoimmune CNS inflammation, inflammatory infiltrates form characteristic perivascular lymphocyte cuffs by mechanisms that are not yet well understood. Here, intravital two-photon imaging of the brain in anesthetized mice, with experimental autoimmune encephalomyelitis, revealed the highly dynamic nature of perivascular immune cells, refuting suggestions that vessel cuffs are the result of limited lymphocyte motility in the CNS. On the contrary, vessel-associated lymphocyte motility is an actively promoted mechanism which can be blocked by CXCR4 antagonism. In vivo interference with CXCR4 in experimental autoimmune encephalomyelitis disrupted dynamic vessel cuffs and resulted in tissue-invasive migration. CXCR4-mediated perivascular lymphocyte movement along CNS vessels was a key feature of CD4(+) T cell subsets in contrast to random motility of CD8(+) T cells, indicating a dominant role of the perivascular area primarily for CD4(+) T cells. Our results visualize dynamic T cell motility in the CNS and demonstrate differential CXCR4-mediated compartmentalization of CD4(+) T-cell motility within the healthy and diseased CNS.