Investigation of the cellular and molecular mechanisms of T cell migration across the BBB in the context of MS has become possible with the development of live cell imaging approaches that record the dynamic interaction with the BBB during EAE. Using a flow chamber setup for brain endothelial cell cultures or a microsurgical window for observation of the spinal cord microvasculature, has enabled the study of dynamic T cell interactions with the BBB under physiological flow both in vitro and in vivo.
The in vitro flow chamber with time-lapse live cell imaging has been used to study the post-arrest dynamic behavior of encephalitogenic CD4+ T cells on the inflamed BBB under flow conditions. The cellular and molecular events underlying the multi-step T cell extravasation across the inflamed BBB in vitro have been studied and the functions of different endothelial adhesion molecules in mediating CD4+ T cell arrest, versus polarization and crawling were delineated. These experiments underline the active role of the BBB endothelium in controlling T cell extravasation during immunosurveillance and inflammation. Results in vitro have been confirmed in vivo by two recent studies investigating T cell extravasation across the spinal cord microvasculature during EAE by two-photon IVM [6, 27], which showed that T cells crawl long distances against the direction of the blood-flow on the spinal cord endothelial surface to find a site permissive for diapedesis using the molecular mechanism found in our studies .
Using high resolution in vitro imaging, we are studying the cellular and molecular mechanisms involved in T cell diapedesis across the BBB under physiological flow to determine if T cells breach the BBB via a transcellular or paracellular route. With pMBMEC preparations of gene-targeted mice and fluorescent-tagged adhesion and junctional molecules, it will be possible to distinguish the molecular events in these processes.
It is important to note that although the flow chamber set-up described here is suitable to study the entire multi-step T cell extravasation across the BBB, the combination with time lapse imaging does not allow fast movements as observed during T cell tethering or rolling on the BBB to be recorded. Whereas T cell rolling along the BBB occurs with velocities of several hundreds of μm per second, T cell polarization and crawling events as described here are much slower and occur at velocities of several μm per minute. Thus investigation of T cell tethering and rolling using such an in vitro flow chamber requires real time imaging at 20 images per second, minimum, or even more than 30 images per second.
In contrast, the IVM real time imaging method described here is optimal to study the initial interaction (rolling/capture), the arrest and the firm adhesion of T cells within the spinal cord microvasculature under physiological flow conditions in vivo. Observation times of one minute suffice to study the initial T cell interaction with the spinal cord microvasculature in vivo and therefore avoid phototoxic effects on the vasculature. Similarly, one minute video sequences of the different FOVs at defined time points after systemic T cell infusion will enable study of T cell adhesion to the BBB in vivo over extended times. Due to the short observation times necessary, we have previously used this imaging approach to successfully study the interaction of human T cells with the spinal cord microvasculature during EAE in vivo in immunocompetent mice, since human integrins engage with mouse endothelial ligands comparable to the human endothelial ligands . In this xenogeneic approach we showed that the anti-α4-integrin antibody natalizumab, used for the treatment of relapsing-remitting MS, specifically blocks T cell adhesion, but not rolling, during EAE in vivo.
The spinal cord window described here is located at the level of cervical spinal cord (C7-C5) and allows direct visualization of both the spinal cord leptomeningeal and white matter microvessels under physiological conditions . During EAE, when inflammatory reactions increase the depth of the leptomeningeal space on the surface of the spinal cord, visualization of white matter microvessels is limited due to the limitation of epifluorescence technique which has a tissue penetration of 50-70 μm. In contrast, the lumbar spinal cord window usually employed for live cell imaging in the spinal cord only allows for observation of the leptomeningeal blood vessels, even when employing 2P-IVM with deeper penetration into the tissue . This might be due to the differences in the angioarchitecture at the different levels of the spinal cord.
The IVM approach introduced here can certainly be extended to study the interaction of immune cell subsets other than T cells with the spinal cord microvasculature in vivo. Using the same experimental approach as described for T cells we were able to show that immature dendritic cells migrate into the CNS during EAE and use α4-integrins to adhere to the inflamed spinal cord microvasculature in vivo. A critical prerequisite to study the interaction of a given immune cell subset with the spinal cord microvasculature using the IVM method described here, is to obtain a highly purified population of the cells of interest. This is due to the fact that only a limited number of cells infused into the systemic blood gain access to the observation window of the spinal cord and even less cells (about 10–20 fluorescent immune cells per field of view (FOV) with 5–6 FOV per spinal cord window) are expected to interact with the endothelium of the exposed spinal cord window microvasculature.
To study CD8+ T cell interaction with the spinal cord microvasculature during EAE we have therefore decided to first investigate CD8+ T cells from a TCR transgenic OT-I mouse. This allowed for a homogeneous ovalbumin-specific T cell activation in vitro which resulted in a population of activated CD8+ T cells with more than 95% purity. Here we demonstrated that activated CD8+ T cells successfully interact with the inflamed spinal cord microvessels during EAE. We therefore asked whether α4-integrins, which are essential for the migration of CD4+ T cells across the BBB, play any role in the multi-step CD8+ T cell extravasation across the BBB in vivo. Here we found that α4β7-, β7- or α4-integrins are not required for the rolling and capture of CD8+ T to the inflamed spinal cord white matter microvasculature. This is in accordance with our previous findings demonstrating that β1-integrin deficient CD4+ and CD8+ T cells have no defect in capturing and rolling on the inflamed BBB during EAE  and that natalizumab fails to interfere with the rolling and capturing of human T cells to the inflamed spinal cord microvessels during EAE . Interestingly, although we initially saw a contribution of α4-integrins in mediating the firm adhesion of CD8+ T cells to the inflamed spinal cord microvasculature, this effect was lost mainly due to the low number of firmly adhering CD8+ T cells observed in the control group over time. These observations therefore suggest that stable adhesion of CD8+ T cells to the inflamed BBB in vivo does not critically rely on α4-integrins. Considering our previous observation that β1-integrin deficient CD8+ T cells fail to enter the CNS parenchyma during EAE , we propose that β1-integrin mediated adhesions might be critical at a later step, namely in CD8+ T cell crossing the endothelial basement membrane.
Although the IVM approach described here allows for live cell imaging of immune cell interactions with the spinal cord microvasculature under physiological and pathological conditions, some limitations apply due to the fact that single-photon excitation used in conventional epifluorescence video microscopy requires short wavelength and therefore high energy excitation light. This results in a disadvantageous high risk of phototoxicity and the restriction of imaging depth to 70 μm. These limitations have been overcome by the introduction of two-photon IVM (2P-IVM) which enables deep tissue penetration with less absorption or scattering of fluorescent light than conventional IVM (for details see ). 2P-IVM has a CNS tissue penetration of 800–1000 μm . It yields time-lapse videos with a high 3D resolution allowing observation of immune cell interactions with the spinal cord microvasculature over a long time period. It is therefore suitable for observing the slow post-arrest immune cell interactions with the spinal cord microvasculature such as T cell polarization, crawling and diapedesis taking place at velocities about 10 μm/min in vivo. In contrast, 2P-IVM is not suited to investigate the molecular mechanisms involved in the fast initial T cell interaction steps with the BBB in vivo taking place at velocities of about 40–100 μm/s.
In summary, combining state-of-the-art live cell imaging approaches with in vitro BBB models and sophisticated surgical window preparations for in vivo observation of the CNS microvasculature, provides a powerful experimental approach to identify the molecular mechanisms employed by the BBB to control immune cell trafficking into the CNS. The identification of some of these traffic signals has proved to be of clinical importance as blocking these molecules reduces the migration of pathogenic immune cells into the CNS and proven beneficial for the treatment of MS. In contrast, induction or enhancement of immune cell trafficking signals on the BBB could be beneficial for the treatment of CNS infections or neoplasia.