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.

In the bone marrow (BM), memory plasma cells (PCs) survive for long time periods in dedicated microenvironmental survival niches, resting in terms of proliferation. Several cell types, such as eosinophils and reticular stromal cells, have been reported to contribute to the survival niche of memory PCs. However, until now it has not been demonstrated whether the niche is formed by a fixed cellular microenvironment. By intravital microscopy, we provide for the first time evidence that the direct contacts formed between PCs and reticular stromal cells are stable in vivo, and thus the PCs are sessile in their niches. The majority (ß80%) of PCs directly contact reticular stromal cells in a non-random fashion. The mesenchymal reticular stromal cells in contact with memory PCs are not proliferating. On the other hand, we show here that eosinophils in the vicinity of longlived PCs are vigorously proliferating cells and represent a dynamic component of the survival niche. In contrast, if eosinophils are depleted by irradiation, newly generated eosinophils localize in the vicinity of radiation-resistant PCs and the stromal cells. These results suggest that memory PC niches may provide attraction for eosinophils to maintain stability with fluctuating yet essential accessory cells. Keywords: Bone marrow r Intravital microscopy r Plasma cells r Stromal cells r Survival niche See accompanying Commentary by Tellier and KalliesAdditional supporting information may be found in the online version of this article at the publisher's web-site IntroductionThe prominent role of the bone marrow (BM) for the maintenance of resting immunological memory is becoming increasingly evident. Several types of memory lymphocytes have been identified which are maintained predominantly in the BM [1]. Longlived, resting memory plasma cells (PCs) reside in the BM [2] and resting professional memory CD4 + T cells have been located to the BM as well [3]. The BM is thought to sustain the survival of Correspondence: Prof. Anja E. Hauser e-mail: hauser@drfz.de these different memory immune cell populations in cell-specific microanatomical niches [4,5]. PCs have been shown to depend on extrinsic survival factors to survive and their colocalization with several cell types which produce these survival factors has been demonstrated by histology. They are in close contact with reticular stromal cells producing chemokine (CXC motif) ligand 12 (CXCL12) [6], a chemokine which has been shown to regulate PC immigration into the BM [7,8], but which also acts as a potent PC survival factor in vitro [9]. Blocking of leukocyte * These authors contributed equally to this work. Results Plasma cells colocalize with stromal cells in the bone marrowPCs of the BM have been described to contact reticular stromal cells, but this has not been quantified so far, because of the difficulty to identify contacts between PCs and dendritic extensions of stromal cells. Here we used genetic staining of stromal cells to better visualize their ramifications and potential contacts to PCs...

Two-photon microscopy is indispensable for deep tissue and intravital imaging. However, current technology based on single-beam point scanning has reached sensitivity and speed limits because higher performance requires higher laser power leading to sample degradation. We utilize a multifocal scanhead splitting a laser beam into a line of 64 foci, allowing sample illumination in real time at full laser power. This technology requires charge-coupled device field detection in contrast to conventional detection by photomultipliers. A comparison of the optical performance of both setups shows functional equivalence in every measurable parameter down to penetration depths of 200 microm, where most actual experiments are executed. The advantage of photomultiplier detection materializes at imaging depths >300 microm because of their better signal/noise ratio, whereas only charge-coupled devices allow real-time detection of rapid processes (here blood flow). We also find that the point-spread function of both devices strongly depends on tissue constitution and penetration depth. However, employment of a depth-corrected point-spread function allows three-dimensional deconvolution of deep-tissue data up to an image quality resembling surface detection.