Biocompatibility of very small superparamagnetic iron oxide nanoparticles in murine organotypic hippocampal slice cultures and the role of microglia

Abstract: Superparamagnetic iron oxide nanoparticles (SPIO) are applied as contrast media for magnetic resonance imaging (MRI) and treatment of neurologic diseases despite the fact that important information concerning their local interactions is still lacking. Due to their small size, SPIO have great potential for magnetically labeling different cell populations, facilitating their MRI tracking in vivo. Before SPIO are applied, however, their effect on cell viability and tissue homoeostasis should be studied thoroughly. We have previously published data showing how citrate-coated very small superparamagnetic iron oxide particles (VSOP) affect primary microglia and neuron cell cultures as well as neuron-glia cocultures. To extend our knowledge of VSOP interactions on the three-dimensional multicellular level, we further examined the influence of two types of coated VSOP (R1 and R2) on murine organotypic hippocampal slice cultures. Our data show that 1) VSOP can penetrate deep tissue layers, 2) long-term VSOP-R2 treatment alters cell viability within the dentate gyrus, 3) during short-term incubation VSOP-R1 and VSOP-R2 comparably modify hippocampal cell viability, 4) VSOP treatment does not affect cytokine homeostasis, 5) microglial depletion decreases VSOP uptake, and 6) microglial depletion plus VSOP treatment increases hippocampal cell death during short-term incubation. These results are in line with our previous findings in cell coculture experiments regarding microglial protection of neurite branching. Thus, we have not only clarified the interaction between VSOP, slice culture, and microglia to a degree but also demonstrated that our model is a promising approach for screening nanoparticles to exclude potential cytotoxic effects

Gadofluorine M-enhanced MRI shows involvement of circumventricular organs in neuroinflammation

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Methods</p> <p>In a longitudinal study we investigated <it>in vivo </it>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.</p> <p>Results</p> <p>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.</p> <p>Conclusion</p> <p>Gf-enhanced MRI provides a novel highly sensitive technique to study cerebral BBB alterations. We demonstrate for the first time <it>in vivo </it>the involvement of CVO during the development of neuroinflammation.</p

In vivo imaging of lymphocytes in the CNS reveals different behaviour of naïve T cells in health and autoimmunity

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Methods</p> <p>We applied <it>ex vivo </it>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.</p> <p>Results</p> <p>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.</p> <p>Conclusions</p> <p>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.</p

Axonal outgrowth in the hippocampal formation during development and after lesion

Titelblatt und Inhaltsverzeichnis Einleitung Material und Methoden Ergebnisse Diskussion Abbildungsverzeichnis Literaturverzeichnis Danksagung Lebenslauf ErklärungWährend der Entwicklung haben die Nervenzellen des Gehirns die bemerkenswerte Fähigkeit, gegenseitige Verbindungen auszubilden, ein Entwicklungsvorgang, der präzises axonales Auswachsen, Wegfindung und korrekte Synapsenbildung voraussetzt. Im Gegensatz dazu, ist die Fähigkeit, neuronale Kontakte nach Läsion wieder herzustellen im ZNS sehr begrenzt. Zunächst untersuchten wir in vitro die späte embryonale und postnatale Entwicklung der hippocampalen Formation mit ihren Projektionen, in für das Homeobox-Gen Emx2-defizienten Mäusen. Da die Emx2-Knockout-Tiere perinatal versterben, haben wir organotypische Schnittkulturen hergestellt. In Schnitten von Knockout-Tieren war die Zytoarchitektur des prospektiven Gyrus dentatus gestört, reiften die Körnerzellen nicht aus und bildeten auch keinen Moosfasertrakt. Diese Befunde zeigen, dass Emx2 essentiell für die endgültige Differenzierung der Körnerzellen und ihrer korrekten Ausbildung der intrinsischen hippocampalen Projektionen ist. Im nächsten Versuchsaufbau konnten wir unter Verwendung der organotypischen Kokultur und anhand von in vivo Experimenten weiterhin in unseren Untersuchungen belegen, dass in den Vorgängen des Auswachsens, der Wegfindung und der synaptischen Zielfindung während der Entwicklung und nach Läsion, die Entität der Polysialinsäure (Polysialic Acid = PSA) involviert ist. Diese stellt eine einzigartige während der Entwicklung regulierte posttranslationale Modifikation des Neuralen Zelladhäsionsmolekül (Neural Cell Adhesion Molecule) NCAM dar. Durch die Verwendung eines chemisch veränderten Sialinsäurevorläufers, konnten wir die Polysialylierung von NCAM zu definierten Entwicklungs- und postläsionalen Zeitpunkten hemmen. Der PSA- Mangel bewirkte nicht nur eine fehlerhafte Moosfaserverteilung in der CA3- Pyramidenzellband, sondern auch das aberrante Eindringen von Moosfasern in die CA1-Region in vivo und förderte das axonalen Auswachsens in vitro. Des Weiteren war die Reinnervation der Moosfasern nach Läsion durch die Inhibition der PSA-Synthese in vitro signifikant erhöht. Basierend auf diesen Ergebnissen schlussfolgern wir, dass PSA notwendig für die axonale Zielfindung während der Ausbildung neuraler Schaltkreise ist und andererseits die Regeneration nach Läsion stört. Dieses Fazit impliziert umgekehrt einen positiven Effekt der pharmakologischen Modifikation von NCAM auf die Regeneration nach axonaler Schädigung, die sich in weiterführenden Experimenten als viel versprechend, in Hinblick auf einen möglichen therapeutischen Nutzen, herausstellen könnte. Um das Phänomen des axonalen Auswachsens besser untersuchen zu können, variierten wir das bereits etablierte Modell der organotypischen Kokultur. In dieser Arbeit nutzten wir die Axonales Auswachsen in der Hippocampusformation 58 organotypische Kokultur mit neugeborener wildtyp und β-Aktin-GFP transgener Maus. Dies erlaubte es uns, die Axone in situ zu markieren und die gesamte Projektion zu verfolgen. In Kokulturen von entorhinalem Cortex und Hippocampus terminierten die GFP-markierten entorhinalen Axone und die kommissuralen Axone in den korrekten hippocampalen Schichten. Ausgehend von diesen Befunden, konnten wir demonstrieren, dass dieser Chimären-Kokultur- Ansatz dazu geeignet ist, die Gesamtheit sich entwickelnder Projektionen darzustellen und die Möglichkeit bietet, direkt das axonale Ansteuern und Auswachsen lebender Neuronen zu beobachten.During development, nerve cells within the brain have the remarkable ability to form mutual interconnections, a process that relies on precisely orchestrated steps of axonal outgrowth, pathfinding, and correct synaptic targeting. In contrast, reestablishing neuronal circuits after lesion in the CNS is extremely limited. First we analysed the late embryonic and postnatal development of the hippocampal formation and its axonal projections in mice lacking the homeobox gene Emx2 expression in vitro. Since the Emx2 mutants die perinatally we used slice cultures of Emx2 mutant hippocampus to circumvent this problem. In mutant cultured hippocampus the presumptive dentate gyrus failed to develop its normal cytoarchitecture and mature dentate granule cells, including the lack of their mossy fiber projection. These data indicate that Emx2 is essential for the terminal differentiation of granule cells and the correct formation of intrinsic hippocampal connections. In another set-up, using the model of the organotypic slice culture as well as in vivo experiments in our investigations we were able to provide confirmation, that the processes of outgrowth, pathfinding, and synaptic targeting during development and following lesion involve the polysialic acid (PSA) moiety, which is a unique, developmentally regulated posttranslational modification of the neural cell adhesion molecule (NCAM). Using a chemically modified sialic acid precursor, we inhibited NCAM polysialylation at selected developmental and postlesional time points. PSA deficiency resulted not only in an abnormal mossy fiber pattern in the CA3 pyramidal layer but also in aberrant invasion of mossy fibers into the CA1 region in vivo and promotion of axonal outgrowth in vitro. Furthermore, the reinnervation by mossy fibers after lesion was significantly enhanced when inhibiting PSA formation in vitro. Based on our data, we conclude that PSA is necessary for correct axonal targeting during neuronal circuit formation, but inhibits regeneration after lesion. Conversely, these results imply that pharmacological modification of NCAM could be beneficial for regeneration following axonal damage, and further experiments may be promising with regard to a potential therapeutical use of this approach. And finally to better investigate the phenomenon of axonal outgrowth, we modified the wellestablished organotypic slice culture approach. Here we used organotypic slice co-cultures from neonate wild-type mice and ß -actin-gfp transgenic mice, which allowed us to prelabel the vital axons and to monitor the complete axonal projection. In co-cultures from entorhinal cortex and hippocampus, gfp-labeled entorhinal axons and commissural projections terminate in their correct hippocampal layers. From our data, we demonstrate that this chimaeric co-culture Axonales Auswachsen in der Hippocampusformation 60 approach is appropriate in tracing entire developing projections and may serve as a tool in directly observing the navigation and axonal elongation of living neurons

In Vivo Imaging of Partially Reversible Th17 Cell-Induced Neuronal Dysfunction in the Course of Encephalomyelitis

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 two-photon 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 Ca2+ 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