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

Martin Pohland,1 Robert Glumm,1,2 Frank Wiekhorst,3 Jürgen Kiwit,4 Jana Glumm1,4 1Institute of Cell Biology and Neurobiology, Center for Anatomy, Charité – Universitätsmedizin Berlin, 2Clinic of Neurology, Jüdisches Krankenhaus, 3Department 8.2 Biosignals, Physikalisch-Technische Bundesanstalt, 4Clinic of Neurosurgery, HELIOS Klinikum Berlin Buch, Berlin, Germany 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. Keywords: VSOP, SPIO, hippocampus, organotypic, viability&nbsp

Studying axonal outgrowth and regeneration of the corticospinal tract in organotypic slice cultures

Studies of axonal outgrowth and regeneration after spinal cord injury are hampered by the complexity of the events involved. Here we present a simple and improved in vitro approach to investigate outgrowth, regeneration of the corticospinal tract and intrinsic parenchymal responses. We prepared organotypic co-cultures using explants from the motor cortex of postnatal donor mice, ubiquitously expressing green fluorescent protein, and cervical spinal cord from wild type pups of the same age. Our data show that a) motor-cortical outgrowth is already detectable after one day in culture and is source specific; b) treatment with neurotrophin-3 and C3 transferase from Clostridium botulinum significantly enhances axonal outgrowth during the course of cultivation; c) outgrowing axons form synaptic connections, as demonstrated by immunohistochemistry and calcium imaging; and d) migrating cells of motor-cortical origin can be reliably identified without previous tracing and are mostly neural precursors that survive and mature in the spinal cord parenchyma. Thus, our model is suitable for screening for candidate substances that enhance outgrowth and regeneration of the corticospinal tract and for studying the role of endogenous neural precursors after lesion

Impaired postnatal development of hippocampal neurons and axon projections in the Emx2-/- mutants

The specification and innervation of cerebral subregions is a complex layer-specific process, primed by region-specific transcription factor expression and axonal guidance cues. In Emx2-/- mice, the hippocampus fails to form a normal dentate gyrus as well as the normal layering of principal neurons in the hippocampus proper. Here, we analyzed the late embryonic and postnatal development of the hippocampal formation and its axonal projections in mice lacking Emx2 expression in vitro. As these mutants die perinatally, we used slice cultures of Emx2 mutant hippocampus to circumvent this problem. In late embryonic Emx2-/- cultivated hippocampi, both the perforant path as well as the distribution of calretinin-positive cells are affected. Traced entorhinal afferents in co-cultures with hippocampus from embryonic Emx2-/- mice terminate diffusely in the prospective dentate gyrus in contrast to the layer-specific termination of co-cultures from wild-type littermates. In addition, in brain slice cultures from null mutants the presumptive dentate gyrus failed to develop its normal cytoarchitecture and mature dentate granule cells, including the lack of their mossy fiber projection. Our data indicate that Emx2 is essential for the terminal differentiation of granular cells and the correct formation of extrinsic and intrinsic hippocampal connections

Impaired postnatal development of hippocampal neurons and axon projections in the Emx2-/-mutants.

The specification and innervation of cerebral subregions is a complex layer-specific process, primed by region-specific transcription factor expression and axonal guidance cues. In Emx2-/- mice, the hippocampus fails to form a normal dentate gyrus as well as the normal layering of principal neurons in the hippocampus proper. Here, we analyzed the late embryonic and postnatal development of the hippocampal formation and its axonal projections in mice lacking Emx2 expression in vitro. As these mutants die perinatally, we used slice cultures of Emx2 mutant hippocampus to circumvent this problem. In late embryonic Emx2-/- cultivated hippocampi, both the perforant path as well as the distribution of calretinin-positive cells are affected. Traced entorhinal afferents in co-cultures with hippocampus from embryonic Emx2-/- mice terminate diffusely in the prospective dentate gyrus in contrast to the layer-specific termination of co-cultures from wild-type littermates. In addition, in brain slice cultures from null mutants the presumptive dentate gyrus failed to develop its normal cytoarchitecture and mature dentate granule cells, including the lack of their mossy fiber projection. Our data indicate that Emx2 is essential for the terminal differentiation of granular cells and the correct formation of extrinsic and intrinsic hippocampal connections

Neues zu Pathogenese, Diagnostik und Therapie der Neuromyelitis optica [Recent findings in pathogenesis, diagnostics and therapy of neuromyelitis optica]

Die Neuromyelitis optica (NMO) ist eine in der westlichen Welt seltene und zumeist schwer verlaufende Autoimmunerkrankung des zentralen Nervensystems (ZNS), die sich klinisch an den Sehnerven und dem Rückenmark manifestiert. Zwar war die NMO urspruenglich von Eugene Devic als eigenständige Entitaet beschrieben worden, jedoch wurde sie lange Zeit als seltene Variante der Multiplen Sklerose (MS) gesehen. Seit Kurzem mehren sich Hinweise, dass es sich bei der NMO um ein gesondertes neuroimmunologisches Krankheitsbild handelt, das sich klinisch, radiologisch, histopathologisch und serologisch von der MS abgrenzen laesst. Die ueberarbeiteten Kriterien zur Diagnose der NMO nach Wingerchuk und Kollegen beinhalten Opticusneuritis, akute Myelitis und zwei der drei folgenden Befunde: langstreckige Signalveraenderung ueber mehr als drei Wirbelsegmente im spinalen Kernspintomogramm, eine fuer die Multiple Sklerose nicht hinreichende zerebrale Magnetresonanz Bildgebung zu Erkrankungsbeginn sowie ein positiver Nachweis von NMO-Antikörpern im Serum. Kuerzlich wurde mit dem Wasserkanal Aquaporin-4 (AQP-4) ein Zielantigen der NMO-Immunglobuline identifiziert. Mittels eines neuen Radioimmunopraezipitations Assay (RIPA), einer hoch-spezifischen, Untersucher unabhaengigen, reproduzierbaren und kostenguenstigen Nachweismethode besteht erstmals die Moeglichkeit einer fruehzeitigen Differenzierung der NMO von der MS in einem grossen Patientenkollektiv und damit kuenftig die zuegigere Einleitung wirksamer Therapien der von schwerer Behinderung bedrohten Patienten

Characterization of natural killer cells in paired CSF and blood samples during neuroinflammation

Natural killer (NK) cells from paired CSF and blood samples of patients with multiple sclerosis (MS), other neuroinflammatory diseases (IND), and non-inflammatory neurological diseases (NIND) were characterized using flow cytometry. NK cell frequency in CSF was overall decreased compared to blood, particularly in MS patients. In contrast to blood NK cells, during neuroinflammation, CSF NK cells display an immature phenotype with bright expression of CD56 and CD27 and reduced CX3CR1 expression. Our findings suggest that, as for central memory T cells, CSF may represent an intermediary compartment for NK cell trafficking and differentiation before entering the CNS parenchyma

CNS-irrelevant T-cells enter the brain, cause blood-brain barrier disruption but no glial pathology

Invasion of autoreactive T-cells and alterations of the blood-brain barrier (BBB) represent early pathological manifestations of multiple sclerosis and its animal model experimental autoimmune encephalomyelitis (EAE). Non-CNS-specific T-cells are also capable of entering the CNS. However, studies investigating the spatial pattern of BBB alterations as well as the exact localization and neuropathological consequences of transferred non-CNS-specific cells have been thus far lacking. Here, we used magnetic resonance imaging and multiphoton microscopy, as well as histochemical and high-precision unbiased stereological analyses to compare T-cell transmigration, localization, persistence, relation to BBB disruption and subsequent effects on CNS tissue in a model of T-cell transfer of ovalbumin (OVA)- and proteolipid protein (PLP)-specific T-cells. BBB alterations were present in both EAE-mice and mice transferred with OVA-specific T-cells. In the latter case, BBB alterations were less pronounced, but the pattern of initial cell migration into the CNS was similar for both PLP- and OVA-specific cells [mean (SEM), 95 x 10(3) (7.6 x 10(3)) and 88 x 10(3) (18 x 10(3)), respectively]. Increased microglial cell density, astrogliosis and demyelination were, however, observed exclusively in the brain of EAE-mice. While mice transferred with non-neural-specific cells showed similar levels of rhodamine-dextran extravasation in susceptible brain regions, EAE-mice presented huge BBB disruption in brainstem and moderate leakage in cerebellum. This suggests that antigen specificity and not the absolute number of infiltrating cells determine the magnitude of BBB disruption and glial pathology