RESUMEN
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.
Asunto(s)
Supervivencia Celular/efectos de los fármacos , Hipocampo/citología , Nanopartículas de Magnetita/administración & dosificación , Ensayo de Materiales/métodos , Microglía/citología , Animales , Células Cultivadas , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Nanopartículas de Magnetita/química , Ratones , Microglía/efectos de los fármacos , Microglía/metabolismo , Técnicas de Cultivo de ÓrganosRESUMEN
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 1 d 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 induction.
Asunto(s)
Axones/efectos de los fármacos , Regeneración Nerviosa/efectos de los fármacos , Tractos Piramidales/crecimiento & desarrollo , ADP Ribosa Transferasas/farmacología , Actinas/genética , Animales , Toxinas Botulínicas/farmacología , Movimiento Celular , Corteza Cerebral/citología , Corteza Cerebral/crecimiento & desarrollo , Proteínas Fluorescentes Verdes , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Corteza Motora/crecimiento & desarrollo , Factores de Crecimiento Nervioso/farmacología , Técnicas de Cultivo de Órganos , Tractos Piramidales/citología , Médula Espinal/crecimiento & desarrollo , Sinapsis/efectos de los fármacosRESUMEN
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.
Asunto(s)
Encefalitis , Células Asesinas Naturales/patología , Esclerosis Múltiple , Adulto , Antígenos CD/metabolismo , Encefalitis/sangre , Encefalitis/líquido cefalorraquídeo , Encefalitis/patología , Femenino , Citometría de Flujo , Humanos , Masculino , Persona de Mediana Edad , Esclerosis Múltiple/sangre , Esclerosis Múltiple/líquido cefalorraquídeo , Esclerosis Múltiple/patología , Linfocitos T/patología , Adulto JovenRESUMEN
During development, axonal projections have a remarkable ability to innervate correct dendritic subcompartments of their target neurons and to form regular neuronal circuits. Altered axonal targeting with formation of synapses on inappropriate neurons may result in neurodevelopmental sequelae, leading to psychiatric disorders. Here we show that altering the expression level of the polysialic acid moiety, which is a developmentally regulated, posttranslational modification of the neural cell adhesion molecule NCAM, critically affects correct circuit formation. Using a chemically modified sialic acid precursor (N-propyl-D: -mannosamine), we inhibited the polysialyltransferase ST8SiaII, the principal enzyme involved in polysialylation during development, at selected developmental time-points. This treatment altered NCAM polysialylation while NCAM expression was not affected. Altered polysialylation resulted in an aberrant mossy fiber projection that formed glutamatergic terminals on pyramidal neurons of the CA1 region in organotypic slice cultures and in vivo. Electrophysiological recordings revealed that the ectopic terminals on CA1 pyramids were functional and displayed characteristics of mossy fiber synapses. Moreover, ultrastructural examination indicated a "mossy fiber synapse"-like morphology. We thus conclude that homeostatic regulation of the amount of synthesized polysialic acid at specific developmental stages is essential for correct synaptic targeting and circuit formation during hippocampal development.
Asunto(s)
Homeostasis , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Ácidos Siálicos/metabolismo , Sinapsis/metabolismo , Animales , Ratones , Ratones Endogámicos C57BLRESUMEN
BACKGROUND: 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. METHODS: We applied ex vivo 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. RESULTS: 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. CONCLUSIONS: 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.
Asunto(s)
Autoinmunidad/inmunología , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/inmunología , Sistema Nervioso Central/citología , Sistema Nervioso Central/inmunología , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/inmunología , Animales , Linfocitos T CD4-Positivos/fisiología , Movimiento Celular/inmunología , Células Cultivadas , Encefalomielitis Autoinmune Experimental/inmunología , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Activación de Linfocitos/inmunología , Tejido Linfoide/citología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Confocal/métodos , Subgrupos de Linfocitos T/fisiologíaRESUMEN
BACKGROUND: 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. METHODS: In a longitudinal study we investigated in vivo 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. RESULTS: 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. CONCLUSION: Gf-enhanced MRI provides a novel highly sensitive technique to study cerebral BBB alterations. We demonstrate for the first time in vivo the involvement of CVO during the development of neuroinflammation.
Asunto(s)
Barrera Hematoencefálica/patología , Encéfalo/anatomía & histología , Encéfalo/patología , Encefalomielitis Autoinmune Experimental/patología , Inflamación/patología , Imagen por Resonancia Magnética/métodos , Compuestos Organometálicos/metabolismo , Animales , Barrera Hematoencefálica/fisiología , Barrera Hematoencefálica/fisiopatología , Encéfalo/fisiopatología , Medios de Contraste/metabolismo , Encefalomielitis Autoinmune Experimental/fisiopatología , Matriz Extracelular/metabolismo , Femenino , Fluorocarburos , Inflamación/fisiopatología , Estudios Longitudinales , Ratones , Monocitos/inmunologíaRESUMEN
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 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.
Asunto(s)
Encefalomielitis Autoinmune Experimental/inmunología , Interleucina-17/fisiología , Neuronas/fisiología , Linfocitos T Colaboradores-Inductores/fisiología , Animales , Apoptosis , Axones/fisiología , Calcio/metabolismo , Comunicación Celular , Movimiento Celular , Células Cultivadas , Ratones , Ratones Endogámicos C57BL , Receptores de N-Metil-D-Aspartato/fisiología , Sinapsis/fisiologíaRESUMEN
OBJECTIVE: Multiple sclerosis (MS) is considered a chronic inflammatory and demyelinating disease of the central nervous system. Evidence that axonal and neuronal pathology contributes to the disease is accumulating, however, the distribution of neuronal injury as well as the underlying mechanisms have not yet been fully clarified. Here, we investigated the role of neuronal cell loss in MS and its animal model, experimental autoimmune encephalomyelitis (EAE). METHODS: We performed electrophysiological investigations in MS patients, including assessment of compound muscle action potentials and motor unit numbers and quantified neuronal cell loss in human MS samples and different EAE models by high-precision stereology. RESULTS: Both electrophysiological and morphological analyses indicated a massive loss of lower motor neurons in MS patients. We regularly found dying spinal motor neurons surrounded by CD3+ (CD4+ as well as CD8+) T cells expressing tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). We observed a similar degree of damage and immune attack in different variants of EAE; the lower motor neurons were preserved in adoptive transfer EAE induced with TRAIL-deficient T lymphocytes. INTERPRETATION: Our study indicates that damage to lower motor neurons and TRAIL-mediated inflammatory neurodegeneration in the spinal cord contribute to MS pathology.
Asunto(s)
Encefalomielitis Autoinmune Experimental/patología , Neuronas Motoras/patología , Esclerosis Múltiple/patología , Traslado Adoptivo , Adulto , Animales , Apoptosis/inmunología , Recuento de Células , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/inmunología , Femenino , Humanos , Activación de Linfocitos/inmunología , Masculino , Persona de Mediana Edad , Esclerosis Múltiple/inmunología , Degeneración Nerviosa/inmunología , Degeneración Nerviosa/patología , Neuronas/inmunología , Neuronas/patología , Médula Espinal/inmunología , Médula Espinal/patología , Linfocitos T/inmunología , Linfocitos T/patología , Ligando Inductor de Apoptosis Relacionado con TNF/inmunologíaRESUMEN
Repair processes that are activated in response to neuronal injury, be it inflammatory, ischaemic, metabolic, traumatic or other cause, are characterized by a failure to replenish neurons and by astrogliosis. The underlying molecular pathways, however, are poorly understood. Here, we show that subtle alterations of the redox state, found in different brain pathologies, regulate the fate of mouse neural progenitor cells (NPCs) through the histone deacetylase (HDAC) Sirt1. Mild oxidation or direct activation of Sirt1 suppressed proliferation of NPCs and directed their differentiation towards the astroglial lineage at the expense of the neuronal lineage, whereas reducing conditions had the opposite effect. Under oxidative conditions in vitro and in vivo, Sirt1 was upregulated in NPCs, bound to the transcription factor Hes1 and subsequently inhibited pro-neuronal Mash1. In utero shRNA-mediated knockdown of Sirt1 in NPCs prevented oxidation-mediated suppression of neurogenesis and caused upregulation of Mash1 in vivo. Our results provide evidence for an as yet unknown metabolic master switch that determines the fate of neural progenitors.
Asunto(s)
Diferenciación Celular/fisiología , Neuronas/fisiología , Sirtuinas/metabolismo , Células Madre/fisiología , Animales , Astrocitos/citología , Astrocitos/fisiología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Encéfalo/citología , Encéfalo/metabolismo , Encéfalo/patología , Linaje de la Célula , Células Cultivadas , Proteínas Co-Represoras , Encefalomielitis Autoinmune Experimental/metabolismo , Encefalomielitis Autoinmune Experimental/patología , Femenino , Regulación del Desarrollo de la Expresión Génica , Histonas/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Morfogénesis/fisiología , Neuronas/citología , Oxidación-Reducción , Embarazo , Regiones Promotoras Genéticas , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Sirtuina 1 , Sirtuinas/genética , Células Madre/citología , Factor de Transcripción HES-1 , Transcripción GenéticaRESUMEN
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.
Asunto(s)
Barrera Hematoencefálica/fisiología , Encéfalo/citología , Encéfalo/fisiología , Neuroglía/fisiología , Linfocitos T/fisiología , Animales , Recuento de Células , Sistema Nervioso Central/citología , Sistema Nervioso Central/fisiología , Interpretación Estadística de Datos , Encefalitis/patología , Femenino , Inmunohistoquímica , Imagen por Resonancia Magnética , Ratones , Ratones Endogámicos C57BL , Microscopía de Fluorescencia por Excitación Multifotónica , Proteína Proteolipídica de la Mielina/inmunología , Ovalbúmina/inmunología , Permeabilidad , Linfocitos T/inmunologíaRESUMEN
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.