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Regional differences in viscoelasticity in normal and inflamed mouse brain
Abstracts
105
Fibroblast growth factor 9 (FGF9) plays important roles in coordinating the proliferation, differentiation and survival of neurons and glia in the developing nervous system, but it is only now becoming apparent that this pleiotropic growth factor may also play an important role in the pathogenesis of multiple sclerosis(MS). Our data demonstrate that FGF9 is up regulated in active MS lesions where it appears to represent a predominately oligodendroglial response to inflammatory demyelination. In vitro studies demonstrate that FGF9 is a powerful inhibitor of (re)myelination, but this is not mediated by a direct effect of FGF9 on cells of the oligodendrocyte lineage, but in response to soluble products secreted by FGF9 signal transduction in astrocytes. Transcriptional profiling of the effects of FGF9 in myelinating cultures demonstrates that it induces complex downstream changes in gene expression that result in a pro-inflammatory signalling environment in which (re)myelination fails. These effects are associated with marked increases in expression Spry2, Spry4, Dusp5, and Dusp6, four well characterised negative feedback inhibitors of FGF signalling. In order to investigate the pathophysiological significance of these in vitro observations we are exploring expression of selected candidates in MS lesions. This confirmed that FGF9 expression is up regulated in oligodendrocytes, and to a lesser extent in astrocytes, in both active acute lesions and at the active rims of chronic active lesions and that this is associated with increased expression of Sprouty2 and Sprouty4 by astrocytes. In contrast Sprouty2/4 expression was minimal in normal appearing white matter adjacent to these lesions and in white matter from healthy controls. These findings provide further evidence that FGF signalling in astrocytes plays a role in the development of MS lesions. Elucidating the function of Spry2/4 during lesion development will help determine how the astrocytic response in MS may be manipulated to promote remyelination; a strategy predicted to restore function and prevent further axonal loss in MS.
previous study in SJL mice, C57Bl/6 mice did not show altered brain viscoelasticity. To establish that stable viscoelastic measurements reflected the reduced brain inflammation in this model compared to the SJL mice, we induced EAE interferon-gamma-deficient C57Bl/6B6 mice, in which a more severe EAE phenotype with increased inflammation in the cerebellum and brainstem has been previously reported. Using this model, we could confirm cerebral inflammation in vivo by the presence of gadolinium-enhancing cerebellar lesions with T1-weighted MRI, and ex vivo, by histopathology. MRE scans of IFNgamma −/− mice showed reduced brain elasticity during EAE, measured at 14 and 21 days post-immunization. Interestingly, elasticity changes occurred first in the anterior brain region and later in the posterior region. Within the timeframe examined in this study, the ‘viscosity’ of the tissue was not altered. Currently, we are examining the histological alteration underlying these findings. Together these results highlight the relationship between alterations in the mechanical properties of the brain and inflammation, and underscore the potential experimental and diagnostic utility of MRE to detect these phenomena during CNS disease.
doi:10.1016/j.jneuroim.2014.08.281
Neuroinflammatory diseases are characterized by infiltration of lymphocytes into the CNS, followed by demyelination and axonal degeneration. Evidence suggests that inflammatory factors released from activated T-cells induce neurotoxicity and impair function of neural stem cells. However, the effect of activated T-cells on the function of oligodendrocyte progenitor cells (OPCs) is uncertain, partly due to the difficulty in obtaining adult human oligodendrocytes. In this study, we used iNSCs to obtain OPCs and determined the effect of inflammatory factors on OPCs. Neural stem cells were induced from adult peripheral CD34 + cells by transduction using Sendai virus encoding transcription factor constructs of sox2, oct3/4, c-myc and klf4 and incubation in neural stem cell medium. The resulting cells were differentiated into OPCs and then exposed to supernatants from activated T-cells. Cell proliferation was determined by EdU incorporation assay and CellQuanti-Blue assay. Cell cycle progression was studied by transfecting OPCs with Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI). VEGF-A and its receptors were studied by RT-PCR and western-blot assay. We found that supernatants from activated T cells increased OPC proliferation by regulating cell cycle progression. While VEGF-A expression was increased in T cells after activation, immunodepletion of VEGF-A from activated T cell supernatants significantly attenuated activated T cells induced OPC proliferation. Furthermore, we found that VEGF receptor 2 (VEGF-R2) was expressed on OPC and specific inhibition of VEGF-R2 with siRNA or inhibitor also attenuated activated T cell supernatants induced OPC proliferation. The effects of T cells and VEGF-A on OPC proliferation were also confirmed in primary human fetal OPC cultures. These results indicate that OPCs generated from CD34-derived neural stem cells behave similarly as primary OPCs in response to treatment by activated T cells and could be used to study the physiological functions and pathogenesis of OPCs, providing a specific advantage especially by using patient specific blood samples. Further, using this model we found that VEGF-A released from activated T cells could enhance OPC proliferation through activating
220 Regional differences in viscoelasticity in normal and inflamed mouse brain Jason Millwarda, Jing Guob, Dominique Berndta, Jürgen Braunb, Ingolf Sackb, Carmen Infante-duartea a Institute for Medical Immunology, Charité — Universitätmedizin Berlin, Berlin, Germany; bDepartment of Radiology, Charité — Universitätmedizin Berlin, Berlin, Germany
Magnetic resonance elastography (MRE) is a novel imaging method that reveals the structural and mechanical properties of tissue, which can be modelled as a combination of ‘elasticity’ and ‘viscosity’ of in response to external forces. Tissue mechanical properties arising from cell and extracellular matrix architecture can be altered during pathological processes such as inflammation. It was recently shown using MRE that brains of multiple sclerosis (MS) patients had reduced viscoelasticity i.e. they were ‘softer’ compared to healthy controls. Our previous study also showed reduced viscoelasticity in EAE in SJL (a model of RRMS). In the present study, we performed MRE in the chronic EAE model in C57Bl/6 mice, and considered distinct brain regions. By performing the scan in the sagittal plane with the 7 Tesla animal MRI scanner, we could analyze the anterior and the posterior brain regions separately. MRE scans of non-EAE and EAE mice revealed that the posterior brain region has a lower viscoelasticity than the anterior region — i.e. the cerebellum/brainstem region is intrinsically ‘softer’ than the rest of the brain. However, during EAE, and in contrast to our
doi:10.1016/j.jneuroim.2014.08.282
527 Activated T cells induce oligodendrocyte progenitor cells proliferation by releasing VEGF-A Elliot Choi, Tongguang Wang, Avindra Nath National Institutes of Health, NINDS, Bethesda, United States
105
Fibroblast growth factor 9 (FGF9) plays important roles in coordinating the proliferation, differentiation and survival of neurons and glia in the developing nervous system, but it is only now becoming apparent that this pleiotropic growth factor may also play an important role in the pathogenesis of multiple sclerosis(MS). Our data demonstrate that FGF9 is up regulated in active MS lesions where it appears to represent a predominately oligodendroglial response to inflammatory demyelination. In vitro studies demonstrate that FGF9 is a powerful inhibitor of (re)myelination, but this is not mediated by a direct effect of FGF9 on cells of the oligodendrocyte lineage, but in response to soluble products secreted by FGF9 signal transduction in astrocytes. Transcriptional profiling of the effects of FGF9 in myelinating cultures demonstrates that it induces complex downstream changes in gene expression that result in a pro-inflammatory signalling environment in which (re)myelination fails. These effects are associated with marked increases in expression Spry2, Spry4, Dusp5, and Dusp6, four well characterised negative feedback inhibitors of FGF signalling. In order to investigate the pathophysiological significance of these in vitro observations we are exploring expression of selected candidates in MS lesions. This confirmed that FGF9 expression is up regulated in oligodendrocytes, and to a lesser extent in astrocytes, in both active acute lesions and at the active rims of chronic active lesions and that this is associated with increased expression of Sprouty2 and Sprouty4 by astrocytes. In contrast Sprouty2/4 expression was minimal in normal appearing white matter adjacent to these lesions and in white matter from healthy controls. These findings provide further evidence that FGF signalling in astrocytes plays a role in the development of MS lesions. Elucidating the function of Spry2/4 during lesion development will help determine how the astrocytic response in MS may be manipulated to promote remyelination; a strategy predicted to restore function and prevent further axonal loss in MS.
previous study in SJL mice, C57Bl/6 mice did not show altered brain viscoelasticity. To establish that stable viscoelastic measurements reflected the reduced brain inflammation in this model compared to the SJL mice, we induced EAE interferon-gamma-deficient C57Bl/6B6 mice, in which a more severe EAE phenotype with increased inflammation in the cerebellum and brainstem has been previously reported. Using this model, we could confirm cerebral inflammation in vivo by the presence of gadolinium-enhancing cerebellar lesions with T1-weighted MRI, and ex vivo, by histopathology. MRE scans of IFNgamma −/− mice showed reduced brain elasticity during EAE, measured at 14 and 21 days post-immunization. Interestingly, elasticity changes occurred first in the anterior brain region and later in the posterior region. Within the timeframe examined in this study, the ‘viscosity’ of the tissue was not altered. Currently, we are examining the histological alteration underlying these findings. Together these results highlight the relationship between alterations in the mechanical properties of the brain and inflammation, and underscore the potential experimental and diagnostic utility of MRE to detect these phenomena during CNS disease.
doi:10.1016/j.jneuroim.2014.08.281
Neuroinflammatory diseases are characterized by infiltration of lymphocytes into the CNS, followed by demyelination and axonal degeneration. Evidence suggests that inflammatory factors released from activated T-cells induce neurotoxicity and impair function of neural stem cells. However, the effect of activated T-cells on the function of oligodendrocyte progenitor cells (OPCs) is uncertain, partly due to the difficulty in obtaining adult human oligodendrocytes. In this study, we used iNSCs to obtain OPCs and determined the effect of inflammatory factors on OPCs. Neural stem cells were induced from adult peripheral CD34 + cells by transduction using Sendai virus encoding transcription factor constructs of sox2, oct3/4, c-myc and klf4 and incubation in neural stem cell medium. The resulting cells were differentiated into OPCs and then exposed to supernatants from activated T-cells. Cell proliferation was determined by EdU incorporation assay and CellQuanti-Blue assay. Cell cycle progression was studied by transfecting OPCs with Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI). VEGF-A and its receptors were studied by RT-PCR and western-blot assay. We found that supernatants from activated T cells increased OPC proliferation by regulating cell cycle progression. While VEGF-A expression was increased in T cells after activation, immunodepletion of VEGF-A from activated T cell supernatants significantly attenuated activated T cells induced OPC proliferation. Furthermore, we found that VEGF receptor 2 (VEGF-R2) was expressed on OPC and specific inhibition of VEGF-R2 with siRNA or inhibitor also attenuated activated T cell supernatants induced OPC proliferation. The effects of T cells and VEGF-A on OPC proliferation were also confirmed in primary human fetal OPC cultures. These results indicate that OPCs generated from CD34-derived neural stem cells behave similarly as primary OPCs in response to treatment by activated T cells and could be used to study the physiological functions and pathogenesis of OPCs, providing a specific advantage especially by using patient specific blood samples. Further, using this model we found that VEGF-A released from activated T cells could enhance OPC proliferation through activating
220 Regional differences in viscoelasticity in normal and inflamed mouse brain Jason Millwarda, Jing Guob, Dominique Berndta, Jürgen Braunb, Ingolf Sackb, Carmen Infante-duartea a Institute for Medical Immunology, Charité — Universitätmedizin Berlin, Berlin, Germany; bDepartment of Radiology, Charité — Universitätmedizin Berlin, Berlin, Germany
Magnetic resonance elastography (MRE) is a novel imaging method that reveals the structural and mechanical properties of tissue, which can be modelled as a combination of ‘elasticity’ and ‘viscosity’ of in response to external forces. Tissue mechanical properties arising from cell and extracellular matrix architecture can be altered during pathological processes such as inflammation. It was recently shown using MRE that brains of multiple sclerosis (MS) patients had reduced viscoelasticity i.e. they were ‘softer’ compared to healthy controls. Our previous study also showed reduced viscoelasticity in EAE in SJL (a model of RRMS). In the present study, we performed MRE in the chronic EAE model in C57Bl/6 mice, and considered distinct brain regions. By performing the scan in the sagittal plane with the 7 Tesla animal MRI scanner, we could analyze the anterior and the posterior brain regions separately. MRE scans of non-EAE and EAE mice revealed that the posterior brain region has a lower viscoelasticity than the anterior region — i.e. the cerebellum/brainstem region is intrinsically ‘softer’ than the rest of the brain. However, during EAE, and in contrast to our
doi:10.1016/j.jneuroim.2014.08.282
527 Activated T cells induce oligodendrocyte progenitor cells proliferation by releasing VEGF-A Elliot Choi, Tongguang Wang, Avindra Nath National Institutes of Health, NINDS, Bethesda, United States