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Find video protocols related to scientific articles indexed in Pubmed.
Transient receptor potential melastatin subfamily member 2 cation channel regulates detrimental immune cell invasion in ischemic stroke.
Stroke
PUBLISHED: 09-18-2014
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Brain injury during stroke results in oxidative stress and the release of factors that include extracellular Ca(2+), hydrogen peroxide, adenosine diphosphate ribose, and nicotinic acid adenine dinucleotide phosphate. These alterations of the extracellular milieu change the activity of transient receptor potential melastatin subfamily member 2 (TRPM2), a nonselective cation channel expressed in the central nervous system and the immune system. Our goal was to evaluate the contribution of TRPM2 to the tissue damage after stroke.
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Evaluation of antiviral efficacy of ribavirin, arbidol, and T-705 (favipiravir) in a mouse model for Crimean-Congo hemorrhagic fever.
PLoS Negl Trop Dis
PUBLISHED: 05-01-2014
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Mice lacking the type I interferon receptor (IFNAR-/- mice) reproduce relevant aspects of Crimean-Congo hemorrhagic fever (CCHF) in humans, including liver damage. We aimed at characterizing the liver pathology in CCHF virus-infected IFNAR-/- mice by immunohistochemistry and employed the model to evaluate the antiviral efficacy of ribavirin, arbidol, and T-705 against CCHF virus.
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Human CLP1 mutations alter tRNA biogenesis, affecting both peripheral and central nervous system function.
Cell
PUBLISHED: 02-21-2014
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CLP1 is a RNA kinase involved in tRNA splicing. Recently, CLP1 kinase-dead mice were shown to display a neuromuscular disorder with loss of motor neurons and muscle paralysis. Human genome analyses now identified a CLP1 homozygous missense mutation (p.R140H) in five unrelated families, leading to a loss of CLP1 interaction with the tRNA splicing endonuclease (TSEN) complex, largely reduced pre-tRNA cleavage activity, and accumulation of linear tRNA introns. The affected individuals develop severe motor-sensory defects, cortical dysgenesis, and microcephaly. Mice carrying kinase-dead CLP1 also displayed microcephaly and reduced cortical brain volume due to the enhanced cell death of neuronal progenitors that is associated with reduced numbers of cortical neurons. Our data elucidate a neurological syndrome defined by CLP1 mutations that impair tRNA splicing. Reduction of a founder mutation to homozygosity illustrates the importance of rare variations in disease and supports the clan genomics hypothesis.
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The extracellular matrix glycoprotein tenascin-R regulates neurogenesis during development and in the adult dentate gyrus of mice.
J. Cell. Sci.
PUBLISHED: 12-11-2013
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Abnormal generation of inhibitory ?-aminobutyric acid synthesizing (GABAergic) neurons is characteristic of neuropsychological disorders. We provide evidence that the extracellular matrix molecule tenascin-R (TNR) - being predominantly expressed, among neurons, by subpopulation of interneurons - plays a role in the generation of GABAergic and granule neurons in the murine dentate gyrus by regulating fate determination of neural stem/progenitor cells (NSCs). During development, absence of TNR in constitutively TNR-deficient (TNR-/-) mice results in increased numbers of dentate gyrus GABAergic neurons, being associated with decreased expression of its receptor ?1 integrin, increased activation of p38 MAPK, and increased expression of the GABAergic specification gene ASCL1. Postnatally, increased GABAergic input to adult hippocampal NSCs in TNR-/- mice is associated not only with increased numbers of GABAergic and, particularly, parvalbumin-immunoreactive neurons, as seen during development, but also with increased numbers of granule neurons, thus contributing to the increased differentiation of NSCs into granule cells. These findings indicate the importance of TNR in the regulation of hippocampal neurogenesis and suggest that TNR acts through distinct direct and indirect mechanisms during development and in the adult.
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Postnatal disruption of the disintegrin/metalloproteinase ADAM10 in brain causes epileptic seizures, learning deficits, altered spine morphology, and defective synaptic functions.
J. Neurosci.
PUBLISHED: 08-09-2013
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The metalloproteinase ADAM10 is of importance for Notch-dependent cortical brain development. The protease is tightly linked with ?-secretase activity toward the amyloid precursor protein (APP) substrate. Increasing ADAM10 activity is suggested as a therapy to prevent the production of the neurotoxic amyloid ? (A?) peptide in Alzheimers disease. To investigate the function of ADAM10 in postnatal brain, we generated Adam10 conditional knock-out (A10cKO) mice using a CaMKII?-Cre deleter strain. The lack of ADAM10 protein expression was evident in the brain cortex leading to a reduced generation of sAPP? and increased levels of sAPP? and endogenous A? peptides. The A10cKO mice are characterized by weight loss and increased mortality after weaning associated with seizures. Behavioral comparison of adult mice revealed that the loss of ADAM10 in the A10cKO mice resulted in decreased neuromotor abilities and reduced learning performance, which were associated with altered in vivo network activities in the hippocampal CA1 region and impaired synaptic function. Histological and ultrastructural analysis of ADAM10-depleted brain revealed astrogliosis, microglia activation, and impaired number and altered morphology of postsynaptic spine structures. A defect in spine morphology was further supported by a reduction of the expression of NMDA receptors subunit 2A and 2B. The reduced shedding of essential postsynaptic cell adhesion proteins such as N-Cadherin, Nectin-1, and APP may explain the postsynaptic defects and the impaired learning, altered network activity, and synaptic plasticity of the A10cKO mice. Our study reveals that ADAM10 is instrumental for synaptic and neuronal network function in the adult murine brain.
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Carcinoembryonic antigen-related cell adhesion molecule 1 inhibits MMP-9-mediated blood-brain-barrier breakdown in a mouse model for ischemic stroke.
Circ. Res.
PUBLISHED: 06-18-2013
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Blood-brain-barrier (BBB) breakdown and cerebral edema result from postischemic inflammation and contribute to mortality and morbidity after ischemic stroke. A functional role for the carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) in the regulation of reperfusion injury has not yet been demonstrated.
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STAT3 silencing inhibits glioma single cell infiltration and tumor growth.
Neuro-oncology
PUBLISHED: 03-13-2013
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Diffuse infiltration remains the fulcrum of glioblastomas incurability, leading inevitably to recurrence. Therefore, uncovering the pathological mechanism is imperative. Because signal transducer and activator of transcription 3 (STAT3) correlates with glioma malignancy and predicts poor clinical outcome, we determined its role in glioma single cell infiltration and tumor growth.
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CLP1 links tRNA metabolism to progressive motor-neuron loss.
Nature
PUBLISHED: 01-18-2013
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CLP1 was the first mammalian RNA kinase to be identified. However, determining its in vivo function has been elusive. Here we generated kinase-dead Clp1 (Clp1(K/K)) mice that show a progressive loss of spinal motor neurons associated with axonal degeneration in the peripheral nerves and denervation of neuromuscular junctions, resulting in impaired motor function, muscle weakness, paralysis and fatal respiratory failure. Transgenic rescue experiments show that CLP1 functions in motor neurons. Mechanistically, loss of CLP1 activity results in accumulation of a novel set of small RNA fragments, derived from aberrant processing of tyrosine pre-transfer RNA. These tRNA fragments sensitize cells to oxidative-stress-induced p53 (also known as TRP53) activation and p53-dependent cell death. Genetic inactivation of p53 rescues Clp1(K/K) mice from the motor neuron loss, muscle denervation and respiratory failure. Our experiments uncover a mechanistic link between tRNA processing, formation of a new RNA species and progressive loss of lower motor neurons regulated by p53.
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Deficiency in serine protease inhibitor neuroserpin exacerbates ischemic brain injury by increased postischemic inflammation.
PLoS ONE
PUBLISHED: 01-01-2013
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The only approved pharmacological treatment for ischemic stroke is intravenous administration of plasminogen activator (tPA) to re-canalize the occluded cerebral vessel. Not only reperfusion but also tPA itself can induce an inflammatory response. Microglia are the innate immune cells of the central nervous system and the first immune cells to become activated in stroke. Neuroserpin, an endogenous inhibitor of tPA, is up-regulated following cerebral ischemia. To examine neuroserpin-dependent mechanisms of neuroprotection in stroke, we studied neuroserpin deficient (Ns(-/-))mice in an animal model of temporal focal ischemic stroke. Infarct size and neurological outcome were worse in neuroserpin deficient mice even though the fibrinolytic activity in the ischemic brain was increased. The increased infarct size was paralleled by a selective increase in proinflammatory microglia activation in Ns(-/-) mice. Our results show excessive microglial activation in Ns(-/-) mice mediated by an increased activity of tPA. This activation results in a worse outcome further underscoring the potential detrimental proinflammatory effects of tPA.
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Dermatan sulfotransferase Chst14/D4st1, but not chondroitin sulfotransferase Chst11/C4st1, regulates proliferation and neurogenesis of neural progenitor cells.
J. Cell. Sci.
PUBLISHED: 12-08-2011
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Chondroitin sulfates (CSs) and dermatan sulfates (DSs) are enriched in the microenvironment of neural stem cells (NSCs) during development and in the adult neurogenic niche, and have been implicated in mechanisms governing neural precursor migration, proliferation and differentiation. In contrast to previous studies, in which a chondroitinaseABC-dependent unselective deglycosylation of both CSs and DSs was performed, we used chondroitin 4-O-sulfotransferase-1 (Chst11/C4st1)- and dermatan 4-O-sulfotransferase-1 (Chst14/D4st1)-deficient NSCs specific for CSs and DSs, respectively, to investigate the involvement of specific sulfation profiles of CS and DS chains, and thus the potentially distinct roles of CSs and DSs in NSC biology. In comparison to wild-type controls, deficiency for Chst14 resulted in decreased neurogenesis and diminished proliferation of NSCs accompanied by increased expression of GLAST and decreased expression of Mash-1, and an upregulation of the expression of the receptors for fibroblast growth factor-2 (FGF-2) and epidermal growth factor (EGF). By contrast, deficiency in Chst11 did not influence NSC proliferation, migration or differentiation. These observations indicate for the first time that CSs and DSs play distinct roles in the self-renewal and differentiation of NSCs.
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Transplanted L1 expressing radial glia and astrocytes enhance recovery after spinal cord injury.
J. Neurotrauma
PUBLISHED: 08-31-2011
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A major obstacle for the transplantation of neural stem cells (NSCs) into the lesioned spinal cord is their predominant astrocytic differentiation after transplantation. We took advantage of this predominant astrocytic differentiation of NSCs and expressed the paradigmatic beneficial neural cell adhesion molecule L1 in radial glial cells and reactive and nonreactive astrocytes as novel cellular vehicles to express L1 under the control of the promoter for the human glial fibrillary acidic protein (GFAP-L1 NSCs). Behavioral analysis and electrophysiological H-reflex recordings revealed that mice transplanted with GFAP-L1 NSCs showed enhanced locomotor recovery in comparison to mice injected with wild type (WT) NSCs or control mice injected with phosphate-buffered saline (PBS). This functional recovery was further accelerated in mice transplanted with L1-expressing radial glial cells that had been immunoisolated from GFAP-L1 NSCs (GFAP-L1-i cells). Morphological analysis revealed that mice grafted with GFAP-L1 NSCs exhibited increased neuronal differentiation and migration of transplanted cells, as well as increased soma size and cholinergic synaptic coverage of host motoneurons and increased numbers of endogenous catecholaminergic nerve fibers caudal to the lesion site. These findings show that L1-expressing astrocytes and radial glial cells isolated from GFAP-L1 NSC cultures represent a novel strategy for improving functional recovery after spinal cord injury, encouraging the use of the human GFAP promoter to target beneficial transgene expression in transplanted stem cells.
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Stress-resistant neural stem cells positively influence regional energy metabolism after spinal cord injury in mice.
J. Mol. Neurosci.
PUBLISHED: 07-13-2011
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The importance of stem cells to ameliorate the devastating consequences of traumatic injuries in the adult mammalian central nervous system calls for improvements in the capacity of these cells to cope, in particular, with the host response to the injury. We have previously shown, however, that in the acutely traumatized spinal cord local energy metabolism led to decreased ATP levels after neural stem cell (NSC) transplantation. As this might counteract NSC-mediated regenerative processes, we investigated if NSC selected for increased oxidative stress resistance are better suited to preserve local energy content. For this purpose, we exposed wild-type (WT) NSC to hydrogen peroxide prior to transplantation. We demonstrate here that transplantation of WT-NSC into a complete spinal cord compression injury model even lowers the ATP content beyond the level detected in spinal cord injury-control animals. Compared to WT-NSC, stress-resistant (SR) NSC did not lead to a further decrease in ATP content. These differences between WT- and SR-NSC were observed 4 h after the lesion with subsequent transplantation. At 24 h after lesioning, these differences were no more as obvious. Thus, in contrast to native NSC, transplantation of NSC selected for oxidative stress resistance can positively influence local energy metabolism in the first hours after spinal cord compression. The functional relevance of this observation has to be tested in further experiments.
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Treatment of glioblastoma with poly(isohexyl cyanoacrylate) nanoparticles.
Int J Pharm
PUBLISHED: 04-08-2011
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Glioblastomas belong to the most devastating cancer diseases. For this reason, polysorbate 80 (Tween 80)-coated poly(isohexyl cyanoacrylate) (PIHCA) (Monorex) nanoparticles loaded with doxorubicin were developed and tested for their use for the treatment of glioblastomas. The preparation of the nanoparticles resulted in spherical particles with high doxorubicin loading. The physico-chemical properties and the release of doxorubicin from the PIHCA-nanoparticles were analysed, and the influence on cell viability of the rat glioblastoma 101/8-cell line was investigated. In vitro, the empty nanoparticles did not show any toxicity, and the anti-cancer effects of the drug-loaded nanoparticles were increased in comparison to doxorubicin solution, represented by IC(50) values. The in vivo efficacy was then tested in intracranially glioblastoma 101/8-bearing rats. Rats were treated with 3 × 1.5mg/kg doxorubicin and were sacrificed 18 days after tumour transplantation. Histological and immunohistochemical analyses were carried out to assess the efficacy of the nanoparticles. Tumour size, proliferation activity, vessel density, necrotic areas, and expression of glial fibrillary acidic protein demonstrated that doxorubicin-loaded PIHCA-nanoparticles were much more efficient than the free drug. The results suggest that poly(isohexyl cyanoacrylate) nanoparticles hold great promise for the non-invasive therapy of human glioblastomas.
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Glycomimetic improves recovery after femoral injury in a non-human primate.
J. Neurotrauma
PUBLISHED: 04-06-2011
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In adult mammals, restoration of function after peripheral nerve injury is often poor and effective therapies are not available. Previously we have shown in mice that a peptide which functionally mimics the human natural killer cell (HNK)-1 trisaccharide epitope significantly improves the outcome of femoral nerve injury. Here we evaluated the translational potential of this treatment using primates. We applied a linear HNK-1 mimetic or a functionally inactive control peptide in silicone cuffs used to reconstruct the cut femoral nerves of adult cynomolgus monkeys (Macaca fascicularis). Functional recovery was evaluated using video-based gait analysis over a 160-day observation period. The final outcome was further assessed using force measurements, H-reflex recordings, nerve histology, and ELISA to assess immunoreactivity to HNK-1 in the treated monkeys. Gait deficits were significantly reduced in HNK-1 mimetic-treated compared with control peptide-treated animals between 60 and 160 days after injury. Better outcome at 160 days after surgery in treated versus control animals was also confirmed by improved quadriceps muscle force, enhanced H-reflex amplitude, decreased H-reflex latency, and larger diameters of regenerated axons. No adverse reactions to the mimetic, in particular immune responses resulting in antibodies against the HNK-1 mimetic or immune cell infiltration into the damaged nerve, were observed. These results indicate the potential of the HNK-1 mimetic as an efficient, feasible, and safe adjunct treatment for nerve injuries requiring surgical repair in clinical settings.
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Lack of a-disintegrin-and-metalloproteinase ADAM10 leads to intracellular accumulation and loss of shedding of the cellular prion protein in vivo.
Mol Neurodegener
PUBLISHED: 04-01-2011
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The cellular prion protein (PrPC) fulfils several yet not completely understood physiological functions. Apart from these functions, it has the ability to misfold into a pathogenic scrapie form (PrPSc) leading to fatal transmissible spongiform encephalopathies. Proteolytic processing of PrPC generates N- and C-terminal fragments which play crucial roles both in the pathophysiology of prion diseases and in transducing physiological functions of PrPC. A-disintegrin-and-metalloproteinase 10 (ADAM10) has been proposed by cell culture experiments to be responsible for both shedding of PrPC and its ?-cleavage. Here, we analyzed the role of ADAM10 in the proteolytic processing of PrPC in vivo.
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Osseous changes in meningioma en plaque.
Anticancer Res.
PUBLISHED: 03-08-2011
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Hyperostosis is the most common skull change associated with meningioma. Five hyperostosis cases of meningioma en plaque infiltrating the skull processed without previous decalcification of the bone tissue were investigated histologically and immunohistochemically with antibodies against somatostatin receptor 2A (SSR2A). Undecalcified bone biopsies embedded in methylmethacrylate and paraffin-embedded extraosseous tumor tissues were analyzed. All five cases were well-differentiated meningotheliomatous meningiomas en plaque according to the WHO classification of tumors and revealed areas of hyperosteoidosis. Furthermore, all five meningiomas en plaque presented strong positive reactions to antibodies against SSR2A in both the intraosseous and extraosseous tumor proliferates. In summary, similar morphological changes characterized by hyperosteoidosis were observed in a small cohort of meningioma en plaque associated with expression of SSR2A and reports in the literature of the histogenesis of hyperostosis in meningioma en plaque are discussed.
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Deposition of hyperphosphorylated tau in cerebellum of PS1 E280A Alzheimers disease.
Brain Pathol.
PUBLISHED: 01-27-2011
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Early-onset familial Alzheimers disease (AD) caused by presenilin-1 mutation E280A (PS1-E280A) presents wide clinical and neuropathological variabilities. We characterized clinically and neuropathologically PS1-E280A focusing in cerebellar involvement and compared it with early-onset sporadic Alzheimers disease (EOSAD). Twelve E280A brains and 12 matched EOSAD brains were analyzed for beta-amyloid and hyperphosphorylated tau (pTau) morphology, beta-amyloid subspecies 1-40, 1-42 levels, pTau levels, and expression of stress kinases in frontal cortex and cerebellum. The data were correlated to clinical and genetic findings. We observed higher beta-amyloid load, beta-amyloid 1-42 and pTau concentrations in frontal cortex of PS1-E280A compared with EOSAD. High beta-amyloid load was found in the cerebellum of PS1-E280A and EOSAD patients. In PS1-E280A, beta-amyloid localized to the molecular and Purkinje cell layers, whereas EOSAD showed them in Purkinje and granular cell layers. Surprisingly, 11 out of 12 PS1-E280A patients showed deposition of pTau in the cerebellum. Also, seven out of 12 PS1-E280A patients presented cerebellar ataxia. We conclude that deposition of beta-amyloid in the cerebellum is prominent in early-onset AD irrespective of genetic or sporadic origin. The presence of pTau in cerebellum in PS1-E280A underscores the relevance of cerebellar involvement in AD and might be correlated to clinical phenotype.
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Efficient chemotherapy of rat glioblastoma using doxorubicin-loaded PLGA nanoparticles with different stabilizers.
PLoS ONE
PUBLISHED: 01-24-2011
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Chemotherapy of glioblastoma is largely ineffective as the blood-brain barrier (BBB) prevents entry of most anticancer agents into the brain. For an efficient treatment of glioblastomas it is necessary to deliver anti-cancer drugs across the intact BBB. Poly(lactic-co-glycolic acid) (PLGA) nanoparticles coated with poloxamer 188 hold great promise as drug carriers for brain delivery after their intravenous injection. In the present study the anti-tumour efficacy of the surfactant-coated doxorubicin-loaded PLGA nanoparticles against rat glioblastoma 101/8 was investigated using histological and immunohistochemical methods.
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Embryonic stem cell-derived L1 overexpressing neural aggregates enhance recovery after spinal cord injury in mice.
PLoS ONE
PUBLISHED: 01-21-2011
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An obstacle to early stem cell transplantation into the acutely injured spinal cord is poor survival of transplanted cells. Transplantation of embryonic stem cells as substrate adherent embryonic stem cell-derived neural aggregates (SENAs) consisting mainly of neurons and radial glial cells has been shown to enhance survival of grafted cells in the injured mouse brain. In the attempt to promote the beneficial function of these SENAs, murine embryonic stem cells constitutively overexpressing the neural cell adhesion molecule L1 which favors axonal growth and survival of grafted and imperiled cells in the inhibitory environment of the adult mammalian central nervous system were differentiated into SENAs and transplanted into the spinal cord three days after compression lesion. Mice transplanted with L1 overexpressing SENAs showed improved locomotor function when compared to mice injected with wild-type SENAs. L1 overexpressing SENAs showed an increased number of surviving cells, enhanced neuronal differentiation and reduced glial differentiation after transplantation when compared to SENAs not engineered to overexpress L1. Furthermore, L1 overexpressing SENAs rescued imperiled host motoneurons and parvalbumin-positive interneurons and increased numbers of catecholaminergic nerve fibers distal to the lesion. In addition to encouraging the use of embryonic stem cells for early therapy after spinal cord injury L1 overexpression in the microenvironment of the lesioned spinal cord is a novel finding in its functions that would make it more attractive for pre-clinical studies in spinal cord regeneration and most likely other diseases of the nervous system.
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Adhesion molecule L1 is down-regulated in malignant peripheral nerve sheath tumors versus benign neurofibromatosis type 1-associated tumors.
Oral Surg Oral Med Oral Pathol Oral Radiol
PUBLISHED: 01-06-2011
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Type 1 neurofibromatosis (NF-1), also known as von Recklinghausen disease, is caused by a disorder of a single gene on chromosome 17 that usually restrains cell division. A sequence that is frequently associated with NF-1 is tumor progression from neurofibromas to malignant peripheral nerve sheath tumors (MPNSTs). The aim of this study was to determine the expression of the neural L1 cell adhesion molecule in dermal-diffuse neurofibromas, plexiform neurofibromas, and MPNSTs of NF-1. We retrospectively analyzed surgically resected primary tumors, including 20 dermal neurofibromas, 23 plexiform neurofibromas, and 17 MPNSTs, by immunohistochemistry in paraffin sections of NF-1 tumors with the use of the L1-specific monoclonal antibody UJ127, which does not cross-react with other members of the L1 family. Immunostainings for CD34 and S100 were included to distinguish and allocate L1-expressing Schwann cells and perineural (specialized) fibroblasts. Our data showed that L1 is highly expressed in all benign NF-1 tumors and in some but not all MPNSTs. Furthermore, we demonstrated a correlation between L1 expression and differentiation grade of MPNSTs. There was a significant trend toward lower or nondetectable expression in the poorly differentiated MPNSTs, in contrast to all other tumor entities so far investigated, in which L1 expression correlated positive with malignancy, except for juvenile but not adult-derived neuroblastomas. Future studies are warranted to elucidate the molecular basis of the varying effects of the degree of L1 expression, receptor, and signal transduction mechanisms in different tumors.
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Dietary curcumin attenuates glioma growth in a syngeneic mouse model by inhibition of the JAK1,2/STAT3 signaling pathway.
Clin. Cancer Res.
PUBLISHED: 12-09-2010
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Glioblastomas are the most common and most deadly primary brain tumors. Here, we evaluated the chemotherapeutic effect of the natural polyphenol curcumin on glioma cells in vitro and in vivo using an immunocompetent orthotopic mouse model.
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The disintegrin/metalloproteinase ADAM10 is essential for the establishment of the brain cortex.
J. Neurosci.
PUBLISHED: 04-08-2010
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The metalloproteinase and major amyloid precursor protein (APP) alpha-secretase candidate ADAM10 is responsible for the shedding of proteins important for brain development, such as cadherins, ephrins, and Notch receptors. Adam10(-/-) mice die at embryonic day 9.5, due to major defects in development of somites and vasculogenesis. To investigate the function of ADAM10 in brain, we generated Adam10 conditional knock-out (cKO) mice using a Nestin-Cre promotor, limiting ADAM10 inactivation to neural progenitor cells (NPCs) and NPC-derived neurons and glial cells. The cKO mice die perinatally with a disrupted neocortex and a severely reduced ganglionic eminence, due to precocious neuronal differentiation resulting in an early depletion of progenitor cells. Premature neuronal differentiation is associated with aberrant neuronal migration and a disorganized laminar architecture in the neocortex. Neurospheres derived from Adam10 cKO mice have a disrupted sphere organization and segregated more neurons at the expense of astrocytes. We found that Notch-1 processing was affected, leading to downregulation of several Notch-regulated genes in Adam10 cKO brains, in accordance with the central role of ADAM10 in this signaling pathway and explaining the neurogenic phenotype. Finally, we found that alpha-secretase-mediated processing of APP was largely reduced in these neurons, demonstrating that ADAM10 represents the most important APP alpha-secretase in brain. Our study reveals that ADAM10 plays a central role in the developing brain by controlling mainly Notch-dependent pathways but likely also by reducing surface shedding of other neuronal membrane proteins including APP.
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Short DNA sequences inserted for gene targeting can accidentally interfere with off-target gene expression.
FASEB J.
PUBLISHED: 01-28-2010
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Targeting of genes in mice, a key approach to study development and disease, often leaves a neo cassette, loxP, or FRT sites inserted in the mouse genome. Insertion of neo can influence the expression of neighboring genes, but similar effects have not been reported for loxP sites. We therefore performed microarray analyses of mice in which the Ncam or the Tnr gene were targeted either by insertion of neo or loxP/FRT sites. In the case of Ncam, neo, but not loxP/FRT insertion, led to a 2-fold reduction in mRNA levels of 3 genes located at distances between 0.2 and 3.1 Mb from the target. In contrast, after introduction of loxP/FRT sites into introns of Tnr, we observed a 2.5- to 4-fold reduction in the transcript level of the Gas5 gene, 1.1 Mb away from Tnr, most probably due to disruption of a conserved regulatory element in Tnr. Insertion of short DNA sequences such as loxP/FRT can thus influence off-target mRNA levels if these sites are accidentally placed into regulatory elements. Our results imply that conditional knockout mice should be analyzed for genomic positional side effects that may influence the animals phenotypes.
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Embryonic stem cell-derived L1 overexpressing neural aggregates enhance recovery in Parkinsonian mice.
Brain
PUBLISHED: 12-07-2009
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Parkinsons disease is the second most common neurodegenerative disease, after Alzheimers disease, and the most common movement disorder. Drug treatment and deep brain stimulation can ameliorate symptoms, but the progressive degeneration of dopaminergic neurons in the substantia nigra eventually leads to severe motor dysfunction. The transplantation of stem cells has emerged as a promising approach to replace lost neurons in order to restore dopamine levels in the striatum and reactivate functional circuits. We have generated substrate-adherent embryonic stem cell-derived neural aggregates overexpressing the neural cell adhesion molecule L1, because it has shown beneficial functions after central nervous system injury. L1 enhances neurite outgrowth and neuronal migration, differentiation and survival as well as myelination. In a previous study, L1 was shown to enhance functional recovery in a mouse model of Huntingtons disease. In another study, a new differentiation protocol for murine embryonic stem cells was established allowing the transplantation of stem cell-derived neural aggregates consisting of differentiated neurons and radial glial cells into the lesioned brain. In the present study, this embryonic stem cell line was engineered to overexpress L1 constitutively at all stages of differentiation and used to generate stem cell-derived neural aggregates. These were monitored in their effects on stem cell survival and differentiation, rescue of endogenous dopaminergic neurons and ability to influence functional recovery after transplantation in an animal model of Parkinsons disease. Female C57BL/6J mice (2 months old) were treated with the mitochondrial toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine intraperitoneally to deplete dopaminergic neurons selectively, followed by unilateral transplantation of stem cell-derived neural aggregates into the striatum. Mice grafted with L1 overexpressing stem cell-derived neural aggregates showed better functional recovery when compared to mice transplanted with wild-type stem cell-derived neural aggregates and vehicle-injected mice. Morphological analysis revealed increased numbers and migration of surviving transplanted cells, as well as increased numbers of dopaminergic neurons, leading to enhanced levels of dopamine in the striatum ipsilateral to the grafted side in L1 overexpressing stem cell-derived neural aggregates, when compared to wild-type stem cell-derived neural aggregates. The striatal levels of gamma-aminobutyric acid were not affected by L1 overexpressing stem cell-derived neural aggregates. Furthermore, L1 overexpressing, but not wild-type stem cell-derived neural aggregates, enhanced survival of endogenous host dopaminergic neurons after transplantation adjacent to the substantia nigra pars compacta. Thus, L1 overexpressing stem cell-derived neural aggregates enhance survival and migration of transplanted cells, differentiation into dopaminergic neurons, survival of endogenous dopaminergic neurons, and functional recovery after syngeneic transplantation in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinsons disease.
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Plasminogen activator inhibitor type 1 up-regulation is associated with skeletal muscle atrophy and associated fibrosis.
Am. J. Pathol.
PUBLISHED: 07-02-2009
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Muscle wasting remains a feature of many diseases and is counteracted by anabolic supplementation or exercise. Persisting atrophy-inducing conditions can be complicated by skeletal muscle fibrosis, which leads to functional impairment. Identification of early mechanisms that initiate atrophy-induced fibrosis may reveal novel targets for therapy or diagnosis. Therefore, we investigated changes in the expression of genes involved in extracellular matrix homeostasis during glucocorticoid-induced atrophy of myotubes and compared them with insulin-like growth factor-1-induced hypertrophy. Obtained results were verified in rat gastrocnemius muscle that was exposed to microgravity by space flight for 2 weeks. Myostatin and atrogin-1 mRNA levels reflected the magnitude of atrophy. Despite differential induction of these negative muscle mass regulators, no major changes in matrix metalloproteinases-2, -9, and -14 mRNAs or their physiological inhibitors could be detected in either atrophy model. In contrast, transcript levels of plasminogen activator inhibitor type 1 (PAI-1) was dramatically increased in atrophic myotubes and microgravity-exposed rat gastrocnemius muscle, while plasminogen activators remained unaltered. In contrast to atrophy, no increase in PAI-1 mRNA levels could be detected in rat hindlimb that was electrically stimulated for 21 days. Furthermore, a strong increase in PAI-1 mRNA levels was identified in skeletal muscle of patients with neurogenic muscle atrophy. Our study suggests that increased PAI-1 expression in atrophic skeletal muscle may lead to muscle fibrosis by reducing plasmin generation.
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Autoantibodies against beta-amyloid are common in Alzheimers disease and help control plaque burden.
Ann. Neurol.
PUBLISHED: 02-06-2009
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Active or passive immunization of Alzheimers disease (AD) patients leads to targeting of beta-amyloid plaques by immunoglobulins (IgG) and their subsequent removal by microglia. Here, we investigate whether naturally occurring autoantibodies to beta-amyloid contribute to beta-amyloid plaque removal in nonimmunized AD patients.
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Heterozygosity for the mutated X-chromosome-linked L1 cell adhesion molecule gene leads to increased numbers of neurons and enhanced metabolism in the forebrain of female carrier mice.
Brain Struct Funct
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Mutations in the X-chromosomal L1CAM gene lead to severe neurological deficits. In this study, we analyzed brains of female mice heterozygous for L1 (L1+/-) to gain insights into the brain structure of human females carrying one mutated L1 allele. From postnatal day 7 onward into adulthood, L1+/- female mice show an increased density of neurons in the neocortex and basal ganglia in comparison to wild-type (L1+/+) mice, correlating with enhanced metabolic parameters as measured in vivo. The densities of astrocytes and parvalbumin immunoreactive interneurons were not altered. No significant differences between L1+/- and L1+/+ mice were seen for cell proliferation in the cortex during embryonic days 11.5-15.5. Neuronal differentiation as estimated by analysis of doublecortin-immunoreactive cortical cells of embryonic brains was similar in L1+/- and L1+/+ mice. Interestingly, at postnatal days 3 and 5, apoptosis was reduced in L1+/- compared to L1+/+ mice. We suggest that reduced apoptosis leads to increased neuronal density in adult L1+/- mice. In conclusion, L1+/- mice display an unexpected phenotype that is not an intermediate between L1+/+ mice and mice deficient in L1 (L1-/y), but a novel phenotype which is challenging to understand regarding its underlying molecular and cellular mechanisms.
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Neutralization of the IL-17 axis diminishes neutrophil invasion and protects from ischemic stroke.
Blood
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The devastating effect of ischemic stroke is attenuated in mice lacking conventional and unconventional T cells, suggesting that inflammation enhances tissue damage in cerebral ischemia. We explored the functional role of ?? and ?? T cells in a murine model of stroke and distinguished 2 different T cell-dependent proinflammatory pathways in ischemia-reperfusion injury. IFN-? produced by CD4(+) T cells induced TNF-? production in macrophages, whereas IL-17A secreted by ?? T cells led to neutrophil recruitment. The synergistic effect of TNF-? and IL-17A on astrocytes resulted in enhanced secretion of CXCL-1, a neutrophil chemoattractant. Application of an IL-17A-blocking antibody within 3 hours after stroke induction decreased infarct size and improved neurologic outcome in the murine model. In autoptic brain tissue of patients who had a stroke, we detected IL-17A-positive lymphocytes, suggesting that this aspect of the inflammatory cascade is also relevant in the human brain. We propose that selective targeting of IL-17A signaling might provide a new therapeutic option for the treatment of stroke.
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In vitro generation of three-dimensional substrate-adherent embryonic stem cell-derived neural aggregates for application in animal models of neurological disorders.
Curr Protoc Stem Cell Biol
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In vitro-differentiated embryonic stem (ES) cells comprise a useful source for cell replacement therapy, but the efficiency and safety of a translational approach are highly dependent on optimized protocols for directed differentiation of ES cells into the desired cell types in vitro. Furthermore, the transplantation of three-dimensional ES cell-derived structures instead of a single-cell suspension may improve graft survival and function by providing a beneficial microenvironment for implanted cells. To this end, we have developed a new method to efficiently differentiate mouse ES cells into neural aggregates that consist predominantly (>90%) of postmitotic neurons, neural progenitor cells, and radial glia-like cells. When transplanted into the excitotoxically lesioned striatum of adult mice, these substrate-adherent embryonic stem cell-derived neural aggregates (SENAs) showed significant advantages over transplanted single-cell suspensions of ES cell-derived neural cells, including improved survival of GABAergic neurons, increased cell migration, and significantly decreased risk of teratoma formation. Furthermore, SENAs mediated functional improvement after transplantation into animal models of Parkinsons disease and spinal cord injury. This unit describes in detail how SENAs are efficiently derived from mouse ES cells in vitro and how SENAs are isolated for transplantation. Furthermore, methods are presented for successful implantation of SENAs into animal models of Huntingtons disease, Parkinsons disease, and spinal cord injury to study the effects of stem cell-derived neural aggregates in a disease context in vivo.
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What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

Video X seems to be unrelated to Abstract Y...

In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.