RESUMEN
Neurogenesis is associated with functional recovery after stroke. However, the underlying molecular mechanisms have not been fully investigated. Using an Ago2-based RNA immunoprecipitation to immunoprecipated Ago2-RNA complexes followed by RNA sequencing (Ago2 RIP-seq) approach, we profiled the miRNomes in neural progenitor cells (NPCs) harvested from the subventricular zone (SVZ) of the lateral ventricles of young adult rats. We identified more than 7 and 15 million reads in normal and ischemic NPC libraries, respectively. We found that stroke substantially changed Ago2-associated miRNA profiles in NPCs compared to those in non-ischemic NPCs. We also discovered a new complex repertoire of isomiRs and multiple miRNA-miRNA* pairs and numerous novel miRNAs in the non-ischemic and ischemic NPCs. Among them, pc-3p-17172 significantly regulated NPC proliferation and neuronal differentiation. Collectively, the present study reveals profiles of Ago2-associated miRNomes in non-ischemic and ischemic NPCs, which provide a molecular basis to further investigate the role of miRNAs in mediating adult neurogenesis under physiological and ischemic conditions.
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Proteínas Argonautas/metabolismo , MicroARNs/metabolismo , Células-Madre Neurales/metabolismo , Neurogénesis/fisiología , Accidente Cerebrovascular/metabolismo , Adulto , Análisis de Varianza , Animales , Proteínas Argonautas/genética , Proliferación Celular , Humanos , Ventrículos Laterales/química , Masculino , MicroARNs/análisis , MicroARNs/genética , Células-Madre Neurales/patología , Cultivo Primario de Células , Ratas , Ratas Wistar , Análisis de Secuencia de ARN , Accidente Cerebrovascular/patología , TranscriptomaRESUMEN
Stroke induces new myelinating oligodendrocytes that are involved in ischemic brain repair. Molecular mechanisms that regulate oligodendrogenesis have not been fully investigated. MicroRNAs (miRNAs) are small non-coding RNA molecules that post-transcriptionally regulate gene expression. MiR-146a has been reported to regulate immune response, but the role of miR-146a in oligodendrocyte progenitor cells (OPCs) remains unknown. Adult Wistar rats were subjected to the right middle cerebral artery occlusion (MCAo). In situ hybridization analysis with LNA probes against miR-146a revealed that stroke considerably increased miR-146a density in the corpus callosum and subventricular zone (SVZ) of the lateral ventricle of the ischemic hemisphere. In vitro, overexpression of miR-146a in neural progenitor cells (NPCs) significantly increased their differentiation into O4+ OPCs. Overexpression of miR-146a in primary OPCs increased their expression of myelin proteins, whereas attenuation of endogenous miR-146a suppressed generation of myelin proteins. MiR-146a also inversely regulated its target gene-IRAK1 expression in OPCs. Attenuation of IRAK1 in OPCs substantially increased myelin proteins and decreased OPC apoptosis. Collectively, our data suggest that miR-146a may mediate stroke-induced oligodendrogenesis.
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MicroARNs/biosíntesis , Oligodendroglía/metabolismo , Accidente Cerebrovascular/metabolismo , Animales , Diferenciación Celular/fisiología , Células Cultivadas , Masculino , Proteínas de la Mielina/biosíntesis , Oligodendroglía/patología , Ratas , Ratas Wistar , Accidente Cerebrovascular/patología , Accidente Cerebrovascular/prevención & controlRESUMEN
We previously demonstrated that treatment of diabetic peripheral neuropathy with the short (4 hours) half-life phosphodiesterase 5 (PDE5) inhibitor, sildenafil, improved functional outcome in diabetic db/db mice. To further examine the effect of PDE5 inhibition on diabetic peripheral neuropathy, we investigated the effect of another potent PDE5 inhibitor, tadalafil, on diabetic peripheral neuropathy. Tadalafil is pharmacokinetically distinct from sildenafil and has a longer half-life (17+hours) than sildenafil. Diabetic mice (BKS.Cg-m+/+Leprdb/J, db/db) at age 20 weeks were treated with tadalafil every 48 hours for 8 consecutive weeks. Compared with diabetic mice treated with saline, tadalafil treatment significantly improved motor and sensory conduction velocities in the sciatic nerve and peripheral thermal sensitivity. Tadalafil treatment also markedly increased local blood flow and the density of FITC-dextran perfused vessels in the sciatic nerve concomitantly with increased intraepidermal nerve fiber density. Moreover, tadalafil reversed the diabetes-induced reductions of axon diameter and myelin thickness and reversed the diabetes-induced increased g-ratio in the sciatic nerve. Furthermore, tadalafil enhanced diabetes-reduced nerve growth factor (NGF) and platelet-derived growth factor-C (PDGF-C) protein levels in diabetic sciatic nerve tissue. The present study demonstrates that tadalafil increases regional blood flow in the sciatic nerve tissue, which may contribute to the improvement of peripheral nerve function and the amelioration of diabetic peripheral neuropathy.
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Diabetes Mellitus Tipo 2/tratamiento farmacológico , Enfermedades del Sistema Nervioso Periférico/tratamiento farmacológico , Nervio Ciático/efectos de los fármacos , Tadalafilo/administración & dosificación , Animales , Glucemia , Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Experimental/fisiopatología , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/fisiopatología , Neuropatías Diabéticas , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Linfocinas/biosíntesis , Linfocinas/genética , Ratones , Ratones Endogámicos NOD/genética , Actividad Motora/efectos de los fármacos , Fibras Nerviosas/efectos de los fármacos , Fibras Nerviosas/patología , Factor de Crecimiento Nervioso/biosíntesis , Factor de Crecimiento Nervioso/genética , Enfermedades del Sistema Nervioso Periférico/genética , Enfermedades del Sistema Nervioso Periférico/fisiopatología , Factor de Crecimiento Derivado de Plaquetas/biosíntesis , Factor de Crecimiento Derivado de Plaquetas/genética , Nervio Ciático/irrigación sanguínea , Nervio Ciático/fisiopatologíaRESUMEN
Animal models of focal cerebral ischemia are well accepted for investigating the pathogenesis and potential treatment strategies for human stroke. Occlusion of the middle cerebral artery (MCA) with an endovascular filament is a widely used model to induce focal cerebral ischemia. However, this model is not amenable to thrombolytic therapies. As thrombolysis with recombinant tissue plasminogen activator (rtPA) is a standard of care within 4.5 h of human stroke onset, suitable animal models that mimic cellular and molecular mechanisms of thrombosis and thrombolysis of stroke are required. By occluding the MCA with a fibrin-rich allogeneic clot, we previously developed an embolic model of MCA occlusion in the rat, which recapitulates the key components of thrombotic development and of thrombolytic therapy of rtPA observed from human ischemic stroke. Here we describe in detail the surgical procedures of our model, including preparing emboli from rat donors. These procedures can be typically completed within â¼30 min, and they are highly adaptable to other strains of rats, as well as mice, in both sexes. Thus, this model provides a powerful tool for translational stroke research.
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Isquemia Encefálica/etiología , Modelos Animales de Enfermedad , Arteria Cerebral Media/cirugía , Animales , Isquemia Encefálica/fisiopatología , Infarto de la Arteria Cerebral Media/etiología , Masculino , Ratas Wistar , Terapia Trombolítica , Dispositivos de Acceso VascularRESUMEN
The unique cellular and vascular architecture of the adult ventricular-subventricular zone (V/SVZ) neurogenic niche plays an important role in regulating neural stem cell function. However, the in vivo identification of neural stem cells and their relationship to blood vessels within this niche in response to stroke remain largely unknown. Using whole-mount preparation of the lateral ventricle wall, we examined the architecture of neural stem cells and blood vessels in the V/SVZ of adult mouse over the course of 3 months after onset of focal cerebral ischemia. Stroke substantially increased the number of glial fibrillary acidic protein (GFAP) positive neural stem cells that are in contact with the cerebrospinal fluid (CSF) via their apical processes at the center of pinwheel structures formed by ependymal cells residing in the lateral ventricle. Long basal processes of these cells extended to blood vessels beneath the ependymal layer. Moreover, stroke increased V/SVZ endothelial cell proliferation from 2% in non-ischemic mice to 12 and 15% at 7 and 14 days after stroke, respectively. Vascular volume in the V/SVZ was augmented from 3% of the total volume prior to stroke to 6% at 90 days after stroke. Stroke-increased angiogenesis was closely associated with neuroblasts that expanded to nearly encompass the entire lateral ventricular wall in the V/SVZ. These data indicate that stroke induces long-term alterations of the neural stem cell and vascular architecture of the adult V/SVZ neurogenic niche. These post-stroke structural changes may provide insight into neural stem cell mediation of stroke-induced neurogenesis through the interaction of neural stem cells with proteins in the CSF and their sub-ependymal neurovascular interaction.
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Isquemia Encefálica/patología , Encéfalo/irrigación sanguínea , Proteínas del Tejido Nervioso/metabolismo , Células-Madre Neurales/metabolismo , Accidente Cerebrovascular/patología , Animales , Proliferación Celular , Células Cultivadas , Modelos Animales de Enfermedad , Células Endoteliales/citología , Proteína Ácida Fibrilar de la Glía , Masculino , Ratones , Ratones Endogámicos C57BL , Neurogénesis , Accidente Cerebrovascular/etiologíaRESUMEN
B7-H1 and B7-H4 are newly discovered members of the B7-CD28 family. They can inhibit T cell activation and proliferation and regulate T cell immune response negatively. Both B7-H1 and B7-H4 are expressed in many tumors and are classified as co-inhibitors of cell-mediated immunity. FOXP3(+) regulatory T cells (Tregs) play an important role in the maintenance of tumor immunity tolerance. However, the implication of B7-H1 and B7-H4 expression and their interaction with Tregs infiltration in colorectal cancer are unknown. The present study aimed to determine the expression of B7-H1 and B7-H4 as well as Tregs infiltration in colorectal cancer and to explore the clinical and pathological implication of suppressor immune cells and molecules. Frozen sections and immunohistochemical assay were undertaken to assess B7-H1, B7-H4 expression and Tregs infiltration in fresh specimens collected from 56 patients with colorectal carcinoma. The results showed that expression of B7-H1 and B7-H4 in colorectal carcinoma tissues was significantly higher than in adjacent normal mucosa (P<0.001). B7-H1 expression was positively correlated to the infiltration depth, lymph node metastasis and advanced Duke's stage (P<0.05, P<0.05 and P<0.05, respectively), whereas B7-H4 expression was positively related to the infiltration depth and lymph node metastasis (P<0.01 and P<0.05, respectively). Furthermore, Tregs infiltration was more frequent in tumor tissue than in adjacent normal mucosa and was associated with poor differentiation and positive lymph node metastasis (P<0.01, and P<0.01, respectively). The statistical analysis indicated a significant correlation between Tregs infiltration and B7-H1 or B7-H4 expression respectively. These results suggest that over-expression of B7-H1 and B7-H4 has stronger prognostic significance and promote tumor tolerance, and they might contribute to Tregs development in the colorectal carcinoma tolerogenic milieu.
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Antígeno B7-H1/metabolismo , Biomarcadores de Tumor/metabolismo , Neoplasias Colorrectales/metabolismo , Linfocitos T Reguladores/inmunología , Inhibidor 1 de la Activación de Células T con Dominio V-Set/metabolismo , Adulto , Anciano , Neoplasias Colorrectales/inmunología , Neoplasias Colorrectales/patología , Femenino , Factores de Transcripción Forkhead/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Linfocitos T Reguladores/metabolismo , Escape del TumorRESUMEN
Cerebral ischemia induces neurogenesis, including proliferation and differentiation of neural progenitor cells and migration of newly generated neuroblasts. MicroRNAs (miRNAs) are small noncoding RNAs that decrease gene expression through mRNA destabilization and/or translational repression. Emerging data indicate that miRNAs have a role in mediating processes of proliferation and differentiation of adult neural progenitor cells. This article reviews recent findings on miRNA profile changes in neural progenitor cells after cerebral infarction and the contributions of miRNAs to their ischemia-induced proliferation and differentiation. We highlight interactions between the miR-124 and the miR17-92 cluster and the Notch and Sonic hedgehog signaling pathways in mediating stroke-induced neurogenesis.
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Isquemia Encefálica/fisiopatología , MicroARNs/metabolismo , Neurogénesis/fisiología , Animales , Movimiento Celular , Humanos , Células-Madre Neurales/metabolismo , Transducción de Señal/fisiologíaRESUMEN
INTRODUCTION: Stroke remains the leading cause of adult disability. Thus, it is imperative to develop restorative therapies for ischemic stroke designed specifically to treat the intact brain tissue to stimulate functional benefit. Therapies targeting amplification of brain repair processes with nitric oxide (NO) donors and phosphodiesterase type 5 (PDE5) inhibitors in preclinical studies are emerging and showing improvement of functional recovery after stroke. AREAS COVERED: This review will mainly cover the effect of NO donors, which produce NO, and PDE5 inhibitors, which elevate cyclic guanosine 3',5'-monophosphate (cGMP), on neural restorative events in ischemic brain and highlight mechanisms underlying their restorative therapeutic activity. EXPERT OPINION: During stroke recovery, interwoven restorative events occur in ischemic brain, which include angiogenesis, neurogenesis, oligodendrogenesis, astrogliosis and neurite outgrowth. Emerging preclinical data indicate that restorative therapies targeting multiple parenchymal cells including neural stem cells, cerebral endothelial cells, astrocytes, oligodendrocytes, neurons would be more effective than agents with a single cell target. Preclinical data suggest that elevated cGMP levels induced by NO donors and PDE5 inhibitors act on cerebral endothelial cells, neural stem cells and oligodendrocyte progenitor cells to enhance stroke-induced angiogenesis, neurogenesis and oligodendrogenesis, respectively. These interacting remodeling events collectively improve neurological function after stroke.
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Isquemia Encefálica/tratamiento farmacológico , GMP Cíclico/metabolismo , Donantes de Óxido Nítrico/uso terapéutico , Óxido Nítrico/metabolismo , Inhibidores de Fosfodiesterasa 5/uso terapéutico , Accidente Cerebrovascular/prevención & control , Animales , Isquemia Encefálica/complicaciones , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patología , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 5/metabolismo , Evaluación Preclínica de Medicamentos , Neovascularización Fisiológica/efectos de los fármacos , Neurogénesis/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/enzimología , Neuronas/metabolismo , Neuronas/patología , Donantes de Óxido Nítrico/administración & dosificación , Inhibidores de Fosfodiesterasa 5/administración & dosificación , Accidente Cerebrovascular/etiología , Accidente Cerebrovascular/metabolismo , Accidente Cerebrovascular/patologíaRESUMEN
Multipotent mesenchymal stromal cells (MSCs) decrease the expression of transforming growth factor ß1 (TGFß1) in astrocytes and subsequently decrease astrocytic plasminogen activator inhibitor 1 (PAI-1) level in an autocrine manner. Since activated microglia/macrophages are also a source of TGFß1 after stroke, we therefore tested whether MSCs regulate TGFß1 expression in microglia/macrophages and subsequently alters PAI-1 expression after ischemia. TGFß1 and its downstream effector phosphorylated SMAD 2/3 (p-SMAD 2/3) were measured in mice subjected to middle cerebral artery occlusion (MCAo). MSC treatment significantly decreased TGFß1 protein expression in both astrocytes and microglia/macrophages in the ischemic boundary zone (IBZ) at day 14 after stroke. However, the p-SMAD 2/3 was only detected in astrocytes and decreased after MSC treatment. In vitro, RT-PCR results showed that the TGFß1 mRNA level was increased in both astrocytes and microglia/macrophages in an astrocyte-microglia/macrophage co-culture system after oxygen-glucose deprived (OGD) treatment. MSCs treatment significantly decreased the above TGFß1 mRNA level under OGD conditions, respectively. OGD increased the PAI-1 mRNA in astrocytes in the astrocyte-microglia/macrophage co-culture system, and MSC administration significantly decreased this level. PAI-1 mRNA was very low in microglia/macrophages compared with that in astrocytes under different conditions. Western blot results also verified that MSC administration significantly decreased p-SMAD 2/3 and PAI-1 level in astrocytes in astrocyte-microglia/macrophage co-culture system under OGD conditions. Our in vivo and in vitro data, in concert, suggest that MSCs decrease TGFß1 expression in microglia/macrophages in the IBZ which contribute to the down-regulation of PAI-1 level in astrocytes.
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Astrocitos/metabolismo , Macrófagos/metabolismo , Trasplante de Células Madre Mesenquimatosas , Microglía/metabolismo , Inhibidor 1 de Activador Plasminogénico/metabolismo , Accidente Cerebrovascular/terapia , Factor de Crecimiento Transformador beta1/metabolismo , Animales , Hipoxia de la Célula , Técnicas de Cocultivo , Regulación hacia Abajo , Glucosa/metabolismo , Infarto de la Arteria Cerebral Media/complicaciones , Ratones , Ratones Endogámicos C57BL , Accidente Cerebrovascular/etiología , Accidente Cerebrovascular/metabolismo , Accidente Cerebrovascular/patologíaRESUMEN
The role of microRNAs (miRNAs) in mediating adult neurogenesis after stroke has not been extensively studied. The present study investigated the function of the miR17-92 cluster in adult neural progenitor cells after experimental stroke. We found that stroke substantially up-regulated miR17-92 cluster expression in neural progenitor cells of the adult mouse. Overexpression of the miR17-92 cluster either in cultured ischemic neural progenitor cells or in the subventricular zone (SVZ) of ischemic animals significantly increased cell proliferation, whereas inhibition of individual members of the miR17-92 cluster, miR-18a and miR-19a, suppressed cell proliferation and increased cell death. The miR17-92 cluster mediated PTEN (phosphatase and tensin homolog) expression, which is a predicted target of the miR17-92 cluster. Addition of Sonic hedgehog (Shh) protein up-regulated miR17-92 expression and elevated c-Myc protein in ischemic neural progenitor cells, whereas blockade of the Shh signaling pathway down-regulated miR17-92 cluster expression and reduced c-Myc levels. Overexpression of c-Myc up-regulated miR17-92 cluster expression. Intraventricular infusion of Shh and a Shh receptor inhibitor, cyclopamine, to ischemic animals further elevated and suppressed, respectively, miR17-92 cluster expression in the SVZ. These data indicate that the miR17-92 cluster plays an important role in mediating neural progenitor cell function and that the Shh signaling pathway is involved in up-regulating miR17-92 cluster expression.
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Proliferación Celular , Regulación de la Expresión Génica , MicroARNs/biosíntesis , Familia de Multigenes , Células-Madre Neurales/metabolismo , Transducción de Señal , Animales , Supervivencia Celular , Modelos Animales de Enfermedad , Proteínas Hedgehog/metabolismo , Masculino , Ratones , Proteínas del Tejido Nervioso/metabolismo , Células-Madre Neurales/patología , Fosfohidrolasa PTEN/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismoRESUMEN
Adult neural stem cells give rise to neurons, oligodendrocytes and astrocytes. Aging reduces neural stem cells. Using an inducible nestin-CreER(T2)/R26R-yellow fluorescent protein (YFP) mouse, we investigated the effect of Sildenafil, a phosphodiesterase type 5 (PDE5) inhibitor, on nestin lineage neural stem cells and their progeny in the ischemic brain of the middle-aged mouse. We showed that focal cerebral ischemia induced nestin lineage neural stem cells in the subventricular zone (SVZ) of the lateral ventricles and nestin expressing NeuN positive neurons and adenomatous polyposis coli (APC) positive mature oligodendrocytes in the ischemic striatum and corpus callosum in the aged mouse. Treatment of the ischemic middle-aged mouse with Sildenafil increased nestin expressing neural stem cells, mature neurons, and oligodendrocytes by 33, 75, and 30%, respectively, in the ischemic brain. These data indicate that Sildenafil amplifies nestin expressing neural stem cells and their neuronal and oligodendrocyte progeny in the ischemic brain of the middle-aged mouse.
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Infarto de la Arteria Cerebral Media/tratamiento farmacológico , Neurogénesis/efectos de los fármacos , Oligodendroglía/efectos de los fármacos , Inhibidores de Fosfodiesterasa 5/farmacología , Piperazinas/farmacología , Sulfonas/farmacología , Animales , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Rastreo Celular , Cuerpo Calloso/efectos de los fármacos , Cuerpo Calloso/metabolismo , Cuerpo Calloso/patología , Proteínas de Unión al ADN , Infarto de la Arteria Cerebral Media/patología , Proteínas de Filamentos Intermediarios/metabolismo , Ventrículos Laterales/efectos de los fármacos , Ventrículos Laterales/metabolismo , Ventrículos Laterales/patología , Proteínas Luminiscentes/biosíntesis , Proteínas Luminiscentes/genética , Masculino , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso/metabolismo , Nestina , Células-Madre Neurales/efectos de los fármacos , Células-Madre Neurales/metabolismo , Células-Madre Neurales/fisiología , Proteínas Nucleares/metabolismo , Oligodendroglía/metabolismo , Oligodendroglía/fisiología , Inhibidores de Fosfodiesterasa 5/uso terapéutico , Piperazinas/uso terapéutico , Purinas/farmacología , Purinas/uso terapéutico , Citrato de Sildenafil , Sulfonas/uso terapéuticoRESUMEN
Peripheral neuropathy is one of the most common complications of diabetes mellitus. Using a mouse model of diabetic peripheral neuropathy, we tested the hypothesis that thymosin ß4 (Tß4) ameliorates diabetes-induced neurovascular dysfunction in the sciatic nerve and promotes recovery of neurological function from diabetic peripheral neuropathy. Tß4 treatment of diabetic mice increased functional vascular density and regional blood flow in the sciatic nerve, and improved nerve function. Tß4 upregulated angiopoietin-1 (Ang1) expression, but suppressed Ang2 expression in endothelial and Schwann cells in the diabetic sciatic nerve. In vitro, incubation of Human Umbilical Vein Endothelial Cells (HUVECs) with Tß4 under high glucose condition completely abolished high glucose-downregulated Ang1 expression and high glucose-reduced capillary-like tube formation. Moreover, incubation of HUVECs under high glucose with conditioned medium collected from Human Schwann Cells (HSCs) treated with Tß4 significantly reversed high glucose-decreased capillary-like tube formation. PI3K/Akt signaling pathway is involved in Tß4-regulated Ang1 expression on endothelial and Schwann cells. These data indicate that Tß4 likely acts on endothelial cells and Schwann cells to preserve and/or restore vascular function in the sciatic nerve which facilitates improvement of peripheral nerve function under diabetic neuropathy. Thus, Tß4 has potential for the treatment of diabetic peripheral neuropathy.
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Neuropatías Diabéticas/metabolismo , Fármacos Neuroprotectores/farmacología , Nervio Ciático/efectos de los fármacos , Timosina/farmacología , Animales , Western Blotting , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/fisiopatología , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/metabolismo , Neuropatías Diabéticas/fisiopatología , Modelos Animales de Enfermedad , Electrofisiología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Humanos , Inmunohistoquímica , Flujometría por Láser-Doppler , Ratones , Reacción en Cadena en Tiempo Real de la Polimerasa , Flujo Sanguíneo Regional , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células de Schwann/efectos de los fármacos , Células de Schwann/metabolismo , Nervio Ciático/irrigación sanguínea , Nervio Ciático/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Serum response factor (SRF) is a transcription factor that transactivates actin-associated genes and has been implicated in oligodendrocyte (OL) differentiation. To date, it has not been investigated in cerebral ischemia. We investigated the dynamics of SRF expression after stroke in vivo and the role of SRF in OL differentiation in vitro. Using immunohistochemistry, we found that SRF was upregulated in OLs and OL precursor cells (OPCs) after stroke. Moreover, upregulation of SRF was concurrent with downregulation of the micro-RNAs (miRNAs) miR-9 and the miR-200 family in the ischemic white matter region, the corpus callosum. Inhibition of SRF activation by CCG-1423, a specific inhibitor of SRF function, blocked OPCs from differentiating into OLs. Overexpression of miR-9 and miR-200 in cultured OPCs suppressed SRF expression and inhibited OPC differentiation. Moreover, co-expression of miR-9 and miR-200 attenuated activity of a luciferase reporter assay containing the Srf 3' untranslated region. Collectively, this study is the first to show that stroke upregulates SRF expression in OPCs and OLs, and that SRF levels are mediated by miRNAs and regulate OPC differentiation.
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Diferenciación Celular/fisiología , MicroARNs/fisiología , Oligodendroglía/fisiología , Factor de Respuesta Sérica/biosíntesis , Células Madre/fisiología , Animales , Células Cultivadas , Masculino , MicroARNs/antagonistas & inhibidores , Oligodendroglía/citología , Ratas , Ratas Wistar , Factor de Respuesta Sérica/fisiología , Células Madre/citología , Accidente Cerebrovascular/metabolismo , Accidente Cerebrovascular/patologíaRESUMEN
OBJECTIVE: To investigate the knowledge and attitude of clinicians in the departments of pediatrics and otolaryngology to pediatric obstructive sleep apnea hypopnea syndrome (OSAHS), since in China, the clinicians in these two departments had closest relationship with the diagnosis and treatment of OSAHS in children. METHODS: A validated questionnaire from USA which was the obstructive sleep apnea knowledge and attitudes questionnaire in children (OSAKA-KIDS) was used and permission by original author. The questionnaire was mailed to ENT doctors and pediatricians in 43 public hospitals in Shandong province. RESULTS: OSA-KIDS in Chinese version was re-tested by 30 physicians, r = 0.92. Totally, 391 valid questionnaires (87.7%) were returned. Average of correct rate (x(-) ± s) in 18 knowledge items was 64.1% ± 19.1%. Cronbach's α coefficient was 0.76. There was no difference between ENT doctors and pediatrics in total knowledge score. However, there was significant difference in below 2 questions: ENT doctors had more correction in answer "nearly 2% of children have OSAHS" and pediatrics had more correction in answer "pediatric OSAHS may be associated with pulmonary hypertension". Only 24.3% clinicians correctly know the degree of snoring (mild to severe) was not correlated with the severity of obstructive apnea in children. Only 16.1% could correctly answer the question about cardio-respiratory monitor could not reliably detect both central and obstructive apnea in infant. Cronbach's α coefficient was 0.72 in 5 items which was about importance of disease and self-evaluation in confidence. While more than 90% clinicians stated that "As a clinical disorder OSAHS is important or very, extremely important". However, among them, only about 36% felt confident in identifying or managing children with OSAHS. Total knowledge score about OSAHS was not different by gender or specialty (P > 0.05), but more knowledge was associated with more positive attitudes overall (P < 0.05) and more elder in age or longer years in practice (r = 0.384, P < 0.0001). CONCLUSIONS: It should be paid more effort to elevate the knowledge and attitude about pediatric OSAHS in pediatricians and otolaryngologists.
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Conocimientos, Actitudes y Práctica en Salud , Médicos , Apnea Obstructiva del Sueño , Encuestas y Cuestionarios , Adulto , Anciano , Niño , Femenino , Humanos , Masculino , Persona de Mediana Edad , Pediatría , Apnea Obstructiva del Sueño/diagnóstico , Apnea Obstructiva del Sueño/terapiaRESUMEN
BACKGROUND: The Notch signaling pathway regulates adult neurogenesis under physiological and pathophysiological conditions. MicroRNAs are small non-coding RNA molecules that regulate gene expression. The present study investigated the effect of miR-124a on the Notch signaling pathway in stroke-induced neurogenesis. METHODOLOGY AND PRINCIPAL FINDINGS: We found that adult rats subjected to focal cerebral ischemia exhibited substantial reduction of miR-124a expression, a neuron specific miRNA, in the neural progenitor cells of the subventricular zone (SVZ) of the lateral ventricle, which was inversely associated with activation of Notch signals. In vitro, transfection of neural progenitor cells harvested from the SVZ of adult rat with miR-124a repressed Jagged-1 (JAG1), a ligand of Notch, in a luciferase construct containing the JAG1 target site. Introduction of miR-124a in neural progenitor cells significantly reduced JAG1 transcript and protein levels, leading to inactivation of Notch signals. Transfection of neural progenitor cells with miR-124a significantly reduced progenitor cell proliferation and promoted neuronal differentiation measured by an increase in the number of Doublecortin positive cells, a marker of neuroblasts. Furthermore, introduction of miR-124a significantly increased p27Kip1 mRNA and protein levels, a downstream target gene of the Notch signaling pathway. CONCLUSIONS: Collectively, our study demonstrated that in vivo, stroke alters miRNA expression in SVZ neural progenitor cells and that in vitro, miR-124a mediates stroke-induced neurogenesis by targeting the JAG-Notch signaling pathway.
Asunto(s)
Ventrículos Cerebrales/metabolismo , Perfilación de la Expresión Génica , MicroARNs/metabolismo , Células-Madre Neurales/metabolismo , Receptores Notch/metabolismo , Accidente Cerebrovascular/genética , Accidente Cerebrovascular/patología , Regiones no Traducidas 3'/genética , Animales , Secuencia de Bases , Diferenciación Celular/genética , Proliferación Celular , Ventrículos Cerebrales/patología , Proteína Doblecortina , Regulación de la Expresión Génica , Infarto de la Arteria Cerebral Media , Masculino , MicroARNs/genética , Datos de Secuencia Molecular , Células-Madre Neurales/patología , Neurogénesis/genética , Ratas , Ratas Wistar , Reproducibilidad de los Resultados , Transducción de Señal/genéticaRESUMEN
Multipotent mesenchymal stromal cells (MSCs) increase tissue plasminogen activator (tPA) activity in astrocytes of the ischemic boundary zone, leading to increased neurite outgrowth in the brain. To probe the mechanisms that underlie MSC-mediated activation of tPA, we investigated the morphogenetic gene, sonic hedgehog (Shh) pathway. In vitro oxygen and glucose deprivation and coculture of astrocytes and MSCs were used to mimic an in vivo ischemic condition. Both real-time-PCR and western blot showed that MSC coculture significantly increased the Shh level and concomitantly increased tPA and decreased plasminogen activator inhibitor 1 (PAI-1) levels in astrocytes. Inhibiting the Shh signaling pathway with cyclopamine blocked the increase of tPA and the decrease of PAI-1 expression in astrocytes subjected to MSC coculture or recombinant mouse Shh (rm-Shh) treatment. Both MSCs and rm-Shh decreased the transforming growth factor-ß1 level in astrocytes, and the Shh pathway inhibitor cyclopamine reversed these decreases. Both Shh-small-interfering RNA (siRNA) and Glil-siRNA downregulated Shh and Gli1 (a key mediator of the Shh transduction pathway) expression in cultured astrocytes and concomitantly decreased tPA expression and increased PAI-1 expression in these astrocytes after MSC or rm-Shh treatment. Our data indicate that MSCs increase astrocytic Shh, which subsequently increases tPA expression and decreases PAI-1 expression after ischemia.
Asunto(s)
Astrocitos/metabolismo , Proteínas Hedgehog/metabolismo , Células Madre Mesenquimatosas/fisiología , Células Madre Multipotentes/fisiología , Serpina E2/biosíntesis , Accidente Cerebrovascular , Activador de Tejido Plasminógeno/biosíntesis , Animales , Astrocitos/citología , Astrocitos/patología , Técnicas de Cultivo de Célula , Hipoxia de la Célula , Técnicas de Cocultivo , Medios de Cultivo , Regulación hacia Abajo , Glucosa/metabolismo , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Ratones , Ratones Endogámicos C57BL , Células Madre Multipotentes/citología , Células Madre Multipotentes/metabolismo , Oxígeno/metabolismo , Receptor Cross-Talk , Transducción de Señal , Accidente Cerebrovascular/metabolismo , Accidente Cerebrovascular/patología , Regulación hacia ArribaRESUMEN
BACKGROUND AND PURPOSE: We tested the effect of bone marrow stromal cells (BMSCs) on neuronal remodeling of the corticospinal tract originating from the contralesional cortex in mice subjected to unilateral pyramidotomy (PT) followed by middle cerebral artery occlusion (MCAO). METHODS: Adult mice with transgenic yellow fluorescent protein labeling in the corticospinal tract were subjected to right hemispheric PT and right permanent or sham MCAO. One day later, the mice were treated intravenously with BMSCs or phosphate-buffered saline. A Foot-Fault test and a single pellet-reaching test were performed before surgery, 3 days after MCAO, and weekly thereafter. Pseudorabies virus-614-monomeric red fluorescent protein was injected into the left forelimb flexor muscles 28 days after surgery (4 days before euthanasia). The brain and cervical cord were processed for fluorescent microscopy to detect red fluorescent protein and yellow fluorescent protein labeling, respectively. RESULTS: Significant functional improvements were evident in PT-MCAO mice treated with BMSCs (n=9) compared with phosphate-buffered saline-treated mice (n=9, P<0.05), but not in mice with PT-sham MCAO treated with either phosphate-buffered saline (n=9) or BMSCs (n=10). Furthermore, in PT-MCAO mice, both corticospinal tract axonal density in the denervated side of the cervical gray matter and red fluorescent protein-labeled pyramidal neurons in the left intact cortex were significantly increased compared with PT-sham MCAO mice (P<0.05). BMSCs significantly enhanced both corticospinal tract density and red fluorescent protein labeling in PT-MCAO mice (P<0.05) only. The behavioral outcome was highly correlated with corticospinal tract density and red fluorescent protein labeling. CONCLUSIONS: BMSCs amplify stroke-induced contralesional neuronal remodeling, which contributes to motor recovery after stroke.
Asunto(s)
Axones/fisiología , Trasplante de Médula Ósea , Isquemia Encefálica/fisiopatología , Destreza Motora/fisiología , Recuperación de la Función/fisiología , Accidente Cerebrovascular/fisiopatología , Factores de Edad , Animales , Isquemia Encefálica/complicaciones , Isquemia Encefálica/cirugía , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Corteza Motora/fisiología , Tractos Piramidales/fisiología , Accidente Cerebrovascular/complicaciones , Accidente Cerebrovascular/cirugía , Células del Estroma/trasplanteRESUMEN
BACKGROUND AND PURPOSE: Bone marrow stromal cells (BMSC) decrease neurological deficits in rodents after stroke and concomitantly induce extensive neurite remodeling in the brain, which highly correlates with the improvement of neurological function. We investigated the effects of endogenous tissue plasminogen activator (tPA) on neurite remodeling after BMSC treatment. METHODS: Adult C57BL/6 wild-type (WT) mice and tPA knockout (tPA(-/-)) mice were subjected to middle cerebral artery occlusion, followed by an injection of 1×10(6) BMSC (n=18) or phosphate-buffered saline (n=18) into the tail vein 24 hours later. Behavioral tests were performed at 3, 7, and 14 days after middle cerebral artery occlusion. Animals were euthanized at 14 days after stroke. RESULTS: The effects of BMSC on functional recovery depended on presence or absence of tPA, even after adjusting for imbalanced stroke severity. BMSC significantly improve functional recovery in WT mice compared to WT controls but show no beneficial effect in the tPA(-/-) mice compared to tPA(-/-) controls. Axonal density and synaptophysin-positive areas along the ischemic boundary zone of the cortex and striatum in WT mice are significantly higher than in the tPA(-/-) mice. BMSC treatment significantly increases tPA protein level and activity only in WT mice. CONCLUSIONS: Our results suggest that endogenous tPA promotes BMSC-induced neurite outgrowth and may contribute to functional recovery after stroke.
Asunto(s)
Trasplante de Médula Ósea , Neuritas/fisiología , Accidente Cerebrovascular/cirugía , Activador de Tejido Plasminógeno/fisiología , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/fisiología , Trasplante de Médula Ósea/métodos , Trasplante de Médula Ósea/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Recuperación de la Función/fisiología , Accidente Cerebrovascular/patología , Células del Estroma/fisiologíaRESUMEN
Neural and oligodendrocyte progenitor cells in the adult brain express Ascl1 (also known as Mash1), a basic helix-loop-helix transcription factor. We examined the progeny and fate of this progenitor population in adult male Ascl1-CreER(TM);R26R-stop-yellow fluorescent protein mice subjected to right middle cerebral occlusion over 60 days after stroke using inducible Cre recombination to label Ascl1-expressing cells at poststroke days 2 to 6 in vivo. Seven days after stroke, a substantial increase in Ascl1 lineage cells was detected in the ipsilateral subventricular zone (SVZ), striatum, and corpus callosum. These cells exhibited proliferating progenitor cell phenotypes (Sox2(+), BrdU(+), and Ki67(+)). Although Ascl1 lineage cells in the ipsilateral SVZ gradually decreased during 14 to 60 days after stroke, Ascl1 lineage cells in the ischemic striatum revealed a remarkable increase during this period. Thirty and sixty days after stroke, Ascl1 lineage cells in the ischemic striatum gave rise to GABAergic neurons and mature oligodendrocytes. In contrast, none of the Ascl1 lineage cells in the contralateral striatum exhibited neuronal and oligodendrocyte phenotypes. Moreover, Ascl1 lineage cells in the corpus callosum were only fated to become mature oligodendrocytes. Our data suggest that Ascl1 lineage cells contribute to stroke-induced neurogenesis and oligodendrogenesis in the adult ischemic brain.