RESUMO
Vascular endothelial growth factor (VEGF), an angiogenic factor produced in response to ischemic injury, promotes vascular permeability (VP). Evidence is provided that Src kinase regulates VEGF-mediated VP in the brain following stroke and that suppression of Src activity decreases VP thereby minimizing brain injury. Mice lacking pp60c-src are resistant to VEGF-induced VP and show decreased infarct volumes after stroke whereas mice deficient in pp59c-fyn, another Src family member, have normal VEGF-mediated VP and infarct size. Systemic application of a Src-inhibitor given up to six hours following stroke suppressed VP protecting wild-type mice from ischemia-induced brain damage without influencing VEGF expression. This was associated with reduced edema, improved cerebral perfusion and decreased infarct volume 24 hours after injury as measured by magnetic resonance imaging and histological analysis. Thus, Src represents a key intermediate and novel therapeutic target in the pathophysiology of cerebral ischemia where it appears to regulate neuronal damage by influencing VEGF-mediated VP.
Assuntos
Acidente Vascular Cerebral/prevenção & controle , Quinases da Família src/fisiologia , Animais , Encéfalo/irrigação sanguínea , Lesões Encefálicas/patologia , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patologia , Permeabilidade Capilar , Infarto Cerebral/patologia , Edema/patologia , Fatores de Crescimento Endotelial/genética , Expressão Gênica , Linfocinas/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fluxo Sanguíneo Regional , Acidente Vascular Cerebral/patologia , Acidente Vascular Cerebral/fisiopatologia , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio Vascular , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/genéticaRESUMO
Bone marrow stromal cells (BMSCs) facilitate functional recovery in rats after focal ischemic attack. Growing evidence suggests that the secretion of various bioactive factors underlies BMSCs' beneficial effects. This study investigates the expression of glial cell derived neurotrophic factor (GDNF) in the ischemic hemisphere with or without BMSC administration. Adult male Wistar rats were subjected to 2 h of middle cerebral artery occlusion followed by an injection of 3 x 10(6) BMSCs (n = 11) or phosphate-buffered saline (n = 10) into the tail vein 24 h later. Animals were sacrificed seven days later. Single and double immunohistochemical staining was performed to measure GDNF, Ki67, doublecortin, and glial fibrillary acidic protein expression as well as the number of apoptotic cells along the ischemic boundary zone (IBZ) and/or in the subventricular zone (SVZ). BMSC treatment significantly increased GDNF expression and decreased the number of apoptotic cells in the IBZ (P < 0.05). GDNF expression was colocalized with GFAP. Meanwhile, BMSCs increased the number of Ki-67 positive cells and the density of DCX positive migrating neuroblasts (P < 0.05). GDNF expression was significantly increased in single astrocytes collected from animals treated with BMSCs, and in astrocytes cocultured with BMSCs after OGD (P < 0.05). Our data suggest that BMSCs increase GDNF levels in the ischemic hemisphere; the major source of GDNF protein is reactive astrocytes. We propose that the increase of GDNF in response to BMSC administration creates a hospitable environment for local cellular repair as well as for migrating neuroblasts from the SVZ, and thus contributes to the functional improvement.
Assuntos
Astrócitos/metabolismo , Transplante de Medula Óssea , Isquemia Encefálica/terapia , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Acidente Vascular Cerebral/terapia , Células Estromais/transplante , Envelhecimento , Animais , Apoptose/fisiologia , Encéfalo/fisiopatologia , Isquemia Encefálica/fisiopatologia , Proteína Duplacortina , Infarto da Artéria Cerebral Média/fisiopatologia , Infarto da Artéria Cerebral Média/terapia , Masculino , Neurônios/fisiologia , Distribuição Aleatória , Ratos , Ratos Wistar , Nicho de Células-Tronco/fisiopatologia , Acidente Vascular Cerebral/fisiopatologiaRESUMO
Arteriogenesis supports restored perfusion in the ischemic brain and improves long-term functional outcome after stroke. We investigate the role of endothelial nitric oxide synthetase (eNOS) and a nitric oxide (NO) donor, (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl) amino] diazen-1-ium-1, 2-diolate (DETA-NONOate), in promoting arteriogenesis after stroke. Adult wild-type (WT, n=18) and eNOS-knockout (eNOS(-/-), n=36) mice were subjected to transient (2.5 h) right middle cerebral artery occlusion (MCAo) and were treated with or without DETA-NONOate (0.4 mg/kg) 24 h after MCAo. Functional evaluation was performed. Animals were sacrificed 3 days after MCAo for arterial cell culture studies, or 14 days for immunohistochemical analysis. Consistent with previous studies, eNOS(-/-) mice exhibited a higher mortality rate (P<0.05, n=18/group) and more severe neurological functional deficit after MCAo than WT mice (P<0.05, n=12/group). Decreased arteriogenesis, was evident in eNOS(-/-) mice compared with WT mice, as demonstrated by reduced vascular smooth muscle cell (VSMC) proliferation, arterial density and diameter in the ischemic brain. eNOS(-/-) mice treated with DETA-NONOate had a significantly decreased mortality rate and improved functional recovery, and exhibited enhanced arteriogenesis identified by increased VSMC proliferation, and upregulated arterial density and diameter compared to eNOS(-/-) mice after stroke (P<0.05, n=12/group). To elucidate the mechanisms underlying eNOS/NO mediated arteriogenesis, VSMC migration was measured in vitro. Arterial cell migration significantly decreased in the cultured common carotid artery (CCA) derived from eNOS(-/-) mice 3 days after MCAo compared to WT arterial cells. DETA-NONOate-treatment significantly attenuated eNOS(-/-)-induced decrease of arterial cell migration compared to eNOS(-/-) control artery (P<0.05; n=6/group). Using VSMC culture, DETA-NONOate significantly increased VSMC migration, while inhibition of NOS significantly decreased VSMC migration (P<0.05; n=6/group). Our data indicated that eNOS not only promotes vascular dilation but also increases VSMC proliferation and migration, and thereby enhances arteriogenesis after stroke. Therefore, increase eNOS may play an important role in regulating of arteriogenesis after stroke.
Assuntos
Infarto da Artéria Cerebral Média/enzimologia , Infarto da Artéria Cerebral Média/fisiopatologia , Neovascularização Fisiológica/fisiologia , Óxido Nítrico Sintase Tipo III/metabolismo , Actinas/metabolismo , Animais , Artéria Carótida Primitiva/citologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Infarto da Artéria Cerebral Média/tratamento farmacológico , Infarto da Artéria Cerebral Média/patologia , Antígeno Ki-67/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miócitos de Músculo Liso/efeitos dos fármacos , NG-Nitroarginina Metil Éster/farmacologia , Neovascularização Fisiológica/efeitos dos fármacos , Exame Neurológico , Doadores de Óxido Nítrico/uso terapêutico , Óxido Nítrico Sintase Tipo III/deficiência , Compostos Nitrosos/uso terapêutico , Recuperação de Função Fisiológica/efeitos dos fármacos , Fatores de TempoRESUMO
Molecular mechanisms by which stroke increases neurogenesis have not been fully investigated. Using neural progenitor cells isolated from the subventricular zone (SVZ) of the adult rat subjected to focal cerebral ischemia, we investigated the Notch pathway in regulating proliferation and differentiation of adult neural progenitor cells after stroke. During proliferation of neural progenitor cells, ischemic neural progenitor cells exhibited substantially increased levels of Notch, Notch intracellular domain (NICD), and hairy enhancer of split (Hes) 1, which was associated with a significant increase of proliferating cells. Blockage of the Notch pathway by short interfering ribonucleic acid (siRNA) against Notch or a gamma secretase inhibitor significantly reduced Notch, NICD and Hes1 expression and cell proliferation induced by stroke. During differentiation of neural progenitor cells, Notch and Hes1 expression was downregulated in ischemic neural progenitor cells, which was coincident with a significant increase of neuronal population. Inhibition of the Notch pathway with a gamma secretase inhibitor further substantially increased neurons, but did not alter astrocyte population in ischemic neural progenitor cells. These data suggest that the Notch signaling pathway mediates adult SVZ neural progenitor cell proliferation and differentiation after stroke.
Assuntos
Células-Tronco Adultas/patologia , Ventrículos Cerebrais/patologia , Infarto da Artéria Cerebral Média/patologia , Neurônios/fisiologia , Receptores Notch/metabolismo , Transdução de Sinais/fisiologia , Análise de Variância , Animais , Bromodesoxiuridina/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Células Cultivadas , Expressão Gênica/efeitos dos fármacos , Fator Inibidor de Leucemia/metabolismo , Masculino , Proteínas do Tecido Nervoso/metabolismo , Neurogênese/fisiologia , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/farmacologia , Ratos , Ratos Wistar , Receptores Notch/genética , Transdução de Sinais/efeitos dos fármacos , Transfecção , Triglicerídeos , Ácido gama-Aminobutírico/análogos & derivadosRESUMO
BACKGROUND: Exosomes from mesenchymal stromal cells (MSCs) are endosome-derived vesicles that have been shown to enhance functional recovery in rodent models of stroke. OBJECTIVE: Building on these findings, we tested exosomes as a treatment in monkeys with cortical injury. METHODS: After being trained on a task of fine motor function of the hand, monkeys received a cortical injury to the hand representation in primary motor cortex. Twenty-four hours later and again 14 days after injury, monkeys received exosomes or vehicle control. Recovery of motor function was followed for 12 weeks. RESULTS: Compared to monkeys that received vehicle, exosome treated monkeys returned to pre-operative grasp patterns and latency to retrieve a food reward in the first three-five weeks of recovery. CONCLUSIONS: These results provide evidence that in monkeys exosomes delivered after cortical injury enhance recovery of motor function.
Assuntos
Exossomos , Córtex Motor/efeitos dos fármacos , Córtex Motor/lesões , Destreza Motora/efeitos dos fármacos , Recuperação de Função Fisiológica/efeitos dos fármacos , Animais , Comportamento Animal/efeitos dos fármacos , Modelos Animais de Doenças , Macaca mulattaRESUMO
Neovascularization may contribute to functional recovery after neural injury. Combination treatment of stroke with a nitric oxide donor, (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl) amino] diazen-1-ium-1, 2-diolate (DETA-NONOate) and bone marrow stromal cells promotes functional recovery. However, the mechanisms underlying functional improvement have not been elucidated. In this study, we tested the hypothesis that combination treatment upregulates angiopoietin-1 and its receptor Tie2 in the ischemic brain and bone marrow stromal cells, thereby enhancing cerebral neovascularization after stroke. Adult wild type male C57BL/6 mice were i.v. administered PBS, bone marrow stromal cells 5x10(5), DETA-NONOate 0.4 mg/kg or combination DETA-NONOate with bone marrow stromal cells (n=12/group) after middle cerebral artery occlusion. Combination treatment significantly upregulated angiopoietin-1/Tie2 and tight junction protein (occludin) expression, and increased the number, diameter and perimeter of blood vessels in the ischemic brain compared with vehicle control (mean+ or -S.E., P<0.05). In vitro, DETA-NONOate significantly increased angiopoietin-1/Tie2 protein (n=6/group) and Tie2 mRNA (n=3/group) expression in bone marrow stromal cells. DETA-NONOate also significantly increased angiopoietin-1 protein (n=6/group) and mRNA (n=3/group) expression in mouse brain endothelial cells (P<0.05). Angiopoietin-1 mRNA (n=3/group) was significantly increased in mouse brain endothelial cells treated with DETA-NONOate in combination with bone marrow stromal cell-conditioned medium compared with cells treated with bone marrow stromal cell-conditioned medium or DETA-NONOate alone. Mouse brain endothelial cell capillary tube-like formation assays (n=6/group) showed that angiopoietin-1 peptide, the supernatant of bone marrow stromal cells and DETA-NONOate significantly increased capillary tube formation compared with vehicle control. Combination treatment significantly increased capillary tube formation compared with DETA-NONOate treatment alone. Inhibition of angiopoietin-1 significantly attenuated combination treatment-induced tube formation. Our data indicated that combination treatment of stroke with DETA-NONOate and bone marrow stromal cells promotes neovascularization, which is at least partially mediated by upregulation of the angiopoietin-1/Tie2 axis.
Assuntos
Angiopoietina-1/metabolismo , Transplante de Medula Óssea/métodos , Neovascularização Fisiológica/efeitos dos fármacos , Compostos Nitrosos/farmacologia , Acidente Vascular Cerebral/tratamento farmacológico , Acidente Vascular Cerebral/cirurgia , Angiopoietina-1/agonistas , Angiopoietina-1/antagonistas & inibidores , Animais , Capilares/efeitos dos fármacos , Capilares/metabolismo , Células Cultivadas , Artérias Cerebrais/efeitos dos fármacos , Artérias Cerebrais/metabolismo , Técnicas de Cocultura , Modelos Animais de Doenças , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Fisiológica/fisiologia , Doadores de Óxido Nítrico/farmacologia , Doadores de Óxido Nítrico/uso terapêutico , Compostos Nitrosos/uso terapêutico , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Receptor TIE-2/genética , Regeneração/efeitos dos fármacos , Regeneração/fisiologia , Células Estromais/transplante , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologiaRESUMO
Transplantation of bone marrow stromal cells (BMSCs) improves animal neurological functional recovery after stroke. To obtain insight into the mechanisms underlying the therapeutic benefit, we directed our attention to the interaction of BMSCs with astrocytes. Astrocytes become reactive (astrogliosis) after a brain injury, such as stroke. Astrogliosis plays both beneficial and detrimental roles in brain recovery. Previously, we have shown that administration of BMSCs to animals with stroke significantly reduces the thickness of the scar wall formed by reactive astrocytes. We tested the influence of mouse bone marrow stromal cell (mBMSC) on astrogliosis under oxygen-glucose deprivation (OGD)/reoxygenation conditions in vitro, employing an anaerobic chamber. Our data indicate that mBMSCs down-regulate glial fibrillary acidic protein (GFAP) expression in astrocytes after 2 h of OGD and an additional 16 h reoxygenation. mBMSCs protected astrocytes from ischemia, maintaining morphological integrity and proliferation. The IL-6/IL-6R/gp130 pathway is associated with astrogliosis in response to CNS (disorders. Therefore, we examined the effects of mBMSC on the IL-6/IL-6R/gp130 pathway as an underlying mechanism of mBMSC-altered astrogliosis. Furthermore, IL-6 siRNA was used to block IL-6 expression in astrocytes to further investigate IL-6 involvement in mBMSC-altered astrogliosis. Our results indicate that the mBMSC-conferred decline of astrogliosis post-ischemia may derive from the down-regulation of the IL-6/IL-6R/gp130 pathway.
Assuntos
Astrócitos/metabolismo , Isquemia Encefálica/fisiopatologia , Gliose/fisiopatologia , Células Estromais/metabolismo , Animais , Astrócitos/patologia , Células da Medula Óssea/citologia , Isquemia Encefálica/patologia , Comunicação Celular , Proliferação de Células , Células Cultivadas , Técnicas de Cocultura , Receptor gp130 de Citocina/genética , Receptor gp130 de Citocina/metabolismo , Citoproteção , Regulação para Baixo/imunologia , Proteína Glial Fibrilar Ácida/metabolismo , Gliose/patologia , Gliose/prevenção & controle , Sobrevivência de Enxerto , Interleucina-6/genética , Interleucina-6/metabolismo , Camundongos , RNA Interferente Pequeno , Receptores de Interleucina-6/genética , Receptores de Interleucina-6/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Células Estromais/citologiaRESUMO
We describe some of our studies on use of neuro-restorative agents for treatment of neural injury. We focus on cell-based therapies and select from a variety of statins. In addition, we show that cell-based and pharmacological-based therapies enhance brain plasticity and promote recovery of function after stroke and intracerebral hemorrhage (ICH). Injured brain recapitulates ontogeny. Cerebral tissue around the infarction expresses developmental genes, many of which are present only during embryonic or neonatal stages of development. Brain response to injury undergoes remodeling with induction of angiogenesis, neurogenesis, and synaptogenesis. The attempt at remodeling, although expressed as a partial improvement in patients with stroke and ICH, is clearly insufficient to promote substantial recovery in many patients. The goal of restorative therapies should be to activate and amplify this endogenous restorative brain plasticity process to potentiate functional recovery. The logic of restorative therapy is to treat intact or marginally compromised tissue and not injured or dying tissue. Thus, these treatments can be made available for all neurological injury. Once demonstrated to be effective for treatment of a large middle cerebral artery occlusion (MCAo), these restorative treatments can be applied to many types of injury, including ICH, traumatic brain injury, and neurodegenerative disease such as experimental autoimmune encephalomyelitis and multiple sclerosis.
Assuntos
Terapia Baseada em Transplante de Células e Tecidos/métodos , Hemorragia Cerebral/terapia , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Acidente Vascular Cerebral/terapia , Humanos , Transplante de Células-TroncoRESUMO
VEGF is a secreted mitogen associated with angiogenesis and is also a potent vascular permeability factor. The biological role of VEGF in the ischemic brain remains unknown. This study was undertaken to investigate whether VEGF enhances cerebral microvascular perfusion and increases blood-brain barrier (BBB) leakage in the ischemic brain. Using magnetic resonance imaging (MRI), three-dimensional laser-scanning confocal microscope, and functional neurological tests, we measured the effects of administrating recombinant human VEGF(165) (rhVEGF(165)) on angiogenesis, functional neurological outcome, and BBB leakage in a rat model of focal cerebral embolic ischemia. Late (48 hours) administration of rhVEGF(165) to the ischemic rats enhanced angiogenesis in the ischemic penumbra and significantly improved neurological recovery. However, early postischemic (1 hour) administration of rhVEGF(165) to ischemic rats significantly increased BBB leakage, hemorrhagic transformation, and ischemic lesions. Administration of rhVEGF(165) to ischemic rats did not change BBB leakage and cerebral plasma perfusion in the contralateral hemisphere. Our results indicate that VEGF can markedly enhance angiogenesis in the ischemic brain and reduce neurological deficits during stroke recovery and that inhibition of VEGF at the acute stage of stroke may reduce the BBB permeability and the risk of hemorrhagic transformation after focal cerebral ischemia.
Assuntos
Barreira Hematoencefálica/efeitos dos fármacos , Isquemia Encefálica/fisiopatologia , Fatores de Crescimento Endotelial/farmacologia , Linfocinas/farmacologia , Neovascularização Patológica/induzido quimicamente , Acidente Vascular Cerebral/tratamento farmacológico , Animais , Processamento de Imagem Assistida por Computador , Imageamento por Ressonância Magnética , Masculino , Microcirculação/efeitos dos fármacos , Ratos , Ratos Wistar , Fluxo Sanguíneo Regional , Resultado do Tratamento , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Stroke stimulates neurogenesis in the adult rodent brain. The molecules that mediate stroke-induced neurogenesis are not definitely known. Using microarrays containing approximately 400 known genes associated with stem cell and angiogenesis, we compared transcriptional profiles of subventricular zone (SVZ) tissue with cultured neural progenitor cells isolated from the SVZ 7 days after ischemic stroke in the adult mouse. In SVZ tissue, we found that stroke upregulated 58 genes which are involved in multiple signaling pathways during embryonic development, suggesting that stroke recaptures embryonic molecular signals. In neural progenitor cells cultured in growth medium, 23 gene expressions were increased after stroke and 8 of 23 genes overlapped with upregulated genes in stroke SVZ tissue. Expression alterations of selected genes were confirmed by real-time RT-PCR and immunohistochemistry. These in vivo and in vitro data provide new insight into the genetic program of adult SVZ neural progenitor cells after stroke and demonstrate gene expression differences between SVZ tissue and cultured SVZ cells.
Assuntos
Encéfalo/fisiologia , Expressão Gênica/fisiologia , Infarto da Artéria Cerebral Média/genética , Infarto da Artéria Cerebral Média/metabolismo , Neurônios/metabolismo , Neurônios/fisiologia , Prosencéfalo/fisiologia , Animais , Contagem de Células , Células Cultivadas , Interpretação Estatística de Dados , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Análise de Sequência com Séries de Oligonucleotídeos , RNA/biossíntese , RNA/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células-Tronco/metabolismoRESUMO
BACKGROUND AND PURPOSE: Recombinant human erythropoietin (rhEPO; Epoetin-alpha; PROCRITtrade mark) has been shown to exert neuroprotective and restorative effects in a variety of CNS injury models. However, limited information is available regarding the dose levels required for these beneficial effects or the neuronal responses that may underlie them. Here we have investigated the dose-response to rhEPO and compared the effects of rhEPO with those of carbamylated rhEPO (CEPO) in a model of cerebral stroke in rats. EXPERIMENTAL APPROACH: Rats subjected to embolic middle cerebral artery occlusion (MCAo) were treated with rhEPO or CEPO, starting at 6 h and repeated at 24 and 48 h, after MCAo. Cerebral infarct volumes were assessed at 28 days and neurological impairment at 7, 14, 21 and 28 days, post-MCAo. KEY RESULTS: rhEPO at dose levels of 500, 1150 or 5000 IU kg(-1) or CEPO at a dose level of 50 microg kg(-1) significantly reduced cortical infarct volume and reduced neurologic impairment. All doses of rhEPO, but not CEPO, produced a transient increase in haematocrit, while rhEPO and CEPO substantially reduced the number of apoptotic cells and activated microglia in the ischemic boundary region. CONCLUSIONS AND IMPLICATIONS: These data indicate that rhEPO and CEPO have anti-inflammatory and anti-apoptotic effects, even with administration at 6 h following embolic MCAo in rats. Taken together, these actions of rhEPO and CEPO are likely to contribute to their reduction of neurologic impairment following cerebral ischemia.
Assuntos
Isquemia Encefálica/tratamento farmacológico , Eritropoetina/análogos & derivados , Eritropoetina/farmacologia , Fármacos Neuroprotetores/farmacologia , Acidente Vascular Cerebral/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Barreira Hematoencefálica , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática , Epoetina alfa , Eritropoetina/administração & dosagem , Eritropoetina/farmacocinética , Hematínicos/administração & dosagem , Hematínicos/farmacocinética , Hematínicos/farmacologia , Hematócrito , Infarto da Artéria Cerebral Média/tratamento farmacológico , Infarto da Artéria Cerebral Média/fisiopatologia , Masculino , Microglia/efeitos dos fármacos , Fármacos Neuroprotetores/administração & dosagem , Fármacos Neuroprotetores/farmacocinética , Ratos , Ratos Wistar , Proteínas RecombinantesRESUMO
Several studies support the hypothesis that after stroke, specific features of brain function revert to those seen at an early stage of development, with the subsequent process of recovery recapitulating ontogeny in many ways. Many clinical characteristics of stroke recovery resemble normal development, particularly in the motor system. Consistent with this, brain-mapping studies after an ischemic insult suggest re-emergence of childhood organizational patterns: recovery being associated with a return to adult patterns. Experimental animal studies demonstrate increased levels of developmental proteins, particularly in the area surrounding an infarct, suggesting an active process of reconditioning in response to cerebral ischemia. Understanding the patterns of similarity between normal development and stroke recovery might be of value in its treatment.
Assuntos
Encéfalo/crescimento & desenvolvimento , Encéfalo/fisiologia , Recuperação de Função Fisiológica/fisiologia , Acidente Vascular Cerebral/fisiopatologia , Adulto , Animais , Criança , Humanos , Reabilitação do Acidente Vascular CerebralRESUMO
The present study investigates the induction of axon and myelin remodeling as a possible mechanism by which treatment of stroke with bone marrow stromal cells improves neurological functional recovery. Adult male Wistar rats were subjected to 2 h of middle cerebral artery occlusion, followed by an injection of 2 x 10(6) rat bone marrow stromal cells or phosphate-buffered saline into the internal carotid artery 24 h later. Animals were killed at 28 days after stroke. Functional tests, histo- and immunohistochemical staining were performed. Significant functional recovery was found after bone marrow stromal cell administration in all the three tests performed (modified neurological severity score, adhesive-removal and corner tests). Bone marrow stromal cell treatment markedly increased vessel sprouting, synaptophysin expression and NG2 positive cell numbers and density in the cortical peri-infarct area. In bone marrow stromal cell-treated rats, the number of Ki-67 positive proliferating cells and oligodendrocyte precursor cells in the corpus callosum increased significantly in concert with the enhancement of the areas of the corpus callosum in both hemispheres. These results suggest that bone marrow stromal cells facilitate axonal sprouting and remyelination in the cortical ischemic boundary zone and corpus callosum, which may underlie neurological functional improvement caused by bone marrow stromal cell treatment.
Assuntos
Transplante de Medula Óssea/métodos , Hipóxia Encefálica/terapia , Regeneração Nervosa/fisiologia , Plasticidade Neuronal/fisiologia , Acidente Vascular Cerebral/terapia , Células Estromais/transplante , Animais , Antígenos/metabolismo , Axônios/fisiologia , Axônios/ultraestrutura , Transplante de Medula Óssea/tendências , Artérias Carótidas , Diferenciação Celular/fisiologia , Modelos Animais de Doenças , Hipóxia Encefálica/fisiopatologia , Infarto da Artéria Cerebral Média/fisiopatologia , Infarto da Artéria Cerebral Média/terapia , Infusões Intra-Arteriais/métodos , Infusões Intra-Arteriais/tendências , Antígeno Ki-67/metabolismo , Masculino , Bainha de Mielina/fisiologia , Bainha de Mielina/ultraestrutura , Neovascularização Fisiológica/fisiologia , Oligodendroglia/citologia , Oligodendroglia/fisiologia , Proteoglicanas/metabolismo , Ratos , Ratos Wistar , Células-Tronco/citologia , Células-Tronco/fisiologia , Acidente Vascular Cerebral/fisiopatologia , Sinaptofisina/metabolismo , Resultado do TratamentoRESUMO
Neurogenesis may contribute to functional recovery after neural injury. Nitric oxide donors such as DETA-NONOate promote functional recovery after stroke. However, the mechanisms underlying functional improvement have not been ascertained. We therefore investigated the effects of DETA-NONOate on neural progenitor/stem cell neurospheres derived from the subventricular zone from young and retired breeder rat brain. Subventricular zone cells were dissociated from normal young adult male Wistar rats (2-3 months old) and retired breeder rats (14 months old), treated with or without DETA-NONOate. Subventricular zone neurosphere formation, proliferation, telomerase activity, and Neurogenin 1 mRNA expression were significantly decreased and glial fibrillary acidic protein expression was significantly increased in subventricular zone neurospheres from retired breeder rats compared with young rats. Treatment of neurospheres with DETA-NONOate significantly decreased neurosphere formation and telomerase activity, and promoted neuronal differentiation and neurite outgrowth concomitantly with increased N-cadherin and beta-catenin mRNA expression in both young and old neurospheres. DETA-NONOate selectively increased Neurogenin 1 and decreased glial fibrillary acidic protein mRNA expression in retired breeder neurospheres. N-cadherin significantly increased Neurogenin 1 mRNA expression in young and old neurospheres. Anti-N-cadherin reversed DETA-NONOate-induced neurosphere adhesion, neuronal differentiation, neurite outgrowth, and beta-catenin mRNA expression. Our data indicate that age has a potent effect on the characteristics of subventricular zone neurospheres; neurospheres from young rats show significantly higher formation, proliferation and telomerase activity than older neurospheres. In contrast, older neurospheres exhibit significantly increased glial differentiation than young neurospheres. DETA-NONOate promotes neuronal differentiation and neurite outgrowth in both young and older neurospheres. The molecular mechanisms associated with the DETA-NONOate modulation of neurospheres from young and older animals as well age dependent effects of neurospheres appear to be controlled by N-cadherin and beta-catenin gene expression, which subsequently regulates the neuronal differentiating factor Neurogenin expression in both young and old neural progenitor cells.
Assuntos
Encéfalo/crescimento & desenvolvimento , Caderinas/metabolismo , Diferenciação Celular/fisiologia , Neurônios/metabolismo , Óxido Nítrico/metabolismo , Células-Tronco/metabolismo , Envelhecimento/fisiologia , Animais , Anticorpos/farmacologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Encéfalo/citologia , Encéfalo/metabolismo , Técnicas de Cultura de Células/métodos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Proteína Glial Fibrilar Ácida/genética , Masculino , Proteínas do Tecido Nervoso/genética , Neuritos/efeitos dos fármacos , Neuritos/metabolismo , Neurônios/efeitos dos fármacos , Doadores de Óxido Nítrico/farmacologia , Compostos Nitrosos/farmacologia , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Esferoides Celulares , Células-Tronco/efeitos dos fármacos , Telomerase/metabolismo , beta Catenina/genéticaRESUMO
Bone morphogenetic proteins play a key role in astrocytic differentiation. Astrocytes express the gap junctional protein connexin-43, which permits exchange of small molecules in brain and enhances synaptic efficacy. Bone marrow stromal cells produce soluble factors including bone morphogenetic protein 2 and bone morphogenetic protein 4 (bone morphogenetic protein 2/4) in ischemic brain. Here, we tested whether intra-carotid infusion of bone marrow stromal cells promotes synaptophysin expression and neurological functional recovery after stroke in rats. Adult male Wistar rats were subjected to 2 h of right middle cerebral artery occlusion. Rats were treated with or without bone marrow stromal cells at 24 h after middle cerebral artery occlusion via intra-arterial injection (n=8/group). A battery of functional tests was performed. Immunostaining of 5-bromo-2-deoxyuridine, Ki67, bone morphogenetic protein 2/4, connexin-43, synaptophysin, glial fibrillary acidic protein, neuronal nuclear antigen, and double staining of 5-bromo-2-deoxyuridine/glial fibrillary acidic protein, 5-bromo-2-deoxyuridine/neuronal nuclear antigen, glial fibrillary acidic protein/bone morphogenetic protein 2/4 and glial fibrillary acidic protein/connexin-43 were employed. Rats treated with bone marrow stromal cells significantly (P<0.05) improved functional recovery compared with the controls. 5-Bromo-2-deoxyuridine and Ki67 positive cells in the ipsilateral subventricular zone were significantly (P<0.05) increased in bone marrow stromal cell treatment group compared with the controls, respectively. Administration of bone marrow stromal cells significantly (P<0.05) promoted the proliferating cell astrocytic differentiation, and increased bone morphogenetic protein 2/4, connexin-43 and synaptophysin expression in the ischemic boundary zone compared with the controls, respectively. Bone morphogenetic protein 2/4 expression correlated with the expression of connexin-43 (r=0.84, P<0.05) and connexin-43 expression correlated with the expression of synaptophysin (r=0.73, P<0.05) in the ischemic boundary zone, respectively. Administration of bone marrow stromal cells via an intra-carotid route increases endogenous brain bone morphogenetic protein 2/4 and connexin-43 expression in astrocytes and promotes synaptophysin expression, which may benefit functional recovery after stroke in rats.
Assuntos
Células da Medula Óssea/fisiologia , Proteínas Morfogenéticas Ósseas/metabolismo , Conexina 43/metabolismo , Células Estromais/fisiologia , Sinaptofisina/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Análise de Variância , Animais , Peso Corporal/fisiologia , Transplante de Medula Óssea/métodos , Proteína Morfogenética Óssea 2 , Proteína Morfogenética Óssea 4 , Bromodesoxiuridina/metabolismo , Diferenciação Celular/fisiologia , Proteína Glial Fibrilar Ácida/metabolismo , Imuno-Histoquímica/métodos , Infarto da Artéria Cerebral Média/metabolismo , Infarto da Artéria Cerebral Média/patologia , Infarto da Artéria Cerebral Média/terapia , Injeções Intra-Arteriais/métodos , Antígeno Ki-67/metabolismo , Masculino , Fosfopiruvato Hidratase/metabolismo , Ratos , Ratos Wistar , Células Estromais/transplante , Regulação para CimaRESUMO
Neurogenesis declines with advancing age. The mammalian achaete-scute homologue-1 encodes a basic helix-loop-helix transcription factor, which controls neuronal differentiation. In this study, we first tested whether atorvastatin treatment enhances neurological functional outcome and neuronal differentiation after stroke in retired breeder 12 month rats. Rats were subjected to middle cerebral artery occlusion and treated with or without atorvastatin (3 mg/kg) for 7 days. Atorvastatin significantly increased expression of mammalian achaete-scute homologue-1, beta-tubulin III, and vascular endothelial growth factor in the ischemic brain, and concomitantly improved functional outcome compared with middle cerebral artery occlusion control rats. Increased neurogenesis significantly correlated with functional recovery after stroke. To further investigate the mechanisms of atorvastatin-induced neuronal differentiation, experiments were performed on neurospheres derived from retired breeder rat subventricular zone cells. Atorvastatin increased neuronal differentiation and upregulated vascular endothelial growth factor and mammalian achaete-scute homologue-1 gene expression in cultured neurospheres. Vascular endothelial growth factor-treated neurospheres significantly increased mammalian achaete-scute homologue-1 and beta-tubulin III expression. Inhibition of vascular endothelial growth factor decreased atorvastatin-induced mammalian achaete-scute homologue-1 and beta-tubulin III expression. These data indicate that atorvastatin increases neuronal differentiation in retired breeder rats. In addition, atorvastatin upregulation of vascular endothelial growth factor expression, influences mammalian achaete-scute homologue-1 transcription factor, which in turn, facilitates an increase in subventricular zone neuronal differentiation. These atorvastatin-mediated molecular events may contribute to the improved functional outcome in retired breeder rats subjected to stroke.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Diferenciação Celular/efeitos dos fármacos , Ácidos Heptanoicos/administração & dosagem , Inibidores de Hidroximetilglutaril-CoA Redutases/administração & dosagem , Infarto da Artéria Cerebral Média , Neurônios/efeitos dos fármacos , Pirróis/administração & dosagem , Fator A de Crescimento do Endotélio Vascular/fisiologia , Análise de Variância , Animais , Anticorpos/farmacologia , Atorvastatina , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Expressão Gênica/efeitos dos fármacos , Imuno-Histoquímica/métodos , Infarto da Artéria Cerebral Média/tratamento farmacológico , Infarto da Artéria Cerebral Média/metabolismo , Infarto da Artéria Cerebral Média/patologia , Infarto da Artéria Cerebral Média/fisiopatologia , Masculino , Atividade Motora/efeitos dos fármacos , Atividade Motora/fisiologia , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Tubulina (Proteína)/metabolismo , Fator A de Crescimento do Endotélio Vascular/imunologiaRESUMO
Doublecortin (DCX) is a microtubule-associated protein expressed in migrating neuroblasts. DCX expression is increased in subventricular zone (SVZ) cells migrating to the boundary of an ischemic lesion after induction of middle cerebral artery occlusion (MCAO) in adult rats and mice. We tested the hypothesis that DCX, in addition to being a marker of migrating neuroblasts, serves to protect neuroblasts from conditions of stress, such as oxygen and glucose deprivation (OGD). Using gene transfer technology, we overexpressed DCX in rat SVZ and U-87 human glioma cells. The cells remained viable against severe OGD, up to 32 h exhibiting 1% apoptosis compared with 100% apoptosis in control. In addition, these genetically modified cells upregulated expression of E-, VE- and N-cadherin, molecules that promote endothelial survival signals via the VE-cadherin/vascular endothelial growth factor receptor-2/phosphoinositide 3-kinase (PI3-K)/AKT/beta-catenin pathway and inactivate the proapoptotic factor Bad. DCX overexpression also significantly increased cell migration in SVZ tissue explants and U-87 cells and significantly upregulated microtubule-associated protein-2 (MAP2) and nestin protein levels in SVZ and U-87 cells compared with wild-type control cells. Knocking down DCX expression in DCX overexpressing SVZ and U-87 cells with DCX small interfering RNA (siRNA), confirmed the specificity of DCX on cell survival against OGD, and the DCX induced upregulation of E-, VE- and N-cadherin, MAP2 and nestin. In NIH3T3 cells, DCX overexpression had no effect on cell survival against OGD, and indicating that the protective effects of DCX was restricted to brain cells e.g. SVZ and U-87 cells. Our data suggest a novel and an important role for DCX as a protective agent for migrating neuroblasts and tumor cells.
Assuntos
Regulação da Expressão Gênica/fisiologia , Glioma/metabolismo , Glucose/deficiência , Hipóxia/metabolismo , Proteínas Associadas aos Microtúbulos/fisiologia , Neuropeptídeos/fisiologia , Células-Tronco/metabolismo , Análise de Variância , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Encéfalo/citologia , Caderinas/classificação , Caderinas/metabolismo , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Células Cultivadas , Proteínas do Domínio Duplacortina , Proteína Duplacortina , Humanos , Marcação In Situ das Extremidades Cortadas/métodos , Proteínas de Filamentos Intermediários/metabolismo , Masculino , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Nestina , Neuropeptídeos/genética , RNA Interferente Pequeno/farmacologia , Ratos , Ratos Wistar , Fatores de Tempo , Transfecção/métodos , beta-Galactosidase/metabolismoRESUMO
AIM: This study was designed to determine any rebleeding after atorvastatin treatment following spontaneous intracerebral hemorrhage (ICH) in a prospective safety trial. PATIENTS: Atorvastatin (80 mg/day) therapy was initiated in 6 patients with primary ICH with admission Glasgow Coma Score (GCS) >5 within 24 hours of ictus and continued for 7 days, with the dose tapered and treatment terminated over the next 5 days. Patients were studied longitudinally by multiparametric magnetic resonance imaging (MRI) at three time points: acute (3 to 5 days), subacute (4 to 6 weeks) and chronic (3 to 4 months). Imaging sequences included T1, T2-weighted imaging (T2WI), diffusion tensor imaging (DTI) and contrast-enhanced MRI measures of cerebral perfusion, blood volume and blood-brain barrier (BBB) permeability. Susceptibility weighted imaging (SWI) was used to identify primary ICH and to check for secondary rebleeding. Final outcome was assessed using Glasgow Outcome Score (GOS) at 3-4 months. RESULTS: Mean admission GCS was 13.2±4.0 and mean GOS at 3 months was 4.5±0.6. Hemorrhagic lesions were segmented into core and rim areas. Mean lesion volumes decreased significantly between the acute and chronic study time points (p=0.008). Average ipsilateral hemispheric tissue loss at 3 to 4 months was 11.4±4.6 cm3. MRI showed acutely reduced CBF (p=0.004) and CBV (p=0.002) in the rim, followed by steady normalization. Apparent diffusion coefficient of water (ADC) in the rim demonstrated no alterations at any of the time points (p>0.2). The T2 values were significantly elevated in the rim acutely (p=0.02), but later returned to baseline. The ICH core showed sustained low CBF and CBV values concurrent with a small reduction in ADC acutely, but significant ADC elevation at the end suggestive of irreversible injury. CONCLUSION: Despite the presence of a small, probably permanent, cerebral lesion in the ICH core, no patients exhibited post-treatment rebleeding. These data suggest that larger, Phase 2 trials are warranted to establish long term clinical safety of atorvastatin in spontaneous ICH.
RESUMO
The mechanisms underlying cerebral microvascular perfusion deficit resulting from occlusion of the middle cerebral artery (MCA) require elucidation. We, therefore, tested the hypothesis that intravascular fibrin deposition in situ directly obstructs cerebral microcirculation and that local changes in type 1 plasminogen activator inhibitor (PAI-1) gene expression contribute to intravascular fibrin deposition after embolic MCA occlusion. Using laser-scanning confocal microscopy (LSCM) in combination with immunofluorescent staining, we simultaneously measured in three dimensions the distribution of microvascular plasma perfusion deficit and fibrin(ogen) immunoreactivity in a rat model of focal cerebral embolic ischemia (n = 12). In addition, using in situ hybridization and immunostaining, we analyzed expression of PAI-1 in ischemic brain (n = 13). A significant (p < 0.05) reduction of cerebral microvascular plasma perfusion accompanied a significant (p < 0.05) increase of intravascular and extravascular fibrin deposition in the ischemic lesion. Microvascular plasma perfusion deficit and fibrin deposition expanded concomitantly from the subcortex to the cortex during 1 and 4 hr of embolic MCA occlusion. Three-dimensional analysis revealed that intravascular fibrin deposition directly blocks microvascular plasma perfusion. Vascular plugs contained erythrocytes, polymorphonuclear leukocytes, and platelets enmeshed in fibrin. In situ hybridization demonstrated induction of PAI-1 mRNA in vascular endothelial cells in the ischemic region at 1 hr of ischemia. PAI-1 mRNA significantly increased at 4 hr of ischemia. Immunohistochemical staining showed the same pattern of increased PAI-1 antigen in the endothelial cells. These data demonstrate, for the first time, that progressive intravascular fibrin deposition directly blocks cerebral microvascular plasma perfusion in the ischemic region during acute focal cerebral embolic ischemia, and upregulation of the PAI-1 gene in the ischemic lesion may foster fibrin deposition through suppression of fibrinolysis.
Assuntos
Isquemia Encefálica/fisiopatologia , Circulação Cerebrovascular , Fibrina/fisiologia , Embolia Intracraniana/fisiopatologia , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Animais , Vasos Sanguíneos/metabolismo , Isquemia Encefálica/metabolismo , Fibrina/metabolismo , Fibrinogênio/metabolismo , Proteína Glial Fibrilar Ácida/metabolismo , Imuno-Histoquímica , Hibridização In Situ , Embolia Intracraniana/metabolismo , Masculino , Microcirculação , Inibidor 1 de Ativador de Plasminogênio/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Fatores de Tempo , Distribuição Tecidual , Regulação para CimaRESUMO
Transplantation of bone marrow stromal cells improves animal neurological functional recovery after stroke. Astrocytes are known to provide structural, trophic and metabolic support for neurons. Thus astrocytes are critical for neural survival during post-ischemia. However, information on the effects of bone marrow stromal cells on astrocytic survival post-ischemia is unavailable. We investigated the influence of rat bone marrow stromal cells on rat astrocytic apoptosis and survival post-ischemia employing an anaerobic chamber. Our data indicate that rat bone marrow stromal cells reduce cell death and apoptosis, and increase the DNA proliferation rate in astrocytes post-ischemia. Mitogen-activated protein kinase kinase/extracellular signal regulated kinase and phosphoinositide 3-kinase/threonine protein kinase pathways are involved in cell survival. Western blot showed that rat bone marrow stromal cells activate these two pathways in astrocytes post-ischemia, and upregulate total extracellular signal regulated kinase 1/2 and threonine protein kinase. Since astrocytes produce various neurotrophic factors, we performed reverse transcription polymerase chain reaction to investigate rat bone marrow stromal cells' effect on astrocyte growth factor gene expression post-ischemia. We observed that brain-derived neurotrophic factor, vascular endothelial growth factor and basic fibroblast growth factor gene expression was enhanced by rat bone marrow stromal cell coculture. These data suggest that bone marrow stromal cells increase astrocytic survival post-ischemic injury. This protective function might involve the activation of mitogen-activated protein kinase kinase/extracellular signal-regulated kinase and phosphoinositide 3-kinase/threonine protein kinase pathways. Upregulation of brain-derived neurotrophic factor, vascular endothelial growth factor and basic fibroblast growth factor may also contribute to astrocyte survival.