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1.
Brain Res ; 1317: 257-67, 2010 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-20043890

RESUMO

Trials of neuroprotection in ischemic stroke mainly concern gray matter protection, with little information on white matter damage. Olfactory ensheathing cells (OECs) are capable of providing continuous neurotrophic factor and of promoting growth and remyelination of damaged axons. We evaluated OECs for the repair of white matter after transplantation in middle cerebral artery occlusion (MCAO) rat. OECs were cultured from the olfactory bulbs of adult green fluorescent protein (GFP)-expressing transgenic rats and transplanted into peri-infarct basal ganglia imminently after reperfusion. The mortality, neurological score, and function were record everyday until 56 days. At 24 h and 56 days, the infarction volume, the Luxol-fast blue (LFB) staining, and Neurofilament (NF) immunohistochemistry and Western blot were performed to assess the demyelination and remyelination in white matter. OECs transplantation reduced the infarct volume, decreased mortality, and improved neurological deficits in 56 days after MCAO. LFB marked myelin, NF-positive immunohistochemistry, and Western blot indicated that the remyelination and axon regeneration in OEC transplanted rat were significant. In conclusion, OECs can protect the white matter from ischemic injury, but the potential mechanisms of transplanted OEC-mediated recovery need further studies to identify.


Assuntos
Infarto da Artéria Cerebral Média/fisiopatologia , Infarto da Artéria Cerebral Média/cirurgia , Fibras Nervosas Mielinizadas/fisiologia , Neuroglia/transplante , Bulbo Olfatório/transplante , Recuperação de Função Fisiológica/fisiologia , Animais , Axônios/patologia , Axônios/fisiologia , Gânglios da Base/patologia , Gânglios da Base/fisiopatologia , Gânglios da Base/cirurgia , Isquemia Encefálica/patologia , Isquemia Encefálica/fisiopatologia , Isquemia Encefálica/cirurgia , Doenças Desmielinizantes/patologia , Doenças Desmielinizantes/fisiopatologia , Doenças Desmielinizantes/cirurgia , Modelos Animais de Doenças , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Infarto da Artéria Cerebral Média/patologia , Masculino , Bainha de Mielina/patologia , Bainha de Mielina/fisiologia , Fibras Nervosas Mielinizadas/patologia , Regeneração Nervosa/fisiologia , Neuroglia/fisiologia , Bulbo Olfatório/fisiologia , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Ratos Transgênicos , Fatores de Tempo , Resultado do Tratamento
2.
Neurobiol Dis ; 33(3): 509-17, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19166937

RESUMO

Hypoxia-inducible factor-1 (HIF-1) plays an essential role in cerebral ischemia as a proapoptotic factor. We hypothesized that HIF-1alpha siRNA can protect the brain from ischemic damage by inhibiting HIF-1alpha induced apoptotic pathway at the RNA level in a rat focal ischemic model. Results showed that treatment with HIF-1alpha siRNA reduced the infarct volume, decreased mortality, improved neurological deficits and reduced Evans blue extravasation. The expression of HIF-1alpha mRNA (Real-Time PCR) and protein were significantly silenced and the immunohistochemistry and Western blot revealed the suppression of HIF-1alpha, VEGF, p53 and Caspase-3. Double fluorescence labeling showed HIF-1alpha positive immunoreactive materials were partly colocalized with NeuN, p53 and Caspase-3 in the injured cerebral cortex. This study showed that HIF-1alpha siRNA may protect the ischemic-reperfused neurons in vivo via inhibition of HIF-1alpha, its downstream VEGF and other apoptotic-related proteins such as p53 and Caspase-3 and may have potentials for the early treatment of ischemic cerebral stroke.


Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Infarto da Artéria Cerebral Média/terapia , Interferência de RNA , Traumatismo por Reperfusão/terapia , Animais , Apoptose , Encéfalo/patologia , Encéfalo/fisiopatologia , Caspase 3/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Infarto da Artéria Cerebral Média/mortalidade , Infarto da Artéria Cerebral Média/patologia , Masculino , Proteínas do Tecido Nervoso/metabolismo , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão/mortalidade , Traumatismo por Reperfusão/patologia , Transfecção , Proteína Supressora de Tumor p53/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
3.
Exp Neurol ; 216(1): 35-46, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19073180

RESUMO

RNA interference appears to have a great potential not only as an in vitro target validation, but also as a novel therapeutic strategy based on the highly specific and efficient silencing of a target gene. We hypothesize that MMP-9 siRNA can be effective as an MMP-9 protein inhibitor in a rat focal ischemia model. Male Sprague-Dawley rats (156) were subjected to 2 h of middle cerebral artery occlusion (by using the suture insertion method) followed by 24 h of reperfusion. In the treatment group, 5 microl MMP-9 siRNA was administrated by intracerebroventricular injection within 60 min after 2 h of focal ischemia. The siRNA transfection was demonstrated by fluorescence conjugated siRNA. Treatment with MMP-9 siRNA produced a significant reduction in the cerebral infarction volume, brain water content, mortality rate and accompanying neurological deficits. The followings were recorded: Evan's blue and IgG extravasation were reduced; the expression of MMP-9 mRNA and protein were significantly silenced; and immunohistochemistry and Western blot analysis revealed that the expression of MMP-9 and VEGF were reduced while occludin and collagen-IV were up-regulated in brain tissues. Our findings provide evidence that a liposomal formulation of siRNA might be used in vivo to silence the MMP-9 gene and could potentially serve as an important therapeutic alternative in patients with cerebral ischemia.


Assuntos
Hipóxia-Isquemia Encefálica/terapia , Infarto da Artéria Cerebral Média/terapia , Inibidores de Metaloproteinases de Matriz , Interferência de RNA , Animais , Encéfalo/irrigação sanguínea , Encéfalo/enzimologia , Encéfalo/fisiopatologia , Edema Encefálico/enzimologia , Edema Encefálico/genética , Edema Encefálico/terapia , Infarto Encefálico/enzimologia , Infarto Encefálico/genética , Infarto Encefálico/terapia , Colágeno Tipo IV/metabolismo , Modelos Animais de Doenças , Regulação para Baixo/genética , Regulação Enzimológica da Expressão Gênica/genética , Hipóxia-Isquemia Encefálica/enzimologia , Hipóxia-Isquemia Encefálica/genética , Infarto da Artéria Cerebral Média/enzimologia , Infarto da Artéria Cerebral Média/genética , Masculino , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Proteínas de Membrana/metabolismo , Ocludina , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Ratos , Ratos Sprague-Dawley , Transfecção/métodos , Regulação para Cima/genética , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo
4.
Chin Med J (Engl) ; 121(5): 414-9, 2008 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-18364113

RESUMO

BACKGROUND: The mechanism of cerebral vasospasm following subarachnoid haemorrhage (SAH) is not understood. Here, we hypothesized that apoptosis of endothelial cells induced by p53 and its target gene em dash p53 upregulated modulator of apoptosis (PUMA) played an important role in development of cerebral vasospasm. We also observed the effects of a p53 inhibitor, pifithrin-alpha (PFT-alpha), on reducing the expression of p53 and PUMA, consequently decreasing the apoptosis of endothelial cells and alleviating cerebral vasospasm. METHODS: Male Sprague-Dawley rats weighing 300-350 g were randomly divided into five groups: a control group (sham surgery), a SAH group, a SAH+dimethyl sulfoxide (DMSO) group, a SAH + PFT-alpha (0.2 mg/kg) group and a SAH + PFT-alpha (2.0 mg/kg) group. PFT-alpha was injected intraperitoneally immediately after SAH. Rats were sacrificed 24 hours after SAH. Western blot and immunohistochemical staining were used to detect the levels of p53, PUMA and caspase-3 protein. In addition, mortality and neurological scores were assessed for each group. Statistical significance was assured by analysis of variance performed in one way ANOVA followed by the Tukey test. The neurological and mortality scores were analyzed by Dunn's method and Fisher exact test, respectively. RESULTS: After SAH, Western blot and immunohistochemical staining showed the levels of p53, PUMA and caspase-3 in the endothelial cells and the numbers of TdT mediated dUTP nick end labelling (TUNEL) positive endothelial cells were all significantly increased in the basilar arteries (P<0.05), but significantly reduced by PFT-alpha (P<0.05). These changes were accompanied by increasing diameters and declining wall thickness of basilar arteries (P<0.05), as well as reduced mortality and neurological deficits of the rats (P<0.05). CONCLUSIONS: PFT-alpha could protect cerebral vessels from development of vasospasm and improve neurological outcome as well as reduce the mortality via suppressing apoptosis induced by p53 in the endothelial cells of cerebral vessels.


Assuntos
Apoptose/efeitos dos fármacos , Benzotiazóis/uso terapêutico , Células Endoteliais/efeitos dos fármacos , Hemorragia Subaracnóidea/tratamento farmacológico , Tolueno/análogos & derivados , Vasoespasmo Intracraniano/prevenção & controle , Animais , Benzotiazóis/farmacologia , Western Blotting , Modelos Animais de Doenças , Células Endoteliais/patologia , Masculino , Ratos , Ratos Sprague-Dawley , Hemorragia Subaracnóidea/complicações , Hemorragia Subaracnóidea/patologia , Hemorragia Subaracnóidea/fisiopatologia , Tolueno/farmacologia , Tolueno/uso terapêutico , Proteína Supressora de Tumor p53/fisiologia
5.
J Neurochem ; 102(6): 1831-1841, 2007 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17532791

RESUMO

Despite 2-methoxyestradiol (2ME2) and tricyclodecan-9-yl-xanthogenate (D609) having multiple effects on cancer cells, mechanistically, both of them down-regulate hypoxia-inducible factor-1alpha (HIF-1alpha) and vascular endothelial growth factor (VEGF). We hypothesize HIF-1alpha plays an essential role in cerebral ischemia as a pro-apoptosis regulator; 2ME2 and D609 decrease the levels of HIF-1alpha and VEGF, that might contribute to protecting brain from ischemia injury. A total of 102 male Sprague-Dawley rats were split into five groups: sham, middle cerebral artery occlusion (MCAO), MCAO + dimethyl sulfoxide, MCAO + 2ME2, and MCAO + D609. 2ME2 and D609 were injected intraperitoneally 1 h after reperfusion. Rats were killed at 24 h and 7 days. At 24 h, 2ME2 and D609 reduce the levels of HIF-1alpha and VEGF (enzyme-linked immunosorbent assay), depress the expression of HIF-1alpha, VEGF, BCL2/adenovirus E1B 19 kDa interacting protein 3 (BNIP3) and cleaved caspase 3 (western blot and immunohistochemistry) in the brain infarct area. Double fluorescence labeling shows HIF-1alpha positive immunoreactive materials are co-localized with BNIP3 and terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling inside the nuclei of neurons. At 7 days, 2ME2 and D609 reduce the infarct volume (2,3,7-triphenyltetrazolium chloride) and blood-brain barrier extravasation, decrease the mortality and improve the neurological deficits. In conclusion, 2ME2 and D609 are powerful agents to protect brain from cerebral ischemic injury by inhibiting HIF-1alpha expression, attenuating the superfluous expression of VEGF to avoid blood-brain barrier disruption and suppressing neuronal apoptosis via BNIP3 pathway.


Assuntos
Apoptose/efeitos dos fármacos , Isquemia Encefálica/tratamento farmacológico , Hidrocarbonetos Aromáticos com Pontes/farmacologia , Estradiol/análogos & derivados , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Fármacos Neuroprotetores/farmacologia , Tionas/farmacologia , 2-Metoxiestradiol , Animais , Apoptose/genética , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/fisiologia , Edema Encefálico/tratamento farmacológico , Edema Encefálico/metabolismo , Edema Encefálico/fisiopatologia , Infarto Encefálico/tratamento farmacológico , Infarto Encefálico/metabolismo , Infarto Encefálico/fisiopatologia , Isquemia Encefálica/metabolismo , Isquemia Encefálica/fisiopatologia , Caspase 3/efeitos dos fármacos , Caspase 3/metabolismo , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Modelos Animais de Doenças , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/fisiologia , Estradiol/farmacologia , Marcação In Situ das Extremidades Cortadas , Infarto da Artéria Cerebral Média/tratamento farmacológico , Infarto da Artéria Cerebral Média/metabolismo , Infarto da Artéria Cerebral Média/fisiopatologia , Masculino , Proteínas de Membrana/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Proteínas Mitocondriais , Norbornanos , Proteínas Proto-Oncogênicas/efeitos dos fármacos , Proteínas Proto-Oncogênicas/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Tiocarbamatos , Resultado do Tratamento , Fator A de Crescimento do Endotélio Vascular/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/metabolismo
6.
Chin Med J (Engl) ; 120(2): 155-8, 2007 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-17335662

RESUMO

BACKGROUND: The fat derived protein adiponectin plays an important role in the regulation of glucose metabolism. The aim of this study was to provide the experimental basis for further investigating on adiponectin (ADPN) function. Its eukaryotic recombinant was constructed and expressed in precursor cells of 3T3-L1 adipocytes. The effects of dexamethasone on peroxisome proliferator activated receptor-gamma (PPAR-gamma) mRNA expression in 3T3-L1 cells with human recombinant adiponectin were assessed. METHODS: The recombinant plasmid pMD18-T-hADPN and eukaryotic expression vector pcDNA3.1(+) were digested by two restrictive endonucleases and adiponectin and linear pcDNA3.1(+) were obtained. Then, they were ligated and translated into JM109. The recombinant pcDNA3.1(+)-hADPN so obtained was identified by digestion by restrictive endonuclease and nucleotide sequencing. The 3T3-L1 precursor cells were transfected using SuperFect Transfection Reagent (Qiagen). Furthermore, 3T3-L1 cells with human recombinant adiponectin incubated with dexamethasone (0.5 mmol/L) for 24 hours, cells were collected and total RNA was extracted. The PPAR-gamma mRNA expression was quantified by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: After eukaryotic recombinant was digested by Hind III and EcoR I, fragments of 800 bp and 5.4 kb were identified by nucleotide sequence scanning and consistent with theoretical values. Electrophoretogram of RT-PCR in 3T3-L1 precursors showed only one band in front of 250 bp, which was consistent with theoretical value 234 bp. In the 3T3-L1 cells, 3T3-L1 cells with plasmid and 3T3-L1 cells human recombinant adiponectin, treatment with dexamethasone (0.5 mmol/L) decreased PPAR-gamma mRNA expression compared to untreated controls (P < 0.01). Effect of dexamethasone on PPAR-gamma mRNA expression in 3T3-L1 cells was reversed by stably transfected human recombinant adiponectin. CONCLUSION: The 3T3-L1 cells stably transfected human recombinant adiponectin had increased PPAR-gamma mRNA expression. Dexamethasone suppressed PPAR-gamma mRNA expression in the 3T3-L1 cells. Effect of dexamethasone on PPAR-gamma mRNA expression in 3T3-L1 cells was reversed by stably transfected human recombinant adiponectin.


Assuntos
Adiponectina/fisiologia , Dexametasona/farmacologia , PPAR gama/genética , RNA Mensageiro/análise , Células 3T3-L1 , Animais , Resistência à Insulina , Camundongos , Reação em Cadeia da Polimerase Via Transcriptase Reversa
7.
Clin Anat ; 20(4): 362-6, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17080461

RESUMO

The microanatomy of the superior orbital fissure (SOF) was studied in 96 sides of cadaver specimens. The SOF is a narrow bony cleft that lies at the apex of the orbit between the greater and lesser wings of the sphenoid. Through this fissure, many important structures enter the orbit from the middle cranial fossa including the third, fourth, sixth cranial nerves, and the ophthalmic branch of the fifth nerve. In addition, the superior opthalmamic vein exits the orbit to drain into the cavernous sinus via the SOF. The fissure can be divided into three anatomical regions by the annulus of Zinn (common annular tendon): the lateral, central, and inferior regions. The lateral wall of the SOF can also be divided between the upper and lower segments, and the angle between them was measured to be 144.27 degrees +/- 20.03 degrees . Defining these regions is useful in describing the course and placement of the nerves and vasculature in the SOF. Managing lesions at the orbital apex requires an extensive knowledge of the cranial base and the intracranial and extracranial relationships of the anatomical structures coursing through the SOF. The goal of this study was to describe the microanatomy of the SOF region in detail and to provide a reference for surgical procedures involving the orbital apex.


Assuntos
Seio Cavernoso/anatomia & histologia , Fossa Craniana Média/anatomia & histologia , Microcirurgia/métodos , Órbita/anatomia & histologia , Seio Cavernoso/inervação , Fossa Craniana Média/irrigação sanguínea , Fossa Craniana Média/inervação , Nervos Cranianos/anatomia & histologia , Humanos , Órbita/irrigação sanguínea , Órbita/inervação , Veias/anatomia & histologia
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