RESUMO
Double-fluorescence staining was combined with confocal laser scanning microscopy to localize fetal liver kinase-1 (Flk-1) and fms-like tyrosine kinase-1 (Flt-1) in the neonatal rat brain. The results showed that Flk-1 and Flt-1 immunostaining was observed in the cells with neuron-specific enolase, a neuronal marker, and with factor VIII (F VIII), an endothelium marker, but not in cells with glial fibrillary acidic protein (GFAP), a glial marker, of brain sections from rats on postnatal day 7 (P7). This indicates that both vascular endothelial growth factor (VEGF) receptors were distributed in the neurons and the vascular endothelium. A regional analysis showed that Flt-1 was distributed most densely in the hippocampus, followed by the retrosplenial agranular cortex and the striatum, and Flk-1 was evenly distributed throughout the brain. In a comparison of the density of immunopositive staining neurons, Flt-1 was much higher than Flk-1 in most of the brain regions. A time-course analysis showed that both Flt-1 and Flk-1 were highly expressed in the cerebral vessel of rats on P1, P7, and P14, and then declined in adults, consistent with the development of angiogenesis in neonates. In the neurons, Flt-1 was highest in the cerebral cortex and hippocampus of P1-P14 rats, and then gradually decreased, whereas Flk-1 abruptly increased and reached its highest level in adults. The results suggest that Flt-1 and Flk-1 are expressed in the neurons with their individual time-dependent manners and regional distribution in the brain. However, the significance of the neuronal distribution of Flt-1 and Flk-1 remains to be determined.
Assuntos
Envelhecimento/metabolismo , Animais Recém-Nascidos/metabolismo , Encéfalo/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Ratos/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Animais Recém-Nascidos/crescimento & desenvolvimento , Imuno-Histoquímica/métodos , Masculino , Microscopia Confocal , Ratos Sprague-Dawley , Coloração e Rotulagem , Distribuição Tecidual , Receptor 1 de Fatores de Crescimento do Endotélio VascularRESUMO
OBJECTIVE: To determine if DNA excision repair enzymes oxoguanine glycosylase 1 (OGG1) and xeroderma pigmentosum group F protein (XPF) are involved in the pathogenesis of Parkinson's disease (PD) in a cell model. METHODS: PC12 cells were treated with 1-Methyl-4-phenylpyridine ion (MPP(+)) for various periods of time to induce oxidative DNA damage. MTT assay was used to determine cell viability. Immunocytochemistry with antibody against 8-hydroxy-2'-deoxyguanosine (8-oxodG) was used to evaluate oxidative DNA damage. Immunoblotting was used to detect the protein levels of OGG1 and XPF. RESULTS: MPP(+) treatment (1 mmol/L) for 18 h and 24 h reduced cell viability to 78.6% and 70.3% of the control, respectively, in a time-dependent way. MPP(+) increased the immunoreactivity of 8-oxodG in the cytoplasm at 3 h and in the nucleus at 24 h of treatment. With the treatment of MPP(+), the expression of OGG1 was significantly increased at 1 h, reaching a peak at 3 h, and then it was decreased at 24 h, as compared to that with vehicle treatment. The same effect was exerted on XPF level, except that the XPF level reached a peak at 18 h of MPP(+) treatment. Moreover, the maximally-increased protein level of OGG1 by MPP(+) was approximately 2-fold higher than that of XPF. CONCLUSION: MPP(+) treatment could time-dependently induce increases in OGG1 and XPF expressions in PC12 cells. Also, this study indicates that the base and nucleotide excision repair pathways may be compensatory activated in the early stage of pathogenesis in the cells after MPP(+) treatment.
Assuntos
1-Metil-4-fenilpiridínio/toxicidade , DNA Glicosilases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Oxidantes/toxicidade , 8-Hidroxi-2'-Desoxiguanosina , Animais , Western Blotting , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Dano ao DNA/efeitos dos fármacos , Desoxiguanosina/análogos & derivados , Desoxiguanosina/metabolismo , Imuno-Histoquímica , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Células PC12 , Doença de Parkinson , Ratos , Fatores de TempoRESUMO
To study the effect of VEGF overexpression on development of cortical newborn neurons in the brains after stroke, we injected human VEGF(165)-expressive plasmids (phVEGF) into the lateral ventricle of rat brains with a transient middle cerebral artery occlusion (MCAO). An injection of phVEGF significantly promoted angiogenesis (BrdU(+)-von Willebrand's factor(+)) and reduced infarct volume in the rat brain after MCAO. Single labeling of 5'-bromodeoxyuridine (BrdU) and double staining of BrdU with lineage-specific neuronal markers were used to indicate the proliferated cells and maturation of newborn neurons in the brain section of rats at 2, 4, and 8 weeks after MCAO. The results showed that BrdU positive (BrdU(+)) cells existed in ipsilateral frontal cortex within 8 weeks after MCAO and reached the maximum at 2 weeks of reperfusion. The phVEGF treatment significantly increased BrdU(+) cells compared with the control plasmid (pEGFP) injection. Cortical neurogenesis was indicated by the presence of newborn immature (BrdU(+)-Tuj1(+)), newborn mature (BrdU(+)-MAP-2(+)), and newborn GABAergic (BrdU(+)-GAD67(+)) neurons. All these neurons declined within 8 weeks after MCAO in the controls. Injection of phVEGF significantly increased BrdU(+)-Tuj1(+) neurons at 2 weeks, and BrdU(+)-MAP-2(+) neurons and BrdU(+)-GAD67(+) neurons at 4 and 8 weeks, respectively after MCAO. Moreover, phVEGF treatment significantly increased neurite length and branch numbers of BrdU(+)-MAP-2(+) newborn neurons compared with pEGFP treatment. These results demonstrate that VEGF enhances maturation of stroke-induced cortical neurogenesis and dendritic formation of newborn neurons in adult mammalian brains.
Assuntos
Isquemia Encefálica/patologia , Córtex Cerebral/citologia , Neuritos/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/farmacologia , Animais , Animais Recém-Nascidos , Antimetabólitos , Western Blotting , Bromodesoxiuridina , Linhagem Celular , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/crescimento & desenvolvimento , DNA Complementar/biossíntese , DNA Complementar/genética , Imunofluorescência , Humanos , Imuno-Histoquímica , Infarto da Artéria Cerebral Média/patologia , Imageamento por Ressonância Magnética , Masculino , Microscopia Confocal , Neuritos/ultraestrutura , Neurônios/ultraestrutura , Plasmídeos/genética , Ratos , Ratos Sprague-Dawley , TransfecçãoRESUMO
To study the regional and cellular distribution of xeroderma pigmentosum group A and B (XPA and XPB) proteins, two nucleotide excision repair (NER) factors, in the mammalian brain we used immunohistochemistry and triple fluorescent immunostaining combined with confocal microscope scanning in brain slices of adult rat brain, including the cerebral cortex, striatum, substantia nigra compacta, ventral tegmental area, red nucleus, hippocampus, and cerebellum. Both XPA and XPB proteins were mainly expressed in neurons, because the XPA- or XPB-immunopositive cells were only costained with NeuN, a specific neuronal marker, but not with glial fibrillary acidic acid, a specific astrocyte marker, in the striatum. Furthermore, XPA- and XPB-positive staining were observed in the neuronal nuclei. Such subcellular distribution was consistent with the location of the NER in the cells. This study provides the first evidence that NER factors XPA and XPB exist in the nuclei of neurons in the brain, suggesting that the NER may play important roles in the process of DNA repair in adult brain neurons.