RESUMO
OBJECTIVE: Mitochondrial depolarization after ATP-sensitive potassium channel activation has been shown to induce cerebral vasodilation by the generation of calcium sparks in smooth muscle. It is unclear, however, whether mitochondrial depolarization in endothelial cells is capable of promoting vasodilation by releasing vasoactive factors. Therefore, we studied the effect of endothelial mitochondrial depolarization by mitochondrial ATP-sensitive potassium channel activators, BMS-191095 (BMS) and diazoxide, on endothelium-dependent vasodilation. APPROACH AND RESULTS: Diameter studies in isolated rat cerebral arteries showed BMS- and diazoxide-induced vasodilations that were diminished by endothelial denudation. Mitochondrial depolarization-induced vasodilation was reduced by inhibition of mitochondrial ATP-sensitive potassium channels, phosphoinositide-3 kinase, or nitric oxide synthase. Scavenging of reactive oxygen species, however, diminished vasodilation induced by diazoxide, but not by BMS. Fluorescence studies in cultured rat brain microvascular endothelial cells showed that BMS elicited mitochondrial depolarization and enhanced nitric oxide production; diazoxide exhibited largely similar effects, but unlike BMS, increased mitochondrial reactive oxygen species production. Measurements of intracellular calcium ([Ca(2+)]i) in cultured rat brain microvascular endothelial cells and arteries showed that both diazoxide and BMS increased endothelial [Ca(2+)]i. Western blot analyses revealed increased phosphorylation of protein kinase B and endothelial nitric oxide synthase (eNOS) by BMS and diazoxide. Increased phosphorylation of eNOS by diazoxide was abolished by phosphoinositide-3 kinase inhibition. Electron spin resonance spectroscopy confirmed vascular nitric oxide generation in response to diazoxide and BMS. CONCLUSIONS: Pharmacological depolarization of endothelial mitochondria promotes activation of eNOS by dual pathways involving increased [Ca(2+)]i as well as by phosphoinositide-3 kinase-protein kinase B-induced eNOS phosphorylation. Both mitochondrial reactive oxygen species-dependent and -independent mechanisms mediate activation of eNOS by endothelial mitochondrial depolarization.
Assuntos
Artérias Cerebrais/metabolismo , Circulação Cerebrovascular , Células Endoteliais/metabolismo , Potencial da Membrana Mitocondrial , Mitocôndrias/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Canais de Potássio/metabolismo , Vasodilatação , Animais , Benzopiranos/farmacologia , Western Blotting , Cálcio/metabolismo , Células Cultivadas , Artérias Cerebrais/efeitos dos fármacos , Circulação Cerebrovascular/efeitos dos fármacos , Diazóxido/farmacologia , Relação Dose-Resposta a Droga , Espectroscopia de Ressonância de Spin Eletrônica , Células Endoteliais/efeitos dos fármacos , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Sequestradores de Radicais Livres/farmacologia , Imidazóis/farmacologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , Fosfatidilinositol 3-Quinase/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio/agonistas , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologiaRESUMO
Transforming growth factor-ßs (TGF-ß1-3) are cytokines that regulate the proliferation, differentiation, and survival of various cell types. The present study describes the induction of TGF-ß1-3 in the rat after focal ischemia at 3 h, 24 h, 72 h and 1 month after transient (1 h) or permanent (24 h) middle cerebral artery occlusion (MCAO) using in situ hybridization histochemistry and quantitative analysis. Double labeling with different markers was used to identify the localization of TGF-ß mRNA relative to the penumbra and glial scar, and the types of cells expressing TGF-ßs. TGF-ß1 expression increased 3 h after MCAO in the penumbra and was further elevated 24 h after MCAO. TGF-ß1 was present mostly in microglial cells but also in some astrocytes. By 72 h and 1 month after the occlusion, TGF-ß1 mRNA-expressing cells also appeared in microglia within the ischemic core and in the glial scar. In contrast, TGF-ß2 mRNA level was increased in neurons but not in astrocytes or microglial cells in layers II, III, and V of the ipsilateral cerebral cortex 24 h after MCAO. TGF-ß3 was not induced in cells around the penumbra. Its expression increased in only a few cells in layer II of the cerebral cortex 24 h after MCAO. The levels of TGF-ß2 and -ß3 decreased at subsequent time points. Permanent MCAO further elevated the levels of all 3 subtypes of TGF-ßs suggesting that reperfusion is not a major factor in their induction. TGF-ß1 did not co-localize with either Fos or ATF-3, while the co-localization of TGF-ß2 with Fos but not with ATF-3 suggests that cortical spreading depolarization, but not damage to neural processes, might be the mechanism of induction for TGF-ß2. The results imply that endogenous TGF-ßs are induced by different mechanisms following an ischemic attack in the brain suggesting that they are involved in distinct spatially and temporally regulated inflammatory and neuroprotective processes.
Assuntos
Encéfalo/metabolismo , Infarto da Artéria Cerebral Média/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Imuno-Histoquímica , Hibridização In Situ , Masculino , Ratos , Ratos Wistar , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo , Fator de Crescimento Transformador beta2/genética , Fator de Crescimento Transformador beta2/metabolismo , Fator de Crescimento Transformador beta3/genética , Fator de Crescimento Transformador beta3/metabolismoRESUMO
Although much is known about the protective effect of acute estrogen therapy in cerebral ischemia, relatively little is known about the importance of apoptosis and cerebral plasticity in this mechanism. In this work 10 min global cerebral ischemia was produced by transient bilateral carotid occlusion in 4-month-old ovariectomized female gerbils. In every of our experimental group (sham for ischemia group, ischemia group and ischemia + a high, single dose 17ß-estradiol pre-treatment group) apoptotic (bcl-Xl, bax) and cerebral plasticity (GAP-43, synapsin-I, nestin) hippocampal genes' expression was measured four days after surgery. Expression of the anti-apoptotic bcl-Xl (p<0.01) and the cerebral plasticity marker synapsin-I and nestin (p<0.01) increased with acute estrogen pretreatment in ischemic animals. No change, however, in bax or GAP-43 expression was detected in estrogen treated animals compared to ischemic gerbils. These results suggest that acute estrogen therapy has anti-apoptotic effect and increases cerebral plasticity, which play an important role in cytoprotection or cerebroprotection.
Assuntos
Apoptose/efeitos dos fármacos , Estradiol/farmacologia , Estradiol/uso terapêutico , Ataque Isquêmico Transitório/tratamento farmacológico , Ataque Isquêmico Transitório/metabolismo , Plasticidade Neuronal/efeitos dos fármacos , Animais , Apoptose/fisiologia , Estenose das Carótidas/complicações , Relação Dose-Resposta a Droga , Feminino , Proteína GAP-43/metabolismo , Gerbillinae , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Hipocampo/patologia , Proteínas de Filamentos Intermediários/metabolismo , Ataque Isquêmico Transitório/etiologia , Modelos Animais , Proteínas do Tecido Nervoso/metabolismo , Nestina , Plasticidade Neuronal/fisiologia , Ovariectomia , Sinapsinas/metabolismo , Proteína X Associada a bcl-2/metabolismo , Proteína bcl-X/metabolismoRESUMO
Permanent bilateral carotid occlusion is a well known cerebral hypoperfusion model in rats. The aim of our study was to investigate the different stages of vascular reaction by detecting changes in the extracellular martix proteins and to examine their relationship to angiogenesis after occlusion. Experiments were performed on adult male rats. Brain samples were investigated from day 1 to day 30 post-surgery. Immunohistochemical analysis was performed on the whole hippocampus and on the adjacent cortex in order to investigate extracellular martix proteins, such as the markers of dystroglycan complex (ß-dystroglycan, α-dystrobrevin and utrophin) and a marker of basal lamina (laminin). The levels of the proteins were estimated by western blot analysis. Vascular density as well as blood-brain barrier permeability were studied on brain slices from the same regions. Our results showed altered laminin and ß-dystroglycan immunoreactivity beginning 2 days after the onset of occlusion followed by an increased utrophin immunoreactivity without blood-brain barrier disruption 5 days later. By day 30 of hypoperfusion, when increased vascular density was detected, all changes returned to baseline levels. Western blot analysis showed significant differences in ß-dystroglycan and utrophin expression. Our results indicate that the different stages of neovascularisation resulting from cerebral hypoperfusion can be well defined by the markers laminin, ß-dystroglycan, and utrophin and that these markers are more likely to correlate with glio-vascular decoupling than does altered blood-brain barrier function.
Assuntos
Isquemia Encefálica/metabolismo , Encéfalo/metabolismo , Complexo de Proteínas Associadas Distrofina/metabolismo , Laminina/metabolismo , Neovascularização Fisiológica/fisiologia , Animais , Barreira Hematoencefálica/fisiologia , Encéfalo/anatomia & histologia , Permeabilidade Capilar/fisiologia , Artérias Carótidas/cirurgia , Córtex Cerebral/metabolismo , Distroglicanas/metabolismo , Proteínas Associadas à Distrofina/metabolismo , Hipocampo/anatomia & histologia , Hipocampo/metabolismo , Masculino , Microvasos/anatomia & histologia , Proteínas do Tecido Nervoso/metabolismo , Ratos , Ratos Wistar , Fatores de Tempo , Utrofina/metabolismoRESUMO
Transforming growth factors-beta1 (TGF-beta1), -2, and -3 form a small group of related proteins involved in the regulation of proliferation, differentiation, and survival of various cell types. Recently, TGF-betas were also demonstrated to be neuroprotective. In the present study, we investigated their distribution in the rat brain as well as their expression following middle cerebral artery occlusion. Probes were produced for all types of TGF-betas, and in situ hybridization was performed. We demonstrated high TGF-beta1 expression in cerebral cortex, hippocampus, central amygdaloid nucleus, medial preoptic area, hypothalamic paraventricular nucleus, substantia nigra, brainstem reticular formation and motoneurons, and area postrema. In contrast, TGF-beta2 was abundantly expressed in deep cortical layers, dentate gyrus, midline thalamic nuclei, posterior hypothalamic area and mamillary body, superior olive, areas of monoaminergic neurons, spinal trigeminal nucleus, dorsal vagal complex, cerebellum, and choroid plexus, and a high level of TGF-beta3 mRNA was found in cerebral cortex, hippocampus, basal amygdaloid nuclei, lateral septal nucleus, several thalamic nuclei, arcuate and supramamillary nuclei, superior colliculus, superior olive, brainstem reticular formation and motoneurons, area postrema, and inferior olive. Focal brain ischemia induced TGF-betas with markedly different expression patterns. TGF-beta1 was induced in the penumbral region of cortex and striatum, whereas TGF-beta2 and -beta3 were induced in different layers of the ipsilateral cortex. The expression of the subtypes of TGF-betas in different brain regions suggests that they are involved in the regulation of different neurons and bind to different latent TGF-beta binding proteins. Furthermore, they might have subtype-specific functions following ischemic attack.
Assuntos
Isquemia Encefálica/metabolismo , Encéfalo , RNA Mensageiro/metabolismo , Fator de Crescimento Transformador beta1 , Fator de Crescimento Transformador beta2 , Fator de Crescimento Transformador beta3 , Animais , Encéfalo/anatomia & histologia , Encéfalo/patologia , Encéfalo/fisiologia , Isquemia Encefálica/patologia , Infarto da Artéria Cerebral Média , Masculino , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ratos , Ratos Wistar , Distribuição Tecidual , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo , Fator de Crescimento Transformador beta2/genética , Fator de Crescimento Transformador beta2/metabolismo , Fator de Crescimento Transformador beta3/genética , Fator de Crescimento Transformador beta3/metabolismoRESUMO
Although much is known about the protective effect of acute estrogen therapy in cerebral ischemia, relatively little is known about its effect on functional outcome at different ages. The impact of age is, however, important on the efficacy of steroids in the central nervous system. We investigated whether a single dose of estradiol pre-treatment would be neuroprotective in young (4 months), middle-aged (9 months) and old (18 months) female gerbils following 10min global brain ischemia. Apoptotic and necrotic cells were labelled and quantified in the affected hippocampus; exploratory activity, attention and memory functions were tested using open field, spontaneous alternation, novel object recognition and hole-board test. Age effect and treatment effect were analysed. High single dose (4mg/kg b.w.) of estradiol pre-treatment exposed a marked neuroprotective effect against hippocampal cell loss in all age groups. In behavioural tests, however, age-related differences could be observed. In middle-aged and old animals the worsening in memory function following ischemia was more prominent compared to that in the young ones. In the Y-maze and the novel object recognition tests the middle-aged, in the hole-board test (investigating working memory and total time) the old gerbils had the worst functional outcome. Only reference memory in hole-board test did not change by age. Estrogen improved memory performances in all the tests at every age. We can conclude that age of experimental animals is a factor worsening the outcome following brain ischemia. A single-dose estrogen therapy prevents the lesion-induced behavioural dysfunctions and the hippocampal cell loss.
Assuntos
Sintomas Comportamentais/etiologia , Isquemia Encefálica/complicações , Estrogênios/farmacologia , Fármacos Neuroprotetores/farmacologia , Fatores Etários , Análise de Variância , Animais , Atenção/efeitos dos fármacos , Sintomas Comportamentais/tratamento farmacológico , Isquemia Encefálica/patologia , Caspase 3/metabolismo , Comportamento Exploratório/fisiologia , Feminino , Gerbillinae/fisiologia , Hipocampo/patologia , Marcação In Situ das Extremidades Cortadas/métodos , Memória/efeitos dos fármacos , Memória/fisiologia , Neurônios/efeitos dos fármacos , Neurônios/patologia , Ovariectomia , Reconhecimento Psicológico/efeitos dos fármacosRESUMO
The anti-apoptotic gene replacements could be an option in preventing hypoxia-induced neuronal loss. In this paper we tested the effect of anti-apoptosis (bcl-2 and bcl-XL) gene transfer on cell plasticity. Nestin, synapsin-1 and c-fos genes and proteins expression were measured in PC12 cells in normal condition, and after hypoxia/re-oxygenation. Gene delivery results a significant increase in both bcl-2 and bcl-XL gene expression. Hypoxia (1h)/re-oxygenation (24h) have a detrimental effect upon cultured cells by increasing the pro-apoptotic, bax gene and protein expression. Bcl-2 or bcl-XL gene delivery resulted in a significant increase in and the cellular levels of the corresponding mRNAs and proteins. Bcl-2 gene augmented the nestin gene and protein expression which has been compromised previously by the hypoxic event. Similarly c-fos mRNA and protein expression decreased significantly after hypoxia, while the anti-apoptotic gene treatment normalized c-fos expression. Synapsin-1 gene or protein expression remained about on the same level under normoxic conditions or following hypoxia after gene treatment. We can conclude that anti-apoptotic gene transfers activate neuronal plasticity proteins nestin and c-fos. This link on anti-apoptotic proteins and cell plasticity is a new finding.