RESUMO
Increased Ca2+ influx through activated N-methyl-D-aspartate (NMDA) receptors and voltage-dependent Ca2+ channels (VDCC) is a major determinant of cell injury following brain ischemia. The activity of these channels is modulated by dynamic changes in the actin cytoskeleton, which may occur, in part, through the actions of the actin filament-severing protein gelsolin. We show that gelsolin-null neurons have enhanced cell death and rapid, sustained elevation of Ca2+ levels following glucose/oxygen deprivation, as well as augmented cytosolic Ca2+ levels in nerve terminals following depolarization in vitro. Moreover, major increases in infarct size are seen in gelsolin-null mice after reversible middle cerebral artery occlusion, compared with controls. In addition, treatment with cytochalasin D, a fungal toxin that depolymerizes actin filaments, reduced the infarct size of both gelsolin-null and control mice to the same final volume. Hence, enhancement or mimicry of gelsolin activity may be neuroprotective during stroke.
Assuntos
Encéfalo/metabolismo , Transtornos Cerebrovasculares/metabolismo , Gelsolina/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Canais de Cálcio/metabolismo , Morte Celular/efeitos dos fármacos , Transtornos Cerebrovasculares/tratamento farmacológico , Transtornos Cerebrovasculares/patologia , Citocalasina D/farmacologia , Citocalasina D/uso terapêutico , Citoesqueleto/metabolismo , Citoesqueleto/patologia , Camundongos , Neurônios/metabolismo , Neurônios/patologia , Inibidores da Síntese de Ácido Nucleico/farmacologia , Inibidores da Síntese de Ácido Nucleico/uso terapêutico , Receptores de N-Metil-D-Aspartato/metabolismoRESUMO
BACKGROUND AND PURPOSE: The 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) lower serum cholesterol and decrease the incidence of stroke and cardiovascular disease. There is growing evidence that statins exert some of their beneficial effects independent of cholesterol lowering. Indeed, we have previously demonstrated that chronic simvastatin administration upregulates endothelial nitric oxide synthase (eNOS), resulting in more functional protein, augmentation of cerebral blood flow, and neuroprotection in a murine model of cerebral ischemia. In this report we examined whether another member of the statin family shared these effects and whether eNOS upregulation is sustained with longer treatment. METHODS: Mevastatin (2 mg/kg or 20 mg/kg per day) was administered to 18- to 22-g male mice for 7, 14, or 28 days before 2-hour middle cerebral artery occlusion with the use of the filament model (n=9 to 12). Neurological deficits and cerebral infarct volumes were assessed at 24 hours. Arterial blood pressure and gases, relative cerebral blood flow, and blood cholesterol levels were monitored in a subset of animals (n=5). Absolute cerebral blood flow was measured by the [(14)C]iodoamphetamine indicator fractionation technique (n=6). eNOS mRNA and protein levels were determined. RESULTS: Mevastatin increased levels of eNOS mRNA and protein, reduced infarct size, and improved neurological deficits in a dose- and time-dependent manner. Greatest protection was seen with 14- and 28-day high-dose treatment (26% and 37% infarct reduction, respectively). Cholesterol levels were reduced only after 28 days of treatment and did not correlate with infarct reduction. Baseline absolute cerebral blood flow was 30% higher after 14-day high-dose treatment. CONCLUSIONS: Chronic prophylactic treatment with mevastatin upregulated eNOS and augmented cerebral blood flow. These changes occurred in the absence of changes in serum cholesterol levels, were sustained for up to 1 month of treatment, and resulted in neuroprotection after middle cerebral artery occlusion.
Assuntos
Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Lovastatina/análogos & derivados , Lovastatina/uso terapêutico , Óxido Nítrico Sintase/metabolismo , Acidente Vascular Cerebral/tratamento farmacológico , Regulação para Cima/efeitos dos fármacos , Animais , Aorta/metabolismo , Aorta/patologia , Velocidade do Fluxo Sanguíneo/efeitos dos fármacos , Gasometria , Pressão Sanguínea/efeitos dos fármacos , Circulação Cerebrovascular/efeitos dos fármacos , Colesterol/sangue , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Immunoblotting , Masculino , Camundongos , Camundongos Endogâmicos , Camundongos Knockout , Óxido Nítrico Sintase/deficiência , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase Tipo II , Óxido Nítrico Sintase Tipo III , RNA Mensageiro/metabolismo , Acidente Vascular Cerebral/genética , Acidente Vascular Cerebral/metabolismo , Resultado do TratamentoRESUMO
The relative importance of hemodynamic factors in the pathogenesis of thrombotic or embolic stroke is unclear. Of particular therapeutic interest are those substances that facilitate vasodilation and the clearance of platelet aggregates in the compromised microvasculature. A likely contributor to these functions is nitric oxide because it is known to inhibit platelet aggregability and promote vascular relaxation. To investigate the involvement of nitric oxide in the hemodynamic changes after experimental ischemia, photochemically induced nonocclusive common carotid artery thrombosis (CCAT) was studied. CCAT is a rat model of unilateral carotid artery stenosis and platelet embolization to the brain. This study characterized the acute hemodynamic consequences of CCAT and the resultant pattern of platelet deposits with and without nitric oxide synthase inhibition by nitro-L-arginine methyl ester (L-NAME). In addition, the subacute local cerebral blood flow changes were studied at 24 hours. Right CCAT was produced in 30 male Wistar rats injected with (111)In-labeled platelets. Between 5 and 15 minutes after thrombosis, rats were treated with either 15 mg/kg of L-NAME (intravenously) or saline vehicle. Hemodynamic changes were studied 30 to 45 minutes after thrombosis using [14C]iodoantipyrine autoradiography. Eight coronal levels were analyzed, and cortical and subcortical regions of interest were defined. Significant increases were observed in total platelets in the ipsilateral hemisphere after L-NAME treatment, and in the distribution of platelets in the anterior frontal and occipital cortices with nitric oxide synthase inhibition, encompassing the anterior and posterior border zone areas of the ipsilateral cortex. Otherwise, foci of labeled platelets were detected throughout the ipsilateral and contralateral hemispheres. Mean local cerebral blood flow images (n = 5) revealed a moderate bilateral global reduction in flow acutely, which normalized in the untreated thrombosed group by 24 hours. In contrast, the L-NAME-treated groups (sham and experimental) had lasting, widespread reductions in flow of approximately 25%. Pairwise comparisons between groups showed that CCAT/L-NAME was significantly different from shams in the corpus callosum and different from L-NAME shams in the internal capsule (P < 0.05) These hemodynamic and platelet accumulation changes may partially account for the aggravation of cognitive and sensorimotor deficits previously reported in this model of thromboembolic stroke.
Assuntos
Plaquetas/fisiologia , Transtornos Cerebrovasculares/fisiopatologia , Embolia e Trombose Intracraniana/fisiopatologia , Óxido Nítrico Sintase/antagonistas & inibidores , Animais , Circulação Cerebrovascular/efeitos dos fármacos , Circulação Cerebrovascular/fisiologia , Inibidores Enzimáticos/farmacologia , Hemodinâmica/efeitos dos fármacos , Hemodinâmica/fisiologia , Masculino , NG-Nitroarginina Metil Éster/farmacologia , Ratos , Ratos Wistar , Fatores de TempoRESUMO
In a process called ischemic preconditioning, a brief, sublethal ischemic insult protects tissue from subsequent, more severe injury. There have been no reports of rapidly induced ischemic preconditioning. The authors sought to develop a model of cerebral ischemic preconditioning in the mouse that can be applied to transgenic and knockout animals. They found that brief middle cerebral artery (MCA) occlusion only minutes before a severe ischemic insult can induce protection from that insult. Here the investigators describe a mouse model of preconditioning using intraluminal MCA occlusion as both the conditioning and the test stimulus. One or three 5-minute episodes of ischemia given 30 minutes before MCA occlusion for 1 or 24 hours (permanent occlusion) confer significant protection as assessed by infarct volume measurements 24 hours later.
Assuntos
Isquemia Encefálica/fisiopatologia , Artérias Cerebrais/patologia , Precondicionamento Isquêmico , Animais , Modelos Animais de Doenças , Camundongos , Camundongos Knockout , Camundongos TransgênicosRESUMO
The hypotheses that cerebral embolic events lead to repetitive episodes of cortical spreading depression (CSD) and that these propagating waves trigger the expression of c-fos, brain-derived neurotrophic factor (BDNF), glial fibrillary acidic protein (GFAP), and heat shock protein 70 (HSP70) mRNA were tested. Wistar rats underwent photochemically induced right common carotid artery thrombosis (CCAT) (n = 18) or sham (n = 8) procedures. In a subgroup of rats (n = 5), laser-Doppler flowmetry probes were placed overlying the right parietal cortex to record CSD-like changes in cortical blood flow during the initial 2-hour postinjury period. Rats were killed by decapitation at 2 or 24 hours after CCAT, and brains were processed for in situ localization of the gene expression. Two to five intermittent transient hyperemic episodes lasting 1 to 2 minutes were recorded ipsilaterally after CCAT. At 2 hours after CCAT, the widespread expression of c-fos and BDNF mRNAs was observed throughout the ipsilateral cerebral cortex. Pretreatment with the N-methyl-D-aspartate receptor blocker MK-801 (2 mg/kg) 1 hour before CCAT reduced the expression of BDNF mRNA expression at 2 hours. At 24 hours after CCAT, increased expression of GFAP mRNA was present in cortical and subcortical regions. In contrast, multifocal regions of HSP70 expression scattered throughout the thrombosed hemisphere were apparent at both 2 and 24 hours after injury. These data indicate that thromboembolic events lead to episodes of CSD and time-dependent alterations in gene expression. The ability of embolic processes to induce widespread molecular responses in neurons and glia may be important in the pathogenesis of transient ischemic attacks and may influence the susceptibility of the postembolic brain to subsequent insults including stroke.
Assuntos
Depressão Alastrante da Atividade Elétrica Cortical , Expressão Gênica , Embolia Intracraniana/genética , Embolia Intracraniana/fisiopatologia , Animais , Autorradiografia , Fator Neurotrófico Derivado do Encéfalo/genética , Circulação Cerebrovascular , Proteína Glial Fibrilar Ácida/genética , Proteínas de Choque Térmico HSP70/genética , Hemodinâmica , Hibridização In Situ , Embolia Intracraniana/metabolismo , Masculino , Proteínas Proto-Oncogênicas c-fos/genética , RNA Mensageiro/metabolismo , Ratos , Ratos WistarRESUMO
A series of molecular pathways have in common a significant role in the pathogenesis and progression of atherosclerosis and cancer. Shared mechanisms implicated for both diseases include oxidative stress and the cellular damage that results from it, toxic metabolites produced by cigarette smoking, and increased dietary fat intake. Atherosclerosis may begin when an injury or infection mutates or transforms a single arterial smooth muscle cell in the progenitor of a proliferative clone, similar to the most widely held carcinogenesis theory. Cell proliferation regulatory pathways have been associated with plaque progression, stenosis, and restenosis after angioplasty and with cancer progression. Alterations in cell adhesion molecules have been linked to plaque formation and thrombosis and to tumor invasion and metastasis. Altered expression of proteases associated with thrombolysis has been implicated in atherosclerotic plaque expansion and hemorrhage and in the invasion and metastasis of malignant neoplasms. Ligand-growth factor receptor interactions have been associated with early atherosclerotic lesions and with cancer development and spread. Nuclear transcription factors have been associated with progression of both diseases. Angiogenesis modulators have been linked to plaque expansion and restenosis of atherosclerotic lesions and to local and metastatic tumor expansion.
Assuntos
Arteriosclerose , Neoplasias , Arteriosclerose/etiologia , Arteriosclerose/genética , Arteriosclerose/patologia , Moléculas de Adesão Celular , Divisão Celular , Gorduras na Dieta/administração & dosagem , Predisposição Genética para Doença , Humanos , NF-kappa B , Neoplasias/etiologia , Neoplasias/genética , Neoplasias/patologia , Neovascularização Patológica , Estresse Oxidativo , Receptores de Fatores de Crescimento , Fumar/efeitos adversos , Fatores de Transcrição , Fator de Crescimento Transformador betaRESUMO
Recent approaches to candidate gene identification and cellular localization have included RNA derived from complex whole tissue profiling on cDNA microarrays followed by in situ hybridization with riboprobes. In this study, the Arcturus PixCell II laser capture microdissection (LCM) system, an argon-based laser-assisted method for the isolation of specific cell types from heterogeneous tissue samples, was used to microdissect the tunica media from normal human arteries and veins (n = 5 in each group). Total RNA was extracted from the sum of 10,000 shots for each blood vessel using the Strataprep MicroKit. RNA was reverse-transcribed, and the resulting cDNA was analyzed using the Applied Biosystems 7700 quantitative PCR system (Q-PCR). Control genes, such as the L-type calcium channel, PECAM (CD-31), and beta-2 microglobulin, were used to assess sample quality and purity. Of 10 laser-captured media samples, five (50%) showed a gene profile that indicated high-quality RNA (abundance of housekeeping genes) and smooth muscle cell enrichment (low levels of PECAM and high levels of the L-type calcium channel). We conclude that the application of the LCM technique to collect smooth muscle cell RNA from the tunica media of human blood vessels can assist in the validation of gene expression and potentially expedite the identification of novel, regulated genes present within vascular smooth muscle.
Assuntos
Artérias/fisiologia , RNA/genética , Veias/fisiologia , Actinas/genética , Artérias/citologia , DNA Complementar/genética , Dissecação/métodos , Técnicas Genéticas , Humanos , Lasers , Molécula-1 de Adesão Celular Endotelial a Plaquetas/genética , Reação em Cadeia da Polimerase , RNA/isolamento & purificação , Túnica Média/fisiologia , Veias/citologiaRESUMO
Recently, a series of shared molecular pathways have emerged that have in common a significant role in the pathogenesis and progression of both atherosclerosis and cancer. Oxidative stress and the cellular damage that results from it have been implicated in a wide variety of disease processes including atherogenesis and neoplasia. Toxic metabolites produced by cigarette smoking and increased dietary fat intake are implicated in the pathogenesis of both diseases. It has been hypothesized that atherosclerosis may begin when an injury or infection mutates or transforms a single arterial smooth muscle cell in the progenitor of a proliferative clone similar to the most widely held theory of carcinogenesis. Cell proliferation regulatory pathways including genes involved in the GIS checkpoint (p53, pRb, p15, p16, and cyclins A, D, E, and cdk 2,4) have been associated with plaque progression, stenosis and restenosis after angioplasty as well as in cancer progression. Alterations in cell adhesion molecules (integrins, cadherin-catenins) have been linked to plaque formation and thrombosis as well as to tumor invasion and metastasis. Altered expression of proteases associated with thrombolysis has been implicated in atherosclerotic plaque expansion and hemorrhage and in the invasion and metastasis of malignancy. Ligand-growth factor receptor interactions (tyrosine kinases) have been associated with early atherosclerotic lesions as well as cancer development and spread. Nuclear transcription factors such as NFkappaB have been associated with progression of both diseases. Angiogenesis modulators have recently been linked to plaque expansion and restenosis of atherosclerotic lesions as well as local and metastatic tumor expansion. Common disease treatments, such as the use of growth factor inhibitors and radiation treatment, established anticancer treatments, were recently introduced into atherosclerosis therapeutic strategies to prevent restenosis after angioplasty and endarterectomy. In conclusion, a series of molecular pathways of disease development and progression common to atherosclerosis and cancer support that the world's two most common diseases are far more closely aligned than previously believed and that emerging anti-inflammatory and antiproliferative therapeutic strategies may ultimately be efficacious in both conditions.
Assuntos
Arteriosclerose/genética , Neoplasias/genética , Arteriosclerose/patologia , Arteriosclerose/fisiopatologia , Divisão Celular , Progressão da Doença , Regulação da Expressão Gênica , Humanos , Neoplasias/patologia , Neoplasias/fisiopatologia , Estresse OxidativoRESUMO
Although nitric oxide (NO) has been shown to play an important role in the pathophysiology of cerebral ischemia, its contribution to the pathogenesis of experimentally induced thromboembolic stroke is unknown. In this study, we pharmacologically manipulated NO levels in the acute post-thrombotic stage and determined the effects on behavior and histopathology. The following drugs were used: nitro-L-arginine-methyl ester (L-NAME), a non-specific endothelial and neuronal nitric oxide synthase (eNOS and nNOS) inhibitor, 3-bromo-7-nitroindazole (7-NI), a specific inhibitor for nNOS, the NO precursor, exogenous L-arginine and the NO-donor, 3-morpholino-sydnonimine (SIN-1). Male Wistar rats (n = 76) were randomly assigned to receive vehicle or drug immediately after common carotid artery thrombosis (CCAT). Regional measurements of cortical NOS activity using the [3H]L-arginine to [3H]L-citrulline conversion assay were decreased 1 h after treatment with L-NAME and 7-NI by 50 and 65%, respectively; hippocampal NOS activity was reduced with L-NAME by 35% and with 7-NI by 65%. L-NAME significantly worsened forelimb placing as compared to other groups. 7-NI accelerated sensorimotor recovery. Water maze retention deficits were noted 48 h after CCAT and these were exacerbated by L-NAME treatment. Histopathological protection was conferred in the hippocampus by 7-NI and SIN-1; conversely, L-NAME increased neuronal injury in the contralateral cortex. L-arginine had no effect on these outcomes. In conclusion, both structural and functional consequences of CCAT can be aggravated by limiting endothelial NO production in the acutely post-thrombotic brain. In contrast, inhibition of nNOS and infusion of an NO donor has a beneficial effect on pathology.
Assuntos
Transtornos Cerebrovasculares/etiologia , Transtornos Cerebrovasculares/fisiopatologia , Embolia e Trombose Intracraniana/complicações , Óxido Nítrico/fisiologia , Tromboembolia/complicações , Animais , Arginina/metabolismo , Comportamento Animal/efeitos dos fármacos , Pressão Sanguínea , Transtornos Cerebrovasculares/psicologia , Citrulina/biossíntese , Transtornos Cognitivos/etiologia , Transtornos Cognitivos/psicologia , Inibidores Enzimáticos/farmacologia , Masculino , Córtex Motor/efeitos dos fármacos , Córtex Motor/fisiopatologia , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Ratos , Ratos Wistar , Córtex Somatossensorial/efeitos dos fármacos , Córtex Somatossensorial/fisiopatologiaRESUMO
Thromboembolic stroke in rats leads to a well-described pattern of histopathological and behavioral abnormalities. However, limited data are available in animal models concerning the response of the white matter to embolic events. The purpose of this study was to document patterns of white matter abnormalities using beta-amyloid precursor protein (betaAPP) immunocytochemistry as a marker of axonal damage. Twelve male Wistar rats underwent photochemically induced right common carotid artery thrombosis (CCAT) or sham procedures. At 3 days after CCAT, rats were perfusion-fixed and sections immunostained for the visualization of betaAPP or stained with hematoxylin and eosin for routine histopathological analysis. As previously described, CCAT produced small ipsilateral embolic infarcts and ischemic cell change within gray matter structures including the medial cerebral cortex, striatum, hippocampus and thalamus. In areas of frank infarction, numerous reactive profiles were observed within borderzones of the damaged site. However, betaAPP immunocytochemistry also revealed reactive axonal profiles within various white matter tracts including the corpus callosum, external capsule and fimbria of the hippocampus. In many cases, the presence of axonal damage could not be appreciated with routine hematoxylin and eosin staining. These data indicate that CCAT leading to platelet embolization to the brain not only produces embolic infarcts but also produces more subtle white matter abnormalities. Previously undetected white matter damage would be expected to participate in the sensorimotor and cognitive behavioral deficits following embolic stroke.