RESUMO
UNLABELLED: The aim of this study was to explore the signaling and neuroprotective effect of transactivator of transcription (TAT) protein transduction of the apoptosis repressor with CARD (ARC) in in vitro and in vivo models of cerebral ischemia in mice. In mice, transient focal cerebral ischemia reduced endogenous ARC protein in neurons in the ischemic striatum at early reperfusion time points, and in primary neuronal cultures, RNA interference resulted in greater neuronal susceptibility to oxygen glucose deprivation (OGD). TAT.ARC protein delivery led to a dose-dependent better survival after OGD. Infarct sizes 72 h after 60 min middle cerebral artery occlusion (MCAo) were on average 30 ± 8% (mean ± SD; p = 0.005; T2-weighted MRI) smaller in TAT.ARC-treated mice (1 µg intraventricularly during MCAo) compared with controls. TAT.ARC-treated mice showed better performance in the pole test compared with TAT.ß-Gal-treated controls. Importantly, post-stroke treatment (3 h after MCAo) was still effective in affording reduced lesion volume by 20 ± 7% (mean ± SD; p < 0.05) and better functional outcome compared with controls. Delayed treatment in mice subjected to 30 min MCAo led to sustained neuroprotection and functional behavior benefits for at least 28 d. Functionally, TAT.ARC treatment inhibited DAXX-ASK1-JNK signaling in the ischemic brain. ARC interacts with DAXX in a CARD-dependent manner to block DAXX trafficking and ASK1-JNK activation. Our work identifies for the first time ARC-DAXX binding to block ASK1-JNK activation as an ARC-specific endogenous mechanism that interferes with neuronal cell death and ischemic brain injury. Delayed delivery of TAT.ARC may present a promising target for stroke therapy. SIGNIFICANCE STATEMENT: Up to now, the only successful pharmacological target of human ischemic stroke is thrombolysis. Neuroprotective pharmacological strategies are needed to accompany therapies aiming to achieve reperfusion. We describe that apoptosis repressor with CARD (ARC) interacts and inhibits DAXX and proximal signals of cell death. In a murine stroke model mimicking human malignant infarction in the territory of the middle cerebral artery, TAT.ARC salvages brain tissue when given during occlusion or 3 h delayed with sustained functional benefits (28 d). This is a promising novel therapeutic approach because it appears to be effective in a model producing severe injury by interfering with an array of proximal signals and effectors of the ischemic cascade, upstream of JNK, caspases, and BIM and BAX activation.
Assuntos
Apoptose , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patologia , Proteínas de Transporte/metabolismo , Proteínas do Citoesqueleto/metabolismo , Produtos do Gene tat/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Animais , Proteínas Correpressoras , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Chaperonas Moleculares , Ligação Proteica , Mapas de Interação de ProteínasRESUMO
BACKGROUND AND PURPOSE: Chronic hypoperfusion in the mouse brain has been suggested to mimic aspects of vascular cognitive impairment, such as white matter damage. Although this model has attracted attention, our group has struggled to generate a reliable cognitive and pathological phenotype. This study aimed to identify neuroimaging biomarkers of brain pathology in aged, more severely hypoperfused mice. METHODS: We used magnetic resonance imaging to characterize brain degeneration in mice hypoperfused by refining the surgical procedure to use the smallest reported diameter microcoils (160 µm). RESULTS: Acute cerebral blood flow decreases were observed in the hypoperfused group that recovered over 1 month and coincided with arterial remodeling. Increasing hypoperfusion resulted in a reduction in spatial learning abilities in the water maze that has not been previously reported. We were unable to observe severe white matter damage with histology, but a novel approach to analyze diffusion tensor imaging data, graph theory, revealed substantial reorganization of the hypoperfused brain network. A logistic regression model from the data revealed that 3 network parameters were particularly efficient at predicting group membership (global and local efficiency and degrees), and clustering coefficient was correlated with performance in the water maze. CONCLUSIONS: Overall, these findings suggest that, despite the autoregulatory abilities of the mouse brain to compensate for a sudden decrease in blood flow, there is evidence of change in the brain networks that can be used as neuroimaging biomarkers to predict outcome.
Assuntos
Encéfalo/diagnóstico por imagem , Circulação Cerebrovascular/fisiologia , Disfunção Cognitiva/diagnóstico por imagem , Modelos Animais de Doenças , Neuroimagem , Animais , Encéfalo/fisiologia , Disfunção Cognitiva/fisiopatologia , Masculino , Aprendizagem em Labirinto/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Vias Neurais/diagnóstico por imagem , Vias Neurais/fisiologia , Neuroimagem/métodos , Valor Preditivo dos TestesRESUMO
BACKGROUND AND PURPOSE: Despite standardization of experimental stroke models, final infarct sizes after middle cerebral artery occlusion (MCAO) vary considerably. This introduces uncertainties in the evaluation of drug effects on stroke. Magnetic resonance imaging may detect variability of surgically induced ischemia before treatment and thus improve treatment effect evaluation. METHODS: MCAO of 45 and 90 minutes induced brain infarcts in 83 mice. During, and 3 and 6 hours after MCAO, we performed multiparametric magnetic resonance imaging. We evaluated time courses of cerebral blood flow, apparent diffusion coefficient (ADC), T1, T2, accuracy of infarct prediction strategies, and impact on statistical evaluation of experimental stroke studies. RESULTS: ADC decreased during MCAO but recovered completely on reperfusion after 45 and partially after 90-minute MCAO, followed by a secondary decline. ADC lesion volumes during MCAO or at 6 hours after MCAO largely determined final infarct volumes for 90 but not for 45 minutes MCAO. The majority of chance findings of final infarct volume differences in random group allocations of animals were associated with significant differences in early ADC lesion volumes for 90, but not for 45-minute MCAO. CONCLUSIONS: The prediction accuracy of early magnetic resonance imaging for infarct volumes depends on timing of magnetic resonance imaging and MCAO duration. Variability of the posterior communicating artery in C57Bl6 mice contributes to differences in prediction accuracy between short and long MCAO. Early ADC imaging may be used to reduce errors in the interpretation of post MCAO treatment effects on stroke volumes.
Assuntos
Encéfalo/patologia , Infarto da Artéria Cerebral Média/patologia , Imageamento por Ressonância Magnética/métodos , Reperfusão , Animais , Isquemia Encefálica/patologia , Circulação Cerebrovascular , Imagem de Difusão por Ressonância Magnética , Modelos Animais de Doenças , Processamento de Imagem Assistida por Computador , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fatores de TempoRESUMO
The functional dynamics and cellular sources of oxidative stress are central to understanding MS pathogenesis but remain elusive, due to the lack of appropriate detection methods. Here we employ NAD(P)H fluorescence lifetime imaging to detect functional NADPH oxidases (NOX enzymes) in vivo to identify inflammatory monocytes, activated microglia, and astrocytes expressing NOX1 as major cellular sources of oxidative stress in the central nervous system of mice affected by experimental autoimmune encephalomyelitis (EAE). This directly affects neuronal function in vivo, indicated by sustained elevated neuronal calcium. The systemic involvement of oxidative stress is mirrored by overactivation of NOX enzymes in peripheral CD11b(+) cells in later phases of both MS and EAE. This effect is antagonized by systemic intake of the NOX inhibitor and anti-oxidant epigallocatechin-3-gallate. Together, this persistent hyper-activation of oxidative enzymes suggests an "oxidative stress memory" both in the periphery and CNS compartments, in chronic neuroinflammation.
Assuntos
Encefalomielite Autoimune Experimental/enzimologia , Esclerose Múltipla/enzimologia , NADPH Oxidases/metabolismo , Estresse Oxidativo/fisiologia , Animais , Antioxidantes/uso terapêutico , Astrócitos/efeitos dos fármacos , Astrócitos/enzimologia , Astrócitos/patologia , Antígeno CD11b/metabolismo , Cálcio/metabolismo , Catequina/análogos & derivados , Catequina/uso terapêutico , Doença Crônica , Progressão da Doença , Encefalomielite Autoimune Experimental/tratamento farmacológico , Encefalomielite Autoimune Experimental/patologia , Inibidores Enzimáticos/uso terapêutico , Acetato de Glatiramer/uso terapêutico , Humanos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia de Fluorescência/métodos , Esclerose Múltipla/tratamento farmacológico , Esclerose Múltipla/patologia , NADPH Oxidases/antagonistas & inibidores , Neurônios/efeitos dos fármacos , Neurônios/enzimologia , Neurônios/patologia , Estresse Oxidativo/efeitos dos fármacosRESUMO
BOLD fMRI localizes activated brain areas by measuring decreases of deoxygenated hemoglobin (deoxy-Hb) caused by neurovascular coupling. To date, it is unclear whether intracranial pressure (ICP) modifies deoxy-Hb signaling for brain mapping. In addition, ICP elevation can test whether the BOLD post-stimulus undershoot, a transient hypo-oxygenation following functional activation, is due to vascular compliance rather than elevated cerebral metabolic rate of oxygen (CMRO(2)). We addressed these questions by studying the effect of ICP elevation on neurovascular coupling. In anesthetized rats, a cranial window was implanted over the somatosensory cortex. Using laser Doppler flowmetry and optical spectroscopy, changes in cerebral blood flow (CBF), cerebral blood volume (CBV) and deoxy-Hb were measured during electrical forepaw stimulation. Neuronal activity was monitored by somatosensory evoked potentials. ICP was elevated by subarachnoideal and intracisternal infusion of artificial cerebrospinal fluid. ICP elevation did not abrogate neurovascular coupling. However, the concomitant deoxy-Hb decrease was reduced (ICP=14mmHg) and reversed (ICP=28mmHg). Therefore, the validity of BOLD fMRI has to be questioned during increased ICP. Moreover, the amplitude of the deoxy-Hb post-stimulus overshoot was reduced with ICP elevation. CMRO(2) was not elevated during the post-stimulus response. Therefore, these data provide experimental evidence that the BOLD post-stimulus undershoot is a passive vascular phenomenon.
Assuntos
Hemoglobinas/metabolismo , Hipertensão Intracraniana/fisiopatologia , Oxigênio/metabolismo , Percepção/fisiologia , Córtex Somatossensorial/fisiopatologia , Anestesia , Animais , Circulação Cerebrovascular/fisiologia , Estimulação Elétrica , Pé/fisiologia , Membro Anterior/fisiologia , Fluxometria por Laser-Doppler , Masculino , Óptica e Fotônica/métodos , Consumo de Oxigênio/fisiologia , Ratos , Ratos Wistar , Fluxo Sanguíneo Regional/fisiologia , Análise Espectral/métodosRESUMO
[This corrects the article DOI: 10.1371/journal.pone.0193961.].
RESUMO
Animal models of disease are an indispensable element in our quest to understand pathophysiology and develop novel therapies. Ex vivo studies have severe limitations, in particular their inability to study individual disease progression over time. In this respect, non-invasive in vivo technologies offer multiple advantages. We here used bilateral common carotid artery occlusion (BCCAO) in mice, an established model for ischemic retinopathy, and performed a multimodal in vivo and ex vivo follow-up. We used scanning laser ophthalmoscopy (SLO), ocular coherence tomography (OCT) and electroretinography (ERG) over 6 weeks followed by ex vivo analyses. BCCAO leads to vascular remodeling with thickening of veins starting at 4 weeks, loss of photoreceptor synapses with concomitant reduced b-waves in the ERG and thinning of the retina. Mononuclear phagocytes showed fluctuation of activity over time. There was large inter-individual variation in the severity of neuronal degeneration and cellular inflammatory responses. Ex vivo analysis confirmed these variable features of vascular remodeling, neurodegeneration and inflammation. In summary, we conclude that multimodal follow-up and subgroup analysis of retinal changes in BCCAO further calls into question the use of ex vivo studies with distinct single end-points. We propose that our approach can foster the understanding of retinal disease as well as the clinical translation of emerging therapeutic strategies.
Assuntos
Arteriopatias Oclusivas/patologia , Doenças das Artérias Carótidas/patologia , Retina/patologia , Vasos Retinianos/patologia , Animais , Artéria Carótida Primitiva/patologia , Modelos Animais de Doenças , Progressão da Doença , Isquemia/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Oftalmoscopia/métodos , Degeneração Retiniana/patologia , Tomografia de Coerência Óptica/métodosRESUMO
Folates are B-vitamins that are vital for normal brain function. Deficiencies in folates either genetic (methylenetetrahydrofolate reductase, MTHFR) or dietary intake of folic acid result in elevated levels of homocysteine. Clinical studies have shown that elevated levels of homocysteine (Hcy) may be associated with the development of dementia, however this link remains unclear. The purpose of this study was to evaluate the impact of increased Hcy levels on a mouse model of vascular cognitive impairment (VCI) produced by chronic hypoperfusion. Male and female Mthfr+/+ and Mthfr+/- mice were placed on either control (CD) or folic acid deficient (FADD) diets after which all animals underwent microcoil implantation around each common carotid artery or a sham procedure. Post-operatively animals were tested on the Morris water maze (MWM), y-maze, and rotarod. Animals had no motor impairments on the rotarod, y-maze, and could learn the location of the platform on the MWM. However, on day 8 of testing of MWM testing during the probe trial, Mthfr+/- FADD microcoil mice spent significantly less time in the target quadrant when compared to Mthfr+/- CD sham mice, suggesting impaired reference memory. All FADD mice had elevated levels of plasma homocysteine. MRI analysis revealed arterial remodeling was present in Mthfr+/- microcoil mice not Mthfr+/+ mice. Acetylcholine and related metabolites were reduced in cortical tissue because of microcoil implantation and elevated levels of homocysteine. Deficiencies in folate metabolism resulting in increased Hcy levels yield a metabolic profile that increases susceptibility to neurodegeneration in a mouse model of VCI.
Assuntos
Acetilcolina/metabolismo , Córtex Cerebral/metabolismo , Demência Vascular/metabolismo , Homocisteína/metabolismo , Memória/fisiologia , Animais , Córtex Cerebral/irrigação sanguínea , Córtex Cerebral/diagnóstico por imagem , Demência Vascular/diagnóstico por imagem , Modelos Animais de Doenças , Feminino , Deficiência de Ácido Fólico/diagnóstico por imagem , Deficiência de Ácido Fólico/metabolismo , Deficiência de Ácido Fólico/psicologia , Masculino , Metilenotetra-Hidrofolato Redutase (NADPH2)/deficiência , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Atividade Motora/fisiologia , Neovascularização Patológica/diagnóstico por imagem , Neovascularização Patológica/metabolismo , Neovascularização Patológica/psicologia , Distribuição AleatóriaRESUMO
Alterations in density and morphology of the cerebral microvasculature have been reported to occur in Alzheimer's disease patients and animal models of the disease. In this study we compared magnetic resonance imaging (MRI) techniques for their utility to detect age-dependent changes of the cerebral vasculature in the arcAß mouse model of cerebral amyloidosis. Dynamic susceptibility contrast (DSC)-MRI was performed by tracking the passage of a superparamagnetic iron oxide nanoparticle in the brain with dynamic gradient echo planar imaging (EPI). From this measurements relative cerebral blood volume [rCBV(DSC)] and relative cerebral blood flow (rCBF) were estimated. For the same animal maps of the relaxation shift index Q were computed from high resolution gradient echo and spin echo data that were acquired before and after superparamagnetic iron oxide (SPIO) nanoparticle injection. Q-values were used to derive estimates of microvessel density. The change in the relaxation rates [Formula: see text] obtained from pre- and post-contrast gradient echo data was used for the alternative determination of rCBV [rCBV([Formula: see text])]. Linear mixed effects modeling found no significant association between rCBV(DSC), rCBV([Formula: see text]), rCBF, and Q with genotype in 13-month old mice [compared to age-matched non-transgenic littermates (NTLs)] for any of the evaluated brain regions. In 24-month old mice there was a significant association for rCBV(DSC) with genotype in the cerebral cortex, and for rCBV([Formula: see text]) in the cerebral cortex and cerebellum. For rCBF there was a significant association in the cerebellum but not in other brain regions. Q-values in the olfactory bulb, cerebral cortex, striatum, hippocampus, and cerebellum in 24-month old mice were significantly associated with genotype. In those regions Q-values were reduced between 11 and 26% in arcAß mice compared to age-matched NTLs. Vessel staining with CD31 immunohistochemistry confirmed a reduction of microvessel density in the old arcAß mice. We further demonstrated a region-specific association between parenchymal and vascular deposition of ß-amyloid and decreased vascular density, without a correlation with the amount of Aß deposition. We found that Q mapping was more suitable than the hemodynamic read-outs to detect amyloid-related degeneration of the cerebral microvasculature.
RESUMO
Dietary deficiencies in folic acid result in elevated levels of plasma homocysteine, which has been associated with the development of dementia and other neurodegenerative disorders. Previously, we have shown that elevated levels of plasma homocysteine in mice deficient for a DNA repair enzyme, uracil-DNA glycosylase (UNG), result in neurodegeneration. The goal of this study was to evaluate how deficiencies in folic acid and UNG along with elevated levels of homocysteine affect vascular cognitive impairment, via chronic hypoperfursion in an animal model. Ung(+/+) and Ung(-/-) mice were placed on either control (CD) or folic acid deficient (FADD) diets. Six weeks later, the mice either underwent implantation of microcoils around both common carotid arteries. Post-operatively, behavioral tests began at 3-weeks, angiography was measured after 5-weeks using MRI to assess vasculature and at completion of study plasma and brain tissue was collected for analysis. Learning impairments in the Morris water maze (MWM) were observed only in hypoperfused Ung(-/-) FADD mice and these mice had significantly higher plasma homocysteine concentrations. Interestingly, Ung(+/+) FADD produced significant remodeling of the basilar artery and arterial vasculature. Increased expression of GFAP was observed in the dentate gyrus of Ung(-/-) hypoperfused and FADD sham mice. Chronic hypoperfusion resulted in increased cortical MMP-9 protein levels of FADD hypoperfused mice regardless of genotypes. These results suggest that elevated levels of homocysteine only, as a result of dietary folic acid deficiency, don't lead to memory impairments and neurobiochemical changes. Rather a combination of either chronic hypoperfusion or UNG deficiency is required.
Assuntos
Transtornos Cerebrovasculares/fisiopatologia , Transtornos Cognitivos/fisiopatologia , Deficiência de Ácido Fólico/fisiopatologia , Homocisteína/sangue , Deficiências da Aprendizagem/fisiopatologia , Uracila-DNA Glicosidase/deficiência , Animais , Artéria Basilar/patologia , Artéria Basilar/fisiopatologia , Encéfalo/irrigação sanguínea , Encéfalo/patologia , Encéfalo/fisiopatologia , Doenças das Artérias Carótidas , Transtornos Cerebrovasculares/patologia , Doença Crônica , Transtornos Cognitivos/patologia , Dieta , Modelos Animais de Doenças , Feminino , Deficiência de Ácido Fólico/patologia , Proteína Glial Fibrilar Ácida , Gliose/patologia , Gliose/fisiopatologia , Deficiências da Aprendizagem/patologia , Masculino , Metaloproteinase 9 da Matriz/metabolismo , Aprendizagem em Labirinto/fisiologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/metabolismo , Distribuição Aleatória , Uracila-DNA Glicosidase/genéticaRESUMO
Our aims were to assess the spatiotemporal development of brain pathology in a mouse model of chronic hypoperfusion using magnetic resonance imaging (MRI), and to test whether the renin-angiotensin system (RAS) can offer therapeutic benefit. For the first time, different patterns of cerebral blood flow alterations were observed in hypoperfused mice that ranged from an immediate and dramatic to a delayed decrease in cerebral perfusion. Diffusion tensor imaging revealed increases in several quantitative parameters in different brain regions that are indicative of white-matter degeneration; this began around 3 weeks after induction of hypoperfusion. While this model may be more variable than previously reported, neuroimaging tools represent a promising way to identify surrogate markers of pathology. Vascular remodelling was observed in hypoperfused mice, particularly in the anterior part of the Circle of Willis. While the angiotensin II receptor type 2 agonist, Compound 21 (C21), did not influence this response, it did promote expansion of the basilar artery in microcoil animals. Furthermore, C21-treated animals exhibited increased brain lymphocyte infiltration, and importantly, C21 had opposing effects on spatial reference memory in hypoperfused and sham mice. These results suggest that the RAS may have a role in vascular cognitive impairment.
Assuntos
Encéfalo/patologia , Circulação Cerebrovascular/fisiologia , Demência Vascular/patologia , Receptor Tipo 2 de Angiotensina/metabolismo , Bloqueadores do Receptor Tipo 2 de Angiotensina II/farmacologia , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Circulação Cerebrovascular/efeitos dos fármacos , Demência Vascular/metabolismo , Modelos Animais de Doenças , Citometria de Fluxo , Processamento de Imagem Assistida por Computador , Imageamento por Ressonância Magnética , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Prefrontocortical dopamine (DA) plays an essential role in the regulation of cognitive functions and behavior. The orbitofrontal cortex (OFC) receives a dopaminergic projection from the ventral tegmental area and is particularly important for goal-directed appetitive behaviors and for the neural representation of reward value. We here examined the effects of DA receptor blockers locally infused into the OFC, on instrumental behavior under a progressive schedule of reinforcement. After continuous reinforcement training (lever pressing for casein pellets) rats received bilateral intra-OFC-infusions of the DA D1-receptor antagonist SCH23390 (3 microg/0.5 microl), the DA D2-receptor antagonist sulpiride (3 microg/0.5 microl), or phosphate buffered saline through chronically indwelling cannulae. Immediately after infusion they were tested under a time-constrained progressive ratio schedule of reinforcement (3, 6, 9, 12, ... lever presses for 1 casein pellet within 180 s). Both SCH23390 and sulpiride led to a significant reduction of the break point (cessation to respond to the increasing criterion of instrumental effort) compared to vehicle infusions. A food preference test revealed no drug effects on the amount of consumed pellets and on the preference of casein pellets over laboratory chow. Leftward shifts of the break point in progressive ratio tasks indicate a disturbance of the mechanisms that translate motivation into appetitive behavior under conditions of increasing instrumental effort. Therefore, our data indicate that orbitofrontal dopamine is necessary for reward-related instrumental behavior.
Assuntos
Condicionamento Operante/fisiologia , Dopamina/fisiologia , Lobo Frontal/fisiologia , Reforço Psicológico , Análise de Variância , Animais , Comportamento Animal , Benzazepinas/farmacologia , Condicionamento Operante/efeitos dos fármacos , Antagonistas de Dopamina/farmacologia , Lobo Frontal/efeitos dos fármacos , Masculino , Ratos , Ratos Wistar , Esquema de Reforço , Sulpirida/farmacologia , Fatores de TempoRESUMO
Astrocytic network alterations have been reported in Alzheimer's disease (AD), but the underlying pathways have remained undefined. Here we measure astrocytic calcium, cerebral blood flow and amyloid-ß plaques in vivo in a mouse model of AD using multiphoton microscopy. We find that astrocytic hyperactivity, consisting of single-cell transients and calcium waves, is most pronounced in reactive astrogliosis around plaques and is sometimes associated with local blood flow changes. We show that astroglial hyperactivity is reduced after P2 purinoreceptor blockade or nucleotide release through connexin hemichannels, but is augmented by increasing cortical ADP concentration. P2X receptor blockade has no effect, but inhibition of P2Y1 receptors, which are strongly expressed by reactive astrocytes surrounding plaques, completely normalizes astrocytic hyperactivity. Our data suggest that astroglial network dysfunction is mediated by purinergic signalling in reactive astrocytes, and that intervention aimed at P2Y1 receptors or hemichannel-mediated nucleotide release may help ameliorate network dysfunction in AD.
Assuntos
Doença de Alzheimer/metabolismo , Astrócitos/metabolismo , Placa Amiloide/metabolismo , Receptores Purinérgicos P2Y1/metabolismo , Trifosfato de Adenosina/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animais , Sinalização do Cálcio , Circulação Cerebrovascular , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Transgênicos , Microscopia de Fluorescência por Excitação Multifotônica , Transdução de SinaisRESUMO
Intravenous administration of iron oxide nanoparticles during the acute stage of experimental stroke can produce signal intensity changes in the ischemic region. This has been attributed, albeit controversially, to the infiltration of iron-laden blood-borne macrophages. The properties of nanoparticles that render them most suitable for phagocytosis is a matter of debate, as is the most relevant timepoint for administration. Both of these questions are examined in the present study. Imaging experiments were performed in mice with 30 minutes of middle cerebral artery occlusion (MCAO). Iron oxide nanoparticles with different charges and sizes were used, and mice received 300 µmol Fe/kg intravenously: either superparamagnetic iron oxide nanoparticles (SPIOs), ultrasmall SPIOs, or very small SPIOs. The particles were administered 7 days before MCAO, at the time of reperfusion, or 72 hours after MCAO. Interestingly, there was no observable signal change in the ischemic brains that could be attributed to iron. Furthermore, no Prussian blue-positive cells were found in the brains or blood leukocytes, despite intense staining in the livers and spleens. This implies that the nanoparticles selected for this study are not phagocytosed by blood-borne leukocytes and do not enter the ischemic mouse brain.
Assuntos
Encéfalo/irrigação sanguínea , Encéfalo/patologia , Meios de Contraste , Infarto da Artéria Cerebral Média/patologia , Imageamento por Ressonância Magnética/métodos , Nanopartículas de Magnetita , Animais , Meios de Contraste/química , Leucócitos/citologia , Leucócitos/patologia , Nanopartículas de Magnetita/química , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Tamanho da Partícula , FagocitoseRESUMO
Cerebral blood flow (CBF) quantification is a valuable tool in stroke research. Mice are of special interest because of the potential of genetic engineering. Magnetic resonance imaging (MRI) provides repetitive, noninvasive CBF quantification. Many MRI techniques require the knowledge of the brain-blood partition coefficient (BBPC) for water. Adopting an MRI protocol described by Roberts et al (1996) in humans, we determined the BBPC for water in 129S6/SvEv mice from proton density measurements of brain and blood, calibrated with deuterium oxide/water phantoms. The average BBPC for water was 0.89 ± 0.03 mL/g, with little regional variation within the mouse brain.
Assuntos
Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Circulação Cerebrovascular , Imageamento por Ressonância Magnética , Água/metabolismo , Animais , Calibragem , Óxido de Deutério/metabolismo , Feminino , Masculino , Camundongos , Camundongos Endogâmicos , Imagens de Fantasmas , PrótonsRESUMO
Assessing neuronal activity by non-invasive functional brain imaging techniques which are based on the hemodynamic response depends totally on the physiological cascade of metabolism and blood flow. At present, functional brain imaging with near infrared spectroscopy (NIRS) or BOLD-fMRI is widely used in cognitive neuroscience in healthy subjects where neurovascular coupling and cerebrovascular reactivity can be assumed to be intact. Local activation studies as well as studies investigating functional connectivity between brain regions of the resting brain provide a rapidly increasing body of knowledge on brain function in humans and animals. Furthermore, functional NIRS and MRI techniques are increasingly being used in patients with severe brain diseases and this use might gain more and more importance for establishing their use in the clinical routine. However, more and more experimental evidence shows that changes in baseline physiological parameters, pharmacological interventions, or disease-related vascular changes may significantly alter the normal response of blood flow and blood oxygenation and thus may lead to misinterpretation of neuronal activity. In this article we present examples of recent experimental findings on pathophysiological changes of neurovascular coupling parameters in animals and discuss their potential implications for functional imaging based on hemodynamic signals such as fNIRS or BOLD-fMRI. To enable correct interpretation of neuronal activity by vascular signals, future research needs to deepen our understanding of the basic mechanisms of neurovascular coupling and the specific characteristics of disturbed neurovascular coupling in the diseased brain.
RESUMO
Neurovascular coupling provides the basis for many functional neuroimaging techniques. Nitric oxide (NO), adenosine, cyclooxygenase, CYP450 epoxygenase, and potassium are involved in dilating arterioles during neuronal activation. We combined inhibition of NO synthase, cyclooxygenase, adenosine receptors, CYP450 epoxygenase, and inward rectifier potassium (Kir) channels to test whether these pathways could explain the blood flow response to neuronal activation. Cerebral blood flow (CBF) and cerebral metabolic rate of oxygen (CMRO(2)) of the somatosensory cortex were measured during forepaw stimulation in 24 rats using a laser Doppler/spectroscopy probe through a cranial window. Combined inhibition reduced CBF responses by two-thirds, somatosensory evoked potentials and activation-induced CMRO(2) increases remained unchanged, and deoxy-hemoglobin (deoxy-Hb) response was abrogated. This shows that in the rat somatosensory cortex, one-third of the physiological blood flow increase is sufficient to prevent microcirculatory increase of deoxy-Hb concentration during neuronal activity. The large physiological CBF response is not necessary to support small changes in CMRO(2). We speculate that the CBF response safeguards substrate delivery during functional activation with a considerable 'safety factor'. Reduction of the CBF response in pathological states may abolish the BOLD-fMRI signal, without affecting underlying neuronal activity.
Assuntos
Circulação Cerebrovascular/fisiologia , Transdução de Sinais/fisiologia , Córtex Somatossensorial/irrigação sanguínea , Córtex Somatossensorial/metabolismo , Animais , Circulação Cerebrovascular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Potenciais Somatossensoriais Evocados/efeitos dos fármacos , Potenciais Somatossensoriais Evocados/fisiologia , Masculino , Canais de Potássio Corretores do Fluxo de Internalização/antagonistas & inibidores , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Córtex Somatossensorial/efeitos dos fármacosRESUMO
Recently, a universal, simple, and fail-safe mechanism has been proposed by which cerebral blood flow (CBF) might be coupled to oxygen metabolism during neuronal activation without the need for any tissue-based mechanism. According to this concept, vasodilation occurs by local erythrocytic release of nitric oxide or ATP wherever and whenever hemoglobin is deoxygenated, directly matching oxygen demand and supply in every tissue. For neurovascular coupling in the brain, we present experimental evidence challenging this view by applying an experimental regime operating without deoxy-hemoglobin. Hyperbaric hyperoxygenation (HBO) allowed us to prevent hemoglobin deoxygenation, as the oxygen that was physically dissolved in the tissue was sufficient to support oxidative metabolism. Regional CBF and regional cerebral blood oxygenation were measured using a cranial window preparation in anesthetized rats. Hemodynamic and neuronal responses to electrical forepaw stimulation or cortical spreading depression (CSD) were analyzed under normobaric normoxia and during HBO up to 4 ATA (standard atmospheres absolute). Inconsistent with the proposed mechanism, during HBO, CBF responses to functional activation or CSD were unchanged. Our results show that activation-induced CBF regulation in the brain does not operate through the release of vasoactive mediators on hemoglobin deoxygenation or through a tissue-based oxygen-sensing mechanism.
Assuntos
Encéfalo/metabolismo , Circulação Cerebrovascular/fisiologia , Hemoglobinas/metabolismo , Neurônios/metabolismo , Oxigênio/metabolismo , Fluxo Sanguíneo Regional/fisiologia , Animais , Astrócitos/metabolismo , Depressão Alastrante da Atividade Elétrica Cortical/fisiologia , Hemodinâmica , Oxigenoterapia Hiperbárica , Masculino , Neurônios/citologia , Óxido Nítrico/metabolismo , Consumo de Oxigênio/fisiologia , Ratos , Ratos Wistar , Vasoconstrição/fisiologiaRESUMO
Neuronal activation is accompanied by a local increase in cerebral blood flow (CBF) and in cerebral metabolic rate of oxygen (CMRO(2)), caused by neurovascular and neurometabolic coupling. Hypothermia is used as a neuroprotective approach in surgical patients and therapeutically after cardiac arrest or stroke. The effect of hypothermia on neurovascular coupling is of interest for evaluating brain function in these patients, but has not been determined so far. It is not clear whether functional hyperaemia actually operates at subnormal temperatures. In addition, decreasing brain temperature reduces spontaneous CMRO(2) following a known quantitative relationship (Q(10)). Q(10) determination may serve to validate a recently introduced CMRO(2) measurement approach relying on optical measurements of CBF and hemoglobin concentration. We applied this method to investigate hypothermia in a functional study of the somatosensory cortex. Anesthetized Wistar rats underwent surgical implantation of a closed cranial window. Using laser Doppler flowmetry and optical spectroscopy, relative changes in CBF and hemoglobin concentration were measured continuously. At the same time, an electroencephalogram (EEG) was recorded from the measurement site. By the application of ice packs, whole-body hypothermia was induced, followed by rewarming. Spontaneous EEG, CBF and CMRO(2) were measured, interleaved by blocks of electrical forepaw stimulation. The Q(10) obtained from spontaneous CMRO(2) changes of 4.4 (95% confidence interval 3.7-5.1) was close to published values, indicating the reliability of the CMRO(2) measurement. Lowering brain temperature decreased functional changes of CBF and CMRO(2) as well as amplitudes of somatosensory evoked potentials (SEP) to the same degree. In conclusion, neurovascular and neurometabolic coupling is preserved during hypothermia.