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Infection during the perinatal period can adversely affect brain development, predispose infants to ischemic stroke and have lifelong consequences. We previously demonstrated that diet enriched in n-3 polyunsaturated fatty acids (n-3 PUFA) transforms brain lipid composition in the offspring and protects the neonatal brain from stroke, in part by blunting injurious immune responses. Critical to the interface between the brain and systemic circulation is the vasculature, endothelial cells in particular, that support brain homeostasis and provide a barrier to systemic infection. Here, we examined whether maternal PUFA-enriched diets exert reprograming of endothelial cell signalling in postnatal day 9 mice after modeling aspects of infection using LPS. Transcriptome analysis was performed on microvessels isolated from brains of pups from dams maintained on 3 different maternal diets from gestation day 1: standard, n-3 enriched or n-6 enriched diets. Depending on the diet, in endothelial cells LPS produced distinct regulation of pathways related to immune response, cell cycle, extracellular matrix, and angiogenesis. N-3 PUFA diet enabled higher immune reactivity in brain vasculature, while preventing imbalance of cell cycle regulation and extracellular matrix cascades that accompanied inflammatory response in standard diet. Cytokine analysis revealed a blunted LPS response in blood and brain of offspring from dams on n-3 enriched diet. Analysis of cerebral vasculature in offspring in vivo revealed no differences in vessel density. However, vessel complexity was decreased in response to LPS at 72 h in standard and n-6 diets. Thus, LPS modulates specific transcriptomic changes in brain vessels of offspring rather than major structural vessel characteristics during early life. N-3 PUFA-enriched maternal diet in part prevents an imbalance in homeostatic processes, alters inflammation and ultimately mitigates changes to the complexity of surface vessel networks that result from infection. Importantly, maternal diet may presage offspring neurovascular outcomes later in life.
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Animais Recém-Nascidos , Ácidos Graxos Ômega-3 , Transcriptoma , Animais , Camundongos , Ácidos Graxos Ômega-3/administração & dosagem , Feminino , Gravidez , Lipopolissacarídeos/toxicidade , Camundongos Endogâmicos C57BL , Efeitos Tardios da Exposição Pré-Natal/metabolismo , Efeitos Tardios da Exposição Pré-Natal/patologia , Inflamação/metabolismo , Inflamação/patologia , Encéfalo/metabolismo , Encéfalo/patologia , Endotoxinas/toxicidadeRESUMO
Human space exploration expansion from Low-Earth Orbit to deep space is accelerating the need to monitor and address the known health concerns related to deep space radiation. The human musculoskeletal system is vulnerable to these risks (alongside microgravity) and its health reflects the well-being of other body systems. Multiparametric magnetic resonance imaging (MRI) is an important approach for assessing temporal physiological changes in the musculoskeletal system. We propose that ultra-low-field MRI provides an optimal low Size Weight and Power (SwaP) solution for non-invasively monitoring muscle and bone changes on the planned Gateway lunar space station. Our proposed ultra-low-field Gateway MRI meets low SWaP design specifications mandated by limited room in the lunar space station. This review summarizes the current state of our knowledge on musculoskeletal consequences of spaceflight, especially with respect to radiation, and then elaborates how MRI can be used to monitor the deleterious effects of space travel and the efficacy of putative countermeasures. We argue that an ultra-low-field MRI in cis-lunar space on the Gateway can provide valuable research and medical insights into the effects of deep space radiation exposure on astronauts. Such an MRI would also allow the development of imaging protocols that would facilitate Earth-bound teams to monitor space personnel musculoskeletal changes during future interplanetary spaceflight. It will especially have a role in monitoring countermeasures, such as the use of melanin, in protecting space explorers.
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Imageamento por Ressonância Magnética , Voo Espacial , Humanos , Imageamento por Ressonância Magnética/métodos , Sistema Musculoesquelético/diagnóstico por imagem , Sistema Musculoesquelético/efeitos da radiação , Astronautas , Ausência de Peso , Radiação Cósmica/efeitos adversosRESUMO
BACKGROUND: Traumatic brain injury (TBI) is a significant risk factor for Alzheimer's disease (AD), and accumulating evidence supports a role for adaptive immune B and T cells in both TBI and AD pathogenesis. We previously identified B cell and major histocompatibility complex class II (MHCII)-associated invariant chain peptide (CLIP)-positive B cell expansion after TBI. We also showed that antagonizing CLIP binding to the antigen presenting groove of MHCII after TBI acutely reduced CLIP + splenic B cells and was neuroprotective. The current study investigated the chronic effects of antagonizing CLIP in the 5xFAD Alzheimer's mouse model, with and without TBI. METHODS: 12-week-old male wild type (WT) and 5xFAD mice were administered either CLIP antagonist peptide (CAP) or vehicle, once at 30 min after either sham or a lateral fluid percussion injury (FPI). Analyses included flow cytometric analysis of immune cells in dural meninges and spleen, histopathological analysis of the brain, magnetic resonance diffusion tensor imaging, cerebrovascular analysis, and assessment of motor and neurobehavioral function over the ensuing 6 months. RESULTS: 9-month-old 5xFAD mice had significantly more CLIP + B cells in the meninges compared to age-matched WT mice. A one-time treatment with CAP significantly reduced this population in 5xFAD mice. Importantly, CAP also improved some of the immune, histopathological, and neurobehavioral impairments in 5xFAD mice over the ensuing six months. Although FPI did not further elevate meningeal CLIP + B cells, it did negate the ability of CAP to reduce meningeal CLIP + B cells in the 5xFAD mice. FPI at 3 months of age exacerbated some aspects of AD pathology in 5xFAD mice, including further reducing hippocampal neurogenesis, increasing plaque deposition in CA3, altering microgliosis, and disrupting the cerebrovascular structure. CAP treatment after injury ameliorated some but not all of these FPI effects.
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Antígenos de Diferenciação de Linfócitos B , Linfócitos B , Lesões Encefálicas Traumáticas , Antígenos de Histocompatibilidade Classe II , Camundongos Transgênicos , Animais , Camundongos , Masculino , Lesões Encefálicas Traumáticas/patologia , Lesões Encefálicas Traumáticas/tratamento farmacológico , Antígenos de Histocompatibilidade Classe II/metabolismo , Linfócitos B/efeitos dos fármacos , Meninges/patologia , Meninges/efeitos dos fármacos , Precursor de Proteína beta-Amiloide/genética , Doença de Alzheimer/patologia , Doença de Alzheimer/tratamento farmacológico , Humanos , Modelos Animais de Doenças , Presenilina-1/genética , Camundongos Endogâmicos C57BLRESUMO
Infection during perinatal period can adversely affect brain development, predispose infants to ischemic stroke and have lifelong consequences. We previously demonstrated that diet enriched in n-3 polyunsaturated fatty acids (PUFA) transforms brain lipid composition and protects from neonatal stroke. Vasculature is a critical interface between blood and brain providing a barrier to systemic infection. Here we examined whether maternal PUFA-enriched diets exert reprograming of endothelial cell signalling in 9-day old mice after endotoxin (LPS)-induced infection. Transcriptome analysis was performed on brain microvessels from pups born to dams maintained on 3 diets: standard, n-3 or n-6 enriched. N-3 diet enabled higher immune reactivity in brain vasculature, while preventing imbalance of cell cycle regulation and extracellular matrix cascades that accompanied inflammatory response in standard diet. LPS response in blood and brain was blunted in n-3 offspring. Cerebral angioarchitecture analysis revealed modified vessel complexity after LPS. Thus, n-3-enriched maternal diet partially prevents imbalance in homeostatic processes and alters inflammation rather than affects brain vascularization during early life. Importantly, maternal diet may presage offspring neurovascular outcomes later in life.
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Introduction: The 5xFAD mouse is a popular model of familial Alzheimer's disease (AD) that is characterized by early beta-amyloid (Aß) deposition and cognitive decrements. Despite numerous studies, the 5xFAD mouse has not been comprehensively phenotyped for vascular and metabolic perturbations over its lifespan. Methods: Male and female 5xFAD and wild type (WT) littermates underwent in vivo 18F-fluorodeoxyglucose (FDG) positron emission tomography (PET) imaging at 4, 6, and 12 months of age to assess regional glucose metabolism. A separate cohort of mice (4, 8, 12 months) underwent "vessel painting" which labels all cerebral vessels and were analyzed for vascular characteristics such as vessel density, junction density, vessel length, network complexity, number of collaterals, and vessel diameter. Results: With increasing age, vessels on the cortical surface in both 5xFAD and WT mice showed increased vessel length, vessel and junction densities. The number of collateral vessels between the middle cerebral artery (MCA) and the anterior and posterior cerebral arteries decreased with age but collateral diameters were significantly increased only in 5xFAD mice. MCA total vessel length and junction density were decreased in 5xFAD mice compared to WT at 4 months. Analysis of 18F-FDG cortical uptake revealed significant differences between WT and 5xFAD mice spanning 4-12 months. Broadly, 5xFAD males had significantly increased 18F-FDG uptake at 12 months compared to WT mice. In most cortical regions, female 5xFAD mice had reduced 18F-FDG uptake compared to WT across their lifespan. Discussion: While the 5xFAD mouse exhibits AD-like cognitive deficits as early as 4 months of age that are associated with increasing Aß deposition, we only found significant differences in cortical vascular features in males, not in females. Interestingly, 5xFAD male and female mice exhibited opposite effects in 18F-FDG uptake. The MCA supplies blood to large portions of the somatosensory cortex and portions of motor and visual cortex and increased vessel length alongside decreased collaterals which coincided with higher metabolic rates in 5xFAD mice. Thus, a potential mismatch between metabolic demand and vascular delivery of nutrients in the face of increasing Aß deposition could contribute to the progressive cognitive deficits seen in the 5xFAD mouse model.
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The amygdaloid complex, including the basolateral nucleus (BLA), contributes crucially to emotional and cognitive brain functions, and is a major target of research in both humans and rodents. However, delineating structural amygdala plasticity in both normal and disease-related contexts using neuroimaging has been hampered by the difficulty of unequivocally identifying the boundaries of the BLA. This challenge is a result of the poor contrast between BLA and the surrounding gray matter, including other amygdala nuclei. Here, we describe a novel diffusion tensor imaging (DTI) approach to enhance contrast, enabling the optimal identification of BLA in the rodent brain from magnetic resonance (MR) images. We employed this methodology together with a slice-shifting approach to accurately measure BLA volumes. We then validated the results by direct comparison to both histological and cellular-identity (parvalbumin)-based conventional techniques for defining BLA in the same brains used for MRI. We also confirmed BLA connectivity targets using DTI-based tractography. The novel approach enables the accurate and reliable delineation of BLA. Because this nucleus is involved in and changed by developmental, degenerative and adaptive processes, the instruments provided here should be highly useful to a broad range of neuroimaging studies. Finally, the principles used here are readily applicable to numerous brain regions and across species.
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Febrile status epilepticus (FSE) is an important risk factor for temporal lobe epilepsy and early identification of those at high risk for epilepsy is vital. In a rat model of FSE, we identified an acute (2 hrs) novel MRI signal where reduced T2 relaxation values in the basolateral amygdala (BLA) predicted epilepsy in adulthood; this T2 signal remains incompletely understood and we hypothesized that it may be influenced by vascular topology. Experimental FSE induced in rat pups reduced blood vessel density of the cortical vasculature in a lateralized manner at 2 hrs post FSE. Middle cerebral artery (MCA) exhibited abnormal topology in FSE pups but not in controls. In the BLA, significant vessel junction reductions and decreased vessel diameter were observed, together with a strong trend for reduced vessel length. Perfusion weighted MRI (PWI) was acutely increased cerebral blood flow (CBF) in cortex, amygdala and hippocampus of FSE pups that correlated to decreased T2 relaxation values compared to controls. This is consistent with increased levels of deoxyhemoglobin associated with increased metabolic demand. In summary, FSE acutely modifies vascular topological and CBF in cortex and BLA that may underlie acute MRI signal changes that predict progression to future epilepsy.
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Epilepsia , Estado Epiléptico , Animais , Ratos , Estado Epiléptico/diagnóstico por imagemRESUMO
Vascular dysfunction and structural abnormalities in Alzheimer's disease (AD) are known to contribute to the progression of the pathology, and studies have tended to ignore the role of the vasculature in AD progression. We utilized the 3xTg-AD mouse model of AD to examine individual cerebral vessels and the cortical vascular network across the lifespan. Our vessel painting approach was used to label the entire cortical vasculature, followed by epifluorescence microscopy. The middle cerebral artery (MCA) tree was assessed with confocal microscopy, and a new method was developed to assess branching patterns as a measure of aging-related changes. We found that vascular remodeling was profoundly altered at 4-6 months of age, when the 3xTg-AD mouse is known to transition to cognitive impairment and Aß deposition in both sexes. Analysis of vascular features (density, junctions, length) of the MCA territory highlighted sex-dependent differences across the 3xTg-AD mouse lifespan, with no alterations in branching patterns. Our current cerebrovascular angioarchitectural analyses demonstrate progressive alterations in individual cortical vessels, as well as in the vascular network of the cortex. These new findings advance our understanding of brain anatomy and physiology in the 3xTg-AD mouse, while potentially identifying unique diagnostic signatures of AD progression.
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Acquired brain injuries due to trauma damage the cortical vasculature, which in turn impairs blood flow to injured tissues. There are reports of vascular morphological recovery following traumatic brain injury, but the remodeling process has not been examined longitudinally in detail after injury in vivo. Understanding the dynamic processes that influence recovery is thus critically important. We evaluated the longitudinal and dynamic microvascular recovery and remodeling up to 2 months post injury using live brain miniscope and 2-photon microscopic imaging. The new imaging approaches captured dynamic morphological and functional recovery processes at high spatial and temporal resolution in vivo. Vessel painting documented the initial loss and subsequent temporal morphological vascular recovery at the injury site. Miniscopes were used to longitudinally image the temporal dynamics of vascular repair in vivo after brain injury in individual mice across each cohort. We observe near-immediate nascent growth of new vessels in and adjacent to the injury site that peaks between 14 and 21 days post injury. 2-photon microscopy confirms new vascular growth and further demonstrates differences between cortical layers after cortical injury: large vessels persist in deeper cortical layers (> 200 µm), while superficial layers exhibit a dense plexus of fine (and often non-perfused) vessels displaying regrowth. Functionally, blood flow increases mirror increasing vascular density. Filopodia development and endothelial sprouting is measurable within 3 days post injury that rapidly transforms regions devoid of vessels to dense vascular plexus in which new vessels become increasingly perfused. Within 7 days post injury, blood flow is observed in these nascent vessels. Behavioral analysis reveals improved vascular modulation after 9 days post injury, consistent with vascular regrowth. We conclude that morphological recovery events are closely linked to functional recovery of blood flow to the compromised tissues, which subsequently leads to improved behavioral outcomes.
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Lesões Encefálicas Traumáticas , Lesões Encefálicas , Animais , Encéfalo/irrigação sanguínea , Circulação Cerebrovascular/fisiologia , Humanos , Camundongos , Recuperação de Função FisiológicaRESUMO
Magnetic resonance imaging (MRI) and positron emission tomography (PET) have made great strides in the diagnosis and our understanding of Alzheimer's Disease (AD). Despite the knowledge gained from human studies, mouse models have and continue to play an important role in deciphering the cellular and molecular evolution of AD. MRI and PET are now being increasingly used to investigate neuroimaging features in mouse models and provide the basis for rapid translation to the clinical setting. Here, we provide an overview of the human MRI and PET imaging landscape as a prelude to an in-depth review of preclinical imaging in mice. A broad range of mouse models recapitulate certain aspects of the human AD, but no single model simulates the human disease spectrum. We focused on the two of the most popular mouse models, the 3xTg-AD and the 5xFAD models, and we summarized all known published MRI and PET imaging data, including contrasting findings. The goal of this review is to provide the reader with broad framework to guide future studies in existing and future mouse models of AD. We also highlight aspects of MRI and PET imaging that could be improved to increase rigor and reproducibility in future imaging studies.
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Chronic pain affects 1.5 billion people worldwide but remains woefully undertreated. Understanding the molecular events leading to its emergence is necessary to discover disease-modifying therapies. Here we show that N-acylethanolamine acid amidase (NAAA) is a critical control point in the progression to pain chronicity, which can be effectively targeted by small-molecule therapeutics that inhibit this enzyme. NAAA catalyzes the deactivating hydrolysis of palmitoylethanolamide, a lipid-derived agonist of the transcriptional regulator of cellular metabolism, peroxisome proliferator-activated receptor-α (PPAR-α). Our results show that disabling NAAA in spinal cord during a 72-h time window following peripheral tissue injury halts chronic pain development in male and female mice by triggering a PPAR-α-dependent reprogramming of local core metabolism from aerobic glycolysis, which is transiently enhanced after end-organ damage, to mitochondrial respiration. The results identify NAAA as a crucial control node in the transition to chronic pain and a molecular target for disease-modifying medicines.
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Stroke is among the top ten causes of death in children but has received disproportionally little attention. Cerebral arteriopathies account for up to 80% of childhood arterial ischemic stroke (CAIS) cases and are strongly predictive of CAIS recurrence and poorer outcomes. The underlying mechanisms of sensitization of neurovasculature by viral infection are undefined. In the first age-appropriate model for childhood arteriopathy-by administration of viral mimetic TLR3-agonist Polyinosinic:polycytidylic acid (Poly-IC) in juvenile mice-we identified a key role of the TLR3-neutrophil axis in disrupting the structural-functional integrity of the blood-brain barrier (BBB) and distorting the developing neurovascular architecture and vascular networks. First, using an array of in-vivo/post-vivo vascular imaging, genetic, enzymatic and pharmacological approaches, we report marked Poly-IC-mediated extravascular leakage of albumin (66kDa) and of a small molecule DiI (â¼934Da) and disrupted tight junctions. Poly-IC also enhanced the neuroinflammatory milieu, promoted neutrophil recruitment, profoundly upregulated neutrophil elastase (NE), and induced neutrophil extracellular trap formation (NETosis). Finally, we show that functional BBB disturbances, NETosis and neuroinflammation are markedly attenuated by pharmacological inhibition of NE (Sivelestat). Altogether, these data reveal NE/NETosis as a novel therapeutic target for viral-induced cerebral arteriopathies in children.
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Artérias Cerebrais/metabolismo , Armadilhas Extracelulares/metabolismo , Elastase de Leucócito , Poli I-C/efeitos adversos , Transdução de Sinais/efeitos dos fármacos , Acidente Vascular Cerebral , Animais , Barreira Hematoencefálica/metabolismo , Artérias Cerebrais/patologia , Criança , Armadilhas Extracelulares/genética , Humanos , Elastase de Leucócito/genética , Elastase de Leucócito/metabolismo , Camundongos , Camundongos Transgênicos , Poli I-C/farmacologia , Transdução de Sinais/genética , Acidente Vascular Cerebral/induzido quimicamente , Acidente Vascular Cerebral/genética , Acidente Vascular Cerebral/metabolismo , Junções Íntimas/genética , Junções Íntimas/metabolismo , Receptor 3 Toll-Like/agonistas , Receptor 3 Toll-Like/genética , Receptor 3 Toll-Like/metabolismoRESUMO
The present study explored the hypothesis that an adverse intrauterine environment caused by maternal undernutrition (MUN) acted through corticosteroid-dependent and -independent mechanisms to program lasting functional changes in the neonatal cerebrovasculature and vulnerability to mild hypoxic-ischemic (HI) injury. From day 10 of gestation until term, MUN and MUN-metyrapone (MUN-MET) group rats consumed a diet restricted to 50% of calories consumed by a pair-fed control; and on gestational day 11 through term, MUN-MET groups received drinking water containing MET (0.5 mg/mL), a corticosteroid synthesis inhibitor. P9/P10 pups underwent unilateral carotid ligation followed 24 h later by 1.5 h exposure to 8% oxygen (HI treatment). An ELISA quantified MUN-, MET-, and HI-induced changes in circulating levels of corticosterone. In P11/P12 pups, MUN programming promoted contractile differentiation in cerebrovascular smooth muscle as determined by confocal microscopy, modulated calcium-dependent contractility as revealed by cerebral artery myography, enhanced vasogenic edema formation as indicated by T2 MRI, and worsened neurobehavior MUN unmasked HI-induced improvements in open-field locomotion and in edema resolution, alterations in calcium-dependent contractility and promotion of contractile differentiation. Overall, MUN imposed multiple interdependent effects on cerebrovascular smooth muscle differentiation, contractility, edema formation, flow-metabolism coupling and neurobehavior through pathways that both required, and were independent of, gestational corticosteroids. In light of growing global patterns of food insecurity, the present study emphasizes that infants born from undernourished mothers may experience greater risk for developing neonatal cerebral edema and sensorimotor impairments possibly through programmed changes in neonatal cerebrovascular function.
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Córtex Cerebral/irrigação sanguínea , Corticosterona/metabolismo , Transtornos da Nutrição Fetal/etiologia , Hipóxia-Isquemia Encefálica/etiologia , Hipóxia-Isquemia Encefálica/metabolismo , Exposição Materna/efeitos adversos , Efeitos Tardios da Exposição Pré-Natal , Animais , Biomarcadores , Corticosterona/sangue , Modelos Animais de Doenças , Suscetibilidade a Doenças , Feminino , Hipóxia-Isquemia Encefálica/diagnóstico por imagem , Hipóxia-Isquemia Encefálica/patologia , Imageamento por Ressonância Magnética , Microscopia Confocal , Gravidez , RatosRESUMO
The DNA vaccine, AV-1959D, targeting N-terminal epitope of Aß peptide, has been proven immunogenic in mice, rabbits, and non-human primates, while its therapeutic efficacy has been shown in mouse models of Alzheimer's disease (AD). Here we report for the first time on IND-enabling biodistribution and safety/toxicology studies of cGMP-grade AV-1959D vaccine in the Tg2576 mouse model of AD. We also tested acute neuropathology safety profiles of AV-1959D in another AD disease model, Tg-SwDI mice with established vascular and parenchymal Aß pathology in a pre-clinical translational study. Biodistribution studies two days after the injection demonstrated high copy numbers of AV-1959D plasmid after single immunization of Tg2576 mice at the injection sites but not in the tissues of distant organs. Plasmids persisted at the injection sites of some mice 60 days after vaccination. In Tg2576 mice with established amyloid pathology, we did not observe short- or long-term toxicities after multiple immunizations with three doses of AV-1959D. Assessment of the repeated dose acute safety of AV-1959D in cerebral amyloid angiopathy (CAA) prone Tg-SwDI mice did not reveal any immunotherapy-induced vasogenic edema detected by magnetic resonance imaging (MRI) or increased microhemorrhages. Multiple immunizations of Tg-SwDI mice with AV-1959D did not induce T and B cell infiltration, glial activation, vascular deposition of Aß, or neuronal degeneration (necrosis and apoptosis) greater than that in the control group determined by immunohistochemistry of brain tissues. Taken together, the safety data from two different mouse models of AD substantiate a favorable safety profile of the cGMP grade AV-1959D vaccine supporting its progression to first-in-human clinical trials.
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Vacinas contra Alzheimer/imunologia , Vacinas de DNA/imunologia , Adjuvantes Imunológicos , Doença de Alzheimer/imunologia , Doença de Alzheimer/prevenção & controle , Peptídeos beta-Amiloides/metabolismo , Animais , Formação de Anticorpos , Angiopatia Amiloide Cerebral/imunologia , Ensaios Clínicos como Assunto , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Transgênicos , Fragmentos de Peptídeos/metabolismoRESUMO
Intranasal recombinant osteopontin (OPN) has been shown to be neuroprotective in different models of acquired brain injury but has never been tested after traumatic brain injury (TBI). We used a model of moderate-to-severe controlled cortical impact in male adult Sprague Dawley rats and tested our hypothesis that OPN treatment would improve neurological outcomes, lesion and brain tissue characteristics, neuroinflammation, and vascular characteristics at 1 day post-injury. Intranasal OPN administered 1 hr after the TBI did not improve neurological score, lesion volumes, blood-brain barrier, or vascular characteristics. When assessing neuroinflammation, we did not observe any effect of OPN on the astrocyte reactivity but discovered an increased number of activated microglia within the ipsilateral hemisphere. Moreover, we found a correlation between edema and heme oxygenase-1 (HO-1) expression which was decreased in OPN-treated animals, suggesting an effect of OPN on the HO-1 response to injury. Thus, OPN may increase or accelerate the microglial response after TBI, and early response of HO-1 in modulating edema formation may limit the secondary consequences of TBI at later time points. Additional experiments and at longer time points are needed to determine if intranasal OPN could potentially be used as a treatment after TBI where it might be beneficial by activating protective signaling pathways.
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Edema Encefálico/tratamento farmacológico , Lesões Encefálicas Traumáticas/tratamento farmacológico , Encéfalo/efeitos dos fármacos , Microglia/efeitos dos fármacos , Fármacos Neuroprotetores/administração & dosagem , Osteopontina/administração & dosagem , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/patologia , Encéfalo/metabolismo , Encéfalo/patologia , Edema Encefálico/metabolismo , Edema Encefálico/patologia , Lesões Encefálicas Traumáticas/metabolismo , Lesões Encefálicas Traumáticas/patologia , Modelos Animais de Doenças , Masculino , Microglia/metabolismo , Microglia/patologia , Fármacos Neuroprotetores/uso terapêutico , Osteopontina/uso terapêutico , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacosRESUMO
Stroke is among the top 10 causes of death in children. The developmental stage of the brain is central to stroke pathophysiology. The incidence of childhood arterial ischemic stroke (CAIS) is lower than of perinatal arterial ischemic stroke but the rate of recurrence is strikingly high. Vascular inflammation is seen as major contributor to CAIS but the mechanisms that govern structural-functional basis of vascular abnormalities remain poorly understood. To identify the contribution of immune-neurovascular interactions to CAIS, we established stroke model in postnatal day 21 (P21) mice. We demonstrate acute functional deficits and histological injury and chronic MRI-identifiable injury, brain atrophy and marked derangements in the vascular network. In contrast to negligible albumin leakage and neutrophil infiltration following acute perinatal stroke, CAIS leads to significantly increased albumin leakage and neutrophil infiltration in injured regions of wild type mice and mice with functional CX3CR1-CCR2 receptors. In mice with dysfunctional CX3CR1-CCR2 signaling, extravascular albumin leakage is significantly attenuated, infiltration of injurious Ccr2+-monocytes essentially aborted, accumulation of Ly6G+ neutrophils reduced and acute injury attenuated. Unique identifiers of microglia and monocytes revealed phenotypic changes in each cell subtype of the monocyte lineage after CAIS. Taken together, CX3CR1-CCR2-dependent microglia-monocyte signaling contributes to cerebrovascular leakage, inflammation and CAIS injury.
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Encéfalo/irrigação sanguínea , Receptor 1 de Quimiocina CX3C/imunologia , Microglia/patologia , Monócitos/patologia , Receptores CCR2/imunologia , Acidente Vascular Cerebral/patologia , Animais , Barreira Hematoencefálica/imunologia , Barreira Hematoencefálica/patologia , Encéfalo/imunologia , Encéfalo/patologia , Permeabilidade Capilar , Células Cultivadas , Criança , Modelos Animais de Doenças , Feminino , Humanos , Inflamação/imunologia , Inflamação/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microglia/imunologia , Monócitos/imunologia , Transdução de Sinais , Acidente Vascular Cerebral/imunologiaRESUMO
We introduce a novel protocol to stain, visualize, and analyze blood vessels from the rat and mouse cerebrum. This technique utilizes the fluorescent dye, DiI, to label the lumen of the vasculature followed by perfusion fixation. Following brain extraction, the labeled vasculature is then imaged using wide-field fluorescence microscopy for axial and coronal images and can be followed by regional confocal microscopy. Axial and coronal images can be analyzed using classical angiographic methods for vessel density, length, and other features. We also have developed a novel fractal analysis to assess vascular complexity. Our protocol has been optimized for adult rat, adult mouse, and neonatal mouse studies. The protocol is efficient, can be rapidly completed, stains cerebral vessels with a bright fluorescence, and provides valuable quantitative data. This method has a broad range of applications, and we demonstrate its use to study the vasculature in assorted models of acquired brain injury.
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We previously reported that traumatic brain injuries (TBI) alter the cerebrovasculature near the injury site in rats, followed by revascularization over a 2-week period. Here, we tested our hypothesis that male and female adult mice have differential cerebrovascular responses following a moderate controlled cortical impact (CCI). Using in vivo magnetic resonance imaging (MRI), a new technique called vessel painting, and immunohistochemistry, we found no differences between males and females in lesion volume, neurodegeneration, blood-brain barrier (BBB) alteration, and microglia activation. However, females exhibited more astrocytic hypertrophy and heme-oxygenase-1 (HO-1) induction at 1 day post-injury (dpi), whereas males presented with increased endothelial activation and expression of ß-catenin, shown to be involved in angiogenesis. At 7 dpi, we observed an increase in the number of vessels and an enhancement in vessel complexity in the injured cortex of males compared with females. Cerebrovasculature recovers differently after CCI, suggesting biological sex should be considered when designing new therapeutic agents.
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Lesões Encefálicas Traumáticas/patologia , Córtex Cerebral/patologia , Caracteres Sexuais , Animais , Córtex Cerebral/irrigação sanguínea , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Aquaporins (AQPs) facilitate water diffusion through the plasma membrane. Brain aquaporin-4 (AQP4) is present in astrocytes and has critical roles in normal and disease physiology. We previously showed that a 24.9% decrease in AQP4 expression after in vivo silencing resulted in a 45.8% decrease in tissue water mobility as interpreted from magnetic resonance imaging apparent diffusion coefficients (ADC). Similar to previous in vitro studies we show decreased expression of the gap junction protein connexin 43 (Cx43) in vivo after intracortical injection of siAQP4 in the rat. Moreover, siAQP4 induced a loss of dye-coupling between astrocytes in vitro, further demonstrating its effect on gap junctions. In contrast, silencing of Cx43 did not alter the level of AQP4 or water mobility (ADC) in the brain. We hypothesized that siAQP4 has off-target effects on Cx43 expression via modification of miRNA expression. The decreased expression of Cx43 in siAQP4-treated animals was associated with up-regulation of miR224, which is known to target AQP4 and Cx43 expression. This could be one potential molecular mechanism responsible for the effect of siAQP4 on Cx43 expression, and the resultant decrease in astrocyte connectivity and dramatic effects on ADC values and water mobility.
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Aquaporina 4/metabolismo , Astrócitos/metabolismo , Encéfalo/metabolismo , Regulação da Expressão Gênica/fisiologia , MicroRNAs/biossíntese , Água/metabolismo , Animais , Astrócitos/citologia , Encéfalo/citologia , Encéfalo/diagnóstico por imagem , Conexina 43/metabolismo , Junções Comunicantes , Imageamento por Ressonância Magnética , Ratos , Ratos Sprague-DawleyRESUMO
Recent data suggest that repairing the cerebral vasculature after traumatic brain injury (TBI) may help to improve functional recovery. The Wnt/ß-catenin signaling pathway promotes blood vessel formation during vascular development, but its role in vascular repair after TBI remains elusive. In this study, we examined how the cerebral vasculature responds to TBI and the role of Wnt/ß-catenin signaling in vascular repair. We induced a moderate controlled cortical impact in adult mice and performed vessel painting to visualize the vascular alterations in the brain. Brain tissue around the injury site was assessed for ß-catenin and vascular markers. A Wnt transgenic mouse line was utilized to evaluate Wnt gene expression. We report that TBI results in vascular loss followed by increases in vascular structure at seven days post injury (dpi). Immature, non-perfusing vessels were evident in the tissue around the injury site. ß-catenin protein expression was significantly reduced in the injury site at 7 dpi. However, there was an increase in ß-catenin expression in perilesional vessels at 1 and 7 dpi. Similarly, we found increased number of Wnt-GFP-positive vessels after TBI. Our findings suggest that Wnt/ß-catenin expression contributes to the vascular repair process after TBI.