RESUMO
BACKGROUND: Intravenous injection of alteplase, a recombinant tPA (tissue-type plasminogen activator) as a thrombolytic agent has revolutionized ischemic stroke management. However, tPA is a more complex enzyme than expected, being for instance able to promote thrombolysis, but at the same time, also able to influence neuronal survival and to affect the integrity of the blood-brain barrier. Accordingly, the respective impact of endogenous tPA expressed/present in the brain parenchyma versus in the circulation during stroke remains debated. METHODS: To address this issue, we used mice with constitutive deletion of tPA (tPANull [tPA-deficient mice]) or conditional deletion of endothelial tPA (VECad [vascular endothelial-Cadherin-Cre-recombinase]-Cre∆tPA). We also developed parabioses between tPANull and wild-type mice (tPAWT), anticipating that a tPAWT donor would restore levels of tPA to normal ones, in the circulation but not in the brain parenchyma of a tPANull recipient. Stroke outcomes were investigated by magnetic resonance imaging in a thrombo-embolic or a thrombotic stroke model, induced by local thrombin injection or FeCl3 application on the endothelium, respectively. RESULTS: First, our data show that endothelial tPA, released into the circulation after stroke onset, plays an overall beneficial role following thrombo-embolic stroke. Accordingly, after 24 hours, tPANull/tPANull parabionts displayed less spontaneous recanalization and reperfusion and larger infarcts compared with tPAWT/tPAWT littermates. However, when associated to tPAWT littermates, tPANull mice had similar perfusion deficits, but less severe brain infarcts. In the thrombotic stroke model, homo- and hetero-typic parabionts did not differ in the extent of brain damages and did not differentially recanalize and reperfuse. CONCLUSIONS: Together, our data reveal that during thromboembolic stroke, endogenous circulating tPA from endothelial cells sustains a spontaneous recanalization and reperfusion of the tissue, thus, limiting the extension of ischemic lesions. In this context, the impact of endogenous parenchymal tPA is limited.
Assuntos
Acidente Vascular Cerebral , AVC Trombótico , Animais , Camundongos , Modelos Animais de Doenças , Células Endoteliais , Endotélio , Camundongos Knockout , Acidente Vascular Cerebral/diagnóstico por imagem , Acidente Vascular Cerebral/patologia , Ativador de Plasminogênio Tecidual/genética , Ativador de Plasminogênio Tecidual/metabolismoRESUMO
Endothelium protection is critical, because of the impact of vascular leakage and edema on pathological conditions such as brain ischemia. Whereas deficiency of class II phosphoinositide 3-kinase alpha (PI3KC2α) results in an increase in vascular permeability, we uncover a crucial role of the beta isoform (PI3KC2ß) in the loss of endothelial barrier integrity following injury. Here, we studied the role of PI3KC2ß in endothelial permeability and endosomal trafficking in vitro and in vivo in ischemic stroke. Mice with inactive PI3KC2ß showed protection against vascular permeability, edema, cerebral infarction, and deleterious inflammatory response. Loss of PI3KC2ß in human cerebral microvascular endothelial cells stabilized homotypic cell-cell junctions by increasing Rab11-dependent VE-cadherin recycling. These results identify PI3KC2ß as a potential new therapeutic target to prevent aggravating lesions following ischemic stroke.
Assuntos
Células Endoteliais , Fosfatidilinositol 3-Quinases , Junções Aderentes/metabolismo , Animais , Antígenos CD/metabolismo , Caderinas/genética , Caderinas/metabolismo , Permeabilidade Capilar , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Camundongos , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismoRESUMO
BACKGROUND: Subarachnoid hemorrhage (SAH) can lead to acute hydrocephalus (AH). AH pathophysiology is classically attributed to an obstruction of the arachnoid granulations by blood. Recent findings in rodents suggest that after intraventricular hemorrhage, AH is related to cerebrospinal fluid (CSF) hypersecretion by the choroid plexus (CP), as it can be reduced by intracerebroventricular (ICV) injection of bumetanide. OBJECTIVE: Here, we investigated if and how CSF hypersecretion and/or CSF outflow disorders contribute to post-SAH hydrocephalus. METHODS: Ninety-four Wistar rats were used. SAH was induced by the endovascular perforation technique. The presence of AH was confirmed by magnetic resonance imaging (MRI), and rats with AH were randomly assigned to 4 groups: control group, superior sagittal sinus (SSS) thrombosis to block CSF reabsorption, ICV injection of saline, and ICV injection of bumetanide to decrease CSF secretion. Clinical outcome was evaluated with a neuroscore. A second MRI was performed 24 h later to evaluate the ventricular volume. RESULTS: Fifty percent of rats that survived SAH induction had AH. Their ventricular volume correlated well to the functional outcome after 24 h (r = 0.803). In rats with AH, 24 h later, ventricular volume remained equally increased in the absence of any further procedure. Similarly, ICV injection of saline or SSS thrombosis had no impact on the ventricular volume. However, ICV injection of bumetanide reduced AH by 35.9% (p = 0.002). CONCLUSION: In rodents, post-SAH hydrocephalus is may be due to hypersecretion of CSF by the CP, as it is limited by ICV injection of bumetanide. However, we cannot exclude other mechanisms involved in post-SAH acute hydrocephalus.
Assuntos
Hidrocefalia , Hemorragia Subaracnóidea , Animais , Bumetanida/farmacologia , Bumetanida/uso terapêutico , Plexo Corióideo , Hidrocefalia/tratamento farmacológico , Hidrocefalia/etiologia , Ratos , Ratos Wistar , Hemorragia Subaracnóidea/complicações , Hemorragia Subaracnóidea/diagnóstico por imagem , Hemorragia Subaracnóidea/tratamento farmacológicoRESUMO
Hyperfibrinolysis is a systemic condition occurring in various clinical disorders such as trauma, liver cirrhosis, and leukemia. Apart from increased bleeding tendency, the pathophysiological consequences of hyperfibrinolysis remain largely unknown. Our aim was to develop an experimental model of hyperfibrinolysis and to study its effects on the homeostasis of the blood-brain barrier (BBB). We induced a sustained hyperfibrinolytic state in mice by hydrodynamic transfection of a plasmid encoding for tissue-type plasminogen activator (tPA). As revealed by near-infrared fluorescence imaging, hyperfibrinolytic mice presented a significant increase in BBB permeability. Using a set of deletion variants of tPA and pharmacological approaches, we demonstrated that this effect was independent of N-methyl-D-aspartate receptor, low-density lipoprotein-related protein, protease-activated receptor-1, or matrix metalloproteinases. In contrast, we provide evidence that hyperfibrinolysis-induced BBB leakage is dependent on plasmin-mediated generation of bradykinin and subsequent activation of bradykinin B2 receptors. Accordingly, this effect was prevented by icatibant, a clinically available B2 receptor antagonist. In agreement with these preclinical data, bradykinin generation was also observed in humans in a context of acute pharmacological hyperfibrinolysis. Altogether, these results suggest that B2 receptor blockade may be a promising strategy to prevent the deleterious effects of hyperfibrinolysis on the homeostasis of the BBB.
Assuntos
Barreira Hematoencefálica/metabolismo , Bradicinina/fisiologia , Permeabilidade Capilar/fisiologia , Fibrinolisina/fisiologia , Fibrinólise/fisiologia , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Bradicinina/metabolismo , Antagonistas de Receptor B2 da Bradicinina/farmacologia , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Permeabilidade Capilar/efeitos dos fármacos , Permeabilidade Capilar/genética , Fibrinolisina/metabolismo , Fibrinólise/efeitos dos fármacos , Fibrinólise/genética , Hidrodinâmica , Camundongos , Camundongos Transgênicos , Receptor B2 da Bradicinina/genética , Receptor B2 da Bradicinina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Ativador de Plasminogênio Tecidual/genética , Ativador de Plasminogênio Tecidual/metabolismoRESUMO
NAD+ depletion is a common phenomenon in neurodegenerative pathologies. Excitotoxicity occurs in multiple neurologic disorders and NAD+ was shown to prevent neuronal degeneration in this process through mechanisms that remained to be determined. The activity of nicotinamide riboside (NR) in neuroprotective models and the recent description of extracellular conversion of NAD+ to NR prompted us to probe the effects of NAD+ and NR in protection against excitotoxicity. Here, we show that intracortical administration of NR but not NAD+ reduces brain damage induced by NMDA injection. Using cortical neurons, we found that provision of extracellular NR delays NMDA-induced axonal degeneration (AxD) much more strongly than extracellular NAD+ Moreover, the stronger effect of NR compared to NAD+ depends of axonal stress since in AxD induced by pharmacological inhibition of nicotinamide salvage, both NAD+ and NR prevent neuronal death and AxD in a manner that depends on internalization of NR. Taken together, our findings demonstrate that NR is a better neuroprotective agent than NAD+ in excitotoxicity-induced AxD and that axonal protection involves defending intracellular NAD+ homeostasis.-Vaur, P., Brugg, B., Mericskay, M., Li, Z., Schmidt, M. S., Vivien, D., Orset, C., Jacotot, E., Brenner, C., Duplus, E. Nicotinamide riboside, a form of vitamin B3, protects against excitotoxicity-induced axonal degeneration.
Assuntos
Axônios/efeitos dos fármacos , Axônios/metabolismo , Niacinamida/análogos & derivados , Animais , Morte Celular/efeitos dos fármacos , Células Cultivadas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , N-Metilaspartato/farmacologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fármacos Neuroprotetores/farmacologia , Niacinamida/farmacologia , Compostos de Piridínio , Reação em Cadeia da Polimerase em Tempo RealRESUMO
SEE SUN ET AL DOI101093/AWW306 FOR A SCIENTIFIC COMMENTARY ON THIS ARTICLE: About 20% of patients with ischaemic stroke have a preceding transient ischaemic attack, which is clinically defined as focal neurological symptoms of ischaemic origin resolving spontaneously. Failure to diagnose transient ischaemic attack is a wasted opportunity to prevent recurrent disabling stroke. Unfortunately, diagnosis can be difficult, due to numerous mimics, and to the absence of a specific test. New diagnostic tools are thus needed, in particular for radiologically silent cases, which correspond to the recommended tissue-based definition of transient ischaemic attack. As endothelial activation is a hallmark of cerebrovascular events, we postulated that this may also be true for transient ischaemic attack, and that it would be clinically relevant to develop non-invasive in vivo imaging to detect this endothelial activation. Using transcriptional and immunohistological analyses for adhesion molecules in a mouse model, we identified brain endothelial P-selectin as a potential biomarker for transient ischaemic attack. We thus developed ultra-sensitive molecular magnetic resonance imaging using antibody-based microparticles of iron oxide targeting P-selectin. This highly sensitive imaging strategy unmasked activated endothelial cells after experimental transient ischaemic attack and allowed discriminating transient ischaemic attack from epilepsy and migraine, two important transient ischaemic attack mimics. We provide preclinical evidence that combining conventional magnetic resonance imaging with molecular magnetic resonance imaging targeting P-selectin might aid in the diagnosis of transient ischaemic attack.
Assuntos
Ataque Isquêmico Transitório/metabolismo , Imageamento por Ressonância Magnética/métodos , Imagem Molecular/métodos , Selectina-P/metabolismo , Acidente Vascular Cerebral/metabolismo , Animais , Biomarcadores/metabolismo , Modelos Animais de Doenças , Células Endoteliais , Ataque Isquêmico Transitório/diagnóstico por imagem , Masculino , Camundongos , Acidente Vascular Cerebral/diagnóstico por imagemRESUMO
In humans, spatial cognition and navigation impairments are a frequent situation during physiological and pathological aging, leading to a dramatic deterioration in the quality of life. Despite the discovery of neurons with location-specific activity in rodents, that is, place cells in the hippocampus and later on grid cells in the entorhinal cortex (EC), the molecular mechanisms underlying spatial cognition are still poorly known. Our present data bring together in an unusual combination 2 molecules of primary biological importance: a major neuronal excitatory receptor, N-methyl-D-aspartate receptor (NMDAR), and an extracellular protease, tissue plasminogen activator (tPA), in the control of spatial navigation. By using tPA-deficient mice and a structure-selective pharmacological approach, we demonstrate that the tPA-dependent NMDAR signaling potentiation in the EC plays a key and selective role in the encoding and the subsequent use of distant landmarks during spatial learning. We also demonstrate that this novel function of tPA in the EC is reduced during aging. Overall, these results argue for the concept that encoding of proximal versus distal landmarks is mediated not only by different anatomical pathways but also by different molecular mechanisms, with the tPA-dependent potentiation of NMDAR signaling in the EC that plays an important role.
Assuntos
Córtex Entorrinal/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Envelhecimento , Animais , Cálcio/metabolismo , Feminino , Hipocampo/metabolismo , Masculino , Camundongos Knockout , Neurônios/metabolismo , Transdução de Sinais/fisiologia , Ativador de Plasminogênio Tecidual/deficiência , Ativador de Plasminogênio Tecidual/metabolismoRESUMO
BACKGROUND AND PURPOSE: Although the mechanisms that contribute to intracranial aneurysm (IA) formation and rupture are not totally elucidated, inflammation and matrix remodeling are incriminated. Because tPA (tissue-type plasminogen activator) controls both inflammatory and matrix remodeling processes, we hypothesized that tPA could be involved in the pathophysiology of IA. METHODS: Immunofluorescence analyses of tPA and its main substrate within the aneurysmal wall of murine and human samples were performed. We then compared the formation and rupture of IAs in wild-type, tPA-deficient and type 1 plasminogen activator inhibitor-deficient mice subjected to a model of elastase-induced IA. The specific contribution of vascular versus global tPA was investigated by performing hepatic hydrodynamic transfection of a cDNA encoding for tPA in tPA-deficient mice. The formation and rupture of IAs were monitored by magnetic resonance imaging tracking for 28 days. RESULTS: Immunofluorescence revealed increased expression of tPA within the aneurysmal wall. The number of aneurysms and their symptomatic ruptures were significantly lower in tPA-deficient than in wild-type mice. Conversely, they were higher in plasminogen activator inhibitor-deficient mice. The wild-type phenotype could be restored in tPA-deficient mice by selectively increasing circulating levels of tPA via hepatic hydrodynamic transfection of a cDNA encoding for tPA. CONCLUSIONS: Altogether, this preclinical study demonstrates that the tPA present in the blood stream is a key player of the formation of IAs. Thus, tPA should be considered as a possible new target for the prevention of IAs formation and rupture.
Assuntos
Aneurisma Roto/metabolismo , Aneurisma Intracraniano/metabolismo , Ativador de Plasminogênio Tecidual/metabolismo , Adulto , Aneurisma Roto/diagnóstico por imagem , Animais , Feminino , Imunofluorescência , Humanos , Imuno-Histoquímica , Aneurisma Intracraniano/diagnóstico por imagem , Imageamento por Ressonância Magnética , Camundongos , Camundongos Knockout , Inibidor 1 de Ativador de Plasminogênio/genética , Ruptura Espontânea , Ativador de Plasminogênio Tecidual/genéticaRESUMO
BACKGROUND AND PURPOSE: The debate over the fact that experimental drugs proposed for the treatment of stroke fail in the translation to the clinical situation has attracted considerable attention in the literature. In this context, we present a retrospective pooled analysis of a large data set from preclinical studies, to examine the effects of early versus late administration of intravenous recombinant tissue-type plasminogen activator. METHODS: We collected data from 26 individual studies from 9 international centers (13 researchers; 716 animals) that compared recombinant tissue-type plasminogen activator with controls, in a unique mouse model of thromboembolic stroke induced by an in situ injection of thrombin into the middle cerebral artery. Studies were classified into early (<3 hours) versus late (≥3 hours) drug administration. Final infarct volumes, assessed by histology or magnetic resonance imaging, were compared in each study, and the absolute differences were pooled in a random-effect meta-analysis. The influence of time of administration was tested. RESULTS: When compared with saline controls, early recombinant tissue-type plasminogen activator administration was associated with a significant benefit (absolute difference, -6.63 mm(3); 95% confidence interval, -9.08 to -4.17; I(2)=76%), whereas late recombinant tissue-type plasminogen activator treatment showed a deleterious effect (+5.06 mm(3); 95% confidence interval, +2.78 to +7.34; I(2)=42%; Pint<0.00001). Results remained unchanged after subgroup analyses. CONCLUSIONS: Our results provide the basis needed for the design of future preclinical studies on recanalization therapies using this model of thromboembolic stroke in mice. The power analysis reveals that a multicenter trial would require 123 animals per group instead of 40 for a single-center trial.
Assuntos
Isquemia Encefálica/tratamento farmacológico , Fibrinolíticos/farmacologia , Acidente Vascular Cerebral/tratamento farmacológico , Ativador de Plasminogênio Tecidual/farmacologia , Animais , Isquemia Encefálica/patologia , Modelos Animais de Doenças , Fibrinolíticos/administração & dosagem , Infarto da Artéria Cerebral Média/tratamento farmacológico , Infarto da Artéria Cerebral Média/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Acidente Vascular Cerebral/patologia , Ativador de Plasminogênio Tecidual/administração & dosagemRESUMO
Interactions between platelet glycoprotein (Gp) IIb/IIIa and plasma proteins mediate platelet cross-linking in arterial thrombi. However, GpIIb/IIIa inhibitors fail to disperse platelet aggregates after myocardial infarction or ischemic stroke. These results suggest that stability of occlusive thrombi involves additional and as-yet-unidentified mechanisms. In the present study, we investigated the mechanisms driving platelet cross-linking during occlusive thrombus formation. Using computational fluid dynamic simulations and in vivo thrombosis models, we demonstrated that the inner structure of occlusive thrombi is heterogeneous and primarily determined by the rheological conditions that prevailed during thrombus growth. Unlike the first steps of thrombus formation, which are GpIIb/IIIa-dependent, our findings reveal that closure of the arterial lumen is mediated by GpIbα-von Willebrand Factor (VWF) interactions. Accordingly, disruption of platelet cross-linking using GpIbα-VWF inhibitors restored vessel patency and improved outcome in a mouse model of ischemic stroke, although the thrombi were resistant to fibrinolysis or traditional antithrombotic agents. Overall, our study demonstrates that disruption of GpIbα-VWF interactions restores vessel patency after occlusive thrombosis by specifically disaggregating the external layer of occlusive thrombi, which is constituted of platelet aggregates formed under very high shear rates.
Assuntos
Plaquetas/patologia , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Trombose/metabolismo , Trombose/patologia , Fator de von Willebrand/metabolismo , Animais , Benzofuranos , Plaquetas/metabolismo , Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/patologia , Hemorreologia , Masculino , Camundongos , Agregação Plaquetária , Mapas de Interação de Proteínas , QuinolinasRESUMO
BACKGROUND: Plasminogen activation is a ubiquitous source of fibrinolytic and proteolytic activity. Besides its role in prevention of thrombosis, plasminogen is involved in inflammatory reactions in the central nervous system. Plasminogen has been detected in the cerebrospinal fluid (CSF) of patients with inflammatory diseases; however, its origin remains controversial, as the blood-CSF barrier may restrict its diffusion from blood. METHODS: We investigated the origin of plasminogen in CSF using Alexa Fluor 488-labelled rat plasminogen injected into rats with systemic inflammation and blood-CSF barrier dysfunction provoked by lipopolysaccharide (LPS). Near-infrared fluorescence imaging and immunohistochemistry fluorescence microscopy were used to identify plasminogen in brain structures, its concentration and functionality were determined by Western blotting and a chromogenic substrate assay, respectively. In parallel, plasminogen was investigated in CSF from patients with Guillain-Barré syndrome (n = 15), multiple sclerosis (n = 19) and noninflammatory neurological diseases (n = 8). RESULTS: Endogenous rat plasminogen was detected in higher amounts in the CSF and urine of LPS-treated animals as compared to controls. In LPS-primed rats, circulating Alexa Fluor 488-labelled rat plasminogen was abundantly localized in the choroid plexus, CSF and urine. Plasminogen in human CSF was higher in Guillain-Barré syndrome (median = 1.28 ng/µl (interquartile range (IQR) = 0.66 to 1.59)) as compared to multiple sclerosis (median = 0.3 ng/µl (IQR = 0.16 to 0.61)) and to noninflammatory neurological diseases (median = 0.27 ng/µl (IQR = 0.18 to 0.35)). CONCLUSIONS: Our findings demonstrate that plasminogen is transported from circulating blood into the CSF of rats via the choroid plexus during inflammation. Our data suggest that a similar mechanism may explain the high CSF concentrations of plasminogen detected in patients with inflammation-derived CSF barrier impairment.
Assuntos
Barreira Hematoencefálica/fisiologia , Inflamação/sangue , Inflamação/líquido cefalorraquidiano , Plasminogênio/líquido cefalorraquidiano , Animais , Western Blotting , Humanos , Masculino , Microscopia de Fluorescência , Ratos , Ratos WistarRESUMO
ABSTRACT: The pharmacological intervention for ischemic stroke hinges on intravenous administration of the recombinant tissue-type plasminogen activator (rtPA, Alteplase/Actilyse) either as a standalone treatment or in conjunction with thrombectomy. However, despite its clinical significance, broader use of rtPA is constrained because of the risk of hemorrhagic transformations (HTs). Furthermore, the presence of diabetes or chronic hyperglycemia is associated with an elevated risk of HT subsequent to thrombolysis. This detrimental impact of tPA on the neurovascular unit in patients with hyperglycemia has been ascribed to its capacity to induce endothelial N-methyl-D-aspartate receptor (NMDAR) signaling, contributing to compromised blood-brain barrier integrity and neuroinflammatory processes. In a mouse model of thromboembolic stroke with chronic hyperglycemia, we assessed the effectiveness of rtPA and N-acetylcysteine (NAC) as thrombolytic agents. We also tested the effect of blocking tPA/NMDAR signaling using a monoclonal antibody, Glunomab. Magnetic resonance imaging, speckle contrast imaging, flow cytometry, and behavioral tasks were used to evaluate stroke outcomes. In hyperglycemic animals, treatment with rtPA resulted in lower recanalization rates and increased HTs. Conversely, NAC treatment reduced lesion sizes while mitigating HTs. After a single administration, either in standalone or combined with rtPA-induced thrombolysis, Glunomab reduced brain lesion volumes, HTs, and neuroinflammation after stroke, translating into improved neurological outcomes. Additionally, we demonstrated the therapeutic efficacy of Glunomab in combination with NAC or as a standalone strategy in chronic hyperglycemic animals. Counteracting tPA-dependent endothelial NMDAR signaling limits ischemic damages induced by both endogenous and exogenous tPA, including HTs and inflammatory processes after ischemic stroke in hyperglycemic animals.
Assuntos
Hiperglicemia , AVC Isquêmico , Acidente Vascular Cerebral , Camundongos , Animais , Humanos , Ativador de Plasminogênio Tecidual/farmacologia , Ativador de Plasminogênio Tecidual/uso terapêutico , Camundongos Obesos , Acidente Vascular Cerebral/tratamento farmacológico , Acidente Vascular Cerebral/etiologia , Hemorragia , Inflamação/tratamento farmacológico , AVC Isquêmico/complicações , AVC Isquêmico/tratamento farmacológico , Hiperglicemia/complicações , Hiperglicemia/tratamento farmacológicoRESUMO
Neuroglia critically shape the brain´s response to ischemic stroke. However, their phenotypic heterogeneity impedes a holistic understanding of the cellular composition of the early ischemic lesion. Here we present a single cell resolution transcriptomics dataset of the brain´s acute response to infarction. Oligodendrocyte lineage cells and astrocytes range among the most transcriptionally perturbed populations and exhibit infarction- and subtype-specific molecular signatures. Specifically, we find infarction restricted proliferating oligodendrocyte precursor cells (OPCs), mature oligodendrocytes and reactive astrocytes, exhibiting transcriptional commonalities in response to ischemic injury. OPCs and reactive astrocytes are involved in a shared immuno-glial cross talk with stroke-specific myeloid cells. Within the perilesional zone, osteopontin positive myeloid cells accumulate in close proximity to CD44+ proliferating OPCs and reactive astrocytes. In vitro, osteopontin increases the migratory capacity of OPCs. Collectively, our study highlights molecular cross talk events which might govern the cellular composition of acutely infarcted brain tissue.
Assuntos
Astrócitos , AVC Isquêmico , Células Precursoras de Oligodendrócitos , Oligodendroglia , Análise de Célula Única , Animais , AVC Isquêmico/genética , AVC Isquêmico/metabolismo , AVC Isquêmico/patologia , Masculino , Camundongos , Análise de Célula Única/métodos , Oligodendroglia/metabolismo , Células Precursoras de Oligodendrócitos/metabolismo , Astrócitos/metabolismo , Neuroglia/metabolismo , Osteopontina/genética , Osteopontina/metabolismo , Transcriptoma , Análise de Sequência de RNA/métodos , Camundongos Endogâmicos C57BL , Encéfalo/metabolismo , Encéfalo/patologia , Ratos , Proliferação de Células , Movimento Celular/genética , Células Mieloides/metabolismo , Modelos Animais de Doenças , Núcleo Celular/metabolismo , Isquemia Encefálica/genética , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patologiaRESUMO
Recanalization is the mainstay of ischemic stroke treatment. However, even with timely clot removal, many stroke patients recover poorly. Leptomeningeal collaterals (LMCs) are pial anastomotic vessels with yet-unknown functions. We applied laser speckle imaging, ultrafast ultrasound, and two-photon microscopy in a thrombin-based mouse model of stroke and fibrinolytic treatment to show that LMCs maintain cerebral autoregulation and allow for gradual reperfusion, resulting in small infarcts. In mice with poor LMCs, distal arterial segments collapse, and deleterious hyperemia causes hemorrhage and mortality after recanalization. In silico analyses confirm the relevance of LMCs for preserving perfusion in the ischemic region. Accordingly, in stroke patients with poor collaterals undergoing thrombectomy, rapid reperfusion resulted in hemorrhagic transformation and unfavorable recovery. Thus, we identify LMCs as key components regulating reperfusion and preventing futile recanalization after stroke. Future therapeutic interventions should aim to enhance collateral function, allowing for beneficial reperfusion after stroke.
Assuntos
Circulação Colateral , AVC Isquêmico , Meninges , Reperfusão , Animais , AVC Isquêmico/fisiopatologia , AVC Isquêmico/terapia , Camundongos , Circulação Colateral/fisiologia , Humanos , Reperfusão/métodos , Meninges/irrigação sanguínea , Masculino , Circulação Cerebrovascular/fisiologia , Camundongos Endogâmicos C57BL , Modelos Animais de Doenças , Encéfalo/irrigação sanguínea , Trombectomia/métodosRESUMO
Background: During the past few decades, several pathophysiological processes contributing to intracranial aneurysm (IA) rupture have been identified, including irregular IA shape, altered hemodynamic stress within the IA, and vessel wall inflammation. The use of preclinical models of IA and imaging tools is paramount to better understand the underlying disease mechanisms. Methods: We used 2 established mouse models of IA, and we analyzed the progression of the IA by magnetic resonance imaging, transcranial Doppler, and histology. Results: In both models of IA, we observed, by transcranial Doppler, a significant decrease of the blood velocities and wall shear stress of the internal carotid arteries. We also observed the formation of tortuous arteries in both models that were correlated with the presence of an aneurysm as confirmed by magnetic resonance imaging and histology. A high grade of tortuosity is associated with a significant decrease of the mean blood flow velocities and a greater artery dilation. Conclusions: Transcranial Doppler is a robust and convenient imaging method to evaluate the progression of IA. Detection of decreased blood flow velocities and increased tortuosity can be used as reliable indicators of IA.
RESUMO
Systemic injection of thrombolytic drugs is the gold standard treatment for non-invasive blood clot resolution. The most serious risks associated with the intravenous injection of tissue plasminogen activator-like proteins are the bleeding complication and the dose related neurotoxicity. Indeed, the drug has to be injected in high concentrations due to its short half-life, the presence of its natural blood inhibitor (PAI-1) and the fast hepatic clearance (0.9 mg/kg in humans, 10 mg/kg in mouse models). Overall, there is a serious need for a dose-reduced targeted treatment to overcome these issues. We present in this article a new acoustic cavitation-based method for polymer MBs synthesis, three times faster than current hydrodynamic-cavitation method. The generated MBs are ultrasound responsive, stable and biocompatible. Their functionalization enabled the efficient and targeted treatment of stroke, without side effects. The stabilizing shell of the MBs is composed of Poly-Isobutyl Cyanoacrylate (PIBCA), copolymerized with fucoidan. Widely studied for its targeting properties, fucoidan exhibit a nanomolar affinity for activated endothelium and activated platelets (P-selectins). Secondly, the thrombolytic agent (rtPA) was loaded onto microbubbles (MBs) with a simple adsorption protocol. Hence, the present study validated the in vivo efficiency of rtPA-loaded Fuco MBs to be over 50 % more efficient than regular free rtPA injection for stroke resolution. In addition, the relative injected rtPA grafted onto targeting MBs was 1/10th of the standard effective dose (1 mg/kg in mouse). As a result, no hemorrhagic event, BBB leakage nor unexpected tissue distribution were observed.
Assuntos
Acidente Vascular Cerebral , Ativador de Plasminogênio Tecidual , Humanos , Animais , Camundongos , Ativador de Plasminogênio Tecidual/uso terapêutico , Microbolhas , Polímeros , Fibrinolíticos/uso terapêutico , Acidente Vascular Cerebral/tratamento farmacológicoRESUMO
Ca2+/calmodulin-dependent protein kinase II alpha (CaMKIIα) is a major contributor to physiological and pathological glutamate-mediated Ca2+ signals, and its involvement in various critical cellular pathways demands specific pharmacological strategies. We recently presented γ-hydroxybutyrate (GHB) ligands as the first small molecules selectively targeting and stabilizing the CaMKIIα hub domain. Here, we report that the cyclic GHB analogue 3-hydroxycyclopent-1-enecarboxylic acid (HOCPCA), improves sensorimotor function after experimental stroke in mice when administered at a clinically relevant time and in combination with alteplase. Further, we observed improved hippocampal neuronal activity and working memory after stroke. On the biochemical level, we observed that hub modulation by HOCPCA results in differential effects on distinct CaMKII pools, ultimately alleviating aberrant CaMKII signalling after cerebral ischemia. As such, HOCPCA normalised cytosolic Thr286 autophosphorylation after ischemia in mice and downregulated ischemia-specific expression of a constitutively active CaMKII kinase proteolytic fragment. Previous studies suggest holoenzyme stabilisation as a potential mechanism, yet a causal link to in vivo findings requires further studies. Similarly, HOCPCA's effects on dampening inflammatory changes require further investigation as an underlying protective mechanism. HOCPCA's selectivity and absence of effects on physiological CaMKII signalling highlight pharmacological modulation of the CaMKIIα hub domain as an attractive neuroprotective strategy.
Assuntos
Oxibato de Sódio , Acidente Vascular Cerebral , Camundongos , Animais , Oxibato de Sódio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , CogniçãoRESUMO
Reactive neuroglia critically shape the brains response to ischemic stroke. However, their phenotypic heterogeneity impedes a holistic understanding of the cellular composition and microenvironment of the early ischemic lesion. Here we generated a single cell resolution transcriptomics dataset of the injured brain during the acute recovery from permanent middle cerebral artery occlusion. This approach unveiled infarction and subtype specific molecular signatures in oligodendrocyte lineage cells and astrocytes, which ranged among the most transcriptionally perturbed cell types in our dataset. Specifically, we characterized and compared infarction restricted proliferating oligodendrocyte precursor cells (OPCs), mature oligodendrocytes and heterogeneous reactive astrocyte populations. Our analyses unveiled unexpected commonalities in the transcriptional response of oligodendrocyte lineage cells and astrocytes to ischemic injury. Moreover, OPCs and reactive astrocytes were involved in a shared immuno-glial cross talk with stroke specific myeloid cells. In situ, osteopontin positive myeloid cells accumulated in close proximity to proliferating OPCs and reactive astrocytes, which expressed the osteopontin receptor CD44, within the perilesional zone specifically. In vitro, osteopontin increased the migratory capacity of OPCs. Collectively, our study highlights molecular cross talk events which might govern the cellular composition and microenvironment of infarcted brain tissue in the early stages of recovery.
RESUMO
BACKGROUND AND PURPOSE: Despite side effects including N-methyl-d-aspartate-mediated neurotoxicity, recombinant tissue-type plasminogen activator (rtPA) remains the only approved acute treatment for ischemic stroke. Memantine, used for treatment of Alzheimer disease, is an antagonist for N-methyl-d-aspartate receptors. We investigated whether memantine could be used as a neuroprotective adjunct therapy for rtPA-induced thrombolysis after stroke. METHODS: In vitro N-methyl-d-aspartate exposure, oxygen and glucose deprivation, and N-methyl-d-aspartate-mediated calcium videomicroscopy experiments were performed on murine cortical neurons in the presence of rtPA and memantine. The therapeutic safety of rtPA and memantine coadministration was evaluated in mouse models of thrombotic stroke and intracerebral hemorrhage. Ischemic and hemorrhagic volumes were assessed by MRI and neurological evaluation was performed by the string test and automated gait analysis. RESULTS: Our in vitro observations showed that memantine was able to prevent the proneurotoxic effects of rtPA in cultured cortical neurons. Although memantine did not alter the fibrinolytic activity of rtPA, our in vivo observations revealed that it blunted the noxious effects of delayed thrombolysis on lesion volumes and neurological deficits after ischemic stroke. In addition, memantine rescued rtPA-induced decrease in survival rate after intracerebral hemorrhage. CONCLUSIONS: Memantine could be used as an adjunct therapy to improve the safety of thrombolysis.
Assuntos
Antagonistas de Aminoácidos Excitatórios/uso terapêutico , Memantina/uso terapêutico , Acidente Vascular Cerebral/tratamento farmacológico , Terapia Trombolítica/métodos , Ativador de Plasminogênio Tecidual/efeitos adversos , Ativador de Plasminogênio Tecidual/uso terapêutico , Animais , Células Cultivadas , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/patologia , Quimioterapia Adjuvante , Antagonistas de Aminoácidos Excitatórios/farmacologia , Fibrinolíticos/efeitos adversos , Fibrinolíticos/farmacologia , Fibrinolíticos/uso terapêutico , Técnicas In Vitro , Imageamento por Ressonância Magnética , Masculino , Memantina/farmacologia , Camundongos , Modelos Animais , N-Metilaspartato/farmacologia , Acidente Vascular Cerebral/patologia , Ativador de Plasminogênio Tecidual/farmacologia , Resultado do TratamentoRESUMO
BACKGROUND: The lack of a relevant stroke model in large nonhuman primates hinders the development of innovative diagnostic/therapeutic approaches concerned with this cerebrovascular disease. Our objective was to develop a novel and clinically relevant model of embolic stroke in the anesthetized monkey that incorporates readily available clinical imaging techniques and that would allow the possibility of drug delivery including strategies of reperfusion. METHODS: Thrombin was injected into the lumen of the middle cerebral artery (MCA) in 12 anesthetized (sevoflurane) male rhesus macaques (Macaca mulatta). Sequential MRI studies (including angiography, FLAIR, PWI, DWI, and gadolinium-enhanced T1W imaging) were performed in a 3T clinical MRI. Physiological and biochemical parameters were monitored throughout the investigations. RESULTS: Once standardized, the surgical procedure induced transient occlusion of the middle cerebral artery in all operated animals. All animals studied showed spontaneous reperfusion, which occurred some time between 2 h and 7 days post-ictus. Eighty percent of the studied animals showed diffusion/perfusion mismatch. The ischemic lesions at 24 h spared both superficial and profound territories of the MCA. Some animals presented hemorrhagic transformation at 7 days post-ictus. CONCLUSION: In this study, we developed a pre-clinically relevant model of embolic stroke in the anesthetized nonhuman primate.