Infarct growth precedes cerebral thrombosis following experimental stroke in mice.
ABSTRACT: Ischemic stroke is among the leading causes of disability and death worldwide. In acute ischemic stroke, successful recanalization of occluded vessels is the primary therapeutic aim, but even if it is achieved, not all patients benefit. Although blockade of platelet aggregation did not prevent infarct progression, cerebral thrombosis as cause of secondary infarct growth has remained a matter of debate. As cerebral thrombi are frequently observed after experimental stroke, a thrombus-induced impairment of the brain microcirculation is considered to contribute to tissue damage. Here, we combine the model of transient middle cerebral artery occlusion (tMCAO) with light sheet fluorescence microscopy and immunohistochemistry of brain slices to investigate the kinetics of thrombus formation and infarct progression. Our data reveal that tissue damage already peaks after 8 h of reperfusion following 60 min MCAO, while cerebral thrombi are only observed at later time points. Thus, cerebral thrombosis is not causative for secondary infarct growth during ischemic stroke.
Project description:OBJECTIVES: We examined the effect of Revacept, an Fc fusion protein which is specifically linked to the extracellular domain of glycoprotein VI (GPVI), on thrombus formation after vessel wall injury and on experimental stroke in mice. BACKGROUND: Several antiplatelet drugs for the treatment of myocardial infarction or ischemic stroke with potent anti-ischemic effects have been developed, but all incur a significant risk of bleeding. METHODS: Platelet adhesion and thrombus formation after endothelial injury was monitored in the carotid artery by intra-vital fluorescence microscopy. The morphological and clinical consequences of stroke were investigated in a mouse model with a one hour-occlusion of the middle cerebral artery. RESULTS: Thrombus formation was significantly decreased after endothelial injury by 1 mg/kg Revacept i.v., compared to Fc only. 1 mg/kg Revacept i.v. applied in mice with ischemic stroke immediately before reperfusion significantly improved functional outcome, cerebral infarct size and edema compared to Fc only. Also treatment with 10 mg/kg rtPA was effective, and functional outcome was similar in both treatment groups. The combination of Revacept with rtPA leads to increased reperfusion compared to treatment with either agent alone. In contrast to rtPA, however, there were no signs of increased intracranial bleeding with Revacept. Both rtPA and Revacept improved survival after stroke compared to placebo treatment. Revacept and vWF bind to collagen and Revacept competitively prevented the binding of vWF to collagen. CONCLUSIONS: Revacept reduces arterial thrombus formation, reduces cerebral infarct size and edema after ischemic stroke, improves functional and prognostic outcome without intracranial bleeding. Revacept not only prevents GPVI-mediated, but probably also vWF-mediated platelet adhesion and aggregate formation. Therefore Revacept might be a potent and safe tool to treat ischemic complications of stroke.
Project description:Serotonin (5-hydroxytryptamin, 5-HT) is an indolamine platelet agonist, biochemically derived from tryptophan. 5-HT is secreted from the enterochromaffin cells into the gastrointestinal tract and blood. Blood 5-HT has been proposed to regulate hemostasis by acting as a vasoconstrictor and by triggering platelet signaling through 5-HT receptor 2A (5HTR2A). Although platelets do not synthetize 5-HT, they take 5-HT up from the blood and store it in their dense granules which are secreted upon platelet activation.To identify the molecular composite of the 5-HT uptake system in platelets and elucidate the role of platelet released 5-HT in thrombosis and ischemic stroke.5-HT transporter knockout mice (5Htt-/-) were analyzed in different in vitro and in vivo assays and in a model of ischemic stroke.In 5Htt-/- platelets, 5-HT uptake from the blood was completely abolished and agonist-induced Ca2+ influx through store operated Ca2+ entry (SOCE), integrin activation, degranulation and aggregation responses to glycoprotein VI (GPVI) and C-type lectin-like receptor 2 (CLEC-2) were reduced. These observed in vitro defects in 5Htt-/- platelets could be normalized by the addition of exogenous 5-HT. Moreover, reduced 5-HT levels in the plasma, an increased bleeding time and the formation of unstable thrombi were observed ex vivo under flow and in vivo in the abdominal aorta and carotid artery of 5Htt-/- mice. Surprisingly, in the transient middle cerebral artery occlusion (tMCAO) model of ischemic stroke 5Htt-/- mice showed nearly normal infarct volume and the neurological outcome was comparable to control mice.Although secreted platelet 5-HT does not appear to play a crucial role in the development of reperfusion injury after stroke, it is essential to amplify the second phase of platelet activation through SOCE and plays an important role in thrombus stabilization.
Project description:Emerging evidence indicates that methamphetamine (MA) abuse can impact cardiovascular disease. In humans, MA abuse is associated with an increased risk of stroke as well as an earlier age at which the stroke occurs. However, little is known about how chronic daily MA exposure can impact ischemic outcome in either humans or animal models. In the present study, mice were injected with MA (10 mg/kg, i.p.) or saline once daily for 10 consecutive days. Twenty-four hours after the final injection, mice were subjected to transient middle cerebral artery occlusion (tMCAO) for one hour followed by reperfusion. Mice were tested for novel object memory at 96 h post-reperfusion, just prior to removal of brains for quantification of infarct volume using 2,3,5-Triphenyltetrazolium Chloride (TTC) staining. Mice treated with MA prior to tMCAO showed decreased object memory recognition and increased infarct volume compared to saline-treated mice. These findings indicate that chronic MA exposure can worsen both cognitive and morphological outcomes following cerebral ischemia.
Project description:Coronavirus disease 2019 (COVID-19) is associated with an increased risk of thrombotic events. Ischemic stroke in COVID-19 patients entails high severity and mortality rates. Here we aimed to analyze cerebral thrombi of COVID-19 patients with large vessel occlusion (LVO) acute ischemic stroke to expose molecular evidence for SARS-CoV-2 in the thrombus and to unravel any peculiar immune-thrombotic features. We conducted a systematic pathological analysis of cerebral thrombi retrieved by endovascular thrombectomy in patients with LVO stroke infected with COVID-19 (n = 7 patients) and non-covid LVO controls (n = 23). In thrombi of COVID-19 patients, the SARS-CoV-2 docking receptor ACE2 was mainly expressed in monocytes/macrophages and showed higher expression levels compared to controls. Using polymerase chain reaction and sequencing, we detected SARS-CoV-2 Clade20A, in the thrombus of one COVID-19 patient. Comparing thrombus composition of COVID-19 and control patients, we noted no overt differences in terms of red blood cells, fibrin, neutrophil extracellular traps (NETs), von Willebrand Factor (vWF), platelets and complement complex C5b-9. However, thrombi of COVID-19 patients showed increased neutrophil density (MPO+ cells) and a three-fold higher Neutrophil-to-Lymphocyte Ratio (tNLR). In the ROC analysis both neutrophils and tNLR had a good discriminative ability to differentiate thrombi of COVID-19 patients from controls. In summary, cerebral thrombi of COVID-19 patients can harbor SARS-CoV2 and are characterized by an increased neutrophil number and tNLR and higher ACE2 expression. These findings suggest neutrophils as the possible culprit in COVID-19-related thrombosis. <h4>Graphical Abstract</h4> <h4>Supplementary Information</h4> The online version contains supplementary material available at 10.1186/s40478-022-01313-y.
Project description:Thrombosis and inflammation are hallmarks of ischemic stroke still unamenable to therapeutic interventions. High-molecular-weight kininogen (KNG) is a central constituent of the contact-kinin system which represents an interface between thrombotic and inflammatory circuits and is critically involved in stroke development. Kng(-/-) mice are protected from thrombosis after artificial vessel wall injury and lack the proinflammatory mediator bradykinin. We investigated the consequences of KNG deficiency in models of ischemic stroke. Kng(-/-) mice of either sex subjected to transient middle cerebral artery occlusion developed dramatically smaller brain infarctions and less severe neurologic deficits without an increase in infarct-associated hemorrhage. This protective effect was preserved at later stages of infarction as well as in elderly mice. Targeting KNG reduced thrombus formation in ischemic vessels and improved cerebral blood flow, and reconstitution of KNG-deficient mice with human KNG or bradykinin restored clot deposition and infarct susceptibility. Moreover, mice deficient in KNG showed less severe blood-brain barrier damage and edema formation, and the local inflammatory response was reduced compared with controls. Because KNG appears to be instrumental in pathologic thrombus formation and inflammation but dispensable for hemostasis, KNG inhibition may offer a selective and safe strategy for combating stroke and other thromboembolic diseases.
Project description:<b>Background and Purpose:</b> Acute ischemic stroke (AIS) with large artery occlusion (LAO) may lead to severe disability or death if not promptly treated. To determine the source of cerebral artery occlusion thrombosis, we studied the pathological components of cerebral artery thrombosis with different etiological classifications to guide clinical formulation of preventive treatment. <b>Materials and Methods:</b> Eighty-eight thrombi from AIS patients with LAO, 12 atrial thrombi from patients with valvular heart disease (VHD), and 11 plaques obtained by carotid endarterectomy (CEA) from patients with carotid artery stenosis were included in this retrospective study. The hematoxylin and eosin-stained specimens were quantitatively analyzed for erythrocytes, white blood cells (WBCs) and fibrin; platelets were shown by immunohistochemistry for CD31. <b>Results:</b> The thrombi of VHD showed the highest percentage of fibrin, followed by those of cardioembolism (CE) and stroke of undetermined etiology (SUE), and these values were higher than those of the other groups. Plaques obtained by CEA showed the highest erythrocyte number, followed by the large artery atherosclerosis (LAA) thrombi, and showed significantly noticeable differences between other stroke subtypes. The proportions of fibrin and erythrocytes in the thrombi of CE and SUE were most similar to those in the thrombi of VHD, and the LAA thrombi were the closest to those obtained by CEA. CE thrombi and CEA plaques had a higher percentage of WBCs than thrombi of other stroke thrombus subtypes and VHD. <b>Conclusions:</b> CE and most cryptogenic thrombi may originate from the heart, and the formation of carotid atherosclerotic plaques may be related to atherosclerotic cerebral embolism. Inflammation may be involved in their formation.
Project description:<h4>Objective</h4>We investigated (1) the associations of pre-stroke aspirin use with thrombus burden, infarct volume, hemorrhagic transformation, early neurological deterioration (END), and functional outcome, and (2) whether stroke subtypes modify these associations in first-ever ischemic stroke.<h4>Methods</h4>This multicenter magnetic resonance imaging (MRI)-based study included 5,700 consecutive patients with acute first-ever ischemic stroke, who did not undergo intravenous thrombolysis or endovascular thrombectomy, from May 2011 through February 2014. Propensity score-based augmented inverse probability weighting was performed to estimate adjusted effects of pre-stroke aspirin use.<h4>Results</h4>The mean age was 67 years (41% women), and 15.9% (n = 907) were taking aspirin before stroke. Pre-stroke aspirin use (vs nonuse) was significantly related to a reduced infarct volume (by 30%), particularly in large artery atherosclerosis stroke (by 45%). In cardioembolic stroke, pre-stroke aspirin use was associated with a ~50% lower incidence of END (adjusted difference = -5.4%, 95% confidence interval [CI] = -8.9 to -1.9). Thus, pre-stroke aspirin use was associated with ~30% higher likelihood of favorable outcome (3-month modified Rankin Scale score < 3), particularly in large artery atherosclerosis stroke and cardioembolic stroke (adjusted difference = 7.2%, 95% CI = 1.8 to 12.5 and adjusted difference = 6.4%, 95% CI = 1.7 to 11.1, respectively). Pre-stroke aspirin use (vs nonuse) was associated with 85% less frequent cerebral thrombus-related susceptibility vessel sign (SVS) in large artery atherosclerosis stroke (adjusted difference = -1.4%, 95% CI = -2.1 to -0.8, p < 0.001) and was associated with ~40% lower SVS volumes, particularly in cardioembolic stroke (adjusted difference = -0.16 cm<sup>3</sup> , 95% CI = -0.29 to -0.02, p = 0.03). Moreover, pre-stroke aspirin use was not significantly associated with hemorrhagic transformation (adjusted difference = -1.1%, p = 0.09).<h4>Interpretation</h4>Pre-stroke aspirin use associates with improved functional independence in patients with first-ever ischemic large arterial stroke by reducing infarct volume and/or END, likely by decreasing thrombus burden, without increased risk of hemorrhagic transformation. ANN NEUROL 2021;90:763-776.
Project description:Computed tomography (CT) is the current standard for time-critical decision-making in stroke patients, informing decisions on thrombolytic therapy with tissue plasminogen activator (tPA), which has a narrow therapeutic index. We aimed to develop a CT-based method to directly visualize cerebrovascular thrombi and guide thrombolytic therapy. Glycol-chitosan-coated gold nanoparticles (GC-AuNPs) were synthesized and conjugated to fibrin-targeting peptides, forming fib-GC-AuNP. This targeted imaging agent and non-targeted control agent were characterized in vitro and in vivo in C57Bl/6 mice (n = 107) with FeCl3-induced carotid thrombosis and/or embolic ischemic stroke. Fibrin-binding capacity was superior with fib-GC-AuNPs compared to GC-AuNPs, with thrombi visualized as high density on microCT (mCT). mCT imaging using fib-GC-AuNP allowed the prompt detection and quantification of cerebral thrombi, and monitoring of tPA-mediated thrombolytic effect, which reflected histological stroke outcome. Furthermore, recurrent thrombosis could be diagnosed by mCT without further nanoparticle administration for up to 3 weeks. fib-GC-AuNP-based direct cerebral thrombus imaging greatly enhance the value and information obtainable by regular CT, has multiple uses in basic / translational vascular research, and will likely allow personalized thrombolytic therapy in clinic by a) optimizing tPA-dosing to match thrombus burden, b) enabling the rational triage of patients to more radical therapies such as endovascular clot-retrieval, and c) potentially serving as a theranostic platform for targeted delivery of concurrent thrombolysis.
Project description:Background Mucosal-associated invariant T (MAIT) cells have been associated with inflammation in several autoimmune diseases. However, their relation to ischemic stroke remains unclear. This study attempted to elucidate the role of MAIT cells in acute ischemic stroke in mice. Methods and Results We used MR1 knockout C57BL/6 (MR1<sup>-/-</sup>) mice and wild-type littermates (MR1<sup>+/+</sup>). After performing a transient middle cerebral artery occlusion (tMCAO), we evaluated the association with inflammation and prognosis in the acute cerebral ischemia. Furthermore, we analyzed the tMCAO C57BL/6 mice administered with the suppressive MR1 ligand and the vehicle control. We also evaluated the infiltration of MAIT cells into the ischemic brain by flow cytometry. Results showed a reduction of infarct volume and an improvement of neurological impairment in MR1<sup>-/-</sup> mice (n=8). There was a reduction in the number of infiltrating microglia/macrophages (n=3-5) and in their activation (n=5) in the peri-infarct area of MR1<sup>-/-</sup> mice. The cytokine levels of interleukin-6 and interleukin-17 at 24 hours after tMCAO (n=3-5), and for interleukin-17 at 72 hours after tMCAO (n=5), were lower in the MR1<sup>-/-</sup> mice. The administration of the suppressive MR1 ligand reduced the infarct volume and improved functional impairment (n=5). Flow cytometric analysis demonstrated there was a reduction of MAIT cells infiltrating into the ischemic brain at 24 hours after tMCAO (n=17). Conclusions Our results showed that MAIT cells play an important role in neuroinflammation after focal cerebral ischemia and the use of MAIT cell regulation has a potential role as a novel neuroprotectant for the treatment of acute ischemic stroke.
Project description:Inflammation and thrombosis currently are recognized as critical contributors to the pathogenesis of ischemic stroke. CD147 (cluster of differentiation 147), also known as extracellular matrix metalloproteinase inducer, can function as a key mediator of inflammatory and immune responses. CD147 expression is increased in the brain after cerebral ischemia, but its role in the pathogenesis of ischemic stroke remains unknown. In this study, we show that CD147 acts as a key player in ischemic stroke by driving thrombotic and inflammatory responses.Focal cerebral ischemia was induced in C57BL/6 mice by a 60-minute transient middle cerebral artery occlusion. Animals were treated with anti-CD147 function-blocking antibody (?CD147) or isotype control antibody. Blood-brain barrier permeability, thrombus formation, and microvascular patency were assessed 24 hours after ischemia. Infarct size, neurological deficits, and inflammatory cells invaded in the brain were assessed 72 hours after ischemia.CD147 expression was rapidly increased in ischemic brain endothelium after transient middle cerebral artery occlusion. Inhibition of CD147 reduced infarct size and improved functional outcome on day 3 after transient middle cerebral artery occlusion. The neuroprotective effects were associated with (1) prevented blood-brain barrier damage, (2) decreased intravascular fibrin and platelet deposition, which in turn reduced thrombosis and increased cerebral perfusion, and (3) reduced brain inflammatory cell infiltration. The underlying mechanism may include reduced NF-?B (nuclear factor ?B) activation, MMP-9 (matrix metalloproteinase-9) activity, and PAI-1 (plasminogen activator inhibitor-1) expression in brain microvascular endothelial cells.Inhibition of CD147 ameliorates acute ischemic stroke by reducing thromboinflammation. CD147 might represent a novel and promising therapeutic target for ischemic stroke and possibly other thromboinflammatory disorders.