fibrin and Infarction--Middle-Cerebral-Artery

Formation of a fibrin clot is mediated by a group of tightly regulated plasma proteases and cofactors. While this system is essential for minimizing blood loss from an injured blood vessel (hemostasis), it also contributes to pathologic fibrin formation and platelet activation that may occlude vessels (thrombosis). Many antithrombotic drugs target key elements of the plasma coagulation mechanism such as thrombin and factor Xa, based on the premise that plasma elements contributing to thrombosis are primarily those involved in hemostasis. Recent studies with genetically altered mice raise questions about this paradigm. Deficiencies of the intrinsic pathway proteases factor XII and factor XI are not associated with abnormal hemostasis in mice, but impair formation of occlusive thrombi in arterial injury models, indicating that pathways not essential for hemostasis participate in arterial thrombosis. If factor XII or factor XI make similar contributions to thrombosis in humans, these proteases could be ideal targets for drugs to treat or prevent thromboembolic disease with minimal risk of therapy-associated bleeding.

Topics: Animals; Anticoagulants; Blood Coagulation; Disease Models, Animal; Factor XI; Factor XI Deficiency; Factor XII; Factor XII Deficiency; Fibrin; Hemostasis; Humans; Infarction, Middle Cerebral Artery; Mice; Mice, Knockout; Protease Inhibitors; Reperfusion Injury; Thromboembolism; Thrombosis

Advances in our understanding of ADAMTS13 structure, and the conformation changes required for full activity, have rejuvenated the possibility of its use as a thrombolytic therapy. We have tested a novel Ala1144Val ADAMTS13 variant (constitutively active [ca] ADAMTS13) that exhibits constitutive activity, characterized using in vitro assays of ADAMTS13 activity, and greatly enhanced thrombolytic activity in 2 murine models of ischemic stroke, the distal FeCl3 middle cerebral artery occlusion (MCAo) model and transient middle cerebral artery occlusion (tMCAO) with systemic inflammation and ischemia/reperfusion injury. The primary measure of efficacy in both models was restoration of regional cerebral blood flow (rCBF) to the MCA territory, which was determined using laser speckle contrast imaging. The caADAMTS13 variant exhibited a constitutively active conformation and a fivefold enhanced activity against fluorescence resonance energy transfer substrate von Willebrand factor 73 (FRETS-VWF73) compared with wild-type (wt) ADAMTS13. Moreover, caADAMTS13 inhibited VWF-mediated platelet capture at subphysiological concentrations and enhanced t-PA/plasmin lysis of fibrin(ogen), neither of which were observed with wtADAMTS13. Significant restoration of rCBF and reduced lesion volume was observed in animals treated with caADAMTS13. When administered 1 hour after FeCl3 MCAo, the caADAMTS13 variant significantly reduced residual VWF and fibrin deposits in the MCA, platelet aggregate formation, and neutrophil recruitment. When administered 4 hours after reperfusion in the tMCAo model, the caADAMTS13 variant induced a significant dissolution of platelet aggregates and a reduction in the resulting tissue hypoperfusion. The caADAMTS13 variant represents a potentially viable therapeutic option for the treatment of acute ischemic stroke, among other thrombotic indications, due to its enhanced in vitro and in vivo activities that result from its constitutively active conformation.

Topics: ADAMTS13 Protein; Animals; Anti-Inflammatory Agents; Fibrin; Fibrinolytic Agents; Infarction, Middle Cerebral Artery; Ischemic Stroke; Mice; Stroke; von Willebrand Factor

To investigate the relationship between fibrin deposition and "no-reflow" within microcirculation after thrombolysis in acute ischemic stroke (AIS).. Experiments were approved by the institutional animal care and use committee. An experimental AIS model was induced in C57BL/6 mice by middle cerebral artery occlusion (MCAO) via the photothrombotic method. Mice were randomly assigned to non-thrombolytic or thrombolytic treated groups (n = 12 per group). The modified Neurological Severity Score and Fast Beam Balance Test were performed by a researcher blinded to the treatment method. MRI was utilized to evaluate all of the mice. An FXIIIa-targeted probe was applied to detect fibrin deposition in acute ischemic brain regions by fluorescence imaging. Necrosis and pathological changes of brain tissue were estimated via Hematoxylin and eosin staining while fibrin deposition was observed by immunohistochemistry.. Thrombolytic therapy improved AIS clinical symptoms. The infarct area of non-thrombolytic treated mice was significantly greater than that of the thrombolytic treated mice (p < .0001). Fluorescent imaging indicated fibrin deposition in ischemic brain tissue in both groups, with less fibrin in non-thrombolytic treated mice than thrombolytic treated mice, though the difference was not significant. Brain cells with abnormal morphology, necrosis, and liquefication were observed in the infarcted area for both groups. Clotted red blood cells (RBCs) and fibrin build-up in capillaries were found near the ischemic area in both non-thrombolytic and thrombolytic treated groups of mice.. Fibrin deposition and stacked RBCs contribute to microcirculation no-reflow in AIS after thrombolytic therapy.

Topics: Animals; Brain Ischemia; Fibrin; Fibrinolytic Agents; Infarction, Middle Cerebral Artery; Ischemic Stroke; Mice; Mice, Inbred C57BL; Molecular Imaging; Stroke; Tissue Plasminogen Activator

Ischemic stroke is characterized by the loss of cerebral blood flow, which frequently leads to neurological deficits. Tissue plasminogen activator is the only therapeutic agent approved to treat ischemic stroke but increases the risk of intracranial hemorrhage and mortality. The fibrinogen-depleting agent lumbrokinase has been used to improve myocardial perfusion in symptomatic stable angina and to prevent secondary ischemic stroke. Lumbrokinase is highly fibrin-specific and only active in the presence of fibrin. Therefore, lumbrokinase has a low risk of hemorrhage due to excessive fibrinolysis. In this study, we aimed to clarify the neuroprotection of lumbrokinase in mice subjected to permanent middle cerebral artery occlusion. Lumbrokinase significantly attenuated infarct volume and improved neurological dysfunction. Lumbrokinase dramatically decreased the expressions of the endoplasmic reticulum (ER) transmembrane receptor protein inositol-requiring enzyme-1 (IRE1) and its downstream transcription factor, XBP-1, caspase-12, and NF-κB activity, thereby significantly inhibiting apoptosis and autophagy and decreasing the NLRP3 inflammasome. Our evidence indicates that post-stroke treatment with lumbrokinase protects against ischemic stroke, thereby regulating ER stress through the collective inhibitory effect of the IRE1 signaling pathways to decrease apoptosis, autophagy, and inflammatory responses. We suggest that lumbrokinase is potential as an adjuvant treatment for ischemic stroke.

Topics: Animals; Apoptosis; Brain Ischemia; Endoplasmic Reticulum Stress; Fibrin; Infarction, Middle Cerebral Artery; Ischemic Stroke; Membrane Proteins; Mice; Protein Serine-Threonine Kinases; Tissue Plasminogen Activator

Endovascular treatment of stroke, although highly effective, may fail to reach complete recanalization in around 20% of cases. The Advanced Thrombectomy System (ANCD) is a novel stroke thrombectomy device designed to reduce clot fragmentation and facilitate retrieval by inducing local flow arrest and allowing distal aspiration in combination with a stent retriever. We aimed to assess the preclinical efficacy of ANCD.. Soft red blood cell (RBC)-rich (n=20/group) and sticky fibrin-rich (n=30/group) clots were used to create middle cerebral artery (MCA) occlusions in two vascular phantoms. Three different treatment strategies were tested: (1) balloon guide catheter + Solitaire (BGC+SR); (2) distal access catheter + SR (DAC+SR); and (3) ANCD+SR, until complete recanalization was achieved or to a maximum of three passes. The recanalization rate was determined after each pass.. After one pass, ANCD+SR resulted in an increased recanalization rate (94%) for all clots together compared with BGC+SR (66%; p<0.01) or DAC+SR (80%; p=0.04). After the final pass the recanalization rate increased in all three groups but remained higher with ANCD+SR (100%) than with BGC+SR (74%; p<0.01) or DAC+SR (90%; p=0.02). The mean number of passes was lower with ANCD+SR (1.06) than with BGC+SR (1.46) or DAC+SR (1.25) (p=0.01). A logistic regression model adjusted for treatment arm, clot type, and model used showed that both RBC-rich clots (OR 8.1, 95% CI 1.6 to 13.5) and ANCD+SR (OR 3.9, 95% CI 1.01 to 15.8) were independent predictors of first-pass recanalization.. In in vitro three-dimensional models replicating MCA-M1 occlusion, ANCD+SR showed significantly better recanalization rates in fewer passes than other commonly used combinations of devices.

Topics: Fibrin; Humans; Infarction, Middle Cerebral Artery; Models, Cardiovascular; Stents; Stroke; Surgical Instruments; Thrombectomy; Thrombosis; Treatment Outcome

The inflammatory process in stroke is the major contributor to blood-brain barrier (BBB) breakdown. Previous studies indicated that semaphorin 4D (Sema4D), an axon guidance molecule, initiated inflammatory microglial activation and disrupted endothelial function in the CNS. However, whether Sema4D disrupts BBB integrity after stroke remains unclear. To study the effect of Sema4D on BBB disruption in stroke, rats were subjected to transient middle cerebral artery occlusion and targeted injection of lentivirus-mediated clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene disruption of PlexinB1. We found that Sema4D synchronously increased with BBB permeability and accumulated in the perivascular area after stroke. Suppressing Sema4D/PlexinB1 signaling in the periinfarct cortex significantly decreased BBB permeability as detected by MRI and fibrin deposition, and thereby improved stroke outcome. In vitro, we confirmed that Sema4D disrupted BBB integrity and endothelial tight junctions. Moreover, we found that Sema4D induced pericytes to acquire a CD11b-positive phenotype and express proinflammatory cytokines. In addition, Sema4D inhibited AUF1-induced proinflammatory mRNA decay effect. Taken together, our data provides evidence that Sema4D disrupts BBB integrity and promotes an inflammatory response by binding to PlexinB1 in pericytes after transient middle cerebral artery occlusion. Our study indicates that Sema4D may be a novel therapeutic target for treatment in the acute phase of stroke.-Zhou, Y.-F., Li, Y.-N., Jin, H.-J., Wu, J.-H., He, Q.-W., Wang, X.-X., Lei, H., Hu, B. Sema4D/PlexinB1 inhibition ameliorates blood-brain barrier damage and improves outcome after stroke in rats.

Topics: Animals; Blood-Brain Barrier; CD11b Antigen; Cells, Cultured; Cytokines; Fibrin; Genetic Therapy; GTPase-Activating Proteins; Infarction, Middle Cerebral Artery; Lentivirus; Male; Pericytes; Rats; Rats, Sprague-Dawley; Receptors, Cell Surface

The efficacy and safety of recombinant tissue plasminogen activator (rtPA) need to be improved due to its low bioavailability and requirement of large dose administration. The purpose of this study was to develop a fibrin-targeted nanoparticle (NP) drug delivery system for thrombosis combination therapy. We conjugated rtPA to poly(ethylene glycol)- poly(e-caprolactone) (PEG-PCL) nanoparticles (rtPA-NP) and investigated its physicochemical characteristics such as particle size, zeta potential, enzyme activity of conjugated rtPA and its storage stability at 4°C. The thrombolytic activity of rtPA-NP was evaluated in vitro and in vivo as well as the half-life of rtPA-NP, the properties to fibrin targeting and its influences on systemic hemostasis in vivo. The results showed that rtPA-NP equivalent to 10% of a typical dose of rtPA could dissolve fibrin clots and were demonstrated to have a neuroprotective effect after focal cerebral ischemia as evidenced by decreased infarct volume and improved neurological deficit (P<0.001). RtPA-NP did not influence the in vivo hemostasis or coagulation system. The half-life of conjugated rtPA was shown to be approximately 18 times longer than that of free rtPA. These experiments suggested that rtPA-conjugated PEG-PCL nanoparticles might be a promising fibrin-targeted delivery system for a combination treatment of thrombosis.

Topics: Animals; Brain Ischemia; Disease Models, Animal; Fibrin; Fibrinolysis; Hemostasis; Infarction, Middle Cerebral Artery; Male; Nanoparticles; Neuroprotection; Particle Size; Rats, Sprague-Dawley; Recombinant Proteins; Static Electricity; Thrombosis; Tissue Plasminogen Activator

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 (αCD147) 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-κB (nuclear factor κB) 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.

Topics: Animals; Antibodies, Blocking; Basigin; Blood Platelets; Blood-Brain Barrier; Brain Ischemia; Fibrin; Infarction, Middle Cerebral Artery; Inflammation; Intracranial Thrombosis; Matrix Metalloproteinase 9; Mice; Mice, Inbred C57BL; Neuroprotective Agents; Stroke; Treatment Outcome

It is known that both platelets and coagulation strongly influence infarct progression after ischemic stroke, but the mechanisms and their interplay are unknown. Our aim was to assess the contribution of the procoagulant platelet surface, and thus platelet-driven thrombin generation, to the progression of thromboinflammation in the ischemic brain.. We present the characterization of a novel platelet and megakaryocyte-specific TMEM16F (anoctamin 6) knockout mouse. Reflecting Scott syndrome, platelets from the knockout mouse had a significant reduction in procoagulant characteristics that altered thrombin and fibrin generation kinetics. In addition, knockout mice showed significant defects in hemostasis and arterial thrombus formation. However, infarct volumes in a model of ischemic stroke were comparable with wild-type mice.. Platelet TMEM16F activity contributes significantly to hemostasis and thrombosis but not cerebral thromboinflammation. These results highlight another key difference between the roles of platelets and coagulation in these processes.

Topics: Animals; Anoctamins; Blood Coagulation; Blood Platelets; Carotid Artery Diseases; Disease Models, Animal; Encephalitis; Fibrin; Hemostasis; Infarction, Middle Cerebral Artery; Kinetics; Megakaryocytes; Mice, Inbred C57BL; Mice, Knockout; Phosphatidylserines; Phospholipid Transfer Proteins; Platelet Activation; Signal Transduction; Thrombin; Thrombosis

Ischaemic stroke is a serious disease with limited therapy options. Glycoprotein (GP)Ib binding to von Willebrand factor (vWF) exposed at vascular injury initiates platelet adhesion and contributes to platelet aggregation. GPIb has been suggested as an effective target for antithrombotic therapy in stroke. Anfibatide is a GPIb antagonist derived from snake venom and we investigated its protective effect on experimental brain ischaemia in mice.. Focal cerebral ischaemia was induced by 90 min of transient middle cerebral artery occlusion (MCAO). These mice were then treated with anfibatide (4, 2, 1 μg·kg(-1) ), injected i.v., after 90 min of MCAO, followed by 1 h of reperfusion. Tirofiban, a GPIIb/IIIα antagonist, was used as a positive control.. Twenty-four hours after MCAO, anfibatide-treated mice showed significantly improved ischaemic lesions in a dose-dependent manner. The mice had smaller infarct volumes, less severe neurological deficits and histopathology of cerebrum tissues compared with the untreated MCAO mice. Moreover, anfibatide decreased the amount of GPIbα, vWF and accumulation of fibrin(ogen) in the vasculature of the ischaemic hemisphere. Tirofiban had similar effects on infarct size and fibrin(ogen) deposition compared with the MCAO group. Importantly, the anfibatide-treated mice showed a lower incidence of intracerebral haemorrhage and shorter tail bleeding time compared with the tirofiban-treated mice.. Our data indicate anfibatide is a safe GPIb antagonist that exerts a protective effect on cerebral ischaemia and reperfusion injury. Anfibatide is a promising candidate that could be beneficial for the treatment of ischaemic stroke.

Topics: Animals; Bleeding Time; Blood Platelets; Brain Ischemia; Cell Count; Cerebral Hemorrhage; Crotalid Venoms; Dose-Response Relationship, Drug; Fibrin; Infarction, Middle Cerebral Artery; Lectins, C-Type; Male; Mice; Platelet Glycoprotein GPIb-IX Complex; Protective Agents; Reperfusion Injury; Stroke; Tirofiban; Tyrosine; von Willebrand Factor

Downstream microvascular thrombosis (DMT) is known to be a contributing factor to incomplete reperfusion in acute ischemic stroke. The aim of this study was to determine the timing of DMT with intravital imaging and to test the hypothesis that intravenous alteplase infusion could reduce DMT in a transient middle cerebral artery occlusion (MCAO) rat stroke model.. Rats were subjected to 60-minute transient MCAO. Alteplase (10 mg/kg) was administered 30 minutes after the beginning of MCAO. Real-time intravital fluorescence microscopy through a dura-sparing craniotomy was used to visualize circulating blood cells and fibrinogen. Cerebral microvessel patency was quantitatively evaluated by fluorescein isothiocyanate-dextran perfusion.. Immediately after MCAO, platelet and leukocyte accumulation were observed mostly in the venous compartment. Within 30 minutes after MCAO, microthrombi and parietal fibrin deposits were detected in postcapillary microvessels. Alteplase treatment significantly (P=0.006) reduced infarct volume and increased the percentage of perfused vessels during MCAO (P=0.02) compared with saline. Plasma levels of fibrinogen from alteplase-treated rats showed a rapid and profound hypofibrinogenemia. In vitro platelet aggregation demonstrated that alteplase reduced platelet aggregation (P=0.0001) and facilitated platelet disaggregation (P=0.001). These effects were reversible in the presence of exogenous fibrinogen.. Our data demonstrate that DMT is an early phenomenon initiated before recanalization. We further show that alteplase-dependent maintenance of downstream perfusion during MCAO improves acute ischemic stroke outcome through a fibrinogen-dependent platelet aggregation reduction. Our results indicate that early targeting of DMT represents a therapeutic strategy to improve the benefit of large artery recanalization in acute ischemic stroke.

Topics: Animals; Blood Platelets; Disease Models, Animal; Fibrin; Fibrinogen; Fibrinolytic Agents; Infarction, Middle Cerebral Artery; Intracranial Thrombosis; Leukocytes; Male; Microscopy, Fluorescence; Microvessels; Platelet Aggregation; Rats; Rats, Sprague-Dawley; Reperfusion; Tissue Plasminogen Activator

The primary limitation of thrombolytic treatment of ischemic stroke with tissue plasminogen activator (tPA) is the hemorrhagic risk. We tested AcSDKP (N-acetyl-seryl-aspartyl-lysyl-proline), as an auxiliary therapeutic agent, to reduce blood-brain barrier (BBB) disruption in a combination tPA thrombolytic treatment of stroke. Wistar rats subjected to embolic stroke were randomly assigned to either the tPA monotherapy group (n=9) or combination of tPA and AcSDKP treatment group (n=9) initiated at 4 h after ischemia. Magnetic resonance imaging (MRI) measurements were performed before and after the treatments. Immunohistochemical staining and measurements were performed to confirm MRI findings. Longitudinal MRI permeability measurements with gadolinium-diethylenetriamine penta-acetic acid (Gd-DTPA) demonstrated that combination treatment of acute embolic stroke with AcSDKP and tPA significantly reduced BBB leakage, compared to tPA monotherapy, at 3 and 6 days (18.3±9.8 mm3 vs. 65.0±21.0 mm3, p<0.001) after the onset of stroke, although BBB leakage was comparable between the two groups prior to the treatments (6.8±4.4 mm3 vs. 4.3±3.3 mm3, p>0.18). The substantial reduction of BBB leakage observed in the combination treatment group was closely associated with reduced ischemic lesions measured by T2 maps (113.6±24.9 mm3 vs. 188.1±60.8 mm3, p<0.04 at 6 days). Histopathological analysis of the same population of rats showed that the combination treatment significantly reduced parenchymal fibrin deposition (0.063±0.059 mm2 vs. 0.172±0.103 mm2, p<0.03) and infarct volume (146.7±35.9 mm3 vs. 199.3±60.4 mm3, p<0.05) compared to the tPA monotherapy at 6days after stroke. MRI provides biological insight into the therapeutic benefit of combination treatment of stroke with tPA and AcSDKP 4h after onset, and demonstrates significantly improved cerebrovascular integrity with neuroprotective effects compared with tPA monotherapy.

Topics: Acute Disease; Animals; Blood-Brain Barrier; Brain; Capillary Permeability; Contrast Media; Disease Models, Animal; Drug Therapy, Combination; Fibrin; Fibrinolytic Agents; Gadolinium DTPA; Immunohistochemistry; Infarction, Middle Cerebral Artery; Longitudinal Studies; Magnetic Resonance Imaging; Male; Neuroprotective Agents; Oligopeptides; Rats, Wistar; Tissue Plasminogen Activator

The purpose of this study was to provide the first correlative study of the hyperdense middle cerebral artery sign (HMCAS) and gradient-echo MRI blooming artifact (BA) with pathology of retrieved thrombi in acute ischemic stroke.. Noncontrast CT and gradient-echo MRI studies before mechanical thrombectomy in 50 consecutive cases of acute middle cerebral artery ischemic stroke were reviewed blinded to clinical and pathology data. Occlusions retrieved by thrombectomy underwent histopathologic analysis, including automated quantitative and qualitative rating of proportion composed of red blood cells (RBCs), white blood cells, and fibrin on microscopy of sectioned thrombi.. Among 50 patients, mean age was 66 years and 48% were female. Mean (SD) proportion was 61% (±21) fibrin, 34% (±21) RBCs, and 4% (±2) white blood cells. Of retrieved clots, 22 (44%) were fibrin-dominant, 13 (26%) RBC-dominant, and 15 (30%) mixed. HMCAS was identified in 10 of 20 middle cerebral artery stroke cases with CT with mean Hounsfield Unit density of 61 (±8 SD). BA occurred in 17 of 32 with gradient-echo MRI. HMCAS was more commonly seen with RBC-dominant and mixed than fibrin-dominant clots (100% versus 67% versus 20%, P=0.016). Mean percent RBC composition was higher in clots associated with HMCAS (47% versus 22%, P=0.016). BA was more common in RBC-dominant and mixed clots compared with fibrin-dominant clots (100% versus 63% versus 25%, P=0.002). Mean percent RBC was greater with BA (42% versus 23%, P=0.011).. CT HMCAS and gradient-echo MRI BA reflect pathology of occlusive thrombus. RBC content determines appearance of HMCAS and BA, whereas absence of HMCAS or BA may indicate fibrin-predominant occlusive thrombi.

Topics: Aged; Aged, 80 and over; Artifacts; Blood Platelets; Erythrocytes; Female; Fibrin; Humans; Infarction, Middle Cerebral Artery; Leukocytes; Magnetic Resonance Imaging; Male; Middle Aged; Retrospective Studies; Stroke; Thrombectomy; Thrombosis; Tomography, X-Ray Computed

Mechanical behavior of the thromboembolus is one of the key factors that determine the efficacy of thrombectomy devices for revascularization in AIS. We characterized the mechanical properties and composition of thromboemboli from clinical cases and compared them with commonly used EAs.. Thromboemboli were obtained from patients with AIS by using aspiration devices and from carotid atherosclerotic plaques harvested during endarterectomy. In the laboratory, common EAs were created by varying blood donor species (human, porcine, and bovine), thrombin concentration, and presence of barium sulfate. Stiffness and elasticity of the specimens were measured with DMA. Scanning electron microscopy and histology were used to investigate the ultrastructure and composition of all specimens.. Red thromboemboli from patients composed mainly of fibrin and erythrocytes were much softer than the calcified and cholesterol-rich material. Of the EAs created in the laboratory, those made from bovine blood presented the highest stiffness that was independent of thrombin concentration. Addition of thrombin increased the stiffness and elasticity of human and porcine EAs (P < .05). The presence of barium sulfate significantly reduced the elasticity of all EAs (P < .05).. Endovascular device testing and development requires realistic EAs. The stiffness and elasticity of the cerebral thromboemboli analyzed in this study were closely matched by recalcified porcine EAs and thrombin-induced human EAs. Stiffness of the thrombus extracted from carotid endarterectomy specimens was similar with that of the thrombin-induced bovine and porcine EAs.

Topics: Acute Disease; Aged; Animals; Barium Sulfate; Biomechanical Phenomena; Brain Ischemia; Cattle; Elasticity; Erythrocytes; Fibrin; Humans; Infarction, Middle Cerebral Artery; Male; Species Specificity; Stress, Mechanical; Swine; Thrombectomy; Thrombin; Thromboembolism

Topics: Acyclovir; Antiviral Agents; Blood Coagulation Disorders; Female; Fibrin; Herpes Zoster; Humans; Hyperlipidemias; Infarction, Middle Cerebral Artery; Lymphangiectasis, Intestinal; Middle Aged; Paraproteinemias; Protein S; Protein-Losing Enteropathies; Urinary Tract Infections

Suppression of platelet activation improves the efficacy of thrombolytic therapy for stroke. Thus, combination treatment with recombinant tissue plasminogen activator (r-tPA) and 7E3 F(ab')2, a GPIIb/IIIa inhibitor that binds the platelet to fibrin, may improve the efficacy of thrombolytic therapy in embolic stroke. Magnetic resonance imaging (MRI) was used to monitor treatment response in rats subjected to embolic middle cerebral artery (MCA) occlusion (MCAo). Animals were randomized into treated (n=12) and control (n=10) groups and received intravenous combination therapy or saline, respectively, 4 hours after MCAo. Magnetic resonance imaging (MRI) measurements performed 1 hour after MCAo showed no difference between groups. However, an increased incidence (50%) of MCA recanalization was found in the treated group at 24 hours compared with 20% in the control group. The area of low cerebral blood flow at 24 and 48 hours was significantly smaller in the combination treatment group, and the lesion size, as indicated from the T2 and T1 maps, differed significantly between groups. Fluorescence microscopy measurements of cerebral microvessels perfused with fluorescein isothiocyanate-dextran and measurements of infarct volume revealed that the combination treatment significantly increased microvascular patency and reduced infarct volume, respectively, compared with the control rats. The efficacy of combination treatment 4 hours after ischemia is reflected by MRI indices of tissue perfusion, MCA recanalization, and reduction of lesion volume. The treatment also reduced secondary microvascular perfusion deficits.

Topics: Animals; Antibodies, Monoclonal; Blood Platelets; Brain; Cerebrovascular Circulation; Dextrans; Drug Therapy, Combination; Echo-Planar Imaging; Fibrin; Fluorescein-5-isothiocyanate; Immunoglobulin Fab Fragments; Infarction, Middle Cerebral Artery; Intracranial Embolism and Thrombosis; Male; Microcirculation; Microscopy, Fluorescence; Middle Cerebral Artery; Plasminogen Activators; Platelet Aggregation; Platelet Glycoprotein GPIIb-IIIa Complex; Radiography; Rats; Rats, Wistar; Recombinant Proteins; Stroke; Tissue Plasminogen Activator

It is known from autopsy studies that thromboembolic stroke can be caused by red, white and mixed clots. We therefore examined whether the efficacy of thrombolysis with recombinant tissue-type plasminogen activator (rt-PA) depends on the proportions of fibrin and erythrocytes within thromboembolic material.. In 23 rabbits intraarterial thrombolysis with 3 mg rt-PA/kg body weight was started 30 minutes after middle cerebral artery occlusion with either red or white autologous emboli 20 hours old. 20 rabbits served as control. Cerebral perfusion was monitored by MRI.. rt-PA enhanced lysis of red but not of white emboli and decreased the infarct volume only if vascular occlusion was due to red emboli (p <.01). Cerebral perfusion improved only in the red treatment group where the normalized first moment (NFM) decreased (p <.05) and the relative regional cerebral blood volume (rrCBV) reached normal values (p <.05).. We suggest that in our animal model the efficacy of thrombolysis increases with the proportion of erythrocytes within thromboembolic material and decreases with its content of fibrin. lf these findings would also be applicable to patients, pretherapeutic estimation of the efficacy of thrombolysis might become feasible because the CT values of red and white thrombi differ.

Topics: Animals; Cerebrovascular Circulation; Data Interpretation, Statistical; Disease Models, Animal; Erythrocytes; Fibrin; Fibrinolytic Agents; Infarction, Middle Cerebral Artery; Intracranial Embolism and Thrombosis; Male; Plasminogen Activators; Prognosis; Rabbits; Stroke; Thrombolytic Therapy; Time Factors; Tissue Plasminogen Activator

We evaluated the neuroprotective effect of UK-279,276 (also referred to as recombinant neutrophil inhibitory factor), a selective CD11b/CD18 antagonist, in combination with thrombolytic therapy on focal cerebral ischemia.. Male Wistar rats (n=88) were subjected to embolic middle cerebral artery occlusion. Animals were randomly assigned to the following groups (n=11 in each group): vehicle treatment alone at 2 or 4 hours, UK-279,276 treatment alone at 2 or 4 hours, recombinant human tissue plasminogen activator (rhtPA) treatment alone at 2 or 4 hours, or the combination of UK-279,276 and rhtPA at 2 or 4 hours. Infarct volume, neurological function, hemorrhagic transformation, neutrophil accumulation, and parenchymal fibrin deposition were measured 7 days after middle cerebral artery occlusion.. Treatment with UK-279,276 significantly (P<0.05) improved neurological severity scores, an index of neurological functional deficit, but had no effect on infarct volume compared with vehicle-treated animals. Treatment with rhtPA alone at 2 but not 4 hours significantly (P<0.05) reduced infarct volume and improved neurological function compared with vehicle-treated animals. Combination treatment with UK-279,276 and rhtPA at 2 or 4 hours significantly (P<0.01) reduced infarct volume and enhanced recovery of neurological function compared with control. Neutrophil accumulation and fibrin deposition in the brain parenchyma of combination-treated rats at 2 and 4 hours after stroke were significantly reduced (P<0.05) compared with corresponding vehicle-treated control groups. The neuroprotective effect of the combined treatments was superior to the additive effects from each treatment of rhtPA or UK-279,276 alone.. These data suggest that the combination treatment with UK-279,276 and rhtPA may extend the window of thrombolytic therapy for the acute treatment of stroke.

Topics: Animals; Body Weight; Brain; CD11b Antigen; Cerebral Hemorrhage; Disease Models, Animal; Fibrin; Glycoproteins; Helminth Proteins; Humans; Infarction, Middle Cerebral Artery; Intracranial Embolism; Male; Membrane Proteins; Neurologic Examination; Neuroprotective Agents; Peroxidase; Rats; Rats, Wistar; Recombinant Proteins; Severity of Illness Index; Stroke; Tissue Plasminogen Activator

Thrombin-induced clots used in experimental thromboembolic stroke differ from clots forming spontaneously under clinical conditions. We investigated whether this difference influences the efficacy and outcome of thrombolytic treatment.. In rats, the middle cerebral artery was occluded by intracarotid injection of fibrin-rich clots, prepared either according to established methods by adding thrombin to freshly drawn arterial blood or by spontaneous coagulation. The mechanical properties of clots were determined in vitro by elasticity and plasticity tests. One hour after embolism, thrombolysis was started by intra-arterial application of recombinant tissue plasminogen activator (rtPA) (10 mg/kg). Treatment efficacy was monitored by MR measurements of blood perfusion, apparent diffusion coefficient (ADC), T2 relaxation time and blood-brain barrier permeability, and by pictorial measurements of ATP and pH.. Thrombin-induced clots were classified as elastic, and spontaneously forming clots were classified as plastic. Middle cerebral artery embolism with thrombin-induced or spontaneously forming clots led to similar reduction of perfusion and ADC, but rtPA treatment efficacy differed greatly. In the spontaneously forming clot group, blood perfusion returned to or above control within 2 hours, ADC and ATP normalized, tissue pH exhibited alkalosis, and T2 and blood-brain barrier permeability did not change. In the thrombin-induced clot group, in contrast, blood reperfusion was delayed, ADC and ATP remained reduced, tissue pH was acidic, and edema developed, as reflected by increased T2 and blood-brain barrier permeability.. rtPA-induced thrombolysis promotes rapid reperfusion and tissue recovery in animals embolized with spontaneously forming clots but not in those embolized with thrombin-induced clots. This difference is explained by the different mechanical and possibly molecular consequences of clot preparation and must be considered for the interpretation of thrombolysis experiments.

Topics: Adenosine Triphosphate; Animals; Blood-Brain Barrier; Brain; Cerebral Hemorrhage; Cerebrovascular Circulation; Disease Models, Animal; Disease Progression; Elasticity; Extravasation of Diagnostic and Therapeutic Materials; Fibrin; Gadolinium DTPA; Infarction, Middle Cerebral Artery; Intracranial Thrombosis; Magnetic Resonance Angiography; Male; Rats; Rats, Wistar; Recombinant Proteins; Reperfusion; Stroke; Thrombin; Thrombolytic Therapy; Time Factors; Tissue Plasminogen Activator; Treatment Outcome

Although thromboembolic stroke is caused by red, white, or mixed clots, the emboli previously used in animal studies on thrombolysis were more often red than white. Because this might be one of the reasons why thrombolysis is less effective in patients than in experimental stroke, we developed a new method of preparing highly standardized red and fibrin-rich white emboli.. The middle cerebral artery of 20 rabbits was embolized with either red or fibrin-rich white autologous emboli. Cerebral perfusion was monitored by MRI.. Red emboli consisted of closely packed erythrocytes within a sparse fibrin net and white emboli of a dense mass of fibrin with only few other blood cells. Infarct volumes were 26+/-4% (mean+/-SD) of the ischemic hemisphere with red and 27+/-6% with white emboli. The relative regional cerebral blood volume dropped below 50% 90 minutes after vascular occlusion with either type of embolus. Late spontaneous lysis and hemorrhagic complications occurred in 37.5% of red but not in white embolus cases.. Emboli prepared by our technique result in standardized cerebral infarctions. Size and composition of the emboli continuously can be adjusted according to the experimental requirements.

Topics: Angiography, Digital Subtraction; Animals; Brain; Cerebral Angiography; Disease Models, Animal; Erythrocytes; Fibrin; Infarction, Middle Cerebral Artery; Intracranial Embolism; Male; Rabbits; Reproducibility of Results; Stroke

Activated protein C (APC) contributes to systemic anticoagulant and anti-inflammatory activities. APC may reduce organ damage by inhibiting thrombin generation and leukocyte activation. Neutrophils and cerebrovascular thrombosis contribute to ischemic neuronal injury, suggesting that APC may be a potential protective agent for stroke.. We examined the effects of APC in a murine model of focal ischemia. After middle cerebral artery occlusion/reperfusion, the average survival time in controls was 13.6 hours. Animals that received purified human plasma-derived APC 2 mg/kg IV either 15 minutes before or 10 minutes after stroke induction survived 24 hours and were killed for neuropathological analysis. APC 2 mg/kg given before or after onset of ischemia restored cerebral blood flow, reduced brain infarct volume (59% to 69%; P:<0.003) and brain edema (50% to 61%; P:<0.05), eliminated brain infiltration with neutrophils, and reduced the number of fibrin-positive cerebral vessels by 57% (P:<0.05) and 25% (nonsignificant), respectively. The neuroprotective effect of APC was dose-dependent and associated with significant inhibition of ICAM-1 expression on ischemic cerebral blood vessels (eg, 61% inhibition with 2 mg/kg APC). Intracerebral bleeding was not observed with APC.. APC exerts anti-inflammatory, antithrombotic, and neuroprotective effects in stroke. Central effects of APC are likely to be related to improved maintenance of the blood-brain barrier to neutrophils and to reduced microvascular obstructions and fibrin deposition.

Topics: Animals; Anti-Inflammatory Agents; Brain Edema; Brain Ischemia; Cerebrovascular Circulation; Chemotaxis, Leukocyte; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme Activation; Fibrin; Fibrinolytic Agents; Hemoglobins; Humans; Infarction, Middle Cerebral Artery; Intercellular Adhesion Molecule-1; Macrophage-1 Antigen; Mice; Mice, Inbred C57BL; Neuroprotective Agents; Neutrophils; Optic Chiasm; Protein C; Psychomotor Performance; Reperfusion Injury; Survival Rate

Argatroban, a specific thrombin inhibitor, has been shown to reduce ischemic lesion size after focal cerebral ischemia in rats. In addition, recombinant tissue plasminogen activator (rtPA) has been shown to reduce ischemic lesion size in both rats and humans if given within 3 hours of symptom onset. We tested the hypothesis that the administration of argatroban with rtPA could extend the treatment window of stroke to 4 hours without increasing gross cerebral hemorrhage rates or reducing efficacy.. Male Wistar rats were subjected to middle cerebral artery (MCA) occlusion by a single fibrin-rich clot. After embolization, rats were administered argatroban at the following dose levels: 2.08, 6.25, and 18.75 microgram . kg(-1). min(-1). In a second experiment, rats received argatroban (6.25 microgram . kg(-1). min(-1)) or argatroban in combination with rtPA 4 hours after MCA occlusion. Tissue sections were then analyzed for lesion volume, gross hemorrhage and fibrin deposition.. The 6.25 microgram. kg(-1). min(-1) dose demonstrated a significant reduction (P<0.05) in lesion volume after 48 hours (27.2+/-6.3%) compared with controls (35.3+/-3.7%). A significant reduction (P<0.05) in lesion volume was observed in the argatroban-plus-rtPA group (17.1+/-10.4%) compared with controls (35.3+/-3.7%). No increase in hemorrhagic transformation was observed. Fibrin deposition in the ipsilateral cortical microvasculature was significantly decreased in the 4-hour combination argatroban-plus-rtPA group compared with the controls (P<0.05).. This study demonstrates that the combination of argatroban and rtPA extends the window of opportunity for treatment of stroke to 4 hours without increasing hemorrhagic transformation.

Topics: Animals; Antithrombins; Arginine; Brain Ischemia; Cerebral Hemorrhage; Dose-Response Relationship, Drug; Fibrin; Incidence; Infarction, Middle Cerebral Artery; Intracranial Embolism; Male; Pipecolic Acids; Rats; Rats, Wistar; Recombinant Proteins; Stroke; Sulfonamides; Time Factors; Tissue Plasminogen Activator