Cerebral Thrombi Research Articles

Assessment of associations between neutrophil extracellular trap biomarkers in blood and thrombi in acute ischemic stroke patients

Inflammation including immunothrombosis by neutrophil extracellular traps (NETs) has important implications in acute ischemic stroke and can affect reperfusion status, susceptibility to stroke associated infections (SAI) as well as functional clinical outcome. NETs were shown to be prevalent in stroke thrombi and NET associated markers were found in stroke patients’ blood. However, little is known whether blood derived NET markers reflect the amount of NETs in thrombi. Conclusions from blood derived markers to thrombus composition might open avenues for novel strategies in diagnostic and therapeutic approaches. We prospectively recruited 166 patients with acute ischemic stroke undergoing mechanical thrombectomy between March 2018 and May 2021. Available thrombi (n = 106) were stained for NET markers DNA-histone-1 complexes and myeloperoxidase (MPO). Cell free DNA (cfDNA), deoxyribonuclease (DNase) activity, MPO-histone complexes and a cytokine-panel were measured before thrombectomy and after seven days. Clinical data, including stroke etiology, reperfusion status, SAI and functional outcome after rehabilitation, were collected of all patients. NET markers were present in all thrombi. At onset the median concentration of cfDNA in blood was 0.19 µg/ml increasing to 0.30 µg/ml at 7 days. Median DNase activity at onset was 4.33 pmol/min/ml increasing to 4.96 pmol/min/ml at 7 days. Within thrombi DNA-histone-1 complexes and MPO correlated with each other (ρ = 0.792; p < 0.001). Moreover, our study provides evidence for an association between the amount of NETs and endogenous DNase activity in blood with amounts of NETs in cerebral thrombi. However, these associations need to be confirmed in larger cohorts, to investigate the potential clinical implications for individualized therapeutic and diagnostic approaches in acute ischemic stroke.Graphical

Coagulation Factor Expression and Composition of Arterial Thrombi in Cancer-Associated Stroke.

Cancer is associated with an increased risk of stroke. Tumor cells activate platelets, induce a coagulation cascade, and generate thrombin. The composition of thrombi may reflect the mechanism of thrombosis, aiding the determination of the treatment strategy. Here, we investigated the composition and expression of coagulation factors in the thrombi of patients with cancer-associated stroke. Patients with stroke who underwent endovascular thrombectomy between September 2014 and June 2020 and whose cerebral thrombi were obtained were divided into those with cancer-associated stroke (cancer group) and propensity score-matched patients without cancer (control group), using 1:1 matching based on age and sex. Immunohistochemistry was performed of the thrombi, and the composition and expression of coagulation factors were compared between groups. Among the 320 patients who underwent endovascular thrombectomy and who had thrombi obtained, this study included 23 patients with cancer and 23 matched controls. In both groups, the median age was 65 years, and 12 patients (52.2%) were men. Platelet composition was significantly higher in the cancer group than in the control group (median [interquartile range], 51.3% [28.0%-61.4%] versus 9.5% [4.8%-14.0%]; P<0.001). Among coagulation factors, thrombin (26.2% [16.2%-52.7%] versus 4.5% [1.3%-7.2%]; P<0.001) and tissue factors (0.60% [0.34%-2.06%] versus 0.37% [0.22%-0.60%]; P=0.024) were higher and factor X was lower (1.25% [0.39%-3.60%] versus 2.33% [1.67%-4.48%]; P=0.034) in the cancer group. There was a positive correlation between thrombin and platelets in the cancer group (r=0.666; P=0.001) but not in the control group (r=-0.167; P=0.627). Cerebral thrombi in patients with cancer-associated stroke showed higher proportions of platelets, thrombin, and tissue factors, suggesting their key roles in arterial thrombosis in cancer and providing a therapeutic perspective for preventing stroke in patients with cancer-associated stroke.

Acute ischemic stroke: how to investigate the association between disease etiology and gene expression profiles

Background: Acute ischemic stroke (AIS) represents one of the principal causes of neurological morbidity and mortality worldwide. For a prompt and efficient cerebral blood restoration, intravenous treatment with rt-PA is often combined with mechanical thrombectomy (MT) which provides cerebral thrombi (CT) as study material, allowing the investigation of its cellular composition, morphological and histopathological features. Indeed, the determination of stroke etiology, typically defined by the TOAST classification, is paramount for prognostic factors, outcome, and management of the event. Aim of the study is therefore to highlight and analyze gene expression profiles in thrombotic tissue and peripheral blood (PB) in the comparison between strokes of cardioembolic (CE) and atherosclerotic (LAA) origin. Methods: We performed gene expression profiles of 92 patients. CT were stored in RNA later and RNA was extracted by PAX gene blood miRNA kit. The global gene expression profile was assessed by Affymetrix technology using GeneChip Human Transcriptome Array 2.0 combined with Affymetrix Transcriptome Analysis Console (TAC) Software. Results: Currently, we focused our attention on CT data analysis. The analysis revealed a significant difference (p-value&lt;0.05 and FoldChange=2 as threshold) in gene expression when comparing LAA and CE stroke. In particular, from CT of atherosclerotic origin emerges an overexpression of 1766 genes. Prominent among them are genes such as MMP-9, TGFB, TGFBR and CXCL1, primarily involved in neutrophil-mediated immunity, Blood-Brain Barrier (BBB) disruption processes, and associated with atherosclerotic plaque instability and related to poor neurological outcome, suggesting a deleterious role in human brain injury. As concerns CE patients, 57 genes mainly involved in transcriptional regulatory processes turn out to be significantly overexpressed. Conclusions: Transcriptome profiling is a powerful weapon for revealing expression patterns associated with complex disorders. The variation of gene expression profiles confirmed and extended several known pathophysiological mechanisms and may be one way of delineating different stroke etiology.

Advancing Stroke Research on Cerebral Thrombi with Omic Technologies.

Cerebrovascular diseases represent a leading cause of disability, morbidity, and death worldwide. In the last decade, the advances in endovascular procedures have not only improved acute ischemic stroke care but also conceded a thorough analysis of patients' thrombi. Although early anatomopathological and immunohistochemical analyses have provided valuable insights into thrombus composition and its correlation with radiological features, response to reperfusion therapies, and stroke etiology, these results have been inconclusive so far. Recent studies applied single- or multi-omic approaches-such as proteomics, metabolomics, transcriptomics, or a combination of these-to investigate clot composition and stroke mechanisms, showing high predictive power. Particularly, one pilot studies showed that combined deep phenotyping of stroke thrombi may be superior to classic clinical predictors in defining stroke mechanisms. Small sample sizes, varying methodologies, and lack of adjustments for potential confounders still represent roadblocks to generalizing these findings. However, these techniques hold the potential to better investigate stroke-related thrombogenesis and select secondary prevention strategies, and to prompt the discovery of novel biomarkers and therapeutic targets. In this review, we summarize the most recent findings, overview current strengths and limitations, and present future perspectives in the field.

Machine Learning Analysis of the Cerebrovascular Thrombi Lipidome in Acute Ischemic Stroke.

OBJECTIVE: The aim of this study was to identify a signature lipid profile from cerebral thrombi in acute ischemic stroke (AIS) patients at the time of ictus. METHODS: We performed untargeted lipidomics analysis using liquid chromatography-mass spectrometry on cerebral thrombi taken from a nonprobability, convenience sampling of adult subjects (≥18 years old, n = 5) who underwent thrombectomy for acute cerebrovascular occlusion. The data were classified using random forest, a machine learning algorithm. RESULTS: The top 10 metabolites identified from the random forest analysis were of the glycerophospholipid species and fatty acids. CONCLUSION: Preliminary analysis demonstrates feasibility of identification of lipid metabolomic profiling in cerebral thrombi retrieved from AIS patients. Recent advances in omic methodologies enable lipidomic profiling, which may provide insight into the cellular metabolic pathophysiology caused by AIS. Understanding of lipidomic changes in AIS may illuminate specific metabolite and lipid pathways involved and further the potential to develop personalized preventive strategies.

Thrombus-associated microbiota in acute ischemic stroke patients.

Background: Despite a reduction in stroke incidence and age-standardized death rates, stroke remains a leading cause of death and disability worldwide. Significant interest in recent years has focused on the microbiota-host interaction because accumulating evidence has revealed myriad ways in which bacteria may contribute to risk of stroke and adverse outcomes after stroke. The emergence of endovascular thrombectomy as a treatment provides a unique opportunity to utilize thrombus retrieved from cerebral arteries to fill knowledge gaps about the influence of bacteria on stroke pathophysiology. While bacterial signatures have been confirmed in cerebral thrombi, the exact nature of the pathogenesis has not been established.Methods: Thrombi were obtained from a cohort of adult ischemic stroke patients during standard of care thrombectomy. After DNA extraction and quantification, thrombi underwent 16S rRNA amplicon-based metagenomic sequencing, followed by bioinformatics processing. Taxonomic identification of bacterial colonies isolated on Agar plates from plated suspension was performed using DNA extraction and full length 16S Sanger sequencing.Results: A broad diversity of bacterial signatures was identified in specimens, primarily of cariogenic origin.Conclusion: In this small study, we demonstrate proof of concept and technical feasibility for amplicon-based metagenomic sequencing of arterial thrombi and briefly discuss preliminary findings, challenges, and near-term translational opportunities for thrombus genomics.

Cohort study on the differential expression of inflammatory and angiogenic factors in thrombi, cerebral and peripheral plasma following acute large vessel occlusion stroke

Inflammation plays an important role in the pathogenesis of stroke. The differential expression of inflammatory and angiogenic factors in thrombi and plasma remain undefined. In this observational cohort study, we evaluated angiogenic factors and inflammatory cytokines, in cerebral thrombi, local cerebral plasma (CP), and peripheral plasma (PP) in patients with acute ischemic stroke. Protein analysis of thrombi, CP and PP were used to measure angiogenic and inflammatory proteins using electrochemiluminescence. Our data indicate that VEGF-A, VEGF-C, bFGF, IL-4, IL-13, IL-1β, IL-2, IL-8, IL-16, IL-6 and IL-12p70 were higher in the thrombi of acute ischemic stroke patients than in the CP and PP of stroke patients. Moreover, the protein levels of GM-CSF were lower in the PP than in the CP and the clot. Moreover, VEGF-D, Flt-1, PIGF, TIE-2, IL-5, TNF-β, IL-15, IL-12/IL-23p40, IFN-γ and IL-17A were higher in PP and CP than in thrombi. Our results show that cytokines mediating the inflammatory response and proteins involved in angiogenesis are differentially expressed in thrombi within the cerebral and peripheral circulations. These data highlight the importance of identifying new biomarkers in different compartments of the circulatory system and in thrombi that may be used for the diagnosis and treatment of stroke patients.

Abstract 356: Phospholipid Profile Of Cerebral Thrombi In Acute Ischemic Stroke

Background and Purpose: The University of Washington Clot Bank (UWCB) has established a tissue banking protocol optimized for “omic” analysis for cerebral thrombi retrieved during standard-of-care thrombectomy. Thrombi from cerebral arteries of acute ischemic stroke (AIS) patients are a novel resource that allow for the evaluation of lipid biological markers present at the time of ictus. The pathophysiological processes following AIS are complex, and involve ischemia, inflammation, and disturbances of lipid metabolism. The purpose of this study was to characterize the lipid profile from cerebral thrombi in AIS patients. Methods: The UWCB study is a non-probability, convenience sampling of adult subjects (&gt; 18 years of age) who undergo thrombectomy for vascular occlusion. Lipidomics analysis was performed using LC-MS of untargeted lipids on thrombi taken from cerebral arteries of AIS subjects who underwent thrombectomy. Random forest, a supervised machine learning method, was employed to identify lipids associated with cerebral thrombi. Results: Five subjects (age = 53 ± 26.4, 3 males) were included from the UWCB registry. The most common comorbid conditions were atrial fibrillation (40%) and hypertension (40%). The top 10 metabolites identified from the random forest analysis, were of phosphatidylethanolamine molecular species. Phospholipids are involved in structural and functional integrity of cellular membranes and mediate host inflammatory responses. They are a key component of bacterial membranes. Changes in the structure or function of membrane phospholipids of host or relevant human microbiota may lead to alterations in membrane permeability and contribute to pathology of diseases such as neurodegeneration. Conclusion: An untargeted LC-MS lipidomics approach was used to identify lipids in cerebral thrombi at the time of mechanical thrombectomy. The top 10 lipids present in cerebral thrombi were identified, which included significant concentrations of phosphatidylethanolamine species. Because phospholipids play a key regulatory role in complex downstream cellular function, these findings may be of great significance in understanding AIS pathophysiology that has the potential to inform personalized preventive strategies.

Infarct growth precedes cerebral thrombosis following experimental stroke in mice

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.

Abstract 10890: Microbial Community Structure of Dental Plaque, Blood and Cerebral Thrombus in Patients with Acute Ischemic Stroke

Introduction: Epidemiological investigations have demonstrated a strong link between periodontal disease and stroke. However, relevant microbiological researches are still lacking at present. This study aimed to investigate the microbial composition of dental plaque, blood and cerebral thrombus in patients with acute ischemic stroke (AIS). Hypothesis: We hypothesized that there was a possible translocation for oral bacteria from periodontal pocket into blood and subsequently involving in the process of cerebral thrombus formation. Methods: Thrombus aspirates, dental plaque and venous blood samples were taken from 13 patients (11 males, 65.5 ± 10.7 years) with AIS. Microbiota was analyzed using high-throughput 16S rRNA gene amplicon sequencing and bioinformatics analyses. Results: Proteobacteria was the most predominant phylum in dental plaque, blood and thrombus. There were no statistic difference in alpha (Shannon and Simpson indexes) and beta diversity (weighted UniFrac distance metric) among dental plaque, blood and thrombus, suggesting a high similarity in their microbial compositions. While, an increased trend of species diversity from supragingival plaque, subgingival plaque, blood to thrombus was observed. Besides, several microbes in thrombus were found to be correlated with plasma coagulation levels, including genera Porphyromonas , which is the common cause of periodontal infection. SourceTracker analysis suggested that about 58% microbial population in blood were oral origin and about 75% microbial population in cerebral thrombi were blood origin. Conclusions: This study described the bacterial profile of dental plaque, blood and thrombus in AIS patients. Our study suggested that some of the bacteria in cerebral thrombus might be derived from the blood and oral cavity and there was a close microbial community compositional relationship among these samples.

The Comparative Study Of Efficiency Of Intravenous Heparin And Oral Aspirin On 48 Hours Beginning Primary Medication In Ischemic Stroke Patients

Objective: This study was done for comparing efficiency of Intravenous Heparin and Oral Aspirin among patients who had cerebral thrombi embolitic vascular accidents. Effectives of Heparin and Aspirin on prognosis and recovery of these patients were evaluated. Method: This study was a clinical randomized trial. Patients were classified randomly in one of the heparin (n=40) and one group Aspirin (n=40) treatment. At first in the Heparin treatment group, patients were medicated by Heparin 100 IU/kg and then it was followed by 1000IU/hour for 48 hours. In the Aspirin treated group, whom took 325mg/day for 48 hours. The effectiveness of both treatments were evaluated after 48 hours on neuro-muscular, speech, vision, and sphincter function.Results: The results showed that both treatments were effective on improvement of neuro-muscular, speech, vision, and sphincter function, while Heparin effectiveness was more than Aspirin. Improvement of Nervous function in the Heparin group was 53.2% in compare of 31.2% for Aspirin (p=0.40). In addition, in the Heparin treatment group 59.6% of muscular dysfunction was attained optimum muscular function in compare of 30.7% with Aspirin (p=0.001). It was found significant relation for improvement of speech function in Heparin treatment group (P=0.01). There were not a significant finding relation for improvement of vision, and sphincter function between Heparin and Aspirin treated groups.Conclusion: Beginning primary medication with Heparin would be effective for achieving optimum function among patients with cerebral thrombi embolitic vascular accidents.

Abstract TP81: Faster Start of IV tPA Does not Decrease the Need for Endovascular Thrombectomy in Acute Ischemic Stroke

Background: Recent patient series have shown IV tPA to more often lyse cerebral thrombi when started sooner after symptom onset in patients with acute ischemic stroke due to large vessel occlusion (AIS-LVO). This association has been attributed to less fibrin-cross-linking and less compaction of thrombi. We sought to determine if this phenomenon would make endovascular thrombectomy less often needed among AIS-LVO patients treated hyperacutely with IV tPA. Methods: In a prospectively maintained registry, we identified patients receiving IV tPA at 2 academic medical centers from March 2005 - May 2015. Inclusion criteria were: 1) LVO seen on CTA or MRA before (or early during) infusion of IV tPA and 2) follow-up vessel imaging within 6h of IV tPA initiation, using CT, MR or catheter angiogram. Degree of thrombus lysis with IV tPA alone was rated using the arterial occlusive lesion (AOL) Scale. Results: Among the 166 patients, average age was 72.4 (±13.4), 52.4% were female and pretreatment NIHSS was 13.9 (±8.4). Onset to needle time (OTN) was median 105 min (IQR 79-129) and door to needle time 44 min (27-65). Initial vessel imaging modality was MRA in 68.7% and CTA in 31.3%. Early post-tPA vessel imaging modality was catheter angiogram in 63.8%, MRA in 33.7% and CTA in 2.4%. Time from tPA initiation to recanalization assessment was faster when post-tPA vessel imaging was catheter angiogram vs MRA/CTA, 72 min (45.5-116.5) vs 232 min (185-283), p&lt;0.001. In cases assessed with early post-treatment catheter angiogram, IV tPA yielded complete recanalization in 17%, partial in 6.6%, and none in 76.4%. In cases assessed with MRA/CTA, IV tPA yielded complete recanalization in 30%, partial in 35%, and none in 35%. Recanalization within the 6h window was visualized more often when imaged with later CTA/MRA than with earlier catheter angiogram (p&lt;0.001). Among patients going directly to catheter angiography, OTN for IV tPA was not different between recanalizers and non-recanalizers, 106 vs 98 min, p = 0.53. Discussion: Among large vessel acute ischemic stroke patients, the rate of complete recanalization with IV tPA alone is only 1 in 6, and faster OTN time is not associated with increased recanalization. All AIS-LVO patients should proceed to thrombectomy as swiftly as possible.

The Role of Iron-Induced Fibrin in the Pathogenesis of Alzheimer’s Disease and the Protective Role of Magnesium

Amyloid hypothesis of Alzheimer’s disease (AD) has recently been challenged by the increasing evidence for the role of vascular and hemostatic components that impair oxygen delivery to the brain. One such component is fibrin clots, which, when they become resistant to thrombolysis, can cause chronic inflammation. It is not known, however, why some cerebral thrombi are resistant to the fibrinolytic degradation, whereas fibrin clots formed at the site of vessel wall injuries are completely, although gradually, removed to ensure proper wound healing. This phenomenon can now be explained in terms of the iron-induced free radicals that generate fibrin-like polymers remarkably resistant to the proteolytic degradation. It should be noted that similar insoluble deposits are present in AD brains in the form of aggregates with Abeta peptides that are resistant to fibrinolytic degradation. In addition, iron-induced fibrin fibers can irreversibly trap red blood cells (RBCs) and in this way obstruct oxygen delivery to the brain and induce chronic hypoxia that may contribute to AD. The RBC-fibrin aggregates can be disaggregated by magnesium ions and can also be prevented by certain polyphenols that are known to have beneficial effects in AD. In conclusion, we argue that AD can be prevented by: (1) limiting the dietary supply of trivalent iron contained in red and processed meat; (2) increasing the intake of chlorophyll-derived magnesium; and (3) consumption of foods rich in polyphenolic substances and certain aliphatic and aromatic unsaturated compounds. These dietary components are present in the Mediterranean diet known to be associated with the lower incidence of AD and other degenerative diseases.

Receptors, Not Clots: Coagulation and Fibrinolytic Enzymes Modulate Stroke Outcome by Targeting Endothelial Cells, Not Cerebral Thrombi

Receptors, Not Clots: Coagulation and Fibrinolytic Enzymes Modulate Stroke Outcome by Targeting Endothelial Cells, Not Cerebral Thrombi

Fetal Thrombotic Vasculopathy in the Placenta: Cerebral Thrombi and Infarcts, Coagulopathies, and Cerebral Palsy

When thrombi form in the fetal circulation of the placenta, they produce a clustering of fibrotic villi, a state termed fetal thrombotic vasculopathy (FTV). This has been related to serious neonatal damage, in particular brain injury. One report found FTV in 11 of 15 cerebral palsy patients. The present study estimated the prevalence of placental FTV in a series of 84 perinatal autopsies and attempted to determine whether it is a valid marker of thrombotic lesions in infants who survive. The autopsies were performed over a 3-year period. Placental lesions of FTV were found in 16 autopsies (19 percent of the total). In all stillbirths, the changes had clearly been present for at least 48 hours. FTV was extensive in all affected babies. One each of the eight mothers tested had combined protein S/antithrombin III deficiency, combined activated protein C resistance/protein S deficiency, an elevated cardiolipin antibody titer, and an elevated antinuclear antibody titer. Another woman with normal coagulation findings had had episodes of deep vein thrombosis and was given heparin during her pregnancy. The only mother tested for the methylenetetrahydrofolate reductase gene mutation was positive. Thrombi in vessels of the brain, kidneys, and lungs exhibited organization, fibrosis, or breakdown. Cerebral infarcts were associated with necrosis, gliosis, and in some cases calcifications. None of the fetuses and infants without FTV had visceral thrombi. The presence of placental FTV signifies a significant risk that thrombi are present in the fetus. They may cause significant neonatal injury, especially cerebral palsy, or perinatal death. It may well be that strokes are a major cause of brain damage for the fetus as they are in adults. Maternal coagulopathy is a significant factor in some of these cases, and this association deserves more study. Hum Pathol 1999;30:759–769