Dai, Daying

Introduction: Numerous endovascular tools exist for the treatment of intracranial aneurysms with an increased use of flow-diverting devices in recent years. However, the biological mechanisms leading to aneurysm healing or potential complications such as delayed aneurysm ruptures after flow-diverter treatment are not yet well established. We used RNA-sequencing technology to identify genes relevant for biological impact of coils and flow-diverters in elastase-induced saccular aneurysms in rabbits and to identify genes and pathways of potential clinical interest. Methods: Elastase-induced saccular aneurysms were created at the origin of the right common carotid artery. Aneurysms were treated with coils (n=5) or flow-diverters (Pipeline Embolic Device, Covidien, CA) (n=4). Untreated aneurysms (n=6) were used as controls. Messenger RNA and microRNA were isolated from the aneurysms at 4 weeks following treatment. RNA samples were processed using RNA-seq technology. RNA-seq results were analyzed by using the Ingenuity Pathway Analysis tool. Results: Using RNA sequencing for coiled versus non-treated aneurysms, 464 genes were found to be differentially expressed with 58 genes down regulated and 406 genes up regulated. Using the same criteria for the flow-diverter treated aneurysms, 177 genes were found to be differentially expressed with 8 genes down regulated and 169 genes up regulated. Comparing coiled versus flow-diverter treated aneurysms, 13 genes were significantly differentially expressed with 8 down regulated and 5 up regulated genes. Genes identified with the highest interest for aneurysms healing were fibroblast growth factor-23, matrix metalloproteinases (MMP)-1, Scinderin and Basigin (implicated in MMP-2 and MMP-9 regulation). Pathway analysis associated these genes with inflammatory response, cellular migration, and coagulation, among other functions. Conclusions: RNA-sequencing analysis of rabbit aneurysms revealed differential regulation of some key pathways, including inflammation and cellular migration that could explain the different biological mechanisms implicated in aneurysms healing either after coiling or flow-diverter treatments and could explain potential device-related complications.

Backgound and Purpose: Mechanisms of both healing and complications, including spontaneous aneurysm rupture, remain unclear following flow diverter treatment. The aim of the current study was to compare the gene expression of various key molecules involved in the healing of aneurysms between aneurysms treated with microcoils and flow diverters. Methods: Saccular aneurysms were created in rabbits (n=12). Aneurysms were either treated with coils (n=6) or with flow diverters (n=6). Aneurysm tissue was harvested at four weeks following treatment and used for gene expression and zymography experiments. Genes with a fold change of 1.2 or greater were considered up regulated, whereas those with a fold change of 0.8 or less were considered down regulated. Results: All coil embolized aneurysms were completely occluded at follow up. Two aneurysms were occluded and the remaining four samples were incompletely occluded in the flow diverter treated group. The following genes were expressed at lower levels in the flow diverter group as compared to the coiled aneurysm group: genes encoding proteinases (matrix metalloproteinases -2 and -9), cellular markers (endothelial nitric oxide synthase and smooth muscle actin), and structural proteins (collagens and fibronectin). Genes related to inflammation (tumor necrosis factor-α and monocyte chemoattractant protein -1) were up regulated in flow diverter treated aneurysms compared to coil embolized aneurysms. Notably, the enzymatic activity of active MMP-9 was high in aneurysms treated with flow diverters. Conclusion: As compared to coiled aneurysms, the molecular milieu surrounding aneurysms treated with flow diverters is characterized by higher levels of activated proteases, elevated expression of genes encoding inflammatory proteins, and diminished expression of genes encoding structural proteins. These data may provide improved understanding of rupture risk and healing following aneurysm treatment, and inform development of therapies aimed at lowering rupture risk and accelerating healing.

Introduction: Mechanical thrombectomy (MT) has become the standard of care for acute ischemic stroke (AIS) patients due to large vessel occlusion (LVO). Clinical outcome of successful recanalization (mTICI 2b or higher) varies greatly and is associated with some baseline characteristics. Many studies have reported the relation of thrombus composition with MT outcomes however the association of thrombus composition with the prognosis of successful recanalization remains to be elucidated. Hypothesis: thrombi retrieved from patients with good clinical outcome may have different compositions compared to that of patients with poor clinical outcome. Methods: Thrombi retrieved from AIS-LVO patients with successful recanalization were included. Patients were divided according to modified Rankin Scale at 90 days (mRS-90days) into good clinical outcome (mRS-90days 0-2) and poor clinical outcome (mRS-90 days 3-6). Representative slide for each thrombus was stained with Martius Scarlet Blue (MSB) for red blood cells (RBCs), white blood cells (WBCs), fibrin (FIB), and platelet (PLT). Immunohistochemistry (IHC) was also performed for NETs (anti-citrullinated H3; CitH3) and vWF in thrombi. The overall mean value of each thrombus composition was calculated. Thrombus was considered rich in given composition if that composition was higher than its overall mean. We compared the thrombus composition and type between patients with good and poor clinical outcomes. Results: A total of 94 patients were included. Fifty-six patients had good clinical outcome. The overall average percentage of RBCs, WBCs, fibrin, platelet, NETs (CitH3), and vWF components in thrombi were 44.3%, 3.6%, 29.3%, 22.8%, 20.3% and 12.5% respectively. There was no association between thrombus type (including RBC-rich, FIB-rich, PLT-rich, vWF-rich, and NETs-rich) and clinical outcomes (P values>0.05). Multivariate logistic regression analysis showed no association between thrombus composition and clinical outcome (good vs poor) while adjusting for age, initial NIHSS score, ASPECT score, pre-MT IV-tPA, and MT technique and number of the device passes. Conclusion: we found no association between thrombus composition and clinical outcome after successful recanalization.

Introduction: Outcomes of mechanical thrombectomy (MT) are influenced by the composition of thrombi. Amongst the risk factors affecting clot composition, diabetes mellitus (DM) may be associated with more thrombus structural and mechanical alterations resulting in fibrinolysis-resistant thrombi. However, extensive analysis of the composition of thrombi obtained from patients is needed to elucidate this relationship. Hypothesis: Thrombi retrieved from patients with DM may have different compositions compared to that of patients with no history of DM. Methods: MT retrieved-thrombi from patients suffering from large vessel occlusion (LVO) stroke were included. Thrombi sections were stained with Martius Scarlet Blue (MSB) for main thrombus components including red blood cells (RBCs), white blood cells (WBCs), fibrin and platelet. Additionally, immunohistochemistry (IHC) was performed to identify neutrophil extracellular traps (NETs) (anti-citrulinated H3; CitH3; NETs-specific marker) and von Willebrand Factor (vWF) in thrombi. Slides were then scanned and analyzed and the thrombus composition was compared between patients with or without a DM diagnosis at the time of stroke presentation. Results: A total of 140 patients were included of which 35 had a diagnosis of DM at stroke presentation. Baseline characteristics (excluding age), IV-tPA use prior to MT, and MT outcomes (number of device passes and final mTICI score) were similar between DM and non DM patients. The overall average percentage of RBCs, WBCs, fibrin, platelet, NETs (CitH3), and vWF components in thrombi were 47.4%, 3.2%, 27.4%, 21.8%, 25.9% and 17.6%, respectively. Thrombi from patients with DM were not statistically significant different from those of patients with no DM in terms of RBCs (44.3 vs 48.4 p=0.37), WBCs (2.7 vs 3.3 p=0.05), fibrin (29.5 vs 26.7 p=0.37), platelet (23.3 vs 21.3 p=0.57), NETs (25.6 vs 22.3 p=0.54), and vWF (17.5 vs 17.7 p=0.96). Conclusion: DM has no effect on the composition of thrombi as no significant difference was found in thrombi obtained from patients with and without DM.

Introduction: Ample evidence exists in support of the anti-inflammatory properties of statins which in turn could affect arterial thrombosis. Compositional analysis of thrombi retrieved by MT may provide us with knowledge useful for evaluating the effect of current Statin therapy on thrombus composition. Methods: Consecutive AIS-LVO patients were included. Histopathological evaluation of thrombi retrieved during MT was performed using Martius Scarlet Blue staining (MSB) and immunohistochemistry for von Willebrand Factor (vWF) and anti-citrulinated H3 (CitH3; NETs marker) and the composition of thrombi was determined using image analysis software. Thrombus was considered rich in a given composition if that composition was higher than its overall mean. We reviewed patient’s medication records and cataloged any statin therapy prior to MT. Results: 117 patients were included in this study. The mean density of red blood cells (RBCs), white blood cells (WBCs), platelets (PLT), fibrin (FIB), vWF and NETs (CitH3) in thrombi was 43.4%, 3.7%, 23.6%, 29.3%, 14.3% and 21.1% respectively. Twenty-seven patients were taking Statins at the time of MT. Statins were not associated with pre-thrombectomy IV-tPA, and stroke etiology (p values: >0.05). There was no association between Statins and thrombi composition and type (p values: >0.05 for all thrombus components and type). Additionally, Statins were not associated with MT outcomes including number of device passes and final mTICI score. Conclusion: Pre-stroke Statin therapy was not associated with differences in thrombus composition.

Background & Purpose It has been hypothesized that circulating neutrophils have a direct correlation with the composition of emboli in acute ischemic stroke (AIS). The aim of this study is to evaluate the association between neutrophil-lymphocyte ratio (NLR) in peripheral blood and the expression of neutrophil extracellular traps (NETs) within stroke emboli. Methods Consecutive patients with acute ischemic stroke (AIS) due to large vessel occlusion (LVO) that underwent mechanical thrombectomy (MT) were included. Patients were divided into two groups based on NLR median value. Retrieved thrombi were histologically analyzed using Martius Scarlett Blue (MSB) for main thrombus components including red blood cells (RBCs), white blood cells (WBCs), fibrin and platelet. Immunohistochemistry staining for von Willebrand Factor (vWF) and anti-citrullinated H3 (H3Cit; NETs marker) was also performed. Results Samples from a total of 84 patients were included. The average percentage of RBCs, WBCs, fibrin, platelet, H3Cit, and vWF components in thrombi were 45.1%, 3.5%, 21.8%, 29.6%, 19.7% and 14.8% respectively. When stratifying by NLR group [low (≤3.94) versus high (>3.95)], high NLR group had significantly more WBCs (4.5%), fibrin (24.2%), H3Cit (22.7%) and vWF (17.1%) thrombus fractions compared to low NLR group. Additionally, RBC content (38.8%) was lower in the high NLR group. Conclusions NLR is correlated with the amounts of WBCs, fibrin, NETs and vWF within the thrombi retrieved from AIS patients due to LVO.

There has been growing interest and insight into the histological composition of retrieved stroke emboli. One of the main focuses of the stroke clot analysis literature has been the implications of clot composition on mechanical thrombectomy procedures. However, the holy grail of clot analysis may not be in the field of clot–device interaction, but rather, in understanding mechanisms of fibrinolysis resistance. The mechanisms underlying the low response to fibrinolytic therapy, even with the newer, more powerful agents, remain poorly understood. While factors such as embolus size, location and collateral status influence alteplase delivery and recanalisation rates; compositional analyses focused on histological and ultrastructural characteristics offer unique insights into mechanisms of alteplase resistance. In this review, we strive to provide comprehensive review of current knowledge on clot composition and ultrastructural analyses that help explain resistance to fibrinolysis.

Background and Purpose: Rabbit aneurysm models are used in the testing of embolization devices as well as elucidating the mechanisms human intracranial aneurysm growth and healing. We employed ribonucleic acid sequencing (RNA-seq) technology to identify genes relevant to induced rabbit aneurysm biology and compare these with genes related to human intracranial aneurysms to identify genes of potential clinical interest. This included sequencing micro RNA, which are important regulatory non-coding RNA. Materials and Methods: Elastase-induced saccular aneurysms were created at the origin of the right common carotid artery (CCA) in 5 rabbits. Messenger ribonucleic acid (mRNA) and micro ribonucleic acid (miRNA) was isolated from the aneurysm and the control unoperated left CCA at 12 weeks and was processed using RNA-seq technology. The results from RNA-seq were analyzed using Ingenuity Pathway Analysis. Results: 9396 genes were analyzed using RNA-seq, of which 649 (6.9%) were found to be significantly differentially expressed between aneurysm and control tissues (p < 0.05, false discovery rate 2). Of these, 500 were up-regulated in the aneurysm and 149 were down-regulated as compared to controls. Using the same criteria, 3 miRNA were identified as down regulated and 5 were identified as up regulated. Pathway analysis associated these genes with inflammatory response, cellular migration, and coagulation, among other functions and pathologies. Conclusion: RNA-seq next-gen analysis of rabbit aneurysms shows similarities to human intracranial aneurysms with respect to regulation of some key pathways.

Introduction: Von Willebrand factor (VWF) is a key component of acute ischemic stroke (AIS) thrombi. The aim of our prospective study was to investigate the immunohistochemical expression of VWF in clots and to evaluate whether VWF is associated with certain subtypes of AIS. Methods: VWF immunostaining was performed on 79 thrombi collected as part of the multi-center Stroke Thromboembolism Registry of Imaging and Pathology (STRIP) registry. The cases were classified according to TOAST criteria. The VWF expression was quantified using Orbit Image Analysis (www.Orbit.bio) machine learning software. IBM SPSS statistics 25 was used to assess the relationship between the VWF levels and different etiology subtypes. Results: A cardioembolic stroke was defined in 39 cases (49.4%) whereas an atherosclerotic origin was identified in 13 patients (16.5%). Other causes accounted for 12 cases (15.1%). Unknown etiology was reported in 15 cases (19%). The mean VWF content in the clots was 12.8%. According to the Mann-Whitney U-test, the level of VWF was significantly higher in the cases with unknown etiology compared to cardioembolic origin (p=0.044). We found also that patients with unknown etiology of stroke had higher VWF expression as compared to the other two subtypes, although this difference was not statistically significant. Conclusions: Among the patients with ischemic stroke included in this study, the VWF expression was significantly increased in those with unknown etiology compared to the group with cardioembolic stroke. Our finding provides new insights into clot composition in cryptogenic stroke and may influence the treatment and secondary prevention in these cases.