Investigating the Role of Brain Natriuretic Peptide (BNP) and N-Terminal-proBNP in Thrombosis and Acute Ischemic Stroke Etiology.

The need for biomarkers for acute ischemic stroke (AIS) to understand the mechanisms implicated in pathological clot formation is critical. The levels of the brain natriuretic peptides known as brain natriuretic peptide (BNP) and NT-proBNP have been shown to be increased in patients suffering from heart failure and other heart conditions. We measured their expression in AIS clots of cardioembolic (CE) and large artery atherosclerosis (LAA) etiology, evaluating their location inside the clots, aiming to uncover their possible role in thrombosis. We analyzed 80 thrombi from 80 AIS patients in the RESTORE registry of AIS clots, 40 of which were of CE and 40 of LAA etiology. The localization of BNP and NT-BNP, quantified using immunohistochemistry and immunofluorescence, in AIS-associated white blood cell subtypes was also investigated. We found a statistically significant positive correlation between BNP and NT-proBNP expression levels (Spearman's rho = 0.668 p < 0.0001 *). We did not observe any statistically significant difference between LAA and CE clots in BNP expression (0.66 [0.13-3.54]% vs. 0.53 [0.14-3.07]%, p = 0.923) or in NT-proBNP expression (0.29 [0.11-0.58]% vs. 0.18 [0.05-0.51]%, p = 0.119), although there was a trend of higher NT-proBNP expression in the LAA clots. It was noticeable that BNP was distributed throughout the thrombus and especially within platelet-rich regions. However, NT-proBNP colocalized with neutrophils, macrophages, and T-lymphocytes, suggesting its association with the thrombo-inflammatory process.

The administration of rtPA before mechanical thrombectomy in acute ischemic stroke patients is associated with a significant reduction of the retrieved clot area but it does not influence revascularization outcome.

Both intravenous thrombolysis (IVT) and mechanical thrombectomy (MT) are evidence-based treatments for acute ischemic stroke (AIS) in selected cases. Recanalization may occur following IVT without the necessity of further interventions or requiring a subsequent MT procedure. IVT prior to MT (bridging-therapy) may be associated with benefits or hazards. We studied the retrieved clot area and degree of recanalization in patients undergoing MT or bridging-therapy for whom it was possible to collect thrombus material. We collected mechanically extracted thrombi from 550 AIS patients from four International stroke centers. Patients were grouped according to the administration (or not) of IVT before thrombectomy and the mechanical thrombectomy approach used. We assessed the number of passes for clot removal and the mTICI (modified Treatment In Cerebral Ischemia) score to define revascularization outcome. Gross photos of each clot were taken and the clot area was measured with ImageJ software. The non-parametric Kruskal-Wallis test was used for statistical analysis. 255 patients (46.4%) were treated with bridging-therapy while 295 (53.6%) underwent MT alone. By analysing retrieved clot area, we found that clots from patients treated with bridging-therapy were significantly smaller compared to those from patients that underwent MT alone (H1 = 10.155 p = 0.001*). There was no difference between bridging-therapy and MT alone in terms of number of passes or final mTICI score. Bridging-therapy was associated with significantly smaller retrieved clot area compared to MT alone but it did not influence revascularization outcome.

Characterization of the 'White' Appearing Clots that Cause Acute Ischemic Stroke.

OBJECTIVES: Most clots retrieved from patients with acute ischemic stroke are 'red' in color. 'White' clots represent a less common entity and their histological composition is less known. Our aim was to investigate the composition, imaging and procedural characteristics of 'white' clots retrieved by mechanical thrombectomy. MATERIALS AND METHODS: Seventy five 'white' thrombi were selected by visual inspection from a cohort of 760 clots collected as part of the RESTORE registry. Clots were evaluated histopathologically. RESULTS: Quantification of Martius Scarlett Blue stain identified platelets/other as the major component in 'white' clots' (mean of 55% of clot overall composition) followed by fibrin (31%), red blood cells (6%) and white blood cells (3%). 'White' clots contained significantly more platelets/other (p<0.001*) and collagen/calcification (p<0.001*) and less red blood cells (p<0.001*) and white blood cells (p=0.018*) than 'red' clots. The mean platelet and von Willebrand Factor expression was 43% and 24%, respectively. Adipocytes were found in four cases. 'White' clots were significantly smaller (p=0.016*), less hyperdense (p=0.005*) on computed tomography angiography/non-contrast CT and were associated with a smaller extracted clot area (p<0.001*) than 'red' clots. They primarily caused the occlusion of middle cerebral artery, were less likely to be removed by aspiration and more likely to require rescue-therapy for retrieval. CONCLUSIONS: 'White' clots represented 14% of our cohort and were platelet, von Willebrand Factor and collagen/calcification-rich. 'White' clots were smaller, less hyperdense, were associated with significantly more distal occlusions and were less successfully removed by aspiration alone than 'red' clots.

Large Artery Atherosclerotic Clots are Larger than Clots of other Stroke Etiologies and have Poorer Recanalization rates.

OBJECTIVES: There is a paucity of knowledge in the literature relating to the extent of clot burden and stroke etiology. In this study, we measured the Extracted Clot Area (ECA) retrieved during endovascular treatment (EVT) and investigated relationships with suspected etiology, administration of intravenous thrombolysis and recanalization. MATERIALS AND METHODS: As part of the multi-institutional RESTORE registry, the ECA retrieved during mechanical thrombectomy was quantified using ImageJ. The effect of stroke etiology (Large-artery atherosclerosis (LAA), Cardioembolism, Cryptogenic and other) and recombinant tissue plasminogen activator (rtPA) on ECA and recanalization outcome (mTICI) was assessed. Successful recanalization was described as mTICI 2c-3. RESULTS: A total of 550 patients who underwent EVT with any clot retrieved were included in the study. The ECA was significantly larger in the LAA group compared to all other etiologies. The average ECA size of each etiology was; LAA=109 mm2, Cardioembolic=52 mm2, Cryptogenic=47 mm2 and Other=52 mm2 (p=0.014*). LAA patients also had a significantly poorer rate of successful recanalization (mTICI 2c-3) compared to all other etiologies (p=0.003*). The administration of tPA was associated with a smaller ECA in both LAA (p=0.007*) and cardioembolic (p=0.035*) groups. CONCLUSION: The ECA of LAA clots was double the size of all other etiologies and this is associated with a lower rate of successful recanalization in LAA stroke subtype. rtPA administration prior to thrombectomy was associated with reduced ECA in LAA and CE clots.

Texture parameters of R2* maps are correlated with iron concentration and red blood cells count in clot analogs: A 7-T micro-MRI study.

BACKGROUND AND PURPOSE: Previous studies have suggested that mechanical revascularization in acute ischemic stroke (AIS) patients could be affected by clot histology. In this 7-T micro-MRI study, we used R2* relaxometry of clot analogs to evaluate the relationship between texture parameters of R2* maps and clot constituents. MATERIALS AND METHODS: Twelve AIS clot analogs were experimentally generated to obtain a wide range of red blood cell concentrations. All clots underwent a MR acquisition using a 7-T micro-MR system. A 3D multi-echo gradient-echo sequence was performed and R2* maps were generated. First order and second order statistics of R2* histograms within the clots were calculated. Iron concentration in clots was measured using absorption spectrometry and red blood cell count (RBC) was obtained by histopathological analysis. RESULTS: RBC count was strongly correlated with iron concentration within clots (r=0.87, P<.001). Higher RBC count and iron concentration were significantly correlated with first order parameters including: (a) global positive shift of the R2* histogram with higher '10th percentile', 'median', 'mean' and '90th percentile'; (b) increase of the global magnitude of voxel values with higher 'total energy' and 'root mean squared'; (c) greater uniformity of the voxel values with higher 'uniformity' and lower 'entropy'. Second order statistical parameters confirmed that higher RBC count and iron concentration correlated with (a) greater concentration of high gray-level values in the image; (b) more "coarse" texture of R2* maps. CONCLUSIONS: Texture analysis of MRI-R2* maps can accurately estimate the red blood cell count and iron content of AIS clot analogs.

Per-Pass Analysis of Thrombus Composition in Patients With Acute Ischemic Stroke Undergoing Mechanical Thrombectomy.

Background and Purpose- Mechanical thrombectomy may involve multiple attempts to retrieve the occluding thrombus. This study examined the composition of thrombus fragments retrieved with each pass of a device during the thrombectomy procedure. Second, the per-pass composition was compared with procedural and clinical data including angiographic outcome and stroke etiology. Methods- Thrombi were retrieved from 60 patients with acute ischemic stroke, where thrombus fragments retrieved in each pass were segregated as individual samples and maintained throughout the histological analysis as independent samples. All samples were stained with hematoxylin and eosin and Martius Scarlet Blue. The relative composition of red blood cells, fibrin, and white blood cells in thrombus fragments from each pass was quantified. Results- Over the 60 cases, thrombus material was retrieved in 106 of 138 passes. The number of passes required to complete the cases ranged from 1 to 6 passes. The analysis of thrombus fragments retrieved in each pass provided a greater insight into the thrombectomy procedure progression than the overall thrombus composition; the red blood cell content of thrombus fragments retrieved in passes 1 and 2 was significantly higher than that retrieved in passes 3 to 6. The removal of thrombus material in a total of 1, 2, or 3 passes was associated with the highest percentage of final modified Thrombolysis in Cerebral Infarction score of 2c-3. There was no association between modified Thrombolysis in Cerebral Infarction score and per-pass thrombus composition. Conclusions- The differentiation achieved through the per-pass analysis of acute ischemic stroke thrombi provides a greater insight into the thrombectomy procedure progression than the combined per-case thrombus analysis. Insights gained may be a useful consideration in determining the treatment strategy as a case evolves and may be useful for the development of new devices to increase rates of 1-pass recanalization.

The application of indirect composite onlays in the restoration of severely broken down posterior teeth.

Increasing interest has developed among dentists regarding alternatives to traditional full-coverage crowns for the restoration of extensively broken-down teeth that are both aesthetic and less destructive of remaining tooth structure. Indirectly fabricated resin composite onlays may offer a viable and cost-effective treatment option in such cases. This paper describes the clinical rationale for resin-based onlays, and includes a case report illustrating the author's experience with the technique to date.

White blood cell subtypes and neutrophil extracellular traps content as biomarkers for stroke etiology in acute ischemic stroke clots retrieved by mechanical thrombectomy.

BACKGROUND: Lymphocytes, macrophages, neutrophils, and neutrophil extracellular traps (NETs) associate with stroke risk factors and form a thrombus through different mechanisms. We investigated the total WBCs, WBC subtypes and NETs composition in acute ischemic stroke (AIS) clots to identify possible etiological differences that could help us further understand the process of thrombosis that leads to AIS. METHODS: AIS clots from 100 cases each of atherothrombotic (AT), cardioembolic (CE) and cryptogenic stroke etiology were collected per-pass as part of the CÚRAM RESTORE registry of AIS clots. Martius Scarlet Blue stain was used to identify the main histological components of the clots. Immunohistochemical staining was used to identify neutrophils, lymphocytes, macrophages, and NETs patterns. The cellular and histological components were quantified using Orbit Image Analysis software. RESULTS: AT clots were larger, with more red blood cells and fewer WBCs than CE clots. AT clots had more lymphocytes and cryptogenic clots had fewer macrophages than other etiologies. Most significantly, CE clots showed higher expression of neutrophils and extracellular web-like NETs compared to AT and cryptogenic clots. There was also a significantly higher distribution of web-like NETs around the periphery of the CE clots while a mixed distribution was observed in AT clots. CONCLUSION: The difference in neutrophil and NETs expression in clots from different etiologies may provide insight into the mechanism of clot formation.

What is a Challenging Clot? : A DELPHI Consensus Statement from the CLOTS 7.0 Summit.

BACKGROUND: Predicting a challenging clot when performing mechanical thrombectomy in acute stroke can be difficult. One reason for this difficulty is a lack of agreement on how to precisely define these clots. We explored the opinions of stroke thrombectomy and clot research experts regarding challenging clots, defined as difficult to recanalize clots by endovascular approaches, and clot/patient features that may be indicative of such clots. METHODS: A modified DELPHI technique was used before and during the CLOTS 7.0 Summit, which included experts in thrombectomy and clot research from different specialties. The first round included open-ended questions and the second and final rounds each consisted of 30 closed-ended questions, 29 on various clinical and clot features, and 1 on number of passes before switching techniques. Consensus was defined as agreement ≥ 50%. Features with consensus and rated ≥ 3 out of 4 on the certainty scale were included in the definition of a challenging clot. RESULTS: Three DELPHI rounds were performed. Panelists achieved consensus on 16/30 questions, of which 8 were rated 3 or 4 on the certainty scale, namely white-colored clots (mean certainty score 3.1), calcified clots under histology (3.7) and imaging (3.7), stiff clots (3.0), sticky/adherent clots (3.1), hard clots (3.1), difficult to pass clots (3.1) and clots that are resistant to pulling (3.0). Most panelists considered switching endovascular treatment (EVT) techniques after 2-3 unsuccessful attempts. CONCLUSION: This DELPHI consensus identified 8 distinct features of a challenging clot. The varying degree of certainty amongst the panelists emphasizes the need for more pragmatic studies to enable accurate a priori identification of such occlusions prior to EVT.

A Review of the Advancements in the in-vitro Modelling of Acute Ischemic Stroke and Its Treatment.

In-vitro neurovascular models of large vessel occlusions (LVOs) causing acute ischemic stroke (AIS) are used extensively for pre-clinical testing of new treatment devices. They enable physicians and engineers to examine device performance and the response of the occlusion to further advance design solutions for current unmet clinical needs. These models also enable physicians to train on basic skills, to try out new devices and new procedural approaches, and for the stroke team to practice workflows together in the comfort of a controlled environment in a non-clinical setting. Removal of the occlusive clot in its entirety is the primary goal of the endovascular treatment of LVOs via mechanical thrombectomy (MT) and the medical treatment via thrombolysis. In MT, recanalization after just one pass is associated with better clinical outcomes than procedures that take multiple passes to achieve the same level of recanalization, commonly known as first pass effect (FPE). To achieve this, physicians and engineers are continually investigating new devices and treatment approaches. To distinguish between treatment devices in the pre-clinical setting, test models must also be optimized and expanded become more nuanced and to represent challenging patient cohorts that could be improved through new technology or better techniques. The aim of this paper is to provide a perspective review of the recent advancements in the in-vitro modeling of stroke and to outline how these models need to advance further in future. This review provides an overview of the various in-vitro models used for the modeling of AIS and compares the advantages and limitations of each. In-vitro models remain an extremely useful tool in the evaluation and design of treatment devices, and great strides have been made to improve replication of physiological conditions. However, further advancement is still required to represent the expanding indications for thrombectomy and thrombolysis, and the generation of new thrombectomy devices, to ensure that smaller treatment effects are captured.

Systematic Review on Endovascular Access to Intracranial Arteries for Mechanical Thrombectomy in Acute Ischemic Stroke.

PURPOSE: In acute ischemic stroke for large vessel occlusions, delayed or failed access to intracranial occlusions has a negative impact on procedural and clinical outcomes. The aim of this review is to identify and quantify access failures and challenges in mechanical thrombectomy. METHODS: A systematic literature review of PubMed and Scopus databases from January 2014 to October 2020 was performed. Articles reporting consecutive patients were used to calculate a crude failure rate of femoral and alternative accesses. RESULTS: A total of 50 articles met the inclusion criteria, totalling 12,838 interventions. Failure to access the occlusion through transfemoral access occurred in 4.4% of patients, most commonly due to challenging supra-aortic vessel anatomy, decreasing to 3.6% when all alternative access routes were attempted. Failed access from alternative routes (direct carotid, radial and brachial approaches) attempted first-line or after failed femoral attempt were reported in 7.3% of patients. The occurrence rate of potentially challenging features (anatomical, diseases or others) ranged from 4.7% to 47.4%, primarily impacting the access time, procedure time, recanalization and clinical outcomes. CONCLUSION: Failure to access the occlusion is a significant contributor to failed recanalization, regardless of access routes. Challenging, but eventually successful access is also a relevant factor in procedural and clinical outcomes; however challenging access requires a universal definition to enable quantification, so that methods for procedural optimization can be critically assessed.

Aspects of ischemic stroke biomechanics derived using ex-vivo and in-vitro methods relating to mechanical thrombectomy.

Establishing the underlying biomechanics of acute ischemic stroke (AIS) and its treatment is fundamental to developing more effective clinical treatments for one of society's most impactful diseases. Recent changes in AIS management, driven by clinical evidence of improved treatments, has already led to a rapid rate of innovation, which is likely to be sustained for many years to come. These unprecedented AIS triage and treatment innovations provide a great opportunity to better understand the disease. In this article we provide a perspective on the recreation of AIS in the laboratory to inform contemporary device design and procedural techniques in mechanical thrombectomy. Presentation of these findings, which have been used to solve the applied problem of designing mechanical thrombectomy devices, is intended to help inform the development of basic biomechanics solutions for AIS.

Combined stent-retriever and aspiration intra-arterial thrombectomy performance for fragmentable blood clots: A proof-of-concept computational study.

Mechanical thrombectomy (MT) treatment of acute ischemic stroke (AIS) patients typically involves use of stent retrievers or aspiration catheters alone or in combination. For in silico trials of AIS patients, it is crucial to incorporate the possibility of thrombus fragmentation during the intervention. This study focuses on two aspects of the thrombectomy simulation: i) Thrombus fragmentation on the basis of a failure model calibrated with experimental tests on clot analogs; ii) the combined stent-retriever and aspiration catheter MT procedure is modeled by adding both the proximal balloon guide catheter and the distal access catheter. The adopted failure criterion is based on maximum principal stress threshold value. If elements of the thrombus exceed this criterion during the retrieval simulation, then they are deleted from the calculation. Comparison with in-vitro tests indicates that the simulation correctly reproduces the procedures predicting thrombus fragmentation in the case of red blood cells rich thrombi, whereas non-fragmentation is predicted for fibrin-rich thrombi. Modeling of balloon guide catheter prevents clot fragments' embolization to further distal territories during MT procedure.

Potential Biomarkers of Acute Ischemic Stroke Etiology Revealed by Mass Spectrometry-Based Proteomic Characterization of Formalin-Fixed Paraffin-Embedded Blood Clots.

Background and Aims: Besides the crucial role in the treatment of acute ischemic stroke (AIS), mechanical thrombectomy represents a unique opportunity for researchers to study the retrieved clots, with the possibility of unveiling biological patterns linked to stroke pathophysiology and etiology. We aimed to develop a shotgun proteomic approach to study and compare the proteome of formalin-fixed paraffin-embedded (FFPE) cardioembolic and large artery atherosclerotic (LAA) clots. Methods: We used 16 cardioembolic and 15 LAA FFPE thrombi from 31 AIS patients. The thrombus proteome was analyzed by label-free quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS). MaxQuant v1.5.2.8 and Perseus v.1.6.15.0 were used for bioinformatics analysis. Protein classes were identified using the PANTHER database and the STRING database was used to predict protein interactions. Results: We identified 1,581 protein groups as part of the AIS thrombus proteome. Fourteen significantly differentially abundant proteins across the two etiologies were identified. Four proteins involved in the ubiquitin-proteasome pathway, blood coagulation or plasminogen activating cascade were identified as significantly abundant in LAA clots. Ten proteins involved in the ubiquitin proteasome-pathway, cytoskeletal remodeling of platelets, platelet adhesion or blood coagulation were identified as significantly abundant in cardioembolic clots. Conclusion: Our results outlined a set of 14 proteins for a proof-of-principle characterization of cardioembolic and LAA FFPE clots, advancing the proteome profile of AIS human thrombi and understanding the pathophysiology of ischemic stroke.

S100b in acute ischemic stroke clots is a biomarker for post-thrombectomy intracranial hemorrhages.

Background and purpose: Post-thrombectomy intracranial hemorrhages (PTIH) are dangerous complications of acute ischemic stroke (AIS) following mechanical thrombectomy. We aimed to investigate if S100b levels in AIS clots removed by mechanical thrombectomy correlated to increased risk of PTIH. Methods: We analyzed 122 thrombi from 80 AIS patients in the RESTORE Registry of AIS clots, selecting an equal number of patients having been pre-treated or not with rtPA (40 each group). Within each subgroup, 20 patients had developed PTIH and 20 patients showed no signs of hemorrhage. Gross photos of each clot were taken and extracted clot area (ECA) was measured using ImageJ. Immunohistochemistry for S100b was performed and Orbit Image Analysis was used for quantification. Immunofluorescence was performed to investigate co-localization between S100b and T-lymphocytes, neutrophils and macrophages. Chi-square or Kruskal-Wallis test were used for statistical analysis. Results: PTIH was associated with higher S100b levels in clots (0.33 [0.08-0.85] vs. 0.07 [0.02-0.27] mm2, H1 = 6.021, P = 0.014*), but S100b levels were not significantly affected by acute thrombolytic treatment (P = 0.386). PTIH was also associated with patients having higher NIHSS at admission (20.0 [17.0-23.0] vs. 14.0 [10.5-19.0], H1 = 8.006, P = 0.005) and higher number of passes during thrombectomy (2 [1-4] vs. 1 [1-2.5], H1 = 5.995, P = 0.014*). S100b co-localized with neutrophils, macrophages and with T-lymphocytes in the clots. Conclusions: Higher S100b expression in AIS clots, higher NIHSS at admission and higher number of passes during thrombectomy are all associated with PTIH. Further investigation of S100b expression in AIS clots by neutrophils, macrophages and T-lymphocytes could provide insight into the role of S100b in thromboinflammation.

Does prior administration of rtPA influence acute ischemic stroke clot composition? Findings from the analysis of clots retrieved with mechanical thrombectomy from the RESTORE registry.

BACKGROUND AND PURPOSE: There is still much debate whether bridging-therapy [intravenous thrombolysis (IVT) prior to mechanical thrombectomy (MT)] might be beneficial compared to MT alone. We investigated the effect of IVT on size and histological composition of the clots retrieved from patients undergoing bridging-therapy or MT alone. METHODS: We collected mechanically extracted thrombi from 1000 acute ischemic stroke (AIS) patients included in RESTORE registry. Patients were grouped according to the administration (or not) of IVT before thrombectomy. Gross photos of each clot were taken and Extracted Clot Area (ECA) was measured using ImageJ software. Martius Scarlett Blue stain was used to characterize the main histological clot components [red blood cells (RBCs), fibrin (FIB), platelets/other (PTL)] and Orbit Image Analysis was used for quantification. Additionally, we calculated the area of each main component by multiplying the component percent by ECA. Chi-squared and Kruskal-Wallis tests were used for statistical analysis. RESULTS: 451 patients (45%) were treated with bridging-therapy while 549 (55%) underwent MT alone. When considering only percent histological composition, we did not find any difference in RBC% (P = 0.895), FIB% (P = 0.458) and PTL% (P = 0.905). However, bridging-therapy clots were significantly smaller than MT-alone clots [32.7 (14.8-64.9) versus 36.8 (20.1-79.8) mm2, N = 1000, H1 = 7.679, P = 0.006*]. A further analysis expressing components per clot area showed that clots retrieved from bridging-therapy cases contained less RBCs [13.25 (4.29-32.06) versus 14.97 (4.93-39.80) mm2, H1 = 3.637, P = 0.056] and significantly less fibrin [9.10 (4.62-17.98) versus 10.54 (5.57-22.48) mm2, H1 = 7.920, P = 0.005*] and platelets/other [5.04 (2.26-11.32) versus 6.54 (2.94-13.79) mm2, H1 = 9.380, P = 0.002*] than MT-alone clots. CONCLUSIONS: Our results suggest that previous IVT administration significantly reduces thrombus size, proportionally releasing all the main histological components.

Blood clot fracture properties are dependent on red blood cell and fibrin content.

Thrombus fragmentation during endovascular stroke treatment, such as mechanical thrombectomy, leads to downstream emboli, resulting in poor clinical outcomes. Clinical studies suggest that fragmentation risk is dependent on clot composition. This current study presents the first experimental characterization of the composition-dependent fracture properties of blood clots, in addition to the development of a predictive model for blood clot fragmentation. A bespoke experimental test-rig and compact tension specimen fabrication has been developed to measure fracture toughness of thrombus material. Fracture tests are performed on three physiologically relevant clot compositions: a high-fibrin clot made from a 5% haematocrit (H) blood mixture, a medium-fibrin clot made form a 20% H blood mixture, a low-fibrin clot made from a 40% H blood mixture. Fracture toughness is observed to significantly increase with increasing fibrin content, i.e. red blood cell-rich clots are more prone to tear during loading compared to the fibrin-rich clots. Results also reveal that the mechanical behaviour of clot analogues is significantly different in compression and tension. Finite element cohesive zone modelling of clot fracture experiments show that fibrin fibres become highly aligned in the direction perpendicular to crack propagation, providing a significant toughening mechanism. The results presented in this study provide the first characterization of the composition-dependent fracture behaviour of blood clots and are of key importance for development of next-generation thrombectomy devices and clinical strategies. STATEMENT OF SIGNIFICANCE: This study provides a characterisation of the composition-dependent fracture toughness of blood clots. This entails the development of novel experimental techniques for fabrication and testing of blood clot compact tension fracture specimens. The study also develops cohesive zone models of fracture initiation and propagation in blood clots. Results reveal that the fracture resistance of fibrin-rich clots is significantly higher than red blood cell rich clots. Simulations also reveal that stretching and realignment of the fibrin network should be included in blood clot material models in order to accurately replicate compression-tension asymmetry and fibrin enhanced fracture toughness. The results of this study have potentially important clinical implications in terms of clot fracture risk and secondary embolization during mechanical thrombectomy procedures.

A new compressible hyperelastic model for the multi-axial deformation of blood clot occlusions in vessels.

Mechanical thrombectomy can be significantly affected by the mechanical properties of the occluding thrombus. In this study, we provide the first characterisation of the volumetric behaviour of blood clots. We propose a new hyperelastic model for the volumetric and isochoric deformation of clot. We demonstrate that the proposed model provides significant improvements over established models in terms of accurate prediction of nonlinear stress-strain and volumetric behaviours of clots with low and high red blood cell compositions. We perform a rigorous investigation of the factors that govern clot occlusion of a tapered vessel. The motivation for such an analysis is twofold: (i) the role of clot composition on the in vivo occlusion location is an open clinical question that has significant implications for thrombectomy procedures; (ii) in vitro measurement of occlusion location in an engineered tapered tube can be used as a quick and simple methodology to assess the mechanical properties/compositions of clots. Simulations demonstrate that both isochoric and volumetric behaviours of clots are key determinants of clot lodgement location, in addition to clot-vessel friction. The proposed formulation is shown to provide accurate predictions of in vitro measurement of clot occlusion location in a silicone tapered vessel, in addition to accurately predicting the deformed shape of the clot.

Investigating the Mechanical Behavior of Clot Analogues Through Experimental and Computational Analysis.

With mechanical thrombectomy emerging as the new standard of care for stroke treatment, clot analogues provide an extremely useful tool in the testing and design of these treatment devices. The aim of this study is to characterise the mechanical behavior of thrombus analogues as a function of composition. Platelet-contracted clot analogues were prepared from blood mixtures of various hematocrits. Mechanical testing was performed whereby clots were subjected to unconfined compression between two rigid plates. Two loading protocols were imposed: cyclic compression for 10 cycles at a constant strain-rate magnitude; stress-relaxation at a constant applied compressive strain. A hyper-viscoelastic constitutive law was identified and calibrated based on the experimental mechanical test data. Scanning electron microscopy (SEM) investigated the clot microstructure at various time-points. Clot analogue composition was found to strongly affect the observed mechanical behavior. The SEM found that the microstructure of the clot analogues was affected by the storage solution and age of the clot. The proposed hyper-viscoelastic constitutive model was found to successfully capture the material test data. The results presented in this study are of key importance to the evaluation and future development mechanical thrombectomy devices and procedures.