Dai, Daying

Introduction: Previous studies have successfully created blood clot analogues for In-vitro testing using animal blood. Blood components vary greatly among species and thus, creating clot analogues with human blood is likely a more accurate representation of thrombi formed in the human vasculature. We present a novel method of creating clot analogues from human blood and platelets that mimic the process by which clots form In-vivo . Methods: Following IRB approval from Mayo Clinic, human whole blood and platelets donations were obtained from the Blood Transfusion service. The whole blood was centrifuged at 1,200RPM for 20 minutes to separate it into its constituents. Plasma was removed and the remaining Red Blood Cells and Buffy Coat were mixed together by inverting. A total of 12 clot analogues were created with varying concentrations of components; Red Blood cells/Buffy Coat, Plasma and Platelets. Thrombin was added first to stimulate platelets activation for a total of 5 mins whilst continuously mixing by inversion. The RBC/WBC mixture was added next followed by CaCl2. The mixture was then quickly drawn into a 3cc syringe and spun overnight at 20RPM at room temperature to mimic dynamic flow conditions. Macro-photographs were taken to display the variation in texture and color between different clot analogue types. The clots were then fixed in 10% neutral buffered formalin for 24 hours prior to being processed. Histopathological analysis was performed using Hematoxylin and Eosin (H&E) and Martius Scarlet Blue (MSB) staining to confirm clot composition. Results: Red Blood cell-rich, Fibrin-rich, Platelet-rich and mixed clot analogues that accurately mimic clots retrieved from Acute Ischemic Stroke Patients were created. The range of histopathological compositions of the clot analogues is similar to that of the clinical samples. Conclusions: The addition and activation of platelets is key to creating accurate clot analogues for In-vitro testing. Spinning the clots is important to prevent natural sedimentation and mimic the In-vivo situation.

Introduction: Characterization of acute ischemic stroke thrombi has typically focused on the two dimensional (2D) histological characterization of the thrombus. Little is known about the three dimensional (3D) structure of these occlusive thrombi. Electron Microscopy (EM) can be used to obtain 3D information about the organization of clot components. Individual structures can be segmented and qualitative and quantitative information obtained from the clots. Methods: Following IRB approval from Mayo Clinic, acute ischemic stroke clot samples were collected from 10 patients treated at Mayo Clinic. Samples were immediately fixed in fresh modified Trumps fixative solution. A representative fragment of clot was taken and split longitudinally into two pieces; one piece was sent for 3D-EM analysis and the other for histological processing and analysis. Hematoxylin and eosin stain (H&E) and Martius Scarlett Blue (MSB) histological stains were used for conventional histopathology evaluation and components analysis. Immunohistochemical (IHC) staining using anti-CD42b (Platelets) was applied to confirm the presence of platelets in each clot. Serial block-face scanning electron microscopy was used to generate high-resolution three-dimensional images. Results: The 2D morphological features of the clot were identified using H&E, MSB and anti-CD42b staining. The 3D organization of thrombi varies greatly, both within and between clots depending on the constituents. Red Blood Cell-rich areas are mainly composed of Red Blood Cells packaged tightly together, with a small number of Fibrin strands interwoven between the cells. Fibrin-rich areas are characterized by dense networks of fibrin strands along with platelets and White Blood Cells with some sparse Red Blood Cells present. Platelet-rich areas are composed of large volumes of de-granulated platelets, some fibrin stands and white blood cells and an absence of Red Blood Cells. Conclusions: 3D electron microscopy can provide important information on the organization of components within thrombi. This could provide key information on the age and etiology of the thrombus. In addition, it may help to inform thrombolytic treatment and medical device design.

Purpose: Endovascular occlusion of aneurysms with the use of detachable platinum coils has proved to be a safe and durable alternative to the traditional surgical clipping. The aim of the present study is to understand the mechanism of apoptosis in aneurysm embolized with platinum coils using rabbit elastase‐induced aneurysm model. Materials & Methods: Elastase‐induced saccular aneurysms were created at the origin of right common carotid artery in thirty rabbits. Aneurysms were allowed to mature for 8 weeks then 20 aneurysms were embolized using platinum coils by endovascular means. Ten uncoiled aneurysms were used as controls. After 2 weeks (n=10) and 4 weeks (n=10) of implantation, aneurysm samples were harvested for apoptotic studies. Results: Expression of pro‐caspases‐3, ‐8 and ‐9 were elevated in coiled aneurysm embolized with platinum coils at both time points when compared with uncoiled aneurysm and control left carotid artery. Cleaved caspases‐3, ‐8 and ‐9 were found to be expressed only at 4 weeks after embolization. Cells within aneurysm cavity were TUNEL positive at 4 weeks only. Expression of tumor necrosis‐α (TNF‐α) was high in coiled aneurysm when compared with controls. There was no significant difference in the expression of Fas‐ligand among groups. Decreased expression of antiapoptotic protein, Bcl‐2, and increased expression of proapoptotic proteins, Bad, Bax and Bid, were observed in coiled aneurysms at both time points. Conclusion: Activation apoptosis in aneurysms after embolization with platinum coils is mediated by both TNF‐α mediated‐extrinsic and Bcl‐2 mediated‐intrinsic pathway.

The purpose of the study was to test the hypothesis that systemic administration of vitamin C, through its action of stimulating collagen synthesis and crosslinking, would improve the healing response in experimental aneurysms treated with platinum coil embolization. Nineteen experimental aneurysms in rabbits were embolized with platinum coils (>30% packing density). Animals were divided into three groups. Group 1 (n=6) rabbits served as controls, Group 2 (n=5) rabbits were fed with a vitamin C‐supplemented feed and Group 3 (n=8) rabbits were medicated with vitamin C pills. Subjects were euthanized at 12 weeks following coil implantation and serum vitamin C levels were measured. Digital subtraction angiography was used to evaluate stability after embolization. Histological samples were examined with a grading system (range, 0–12) based on the neck and dome features. Masson Trichrome was employed to evaluate the collagen deposition. One‐way analysis of variance was used to test the significance in histological healing and angiographic measurements between groups. There were no significant differences between groups in aneurysm size or angiographic findings. Mean serum vitamin C concentration was significantly higher in Group 3 (45.4±9.0 mM) and Group 2 (38.6±8.9 mM) compared to Group 1 (21.1±3.6 mM). There was no statistically significant change in the level of vitamin C between Group 2 and Group 3. There was no significant difference in histological scores or collagen deposition among groups. Our results suggest that vitamin C supplementation following platinum coil embolization may not improve long‐term cerebral aneurysm. This study was supported by the American Heart Association (09SDG2160146‐01).

Background Flow diverters (FDs) are an effective treatment for intracranial aneurysms, though not free from hemorrhagic complications. A previous study demonstrated increased vascular contractility after FD-implantation as a potential mechanism of distal complications. Our study aimed to investigate whether L-arginine medication affects vascular contractility following FD deployment in a rabbit model. Methods FDs were implanted in the aorta of normal rabbits (+FD, n = 10), with sham-operated aorta as controls (n = 5). L-Arginine was given in the drinking water (2.25% L-arginine hydrochloride) of half of the +FD animals (+FD/+Arg). Force contraction vascular contractility studies were performed on the aortic rings proximal and distal to the FD using an organ bath. Total eNOS, eNOS(pS1177), eNOS(pT495), COX-2, and S100A4 were quantified by western analysis on total protein lysates from aortic segments, normalizing to GAPDH. Results Mean vascular contractility was 53% higher in distal relative to proximal aortic segments (P = 0.0038) in +FD animals, but were not significantly different in +FD/+Arg animals, or in sham-operated controls. The +FD animals expressed significantly reduced levels of eNOS(pS1177) than sham-operated controls (P = 0.0335), while both the +FD and +FD/+Arg groups had reduced levels of eNOS(pT495) relative to sham-operated controls (P = 0.0331 and P = 0.0311, respectively). Conclusion These results suggest that L-arginine medication reduces distal vascular contractility after FD treatment via nitric oxide production and thus might mitigate risk for downstream complications.

Background Several animal studies have demonstrated that mechanical thrombectomy (MT) for acute ischemic stroke (AIS) may cause vessel wall injury (VWI). However, the histological changes in human cerebral arteries following MT are difficult to determine. Objective To investigate the occurrence of VWI during MT by histological and immunohistochemical evaluation of AIS clots. Methods As part of the multicenter STRIP registry, 277 clots from 237 patients were analyzed using Martius Scarlett Blue stain and immunohistochemistry for CD34 (endothelial cells) and smooth muscle actin (smooth muscle cells). Results MT devices used were aspiration catheters (100 cases), stentriever (101 cases), and both (36 cases). VWI was found in 33/277 clots (12%). There was no significant correlation between VWI and MT device. The degree of damage varied from grade I (mild intimal damage, 24 clots), to grade II (relevant intimal and subintimal damage, 3 clots), and III (severe injury, 6 clots). VWI clots contained significantly more erythrocytes (p=0.006*) and less platelets/other (p=0.005*) than non-VWI clots suggesting soft thrombus material. Thrombolysis correlated with a lower rate of VWI (p=0.04*). VWI cases showed a significantly higher number of passes (2 [1–4] vs 1 [1–3], p=0.028*) and poorer recanalization outcome (p=0.01*) than cases without VWI. Conclusions Histological markers of VWI were present in 12% of AIS thrombi, suggesting that VWI might be related to MT. VWI was associated with soft thrombus consistency, higher number of passes and poorer revascularization outcome. There was no significant correlation between VWI and MT device.

Background and aim Mechanical thrombectomy (MT) for large vessel occlusion often requires multiple passes to retrieve the entire thrombus load. In this multi-institutional study we sought to examine the composition of thrombus fragments retrieved with each pass during MT. Methods Patients who required multiple passes during thrombectomy were included. Histopathological evaluation of thrombus fragments retrieved from each pass was performed using Martius Scarlet Blue staining and the composition of each thrombus component including RBC, fibrin and platelet was determined using image analysis software. Results 154 patients underwent MT and 868 passes was performed which resulted in 263 thrombus fragments retrieval. The analysis of thrombus components per pass showed higher RBC, lower fibrin and platelet composition in the pass 1 and 2 when compared to pass 3 and passes 4 or more combined (P values <0.05). There were no significant differences between thrombus fragments retrieved in pass 1 and pass 2 in terms of RBC, WBC, fibrin, and platelet composition (P values >0.05). Similarly, when each composition of thrombus fragments retrieved in pass 3 and passes 4 or more combined were compared with each other, no significant difference was noted (P values >0.05). Conclusion Our findings confirm that thrombus fragments retrieved with each pass differed significantly in histological content. Fragments in the first passes were associated with lower fibrin and platelet composition compared to fragments retrieved in passes three and four or higher. Also, thrombus fragments retrieved after failed pass were associated with higher fibrin and platelet components.

Abstract Objective Therapeutic hypothermia is a potentially powerful and controversial clinical tool for neuroprotection following acute neurologic pathology, particularly vascular injury. Indeed, therapeutic hypothermia remains a standard of care for postcardiac arrest ischemia and acute neonatal hypoxic-ischemic encephalopathy, improving both survival and outcomes. Although therapeutic hypothermia remains promising for cellular and systems-based neuronal protection in other neurologic injury states, the systemic side effects have limited clinical utility, confounded analysis of potential neurologic benefits, and precluded the completion of meaningful clinical trials. Methods To address such limitations, we developed and tested a novel, minimally invasive, neurocritical care device that employs continuous circulation of cold saline through the pharyngeal region to deliver focal cerebrovascular cooling. We conducted a preclinical safety and efficacy trial in six adult porcine animals to assess the validity and functionality of the NeuroSave device, and assess cooling potential following middle cerebral artery occlusion (n = 2). Results NeuroSave consistently lowered brain parenchymal temperature by a median of 9°C relative to core temperature within 60 minutes of initiation, including in ischemic cerebral parenchyma. The core body temperature experienced a maximal reduction of 2°C, or 5% of body temperature, with no associated adverse effects identified. Conclusion The present study uses a large animal preclinical model to demonstrate the safety and efficacy of a novel, noninvasive device for the induction of robust and systemically safe hypothermia within the brain.

Background To develop a preclinical thromboembolic occlusion model for studying revascularization strategies. Methods Clot analog with barium sulfate was injected into the distal aorta in 9 New Zealand white rabbits. The situation of aorta occlusion was compared among fibrin-rich (n=4), red blood cell (RBC)-rich (n=3), and whole blood clot analogs (n=2) using digital subtraction angiography. Arterial geometries, histologic features and circumferential stretch of the distal aorta in rabbits were compared with the common carotid artery in swine and the distal internal carotid artery (ICA) in humans. Aspiration thrombectomy and mechanical thrombectomy using a stent retriever were performed in two rabbits. Results The aortic bifurcation was occluded after a single delivery of clot in 4 cases. It was occluded after the second clot injection in the 5 remaining rabbits. Fragmentation of RBC-rich clots occurred during clot injection in 2 cases. The mean diameters of the distal aorta and right common iliac artery in rabbits were 3.7±0.4 and 2.8±0.3 mm, respectively; the mean diameters of human ICA, and first and second segments of the middle cerebral artery (M1, M2) were 3.6±0.4, 3.1±0.4, and 2.4±0.4 mm, respectively. Arterial revascularization was achieved in both rabbits. Geometric, mechanical and histological factors of the distal aorta in rabbit were more close to human distal ICA than swine carotid artery. Conclusion Arterial occlusion can be achieved at the aortic bifurcation in rabbits, which is comparable to human ICA bifurcation. This thrombectomy model has the potential to be used for testing of thrombectomy devices.

Background Characterization of acute ischemic stroke (AIS) clots has typically focused on two-dimensional histological analysis of the thrombus. The three-dimensional (3D) architecture and distribution of components within emboli have not been fully investigated. The aim of this study was to examine the composition and microstructure of AIS clots using histology and serial block-face scanning electron microscopy (SBFSEM). Methods As part of the multi-institutional STRIP registry, 10 consecutive AIS emboli were collected from 10 patients treated by mechanical thrombectomy. Histological and immunohistochemical analysis was performed to determine clot composition. SBFSEM was used to assess the ultrastructural organization of the clots and specific features of individual components. Results Quantification of Martius Scarlett Blue stain identified fibrin (44.4%) and red blood cells (RBCs, 32.6%) as the main components. Immunohistochemistry showed a mean platelet and von Willebrand factor content of 23.9% and 11.8%, respectively. The 3D organization of emboli varied greatly depending on the region analyzed. RBC-rich areas were composed mainly of tightly packed RBCs deformed into polyhedrocytes with scant fibrin fibers interwoven between cells. The regions with mixed composition showed thick fibrin fibers along with platelets, white blood cells and RBC clusters. Fibrin-rich areas contained dense fibrin masses with sparse RBC. In three cases, the fibrin formed a grid-like or a sponge-like pattern, likely due to thrombolytic treatment. Segmentation showed that fibrin fibers were thinner and less densely packed in these cases. Conclusions 3D-SEM provides novel and potentially clinically relevant information on clot components and ultrastructure which may help to inform thrombolytic treatment and medical device design.