Thromboembolic Ischemic Stroke Research Articles

Abstract TP274: Inactivation of Serpinf2 Saves Lives, Prevents Disability and Hemorrhage in Experimental Thromboembolic Ischemic Stroke

More than 15 million individuals suffer ischemic stroke each year. High levels of serpinf2 (Sf2, a2-plasmin inhibitor, a2-antiplasmin) have been identified as a potential risk factor for human ischemic stroke. Methods: We examined the integrated effects of sf2 in a thromboembolic MCA stroke model with translational relevance to human disease. Using a blinded observer, neuronal cell death was quantified by TTC staining; hemorrhage and brain swelling were determined by planimetry. Immunohistochemistry was used to assess apoptosis, matrix metalloproteinase 9 expression and breakdown of the blood brain barrier. Results: Increasing the levels of sf2 caused larger infarctions by comparison to control mice (51% larger, p<0.002). Sf2 administration also markedly worsened brain swelling (p<0.05) but it did not cause hemorrhage. Conversely, when mice were given an sf2 inactivator (sf2-i), it markedly reduced stroke infarct size (95%) by comparison to control (p<0.001) and TPA-treated mice (p<0.001). Treatment of mice with an sf2-I also markedly reduced mortality rates by comparison to TPA-treated mice (p<0.0005) or control mice (p<0.004). The survival effect was dose-dependent: lower doses of the sf2-I were less effective than higher doses (p≤ 0.05) but still reduced mortality by comparison to control and TPA (p≤ 0.01). Microscopic examination of the brains of mice surviving the initial stroke period (> 12 hrs.) showed that sf2-i reduced stroke infarct size by comparison to control mice or TPA treated mice (p<0.001). There was less hemorrhage in sf2-i treated mice than occurred in control mice (p<0.001) or than in those receiving TPA (p<0.01). Inactivation of sf2 markedly reduced brain swelling by comparison to controls (p<0.001) and TPA-treated mice (p<0.05). Sf2-i treated mice showed no significant disability in Rotarod behavioral testing when compared to sham mice without strokes. Conclusions: Sf2 significantly enhances brain injury in ischemic stroke. Since inactivation of Sf2 prevents disability, hemorrhage and death it may prove to be a safe and effective strategy for treating acute ischemic stroke.

Novel Use of Scanning Electron Microscopy for Detection of Iron‐Induced Morphological Changes in Human Blood

Fibrinogen is key to the maintenance of hemostasis and is an acute phase protein that is part of the coagulation cascade of proteins. It plays a fundamental role in inflammation, particularly as indicator for a proinflammatory state and is a prominent marker for developing vascular inflammatory diseases. The ultrastructure of fibrin nets can be studied using scanning electron microscopy (SEM) with the addition of thrombin to plasma. In inflammatory conditions such as thromboembolic ischemic stroke and diabetes, the fibrin networks are changed to from dense matted fibrin deposits (DMDs) instead of typical netlike appearance. Similar DMDs can also be induced with the addition of FeCl(2) and FeCl(3). Importantly, the iron-induced DMDs look similar to those from patients with prothrombotic conditions. Excessive or misplaced tissue iron now is recognized to pose a substantial health risk. The current research therefore investigates the establishment of a laboratory fibrinogen model to study that might mimic fibrin fiber generation that is achieved using plasma from healthy and diseased individuals. Furthermore, to determine whether the addition of iron to purified fibrinogen will show DMDs and whether hydrophilic agents can prevent them. We conclude that SEM is a very effective tool for the visualization of circulatory consequences of the interaction of iron-induced hydroxyl radicals with human fibrinogen. Furthermore, this novel fibrinogen model provides a convenient method to study the interactions of the intramolecular and intermolecular hydrophobic forces responsible for the maintenance of the tertiary structure of native fibrin(ogen) and the prevention of iron-induced DMDs formation by hydrophilic agents.

Abstract 208: Detailed Analysis of the 30-day Outcomes in the Stenting Arm of the SAMMPRIS Trial

Background: Enrollment in first multicenter, randomized trial of Stenting and Aggressive Medical Management for Prevention of Recurrent Ischemic Stroke (SAMMPRIS) was stopped early owing to an excess 30-day event rate in the stenting group relative to the medical arm and a futility analysis. The purpose of this study was to carefully examine the peri-procedural events that occurred in the trial and to correlate them with any baseline or procedural factors that might be associated with an increased risk for an adverse outcome. Methods: 224 of 451 patients were randomized to the stent arm. 33 suffered an independently-adjudicated endpoint event (stroke or death) within 30 days. The present analysis was limited to patients that underwent the complete per-protocol stenting procedure (n=209). 30 total endpoint events occurred in these 209 patients. Two asymptomatic hemorrhages also occurred and were included in the analysis of peri-procedural complications. Study records, including case report forms, procedural reports, and baseline and procedural imaging for these 32 patients were reviewed. 30-day events were further categorized as wire perforation, reperfusion hemorrhage, and thrombo-embolic ischemic stroke. The ischemic strokes were further categorized as perforator or embolic, based on imaging findings. Baseline and procedural factors in these patients were compared to the remaining 177 patients for predictors of the 30-day events. Results: There were seven wire perforations, six resulting in an endpoint. Five were subarachnoid hemorrhages (SAH). One was treated with nBCA and resulted in an ischemic stroke. One additional asymptomatic SAH occurred as a result of a wire perforation. Six intra-parenchymal hemorrhages (IPH) occurred. Five of the six were definitely delayed hours or days after a successful revascularization procedure, likely representing reperfusion hemorrhage. One was asymptomatic. The remaining 19 30-day endpoints were ischemic strokes. 12 were perforator strokes and seven were distal embolic. Two of these were related to delayed stent thrombosis days after the procedure. No correlation between operator experience or volume in the trial and 30-day events was found. Baseline risk factors and procedural factors were also not correlated with the risk for a procedural complication. Conclusions: The 30-day events in the SAMMPRIS trial were multi-factorial, with no single factor accounting for the majority of events. Further research on angioplasty and stenting for intracranial stenosis will need to address several different mechanisms for this procedure to be proven effective.

Thromboembolic Ischemic Stroke and the Presence of Necrotic Platelets: A Scanning Electron Microscopy Investigation

Stroke is one of the most debilitating diseases worldwide, with its occurrence increasing in Western societies. Central to the pathogenesis of thromboembolic stroke is the involvement of platelets. During thromboembolic events, nucleated cells undergo cell death, and platelets are also affected by parameters causing these incidents. Particularly, initiation of necrotic cell death at sites of vascular injury may play an important role in inducing inflammatory and repair processes. In the current research, the authors investigate whether a changed platelet ultrastructure is visible in thromboembolic stroke and whether it might be visible in platelets as apoptosis or necrosis. Therefore, in the current investigation, the authors study smears of platelet-rich plasma (PRP) from thromboembolic ischemic stroke patients to investigate whether a changed morphology is visible in distressed platelets. Scanning electron microscopy is used and platelets are viewed at up to 200,000× machine magnification. Results indicate that thromboembolic ischemic stroke causes membrane tears and swollen platelets, and this is indicative of necrosis. It is therefore concluded that this morphology might be due to the pro-coagulant activity characteristic of the disease.

Differences in fibrin fiber diameters in healthy individuals and thromboembolic ischemic stroke patients

Cerebrovascular disease is one of the leading causes of death and the cause of long-term adult disability. An important characteristic of thromboembolic ischemic stroke is a prothrombotic or hypercoagulable state and altered fibrin clot structure, whereas a resistance to fibrinolysis is also present. An expansive fibrin network is created when adding thrombin, and in stroke, the network appears thickened, netted and matted, compared with that of healthy individuals. Although this is clearly visible in micrographs of patients, there is a need to quantify the changes. The current study, therefore, investigates fibrin fiber diameters in stroke patients and compares it to healthy individuals. The fiber diameters were measured in nanometres, with University of Texas Health Science Center at San Antonio (UTHSCSA) Image Tool. A total of 100 measurements were done for each of the 12 patients in the healthy control group, and the same number of measurements was done for 12 stroke patients. These measurements were statistically analysed with NCSS 2007, using a significance level of 0.05. Normality was assessed with the Shapiro-Wilk W test and the thickest and thinnest fiber of each individual in the two groups was quantified and differences between groups were assessed with the Student's t-test. Results showed that there is a statistical difference in fibrin fiber thickness during thromboembolic ischemic stroke. We conclude that the changed coagulation and hemostasis, typically associated with stroke, causes a statistically relevant change in fibrin thickness, and that this netted and matted network is more resistant to lyses.

Scanning electron microscopy analysis of erythrocytes in thromboembolic ischemic stroke

Erythrocytes play an important role in hemostasis and disease conditions. During ischemic stroke, erythrocytes undergo oxidative and proteolytic changes resulting in a changed cellular rheology. Blood samples were obtained from controls and thromboembolic ischemic stroke patients (within 48 h of stroke). The ultrastructure of erythrocytes was compared, using a scanning electron microscope (SEM). Abnormal morphology included codocytes, knizocytes, stomatocytes, and echinocytes. Percentage of abnormal cells was calculated, and the analyses were performed using the statistical program NCSS with the level of significance set at 0.05. A t-test was carried out to compare the data from the erythrocyte counts of stroke patients with that of the control subjects. Ultrastructural SEM results showed that there are a large percentage of erythrocytes in healthy individuals that do not have a typical discoid shape, when studying the cells using a high magnification electron microscope. Furthermore, analysis showed that variation in shape is so subtle that it is not clearly visible using a typical light microscopy blood smear analysis. Thromboembolic ischemic stroke patients presented with a significant amount of erythrocytes with abnormal morphology. We suggest that in healthy individuals, a typical smear would contain several nondiscoid-shaped erythrocytes, only clearly visible at high magnification. However, thromboembolic ischemic stroke does significantly impact erythorcyte shape, and this change in morphology may result in an impaired microcirculation, as well as impaired oxygen carrying capacity. This changed morphology may further complicate the restoring of homeostasis caused by acute thromboembolic stroke.

Interactions between Nitrous Oxide and Tissue Plasminogen Activator in a Rat Model of Thromboembolic Stroke

Preclinical evidence in rodents has suggested that inert gases, such as xenon or nitrous oxide, may be promising neuroprotective agents for treating acute ischemic stroke. This has led to many thinking that clinical trials could be initiated in the near future. However, a recent study has shown that xenon interacts with tissue-type plasminogen activator (tPA), a well-recognized approved therapy of acute ischemic stroke. Although intraischemic xenon inhibits tPA-induced thrombolysis and subsequent reduction of brain damage, postischemic xenon virtually suppresses both ischemic brain damage and tPA-induced brain hemorrhages and disruption of the blood-brain barrier. The authors investigated whether nitrous oxide could also interact with tPA. The authors performed molecular modeling of nitrous oxide binding on tPA, characterized the concentration-dependent effects of nitrous oxide on tPA enzymatic and thrombolytic activity in vitro, and investigated the effects of intraischemic and postischemic nitrous oxide in a rat model of thromboembolic acute ischemic stroke. The authors demonstrate nitrous oxide is a tPA inhibitor, intraischemic nitrous oxide dose-dependently inhibits tPA-induced thrombolysis and subsequent reduction of ischemic brain damage, and postischemic nitrous oxide reduces ischemic brain damage, but in contrast with xenon, it increases brain hemorrhages and disruption of the blood-brain barrier. In contrast with previous studies using mechanical acute stroke models, these data obtained in a clinically relevant rat model of thromboembolic stroke indicate that nitrous oxide should not be considered a good candidate agent for treating acute ischemic stroke compared with xenon.

The Use of a Desktop Scanning Electron Microscope as a Diagnostic Tool in Studying Fibrin Networks of Thrombo-embolic Ischemic Stroke

Proneness to the formation of tight and rigid fibrin networks has been shown to be independently associated with thrombotic disease. These changes may be visible long before the actual event. Previous research has shown that there is a fundamental difference between fibrin network architecture of controls compared to fibrin networks of patients 48 h post-thrombo-embolic ischemic stroke. This conclusion was made using a high-tech scanning electron microscope (SEM). Here the authors investigate whether ultrastructure of these networks can be successfully analyzed when using a smaller, desktop SEM. Such a screening procedure would not only be inexpensive, but could potentially warn patients of a possible thrombotic event long before any symptoms are prevalent. Platelet-rich plasma, obtained from healthy volunteers and thrombo-embolic ischemic stroke patients (48 h poststroke), was activated by the addition of thrombin. Fibrin networks were compared using a Zeiss ULTRA plus FEG-SEM with InLens and a desktop portable ZEOL SEM (ZEOLNeoScope). This desktop version produces micrographs that may easily be analyzed, and the information gained by studying the micrographs was comparable to that of the Zeiss ULTRA plus FEG-SEM. Such a desktop machine might be used as a screening tool or to identify individuals with risk, before the actual event. In addition, it may provide valuable information in recovering stroke patients.

A descriptive investigation of the ultrastructure of fibrin networks in thrombo-embolic ischemic stroke

Stroke is one of the leading causes of death worldwide. Formation of a fibrin clot is controlled by a group of tightly regulated plasma proteases and cofactors and a change in the fibrin fiber formation causes an alteration in clot morphology. This plays an important role during thrombotic events. In the current study we investigated the ultrastructure of fibrin networks from fifteen ischemic stroke patients by using scanning electron microscopy. Clot morphology was investigated with and without the addition of human thrombin to the platelet rich plasma. Previously it was shown that, when studying the ultrastructure of fibrin networks, the addition of thrombin is necessary to form an expansive, fully coagulated layer of fibers. Results from the addition of thrombin to the plasma showed thick, matted fibrin fibers and a net covering some of the major fibers in stroke patients. Typical control morphology with major thick fibers and minor thin fibers could be seen in some areas in the stroke patients. In stroke patients, without the addition of thrombin, a matted fibrin network still formed, indicating that the factors responsible for the abnormal fibrin morphology are present in the circulating plasma and is the cause of the observed matted, layered morphology. This is not present in healthy individuals. From the results obtained we suggest that this changed morphology might be useful in a screening regime to identify the possibility of a stroke or even to follow the progress of stroke patients after treatment.

Acute changes of coagulation and fibrinolysis parameters after experimental thromboembolic stroke and thrombolytic therapy

Thrombolysis is the only effective pharmaceutical therapy in acute ischemic stroke in humans but has a high risk of intracerebral hemorrhage. We aimed to establish an animal model to study changes of coagulation and fibrinolytic parameters during thromboembolic ischemic stroke and thrombolysis with recombinant tissue plasminogen activator (rt-PA). We used a thromboembolic stroke model in the rat. Animals were treated with rt-PA thrombolysis (n=10) and compared with untreated (n=10), sham operated (n=10) and control animals (n=20). Coagulation parameters (APTT, PT, TT, fibrinogen, AT III, TAT) and fibrinolytic parameters (t-PA antigen concentration, t-PA activity, PAI-1 concentration, PAI activity, plasminogen, antiplasmin) were measured at two time points (2.5 and 5h after stroke induction) with a battery of commercially available test kits. We observed an (1) initiation of coagulation and inhibition of fibrinolysis by the operation procedure itself, (2) simultaneous activation of fibrinolysis and its inhibitors after stroke induction and (3) potent initiation of fibrinolysis and consumption of fibrinolysis inhibitors after rt-PA therapy of stroke. We established a model system to monitor coagulation and fibrinolysis during thrombolytic therapy of stroke in the rat. This model may be used to study the influence of these parameters on hemorrhagic stroke transformation and outcome in experimental stroke in future.

Access to state-of-the-art healthcare: a missing dynamic in consumer selection of a retirement community.

The top ten retirement destinations in the United States are compared to the State of Michigan with regard to the quality of medical care that can be readily obtained in the event of a medical emergency. Access to care during the following three medical emergencies was analyzed: cardiac arrest, thromboembolic (ischemic) stroke, and severe traumatic injury. In all instances, successful treatment is dependent on timely access to care. The authors report that, when combined with the health status of baby boomers and their migratory trends on retirement, access to tertiary medical care for the treatment of cardiac arrest, certified primary stroke centers, and level I trauma centers is the missing dynamic in the choice of retirement community among today's prospective retirees. They recommend that physicians encourage this patient population to consider such factors when choosing a retirement community.

Safety and efficacy of intravenous enoxaparin for carotid endarterectomy: A prospective randomized pilot trial

This prospective, randomized, single-center, open-label pilot study evaluated the safety and efficacy in carotid surgery of a single intraoperative bolus of body weight-adjusted enoxaparin compared with unfractionated heparin. Symptomatic and asymptomatic patients with high-grade internal carotid artery stenosis were included. The primary objective was to evaluate perioperative efficacy (incidence of thromboembolic ischemic stroke). The secondary objective was to evaluate safety, including avoidance of hematoma at the site of surgery, gastrointestinal bleeding, rate of blood transfusions, and occurrence of heparin-induced thrombocytopenia. From July 2005 to June 2006, 338 consecutive patients undergoing carotid endarterectomy were enrolled; of these, 115 patients did not fulfill inclusion criteria, and 63 patients refused to participate. The remaining 160 patients were assigned in a 3:1 randomization to receive enoxaparin (0.5 mg/kg) or unfractionated heparin (5000 IU) intraoperatively as an intravenous bolus (120 and 40 patients, respectively). The mean patient age was 70.3 years (range, 43.3-94.7 years), and 54 were women. Internal carotid artery stenosis was asymptomatic in 55% and symptomatic in 45%. The difference in baseline characteristics between these groups was not significant. The rate of cerebral embolic events was 0.8% in the enoxaparin group (n = 1) and 2.5% in the unfractionated heparin group (n = 1). The rate of severe bleeding complications was 1.7% in the enoxaparin group (n = 2) and 5% in the unfractionated heparin group (n = 2; P > .05). No case of heparin-induced thrombocytopenia was observed. This pilot study found no difference between enoxaparin and unfractionated heparin during carotid endarterectomy in perioperative bleeding or embolic events. A large multicenter trial seems to be warranted.