Angelika Alonso

Molecular Imaging of Human Thrombus With Novel Abciximab Immunobubbles and Ultrasound

Background and Purpose— Molecular imaging of therapeutic interventions with targeted agents that simultaneously carry drugs or genes for local delivery is appealing. We investigated the ability of a novel microbubble carrier (immunobubble) for abciximab, a glycoprotein IIb/IIIa receptor inhibitor, for ultrasonographic molecular imaging of human clots. Methods— Human thrombi were incubated with immunobubbles conjugated with abciximab. Control clots were incubated in either saline or with immunobubbles conjugated with nonspecific antibody. We evaluated immunobubble suspensions with variable concentrations of encapsulated gas and measured mean acoustic intensity of the incubated clots. In vivo molecular imaging of human thrombi with abciximab immunobubbles was evaluated in a rat model of carotid artery occlusion. Results— Mean acoustic intensity was significantly higher for abciximab immunobubbles as compared with control immunobubbles under all conditions tested with maximum difference in intensity at a gas volume of 0.2 &mgr;L (P=0.0013 for mechanical index 0.05, P=0.0001 for mechanical index 0.7). Binding of abciximab immunobubbles to clots in vitro led to enhanced echogenicity dependent on bubble concentration. In vivo ultrasonic detectability of carotid thrombi was significantly higher for clots targeted with abciximab immunobubbles (P<0.05). Quantification of in vivo contrast enhancement displayed a highly significant increment for abciximab immunobubble-targeted clots compared with nonspecific immunobubble-targeted clots (P<0.0001) and to native clots (P<0.0001). Conclusions— This study demonstrates the feasibility of using a therapeutic agent for selective targeting in vascular imaging. Abciximab immunobubbles improve visualization of human clots both in vitro and in an in vivo model of acute arterial thrombotic occlusion.

In vivo clot lysis of human thrombus with intravenous abciximab immunobubbles and ultrasound

Abciximab immunobubbles have been introduced recently for ultrasonographic molecular imaging of human thrombus. This study investigates the potential of using these novel bubbles for enhancing sonothrombolysis. In particular, it addresses the question of whether ligand targeting of bubbles with abciximab improves the effectiveness of lysis with ultrasound. A partial thrombotic occlusion of the right common carotid artery of 16 rats was produced by insertion of human clot material via an external carotid artery catheter. Rats received abciximab immunobubbles, non-specific control immunobubbles or saline intravenously over 30 minutes in combination with pulsed 2 MHz ultrasound. Blood samples were taken at baseline and 5, 10, 20, 30 and 60 minutes after beginning treatment. Human D-dimer levels for quantification of thrombolysis were analysed by ELISA. Only animals treated with abciximab immunobubbles and ultrasound showed a significant increase of D-dimer levels over time (p = 0.043, linear trend p = 0.037), whereas in the other two groups, no significant increase over time was found. Overall, animals in the abciximab immunobubbles group showed higher plasma D-dimer levels than animals treated with non-specific immunobubbles (p = 0.049) and animals treated with ultrasound alone (p = 0.017). In histological sections, thrombi treated with abciximab immunobubbles and ultrasound showed clear signs of disintegration in contrast to thrombi in both control groups. 2 MHz ultrasound in combination with abciximab immunobubbles induces thrombolysis without lytic agents that is superior to insonation of non-specific immunobubbles.

Progress in Sonothrombolysis for the Treatment of Stroke

In 1974, Sobbe et al1 applied 26.5-kHz ultrasound (US) to recanalize thrombosed iliofemoral arteries in dogs with minimal complications. These pioneer efforts were followed by studies showing that catheter-based or transcutaneous US can enhance the effect of fibrinolytic agents in recanalizing thrombosed arteries,2–8 thus paving the way for first clinical studies evaluating the adjunct effect of US in treating patients with ischemic stroke. ### Mechanisms #### US Thrombolysis Despite numerous studies documenting a thrombolytic effect of US, the mechanisms remain poorly understood. Inertial cavitation (ie, the formation and violent collapse of gas-filled bubbles in a fluid exposed to US) gave rise to transient microjets that disintegrate thrombus mechanically.9 Stable cavitation (ie, sustainable nonlinear periodic contraction or expansion of a gas body or bubble) may be more effective than inertial cavitation in clot lysis.10 US also facilitates penetration of fibrinolytic drugs into the thrombus and binding to fibrin.11 This is because US promotes the motion of fluids around the clot surface through a process called microstreaming. Moreover, pressure waves may increase the permeation of tissue-type plasminogen activator (tPA) into the interior of the fibrin network.12 Heating is uniformly present in tissue exposed to US but has been deemed too mild to explain thrombolytic effects. #### Microbubble-Enhanced Thrombolysis With tPA Significant amplification of lysis occurs with the addition of microbubbles to the combination of thrombolytic drug and US.13–15 Microbubbles, composed of lipid, albumin, or galactose shells and ranging in size from 0.5 to 5 μm, lower the threshold for thrombolysis by providing a pre-existing bubble that easily can be made to cavitate by US. Stable cavitation can produce microstreaming in the area and dramatically enlarge the bubble momentarily. This will cause localized mechanical stress on the adjacent clot. The surface of the clot will erode, and even penetration and numerous microscopic …

Reorganization of gap junctions after focused ultrasound blood-brain barrier opening in the rat brain

Ultrasound-induced opening of the blood–brain barrier (BBB) is an emerging technique for targeted drug delivery to the central nervous system. Gap junctions allow transfer of information between adjacent cells and are responsible for tissue homeostasis. We examined the effect of ultrasound-induced BBB opening on the structure of gap junctions in cortical neurons, expressing Connexin 36, and astrocytes, expressing Connexin 43, after focused 1-MHz ultrasound exposure at 1.25 MPa of one hemisphere together with intravenous microbubble (Optison, Oslo, Norway) application. Quantification of immunofluorescence signals revealed that, compared with noninsonicated hemispheres, small-sized Connexin 43 and 36 gap-junctional plaques were markedly reduced in areas with BBB breakdown after 3 to 6 hours (34.02±6.04% versus 66.49±2.16%, P=0.02 for Connexin 43; 33.80±1.24% versus 36.77±3.43%, P=0.07 for Connexin 36). Complementing this finding, we found significant increases in large-sized gap-junctional plaques (5.76±0.96% versus 1.02±0.84%, P=0.05 for Connexin 43; 5.62±0.22% versus 4.65±0.80%, P=0.02 for Connexin 36). This effect was reversible at 24 hours after ultrasound exposure. Western blot analyses did not show any change in the total connexin amount. These results indicate that ultrasound-induced BBB opening leads to a reorganization of gap-junctional plaques in both neurons and astrocytes. The plaque-size increase may be a cellular response to imbalances in extracellular homeostasis after BBB leakage.

Focal Delivery of AAV2/1-transgenes Into the Rat Brain by Localized Ultrasound-induced BBB Opening

Delivery of drugs and macromolecules to the central nervous system (CNS) is hindered by the blood–brain barrier (BBB). Several approaches have been used to overcome this hindrance to facilitate the treatment of various CNS diseases. We now present results showing that chimeric adeno-associated virus 2/1 (AAV2/1) particles containing the coding region for the LacZ gene are efficiently delivered into the rat brain upon intravenous (IV) administration after BBB opening by focused ultrasound in the presence of vascular acoustic resonators. We show that the transgene is correctly and efficiently expressed in cells located in the neighborhood of the insonated focus, especially in the vicinity of small vessels and capillaries. Histochemical LacZ staining allows the identification of large amounts of cells expressing the enzymatically active protein. Using double immunofluorescence (IF) with antibodies against tubulinIII and bacterial LacZ, we identified these cells to be mostly neurons. A small proportion of the transduced cells was recognized as glial cells, reacting positive in the IF with antibodies against astrocytic markers. These results demonstrate that our approach allows a very specific, localized, and efficient expression of intravenously administered transgenes in the brain of rats upon ultrasound-induced BBB opening.

What does elevated high-sensitive troponin I in stroke patients mean: concomitant acute myocardial infarction or a marker for high-risk patients?

Background: Acute ischemic stroke patients may occasionally suffer from concomitant acute coronary syndrome (ACS). Troponin I and T are established biomarkers to detect ACS. Recently introduced high-sensitive cardiac troponin (hs-TNI and hs-TNT) assays are increasingly used to identify ACS in stroke patients even without signs or symptoms of ACS. These new test systems very often detect elevated values of hs-troponin, although clinical relevance and consequences of elevated hs-TNI values in these patients are unclear so far. Patients and Methods: We examined hs-TNI values in 834 consecutive ischemic stroke patients admitted to our Comprehensive Stroke Center during a 1-year period. hs-TNI was measured immediately after admission and after 3 h if initial hs-TNI was elevated above the 99th percentile of normal values (>0.045 ng/ml). Patients with elevated values were divided into two groups: (1) constant and (2) dynamic hs-TNI values. The dynamic approach was defined as a 30% rise or fall of the hs-TNI value above the critical value within 3 h. All patients received stroke diagnostic and continuous monitoring according to international stroke unit standards, including a 12-lead ECG, blood pressure, body temperature and continuous ECG monitoring, as well as regular 6-hourly neurological and general physical examination (including NIHSS scores). The cardiologists - as members of the Stroke Unit team - evaluated clinical symptoms/examination, as well as laboratory, echocardiographic and ECG findings for the diagnosis of ACS. Results: 172/834 (20.6%) patients showed elevated hs-TNI levels on admission. Patients with elevated hs-TNI values exhibited a significantly (p < 0.001) increased rate of hypertension (89 vs. 77.2%), history of stroke (24.4 vs. 14.8%), history of coronary artery disease (65.7 vs. 34.1%), history of myocardial infarction (22.1 vs. 7.6%), heart failure (12.8 vs. 5.7%) and atrial fibrillation (44.2 vs. 23.6%). 82/136 patients showed constant and 54/136 patients dynamic hs-TNI values: among the latter, 5 patients were diagnosed with ST segment elevation myocardial infarction (STEMI) and 24 with non-STEMI (NSTEMI). Conclusion: Our data demonstrate that hs-TNI was elevated in about 20.6% of acute ischemic stroke patients but therapeutically relevant ACS was diagnosed only in the dynamic group. hs-TNI elevations without dynamic changes may occur in stroke patients without ACS due to different reasons that stress the heart. Therefore, we suppose that hs-TNI is a sensitive marker to detect high-risk patients but serial measurements are mandatory and expert cardiological workup is essential for best medical treatment and to accurately diagnose ACS in acute ischemic stroke patients.

Brain Temperature During 340-kHz Pulsed Ultrasound Insonation A Safety Study for Sonothrombolysis

Background and Purpose— Because ultrasound is used for improving thrombolysis of cerebral infarction but continuous ultrasound insonation also has significant thermal effects, we evaluated brain temperature increase and tissue destruction during pulsed ultrasound emission. Methods— We examined 340-kHz pulsed ultrasound effects in male Wistar rats. Ultrasound was applied transcranially for 30 minutes on different power levels (1 to 7 W/cm2). Temperature was measured at different locations (brain, in the focus of ultrasound beam, inner ear, temporalis muscle, and rectum). The cooling time after 30-minute insonation for every power level was recorded, and animals were examined by postmortem brain histology (TUNEL and hematoxylin/eosin). Results— Brain temperature increased within 2 to 5 minutes of insonation. Brain temperature increase and cooling time were in proportion to power level, and even with the highest intensity of 7 W/cm2 for 30 minutes, the maximum elevation of mean brain temperature was 0.9°C, with the highest cooling time of 40 minutes. No deleterious side effects of this treatment could be found in histological examination. Conclusions— Using a pulsed ultrasound design, only a moderate temperature increase could be observed with no histopathological abnormalities. Deleterious side effects of mid-kilohertz ultrasound (eg, intracerebral hemorrhage) are therefore not a consequence of local brain temperature increase.

Comparison of the new ASCO classification with the TOAST classification in a population with acute ischemic stroke

Precise analysis of stroke subtypes is important for clinical treatment decisions, the prognostic evaluation of patients, as well as defining stroke populations in clinical studies. The TOAST classification is the most widely used and approved form for etiologic subtyping. Increasing knowledge about stroke mechanisms and the introduction of new diagnostic techniques have supported the promotion of the new ASCO phenotypic classification, which aims to characterize patients using different grades of evidence for stroke subtypes. We prospectively assigned 103 consecutive patients from our stroke center for subtype classification using ASCO and TOAST. Clinical features and complementary investigations were recorded according to our standardized acute stroke care protocol. Evidence grade 1 with ASCO was assessed in 12.62% for large artery disease (A), 23.30% small-vessel disease (S), 36.89% cardiac source (C) and 1.94% another cause (O). Evidence grades 1–3 were identified in 60.19% A, 75.73% S, 49.51% C, and 3.88% O. A total of 68.93% of the patients were classified in more than one category, and only 3.88% remained completely undetermined. The κ value for inter-rater agreement was 0.92–1. Using TOAST, the distribution was 9.71% A, 23.30% S, 34.95% C, 1.94% O, and 30.10% undetermined. The ASCO classification showed a good concordance with TOAST. The inter-rater agreement was high. The comprehensive character of ASCO allows the recording of important additional information. This may be helpful for a specific treatment adaptation in each individual patient and creation of different etiological profiles in view of adapted clinical trials.

Effect of combined ultrasound and microbubbles treatment in an experimental model of cerebral ischemia.

Combined 2-MHz ultrasound (US) and second-generation, sulfur hexafluoride microbubbles (MB) treatment (US+MB) was performed in a permanent middle cerebral artery (MCA) occlusion model in rats to evaluate possible effects on the ischemic cascade. We used 16 Wistar rats and the MCA occlusion model for stroke induction. Glutamate, pyruvate, lactate and glycerol levels were measured by intracerebral microdialysis before and after stroke induction and after US+MB application (n = 8) for 20 h. After 24 h, brain infarct volume, apoptosis and IL-6 and TNF-alpha levels were evaluated. The infarct volume was significantly reduced (p < 0.05) in the US+MB-treated group compared with control animals. In additional, glutamate levels were significantly lower in US+MB-treated animals, and these animals showed a higher rate of apoptotic cell death in the infarcted area. The levels of IL-6 and TNF-alpha concentrations were not different in both groups, and there was no apoptotic cell death outside the infarction in animals treated with US+MB. The results demonstrate that US+MB with second generation microbubbles does not have a harmful effect on ischemic stroke in an MCA occlusion model of the rat.

Single-Nucleotide Polymorphisms of MMP-2 Gene in Stroke Subtypes

Background: Matrix metalloproteinases (MMP) are expressed after ischemic stroke. These proteases are responsible for a higher incidence of hemorrhages, are correlated to size of infarction and influence the effects of recombinant tissue plasminogen activator treatment. We therefore evaluated single nucleotide polymorphisms (SNP) of MMP-2 in different subtypes of stroke patients in an association study using a case-control design. Methods: 197 stroke patients were divided according to modified TOAST criteria (small vessel disease, large vessel disease, hemorrhagic stroke and asymptomatic carotid artery stenosis) and compared to 143 controls. Clinical data like age, sex, risk factors and diagnostic results including MRI or cranial CT scans and ultrasound evaluations of intra- and extracranial arteries were obtained. Genotypes of MMP-2 (12 SNP) were compared to controls and DNA samples were analyzed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) analysis. Logistic regression analysis was performed for small vessel disease to test for interactions between markers and defined clinical risk factors. Additionally, MMP-2 serum levels obtained in the first 24 h after stroke were measured. Results: From the MMP-2 gene, 5 markers (rs1030868, rs2241145, rs2287074, rs2287076, rs7201) showed a significant association with small vessel infarcts (p < 0.05) and rs7201:g.C was identified as an independent risk factor by multivariable logistic regression analysis. MMP-2 protein levels were significantly lower in this group (174 ± 48 ng/dl) versus controls (214 ± 56 ng/dl). For other stroke subtypes, no significant association with MMP-2 SNP could be found. Conclusion: Our study demonstrates an association of the MMP-2 gene with the development of lacunar stroke, and no association of MMP-2 with other stroke subtypes.