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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.

Background: Both ischemic stroke and intracerebral hemorrhage are associated with altered expression and activation of matrix metalloproteinases (MMPs). Particularly relevant are MMP-2 and MMP-9. This proteolytic effect is dampened by tissue inhibitors of metalloproteinases (TIMPs). TIMP-2 is an important endogenous inhibitor of MMP-2. Alterations in the TIMP-2 gene expression may contribute to the incidence of ischemic stroke and intracerebral hemorrhage., Methods: TIMP-2 gene SNP -261G/A was genotyped from sequentially recruited stroke patients (n = 356, f/m 151/205, mean age 68.2 years, range 19-100 years) and gender and age matched controls (n = 253, f/m 114/139, mean age 68.5 years, range 32-92 years). The SNP -261G/A was detected after gene sequencing of 95 patients and controls. Furthermore, in a subgroup of 93 patients the serum levels of TIMP-2 were measured during the first 7 days after stroke onset and compared to the genotype., Results: SNP -261G/A in the TIMP-2 gene shows an allele frequency of approximately 39.14%. Homozygosity for allele A is associated significantly with the development of ICH (p = 0.025, OR = 2.020, CI = 1.115-3.661) as compared to heterozygosity and homozygosity for allele G (recessive genotypic model). Concordantly, the serum levels of TIMP-2 showed a nonsignificant decreases, depending on the genotype (p = 0.111)., Conclusion: We investigated a SNP 261 base pairs upstream of the start codon in exon 1 of TIMP-2. Our data suggest that carriers of homozygosity for allele A are at increased risk of developing intracerebral hemorrhage., (Copyright (c) 2009 S. Karger AG, Basel.)

Stem cell therapy seems promising in reducing deficits after focal cerebral ischemia. As stroke may result from intracerebral hemorrhage (ICH) in up to 20% we investigated whether human processed lipoaspirate mesenchymal stem cells (PLA-MSC) influence the functional outcome, migration behavior and the activation of endogenous progenitor cells. Experimental ICH was induced by stereotactic administration of collagenase in rats randomly assigned to the control or treatment group. The latter received 3 x 10(6) PLA-MSC by intravenous (i.v.) injection 24h after ICH induction. The outcome was continuously monitored using the RotaRod test over a period of 4 weeks. Morphometric analysis of ICH was performed consecutively by magnetic resonance imaging (MRI) studies and immunohistochemical analysis. The RotaRod test revealed a significant 1.5-fold improvement (p<0.005) in functional outcome for the PLA-MSC treated group after 4 weeks compared to controls. Histological and MRI assessment of lesion size showed no difference between the two groups. Although i.v. injected human cells could not be detected in the post mortem brain, evaluation of the number of endogenous progenitor cells revealed a twofold increase in the treated animals compared to controls. Treatment with PLA-MSC improved the functional outcome significantly in an experimental ICH model. This effect was achieved by stimulation of endogenous progenitor cells rather than integration and differentiation of the infused PLA-MSC.

Background: Ultrasound perfusion imaging (UPI) is a new approach for the assessment of brain perfusion. In contrast to the increasing experience with this method in patients with ischemic stroke, data on the value of UPI for the diagnosis of intracerebral hemorrhage (ICH) are lacking., Methods: We investigated 12 consecutive patients with sufficient temporal bone windows and a CT diagnosis of acute supratentorial ICH (basal ganglia n = 9 and lobar n = 3). Native transcranial B-mode ultrasound and UPI studies with echo contrast agents were performed on day 1 and on days 5-7 including volume measurements using the maximum extension on transverse and coronal ultrasound planes., Results: ICH was identified as hyperechogenic mass on B-mode ultrasound in 11/12 patients, but the correlation with CT volume measurements was poor (day 1: r = 0.4, 95% confidence interval, CI: -0.23-0.79; p = 0.1; follow-up: r = 0.58, 95% CI: 0.04-0.86; p = 0.21). Volume measurement was more precise using UPI with a significant correlation on day 1 (r = 0.8, 95% CI: 0.47-0.94; p < 0.001) and during the follow-up (r = 0.94, 95% CI: 0.81-0.98; p < 0.001). Using UPI the typical finding was a focal reduction of contrast agent arrival in the ICH core which led to better delineation of the lesion borders from adjacent tissue. Depiction of lobar ICH was difficult with ultrasound, and lesion sizes tended to be underestimated., Conclusions: This study supports earlier work demonstrating the usefulness of native transcranial ultrasound for the diagnosis of ICH. UPI further improves diagnostic reliability and allows very precise ICH volume measurements. If confirmed in larger studies, this approach may be useful for bedside monitoring of ICH progression and regression., (Copyright 2008 S. Karger AG, Basel.)

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., ((c) 2008 S. Karger AG, Basel.)

Biological markers play an evolving role in the diagnosis of Alzheimer disease (AD). We compare conventional measurements of cerebrospinal fluid (CSF) tau and beta-amyloid(1-42) proteins to a novel approach - Fourier transformed infrared (FT-IR) spectroscopy - a simple technique derived from chemical and physical sciences that characterizes intramolecular bonds. For automatic diagnostic analysis, we developed an artificial neural network (ANN). We examined 71 patients with a clinical diagnosis of AD and 66 controls. beta-Amyloid(1-42) was decreased (sensitivity 80% and specificity 78%); tau was elevated (sensitivity 76% and specificity 88%) in CSF of AD patients. The combined tau/beta-amyloid(1-42) quotient was able to distinguish healthy from diseased subjects with 99% sensitivity and 86% specificity. The ANN could separate FT-IR spectroscopy data with 88.5% sensitivity and 80% specificity. FT-IR spectroscopy proved to be cost-effective and simple to perform. Diagnostic sensitivity and specificity is in the range of CSF tau and beta-amyloid(1-42) protein analysis. Larger sample numbers for ANN training and validation could increase diagnostic accuracy and thus prove to be a useful screening tool.

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 microL (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.

Objectives: Cell tracking using ultrasmall iron particles is well established in magnetic resonance imaging (MRI). However, in experimental models, intrinsic iron signals derived from erythrocytes mask the labeled cells. Therefore, we evaluated Gadofluorine M with other gadolinium chelates for a T1-weighted positive enhancement for cell tracking in vitro. In addition, Gadofluorine M was tested in vivo., Material and Methods: Gadofluorine M and other gadolinium chelates were used to label stem cells with and without uptake-mediating agents in vitro and in vivo using a 1.5 T MRI. In addition, histology and molecular modeling was investigated., Results: Gadofluorine M revealed comparable properties to an uptake mediating agent in molecular modeling. Without an uptake-mediating agent Gadofluorine M-labeled cells were detected as a T1-weighted positive contrast in vitro and in vivo. Histology confirmed a 100% success rate for intracellular labeling., Conclusion: This study describes a novel contrast agent with the capability of intracellular accumulation without an uptake mediator providing a T1-positive MRI signal at 1.5 T and may be suitable for cell tracking in animal models with intraparenchymal hemorrhages such as stroke or malignant tumors.

Microbubble-enhanced sonothrombolysis (MEST) may be an alternative therapeutic option in ischemic stroke. Clinical study of the efficacy of MEST as an adjunct stroke therapy, before imaging with CT or MRI, requires experimental data on the safety of this approach in the presence of hemorrhagic stroke. We, therefore, investigated the effect of diagnostic transcranial ultrasound combined with microbubbles (US + MB) in an experimental animal model of intracerebral hemorrhage (ICH). ICH was induced in anesthetized rats by intracerebral collagenase injection. Transcranial ultrasound (2 MHz, mechanical index 1.3, 1051 kPa) was applied 3 h after ICH induction to rat brains for 30 min during a continuous IV infusion of sulfur hexafluoride microbubbles (SonoVue). The size of cerebral hemorrhage, the extent of brain edema, and the amount of apoptosis were compared with those from control rats with ICH but without US + MB. Results showed no significant effect of US + MB on hemorrhage size (control 23.3 +/- 10.7 mm(3), US + MB 20.3 +/- 5.8 mm(3)), on the extent of brain edema (control 3.3 +/- 2.0%, US +MB 3.5 +/- 1.9%), or on the rate of apoptosis (control 5.2 +/- 1.5%, US + MB 5.2 +/- 1.0%). We conclude that diagnostic ultrasound in combination with microbubbles does not cause additional damage to the rat brain during ICH in our experimental set-up. This finding provides support for the use of MEST as an early stroke therapy.

Objective: The aim of this study was to evaluate number and kind of neurological patients in comparison with other patients on a medical ICU., Methods: Over a period of one year, all neurological intensive care patients on a medical ICU were evaluated according to age, sex, diagnosis, mortality, diagnostic methods, ventilation and referral to other hospitals and general wards., Results: Comparable to a specialist neurological ICU a wide spectrum of neurological diseases could be observed on an interdisciplinary ICU. In comparison to other patient groups, patients with neurological disease had a higher rate of ventilation, a longer hospital stay and a higher mortality., Conclusion: Our data also demonstrate the relevant amount of neurological patients (19 % measured by bed assignment) in comparison to all patients, and the specific neurological procedures were applicable on a medical/interdisciplinary ICU. A higher interest for neurological patient on a medical ICU would therefore be essential.

The assessment of S-100B in acute neurological disorders such as global hypoxia, ischaemic or haemorrhagic stroke and traumatic brain injury reflects severity of symptoms and outcome. However, the temporal profile of S-100B release depends on topography, intensity and pathophysiology of the damage e.g. immediate release after traumatic brain injury following the acute destruction of neuronal tissue or delayed release after ischaemic stroke in which gradual breakdown of the blood-brain barrier plays a crucial role. In chronic brain diseases, knowledge about the clinical value of quantification of S-100B is scarce and further evaluations are needed. This review considers both conditions for S-100B measurement and illustrates advantages and limitations in comparison with clinical and neuroimaging data.

Matrix metalloproteinases are important factors for tissue remodelling and are activated during several physiological and pathological conditions, including cerebrovascular diseases. We give an overview of the structure, production and physiological effects of these widely distributed proteases and describe the genetic background and regulation pathways. In particular, we discuss the role of matrix metalloproteinases in vascular remodelling concerning ischaemic stroke, brain haemorrhage, vascular dementia, carotid artery plaques and cerebral aneurysms., (Copyright (c) 2005 S. Karger AG, Basel.)

Re-opening of the occluded artery is the primary therapeutic goal in hyper-acute ischemic stroke. Systemic treatment with IV rt-PA has been shown to be beneficial at least in a 3 h 'door to needle' window and is approved within that interval in many countries. Trials of thrombolytic therapy with rt-PA demonstrated a small, but significant improvement in neurological outcome in selected patients. As recently shown, intra-arterial application of rt-PA is effective and opens the therapeutical window to 6 h, but requires invasive intra-arterial angiographic intervention in a high number of patients, who do not finally achieve thrombolysis. Ultrasound (US) is known to have several biological effects depending on the emission characteristics. At higher energy levels US alone has a thrombolytic effect. That effect is already used for clinical purposes in interventional therapy using US catheters. Recently, there is growing evidence that US at lower energy levels (<2 W/cm(2)) facilitates enzymatic mediated thrombolysis, most probably by breaking molecular linkages of fibrin polymers and therefore, increasing the working surface for the thrombolytic drug. Different in-vitro and in-vivo experiments have shown increased clot lysis as well as accelerated recanalization of occluded peripheral, coronary vessels and most recently also intracerebral arteries. Sonothrombolysis at low energy levels, however, is of great interest because of the low risk for collateral tissue damage, enabling external insonation without the need for local catheterization. Whereas little or no attenuation of US can be expected through skin and chest, intensity will be significantly attenuated if penetration of bones, particularly the skull, is required. That effect, however, is frequency dependent. Whereas >90% of intracerebral US intensity is lost (of the output power) in frequencies currently used for diagnostic purposes (mostly 2 MHz and up), that ratio is nearly reversed in the lower KHz range (<300 kHz). US at these low frequencies, however, is efficient for accelerating enzymatic thrombolysis in-vitro as well as in vivo within a wide range of intensities, from 0.5 W/cm(2) (MI approximately 0.3) to several W/cm(2). Since the emitted US beam widens with decreasing frequency, low-frequency US can insonate the entire intracerebral vasculature. That may overcome the limitation of US in the MHz range being restricted to insonation of the MCA mainstem. There are no reports in the preclinical literature about intracerebral bleeding or relevant cerebral cellular damage (either signs of necrosis or apoptosis) for US energy levels up to 1 W/cm(2). Moreover, recent investigations showed no break-down of the blood brain barrier. Safety of US exposure of the brain for therapeutic purposes has to address heating. Heating depends critically on the characteristics of the US. The most significant heating of the brain tissue itself is >1 degrees C/h using a continuous wave (CW) 2 W/cm(2) probe, whereas no significant heating could be found when using an intermittent (pulsed) emission protocol. The experimental data so far help to characterize the optimal US settings for sonothrombolysis and support the hypothesis that this combined treatment is a prospective advance in optimizing thrombolytic therapy in acute stroke.

Objective: An inflammatory host response in the ischemically injured brain is well documented. However, its pathophysiological relevance is uncertain. We investigated whether inflammatory leukocyte response in the ischemic brain alters infarct size., Methods: The cellular inflammatory response to cerebral ischemia in Wistar-derived rats induced by the transient occlusion of the middle cerebral artery with a thread was pharmacologically upmodulated by lipopolysaccharide (LPS) or downmodulated by continuous infusion of carboxylated sialyl Lewis(x) (sLex). The effects of such experimental modulation of focal cerebral leukocyte recruitment on the extent of the resulting infarction were assessed., Results: Compared to control treatments, LPS strongly enhanced (540.5 +/- 504.8 vs. 94.6 +/- 60.6, p < 0.01) and sLex decreased (32.8 +/- 29.1 vs. 97.0 +/- 49.7, p < 0.05) the numbers of neutrophils at the investigated sites in cerebral ischemia. Unexpectedly, despite such marked experimental modulation of leukocyte infiltration in the ischemic brain, the extent of the resulting cerebral infarction (percent of total hemisphere) remained unchanged under these different conditions (54.5 +/- 10.8 vs. 53.0 +/- 19.1, n.s. and 50.3 +/- 18.0 vs. 57.2 +/- 10.0, n.s., respectively)., Conclusions: The striking dissociation between the massively altered inflammatory leukocyte infiltration in the ischemic brain and the unchanged infarct outcome indicates that intracerebral inflammatory leukocyte recruitment is not a major pathogenic factor in the development of ischemic tissue damage., (Copyright 2002 S. Karger AG, Basel)