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Background: Coronavirus disease 2019 (COVID-19) has placed a tremendous strain on healthcare services. This study, prepared by a large international panel of stroke experts, assesses the rapidly growing research and personal experience with COVID-19 stroke and offers recommendations for stroke management in this challenging new setting: modifications needed for prehospital emergency rescue and hyperacute care; inpatient intensive or stroke units; posthospitalization rehabilitation; follow-up including at-risk family and community; and multispecialty departmental developments in the allied professions., Summary: The severe acute respiratory syndrome coronavirus 2 uses spike proteins binding to tissue angiotensin-converting enzyme (ACE)-2 receptors, most often through the respiratory system by virus inhalation and thence to other susceptible organ systems, leading to COVID-19. Clinicians facing the many etiologies for stroke have been sobered by the unusual incidence of combined etiologies and presentations, prominent among them are vasculitis, cardiomyopathy, hypercoagulable state, and endothelial dysfunction. International standards of acute stroke management remain in force, but COVID-19 adds the burdens of personal protections for the patient, rescue, and hospital staff and for some even into the postdischarge phase. For pending COVID-19 determination and also for those shown to be COVID-19 affected, strict infection control is needed at all times to reduce spread of infection and to protect healthcare staff, using the wealth of well-described methods. For COVID-19 patients with stroke, thrombolysis and thrombectomy should be continued, and the usual early management of hypertension applies, save that recent work suggests continuing ACE inhibitors and ARBs. Prothrombotic states, some acute and severe, encourage prophylactic LMWH unless bleeding risk is high. COVID-19-related cardiomyopathy adds risk of cardioembolic stroke, where heparin or warfarin may be preferable, with experience accumulating with DOACs. As ever, arteritis can prove a difficult diagnosis, especially if not obvious on the acute angiogram done for clot extraction. This field is under rapid development and may generate management recommendations which are as yet unsettled, even undiscovered. Beyond the acute management phase, COVID-19-related stroke also forces rehabilitation services to use protective precautions. As with all stroke patients, health workers should be aware of symptoms of depression, anxiety, insomnia, and/or distress developing in their patients and caregivers. Postdischarge outpatient care currently includes continued secondary prevention measures. Although hoping a COVID-19 stroke patient can be considered cured of the virus, those concerned for contact safety can take comfort in the increasing use of telemedicine, which is itself a growing source of patient-physician contacts. Many online resources are available to patients and physicians. Like prior challenges, stroke care teams will also overcome this one. Key Messages: Evidence-based stroke management should continue to be provided throughout the patient care journey, while strict infection control measures are enforced., (© 2021 S. Karger AG, Basel.)

Background: Diffusion magnetic resonance imaging (MRI) is the current-state-of-the-art technique to clinically investigate acute (0-24 h) ischemic stroke tissue. However, reduced apparent diffusion coefficient (ADC)-considered a marker of tissue damage-was observed to reverse spontaneously during the subacute stroke phase (24-72 h) which means that low ADC cannot be used to reflect the damaged tissue after 24 h in experimental and clinical studies. One reason for the change in ADC is that ADC values drop with cytotoxic edema (acute phase) and rise when vasogenic edema begins (subacute phase). Recently, combined 1 H- and 23 Na-MRI was proposed as a more accurate approach to improve delineation between reversible (penumbra) and irreversible ischemic injury (core). The aim of this study was to investigate the effects of reperfusion on the ADC and the sodium MRI signal after experimental ischemic stroke in rats in well-defined areas of different viability levels of the cerebral lesion, i.e. core and penumbra as defined via perfusion and histology. Transient middle cerebral artery occlusion was induced in male rats by using the intraluminal filament technique. MRI sodium, perfusion and diffusion measurement was recorded before reperfusion, shortly after reperfusion and 24 h after reperfusion. The animals were reperfused after 90 min of ischemia., Results: Sodium signal in core did not change before reperfusion, increased after reperfusion while sodium signal in penumbra was significantly reduced before reperfusion, but showed no changes after reperfusion compared to control. The ADC was significantly decreased in core tissue at all three time points compared to contralateral side. This decrease recovered above commonly applied viability thresholds in the core after 24 h., Conclusions: Reduced sodium-MRI signal in conjunction with reduced ADC can serve as a viability marker for penumbra detection and complement hydrogen diffusion- and perfusion-MRI in order to facilitate time-independent assessment of tissue fate and cellular bioenergetics failure in stroke patients.

Background and Purpose: The debate over the fact that experimental drugs proposed for the treatment of stroke fail in the translation to the clinical situation has attracted considerable attention in the literature. In this context, we present a retrospective pooled analysis of a large data set from preclinical studies, to examine the effects of early versus late administration of intravenous recombinant tissue-type plasminogen activator., Methods: We collected data from 26 individual studies from 9 international centers (13 researchers; 716 animals) that compared recombinant tissue-type plasminogen activator with controls, in a unique mouse model of thromboembolic stroke induced by an in situ injection of thrombin into the middle cerebral artery. Studies were classified into early (<3 hours) versus late (≥3 hours) drug administration. Final infarct volumes, assessed by histology or magnetic resonance imaging, were compared in each study, and the absolute differences were pooled in a random-effect meta-analysis. The influence of time of administration was tested., Results: When compared with saline controls, early recombinant tissue-type plasminogen activator administration was associated with a significant benefit (absolute difference, -6.63 mm(3); 95% confidence interval, -9.08 to -4.17; I(2)=76%), whereas late recombinant tissue-type plasminogen activator treatment showed a deleterious effect (+5.06 mm(3); 95% confidence interval, +2.78 to +7.34; I(2)=42%; Pint<0.00001). Results remained unchanged after subgroup analyses., Conclusions: Our results provide the basis needed for the design of future preclinical studies on recanalization therapies using this model of thromboembolic stroke in mice. The power analysis reveals that a multicenter trial would require 123 animals per group instead of 40 for a single-center trial., (© 2016 American Heart Association, Inc.)

Medical treatment options for central nervous system (CNS) diseases are limited due to the inability of most therapeutic agents to penetrate the blood-brain barrier (BBB). Although a variety of approaches have been investigated to open the BBB for facilitation of drug delivery, none has achieved clinical applicability. Mounting evidence suggests that ultrasound in combination with microbubbles might be useful for delivery of drugs to the brain through transient opening of the BBB. This technique offers a unique non-invasive avenue to deliver a wide range of drugs to the brain and promises to provide treatments for CNS disorders with the advantage of being able to target specific brain regions without unnecessary drug exposure. If this method could be applied for a range of different drugs, new CNS therapeutic strategies could emerge at an accelerated pace that is not currently possible in the field of drug discovery and development. This article reviews both the merits and potential risks of this new approach. It assesses methods used to verify disruption of the BBB with MRI and examines the results of studies aimed at elucidating the mechanisms of opening the BBB with ultrasound and microbubbles. Possible interactions of this novel delivery method with brain disease, as well as safety aspects of BBB disruption with ultrasound and microbubbles are addressed. Initial translational research for treatment of brain tumors and Alzheimer's disease is presented.

Clinical studies demonstrated favorable effects of statins in stroke beyond lipid-lowering effects. In acute stroke, the disruption of the blood-brain barrier (BBB) is mediated by matrix metalloproteinases (MMPs). A modified MMP metabolism may account for the beneficial effects of statins. Cultured human brain microvascular endothelial cells (BMECs) were pretreated with simvastatin and subjected to oxygen glucose deprivation (OGD). Gene expression and protein secretion of MMP-2 and MMP-9 and the tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 were measured by quantitative real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). Simvastatin significantly dampened the expression but not secretion of MMP-2 under OGD. MMP-9 synthesis rate was low and unaffected by simvastatin treatment, while the gene expression and protein secretion of TIMP-1 and TIMP-2 were both strongly induced. Our results provide evidence for a positive effect of simvastatin on the MMP metabolism in human BMECs and experimental stroke mainly by means of the increased expression and secretion of TIMP-1 and TIMP-2.

Ultrasound (US) applied as an adjunct to thrombolytic therapy improves the recanalization of occluded vessels, and microbubbles can amplify this effect. New data suggests that the combination of US and microbubbles without tissue plasminogen activator may achieve recanalization with a lower risk of hemorrhage. Further possibilities include specific targeting of thrombus with immunobubbles as well as local drug delivery with US-sensitive liposomes. Clinical studies support the use of US for ischemic stroke therapy, and the first trials of enhancing sonothrombolysis with microbubbles have been encouraging. One emerging clinical application is sonothrombolysis of intracranial hemorrhages for clot evacuation. Microcirculation, irrespective of recanalization, may also be improved by US and microbubbles, and this effect may open new opportunities for the application of sonothrombolysis in acute ischemic stroke. Understanding the mechanisms of therapeutic action and relating this knowledge to issues of efficacy and safety are important objectives of ongoing research. This review will discuss the translational capacities of in vitro studies and preclinical research and will assess the first clinical studies of this promising therapeutic strategy., (© 2015 S. Karger AG, Basel.)

In the last several years, great progress has been made in ultrasound perfusion imaging of the brain. Different approaches have been assessed and shown to be capable of the early detection of cerebral perfusion deficits in stroke patients. Real-time low-mechanical index imaging simplifies the acquisition of perfusion parameters and alleviates many of the previous imaging problems related to shadowing, uniplanar analysis, and temporal resolution. With the advent of this new, highly sensitive contrast-specific imaging technique, new possibilities of the real-time visualization of brain infarctions and cerebral hemorrhages have emerged. This review will detail the methodology of ultrasound perfusion imaging, discuss aspects of its safety and present the emerging clinical applications of brain perfusion assessment with ultrasound in acute stroke patients., (© 2015 S. Karger AG, Basel.)

Background and Purpose: Intracerebral hemorrhage (ICH) is the most adverse event of thrombolysis in ischemic stroke. Cerebral amyloid angiopathy increases the risk for spontaneous lobar ICH. Although thrombolysis may be performed in cerebral amyloid angiopathy-affected patients, there is still little knowledge available on the risk for secondary ICH., Methods: We investigated the effect of recombinant tissue-type plasminogen activator on experimental ischemic stroke in APP23 transgenic mice (n=18) and wild-type littermates (n=15). Focal ischemic stroke was induced in 26-month-old mice by temporal middle cerebral artery occlusion (filament model), followed by treatment with 10 mg/kg recombinant tissue-type plasminogen activator. Twenty-four hours later, a functional score was assessed and the mice were euthanized for histological analysis. ICH was classified as grades 1 to 3 depending on severity., Results: The groups did not differ regarding mortality (P=0.67) and functional deficit (P=0.18). Compared with wild-type mice, the APP23 genotype was associated with a higher appearance for ICH in the infarct area (P=0.05). ICH severity grades 2 and 3 correlated significantly with infarct size (P=0.004 and 0.008, respectively)., Conclusions: The APP23 genotype was not associated with increased mortality or worse functional outcome. Our results suggest an increased risk for ICH in the cerebral amyloid angiopathy-affected brain; however, no ICH was observed outside the ischemic area., (© 2014 American Heart Association, Inc.)

This study characterizes a new model of thromboembolic stroke of the middle cerebral artery in C57 black/6J mice, thus offering an opportunity to use the model for studying ischemic stroke in transgenic mice. Thromboembolic stroke was induced by local injection of either 1.5 or 3.0 UI of thrombin directly into the right MCA of C57 black/6J mice. Cerebral blood flow (CBF) velocity was measured continuously by laser Doppler flowmetry, which allowed documentation of both MCA occlusion and of spontaneous recanalization. After 24 h, all animals were euthanized. Cryosections were cut at 400-μm intervals and silver stained with the high-contrast method for volumetric assessment of infarct size. Interleukin (IL)-6, tumor necrosis factor-alpha (TNF-α), caspase-3 and hsp 70 protein levels were investigated by immunofluorescence. Thrombin injection resulted in clot formation in all animals. Cortical infarction occurred in 63% of the mice while 37% had a spontaneous MCA recanalization during the first 20 min following thrombin injection. In cases of successful MCA occlusion with consequent infarction, the clot was stable up to 2 h after formation. Subsequently, 20% recanalized spontaneously. Infarctions were restricted to the cortex with a mean lesion volume of 36 ± 5 for 1.5 UI and 56 ± 8 for 3.0 UI thrombin. Protein levels of IL-6, TNF-α, caspase-3, and hsp 70 were significantly increased after MCAO. The results demonstrate that the mouse thromboembolic stroke model produces cortical infarctions of consistent size in C57 black/6J mice, which is dependent upon the amount of thrombin used for clot formation. Spontaneous MCA recanalization occurs after 2 h of ischemia in 20% of mice. Thus, the thromboembolic model is an applicable stroke model for C57 black/6J mice, which mimics many of the features of human stroke, including spontaneous recanalization. However, strain differences between Swiss and C57 black/6J mice must be taken into account when using the model.

Background: A recent study suggests that patients with persistent occlusion of the middle cerebral artery (MCA) following treatment with recombinant tissue plasminogen activator (rt-PA) have better outcomes than patients with MCA occlusion not receiving rt-PA. We performed a study to elucidate possible mechanisms of this finding in a new model of thromboembolic stroke closely mimicking human pathophysiology., Methods: Thromboembolic stroke was induced by local injection of thrombin directly into the right MCA of C57 black/6J mice. Rt-PA was administered 20 and 40 min after clot formation. The efficiency of rt-PA to induce thrombolysis was measured by laser Doppler. After 24 h, all animals were euthanized and interleukin (IL)-6, tumor necrosis factor-alpha (TNF-α), matrix metalloproteinase (MMP)-9, Caspase-3, hsp 32 and hsp 70 protein levels were investigated by immunofluorescence. Presence of hemorrhage was verified and infarct volume was measured using histology., Results: Thrombin injection resulted in clot formation giving rise to cortical brain infarction. Early rt-PA treatment starting at 20 min after the clot formation resulted in 100% recanalization. However, rt-PA-induced thrombolysis dissolved the clot in only 38% of the animals when administered 40 min after clot formation. Protein levels of IL-6, TNF-α, MMP-9, Caspase-3, hsp 32 and hsp 70 were increased after MCAO, whereas treatment with rt-PA attenuated the expressions of inflammatory markers in those animals where the thrombolysis was successful. In addition, the infarct size was significantly reduced with rt-PA treatment compared to non-treated MCAO, regardless of whether MCA thrombolysis was successful., Conclusions: The present study demonstrates a clear correlation of the protein expression of inflammatory mediators, apoptosis and stress genes with the recanalization data after rt-PA treatment. In this model rt-PA treatment decreases the infarct size regardless of whether vessel recanalization is successful.

We investigated whether real-time ultrasound perfusion imaging (rt-UPI) is able to detect perfusion changes related to arterial recanalization in the acute phase of middle cerebral artery (MCA) stroke. Twenty-four patients with acute territorial MCA stroke were examined with rt-UPI and transcranial color-coded duplex ultrasound (TCCD). Ultrasound studies were consecutively performed within 24 h and 72-96 h after stroke onset. Real-time UPI parameters of bolus kinetics (time to peak, rt-TTP) and of refill kinetics (plateau A and slope β of the exponential replenishment curve) were calculated from regions of interest of ischemic versus normal brain tissue; these parameters were compared between early and follow-up examinations in patients who recanalized. At the early examination, there was a delay of rt-TTP in patients with MCA occlusion (rt-TTP [s]: 13.09 ± 3.21 vs. 10.16 ± 2.6; p = 0.01) and a lower value of the refill parameter β (β [1/s]: 0.62 ± 0.34 vs. 1.09 ± 0.58; p = 0.01) in ischemic compared with normal brain tissue, whereas there were no differences of the parameters A and Axβ. At follow-up, the delay of rt-TTP was reversible once recanalization of an underlying MCA obstruction was demonstrated: rt-TTP [s], 13.09 ± 3.21 at 24 h versus 10.95 ± 1.5 at 72-96 h (p = 0.03). Correspondingly, β showed a higher slope than at the first examination: β [1/s]: 0.55 ± 0.29 at 24 h versus 0.71 ± 0.27 at 72-96 h (p = 0.04). We conclude that real-time UPI can detect hemodynamic impairment in acute MCA occlusion and subsequent improvement following arterial recanalization. This offers the chance for bedside monitoring of the hemodynamic compromise (e.g. during therapeutic interventions such as systemic thrombolysis)., (Copyright © 2013 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.)

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.Molecular Therapy - Nucleic Acids (2013) 2, e73; doi:10.1038/mtna.2012.64; published online 19 February 2013.

Background: Matrix metalloproteinases (MMPs) are key players in proteolytic blood-brain barrier (BBB) disruption during ischemic stroke, leading to vascular edema, hemorrhagic transformation and infiltration by leukocytes. Their effect is dampened by the endogenous tissue inhibitors of metalloproteinases (TIMPs). The respective cellular source of specific MMPs and TIMPs during BBB breakdown is still under investigation., Methods: We analyzed the MMP and TIMP release of human brain microvascular endothelial cells (BMECs) under oxygen glucose deprivation (OGD). Cultured human BMECs (the hCMEC/D3 cell line) were subjected to OGD (6, 12, 18 and 24 h). Gene expression of MMP-2, MMP-9, TIMP-1 and TIMP-2 were serially measured by quantitative real time-PCR and compared to ELISA-detected cell culture medium levels., Results: OGD induced a significant and long-lasting increase in MMP-2 gene expression, reaching a plateau after 12 h. Medium protein levels of MMP-2 were correspondingly elevated at 12 h of OGD. The MMP-9 synthesis rate was detectable at very low levels and remained unaffected by OGD. TIMP-1 gene expression and secretion declined under OGD, whereas both expression and secretion of TIMP-2 remained stable. Contrary to the respective gene expression rate, medium levels of MMP-2, TIMP-1 and TIMP-2 started a simultaneous decline after 12 h of OGD. This is most likely due to an impaired synthesis and enhanced consumption rate under OGD., Conclusions: The objective of our study was to determine the contribution of human BMECs to the MMP metabolism under in vitro OGD conditions simulating ischemic stroke. Our results suggest that human BMECs switch to a proinflammatory state by means of an enhanced production of MMP-2, attenuated release of TIMP-1, and unaffected production of TIMP-2. Thus, human BMECs might participate in the MMP-mediated BBB breakdown during ischemic stroke. However, our data does not support human BMECs to be a source of MMP-9., (Copyright © 2013 S. Karger AG, Basel.)

Background: A new thromboembolic animal model showed beneficial effects of t-PA with an infarct volume reduction of 36.8% in swiss mice. Because knock-out animal experiments for stroke frequently used C57BL76 mice we evaluated t-PA effects in this mouse strain and measured infarct volume and vascular recanalisation in-vivo by using high-field 9.4 T MRI and a 1H surface cryo coil., Methods: Clot formation was triggered by microinjection of murine thrombin into the right middle cerebral artery (MCA). Animals (n = 28) were treated with 10 mg/kg, 5 mg/kg or no tissue plasminogen activator (t-PA) 40 min after MCA occlusion. For MR-imaging a Bruker 9.4 T animal system with a 1H surface cryo probe was used and a T2-weighted RARE sequence, a diffusion weighted multishot EPI sequence and a 3D flow-compensated gradient echo TOF angiography were performed., Results: The infarct volume in animals treated with t-PA was significantly reduced (0.67 ± 1.38 mm3 for 10 mg/kg and 10.9 ± 8.79 mm3 for 5 mg/kg vs. 19.76 ± 2.72 mm3 ; p < 0.001) compared to untreated mice. An additional group was reperfused with t-PA inside the MRI. Already ten minutes after beginning of t-PA treatment, reperfusion flow was re-established in the right MCA. However, signal intensity was lower than in the contralateral MCA. This reduction in cerebral blood flow was attenuated during the first 60 minutes after reperfusion. 24 h after MCA occlusion and reperfusion, no difference in signal intensity of the contralateral and ipsilateral MCAs was observed., Conclusions: We confirm a t-Pa effect using this stroke model in the C57BL76 mouse strain and demonstrate a chronological sequence MRI imaging after t-PA using a 1H surface cryo coil in a 9.4 T MRI. This setting will allow testing of new thrombolytic strategies for stroke treatment in-vivo in C57BL76 knock-out mice.

Rapid advances in microbubble pharmacology together with novel ultrasound technologies for contrast-specific imaging of the macro- and microcirculation have led to a number of new applications for assessment of stroke patients. In particular, ultrasound perfusion imaging has added new perspectives for diagnosis and monitoring of both ischemic and hemorrhagic stroke. Recently, real-time brain perfusion imaging of middle cerebral artery infarctions has been introduced and new quantitative algorithms for evaluation of regional cerebral blood flow are being applied for the first time in humans. Microbubbles enable visualization of carotid artery plaque neovascularization to detect plaque vulnerability. There is growing interest in therapeutic applications of ultrasound, particularly in the field of sonothrombolysis. The treatment of acute ischemic stroke can be improved by ultrasound and microbubbles in combination with thrombolytic drugs. Excitingly, ultrasound and microbubbles may be effective in clot lysis of ischemic stroke even without additional thrombolytic drugs. New therapeutic avenues include opening of the blood-brain barrier (BBB) with ultrasound and microbubbles to enable novel drug delivery to the brain. Microbubbles are also assuming a central role in ultrasound molecular imaging with many targets of interest for evaluating pathophysiologic processes involved in cerebrovascular disease including angiogenesis, inflammation, and thrombus formation.

Microbubbles are Food and Drug Administration (FDA) approved contrast agents for ultrasound imaging. It has been reported that applying ultrasound on drug-loaded microbubbles facilitates drug uptake by cells, due to so-named sonoporation. However, the biophysics behind sonoporation are not fully understood. It is believed that sonoporation results in a "direct" delivery of drugs in the cytoplasm of cells, though it has been suggested as well that sonoporation facilitates endocytosis which would improve the internalization of drugs by cells. To get a better understanding of sonoporation, this study reports on the ultrasound assisted delivery of adeno-associated virus (AAV) loaded on the surface of microbubbles. AAVs rely on endocytosis for efficient transduction of cells and are, consequently, an elegant tool to evaluate whether endocytosis is involved in ultrasound-induced sonoporation. Applying ultrasound on AAV-loaded microbubbles clearly improved the internalization of AAVs by cells, though transduction of the cells did not occur, indicating that by sonoporation substances become directly delivered in the cytosol of cells.

Purpose: To estimate changes in the (23)Na density and in the (23)Na relaxation time T(2) * in the anatomically small murine brain after stroke., Materials and Methods: Three-dimensional acquisition weighted chemical shift imaging at a resolution of 0.6 × 0.6 × 1.2 mm(3) was used for sodium imaging and relaxation parameter mapping. In vivo measurements of the mouse brain (n = 4) were performed 24 hours after stroke, induced by microinjection of purified murine thrombin into the right middle cerebral artery. The measurement time was 14 minutes in one mouse and 65 minutes in the other three. An exponential fit estimation of the free induction decay was calculated for each voxel enabling the reconstruction of locally resolved relaxation parameter maps., Results: The infarcted areas showed an increase in sodium density between 160% and 250%, while the T(2) * relaxation time increased by 5%-72% compared to unaffected contralateral brain tissue., Conclusion: (23)Na chemical shift imaging at a resolution of 0.6 × 0.6 × 1.2 mm(3) enabled sodium imaging of the anatomical small mouse brain and the acquired data allowed calculating relaxation parameter maps and hence a more exact evaluation of sodium signal changes after stroke., (Copyright © 2011 Wiley-Liss, Inc.)

Ultrasound-mediated opening of the blood-brain barrier (BBB) in the presence of gas-filled microbubbles is a potential strategy for drug delivery across the blood-brain barrier to promote regeneration after ischemic stroke. However, related bioeffects and potential side-effects that could limit a translation into clinical application are poorly understood so far. We therefore examined the clearance of extravasated albumin following ultrasound-mediated BBB opening. Autofluorescence of albumin-bound Evans Blue dye indicated cellular albumin uptake as soon as 30min after insonation (2±0.72 cells/optical field). Cellular albumin uptake increased constantly over 24h (22±3.33 cells/optical field, p<0.05). Initially, the majority of albumin-positive cells were located in the periphery of brain capillaries. Most albumin phagocyting cells stained positive for CD163 and Iba-1, identifying them as activated microglia. Further, a small fraction of albumin-positive cells stained positive for the astroglial markers GFAP/S100B. Some perivascular cells with intracellular albumin were shown to express the endothelial marker protein EN4. Albumin uptaking cells stained negative for the neuronal TubulinIII. Thus, ultrasound-induced BBB opening leads to albumin extravasation which is phagocytized predominantly by activated microglia, astrocytes and endothelial cells. As albumin uptake into neurons has been shown to be neurotoxic, rapid albumin clearance by microglia might prevent neuronal cell death., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Real-time ultrasound perfusion imaging (rt-UPI) allows visualization of microbubbles flowing through the cerebral microvasculature. We hypothesized that analysis of microbubble tissue replenishment would enable for characterization of perfusion deficits in acute middle cerebral artery (MCA) territory stroke. Twenty-three patients (mean age 70.2 ± 13.2 years, 9 weeks) were included. Sequential images of bubble replenishment were acquired by transcranial rt-UPI at low mechanical index immediately after microbubble destruction. Different parameters were calculated from regions of interest (ROIs): real-time time to peak (rt-TTP), rise rate (β), and plateau (A) of acoustic intensity, and A × β was used as an index of blood flow. Results were compared with diffusion-weighted and perfusion magnetic resonance imaging. Parameters of rt-UPI had lower values in ROIs of ischemic as compared with normal tissue (β=0.58 ± 0.40 versus 1.25 ± 0.83; P=0.001; A=1.44 ± 1.75 versus 2.63 ± 2.31; P=0.05; A × β=1.14 ± 2.25 versus 2.98 ± 2.70; P=0.01). Real-time time to peak was delayed in ischemic tissue (11.43 ± 2.67 versus 8.88 ± 1.66 seconds; P<0.001). From the analysis of receiver operating characteristic curves, β and A × β had the largest areas under the curve with optimal cutoff values of β<0.76 and A × β<1.91. We conclude that rt-UPI with analysis of microbubble replenishment correctly identifies ischemic brain tissue in acute MCA stroke.

Liposome-loaded microbubbles have been recently introduced as a promising drug delivery platform for ultrasound guided drug delivery. In this paper we design liposome-loaded (lipid-shelled) microbubbles through the simple self-assembly of the involved compounds in a single step process. We thoroughly characterized the liposome-loading of the microbubbles and evaluated the cell killing efficiency of this material using doxorubicin (DOX) as a model drug. Importantly, we observed that the DOX liposome-loaded microbubbles allowed killing of melanoma cells even at very low doses of DOX. These findings clearly prove the potential of liposome-loaded microbubbles for ultrasound targeted drug delivery to cancer tissues., (Crown Copyright © 2011. Published by Elsevier B.V. All rights reserved.)

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 non-insonicated 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.

Two clinical trials have used ultrasound to improve tPA thrombolysis in patients with acute ischemic stroke. The Combined Lysis of Thrombus in Brain Ischemia Using Transcranial Ultrasound and Systemic tPA (CLOTBUST) trial reported accelerated recanalisation of the middle cerebral artery (MCA) in patients with symptoms of MCA infarction, which were monitored with 2-MHz transcranial Doppler. In CLOTBUST, there was no increased bleeding as evidenced by cranial computed tomography. The Transcranial Low-Frequency Ultrasound-Mediated Thrombolysis in Brain Ischemia (TRUMBI) trial, which employed magnetic resonance imaging (MRI) before and after tPA thrombolysis, was discontinued prematurely because of an increased number of secondary hemorrhages, possibly related to the use of low frequency 300-kHz ultrasound. The purpose of our work is to help identify possible mechanisms of intracerebral hemorrhage resulting from sonothrombolysis by applying a simulation tool that estimates the pressure levels in the human brain that are produced with different sonothrombolysis devices. A simulation software based on a finite difference time domain (FDTD) three-dimensional (3D) scheme was developed to predict acoustic pressures in the brain. This tool numerically models the wave propagation through the skull and reproduces both ultrasound protocols of CLOTBUST and TRUMBI for analysis of the distribution of acoustic pressure in the brain during stroke treatment. For the simulated TRUMBI trial, we analyzed both a "high" and "low" hypothesis according to published parameters (for high and low amplitude excitations). For these hypotheses, the mean peak rarefactional pressures in the brain were 0.26 +/- 0.2 MPa (high hypothesis) and 0.06 +/- 0.05 MPa (low hypothesis), with maximal local values as high as 1.2 MPa (high hypothesis) and 0.27 MPa (low hypothesis) for configurations modelled in this study. The peak rarefactional pressure was thus higher than the inertial acoustic cavitation threshold in the presence of a standing wave in large areas of the brain, even outside the targeted clot. For the simulated CLOTBUST trial, the maximum peak negative pressure was less than 0.07 MPa. This simulated pressure is below the threshold for both inertial and stable acoustic cavitation but likewise lower than any acoustic pressure that has been reported as sufficient for effective sonothrombolysis. Simulating the pressure field of ultrasound protocols for clinical trials of sonothrombolysis may help explain mechanisms of adverse effects. Such simulations could prove useful in the initial design and optimization of future protocols for this promising therapy of ischemic stroke.

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.

Improved treatment of acute ischemic stroke with ultrasound and microbubbles in combination with thrombolytic drugs shows great promise, but the optimal techniques, indications, and contraindications have not yet been well defined. Moreover, details such as microbubble dosage, delivery, required thrombolytic drug dosage, and optimal ultrasound characteristics all remain uncertain. Recent results suggest that ultrasound and microbubbles may be effective in clot lysis of ischemic stroke without additional thrombolytic drugs. Moreover, targeting thrombus with specific immunobubbles may improve the efficacy of sonothrombolysis. Safety remains a major concern in the further development of ultrasound-enhanced thrombolysis and extensive animal work is required to define the most promising methods to translate into human application., (Copyright 2009 S. Karger AG, Basel.)

The general indication for the use of ultrasound contrast agents in neurosonologic applications is an insufficient signal-to-noise ratio when investigating the cerebral macro- and microcirculation. Clinical problems that are often encountered in native sonography are 'no flow, slow flow and low flow' phenomena. In these cases, ultrasound contrast agents are used to differentiate between vessel occlusion and insufficient insonation conditions, as well as for the detection of very slow blood flow velocities and low flow volumes. Echo-contrast agents significantly increase the success rate of transcranial color duplex examinations of patients with acute cerebrovascular disease. Ultrasound contrast imaging also allows assessment of the cerebral microcirculation. There are a number of techniques available for performing perfusion studies. These generally utilize high mechanical index imaging, since until recently lower acoustic outputs were unable to detect microbubbles in the brain. New-generation microbubbles in combination with very sensitive contrast-specific ultrasound techniques now enable real-time visualization of stroke. Moreover, destruction sequences with assessment of microbubble replenishment using real-time, low mechanical index imaging are now available. This article reviews of state-of-the-art contrast-specific imaging techniques for ultrasound evaluation of acute stroke patients., (Copyright 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.)

The field of neurovascular ultrasound is expanding rapidly with exciting new applications. While ultrasound contrast agents were initially used to overcome insufficient transcranial bone windows for identification of the basal cerebral arteries, new-generation microbubbles in combination with very sensitive contrast-specific ultrasound techniques now enable real-time visualization of stroke. This article will provide a review of recent and emerging developments in ultrasound technology and contrast-specific imaging techniques for evaluation of acute stroke patients., (Copyright 2008 S. Karger AG, Basel.)

Stroke poses a massive burden of disease, yet we have few effective therapies. The paucity of therapeutic options stands contrary to intensive research efforts. The failure of these past investments demands a thorough re-examination of the pathophysiology of ischaemic brain injury. Several critical areas hold the key to overcoming the translational roadblock: (1) vascular occlusion: current recanalization strategies have limited effectiveness and may have serious side effects; (2) complexity of stroke pathobiology: therapy must acknowledge the 'Janus-faced' nature of many stroke targets and must identify endogenous neuroprotective and repair mechanisms; (3) inflammation and brain-immune-system interaction: inflammation contributes to lesion expansion, but is also instrumental in lesion containment and repair; stroke outcome is modulated by the interaction of the injured brain with the immune system; (4) regeneration: the potential of the brain for reorganization, plasticity and repair after injury is much greater than previously thought; (5) confounding factors, long-term outcome and predictive modelling. These 5 areas are linked on all levels and therefore need to be tackled by an integrative approach and innovative therapeutic strategies.

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.

The blood-brain barrier (BBB) is a specialized system of capillary endothelial cells that protects the brain from harmful substances in the blood stream, while supplying the brain with the required nutrients for proper function. The BBB controls transport through both tight junctions and metabolic barriers and is often a rate-limiting factor in determining permeation of therapeutic drugs into the brain. It is a significant obstacle for delivery of both small molecules and macromolecular agents. Although many drugs could be potentially used to treat brain disease, there has been no method that allows non-invasive-targeted delivery through the BBB. Recently, promising studies indicate that ultrasound can be used to locally deliver a drug or gene to a specific region of interest in the brain. If microbubbles are combined with ultrasound exposure, the effects of ultrasound can be focused upon the vasculature to reduce the acoustic intensity needed to produce BBB opening. Several avenues of transcapillary passage after ultrasound sonication have been identified including transcytosis, passage through endothelial cell cytoplasmic openings, opening of tight junctions and free passage through injured endothelium. This article reviews the topic of transient disruption of the BBB with ultrasound and microbubbles and addresses related safety issues. It also discusses possible roles of the BBB in brain disease and potential interactions with ultrasound and microbubbles in such disease states.

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.

Object: Moyamoya is a rare, chronic disease that leads to the progressive narrowing and/or occlusion of the distal internal carotid and proximal cerebral arteries. Chronic cerebral ischemia ensues due to insufficient collateral blood supply. One potential treatment consists of the restoration of regional cerebral blood flow by direct or indirect revascularization surgery. The extent of neovascularization, especially in indirect procedures such as encephalomyosynangiosis (EMS), is currently evaluated with conventional angiography. Because this method is invasive and carries some risks, the authors investigated power Doppler imaging as an alternative noninvasive bedside procedure that can be used to assess surgically induced indirect revascularization in adult patients with moyamoya disease., Methods: Twelve symptomatic patients with adult moyamoya disease (seven women and five men, mean age 38 +/- 17 years) underwent combined (direct and indirect) revascularization. They were then examined using conventional angiography and power Doppler imaging to assess the extent of revascularization within 120 days postsurgery. According to the number of intracranial vessels demonstrating opacification on conventional angiography and power Doppler imaging studies, EMS was graded as follows: 1, absent (0 vessels); 2, moderate (one-four vessels); and 3, extensive (> four vessels) for both methods. Examiners were blinded to the classification results for the procedure that they did not grade. All 24 hemispheres were examined. The visual grading of EMS revealed a highly significant agreement between conventional angiography and power Doppler imaging (Spearman rank coefficient, r = 0.92; p < 0.001) and there was 100% agreement of patency of the bypass between the direct and indirect methods., Conclusions: The authors found excellent agreement between the two methods. Therefore, power Doppler imaging is a valid noninvasive alternative to carotid artery angiography in evaluating direct and indirect revascularization.

Purpose: Correlation of duplex ultrasonographic grading of unilateral internal carotid artery (ICA) stenosis and ICA blood volume flow (BVF) quantification., Materials and Methods: Using a 2D cine phase-contrast MR technique, 62 patients with unilateral ICA stenosis at the level of the bifurcation between 50 % and 98 % and 20 age-matched normal controls were examined. BVF was measured in the stenosed ICA. Ultrasonographic grading of stenoses was based on cross-sectional duplex sonography (color Doppler flow imaging [CDFI], real-time compound imaging) and compared to the changes in BVF in the stenosed ICA., Results: There was no statistically significant difference in BVF in stenoses of the ICA up to 70 % and in normal controls. ICA stenoses greater 70 % began to be hemodynamically relevant. With increasing stenosis, a decrease in BVF in the ipsilateral ICA was determined with a high and linear correlation of r = - 0.83. Normal controls showed a BVF in an ICA of 247.0 +/- 32.0 ml/min, patients with 70 % stenosis a mean BVF of 225.3 +/- 32.2 ml/min (P = 0.4) without significant reduction, patients with 80 % stenosis a significant reduction of BVF to a mean flow of 184.0 +/- 53.8 ml/min (P < 0.005), patients with 90 % stenosis a reduction of the mean BVF in the stenosed ICA to 84.6 +/- 41.9 ml/min (P < 0.0005) and patients with stenoses > 95 % a mean BVF of only 26.0 +/- 4.0 ml/min (P < 0.0005). In patients with unilateral ICA stenosis greater than 81 %, a significant decrease of BVF in the stenosed ICA was documented., Conclusion: Comparison of ultrasonographic grading of unilateral ICA stenosis and BVF determination in patients with ICA stenoses demonstrate a high correlation between increase in the stenosis and decrease in the ipsilateral blood flow beginning at 70 % stenosis. ICA stenoses greater than 80 % are significantly hemodynamically relevant.