Microbubbles combined with ultrasound therapy in ischemic stroke: A systematic review of in-vivo preclinical studies

PLOS ONE, Feb 2018

Background Microbubbles (MBs) combined with ultrasound sonothrombolysis (STL) appears to be an alternative therapeutic strategy for acute ischemic stroke (IS), but clinical results remain controversial. Objective The aim of this systematic review is to identify the parameters tested; to assess evidence on the safety and efficacy on preclinical data on STL; and to assess the validity and publication bias. Methods Pubmed® and Web of ScienceTM databases were systematically searched from January 1995 to April 2017 in French and English. We included studies evaluating STL on animal stroke model. This systematic review was conducted in accordance with the PRISMA guidelines. Data were extracted following a pre-defined schedule by two of the authors. The CAMARADES criteria were used for quality assessment. A narrative synthesis was conducted. Results Sixteen studies met the inclusion criteria. The result showed that ultrasound parameters and types of MBs were heterogeneous among studies. Numerous positive outcomes on efficacy were found, but only four studies demonstrated superiority of STL versus recombinant tissue-type plasminogen activator on clinical criteria. Data available on safety are limited. Limitations Quality assessment of the studies reviewed revealed a number of biases. Conclusion Further in vivo studies are needed to demonstrate a better efficacy and safety of STL compared to currently approved therapeutic options. Systematic review registration http://syrf.org.uk/protocols/

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Microbubbles combined with ultrasound therapy in ischemic stroke: A systematic review of in-vivo preclinical studies

February Microbubbles combined with ultrasound therapy in ischemic stroke: A systematic review of in-vivo preclinical studies Laurent Auboire 0 1 Charles A. Sennoga 0 1 Jean-Marc Hyvelin 0 Fre deric Ossant 0 1 Jean- Michel Escoffre 0 1 FrancË ois Tranquart 0 Ayache Bouakaz 0 1 0 Editor: Paul A. Lapchak, Cedars-Sinai Medical Center , UNITED STATES 1 UMR Imagerie et Cerveau, Inserm U930, Universite FrancËois-Rabelais de Tours, France, 2 CHRU de Tours, Service d'e chographie-Doppler , Tours, France, 3 Bracco Suisse SA, Geneva, Switzerland, 4 CHRU de Tours, CIC-IT, Tours, France, 5 Advice-US Consulting, Nernier , France Methods conducted. - OPEN ACCESS Data Availability Statement: All relevant data are within the paper and its Supporting Information files. The protocol is available at http://syrf.org.uk/ protocols/. Funding: This work was funded by INSERM. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. This does not alter our adherence to PLOS ONE policies on sharing data and materials. Background controversial. Objective bias. Results Sixteen studies met the inclusion criteria. The result showed that ultrasound parameters and types of MBs were heterogeneous among studies. Numerous positive outcomes on efficacy were found, but only four studies demonstrated superiority of STL versus recombinant tissue-type plasminogen activator on clinical criteria. Data available on safety are limited. Limitations Quality assessment of the studies reviewed revealed a number of biases. Competing interests: Bracco Suisse SA provided support in the form of salaries for author [JMH], Advice-US consulting provided support in the form of salaries for author [FT]. Bracco Suisse SA and Advice-US consulting did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the `author contributions' section. This does not alter our adherence to PLOS ONE policies on sharing data and materials. Conclusion Further in vivo studies are needed to demonstrate a better efficacy and safety of STL compared to currently approved therapeutic options. Systematic review registration http://syrf.org.uk/protocols/ Introduction Ischemic stroke (IS), described as death of neuronal cells due to arrest of regional cerebral blood flow by intra-arterial thrombi, is the second most common cause of death and the first leading cause of disability worldwide [ 1 ]. Although intravenous (IV) administration of recombinant tissue-type plasminogen activator (rtPA), the only thrombolytic agent approved for the reperfusion of occluded arteries, shows improved outcomes in terms of mortality and disability in IS patients, its use is characterized by increased incidences of hemorrhage [ 2 ]. Patients ineligible for rtPA or failing to recanalize with rtPA are now treatable using thrombectomy. However, thrombectomy is presently used only at comprehensive stroke centers and limited to an artery with a diameter > 2 mm [ 3, 4 ]. Use of other thrombolytic drugs (e.g., tenecteplase) were previously trialled, but showed no beneficial improvements in treatment outcomes when compared with rtPA [5]. Sonothrombolysis (STL), involving the use of microbubbles (MBs) combined with transcranial ultrasound (US) to potentiate clot dissolution with or without administration of rtPA, is under clinical evaluation as a method for the recanalization of occluded cerebral arteries [6±8]. A metaanalysis of clinical STL shows no beneficial treatment outcome in terms of mortality and disability at 3 months versus approved treatment (rtPA). Patients also experience a higher rate of cerebral hemorrhage [ 9 ]. Another meta-analysis of randomized controlled trials and case-control studies concluded that sonothrombolysis with microbubbles is a safe and effective treatment in ischemic stroke [ 10 ]. A more recent clinical trial, NOR-SASS (Norwegian Sonothrombolysis in Acute Stroke Study), which was prematurely interrupted (lack of funding), concluded that the use of STL is safe as a thrombolytic therapy and with no significant adverse effects [ 8 ]. Twenty years after the demonstration by Tachibana et al. that STL with microbubbles has a thrombolytic effect, numerous studies have been performed in Vivo [ 11 ]. If comprehensive review on the subject are available [12±15], no systematic review on in-vivo studies has been performed yet. Such review is required to summarize the evidence accumulated, to provide transparency on the quality of these studies and help to design new studies, which can ease clinical translation. This systematic review will focus on the association of microbubbles and ultrasound for sonothrombolysis. Other approaches such as histotripsy or sonothrombolysis without microbubbles will not be included in this review to maintain a homogeneity in the parameters tested. The purpose of this systematic review is (i) to identify the parameters tested and their results, (ii) to assess evidence on the safety and efficacy from preclinical data on STL, and (iii) to assess the validity of the reviewed study and identify publication bias. Methods Pubmed1 and Web of ScienceTM electronic databases were screened by two of the authors (LA, CAS) using pre-defined search terms (January 1995±April 2017) for preclinical in vivo 2 / 19 reports employing STL or STL combined with rtPA (rtPA-STL) in IS. The search terms ((stroke [MeSH Terms] OR fibrinolytic drugs [MeSh Terms] OR thrombolytic OR thrombolytic agents [MeSH Terms] OR thrombolytic drugs [MeSH Terms]) AND (microbubbles [MeSH Terms] OR microspheres [MeSH Terms]) AND ("French" [language] OR "English" [language])) OR ªsonothrombolysisº were used on the Pubmed1 database. The search terms (ªthrombolytic therapy OR strokeº) AND (ªmicrobubbles OR microspheresº) OR ªsonothrombolysisº, (ªultrasound contrast agents AND strokeº) were used on the Web of ScienceTM database. The search term ªultrasound AND thrombolysisº was used on both databases. The full electronic search strategy for the two databases are available in S1 File. The inclusion and exclusion criteria are summarized in Table 1. Data were extracted by two of the authors (LA, CAS) following a pre-defined schedule including: species, stroke model, type of clot, duration of clotting, duration of ischemia, ultrasound parameters (frequency, duty cycle, duration of insonation, position of the ultrasound transducer, acoustic power, and peak negative pressure), microbubbles characteristics (type, route of administration, thrombolytic drug associated, volume of injection, and concentration of microbubbles per kilogram), significant outcomes, and quality evaluation. The Collaborative Approach to Meta-Analysis and Review of Animal Data from Experimental Studies (CAMARADES) was used to assess the quality and risk of bias of the studies included [ 16 ]. This systematic review was written in accordance with the PRISMA guidelines [ 17 ]. The protocol was registered on the CAMARADES-NC3Rs Preclinical Systematic Review & Meta-analysis Facility (SyRF) and is available online (https://drive.google.com/ file/d/0B7Z0eAxKc8ApWi1HY3lvNU1pU2M/view). Results Studies selected Sixteen studies met the inclusion criteria (Table 2). The selection schedule used is illustrated in Fig 1. Animal models and clots Thrombi (0.1 mL±1 mL) were delivered into the ascending pharyngeal artery and rete mirabile using femoral catheterization in a swine model [18±20] and arterial occlusions assessed using angiography [ 18, 19 ] or MRI [20]. Gao et al. [ 20 ] achieved bilateral arterial occlusions, because the right and the left ascending pharyngeal arteries supply the rete mirabile. Acute arterial occlusions, induced by blocking the right middle cerebral artery with autologous blood clots delivered by femoral catheterization, were used in a rabbit model [21±26]. Finally, five studies used a rat model of stroke with varying modifications [27±31]. Alonso et al. [ 27 ] injected 4 mm 3 / 19 5 studies focused only on STL treatment efficacy 4 studies focused on safety and efficacy of STL 2 studies focused on rtPA-STL treatment efficacy 5 Studies focused on efficacy and safety of rtPA-STL 1. Moumouh et al. (2009), with microbubbles (SonoVue1) alone 2. Culp et al. (2003), with lipid shelled microbubbles alone 3. Wang et al. (2008), with targeted microbubbles (eptifibatide) alone 4. Alonso et al. (2009), with targeted microbubbles (abciximab) alone 5. Culp et al. (2004), with targeted microbubbles (eptifibatide) alone 1. Culp et al. (2011), with targeted or non-targeted microbubbles (Definity1, or custom 3 μm microbubbles), or targeted microbubbles (eptifibatide) alone 2. Liu et al. (2012), with microbubbles (SonoVue1) with Urokinase 3. Ren et al. (2012), with targeted microbubbles (eptifibafide) alone 4. Gao et al. (2014) with microbubbles (Definity1) alone, with the objective of determining the type and level of cavitation required to dissolve thrombi and improve cerebral blood flow 1. Tomkins et al. (2015) with lipid shelled microbubbles (BR-381) and rtPA on a new in-vivo model using platelet rich plasma clot 2. Ren et al. (2015), tested the efficacy of rtPA-STL for thrombolysis. 1. Brown et al. (2011), with Definity1 or albumin/dextrose microbubbles and low dose of rtPA, addressed the possibility of reducing the dose of rtPA and decreasing the rate of intracerebral hemorrhages 2. Flores et al. (2013), with microbubbles (Definity1) alone and microbubbles with rtPA 3. Lu et al. (2016), attempted to determine whether STL or rtPA-STL could dissolve platelet rich or erythrocyte rich microthrombi. 4. Schleicher et al. (2016) and Nedelmann et al. (2010) tested the ability of STL or rtPA-STL to improve microvascular patency after a transient vascular occlusion. long human blood clots through the external carotid artery (ECA) and delivered them into a pre-ligated common carotid artery (CCA). Moumouh et al. [ 29 ] injected 500 μm of autologous blood clots directly into the CCA. Tomkins et al. [ 31 ] administered 30 mm plasma-rich clots into the internal carotid artery via the ECA and assessed vascular occlusion using laser Doppler velocimetry. Ren et al. [ 30 ] introduced 0.6 mm × 0.08 cm autologous clots into the left CCA. Lu et al. [ 28 ] injected white or red micro-thrombi into a distally ligated ECA after clamping the CCA. The clamp was subsequently removed and microthrombi were liberated into the cerebral vasculature. Nedelmann et al. and Schleicher et al. [ 32, 33 ] induced a transient vascular occlusion by introducing a filament through the carotid artery in the middle cerebral artery. Table 3 summarizes the clot compositions, clotting time prior to injection, duration following in vivo vessel occlusion, and initiation of thrombolytic treatment employed. Sonothrombolysis Thrombolytic drugs co-administered with the microbubbles Fig 1. PRISMA 2009 flow diagram. Extracting schedule of the studies. Evaluation criteria Efficacy. TIMI (Thrombolysis in Myocardial Infarction), TIBI (Thrombolysis in Brain Infarction), custom patency, or de-clotting scores were used to assess the quality of arterial recanalization with angiography [18, 19, 24±26]. Gao et al. [ 20 ] used MRI to assess restoration of blood flow. Ren et al. [ 30 ] used US imaging and pulsed wave Doppler to assess recanalization. Other coagulation parameters used included activated partial thromboplastin time [ 26 ], activated clotting time [ 19 ], prothrombin time [ 26 ], fibrinogen [ 26, 27 ] and D-dimer [26]. Alonso et al. and Tomkins et al. [ 27, 31 ] examined thrombi in treated arteries using histology. 5 / 19 Species Clot type Clotting time before injection (min) 217 276 NA 120 30 Time between injection of blood clot and the start of treatment (min) Immediately Immediately 15 Immediately 240 Liu et al. [ 24 ] determined changes in S100B astroglial protein levels in blood were a marker of cerebral damage [ 34 ]. Diffusion-weighted sequences and phosphorus spectroscopy were used by Moumouh et al. [ 29 ] to assess and to quantify the extent of cerebral infarct. Nedelmann et al. [ 32 ] used MRI T2 relaxation time as indicator of edema formation. Cerebral infarct volume was assessed by eight studies as a treatment outcome [21±25, 28, 31, 32]. Nedelmann et al. and Schleicher et al. [ 32, 33 ] evaluated vascular volumes with microcomputed tomography. Finally, four studies [ 21, 22, 28, 32 ] used functional tests (as a marker of neurological improvement) after STL or rtPA-STL treatment. Safety. Nine studies used histological analysis to assess intracranial hemorrhage (ICH) [20±25, 28, 32, 33]. Treatment outcomes s in le .la )6 gou dn lto od z gn e C n1 i)a teLu (012 ltoou read itceh tram 2HM iagm robp (14V scou eqou I A s A w a . s la ) ou a t l z rce 2 te 012 lgo itn ibb eod 0kH sdu .c8m en (2 to lo ra m 0 n 2 R u c 8 tra fo A . l 5 l a ( a te )1 an an it le z d ) irc 2 se 10 m it bb od H se m d am cm lro (2 uH lco ra m 1M cou m liyn eb 01 F F c )1 (5 la n 01 an an it le z d ) irc 2 w 2 m it bb od H se m d am cm roB .l(a uH lco ra m 1M cou m liyn eb 10 te F c .l s (5 la a ) ou a t l c 2 t 1 n i e z d ) ir leup (201 ltgoou litco rabb odm 1HM sceou mm liydn eabm c10m C A F c nn )10 t in le lau ten zH rebo ;00 ) a r a 0 en n od sca rm p 75 s 1 .ised leedNm .lt(ea2 liaFm lisccou tram ircvo iiapm t31oM iaggn sSoon liiphP o m m ( I N N 4 9 . 2 s a e P t u A A M .7 A A in 1 N N N N 8 6 . 2 A A M .9 A A 1 N N N N 4 9 , 2 A A M .7 A A 1 N N a l a iod ghu anm raopm eobn AN AN 9M .9 A A t P ra ro u 0 N N . 0 4 9 . 2 a A A o P .7 A A N N t M 1 N N u t s e h t a r h t u S . s e 5 c le ren b e f a e T R A N l a r o p m e T e m th ll m ev ku 0 o s 4 b a l a ro e p n o m b e T d i t o r a c n O l a ro e p n o m b e T l a ro e p n o m b e T l a ro e p n o m b e T A N l ta e ie n r o a b P d i t o r a c n O e h t h e B d e in ey l a ro e p n o m b e T l a r o e p n o m b e T f o e n b o o it r is p o S n i m 0 3 n i m 0 2 n i m 0 6 n i m 0 3 n i n i m 0 3 n i m 0 6 n i m 0 6 n i m 0 6 n i m 0 6 n i m 0 3 n i m : a 5 t 0 sa t0 I . ; 8 e 8 l 7 b 1 a l 9 i 1 a With regard to brain damage, Brown et al. [ 21 ] noted a significant reduction in infarct brain volume for rtPA-STL when compared to STL or control animals. Similarly, Lu et al. [ 28 ] reported a reduction in infarct brain volume for STL and rtPA-STL on white microthrombi compared to rtPA alone and rtPA-STL on red microthrombi compared to rtPA alone. Moreover, Liu et al. [ 24 ] described a significant reduction in infarct brain volume for STL + urokinase when compared to urokinase treatment alone (p = 0.025). Nedelmann et al. [ 32 ] showed a lower infarct volume with a lower edema volume using rtPA-STL compared to rtPA treatment alone. In addition, Schleicher et al. [ 33 ] reported also a lower infarct volume using rtPA-STL compared to rtPA alone. Ren et al. [ 30 ] reported improved recanalization rates with - IA TSTL> IV TSTL (declotting time, success rate) - IA or IV TSTL > US (declotting time, and TIMI) - IA or IV TSTL > IA saline control - IV TSTL > IV STL (declotting time, TIMI) - STL > RtPA (Pcr/Pi (estimation of the oxidative phosphorylation metabolism)) - IV STL with TMB > IV STL (D-Dimer) - IV STL with TMB > US (D-Dimer) - IA STL > IA Saline/US (declotting score, flow scores, success at 24 min) - IV TSTL > IV eptifibatide/US (angiographic success, declotting score) - IV TSTL > IV saline/US (angiographic success, declotting score) No recanalization - After 10 min of treatment: RtPA-STL > contrl; RtPA; STL (Recanalization rate) - After 20 min of treatment: All conditions (except STL) induced a significant recanalization versus control (recanalization rate) - All MB STL > control, rtPA (infarct volume, SB100 rate), Pooled analysis with all type of MB (Lipid, Albumin, TMB) - Urokinase-STL > Urokinase (infarct volume) - Urokinase-STL > Urokinase (mean flow velocity at 120 min) - IA TSTL > IA Saline/US (patency score, TIBI) - IA TSTL > IA STL (patency score, TIBI) - IA TSTL > IA Saline/US (recanalization rate) - IA rtPA > IA Saline/US (recanalization rate) - IA TSTL > IA STL (recanalization rate) - (1) 1.7MHz, 1.7 MI, - (2)>(3) (after 24H, relatives changes in cerebral 20 μs pulse STL blood flow) - (2) 1.6MHz, 2.4 MI, 5 μs - (2)>(1) (complete recanalization of ipsi and pulse STL contralateral internal carotid and middle cerebral - (3) 1.6MHz, 2.4 MI, 5 μs artery) pulse US 9 / 19 STL efficacy Efficacy of rtPA-STL Safety and efficacy of STL Culp et al. (2011) Wang et al. (2008) Moumouh et al. (2010) PLOS ONE | https://doi.org/10.1371/journal.pone.0191788 10 / 19 rtPA-STL as compared to STL alone or rtPA alone after 10 minutes of treatment. However, Tomkins et al. [ 31 ] reported a complete absence of recanalization for rtPA-STL treatment on platelet-rich thrombi. In addition, functional tests performed in the studies by Brown et al., Culp et al., and Lu et al. [ 21, 22, 28 ] showed no noticeable differences between treatment regimens. Nevertheless, Lu et al. [ 28 ] reported better neurological scores for STL when compared to rtPA alone for the treatment of white microthrombi emboli. Safety. Flores et al. [ 23 ] reported fewer hemorrhages outside the infarct zone for STL when compared to rtPA-STL, US + rtPA, rtPA alone, and US alone (p < 0.004). Brown et al. [ 21 ] found lower incidences of ICH not only for rtPA-STL compared to rtPA alone but also for the rtPA concentration of 0.8±0.9 mg/kg. The authors also reported reduced incidence of ICH outside of stroke zones when using rtPA-STL in comparison to rtPA (p = 0.005) alone, when the results obtained with all concentrations of rtPA were combined. All other studies showed no noticeable differences in the incidence of cerebral hemorrhage using rtPA-STL, STL alone, and rtPA alone. Quality assessment The average CAMARADES criteria were 4.68 (± 1.08) for the 10 criteria tested, as summarized in Table 7. Owing to considerable heterogeneity in the reported methods and data, meta-analysis was not feasible. As a result, no statistical examination was performed. Discussion Efficacy This systematic review highlights a number of positive outcomes in terms of efficacy. Numerous criteria are evaluated throughout the studies. Half of the studies (8 of 16) assess cerebral infarct volume, while four studies use functional recovery tests and neurological scores. The most commonly used criteria are the recanalization rate and the quality of the vascular flow stream. However, discordances have been reported between apparent positive recanalization and favorable outcomes. Ren et al. [ 25 ] highlight the limit of this pure blood flow restoration assessment by showing a significant difference in recanalization scores and quality of the flow stream, but not in the infarcted brain volume. Infarcted brain volume and neurological score are the two main outcomes used in clinical trials to assess thrombolytic therapy [2±4]. Similar results are described in humans in the CLOTBUST (Combined Lysis Of Thrombus in Brain ischemia using transcranial Ultrasound and Systemic TPA) study [ 35 ], which has raised the need to consider clinical outcome as the pivotal end-point for all studies. This observation is in line with the Stroke Therapy Academic Industry Roundtable (STAIR) group recommendations [ 36 ]. Different stroke models are used to investigate the efficacy of STLÐ13 studies assess it on a thromboembolic model with a blood clot, and 3 studies assess it on microvasculature impairment (transient occlusion or microthrombi emboli). It appears that a clear benefit exists with the use of rtPA-STL in microvasculature impairment (transient occlusion and red or white micro-thrombi) compared to control or rtPA alone. White thrombi seem to be more resistant to STL or rtPA-STL [ 31 ]. Studies involving a thromboembolic model, present more heterogeneous results, as only 3 studies demonstrated a benefit of STL or rtPA-STL versus rtPA alone on clinical criteria. These discrepancies can be partly due to the variation in treatment initiation times highlighted in this review. Indeed, ischemia induce an inflammatory response in the salvageable penumbra area which is responsible for the secondary necrosis of this area [ 37 ]. Furthermore, this response could also lead to a reperfusion injury syndrome 11 / 19 Sonothrombolysis with microbubbles in ischemicstroke - an assessment of pre-clinicalevidence N N Y Y 6 N N Y Y 4 N N N N 3 Y N Y Y 6 N N Y N 4 N N Y Y 4 N N Y Y 6 N N Y N N N Y N 3 5 N N Y Y 4 N N Y Y 6 N N Y N 4 5 7 0 0 .t 8 8 7 1 4 9 inducing oedema and hemorrhages [ 38 ]. These phenomenon are presumably absent immediately after vascular occlusion [ 39 ]. In terms of MBs used and US parameters, TMBs (Targeted Microbubbles) are more efficient than MBs, but none of the studies have tested the association of rtPA-TMBs (rtPA associated with TMB) versus rtPA. With the data available, one cannot conclude on the clinical interest of TMBs. Further investigations are required to evaluate the therapeutic efficacy of TMBs for the STL. The heterogeneity of US parameters used does not allow us to conclude which combination is the more efficient or if a specific combination of US parameters is giving better benefit to risk ratio in the specific pathophysiology of stroke. The only parameter between studies that seems to be consensual is the use of an US frequency predominantly in the Megahertz (MHz) range, probably to avoid standing waves [ 40 ] which has been supposed to be responsible of a high rate of ICH through an augmentation of peak negative pressure in the Transcranial Low-Frequency Ultrasound-Mediated Thrombolysis in Brain Ischemia (TRUMBI) study with the use of a US frequency of 300 kiloHertz (kHz) wi wi thout MBs in humans [ 41 ]. Safety Existing clinical STL data has shown that cerebral hemorrhage is the major side effect in terms of safety. Two studies report that rtPA-STL leads to fewer cerebral hemorrhages outside the infarcted zone. These two studies show no significant efficacy of rtPA-STL versus rtPA alone [ 21, 23 ]. Based on these limited results, it may be inappropriate to draw conclusions on the safety of STL or rtPA-STL versus rtPA alone; and more generally on the benefit to risk ratio of this therapeutic approach. Compared to clinical trials, the results reported in this review are surprising, as none of the studies revealed a higher rate of cerebral hemorrhage versus control, even with the use of rtPA alone, which is known to significantly increase the rates of cerebral hemorrhage in clinical studies [2]. Some hypotheses can be made to explain the following: publication bias (e.g., the absence of the publication of studies showing a higher rate of cerebral hemorrhage with STL), the use of animal models with no risk factor of bleeding (e.g., aging or hypertension), and the absence of ischemia before treatment. Clinical translation/perspectives The studies reviewed in this report use a thromboembolic stroke model or a microvasculature impairment model (by transient ischemia or deployment of microthrombi). Stroke in humans is due to different pathophysiological mechanisms associated with cardiovascular (CV) risk factors that are summarized in the TOAST (Trial of ORG 10172 in Acute Stroke Treatment) classification [ 42 ]. The use of an animal model closer phylogenetically to humans, such as gyrencephalic species, associated with CV risk factors (e.g., aging and hypertension) could bring more consistent data, as suggested by the recommendations for animal testing in stroke [ 36 ]. Some specificity of the human pathophysiology (e.g., the human skull) may be considered to ensure a clinical translation. Indeed, the interactions between the human skull and US beam are responsible for acoustic processes including standing waves, degradation of US focalization, and degradation of beam shape. High frequency ultrasound will also lead to more attenuation and heating [ 43 ]. These processes depend on the frequency used, the position of the US transducer, and the length of the US wave [ 44 ]. The TRUMBI trial [ 41 ] reports higher rates of cerebral hemorrhage, which are probably caused by standing waves. Standing waves field happens mainly with the use of low frequency US, and lower the cavitation threshold in comparison with a progressive wave field [ 45 ]. In STL, MBs act as nuclei in the medium and can induce different effects depending on the acoustic conditions. While MB can be used to induce 13 / 19 stable cavitation, which enhance rtPA thrombolysis without disrupting the blood brain barrier [ 46, 47 ], MB also lower the threshold of inertial cavitation which is able to induce a putative cerebral hemorrhage. Blood brain barrier disruption is induced by STL in certain acoustic conditions, which can also explain ICH (specifically in presence of rtPA, which is known to be neurotoxic and to cause hemorrhage) [ 44, 48, 49 ]. To avoid these adverse effects, some solutions have been developed to create a controlled intracranial US field that have not yet been tested in STL for IS. For example, it is now possible to monitor inertial cavitation in the brain in real time by using a passive cavitation detector [50]. Wright et al. were able to monitor cavitation during sonothrombolysis with high-intensity focused ultrasound in a rabbit femoral artery [ 51 ]. Frequency modulation is a solution to lower the occurrence of standing waves [ 52 ] through the use of higher US frequencies and short emission duration [ 44 ]. Associating STL with thrombolytic drugs or with mechanical thrombi retrieval is not well defined and needs to be fully explored [ 4 ]. Similarly, the question of whether STL without thrombolytic drugs or STL with reduced MBs dose leads to better safety ratios in certain clinical situations (e.g., when rtPA is contraindicated) needs to be addressed. Furthermore, the CLOTBUST clinical trial (using US alone) has pointed out that insonation remains operator dependent. A couple of solutions existÐthe first one is the MRI-guidance (MRI = Magnetic resonance Imaging) of US [ 53, 54 ] and, the second one is the use of an operator independent US device (as reported in the CLOTBUST-HF trial) [55]. At this time, none of these solutions have been evaluated with STL in IS. The use of TMBs seems promising, but TMBs are not yet validated for clinical use even for diagnostic purpose. In addition, regulatory complications that have to be overcome to get clinical approval of a new type of MBs agent will delay the clinical translation of this therapeutic approach. The concentration of MBs to be used is not consensual and appears as a critical safety element. Indeed, Transcranial ultrasound in clinical sonothrombolysis (TUCSON) trial [ 7 ] indicate that an association between the concentration of MBs and the rate of cerebral hemorrhage could exist (for the same US parameters). Recently McMahon et al. demonstrated that acute inflammatory response (which lead to microhemorrhage) following blood-brain-barrier opening with focused ultrasound and microbubbles is dependent on microbubble dose [ 56 ]. Limitations The present review represents the first systematic evaluation of in vivo preclinical studies with respect to STL treatment of experimental IS. Although our systematic screening of the relevant databases identified > 1000 articles on STL, only 16 studies met the inclusion criteria. We decide to exclude studies involving coronary or peripheral arteries, as brain has a specific vulnerability to ischemia and hemorrhage that is not present in cardiac muscle or other organs. We regroup the microthrombi spreading and transient vascular occlusion under the term ªmicrovasculature impairment modelº, because authors stated that these models refer to the human ªno reflow phenomenonº (where the distal arterioles stay occluded after a large vessel recanalization). Meta-analysis is not feasible because of the heterogeneity of the studies reviewed. Our quality evaluation reveals biases, which can be easily corrected in future investigations. 3 studies were published before or the year after the publication of the CAMARADES criteria. The absence of power calculations of the sample sizes in all studies reviewed means that some of the negative results reported may be linked to a default in statistical power. Information such as temperature of the animal is not always monitored, while hypothermia can lead to neuroprotection. Similarly, free neuroprotective anaesthetics can be implemented because their effects can falsely influence treatment outcomes. 14 / 19 Conclusions STL is a complex treatment requiring a multidisciplinary scientific approach. Nevertheless, selected preclinical studies have shown that STL has a substantial thrombolytic effect. Data available on safety are limited and more preclinical evaluation is needed to demonstrate a clear benefit to risk ratio on clinical endpoints, as compared to approved treatment. We suggest testing frequencies in the range from sub-MHz to 1 MHz, with intensities below the threshold of inertial cavitation, so to favor stable cavitation of microbubbles. The use of frequency modulation and short pulse durations should lessen the probability of the generation of standing wave. While TMB demonstrated a better efficacy than microbubbles, the use of clinicallyapproved microbubble may accelerate clinical translation. Further investigations on the doses of microbubbles and their association with thrombolytic drugs (mainly rtPA) are needed to improve the therapeutic benefit of STL. Although this therapeutic option could be used for large artery occlusion, this approach should also be beneficial in the situation of microcirculation impairment (e.g., after thrombectomy). Moreover, the future explorations on the safety of STL are essential. To achieve this objective, animal models, which reproduce the human pathophysiology (i.e., older animals, cardio-vascular risk factors, significant duration of ischemia) should be used. Finally, MRI guidance and cavitation detection have not been evaluated in ischemic stroke yet but their use in STL look promising to guarantee a good efficacy/safety profile. Supporting information S1 PRISMA Checklist. PRISMA checklist of the systematic review following the preferred reporting items for systematic reviews and meta-analyses. [ 17 ]. (DOC) S1 File. S1 File contains the full electronic search strategy for the two electronical databases. (DOCX) Author Contributions Conceptualization: Laurent Auboire, Ayache Bouakaz. Data curation: Laurent Auboire, Charles A. Sennoga. Formal analysis: Laurent Auboire. Investigation: Laurent Auboire. Methodology: Laurent Auboire. Project administration: Laurent Auboire, Ayache Bouakaz. Supervision: FrancËois Tranquart, Ayache Bouakaz. Validation: Charles A. Sennoga, Jean-Michel Escoffre, Ayache Bouakaz. Writing ± original draft: Laurent Auboire, Charles A. Sennoga, Jean-Michel Escoffre, FrancËois Tranquart, Ayache Bouakaz. Writing ± review & editing: Laurent Auboire, Charles A. Sennoga, Jean-Marc Hyvelin, FreÂderic Ossant, Jean-Michel Escoffre, FrancËois Tranquart, Ayache Bouakaz. 15 / 19 16 / 19 17 / 19 18 / 19 1. Feigin VL , Forouzanfar MH , Krishnamurthi R , Mensah GA , Connor M , Bennett DA , et al. Global and regional burden of stroke during 1990±2010: findings from the Global Burden of Disease Study 2010 . Lancet. 2014 ; 383 ( 9913 ): 245 ± 54 . Epub 2014/01/23. https://doi.org/10.1016/S0140- 6736 ( 13 ) 61953 - 4 PMID: 24449944; PubMed Central PMCID : PMCPMC4181600 . 2. National Institute of Neurological Disorders and Stroke rt-PA Stroke Study Group . Tissue plasminogen activator for acute ischemic stroke . N Engl J Med . 1995 ; 333 ( 24 ): 1581 ± 7 . Epub 1995/12/14. https://doi. org/10.1056/NEJM199512143332401 PMID: 7477192 . 3. Campbell BC , Mitchell PJ , Kleinig TJ , Dewey HM , Churilov L , Yassi N , et al. Endovascular therapy for ischemic stroke with perfusion-imaging selection . N Engl J Med . 2015 ; 372 ( 11 ): 1009 ± 18 . Epub 2015/ 02/12. https://doi.org/10.1056/NEJMoa1414792 PMID: 25671797 . 4. Berkhemer OA , Fransen PS , Beumer D , van den Berg LA , Lingsma HF , Yoo AJ , et al. A randomized trial of intraarterial treatment for acute ischemic stroke . N Engl J Med . 2015 ; 372 ( 1 ): 11 ± 20 . Epub 2014/ 12/18. https://doi.org/10.1056/NEJMoa1411587 PMID: 25517348 . 5. Huang X , Cheripelli BK , Lloyd SM , Kalladka D , Moreton FC , Siddiqui A , et al. Alteplase versus tenecteplase for thrombolysis after ischaemic stroke (ATTEST): a phase 2, randomised, open-label, blinded endpoint study . Lancet Neurol . 2015 ; 14 ( 4 ): 368 ± 76 . Epub 2015/03/03. https://doi.org/10.1016/S1474- 4422 ( 15 ) 70017 - 7 PMID: 25726502 . 6. Larrue V , Viguier A , Arnaud C , Cognard C , Petit R , Rigal M , et al. Transcranial ultrasound combined with intravenous microbubbles and Tissue Plasminogen Activator for Acute Ischemic Stroke: A Randomized Controlled Study . Stroke . 2007 ; 38 : 472 . 7. Molina CA , Barreto AD , Tsivgoulis G , Sierzenski P , Malkoff MD , Rubiera M , et al. Transcranial ultrasound in clinical sonothrombolysis (TUCSON) trial . Ann Neurol. 2009 ; 66 ( 1 ): 28 ± 38 . Epub 2009/08/12. https://doi.org/10.1002/ana.21723 PMID: 19670432 . 8. Nacu A , Kvistad CE , Naess H , Oygarden H , Logallo N , Assmus J , et al. NOR-SASS (Norwegian Sonothrombolysis in Acute Stroke Study): Randomized Controlled Contrast-Enhanced Sonothrombolysis in an Unselected Acute Ischemic Stroke Population . Stroke . 2017 ; 48 ( 2 ): 335 ± 41 . Epub 2016/12/17. https://doi.org/10.1161/STROKEAHA.116.014644 PMID: 27980128; PubMed Central PMCID : PMCPMC5266415 . 9. Ricci S , Dinia L , Del Sette M , Anzola P , Mazzoli T , Cenciarelli S , et al. Sonothrombolysis for acute ischaemic stroke . Cochrane Database Syst Rev . 2012 ; 6 ( 6 ): CD008348 . Epub 2012/06/15. https://doi. org/10.1002/14651858.CD008348.pub2 PMID: 22696378 . 10. Saqqur M , Tsivgoulis G , Nicoli F , Skoloudik D , Sharma VK , Larrue V , et al. The role of sonolysis and sonothrombolysis in acute ischemic stroke: a systematic review and meta-analysis of randomized controlled trials and case-control studies . J Neuroimaging . 2014 ; 24 ( 3 ): 209 ± 20 . Epub 2013/04/24. https:// doi.org/10.1111/jon.12026 PMID: 23607713 . 11. Tachibana K , Tachibana S . Albumin microbubble echo-contrast material as an enhancer for ultrasound accelerated thrombolysis . Circulation . 1995 ; 92 ( 5 ): 1148 ± 50 . Epub 1995/09/01. PMID: 7648659 . 12. Meairs S. Contrast-enhanced ultrasound perfusion imaging in acute stroke patients . Eur Neurol . 2008 ; 59 Suppl 1 : 17 ± 26 . Epub 2008/04/18. https://doi.org/10.1159/000114456 PMID: 18382109 . 13. Meairs S , Alonso A , Hennerici MG . Progress in sonothrombolysis for the treatment of stroke . Stroke . 2012 ; 43 ( 6 ): 1706 ± 10 . Epub 2012/04/27. https://doi.org/10.1161/STROKEAHA.111.636332 PMID: 22535275 . 14. Bader KB , Bouchoux G , Holland CK . Sonothrombolysis. In: Escoffre JM , Bouakaz A , editors. Therapeutic Ultrasound2016 . p. 339 ± 62 . 15. Stride E. Physical Principles of Microbubbles for Ultrasound Imaging and Therapy . In: Alonso A , Hennerici MG , Meairs S , editors. Translational Neurosonology2015 . p. 11 ± 22 . 16. Sena E , van der Worp HB , Howells D , Macleod M. How can we improve the pre-clinical development of drugs for stroke? Trends Neurosci . 2007 ; 30 ( 9 ): 433 ± 9 . Epub 2007/09/04. https://doi.org/10.1016/j.tins. 2007 . 06 .009 PMID: 17765332 . 17. Moher D , Liberati A , Tetzlaff J , Altman DG , Group P. Preferred reporting items for systematic reviews and meta-analyses: the PRISMA statement . J Clin Epidemiol . 2009 ; 62 ( 10 ): 1006 ± 12 . Epub 2009/07/ 28. https://doi.org/10.1016/j.jclinepi. 2009 . 06 .005 PMID: 19631508 . 18. Culp WC , Erdem E , Roberson PK , Husain MM . Microbubble potentiated ultrasound as a method of stroke therapy in a pig model: preliminary findings . J Vasc Interv Radiol . 2003 ; 14 ( 11 ): 1433 ± 6 . Epub 2003/11/08. https://doi.org/10.1097/01.RVI. 0000096767 .47047.FA PMID: 14605109 . 19. Culp WC , Porter TR , Lowery J , Xie F , Roberson PK , Marky L . Intracranial clot lysis with intravenous microbubbles and transcranial ultrasound in swine . Stroke . 2004 ; 35 ( 10 ): 2407 ± 11 . Epub 2004/08/24. https://doi.org/10.1161/01.STR. 0000140890 .86779.79 PMID: 15322299 . 20. Gao S , Zhang Y , Wu J , Shi WT , Lof J , Vignon F , et al. Improvements in cerebral blood flow and recanalization rates with transcranial diagnostic ultrasound and intravenous microbubbles after acute cerebral emboli . Invest Radiol . 2014 ; 49 ( 9 ): 593 ± 600 . Epub 2014/04/03. https://doi.org/10.1097/RLI. 0000000000000059 PMID: 24691139 . 21. Brown AT , Flores R , Hamilton E , Roberson PK , Borrelli MJ , Culp WC . Microbubbles improve sonothrombolysis in vitro and decrease hemorrhage in vivo in a rabbit stroke model . Invest Radiol . 2011 ; 46 ( 3 ): 202 ± 7 . Epub 2010/12/15. https://doi.org/10.1097/RLI.0b013e318200757a PMID: 21150788; PubMed Central PMCID : PMCPMC3845811 . 22. Culp WC , Flores R , Brown AT , Lowery JD , Roberson PK , Hennings LJ , et al. Successful microbubble sonothrombolysis without tissue-type plasminogen activator in a rabbit model of acute ischemic stroke . Stroke . 2011 ; 42 ( 8 ): 2280 ± 5 . Epub 2011/06/28. https://doi.org/10.1161/STROKEAHA.110.607150 PMID: 21700942; PubMed Central PMCID : PMCPMC3266124 . 23. Flores R , Hennings LJ , Lowery JD , Brown AT , Culp WC . Microbubble-augmented ultrasound sonothrombolysis decreases intracranial hemorrhage in a rabbit model of acute ischemic stroke . Invest Radiol . 2011 ; 46 ( 7 ): 419 ± 24 . Epub 2011/02/24. https://doi.org/10.1097/RLI.0b013e31820e143a PMID: 21343824; PubMed Central PMCID : PMCPMC3109116 . 24. Liu WS , Huang ZZ , Wang XW , Zhou J. Effects of microbubbles on transcranial Doppler ultrasoundassisted intracranial urokinase thrombolysis . Thromb Res . 2012 ; 130 ( 3 ): 547 ± 51 . Epub 2012/07/25. https://doi.org/10.1016/j.thromres. 2012 . 06 .020 PMID: 22823944 . 25. Ren ST , Long LH , Wang M , Li YP , Qin H , Zhang H , et al. Thrombolytic effects of a combined therapy with targeted microbubbles and ultrasound in a 6 h cerebral thrombosis rabbit model . J Thromb Thrombolysis . 2012 ; 33 ( 1 ): 74 ± 81 . Epub 2011/10/22. https://doi.org/10.1007/s11239-011-0644-z PMID: 22016106 . 26. Wang B , Wang L , Zhou XB , Liu YM , Wang M , Qin H , et al. Thrombolysis effect of a novel targeted microbubble with low-frequency ultrasound in vivo . Thromb Haemost . 2008 ; 100 ( 2 ): 356 ± 61 . Epub 2008/08/12. https://doi.org/10.1160/TH07-09-0583 PMID: 18690359 . 27. Alonso A , Dempfle CE , Della Martina A , Stroick M , Fatar M , Zohsel K , et al. In vivo clot lysis of human thrombus with intravenous abciximab immunobubbles and ultrasound . Thromb Res . 2009 ; 124 ( 1 ): 70 ± 4 . Epub 2009/04/08. https://doi.org/10.1016/j.thromres. 2008 . 11 .019 PMID: 19349068 . 28. Lu Y , Wang J , Huang R , Chen G , Zhong L , Shen S , et al. Microbubble-Mediated Sonothrombolysis Improves Outcome After Thrombotic Microembolism-Induced Acute Ischemic Stroke . Stroke . 2016 ; 47 ( 5 ): 1344 ± 53 . Epub 2016/04/07. https://doi.org/10.1161/STROKEAHA.115.012056 PMID: 27048701 . 29. Moumouh A , Barentin L , Tranquart F , Serrierre S , Bonnaud I , Tasu JP . Fibrinolytic effects of transparietal ultrasound associated with intravenous infusion of an ultrasound contrast agent: study of a rat model of acute cerebral stroke . Ultrasound Med Biol . 2010 ; 36 ( 1 ): 51 ± 7 . Epub 2009/10/27. https://doi.org/10. 1016/j.ultrasmedbio. 2009 . 06 .1103 PMID: 19854567 . 30. Ren X , Wang Y , Wang Y , Chen H , Chen L , Liu Y , et al. Thrombolytic therapy with rt-PA and transcranial color Doppler ultrasound (TCCS) combined with microbubbles for embolic thrombus . Thromb Res . 2015 ; 136 ( 5 ): 1027 ± 32 . Epub 2015/09/22. https://doi.org/10.1016/j.thromres. 2015 . 08 .027 PMID: 26388121 . 31. Tomkins AJ , Schleicher N , Murtha L , Kaps M , Levi CR , Nedelmann M , et al. Platelet rich clots are resistant to lysis by thrombolytic therapy in a rat model of embolic stroke . Exp Transl Stroke Med . 2015 ; 7:2 . Epub 2015/02/07. https://doi.org/10.1186/s13231-014-0014-y PMID: 25657829 ; PubMed Central PMCID : PMCPMC4318170 . 32. Nedelmann M , Ritschel N , Doenges S , Langheinrich AC , Acker T , Reuter P , et al. Combined contrastenhanced ultrasound and rt-PA treatment is safe and improves impaired microcirculation after reperfusion of middle cerebral artery occlusion . J Cereb Blood Flow Metab . 2010 ; 30 ( 10 ): 1712 ± 20 . Epub 2010/ 06/10. https://doi.org/10.1038/jcbfm. 2010 .82 PMID: 20531462; PubMed Central PMCID : PMCPMC3023400 . 33. Schleicher N , Tomkins AJ , Kampschulte M , Hyvelin JM , Botteron C , Juenemann M , et al. Sonothrombolysis with BR38 Microbubbles Improves Microvascular Patency in a Rat Model of Stroke . PLoS One . 2016 ; 11 ( 4 ): e0152898 . Epub 2016 /04/15. https://doi.org/10.1371/journal.pone.0152898 PMID: 27077372; PubMed Central PMCID : PMCPMC4831751 . 34. Woods SD , Flores R , Roberson PK , Lowery JD , Skinner RD , Culp WC . Decreased Serum Levels of S100B Protein Reflect Successful Treatment Effects in a Rabbit Model of Acute Ischemic Stroke . Open Neurol J. 2011 ; 5 : 55 ± 7 . Epub 2011/07/16. https://doi.org/10.2174/1874205X01105010055 PMID: 21760859; PubMed Central PMCID : PMCPMC3134987 . 35. Alexandrov AV , Demchuk AM , Burgin WS , Robinson DJ , Grotta JC , Investigators C . Ultrasound-enhanced thrombolysis for acute ischemic stroke: phase I. Findings of the CLOTBUST trial . J Neuroimaging . 2004 ; 14 ( 2 ): 113 ± 7 . Epub 2004/04/21. https://doi.org/10.1177/1051228403261462 PMID: 15095555 . 36. Fisher M , Feuerstein G , Howells DW , Hurn PD , Kent TA , Savitz SI , et al. Update of the stroke therapy academic industry roundtable preclinical recommendations . Stroke . 2009 ; 40 ( 6 ): 2244 ± 50 . Epub 2009/ 02/28. https://doi.org/10.1161/STROKEAHA.108.541128 PMID: 19246690; PubMed Central PMCID : PMCPMC2888275 . 37. Fisher M. The ischemic penumbra: identification, evolution and treatment concepts . Cerebrovasc Dis . 2004 ; 17 Suppl 1 : 1 ± 6 . Epub 2003/12/25. https://doi.org/10.1159/000074790 PMID: 14694275 . 38. Pan J , Konstas AA , Bateman B , Ortolano GA , Pile-Spellman J . Reperfusion injury following cerebral ischemia: pathophysiology, MR imaging, and potential therapies . Neuroradiology . 2007 ; 49 ( 2 ): 93 ± 102 . Epub 2006/12/21. https://doi.org/10.1007/s00234-006-0183-z PMID: 17177065; PubMed Central PMCID : PMCPMC1786189 . 39. Wang Q , Tang XN , Yenari MA . The inflammatory response in stroke . J Neuroimmunol . 2007 ; 184 ( 1 ± 2): 53 ± 68 . Epub 2006/12/26. https://doi.org/10.1016/j.jneuroim. 2006 . 11 .014 PMID: 17188755; PubMed Central PMCID : PMCPMC1868538 . 40. O 'Reilly MA , Huang Y , Hynynen K. The impact of standing wave effects on transcranial focused ultrasound disruption of the blood-brain barrier in a rat model . Phys Med Biol . 2010 ; 55 ( 18 ): 5251 ± 67 . Epub 2010/08/20. https://doi.org/10.1088/ 0031 -9155/55/18/001 PMID: 20720286; PubMed Central PMCID : PMCPMC3207361 . 41. Daffertshofer M , Gass A , Ringleb P , Sitzer M , Sliwka U , Els T , et al. Transcranial low-frequency ultrasound-mediated thrombolysis in brain ischemia: increased risk of hemorrhage with combined ultrasound and tissue plasminogen activator: results of a phase II clinical trial . Stroke . 2005 ; 36 ( 7 ): 1441 ± 6 . Epub 2005/06/11. https://doi.org/10.1161/01.STR. 0000170707 .86793.1a PMID: 15947262 . 42. Adams HP , Jr. , Bendixen BH , Kappelle LJ , Biller J , Love BB , Gordon DL , et al. Classification of subtype of acute ischemic stroke. Definitions for use in a multicenter clinical trial . TOAST. Trial of Org 10172 in Acute Stroke Treatment. Stroke . 1993 ; 24 ( 1 ): 35 ± 41 . Epub 1993/01/01. PMID: 7678184 . 43. Ammi AY , Mast TD , Huang IH , Abruzzo TA , Coussios CC , Shaw GJ , et al. Characterization of ultrasound propagation through ex-vivo human temporal bone . Ultrasound Med Biol . 2008 ; 34 ( 10 ): 1578 ± 89 . Epub 2008/05/06. https://doi.org/10.1016/j.ultrasmedbio. 2008 . 02 .012 PMID: 18456391; PubMed Central PMCID : PMCPMC4921610 . 44. Baron C , Aubry JF , Tanter M , Meairs S , Fink M. Simulation of intracranial acoustic fields in clinical trials of sonothrombolysis . Ultrasound Med Biol . 2009 ; 35 ( 7 ): 1148 ± 58 . Epub 2009/04/28. https://doi.org/10. 1016/j.ultrasmedbio. 2008 . 11 .014 PMID: 19394756 . 45. Azuma T , Kawabata K-i, Umemura S-i, Ogihara M , Kubota J , Sasaki A , et al. Bubble Generation by Standing Wave in Water Surrounded by Cranium with Transcranial Ultrasonic Beam . Jpn J Appl Phys . 2005 ; 44 ( 6B ): 4625 ± 30 . https://doi.org/10.1143/jjap.44.4625 46. Bader KB , Gruber MJ , Holland CK . Shaken and stirred: mechanisms of ultrasound-enhanced thrombolysis . Ultrasound Med Biol . 2015 ; 41 ( 1 ): 187 ± 96 . Epub 2014/12/03. https://doi.org/10.1016/j. ultrasmedbio. 2014 . 08 .018 PMID: 25438846; PubMed Central PMCID : PMCPMC4258471 . 47. O 'Reilly MA , Hynynen K. Blood-brain barrier: real-time feedback-controlled focused ultrasound disruption by using an acoustic emissions-based controller . Radiology . 2012 ; 263 ( 1 ): 96 ± 106 . Epub 2012/02/ 15. https://doi.org/10.1148/radiol.11111417 PMID: 22332065; PubMed Central PMCID : PMCPMC3309801 . 48. Kaur J , Zhao Z , Klein GM , Lo EH , Buchan AM . The neurotoxicity of tissue plasminogen activator? J Cereb Blood Flow Metab . 2004 ; 24 ( 9 ): 945 ± 63 . Epub 2004/09/10. https://doi.org/10.1097/01.WCB. 0000137868 .50767.E8 PMID: 15356416 . 49. Benchenane K , Lopez-Atalaya JP , Fernandez-Monreal M , Touzani O , Vivien D. Equivocal roles of tissue-type plasminogen activator in stroke-induced injury . Trends Neurosci . 2004 ; 27 ( 3 ): 155 ± 60 . Epub 2004/03/24. https://doi.org/10.1016/j.tins. 2003 . 12 .011 PMID: 15036881 . 50. Wu SY , Tung YS , Marquet F , Downs M , Sanchez C , Chen C , et al. Transcranial cavitation detection in primates during blood-brain barrier openingÐa performance assessment study . IEEE Trans Ultrason Ferroelectr Freq Control . 2014 ; 61 ( 6 ): 966 ± 78 . Epub 2014/05/27. https://doi.org/10.1109/TUFFC. 2014 . 2992 PMID: 24859660; PubMed Central PMCID : PMCPMC4034133 . 51. Wright C , Hynynen K , Goertz D. In vitro and in vivo high-intensity focused ultrasound thrombolysis . Invest Radiol . 2012 ; 47 ( 4 ): 217 ± 25 . Epub 2012/03/01. https://doi.org/10.1097/RLI.0b013e31823cc75c PMID: 22373533; PubMed Central PMCID : PMCPMC3302946 . 52. Tang SC , Clement GT . Standing-wave suppression for transcranial ultrasound by random modulation . IEEE Trans Biomed Eng . 2010 ; 57 ( 1 ): 203 ± 5 . Epub 2009/08/22. https://doi.org/10.1109/TBME. 2009 . 2028653 PMID: 19695991; PubMed Central PMCID : PMCPMC2887681 . 53. Clement GT , Hynynen K. A non-invasive method for focusing ultrasound through the human skull . Phys Med Biol . 2002 ; 47 ( 8 ): 1219 ± 36 . Epub 2002/05/28. PMID: 12030552 . 54. Ghanouni P , Pauly KB , Elias WJ , Henderson J , Sheehan J , Monteith S , et al. Transcranial MRI-Guided Focused Ultrasound: A Review of the Technologic and Neurologic Applications . AJR Am J Roentgenol . 2015 ; 205 ( 1 ): 150 ± 9 . Epub 2015/06/24. https://doi.org/10.2214/AJR.14.13632 PMID: 26102394; PubMed Central PMCID : PMCPMC4687492 . 55. Barreto AD , Alexandrov AV , Shen L , Sisson A , Bursaw AW , Sahota P , et al. CLOTBUST-Hands Free: pilot safety study of a novel operator-independent ultrasound device in patients with acute ischemic stroke . Stroke . 2013 ; 44 ( 12 ): 3376 ± 81 . Epub 2013/10/26. https://doi.org/10.1161/STROKEAHA.113. 002713 PMID: 24159060 . 56. McMahon D , Hynynen K. Acute Inflammatory Response Following Increased Blood-Brain Barrier Permeability Induced by Focused Ultrasound is Dependent on Microbubble Dose . Theranostics. 2017 ; 7 ( 16 ): 3989 ± 4000 . Epub 2017/11/08. https://doi.org/10.7150/thno.21630 PMID: 29109793; PubMed Central PMCID : PMCPMC5667420 .


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Laurent Auboire, Charles A. Sennoga, Jean-Marc Hyvelin, Fréderic Ossant, Jean-Michel Escoffre, François Tranquart, Ayache Bouakaz. Microbubbles combined with ultrasound therapy in ischemic stroke: A systematic review of in-vivo preclinical studies, PLOS ONE, 2018, DOI: 10.1371/journal.pone.0191788