Microbubbles combined with ultrasound therapy in ischemic stroke: A systematic review of in-vivo preclinical studies
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
FreÂ 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, UniversiteÂ FrancËois-Rabelais de Tours, France, 2 CHRU de Tours, Service d'eÂ chographie-Doppler , Tours, France, 3 Bracco Suisse SA, Geneva, Switzerland, 4 CHRU de Tours, CIC-IT, Tours, France, 5 Advice-US Consulting, Nernier , France
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
files. The protocol is available at http://syrf.org.uk/
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
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.
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.
Further in vivo studies are needed to demonstrate a better efficacy and safety of STL
compared to currently approved therapeutic options.
Systematic review registration
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 [
]. 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 [
]. 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
]. Use of other thrombolytic drugs (e.g., tenecteplase) were previously trialled, but
showed no beneficial improvements in treatment outcomes when compared with rtPA .
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
]. Another meta-analysis of randomized controlled trials and case-control studies
concluded that sonothrombolysis with microbubbles is a safe and effective treatment in ischemic
]. 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 [
Twenty years after the demonstration by Tachibana et al. that STL with microbubbles has a
thrombolytic effect, numerous studies have been performed in Vivo [
]. 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
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.
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 [
]. This systematic review was written in accordance with the PRISMA
]. The protocol was registered on the CAMARADES-NC3Rs Preclinical
Systematic Review & Meta-analysis Facility (SyRF) and is available online (https://drive.google.com/
Sixteen studies met the inclusion criteria (Table 2). The selection schedule used is illustrated in
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
] or MRI . Gao et al. [
] 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. [
] injected 4 mm
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5 studies focused only on STL
4 studies focused on safety and
efficacy of STL
2 studies focused on rtPA-STL
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)
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
long human blood clots through the external carotid artery (ECA) and delivered them into a
pre-ligated common carotid artery (CCA). Moumouh et al. [
] injected 500 μm of autologous
blood clots directly into the CCA. Tomkins et al. [
] 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. [
] introduced 0.6 mm × 0.08 cm autologous clots into the left CCA. Lu
et al. [
] 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. [
] 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.
Thrombolytic drugs co-administered with the microbubbles
Fig 1. PRISMA 2009 flow diagram. Extracting schedule of the studies.
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. [
] used MRI to assess restoration
of blood flow. Ren et al. [
] used US imaging and pulsed wave Doppler to assess
recanalization. Other coagulation parameters used included activated partial thromboplastin time [
activated clotting time [
], prothrombin time [
], fibrinogen [
] and D-dimer .
Alonso et al. and Tomkins et al. [
] examined thrombi in treated arteries using histology.
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Species Clot type
Clotting time before injection (min)
Time between injection of blood clot and the start of treatment (min)
Liu et al. [
] determined changes in S100B astroglial protein levels in blood were a marker of
cerebral damage [
]. Diffusion-weighted sequences and phosphorus spectroscopy were used
by Moumouh et al. [
] to assess and to quantify the extent of cerebral infarct. Nedelmann
et al. [
] 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. [
] 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].
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With regard to brain damage, Brown et al. [
] noted a significant reduction in infarct
brain volume for rtPA-STL when compared to STL or control animals. Similarly, Lu et al. [
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. [
] described a significant reduction in infarct brain volume for STL +
urokinase when compared to urokinase treatment alone (p = 0.025). Nedelmann et al. [
a lower infarct volume with a lower edema volume using rtPA-STL compared to rtPA
treatment alone. In addition, Schleicher et al. [
] reported also a lower infarct volume using
rtPA-STL compared to rtPA alone. Ren et al. [
] reported improved recanalization rates with
- IA TSTL> IV TSTL (declotting time, success
- 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
- 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,
- 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)
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Efficacy of rtPA-STL
Safety and efficacy of STL
Culp et al.
Wang et al.
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. [
] 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. [
] reported better neurological scores for STL when compared
to rtPA alone for the treatment of white microthrombi emboli.
Safety. Flores et al. [
] 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.
] 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.
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.
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. [
] 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 [
], 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
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 [
]. 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 [
]. Furthermore, this response could also lead to a reperfusion injury syndrome
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Sonothrombolysis with microbubbles in ischemicstroke - an assessment of pre-clinicalevidence
N N Y
N N Y
N N N
N N Y
N N Y
N N Y
N N Y
N N Y
N N Y
N N Y
N N Y
inducing oedema and hemorrhages [
]. These phenomenon are presumably absent
immediately after vascular occlusion [
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 [
] 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
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
]. 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 . 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.
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)
]. 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 [
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 [
]. These processes depend on the frequency used, the position of the US
transducer, and the length of the US wave [
]. The TRUMBI trial [
] 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 [
]. 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
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stable cavitation, which enhance rtPA thrombolysis without disrupting the blood brain barrier
], 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 . Wright et al. were able to monitor
cavitation during sonothrombolysis with high-intensity focused ultrasound in a rabbit femoral
]. Frequency modulation is a solution to lower the occurrence of standing waves [
through the use of higher US frequencies and short emission duration [
Associating STL with thrombolytic drugs or with mechanical thrombi retrieval is not well
defined and needs to be fully explored [
]. 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 [
] and, the second one is the use of an operator independent
US device (as reported in the CLOTBUST-HF trial) . 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 [
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 [
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
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
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
S1 PRISMA Checklist. PRISMA checklist of the systematic review following the preferred
reporting items for systematic reviews and meta-analyses. [
S1 File. S1 File contains the full electronic search strategy for the two electronical
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.
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