Microbubble-Mediated Sonothrombolysis Improves

Outcome After Thrombotic Microembolism-Induced

Acute Ischemic Stroke
Yongkang Lu, MD, PhD*; Junfen Wang, MD, PhD*; Ruizhu Huang, MD; Gangbin Chen, MD;
Lintao Zhong, MD; Shuxin Shen, MD, PhD; Chuanxi Zhang, MD; Xinzhong Li, MD;
Shiping Cao, MD, PhD; Wangjun Liao, MD, PhD; Yulin Liao, MD, PhD; Jianping Bin, MD, PhD

Background and PurposeMicrothrombi originating from disintegrated clots or formed in situ may account for the
poor clinical improvement of acute ischemic stroke after recanalization therapy. We attempted to determine whether
microbubble-mediated sonothrombolysis could dissolve platelet-rich and erythrocyte-rich microthrombi, thereby
reducing their brain injury-causing potential.
MethodsPlatelet- and erythrocyte-rich microthrombosis were induced by periadventitial application of 5% ferric chloride
or thrombin to mesenteric microvessels in 75 SpragueDawley rats. Acute ischemic stroke was induced by intracarotid
injection of platelet- or erythrocyte-rich microthrombi in another 50 rats. Rats were randomly divided into control (CON),
ultrasound (US), ultrasound and microbubble (US+MB), recombinant tissue-type plasminogen activator (r-tPA), and
US+MB+r-tPA groups. The post-treatment mesenteric microvessel recanalization rates, cerebral infarct volumes, and
neurological scores were determined.
ResultsThe recanalization rates of platelet- and erythrocyte-rich microthrombi in mesenteric microvessels were higher
(P<0.05), and the cerebral infarct volumes and neurological scores of rats with either microthrombi were lower in the
US+MB group than in the CON group (P<0.01). The infarct volumes and neurological scores were greater in the r-tPA
group than in the US+MB and US+MB+r-tPA groups after treatment of rats with platelet-rich microthrombi (P<0.05).
In contrast, after treatment of rats with erythrocyte-rich microthrombi, the infarct volumes and neurological scores were
similar in the r-tPA and US+MB groups, but smaller in the US+MB+r-tPA group (P<0.05).
ConclusionsMicrobubble-mediated sonothrombolysis improved the outcomes of microthrombi-induced acute ischemic
stroke. Thus, this method may serve as an attractive adjunct to recanalization therapy for acute ischemic stroke.
(Stroke. 2016;47:1344-1353. DOI: 10.1161/STROKEAHA.115.012056.)
Key Words: microbubbles thrombolytic therapy thrombus stroke ultrasonics

A lthough early recanalization of the major occluded

brain arteries has become an essential part of ther-
apy after acute ischemic stroke, a subset of patients fails to
stroke outcomes have not been well established. Despite the
use of the latest thrombectomy devices, microemboli may still
be found in cerebral arterioles downstream from the treated
achieve clinical improvement despite recanalization.1 One vessel.12,13 Intravenous antiplatelet agents may suppress local
important reason for this failure might be microcirculation microthrombus and microembolus formation, but clinical trial
no-reflow, a phenomenon in which major vessel recanaliza- results have not demonstrated improved outcomes. Rather,
tion does not result in adequate microvascular reperfusion.25 they have highlighted an increased risk of intracranial hemor-
Microthrombi, originating from upstream clot fragmentation rhage after the combined use of antiplatelet agents and phar-
or formed in situ, have been shown to play pivotal roles in macological fibrinolysis.1416
the development of the no-reflow phenomenon.68 These Microbubble-mediated sonothrombolysis is a promis-
microthrombi can exhibit a range of histological patterns and ing treatment for cerebral microthrombi and is based on
may be platelet or erythrocyte rich.911 Until now, efficacious ultrasound driven cavitation of microbubbles that accelerate
therapies for reducing cerebral microthrombi and improving thrombolysis via localized mechanical stress on the thrombi.17

Received November 5, 2015; final revision received March 3, 2016; accepted March 4, 2016.
From the State Key Laboratory of Organ Failure Research, Department of Cardiology (Y. Lu, J.W., R.H., G.C., L.Z., S.S., C.Z., X.L., S.C., Y. Liao,
J.B.) and Department of Oncology (W.L.), Nanfang Hospital, Southern Medical University, Guangzhou, China; and Department of Cardiology, the 458th
Hospital of the Chinese Peoples Liberation Army, Guangzhou, China (R.H.).
*Drs Lu and Wang contributed equally.
The online-only Data Supplement is available with this article at http://stroke.ahajournals.org/lookup/suppl/doi:10.1161/STROKEAHA.
115.012056/-/DC1.
Correspondence to Jianping Bin, MD, PhD, State Key Laboratory of Organ Failure Research, Department of Cardiology, Nanfang Hospital, Southern
Medical University, 1838 Guangzhou Avenue North, Guangzhou, 510515, China. E-mail [email protected] or [email protected]
2016 American Heart Association, Inc.
Stroke is available at http://stroke.ahajournals.org DOI: 10.1161/STROKEAHA.115.012056

1344
 Lu et al Sonothrombolysis Reduces Cerebral Microemboli 1345

Multiple animal studies have demonstrated that ultrasound in In Vitro Clot Preparation and Experimental Setup
combination with microbubbles can recanalize acute intravas- Platelet- and erythrocyte-rich thrombi, also known as white and red
cular thrombi in large vessels, including the iliofemoral arter- thrombi, were prepared as previously described27 and formed in the
rubber tubes (diameter, 5 mm; Figure1A). A closed-loop flow system
ies,18,19 arteriovenous grafts,20,21 central venous catheters,22 was filled with phosphate buffered saline (45 mL) and kept at 37C
coronary arteries,23 and intracranial arteries,24,25 underlining (Figure1B). The thrombus was attached to a cotton thread and sus-
the potential of the treatment for dissolving microvascu- pended in the flow system. A peristaltic pump was used to maintain
lar thrombi. However, data on the effects of ultrasound and a flow rate of 5 mL/min through the system (Methods in the online-
only Data Supplement).
microbubble (US+MB) treatment on the thrombolysis of
microthrombi are lacking, and it is unclear whether US+MB
treatment may improve the outcomes of acute ischemic stroke
In Vitro Thrombolysis
White and red thrombi were randomly divided into 5 treatment
induced by cerebral microthrombi. Only one study has evalu- groups: control (CON), ultrasound (US), US+MB, recombinant tis-
ated the efficacy of the treatment for dissolving erythrocyte- sue-type plasminogen activator (r-tPA), and US+MB+r-tPA groups
rich microthrombi in a rat hindlimb model.26 Animal studies (n=5 per group). In the CON group, each thrombus was suspended
have not addressed the utility of US+MB treatment for dis- in the flow system but was left untreated. In the US and US+MB
groups, ultrasound treatment was conducted using a Siemens
solving platelet-rich microthrombi, which account for the Acuson Sequoia with 4V1c sector-array transducer (frequency
largest proportion of thromboemboli in stroke.9 Furthermore, range, 14.5 MHz; diameter in azimuthal direction, 32 mm; eleva-
neither platelet- nor erythrocyte-rich microthrombi have been tion plane, 5 mm) by use of contrast pulse sequencing (Siemens
used to evaluate the stroke outcomes after the treatment. Medical Systems). The transducer was immersed in the water and
positioned at a 35-mm distance to the center of clot. Ultrasound
In this study, we hypothesized that microbubble-mediated insonation was performed at a mechanical index of 1.9 and a fre-
sonothrombolysis could dissolve both platelet- and erythro- quency of 2 MHz, and lasted for 30 minutes. For US+MB treat-
cyte-rich microthrombi, consequently improving the out- ment, lipid-shelled perfluoropropane microbubbles were prepared
comes of microthrombi-induced acute ischemic strokes. To as previously described,28 added into the flow system (0.008 mL/
min), and combined with ultrasound treatment. In the r-tPA group,
verify this hypothesis, we evaluated the lytic efficiency of r-tPA (0.02 mg/mL; Actilyse, Boehringer Ingelheim) was added to
US+MB treatment on both types of thrombi in vitro and in the PBS. In the US+MB+r-tPA group, microbubbles (0.008 mL/min)
rat models of mesenteric microthrombosis. Furthermore, we and half-dose r-tPA (0.01 mg/mL) were added into the flow system
investigated the therapeutic effect of the treatment in rats with and combined with ultrasound treatment. The diagram of in vitro
thrombolysis is shown in Figure1B.
acute ischemic stroke, induced using thrombotic microemboli. After the US+MB treatment, liquid waste from the flow system
was collected and the diameters of residues were measured using a
Materials and Methods Coulter Counter (Multisizer 3, Beckman Coulter).
The animal studies were approved by the Animal Research Committee
of Southern Medical University (Guangzhou, China), and the inves- Mesenteric Microvascular Thrombosis Model
tigation conformed to the Guide for the Care and Use of Laboratory Mesenteric microvascular thrombosis was induced by periadventitial
Animals published by the US National Institutes of Health (NIH application of 5% ferric chloride (platelet-rich) or thrombin (eryth-
Publication No. 8523, revised in 1996). rocyte-rich) to the selected microvessels (diameter, 70100 m) in

A
B
White thrombi
Red thrombi

C White thrombi Red thrombi

30m 10m
10m 30m 10m

Figure 1. In vitro model for thrombolysis and thrombi characteristics. A, Appearance of white and red thrombi formed in rubber tubing. B,
Diagram of experimental setup. C, Representative hematoxylin and eosinstained and scanning electron microscopy images of white and
red thrombi.
1346StrokeMay 2016

SpragueDawley rats (Figure I, Movies I and II, detailed Methods in Magnetic Resonance Imaging and
the online-only Data Supplement). Triphenyltetrazolium Chloride Staining
Cerebral magnetic resonance imaging (MRI) was performed in rats
Cerebral Thrombotic Microembolism Model before and 24 hours after surgery, using a 3.0 T system (3T Trio-
White and red thrombi were prepared as described above and fro- Tim; Siemens) and a locally made radiofrequency coil (online-only
zen in liquid nitrogen, followed by mechanical comminution. The Data Supplement). The brains were then harvested and sectioned into
resultant microthrombi that were able to pass through a 100-m 1.5-mm-thick coronal slices for 2,3,5-triphenyltetrazolium chloride
mesh, but were retained on a 70-m mesh, were resuspended in (TTC; Sigma) staining.29 Macroscopic evidence of cerebral infarc-
normal saline. Acute ischemic stroke was induced by intracarotid tion was compared with MRI findings for the corresponding sections.
injection of platelet- or erythrocyte-rich microthrombi. Briefly, the Images were measured by observers blinded to the treatment para-
right distal external carotid artery was ligated and the right common digm. To eliminate error introduced by brain edema, infarct volumes
carotid artery was temporarily clamped. Subsequently, suspended were calculated using an indirect method.30 The infarct volume was
microthrombi (0.1 mL) were gently injected into the external carotid calculated as the volume of the contralateral hemisphere minus the
artery. Immediately thereafter, the external carotid artery was ligated noninfarcted volume of the ipsilateral hemisphere and expressed as
proximal to the injection site, and the common carotid artery clamp a volume percentage of the infarct compared with the contralateral
hemisphere.
was released.

In Vivo Thrombolysis Neurological Score

Neurological functions, at baseline and 24 hours after embolization,
The animal grouping and ultrasound parameters were the same
were studied by an observer blinded to the treatment paradigm using
as those described for the in vitro thrombolysis. In the mesenteric
a Bederson behavior test.31 The neurological scores ranged from 0
microvascular thrombosis model, after the selected microves-
to 3, with higher scores indicating more serious neurological injury
sel occluded, as evidenced by thrombus formation and blood flow
(Movies IIIVI in the online-only Data Supplement).
decrease, an interval of 5 minutes was required before the treatment
to ensure that the thrombus was stable and unlikely to be washed
away by the bloodstream. During treatment, the ultrasound probe Statistical Analysis
was positioned 4 cm above the thrombosed microvessel, and the dis- Statistical analyses were performed using SPSS 16.0 software.
tance was bridged with a gel-filled spacer. In the cerebral thrombotic Multiple comparisons were performed by 1-way ANOVA with the
microembolism model, after intracarotid injection of microthrombi, Bonferroni multiple comparison test. The Fisher exact test was used
an interval of 5 minutes was required before the treatment to allow to test for significant differences in the post-treatment recanalization
for ligation of external carotid artery and closure of cervical incision. rates of the thrombosed mesenteric microvessels between groups. The
During treatment, the probe was placed 0.5 cm above a dehaired neurological scores of rats were evaluated by using KruskalWallis
area behind the rats eye on the ipsilateral side of the head and was and Nemenyi tests. All tests were 2 sided. The results are expressed
coupled to the scalp with ultrasound gel. The sound beam was aimed as meanSD; P<0.05 was considered significant.
to expose the whole hemisphere of the brain by manually swinging
the probe. The ultrasound treatment lasted for 30 minutes. In the
US+MB group, microbubbles were injected (0.008 mL/min) via a Results
tail vein catheter. In the r-tPA group, r-tPA (10 mg/kg) was admin- In Vitro Thrombolytic Effects of Microbubble-
istered through the tail vein; 10% was administered as a bolus and
the remainder as a 30-minute infusion. In the US+MB+r-tPA group,
Mediated Sonothrombolysis
microbubbles (0.008 mL/min) and half-dose r-tPA (5 mg/kg) were As shown in Figure1A, in vitro thrombi were 0.370.09
administered through different tail veins. cm in diameter and 2.850.47 cm in length. H&E staining
and scanning electron microscopy showed that platelet-rich
B-Mode and Doppler Ultrasonic Imaging thrombi (white thrombi) exhibited a compact fibrinplatelet
Ultrasonic evaluation of in vitro thrombolytic effect was performed mesh pattern, interspersed with limited deposits of nucleated
using a Sequoia ultrasound system with a linear array ultrasound cells and erythrocytes (Figure1C). In contrast, erythrocyte-
transducer (15L8-S; mechanical index, 0.17; frequency, 14 MHz; rich thrombi (red thrombi) were composed of a mass of eryth-
Siemens Medical Systems). The transverse and longitudinal areas of
rocytes trapped in the fibrin network, with nucleated cells
the tubing lumens and the flow velocity, at the points of thrombus
suspension, were measured using B-mode and Doppler ultrasonic dispersed throughout.
imaging, respectively. The total duration of ultrasound exposure for After treatment of the white and red thrombi, the increase
imaging in each group was 3 minutes. in both the transverse and the longitudinal areas of the tubing
lumens, at the points of thrombus suspension, was greater
Histological Examinations in the US+MB group than in the CON group (P<0.05;
To confirm the presence of mesenteric and cerebral microvascu- Figure2A). Similarly, the reduction of flow velocities at the
lar thromboses/embolisms, specimens were paraffin embedded sites of the thrombi and the loss of clot masses were also
and stained with hematoxylin-eosin (H&E), anti-CD41 antibody greater in the US+MB group than in the CON group (P<0.05;
(Abcam) and antifibrinopeptide A antibody (Abcam). Anti-CD41
Figure2B and 2C). After treatment of the white thrombi, the
and antifibrinopeptide A immunohistochemistry indicated the
presence of platelets and fibrin, respectively. To determine the pres- changes in lumen areas, flow velocities, and clot masses were
ence of cerebral infarction and hemorrhage, H&E staining was per- significantly smaller in the r-tPA group than in the US+MB
formed 24 hours after emboli injection. For evaluation of possible group (P<0.05), whereas they were similar in the US+MB
cell or tissue damage, H&E staining was performed immediately and US+MB+r-tPA groups. In contrast, after treatment of
and at 6 hours after US and US+MB treatment (online-only Data the red thrombi, the changes in lumen areas, flow veloci-
Supplement).
For the ultrastructural observations, thrombi were prepared follow- ties, and clot masses were similar in the r-tPA and US+MB
ing a standard procedure, and slides were visualized under a scanning groups, but significantly larger in the US+MB+r-tPA group
electron microscope (HITACHI, S-3000 N). (P<0.05). The changes in lumen areas, flow velocities, and
 Lu et al Sonothrombolysis Reduces Cerebral Microemboli 1347

B C

Figure 2. Effects of microbubble-mediated sonothrombolysis on in vitro thrombi. A, Representative B-mode ultrasonic images of trans-
verse sections of tubing lumens before and after treatment, and quantification of transverse and longitudinal area changes. B and C,
Quantitative analysis of flow velocity changes (B) and clot mass losses (C). D, Coulter particle analysis of the diameter of residues within
the flow system after ultrasound and microbubble (US+MB) treatment. *P<0.05 vs control group, #P<0.05 recombinant tissue-type plas-
minogen activator (r-tPA) vs US+MB group, P<0.05 US+MB+r-tPA vs US+MB group, &P<0.05 US+MB+r-tPA vs r-tPA group, P<0.05
internal group. n=5 per group.

clot masses of the white thrombi were significantly smaller In Vivo Thrombolytic Effects of Microbubble-
than those of the red thrombi in the r-tPA and US+MB+r-tPA Mediated Sonothrombolysis
groups (P<0.05). Intravital microscopy showed that thromboses were success-
Coulter particle analysis showed that the white and red fully induced in mesenteric microvessels (Figure3A). H&E
thrombi debris, after US+MB treatment, was <8 m in diam- staining showed that the white microthrombi in the mesen-
eter (Figure2D), suggesting that they were unlikely to cause teric microvessels contained a fibrinplatelet network, with
distal embolization. a few nucleated cells and erythrocytes dispersed within it
1348StrokeMay 2016

B C

Figure 3. Effects of microbubble (MB) mediated sonothrombolysis on mesenteric microvascular microthrombi. A, Representative intravital
microscopy images of mesenteric microvessels in different conditions. Arrows denote the locations of intravascular microthrombi. B, Rep-
resentative images of white and red microthrombi in mesenteric microvessels as visualized by hematoxylin and eosin (H&E) staining, or
labeled with antibodies against CD41 and fibrinopeptide A (FPA). C, Quantification of the recanalization rates of thrombosed mesenteric
microvessels. *P<0.01, #P<0.05 vs control group. r-tPA indicates recombinant tissue-type plasminogen activator; and US, ultrasound.

(Figure3B). In contrast, the red microthrombi were mainly Stroke Outcomes

composed of clustered erythrocytes. Immunohistochemical Microthrombi prepared for embolization exhibited a homo-
staining showed that both CD41 (platelets) and fibrino- geneous morphology, with diameters of 70 to 100 m
peptide A (fibrin) were expressed in the white and red (Figure4A). Their components and structures were similar
microthrombi. to those of the large in vitro thrombi. Thirty minutes after
In the CON group, the white and red microthrombi grew microemboli injection, the rats were euthanized and histologi-
larger and blood flow slowed within a few minutes of estab- cal evidence of intravascular microthrombi in the brain was
lishing the thrombosis models (Figure3A). After US+MB obtained. H&E and immunohistochemical staining showed
treatment, both microthrombi types dissolved, blood flow was that the cerebral arteriole lumens (diameter, 100 m) were
restored, and occlusion did not recur within 30 minutes. For obstructed by white or red microthrombi, which contained
both types of mesenteric microvessel microthrombi, the recan- platelets (CD41) and fibrin (fibrinopeptide A; Figure4B).
alization rates were higher in the US+MB and US+MB+r- Twenty-four hours after embolization, H&Estained coronal
tPA groups than in the CON group (P<0.05; Figure3C). The sections of the brains showed multiple small infarctions in the
recanalization rate was higher in the r-tPA group than in the form of poorly stained regions involving the cerebral cortex,
CON group after treatment of the red microthrombi (P<0.01), hippocampus, caudate-putamen, and thalamus, predominantly
whereas there was no significant difference between the in the cortex (Figure4C). In vivo ultrasound treatment was
groups after treatment of the white microthrombi. performed as shown in Figure4D.
 Lu et al Sonothrombolysis Reduces Cerebral Microemboli 1349

A White microthrombi Red microthrombi

50m 200m 50m 200m

30m 5m 30m 5m

B
H&E

400m 100m 400m 100m

CD41
FPA

1mm 100m 1mm 100m

Figure 4. Thrombotic microembolism-induced acute ischemic stroke models. A, Representative hematoxylin and eosin (H&E) stained and
scanning electron microscopy images of in vitro white and red microthrombi. B, Representative images of white and red microthrombi in
cerebral arterioles as visualized by H&E staining, or labeled with antibodies against CD41 and fibrinopeptide A (FPA). C, H&E staining of
coronal brain sections 24 h after embolization showing multiple poorly stained small infarcts. D, Diagram of in vivo sonothrombolysis.

MRI showed that the rats used in the experiment did not the cortex and few in the subcortical regions, in consistent
demonstrate any signs of cerebral infarction or hemorrhage with histological findings. The cerebral infarct volumes
before microemboli injection (data not shown). Twenty- were smaller in the US+MB group than in the CON group
four hours after the rats were embolized with the white or (white microthrombi: 9.522.99% versus 26.883.10%
red microthrombi, MRI and TTC-stained sections showed for MRI, 8.462.92% versus 25.683.38% for TTC; red
that the infarcts were multiple with most distributed in microthrombi: 8.642.82% versus 24.603.13% for MRI,
1350StrokeMay 2016

8.022.83% versus 22.983.92% for TTC; all P<0.01; the neurological scores of rats were 2 or 3 in the CON group,
Figures5A and 5B and 6A and 6B). After treatment of rats but were lower in the US+MB group (both P<0.01; Figures5C
with white microthrombi, the infarct volumes were larger in and 6C). After treatment of rats with white microthrombi,
the r-tPA group than in the US+MB group (17.122.98% ver- neurological scores were higher in the r-tPA group than in
sus 9.522.99% for MRI, 16.083.20% versus 8.462.92% the US+MB group (P<0.05), whereas they were similar in the
for TTC; P<0.01), whereas they were similar in the US+MB US+MB and US+MB+r-tPA groups. In contrast, after treat-
and US+MB+r-tPA groups. After treatment of rats with red ment of rats with red microthrombi, there was no significant
microthrombi, the infarct volumes were similar in the r-tPA difference of neurological scores among the r-tPA, US+MB,
and US+MB groups, but smaller in the US+MB+r-tPA group and US+MB+r-tPA groups.
than in the r-tPA group (3.961.43% versus 9.462.94% for
MRI; P<0.05). H&E staining showed no intracranial hemor- Discussion
rhage in rats with microthrombi after US+MB or US+MB+r- In this study, we demonstrated the efficacy of US+MB treat-
tPA treatment (Figure II in the online-only Data Supplement). ment in dissolving platelet- and erythrocyte-rich microthrombi
In addition, there was also no evidence of intraparenchymal in vivo. We found that microbubble-mediated sonothromboly-
or perivascular hemorrhage, as well as degeneration or necro- sis significantly reduced cerebral infarction and improved
sis of cells in the brain and scalp of healthy rats after US neurological deficits in microemboli-induced acute ischemic
or US+MB treatment (Figure III in the online-only Data strokes through dissolution of the microthrombi, includ-
Supplement). ing the platelet-rich ones that are resistant to r-tPAinduced
Before microemboli injection, the neurological scores of all thrombolysis.
rats were 0 according to Bederson behavior test. Twenty-four To investigate the therapeutic effect of US+MB treat-
hours after embolization with the white or red microthrombi, ment for acute ischemic strokes induced by thrombotic

A White microthrombi
Control

B 30
MRI TTC
Cerebral infarction (%)

* *
Ultrasound

20

*
* *
10 *

0
US+MB

C 100 *& *&

3
Neurological score (%)

80 2
1
60
0
40
r-tPA

20

0
US+MB+r-tPA

Figure 5. Effects of microbubble (MB) mediated sonothrombolysis on cerebral infarction and neurological deficits induced by white micro-
thrombi. A, Magnetic resonance imaging (MRI) and 2,3,5-triphenyltetrazolium chloride (TTC) staining of coronal brain sections 24 h after
embolization. B, Quantification of infarct volumes determined using MRI and TTC staining. The infarct volumes were expressed as a vol-
ume percentage of the infarct compared with the contralateral hemispheres (see text for details). C, Neurological scores 24 h after embo-
lization. *P<0.01 vs control group. P<0.01, &P<0.05 vs recombinant tissue-type plasminogen activator (r-tPA) group. n=5 per group. US
indicates ultrasound.
 Lu et al Sonothrombolysis Reduces Cerebral Microemboli 1351

Figure 6. Effects of microbubble (MB) mediated sonothrombolysis on cerebral infarction and neurological deficits induced by red micro-
thrombi. A, Magnetic resonance imaging (MRI) and 2,3,5-triphenyltetrazolium chloride (TTC) staining of coronal brain sections 24 h after
embolization. B, Quantification of infarct volumes determined by MRI and TTC staining. C, Neurological scores 24 h after embolization.
*P<0.01 vs control group. &P<0.05 vs recombinant tissue-type plasminogen activator (r-tPA) group. n=5 per group. US indicates ultrasound.

microemboli, we established a rat stroke model by intracarotid after reopening of occluded cerebral artery, which is usually
injection of either platelet- or erythrocyte-rich microemboli. associated with poor clinic outcomes.32,33 We also found that
This model mimics the clinical situation in which microem- the thrombolytic effect of r-tPA was augmented in combina-
boli originate from a disintegrating thrombus, migrate down- tion with US+MB treatment, whereas the incidence of intra-
stream, and obstruct the distal arterioles. The model possesses cerebral hemorrhage was not increased. Hence, US+MB
several characteristics of clinical cerebral microembolism, treatment could be performed along with r-tPA administration.
including (1) microemboli (diameter, 100 m) found in the Consistent with the brain results, US+MB treatment was
arterioles of the leptomeningeal space or cerebral cortex, simi- demonstrated to dissolve both the platelet- and erythrocyte-
lar to autopsy findings in patients with stroke;11 (2) microem- rich microthrombi in mesenteric microvessels, whereas r-tPA
boli comprising fibrin/platelets, erythrocytes, and nucleated was only efficient in dissolving erythrocyte-rich micro-
cells arranged in a platelet-rich or erythrocyte-rich pattern; (3) thrombi. It should be noted that the penetration of ultrasound
small cerebral infarcts and neurological deficits noted 24 hours waves through the skull was less efficient than in the mes-
after embolization; and (4) multiple microinfarcts with most enteric thrombosis model. However, in this study, ultrasound
distributed in the cortex and few in the subcortical regions, treatment was performed at a frequency of 2 MHz, which is
findings consistent with those of autopsy cases11 and possibly widely used in transcranial Doppler devices for the evaluation
associated with the size, distribution, and hemodynamics of of cerebral hemodynamics. A wealth of clinical data has indi-
cerebral vessels. Our results indicate that US+MB treatment, cated that 2-MHz ultrasound penetrates the skull adequately
which decreased infarct volumes and improved neurological to augment r-tPAinduced thrombolysis and provides suffi-
deficits in rats with either platelet- or erythrocyte-rich micro- cient power for microbubble-mediated sonothrombolysis.3436
emboli, would provide a potential adjunct to recanalization Furthermore, we demonstrated that the diameter, histological
therapy for improving outcomes of acute ischemic stroke, par- components, and patterns of the microthrombi in the mesen-
ticularly in the case that microvascular flow was not restored teric microvessels were similar to those injected into the brain.
1352StrokeMay 2016

These findings, therefore, indicate that dissolution of cere- thrombi are known to be more vulnerable to sonothrombolysis
bral microthrombi by US+MB treatment contributed to the than older ones.43 Thus, the treatment efficiency against older
improved outcomes of microemboli-induced acute ischemic thrombi is uncertain. However, the present results provide
strokes. We noticed that there was a discrepancy in the throm- reference information for clinical acute stroke patients who
bolytic efficacy in vivo and in vitro in each treatment group, are hospitalized within several hours of the ictus. Second, we
which may be attributed to the different size of thrombi used in did not evaluate the progression of intracranial hemorrhage
the experiments. The lack of plasminogen in the in vitro flow beyond 24 hours. The risk of hemorrhage in stroke patients is
system may also account for the discrepancy of r-tPAinduced known to persist for several days or weeks and increase again
clot lysis, but this effect should be minor because r-tPA prefer- during the healing phase.44 Nevertheless, late-onset intracra-
entially activated plasminogen on the fibrin surface. nial hemorrhage is considered to be more related to the degree
Intracranial hemorrhage, one of the most common and dev- of ischemia and the development of collaterals rather than to
astating treatment side effects, was encountered in patients with the treatment methods applied.45,46 Therefore, an evaluation
stroke after treatment with 300-kHz ultrasound and normal doses of the hemorrhagic complications of microbubble-mediated
of r-tPA.37 However, we did not find any evidence of intracranial sonothrombolysis within 24 hours is reasonable.
hemorrhage after US+MB treatment in the present study. This In conclusion, microbubble-mediated sonothrombolysis
discrepancy may be ascribed to the administration of r-tPA and was demonstrated to be capable of dissolving both plate-
the different ultrasound parameters applied. We used a 2-MHz let- and erythrocyte-rich microthrombi, resulting in recana-
ultrasound beam emitted by a diagnostic ultrasound device, lization of occluded cerebral arterioles and, consequently,
which has been demonstrated to not increase bleeding.34,38,39 alleviating brain injury. Therefore, microbubble-mediated
Arterial reocclusion and distal embolization are also potential sonothrombolysis, as an adjunct to recanalization therapy,
treatment side effects. Ultrasound application (6.3 W/cm2) was may offer a new approach to improve outcomes of acute isch-
shown to increase the rate of arterial reocclusion after initial emic stroke.
recanalization due to platelet activation.40 Meanwhile, US+MB
treatment exerts its thrombolytic effects by mechanically disin- Sources of Funding
tegrating clots, the fragments of which may result in downstream This work was supported by grants to Jianping Bin from the
capillary embolization. However, we did not observe post-treat- National Basic Research Program of China (973 Program) (No.
ment mesenteric microvascular reocclusion, possibly because of 2013CB733804), National Natural Science Foundation of China
(No. 81571698, No. 81227801 and No. 81271640), and the Team
the low ultrasound intensity applied in this study. In addition, our Program of Natural Science Foundation of Guangdong Province,
in vitro experiments showed that the post-treatment clot debris China (S2011030003134).
was <8 m in diameter and unlikely to cause distal emboliza-
tion. In this study, we also found that ultrasound insonation at Disclosures
a mechanical index of 1.9 did not cause healthy brain or scalp None.
damage. Given these observations, microbubble-mediated
sonothrombolysis might avoid the adverse effects exerted on an References
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