Engineering, 2013, 5, 352-356 Published Online Octob er 2013 (
Copyright © 2013 SciRes. ENG
Sonothromblysis in Combination w ith Thrombolytic Drugs
in a Rabbit Model Using MRI-Guidance
Christakis Damianou1, Nicos Mylonas2, Kleanthis Ioannides3
1Cyprus University of Technology, Cyprus
2Frederick University Cyprus, Limassol, Cyprus
3Polikliniki Ygia, Limassol, Cyprus
Received June 2013
The potential of MRI-guided focused ultrasound (MRgFUS) combined with the thrombolytic drug recombinant tissue
plas mino gen a ctiva to r (r t-PA), to d issolve clots in the carotid of a New Zealand rab bit in vivo is evaluated. A spherical-
ly-focused tr a nsd uce r s of 5 cm diameter; focusing at 10 cm and operating at 1 MHz was used. A pulsed ultrasound pro-
tocol was used that maintains a tissue temperature increase of less than 1˚C in the clot (called safe temperature).
MRgFUS has t he potentials to dissolve clots t hat are injected in the carotid of rabbits in vivo. It was found that the ti me
needed for opening the carotid artery using ultrasound and rt-PA was decreased compared to just using rt-P A. The pro-
posed protocol was monitored using Magnetic Resonance Angiography (MRA) ever y 1 min.
Keywords: Ultrasound; MRI; Stroke; Thrombolysis
1. Introduction
The feasibility of ultrasound to enhance thrombolysis
was reported in the mid 70s [1,2]. In the foll owin g ye ars,
several in vitro studies (Kimura et al. 1994 [3] and
Spengos K et al. 2000 [4]) have confirmed the above
results. In the former studies, the range of intensity va-
ried from 0.2 - 2.0 W/cm2 (spatial peak temporal average
intensity) and the freque ncy from 20 kHz to 2 MHz using
unfocused ultrasound.
It is speculated that ultrasound accelerates enzymatic
fibrinolysis primarily through mechanical mechanisms,
by enhancing the effectiveness of thrombolytic drugs,
possibly by exposing more binding sites on the fibrin to
the r ecombi nant ti ssue p lasmi nogen a ctivato r (r t-PA) [5].
Other mechanical effects of ultrasound, such as cavita-
tion and radiation force, are possibly influencing drug
transport [6,7]. Acoustic cavitation plays a very signifi-
cant r ole in ul trasound -accelerated fibrinolysis [6]. Other
theories revolve around the fact that ultrasound promotes
motion of fluid around the clot, an effect called micro-
streaming [7].
Birnbaum et al. in 1998 [8], reported that in vivo ar-
terial clot dissolution can be achieved with intravenous
microbubbles and transcutaneous ultrasound delivery
alone. Moreover, a study has shown the effectiveness of
transcutaneous ultrasound and intravascular microbub-
bles in lysing intracrania l c lot in pigs [9].
Administration of gaseous microspheres dramatically
lowers the threshold for cavitation and increases the lytic
activity of ultrasound (Holland CK and Aplef RE
1990[10]). Because the bubbles are destroyed in the
process, they must be constantly injected for complete
clot dissolution. Recent studies have demonstrated that
microbubbles can be concentrated at the surface of clot
by attaching a glycoprotein receptor antagonist, which
increases their adherence to acute clot, to the bubbles
The use of Focused ultrasound for destroying clots has
received attention lately (Frenkel V et al. 2006 [12];
Shawa G et al. 2009 [13]; Hölscher T et al. 2011[14] and
Laing S et al. 20 11[ 15]). In the stud y b y Frenke l V et al.
2006 [12], clots from humans in vitro were ablated using
pulse ultrasound with a 1 MHz single element transducer
in syne rg y with r t -PA. In t hi s stud y the c oncl us ion is t ha t
by using rt-PA and focused ultrasound, improved clot
destruction rates were achieved. In another study, using
unfocused ultrasound at 120 KHz [12], showed that ul-
trasound has the potential of destroying clots in vitro in
combination with Liposomes loaded with rt-PA. Human
whole blood clots were ablated though the skull using a
hemispheric phased array transducer with 1,000 single
piezo elements [13] using pulse ultrasound. The conclu-
sion of this study is that ultrasound alone can be utilized
to destro y clots.
In addition to these in vitro studies, there are some in-
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teresting in vivo studies confirming the therapeutic effect
of pulse ultrasound (Hölscher T. et al. 2011 [14] and
Laing S et al. 2011 [15]). In one study (Laing S et al.
2011 [15]), a rabbit aorta model was used in order to
ablate using pulse ultrasound in combination with Lipo-
somes loaded with rt-PA. In another study (Hölscher T.
et al. 2011 [14]), clots that were formed in the rabbit
marginal ear vein were ablated using pulse ultrasound
with a 1 MHz single element transducer in synergy with
recombinant tissue plasminog e n activator (rt-PA).
Currently there are few but sig ni ficant c linical trials: a)
The CLOTBUST (Combined Lysis of Thrombus in Brain
ischemia using transcranial Ultrasound and Systemic
Recombinant Tissue-Type Plasminogen Activator (rt-
PA)) is a Phase II randomized multi-center international
clinical trial [16]; b) The EKOS clinical trial [17] which
involves the inser tion of a catheter is now being tested in
phase II-III Interventional Management of Stroke (IMS)
trials; c) The TRUMBI clinical trial (Daffertshofer M et
al. 2005 [18]), using transcranial low-frequency ultra-
sound-mediated thrombolysis in brain ischemia in com-
bination with intravenous administration of tPA; and d)
The TUCSON clinical trial (Barreto A et al. 2009 [19]),
a phase I-II randomized placebo-controlled, international
multi-center study, using perfultr en-lipid microsp heres.
In this paper, the therapeutic effect of focused ultra-
sound and administration of rt-PA to dissolve artificial
clot that was injected in the carotid artery of a New
Zealand rabbit was examined. In the current study, high-
er intensities were used than what was proposed in a
study by Alexandrov AV et al. in 2004 [20]. We antic-
ipate that by using higher intensities, the rate of clot de-
struction will be accelerated. The previous studies mostly
make use of unfocused ultrasound (Holland CK et al.
2008 [21]; S aguchi T et al. 200 8 [22 ] and Jür gen E et al.
2008 [23]). Thus, the intensity levels that could be used
were limited. I n this stud y, focused ultrasound is investi-
gated and therefore higher intensities are utilized pro-
vided that thermal effects are avoided. Focused ultra-
sound targeting the clot is used with spatial average
temporal average (SATA) in situ intensity of 20 W/cm2.
2. Materials and Methods
2.1. HIFU /MRI System
Figure 1 shows the block diagram of the MRgFUS which
includes the following subsystems: 1) Focused ultra-
sound system, 2) MRI system, 3) Transducer holder and
4) Temperature measurement. Since thi s system e ventually
will be utilized in conjunction with MRI the transducer
and transducer holder are both designed to be MRI com-
patible. We have chosen to use MRI, because regarding
vascular imaging is considered the gold standard.
RF amplifiers
Signal generator
Figure 1 . MRgFUS system for in vitro sonothrombolysis.
2.2. Focused Ultrasound System—MR Imaging
The ultra sound syste m co nsi sts o f a ra dio fr eque ncy (RF )
generator/amplifier (1000 W, JJ&A Instruments, Duvall,
WA, USA), and a spherically shaped transducer made
from piezoelectric ceramic of low magnetic susceptibility
(Piezotechnologies, Etalon, Lebanon, IN, USA). The
transducer used operates at 1 MHz. The transducer has a
focal length of 10 cm and a diameter of 5 cm. The trans-
ducer is coupled to the artery using a special designed
plastic holder (MEDSONIC, Limassol, Cyprus). The
transducer and transducer holder were placed inside an
MRI scanner (Signa 1.5 T, by General Electric, Fairfield,
2.3. Production of Sample Clots
Blood clots were obtained by natural coagulation of ani-
mal blood samples from healthy cows. The animal expe-
riments protocol was approved by the national body in
Cyprus responsible for animal studies (Ministry of Agri-
culture, Animal Services). Blood was drawn into small
containers and placed in a 37˚C water bath for 3 h and
then stored in a refrigerator at a temperature of 5˚C for at
least 72 h before use in the experiments to allow com-
plete clot r e traction (Holland CK et al. 2008 [21]).
2.4. Preparation of rt-PA
The rt-PA was obtained as a lyophilized powder (rt-PA,
Actilase, Genentech, San Francisco, CA, USA) mixed
with sterile water as per manufacturer’s instructions. A
dose of 1 mg/mL was administered.
2.5. MRI Parameters
For T2-W FSE the following parameters were used: TR
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= 2500 ms, echo time (TE) was variable from 10 ms to
160 ms, slice thickness = 3 mm (gap 0.3 mm), matrix =
256 × 256, FOV = 16 cm, NEX = 1, and ETL = 8. For
the MRI sequence of MRA the following parameters
were used: rep etition time (TR) = 40 ms, echo time (TE)
= 2.7 ms, Field of View (FOV) = 16 cm, matr ix = 256 ×
256, flip angle = 10˚, Ban dwidth = 15.6 KHz, Number of
excitations (NEX) = 1. A spinal coil (USA instruments,
Cleveland, OH, USA) was used to acquire the MRI sig-
2.6. In Vi v o Experiments
For the in vivo experiments, 16 New Zealand adult rab-
bits were used weighting approximately 3.5 - 4 kg. The
rabbits were anaesthetized using a mixture of 500 mg of
ketamine (100 mg/mL, Aveco, Ford Dodge, IA), 160 mg
of xylazine (20 mg/mL, Loyd Laboratories, Shenandoah,
IA), and 20 mg of acepromazine (10 mg/mL, Aveco,
Ford Dodge, IA) at a dose of 1 mL/ kg. T he ani mal exp e-
riments protocol was approved by the national body in
Cyprus responsible for animal studies (Ministry of Agri-
culture, Animal Services).
For the in vivo experi ments, the clot was injected in
the carotid artery using a thin needle. Once the blockage
of the artery was confirmed, ultrasound and rt-PA thera-
py was applied. A1 mg/ml/kg rt-PA was injected in the
jugular vein. Prior to the application of ultrasound a bo-
lus of an ultrasound contrast agent (SonoVue; Bracco
SpA, Milan, Ita ly) was injected intravenously through the
ear vein at a dose of 0.02 mL/kg.
3. Resul ts
Figure 2(a) shows the coupling of the transducer to the
carotid artery and Figure 2(b) shows the MRI image
using T2-W of the coupling of the ultrasonic transducer
to the carotid artery of the rabbit. The intense signal in-
dicates the water of the transducer holder which makes
an excellent coupling to the ra bbit.
Figure 4(a) shows the carotid artery using MRA be-
fore the i nj ec ti on of t he t hr ombus. Figure 4(b) shows t he
MRA image of the carotid artery immediately after the
injection of the thrombus. Figure 4(c) shows the MRA
image of the carotid artery after app lying Ultrasound (f =
1 M Hz, SAT A intensity = 20 W/cm2, Dut y factor = 10%,
pulse repetitio n rate = 10 Hz,) and rt-PA for 70 mins (the
artery is completely opened).
4. Discussion
The results in this study demonstrate the ability of
MRgFUS in combination with rt-PA to dissolve clots in
an in vivo model. We have proved the capability of the
clot model ( thrombus) to blo c k the car otid artery and also
Figure 2. (a). Transducer coupling to the artery (b) MR
image using T2-W FSE for the coupling of the ultrasonic
transducer to the neck of th e r abbit.
Figure 3. Show s the coupli ng of the transducer to the car o-
tid artery .
(a) (b) (c)
Figure 4. (a) M RA image of carotid art ery before the injec-
tion of the thrombus; (b) MRA i mage of carotid artery im-
mediately after the injection of the thrombus; (c) MRA
image of the carotid artery after applying Ultrasound and
rt-PA for 70 min.
proved the ability of therapeutic ultrasound in synergy
with rt-PA to dissolve clots and at the same time moni-
tor ing the whole p ro cess usi ng M R A. We have cho se n to
use the carotid artery because most of the arteries in such
a small animal such us the rabbit cannot be visualized
with MRI due to spatial resolution problems (for exam-
ple ear, femoral, middle cerebral artery etc).
The results of this study clearly show that focused ul-
trasound has the potential to accelerate the treatment of
vascular occlusions (potential application could be
ischemic stro ke) b y reducing the treat ment time whic h is
crucial for future clinical treatments (ischemic stroke for
Copyright © 2013 SciRes. ENG
example). By now, the administration of rt-PA is an ef-
fective treatment for acute ischemic stroke, but it i s as so-
ciated with an increased risk of intracranial haemorrhage.
Therefore, a potential benefit from the application of
focused ultrasound is to use lower rt-PA dose without
affecting the functionality of the treatment, and ensuring
no haemorrhage is cause d due to rt-PA.
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