Sonothromblysis in Combination with Thrombolytic Drugs in a Rabbit Model Using MRI-Guidance

doi:10.4236/eng.2013.510B071

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Engineering, 2013, 5, 352-356

http://dx.doi.org/10.4236/eng.2013.510B071 Published Online Octob er 2013 (http://www.scirp.org/journal/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

Email: [email protected], [email protected]

Received June 2013

ABSTRACT

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

[11].

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-

C. DAMIANOU ET AL.

353

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.

MRI

compatible

Holder

Transducer

RF amplifiers

Signal generator

Temperature

reader

MRI

Rabbit

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,

CT, USA).

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

C. DAMIANOU ET AL.

354

= 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-

nal.

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

TRANSDUCE

CAROTID

C. DAMIANOU ET AL.

355

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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