From the Divisions of Cardiology (Y.B., H.L., T.N., R.J.S.) and Anatomic
Pathology (M.C.F.), Cedars-Sinai Medical Center, Los Angeles, Calif, and the
Section of Cardiology (S.L., D.K., T.R.P.), University of Nebraska Medical
Center, Omaha, Neb.
Correspondence to Robert J. Siegel, MD, Division of Cardiology, Room 5335, 8700 Beverly Blvd, Los Angeles, CA 90048. E-mail siegel{at}csmc.edu
Methods and Results—The objective of this study was to examine
the efficacy of arterial clot disruption by a noninvasive,
nonlytic approach with intravenous administration of
perfluorocarbon-exposed sonicated dextrose albumin (PESDA) and
transcutaneous delivery of ultrasound alone. Pairs of iliofemoral
arteries in 10 rabbits were randomized to receive transcutaneous
ultrasound treatment or no ultrasound treatment after an acute artery
thrombotic occlusion and intravenous PESDA infusion. Five
arteries from 3 additional rabbits served as controls (ultrasound
alone). All 10 iliofemoral arteries treated with PESDA+ultrasound were
recanalized by angiography after ultrasound treatment. None of the 10
contralateral arteries treated with PESDA alone and none of the 5
arteries treated with ultrasound alone were patent after 1 hour.
D-Dimer levels did not change after intravenous
PESDA+ultrasound–mediated reperfusion.
Conclusions—In vivo arterial clot dissolution can be
achieved with intravenous microbubbles and transcutaneous
ultrasound delivery alone. This technique has potential for clinical
application in patients with acute arterial and venous
thrombotic occlusions.
Recently, it has been shown that perfluorocarbon-exposed sonicated
dextrose albumin (PESDA) microbubbles alone enhance clot
disruption with ultrasound in an in vitro model.1 In the
present study, we evaluated a noninvasive approach to recanalize in
vivo thrombosed arteries by using a combination of transcutaneous
ultrasound with intravenous administration of PESDA
microbubbles, avoiding the administration of a fibrinolytic agent.
The method used to induce arterial thrombus has been
previously described.2 Occlusion was induced in both
iliofemoral arteries in a random order.
Experimental Protocol
Intravascular ultrasound imaging (3.5F catheter, 30-MHz transducer;
Boston Scientific Corp and Hewlett-Packard Sonos Intravascular Imaging
System) was performed in one rabbit treated with PESDA to assess (1) if
intravenously administered PESDA microbubbles cross the
lungs and reach the arterial circulation and (2) how long
after injection the microbubbles can be visualized in the
arterial circulation (aorta). Images were recorded at
baseline, before administration of PESDA, every 1 minute for 10 seconds
during the first 15-minute ultrasound delivery, and at the beginning of
the second 15-minute ultrasound delivery.
Plasma D-dimer levels (Diagnostica Stago) were
measured in 5 rabbits. Samples were obtained after thrombus induction,
after ultrasound and PESDA therapy resulted in angiographic
arterial patency, and 1 hour after 1000 units of heparin
was administered intravenously.
Perfluorocarbon-Exposed Sonicated Dextrose Albumin
Ultrasound Device
Pathological Studies
Statistical Analysis
Angiographic Results
After the initial recanalization of the
ultrasound-treated arteries, heparin was administered
intravenously and arterial patency was assessed
by repeated angiography every 15 minutes for a total of 1 hour. All of
the 10 arteries that recanalized after ultrasound therapy remained
patent during this period. However, 3 of the arteries treated with
PESDA without ultrasound recanalized after administration of heparin: 1
artery 15 minutes after heparin administration and 2 arteries 30
minutes after heparin was infused.
In one rabbit in the PESDA group, distal embolization with an occlusion
of a side branch was evident after ultrasound reperfusion. In this
rabbit, the contralateral artery recanalized after heparin
administration; angiography also revealed distal embolization in this
artery.
Intravascular Ultrasound Imaging
Plasma D-Dimer Levels
Histopathology
Since Tachibana and Tachibana6 demonstrated in vitro that
the room air-filled albumin microbubbles (Albunex, Molecular
Biosystems, Inc.) accelerate the clot-dissolving effects of ultrasound
energy, the clinical potential for microbubbles to produce clot
dissolution has been further elucidated. Nishioka et al,5
using a similar rabbit iliofemoral artery model and a 20-kHz ultrasound
transducer, were able to reperfuse (TIMI flow grade II or III) 13 out
of 17 (76%) of the arteries with a combination of transcutaneous
ultrasound and direct injection of dodecafluoropentane (Sonus
Pharmaceuticals, Inc) emulsion into the iliofemoral artery. However,
thermal damage induced by the ultrasound exposure to the overlying skin
and subcu-taneous soft tissues was a major problem. Moreover, in that
experiment, the microbubbles were delivered by a more invasive
intra-arterial injection. In the present study the
microbubbles were administered into a peripheral vein
without the loss of their effectiveness in enhancing clot
disruption.
The mechanism of clot dissolution with ultrasound and PESDA is still
unclear. One possibility is that the microbubbles provide a nucleus for
cavitation. This would lead to microstreaming,1 5 which
could produce a "shearing away" of the thrombus. This possibility
is supported by the observation that D-dimer levels did not
change after ultrasound and PESDA, indicating that
fibrinolysis was not induced. This could also explain
why microbubbles enhance the effect of a thrombolytic agent in the
presence of ultrasound. By shearing away the clot, more fibrin is
exposed to the lytic agent. A direct effect of the dextrose and
albumin solution is unlikely because none of the arteries
treated with PESDA alone recanalized.
This new method of treating arterial thrombosis also
appears to be safe. The ultrasound device used in this study emits a
concentrated ultrasound beam, in a pulsed mode, at 37 kHz, and has a
cooling manifold over the skin surface. There was no
histological evidence of ultrasound-mediated damage to
overlying skin or soft tissues, as was observed in previous
studies.5 6 In this model, we did not examine the
long-term effects of transcutaneous ultrasound on the tissues. Although
pathological changes were not detected by light microscopy,
ultrastructural changes cannot be ruled out. Distal embolization was
evident in only one ultrasound-treated artery. In this rabbit, distal
embolization was also present in the contralateral artery that
reperfused after heparin administration.
Conclusions
Received August 14, 1997;
revision received October 31, 1997;
accepted November 3, 1997.
2.
Steffen W, Fishbein MC, Luo H, Lee D-Y, Nita H,
Cumberland DC, Tabak SW, Carbonne M, Maurer G, Siegel RJ. High
intensity, low frequency catheter-delivered ultrasound dissolution of
occlusive coronary artery thrombi: an in vitro and in vivo
study. J Am Coll Cardiol.. 1994;24:1571-1579.[Abstract]
3.
Porter TR, Xie F, Kilzer K.
Intravenous perfluoropropane-exposed sonicated
dextrose albumin produces myocardial contrast which correlates
with coronary blood flow. J Am Soc
Echocardiogr.. 1995;8:710-718.[Medline]
[Order article via Infotrieve]
4.
Luo H, Nishioka T, Fishbein MC, Cercek B, Forrester
JS, Kim CJ, Berglund H, Siegel RJ. Transcutaneous ultrasound
augments lysis of arterial thrombi in vivo.
Circulation.. 1996;94:775-778.
5.
Nishioka T, Luo H, Fishbein MC, Cercek B, Forrester
JS, Kim CJ, Berglund H, Siegel RJ. Dissolution of thrombotic
arterial occlusion by high intensity low frequency
ultrasound and dodecafluoropentane emulsion: in vitro and in vivo
study. J Am Coll Cardiol.. 1997;30:561-568.[Abstract]
6.
Tachibana K, Tachibana S. Albumin
microbubble echo-contrast material as an enhancer for ultrasound
accelerated thrombolysis.
Circulation.. 1995;92:1148-1150.
© 1998 American Heart Association, Inc.
Brief Rapid Communications
Noninvasive In Vivo Clot Dissolution Without a Thrombolytic Drug
Recanalization of Thrombosed Iliofemoral Arteries by Transcutaneous Ultrasound Combined With Intravenous Infusion of Microbubbles
Abstract
Top
Abstract
Introduction
Methods
Results
Discussion
References
Background—Previous in vivo studies have shown that
microbubbles not only enhance the effectiveness of
thrombolytic agents in the presence of ultrasound but
may also augment clot dissolution without thrombolytic
drugs.
Key Words: thrombosis • ultrasonics • occlusion
Introduction
Top
Abstract
Introduction
Methods
Results
Discussion
References
Although
thrombolytic therapy is effective, there is a need for
simpler, safer, more inexpensive, and noninvasive methods for rapid and
effective recanalization of thrombosed arteries
that can be initiated immediately upon arrival to the hospital or even
en route.
Methods
Top
Abstract
Introduction
Methods
Results
Discussion
References
Animal Preparation
Thirteen adult New Zealand White rabbits weighing 3.2 to 5.3 kg
were anesthetized and maintained with ketamine (20
mg/kg) and xylazine (3.0 mg/kg) administrated
intravenously. The right carotid artery was canulated with
a 5F arterial sheath; a 3.5F coronary Tracker
catheter over a coronary guidewire was
inserted through the carotid arterial sheath to the
iliofemoral artery. Selective angiography was performed at baseline,
using manual injections of 1 to 2 mL of Omnipaque (Sanofi Winthrop
Pharmaceuticals).
After angiographic documentation of bilateral thrombotic
occlusions of the iliofemoral arteries in 10 rabbits, PESDA was
administered intravenously and on one side the artery was
randomized to ultrasound therapy. The contralateral artery served as a
control (exposed to PESDA without ultrasound). Transcutaneous
ultrasound alone (a control without administration of PESDA) was
applied to 3 rabbits with 5 iliofemoral artery thrombotic occlusions.
After 15 minutes of unilateral transcutaneous ultrasound exposure,
angiography was repeated for documentation of patency of each
iliofemoral artery. Each angiogram was done after
intraarterial injection of 100 µg of
nitroglycerin to exclude arterial spasm. If
the ultrasound-treated artery was still occluded, an additional 15
minutes of transcutaneous ultrasound was repeated (up to a total of 4
periods of 15 minutes). After recanalization was
documented, heparin (1000 U) was administered
intravenously, and the rabbit was followed with repeated
bilateral angiography every 15 minutes up to 60 minutes to assess
patency of each artery. In the transcutaneous ultrasound control group
(no PESDA), after 60 minutes of ultrasound delivery to the first
artery, the contralateral artery was exposed to ultrasound in the same
fashion. Each angiogram was analyzed by the consensus of four
reviewers for the presence or absence of occlusion, vessel spasm, and
distal or side branch embolization.
The preparation of PESDA microbubbles was described
previously.1 3 One milliliter of PESDA was infused slowly
over 5 minutes through the ear vein catheter at the beginning of the
first 15 minutes of ultrasound delivery.
An ultrasonic generator (Cybersonics) was used that
operates at 
37 kHz and uses both pulse and sweep frequencies, 91 Hz
and ±1 kHz, respectively. The power can range to 160 W and the peak
negative pressure is 103 kPa (1.03 bAr). The ultrasound transducer was
applied transcutaneously over the arterial occlusion site,
which was marked on the skin with a metallic marker that was positioned
at the time of angiography.
At the end of each experiment, rabbits were euthanized by an
intravenous injection of KCl. The iliofemoral arteries,
ultrasound-exposed skin, and soft tissues in all rabbits were excised,
examined grossly, and then fixed in 10% neutral buffered formalin for
24 to 72 hours. The iliofemoral arteries were then cut transversely
every 2 mm for the length of the vessel and processed as
previously described.4 5 The arteries were examined in
random order, with the examiner blinded to whether the artery was
treated with ultrasound.
Data are given as mean value±SD. The 
2 test was
used to compare the angiographic patency rate among the different
treatment groups. A value of P
.05 was considered
statistically significant.
Results
Top
Abstract
Introduction
Methods
Results
Discussion
References
Thirteen rabbits were included in the study, 10 in the
intravenous PESDA+unilateral ultrasound exposure group and
3 in the ultrasound-alone control group.
All 10 arteries treated with ultrasound and PESDA reperfused by
angiography within 60 minutes of ultrasound delivery, 3 of them (30%)
were recanalized after one 15-minute treatment period, 2 more arteries
(20%) reperfused after the second 15-minute of (30 minutes) ultrasound
therapy, and an additional 2 after the third 15-minute (45 minutes)
ultrasound therapy period. The remaining 3 arteries recanalized after a
cumulative time period of 60 minutes. In contrast, none of the
contralateral arteries exposed to PESDA without ultrasound recanalized
during the same period (P=.00006). None of the 5 control
arteries that were treated with 60 minutes of transcutaneous ultrasound
without PESDA reperfused (P<.000004 for the difference
among groups). Fig 1
shows an example of
an angiogram of rabbit iliofemoral artery treated with PESDA and
ultrasound after induction of a thrombotic occlusion (a) and after
recanalization (c).
View larger version (172K):
[in a new window]
Figure 1. Case example of repeated angiography of the both
iliofemoral arteries. a and b, Bilateral thrombotic occlusions of the
arteries; c, artery treated with intravenous
perfluorocarbon-exposed sonicated dextrose albumin (PESDA) and
transcutaneous ultrasound (recanalized after 30 minutes of therapy),
which remained patent 60 minutes after intravenous heparin;
and d, control side, treated with PESDA alone, remained
occluded.
After intravenous injection of PESDA microbubbles,
there was opacification of the aorta for >10 minutes, confirming that
the PESDA crossed the lungs, recirculated in the blood, and was
present at the site of thrombotic occlusion.
After induction of bilateral thrombotic iliofemoral
arterial occlusions, the mean D-dimer level was
5500±3535 ng/mL; after combined transcutaneous ultrasound and PESDA
induction of unilateral arterial patency, the mean
D-dimer level was 2650±3008 ng/mL, and 1 hour after a
bolus of 1000 units of heparin and monitoring to confirm
arterial patency, the mean D-dimer level fell
to 800±410 ng/mL.
Fig 2A demonstrates a patent
iliofemoral artery after exposure to ultrasound+PESDA.
Histological evaluation revealed that 9 of 10 arteries
treated with ultrasound+PESDA were patent; these 9 arteries had minimal
residual thrombus (25% cross-sectional area obstruction by
thrombus). However, 1 ultrasound+PESDA-treated artery was occluded by
thrombus. As shown in the Table, this
artery only had 15 minutes of exposure to ultrasound, whereas all the
other arteries exposed to ultrasound+PESDA had 25% of the
arterial cross-sectional area occupied by residual
thrombus. Three arteries exposed to PESDA alone that recanalized by
angiography after heparin administration were patent by microscopy. The
other 7 arteries exposed to PESDA alone remained occluded. Fig 2B
demonstrates a thrombosed iliofemoral artery that had been exposed to
PESDA alone, and it failed to recanalize. Micro-scopic areas of
necrosis were found in the arterial wall in all vessels.
The magnitude of vessel injury was the same in the ultrasound-treated
arteries as in the control arteries. There was no evidence of damage or
inflammation in the skin or soft tissues overlying the
ultrasound-treated iliofemoral arteries.
View larger version (119K):
[in a new window]
Figure 2. Microscopic findings. A, Patent iliofemoral
artery after exposure to ultrasound plus perfluorocarbon-exposed
sonicated dextrose albumin (PESDA); B, artery exposed to PESDA
alone, which failed to recanalize.
View this table:
[in a new window]
Table 1. Pathological Findings
Discussion
Top
Abstract
Introduction
Methods
Results
Discussion
References
In this rabbit study of a thrombotically occluded iliofemoral
artery, all arteries treated with the combination of PESDA and
ultrasound recanalized angiographically within 60 minutes of therapy.
Ultrasound and PESDA were both necessary, since none of the arteries
exposed to ultrasound alone or PESDA alone recanalized within this
period. Furthermore, ultrasound-induced thrombus disruption was not
associated with reocclusion (as documented by repeated angiography)
after recanalization and did not result in an
elevation of D-dimer levels, and therefore was not
associated with activation of the fibrinolytic system.
This is the first study to demonstrate in vivo a noninvasive,
nonlytic approach for clot dissolution with the use of
intravenous microbubbles and transcutaneous ultrasound. Our
findings show that transcutaneous ultrasound combined with PESDA is an
efficient, rapid, and safe method for clot dissolution. We conclude
that transcutaneous ultrasound in combination with PESDA could be an
alternative to systemic lytic therapy in patients with acute
arterial and venous thrombotic occlusions.
Acknowledgments
This work was supported in part by the Lee E. Siegel MD Memorial
Fund, the Save a Heart Foundation, and the Western Cardiac
Foundation.
References
Top
Abstract
Introduction
Methods
Results
Discussion
References
1.
Porter TR, LeVeen RF, Fox R, Kricsfeld A, Xie F.
Thrombolytic enhancement with
perfluorocarbon-exposed sonicated dextrose albumin
microbubbles. Am Heart J.. 1996;132:964-968.[Medline]
[Order article via Infotrieve]
This article has been cited by other articles:
 |
|
 |
|
  T. R. Porter The utilization of ultrasound and microbubbles for therapy in acute coronary syndromes Cardiovasc Res, July 9, 2009; (2009) cvp206v2. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Xie, J. Lof, T. Matsunaga, R. Zutshi, and T. R. Porter Diagnostic Ultrasound Combined With Glycoprotein IIb/IIIa-Targeted Microbubbles Improves Microvascular Recovery After Acute Coronary Thrombotic Occlusions Circulation, March 17, 2009; 119(10): 1378 - 1385. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. B. Walton and R. V. Shohet Tiny Bubbles and Endocytosis? Circ. Res., March 13, 2009; 104(5): 563 - 565. [Full Text] [PDF]
|
|
|
|
 |
|
 A. Alonso, A. Della Martina, M. Stroick, M. Fatar, M. Griebe, S. Pochon, M. Schneider, M. Hennerici, E. Allemann, and S. Meairs Molecular Imaging of Human Thrombus With Novel Abciximab Immunobubbles and Ultrasound Stroke, May 1, 2007; 38(5): 1508 - 1514. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. C. Culp, T. R. Porter, J. Lowery, F. Xie, P. K. Roberson, and L. Marky Intracranial Clot Lysis With Intravenous Microbubbles and Transcranial Ultrasound in Swine Stroke, October 1, 2004; 35(10): 2407 - 2411. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P.A Dijkmans, L.J.M Juffermans, R.J.P Musters, A van Wamel, F.J ten Cate, W van Gilst, C.A Visser, N de Jong, and O Kamp Microbubbles and ultrasound: from diagnosis to therapy Eur J Echocardiogr, August 1, 2004; 5(4): 245 - 246. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. W Francis and V. N Suchkova Ultrasound and thrombolysis Vascular Medicine, August 1, 2001; 6(3): 181 - 187. [Abstract] [PDF]
|
|
|
|
|
|
U. Rosenschein, V. Furman, E. Kerner, I. Fabian, J. Bernheim, and Y. Eshel Ultrasound Imaging-Guided Noninvasive Ultrasound Thrombolysis : Preclinical Results Circulation, July 11, 2000; 102(2): 238 - 245. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. N. Suchkova, R. B. Baggs, and C. W. Francis Effect of 40-kHz Ultrasound on Acute Thrombotic Ischemia in a Rabbit Femoral Artery Thrombosis Model : Enhancement of Thrombolysis and Improvement in Capillary Muscle Perfusion Circulation, May 16, 2000; 101(19): 2296 - 2301. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. J. Siegel, S. Atar, M. C. Fishbein, A. V. Brasch, T. M. Peterson, T. Nagai, D. Pal, T. Nishioka, J.-S. Chae, Y. Birnbaum, et al. Noninvasive, Transthoracic, Low-Frequency Ultrasound Augments Thrombolysis in a Canine Model of Acute Myocardial Infarction Circulation, May 2, 2000; 101(17): 2026 - 2029. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. A. Strickberger, T. Tokano, J.-U. A. Kluiwstra, F. Morady, and C. Cain Extracardiac Ablation of the Canine Atrioventricular Junction by Use of High-Intensity Focused Ultrasound Circulation, July 13, 1999; 100(2): 203 - 208. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Suchkova, F. N. Siddiqi, E. L. Carstensen, D. Dalecki, S. Child, and C. W. Francis Enhancement of Fibrinolysis With 40-kHz Ultrasound Circulation, September 8, 1998; 98(10): 1030 - 1035. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 ON THE HORIZON: CLOT DISSOLUTION WITHOUT THROMBOLYTICS Journal Watch (General), January 30, 1998; 1998(130): 7 - 7. [Full Text]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||