(Circulation. 1995;92:1148-1150.)
© 1995 American Heart Association, Inc.
Articles |
Albumin Microbubble Echo-Contrast Material as an Enhancer for Ultrasound Accelerated Thrombolysis
From the First Department of Internal Medicine, Fukuoka University School of Medicine (K.T.) and the Department of Advanced Drug Delivery Systems, Wakasugi Medical Research Institute and Hospital (S.T.), Fukuoka, Japan.
Correspondence to Katsuro Tachibana, MD, First Department of Internal Medicine, Fukuoka University School of Medicine, 7-45-1 Nanakuma, Fukuoka 814-80, Japan.
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Abstract |
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 Top Abstract IntroductionMethods Results Discussion References |
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Background Recent findings suggest that acoustic cavitation is responsible for acceleration of thrombolysis by ultrasound (US) energy. It is known that albumin microbubbles lower the threshold of acoustic cavitation production.
Methods and Results The present study was designed to determine whether the presence of albumin microbubbles used for echo-contrast material (Albunex) can further accelerate fibrinolysis by US. Artificial thrombus was produced by Chandler's loop method with blood extracted from a healthy subject. Urokinase (UK, 1200 IU/mL) was added to the artificial thrombi placed in test tubes. Each thrombus was exposed to US (170 kHz) at a distance of 1.2 cm for a total of 60 seconds at an intensity of 0.5 W/cm2 at intervals of 2 seconds on and 4 seconds off. Echo-contrast material (0.6x106 microspheres per mL) or 5% albumin (for control) was circulated near the thrombus at a rate of 1 mL/min during the US exposure. Fibrinolysis was later determined by percentage of weight loss of thrombus after 60 minutes of incubation (n=15). Fibrinolysis with UK alone was 26.6±4.8%. Fibrinolysis with UK+US treatment was 33.3±5.8%. Further increase of fibrinolysis to 51.3±7.7% occurred in the presence of Albunex (UK+US+Albunex). Statistical differences were obtained between all these groups (ANOVA).
Conclusions The presence of the echo-contrast agent induced further acceleration of thrombolysis by US energy. It is suggested that this diagnostic echo-contrast material can be used as an alternative therapeutic US drug enhancer for thrombolysis.
Key Words: contrast media • pharmacokinetics • thrombolysis • ultrasonics • UK
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Introduction |
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TopAbstractIntroductionMethods Results Discussion References |
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A number of researchers have recently reported acceleration of fibrinolysis by ultrasound (US) energy in combination with fibrinolytic agents.1 2 3 4 5 6 7 Although US intensity, frequency, and methods of exposure have varied among the experiments, the efficiencies of fibrinolytic agents were clearly increased by nonthermal US energy. It was hypothesized that acoustic cavitation played a major role in increasing the bioavailability of fibrinolytic agents at the surface of the thrombus.8 Cavitation, which can be defined as the formation and collapse of bubbles in liquids, can generate high-velocity jets or a steady flow of fluid known as microstreaming. In addition, countless microscopic bubbles oscillate in size near the thrombus during US exposure, which may increase penetration of fibrinolytic agents into the thrombus and thus accelerate fibrinolysis. Holland and Apfel9 recently reported that microscopic albumin bubbles used for diagnostic echo enhancement can lower acoustic cavitation production thresholds to as low as one third the energy. This phenomenon opens new possibilities for applying this material for therapeutic means to induce cavitation with a lower US energy level.
The object of the present study was to determine whether the presence of albumin microbubbles used for diagnostic echo contrast can increase acceleration of fibrinolysis in vitro during therapeutic exposure of thrombus to US with urokinase (UK).
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Methods |
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TopAbstractIntroductionMethods Results Discussion References |
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Artificial thrombi were produced by the Chandler loop method described elsewhere.1 10 Briefly, after approval from the institutional ethical committee, citrated whole venous blood was extracted from a healthy 29-year-old male volunteer. The experiment protocol was carried out on three separate occasions, each at least 3 weeks apart. All experiments were performed on the day of blood collection. Blood (1 mL) was pipetted into a silicone tube (diameter, 3 mm; length, 265 mm); 0.1 mL of 0.25 mol/L CaCl2 was added before the tube was closed into a circle with a plastic collar. These loops were then rotated at 12 rpm for 20 minutes at 37°C to form artificial clots. Each thrombus and the whole blood within the tube were then transferred to a Pyrex test tube (diameter, 1.4 cm; length, 10 cm). A layer of 2% agar was made beforehand at the bottom of all test tubes to prevent direct contact of the thrombus with the Pyrex wall (Fig 1
).
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A US piezoceramic element (5x5x1 mm) was inserted horizontally
into
the test tube containing the artificial thrombus. The distance from the
US-emitting element and the thrombus was 12 mm. The US-emitting element
was connected to a power amplifier (Electronic Navigation Industries)
and a US audio generator (Wandel and Goltermann). Driving signals were
monitored by an oscilloscope (type 502A dual beam, Tektronix Inc). The
test tube and the US-emitting element were positioned by a specially
modified holder originally used for neurostereotaxic
surgery (Mechanical Developments Co). The location of the test tube and
the US catheter were independently specified at an accuracy of 0.1 mm.
An inlet for UK (Green Cross Co) and/or the albumin microbubble
(Albunex, ABX; Molecular Biosystems Inc) solution circulation system
was placed near the bottom of the test tube. An outlet was fixed near
the surface of the solution. UK and/or ABX solution was continuously
circulated within the test tube at a rate of 1.0 mL/min by an infusion
pump (type A-II multipurpose dual-syringe pressure-vacuum pump, Eiko
Co) during treatment of the thrombus. The solution was drained out at
the same flow rate from the outlet with the same pump device. UK
concentration was 1200 IU/mL. ABX was diluted to a final concentration
of 
0.6x106 microspheres/mL. The thrombus
was exposed to US energy delivered in 2-second-on/4-second-off pulses
for a duration of 3 minutes. US frequency was fixed at 170 kHz, with US
intensity of 0.5 W/cm2. Change of temperature during US
exposure within the test tube was measured by a needle thermometer
(Tel-Thermometer, Yellow Springs Instrument Co Inc).
The US element was removed from the test tube after US exposure, and
the thrombi were incubated at 37°C for a duration of 30, 60, 90, or
120 minutes. The thrombi were then gently removed from the test tube
and washed with saline two to three times before being fixed in Bouin
solution overnight. Each thrombus was dried the next day on filter
paper (Qualitive 2, Toyo Roshi Co) for 30 minutes before measurement of
the dry weight (model H54AR balance, Mettler Instruments).
Fibrinolysis was calculated as the percentage of the
weight loss of the thrombus as follows:
[(WC-WT)/WC]x100, where
WC is the weight of the control thrombus and WT
is the weight of the treated thrombus. The weight of thrombi without
treatment with US or fibrinolytic agents was considered to be the
initial control thrombus weight. To determine the effects of US alone,
groups of thrombi were circulated with 5% albumin or ABX,
neither of which contained fibrinolytic agents during US exposure
(Table). Albumin (5%) was prepared for controls
because the ABX (albumin microspheres) is the same
substance. All the above experiments were performed separately with a
single thrombus in each test tube, a total of 15 samples for each
condition. Mean±SD was calculated from the percentage of
fibrinolysis of all experiments performed. Differences
between multiple groups were assessed by two-way ANOVA for repeated
measures. A value of P<.05 was considered to be
statistically significant. Calculations were performed with the
STATVIEW II statistics package (Abacus Concepts Inc) on
an Apple Macintosh Computer.
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Results |
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TopAbstractIntroductionMethods Results Discussion References |
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The Table
shows fibrinolysis percentages of
thrombi exposed to US with or without UK. Thrombi exposed to US without
UK and incubated for 120 minutes did not result in significant
differences in fibrinolysis compared with control
thrombi (P<.05). Thrombi exposed to US in the presence of
ABX without UK also showed no significant differences in
fibrinolysis compared with control thrombi. No
significant differences in fibrinolysis were seen in
samples exposed to ABX but unexposed to US.
Fibrinolysis increased with longer incubation duration
for all UK-exposed thrombi (Fig 2). No significant
differences were obtained between the three groups, UK, UK+US, and
UK+US+ABX, when they were incubated for 30 minutes. In contrast,
fibrinolysis was significantly different
(P<.05) compared between the groups, UK+US+ABX,
UK+US, and
UK, when they were incubated for 60, 90, and 120 minutes.
Fibrinolysis after 60 minutes of incubation was
51.3±7.7% (mean±SD) for thrombi exposed to US in the presence of
ABX
and UK (UK+US+ABX), whereas thrombi exposed to US and UK
(UK+US) showed
33.3±5.8% fibrinolysis. Fibrinolysis
of thrombi by UK alone (UK) was 26.6±4.8%. The temperature increase
within the test tube during US exposure was <0.2°C in all
experiments.
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Discussion |
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TopAbstractIntroductionMethods Results Discussion References |
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Numerous articles on US-accelerated thrombolysis have appeared in recent years.1 2 3 4 5 6 7 Experiments have been performed with various US methods and conditions. US exposure frequencies have ranged from 50 kHz to 3 MHz, and intensity levels of 30 mW/cm2 to as high as 8 W/cm2 have been applied. The most appropriate US frequency and minimum intensity needed to enhance fibrinolysis have not been determined. However, excessive exposure of the tissue to US will produce unnecessary heating11 12 or chemical reactions13 that could damage vessels and surrounding tissues. To minimize unwanted side effects, either the US sensitivity of fibrinolytic agents must be increased or US energy must be extremely localized at the required site. As previously mentioned, Holland and Apfel9 reported a lowering, in the presence of albumin microspheres, of the threshold of cavitation production currently used for diagnostic echo contrast. The size of the albumin microspheres in ABX ranges from 0.5 to 10 µm, with a mean diameter of 4.0 µm. Since acoustic cavitation is postulated to be one of the factors for inducing US-accelerated thrombolysis, it was hypothesized that microbubbles, such as ABX, can be used to further enhance fibrinolysis by US. Results from the present preliminary study with artificial thrombi clearly indicated a significant increase of fibrinolysis in the presence of ABX.
The mechanism of increased fibrinolysis in the presence
of ABX is unclear; however, previous studies have suggested that
nonthermal effects of US, such as acoustic cavitation, are responsible
for acceleration of
fibrinolysis.1 2 3 4 5 6 7 8
Cavitation or collapse of ABX microspheres can produce
microstreaming, which may drive fibrinolytic agents into the fibrin net
structure of a thrombus, thus increasing the bioavailability of drugs.
It is therefore postulated that the amount of cavitation produced may
reflect the rate of fibrinolysis. Numerous factors,
such as the viscosity, temperature, and amount of dissolved gas, can
change cavitation thresholds. In addition, the presence of such
materials in the medium as ethanol, red blood cells, and echo-contrast
agents has also been reported to alter cavitation
thresholds.9 The existence of ABX around the thrombus in
the present study may have increased the amount of cavitation
production so as to increase the acceleration of
fibrinolysis. To access the status of ABX
microspheres during US exposure, an additional experiment was
devised to visually observe the material by diluting the ABX with
saline instead of whole blood, as in the main study. ABX visually
appears to be a white nontransparent solution due to the countless
microscopic bubbles; however, after initiation of US at the same
intensity and frequency as previously described, a transparent area was
suddenly observed near the US element. US intensity near the element
probably exceeded the threshold level of ABX collapse, thus resulting
in "clearing" of the solution. It is expected that a similar
phenomenon occurred near the thrombus in the main experiment. However,
the induction of acoustic cavitation is apt to change in different
situations, for example, in the presence of red blood cells. The main
experiments were carried out with whole blood, and thus, ABX collapse
could not be visually confirmed; however, echographic observations in a
separate experimental system (not described) have shown similar
disappearance of ABX near the US element in ABX included in citrated
whole blood (Fig 3). Mor-Avi et al14 also
quantitatively measured a similar decrease of videointensity after
exposure of ABX to high-intensity US with a similar method. These
observations strongly support the hypothesis that collapsing ABX
microspheres promote an increase in the efficiency of
fibrinolytic agents at the US frequencies and intensity levels used in
the present study. Additionally, no indication of fibrinolytic
effects by ABX in samples without UK in the present study suggests
that mechanical destruction of the thrombus by US was minimal.
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The major benefits of ABX administration are that (1) maximum effects of cavitation can be localized within the blood vessel where ABX exists during US exposure and (2) localization of therapeutic US energy can be monitored simultaneously by observing hypoechoic images during ABX enhancement with the assistance of a diagnostic echo device. However, intravenous administration of ABX results in loss or breakdown of the material in several minutes during passage through the lungs. This may limit full access of the material to the targeted thrombus. Thus, to obtain maximum therapeutic effects of ABX, local delivery of the drug near the location of the thrombus may be needed. Nevertheless, additional evaluation is needed to access the potential benefits of using ABX for US fibrinolysis in vivo.
The present study was done primarily to determine whether ABX has an effect on US-accelerated thrombolysis and to gain insights into the mechanism. It is concluded that the echo-contrast agent ABX further increased acceleration of thrombolysis at this US frequency and intensity. ABX may prove to be a useful therapeutic promoter for US thrombolysis as well as an echo-enhancing agent. However, more in vivo experiments must be undertaken to specify the appropriate US exposure method, intensity, and frequency and the ABX dosage for US-accelerated thrombolysis therapy in combination with ABX.
Received October 13, 1994; revision received March 13, 1995; accepted March 17, 1995.
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