Ultrasound, microbubbles, and thrombolysis

Ultrasound, Microbubbles, and Thrombolysis Thomas R. Porter and Feng Xie

Although dissolution of thrombus using ultrasound has been attempted for over 25 years, the clinical use of this technique remains limited. The ability of microbubbles to potentiate ultrasound-induced thrombolysis has renewed interest in this technique, which recanalizes occluded vessels without the need for fibrinolytic therapy. In this article, the potential mechanisms by which ultrasound and microbubbles produce thrombus dissolution are explored. In vitro and in vivo studies using ultrasound alone and ultrasound in combination with microbubbles to cause thrombolysis are reviewed. Potential clinical implications of more recent findings are explored. Copyright © 2001 by W.B. Saunders Company

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lthough pharmacologic approaches to achieving reperfusion in acute coronary thrombotic syndromes continue to evolve, at least 3 clinically significant problems with this approach have been described.1 First, there is a consistent 20% of patients who do not achieve angiographic recanalization despite the early (⬍6 hours) use of both fibrinolytic agents and platelet aggregation inhibitors. Secondly, despite recanalization, there is a persistent high-grade stenosis in a majority of patients that most likely contains a significant amount of residual thrombus and often requires a percutaneous revascularization technique. Thirdly, the process of reperfusion is usually accompanied by injury, which can damage the previously ischemic myocardium and lead to severe regional dysfunction. Additional measures that will address these thrombosisrelated problems and complications are still needed. One potential supplemental treatment is therapeutic ultrasound. The potential for high-frequency ultrasound to dissolve intra-arterial thrombi was first reported by Trubestein et al2,3 in 1976. In these

preliminary studies, a 26.5-kHz ultrasound transducer was found to successfully recanalize thrombosed iliofemoral arteries in dogs with minimal complications. Subsequent studies in the next 25 years using catheter-based or transcutaneous ultrasound have been supportive of these original studies, but have focused mainly on the ability of ultrasound to enhance the effect of fibrinolytic agents in recanalizing acutely thrombosed arteries.4-12 More recently, animal studies have suggested that intravenously injected microbubbles may enhance the effects of ultrasound, and may even produce recanalization of acutely thrombosed arteries in the presence of ultrasound without the need of fibrinolytic agents.13,14 Although in vitro studies have observed microfragmentation of thrombi in the presence of microbubbles and ultrasound,15 the precise mechanism by which ultrasound and microbubbles induce thrombus dissolution remains unknown. The purpose of this article is to examine potential mechanisms for ultrasound-induced thrombolysis and to explore the future potential of this technique as either an adjuvant or an alternative to current pharmacologic techniques in noninvasively achieving coronary and peripheral artery reperfusion.

Rationale for Ultrasound-Induced Thrombus Dissolution There are several proposed explanations for why ultrasound can result in intravascular thrombus

From the Department of Internal Medicine, Section of Cardiology, University of Nebraska Medical Center, Omaha, NE. Address reprint requests to Thomas R. Porter, MD, University of Nebraska Medical Center, 981165 Nebraska Medical Center, Omaha, NE 68198-1165. Copyright © 2001 by W.B. Saunders Company 0033-0620/01/4402-0002$35.00/0 doi:10.1053/pcad.2001.26441

Progress in Cardiovascular Diseases, Vol. 44, No. 2, (September/October) 2001: pp 101-110

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102 dissolution. First, ultrasound induces axial fluid acceleration that results in the phenomenon of acoustic streaming.16 Streaming creates highvelocity gradients at the thrombus surface, which could then mechanically remove the thrombus as well as increase the exposure of fibrin to fibrinolytic agents. A second potential explanation is cavitation-induced microstreaming. Cavitation refers to the growth and collapse of bubbles in a sound field, and several investigators have shown with megahertz frequencies that cavitation results in microstreaming vortices of sufficient magnitude to lyse blood cells.17,18 Cavitation-induced microstreaming would not require activation of fibrinolytic pathways to be effective, because the streaming results in a mechanical shearing of the outer surface of the thrombus. In vitro and in vivo observations seem to support a cavitation-induced microstreaming mechanism. First, there is no evidence of enhanced fibrinolysis when using ultrasound alone or ultrasound and microbubbles to dissolve thrombi.13,19,20 Secondly, microbubbles, which lower the threshold for inducing cavitation, have consistently been shown to enhance the effects of ultrasound in causing clot lysis. Other possible mechanisms for ultrasound-induced thrombolysis include increased fibrinolytic activity as a result of thermal energy. However, there are no in vitro or in vivo studies that have shown that temperature elevation is required to achieve thrombus dissolution. Ultrasoundinduced alterations in the viscous properties of tissue and ultrasound-induced increases in membrane permeability have been suggested as mechanisms,21 but no studies have specifically looked at the role of these variables in causing thrombus dissolution.

Effectiveness of Ultrasound Alone in Thrombus Dissolution Ultrasound at low frequencies has been delivered either transcutaneously or via a transcatheter route to recanalize thrombotic arterial obstructions. A small number of in vivo studies using intra-arterial catheter-based ultrasound delivery techniques have uniformly been successful in recanalizing obstructed coronary arteries or even saphenous vein grafts using low-frequency (20- to 45- kHz) transducers.4,9,10 However, because ultrasound at this frequency range shows only minor attenuation, the majority of animal studies

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have focused on transcutaneous instead of transcatheter ultrasound to enhance fibrinolysis. Table 1 shows the different in vitro, animal, and human studies that have used ultrasound to dissolve thrombi. As can be seen, the majority of studies enhanced the effectiveness of fibrinolytic agents. A wide range of frequencies (20 kHz to 1.5 mHz) and power outputs (0.25 to 40 W/cm2) were used, with both continuous- and pulsed-wave ultrasound delivery. Despite this wide range of frequencies, power outputs, and modes of delivery, enhanced fibrinolytic activity was consistently observed. The findings from in vivo studies indicate that the specific type of ultrasound used may play a role in effectiveness. Luo et al11 used intravenous streptokinase combined with transcutaneous 26 kHz ultrasound at a continuous wave output of 18 W/cm2 in an attempt to recanalize acutely thrombosed femoral arteries in rabbits. The addition of transcutaneous ultrasound (TCUS) to intravenous streptokinase increased the femoral artery recanalization rate from 6% in rabbits treated with streptokinase alone to 59% in rabbits treated with streptokinase and TCUS. Subsequent studies by this same group using this same rabbit iliofemoral thrombosis model have shown greater than 90% patency rates using intravenous streptokinase and a transcutaneous 37 kHz transducer.6 However, Kornowski et al22 examined the ability of 1-mHz ultrasound to enhance the thrombolytic effectiveness of recombinant tissue-type plasminogen activator (TPA). The power output of the transcutaneous ultrasound transducer was continuous wave at 6.3 W/cm2. TPA alone without ultrasound was able to recanalize 12 of 13 thrombosed femoral arteries, whereas the addition of ultrasound increased the incidence of thrombotic reocclusion.22 Therefore, higher-frequency transducers may be more prone to induce an opposing prothrombotic effect. Others have shown that higher-power output settings at the same frequency can lead to enhanced platelet deposition and fibrin formation. Nilsson et al23 showed that exposing human blood clots to streptokinase or TPA while being insonified at peak intensities ranging from 1.1 to 3.2 W/cm2 can lead to an entire spectrum of results. The investigators found no significant difference in the amount of clot lysis between frequencies ranging from 0.5 to 2.3 mHz, but did observe that the greatest degree of clot lysis occurred in the 0.5 and 1.0 W/cm2 outputs. Beyond this, there was no

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Table 1. In Vivo and In Vitro Studies Since 1992 That Have Examined the Effectiveness of Ultrasound Alone or in Combination With Microbubbles to Produce Thrombolysis Reference

Year

PW/CW

US Frequency

W/cm2

MB

In Vitro/In Vivo

Catheter/ Transcutaneous

11

Yes

In vitro

Catheter

In vitro In vitro In vitro Human, SVG In vitro Rabbit, iliofemoral artery In vitro/rabbit, iliofemoral artery In vitro Rabbit, retinal vein Rabbit, iliofemoral artery In vitro In vitro

38

1999 PW

19.5 kHz

39 37 40 4 31 13

1999 1999 1999 1999 1999 1998

1.1 mHz 33/71 kHz 10 mHz 41 kHz 10.0 mHz 37 kHz

560–2360 0.5/3.4 0.5–1.0 18 1.02 n/a

Yes No Yes No Yes Yes

14

1998 CW

20 kHz/24 kHz

1.5/2.9

Yes

41 5 6

1998 CW 1998 PW 1998 PW

212 kHz/1.0 mHz 1 mHz 37 kHz

0.25/1.0 1.0 Up to 160

No No No

42 30

1998 CW 1998 CW

22.5 kHz 1.0 mHz/20 kHz

No Yes

43 7

1997 PW 1997 Unknown

1.3 mHz 1.0 mHz

30–36 420 kPa/ 0.9–5.0 kPa 0.3 2

8 9 44 32 11

1997 1997 1996 1996 1996

PW n/a PW CW CW

19.5 kHz 45 kHz 640 kHz 20 kHz 26 kHz

29 45 23 23 23 12 46 22

1995 1995 1995 1995 1995 1994 1994 1994

PW PW PW PW CW CW CW

170 kHz 1.0 mHz 0.5, 1.0, 2.3 mHz 0.5, 1.0, 2.3 mHz 0.5, 1.0, 2.3 mHz 1 mHz 300 kHz/1.0 mHz 1.0 mHz

21 47 19 48 49

1994 1993 1993 1993 1992

PW CW PW CW PW

0.17, 1.0 mHz 20 kHz 1.0 mHz 0.5 mHz 1.0 mHz

PW n/a PW/CW n/a CW PW

11 18 — 40 18 0.5 4 0.5 0.5–1.5 ⬎4 2.5 0.07–0.4 6.3 1 1–2 1–2.2 8.0 1.75

No No No No No Yes No Yes No No No No No No No No No No No No

In vitro Rabbit, femoral artery Human, coronary Human, coronary In vitro In vitro Rabbit, iliofemoral artery In vitro In vitro In vitro In vitro In vitro In vitro In vitro Rabbit, femoral artery In vitro In vitro In vitro In vitro In vitro

Agents

Outcome ⫹

Transcutaneous Transcutaneous Catheter Catheter Catheter Transcutaneous

Heparin, tPA, Tirofiban* No tPA tPA No tPA No

⫹ ⫹ ⫹ ⫹ ⫹ ⫹

Transcutaneous

No

⫹

Transcutaneous Transcutaneous Transcutaneous

Urokinase Streptokinase Streptokinase

⫹ ⫹ ⫹

Catheter Transcutaneous

No Urokinase

⫹ ⫹

Catheter Transcutaneous

Urokinase Streptokinase

⫹ ⫹

Catheter Catheter Catheter Transcutaneous Transcutaneous

Heparin Heparin/aspirin Urokinase Urokinase Streptokinase

⫹ ⫹ ⫹ ⫹ ⫹

Transcutaneous Transcutaneous Transcutaneous Transcutaneous Transcutaneous Transcutaneous Transcutaneous Transcutaneous

Urokinase tPA Streptokinase Streptokinase Streptokinase Urokinase tPA tPA, aspirin

⫹ ⫹ ⫺ ⫹ ⫺ ⫹ ⫹ ⫺

Transcutaneous Catheter Transcutaneous Transcutaneous Transcutaneous

Streptokinase Urokinase Streptokinase tPA tPA

⫹ ⫹ ⫹ ⫹ ⫹

*Merck, Sharp, & Dohme, White House Station, NJ. Abbreviations: PW, pulse wave; CW, continuous wave; MB, microbubble; SVG, saphenous vein graft; tPA, tissue-type plasminogen activator.

additional effect until they reached 4 W/cm2, at which point a detrimental effect was observed. At this output, the clot lysis achieved with ultrasound combined with fibrinolytic agents was less than with fibrinolytic agents alone (Fig 1). In vitro studies have also confirmed that higher-power outputs at the 1 mHz frequency can paradoxically prevent recanalization of thrombosed vessels (Table 2). The mechanism for this prothrombotic effect at higher outputs has been attributed to either stimulation of platelet aggregation or fibrin deposition. Riggs et al24 have shown in vivo that the addition of continuous-wave 1-mHz ultrasound

at 2 W/cm2 will increase platelet accumulation on acute femoral artery thrombi.24 High-intensity ultrasound energies at slightly higher frequencies have actually been used to promote hemostasis of punctured blood vessels.25-27 In these studies, ultrasound transducer frequencies between 2 and 3.5 mHz were used to close punctures or incisions made in vessels or tissue. Microscopic examination of vessels treated with ultrasound at these high intensities showed fibrin deposition. On review of Table 1, it is apparent that lower frequencies and lower power outputs resulted in better TCUS-mediated thrombus dissolution in vivo.

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Fig 1. An example of the effect of different ultrasound power outputs on the enhancement of thrombolysis in vitro. Note that at higher power outputs there is an actual potentiation of thrombus formation. (*P < .05, **P < .01, ***P < .001) (Reprinted with permission of Elsevier Science from Pro- and antifibrinolytic effects of ultrasound on streptokinase-induced thrombolysis by Nilsson AM, Odselius R, Roijer A, et al, Ultrasound in Medicine and Biology, Vol 21, pp 833-840, Copyright 1995 by World Federation of Ultrasound in Medicine and Biology.23)

Effectiveness of Microbubbles in Enhancing Ultrasound-Induced Thrombolysis Microbubbles have been shown to lower the threshold for ultrasound-induced cavitation in tissue.28 Because one of the most plausible mechanisms for ultrasound-induced clot dissolution is cavitation, the addition of microbubbles should enhance this phenomenon and lower the energy requirements for producing ultrasound-mediated thrombolysis. This enhancement has been shown in several in vitro studies using room-air-filled microbubbles,29 as well as perfluorocarbon-containing microbubbles and emulsions.30-32 These studies not only showed an enhancing effect, but also for the first time were able to show that ultrasound and microbubbles could by themselves enhance thrombus removal in a manner equivalent to that of a fibrinolytic agent (Fig 2). The mechanism for ultrasound-induced lysis in

the presence of microbubbles appears to be nonenzymatic.33 We have measured D-dimer levels and residual microparticle size after low-frequency ultrasound treatment (20 kHz) of venous thrombi treated with perfluorocarbon-containing microbubbles (PESDA). These studies showed that ultrasound plus PESDA produced equivalent or even greater clot dissolution than ultrasound plus urokinase, but D-dimer levels were increased only in the presence of urokinase. Residual particle size, however, was similar to that achieved with fibrinolysis.33 The absence of any increase in D-dimer activity after ultrasound and microbubble-induced vascular recanalization has also been observed in vivo.13 In vitro studies indicate that the potentiation of ultrasound-induced thrombolysis with microbubbles is caused by cavitation. Tachibana et al have shown a distinct clearing of albumin-coated microbubbles in the region where ultrasound was

Table 2. Effect of Different Ultrasound Outputs and Frequencies on the Time Required to Reperfuse a Clotted Vessel In Vitro F (mHz)

LA (W/cm2)

Isata Mode

1 1 1 1 0.170 0.170

1.0 1.0 5.0 5.0 0.5 0.75

continuous burst continuous burst continuous burst

(W/cm )

RT, Control (min)

RT, Ultrasound (min)

% Change

1.0 0.01 5.0 0.05 0.5 0.0075

39 ⫾ 20 (n ⫽ 15) 61 ⫾ 24 (n ⫽ 9) 39 ⫾ 19 (n ⫽ 9) 69 ⫾ 17 (n ⫽ 8) 65 ⫾ 16 (n ⫽ 16) 62 ⫾ 21 (n ⫽ 14)

20 ⫾ 7 (n ⫽ 15) 28 ⫾ 9 (n ⫽ 18) ⬎116 ⫾ 3 (n ⫽ 13) 55 ⫾ 9 (n ⫽ 11) 34 ⫾ 7 (n ⫽ 12) 31 ⫾ 9 (n ⫽ 20)

⫺49% ⫺54% ⬎197% ⫺20% ⫺47% ⫺51%

2

Significance P P P P P P

⬍ ⬍ ⬍ ⫽ ⬍ ⬍

.01 .01 .01 .05 .01 .01

Effective Energy (J/cm2) 1200 16.8 — 165 780 14

NOTE: The average sound intensities over one ultrasound cycle (IA) and spatial average temporal average intensity (ISATA) are shown. Reperfusion time (RT) is in minutes. At any one frequency, the intensity of ultrasound delivered has a large impact on effectiveness of clot lysis. Reprinted with permission of Elsevier Science from Enhancement of thrombolysis by ultrasound, by Olsson SB, Johansson B, Nilsson AM, et al, Ultrasound in Medicine and Biology, Vol 20, pp 375-382, Copyright 1994 by World Federation of Ultrasound in Medicine and Biology.21

ULTRASOUND, MICROBUBBLES, AND THROMBOLYSIS

Fig 2. An example of the ability of low-frequency ultrasound and microbubbles to induce thrombus dissolution in the absence of fibrinolytic agents. Note that perfluorocarbon-containing microbubbles and ultrasound produced thrombolysis without the need for fibrinolytic agents. (Reprinted with permission.32)

being applied to a thrombus in vitro. The investigators proposed that the clearing was caused by microbubble destruction by cavitation, and found that the degree of clearing correlated with the degree of enhanced fibrinolytic drug effectiveness.29 Visualization of thrombi after exposure to ultrasound plus PESDA microbubbles has provided evidence that a mechanical shearing of the thrombus is occurring (Fig 3). Such a phenomena could be explained by acoustic microstreaming of the microbubbles within the field of insonation.

In Vivo Studies of MicrobubbleMediated Thrombus Removal To date, there are only 2 published in vivo studies in which transcutaneous ultrasound and intravenous microbubbles alone have produced arterial thrombus dissolution.13-14 Both of these studies were performed in the absence of fibrinolytic agents, and antithrombotic drugs were not administered until recanalization had been achieved with transcutaneous ultrasound (25 to 37 kHz) and intravenous microbubbles (dodecafluoropentane emulsion or PESDA microbubbles). Both were performed after iliofemoral thrombotic occlusions in a rabbit model, and the thrombi were less than 1 hour old at the time of treatment. Recanalization success rates were 100% with intravenous PESDA and 82% with dodecafluoropentane emulsions after 1 hour of treatment (Fig 4), compared with a ⬍10% recanalization rate with transcutaneous ultrasound alone. In essence, intravenous perfluorocarbon-containing micro-

105 bubbles were equivalent to a fibrinolytic agent in terms of effectiveness. Unlike fibrinolytic agents, there was no increase in D-dimer activity after ultrasound and microbubble treatment, a factor that indicates that ultrasound and microbubbles may have a greater clinical applicability than fibrinolytic agents. Although these approaches appear promising, the use of a very-low-frequency probe may induce thermal effects. Without cooling manifolds around these probes, temperature elevations of up to 12°C have been observed at the transducer tip and were associated with coagulative necrosis and hemorrhage of the dermis and subcutaneous tissue.14 Newer ultrasound transducers have incorporated a cooling manifold at the transducer tip and have not been shown to induce these skin lesions.6,13 Preliminary in vivo studies using intravenous microbubbles and transthoracic ultrasound to treat acute coronary thromboses in large animal models have been less successful. In a preliminary study, recanalization of acute coronary thrombosis in pigs was achieved in only 7 of 14 (50%) after treatment with intravenous PESDA microbubbles and transthoracic 1 mHz ultrasound (Fig 5). Although this success rate is less than that seen with peripheral arterial thromboses, treatment with ultrasound and PESDA seemed to have beneficial effects that were independent of whether vessel recanalization occurred.34,35 First, pigs treated with ultrasound and intravenous PESDA had significantly greater improvements in ST segments over the 30-minute treatment period when compared with pigs treated with ultrasound alone or with control (Fig 6). Secondly, wall thickening within the risk area improved significantly more than wall thickening in the control groups. Finally, there was a significantly smaller myocardial contrast defect size after treatment with ultrasound and PESDA for 30 minutes (Fig 7 and 8). All of these changes were seen with equal magnitude in pigs that did and did not achieve angiographic recanalization after treatment with ultrasound and PESDA.35 One possible explanation is that the use of intravenous microbubbles and ultrasound is improving myocardial blood flow to the risk area via collaterals. Further investigation is underway to examine this possibility.

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Fig 3. In vitro changes in thrombus size and shape after treatment with 1-mHz ultrasound and PESDA at a tissue depth of 6 cm. Note that after treatment, thrombus size is smaller because of lateral indentations. This was evident both at 0° and 90° projections.

Potential Human Investigations Using Intravenous PerfluorocarbonContaining Microbubbles in Acute Arterial Thromboses Based on the animal studies performed thus far, there seems to be a potential role for the use of intravenous perfluorocarbon-containing microbubbles and TCUS for peripheral arterial occlusions that are caused by emboli or thrombosed arteriovenous grafts used for hemodialysis. In acute coronary syndromes, however, there are several important factors to consider. First, if it were to add to current fibrinolytic and antiplatelet regimens, ultrasound and microbubbles would need to achieve over a 95% success rate in epicardial recanalization in acute myocardial infarction. Secondly, this therapy should be able to cause a significant reduction in the degree of reperfusion injury by enhancing oxygen delivery to ischemic myocardium before and after reperfusion.36 Thirdly, such a treatment regimen

should also cause a significant reduction in the rates of percutaneous or surgical revascularization after acute coronary syndromes because of more effective clearing of residual thrombus. Finally, all of these additive treatments should result in a better longterm result measured by left ventricular regional and global systolic function. Further large animal studies and phase I human studies are anticipated within the next 2 years, and will determine whether this new therapeutic approach can achieve these goals. Low-frequency ultrasound combined with intravenous microbubbles may also have a role in acute stroke. Recent in vitro studies have shown that very-low-frequency ultrasound transducers (33 to 71 kHz) can significantly potentiate the effect of TPA-mediated thrombolysis when delivered through the temporal bone.37 These findings raise the possibility that transtemporal ultrasound combined with intravenous microbubbles may on their own produce recanalization of thrombosed

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ULTRASOUND, MICROBUBBLES, AND THROMBOLYSIS

Fig 4. Angiographs showing how transcutaneous ultrasound and intravenous PESDA resulted in recanalization of a thrombosed iliac artery in a rabbit. The contralateral iliac artery, which received intravenous PESDA, only is shown on the right. (Reprinted with permission from Birnbaum Y, Luo H, Nagai T, et al, Non-invasive in vitro clot dissolution without a thrombolytic drug: Recanalization of thrombosed iliofemoral arteries by transcutaneous ultrasound combined with intravenous infusion of perfluorocarbon-exposed sonicated dextrose albumin microbubbles (PESDA), Circulation, Vol 97, pp 130-134.13)

Image Unavailable. Please See Print Journal.

Fig 5. Coronary angiogram of a thrombosed left anterior descending artery treated with intravenous PESDA and transthoracic 1-mHz ultrasound (1.1 mPa peak negative pressure; 1.0 W/cm2).

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Fig 6. Electrocardiographic evidence of improvements in the ST segment that occurred after treatment with transthoracic ultrasound and PESDA in the absence of recanalization.

cerebral vessels, similar to what has been observed in peripheral vessels. In vivo large animal studies will be required to confirm these in vitro findings.

Conclusions Although ultrasound alone appears to potentiate the beneficial effects of fibrinolytic agents, the addition

of microbubbles has resulted in effective ultrasoundinduced thrombus dissolution without the need of fibrinolytic agents. Human trials are now needed to determine the effectiveness of transthoracic ultrasound and intravenous microbubbles in supplementing current treatment regimens and whether they can be a safe alternative in patients who have a contraindication to fibrinolytic agents.

Fig 7. Myocardial contrast evidence of improvement in perfusion to the left anterior descending perfusion bed after treatment with intravenous PESDA and transthoracic 1-mHz ultrasound. There was no evidence of angiographic recanalization.

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ULTRASOUND, MICROBUBBLES, AND THROMBOLYSIS

Fig 8. Myocardial contrast evidence of improvement in perfusion to the left circumflex perfusion bed after treatment with intravenous PESDA and transthoracic 1-mHz ultrasound. There was no evidence of angiographic recanalization.

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