Cardiovascular Sonothrombolysis-Therapeutic Application of Ultrasound

US energy has been used for almost 3 decades in interventional medicine. Formerly considered only for diagnosis, it has been lately used as promising therapeutic method. In diagnostic field, the US ability was enhanced with the combination of US contrast agents, which improves image delineation of organs, including chambers of the heart, assists in imaging blood flow, including blood flow deficiencies (organ perfusion); and also can be applied for the evaluation of patency of anatomical structures (e.g. fallopian tubes). As therapeutic method, US has been used alone or as adjuvant to facilitate artery angioplasty. Lately, its efficacy as treatment of thrombosed arteries in AMI has been demonstrated both in percutaneous transluminal and in transtoracic applications. Its action is mainly through IC of systemically administered US contrast agents and this mechanism will be also discussed in this review. Received: September 13, 2019

Despite the effectiveness of previous treatments for peripheral or coronary thromboembolism namely direct surgical embolectomy and/or arterial bypass, percutaneous balloon catheter extraction, and local or systemic fibrinolytic therapy, side effects and complications cannot be discharge. Several studies demonstrated increased risk for operative morbidity, bleeding complications secondary to fibrinolytic therapy, arterial perforation, intimal injury, avulsion of atherosclerotic plaque, arteriovenous fistulas, impaction of an embolus or thrombus into the distal arterial tree, or shifting of the thrombus [1][2][3] In this context, US appears as a method with minimal chance of undesired events. The new concept of blood vessel thrombi dissolution by US was initially described by Trubestein et al. [4,5] and was latter demonstrated in vitro, [6][7][8][9] in vivo and in animal [7][8][9][10] and human studies [11][12][13][14].
In the previous classical studies, the authors demonstrated the potential of US for destroying artificial thrombi in peripheral  [8] using an experimental ultrasonic angioplasty device, with a reduction of the obstruction from 93% to 18% without arterial wall injury. In addition, these authors demonstrated that sonication was also effective to reduce the size of atherosclerotic plaques (in vitro aortic segments) without damage to the media or adventitia, demonstrating the potential of US for angioplasty. More than 1 decade later, the effectiveness of percutaneous catheterdelivery US was demonstrated in clinical studies of vascular disease without evidence of clinical complications, such as arterial emboli, dissection, spasm, or perforation [15,16]. In addition, Siegel et al. [10] observed the opening of completely obstructed xenograft implantation of atherosclerotic human vessels in 12 dog arteries (carotid, aortic, iliac and femoral) by applying US energy through a catheter-delivery system creating an appropriated lumen for subsequent balloon dilation. Further, they found that calcific arterial obstructions do not impede US recanalization. In the vessels with xenograft implant, there was no angiographic evidence of US-induced vasospasm, thrombosis or arterial dissection. In other study, Siegel et al. [17] demonstrated, in patients undergoing percutaneous US angioplasty, 86% of recanalization in arterial lesions including 34% with calcification. These authors observed decrease in the stenosis from 94% to 55% after US angioplasty and to 12% after balloon angioplasty and restenosis rate was of only 20% at 6 to 12 months follow-up.
The studies were not only limited to the potential use of US in peripheral vessels but also conducted as an angioplasty and clot-dissolving device in thrombosed coronary arteries. This technique differs from balloon angioplasty, as it has been shown to provoke dissolution of calcific and fibrotic atherosclerotic plaques, thrombus and arterial vasodilatation. Steffen et al.9 showed that catheter-delivered US dissolved blood clots in vitro and in vivo dog coronary arteries. Angiography revealed widely patent coronary arteries in 87% of the dogs and there was no histologic evidence of US-mediated vessel damage. In the following year, the results of The European Multicenter Experience with therapeutic US coronary angioplasty in symptomatic patients were reported [18].
US reduced the mean arterial stenosis from 86 to 71% in 136 of 163 patients who presented lesions with calcification or thrombus.
Siegel et al. [19] demonstrated, in the clinical part of their study, in patients, that catheter-delivered high-intensity, low-frequency US (intracoronary US) decreased the coronary arterial stenosis from 80% to 60% after US and to 26% after adjunctive balloon angioplasty. Using postmortem occluded coronary, they showed 100% recanalization and very small particulate debris (99% were lower than 10 microns in diameter). These previous studies confirmed the usefulness of US to dissolve thrombi, increase arterial distensibility and modify atherosclerotic plaques without adverse reaction or with only small damage to US exposure.
The results of percutaneous US thrombolysis were also positive in human studies of AMI as reperfusion therapy [11,14,20,21].
Hamm et al. [14] reported a case of successful US-mediated intracoronary thrombus dissolution in a patient with AMI. The same group21 treated 11 patients with US and observed removing of angiographically visible clots. TIMI flow 3 and 2 were observed in 64% of patients and after adjunctive percutaneous transluminal coronary angioplasty, TIMI 3 flow was present in 91% of the patients. Two years later, the researchers20 tested the effect of intracoronary US thrombolysis as initial therapy in patients with AMI. US was effective and safe to recanalize thrombotically occluded coronary arteries. Angiographically visible thrombi were dissolved without US related morbidity or adverse effects.
Rosenschein et al. [11] corroborated the clinical feasibility of percutaneous transluminal coronary US thrombolysis in 15 AMI patients, demonstrating successful reperfusion (TIMI grade 3 flow) in 87% of the patients without adverse angiographic signs or clinical effects during procedure.
All these results together suggest US as an effective and safe treatment for peripheral and coronary arterial thrombosis since its efficacy to dissolve tissues with low elasticity, such as atheroma and thrombi, was demonstrated. Of note, the US application technique progressed in order to prevent possible damage to normal tissues near the sites of occlusions, beginning with direct in vitro application, delivered through catheters within the vessels and ultimately, with transcutaneous or transthoracic application, as described hereafter in this review.

Ultrasound Associated with Fibrinolytic Therapy
Thrombolytic therapy presents several limitations, such as, low rate of TIMI grade 3 reflow in AMI patients, intermittent patency and reocclusion of recanalized artery or clinical complications, such as cerebral and gastrointestinal bleeding. In an attempt to overcome these limitations, to shorten occlusion time of arteries, and to reduce the total dose of thrombolytic agents, a number of researchers have reported acceleration of thrombolysis by US energy application in association with fibrinolytic agents [22][23][24] Kudo25 published the first studies describing the use of US to increase the efficacy of systemic tissue-type plasminogen activator Despite different characteristics of US technique applied, these above data suggest that non-invasive intermittent US acts as an adjunct to thrombolytic therapy enhancing both efficacy and rate of thrombolysis. It seems that US enhance the efficacy of some drugs to produce lysis through perturbation of the clot thereby allowing increased binding of the thrombolytic agent to fibrin. This hypothesis is based on mechanical effect of US: cavitation, which can generate high-velocity jets or a steady flow of fluid known as microstreaming. In addition, countless microscopic bubbles that oscillate in size near the thrombus during US exposure, may influence the local fluid flow and, therefore, enhance transport and penetration of endogenous or exogenous lytic agents into the thrombus and thus accelerate fibrinolysis [26,27].

Mechanism of Ultrasound and Ultrasound Associated with Microbubbles as Therapy
US has been generally used for diagnostic imaging using MHz frequencies. As a therapeutic tool, it was developed to direct mechanical energy effects of low-frequency (kHz) by virtue of its ability to exert mechanical forces, stresses, and torques, as well as displacements and flow. Low-frequency US energy accelerates thrombolysis by itself or when used in conjunction with a thrombolytic agent in vitro and in vivo or when adding US contrast agents. Basically, mechanical effects induced by lowfrequency US, such as, cavitation, precavitation which leads to microstreaming and radiation force [6,8,9,23,[28][29][30][31][32][33][34][35] are the main mechanisms to explain thrombi and atherosclerotic plaques disruption. Cavitation is a phenomenon by which small gas filled cavities, called microbubbles, exposed to an ultrasonic field grow, oscillate, collapse and disrupt, exerting various physical stresses in a liquid medium, resulting in tissue alteration. Both the vibration of microbubbles and their rapid collapse in the acoustic field result in local pressures of up to 20 000 atm [36].
This mechanical shock of bubble collapse is felt at a distance of a few microns, resulting in intense shear stress that could break fibrin bonds of thrombi and induce thrombus fragmentation.
Acoustic cavitation is presented in two forms:

1.
Non-inertial -described as the stable oscillation of gas- Bjerknes forces may play in sonothrombolysis. In fact, Hong et al. [6] suggested mechanical and cavitational energies as mechanisms to explain ultrasonic clot disintegration instead of biochemical activation of the fibrinolytic cascade. This hypothesis was based on the fact that US alone produces eight-to 16-fold fewer D-dimers than in streptokinase-exposed samples, indicating the lack of fibrinolytic activation by the wire probe. Besides the demonstration that cavitation effect plays an important role in the mechanism of US thrombolysis, Rosenschein et al. [29] also showed that tissues presenting low levels of elasticity, as thrombi, are highly sensitive to US ablation. In contrast, tissues containing a heavy matrix of collagen and elastin, such as arterial wall, bladder, or heart valves, are resistant to US.  which causes bone marrow-derived stem cell mobilization, which in turn contribute to cardiac repair after acute myocardial injury, [43,44] is associated with an improvement in LV ejection fraction.
Thus, it was hypothesized that the association of myocardial contrast echocardiography could enhance the bone marrowderived cells homing in the myocardium and determines superior improvement in regional and global contractile function, myocardial perfusion and infarct extension in patients with large ST-Segment Elevation Myocardial Infarction (STEMI). This phenomena has already been observed by these authors (unpublished data) [42] and demonstrated in animal studies [45,46] and is still under investigation.
Interesting to know that improvement in microvascular blood flow could even occur in the absence of epicardial recanalization and US-induced coronary artery vasodilatation is a possible explanation for this finding. Low-frequency US applied alone resulted in vasodilation in canine coronary artery (transthoracic) [47] and human brachial arteries (transcutaneous) [48] It has been showed that acute arterial thrombi still have small microchannels that cannot be seen with angiography, but which permit microbubbles to penetrate the interior of a thrombus [39] Thus, an increase in coronary diameter induced by US could improve local delivery of both microbubbles and/or lytic agent through micro-channels, resulting in increased flow in the downstream microvasculature, even without angiographic evidence of coronary artery reperfusion.

Ultrasound Associated with Pharmacological Agents -Microbubbles Echocardiography Contrast
Once microbubbles are naturally present in a liquid medium or can be spontaneously formed or be exogenously injected in the blood streaming; the acoustic cavitation process displayed by US become one important non-invasive method in clinical practice for diagnostic purpose and therapeutic purpose, such as thrombolysis [26,30]. In this regard, various transpulmonary echo contrast agents, detecting harmonics or overtones produced by microbubbles, and enhance microbubbles signal [49]. The methods for quantifying tissue blood flow consider the similar behaviour of microbubbles in the circulation as compared with that of the red blood cells [50] and the fact that microbubbles can be destroyed at high acoustic powers. Briefly, the method calculate microvascular perfusion from the product of microvascular red blood cell velocity, measured by the rate of increase in acoustic signal (rate of replenishment) after microbubbles destruction by high power US, and microvascular blood volume, estimated from acoustic intensity from microbubbles, or amount of contrast enhancement [49,51] Myocardial contrast US is mainly helpful to diagnose AMI and to detect coronary artery disease [52] and provides information on perfusion at capillary level.
The first air-filled microbubbles, using agitated saline solution, produced bright eco enhancement of the blood, despite the very low stability and short duration [53] The action of microbubbles as US contrast is based on their acoustic wave scattering. When a pulse of US excited a gas microbubble, with a diameter as low as several microns, the bubble oscillates compressing and expanding during high-and low-pressure phases, respectively and emitting secondary US waves of high intensity in all directions. This high reflectance of microbubbles provides bright blood pool contrast from vessels or cardiac chambers. Thus, gas bubbles generate a backscatter signal which is stronger by several orders of magnitude, justifying the use of gas as the core of the US contrast materials [54] The next challenging was to cover the gas core with a shell polymer-based, [55] protein-based, [56] or lipid-based surfactant [57] which would increase the lasting of the gas bubble. Thus, the first generation of US contrast agents (air-filled, shell-coated microbubbles) with extended storage stability and standardization of size was created.
The encapsulation process provided microbubbles size standardization and increased their stability, becoming possible intravenous application.  [58].
The production of microbubbles contrast agents must consider many factors that has influence on their signal and action, such as compressibility and density of the gas, viscosity and density of the surrounding medium, frequency and power of US applied, bubble size, viscous and elastic damping effects of a shell and the capability of destruction of microbubbles [49,[59][60][61][62]. The later feature is important for both perfusion imaging protocols and for therapeutic applications [49]. Therefore, an alternative to improve the stability of preformed microbubbles was the modification of the microbubble shell and/or gas content. Authors suggested the development of microbubbles with optimal shell viscoelastic properties to provide better signal-to-noise with low-MI imaging [62]. The application of organic gases, as fluorocarbons and sulphur hexafluoride, which are poorly soluble in biological fluids, presented enhanced stability in vivo. Encapsulation of high-molecularmass gases with lipids, proteins or biopolymers is still used to control their size distribution, and to further improve stability by reducing diffusion and surface tension. The second generation of commercial microbubbles-based US contrast agents is generally filled with perfluorocarbon gas, which present increased lifespan in vivo and provides enough time to acquire heart and peripheral organs images [63]. Porter et al. [30] demonstrated the potential of the US contrast agent perfluorocarbon-exposed sonicated dextrose albumin microbubbles to enhance the thrombolysis effectiveness of low-frequency US in vitro and to provide a reduction in the dosage of thrombolytic agents for treating thrombosis.

Weight reduction of the thrombus was greatest in the DDFP
Group. This group also presented the longest persistence of the microbubbles and greatest change in multiple cavity formation, observed histopathologically. Interestingly, the efficacy of DDFP and describes 3 commercially available US enhancing agents approved by the United States food and drug administration: Lumason (sulfur hexafluoride lipid-type A microspheres); Definity (perflutren lipid microsphere) and Optison (perflutren protein type-A microspheres). All these microbubbles contain an inert biocompatible high-molecular weight gas, and are encapsulated with a lipid or protein shell and are designed for Left Ventricular (LV) cavity opacification during echocardiography. According to the American Society of Echocardiography Guideline, microbubbles allow to better define the blood pool improving the identification of endocardial borders for assessment of LV systolic function and regional wall motion [66].
In addition, based on significant scientific literature support, its use has been also recommended for myocardial perfusion, to assess tissue blood flow at the microvascular level and coronary blood flow reserve, pediatric and vascular applications, and during stress echocardiography [66][67][68][69]. Microbubbles persist exclusively within the intravascular space, and their existence within any myocardial territory denotes the status of microvascular perfusion within that area [70]. Thus, in cardiovascular practice, microbubbles associated with US have been applied to non-invasively detect LV systolic function, coronary artery disease, to diagnose AMI and to differentiate viable myocardium from scar [69,[71][72][73].

Clinical Results for Ultrasound Associated with Microbubbles as Therapeutic Strategy
AMI is still an important public health problem worldwide and STEMI constitute a high part of the all AMI. In this matter, it is highly recommended to find new strategies to lead to a better outcome in patients suffering of heart disease or presenting for the first time a cardiac event. First, intravenous injections or infusions of perfluorocarbon-exposed sonicated dextrose albumin microbubbles were used in patients with AMI and demonstrated to be very effective to show a persistent contrast defect in the infarct zone following restoration of TIMl grade 3 flow in the infarct vessel which identified patients likely to have deterioration in both regional and global systolic functions [74]. After that, the studies evidenced the success of the US contrast agent technique to open vessels in animal models of acutely thrombosed vessels and AMI [38,39,75]. More recently, this intervention has been for the first time applied for clinical investigation in human. Mathias et al. [12] showed that transthoracic diagnostic high-Mechanical Index (MI) US associated with microbubbles applied in patients with STEMI before invasive percutaneous coronary intervention (PCI) increased the early recanalization rate of the culprit coronary artery to nearly 60% and led to a medium term (6 months) lower LV dysfunction when compared with patients who received PCI only. Recently, the same group conducted the first prospective randomized human study examining the effect of high-MI impulses during a microbubble infusion (sonothrombolysis) in STEMI patients treated with PCI. 13 The authors observed angiographic recanalization in 48% of high-MI PCI patients compared with 20% in PCI only patients. Importantly, infarct size measured by magnetic resonance imaging and LV ejection fraction increased immediately after PCI, remaining higher at six months in the high-MI patients. The clinical application of transthoracic US contrast or sonothrombolysis was recently extended for the first time to treat pulmonary embolism and successful recanalization of thrombotic occlusion in pulmonary artery stent in human was reported [78].

Ultrasound Characteristics
The description of ultrasonic frequency and its desired bioeffect when used for therapy without microbubbles infusion was well presented by Siegel et al. [35] Basically, the ultrasonic frequency range stretches from around 20 kHz (0.02 MHz) up to many megahertz, varying from about 20 kHz (0.02 MHz) to 3 MHz.
In the higher (MHz) range spectrum, low power sound (low energy) will be generated and no chemical reactions are induced. Thus, low-power US in the 2-10 MHz range is employed for diagnostic purpose because sound of such low intensity does not cause any permanent chemical changes in the medium through which it passes. On contrary, at the lower frequencies (just above 0.02 MHz) much higher power can be generated, leading to cavitation in the medium through which it passes, and consequently the potential to cause chemical changes [35].
Thus, the traditional definition of high-power US relies on US frequency range between 0.02 and 0.1 MHz (20 and 100 KHz) and was the range of operation of the majority US generators and was used for treatment of arterial thrombi in recent past. Moreover, the size of microbubbles generated by US during cavitation is inversely proportional to US frequency. Low-frequency US produces large bubbles that in turn produce greater force during their vibration and implosion compared to small bubbles produced at high-frequency US. As US frequency is increased, more power is necessary to produce cavitation [31]. The intensity of the collapsing force is greatly diminished at >1 MHz, and cavitation cannot be produced at all at >2.5 MHz. When higher frequency are used as did in the study of MIZUSHIGE et al. [64] (10-MHz frequency and 1.02 W/ cm2 intensity) compared with others [11,24,34] (45 kHz-1 MHz), smaller change on acoustic pressure will occur and would reduce the US-induced cavitation phenomenon and mechanical damage produced by microbubble streaming will play more important role.
More recently, with the use of microbubbles echocardiography contrast agents, the US frequency range is around 1 to 2 MHz and it is also important consider the combination of the Mechanical Index (MI) that is the power of the US wave. On Table 1  107 and 2 x 108 MBs/mL). This study was conducted motived by the findings that long US tone bursts (up to 5,000 cycles) at high acoustic pressures enhanced the thrombolytic effect in an in vitro flow model [80]. Cavitation detection confirmed continued, albeit diminishing, acoustic activity throughout the 5 ms US excitation.  Of importance is the fact that unexpected high incidence of coronary vasoconstriction was observed using sonolysis of long pulse duration and high-MI to treat STEMI patients [82]. This trial (ROMIUS) was aborted after inclusion of 6 patients because three patients experienced coronary vasoconstriction distal to the culprit artery who were unresponsive to nitroglycerin. Thus, the biosafety for the treatment of coronary thrombose applying long pulse duration is still controversy despite the improved microvascular recovery.
Related to the desired MI to enhance microbubbles cavitation and produce thrombolysis, it is already known the linear relationship between the peak negative pressure (kPa) and mechanical indexes.
As higher peak negative pressure leads to higher microbubbles cavitation, Xie F. et al. [39] tested whether intermittent high-MI impulses from diagnostic US could dissolve intravascular thrombi using intravenous microbubbles in a canine model. For that, they recorded cavitational activity all the way down from 2.0 to 0.5 MI and observed a fall when an MI of < 0.5 was applied. The authors concluded that intravascular cavitational activity occurs within a deeply located thrombosed vessel when high-MI impulses were applied after the replenishment of the insonified field with microbubbles. The angiographic success rate with intermittent high-MI US was of 79% at 45 min, compared with 30% at this time in the low-MI US alone. More importantly, they showed that a diagnostic transducer has the capability of simultaneously being both the guiding transducer (at a low-MI) and the therapeutic transducer (brief 1.9-MI impulses).
As a final point, intermittent therapeutic US mode will be preferred considering that studies applying therapeutic US in an uninterrupted fashion, [83,84] may have reduced the effectiveness of the microbubbles by prohibiting replenishment of microbubbles within the field of interest that would serve as nuclei for the desired cavitation effect. Considering the results of the studies and although knowing that modification of some characteristics of US and even of the microbubbles may potentiate the effect of sonotrombolysis we must keep in mind that undesirable bio-effects should be avoided in the treatment of patients. It seems that additional studies may still bring relevant information to find the ideal pulse duration or MI or other characteristic in order to maximize the bio-effects without causing serious harm to the patient.