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Therapeutic Use of Microbubbles and Ultrasound in Acute Peripheral Arterial Thrombosis: A Systematic Review

Open AccessPublished:July 13, 2021DOI:https://doi.org/10.1016/j.ultrasmedbio.2021.06.001

      Abstract

      Catheter-directed thrombolysis (CDT) for acute peripheral arterial occlusion is time consuming and carries a risk of major hemorrhage. Contrast-enhanced sonothrombolysis (CEST) might enhance outcomes compared with standard CDT. In the study described here, we systematically reviewed all in vivo studies on contrast-enhanced sonothrombolysis in a setting of arterial thrombosis. A systematic search of the PubMed, Embase, Cochrane Library and Web of Science databases was conducted. Two reviewers independently performed the study selection, quality assessment and data extraction. Primary outcomes were recanalization rate and thrombus weight. Secondary outcome was any possible adverse event. The 35 studies included in this review were conducted in four different (pre)clinical settings: ischemic stroke, myocardial infarction, (peripheral) arterial thrombosis and arteriovenous graft occlusion. Because of the high heterogeneity among the studies, it was not possible to conduct a meta-analysis. In almost all studies, recanalization rates were higher in the group that underwent a form of CEST. One study was terminated early because of a higher incidence of intracranial hemorrhage. Studies on CEST suggest that adding microbubbles and ultrasound to standard intra-arterial CDT is safe and might improve outcomes in acute peripheral arterial thrombosis. Further research is needed before CEST can be implemented in daily practice.

      Key Words

      Introduction

      Peripheral arterial disease represents an affliction in which one or more non-coronary arteries are partially or completely occluded, leading to compromised blood flow and eventually ischemia (
      • Morcos R
      • Louka B
      • Tseng A
      • Misra S
      • McBane R
      • Esser H
      • Shamoun F.
      The evolving treatment of peripheral arterial disease through guideline-directed recommendations.
      ). With rare exceptions, atherosclerosis remains the most common pathology underlying this affliction. The estimated number of people that are affected ranges from 30–200 million worldwide (
      • Yurtkuran AJ
      • Tok M
      • Emel E.
      A clinical decision support system for femoral peripheral arterial disease treatment.
      ;
      • Morcos R
      • Louka B
      • Tseng A
      • Misra S
      • McBane R
      • Esser H
      • Shamoun F.
      The evolving treatment of peripheral arterial disease through guideline-directed recommendations.
      ). Peripheral arterial disease in an advanced stage can cause acute limb ischemia (ALI), usually caused by a thrombus that completely occludes a blood vessel. ALI is a life-threatening condition that may result in amputation or even death. Advancing age, chronic kidney disease and diabetes are all known risk factors for development of atherosclerosis. With an aging world population and a rising incidence of both chronic kidney disease and diabetes, peripheral arterial disease—and with it ALI—is likely to dramatically increase in the near future (
      • Falluji N
      • Mukherjee D.
      Critical and acute limb ischemia: An overview.
      ). Rapid treatment is essential to restore blood flow to the extremity (
      • Santistevan JR.
      Acute limb ischemia: An emergency medicine approach.
      ).
      In the majority of cases of ALI, treatment will consist of catheter-directed thrombolysis (CDT), with only a small percentage of patients (13.1% in a study by
      • Davis FM
      • Albright J
      • Gallagher KA
      • Gurm HS
      • Koenig GC
      • Schreiber T
      • Grossman PM
      • Henke PK.
      Early outcomes following endovascular, open surgical, and hybrid revascularization for lower extremity acute limb ischemia.
      ) still needing primary surgical revascularization. With CDT, an intra-arterial catheter is guided to the site of the occlusion and used to administer the thrombolytic agent locally. Although CDT has the benefit of being minimally invasive, procedures are time consuming and expensive and carry a risk of major bleeding (
      • Falluji N
      • Mukherjee D.
      Critical and acute limb ischemia: An overview.
      ). Current thrombolytic therapy is effective in many cases, but a simpler, safer and non-invasive treatment is urgently required to improve on patient outcomes and lower the burden for both the patient and the health care system. A new treatment currently being researched is the addition of ultrasound (US)-induced microbubble cavitation to standard CDT (sCDT), that is, contrast-enhanced sonothrombolysis (CEST) (
      • Unger E
      • Porter T
      • Lindner J
      • Grayburn P.
      Cardiovascular drug delivery with ultrasound and microbubbles.
      ). Microbubbles (MBs), also known as US contrast agents, oscillate and collapse under the influence of US. The oscillations and cavitation cause highly energetic microflows near the thrombus, thereby destroying the (surface) structure of the thrombus. This leads to greater exposure of the thrombus surface to the lytic agent and, consequently, can accelerate thrombolysis. Another development in CEST is the ability to load the microbubble with a lytic agent, such as tissue plasminogen activator (tPA) or urokinase. In addition, arginine–glycine–aspartic acid–serine peptide (RGDS) can be added to the surface of the microbubble as a targeting ligand for thrombus (
      • Nederhoed JH
      • Ebben HP
      • Slikkerveer J
      • Hoksbergen AWJ
      • Kamp O
      • Tangelder GJ
      • Wisselink W
      • Musters RJP
      • Yeung KK.
      Intravenous targeted microbubbles carrying urokinase versus urokinase alone in acute peripheral arterial thrombosis in a porcine model.
      ). Both developments might increase the efficacy of CEST even further, as the MBs will adhere to the surface of the thrombus and the lytic agent can be released locally. Moreover, these factors might also reduce bleeding risks and cost of the treatment. CEST holds considerable promise for therapeutic use in acute peripheral arterial thrombosis. Several studies have been conducted on recanalization using microbubbles (MBs) and US; however, their therapeutic use in peripheral arterial thrombosis has been reported in only a few studies (
      • Ebben HP
      • Nederhoed JH
      • Slikkerveer J
      • Kamp O
      • Tangelder GWJM
      • Musters RJP
      • Wisselink W
      • Yeung KK.
      Therapeutic application of contrast-enhanced ultrasound and low-dose urokinase for thrombolysis in a porcine model of acute peripheral arterial occlusion.
      ;
      • Nederhoed JH
      • Ebben HP
      • Slikkerveer J
      • Hoksbergen AWJ
      • Kamp O
      • Tangelder GJ
      • Wisselink W
      • Musters RJP
      • Yeung KK.
      Intravenous targeted microbubbles carrying urokinase versus urokinase alone in acute peripheral arterial thrombosis in a porcine model.
      ). As very little research has been done on CEST in the setting of peripheral arterial thrombosis, we investigated the effectiveness and safety of CEST in any setting of arterial thrombosis, excluding articles on microvascular arterial thrombosis. The main objective of this systematic review is to give an overview of all relevant in vivo studies on the subject of CEST in arterial thrombosis and to raise awareness of the possibility of CEST among specialists treating acute peripheral arterial thrombosis. Our primary outcomes are recanalization rate and thrombus weight. Our secondary outcome is any adverse event that might be related to the given treatment.

      Methods

      Search strategy and selection criteria

      This study was performed in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) statement (
      • Liberati A
      • Altman DG
      • Tetzlaff J
      • Mulrow C
      • Gøtzsche PC
      • Ioannidis JP
      • Clarke M
      • Devereaux PJ
      • Kleijnen J
      • Moher D.
      The PRISMA statement for reporting systematic reviews and meta-analyses of studies that evaluate health care interventions: Explanation and elaboration.
      ). To identify all relevant publications on the therapeutic use of MBs in peripheral arterial thrombosis, three authors (one of whom is an experienced research librarian) systematically searched the bibliographic databases PubMed, Embase, Cochrane Library (via Wiley) and Web of Science for publications up to February 3, 2020. Search terms expressing “microbubbles” were used in combination with search terms comprising “thrombosis,” and both controlled terms (MeSH in PubMed and Emtree in Embase) and free text terms (The Cochrane Library) were used. After the initial search, the title and abstract of all citations were independently screened by two of the authors. Included were all studies that investigated therapeutic use of MBs and US in arterial thrombosis in an in vivo setting (human or animal) and that reported the outcome measures recanalization rate and/or thrombus weight. There were no limitations on language. Articles were excluded if they did not report original data or consisted only of case reports or where the full text was not available. As the focus of this review is CEST in peripheral arterial thrombosis, we also excluded articles in which the therapeutic US was not directed at the occluded vessel, but at the outflow territory. All articles were screened for statements on approval by a local ethics committee, adherence to guidelines for animal care and/or obtaining informed consent. As articles that had no such statement might still contain valuable data, this was not used as an exclusion criterion, but an annotation was added in the Results section. For the complete search strategy see the appendix.

      Quality assessment

      Quality assessment of the included studies was done independently by the two previously mentioned authors, using the SYstematic Review Centre for Laboratory animal Experimentation (SYRCLE) tool for animal studies, the Methodological Index for Non-Randomized Studies (MINORS) criteria for non-randomized studies in humans and the Cochrane Risk of Bias tool for randomized trials in a clinical setting (
      • Slim K
      • Nini E
      • Forestier D
      • Kwiatkowski F
      • Panis Y
      • Chipponi J.
      Methodological Index for Non-Randomized Studies (MINORS): Development and validation of a new instrument.
      ;
      • Hooijmans CR
      • Rovers MM
      • de Vries RBM
      • Leenaars M
      • Ritskes-Hoitinga M
      • Langendam MW.
      SYRCLE's risk of bias tool for animal studies.
      ;
      • Sterne JAC
      • Savović J
      • Page MJ
      • Elbers RG
      • Blencowe NS
      • Boutron I
      • Cates CJ
      • Cheng HY
      • Corbett MS
      • Eldridge SM
      • Emberson JR
      • Hernán MA
      • Hopewell S
      • Hróbjartsson A
      • Junqueira DR
      • Jüni P
      • Kirkham JJ
      • Lasserson T
      • Li T
      • McAleenan A
      • Reeves BC
      • Shepperd S
      • Shrier I
      • Stewart LA
      • Tilling K
      • White IR
      • Whiting PF
      • Higgins JPT
      RoB 2: A revised tool for assessing risk of bias in randomised trials.
      ). Disagreements between the two reviewers were resolved via consensus meetings.

      Data extraction

      Two authors extracted the data following a pre-defined schedule that included study design, subject studied (human or animal), number of subjects, location and duration of arterial thrombosis, type of microbubble used, dosage of MBs, site of injection (intravenous or intra-arterial), type of fibrinolytic agent used including dosage and site of injection, US settings, duration of therapy given, outcomes (recanalization rate and/or thrombus weight) and possible adverse events. When looking at the recanalization rate (primary outcome), the method for determining recanalization was noted, as was the definition of successful recanalization. Mortality and intracranial hemorrhage (ICH) were scored separately as possible adverse events (secondary outcome), and whether ICH was symptomatic (sICH) was noted. Treatment protocols that combined the use of MB, US and a fibrinolytic agent were scored as the CEST arm of the study.

      Results

      After removal of duplicates, 6953 records were identified for screening. On the basis of our inclusion and exclusion criteria, 35 studies were included in this review: 26 pre-clinical and 9 clinical trials, 3 of which were randomized. See Figure 1 for the PRISMA flow diagram and Table 14 for an overview of the selected studies.
      Fig 1
      Fig. 1Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) flow diagram.
      Table 1Overview of included trials: Pre-clinical, Part 1
      No.StudyObjectiveConclusion
      1
      • 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: An in vitro and in vivo study.
      To investigate the enhancing effect of MBs on the in vivo recanalization rate of thrombotically occluded rabbit iliofemoral arteries by transcutaneous US exposureDodecafluoropentane emulsion significantly enhances the clot-disrupting effect of low-frequency US.
      2
      • Birnbaum Y
      • Luo H
      • Nagai T
      • Fishbein FC
      • Peterson TM
      • Li S
      • Kricsfeld D
      • Porter TR
      • Siegel RJ.
      Noninvasive in vivo clot dissolution without a thrombolytic drug recanalization of thrombosed iliofemoral arteries by transcutaneous ultrasound combined with intravenous infusion of microbubbles.
      To examine the efficacy of arterial clot disruption using a non-invasive, non-lytic approach with intravenous administration of PESDA and transcutaneous delivery of US aloneIn vivo arterial clot dissolution can be achieved with intravenous MBs and transcutaneous US delivery alone.
      3
      • Culp WC
      • Porter TR
      • Xie F
      • Goertzen TC
      • McCowan TC
      • Vonk BN
      • Baxter BT.
      Microbubble potentiated ultrasound as a method of declotting thrombosed dialysis grafts: Experimental study in dogs.
      To develop a method to de-clot full-size arteriovenous dialysis grafts using intravenous PESDA MBs and LFUSDirect injection of PESDA with transcutaneous LFUS succeeds in lysing moderate-size clots and recanalizing thrombosed fistulas.
      4
      • Porter TR
      • Kricsfeld D
      • Lof J
      • Everbach EC
      • Xie F.
      Effectiveness of transcranial and transthoracic ultrasound and microbubbles in dissolving intravascular thrombi.
      To examine the effectiveness of different US frequencies in recanalizing thrombosed vessels in a simple attenuation modelTranscranial and transthoracic US in the presence of intravenous MBs can improve flow to ischemic regions.
      5
      • Culp WC
      • Porter TR
      • McCowan TC
      • Roberson PK
      • James CA
      • Jean Matchett W
      • Moursi M
      Microbubble-augmented ultrasound declotting of thrombosed arteriovenous dialysis grafts in dogs.
      To determine the utility of Food and Drug Administration-approved MBs for arteriovenous graft de-clotting, establish efficacy levels and assess US parameters including intensity and wave mode applications for this purposeLFUS with direct injection of MBs is effective in lysing moderate-sized clots and recanalizing thrombosed arteriovenous grafts. It best succeeds at the higher range of intensity settings tested in pulsed wave mode.
      6
      • Culp WC
      • Porter TR
      • Lowery J
      • Xie F
      • Roberson PK
      • Marky L.
      Intracranial clot lysis with intravenous microbubbles and transcranial ultrasound in swine.
      To determine that transtemporal LFUS used with MBs tagged with a low-dose glycoprotein 2b/3a receptor antagonist can recanalize acutely thrombosed intracranial vesselsIntravenous platelet-targeted MBs combined with transcranial LFUS can rapidly open acute intracranial thrombotic occlusions.
      7
      • Xie F
      • Tsutsui JM
      • Lof J
      • Unger EC
      • Johanning J
      • Culp WC
      • Matsunaga T
      • Porter TR.
      Effectiveness of lipid microbubbles and ultrasound in declotting thrombosis.
      To determine the effectiveness of lipid-encapsulated MBs and US in recanalizing arteriovenous graft thrombi and the effect that tissue attenuation has on the success rateUS and MBs are capable of recanalizing acute arteriovenous graft thromboses. Higher intensities may be needed in the presence of tissue attenuation.
      8
      • Tsutsui JM
      • Xie F
      • Johanning J
      • Lof J
      • Cory B
      • He A
      • Thomas L
      • Matsunaga T
      • Unger E
      • Porter TR
      Treatment of deeply located acute intravascular thrombi with therapeutic ultrasound guided by diagnostic ultrasound and intravenous microbubbles.
      To determine the added value of simultaneous imaging of intravenously infused MBs that are being used to dissolve an intravascular thrombus with therapeutic USThe use of therapeutic US with intravenous MBs has a high success rate in recanalizing deeply located thrombosed arteriovenous grafts when performed with DUS guidance.
      9
      • Xie F
      • Lof J
      • Everbach EC
      • He A
      • Bennett RM
      • Matsunaga T
      • Johanning J
      • Porter TR.
      Treatment of acute intravascular thrombi with diagnostic ultrasound and intravenous microbubbles.
      To determine whether diagnostic US, in the presence of MBs, would be capable of recanalizing deeply located intravascular thrombi without the need for fibrinolysis or systemic anticoagulationDiagnostic US can recanalize thrombosed vessels without a fibrinolytic agent. High-MI US has a higher recanalization rate than low-MI US.
      10
      • Xie F
      • Lof J
      • Matsunaga T
      • Zutshi R
      • Porter TR.
      Diagnostic ultrasound combined with glycoprotein IIb/IIIa–targeted microbubbles improves microvascular recovery after acute coronary thrombotic occlusions.
      To determine whether a low-MI MB-sensitive imaging system could be used to guide high-MI impulses from the same diagnostic transducer during a platelet-targeted MB infusion and improve both epicardial and microvascular flow in a pig model of acute coronary thrombosisIntravenous platelet-targeted MBs combined with brief high-MI diagnostic US impulses guided by contrast pulse sequencing improve both epicardial recanalization rates and microvascular recovery.
      11Laing et al. 2011To test whether TELIPs with US confer thrombolytic efficacy similar (or superior) to that of other clinically used or proposed tPA treatment modalities for ischemic strokesThe thrombolytic efficacy of TELIPs is comparable to that of other clinically described effective treatment protocols, while offering the advantages of US monitoring and enhanced thrombolysis from a site-specific delivery agent.
      12
      • Ren ST
      • Long LH
      • Wang M
      • Li YP
      • Qin H
      • Zhang H
      • Jing BB
      • Li YX
      • Zang WJ
      • Wang B
      • Shen XL.
      Thrombolytic effects of a combined therapy with targeted microbubbles and ultrasound in a 6 h cerebral thrombosis rabbit model.
      To address whether P1 polypeptide-loaded TMBs can prolong the 3-h therapeutic window in cerebral thrombosis and achieve a higher recanalization rate with a lower risk of cerebral hemorrhageTMBs/LFUS is an effective and safe therapy for thrombolysis in a 6-h cerebral thrombosis rabbit model.
      13
      • Liu WS
      • Huang ZZ
      • Wang XW
      • Zhou J.
      Effects of microbubbles on transcranial Doppler ultrasound-assisted intracranial urokinase thrombolysis.
      To evaluate the efficacy of MBs in TCD-assisted urokinase thrombolysisThe addition of MBs enhanced the effects of TCD-assisted urokinase thrombolysis.
      14
      • Xie F
      • Gao S
      • Wu J
      • Lof J
      • Radio S
      • Vignon F
      • Shi W
      • Powers J
      • Unger E
      • Everbach EC
      • Liu J
      • Porter TR.
      Diagnostic ultrasound induced inertial cavitation to non-invasively restore coronary and microvascular flow in acute myocardial infarction.
      To determine the type of cavitation required for successfully dissolving intravascular and microvascular thrombi in acute myocardial infarction and whether longer-pulse-duration therapeutic impulses (sustaining the duration of cavitation) could restore both microvascular and epicardial flow with this technique.Although short-pulse-duration guided therapeutic impulses from a diagnostic transducer transiently improve microvascular flow, long-pulse-duration therapeutic impulses produce sustained epicardial and microvascular re-flow in acute myocardial infarction
      15
      • Hagisawa K
      • Nishioka T
      • Suzuki R
      • Maruyama K
      • Takase B
      • Ishihara M
      • Kurita A
      • Yoshimoto N
      • Nishida Y
      • Iida K
      • Luo H
      • Siegel RJ.
      Thrombus-targeted perfluorocarbon-containing liposomal bubbles for enhancement of ultrasonic thrombolysis: in vitro and in vivo study.
      To examine the enhancing effect of thrombus-targeted BLs developed for fresh thrombus imaging during ultrasonic thrombolysis.TIMI grade 3 flow was present in a significantly larger number of rabbits with USD and targeted BLs than rabbits with USD and non-targeted BLs or with rtPA monotherapy.
      16
      • Gao S
      • Zhang Y
      • Wu J
      • Shi WT
      • Lof J
      • Vignon F
      • Drvol L
      • Xie F
      • Muirhead D
      • Powers JE
      • High R
      • White ML
      • Porter TR.
      Improvements in cerebral blood flow and recanalization rates with transcranial diagnostic ultrasound and intravenous microbubbles after acute cerebral emboli.
      To determine whether skull attenuation would limit the ability of US alone to induce the type and level of cavitation required to dissolve thrombi and improve CBF in acute ischemic strokeGuided high-MI impulses from a US imaging system produce sustained improvements in ipsilateral and contralateral CBF after acute cerebral emboli.
      17
      • Hua X
      • Zhou L
      • Liu P
      • He Y
      • Tan K
      • Chen Q
      • Gao Y
      • Gao Y.
      In vivo thrombolysis with targeted microbubbles loading tissue plasminogen activator in a rabbit femoral artery thrombus model.
      To investigate the thrombolytic efficacy of a novel type of MB carrying tPA that can bind to a fresh thrombus via the arginine–glycine–aspartic acid–serine polypeptide in vivo under US exposureUS-Induced targeted tPA-loaded MB release is a promising thrombolytic method with satisfactory thrombolytic efficacy, lowered tPA dose and potentially decreased hemorrhagic risk.
      18
      • Ebben HP
      • Nederhoed JH
      • Slikkerveer J
      • Kamp O
      • Tangelder GWJM
      • Musters RJP
      • Wisselink W
      • Yeung KK.
      Therapeutic application of contrast-enhanced ultrasound and low-dose urokinase for thrombolysis in a porcine model of acute peripheral arterial occlusion.
      To investigate the effect of additional US and MBs on standard low-dose intra-arterial thrombolysis in a porcine model of extensive peripheral arterial occlusionThe addition of contrast-enhanced US accelerated the thrombolytic effect of low-dose intra-arterial thrombolysis in peripheral arterial occlusions
      19
      • Wu J
      • Xie F
      • Lof J
      • Sayyed S
      • Porter TR.
      Utilization of modified diagnostic ultrasound and microbubbles to reduce myocardial infarct size.
      To determine whether guided high-MI impulses from a diagnostic US transducer during an intravenous MB infusion could augment low-dose fibrinolytic therapy in treating acute myocardial infarction (ST-segment elevation myocardial infarction)Guided high-MI-induced MB cavitation from a diagnostic transducer added to low-dose tPA can immediately improve regional function and reduce infarct size in acute ST-segment elevation myocardial infarction.
      20
      • Tomkins AJ
      • Schleicher N
      • Murtha L
      • Kaps M
      • Levi CR
      • Nedelmann M
      • Spratt NJ.
      Platelet rich clots are resistant to lysis by thrombolytic therapy in a rat model of embolic stroke.
      To investigate the effect on recanalization rates of tPA therapy alone or in conjunction with US and a new MB formulation (BR38) in a model with platelet-rich clotsThese platelet-rich clots were highly resistant to tPA with or without MB-enhanced sonothrombolysis
      21
      • Ren X
      • Wang Y
      • Wang Y
      • Chen H
      • Chen L
      • Liu Y
      • Xue C.
      Thrombolytic therapy with rt-PA and transcranial color Doppler ultrasound (TCCS) combined with microbubbles for embolic thrombus.
      To investigate the efficacy of TCCS combined with MBs and rtPA for thrombolysis in vivoTCCS + MBs combined with rtPA is a relatively effective approach for ischemic arterial thrombosis with an additive or synergistic effect.
      22
      • Zhu Y
      • Guan L
      • Mu Y.
      Combined low-frequency ultrasound and urokinase-containing microbubbles in treatment of femoral artery thrombosis in a rabbit model.
      To study the thrombolytic effect of low-frequency US combined with targeted urokinase-containing MB contrast agents on treatment of thrombosis in rabbit femoral artery and to determine the optimal combination of parameters for achieving thrombolysis in this modeThe optimal parameters for thrombolysis were determined to be (i) a US frequency of 2.2 MHz and (ii) a 90,000 IU/kg dose of urokinase. US exposure time (30 min vs. 60 min) had no significant effect on the thrombolytic effects.
      23
      • Nederhoed JH
      • Ebben HP
      • Slikkerveer J
      • Hoksbergen AWJ
      • Kamp O
      • Tangelder GJ
      • Wisselink W
      • Musters RJP
      • Yeung KK.
      Intravenous targeted microbubbles carrying urokinase versus urokinase alone in acute peripheral arterial thrombosis in a porcine model.
      To compare intravenously administered targeted MB incorporating urokinase and locally applied US, with intravenous urokinase and US aloneMinimally invasive thrombolysis using intravenously targeted MBs carrying urokinase combined with US is feasible and might accelerate thrombolysis compared with treatment with urokinase and US alone.
      24
      • Porter TR
      • Xie F
      • Lof J
      • Powers J
      • Vignon F
      • Shi W
      • White M.
      The thrombolytic effect of diagnostic ultrasound induced microbubble cavitation in acute carotid thromboembolism.
      To examine the effect of different forms of DUS-induced cavitation in restoring branch flow following carotid artery thrombosis in a porcine model of common carotid stenosis and thromboembolismHigh-MI 20-μs pulse duration impulses during a commercial MB infusion can be used to recanalize acutely thrombosed carotid arteries and restore downstream flow without anticoagulants. However, this effect is seen only with stable cavitation-inducing impulses and not at higher mechanical indices, when a paradoxical reversal of the thrombolytic effect is observed.
      25
      • Cui H
      • Zhu Q
      • Gao Y
      • Xia H
      • Tan K
      • He Y
      • Liu Z
      • Xu Y.
      Ultrasound mediated microbubbles destruction augmented sonolysis: An in vitro and in vivo study.
      To determine whether combining TUS with MBs could accelerate thrombolysis in in vitro and in vivo studies without the use of fibrinolytic drugsRecanalization rates and flow scores in the TUS + MB group were significantly higher than those in the control and TUS groups.
      26
      • Chen X
      • Wu W
      • Wang W
      • Zhong J
      • Moumin Djama N
      • Wei G
      • Lai Y
      • Si X
      • Cao S
      • Liao W
      • Liao Y
      • Li H
      • Bin J
      Magnetic targeting improves the therapeutic efficacy of microbubble-mediated obstructive thrombus sonothrombolysis.
      To investigate the feasibility and efficacy of magnetically targeted MB-mediated sonothrombolysis for the treatment of obstructive thrombiThe recanalization rate, average blood flow velocity, and hindlimb perfusion in the red and white thromboembolic models were all significantly higher in the US + magnetic MBs and US + magnetic MBs + rtPA groups than in the control and US + control MB groups.
      No statement was included on adherence to guidelines for animal care or obtaining permission from a local ethics committee.
      BLs = bubble liposomes; CBF = cerebral blood flow; DUS = diagnostic ultrasound; LFUS = low-frequency ultrasound; MBs = microbubbles; MI = mechanical index; PESDA = perfluorocarbon-exposed sonicated dextrose albumin; rtPA = recombinant tissue plasminogen activator; tPA = tissue plasminogen activator; TCD = transcranial Doppler ultrasound; TMBs = targeted microbubbles; TELIPs = tissue plasminogen activator-loaded echogenic liposomes; TIMI = thrombolysis in myocardial infarction; TUS = therapeutic ultrasound; US = ultrasound.
      Table 2Overview of included trials: Preclinical, Part 2
      No.No. of treatments (subjects)Location of thrombosisAge of clot (min)MicrobubblesFibrinolytic agentReported outcome
      Recanalization rateThrombus weightSafety
      134 (17)

      Rabbit
      IFA<30EchoGen (dodecafluoro-pentane)NoneX..X
      225 (13)

      Rabbit
      IFA<30PESDANoneX..X
      326 (3)

      Canine
      FAVG≥60PESDANoneX..X
      415 (17)

      Porcine
      CA (LCX )<30PESDANoneX..X
      5104 (5)

      Canine
      FAVG≥60OptisonNoneX..X
      623 (15)

      Porcine
      APA120–420PESDANoneX....
      755 (4)

      Canine
      FAVG240DefinityNoneX....
      824 (1)

      Canine
      FAVG240MRX815NoneX..X
      924 (2)

      Canine
      FAVG240MRX-801NoneX....
      1045

      Porcine
      CA (LAD)≥20MRX-835 + MRX-802Pro-urokinaseX..X
      1159

      Rabbit
      AA10Intrinsically echogenic lipid microbubbles and DefinitytPAX....
      1247 (52)

      Rabbit
      CCA360DPPG/DSPE-PEG-BTC microbubbletPAX..X
      1332

      Rabbit
      MCA60SonoVueUrokinaseX..X
      1436

      Porcine
      CA (LAD)≥20MRX-801tPAX....
      1554

      Rabbit
      IFA<30Perfluorocarbon filled lipid MBstPAX....
      1624

      Porcine
      ICA, APA and rete255DefinityNoneX..X
      1770 (40)

      Rabbit
      IFA30HM perfluoropropane/polyethylene glycoltPAX....
      1810

      Porcine
      IFA100SonoVueUrokinaseXXX
      1932

      Porcine
      CA (LAD)≥20MRX-801tPAX..X
      2030

      Rodent
      MCA>480BR38tPAX..X
      2130

      Rodent
      CCA180-240SonoVuetPAX....
      2272

      Rabbit
      IFA≥20HM biotinylated urokinase and RGDS Targestar SAUrokinaseX....
      239

      Porcine
      IFA100SonoVue (RGDS-targeted MBs)UrokinaseXXX
      2438 (21)

      Porcine
      CCA20DefinityNoneX..X
      2536

      Rabbit
      IFA120Perfluorocarbon-filled lipid MBsNoneX....
      2640 (20)

      Rodent
      IFA60Magnetic (streptavidin beads to lipid shell) and normal lipid MBstPAX....
      AA = abdominal aorta; APA = ascending pharyngeal artery; BA = basilar artery; BLs = bubble liposomes; CA = coronary artery; CCA = common carotid artery; DPPG/DSPE-PEG-BTC = dipalmitoylphosphatidylethanolamine/distearoylphosphatidylethanolamine- polyethylene glycol-benzotriazole carbonate (home made); FAVG = femoral arteriovenous graft; ICA = internal carotid artery; IFA = iliofemoral artery; LAD = left anterior descending; LCX = left circumflex; MBs = microbubbles; MCA = middle cerebral artery; PCA = posterior cerebral artery; PESDA = perfluorocarbon-exposed sonicated dextrose albumin; RCA = right coronary artery; RGDS = arginine–glycine–aspartic acid–serine peptide; .. = not reported.
      Table 3Overview of included trials: Clinical, Part 1
      No.StudyObjectiveConclusion
      1
      • Molina CA
      • Ribo M
      • Rubiera M
      • Montaner J
      • Santamarina E
      • Delgado-Mederos R
      • Arenillas JF
      • Huertas R
      • Purroy F
      • Delgado P
      • Alvarez-Sabín J.
      Microbubble administration accelerates clot lysis during continuous 2-MHz ultrasound monitoring in stroke patients treated with intravenous tissue plasminogen activator.
      To evaluate the effects of administration of MBs on the beginning, speed and degree of MCA recanalization during systemic thrombolysis and continuous 2-MHz pulsed-wave TCD monitoringAdministration of MBs induces further acceleration of US-enhanced thrombolysis in acute stroke, leading to a more complete recanalization and to a trend toward better short- and long-term outcome
      2
      • Perren F
      • Loulidi J
      • Poglia D
      • Landis T
      • Sztajzel R.
      Microbubble potentiated transcranial duplex ultrasound enhances IV thrombolysis in acute stroke.
      To study whether TCCD US, combined with a second-generation phospholipid ECA, accelerates intravenous rtPA-thrombolysis in the acute phase of MCA stroke more than TCCD US monitoring aloneECA-Enhanced TCCD-monitored rtPA thrombolysis is superior to TCCD-monitored rtPA thrombolysis in terms of residual flow improvement as measured by TIBI, especially during the first 30 min. It is also superior in the immediate (24 h) clinical improvement as measured with the National Institutes of Health Stroke Scale/Score. The rate of hemorrhagic transformation did not differ between the two groups.
      3
      • Pagola J
      • Ribo M
      • Alvarez-Sabín J
      • Lange M
      • Rubiera M
      • Molina CA.
      Timing of recanalization after microbubble-enhanced intravenous thrombolysis in basilar artery occlusion.
      To determine the timing of recanalization in basilar artery occlusion treated with systemic thrombolysis, MBs and continuous TCD monitoringCombined treatment with intravenous tPA, MBs and continuous US in acute basilar artery occlusion leads to early recanalization in a significant number of patients; this is associated with favorable outcomes.
      4
      • Alexandrov AV
      • Mikulik R
      • Ribo M
      • Sharma VK
      • Lao AY
      • Tsivgoulis G
      • Sugg RM
      • Barreto A
      • Sierzenski P
      • Malkoff MD
      • Grotta JC.
      A pilot randomized clinical safety study of sonothrombolysis augmentation with ultrasound-activated perflutren-lipid microspheres for acute ischemic stroke.
      To test the feasibility and safety of novel lipid-coated MBs containing perflutren, which are consistent in size (1 to 2 μm) and more stable in saline solutionPerflutren MBs reached and permeated beyond intracranial occlusions with no increase in symptomatic intracranial hemorrhage after systemic thrombolysis, suggesting the feasibility of further MB dose-escalation studies and development of drug delivery to tissues with compromised perfusion.
      5
      • Rubiera M
      • Ribo M
      • Delgado-Mederos R
      • Santamarina E
      • Maisterra O
      • Delgado P
      • Montaner J
      • Alvarez-Sabín J
      • Molina CA.
      Do bubble characteristics affect recanalization in stroke patients treated with microbubble-enhanced sonothrombolysis?.
      To compare the effect of galactose-based air-filled MBs (Levovist) and sulfur hexafluoride-filled MBs (Sonovue) on recanalization and clinical outcome in sonothrombolysis for acute ischemic strokeMB administration during sonothrombolysis is associated with a high recanalization rate. Recanalization rates, clinical course and long-term outcome are comparable when administering galactose-based air-filled MBs or sulfur hexafluoride-filled MBs.
      6
      • Dinia L
      • Rubiera M
      • Ribo M
      • Maisterra O
      • Ortega G
      • del Sette M
      • Alvarez-Sabín J
      • Molina CA.
      Reperfusion after stroke sonothrombolysis with microbubbles may predict intracranial bleeding.
      To investigate the risk of hemorrhagic transformation after MB-enhanced sonothrombolysis in acute strokeMicrobubble administration was associated with early recanalization and a high rate of hemorrhagic transformation but does not seem to increase the risk of symptomatic intracranial hemorrhage
      7
      • Molina CA
      • Barreto AD
      • Tsivgoulis G
      • Sierzenski P
      • Malkoff MD
      • Rubiera M
      • Gonzales N
      • Mikulik R
      • Pate G
      • Ostrem J
      • Singleton W
      • Manvelian G
      • Unger EC
      • Grotta JC
      • Schellinger PD
      • Alexandrov AV.
      Transcranial Ultrasound in Clinical Sonothrombolysis (TUCSON) trial.
      To study the safety, tolerability, and activity of perflutren-lipid MBs and MRX-801 2-MHz TCD insonation as an adjuvant to intravenous tPA, and to conduct a phase I–II safety dose escalation studyPerflutren–lipid MBs can be safely combined with systemic tPA and US at a dose of 1.4 mL. Safety concerns in the second dose tier may necessitate extended enrollment and further experiments to determine the mechanisms by which MBs interact with tissues
      8
      • Ribo M
      • Molina CA
      • Alvarez B
      • Rubiera M
      • Alvarez-Sabin J
      • Matas M.
      Intra-arterial administration of microbubbles and continuous 2-MHz ultrasound insonation to enhance intra-arterial thrombolysis.
      To evaluate the safety and efficacy of local MB administration in MCA recanalization during intra-arterial thrombolysis and continuous TCD monitoringThe combination of ultrasound and intra-arterial MBs and tPA may be a strategy to enhance the thrombolytic effect and increase recanalization rates
      9
      • Slikkerveer J
      • Kleijn SA
      • Appelman Y
      • Porter TR
      • Veen G
      • van Rossum AC
      • Kamp O.
      Ultrasound enhanced prehospital thrombolysis using microbubbles infusion in patients with acute ST elevation myocardial infarction: Pilot of the Sonolysis study.
      To determine the safety and feasibility of treatment with low-dose thrombolytics in combination with ultrasound and microbubbles prior to percutaneous coronary intervention to increase the epicardial recanalization rates in patients with a first acute ST-segment-elevated myocardial infarctionNo significant differences between the treatment and control groups in safety (minor adverse events 2/5 vs. 2/5, p = NS) and outcome (TIMI III flow 3/5 vs. 1/5 respectively, p = 0.23) were recorded. These results demonstrate that the study protocol is feasible in the acute cardiac care setting and safe during treatment and follow-up.
      MBs = microbubbles; MCA = middle cerebral artery; NS = non-significant; TCD = transcranial Doppler; TCCD – transcranial color-coded duplex sonography; tPA = tissue plasminogen activator; US = ultrasound.
      Table 4Overview of included trials: Clinical, Part 2
      Reported outcomes
      No.No. of patientsLocation of thrombosisTime to treatment (min)MicrobubblesFibrinolytic agentRecanalization rateThrombus weightSafety
      1111MCA156LevovisttPAX..X
      226MCA<180SonoVuetPAX..X
      320BA180 (<720)LevovisttPAX..X
      415MCA<180Perflutren–lipidtPAX..X
      5138MCA178 (mean)Levovist and SonoVuetPAX..X
      6286ICA (38), MCA (248)158 (<180)Galactose-basedtPAX..X
      735MCA (33), PCA (2)126–139 (mean)MRX-801tPAX..X
      818MCA175 (median)LevovisttPAX..X
      910CA (LAD 7, RCA 3)134–177LuminitytPAX..X
      AA = abdominal aorta; APA = ascending pharyngeal artery; BA = basilar artery; BLs = bubble liposomes; CA = coronary artery; CCA = common carotid artery; FAVG = femoral arteriovenous graft; ICA = internal carotid artery; IFA = iliofemoral artery; LAD = left anterior descending; LCX = left circumflex; MB = microbubbles; MCA = middle cerebral artery; PCA = posterior cerebral artery; RCA = right coronary artery; tPA = tissue plasminogen activator.
      *No statement was included on adherence to guidelines for animal care or obtaining permission from a local ethics committee.
      †No statement was given on approval of a local ethics committee or obtaining informed consent. Approval was given by a local ethics committee, but no statement on obtaining informed consent was included in the article.

      Quality assessment

      Fig. 2, Fig. 3, Fig. 4 summarize the results of quality assessment. The pre-clinical studies all had a risk of bias because of problems in allocation generation and/or concealment. Baseline characteristics were unclear in most of these trials and, save for one study, caregivers and investigators were not blinded to the treatment given. Most studies did not report on the housing of the animals, but as these experiments were short, this would have had a minimal impact on the outcomes.
      Fig 2
      Fig. 2Results of quality assessment: Systematic Review Center for Laboratory Animal Experimentation (SYRCLE)’s tool.
      Fig 3
      Fig. 3Results of quality assessment: Methodological Index for Non-Randomized Studies (MINORS) criteria.
      Fig 4
      Fig. 4Results of quality assessment: Cochrane risk of bias tool.
      The pre-clinical models used for arterial thrombosis were created to approximate four different clinical settings: ischemic stroke (n = 8), myocardial infarction (n = 4), arterial thrombosis (n = 8) and arteriovenous graft occlusion (n = 6). The clinical trials comprised two clinical settings: ischemic stroke (n = 8) and acute myocardial infarction (n = 2). There was high heterogeneity in the selected studies. They not only varied in type of subject used for the experiments and location of thrombus formation, but also in type of MBs used, site of microbubble injection (intravenous or intra-arterial), duration of experiments, US settings, use of a fibrinolytic agent and, if so, which fibrinolytic agent, site of injection of the fibrinolytic agent, the method for monitoring outcomes and definition of success. It was therefore not possible to perform a meta-analysis on any of the pre-defined outcomes.

      Primary outcomes

      Recanalization rate

      Definition of successful recanalization varied between the studies. Some defined success as (a variation of) any indication of flow in the previous occluded artery (10 studies), some used a complete return of flow or varying degrees of thrombolysis in myocardial infarction (TIMI)/thrombolysis in brain ischemia (TIBI) or thrombolysis in cerebral infarction (TICI) scores (Table 5).
      Table 5Overview of classifications for flow and recanalization
      GradeDefinition
      TIMI
      Adapted from the TIMI Study Group (1985).
      0No perfusionNo penetration of contrast past the clot in the obstructed artery
      (angiography)1Penetration without perfusionContrast passes the occlusion, but there is no perfusion of the target area
      2Partial perfusionContrast passes the obstruction and reaches the target area, but more slowly than adjacent normal vessels
      3Normal perfusionNormal flow through obstructed artery in comparison to adjacent vessels
      TIBI
      Demchuk et al. (2001).
      0AbsentLack of regular pulsatile flow signals despite varying degrees of background noise
      (Transcranial Doppler)1MinimalSystolic spikes of variable velocity and duration, absent flow during all cardiac cycles, reverberating flow
      2BluntedFlattened systolic flow acceleration of variable duration compared with control, positive end-diastolic velocity and pulsatility index <1.2
      3DampenedNormal systolic flow acceleration, positive end-diastolic velocity, decreased MFVs by >30% compared with control
      4StenoticMFV >80 cm/s and velocity difference >30% compared with control OR if both affected and comparison sides have MFV <80 cm/s owing to low end-diastolic velocities: MFV >30% compared with the control side and signs of turbulence
      5Normal<30% mean velocity difference compared with control, similar waveform shapes compared with control
      TICI
      Higashida et al. (2003).
      0No perfusionNo antegrade flow beyond the point of occlusion
      (Angiography)1Penetration with minimal perfusionThe contrast material passes beyond the area of the obstruction but fails to opacify the entire cerebral bed distal to the obstruction for the duration of the angiographic run.
      2Partial perfusionThe contrast material passes beyond the obstruction and opacifies the arterial bed distal to the obstruction. However, the rate of entry of contrast into the vessel distal to the obstruction and/or its rate of clearance from the distal bed are perceptibly slower than its entry into and/or clearance from comparable areas not perfused by the previously occluded vessel (e.g., the opposite cerebral artery or the arterial bed proximal to the obstruction)
      2aOnly partial filling (<2/3) of the entire vascular territory is visualized
      2bComplete filling of all of the expected vascular territory is visualized, but the filling is slower than normal
      3Complete perfusionAntegrade flow into the bed distal to the obstruction occurs as promptly as into the obstruction and clearance of contrast material from the involved bed is as rapid as from an uninvolved other bed of the same vessel or the opposite cerebral artery
      MFV = mean flow velocity; TIBI = thrombolysis in brain ischemia; TICI = thrombolysis in cerebral ischemia; TIMI = thrombolysis in myocardial infarction.
      low asterisk Adapted from the
      Thrombosis in Myocardial Infarction (TIMI) Study Group
      The Thrombolysis in Myocardial Infarction (TIMI) trial: Phase I findings.
      .
      • Demchuk AM
      • Burgin WS
      • Christou I
      • Felberg RA
      • Barber PA
      • Hill MD
      • Alexandrov AV.
      Thrombolysis in brain ischemia (TIBI) transcranial Doppler flow grades predict clinical severity, early recovery, and mortality in patients treated with intravenous tissue plasminogen activator.
      .
      • Higashida RT
      • Furlan AJ
      • Roberts H
      • Tomsick T
      • Connors B
      • Barr J
      • Dillon W
      • Warach S
      • Broderick J
      • Tilley B
      • Sacks D.
      Trial design and reporting standards for intra-arterial cerebral thrombolysis for acute ischemic stroke.
      .
      In the pre-clinical trials, 12 studies investigated a combination of MBs, US and a fibrinolytic agent (CEST). In 10 of the 12 studies, the rate of recanalization using CEST was higher than in any other tested arm (Table 6 and Table 7). In the other 2 studies the recanalization rate was similar to the best results in another arm tested. One of these, the study by
      • Tomkins AJ
      • Schleicher N
      • Murtha L
      • Kaps M
      • Levi CR
      • Nedelmann M
      • Spratt NJ.
      Platelet rich clots are resistant to lysis by thrombolytic therapy in a rat model of embolic stroke.
      , was the only study in which no recanalization was found in the 10 occlusions studied with the combination of MB, US and a fibrinolytic agent. The investigators used platelet-rich clots that seem to be highly resistant to thrombolysis in general. The other study,
      • Laing ST
      • Moody MR
      • Kim H
      • Smulevitz B
      • Huang SL
      • Holland CK
      • McPherson DD
      • Klegerman ME.
      Thrombolytic efficacy of tissue plasminogen activator-loaded echogenic liposomes in a rabbit thrombus model.
      , had similar recanalization rates for CEST and was the best compared alternative. The thrombolytic effect of CEST was compared with that of thrombus-targeted, tPA-loaded echogenic liposomes and the recanalization rates were similar at 60% in both treatment arms.
      Table 6Recanalization rates for CEST versus best alternative arm in same study: Pre-clinical, Part 1
      No.AuthorDesignNo. of occlusionsUltrasound settingClot age (min)Duration of therapy (min)
      1
      • Xie F
      • Lof J
      • Matsunaga T
      • Zutshi R
      • Porter TR.
      Diagnostic ultrasound combined with glycoprotein IIb/IIIa–targeted microbubbles improves microvascular recovery after acute coronary thrombotic occlusions.
      Pro-urokinase i.v. vs. low-MI US guided high-MI pulses + prourokinase i.v. + TMBs i.v. vs low-MI US guided high-MI pulses + pro-urokinase i.v. + NTMBs i.v.45Low MI (0.2) 1.5 MHz pulsed; high MI (1.9) 1.5 MHz pulsed≥2030 (+ 60 follow-up)
      2
      • Laing ST
      • Moody MR
      • Kim H
      • Smulevitz B
      • Huang SL
      • Holland CK
      • McPherson DD
      • Klegerman ME.
      Thrombolytic efficacy of tissue plasminogen activator-loaded echogenic liposomes in a rabbit thrombus model.
      i.a. saline vs. i.a. free tPA vs. i.a. tPA mixed with Definity vs. tPA-loaded echogenic liposomes; randomization within these groups for high- vs. low-MI US or no US596-MHz pulses high MI (0.4) 2 min vs. 6 MHz intermittently low MI (0.2) 30 min102 vs. 30
      3
      • Liu WS
      • Huang ZZ
      • Wang XW
      • Zhou J.
      Effects of microbubbles on transcranial Doppler ultrasound-assisted intracranial urokinase thrombolysis.
      Urokinase i.v. + US vs. urokinase i.v. + MBs i.v. + US322-MHz pulse 0.252 W/cm26020 (+ 100 follow-up)
      4
      • Xie F
      • Gao S
      • Wu J
      • Lof J
      • Radio S
      • Vignon F
      • Shi W
      • Powers J
      • Unger E
      • Everbach EC
      • Liu J
      • Porter TR.
      Diagnostic ultrasound induced inertial cavitation to non-invasively restore coronary and microvascular flow in acute myocardial infarction.
      rtPA i.v. vs rtPA i.v. + MB i.v. + high MI short pulse US vs rtPA i.v. + MB i.v. + guided low MI long pulse US361.6-MHz-long pulses; high MI (2.0), pulse length 5 µs vs. low MI (1.0), pulse length 20 µs≥2030 (+ 60 min follow-up)
      5
      • Hua X
      • Zhou L
      • Liu P
      • He Y
      • Tan K
      • Chen Q
      • Gao Y
      • Gao Y.
      In vivo thrombolysis with targeted microbubbles loading tissue plasminogen activator in a rabbit femoral artery thrombus model.
      no treatment vs tPA i.v. vs TUS vs TUS + tPA i.v. vs TUS + NTMBs i.v. + tPA i.v. vs. TUS + tPA-loaded TMBs i.v. vs. DUS + tPA-loaded tMB i.v.702 MHz, intensity of 1.8 W/cm2 TUS; 2 MHz, MI 1.4 DUS3030
      6
      • Ebben HP
      • Nederhoed JH
      • Slikkerveer J
      • Kamp O
      • Tangelder GWJM
      • Musters RJP
      • Wisselink W
      • Yeung KK.
      Therapeutic application of contrast-enhanced ultrasound and low-dose urokinase for thrombolysis in a porcine model of acute peripheral arterial occlusion.
      Urokinase i.a. vs urokinase i.a. + MB i.v. + US101.6 MHz, high-MI (1.2) pulses (5 s off, 1 s on)10060 UK ± MB followed by 120 UK alone
      7
      • Wu J
      • Xie F
      • Lof J
      • Sayyed S
      • Porter TR.
      Utilization of modified diagnostic ultrasound and microbubbles to reduce myocardial infarct size.
      1/2 dose tPA i.v. vs. full dose tPA i.v. vs. 1/2 dose tPA i.v. + MBs iv + high-MI US vs. MBs iv + high-MI US32Intermittent high MI (2.0)≥2030 (+ 60 min follow-up)
      8
      • Tomkins AJ
      • Schleicher N
      • Murtha L
      • Kaps M
      • Levi CR
      • Nedelmann M
      • Spratt NJ.
      Platelet rich clots are resistant to lysis by thrombolytic therapy in a rat model of embolic stroke.
      tPA i.v. vs. tPA i.v. + US + MBs i.v. vs. saline i.v.303 MHz, continuous, MI 1.7>48060 (+ 70 min follow-up)
      9
      • Ren X
      • Wang Y
      • Wang Y
      • Chen H
      • Chen L
      • Liu Y
      • Xue C.
      Thrombolytic therapy with rt-PA and transcranial color Doppler ultrasound (TCCS) combined with microbubbles for embolic thrombus.
      No treatment vs. rtPA i.v. vs. TCCS + MB i.v. vs. TCCS + MB. i.v. + full dose rtPA i.v. vs. TCCS + MB. i.v. + ½ dose rtPA i.v.301-MHz transcranial color Doppler, MI 0.9180–24020?
      10
      • Zhu Y
      • Guan L
      • Mu Y.
      Combined low-frequency ultrasound and urokinase-containing microbubbles in treatment of femoral artery thrombosis in a rabbit model.
      Urokinase-loaded TMBs i.v. in different settings
      A 3 × 2 × 2 factorial table was applied to create different combinations of ultrasonic frequencies (factor A: 1.6 MHz, 2.2 MHz, 2.8 MHz), doses of urokinase i.v. (factor B: 90,000 IU/kg, 180,000 IU/kg) and ultrasound exposure time (factor C: 30 min, 60 min).
      721.6 MHz vs. 2.2 MHz vs. 2.8 MHz≥2030/60 (+ 120 min follow-up)
      11
      • Nederhoed JH
      • Ebben HP
      • Slikkerveer J
      • Hoksbergen AWJ
      • Kamp O
      • Tangelder GJ
      • Wisselink W
      • Musters RJP
      • Yeung KK.
      Intravenous targeted microbubbles carrying urokinase versus urokinase alone in acute peripheral arterial thrombosis in a porcine model.
      Urokinase-loaded TMBs i.v. + US vs. urokinase i.v. + US91.6 MHz, 3-µs pulse duration, 24-kHz pulse repetition frequency, MI 1.110060 (+ 120 min follow up)
      12
      • Chen X
      • Wu W
      • Wang W
      • Zhong J
      • Moumin Djama N
      • Wei G
      • Lai Y
      • Si X
      • Cao S
      • Liao W
      • Liao Y
      • Li H
      • Bin J
      Magnetic targeting improves the therapeutic efficacy of microbubble-mediated obstructive thrombus sonothrombolysis.
      Saline i.v. vs. US + MBs i.v. vs. US + magnetic MBs i.v. vs. US + magnetic MBs i.v. + rtPA i.v.402 MHz, MI 1.96030
      CEST = contrast-enhanced sonothrombolysis; MBs = microbubbles; MI = mechanical index; NR = not reported; NS = not significant; NTMBs = non-targeted microbubbles; rtPA = recombinant tissue plasminogen activator; TMBs = targeted microbubbles; tPA = tissue plasminogen activator; TCD = transcranial Doppler ultrasound; TIBI = thrombus in brain ischemia.
      low asterisk A 3 × 2 × 2 factorial table was applied to create different combinations of ultrasonic frequencies (factor A: 1.6 MHz, 2.2 MHz, 2.8 MHz), doses of urokinase i.v. (factor B: 90,000 IU/kg, 180,000 IU/kg) and ultrasound exposure time (factor C: 30 min, 60 min).
      Table 7Recanalization rates for CEST versus best alternative arm in same study: Pre-clinical, Part 2
      No.Determination of recanalizationDefinition of recanalizationRecanalization ratep
      Best result CESTBest alternative arm
      1AngiographyEvidence of contrast flow through the site of occlusion and normal runoff of flow distal to occlusion60% (TMBs)20%NS
      2Pulsed spectral Doppler≥95% recanalization60% (tPA mixed with Definity, high-MI US)60% (targeted echogenic liposomes)NR
      3TCDIf blood flow velocity was improved >30% or waveform improvement was greater than 1 grade according to TIBI grading system56%31%0.154
      4AngiographyNot defined83% (1.0-MI long-pulse duration)45%<0.05
      5B-Flow imaging (non-Doppler US)Re-appearance of signal post-treatment80% (NTMBs, fibrinolytic and US)50% (fibrinolytic and US)<0.05
      6Ultrasonic perivascular flow probeChanges in flow (mL/min) as percentage of baseline (= flow after creating stenosis; formula: change in flow/baseline flow × 100%)27%
      If success were defined as “any recanalization,” the result would be 67% for CEST and 25% for the alternative arm. If defined as “recanalization over 15% of baseline flow,” the result would be 50% for CEST, 0% for the alternative arm.
      0%NR
      7AngiographyNot defined83%50% (in 2 arms: full dose tPA and MBs + US)NR
      8Laser Doppler flowReturn to ≥100% of baseline regional cerebral blood flow0%0%NS
      9Ultrasound + color Doppler flow imagingNot defined, but based on result section any grade ≥1 in author-created grading system100% (full dose tPA)50% (tPA alone)0.046
      10Pulsed Doppler flowmetryRecanalization >15% of baseline flow93% (2.2 MHz, 90,000 U, 30 min)NA
      11Ultrasonic flow probeAny recanalization80%25%NR
      12Doppler US (MI 0.18, frequency 14 MHz)Not defined100%95% (magnetic MBs; 40% for regular MBs)NS (<0.05 compared with regular MBs)
      CEST = contrast-enhanced sonothrombolysis; MB = microbubble; MI = mechanical index; NR = not reported; NS = not significant; rtPA = recombinant tissue plasminogen activator; tPA = tissue plasminogen activator; TCD = transcranial Doppler ultrasound; TIBI = thrombus in brain ischemia; TMBs = targeted microbubbles.
      low asterisk If success were defined as “any recanalization,” the result would be 67% for CEST and 25% for the alternative arm. If defined as “recanalization over 15% of baseline flow,” the result would be 50% for CEST, 0% for the alternative arm.
      The maximum recanalization rate in the groups combining MB, US and a fibrinolytic agent (CEST), except for the study by Tomkins et al., varied between 56% and 100% (Table 8). In contrast, in studies investigating the possibility of arterial recanalization using MBs alone (n = 2), the recanalization rate was 0% (0/6 and 0/10). The effect of US alone was investigated in 13 studies. Recanalization rates varied from 0% (5 studies) to 63% (5/8 occlusions, 4 complete) in the study by
      • Gao S
      • Zhang Y
      • Wu J
      • Shi WT
      • Lof J
      • Vignon F
      • Drvol L
      • Xie F
      • Muirhead D
      • Powers JE
      • High R
      • White ML
      • Porter TR.
      Improvements in cerebral blood flow and recanalization rates with transcranial diagnostic ultrasound and intravenous microbubbles after acute cerebral emboli.
      . In this study, US was applied with a high mechanical index (MI) of 2.4 and a short pulse duration of 5 µs. Ten studies tested the use of a fibrinolytic agent alone. The recanalization rates reported in these studies varied between 0% (2 studies) and 50% (1 study), with the majority of the studies reporting recanalization rates between 40% and 50% (6/10). The combination of US and a fibrinolytic agent, tested in 4 studies, had recanalization rates between 20% and 50%. The combination of MBs and a fibrinolytic agent, without US, was tested only in the trial by
      • Laing ST
      • Moody MR
      • Kim H
      • Smulevitz B
      • Huang SL
      • Holland CK
      • McPherson DD
      • Klegerman ME.
      Thrombolytic efficacy of tissue plasminogen activator-loaded echogenic liposomes in a rabbit thrombus model.
      , previously mentioned. The protocol that was most frequently compared with CEST (17 studies) was the combination of US and MBs. Looking at the 39 different protocols investigated in these 17 studies, recanalization rates varied from 0 (2 study arms) to 100% (5 study arms). The best recanalization rates of these studies varied between 29% and 100%.
      Table 8Range of recanalization rates for different study arms
      Study arm (No. of studies)Recanalization rate range (%)
      CEST (12)56–100
      Placebo + no US (7)0–10
      MBs alone (2)0
      US alone (13)0–63
      Fibrinolytic agent alone (10)0–50
      US + MBs (17)0–100
      US + fibrinolytic agent (4)20–50
      MBs + fibrinolytic agent (1)50
      CEST = contrast-enhanced sonothrombolysis; MBs = microbubbles; MI = mechanical index; PCI = percutaneous coronary intervention; tPA = tissue plasminogen activator; US = ultrasound.
      Of the 12 pre-clinical trials that tested a form of CEST, only 2 administered the fibrinolytic agent intra-arterially as is common in sCDT for peripheral arterial thrombosis. The first of these was the study by
      • Laing ST
      • Moody MR
      • Kim H
      • Smulevitz B
      • Huang SL
      • Holland CK
      • McPherson DD
      • Klegerman ME.
      Thrombolytic efficacy of tissue plasminogen activator-loaded echogenic liposomes in a rabbit thrombus model.
      , mentioned previously. Their definition of success was a return to at least 95% of baseline flow, and they obtained a recanalization rate of 60% for both the group with echogenic liposomes and the high-MI US CEST group.
      • Ebben HP
      • Nederhoed JH
      • Slikkerveer J
      • Kamp O
      • Tangelder GWJM
      • Musters RJP
      • Wisselink W
      • Yeung KK.
      Therapeutic application of contrast-enhanced ultrasound and low-dose urokinase for thrombolysis in a porcine model of acute peripheral arterial occlusion.
      studied sCDT versus CEST in a porcine model of peripheral arterial thrombosis. They found median increases in flow of 27% in the CEST group and 0% in the sCDT group. If the same definition of success is used as in the study by
      • Laing ST
      • Moody MR
      • Kim H
      • Smulevitz B
      • Huang SL
      • Holland CK
      • McPherson DD
      • Klegerman ME.
      Thrombolytic efficacy of tissue plasminogen activator-loaded echogenic liposomes in a rabbit thrombus model.
      , their recanalization rate in the CEST group is 33% versus 0% using sCDT.
      Clinical trials concerning CEST were performed in patients with ischemic stroke (8 studies) and myocardial infarction (1 study) (Table 9). There was a higher recanalization rate of the affected artery in the CEST group in all trials in which CEST was compared with standard care in either another study arm or in previously reported data (Table 10) . All clinical trials investigated the use of an intravenous fibrinolytic agent as part of their thrombolytic therapy. Only
      • Ribo M
      • Molina CA
      • Alvarez B
      • Rubiera M
      • Alvarez-Sabin J
      • Matas M.
      Intra-arterial administration of microbubbles and continuous 2-MHz ultrasound insonation to enhance intra-arterial thrombolysis.
      investigated CEST using intra-arterial tPA when recanalization did not occur with intravenous tPA alone (n = 7 of 16 patients) or when there was a contraindication to intravenous thrombolysis (n = 2). They achieved in-procedure recanalization in 7 of these 9 patients (2 complete, 5 partial).
      Table 9Recanalization rates for CEST versus best alternative arm in same study: Clinical, Part 1
      No.AuthorIndicationDesign (No. of patients/group)Randomization factorUltrasound settingDuration of therapy (min)
      1
      • Molina CA
      • Ribo M
      • Rubiera M
      • Montaner J
      • Santamarina E
      • Delgado-Mederos R
      • Arenillas JF
      • Huertas R
      • Purroy F
      • Delgado P
      • Alvarez-Sabín J.
      Microbubble administration accelerates clot lysis during continuous 2-MHz ultrasound monitoring in stroke patients treated with intravenous tissue plasminogen activator.
      Ischemic stroketPA i.v. + US + MBs i.v. (38) vs. tPA i.v. + US (37) vs. tPA i.v. (36)Not randomizedContinuous 2-MHz TCD120
      2Perren et al. 2007Ischemic stroketPA i.v. + US + MBs i.v. (9) vs. tPA i.v. + US (15)Not randomized2-MHz TCCD, pulsed-wave mode, 189 mW/cm260
      3
      • Pagola J
      • Ribo M
      • Alvarez-Sabín J
      • Lange M
      • Rubiera M
      • Molina CA.
      Timing of recanalization after microbubble-enhanced intravenous thrombolysis in basilar artery occlusion.
      Ischemic stroketPA i.v. + MBs i.v. + US (20)Not randomizedContinuous 2-MHz TCD120
      4
      • Alexandrov AV
      • Mikulik R
      • Ribo M
      • Sharma VK
      • Lao AY
      • Tsivgoulis G
      • Sugg RM
      • Barreto A
      • Sierzenski P
      • Malkoff MD
      • Grotta JC.
      A pilot randomized clinical safety study of sonothrombolysis augmentation with ultrasound-activated perflutren-lipid microspheres for acute ischemic stroke.
      Ischemic stroketPA i.v. + US + MBs i.v. (12) vs. tPA i.v. + US (3)*MBs vs. no MBs, ratio 3:12 MHz, power outputs <720 mW120
      5
      • Rubiera M
      • Ribo M
      • Delgado-Mederos R
      • Santamarina E
      • Maisterra O
      • Delgado P
      • Montaner J
      • Alvarez-Sabín J
      • Molina CA.
      Do bubble characteristics affect recanalization in stroke patients treated with microbubble-enhanced sonothrombolysis?.
      Ischemic stroketPA i.v. + Levovist MBs i.v. + US (91) vs. tPA i.v. + Sonovue MBs i.v. + US (47)Not randomized1.97-MHz pulsed wave, 8-kHz pulse repetition frequency, 385 mW/cm2 spatial peak temporal intensity, MI 0.24120
      6
      • Dinia L
      • Rubiera M
      • Ribo M
      • Maisterra O
      • Ortega G
      • del Sette M
      • Alvarez-Sabín J
      • Molina CA.
      Reperfusion after stroke sonothrombolysis with microbubbles may predict intracranial bleeding.
      Ischemic stroketPA i.v. + MBs i.v. (188) + US vs. tPA i.v. + US (98; historical control group)Not randomized2-MHz pulsed-wave diagnostic transducers, 750 mW120
      7
      • Molina CA
      • Barreto AD
      • Tsivgoulis G
      • Sierzenski P
      • Malkoff MD
      • Rubiera M
      • Gonzales N
      • Mikulik R
      • Pate G
      • Ostrem J
      • Singleton W
      • Manvelian G
      • Unger EC
      • Grotta JC
      • Schellinger PD
      • Alexandrov AV.
      Transcranial Ultrasound in Clinical Sonothrombolysis (TUCSON) trial.
      Ischemic stroketPA i.v. (12) vs. tPA i.v. + 1.4 mL MBs i.v. (12) vs. tPA i.v. + 2.8 mL MBs (11) i.v. vs. tPA i.v. + 5.6 mL MBs i.v. (0) vs. tPA i.v. + 11.2 mL MBs i.v. (0)MBs + US vs no MBs + US, ratio 2:1Continuous 2-MHz TCD, maximum 606 ±19-mW output90
      8
      • Ribo M
      • Molina CA
      • Alvarez B
      • Rubiera M
      • Alvarez-Sabin J
      • Matas M.
      Intra-arterial administration of microbubbles and continuous 2-MHz ultrasound insonation to enhance intra-arterial thrombolysis.
      Ischemic stroketPA i.v. + US (16) vs. tPA i.a. + MB i.a. + US (7 after first receiving tPA i.v. with no improvement of flow, 2 direct i.a.Not randomizedContinuous 2-MHz pulsed-wave diagnostic transducer, <750 mW60 min tPA i.v., <60 min i.a.
      9
      • Slikkerveer J
      • Kleijn SA
      • Appelman Y
      • Porter TR
      • Veen G
      • van Rossum AC
      • Kamp O.
      Ultrasound enhanced prehospital thrombolysis using microbubbles infusion in patients with acute ST elevation myocardial infarction: Pilot of the Sonolysis study.
      Myocardial infarctionMBs i.v. + US + tPA i.v. ± PCI (5) vs. saline i.v. + tPA i.v. ± PCI (5)MBs + US vs. no MBs + USIntermittent high-MI impulses (5 s on, 5 s off), 28-Hz pulse repetition period, 1.25-ms pulse duration, 1.5-MPa peak rare faction pressure, 26 mW/cm2 pulse average intensity at maximum MI15
      CEST = contrast-enhanced sonothrombolysis; MBs = microbubbles; MI = mechanical index; PCI = percutaneous coronary intervention; TCD = transcranial Doppler; TCCD = transcranial color coded duplex sonography; tPA = tissue plasminogen activator; US = ultrasound.
      Table 10Recanalization rates for CEST versus best alternative arm in same study: Clinical, Part 2
      No.Determination of recanalizationDefinition of successRecanalization ratep
      Best result CESTBest alternative arm
      1TCDPartial when blunted or dampened signals appeared in a previously absent or minimal flow and complete if the end-diastolic flow velocity improved to normal or elevated values71% after 2 h (54% complete)68% after 2 h (41% complete; tPA i.v. + US)0.65
      2TCCDPartial when the TIBI grade evolved from 0–1 to 2–3 and complete with a final TIBI grade of 4 or 563.6% after 1 h (all complete)53.3% after 1 h (complete)NR
      3TCDPartial when dampened signals appeared in a previously demonstrated absent or minimal flow, complete if the end-diastolic flow velocity improved to normal or elevated values20% after 1 h (10% complete), 50% after 24 h (all complete)NA
      4TCDPartial if the affected MCA segment with the worst residual flow grade pre-treatment improved by 1 TIBI grade or more to TIBI grade 2 or 3, complete if TIBI flow grades recovered to grade 4 or 5 within 2 h83% after 2 h (50% complete)66.7% after 2 h (none complete)NR
      5TCDPartial when TIBI 2 or 3 appeared in a previously demonstrated TIBI 1 pattern, complete if TIBI 4 or 5 was achieved32.2% LV/35.6% SV after 1 h, 50.0% LV/46.7% SV after 2 h, 63.8% LV/54.5% SV after 6 h (% complete not specified)NA
      6TCDPartial when blunted or dampened signals appeared in a previously demonstrated absent or minimal flow, complete if the end-diastolic flow velocity improved to normal or elevated values32.2% after 1 h, 50% after 2 h, 63.8% after 6 h, 74.3% after 12 h (% complete not specified)21% after 1 h, 36.7% after 2 h, 44.5% after 6 h, 56.2% after 12 h (% complete not specified)<0.05
      7TCDPartial if abnormal signals were still seen at the distal portion (TIMI grade II flow equivalent), complete when a normal waveform or a low-resistance stenotic signal appeared at the selected depth of insonation (TIMI grade III flow)84% 1.4-mL group after 2 h (67% complete), 45% 2.8-ml group after 2 h (all complete)58% after 2 h (33% complete)NR (0.255 for complete recanalization)
      8TCD and angiographyPartial if TICI II, complete if TICI III78% after i.a. treatment (22% complete), 78% after 12 h (56% complete)56% after 1 h (25% complete)NR
      9AngiographyTIMI grade III in culprit vessel at PCI
      No documentation of TIMI grade previous to intervention.
      60%20%0.23
      MCA = middle cerebral artery; NR = not reported; PCI = percutaneous coronary intervention; TCD = transcranial Doppler; TCCD = transcranial color coded duplex sonography; TIBI = thrombolysis in brain ischemia; TICI = thrombolysis in cerebral ischemia; TIMI = thrombolysis in myocardial infarction; tPA = tissue plasminogen activator; US = ultrasound.
      *Study was terminated early for administrative reasons and, thus, did not reach a predetermined sample size of 40.
      †Study was terminated early because of high rate of ICH in the 2.4-mL MB dose group.
      No documentation of TIMI grade previous to intervention.
      • Perren F
      • Loulidi J
      • Poglia D
      • Landis T
      • Sztajzel R.
      Microbubble potentiated transcranial duplex ultrasound enhances IV thrombolysis in acute stroke.
      compared the effect of MBs combined with a 2-MHz transcranial color-coded duplex (TCCD) US and intravenous tPA (n=11) with TCCD US and tPA alone (n = 15) in patients with acute middle cerebral artery (MCA) stroke. They obtained a complete recanalization rate in 7 of 11 patients in the CEST group (64%) at 1 h treatment versus 8 of 15 patients in the control group (53%). They observed significantly greater improvement in clinical outcome in the CEST group versus the control group (p = 0.05). In the study by
      • Dinia L
      • Rubiera M
      • Ribo M
      • Maisterra O
      • Ortega G
      • del Sette M
      • Alvarez-Sabín J
      • Molina CA.
      Reperfusion after stroke sonothrombolysis with microbubbles may predict intracranial bleeding.
      , 188 stroke patients that received CEST were compared with a historic group of 98 stroke patients that received tPA + transcranial Doppler (TCD). They obtained significantly higher recanalization rates in the CEST group at all measurement windows (p < 0.05), as well as a larger proportion of patients achieving independence at 3 mo in the CEST group versus the control group (51% vs. 37%, p = 0.052). In the TUSCON (Transcranial Ultrasound in Clinical Sonothrombolysis) trial by
      • Molina CA
      • Barreto AD
      • Tsivgoulis G
      • Sierzenski P
      • Malkoff MD
      • Rubiera M
      • Gonzales N
      • Mikulik R
      • Pate G
      • Ostrem J
      • Singleton W
      • Manvelian G
      • Unger EC
      • Grotta JC
      • Schellinger PD
      • Alexandrov AV.
      Transcranial Ultrasound in Clinical Sonothrombolysis (TUCSON) trial.
      , the aim was to test the safety, tolerability and activity of various doses of perflutren–lipid MBs combined with TCD and intravenous tPA. It was a multicenter randomized trial that was terminated early because of safety issues (see Secondary Outcomes section). A total of 35 patients were randomized: 12 received 1.4 mL of MBs with TCD and tPA, 11 received 2.8 mL of MBs and 12 received tPA with only brief TCD assessment (control group). Sustained complete recanalization at the end of the treatment was 67% for the 1.4-mL CEST group, 46% for the 2.8-mL CEST group and 33% for the control group (p = 0.255). Functional independence at 3 mo was 75% in the 1.4-mL CEST group, 50% in the 2.8-mL CEST group and 36% in the control group (p = 0.167).
      One study compared two different types of MBs (galactose-based air-filled vs. sulfur hexafluoride based) combined with tPA and TCD in 138 MCA stroke patients (
      • Rubiera M
      • Ribo M
      • Delgado-Mederos R
      • Santamarina E
      • Maisterra O
      • Delgado P
      • Montaner J
      • Alvarez-Sabín J
      • Molina CA.
      Do bubble characteristics affect recanalization in stroke patients treated with microbubble-enhanced sonothrombolysis?.
      ). There were no differences in recanalization rate or clinical outcome.
      • Slikkerveer J
      • Kleijn SA
      • Appelman Y
      • Porter TR
      • Veen G
      • van Rossum AC
      • Kamp O.
      Ultrasound enhanced prehospital thrombolysis using microbubbles infusion in patients with acute ST elevation myocardial infarction: Pilot of the Sonolysis study.
      conducted a pilot safety and feasibility study of low-dose tPA combined with US and MBs before percutaneous coronary intervention (PCI) in patients with acute myocardial infarction. All patients (n = 10) received a single bolus of 50 mg of tPA in the ambulance. At presentation at the emergency room, they were randomized to receive either 1 vial of perflutren-containing lipid MBs and high-MI, short-pulse-duration US or a placebo. After 15 min, all patients underwent an emergency coronary angiogram and, when necessary, intervention. At angiography, 3 of 5 patients in the CEST group had TIMI flow III versus 1 of 5 in the control group (p = 0.23).

      Thrombus weight

      Only two studies reported on thrombus weight as an outcome of the therapy, Ebben et al. in 2015 and Nederhoed et al in 2017. In the first study, standard intra-arterial CDT was compared with intra-arterial CDT enhanced with intravenous MBs and locally applied US. CEST resulted in a significantly lower thrombus weight at the end of therapy in this study (1.1g vs. 1.6 g, p = 0.01). In the second study, the MBs were loaded with urokinase and targeted to the thrombus by adding RGDS to the microbubble surface. The targeted, urokinase-loaded MBs were administered intravenously and high-MI US (MI = 1.1) was applied at the site of the occlusion. This form of CEST was compared with intravenously administered urokinase, combined with the same regimen of US. Thrombus weight at the end of therapy was significantly lower in the CEST group (0.94 g vs. 1.54 g, p = 0.017).

      Secondary outcomes

      Out of the 26 pre-clinical studies, 11 did not report safety outcomes. Of the 15 studies that did report safety outcomes, only 4 found possible adverse events:
      • Ebben HP
      • Nederhoed JH
      • Slikkerveer J
      • Kamp O
      • Tangelder GWJM
      • Musters RJP
      • Wisselink W
      • Yeung KK.
      Therapeutic application of contrast-enhanced ultrasound and low-dose urokinase for thrombolysis in a porcine model of acute peripheral arterial occlusion.
      reported 1 distal embolization in the group that received MBs.
      • Wu J
      • Xie F
      • Lof J
      • Sayyed S
      • Porter TR.
      Utilization of modified diagnostic ultrasound and microbubbles to reduce myocardial infarct size.
      found histologic evidence of focal hemorrhage in the infarct area in 3 of 18 pigs, but there was no difference in incidence between the groups.
      • Tomkins AJ
      • Schleicher N
      • Murtha L
      • Kaps M
      • Levi CR
      • Nedelmann M
      • Spratt NJ.
      Platelet rich clots are resistant to lysis by thrombolytic therapy in a rat model of embolic stroke.
      reported 2 cases of subarachnoid hemorrhage caused by incorrect catheter placement and 1 catheter dislodgement. Finally,
      • Nederhoed JH
      • Ebben HP
      • Slikkerveer J
      • Hoksbergen AWJ
      • Kamp O
      • Tangelder GJ
      • Wisselink W
      • Musters RJP
      • Yeung KK.
      Intravenous targeted microbubbles carrying urokinase versus urokinase alone in acute peripheral arterial thrombosis in a porcine model.
      reported 1 re-occlusion during therapy in the CEST group, possibly caused by distal embolization.
      In the clinical studies, the main adverse event reported was ICH (Table 11 and Table 12). Some studies reported only on symptomatic ICH, that is, clinically important ICH. The TUSCON trial was a prospective randomized multicenter phase II trial of MB dose escalation combined with systemic thrombolysis in patients with ischemic stroke (
      • Molina CA
      • Barreto AD
      • Tsivgoulis G
      • Sierzenski P
      • Malkoff MD
      • Rubiera M
      • Gonzales N
      • Mikulik R
      • Pate G
      • Ostrem J
      • Singleton W
      • Manvelian G
      • Unger EC
      • Grotta JC
      • Schellinger PD
      • Alexandrov AV.
      Transcranial Ultrasound in Clinical Sonothrombolysis (TUCSON) trial.
      ). The aim was to include 72 patients (48 targets and 24 controls), but the study was terminated after 35 inclusions because of a higher incidence of sICH in the 2.8-mL MB dose group (3/11 patients in this group vs. 0/12 in the other two groups, p = 0.028). There were no cases of sICH in the 12 patients receiving the first tier of the dose escalation study, that is, 1.4 mL of MBs.
      Table 11Adverse events in clinical trials: Part 1
      No.StudyClinical conditionTreatmentNo. of patientsCESTUS and MBs
      sICH (%)ICH (%)Mortality (%)sICH (%)ICH (%)Mortality (%)
      1
      • Molina CA
      • Ribo M
      • Rubiera M
      • Montaner J
      • Santamarina E
      • Delgado-Mederos R
      • Arenillas JF
      • Huertas R
      • Purroy F
      • Delgado P
      • Alvarez-Sabín J.
      Microbubble administration accelerates clot lysis during continuous 2-MHz ultrasound monitoring in stroke patients treated with intravenous tissue plasminogen activator.
      Ischemic strokeCEST vs. sono-thrombolysis vs. thrombolysis alone1111 (2.6)9 (23)........
      2Perren et al. 2007Ischemic strokeCEST vs. sono-thrombolysis261 (9.1)..........
      3
      • Pagola J
      • Ribo M
      • Alvarez-Sabín J
      • Lange M
      • Rubiera M
      • Molina CA.
      Timing of recanalization after microbubble-enhanced intravenous thrombolysis in basilar artery occlusion.
      Ischemic strokeCEST200..7 (35)......
      4
      • Alexandrov AV
      • Mikulik R
      • Ribo M
      • Sharma VK
      • Lao AY
      • Tsivgoulis G
      • Sugg RM
      • Barreto A
      • Sierzenski P
      • Malkoff MD
      • Grotta JC.
      A pilot randomized clinical safety study of sonothrombolysis augmentation with ultrasound-activated perflutren-lipid microspheres for acute ischemic stroke.
      Ischemic strokeCEST vs. sono-thrombolysis1503(25)4 (33)......
      5
      • Rubiera M
      • Ribo M
      • Delgado-Mederos R
      • Santamarina E
      • Maisterra O
      • Delgado P
      • Montaner J
      • Alvarez-Sabín J
      • Molina CA.
      Do bubble characteristics affect recanalization in stroke patients treated with microbubble-enhanced sonothrombolysis?.
      Ischemic strokeCEST (Levovist vs. Sonovue)1384 (2.9)28 (29.2)13(9.4)......
      6
      • Dinia L
      • Rubiera M
      • Ribo M
      • Maisterra O
      • Ortega G
      • del Sette M
      • Alvarez-Sabín J
      • Molina CA.
      Reperfusion after stroke sonothrombolysis with microbubbles may predict intracranial bleeding.
      Ischemic strokeCEST vs. sonothrombolysis2865 (2.9)48 (26)23 (12)......
      7
      • Molina CA
      • Barreto AD
      • Tsivgoulis G
      • Sierzenski P
      • Malkoff MD
      • Rubiera M
      • Gonzales N
      • Mikulik R
      • Pate G
      • Ostrem J
      • Singleton W
      • Manvelian G
      • Unger EC
      • Grotta JC
      • Schellinger PD
      • Alexandrov AV.
      Transcranial Ultrasound in Clinical Sonothrombolysis (TUCSON) trial.
      Ischemic strokeCEST (various doses of MBs) vs. thrombolysis353 (13)
      All sICHs occurred in the 2.8-mL dose MB group.
      3 (13)
      Two of these ICHs occurred in the 1.4-mL dose group (17%) and one in the 2.8-mL dose group (9%).
      3 (13)
      All 3 deaths were in the 2.8-mL dose group (30%). Two deaths were attributed to sICH, and one to progression of the ischemic stroke.
      ......
      8
      • Ribo M
      • Molina CA
      • Alvarez B
      • Rubiera M
      • Alvarez-Sabin J
      • Matas M.
      Intra-arterial administration of microbubbles and continuous 2-MHz ultrasound insonation to enhance intra-arterial thrombolysis.
      Ischemic strokeCEST
      Thrombolysis was given intravenously in 16 of 18 patients, 7 of whom then proceeded to intra-arterial CEST as there was no recanalization. The remaining 2 patients had a contraindication to intravenous thrombolysis and received primary intra-arterial CEST.
      vs. thrombolysis
      181(11)..1(11)......
      9
      • Slikkerveer J
      • Kleijn SA
      • Appelman Y
      • Porter TR
      • Veen G
      • van Rossum AC
      • Kamp O.
      Ultrasound enhanced prehospital thrombolysis using microbubbles infusion in patients with acute ST elevation myocardial infarction: Pilot of the Sonolysis study.
      Myo-cardial infarctionCEST vs. thrombolysis100..0......
      CEST = contrast-enhanced sonothrombolysis; ICH = intracranial hemorrhage; MBs = microbubbles; NR = not reported; sICH = symptomatic intracranial hemorrhage; US = ultrasound.
      low asterisk All sICHs occurred in the 2.8-mL dose MB group.
      Two of these ICHs occurred in the 1.4-mL dose group (17%) and one in the 2.8-mL dose group (9%).
      All 3 deaths were in the 2.8-mL dose group (30%). Two deaths were attributed to sICH, and one to progression of the ischemic stroke.
      § Thrombolysis was given intravenously in 16 of 18 patients, 7 of whom then proceeded to intra-arterial CEST as there was no recanalization. The remaining 2 patients had a contraindication to intravenous thrombolysis and received primary intra-arterial CEST.
      Table 12Adverse events in clinical trials: Part 2
      No.SonothrombolysisThrombolysisNo thrombolytic therapy
      sICH (%)ICH (%)Mortality (%)sICH (%)ICH (%)Mortality (%)sICH (%)ICH (%)Mortality (%)Follow-up
      11(2,7)7(19)..2(5.5)1 (16)........3 mo
      21 (6,7)................15 d
      3..................3 mo
      401 (33)1 (33)............3 mo
      5..................3 mo
      63 (2,1)17 (19)15 (15)............24 mo
      7......000......3 mo
      8......NRNRNR......3 mo
      9......0..0......4 mo
      CEST = contrast-enhanced sonothrombolysis; ICH = intracranial hemorrhage; MB = microbubble; NR = not reported; sICH = symptomatic intracranial hemorrhage.
      The only other adverse events were reported by
      • Slikkerveer J
      • Kleijn SA
      • Appelman Y
      • Porter TR
      • Veen G
      • van Rossum AC
      • Kamp O.
      Ultrasound enhanced prehospital thrombolysis using microbubbles infusion in patients with acute ST elevation myocardial infarction: Pilot of the Sonolysis study.
      , who tested the feasibility of treatment with low-dose thrombolytics in combination with US and MBs before primary PCI in patients with a first acute ST-elevation myocardial infarction. They reported two cases of minor leakage at the injection site of the catheters, one in each treatment arm of the study and one non-sustained ventricular tachycardia in each treatment arm.

      Discussion

      This systematic review highlights a number of positive outcomes in terms of the efficacy and safety of CEST, but much remains unknown. The studies suffered from low numbers, lack of randomization, differences in CEST protocols including variation in US settings, location of arterial thrombosis, duration of therapy and fibrinolytic agent used. The outcomes and modes of determining outcome studied, as well as definitions of success, varied widely across the studies. Therefore, comparisons among the studies are challenging, and conclusions on what CEST protocol would be best for patients with acute peripheral arterial occlusion are difficult to draw. Despite the heterogeneity of the included studies, among the pre-clinical trials, only one study failed to observe recanalization in any of the treatment arms, and in one study, US did not seem to have an influence when thrombus-targeted echogenic liposomes were used. In all other pre-clinical studies in which CEST was investigated, recanalization rates were higher than in any of the control arms. All clinical trials that compared CEST with standard care, whether in their study or in previously reported data, had a higher recanalization rate of the affected artery in the CEST group. Where clinical outcomes are reported, they are better than in the standard group. This was even true for the study arm of the TUCSON trial that received the 2.8-mL dose of MBs and had a higher incidence of ICH (
      • Molina CA
      • Barreto AD
      • Tsivgoulis G
      • Sierzenski P
      • Malkoff MD
      • Rubiera M
      • Gonzales N
      • Mikulik R
      • Pate G
      • Ostrem J
      • Singleton W
      • Manvelian G
      • Unger EC
      • Grotta JC
      • Schellinger PD
      • Alexandrov AV.
      Transcranial Ultrasound in Clinical Sonothrombolysis (TUCSON) trial.
      ).
      Although few adverse events were reported that might be attributed to the addition of MBs and US to standard thrombolysis in the included studies, adverse events have been reported in other studies.
      • Roos ST
      • Juffermans LJM
      • van Royen N
      • van Rossum AC
      • Xie F
      • Appelman Y
      • Porter TR
      • Kamp O.
      Unexpected high incidence of coronary vasoconstriction in the Reduction of Microvascular Injury Using Sonolysis (ROMIUS) Trial.
      reported an unexpected high incidence of coronary vasoconstriction in their study on the role of sonolysis in reducing microvascular injury in patients with myocardial infarction. In this study, patients with acute myocardial infarction received an intravenous infusion of the Definity MBs at 1.3 mL/min. Diagnostic US (MI = 0.18) was used to guide therapeutic high-MI, long-pulse-duration US (MI = 1.3, pulse duration = 20 µs) to the site of the myocardial perfusion defect. The treatment was given for a maximum of 15 min before PCI, during which diagnostic and therapeutic US regimens were alternated at the rate of 15 s per imaging mode. The inclusion target for this safety and feasibility study was set at 20, but the trial was aborted after 3 of the first 6 patients manifested vasoconstriction unresponsive to nitroglycerine in the affected artery on angiography. None of the patients that manifested vasoconstriction had any lasting noticeable effects. A subsequent study by the authors using a porcine model for acute myocardial infarction revealed a decrease in coronary artery diameter distal to the injury site after application of US. The authors conclude that long-pulse-duration US might cause coronary vasoconstriction distal to the location of the culprit vessel. In a study by
      • Mathias Jr, W
      • Tsutsui JM
      • Tavares BG
      • Fava AM
      • Aguiar MOD
      • Borges BC
      • Oliveira Jr, MT
      • Soeiro A
      • Nicolau JC
      • Ribeiro HB
      • Chiang HP
      • Sbano JCN
      • Morad A
      • Goldsweig A
      • Rochitte CE
      • Lopes BBC
      • Ramirez JAF
      • Kalil Filho R
      • Porter TR
      Sonothrombolysis in ST-segment elevation myocardial infarction treated with primary percutaneous coronary intervention.
      , 100 first ST-segment-elevated myocardial infarction patients were randomized to receive either short-pulse-duration (<5 µs) high-MI US directed at different myocardial segments combined with infusion of Definity or only very-low-MI diagnostic US combined with Definity. They obtained a significantly higher rate of pre-PCI recanalization of the infarct vessel in the high-MI US group (24/50 vs. 10/50, p < 0.001) and reported no adverse events. This supports the theory that the vasoconstriction found in the study by
      • Roos ST
      • Juffermans LJM
      • van Royen N
      • van Rossum AC
      • Xie F
      • Appelman Y
      • Porter TR
      • Kamp O.
      Unexpected high incidence of coronary vasoconstriction in the Reduction of Microvascular Injury Using Sonolysis (ROMIUS) Trial.
      might have been caused by the long-pulse-duration US. Because affected vessels in peripheral arterial disease have a larger diameter and tissues distal to the affected vessels are more resilient to ischemic conditions, possible temporary vasoconstriction resulting from US is not expected to cause a clinical problem in the setting of peripheral arterial thrombosis.

      Future perspectives

      This systematic review was conducted to assess the safety and efficacy of CEST in peripheral arterial thrombosis. As there are so few studies on this subject, we added all in vivo studies that tested a form of CEST in combination with an arterial thrombosis. This led to the current overview, in which most of the studies were conducted in a setting of small cerebral or coronary artery occlusions. Although we cannot extrapolate the data directly to patients with peripheral arterial disease, this overview points us in the direction of future studies and, it is hoped, improved treatment for our patients. For future clinical studies, we suggest infusing the MBs and applying local US during the first hour of treatment, as this was responsible for success in many of the trials in this review. A longer duration of US would burden both the patient and the care system. Furthermore, we suggest that US with a high MI of around 1 and switching between “on” and “off” settings every few seconds be used to allow the MBs to re-enter the area of the thrombus. We have conducted the MUST (Microbubbles and UltraSound-accelerated Thrombolysis for peripheral arterial occlusions) trial, a clinical safety and applicability trial for CEST in acute peripheral arterial thrombosis, using these settings (
      • Ebben HP
      • Nederhoed JH
      • Lely RJ
      • Wisselink W
      • Yeung KK
      MUST collaborators
      Microbubbles and Ultrasound-Accelerated Thrombolysis (MUST) for peripheral arterial occlusions: A phase-II single arm trial.
      ). Inclusion has just been completed, and preliminary data indicate promising results without major complications (
      • Evers JM
      • Ebben HP
      • Nederhoed J H
      • Lely RJ
      • Wisselink W
      • Yeung KK.
      Contrast-enhanced sonothrombolysis to accelerate treatment of acute peripheral arterial occlusions: The Preliminary Results of the MUST trial (a feasibility and safety phase II trial).
      ). We need to wait for definitive data, however, to draw more solid conclusions. Another aspect to take into account when determining US settings for clinical use of CEST is the vasoconstriction mentioned previously when long-pulse-duration US was used. This might not be a problem with peripheral arterial occlusion, but use of a short pulse duration of <5 µs can be explored. Depending on the definitive results of the MUST trial, a large multicenter randomized trial comparing standard intra-arterial CDT and CEST will be set up in the near future. End points will include time to reperfusion of the affected limb, major and minor amputations, duration of hospitalization and any adverse event with emphasis on intracranial hemorrhage. Long-term follow-up is needed to detect any possible long-term side effects of the experimental treatment. For the more distant future, aims might include not only a better clinical outcome and shorter hospitalization times, but also less invasive treatment for the patient with the acutely ischemic limb. As discussed before, MBs can be used as a vehicle for medication. They can be targeted to adhere to a thrombus and, with the most recent developments, can now also be directed magnetically (
      • Chen X
      • Wu W
      • Wang W
      • Zhong J
      • Moumin Djama N
      • Wei G
      • Lai Y
      • Si X
      • Cao S
      • Liao W
      • Liao Y
      • Li H
      • Bin J
      Magnetic targeting improves the therapeutic efficacy of microbubble-mediated obstructive thrombus sonothrombolysis.
      ). Combining these attributes, it seems a catheter-free intra-arterial form of thrombolysis might very well be possible in the future. More research is needed, however, before these theories can be translated to the clinical setting. As mentioned previously, we excluded articles in which the therapeutic US was not directed at the occluded vessel, but at the outflow territory. The reason for not including studies that focused on outflow territory is that the focus of this review is on revascularization of the ischemic limb using CEST, and it does not seem feasible to treat the arterial occlusion by directing therapeutic US at such a large outflow territory that is also far from the occlusion. Treating the microvasculature in peripheral arterial occlusion with a combination of MBs and US might still be beneficial though. Several studies reported better microcirculatory perfusion after using MBs, which could help in prevention of the no-reflow phenomenon after revascularization of an acutely ischemic limb (
      • Xie F
      • Lof J
      • Matsunaga T
      • Zutshi R
      • Porter TR.
      Diagnostic ultrasound combined with glycoprotein IIb/IIIa–targeted microbubbles improves microvascular recovery after acute coronary thrombotic occlusions.
      ;
      • Leeman JE
      • Kim JS
      • Yu FT
      • Chen X
      • Kim K
      • Wang J
      • Chen X
      • Villanueva FS
      • Pacella JJ.
      Effect of acoustic conditions on microbubble-mediated microvascular sonothrombolysis.
      ). We speculate that MBs might also play a role in diabetic foot ulcers, as a loss of microcirculation is one of the main factors contributing to both the development of ulcers and the difficult healing process after macrovascular optimization of blood flow.

      Conclusions

      Studies on CEST reveal promising results with respect to recanalization rate and clinical outcome in acute arterial thrombosis. It was not possible to perform a meta-analysis because of the high heterogeneity among the studies. Nonetheless, the data suggest that adding MBs and US to standard intra-arterial CDT might be a safe way to improve outcomes for patients with acute peripheral arterial thrombosis. More research on safety and applicability in a clinical setting is needed before it can be implemented in daily practice.

      Conflict of interest disclosure

      The authors declare that they have no affiliations that could inappropriately influence (bias) their work.

      Appendix. Supplementary materials

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