Frequency-sum beamforming for passive cavitation imaging.

Beamforming includes a variety of spatial filtering techniques that may be used for determining sound source locations from near-field sensor array recordings. For this scenario, beamforming resolution depends on the acoustic frequency, array geometry, and target location. Random scattering in the medium between the source and the array may degrade beamforming resolution with higher frequencies being more susceptible to degradation. The performance of frequency-sum (FS) beamforming for reducing such sensitivity to mild scattering while increasing resolution is reported here. FS beamforming was used with a data-dependent [minimum variance (MV)] or data-independent (delay-and-sum, DAS) weight vector to produce higher frequency information from lower frequency signal components via a quadratic product of complex signal amplitudes. The current findings and comparisons are based on simulations and passive cavitation imaging experiments using 3 MHz and 6 MHz emissions recorded by a 128-element linear array. FS beamforming results are compared to conventional DAS and MV beamforming using four metrics: point spread function (PSF) size, axial and lateral contrast, and computation time. FS beamforming produces a smaller PSF than conventional DAS beamforming with less computation time than MV beamforming in free space and mild scattering environments. However, it may fail when multiple unknown sound sources are present.

Ultrasound-enhanced bevacizumab release from echogenic liposomes for inhibition of atheroma progression.

CONTEXT: Bevacizumab (BEV) is a monoclonal antibody to vascular endothelial growth factor (VEGF) that ameliorates atheroma progression by inhibiting neovascularization. OBJECTIVE: We aimed to determine whether BEV release from echogenic liposomes (BEV-ELIP) could be enhanced by color Doppler ultrasound (US) and whether the released BEV inhibits VEGF expression by endothelial cells in vitro. MATERIALS AND METHODS: BEV-ELIP samples were subjected to 6 MHz color Doppler ultrasound (MI = 0.4) for 5 min. We assessed release of BEV with a direct ELISA and with fluoresceinated BEV (FITC-BEV) loaded into ELIP by the same method. Human umbilical vein endothelial cell (HUVEC) cultures were stimulated to express VEGF by 10 nM phorbol-12-myristate 13-acetate (PMA). Cell-associated VEGF levels were determined using a cell-based ELISA. RESULTS: Overall, US caused an additional 100 µg of BEV to be released or exposed per BEV-ELIP aliquot within 60 min BEV-ELIP treated with US inhibited VEGF expression by 90% relative to non-treated controls and by 70% relative to BEV-ELIP without US. Also, US-treated BEV-ELIP inhibited HUVEC proliferation by 64% relative to untreated controls and by 45% relative to BEV-ELIP without US. DISCUSSION AND CONCLUSION: We have demonstrated that BEV-ELIP retains its VEGF-binding activity in a liposomal formulation and that clinical Doppler US can significantly increase that activity, both by releasing free BEV and by enhancing the surface exposure of the immunoreactive antibody.

Size-isolation of ultrasound-mediated phase change perfluorocarbon droplets using differential centrifugation.

Perfluorocarbon droplets that are capable of an ultrasound-mediated phase transition have applications in diagnostic and therapeutic ultrasound. Techniques to modify the droplet size distribution are of interest because of the size-dependent acoustic response of the droplets. Differential centrifugation has been used to isolate specific sizes of microbubbles. In this work, differential centrifugation was employed to isolate droplets with diameters between 1 and 3 µm and 2 and 5 µm from an initially polydisperse distribution. Further, an empirical model was developed for predicting the droplet size distribution following differential centrifugation and to facilitate the selection of centrifugation parameters for obtaining desired size distributions.

Histotripsy and Catheter-Directed Lytic: Efficacy in Highly Retracted Porcine Clots In Vitro.

OBJECTIVE: Standard treatment for deep vein thrombosis (DVT) involves catheter-directed anticoagulants or thrombolytics, but the chronic thrombi present in many DVT cases are often resistant to this therapy. Histotripsy has been found to be a promising adjuvant treatment, using the mechanical action of cavitating bubble clouds to enhance thrombolytic activity. The objective of this study was to determine if histotripsy enhanced recombinant tissue plasminogen activator (rt-PA) thrombolysis in highly retracted porcine clots in vitro in a flow model of occlusive DVT. METHODS: Highly retracted porcine whole blood clots were treated for 1 h with either catheter-directed saline (negative control), rt-PA (lytic control), histotripsy, DEFINITY and histotripsy or the combination of rt-PA and histotripsy with or without DEFINITY. Five-cycle, 1.5 MHz histotripsy pulses with a peak negative pressure of 33.2 MPa and pulse repetition frequency of 40 Hz were applied along the clot. B-Mode and passive cavitation images were acquired during histotripsy insonation to monitor bubble activity. RESULTS: Clots subjected to histotripsy with and without rt-PA exhibited greater thrombolytic efficacy than controls (7.0% flow recovery or lower), and histotripsy with rt-PA was more efficacious than histotripsy with saline (86.1 ± 10.2% compared with 61.7 ± 19.8% flow recovery). The addition of DEFINITY to histotripsy with or without rt-PA did not enhance either thrombolytic efficacy or cavitation dose. Cavitation dose generally did not correlate with thrombolytic efficacy. CONCLUSION: Enhancement of thrombolytic efficacy was achieved using histotripsy, with and without catheter-directed rt-PA, in the presence of physiologic flow. This suggests these treatments may be effective as therapy for DVT.

Quantifying the Effect of Acoustic Parameters on Temporal and Spatial Cavitation Activity: Gauging Cavitation Dose.

OBJECTIVE: Cavitation-enhanced delivery of therapeutic agents is under development for the treatment of cancer and neurodegenerative and cardiovascular diseases, including sonothrombolysis for deep vein thrombosis. The objective of this study was to quantify the spatial and temporal distribution of cavitation activity nucleated by Definity infused through the EKOS catheter over a range of acoustic parameters controlled by the EKOS endovascular system. METHODS: Three insonation protocols were compared in an in vitro phantom mimicking venous flow to measure the effect of peak rarefactional pressure, pulse duration and pulse repetition frequency on cavitation activity energy, location and duration. Inertial and stable cavitation activity was quantified using passive cavitation imaging, and a metric of cavitation dose based on energy density was defined. RESULTS: For all three insonation protocols, cavitation was sustained for the entire 30 min Definity infusion. The evolution of cavitation energy during each pulse duration was similar for all three protocols. For insonation protocols with higher peak rarefactional acoustic pressures, inertial and stable cavitation doses also increased. A complex relationship between the temporal behavior of cavitation energy within each pulse and the pulse repetition frequency affected the cavitation dose for the three insonation protocols. The relative predominance of stable or inertial cavitation dose varied according to insonation schemes. Passive cavitation images revealed the spatial distribution of cavitation activity. CONCLUSION: Our cavitation dose metric based on energy density enabled the impact of different acoustic parameters on cavitation activity to be measured. Depending on the type of cavitation to be promoted or suppressed, particular pulsing schemes could be employed in future studies, for example, to correlate cavitation dose with sonothrombolytic efficacy.

Passive Cavitation Imaging Artifact Reduction Using Data-Adaptive Spatial Filtering.

Passive cavitation imaging (PCI) with a clinical diagnostic array results in poor axial localization of bubble activity due to the size of the point spread function (PSF). The objective of this study was to determine if data-adaptive spatial filtering improved PCI beamforming performance relative to standard frequency-domain delay, sum, and integrate (DSI) or robust Capon beamforming (RCB). The overall goal was to improve source localization and image quality without sacrificing computation time. Spatial filtering was achieved by applying a pixel-based mask to DSI- or RCB-beamformed images. The masks were derived from DSI, RCB, or phase or amplitude coherence factors (ACFs) using both receiver operating characteristic (ROC) and precision-recall (PR) curve analyses. Spatially filtered passive cavitation images were formed from cavitation emissions based on two simulated sources densities and four source distribution patterns mimicking cavitation emissions induced by an EkoSonic catheter. Beamforming performance was assessed via binary classifier metrics. The difference in sensitivity, specificity, and area under the ROC curve (AUROC) differed by no more than 11% across all algorithms for both source densities and all source patterns. The computational time required for each of the three spatially filtered DSIs was two orders of magnitude less than that required for time-domain RCB and thus this data-adaptive spatial filtering strategy for PCI beamforming is preferable given the similar binary classification performance.

Initiating and imaging cavitation from infused echo contrast agents through the EkoSonic catheter.

Ultrasound-enhanced delivery of therapeutic-loaded echogenic liposomes is under development for vascular applications using the EkoSonic Endovascular System. In this study, fibrin-targeted echogenic liposomes loaded with an anti-inflammatory agent were characterized before and after infusion through an EkoSonic catheter. Cavitation activity was nucleated by Definity or fibrin-targeted, drug-loaded echogenic liposomes infused and insonified with EkoSonic catheters. Passive cavitation imaging was used to quantify and map bubble activity in a flow phantom mimicking porcine arterial flow. Cavitation was sustained during 3-min infusions of Definity or echogenic liposomes along the distal 6 cm treatment zone of the catheter. Though the EkoSonic catheter was not designed specifically for cavitation nucleation, infusion of drug-loaded echogenic liposomes can be employed to trigger and sustain bubble activity for enhanced intravascular drug delivery.

Research Initiative Supporting Excellence at the University of Cincinnati (RISE-UC): A Program to Develop and Support Research-Active Faculty Members.

A combination of forces have markedly increased challenges to research-active faculty achieving sustained success. This article describes how one department at the University of Cincinnati College of Medicine (UCCOM) implemented a strategic plan, the Research Initiative Supporting Excellence at the University of Cincinnati (RISE-UC), to promote the research activity of its research-active faculty, fiscal year (FY) 2011-FY 2021. RISE-UC was implemented and regularly updated to address evolving needs. RISE-UC supported faculty members pursuing research via fiscal and administrative services to grow a critical mass of investigators; establish a shared governance model; create pathways for developing physician-scientists; develop discrete and targeted internal research funding; establish an Academic Research Service (ARS) unit (as infrastructure to support research); enhance faculty member mentorship; and recognize, celebrate, and reward research success. RISE-UC was informed by shared governance and resulted in substantial increases in total size of the faculty and external funding. More than 50% of Physician-Scientist Training Program graduates are active researchers at UCCOM. The internal awards program realized a return on investment of ~16.4-fold, and total external direct cost research funds increased from ~$55,400,000 (FY 2015) to ~$114,500,000 (FY 2021). The ARS assisted in the submission of 57 grant proposals and provided services faculty members generally found very helpful or helpful. The peer-mentoring group for early-career faculty members resulted in 12 of 23 participants receiving major grant funding (≥ $100,000; spring 2017-spring 2021) from sources including National Institutes of Health awards, Department of Defense funding, Veterans Affairs funding, and foundation awards. Research recognition included ~$77,000/year in incentive payments to faculty members for grant submissions and grants awarded. RISE-UC is an example of a comprehensive approach to promote research faculty member success and may serve as a model for other institutions with similar aspirations.

Advances in Focused Ultrasound for the Treatment of Brain Tumors.

Employing the full arsenal of therapeutics to treat brain tumors is limited by the relative impermeability of the blood-brain and blood-tumor barriers. In physiologic states, the blood-brain barrier serves a protective role by passively and actively excluding neurotoxic compounds; however, this functionality limits the penetrance of therapeutics into the tumor microenvironment. Focused ultrasound technology provides a method for overcoming the blood-brain and blood-tumor barriers through ultrasound frequency to transiently permeabilize or disrupt these barriers. Concomitant delivery of therapeutics has allowed for previously impermeable agents to reach the tumor microenvironment. This review details the advances in focused ultrasound in both preclinical models and clinical studies, with a focus on its safety profile. We then turn towards future directions in focused ultrasound-mediated therapies for brain tumors.

Demonstration of ultrasound-mediated therapeutic delivery of fibrin-targeted pioglitazone-loaded echogenic liposomes into the arterial bed for attenuation of peri-stent restenosis.

Peri-stent restenosis following stent implantation is a major clinical problem. We have previously demonstrated that ultrasound-facilitated liposomal delivery of pioglitazone (PGN) to the arterial wall attenuated in-stent restenosis. To evaluate ultrasound mediated arterial delivery, in Yucatan miniswine, balloon inflations were performed in the carotid and subclavian arteries to simulate stent implantation and induce fibrin formation. The fibrin-binding peptide, GPRPPGGGC, was conjugated to echogenic liposomes (ELIP) containing dinitrophenyl-L-alanine-labelled pioglitazone (DNP-PGN) for targeting purposes. After pre-treating the arteries with nitroglycerine, fibrin-binding peptide-conjugated PGN-loaded ELIP (PAFb-DNP-PGN-ELIP also termed atheroglitatide) were delivered to the injured arteries via an endovascular catheter with an ultrasound core, either with or without ultrasound application (EKOSTM Endovascular System, Boston Scientific). In arteries treated with atheroglitatide, there was substantial delivery of PGN into the superficial layers (5 µm from the lumen) of the arteries with and without ultrasound, [(1951.17 relative fluorescence units (RFU) vs. 1901.17 RFU; P-value = 0.939)]. With ultrasound activation there was increased penetration of PGN into the deeper arterial layers (up to 35 µm from the lumen) [(13195.25 RFU vs. 7681.00 RFU; P-value = 0.005)]. These pre-clinical data demonstrate ultrasound mediated therapeutic vascular delivery to deeper layers of the injured arterial wall. This model has the potential to reduce peri- stent restenosis.

Passive imaging with pulsed ultrasound insonations.

Previously, passive cavitation imaging has been described in the context of continuous-wave high-intensity focused ultrasound thermal ablation. However, the technique has potential use as a feedback mechanism for pulsed-wave therapies, such as ultrasound-mediated drug delivery. In this paper, results of experiments and simulations are reported to demonstrate the feasibility of passive cavitation imaging using pulsed ultrasound insonations and how the images depend on pulsed ultrasound parameters. The passive cavitation images were formed from channel data that was beamformed in the frequency domain. Experiments were performed in an invitro flow phantom with an experimental echo contrast agent, echogenic liposomes, as cavitation nuclei. It was found that the pulse duration and envelope have minimal impact on the image resolution achieved. The passive cavitation image amplitude scales linearly with the cavitation emission energy. Cavitation images for both stable and inertial cavitation can be obtained from the same received data set.

Impact of Perfluoropentane Microdroplets Diameter and Concentration on Acoustic Droplet Vaporization Transition Efficiency and Oxygen Scavenging.

Acoustic droplet vaporization is the ultrasound-mediated phase change of liquid droplets into gas microbubbles. Following the phase change, oxygen diffuses from the surrounding fluid into the microbubble. An in vitro model was used to study the effects of droplet diameter, the presence of an ultrasound contrast agent, ultrasound duty cycle, and droplet concentration on the magnitude of oxygen scavenging in oxygenated deionized water. Perfluoropentane droplets were manufactured through a microfluidic approach at nominal diameters of 1, 3, 5, 7, 9, and 12 µm and studied at concentrations varying from 5.1 × 10-5 to 6.3 × 10-3 mL/mL. Droplets were exposed to an ultrasound transduced by an EkoSonicTM catheter (2.35 MHz, 47 W, and duty cycles of 1.70%, 2.34%, or 3.79%). Oxygen scavenging and the total volume of perfluoropentane that phase-transitioned increased with droplet concentration. The ADV transition efficiency decreased with increasing droplet concentration. The increasing duty cycle resulted in statistically significant increases in oxygen scavenging for 1, 3, 5, and 7 µm droplets, although the increase was smaller than when the droplet diameter or concentration were increased. Under the ultrasound conditions tested, droplet diameter and concentration had the greatest impact on the amount of ADV and subsequent oxygen scavenging occurred, which should be considered when using ADV-mediated oxygen scavenging in therapeutic ultrasounds.

Effect of Thrombin and Incubation Time on Porcine Whole Blood Clot Elasticity and Recombinant Tissue Plasminogen Activator Susceptibility.

Deep vein thrombosis is a major source of morbidity and mortality worldwide. Catheter-directed thrombolytics are the frontline approach for vessel recanalization, though fibrinolytic efficacy is limited for stiff, chronic thrombi. Although thrombin has been used in preclinical models to induce thrombosis, the effect on lytic susceptibility and clot stiffness is unknown. The goal of this study was to explore the effect of bovine thrombin concentration and incubation time on lytic susceptibility and stiffness of porcine whole blood clots in vitro. Porcine whole blood was allowed to coagulate at 37°C in glass pipets primed with 2.5 or 15 U/mL thrombin for 15 to 120 min. Lytic susceptibility to recombinant tissue plasminogen activator (rt-PA, alteplase) over a range of concentrations (3.15-107.00 µg/mL) was evaluated using percentage clot mass loss. The Young's moduli and degrees of retraction of the clots were estimated using ultrasound-based single-track-location shear wave elasticity and B-mode imaging, respectively. Percentage mass loss decreased and clot stiffness increased with the incubation period. Clots formed with 15 U/mL and incubated for 2 h exhibited properties similar to those of highly retracted clots: Young's modulus of 2.39 ± 0.36 kPa and percentage mass loss of 8.69 ± 2.72% when exposed to 3.15 µg/mL rt-PA. The histological differences between thrombin-induced porcine whole blood clots in vitro and thrombi in vivo are described.

Advances in Immunotherapy for the Treatment of Adult Glioblastoma: Overcoming Chemical and Physical Barriers.

Glioblastoma, or glioblastoma multiforme (GBM, WHO Grade IV), is a highly aggressive adult glioma. Despite extensive efforts to improve treatment, the current standard-of-care (SOC) regimen, which consists of maximal resection, radiotherapy, and temozolomide (TMZ), achieves only a 12-15 month survival. The clinical improvements achieved through immunotherapy in several extracranial solid tumors, including non-small-cell lung cancer, melanoma, and non-Hodgkin lymphoma, inspired investigations to pursue various immunotherapeutic interventions in adult glioblastoma patients. Despite some encouraging reports from preclinical and early-stage clinical trials, none of the tested agents have been convincing in Phase III clinical trials. One, but not the only, factor that is accountable for the slow progress is the blood-brain barrier, which prevents most antitumor drugs from reaching the target in appreciable amounts. Herein, we review the current state of immunotherapy in glioblastoma and discuss the significant challenges that prevent advancement. We also provide thoughts on steps that may be taken to remediate these challenges, including the application of ultrasound technologies.

Design and Characterization of an Ultrasound Transducer for Combined Histotripsy-Thrombolytic Therapy.

Chronic thrombi of the deep veins of the leg are resistant to dissolution or removal by current interventions and can act as thrombogenic sources. Histotripsy, a focused ultrasound therapy, uses the mechanical activity of bubble clouds to liquefy target tissues. In vitro experiments have shown that histotripsy enhances thrombolytic agent recombinant tissue plasminogen activator in a highly retracted clot model resistant to lytic therapy alone. Although these results are promising, further refinement of the acoustic source is necessary for in vivo studies and clinical translation. The source parameters for use in vivo were defined, and a transducer was fabricated for transcutaneous exposure of porcine and human iliofemoral deep-vein thrombosis (DVT) as the target. Based on the design criteria, a 1.5-MHz elliptical source with a 6-cm focal length and a focal gain of 60 was selected. The source was characterized by fiber-optic hydrophone and holography. High-speed photography showed that the cavitation cloud could be confined to dimensions smaller than the specified vessel lumen. The source was also demonstrated in vitro to create confined lesions within clots. The results support that this design offers an appropriate clinical prototype for combined histotripsy-thrombolytic therapy.

Acoustic characterization of echogenic liposomes: frequency-dependent attenuation and backscatter.

Ultrasound contrast agents (UCAs) are used clinically to aid detection and diagnosis of abnormal blood flow or perfusion. Characterization of UCAs can aid in the optimization of ultrasound parameters for enhanced image contrast. In this study echogenic liposomes (ELIPs) were characterized acoustically by measuring the frequency-dependent attenuation and backscatter coefficients at frequencies between 3 and 30 MHz using a broadband pulse-echo technique. The experimental methods were initially validated by comparing the attenuation and backscatter coefficients measured from 50-µm and 100-µm polystyrene microspheres with theoretical values. The size distribution of the ELIPs was measured and found to be polydisperse, ranging in size from 40 nm to 6 µm in diameter, with the highest number observed at 65 nm. The ELIP attenuation coefficients ranged from 3.7 ± 1.0 to 8.0 ± 3.3 dB/cm between 3 and 25 MHz. The backscatter coefficients were 0.011 ± 0.006 (cm str)(-1) between 6 and 9 MHz and 0.023 ± 0.006 (cm str)(-1) between 13 and 30 MHz. The measured scattering-to-attenuation ratio ranged from 8% to 22% between 6 and 25 MHz. Thus ELIPs can provide enhanced contrast over a broad range of frequencies and the scattering properties are suitable for various ultrasound imaging applications including diagnostic and intravascular ultrasound.

Controlling Reperfusion Injury With Controlled Reperfusion: Historical Perspectives and New Paradigms.

Cardiac reperfusion injury is a well-established outcome following treatment of acute myocardial infarction and other types of ischemic heart conditions. Numerous cardioprotection protocols and therapies have been pursued with success in pre-clinical models. Unfortunately, there has been lack of successful large-scale clinical translation, perhaps in part due to the multiple pathways that reperfusion can contribute to cell death. The search continues for new cardioprotection protocols based on what has been learned from past results. One class of cardioprotection protocols that remain under active investigation is that of controlled reperfusion. This class consists of those approaches that modify, in a controlled manner, the content of the reperfusate or the mechanical properties of the reperfusate (e.g., pressure and flow). This review article first provides a basic overview of the primary pathways to cell death that have the potential to be addressed by various forms of controlled reperfusion, including no-reflow phenomenon, ion imbalances (particularly calcium overload), and oxidative stress. Descriptions of various controlled reperfusion approaches are described, along with summaries of both mechanistic and outcome-oriented studies at the pre-clinical and clinical phases. This review will constrain itself to approaches that modify endogenously-occurring blood components. These approaches include ischemic postconditioning, gentle reperfusion, controlled hypoxic reperfusion, controlled hyperoxic reperfusion, controlled acidotic reperfusion, and controlled ionic reperfusion. This review concludes with a discussion of the limitations of past approaches and how they point to potential directions of investigation for the future.

Clot Degradation Under the Action of Histotripsy Bubble Activity and a Lytic Drug.

Deep vein thrombosis is a major source of morbidity worldwide. For critical obstructions, catheter-directed thrombolytics are the frontline therapy to achieve vessel recanalization. Techniques that aid lytic therapy are under development to improve treatment efficacy and reduce procedure-related complications. Histotripsy is one such adjuvant under development that relies on focused ultrasound for in situ nucleation of bubble clouds. Prior studies have demonstrated synergistic effects for clot dissolution when histotripsy is combined with lytic therapy. The success of this combination approach is hypothesized to promote thrombolytic efficacy via two mechanisms: erythrocyte fractionation (hemolysis) and increased lytic activity (fibrinolysis). In this study, the contributions of hemolysis and fibrinolysis to clot degradation under histotripsy and a lytic were quantified with measurements of hemoglobin and D-dimer, respectively. A linear regression analysis was used to determine the relationship between hemoglobin, D-dimer, and the overall treatment efficacy (clot mass loss). A similar analysis was conducted to gauge the role of bubble activity, which was assessed with passive cavitation imaging, on hemolysis and fibrinolysis. Tabulation of these data demonstrated hemolysis and fibrinolysis contributed equally to clot mass loss. Furthermore, bubble cloud activity promoted the generation of hemoglobin and D-dimer in equal proportion. These studies indicate a multifactorial process for clot degradation under the action of histotripsy and a lytic therapy.

Cavitation Emissions Nucleated by Definity Infused through an EkoSonic Catheter in a Flow Phantom.

The EkoSonic endovascular system has been cleared by the U.S. Food and Drug Administration for the controlled and selective infusion of physician specified fluids, including thrombolytics, into the peripheral vasculature and the pulmonary arteries. The objective of this study was to explore whether this catheter technology could sustain cavitation nucleated by infused Definity, to support subsequent studies of ultrasound-mediated drug delivery to diseased arteries. The concentration and attenuation spectroscopy of Definity were assayed before and after infusion at 0.3, 2.0 and 4.0 mL/min through the EkoSonic catheter. PCI was used to map and quantify stable and inertial cavitation as a function of Definity concentration in a flow phantom mimicking the porcine femoral artery. The 2.0 mL/min infusion rate yielded the highest surviving Definity concentration and acoustic attenuation. Cavitation was sustained throughout each 15 ms ultrasound pulse, as well as throughout the 3 min infusion. These results demonstrate a potential pathway to use cavitation nucleation to promote drug delivery with the EkoSonic endovascular system.

In Vitro Thrombolytic Efficacy of Single- and Five-Cycle Histotripsy Pulses and rt-PA.

Although primarily known as an ablative modality, histotripsy can increase the efficacy of lytic therapy in a retracted venous clot model. Bubble cloud oscillations are the primary mechanism of action for histotripsy, and the type of bubble activity is dependent on the pulse duration. A retracted human venous clot model was perfused with and without the thrombolytic recombinant tissue plasminogen activator (rt-PA). The clot was exposed to histotripsy pulses of single- or five-cycle duration and peak negative pressures of 0-30 MPa. Bubble activity within the clot was monitored via passive cavitation imaging. The combination of histotripsy and rt-PA was more efficacious than rt-PA alone for single- and five-cycle pulses with peak negative pressures of 25 and 20 MPa, respectively. For both excitation schemes, the detected acoustic emissions correlated with the degree of thrombolytic efficacy. These results indicate that rt-PA and single- or multicycle histotripsy pulses enhance thrombolytic therapy.