Histotripsy induces apoptosis and reduces hypoxia in a neuroblastoma xenograft model.

BACKGROUND: Neuroblastoma (NB) is the most common extracranial solid tumor of childhood, and high-risk disease is resistant to intensive treatment. Histotripsy is a focused ultrasound therapy under development for tissue ablation via bubble activity. The goal of this study was to assess outcomes of histotripsy ablation in a xenograft model of high-risk NB. METHODS: Female NCr nude mice received NGP-luciferase cells intrarenally. Under ultrasound image guidance, histotripsy pulses were applied over a distance of 4-6 mm within the tumors. Bioluminescence indicative of tumor viability was quantified before, immediately after, and 24 h after histotripsy exposure. Tumors were immunostained to assess apoptosis (TUNEL), endothelium (endomucin), pericytes (αSMA), hypoxia (pimonidazole), vascular endothelial growth factor A (VEGFA), and platelet-derived growth factor-B (PDGF-B). The apoptotic cytokine TNFα and its downstream effector cleaved caspase-3 (c-casp-3) were assessed with SDS-PAGE. RESULTS: Histotripsy induced a 50% reduction in bioluminescence compared to untreated controls, with an absence of nuclei in the treatment core surrounded by a dense rim of TUNEL-positive cells. Tumor regions not targeted by histotripsy also showed an increase in TUNEL staining density. Increased apoptosis in histotripsy samples was consistent with increases in TNFα and c-casp-3 relative to controls. Treated tumors exhibited a decrease in hypoxia, VEGF, PDGF-B, and pericyte coverage of vasculature compared to control samples. Further, increases in vasodilation were found in histotripsy-treated specimens. CONCLUSIONS: In addition to ablative effects, histotripsy was found to drive tumor apoptosis through intrinsic pathways, altering blood vessel architecture, and reducing hypoxia.

Ultrasound for Aesthetic Applications: A Review of Biophysical Mechanisms and Safety.

Aesthetic ultrasound is used for fat reduction and to improve skin appearance. In this review, the fundamental mechanisms by which ultrasound can alter tissue are outlined. The technologies that are commercially available or under development are discussed. Finally, recommendations are made for safe and effective use of aesthetic ultrasound.

The influence of gas diffusion on bubble persistence in shock-scattering histotripsy.

Bubble cloud persistence reduces the efficacy of mechanical liquefaction with shock-scattering histotripsy. In this study, the contribution of gas transfer to bubble longevity was investigated in silico by solving the equations for bubble oscillations and diffusion in parallel. The bubble gas content increased more than 5 orders of magnitude during the expansion phase, arresting the inertial collapse. The residual gas bubble required more than 15 ms for passive dissolution post excitation, consistent with experimental observation. These results demonstrate gas diffusion is an important factor in the persistence of histotripsy-induced cavitation.

Sonothrombolysis.

Thrombo-occlusive disease is a leading cause of morbidity and mortality. In this chapter, the use of ultrasound to accelerate clot breakdown alone or in combination with thrombolytic drugs will be reported. Primary thrombus formation during cardiovascular disease and standard treatment methods will be discussed. Mechanisms for ultrasound enhancement of thrombolysis, including thermal heating, radiation force, and cavitation, will be reviewed. Finally, in-vitro, in-vivo and clinical evidence of enhanced thrombolytic efficacy with ultrasound will be presented and discussed.

Thrombolytic efficacy and enzymatic activity of rt-PA-loaded echogenic liposomes.

Echogenic liposomes (ELIP), that can encapsulate both recombinant tissue-type plasminogen activator (rt-PA) and microbubbles, are under development to improve the treatment of thrombo-occlusive disease. However, the enzymatic activity, thrombolytic efficacy, and stable cavitation activity generated by this agent has yet to be evaluated and compared to another established ultrasound-enhanced thrombolytic scheme. A spectrophotometric method was used to compare the enzymatic activity of the rt-PA incorporated into ELIP (t-ELIP) to that of rt-PA. An in vitro flow model was employed to measure the thrombolytic efficacy and dose of ultraharmonic emissions from stable cavitation for 120-kHz ultrasound exposure of three treatment schemes: rt-PA, rt-PA and the perfluorocarbon-filled microbubble Definity(®), and t-ELIP. The enzymatic activity of rt-PA incorporated into t-ELIP was 28 % that of rt-PA. Thrombolytic efficacy of t-ELIP or rt-PA and Definity(®) was equivalent when the dose of t-ELIP was adjusted to produce comparable enzymatic activity. Sustained bubble activity was nucleated from Definity but not from t-ELIP exposed to 120-kHz ultrasound. These results emphasize the advantages of encapsulating a thrombolytic and the importance of incorporating an insoluble gas required to promote sustained, stable cavitation activity.

Cavitation thresholds of contrast agents in an in vitro human clot model exposed to 120-kHz ultrasound.

Ultrasound contrast agents (UCAs) can be employed to nucleate cavitation to achieve desired bioeffects, such as thrombolysis, in therapeutic ultrasound applications. Effective methods of enhancing thrombolysis with ultrasound have been examined at low frequencies (<1 MHz) and low amplitudes (<0.5 MPa). The objective of this study was to determine cavitation thresholds for two UCAs exposed to 120-kHz ultrasound. A commercial ultrasound contrast agent (Definity(®)) and echogenic liposomes were investigated to determine the acoustic pressure threshold for ultraharmonic (UH) and broadband (BB) generation using an in vitro flow model perfused with human plasma. Cavitation emissions were detected using two passive receivers over a narrow frequency bandwidth (540-900 kHz) and a broad frequency bandwidth (0.54-1.74 MHz). UH and BB cavitation thresholds occurred at the same acoustic pressure (0.3 ± 0.1 MPa, peak to peak) and were found to depend on the sensitivity of the cavitation detector but not on the nucleating contrast agent or ultrasound duty cycle.

Development of Convolutional Neural Network to Segment Ultrasound Images of Histotripsy Ablation.

OBJECTIVE: Histotripsy is a focused ultrasound therapy that ablates tissue via the action of bubble clouds. It is under investigation to treat a number of ailments, including renal tumors. Ultrasound imaging is used to monitor histotripsy, though there remains a lack of definitive imaging metrics to confirm successful treatment outcomes. In this study, a convolutional neural network (CNN) was developed to segment ablation on ultrasound images. METHODS: A transfer learning approach was used to replace classification layers of the residual network ResNet-18. Inputs to the classification layers were based on ultrasound images of ablated red blood cell phantoms. Digital photographs served as the ground truth. The efficacy of the CNN was compared to subtraction imaging, and manual segmentation of images by two board-certified radiologists. RESULTS: The CNN had a similar performance to manual segmentation, though was improved relative to segmentation with subtraction imaging. Predictions of the network improved over the course of treatment, with the Dice similarity coefficient less than 20% for fewer than 500 applied pulses, but 85% for more than 750 applied pulses. The network was also applied to ultrasound images of ex vivo kidney exposed to histotripsy, which indicated a morphological shift in the treatment profile relative to the phantoms. These findings were consistent with histology that confirmed ablation of the targeted tissue. CONCLUSION: Overall, the CNN showed promise as a rapid means to assess outcomes of histotripsy and automate treatment. SIGNIFICANCE: Data collected in this study indicate integration of CNN image segmentation to gauge outcomes for histotripsy ablation holds promise for automating treatment procedures.

In silico assessment of histotripsy-induced changes in catheter-directed thrombolytic delivery.

Introduction: For venous thrombosis patients, catheter-directed thrombolytic therapy is the standard-of-care to recanalize the occluded vessel. Limitations with thrombolytic drugs make the development of adjuvant treatments an active area of research. One potential adjuvant is histotripsy, a focused ultrasound therapy that lyses red blood cells within thrombus via the spontaneous generation of bubbles. Histotripsy has also been shown to improve the efficacy of thrombolytic drugs, though the precise mechanism of enhancement has not been elucidated. In this study, in silico calculations were performed to determine the contribution of histotripsy-induced changes in thrombus diffusivity to alter catheter-directed therapy. Methods: An established and validated Monte Carlo calculation was used to predict the extent of histotripsy bubble activity. The distribution of thrombolytic drug was computed with a finite-difference time domain (FDTD) solution of the perfusion-diffusion equation. The FDTD calculation included changes in thrombus diffusivity based on outcomes of the Monte Carlo calculation. Fibrin degradation was determined using the known reaction rate of thrombolytic drug. Results: In the absence of histotripsy, thrombolytic delivery was restricted in close proximity to the catheter. Thrombolytic perfused throughout the focal region for calculations that included the effects of histotripsy, resulting in an increased degree of fibrinolysis. Discussion: These results were consistent with the outcomes of in vitro studies, suggesting histotripsy-induced changes in the thrombus diffusivity are a primary mechanism for enhancement of thrombolytic drugs.

Contrast-Enhanced Imaging of Histotripsy Bubble Clouds Using Chirp-Coded Excitation and Volterra Filtering.

Histotripsy is a focused ultrasound therapy that ablates tissue via bubble cloud activity. Real-time ultrasound image guidance is used to ensure safe and effective treatment. Plane-wave imaging enables tracking of histotripsy bubble clouds at a high frame rate but lacks adequate contrast. Furthermore, bubble cloud hyperechogenicity is reduced in abdominal targets, making the development of contrast-specific sequences for deep-seated targets an active area of research. Chirp-coded subharmonic imaging was reported previously to enhance histotripsy bubble cloud detection by a modest 4-6 dB compared to the conventional sequence. Incorporating additional steps into the signal processing pipeline could enhance bubble cloud detection and tracking. In this study, we evaluated the feasibility of combining chirp-coded subharmonic imaging with Volterra filtering for enhancing bubble cloud detection in vitro. Chirped imaging pulses were used to track bubble clouds generated in scattering phantoms at a 1-kHz frame rate. Fundamental and subharmonic matched filters were applied to the received radio frequency signals, followed by a tuned Volterra filter to extract bubble-specific signatures. For subharmonic imaging, the application of the quadratic Volterra filter improved the contrast-to-tissue ratio from 5.18 ± 1.29 to 10.90 ± 3.76 dB, relative to the application of the subharmonic matched filter. These findings demonstrate the utility of the Volterra filter for histotripsy image guidance.

The effect of static pressure on the strength of inertial cavitation events.

Recent investigations of cavitation in fluids pressurized up to 30 MPa found that the intensity of light emissions increased by 1000-fold over that measured for single bubble sonoluminescence. A series of measurements is reported here to extend this original work by resolving the static pressure dependence of the shock wave and light emissions from the first and the most energetic collapses, along with the total shock wave energy and light emissions for the event. Each of these parameters was found to increase with the static pressure of the fluid. Furthermore, the energy of these shock wave and light emissions was found to increase in proportion to the stored acoustic energy in the system. These findings were corroborated using the Gilmore equation to numerically compute the work done by the liquid during the bubble collapse. The overall findings suggest that the increased collapse strength at high static pressure is due to the increased tension required to generate inertial cavitation, and not an increased pressure gradient between the interior of the vaporous bubble and the surrounding liquid.

The effect of static pressure on the inertial cavitation threshold.

The amplitude of the acoustic pressure required to nucleate a gas or vapor bubble in a fluid, and to have that bubble undergo an inertial collapse, is termed the inertial cavitation threshold. The magnitude of the inertial cavitation threshold is typically limited by mechanisms other than homogeneous nucleation such that the theoretical maximum is never achieved. However, the onset of inertial cavitation can be suppressed by increasing the static pressure of the fluid. The inertial cavitation threshold was measured in ultrapure water at static pressures up to 30 MPa (300 bars) by exciting a radially symmetric standing wave field in a spherical resonator driven at a resonant frequency of 25.5 kHz. The threshold was found to increase linearly with the static pressure; an exponentially decaying temperature dependence was also found. The nature and properties of the nucleating mechanisms were investigated by comparing the measured thresholds to an independent analysis of the particulate content and available models for nucleation.

Assessment of bubble activity generated by histotripsy combined with echogenic liposomes.

Objective.Histotripsy is a form of focused ultrasound therapy that uses the mechanical activity of bubbles to ablate tissue. While histotripsy alone degrades the cellular content of tissue, recent studies have demonstrated it effectively disrupts the extracellular structure of pathologic conditions such as venous thrombosis when combined with a thrombolytic drug. Rather than relying on standard administration methods, associating thrombolytic drugs with an ultrasound-triggered echogenic liposome vesicle will enable targeted, systemic drug delivery. To date, histotripsy has primarily relied on nano-nuclei inherent to the medium for bubble cloud generation, and microbubbles associated with echogenic liposomes may alter the histotripsy bubble dynamics. The objective of this work was to investigate the interaction of histotripsy pulse with echogenic liposomes.Approach.Bubble clouds were generated using a focused source in anin vitromodel of venous flow. Acoustic emissions generated during the insonation were passively acquired to assess the mechanical activity of the bubble cloud. High frame rate, pulse inversion imaging was used to track the change in echogenicity of the liposomes following histotripsy exposure.Main results.For peak negative pressures less than 20 MPa, acoustic emissions indicative of stable and inertial bubble activity were observed. As the peak negative pressure of the histotripsy excitation increased, harmonics of the excitation were observed in OFP t-ELIP solutions and plasma alone. Additional observations with high frame rate imaging indicated a transition of bubble behavior as the pulse pressure transitioned to shock wave formation.Significance.These observations suggest that a complex interaction between histotripsy pulses and echogenic liposomes that may be exploited for combination treatment approaches.

Histotripsy Bubble Cloud Contrast With Chirp-Coded Excitation in Preclinical Models.

Histotripsy is a focused ultrasound therapy for tissue ablation via the generation of bubble clouds. These effects can be achieved noninvasively, making sensitive and specific bubble imaging essential for histotripsy guidance. Plane-wave ultrasound imaging can track bubble clouds with an excellent temporal resolution, but there is a significant reduction in echoes when deep-seated organs are targeted. Chirp-coded excitation uses wideband, long-duration imaging pulses to increase signals at depth and promote nonlinear bubble oscillations. In this study, we evaluated histotripsy bubble contrast with chirp-coded excitation in scattering gel phantoms and a subcutaneous mouse tumor model. A range of imaging pulse durations were tested, and compared to a standard plane-wave pulse sequence. Received chirped signals were processed with matched filters to highlight components associated with either fundamental or subharmonic (bubble-specific) frequency bands. The contrast-to-tissue ratio (CTR) was improved in scattering media for subharmonic contrast relative to fundamental contrast (both chirped and standard imaging pulses) with the longest-duration chirped-pulse tested (7.4 [Formula: see text] pulse duration). The CTR was improved for subharmonic contrast relative to fundamental contrast (both chirped and standard imaging pulses) by 4.25 dB ± 1.36 dB in phantoms and 3.84 dB ± 6.42 dB in vivo. No systematic changes were observed in the bubble cloud size or dissolution rate between sequences, indicating image resolution was maintained with the long-duration imaging pulses. Overall, this study demonstrates the feasibility of specific histotripsy bubble cloud visualization with chirp-coded excitation.

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.

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.

(More than) doubling down: Effective fibrinolysis at a reduced rt-PA dose for catheter-directed thrombolysis combined with histotripsy.

Deep vein thrombosis is a major source of morbidity and mortality worldwide. For acute proximal deep vein thrombosis, catheter-directed thrombolytic therapy is an accepted method for vessel recanalization. Thrombolytic therapy is not without risk, including the potential for hemorrhagic bleeding that increases with lytic dose. Histotripsy is a focused ultrasound therapy that generates bubble clouds spontaneously in tissue at depth. The mechanical activity of histotripsy increases the efficacy of thrombolytic therapy at doses consistent with current pharmacomechanical treatments for venous thrombosis. The objective of this study was to determine the influence of lytic dose on histotripsy-enhanced fibrinolysis. Human whole blood clots formed in vitro were exposed to histotripsy and a thrombolytic agent (recombinant tissue plasminogen activator, rt-PA) in a venous flow model perfused with plasma. Lytic was administered into the clot via an infusion catheter at concentrations ranging from 0 (control) to 4.54 µg/mL (a common clinical dose for catheter-directed thrombolysis). Following treatment, perfusate samples were assayed for markers of fibrinolysis, hemolysis, and intact red blood cells and platelets. Fibrinolysis was equivalent between the common clinical dose of rt-PA (4.54 µg/mL) and rt-PA at a reduction to one-twentieth of the common clinical dose (0.23 µg/mL) when combined with histotripsy. Minimal changes were observed in hemolysis for treatment arms with or without histotripsy, potentially due to clot damage from insertion of the infusion catheter. Likewise, histotripsy did not increase the concentration of red blood cells or platelets in the perfusate following treatment compared to rt-PA alone. At the highest lytic dose, a refined histotripsy exposure scheme was implemented to cover larger areas of the clot. The updated exposure scheme improved clot mass loss and fibrinolysis relative to administration of lytic alone. Overall, the data collected in this study indicate the rt-PA dose can be reduced by more than a factor of ten and still promote fibrinolysis when combined with histotripsy.

An In vitro System to Gauge the Thrombolytic Efficacy of Histotripsy and a Lytic Drug.

Deep vein thrombosis (DVT) is a global health concern. The primary approach to achieve vessel recanalization for critical obstructions is catheter-directed thrombolytics (CDT). To mitigate caustic side effects and the long treatment time associated with CDT, adjuvant and alternative approaches are under development. One such approach is histotripsy, a focused ultrasound therapy to ablate tissue via bubble cloud nucleation. Pre-clinical studies have demonstrated strong synergy between histotripsy and thrombolytics for clot degradation. This report outlines a benchtop method to assess the efficacy of histotripsy-aided thrombolytic therapy, or lysotripsy. Clots manufactured from fresh human venous blood were introduced into a flow channel whose dimensions and acousto-mechanical properties mimic an iliofemoral vein. The channel was perfused with plasma and the lytic recombinant tissue-type plasminogen activator. Bubble clouds were generated in the clot with a focused ultrasound source designed for the treatment of femoral venous clots. Motorized positioners were used to translate the source focus along the clot length. At each insonation location, acoustic emissions from the bubble cloud were passively recorded, and beamformed to generate passive cavitation images. Metrics to gauge treatment efficacy included clot mass loss (overall treatment efficacy), and the concentrations of D-dimer (fibrinolysis) and hemoglobin (hemolysis) in the perfusate. There are limitations to this in vitro design, including lack of means to assess in vivo side effects or dynamic changes in flow rate as the clot lyses. Overall, the setup provides an effective method to assess the efficacy of histotripsy-based strategies to treat DVT.

Assessment of Collaborative Robot (Cobot)-Assisted Histotripsy for Venous Clot Ablation.

OBJECTIVE: The application of bubble-based ablation with the focus ultrasound therapy histotripsy is gaining traction for the treatment of venous thrombosis, among other pathologies. For extensive clot burden, the histotripsy source must be translated to ensure uniform bubble activity throughout the vascular obstruction. The purpose of this study was to evaluate the targeting accuracy of a histotripsy system comprised of a focused source, ultrasound image guidance, and a collaborative robot (cobot) positioner. The system was designed with a primary emphasis for treating deep vein thrombosis. METHODS: Studies to test treatment planning and targeting bubble activity with the histotripsy-cobot system were conducted in an in vitro clot model. A tissue-mimicking phantom was also targeted with the system, and the predicted and actual areas of liquefaction were compared to gauge the spatial accuracy of ablation. RESULTS: The system provided submillimeter accuracy for both tracking along an intended path (within 0.6 mm of a model vessel) and targeting bubble activity within the venous clot model (0.7 mm from the center of the clot). Good correlation was observed between the planned and actual liquefaction locations in the tissue phantom, with an average Dice similarity coefficient of 77.8%, and average Hausdorff distance of 1.6 mm. CONCLUSION: Cobots provide an effective means to apply histotripsy pulses over a treatment volume, with the ablation precision contingent on the quality of image guidance. SIGNIFICANCE: Overall, these results demonstrate cobots can be used to guide histotripsy ablation for targets that extend beyond the natural focus of the transducer.

Estimating the mechanical energy of histotripsy bubble clouds with high frame rate imaging.

Mechanical ablation with the focused ultrasound therapy histotripsy relies on the generation and action of bubble clouds. Despite its critical role for ablation, quantitative metrics of bubble activity to gauge treatment outcomes are still lacking. Here, plane wave imaging was used to track the dissolution of bubble clouds following initiation with the histotripsy pulse. Information about the rate of change in pixel intensity was coupled with an analytic diffusion model to estimate bubble size. Accuracy of the hybrid measurement/model was assessed by comparing the predicted and measured dissolution time of the bubble cloud. Good agreement was found between predictions and measurements of bubble cloud dissolution times in agarose phantoms and murine subcutaneous SCC VII tumors. The analytic diffusion model was extended to compute the maximum bubble size as well as energy imparted to the tissue due to bubble expansion. Regions within tumors predicted to have undergone strong bubble expansion were collocated with ablation. Further, the dissolution time was found to correlate with acoustic emissions generated by the bubble cloud during histotripsy insonation. Overall, these results indicate a combination of modeling and high frame rate imaging may provide means to quantify mechanical energy imparted to the tissue due to bubble expansion for histotripsy.

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.