Kenneth B. Bader

Shaken and Stirred: Mechanisms of Ultrasound-Enhanced Thrombolysis

The use of ultrasound and microbubbles as an effective adjuvant to thrombolytics has been reported in vitro, ex vivo and in vivo. However, the specific mechanisms underlying ultrasound-enhanced thrombolysis have yet to be elucidated. We present visual observations illustrating two mechanisms of ultrasound-enhanced thrombolysis: acoustic cavitation and radiation force. An in vitro flow model was developed to observe human whole blood clots exposed to human fresh-frozen plasma, recombinant tissue-type plasminogen activator (0, 0.32, 1.58 or 3.15 μg/mL) and the ultrasound contrast agent Definity (2 μL/mL). Intermittent, continuous-wave ultrasound (120 kHz, 0.44 MPa peak-to-peak pressure) was used to insonify the perfusate. Ultraharmonic emissions indicative of stable cavitation were monitored with a passive cavitation detector. The clot was observed with an inverted microscope, and images were recorded with a charge-coupled device camera. The images were post-processed to determine the time-dependent clot diameter and root-mean-square velocity of the clot position. Clot lysis occurred preferentially surrounding large, resonant-sized bubbles undergoing stable oscillations. Ultraharmonic emissions from stable cavitation were found to correlate with the lytic rate. Clots were observed to translate synchronously with the initiation and cessation of the ultrasound exposure. The root-mean-square velocity of the clot correlated with the lytic rate. These data provide visual documentation of stable cavitation activity and radiation force during sub-megahertz sonothrombolysis. The observations of this study suggest that the process of clot lysis is complex, and both stable cavitation and radiation force are mechanistically responsible for this beneficial bio-effect in this in vitro model.

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.

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.

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.

Predicting the growth of nanoscale nuclei by histotripsy pulses.

Histotripsy is a focused ultrasound therapy that ablates tissue through the mechanical action of cavitation. Histotripsy-initiated cavitation activity is generated from shocked ultrasound pulses that scatter from incidental nuclei (shock scattering histotripsy), or purely tensile ultrasound pulses (microtripsy). The Yang/Church model was numerically integrated to predict the behavior of the cavitation nuclei exposed to measured shock scattering histotripsy pulses. The bubble motion exhibited expansion only behavior, suggesting that the ablative action of a histotripsy pulse is related to the maximum size of the bubble. The analytic model of Holland and Apfel was extended to predict the maximum size of cavitation nuclei for both shock scattering histotripsy and microtripsy excitations. The predictions of the analytic model and the numerical model agree within 2% for fully developed shock scattering histotripsy pulses (>72 MPa peak positive pressure). For shock scattering histotripsy pulses that are not fully developed (<72 MPa), the analytic model underestimated the maximum size by less than 5%. The analytic model was also used to predict bubble growth nucleated from microtripsy insonations, and was found to be consistent with experimental observations. Based on the extended analytic model, metrics were developed to predict the extent of the treatment zone from histotripsy pulses.

Efficacy of histotripsy combined with rt-PA in vitro

Histotripsy, a form of therapeutic ultrasound that uses the mechanical action of microbubble clouds for tissue ablation, is under development to treat chronic deep vein thrombosis (DVT). We hypothesize that combining thrombolytic agents with histotripsy will enhance clot lysis. Recombinant tissue plasminogen activator (rt-PA) and rt-PA-loaded echogenic liposomes that entrain octafluoropropane microbubbles (OFP t-ELIP) were used in combination with highly shocked histotripsy pulses. Fully retracted porcine venous clots, with similar features of DVT occlusions, were exposed either to histotripsy pulses alone (peak negative pressures of 7-20 MPa), histotripsy and OFP t-ELIP, or histotripsy and rt-PA. Microbubble cloud activity was monitored with passive cavitation imaging during histotripsy exposure. The power levels of cavitation emissions from within the clot were not statistically different between treatment types, likely due to the near instantaneous rupture and destruction of OFP t-ELIP. The thrombolytic efficacy was significantly improved in the presence of rt-PA. These results suggest the combination of histotripsy and rt-PA could serve as a potent therapeutic strategy for the treatment of DVT.

In vitro thrombolytic efficacy of echogenic liposomes loaded with tissue plasminogen activator and octafluoropropane gas

Echogenic liposomes loaded with the thrombolytic recombinant tissue-type plasminogen activator (rt-PA) are under development for the treatment of ischemic stroke. These agents are designed to co-encapsulate cavitation nuclei to promote bubble activity in response to ultrasound exposure, and to enable localized delivery of thrombolytic. Stable cavitation improves the efficacy of the thrombolytic through enhanced fluid mixing. Echogenic liposomes that encapsulate air-filled microbubbles nucleate scant stable cavitation activity in response to 120 kHz intermittent ultrasound exposure, and have demonstrated thrombolytic efficacy equivalent to rt-PA alone. It was hypothesized that encapsulating octafluoropropane (OFP) gas within rt-PA-loaded liposomes instead of air will enhance ultrasound-mediated stable cavitation activity and increase thrombolytic efficacy compared to previous studies. The thrombolytic efficacy and cavitation activity nucleated from liposomes that encapsulate OFP microbubbles and rt-PA (OFP t-ELIP) was evaluated in vitro. Human whole blood clots were exposed to human fresh-frozen plasma alone, rt-PA (0, 0.32, 1.58, and 3.15 µg ml-1), or OFP t-ELIP at equivalent enzymatic activity, with and without exposure to intermittent ultrasound. Further, numerical simulations were performed to gain insight into the mechanisms of cavitation nucleation. Sustained ultraharmonic activity was nucleated from OFP t-ELIP when exposed to ultrasound. Furthermore, the thrombolytic efficacy was enhanced compared to rt-PA alone at concentrations of 1.58 µg ml-1 and 3.15 µg ml-1 (p  <  0.05). These results indicate that OFP t-ELIP can nucleate sustained stable cavitation activity and enhance the efficacy of thrombolysis.

Quantitative Frequency-Domain Passive Cavitation Imaging

Passive cavitation detection has been an instrumental technique for measuring cavitation dynamics, elucidating concomitant bioeffects, and guiding ultrasound therapies. Recently, techniques have been developed to create images of cavitation activity to provide investigators with a more complete set of information. These techniques use arrays to record and subsequently beamform received cavitation emissions, rather than processing emissions received on a single-element transducer. In this paper, the methods for performing frequency-domain delay, sum, and integrate passive imaging are outlined. The method can be applied to any passively acquired acoustic scattering or emissions, including cavitation emissions. To compare data across different systems, techniques for normalizing Fourier transformed data and converting the data to the acoustic energy received by the array are described. A discussion of hardware requirements and alternative imaging approaches is additionally outlined. Examples are provided in MATLAB.

The effect of static pressure on the inertial cavitation threshold and collapse strength.

The conditions within an inertially collapsing bubble are known to produce high temperatures and pressures. Previous investigators have proposed various mechanisms for driving the collapse in order to maximize the energy density of the bubble’s contents. Working in fluids at high static pressures (up to 30 MPa) has shown some promising results. Preliminary data indicate that the strength of the collapse scales with the static pressure in the fluid. We report here the results of a series of rigorous experiments designed to better define this relation at the threshold of inertial cavitation. The hydrostatic pressure dependence of the inertial cavitation threshold in ultrapure water was measured in a radially symmetric standing wave field in a spherical resonator driven at 26 kHz. At this threshold, the collapse strength will be reported in terms of the resulting photon radiance below 400 nm and the relative shock wave amplitude. These experimental results will be compared to predictions of collapse strength...

Inertial cavitation threshold dependence on static pressures

A bubble will be nucleated in a liquid if the tension (or negative pressure) overcomes the forces that oppose the bubbles growth (static pressure and surface tension). The magnitude of the acoustic pressure required to nucleate a bubble capable of producing macroscopically measurable phenomena (e.g. light or shock waves) is the termed the inertial cavitation threshold. The hydrostatic pressure dependence of the inertial cavitation threshold in water was measured using standing waves in a spherical resonator. The resonator was driven at a radially symmetric mode with resonant frequency of 25.6 kHz at 1 atmosphere. The preliminary experimental measurements, reported here, showed the inertial cavitation threshold to be linear (r2= 0.98) over the range of static pressures measured, 10-250 bar. This is consistent with data reported for threshold reported at modest static pressures.