Destruction thresholds of echogenic liposomes with clinical diagnostic ultrasound

Echogenic liposomes (ELIP) are submicron-sized phospholipid vesicles that contain both gas and fluid. With antibody conjugation and drug incorporation, these liposomes can be used as novel targeted diagnostic and therapeutic ultrasound contrast agents. The utility of liposomes for contrast depends upon their stability in an acoustic field, whereas the use of liposomes for drug delivery requires the liberation of encapsulated gas and drug payload at the desired treatment site. The objective of this study was twofold: (1) to characterize the stability of liposome echogenicity after reconstitution and (2) to quantitate the acoustic destruction thresholds of liposomes as a function of peak rarefactional pressure (P(r)), pulse duration (PD) and pulse repetition frequency (PRF). The liposomes were insonified in an anechoic sample chamber using a Philips HDI 5000 diagnostic ultrasound scanner with a L12-5 linear array. Liposome stability was evaluated with 6.9-MHz fundamental and 4.5-MHz harmonic B-mode pulses at various P(r) at a fixed PRF. Liposome destruction thresholds were determined using 6.0-MHz Doppler pulses, by varying the PD with a fixed PRF of 1.25 kHz and by varying the PRF with a fixed PD of 3.33 micros. Videos or freeze-captured images were acquired during each insonation experiment and analyzed for echogenicity in a fixed region of interest as a function of time. An initial increase in echogenicity was observed for fundamental and harmonic B-mode imaging pulses. The threshold for acoustically driven diffusion of gas out of the liposomes using 6.0-MHz Doppler pulses was weakly dependent upon PRF and PD. The rapid fragmentation thresholds, however, were highly dependent upon PRF and PD. The quantification of acoustic destruction thresholds of ELIP is an important first step in their development as diagnostic and drug delivery agents.     

Ultrasound-Triggered Release of Recombinant Tissue-Type Plasminogen Activator from Echogenic Liposomes

Echogenic liposomes (ELIP) were developed as ultrasound-triggered targeted drug or gene delivery vehicles (Lanza et al. 1997; Huang et al. 2001). Recombinant tissue-type plasminogen activator (rt-PA), a thrombolytic, has been loaded into ELIP (Tiukinhoy-Laing et al. 2007). These vesicles have the potential to be used for ultrasound-enhanced thrombolysis in the treatment of acute ischemic stroke, myocardial infarction, deep vein thrombosis or pulmonary embolus. A clinical diagnostic ultrasound scanner (Philips HDI 5000; Philips Medical Systems, Bothell, WA, USA) equipped with a linear array transducer (L12-5) was employed for in vitro studies using rt-PA-loaded ELIP (T-ELIP). The goal of this study was to quantify ultrasound-triggered drug release from rt-PA-loaded echogenic liposomes. T-ELIP samples were exposed to 6.9-MHz B-mode pulses at a low pressure amplitude (600 kPa) to track the echogenicity over time under four experimental conditions: (1) flow alone to monitor gas diffusion from the T-ELIP, (2) pulsed 6.0-MHz color Doppler exposure above the acoustically driven threshold (0.8 MPa) to force gas out of the liposome gently, (3) pulsed 6.0-MHz color Doppler above the rapid fragmentation threshold (2.6 MPa) or (4) Triton X-100 to rupture the T-ELIP chemically as a positive control. Release of rt-PA for each ultrasound exposure protocol was assayed spectrophotometrically. T-ELIP were echogenic in the flow model (5 mL/min) for 30 min. The thrombolytic drug remained associated with the liposome when exposed to low-amplitude B-mode pulses over 60 min and was released when exposed to color Doppler pulses or Triton X-100. The rt-PA released from the liposomes had similar enzymatic activity as the free drug. These T-ELIP are robust and echogenic during continuous fundamental 6.9-MHz B-mode imaging at a low exposure output level (600 kPa). Furthermore, a therapeutic concentration of rt-PA can be released by fragmenting the T-ELIP with pulsed 6.0-MHz color Doppler ultrasound above the rapid fragmentation threshold (1.59 MPa). (E-mail: denise.smith@uc.edu)     

On the usefulness of the mechanical index displayed on clinical ultrasound scanners for predicting contrast microbubble destruction.

OBJECTIVE: The purpose of this study was to evaluate the mechanical index (MI) displayed on clinical ultrasound scanners as a predictor of exposure conditions related to the destruction of sonographic microbubble contrast agents. METHODS: Sonazoid (GE Healthcare, Oslo, Norway) and Optison (GE Healthcare, Princeton, NJ) microbubbles were injected into a tissue-mimicking flow phantom. Gray scale imaging was performed with 4 different scanners and 3 different transducers (3.5 MHz curved linear, 2.5 MHz convex, and 7.5 MHz linear array), and the MI displayed by the scanner was varied from 0.2 to 1.5 by changing the system output power. All other scanning parameters were kept constant. Downstream changes in echogenicity were monitored with a PowerVision 7000 scanner (Toshiba America Medical Systems, Tustin, CA) as an indirect measure of bubble destruction. Video intensity changes within the flow tube were determined as a function of MI for the different scanner/transducer combinations, and the best linear fit was determined. RESULTS: At a displayed MI of 0.7, different scanner/transducer combinations exhibited a range in video intensity from +16% to -3% of baseline for Sonazoid and from +8% to -71% for Optison. At an MI of 0.3, reductions in video intensity of up to 32% were produced. These results indicate a wide range in bubble destruction at identical MI values. Likewise, regression analysis found no linear fits for all scanner/transducer combinations (r2 < 0.046). CONCLUSIONS: The MI displayed on clinical ultrasound scanners does not predict the degree of microbubble destruction and should not be used by itself to define exposure conditions for destruction of microbubble contrast agents.     

Examination of inertial cavitation of Optison in producing sonoporation of chinese hamster ovary cells

The objective of this project was to elucidate the relationship between ultrasound contrast agents (UCAs) and sonoporation. Sonoporation is an ultrasound-induced, transient cell membrane permeability change that allows for the uptake of normally impermeable macromolecules. Specifically, this study will determine the role that inertial cavitation plays in eliciting sonoporation. The inertial cavitation thresholds of the UCA, Optison, are compared directly with the results of sonoporation to determine the involvement of inertial cavitation in sonoporation. Chinese hamster ovary (CHO) cells were exposed as a monolayer in a solution of Optison, 500,000 Da fluorescein isothiocyanate-dextran (FITC-dextran), and phosphate-buffered saline (PBS) to 30 s of pulsed ultrasound at 3.15-MHz center frequency, 5-cycle pulse duration and 10-Hz pulse repetition frequency. The peak rarefactional pressure (P(r)) was varied over a range from 120 kPa-3.5 MPa, and five independent replicates were performed at each pressure. As the P(r) was increased, from 120 kPa-3.5 MPa, the fraction of sonoporated cells among the total viable population increased from 0.63-10.21%, with the maximum occurring at 2.4 MPa. The inertial cavitation threshold for Optison at these exposure conditions has previously been shown to be in the range 0.77-0.83 MPa, at which sonoporation activity was found to be 50% of its maximum level. Furthermore, significant sonoporation activity was observed at pressure levels below the threshold for inertial cavitation of Optison. Above 2.4 MPa, a significant drop in sonoporation activity occurred, corresponding to pressures where >95% of the Optison was collapsing. These results demonstrate that sonoporation is not directly a result of inertial cavitation of the UCA, rather that the effect is related to linear and/or nonlinear oscillation of the UCA occurring at pressure levels below the inertial cavitation threshold.     

The disappearance of ultrasound contrast bubbles: observations of bubble dissolution and cavitation nucleation

The destruction process of biSphere and Optison ultrasound (US) contrast microbubbles were studied at 1.1 MHz. High-amplitude tone bursts caused shell disruption and/or fragmentation of the microbubbles, leading to dissolution of the freed gas. The bubble destruction and subsequent dissolution process was imaged with a high pulse-repetition frequency (PRF) 10-cycle, 5-MHz bistatic transducer configuration. Three types of dissolution profiles were measured: In one case, biSphere microbubbles showed evidence of dissolution through resonance, during which a temporary increase in the scattering amplitude was observed. In another case, both biSphere and Optison microbubbles showed evidence of fragmentation, during which the scattering amplitude decreased rapidly. Finally, in some cases, we observed the impulsive growth and subsequent rapid decay of signals that appear to be due to cavitation nucleation. Simulations of bubble dissolution curves show good agreement with experiments.     

Inertial cavitation dose produced in ex vivo rabbit ear arteries with Optison by 1-MHz pulsed ultrasound

Previous in vitro studies have shown that ultrasound-induced mechanical bioeffects with contrast agents present are highly correlated with inertial cavitation (IC) "dose" (Chen et al. 2003a, 2003c). The ex vivo experiments conducted here addressed the following hypotheses: 1. IC activity can be generated by insonating perfused rabbit ear blood vessel, and 2. the IC "dose" developed during insonation treatment can be reliably measured and will vary with varying acoustic parameters and Optison concentration. Ex vivo rabbit auricular arteries were perfused with Optison suspensions and then exposed to 1.1-MHz pulsed focused ultrasound. Experimental variables included peak negative acoustic pressure (0.2 MPa to 5.2 MPa), pulse-repetition frequency (5, 50 or 500 Hz), pulse length (50, 100, 500 or 1000 cycles), and Optison volume concentration (0, 0.2, 0.5 or 1%). Cavitation activity was quantified as IC dose, based on passive cavitation detection measurements. The results show that: 1. The IC pressure threshold decreases with higher concentrations of Optison, and 2. IC dose increases significantly with increasing acoustic pressure, Optison concentration, pulse length or with decreasing pulse-repetition frequency.     

Lack of bioeffects of ultrasound energy after intravenous administration of FS069 (Optison) in the anesthetized rabbit.

The current study was designed to provide a sensitive in vivo model to maximize the potential bioeffects (measured by hemolysis) of B-mode ultrasound energy in combination with FS069 (Optison). B-mode ultrasound energy was delivered to anesthetized male New Zealand white rabbits with a phased array 5 MHz transducer on a Hewlett-Packard Sonos 1500 ultrasonograph, with transmit level set to maximum (40 dB, approx 135 W/cm2). FS069 (Optison), latex particles in human albumin, or human albumin alone (vehicle) was infused via an ear vein at 0.6 mL/kg. No statistically significant changes were noted in serum free hemoglobin or lactate dehydrogenase either over time or between groups.     

Membrane damage thresholds for pulsed or continuous ultrasound in phagocytic cells loaded with contrast agent gas bodies

Cell membrane damage induced by pulsed or continuous ultrasound (US) activation of attached contrast agent gas bodies was examined in an in vitro model system. Monolayers of mouse macrophage-like cells were cultured on the inside of one window of an exposure chamber. The monolayers were incubated with Optison (Amersham Health Inc., Princeton, NJ) or Definity (Bristol-Myers Squib Medical Imaging, North Billerica, MA) and then rinsed to remove unattached gas bodies. A 3.5-MHz focused transducer was aimed at the chamber 3.7 cm away in a 37 degrees C water bath. The cells were scored for Trypan blue dye exclusion, with stained nuclei indicative of cell membrane damage. Exposure-response functions were approximated with exposure levels spaced 3-dB apart. Thresholds were located between the lowest exposure with statistically significant counts of blue-stained cells relative to sham exposures, and the next lower level. Thresholds with Optison included 0.05 MPa for 60-s continuous exposure duration, and 0.21 MPa for 0.6-micros pulses with 60-micros repetition period for 60-s pulsed exposure duration. Results were similar for Definity. Thresholds changed slowly with changes in timing parameters; for example, the threshold for a 0.6-micros continuous exposure (i.e., one pulse) was 0.84 MPa. Compared to 60-s exposure, this represents a factor of 16.8 increase in threshold for a factor of 10(8) decrease in exposure duration. The thresholds are less than the pressure amplitudes needed for nucleation of inertial cavitation, which suggests classification of the phenomenon as a form of gas body activation.     

DNA transfer and cell killing in epidermoid cells by diagnostic ultrasound activation of contrast agent gas bodies in vitro

DNA transfer by sonoporation and cell killing in monolayer cells were examined by contrast-aided low-power diagnostic ultrasound (US). Culture chambers with epidermoid cell monolayers were scanned at about 1 mm/s with a 1.5-MHz scan head aimed upward at the chamber in a 37 degrees C water bath. For DNA transfer tests, plasmids coding for green fluorescent protein (GFP) were added to the medium, and GFP expression was assessed by flow cytometry after 2 days. In separate tests, cell killing was determined immediately after treatment. GFP-positive cell counts were 0.4% (0.7% SD) for shams and 3.7% (1.2% SD) of cells for exposure at 2.3 MPa with 2% Optison contrast agent. The fraction of dead cells was 3.4% (1.7% SD) in shams and 28.6% (6.3% SD) in exposed chambers. Both effects increased for increasing Optison concentration and increasing peak rarefactional pressure amplitude. Contrast-aided diagnostic US has a potential therapeutic application for gene transfer, but a trade-off appears to exist with cell killing.     

Membrane damage thresholds for 1- to 10-MHz pulsed ultrasound exposure of phagocytic cells loaded with contrast agent gas bodies in vitro

Monolayers of mouse macrophage-like cells provide a model system for the study of bioeffects of pulsed ultrasound (US) activation of contrast agent gas bodies. In this study, the dependence of membrane damage on ultrasonic frequency was examined for gas bodies attached to the cells. The monolayers cultured on the inside of one window of an exposure chamber were incubated with 2% Optison (Amersham Health Inc., Princeton, NJ) and then rinsed to remove unattached gas bodies. The chamber was filled with culture medium plus 20% trypan blue stain solution and was mounted at the 3.8-cm focus of an US transducer in a 37 degrees C water bath. Transducers were used with center frequencies of 1.0, 2.25, 3.5, 5.0, 7.5 and 10.0 MHz. The 1-min pulsed exposures utilized two-cycle excitation with 1% duty cycle. After exposure, cells in the focal zone were scored for trypan blue dye exclusion, with stained nuclei indicative of cell membrane damage. Exposure-response functions were approximated by performing a series of exposures with peak rarefactional pressure amplitudes differing by a factor of radical 2 (i.e., 3 dB apart). Linear regressions were performed on selected data to determine a threshold pressure amplitude at each frequency. Thresholds ranged from 0.066 MPa at 1.0 MHz to 0.62 MPa at 10 MHz and were approximately proportional to the frequency. These thresholds are less than the pressure amplitudes needed for nucleation of inertial cavitation and have a different frequency dependence than the general Mechanical Index.     

The potential for enhancement of mouse melanoma metastasis by diagnostic and high-amplitude ultrasound

The potential for enhancement of the metastatic spread of cells from mouse melanoma tumors was examined for exposure to diagnostic ultrasound (DUS) and high-amplitude ultrasound (HAUS) without and with ultrasound (US) contrast agent. The melanoma cell line B16-D5, which is metastatic specifically to lung, was cultured and implanted on a hind leg of female C57/bl6 mice. For DUS, tumors were scanned using 1.5-MHz harmonic B-mode imaging with 1-Hz intermittent frame triggering at 2.1 MPa (equivalent MI = 1.7) in a 37 degrees C water bath. For HAUS, a 1.35-MHz focused transducer directed 1-ms bursts at 5 MPa to the tumor at a 1-Hz rate. A total dose of 1 mL/kg Optison was injected during exposures. Exposure without contrast agent received the same exposure followed by the contrast agent with the US off. The primary tumor was removed surgically one day after US. Lungs were removed after four weeks for evaluation of metastases. Experiments involved exposure without and with contrast agent in groups of 20 mice. For DUS, mean counts of 0.8 +/- 0.3 (standard error) and 1.3 +/- 0.9 (P = 0.62) metastases were found for groups exposed without and with contrast, respectively. For HAUS, mean counts of 3.4 +/- 1.2 and 5.9 +/- 1.7 (P = 0.35) metastases were found for groups exposed without and with contrast, respectively. The lack of effect of DUS exposure with contrast confirms a previous finding. However, the HAUS counts and incidence were significantly larger than the DUS results (P < 0.05) in a two-way analysis of variance. This indicates a potential for HAUS to enhance metastasis.     

Improved visualization of high-intensity focused ultrasound lesions

Spectral parameter imaging in both the fundamental and harmonic of backscattered radio-frequency (RF) data were used for immediate visualization of high-intensity focused ultrasound (HIFU) lesion sites. A focused 5-MHz HIFU transducer with a coaxial 9-MHz focused single-element diagnostic transducer was used to create and scan lesions in chicken breast and freshly excised rabbit liver. B-mode images derived from the backscattered RF signal envelope were compared with midband fit (MBF) spectral parameter images in the fundamental (9-MHz) and harmonic (18-MHz) bands of the diagnostic probe. Images of HIFU-induced lesions derived from the MBF to the calibrated spectrum showed improved contrast (approximately 3 dB) of tumor margins versus surround compared with images produced from the conventional signal envelope. MBF parameter images produced from the harmonic band showed higher contrast in attenuated structures (core, shadow) compared with either the conventional envelope (3.3 dB core; 11.6 dB shadow) or MBF images of the fundamental band (4.4 dB core; 7.4 dB shadow). The gradient between the lesion and surround was 3.4 dB/mm, 6.9 dB/mm and 17.2 dB/mm for B-mode, MBF-fundamental mode and MBF-harmonic mode, respectively. Images of threshold and "popcorn" lesions produced in freshly excised rabbit liver were most easily visualized and boundaries best-defined using MBF-harmonic mode.     

The impact of emission power on the destruction of echo contrast agents and on the origin of tissue harmonic signals using power pulse-inversion imaging.

The purpose of this study was to determine the impact of emission power on ultrasound (US)-induced destruction of echocontrast microbubbles during real-time power pulse inversion imaging (PPI) in myocardial contrast echocardiography (MCE) and to evaluate the magnitude of noncontrast PPI signals arising from myocardial tissue at variable emission power to define the cut-off emission power for optimal MCE using low power technologies. In vitro studies were performed in a flow phantom using Optison, Definity and AFO 150. PPI signal intensity during real-time imaging at 27 Hz was compared with intermittent imaging at 0.1 Hz to evaluate bubble destruction at variable emission power (MI: 0.09 to 1.3). In healthy volunteers, PPI signal intensities during constant infusion of Optison(R) was studied in real-time PPI 22 HZ and during intermittent imaging triggered end-systolic frames every, every 3rd and every 5th cardiac cycle. In addition, the impact of emission power on nonlinear PPI signals from myocardial structures was studied. In vitro, there was a 40% decrease of real-time PPI signal intensity for Optison and AFO 150 at lowest emission power (0.09), whereas no signal loss was observed for Definity. Increase of emission power resulted in a faster decay for Optison(R) and AFO 150 as compared to Definity. In vivo, real-time PPI during continuous infusion of Optison(R) resulted in a 40% decrease of myocardial signal intensity as compared to intermittent imaging every 5th cardiac cycle, even at lowest possible emission power (mechanical index = 0.09). There was a strong positive relationship between MI and noncontrast myocardial PPI signals in all myocardial segments. PPI signal intensity was found to be lower than 1 dB only for extremely low emission power (MI < 0.2). Destruction of microbubbles during real-time imaging by use of PPI at low emission power varies considerably for different echo contrast agents. However, bubble destruction and the onset of tissue harmonic signals focus the use of real-time perfusion imaging to very low emission power.     

Ultrasound-mediated cavitation thresholds of liquid perfluorocarbon droplets in vitro

This study was undertaken to measure the ultrasound (US)-mediated cavitation threshold of microdroplets as a function of its content and US parameters (frequency, amplitude and burst length). Albumin-coated droplets were prepared with perfluoropropane, perfluorohexane or perfluoromethylcyclohexane contents. The filtered suspensions were diluted to 1:1000 (v) and compared with Optison. The formulations were injected into an acoustically transparent vessel and sonicated with a single focused transducer. The frequencies employed were 0.74, 1.1, 2.18 and 3.3 MHz and the burst length and acoustic pressure were varied. The inertial cavitation threshold for each experiment was monitored through passive acoustic detection. The formation of droplet emulsion of the perfluorocarbon increased the natural boiling point of the perfluorocarbon. However, perfluorocarbon droplets having contents with higher molecular weights and boiling points did not have detectably higher inertial cavitation thresholds and, thus, the droplets do not need to be in a superheated state to be cavitated by US bursts. Therefore, higher boiling point perfluorocarbons should be investigated for this purpose and may prove to be useful for both imaging and therapy. The inertial cavitation threshold of perfluorocarbon droplets increases with frequency, and was approximately 0.7 MPa at 0.74 MHz and 1.75 MPa at 3.3 MHz. Optison, already in a gaseous state, has the lowest cavitation threshold of all formulations studied. Results show that, for the frequencies tested, there is no dependence between inertial cavitation threshold and burst lengths between 20 and 100 ms. As a conclusion, the inertial cavitation threshold of albumin-coated microdroplets of several perfluorocarbons was determined in vitro. The results indicate that the physical properties of these droplets are such that they may be useful for localized US therapies.     

Sonoporation of monolayer cells by diagnostic ultrasound activation of contrast-agent gas bodies

Human (A431 epidermoid carcinoma) cells were grown as monolayers on 5 microm thick Mylar sheets, which formed the upper window for a 1-mm thick, 23-mm diameter disc-shaped exposure chamber. A 3.5-MHz curved linear-array transducer was aimed upward at the chamber, 7 cm away, in a 37 degrees C water bath. The chamber contained phosphate-buffered saline (PBS) with 10 mg/mL fluorescent dextran and 1% Optison ultrasound (US) contrast agent. Significant fluorescent cell counts, indicative of membrane damage (i.e., sonoporation), up to about 10% of cells within a 1-mm diameter field of view, were noted for spectral Doppler and two-dimensional (2-D) scan mode with or without a tissue-mimicking phantom. The effect was only weakly dependent on pulse-repetition frequency or exposure duration, but was strongly dependent on contrast agent concentration below 2%. Thus, diagnostic US activation of contrast-agent gas bodies can produce cell membrane damage.     

Threshold estimates and superthreshold behavior of ultrasound-induced lung hemorrhage in adult rats: role of pulse duration

The study objective was to estimate the pressure threshold (ED(05), effective dose, or in situ peak rarefactional pressure associated with 5% probability of lesions) of ultrasound (US)-induced lung hemorrhage as a function of pulse duration (PD) in adult rats. A total of 220 10- to 11-week-old 250-g female Sprague-Dawley rats (Harlan) were randomly divided into 20 ultrasonic exposure groups (10 rats/group) and one sham group (20 rats). The 20 ultrasonic exposure groups (2.8-MHz; 10-s exposure duration; 1-kHz PRF; -6-dB pulse-echo focal beam width of 470 microm) were divided into four PD groups (1.3, 4.4, 8.2 and 11.6 micros) and, for each PD group, there were five in situ peak rarefactional pressures (range between 4 and 9 MPa). Rats were weighed, anesthetized, depilated, exposed, and euthanized under anesthesia. The left lung was removed and scored for the occurrence of hemorrhage. If hemorrhage was present, the lesion surface area and depth were measured. Individuals involved in animal handling, exposure and lesion scoring were "blinded" to the exposure conditions. Logistic regression analysis was used to examine the dependence of the lesion occurrences, and Gaussian tobit regression analysis was used to examine the dependence of the lesion surface areas and depths on in situ peak rarefactional pressure and PD. Threshold results are reported in terms of ED(05). For PDs of 1.3, 4.4, 8.2 and 11.6 micros, respectively, lesion occurrence ED(05)s were 3.1, 2.8, 2.3 and 2.0 MPa with standard errors around 0.6 MPa. Lesion size ED(05)s showed similar values. A mechanical index (MI) of 1.9, the US Food and Drug Administration (FDA) regulatory limit of diagnostic US equipment, is equivalent to the adult rat's in situ peak rarefactional pressure of 4.0 MPa. PDs of 8.2 and 11.6 micros had ED(05)s more than 2 standard errors below 4.0 MPa, indicating that the ED(05)s of these two PDs are statistically significantly different from 4.0 MPa. The ED(05) threshold levels for a PD of 1.3 micros are consistent with previous US-induced lung hemorrhage studies. As the PD increases, the ED(05) levels decrease, suggesting greater likelihood of lung damage as the PD increases. All of the ED(05)s are less than the FDA limit.     

The Influence of Xylazine and Clonidine on Lung Ultrasound–Induced Pulmonary Capillary Hemorrhage in Spontaneously Hypertensive Rats

Induction of pulmonary capillary hemorrhage (PCH) by lung ultrasound (LUS) depends not only on physical exposure parameters but also on physiologic conditions and drug treatment. We studied the influence of xylazine and clonidine on LUS-induced PCH in spontaneously hypertensive and normotensive rats using diagnostic B-mode ultrasound at 7.3 MHz. Using ketamine anesthesia, rats receiving saline, xylazine, or clonidine treatment were tested with different pulse peak rarefactional pressure amplitudes in 5 min exposures. Results with xylazine or clonidine in spontaneously hypertensive rats were not significantly different at the three exposure pulse peak rarefactional pressure amplitudes, and thresholds were lower (2.2 MPa) than with saline (2.6 MPa). Variations in LUS PCH were not correlated with mean systemic blood pressure. Similar to previous findings for dexmedetomidine, the clinical drug clonidine tended to increase susceptibility to LUS PCH.     

Characterization of Bioeffects on Endothelial Cells under Acoustic Droplet Vaporization

Gas embolotherapy is achieved by locally vaporizing microdroplets through acoustic droplet vaporization, which results in bubbles that are large enough to occlude blood flow directed to tumors. Endothelial cells, lining blood vessels, can be affected by these vaporization events, resulting in cell injury and cell death. An idealized monolayer of endothelial cells was subjected to acoustic droplet vaporization using a 3.5-MHz transducer and dodecafluoropentane droplets. Treatments included insonation pressures that varied from 2 to 8 MPa (rarefactional) and pulse lengths that varied from 4 to 16 input cycles. The bubble cloud generated was directly dependent on pressure, but not on pulse length. Cellular damage increased with increasing bubble cloud size, but was limited to the bubble cloud area. These results suggest that vaporization near the endothelium may impact the vessel wall, an effect that could be either deleterious or beneficial depending on the intended overall therapeutic application.     

Pulmonary Capillary Hemorrhage Induced by Different Imaging Modes of Diagnostic Ultrasound

The induction of pulmonary capillary hemorrhage (PCH) is a well-established non-thermal biological effect of pulsed ultrasound in animal models. Typically, research has been done using laboratory pulsed ultrasound systems with a fixed beam and, recently, by B-mode diagnostic ultrasound. In this study, a GE Vivid 7 Dimension ultrasound machine with 10?L linear array probe was used at 6.6?MHz to explore the relative PCH efficacy of B-mode imaging, M-mode (fixed beam), color angio mode Doppler imaging and pulsed Doppler mode (fixed beam). Anesthetized rats were scanned in a warmed water bath, and thresholds were determined by scanning at different power steps, 2?dB apart, in different groups of six rats. Exposures were performed for 5?min, except for a 15-s M-mode group. Peak rarefactional pressure amplitude thresholds were 1.5?MPa for B-mode and 1.1?MPa for angio Doppler mode. For the non-scanned modes, thresholds were 1.1?MPa for M-mode and 0.6?MPa for pulsed Doppler mode with its relatively high duty cycle (7.7?×?10^?3 vs. 0.27?×?10^?3 for M-mode). Reducing the duration of M-mode to 15?s (from 300?s) did not significantly reduce PCH (area, volume or depth) for some power settings, but the threshold was increased to 1.4?MPa. Pulmonary sonographers should be aware of this unique adverse bio-effect of diagnostic ultrasound and should consider reduced on-screen mechanical index settings for potentially vulnerable patients.     

Inertial cavitation dose and hemolysis produced in vitro with or without Optison

Gas-based contrast agents (CAs) increase ultrasound (US)-induced bioeffects, presumably via an inertial cavitation (IC) mechanism. The relationship between IC dose (ICD) (cumulated root mean squared [RMS] broadband noise amplitude; frequency domain) and 1.1-MHz US-induced hemolysis in whole human blood was explored with Optison; the hypothesis was that hemolysis would correlate with ICD. Four experimental series were conducted, with variable: 1. peak negative acoustic pressure (P-), 2. Optison concentration, 3. pulse duration and 4. total exposure duration and Optison concentration. P- thresholds for hemolysis and ICD were approximately 0.5 MPa. ICD and hemolysis were detected at Optison concentrations >/= 0.01 V%, and with pulse durations as low as four or two cycles, respectively. Hemolysis and ICD evolved as functions of time and Optison concentration; final hemolysis and ICD values depended on initial Optison concentration, but initial rates of change did not. Within series, hemolysis was significantly correlated with ICD; across series, the correlation was significant at p < 0.001.