THE EFFECT OF AN OVERPRESSURE OF 90 mmHg ON THE CAVITATION THRESHOLD AND ECHOGENICITY OF DEFINITY® Tyler Fosnight, Kirthi Radhakrishnan, and Kevin Haworth School of Energy, Environmental, Biological and Medical Engineering, University of Cincinnati, Cincinnati, OH Abstract- Cavitation of ultrasound contrast agents is currently being investigated as anultrasound mediated therapeutic mechanism. In this in vitro study, cavitation thresholds andechogenicity change thresholds were measured for the commercial ultrasound contrast agentDefinity® insonified by a clinical transducer operated in pulsed Doppler (center frequency 6MHz, pulse repetition frequency 1250 Hz, mechanical indexes ranging from 0.0 to 0.56 (i.e. peakrarefactional pressure (PRP) 0 to 1.43 MPa)). Definity® was diluted in 0.5% (w/v) bovine serumalbumin and flowed through a latex tube at a flow rate of 5 ml/min with the overpressures, withrespect to atmospheric, set to 0 mmHg or 90 mmHg (0 kPa and 12 kPa, respectively).Subharmonic and broadband emissions acquired using a 10 MHz spherically focused passivecavitation detector (PCD) were used to define the stable and inertial cavitation thresholds,respectively. Images of Definity® were acquired by operating the clinical transducer in B-mode.Single frames before insonification with pulsed Doppler and after insonification with pulsedDoppler were acquired. Mean grayscale values from captured single frames were used to definean echogenicity loss threshold. For the UCA insonified with 6 MHz pulsed Doppler, the onset ofechogenicity loss was at a higher MI with an overpressure of 0 mmHg than 90 mmHg. The stableand inertial cavitation thresholds and bubble activity were approximately equal, for bothoverpressures of 0 and 90 mmHg. The independence of cavitation thresholds and the cavitationemission energy on the overpressure suggests that these thresholds may be considered applicableto in vivo environments as well.Introduction The use of ultrasound to enhance drug and gene delivery has many potential benefits inthe treatment of diseases like ischemic stroke (Datta, 2008; Smith, 2010), myocardial infarctions(Christiansen, 2002), and deep vein thrombosis (Datta, 2008). Several studies have demonstratedthat ultrasound mediated enhancement of drug and gene delivery are correlated with some formof bubble activity, as measured by the power in certain frequency bands of the signal, in thepresence of ultrasound. Cavitation can be defined as the formation and/or oscillation of a gasphase (i.e. bubble) in a liquid phase due to changes in pressure. For therapeutic applications,stable or inertial cavitation are the two areas most studied. During stable cavitation, the bubbleexperiences nonlinear oscillations resulting in sub- and ultra-harmonic acoustic emissions(Leighton, 1997). Inertial cavitation takes place when sufficient acoustical pressure is reachedcausing the bubble to reach a critical radius thus resulting in the violent collapse of the bubbleindicated by the presence of broadband emissions from the bubble (Holland & Apfel, 1989).Datta et al. (2008) showed that stable cavitation activity correlated well with enhanced lysis of 1
2clots. On the other hand Lai et al. (2006) showed that delivery of genes into cells wereconcomitant with inertial cavitation. Datta et al. (2008) showed that the presence of pre-existing nuclei in the form of acontrast agent, such as Definity® lowered the thresholds of cavitation and enhanced clot massloss. Definity®, is a lipid-shelled ultrasound contrast agent (UCA) containing octafluoropropanegas which produces enhanced echogenicity or contrast because of the difference in the acousticalimpedance between the encapsulated gas and the surrounding blood pool and tissue (Kremkau,2006). Intravenous administration of Definity® is currently clinically approved for use only toimprove delineation of the endocardial border in previously suboptimal echocardiograms(Kremkau, 2006). However, in order to use Definity® and other UCAs as cavitation nucleatingagents as adjuvants in ultrasound mediated therapy in clinical settings, a rigorous study of theircavitation thresholds under physiologic conditions is required primarily because overpressuresabove atmospheric levels have been shown to suppress subharmonic emissions and cavitationfrom contrast agents. Adam (2001), observed this phenomenon for Optison®, a suspension ofhuman serum-albumin coated microspheres filled with octafluoropropane. Specifically, Adam(2001) reported a subharmonic amplitude that decreased over time for overpressures of 70, 140,and 210 mmHg (9, 19, and 28 kPa, respectively) in an in vitro system while insonifing at 2 MHz. Processing cavitation signals to identify different types of cavitation can be acomputationally intensive process. Also, cavitation monitoring requires additional equipmentsuch as hydrophones, preamplifiers, and oscilloscopes. Therefore, it would be desirable todemonstrate a correlation between cavitation activity and changes in loss of echogenicity onstandard ultrasound B-mode images. This would allow for a more clinically relevant approach tomonitor cavitation during therapy (Porter, 2006; Smith, 2007). The objective of this study was to determine the effects of an overpressure of 90 mmHgon the cavitation threshold and echogenicity of Definity®. The hypotheses tested in this studyare (a) the presence of a mean overpressure, with respect to atmospheric, of 90 mmHg wouldresult in the delayed onset of both stable and inertial cavitation activity for a sample volume ofDefinity® being exposed to 6 MHz pulsed Doppler with a pulse duration of 5.83 µs and pulserepetition frequency of 1250 Hz, and (b) the change of onset in echogenicity of Definity® underthese conditions correlates with the change of onset in stable cavitation emissions.Materials and MethodsFlow phantom The flow phantom, figure 1, consisted of a latex tube with an inner diameter of 3 mm anda wall thickness of 400 microns. A peristaltic pump (Rainin Instrument Rabbit Peristatic Pump,Oakland, CA, USA), was used to maintain a flow rate of 5 ml/min within the tube. A meanoverpressure, with respect to atmospheric, of 0 mmHg or 90 mmHg was introduced using agravity-mediated afterload reservoir. The overpressures were measured using a pressuretransducer and pressure monitor (Hugo Sachs Elektronik 73-0045, March-Hugstetten, Germany).Pressure measurements were recorded every 0.016 s and the files were saved to a personnel
3computer. The latex tubing was immersed in a Lucite tank filled with degassed water, maintainedat 37˚C, with a dissolved oxygen content at 40% saturation as measured by a dissolved oxygenmeter (Oakton DO100, Vernon Hills, IL, USA). The UCA was pumped from the UCA reservoirmaintained at room temperature.Fig. 1. Experimental setup for 6 MHz pulsed Doppler exposure of a flow phantom containing Definity® and bovine albumin serum (BSA). A focused 10 MHz PCD, aligned confocally with and orthogonally to the acoustical axis of the L12-5 transducer Doppler sample volume.Contrast agent preparation According to manufacturer instructions, Definity® vials were activated at roomtemperature using the Vialmix ™ (Bristol-Myers Squibb Medical Imaging, MA, USA). After theactivation period of 45 s the vial was left on the bench to return to room temperature. The agentwas drawn from the vial using a 20 gage needle while allowing venting to the atmosphere usinga second 20 gage needle with a 0.2 µm filter. A 0.05 mL volume of the agent was drawn andthen diluted into 0.5% (w/v) bovine serum albumin (BSA) in deionized water to a concentrationof 0.31 µm/mL at room temperature. Fifty milliliters of this dilution was transferred into theUCA reservoir seen in figure 1.Insonation parameters and echogencity measurements The L12-5, an imaging transducer used with the Philips HDI-5000 clinical ultrasoundscanner (Philips HDI 5000, Bothell, WA, USA), was used to both record B-mode images forechogenicity measurements and to insonate the Definity® using a 6 MHz pulsed Doppler signal.“2-D Live” mode was used for this study as it allowed the use of pulsed Doppler or B-modeseparately. To acquire images the B-mode was turned on for 1 s to acquire an image of the UCAwith the flow turned off (i.e. 0 mL/min) as a representative image of Definity® prior to exposureto Doppler pulses. The transducer was then switched to Doppler mode at the appropriatemechanical index (MI), and flow was turned on. As the Definity® flowed through the pulsedDoppler sample volume located at the center of the B-mode image, as shown in figure 2, theywere exposed to Doppler pulses, cavitated, and subsequently lost echogenicity. To acquire animage of the Definity® exposed to Doppler pulses, both the transducer and flow were turned off
4and the transducer was set again in B-mode to acquire an image. The B-mode MI was set to 0.04(95 kPa) and was left on for no more than 1 s in order to minimize any destruction that may becaused by B-mode pulses. Fig. 2A representative image captured after Definity® exposure to Doppler pulses at an MI of 0.56 (1.43 MPa). The image depicts the regions of interest (ROIs) that were used for obtaining the echogenicity. The pulsed Doppler sample volume was centered in the azimuthal direction of the B-mode imaging plane and placed at a depth of 1 cm. Previous calibrations showed that thisplacement results in a 0.8 mm -3 dB lateral beamwidth. For the experiments described in thisstudy, the L12-5 transducer in pulsed Doppler mode produced a pulse with a 5.83 µs pulseduration at a 1250 Hz pulse repetition frequency.Cavitation detection The passive cavitation detector (PCD) (Valpey Fisher, Hopkinton, MA , USA) was acircular, spherically focused, single-element, 10 MHz transducer with a -3 dB axial beamwidthof 2 cm, lateral beamwidth of 1 mm, and a focal distance of 2 cm. An illustration of theexperimental set up and alignment is shown in figure 1. The PCD was placed 90° to the acousticaxis of the L12-5 transducer and confocally aligned with the pulsed Doppler sample volumeusing two micrometer three-axis translation stages (Newport 423, Irvine, CA, USA). To obtainthis alignment, the latex tube was filled with degassed water and then insonified with the pulsedDoppler signal. The PCD was moved until the signal was maximized indicating that the PCDwas aligned to both the top of the tube and confocal with the clinical transducer along the axis ofthe tube. Subsequently, the PCD was driven with a pulser-receiver (Panametrics, SquarewavePulser/Receiver 5077PR, Waltham, MA, USA) at 10-MHz and a pulse repetition frequency of100 Hz. The PCD was then moved down by 1.5 mm, half the inner diameter of the latex tube, sothat it was aligned to the center of the latex tube and the echoes from the pulser-receiver weremaximized and corresponded to the correct inner diameter of the latex tube. After alignment, either degassed water or Definity® was flowed through the system andinsonified at varying pressures (Table 1) at the desired overpressure of either 0 mmHg or 90mmHg. The cavitation emissions were detected with the PCD, amplified 46 dB by a poweramplifier (Amplifier Research 50A15), and stored using a digital oscilloscope (LeCroyWaverunner LTS84, Chestnut Ridge, NY, USA). The oscilloscope was operated in sequence
5mode with a sampling rate of 50 MHz for each sequence. Each segment contained 250 tracescorresponding to 250 consecutive Doppler firings. A total of 1000 time domain traces wererecorded. Files from the oscilloscope were then transferred to a personnel computer for later postprocessing in MATLAB (The MathWorks Inc., Natick, MA, USA).Table 1 Peak Rarefraction Pressure [MPa]0.00 0.09 0.11 0.13 0.16 0.18 0.20 0.22 0.25 0.28 0.31 0.35 0.39 0.45 0.50 0.55 0.62 0.72 0.80 0.91 1.03 1.15 1.27 1.43 Mechanical Index (MI) 0 0.04 0.05 0.06 0.07 0.08 0.09 0.1 0.11 0.13 0.14 0.16 0.18 0.2 0.22 0.24 0.27 0.3 0.33 0.37 0.41 0.46 0.5 0.56Peak rarefactional pressures (and corresponding MIs) used to insonate both degassed water and Definity® with over pressures of 0 mmHg or 90 mmHg.Image and cavitation analysis For each MI setting, B-mode images of Definity® were saved before and after exposureto Doppler as described above. On each image two regions of interest were defined upstream anddownstream of the Doppler sample volume as shown in figure 2. The ROIs had a height equal tothe tubing image inner diameter and a length that was approximately one quarter of the tubingimage length. The mean gray scale value (MGSV) of the ROI was computed. The averageMGSV was then calculated over eight trials at each MI to determine echogenicity thresholds. At each MI, the average time-domain degassed water cases were subtracted from theindividual Definity® time domain traces to remove the coherent scattering from the tubing. ATukey window (r = 0.5) was applied to the subtracted time-domain signal to reduce artifactualsignal components. These windowed time-domain traces were converted into power spectra byapplying a 2048-point fast Fourier transform in MATLAB. Averaged power spectra werecomputed from the 1000 traces captured at each MI. The mean subharmonic power wascomputed as the mean power in the subharmonic band centered at 3 MHz with a 1 MHzbandwidth. This bandwidth is the same as the bandwidth of the main lobe at 6 MHz. The meansubharmonic power is indicative of stable cavitation activity. A comb filter was used to extractthe broadband components from the frequency ranges 9.5-10.5 MHz, 13.5-14.5 MHz and 15.5-16.5 MHz, which lie outside the harmonic and ultraharmonic frequency bands. To determine thecavitation threshold, the logarithms of the subharmonic and broadband powers were computedfrom four trials and were then plotted against the peak rarefactional pressures (PRP).ResultsEchogenicity The MGSV versus the peak rarefactional pressure for 0 mmHg and 90 mmHg are shown,respectively, in figures 3 and 4. Both figures show the MGSV for the upstream and downstreamROIs both before and after pulsed Doppler insonation. One does not see any significant changesin echogenicity for either ROI in the before insonation cases (figures 3a and 4a). This is expectedand serves as a check that the echogenicity is on average uniform across experimental runs. Thissuggests that the statistical measures of the bubble populations themselves remain stationary.Each data point is an average of eight samples, and the error bars represent the standarddeviation of the measurements. In comparision, the echogenicity changes were seen in the
6downstream ROIs for Definity® exposed to 6 MHz pulsed Doppler (figure 3b and 4b) at higherMIs. (a) 50 45 40 35 30 Down stream MGSV 25 Up stream 20 15 10 5 0 0.000 0.200 0.400 0.600 0.800 1.000 1.200 1.400 1.600 Peak rarefactional pressure (MPa) (b) 50 45 40 35 30 Down Stream MGSV 25 Up stream 20 15 10 5 0 0.000 0.200 0.400 0.600 0.800 1.000 1.200 1.400 1.600 Peak rarefactional pressure (MPa)Fig. 3. Echogenicity, as measured by MGSV, as a function of peak rarefactional pressure under an overpressure of 0 mmHg for (a) ROIs downstream and upstream of the pulsed Doppler sample volume before pulsed Dopplerinsonation was turned on, and (b) ROIs downstream and upstream of the pulsed Doppler sample volume after pulsed Doppler insonation was turned on. A sample size of eight was used for each set of MIs, and error bars represent the standard deviation of the measurements.
7 (a) 50 45 40 35 Down stream 30 Up stream MGSV 25 20 15 10 5 0 0.000 0.200 0.400 0.600 0.800 1.000 1.200 1.400 1.600 Peak rarefactional pressure (MPa) (b) 50 45 40 35 30 Down Stream MGSV 25 Up Stream 20 15 10 5 0 0.000 0.200 0.400 0.600 0.800 1.000 1.200 1.400 1.600 Peak rarefactional pressure (MPa) Fig. 4. Change in echogenicity, as measured as the MGSV, as a function of peak rarefactional pressure under an overpressure of 90 mmHg for (a) ROIs downstream and upstream of the pulsed Doppler sample volume with thepulsed Doppler insonation off; and (b) ROIs downstream and upstream of the pulsed Doppler sample volume withthe pulsed Doppler insonation on. A sample size of eight was used for each set of MIs, and error bars represent the standard deviation of the measurements.
8Cavitation The logarithm of the mean subharmonic power and the broadband power plotted againstthe peak rarefactional pressure correlating to stable and inertial cavitation activity are shown infigure 5a and b respectively. Each data point is the average of the power from four samples, andthe error bars are the standard deviation of the measurements. For the stable cavitation activity, itis evident that both thresholds were equal for both overpressures of 0 and 90 mmHg.Furthermore, the stable cavitation bubble activity, represented by the subharmonic power, is alsoapproximately the same for both cases. For inertial cavitation, both overpressures show athreshold that is approximately equal. The inertial bubble activity also remains approximatelyequal for both overpressures. (a) -2.50 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 Log(normalized subharmonic power) -3.00 Def inity at 0 mmHg -3.50 Degassed Water at 0 mmHg -4.00 Def inity at 90 mmHg Degassed H2O at 90 -4.50 mmHg -5.00 -5.50 -6.00 -6.50 Peak rarefactional pressure (MPa)
9 (b) -3.00 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 Log(mean broadband power) -3.50 Definity at 0 mmHg Degassed Water 0 -4.00 mmHg Definity at 90 mmHg Degassed Water at 90 mmHg -4.50 -5.00 -5.50 Peak Rarefactional Pressure (MPa)Fig. 5. The logarithm of the mean (a) subharmonic and (b) broadband power as a function of the peak rarefactional pressure (MPa) for Definity® and degassed water at 0 mmHg, and Definity® and degassed water at 90 mmHg.Discussion The effect of an overpressure above atmospheric on cavitation and echogenicitythresholds were explored in this study. This study also looked to see if the cavitation activitycould be correlated with the echogenicity loss. Correlation of echogenicity changes andcavitation activity could help to simplify the monitoring of drug and gene delivery in the clinicalsetting. Correlation of the thresholds for cavitation onset and echogenicity loss did not appear tobe concomitant. For echogenicity loss, the threshold for change appears to decrease slightly (though arobust statistical method was not used to define a precise threshold) at the higher overpressure.This may be due to the additional pressure on the bubble making it more susceptible todissolution. It is interesting to note, the pressure at which echogenicity loss occurred forDefinity®, is close to the value seen by Porter et al. (2006) for Optison®. Porter et al. attributedthis loss to a process termed acoustically driven diffusion. While the experimental protocols aredifferent between the work of Porter et al. and the one described in this study, it may be that asimilar process is causing echogenicity loss as was observed here. Further, the results indicate that the cavitation thresholds for Definity® are independentof overpressures at 0 mmHg and 90 mmHg above atmospheric for both stable and inertialcavitation. Earlier, Adam (2001) observed the amplitude of the first, second, and sub-harmonicfor the sterile suspension of human serum-albumin coated microspheres filled withoctafluoropropane UCA, (Optison®), decreased over time for overpressures of 70, 140, 210mmHg (9, 19, and 28 kPa, respectively) in an in vitro system while insonifing at 2 MHz. Alongthe same lines, Anderson et al. (2010) compared the energy ratio of the subharmonic andfundamental to overpressures of 0 to 187 mmHg (0 and 25 kPa, respectively) for the UCA
10SonoVue®, a phospholipid membrane containing sulfur hexafluoride. The UCA was insonifiedat 4 MHz in a static chamber while constantly mixing the UCA with a magnetic stir bar. Theyreported a decrease in the energy ratio as a function of the overpressure. Shi et al. (1999)demonstrated in vitro, for overpressures of 0 to186 mmHg (0 and 25 kPa, respectively), that theUCA Levovist®, a glactose/palamitic acid shell encapsulating air, insonated at 2 MHz had areduction of amplitude of the first, second, and sub-harmonics by 2.4, 1.8, and 9.6 dB,respectively, for a constant acoustic pressure of 0.39 MPa. Another study (Bailey, 2001), thatfocused on reducing distortion of the shape and location of high intensity focused ultrasound(HIFU) lesions in tissue by introducing an overpressure (5.6 MPa) to suppress gas or vapor waterbubbles resulting from heating, saw similar results in reducing bubble activity. These studieshave demonstrated the effect of an overpressure on bubble activity on various UCAs. Thenovelty of this study is in investigating the cavitation thresholds and bubble activity forDefinity® under physiologically relevant overpressures, which hasnt been done in previousstudies. Further, in sharp contrast to the results reported in earlier studies we found that anoverpressure of 90 mmHg did not affect the cavitation threshold or bubble activity for stable orinertial cavitation in Definity®. Further investigation of this result needs to be carried out todetermine the reason. It may be due to Definity® having a different shell composition than theother UCAs, or the dependence of cavitation activity on the flow (i.e. with flow, without flow, orconstant agitation in a static chamber).Conclusions Echogenicity loss and subharmonic emissions can be used to monitor and measurecavitation activity, so a clear understanding of the dependence of cavitation thresholds andbubble activity with respect to overpressures is needed to move these methods into in vivosystems. In this study, no loss of echogenicity was observed due to overpressures alone. Whiledifferences in the onset of echogenicity loss for the overpressures of 0 and 90 mmHg could be aresult of dissolution of the UCA. Stable and inertial cavitation thresholds and bubble activity forboth 0 and 90 mmHg overpressures were approximately equal thus supporting the use of thesecavitation thresholds from this in vitro study for future in vivo studies.Future Work Future work could include repeating the protocol described in this study with otherUCAs, such as Optison®, that were used in the before mentioned reports to see if their resultscan be repeated. This would allow the exploration of the dependence of cavitation activity andechogenicity on the UCA composition. Other future work could be concentrated on studying theeffect of varying pulse repetition frequency (PRF) and pulse duration (PD) on cavitation activityand echogenicity for Definity®. Data might be collected for the parameter sets listed in Table 2(numbers in nonshaded cells indicate the maximum MI available). Within this study a statisticalmethod to derive a threshold for echogenicity should be investigated in order to morequantitatively compare echogenicity changes with cavitation activity.
11Table 2. Suggested combinations of PRF and PD. The numbers in non-shaded cells indicate the maximum MI forthe Philips HDI-5000 clinical ultrasound scanner for the corresponding PRF/PD combinations. Pulse Duration (PD) [µs] 1.67 3.33 5.83 8.33 16.7 P 1250 0.9 0.7 0.56 0.47 0.31 R 2500 0.53 F 5000 0.39 [H 8333 0.32 z] 0.27 11905Acknowledgements- I gratefully acknowledge the assistance of Kirthi Radhakrishnan and KevinHaworth for their help in completing this study, specifically the analysis of the cavitation data byKirthi, and for the valuable discussions. I also gratefully acknowledge all other valuablediscussions with the rest of the members in the Holland Lab.References Adam, D., & Burla, E. (2001). Study of pressure dependence of signals from ultrasound contrast agents. Paper presented at the, 4 3370-3373. Andersen, K. S., & Jensen, J. A. (2010). Impact of acoustic pressure on ambient pressure estimation using ultrasound contrast agent. Ultrasonics, 50(2), 294-299. Bailey, M. R., Couret, L. N., Sapozhnikov, O. A., Khokhlova, V. A., Ter Haar, G., Vaezy, S., et al. (2001). Use of overpressure to assess the role of bubbles in focused ultrasound lesion shape in vitro. Ultrasound in Medicine and Biology, 27(5), 695-708. Christiansen, J. P., Leong-Poi, H., Klibanov, A. L., Kaul, S., & Lindner, J. R. (2002). Noninvasive imaging of myocardial reperfusion injury using leukocyte-targeted contrast echocardiography. Circulation, 105(15), 1764-1767. Datta, S., Coussios, C., Ammi, A. Y., Mast, T. D., de Courten-Myers, G. M., & Holland, C. K. (2008). Ultrasound-enhanced thrombolysis using definity® as a cavitation nucleation agent. Ultrasound in Medicine and Biology, 34(9), 1421-1433. Denise A. B. Smith, Sampada S. Vaidya, Jonathan A. Kopechek, Shao-Ling Huang, Melvin E. Klegerman, David D. McPherson, and Christy K. Holland. Ultrasound-triggered release of re- combinant tissue-type plasminogen activator frome echogenic liposomes. Ultrasound in Medicine and Biology, 36(1):145–157, Januray 2010.
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