Abstract
OBJECTIVES
The authors investigated ideal acoustic conditions on a clinical scanner custom-programmed for ultrasound (US) cavitation-mediated flow augmentation in preclinical models. We then applied these conditions in a first-in-human study to test the hypothesis that contrast US can increase limb perfusion in normal subjects and patients with peripheral artery disease (PAD).
BACKGROUND
US-induced cavitation of microbubble contrast agents augments tissue perfusion by convective shear and secondary purinergic signalling that mediates release of endogenous vasodilators.
METHODS
In mice, unilateral exposure of the proximal hindlimb to therapeutic US (1.3 MHz, mechanical index 1.3) was performed for 10 min after intravenous injection of lipid microbubbles. US varied according to line density (17, 37, 65 lines) and pulse duration. Microvascular perfusion was evaluated by US perfusion imaging, and in vivo adenosine triphosphate (ATP) release was assessed using in vivo optical imaging. Optimal parameters were then used in healthy volunteers and patients with PAD where calf US alone or in combination with intravenous microbubble contrast infusion was performed for 10 min.
RESULTS
In mice, flow was augmented in the US-exposed limb for all acoustic conditions. Only at the lowest line density was there a stepwise increase in perfusion for longer (40-cycle) versus shorter (5-cycle) pulse duration. For higher line densities, blood flow consistently increased by 3-fold to 4-fold in the US-exposed limb irrespective of pulse duration. High line density and long pulse duration resulted in the greatest release of ATP in the cavitation zone. Application of these optimized conditions in humans together with intravenous contrast increased calf muscle blood flow by >2-fold in both healthy subjects and patients with PAD, whereas US alone had no effect.
CONCLUSIONS
US of microbubbles when using optimized acoustic environments can increase perfusion in limb skeletal muscle, raising the possibility of a therapy for patients with PAD. (Augmentation of Limb Perfusion With Contrast Ultrasound; NCT03195556)
Keywords: cavitation, microcirculation, peripheral artery disease, muscle perfusion, ultrasound
Ultrasound (US) has a wide variety of biological effects. The ability of US to increase tissue blood flow in a non-thermal manner occurs, in part, through acoustically driven vascular shear and convective motion, thereby initiating endogenous endothelial-dependent vasodilatory pathways (1,2). Encapsulated microbubble (MB) contrast agents exposed to US undergo volumetric oscillation, called cavitation, which produces high local shear forces. Accordingly, MB inertial cavitation (destruction) increases tissue perfusion to a much greater extent than US alone (3,4). The molecular mechanism by which contrast-enhanced US (CEU) augments flow is through release of adenosine triphosphate (ATP) and subsequent purinergic signaling that leads to production of nitric oxide, prostaglandins, and adenosine (5).
The therapeutic potential of MB cavitation using US frequencies and pressures within the diagnostic range has been demonstrated in animal models of acute myocardial infarction and peripheral artery disease (PAD) where tissue ischemia could be either fully or partially reversed (3,5). It has been observed that this beneficial effect can last long after completion of CEU, which is thought to represent a sustained ATP release that occurs from not only acute cellular microporation but also from purinergic channel activation (5). In this study, we investigated how limb flow augmentation and ATP release in mice could be optimized by modifying US transmit settings (line density, pulse duration) that influence the pressure and shear conditions during MB inertial cavitation. We then applied this knowledge in a first-in-human trial to test the hypothesis that augmentation in limb blood flow could be achieved by CEU performed with short-duration MB cavitation protocols in normal human subjects and in patients with PAD. We also tested whether flow augmentation occurs remotely from the US-exposed region based on the potential of cavitation to increase systemic concentrations of ATP or vasodilators.
METHODS
ULTRASOUND BEAM SETTINGS.
Modifications of the US field for murine experiments were performed by programming a commercial imaging system (EPIQ 7, Philips Healthcare, Andover, Massachusetts) to deliver customized pulse sequences on a matrix-array transducer (X5–1). Experiments were performed with a 2-dimensional beam at 1.3 MHz and a mechanical index of 1.3. Pulse duration was varied by generating 5-cycle or 40-cycle sinusoidal pulses per line, or a “power-Doppler” scheme composed of 8 5-cycle pulses with a pulse-repetition frequency of 10 kHz. The second modified variable was US line density, which, as described in Figure 1A, can influence MB inertial cavitation pressure. Beams were transmitted in sequence spanning a 64-degree arc and beam overlap was modified by altering the angle between neighboring beams to be 1, 2, or 4 degrees, producing a sector of 65, 33, and 17 lines, respectively. All piezoelectric elements were programmed to be active on transmit with a rectangular apodization across the elements laterally and in elevation to create as narrow a beam width as possible at the focus.
FIGURE 1. Ultrasound Schemes for Producing Cavitation.
(A) Schematic showing that at low line density, microbubble cavitation within each electronically focused line occurs at the intended high acoustic pressure, yet between-line gaps can result in regions where high-pressure cavitation does not occur. At high line density, gaps are eliminated, but microbubble inertial cavitation from pressures above the CT can occur from neighboring lines at a lower than intended pressure. (B) Relative pressure measurements from calibrated hydrophone and those generated by the simulation program illustrating close correlation with regard to pressure distribution in the lateral dimension for a 3-line frame. (C) Simulated line profiles in the axial-lateral dimensions for the 17-, 33-, and 65-line settings; elevational pressure dimensions. Side-lobes or “grating lobes” are detected near the focus. Relative pressure scale is shown at left. CT = cavitation threshold.
ACOUSTIC FIELD TESTING AND BEAM PROFILING.
Acoustic pressures produced using the X5–1 transducer were measured as a function of the applied output voltage using a calibrated capsule hydrophone (HGL-0200/AH-2010, ONDA Corp., Sunnyvale, California) submerged in a degassed water tank. The hydrophone was positioned at the location of maximum US intensity, and signals from the hydrophone were captured in MATLAB (The Mathworks, Inc., Natick, Maryland) for offline analysis. The 3-dimensional pressure fields produced from the custom pulsing sequence were mapped by translating the hydrophone using a 3-axis motorized positioning system. Hydrophone signals were captured in MATLAB at each position within the volume of interest and analyzed offline. Volumes of interest were typically sampled in step sizes of 2 mm axially, 0.5 mm laterally, and 0.5 mm elevationally. Hydrophone data were up-sampled 10-fold using cubic interpolation for final graphical representation. To validate beam transmit design, custom scripts were developed to simulate the beam patterns of the X5–1 matrix transducer using the Field-II Simulation Program (6), and incorporating the transducer geometry and pulsing conditions described above. Specifically, the simulation was compared with hydrophone data to determine how accurately simulations represented overlap conditions and the general lateral and elevational beam width.
MICROBUBBLES.
For murine and in vitro experiments, lipid-shelled decafluorobutane MBs were prepared using sonication of a gas-saturated aqueous suspension of distearoylphosphatidylcholine (2 mg/ml) and polyoxyethylene-40-stearate (1 mg/ml). MB concentrations and size distributions were measured using electrozone sensing (Multisizer III, Beckman Coulter, Brea, California).
CAVITATION FIELD.
To spatially evaluate the MB cavitation profile, a phantom that fixed MBs in place was created. A mixture of degassed US gel and water (2:1 vol:vol) was combined with MBs for a final concentration of 1.7 × 105 ml−1. The MB suspension was placed in 35-mm culture dishes and covered with clear paraffin film. The gel phantom was suspended in degassed deionized water in the path of the US transducer at a distance equal to the acoustic focus (4 cm). Therapeutic US using the conditions above were generated at a frame rate of 1 Hz. The spatial assessment of contrast was visually assessed every 5 frames for 30 frames by using low-mechanical index (0.16) contrast-specific CEU imaging at 7 MHz (Sequoia 512, Siemens Medical, Mountain View, California) with linear-array transducer oriented orthogonally to the therapeutic transducer to image the entire circular area of the dish.
US FLOW AUGMENTATION IN MICE.
The study was approved by the Animal Care and Use Committee of the Oregon Health & Science University. Wild-type C57Bl/6 mice (n = 50) were studied at 10 to 20 weeks of age. Mice were anesthetized for study procedures with inhaled isofluorane (1.0% to 1.5%) mixed with room air and kept euthermic with external heating sources. A jugular vein was cannulated for intravenous access for administration of MBs and luciferase. The proximal left hindlimb was exposed to therapeutic US for 10 min. The X5–1 transducer was placed 4 cm from the mid-portion of the muscle using a fixed transverse imaging plane and the acoustic focus was placed at the mid-muscle depth. A bolus injection of lipid MBs (2 × 108) was given over 1 min while therapeutic US was performed for 10 min at 5-s frame intervals. US conditions were varied according to a 3 × 3 matrix for pulse duration and line density (Supplemental Figure 1). After therapeutic US (starting within 5 min), either microvascular perfusion imaging or in vivo optical imaging for ATP was performed.
MURINE MICROVASCULAR PERFUSION IMAGING.
CEU perfusion imaging of the proximal hindlimb adductor muscles of the therapeutic US-exposed and contralateral control limb was performed within 10 min of therapeutic US. Contrast-specific phase-inversion and amplitude-modulation (Sequoia 512, Siemens Medical Systems) was performed at 7 MHz and a mechanical index (MI) of 0.18 with a linear-array transducer. This form of low-power imaging has been shown not to produce any detectable changes in perfusion (4). MBs were infused at a rate of 1 ×107 min−1. Time-intensity data at a frame rate of 5 Hz were acquired after a high-power (MI 1.0) 5-frame sequence and were fit to the function: y = A(1-eβt); using non-linear least-squares regression, where y is intensity at time t, A is the plateau intensity representing relative microvascular blood volume, and the rate constant β is the microvascular flux rate (7). Microvascular blood flow was quantified by the product of A and β (7).
IN VIVO IMAGING OF ATP RELEASE.
Optical imaging of luciferin-luciferase activity was performed to temporally and spatially assess intravascular ATP release. Mice were injected intraperitoneally with 3 mg D-luciferin (Thermo Fisher, Waltham, Massachusetts) by intraperitoneal route immediately before therapeutic US with MB cavitation. Firefly luciferase (25 μg, Thermo Fisher) was injected by intravenous route 1 min prior to completion of the 10 min US exposure. Optical imaging (IVIS Spectrum, Caliper Life Sciences Hopkinton, Massachusetts) was performed at 5, 10, 15, and 20 min after completion of US using medium binning. Data were expressed as photons/s/cm2.
US FLOW AUGMENTATION IN HUMANS.
The study was approved by the Institutional Review Board of Oregon Health & Science University, and registered with ClinicalTrials.gov ( NCT03195556). Ten healthy adult volunteers and 10 patients with PAD were recruited. Patient inclusion criteria were a history of unilateral or bilateral PAD diagnosed based on moderate to severe symptoms (Rutherford symptom class 2 to 6) and diagnostic confirmation based on reduced ankle-brachial index (<0.9) or angiography. Subjects were excluded for medical illnesses affecting limb perfusion, severe (New York Heart Association functional class IV) heart failure, hemodynamic instability, pregnancy, lactation, or hypersensitivity to US contrast agents. Therapeutic CEU was performed during a continuous intravenous infusion of perflutren lipid MBs (DEFINITY, Lantheus Medical Imaging, North Billerica, Massachusetts). One vial diluted to 30 ml in normal saline was infused at 2 ml/min. Therapeutic CEU of the calf muscle from a single limb, which for subjects with PAD was the limb with the lowest ankle-brachial index, was performed for 9 min. US was performed using harmonic power Doppler (Sonos 5500, Philips Ultrasound) at 1.3 MHz with a pulse repetition frequency of 9.3 kHz, an MI of 1.3, and the acoustic focus set at the level of the mid soleus (Supplemental Figure 1). Use of this system for human studies was based on it being a Food and Drug Administration–approved system capable of providing acoustic conditions (64 lines, 8 × 5-cycle pulses) closest to the experimentally programmed condition used in mice. Four adjacent short-axis elevational planes of the calf separated by 1 cm were exposed. The frame rate was set at 0.5 Hz (pulsing interval of 2 s) and 2 frames were used for each plane to ensure complete MB cavitation before advancing to the next imaging plane in a proximal to distal sequence before returning to the proximal plane. This paradigm allowed for a 7-s interval for complete MB replenishment between each 2-frame bundle. Healthy volunteers returned on a separate day to assess the effects of US alone without MBs. The leg contralateral to the CEU-exposed limb was imaged using identical settings except US was performed with continuous imaging (10 to 15 Hz) for 2 s in each adjacent plane because pulsing intervals are required only for MB refill. Muscle perfusion was assessed using CEU imaging performed bilaterally before, immediately after, and 1 h after therapeutic US. Pre-therapy perfusion imaging was not performed for US-alone conditions to avoid the presence of MBs during therapy.
LIMB PERFUSION IMAGING IN HUMANS.
Diagnostic CEU perfusion imaging was performed with a phased-array transducer (IE33, Philips Ultrasound) using power-modulation imaging at 1.8 MHz and a MI of 0.18. Imaging of the calf was performed bilaterally using trans-axial imaging planes that, for the therapy leg, was located in the center of the 4-plane region. Lipid MB dosing was identical to that performed during therapeutic CEU. End-diastolic frames were obtained after a 5-frame high-power (MI >1.0) pulse sequence. Image analysis was performed similar to that described for murine studies.
STATISTICAL ANALYSIS.
Data were analyzed using Prism (version 5.0, GraphPad Software). Group-wise differences were assessed using unpaired Student’s t-test, or using Mann-Whitney U test for data that were determined to be non-normally distributed using D’Agostino and Pearson omnibus test. Comparisons within a group (i.e., change in perfusion from baseline) was evaluated using paired Student’s t-test or Wilcoxon test. Bonferroni’s correction was used for comparisons for more than 2 conditions. Differences were considered significant at p < 0.05 (2-sided).
RESULTS
PROGRAMMED US FIELDS.
Calibrated hydrophone measurements demonstrated that the clinical imaging probe could achieve peak negative acoustic pressures ranging from 20 kPa to 4.0 MPa (MI 0.03 to 3.44), and were largely independent of pulse length. Output conditions were then set to achieve a peak negative acoustic pressure of 1.5 MPa to approximate that previously used to augment tissue flow in mice (8). Computer-simulated field assessment for the different programmed line densities showed excellent agreement with hydrophone data, with beam widths measured to within 5% agreement (Figure 1B). Simulations produced an estimated beam width of 2.6 mm in the lateral dimension and 4.1 mm in elevation (with side-lobes noted) at the narrowest point in the axial-lateral plane, and varying levels of overlap between neighboring beams (Figure 1C). The ratio of the pressure amplitude at the region of line overlap relative to maximal acoustic pressure at the acoustic focus was 0.56, 0.87, and 0.96 for the 17-line, 33-line, and 65-line transmission, respectively.
SPATIAL ASSESSMENT OF CAVITATION.
Experiments using stationary MBs suspended in gel revealed modest differences in the spatial-temporal pattern of inertial cavitation using a 5-cycle pulse according to line density (Figure 2). Analysis for regions-of-interest that included either the primary beam elevation (Figures 2A and 2C) or that were expanded to include side-lobes (Figures 2B and 2D) demonstrated that increasing line density produced a more complete, more rapid, and slightly larger area of inertial cavitation. These data also demonstrate that any potential three-dimensional (3-D) exposure would best be suited with approximately 1 cm rastering in the elevational direction to avoid unnecessary overlap of cavitation fields.
FIGURE 2. Spatial Assessment of Microbubble Cavitation.
(A) Example of images obtained using contrast-enhanced ultrasound imaging showing the spatial pattern of inertial cavitation (destruction) of stationary microbubbles in a gel phantom with the therapeutic probe oriented at orthogonal plane. Images are shown before exposure and every 5 frames. Therapeutic beam elevational plane is represented by the vertical dimension of microbubble clearing with thin flanking elevational side-lobes. The dashed and dotted lines illustrate ROI to span the elevational plane with and without side-lobes. Graphs illustrate mean (±SEM) normalized contrast intensity in the elevational plane ROI without (B) and with side-lobes (C); and the contrast-free area in the elevational plane with (D) and without (E) side-lobes. ROI = regions of interest.
LIMB PERFUSION AND ATP RELEASE IN MICE.
After 10 min of high-power US exposure to produce MB inertial cavitation, CEU perfusion imaging revealed a significant increase in limb perfusion in the US-exposed versus contralateral control limb for all conditions irrespective of line density and pulse length (Figure 3). At the lowest line density (17 lines), there was a stepwise increase in perfusion in the US-exposed limb for the 5-cycle, the 8 × 5-cycle, and the 40-cycle pulse scheme. In contrast, pulse duration did not significantly influence the degree of flow augmentation when using 33- or 65-line densities. In vivo optical imaging of ATP release after limb CEU demonstrated a trend for greater ATP signal with longer pulse duration. The highest signal was observed with high line density and long pulse duration condition (Figure 4). ATP release data taken in context of changes in blood flow, however, reveal that ATP release may not be linearly related to changes in blood flow, and instead there is likely to be a threshold response.
FIGURE 3. Flow Augmentation in the Murine Hindlimb According to Line Density and Pulse Duration.
(A) Example of time-intensity data (graph) and corresponding background-subtracted color-coded contrast-enhanced ultrasound images at various time intervals (seconds) after the destructive pulse-sequence illustrating flow augmentation in the hindlimb exposed to US (33 line-density; 40-cycle pulse) compared with the contralateral control leg. The regions of interest are shown by dashed lines in the first frame. The bar graphs illustrate mean (±SEM) microvascular flux rate (β-value) for (B) 17-line, (C) 33-line, and (D) 65-line density settings using different pulse durations. *p < 0.05 versus corresponding control limb; †p < 0.05 versus 5-cycle. US = ultrasound.
FIGURE 4. Optical Imaging of Adenosine Triphosphate After Limb Contrast US.
Mean (±SEM) photon flux in the US-exposed and contralateral limb measured 5 min (A) and 20 min (B) after contrast-enhanced ultrasound. *p < 0.01 versus contralateral. Inset images show examples from mice treated with 17-line, 40-cycle conditions at each time interval. Abbreviation as in Figure 3.
FLOW AUGMENTATION IN HUMANS.
Mean age for healthy volunteers was 38 ± 11 years; demographic and clinical data for study patients with PAD are presented in Table 1. In healthy control subjects, resting perfusion at baseline was similar between legs (Figure 5). After 10 min exposure to continuous US alone, there were no significant changes in perfusion in the US-exposed or contralateral limb. On a separate day, US cavitation of MBs produced on average a 2.5-fold increase in muscle perfusion, which completely resolved by 1 h. Modest flow augmentation was also observed in the contralateral calf, although this change did not reach statistical significance. In patients with PAD, baseline perfusion was not substantially different between calves and MB cavitation produced on average a >2-fold increase in tissue perfusion, which did not completely return to baseline at 1 h. Again, a modest but statistically insignificant increase in muscle perfusion was seen in the contralateral leg. There were no reports of adverse events associated with contrast administration or limb cavitation.
TABLE 1.
Clinical Characteristics of the Patients With PAD
| Age (yrs) | 73 (62–75) |
| Male | 9(82) |
| BMI (kg/m2) | 27 ± 5 |
| Prior revascularization | 5 (50) |
| Co-morbidities | |
| Hypertension | 9 (90) |
| Hyperlipidemia | 8 (80) |
| Diabetes | 3 (30) |
| Tobacco use | |
| Current | 3 (30) |
| Former | 7 (70) |
| Medications | |
| Aspirin | 9 (90) |
| Clopidogrel | 3 (30) |
| Oral anticoagulant | 3 (30) |
| Beta-blocker | 2 (20) |
| Statin | 10 (100) |
| Insulin | 1 (10) |
| SGLT-2 inhibitors | 0 (0) |
| GLP-1 agonists | 0 (0) |
| Rutherford category* | |
| 2 Moderate claudication | 5 (50) |
| 3 Severe claudication | 3 (30) |
| 4 Ischemic rest pain | 2 (20) |
| 5 Minor tissue loss | 0 (0) |
| HR (beats/min) | 69 ±15 |
| Systolic BP (mm Hg) | 129 ±14 |
| Diastolic BP (mm Hg) | 66 ± 6 |
| ABI (most symptomatic limb) | 0.53 ± 0.17 |
Values are median (interquartile range) or n (%).
ABI = ankle-brachial index; BMI = body mass index; BP = blood pressure; GLP = glucogon-like peptide; HR = heart rate; PAD = peripheral artery disease; SGLT = sodium glucose cotransporter.
FIGURE 5. Augmentation in Limb Skeletal Muscle Perfusion in Humans.
Examples of (A) contrast-enhanced ultrasound images of the calf after a destructive pulse sequence, and (B) corresponding time-intensity curves from a healthy subject before and after therapeutic cavitation with contrast-enhanced ultrasound. An increase in flow (A×β) was attributable to an increase in flux rate (β-value) and microvascular blood volume (plateau intensity). Graphs display MBF in the US-exposed and contralateral calf before, immediately after, and 1 h after exposure to (C) US alone in healthy controls; (D) CEU in healthy controls; and (E) contrast-enhanced ultrasound in subjects with peripheral artery disease. *p < 0.05 versus baseline. BG = background; MB = microbubbles; MBF = microvascular blood flow. Other abbreviation as in Figure 3.
DISCUSSION
Of the non-thermal bioeffects produced by US, shear forces that occur from convective motion, micro-streaming, or shock wave formation are thought to be important for therapeutic augmentation of tissue perfusion. The effects of US on microvascular resistance are amplified several-fold by the presence of MB contrast agents, including those that are approved for diagnostic use in humans (4,9). In this study, we have tested the vascular effects of different acoustic settings within regulatory pressure limits that can be implemented on a clinical scanner and tested efficacy in humans to show that a >2 fold increase in limb blood flow is achievable in normal subjects and patients with PAD.
Our preclinical studies focused entirely on MB cavitation because we have previously demonstrated that limb flow augmentation is much less with US alone compared with CEU, despite a duty factor (duration of US exposure) that is several orders of magnitude higher (4). It is also known that limb flow augmentation produced by inertial cavitation increases with the acoustic pressures up to the limits of a clinical scanner without causing adverse bioeffects (4). Accordingly, the examination of line density in the current study was intended to find a balance between: 1) avoiding “between-line gaps” at low line density, which results in a situation where some MBs are not exposed to high acoustic pressures; and 2) avoiding MB cavitation by a neighboring line at high line density, which would destroy MBs before exposure to the intended high acoustic pressures (Figure 1A). Pulse duration was also studied based on the knowledge that MBs can undergo sustained cavitation with long pulses, probably through the continued acoustic activity of free gas released from the shell boundaries (10,11). For the long pulse durations, we compared continuous 40-cycle to segmented 8 × 5-cycle scheme because previous studies have been performed with the latter and yet sustained cavitation without cycle interruption may be more effective. Our results indicated that higher line densities, up to 65 lines in a standard sector size, provide the most reproducible results that were not influenced by pulse duration, without evidence of loss of effect from line overlap. The notion that low line density results in between-line pressure nadirs was supported by the more gradual clearance of MBs in the gel phantoms.
In vivo studies have indicated that the release of ATP is a key process in CEU-mediated flow augmentation (Central Illustration), acting secondarily through a variety of vasodilators including nitric oxide, adenosine, and prostanoids (5). In the current study, settings that were most reliable for increasing flow also produced the greatest ATP signal at the US-exposed site. However, our findings also indicate that ATP signal is not proportionally related to flow augmentation. This phenomenon could be explained by existence of a threshold concentration over which ATP does not further increase flow, or because purinegic signaling is likely to be dominated by ATP transiently released from shear forces on red blood cells during US (5,12). Also, signal on optical imaging is unlikely to be only from intravascular ATP, which mediates vasodilation, but can also reflect interstitial ATP, which can produce neurogenic vasoconstriction (13,14).
CENTRAL ILLUSTRATION. Ultrasound Exposure of the Calf Skeletal Muscle During Infusion of Ultrasound Contrast Agents Results in Microbubble Inertial Cavitation.
The shear-mediated release of ATP results in production of various vasodilator compounds including NO, prostanoids, and adenosine, all of which produce regional vasodilation. ATP = adenosine triphosphate; NO = nitric oxide.
The first evidence that limb blood flow in humans could be increased by MB cavitation was described in a study where CEU was being used to assess skeletal muscle perfusion in patients with sickle cell disease (5). These studies were neither intended nor designed to evaluate therapeutic CEU, yet an almost 2-fold increase in forearm skeletal muscle flow was detected after approximately 10 min of “diagnostic” imaging acquisitions using CEU at sufficiently high mechanical index to produce inertial cavitation. In the current study, it was necessary to use a clinical scanner in humans that could approximate but not necessarily reproduce the acoustic environment produced in the preclinical experiments. The time-based sequential changes in transducer position (Supplemental Figure 1) assured both complete cavitation and complete replenishment of MBs into the sector. These studies demonstrated a >2-fold increase in muscle blood flow in normal subjects and those with PAD. This finding is remarkable because only a small volume of the calf was exposed to US. Unlike in murine models of PAD where 10 min CEU exposure can reverse ischemia for >24 h (5), the duration of effect was limited in humans. This finding likely reflects major differences in the proportion of the limb that was exposed to US. We believe therapeutic effect will be augmented by 3-D US that can be programmed on the existing system, using spatial data on cavitation on this study to design elevational spacing.
Although the study was performed in patients primarily with intermittent claudication, the implication of our results is that therapeutic CEU could be used in those with more severe disease to accelerate wound healing or for limb salvage in those with critical limb-threatening ischemia who require a delay in definitive therapy. Successful implementation of such a strategy will require further investigation into protocol changes that could produce a longer duration of effect. According to murine data, this may be possible with greater spatial extent of cavitation, or with MB agents that circulate for a long duration before clearance, thereby increasing the potential duration of therapy. Cavitation of MBs is also being explored for its ability to accelerate clot lysis in acute MI and in peripheral thrombotic events (15–17). The ability to increase tissue perfusion independent of sonothrombolysis needs to be considered when examining the efficacy of these therapies.
STUDY LIMITATIONS.
There are several limitations of this study to be acknowledged. We did not assess spatial extent of perfusion augmentation. These studies are planned in the course of evaluating the effect of rastered elevational planes, which is possible with the 3-D array probe used in our preclinical portion of this study. Neither MB dose-response nor time-response were tested. Instead, we elected to evaluate dosing strategies that conformed to Food and Drug Administration–approved limits and to use US durations in humans that are effective in mice and in the subjects with sickle cell disease (5). Because we studied patients with claudication in whom resting blood flow by CEU was not substantially impaired, we did not evaluate changes in symptoms. We believe that evaluation of clinical effects should be performed once full optimization is complete and large-volume tissue exposure with the optimized system is possible. Studies in PAD subjects instead were intended to evaluate changes in microvascular tone in the clinical setting of atherosclerosis and comorbidities such as diabetes and hyperlipidemia.
CONCLUSIONS
This study we have examined acoustic variables that are important for optimizing flow augmentation with CEU and can be implemented using current clinical scanning technology. We have also demonstrated that MB cavitation can produce regional flow augmentation that can be achieved in normal subjects and patients with PAD despite using a very limited spatial extent of exposure. Further clinical translation will require modifications in transducer technology, scanning schemes, and possibly contrast agent composition that will permit large-volume therapy and longer duration of effect.
Supplementary Material
COMPETENCY IN MEDICAL KNOWLEDGE:
Ischemic symptoms in patients with PAD can be reduced by non-pharmacological methods that improve limb blood flow.
TRANSLATIONAL OUTLOOK:
US cavitation of MBs, and other mechanical methods that increase microvascular shear, can increase tissue perfusion and should be investigated for their role to improve wound healing and reduce symptoms in patients with severe PAD.
Acknowledgments
Dr. Lindner is supported by grants R01-HL078610 and R01-HL130046, and Dr. Mason is supported by grant T32-HL094294 from the National Institutes of Health. The study was supported by a material support grant for contrast agent from Lantheus Medical Imaging, Inc., N. Billerica, Massachusetts. Drs. Sheeran and Powers are employees of Philips Healthcare, Bothell, Washington; and Dr. Sutton is an employee of Philips Research, Cambridge, Massachusetts. Dr. Sheeran’s position at Philips is funded through grant R01-HL130046 from the National Institutes of Health (Principal Investigator: Dr. Lindner). All other authors have reported that they have no relationships relevant to the contents of this paper to disclose.
ABBREVIATIONS AND ACRONYMS
- 3-D
3-dimensional
- ATP
adenosine triphosphate
- CEU
contrast-enhanced ultrasound
- MB
microbubble
- MI
mechanical index
- PAD
peripheral artery disease
- US
ultrasound
Footnotes
APPENDIX For a supplemental figure, please see the online version of this paper.
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