Augmentation of Muscle Blood Flow by Ultrasound Cavitation Is Mediated by ATP and Purinergic Signaling.

BACKGROUND: Augmentation of tissue blood flow by therapeutic ultrasound is thought to rely on convective shear. Microbubble contrast agents that undergo ultrasound-mediated cavitation markedly amplify these effects. We hypothesized that purinergic signaling is responsible for shear-dependent increases in muscle perfusion during therapeutic cavitation. METHODS: Unilateral exposure of the proximal hindlimb of mice (with or without ischemia produced by iliac ligation) to therapeutic ultrasound (1.3 MHz, mechanical index 1.3) was performed for 10 minutes after intravenous injection of 2×108 lipid microbubbles. Microvascular perfusion was evaluated by low-power contrast ultrasound perfusion imaging. In vivo muscle ATP release and in vitro ATP release from endothelial cells or erythrocytes were assessed by a luciferin-luciferase assay. Purinergic signaling pathways were assessed by studying interventions that (1) accelerated ATP degradation; (2) inhibited P2Y receptors, adenosine receptors, or KATP channels; or (3) inhibited downstream signaling pathways involving endothelial nitric oxide synthase or prostanoid production (indomethacin). Augmentation in muscle perfusion by ultrasound cavitation was assessed in a proof-of-concept clinical trial in 12 subjects with stable sickle cell disease. RESULTS: Therapeutic ultrasound cavitation increased muscle perfusion by 7-fold in normal mice, reversed tissue ischemia for up to 24 hours in the murine model of peripheral artery disease, and doubled muscle perfusion in patients with sickle cell disease. Augmentation in flow extended well beyond the region of ultrasound exposure. Ultrasound cavitation produced an ≈40-fold focal and sustained increase in ATP, the source of which included both endothelial cells and erythrocytes. Inhibitory studies indicated that ATP was a critical mediator of flow augmentation that acts primarily through either P2Y receptors or adenosine produced by ectonucleotidase activity. Combined indomethacin and inhibition of endothelial nitric oxide synthase abolished the effects of therapeutic ultrasound, indicating downstream signaling through both nitric oxide and prostaglandins. CONCLUSIONS: Therapeutic ultrasound using microbubble cavitation to increase muscle perfusion relies on shear-dependent increases in ATP, which can act through a diverse portfolio of purinergic signaling pathways. These events can reverse hindlimb ischemia in mice for >24 hours and increase muscle blood flow in patients with sickle cell disease. CLINICAL TRIAL REGISTRATION: URL: http://clinicaltrials.gov. Unique identifier: NCT01566890.

Ultrasound stimulates formation and release of vasoactive compounds in brain endothelial cells.

Stroke outcome is improved by therapeutic ultrasound. This benefit is presumed to be principally from ultrasound-mediated thrombolysis. We hypothesized that the therapeutic benefit of ultrasound in stroke may, in part, be mediated by the release of beneficial vasoactive substances. Accordingly, we investigated the effect of ultrasound on levels of cytochrome P-450, lipoxygenase, and cyclooxygenase metabolites of arachidonic acid as well as adenosine release and endothelial nitric oxide synthase (eNOS) phosphorylation in primary brain endothelial cells in vitro. Brain endothelial cells were exposed to 1.05-MHz ultrasound at peak rarefactional acoustic pressure amplitudes of 0.35, 0.55, 0.90, and 1.30 MPa. Epoxyeicosatrienoic acids (EETs), hydroxyeicosatetraenoic acids (HETEs), PGE2, adenosine, nitrate/nitrite, and eNOS phosphorylation were measured after ultrasound exposure. Levels of 8,9-EET, 11,12-EET, and 14,15-EET increased by 230 ± 28%, 240 ± 30%, and 246 ± 31% (P < 0.05), respectively, whereas 5-HETE and 15-HETE levels were reduced to 24 ± 14% and 10 ± 3% (P < 0.05), respectively, compared with cells not exposed to ultrasound. PGE2 levels were reduced to 56 ± 14% of control. Adenosine increased more than sixfold after ultrasound exposure compared with unstimulated cells (1.36 ± 0.22 vs. 0.37 ± 0.10 ng/ml, P < 0.05), nitrate/nitrite was below levels of quantification, and eNOS phosphorylation was not altered significantly. Our results suggest that ultrasound may enhance tissue perfusion during stroke by augmenting the generation of vasodilator compounds and inhibiting that of vasoconstrictors. Such regulation supports a beneficial role for therapeutic ultrasound in stroke independent of its effect on the occlusive thrombus.

Low-Intensity Ultrasound Reduces Brain Infarct Size by Upregulating Phosphorylated Endothelial Nitric Oxide in Mouse Model of Middle Cerebral Artery Occlusion.

OBJECTIVE: There have been attempts to use therapeutic ultrasound (US) for the treatment of both experimental and clinical stroke. We hypothesized that low-intensity US has direct beneficial effects on the brain independent of cerebral blood flow (CBF) during middle cerebral artery occlusion (MCAO). METHODS: Three groups of mice were studied. Group I included 84 mice with MCAO undergoing US treatment/no treatment at two US frequencies (0.25 and 1.05 MHz) with three different acoustic pressures at each frequency in which infarct size (IS) was measured 24 h later. Group II included 11 mice undergoing treatment based on best US results from group I animals in which the IS/risk area (RA) ratio was measured 24 h later. Group III included 38 normal mice undergoing US treatment/no treatment for assessment of CBF, tissue metabolite and protein expression and histopathology. DISCUSSION: Ultrasound at both frequencies and most acoustic pressures resulted in reduction in IS in group I animals, with the best results obtained with 0.25 MHz at 2.0 MPa: IS was reduced 4-fold in the cerebral cortex, 1.5-fold in the caudate putamen and 3.5-fold in the cerebral hemisphere compared with control. US application in group III animals elicited only a marginal increase in CBF despite a 2.6-fold increase in phosphorylated endothelial nitric oxide synthase (p-eNOS)-S1177 and a corresponding decrease in p-eNOS-T494. Histopathology revealed no evidence of hemorrhage, inflammation or necrosis. CONCLUSION: Low-intensity US at specific frequencies and acoustic pressures results in marked neuroprotection in a mouse model of stroke by modulation of p-eNOS independent of its effect on CBF.

Quantification of microbubble-induced sonothrombolysis in an ex vivo non-human primate model.

BACKGROUND: In vitro studies with ultrasound (US) and microbubbles (MB) have reported that sono-thrombolysis can be achieved at high peak rarefactional acoustic pressure amplitudes (PRAPAs) using 0.25 and 1.05 MHz US frequencies. OBJECTIVE: The aim of the current study was to determine if these parameters work on an ex vivo physiological model of thrombosis. METHODS: A thrombogenic device was placed in an ex vivo chronic arteriovenous shunt in juvenile baboons. Platelet accumulation was measured by dynamic imaging of the device and the 10 cm thrombus tail with 111 In-labeled platelets. After 15 minutes of thrombus formation, treatment with either low-dose recombinant tissue plasminogen activator (rtPA) or low-dose rtPA + MB+US was performed for 20 minutes. Four US settings at 0.25% duty cycle were used: 0.25 MHz at PRAPAs of 1.20 and 2.20 MPa, and 1.05 MHz at 1.75 and 4.75 MPa. RESULTS: Platelet accumulation was not inhibited by low-dose rtPA or MB with US alone. Platelet accumulation was significantly reduced with 0.25 MHz US at 2.20 PRAPA (P < .001) and with 1.05 MHz at 1.75 MPa and 4.75 MPa (P < .05) when used with MB and low-dose rtPA. Although this approach prevented platelet accumulation it did not cause thrombolysis on the device. CONCLUSIONS: rtPA + MB + US (0.25 and 1.05 MHz) resulted in inhibition of platelet accumulation on the thrombogenic device when moderately high PRAPAs (≥1.75 MPa) were used. These results taken in context with lytic effects of US on myocardial microthrombi and direct effect on myocardial blood flow and function provide direction for the use of therapeutic US in acute coronary syndromes.

Ultrasound therapy for treatment of lower extremity intermittent claudication.

BACKGROUND: While often thought of as a diagnostic tool, ultrasound (US) can also potentially be used as a therapeutic modality. US applies mechanical stress on endothelial cells and induces nitric oxide synthase, which regulates the secretion of nitric oxide, a potent vasodilator. In animal ischemic models, US has been shown to improve hindlimb, myocardial, and cerebral perfusion. We performed a pilot trial of US therapy in the lower extremities of human subjects with intermittent claudication. METHODS: 10 subjects (5 male, 5 female, mean age 69.7 ± 10.3) with intermittent claudication were recruited. Both legs were placed in a specially designed boot with a water interface between US transducers and the legs. Subjects underwent pulsed US therapy at 250 kHz frequency for 30 min for three treatments a week for six weeks. Pre and post treatment ankle:brachial index (ABI), 6-min walk (6 MW), Walking Impairment Questionnaire (WIQ), and Short Form 36 (SF36) were performed. Pre and post-treatment results were compared with paired t-test. RESULTS: Six minute walking distance at baseline was 352 ± 70 m, after one treatment session 353 ± 70 m (p = 0.99), and at completion 372 ± 71 m (p = 0.015). There was a trend toward improved ABI after 6 weeks of treatment (0.53 ± 0.17 vs 0.64 ± 0.12, p = 0.083). After six weeks, significant improvements were noted in overall WIQ score (2.00 ± 1.48 vs 2.63 ± 1.38, p = 0.0001), WIQ (distance) 2.07 ± 1.54 vs 2.73 ± 1.42 (p = 0.036), and WIQ (stair) 2.00 ± 1.67 vs 2.62 ± 1.24, p = 0.034, with a trend in WIQ (speed), 1.89 ± 1.26 vs 2.46 ± 1.43, p = 0.069. In the SF-36, significant improvements were noted in the domains of physical functioning (44.0 ± 41.6 vs 50.5 ± 41.1, p = 0.009) and role limitations - physical (35.0 ± 48.3 vs 60.0 ± 49.6, p = 0.006) after six weeks. CONCLUSIONS: Therapeutic US is a potential noninvasive treatment for intermittent claudication. Pilot study patients noted significant improvements in 6 MW and WIQ results after 6 weeks of treatment. A nonsignificant improvement in ABI was noted. Further research will be needed to clarify optimal treatment frequency and duration.

Phosphoproteomic response of cardiac endothelial cells to ischemia and ultrasound.

Myocardial infarction and subsequent therapeutic interventions activate numerous intracellular cascades in every constituent cell type of the heart. Endothelial cells produce several protective compounds in response to therapeutic ultrasound, under both normoxic and ischemic conditions. How endothelial cells sense ultrasound and convert it to a beneficial biological response is not known. We adopted a global, unbiased phosphoproteomics approach aimed at understanding how endothelial cells respond to ultrasound. Here, we use primary cardiac endothelial cells to explore the cellular signaling events underlying the response to ischemia-like cellular injury and ultrasound exposure in vitro. Enriched phosphopeptides were analyzed with a high mass accuracy liquid chromatrography (LC) - tandem mass spectrometry (MS/MS) proteomic platform, yielding multiple alterations in both total protein levels and phosphorylation events in response to ischemic injury and ultrasound. Application of pathway algorithms reveals numerous protein networks recruited in response to ultrasound including those regulating RNA splicing, cell-cell interactions and cytoskeletal organization. Our dataset also permits the informatic prediction of potential kinases responsible for the modifications detected. Taken together, our findings begin to reveal the endothelial proteomic response to ultrasound and suggest potential targets for future studies of the protective effects of ultrasound in the ischemic heart.

(Phospho)Proteomic dataset of ischemia- and ultrasound- stimulated mouse cardiac endothelial cells in vitro.

Cardiac endothelial cells respond to both ischemia and therapeutic ultrasound; the proteomic changes underlying these responses are unknown. This data article provides raw and processed data resulting from our global, unbiased phosphoproteomics investigation conducted on primary mouse cardiac endothelial cells exposed to ischemia (2-hour oxygen glucose deprivation) and ultrasound (250 kHz, 1.2 MPa) in vitro [1]. Proteins were extracted from cell lysates and enriched phosphopeptides were analyzed with a high mass accuracy liquid chromatrography (LC) - tandem mass spectrometry (MS/MS) proteomic platform, yielding multiple alterations in both total protein levels and phosphorylation events in response to ischemic injury and ultrasound. This dataset can be used as a reference for future studies on the cardiac endothelial response to ischemia and the mechanistic underpinnings of the cellular response to ultrasound, with the potential to yield clinically relevant therapeutic targets.

Flow Augmentation in the Myocardium by Ultrasound Cavitation of Microbubbles: Role of Shear-Mediated Purinergic Signaling.

BACKGROUND: Ultrasound-mediated cavitation of microbubble contrast agents produces high intravascular shear. We hypothesized that microbubble cavitation increases myocardial microvascular perfusion through shear-dependent purinergic pathways downstream from ATP release that is immediate and sustained through cellular ATP channels such as Pannexin-1. METHODS: Quantitative myocardial contrast echocardiography perfusion imaging and in vivo optical imaging of ATP was performed in wild-type and Pannexin-1-deficient (Panx1-/-) mice before and 5 and 30 minutes after 10 minutes of ultrasound-mediated (1.3 MHz, mechanical index 1.3) myocardial microbubble cavitation. Flow augmentation in a preclinical model closer to humans was evaluated in rhesus macaques undergoing myocardial contrast echocardiography perfusion imaging after high-power cavitation in the apical four-chamber plane for 10 minutes. RESULTS: Microbubble cavitation in wild-type mice (n = 7) increased myocardial perfusion by 64% ± 25% at 5 minutes and 95% ± 55% at 30 minutes compared with baseline (P < .05). In Panx1-/- mice (n = 5), perfusion increased by 28% ± 26% at 5 minutes (P = .04) but returned to baseline at 30 minutes. Myocardial ATP signal in wild-type (n = 7) mice undergoing cavitation compared with sham-treated controls (n = 3) was 450-fold higher at 5 minutes and 90-fold higher at 30 minutes after cavitation (P < .001). The ATP signal in Panx1-/- mice (n = 4) was consistently 10-fold lower than that in wild-type mice and was similar to sham controls at 30 minutes. In macaques (n = 8), myocardial perfusion increased twofold in the cavitation-exposed four-chamber plane, similar in degree to that produced by adenosine, but did not increase in the control two-chamber plane. CONCLUSIONS: Cavitation of microbubbles in the myocardial microcirculation produces an immediate release of ATP, likely from cell microporation, as well as sustained release, which is channel dependent and responsible for persistent flow augmentation. These findings provide mechanistic insight by which cavitation improves perfusion and reduces infarct size in patients with myocardial infarction.

Therapeutic Ultrasound Improves Myocardial Blood Flow and Reduces Infarct Size in a Canine Model of Coronary Microthromboembolism.

BACKGROUND: Therapeutic ultrasound (TUS) has been used to lyse infarct-related coronary artery thrombus. There has been no study examining the effect of TUS specifically on myocardial microthromboemboli seen in acute myocardial infarction and acute coronary syndromes. The aim of this study was to test the hypothesis that TUS improves myocardial blood flow (MBF) and reduces infarct size (IS) in this situation by dissolving myocardial microthrombi. METHODS: An open-chest canine model of myocardial microthromboembolism was created by disrupting a thrombus in the left anterior descending coronary artery, and 1.05- and 0.25-MHz TUS (n = 7 each) delivered epicardially for 30 min was compared with control (n = 6). MBF and IS (as a percentage of left anterior descending coronary artery perfusion bed size) were measured 60 min after treatment. In addition, immunohistochemistry was performed to assess microthrombi, and histopathology was performed to define inflammation. RESULTS: Transmural, epicardial, and endocardial myocardial blood volume and MBF (measured using myocardial contrast echocardiography) and percentage wall thickening were significantly higher 60 min after receiving TUS compared with control. The ratio of IS to left anterior descending coronary artery perfusion bed size was significantly smaller (P = .03) in the 1.05-MHz TUS group (0.14 ± 0.04) compared with the control (0.31 ± 0.06, P = .04) and 0.25-MHz (0.36 ± 0.08) groups. MBF versus percentage wall thickening exhibited a linear relation (r = 0.65) in the control and 1.05-MHz TUS groups but not in the 0.25-MHz TUS group (r = 0.29). The presence of myocardial microemboli in vessels >10 µm in diameter was significantly reduced in the 1.05-MHz TUS group compared with the other two groups. The distribution and intensity of inflammation was higher in the 0.25-MHz TUS group compared with the other groups. CONCLUSIONS: TUS at 1.05 MHz is effective in restoring myocardial blood volume and MBF, thus reducing IS by clearing the microcirculation of microthrombi. IS reduction is not seen at 0.25 MHz, despite improvement in MBF, which may be related to the increased inflammation noted at this frequency. Because both acute myocardial infarction and acute coronary syndromes are associated with microthromboembolism, these results suggest that TUS could have a potential adjunctive role in the treatment of both conditions.

Therapeutic Ultrasound Increases Myocardial Blood Flow in Ischemic Myocardium and Cardiac Endothelial Cells: Results of In Vivo and In Vitro Experiments.

BACKGROUND: Therapeutic ultrasound can reduce infarct size in a model of coronary thrombosis even when sonothrombolysis is ineffective. The aim of this study was to test the hypothesis that ultrasound-induced cardioprotection is mediated by molecules released from the vascular endothelium that increase myocardial blood flow (MBF) and also have direct tissue-salvaging effects. METHODS: In vivo and in vitro experiments were performed using a 1.05-MHz transducer. For the in vivo experiments 10 control and 10 ultrasound-treated dogs undergoing occlusion of the left anterior descending coronary artery were studied. MBF was measured using myocardial contrast echocardiography. For the in vitro experiments, primary mouse cardiac endothelial cells were exposed to ultrasound at baseline or following oxygen-glucose deprivation and endothelial nitric oxide synthase phosphorylation as well as adenosine and the eicosanoids epoxyeicosatrienoic acids, dihydroxyeicosatrienoic acids, and hydroxyl-eicosatetraenoic acids were measured. RESULTS: In vivo, ultrasound treatment caused higher MBF (20 ± 10 vs 10 ± 8, P = .03) and higher wall thickening (3 ± 3% vs 1 ± 0.4%, P = .01) in the collateral-derived border zone compared with control. Epicardial MBF in the left anterior descending coronary artery bed also tended to be higher (17 ± 17 vs 5 ± 4, P = .05) in ultrasound-treated versus control animals; however, endocardial MBF in this region was similar to that in controls (13 ± 14 vs 14 ± 7). In vitro, phosphorylated endothelial nitric oxide synthase and adenosine increased (by 129 ± 11% and 286 ± 63%, respectively, P < .01) with ultrasound compared with unstimulated cells. Similar results were obtained with epoxyeicosatrienoic acids. After oxygen-glucose deprivation, phosphorylated endothelial nitric oxide synthase decreased and was restored with application of ultrasound. Similar changes were noted with epoxyeicosatrienoic acids. Cell viability decreased with oxygen-glucose deprivation and returned to near baseline with ultrasound. CONCLUSIONS: Ultrasound increases MBF in ischemic tissue in vivo. This effect is likely mediated by the release of a plethora of coronary vasodilators during ultrasound treatment that also have direct tissue-salvaging effects. Therapeutic ultrasound, therefore, has potential for treatment of acute and chronic myocardial ischemia independent of its effect on thrombolysis.

Ultrasound-enhanced thrombolysis using Definity as a cavitation nucleation agent.

Ultrasound has been shown previously to act synergistically with a thrombolytic agent, such as recombinant tissue plasminogen activator (rt-PA) to accelerate thrombolysis. In this in vitro study, a commercial contrast agent, Definity, was used to promote and sustain the nucleation of cavitation during pulsed ultrasound exposure at 120 kHz. Ultraharmonic signals, broadband emissions and harmonics of the fundamental were measured acoustically by using a focused hydrophone as a passive cavitation detector and used to quantify the level of cavitation activity. Human whole blood clots suspended in human plasma were exposed to a combination of rt-PA, Definity and ultrasound at a range of ultrasound peak-to-peak pressure amplitudes, which were selected to expose clots to various degrees of cavitation activity. Thrombolytic efficacy was determined by measuring clot mass loss before and after the treatment and correlated with the degree of cavitation activity. The penetration depth of rt-PA and plasminogen was also evaluated in the presence of cavitating microbubbles using a dual-antibody fluorescence imaging technique. The largest mass loss (26.2%) was observed for clots treated with 120-kHz ultrasound (0.32-MPa peak-to-peak pressure amplitude), rt-PA and stable cavitation nucleated by Definity. A significant correlation was observed between mass loss and ultraharmonic signals (r = 0.85, p < 0.0001, n = 24). The largest mean penetration depth of rt-PA (222 microm) and plasminogen (241 microm) was observed in the presence of stable cavitation activity. Stable cavitation activity plays an important role in enhancement of thrombolysis and can be monitored to evaluate the efficacy of thrombolytic treatment.

Rest-Stress Limb Perfusion Imaging in Humans with Contrast Ultrasound Using Intermediate-Power Imaging and Microbubbles Resistant to Inertial Cavitation.

BACKGROUND: Contrast-enhanced ultrasound (CEU) limb perfusion imaging is a promising approach for evaluating peripheral artery disease (PAD). However, low signal enhancement in skeletal muscle has necessitated high-power intermittent imaging algorithms, which are not clinically feasible. We hypothesized that CEU using a combination of intermediate power and a contrast agent resistant to inertial cavitation would allow real-time limb stress perfusion imaging. METHODS: In normal volunteers, CEU of the calf skeletal muscle was performed on separate days with Sonazoid, Optison, or Definity. Progressive reduction in the ultrasound pulsing interval was used to assess the balance between signal enhancement and agent destruction at escalating mechanical indices (MI, 0.1-0.4). Real-time perfusion imaging at MI 0.1-0.4 using postdestructive replenishment kinetics was performed at rest and during 25 W plantar flexion contractile exercise. RESULTS: For Optison, limb perfusion imaging was unreliable at rest due to very low signal enhancement generated at all MIs and was possible during exercise-induced hyperemia only at MI 0.1 due to agent destruction at higher MIs. For Definity, signal intensity progressively increased with MI but was offset by microbubble destruction, which resulted in modest signal enhancement during CEU perfusion imaging and distortion of replenishment curves at MI ≥ 0.2. For Sonazoid, there strong signal enhancement at MI ≥ 0.2, with little destruction detected only at MI 0.4. Accordingly, high signal intensity and nondistorted perfusion imaging was possible at MI 0.2-0.3 and detected an 8.0- ± 5.7-fold flow reserve. CONCLUSIONS: Rest-stress limb perfusion imaging in humans with real-time CEU, which requires only seconds to perform, is possible using microbubbles with viscoelastic properties that produce strong nonlinear signal generation without destruction at intermediate acoustic pressures.

Ultrasonic contrast agent shell rupture detected by inertial cavitation and rebound signals.

Determining the rupture pressure threshold of ultrasound contrast agent microbubbles has significant applications for contrast imaging, development of therapeutic agents, and evaluation of potential bioeffects. Using a passive cavitation detector, this work evaluates rupture based on acoustic emissions from single, encapsulated, gas-filled microbubbles. Sinusoidal ultrasound pulses were transmitted into weak solutions of Optison at different center frequencies (0.9, 2.8, and 4.6 MHz), pulse durations (three, five, and seven cycles of the center frequencies), and peak rarefactional pressures (0.07 to 5.39 MPa). Pulse repetition frequency was 10 Hz. Signals detected with a 13-MHz, center-frequency transducer revealed postexcitation acoustic emissions (between 1 and 5 micros after excitation) with broadband spectral content. The observed acoustic emissions were consistent with the acoustic signature that would be anticipated from inertial collapse followed by "rebounds" when a microbubble ruptures and thus generates daughter/free bubbles that grow and collapse. The peak rarefactional pressure threshold for detection of these emissions increased with frequency (e.g., 0.53, 0.87, and 0.99 MPa for 0.9, 2.8, and 4.6 MHz, respectively; five-cycle pulse duration) and decreased with pulse duration. The emissions identified in this work were separated from the excitation in time and spectral content, and provide a novel determination of microbubble shell rupture.

Influence of DNA-Microbubble Coupling on Contrast Ultrasound-Mediated Gene Transfection in Muscle and Liver.

BACKGROUND: Contrast ultrasound-mediated gene delivery (CUMGD) is a promising approach for enhancing gene therapy that relies on microbubble (MB) cavitation to augment complementary deoxyribonucleic acid (cDNA) transfection. The aims of this study were to determine optimal conditions for charge-coupling cDNA to MBs and to evaluate the advantages of surface loading for gene transfection in muscle and liver. METHODS: Charge coupling of fluorescently labeled cDNA to either neutral MBs (MBN) or cationic MBs (MB+) in low- to high-ionic conditions (0.3%-1.8% NaCl) was assessed by flow cytometry. MB aggregation from cDNA coupling was determined by electrozone sensing. Tissue transfection of luciferase in murine hindlimb skeletal muscle and liver was made by CUMGD with MBN or MB+ combined with subsaturated, saturated, or supersaturated cDNA concentrations (2.5, 50, and 200 µg/10(8) MBs). RESULTS: Charge-coupling of cDNA was detected for MB+ but not MBN. Coupling occurred over almost the entire range of ionic conditions, with a peak at 1.2% NaCl, although electrostatic interference occurred at >1.5% NaCl. DNA-mediated aggregation of MB+ was observed at ≤0.6% NaCl but did not reduce the ability to produce inertial cavitation. Transfection with CUMGD in muscle and liver was low for both MBs at subsaturation concentrations. In muscle, higher cDNA concentrations produced a 10-fold higher degree of transfection with MB+, which was approximately fivefold higher (P < .05) than that for MBN. There was no effect of DNA supersaturation. The same pattern was seen for liver except that supersaturation further increased transfection with MBN equal to that of MB+. CONCLUSIONS: Efficient charge-coupling of cDNA to MB+ but not MBN occurs over a relatively wide range of ionic conditions without aggregation. Transfection with CUMGD is much more efficient with charge-coupling of cDNA to MBs and is not affected by supersaturation except in the liver, which is specialized for macromolecular and cDNA uptake.

Efficacy and spatial distribution of ultrasound-mediated clot lysis in the absence of thrombolytics.

Ultrasound and microbubble (MB) contrast agents accelerate clot lysis, yet clinical trials have been performed without defining optimal acoustic conditions. Our aim was to assess the effect of acoustic pressure and frequency on the extent and spatial location of clot lysis. Clots from porcine blood were created with a 2-mm central lumen for infusion of lipid-shelled perfluorocarbon MBs (1×10(7) ml(-1)) or saline. Therapeutic ultrasound at 0.04, 0.25, 1.05, or 2.00 MHz was delivered at a wide range of peak rarefactional acoustic pressure amplitudes (PRAPAs). Ultrasound was administered over 20 minutes grouped on-off cycles to allow replenishment of MBs. The region of lysis was quantified using contrast-enhanced ultrasound imaging. In the absence of MBs, sonothrombolysis did not occur at any frequency. Sonothrombolysis was also absent in the presence of MBs despite their destruction at 0.04 and 2.00 MHz. It occurred at 0.25 and 1.05 MHz in the presence of MBs for PRAPAs > 1.2 MPa and increased with PRAPA. At 0.25 MHz the clot lysis was located in the far wall. At 1.05 MHz, however, there was a transition from far to near wall as PRAPA was increased. The area of clot lysis measured by ultrasound imaging correlated with that by micro-CT and quantification of debris in the effluent. In conclusion, sonothrombolysis with MBs was most efficient at 0.25 MHz. The spatial location of sonothrombolysis varies with pressure and frequency indicating that the geometric relation between therapeutic probe and vascular thrombosis is an important variable for successful lysis clinically.

Augmentation of limb perfusion and reversal of tissue ischemia produced by ultrasound-mediated microbubble cavitation.

BACKGROUND: Ultrasound can increase tissue blood flow, in part, through the intravascular shear produced by oscillatory pressure fluctuations. We hypothesized that ultrasound-mediated increases in perfusion can be augmented by microbubble contrast agents that undergo ultrasound-mediated cavitation and sought to characterize the biological mediators. METHODS AND RESULTS: Contrast ultrasound perfusion imaging of hindlimb skeletal muscle and femoral artery diameter measurement were performed in nonischemic mice after unilateral 10-minute exposure to intermittent ultrasound alone (mechanical index, 0.6 or 1.3) or ultrasound with lipid microbubbles (2×10(8) IV). Studies were also performed after inhibiting shear- or pressure-dependent vasodilator pathways, and in mice with hindlimb ischemia. Ultrasound alone produced a 2-fold increase (P<0.05) in muscle perfusion regardless of ultrasound power. Ultrasound-mediated augmentation in flow was greater with microbubbles (3- and 10-fold higher than control for mechanical index 0.6 and 1.3, respectively; P<0.05), as was femoral artery dilation. Inhibition of endothelial nitric oxide synthase attenuated flow augmentation produced by ultrasound and microbubbles by 70% (P<0.01), whereas inhibition of adenosine-A2a receptors and epoxyeicosatrienoic acids had minimal effect. Limb nitric oxide production and muscle phospho-endothelial nitric oxide synthase increased in a stepwise fashion by ultrasound and ultrasound with microbubbles. In mice with unilateral hindlimb ischemia (40%-50% reduction in flow), ultrasound (mechanical index, 1.3) with microbubbles increased perfusion by 2-fold to a degree that was greater than the control nonischemic limb. CONCLUSIONS: Increases in muscle blood flow during high-power ultrasound are markedly amplified by the intravascular presence of microbubbles and can reverse tissue ischemia. These effects are most likely mediated by cavitation-related increases in shear and activation of endothelial nitric oxide synthase.

Real-time contrast ultrasound muscle perfusion imaging with intermediate-power imaging coupled with acoustically durable microbubbles.

BACKGROUND: There is growing interest in limb contrast-enhanced ultrasound (CEU) perfusion imaging for the evaluation of peripheral artery disease. Because of low resting microvascular blood flow in skeletal muscle, signal enhancement during limb CEU is prohibitively low for real-time imaging. The aim of this study was to test the hypothesis that this obstacle can be overcome by intermediate- rather than low-power CEU when performed with an acoustically resilient microbubble agent. METHODS: Viscoelastic properties of Definity and Sonazoid were assessed by measuring bulk modulus during incremental increases in ambient pressure to 200 mm Hg. Comparison of in vivo microbubble destruction and signal enhancement at a mechanical index (MI) of 0.1 to 0.4 was performed by sequential reduction in pulsing interval from 10 to 0.05 sec during limb CEU at 7 MHz in mice and 1.8 MHz in dogs. Destruction was also assessed by broadband signal generation during passive cavitation detection. Real-time CEU perfusion imaging with destruction-replenishment was then performed at 1.8 MHz in dogs using an MI of 0.1, 0.2, or 0.3. RESULTS: Sonazoid had a higher bulk modulus than Definity (66 ± 12 vs 29 ± 2 kPa, P = .02) and exhibited less inertial cavitation (destruction) at MIs ≥ 0.2. On in vivo CEU, maximal signal intensity increased incrementally with MI for both agents and was equivalent between agents except at an MI of 0.1 (60% and 85% lower for Sonazoid at 7 and 1.8 MHz, respectively, P < .05). However, on progressive shortening of the pulsing interval, Definity was nearly completely destroyed at MIs ≥ 0.2 at 1.8 and 7 MHz, whereas Sonazoid was destroyed only at 1.8 MHz at MIs ≥ 0.3. As a result, real-time CEU perfusion imaging demonstrated approximately fourfold greater enhancement for Sonazoid at an MI of 0.3 to 0.4. CONCLUSIONS: Robust signal enhancement during real-time CEU perfusion imaging of the limb is possible when using intermediate-power imaging coupled with a durable microbubble contrast agent.

Ultrasound-mediated vascular gene transfection by cavitation of endothelial-targeted cationic microbubbles.

OBJECTIVES: Ultrasound-mediated gene delivery can be amplified by acoustic disruption of microbubble carriers that undergo cavitation. We hypothesized that endothelial targeting of microbubbles bearing cDNA is feasible and, through optimizing proximity to the vessel wall, increases the efficacy of gene transfection. BACKGROUND: Contrast ultrasound-mediated gene delivery is a promising approach for site-specific gene therapy, although there are concerns with the reproducibility of this technique and the safety when using high-power ultrasound. METHODS: Cationic lipid-shelled decafluorobutane microbubbles bearing a targeting moiety were prepared and compared with nontargeted microbubbles. Microbubble targeting efficiency to endothelial adhesion molecules (P-selectin or intercellular adhesion molecule [ICAM]-1) was tested using in vitro flow chamber studies, intravital microscopy of tumor necrosis factor-alpha (TNF-α)-stimulated murine cremaster muscle, and targeted contrast ultrasound imaging of P-selectin in a model of murine limb ischemia. Ultrasound-mediated transfection of luciferase reporter plasmid charge coupled to microbubbles in the post-ischemic hindlimb muscle was assessed by in vivo optical imaging. RESULTS: Charge coupling of cDNA to the microbubble surface was not influenced by the presence of targeting ligand, and did not alter the cavitation properties of cationic microbubbles. In flow chamber studies, surface conjugation of cDNA did not affect attachment of targeted microbubbles at microvascular shear stresses (0.6 and 1.5 dyne/cm(2)). Attachment in vivo was also not affected by cDNA according to intravital microscopy observations of venular adhesion of ICAM-1-targeted microbubbles and by ultrasound molecular imaging of P-selectin-targeted microbubbles in the post-ischemic hindlimb in mice. Transfection at the site of high acoustic pressures (1.0 and 1.8 MPa) was similar for control and P-selectin-targeted microbubbles but was associated with vascular rupture and hemorrhage. At 0.6 MPa, there were no adverse bioeffects, and transfection was 5-fold greater with P-selectin-targeted microbubbles. CONCLUSIONS: We conclude that ultrasound-mediated transfection at safe acoustic pressures can be markedly augmented by endothelial juxtaposition.

Temporal characterization of the functional density of the vasa vasorum by contrast-enhanced ultrasonography maximum intensity projection imaging.

OBJECTIVES: We sought to determine whether contrast-enhanced ultrasound (CEU) microangiography with maximum intensity projection (MIP) processing could temporally evaluate proliferation of the vasa vasorum (VV) in a model of mural hemorrhage. BACKGROUND: Expansion of the VV and plaque neovascularization contributes to plaque growth and instability and may be triggered by a variety of stimuli, including vascular hemorrhage. However, quantitative in vivo methods for temporal assessment of VV remodeling are lacking. METHODS: In 24 rabbits fed a high-fat diet, either autologous whole blood or saline was percutaneously injected into the media-adventitia of the femoral artery using ultrahigh-frequency ultrasound guidance. Functional VV density at the injection site and contralateral control artery was assessed 1, 2, and 6 weeks after injection with CEU imaging with MIP processing. In vitro studies with renathane microtubes were also performed to validate linear density measurement with CEU and MIP processing. RESULTS: In vitro studies demonstrated that MIP processing of CEU data reflected the relative linear density of vessels in a manner that was relatively independent of contrast concentration or microtube flow rate. On CEU with MIP, there was a 3-fold increase in femoral artery VV microvascular density at 1 and 2 weeks after blood injection (p < 0.01 vs. contralateral control), whereas VV density increased minimally after saline injection. At 6 weeks, VV vascular density decreased in blood-treated vessels and was not different from saline-injected or contralateral control vessels. CONCLUSIONS: CEU with MIP processing can provide quantitative data on temporal changes in the functional density of the VV. This method may be useful for evaluating high-risk features of plaque neovascularization or response to therapies aimed at plaque neovessels.

Characterization of ultrasound propagation through ex-vivo human temporal bone.

Adjuvant therapies that lower the thrombolytic dose or increase its efficacy would represent a significant breakthrough in the treatment of patients with ischemic stroke. The objective of this study was to perform intracranial measurements of the acoustic pressure field generated by 0.12, 1.03 and 2.00-MHz ultrasound transducers to identify optimal ultrasound parameters that would maximize penetration and minimize aberration of the beam. To achieve this goal, in vitro experiments were conducted on five human skull specimens. In a water-filled tank, two unfocused transducers (0.12 and 1.03 MHz) and one focused transducer (2.00 MHz) were consecutively placed near the right temporal bone of each skull. A hydrophone, mounted on a micropositioning system, was moved to an estimated location of the middle cerebral artery (MCA) origin, and measurements of the surrounding acoustic pressure field were performed. For each measurement, the distance from the position of maximum acoustic pressure to the estimated origin of the MCA inside the skulls was quantified. The -3 dB depth-of-field and beamwidth in the skull were also investigated as a function of the three frequencies. Results show that the transducer alignment relative to the skull is a significant determinant of the detailed behavior of the acoustic field inside the skull. For optimal penetration, insonation normal to the temporal bone was needed. The shape of the 0.12-MHz intracranial beam was more distorted than those at 1.03 and 2.00 MHz because of the large aperture and beamwidth. However, lower ultrasound pressure reduction was observed at 0.12 MHz (22.5%). At 1.03 and 2.00 MHz, two skulls had an insufficient temporal bone window and attenuated the beam severely (up to 96.6% pressure reduction). For all frequencies, constructive and destructive interference patterns were seen near the contralateral skull wall at various elevations. The 0.12-MHz ultrasound beam depth-of-field was affected the most when passing through the temporal bone and showed a decrease in size of more than 55% on average. The speed of sound in the temporal bone of each skull was estimated at 1.03 MHz and demonstrated a large range (1752.1 to 3285.3 m/s). Attenuation coefficients at 1.03 and 2.00 MHz were also derived for each of the five skull specimens. This work provides needed information on ultrasound beam shapes inside the human skull, which is a necessary first step for the development of an optimal transcranial ultrasound-enhanced thrombolysis device.