Azzdine Y. Ammi

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.

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.

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 (2×108 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.

Monitoring and simulating stable cavitation during ultrasound‐enhanced thrombolysis

The presence of stable cavitation has been shown to be highly correlated with thrombolytic efficacy for recombinant tissue plasminogen activator (rt‐PA) mediated thrombolysis. A commercial contrast agent, Definity, was used with 120 kHz pulsed ultrasound to nucleate, promote, and sustain stable cavitation. The effect of stable cavitation on increased penetration of rt‐PA into the clots is discussed. Also, the possibility of lowering the rt‐PA dose using sustained stable cavitation adjuvant to thrombolytics is presented. To understand the bubble dynamics involved, the bubble response was studied using the Keller‐Miksis model and stable and inertial cavitation thresholds were studied as a function of bubble radius. The largest mass loss (26.2%) was observed for clots treated with 120 kHz ultrasound (0.32 MPa peak‐to‐peak pressure amplitude, 80% duty cycle), rt‐PA (96 μg/ml) and stable cavitation nucleated by Definity. A comparable mass loss of 22% was observed at a much lower concentration of 11 μg/ml in th...

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.

120 kilohertz ultrasound‐enhanced thrombolysis in a porcine intracerebral hemorrhage model.

Ultrasound acts synergistically with thrombolytic agents, such as recombinant tissue plasminogen activator (rt‐PA), to accelerate thrombolysis. The aim of the study was to demonstrate the efficacy of 120‐kHz ultrasound‐enhanced rt‐PA thrombolysis in a porcine hemorrhagic stroke model in vivo. Clots were formed by infusing 3 ml of autologous blood into the frontal white matter of 30 mixed‐bred Yorkshire pigs (20.5–3.1 kg) and incubated for 3 h. For these nonsurvival studies, six pigs received rt‐PA alone (0.3 cc of 0.107 mg/ml), six received ultrasound alone, six received rt‐PA plus ultrasound, six were sham‐exposed (saline only), and six were controls (no ultrasound or rt‐PA treatment). The clots receiving ultrasound treatment were insonified with a peak‐to‐peak pressure of 0.48 MPa in situ (80% duty cycle, and PRF of 1.7 kHz) for 30 min. Clots treated with rt‐PA alone exhibited a volume loss of 55.0% and clots treated with rt‐PA and 120‐kHz ultrasound had a significantly higher volume loss of 75.2% and a...

1-MHz ultrasound stimulates protective mechanisms in cardiac endothelial cells during oxygen and glucose deprivation

Ultrasound improves myocardial perfusion during coronary occlusion. Our aim was to study the production of vasodilatory compounds by primary mouse cardiac endothelial cells (ECs) exposed to ultrasound after a 2-hour oxygen and glucose deprivation (OGD). A 1.05-MHz transducer was used to insonify ECs with a 50-cycle tone burst at a peak rarefactional pressure of 0.5 MPa and a PRF of 50 Hz. US exposure of ECs after OGD increased the adenosine release to 168±16% of control (n = 11, p<0.05). It also resulted in an increase in 8,9-, 11,12- and 14,15-EETs reaching 125±19%, 123±17%, and 118±15% of control (n = 7, p<0.05), respectively. US caused an increase of 5,6-, 8,9-, 11,12-, and 14,15-DHETs to 135±16%, 138±17%, 133±15%, and 133±14% of control, respectively. It also caused levels of 18-, 19-, and 20-HETEs to be statistically different when compared to control and OGD alone (n = 7, p<0.05). eNOS phosphorylation level was not statistically significant to either control or OGD alone. Higher level of cell viabil...

1-MHz ultrasound stimulates in vitro production of cardiac and cerebrovascular endothelial cell vasodilators

Ultrasound exposure of the heart and brain during vessel occlusion reduces infarct size. Our aim was to study the production of vasodilatory compounds by endothelial cells after ultrasound stimulation. A 1.05-MHz single element transducer was used to insonify primary mouse endothelial cells (ECs) from heart and brain with a 50-cycle tone burst at a pulse repetition frequency of 50 Hz. Two time points were studied after ultrasound exposure: 15 and 45 minutes. In heart ECs, EETs levels increased significantly with 0.5 MPa (139 ± 16%, p<0.05) and 0.3 MPa (137 ± 15%, p<0.05) at 15 and 45 min post stimulation, respectively. HETEs and DHETs did not change significantly. There was a trend toward increased adenosine, with maximum release at 0.5 MPa (332 ± 73% vs. 100% control, p<0.05). The trend toward increased eNOS phosphorylation was greater at 15 than 45 min. In brain ECs adenosine release was increased, however increased eNOS phosphorylation was not significant. 11, 12- and 14, 15- EETs were increased while ...

Ultrasound-enhanced thrombolysis in porcine clots in a flow system

Ultrasound and ultrasound contrast agent microbubbles (UCAM) are able to mechanically induce clot reduction. The aim of the study was to demonstrate the efficacy of various ultrasound conditions to enhanced thrombolysis in porcine clots inside a flow system. Clots were formed by infusing 4.1 ml of blood in transfer pipets. The pipets contained Dacron grafts to anchor the clot and initiated its formation. A 14-gage needle was placed at the center of the pipet and removed after clot formation to allow flow inside the clot. The clots were treated for 20 min with ultrasound and homemade UCAM at a concentration of 107 microbubbles/ml (flow rate 0.9 ml/min). Thrombolysis was monitored using an ultrasound scanner in pulse inversion mode. Results show that the radiation force causes the microbubbles to be pushed against the inner clot wall and cavitation induced lysis. The portion of the clot closest to the transducer was not affected by the therapy as the microbubbles were pushed in the direction of propagation....

Characterization of Ultrasound Propagation Through Ex‐vivo Human Temporal Bone

Knowledge of cranial and intracranial ultrasonic properties is essential for optimal results in brain vasculature imaging and therapy. The aims of this study were to perform measurements of the intracranial acoustic pressure field, to identify ultrasound parameters that maximize penetration and minimize beam aberration, and to estimate the speed of sound and the attenuation per unit length in the temporal bone (TB). In vitro experiments were conducted on five human skulls. In a water‐filled tank, two unfocused (0.12 and 1.03 MHz) and one focused (2.00 MHz) transducers were consecutively placed near the TB of each skull. The acoustic pressure field was measured in a volume estimated to encompass the middle cerebral artery (MCA). For each measurement, the intracranial distance from the position of maximum acoustic pressure to the estimated MCA origin was quantified. The pressure reductions at these locations relative to the free field were also estimated. The intracranial ‐3 dB depth of field and beam width...