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

ABSTRACT: 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.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.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.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.URL: http://clinicaltrials.gov. Unique identifier: NCT01566890.

SUBMITTER: Belcik JT

PROVIDER: S-EPMC5373943 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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

Belcik J Todd JT Davidson Brian P BP Xie Aris A Wu Melinda D MD Yadava Mrinal M Qi Yue Y Liang Sherry S Chon Chae Ryung CR Ammi Azzdine Y AY Field Joshua J Harmann Leanne L Chilian William M WM Linden Joel J Lindner Jonathan R JR

Circulation 20170207 13

<h4>Background</h4>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.<h4>Methods</h4>Unilateral exposure of the proximal hindlimb of mice (with or without ischemia produced by iliac ligation) to therapeutic ul ...[more]

PMID: 28174191

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Project description:BackgroundUltrasound-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.MethodsQuantitative 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.ResultsMicrobubble 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.ConclusionsCavitation 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.

Project description:New findingsWhat is the central question of this study? Contrast-enhanced ultrasound (CEUS) can be used to directly assess skeletal muscle perfusion but its day-to-day repeatability over time has not yet been validated: is CEUS a repeatable method for the measurement of skeletal muscle microvascular blood flow (MBF) at rest and in response to exercise, across independent assessment sessions? What is the main finding and its importance? A strong agreement between CEUS MBF measures across sessions suggests it is a repeatable method for assessing skeletal muscle perfusion over time. This validation provides confidence for incorporating these measures into longitudinal studies such as a chronic intervention or disease progression to gain further knowledge of skeletal muscle microvascular function.AbstractContrast-enhanced ultrasound (CEUS) can be used to directly assess skeletal muscle perfusion. However, its repeatability over time has not yet been validated and therefore its use in longitudinal measures (i.e., exploring the impact of a chronic intervention or disease progression) is limited. This study aimed to determine the repeatability of CEUS for the measurement of skeletal muscle microvascular blood flow (MBF) at baseline and in response to exercise, across independent assessment sessions. Ten healthy volunteers (five female; 30 ± 6 years) had CEUS of the right vastus lateralis recorded in two separate sessions, 14 days apart. Measurements were taken at baseline, during an isometric leg extension and during recovery. Acoustic intensity data from a region of interest were plotted as a replenishment curve to obtain blood volume (A) and flow velocity (β) values from a one-phase association non-linear regression of mean tissue echogenicity. Linear regression and Bland-Altman analyses of A and β values were performed, with significance assumed as P < 0.05. Strong positive correlations were observed across sessions for all A and β values (both P < 0.0001). Bland-Altman analysis showed a bias (SD) of -0.013 ± 1.24 for A and -0.014 ± 0.31 for β. A bias of 0.201 ± 0.770 at baseline, 0.527 ± 1.29 during contraction and -0.203 ± 1.29 at recovery was observed for A, and -0.0328 ± 0.0853 (baseline), -0.0446 ± 0.206 (contraction) and 0.0382 ± 0.233 (recovery) for β. A strong agreement between CEUS MBF measures across independent sessions suggests it to be a repeatable method for assessing skeletal muscle perfusion over time, and therefore facilitates wider use in longitudinal studies.

Project description:Focused ultrasound (FUS) in combination with systemically injected microbubbles can be used to non-invasively open the blood-brain barrier (BBB) in targeted regions for a variety of therapeutic applications. Over the past two decades, preclinical research into the safety and efficacy of FUS-induced BBB opening has proven this technique to be transient and efficacious, propelling FUS-induced BBB opening into several clinical trials in recent years. However, as clinical trials further progress, the neuroinflammatory response to FUS-induced BBB opening needs to be better understood. In this study, we provide further insight into the relationship of microbubble cavitation and the resulting innate immune response to FUS-induced BBB opening. By keeping ultrasound parameters fixed (i.e. frequency, pressure, pulse length, etc.), three groups of mice were sonicated using a real-time cavitation controller until a target cavitation dose was reached (1 x 107 V2•s, 5 x 107 V2•s, 1 x 108 V2•s). The change in relative gene expression of the mouse inflammatory cytokines and receptors were evaluated at three different time-points (6 h, 24 h, and 72 h) after FUS. At both 6 and 24 h time-points, significant changes in relative gene expression of inflammatory cytokines and receptors were observed across all cavitation groups. However, the degree of changes in relative expression levels and the number of genes with significant changes in expression varied across the cavitation groups. Groups with a higher cavitation dose exhibited both greater changes in relative expression levels and greater number of significant changes. By 72 h post-opening, the gene expression levels returned to baseline in all cavitation dose groups, signifying a transient inflammatory response to FUS-induced BBB opening at the targeted cavitation dose levels. Furthermore, the real-time cavitation controller was able to produce consistent and significantly different BBB permeability enhancement volumes across the three different cavitation dose groups. These results indicate that cavitation monitoring and controlling during FUS-induced BBB opening can be used to potentially modulate or limit the degree of neuroinflammation, further emphasizing the importance of implementing cavitation controllers as FUS-induced BBB opening is translated into the clinic.

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

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

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