Augmentation of Limb Perfusion and Reversal of Tissue Ischemia Produced by Ultrasound-Mediated Microbubble Cavitation

Abstract

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 biologic mediators. Methods and Results Contrast ultrasound perfusion imaging of hindlimb skeletal muscle and femoral artery diameter measurement were performed in non-ischemic mice after unilateral 10 min exposure to intermittent ultrasound alone (mechanical index [MI] 0.6 or 1.3) or ultrasound with lipid microbubbles (2×108 I.V.). 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-fold and 10-fold higher than control for MI 0.6 and 1.3, respectively; p<0.05), as was femoral artery dilation. Inhibition of endothelial nitric oxide synthase (eNOS) 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 (NO) production and muscle phospho-eNOS increased in a stepwise fashion by ultrasound and ultrasound with microbubbles. In mice with unilateral hindlimb ischemia (40–50% reduction in flow), ultrasound (MI 1.3) with microbubbles increased perfusion by 2-fold to a degree that was greater than the control non-ischemic 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 eNOS.

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

“…1a). After high-power US exposure, CEU perfusion imaging performed at very low power which has no effects on tissue blood flow 8 revealed a 6-fold increase in perfusion in the muscle exposed to US (Fig. 1b to f).…”

“…First, wild-type mice were pre-treated with both apyrase to promote ATP degradation and ZM241385 to inhibit adenosine A 2A and A 2B receptors, the latter of which has been shown alone to not influence flow augmentation. 8 Second, mice deficient for the major P 2 Y endothelial receptor in skeletal muscle microvasculature (P2Y 2 −/− ) were studied after treatment with ZM241385. These two approaches resulted in a similar and substantial reduction in perfusion in the US-treated limb (Fig 2a and Supplemental Fig 4), indicating that vasodilation in response to US-mediated cavitation is largely mediated by a combination of purinergic pathways.…”

“…8,42 For limb perfusion, pre-clinical studies have demonstrated that addition of microbubbles to ultrasound alone results in a >5-fold greater effect on muscle blood flow and a greater increase in arterial dilation. 8 The effects of CEU on perfusion are greatest when the acoustic power is high enough to produce inertial cavitation (exaggerated microbubble oscillation and destruction), and there is also sufficient time between ultrasound exposures to allow refill of microbubbles into the ultrasound sector.…”

“…This form of low-power imaging has been shown not to produce any detectable changes in perfusion. 8 Microbubbles were infused at a rate of 1×10 7 min −1 . Time-intensity data at a frame rate of 5 Hz were acquired after a high-power (MI 0.98) 5-frame sequence and were fit to the function: y=A(1-e βt ); 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.…”

“…5,6 The presence of gas bodies within the microcirculation in the form of encapsulated microbubble contrast agents that undergo acoustic cavitation can produce very high focal shear and markedly augment the effects of US on blood flow. 7,8 Contrast-enhanced ultrasound (CEU) with microbubbles is being investigated as a promising therapy for acute ischemic syndromes.…”