To investigate the effects of sonoporation, spatiotemporal evolution of ultrasound-induced changes in intracellular calcium ion concentration ([Ca 2+ ] i ) was determined using real time fura-2AM fluorescence imaging. Monolayers of Chinese hamster ovary (CHO) cells were exposed to 1-MHz ultrasound tone burst (0.2 s, 0.45 MPa) in the presence of Optison ™ microbubbles. At extracellular [Ca 2+ ] o of 0.9 mM, ultrasound application generated both non-oscillating and oscillating (periods 12-30 s) transients (changes of [Ca 2+ ] i in time) with durations of 100-180 s. Immediate [Ca 2+ ] i transients after ultrasound application were induced by ultrasound-mediated microbubble-cell interactions. In some cases, the immediately-affected cells did not return to pre-ultrasound equilibrium [Ca 2+ ] i levels, thereby indicating irreversible membrane damage. Spatial evolution of [Ca 2+ ] i in different cells formed a calcium wave and was observed to propagate outward from the immediately-affected cells at 7-20 μm/s over a distance greater than 200 μm, causing delayed transients in cells to occur sometimes 60 s or more after ultrasound application. In calcium-free solution, ultrasound-affected cells did not recover, consistent with the requirement of extracellular Ca 2+ for cell membrane recovery subsequent to sonoporation. In summary, ultrasound application in the presence of Optison ™ microbubbles can generate transient [Ca 2+ ] i changes and oscillations at a focal site and in surrounding cells via calcium waves that last longer than the ultrasound duration and spread beyond the focal site. These results demonstrate the complexity of downstream effects of sonoporation beyond the initial pore formation and subsequent diffusion-related transport through the cellular membrane.

This study investigated the effects of ultrasound on the intracellular [Ca(2+)] of Chinese hamster ovary cells in the presence of albumin-encapsulated Optison microbubbles. Cells were exposed to 1 MHz ultrasound (tone burst of 0.2 s duration, 0.45 MPa peak pressure) while immersed in solution of 0.9 mM Ca(2+). Calcium imaging of the cells was performed using digital video fluorescence microscopy and Ca(2+)-indicator dye fura-2AM. Experimental evidence indicated that ultrasound caused a direct microbubble-cell interaction resulting in the breaking and eventual dissolution of the microbubble and concomitant permeabilization of the cells to Ca(2+). These cells exhibited a large influx of Ca(2+) over 3-4 s and did not return to their equilibrium levels. Subsequently, some cells exhibited one or more Ca(2+) oscillations with the onset of oscillations delayed by 10-80 s after the ultrasound pulse. A variety of oscillations were observed including decaying oscillations returning to the baseline value over 35-100 s, oscillations superimposed on a more gradual recovery over 150-200 s, and oscillations continued with increased amplitude caused by a second ultrasound tone burst. The delays in onset appeared to result from calcium waves that propagated across the cells after the application of the ultrasound pulse.

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