Abstract
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 a 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 nonoscillating and oscillating (periods 12 to 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 that was observed to propagate outward from the immediately affected cells at 7-20 microm/s over a distance >200 microm, 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.