Modulation of intracellular Ca2+ concentration in brain microvascular endothelial cells in vitro by acoustic...

Localized delivery of therapeutic agents through the blood-brain barrier (BBB) is a clinically significant task that remains challenging. Ultrasound (US) application after intravenous administration of microbubbles has been shown to generate localized BBB opening in animal models but the detailed mechanisms are not yet fully described. The current study investigates the effects of US-stimulated microbubbles on in vitro murine brain microvascular endothelial (bEnd.3) cells by monitoring sonoporation and changes in intracellular calcium concentration ([Ca(2+)](i)) using real-time fluorescence and high-speed brightfield microscopy. Cells seeded in microchannels were exposed to a single US pulse (1.25 MHz, 10 cycles, 0.24 MPa peak negative pressure) in the presence of Definity microbubbles and extracellular calcium concentration [Ca(2+)](o) = 0.9 mM. Disruption of the cell membrane was assessed using propidium iodide (PI) and change in the [Ca(2+)](i) was measured using fura-2. Cells adjacent to a microbubble exhibited immediate [Ca(2+)](i) changes after US pulse with and without PI uptake and the [Ca(2+)](i) changes were twice as large in cells with PI uptake. Cell viability assays showed that sonoporated cells could survive with modulation of [Ca(2+)](i) and uptake of PI. Cells located near sonoporated cells were observed to exhibit changes in [Ca(2+)](i) that were delayed from the time of US application and without PI uptake. These results demonstrate that US-stimulated microbubbles not only directly cause changes in [Ca(2+)](i) in brain endothelial cells in addition to sonoporation but also generate [Ca(2+)](i) transients in cells not directly interacting with microbubbles, thereby affecting cells in larger regions beyond the cells in contact with microbubbles.