Direct and safe manipulation of neurons by external means is an increasingly studied therapeutic modality with the potential to treat many neurological diseases. Anticipating such future applications, we investigated reversible bioeffects of very low dose focused ultrasound on neuronal cell morphology and function in vitro. To test morphological changes, undifferentiated PC12 cells were serum-cultured. The culture plates were placed on an inverted optical microscope. An f/1.1 ultrasound transducer with a water-filled coupling cone was focused on the culture and excited with 30-ms 4.67-MHz 100-kPa pulses. To test functional changes, rat hippocampal slices were cultured and individually transferred to the well of a 60-channel multi electrode array. An f/2.1 ultrasound transducer with a water-filled coupling cone was focused on a culture and excited with 100-Ps 4.04-MHz 77-kPa pulses. The culture was stimulated before and after the ultrasonic stimulus with a 100-Ps 100-PA biphasic electrical stimulus. Optical microscopy of PC12 cultures under insonification revealed that cells that were clustered near the ultrasound focal region elongated by approximately 2 Pm during insonification and returned to approximately their original shapes following insonification. We conclude that the acoustic radiation force is capable of reversibly deforming cultured cells. In the rat hippocampal cultures, the ultrasonically and electrically evoked responses exhibited similar biphasic waveforms. In addition, robust electrically evoked responses following insonification indicated that the insonified cultures remained viable. We conclude that low-dose ultrasound can stimulate neurons; the mechanism is currently under investigation.

To understand and prevent brain injuries from head trauma, researchers study mechanically stressed neuronal tissue. To anticipate future application of controllable ultrasonic in-vivo stress, we investigated the effects of acoustic radiation force on in-vitro PC12 cells. Undifferentiated PC12 cells were serum-cultured in DMEM/F12 on poly-L-lysine-coated polystyrene. Some cultures were DAPI stained. The culture plates were placed on an inverted phase-contrast microscope. An f/1.1 ultrasound transducer with a water-filled coupling cone was focused on the culture at a 45-degree angle-of-incidence, and excited with 30-ms 4.7-MHz pulses. Acoustic power was 8 W, and peak pressure was estimated at 300 kPa. Digital images were recorded before, during, and after insonification. Incident-light and fluorescence images revealed three populations: cells that were stationary (apparently outside the effective force field region), cells that elongated about 2 µm under radiation force and returned to approximately their original shapes when the force was removed (apparently adhered to the substrate), and cells that moved about 50 µm with each pulse and did not return (apparently free-floating). We conclude that cell morphology can be influenced reversibly with acoustic radiation force.