Ultrasound can Modulate Neuronal Development: Impact on Neurite Growth and Cell Body Morphology
Yaxin Hu, Wenjing Zhong, Jennifer M.F. Wan, Alfred C.H. Yu
Ultrasound in Medicine & Biology 2013, 39, 915-925 · 10.1016/j.ultrasmedbio.2012.12.003
Abstract
Neuronal development is known to be a dynamic process that can be modulated by presenting guidance cues to neuronal cells. We show that ultrasound, when applied at pulsed settings and with intensities slightly greater than clinical diagnosis levels, can potentially act as a repulsive cue for modulating neuronal growth dynamics. Using differentiated Neuro-2a cells as the model, we have examined in vitro how neuronal development can change during and after exposure to 1-MHz ultrasound for different acoustic settings. Neurite retraction and cell body shrinkage were found in neuronal cells over a 10-min exposure period with 1.168 W/cm(2) spatial-peak, time-averaged intensity (based on 0.84 MPa peak acoustic pressure, 100-cycle pulse duration, and 500-Hz pulse repetition frequency). These effects were found to result in instances of neuronal cell body displacement. The extent of the effects was dependent on acoustic intensity, with peak acoustic pressure being a more important contributing factor compared with pulse duration. The morphological changes were found to be non-destructive, in that post-exposure neurite outgrowth and neuritogenesis were respectively observed in neurite-bearing and neurite-less neuronal cells. Our results also showed that mechanotransduction might be involved in mediating ultrasound-neuron interactions, as the morphological changes were suppressed if stretch-activated ion channels were blocked or if calcium messenger ions were chelated. Overall, these findings suggest that ultrasound can potentially influence how neuronal cells develop through modifying their cytomechanical characteristics.
Abstract via europepmc.
Exposures
Exposure 1: 1 MHz pulsed ultrasound exposure of differentiated Neuro-2a cells
Target: cultured neurons — “differentiated Neuro-2a (N2a) neuroblastoma cells”
Device: custom-built · Wuxi Beisheng Technology ✓
| Waveform | pulsed | |
|---|---|---|
| Fundamental frequency (kHz) | 1,000 | ✓✓✓ |
| Pulse duration (ms) | 0.02, 0.1swept | ✓✓✓ |
| Pulse repetition frequency (Hz) | 500, 2,500swept | ✓✓✓ |
| Duty cycle (%) | 1, 5swept | ✓✓✓ |
| Sonication duration (s) | 300, 600swept | ✓✓✓⚑ |
| Free-field pressure (kPa) | 130, 400, 840swept | ✓✓✓ |
|---|---|---|
| Free-field Isppa (W/cm²) | not reported | |
| Free-field Ispta (W/cm²) | 0.028, 0.234, 0.266, 1.17swept | ✓✓✓⚑ |
| In-situ estimate | not applicable | |
| In-situ pressure (kPa) | not applicable | |
| In-situ Isppa (W/cm²) | not applicable | |
| In-situ Ispta (W/cm²) | not applicable |
Cell dishes were exposed to 1-MHz pulsed ultrasound at a 45-degree incidence angle for 10 min per acoustic setting (main experiments) or 5 min (ion-channel/calcium mechanism experiments), using three peak rarefactional pressures (0.13, 0.40, 0.84 MPa), pulse durations of 20 or 100 cycles and PRFs of 500 or 2500 Hz, giving duty cycles of 1% or 5% and SPTA intensities between 0.028 and 1.168 W/cm2; imaging was recorded from the start of exposure to 100 min after the end of exposure.
Flags from extraction
n_subjects— Paper reports N=4 independent cell-culture groups of 250 cells per acoustic condition, but many different acoustic conditions (intensity, PD/PRF, mechanism-blocking) were each tested with their own N=4 groups; no single study-wide total number of culture replicates is given.n_sessions_per_subject— Not explicitly stated; some cell groups received a single exposure while at least one group (Fig. 4) was sonicated sequentially at two intensities in the same session, so a single session count could not be determined.sham_type— Figure 5 legend refers to a 'sham control' but the Methods do not describe how the sham/no-ultrasound control condition was implemented.exposures[0].timing.sonication_duration_s— Most experiments used a 10-min exposure; a shorter 5-min exposure duration was used specifically for the stretch-activated channel/calcium mechanism experiments (Fig. 7).exposures[0].free_field.ispta_w_cm2— 0.028 and 1.168 W/cm2 are the stated overall range; 0.234 and 0.266 W/cm2 are additional specific SPTA values used in Figure 5b/4 for comparing pressure vs. PRF/PD contributions.exposures[0].in_situ— This is an in vitro dish exposure with no skull or tissue path; in_situ fields are set to null per protocol even though the paper separately characterizes an 'in situ reflected pressure' as part of acoustic field calibration (a reflection artifact check, not a reported delivered dose).