Unmyelinated Peripheral Nerves Can Be Stimulated in Vitro Using Pulsed Ultrasound
Christopher J. Wright, Seyyed R. Haqshenas, John Rothwell, Nader Saffari
Ultrasound in Medicine & Biology 2017, 43, 2269-2283 · 10.1016/j.ultrasmedbio.2017.05.008
Abstract
Appreciation for the medical and research potential of ultrasound neuromodulation is growing rapidly, with potential applications in non-invasive treatment of neurodegenerative disease and functional brain mapping spurring recent progress. However, little progress has been made in our understanding of the ultrasound-tissue interaction. The current study tackles this issue by measuring compound action potentials (CAPs) from an ex vivo crab walking leg nerve bundle and analysing the acoustic nature of successful stimuli using a passive cavitation detector (PCD). An unimpeded ultrasound path, new acoustic analysis techniques and simple biological targets are used to detect different modes of cavitation and narrow down the candidate biological effectors with high sensitivity. In the present case, the constituents of unmyelinated axonal tissue alone are found to be sufficient to generate de novo action potentials under ultrasound, the stimulation of which is significantly correlated to the presence of inertial cavitation and is never observed in its absence.
Abstract via europepmc.
Exposures
Exposure 1: 0.67 MHz pulsed ultrasound to crab nerve bundle (8 ms, 100 ms and 300 ms protocols)
Target: excised nerve — “crab walking leg nerve bundle (unmyelinated axons)”
Device: other named manufacturer · Precision Acoustics · PA409 ✓
| Waveform | pulsed | |
|---|---|---|
| Fundamental frequency (kHz) | 670 | ✓✓✓ |
| Pulse duration (ms) | not reported | ⚑ |
| Pulse repetition frequency (Hz) | 10,000, 500swept | ✓✓✓ |
| Duty cycle (%) | 50 | ✓✓✓ |
| Sonication duration (s) | 0.008, 0.1, 0.3swept | ✓✓✓⚑ |
| Free-field pressure (kPa) | 800, 1,600, 1,800, 2,200, 2,400, 2,900, 3,200, 3,600, 4,000, 4,300, 4,700swept | ✓?⚑ |
|---|---|---|
| Free-field Isppa (W/cm²) | 20, 76, 100, 140, 169, 230, 274, 352, 419, 485, 562swept | ✓✓✓ |
| Free-field Ispta (W/cm²) | not reported | |
| 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 |
The primary stimulus protocol was 80 pulses of 0.67-MHz driving frequency at 10-kHz PRF, 50% duty cycle, over an 8-ms total stimulus duration (TSD). A second set of experiments used the same parameters but with a 100-ms TSD (1000 pulses) to test longer exposures. A third variant reproduced parameters from an in vivo CNS neurostimulation study (250-kHz fundamental, 500-Hz PRF, 50% duty cycle, 300 ms duration, 3 s between stimuli) at 0.67 MHz; this did not evoke responses until intensity was raised to the same 169 W/cm2 threshold as the 100-ms protocol.
Exposure 2: 1.1 MHz pulsed ultrasound to crab nerve bundle, radiation-force matched to 0.67 MHz
Target: excised nerve — “crab walking leg nerve bundle (unmyelinated axons)”
Device: Sonic Concepts · Sonic Concepts · H-101-MR ✓
| Waveform | pulsed | |
|---|---|---|
| Fundamental frequency (kHz) | 1,100 | ✓✓✓ |
| Pulse duration (ms) | not reportedimplied by duty cycle ÷ PRF: 0.05 ms (not stated by the paper) | |
| Pulse repetition frequency (Hz) | 10,000 | ✓✓✓ |
| Duty cycle (%) | 50 | ✓✓✓ |
| Sonication duration (s) | 0.008 | ✓✓✓ |
| Free-field pressure (kPa) | 700, 1,700, 2,200, 2,800, 3,600, 4,200swept | ✓?⚑ |
|---|---|---|
| Free-field Isppa (W/cm²) | 17, 84, 143, 232, 353, 475swept | ✓✓✓ |
| Free-field Ispta (W/cm²) | not reported | |
| 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 |
The 1.1-MHz exposure used the same 10-kHz PRF, 50% duty cycle, 8-ms TSD protocol as the primary 0.67-MHz stimulus, with intensities recalculated to match the acoustic radiation forces produced at 0.67 MHz. No nerve responses were observed at any of these intensities.
Exposure 3: 2 MHz pulsed ultrasound to crab nerve bundle, radiation-force matched to 0.67 MHz
Target: excised nerve — “crab walking leg nerve bundle (unmyelinated axons)”
Device: Sonic Concepts · Sonic Concepts · H-106 ✓
| Waveform | pulsed | |
|---|---|---|
| Fundamental frequency (kHz) | 2,000 | ✓✓✓ |
| Pulse duration (ms) | not reportedimplied by duty cycle ÷ PRF: 0.05 ms (not stated by the paper) | |
| Pulse repetition frequency (Hz) | 10,000 | ✓✓✓ |
| Duty cycle (%) | 50 | ✓✓✓ |
| Sonication duration (s) | 0.008 | ✓✓✓ |
| Free-field pressure (kPa) | 500, 1,300, 1,800, 2,400, 3,000, 3,600swept | ✓?⚑ |
|---|---|---|
| Free-field Isppa (W/cm²) | 12, 61, 103, 167, 255, 343swept | ✓✓✓ |
| Free-field Ispta (W/cm²) | not reported | |
| 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 |
The 2-MHz exposure used the same 10-kHz PRF, 50% duty cycle, 8-ms TSD protocol as the primary 0.67-MHz stimulus, with intensities recalculated to match the acoustic radiation forces produced at 0.67 MHz. No nerve responses were observed at any of these intensities.
Flags from extraction
exposures[0].timing.pulse_duration_ms— PRF (10 kHz) and duty cycle (50%) imply an individual pulse width of ~0.05 ms, but a figure caption (Fig. 4) separately describes '0.5-ms pulses at 50% DC', an order of magnitude different; the discrepancy could not be resolved from the main text, so pulse duration is left as not_reported.exposures[0].free_field.pressure_kpa— Table 2 reports separate peak negative and peak positive pressures at each intensity; only the peak positive pressure is recorded here as pressure_kpa.exposures[1].free_field.pressure_kpa— Table 2 reports separate peak negative and peak positive pressures at each intensity; only the peak positive pressure is recorded here as pressure_kpa.exposures[2].free_field.pressure_kpa— Table 2 reports separate peak negative and peak positive pressures at each intensity; only the peak positive pressure is recorded here as pressure_kpa.sham_type— Two distinct control experiments are described: an air-gap block of the acoustic path (raised cone) and separate nerves never exposed to ultrasound; both are grouped here as inactive_transducer.exposures[0].timing.sonication_duration_s— The 300-ms/500-Hz-PRF variant reproducing Lee et al. (2015) parameters is included as a third list value even though its PRF (500 Hz) differs from the 10-kHz PRF used for the 8-ms and 100-ms protocols; see protocol_description.