Transcranial Low-Intensity Pulsed Ultrasound Stimulation Induces Neuronal Autophagy
Xiaowei Huang, Lili Niu, Long Meng, Zhengrong Lin, Wei Zhou, Xiufang Liu, Jiqing Huang, Derek Abbott, Hairong Zheng
IEEE Transactions on Ultrasonics, Ferroelectrics, and Frequency Control 2021 · 10.1109/tuffc.2020.3028619
Abstract
Autophagy, or cellular self-digestion, is an essential process for eliminating abnormal protein in mammalian cells. Accumulating evidence indicates that increased neuronal autophagy has a protective effect on neurodegenerative disorders. It has been reported that low-intensity pulsed ultrasound (LIPUS) can noninvasively modulate neural activity in the brain. Yet, the effect of LIPUS on neuronal autophagy is still unclear. The objective of this study was to examine whether LIPUS stimulation could induce neuronal autophagy. Primary neurons were treated by LIPUS with a frequency of 0.68 MHz, a pulse repetition frequency (PRF) of 500 Hz, a spatial peak temporal-average intensities ( [Formula: see text]) of 70 and 165 mW/cm 2 . Then, the immunofluorescent analysis of LC3B was carried out for evaluating neuronal autophagy. Furthermore, 0.5-MHz LIPUS was noninvasively delivered to the cortex and hippocampus of adult mice ( n = 16 ) with PRF of 500 Hz and [Formula: see text] of 235 mW/cm 2 . The LC3BII/LC3BI ratio and p62 (autophagic markers) were measured by western blot analysis. In the in vitro study, the expression of LC3B in primary neurons was statistically improved after LIPUS stimulation was implemented for 4 h ( ). With the increase in the irradiation duration or acoustic intensity of LIPUS stimulation, the expression of LC3B in primary neurons was increased. Furthermore, transcranial LIPUS stimulation increased the LC3BII/LC3BI ratio ( ) and decreased the expression of p62 ( ) in the cortex and hippocampus. We concluded that LIPUS provides a safe and capable tool for activating neuronal autophagy in vitro and in vivo.
Abstract via europepmc.
Exposures
Exposure 1: Cultured neurons, 0.68 MHz
Target: cultured neurons — “primary cultured neurons”
Device: custom-built
| Waveform | pulsed | |
|---|---|---|
| Fundamental frequency (kHz) | 680 | ✓✓✓ |
| Pulse duration (ms) | not reportedimplied by duty cycle ÷ PRF: 0.1 ms (not stated by the paper) | ⚑ |
| Pulse repetition frequency (Hz) | 500 | ✓✓✓ |
| Duty cycle (%) | 5 | ✓✓✓ |
| Sonication duration (s) | 1 | ✓✓✓ |
| Free-field pressure (kPa) | not reported | |
|---|---|---|
| Free-field Isppa (W/cm²) | not reported | |
| 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 | |
| Ispta, domain unspecified (W/cm²) | 0.07, 0.165swept | ✓✓✓⚑ |
Sonication duration of 1 s, interstimulus interval of 1 s, duty cycle 5%, PRF 500 Hz, delivered through the bottom of the culture chamber in a 37 C incubator for 4 or 6 h.
Exposure 2: Mouse cortex and hippocampus, 0.5 MHz
Target: cerebral cortex, hippocampus — “mice cortex and hippocampus”
Device: other named manufacturer · NdtXducer, Northborough, MA, USA · IBP0.506 ✓
| Waveform | pulsed | |
|---|---|---|
| Fundamental frequency (kHz) | 500 | ✓✓✓ |
| Pulse duration (ms) | not reportedimplied by duty cycle ÷ PRF: 0.1 ms (not stated by the paper) | ⚑ |
| Pulse repetition frequency (Hz) | 500 | ✓✓✓ |
| Duty cycle (%) | 5 | ✓✓✓ |
| Sonication duration (s) | 1 | ✓✓✓⚑ |
| Free-field pressure (kPa) | not reported | |
|---|---|---|
| Free-field Isppa (W/cm²) | not reported | |
| Free-field Ispta (W/cm²) | not reported | |
| In-situ estimate | not reportedsingle value | |
| In-situ pressure (kPa) | 340 | ✓✓✓⚑ |
| In-situ Isppa (W/cm²) | not reported | |
| In-situ Ispta (W/cm²) | 0.235 | ✓✓✓ |
Sonication duration of 1 s, interstimulus interval of 1 s, duty cycle 5%, PRF 500 Hz, transcranially for 4 h (40 min for the c-Fos experiment). Sham mice had the same treatment with the ultrasound switched off.
Flags from extraction
n_subjects— 28 mice in sham and LIPUS groups; number of cultures for the in vitro experiments is not stated.exposures[0].timing.pulse_duration_ms— Pulse duration not stated; not computed.exposures[1].timing.pulse_duration_ms— Pulse duration not stated; not computed.exposures[1].in_situ.method— Intensity is given after skull attenuation of about 35%, but how it was obtained (measurement or derating) is not said.exposures[1].in_situ.pressure_kpa— 0.34 MPa peak negative pressure is described as corresponding to the 235 mW/cm2 in-skull intensity.exposures[0].unspecified_domain.ispta_w_cm2— Intensities (70 and 165 mW/cm2) are stated after about 10% chamber-bottom attenuation; converted to W/cm2.exposures[0].device.family— Transducer fabricated in the authors' lab; no maker named.exposures[1].timing.sonication_duration_s— Per-sonication 1 s with 1 s interstimulus interval, total 4 h session.