Very Low‐Intensity Ultrasound Facilitates Glymphatic Influx and Clearance via Modulation of the TRPV4‐AQP4 Pathway
Chueh‐Hung Wu, Wei‐Hao Liao, Ya‐Cherng Chu, Ming‐Yen Hsiao, Yi Kung, Jaw‐Lin Wang, Wen‐Shiang Chen
Advanced Science 2024 · 10.1002/advs.202401039
Abstract
Recently, the glymphatic system has been proposed as a mechanism for waste clearance from the brain parenchyma. Glymphatic dysfunction has previously been shown to be associated with several neurological diseases, including Alzheimer's disease, traumatic brain injury, and stroke. As such, it may serve as an important target for therapeutic interventions. In the present study, very low-intensity ultrasound (VLIUS) (center frequency, 1 MHz; pulse repetition frequency, 1 kHz; duty factor, 1%; spatial peak temporal average intensity [I spta ] = 3.68 mW cm 2 ; and duration, 5 min) is found to significantly enhance the influx of cerebrospinal fluid tracers into the paravascular spaces of the brain, and further facilitate interstitial substance clearance from the brain parenchyma, including exogenous β-amyloid. Notably, no evidence of brain damage is observed following VLIUS stimulation. VLIUS may enhance glymphatic influx via the transient receptor potential vanilloid-4-aquaporin-4 pathway in astrocytes. This mechanism may provide insights into VLIUS-regulated glymphatic function that modifies the natural course of central nervous system disorders related to waste clearance dysfunction.
Abstract via europepmc.
Exposures
Exposure 1: transcranial VLIUS of mouse brain (in vivo)
Target: whole brain or unfocused — “mouse brain (planar transducer at center of brain)”
Device: Olympus / Panametrics · Olympus, Tokyo, Japan · C539-SM ✓
| Waveform | pulsed | |
|---|---|---|
| Fundamental frequency (kHz) | 1,000 | ✓✓✓ |
| Pulse duration (ms) | 0.01 | ✓✓✓⚑ |
| Pulse repetition frequency (Hz) | 1,000 | ✓✓✓ |
| Duty cycle (%) | 1pulse duration × PRF gives 1% | ✓✓✓ |
| Sonication duration (s) | 300 | ✓✓✓ |
| Free-field pressure (kPa) | not reported | |
|---|---|---|
| Free-field Isppa (W/cm²) | not reported | |
| Free-field Ispta (W/cm²) | not reported | |
| In-situ estimate | not reported | |
| In-situ pressure (kPa) | not reported | |
| In-situ Isppa (W/cm²) | not reported | |
| In-situ Ispta (W/cm²) | not reported | |
| Pressure, domain unspecified (kPa) | 98 | ✓✓✓⚑ |
| Ispta, domain unspecified (W/cm²) | 0.00092, 0.00368, 0.00585swept | ✓✓✓ |
Following intracisternal (or intrastriatal) injection, mice were immediately stimulated with VLIUS for 5 min via a planar transducer coupled to the scalp with ultrasound gel. Three intensities (Ispta 0.92, 3.68, 5.85 mW/cm2) were screened; 3.68 mW/cm2 (pressure ~98 kPa) was optimal and used for subsequent experiments. The paper notes the short pulse repetition period made the exposure appear similar to continuous wave macroscopically despite the 1% duty cycle.
Exposure 2: VLIUS of C6 glioma cells (in vitro)
Target: cultured glia — “C6 glioma cells (astrocyte-like)”
Device: Olympus / Panametrics · Olympus, Tokyo, Japan · C539-SM ✓
| Waveform | pulsed | |
|---|---|---|
| Fundamental frequency (kHz) | 1,000 | ✓✓✓ |
| Pulse duration (ms) | 0.01 | ✓✓✓ |
| Pulse repetition frequency (Hz) | 1,000 | ✓✓✓ |
| Duty cycle (%) | 1pulse duration × PRF gives 1% | ✓✓✓ |
| Sonication duration (s) | 60 | ✓✓✓ |
| 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 | |
| Pressure, domain unspecified (kPa) | 98 | ✓✓✓ |
| Ispta, domain unspecified (W/cm²) | 0.00368 | ✓✓✓ |
A planar transducer was placed directly above C6 cells in a 24-well plate; the VLIUS setting was the same as the in vivo protocol except the duration was changed to 1 min. A separate micropipette-guided ultrasound system (not further parameterised here) was also used for live-cell calcium imaging, applying a 3 s ultrasound stimulus after 10 s pretreatment.
Flags from extraction
n_subjects— Group sizes vary per experiment/figure (e.g. n=5-9 mice per arm across Figures 1-6) and the Figure 1 legend n-value ('n = 70 mice/group; two independent repeats of n = 5 mice per group') appears internally inconsistent (likely an OCR/typesetting error for n=10); no single overall total for VLIUS-exposed mice is given.exposures[0].timing.pulse_duration_ms— Pulse duration (0.01 ms = 10 microseconds) derived from the paper's explicit statement of '10 cycles/pulse' at the 1 MHz fundamental frequency (cycles per pulse ÷ frequency), an allowed synonym conversion, not from dividing duty cycle by PRF.exposures[0].unspecified_domain.pressure_kpa— Domain (free-field vs in-situ) not stated; paper elsewhere notes ~20% energy loss traversing the mouse skull, suggesting the 98 kPa value may be an unattenuated/output figure, but this is not stated explicitly.exposures[1]— A separate micropipette-guided ultrasound device (cited to Chu et al.) was used for the live-cell calcium imaging assay with a 3 s exposure; its frequency/pressure are not restated in this paper, so it is described only in protocol_description rather than as a third exposure entry.sham_type— Control mice were 'treated with or without VLIUS stimulation'; the mechanism of the control condition (transducer present but off, vs. no device at all) is not described.