Focused Ultrasound Enhances Glymphatic Transport Robustly Across Anesthesia Levels
Haijun Xiao, Abhijith Sreejith, Iylan Howson, Alexander Aviles Cruz, Taylor Key, Jeffrey J. Iliff, Muna Aryal
Ultrasound in Medicine & Biology 2025, 51, 1701-1709 · 10.1016/j.ultrasmedbio.2025.06.009
Abstract
Objective We recently discovered that low-intensity transcranial focused ultrasound (FUS) can enhance glymphatic influx, the process by which the brain clears metabolic waste, in a rat model. The hypothesis is that ultrasound influences convective forces from arterial pulsations, aiding glymphatic transport. The initial study was conducted under approximately 2.5% isoflurane anesthesia, but it remains unclear how different anesthesia levels affect ultrasonic glymphatic influx as glymphatic function varies with physiological states. Hence, establishing standardized protocols for ultrasonic enhancement of glymphatic influx is crucial for ensuring consistent, reliable results across research labs during preclinical development. We aimed to investigate how different levels of isoflurane anesthesia affect ultrasonic enhancement of glymphatic influx for a model tracer. Methods FUS (650 kHz, 0.2 MPa, duty cycle 7.7%, brain-wide for 10 min) was applied to rats under anesthesia levels ranging from 1.5% to 3.0%. A model tracer, 1 kDa-IRDye800 was used to assess glymphatic influx in ex-vivo brain. Results Results demonstrated a significant main effect of FUS treatment (p = 7.70 × 10⁻⁷), indicating that FUS significantly enhanced glymphatic transport acoss all anesthesia levels. Anesthesia level also had a significant effect on glymphatic clearance (p = 7.45 × 10⁻¹¹), consistent with prior reports linking anesthetic depth to cerebrospinal fluid dynamics. However, the interaction between treatment and anesthesia was not statistically significant (p = 0.106), suggesting that the efficacy of FUS was consistent across anesthesia levels. Conclusion These findings support the robustness of brain-wide FUS-induced glymphatic enhancement and highlight its potential as a modifiable therapeutic tool, independent of anesthetic conditions.
Abstract via europepmc.
Exposures
Exposure 1: Brain-wide grid-pattern FUS for glymphatic transport enhancement
Target: whole brain or unfocused — “entire brain (non-targeted, brain-wide grid-pattern sonication)”
Device: other named manufacturer · Image Guided Therapy ✓
| Waveform | pulsed | |
|---|---|---|
| Fundamental frequency (kHz) | 650 | ✓✓✓ |
| Pulse duration (ms) | not reported | |
| Pulse repetition frequency (Hz) | not reported | |
| Duty cycle (%) | 7.7 | ✓✓✓ |
| Sonication duration (s) | 840 | ✓✓✓⚑ |
| Free-field pressure (kPa) | not reported | |
|---|---|---|
| Free-field Isppa (W/cm²) | not reported | |
| Free-field Ispta (W/cm²) | not reported | |
| In-situ estimate | deratingsingle value | |
| In-situ pressure (kPa) | 200 | ✓✓✓ |
| In-situ Isppa (W/cm²) | not reported | |
| In-situ Ispta (W/cm²) | not reported |
A 650 kHz spherically focused transducer was swept across the entire rat brain in a grid pattern (six 3 mm x 14 mm sub-loops, START (0,0) to STOP (18,14)), with each loop taking ~27.12 s plus a 50 ms pause, for a total of 30 loops per rat. Sham procedures used the identical trajectory with zero input power. Rats were under 1.5%, 2%, or 3% isoflurane anesthesia during sonication, beginning 10 min after intrathecal tracer injection.
Exposure 2: In-vivo calibration: focal vascular barrier opening validation
Target: other — “focal targeted brain region used to validate transducer targeting/coupling (region not further specified)”
Device: other named manufacturer · Image Guided Therapy ✓
| Waveform | pulsed | |
|---|---|---|
| Fundamental frequency (kHz) | 650 | ✓✓✓ |
| Pulse duration (ms) | not reported | |
| Pulse repetition frequency (Hz) | 1 | ✓✓✓⚑ |
| Duty cycle (%) | not reported | |
| 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 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) | 350 | ✓✓✓ |
To confirm the ultrasound field's in-vivo targeting/coupling, a vascular barrier opening assay was performed with an established ultrasound pulse (650 kHz, 1 PRF, 1 min, 0.35 MPa) combined with an ultrasound contrast agent (Optison), in 5 rats designated for calibration.
Flags from extraction
exposures[0].timing.sonication_duration_s— Abstract/results state the ultrasound exposure lasted 10 min, but the methods describe the grid-sweep trajectory (30 loops) taking approximately 14 min to complete; both durations are recorded as a list due to this text-text discrepancy.exposures[0].in_situ.method— The 0.2 MPa in-situ pressure is based on an assumed 30% pressure insertion loss for skull attenuation rather than a direct measurement through skull in this cohort; classified as derating.n_subjects— Represents the four groups (3 anesthesia-level glymphatic FUS+ groups of n=5 each, plus n=5 calibration animals) that received active ultrasound; paper gives only group sizes (Table 1), not an overall total exposed to ultrasound.exposures[1].timing.pulse_repetition_frequency_hz— Text states '1PRF' for the calibration pulse; interpreted as PRF = 1 Hz, but this notation is ambiguous.exposures[1].target— The specific brain region targeted for the vascular-barrier-opening calibration assay is not explicitly named in the text (only 'targeted brain regions').