Focused Ultrasound Enhances Glymphatic Transport Robustly Across Anesthesia Levels.

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. 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 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. 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.