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High-resolution fluorescence-guided transcranial ultrasound mapping in the live mouse brain

Hector Estrada, Justine Robin, Ali Özbek, Zhenyue Chen, Anne Marowsky, Quanyu Zhou, Daniel Beck, Beau le Roy, Michael Arand, Shy Shoham, Daniel Razansky

Science Advances 2021, 7 · 10.1126/sciadv.abi5464

rodenthealthycellular imaginghistology molecular

Abstract

Understanding the physiological impact of transcranial ultrasound in rodent brains may offer an important preclinical model for human scale magnetic resonance–guided focused ultrasound methods. However, precision tools for high-resolution transcranial ultrasound targeting and real-time in vivo tracking of its effects at the mouse brain scale are currently lacking. We report a versatile bidirectional hybrid fluorescence-ultrasound (FLUS) system incorporating a 0.35-mm precision spherical-phased array ultrasound emission with a fiberscope-based wide-field fluorescence imaging. We show how the marriage between cortex-wide functional imaging and targeted ultrasound delivery can be used to transcranially map previously undocumented localized fluorescence events caused by reversible thermal processes and perform high-speed large-scale recording of neural activity induced by focused ultrasound. FLUS thus naturally harnesses the extensive toolbox of fluorescent tags and ultrasound’s localized bioeffects toward visualizing and causally perturbing a plethora of normal and pathophysiological processes in the living murine brain.

Abstract via europepmc.

Speciesmouse (C57BL/6J-Tg(Thy1-GCaMP6f))
Subjects21 animals
Sessions per subjectnot reported
Randomisednot reported
Blindingnot reported
Sham / controlnone
Auditory controlnot reported
Readout timingboth
Anaesthesiaanaesthetised
Readoutscellular imaging, histology molecularwide-field epifluorescence calcium imaging (GCaMP6f); functional connectivity analysis; DAPI, TUNEL, Fluoro-Jade C, c-Fos, CD68, GFAP/podocalyxin immunohistochemistry
Direction of effectmixed or unclearFluorescence dips reflected a local thermal effect, not neural activity; functional connectivity was unchanged after 20 sonications at 3 MPa (sequence 1) but decreased after a higher-pressure sonication (3.9 MPa) to n=3 mice, associated with blood-brain-barrier disruption; higher pressures also triggered cortical spreading depolarization.
Adverse eventsobservedNo signs of brain damage were found for 3-MPa focal pressures (sequence 1); at higher pressures histology showed enhanced IgG immunolabeling, perivascular microglia and astrocytic gliosis indicating blood-brain-barrier disruption and local inflammation, and cortical spreading depolarization was triggered.

Exposures

Exposure 1: Transcranial FUS to mouse cortex (sequences 1-3)

Target: cerebral cortex — “cortical locations (mouse cortex)
Device: IGT / Imasonic · Imasonic

Pulse timing
Waveformcontinuous
Fundamental frequency (kHz)3,000✓✓
Pulse duration (ms)not applicable
Pulse repetition frequency (Hz)not applicable
Duty cycle (%)not applicable
Sonication duration (s)0.1, 0.15, 0.3swept?
Pressure and intensity, by domain
Free-field pressure (kPa)not reported
Free-field Isppa (W/cm²)not reported
Free-field Ispta (W/cm²)not reported
In-situ estimatemeasurementmean or range across subjects
In-situ pressure (kPa)3,000, 4,800swept✓✓
In-situ Isppa (W/cm²)284, 724swept✓✓
In-situ Ispta (W/cm²)not reported
Protocol, in the paper’s words

Continuous transcranial sonication delivered sequentially to cortical locations. Sequence 1: 150 ms, 3 MPa, delivered 250 times across 12 mice (0% CSD probability). Sequence 2: 100-300 ms, 3-3.9 MPa, delivered 23 times across 10 mice (8.7% CSD probability). Sequence 3: 150 ms, 4.8 MPa, delivered 5 times across 3 mice (100% CSD probability). Two mice received 150-ms pulses delivered sequentially every 11 s to two cortical locations 1 mm apart; 19 other mice received the sequences in Table 1 under isoflurane anesthesia.

Flags from extraction

  • exposures[0].timing.sonication_duration_sTable 1 pairs pulse duration with pressure across three sequences (150ms/3MPa; 100-300ms/3-3.9MPa; 150ms/4.8MPa); flattening into lists loses this pairing and the CSD-probability association; consult Table 1 directly.
  • exposures[0].in_situ.pressure_kpaPressure/intensity were measured with a hydrophone through an excised skull sample; treated here as in_situ though it is an ex vivo skull measurement rather than an in vivo measurement.
  • n_sessions_per_subjectNot clearly stated how many separate sonication sessions each individual mouse received.
  • direction_of_effectThis is a bioeffects/thermal characterization study rather than a neuromodulation study; excitatory/inhibitory classification does not map cleanly.