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Noninvasive Ultrasound Stimulation of Ventral Tegmental Area Induces Reanimation from General Anaesthesia in Mice

Tianyuan Bian, Wen Meng, Meihong Qiu, Zhigang Zhong, Zhengrong Lin, Junjie Zou, Yibo Wang, Xiaowei Huang, Lisheng Xu, Tifei Yuan, Zhili Huang, Lili Niu, Long Meng, Hairong Zheng

Research 2021, 2021 · 10.34133/2021/2674692

rodentdisorders of consciousnesstraumatic brain injurybehaviourhistology molecular

Abstract

Evidence in animals suggests that deep brain stimulation or optogenetics can be used for recovery from disorders of consciousness (DOC). However, these treatments require invasive procedures. This report presents a noninvasive strategy to stimulate central nervous system neurons selectively for recovery from DOC in mice. Through the delivery of ultrasound energy to the ventral tegmental area, mice were aroused from an unconscious, anaesthetized state in this study, and this process was controlled by adjusting the ultrasound parameters. The mice in the sham group under isoflurane-induced, continuous, steady-state general anaesthesia did not regain their righting reflex. On insonation, the emergence time from inhaled isoflurane anaesthesia decreased (sham: 13.63 ± 0.53 min, ultrasound: 1.5 ± 0.19 min, p p p < 0.01). This noninvasive strategy could be used on demand to promote emergence from DOC and may be a potential treatment for such disorders.

Abstract via europepmc.

Speciesmouse (C57BL/6J)
Subjects8, 6, 6, 4, 8swept animals
Sessions per subjectnot reported
Randomisedyes
Blindingsingle
Sham / controlinactive transducer
Auditory controldeafened subjects
Readout timingonline
Anaesthesiaboth
Readoutsbehaviour, histology moleculararousal behavioral scoring (head/whisker/leg/tail movement, righting reflex), open field test, c-Fos/tyrosine hydroxylase immunohistochemistry, H&E and Nissl staining
Direction of effectexcitatoryUltrasound stimulation of the VTA increased c-Fos expression, induced arousal/righting reflex recovery, shortened emergence time from isoflurane anaesthesia, and reduced recovery time after TBI; the D1 antagonist SCH-23390 blocked the arousal response, indicating a dopaminergic excitatory mechanism.
Adverse eventsnone observedH&E and Nissl staining showed no abnormal findings (haemorrhaging or tissue damage) in the VTA of the ultrasound group compared with sham; mechanical index was about 0.3 and estimated brain temperature rise only 0.1°C, within clinical ultrasound imaging safety guidelines.

Exposures

Exposure 1: VTA sonication (arousal/reanimation experiments)

Target: ventral tegmental area — “Collimators to focus ultrasound to the VTA
Device: custom-built

Pulse timing
Waveformpulsed
Fundamental frequency (kHz)3,300✓✓
Pulse duration (ms)1✓✓
Pulse repetition frequency (Hz)500✓✓
Duty cycle (%)50pulse duration × PRF gives 50%✓✓
Sonication duration (s)1✓✓
Pressure and intensity, by domain
Free-field pressure (kPa)360, 586, 758swept✓✓
Free-field Isppa (W/cm²)not reported
Free-field Ispta (W/cm²)not reported
In-situ estimatenot reported
In-situ pressure (kPa)not reported
In-situ Isppa (W/cm²)not reported
In-situ Ispta (W/cm²)not reported
Isppa, domain unspecified (W/cm²)7.94✓✓
Protocol, in the paper’s words

Ultrasound stimulation was initiated after 40 min of anaesthesia and continued for two doses of 30 min or until the righting reflex behavior returned, with an interstimulus interval (ISI) of 2 s. In the TBI experiment, 15 min of US stimulation was given to each injured mouse 15 min after injury.

Exposure 2: V1 sonication (control target)

Target: primary visual cortex — “V1 (related to the bregma: 1.10 mm anterior/posterior, −1.50 mm medial/lateral, and −0.50 mm dorsal/ventral)
Device: custom-built

Pulse timing
Waveformpulsed
Fundamental frequency (kHz)3,300✓✓
Pulse duration (ms)1✓✓
Pulse repetition frequency (Hz)500✓✓
Duty cycle (%)50pulse duration × PRF gives 50%✓✓
Sonication duration (s)1✓✓
Pressure and intensity, by domain
Free-field pressure (kPa)586✓✓
Free-field Isppa (W/cm²)not reported
Free-field Ispta (W/cm²)not reported
In-situ estimatenot reported
In-situ pressure (kPa)not reported
In-situ Isppa (W/cm²)not reported
In-situ Ispta (W/cm²)not reported
Protocol, in the paper’s words

We repeated the arousal experiment with US stimulation of the primary visual cortex (V1) to demonstrate the specificity of US stimulation, using the same stimulation protocol as for the VTA.

Flags from extraction

  • n_subjectsMultiple sub-experiments report different ultrasound-exposed group sizes (c-Fos n=6, dose-response n=8 per pressure, antagonist NS-US n=4, V1-comparison n=6, TBI+US n=8); the paper never states a combined total and cohort overlap across experiments is unclear, so group sizes are listed rather than summed.
  • exposures[0].free_field.pressure_kpaThree acoustic pressures (360, 586, 758 kPa) were tested to find behavioral thresholds; 586 kPa was used for the main follow-on experiments.
  • exposures[0].unspecified_domain.isppa_w_cm2This ISPPA value (7.94 W/cm2) is mentioned in the Discussion as the intensity used in the study compared to a regulatory limit, but is not clearly labeled free-field or in-situ.
  • exposures[0].in_situ.pressure_kpaPaper reports that skull/dura attenuation was about 54% and gives transcranial focus dimensions, but never states a resulting derated in-situ pressure value in kPa.
  • device.manufacturerThe custom wearable ultrasound transducer's manufacturer/model is not given; only the driving function generator and amplifier models are specified.