Focused Ultrasound Combined With Microbubbles Attenuate Symptoms in Heroin-Addicted Mice

Yuran Feng, Jiaxue Sun, Tao Wang, Yu Zheng, Yi Zhao, Youzhuo Li, Shishi Lai, Yu Xu, Mei Zhu

Ultrasound in Medicine & Biology 2024 · 10.1016/j.ultrasmedbio.2024.06.014

rodentsubstance use disorderbehaviourhistology molecularother

Abstract

Objective To explore the efficacy and mechanisms of stimulating the nucleus accumbens (NAc) in heroin-addicted mice using focused ultrasound and microbubbles (MBs). Methods The conditioned place preference (CPP) method was employed to establish a heroin-addicted mice model. Mice were randomized into control (C), heroin (H), heroin + ultrasound (H + U) and H + U + MBs. Ultrasound (2 MHz fundamental frequency, 1.34 MPa peak-negative pressure, 1 MHz pulse repetition frequency, 5% duty cycle, 15 min/d, over 2 d) was applied to stimulate the NAc in the latter 2 groups. Whereas H + U + MBs received an injection of sulfur hexafluoride MBs during the stimulation. Subsequently, CPP scores, open-field test (OFT), and elevated plus-maze test (EPMT) were conducted to assess behavioral changes in addiction memory, anxiety and exercise status. HE staining was performed to detect pathological structures. Neurotransmitters such as dopamine (DA), serotonin (5-HT) and glutamate (Glu) were detected using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Transmission electron microscopy (TEM) was used to observe ultrastructural changes of synapses in NAc. Immunohistochemistry (IHC) was utilized to detect Cleaved Caspase-3 in the NAc region. Western blotting (WB) was used to detect the protein expression of Cleaved Caspase-3, Bax and Bcl-2 in NAc. Results HE staining showed small patches of erythrocyte exudation were observed in the NAc and adjacent areas in H + U + MBs. The CPP scores of H + U + MBs were lower (p Conclusion Focused ultrasound combined with MBs stimulate the NAc can weaken the addictive memory and improve anxiety of heroin-related mice. The mechanical effect of ultrasound combined with the cavitation effect may be a potential treatment for addiction.

Abstract via europepmc.

Speciesmouse (C57BL/6J)

Subjects10 animals

Sessions per subject2

Randomisedyes

Blindingnot reported

Sham / controlinactive transducer

Auditory controlnot reported

Readout timingoffline

Anaesthesiaanaesthetised

Readoutsbehaviour, histology molecular, other — Conditioned place preference, open-field test, elevated plus maze; UPLC-MS/MS of DA, 5-HT, glutamate; HE staining, TEM, cleaved caspase-3 IHC, western blot

Direction of effectno effectUltrasound alone (H + U) produced no significant change in conditioned place preference, anxiety-like behaviour or NAc neurotransmitter levels; the effects the paper reports are from the microbubble arm, which is not recorded.

Adverse eventsnone observedHE staining showed no abnormality in the H + U group; no deaths or gait abnormalities. Erythrocyte extravasation, neuropil vacuolation and synapse loss were confined to the microbubble arm.

Exposures

Exposure 1: Focused ultrasound to left NAc (H + U group, ultrasound only)

Target: nucleus accumbens — “left NAc”
Device: other named manufacturer · Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences · portable ultrasound stimulation system with 2 MHz focused transducer ✓

Pulse timing
Waveform	pulsed
Fundamental frequency (kHz)	2,000	✓✓✓
Pulse duration (ms)	not reportedimplied by duty cycle ÷ PRF: 0.0001 ms (not stated by the paper)
Pulse repetition frequency (Hz)	1,000,000	✓✓✓⚑
Duty cycle (%)	5	✓✓✓
Sonication duration (s)	900	✓✓✓⚑

Pressure and intensity, by domain
Free-field pressure (kPa)	1,340	✓✓✓⚑
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

Protocol, in the paper’s words

2 MHz fundamental, 1.34 MPa peak-negative pressure, PRF stated as 1 MHz, 5% duty cycle, 15 min per day over 2 days under 1.5% isoflurane, with 0.2 mL saline given intravenously during stimulation. Only this ultrasound-only arm is recorded; the H + U + MBs arm received SonoVue microbubbles and fails condition D.

Flags from extraction

exposures[0].timing.pulse_repetition_frequency_hz — PRF stated as '1 MHz' (equal to 1,000,000 Hz) alongside 5% duty cycle; likely a typo for kHz or Hz. Recorded as written, converted to Hz. Pulse duration cannot be resolved.
exposures[0].free_field.pressure_kpa — 1.34 MPa PNP; hydrophone measurement in degassed water without derating is described for the sound field data, so placed in free_field.
n_subjects — n = 10 per group for behaviour, listed for the two ultrasound groups; n = 4 for HE and n = 6 for neurotransmitters, so animals analysed differ by assay.
exposures[0].device.family — Transducer bought from SIAT as an ultrasonic neurostimulator; recorded as other_manufacturer.
exposures[0].timing.sonication_duration_s — 15 min per day recorded as 900 s.

Notes: Judged out of scope: the aim is to damage the NAc using microbubbles (condition D, E4); the H+U arm without microbubbles is stimulation, but the paper's treatment arm is the microbubble group.