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Neuroprotective Effect of Low-Intensity Pulsed Ultrasound on the Mouse MPTP/MPP+ Model of Dopaminergic Neuron Injury

Xueying Chen, Dong Wang, Liang Zhang, Huan Yao, Hui Zhu, Nvjun Zhao, Xiaoqiong Peng, Ke Yang

Ultrasound in Medicine & Biology 2021, 47, 2321-2330 · 10.1016/j.ultrasmedbio.2021.03.034

rodentin vitro cellparkinsons diseasebehaviourhistology molecularcellular imagingother

Abstract

Ultrasound mediated neuromodulation has been demonstrated to a safe treatment strategy in the field of neuroscience. In this study, low-intensity pulsed ultrasound (LIPUS) was used to treat Parkinson's disease (PD) models induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 1-methyl-4-phenylpyridinium (MPP + ) to explore the possibility of ultrasound neuroprotective effect on PD. The results demonstrated that LIPUS treatment can attenuate the central neurotoxicity of MPTP in mice, reduce the loss of tyrosine hydroxylase positive neurons in the substantia nigra pars compacta and decrease the apoptosis in the section of substantia nigra. The movement and balance dysfunctions in PD mice were improved with LIPUS treatment. In addition, we demonstrated that LIPUS can inhibit the decreased activity and increased apoptosis of dopaminergic neurons induced by MPP + , restrain the accumulation of reactive oxygen species (ROS) and decrease of mitochondrial membrane potential caused by MPP + . Moreover, LIPUS stimulation alone did not cause any cytotoxicity and tissue damage in our study. Taken together, the protective and regulatory effects of LIPUS on dopaminergic neurons make it possible as a new, safe and noninvasive treatment for PD.

Abstract via europepmc.

Speciesmouse (C57BL/6); N2a mouse neuroblastoma cell line
Subjects5, 5swept animals
Sessions per subject5
Randomisedyes
Blindingnot reported
Sham / controlnone
Auditory controlnot reported
Readout timingoffline
Anaesthesianot reported
Readoutsbehaviour, histology molecular, cellular imaging, otherrotarod test; pole test; tyrosine-hydroxylase and TUNEL immunohistochemistry of substantia nigra; Nissl/H&E staining; CCK-8 cell viability assay; Annexin V-FITC/PI flow cytometry apoptosis assay; DCFH-DA ROS fluorescence assay; JC-1 mitochondrial membrane potential assay; TRPM7 mRNA expression
Direction of effectnot assessedStudy assessed neuroprotection (reduced dopaminergic neuron loss/apoptosis, improved motor function) rather than a direct excitatory/inhibitory neuromodulatory effect.
Adverse eventsnone observedLIPUS alone did not cause cytotoxicity in N2a cells or tissue damage in mouse brain (H&E, Nissl staining); however, in a separate preliminary experiment mice exposed at a much higher intensity (Grade 5, ~355-427 mW/cm2) died on the third day of treatment.

Exposures

Exposure 1: In vivo mouse head LIPUS irradiation (Grade 2 intensity), MPTP model

Target: whole brain or unfocused — “mouse head (non-focusing cylindrical transducer aligned to head), assessed effect on substantia nigra pars compacta dopaminergic neurons
Device: other named manufacturer · Institute of Ultrasound Imaging of Chongqing Medical University · Ultrasound Gene Transfection Treatment Meter (UGT 1025)

Pulse timing
Waveformpulsed
Fundamental frequency (kHz)1,000✓✓
Pulse duration (ms)not reportedimplied by duty cycle ÷ PRF: 0.2 ms (not stated by the paper)
Pulse repetition frequency (Hz)1,000✓✓
Duty cycle (%)20✓✓
Sonication duration (s)600✓✓
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 estimatenot reported
In-situ pressure (kPa)not reported
In-situ Isppa (W/cm²)not reported
In-situ Ispta (W/cm²)not reported
Ispta, domain unspecified (W/cm²)0.111, 0.123swept✓✓
Protocol, in the paper’s words

10 min of ultrasonic irradiation delivered once every 24 h for 5 sessions total, in mice receiving MPTP (or saline) 4 days before LIPUS onset.

Exposure 2: N2a cell culture LIPUS irradiation (Grade 1 intensity), MPP+ model

Target: other — “N2a cells (mouse neuroblastoma cell line) in culture
Device: other named manufacturer · Institute of Ultrasound Imaging of Chongqing Medical University · Ultrasound Gene Transfection Treatment Meter (UGT 1025)

Pulse timing
Waveformpulsed
Fundamental frequency (kHz)1,000✓✓
Pulse duration (ms)not reportedimplied by duty cycle ÷ PRF: 0.2 ms (not stated by the paper)
Pulse repetition frequency (Hz)1,000✓✓
Duty cycle (%)20✓✓
Sonication duration (s)600✓✓
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 estimatenot applicable
In-situ pressure (kPa)not applicable
In-situ Isppa (W/cm²)not applicable
In-situ Ispta (W/cm²)not applicable
Ispta, domain unspecified (W/cm²)0.0403, 0.0405swept✓✓
Protocol, in the paper’s words

N2a cells were treated with LIPUS for 10 min, 4 h after MPP+ (0.75 mM) treatment; non-focusing cylindrical transducer placed under the culture plate.

Flags from extraction

  • n_subjectsPaper gives per-group n (5 animals/group, 4 groups); only the LIPUS-exposed groups (LIPUS-only and MPTP+LIPUS) are counted here as [5,5]; total not stated.
  • exposures[0].unspecified_domain.isppa_w_cm2Paper reports 'intensity of Grade 2' as measured by an ultrasound power meter without specifying whether this is ISPPA or ISPTA, and without specifying free-field vs in-situ domain.
  • exposures[1].unspecified_domain.isppa_w_cm2Same ambiguity as Grade 2 (ISPPA/ISPTA and domain not specified) for the Grade 1 intensity used on N2a cells.
  • exposures[0].timing.pulse_duration_msPRF (1000 Hz) and duty cycle (20%) are stated but pulse duration is never given directly; computing it would require arithmetic on the stated values, so it is left not_reported.
  • safety_notesA separate, higher-intensity 'Grade 5' condition (~355-427 mW/cm2) caused death of mice by day 3; this was a preliminary safety observation, not part of the formally analysed Grade 1/2 exposures.
  • exposures[1].in_situN2a cells were cultured in a well plate (dish) with no skull/tissue path; fields set to not_reported (rather than null) because the overall study also includes in vivo rodent exposures.