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Whole-brain low-intensity pulsed ultrasound therapy markedly improves cognitive dysfunctions in mouse models of dementia - Crucial roles of endothelial nitric oxide synthase

Kumiko Eguchi, Tomohiko Shindo, Kenta Ito, Tsuyoshi Ogata, Ryo Kurosawa, Yuta Kagaya, Yuto Monma, Sadamitsu Ichijo, Sachie Kasukabe, Satoshi Miyata, Takeo Yoshikawa, Kazuhiko Yanai, Hirofumi Taki, Hiroshi Kanai, Noriko Osumi, Hiroaki Shimokawa

Brain Stimulation 2018, 11, 959-973 · 10.1016/j.brs.2018.05.012

rodentvascular dementiaalzheimers diseasebehaviourhistology molecularcerebral haemodynamics

Abstract

Background Therapeutic focused-ultrasound to the hippocampus has been reported to exert neuroprotective effects on dementia. In the present study, we examined whether the whole-brain LIPUS (low-intensity pulsed ultrasound) therapy is effective and safe in 2 mouse models of dementia (vascular dementia, VaD and Alzheimer's disease, AD), and if so, to elucidate the common underlying mechanism(s) involved. Methods We used bilateral carotid artery stenosis (BCAS) model with micro-coils in male C57BL/6 mice as a VaD model and 5XFAD transgenic mice as an AD model. We applied the LIPUS therapy (1.875 MHz, 6.0 kHz, 32cycles) to the whole brain. Results In both models, the LIPUS therapy markedly ameliorated cognitive impairments (Y-maze test and/or passive avoidance test) associated with improved cerebral blood flow (CBF). Mechanistically, the LIPUS therapy significantly increased CD31-positive endothelial cells and Olig2-positive oligodendrocyte precursor cells (OPCs) in the VaD model, while it reduced Iba-1-positive microglias and amyloid-β (Aβ) plaque in the AD model. In both models, endothelium-related genes were significantly upregulated in RNA-sequencing, and expressions of endothelial nitric oxide synthase (eNOS) and neurotrophins were upregulated in Western blotting. Interestingly, the increases in glia cells and neurotrophin expressions showed significant correlations with eNOS expression. Importantly, these beneficial effects of LIPUS were absent in eNOS-knockout mice. Conclusions These results indicate that the whole-brain LIPUS is an effective and non-invasive therapy for dementia by activating specific cells corresponding to each pathology, for which eNOS activation plays an important role as a common mechanism.

Abstract via europepmc.

Speciesmouse
Subjectsnot reported animals
Sessions per subjectnot reported
Randomisednot reported
Blindingnot reported
Sham / controlundescribed
Auditory controlnot reported
Readout timingoffline
Anaesthesianot reported
Readoutsbehaviour, histology molecular, cerebral haemodynamicsY-maze, passive avoidance, and novel object recognition tests; cerebral blood flow by laser speckle imaging; immunohistochemistry (GST-p, Klüver-Barrera, CD31, GFAP, Iba-1, DCX, Ki67, amyloid-beta); RNA-sequencing, RT-qPCR, Western blotting, ELISA (eNOS, neurotrophins, APP/BACE-1, etc.)
Direction of effectnot assessedWhole-brain LIPUS therapy improved cognitive function, cerebral blood flow, and reduced pathology (white matter lesions in the VaD model; amyloid-beta plaque and microgliosis in the AD model) via eNOS-dependent mechanisms; the paper does not assess acute excitatory/inhibitory neuromodulation.
Adverse eventsnone observedLIPUS-treated mice showed no signs of cramps, paralysis, cerebral hemorrhage, hypothermia, hyperthermia, or increased mortality compared with control mice, and no effects on body weight or systolic blood pressure, for up to 3 months.

Exposures

Exposure 1: Whole-brain LIPUS therapy

Target: whole brain or unfocused — “whole brain
Device: not reported

Pulse timing
Waveformpulsed
Fundamental frequency (kHz)1,875✓✓
Pulse duration (ms)0.0171✓✓
Pulse repetition frequency (Hz)6,000✓✓
Duty cycle (%)not reportedpulse duration × PRF gives 10.24%
Sonication duration (s)not reported
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
Protocol, in the paper’s words

Whole-brain LIPUS therapy (1.875 MHz, 6.0 kHz, 32 cycles per pulse) was applied to mouse models of vascular dementia (BCAS) and Alzheimer's disease (5XFAD), including eNOS-knockout mice; full device, session number, and treatment-schedule details are given only in Supplementary Methods, which were not available in the main text.

Flags from extraction

  • exposures[0].timing.pulse_repetition_frequency_hzThe abstract lists three numbers (1.875 MHz, 6.0 kHz, 32 cycles) without labeling them; 6.0 kHz is interpreted as the pulse repetition frequency based on common usage in this parameter format, but the paper does not explicitly name it as PRF.
  • exposures[0].timing.pulse_duration_msPulse duration was derived from the stated 32 cycles per pulse divided by the 1.875 MHz fundamental frequency (32/1,875,000 Hz = 0.017067 ms), per the allowed cycles-per-pulse-over-frequency conversion; the paper does not itself state a pulse duration in ms.
  • n_subjectsFull methods (including group sizes) are in Supplementary Methods, not provided; main text gives many different per-figure n's (e.g., n=12-17, n=5-12, n=6, n=9-10, n=20-25) across different endpoints and models, with no single total.
  • sham_typeControl mice 'underwent the same procedure without the LIPUS treatment', but the paper does not describe whether a transducer was present but inactive, or whether ultrasound was simply not applied; coded as 'other'.
  • anaesthesiaMethods for whole-brain LIPUS delivery (including whether mice were anaesthetised or awake during sonication) are in Supplementary Methods, not provided in the main text.