Transcranial Low-Intensity Pulsed Ultrasound Modulates Structural and Functional Synaptic Plasticity in Rat Hippocampus

Xiaowei Huang, Hairong Zheng, Zhengrong Lin, Kaiyue Wang, Xiufang Liu, Wei Zhou, Long Meng, Jiqing Huang, Kexin Yuan, Lili Niu

IEEE Transactions on Ultrasonics, Ferroelectrics, and Frequency Control 2019, 66, 930-938 · 10.1109/tuffc.2019.2903896

rodenthealthyinvasive electrophysiologyhistology molecular

Abstract

Plasticity of synaptic structure and function play an essential role in neuronal development, cognitive functions, and degenerative diseases. Recently, low-intensity pulsed ultrasound (LIPUS) stimulation has been reported as a promising technology for neuromodulation. However, the effect of LIPUS stimulation on the structural and functional synaptic plasticity in rat hippocampus has not yet been addressed. The aim of this study was to investigate whether LIPUS stimulation could affect the dendritic structure, electrophysiological properties, and expression level of glutamate receptors GluN2A, GluN2B, and GluR1 subunits in rat hippocampus. Transcranial LIPUS was delivered to CA1 of the intact hippocampus of rats ( n = 40 ) for 10 days (10 min/day) with the following parameters: fundamental frequency of 0.5 MHz, pulse repetition frequency (PRF) of 500 Hz, peak negative pressure of 0.42 MPa, and I spta of 360 mW/cm 2 . The effect of LIPUS on dendritic structure, electrophysiological properties, and the expression of neurotransmitter receptors was measured using Golgi staining, electrophysiological recording, and western blotting, respectively. Golgi staining and electrophysiological recordings showed that LIPUS stimulation significantly increased the density of dendritic spines (0.72 ± 0.17 versus 0.94 ± 0.19 spines/ [Formula: see text], ) and the frequency of spontaneous excitatory postsynaptic current (0.37 ± 0.14 versus 1.77 ± 0.37 Hz, ) of CA1 hippocampal neurons. Furthermore, the western blotting analysis demonstrated a significant increase in the expression level of GluN2A ( ). The results illustrated the effect of LIPUS on the dendritic structure, function, and neurotransmitter receptors, which may provide a powerful tool for treating neurodegenerative diseases.

Abstract via europepmc.

Speciesrat (Sprague-Dawley)

Subjects22 animals

Sessions per subject10

Randomisedyes

Blindingsingle

Sham / controlundescribed

Auditory controlnot reported

Readout timingoffline

Anaesthesiaawake

Readoutsinvasive electrophysiology, histology molecular — c-Fos immunofluorescence; Golgi staining of dendritic spine density; whole-cell patch-clamp recording of spontaneous EPSCs; western blot of GluN2A, GluN2B, GluR1; H&E and Nissl histology

Direction of effectexcitatory10 days of transcranial LIPUS to CA1 increased c-Fos expression, dendritic spine density, spontaneous EPSC frequency, and GluN2A protein expression in CA1 hippocampal neurons relative to control, with no significant change in GluN2B, GluR1, or EPSC amplitude/kinetics.

Adverse eventsnone observedH&E staining showed no hemorrhaging or tissue damage and Nissl staining showed no apparent neuronal damage in cortex or CA1 hippocampus after LIPUS; temperature rise in hippocampal tissue during stimulation was less than 0.3 degrees C.

Exposures

Exposure 1: Transcranial LIPUS to hippocampal CA1 in neonatal rats, 10 min/day for 10 days

Target: CA1 — “CA1 of the intact hippocampus”
Device: other named manufacturer · NdtXducer

Pulse timing
Waveform	pulsed
Fundamental frequency (kHz)	500	✓✓✓
Pulse duration (ms)	0.1	✓✓✓⚑
Pulse repetition frequency (Hz)	500	✓✓✓
Duty cycle (%)	5pulse duration × PRF gives 5%	✓✓✓
Sonication duration (s)	600	✓✓✓

Pressure and intensity, by domain
Free-field pressure (kPa)	420	✓✓✓
Free-field Isppa (W/cm²)	7.2	✓✓✓
Free-field Ispta (W/cm²)	0.36	✓✓✓
In-situ estimate	measurementsingle value
In-situ pressure (kPa)	350	✓✓✓⚑
In-situ Isppa (W/cm²)	5.1	✓✓✓
In-situ Ispta (W/cm²)	0.255	✓✓✓

Protocol, in the paper’s words

Transcranial LIPUS was delivered to CA1 of the intact hippocampus of rats (n=40) for 10 days (10 min/day) with the following parameters: fundamental frequency of 0.5 MHz, pulse repetition frequency (PRF) of 500 Hz, peak negative pressure of 0.42 MPa, and Ispta of 360 mW/cm2. All rats received LIPUS treatment for 10 min/day for 10 days. Before LIPUS stimulation, depilatory paste was used to remove hair from the scalp and acoustic coupling gel was applied; the focused transducer was mounted on a custom acoustic collimator cone filled with degassed water. A separate immunofluorescence cohort was stimulated for 40 min to assess c-Fos expression.

Flags from extraction

sham_type — Control group (n=22) received no ultrasound but the paper never describes whether a transducer was present/inactive or any other sham mechanism.
exposures[0].timing.pulse_duration_ms — Fig. 1 caption states '50 cycles per pulses' (would give ~0.1 ms pulse duration at 0.5 MHz, consistent with 5% duty cycle at 500 Hz PRF), but this number appears only in a figure legend, not the main text, so it was not extracted as a value.
exposures[0].in_situ.pressure_kpa — The abstract states treatment parameters as 0.42 MPa / 360 mW/cm2 (the free-field water-tank values), while the Discussion states the study was actually performed with 0.35 MPa / 255 mW/cm2 (the through-skull measured values); both are recorded in their respective domains as stated.