Non-invasive Low-Intensity Pulsed Ultrasound Modulates Primary Cilia of Rat Hippocampal Neurons

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

Ultrasound in Medicine & Biology 2019 · 10.1016/j.ultrasmedbio.2018.12.012

rodentin vitro cellhealthycellular imaginghistology molecular

Abstract

Transcranial modulation of primary cilia may provide new opportunities in the treatment of neurodegenerative diseases. This study investigates the effect of non-invasive low-intensity pulsed ultrasound (LIPUS) stimulation on primary cilia of rat hippocampal neurons. Three hours of LIPUS stimulation significantly reduced the incidence rate and length of cilia on cultured neurons (p < 0.01). Similarly, increasing the duration and intensity of LIPUS stimulation decreased the incidence and length of cilia. LIPUS stimulation improved c-fos expression when it was delivered to CA1 of the intact hippocampus of rats. And prolonged LIPUS stimulation (frequency: 0.5 MHz, pulse repetition frequency: 500 Hz, duty cycle: 5%, Ispta: 255 mW/cm2, 10 min/d, 10 d) caused a statistically significant reduction in the incidence rate (p < 0.05) and length of primary cilia (p < 0.01) of neurons in rat CA1 hippocampus. These results indicate the promising potential of LIPUS stimulation in the treatment of primary cilium-related brain diseases.

Abstract via pubmed.

Speciesrat (Sprague-Dawley)

Subjects10 animals

Sessions per subject10

Randomisedyes

Blindingnot reported

Sham / controlinactive transducer

Auditory controlnot reported

Readout timingoffline

Anaesthesianot reported

Readoutscellular imaging, histology molecular — primary cilia incidence/length via immunofluorescence (anti-ACIII); c-fos immunostaining; Western blot for ACIII protein; H&E and Nissl staining

Direction of effectexcitatoryLIPUS stimulation increased c-fos expression in CA1 hippocampal neurons (indicative of excitatory activation), while it decreased the incidence rate and length of neuronal primary cilia in both cultured hippocampal neurons and intact rat CA1 hippocampus, in a duration- and intensity-dependent manner.

Adverse eventsnone observedH&E staining revealed that LIPUS stimulation had no damaging effect on cortex and CA1 hippocampus, and Nissl staining showed neuronal density appeared similar throughout the brain; the temperature of the hippocampus rose less than 0.3 degC after transcranial LIPUS stimulation.

Exposures

Exposure 1: Cultured rat hippocampal neurons (in vitro)

Target: cultured neurons — “cultured rat hippocampal neurons”
Device: custom-built · 0.68-MHz plane transducer (8 mm diameter), lab-fabricated

Pulse timing
Waveform	pulsed
Fundamental frequency (kHz)	680	✓✓✓
Pulse duration (ms)	not reportedimplied by duty cycle ÷ PRF: 0.1 ms (not stated by the paper)	⚑
Pulse repetition frequency (Hz)	500	✓✓✓
Duty cycle (%)	5	✓✓✓
Sonication duration (s)	10,800, 21,600swept	✓✓✓

Pressure and intensity, by domain
Free-field pressure (kPa)	not reported
Free-field Isppa (W/cm²)	not reported
Free-field Ispta (W/cm²)	0.065, 0.21swept	✓✓✓
In-situ estimate	not applicable
In-situ pressure (kPa)	not applicable
In-situ Isppa (W/cm²)	not applicable
In-situ Ispta (W/cm²)	not applicable

Protocol, in the paper’s words

LIPUS was generated with a 0.68-MHz plane transducer and transmitted to the bottom of the culture chambers for 3 and 6 h; acoustic intensity attenuation through the bottom of the culture dish was about 15%.

Exposure 2: Intact hippocampus (CA1), in vivo

Target: CA1 — “CA1 of the intact hippocampus”
Device: other named manufacturer · NdtXducer · 0.5-MHz focused transducer (19 mm diameter) ✓

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, 2,400swept	✓✓✓

Pressure and intensity, by domain
Free-field pressure (kPa)	not reported
Free-field Isppa (W/cm²)	not reported
Free-field Ispta (W/cm²)	0.255	✓✓✓
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

Each rat was treated with LIPUS stimulations (10 min/d) daily for 10 d for the cilia study; for the c-fos experiment, immunofluorescence staining was performed after LIPUS stimulation for 40 min. Acoustic intensity attenuation through the rat skull was about 30%; hippocampal temperature rose less than 0.3 degC.

Flags from extraction

n_subjects — Sub-experiment cohort sizes reported elsewhere in Results (e.g. n=8 rats for the in vivo cilia assay, n=4 for c-fos) appear to be subsets/combinations of the overall 10 sham + 10 LIPUS rats stated in Methods, but exact overlap is not made explicit.
anaesthesia — Isoflurane anaesthesia is mentioned only for terminal perfusion/decapitation procedures; it is not stated whether animals were anaesthetised or awake during the repeated daily LIPUS stimulation sessions.
exposures[0].timing.pulse_duration_ms — Fig. 1 gives a 0.1 ms tone-burst duration explicitly for the in vivo (500 kHz) system; no equivalent tone-burst duration is stated for the in vitro 0.68 MHz system, so left not_reported rather than assumed equal.
exposures[*].free_field — Domain assigned as free_field based on the general statement that acoustic parameters were measured with a calibrated hydrophone and that skull/dish attenuation is reported separately as a percentage loss from these values; not explicitly labelled per number.