Noninvasive Transcranial Stimulation of Rat Abducens Nerve by Focused Ultrasound

Hyungmin Kim, Seyed Javid Taghados, Krisztina Fischer, Lee-So Maeng, Shinsuk Park, Seung-Schik Yoo

Ultrasound in Medicine & Biology 2012, 38, 1568-1575 · 10.1016/j.ultrasmedbio.2012.04.023

rodenthealthybehaviourhistology molecular

Abstract

Nonpharmacologic and nonsurgical transcranial modulation of the nerve function may provide new opportunities in evaluation and treatment of cranial nerve diseases. This study investigates the possibility of using low-intensity transcranial focused ultrasound (FUS) to selectively stimulate the rat abducens nerve located above the base of the skull. FUS (frequencies of 350 kHz and 650 kHz) operating in a pulsed mode was applied to the abducens nerve of Sprague-Dawley rats under stereotactic guidance. The abductive eyeball movement ipsilateral to the side of sonication was observed at 350 kHz, using the 0.36-msec tone burst duration (TBD), 1.5-kHz pulse repetition frequency (PRF), and the overall sonication duration of 200 msec. Histologic and behavioral monitoring showed no signs of disruption in the blood brain barrier (BBB), as well as no damage to the nerves and adjacent brain tissue resulting from the sonication. As a novel functional neuro-modulatory modality, the pulsed application of FUS has potential for diagnostic and therapeutic applications in diseases of the peripheral nervous system.

Abstract via europepmc.

Speciesrat

Subjects22 animals

Sessions per subjectnot reported

Randomisednot reported

Blindingnot reported

Sham / controlnone

Auditory controlnot reported

Readout timingonline

Anaesthesiaanaesthetised

Readoutsbehaviour, histology molecular — Video-based frame-by-frame quantification of abductive eyeball movement; H&E histology of nerve/brain tissue; Trypan Blue extravasation for blood-brain-barrier integrity; thermocouple temperature measurement in a tissue-mimicking gel phantom and excised cranial tissue (safety assay, not a neural readout)

Direction of effectexcitatoryPulsed FUS at 350 kHz to the abducens nerve elicited discrete, stepwise ipsilateral abductive eyeball movement that increased with repeated 1-s-interval sonications and reversed after sonication stopped, indicating excitatory nerve stimulation; the same waveform parameters at 650 kHz produced no eye movement in any animal across the full intensity range tested.

Adverse eventsnone observedNo evidence of blood-brain-barrier disruption (Trypan Blue) or acute nerve/brain tissue damage (H&E) was found after sonication; surviving rats (up to 49 days) showed normal behaviour including normal eye tracking; in vitro/ex vivo thermal measurements detected no temperature change beyond instrumental fluctuation (<0.1 C); the peak negative pressure used (0.54 MPa) and mechanical index (0.9) were far below thresholds associated with cavitation-related tissue damage (40 MPa) and the diagnostic-imaging MI limit (1.9).

Exposures

Exposure 1: 350 kHz FUS to abducens nerve (effective stimulation)

Target: peripheral nerve — “abducens nerve (cranial nerve VI)”
Device: custom-built

Pulse timing
Waveform	pulsed
Fundamental frequency (kHz)	350	✓✓✓
Pulse duration (ms)	0.36	✓✓✓
Pulse repetition frequency (Hz)	1,500	✓✓✓
Duty cycle (%)	54pulse duration × PRF gives 54%	✓✓✓
Sonication duration (s)	0.2	✓✓✓

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 estimate	deratingsingle value
In-situ pressure (kPa)	540	✓✓✓⚑
In-situ Isppa (W/cm²)	8.6	✓✓✓
In-situ Ispta (W/cm²)	4.6	✓✓✓

Protocol, in the paper’s words

For the 350 kHz condition, the same intensity range (0.5-20 W/cm2 Isppa, 0.5 W/cm2 steps) was first tested in a separate group (n=3); Isppa=8.6 W/cm2, the minimum intensity giving successful stimulation, was then used throughout the rest of the experiment (n=16). Sonication was given in 1-s intervals, 10 times per trial; the eyeball movement increased stepwise during the repetitive sonication, reaching its maximum around 7 s after onset, and rolled back to baseline in 6.9+-0.4 s after sonication stopped.

Exposure 2: 650 kHz FUS to abducens nerve (no effect)

Target: peripheral nerve — “abducens nerve (cranial nerve VI)”
Device: custom-built

Pulse timing
Waveform	pulsed
Fundamental frequency (kHz)	650	✓✓✓
Pulse duration (ms)	0.36	✓✓✓
Pulse repetition frequency (Hz)	1,500	✓✓✓
Duty cycle (%)	54pulse duration × PRF gives 54%	✓✓✓
Sonication duration (s)	0.2	✓✓✓

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 estimate	not reported
In-situ pressure (kPa)	not reported
In-situ Isppa (W/cm²)	0.5, 20swept	✓✓✓
In-situ Ispta (W/cm²)	not reported	⚑

Protocol, in the paper’s words

Acoustic intensities from 0.5 up to 20 W/cm2 Isppa (0.5 W/cm2 steps) were applied to a separate group of rats (n=3) at 650 kHz using the same pulsing parameters (TBD=0.36 msec, PRF=1.5 kHz, overall sonication duration 200 msec) as the 350 kHz condition; no eyeball movement was elicited at any tested intensity.

Flags from extraction

exposures[*].target — The abducens nerve (cranial nerve VI) has no dedicated id in the target vocabulary; mapped to the generic 'peripheral_nerve' parent term.
exposures[0].in_situ.pressure_kpa — Methods section gives a garbled figure ('peak negative pressure 20.53 MPa'); the Discussion states a clean value of 0.54 MPa, which was used here (540 kPa).
exposures[1].timing — Pulse duration, PRF, duty factor and overall sonication duration are stated in Methods as shared parameters used 'at each fundamental frequency' (350 and 650 kHz); applied to both exposures on that basis.
exposures[1].in_situ.ispta_w_cm2 — No Ispta or pressure value is given specifically for the (ineffective) 650 kHz condition; only the tested Isppa range is stated.
sham_type — No sham or inactive-transducer control condition is described; only different frequencies/intensities are compared.