Assessment of focused ultrasound stimulation to induce peripheral nerve activity and potential damage in vivo

Bruno Rodríguez-Meana, Eva Santos-Nogueira, Sònia Trujillo-Vázquez, Anette Jakob, Esther Udina, Marc Fournelle, Xavier Navarro

Frontiers in Neurology 2024 · 10.3389/fneur.2024.1346412

rodentex vivo tissuehealthyemg mepinvasive electrophysiologybehaviourhistology molecular

Abstract

Introduction Peripheral neuroprostheses are aimed to restore loss of sensory and motor functions by interfacing axons in the peripheral nerves. Most common interfaces in neuroprostheses are electrodes that establish electrical connection with peripheral axons. However, some challenges arise related to long-term functionality, durability, and body response. Recently, focused ultrasound stimulation (FUS) has emerged as a non-invasive approach to modulate the nervous system. However, it is controversial whether FUS can induce axon depolarization. Methods We have assessed FUS applied in vivo to the rat peripheral nerve, with two objectives: first, to test whether FUS activates peripheral nerves under different stimulation conditions, and second, to evaluate if FUS inflicts damage to the nerve. FUS was delivered with three ultrasound transducers (Sonic Concept H115, H107, and H102) covering the largest set of parameters examined for FUS of peripheral nerves so far. Results We did not obtain reliable evoked action potentials in either nerves or muscles, under any FUS condition applied, neither over the skin nor directly to the nerve exposed. Additional experiments ex vivo and in vivo on mice, confirmed this conclusion. When FUS stimulation was applied directly to the exposed sciatic nerve, neuromuscular function decreased significantly, and recovered one week later, except for FUS at 0.25 MHz. Histologically, degenerating nerve fibers were observed, with a tendency to be higher with the lower FUS frequency. Discussion Past reports on the ability of ultrasound to stimulate the peripheral nerve are controversial. After testing a wide range of FUS conditions, we conclude that it is not a reliable and safe method for stimulating the peripheral nerve. Special consideration should be taken, especially when low-frequency FUS is applied, as it may lead to nerve damage.

Abstract via europepmc.

Speciesrat (Sprague-Dawley); mouse (Balb/c)

Subjects22, 4swept animals

Sessions per subject1

Randomisednot reported

Blindingnot reported

Sham / controlnone

Auditory controlnot reported

Readout timingboth

Anaesthesiaanaesthetised

Readoutsemg mep, invasive electrophysiology, behaviour, histology molecular — CMAP and CNAP recording; Sciatic Functional Index (walking track); toluidine blue semithin sections; RT97 and Iba1 immunostaining

Direction of effectno effectNo reliable evoked nerve or muscle action potentials under any FUS condition (skin, exposed sciatic nerve, plantar skin; also mouse and ex vivo). Direct nerve exposure reduced electrically evoked responses and caused axonal degeneration, worst at 0.25 MHz.

Adverse eventsobservedSkin irritation at 0.25 and 0.5 MHz, withdrawal responses at the paw, functional loss and nerve fibre degeneration after direct nerve exposure, most severe at 0.25 MHz (SFI -63 +/- 3).

Exposures

Exposure 1: Sciatic nerve (through skin, exposed nerve) and hindpaw, five transducer frequencies

Target: sciatic nerve — “sciatic nerve”
Device: Sonic Concepts · Sonic Concepts · H115, H107, H102 (H107 and H102 also at 3rd harmonic)

Pulse timing
Waveform	pulsed
Fundamental frequency (kHz)	250, 500, 1,120, 1,630, 3,580swept	✓✓✓⚑
Pulse duration (ms)	1, 10, 50, 100, 200swept	✓✓✓
Pulse repetition frequency (Hz)	not reported	⚑
Duty cycle (%)	not reported
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 estimate	not reported
In-situ pressure (kPa)	not reported
In-situ Isppa (W/cm²)	not reported
In-situ Ispta (W/cm²)	not reported
Pressure, domain unspecified (kPa)	100, 5,000swept	✓?⚑

Protocol, in the paper’s words

5 sequences of 1, 3 or 10 pulses of 1, 10, 50, 100 or 200 ms with an interstimulus interval of 100 ms; intersequence interval 1 s; pressure stepped 0, 1.25, 2.5, 3.75 and 5 MPa on each sequence.

Flags from extraction

n_subjects — 22 rats and 4 mice stated; total not stated separately and one ex vivo experiment used a rat that may be among the 22.
exposures[0].fundamental_frequency_khz — Five frequencies (H115 0.25, H107 0.5, H102 1.12 MHz and 3rd harmonics 1.63 and 3.58 MHz) recorded as a list in one exposure; focal depth and parameters otherwise shared.
exposures[0].unspecified_domain.pressure_kpa — Pressure range 0.1-5 MPa described as 'effective pressure'; transducers were calibrated in water under free-field conditions but the paper does not say the stated pressures are free-field values. Range also read from Table 1.
exposures[0].timing.pulse_repetition_frequency_hz — Only an interstimulus interval of 100 ms between pulses is given; PRF, duty cycle and sonication duration are not stated.
sham_type — No FUS sham; electrical stimulation used as a control for recording and nerve function.