Interaction between acoustic intensity and focal depth in low-intensity focused ultrasound analgesia: A 2×2 factorial study in a rat model of inflammatory pain

Fengjun Ni, Yong Yao, Xiaochen Weng, Huayuan Yang

2026 · 10.21203/rs.3.rs-10446991/v1

rodentchronic painbehaviourhistology molecular

Abstract

Objective In traditional acupuncture, the depth of needle insertion and the intensity of stimulation are operator-dependent and lack objective quantification. This study aimed to determine whether focal depth and acoustic intensity affect analgesic efficacy and whether there is an interaction between them. Methods We developed a focused low-intensity pulsed ultrasound (FLIPUS) device, named Ultrasound Needle‑Free Acupuncture (UNFA), which allows for the independent control of acoustic intensity (0.03–3.0 W/cm²) and focal depth (3 or 5 mm). Using a 2×2 factorial design in a rat model of complete Freund's adjuvant (CFA)-induced inflammatory pain (n = 8 per group), we tested the main effects and interaction of intensity (0.03 vs. 1.0 W/cm²) and depth (3 vs. 5 mm) on mechanical and thermal pain thresholds, paw edema, and open field centre distance. Results There was a significant main effect of focal depth on all four outcomes (p = 0.008, 0.038, 0.0037, and 0.0016, respectively), with shallow (3 mm) stimulation consistently outperforming deep (5 mm) stimulation. There was a trend favouring low-intensity stimulation over high-intensity stimulation. No significant interaction between intensity and depth was detected. The optimal parameter set was the US1 combination (shallow depth + low intensity). Furthermore, this significantly reversed the upregulation of key pain-related proteins (PLA2G2A, NTRK1, and BDKRB2) in the spinal cord, providing molecular mechanistic support for the behavioural findings. Conclusion Focal depth is the primary determinant of the analgesic efficacy of ultrasound-based acupuncture (UNFA). These findings provide a parametric benchmark for ultrasound-based acupuncture standardisation. Based on the observation that specific parameter combinations (shallow depth and low intensity) produce distinct behavioural and molecular outcomes, future studies could explore whether quantifiable physical parameters of acupoint stimulation can be systematically mapped to subjective sensations, such as de qi (needle sensation).

Abstract via crossref.

Speciesrat (Sprague-Dawley)

Subjects32 animals

Sessions per subject4

Randomisedyes

Blindingsingle

Sham / controlnone

Auditory controlnot reported

Readout timingoffline

Anaesthesiaanaesthetised

Readoutsbehaviour, histology molecular — von Frey filament mechanical pain threshold; Hargreaves radiant-heat thermal pain threshold (paw withdrawal latency); paw thickness (edema) by digital calliper; open field test centre distance; Western blot of PLA2G2A, NTRK1, BDKRB2 in spinal dorsal horn

Direction of effectinhibitoryAll four intensity/depth combinations raised mechanical and thermal pain thresholds and reduced paw edema versus the CFA-inflamed baseline, with shallow depth (3 mm) consistently outperforming deep depth (5 mm) on all four outcomes; the shallow+low-intensity (US1) combination was most effective and reversed CFA-induced upregulation of PLA2G2A, NTRK1 and BDKRB2 in the spinal dorsal horn. No sham/placebo arm was included for direct comparison.

Adverse eventsnot reported

Exposures

Exposure 1: UNFA (ultrasound needle-free acupuncture) at bilateral Zusanli (ST36) acupoint, 2x2 intensity x depth sweep

Target: other — “bilateral Zusanli acupoint (ST36)”
Device: custom-built · Zanshutech · UNFA-A ✓

Pulse timing
Waveform	pulsed
Fundamental frequency (kHz)	600	✓✓✓
Pulse duration (ms)	not reported	⚑
Pulse repetition frequency (Hz)	not reported
Duty cycle (%)	20	✓✓✓
Sonication duration (s)	300	✓✓✓

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
Isppa, domain unspecified (W/cm²)	0.03, 1swept	✓✓✓⚑

Protocol, in the paper’s words

The stimulation parameters were set to a fundamental frequency of 0.6 MHz and a duty cycle of 20%. Each 5-minute session was administered once every other day for a total of four sessions (7 days). Focal depth was independently set to 3 mm (shallow) or 5 mm (deep) using a 2x2 factorial design combined with intensity (0.03 vs. 1.0 W/cm^2), giving four groups: US1 (3 mm, 0.03 W/cm^2), US2 (5 mm, 0.03 W/cm^2), US3 (3 mm, 1.0 W/cm^2), US4 (5 mm, 1.0 W/cm^2).

Flags from extraction

target.terms — Target is the Zusanli (ST36) acupoint, an acupuncture point on the hindlimb rather than a named nerve; no target vocabulary term matches directly, so 'other' was used. The discussion mentions nearby deep peroneal nerve branches but the paper does not state the peroneal nerve itself was targeted.
exposures[0].unspecified_domain.isppa_w_cm2 — Paper reports 'acoustic intensity' in W/cm^2 without specifying whether this is Isppa or Ispta, or whether it is a free-field or in-situ value; placed in unspecified_domain.isppa_w_cm2 as the closest matching field.
n_subjects — An additional cohort of rats (US1-treated, n=4) was used for Western blot molecular analysis in Fig. 3, separate from the 32 rats in the main 2x2 behavioural cohort; this additional group is not included in the n_subjects count.
exposures[0].timing.pulse_duration_ms — Duty cycle (20%) is stated but no pulse repetition frequency is given, so pulse duration cannot be determined and is left not_reported per the burst rule; focal depth (3 vs 5 mm) also varies within this exposure but has no dedicated schema field, so it is described in protocol_description only.