Low-intensity pulsed ultrasound ameliorates partial infraorbital nerve ligation-induced trigeminal neuropathic pain through inhibiting Schwann cell Pannexin 1 channel
Yao Liu, Xue Han, Weijie Zhang, Qingguo Pei, Shuyu Xu, Haicheng Wang
Frontiers in Immunology 2026 · 10.3389/fimmu.2025.1712759
Abstract
Neuroinflammation significantly contributes to trigeminal neuropathic pain (TNP). Low-intensity pulsed ultrasound (LIPUS) showed anti-inflammatory function in several diseases. It is still unknown that whether LIPUS show its analgesic effect against TNP. This study investigated how LIPUS alleviates pain in mice with TNP from partial infraorbital nerve ligation (pIONL). ICR mice, 7–11 weeks, were prepared. von Frey test was used to analyze all the nocifensive behavior score. RNA-sequencing was performed on the infraorbital nerve (ION) three days post-pIONL and on 24-hour cultured Schwann cells to identify inflammation-related genes and pathways. RT-qPCR, western blotting, immunofluorescent staining was used to analyze the expressions of Pannexin 1 channel and pro-inflammatory cytokines in vivo and in vitro studies. pIONL induced persistent neuroinflammatory responses and mechanical allodynia, which were ameliorated by LIPUS treatment. Panx 1 was highly expressed after pIONL., LIPUS treatment inhibited pIONL-induced neuroinflammation in ION, trigeminal ganglion, and spinal cord tissue. Inhibition of Panx 1 via siRNA significantly attenuated the mechanical allodynia. Several cytokines were inhibited by Panx 1-siRNA, both Panx 1-siRNA and LIPUS treatment suppressed several cytokines. Next, we cultured Schwann cells with TNF-α (200ng/ml). We found LIPUS effectively downregulated the expression of Panx 1 and pro-inflammatory cytokines in Vitro . Intra-ION injection of BzATP induced TNP, which was ameliorated by LIPUS along with downregulation of Panx 1 and pro-inflammatory cytokines,. RNA-sequencing analysis revealed that LIPUS downregulates pathways related to inflammation, ion channels, and metabolism in Schwann cells. This study demonstrates that LIPUS exerts an analgesic effect by targeting Panx1 in Schwann cells of the peripheral nervous system, thereby ameliorating neuroinflammation and providing sustained relief from TNP.
Abstract via crossref.
Exposures
Exposure 1: LIPUS to injured whisker pad / infraorbital nerve after partial infraorbital nerve ligation (pIONL)
Target: trigeminal nerve — “infraorbital nerve (ION) / whisker pad”
Device: other named manufacturer · ITO Co. · Osteotron V ✓
| Waveform | pulsed | |
|---|---|---|
| Fundamental frequency (kHz) | 1,500 | ✓✓✓ |
| Pulse duration (ms) | 200 | ✓✓✓⚑ |
| Pulse repetition frequency (Hz) | 1,500 | ✓✓✓ |
| Duty cycle (%) | 20pulse duration × PRF gives 30000%, which disagrees with the stated value | ✓✓✓ |
| Sonication duration (s) | 1,200 | ✓✓✓ |
| 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 | |
| Ispta, domain unspecified (W/cm²) | 0.03 | ✓✓✓⚑ |
Following the pIONL protocol, the LIPUS device was utilized on the injured whisker pad with a frequency of four or eight treatments, spanning days 0 to 3 and days 0 to 7, used for 20 minutes daily. The circular transducer has a cross-sectional area of 5.0 cm2, an active emitting surface of 4.1 cm2, and a beam non-uniformity ratio of 3.6.
Consistency checks: pd exceeds period.
Exposure 2: LIPUS stimulation of TNF-a-stimulated cultured Schwann cells
Target: other — “cultured Schwann cells (ATCC CRL-2766)”
Device: other named manufacturer · ITO Co. · Osteotron V
| Waveform | not reported | |
|---|---|---|
| Fundamental frequency (kHz) | 1,500 | ✓✓✓ |
| Pulse duration (ms) | not reported | |
| Pulse repetition frequency (Hz) | not reported | |
| Duty cycle (%) | not reported | |
| Sonication duration (s) | 1,200 | ✓✓✓ |
| Free-field pressure (kPa) | not reported | |
|---|---|---|
| Free-field Isppa (W/cm²) | not reported | |
| Free-field Ispta (W/cm²) | not reported | |
| 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 | |
| Ispta, domain unspecified (W/cm²) | 0.03 | ✓✓✓ |
Following a 24-hour incubation period in serum-free DMEM containing 200 ng/mL TNF-a, either with or without LIPUS stimulation (continuous 2-day treatment, day 0 to day 1, 1.5MHz, 30mW/cm2, 20min/day), the cells were extracted for gene analysis.
Flags from extraction
exposures[0].timing.pulse_duration_ms— Stated pulse width of 200 ms is inconsistent with the stated PRF of 1.5 kHz (period 0.667 ms) and duty cycle of 20% (implied pulse duration ~0.133 ms); likely a units typo (200 µs), but recorded as stated in the text per protocol.exposures[0].unspecified_domain.ispta_w_cm2— Paper reports 'spatial average temporal average intensity' (SATA), not spatial-peak temporal-average (ISPTA); recorded under ispta_w_cm2 as the closest schema field but the domains differ (spatial average vs spatial peak).n_subjects— Multiple experiments report different group sizes (e.g. n=5, n=5-8, n=3 per group across figures); no single overall total of LIPUS-exposed animals is stated.n_sessions_per_subject— In vivo LIPUS was given daily for either 4 or 8 treatments (days 0-3 or days 0-7) depending on sub-experiment/sacrifice timepoint; no single value applies to all subjects.exposures[1].timing— PRF, pulse duration and duty cycle are not restated for the in vitro Schwann cell experiments; only frequency, SATA intensity and daily duration are repeated from the in vivo protocol.