Therapeutic Effects and Mechanistic Insights of Low-Intensity Focused Ultrasound in Peripheral Neuropathic Pain

Abstract

Objective

To investigate the therapeutic effects and underlying mechanisms of low-intensity focused ultrasound (LIFU) in peripheral neuropathic pain (NP).

Methods

Eight-week-old Sprague-Dawley rats were randomly assigned to four groups: Normal (no treatment), Sham (sciatic nerve exposure only), chronic constriction injury (CCI), and CCI + LIFU (LIFU applied after injury). Behavioral tests were conducted 7–9 days post-intervention. Rats were then anesthetized and sacrificed for dorsal root ganglion (DRG) collection. RNA sequencing (RNA-seq) was performed to identify differentially expressed genes. Enzyme-linked immunosorbent assay (ELISA) and immunofluorescence were used to assess inflammatory cytokines. Electrophysiological changes were evaluated using electromyography and patch-clamp techniques at both nerve fiber and cellular levels.

Results

Compared with the CCI group, rats in the CCI + LIFU group showed significantly increased mechanical and thermal pain thresholds (n = 7, P < 0.05), with no difference between Sham and Normal groups. Electrophysiological analysis indicated reduced nerve fiber excitability and suppressed inward sodium currents following LIFU treatment (n = 5, P < 0.05). RNA-seq demonstrated downregulation of inflammation-related genes in the CCI + LIFU group. Consistently, ELISA and immunofluorescence revealed significantly decreased expression of tumor necrosis factor-α, interleukin-1β, and calcitonin gene-related peptide compared with the CCI group.

Conclusion

LIFU can alleviate peripheral NP by inhibiting the transmission of pain signals from peripheral injury sites to the DRG, thereby achieving sustained blockade of nociceptive signaling and effectively reducing peripheral sensitization.

Introduction

Neuropathic pain (NP) is a chronic secondary pain disorder caused by primary or secondary damage or dysfunction of the central or peripheral nervous system.¹ The prevalence of chronic pain with neuropathic characteristics is estimated to range from 7% to 10%.² NP conditions are generally classified into two major categories: central and peripheral NP. Central NP includes pain resulting from spinal cord or brain injury, post-stroke pain, and pain associated with multiple sclerosis. Peripheral NP includes trigeminal neuralgia, peripheral nerve injury, painful polyneuropathy, postherpetic neuralgia, and painful radiculopathy.³

The mechanisms underlying NP are complex and remain incompletely understood. Multiple factors, including activation of inflammatory cells, peripheral and central sensitization, and oxidative stress, have been reported to contribute to its development.^4–6 Owing to the complex etiology of NP, current clinical treatments remain unsatisfactory. Existing pharmacological therapies are often unable to provide adequate pain relief, which severely affects patients’ quality of life.^7,8

At present, the main therapeutic approaches for NP include pharmacological therapy, interventional treatment, and psychological therapy. Pharmacological treatments mainly involve opioids, N-methyl-D-aspartate receptor antagonists, anticonvulsants, antidepressants, calcitonin, and anesthetics. However, the long-term clinical effectiveness of these treatments remains uncertain, and they are often associated with considerable adverse effects.⁹ For patients with NP who respond poorly to pharmacological therapy, alone or in combination with non-pharmacological treatments, interventional therapy is usually considered; however, its efficacy is often limited. Meanwhile, there is insufficient evidence supporting the effectiveness and safety of psychological interventions for chronic NP.¹⁰ Therefore, a deeper understanding of NP and the development of novel, safer, and more effective therapeutic strategies based on its underlying mechanisms are urgently needed.

Focused ultrasound (FU) provides a foundation for therapeutic ultrasound applications in clinical practice due to its unique mechanical and thermal effects.¹¹ The thermal effect refers to the physical heating of target tissues caused by the absorption of ultrasonic energy. At relatively low deposited energy doses (<55°C), the induced hyperthermia can increase cellular permeability, thereby facilitating drug delivery. At higher deposited energy doses (>55°C), coagulative necrosis occurs, leading to cell death. The mechanical effects include radiation force, increased pressure, and acoustic cavitation. Acoustic cavitation refers to the oscillation and collapse of gas-containing bubbles generated in tissues under FU exposure, which can increase cellular permeability and promote intercellular transport of substances.^12,13

A growing number of animal studies have reported the neuromodulatory potential of FU.^14,15 Therefore, in the present study, we attempted to apply FU in the treatment of chronic NP to evaluate its therapeutic effects and explore the underlying mechanisms.

Materials and Methods

Experimental Animals

Sixty 8-week-old Sprague–Dawley (SD) rats were purchased from the Experimental Animal Center of Chongqing Medical University. All animal experiments were approved by the Institutional Animal Care and Use Committee and were conducted in accordance with established guidelines for the ethical use of laboratory animals.

Animal Model Establishment

Eight-week-old female SD rats weighing 200–250 g were randomly divided into four groups: the normal group (Normal), which received no intervention; the sham group (Sham), in which the left sciatic nerve was exposed without further manipulation; the chronic constriction injury group (CCI), in which the left sciatic nerve was exposed and ligated; and the intervention group (CCI + low-intensity focused ultrasound [LIFU]), in which LIFU was applied to the injured sciatic nerve after ligation. Following CCI induction, rats with comparable pain sensitivity, as assessed by the von Frey test, were selected and randomly allocated to the CCI control group or the CCI + LIFU treatment group.

For the CCI procedure, rats were anesthetized using an animal anesthesia machine (R620-S1-IECP; RWD, China). Anesthesia was induced and maintained with 2% isoflurane administered via inhalation using an anesthesia machine. The left sciatic nerve was carefully exposed and loosely ligated with 4–0 silk sutures. The ligature tightness was adjusted until a slight twitch of the gastrocnemius muscle was observed, confirming appropriate nerve constriction. The overlying muscle and skin were then carefully sutured, and the animals were allowed to recover after surgery.

For LIFU intervention, the incision was completely healed after CCI surgery, and stimulation was performed transcutaneously. A B-mode color Doppler ultrasound system (DC-8; Mindray, China) was used to determine the depth and location of the sciatic nerve before LIFU stimulation. Ultrasound stimulation was delivered using an FU probe (SY23WX002; Chongqing Haifu, China) connected to an ultrasound generator (Waveform Generator; Chongqing Haifu). An appropriate probe was selected to ensure that the sciatic nerve trunk was positioned within the ultrasound focal zone. Based on previously reported parameters,¹⁶ ultrasound stimulation was applied for 3 min with a center frequency of 3.745 MHz, pulse repetition frequency of 0.5 Hz, acoustic pressure of 0.75 MPa, and duty cycle of 5%. After behavioral differences were observed (7–9 days after intervention), rats were anesthetized with isoflurane (2% for induction followed by 5% for deep anesthesia) delivered via inhalation until respiratory arrest, and subsequently euthanized by cervical dislocation in accordance with the AVMA Guidelines for the Euthanasia of Animals. Transcardial perfusion with phosphate-buffered saline (PBS) was then performed, and dorsal root ganglia (DRG) were rapidly harvested for subsequent experiments.

Behavioral Assessment

Seven rats per group were used for behavioral testing.

Von Frey test: Mechanical sensitivity was evaluated using a von Frey filament apparatus (Aesthesio, USA) with nine filaments of graded forces. The median filament was initially applied to the mid-plantar surface of the hind paw. Each filament was applied until it bent into a “C” or “S” shape and maintained for 6–8s. Paw withdrawal responses were recorded. Each filament was tested five times with a 15-s interval. A positive response was defined as at least three withdrawals out of five stimulations. The up–down method was used to calculate the 50% paw withdrawal threshold.

Thermal withdrawal test: Thermal nociception was assessed using an intelligent hot plate analyzer (SY-YLS-6BS; Shanghai Shaoyi Biotechnology, China). The hot plate was maintained at 50°C. Rats were placed on the heated surface, and the latency to paw withdrawal or paw licking was recorded as the thermal withdrawal latency.^17,18 The hot plate test was not performed repeatedly at consecutive time points because it involves noxious thermal stimulation that may cause additional stress or discomfort to the animals. Therefore, the number of hot plate assessments was minimized to reduce unnecessary stimulation while obtaining reliable behavioral measurements. During the behavioral experiments, the investigators responsible for testing were blinded to the group allocation and treatment conditions of each animal.

Electrophysiological Recording

Electromyography: In the control group, the sciatic nerve was exposed without further treatment. In the experimental group (LIFU), the exposed sciatic nerve was irradiated using FU delivered by the Chongqing Haifu ultrasound system with a transducer center frequency of 3.745 MHz, pulse repetition frequency of 0.5 Hz, acoustic pressure of 0.75 MPa, and duty cycle of 5% for 1 min. Electrical stimulation (0.4 mA, 1 Hz) was applied to the sciatic nerve, and distal compound muscle action potentials (CMAPs) were recorded using an electromyography monitoring system (MP160; Puseng Technology, China).¹⁹ The average peak amplitude of a stable positive waveform segment was used for statistical analysis.

Patch-clamp recording: DRG from SD rats were isolated for primary neuronal culture and randomly assigned to experimental groups. Whole-cell recordings were performed using a patch-clamp system (EPC 10 USB; HEKA, Germany). In the control group, neurons received no treatment. In the LIFU group, neurons were exposed to FU stimulation with a transducer center frequency of 1 MHz, acoustic intensity of 0.05 W/cm², pulse repetition frequency of 1 Hz, and duty cycle of 20% for 30s. In the LIFU + 1 h group and LIFU + 2 h group, neurons were incubated for an additional 1 h or 2 h after ultrasound exposure before electrophysiological recording.

RNA Sequencing

DRG tissues were collected as described above, immediately frozen in liquid nitrogen, and subsequently sent to Beijing Genomics Institute sequencing services for transcriptomic analysis.

Enzyme-Linked Immunosorbent Assay

DRG tissues were collected as described above. Tissues were rinsed with precooled PBS, weighed, and minced. Samples were homogenized in PBS at a ratio of 1:9 (g/mL) using ultrasonic disruption. After centrifugation at 5000 g for 10 min at 4°C, the supernatant was collected. Levels of inflammatory cytokines were measured using enzyme-linked immunosorbent assay kits (Jingmei, China) according to the manufacturer’s instructions.

Immunofluorescence

DRG tissues were collected as described above and fixed in 4% paraformaldehyde (BL539A; Biosharp, China) or specialized neural tissue fixative for 24–72 h. Tissues were then processed for embedding, sectioning, antigen retrieval, permeabilization, blocking, primary antibody incubation, washing, secondary antibody incubation, nuclear staining, and mounting. The following primary antibodies were used: interleukin (IL)-1β antibody (YM-A05643, China), tumor necrosis factor (TNF)-α antibody (AB307164; Abcam, UK), glial fibrillary acidic protein (GFAP) antibody (GB11096-100; Servicebio, China), and calcitonin gene-related peptide (CGRP) antibody (14959; Cell Signaling Technology, USA). Fluorescence images were captured using fluorescence microscopy.

Statistical Analysis

Statistical analyses were performed using GraphPad Prism (version XX). Data are presented as mean ± standard deviation (SD). Comparisons between two groups were performed using an unpaired two-tailed Student’s t-test. Comparisons among multiple groups were performed using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test. For experiments involving repeated measurements over time, two-way ANOVA followed by appropriate post hoc tests was applied. A P value < 0.05 was considered statistically significant.

Results

Focused Ultrasound Mitigates Chronic Constriction Injury-Induced Pain

Previous studies have reported that FU can inhibit the peripheral nervous system and has potential as a tool for pain management,^14,15 although the difficulty of focusing ultrasound on small targets has also been noted. Therefore, we used B-mode ultrasound to locate the depth and position of the sciatic nerve and selected an appropriate ultrasound probe to ensure that the sciatic nerve was positioned as close as possible to the focal region of the transducer, thereby substantially improving the probability of successful intervention. Prior to FU treatment, no significant difference in mechanical pain threshold was observed between Sham and Normal. In contrast, rats subjected to sciatic nerve ligation showed a marked reduction in pain threshold, indicating the development of pain sensitization and confirming the successful establishment of the CCI NP model. After FU intervention, the treatment group (CCI + LIFU) exhibited behavioral improvement in pain hypersensitivity compared with the untreated CCI group (Figure 1).

Figure 1.

Experimental procedure and behavioral assessments. (a) Schematic illustration of the experimental procedure: (i) establishment of the chronic constriction injury (CCI) model; (ii) ultrasound localization of the sciatic nerve; and (iii) focused ultrasound (FU) treatment (0.75 MPa, 0.5 Hz, 4 min). (b) Behavioral testing workflow. (c) Hot plate test showing thermal withdrawal latency. Data were analyzed using a t-test (n = 7). ns P > 0.05. **P < 0.01. (d) Changes in mechanical pain threshold over the treatment period (n = 7). LIFU, CCI rats treated with FU; CCI, CCI rats without intervention; Sham, sham-operated rats with sciatic nerve exposure without ligation; Normal, untreated rats.

Abbreviation: LIFU, low-intensity focused ultrasound.

Focused Ultrasound Reduces Nerve Fiber Excitability

This experiment was performed according to procedures described in previous studies to further validate the inhibitory effect of FU on nerve fibers.¹⁶ The exposed sciatic nerve was irradiated with FU (0.75 MPa, 0.5 Hz, 1 min). Electrical stimulation (0.4 mA, 1 Hz) was then applied to the sciatic nerve, and distal CMAPs were recorded using an electromyography monitoring system. The peak amplitude of the positive waveform was measured for quantitative analysis (Figure 2).

Figure 2.

Effects of focused ultrasound on sciatic nerve electrophysiological activity. (a) Experimental setup: electrical stimulation (0.4 mA, 1 Hz) was applied to the sciatic nerve following focused ultrasound (FU) intervention (0.75 MPa, 0.5 Hz, 1 min), and electromyographic (EMG) signals were recorded. (b) Representative EMG waveforms from the control group and the FU intervention group. Positive deflections above the baseline represent the positive amplitude. (c) The average value of the positive amplitudes from eight consecutive and stable waveforms was calculated as one data point. (d) Statistical comparison between the control and intervention groups. Data were analyzed using a t-test (n = 5). ****P < 0.0001.

Abbreviation: LIFU, low-intensity focused ultrasound.

Focused Ultrasound Suppresses Inward Sodium Currents

To further investigate the effects of FU on neuronal excitability, primary neurons derived from DRG were isolated and cultured. After incubation for 3–4 hours, neurons in the control group received no treatment, whereas neurons in the LIFU group were exposed to FU stimulation (0.05 W/cm², 1 Hz, 30s). In the LIFU + 1 h and LIFU + 2 h groups, neurons were incubated for an additional 1 h or 2 h after ultrasound exposure before electrophysiological recording. The results showed that FU significantly suppressed inward sodium currents. Notably, under relatively low-stimulation parameters, this change appeared to be reversible. We speculate that the mechanical energy generated by FU may alter the morphology of the cell membrane and the conformation of ion channels, thereby affecting sodium ion conductance (Figure 3).

Figure 3.

Effects of focused ultrasound on neuronal electrophysiological activity. (a) Neurons were subjected to focused ultrasound stimulation (0.05 W/cm², 1 Hz, 30s) prior to electrophysiological recording. (b) Representative traces of continuous recordings. (c) Experimental groups: Control, untreated neurons; LIFU, neurons immediately recorded after ultrasound stimulation; LIFU + 1 h, neurons recorded after 1 h incubation following the same ultrasound treatment; LIFU + 2 h, neurons recorded after 2 h incubation following the same ultrasound treatment. (d) Quantitative analysis of electrophysiological data. Data were analyzed using a t-test (n = 5). ns P > 0.05 **P < 0.01, ****P < 0.0001.

Abbreviation: LIFU, low-intensity focused ultrasound.

RNA-Sequencing Suggests Downregulation of Inflammation-Related Genes in the Dorsal Root Ganglia Following Focused Ultrasound Intervention

To further identify biologically relevant and statistically significant terms, GO functional enrichment analysis was conducted based on the differentially expressed genes. The analysis revealed a relative downregulation of chemokine activity-related pathways in the CCI + LIFU group. In addition, a substantial proportion of inflammation-related differentially expressed genes were downregulated following LIFU treatment, including GFAP, C-C motif chemokine ligand (CCL)2, and CCL7. These findings suggest that FU may alleviate peripheral NP by blocking or attenuating the transmission of nociceptive signals from the peripheral injury site to the DRG. The reduction in persistent nociceptive input may subsequently lead to alterations in gene expression profiles (Figure 4).

Figure 4.

Transcriptomic analysis of gene expression changes. (a) Bubble plot of GO functional enrichment analysis. (b) Bar plot of GO functional enrichment analysis for molecular function (MF) terms. (c) Heatmap showing hierarchical clustering of differentially expressed genes. (d) Relative downregulation of genes associated with satellite glial cell activation and inflammation. (e) Volcano plot showing differentially expressed genes.

Focused Ultrasound Alleviates Peripheral Sensitization in Chronic Constriction Injury Model Rats

Based on the RNA sequencing (RNA-seq) results, the local microenvironment of the DRG in the CCI and CCI + LIFU groups may differ in terms of inflammatory infiltration. On the one hand, activation of GFAP in satellite glial cells within the DRG is regulated by multiple signaling pathways (such as nuclear factor-kappa B and P2 receptors). Increased GFAP expression promotes glial cell activation, inflammatory responses, and neuronal dysfunction, thereby contributing to the pathophysiological processes underlying chronic pain and neuropathy.^20,21 On the other hand, substantial evidence suggests that inflammation itself is a key driver of peripheral sensitization.⁵ Therefore, we further evaluated the expression of GFAP, inflammatory cytokines (TNF-α and IL-1β), and the nociceptive mediator CGRP to investigate differences in peripheral sensitization between the two groups (Figures 5 and 6).

Figure 5.

Immunohistochemical analysis of glial fibrillary acidic protein expression. (a) Representative images of glial fibrillary acidic protein immunohistochemical staining (Scale bar: 100 μm). (b) Three representative regions were selected for analysis, and the mean optical density (MOD) was calculated and averaged. (c) Quantitative analysis of MOD values (n = 5). Data were analyzed using a t-test. **P < 0.01.

Abbreviations: CCI, chronic constriction injury; LIFU, low-intensity focused ultrasound.

Figure 6.

Immunofluorescence and inflammatory cytokine analysis. (a) Representative images of triple-label immunofluorescence staining (Scale bar: 20um). (b) Quantitative analysis of mean fluorescence intensity (n = 4). Data were analyzed using a t-test. *P < 0.05, **P < 0.01, (c) Enzyme-linked immunosorbent assay results showing cytokine levels (n = 4). Data were analyzed using a t-test. ***P < 0.001. ****P < 0.0001.

Abbreviations: TNF, tumor necrosis factor; IL, interleukin; CGRP, calcitonin gene-related peptide; LIFU, low-intensity focused ultrasound; CCI, chronic constriction injury.

Discussion

In this study, we investigated the neuromodulatory effects of LIFU in a CCI rat model of NP. Behavioral results showed that LIFU significantly increased the pain threshold and alleviated pain hypersensitivity in CCI rats. Electrophysiological recordings demonstrated that LIFU reduced nerve fiber excitability and suppressed inward sodium currents in DRG neurons. RNA-seq analysis further revealed that LIFU was associated with the downregulation of several inflammation-related genes in the DRG. Consistently, subsequent molecular analyses suggested that LIFU attenuated peripheral sensitization by modulating inflammatory responses and nociceptive signaling in the DRG. Collectively, these findings indicate that LIFU may relieve NP through both the inhibition of neuronal excitability and the regulation of local inflammatory processes.

FU can produce a variety of biological effects, including neuromodulation and anti-inflammatory responses.²² Previous studies have frequently suggested that the nerve-blocking effect of FU may be related to its thermal effects.¹⁵ However, based on our observations, this characteristic may be more closely associated with its mechanical effects. On the one hand, ultrasound, as a mechanical wave, exerts mechanical forces on the target tissue, which are inherently accompanied by a certain degree of temperature elevation.²³ In the early stage of our experiments, we indeed observed skin burns when the irradiation duration was prolonged. However, as the acoustic energy attenuates with depth, no additional damage to deeper tissues was detected beyond the original injury. On the other hand, in patch-clamp experiments, FU stimulation did not cause a detectable increase in the temperature of the culture medium, yet a reversible inhibitory effect on cellular activity was still observed. Previous studies have also reported that FU can affect cell membrane morphology,²⁴ demonstrating that ultrasound exposure may induce structural alterations of the cell membrane, with the extent of these changes depending on membrane composition. Therefore, we speculate that mechanical effects may play a dominant role, whereas temperature changes may represent a secondary phenomenon. Nevertheless, it should be noted that elevated environmental temperature alone may also influence neuronal function under certain conditions.²⁵

We propose that FU can exert a nerve-blocking effect; however, the underlying mechanism may involve a “disruptive” action characterized by alterations in cell membrane morphology and suppression of ion channel function. In this regard, the mechanism shares certain similarities with that of local anesthetics used for nerve blockade. Local anesthetics block signal conduction in nerve fibers, thereby reducing or preventing the transmission of harmful signals to the dorsal root ganglion,²⁶ which represents a protective process. Experimental and clinical evidence has shown that early blockade of injured primary sensory afferents can reduce the activation of satellite glial cells.²⁷ Since activation of satellite glial cells contributes to inflammatory responses and persistent pain sensitization,²¹ FU, given its high targeting precision and noninvasive nature, may have considerable therapeutic potential for the treatment of NP when applied with appropriate parameters.

Conclusions

LIFU alleviates NP in the CCI model by reducing neuronal excitability and modulating inflammatory responses in the DRG. These findings suggest that FU may represent a promising noninvasive neuromodulation strategy for the treatment of NP.

Funding Statement

This study was supported by a horizontal project of Nanchong Guocheng Orthopedic Hospital in Sichuan Province (contract number 2021090010).

Data Sharing Statement

The datasets generated and analyzed in the present study are available from the corresponding author, Min Feng, upon reasonable request.

Ethics Approval and Informed Consent

All animal experiments were approved by the Institutional Animal Care and Use Committee of Chongqing Medical University (reference number: IACUC-CQMU-2025-11013; study-specific approval number: IACUC-CQMU-2026-0320), and conducted under animal experiment permit number SYXK (CQ) 2022-0016. All procedures involving laboratory animals were performed in accordance with the Laboratory Animal—Guideline for Ethical Review of Animal Welfare (GB/T 35892-2018), as well as the ethical standards approved by the Institutional Animal Care and Use Committee of Chongqing Medical University.

Author Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Disclosure

The authors declare that they have no competing interests.