Introduction

Neuropathic pain, caused by lesions or diseases in the somatosensory system, is classified as peripheral (e.g., trigeminal neuralgia, postherpetic neuralgia) or central (e.g., spinal cord injury, stroke-related pain) based on affected nervous system regions1. It presents with spontaneous burning, electric-shock-like, or tingling sensations and evoked symptoms like allodynia and hyperalgesia2. Patients often experience comorbidities such as sleep disturbances, anxiety, and depression, severely impacting quality of life. Rising global prevalence is linked to aging populations, increased diabetes, and cancer survivorship3. Despite advances in understanding its pathophysiology, treatment remains challenging due to complex maladaptive plasticity in pain pathways, sustaining its status as a key focus in pain research.

Accumulating evidence highlights the pivotal role of neuroinflammation in both the initiation and perpetuation of neuropathic pain4,5. This pathological process is mediated by glial cell activation and immune cell recruitment within the peripheral and central nervous systems6,7. Microglia, the principal immunocompetent cells orchestrating neural homeostasis in the CNS, exhibit particular significance in this context. Emerging evidence indicates that activated microglia in the spinal dorsal horn critically contribute to neuropathic pain development following neural injury8. Following activation, these cells initiate a proinflammatory cascade through sustained release of cytokines (IL-1β, IL-6, TNF-α), which directly sensitize spinal nociceptive neurons and potentiate pain signal transmission, thereby exacerbating neuropathic pain states9,10.

The NF-κB signaling pathway is recognized as a pivotal pro-inflammatory signaling cascade, primarily attributed to its activation by pro-inflammatory cytokines and its regulatory role in the expression of various inflammatory mediators including cytokines, chemokines, and adhesion molecules11. Under physiological conditions, NF-κB remains sequestered in the cytoplasm through its interaction with the inhibitory protein IκB. Upon stimulation by inflammatory, oxidative, or mechanical stressors, IκB undergoes phosphorylation by IκB kinase (IKK), followed by ubiquitination and proteasomal degradation. This degradation process facilitates NF-κB phosphorylation, subsequent nuclear translocation, and binding to specific DNA consensus sequences, thereby initiating transcription of target genes such as tumor necrosis factor-alpha (TNF-α)12. Current research demonstrates that NF-κB pathway activation is significantly associated with neuropathic pain pathogenesis9, Importantly, pharmacological inhibition of this pathway has shown therapeutic efficacy in alleviating neuropathic pain symptoms13,14.

Current pharmacological management remains the cornerstone of neuropathic pain treatment, yet clinically available agents often fail to provide satisfactory pain relief while inducing dose-limiting adverse effects. Commonly prescribed agents (anticonvulsants, antidepressants) frequently induce adverse neurological and cardiovascular complications including dizziness, somnolence, severe cephalalgia, and hypertension, substantially compromising treatment adherence and clinical efficacy15. This therapeutic impasse underscores the urgent need for developing safer and more effective analgesic strategies. Ropivacaine, a long-acting amide-type local anesthetic, has gained prominence in perioperative analgesia due to its superior sensory blockade profile and enhanced central nervous/cardiovascular safety margin. Emerging evidence confirms its therapeutic potential in neuropathic pain management, with demonstrated capacity to suppress NF-κB pathway activation and subsequent proinflammatory cytokine release16,17. Notably, the precise mechanistic relationship between ropivacaine’s antinociceptive effects and NF-κB modulation in neuropathic pain pathogenesis remains uncharacterized.

To address this knowledge gap, we employed a rat peripheral nerve injury model to investigate ropivacaine-NF-κB signaling interactions systematically. Our findings are anticipated to elucidate the neuroimmunological mechanisms underlying ropivacaine-mediated pain alleviation, thereby providing novel therapeutic insights for optimizing neuropathic pain management protocols.

Materials and methods

Animals and ethics statement

Forty male Sprague-Dawley (SD) rats (6–8 weeks old, weighing 210–250 g) were obtained from Jinan Pengyue Experimental Animal Breeding Co., Ltd. The animals were maintained under controlled environmental conditions with temperature regulated at 22 ± 0.5 °C, relative humidity of 60% ± 5%, and a standardized 12-hour light/dark cycle. All subjects were provided ad libitum access to food and water throughout the experimental period. The design, conduct, and reporting of this study were performed in strict accordance with the ARRIVE 2.0 guidelines (Animal Research: Reporting of In Vivo Experiments). The study protocol received formal approval from the ethics committee of Qingdao Hospital of Traditional Chinese Medicine (Qingdao Hiser Hospital) (Approval No. 2022HC10LS007), with strict adherence to the ethical guidelines for experimental animals established by the International Association for the Study of Pain (IASP).

Grouping and model

Animal Grouping and Surgical Procedures: Forty male Sprague-Dawley rats were randomly assigned to four groups (n = 10/group): Sham control, spared nerve injury (SNI) model, SNI + normal saline (NS), and SNI + ropivacaine (Ropi).

Intrathecal Catheterization: PE-10 polyethylene catheters (14 cm length) were pre-treated by wrapping 4 − 0 silk sutures at 7.5 cm from the distal end to mark insertion depth, sterilized in 75% ethanol for 15 min, and rinsed with sterile saline. Rats were anesthetized intraperitoneally with 40 mg/kg pentobarbital sodium and secured in a stereotaxic frame. Following cervical disinfection, a 2–3 cm midline incision was made to expose the atlanto-occipital membrane. With the head flexed 30° ventrally, the membrane was punctured using an ophthalmic needle. Cerebrospinal fluid (CSF) efflux confirmed successful dural penetration, whereupon the catheter was advanced intrathecally until CSF reflux was observed. The catheter was secured by suturing deep and superficial cervical muscles, with the external orifice sealed for subsequent drug administration.

SNI Model Establishment (1 Week Post-catheterization): Under pentobarbital anesthesia (40 mg/kg i.p.), the sciatic nerve and its branches (sural, common peroneal, and tibial nerves) were exposed. The common peroneal and tibial nerves were tightly ligated at their trifurcation and transected distal to the ligature, with 3–5 mm of nerve tissue excised. The sural nerve remained intact. Sham controls underwent nerve exposure without ligation. Postoperative penicillin (40,000 U/day) was administered for 3 days to prevent infection.

Drug Administration: From postoperative day 1, the SNI + Ropi group received daily intrathecal injections of 0.5% ropivacaine (0.12 µl/100 g), while the SNI + NS group received equivalent volumes of normal saline, both continuing for 7 consecutive days.

Threshold testing of paw withdrawal response to mechanical stimulation

All experimental time points were designated as Day 0 (preoperative baseline) followed by postoperative days 1, 3, 7, 10, and 14 for behavioral assessments. Prior to testing, animals were acclimated to the experimental environment for 30 min. Mechanical withdrawal thresholds were determined using calibrated Von Frey filaments (0.2–25 g range) through the up-down method as described by Chaplan et al.18 The 50% paw withdrawal threshold was calculated using their established formula. To maintain blinding, all behavioral assessments were conducted by a single investigator (LW) who remained unaware of group assignments throughout the study period.

Immunofluorescence staining technique

Following anesthesia, rats underwent transcardial perfusion with 300 mL of 0.9% saline followed by 250 mL of 4% paraformaldehyde. The L4–L6 spinal cord segment was isolated for subsequent processing. After overnight post-fixation at 4 °C, tissues were cryoprotected in 0.1 M phosphate-buffered saline (PBS) containing 30% sucrose at 4 °C. Transverse spinal cord Sect. (20 μm thickness) were prepared using a LEICA CM1900 UV cryostat. Primary antibodies for immunofluorescence included: Iba-1 (1:400; rabbit polyclonal; ABclonal).Imaging was performed using a Sony digital camera (E3ISPM) with consistent 20× magnification. Fluorescence intensity quantification was conducted using ImageJ software (National Institutes of Health) after background subtraction. For Iba-1 analysis, three randomly selected L5 spinal cord sections per animal were imaged, focusing on the dorsal horn regions. Intensity values were acquired and calculated using the ImageJ computer-assisted imaging analysis system.

Western blot (WB)

Spinal cord segments L4–L6 were used for subsequent experiments. Protein quantification was performed using a BCA Protein Assay Kit (Solarbio, Beijing, China). For each sample, 20 µg of protein was loaded per lane and separated on 10% or 12.5% SDS-PAGE gels before electrophoretic transfer onto 0.45 μm nitrocellulose membranes.Membranes were incubated with the following primary antibodies: TRAF2 (1:10,000), Akt (1:5000), p-Akt (1:1000), IκBα (1:1000), p-IκBα (1:1000), p65 (1:5000), p-p65 (1:5000), and β-actin (1:10,000)—all rabbit polyclonal antibodies from ABclonal (Wuhan, China). Following primary antibody incubation, membranes were probed with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:5000, ABclonal). Protein signals were visualized using an enhanced chemiluminescence (ECL) detection system (Pierce Biotechnology, Rockford, IL, USA). To normalize protein loading and transfer efficiency, β-actin (1:10,000, ABclonal) was used as an internal reference. Quantitative analysis of band intensity was performed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Quantitative real-time PCR (qRT-PCR)

Spinal cord segments L4-L6 were used for subsequent experiments. Total RNA was extracted using the AFTSpin Tissue/Cell Fast RNA Extraction Kit for Animal (ABclonal), followed by cDNA synthesis with the ABScript III RT Master Mix (ABclonal). qRT-PCR analysis was performed on the Mx3000P SYBR Green system using 2 × SYBR Green qPCR Master Mix (ABclonal). The primer sequences were as follows: GAPDH (Forward: 5′-TGAAGGTCGGTGTCAACGGATTTGGC-3′; Reverse: 5′-CATGTAGGCCATGAGGTCCACCAC-3′); TNF-α (Forward: 5′-TACTGAACTTCGGGGTGATTGGTCC-3′; Reverse: 5′-CAGCCTTGTCCCTTGAAGAGAACC-3′); IL-1β (Forward: 5′-CCTTGTGCAAGTGTCTGAAGC-3′; Reverse: 5′-CCCAAGTCAAGGGCTTGGAA-3′); IL-6 (Forward: 5′-CCAAGAGGTGAGTGCTTCCC-3′; Reverse: 5′-CTGTTGTTCAGACTCTCTCCCT-3′). Data were expressed as relative Ct values, with relative expression levels calculated using the 2−ΔΔCt method.

Statistical analysis

Statistical analyses were performed using GraphPad Prism 10. Data are expressed as mean ± standard deviation (SD). Behavioral assessments were analyzed by two-way ANOVA followed by Tukey’s post hoc test for intergroup comparisons at identical time points. Comparisons of fluorescence intensity, WB data, and qRT-PCR results were conducted using Student’s t test. A p value < 0.05 was considered statistically significant.

Results

The SNI model was successfully constructed

The spared nerve injury (SNI) model was surgically induced via selective ligation and transection of the tibial and common peroneal nerves while preserving the sural nerve. Mechanical withdrawal thresholds of the ipsilateral hindpaw were quantitatively assessed using von Frey filaments at baseline (preoperative day) and postoperative days 1, 3, 7, 10, and 14. Compared with the Sham group, the SNI group exhibited significant mechanical hypersensitivity from postoperative day, with sustained allodynic responses persisting through day 14 (Fig. 1). These characteristic neuropathic pain manifestations confirmed successful model establishment.

Fig. 1
figure 1

Changes in 50% PWMT across experimental groups. Data are expressed as mean ± SD (n = 10). *p < 0.05 compared with the Sham group. Statistical significance was determined using two-way ANOVA with Tukey’s post hoc analysis.

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Intrathecal administration of ropivacaine alleviates SNI-induced neuropathic pain

Postoperatively, the SNI + Ropi group received daily intrathecal injections of 0.5% ropivacaine at a dose of 0.12 µl/100 g body weight, while the SNI + NS group was administered equivalent volumes of normal saline. Comparative analysis revealed that the SNI + Ropi group exhibited significant attenuation of mechanical allodynia induced by SNI compared to the SNI + NS group (Fig. 2). Pharmacodynamic evaluation demonstrated a sustained analgesic effect lasting approximately 4 h post-administration, with peak efficacy observed at 2 h (Fig. 3).

Fig. 2
figure 2

Changes in 50% PWMT across experimental groups. Data are expressed as mean ± SD (n = 10). #p < 0.05 compared with the SNI + NS group. Statistical significance was determined using two-way ANOVA with Tukey’s post hoc analysis.

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Fig. 3
figure 3

Temporal changes in 50% PWMT of the SNI + Ropi group pre- and post-administration. Data are expressed as mean ± SD (n = 10). Asterisks (*) denote statistically significant differences (p < 0.05) at 1, 2, 3, and 4 h post-administration compared with baseline (0 h). Statistical significance was determined using two-way ANOVA followed by Tukey’s post hoc test.

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Intrathecal administration of ropivacaine significantly suppressed SNI-induced spinal microglial activation and reduced pro-inflammatory cytokine levels

Given the pivotal role of neuroinflammation in neuropathic pain progression, we assessed microglial activation (the primary immunocompetent cells in CNS) by quantifying Iba-1 expression in ipsilateral spinal dorsal horns using immunofluorescence. Quantitative analysis revealed marked microglial proliferation and activation in SNI group compared to Sham controls, accompanied by significantly elevated mRNA expression of TNF-α, IL-1β, and IL-6 (Fig. 4), confirming SNI-induced neuroinflammatory responses. Notably, ropivacaine treatment attenuated microglial hyperactivation in SNI + Ropi group versus SNI + NS controls, with concurrent downregulation of these pro-inflammatory cytokines (Fig. 5). These findings demonstrate that ropivacaine mitigates neuroinflammation through mechanisms: inhibiting microglial activation and suppressing pro-inflammatory mediator release.

Fig. 4
figure 4

Following behavioral testing on day 14 post-surgery, rat spinal cord tissues were collected for: A immunofluorescence staining of Iba-1 in the dorsal horn (× 20 magnification; scale bar: 50 μm), and B qRT-PCR analysis of TNF-α, IL-1β, and IL-6 mRNA levels. Bar graphs represent mean ± SD (n = 3). *p < 0.05 vs. Sham group. Statistical significance was determined using Student’s t test.

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Fig. 5
figure 5

Following behavioral testing on day 14 post-surgery, rat spinal cord tissues were collected for: A immunofluorescence staining of Iba-1 in the dorsal horn (× 20 magnification; scale bar: 50 μm). B qRT-PCR analysis of TNF-α, IL-1β, and IL-6 mRNA levels bar graphs represent mean ± SD (n = 3). #p < 0.05 vs. SNI + NS group. Statistical significance was determined using Student’s t test.

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Ropivacaine alleviates SNI-induced neuroinflammation by suppressing NF-κB activation

The experimental results demonstrated that ropivacaine effectively attenuated SNI-induced neuropathic pain and concurrently inhibited SNI-triggered neuroinflammatory responses. We hypothesized that this effect might be associated with suppression of NF-κB activation, as prior evidence indicates ropivacaine’s ability to inhibit TNF-α-stimulated NF-κB activation in cellular models17.

To validate this mechanism, we further investigated whether ropivacaine’s anti-neuroinflammatory action involves modulation of the NF-κB signaling pathway. Previous studies established that TNF receptor 2 (TNFR2) is preferentially activated by transmembrane TNF-α, leading to recruitment of TNF receptor-associated factor 2 (TRAF2) to intracellular TRAF-binding domains. Subsequent TRAF2 activation stimulates the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) cascade, thereby inducing IκB phosphorylation and subsequent NF-κB nuclear translocation19.

Quantitative analysis of protein expression in ipsilateral spinal cord tissue revealed significant upregulation of TRAF2, phosphorylated Akt (p-Akt), phosphorylated IκBα (p-IκBα), and phosphorylated p65 (p-p65) in SNI-operated rats compared to sham controls. Given that p-IκBα degradation and p-p65 nuclear translocation are canonical hallmarks of NF-κB pathway activation11, these findings confirm NF-κB signaling activation during SNI-induced neuroinflammation. Notably, intrathecal administration of ropivacaine (SNI + Ropi group) substantially reduced the expression levels of these phosphorylated signaling molecules relative to the NS-treated SNI group (SNI + NS), demonstrating ropivacaine’s potent inhibitory effect on NF-κB activation (Fig. 6).

Fig. 6
figure 6

Following behavioral testing on day 14 post-surgery, rat spinal cord tissues were collected for Western blot (WB) analysis. A Protein levels of AKT, p-AKT, IκB, p-IκB, p65, p-p65, and TRAF2 in spinal cords from the four groups: Sham, SNI, SNI + NS, and SNI + Ropi. B Quantified Western blots of p-AKT/AKT, p-IκB/IκB, p-p65/p65, and TRAF2/β-actin ratios in the Sham group versus the SNI group. C Quantified Western blots of p-AKT/AKT, p-IκB/IκB, p-p65/p65, and TRAF2/β-actin ratios in the SNI + NS group versus the SNI + Ropi group. Quantitative assessment of protein levels is presented as mean ± SD (n = 3). Statistical significance (*p < 0.05) was determined relative to the Sham group, while (#p < 0.05) denotes comparison with the SNI + NS group. A Student’s t-test was employed for statistical analysis.

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Discussion

The analgesic efficacy of ropivacaine in neuropathic pain management has been well-documented16,20,21, with accumulating evidence supporting its anti-inflammatory properties22,23. However, the precise molecular mechanisms underlying its therapeutic effects, particularly in mitigating neuroinflammation, remain incompletely elucidated. Our study revealed that intrathecal ropivacaine rapidly and durably alleviated SNI-induced neuropathic pain while suppressing spinal pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) and microglial activation in the dorsal horn, indicating anti-neuroinflammatory effects. Mechanistically, ropivacaine inhibited the TRAF2/PI3K/Akt/NF-κB signaling pathway, with this work being the first to identify this pathway as a key mediator of its analgesic action, offering new insights into its therapeutic mechanism for neuropathic pain.

Our findings demonstrated that a single intrathecal injection of ropivacaine provides rapid analgesic effects. Research indicates that ropivacaine exerts its analgesic properties through reversible inhibition of sodium ion influx in nerve fibers, thereby blocking action potential propagation24., a mechanism strongly associated with its immediate pain-relieving efficacy. Our data revealed that this effect persists for approximately 4 h, reaching peak intensity at 2 h post-administration. Notably, consecutive daily intrathecal injections of ropivacaine for 7 days resulted in sustained alleviation of mechanical allodynia in the treatment group, persisting for at least 14 days post-surgery despite treatment cessation. Given the transient nature of sodium channel blockade by ropivacaine, we hypothesize that the prolonged analgesic effect following treatment discontinuation may be attributed to its anti-neuroinflammatory properties.

Previous studies have established that neural tissue injury triggers continuous infiltration of immune cells (including neutrophils, macrophages, and T lymphocytes) into damaged regions. These activated immune cells release pro-inflammatory cytokines and chemokines that bind to cytokine/chemokine receptors on nociceptors, thereby initiating pain signaling. While acute inflammation serves to eliminate pathogens and promote tissue repair, unresolved inflammatory processes contribute to chronic pain development25. Emerging evidence implicates both peripheral and central nervous system inflammation in maintaining nociceptor sensitization, which perpetuates chronic pain states26. Therapeutic strategies targeting neuroinflammatory pathways may therefore enhance chronic pain management. Consistent with existing evidence of ropivacaine’s anti-inflammatory effects, our experimental results showed significant suppression of SNI-induced upregulation of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) in the ipsilateral spinal cord.

Furthermore, we investigated microglial activation in the ipsilateral spinal dorsal horn. As resident immune cells of the CNS, microglia undergo morphological changes (hypertrophy and amoeboid transformation) and proliferate following peripheral nerve injury, with increased expression of the microglial marker Iba-1 being strongly associated with neuropathic pain pathogenesis27. Our observations revealed that ropivacaine treatment effectively reduced both microglial density and Iba-1 expression in the spinal dorsal horn, suggesting its potent inhibitory effect on neuroinflammation during neuropathic pain progression. These collective findings provide mechanistic insights into ropivacaine’s dual role in acute analgesia and long-term neuroinflammatory modulation.

The activation of the NF-κB pathway in microglia promotes the expression of pro-inflammatory cytokines required for the development of hyperalgesia in the spinal dorsal horn following neural injury8. Studies have demonstrated that suppressing NF-κB pathway activation alleviates neuroinflammation and neuropathic pain13. To further elucidate the mechanism underlying ropivacaine’s anti-neuroinflammatory effects, we investigated its regulatory role in NF-κB activation. Previous research indicates that ropivacaine inhibits TNF-α-induced phosphorylation of IκBα and nuclear translocation of p65 in mesenchymal stem cells28.

In this study, ropivacaine attenuated SNI-induced upregulation of p65, IκBα, and phosphorylated p65 protein levels, consistent with existing evidence that ropivacaine suppresses NF-κB activation during neuropathic pain progression. Furthermore, Exploration of upstream signaling pathways revealed that ropivacaine inhibited SNI-triggered activation of the TRAF2/PI3K/Akt cascade. Coupled with the observed suppression of p-IκBα and p-p65 (Fig. 6), these findings collectively demonstrate that ropivacaine mitigates neuroinflammation and alleviates neuropathic pain by targeting the TRAF2/PI3K/Akt/NF-κB signaling axis.

This study has several important limitations. First, the absence of an NF-κB pathway inhibitor control group precludes direct evidence for ropivacaine’s therapeutic efficacy in neuropathic pain through NF-κB suppression, as our findings only established a correlative rather than causative relationship between these mechanisms. Second, the use of a single concentration and dosage of ropivacaine limits the ability to determine its optimal therapeutic concentration and dosage range, particularly regarding NF-κB pathway inhibition in neuropathic pain management. Therefore, further studies are warranted to validate the critical role of ropivacaine-mediated NF-κB suppression in attenuating neuropathic pain, particularly to establish the causal relationship between NF-κB pathway inhibition and pain relief. This would further clarify its mechanistic significance in neuroinflammatory modulation and therapeutic interventions.

Conclusion

In summary, through the establishment of a rat model of nerve injury, we demonstrated that continuous intrathecal administration of ropivacaine produces sustained analgesic effects against neuropathic pain by suppressing neuroinflammation. The study revealed that ropivacaine significantly inhibits the activation of the TRAF2/PI3K/Akt/NF-κB signaling pathway induced by neural damage, while concurrently reducing microglial activation and downregulating the expression of the pro-inflammatory cytokine in the spinal dorsal horn. This research provides the first experimental evidence delineating the relationship between ropivacaine and NF-κB signaling in the context of neuropathic pain, offering novel mechanistic insights to support its clinical application for alleviating neuropathic pain through anti-inflammatory pathways.