IRAK-M attenuates pain hypersensitivity and anxiety-like behaviors in a nitroglycerin-induced chronic migraine mouse model with concomitant reductions in microglial activation and neuroinflammation

Abstract

Background

Chronic migraine (CM) is a disabling neurological disorder in which neuroinflammatory mechanisms and central sensitization are thought to contribute to disease pathophysiology. Interleukin-1 receptor-associated kinase M (IRAK-M), which is predominantly expressed in microglia in the central nervous system (CNS), is an endogenous negative regulator of innate immune signaling. Previous studies have linked IRAK-M to the restraint of Toll-like receptor 4 (TLR4) signaling and NLR family pyrin domain-containing 3 (NLRP3) inflammasome-related responses. However, the role of IRAK-M in CM-related pathophysiology and associated neuropsychiatric comorbidities remains unclear. This study aimed to investigate the function of IRAK-M and its underlying molecular mechanisms in a mouse model of chronic migraine.

Methods

Mice were repeatedly given intraperitoneal injections of nitroglycerin (NTG; 10 mg/kg) to create a chronic migraine model. Mechanical and thermal sensitivity were assessed using von Frey filaments and the hot-plate test, respectively; light aversion and anxiety-like behaviors were evaluated using the light-aversion test, open-field test, and elevated plus-maze test. To further clarify the role of IRAK-M, both transgenic genetic engineering approaches and adeno-associated virus (AAV)-mediated expression strategies were employed. The underlying molecular mechanisms were further investigated using quantitative PCR, immunoblotting, immunofluorescence, and three-dimensional reconstruction.

Results

Repeated administration of NTG increased IRAK-M protein in the trigeminal nucleus caudalis (TNC). IRAK-M deficiency exacerbated mechanical and thermal hyperalgesia, increased c-Fos and CGRP expression, and enhanced microglial activation; these changes were accompanied by increased TLR4/NF-κB-related signaling, NLRP3 inflammasome activation, and GSDMD cleavage. Conversely, TNC-targeted IRAK-M overexpression attenuated pain hypersensitivity and anxiety-like behavioral alterations and was accompanied by corresponding reductions in neuroinflammatory molecular and cellular readouts. These findings support IRAK-M as an important regulator of central sensitization and neuroinflammatory responses in a chronic migraine model.

Conclusions

Our findings identify IRAK-M as an important preclinical regulator of microglial reactivity and neuroinflammatory responses in the NTG-induced chronic migraine model. IRAK-M manipulation was accompanied by bidirectional changes in TLR4/NF-κB- and NLRP3/GSDMD-related signaling, pain hypersensitivity, and anxiety-like behavioral alterations. These findings provide a preclinical rationale for further investigation of IRAK-M-related neuroimmune signaling in migraine.

Clinical trial number

Not applicable.

Graphical Abstract

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s10194-026-02457-1.

Introduction

Migraine is a common neurological disorder characterized by recurrent and disabling headache attacks that are frequently accompanied by photophobia, phonophobia, and nausea [1, 2]. Chronic migraine (CM), often arising as a progression from episodic migraine (EM), affects approximately 1–2% of the general population and shows a higher prevalence of neuropsychiatric comorbidities such as anxiety and depression [3, 4]. These comorbidities, in turn, exacerbate migraine by lowering pain thresholds, increasing attack frequency, and reducing treatment efficacy through shared neurobiological and inflammatory mechanisms [5, 6].

Central sensitization is considered a major mechanism underlying the development and persistence of CM and is characterized by an abnormal increase in the excitability and responsiveness of neurons in the central nervous system (CNS) to sensory inputs [7–10]. Recurrent migraine attacks are proposed to promote hyperactivation of the trigeminovascular system, resulting in sensitization of neurons within the trigeminal nucleus caudalis (TNC) [11, 12]. Neuroinflammatory processes, particularly those involving microglial activation and the release of pro-inflammatory molecules, have been suggested to further perpetuate central sensitization by sustaining neuronal hyperexcitability within the TNC and other central pain-processing regions [13–15]. Consistent with this concept, pharmacological attenuation of microglial activation with minocycline has been shown to alleviate central sensitization and migraine-like symptoms in preclinical models [16].

Among the signaling pathways linking microglial reactivity to neuroinflammation, Toll-like receptor 4 (TLR4)-dependent signaling has been implicated in preclinical models of migraine [17–19]. TLR4 is a pattern-recognition receptor that recruits the adaptor molecule myeloid differentiation primary response 88 (MyD88), leading to activation of nuclear factor-κB (NF-κB) signaling and the production of pro-inflammatory cytokines and chemokines. Intracisternal administration of OxPAPC, an inhibitor of TLR2/4 signaling, has been reported to reduce cortical microglial activation in response to spreading depolarization (SD), an electrophysiological event associated with migraine aura [20]. Despite these preclinical findings, the clinical translation of glia-targeted approaches remains uncertain. For example, ibudilast, a putative glial modulator, did not demonstrate clear efficacy in pilot studies involving patients with CM or medication-overuse headache [21, 22]. These limitations highlight the need to identify more specific endogenous regulators of neuroinflammatory signaling.

Interleukin-1 receptor-associated kinase M (IRAK-M), an endogenous negative regulator of MyD88-dependent innate immune signaling, is enriched in microglia within the CNS [23]. By preventing the dissociation of IRAK1 and IRAK4 from the MyD88 signaling complex, IRAK-M restricts downstream NF-κB activation and pro-inflammatory cytokine production [23, 24]. IRAK-M has been reported to restrain pro-inflammatory microglial polarization by suppressing NF-κB signaling and signal transducer and activator of transcription 1 (STAT1) activation, which may contribute to the maintenance of a less reactive microglial morphology [23, 25].

Excessive TLR4-dependent inflammatory signaling may also facilitate the assembly and activation of the NLR family pyrin domain-containing 3 (NLRP3) inflammasome [26–28]. Rodent models of migraine show microglial NLRP3 inflammasome activation, leading to caspase-1 cleavage and subsequent maturation of IL-1β and IL-18 [29–34]. This inflammatory cascade may not only amplify neuroinflammation but also contribute to neuronal dysfunction and central sensitization through processes that include GSDMD-mediated pyroptotic signaling [35, 36]. Consequently, the microglial NLRP3 inflammasome has emerged as a potential pro-inflammatory amplifier in preclinical models of CM.

Given the known immunoregulatory role of IRAK-M and the suggested involvement of the NLRP3 inflammasome in preclinical models of CM, we hypothesized that IRAK-M may attenuate CM-like pathophysiology and be associated with reduced microglial inflammasome- and pyroptosis-related responses. Using a well-established mouse model of CM, we examined the effects of IRAK-M deficiency and TNC-targeted IRAK-M overexpression on CM-like phenotypes, microglial reactivity, TLR4/NF-κB-related signaling proteins, NLRP3 inflammasome components, and pyroptosis-associated markers. These findings provide a preclinical rationale for further investigating IRAK-M as a potential therapeutic target for CM.

Methods

Animals

Male C57BL/6J mice (5–10 weeks old, 18–26 g) were obtained from the Experimental Animal Center of Southern Medical University (Guangzhou, China). The animals were housed under specific pathogen-free conditions at 23 ± 1 °C and 50–60% relative humidity under a 12-h light/dark cycle (lights on from 6:00 a.m. to 6:00 p.m.), with ad libitum access to food and water. Mice were group-housed four per cage throughout the study and randomly assigned to experimental groups at the cage level, with all animals within the same cage receiving the same treatment. Data analyses were performed by an investigator blinded to group allocation. A total of 162 mice were used, of which three were excluded from the final analyses because of anesthesia-related death or excessive bleeding during stereotaxic surgery. All experimental procedures were approved by the Institutional Animal Care and Use Committee of Southern Medical University (approval no. L-202406032), and every effort was made to minimize animal suffering and reduce the number of animals used.

IRAK-M knockout mice (B6.129S1-Irak3^tm1Flv/J; The Jackson Laboratory, Bar Harbor, ME, USA) and corresponding wild-type (WT) littermates were generated through heterozygous crosses. Genotyping was conducted by PCR using the following primer sequences: (1) wild type forward, GCC AGA AGA ATA CAT CAG ACA GGG; (2) wild type reverse, TGT TTC GGG TCA TCC AGC AC; (3) mutant forward, CGT TCC ATA ACA CAC CTC TCT GC; and (4) mutant reverse, TTC TAT CGC CTT CTT GAC GAG TTC. PCR amplicons were separated on 1.5% agarose gels, and genotypes were confirmed by bands of the expected sizes (Supplementary Fig. 1).

NTG-induced chronic migraine model

A validated CM model was induced by administering NTG (10 mg/kg, intraperitoneally) every other day for a total of five injections [37]. The NTG stock solution (5.0 mg/ml, Beijing Yimin, H11020289) was supplied in a mixed solvent containing 30% ethanol, 30% propylene glycol, and water and was freshly diluted with sterile 0.9% saline to a final working concentration of 1.0 mg/ml immediately before use. At this dilution, the final concentrations of ethanol and propylene glycol were considered negligible for inducing behavioral or inflammatory effects [38]. Control animals received an equal volume of 0.9% saline. Preliminary control experiments further confirmed that there were no significant differences in behavioral outcomes between the saline group and the vehicle control group (Supplementary Fig. 2).

Pain thresholds were evaluated 30 min prior to (basal pain response) and 2 h following each injection (acute/post-injection pain response).

Stereotaxic viral injection

Stereotaxic viral injection was conducted following our previous report, with minor modifications as noted below. Mice were anesthetized with isoflurane delivered in oxygen through a precision vaporizer (3% for induction and 1.5–2.0% for maintenance). After an adequate depth of anesthesia had been confirmed by the absence of the pedal withdrawal reflex, the mice were secured in a stereotaxic frame, and anesthesia was continuously maintained through a nose cone. Using a 0.5 µL microsyringe (Gaoge, China) connected to a microsyringe pump (Longer, China), viral vectors were infused into the TNC (anteroposterior, − 7.3 mm; mediolateral, ± 1.6 mm; dorsoventral, − 5.0 mm). Each site received 200 nL of the AAV9-IRAK-M overexpression vector (1 × 10¹⁰ vg in PBS, serotype 9) at a rate of 0.05 µL/min over 4 min. The injection needle was retained in place for 10 min after infusion and then withdrawn slowly to minimize reflux. Mice were allowed 3 weeks for transgene expression before subsequent experiments, and IRAK-M overexpression was confirmed by RT-qPCR.

Behavioral testing paradigms

To reduce stress associated with human contact, mice were gently handled for three consecutive days before behavioral testing. All behavioral assessments were conducted in a quiet, standardized testing room physically separated from the housing facility, with at least 2 h of room acclimatization before each session. The experimenter was blinded to group allocation during testing and subsequent data analysis.

Automated behavioral tracking

Behavioral testing was performed between 9:00 a.m. and 6:00 p.m. Animal behavior was recorded and analyzed using SMART video-tracking software, version 3.0 (Panlab S.L.U., Barcelona, Spain), equipped with infrared CCD imaging. This system automatically quantified locomotor and exploratory parameters. The testing apparatus was carefully cleaned with alcohol after each animal trial to eliminate residual olfactory cues (e.g., urine, feces) that could influence subsequent test subjects.

Mechanical sensitivity assessment

Mechanical sensitivity was assessed as previously described [39, 40] using calibrated von Frey filaments (Danmic Aesthesio, USA). Each mouse was placed individually in a transparent acrylic chamber on an elevated wire mesh floor and allowed to acclimatize for 30 min. Filaments ranging from 0.008 to 2.0 g were applied to the plantar surface of the hindpaw for 5–6 s, with an interval of at least 30 s between applications. The 50% paw withdrawal threshold (PWT) was calculated according to the up–down paradigm [41]. Basal thresholds were measured before each NTG or saline injection, and post-injection thresholds were assessed 2 h after injection. Three replicate measurements were obtained per mouse at each time point, separated by intervals of at least 5 min, and averaged for analysis.

Thermal sensitivity assessment

Thermal nociception was measured using the hot plate test as previously described [42, 43]. The surface temperature was maintained at 55.0 ± 0.2 °C. Each mouse was gently placed on the heated surface, and the latency to hindpaw licking or jumping was recorded. A cutoff time of 60 s was applied to prevent tissue injury. Basal withdrawal latency was measured 30 min before each NTG or saline injection, and the post-injection response was assessed 2 h after injection.

Light aversion test

As previously described [10], light aversion was assessed using a light–dark box consisting of two equally sized compartments connected by a central opening. The light compartment was brightly lit (1000 lx), while the other compartment remained dark. To minimize the potential confounding effects of novelty- and apparatus-related anxiety, mice were repeatedly habituated to the apparatus during the modeling period before formal testing. On the day of the fifth NTG injection, mice were tested twice: once 1.5 h before injection to evaluate the basal response and again 1.5 h after injection to assess the post-injection response. During each session, mice were placed at the central opening facing the light compartment and allowed to explore freely for 20 min. Time spent in the light compartment was automatically recorded using SMART 3.0 software.

Open field test

As previously described [44], each mouse was placed in the central zone of the arena and allowed to explore for 5 min. SMART 3.0 software automatically recorded the total distance traveled and the time spent in the central zone. The open-field test was performed at three time points: before the first NTG injection (baseline), 2 h before the fifth NTG injection (basal response), and 2 h after the fifth NTG injection (post-injection response).

Elevated plus maze

As previously described [44], each mouse was placed in the center of the maze facing an open arm and allowed to explore for 5 min. SMART 3.0 software was used to quantify the time spent in the open arms and the number of open-arm entries. The elevated plus maze was performed at three time points: before the first NTG injection (baseline), 2 h before the fifth NTG injection (basal response), and 2 h after the fifth NTG injection (post-injection response).

Tissue collection

Unless otherwise indicated, mice were euthanized on day 10. For fresh tissue collection, mice were deeply anesthetized with 5% isoflurane until a surgical plane of anesthesia was achieved, as confirmed by the absence of a pedal withdrawal response to a firm toe pinch, and were then euthanized by decapitation. The brainstem, including the cervicomedullary junction, was rapidly removed. The trigeminal nucleus caudalis (TNC), corresponding to the caudal part of the spinal trigeminal nucleus (SPVC/Sp5C), was identified bilaterally in the dorsolateral caudal medulla. For biochemical analyses, anatomically matched bilateral tissue blocks were dissected from coronal levels approximately 7.5–8.2 mm caudal to bregma, and the nuclear boundaries were confirmed with reference to the Allen Mouse Brain Reference Atlas (Allen Institute for Brain Science, Seattle, WA, USA). Care was taken to minimize the inclusion of the rostrally adjacent spinal trigeminal interpolar nucleus and medially located nucleus of the solitary tract. Because the TNC transitions caudally into the upper cervical dorsal horn without a sharp anatomical boundary, the caudal sampling limit was standardized at the cervicomedullary junction across all animals. The dissected tissue was immediately transferred to precooled cryotubes, snap-frozen in liquid nitrogen, and stored at − 80 °C until analysis. When required, tissues were collected at the specific time points described in the relevant Methods sections or figure legends.

Real-time quantitative polymerase chain reaction (RT‒qPCR)

RT-qPCR was performed as previously reported [45], with minor modifications. Total RNA was extracted from TNC tissues using TRIzol reagent (Invitrogen, USA) according to the manufacturer’s instructions. Total RNA (1 µg) was reverse-transcribed using the SevenFast^® Two Step RT&qPCR Kit (Seven Biotech, China), with simultaneous removal of genomic DNA. Reverse transcription was performed at 50 °C for 5–8 min, followed by 85 °C for 5 s. RT-qPCR was conducted on a LightCycler^® 96 System (Roche, Switzerland) using SevenFast^® 2×SYBR Green qPCR Mix. Each 20-µL reaction contained 10 µL of qPCR mix, 0.4 µL of each primer, 1 µL of cDNA, and nuclease-free water. Amplification consisted of 95 °C for 30 s, followed by 35–45 cycles of 95 °C for 10–20 s, 60 °C for 10–20 s, and 72 °C for 20–30 s, followed by melting-curve analysis. Primer sequences (supplied by Beijing Qingke Biotechnology Co., Ltd., China) are shown in Supplementary Material 1.

Western blotting

TNC tissues were lysed in RIPA buffer (Beyotime, China) supplemented with phosphatase and protease inhibitors (Beyotime, China) on ice for 30 min. After centrifugation at 12,000 × g for 15 min at 4 °C, the supernatants were collected, and protein concentrations were determined using a BCA protein assay kit (Beyotime, China). Equal amounts of protein (40 µg per lane) were separated by 10% SDS–PAGE (EpiZyme, China) and transferred onto PVDF membranes (Millipore, USA). Membranes were blocked with 5% BSA (Biofroxx, Germany) and then incubated with the indicated primary antibodies, followed by HRP-conjugated secondary antibodies (see Supplementary Material 1). β-actin was used as the loading control. Expected molecular weights and original uncropped blot images are provided in Supplementary Material 2.

Immunofluorescence staining

Following deep anesthesia, mice were transcardially perfused with ice-cold PBS. The entire brain was removed and immersion-fixed in 4% paraformaldehyde (Leagene, China) for 24 h, followed by sequential cryoprotection in 15% and 30% sucrose solutions. Coronal Sections (30 μm) were prepared and stored at − 80 °C. Free-floating sections were washed three times with PBS, permeabilized with 0.1% Triton X-100 in PBS for 15 min, and blocked with 2.5% normal goat serum for 1.5 h at room temperature. Sections were incubated overnight at 4 °C with primary antibodies against c-Fos (1:3000), CGRP (1:200), Iba1 (1:300), NLRP3 (1:50), ASC (1:200), caspase-1 (1:50), and GSDMD (1:50) (Supplementary Material 1). After washing, sections were incubated with Alexa Fluor 488- or 594-conjugated secondary antibodies (1:500) for 1–2 h at room temperature. Coverslips were mounted using antifade mounting medium containing DAPI (Solarbio, Beijing, China). Images were acquired using a Zeiss LSM 980 confocal microscope with Airyscan 2 (Zeiss, Germany).

Immunofluorescence analyses included six mice per group. For each mouse, three anatomically matched, nonadjacent coronal TNC sections separated by at least 120 μm were analyzed. One standardized field from each side of the TNC was acquired from each section using identical imaging settings across groups, yielding six fields per mouse. Regions of interest were defined according to Sp5C boundaries in the Allen Mouse Brain Reference Atlas. c-Fos-positive cells and Iba1-positive microglia were counted using fixed threshold and size criteria, whereas CGRP and inflammasome/pyroptosis-related signals were quantified as background-subtracted mean fluorescence intensity. Values from all fields and sections were averaged to generate one biological replicate per mouse. Image acquisition and quantification were performed by an investigator blinded to group allocation using Fiji.

Three-dimensional (3D) reconstruction and morphometric analysis

High-resolution z-stack images of Iba1-immunostained microglia in the TNC were acquired via a Zeiss LSM 980 confocal microscope with Airyscan 2 superresolution technology. Serial optical sections were captured at intervals sufficient for accurate 3D rendering. Microglial morphology was reconstructed and analyzed via Imaris 9.0.0 software (Bitplane, Switzerland). The AnalyseSkeleton plugin skeletonizes Iba1 + cells to quantify their morphological complexity (e.g., total process length, branch points).

Statistical analysis

Statistical analyses were conducted using IBM SPSS Statistics, version 20.0 (IBM Corp., Armonk, NY, USA), and GraphPad Prism, version 8.0 (GraphPad Software, LLC, San Diego, CA, USA). Data are presented as mean ± SEM unless otherwise specified. Sample sizes were determined on the basis of preliminary experiments: behavioral tests generally included 12 mice per group (e.g., Fig. 1G, H), whereas molecular and immunohistochemical analyses generally included 6 mice per group (e.g., Fig. 2E–H). Morphometric analyses, including microglial Sholl and FracLac analyses (e.g., Fig. 4B, C, and E), were performed using ImageJ plugins. Normality and homogeneity of variance were assessed before statistical testing using the Shapiro–Wilk and Levene tests, respectively. Comparisons between two groups were performed using an unpaired two-tailed Student’s t test. Nonlongitudinal data involving multiple groups were analyzed using one-way or two-way ANOVA followed by Dunnett’s or Bonferroni-adjusted multiple-comparisons tests, as appropriate. Outcomes measured repeatedly in the same animals were analyzed using two-way repeated-measures ANOVA, with group as the between-subject factor and time or radial distance as the within-subject factor. The models evaluated the main effects and the group × within-subject-factor interaction. The Geisser–Greenhouse correction was applied to radial-distance and interaction effects in Sholl analyses. Prespecified comparisons were evaluated using Bonferroni-adjusted multiple comparisons. Mean differences, 95% confidence intervals, and adjusted p-values for prespecified comparisons are reported in Supplementary Table S3. p < 0.05 was considered statistically significant (*p < 0.05, **p < 0.01).

Fig. 1.

Repeated nitroglycerin administration induces chronic migraine-like hyperalgesia and anxiety-like behavioral alterations in mice. (A) Schematic overview of the experimental protocol. (B–C) Mechanical withdrawal thresholds measured before and after saline or NTG injection using von Frey filaments. (D–E) Thermal withdrawal latencies measured before and after saline or NTG injection using the hot-plate test. (F) Schematic diagram and representative movement trajectories from the light-aversion test (LAT). (G–H) Time spent in the light compartment and number of entries into the light compartment at baseline and before and after the fifth saline or NTG injection. (I) Representative movement trajectories and occupancy heat maps from the open-field test (OFT). (J–K) Total distance traveled and time spent in the central zone at baseline and before and after the fifth saline or NTG injection. (L) Representative movement trajectories from the elevated plus-maze test (EPM). (M–N) Number of open-arm entries and percentage of time spent in the open arms at baseline and before and after the fifth saline or NTG injection. n = 12 mice per group. Data are presented as mean ± SEM. Two-way repeated-measures ANOVA followed by Bonferroni-adjusted multiple comparisons was used for (B–E, G–H, J–K, and M–N). *p < 0.05, **p < 0.01; ns, not significant

Fig. 2.

Compensatory upregulation of IRAK-M and engagement of TLR4/NF-κB-related signaling in the TNC during chronic migraine modeling. (A) Schematic overview of TNC tissue collection during repeated NTG administration. Tissues were collected 2 h after NTG injection on days 1, 3, 5, 7, and 9. (B) Representative immunoblots showing IRAK-M protein expression at the indicated time points. (C) Quantitative line graph summarizing IRAK-M protein expression dynamics (n = 3 mice per time point). (D) Representative immunoblots showing TLR4, IRAK1, TRAF6, phosphorylated p65 (p-p65), total p65, and β-actin expression in the TNC of saline- and NTG-treated WT mice. (E–H) Quantification of TLR4, IRAK1, and TRAF6 protein expression and the p-p65/p65 ratio (n = 6 mice per group). Expected molecular weights are indicated adjacent to the representative blots. Data are presented as mean ± SEM. One-way ANOVA followed by Dunnett’s multiple-comparisons test was used for (C), with Day 1 as the reference group, *p < 0.05 and **p < 0.01 versus Day 1. An unpaired two-tailed Student’s t test was used for (E–H), **p < 0.01 versus saline-treated WT mice

Fig. 4.

IRAK-M deficiency is associated with enhanced microglial reactivity and TLR4/NF-κB-related neuroinflammatory readouts. (A) Representative immunofluorescence images showing Iba1-positive microglia in the TNC. Scale bar = 50 μm. (B–C) Quantification of Iba1-positive cell number and total microglial process length (n = 6 mice per group). (D) Representative images of individual microglia and corresponding Sholl analysis overlays. Scale bars = 5 μm. (E) Sholl analysis showing the number of intersections as a function of radial distance from the soma (n = 6 mice per group). (F) Representative immunoblots showing TLR4, IRAK1, TRAF6, phosphorylated p65 (p-p65), total p65, and β-actin expression in the TNC. (G–J) Quantification of TLR4, IRAK1, and TRAF6 protein expression and the p-p65/p65 ratio (n = 6 mice per group). (K–N) Relative mRNA level of IL-1β, IL-6, TNF-α, and IFN-γ (n = 4 mice per group). Data are presented as mean ± SEM. One-way ANOVA followed by Dunnett’s multiple-comparisons test was used for (B, C, and G–N). Sholl data in (E) were analyzed using two-way repeated-measures ANOVA, with group as the between-subject factor and radial distance as the within-subject factor, and Geisser–Greenhouse correction was applied to radial-distance and interaction effects, followed by Bonferroni-adjusted prespecified comparisons. *p < 0.05, **p < 0.01; ns, not significant

Results

Intermittent nitroglycerin administration induces pain hypersensitivity and anxiety-like behaviors in mice

Mechanical and thermal sensitivity were assessed 30 min before and 2 h after each injection using von Frey filaments and the hot-plate test, respectively (Fig. 1A). Consistent with previous reports [10, 31], acute reductions in pain thresholds were observed after each NTG injection (Fig. 1C, E). Basal pain thresholds became significantly lower after the second injection (Fig. 1B, D), indicating the development of sustained hyperalgesia.

By the fifth NTG administration, photophobia-like and anxiety-like behavioral alterations were evaluated using the light aversion test, open field test, and elevated plus maze test (Fig. 1A). In the light-aversion test, NTG-treated WT mice made fewer entries into the light compartment and spent less time there than saline-treated controls (Fig. 1F–H). In the open field test, NTG-treated WT mice traveled a shorter total distance and spent less time in the central zone (Fig. 1I–K). In the elevated plus maze test, NTG-treated WT mice made fewer entries into the open arms and spent less time there than saline-treated controls (Fig. 1L–N). Together, these findings indicate that repeated NTG exposure induces mechanical and thermal hypersensitivity and increases light aversion behavior. NTG-treated mice also exhibited anxiety-like behavioral alterations; however, the concurrent reduction in locomotor activity may have contributed to some of these behavioral readouts, particularly those based on exploration and compartment entries. Collectively, these results support the utility of this model for investigating migraine-related behaviors and their underlying neurobiological mechanisms in mice.

Dynamic upregulation of IRAK-M and engagement of TLR4/NF-κB-related signaling in the TNC during chronic migraine modeling

To examine the dynamic expression pattern of IRAK-M in the NTG-induced CM model, we assessed IRAK-M protein levels in the TNC at a fixed time point (2 h) after each NTG injection throughout the repeated administration protocol (Fig. 2A). Immunoblotting showed that IRAK-M expression progressively increased following repeated NTG exposure, which may reflect an inducible compensatory anti-inflammatory response in the TNC (Fig. 2B, C).

Given the reported inhibitory role of IRAK-M in TLR4/NF-κB signaling, we next examined key pathway-related proteins in the TNC at the experimental endpoint (day 10, one day after the fifth NTG injection). Compared with saline-treated controls, NTG-treated WT mice exhibited higher levels of TLR4, IRAK1, and TRAF6 and an increased p-p65/p65 ratio (Fig. 2D–H). Thus, repeated NTG administration was associated with increased TLR4/NF-κB-related inflammatory readouts in the TNC.

IRAK-M deficiency exacerbates pain hypersensitivity and neuronal activation in the NTG-induced chronic migraine model

To investigate the role of IRAK-M in migraine-related pathophysiology, IRAK-M-knockout (KO) mice and wild-type (WT) controls received repeated intraperitoneal injections of NTG or saline. Compared with NTG-treated WT mice, NTG-treated KO mice developed more severe pain hypersensitivity, as evidenced by a progressive reduction in both mechanical and thermal pain thresholds, with significant differences emerging after the third NTG injection (Fig. 3A–D). Immunofluorescence analysis further showed higher c-Fos and CGRP expression in the TNC of NTG-treated KO mice than in NTG-treated WT mice (Fig. 3E–H). No significant differences in pain thresholds or neuronal activation were detected between saline-treated KO and WT mice. These findings indicate that IRAK-M deficiency exacerbates pain hypersensitivity and nociceptive neuronal activation in the NTG-induced CM model.

Fig. 3.

IRAK-M deficiency exacerbates NTG-induced pain hypersensitivity and nociceptive neuronal activation. (A–B) Mechanical withdrawal thresholds in wild-type (WT) and IRAK-M-knockout (KO) mice following repeated saline or NTG administration (n = 12 mice per group). (C–D) Thermal withdrawal latencies in the indicated experimental groups (n = 12 mice per group). (E) Representative immunofluorescence images showing c-Fos expression in the TNC for different experimental groups. Scale bar = 50 μm. (F) Quantification of c-Fos-positive cells (n = 6 mice per group). (G) Representative immunofluorescence images showing CGRP expression in the TNC. Scale bar = 200 μm. (H) Quantification of CGRP fluorescence intensity (n = 6 mice per group). Data are presented as mean ± SEM. One-way ANOVA followed by Dunnett’s multiple-comparisons test was used for (F, H), and two-way repeated-measures ANOVA followed by Bonferroni-adjusted multiple comparisons was used for (A–D). For the longitudinal analyses in (A–D), the prespecified pairwise comparison was between NTG-treated WT mice and NTG-treated IRAK-M-KO mice at each corresponding time point. *p < 0.05 and **p < 0.01 indicate significant differences between these two groups

IRAK-M deficiency is associated with enhanced microglial activation and neuroinflammatory responses in the NTG-induced chronic migraine model

Morphological analysis of Iba1-positive microglia revealed clear group-dependent differences in microglial abundance and morphology. Compared with saline-treated WT mice, NTG-treated WT mice showed an increased number of Iba1-positive cells in the TNC and shorter microglial processes, consistent with increased microglial reactivity. NTG-treated KO mice exhibited a further increase in Iba1-positive cell number compared with NTG-treated WT mice (Fig. 4A–E). Although the number of Sholl intersections was numerically lower in NTG-treated KO mice than in NTG-treated WT mice, the overall difference was not statistically significant after Bonferroni correction. Thus, IRAK-M deficiency was associated primarily with a greater increase in microglial abundance, whereas evidence for an additional effect on microglial morphology was limited. Immunoblot analysis showed that TLR4, IRAK1, and TRAF6 levels and the p-p65/p65 ratio were significantly higher in NTG-treated KO mice than in NTG-treated WT mice (Fig. 4F–J). These findings indicate that IRAK-M deficiency is associated with increased TLR4/NF-κB-related inflammatory signaling in the NTG-induced CM model.

Consistent with these findings, RT-qPCR analysis showed that the mRNA levels of IL-1β, TNF-α, IL-6, and IFN-γ were significantly higher in NTG-treated WT mice than in saline-treated WT mice. These cytokine transcripts were further increased in NTG-treated KO mice, supporting an association between IRAK-M deficiency and an intensified neuroinflammatory response to repeated NTG exposure (Fig. 4K–N).

Collectively, these findings indicate that loss of IRAK-M is associated with increased microglial reactivity, enhanced TLR4/NF-κB-related signaling, and elevated proinflammatory cytokine expression in the NTG-induced CM model.

IRAK-M deficiency is associated with enhanced NLRP3 inflammasome activation and pyroptosis-related signaling in the NTG-induced chronic migraine model

The NLRP3 inflammasome and GSDMD-mediated pyroptotic signaling have been implicated in neuroinflammatory responses. To examine the cellular distribution of inflammasome- and pyroptosis-related proteins in the NTG-induced CM model, we performed immunofluorescence staining in the TNC. Saline-treated WT mice exhibited low immunoreactivity for NLRP3, ASC, GSDMD, and caspase-1, whereas repeated NTG administration markedly increased these signals. A substantial proportion of the increased immunoreactivity spatially overlapped with Iba1-positive microglia in the TNC (Fig. 5A–H), supporting microglial involvement in inflammasome- and pyroptosis-related responses. However, because neuronal and astrocytic markers were not examined in parallel, these data do not establish the relative contribution of microglia compared with other CNS cell types.

Fig. 5.

IRAK-M deficiency is associated with enhanced NLRP3 inflammasome activation and pyroptosis-related signaling. (A–D) Representative immunofluorescence images showing NLRP3, ASC, GSDMD, and caspase-1 immunoreactivity together with Iba1-positive microglia in the TNC of saline- and NTG-treated WT mice. Scale bars = 50 μm. (E–H) Quantification of NLRP3, ASC, GSDMD, and caspase-1 fluorescence intensity (n = 6 mice per group). (I) Representative immunoblots showing NLRP3, ASC, full-length and cleaved GSDMD, pro- and cleaved caspase-1, pro- and mature IL-1β, IL-18, and β-actin in the indicated groups. (J–O) Quantification of NLRP3 and ASC protein expression, the cleaved GSDMD/full-length GSDMD ratio, the cleaved caspase-1/pro-caspase-1 ratio, the mature IL-1β/pro-IL-1β ratio, and IL-18 protein expression (n = 6 mice per group). Data are presented as mean ± SEM. An unpaired two-tailed Student’s t test was used for (E–H), and one-way ANOVA followed by Dunnett’s multiple-comparisons test was used for (J–O). *p < 0.05, **p < 0.01

We next assessed whether IRAK-M deficiency was associated with altered inflammasome- and pyroptosis-related signaling in the TNC. Western blot analysis showed that, compared with NTG-treated WT mice, NTG-treated KO mice exhibited higher levels of NLRP3 and ASC, increased cleavage of caspase-1 and GSDMD, and elevated expression of mature IL-1β and IL-18 (Fig. 5I–O). These findings indicate that IRAK-M deficiency is associated with enhanced NLRP3 inflammasome activation and pyroptosis-related signaling in the NTG-induced CM model.

IRAK-M overexpression alleviates pain hypersensitivity and reduces neuronal activation in the NTG-induced chronic migraine model

IRAK-M overexpression alone did not induce detectable changes in basal nociceptive thresholds or inflammatory cytokine expression under control conditions, as no significant differences were observed between saline-treated AAV^−control and AAV^−IRAK−M mice (Supplementary Fig. 5A–F). To further investigate its role in chronic migraine, mice received TNC-targeted AAV-mediated IRAK-M overexpression followed by repeated intraperitoneal NTG injections. Compared with NTG-treated WT mice, NTG-treated AAV^−IRAK−M mice exhibited attenuated mechanical and thermal pain hypersensitivity, with significant differences in most pain-threshold measures emerging by the third NTG injection (Fig. 6A–D).

Fig. 6.

IRAK-M overexpression attenuates central sensitization and suppresses neuronal activation. (A–B) Mechanical withdrawal thresholds in WT + saline, WT + NTG, AAV^−control + NTG, and AAV^−IRAK−M + NTG mice during repeated saline or NTG administration (n = 12 mice per group). (C–D) Thermal withdrawal latencies in the indicated groups (n = 12 mice per group). (E) Representative immunofluorescence images showing c-Fos expression in the TNC. Scale bar = 50 μm. (F) Quantification of c-Fos-positive cells (n = 6 mice per group). (G) Representative immunofluorescence images showing CGRP expression in the TNC. Scale bar = 200 μm. (H) Quantification of CGRP fluorescence intensity (n = 6 mice per group). Data are presented as mean ± SEM. One-way ANOVA followed by Dunnett’s multiple-comparisons test was used for (F, H), and two-way repeated-measures ANOVA followed by Bonferroni-adjusted multiple comparisons was used for (A–D). For the longitudinal analyses in (A–D), the prespecified comparison was between NTG-treated WT mice and NTG-treated AAV^−IRAK−M mice at each corresponding time point. *p < 0.05 and **p < 0.01 indicate significant differences between these two groups

Consistent with these behavioral improvements, IRAK-M overexpression significantly reduced the number of c-Fos-positive cells and CGRP fluorescence intensity in the TNC compared with the NTG-treated WT mice (Fig. 6E–H). These findings indicate that TNC-targeted IRAK-M overexpression attenuates NTG-induced pain hypersensitivity and reduces nociceptive neuronal activation.

IRAK-M overexpression reduces microglial activation and neuroinflammation-related signaling in the NTG-induced chronic migraine model

Immunofluorescence and morphometric analyses showed that NTG-treated AAV^−IRAK−M mice had fewer Iba1-positive microglia, longer microglial processes, and more Sholl intersections than NTG-treated WT mice (Fig. 7A–E), indicating reduced microglial reactivity and increased branching complexity following IRAK-M overexpression.

Fig. 7.

IRAK-M overexpression is associated with reduced microglial reactivity and TLR4/NF-κB-related neuroinflammatory readouts. (A) Representative immunofluorescence images showing Iba1-positive microglia in the TNC. Scale bar = 50 μm. (B–C) Quantification of Iba1-positive cell number and total microglial process length (n = 6 mice per group). (D) Representative images of individual microglia and corresponding Sholl analysis overlays. Scale bars = 5 μm. (E) Sholl analysis showing the number of intersections as a function of radial distance from the soma (n = 6 mice per group). (F) Representative immunoblots showing TLR4, IRAK1, TRAF6, phosphorylated p65 (p-p65), total p65, and β-actin expression in the TNC. (G–J) Quantification of TLR4, IRAK1, and TRAF6 protein expression and the p-p65/p65 ratio (n = 6 mice per group). (K–N) Relative mRNA level of IL-1β, IL-6, TNF-α, and IFN-γ (n = 4 mice per group). Data are presented as mean ± SEM. One-way ANOVA followed by Dunnett’s multiple-comparisons test was used for (B, C, and G–N). Sholl data in (E) were analyzed using two-way repeated-measures ANOVA, with group as the between-subject factor and radial distance as the within-subject factor, and Geisser–Greenhouse correction was applied to radial-distance and interaction effects, followed by Bonferroni-adjusted prespecified comparisons. *p < 0.05, **p < 0.01; ns, not significant

Immunoblot analysis further showed lower TLR4, IRAK1, and TRAF6 levels and a reduced p-p65/p65 ratio in NTG-treated AAV^−IRAK−M mice than in NTG-treated WT mice (Fig. 7F–J). The mRNA levels of the proinflammatory cytokines IL-1β, TNF-α, IL-6, and IFN-γ were also lower after IRAK-M overexpression (Fig. 7K–N).

Together, these findings indicate that IRAK-M overexpression is associated with reduced microglial reactivity, lower proinflammatory cytokine expression, and decreased TLR4/NF-κB-related inflammatory readouts in the NTG-induced CM model.

IRAK-M overexpression attenuates NLRP3 inflammasome activation and pyroptosis-related signaling in the NTG-induced chronic migraine model

Western blot analysis showed that, compared with NTG-treated WT mice, NTG-treated AAV^−IRAK−M mice exhibited lower levels of NLRP3 and ASC, reduced cleavage of caspase-1 and GSDMD, and decreased expression of mature IL-1β and IL-18 (Fig. 8A–G). These molecular changes indicate that IRAK-M overexpression was associated with attenuated inflammasome activation and pyroptosis-related signaling in the TNC.

Fig. 8.

IRAK-M overexpression is associated with reduced NLRP3 inflammasome activation and pyroptosis-related signaling. (A) Representative immunoblots showing NLRP3, ASC, full-length and cleaved GSDMD, pro- and cleaved caspase-1, pro- and mature IL-1β, IL-18, and β-actin expression in the TNC of WT + saline, WT + NTG, AAV^−control + NTG, and AAV^−IRAK−M + NTG mice. (B–G) Quantification of NLRP3 and ASC protein expression, the cleaved GSDMD/full-length GSDMD ratio, the cleaved caspase-1/pro-caspase-1 ratio, the mature IL-1β/pro-IL-1β ratio, and IL-18 protein expression. (n = 6 mice per group). Data are presented as mean ± SEM. One-way ANOVA followed by Dunnett’s multiple-comparisons test was used for (B–G). **p < 0.01

When considered together with the corresponding changes observed in IRAK-M-knockout mice, the reciprocal effects of IRAK-M loss and overexpression support a regulatory role for IRAK-M in neuroinflammatory responses during chronic migraine modeling. Nevertheless, pathway-specific intervention studies are required to determine whether the NLRP3/caspase-1/GSDMD axis directly mediates the behavioral effects of IRAK-M.

IRAK-M overexpression reduces light aversion and improves anxiety-like behavioral measures in the NTG-induced chronic migraine model

To determine whether IRAK-M overexpression modulates migraine-related behavioral abnormalities, light aversion was first assessed using the light-aversion test. Compared with NTG-treated WT mice, NTG-treated AAV^−IRAK−M mice spent more time in the light compartment at both the basal and post-injection response on the day of the fifth NTG injection. Although entries into the light compartment also increased after the fifth NTG injection, the difference was not statistically significant (Fig. 9A–C).

Fig. 9.

IRAK-M overexpression improves light aversion and anxiety-like behavioral measures in the NTG-induced CM model. (A) Schematic of the light-aversion test. (B–C) Time spent in the light compartment and number of entries measured before and after saline or NTG injection in WT + saline, WT + NTG, AAV^−control + NTG, and AAV^−IRAK−M + NTG mice (n = 12 per group). (D) Representative movement trajectories from the open-field test. (E) Representative movement trajectories from the elevated plus-maze test. (F–G) Total distance traveled and percentage of time spent in the central zone at baseline and before and after the fifth saline or NTG injection (n = 12 mice per group). (H–I) Percentage of time spent in the open arms and number of open-arm entries at baseline and before and after the fifth saline or NTG injection (n = 12 mice per group). Data are presented as mean ± SEM. Two-way repeated-measures ANOVA followed by Bonferroni-adjusted multiple comparisons was used for (B, C, and F–I). The indicated comparisons were between NTG-treated WT mice and NTG-treated AAV^−IRAK−M mice at the corresponding assessment time points. *p < 0.05, **p < 0.01; ns, not significant

Anxiety-like behavioral measures were then evaluated using the open-field and elevated plus-maze tests. In the open field test, NTG-treated AAV^−IRAK−M mice spent more time in the center zone and traveled greater distances than NTG-treated WT mice (Fig. 9D, F, and G). In the elevated plus-maze test, NTG-treated AAV^−IRAK−M mice made more entries into the open arms and spent more time there than NTG-treated WT mice (Fig. 9E, H, and I).

Taken together, these findings suggest that IRAK-M overexpression attenuates light aversion and improves anxiety-like behavioral measures in the NTG-induced chronic migraine model. However, the accompanying increase in locomotor activity should be considered when interpreting outcomes that depend on exploratory activity.

Discussion

Here, we identify IRAK-M as an important endogenous regulator of neuroinflammatory responses in an NTG-induced chronic migraine model. Using the repeated NTG paradigm, we found that IRAK-M expression in the TNC progressively increased during chronic migraine modeling. Genetic deletion of IRAK-M aggravated mechanical and thermal hyperalgesia and enhanced nociceptive neuronal activation, as reflected by increased c-Fos and CGRP expression, whereas TNC-targeted AAV-mediated IRAK-M overexpression attenuated these phenotypes. These bidirectional effects were accompanied by corresponding changes in microglial reactivity, TLR4/NF-κB-related signaling, NLRP3 inflammasome-associated proteins, and GSDMD cleavage. Collectively, these findings support a regulatory role for IRAK-M in limiting neuroinflammatory and pyroptosis-related responses associated with central sensitization in the NTG-induced chronic migraine model.

Neuroimmune interactions have been implicated in preclinical and translational studies of migraine, although their causal and therapeutic relevance in humans remains incompletely established [46–48]. Clinically, CM patients often present with abnormal pain perception of the head and skin, which is strongly related to enhanced responsiveness of second-order neurons within the TNC [49–51]. c-Fos, an immediate early gene, is commonly employed as a marker of neuronal activation because of its rapid and transient expression after stimulation. CGRP is a key endogenous mediator of migraine and may contribute to attack chronification, with some studies suggesting that its expression may be associated with attack frequency [52]. In our study, repeated NTG administration induced progressive mechanical and thermal hypersensitivity and increased c-Fos and CGRP expression in the TNC. NTG-treated mice also exhibited increased light aversion and anxiety-like behavioral alterations, supporting the utility of this model for investigating migraine-related behaviors and their associated neurobiological changes.

Microglia are intimately involved in pain processing and contribute to the pathogenesis of neurological disorders through the regulation of neuroinflammation [53–56]. Even under physiological conditions, microglia are not quiescent but continuously survey the local microenvironment and interact dynamically with neighboring cells. In response to persistent or excessive stimulation, they may transition from a homeostatic surveillance state to a reactive, pro-inflammatory phenotype characterized by morphological remodeling and increased expression of M1-associated markers, including CD86 and iNOS [31, 57].

TLR4 represents a key mediator in initiating and sustaining microglial activation as well as neuropathic pain [58–60]. Evidence from animal models indicates that TLR4 deletion or knockdown attenuates microglial activation, suppresses proinflammatory cytokine production, and alleviates pain hypersensitivity [53, 59]. NF-κB signaling is a central mediator of these processes, orchestrating inflammatory responses and driving both macrophage and microglial polarization [61–63]. Inhibition of NF-κB favors M1-to-M2 phenotypic polarization of microglia [23, 31]. Similarly, members of the STAT family contribute to M1/M2 regulation in both the peripheral immune system and the CNS [23, 64, 65]. In particular, the NF-κB/STAT1 signaling axis enhances interferon- and TLR-mediated responses, thereby reinforcing M1 polarization [23, 66, 67].

Given the central role of TLR4/NF-κB signaling, increasing attention has focused on its endogenous regulatory mechanisms. In microglia, IRAK-M limits TLR signaling by stabilizing the IRAK1/IRAK4–MyD88 complex, thus preventing NF-κB activation [68]. IRAK-M expression is upregulated in response to inflammation in conditions such as epilepsy and experimental autoimmune encephalomyelitis (EAE) [23–25]. These studies suggest that IRAK-M functions as an intrinsic negative feedback regulator that suppresses neuroinflammatory cascades. In addition, IRAK-M modulates microglial and macrophage polarization: microglia derived from IRAK-M knockout (IRAK-M⁻/⁻) mice display a marked shift toward the M1 phenotype [69]. Mechanistically, IRAK-M limits M1 polarization through suppression of the NF-κB and STAT1 pathways [23, 25].

In our study, repeated NTG administration was accompanied by progressive upregulation of IRAK-M and activation of TLR4/NF-κB/STAT1-related signaling in the TNC. Notably, IRAK-M deletion further intensified the inflammatory phenotype, as reflected by greater microglial reactivity, increased expression of the pro-inflammatory microglial markers CD86 and iNOS (Supplementary Fig. 4), and higher levels of pro-inflammatory mediators, including IL-1β, IL-6, TNF-α, and IFN-γ. These findings suggest that IRAK-M upregulation during chronic migraine modeling may represent an endogenous counter-regulatory response that limits excessive microglial activation and neuroinflammation. Consistent with this interpretation, IRAK-M overexpression produced reciprocal effects, including reduced microglial reactivity and lower levels of neuroinflammation-related signaling in the TNC.

An important finding of this study is that IRAK-M was closely linked to NLRP3 inflammasome- and pyroptosis-related responses in the NTG-induced CM model. TLR4/NF-κB signaling provides the transcriptional priming required for IL-1β production, whereas NLRP3-mediated caspase-1 activation promotes its maturation and release [70, 71]. IL-1β may subsequently enhance excitatory synaptic transmission by promoting NMDAR phosphorylation while weakening GABAergic and glycinergic inhibition, thereby favoring neuronal hyperexcitability [72–74]. In the TNC, NLRP3, ASC, GSDMD, and caspase-1 signals showed substantial overlap with Iba1-positive microglia, supporting the involvement of microglia in inflammasome- and pyroptosis-related responses during chronic migraine modeling. Importantly, IRAK-M deficiency was accompanied by increased expression of inflammasome components, enhanced caspase-1 and GSDMD cleavage, and elevated levels of mature inflammatory cytokines, whereas TNC-targeted IRAK-M overexpression produced the opposite pattern. Taken together, these bidirectional changes suggest that IRAK-M may act as an endogenous brake on excessive inflammasome activation and pyroptosis-related inflammation in the TNC. One plausible interpretation is that IRAK-M limits both NF-κB-dependent inflammatory priming and the subsequent amplification of NLRP3/caspase-1/GSDMD-related responses, although the direct contribution of this pathway to the behavioral effects of IRAK-M remains to be established.

Beyond its effects on nociceptive hypersensitivity, IRAK-M overexpression was also associated with improved outcomes in open-field and elevated plus-maze assays, which are commonly used to assess anxiety-like behavioral measures in rodents. This observation is relevant because chronic migraine is frequently accompanied by psychiatric comorbidities, particularly anxiety and depression [6, 75, 76]. In preclinical headache research, anxiety-like behaviors are commonly evaluated using the open-field, elevated plus-maze, and light aversion tests; however, interpretation of these behavioral paradigms requires caution, especially in migraine models, because pain, photophobia, locomotor suppression, and exploratory avoidance may produce overlapping behavioral outcomes [77]. The open-field test, in particular, reflects both general locomotor activity and anxiety-related exploratory behavior, whereas the elevated plus-maze is a widely used assay for anxiety-related behavior in rodents [78, 79].

In the present study, NTG-treated AAV^−IRAK−M mice showed greater center-zone exploration in the open-field test and greater open-arm exploration in the elevated plus-maze test. These changes may reflect reduced anxiety-like avoidance, alleviation of pain-related behavioral suppression, or both. Importantly, IRAK-M overexpression also increased total locomotor activity, which may have contributed to the increased exploratory behavior observed in these tests. Therefore, although the behavioral data support an improvement in anxiety-like behavioral measures associated with the chronic migraine model, they do not establish an independent anxiolytic effect of IRAK-M overexpression. Future studies using behavioral paradigms less dependent on spontaneous locomotion will be required to determine whether IRAK-M directly modulates anxiety-related mechanisms.

Nonetheless, several limitations should be acknowledged. First, although IRAK-M knockout and overexpression produced reciprocal changes in TLR4/NF-κB- and NLRP3/GSDMD-related readouts, no pathway-specific pharmacological inhibition, genetic epistasis, or rescue assay was performed. Therefore, the present data do not establish direct mediation through these pathways. Second, the use of global IRAK-M knockout mice does not allow a definitive separation of central and peripheral effects, as IRAK-M deficiency may influence multiple cell types systemically. Although TNC-targeted AAV-mediated IRAK-M overexpression supports an important role for central IRAK-M signaling in the CM model, the precise cell type-specific mechanisms remain to be defined using conditional strategies. Third, mechanical sensitivity was assessed only in the hindpaw, which reflects generalized pain sensitization but does not directly capture cephalic hypersensitivity relevant to migraine. Fourth, interpretation of the open-field findings is potentially confounded by changes in locomotor activity. Additional behavioral paradigms less dependent on spontaneous movement will be required to distinguish anxiolytic-like effects from behavioral improvements secondary to pain relief. Fifth, because microglia- and TLR4-dependent pain mechanisms are sexually dimorphic, the present findings should be interpreted as applying to male mice only [80–83]. Finally, the clinical relevance of glial-targeted interventions remains uncertain; prior pilot studies of ibudilast in chronic migraine and medication-overuse headache did not show clear benefit [21, 22]. These limitations warrant caution when translating the present preclinical findings to human migraine. Future work should include cephalic sensory assays, female animals, cell type-specific manipulation, and pathway-specific rescue experiments.

Conclusion

In conclusion, our findings support IRAK-M as an important endogenous regulator of microglial reactivity and neuroinflammatory responses in the NTG-induced chronic migraine model. IRAK-M deficiency exacerbated pain hypersensitivity and was accompanied by increased microglial reactivity and enhanced inflammasome- and pyroptosis-related inflammatory signaling. Conversely, TNC-targeted IRAK-M overexpression attenuated pain hypersensitivity and improved anxiety-related behavioral measures, together with corresponding reductions in these molecular and cellular alterations. The bidirectional effects of IRAK-M manipulation support a protective role for IRAK-M in CM-like pathophysiology, associated with reduced TLR4/NF-κB-related signaling, NLRP3 inflammasome activation, and GSDMD-associated pyroptotic responses. These findings provide a preclinical basis for further investigating IRAK-M-related neuroimmune pathways as potential targets for chronic migraine and associated anxiety-like behavioral alterations.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

CM

chronic migraine

NTG

nitroglycerin

IRAK-M

interleukin-1 receptor-associated kinase M

TLR4

Toll-like receptor 4

NF-κB

nuclear factor-κB

MyD88

myeloid differentiation primary response 88

NLRP3

NLR family pyrin domain-containing 3

AAV

adeno-associated virus

TNC

trigeminal nucleus caudalis

SD

spreading depolarization

CNS

central nervous system

PWT

paw withdrawal threshold

LAT

light-aversion test

OFT

open-field test

EPM

elevated plus maze

WT

wild-type

KO

knockout

Sp5C

spinal trigeminal nucleus, caudal part

EAE

experimental autoimmune encephalomyelitis

Author contributions

X.-T. L., S.-R. X., and X.-S. L. drafted the initial version of the manuscript. X.-T. L. conducted the behavioral tests, viral injection, immunofluorescence staining, and western blotting, with assistance from X.-Y. Z., Y.-H. Z., X.-M. L. Data analysis was performed by Y. H., Z.-Y. W., and Y.-F. X. Y.-Y. Z., Y.-W. D., X.-S. L., and W. X. conceived and designed the study, contributed to manuscript preparation, and supervised all aspects of the project. All authors reviewed and approved the final version of the manuscript.

Funding

This work was supported by the Major and Refractory Disease Clinical Collaboration Program of Integrated Traditional Chinese and Western Medicine (ZDYN-2024-A-120); the National Natural Science Foundation of China (No. 82405378); the China Postdoctoral Science Foundation (No. 2024M761351, 2025T181075); the Guangdong Basic and Applied Basic Research Foundation (Grant No. 2023A1515220030); the Key Discipline Construction Project of Traditional Chinese Medicine of Guangdong Province (Grant No. 20220105); and the second national famous Traditional Chinese Medicine Inheritance Studio (Grant No. G724290126).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethical approval

All animal experiments were conducted in accordance with protocols approved by the Institutional Animal Care and Use Committee of Southern Medical University (approval no. L202406032, Guangdong, China).

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.