Neuronal GLUT3 alleviates hyperalgesia and neuroinflammation in chronic migraine mice via the TCA cycle

Neuronal GLUT3 alleviates hyperalgesia and neuroinflammation in chronic migraine mice via the TCA cycle | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Neuronal GLUT3 alleviates hyperalgesia and neuroinflammation in chronic migraine mice via the TCA cycle JingYuan Zheng, ZiJuan Guo, Xiao Ren, XingYang Liu, Ke Wang, KaiYan Lin, and 14 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8411532/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Chronic migraine (CM) is closely linked to systemic metabolic disorders and neuroinflammation, but the molecular mechanisms connecting energy metabolism dysfunction to pain-related inflammation remain unclear. Methods Plasma samples from CM patients were analyzed using transcriptomics and metabolomics. A CM mouse model was induced by administration of nitroglycerin (NTG). Behavioral evaluations were conducted using von Frey filaments and hot plate tests. Western blotting, immunofluorescence, transcriptome, targeted metabolomics techniques were employed to investigate the molecular mechanisms. 18 F-FDG-PET/CT and 2-NBDG staining were employed to assess glucose uptake. Neuron-specific GLUT3 overexpression and intervention with 20 mg/kg 3-nitropropionic acid (3-NPA), a tricarboxylic acid (TCA) cycle inhibitor, were performed to verify the regulatory loop’s specificity and clinical therapeutic potential, as well as 100 mg/kg Idebenone. Results Patients with CM exhibited energy metabolism abnormalities including dysregulated oxidative phosphorylation pathway-related genes and reduced plasma levels of TCA cycle-related metabolites. NTG-induced mice exhibited decreased glucose uptake in the trigeminal nucleus caudalis (TNC) and neuron-specific GLUT3 downregulation, rather than glial cells. Neuron-specific GLUT3 overexpression enhanced TCA cycle function, increased mechanical pain thresholds, and inhibited central sensitization by downregulating the expression of c-Fos and calcitonin gene-related peptide (CGRP), accompanied by an inhibition of NF-κB pathway. Furthermore, 3-NPA reversed the inhibitory effect of neuron-specific GLUT3 overexpression on the NF-κB pathway. Idebenone, a clinically approved mitochondrial protective agent, alleviated CM-related mechanical allodynia and reversed the upregulation of c-Fos/CGRP in the TNC of NTG-induced mice. Conclusions These results identify the neuronal GLUT3-TCA cycle-NF-κB axis as a key regulatory pathway in CM, bridging metabolic dysfunction and neuroinflammation. Notably, the clinically available mitochondrial protective agent Idebenone mimics these benefits via TCA cycle targeting, underscoring its application potential in the cilinic. These findings offer novel mechanistic insights and metabolic-targeted therapeutic strategies for CM. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Introduction Chronic Migraine (CM), one of the most prevalent neurovascular disorders globally, has an epidemiological prevalence of 1.4%-2.2% worldwide[ 1 ]. Approximately 80% of patients experience ≥ 15 headache days per month, accompanied by severe symptoms such as nausea, vomiting, photophobia, and phonophobia, leading to significant disability and reduced quality of life[ 2 ]. Neuroinflammation and trigeminal vascular system sensitization are well-recognized key downstream events in CM pathogenesis. Clinically, triptans and calcitonin gene-related peptide (CGRP) antagonists are the mainstay treatments; however, 30%-40% of patients exhibit drug resistance, 60% fail to achieve effective symptom control, with long-term use causing complications like medication-overuse headache[ 3 , 4 ]. Notably, emerging evidence suggests that metabolic dysfunction is also closely implicated in CM development, potentially interacting with neuroinflammation to drive disease progression. The core challenge underlying this predicament lies in the incomplete understanding of CM’s pathogenesis, while neuroinflammation and trigeminal sensitization are well-recognized key downstream events, the identification of novel upstream regulatory targets remains urgently needed[ 5 , 6 , 7 , 8 ]. Neurons, as the core units of brain energy metabolism and signal transduction, represent the initial site of functional imbalance in cerebral metabolic disorders. Glucose transporter 3 (GLUT3), a neuron-specific glucose transporter with 12 transmembrane domains and high glucose affinity, maintains neuronal energy supply under low glucose conditions and provides critical metabolic substrates[ 9 , 10 ]. Previous studies indicate that GLUT3 knockout lowers glucose supply, increases oxidative stress, and aggravates hippocampal neuronal death and cognitive impairment under hypoxic conditions[ 11 , 12 , 13 ], whereas its overexpression raises neuronal glucose uptake, up-regulates Aβ-degrading enzymes, suppresses the integrated stress response, and thereby improves cognitive performance in Alzheimer’s disease models[ 14 , 15 ]. The tricarboxylic acid (TCA) cycle—the hub of mitochondrial energy metabolism—depends on glucose-derived pyruvate to maintain flux and to generate ATP, NADH and FADH₂, any GLUT3-mediated reduction in intracellular glucose will decrease pyruvate delivery to mitochondria, slow TCA turnover, and lower the NAD⁺/NADH ratio. The resulting redox imbalance impairs NAD⁺-dependent anti-oxidative enzymes, increases reactive oxygen species (ROS), and activates pro-inflammatory pathways such as NF-κB, thus driving neuroinflammation[ 16 , 17 ]. Despite the accumulating evidence pointing to the role of GLUT3 in neuronal metabolism and inflammation regulation, its functions in CM development and progression, as well as its involvement in CM-related metabolic and inflammatory dysregulation, have not been well studied. In the present study, we aimed to elucidate the roles of GLUT3 in CM pathogenesis and progression. Integrating transcriptomic and metabolomic analyses, we observed that neuronal GLUT3 overexpression enhanced glucose uptake, restored TCA cycle function, and thereby inhibited the NF-κB pathway to alleviate central sensitization and delay CM progression. Notably, our findings highlight GLUT3 as a promising therapeutic target for CM management, while positioning Idebenone, a clinically actionable agent known to directly enhance TCA cycle activity, as a potential therapeutic drug that holds promise for alleviating CM pathology. Methods Animal Eight-week-old male C57BL/6 mice, weighing 20–25 g, were purchased from the Laboratory Animal Center of Xiamen University. All animal experiments were approved by the Animal Ethics Committee of Xiamen University. All mice were housed under specific pathogen-free (SPF) conditions with a controlled temperature of 22 ± 1°C, relative humidity of 50%-60%, and a 12-hour light/dark cycle. Ad libitum access to standard laboratory chow and sterile water was provided throughout the experimental period. Mouse model of chronic migraine The CM model was established with minor modifications to a previous protocol19]. Nitroglycerin (NTG; Baiyunshan, China) stock solution (5 mg/mL) was prepared in 30% ethanol/30% propylene glycol/40% ultrapure water, and diluted to 1 mg/mL with sterile saline immediately before use to avoid degradation. Mice in the CM group received intraperitoneal NTG (10 mg/kg) every other day for 5 injections; controls received an equal volume of sterile saline. Adeno-associated virus (AAV) and Drug Administration GLUT3 was encoded by gene Slc2a3. Viral solution is thawed on an ice bath (≤ 2 freeze-thaw cycles), centrifuged at 500×g for 1 min (4°C), and then stereotaxically injected into the lateral ventricle (AP: -7.5 mm, ML: ±1.7 mm, DV: -4.4 mm, relative to the xiphoid process). The injection rate was 0.02 µL/min (total 100 nL). To minimize backflow, the needle was held stationary for 10 min post-injection. Mice were anesthetized with 1%-1.5% isoflurane during surgery. Postoperatively, analgesia was provided with buprenorphine (2 mg/kg, intraperitoneal injection), followed by 15 min of recovery at 37°C. Transfection efficiency ≥ 60% was verified by immunofluorescence 3 weeks post-injection. Mice received intraperitoneal injections of 3-NPA (20 mg/kg, diluted in 0.9% saline) or intragastric administration of Idebenone (200 mg/kg, dissolved in corn oil) 60 min prior to each NTG injection[ 20 ]. These treatments were administered every other day for a total of five doses. Control mice received an equal volume of 0.9% saline (to match the 3-NPA vehicle) and corn oil (to match the idebenone vehicle) via the corresponding routes. The dosages of AAV and pharmacological agents were selected based on previously published literature. Animal Behavioral Experiments All behavioral tests were conducted by operators blinded to group assignments during both testing and data analysis, from 9:00 AM to 7:00 PM. Prior to the experiment, mice underwent 3 consecutive days of acclimation to the experimenters (10 min daily) and were acclimated for 30 min in a quiet acrylic cage with a wire mesh flooring to familiarize them with the general testing environment. For each test session, mice were further acclimated to the specific testing apparatus for 30 min prior to assessment: for hindlimb mechanical testing, this involved adaptation in a glass chamber with a wire mesh floor; for periorbital mechanical testing, mice were confined in an enclosure with only their head exposed to prevent movement. The testing room maintained a constant temperature of 22 ± 1°C, relative humidity of 50%–60%, and no noise interference; all testing equipment was disinfected with 75% ethanol before use and after each mouse was tested. Mechanical pain thresholds were assessed prior to and within a 2-hour timeframe following each administration. For hindlimb mechanical pain threshold measurement, von Frey filaments (Stoelting, 0.008–2 g) were applied vertically to the fat pad-avoiding central region of the hindlimb. A positive response was defined as paw withdrawal, trembling, or licking following stimulation. The 50% withdrawal threshold was determined using the “up-and-down method”, with three repetitions performed per test and the mean value calculated for final analysis. For periorbital mechanical sensitivity assessment, the same von Frey filaments were used, with filaments applied to the periorbital region with gradually increasing intensity. A positive response was characterized by immediate head withdrawal or forelimb scratching of the stimulated periorbital area. Consistent with hindlimb testing, three repetitions were conducted, and the mean value was used for subsequent analysis. 18 F-FDG-PET/CT Mice were subjected to 16 h overnight fasting with free access to water to minimize blood glucose-related interference on 18 F-FDG uptake, and maintained at 22 ± 1℃ during fasting to avoid stress-induced glycemic fluctuations. Prior to imaging, mice were anesthetized via isoflurane inhalation with induction parameters of 2–3% isoflurane in 1 L/min O₂ and maintenance parameters of 1%-1.5% isoflurane in 1 L/min O₂ until loss of corneal reflex and stable muscle relaxation. Animals were then immobilized prone on a custom imaging bed, weighed accurately, and 5 µL of tail vein blood was collected for glucose measurement using a portable Accu-Chek glucometer (Roche). Only mice with blood glucose levels of 3.9–7.8 mmol/L were included. 18 F-FDG with radiochemical purity > 95% and calibrated via a dose calibrator was administered via tail vein injection at 10 µCi/g body weight at a slow rate to prevent extravasation. Post-injection, the injection site was visually inspected for leakage, and mice with evident extravasation were excluded from subsequent analysis. The imaging bed was positioned to center the mouse head on the detector, with body temperature maintained at 37 ± 0.5℃ via a feedback-controlled heating pad and respiratory rate continuously monitored at ≥ 60 breaths/min through a pressure-sensitive sensor to ensure anesthetic stability. CT scanning with 80 kV voltage, 500 µA current, 0.2 mm slice thickness, and 80×80 mm field of view was performed first for anatomical localization and PET attenuation correction. Immediately following CT acquisition, a 60-min dynamic PET scan with a 128×128 matrix and list-mode acquisition was initiated, reconstructed via ordered subsets expectation maximization with point spread function correction (OSEM-PSF) using 11 iterations and 16 subsets. Image analysis was conducted with PMOD 3.0 software: PET and CT images were spatially co-registered, followed by alignment to the Allen Reference Atlas (ARA) Common Coordinate Framework (CCFV3) for automated anatomical segmentation. Standardized uptake values (SUVs) were calculated as (tissue radioactivity concentration in Bq/mL × body weight in g) / injected dose in Bq, with SUVmean as the primary quantitative indicator and SUVmax as a supplementary parameter. Target brain regions included the hippocampus, cerebral cortex, cerebellum, hypothalamus, and medulla oblongata, and region-of-interest (ROI) delineation was manually verified for ambiguous anatomical boundaries. Sample Collection and Frozen Section Preparation Mice were deeply anesthetized with sodium pentobarbital (60 mg/kg, intraperitoneal injection). The trigeminal nucleus caudalis (TNC) tissue was rapidly dissected on ice and immediately flash-frozen in liquid nitrogen, reserved for metabolomic, transcriptomic, and Western blotting (WB) analyses without fixation to preserve molecular integrity. For the remaining TNC tissue, transcardial perfusion was performed via the left ventricle with 30 mL 4°C prechilled 0.01 M PBS at a flow rate of 4–5 mL/min until the liver and spleen turned pale, followed by 10 mL 4°C prechilled 4% paraformaldehyde (PFA) for fixation. The fixed TNC tissue was retrieved on ice, post-fixed in 4% PFA at 4°C for 24–36 h, then dehydrated in 30% sucrose solution at 4°C for 48 h with daily solution renewal. Subsequently, the tissue was embedded in Sakura OCT compound and frozen at -80°C for at least 2 h. Coronal sections (30 µm) containing the TNC region were cut using a Leica CM1950 cryostat set at -20°C for immunofluorescence staining, and stored in 0.01 M PBS at 4°C protected from light. Immunofluorescence (IF) 30 µm TNC sections were washed with 0.01 M PBS (pH 7.4) for 3 × 3 min to remove residual storage buffer. Subsequently, primary antibodies (Table 1 ) were diluted in 0.01 M PBS containing 0.3% Triton X-100 and 1% bovine serum albumin (BSA) (blocking buffer), and free-floating sections were incubated overnight at 4°C in the dark to prevent fluorophore quenching. The next day, sections were washed with 0.01 M PBS for 3 × 5 min to remove unbound primary antibodies, then incubated with corresponding fluorophore-conjugated secondary antibodies at room temperature for 2 h in the dark. After secondary antibody incubation, sections were washed with 0.01 M PBS for 3 × 5 min, followed by nuclear counterstaining with 4’,6-diamidino-2-phenylindole (DAPI) (Yeasen Biotech, China, 40728ES03) for 10 min in the dark. Sections were rinsed with pre-chilled 0.01 M PBS for 3 × 5 min, mounted with anti-fade mounting medium, and sealed with nail polish. Confocal images were captured using a Zeiss LSM800 inverted laser scanning confocal microscope, with uniform gain and exposure time applied across all samples to ensure comparability. Table 1 The antibodies used in the immunofluorescence staining. NeuN Polyclonal antibody Abcam, 1:500, ab177487 Iba1 Polyclonal antibody Abcam, 1:500, ab5076 GFAP Polyclonal antibody Abcam, 1:500, c-Fos Polyclonal antibody CST, 1:500, 2250 CGRP Polyclonal antibody SCBT, 1:1000, sc-57053 TOM20 Polyclonal antibody Proteintech,1:500,11802-1-AP 2-NBDG Glucose Uptake Assay Mice were subjected to a 16-hour fasting period with free access to water to eliminate feeding-induced glucose fluctuations prior to tail vein injection of 2-NBDG (MCE, USA, HY-116215) at a dose of 375 µg per mouse (stock solution concentration: 2.5 mg/mL; injection volume: 150 µL per mouse)[ 21 , 22 ]. Post-injection leakage or bleeding was monitored, and one hour later, mice were deeply anesthetized and perfused with pre-cooled 0.01 M PBS (pH 7.4) until organ blanching, followed by 4% PFA fixation for subsequent sectioning and fluorescence detection (excitation/emission: 488 nm/520 nm). For validation of GLUT-mediated 2-NBDG uptake, Cytochalasin B (Cyto B, 0.5 mM, 0.5 µL per side) and 4,6-O-methyl-D-glucose (4,6-O, 25 mM, 0.5 µL per side), GLUT inhibitors, were stereotaxically injected into the bilateral TNC prior to 2-NBDG administration. IRDye 800CW 2-DG Glucose Uptake Assay IRDye 800CW 2-DG (Licor, USA, 926–08946), a near-infrared fluorescent glucose analog, was reconstituted in sterile 1× PBS to 0.1 nmol/µL and filter-sterilized through a 0.2 µm filter if needed. Under gentle restraint, mice were injected with 10 nmol of the probe via tail vein, with post-injection leakage or bleeding monitored[ 23 ]. Twenty-four hours later (per manufacturer’s recommendation for optimal signal-to-noise ratio), mice were deeply anesthetized and perfused with pre-cooled 0.01 M PBS (pH 7.4) until organ blanching, followed by 4% PFA fixation for subsequent sectioning and fluorescence detection (excitation/emission: 774 nm/791 nm). Detection of Reactive Oxygen Species (ROS) TNC tissues were homogenized in ice-cold RIPA lysis buffer, centrifuged (12,000×g, 20 min, 4℃) to collect the lysate supernatant, and protein concentration was quantified via a BCA protein assay kit. For each sample, a 200 µL reaction system (20 µL lysate + 170 µL PBS [pH 7.4] + 10 µL DHE, final concentration 10 µM) was prepared, alongside a blank control (190 µL PBS + 10 µL sterile water) and a negative control (180 µL PBS + 10 µL DHE) to correct for DHE auto-oxidation. All mixtures were incubated at 37℃ for 30 min in the dark, then transferred to a black 96-well plate (100 µL/well, triplicates) to measure fluorescence (Ex/Em = 530/590 nm) via a microplate reader. Relative sample fluorescence was calculated by subtracting the negative control’s intensity from raw values. Western Blotting (WB) Total proteins were extracted from frozen brain tissue, which was thawed on ice and homogenized at a 1:10 weight-to-volume ratio in pre-chilled RIPA lysis buffer for 2 min at 120 Hz, followed by ultrasonication on ice for 30 s at 300 W. The homogenate was centrifuged at 12,000×g for 30 min at 4°C, and the supernatant was collected as total protein. Protein concentration was determined using the BCA method with a bovine serum albumin standard curve (0-400 µg/mL). Equal amounts of protein were mixed with 5× loading buffer and boiled at 95°C for 10 min, with membrane proteins excluded from heating. Proteins were separated by 10% or 15% SDS-PAGE at 80 V for 30 min, followed by 120 V for 60–90 min. Polyvinylidene fluoride (PVDF) membranes (Millipore, USA) were activated with methanol for 10 s prior to protein transfer, which was performed at 300 mA with the temperature maintained below 40°C for 60 min. The membranes were blocked with 5% skim milk powder in TBST for 2 h at room temperature, then incubated overnight at 4°C with primary antibodies (Table 2 ) diluted in 5% BSA. Subsequently, membranes were washed three times with TBST (10 min each) and incubated in the dark with HRP-conjugated secondary antibodies (1:10,000 dilution in 5% nonfat milk powder) for 1 hour at room temperature. After final washes with TBST and distilled water, protein bands were visualized using a fluorescence imager and saved as TIFF files. Band intensities were quantified using ImageJ software (NIH, USA). Table 2 The antibodies used in the immunoblot analysis. GAPDH Polyclonal antibody Proteintech,1:10000, 10494-1-AP Beta Tubulin Polyclonal antibody Proteintech,1:10000,10094-1-AP NF-kappaB p65 Monoclonal Antibody CST, 1:10000, 8242 Phospho-NF-kappaB p65 (Ser536) Monoclonal Antibody CST, 1:10000, 3033 IkappaB alpha Monoclonal Antibody CST, 1:10000, 4812 Phospho-IkappaB alpha (Ser32) Monoclonal Antibody CST, 1:10000, 2859 TNF-alpha Polyclonal antibody Proteintech,1:10000, 17590-1-AP IL-1 beta Polyclonal antibody Proteintech,1:10000, 16806-1-AP IL-6 Polyclonal antibody Proteintech,1:10000, 21865-1-AP GLUT3 Polyclonal antibody Proteintech,1:10000, 20403-1-AP Transmission Electron Microscopy (TEM) Mice were decapitated under deep isoflurane anesthesia, and brain tissue was harvested on ice within 30 s. The TNC region was dissected into 1 mm³ cubes and fixed in pre-chilled TEM fixative (2.5% glutaraldehyde, 2% PFA in 0.1 M phosphate buffer, pH 7.4) at 4°C for 24 h. Tissues were rinsed with 0.1 M phosphate-buffered saline (3 × 15 min), post-fixed with 1% osmium tetroxide solution (room temperature, 1–2 h, protected from light), and rinsed again (3 × 15 min). Gradient dehydration was performed using ethanol solutions (30%, 50%, 70% for 15 min each; 80%, 90%, 95%, 100% for 15 min each) and acetone (2×30 min). Tissues were infiltrated with Epon 812 embedding medium (8:1:1 = Epon 812: hardener: accelerator), polymerized at 37°C (12 h) -45°C (12 h) − 60°C (24 h), and sectioned into 60–80 nm ultrathin slices. Slices were stained with 2% uranyl acetate (15 min, dark) and 0.3% lead citrate (8 min, CO₂-free environment), then imaged using a TEM (500–1000× for screening, 5000–50000× for organelle observation; 5–8 fields/sample, 2048×2048 pixels, TIFF format). Human Plasma Samples Informed consent was obtained from all participants, and the study was approved by the Ethics Committee of Xiamen University. Plasma samples were collected from 359 migraine patients and 140 healthy controls at the First Affiliated Hospital of Xiamen University, China, following written informed consent. Blood samples were centrifuged at 3,000 × g for 15 min at 4°C to isolate plasma, which was then aliquoted and stored at -80°C until metabolomic and transcriptomic analyses. Metabolomics Frozen mouse TNC brain tissues and human plasma samples were processed separately for metabolomic analysis. For metabolite extraction, samples were thawed at 4°C, mixed with pre-cooled methanol-acetonitrile-water (2:2:1, v/v), vortexed, subjected to low-temperature sonication for 30 min, incubated at -20°C for 10 min, centrifuged at 14,000×g, 4°C for 20 min, and supernatants were vacuum-dried; residues were reconstituted in 100 µL acetonitrile-water (1:1, v/v), vortexed, re-centrifuged at 14,000×g, 4°C for 15 min, and the resulting supernatants were used for injection. Chromatographic separation was performed using an Agilent 1290 Infinity LC system with two columns: For the HILIC column, we set a column temperature of 55°C, flow rate of 0.3 mL/min, and injection volume of 2 µL; mobile phases were A (90% water + 2 mM ammonium formate + 10% acetonitrile) and B (acetonitrile + 0.4% formic acid), with the gradient: 85% B 0–1.0 min, linear decrease to 80% B 1.0–3.0 min, 80% B 3.0–4.0 min, linear decrease to 70% B 4.0–6.0 min, linear decrease to 50% B 6.0–10.0 min, 50% B 10-15.5 min, and linear increase to 85% B 15.5–23 min. For the C18 column, column temperature was 40°C, flow rate 0.4 mL/min, and injection volume 2 µL; mobile phases were A water + 5 mM ammonium acetate and B 99.5% acetonitrile, with the gradient: 5% increased linearly to 60% B over 0–5 min, 60% increased linearly to 100% B over 5–11 min, maintained at 100% B for 11–13 min, decreased linearly to 5% B over 13-13.1 min, and held constant at 5% B for 13.1–16 min. Samples were held in a 4°C autosampler and analyzed randomly, with quality control samples inserted into the sequence to monitor system stability. Mass spectrometry detection used an AB 6500 + QTRAP instrument (AB SCIEX) with an ESI source: source temperature 580°C, ion source gas 1 (GS1) 45, ion source gas 2 (GS2) 60, curtain gas (CUR) 35, ion spray voltage ± 4500 V (positive/negative modes), and operation in multiple reaction monitoring (MRM) mode. Raw MRM data were processed via MultiQuant software for peak area extraction; analyte concentrations were quantified using the ratio of each analyte’s peak area to the internal standard’s peak area combined with calibration curves, and acquired data underwent quality assessment prior to subsequent analyses. RNA-seq and data processing Total RNA was extracted from frozen mouse TNC brain tissues and human plasma samples: mouse TNC tissues and human plasma samples were processed using TRIzol reagent and a commercial plasma RNA extraction kit, respectively, following the manufacturers’ instructions. After RNA extraction, RNA purity and integrity were evaluated by NanoDrop 2000 and agarose gel electrophoresis for quality control. Libraries were constructed with the TruSeq Stranded mRNA Library Prep Kit (Illumina) and sequenced on an Illumina NovaSeq 6000 platform. Raw reads were filtered to remove low-quality reads and adapters; clean reads from mouse TNC tissues were aligned to the mouse reference genome (GRCm39), while those from human plasma were aligned to the human reference genome (GRCh38), both via HISAT2. To measure differentially expressed genes (DEGs), DESeq2 (version 1.40.2) was used with the criteria of |log2 fold change| > 1 and P < 0.05. Gene Ontology (GO) and KEGG pathway enrichment analyses were conducted using the R package clusterProfiler (version 4.8.3) for functional annotation of DEGs, with p < 0.05 considered statistically significant. Statistical Analysis GraphPad Prism 9.0 was used to perform statistical tests and all data are shown as the mean ± SEM. Unpaired two-tailed Student’s t test (for two group comparisons) and one-way or two-way analysis of variance (ANOVA) (for multiple group comparisons) followed by Tukey’s post-hoc test. A significance level of p < 0.05 was considered statistically significant. Results 1. Abnormal glucose metabolism in CM patients To explore the systemic metabolic and inflammatory perturbations underlying CM, we collected plasma samples and clinical data from 359 CM patients and 140 healthy controls, followed by metabolomic and transcriptomic profiling and integrated multi-omics analysis as outlined in the study workflow (Fig. 1 A). Multi-omics co-enrichment analysis for CM versus Control identified concurrent dysregulation of glucose metabolism, with core energy metabolic cascades such as the TCA cycle and OXPHOS being suppressed (Fig. 1 B). Transcriptomic analysis further delineated pathway alterations at the transcriptional level: Top 30 Gene Ontology (GO) term enrichment analysis (Fig. 1 C) revealed that downregulated transcripts were enriched in energy metabolism-related terms across multiple GO categories. Specifically, under Biological Process, these included “mitochondrial electron transport, NADH to ubiquinone” and “mitochondrial respiratory chain complex I assembly”; under Cellular Component, they covered “respiratory chain complex IV” and “mitochondrial respiratory chain complex I”; while under Molecular Function, terms such as “NADH dehydrogenase activity” and “NADH dehydrogenase (ubiquinone) activity” were included. Consistent with GO results, top 20 KEGG pathway enrichment dot plots for transcriptomic data revealed downregulated transcripts clustered in energy metabolism pathways such as oxidative phosphorylation, whereas upregulated transcripts concentrated in inflammatory pathways including the NF-κB signaling pathway and TNF signaling pathway (Fig. 1 D). Metabolomic analysis (Fig. 1 E) validated and complemented the transcriptomic findings that focused pathway enrichment plots showed the top suppressed metabolic pathways corresponded to core glucose metabolic cascades such as the TCA cycle, while the top elevated metabolic pathways such as caffeine metabolism. 2. Global cerebral glucose metabolism changes in NTG-induced CM mice The success of the NTG-induced chronic migraine mouse model was first confirmed by assessing the Paw Withdrawal Threshold and periorbital Withdrawal Threshold, a core behavioral phenotype of CM, using von Frey filaments. The model was established by administering intraperitoneal NTG at 10 mg/kg every other day for 5 total doses, spanning 9 days; VEH control group received equal volumes of sterile saline. To explore cerebral energy metabolism changes in this CM model, we performed macroscale detection of whole-brain glucose uptake using 18 F-FDG-PET/CT under anesthesia (Fig. 2 A). Results showed that NTG-treated mice had reduction in global cerebral glucose uptake rate that assessed by mean standardized uptake value (SUVmean) compared to VEH controls (Fig. 2 B-C). Among the key brain regions—including the hippocampus, cerebral cortex, cerebellum, and hypothalamus—the medulla oblongata emerged as the site of most significant metabolic alteration (Fig. 2 D). Specifically, as a pivotal relay station for migraine pain signaling located within the medulla oblongata, the TNC, a key brain region in CM, demonstrated a substantial decrease in glucose uptake relative to the VEH group (Fig. 2 E). To identify the cell type responsible for TNC metabolic abnormalities, we used 2-NBDG, a 488 nm-excitable fluorescent glucose analog, and IRDye 800CW 2-DG, a near-infrared fluorescent glucose analog, to assess cellular glucose uptake. We performed immunofluorescence co-staining with neuron-specific marker NeuN, microglia-specific marker Iba1, and astrocyte-specific marker GFAP to localize glucose uptake (Fig. 2 E, S1 A). Consistent results were obtained with both probes: NeuN + neurons accounted for the vast majority of glucose uptake in the TNC under both VEH and NTG conditions, exceeding that of Iba1 + microglia and GFAP + astrocytes (Fig. 2 F, S1 B). When analysing glucose uptake intensity across cell types, we similarly observed significantly reduced glucose uptake in NeuN + neurons from CM mice compared to VEH controls (Fig. 2 G, S1 C). Conversely, glucose uptake by Iba1 + microglia and GFAP + astrocytes showed no significant alterations (Fig. 2 H-I, S1 D-E). This indicated that TNC metabolic dysfunction in CM is neuron-specific, highlighting the clinical relevance of targeting neuronal glucose uptake for CM intervention. 2-NBDG fluorescence intensity in TNC neurons was significantly reduced by 4,6-O and Cyto B, GLUT inhibitors, thus confirming 2-NBDG uptake in TNC neurons is dependent on GLUT and validating it as a reliable marker for assessing GLUT-mediated glucose uptake (Fig. S2A-B). GLUTs are rate-limiting for cerebral glucose uptake, and neurons predominantly express GLUT1, GLUT3, and GLUT4[ 24 ]. To identify the molecular driver of impaired neuronal glucose uptake, we detected the expression of these GLUTs in the TNC using WB after the last NTG injection. WB results showed that GLUT3 protein levels were reduced in CM mice compared to VEH mice (Fig. 2 G-H), while GLUT1 and GLUT4 protein expression remained unchanged (Fig. 2 I-J). These data collectively indicate that neuronal GLUT3 downregulation is the core mechanism underlying abnormal TNC glucose uptake in CM. 3. Neuronal GLUT3 overexpression alleviates hyperalgesia and central sensitization in CM mice To elucidate the role of neuronal GLUT3 in CM, we performed stereotaxic injection of adeno-associated virus (AAV) vectors targeting the TNC. The experimental group received AAV-hSyn-GLUT3 for neuron-specific GLUT3 overexpression, while the control group was injected with AAV-hSyn-mCherry as the empty vector control. Three weeks after viral injection, procedures for establishing the chronic migraine model were initiated (Fig. S3A-B). Subsequently, WB analysis quantitatively measured GLUT3 protein levels to confirm transfection efficiency (Fig. S3C-D). Immunofluorescence co-staining of GLUT3 with NeuN, Iba1, and GFAP was performed to verify transfection specificity. Results showed that GLUT3 expression was neuron-specific (Fig. S3E-F). Behavioral tests using von Frey filaments demonstrated that neuronal GLUT3 overexpression significantly enhanced both plantar and periorbital pain thresholds, effectively alleviating NTG-induced basal and acute mechanical allodynia in CM mice (Fig. 3 A-D). c-Fos is an immediate early gene marker of neuronal activation, and CGRP is a key neuropeptide that drives migraine central sensitization[ 26 ]. To assess neuronal activation and central sensitization in the TNC, we performed immunofluorescence staining for these two markers. Quantitative analysis confirmed that relative to the VEH group, the CM group exhibited marked elevations in TNC c-Fos + cell counts and CGRP fluorescence intensity. Notably, neuronal GLUT3 overexpression robustly reversed these pro-sensitization alterations, reducing c-Fos + cell numbers and CGRP fluorescence intensity in the TNC of CM mice to levels comparable to the VEH group. These molecular changes correlated with the restored mechanical pain thresholds observed in the GLUT3 overexpression group (Fig. 3 E-H). 4. Neuronal GLUT3 overexpression restores neuronal glucose metabolism and suppresses microglia activation in CM mice As a high-affinity glucose transporter, GLUT3 overexpression enhances neuronal glucose uptake efficiency, fueling mitochondrial energy production pathways (Fig. 4 A-B, S1 F-G). Given that mitochondrial structural integrity is tightly linked to functional capacity, we used transmission electron microscopy (TEM) to observe the ultrastructure of TNC neurons. In CM mice, neurons exhibited prominent mitochondrial swelling, fragmented and vacuolated cristae, and a reduced number of mitochondria compared to VEH group. Strikingly, GLUT3 overexpression significantly ameliorated these pathological changes, restoring mitochondrial morphology and quantity (Fig. 4 C-D). TOM20 is a key marker of mitochondrial biogenesis and membrane transport capacity[ 27 , 28 ]. To further assess mitochondrial function, we examined it via immunofluorescence and WB. The results showed that TOM20 fluorescence intensity in the TNC of CM mice was markedly decreased, indicating impaired mitochondrial biogenesis and function. In contrast, GLUT3 overexpression robustly restored TOM20 expression at both protein and fluorescence levels (Fig. 4 E-H). These findings collectively demonstrate that neuronal GLUT3 overexpression improves mitochondrial structural integrity, enhances membrane potential stability, and restores membrane protein transport function, thereby reversing cerebral energy metabolism dysfunction in CM mice. To characterize microglial activation, we performed Iba1 immunofluorescence staining in the TNC (Fig. 4 I). Relative to the NTG + AAV-NC group, the NTG + AAV-Slc2a3 + group showed significantly reduced Iba1 + cell numbers and fluorescence intensity, verifying GLUT3 overexpression suppresses microglial activation (Fig. 4 J-K). 5. Neuronal GLUT3 overexpression modulates the TCA cycle and reduced overproduction of ROS in CM mice To characterize GLUT3-mediated metabolic regulation in CM, we performed targeted metabolomic profiling of TNC tissues from VEH + AAV-NC, NTG + AAV-NC, and NTG + AAV-Slc2a3 + groups. A principal component analysis (PCA) score plot showed distinct separation between the NTG + AAV-NC and VEH + AAV-NC groups, while the NTG + AAV-Slc2a3 + group clustered closely with VEH + AAV-NC—confirming that GLUT3 overexpression reverses NTG-induced metabolic dysregulation (Fig. 5 A). To characterize metabolic perturbations and the regulatory role of GLUT3 in CM, we first identified differential metabolites across experimental groups. Venn diagram analysis (Fig. 5 B-C) revealed that 145 metabolites were elevated in the VEH + AAV-NC vs NTG + AAV-NC group, while 26 metabolites were downregulated in the NTG + AAV-NC vs NTG + AAV-Slc2a3 + group. Notably, 14 of these metabolites exhibited reversed expression trends following GLUT3 overexpression. 88 metabolites were reduced in the VEH + AAV-NC vs NTG + AAV-NC group, alongside 45 downregulated metabolites in the NTG + AAV-NC vs NTG + AAV-Slc2a3 + group. Among these, 19 metabolites showed reversed expression trends upon GLUT3 overexpression. KEGG pathway enrichment of differential metabolites (Fig. 5 D) and subgroup comparisons (Fig. S3A-B) highlighted glucose metabolism-related cascades as the most significantly enriched in NTG-induced dysregulation and further showed that central carbon metabolism, glucagon signaling and the TCA cycle were functionally reversed by GLUT3 overexpression. A heatmap of key metabolites in glycolysis, the pentose phosphate pathway (PPP), and the TCA cycle revealed pathological metabolic remodeling in NTG + AAV-NC mice: upstream glycolysis/PPP metabolites were elevated, while downstream glycolysis/TCA/OXPHOS metabolites were reduced. Notably, GLUT3 overexpression selectively restored TCA cycle metabolites, emphasizing its role in repairing core glucose metabolic networks (Fig. 5 C-D). Quantification of TCA intermediates confirmed that NTG reduced cis-aconitic acid, isocitric acid, fumaric acid, and malic acid, while changes reversed by GLUT3 overexpression (Fig. 5 G-J). Consistent with metabolic restoration, GLUT3 overexpression also attenuated NTG-induced ROS elevation (Fig. 5 K). In glycolysis pathway, NTG + AAV-NC mice showed elevated levels of upstream metabolites including fructose 6-phosphate, fructose 1,6-bisphosphate and glucose 6-phosphate (Fig. S4C-F), alongside reduced concentrations of mid-late metabolites such as 2-phosphoglyceric acid, phosphoenolpyruvic acid and lactate (Fig. S4G-L). Additionally, in the OXPHOS, NTG + AAV-NC mice showed levels of nicotinamide (Fig. S4M) and nicotinamide ribotide (Fig. S4N) remained unchanged, while energy intermediates were disrupted—characterized by elevated AMP and AMP/ADP ratio, and decreased ADP (Fig. S4J-L). In PPP pathway, NTG + AAV-NC mice exhibited a state of pathway activation characterized by elevated levels of metabolites including D-sedoheptulose 7-phosphate, D-ribulose 5-phosphate, D-ribose, Glucosamine 6-phosphate and N-acetylglucosamine 6-phosphate (Fig. S4O-S). This compensatory activation, presumably driven by oxidative stress, was not significantly modulated by GLUT3 overexpression. In contrast, GLUT3 overexpression did not exert significant modulatory effects on the glycolysis pathway or the compensatory activation of PPP driven by oxidative stress in CM mice. These findings collectively demonstrate that GLUT3 ameliorates CM via selective restoration of the TCA cycle rather than modulation of glycolysis or PPP pathways. 6. Neuronal GLUT3 overexpression ameliorates chronic migraine via the NF-κB pathway To profile transcriptomic changes in the TNC, RNA sequencing was performed. In the comparison between the VEH + AAV-NC group and the NTG + AAV-NC group, 365 genes were upregulated and 587 genes were downregulated in the VEH + AAV-NC group. In the comparison between the NTG + AAV-NC group and the NTG + AAV-Slc2a3 + group, 382 genes were upregulated and 716 genes were downregulated in the NTG + AAV-NC group (Fig. 6 A-B). Venn diagrams showed that 214 and 187 differentially expressed genes (DEGs) reversed their expression trends following GLUT3 overexpression (Fig. 6 C-D). KEGG pathway enrichment analysis of the VEH + AAV-NC vs NTG + AAV-NC comparison (Fig. 6 E) showed that NTG treatment altered key pathway profiles: glucose metabolism-related processes, including Glycolysis/Gluconeogenesis and the Pentose phosphate pathway, were among the upregulated pathways, while downregulated pathways were enriched in neuroinflammation and sensory signaling-related cascades, such as the NF-κB signaling pathway and the Inflammatory mediator regulation of TRP channels. In contrast, in the NTG + AAV-NC vs NTG + AAV-Slc2a3 + comparison, GLUT3 overexpression mainly suppressed the NF-κB pathway (Fig. 6 F). These findings align with our prior functional and molecular observations, further emphasizing the role of GLUT3 in regulating NF-κB-mediated neuroinflammation. 7. Neuronal GLUT3 overexpression alleviates hyperalgesia in chronic migraine mice via the TCA cycle To verify whether GLUT3 alleviates CM via the TCA cycle, relevant experimental operations were carried out. 3-NPA, a specific inhibitor of SDH, a rate-limiting enzyme in the TCA cycle, was used to block TCA flux30]. Behavioral tests using von Frey filaments evaluated mechanical allodynia at baseline and 2 h post-each NTG injection. Results showed that the AAV-Slc2a3 + group exhibited significantly elevated PWTs at both basal and acute phases, effectively alleviating NTG-induced mechanical allodynia. Notably, co-administration of 3-NPA substantially reversed the analgesic effect of GLUT3 overexpression (Fig. 7 F-I), demonstrating that blocking the TCA cycle abrogates GLUT3 overexpression-mediated pain relief. To assess the impact on central sensitization, we performed immunofluorescence staining for c-Fos and CGRP in the TNC and found that c-Fos + cell numbers and CGRP fluorescence intensity in the NTG + AAC-Slc2a3 + +3-NPA group were significantly higher than those in the NTG + AAC-Slc2a3 + group and indistinguishable from the CM group (Fig. 7 J-M)​. 8. Neuronal GLUT3 overexpression mitigates neuroinflammation in chronic migraine via the TCA cycle Immunoblot of NF-κB pathway components and downstream pro-inflammatory factors (Fig. 8 A) showed that in the TNC of NTG-induced CM mice, the ratios of phosphorylated p65 (p-p65)/total p65 and phosphorylated IκBα (p-IκBα)/total IκBα were significantly elevated (Fig. 8 B-C). Concurrently, protein levels of pro-inflammatory cytokines TNF-α, IL-6, and IL-1β were markedly upregulated (Fig. 8 D-F), indicating excessive NF-κB pathway activation and prominent neuroinflammation in the TNC under CM conditions. In the GLUT3 overexpression group, the p-p65/p65 ratio, p-IκBα/IκBα ratio, and expression of TNF-α, IL-6, and IL-1β were significantly reduced relative to the NTG group, demonstrating that GLUT3 effectively inhibits NF-κB-mediated neuroinflammation. Notably, co-administration of 3-NPA elevated all indices back, confirming the TCA cycle is an essential link for GLUT3’s regulation of NF-κB neuroinflammation (Fig. 8 B-F). To characterize microglial activation, we performed Iba1 immunofluorescence staining in the TNC (Fig. 8 G). Relative to the NTG + AAV-NC group, the NTG + AAV-Slc2a3 + group showed significantly reduced Iba1 + cell numbers and fluorescence intensity, verifying GLUT3 overexpression suppresses microglial activation. However, Iba1 + cell counts and intensity in the NTG + AAV-Slc2a3 + +3-NPA group were elevated back (Fig. 8 H-I). As shown in Fig. 8 J, the TNC tissue ROS level in the NTG + AAV-NC group was significantly elevated relative to the VEH + AAV-NC control group, indicating excessive ROS accumulation in the TNC under CM conditions. In contrast, the NTG + AAV-Slc2a3 + group exhibited a marked reduction in ROS level compared to the NTG + AAV-NC group, demonstrating that GLUT3 overexpression effectively suppresses ROS production in CM mice. Notably, the ROS level in the NTG + AAV-Slc2a3 + +3-NPA group was significantly increased relative to the NTG + AAV-Slc2a3 + group, verifying that GLUT3 inhibits ROS accumulation via the TCA cycle (Fig. 8 J). Collectively, these results indicate GLUT3 suppresses microglial activation and NF-κB-mediated neuroinflammation via TCA cycle, reinforcing the TCA cycle’s critical role as a downstream mediator of GLUT3’s anti-inflammatory effects in CM.​ 9. Idebenone alleviates hyperalgesia in chronic migraine mice Given our prior finding that the TCA cycle serves as a critical downstream mediator of GLUT3’s anti-migraine effects, we explored Idebenone, a clinically used mitochondrial protector that specifically enhances TCA cycle flux, as a potential therapeutic agent, while validating the central regulatory role of the TCA cycle in CM pathogenesis. Behavioral results showed NTG-induced CM mice exhibited notably reduced periorbital/plantar mechanical withdrawal thresholds, indicative of hyperalgesia. Idebenone reversed this threshold reduction in both basal (Fig. 9 A, C) and post-treatment phases (Fig. 9 B, D) relative to the NTG group. Immunofluorescence analysis revealed that, compared with the NTG group, Idebenone decreased c-Fos + cell counts and CGRP intensity in the TNC, thereby inhibiting neuronal overactivation and central sensitization (Fig. 9 E–H). These findings indicate that Idebenone plays a critical role in regulating central sensitization in CM mice. As a clinically safe agent, it holds translational potential for CM treatment. Discussion This study is the first to investigate how energy metabolism dysfunction impacts chronic migraine. Clinically, we detected abnormal expression of key OXPHOS pathway genes and reduced levels of tricarboxylic acid cycle metabolites in the plasma of CM patients for the first time, confirming the inherent systemic metabolic imbalance in CM. In an NTG-induced CM mouse model, we confirmed significantly reduced glucose uptake in TNC and neuronal-specific downregulation of GLUT3 expression, while no significant changes were observed in glial cells. Notably, neuron-specific overexpression of GLUT3 in TNC enhanced TCA cycle function, elevated mechanical pain thresholds, suppressed central sensitization by downregulating c-Fos and CGRP, and blocked NF-κB pathway activation. The TCA cycle inhibitor 3-NPA reversed GLUT3's suppression of the NF-κB pathway and its analgesic effects in CM mice, demonstrating that GLUT3 modulates CM-associated phenotypes by regulating TCA cycle function, which in turn influences NF-κB pathway activity. Furthermore, Idebenone significantly downregulated abnormal expression of c-Fos and CGRP in the TNC. These findings provide novel mechanistic insights and potential targets for metabolic interventions in CM (Fig. 10 ). Numerous studies have demonstrated that patients with CM exhibit systemic metabolic disorders, with energy fluctuation-related factors such as fasting and rapid blood glucose changes identified as important triggers for CM attacks[ 31 , 32 , 33 , 34 ]. Consistent with these clinical phenomena, we detect abnormal expression of key OXPHOS genes and significantly reduced levels of TCA cycle metabolites in the plasma of CM patients. In NTG-induced CM mouse models, previous studies have observed abnormalities in cerebral energy metabolites. For instance, Gao et al. found decreased levels of ATP-related metabolites including NAD, ADP, and AMP in the brain tissue of NTG -induced CM mice[ 35 ]; however, these studies failed to clarify specific abnormal metabolic pathways or brain region specificity, whether improving cerebral energy metabolism can ameliorate CM, resulting in a lack of precise targets for mechanistic dissection and an unclear therapeutic potential of metabolic modulation in CM. By integrating whole-brain metabolic imaging with targeted metabolomics, our study yielded three key findings: First, we demonstrated that glucose metabolic abnormalities are most prominent in the medullary region containing the TNC during acute CM. Compared to non-pain-related brain regions such as the hippocampus and cortex, NTG-induced CM mice exhibited significantly reduced glucose uptake in this medullary region. This result is consistent with the physiological role of the TNC as a relay hub for migraine pain signals, providing experimental evidence for the "pain-specific brain region localization" of metabolic abnormalities. Second, we confirmed that cerebral glucose uptake is primarily localized to TNC neurons, with no significant alterations observed in glial cells, and underscoring the pivotal role of intrinsic neuronal glucose transport deficits. Furthermore, the neuron-specific glucose transporter GLUT3 is also downregulated in the TNC, which may serve as a key initiator of local metabolic dysfunction. GLUT3, a neuron-specific glucose transporter that mediates cerebral glucose uptake and maintains neuronal energy homeostasis, has been extensively investigated in various neurological disorders[ 36 , 37 , 38 , 39 ]. However, its role and underlying regulatory mechanisms in the pathophysiology of CM remain poorly defined. Through multi-omics analyses and functional interventions, this study verified that NTG-induced CM mice exhibit concomitant phenotypes of reduced TCA cycle metabolites and NF-κB pathway activation, which is consistent with previous clinical and basic research findings. Further investigations revealed that neuron-specific GLUT3 overexpression concurrently improved glucose uptake, restored TCA cycle function, inhibited microglia activation and NF-κB pathway activation as well as pain phenotypes. This result, for the first time, identifies GLUT3 as a key upstream metabolic target mediating metabolic-inflammatory crosstalk in CM. The NF-κB pathway, as a central regulatory axis of neuroinflammation, sustained activation of which drives central sensitization and CM chronification. In the NTG-induced chronic migraine-like pain mouse model, microglia in the TNC region can be rapidly activated and polarized to a pro-inflammatory phenotype. These activated microglia form a positive feedback loop of “inflammatory factor release-increased neuronal excitability” by releasing pro-inflammatory cytokines such as IL-1β and IL-6, while upregulating the expression of neuropeptides such as CGRP, thereby exacerbating pain signal amplification and central sensitization processes[ 42 , 43 ]. Previous studies have confirmed that TCA cycle dysfunction can activate NF-κB pathway via the accumulation of ROS[ 44 , 45 ]. Our results showed that 3-NPA completely abrogated the NF-κB inhibitory effect and analgesic action mediated by GLUT3 overexpression, directly verifying that the TCA cycle serves as an indispensable link connecting GLUT3-dependent glucose uptake and NF-κB-driven inflammatory activation. Building on the aforementioned mechanistic insights, we further conducted confirmatory experiments targeting the TCA cycle, employing Idebenone, a well-characterized mitochondrial protector and TCA cycle optimizer, to validate the clinical translational potential of the core mechanism[ 46 , 47 ]. Idebenone is known to specifically enhance succinate dehydrogenase (SDH) activity, optimize TCA cycle flux, improve mitochondrial function and promote ATP production[ 48 , 49 ]. Its target of action highly aligns with the key regulatory nodes of the TCA cycle identified in this study; thus, we hypothesized that it could alleviate CM by targeting the TCA cycle. Experimental results showed that intraperitoneal administration of Idebenone to NTG-induced CM mice significantly increased the periorbital and plantar mechanical pain thresholds, effectively suppressed excessive activation of TNC neurons and downregulated CGRP levels. Notably, as a clinically used compound with favorable oral bioavailability, Idebenone has established safety profiles in neurological diseases such as Friedreich's ataxia and cerebral ischemia-reperfusion[ 50 , 51 , 52 , 53 ]. It provided a feasible clinical translation direction for metabolic-targeted therapy in CM and accelerating the conversion of our mechanistic discoveries into clinical applications. Nevertheless, it is important to note that this study has several limitations. Given that this study only employed the NTG-induced chemical CM mouse model, while this model can simulate some pain phenotypes and short-term metabolic abnormalities of idiopathic CM, it may not fully recapitulate the long-term chronic metabolic disorder characteristics observed in refractory human CM. To avoid the impact of estrogen on pain neurotransmitter transmission, male C57BL/6 mice were used to establish the CM model in this experiment, and the role of the GLUT3 in female CM models remains to be explored. Furthermore, the specific mechanisms underlying the therapeutic effects of Idebenone have not been fully elucidated, and more detailed mechanistic studies are required in the future. Conclusion In summary, our research has underscored the pivotal role of neuron-specific GLUT3 in mediating metabolic dysfunction and subsequent neuroinflammation in CM. Clinically, CM patients exhibit systemic metabolic imbalance. In NTG-induced CM mice, TNC neurons display decreased glucose uptake and specific downregulation of GLUT3, whereas GLUT3 overexpression alleviates CM-related phenotypes by enhancing TCA cycle function, suppressing the expression of c-Fos and CGRP, and inhibiting NF-κB pathway activation. Notably, these protective effects are fully reversed by the TCA cycle inhibitor 3-NPA, confirming that the TCA cycle acts as an indispensable downstream mediator of GLUT3-dependent regulation. Additionally, Idebenone, a clinically approved agent and TCA cycle modulator, also alleviates CM-related phenotypes. Collectively, these findings indicate that targeting GLUT3 or its downstream TCA cycle could be a promising therapeutic strategy for CM. This discovery not only deepens our understanding of the molecular link between neuronal glucose metabolism and neuroinflammation in CM but also paves the way for developing novel metabolism-targeted treatments. Abbreviations AAV Adeno-associated virus; CM Chronic migraine; NTG Nitroglycerin; CGRP Calcitonin gene-related peptide; TNC Trigeminal nucleus caudalis; GLUT3 Glucose transporter 3; RAPA Rapamycin; WB Western blot; IF Immunofluorescence; 3-NPA 3-nitropropionic acid; 2-NBDG 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxy-D-glucose; TCA tricarboxylic acid; PPP pentose phosphate pathway; OXPHOS oxidative phosphorylation; GO Gene Ontology; KEGG Kyoto Encyclopedia of Genes and Genomes; 18F-FDG-PET/CT 2-deoxy-2-[18F]fluoro-D-glucose positron emission tomography/computed tomography; DEGs differentially expressed genes; TNF-α tumor necrosis factor-α; NF-κB nuclear factor kappa B; p-NF-κB Phosphorylated Nuclear Factor Kappa B; IκBα Inhibitor of nuclear factor kappa B alpha; p-IκBα phosphorylated Inhibitor of nuclear factor kappa B alpha; ATP Adenosine triphosphate; ADP Adenosine diphosphate; AMP Adenosine monophosphate; ROS reactive oxygen species; NADPH Nicotinamide adenine dinucleotide phosphate hydrogen; NAD⁺/NADH Nicotinamide adenine dinucleotide (oxidized/reduced); FADH₂ Flavin adenine dinucleotide hydride. Declarations Ethical Approval and Consent to participate Ethical approval for this study was obtained from the First Affiliated Hospital of Xiamen University (Approval Number: [2020] Scientific Research Ethics Review No. (060)]; Approval Date: January 28, 2021]). All procedures were performed in accordance with the principles of the Declaration of Helsinki (2024 revised version) and relevant national ethical regulations. Informed consent was obtained from all individual participants prior to their involvement in the study, using the informed consent form (Version No.: 2.0; Version Date: January 25, 2021) approved by the ethics committee. All participants were fully informed of the study purpose, procedures, potential risks, benefits, and the right to withdraw at any time without prejudice to their medical care. Consent for publication Not applicable. Competing interests The authors declare no conflict of interest. Funding This work is financially supported by the National Natural Science Foundation of China (32300823 and 625B2156), China Postdoctoral Science Foundation (2024M761265), Jiangxi Provincial Natural Science Foundation (20252BAC200488), Integration of Medicine and Engineering Project of Xiamen (3502Z20244ZD2003), Neurological Diseases and Nutritional Health Project of National Health Commission Capacity Building and Continuing Education Center (W2024SNKT43). Author Contribution J.Y.Z., Z.J.G., X.R., and X.Y.L. conceived and designed the experiments. K.Y.L., W.X.W., H.P.Y., Y.H.W., and Z.L.X. collected clinical information and samples. T.T.C., T.H.F., Y.H.X., T.X.W., and X.D.L. performed the experiments. C.P., K.W., and J.J.Z. analyzed the data. J.F., Q.L.M., and Y.G.W. supervised data collection. All authors have read and agreed to the published version of the manuscript. Acknowledgement We acknowledge the financial support by the National Natural Science Foundation of China, China Postdoctoral Science Foundation, Jiangxi Provincial Natural Science Foundation, Integration of Medicine and Engineering Project of Xiamen, Neurological Diseases and Nutritional Health Project of National Health Commission Capacity Building and Continuing Education Center. Data Availability The data will be made available upon reasonable request to the corresponding authors. References Headache Classification Committee of the International Headache Society (IHS) The International Classification of Headache Disorders, 3rd edition, Cephalalgia Int. J. Headache, vol. 38, no. 1, pp. 1–211. Jan. 2018, 10.1177/0333102417738202 Ashina M, et al. Migraine: disease characterisation, biomarkers, and precision medicine. Lancet. Apr. 2021;397(10283):1496–504. 10.1016/S0140-6736(20)32162-0 . Killoran PM et al. 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The putative role of neuroinflammation in the complex pathophysiology of migraine: From bench to bedside. Neurobiol Dis. May 2023;180:106072. 10.1016/j.nbd.2023.106072 . Thuraiaiyah J, Erritzøe-Jervild M, Al-Khazali HM, Schytz HW, Younis S. The role of cytokines in migraine: A systematic review, Cephalalgia Int. J. Headache , vol. 42, no. 14, pp. 1565–1588, Dec. 2022, 10.1177/03331024221118924 Tai Y-H et al. Targeting the TCA cycle can ameliorate widespread axonal energy deficiency in neuroinflammatory lesions, Nat. Metab. , vol. 5, no. 8, pp. 1364–1381, 2023, 10.1038/s42255-023-00838-3 Martínez-Reyes I, Chandel NS. Mitochondrial TCA cycle metabolites control physiology and disease. Nat Commun. Jan. 2020;11:102. 10.1038/s41467-019-13668-3 . Yi B, et al. Idebenone: Clinical Potential Beyond Neurological Diseases. Drug Des Devel Ther. 2025;19:7929–46. 10.2147/DDDT.S515053 . Xu Z, et al. 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Eur J Med Chem. Oct. 2025;296:117873. 10.1016/j.ejmech.2025.117873 . Peng J et al. Jul., Idebenone attenuates cerebral inflammatory injury in ischemia and reperfusion via dampening NLRP3 inflammasome activity, Mol. Immunol. , vol. 123, pp. 74–87, 2020, 10.1016/j.molimm.2020.04.013 Li M, et al. A mitochondria-targeted idebenone derivative W1a mitigates cerebral ischemia-reperfusion injury by preserving mitochondrial integrity and suppressing neuroinflammation. Bioorg Chem. Dec. 2025;168:109356. 10.1016/j.bioorg.2025.109356 . Additional Declarations No competing interests reported. Supplementary Files OriginalWBData.docx FigureS1.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8411532","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":599649700,"identity":"31c15979-b437-4894-a893-a0037859267d","order_by":0,"name":"JingYuan Zheng","email":"","orcid":"","institution":"Xiamen University","correspondingAuthor":false,"prefix":"","firstName":"JingYuan","middleName":"","lastName":"Zheng","suffix":""},{"id":599649701,"identity":"a94b7d24-630f-4410-b110-e412b6d9d9a5","order_by":1,"name":"ZiJuan Guo","email":"","orcid":"","institution":"Fujian Medical 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10:38:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8411532/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8411532/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104177894,"identity":"96b1c623-c53d-4468-b5ae-52823fcca6c2","added_by":"auto","created_at":"2026-03-08 16:50:28","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":924981,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIntegrated multi-omics profiling reveals suppressed energy metabolism in CM patients.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Study workflow. Plasma samples were collected from 359 CM patients and 140 healthy controls, followed by metabolomic and transcriptomic profiling, and integrated multi-omics analysis.(CM patients: n=359; healthy controls: n=140). \u003cstrong\u003e(B)\u003c/strong\u003e Multi-omics co-enrichment plot comparing CM patients with controls. \u003cstrong\u003e(C)\u003c/strong\u003e Transcriptomic GO term enrichment analyses of differentially expressed molecules between CM patients and controls. \u003cstrong\u003e(D)\u003c/strong\u003e Transcriptomic KEGG pathway enrichment dot plots comparing CM patients with controls. \u003cstrong\u003e(E)\u003c/strong\u003eMetabolomic focused pathway enrichment plots showing key differentially regulated pathways between CM patients and controls.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8411532/v1/1a9932f35e8fafbc4b2953dc.png"},{"id":104404875,"identity":"e8ac21b3-e789-4448-a16e-30942103889a","added_by":"auto","created_at":"2026-03-11 12:21:16","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1543599,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBrain glucose uptake dysfunction and reduced neuronal GLUT3 expression in NTG-induced CM mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Experimental timeline. Mice were administered NTG or saline (VEH) over 9 days, with behavioral tests conducted on days 1, 3, 5, 7, and 9; \u003csup\u003e18\u003c/sup\u003eF-FDG-PET/CT imaging were performed prior to sacrifice. Following TNC tissue collection, transcriptomic, metabolomic, and WB analyses were conducted. \u003cstrong\u003e(B)\u003c/strong\u003e Representative \u003csup\u003e18\u003c/sup\u003eF-FDG-PET/CT SUV heatmaps. \u003cstrong\u003e(C)\u003c/strong\u003e Quantitative analysis of mean whole-brain SUV revealed decreased whole-brain glucose uptake in NTG group compared to VEH controls. \u003cstrong\u003e(D)\u003c/strong\u003e Regional glucose uptake, expressed as a percentage of injected dose, in the hypothalamus, hippocampus, cerebral cortex, cerebellum, and medulla oblongata; the most pronounced reduction was detected in the medulla oblongata (n = 4 mice/group). \u003cstrong\u003e(E)\u003c/strong\u003e Immunofluorescence co-localization images showing 2-NBDG (glucose uptake probe, green) with NeuN, Iba1, or GFAP (red) in the TNC. Scale bar = 100 μm, 30 μm (enlarged images). \u003cstrong\u003e(F\u003c/strong\u003e) Percentage distribution of total 2-NBDG fluorescence signal among NeuN⁺ neurons, Iba1⁺ microglia, and GFAP⁺ astrocytes in the TNC. In both VEH and NTG groups, neurons accounted for the majority of glucose uptake, with minimal changes in the proportion of glucose uptake by microglia and astrocytes (n = 5 mice/group). \u003cstrong\u003e(G-I\u003c/strong\u003e) Quantitative analyses of 2-NBDG immunofluorescence intensity in NeuN⁺ neurons, Iba1⁺ microglia, and GFAP⁺ astrocytes, respectively. NTG treatment significantly decreased glucose uptake in TNC neurons, whereas glucose uptake in microglia and astrocytes showed no statistically significant difference between groups. \u003cstrong\u003e(J)\u003c/strong\u003e Representative WB blots of GLUT1, GLUT3, GLUT4. \u003cstrong\u003e(K–M)\u003c/strong\u003e Quantitative analyses of WB results demonstrated significant GLUT3 downregulation in the NTG group, whereas GLUT1 and GLUT4 expression remained unchanged (n = 4 mice/group). All data are presented as mean ± SEM; ns, not significant; \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01画. Statistical significance was determined by unpaired two-tailed t-test.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8411532/v1/4bcbce929c0c4e78ab198884.png"},{"id":104177905,"identity":"8e95622f-d8a7-4f80-8ad9-7207cabb3b6f","added_by":"auto","created_at":"2026-03-08 16:50:28","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":691259,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNeuronal GLUT3 overexpression alleviates pain and central sensitization in NTG-induced CM mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A-B)\u003c/strong\u003e GLUT3 reversed the NTG-induced decrease in periorbital mechanical thresholds in both basal\u003cstrong\u003e (A)\u003c/strong\u003e and post-treatment \u003cstrong\u003e(B)\u003c/strong\u003e phases (n = 10 mice/group). \u003cstrong\u003e(C-D) \u003c/strong\u003eGLUT3 reversed the NTG-induced decrease in paw mechanical thresholds in basal \u003cstrong\u003e(C) \u003c/strong\u003eand post-treatment\u003cstrong\u003e (D) \u003c/strong\u003ephases (n = 10 mice/group). \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, \u003csup\u003e***\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 compared with the VEH group; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, \u003csup\u003e###\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 compared with the NTG group. \u003cstrong\u003e(E)\u003c/strong\u003e Immunofluorescence staining of c-Fos. scale bar = 100 μm. \u003cstrong\u003e(F)\u003c/strong\u003e Quantification of c-Fos\u003csup\u003e+\u003c/sup\u003e cell counts showed NTG treatment increased c-Fos\u003csup\u003e+\u003c/sup\u003e cell numbers in the TNC, while GLUT3 overexpression attenuated this elevation (n = 6 mice/group). \u003cstrong\u003e(G) \u003c/strong\u003eImmunofluorescence staining of CGRP in the TNC. scale bar = 400 μm. \u003cstrong\u003e(H)\u003c/strong\u003e Quantification of CGRP fluorescence intensity revealed NTG elevated CGRP intensity, which was reversed by GLUT3 overexpression (n = 5 mice/group). All data are presented as the mean ± SEM. A-D: two-way analysis of variance followed by Tukey’s post hoc test; F, H: one-way analysis of variance followed by Tukey’s post-hoc test. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, \u003csup\u003e***\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8411532/v1/fda5fed8a1530958b335393b.png"},{"id":104177897,"identity":"c47f45f4-9178-48cb-82fa-72d7882431bc","added_by":"auto","created_at":"2026-03-08 16:50:28","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2921953,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGLUT3 overexpression restores neuronal glucose metabolism and suppresses microglia activation in TNC of NTG-induced CM mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Immunofluorescence images of 2-NBDG (glucose uptake probe, green) and NeuN (red). scale bar = 100 μm, 30 μm (enlarged images). \u003cstrong\u003e(B)\u003c/strong\u003e Quantitative analysis of 2-NBDG fluorescence intensity in NeuN\u003csup\u003e+\u003c/sup\u003e neurons, confirming that NTG-induced reduction in neuronal glucose uptake was reversed by GLUT3 overexpression (n = 5 mice/group). \u003cstrong\u003e(C)\u003c/strong\u003e Transmission electron microscopy (TEM) images of mitochondria in TNC neurons revealed the NTG group exhibited swollen mitochondria with fragmented cristae, vacuolization, and reduced abundance, while neuronal GLUT3 overexpression showed restored mitochondrial morphology and quantity. Scale bar = 500 nm, 250 nm (enlarged images). \u003cstrong\u003e(D)\u003c/strong\u003e Quantitative analysis of mitochondrial number per NeuN\u003csup\u003e+\u003c/sup\u003e neuron, revealing a marked reduction in the NTG+AAV-NC group and recovery in the NTG+AAV-Slc2a3\u003csup\u003e+\u003c/sup\u003e group (n = 3 mice/group). \u003cstrong\u003e(E)\u003c/strong\u003e Immunofluorescence images of TOM20 (mitochondrial outer membrane marker, red) and NeuN (green). scale bar = 50 μm. \u003cstrong\u003e(F)\u003c/strong\u003e Quantitative analysis of TOM20 fluorescence intensity showed NTG treatment decreased TOM20 signal, which was restored by neuronal GLUT3 overexpression (n = 5 mice/group).\u003cstrong\u003e (G)\u003c/strong\u003e Representative WB bands of TOM20. \u003cstrong\u003e(H)\u003c/strong\u003e Quantitative analysis verifying significant downregulation of TOM20 in the NTG+AAV-NC group and reversal in the NTG+AAV-Slc2a3\u003csup\u003e+\u003c/sup\u003e group. \u003cstrong\u003e(I)\u003c/strong\u003e Immunofluorescence staining of Iba1 (n = 6 mice/group). scale bar = 300 μm, 100 μm (enlarged images). \u003cstrong\u003e(J)\u003c/strong\u003e Quantification of Iba1\u003csup\u003e+\u003c/sup\u003e cell counts showed that NTG elevated Iba1\u003csup\u003e+\u003c/sup\u003e cell numbers, which was attenuated by GLUT3 overexpression. \u003cstrong\u003e(K)\u003c/strong\u003e Quantification of Iba1 immunofluorescence intensity revealed that NTG elevated Iba1 intensity, while GLUT3 overexpression reduced this intensity. All data are presented as mean ± SEM (n = 6 mice/group). Statistical significance was determined by one-way analysis of variance followed by Tukey’s post-hoc test. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, \u003csup\u003e***\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003cstrong\u003e\u003cbr\u003e\n\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8411532/v1/5f0d3323ac10ff351ff23bde.png"},{"id":104779615,"identity":"97b17bca-0d87-4863-8cd0-0a6831a1c013","added_by":"auto","created_at":"2026-03-17 07:43:17","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":519899,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGLUT3 overexpression reverses TCA cycle dysregulation in TNC of NTG-induced CM mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e PCA score plot of metabolite profiles. \u003cstrong\u003e(B)\u003c/strong\u003e Venn diagrams of differential metabolites revealed 145 upregulated metabolites in the VEH+AAV-NC vs NTG+AAV-NC comparison (left panel) and 26 downregulated metabolites in the NTG+AAV-NC vs NTG+AAV-Slc2a3\u003csup\u003e+\u003c/sup\u003e comparison (right panel), with 14 differential metabolites exhibiting reversed expression trends rescued by GLUT3 overexpression. \u003cstrong\u003e(C)\u003c/strong\u003e Venn diagrams of differential metabolites revealed 88 downregulated metabolites in the VEH+AAV-NC vs NTG+AAV-NC comparison (left panel) and 45 downregulated metabolites in the NTG+AAV-NC vs NTG+AAV-Slc2a3\u003csup\u003e+\u003c/sup\u003e comparison (right panel), with 19 differential metabolites showing reversed expression trends rescued by GLUT3 overexpression. Differential metabolites between each pair of groups via two-tailed t-tests. \u003cstrong\u003e(D)\u003c/strong\u003e KEGG pathway enrichment of differential metabolites. \u003cstrong\u003e(E)\u003c/strong\u003e Schematic of glucose metabolism networks (glycolysis, pentose phosphate pathway [PPP], TCA cycle). \u003cstrong\u003e(F)\u003c/strong\u003e Heatmap of key metabolites in glycolysis, PPP, and TCA cycle showed the NTG group elevated upstream glycolysis/PPP metabolites and reduced downstream glycolysis/TCA/OXPHOS metabolites, while GLUT3 overexpression mainly restored TCA cycle. \u003cstrong\u003e(G-J)\u003c/strong\u003e Quantitative analysis of TCA cycle metabolites revealed NTG reduced concentrations of cis-Aconitic acid \u003cstrong\u003e(G)\u003c/strong\u003e, Isocitric acid \u003cstrong\u003e(H)\u003c/strong\u003e, Fumaric acid\u003cstrong\u003e (I)\u003c/strong\u003e, and Malic acid \u003cstrong\u003e(J)\u003c/strong\u003e, which were restored in the NTG+AAV-Slc2a3\u003csup\u003e+\u003c/sup\u003e group (n = 4 mice/group). \u003cstrong\u003e(K)\u003c/strong\u003e Relative fluorescence intensity of ROS showed NTG elevated ROS levels, while GLUT3 overexpression attenuated this elevation (n = 5 mice/group). All data are presented as mean ± SEM. Statistical significance was determined by one-way analysis of variance followed by Tukey’s post-hoc test. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, \u003csup\u003e***\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-8411532/v1/17b68610b2dc3825a3b0c5d2.png"},{"id":104177896,"identity":"5b7e4b5c-a808-41f9-847f-eee00869fb2b","added_by":"auto","created_at":"2026-03-08 16:50:28","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":427129,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGLUT3 overexpression reverses NF-κB-mediated neuroinflammatory signaling in TNC of NTG-induced CM mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Volcano plot (VEH+AAV-NC vs NTG+AAV-NC) showed 365 upregulated genes and 587 downregulated genes, reflecting NTG-induced transcriptomic dysregulation. \u003cstrong\u003e(B)\u003c/strong\u003e Volcano plot (NTG+AAV-NC vs NTG+AAV-Slc2a3\u003csup\u003e+\u003c/sup\u003e) revealed 382 upregulated genes and 719 downregulated genes, demonstrating GLUT3-mediated reversal of NTG-induced DEGs. \u003cstrong\u003e(C) \u003c/strong\u003eVenn diagrams of differentially expressed genes (DEGs) revealed two subsets of DEGs: 382 downregulated genes in the VEH+AAV-NC vs NTG+AAV-NC comparison (left panel) and 587 upregulated genes in the NTG+AAV-NC vs NTG+AAV-Slc2a3\u003csup\u003e+\u003c/sup\u003e comparison (right panel), with 214 DEGs exhibiting reversed expression trends rescued by GLUT3 overexpression. \u003cstrong\u003e(D)\u003c/strong\u003e Venn diagrams of DEGs further identified two additional subsets: 719 upregulated genes in the VEH+AAV-NC vs NTG+AAV-NC comparison (left panel) and 365 downregulated genes in the NTG+AAV-NC vs NTG+AAV-Slc2a3\u003csup\u003e+\u003c/sup\u003e comparison (right panel), with 187 DEGs showing reversed expression trends rescued by GLUT3 overexpression. \u003cstrong\u003e(E)\u003c/strong\u003e KEGG pathway enrichment analysis of DEGs in the TNC between VEH+AAV-NC and NTG+AAV-NC groups. \u003cstrong\u003e(F)\u003c/strong\u003e KEGG pathway enrichment analysis of DEGs in the TNC between NTG+AAV-NC and NTG+AAV-Slc2a3\u003csup\u003e+\u003c/sup\u003e groups (n = 3 mice/group for VEH+AAV-NC and NTG+AAV-NC; n = 5 mice for NTG+AAV-Slc2a3 group). All data are presented as mean ± SEM.\u003cbr\u003e\n\u003c/p\u003e","description":"","filename":"floatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-8411532/v1/1e6d07e9701f20c6c8743963.png"},{"id":104177898,"identity":"bd2f956a-2af7-4c8c-9bb8-006598206132","added_by":"auto","created_at":"2026-03-08 16:50:28","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1201985,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBlocking TCA cycle abrogates GLUT3’s analgesic in NTG-induced CM mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Experimental timeline. AAVs were stereotaxically injected into the TNC 28 days prior to daily NTG administration; 20 mg/kg 3-NPA was administered 2 days before NTG initiation, and behavioral assessments were performed during NTG infusion. \u003cstrong\u003e(B–E)\u003c/strong\u003e Mechanical pain thresholds of the periorbital (\u003cstrong\u003eB\u003c/strong\u003e: basal phase; \u003cstrong\u003eC\u003c/strong\u003e: post-treatment phase) and paw (\u003cstrong\u003eD\u003c/strong\u003e: basal phase; \u003cstrong\u003eE\u003c/strong\u003e: post-treatment phase) regions over 9 days of infusion. Results showed that NTG induced a significant reduction in mechanical thresholds, which was reversed by GLUT3 overexpression; however, this analgesic effect of GLUT3 overexpression was abrogated by 3-NPA treatment (n = 6 mice/group).\u003cstrong\u003e \u003c/strong\u003e\u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, \u003csup\u003e***\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 compared with the VEH+AAV-NC group; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01, \u003csup\u003e###\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 compared with the NTG+AAV-NC group; \u003csup\u003e\u0026amp;\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e\u0026amp;\u0026amp;\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, \u003csup\u003e\u0026amp;\u0026amp;\u0026amp;\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 compared with the NTG+AAV-Slc2a3\u003csup\u003e+\u003c/sup\u003e group. \u003cstrong\u003e(F) \u003c/strong\u003eImmunofluorescence staining of c-Fos in the TNC; scale bar = 100 μm. (G) Quantification of c-Fos\u003csup\u003e+\u003c/sup\u003e cell counts showed that NTG treatment increased c-Fos\u003csup\u003e+\u003c/sup\u003e cell numbers in the TNC, while GLUT3 overexpression attenuated this elevation; 3-NPA administration reversed the inhibitory effect of GLUT3, leading to a rebound in c-Fos\u003csup\u003e+\u003c/sup\u003e cell counts (n = 6 mice/group). \u003cstrong\u003e(H)\u003c/strong\u003e Immunofluorescence staining of CGRP in the TNC; scale bar = 400 μm. \u003cstrong\u003e(I)\u003c/strong\u003e Quantification of CGRP immunofluorescence intensity revealed that NTG elevated CGRP levels in the TNC, which was reversed by GLUT3 overexpression; 3-NPA treatment abrogated this effect, resulting in increased CGRP intensity again (n = 6 mice/group). All data are presented as the mean ± SEM. B-E: two-way analysis of variance followed by Tukey’s post hoc test; G, I: one-way analysis of variance followed by Tukey’s post-hoc test; \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, \u003csup\u003e***\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage11.png","url":"https://assets-eu.researchsquare.com/files/rs-8411532/v1/f2a30675f0d26b3616afdd0a.png"},{"id":104177904,"identity":"9e0009d5-3385-476a-ac7f-ac50cdd27959","added_by":"auto","created_at":"2026-03-08 16:50:28","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1716427,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBlocking TCA cycle abrogates GLUT3’s anti-inflammatory effects in NTG-induced CM mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eRepresentative Western blot bands of NF-κB pathway components (phosphorylated p65 [p-p65], total p65, phosphorylated IκBα [p-IκBα], total IκBα) and proinflammatory cytokines (TNF-α, IL-6, IL-1β); β-actin served as the loading control (n = 6 mice/group). \u003cstrong\u003e(B)\u003c/strong\u003e Quantitative analysis of p-p65 protein expression (normalized to total p65) showed that NTG elevated the p-p65/p65 ratio, which was reduced by GLUT3 overexpression; this inhibitory effect of GLUT3 was abrogated by 3-NPA. \u003cstrong\u003e(C)\u003c/strong\u003e Quantitative analysis of p-IκBα protein expression (normalized to total IκBα) revealed that NTG increased the p-IκBα/IκBα ratio, while GLUT3 overexpression lowered this ratio; 3-NPA reversed GLUT3’s effect. \u003cstrong\u003e(D) \u003c/strong\u003eQuantitative analysis of TNF-α protein expression (normalized to β-actin) showed that NTG elevated TNF-α levels, which were reduced by GLUT3 overexpression; 3-NPA abrogated this reduction.\u003cstrong\u003e (E) \u003c/strong\u003eQuantitative analysis of IL-6 protein expression (normalized to β-actin) revealed that NTG increased IL-6 levels, while GLUT3 overexpression attenuated these levels; 3-NPA reversed GLUT3’s inhibitory effect. \u003cstrong\u003e(F)\u003c/strong\u003e Quantitative analysis of IL-1β protein expression (normalized to β-actin) showed that NTG elevated IL-1β levels, which were reduced by GLUT3 overexpression; 3-NPA abrogated this effect. \u003cstrong\u003e(G)\u003c/strong\u003e Immunofluorescence staining of Iba1 in the TNC; scale bar = 300 μm,100 μm (enlarged images) (n = 6 mice/group). \u003cstrong\u003e(H)\u003c/strong\u003e Quantification of Iba1\u003csup\u003e+\u003c/sup\u003e cell numbers showed that NTG elevated Iba1+ cell counts, which was attenuated by GLUT3 overexpression; 3-NPA reversed this attenuation. \u003cstrong\u003e(I)\u003c/strong\u003e Quantification of Iba1 immunofluorescence intensity revealed that NTG elevated Iba1 intensity, while GLUT3 overexpression reduced this intensity; 3-NPA abrogated GLUT3’s effect.\u003cstrong\u003e (J)\u003c/strong\u003e Quantification of TNC tissue ROS levels, presented as ROS fluorescence intensity per mg protein. NTG treatment elevated ROS levels, which were reduced by GLUT3 overexpression; 3-NPA reversed this reduction. All data are presented as mean ± SEM. Statistical significance was determined by one-way analysis of variance followed by Tukey’s post-hoc test. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"floatimage12.png","url":"https://assets-eu.researchsquare.com/files/rs-8411532/v1/8716513a9a8d1427a0dd0308.png"},{"id":104177901,"identity":"b1e851d7-5d50-45e4-b530-f1457af609f8","added_by":"auto","created_at":"2026-03-08 16:50:28","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":726485,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIdebenone alleviates pain and central sensitization in NTG-induced CM mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A-B)\u003c/strong\u003e Idebenone reversed the NTG-induced decrease in periorbital mechanical thresholds in both basal \u003cstrong\u003e(A) \u003c/strong\u003eand post-treatment \u003cstrong\u003e(B) \u003c/strong\u003ephases (n = 10 mice/group). \u003cstrong\u003e(C-D) \u003c/strong\u003eIdebenone reversed the NTG-induced decrease in paw mechanical thresholds in basal\u003cstrong\u003e (C)\u003c/strong\u003e and post-treatment\u003cstrong\u003e (D)\u003c/strong\u003e phases (n = 10 mice/group). \u003cstrong\u003e(E)\u003c/strong\u003e Immunofluorescence staining of c-Fos. scale bar = 100 μm. \u003cstrong\u003e(F)\u003c/strong\u003e Quantification of c-Fos\u003csup\u003e+\u003c/sup\u003e cell counts showed NTG treatment increased c-Fos\u003csup\u003e+\u003c/sup\u003e cell numbers, while Idebenone attenuated this elevation. n = 6 mice/group. (G) Immunofluorescence staining of CGRP in the TNC. scale bar = 400 μm. (H) Quantification of CGRP fluorescence intensity revealed NTG elevated CGRP intensity, which was reversed by Idebenone. n = 6 mice/group. All data are presented as the mean ± SEM. A-D: two-way analysis of variance followed by Tukey’s post hoc test; \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, \u003csup\u003e***\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 compared with the VEH group; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, \u003csup\u003e###\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 compared with the NTG group. F, H: one-way analysis of variance followed by Tukey’s post-hoc test. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, \u003csup\u003e***\u003c/sup\u003e\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage13.png","url":"https://assets-eu.researchsquare.com/files/rs-8411532/v1/3459ceadf53a03eaa95253f5.png"},{"id":104177906,"identity":"84d4f743-1077-4e20-923a-2a7e45514432","added_by":"auto","created_at":"2026-03-08 16:50:28","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":815536,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNeuronal GLUT3 overexpression alleviates hyperalgesia and suppresses inflammatory responses via enhancing TCA cycle in CM.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis figure contrasts the regulatory differences between the chronic migraine (CM) state (left panel) and the neuronal GLUT3 overexpression state (right panel): In the left panel (corresponding to the CM state), GLUT3-mediated glucose uptake is reduced, and the TCA cycle is suppressed. This subsequently triggers elevated ROS levels, enhanced microglial activation, activation of the NF-κB pathway, and massive release of pro-inflammatory cytokines, including TNF-α, IL-1β, and IL-6, ultimately resulting in the discomfort phenotype associated with chronic migraine. In the right panel (neuronal GLUT3 overexpression state), GLUT3 overexpression increases glucose uptake and improves mitochondrial TCA cycle function, leading to a corresponding reduction in inflammatory responses as reflected by decreased ROS levels, attenuated microglial activation, diminished NF-κB pathway activity, and lowered pro-inflammatory cytokine levels, with the phenotype manifesting as a favorable state with improved metabolism and inflammation. However, 3-NPA exerts an inhibitory effect on the TCA cycle, which reverses the ameliorative effect of GLUT3 overexpression on chronic migraine; in contrast, Idebenone can target and enhance the mitochondrial TCA cycle, helping to alleviate chronic migraine. This figure visually illustrates how GLUT3 overexpression influences inflammatory responses and the chronic migraine phenotype by regulating glucose metabolism and mitochondrial function, while also clarifying the interventional effects of 3-NPA and Idebenone on this process.\u003c/p\u003e","description":"","filename":"floatimage14.png","url":"https://assets-eu.researchsquare.com/files/rs-8411532/v1/3d55af558cbb967b4e3e9de1.png"},{"id":105903818,"identity":"b9e12000-6bd4-4de8-b037-3e9c7cdc6ca6","added_by":"auto","created_at":"2026-04-01 09:54:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":10958121,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8411532/v1/757fdf22-5234-44e5-86a5-cb6b501ba535.pdf"},{"id":104404023,"identity":"172bb2af-1761-41b2-8430-5a4ad2f5580a","added_by":"auto","created_at":"2026-03-11 12:19:37","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1337891,"visible":true,"origin":"","legend":"","description":"","filename":"OriginalWBData.docx","url":"https://assets-eu.researchsquare.com/files/rs-8411532/v1/8413b0d5678809f3ebab083a.docx"},{"id":104177900,"identity":"a4262d1b-a2c3-497c-a079-dfbd846f54d2","added_by":"auto","created_at":"2026-03-08 16:50:28","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":4649468,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8411532/v1/6102b673ce75edf48307bb0d.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Neuronal GLUT3 alleviates hyperalgesia and neuroinflammation in chronic migraine mice via the TCA cycle","fulltext":[{"header":"Introduction","content":"\u003cp\u003eChronic Migraine (CM), one of the most prevalent neurovascular disorders globally, has an epidemiological prevalence of 1.4%-2.2% worldwide[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Approximately 80% of patients experience\u0026thinsp;\u0026ge;\u0026thinsp;15 headache days per month, accompanied by severe symptoms such as nausea, vomiting, photophobia, and phonophobia, leading to significant disability and reduced quality of life[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Neuroinflammation and trigeminal vascular system sensitization are well-recognized key downstream events in CM pathogenesis. Clinically, triptans and calcitonin gene-related peptide (CGRP) antagonists are the mainstay treatments; however, 30%-40% of patients exhibit drug resistance, 60% fail to achieve effective symptom control, with long-term use causing complications like medication-overuse headache[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Notably, emerging evidence suggests that metabolic dysfunction is also closely implicated in CM development, potentially interacting with neuroinflammation to drive disease progression. The core challenge underlying this predicament lies in the incomplete understanding of CM\u0026rsquo;s pathogenesis, while neuroinflammation and trigeminal sensitization are well-recognized key downstream events, the identification of novel upstream regulatory targets remains urgently needed[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNeurons, as the core units of brain energy metabolism and signal transduction, represent the initial site of functional imbalance in cerebral metabolic disorders. Glucose transporter 3 (GLUT3), a neuron-specific glucose transporter with 12 transmembrane domains and high glucose affinity, maintains neuronal energy supply under low glucose conditions and provides critical metabolic substrates[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Previous studies indicate that GLUT3 knockout lowers glucose supply, increases oxidative stress, and aggravates hippocampal neuronal death and cognitive impairment under hypoxic conditions[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], whereas its overexpression raises neuronal glucose uptake, up-regulates Aβ-degrading enzymes, suppresses the integrated stress response, and thereby improves cognitive performance in Alzheimer\u0026rsquo;s disease models[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The tricarboxylic acid (TCA) cycle\u0026mdash;the hub of mitochondrial energy metabolism\u0026mdash;depends on glucose-derived pyruvate to maintain flux and to generate ATP, NADH and FADH₂, any GLUT3-mediated reduction in intracellular glucose will decrease pyruvate delivery to mitochondria, slow TCA turnover, and lower the NAD⁺/NADH ratio. The resulting redox imbalance impairs NAD⁺-dependent anti-oxidative enzymes, increases reactive oxygen species (ROS), and activates pro-inflammatory pathways such as NF-κB, thus driving neuroinflammation[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Despite the accumulating evidence pointing to the role of GLUT3 in neuronal metabolism and inflammation regulation, its functions in CM development and progression, as well as its involvement in CM-related metabolic and inflammatory dysregulation, have not been well studied.\u003c/p\u003e \u003cp\u003eIn the present study, we aimed to elucidate the roles of GLUT3 in CM pathogenesis and progression. Integrating transcriptomic and metabolomic analyses, we observed that neuronal GLUT3 overexpression enhanced glucose uptake, restored TCA cycle function, and thereby inhibited the NF-κB pathway to alleviate central sensitization and delay CM progression. Notably, our findings highlight GLUT3 as a promising therapeutic target for CM management, while positioning Idebenone, a clinically actionable agent known to directly enhance TCA cycle activity, as a potential therapeutic drug that holds promise for alleviating CM pathology.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimal\u003c/h2\u003e \u003cp\u003eEight-week-old male C57BL/6 mice, weighing 20\u0026ndash;25 g, were purchased from the Laboratory Animal Center of Xiamen University. All animal experiments were approved by the Animal Ethics Committee of Xiamen University. All mice were housed under specific pathogen-free (SPF) conditions with a controlled temperature of 22\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C, relative humidity of 50%-60%, and a 12-hour light/dark cycle. Ad libitum access to standard laboratory chow and sterile water was provided throughout the experimental period.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMouse model of chronic migraine\u003c/h3\u003e\n\u003cp\u003eThe CM model was established with minor modifications to a previous protocol19]. Nitroglycerin (NTG; Baiyunshan, China) stock solution (5 mg/mL) was prepared in 30% ethanol/30% propylene glycol/40% ultrapure water, and diluted to 1 mg/mL with sterile saline immediately before use to avoid degradation. Mice in the CM group received intraperitoneal NTG (10 mg/kg) every other day for 5 injections; controls received an equal volume of sterile saline.\u003c/p\u003e\n\u003ch3\u003eAdeno-associated virus (AAV) and Drug Administration\u003c/h3\u003e\n\u003cp\u003eGLUT3 was encoded by gene Slc2a3. Viral solution is thawed on an ice bath (\u0026le;\u0026thinsp;2 freeze-thaw cycles), centrifuged at 500\u0026times;g for 1 min (4\u0026deg;C), and then stereotaxically injected into the lateral ventricle (AP: -7.5 mm, ML: \u0026plusmn;1.7 mm, DV: -4.4 mm, relative to the xiphoid process). The injection rate was 0.02 \u0026micro;L/min (total 100 nL). To minimize backflow, the needle was held stationary for 10 min post-injection. Mice were anesthetized with 1%-1.5% isoflurane during surgery. Postoperatively, analgesia was provided with buprenorphine (2 mg/kg, intraperitoneal injection), followed by 15 min of recovery at 37\u0026deg;C. Transfection efficiency\u0026thinsp;\u0026ge;\u0026thinsp;60% was verified by immunofluorescence 3 weeks post-injection.\u003c/p\u003e \u003cp\u003eMice received intraperitoneal injections of 3-NPA (20 mg/kg, diluted in 0.9% saline) or intragastric administration of Idebenone (200 mg/kg, dissolved in corn oil) 60 min prior to each NTG injection[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. These treatments were administered every other day for a total of five doses. Control mice received an equal volume of 0.9% saline (to match the 3-NPA vehicle) and corn oil (to match the idebenone vehicle) via the corresponding routes. The dosages of AAV and pharmacological agents were selected based on previously published literature.\u003c/p\u003e\n\u003ch3\u003eAnimal Behavioral Experiments\u003c/h3\u003e\n\u003cp\u003eAll behavioral tests were conducted by operators blinded to group assignments during both testing and data analysis, from 9:00 AM to 7:00 PM. Prior to the experiment, mice underwent 3 consecutive days of acclimation to the experimenters (10 min daily) and were acclimated for 30 min in a quiet acrylic cage with a wire mesh flooring to familiarize them with the general testing environment. For each test session, mice were further acclimated to the specific testing apparatus for 30 min prior to assessment: for hindlimb mechanical testing, this involved adaptation in a glass chamber with a wire mesh floor; for periorbital mechanical testing, mice were confined in an enclosure with only their head exposed to prevent movement. The testing room maintained a constant temperature of 22\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C, relative humidity of 50%\u0026ndash;60%, and no noise interference; all testing equipment was disinfected with 75% ethanol before use and after each mouse was tested.\u003c/p\u003e \u003cp\u003eMechanical pain thresholds were assessed prior to and within a 2-hour timeframe following each administration. For hindlimb mechanical pain threshold measurement, von Frey filaments (Stoelting, 0.008\u0026ndash;2 g) were applied vertically to the fat pad-avoiding central region of the hindlimb. A positive response was defined as paw withdrawal, trembling, or licking following stimulation. The 50% withdrawal threshold was determined using the \u0026ldquo;up-and-down method\u0026rdquo;, with three repetitions performed per test and the mean value calculated for final analysis.\u003c/p\u003e \u003cp\u003eFor periorbital mechanical sensitivity assessment, the same von Frey filaments were used, with filaments applied to the periorbital region with gradually increasing intensity. A positive response was characterized by immediate head withdrawal or forelimb scratching of the stimulated periorbital area. Consistent with hindlimb testing, three repetitions were conducted, and the mean value was used for subsequent analysis.\u003c/p\u003e \u003cp\u003e \u003csup\u003e \u003cb\u003e18\u003c/b\u003e \u003c/sup\u003e \u003cb\u003eF-FDG-PET/CT\u003c/b\u003e \u003c/p\u003e \u003cp\u003eMice were subjected to 16 h overnight fasting with free access to water to minimize blood glucose-related interference on \u003csup\u003e18\u003c/sup\u003eF-FDG uptake, and maintained at 22\u0026thinsp;\u0026plusmn;\u0026thinsp;1℃ during fasting to avoid stress-induced glycemic fluctuations. Prior to imaging, mice were anesthetized via isoflurane inhalation with induction parameters of 2\u0026ndash;3% isoflurane in 1 L/min O₂ and maintenance parameters of 1%-1.5% isoflurane in 1 L/min O₂ until loss of corneal reflex and stable muscle relaxation. Animals were then immobilized prone on a custom imaging bed, weighed accurately, and 5 \u0026micro;L of tail vein blood was collected for glucose measurement using a portable Accu-Chek glucometer (Roche). Only mice with blood glucose levels of 3.9\u0026ndash;7.8 mmol/L were included. \u003csup\u003e18\u003c/sup\u003eF-FDG with radiochemical purity\u0026thinsp;\u0026gt;\u0026thinsp;95% and calibrated via a dose calibrator was administered via tail vein injection at 10 \u0026micro;Ci/g body weight at a slow rate to prevent extravasation. Post-injection, the injection site was visually inspected for leakage, and mice with evident extravasation were excluded from subsequent analysis. The imaging bed was positioned to center the mouse head on the detector, with body temperature maintained at 37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5℃ via a feedback-controlled heating pad and respiratory rate continuously monitored at \u0026ge;\u0026thinsp;60 breaths/min through a pressure-sensitive sensor to ensure anesthetic stability.\u003c/p\u003e \u003cp\u003eCT scanning with 80 kV voltage, 500 \u0026micro;A current, 0.2 mm slice thickness, and 80\u0026times;80 mm field of view was performed first for anatomical localization and PET attenuation correction. Immediately following CT acquisition, a 60-min dynamic PET scan with a 128\u0026times;128 matrix and list-mode acquisition was initiated, reconstructed via ordered subsets expectation maximization with point spread function correction (OSEM-PSF) using 11 iterations and 16 subsets. Image analysis was conducted with PMOD 3.0 software: PET and CT images were spatially co-registered, followed by alignment to the Allen Reference Atlas (ARA) Common Coordinate Framework (CCFV3) for automated anatomical segmentation. Standardized uptake values (SUVs) were calculated as (tissue radioactivity concentration in Bq/mL \u0026times; body weight in g) / injected dose in Bq, with SUVmean as the primary quantitative indicator and SUVmax as a supplementary parameter. Target brain regions included the hippocampus, cerebral cortex, cerebellum, hypothalamus, and medulla oblongata, and region-of-interest (ROI) delineation was manually verified for ambiguous anatomical boundaries.\u003c/p\u003e\n\u003ch3\u003eSample Collection and Frozen Section Preparation\u003c/h3\u003e\n\u003cp\u003eMice were deeply anesthetized with sodium pentobarbital (60 mg/kg, intraperitoneal injection). The trigeminal nucleus caudalis (TNC) tissue was rapidly dissected on ice and immediately flash-frozen in liquid nitrogen, reserved for metabolomic, transcriptomic, and Western blotting (WB) analyses without fixation to preserve molecular integrity. For the remaining TNC tissue, transcardial perfusion was performed via the left ventricle with 30 mL 4\u0026deg;C prechilled 0.01 M PBS at a flow rate of 4\u0026ndash;5 mL/min until the liver and spleen turned pale, followed by 10 mL 4\u0026deg;C prechilled 4% paraformaldehyde (PFA) for fixation.\u003c/p\u003e \u003cp\u003eThe fixed TNC tissue was retrieved on ice, post-fixed in 4% PFA at 4\u0026deg;C for 24\u0026ndash;36 h, then dehydrated in 30% sucrose solution at 4\u0026deg;C for 48 h with daily solution renewal. Subsequently, the tissue was embedded in Sakura OCT compound and frozen at -80\u0026deg;C for at least 2 h. Coronal sections (30 \u0026micro;m) containing the TNC region were cut using a Leica CM1950 cryostat set at -20\u0026deg;C for immunofluorescence staining, and stored in 0.01 M PBS at 4\u0026deg;C protected from light.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence (IF)\u003c/h2\u003e \u003cp\u003e30 \u0026micro;m TNC sections were washed with 0.01 M PBS (pH 7.4) for 3 \u0026times; 3 min to remove residual storage buffer. Subsequently, primary antibodies (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) were diluted in 0.01 M PBS containing 0.3% Triton X-100 and 1% bovine serum albumin (BSA) (blocking buffer), and free-floating sections were incubated overnight at 4\u0026deg;C in the dark to prevent fluorophore quenching. The next day, sections were washed with 0.01 M PBS for 3 \u0026times; 5 min to remove unbound primary antibodies, then incubated with corresponding fluorophore-conjugated secondary antibodies at room temperature for 2 h in the dark. After secondary antibody incubation, sections were washed with 0.01 M PBS for 3 \u0026times; 5 min, followed by nuclear counterstaining with 4\u0026rsquo;,6-diamidino-2-phenylindole (DAPI) (Yeasen Biotech, China, 40728ES03) for 10 min in the dark. Sections were rinsed with pre-chilled 0.01 M PBS for 3 \u0026times; 5 min, mounted with anti-fade mounting medium, and sealed with nail polish. Confocal images were captured using a Zeiss LSM800 inverted laser scanning confocal microscope, with uniform gain and exposure time applied across all samples to ensure comparability.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe antibodies used in the immunofluorescence staining.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNeuN Polyclonal antibody\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAbcam, 1:500, ab177487\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIba1 Polyclonal antibody\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAbcam, 1:500, ab5076\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGFAP Polyclonal antibody\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAbcam, 1:500,\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ec-Fos Polyclonal antibody\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCST, 1:500, 2250\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCGRP Polyclonal antibody\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSCBT, 1:1000, sc-57053\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTOM20 Polyclonal antibody\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProteintech,1:500,11802-1-AP\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e2-NBDG Glucose Uptake Assay\u003c/b\u003e \u003c/p\u003e \u003cp\u003eMice were subjected to a 16-hour fasting period with free access to water to eliminate feeding-induced glucose fluctuations prior to tail vein injection of 2-NBDG (MCE, USA, HY-116215) at a dose of 375 \u0026micro;g per mouse (stock solution concentration: 2.5 mg/mL; injection volume: 150 \u0026micro;L per mouse)[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Post-injection leakage or bleeding was monitored, and one hour later, mice were deeply anesthetized and perfused with pre-cooled 0.01 M PBS (pH 7.4) until organ blanching, followed by 4% PFA fixation for subsequent sectioning and fluorescence detection (excitation/emission: 488 nm/520 nm). For validation of GLUT-mediated 2-NBDG uptake, Cytochalasin B (Cyto B, 0.5 mM, 0.5 \u0026micro;L per side) and 4,6-O-methyl-D-glucose (4,6-O, 25 mM, 0.5 \u0026micro;L per side), GLUT inhibitors, were stereotaxically injected into the bilateral TNC prior to 2-NBDG administration.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eIRDye 800CW 2-DG Glucose Uptake Assay\u003c/h3\u003e\n\u003cp\u003eIRDye 800CW 2-DG (Licor, USA, 926\u0026ndash;08946), a near-infrared fluorescent glucose analog, was reconstituted in sterile 1\u0026times; PBS to 0.1 nmol/\u0026micro;L and filter-sterilized through a 0.2 \u0026micro;m filter if needed. Under gentle restraint, mice were injected with 10 nmol of the probe via tail vein, with post-injection leakage or bleeding monitored[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Twenty-four hours later (per manufacturer\u0026rsquo;s recommendation for optimal signal-to-noise ratio), mice were deeply anesthetized and perfused with pre-cooled 0.01 M PBS (pH 7.4) until organ blanching, followed by 4% PFA fixation for subsequent sectioning and fluorescence detection (excitation/emission: 774 nm/791 nm).\u003c/p\u003e\n\u003ch3\u003eDetection of Reactive Oxygen Species (ROS)\u003c/h3\u003e\n\u003cp\u003eTNC tissues were homogenized in ice-cold RIPA lysis buffer, centrifuged (12,000\u0026times;g, 20 min, 4℃) to collect the lysate supernatant, and protein concentration was quantified via a BCA protein assay kit. For each sample, a 200 \u0026micro;L reaction system (20 \u0026micro;L lysate\u0026thinsp;+\u0026thinsp;170 \u0026micro;L PBS [pH 7.4]\u0026thinsp;+\u0026thinsp;10 \u0026micro;L DHE, final concentration 10 \u0026micro;M) was prepared, alongside a blank control (190 \u0026micro;L PBS\u0026thinsp;+\u0026thinsp;10 \u0026micro;L sterile water) and a negative control (180 \u0026micro;L PBS\u0026thinsp;+\u0026thinsp;10 \u0026micro;L DHE) to correct for DHE auto-oxidation. All mixtures were incubated at 37℃ for 30 min in the dark, then transferred to a black 96-well plate (100 \u0026micro;L/well, triplicates) to measure fluorescence (Ex/Em\u0026thinsp;=\u0026thinsp;530/590 nm) via a microplate reader. Relative sample fluorescence was calculated by subtracting the negative control\u0026rsquo;s intensity from raw values.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eWestern Blotting (WB)\u003c/h2\u003e \u003cp\u003eTotal proteins were extracted from frozen brain tissue, which was thawed on ice and homogenized at a 1:10 weight-to-volume ratio in pre-chilled RIPA lysis buffer for 2 min at 120 Hz, followed by ultrasonication on ice for 30 s at 300 W. The homogenate was centrifuged at 12,000\u0026times;g for 30 min at 4\u0026deg;C, and the supernatant was collected as total protein. Protein concentration was determined using the BCA method with a bovine serum albumin standard curve (0-400 \u0026micro;g/mL). Equal amounts of protein were mixed with 5\u0026times; loading buffer and boiled at 95\u0026deg;C for 10 min, with membrane proteins excluded from heating. Proteins were separated by 10% or 15% SDS-PAGE at 80 V for 30 min, followed by 120 V for 60\u0026ndash;90 min. Polyvinylidene fluoride (PVDF) membranes (Millipore, USA) were activated with methanol for 10 s prior to protein transfer, which was performed at 300 mA with the temperature maintained below 40\u0026deg;C for 60 min. The membranes were blocked with 5% skim milk powder in TBST for 2 h at room temperature, then incubated overnight at 4\u0026deg;C with primary antibodies (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) diluted in 5% BSA. Subsequently, membranes were washed three times with TBST (10 min each) and incubated in the dark with HRP-conjugated secondary antibodies (1:10,000 dilution in 5% nonfat milk powder) for 1 hour at room temperature. After final washes with TBST and distilled water, protein bands were visualized using a fluorescence imager and saved as TIFF files. Band intensities were quantified using ImageJ software (NIH, USA).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe antibodies used in the immunoblot analysis.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGAPDH Polyclonal antibody\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProteintech,1:10000, 10494-1-AP\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBeta Tubulin Polyclonal antibody\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProteintech,1:10000,10094-1-AP\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNF-kappaB p65 Monoclonal Antibody\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCST, 1:10000, 8242\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePhospho-NF-kappaB p65 (Ser536) Monoclonal Antibody\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCST, 1:10000, 3033\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIkappaB alpha Monoclonal Antibody\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCST, 1:10000, 4812\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePhospho-IkappaB alpha (Ser32) Monoclonal Antibody\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCST, 1:10000, 2859\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTNF-alpha Polyclonal antibody\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProteintech,1:10000, 17590-1-AP\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIL-1 beta Polyclonal antibody\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProteintech,1:10000, 16806-1-AP\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIL-6 Polyclonal antibody\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProteintech,1:10000, 21865-1-AP\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGLUT3 Polyclonal antibody\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProteintech,1:10000, 20403-1-AP\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eTransmission Electron Microscopy (TEM)\u003c/h2\u003e \u003cp\u003eMice were decapitated under deep isoflurane anesthesia, and brain tissue was harvested on ice within 30 s. The TNC region was dissected into 1 mm\u0026sup3; cubes and fixed in pre-chilled TEM fixative (2.5% glutaraldehyde, 2% PFA in 0.1 M phosphate buffer, pH 7.4) at 4\u0026deg;C for 24 h. Tissues were rinsed with 0.1 M phosphate-buffered saline (3 \u0026times; 15 min), post-fixed with 1% osmium tetroxide solution (room temperature, 1\u0026ndash;2 h, protected from light), and rinsed again (3 \u0026times; 15 min). Gradient dehydration was performed using ethanol solutions (30%, 50%, 70% for 15 min each; 80%, 90%, 95%, 100% for 15 min each) and acetone (2\u0026times;30 min). Tissues were infiltrated with Epon 812 embedding medium (8:1:1\u0026thinsp;=\u0026thinsp;Epon 812: hardener: accelerator), polymerized at 37\u0026deg;C (12 h) -45\u0026deg;C (12 h)\u0026thinsp;\u0026minus;\u0026thinsp;60\u0026deg;C (24 h), and sectioned into 60\u0026ndash;80 nm ultrathin slices. Slices were stained with 2% uranyl acetate (15 min, dark) and 0.3% lead citrate (8 min, CO₂-free environment), then imaged using a TEM (500\u0026ndash;1000\u0026times; for screening, 5000\u0026ndash;50000\u0026times; for organelle observation; 5\u0026ndash;8 fields/sample, 2048\u0026times;2048 pixels, TIFF format).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eHuman Plasma Samples\u003c/h2\u003e \u003cp\u003e \u003cstrong\u003eInformed consent\u003c/strong\u003e \u003cp\u003e was obtained from all participants, and the study was approved by the Ethics Committee of Xiamen University. Plasma samples were collected from 359 migraine patients and 140 healthy controls at the First Affiliated Hospital of Xiamen University, China, following written informed consent. Blood samples were centrifuged at 3,000 \u0026times; g for 15 min at 4\u0026deg;C to isolate plasma, which was then aliquoted and stored at -80\u0026deg;C until metabolomic and transcriptomic analyses.\u003c/p\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eMetabolomics\u003c/h2\u003e \u003cp\u003eFrozen mouse TNC brain tissues and human plasma samples were processed separately for metabolomic analysis. For metabolite extraction, samples were thawed at 4\u0026deg;C, mixed with pre-cooled methanol-acetonitrile-water (2:2:1, v/v), vortexed, subjected to low-temperature sonication for 30 min, incubated at -20\u0026deg;C for 10 min, centrifuged at 14,000\u0026times;g, 4\u0026deg;C for 20 min, and supernatants were vacuum-dried; residues were reconstituted in 100 \u0026micro;L acetonitrile-water (1:1, v/v), vortexed, re-centrifuged at 14,000\u0026times;g, 4\u0026deg;C for 15 min, and the resulting supernatants were used for injection. Chromatographic separation was performed using an Agilent 1290 Infinity LC system with two columns: For the HILIC column, we set a column temperature of 55\u0026deg;C, flow rate of 0.3 mL/min, and injection volume of 2 \u0026micro;L; mobile phases were A (90% water\u0026thinsp;+\u0026thinsp;2 mM ammonium formate\u0026thinsp;+\u0026thinsp;10% acetonitrile) and B (acetonitrile\u0026thinsp;+\u0026thinsp;0.4% formic acid), with the gradient: 85% B 0\u0026ndash;1.0 min, linear decrease to 80% B 1.0\u0026ndash;3.0 min, 80% B 3.0\u0026ndash;4.0 min, linear decrease to 70% B 4.0\u0026ndash;6.0 min, linear decrease to 50% B 6.0\u0026ndash;10.0 min, 50% B 10-15.5 min, and linear increase to 85% B 15.5\u0026ndash;23 min. For the C18 column, column temperature was 40\u0026deg;C, flow rate 0.4 mL/min, and injection volume 2 \u0026micro;L; mobile phases were A water\u0026thinsp;+\u0026thinsp;5 mM ammonium acetate and B 99.5% acetonitrile, with the gradient: 5% increased linearly to 60% B over 0\u0026ndash;5 min, 60% increased linearly to 100% B over 5\u0026ndash;11 min, maintained at 100% B for 11\u0026ndash;13 min, decreased linearly to 5% B over 13-13.1 min, and held constant at 5% B for 13.1\u0026ndash;16 min. Samples were held in a 4\u0026deg;C autosampler and analyzed randomly, with quality control samples inserted into the sequence to monitor system stability. Mass spectrometry detection used an AB 6500\u0026thinsp;+\u0026thinsp;QTRAP instrument (AB SCIEX) with an ESI source: source temperature 580\u0026deg;C, ion source gas 1 (GS1) 45, ion source gas 2 (GS2) 60, curtain gas (CUR) 35, ion spray voltage\u0026thinsp;\u0026plusmn;\u0026thinsp;4500 V (positive/negative modes), and operation in multiple reaction monitoring (MRM) mode. Raw MRM data were processed via MultiQuant software for peak area extraction; analyte concentrations were quantified using the ratio of each analyte\u0026rsquo;s peak area to the internal standard\u0026rsquo;s peak area combined with calibration curves, and acquired data underwent quality assessment prior to subsequent analyses.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eRNA-seq and data processing\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from frozen mouse TNC brain tissues and human plasma samples: mouse TNC tissues and human plasma samples were processed using TRIzol reagent and a commercial plasma RNA extraction kit, respectively, following the manufacturers\u0026rsquo; instructions. After RNA extraction, RNA purity and integrity were evaluated by NanoDrop 2000 and agarose gel electrophoresis for quality control. Libraries were constructed with the TruSeq Stranded mRNA Library Prep Kit (Illumina) and sequenced on an Illumina NovaSeq 6000 platform. Raw reads were filtered to remove low-quality reads and adapters; clean reads from mouse TNC tissues were aligned to the mouse reference genome (GRCm39), while those from human plasma were aligned to the human reference genome (GRCh38), both via HISAT2. To measure differentially expressed genes (DEGs), DESeq2 (version 1.40.2) was used with the criteria of |log2 fold change| \u0026gt; 1 and P\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Gene Ontology (GO) and KEGG pathway enrichment analyses were conducted using the R package clusterProfiler (version 4.8.3) for functional annotation of DEGs, with \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 considered statistically significant.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eGraphPad Prism 9.0 was used to perform statistical tests and all data are shown as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Unpaired two-tailed Student\u0026rsquo;s t test (for two group comparisons) and one-way or two-way analysis of variance (ANOVA) (for multiple group comparisons) followed by Tukey\u0026rsquo;s post-hoc test. A significance level of \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003e1. Abnormal glucose metabolism in CM patients\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo explore the systemic metabolic and inflammatory perturbations underlying CM, we collected plasma samples and clinical data from 359 CM patients and 140 healthy controls, followed by metabolomic and transcriptomic profiling and integrated multi-omics analysis as outlined in the study workflow (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Multi-omics co-enrichment analysis for CM versus Control identified concurrent dysregulation of glucose metabolism, with core energy metabolic cascades such as the TCA cycle and OXPHOS being suppressed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Transcriptomic analysis further delineated pathway alterations at the transcriptional level: Top 30 Gene Ontology (GO) term enrichment analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC) revealed that downregulated transcripts were enriched in energy metabolism-related terms across multiple GO categories. Specifically, under Biological Process, these included \u0026ldquo;mitochondrial electron transport, NADH to ubiquinone\u0026rdquo; and \u0026ldquo;mitochondrial respiratory chain complex I assembly\u0026rdquo;; under Cellular Component, they covered \u0026ldquo;respiratory chain complex IV\u0026rdquo; and \u0026ldquo;mitochondrial respiratory chain complex I\u0026rdquo;; while under Molecular Function, terms such as \u0026ldquo;NADH dehydrogenase activity\u0026rdquo; and \u0026ldquo;NADH dehydrogenase (ubiquinone) activity\u0026rdquo; were included. Consistent with GO results, top 20 KEGG pathway enrichment dot plots for transcriptomic data revealed downregulated transcripts clustered in energy metabolism pathways such as oxidative phosphorylation, whereas upregulated transcripts concentrated in inflammatory pathways including the NF-κB signaling pathway and TNF signaling pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Metabolomic analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE) validated and complemented the transcriptomic findings that focused pathway enrichment plots showed the top suppressed metabolic pathways corresponded to core glucose metabolic cascades such as the TCA cycle, while the top elevated metabolic pathways such as caffeine metabolism.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e2. Global cerebral glucose metabolism changes in NTG-induced CM mice\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe success of the NTG-induced chronic migraine mouse model was first confirmed by assessing the Paw Withdrawal Threshold and periorbital Withdrawal Threshold, a core behavioral phenotype of CM, using von Frey filaments. The model was established by administering intraperitoneal NTG at 10 mg/kg every other day for 5 total doses, spanning 9 days; VEH control group received equal volumes of sterile saline. To explore cerebral energy metabolism changes in this CM model, we performed macroscale detection of whole-brain glucose uptake using \u003csup\u003e18\u003c/sup\u003eF-FDG-PET/CT under anesthesia (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Results showed that NTG-treated mice had reduction in global cerebral glucose uptake rate that assessed by mean standardized uptake value (SUVmean) compared to VEH controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-C). Among the key brain regions\u0026mdash;including the hippocampus, cerebral cortex, cerebellum, and hypothalamus\u0026mdash;the medulla oblongata emerged as the site of most significant metabolic alteration (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Specifically, as a pivotal relay station for migraine pain signaling located within the medulla oblongata, the TNC, a key brain region in CM, demonstrated a substantial decrease in glucose uptake relative to the VEH group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). To identify the cell type responsible for TNC metabolic abnormalities, we used 2-NBDG, a 488 nm-excitable fluorescent glucose analog, and IRDye 800CW 2-DG, a near-infrared fluorescent glucose analog, to assess cellular glucose uptake. We performed immunofluorescence co-staining with neuron-specific marker NeuN, microglia-specific marker Iba1, and astrocyte-specific marker GFAP to localize glucose uptake (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA). Consistent results were obtained with both probes: NeuN\u003csup\u003e+\u003c/sup\u003e neurons accounted for the vast majority of glucose uptake in the TNC under both VEH and NTG conditions, exceeding that of Iba1\u003csup\u003e+\u003c/sup\u003e microglia and GFAP\u003csup\u003e+\u003c/sup\u003e astrocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB). When analysing glucose uptake intensity across cell types, we similarly observed significantly reduced glucose uptake in NeuN\u003csup\u003e+\u003c/sup\u003e neurons from CM mice compared to VEH controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC). Conversely, glucose uptake by Iba1\u003csup\u003e+\u003c/sup\u003e microglia and GFAP\u003csup\u003e+\u003c/sup\u003e astrocytes showed no significant alterations (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH-I, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003eS1\u003c/span\u003eD-E). This indicated that TNC metabolic dysfunction in CM is neuron-specific, highlighting the clinical relevance of targeting neuronal glucose uptake for CM intervention. 2-NBDG fluorescence intensity in TNC neurons was significantly reduced by 4,6-O and Cyto B, GLUT inhibitors, thus confirming 2-NBDG uptake in TNC neurons is dependent on GLUT and validating it as a reliable marker for assessing GLUT-mediated glucose uptake (Fig. S2A-B). GLUTs are rate-limiting for cerebral glucose uptake, and neurons predominantly express GLUT1, GLUT3, and GLUT4[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. To identify the molecular driver of impaired neuronal glucose uptake, we detected the expression of these GLUTs in the TNC using WB after the last NTG injection. WB results showed that GLUT3 protein levels were reduced in CM mice compared to VEH mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG-H), while GLUT1 and GLUT4 protein expression remained unchanged (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI-J). These data collectively indicate that neuronal GLUT3 downregulation is the core mechanism underlying abnormal TNC glucose uptake in CM.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e3. Neuronal GLUT3 overexpression alleviates hyperalgesia and central sensitization in CM mice\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo elucidate the role of neuronal GLUT3 in CM, we performed stereotaxic injection of adeno-associated virus (AAV) vectors targeting the TNC. The experimental group received AAV-hSyn-GLUT3 for neuron-specific GLUT3 overexpression, while the control group was injected with AAV-hSyn-mCherry as the empty vector control. Three weeks after viral injection, procedures for establishing the chronic migraine model were initiated (Fig. S3A-B). Subsequently, WB analysis quantitatively measured GLUT3 protein levels to confirm transfection efficiency (Fig. S3C-D). Immunofluorescence co-staining of GLUT3 with NeuN, Iba1, and GFAP was performed to verify transfection specificity. Results showed that GLUT3 expression was neuron-specific (Fig. S3E-F).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBehavioral tests using von Frey filaments demonstrated that neuronal GLUT3 overexpression significantly enhanced both plantar and periorbital pain thresholds, effectively alleviating NTG-induced basal and acute mechanical allodynia in CM mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-D). c-Fos is an immediate early gene marker of neuronal activation, and CGRP is a key neuropeptide that drives migraine central sensitization[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. To assess neuronal activation and central sensitization in the TNC, we performed immunofluorescence staining for these two markers. Quantitative analysis confirmed that relative to the VEH group, the CM group exhibited marked elevations in TNC c-Fos\u003csup\u003e+\u003c/sup\u003e cell counts and CGRP fluorescence intensity. Notably, neuronal GLUT3 overexpression robustly reversed these pro-sensitization alterations, reducing c-Fos\u003csup\u003e+\u003c/sup\u003e cell numbers and CGRP fluorescence intensity in the TNC of CM mice to levels comparable to the VEH group. These molecular changes correlated with the restored mechanical pain thresholds observed in the GLUT3 overexpression group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eE-H).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e4. Neuronal GLUT3 overexpression restores neuronal glucose metabolism and suppresses microglia activation in CM mice\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAs a high-affinity glucose transporter, GLUT3 overexpression enhances neuronal glucose uptake efficiency, fueling mitochondrial energy production pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-B, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003eS1\u003c/span\u003eF-G). Given that mitochondrial structural integrity is tightly linked to functional capacity, we used transmission electron microscopy (TEM) to observe the ultrastructure of TNC neurons. In CM mice, neurons exhibited prominent mitochondrial swelling, fragmented and vacuolated cristae, and a reduced number of mitochondria compared to VEH group. Strikingly, GLUT3 overexpression significantly ameliorated these pathological changes, restoring mitochondrial morphology and quantity (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eC-D). TOM20 is a key marker of mitochondrial biogenesis and membrane transport capacity[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. To further assess mitochondrial function, we examined it via immunofluorescence and WB. The results showed that TOM20 fluorescence intensity in the TNC of CM mice was markedly decreased, indicating impaired mitochondrial biogenesis and function. In contrast, GLUT3 overexpression robustly restored TOM20 expression at both protein and fluorescence levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eE-H). These findings collectively demonstrate that neuronal GLUT3 overexpression improves mitochondrial structural integrity, enhances membrane potential stability, and restores membrane protein transport function, thereby reversing cerebral energy metabolism dysfunction in CM mice. To characterize microglial activation, we performed Iba1 immunofluorescence staining in the TNC (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eI). Relative to the NTG\u0026thinsp;+\u0026thinsp;AAV-NC group, the NTG\u0026thinsp;+\u0026thinsp;AAV-Slc2a3\u003csup\u003e+\u003c/sup\u003e group showed significantly reduced Iba1\u003csup\u003e+\u003c/sup\u003e cell numbers and fluorescence intensity, verifying GLUT3 overexpression suppresses microglial activation (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ-K).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e5. Neuronal GLUT3 overexpression modulates the TCA cycle and reduced overproduction of ROS in CM mice\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo characterize GLUT3-mediated metabolic regulation in CM, we performed targeted metabolomic profiling of TNC tissues from VEH\u0026thinsp;+\u0026thinsp;AAV-NC, NTG\u0026thinsp;+\u0026thinsp;AAV-NC, and NTG\u0026thinsp;+\u0026thinsp;AAV-Slc2a3\u003csup\u003e+\u003c/sup\u003e groups. A principal component analysis (PCA) score plot showed distinct separation between the NTG\u0026thinsp;+\u0026thinsp;AAV-NC and VEH\u0026thinsp;+\u0026thinsp;AAV-NC groups, while the NTG\u0026thinsp;+\u0026thinsp;AAV-Slc2a3\u003csup\u003e+\u003c/sup\u003e group clustered closely with VEH\u0026thinsp;+\u0026thinsp;AAV-NC\u0026mdash;confirming that GLUT3 overexpression reverses NTG-induced metabolic dysregulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). To characterize metabolic perturbations and the regulatory role of GLUT3 in CM, we first identified differential metabolites across experimental groups. Venn diagram analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eB-C) revealed that 145 metabolites were elevated in the VEH\u0026thinsp;+\u0026thinsp;AAV-NC vs NTG\u0026thinsp;+\u0026thinsp;AAV-NC group, while 26 metabolites were downregulated in the NTG\u0026thinsp;+\u0026thinsp;AAV-NC vs NTG\u0026thinsp;+\u0026thinsp;AAV-Slc2a3\u003csup\u003e+\u003c/sup\u003e group. Notably, 14 of these metabolites exhibited reversed expression trends following GLUT3 overexpression. 88 metabolites were reduced in the VEH\u0026thinsp;+\u0026thinsp;AAV-NC vs NTG\u0026thinsp;+\u0026thinsp;AAV-NC group, alongside 45 downregulated metabolites in the NTG\u0026thinsp;+\u0026thinsp;AAV-NC vs NTG\u0026thinsp;+\u0026thinsp;AAV-Slc2a3\u003csup\u003e+\u003c/sup\u003e group. Among these, 19 metabolites showed reversed expression trends upon GLUT3 overexpression. KEGG pathway enrichment of differential metabolites (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eD) and subgroup comparisons (Fig. S3A-B) highlighted glucose metabolism-related cascades as the most significantly enriched in NTG-induced dysregulation and further showed that central carbon metabolism, glucagon signaling and the TCA cycle were functionally reversed by GLUT3 overexpression. A heatmap of key metabolites in glycolysis, the pentose phosphate pathway (PPP), and the TCA cycle revealed pathological metabolic remodeling in NTG\u0026thinsp;+\u0026thinsp;AAV-NC mice: upstream glycolysis/PPP metabolites were elevated, while downstream glycolysis/TCA/OXPHOS metabolites were reduced. Notably, GLUT3 overexpression selectively restored TCA cycle metabolites, emphasizing its role in repairing core glucose metabolic networks (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eC-D). Quantification of TCA intermediates confirmed that NTG reduced cis-aconitic acid, isocitric acid, fumaric acid, and malic acid, while changes reversed by GLUT3 overexpression (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eG-J). Consistent with metabolic restoration, GLUT3 overexpression also attenuated NTG-induced ROS elevation (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eK).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn glycolysis pathway, NTG\u0026thinsp;+\u0026thinsp;AAV-NC mice showed elevated levels of upstream metabolites including fructose 6-phosphate, fructose 1,6-bisphosphate and glucose 6-phosphate (Fig. S4C-F), alongside reduced concentrations of mid-late metabolites such as 2-phosphoglyceric acid, phosphoenolpyruvic acid and lactate (Fig. S4G-L). Additionally, in the OXPHOS, NTG\u0026thinsp;+\u0026thinsp;AAV-NC mice showed levels of nicotinamide (Fig. S4M) and nicotinamide ribotide (Fig. S4N) remained unchanged, while energy intermediates were disrupted\u0026mdash;characterized by elevated AMP and AMP/ADP ratio, and decreased ADP (Fig. S4J-L). In PPP pathway, NTG\u0026thinsp;+\u0026thinsp;AAV-NC mice exhibited a state of pathway activation characterized by elevated levels of metabolites including D-sedoheptulose 7-phosphate, D-ribulose 5-phosphate, D-ribose, Glucosamine 6-phosphate and N-acetylglucosamine 6-phosphate (Fig. S4O-S). This compensatory activation, presumably driven by oxidative stress, was not significantly modulated by GLUT3 overexpression. In contrast, GLUT3 overexpression did not exert significant modulatory effects on the glycolysis pathway or the compensatory activation of PPP driven by oxidative stress in CM mice. These findings collectively demonstrate that GLUT3 ameliorates CM via selective restoration of the TCA cycle rather than modulation of glycolysis or PPP pathways.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e6. Neuronal GLUT3 overexpression ameliorates chronic migraine via the NF-κB pathway\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo profile transcriptomic changes in the TNC, RNA sequencing was performed. In the comparison between the VEH\u0026thinsp;+\u0026thinsp;AAV-NC group and the NTG\u0026thinsp;+\u0026thinsp;AAV-NC group, 365 genes were upregulated and 587 genes were downregulated in the VEH\u0026thinsp;+\u0026thinsp;AAV-NC group. In the comparison between the NTG\u0026thinsp;+\u0026thinsp;AAV-NC group and the NTG\u0026thinsp;+\u0026thinsp;AAV-Slc2a3\u003csup\u003e+\u003c/sup\u003e group, 382 genes were upregulated and 716 genes were downregulated in the NTG\u0026thinsp;+\u0026thinsp;AAV-NC group (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-B). Venn diagrams showed that 214 and 187 differentially expressed genes (DEGs) reversed their expression trends following GLUT3 overexpression (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eC-D). KEGG pathway enrichment analysis of the VEH\u0026thinsp;+\u0026thinsp;AAV-NC vs NTG\u0026thinsp;+\u0026thinsp;AAV-NC comparison (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eE) showed that NTG treatment altered key pathway profiles: glucose metabolism-related processes, including Glycolysis/Gluconeogenesis and the Pentose phosphate pathway, were among the upregulated pathways, while downregulated pathways were enriched in neuroinflammation and sensory signaling-related cascades, such as the NF-κB signaling pathway and the Inflammatory mediator regulation of TRP channels. In contrast, in the NTG\u0026thinsp;+\u0026thinsp;AAV-NC vs NTG\u0026thinsp;+\u0026thinsp;AAV-Slc2a3\u003csup\u003e+\u003c/sup\u003e comparison, GLUT3 overexpression mainly suppressed the NF-κB pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eF). These findings align with our prior functional and molecular observations, further emphasizing the role of GLUT3 in regulating NF-κB-mediated neuroinflammation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e7. Neuronal GLUT3 overexpression alleviates hyperalgesia in chronic migraine mice via the TCA cycle\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo verify whether GLUT3 alleviates CM via the TCA cycle, relevant experimental operations were carried out. 3-NPA, a specific inhibitor of SDH, a rate-limiting enzyme in the TCA cycle, was used to block TCA flux30]. Behavioral tests using von Frey filaments evaluated mechanical allodynia at baseline and 2 h post-each NTG injection. Results showed that the AAV-Slc2a3\u003csup\u003e+\u003c/sup\u003e group exhibited significantly elevated PWTs at both basal and acute phases, effectively alleviating NTG-induced mechanical allodynia. Notably, co-administration of 3-NPA substantially reversed the analgesic effect of GLUT3 overexpression (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e7\u003c/span\u003eF-I), demonstrating that blocking the TCA cycle abrogates GLUT3 overexpression-mediated pain relief. To assess the impact on central sensitization, we performed immunofluorescence staining for c-Fos and CGRP in the TNC and found that c-Fos\u003csup\u003e+\u003c/sup\u003e cell numbers and CGRP fluorescence intensity in the NTG\u0026thinsp;+\u0026thinsp;AAC-Slc2a3\u003csup\u003e+\u003c/sup\u003e+3-NPA group were significantly higher than those in the NTG\u0026thinsp;+\u0026thinsp;AAC-Slc2a3\u003csup\u003e+\u003c/sup\u003e group and indistinguishable from the CM group (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e7\u003c/span\u003eJ-M)​.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e8. Neuronal GLUT3 overexpression mitigates neuroinflammation in chronic migraine via the TCA cycle\u003c/b\u003e \u003c/p\u003e \u003cp\u003eImmunoblot of NF-κB pathway components and downstream pro-inflammatory factors (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e8\u003c/span\u003eA) showed that in the TNC of NTG-induced CM mice, the ratios of phosphorylated p65 (p-p65)/total p65 and phosphorylated IκBα (p-IκBα)/total IκBα were significantly elevated (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e8\u003c/span\u003eB-C). Concurrently, protein levels of pro-inflammatory cytokines TNF-α, IL-6, and IL-1β were markedly upregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e8\u003c/span\u003eD-F), indicating excessive NF-κB pathway activation and prominent neuroinflammation in the TNC under CM conditions. In the GLUT3 overexpression group, the p-p65/p65 ratio, p-IκBα/IκBα ratio, and expression of TNF-α, IL-6, and IL-1β were significantly reduced relative to the NTG group, demonstrating that GLUT3 effectively inhibits NF-κB-mediated neuroinflammation. Notably, co-administration of 3-NPA elevated all indices back, confirming the TCA cycle is an essential link for GLUT3\u0026rsquo;s regulation of NF-κB neuroinflammation (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e8\u003c/span\u003eB-F).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo characterize microglial activation, we performed Iba1 immunofluorescence staining in the TNC (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e8\u003c/span\u003eG). Relative to the NTG\u0026thinsp;+\u0026thinsp;AAV-NC group, the NTG\u0026thinsp;+\u0026thinsp;AAV-Slc2a3\u003csup\u003e+\u003c/sup\u003e group showed significantly reduced Iba1\u003csup\u003e+\u003c/sup\u003e cell numbers and fluorescence intensity, verifying GLUT3 overexpression suppresses microglial activation. However, Iba1\u003csup\u003e+\u003c/sup\u003e cell counts and intensity in the NTG\u0026thinsp;+\u0026thinsp;AAV-Slc2a3\u003csup\u003e+\u003c/sup\u003e+3-NPA group were elevated back (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e8\u003c/span\u003eH-I). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e8\u003c/span\u003eJ, the TNC tissue ROS level in the NTG\u0026thinsp;+\u0026thinsp;AAV-NC group was significantly elevated relative to the VEH\u0026thinsp;+\u0026thinsp;AAV-NC control group, indicating excessive ROS accumulation in the TNC under CM conditions. In contrast, the NTG\u0026thinsp;+\u0026thinsp;AAV-Slc2a3\u003csup\u003e+\u003c/sup\u003e group exhibited a marked reduction in ROS level compared to the NTG\u0026thinsp;+\u0026thinsp;AAV-NC group, demonstrating that GLUT3 overexpression effectively suppresses ROS production in CM mice. Notably, the ROS level in the NTG\u0026thinsp;+\u0026thinsp;AAV-Slc2a3\u003csup\u003e+\u003c/sup\u003e+3-NPA group was significantly increased relative to the NTG\u0026thinsp;+\u0026thinsp;AAV-Slc2a3\u003csup\u003e+\u003c/sup\u003e group, verifying that GLUT3 inhibits ROS accumulation via the TCA cycle (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e8\u003c/span\u003eJ). Collectively, these results indicate GLUT3 suppresses microglial activation and NF-κB-mediated neuroinflammation via TCA cycle, reinforcing the TCA cycle\u0026rsquo;s critical role as a downstream mediator of GLUT3\u0026rsquo;s anti-inflammatory effects in CM.​\u003c/p\u003e \u003cp\u003e \u003cb\u003e9. Idebenone alleviates hyperalgesia in chronic migraine mice\u003c/b\u003e \u003c/p\u003e \u003cp\u003eGiven our prior finding that the TCA cycle serves as a critical downstream mediator of GLUT3\u0026rsquo;s anti-migraine effects, we explored Idebenone, a clinically used mitochondrial protector that specifically enhances TCA cycle flux, as a potential therapeutic agent, while validating the central regulatory role of the TCA cycle in CM pathogenesis. Behavioral results showed NTG-induced CM mice exhibited notably reduced periorbital/plantar mechanical withdrawal thresholds, indicative of hyperalgesia. Idebenone reversed this threshold reduction in both basal (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e9\u003c/span\u003eA, C) and post-treatment phases (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e9\u003c/span\u003eB, D) relative to the NTG group. Immunofluorescence analysis revealed that, compared with the NTG group, Idebenone decreased c-Fos\u003csup\u003e+\u003c/sup\u003e cell counts and CGRP intensity in the TNC, thereby inhibiting neuronal overactivation and central sensitization (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e9\u003c/span\u003eE\u0026ndash;H). These findings indicate that Idebenone plays a critical role in regulating central sensitization in CM mice. As a clinically safe agent, it holds translational potential for CM treatment.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study is the first to investigate how energy metabolism dysfunction impacts chronic migraine. Clinically, we detected abnormal expression of key OXPHOS pathway genes and reduced levels of tricarboxylic acid cycle metabolites in the plasma of CM patients for the first time, confirming the inherent systemic metabolic imbalance in CM. In an NTG-induced CM mouse model, we confirmed significantly reduced glucose uptake in TNC and neuronal-specific downregulation of GLUT3 expression, while no significant changes were observed in glial cells. Notably, neuron-specific overexpression of GLUT3 in TNC enhanced TCA cycle function, elevated mechanical pain thresholds, suppressed central sensitization by downregulating c-Fos and CGRP, and blocked NF-κB pathway activation. The TCA cycle inhibitor 3-NPA reversed GLUT3's suppression of the NF-κB pathway and its analgesic effects in CM mice, demonstrating that GLUT3 modulates CM-associated phenotypes by regulating TCA cycle function, which in turn influences NF-κB pathway activity. Furthermore, Idebenone significantly downregulated abnormal expression of c-Fos and CGRP in the TNC. These findings provide novel mechanistic insights and potential targets for metabolic interventions in CM (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e10\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNumerous studies have demonstrated that patients with CM exhibit systemic metabolic disorders, with energy fluctuation-related factors such as fasting and rapid blood glucose changes identified as important triggers for CM attacks[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Consistent with these clinical phenomena, we detect abnormal expression of key OXPHOS genes and significantly reduced levels of TCA cycle metabolites in the plasma of CM patients. In NTG-induced CM mouse models, previous studies have observed abnormalities in cerebral energy metabolites. For instance, Gao et al. found decreased levels of ATP-related metabolites including NAD, ADP, and AMP in the brain tissue of NTG -induced CM mice[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]; however, these studies failed to clarify specific abnormal metabolic pathways or brain region specificity, whether improving cerebral energy metabolism can ameliorate CM, resulting in a lack of precise targets for mechanistic dissection and an unclear therapeutic potential of metabolic modulation in CM. By integrating whole-brain metabolic imaging with targeted metabolomics, our study yielded three key findings: First, we demonstrated that glucose metabolic abnormalities are most prominent in the medullary region containing the TNC during acute CM. Compared to non-pain-related brain regions such as the hippocampus and cortex, NTG-induced CM mice exhibited significantly reduced glucose uptake in this medullary region. This result is consistent with the physiological role of the TNC as a relay hub for migraine pain signals, providing experimental evidence for the \"pain-specific brain region localization\" of metabolic abnormalities. Second, we confirmed that cerebral glucose uptake is primarily localized to TNC neurons, with no significant alterations observed in glial cells, and underscoring the pivotal role of intrinsic neuronal glucose transport deficits. Furthermore, the neuron-specific glucose transporter GLUT3 is also downregulated in the TNC, which may serve as a key initiator of local metabolic dysfunction.\u003c/p\u003e \u003cp\u003eGLUT3, a neuron-specific glucose transporter that mediates cerebral glucose uptake and maintains neuronal energy homeostasis, has been extensively investigated in various neurological disorders[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. However, its role and underlying regulatory mechanisms in the pathophysiology of CM remain poorly defined. Through multi-omics analyses and functional interventions, this study verified that NTG-induced CM mice exhibit concomitant phenotypes of reduced TCA cycle metabolites and NF-κB pathway activation, which is consistent with previous clinical and basic research findings. Further investigations revealed that neuron-specific GLUT3 overexpression concurrently improved glucose uptake, restored TCA cycle function, inhibited microglia activation and NF-κB pathway activation as well as pain phenotypes. This result, for the first time, identifies GLUT3 as a key upstream metabolic target mediating metabolic-inflammatory crosstalk in CM. The NF-κB pathway, as a central regulatory axis of neuroinflammation, sustained activation of which drives central sensitization and CM chronification. In the NTG-induced chronic migraine-like pain mouse model, microglia in the TNC region can be rapidly activated and polarized to a pro-inflammatory phenotype. These activated microglia form a positive feedback loop of \u0026ldquo;inflammatory factor release-increased neuronal excitability\u0026rdquo; by releasing pro-inflammatory cytokines such as IL-1β and IL-6, while upregulating the expression of neuropeptides such as CGRP, thereby exacerbating pain signal amplification and central sensitization processes[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Previous studies have confirmed that TCA cycle dysfunction can activate NF-κB pathway via the accumulation of ROS[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Our results showed that 3-NPA completely abrogated the NF-κB inhibitory effect and analgesic action mediated by GLUT3 overexpression, directly verifying that the TCA cycle serves as an indispensable link connecting GLUT3-dependent glucose uptake and NF-κB-driven inflammatory activation.\u003c/p\u003e \u003cp\u003eBuilding on the aforementioned mechanistic insights, we further conducted confirmatory experiments targeting the TCA cycle, employing Idebenone, a well-characterized mitochondrial protector and TCA cycle optimizer, to validate the clinical translational potential of the core mechanism[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Idebenone is known to specifically enhance succinate dehydrogenase (SDH) activity, optimize TCA cycle flux, improve mitochondrial function and promote ATP production[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Its target of action highly aligns with the key regulatory nodes of the TCA cycle identified in this study; thus, we hypothesized that it could alleviate CM by targeting the TCA cycle. Experimental results showed that intraperitoneal administration of Idebenone to NTG-induced CM mice significantly increased the periorbital and plantar mechanical pain thresholds, effectively suppressed excessive activation of TNC neurons and downregulated CGRP levels. Notably, as a clinically used compound with favorable oral bioavailability, Idebenone has established safety profiles in neurological diseases such as Friedreich's ataxia and cerebral ischemia-reperfusion[\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. It provided a feasible clinical translation direction for metabolic-targeted therapy in CM and accelerating the conversion of our mechanistic discoveries into clinical applications.\u003c/p\u003e \u003cp\u003eNevertheless, it is important to note that this study has several limitations. Given that this study only employed the NTG-induced chemical CM mouse model, while this model can simulate some pain phenotypes and short-term metabolic abnormalities of idiopathic CM, it may not fully recapitulate the long-term chronic metabolic disorder characteristics observed in refractory human CM. To avoid the impact of estrogen on pain neurotransmitter transmission, male C57BL/6 mice were used to establish the CM model in this experiment, and the role of the GLUT3 in female CM models remains to be explored. Furthermore, the specific mechanisms underlying the therapeutic effects of Idebenone have not been fully elucidated, and more detailed mechanistic studies are required in the future.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, our research has underscored the pivotal role of neuron-specific GLUT3 in mediating metabolic dysfunction and subsequent neuroinflammation in CM. Clinically, CM patients exhibit systemic metabolic imbalance. In NTG-induced CM mice, TNC neurons display decreased glucose uptake and specific downregulation of GLUT3, whereas GLUT3 overexpression alleviates CM-related phenotypes by enhancing TCA cycle function, suppressing the expression of c-Fos and CGRP, and inhibiting NF-κB pathway activation. Notably, these protective effects are fully reversed by the TCA cycle inhibitor 3-NPA, confirming that the TCA cycle acts as an indispensable downstream mediator of GLUT3-dependent regulation. Additionally, Idebenone, a clinically approved agent and TCA cycle modulator, also alleviates CM-related phenotypes. Collectively, these findings indicate that targeting GLUT3 or its downstream TCA cycle could be a promising therapeutic strategy for CM. This discovery not only deepens our understanding of the molecular link between neuronal glucose metabolism and neuroinflammation in CM but also paves the way for developing novel metabolism-targeted treatments.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAAV Adeno-associated virus; CM Chronic migraine; NTG Nitroglycerin; CGRP Calcitonin gene-related peptide; TNC Trigeminal nucleus caudalis; GLUT3 Glucose transporter 3; RAPA Rapamycin; WB Western blot; IF Immunofluorescence; 3-NPA 3-nitropropionic acid; 2-NBDG 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxy-D-glucose; TCA tricarboxylic acid; PPP pentose phosphate pathway; OXPHOS oxidative phosphorylation; GO Gene Ontology; KEGG Kyoto Encyclopedia of Genes and Genomes; 18F-FDG-PET/CT 2-deoxy-2-[18F]fluoro-D-glucose positron emission tomography/computed tomography; DEGs differentially expressed genes; TNF-α tumor necrosis factor-α; NF-κB nuclear factor kappa B; p-NF-κB Phosphorylated Nuclear Factor Kappa B; IκBα Inhibitor of nuclear factor kappa B alpha; p-IκBα phosphorylated Inhibitor of nuclear factor kappa B alpha; ATP Adenosine triphosphate; ADP Adenosine diphosphate; AMP Adenosine monophosphate; ROS reactive oxygen species; NADPH Nicotinamide adenine dinucleotide phosphate hydrogen; NAD⁺/NADH Nicotinamide adenine dinucleotide (oxidized/reduced); FADH₂ Flavin adenine dinucleotide hydride.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eEthical Approval and Consent to participate\u003c/h2\u003e \u003cp\u003e Ethical approval for this study was obtained from the First Affiliated Hospital of Xiamen University (Approval Number: [2020] Scientific Research Ethics Review No. (060)]; Approval Date: January 28, 2021]). All procedures were performed in accordance with the principles of the Declaration of Helsinki (2024 revised version) and relevant national ethical regulations.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eInformed consent\u003c/strong\u003e \u003cp\u003ewas obtained from all individual participants prior to their involvement in the study, using the informed consent form (Version No.: 2.0; Version Date: January 25, 2021) approved by the ethics committee. All participants were fully informed of the study purpose, procedures, potential risks, benefits, and the right to withdraw at any time without prejudice to their medical care.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work is financially supported by the National Natural Science Foundation of China (32300823 and 625B2156), China Postdoctoral Science Foundation (2024M761265), Jiangxi Provincial Natural Science Foundation (20252BAC200488), Integration of Medicine and Engineering Project of Xiamen (3502Z20244ZD2003), Neurological Diseases and Nutritional Health Project of National Health Commission Capacity Building and Continuing Education Center (W2024SNKT43).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eJ.Y.Z., Z.J.G., X.R., and X.Y.L. conceived and designed the experiments. K.Y.L., W.X.W., H.P.Y., Y.H.W., and Z.L.X. collected clinical information and samples. T.T.C., T.H.F., Y.H.X., T.X.W., and X.D.L. performed the experiments. C.P., K.W., and J.J.Z. analyzed the data. J.F., Q.L.M., and Y.G.W. supervised data collection. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe acknowledge the financial support by the National Natural Science Foundation of China, China Postdoctoral Science Foundation, Jiangxi Provincial Natural Science Foundation, Integration of Medicine and Engineering Project of Xiamen, Neurological Diseases and Nutritional Health Project of National Health Commission Capacity Building and Continuing Education Center.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe data will be made available upon reasonable request to the corresponding authors.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHeadache Classification Committee of the International Headache Society (IHS) The International Classification of Headache Disorders, 3rd edition, Cephalalgia Int. J. Headache, vol. 38, no. 1, pp. 1\u0026ndash;211. 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Dec. 2025;168:109356. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.bioorg.2025.109356\u003c/span\u003e\u003cspan address=\"10.1016/j.bioorg.2025.109356\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-8411532/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8411532/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eChronic migraine (CM) is closely linked to systemic metabolic disorders and neuroinflammation, but the molecular mechanisms connecting energy metabolism dysfunction to pain-related inflammation remain unclear.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003ePlasma samples from CM patients were analyzed using transcriptomics and metabolomics. A CM mouse model was induced by administration of nitroglycerin (NTG). Behavioral evaluations were conducted using von Frey filaments and hot plate tests. Western blotting, immunofluorescence, transcriptome, targeted metabolomics techniques were employed to investigate the molecular mechanisms. \u003csup\u003e18\u003c/sup\u003eF-FDG-PET/CT and 2-NBDG staining were employed to assess glucose uptake. Neuron-specific GLUT3 overexpression and intervention with 20 mg/kg 3-nitropropionic acid (3-NPA), a tricarboxylic acid (TCA) cycle inhibitor, were performed to verify the regulatory loop\u0026rsquo;s specificity and clinical therapeutic potential, as well as 100 mg/kg Idebenone.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003ePatients with CM exhibited energy metabolism abnormalities including dysregulated oxidative phosphorylation pathway-related genes and reduced plasma levels of TCA cycle-related metabolites. NTG-induced mice exhibited decreased glucose uptake in the trigeminal nucleus caudalis (TNC) and neuron-specific GLUT3 downregulation, rather than glial cells. Neuron-specific GLUT3 overexpression enhanced TCA cycle function, increased mechanical pain thresholds, and inhibited central sensitization by downregulating the expression of c-Fos and calcitonin gene-related peptide (CGRP), accompanied by an inhibition of NF-κB pathway. Furthermore, 3-NPA reversed the inhibitory effect of neuron-specific GLUT3 overexpression on the NF-κB pathway. Idebenone, a clinically approved mitochondrial protective agent, alleviated CM-related mechanical allodynia and reversed the upregulation of c-Fos/CGRP in the TNC of NTG-induced mice.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThese results identify the neuronal GLUT3-TCA cycle-NF-κB axis as a key regulatory pathway in CM, bridging metabolic dysfunction and neuroinflammation. Notably, the clinically available mitochondrial protective agent Idebenone mimics these benefits via TCA cycle targeting, underscoring its application potential in the cilinic. These findings offer novel mechanistic insights and metabolic-targeted therapeutic strategies for CM.\u003c/p\u003e","manuscriptTitle":"Neuronal GLUT3 alleviates hyperalgesia and neuroinflammation in chronic migraine mice via the TCA cycle","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-08 16:50:23","doi":"10.21203/rs.3.rs-8411532/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d4fab615-0c65-4579-9a1f-2c4f3ce0e131","owner":[],"postedDate":"March 8th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-28T18:54:11+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-08 16:50:23","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8411532","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8411532","identity":"rs-8411532","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}