IGFBPL1 in DRG Nociceptors Drives Neuropathic Pain and Neuroimmune Crosstalk via IGF1R–ERK Signaling

IGFBPL1 in DRG Nociceptors Drives Neuropathic Pain and Neuroimmune Crosstalk via IGF1R–ERK Signaling | 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 Article IGFBPL1 in DRG Nociceptors Drives Neuropathic Pain and Neuroimmune Crosstalk via IGF1R–ERK Signaling Euiheon Chung, Emmanuel Acquah, An Nazmus Sakib, SangSeong Kim, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7783701/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 Neuronal hyperexcitability and neuroimmune dysregulation are central hallmarks of neuropathic pain. IGFBPL1, a secreted glycoprotein and classical member of the insulin-like growth factor binding protein (IGFBP) family, has been implicated in GABAergic circuits, neurite outgrowth, and immune modulation in the CNS; however, its functional role in the peripheral nervous system (PNS), particularly in the somatosensory system, remains unexplored. Here, we report that IGFBPL1 is increased in the dorsal root ganglion (DRG) following peripheral nerve injury, and that this increase contributes to the development and maintenance of neuropathic pain. We show that IGFBPL1 upregulation in DRG sensory neurons induces pain behaviors and neuronal hyperexcitability through IGF1R–ERK signaling and drives macrophage recruitment. Conversely, Igfbpl1-specific knockdown or pharmacological inhibition alleviates pain hypersensitivity, normalizes neuronal excitability, and reduces macrophage infiltration and neuroimmune crosstalk. Notably, Igfbpl1-specific knockdown also improved gait performance in chronic constriction injury (CCI) mice. Our findings identify IGFBPL1 as a critical regulator of DRG pathophysiology, linking growth factor signaling, sensory neuron plasticity, and neuroimmune interactions in neuropathic pain. Biological sciences/Neuroscience/Somatosensory system/Pain/Chronic pain Health sciences/Diseases/Neurological disorders/Neuropathic pain Biological sciences/Neuroscience/Neuronal physiology/Excitability Health sciences/Health care/Therapeutics/Drug therapy/Molecularly targeted therapy Biological sciences/Neuroscience/Peripheral nervous system/Somatic system Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Peripheral neuropathic pain (PNP), a significant global public health issue, arises from damage to or dysfunction of peripheral somatosensory nerves and severely impacts quality of life due to its high prevalence (6.9–10% of the population) 1 , 2 , 3 , debilitating effects, and substantial socioeconomic burden 4 , 5 , 6 . Individuals with PNP typically present with aberrant sensory phenotypes, including hyperalgesia, allodynia, and spontaneous pain 7 , 8 . Despite a broadening pharmacological armamentarium–encompassing serotonin-norepinephrine reuptake inhibitors, tricyclic antidepressants, and anticonvulsants–treatment efficacy remains limited and frequently inadequate 9 , 10 , 11 . Therefore, a deeper understanding of the molecular and cellular mechanisms that drive peripheral sensitization after nerve injury is essential for advancing our knowledge of neuropathic pain pathogenesis and for enabling the development of targeted and more effective analgesics. Peripheral nerve injury profoundly alters transcriptional programs in primary sensory neurons within the dorsal root ganglion (DRG) 12 , 13 , a critical hub for the initiation and maintenance of neuropathic pain 14 , 15 , 16 , 17 , 18 . Injury-induced changes in ion channel expression, neuronal excitability, and cytokine release are further amplified through reciprocal signaling with glia and infiltrating immune cells, ultimately leading to persistent nociceptor sensitization 19 , 20 . However, the molecular mechanisms that regulate somatosensory function in neuropathic pain remain incompletely defined. Growth factor signaling constitutes a fundamental component of the cellular response to nerve injury, regulating processes that span axonal regeneration, immune reactivity, neuronal excitability, and synaptic remodeling 21 , 22 , 23 , 24 . Among these pathways, the insulin-like growth factor (IGF) axis is well established as a regulator of neuronal survival, maturation, and activity-dependent synaptic plasticity 25 , 26 . Traditionally, IGF bioactivity is modulated by a family of high-affinity binding proteins that control its tissue distribution and receptor accessibility 27 , 28 , 29 . Notably, however, several IGF-binding proteins also execute IGF-independent functions through interactions with extracellular matrix proteins, integrins, and diverse cell-surface receptors 30 , 31 , 32 . Insulin-like growth factor binding protein-like 1 (IGFBPL1) is a secreted glycoprotein of approximately 25–30 kDa, structurally related to the canonical insulin-like growth factor-binding protein (IGFBP) superfamily, and functions as a modulatory factor in extracellular signaling. IGFBPL1 has been implicated in diverse biological processes, including tissue remodeling, immune regulation, and tumor suppression 33 , 34 . IGFBPL1 stabilizes microglia under neurodegenerative conditions, fostering anti-inflammatory and neuroprotective phenotypes 35 , and modulates lipid metabolism in peripheral macrophages via IGF1R–dependent mechanisms 36 . In the nervous system, IGFBPL1 facilitates axonal elongation through IGF1 signaling, as demonstrated in developing retinal neurons and embryonic circuits 37 , 38 , and its loss in the striatum results in synaptic deficits, neuronal degeneration, and sustained inflammation 35 , 39 . While these studies highlight IGFBPL1 as a key regulator of CNS homeostasis and neuroregeneration, its specific role in adult dorsal root ganglion (DRG) neurons and in neuropathic pain remains largely unknown. Whether IGFBPL1 is reactivated in sensory neurons or associated immune cells after peripheral nerve injury, and whether it contributes to maladaptive processes driving chronic pain, remains unresolved. Here we show that IGFBPL1 functions as a pivotal regulator at the intersection of growth factor signaling, sensory neuron excitability, and neuroinflammation in the injured DRG. Specifically, we show that IGFBPL1 upregulation after nerve injury engages the IGF1R–ERK signaling cascade, thereby inducing proinflammatory gene expression and driving neuronal hyperexcitability. Moreover, IGFBPL1 facilitates macrophage infiltration and amplifies neuroimmune crosstalk, thereby escalating inflammatory signaling within the injured DRG. Together, these processes contribute to the onset and persistence of neuropathic pain, positioning IGFBPL1 as a critical nexus integrating growth factor signaling with neuroimmune dynamics in sensory neurons. Intriguingly, CCI mice also demonstrated significant improvement in locomotor performance following IGFBPL1 inhibition. Overall, our findings define IGFBPL1 as a central mediator of maladaptive plasticity, linking sensory pain processing with motor dysfunction, and establish its potential as a therapeutic target. Materials and Methods Animals All animal care and experimental procedures were approved by the Gwangju institute of science and technology (GIST) Laboratory Animal Research Center (LARC). Adult male C57BL/6 mice (8–10 weeks old, 24–28 g) were housed with food and water ad libitum under a standard 12-hour light/dark cycle. All mice were bred at the GIST animal facility. Stereotaxic surgery and intra-DRG microinjection of siRNA, drugs and neutralizing antibodies After anesthesia with isoflurane, animals were secured to the stereotaxic instrument (RWD Model 68000). A midline incision in the lower lumbar back was made, and the lumbar articular process was exposed and removed. The DRG was injected with 5 µg in 5 µl of specific (IGFBPL1, IGF1R) or non-specific (scrambled control) siRNAs complexed with 9 µl polyethyl ethyleneimine (jetPEI, 10 mM) 10 µl Hamilton microsyringe (Model 1701N) connected to a WPI microsyringe pump. The needle remained in place for 10 min post-injection. In vivo siRNAs were modified with 2’-OME and 5’-Chol (GenePharma). The siRNA sequences used in this study are listed in Table S1 . NBI-31772 (5 mg/kg, Tocris), recombinant mouse IGFBPL1 (JB1; 10 µg per injection, Cat No. 4130-BL, R&D Systems), anti-IGFBPL1 neutralizing antibody (10 µg per injection, MAB391), anti-IGF1R neutralizing antibody (10 µg per injection), IgG isotype control, MEK inhibitor U0126 (10 µg per injection), and AKT inhibitor MK-2206 (10 µg per injection) and were administered via intra-DRG injection following the same procedure. Animals showing signs of paresis or other abnormalities were excluded from analysis. Pain models Chronic constriction injury (CCI) pain model A 1.5 cm lateral incision was made on the right hind limb after the animals were anesthetized with 1% Zoletil (intraperitoneal) The muscle was bluntly separated to expose the sciatic nerve trunk, which was then loosely ligated with three 6 − 0 silk sutures placed 1–2 mm apart proximal to the trifurcation. The sutures were gently tightened until a brisk twitch was observed in the right hind limb. Finally, the skin and muscles were sutured in layers. Sham-operated mice underwent identical procedures except without nerve ligature. Sciatic nerve crush (SNC) pain model Animals were anesthetized with Zoletil (10 mg/kg, intraperitoneal). Using fine jeweler’s forceps, the sciatic nerve at mid-thigh level was crushed for 30 seconds at two adjacent sites. The crush injury was performed only on the right side, with the contralateral side serving as control. After the procedure, layered skin closure was performed. SNC mice were placed on a warm pad for 5–10 min before being returned to their home cages. Spared nerve injury (SNI) pain model For the SNI model, the common peroneal and tibial nerves were isolated, ligated with 6 − 0 silk sutures, and then cut distal to the ligation. The sural nerve was carefully preserved. Sham-operated mice underwent the same procedure without ligation or transection of the sciatic nerve branches. Behavioral assessments C57BL/6 mice aged 8–10 weeks, weighing 20–25 g were used for the mechanical and thermal behavioral tests. All animals were acclimatized to the testing room or apparatus for at least 1 hour before behavioral assessments. Blind scoring was performed to ensure that observers were unaware of treatments. von Frey filament assay For mechanical allodynia, mice were placed in individual Plexiglas chambers on an elevated wire mesh surface and acclimatized for 1 hour. The mid-plantar surface of the left and right hind paws was stimulated with a series of von Frey filaments applied vertically (0.02 to 2.56 g, Stoelting, Wood Dale, IL). The 50% paw withdrawal threshold (PWT) was determined by Dixon’s up-down method. A positive response was defined as paw withdrawal, flinching, shaking, or licking following stimulation. Hargreaves assay For thermal hyperalgesia, nociceptive responses to thermal stimulation were measured using a Model 336 Analgesia Meter (IITC Inc. Life Science Instruments. Woodland Hills, CA). Mice were placed in a chamber with a transparent glass bottom and habituated for 1 hour. A radiant heat source was focused on the mid-plantar surface of the hind paw. Paw withdrawal latency (PWL) was recorded as the time duration between the onset of the stimulus and paw withdrawal. To prevent tissue damage, a cut-off time of 20 seconds was applied. Each test was repeated three times with 10-minute intervals for both hind paws. RNA extraction and qPCR in mouse DRGs Bilateral L4 and L5 DRGs were collected at different time points post-operative days and stored at -80°C. RNA extraction was performed using RNAesay kit following the manufacturer’s instructions and reversed-transcribed using RT Master Mix (AbmGold). Quantitative polymerase chain reaction (qPCR) was then performed using SYBR™ Green Universal Master Mix (Applied Biosystems). Reactions were carried out in a BIO-RAD CFX96 real-time PCR system. GAPDH was used as an internal control for normalization, as it has been demonstrated to be stable even after peripheral nerve injury 84 . Sequences of the primer pairs used for qPCR are shown in Table S2. Western blot Unilateral L4 and L5 DRGs were collected, pooling four DRGs per sample to obtain sufficient protein concentration. Tissues and primary neurons were homogenized in ice-cold RIPA lysis buffer containing protease, phosphatase, and RNase inhibitors. After centrifugation at 12,000 g for 15 min at 4°C, protein concentrations were measured using a BCA assay. Samples were heated at 95°C for 5 min, separated on 8–12% SDS-PAGE, and transferred onto nitrocellulose membranes. Next, membranes were blocked with 5% nonfat dried milk in TBS-T (Tris-buffered saline containing 0.1% Tween-20) for 1 hour, followed by overnight incubation at 4°C with the following primary antibodies: goat anti-IGFBPL1(1:1000, R&D Systems), mouse anti-IGF-1(1:500, R&D Systems), mouse anti-IGF-1R(1:1000, R&D Systems), rabbit anti-ERK (1:1000, Cell Signaling Technology), rabbit anti-phospho-ERK (1:1000, Cell Signaling Technology), rabbit anti-AKT (1: 1000, Cell Signaling Technology), ), rabbit anti-phospho-AKT (1: 2000, Cell Signaling Technology), mouse anti- β-actin (1:2500, Cell Signaling Technology). Secondary antibodies (horseradish peroxidase-conjugated-anti-mouse, anti-rabbit, or anti-goat) were incubated for 1 hour at room temperature after TBS-T washes. Protein bands were visualized using ECL substrate and detected with the ChemiDoc XRS System. Band intensities were quantified by densitometry using ImageJ/FIJI software (Bio-Rad). Primary DRG neuronal culture and transfection Adult mice (8 to 10 weeks old) lumbar DRGs were collected and dissociated with 100 U papain (P4762, Sigma-Aldrich) followed by collagenase II (1 mg/ml; 11179179001, Roche) and dispase II (1.2 mg/ml; 04942078001, Roche) for 1 hour at 37°C. The ganglia were then triturated in Hanks’ balanced salt solution containing 10 mM glucose and 5 mM HEPES (pH 7.35) using a fire-polished Pasteur pipette to obtain a single-cell suspension. Dissociated DRG neuronal cells were filtered through a 40-µm strainer, collected by centrifugation at 1000 g for 8 min, and resuspended in Neurobasal medium supplemented with B27 (Thermo Fisher Scientific) and l-glutamine (Thermo Fisher Scientific). Cells were then seeded on poly-l-ornithine (P4832, Sigma-Aldrich)– and laminin (23017-015, Sigma-Aldrich)–coated six-well plates in DMEM supplemented with 10% fetal bovine serum, penicillin (20 U/ml), and streptomycin (0.2 mg/ml), and maintained at 37°C in a humidified incubator with 95% O 2 and 5% CO 2 . DRG neurons were transfected with either 0.6 µg of 6-FAM–-modified siRNA or negative control siRNA at a final concentration of 100 nM using the Oligofectamine™ Transfection Reagent (12252011, Invitrogen) according to the manufacturer’s instructions. DRGs were harvested 48 hours post-transfection for calcium imaging, western blot, and immunocytochemistry. Calcium imaging siRNA-transfected or control primary DRG neurons were loaded with 1 µM Fluo-3 AM (F14201, Invitrogen, Thermo Fisher Scientific) in extracellular solution (ECS: 150 mM NaCl, 5 mM KCl, 2.5 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 10 mM D-Glucose, pH adjusted to 7.4 with NaOH) for 30 min at room temperature, followed by three washes and a 15 min de-esterification period in ECS. Cells were images using a Zeiss LSM 710 microscope with ×20 and ×40 objectives. Neurons were illuminated at 488 nm, and time-lapse images were acquired for 4 min per region of interest (ROI). Fluorescence intensity was quantified using ZEN software (Zeiss) and ImageJ. Intracellular calcium dynamics were expressed as ΔF/F₀, where F₀ denotes baseline fluorescence. Immunocytochemistry Cultured cells were washed in PBS, fixed in 4% paraformaldehyde (PFA) for 20 min, and permeabilized with 0.1% Triton X-100. After blocking in 5% donkey serum, bovine serum albumin (1 mg/ml; BSA), and 0.2% Triton X-100 in PBS for 1 hour at room temperature, cells were incubated with primary antibodies in 5% donkey serum and BSA (1 mg/ml) in PBS overnight at 4°C. Secondary antibodies (raised in donkey, 1:1000, Jackson ImmunoResearch) were applied in for 1 hour at room temperature. Coverslips were mounted with Fluoromount Aqueous Mounting Medium (F4680, Sigma-Aldrich). Sample images were acquired using an Olympus FV1000 confocal laser-scanning microscope at ×60 magnification with oil-immersion objective [Olympus UPLSAPO, NA 1.35]. Immunofluorescence Anesthetized mice were fixed via transcardial perfusion with PBS followed by 4% (w/v) paraformaldehyde (PFA, RNase-free; Bioss, C2055). DRG tissues were freshly dissected and post-fixed in 4% PFA at 4°C overnight. Tissues were cryoprotected in 30% (w/v) sucrose (Sigma-Aldrich, S8501) in PBS for 48 hours, embedded in Tissue-Tek O.C.T. compound (Sakura, 4583), and sectioned at 10 µm using a Leica cryostat (Leica CM1850). Cryo-sections were blocked in 5% donkey serum and 0.2% Triton X-100 in PBS for 1 hours at room temperature. Sections were incubated in primary antibodies (diluted in 5% donkey serum and 0.3% Triton X-100 in PBS) overnight at 4°C, followed by incubation with secondary antibodies for 1 hours at room temperature. Primary antibodies used: goat anti-IGFBPL1(1:500, R&D Systems), mouse anti-IGFBPL1(1:500, Santa Cruz Biotechnology), Guinea pig anti-TRPV1 (1:200, Alomone Labs), rabbit anti-PIEZO2 (1:200, Alomone Labs). Slides were mounted with Prolong Gold antifade reagent containing DAPI (Invitrogen, P36931) and imaged using OlyVIA research laser scanner (Olympus) and an Olympus FV1000 confocal microscope. For quantification, four random sections were selected per mouse and averaged to determine the number of positive cells. RNAscope in situ hybridization RNAscope in situ hybridization was performed using the RNAscope 2.5 HD RED Kit (ACDBio, 322350, ACDBio, CA) according to the manufacturer’s protocol. Mice were transcardially perfused with 0.1 M PBS followed by 4% paraformaldehyde containing 0.1% diethyl pyrocarbonate. DRGs were dissected, post-fixed, cryoprotected, and sectioned. Frozen tissue sections were treated with hydrogen peroxide and Protease Plus (ACDBio) for 2 min at room temperature, followed by hybridization with probes targeting Igfbpl1 (Cat No. 488851, ACDBio, CA) or Igf1r (Cat No. 417561, ACDBio, CA) for 2 h at 40°C. Signal amplification steps (Amp1–Amp6) were performed per manufacturer’s instructions, with washes between steps using RNAscope wash buffer. Next, slides were washed three times in third-grade distilled water (TDW) and processed for additional immunofluorescence. Blocking was performed in 5% donkey serum and 0.2% Triton X-100 in PBS for 1 h at room temperature, followed by overnight incubation with primary antibodies at 4°C. After three washes (10 min each) in 0.3% PBST, sections were incubated with secondary antibodies for 1 hours at room temperature. Sections were washed thrice, air dried, and mounted with ProLong Gold Antifade mounting medium (P0131, Beyotime Biotechnology, China) before imaging. Flow Cytometry CCI–si Igfbpl1– and scramble-treated treated mice were anesthetized and perfused with ice-cold PBS to remove circulating blood. Lumbar DRGs were dissected and digested with 100 U papain (P4762, Sigma-Aldrich), followed by collagenase II (1 mg/ml; 11179179001, Roche) and dispase II (1.2 mg/ml; 04942078001, Roche) for 1hr at 37°C. Triturated single-cell suspensions were filtered through a 40-µm strainer, and centrifuged at 500 g for 5 min at 4°C. Cells were blocked with anti-CD16/32 antibody for 10 min and stained with fluorophore-conjugated antibodies against CD11b, CD45, and F4/80 for 1 hour at 4°C. Following antibody staining, cells were incubated with DAPI to exclude nonviable cells. After washing, cells were resuspended in PBS containing 2% FBS, and flow cytometry data were acquired on a BD FACS Canto II flow cytometer with BD FACS Diva 8 software (BD Biosciences). Data were processed using FlowJo software (Tree Star), and CD11b⁺CD45 high F4/80⁺ cells were quantified as macrophages. Gait assay and stride analysis Gait assay and stride analysis was conducted as previously described by Tiwari et al. , 2024. In brief, the hind paws of mice were lightly coated with water-based dyes before being released at one end of a chamber measuring 45 cm (L) × 5.0 cm (W) × 12.0 cm (H) to walk freely to the other end. Paw prints were recorded on standard printing paper placed beneath the chamber. Stride length was defined as the distance between two consecutive prints from the same toe. Stride lengths from the same mouse were averaged to produce a single value for statistical analysis. All procedures and analyses were performed consistently across experimental groups. Quantification and statistical analysis Data analysis was performed using ImageJ/FIJI, OlyVia, SW software, with statistical analyses conducted in GraphPad Prism 9. Results are presented as mean ± SEM. Statistical tests, including one-way ANOVA, two-way ANOVA, or Student’s t -test, were applied to behavioral, biochemical, and morphological data depending on the experimental design and number of variables. Detailed statistical methods are provided in the figure legends. Differences were considered statistically significant at p < 0.05. RESULTS IGFBPL1 upregulation in DRG after nerve injury contributes to neuropathic pain To identify candidate mediators of neuropathic pain, we analyzed previously published RNA-seq datasets of dorsal root ganglia (DRG) after peripheral nerve injury 40, 41, 42 . Among the transcripts most strongly induced across datasets, we consistently found increased expression of Igfbpl1 , indicating a potential role in sensory neuron plasticity and pain processing. To study the role of IGFBPL1 in peripheral neuropathic pain (PNP), we established the chronic constriction injury (CCI) model of persistent peripheral neuropathic pain 43 (Fig. 1a) and examined the temporal expression pattern of IGFBPL1 in the DRG. RNAscope in situ hybridization demonstrated marked upregulation of Igfbpl1 expression in DRG tissue of CCI mice compared with Sham mice (Fig. 1b,c; Supplementary Fig. 1a–c). Following peripheral nerve injury, Igfbpl1 mRNA and IGFBPL1 protein were significantly upregulated in a time-dependent manner, peaking at day 7 and persisting through day 21, as shown by RT-qPCR and immunofluorescence (Fig. 1d–f). We observed a similar increase in Igfbpl1 mRNA and IGFBPL1 protein in the sciatic nerve crush (SNC) model, another well-described paradigm of neuropathic pain in rodents 44 (Supplementary Fig. 1d–f). Given the nerve injury-dependent up-regulation of IGFBPL1 in the DRG, we next asked whether IGFBPL1 is functionally required for pain pathogenesis. To test this, we performed knockdown using intraganglionic delivery of IGFBPL1-specific siRNA (si Igfbpl1 ) or scrambled control siRNA into the ipsilateral DRG of CCI and sham mice (Fig. 1g). The efficiency and specificity of IGFBPL1 silencing was validated in DRG tissue and primary cultured DRG neurons by immunoblotting and RT-qPCR (Fig. 1h; Supplementary Fig. 2a–c). Notably, two different siRNAs targeting either the open reading frame of the 3’ UTR of Igfbpl1 achieved 75-80% knockdown efficiency (Supplementary Fig. 2a–c). Behaviorally, Igfbpl1 knockdown significantly attenuated CCI-induced mechanical allodynia and thermal hyperalgesia (Fig. 1i,j), without affecting baseline nociceptive thresholds on the contralateral side or in sham controls (Supplementary Fig. 3a–d). Moreover, intraganglionic injection of IGFBPL1-neutralizing antibody (IGFBPL1 Ab), or administration of the IGFBP inhibitor, NIB31772, recapitulated these analgesic effects in a dose- and time-dependent manner when administered during both the early and late phases of CCI (Fig. 1k, n; Supplementary Fig. 4, a–f), indicating a sustained role of IGFBPL1 in neuropathic pain hypersensitivity. To further assess whether IGFBPL1 contributes to neuropathic pain across models, we used the spared nerve injury (SNI) paradigm (Fig. 1o). Similar to CCI, DRG-targeted si Igfbpl1 in SNI mice robustly attenuated mechanical and thermal hypersensitivity from day 3 through day 42 post-injury (Fig. 1p,q). Together, these findings demonstrate that IGFBPL1 upregulation in DRG neurons is a key driver of the onset and persistence of neuropathic pain. The cellular localization and expression of IGFBPL1 in human and mouse DRG We next examined the cellular source of IGFBPL1 in the injured DRG by performing co-immunostaining of L5 DRG and sciatic nerve sections from CCI and sham mice using antibodies against IGFBPL1 and canonical cell-type markers: NeuN (neurons), GFAP (astrocytes), and Iba1 (microglia). IGFBPL1 was strongly enriched in NeuN-positive sensory neurons, with no detectable co-localization with astrocytic or microglial markers (Supplementary Fig. 5a–c). IGFBPL1 was also markedly elevated in sciatic nerve axons (Fig. 2d,e), consistent with anterograde transport 45 . Moreover, IGFBPL1 colocalized with ATF3, a canonical marker of axonal injury (Supplementary Fig. 5c,d). To further define neuronal subtypes expressing IGFBPL1, we performed double-labeling of IGFBPL1 with subtype-specific markers. IGFBPL1 was predominantly localized to medium- to large-diameter NF200⁺ myelinated A-fiber mechanosensitive neurons and CGRP⁺ peptidergic nociceptors, with weaker expression in IB4⁺ non-peptidergic neurons, and minimal detection in GS⁺ satellite glial cells (Fig. 2a–d). We next validated these findings using published scRNA-seq datasets from mouse and human DRGs 46 . At baseline, Igfbpl1 transcript levels were broadly distributed within Calca (CGRP+) cluster subtypes but at low abundance. In contrast, Igf1r (Igfbpl1 signaling receptor) was expressed at relatively high levels across neuronal subtypes in both species, whereas Igf1 was detected at lower levels (Supplementary Fig. 6a–h). In another reanalyzed murine DRG dataset after CCI 47 , Igfbpl1 expression was concentrated in four clusters of peptidergic nociceptors (PEP1–4) and, to a lesser extent, detected in non-peptidergic (NP) and non-nociceptor neurons or glial populations–consistent with our immunostaining results (Fig. 2d,e). By comparison, Igf1 showed only modest upregulation in peptidergic neurons (PEP), NF neurons, and glia, whereas Igf1r remained broadly and highly expressed across all neuronal subtypes and glia clusters. Additionally, Trpv1 (a well-established mediator of thermal nociception) and Piezo2 (a mechanosensitive ion channel required for touch and proprioceptive) were enriched within the same pain-related subsets (Supplementary Fig. 7a–f). Together, these results (including the spatial enrichment) establish IGFBPL1 as a sensory neuron–specific, injury-inducible gene in the DRG, with preferential expression in peptidergic and myelinated nociceptors–aligning with its role in peripheral pain signaling after nerve injury. IGFBPL1 promotes TRPV1 activity, driving DRG hyperexcitability and neuropathic pain Neuronal hyperexcitability in primary sensory neurons of the dorsal root ganglion (DRG) is a hallmark of chronic neuropathic pain 48, 49, 50 . Given prior evidence that IGFBPL1 facilitates IGF-1–induced Ca²⁺ signaling required for axonal growth in retinal ganglion cells 37 , we hypothesized that IGFBPL1 may similarly promote Ca²⁺ influx in sensory neurons by acting upstream of TRPV1 to drive injury-induced excitability. To probe this, we conducted calcium imaging using primary cultured DRG neurons isolated from CCI mice (Fig. 3a). CAP (100 nM) application elicited robust Ca²⁺ responses in control DRG neurons, while Igfbpl1 knockdown markedly reduced these responses (Fig. 3b,c). Rescue with recombinant IGFBPL1 (rIGFBPL1) restored CAP-evoked Ca²⁺ influx to control levels, demonstrating that endogenous IGFBPL1 is required for TRPV1-mediated Ca²⁺ signaling (Fig. 3b,c). Furthermore, treatment with the TRPV1 antagonist capsazepine (CPZ, 10 µM) abolished CAP responses, confirming assay specificity (Fig. 3b–d). The distribution of CAP-responsive cells showed that IGFBPL1 depletion shifted the majority of neurons to an unresponsive phenotype, while rIGFBPL1 rescue restored responsiveness (Fig. 3e). Post-hoc co-immunostaining of calcium-imaged cultures confirmed co-localization of IGFBPL1 with TRPV1 and the pan-neuronal marker TUJ1, indicating that TRPV1-expressing nociceptors are the primary responsive population (Fig. 3f–g). Finally, to determine whether IGFBPL1 is sufficient to drive pain hypersensitivity in vivo, we administered recombinant IGFBPL1 (hereafter used as rIGFBPL1) via intraganglionic injection into L5 DRGs of CCI mice. Both low (0.05 µg) and high (1.0 µg) doses of rIGFBPL1 significantly lowered paw withdrawal thresholds (PWTs) compared with PBS-treated controls (Fig. 3h,j), and the normalized area under the curve (AUC) confirmed robust increases in mechanical and thermal hypersensitivity (Fig. 3i,k). These results demonstrate that IGFBPL1 potentiates TRPV1-dependent neuronal hyperexcitability and directly contributes to nociceptive behaviors in CCI mice. IGFBPL1-driven pain sensitization is mediated via IGF1R-dependent signaling Having established a functional role of IGFBPL1 in neuropathic pain, we next sought to identify the downstream effector mechanisms. Given that IGFBPL1 has been shown to modulate IGF1 bioavailability and that IGF1 receptor (IGF1R) is a key transducer of IGF1-dependent signaling 37, 51 , we hypothesized that IGFBPL1 drives neuropathic pain through IGF1R activation. Consistent with our reanalyzed public scRNA-seq dataset showing elevated Igf1r expression in mouse DRGs after CCI, quantitative PCR revealed a progressive, time-dependent increase in Igf1r mRNA in the ipsilateral DRG of CCI mice but not in sham controls (Fig. 4a). RNAscope in situ hybridization and immunostaining further demonstrated a robust increase in Igf1r mRNA and IGF1R protein levels peaking at post-injury day 14 (Fig. 4, b–e). Together, these findings indicate that IGF1R upregulation parallels IGFBPL1 induction after nerve injury, suggesting a potential IGFBPL1–IGF1R signaling axis in neuropathic pain. We next assessed the role of IGF1R in peripheral neuropathic pain by confirming its knockdown (65.5%) via intraganglionic injection of si Igf1r or scrambled control on day 7 after CCI surgery (Fig. 4f). Behavioral assays revealed that Igf1r -specific knockdown significantly and persistently alleviated mechanical allodynia (days 7–42) and thermal hyperalgesia (days 3–42) (Fig. 4g,h), demonstrating that IGF1R is required for both the development and persistence of neuropathic pain. To determine whether IGFBPL1’s pronociceptive effects are mediated by IGF1R, we administered rIGFBPL1 followed by intraganglionic injection of either PBS or the highly selective IGF1R antagonist JB1. While rIGFBPL1 + PBS significantly exacerbated mechanical allodynia and thermal hyperalgesia, rIGFBPL1 + JB1 almost completely prevented these pain-like behaviors in both acute (Fig. 4i,j) and chronic (Fig. 4k,l) phases of neuropathic pain, demonstrating that IGFBPL1 induces nociception through IGF1R activation. IGFBPL1/IGF1R signaling induces the activation of pERK in DRG sensory neurons To delineate the underlying mechanisms of IGFBPL1–IGF1R in pain processing, we examined intracellular signaling pathways downstream of IGF1R that are crucial in neuropathic pain. IGF1R activation triggers phosphorylations cascades involving p38 MAPK, ERK/MAPK, PKB/AKT, and PI3K 52, 53, 54, 55 . Immunostaining of DRG sections revealed that CCI markedly increased phosphorylated ERK (pERK), which was significantly reduced by Igfbpl1 knockdown (Fig. 5a). Notably, pAKT was unaffected by IGFBPL1 silencing (Fig. 5b) or overexpression in DRG tissue and primary DRG neurons (Fig. 5b; Supplementary Fig. 9a,b). Both pERK and pAKT colocalized with TUJ1 (Fig 5c,d). Consistent with these findings, intraganglionic injection of rIGFBPL1 elevated pERK without altering pAKT (Fig 5a,b), indicating selective engagement of the ERK pathway. Pharmacological inhibition with MEK inhibitor U0126 or the AKT inhibitor MK-2206 abolished IGFBPL1-induced upregulation of pERK and AKT, respectively (Fig 5a,b). Administration of U0126 markedly reduced pERK in primary cultured DRG neurons (Fig. 5e,f). Intraganglionic injection of U0126 also reversed IGFBPL1-induced neuropathic pain behaviors in vivo (Fig. 5g,h). In contrast, MK-2206, failed to reduce pAKT levels (Fig. 5f) following IGFBPL1 overexpression and had no effect on rIGFBPL1-driven responses (Fig. 5i,j). These results indicate that IGFBPL1 drives neuropathic pain via an ERK-dependent but AKT-independent pathway. Furthermore, CCI mice treated with JB1 exhibited reduced rIGFBPL1-induced pERK but not AKT compared with PBS-treatment mice (Fig. 5k,l,m), demonstrating that IGFBPL1-driven ERK activation requires upstream IGF1R. Together, these findings identify ERK as a selective and essential downstream effector of IGFBPL1–IGF1R signaling in sensory neurons. IGFBPL1 inhibition reduces peripheral inflammation and macrophage recruitment in DRG Peripheral sensory neurons and immune cells engage in bidirectional communication that shapes neuropathic pain 56, 57, 58, 59 . Given that neuronal hyperactivity and ERK activation can induce cytokine and chemokine release, we investigated whether IGFBPL1-mediated nociceptor sensitization influences macrophage recruitment in the injured DRG. To this end, DRG neurons were acutely isolated 7 days after CCI following intraganglionic injection of either si Igfbpl1 or scrambled control. IGFBPL1 knockdown markedly reduced F4/80⁺ macrophages and CD11b⁺/CD68⁺ populations in the DRG (Fig. 6a,b). We also observed reduced macrophage infiltration in the sciatic nerve (Fig. 6c) and fewer neuron–macrophage contacts (Fig. 6d). Correspondingly, the expression levels of proinflammatory genes ( Tnf-α , Il-1β , Il-6 , and Ccr2 ) were significantly suppressed (Fig. 6e). To determine whether macrophages are necessary for IGFBPL1-driven pain, mice underwent clodronate-mediated macrophage depletion followed by intraganglionic injection of recombinant IGFBPL1 (rIGFBPL1) or vehicle. In macrophage-depleted mice, rIGFBPL1 failed to elicit mechanical allodynia or thermal hyperalgesia, whereas vehicle-treated controls exhibited robust pain responses (Fig. 6g–j). Consistently, calcium imaging of primary DRG neurons showed that rIGFBPL1-induced Ca²⁺ influx was markedly reduced following macrophage depletion (Fig. 6k–m). These findings demonstrate that neuronal IGFBPL1 drives persistent neuropathic pain primarily through TRPV1-dependent Ca²⁺ influx and ERK activation in DRG sensory neurons, which secondarily recruits macrophages and amplifies inflammatory signaling. Macrophage infiltration thus functions as an amplification mechanism rather than a direct target of IGFBPL1, linking neuronal sensitization to the broader neuroimmune response. IGFBPL1 downregulation improves gait performance following peripheral nerve injury After establishing the role of IGFBPL1 in nociceptor sensitization and pain hypersensitivity, we next asked whether IGFBPL1 inhibition also influences non-reflexive pain-related behaviors. To this end, we evaluated locomotor function by performing gait analysis in CCI and sham mice treated with intraganglionic injections of si Igfbpl1 or scrambled control into the ipsilateral L5 DRG from days 0 to 4 post-surgery. On day 7, following CCI or sham surgery, we analyzed the footprint patterns of the mice 60 (Figure 7a,b). Marked improvements in gait parameters were observed in si Igfbpl1 -treated mice. Specifically, the stride length of the CCI and CCI + Scrambled groups was significantly shorter compared with the sham and CCI + si Igfbpl1 groups (Figure 7c). Similarly, CCI and CCI + Scramble mice exhibited increased stride width (Figure 7d) and decreased paw rotation (Figure 7e), whereas no such abnormalities were detected in the CCI + si Igfbpl1 group. These findings indicate that IGFBPL1 downregulation substantially improves gait dynamics in CCI-induced neuropathic pain. Taken together, our results demonstrate that IGFBPL1 downregulation in peripheral sensory neurons alleviates both reflexive hypersensitivity and locomotor abnormalities following nerve injury. DISCUSSION The dorsal root ganglion (DRG) serves as a critical hub for transmitting peripheral injury signals to spinal and supraspinal circuits, generating both the sensory-discriminative and affective components of pain 19 , 61 , 62 , 63 . Increasing evidence highlights growth factors and neurotrophins as key modulators of chronic neuropathic pain and of ion channel activity in DRG neurons 21 , 49 , 52 , 53 , 55 , 64 , 65 , 66 . IGFBPL1, a secreted member of the IGFBP family, is linked to neurodevelopmental and regenerative roles including axon growth and survival, with recent work implicating it in GABAergic circuitry, neurite outgrowth, and immune modulation in the CNS. However, its functional role in peripheral sensory neurons and in neuropathic pain remained undefined. Here, using complementary genetic, pharmacological, and behavioral approaches, we demonstrate that IGFBPL1 is selectively upregulated in injured DRG neurons and drives persistent pain by coupling IGF1R–ERK signaling to TRPV1-dependent hyperexcitability and local neuroimmune activation. To our knowledge, this is the first study to show both the expression and functional role of IGFBPL1 in the peripheral nervous system and its contribution to neuropathic pain. Nociceptive sensory neurons, particularly peptidergic C-fibers and subsets of myelinated A-fiber nociceptors, are well-established drivers of pain hypersensitivity during tissue injury, inflammation, and neuropathy 67 , 68 , 69 , 70 . Reanalyzed public scRNA-seq datasets from human and mouse DRGs, together with histological validation, revealed that IGFBPL1 expression is largely restricted to CGRP⁺ peptidergic C-fibers and subsets of NF200⁺ myelinated A-fiber nociceptors, with moderate expression in IB4⁺ non-peptidergic C-fibers. This distribution places IGFBPL1 within pain-relevant neuronal subsets 67 , 70 , 71 and suggests a role in the molecular reprogramming of nociceptors that underlies neuropathic pain 72 , 73 . Neuropathic pain is characterized by the hyperexcitability of injured sensory neurons, which underlies mechanical and thermal hypersensitivity 13 , 19 , 24 , 74 , 75 , 76 . Although IGFBPL1 has previously been shown to enable IGF1-dependent calcium signaling and downstream events important for axonal growth 35 , its contribution to afferent hyperexcitability was unexplored. In our functional assays, we found that recombinant IGFBPL1 potentiated TRPV1-mediated calcium influx in sensory neurons and elicited sustained mechanical and thermal hypersensitivity in vivo, whereas DRG-targeted knockdown of Igfbpl1 attenuated nociceptive behaviors, confirming a causal role in nociceptor sensitization. Together, these findings extend the role of IGFBPL1 in ion channel regulation and establish it as both necessary and sufficient for sustaining nociceptor sensitization in neuropathic pain. At the cellular level, IGFBPL1 engaged IGF1R with preferential activation of ERK signaling, while AKT remained largely unaffected, consistent with prior reports that IGF1R can selectively couple to downstream pathways depending on ligand context, receptor density, and cofactor availability 64 , 77 , 78 . Additionally, IGF1R activation has been linked to both AKT and ERK cascades in inflammatory and nerve injury–induced pain 79 , 80 . Pharmacological inhibition of ERK with U0126 abolished IGFBPL1-driven hypersensitivity, whereas AKT inhibition (MK-2206) had no effect, further supporting selective engagement of the ERK pathway in pain-relevant signaling 81 . These findings indicate that IGFBPL1 functions as a context-dependent amplifier of IGF1R–ERK signaling in sensory neurons, selectively enhancing excitability-related pathways without broadly activating trophic signaling. Beyond neuronal excitability, IGFBPL1 orchestrates neuroimmune interactions critical for pain maintenance. IGFBPL1 knockdown suppressed expression of pro-inflammatory cytokines and chemokines, including Tnf-a , Il-1β , Il-6 , and Ccr2 , in DRG tissues and cultured neurons, while limiting macrophage infiltration into DRG and sciatic nerves. Macrophage depletion abolished IGFBPL1-induced hypersensitivity and attenuated intracellular calcium responses, indicating that immune cell recruitment acts as an amplification mechanism rather than a direct target of IGFBPL1. These findings highlight how neuronal sensitization engages the broader neuroimmune environment to sustain chronic pain, consistent with growing evidence of neuron–immune crosstalk as a critical driver of neuropathic pain 56 , 81 , 82 , 83 . Whether these cytokine changes reflect a consequence of altered excitability or represent an independent regulatory effect of IGFBPL1 requires further study. Finally, our results show that IGFBPL1 contributes not only to pain hypersensitivity but also to gait abnormalities following nerve injury, indicating broader circuit-level effects 37 , 45 . Dysfunction in DRG sensory neurons can propagate through spinal and supraspinal networks, disrupting sensorimotor integration 61 , 62 , 63 . IGFBPL1 knockdown improved gait parameters, suggesting that the IGFBPL1–IGF1R–ERK axis may modulate both peripheral and central processing of nociceptive signals. These observations are consistent with prior work showing that perturbation of IGF1 signaling can affect locomotion (Santucci et al., 2005), supporting the idea that IGFBPL1 influence on non-reflexive behavior may be mediated via IGF1R-dependent pathways. Whether these effects arise from retrograde signaling to central circuits or from local DRG-to-spinal interactions remains to be determined. Our study has several limitations. First, while knockdown allowed selective reduction of IGFBPL1 in adult DRG neurons, conditional knockout models are needed to dissect its roles across developmental stages and non-neuronal cell types, as well as in other neuropathy models, including diabetic and chemotherapy-induced forms. Second, all experiments were conducted in male mice to reduce variability and focus on mechanisms; whether IGFBPL1 functions similarly in females remains to be determined, given well documented sex-specific differences in neuropathic pain. Third, IGFBPL1 may modulate nociceptor excitability and macrophage recruitment through autocrine or paracrine mechanisms, and the relative contributions of these pathways in vivo warrants further investigation. Taken together, our findings identify IGFBPL1 as a previously unrecognized neuronal regulator of peripheral neuropathic pain via selective activation of IGF1R–ERK signaling, linking nociceptor hyperexcitability with neuron-macrophage crosstalk, and highlight it as a promising therapeutic target for peripheral neuropathic pain. Declarations Acknowledgments We thank Professors Chul-Kyu Park at Gachon University College of Medicine and Hyunsoo Shawn. JE at Duke-NUS Graduate Medical School for their helpful feedback on the manuscript. We also appreciate Professor Seog Bae Oh at Department of Neurobiology and Physiology, School of Dentistry, and Dental Research Institute, Seoul National University for his insightful comments and review during the manuscript preparation. We further thank Laxman Manandhar (GIST, Korea) for assistance with western blot experiments. Funding: This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (RS-2023-00264409, RS-2023-00302281, RS-2025-00522868, RS-2025-00573499). Authors and Affiliations Department of Biomedical Science and Engineering, Institute of Integrated Technology (GIST), Gwangju 61005, Republic of Korea Emmanuel Acquah, An Nazmus Sakib, Sang Seong Kim, Hyuk Sang Kwon & Euiheon Chung Athinoula A. Martinos Center for Biomedical Imaging, Massachusetts General Hospital, Charlestown, Massachusetts United States Young Ro Kim Department of Radiology, Harvard Medical School, Boston, Massachusetts, United States Young Ro Kim AI Graduate School, Gwangju Institute of Science and Technology (GIST), Republic of Korea Euiheon Chung Contributions: Conceptualization, E.A., and E.C.; methodology, E.A.; investigation, E.A., A.N.S., and E.C.; visualization, E.A., A.N.S., S.S.K, H.S.K, Y.R.K., and E.C.; funding acquisition, E.C.; supervision, E.C.; writing—original draft, E.A.; writing—review and editing, E.A., S.S.K, and E.C. Corresponding authors Correspondence to Euiheon Chung. Competing interests: The authors declare no competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. References Van Hecke O, Austin SK, Khan RA, Smith BH, Torrance N. Neuropathic pain in the general population: a systematic review of epidemiological studies. PAIN® 155 , 654-662 (2014). Smith BH, Torrance N, Bennett MI, Lee AJ. Health and quality of life associated with chronic pain of predominantly neuropathic origin in the community. The Clinical journal of pain 23 , 143-149 (2007). Colloca L , et al. Neuropathic pain. Nature reviews Disease primers 3 , 1-19 (2017). Breivik H, Collett B, Ventafridda V, Cohen R, Gallacher D. Survey of chronic pain in Europe: prevalence, impact on daily life, and treatment. European journal of pain 10 , 287-333 (2006). Bouhassira D, Lantéri-Minet M, Attal N, Laurent B, Touboul C. Prevalence of chronic pain with neuropathic characteristics in the general population. Pain 136 , 380-387 (2008). Gustorff B , et al. Prevalence of self‐reported neuropathic pain and impact on quality of life: a prospective representative survey. Acta Anaesthesiologica Scandinavica 52 , 132-136 (2008). Basbaum AI, Bautista DM, Scherrer G, Julius D. Cellular and molecular mechanisms of pain. Cell 139 , 267-284 (2009). Finnerup NB, Kuner R, Jensen TS. Neuropathic pain: from mechanisms to treatment. Physiological reviews , (2020). Sindrup SH, Otto M, Finnerup NB, Jensen TS. Antidepressants in the treatment of neuropathic pain. Basic & clinical pharmacology & toxicology 96 , 399-409 (2005). Jang HN, Oh TJ. Pharmacological and nonpharmacological treatments for painful diabetic peripheral neuropathy. Diabetes & metabolism journal 47 , 743-756 (2023). Soliman N , et al. Pharmacotherapy and non-invasive neuromodulation for neuropathic pain: a systematic review and meta-analysis. The Lancet Neurology 24 , 413-428 (2025). Yuan J , et al. Contribution of dorsal root ganglion octamer transcription factor 1 to neuropathic pain after peripheral nerve injury. Pain 160 , 375-384 (2019). Costigan M, Scholz J, Woolf CJ. Neuropathic pain: a maladaptive response of the nervous system to damage. Annual review of neuroscience 32 , 1-32 (2009). Li Z , et al. Dorsal root ganglion myeloid zinc finger protein 1 contributes to neuropathic pain after peripheral nerve trauma. Pain 156 , 711-721 (2015). Yu X , et al. Dorsal root ganglion macrophages contribute to both the initiation and persistence of neuropathic pain. Nature communications 11 , 264 (2020). Maruyama M , et al. Neat1 lncRNA organizes the inflammatory gene expressions in the dorsal root ganglion in neuropathic pain caused by nerve injury. Frontiers in Immunology 14 , 1185322 (2023). Hayward R , et al. Transcriptional reprogramming post-peripheral nerve injury: A systematic review. Neurobiology of Disease 200 , 106624 (2024). Denk F, McMahon SB, Tracey I. Pain vulnerability: a neurobiological perspective. Nature neuroscience 17 , 192-200 (2014). Jang K, Garraway SM. A review of dorsal root ganglia and primary sensory neuron plasticity mediating inflammatory and chronic neuropathic pain. Neurobiology of Pain 15 , 100151 (2024). Liu J, Chen Y, Chen G. The Role and Mechanisms of Macrophages in Chronic Pain: A Peripheral-to-Central Perspective. Brain Research Bulletin , 111470 (2025). Turner CA, Eren-Koçak E, Inui EG, Watson SJ, Akil H. Dysregulated fibroblast growth factor (FGF) signaling in neurological and psychiatric disorders. In: Seminars in cell & developmental biology ). Elsevier (2016). Harrington AW, Ginty DD. Long-distance retrograde neurotrophic factor signalling in neurons. Nature Reviews Neuroscience 14 , 177-187 (2013). Li R, Li D-h, Zhang H-y, Wang J, Li X-k, Xiao J. Growth factors-based therapeutic strategies and their underlying signaling mechanisms for peripheral nerve regeneration. Acta Pharmacologica Sinica 41 , 1289-1300 (2020). Wang H , et al. Brain-derived neurotrophic factor stimulation of T-type Ca2+ channels in sensory neurons contributes to increased peripheral pain sensitivity. Science signaling 12 , eaaw2300 (2019). Liu Y , et al. IGF-1 signaling pathway activation promotes axonal regeneration and repair: A mechanism study on catalpol-induced functional recovery after ischemic stroke. Journal of Ethnopharmacology 348 , 119808 (2025). Tu X, Jain A, Parra Bueno P, Decker H, Liu X, Yasuda R. Local autocrine plasticity signaling in single dendritic spines by insulin-like growth factors. Science Advances 9 , eadg0666 (2023). Cai Z, Chen H, Boyle B, Rupp F, Funk W, Dedera D. Identification of a novel insulin-like growth factor binding protein gene homologue with tumor suppressor like properties. Biochemical and biophysical research communications 331 , 261-266 (2005). Kashyap MK. Role of insulin-like growth factor-binding proteins in the pathophysiology and tumorigenesis of gastroesophageal cancers. Tumor Biology 36 , 8247-8257 (2015). Firth SM, Baxter RC. Cellular actions of the insulin-like growth factor binding proteins. Endocrine reviews 23 , 824-854 (2002). Baxter RC. Signaling pathways of the insulin-like growth factor binding proteins. Endocrine reviews 44 , 753-778 (2023). Oh Y, Müller HL, Lamson G, Rosenfeld RG. Insulin-like growth factor (IGF)-independent action of IGF-binding protein-3 in Hs578T human breast cancer cells. Cell surface binding and growth inhibition. Journal of Biological Chemistry 268 , 14964-14971 (1993). Lewitt M, Boyd G. The role of Insulin-like Growth Factors (IGFs) and IGF-binding proteins in the nervous system. Biochemistry Insights 12 , (2019). Li Y, Zhao E, Chen DF. Emerging roles for insulin-like growth factor binding protein like protein 1. Neural Regeneration Research 14 , 258-259 (2019). Smith P , et al. Epigenetic inactivation implies independent functions for insulin-like growth factor binding protein (IGFBP)-related protein 1 and the related IGFBPL1 in inhibiting breast cancer phenotypes. Clinical cancer research 13 , 4061-4068 (2007). Pan L , et al. IGFBPL1 is a master driver of microglia homeostasis and resolution of neuroinflammation in glaucoma and brain tauopathy. Cell reports 42 , (2023). Hou L , et al. IGFBPL1 inhibits macrophage lipid accumulation by enhancing the activation of IGR1R/LXRα/ABCG1 pathway. Aging (Albany NY) 15 , 14791 (2023). Guo C , et al. IGFBPL1 regulates axon growth through IGF-1-mediated signaling cascades. Scientific reports 8 , 2054 (2018). Gonda Y, Sakurai H, Hirata Y, Tabata H, Ajioka I, Nakajima K. Expression profiles of Insulin-like growth factor binding protein-like 1 in the developing mouse forebrain. Gene expression patterns 7 , 431-440 (2007). Butti E , et al. Neural precursor cells tune striatal connectivity through the release of IGFBPL1. Nature communications 13 , 7579 (2022). Qu Y, Cai R, Li Q, Wang H, Lu L. Neuroinflammation signatures in dorsal root ganglia following chronic constriction injury. Heliyon 10 , (2024). Stephens KE , et al. Sex differences in gene regulation in the dorsal root ganglion after nerve injury. BMC genomics 20 , 147 (2019). Cobos EJ , et al. Mechanistic differences in neuropathic pain modalities revealed by correlating behavior with global expression profiling. Cell reports 22 , 1301-1312 (2018). Bennett GJ, Xie Y-K. A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man. Pain 33 , 87-107 (1988). Kim KJ, Chung JM. Comparison of three rodent neuropathic pain models. Experimental brain research 113 , 200-206 (1997). Chen G , et al. PD-L1 inhibits acute and chronic pain by suppressing nociceptive neuron activity via PD-1. Nature neuroscience 20 , 917-926 (2017). Bhuiyan SA , et al. Harmonized cross-species cell atlases of trigeminal and dorsal root ganglia. Science Advances 10 , eadj9173 (2024). Zhang C , et al. scRNA-sequencing reveals subtype-specific transcriptomic perturbations in DRG neurons of PirtEGFPf mice in neuropathic pain condition. Elife 11 , e76063 (2022). Li L , et al. Peripheral CCL2 induces inflammatory pain via regulation of I h currents in small diameter DRG neurons. Frontiers in Molecular Neuroscience 16 , 1144614 (2023). Deng Y-T , et al. Amphiregulin contributes to neuropathic pain by enhancing glycolysis that stimulates histone lactylation in sensory neurons. Science Signaling 18 , eadr9397 (2025). Hu Q , et al. TRPV1 channel contributes to the behavioral hypersensitivity in a rat model of complex regional pain syndrome type 1. Frontiers in Pharmacology 10 , 453 (2019). Bleach R, Sherlock M, O’Reilly MW, McIlroy M. Growth hormone/insulin growth factor axis in sex steroid associated disorders and related cancers. Frontiers in cell and developmental biology 9 , 630503 (2021). Miura M , et al. Peripheral sensitization caused by insulin-like growth factor 1 contributes to pain hypersensitivity after tissue injury. PAIN® 152 , 888-895 (2011). Zhang Y , et al. Peripheral pain is enhanced by insulin-like growth factor 1 through a G protein–mediated stimulation of T-type calcium channels. Science signaling 7 , ra94-ra94 (2014). Dai W , et al. Calcium deficiency-induced and TRP channel-regulated IGF1R-PI3K-Akt signaling regulates abnormal epithelial cell proliferation. Cell Death & Differentiation 21 , 568-581 (2014). Ma L , et al. IGF/IGF-1R signal pathway in pain: a promising therapeutic target. International Journal of Biological Sciences 19 , 3472 (2023). Tauber M, Wang F, Kim B, Gaudenzio N. Bidirectional sensory neuron–immune interactions: a new vision in the understanding of allergic inflammation. Current Opinion in Immunology 72 , 79-86 (2021). Fattori V , et al. Nociceptor-to-macrophage communication through CGRP/RAMP1 signaling drives endometriosis-associated pain and lesion growth in mice. Science Translational Medicine 16 , eadk8230 (2024). Kim BS, Artis D. The sensory neuroimmune frontier. Immunity 58 , 1033-1039 (2025). Laumet G. Peripheral somatosensory neurons listen and orchestrate the immune response. Journal of Allergy and Clinical Immunology 153 , 977-979 (2024). Tiwari N, Smith C, Sharma D, Shen S, Mehta P, Qiao LY. Plp1-expresssing perineuronal DRG cells facilitate colonic and somatic chronic mechanical pain involving Piezo2 upregulation in DRG neurons. Cell reports 43 , (2024). Rustioni S , et al. A comprehensive review of the supraspinal mechanisms of spinal cord stimulation on chronic pain and cognition. Frontiers in Pain Research 6 , 1589723 (2025). Abd-Elsayed A, Vardhan S, Aggarwal A, Vardhan M, Diwan SA. Mechanisms of action of dorsal root ganglion stimulation. International journal of molecular sciences 25 , 3591 (2024). Stachowski NJ, Dougherty KJ. Spinal inhibitory interneurons: gatekeepers of sensorimotor pathways. International journal of molecular sciences 22 , 2667 (2021). Girnita L, Worrall C, Takahashi S-I, Seregard S, Girnita A. Something old, something new and something borrowed: emerging paradigm of insulin-like growth factor type 1 receptor (IGF-1R) signaling regulation. Cellular and molecular life sciences 71 , 2403-2427 (2014). Thakkar B, Acevedo EO. BDNF as a biomarker for neuropathic pain: Consideration of mechanisms of action and associated measurement challenges. Brain and Behavior 13 , e2903 (2023). Xiong H-Y , et al. The role of the brain-derived neurotrophic factor in chronic pain: links to central sensitization and neuroinflammation. Biomolecules 14 , 71 (2024). Schou WS, Ashina S, Amin FM, Goadsby PJ, Ashina M. Calcitonin gene-related peptide and pain: a systematic review. The journal of headache and pain 18 , 34 (2017). Assas BM, Pennock JI, Miyan JA. Calcitonin gene-related peptide is a key neurotransmitter in the neuro-immune axis. Frontiers in neuroscience 8 , 23 (2014). Benemei S, Nicoletti P, Capone JG, Geppetti P. CGRP receptors in the control of pain and inflammation. Current opinion in pharmacology 9 , 9-14 (2009). Wattiez A-S, Sowers LP, Russo AF. Calcitonin gene-related peptide (CGRP): role in migraine pathophysiology and therapeutic targeting. Expert opinion on therapeutic targets 24 , 91-100 (2020). Middleton SJ , et al. Studying human nociceptors: from fundamentals to clinic. Brain 144 , 1312-1335 (2021). Ghitani N , et al. Specialized mechanosensory nociceptors mediating rapid responses to hair pull. Neuron 95 , 944-954. e944 (2017). Wang Y , et al. TRPV1 SUMOylation regulates nociceptive signaling in models of inflammatory pain. Nature communications 9 , 1529 (2018). Fazen LE, Ringkamp M. The pathophysiology of neuropathic pain: a review of current research and hypotheses. Neurosurgery Quarterly 17 , 245-262 (2007). Yu X , et al. ACVR1-activating mutation causes neuropathic pain and sensory neuron hyperexcitability in humans. Pain 164 , 43-58 (2023). Ratte S, Prescott SA. Afferent hyperexcitability in neuropathic pain and the inconvenient truth about its degeneracy. Current opinion in neurobiology 36 , 31-37 (2016). Larsson O, Girnita A, Girnita L. Role of insulin-like growth factor 1 receptor signalling in cancer. British journal of cancer 92 , 2097-2101 (2005). Sarfstein R, Nagaraj K, LeRoith D, Werner H. Differential effects of insulin and IGF1 receptors on ERK and AKT subcellular distribution in breast cancer cells. Cells 8 , 1499 (2019). Zhou L , et al. IGF-1R promotes pain-like behavior in rats with myofascial pain syndrome via the PI3K/AKT pathway. Scientific Reports 15 , 29050 (2025). Jiang B-C , et al. Follistatin drives neuropathic pain in mice through IGF1R signaling in nociceptive neurons. Science translational medicine 16 , eadi1564 (2024). Liao Y , et al. Interleukin-6 signaling mediates cartilage degradation and pain in posttraumatic osteoarthritis in a sex-specific manner. Science signaling 15 , eabn7082 (2022). Nie H , et al. Neuronal Reg3β/macrophage TNF-α–mediated positive feedback signaling contributes to pain chronicity in a rat model of CRPS-I. Science Advances 11 , eadu4270 (2025). Zheng K , et al. Chemokine CXCL13–CXCR5 signaling in neuroinflammation and pathogenesis of chronic pain and neurological diseases. Cellular & Molecular Biology Letters 29 , 134 (2024). Zhao X , et al. A long noncoding RNA contributes to neuropathic pain by silencing Kcna2 in primary afferent neurons. Nature neuroscience 16 , 1024-1031 (2013). Additional Declarations There is no conflict of interest Supplementary Files SupplementaryInformation.pdf Supplemmentary Information 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-7783701","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":542404352,"identity":"ea32425e-f5b3-41f1-8d6a-1c5646e07899","order_by":0,"name":"Euiheon Chung","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5UlEQVRIiWNgGAWjYLACxj8ScgzMUI4EcVoabIyBWhgbSNGSltjAQKwWfv7Fxx5+3XE4fb47+/MHDDV2DJKzD+DXIjnjWbqx7JnDuRsP8xg2MBxLZpDmS8CvxeDGGTNpCTaglmYeoMPYDjDI8RBwGExLumEz+8MGhn/EaDnfYyb5sS0tQZ6ZwbCBse0AgzQhLZIz2NKkGc7YGG5g5jGckdiXzCPZQ0ALP//hY5I/KiTk5fuPP/jw4ZudnMQZAloYJBIYmEFOMTgAJBIYGAg5C2TNAQbGH0BavoGw2lEwCkbBKBihAAAMPD+PSV4Y/gAAAABJRU5ErkJggg==","orcid":"","institution":"Gwangju Institute of Science and Technology","correspondingAuthor":true,"prefix":"","firstName":"Euiheon","middleName":"","lastName":"Chung","suffix":""},{"id":542404353,"identity":"bed6a9ef-95ac-46a4-9db0-03c1e435e2a5","order_by":1,"name":"Emmanuel Acquah","email":"","orcid":"","institution":"Gwangju Institute of Science and Technology (GIST)","correspondingAuthor":false,"prefix":"","firstName":"Emmanuel","middleName":"","lastName":"Acquah","suffix":""},{"id":542404354,"identity":"aeceb43b-ce79-4ba8-904a-d563fa224081","order_by":2,"name":"An Nazmus Sakib","email":"","orcid":"https://orcid.org/0009-0004-7127-3089","institution":"Gwangju Institute of Science and Technology (GIST)","correspondingAuthor":false,"prefix":"","firstName":"An","middleName":"Nazmus","lastName":"Sakib","suffix":""},{"id":542404355,"identity":"b680a715-3f85-45c1-81c7-98a9e8c1ff0d","order_by":3,"name":"SangSeong Kim","email":"","orcid":"","institution":"Gwangju Institute of Science and Technology (GIST)","correspondingAuthor":false,"prefix":"","firstName":"SangSeong","middleName":"","lastName":"Kim","suffix":""},{"id":542404356,"identity":"a4872e3f-2c97-4e7e-b18e-3875898e1189","order_by":4,"name":"Hyuk Sang Kwon","email":"","orcid":"","institution":"Gwangju Institute of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Hyuk","middleName":"Sang","lastName":"Kwon","suffix":""},{"id":542404357,"identity":"752161e0-89fc-4616-8640-1c8ba06809c1","order_by":5,"name":"Young Ro Kim","email":"","orcid":"","institution":"Massachusetts General Hospital","correspondingAuthor":false,"prefix":"","firstName":"Young","middleName":"Ro","lastName":"Kim","suffix":""}],"badges":[],"createdAt":"2025-10-05 08:45:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7783701/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7783701/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":96367538,"identity":"d923ad65-8ecb-4e4e-9cfe-da4d8aa466a8","added_by":"auto","created_at":"2025-11-20 10:13:07","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":127180,"visible":true,"origin":"","legend":"","description":"","filename":"MainManuscript.docx","url":"https://assets-eu.researchsquare.com/files/rs-7783701/v1/a3b1a3310b644e85342224ae.docx"},{"id":96321392,"identity":"4c184160-7429-4106-b1fc-daaa17c09bf3","added_by":"auto","created_at":"2025-11-19 19:12:08","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":3444345,"visible":true,"origin":"","legend":"","description":"","filename":"MainFigure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7783701/v1/a4fa9a994faa0baf01163d33.png"},{"id":96321396,"identity":"cce2c7d5-2e32-40a6-9202-62da236ba868","added_by":"auto","created_at":"2025-11-19 19:12:08","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":4122902,"visible":true,"origin":"","legend":"","description":"","filename":"MainFigure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7783701/v1/5c45222bbab2fb694b66a65c.png"},{"id":96365974,"identity":"3e68b4d0-a374-4643-bda5-d7a0b41fecc9","added_by":"auto","created_at":"2025-11-20 10:11:00","extension":"png","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2075103,"visible":true,"origin":"","legend":"","description":"","filename":"MainFigure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7783701/v1/32fe870c5ceaabcc3d972869.png"},{"id":96365254,"identity":"29e85bc3-95e8-4935-87f8-b21e6ad22f5d","added_by":"auto","created_at":"2025-11-20 10:10:10","extension":"png","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":5609972,"visible":true,"origin":"","legend":"","description":"","filename":"MainFigure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7783701/v1/0d37e8787f46326924824b8e.png"},{"id":96365206,"identity":"a1570794-9211-4e23-a5bb-b6e3d6a6111c","added_by":"auto","created_at":"2025-11-20 10:10:06","extension":"png","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":6707308,"visible":true,"origin":"","legend":"","description":"","filename":"MainFigure5.png","url":"https://assets-eu.researchsquare.com/files/rs-7783701/v1/02c02b122ed93f2e2c394252.png"},{"id":96366136,"identity":"00a26215-3fa2-4769-9a58-ac5007d93583","added_by":"auto","created_at":"2025-11-20 10:11:14","extension":"png","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2595770,"visible":true,"origin":"","legend":"","description":"","filename":"MainFigure6.png","url":"https://assets-eu.researchsquare.com/files/rs-7783701/v1/1e307aafe53cc7d96edd192f.png"},{"id":96364930,"identity":"4297e0cb-66f7-484a-9aa7-a8c5507f22e7","added_by":"auto","created_at":"2025-11-20 10:09:48","extension":"png","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1468512,"visible":true,"origin":"","legend":"","description":"","filename":"MainFigure7.png","url":"https://assets-eu.researchsquare.com/files/rs-7783701/v1/f70b967a21187c3438e40e2d.png"},{"id":96321398,"identity":"d9456422-6936-4d9b-825e-3f714fa7cc43","added_by":"auto","created_at":"2025-11-19 19:12:08","extension":"png","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1782461,"visible":true,"origin":"","legend":"","description":"","filename":"MainFigure8.png","url":"https://assets-eu.researchsquare.com/files/rs-7783701/v1/432badeb87edeb0b19519df4.png"},{"id":96321400,"identity":"e0582763-1f57-46a4-bb09-5b387235de78","added_by":"auto","created_at":"2025-11-19 19:12:08","extension":"json","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":7826,"visible":true,"origin":"","legend":"","description":"","filename":"EMM20252472.json","url":"https://assets-eu.researchsquare.com/files/rs-7783701/v1/6f8b4bf94382843d26e9ae68.json"},{"id":96321418,"identity":"0991132f-2de4-4b85-a515-16811d54f711","added_by":"auto","created_at":"2025-11-19 19:12:09","extension":"pdf","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2003568,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7783701/v1/f48a03df4c8e3ec2d65858ad.pdf"},{"id":96321407,"identity":"f5caa09e-a454-4e86-984e-742393f3016f","added_by":"auto","created_at":"2025-11-19 19:12:08","extension":"xml","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":189202,"visible":true,"origin":"","legend":"","description":"","filename":"EMM202524720enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-7783701/v1/3824fd680967f1d077527201.xml"},{"id":96321423,"identity":"82e216d7-312d-44b7-bae8-28322ec0f5d3","added_by":"auto","created_at":"2025-11-19 19:12:09","extension":"png","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":3444345,"visible":true,"origin":"","legend":"","description":"","filename":"MainFigure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7783701/v1/e352859b98e37819417c09ec.png"},{"id":96321413,"identity":"3dd1ee9e-efc1-4bd0-8089-ebb449fc4865","added_by":"auto","created_at":"2025-11-19 19:12:09","extension":"png","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":4122902,"visible":true,"origin":"","legend":"","description":"","filename":"MainFigure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7783701/v1/d24cdd0630a895733e8084b5.png"},{"id":96366506,"identity":"83f1d85a-d8e0-4c96-aa28-de7f8ad97838","added_by":"auto","created_at":"2025-11-20 10:11:31","extension":"png","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2075103,"visible":true,"origin":"","legend":"","description":"","filename":"MainFigure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7783701/v1/fadada2286a4f29c556fd8b3.png"},{"id":96366046,"identity":"d7f0dc7d-c5cb-4814-b517-7cf95a0cdcec","added_by":"auto","created_at":"2025-11-20 10:11:05","extension":"png","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":5609972,"visible":true,"origin":"","legend":"","description":"","filename":"MainFigure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7783701/v1/f99ef295d9eb9d1a1914366a.png"},{"id":96321416,"identity":"04e88b49-8a72-4366-a296-9e4e61290785","added_by":"auto","created_at":"2025-11-19 19:12:09","extension":"png","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":6707308,"visible":true,"origin":"","legend":"","description":"","filename":"MainFigure5.png","url":"https://assets-eu.researchsquare.com/files/rs-7783701/v1/38b63ce4f66f9680022dd381.png"},{"id":96321417,"identity":"11f02e4e-b4f3-4e20-9178-667a4711e32d","added_by":"auto","created_at":"2025-11-19 19:12:09","extension":"png","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2595770,"visible":true,"origin":"","legend":"","description":"","filename":"MainFigure6.png","url":"https://assets-eu.researchsquare.com/files/rs-7783701/v1/c1aafc713c450bac12172170.png"},{"id":96321405,"identity":"5eb538ce-1738-4625-943f-1646e8fbd0bf","added_by":"auto","created_at":"2025-11-19 19:12:08","extension":"png","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1468512,"visible":true,"origin":"","legend":"","description":"","filename":"MainFigure7.png","url":"https://assets-eu.researchsquare.com/files/rs-7783701/v1/4331cce9ab499dd43d33def8.png"},{"id":96321404,"identity":"310267b7-3ffd-45ca-8835-6340861050f1","added_by":"auto","created_at":"2025-11-19 19:12:08","extension":"png","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1782461,"visible":true,"origin":"","legend":"","description":"","filename":"MainFigure8.png","url":"https://assets-eu.researchsquare.com/files/rs-7783701/v1/e834cf5313a0ef873a7a7ff6.png"},{"id":96365735,"identity":"23c61ec4-15ad-4652-8ffe-ffef55747987","added_by":"auto","created_at":"2025-11-20 10:10:44","extension":"png","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":596410,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineMainFigure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7783701/v1/05f89c9c97adfb04498cb11c.png"},{"id":96321409,"identity":"008bb81f-9a53-4420-b127-a6b32144822c","added_by":"auto","created_at":"2025-11-19 19:12:08","extension":"png","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":846324,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineMainFigure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7783701/v1/9e876a0704b26d84594c5b6c.png"},{"id":96321402,"identity":"af9bcf52-73d6-4a62-af4d-2a2aa070dc97","added_by":"auto","created_at":"2025-11-19 19:12:08","extension":"png","order_by":22,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":444426,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineMainFigure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7783701/v1/0a9ba9998560edd74416d231.png"},{"id":96365728,"identity":"58988415-1e24-4e4f-a73c-d9f09cee1517","added_by":"auto","created_at":"2025-11-20 10:10:44","extension":"png","order_by":23,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":805902,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineMainFigure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7783701/v1/55b7f1663426e8dd9ac7adb9.png"},{"id":96321415,"identity":"4c55e9f2-cead-4b6a-94dc-93decc35ac5b","added_by":"auto","created_at":"2025-11-19 19:12:09","extension":"png","order_by":24,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":764387,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineMainFigure5.png","url":"https://assets-eu.researchsquare.com/files/rs-7783701/v1/29c18f750e9a4b7837fd2869.png"},{"id":96365257,"identity":"b44136ab-0683-4bc8-9184-174c0236595f","added_by":"auto","created_at":"2025-11-20 10:10:11","extension":"png","order_by":25,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":536357,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineMainFigure6.png","url":"https://assets-eu.researchsquare.com/files/rs-7783701/v1/338f45e142fbddade5bb84d9.png"},{"id":96321412,"identity":"c694aa98-fbf3-4da0-af84-49765a1ea963","added_by":"auto","created_at":"2025-11-19 19:12:08","extension":"png","order_by":26,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":206448,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineMainFigure7.png","url":"https://assets-eu.researchsquare.com/files/rs-7783701/v1/357770de1ff3fd3d4f37200a.png"},{"id":96321425,"identity":"40150392-05cf-4cf2-8760-2126c48def2d","added_by":"auto","created_at":"2025-11-19 19:12:09","extension":"png","order_by":27,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":301496,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineMainFigure8.png","url":"https://assets-eu.researchsquare.com/files/rs-7783701/v1/b77ddd5127f9cf58d2246bdb.png"},{"id":96367096,"identity":"a17daec8-266b-473c-9a86-715ed1054243","added_by":"auto","created_at":"2025-11-20 10:12:10","extension":"xml","order_by":28,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":186894,"visible":true,"origin":"","legend":"","description":"","filename":"EMM202524720structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7783701/v1/8d2a1f34488ed36d2dc0a438.xml"},{"id":96321419,"identity":"a53f3b1b-0b6d-4bed-98b0-0a5df45dae15","added_by":"auto","created_at":"2025-11-19 19:12:09","extension":"html","order_by":29,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":207790,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7783701/v1/893646914f0778424b0eccb6.html"},{"id":96321388,"identity":"28783720-d51e-4102-a488-898ff1c06cff","added_by":"auto","created_at":"2025-11-19 19:12:08","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3444345,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIGFBPL1 upregulation in the DRG drives neuropathic pain in mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e Experimental schematic and timeline after Sham-operated and chronic constriction injury (CCI) surgery. (\u003cstrong\u003eb\u003c/strong\u003e,\u003cstrong\u003ec\u003c/strong\u003e) In situ hybridization and quantification showing increased \u003cem\u003eIgfbpl1\u003c/em\u003emRNA in the ipsilateral DRG of Sham and CCI mice 7d post-surgery.\u003cem\u003e n\u003c/em\u003e = 4 mice per group. Scale bar, 50 μm. (\u003cstrong\u003ed\u003c/strong\u003e–\u003cstrong\u003ef\u003c/strong\u003e) Time course analysis and quantification of changes in \u003cem\u003eIgfbpl1\u003c/em\u003emRNA (\u003cstrong\u003ed\u003c/strong\u003e) and IGFBPL1 protein (\u003cstrong\u003ee\u003c/strong\u003e,\u003cstrong\u003ef\u003c/strong\u003e) levels in the ipsilateral DRG of naive, sham-operated and CCI mice by qRT-PCR and immunofluorescence. \u003cem\u003en\u003c/em\u003e = 4 mice per group. (\u003cstrong\u003eg\u003c/strong\u003e,\u003cstrong\u003eh\u003c/strong\u003e) Schematic and \u003cem\u003eIgfbpl1\u003c/em\u003e mRNA level assessed by qRT-PCR after DRG-targeted injection of si\u003cem\u003eIgfbpl1\u003c/em\u003eor Scramble in CCI mice. \u003cem\u003en\u003c/em\u003e = 3 mice per group. (\u003cstrong\u003ei\u003c/strong\u003e,\u003cstrong\u003ej\u003c/strong\u003e) DRG-targeted injection of si\u003cem\u003eIgfbpl1\u003c/em\u003eprevented CCI-induced mechanical allodynia (\u003cstrong\u003ei\u003c/strong\u003e) and thermal hyperalgesia (\u003cstrong\u003ej\u003c/strong\u003e). \u003cem\u003en\u003c/em\u003e = 6 mice per group. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 versus CCI + Scramble; #\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, ##\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01 and ###\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001 versus Sham + Scramble. (\u003cstrong\u003ek\u003c/strong\u003e,\u003cstrong\u003el\u003c/strong\u003e) CCI-induced mechanical and thermal hyperalgesia in mice after intraganglionic injection of anti-IGFBPL1 or anti-IgG on post-CCI day 3. \u003cem\u003en\u003c/em\u003e = 12 per group. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 versus CCI + anti-IgG. (\u003cstrong\u003em\u003c/strong\u003e,\u003cstrong\u003en\u003c/strong\u003e) CCI-induced mechanical and thermal hyperalgesia in mice after intraganglionic injection of anti-IGFBPL1 or anti-IgG on post-CCI day 28. \u003cem\u003en\u003c/em\u003e = 12 mice per group. \u003cstrong\u003eo\u003c/strong\u003e Experimental schematic of spared nerve injury (SNI) surgery procedure. (\u003cstrong\u003ep\u003c/strong\u003e,\u003cstrong\u003eq\u003c/strong\u003e) SNI-induced mechanical (\u003cstrong\u003ep\u003c/strong\u003e) and thermal hyperalgesia (\u003cstrong\u003eq\u003c/strong\u003e) in mice after intraganglionic injection of si\u003cem\u003eIgfbpl1\u003c/em\u003eor Scramble. \u003cem\u003en\u003c/em\u003e = 12 mice per group. *\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 versus SNI + Scramble; #\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, ##\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01 and ###\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001 versus Sham + Scramble. All data are presented as mean ± SEM. Two-way repeated-measures ANOVA with post hoc Bonferroni’s test for (\u003cstrong\u003ei\u003c/strong\u003e–\u003cstrong\u003en\u003c/strong\u003e,\u003cstrong\u003ep\u003c/strong\u003e,\u003cstrong\u003eq\u003c/strong\u003e). One-way ANOVA with Dunnett’s post hoc test (\u003cstrong\u003ef\u003c/strong\u003e) and Student t test for (\u003cstrong\u003ec\u003c/strong\u003e, \u003cstrong\u003ed\u003c/strong\u003e,\u003cstrong\u003eh\u003c/strong\u003e). BL, baseline.\u003c/p\u003e","description":"","filename":"MainFigure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7783701/v1/541caa6389ca27715d2a8442.png"},{"id":96365362,"identity":"58cdc65d-bef8-4a9f-b085-d4adb069b45c","added_by":"auto","created_at":"2025-11-20 10:10:17","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":4122902,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIGFBPL1 is expressed in mouse DRG nociceptive neurons.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a\u003c/strong\u003e–\u003cstrong\u003ec\u003c/strong\u003e) Representative immunofluorescence images showing co-localization of IGFBPL1 (red) with neuronal and glial markers in DRG sections: (\u003cstrong\u003ea\u003c/strong\u003e) NF200 (myelinated A-fiber marker), (\u003cstrong\u003eb\u003c/strong\u003e) IB4 (non-peptidergic C-fiber marker), (\u003cstrong\u003ec\u003c/strong\u003e) CGRP (peptidergic C-fiber marker), and (\u003cstrong\u003ed\u003c/strong\u003e) GS (satellite glial cell marker). Scale bars, 50 μm. (\u003cstrong\u003ee\u003c/strong\u003e) Cell size distribution of IGFBPL1⁺ and NF200⁺ neurons. A total of 3417 IGFBPL1⁺ neurons from 3 mice were analyzed. (\u003cstrong\u003ef\u003c/strong\u003e) Percentage of IGFBPL1⁺ neurons co-expressing NF200, IB4, or CGRP. A total of 5673 IGFBPL1⁺ neurons from 5 mice were counted. \u003cstrong\u003eg\u003c/strong\u003e Double immunostaining of IGFBPL1 (red) and TUJ1 (green, pan-neuronal marker) in sciatic nerve sections from sham and CCI mice. Nuclei are counterstained with DAPI (blue). IGFBPL1 immunoreactivity is colocalized with TUJ1-positive axons, and CCI nerves exhibit increased thickness. Scale bars, 50 μm.\u003c/p\u003e","description":"","filename":"MainFigure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7783701/v1/c1fdecf703c47016604db55b.png"},{"id":96321387,"identity":"7fe8c989-7d2c-4b66-84bd-acb0dfe632e0","added_by":"auto","created_at":"2025-11-19 19:12:08","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2075103,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIGFBPL1 positively regulates TRPV1 activity and pain hypersensitivity.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003eSchematic of experimental design. DRG neurons were dissociated, loaded with Fluo-4 AM, and subjected to calcium imaging after treatment with capsaicin (CAP), recombinant IGFBPL1 (rIGFBPL1), or capsazepine (CPZ, 10 μM). \u003cstrong\u003eb\u003c/strong\u003e Representative calcium imaging traces before (Pre) and after (Post) CAP (1 μM) application. Neurons were treated with Scramble, si\u003cem\u003eIgfbpl1\u003c/em\u003e, si\u003cem\u003eIgfbpl1\u003c/em\u003e + rIGFBPL1 (rescue), or Scramble + CPZ. Arrows indicate CAP-responsive neurons. Scale bar, 50 μm. \u003cstrong\u003ec\u003c/strong\u003e Heat maps and representative traces of CAP-induced calcium transients (ΔF/F₀). rIGFBPL1 enhanced Ca²⁺ influx, si\u003cem\u003eIgfbpl1\u003c/em\u003ereduced responses, and si\u003cem\u003eIgfbpl1\u003c/em\u003e + rIGFBPL1 partially restored activity. CPZ abolished CAP responses. Bar graph shows distribution of CAP-responsive cells across conditions (n = 20–30 neurons from ≥ 3 mice per group). (\u003cstrong\u003ed\u003c/strong\u003e,\u003cstrong\u003ee\u003c/strong\u003e) Quantification of calcium responses. Time-course analysis and normalized area under the curve (AUC). *\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001. (\u003cstrong\u003ef\u003c/strong\u003e,\u003cstrong\u003eg\u003c/strong\u003e) Post-hoc immunofluorescence staining of DRG sections showing co-localization of IGFBPL1 (red), TUJ1 (green), and TRPV1 (magenta). Pie chart quantifies the proportion of IGFBPL1⁺/TRPV1⁺ neurons among all sensory neurons (TUJ1⁺). Scale bar, 20 μm. n = 4 mice per group. One-way ANOVA with Tukey’s post hoc test. (\u003cstrong\u003eh\u003c/strong\u003e–\u003cstrong\u003ek\u003c/strong\u003e) Gain-of-function studies. Intrathecal injection of rIGFBPL1 (0.05 or 1.0 μg) increased paw withdrawal thresholds (PWTs) in CCI mice, reversing mechanical allodynia (\u003cstrong\u003eh\u003c/strong\u003e,\u003cstrong\u003ei\u003c/strong\u003e) and thermal hyperalgesia (\u003cstrong\u003ej\u003c/strong\u003e,\u003cstrong\u003ek\u003c/strong\u003e). \u003cem\u003en\u003c/em\u003e= 10–12 mice per group. All data are presented as mean ± SEM. Two-way repeated-measures ANOVA with Bonferroni’s post hoc test for (\u003cstrong\u003eh\u003c/strong\u003e,\u003cstrong\u003ej\u003c/strong\u003e) and one-way ANOVA with Tukey’s post hoc test for (\u003cstrong\u003ei\u003c/strong\u003e,\u003cstrong\u003ek\u003c/strong\u003e). *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 versus CCI + PBS. ECS: external recording solution.\u003c/p\u003e","description":"","filename":"MainFigure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7783701/v1/076032ad233c558154303165.png"},{"id":96365595,"identity":"764c53c2-b785-43ad-833a-f084f89cb5f1","added_by":"auto","created_at":"2025-11-20 10:10:33","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":5609972,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIGF1R upregulation in the DRG is required for CCI-induced neuropathic pain.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003eTime-course analysis of \u003cem\u003eIgf1r\u003c/em\u003e mRNA expression in the ipsilateral DRG of sham and CCI mice by qRT–PCR. (\u003cstrong\u003eb\u003c/strong\u003e,\u003cstrong\u003ec\u003c/strong\u003e) RNAscope in situ hybridization showing increased \u003cem\u003eIgf1r\u003c/em\u003e mRNA expression in CCI compared to sham. Scale bar, 50 μm. (\u003cstrong\u003ed\u003c/strong\u003e,\u003cstrong\u003ee\u003c/strong\u003e) Representative immunofluorescence images and quantification of IGF1R protein expression in the DRG at different time points after CCI. Scale bar, 50 μm. \u003cem\u003en\u003c/em\u003e = 5 mice per group. (\u003cstrong\u003eF\u003c/strong\u003e)\u003cem\u003e \u003c/em\u003eValidation of \u003cem\u003eIgf1r\u003c/em\u003e knockdown in the DRG by qRT–PCR following intraganglionic injection of si\u003cem\u003eIgf1r \u003c/em\u003eor Scramble. (\u003cstrong\u003eg\u003c/strong\u003e,\u003cstrong\u003eh\u003c/strong\u003e) Behavioral assays showing attenuation of CCI-induced mechanical allodynia (\u003cstrong\u003eg\u003c/strong\u003e) and thermal hyperalgesia (\u003cstrong\u003eh\u003c/strong\u003e) after intraganglionic si\u003cem\u003eIgf1r\u003c/em\u003einjection. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 versus CCI + Scramble; ####\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.0001 versus Sham + Scramble. (\u003cstrong\u003ei\u003c/strong\u003e,\u003cstrong\u003ej\u003c/strong\u003e) CCI-induced mechanical (\u003cstrong\u003ei\u003c/strong\u003e) and thermal (\u003cstrong\u003ej\u003c/strong\u003e) hypersensitivity following intraganglionic injection of the selective IGF1R antagonist JB1 or PBS on post-CCI day 3. (\u003cstrong\u003ek\u003c/strong\u003e,\u003cstrong\u003el\u003c/strong\u003e) Effects of JB1 versus PBS on mechanical (\u003cstrong\u003ek\u003c/strong\u003e) and thermal (\u003cstrong\u003el\u003c/strong\u003e) hypersensitivity when administered on post-CCI day 21. (\u003cstrong\u003em\u003c/strong\u003e,\u003cstrong\u003en\u003c/strong\u003e) Co-immunostaining of IGF1R (green) with NeuN (\u003cstrong\u003em\u003c/strong\u003e, neuronal marker) or GS (\u003cstrong\u003en\u003c/strong\u003e, satellite glial cell marker) in DRG sections. IGF1R is enriched in NeuN-positive sensory neurons but largely absent in GS-positive satellite glial cells. Nuclei are counterstained with DAPI. Scale bar, 50 μm. All data are presented as mean ± SEM. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001. Student’s t test for (\u003cstrong\u003ea\u003c/strong\u003e,\u003cstrong\u003ec\u003c/strong\u003e,\u003cstrong\u003ef\u003c/strong\u003e). One-way ANOVA with Dunnett’s post-hoc test for (\u003cstrong\u003ee\u003c/strong\u003e) and two-way repeated-measures ANOVA followed by post hoc Bonferroni’s test for (\u003cstrong\u003eg\u003c/strong\u003e–\u003cstrong\u003el\u003c/strong\u003e). BL, baseline.\u003c/p\u003e","description":"","filename":"MainFigure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7783701/v1/8175510282f31077b3591ac4.png"},{"id":96321394,"identity":"1b624970-a42e-407c-9347-a95f625e4ded","added_by":"auto","created_at":"2025-11-19 19:12:08","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":6707308,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInvolvement of IGF1R/ERK signaling pathway in IGFBPL1-mediated pain behaviors.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e,\u003cstrong\u003eb\u003c/strong\u003e) Representative immunostaining and quantifications of pERK (\u003cstrong\u003ea\u003c/strong\u003e) and pAKT (\u003cstrong\u003eb\u003c/strong\u003e) expression in DRGs from mice with \u003cem\u003eIgfbpl1\u003c/em\u003e-specific knockdown or recombinant IGFBPL1 (rIGFBPL1) overexpression, with pERK inhibitor (U0126) or pAKT inhibitor (MK-2206) treatments. \u003cem\u003en\u003c/em\u003e= 10–18 sections from 6–8 mice per group. (\u003cstrong\u003ec\u003c/strong\u003e, \u003cstrong\u003ed\u003c/strong\u003e) Double immunofluorescence staining of pERK (\u003cstrong\u003ec\u003c/strong\u003e) or pAKT (\u003cstrong\u003ed\u003c/strong\u003e) and TUJ1 from DRG sections. \u003cem\u003en\u003c/em\u003e = 4 mice. \u003cstrong\u003ee\u003c/strong\u003e Representative immunoblot and quantification shows pERK protein expression in DRG neurons after U0126 and rIGFBPL1 injection in CCI mice. \u003cem\u003en\u003c/em\u003e = 4 mice per group. \u003cstrong\u003ef\u003c/strong\u003e Immunoblot and quantification showing pAKT protein expression in DRG neurons after MK-2206 and rIGFBPL1 injection in CCI mice. \u003cem\u003en\u003c/em\u003e = 4 mice per group. (\u003cstrong\u003eg\u003c/strong\u003e,\u003cstrong\u003eh\u003c/strong\u003e) Mechanical allodynia (\u003cstrong\u003eg\u003c/strong\u003e) and thermal hyperalgesia (\u003cstrong\u003eh\u003c/strong\u003e) in CCI mice after intraganglionic injection of U0126 and rIGFBPL1. \u003cem\u003en\u003c/em\u003e = 8 mice per group. (\u003cstrong\u003ei\u003c/strong\u003e,\u003cstrong\u003ej\u003c/strong\u003e) Mechanical allodynia (\u003cstrong\u003ei\u003c/strong\u003e) and thermal hyperalgesia (\u003cstrong\u003ej\u003c/strong\u003e) in CCI mice after intraganglionic injection of MK-2206 and rIGFBPL1. n = 8 mice per group. (\u003cstrong\u003ek\u003c/strong\u003e,\u003cstrong\u003el\u003c/strong\u003e) IHC (\u003cstrong\u003ek\u003c/strong\u003e) and western blot (\u003cstrong\u003el\u003c/strong\u003e) analyses of pERK expression in the DRG sections and cultured DRG neurons after intraganglionic injection of JB1 and rIGFBPL1. \u003cem\u003en\u003c/em\u003e = 4 or 8 mice per group. (\u003cstrong\u003em\u003c/strong\u003e,\u003cstrong\u003en\u003c/strong\u003e) CCI-induced mechanical (\u003cstrong\u003em\u003c/strong\u003e) and thermal hyperalgesia(\u003cstrong\u003en\u003c/strong\u003e) in mice after intraganglionic JB1 and rIGFBPL1 injection. \u003cem\u003en\u003c/em\u003e = 12 mice per group. All data are presented as mean ± SEM. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 and ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001. ns, non-significant. One-way ANOVA followed by Tukey’s multiple comparison test for (A and B). Student’s t test for (\u003cstrong\u003ee\u003c/strong\u003e,\u003cstrong\u003ef\u003c/strong\u003e,\u003cstrong\u003ek\u003c/strong\u003e,\u003cstrong\u003el\u003c/strong\u003e) and two-way repeated-measures ANOVA followed by post hoc Bonferroni’s test for (\u003cstrong\u003eg\u003c/strong\u003e–\u003cstrong\u003ej\u003c/strong\u003e, \u003cstrong\u003em\u003c/strong\u003e,\u003cstrong\u003en\u003c/strong\u003e). BL, baseline. Blue DAPI staining shows all cell nuclei in DRG sections.\u003c/p\u003e","description":"","filename":"MainFigure5.png","url":"https://assets-eu.researchsquare.com/files/rs-7783701/v1/4b1a21719c1b0ae704e44a4f.png"},{"id":96321397,"identity":"13ca82e0-cf9a-46e7-b34d-0c5862b07185","added_by":"auto","created_at":"2025-11-19 19:12:08","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2595770,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInhibition of IGFBPL1 or macrophage depletion reduces macrophage infiltration and inflammation, relieving persistent pain.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e Representative immunostaining and quantification of F4/80\u003csup\u003e+\u003c/sup\u003e cells in the DRG in Sham-operated or CCI mice after si\u003cem\u003eIgfbpl1 \u003c/em\u003einjection. \u003cem\u003en\u003c/em\u003e = 5 independent experiments.\u0026nbsp; \u003cstrong\u003eb\u003c/strong\u003e Flow cytometry analysis of CD11b\u003csup\u003e+\u003c/sup\u003e and CD68\u003csup\u003e+\u003c/sup\u003e cells from the DRGs of CCI mice after si\u003cem\u003eIgfbpl1 \u003c/em\u003einjection. \u003cem\u003en\u003c/em\u003e = 3 independent experiments. \u003cstrong\u003ec\u003c/strong\u003e Representative images of F4/80 cells in the sciatic nerves in Sham-operated or CCI mice after si\u003cem\u003eIgfbpl1 \u003c/em\u003einjection. \u003cem\u003en\u003c/em\u003e = 5 independent experiments. \u003cstrong\u003ed\u003c/strong\u003e Representative immunostaining of TUJ1⁺ neuronal processes (green), CD68⁺ macrophages (red), and nuclei (blue) shows the proximity of neurons and macrophages in the DRG after intraganglionic injection of si\u003cem\u003eIgfbpl1\u003c/em\u003e. \u003cem\u003en\u003c/em\u003e = 6 mice per group. (Scale bar, 100 μm). \u003cem\u003eIgfbpl1\u003c/em\u003e knockdown reduces macrophage infiltration and neuron–macrophage contacts. \u003cstrong\u003ee\u003c/strong\u003e The mRNA expression of \u003cem\u003eTnf-a\u003c/em\u003e,\u003cem\u003e Il-1b\u003c/em\u003e,\u003cem\u003e Il-6 \u003c/em\u003eand\u003cem\u003e Ccr2\u003c/em\u003e in the DRG after si\u003cem\u003eIgfbpl1\u003c/em\u003e injection in CCI mice. **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 versus CCI + Scramble. \u003cem\u003en\u003c/em\u003e = 3 mice per group. (\u003cstrong\u003ef\u003c/strong\u003e) Experimental schematic for Chlodronate (Chlod.)-mediated ablation of macrophages and timeline of behavior assays. (\u003cstrong\u003eg\u003c/strong\u003e–\u003cstrong\u003ej\u003c/strong\u003e) Macrophage ablation upon rIGFBPL1 injection attenuated mechanical allodynia (\u003cstrong\u003eg\u003c/strong\u003e,\u003cstrong\u003eh\u003c/strong\u003e) and thermal hyperalgesia (\u003cstrong\u003ei\u003c/strong\u003e,\u003cstrong\u003ej\u003c/strong\u003e) in CCI mice. \u003cem\u003en\u003c/em\u003e = 6 mice per group.\u0026nbsp; (\u003cstrong\u003ek\u003c/strong\u003e–\u003cstrong\u003em\u003c/strong\u003e) Changes in [Ca\u003csup\u003e2+\u003c/sup\u003e]\u003csub\u003ei\u003c/sub\u003e\u0026nbsp;levels and time course calcium imaging in primary cultured DRG neurons from CCI mice after rIGFBPL1 injection with or without macrophage ablation. (\u003cem\u003en\u003c/em\u003e = 20 – 44 cells from 4 cultures per group). \u003cem\u003en\u003c/em\u003e = 8 mice per group. DRG/sciatic nerve tissues were stained with DAPI. All data are presented as mean ± SEM. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 and ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001. One-way ANOVA with Bonferroni’s post hoc test for (\u003cstrong\u003ea\u003c/strong\u003e,\u003cstrong\u003ec\u003c/strong\u003e,\u003cstrong\u003ee\u003c/strong\u003e,\u003cstrong\u003eh\u003c/strong\u003e,\u003cstrong\u003ej\u003c/strong\u003e,\u003cstrong\u003em\u003c/strong\u003e) and two-way repeated-measures ANOVA with Bonferroni’s post hoc test for (\u003cstrong\u003eg\u003c/strong\u003e,\u003cstrong\u003ei\u003c/strong\u003e). Schematic diagram in (\u003cstrong\u003ef\u003c/strong\u003e) was created using BioRender (https://biorender.com). BL, baseline. AUC, area under the curve.\u003c/p\u003e","description":"","filename":"MainFigure6.png","url":"https://assets-eu.researchsquare.com/files/rs-7783701/v1/c22ceedde72fc1a5d601ad2b.png"},{"id":96365388,"identity":"fc23f9ad-f375-4810-a0f5-e0310006d946","added_by":"auto","created_at":"2025-11-20 10:10:19","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1468512,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIGFBPL1 inhibition improves gait deficits (locomotion) in CCI mice\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003eDepiction of gait assay (left) and definitions of stride length, stride width and paw rotation (right). \u003cstrong\u003eb\u003c/strong\u003eRepresentative gait assay of each experimental group. The hindpaws of individual mice were coated with light water-based dyes and the animals allowed to walk freely across a white sheet of paper lining the chamber. (\u003cstrong\u003ec\u003c/strong\u003e–\u003cstrong\u003ee\u003c/strong\u003e) CCI and CCI + Scramble groups exhibited shorter stride length (\u003cstrong\u003ec\u003c/strong\u003e) and wider stride width (\u003cstrong\u003ed\u003c/strong\u003e), and greater paw rotation (\u003cstrong\u003ee\u003c/strong\u003e) compared to Sham or CCI + si\u003cem\u003eIgfbpl1 \u003c/em\u003egroups. \u003cem\u003en\u003c/em\u003e = 6 mice per group. All data are presented as mean ± SEM. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; ns, not significant.\u003c/p\u003e","description":"","filename":"MainFigure7.png","url":"https://assets-eu.researchsquare.com/files/rs-7783701/v1/49535063634258ed5c2ecce4.png"},{"id":96365242,"identity":"4a84a32d-0aad-4122-b1c3-aef3831c8c6b","added_by":"auto","created_at":"2025-11-20 10:10:09","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1272869,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eWorking model of IGFBPL1-mediated peripheral neuropathic pain.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNerve injury induces IGFBPL1 upregulation in the DRG neurons, leading to IGF1R–ERK activation that drives neuron hyperexcitability, TRPV1 sensitization, and proinflammatory cytokine release (\u003cem\u003eTnf-α\u003c/em\u003e, \u003cem\u003eIl-6\u003c/em\u003e, \u003cem\u003eIl-1β\u003c/em\u003e, \u003cem\u003eCcr2\u003c/em\u003e). Recruited macrophages amplify inflammation and sustain nociceptor hyperexcitability, forming a self-perpetuating neuroimmune pain loop. Inhibition of IGFBPL1 (siRNA, neutralizing antibody, or IGF1R/ERK inhibitors) disrupts this loop, reduces macrophage infiltration, and restores pain thresholds.\u003c/p\u003e","description":"","filename":"MainFigure8.png","url":"https://assets-eu.researchsquare.com/files/rs-7783701/v1/24baa7bc4116db471148868e.png"},{"id":98778097,"identity":"e7ecee61-c242-40a4-92c9-35d17139d667","added_by":"auto","created_at":"2025-12-22 12:28:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":17179177,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7783701/v1/c4b687dd-86fc-49b2-b990-dbd715374fdd.pdf"},{"id":96321390,"identity":"2508eb96-62db-4a99-afd1-a879e9c859d4","added_by":"auto","created_at":"2025-11-19 19:12:08","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2003568,"visible":true,"origin":"","legend":"Supplemmentary Information","description":"","filename":"SupplementaryInformation.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7783701/v1/e9ae164ac421c8f3ecb07ffe.pdf"}],"financialInterests":"There is no conflict of interest","formattedTitle":"IGFBPL1 in DRG Nociceptors Drives Neuropathic Pain and Neuroimmune Crosstalk via IGF1R–ERK Signaling","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePeripheral neuropathic pain (PNP), a significant global public health issue, arises from damage to or dysfunction of peripheral somatosensory nerves and severely impacts quality of life due to its high prevalence (6.9\u0026ndash;10% of the population)\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e, debilitating effects, and substantial socioeconomic burden\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Individuals with PNP typically present with aberrant sensory phenotypes, including hyperalgesia, allodynia, and spontaneous pain\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Despite a broadening pharmacological armamentarium\u0026ndash;encompassing serotonin-norepinephrine reuptake inhibitors, tricyclic antidepressants, and anticonvulsants\u0026ndash;treatment efficacy remains limited and frequently inadequate\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Therefore, a deeper understanding of the molecular and cellular mechanisms that drive peripheral sensitization after nerve injury is essential for advancing our knowledge of neuropathic pain pathogenesis and for enabling the development of targeted and more effective analgesics.\u003c/p\u003e\u003cp\u003ePeripheral nerve injury profoundly alters transcriptional programs in primary sensory neurons within the dorsal root ganglion (DRG)\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e, a critical hub for the initiation and maintenance of neuropathic pain\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Injury-induced changes in ion channel expression, neuronal excitability, and cytokine release are further amplified through reciprocal signaling with glia and infiltrating immune cells, ultimately leading to persistent nociceptor sensitization\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. However, the molecular mechanisms that regulate somatosensory function in neuropathic pain remain incompletely defined.\u003c/p\u003e\u003cp\u003eGrowth factor signaling constitutes a fundamental component of the cellular response to nerve injury, regulating processes that span axonal regeneration, immune reactivity, neuronal excitability, and synaptic remodeling\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Among these pathways, the insulin-like growth factor (IGF) axis is well established as a regulator of neuronal survival, maturation, and activity-dependent synaptic plasticity\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Traditionally, IGF bioactivity is modulated by a family of high-affinity binding proteins that control its tissue distribution and receptor accessibility\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Notably, however, several IGF-binding proteins also execute IGF-independent functions through interactions with extracellular matrix proteins, integrins, and diverse cell-surface receptors\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eInsulin-like growth factor binding protein-like 1 (IGFBPL1) is a secreted glycoprotein of approximately 25\u0026ndash;30 kDa, structurally related to the canonical insulin-like growth factor-binding protein (IGFBP) superfamily, and functions as a modulatory factor in extracellular signaling. IGFBPL1 has been implicated in diverse biological processes, including tissue remodeling, immune regulation, and tumor suppression\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIGFBPL1 stabilizes microglia under neurodegenerative conditions, fostering anti-inflammatory and neuroprotective phenotypes\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e, and modulates lipid metabolism in peripheral macrophages via IGF1R\u0026ndash;dependent mechanisms\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. In the nervous system, IGFBPL1 facilitates axonal elongation through IGF1 signaling, as demonstrated in developing retinal neurons and embryonic circuits\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e, and its loss in the striatum results in synaptic deficits, neuronal degeneration, and sustained inflammation\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. While these studies highlight IGFBPL1 as a key regulator of CNS homeostasis and neuroregeneration, its specific role in adult dorsal root ganglion (DRG) neurons and in neuropathic pain remains largely unknown. Whether IGFBPL1 is reactivated in sensory neurons or associated immune cells after peripheral nerve injury, and whether it contributes to maladaptive processes driving chronic pain, remains unresolved.\u003c/p\u003e\u003cp\u003eHere we show that IGFBPL1 functions as a pivotal regulator at the intersection of growth factor signaling, sensory neuron excitability, and neuroinflammation in the injured DRG. Specifically, we show that IGFBPL1 upregulation after nerve injury engages the IGF1R\u0026ndash;ERK signaling cascade, thereby inducing proinflammatory gene expression and driving neuronal hyperexcitability. Moreover, IGFBPL1 facilitates macrophage infiltration and amplifies neuroimmune crosstalk, thereby escalating inflammatory signaling within the injured DRG. Together, these processes contribute to the onset and persistence of neuropathic pain, positioning IGFBPL1 as a critical nexus integrating growth factor signaling with neuroimmune dynamics in sensory neurons. Intriguingly, CCI mice also demonstrated significant improvement in locomotor performance following IGFBPL1 inhibition. Overall, our findings define IGFBPL1 as a central mediator of maladaptive plasticity, linking sensory pain processing with motor dysfunction, and establish its potential as a therapeutic target.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eAnimals\u003c/p\u003e\u003cp\u003e All animal care and experimental procedures were approved by the Gwangju institute of science and technology (GIST) Laboratory Animal Research Center (LARC). Adult male C57BL/6 mice (8\u0026ndash;10 weeks old, 24\u0026ndash;28 g) were housed with food and water \u003cem\u003ead libitum\u003c/em\u003e under a standard 12-hour light/dark cycle. All mice were bred at the GIST animal facility.\u003c/p\u003e\u003cp\u003eStereotaxic surgery and intra-DRG microinjection of siRNA, drugs and neutralizing antibodies\u003c/p\u003e\u003cp\u003eAfter anesthesia with isoflurane, animals were secured to the stereotaxic instrument (RWD Model 68000). A midline incision in the lower lumbar back was made, and the lumbar articular process was exposed and removed. The DRG was injected with 5 \u0026micro;g in 5 \u0026micro;l of specific (IGFBPL1, IGF1R) or non-specific (scrambled control) siRNAs complexed with 9 \u0026micro;l polyethyl ethyleneimine (jetPEI, 10 mM) 10 \u0026micro;l Hamilton microsyringe (Model 1701N) connected to a WPI microsyringe pump. The needle remained in place for 10 min post-injection. In vivo siRNAs were modified with 2\u0026rsquo;-OME and 5\u0026rsquo;-Chol (GenePharma). The siRNA sequences used in this study are listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003eNBI-31772 (5 mg/kg, Tocris), recombinant mouse IGFBPL1 (JB1; 10 \u0026micro;g per injection, Cat No. 4130-BL, R\u0026amp;D Systems), anti-IGFBPL1 neutralizing antibody (10 \u0026micro;g per injection, MAB391), anti-IGF1R neutralizing antibody (10 \u0026micro;g per injection), IgG isotype control, MEK inhibitor U0126 (10 \u0026micro;g per injection), and AKT inhibitor MK-2206 (10 \u0026micro;g per injection) and were administered via intra-DRG injection following the same procedure. Animals showing signs of paresis or other abnormalities were excluded from analysis.\u003c/p\u003e\u003cp\u003ePain models\u003c/p\u003e\u003cp\u003eChronic constriction injury (CCI) pain model\u003c/p\u003e\u003cp\u003eA 1.5 cm lateral incision was made on the right hind limb after the animals were anesthetized with 1% Zoletil (intraperitoneal) The muscle was bluntly separated to expose the sciatic nerve trunk, which was then loosely ligated with three 6\u0026thinsp;\u0026minus;\u0026thinsp;0 silk sutures placed 1\u0026ndash;2 mm apart proximal to the trifurcation. The sutures were gently tightened until a brisk twitch was observed in the right hind limb. Finally, the skin and muscles were sutured in layers. Sham-operated mice underwent identical procedures except without nerve ligature.\u003c/p\u003e\u003cp\u003eSciatic nerve crush (SNC) pain model\u003c/p\u003e\u003cp\u003eAnimals were anesthetized with Zoletil (10 mg/kg, intraperitoneal). Using fine jeweler\u0026rsquo;s forceps, the sciatic nerve at mid-thigh level was crushed for 30 seconds at two adjacent sites. The crush injury was performed only on the right side, with the contralateral side serving as control. After the procedure, layered skin closure was performed. SNC mice were placed on a warm pad for 5\u0026ndash;10 min before being returned to their home cages.\u003c/p\u003e\u003cp\u003eSpared nerve injury (SNI) pain model\u003c/p\u003e\u003cp\u003eFor the SNI model, the common peroneal and tibial nerves were isolated, ligated with 6\u0026thinsp;\u0026minus;\u0026thinsp;0 silk sutures, and then cut distal to the ligation. The sural nerve was carefully preserved. Sham-operated mice underwent the same procedure without ligation or transection of the sciatic nerve branches.\u003c/p\u003e\u003cp\u003eBehavioral assessments\u003c/p\u003e\u003cp\u003eC57BL/6 mice aged 8\u0026ndash;10 weeks, weighing 20\u0026ndash;25 g were used for the mechanical and thermal behavioral tests. All animals were acclimatized to the testing room or apparatus for at least 1 hour before behavioral assessments. Blind scoring was performed to ensure that observers were unaware of treatments.\u003c/p\u003e\u003cp\u003evon Frey filament assay\u003c/p\u003e\u003cp\u003eFor mechanical allodynia, mice were placed in individual Plexiglas chambers on an elevated wire mesh surface and acclimatized for 1 hour. The mid-plantar surface of the left and right hind paws was stimulated with a series of von Frey filaments applied vertically (0.02 to 2.56 g, Stoelting, Wood Dale, IL). The 50% paw withdrawal threshold (PWT) was determined by Dixon\u0026rsquo;s up-down method. A positive response was defined as paw withdrawal, flinching, shaking, or licking following stimulation.\u003c/p\u003e\u003cp\u003eHargreaves assay\u003c/p\u003e\u003cp\u003eFor thermal hyperalgesia, nociceptive responses to thermal stimulation were measured using a Model 336 Analgesia Meter (IITC Inc. Life Science Instruments. Woodland Hills, CA). Mice were placed in a chamber with a transparent glass bottom and habituated for 1 hour. A radiant heat source was focused on the mid-plantar surface of the hind paw. Paw withdrawal latency (PWL) was recorded as the time duration between the onset of the stimulus and paw withdrawal. To prevent tissue damage, a cut-off time of 20 seconds was applied. Each test was repeated three times with 10-minute intervals for both hind paws.\u003c/p\u003e\u003cp\u003eRNA extraction and qPCR in mouse DRGs\u003c/p\u003e\u003cp\u003eBilateral L4 and L5 DRGs were collected at different time points post-operative days and stored at -80\u0026deg;C. RNA extraction was performed using RNAesay kit following the manufacturer\u0026rsquo;s instructions and reversed-transcribed using RT Master Mix (AbmGold). Quantitative polymerase chain reaction (qPCR) was then performed using SYBR\u0026trade; Green Universal Master Mix (Applied Biosystems). Reactions were carried out in a BIO-RAD CFX96 real-time PCR system. GAPDH was used as an internal control for normalization, as it has been demonstrated to be stable even after peripheral nerve injury \u003csup\u003e\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e\u003c/sup\u003e. Sequences of the primer pairs used for qPCR are shown in Table S2.\u003c/p\u003e\u003cp\u003eWestern blot\u003c/p\u003e\u003cp\u003eUnilateral L4 and L5 DRGs were collected, pooling four DRGs per sample to obtain sufficient protein concentration. Tissues and primary neurons were homogenized in ice-cold RIPA lysis buffer containing protease, phosphatase, and RNase inhibitors. After centrifugation at 12,000 g for 15 min at 4\u0026deg;C, protein concentrations were measured using a BCA assay. Samples were heated at 95\u0026deg;C for 5 min, separated on 8\u0026ndash;12% SDS-PAGE, and transferred onto nitrocellulose membranes. Next, membranes were blocked with 5% nonfat dried milk in TBS-T (Tris-buffered saline containing 0.1% Tween-20) for 1 hour, followed by overnight incubation at 4\u0026deg;C with the following primary antibodies: goat anti-IGFBPL1(1:1000, R\u0026amp;D Systems), mouse anti-IGF-1(1:500, R\u0026amp;D Systems), mouse anti-IGF-1R(1:1000, R\u0026amp;D Systems), rabbit anti-ERK (1:1000, Cell Signaling Technology), rabbit anti-phospho-ERK (1:1000, Cell Signaling Technology), rabbit anti-AKT (1: 1000, Cell Signaling Technology), ), rabbit anti-phospho-AKT (1: 2000, Cell Signaling Technology), mouse anti- β-actin (1:2500, Cell Signaling Technology). Secondary antibodies (horseradish peroxidase-conjugated-anti-mouse, anti-rabbit, or anti-goat) were incubated for 1 hour at room temperature after TBS-T washes. Protein bands were visualized using ECL substrate and detected with the ChemiDoc XRS System. Band intensities were quantified by densitometry using ImageJ/FIJI software (Bio-Rad).\u003c/p\u003e\u003cp\u003ePrimary DRG neuronal culture and transfection\u003c/p\u003e\u003cp\u003e Adult mice (8 to 10 weeks old) lumbar DRGs were collected and dissociated with 100 U papain (P4762, Sigma-Aldrich) followed by collagenase II (1 mg/ml; 11179179001, Roche) and dispase II (1.2 mg/ml; 04942078001, Roche) for 1 hour at 37\u0026deg;C. The ganglia were then triturated in Hanks\u0026rsquo; balanced salt solution containing 10 mM glucose and 5 mM HEPES (pH 7.35) using a fire-polished Pasteur pipette to obtain a single-cell suspension. Dissociated DRG neuronal cells were filtered through a 40-\u0026micro;m strainer, collected by centrifugation at 1000 g for 8 min, and resuspended in Neurobasal medium supplemented with B27 (Thermo Fisher Scientific) and l-glutamine (Thermo Fisher Scientific). Cells were then seeded on poly-l-ornithine (P4832, Sigma-Aldrich)\u0026ndash; and laminin (23017-015, Sigma-Aldrich)\u0026ndash;coated six-well plates in DMEM supplemented with 10% fetal bovine serum, penicillin (20 U/ml), and streptomycin (0.2 mg/ml), and maintained at 37\u0026deg;C in a humidified incubator with 95% O\u003csub\u003e2\u003c/sub\u003e and 5% CO\u003csub\u003e2\u003c/sub\u003e. DRG neurons were transfected with either 0.6 \u0026micro;g of 6-FAM\u0026ndash;-modified siRNA or negative control siRNA at a final concentration of 100 nM using the Oligofectamine\u0026trade; Transfection Reagent (12252011, Invitrogen) according to the manufacturer\u0026rsquo;s instructions. DRGs were harvested 48 hours post-transfection for calcium imaging, western blot, and immunocytochemistry.\u003c/p\u003e\u003cp\u003eCalcium imaging\u003c/p\u003e\u003cp\u003esiRNA-transfected or control primary DRG neurons were loaded with 1 \u0026micro;M Fluo-3 AM (F14201, Invitrogen, Thermo Fisher Scientific) in extracellular solution (ECS: 150 mM NaCl, 5 mM KCl, 2.5 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 10 mM D-Glucose, pH adjusted to 7.4 with NaOH) for 30 min at room temperature, followed by three washes and a 15 min de-esterification period in ECS. Cells were images using a Zeiss LSM 710 microscope with \u0026times;20 and \u0026times;40 objectives. Neurons were illuminated at 488 nm, and time-lapse images were acquired for 4 min per region of interest (ROI). Fluorescence intensity was quantified using ZEN software (Zeiss) and ImageJ. Intracellular calcium dynamics were expressed as ΔF/F₀, where F₀ denotes baseline fluorescence.\u003c/p\u003e\u003cp\u003eImmunocytochemistry\u003c/p\u003e\u003cp\u003eCultured cells were washed in PBS, fixed in 4% paraformaldehyde (PFA) for 20 min, and permeabilized with 0.1% Triton X-100. After blocking in 5% donkey serum, bovine serum albumin (1 mg/ml; BSA), and 0.2% Triton X-100 in PBS for 1 hour at room temperature, cells were incubated with primary antibodies in 5% donkey serum and BSA (1 mg/ml) in PBS overnight at 4\u0026deg;C. Secondary antibodies (raised in donkey, 1:1000, Jackson ImmunoResearch) were applied in for 1 hour at room temperature. Coverslips were mounted with Fluoromount Aqueous Mounting Medium (F4680, Sigma-Aldrich). Sample images were acquired using an Olympus FV1000 confocal laser-scanning microscope at \u0026times;60 magnification with oil-immersion objective [Olympus UPLSAPO, NA 1.35].\u003c/p\u003e\u003cp\u003eImmunofluorescence\u003c/p\u003e\u003cp\u003eAnesthetized mice were fixed via transcardial perfusion with PBS followed by 4% (w/v) paraformaldehyde (PFA, RNase-free; Bioss, C2055). DRG tissues were freshly dissected and post-fixed in 4% PFA at 4\u0026deg;C overnight. Tissues were cryoprotected in 30% (w/v) sucrose (Sigma-Aldrich, S8501) in PBS for 48 hours, embedded in Tissue-Tek O.C.T. compound (Sakura, 4583), and sectioned at 10 \u0026micro;m using a Leica cryostat (Leica CM1850). Cryo-sections were blocked in 5% donkey serum and 0.2% Triton X-100 in PBS for 1 hours at room temperature. Sections were incubated in primary antibodies (diluted in 5% donkey serum and 0.3% Triton X-100 in PBS) overnight at 4\u0026deg;C, followed by incubation with secondary antibodies for 1 hours at room temperature. Primary antibodies used: goat anti-IGFBPL1(1:500, R\u0026amp;D Systems), mouse anti-IGFBPL1(1:500, Santa Cruz Biotechnology), Guinea pig anti-TRPV1 (1:200, Alomone Labs), rabbit anti-PIEZO2 (1:200, Alomone Labs). Slides were mounted with Prolong Gold antifade reagent containing DAPI (Invitrogen, P36931) and imaged using OlyVIA research laser scanner (Olympus) and an Olympus FV1000 confocal microscope. For quantification, four random sections were selected per mouse and averaged to determine the number of positive cells.\u003c/p\u003e\u003cp\u003eRNAscope \u003cem\u003ein situ\u003c/em\u003e hybridization\u003c/p\u003e\u003cp\u003eRNAscope \u003cem\u003ein situ\u003c/em\u003e hybridization was performed using the RNAscope 2.5 HD RED Kit (ACDBio, 322350, ACDBio, CA) according to the manufacturer\u0026rsquo;s protocol. Mice were transcardially perfused with 0.1 M PBS followed by 4% paraformaldehyde containing 0.1% diethyl pyrocarbonate. DRGs were dissected, post-fixed, cryoprotected, and sectioned. Frozen tissue sections were treated with hydrogen peroxide and Protease Plus (ACDBio) for 2 min at room temperature, followed by hybridization with probes targeting \u003cem\u003eIgfbpl1\u003c/em\u003e (Cat No. 488851, ACDBio, CA) or \u003cem\u003eIgf1r\u003c/em\u003e (Cat No. 417561, ACDBio, CA) for 2 h at 40\u0026deg;C. Signal amplification steps (Amp1\u0026ndash;Amp6) were performed per manufacturer\u0026rsquo;s instructions, with washes between steps using RNAscope wash buffer. Next, slides were washed three times in third-grade distilled water (TDW) and processed for additional immunofluorescence. Blocking was performed in 5% donkey serum and 0.2% Triton X-100 in PBS for 1 h at room temperature, followed by overnight incubation with primary antibodies at 4\u0026deg;C. After three washes (10 min each) in 0.3% PBST, sections were incubated with secondary antibodies for 1 hours at room temperature. Sections were washed thrice, air dried, and mounted with ProLong Gold Antifade mounting medium (P0131, Beyotime Biotechnology, China) before imaging.\u003c/p\u003e\u003cp\u003eFlow Cytometry\u003c/p\u003e\u003cp\u003eCCI\u0026ndash;si\u003cem\u003eIgfbpl1\u0026ndash;\u003c/em\u003e and scramble-treated treated mice were anesthetized and perfused with ice-cold PBS to remove circulating blood. Lumbar DRGs were dissected and digested with 100 U papain (P4762, Sigma-Aldrich), followed by collagenase II (1 mg/ml; 11179179001, Roche) and dispase II (1.2 mg/ml; 04942078001, Roche) for 1hr at 37\u0026deg;C. Triturated single-cell suspensions were filtered through a 40-\u0026micro;m strainer, and centrifuged at 500 g for 5 min at 4\u0026deg;C. Cells were blocked with anti-CD16/32 antibody for 10 min and stained with fluorophore-conjugated antibodies against CD11b, CD45, and F4/80 for 1 hour at 4\u0026deg;C. Following antibody staining, cells were incubated with DAPI to exclude nonviable cells. After washing, cells were resuspended in PBS containing 2% FBS, and flow cytometry data were acquired on a BD FACS Canto II flow cytometer with BD FACS Diva 8 software (BD Biosciences). Data were processed using FlowJo software (Tree Star), and CD11b⁺CD45\u003csup\u003ehigh\u003c/sup\u003eF4/80⁺ cells were quantified as macrophages.\u003c/p\u003e\u003cp\u003eGait assay and stride analysis\u003c/p\u003e\u003cp\u003eGait assay and stride analysis was conducted as previously described by Tiwari \u003cem\u003eet al.\u003c/em\u003e, 2024. In brief, the hind paws of mice were lightly coated with water-based dyes before being released at one end of a chamber measuring 45 cm (L) \u003cb\u003e\u0026times;\u003c/b\u003e 5.0 cm (W) \u003cb\u003e\u0026times;\u003c/b\u003e 12.0 cm (H) to walk freely to the other end. Paw prints were recorded on standard printing paper placed beneath the chamber. Stride length was defined as the distance between two consecutive prints from the same toe. Stride lengths from the same mouse were averaged to produce a single value for statistical analysis. All procedures and analyses were performed consistently across experimental groups.\u003c/p\u003e\u003cp\u003eQuantification and statistical analysis\u003c/p\u003e\u003cp\u003eData analysis was performed using ImageJ/FIJI, OlyVia, SW software, with statistical analyses conducted in GraphPad Prism 9. Results are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Statistical tests, including one-way ANOVA, two-way ANOVA, or Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test, were applied to behavioral, biochemical, and morphological data depending on the experimental design and number of variables. Detailed statistical methods are provided in the figure legends. Differences were considered statistically significant at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cstrong\u003eIGFBPL1 upregulation in DRG after nerve injury contributes to neuropathic pain\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo identify candidate mediators of neuropathic pain, we analyzed previously published RNA-seq datasets of dorsal root ganglia (DRG) after peripheral nerve injury \u003csup\u003e40, 41, 42\u003c/sup\u003e. Among the transcripts most strongly induced across datasets, we consistently found increased expression of \u003cem\u003eIgfbpl1\u003c/em\u003e, indicating a potential role in sensory neuron plasticity and pain processing. To study the role of IGFBPL1 in peripheral neuropathic pain (PNP), we established the chronic constriction injury (CCI) model of persistent peripheral neuropathic pain\u003csup\u003e43\u003c/sup\u003e (Fig. 1a) and examined the temporal expression pattern of IGFBPL1 in the DRG. RNAscope in situ hybridization demonstrated marked upregulation of \u003cem\u003eIgfbpl1\u0026nbsp;\u003c/em\u003eexpression in DRG tissue of CCI mice compared with Sham mice (Fig. 1b,c; Supplementary Fig. 1a\u0026ndash;c). Following peripheral nerve injury, \u003cem\u003eIgfbpl1\u003c/em\u003e mRNA and IGFBPL1 protein were significantly upregulated in a time-dependent manner, peaking at day 7 and persisting through day 21, as shown by RT-qPCR and immunofluorescence (Fig. 1d\u0026ndash;f). We observed a similar increase in \u003cem\u003eIgfbpl1\u003c/em\u003e mRNA and IGFBPL1 protein in the sciatic nerve crush (SNC) model, another well-described paradigm of neuropathic pain in rodents\u003csup\u003e44\u003c/sup\u003e (Supplementary Fig. 1d\u0026ndash;f).\u003c/p\u003e\n\u003cp\u003eGiven the nerve injury-dependent up-regulation of IGFBPL1 in the DRG, we next asked whether IGFBPL1 is functionally required for pain pathogenesis. To test this, we performed knockdown using intraganglionic delivery of IGFBPL1-specific siRNA (si\u003cem\u003eIgfbpl1\u003c/em\u003e) or scrambled control siRNA into the ipsilateral DRG of CCI and sham mice (Fig. 1g). The efficiency and specificity of IGFBPL1 silencing was validated in DRG tissue and primary cultured DRG neurons by immunoblotting and RT-qPCR (Fig. 1h; Supplementary Fig. 2a\u0026ndash;c). Notably, two different siRNAs targeting either the open reading frame of the 3\u0026rsquo; UTR of \u003cem\u003eIgfbpl1\u003c/em\u003e achieved 75-80% knockdown efficiency (Supplementary Fig. 2a\u0026ndash;c).\u003c/p\u003e\n\u003cp\u003eBehaviorally, \u003cem\u003eIgfbpl1\u003c/em\u003e knockdown significantly attenuated CCI-induced mechanical allodynia and thermal hyperalgesia (Fig. 1i,j), without affecting baseline nociceptive thresholds on the contralateral side or in sham controls (Supplementary Fig. 3a\u0026ndash;d). Moreover, intraganglionic injection of IGFBPL1-neutralizing antibody (IGFBPL1 Ab), or administration of the IGFBP inhibitor, NIB31772, recapitulated these analgesic effects in a dose- and time-dependent manner when administered during both the early and late phases of CCI (Fig. 1k, n; Supplementary Fig. 4, a\u0026ndash;f), indicating a sustained role of IGFBPL1 in neuropathic pain hypersensitivity. To further assess whether IGFBPL1 contributes to neuropathic pain across models, we used the spared nerve injury (SNI) paradigm (Fig. 1o). Similar to CCI, DRG-targeted si\u003cem\u003eIgfbpl1\u003c/em\u003e in SNI mice robustly attenuated mechanical and thermal hypersensitivity from day 3 through day 42 post-injury (Fig. 1p,q). Together, these findings demonstrate that IGFBPL1 upregulation in DRG neurons is a key driver of the onset and persistence of neuropathic pain.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe cellular localization and expression of IGFBPL1 in human and mouse DRG\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe next examined the cellular source of IGFBPL1 in the injured DRG by performing co-immunostaining of L5 DRG and sciatic nerve sections from CCI and sham mice using antibodies against IGFBPL1 and canonical cell-type markers: NeuN (neurons), GFAP (astrocytes), and Iba1 (microglia). IGFBPL1 was strongly enriched in NeuN-positive sensory neurons, with no detectable co-localization with astrocytic or microglial markers (Supplementary Fig. 5a\u0026ndash;c). IGFBPL1 was also markedly elevated in sciatic nerve axons (Fig. 2d,e), consistent with anterograde transport\u003csup\u003e45\u003c/sup\u003e. Moreover, IGFBPL1 colocalized with ATF3, a canonical marker of axonal injury (Supplementary Fig. 5c,d). To further define neuronal subtypes expressing IGFBPL1, we performed double-labeling of IGFBPL1 with subtype-specific markers. IGFBPL1 was predominantly localized to medium- to large-diameter NF200⁺\u0026nbsp;myelinated A-fiber mechanosensitive neurons and CGRP⁺\u0026nbsp;peptidergic nociceptors, with weaker expression in IB4⁺\u0026nbsp;non-peptidergic neurons, and minimal detection in GS⁺\u0026nbsp;satellite glial cells (Fig. 2a\u0026ndash;d).\u003c/p\u003e\n\u003cp\u003eWe next validated these findings using published scRNA-seq datasets from mouse and human DRGs\u003csup\u003e46\u003c/sup\u003e. At baseline, \u003cem\u003eIgfbpl1\u003c/em\u003e transcript levels were broadly distributed within Calca (CGRP+) cluster subtypes but at low abundance. In contrast, \u003cem\u003eIgf1r (Igfbpl1\u0026nbsp;\u003c/em\u003esignaling receptor) was expressed at relatively high levels across neuronal subtypes in both species, whereas \u003cem\u003eIgf1\u003c/em\u003e was detected at lower levels (Supplementary Fig. 6a\u0026ndash;h). In another reanalyzed murine DRG dataset after CCI\u003csup\u003e47\u003c/sup\u003e, \u003cem\u003eIgfbpl1\u003c/em\u003e expression was concentrated in four clusters of peptidergic nociceptors (PEP1\u0026ndash;4) and, to a lesser extent, detected in non-peptidergic (NP) and non-nociceptor neurons or glial populations\u0026ndash;consistent with our immunostaining results (Fig. 2d,e). By comparison, \u003cem\u003eIgf1\u003c/em\u003e showed only modest upregulation in peptidergic neurons (PEP), NF neurons, and glia, whereas \u003cem\u003eIgf1r\u003c/em\u003e remained broadly and highly expressed across all neuronal subtypes and glia clusters. Additionally, \u003cem\u003eTrpv1\u0026nbsp;\u003c/em\u003e(a well-established mediator of thermal nociception) and \u003cem\u003ePiezo2\u003c/em\u003e (a mechanosensitive ion channel required for touch and proprioceptive) were enriched within the same pain-related subsets (Supplementary Fig. 7a\u0026ndash;f). Together, these results (including the spatial enrichment) establish IGFBPL1 as a sensory neuron\u0026ndash;specific, injury-inducible gene in the DRG, with preferential expression in peptidergic and myelinated nociceptors\u0026ndash;aligning with its role in peripheral pain signaling after nerve injury.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIGFBPL1 promotes TRPV1 activity, driving DRG hyperexcitability and neuropathic pain\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNeuronal hyperexcitability in primary sensory neurons of the dorsal root ganglion (DRG) is a hallmark of chronic neuropathic pain\u003csup\u003e48, 49, 50\u003c/sup\u003e. Given prior evidence that IGFBPL1 facilitates IGF-1\u0026ndash;induced Ca\u0026sup2;⁺\u0026nbsp;signaling required for axonal growth in retinal ganglion cells\u003csup\u003e37\u003c/sup\u003e, we hypothesized that IGFBPL1 may similarly promote Ca\u0026sup2;⁺ influx in sensory neurons by acting upstream of TRPV1 to drive injury-induced excitability. To probe this, we conducted calcium imaging using primary cultured DRG neurons isolated from CCI mice (Fig. 3a). CAP (100 nM) application elicited robust Ca\u0026sup2;⁺ responses in control DRG neurons, while \u003cem\u003eIgfbpl1\u003c/em\u003e knockdown markedly reduced these responses (Fig. 3b,c). Rescue with recombinant IGFBPL1 (rIGFBPL1) restored CAP-evoked Ca\u0026sup2;⁺ influx to control levels, demonstrating that endogenous IGFBPL1 is required for TRPV1-mediated Ca\u0026sup2;⁺ signaling (Fig. 3b,c). Furthermore, treatment with the TRPV1 antagonist capsazepine (CPZ, 10 \u0026micro;M) abolished CAP responses, confirming assay specificity (Fig. 3b\u0026ndash;d). The distribution of CAP-responsive cells showed that IGFBPL1 depletion shifted the majority of neurons to an unresponsive phenotype, while rIGFBPL1 rescue restored responsiveness (Fig. 3e). Post-hoc co-immunostaining of calcium-imaged cultures confirmed co-localization of IGFBPL1 with TRPV1 and the pan-neuronal marker TUJ1, indicating that TRPV1-expressing nociceptors are the primary responsive population (Fig. 3f\u0026ndash;g). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFinally, to determine whether IGFBPL1 is sufficient to drive pain hypersensitivity in vivo, we administered recombinant IGFBPL1 (hereafter used as rIGFBPL1) via intraganglionic injection into L5 DRGs of CCI mice. Both low (0.05 \u0026micro;g) and high (1.0 \u0026micro;g) doses of rIGFBPL1 significantly lowered paw withdrawal thresholds (PWTs) compared with PBS-treated controls (Fig. 3h,j), and the normalized area under the curve (AUC) confirmed robust increases in mechanical and thermal hypersensitivity (Fig. 3i,k). These results demonstrate that IGFBPL1 potentiates TRPV1-dependent neuronal hyperexcitability and directly contributes to nociceptive behaviors in CCI mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIGFBPL1-driven pain sensitization is mediated via IGF1R-dependent signaling\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHaving established a functional role of IGFBPL1 in neuropathic pain, we next sought to identify the downstream effector mechanisms. Given that IGFBPL1 has been shown to modulate IGF1 bioavailability and that IGF1 receptor (IGF1R) is a key transducer of IGF1-dependent signaling\u003csup\u003e37, 51\u003c/sup\u003e, we hypothesized that IGFBPL1 drives neuropathic pain through IGF1R activation. Consistent with our reanalyzed public scRNA-seq dataset showing elevated \u003cem\u003eIgf1r\u003c/em\u003e expression in mouse DRGs after CCI, quantitative PCR revealed a progressive, time-dependent increase in \u003cem\u003eIgf1r\u003c/em\u003e mRNA in the ipsilateral DRG of CCI mice but not in sham controls (Fig. 4a). RNAscope in situ hybridization and immunostaining further demonstrated a robust increase in \u003cem\u003eIgf1r\u003c/em\u003e mRNA and IGF1R protein levels peaking at post-injury day 14 (Fig. 4, b\u0026ndash;e). Together, these findings indicate that IGF1R upregulation parallels IGFBPL1 induction after nerve injury, suggesting a potential IGFBPL1\u0026ndash;IGF1R signaling axis in neuropathic pain.\u003c/p\u003e\n\u003cp\u003eWe next assessed the role of IGF1R in peripheral neuropathic pain by confirming its knockdown (65.5%) via intraganglionic injection of si\u003cem\u003eIgf1r\u003c/em\u003e or scrambled control on day 7 after CCI surgery (Fig. 4f). Behavioral assays revealed that \u003cem\u003eIgf1r\u003c/em\u003e-specific knockdown significantly and persistently alleviated mechanical allodynia (days 7\u0026ndash;42) and thermal hyperalgesia (days 3\u0026ndash;42) (Fig. 4g,h), demonstrating that IGF1R is required for both the development and persistence of neuropathic pain.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo determine whether IGFBPL1\u0026rsquo;s pronociceptive effects are mediated by IGF1R, we administered rIGFBPL1 followed by intraganglionic injection of either PBS or the highly selective IGF1R antagonist JB1. While rIGFBPL1 + PBS significantly exacerbated mechanical allodynia and thermal hyperalgesia, rIGFBPL1 + JB1 almost completely prevented these pain-like behaviors in both acute (Fig. 4i,j) and chronic (Fig. 4k,l) phases of neuropathic pain, demonstrating that IGFBPL1 induces nociception through IGF1R activation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIGFBPL1/IGF1R signaling induces the activation of pERK in DRG sensory neurons\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo delineate the underlying mechanisms of IGFBPL1\u0026ndash;IGF1R in pain processing, we examined intracellular signaling pathways downstream of IGF1R that are crucial in neuropathic pain. IGF1R activation triggers phosphorylations cascades involving p38 MAPK, ERK/MAPK, PKB/AKT, and PI3K\u003csup\u003e52, 53, 54, 55\u003c/sup\u003e. Immunostaining of DRG sections revealed that CCI markedly increased phosphorylated ERK (pERK), which was significantly reduced by \u003cem\u003eIgfbpl1\u003c/em\u003e knockdown (Fig. 5a). Notably, pAKT was unaffected by IGFBPL1 silencing (Fig. 5b) or overexpression in DRG tissue and primary DRG neurons (Fig. 5b; Supplementary Fig. 9a,b). \u0026nbsp;Both pERK and pAKT colocalized with TUJ1 (Fig 5c,d). Consistent with these findings, intraganglionic injection of rIGFBPL1 elevated pERK without altering pAKT (Fig 5a,b), indicating selective engagement of the ERK pathway. Pharmacological inhibition with MEK inhibitor U0126 or the AKT inhibitor MK-2206 abolished IGFBPL1-induced upregulation of pERK and AKT, respectively (Fig 5a,b).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAdministration of U0126 markedly reduced pERK in primary cultured DRG neurons (Fig. 5e,f). Intraganglionic injection of U0126 also reversed IGFBPL1-induced neuropathic pain behaviors \u003cem\u003ein vivo\u003c/em\u003e (Fig. 5g,h). In contrast, MK-2206, failed to reduce pAKT levels (Fig. 5f) following IGFBPL1 overexpression and had no effect on rIGFBPL1-driven responses (Fig. 5i,j). These results indicate that IGFBPL1 drives neuropathic pain via an ERK-dependent but AKT-independent pathway. Furthermore, CCI mice treated with JB1 exhibited reduced rIGFBPL1-induced pERK but not AKT compared with PBS-treatment mice (Fig. 5k,l,m), demonstrating that IGFBPL1-driven ERK activation requires upstream IGF1R. Together, these findings identify ERK as a selective and essential downstream effector of IGFBPL1\u0026ndash;IGF1R signaling in sensory neurons.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIGFBPL1 inhibition reduces peripheral inflammation and macrophage recruitment in DRG\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePeripheral sensory neurons and immune cells engage in bidirectional communication that shapes neuropathic pain\u003csup\u003e56, 57, 58, 59\u003c/sup\u003e. Given that neuronal hyperactivity and ERK activation can induce cytokine and chemokine release, we investigated whether IGFBPL1-mediated nociceptor sensitization influences macrophage recruitment in the injured DRG. To this end, DRG neurons were acutely isolated 7 days after CCI following intraganglionic injection of either si\u003cem\u003eIgfbpl1\u003c/em\u003e or scrambled control. IGFBPL1 knockdown markedly reduced F4/80⁺ macrophages and CD11b⁺/CD68⁺ populations in the DRG (Fig. 6a,b). We also observed reduced macrophage infiltration in the sciatic nerve (Fig. 6c) and fewer neuron\u0026ndash;macrophage contacts (Fig. 6d). Correspondingly, the expression levels of proinflammatory genes (\u003cem\u003eTnf-\u0026alpha;\u003c/em\u003e, \u003cem\u003eIl-1\u0026beta;\u003c/em\u003e, \u003cem\u003eIl-6\u003c/em\u003e, and \u003cem\u003eCcr2\u003c/em\u003e) were significantly suppressed (Fig. 6e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo determine whether macrophages are necessary for IGFBPL1-driven pain, mice underwent clodronate-mediated macrophage depletion followed by intraganglionic injection of recombinant IGFBPL1 (rIGFBPL1) or vehicle. In macrophage-depleted mice, rIGFBPL1 failed to elicit mechanical allodynia or thermal hyperalgesia, whereas vehicle-treated controls exhibited robust pain responses (Fig. 6g\u0026ndash;j). Consistently, calcium imaging of primary DRG neurons showed that rIGFBPL1-induced Ca\u0026sup2;⁺ influx was markedly reduced following macrophage depletion (Fig. 6k\u0026ndash;m). These findings demonstrate that neuronal IGFBPL1 drives persistent neuropathic pain primarily through TRPV1-dependent Ca\u0026sup2;⁺ influx and ERK activation in DRG sensory neurons, which secondarily recruits macrophages and amplifies inflammatory signaling. Macrophage infiltration thus functions as an amplification mechanism rather than a direct target of IGFBPL1, linking neuronal sensitization to the broader neuroimmune response.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIGFBPL1 downregulation improves gait performance following peripheral nerve injury\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter establishing the role of IGFBPL1 in nociceptor sensitization and pain hypersensitivity, we next asked whether IGFBPL1 inhibition also influences non-reflexive pain-related behaviors. To this end, we evaluated locomotor function by performing gait analysis in CCI and sham mice treated with intraganglionic injections of si\u003cem\u003eIgfbpl1\u003c/em\u003e or scrambled control into the ipsilateral L5 DRG from days 0 to 4 post-surgery. On day 7, following CCI or sham surgery, we analyzed the footprint patterns of the mice\u003csup\u003e60\u003c/sup\u003e (Figure 7a,b). Marked improvements in gait parameters were observed in si\u003cem\u003eIgfbpl1\u003c/em\u003e-treated mice. Specifically, the stride length of the CCI and CCI + Scrambled groups was significantly shorter compared with the sham and CCI + si\u003cem\u003eIgfbpl1\u003c/em\u003e groups (Figure 7c). Similarly, CCI and CCI + Scramble mice exhibited increased stride width (Figure 7d) and decreased paw rotation (Figure 7e), whereas no such abnormalities were detected in the CCI + si\u003cem\u003eIgfbpl1\u003c/em\u003e group. These findings indicate that IGFBPL1 downregulation substantially improves gait dynamics in CCI-induced neuropathic pain. Taken together, our results demonstrate that IGFBPL1 downregulation in peripheral sensory neurons alleviates both reflexive hypersensitivity and locomotor abnormalities following nerve injury.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe dorsal root ganglion (DRG) serves as a critical hub for transmitting peripheral injury signals to spinal and supraspinal circuits, generating both the sensory-discriminative and affective components of pain\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e. Increasing evidence highlights growth factors and neurotrophins as key modulators of chronic neuropathic pain and of ion channel activity in DRG neurons\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e. IGFBPL1, a secreted member of the IGFBP family, is linked to neurodevelopmental and regenerative roles including axon growth and survival, with recent work implicating it in GABAergic circuitry, neurite outgrowth, and immune modulation in the CNS. However, its functional role in peripheral sensory neurons and in neuropathic pain remained undefined. Here, using complementary genetic, pharmacological, and behavioral approaches, we demonstrate that IGFBPL1 is selectively upregulated in injured DRG neurons and drives persistent pain by coupling IGF1R\u0026ndash;ERK signaling to TRPV1-dependent hyperexcitability and local neuroimmune activation. To our knowledge, this is the first study to show both the expression and functional role of IGFBPL1 in the peripheral nervous system and its contribution to neuropathic pain.\u003c/p\u003e\u003cp\u003eNociceptive sensory neurons, particularly peptidergic C-fibers and subsets of myelinated A-fiber nociceptors, are well-established drivers of pain hypersensitivity during tissue injury, inflammation, and neuropathy\u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e, \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e. Reanalyzed public scRNA-seq datasets from human and mouse DRGs, together with histological validation, revealed that IGFBPL1 expression is largely restricted to CGRP⁺ peptidergic C-fibers and subsets of NF200⁺ myelinated A-fiber nociceptors, with moderate expression in IB4⁺ non-peptidergic C-fibers. This distribution places IGFBPL1 within pain-relevant neuronal subsets\u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e, \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e, \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u003c/sup\u003e and suggests a role in the molecular reprogramming of nociceptors that underlies neuropathic pain\u003csup\u003e\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e, \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eNeuropathic pain is characterized by the hyperexcitability of injured sensory neurons, which underlies mechanical and thermal hypersensitivity\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e, \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e, \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e\u003c/sup\u003e. Although IGFBPL1 has previously been shown to enable IGF1-dependent calcium signaling and downstream events important for axonal growth\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e, its contribution to afferent hyperexcitability was unexplored. In our functional assays, we found that recombinant IGFBPL1 potentiated TRPV1-mediated calcium influx in sensory neurons and elicited sustained mechanical and thermal hypersensitivity in vivo, whereas DRG-targeted knockdown of \u003cem\u003eIgfbpl1\u003c/em\u003e attenuated nociceptive behaviors, confirming a causal role in nociceptor sensitization. Together, these findings extend the role of IGFBPL1 in ion channel regulation and establish it as both necessary and sufficient for sustaining nociceptor sensitization in neuropathic pain.\u003c/p\u003e\u003cp\u003eAt the cellular level, IGFBPL1 engaged IGF1R with preferential activation of ERK signaling, while AKT remained largely unaffected, consistent with prior reports that IGF1R can selectively couple to downstream pathways depending on ligand context, receptor density, and cofactor availability\u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e, \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e, \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e\u003c/sup\u003e. Additionally, IGF1R activation has been linked to both AKT and ERK cascades in inflammatory and nerve injury\u0026ndash;induced pain\u003csup\u003e\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e, \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e\u003c/sup\u003e. Pharmacological inhibition of ERK with U0126 abolished IGFBPL1-driven hypersensitivity, whereas AKT inhibition (MK-2206) had no effect, further supporting selective engagement of the ERK pathway in pain-relevant signaling\u003csup\u003e\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e\u003c/sup\u003e. These findings indicate that IGFBPL1 functions as a context-dependent amplifier of IGF1R\u0026ndash;ERK signaling in sensory neurons, selectively enhancing excitability-related pathways without broadly activating trophic signaling.\u003c/p\u003e\u003cp\u003eBeyond neuronal excitability, IGFBPL1 orchestrates neuroimmune interactions critical for pain maintenance. IGFBPL1 knockdown suppressed expression of pro-inflammatory cytokines and chemokines, including \u003cem\u003eTnf-a\u003c/em\u003e, \u003cem\u003eIl-1β\u003c/em\u003e, \u003cem\u003eIl-6\u003c/em\u003e, and \u003cem\u003eCcr2\u003c/em\u003e, in DRG tissues and cultured neurons, while limiting macrophage infiltration into DRG and sciatic nerves. Macrophage depletion abolished IGFBPL1-induced hypersensitivity and attenuated intracellular calcium responses, indicating that immune cell recruitment acts as an amplification mechanism rather than a direct target of IGFBPL1. These findings highlight how neuronal sensitization engages the broader neuroimmune environment to sustain chronic pain, consistent with growing evidence of neuron\u0026ndash;immune crosstalk as a critical driver of neuropathic pain\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e, \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e, \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e\u003c/sup\u003e. Whether these cytokine changes reflect a consequence of altered excitability or represent an independent regulatory effect of IGFBPL1 requires further study.\u003c/p\u003e\u003cp\u003eFinally, our results show that IGFBPL1 contributes not only to pain hypersensitivity but also to gait abnormalities following nerve injury, indicating broader circuit-level effects\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. Dysfunction in DRG sensory neurons can propagate through spinal and supraspinal networks, disrupting sensorimotor integration\u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e. IGFBPL1 knockdown improved gait parameters, suggesting that the IGFBPL1\u0026ndash;IGF1R\u0026ndash;ERK axis may modulate both peripheral and central processing of nociceptive signals. These observations are consistent with prior work showing that perturbation of IGF1 signaling can affect locomotion (Santucci et al., 2005), supporting the idea that IGFBPL1 influence on non-reflexive behavior may be mediated via IGF1R-dependent pathways. Whether these effects arise from retrograde signaling to central circuits or from local DRG-to-spinal interactions remains to be determined.\u003c/p\u003e\u003cp\u003eOur study has several limitations. First, while knockdown allowed selective reduction of IGFBPL1 in adult DRG neurons, conditional knockout models are needed to dissect its roles across developmental stages and non-neuronal cell types, as well as in other neuropathy models, including diabetic and chemotherapy-induced forms. Second, all experiments were conducted in male mice to reduce variability and focus on mechanisms; whether IGFBPL1 functions similarly in females remains to be determined, given well documented sex-specific differences in neuropathic pain. Third, IGFBPL1 may modulate nociceptor excitability and macrophage recruitment through autocrine or paracrine mechanisms, and the relative contributions of these pathways in vivo warrants further investigation.\u003c/p\u003e\u003cp\u003eTaken together, our findings identify IGFBPL1 as a previously unrecognized neuronal regulator of peripheral neuropathic pain via selective activation of IGF1R\u0026ndash;ERK signaling, linking nociceptor hyperexcitability with neuron-macrophage crosstalk, and highlight it as a promising therapeutic target for peripheral neuropathic pain.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Professors Chul-Kyu Park at Gachon University College of Medicine and Hyunsoo Shawn. JE at Duke-NUS Graduate Medical School for their helpful feedback on the manuscript. We also appreciate Professor Seog Bae Oh at Department of Neurobiology and Physiology, School of Dentistry, and Dental Research Institute, Seoul National University for his insightful comments and review during the manuscript preparation. We further thank Laxman Manandhar (GIST, Korea) for assistance with western blot experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (RS-2023-00264409, RS-2023-00302281, RS-2025-00522868, RS-2025-00573499).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors and Affiliations\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDepartment of Biomedical Science and Engineering, Institute of Integrated Technology (GIST), Gwangju 61005, Republic of Korea\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEmmanuel Acquah, An Nazmus Sakib, Sang Seong Kim, Hyuk Sang Kwon \u0026amp; Euiheon Chung\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAthinoula A. Martinos Center for Biomedical Imaging, Massachusetts General Hospital, Charlestown, Massachusetts United States\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYoung Ro Kim\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDepartment of Radiology, Harvard Medical School, Boston, Massachusetts, United States\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYoung Ro Kim\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAI Graduate School, Gwangju Institute of Science and Technology (GIST), Republic of Korea\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEuiheon Chung\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions:\u003c/strong\u003e Conceptualization, E.A., and E.C.; methodology, E.A.; investigation, E.A., A.N.S., and E.C.; visualization, E.A., A.N.S., S.S.K, H.S.K, Y.R.K., and E.C.; funding acquisition, E.C.; supervision, E.C.; writing\u0026mdash;original draft, E.A.; writing\u0026mdash;review and editing, E.A., S.S.K, and E.C.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding authors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondence to Euiheon Chung.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u003c/strong\u003e The authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData and materials availability:\u003c/strong\u003e All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eVan Hecke O, Austin SK, Khan RA, Smith BH, Torrance N. Neuropathic pain in the general population: a systematic review of epidemiological studies. \u003cem\u003ePAIN\u0026reg;\u003c/em\u003e \u003cstrong\u003e155\u003c/strong\u003e, 654-662 (2014).\u003c/li\u003e\n\u003cli\u003eSmith BH, Torrance N, Bennett MI, Lee AJ. Health and quality of life associated with chronic pain of predominantly neuropathic origin in the community. \u003cem\u003eThe Clinical journal of pain\u003c/em\u003e \u003cstrong\u003e23\u003c/strong\u003e, 143-149 (2007).\u003c/li\u003e\n\u003cli\u003eColloca L\u003cem\u003e, et al.\u003c/em\u003e Neuropathic pain. \u003cem\u003eNature reviews Disease primers\u003c/em\u003e \u003cstrong\u003e3\u003c/strong\u003e, 1-19 (2017).\u003c/li\u003e\n\u003cli\u003eBreivik H, Collett B, Ventafridda V, Cohen R, Gallacher D. Survey of chronic pain in Europe: prevalence, impact on daily life, and treatment. \u003cem\u003eEuropean journal of pain\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 287-333 (2006).\u003c/li\u003e\n\u003cli\u003eBouhassira D, Lant\u0026eacute;ri-Minet M, Attal N, Laurent B, Touboul C. Prevalence of chronic pain with neuropathic characteristics in the general population. \u003cem\u003ePain\u003c/em\u003e \u003cstrong\u003e136\u003c/strong\u003e, 380-387 (2008).\u003c/li\u003e\n\u003cli\u003eGustorff B\u003cem\u003e, et al.\u003c/em\u003e Prevalence of self‐reported neuropathic pain and impact on quality of life: a prospective representative survey. \u003cem\u003eActa Anaesthesiologica Scandinavica\u003c/em\u003e \u003cstrong\u003e52\u003c/strong\u003e, 132-136 (2008).\u003c/li\u003e\n\u003cli\u003eBasbaum AI, Bautista DM, Scherrer G, Julius D. Cellular and molecular mechanisms of pain. \u003cem\u003eCell\u003c/em\u003e \u003cstrong\u003e139\u003c/strong\u003e, 267-284 (2009).\u003c/li\u003e\n\u003cli\u003eFinnerup NB, Kuner R, Jensen TS. Neuropathic pain: from mechanisms to treatment. \u003cem\u003ePhysiological reviews\u003c/em\u003e, (2020).\u003c/li\u003e\n\u003cli\u003eSindrup SH, Otto M, Finnerup NB, Jensen TS. Antidepressants in the treatment of neuropathic pain. \u003cem\u003eBasic \u0026amp; clinical pharmacology \u0026amp; toxicology\u003c/em\u003e \u003cstrong\u003e96\u003c/strong\u003e, 399-409 (2005).\u003c/li\u003e\n\u003cli\u003eJang HN, Oh TJ. Pharmacological and nonpharmacological treatments for painful diabetic peripheral neuropathy. \u003cem\u003eDiabetes \u0026amp; metabolism journal\u003c/em\u003e \u003cstrong\u003e47\u003c/strong\u003e, 743-756 (2023).\u003c/li\u003e\n\u003cli\u003eSoliman N\u003cem\u003e, et al.\u003c/em\u003e Pharmacotherapy and non-invasive neuromodulation for neuropathic pain: a systematic review and meta-analysis. \u003cem\u003eThe Lancet Neurology\u003c/em\u003e \u003cstrong\u003e24\u003c/strong\u003e, 413-428 (2025).\u003c/li\u003e\n\u003cli\u003eYuan J\u003cem\u003e, et al.\u003c/em\u003e Contribution of dorsal root ganglion octamer transcription factor 1 to neuropathic pain after peripheral nerve injury. \u003cem\u003ePain\u003c/em\u003e \u003cstrong\u003e160\u003c/strong\u003e, 375-384 (2019).\u003c/li\u003e\n\u003cli\u003eCostigan M, Scholz J, Woolf CJ. Neuropathic pain: a maladaptive response of the nervous system to damage. \u003cem\u003eAnnual review of neuroscience\u003c/em\u003e \u003cstrong\u003e32\u003c/strong\u003e, 1-32 (2009).\u003c/li\u003e\n\u003cli\u003eLi Z\u003cem\u003e, et al.\u003c/em\u003e Dorsal root ganglion myeloid zinc finger protein 1 contributes to neuropathic pain after peripheral nerve trauma. \u003cem\u003ePain\u003c/em\u003e \u003cstrong\u003e156\u003c/strong\u003e, 711-721 (2015).\u003c/li\u003e\n\u003cli\u003eYu X\u003cem\u003e, et al.\u003c/em\u003e Dorsal root ganglion macrophages contribute to both the initiation and persistence of neuropathic pain. \u003cem\u003eNature communications\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 264 (2020).\u003c/li\u003e\n\u003cli\u003eMaruyama M\u003cem\u003e, et al.\u003c/em\u003e Neat1 lncRNA organizes the inflammatory gene expressions in the dorsal root ganglion in neuropathic pain caused by nerve injury. \u003cem\u003eFrontiers in Immunology\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 1185322 (2023).\u003c/li\u003e\n\u003cli\u003eHayward R\u003cem\u003e, et al.\u003c/em\u003e Transcriptional reprogramming post-peripheral nerve injury: A systematic review. \u003cem\u003eNeurobiology of Disease\u003c/em\u003e \u003cstrong\u003e200\u003c/strong\u003e, 106624 (2024).\u003c/li\u003e\n\u003cli\u003eDenk F, McMahon SB, Tracey I. Pain vulnerability: a neurobiological perspective. \u003cem\u003eNature neuroscience\u003c/em\u003e \u003cstrong\u003e17\u003c/strong\u003e, 192-200 (2014).\u003c/li\u003e\n\u003cli\u003eJang K, Garraway SM. A review of dorsal root ganglia and primary sensory neuron plasticity mediating inflammatory and chronic neuropathic pain. \u003cem\u003eNeurobiology of Pain\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, 100151 (2024).\u003c/li\u003e\n\u003cli\u003eLiu J, Chen Y, Chen G. The Role and Mechanisms of Macrophages in Chronic Pain: A Peripheral-to-Central Perspective. \u003cem\u003eBrain Research Bulletin\u003c/em\u003e, 111470 (2025).\u003c/li\u003e\n\u003cli\u003eTurner CA, Eren-Ko\u0026ccedil;ak E, Inui EG, Watson SJ, Akil H. Dysregulated fibroblast growth factor (FGF) signaling in neurological and psychiatric disorders. In: \u003cem\u003eSeminars in cell \u0026amp; developmental biology\u003c/em\u003e). Elsevier (2016).\u003c/li\u003e\n\u003cli\u003eHarrington AW, Ginty DD. Long-distance retrograde neurotrophic factor signalling in neurons. \u003cem\u003eNature Reviews Neuroscience\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 177-187 (2013).\u003c/li\u003e\n\u003cli\u003eLi R, Li D-h, Zhang H-y, Wang J, Li X-k, Xiao J. Growth factors-based therapeutic strategies and their underlying signaling mechanisms for peripheral nerve regeneration. \u003cem\u003eActa Pharmacologica Sinica\u003c/em\u003e \u003cstrong\u003e41\u003c/strong\u003e, 1289-1300 (2020).\u003c/li\u003e\n\u003cli\u003eWang H\u003cem\u003e, et al.\u003c/em\u003e Brain-derived neurotrophic factor stimulation of T-type Ca2+ channels in sensory neurons contributes to increased peripheral pain sensitivity. \u003cem\u003eScience signaling\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, eaaw2300 (2019).\u003c/li\u003e\n\u003cli\u003eLiu Y\u003cem\u003e, et al.\u003c/em\u003e IGF-1 signaling pathway activation promotes axonal regeneration and repair: A mechanism study on catalpol-induced functional recovery after ischemic stroke. \u003cem\u003eJournal of Ethnopharmacology\u003c/em\u003e \u003cstrong\u003e348\u003c/strong\u003e, 119808 (2025).\u003c/li\u003e\n\u003cli\u003eTu X, Jain A, Parra Bueno P, Decker H, Liu X, Yasuda R. Local autocrine plasticity signaling in single dendritic spines by insulin-like growth factors. \u003cem\u003eScience Advances\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, eadg0666 (2023).\u003c/li\u003e\n\u003cli\u003eCai Z, Chen H, Boyle B, Rupp F, Funk W, Dedera D. Identification of a novel insulin-like growth factor binding protein gene homologue with tumor suppressor like properties. \u003cem\u003eBiochemical and biophysical research communications\u003c/em\u003e \u003cstrong\u003e331\u003c/strong\u003e, 261-266 (2005).\u003c/li\u003e\n\u003cli\u003eKashyap MK. Role of insulin-like growth factor-binding proteins in the pathophysiology and tumorigenesis of gastroesophageal cancers. \u003cem\u003eTumor Biology\u003c/em\u003e \u003cstrong\u003e36\u003c/strong\u003e, 8247-8257 (2015).\u003c/li\u003e\n\u003cli\u003eFirth SM, Baxter RC. Cellular actions of the insulin-like growth factor binding proteins. \u003cem\u003eEndocrine reviews\u003c/em\u003e \u003cstrong\u003e23\u003c/strong\u003e, 824-854 (2002).\u003c/li\u003e\n\u003cli\u003eBaxter RC. Signaling pathways of the insulin-like growth factor binding proteins. \u003cem\u003eEndocrine reviews\u003c/em\u003e \u003cstrong\u003e44\u003c/strong\u003e, 753-778 (2023).\u003c/li\u003e\n\u003cli\u003eOh Y, M\u0026uuml;ller HL, Lamson G, Rosenfeld RG. Insulin-like growth factor (IGF)-independent action of IGF-binding protein-3 in Hs578T human breast cancer cells. Cell surface binding and growth inhibition. \u003cem\u003eJournal of Biological Chemistry\u003c/em\u003e \u003cstrong\u003e268\u003c/strong\u003e, 14964-14971 (1993).\u003c/li\u003e\n\u003cli\u003eLewitt M, Boyd G. The role of Insulin-like Growth Factors (IGFs) and IGF-binding proteins in the nervous system. \u003cem\u003eBiochemistry Insights\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, (2019).\u003c/li\u003e\n\u003cli\u003eLi Y, Zhao E, Chen DF. Emerging roles for insulin-like growth factor binding protein like protein 1. \u003cem\u003eNeural Regeneration Research\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 258-259 (2019).\u003c/li\u003e\n\u003cli\u003eSmith P\u003cem\u003e, et al.\u003c/em\u003e Epigenetic inactivation implies independent functions for insulin-like growth factor binding protein (IGFBP)-related protein 1 and the related IGFBPL1 in inhibiting breast cancer phenotypes. \u003cem\u003eClinical cancer research\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 4061-4068 (2007).\u003c/li\u003e\n\u003cli\u003ePan L\u003cem\u003e, et al.\u003c/em\u003e IGFBPL1 is a master driver of microglia homeostasis and resolution of neuroinflammation in glaucoma and brain tauopathy. \u003cem\u003eCell reports\u003c/em\u003e \u003cstrong\u003e42\u003c/strong\u003e, (2023).\u003c/li\u003e\n\u003cli\u003eHou L\u003cem\u003e, et al.\u003c/em\u003e IGFBPL1 inhibits macrophage lipid accumulation by enhancing the activation of IGR1R/LXR\u0026alpha;/ABCG1 pathway. \u003cem\u003eAging (Albany NY)\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, 14791 (2023).\u003c/li\u003e\n\u003cli\u003eGuo C\u003cem\u003e, et al.\u003c/em\u003e IGFBPL1 regulates axon growth through IGF-1-mediated signaling cascades. \u003cem\u003eScientific reports\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 2054 (2018).\u003c/li\u003e\n\u003cli\u003eGonda Y, Sakurai H, Hirata Y, Tabata H, Ajioka I, Nakajima K. Expression profiles of Insulin-like growth factor binding protein-like 1 in the developing mouse forebrain. \u003cem\u003eGene expression patterns\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e, 431-440 (2007).\u003c/li\u003e\n\u003cli\u003eButti E\u003cem\u003e, et al.\u003c/em\u003e Neural precursor cells tune striatal connectivity through the release of IGFBPL1. \u003cem\u003eNature communications\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 7579 (2022).\u003c/li\u003e\n\u003cli\u003eQu Y, Cai R, Li Q, Wang H, Lu L. Neuroinflammation signatures in dorsal root ganglia following chronic constriction injury. \u003cem\u003eHeliyon\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, (2024).\u003c/li\u003e\n\u003cli\u003eStephens KE\u003cem\u003e, et al.\u003c/em\u003e Sex differences in gene regulation in the dorsal root ganglion after nerve injury. \u003cem\u003eBMC genomics\u003c/em\u003e \u003cstrong\u003e20\u003c/strong\u003e, 147 (2019).\u003c/li\u003e\n\u003cli\u003eCobos EJ\u003cem\u003e, et al.\u003c/em\u003e Mechanistic differences in neuropathic pain modalities revealed by correlating behavior with global expression profiling. \u003cem\u003eCell reports\u003c/em\u003e \u003cstrong\u003e22\u003c/strong\u003e, 1301-1312 (2018).\u003c/li\u003e\n\u003cli\u003eBennett GJ, Xie Y-K. A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man. \u003cem\u003ePain\u003c/em\u003e \u003cstrong\u003e33\u003c/strong\u003e, 87-107 (1988).\u003c/li\u003e\n\u003cli\u003eKim KJ, Chung JM. Comparison of three rodent neuropathic pain models. \u003cem\u003eExperimental brain research\u003c/em\u003e \u003cstrong\u003e113\u003c/strong\u003e, 200-206 (1997).\u003c/li\u003e\n\u003cli\u003eChen G\u003cem\u003e, et al.\u003c/em\u003e PD-L1 inhibits acute and chronic pain by suppressing nociceptive neuron activity via PD-1. \u003cem\u003eNature neuroscience\u003c/em\u003e \u003cstrong\u003e20\u003c/strong\u003e, 917-926 (2017).\u003c/li\u003e\n\u003cli\u003eBhuiyan SA\u003cem\u003e, et al.\u003c/em\u003e Harmonized cross-species cell atlases of trigeminal and dorsal root ganglia. \u003cem\u003eScience Advances\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, eadj9173 (2024).\u003c/li\u003e\n\u003cli\u003eZhang C\u003cem\u003e, et al.\u003c/em\u003e scRNA-sequencing reveals subtype-specific transcriptomic perturbations in DRG neurons of PirtEGFPf mice in neuropathic pain condition. \u003cem\u003eElife\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, e76063 (2022).\u003c/li\u003e\n\u003cli\u003eLi L\u003cem\u003e, et al.\u003c/em\u003e Peripheral CCL2 induces inflammatory pain via regulation of I h currents in small diameter DRG neurons. \u003cem\u003eFrontiers in Molecular Neuroscience\u003c/em\u003e \u003cstrong\u003e16\u003c/strong\u003e, 1144614 (2023).\u003c/li\u003e\n\u003cli\u003eDeng Y-T\u003cem\u003e, et al.\u003c/em\u003e Amphiregulin contributes to neuropathic pain by enhancing glycolysis that stimulates histone lactylation in sensory neurons. \u003cem\u003eScience Signaling\u003c/em\u003e \u003cstrong\u003e18\u003c/strong\u003e, eadr9397 (2025).\u003c/li\u003e\n\u003cli\u003eHu Q\u003cem\u003e, et al.\u003c/em\u003e TRPV1 channel contributes to the behavioral hypersensitivity in a rat model of complex regional pain syndrome type 1. \u003cem\u003eFrontiers in Pharmacology\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 453 (2019).\u003c/li\u003e\n\u003cli\u003eBleach R, Sherlock M, O\u0026rsquo;Reilly MW, McIlroy M. Growth hormone/insulin growth factor axis in sex steroid associated disorders and related cancers. \u003cem\u003eFrontiers in cell and developmental biology\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 630503 (2021).\u003c/li\u003e\n\u003cli\u003eMiura M\u003cem\u003e, et al.\u003c/em\u003e Peripheral sensitization caused by insulin-like growth factor 1 contributes to pain hypersensitivity after tissue injury. \u003cem\u003ePAIN\u0026reg;\u003c/em\u003e \u003cstrong\u003e152\u003c/strong\u003e, 888-895 (2011).\u003c/li\u003e\n\u003cli\u003eZhang Y\u003cem\u003e, et al.\u003c/em\u003e Peripheral pain is enhanced by insulin-like growth factor 1 through a G protein\u0026ndash;mediated stimulation of T-type calcium channels. \u003cem\u003eScience signaling\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e, ra94-ra94 (2014).\u003c/li\u003e\n\u003cli\u003eDai W\u003cem\u003e, et al.\u003c/em\u003e Calcium deficiency-induced and TRP channel-regulated IGF1R-PI3K-Akt signaling regulates abnormal epithelial cell proliferation. \u003cem\u003eCell Death \u0026amp; Differentiation\u003c/em\u003e \u003cstrong\u003e21\u003c/strong\u003e, 568-581 (2014).\u003c/li\u003e\n\u003cli\u003eMa L\u003cem\u003e, et al.\u003c/em\u003e IGF/IGF-1R signal pathway in pain: a promising therapeutic target. \u003cem\u003eInternational Journal of Biological Sciences\u003c/em\u003e \u003cstrong\u003e19\u003c/strong\u003e, 3472 (2023).\u003c/li\u003e\n\u003cli\u003eTauber M, Wang F, Kim B, Gaudenzio N. Bidirectional sensory neuron\u0026ndash;immune interactions: a new vision in the understanding of allergic inflammation. \u003cem\u003eCurrent Opinion in Immunology\u003c/em\u003e \u003cstrong\u003e72\u003c/strong\u003e, 79-86 (2021).\u003c/li\u003e\n\u003cli\u003eFattori V\u003cem\u003e, et al.\u003c/em\u003e Nociceptor-to-macrophage communication through CGRP/RAMP1 signaling drives endometriosis-associated pain and lesion growth in mice. \u003cem\u003eScience Translational Medicine\u003c/em\u003e \u003cstrong\u003e16\u003c/strong\u003e, eadk8230 (2024).\u003c/li\u003e\n\u003cli\u003eKim BS, Artis D. The sensory neuroimmune frontier. \u003cem\u003eImmunity\u003c/em\u003e \u003cstrong\u003e58\u003c/strong\u003e, 1033-1039 (2025).\u003c/li\u003e\n\u003cli\u003eLaumet G. Peripheral somatosensory neurons listen and orchestrate the immune response. \u003cem\u003eJournal of Allergy and Clinical Immunology\u003c/em\u003e \u003cstrong\u003e153\u003c/strong\u003e, 977-979 (2024).\u003c/li\u003e\n\u003cli\u003eTiwari N, Smith C, Sharma D, Shen S, Mehta P, Qiao LY. Plp1-expresssing perineuronal DRG cells facilitate colonic and somatic chronic mechanical pain involving Piezo2 upregulation in DRG neurons. \u003cem\u003eCell reports\u003c/em\u003e \u003cstrong\u003e43\u003c/strong\u003e, (2024).\u003c/li\u003e\n\u003cli\u003eRustioni S\u003cem\u003e, et al.\u003c/em\u003e A comprehensive review of the supraspinal mechanisms of spinal cord stimulation on chronic pain and cognition. \u003cem\u003eFrontiers in Pain Research\u003c/em\u003e \u003cstrong\u003e6\u003c/strong\u003e, 1589723 (2025).\u003c/li\u003e\n\u003cli\u003eAbd-Elsayed A, Vardhan S, Aggarwal A, Vardhan M, Diwan SA. Mechanisms of action of dorsal root ganglion stimulation. \u003cem\u003eInternational journal of molecular sciences\u003c/em\u003e \u003cstrong\u003e25\u003c/strong\u003e, 3591 (2024).\u003c/li\u003e\n\u003cli\u003eStachowski NJ, Dougherty KJ. Spinal inhibitory interneurons: gatekeepers of sensorimotor pathways. \u003cem\u003eInternational journal of molecular sciences\u003c/em\u003e \u003cstrong\u003e22\u003c/strong\u003e, 2667 (2021).\u003c/li\u003e\n\u003cli\u003eGirnita L, Worrall C, Takahashi S-I, Seregard S, Girnita A. Something old, something new and something borrowed: emerging paradigm of insulin-like growth factor type 1 receptor (IGF-1R) signaling regulation. \u003cem\u003eCellular and molecular life sciences\u003c/em\u003e \u003cstrong\u003e71\u003c/strong\u003e, 2403-2427 (2014).\u003c/li\u003e\n\u003cli\u003eThakkar B, Acevedo EO. BDNF as a biomarker for neuropathic pain: Consideration of mechanisms of action and associated measurement challenges. \u003cem\u003eBrain and Behavior\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, e2903 (2023).\u003c/li\u003e\n\u003cli\u003eXiong H-Y\u003cem\u003e, et al.\u003c/em\u003e The role of the brain-derived neurotrophic factor in chronic pain: links to central sensitization and neuroinflammation. \u003cem\u003eBiomolecules\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 71 (2024).\u003c/li\u003e\n\u003cli\u003eSchou WS, Ashina S, Amin FM, Goadsby PJ, Ashina M. Calcitonin gene-related peptide and pain: a systematic review. \u003cem\u003eThe journal of headache and pain\u003c/em\u003e \u003cstrong\u003e18\u003c/strong\u003e, 34 (2017).\u003c/li\u003e\n\u003cli\u003eAssas BM, Pennock JI, Miyan JA. Calcitonin gene-related peptide is a key neurotransmitter in the neuro-immune axis. \u003cem\u003eFrontiers in neuroscience\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 23 (2014).\u003c/li\u003e\n\u003cli\u003eBenemei S, Nicoletti P, Capone JG, Geppetti P. CGRP receptors in the control of pain and inflammation. \u003cem\u003eCurrent opinion in pharmacology\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 9-14 (2009).\u003c/li\u003e\n\u003cli\u003eWattiez A-S, Sowers LP, Russo AF. Calcitonin gene-related peptide (CGRP): role in migraine pathophysiology and therapeutic targeting. \u003cem\u003eExpert opinion on therapeutic targets\u003c/em\u003e \u003cstrong\u003e24\u003c/strong\u003e, 91-100 (2020).\u003c/li\u003e\n\u003cli\u003eMiddleton SJ\u003cem\u003e, et al.\u003c/em\u003e Studying human nociceptors: from fundamentals to clinic. \u003cem\u003eBrain\u003c/em\u003e \u003cstrong\u003e144\u003c/strong\u003e, 1312-1335 (2021).\u003c/li\u003e\n\u003cli\u003eGhitani N\u003cem\u003e, et al.\u003c/em\u003e Specialized mechanosensory nociceptors mediating rapid responses to hair pull. \u003cem\u003eNeuron\u003c/em\u003e \u003cstrong\u003e95\u003c/strong\u003e, 944-954. e944 (2017).\u003c/li\u003e\n\u003cli\u003eWang Y\u003cem\u003e, et al.\u003c/em\u003e TRPV1 SUMOylation regulates nociceptive signaling in models of inflammatory pain. \u003cem\u003eNature communications\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 1529 (2018).\u003c/li\u003e\n\u003cli\u003eFazen LE, Ringkamp M. The pathophysiology of neuropathic pain: a review of current research and hypotheses. \u003cem\u003eNeurosurgery Quarterly\u003c/em\u003e \u003cstrong\u003e17\u003c/strong\u003e, 245-262 (2007).\u003c/li\u003e\n\u003cli\u003eYu X\u003cem\u003e, et al.\u003c/em\u003e ACVR1-activating mutation causes neuropathic pain and sensory neuron hyperexcitability in humans. \u003cem\u003ePain\u003c/em\u003e \u003cstrong\u003e164\u003c/strong\u003e, 43-58 (2023).\u003c/li\u003e\n\u003cli\u003eRatte S, Prescott SA. Afferent hyperexcitability in neuropathic pain and the inconvenient truth about its degeneracy. \u003cem\u003eCurrent opinion in neurobiology\u003c/em\u003e \u003cstrong\u003e36\u003c/strong\u003e, 31-37 (2016).\u003c/li\u003e\n\u003cli\u003eLarsson O, Girnita A, Girnita L. Role of insulin-like growth factor 1 receptor signalling in cancer. \u003cem\u003eBritish journal of cancer\u003c/em\u003e \u003cstrong\u003e92\u003c/strong\u003e, 2097-2101 (2005).\u003c/li\u003e\n\u003cli\u003eSarfstein R, Nagaraj K, LeRoith D, Werner H. Differential effects of insulin and IGF1 receptors on ERK and AKT subcellular distribution in breast cancer cells. \u003cem\u003eCells\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 1499 (2019).\u003c/li\u003e\n\u003cli\u003eZhou L\u003cem\u003e, et al.\u003c/em\u003e IGF-1R promotes pain-like behavior in rats with myofascial pain syndrome via the PI3K/AKT pathway. \u003cem\u003eScientific Reports\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, 29050 (2025).\u003c/li\u003e\n\u003cli\u003eJiang B-C\u003cem\u003e, et al.\u003c/em\u003e Follistatin drives neuropathic pain in mice through IGF1R signaling in nociceptive neurons. \u003cem\u003eScience translational medicine\u003c/em\u003e \u003cstrong\u003e16\u003c/strong\u003e, eadi1564 (2024).\u003c/li\u003e\n\u003cli\u003eLiao Y\u003cem\u003e, et al.\u003c/em\u003e Interleukin-6 signaling mediates cartilage degradation and pain in posttraumatic osteoarthritis in a sex-specific manner. \u003cem\u003eScience signaling\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, eabn7082 (2022).\u003c/li\u003e\n\u003cli\u003eNie H\u003cem\u003e, et al.\u003c/em\u003e Neuronal Reg3\u0026beta;/macrophage TNF-\u0026alpha;\u0026ndash;mediated positive feedback signaling contributes to pain chronicity in a rat model of CRPS-I. \u003cem\u003eScience Advances\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, eadu4270 (2025).\u003c/li\u003e\n\u003cli\u003eZheng K\u003cem\u003e, et al.\u003c/em\u003e Chemokine CXCL13\u0026ndash;CXCR5 signaling in neuroinflammation and pathogenesis of chronic pain and neurological diseases. \u003cem\u003eCellular \u0026amp; Molecular Biology Letters\u003c/em\u003e \u003cstrong\u003e29\u003c/strong\u003e, 134 (2024).\u003c/li\u003e\n\u003cli\u003eZhao X\u003cem\u003e, et al.\u003c/em\u003e A long noncoding RNA contributes to neuropathic pain by silencing Kcna2 in primary afferent neurons. \u003cem\u003eNature neuroscience\u003c/em\u003e \u003cstrong\u003e16\u003c/strong\u003e, 1024-1031 (2013).\u003c/li\u003e\n\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-7783701/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7783701/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eNeuronal hyperexcitability and neuroimmune dysregulation are central hallmarks of neuropathic pain. IGFBPL1, a secreted glycoprotein and classical member of the insulin-like growth factor binding protein (IGFBP) family, has been implicated in GABAergic circuits, neurite outgrowth, and immune modulation in the CNS; however, its functional role in the peripheral nervous system (PNS), particularly in the somatosensory system, remains unexplored. Here, we report that IGFBPL1 is increased in the dorsal root ganglion (DRG) following peripheral nerve injury, and that this increase contributes to the development and maintenance of neuropathic pain. We show that IGFBPL1 upregulation in DRG sensory neurons induces pain behaviors and neuronal hyperexcitability through IGF1R\u0026ndash;ERK signaling and drives macrophage recruitment. Conversely, Igfbpl1-specific knockdown or pharmacological inhibition alleviates pain hypersensitivity, normalizes neuronal excitability, and reduces macrophage infiltration and neuroimmune crosstalk. Notably, Igfbpl1-specific knockdown also improved gait performance in chronic constriction injury (CCI) mice. Our findings identify IGFBPL1 as a critical regulator of DRG pathophysiology, linking growth factor signaling, sensory neuron plasticity, and neuroimmune interactions in neuropathic pain.\u003c/p\u003e","manuscriptTitle":"IGFBPL1 in DRG Nociceptors Drives Neuropathic Pain and Neuroimmune Crosstalk via IGF1R–ERK Signaling","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-19 19:12:03","doi":"10.21203/rs.3.rs-7783701/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":"0a98f70f-1c84-4c22-979c-d9d2368869c3","owner":[],"postedDate":"November 19th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":57698507,"name":"Biological sciences/Neuroscience/Somatosensory system/Pain/Chronic pain"},{"id":57698508,"name":"Health sciences/Diseases/Neurological disorders/Neuropathic pain"},{"id":57698509,"name":"Biological sciences/Neuroscience/Neuronal physiology/Excitability"},{"id":57698510,"name":"Health sciences/Health care/Therapeutics/Drug therapy/Molecularly targeted therapy"},{"id":57698511,"name":"Biological sciences/Neuroscience/Peripheral nervous system/Somatic system"}],"tags":[],"updatedAt":"2025-12-22T07:20:37+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-19 19:12:03","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7783701","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7783701","identity":"rs-7783701","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}