GluR2 overexpression in ACC glutamatergic neurons alleviates cancer-induced bone pain in rats

GluR2 overexpression in ACC glutamatergic neurons alleviates cancer-induced bone pain in rats | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article GluR2 overexpression in ACC glutamatergic neurons alleviates cancer-induced bone pain in rats Futing Ba, Jinrong Wei, Qi-Yan Feng, Chen-Yang Yu, Meng-Xue Song, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4820333/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 07 Apr, 2025 Read the published version in Molecular Medicine → Version 1 posted 13 You are reading this latest preprint version Abstract Background Cancer-induced bone pain (CIBP) is a complex chronic pain with poorly understood mechanisms. The anterior cingulate cortex (ACC) plays a critical role in processing and modulating chronic pain. This study investigates how the GluR2 receptors (calcium impermeable AMPA receptors) in ACC glutamatergic neurons regulate CIBP. Methods The CIBP models were established by injecting Walker 256 cells into the tibia of SD rats. Paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) were used as indicators of hyperalgesia. The immunofluorescence staining was employed to detect the expression of c-Fos in ACC and identify the subtypes of co-labeled c-Fos + neurons. Real-time monitoring of calcium activity in ACC glutamatergic neurons was achieved through the fiber photometry. The excitability of glutamatergic neurons in ACC was modulated using chemicalgenetics and optogenetics techniques. The expression of GluR2 at the mRNA and protein level in ACC were assessed using RT-qPCR and Western blotting. Results There were significant reductions in PWT and PWL of CIBP rats after Walker 256 cell injection. The ACC of CIBP rats showed increased c-Fos expression compared to sham rats, with mainly activated c-Fos co-localized with glutamatergic neurons. Optogenetic or chemogenetic activation of ACC glutamatergic neurons led to increased hyperalgesia in sham rats, while suppression of their activity alleviated hyperalgesia in CIBP rats. Calcium activity in ACC glutamatergic neurons of CIBP rats was increased with suprathreshold stimulation of von Frey filament. Notably, surface GluR2 protein and mRNA were reduced in ACC of CIBP rats. Furthermore, overexpression of GluR2 by AAV-CaMKIIα-GluR2 injection was decreased c-Fos expression in ACC and alleviated hyperalgesia in CIBP rats. Conclusions These findings suggest that decreased surface GluR2 receptors in ACC glutamatergic neurons contribute to calcium activity and excessive excitability, thereby inducing CIBP in rats. Conversely, GluR2 overexpression in ACC glutamatergic neurons alleviates CIBP in rats. This study provides a new potential therapeutic approach for targeting the GluR2 receptor to alleviate CIBP for cancer patients. Cancer-induced bone pain Anterior cingulate cortex Glutamatergic neurons AMPA receptors GluR2 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Cancer-induced bone pain (CIBP) is a persistent and worsening pain resulting from the cancer metastasis to the spinal cord cavity or bone tissues [ 1 ]. In breast cancer cases, about 75% involve bone metastasis, with a 5-year survival rate of 22.8% [ 2 ]. Skeletal-related events (SREs), including CIBP, affect up to 43% of patients with bone metastases, with 75% of advanced breast cancer patients experiencing SREs [ 3 ]. Patients with CIBP often face a persistent pain that worsens gradually, with unpredictable episodes of breakthrough pain, especially during activities like walking, exercise, and at night [ 4 ], This pain significantly impacts their quality of life and can worsen symptoms of depression and anxiety [ 5 ]. Despite systemic treatment, around 50% of CIBP patients still struggle with inadequate pain control and poor response to therapy [ 6 , 7 ]. The traditional 'step analgesia' approach, while effective in managing pain, does not prevent fractures, maintain mobility, or extend survival, and can have side effects [ 8 , 9 ]. Therefore, there is a critical need for new therapies to provide palliative care for cancer patients, easing pain and improving their quality of life. The Anterior cingulate cortex (ACC), which encompasses Brodmann areas 24, 32, and 33, is a critical component of the limbic system in the brain [ 10 ]. It functions as a central hub for pain regulation [ 11 ] and is involved in emotional processes like fear [ 12 ], empathy [ 13 , 14 ], cognition [ 15 ], and reward behavior [ 16 ]. Resting-state functional magnetic resonance imaging (rs-fMRI) has revealed abnormal activation in the cingulate, prefrontal cortex, and ventral striatum as part of the central nervous system's regulation of CIBP [ 17 ]. Recent studies have revealed that the ACC-NAc pathway is specifically linked to empathic responses related to pain and analgesia, emphasizing the connection between the ACC and human pain empathy [ 18 ]. Nevertheless, the precise mechanism by which pain stimulation triggers neuronal activity in the ACC and perpetuates the development and progression of CIBP remains unclear. Ionotropic glutamate receptors (iGluRs) are widely distributed in the brain, spinal cord, and peripheral nervous system. They act as ligand-gated ion channels, responding to glutamate release from presynaptic terminals. These receptors are crucial for excitatory synaptic transmission and synaptic plasticity in the central nervous system, contributing to various neurological disorders [ 19 ]. iGluRs are categorized into N-methyl-D-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA), and kainate (KA) receptors [ 20 ]. The AMPA receptors consist of subunits GluR1, GluR3, and GluR4 with Ca 2+ permeability and GluR2 lacking Ca 2+ permeability [ 21 ]. Following peripheral nerve injury, there are lasting changes in excitatory synaptic transmission in ACC glutamatergic neurons, leading to increased presynaptic glutamate release and enhanced postsynaptic glutamatergic AMPA receptor response, these changes are implicated in the maintenance of pain [ 22 ]. In a model of peripheral inflammatory pain hypersensitivity, it was observed that GluR2-containing AMPA receptors transitioned to a GluR2-lacking form, resulting in heightened Ca 2+ permeability mediated by AMPA receptors at synapses in spinal dorsal horn neurons, this process contributes to the development and persistence of peripheral inflammatory pain hypersensitivity [ 23 ]. These findings suggest that alterations in AMPA receptor subunits on postsynaptic neurons may be involved in maintaining pain sensitization. However, the specific mechanism by which GluR2-containing AMPA receptors regulate and sustain CIBP through ACC neurons remains unclear. Based on previous studies, we hypothesized that CIBP would activate ACC glutamatergic neurons, causing downregulation of AMPA receptors containing GluR2 on the postsynaptic membrane. This downregulation would result in increased permeability of the postsynaptic membrane to Ca 2+ , excessive excitability and hyperalgesia in rats with CIBP. The study may contribute to a better understanding of the mechanisms underlying hyperalgesia in CIBP, and potentially identify a clinical therapeutic target for managing cancer patients for pain relief. 2. Materials and methods 2.1. Animals Female Sprague-Dawley rats weighing 180–200 g were housed in groups of four per cage in temperature- (24 ± 1°C) and light-controlled (12-h light/dark cycle) room with access to standard rodent chow and water ad libitum . Animals were randomly assigned to different groups, and experiments were performed in blind manner. All procedures were approved by the Institutional Animal Care and Use Committee of Soochow University (SYXK 2022-0043). Animal experiments were conducted in strict accordance with the International Association for the Study of Pain (IASP). 2.2. Rat model of cancer-induced bone pain Implantation of Walker 256 cells was performed as previously described [ 24 – 27 ]. Briefly, Walker 256 cells were grown in SD rats (60–80 g) by intraperitoneal injection of 2×10 7 cells. One week later, the ascites was collected and resuspended with normal saline (1×10 8 cells/mL). After establishing isoflurane anesthesia, a small incision was made in the right leg of SD rats exposing the tibial plateau. A hole was drilled into the tibia cavity, and 10 µL tumor cells or NS was slowly injected into the hole using a 10 µL microinjection syringe. To prevent leakage of cells outside the bone, the injection site was closed with bone wax. The incision was then sutured, and the rats were transferred to a warm pad to wake. 2.3. Pain behavioral assessments The measurement of paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) described by our previous report [ 24 – 26 ]. In brief, the rats were habituated to a transparent Plexiglas box for 30 minutes before testing. PWT was assessed using the von Frey up-down method. The rat’s foot withdrawal in response to tactile stimulus was used to detect tactile allodynia using a series of calibrated nylon von Frey filaments. PWL was measured by heat radiation method for the thermal hyperalgesia of rats. The interval between the two tests was more than 5 minutes, and the test was repeated 5 times for each rat. 2.4. Immunofluorescence staining Rats were transcardially perfused with 0.9% normal saline and 4% paraformaldehyde (PFA) under deep anesthesia at 14 days after induction of the CIBP model. The brains were removed and immersed for post-fixation in PFA at 4℃ for 12 hours. After dehydration with sucrose gradient, brains were embedded in OCT and then cut into sections with 25–30 µm thickness. Brain sections were washed with PBS and blocked with blocking buffer containing 7% normal donkey serum, 0.3% Triton X-100, and 0.05% sodium azide for 1 hour at room temperature. After blocking, brain sections were incubated with primary antibodies solution diluted with donkey serum blocking solution overnight at 4℃. The following primary antibodies were purchased from commercial suppliers: anti-Mouse-c-Fos (Santa Cruz, 1: 200), anti-Rabbit-Glutamate (Cell Signaling Technology, 1: 100), anti-Mouse-CaMKIIα (Cell Signaling Technology, 1: 100), anti-Rabbit-GABA (Santa Cruz, 1: 200), anti-Mouse-NeuN (Millipore, 1: 50), anti-Rabbit-GluR2 (Abmart, 1: 200), anti-Mouse-GFAP (Cell Signaling Technology, 1: 100), anti-Goat-Ib1 (Abcam, 1: 200). After washing with PBS 3 times (10 minutes each time), the brain sections were incubated with secondary antibodies for 1 hour at room temperature. The following secondary antibodies were purchased from commercial suppliers: Alexa Fluor TM 488 Donkey Anti-Rabbit IgG (Thermo Fisher Scientific, 1: 500), Alexa Fluor TM 555 Donkey Anti-Mouse IgG (Thermo Fisher Scientific, 1: 200), Alexa Fluor TM 555 Donkey Anti-Goat IgG (Thermo Fisher Scientific, 1: 200). 2.5. Stereotaxic virus injection and optical fiber implantation Following deep anesthesia, rats were head fixed on a stereotaxic apparatus (RWD Life Science, China) with a bite bar and ear bars. After disinfection with iodophor solution, rat scalps were cut with a longitudinal midline incision to expose the skull. The skull above the target left ACC was removed carefully with a skull drill (RWD Life Science, China). The virus injection was made via a micro syringe (Gaoge, China) modified with glass micropipettes pulling by a Sutter Instrument P-97 micropipette puller. 300 nL of the virus was injected into the left ACC of the rat brain using a microsyringe pump (Longer Pump, China) at a rate of 30 nL/min. Front fontanelle as the origin of coordinates, virus injection coordinates are AP, + 1.0 mm; ML, + 0.5 mm; DV, -2.5 mm. To prevent virus backflow, the micropipette was left in place for approximately 10 minutes after virus injection and then retracted from the brain slowly. Optical fiber implantation was conducted immediately after virus injection. The optical fiber (diameter, 400 µm, Newdoon, China) were secured to the skull of each rat using 3–4 screws and dental cement. 2.6. Optical Stimulation One week prior to modeling, the CIBP group had been injected with AAV2/9-CaMKIIα-eNPHR-eGFP or AAV2/9-CaMKIIα-eGFP (from Gene Biotechnology, China, titer: 6.27×10 12 genome copies/ml) into the left ACC, with CON group injected with AAV2/9-CaMKIIα-ChR2-mCherry or AAV2/9-CaMKIIα-mCherry (from Gene Biotechnology, China, titer: 5.2×10 12 genome copies/ml). The behavioral tests were performed at 14 days after induction of the CIBP model. The delivery of a 20-s pulse of blue (473 nm, 2–5 mW, 20-ms pulses, 10 Hz) or yellow (594 nm, 3–5 mW, constant) light was controlled by optogenetic system (Alpha Omega Engineering, Israel). 2.7. Chemogenetic Manipulations One week prior to modeling, the CIBP group had been injected with AAV-CaMKIIα-hM4D(Gi)-mCherry (from BrainVTA, China, titer: 2.44×10 12 genome copies/ml) into the left ACC, with CON group injected with AAV-CaMKIIα-hM3D(Gq)-mCherry (from BrainVTA, China, titer: 5.40×10 12 genome copies/ml). At 14 days after induction of the CIBP model, the behavioral tests were measured before and at various time points (0.5, 1, 2, 4, 6, and 8h) after interperitoneal injection of clozapine-N-oxide (CNO, 3mg/kg, BrainVTA, China). 2.8. Fiber photometry system AAV-CaMKIIα-GCaMP6f (from Taitool Bioscience, China, titer: 3.6×10 12 genome copies/ml) was injected into the left ACC of the SD rats. GCaMP consists of an enhanced green fluorescent protein (eGFP) fused to calmodulin (CaM) and myosin light-chain kinase (M13) [ 28 ]. CaM is a target of Ca2 + within the cell can bind to Ca 2+ [ 29 ].This binding is believed to cause a conformational change in eGFP, transforming eGFP into a more efficient configuration. Fiber photometry system (ThinkerTech Nanjing Bioscience Inc.) allows for real-time excitation and recording of fluorescence from GCaMP in freely moving rats. Two excitation wavelengths, 405 and 470 nm, were used in this system, coupled into a 400 µm optical fiber by a commutator. The laser intensity was adjusted at the tip of the optical fiber to 10 to 20 mW. The photometry data were analyzed with custom-written MATLAB codes (MATLAB R2017b, MathWorks). 2.9. Real-time quantitative polymerase chain reaction for mRNA analysis Total RNA was extracted from the left ACC from the CON and CIBP rats using TRIzol (Ambion), and cDNA was synthesized from total RNA using an EasyScript One-Step gDNA Removal and cDNA Synthesis SuperMix kit (TransGen Biotech, China) following the manufacturer’s instructions. The expression level of mRNAs was normalized by the Ct value of GAPDH using the 2 ∆∆Ct relative quantification method. The mRNAs of GluR1, GluR2, GluR3, GluR4 and GAPDH (internal control) were measured in the quantitative polymerase chain reaction using the following primers: GluR1 forward primer: 5’-AATGTGGCAGGCGTGTTCTA-3’, reverse primer: 5’-GGATTGCATGGACTTGGGGA-3’; GluR2 forward primer: 5’-GCCAGAGTCCGGAAATCCAA-3’, reverse primer: 5’-CCGCACTCTCCTTTGTCGTA-3’; GluR3 forward primer: 5’-AGCCGTGCGATACGATGAAA-3’, reverse primer: 5’-ATAGAACACGCCTGCCACAT-3’; GluR4 forward primer: 5’-TACGACAAAGGAGAATGTGGCAG-3’, reverse primer: 5’-CAATGACAGCCAATCCCGAA-3’; GAPDH forward primer: 5’-TGGAGTCTACTGGCGTCTT-3’, reverse primer: 5’-TGTCATATTTCTCGTGGTTCA-3’. 2.10. Extraction of membrane protein and Western blotting Expressions of GluR1, GluR2, GluR3, GluR4, GAPDH and Na + -K + -ATPase in left ACC from CON and CIBP rats were measured using western blotting. According to the manufacturer’s instructions, membrane proteins were extracted by the Mem-PER™ Plus Kit (Thermo Fisher Scientific, USA). Supernatants of ultrasonic disruption were carefully prepared and the protein concentration was measured using a BCA Protein Quantitation Kit (Beyotime, China). The primary antibodies in the present study included anti-Rabbit-GAPDH (Goodhere, China, 1: 1000), anti-Rabbit-GluR1 (Abmart, China, 1: 2000), anti-Rabbit-GluR2 (Abmart, China, 1: 2000), anti-Rabbit-GluR3 (Abmart, China, 1: 2000), anti-Rabbit-GluR4 (Abmart, China, 1: 2000), Anti-Sodium Potassium ATPase (Abcam, USA, 1: 2000). The secondary antibody in the present study included anti-rabbit peroxidase-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, USA, 1: 2000). The densities of protein bands were analyzed using Image J (National Institutes of Health, Betheseda, MD). 2.11. Data analysis All data were analyzed using GraphPad Prism 8.0 and MATLAB software. All data are presented as mean values ± SEM. Normality was checked for all data before comparison. The t -test was used to determine significance of changes between 2 groups. Two-way repeated-measures analysis of variance (ANOVA) followed by Sidak’s multiple comparison test were performed where appropriate. P < 0.05 was regarded as statistically significant. 3. Results 3.1. Injection of Walker 256 cells into the tibia cavity induced mechanical and thermal hyperalgesia accompanied by an increased in c-Fos expression in ACC The paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) were measured before modeling (pre), 7 d, 14 d and 21 d after modeling to examine whether Walker 256 cell injection induces pain hyperalgesia. The results showed that the PWT and PWL in the ipsilateral hind-paw of the CIBP group were lower than that in the CON group at 7 d, 14 d and 21 d after modeling (Fig. 1 A, B). However, there was no significant difference in the contralateral PWT and PWL between the CON and CIBP groups (Fig. 1 C, D). These data showed that injection of Walker 256 cells into the tibia cavity induced mechanical and thermal hyperalgesia in rats, consistent with previous studies [ 24 – 26 ]. To investigate whether the ACC is involved in CIBP, we compared the expression of c-Fos in the ACC after rats were given suprathreshold stimulation using von Frey filament. The steps of suprathreshold stimulation are shown in Fig. 1 E. The results showed that the number of c-Fos + neurons was dramatically increased in the ACC of CIBP group as compared with CON group (Fig. 1 F, G). These results suggested that increasing neuronal activity in the ACC may be involved in the development and maintenance of CIBP in rats. 3.2. ACC glutamatergic neurons were the main type of neurons involved in CIBP To explore which type of neurons in the ACC were involved in CIBP, we double-labeled c-Fos + neurons with the glutamatergic neuron marker Glutamate and GABAergic neuron marker GABA. Immunostaining of c-Fos two week after CIBP showed that c-Fos + cells activated by CIBP were mainly co-localized with glutamate (Fig. 2 A), whereas only a few c-Fos + neurons were co-labeled with GABA (Fig. 2 C). The ratio of Glutamate + c-Fos to c-Fos was 81.75% (Fig. 2 B), and the ratio of GABA + c-Fos to c-Fos was 11.83% in ACC of CIBP rats (Fig. 2 D). These data indicated that glutamatergic neurons, but not GABAergic neurons were the main type of neurons involved in CIBP. 3.3. Optogenetic or chemogenetic inhibition of ACC glutamatergic neurons attenuated mechanical hyperalgesia in CIBP rats, whereas optogenetic or chemogenetic activation of ACC glutamatergic neurons promoted mechanical hyperalgesia in sham rats To further verify the role of ACC glutamatergic neurons in CIBP, we used optogenetic and chemogenetic technologies in vivo to selectively and temporarily inhibit glutamatergic neurons. The experimental procedure is depicted in Fig. 3 A and D. We injected AAV2/9-CaMKIIα-eNPHR-eGFP or AAV2/9-CaMKIIα-eGFP as a control into the ACC of CIBP rats to inhibit glutamatergic neurons (Fig. 3 B). The results showed that inhibition of ACC glutamatergic neurons by yellow-light significantly produced an acute and rapid attenuated in mechanical hyperalgesia in CIBP rats (Fig. 3 C). Meanwhile, we injected AAV-CaMKIIα-hM4D(Gi)-mCherry into the ACC of CIBP rats to inhibit glutamatergic neurons (Fig. 3 E). We found that 1h, 2h and 4h after CNO injection significantly attenuated mechanical hyperalgesia in CIBP rats (Fig. 3 G). However, there was no significant effect in thermal hyperalgesia (Fig. 3 H). Furthermore, we injected AAV2/9-CaMKIIα-ChR2-mCherry or AAV2/9-CaMKIIα-mCherry as a control into the ACC of sham rats to activate glutamatergic neurons (Fig. 3 B). The results showed that blue-light activation of the ACC glutamatergic neurons expressing ChR2-mCherry, but not those expressing mCherry, induced marked mechanical hyperalgesia, as manifested by a drastic decrease in PWT in sham rats (Fig. 3 F). At the same time, we injected AAV-CaMKIIα-hM3D(Gq)-mCherry into the ACC of sham rats to activate glutamatergic neurons (Fig. 3 E). We found that PWT was decreased from 1h to 4h after CNO injection (Fig. 3 I). However, there was no significant effect in thermal hyperalgesia (Fig. 3 J). Consequently, these data indicated that ACC glutamatergic neurons contributed to mechanical hyperalgesia in CIBP rats. 3.4. Calcium activity of ACC glutamatergic neurons was increased in CIBP rats To investigate dynamic activity of glutamatergic neurons in ACC of CIBP rats, we recorded their Ca 2+ levels in awake behaving rats using in vivo fibro photometry. Rats were stereotactically injected with AAV-CaMKIIα-GCaMP6f into ACC. One weeks after virus injection, rats were subjected to CIBP or sham-operation (Fig. 4 A, B). We found that a suprathreshold stimulation by von Frey filament stimulation induced a dynamic Ca 2+ fluctuation in both CIBP and sham rats (Fig. 4 C, D). However, the same suprathreshold stimulus induced dramatic Ca 2+ elevation in ACC glutamatergic neurons of CIBP rats (Fig. 4 E-G). These results confirmed that Ca 2+ activity in ACC glutamatergic neurons were selectively activated by CIBP. 3.5. Surface GluR2 receptors were decreased in the ACC of CIBP rats The levels of GluR1-4 mRNA in ACC of CIBP rats were detected by qPCR. Compared with the CON rats, the GluR2 mRNA in the left ACC of CIBP rats was reduced (Fig. 5 A). At the level of total protein, there was no significant alteration observed in the expression of GluR1-4 receptors in the left ACC between the CIBP and CON groups (Fig. 5 C-F). However, at the level of membrane proteins, the expression of GluR2 receptor in the left ACC was reduced in the CIBP group compared to the CON group (Fig. 5 B), while there were no significant changes observed in the expression of GluR1, GluR3 and GluR4 receptors (Fig. 5 G-I). 3.6. Overexpression of GluR2 alleviated mechanical hypersensitivity in CIBP rats To validate the impact of GluR2 on CIBP, we injected a virus that overexpresses GluR2 or a control virus into the left ACC of the rats. Compared to the AAV-CaMKIIα-NC group, the AAV-CaMKIIα-GluR2 group exhibited a significant upregulation in the mRNA level of GluR2, thereby indicating the efficacy of GluR2-overexpressing virus (Fig. 6 D). The experimental results demonstrated that there was no significant change in the PWT of rats in the AAV-CaMKIIα-GluR2 group on 7d, compares to the AAV-CaMKIIα-NC group; however, a significant increase was observed on 14d (Fig. 6 B). Furthermore, the PWL remained unchanged in the AAV-CaMKIIα-GluR2 group at both 7d and 14d (Fig. 6 C). Meanwhile, in comparison to the AAV-CaMKIIα-NC group, the AAV-CaMKIIα-GluR2 group exhibited a significant reduction in the number of c-Fos + cells in the left ACC (Fig. 6 A, E, F). These results indicate that decreased expression of the GluR2 receptors contributed to mechanical hyperalgesia, and GluR2 overexpression alleviated mechanical hypersensitivity in CIBP rats. 3.7. GluR2 receptors were mainly expressed in ACC glutamatergic neurons and negatively regulated mechanical hypersensitivity We investigated the distribution of GluR2 receptors in the ACC of normal rats by examining their co-localization with various cellular markers. The results showed that the GluR2 receptors were mainly co-expressed with NeuN (Neuronal nuclei) and glutamatergic neuron marker CaM-dependent protein kinase Ⅱ (CaMKⅡ), but they were not associated with Glial fibrillary acidic protein (GFAP) in astrocyte, and Ionized calcium binding adapter molecule 1 (Iba1) in microglia (Fig. 7 A, B). These results suggested that GluR2 receptors were mainly expressed in ACC glutamatergic neurons. In order to further verify the effects of GluR2 receptors in ACC glutamatergic neurons on PWT and PWL in CIBP rats, we injected GluR2 overexpression virus and AAV-CaMKIIα-hM3D(Gq)-mCherry virus (AAV-CaMKIIα-GluR2 + hM3D group) into the left ACC of rats (Fig. 7 C). The results showed that the PWT of CIBP rats in AAV-CaMKIIα-GluR2 + hM3D group was reduced at 1 h, 2 h and 4 h after the administration of CNO-activated glutamatergic neurons (Fig. 7 D), but the PWL had no significant change (Fig. 7 E). 4. Discussion Cancer-induced bone pain (CIBP) is a severe clinical condition that occurs when cancer cells spread to the spinal cord and bone tissue [ 30 ]. The pathophysiology of CIBP is complex, involving inflammatory factors, neuropathic components, and cancer-specific mediators [ 31 ]. Current pharmacological treatments for CIBP encompass nonsteroidal anti-inflammatory drugs (NSAIDs), potent opioid analgesics, bisphosphonates, and monoclonal antibodies targeting osteoclast activity [ 32 ]. While opioids are recommended as the first-line therapy by the World Health Organization (WHO), their use can be limited by adverse effects like gastrointestinal symptoms, fatigue, decreased consciousness, and respiratory depression [ 33 ]. Even with adherence to WHO guidelines, around 10–20% of CIBP patients still do not experience relief [ 34 ]. Therefore, understanding the pathophysiological mechanisms of CIBP and identifying new treatment targets are crucial. This study established a CIBP model in rats by injecting Walker 256 cells into the tibial bone marrow cavity, closely mimicking bone cancer pain from human breast cancer bone metastasis [ 35 – 39 ]. The results of PWT and PWL in the ipsilateral hind-paw of the CIBP group showed a gradual decrease as CIBP progressed. while no significant changes were observed in the contralateral hind-paw, the results indicate that the CIBP model has been successfully established. The ACC has been implicated in sensory processing, including pain perception [ 40 ], as well as motor and cognitive functions [ 41 ]. Early study revealed that cingulotomy provided pain relief to 60% of patients with advanced cancer or non-malignant tumors [ 42 ]. However, the specific role of ACC in various pain states, particularly CIBP, remains underexplored. By utilizing the immunofluorescence technique, a significant augmentation in the quantity of c-Fos + neurons within the ACC were observed in rats experiencing CIBP compared to those in the control group. The c-Fos + neurons were found to be co-localized with Glutamate-positive neurons This suggests a pivotal role of ACC glutamatergic neurons in CIBP regulation. Further validation was done through optogenetic and chemogenetic techniques, showing that inhibition of glutamatergic neurons in the ACC led to pain relief in CIBP rats, while activation resulted in increased pain perception in control rats. Additionally, in vivo fibro photometry revealed a significant increase in calcium activity of ACC glutamatergic neurons in CIBP rats. These findings collectively demonstrate ACC glutamatergic neurons contribute CIBP and their significant role in pain modulation. Glutamate is the primary excitatory neurotransmitter in the mammalian cerebral cortex [ 43 ]. Glutamate receptors are classified into two main categories: ionotropic glutamate receptors (iGluRs) and metabotropic glutamate receptors (mGluRs) [ 44 ]. AMPA receptors, a type of iGluR, have been shown through pharmacological, electrophysiological, and behavioral studies to play a vital role in regulating neuronal excitability and synaptic transmission in the central nervous system [ 45 ]. Specifically, AMPA receptors are crucial in transmitting pain signals. These receptors consist of subunits GluR1, GluR2, GluR3, and GluR4 [ 46 ]. The GluR2 subunit is particularly important, influencing the assembly, transport, and various forms of long-term potentiation (LTP) [ 47 ]. In the brain, most GluR2 exists in a Q/R-edited form that blocks Ca 2+ permeation [ 48 ]. Thus, AMPA receptors containing edited GluR2 subunits exhibit impermeability to Ca 2+ , whereas those lacking GluR2 or possessing unedited GluR2 are permeable to Ca 2+ [ 49 ]. Studies have linked GluR2 receptors at synapses to the development and progression of neuropathic pain, aversion, and depression following spinal cord nerve ligation (SNL) in rats [ 50 ]. The study examined the potential involvement of AMPA receptors in the development and maintenance of CIBP. RT-qPCR technology was used to detect the mRNA level of AMPA receptors in the left ACC of rats with CIBP. The results showed a significant decrease in GluR2 receptor mRNA levels, which was further confirmed by Western blotting demonstrating a reduction in GluR2 receptor membrane protein levels. These findings suggest that GluR2 receptors negatively regulate the occurrence and progression of CIBP. Immunofluorescence staining was then utilized to show the co-expression of GluR2 receptors with glutamatergic neurons, indicating that the downregulation of GluR2 receptors in ACC glutamatergic neurons' postsynaptic membranes may enhance Ca 2+ permeability and increase synaptic excitability, ultimately leading to heightened pain sensitivity in rats with CIBP. To further investigate the impact of GluR2 receptor on CIBP, we conducted experiments where AAV virus overexpressing GluR2 receptor was administered into the left ACC of CIBP rats. The results showed a significant decrease in c-Fos expression within their ACC. Behavioral tests revealed a notable increase in PWT following the microinjection of AAV-CaMKIIα-GluR2 into the left ACC, although no significant change was observed in PWL. Subsequent chemogenetic techniques indicated that activating glutamatergic neurons in the left ACC of CIBP rats could reverse the heightened PWT induced by AAV-CaMKIIα-GluR2 overexpression. These findings suggested that GluR2 overexpression in ACC glutamatergic neurons alleviates cancer-induced bone pain. Conclusions This study demonstrates that reducing surface GluR2 receptors in ACC glutamatergic neurons contribute to pain hypersensitivity in rats with CIBP, while overexpressing GluR2 receptors in ACC glutamatergic neurons could alleviate pain hypersensitivity in rats with CIBP. These findings provide a new theoretical basis for targeted treatment involving the GluR2 receptors and propose a potential clinical approach to alleviate CIBP for cancer patients. Declarations Acknowledgements The authors wish to thank all study participants, researchers, technicians, administrative staff, editors and reviewers who contributed to this study. Availability of data and materials All data generated or analyzed during this study are included in this published article. The datasets used or analyzed during the current study are available from the corresponding author upon reasonable request. Author Contributions F.B. and J.W. performed experiments, analyzed data, prepared figures, and wrote the manuscript. Q-Y.F., C-Y.Y. and M-X.S. performed experiments. S.H. and G-Y.X. edited the manuscript. G-Q.J. and H-L.Z. designed the study, supervised the work, and finalized the paper. All the authors have read and approved the paper. Funding This work was supported by grants from the National Natural Science Foundation of China (81873730 and 82201575). Ethics approval and consent to participate All procedures were approved by the Institutional Animal Care and Use Committee of Soochow University (SYXK 2022-0043). Animal experiments were conducted in strict accordance with the International Association for the Study of Pain (IASP). 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CIBP, n = 11, *\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.001, two-way ANOVA followed by Sidak’s multiple comparison test). \u003cstrong\u003e(C, D) \u003c/strong\u003eCompared with CON group, PWT and PWL in the contralateral hind-paw of the CIBP group were not different at 7 d, 14 d and 21 d after modeling (CON, n = 8; CIBP, n = 11, \u003cem\u003ep \u003c/em\u003e\u0026gt; 0.05, two-way ANOVA followed by Sidak’s multiple comparison test). \u003cstrong\u003e(E)\u003c/strong\u003e Schematic representation of c-Fos expression evoked by von Frey filament stimulation. \u003cstrong\u003e(F) \u003c/strong\u003eRepresentative images of c-Fos\u003csup\u003e+\u003c/sup\u003e cells in left ACC after von Frey filament stimulation, Scale bar: 100 μm. \u003cstrong\u003e(G)\u003c/strong\u003e Number of c-Fos\u003csup\u003e+ \u003c/sup\u003ecells in left ACC after von Frey filament stimulation (n = 4 brain sections from 4 rats for each group, *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, two-tailed t-test).\u003c/p\u003e","description":"","filename":"Slide1.png","url":"https://assets-eu.researchsquare.com/files/rs-4820333/v1/102cc28d38017a762d077e15.png"},{"id":64164009,"identity":"92c54531-7790-4741-9751-7a9547f335ee","added_by":"auto","created_at":"2024-09-09 08:45:33","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":431914,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCIBP mainly activated ACC glutamatergic neurons.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e Representative images of c-Fos (red) and Glutamate (green) co-expression in ACC of CIBP rats, Scale bar: 100 μm.\u003cstrong\u003e (B) \u003c/strong\u003ePercentage of co-expression of glutamate and c-Fos in ACC (n = 4 brain sections from 4 rats). \u003cstrong\u003e(C)\u003c/strong\u003e Representative images of c-Fos (red) and GABA (green) co-expression in ACC of CIBP rats, Scale bar: 100 μm.\u003cstrong\u003e (D)\u003c/strong\u003e Percentage of co-expression of GABA and c-Fos in ACC (n = 3 brain sections from 3 rats).\u003c/p\u003e","description":"","filename":"Slide2.png","url":"https://assets-eu.researchsquare.com/files/rs-4820333/v1/a0599301e429f31d00632fb8.png"},{"id":64163246,"identity":"5a1d237e-abef-42e0-9657-af850a54ec94","added_by":"auto","created_at":"2024-09-09 08:37:33","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":246118,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOptogenetic or chemogenetic inhibition of ACC glutamatergic neurons relieved mechanical hyperalgesia in CIBP rats, optogenetic or chemogenetic activation of ACC glutamatergic neurons induced mechanical hyperalgesia in sham rats.\u003c/strong\u003e \u003cstrong\u003e(A, D)\u003c/strong\u003e Schematic representation of optogenetic or chemogenetic experiment design. \u003cstrong\u003e(B)\u003c/strong\u003e Schematic diagram of the stereotaxic delivery of AAV and fiber into the ACC of CIBP rats and representative images of AAV2/9-CaMKIIα-eNPHR-eGFP (left) or AAV2/9-CaMKIIα-ChR2-mCherry (right) expression in the ACC, Scale bar: 100 μm. \u003cstrong\u003e(C) \u003c/strong\u003eYellow-light significantly elevated the PWT in CIBP rats transfected with eNPHR-eGFP, but not in rats transfected with eGFP in glutamatergic neurons (n = 6, *\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, two-way ANOVA followed by Sidak’s multiple comparison test).\u003cstrong\u003e (E)\u003c/strong\u003e Schematic diagram of the stereotaxic delivery of AAV into the ACC of CIBP rats and representative images of AAV-CaMKIIα-hM4D(Gi)-mCherry (left) or AAV-CaMKIIα-hM3D(Gq)-mCherry (right) expression in the ACC, Scale bar: 100 μm. \u003cstrong\u003e(F)\u003c/strong\u003eBlue-light significantly lowered the PWT in sham rats transfected with ChR2-mCherry, but not in rats transfected with mCherry in glutamatergic neurons (n = 6, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, two-way ANOVA followed by Sidak’s multiple comparison test).\u003cstrong\u003e (G, I)\u003c/strong\u003e The effects of PWT after 0.5h, 1h, 2h, 4h, 6h and 8h after CNO injection in rats that received injections of AAV-CaMKIIα-hM4Di-mCherry or AAV-CaMKIIα-hM3D(Gq)-mCherry in the ACC (n = 9, **\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, two-tailed \u003cem\u003et\u003c/em\u003e-test). \u003cstrong\u003e(H, J)\u003c/strong\u003e The effects of PWL after 0.5h, 1h, 2h, 4h, 6h and 8h after CNO injection in rats that received injections of AAV-CaMKIIα-hM4Di-mCherry or AAV-CaMKIIα-hM3D(Gq)-mCherry in the ACC (n = 9, \u003cem\u003ep \u003c/em\u003e\u0026gt; 0.05, two-tailed \u003cem\u003et\u003c/em\u003e-test).\u003c/p\u003e","description":"","filename":"Slide3.png","url":"https://assets-eu.researchsquare.com/files/rs-4820333/v1/48e5efcbc2e2a03c2867b7e5.png"},{"id":64163245,"identity":"62f2ae52-ae4d-4306-8ddb-6d7ca8b923ad","added_by":"auto","created_at":"2024-09-09 08:37:33","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":193072,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCalcium activity in ACC glutamatergic neurons was activated by CIBP.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e Schematic representation of in vivo fibro photometry experiment design.\u003cstrong\u003e (B)\u003c/strong\u003e Schematic of the stereotaxic delivery of AAV and fiber into the ACC (left) and representative images of AAV-CaMKIIα-GCaMP6f expression in the ACC (right), Scale bar: 100 μm.\u003cstrong\u003e (C, D)\u003c/strong\u003e Heatmap (left) and average Ca\u003csup\u003e2+ \u003c/sup\u003esignals (ΔF/F) (right) of ACC glutamatergic neurons in Sham (upper) and CIBP (lower) rats. \u003cstrong\u003e(E) \u003c/strong\u003eAverage Ca\u003csup\u003e2+ \u003c/sup\u003esignals (ΔF/F) of ACC glutamatergic neurons in Sham (blue) and CIBP (red) rats (Sham, n = 6; CIBP, n = 7).\u003cstrong\u003e (F)\u003c/strong\u003e Average area under the curve (AUC) of calcium activity of ACC glutamatergic neurons in Sham and CIBP rats (Sham, n = 6; CIBP, n = 7, ***\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.001, two-tailed\u003cem\u003e t\u003c/em\u003e-test). \u003cstrong\u003e(G)\u003c/strong\u003e Averaged peak ΔF/F of calcium activity of ACC glutamatergic neurons in Sham and CIBP rats (Sham, n = 6; CIBP, n = 7, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, two-tailed \u003cem\u003et\u003c/em\u003e-test).\u003c/p\u003e","description":"","filename":"Slide4.png","url":"https://assets-eu.researchsquare.com/files/rs-4820333/v1/d789efc8090086eef577e08b.png"},{"id":64163248,"identity":"6e1178c4-3c41-4182-af80-45dba12c8d4e","added_by":"auto","created_at":"2024-09-09 08:37:33","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":181219,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAlteration of surface AMPAR subunits in ACC of CIBP rats.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e The mRNA level of GluR1-4 in left ACC of CIBP rats (n = 4 for each group, *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, two-tailed\u003cem\u003e t\u003c/em\u003e-test).\u003cstrong\u003e (B)\u003c/strong\u003e The membrane protein expression of GluR2 in left ACC of CIBP rats (n = 4 for each group, *\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, two-tailed\u003cem\u003e t\u003c/em\u003e-test). \u003cstrong\u003e(C-F)\u003c/strong\u003e The total protein expression of GluR1, GluR2, GluR3 and GluR4 in left ACC of CIBP rats (n = 4 for each group, \u003cem\u003ep \u003c/em\u003e\u0026gt; 0.05, two-tailed \u003cem\u003et\u003c/em\u003e-test). \u003cstrong\u003e(G-I)\u003c/strong\u003e The membrane protein expression of GluR1, GluR3 and GluR4 in left ACC of CIBP rats (n = 4 for each group, \u003cem\u003ep \u003c/em\u003e\u0026gt; 0.05, two-tailed \u003cem\u003et\u003c/em\u003e-test). The left is representative of Western blots and the right is statistical plots for the relative intensity, GAPDH or Na\u003csup\u003e+\u003c/sup\u003e-K\u003csup\u003e+\u003c/sup\u003e-ATPase is used as an internal control.\u003c/p\u003e","description":"","filename":"Slide5.png","url":"https://assets-eu.researchsquare.com/files/rs-4820333/v1/5730c5b783fb73f5bb8250c0.png"},{"id":64163247,"identity":"7e1be732-2aea-4781-b7f5-a9c916af7691","added_by":"auto","created_at":"2024-09-09 08:37:33","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":166014,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGluR2 overexpression in ACC glutamatergic neurons attenuated mechanical hyperalgesia in CIBP rats. (A)\u003c/strong\u003e Schematic representation of c-Fos expression evoked by von Frey filament stimulation. \u003cstrong\u003e(B)\u003c/strong\u003e Compared with AAV-CaMKIIα-NC group, PWT in the ipsilateral hind-paw of the AAV-CaMKIIα-GluR2 group was increased at 14 d after modeling (n = 8 for each group, *\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, two-way ANOVA followed by Sidak’s multiple comparison test).\u003cstrong\u003e (C)\u003c/strong\u003e Compared with AAV-CaMKIIα-NC group, PWL in the ipsilateral hind-paw of the AAV-CaMKIIα-GluR2 group was not different at 7 d, 14 d after modeling (n = 8 for each group, \u003cem\u003ep \u003c/em\u003e\u0026gt; 0.05, two-way ANOVA followed by Sidak’s multiple comparison test).\u003cstrong\u003e (D)\u003c/strong\u003e GluR2 mRNA level in ACC of AAV-CaMKIIα-GluR2 group was upregulated compared with AAV-CaMKIIα-NC group (n = 4 for each group, *\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, two-tailed \u003cem\u003et\u003c/em\u003e-test).\u003cstrong\u003e (E)\u003c/strong\u003e Representative images of c-Fos\u003csup\u003e+\u003c/sup\u003e neurons in left ACC after von Frey filament stimulation. Scale bar:100 μm.\u003cstrong\u003e (F)\u003c/strong\u003e Number of c-Fos\u003csup\u003e+ \u003c/sup\u003ecells in ACC after von Frey filament stimulation (n = 3 for each group, ***\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.001, two-tailed \u003cem\u003et\u003c/em\u003e-test).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"Slide6.png","url":"https://assets-eu.researchsquare.com/files/rs-4820333/v1/6995ea6244a169f2714dd7d9.png"},{"id":64163249,"identity":"8d7fef69-2713-461c-ae09-1f67f044d18f","added_by":"auto","created_at":"2024-09-09 08:37:33","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":429357,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGluR2 receptors were mainly expressed in ACC glutamatergic neurons and negatively regulated mechanical hyperalgesia. (A) \u003c/strong\u003eRepresentative images of co-localization of GluR2 (green) with NeuN (red), CaMKⅡ (red), GFAP (red), or Iba1 (red) in ACC of normal rats, Scale bar:100 μm.\u003cstrong\u003e (B)\u003c/strong\u003e Quantified analysis showed GluR2 was mainly present in NeuN-positive neurons and CaMKⅡ-positive neurons with a very small amount in GFAP-positive astrocytes and Iba1-positive microglial cells in ACC.\u003cstrong\u003e(C) \u003c/strong\u003eSchematic representation of experiment design.\u003cstrong\u003e (D)\u003c/strong\u003e The effects of PWT after 0.5h, 1h, 2h, 4h, 6h and 8h after CNO injection in AAV-CaMKIIα-GluR2 + hM3D group that received injections of AAV-CaMKIIα-hM3D(Gq)-mCherry and AAV-CaMKIIα-GluR2 in the ACC (n = 8, *\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, *** \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.001, two-tailed\u003cem\u003e t\u003c/em\u003e-test). \u003cstrong\u003e(E)\u003c/strong\u003e The effects of PWL after 0.5h, 1h, 2h, 4h, 6h and 8h after CNO injection in AAV-CaMKIIα-GluR2 + hM3D group that received injections of AAV-CaMKIIα-hM3D(Gq)-mCherry and AAV-CaMKIIα-GluR2 in the ACC (n = 8, \u003cem\u003ep \u003c/em\u003e\u0026gt; 0.05, two-tailed \u003cem\u003et\u003c/em\u003e-test).\u003c/p\u003e","description":"","filename":"Slide7.png","url":"https://assets-eu.researchsquare.com/files/rs-4820333/v1/16c8b2550c12574595b5bc4f.png"},{"id":80558258,"identity":"c7d38984-6f17-4dc0-a2da-2eae52545b3a","added_by":"auto","created_at":"2025-04-14 16:14:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3333724,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4820333/v1/07d33cfe-40e0-4237-98a8-5d5477c69386.pdf"},{"id":64163242,"identity":"c16b62f6-b693-4010-80a4-217c1bd3993e","added_by":"auto","created_at":"2024-09-09 08:37:33","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":72143,"visible":true,"origin":"","legend":"","description":"","filename":"OriginalImagesforBlots.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4820333/v1/1e03dac4d31284078b0cee38.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"GluR2 overexpression in ACC glutamatergic neurons alleviates cancer-induced bone pain in rats","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eCancer-induced bone pain (CIBP) is a persistent and worsening pain resulting from the cancer metastasis to the spinal cord cavity or bone tissues [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In breast cancer cases, about 75% involve bone metastasis, with a 5-year survival rate of 22.8% [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Skeletal-related events (SREs), including CIBP, affect up to 43% of patients with bone metastases, with 75% of advanced breast cancer patients experiencing SREs [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Patients with CIBP often face a persistent pain that worsens gradually, with unpredictable episodes of breakthrough pain, especially during activities like walking, exercise, and at night [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], This pain significantly impacts their quality of life and can worsen symptoms of depression and anxiety [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Despite systemic treatment, around 50% of CIBP patients still struggle with inadequate pain control and poor response to therapy [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The traditional 'step analgesia' approach, while effective in managing pain, does not prevent fractures, maintain mobility, or extend survival, and can have side effects [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Therefore, there is a critical need for new therapies to provide palliative care for cancer patients, easing pain and improving their quality of life.\u003c/p\u003e \u003cp\u003eThe Anterior cingulate cortex (ACC), which encompasses Brodmann areas 24, 32, and 33, is a critical component of the limbic system in the brain [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. It functions as a central hub for pain regulation [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] and is involved in emotional processes like fear [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], empathy [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], cognition [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], and reward behavior [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Resting-state functional magnetic resonance imaging (rs-fMRI) has revealed abnormal activation in the cingulate, prefrontal cortex, and ventral striatum as part of the central nervous system's regulation of CIBP [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Recent studies have revealed that the ACC-NAc pathway is specifically linked to empathic responses related to pain and analgesia, emphasizing the connection between the ACC and human pain empathy [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Nevertheless, the precise mechanism by which pain stimulation triggers neuronal activity in the ACC and perpetuates the development and progression of CIBP remains unclear.\u003c/p\u003e \u003cp\u003eIonotropic glutamate receptors (iGluRs) are widely distributed in the brain, spinal cord, and peripheral nervous system. They act as ligand-gated ion channels, responding to glutamate release from presynaptic terminals. These receptors are crucial for excitatory synaptic transmission and synaptic plasticity in the central nervous system, contributing to various neurological disorders [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. iGluRs are categorized into N-methyl-D-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA), and kainate (KA) receptors [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The AMPA receptors consist of subunits GluR1, GluR3, and GluR4 with Ca\u003csup\u003e2+\u003c/sup\u003e permeability and GluR2 lacking Ca\u003csup\u003e2+\u003c/sup\u003e permeability [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Following peripheral nerve injury, there are lasting changes in excitatory synaptic transmission in ACC glutamatergic neurons, leading to increased presynaptic glutamate release and enhanced postsynaptic glutamatergic AMPA receptor response, these changes are implicated in the maintenance of pain [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In a model of peripheral inflammatory pain hypersensitivity, it was observed that GluR2-containing AMPA receptors transitioned to a GluR2-lacking form, resulting in heightened Ca\u003csup\u003e2+\u003c/sup\u003e permeability mediated by AMPA receptors at synapses in spinal dorsal horn neurons, this process contributes to the development and persistence of peripheral inflammatory pain hypersensitivity [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. These findings suggest that alterations in AMPA receptor subunits on postsynaptic neurons may be involved in maintaining pain sensitization. However, the specific mechanism by which GluR2-containing AMPA receptors regulate and sustain CIBP through ACC neurons remains unclear.\u003c/p\u003e \u003cp\u003eBased on previous studies, we hypothesized that CIBP would activate ACC glutamatergic neurons, causing downregulation of AMPA receptors containing GluR2 on the postsynaptic membrane. This downregulation would result in increased permeability of the postsynaptic membrane to Ca\u003csup\u003e2+\u003c/sup\u003e, excessive excitability and hyperalgesia in rats with CIBP. The study may contribute to a better understanding of the mechanisms underlying hyperalgesia in CIBP, and potentially identify a clinical therapeutic target for managing cancer patients for pain relief.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Animals\u003c/h2\u003e \u003cp\u003eFemale Sprague-Dawley rats weighing 180\u0026ndash;200 g were housed in groups of four per cage in temperature- (24\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C) and light-controlled (12-h light/dark cycle) room with access to standard rodent chow and water \u003cem\u003ead libitum\u003c/em\u003e. Animals were randomly assigned to different groups, and experiments were performed in blind manner. All procedures were approved by the Institutional Animal Care and Use Committee of Soochow University (SYXK 2022-0043). Animal experiments were conducted in strict accordance with the International Association for the Study of Pain (IASP).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Rat model of cancer-induced bone pain\u003c/h2\u003e \u003cp\u003eImplantation of Walker 256 cells was performed as previously described [\u003cspan additionalcitationids=\"CR25 CR26\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Briefly, Walker 256 cells were grown in SD rats (60\u0026ndash;80 g) by intraperitoneal injection of 2\u0026times;10\u003csup\u003e7\u003c/sup\u003e cells. One week later, the ascites was collected and resuspended with normal saline (1\u0026times;10\u003csup\u003e8\u003c/sup\u003e cells/mL). After establishing isoflurane anesthesia, a small incision was made in the right leg of SD rats exposing the tibial plateau. A hole was drilled into the tibia cavity, and 10 \u0026micro;L tumor cells or NS was slowly injected into the hole using a 10 \u0026micro;L microinjection syringe. To prevent leakage of cells outside the bone, the injection site was closed with bone wax. The incision was then sutured, and the rats were transferred to a warm pad to wake.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Pain behavioral assessments\u003c/h2\u003e \u003cp\u003eThe measurement of paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) described by our previous report [\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In brief, the rats were habituated to a transparent Plexiglas box for 30 minutes before testing. PWT was assessed using the von Frey up-down method. The rat\u0026rsquo;s foot withdrawal in response to tactile stimulus was used to detect tactile allodynia using a series of calibrated nylon von Frey filaments. PWL was measured by heat radiation method for the thermal hyperalgesia of rats. The interval between the two tests was more than 5 minutes, and the test was repeated 5 times for each rat.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Immunofluorescence staining\u003c/h2\u003e \u003cp\u003eRats were transcardially perfused with 0.9% normal saline and 4% paraformaldehyde (PFA) under deep anesthesia at 14 days after induction of the CIBP model. The brains were removed and immersed for post-fixation in PFA at 4℃ for 12 hours. After dehydration with sucrose gradient, brains were embedded in OCT and then cut into sections with 25\u0026ndash;30 \u0026micro;m thickness. Brain sections were washed with PBS and blocked with blocking buffer containing 7% normal donkey serum, 0.3% Triton X-100, and 0.05% sodium azide for 1 hour at room temperature. After blocking, brain sections were incubated with primary antibodies solution diluted with donkey serum blocking solution overnight at 4℃. The following primary antibodies were purchased from commercial suppliers: anti-Mouse-c-Fos (Santa Cruz, 1: 200), anti-Rabbit-Glutamate (Cell Signaling Technology, 1: 100), anti-Mouse-CaMKIIα (Cell Signaling Technology, 1: 100), anti-Rabbit-GABA (Santa Cruz, 1: 200), anti-Mouse-NeuN (Millipore, 1: 50), anti-Rabbit-GluR2 (Abmart, 1: 200), anti-Mouse-GFAP (Cell Signaling Technology, 1: 100), anti-Goat-Ib1 (Abcam, 1: 200). After washing with PBS 3 times (10 minutes each time), the brain sections were incubated with secondary antibodies for 1 hour at room temperature. The following secondary antibodies were purchased from commercial suppliers: Alexa Fluor TM 488 Donkey Anti-Rabbit IgG (Thermo Fisher Scientific, 1: 500), Alexa Fluor TM 555 Donkey Anti-Mouse IgG (Thermo Fisher Scientific, 1: 200), Alexa Fluor TM 555 Donkey Anti-Goat IgG (Thermo Fisher Scientific, 1: 200).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Stereotaxic virus injection and optical fiber implantation\u003c/h2\u003e \u003cp\u003eFollowing deep anesthesia, rats were head fixed on a stereotaxic apparatus (RWD Life Science, China) with a bite bar and ear bars. After disinfection with iodophor solution, rat scalps were cut with a longitudinal midline incision to expose the skull. The skull above the target left ACC was removed carefully with a skull drill (RWD Life Science, China). The virus injection was made via a micro syringe (Gaoge, China) modified with glass micropipettes pulling by a Sutter Instrument P-97 micropipette puller. 300 nL of the virus was injected into the left ACC of the rat brain using a microsyringe pump (Longer Pump, China) at a rate of 30 nL/min. Front fontanelle as the origin of coordinates, virus injection coordinates are AP, +\u0026thinsp;1.0 mm; ML, +\u0026thinsp;0.5 mm; DV, -2.5 mm. To prevent virus backflow, the micropipette was left in place for approximately 10 minutes after virus injection and then retracted from the brain slowly. Optical fiber implantation was conducted immediately after virus injection. The optical fiber (diameter, 400 \u0026micro;m, Newdoon, China) were secured to the skull of each rat using 3\u0026ndash;4 screws and dental cement.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Optical Stimulation\u003c/h2\u003e \u003cp\u003eOne week prior to modeling, the CIBP group had been injected with AAV2/9-CaMKIIα-eNPHR-eGFP or AAV2/9-CaMKIIα-eGFP (from Gene Biotechnology, China, titer: 6.27\u0026times;10\u003csup\u003e12\u003c/sup\u003e genome copies/ml) into the left ACC, with CON group injected with AAV2/9-CaMKIIα-ChR2-mCherry or AAV2/9-CaMKIIα-mCherry (from Gene Biotechnology, China, titer: 5.2\u0026times;10\u003csup\u003e12\u003c/sup\u003e genome copies/ml). The behavioral tests were performed at 14 days after induction of the CIBP model. The delivery of a 20-s pulse of blue (473 nm, 2\u0026ndash;5 mW, 20-ms pulses, 10 Hz) or yellow (594 nm, 3\u0026ndash;5 mW, constant) light was controlled by optogenetic system (Alpha Omega Engineering, Israel).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Chemogenetic Manipulations\u003c/h2\u003e \u003cp\u003eOne week prior to modeling, the CIBP group had been injected with AAV-CaMKIIα-hM4D(Gi)-mCherry (from BrainVTA, China, titer: 2.44\u0026times;10\u003csup\u003e12\u003c/sup\u003e genome copies/ml) into the left ACC, with CON group injected with AAV-CaMKIIα-hM3D(Gq)-mCherry (from BrainVTA, China, titer: 5.40\u0026times;10\u003csup\u003e12\u003c/sup\u003e genome copies/ml). At 14 days after induction of the CIBP model, the behavioral tests were measured before and at various time points (0.5, 1, 2, 4, 6, and 8h) after interperitoneal injection of clozapine-N-oxide (CNO, 3mg/kg, BrainVTA, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Fiber photometry system\u003c/h2\u003e \u003cp\u003eAAV-CaMKIIα-GCaMP6f (from Taitool Bioscience, China, titer: 3.6\u0026times;10\u003csup\u003e12\u003c/sup\u003e genome copies/ml) was injected into the left ACC of the SD rats. GCaMP consists of an enhanced green fluorescent protein (eGFP) fused to calmodulin (CaM) and myosin light-chain kinase (M13) [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. CaM is a target of Ca2\u003csup\u003e+\u003c/sup\u003e within the cell can bind to Ca\u003csup\u003e2+\u003c/sup\u003e [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].This binding is believed to cause a conformational change in eGFP, transforming eGFP into a more efficient configuration. Fiber photometry system (ThinkerTech Nanjing Bioscience Inc.) allows for real-time excitation and recording of fluorescence from GCaMP in freely moving rats. Two excitation wavelengths, 405 and 470 nm, were used in this system, coupled into a 400 \u0026micro;m optical fiber by a commutator. The laser intensity was adjusted at the tip of the optical fiber to 10 to 20 mW. The photometry data were analyzed with custom-written MATLAB codes (MATLAB R2017b, MathWorks).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Real-time quantitative polymerase chain reaction for mRNA analysis\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from the left ACC from the CON and CIBP rats using TRIzol (Ambion), and cDNA was synthesized from total RNA using an EasyScript One-Step gDNA Removal and cDNA Synthesis SuperMix kit (TransGen Biotech, China) following the manufacturer\u0026rsquo;s instructions. The expression level of mRNAs was normalized by the Ct value of GAPDH using the 2\u003csup\u003e∆∆Ct\u003c/sup\u003e relative quantification method. The mRNAs of GluR1, GluR2, GluR3, GluR4 and GAPDH (internal control) were measured in the quantitative polymerase chain reaction using the following primers:\u003c/p\u003e \u003cp\u003eGluR1 forward primer: 5\u0026rsquo;-AATGTGGCAGGCGTGTTCTA-3\u0026rsquo;,\u003c/p\u003e \u003cp\u003ereverse primer: 5\u0026rsquo;-GGATTGCATGGACTTGGGGA-3\u0026rsquo;;\u003c/p\u003e \u003cp\u003eGluR2 forward primer: 5\u0026rsquo;-GCCAGAGTCCGGAAATCCAA-3\u0026rsquo;,\u003c/p\u003e \u003cp\u003ereverse primer: 5\u0026rsquo;-CCGCACTCTCCTTTGTCGTA-3\u0026rsquo;;\u003c/p\u003e \u003cp\u003eGluR3 forward primer: 5\u0026rsquo;-AGCCGTGCGATACGATGAAA-3\u0026rsquo;,\u003c/p\u003e \u003cp\u003ereverse primer: 5\u0026rsquo;-ATAGAACACGCCTGCCACAT-3\u0026rsquo;;\u003c/p\u003e \u003cp\u003eGluR4 forward primer: 5\u0026rsquo;-TACGACAAAGGAGAATGTGGCAG-3\u0026rsquo;,\u003c/p\u003e \u003cp\u003ereverse primer: 5\u0026rsquo;-CAATGACAGCCAATCCCGAA-3\u0026rsquo;;\u003c/p\u003e \u003cp\u003eGAPDH forward primer: 5\u0026rsquo;-TGGAGTCTACTGGCGTCTT-3\u0026rsquo;,\u003c/p\u003e \u003cp\u003ereverse primer: 5\u0026rsquo;-TGTCATATTTCTCGTGGTTCA-3\u0026rsquo;.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10. Extraction of membrane protein and Western blotting\u003c/h2\u003e \u003cp\u003eExpressions of GluR1, GluR2, GluR3, GluR4, GAPDH and Na\u003csup\u003e+\u003c/sup\u003e-K\u003csup\u003e+\u003c/sup\u003e-ATPase in left ACC from CON and CIBP rats were measured using western blotting. According to the manufacturer\u0026rsquo;s instructions, membrane proteins were extracted by the Mem-PER\u0026trade; Plus Kit (Thermo Fisher Scientific, USA). Supernatants of ultrasonic disruption were carefully prepared and the protein concentration was measured using a BCA Protein Quantitation Kit (Beyotime, China). The primary antibodies in the present study included anti-Rabbit-GAPDH (Goodhere, China, 1: 1000), anti-Rabbit-GluR1 (Abmart, China, 1: 2000), anti-Rabbit-GluR2 (Abmart, China, 1: 2000), anti-Rabbit-GluR3 (Abmart, China, 1: 2000), anti-Rabbit-GluR4 (Abmart, China, 1: 2000), Anti-Sodium Potassium ATPase (Abcam, USA, 1: 2000). The secondary antibody in the present study included anti-rabbit peroxidase-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, USA, 1: 2000). The densities of protein bands were analyzed using Image J (National Institutes of Health, Betheseda, MD).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11. Data analysis\u003c/h2\u003e \u003cp\u003eAll data were analyzed using GraphPad Prism 8.0 and MATLAB software. All data are presented as mean values\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Normality was checked for all data before comparison. The \u003cem\u003et\u003c/em\u003e-test was used to determine significance of changes between 2 groups. Two-way repeated-measures analysis of variance (ANOVA) followed by Sidak\u0026rsquo;s multiple comparison test were performed where appropriate. \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was regarded as statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003e \u003cb\u003e3.1. Injection of Walker 256 cells into the tibia cavity induced mechanical and thermal hyperalgesia accompanied by an increased in c-Fos expression in ACC\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) were measured before modeling (pre), 7 d, 14 d and 21 d after modeling to examine whether Walker 256 cell injection induces pain hyperalgesia. The results showed that the PWT and PWL in the ipsilateral hind-paw of the CIBP group were lower than that in the CON group at 7 d, 14 d and 21 d after modeling (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B). However, there was no significant difference in the contralateral PWT and PWL between the CON and CIBP groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, D). These data showed that injection of Walker 256 cells into the tibia cavity induced mechanical and thermal hyperalgesia in rats, consistent with previous studies [\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. To investigate whether the ACC is involved in CIBP, we compared the expression of c-Fos in the ACC after rats were given suprathreshold stimulation using von Frey filament. The steps of suprathreshold stimulation are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE. The results showed that the number of c-Fos\u003csup\u003e+\u003c/sup\u003e neurons was dramatically increased in the ACC of CIBP group as compared with CON group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF, G). These results suggested that increasing neuronal activity in the ACC may be involved in the development and maintenance of CIBP in rats.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.2. ACC glutamatergic neurons were the main type of neurons involved in CIBP\u003c/h2\u003e \u003cp\u003eTo explore which type of neurons in the ACC were involved in CIBP, we double-labeled c-Fos\u003csup\u003e+\u003c/sup\u003e neurons with the glutamatergic neuron marker Glutamate and GABAergic neuron marker GABA. Immunostaining of c-Fos two week after CIBP showed that c-Fos\u003csup\u003e+\u003c/sup\u003e cells activated by CIBP were mainly co-localized with glutamate (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), whereas only a few c-Fos\u003csup\u003e+\u003c/sup\u003e neurons were co-labeled with GABA (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). The ratio of Glutamate\u0026thinsp;+\u0026thinsp;c-Fos to c-Fos was 81.75% (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), and the ratio of GABA\u0026thinsp;+\u0026thinsp;c-Fos to c-Fos was 11.83% in ACC of CIBP rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). These data indicated that glutamatergic neurons, but not GABAergic neurons were the main type of neurons involved in CIBP.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e3.3. Optogenetic or chemogenetic inhibition of ACC glutamatergic neurons attenuated mechanical hyperalgesia in CIBP rats, whereas optogenetic or chemogenetic activation of ACC glutamatergic neurons promoted mechanical hyperalgesia in sham rats\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo further verify the role of ACC glutamatergic neurons in CIBP, we used optogenetic and chemogenetic technologies in vivo to selectively and temporarily inhibit glutamatergic neurons. The experimental procedure is depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and D. We injected AAV2/9-CaMKIIα-eNPHR-eGFP or AAV2/9-CaMKIIα-eGFP as a control into the ACC of CIBP rats to inhibit glutamatergic neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). The results showed that inhibition of ACC glutamatergic neurons by yellow-light significantly produced an acute and rapid attenuated in mechanical hyperalgesia in CIBP rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Meanwhile, we injected AAV-CaMKIIα-hM4D(Gi)-mCherry into the ACC of CIBP rats to inhibit glutamatergic neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). We found that 1h, 2h and 4h after CNO injection significantly attenuated mechanical hyperalgesia in CIBP rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). However, there was no significant effect in thermal hyperalgesia (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurthermore, we injected AAV2/9-CaMKIIα-ChR2-mCherry or AAV2/9-CaMKIIα-mCherry as a control into the ACC of sham rats to activate glutamatergic neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). The results showed that blue-light activation of the ACC glutamatergic neurons expressing ChR2-mCherry, but not those expressing mCherry, induced marked mechanical hyperalgesia, as manifested by a drastic decrease in PWT in sham rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). At the same time, we injected AAV-CaMKIIα-hM3D(Gq)-mCherry into the ACC of sham rats to activate glutamatergic neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). We found that PWT was decreased from 1h to 4h after CNO injection (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI). However, there was no significant effect in thermal hyperalgesia (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ). Consequently, these data indicated that ACC glutamatergic neurons contributed to mechanical hyperalgesia in CIBP rats.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Calcium activity of ACC glutamatergic neurons was increased in CIBP rats\u003c/h2\u003e \u003cp\u003eTo investigate dynamic activity of glutamatergic neurons in ACC of CIBP rats, we recorded their Ca\u003csup\u003e2+\u003c/sup\u003e levels in awake behaving rats using in vivo fibro photometry. Rats were stereotactically injected with AAV-CaMKIIα-GCaMP6f into ACC. One weeks after virus injection, rats were subjected to CIBP or sham-operation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B). We found that a suprathreshold stimulation by von Frey filament stimulation induced a dynamic Ca\u003csup\u003e2+\u003c/sup\u003e fluctuation in both CIBP and sham rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, D). However, the same suprathreshold stimulus induced dramatic Ca\u003csup\u003e2+\u003c/sup\u003e elevation in ACC glutamatergic neurons of CIBP rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE-G). These results confirmed that Ca\u003csup\u003e2+\u003c/sup\u003e activity in ACC glutamatergic neurons were selectively activated by CIBP.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Surface GluR2 receptors were decreased in the ACC of CIBP rats\u003c/h2\u003e \u003cp\u003eThe levels of GluR1-4 mRNA in ACC of CIBP rats were detected by qPCR. Compared with the CON rats, the GluR2 mRNA in the left ACC of CIBP rats was reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). At the level of total protein, there was no significant alteration observed in the expression of GluR1-4 receptors in the left ACC between the CIBP and CON groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC-F). However, at the level of membrane proteins, the expression of GluR2 receptor in the left ACC was reduced in the CIBP group compared to the CON group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB), while there were no significant changes observed in the expression of GluR1, GluR3 and GluR4 receptors (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG-I).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.6. Overexpression of GluR2 alleviated mechanical hypersensitivity in CIBP rats\u003c/h2\u003e \u003cp\u003eTo validate the impact of GluR2 on CIBP, we injected a virus that overexpresses GluR2 or a control virus into the left ACC of the rats. Compared to the AAV-CaMKIIα-NC group, the AAV-CaMKIIα-GluR2 group exhibited a significant upregulation in the mRNA level of GluR2, thereby indicating the efficacy of GluR2-overexpressing virus (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). The experimental results demonstrated that there was no significant change in the PWT of rats in the AAV-CaMKIIα-GluR2 group on 7d, compares to the AAV-CaMKIIα-NC group; however, a significant increase was observed on 14d (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Furthermore, the PWL remained unchanged in the AAV-CaMKIIα-GluR2 group at both 7d and 14d (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Meanwhile, in comparison to the AAV-CaMKIIα-NC group, the AAV-CaMKIIα-GluR2 group exhibited a significant reduction in the number of c-Fos\u003csup\u003e+\u003c/sup\u003e cells in the left ACC (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, E, F). These results indicate that decreased expression of the GluR2 receptors contributed to mechanical hyperalgesia, and GluR2 overexpression alleviated mechanical hypersensitivity in CIBP rats.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.7. GluR2 receptors were mainly expressed in ACC glutamatergic neurons and negatively regulated mechanical hypersensitivity\u003c/h2\u003e \u003cp\u003eWe investigated the distribution of GluR2 receptors in the ACC of normal rats by examining their co-localization with various cellular markers. The results showed that the GluR2 receptors were mainly co-expressed with NeuN (Neuronal nuclei) and glutamatergic neuron marker CaM-dependent protein kinase Ⅱ (CaMKⅡ), but they were not associated with Glial fibrillary acidic protein (GFAP) in astrocyte, and Ionized calcium binding adapter molecule 1 (Iba1) in microglia (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, B). These results suggested that GluR2 receptors were mainly expressed in ACC glutamatergic neurons. In order to further verify the effects of GluR2 receptors in ACC glutamatergic neurons on PWT and PWL in CIBP rats, we injected GluR2 overexpression virus and AAV-CaMKIIα-hM3D(Gq)-mCherry virus (AAV-CaMKIIα-GluR2\u0026thinsp;+\u0026thinsp;hM3D group) into the left ACC of rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). The results showed that the PWT of CIBP rats in AAV-CaMKIIα-GluR2\u0026thinsp;+\u0026thinsp;hM3D group was reduced at 1 h, 2 h and 4 h after the administration of CNO-activated glutamatergic neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD), but the PWL had no significant change (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eCancer-induced bone pain (CIBP) is a severe clinical condition that occurs when cancer cells spread to the spinal cord and bone tissue [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The pathophysiology of CIBP is complex, involving inflammatory factors, neuropathic components, and cancer-specific mediators [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Current pharmacological treatments for CIBP encompass nonsteroidal anti-inflammatory drugs (NSAIDs), potent opioid analgesics, bisphosphonates, and monoclonal antibodies targeting osteoclast activity [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. While opioids are recommended as the first-line therapy by the World Health Organization (WHO), their use can be limited by adverse effects like gastrointestinal symptoms, fatigue, decreased consciousness, and respiratory depression [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Even with adherence to WHO guidelines, around 10–20% of CIBP patients still do not experience relief [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Therefore, understanding the pathophysiological mechanisms of CIBP and identifying new treatment targets are crucial. This study established a CIBP model in rats by injecting Walker 256 cells into the tibial bone marrow cavity, closely mimicking bone cancer pain from human breast cancer bone metastasis [\u003cspan additionalcitationids=\"CR36 CR37 CR38\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e–\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe results of PWT and PWL in the ipsilateral hind-paw of the CIBP group showed a gradual decrease as CIBP progressed. while no significant changes were observed in the contralateral hind-paw, the results indicate that the CIBP model has been successfully established. The ACC has been implicated in sensory processing, including pain perception [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], as well as motor and cognitive functions [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Early study revealed that cingulotomy provided pain relief to 60% of patients with advanced cancer or non-malignant tumors [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. However, the specific role of ACC in various pain states, particularly CIBP, remains underexplored. By utilizing the immunofluorescence technique, a significant augmentation in the quantity of c-Fos\u003csup\u003e+\u003c/sup\u003e neurons within the ACC were observed in rats experiencing CIBP compared to those in the control group. The c-Fos\u003csup\u003e+\u003c/sup\u003e neurons were found to be co-localized with Glutamate-positive neurons This suggests a pivotal role of ACC glutamatergic neurons in CIBP regulation. Further validation was done through optogenetic and chemogenetic techniques, showing that inhibition of glutamatergic neurons in the ACC led to pain relief in CIBP rats, while activation resulted in increased pain perception in control rats. Additionally, in vivo fibro photometry revealed a significant increase in calcium activity of ACC glutamatergic neurons in CIBP rats. These findings collectively demonstrate ACC glutamatergic neurons contribute CIBP and their significant role in pain modulation.\u003c/p\u003e \u003cp\u003eGlutamate is the primary excitatory neurotransmitter in the mammalian cerebral cortex [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Glutamate receptors are classified into two main categories: ionotropic glutamate receptors (iGluRs) and metabotropic glutamate receptors (mGluRs) [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. AMPA receptors, a type of iGluR, have been shown through pharmacological, electrophysiological, and behavioral studies to play a vital role in regulating neuronal excitability and synaptic transmission in the central nervous system [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Specifically, AMPA receptors are crucial in transmitting pain signals. These receptors consist of subunits GluR1, GluR2, GluR3, and GluR4 [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. The GluR2 subunit is particularly important, influencing the assembly, transport, and various forms of long-term potentiation (LTP) [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. In the brain, most GluR2 exists in a Q/R-edited form that blocks Ca\u003csup\u003e2+\u003c/sup\u003e permeation [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Thus, AMPA receptors containing edited GluR2 subunits exhibit impermeability to Ca\u003csup\u003e2+\u003c/sup\u003e, whereas those lacking GluR2 or possessing unedited GluR2 are permeable to Ca\u003csup\u003e2+\u003c/sup\u003e [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Studies have linked GluR2 receptors at synapses to the development and progression of neuropathic pain, aversion, and depression following spinal cord nerve ligation (SNL) in rats [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. The study examined the potential involvement of AMPA receptors in the development and maintenance of CIBP. RT-qPCR technology was used to detect the mRNA level of AMPA receptors in the left ACC of rats with CIBP. The results showed a significant decrease in GluR2 receptor mRNA levels, which was further confirmed by Western blotting demonstrating a reduction in GluR2 receptor membrane protein levels. These findings suggest that GluR2 receptors negatively regulate the occurrence and progression of CIBP. Immunofluorescence staining was then utilized to show the co-expression of GluR2 receptors with glutamatergic neurons, indicating that the downregulation of GluR2 receptors in ACC glutamatergic neurons' postsynaptic membranes may enhance Ca\u003csup\u003e2+\u003c/sup\u003e permeability and increase synaptic excitability, ultimately leading to heightened pain sensitivity in rats with CIBP.\u003c/p\u003e \u003cp\u003eTo further investigate the impact of GluR2 receptor on CIBP, we conducted experiments where AAV virus overexpressing GluR2 receptor was administered into the left ACC of CIBP rats. The results showed a significant decrease in c-Fos expression within their ACC. Behavioral tests revealed a notable increase in PWT following the microinjection of AAV-CaMKIIα-GluR2 into the left ACC, although no significant change was observed in PWL. Subsequent chemogenetic techniques indicated that activating glutamatergic neurons in the left ACC of CIBP rats could reverse the heightened PWT induced by AAV-CaMKIIα-GluR2 overexpression. These findings suggested that GluR2 overexpression in ACC glutamatergic neurons alleviates cancer-induced bone pain.\u003c/p\u003e "},{"header":"Conclusions","content":"\u003cp\u003eThis study demonstrates that reducing surface GluR2 receptors in ACC glutamatergic neurons contribute to pain hypersensitivity in rats with CIBP, while overexpressing GluR2 receptors in ACC glutamatergic neurons could alleviate pain hypersensitivity in rats with CIBP. These findings provide a new theoretical basis for targeted treatment involving the GluR2 receptors and propose a potential clinical approach to alleviate CIBP for cancer patients.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors wish to thank all study participants, researchers, technicians, administrative staff, editors and reviewers who contributed to this study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article. The datasets used or analyzed during the current study are available from the corresponding author upon reasonable request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eF.B. and J.W. performed experiments, analyzed data, prepared figures, and wrote the manuscript. Q-Y.F., C-Y.Y. and M-X.S. performed experiments. S.H. and G-Y.X. edited the manuscript. G-Q.J. and H-L.Z. designed the study, supervised the work, and finalized the paper. All the authors have read and approved the paper.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants from the National Natural Science Foundation of China (81873730 and 82201575).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll procedures were approved by the Institutional Animal Care and Use Committee of Soochow University (SYXK 2022-0043). Animal experiments were conducted in strict accordance with the International Association for the Study of Pain (IASP).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declared no potential conflicts of interest with respect to the research, authorship, and publication of this article.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eYang, Y., et al., \u003cem\u003ePeripheral Mechanism of Cancer-Induced Bone Pain.\u003c/em\u003e Neuroscience Bulletin, 2023.\u003c/li\u003e\n\u003cli\u003eMacedo, F., et al., \u003cem\u003eBone Metastases: An Overview.\u003c/em\u003e Oncology Reviews, 2017. \u003cstrong\u003e11\u003c/strong\u003e(1): p. 321.\u003c/li\u003e\n\u003cli\u003eGillespie, E.F., et al., \u003cem\u003eProphylactic Radiation Therapy Versus Standard of Care for Patients With High-Risk Asymptomatic Bone Metastases: A Multicenter, Randomized Phase II Clinical Trial.\u003c/em\u003e Journal of Clinical Oncology : Official Journal of the American Society of Clinical Oncology, 2023: p. 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The anterior cingulate cortex (ACC) plays a critical role in processing and modulating chronic pain. This study investigates how the GluR2 receptors (calcium impermeable AMPA receptors) in ACC glutamatergic neurons regulate CIBP.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThe CIBP models were established by injecting Walker 256 cells into the tibia of SD rats. Paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) were used as indicators of hyperalgesia. The immunofluorescence staining was employed to detect the expression of c-Fos in ACC and identify the subtypes of co-labeled c-Fos\u003csup\u003e+\u003c/sup\u003e neurons. Real-time monitoring of calcium activity in ACC glutamatergic neurons was achieved through the fiber photometry. The excitability of glutamatergic neurons in ACC was modulated using chemicalgenetics and optogenetics techniques. The expression of GluR2 at the mRNA and protein level in ACC were assessed using RT-qPCR and Western blotting.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThere were significant reductions in PWT and PWL of CIBP rats after Walker 256 cell injection. The ACC of CIBP rats showed increased c-Fos expression compared to sham rats, with mainly activated c-Fos co-localized with glutamatergic neurons. Optogenetic or chemogenetic activation of ACC glutamatergic neurons led to increased hyperalgesia in sham rats, while suppression of their activity alleviated hyperalgesia in CIBP rats. Calcium activity in ACC glutamatergic neurons of CIBP rats was increased with suprathreshold stimulation of von Frey filament. Notably, surface GluR2 protein and mRNA were reduced in ACC of CIBP rats. Furthermore, overexpression of GluR2 by AAV-CaMKIIα-GluR2 injection was decreased c-Fos expression in ACC and alleviated hyperalgesia in CIBP rats.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThese findings suggest that decreased surface GluR2 receptors in ACC glutamatergic neurons contribute to calcium activity and excessive excitability, thereby inducing CIBP in rats. Conversely, GluR2 overexpression in ACC glutamatergic neurons alleviates CIBP in rats. This study provides a new potential therapeutic approach for targeting the GluR2 receptor to alleviate CIBP for cancer patients.\u003c/p\u003e","manuscriptTitle":"GluR2 overexpression in ACC glutamatergic neurons alleviates cancer-induced bone pain in rats","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-09-09 08:37:28","doi":"10.21203/rs.3.rs-4820333/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-01-23T17:19:24+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-01-22T12:22:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"68000386155676108090834357103889709413","date":"2025-01-13T13:08:26+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-01-09T21:12:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"158500805916902707487213550841231848174","date":"2024-12-18T20:40:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"41564420477406727757756091965570125318","date":"2024-10-27T01:17:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"290451899075697460822670970848054371165","date":"2024-10-04T08:29:30+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-19T16:05:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"62256878780638426204720279895536700632","date":"2024-08-08T18:06:31+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-08-08T16:53:27+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-08-03T03:28:07+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-08-03T03:26:56+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Medicine","date":"2024-07-29T08:35:12+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mome","sideBox":"Learn more about [Molecular Medicine](https://molmed.biomedcentral.com)","snPcode":"10020","submissionUrl":"https://submission.springernature.com/new-submission/10020/3","title":"Molecular Medicine","twitterHandle":"@MolecularMedic1","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d8956044-54b7-4a4f-bba4-5a2f571fe9d8","owner":[],"postedDate":"September 9th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-04-14T16:07:34+00:00","versionOfRecord":{"articleIdentity":"rs-4820333","link":"https://doi.org/10.1186/s10020-025-01183-9","journal":{"identity":"molecular-medicine","isVorOnly":false,"title":"Molecular Medicine"},"publishedOn":"2025-04-07 16:04:53","publishedOnDateReadable":"April 7th, 2025"},"versionCreatedAt":"2024-09-09 08:37:28","video":"","vorDoi":"10.1186/s10020-025-01183-9","vorDoiUrl":"https://doi.org/10.1186/s10020-025-01183-9","workflowStages":[]},"version":"v1","identity":"rs-4820333","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4820333","identity":"rs-4820333","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}