Background & Aims
Repetitive transcranial magnetic stimulation (rTMS) has emerged as a promising intervention for neuropathic pain (NP) treatment; however, the underlying mechanisms contributing to its efficacy remain poorly understood. Our previous study demonstrated that inhibiting P2X7R in the amygdala effectively relieves NP. This investigation aims to identify the key differentially expressed genes in the amygdala of NP rats and elucidate their regulatory roles with P2X7 receptor (P2X7R) in response to rTMS treatment. Our objective is to shed light on potential molecular mechanisms and offer clinical support for rTMS treatment.
Methods
We established a rat model of NP using the sciatic nerve chronic constriction injury (CCI) method and employed 0.5 Hz rTMS for treatment. Pain-related behavior and abnormal remodeling of dendritic spines in the amygdala were assessed to evaluate treatment effects. Transcriptome sequencing was used to identify key differentially expressed genes in the amygdala after rTMS treatment. Co-immunoprecipitation (Co-IP) experiments were performed to confirm the interaction between the key gene and P2X7R. The regulatory mechanisms of the key gene and its interaction with P2X7R/ NLRP3 inflammatory signaling pathway response to rTMS treatment were validated by induce gene overexpress and silencing through lentivirus, RT-qPCR, Western blot, and immunohistochemistry.
Results
rTMS effectively alleviated pain-related behavior and improved abnormal remodeling of dendritic spines in the amygdala of NP rats. RNA transcriptome sequencing identified integrin ?v?3 as a key gene in rTMS treatment for NP. Expression of integrin ?v?3 in amygdala tissue was increased in NP rats, and rTMS reversed this effect. Co-IP experiments demonstrated a definitive interaction between integrin ?v?3 and its receptor P2X7R in the amygdala of NP rats, which was blocked by rTMS treatment. Both overexpression and silencing of integrin ?v?3 and P2X7R regulates pain-related behavior and abnormal remodeling of dendritic spines in the amygdala of NP rats. Notably, the overexpression of integrin ?v?3 and P2X7R counteracted the therapeutic effect of rTMS. Furthermore, the inhibitor of the NLRP3 inflammatory signaling pathway partially countered the overexpression of P2X7R in pain-related behavior and inflammatory expression.
Conclusions
Our study demonstrates the efficacy of rTMS in improving pain-related behaviors and abnormal remodeling of dendritic spines in the amygdala of NP rats. We identify a potential link between amygdala neural activity and integrin ?v?3, unveil a significant interaction between integrin ?v?3 and P2X7R in the amygdala, suggesting their involvement in the response to rTMS treatment. Further investigation into P2X7R downstream signaling pathways reveals the activation of the NLRP3 inflammatory signaling pathway as an intrinsic mechanism underlying rTMS efficacy. These results imply a potential mechanism of rTMS treatment for NP by blocking the interaction between integrin ?v?3 and P2X7R in the amygdala, consequently inhibiting the NLRP3 inflammatory pathway.
References
1. Zhou W, Xie Z, Li C, Xing Z, Xie S, Li M, et al. Driving effect of BDNF in the spinal dorsal horn on neuropathic pain. Neurosci Lett 2021;756:135965. doi: 10.1016/j.neulet.2021.135965.
2. Baron R, Binder A, Wasner G. Neuropathic pain: diagnosis, pathophysiological mechanisms, and treatment. Lancet Neurol 2010;9:807-819. doi: 10.1016/S1474-4422(10)70143-5.
3. Moisset X, Page MG. Interest of registries in neuropathic pain research. Rev Neurol (Paris) 2021;177:843-848. doi: 10.1016/j.neurol.2021.07.011.
4. Drat-Gzubicka J, Pyszora A, Budzynski J, Currow D, Krajnik M. Is Neuropathic Pain a Good Marker of Peripheral Neuropathy in Hospice Patients with Advanced Cancer? The Single Center Pilot Study. Diagnostics (Basel) 2021;11. doi: 10.3390/diagnostics11081377.
5. Dowell D, Ragan KR, Jones CM, Baldwin GT, Chou R. CDC Clinical Practice Guideline for Prescribing Opioids for Pain – United States, 2022. MMWR Recomm Rep 2022;71:1-95. doi: 10.15585/mmwr.rr7103a1.
6. Giusti EM, Castelnuovo G, Molinari E. Differences in Multidisciplinary and Interdisciplinary Treatment Programs for Fibromyalgia: A Mapping Review. Pain Res Manag 2017;2017:7261468. doi: 10.1155/2017/7261468.
7. Bates D, Schultheis BC, Hanes MC, Jolly SM, Chakravarthy KV, Deer TR, et al. A Comprehensive Algorithm for Management of Neuropathic Pain. Pain Med 2019;20:S2-S12. doi: 10.1093/pm/pnz075.
8. Varshney V, Osborn J, Chaturvedi R, Shah V, Chakravarthy K. Advances in the interventional management of neuropathic pain. Ann Transl Med 2021;9:187. doi: 10.21037/atm-20-6190.
9. Cavalli E, Mammana S, Nicoletti F, Bramanti P, Mazzon E. The neuropathic pain: An overview of the current treatment and future therapeutic approaches. Int J Immunopathol Pharmacol 2019;33:2058738419838383. doi: 10.1177/2058738419838383.
10. Fornasari D. Pharmacotherapy for Neuropathic Pain: A Review. Pain Ther 2017;6:25-33. doi: 10.1007/s40122-017-0091-4.
11. Attia M, McCarthy D, Abdelghani M. Repetitive Transcranial Magnetic Stimulation for Treating Chronic Neuropathic Pain: a Systematic Review. Curr Pain Headache Rep 2021;25:48. doi: 10.1007/s11916-021-00960-5.
12. Zang Y, Zhang Y, Lai X, Yang Y, Guo J, Gu S, et al. Evidence Mapping Based on Systematic Reviews of Repetitive Transcranial Magnetic Stimulation on the Motor Cortex for Neuropathic Pain. Front Hum Neurosci 2021;15:743846. doi: 10.3389/fnhum.2021.743846.
13. Kim JK, Park HS, Bae JS, Jeong YS, Jung KJ, Lim JY. Effects of multi-session intermittent theta burst stimulation on central neuropathic pain: A randomized controlled trial. NeuroRehabilitation 2020;46:127-134. doi: 10.3233/NRE-192958.
14. Finnerup NB, Kuner R, Jensen TS. Neuropathic Pain: From Mechanisms to Treatment. Physiol Rev 2021;101:259-301. doi: 10.1152/physrev.00045.2019.
15. Klomjai W, Katz R, Lackmy-Vallee A. Basic principles of transcranial magnetic stimulation (TMS) and repetitive TMS (rTMS). Ann Phys Rehabil Med 2015;58:208-213. doi: 10.1016/j.rehab.2015.05.005.
16. Tomeh A, Yusof Khan AHK, Inche Mat LN, Basri H, Wan Sulaiman WA. Repetitive Transcranial Magnetic Stimulation of the Primary Motor Cortex beyond Motor Rehabilitation: A Review of the Current Evidence. Brain Sci 2022;12. doi: 10.3390/brainsci12060761.
17. Pei Q, Wu B, Tang Y, Yang X, Song L, Wang N, et al. Repetitive Transcranial Magnetic Stimulation at Different Frequencies for Postherpetic Neuralgia: A Double-Blind, Sham-Controlled, Randomized Trial. Pain Physician 2019;22:E303-E313. doi.
18. Xing Y, Zhang Y, Li C, Luo L, Hua Y, Hu J, et al. Repetitive Transcranial Magnetic Stimulation of the Brain After Ischemic Stroke: Mechanisms from Animal Models. Cell Mol Neurobiol 2023;43:1487-1497. doi: 10.1007/s10571-022-01264-x.
19. Nurmikko T, MacIver K, Bresnahan R, Hird E, Nelson A, Sacco P. Motor Cortex Reorganization and Repetitive Transcranial Magnetic Stimulation for Pain-A Methodological Study. Neuromodulation 2016;19:669-678. doi: 10.1111/ner.12444.
20. Mori N, Hosomi K, Nishi A, Oshino S, Kishima H, Saitoh Y. Analgesic Effects of Repetitive Transcranial Magnetic Stimulation at Different Stimulus Parameters for Neuropathic Pain: A Randomized Study. Neuromodulation 2022;25:520-527. doi: 10.1111/ner.13328.
21. Zhao X, Li Y, Tian Q, Zhu B, Zhao Z. Repetitive transcranial magnetic stimulation increases serum brain-derived neurotrophic factor and decreases interleukin-1beta and tumor necrosis factor-alpha in elderly patients with refractory depression. J Int Med Res 2019;47:1848-1855. doi: 10.1177/0300060518817417.
22. Toledo RS, Stein DJ, Sanches PRS, da Silva LS, Medeiros HR, Fregni F, et al. rTMS induces analgesia and modulates neuroinflammation and neuroplasticity in neuropathic pain model rats. Brain Res 2021;1762:147427. doi: 10.1016/j.brainres.2021.147427.
23. Kim JY, Choi GS, Cho YW, Cho H, Hwang SJ, Ahn SH. Attenuation of spinal cord injury-induced astroglial and microglial activation by repetitive transcranial magnetic stimulation in rats. J Korean Med Sci 2013;28:295-299. doi: 10.3346/jkms.2013.28.2.295.
24. Luo J, Feng Y, Li M, Yin M, Qin F, Hu X. Repetitive Transcranial Magnetic Stimulation Improves Neurological Function and Promotes the Anti-inflammatory Polarization of Microglia in Ischemic Rats. Front Cell Neurosci 2022;16:878345. doi: 10.3389/fncel.2022.878345.
25. Starobova H, S WAH, Lewis RJ, Vetter I. Transcriptomics in pain research: insights from new and old technologies. Mol Omics 2018;14:389-404. doi: 10.1039/c8mo00181b.
26. Athie MCP, Vieira AS, Teixeira JM, Dos Santos GG, Dias EV, Tambeli CH, et al. Transcriptome analysis of dorsal root ganglia’s diabetic neuropathy reveals mechanisms involved in pain and regeneration. Life Sci 2018;205:54-62. doi: 10.1016/j.lfs.2018.05.016.
27. Jeong H, Na YJ, Lee K, Kim YH, Lee Y, Kang M, et al. High-resolution transcriptome analysis reveals neuropathic pain gene-expression signatures in spinal microglia after nerve injury. Pain 2016;157:964-976. doi: 10.1097/j.pain.0000000000000470.
28. Ray PR, Khan J, Wangzhou A, Tavares-Ferreira D, Akopian AN, Dussor G, et al. Transcriptome Analysis of the Human Tibial Nerve Identifies Sexually Dimorphic Expression of Genes Involved in Pain, Inflammation, and Neuro-Immunity. Front Mol Neurosci 2019;12:37. doi: 10.3389/fnmol.2019.00037.
29. Ray P, Torck A, Quigley L, Wangzhou A, Neiman M, Rao C, et al. Comparative transcriptome profiling of the human and mouse dorsal root ganglia: an RNA-seq-based resource for pain and sensory neuroscience research. Pain 2018;159:1325-1345. doi: 10.1097/j.pain.0000000000001217.
30. Kasai S, Ikeda K. Reduced supraspinal nociceptive responses and distinct gene expression profile in CXBH recombinant inbred mice. J Pain 2013;14:648-661. doi: 10.1016/j.jpain.2013.01.773.
31. Zeisel A, Munoz-Manchado AB, Codeluppi S, Lonnerberg P, La Manno G, Jureus A, et al. Brain structure. Cell types in the mouse cortex and hippocampus revealed by single-cell RNA-seq. Science 2015;347:1138-1142. doi: 10.1126/science.aaa1934.
32. Farmer MA, Baliki MN, Apkarian AV. A dynamic network perspective of chronic pain. Neurosci Lett 2012;520:197-203. doi: 10.1016/j.neulet.2012.05.001.
33. Martins D, Dipasquale O, Veronese M, Turkheimer F, Loggia ML, McMahon S, et al. Transcriptional and cellular signatures of cortical morphometric remodelling in chronic pain. Pain 2022;163:e759-e773. doi: 10.1097/j.pain.0000000000002480.
34. Hwang W, Choi JK, Bang MS, Park WY, Oh BM. Gene Expression Profile Changes in the Stimulated Rat Brain Cortex After Repetitive Transcranial Magnetic Stimulation. Brain Neurorehabil 2022;15:e27. doi: 10.12786/bn.2022.15.e27.
35. Liang SH, Zhao WJ, Yin JB, Chen YB, Li JN, Feng B, et al. A Neural Circuit from Thalamic Paraventricular Nucleus to Central Amygdala for the Facilitation of Neuropathic Pain. J Neurosci 2020;40:7837-7854. doi: 10.1523/JNEUROSCI.2487-19.2020.
36. Huang J, Gadotti VM, Chen L, Souza IA, Huang S, Wang D, et al. A neuronal circuit for activating descending modulation of neuropathic pain. Nat Neurosci 2019;22:1659-1668. doi: 10.1038/s41593-019-0481-5.
37. Dalhuisen I, Ackermans E, Martens L, Mulders P, Bartholomeus J, de Bruijn A, et al. Longitudinal effects of rTMS on neuroplasticity in chronic treatment-resistant depression. Eur Arch Psychiatry Clin Neurosci 2021;271:39-47. doi: 10.1007/s00406-020-01135-w.
38. Tjalkens RB, Popichak KA, Kirkley KA. Inflammatory Activation of Microglia and Astrocytes in Manganese Neurotoxicity. Adv Neurobiol 2017;18:159-181. doi: 10.1007/978-3-319-60189-2_8.
39. Lu HJ, Gao YJ. Astrocytes in Chronic Pain: Cellular and Molecular Mechanisms. Neurosci Bull 2023;39:425-439. doi: 10.1007/s12264-022-00961-3.
40. Li T, Chen X, Zhang C, Zhang Y, Yao W. An update on reactive astrocytes in chronic pain. J Neuroinflammation 2019;16:140. doi: 10.1186/s12974-019-1524-2.
41. Lin JP, Chen CQ, Huang LE, Li NN, Yang Y, Zhu SM, et al. Dexmedetomidine Attenuates Neuropathic Pain by Inhibiting P2X7R Expression and ERK Phosphorylation in Rats. Exp Neurobiol 2018;27:267-276. doi: 10.5607/en.2018.27.4.267.
42. Qin B, Luo N, Li Y, Gong D, Zheng J, Tan X, et al. Protective effect of gastrodin on peripheral neuropathy induced by anti-tumor treatment with vincristine in rat models. Drug Chem Toxicol 2021;44:84-91. doi: 10.1080/01480545.2018.1547739.
43. Bernier LP, Ase AR, Seguela P. P2X receptor channels in chronic pain pathways. Br J Pharmacol 2018;175:2219-2230. doi: 10.1111/bph.13957.
44. Yang J, Park KS, Yoon JJ, Bae HB, Yoon MH, Choi JI. Anti-allodynic effect of intrathecal processed Aconitum jaluense is associated with the inhibition of microglial activation and P2X7 receptor expression in spinal cord. BMC Complement Altern Med 2016;16:214. doi: 10.1186/s12906-016-1201-2.
45. Chessell IP, Hatcher JP, Bountra C, Michel AD, Hughes JP, Green P, et al. Disruption of the P2X7 purinoceptor gene abolishes chronic inflammatory and neuropathic pain. Pain 2005;114:386-396. doi: 10.1016/j.pain.2005.01.002.
46. Hu X, Liu Y, Wu J, Liu Y, Liu W, Chen J, et al. Inhibition of P2X7R in the amygdala ameliorates symptoms of neuropathic pain after spared nerve injury in rats. Brain Behav Immun 2020;88:507-514. doi: 10.1016/j.bbi.2020.04.030.
47. Wu Q, Yue J, Lin L, Yu X, Zhou Y, Ying X, et al. Electroacupuncture may alleviate neuropathic pain via suppressing P2X7R expression. Mol Pain 2021;17:1744806921997654. doi: 10.1177/1744806921997654.
48. Wu Z, Liu Q. Effects of repetitive transcranial magnetic stimulation combined with acupuncture on NLRP3 inflammasome and protease levels in patients with neuropathic pain. Am J Transl Res 2023;15:4699-4708. doi.
49. Sun W, Zhang N, Liu B, Yang J, Loers G, Siebert HC, et al. HDAC3 Inhibitor RGFP966 Ameliorated Neuroinflammation in the Cuprizone-Induced Demyelinating Mouse Model and LPS-Stimulated BV2 Cells by Downregulating the P2X7R/STAT3/NF-kappaB65/NLRP3 Activation. ACS Chem Neurosci 2022;13:2579-2598. doi: 10.1021/acschemneuro.1c00826.
50. Yang L, Zhou G, Chen J, Zhang S. Gelsemine relieves the neuropathic pain by down-regulating DPP4 level in rats. Neurosci Lett 2023;792:136961. doi: 10.1016/j.neulet.2022.136961.
51. Yu H, Zhang Z, Wei F, Hou G, You Y, Wang X, et al. Hydroxytyrosol Ameliorates Intervertebral Disc Degeneration and Neuropathic Pain by Reducing Oxidative Stress and Inflammation. Oxid Med Cell Longev 2022;2022:2240894. doi: 10.1155/2022/2240894.
52. Chen L, Wang H, Xing J, Shi X, Huang H, Huang J, et al. Silencing P2X7R Alleviates Diabetic Neuropathic Pain Involving TRPV1 via PKCepsilon/P38MAPK/NF-kappaB Signaling Pathway in Rats. Int J Mol Sci 2022;23. doi: 10.3390/ijms232214141.
53. Yang HY, Wu J, Lu H, Cheng ML, Wang BH, Zhu HL, et al. Emodin suppresses oxaliplatin-induced neuropathic pain by inhibiting COX2/NF-kappaB mediated spinal inflammation. J Biochem Mol Toxicol 2023;37:e23229. doi: 10.1002/jbt.23229.
54. Feng L, Yang Z, Li Y, Pan Q, Zhang X, Wu X, et al. MicroRNA-378 contributes to osteoarthritis by regulating chondrocyte autophagy and bone marrow mesenchymal stem cell chondrogenesis. Mol Ther Nucleic Acids 2022;28:328-341. doi: 10.1016/j.omtn.2022.03.016.
55. Zhang C, Huang Y, Ouyang F, Su M, Li W, Chen J, et al. Extracellular vesicles derived from mesenchymal stem cells alleviate neuroinflammation and mechanical allodynia in interstitial cystitis rats by inhibiting NLRP3 inflammasome activation. J Neuroinflammation 2022;19:80. doi: 10.1186/s12974-022-02445-7.
56. Tan Y, Wang Z, Liu T, Gao P, Xu S, Tan L. RNA interference-mediated silencing of DNA methyltransferase 1 attenuates neuropathic pain by accelerating microglia M2 polarization. BMC Neurol 2022;22:376. doi: 10.1186/s12883-022-02860-6.
57. Zhang D, Sun J, Yang B, Ma S, Zhang C, Zhao G. Therapeutic Effect of Tetrapanax papyriferus and Hederagenin on Chronic Neuropathic Pain of Chronic Constriction Injury of Sciatic Nerve Rats Based on KEGG Pathway Prediction and Experimental Verification. Evid Based Complement Alternat Med 2020;2020:2545806. doi: 10.1155/2020/2545806.
58. Takada Y, Ye X, Simon S. The integrins. Genome Biol 2007;8:215. doi: 10.1186/gb-2007-8-5-215.
59. Lagos-Cabre R, Alvarez A, Kong M, Burgos-Bravo F, Cardenas A, Rojas-Mancilla E, et al. alpha(V)beta(3) Integrin regulates astrocyte reactivity. J Neuroinflammation 2017;14:194. doi: 10.1186/s12974-017-0968-5.
60. Cheng T, Xu Z, Ma X. The role of astrocytes in neuropathic pain. Front Mol Neurosci 2022;15:1007889. doi: 10.3389/fnmol.2022.1007889.
61. Liu L, Hu H, Wu J, Koleske AJ, Chen H, Wang N, et al. Integrin alpha3 is required for high-frequency repetitive transcranial magnetic stimulation-induced glutamatergic synaptic transmission in mice with ischemia. CNS Neurosci Ther 2023. doi: 10.1111/cns.14498.
62. Alvarez A, Lagos-Cabre R, Kong M, Cardenas A, Burgos-Bravo F, Schneider P, et al. Integrin-mediated transactivation of P2X7R via hemichannel-dependent ATP release stimulates astrocyte migration. Biochim Biophys Acta 2016;1863:2175-2188. doi: 10.1016/j.bbamcr.2016.05.018.
63. Henriquez M, Herrera-Molina R, Valdivia A, Alvarez A, Kong M, Munoz N, et al. ATP release due to Thy-1-integrin binding induces P2X7-mediated calcium entry required for focal adhesion formation. J Cell Sci 2011;124:1581-1588. doi: 10.1242/jcs.073171.
64. Karavis MY, Siafaka I, Vadalouca A, Georgoudis G. Role of Microglia in Neuropathic Pain. Cureus 2023;15:e43555. doi: 10.7759/cureus.43555.
65. Di Virgilio F, Dal Ben D, Sarti AC, Giuliani AL, Falzoni S. The P2X7 Receptor in Infection and Inflammation. Immunity 2017;47:15-31. doi: 10.1016/j.immuni.2017.06.020.
66. Wang Y, Shan Z, Zhang L, Fan S, Zhou Y, Hu L, et al. P2X7R/NLRP3 signaling pathway-mediated pyroptosis and neuroinflammation contributed to cognitive impairment in a mouse model of migraine. J Headache Pain 2022;23:75. doi: 10.1186/s10194-022-01442-8.
67. Yue N, Huang H, Zhu X, Han Q, Wang Y, Li B, et al. Activation of P2X7 receptor and NLRP3 inflammasome assembly in hippocampal glial cells mediates chronic stress-induced depressive-like behaviors. J Neuroinflammation 2017;14:102. doi: 10.1186/s12974-017-0865-y.
68. Leung A, Donohue M, Xu R, Lee R, Lefaucheur JP, Khedr EM, et al. rTMS for suppressing neuropathic pain: a meta-analysis. J Pain 2009;10:1205-1216. doi: 10.1016/j.jpain.2009.03.010.
69. Quesada C, Pommier B, Fauchon C, Bradley C, Creac’h C, Vassal F, et al. Robot-Guided Neuronavigated Repetitive Transcranial Magnetic Stimulation (rTMS) in Central Neuropathic Pain. Arch Phys Med Rehabil 2018;99:2203-2215 e2201. doi: 10.1016/j.apmr.2018.04.013.
70. Ma SM, Ni JX, Li XY, Yang LQ, Guo YN, Tang YZ. High-Frequency Repetitive Transcranial Magnetic Stimulation Reduces Pain in Postherpetic Neuralgia. Pain Med 2015;16:2162-2170. doi.
71. Stratton HJ, Khanna R. Sculpting Dendritic Spines during Initiation and Maintenance of Neuropathic Pain. J Neurosci 2020;40:7578-7589. doi: 10.1523/JNEUROSCI.1664-20.2020.
Presenting Author
Fengrui Yang
Poster Authors
Fengrui Yang
Dr
Lead Author
Topics
- Treatment/Management: Interventional Therapies – Neuromodulation