Skip to main content

Advertisement

Log in

Microvesicles shed from microglia activated by the P2X7-p38 pathway are involved in neuropathic pain induced by spinal nerve ligation in rats

  • Original Article
  • Published:
Purinergic Signalling Aims and scope Submit manuscript

Abstract

Microglia are critical in the pathogenesis of neuropathic pain. In this study, we investigated the role of microvesicles (MVs) in neuropathic pain induced by spinal nerve ligation (SNL) in rats. First, we found that MVs shed from microglia were increased in the cerebrospinal fluid and dorsal horn of the spinal cord after SNL. Next, MVs significantly reduced paw withdrawal threshold (PWT) and paw withdrawal latency (PWL). In addition, the P2X7-p38 pathway was related to the bleb of MVs after SNL. Interleukin (IL)-1β was found to be significantly upregulated in the package of MVs, and PWT and PWL increased following inhibition with shRNA-IL-1β. Finally, the amplitude and frequency of spontaneous excitatory postsynaptic currents increased following stimulation with MVs. Our results indicate that the P2X7-p38 pathway is closely correlated with the shedding of MVs from microglia in neuropathic pain, and MVs had a significant effect on neuropathic pain by participating in the interaction between microglia and neurons.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

Abbreviations

ANOVA:

Analysis of variance

BCA:

Bicinchoninic acid

CNS:

Central nervous system

CSF:

Cerebrospinal fluid

ELISA:

Enzyme-linked immunosorbent assay

EM:

Electron microscopy

MVs:

Microvesicles

PBS:

Phosphate buffered saline

PFA:

Paraformaldehyde

PWT:

Paw withdrawal threshold

PWL:

Paw withdrawal latency

SEM:

Standard error

SNL:

Spinal nerve ligation

sEPSC:

Spontaneous excitatory postsynaptic currents.

References

  1. Gu N, Eyo UB, Murugan M et al (2016) Microglial P2Y12 receptors regulate microglial activation and surveillance during neuropathic pain. Brain Behav Immun 55:82–92

    Article  CAS  PubMed  Google Scholar 

  2. Baron R (2006) Mechanisms of disease: neuropathic pain—a clinical perspective. Nat Clin Pract Neurol 2(2):95–106

    Article  PubMed  Google Scholar 

  3. Scholz J, Woolf CJ (2007) The neuropathic pain triad: neurons, immune cells and glia. Nat Neurosci 10:1361–1368

    Article  CAS  PubMed  Google Scholar 

  4. Calvo M, Dawes JM, Bennett DL (2012) The role of the immune system in the generation of neuropathic pain. Lancet Neurol 11:629–642

    Article  CAS  PubMed  Google Scholar 

  5. Bele T, Fabbretti E (2015) P2X receptors, sensory neurons and pain. Curr Med Chem 22:845–850

    Article  CAS  PubMed  Google Scholar 

  6. Melli G, Keswani SC, Fischer A, Chen W, Höke A (2006) Spatially distinct and functionally independent mechanisms of axonal degeneration in a model of HIV-associated sensory neuropathy. Brain 129:1330–1338

    Article  PubMed  Google Scholar 

  7. Keswani SC, Polley M, Pardo CA, Griffin JW, McArthur JC, Hoke A (2003) Schwann cell chemokine receptors mediate HIV-1 gp120 toxicity to sensory neurons. Ann Neurol 54:287–296

    Article  CAS  PubMed  Google Scholar 

  8. Sommer C, Schäfers M (1998) Painful mononeuropathy in C57BL/Wld mice with delayed wallerian degeneration: differential effects of cytokine production and nerve regeneration on thermal and mechanical hypersensitivity. Brain Res 784:154–162

    Article  CAS  PubMed  Google Scholar 

  9. Winkelstein BA, Rutkowski MD, Sweitzer SM, Pahl JL, DeLeo JA (2001) Nerve injury proximal or distal to the DRG induces similar spinal glial activation and selective cytokine expression but differential behavioral responses to pharmacologic treatment. J Comp Neurol 439:127–139

    Article  CAS  PubMed  Google Scholar 

  10. Budnik V, Ruiz-Cañada C, Wendler F (2016) Extracellular vesicles round off communication in the nervous system. Nat Rev Neurosci 17:160–172

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Bianco F, Perrotta C, Novellino L, Francolini M, Riganti L, Menna E, Saglietti L, Schuchman EH, Furlan R, Clementi E, Matteoli M, Verderio C (2009) Acid sphingomyelinase activity triggers microparticle release from glial cells. EMBO J 28:1043–1054

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Fukuoka T, Tokunaga A, Tachibana T, Dai Y, Yamanaka H, Noguchi K (2002) VR1, but not P2X(3), increases in the spared L4 DRG in rats with L5 spinal nerve ligation. Pain 99:111–120

    Article  CAS  PubMed  Google Scholar 

  13. Peng HY, Chen GD, Tung KC, Chien YW, Lai CY, Hsieh MC, Chiu CH, Lai CH, Lee SD, Lin TB (2009) Estrogen-dependent facilitation on spinal reflex potentiation involves the Cdk5/ERK1/2/NR2B cascade in anesthetized rats. Am J Physiol Endocrinol Metab 297:E416–E426

    Article  CAS  PubMed  Google Scholar 

  14. Marzesco AM, Janich P, Wilsch-Bräuninger M, Dubreuil V, Langenfeld K, Corbeil D, Huttner WB (2005) Release of extracellular membrane particles carrying the stem cell marker prominin-1 (CD133) from neural progenitors and other epithelial cells. J Cell Sci 118:2849–2858

    Article  CAS  PubMed  Google Scholar 

  15. Zhou D, Chen ML, Zhang YQ, Zhao ZQ (2010) Involvement of spinal microglial P2X7 receptor in generation of tolerance to morphine analgesia in rats. J Neurosci 30:8042–8047

    Article  CAS  PubMed  Google Scholar 

  16. MacKenzie A, Wilson HL, Kiss-Toth E, Dower SK, North RA, Surprenant A (2001) Rapid secretion of interleukin-1beta by microvesicle shedding. Immunity 15:825–835

    Article  CAS  PubMed  Google Scholar 

  17. Liu X, Liu H, Xu S, Tang Z, Xia W, Cheng Z, Li W, Jin Y (2016) Spinal translocator protein alleviates chronic neuropathic pain behavior and modulates spinal astrocyte-neuronal function in rats with L5 spinal nerve ligation model. Pain 157:103–116

    Article  CAS  PubMed  Google Scholar 

  18. Shefler I, Pasmanik-Chor M, Kidron D, Mekori YA, Hershko AY (2014) T cell-derived microvesicles induce mast cell production of IL-24: relevance to inflammatory skin diseases. J Allergy Clin Immunol 133:217–224.e1-3

    Article  CAS  PubMed  Google Scholar 

  19. Kim SH, Chung JM (1992) An experimental model for peripheral neuropathy produced by segmental spinal nerve ligation in the rat. Pain 50:355–363

    Article  CAS  PubMed  Google Scholar 

  20. Zhuang ZY, Wen YR, Zhang DR, Borsello T, Bonny C, Strichartz GR, Decosterd I, Ji RR (2006) A peptide c-Jun N-terminal kinase (JNK) inhibitor blocks mechanical allodynia after spinal nerve ligation: respective roles of JNK activation in primary sensory neurons and spinal astrocytes for neuropathic pain development and maintenance. J Neurosci 26:3551–3560

    Article  CAS  PubMed  Google Scholar 

  21. Ahmed Z, Shaw G, Sharma VP, Yang C, McGowan E, Dickson DW (2007) Actin-binding proteins coronin-1a and IBA-1 are effective microglial markers for immunohistochemistry. J Histochem Cytochem 55:687–700

    Article  CAS  PubMed  Google Scholar 

  22. Ji RR, Suter MR (2007) p38 MAPK, microglial signaling, and neuropathic pain. Mol Pain 3:33

    PubMed  PubMed Central  Google Scholar 

  23. Beggs S, Salter MW (2013) The known knowns of microglia-neuronal signalling in neuropathic pain. Neurosci Lett 557(Pt A):37–42

    Article  CAS  PubMed  Google Scholar 

  24. Verderio C, Muzio L, Turola E, Bergami A, Novellino L, Ruffini F, Riganti L, Corradini I, Francolini M, Garzetti L, Maiorino C, Servida F, Vercelli A, Rocca M, Dalla LD, Martinelli V, Comi G, Martino G, Matteoli M, Furlan R (2012) Myeloid microvesicles are a marker and therapeutic target for neuroinflammation. Ann Neurol 72:610–624

    Article  CAS  PubMed  Google Scholar 

  25. Bianco F, Pravettoni E, Colombo A, Schenk U, Möller T, Matteoli M, Verderio C (2005) Astrocyte-derived ATP induces vesicle shedding and IL-1 beta release from microglia. J Immunol 174:7268–7277

    Article  CAS  PubMed  Google Scholar 

  26. Sommer C, Kress M (2004) Recent findings on how proinflammatory cytokines cause pain: peripheral mechanisms in inflammatory and neuropathic hyperalgesia. Neurosci Lett 361:184–187

    Article  CAS  PubMed  Google Scholar 

  27. Vezzani A, Ravizza T, Balosso S, Aronica E (2008) Glia as a source of cytokines: implications for neuronal excitability and survival. Epilepsia 49(Suppl 2):24–32

    Article  CAS  PubMed  Google Scholar 

  28. Viviani B, Gardoni F, Marinovich M (2007) Cytokines and neuronal ion channels in health and disease. Int Rev Neurobiol 82:247–263

    Article  CAS  PubMed  Google Scholar 

  29. Turola E, Furlan R, Bianco F, Matteoli M, Verderio C (2012) Microglial microvesicle secretion and intercellular signaling. Front Physiol 3:149

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Schiera G, Proia P, Alberti C, Mineo M, Savettieri G, Di LI (2007) Neurons produce FGF2 and VEGF and secrete them at least in part by shedding extracellular vesicles. J Cell Mol Med 11:1384–1394

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Gosselin RD, Meylan P, Decosterd I (2013) Extracellular microvesicles from astrocytes contain functional glutamate transporters: regulation by protein kinase C and cell activation. Front Cell Neurosci 7:251

    PubMed  PubMed Central  Google Scholar 

  32. Tsuda M, Shigemoto-Mogami Y, Koizumi S, Mizokoshi A, Kohsaka S, Salter MW, Inoue K (2003) P2X4 receptors induced in spinal microglia gate tactile allodynia after nerve injury. Nature 424:778–783

    Article  CAS  PubMed  Google Scholar 

  33. Thacker MA, Clark AK, Bishop T, Grist J, Yip PK, Moon LD, Thompson SW, Marchand F, McMahon SB (2009) CCL2 is a key mediator of microglia activation in neuropathic pain states. Eur J Pain 13:263–272

    Article  CAS  PubMed  Google Scholar 

  34. Beggs S, Salter MW (2007) Stereological and somatotopic analysis of the spinal microglial response to peripheral nerve injury. Brain Behav Immun 21:624–633

    Article  CAS  PubMed  Google Scholar 

  35. Echeverry S, Shi XQ, Zhang J (2008) Characterization of cell proliferation in rat spinal cord following peripheral nerve injury and the relationship with neuropathic pain. Pain 135:37–47

    Article  CAS  PubMed  Google Scholar 

  36. Zhang J, Shi XQ, Echeverry S, Mogil JS, De Koninck Y, Rivest S (2007) Expression of CCR2 in both resident and bone marrow-derived microglia plays a critical role in neuropathic pain. J Neurosci 27:12396–12406

    Article  CAS  PubMed  Google Scholar 

  37. Narita M, Yoshida T, Nakajima M, Narita M, Miyatake M, Takagi T, Yajima Y, Suzuki T (2006) Direct evidence for spinal cord microglia in the development of a neuropathic pain-like state in mice. J Neurochem 97:1337–1348

    Article  CAS  PubMed  Google Scholar 

  38. Inoue K, Tsuda M (2009) Microglia and neuropathic pain. Glia 57:1469–1479

    Article  PubMed  Google Scholar 

  39. Woolf CJ, Salter MW (2000) Neuronal plasticity: increasing the gain in pain. Science 288:1765–1769

    Article  CAS  PubMed  Google Scholar 

  40. Scholz J, Woolf CJ (2002) Can we conquer pain. Nat Neurosci 5(Suppl):1062–1067

    Article  CAS  PubMed  Google Scholar 

  41. Tsuda M, Inoue K, Salter MW (2005) Neuropathic pain and spinal microglia: a big problem from molecules in “small” glia. Trends Neurosci 28:101–107

    Article  CAS  PubMed  Google Scholar 

  42. Piccin A, Murphy WG, Smith OP (2007) Circulating microparticles: pathophysiology and clinical implications. Blood Rev 21:157–171

    Article  CAS  PubMed  Google Scholar 

  43. Pap E, Pállinger E, Pásztói M, Falus A (2009) Highlights of a new type of intercellular communication: microvesicle-based information transfer. Inflamm Res 58:1–8

    Article  CAS  PubMed  Google Scholar 

  44. Stock C, Schilling T, Schwab A, Eder C (2006) Lysophosphatidylcholine stimulates IL-1beta release from microglia via a P2X7 receptor-independent mechanism. J Immunol 177:8560–8568

    Article  CAS  PubMed  Google Scholar 

  45. Shieh CH, Heinrich A, Serchov T, van Calker D, Biber K (2014) P2X7-dependent, but differentially regulated release of IL-6, CCL2, and TNF-α in cultured mouse microglia. Glia 62:592–607

    Article  PubMed  Google Scholar 

  46. Apkarian AV, Lavarello S, Randolf A, Berra HH, Chialvo DR, Besedovsky HO, del RA (2006) Expression of IL-1beta in supraspinal brain regions in rats with neuropathic pain. Neurosci Lett 407:176–181

    Article  PubMed  Google Scholar 

  47. del RA, Apkarian AV, Martina M, Besedovsky HO (2012) Chronic neuropathic pain-like behavior and brain-borne IL-1β. Ann N Y Acad Sci 1262:101–107

    Article  Google Scholar 

  48. Nadeau S, Filali M, Zhang J, Kerr BJ, Rivest S, Soulet D, Iwakura Y, de Rivero Vaccari JP, Keane RW, Lacroix S (2011) Functional recovery after peripheral nerve injury is dependent on the pro-inflammatory cytokines IL-1β and TNF: implications for neuropathic pain. J Neurosci 31:12533–12542

    Article  CAS  PubMed  Google Scholar 

  49. Li P, Wilding TJ, Kim SJ, Calejesan AA, Huettner JE, Zhuo M (1999) Kainate-receptor-mediated sensory synaptic transmission in mammalian spinal cord. Nature 397:161–164

    Article  CAS  PubMed  Google Scholar 

  50. Antonucci F, Turola E, Riganti L, Caleo M, Gabrielli M, Perrotta C, Novellino L, Clementi E, Giussani P, Viani P, Matteoli M, Verderio C (2012) Microvesicles released from microglia stimulate synaptic activity via enhanced sphingolipid metabolism. EMBO J 31:1231–1240

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

This work was funded by the National Natural Science Foundation of China (81371253), the Shanghai Municipal Education Commission (14ZZ083), and the Shanghai Changzheng Hospital (2015CZQN01).

Author information

Authors and Affiliations

Authors

Corresponding authors

Correspondence to Zhenghua Xiang or Hongbin Yuan.

Ethics declarations

All animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee of the Second Military Medical University and conformed to the American Physiological Society’s Guiding Principles in the Care and Use of Animals.

Conflict of interest

The authors declared no conflicts of interest with respect to the research, authorship, and/or publication of this article.

Additional information

Jian Li, Xiangnan Li, and Xin Jiang contributed equally to this work and should be considered as co-first authors.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Li, J., Li, X., Jiang, X. et al. Microvesicles shed from microglia activated by the P2X7-p38 pathway are involved in neuropathic pain induced by spinal nerve ligation in rats. Purinergic Signalling 13, 13–26 (2017). https://doi.org/10.1007/s11302-016-9537-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11302-016-9537-0

Keywords

Navigation