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Localized Inflammatory Irritation of the Lumbar Ganglia: An Animal Model of Chemogenic Low Back Pain and Radiculopathy

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Part of the book series: Neuromethods ((NM,volume 49))

Abstract

In this chapter, we describe a newly developed rodent model of chemogenic pain involving the inflammation of one or two lumbar sensory ganglia using the immune activator, zymosan. Using this model, we have investigated cellular, molecular, and ionic mechanisms of inflammatory responses within the dorsal root ganglion (DRG) and their contribution to the development of chemogenic pathological pain. DRG inflammation was induced by a single deposit of zymosan in the epidural space near the L5 DRG via a small hole drilled through the transverse process. After a single zymosan injection, rats developed bilateral mechanical hyperalgesia and allodynia which began by day 1 after surgery, peaked at days 3–7, and lasted up to 28 days. Robust satellite glial activation was observed in inflamed ganglia. Cytokine profile analysis using a multiplexing protein array system showed that local inflammatory irritation selectively increased proinflammatory cytokines/chemokines such as IL-1β, IL-6, IL-18, MCP-1, and GRO/KC up to 17-fold, and decreased anti-inflammatory cytokines such as IL-2 and IL-12 (p70) up to threefold. Inflaming the DRG also remarkably increased the incidence of spontaneous activity of A- and C-fibers recorded in the dorsal root. Many of the spontaneously active A-fibers exhibited a short-bursting discharge pattern. Changes in cytokines and spontaneous activity correlated with the time course of pain behaviors, especially light stroke-evoked tactile allodynia. Local inflammation induced extensive sprouting of sympathetic fibers, extending from vascular processes within the inflamed DRG. Finally, patch clamp recording of the acutely dissociated DRG neurons revealed a significant upregulation of sodium and potassium channels. These results demonstrate the feasibility of inducing chronic localized inflammatory responses in the DRG in the absence of traumatic nerve damage and highlight the possible contribution of several inflammatory cytokines/chemokines to the generation of spontaneous activity and the development and persistence of chemogenic pathologic pain.

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References

  1. DeLeo, J. A., Colburn, R. W., Nichols, M., and Malhotra, A. (1996) Interleukin-6-mediated hyperalgesia/allodynia and increased spinal IL-6 expression in a rat mononeuropathy model, J Interferon Cytokine Res 16, 695–700.

    Article  PubMed  CAS  Google Scholar 

  2. Briggs, C. A. and Chandraraj, S. (1995) Variations in the lumbosacral ligament and associated changes in the lumbosacral region resulting in compression of the fifth dorsal root ganglion and spinal nerve, Clin Anat 8, 339–346.

    Article  PubMed  CAS  Google Scholar 

  3. Hoch, R. C., Rodriguez, R., Manning, T., Bishop, M., Mead, P., Shoemaker, W. C., and Abraham, E. (1993) Effects of accidental trauma on cytokine and endotoxin production, Crit Care Med 21, 839–845.

    Article  PubMed  CAS  Google Scholar 

  4. Poletti, C. E. (1996) Third cervical nerve root and ganglion compression: clinical syndrome, surgical anatomy, and pathological findings, Neurosurgery 39, 941–948; discussion 948–949.

    PubMed  CAS  Google Scholar 

  5. Sato, K. and Kikuchi, S. (1993) An anatomic study of foraminal nerve root lesions in the lumbar spine, Spine 18, 2246–2251.

    Article  PubMed  CAS  Google Scholar 

  6. Saal, J. S., Franson, R. C., Dobrow, R., Saal, J. A., White, A. H., and Goldthwaite, N. (1990) High levels of inflammatory phospholipase A2 activity in lumbar disc herniations, Spine 15, 674–678.

    Article  PubMed  CAS  Google Scholar 

  7. Kang, J. D., Georgescu, H. I., McIntyre-Larkin, L., Stefanovic-Racic, M., and Evans, C. H. (1995) Herniated cervical intervertebral discs spontaneously produce matrix metalloproteinases, nitric oxide, interleukin-6, and prostaglandin E2, Spine 20, 2373–2378.

    Article  PubMed  CAS  Google Scholar 

  8. Yamagishi, M., Nemoto, O., and Kikuchi, T. (1992) Ruputured human disc tissues produce metalloproteinase and interleukin-1, Trans Orthop Res Soc 17, 190.

    Google Scholar 

  9. Olmarker, K. and Myers, R. R. (1998) Pathogenesis of sciatic pain: role of herniated nucleus pulposus and deformation of spinal nerve root and dorsal root ganglion, Pain 78, 99–105.

    Article  Google Scholar 

  10. McCarron, R. F., Wimpee, M. W., Hudkins, P. G., and Laros, G. S. (1987) The inflammatory effect of nucleus pulposus. A possible element in the pathogenesis of low-back pain, Spine 12, 760–764.

    Article  PubMed  CAS  Google Scholar 

  11. Cavanaugh, J. M., Ozaktay, A. C., and Vaidyanathan, S. (1994) Mechano- and chemosensitivity of lumbar dorsal roots and dorsal root ganglia: an in vitro study, Trans Orthop Res Soc 1994, 109.

    Google Scholar 

  12. Hu, S. J. and Xing, J. L. (1998) An experimental model for chronic compression of dorsal root ganglion produced by intervertebral foramen stenosis in the rat, Pain 77, 15–23.

    Article  PubMed  CAS  Google Scholar 

  13. Song, X. J., Hu, S. J., Greenquist, K. W., Zhang, J.-M., and LaMotte, R. H. (1999) Mechanical and thermal hyperalgesia and ectopic neuronal discharge after chronic compression of dorsal root ganglia, J Neurophysiol 82, 3347–3358.

    PubMed  CAS  Google Scholar 

  14. Xie, W. R., Deng, H., Li, H., Bowen, T. L., Strong, J. A., and Zhang, J.-M. (2006) Robust increase of cutaneous sensitivity, cytokine production and sympathetic sprouting in rats with localized inflammatory irritation of the spinal ganglia, Neuroscience 142, 809–822.

    Article  PubMed  CAS  Google Scholar 

  15. Zhang, J.-M., Homma, Y., Ackerman, W. E., and Brull, S. J. (2001) Topical application of acidic bupivacaine to the lumbar ganglion induces mechanical hyperalgesia in the rat, Anesth Analg 93, 466–471.

    PubMed  CAS  Google Scholar 

  16. Watkins, L. R. and Maier, S. F. (2002) Beyond neurons: Evidence that immune and glial cells contribute to pathological pain states [Review], Physiol Rev 82, 981–1011.

    PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  18. Qin, X., Wan, Y., and Wang, X. (2005) CCL2 and CXCL1 trigger calcitonin gene-related peptide release by exciting primary nociceptive neurons, J Neurosci Res 82, 51–62.

    Article  PubMed  CAS  Google Scholar 

  19. Losy, J., Zaremba, J., and Skrobanski, P. (2005) CXCL1 (GRO-alpha) chemokine in acute ischaemic stroke patients, Folia Neuropathol 43, 97–102.

    PubMed  CAS  Google Scholar 

  20. Valles, A., Grijpink-Ongering, L., de Bree, F. M., Tuinstra, T., and Ronken, E. (2006) Differential regulation of the CXCR2 chemokine network in rat brain trauma: Implications for neuroimmune interactions and neuronal survival, Neurobiol Dis 22, 312–322.

    Article  PubMed  CAS  Google Scholar 

  21. Xia, M. and Hyman, B. T. (2002) GROalpha/KC, a chemokine receptor CXCR2 ligand, can be a potent trigger for neuronal ERK1/2 and PI-3 kinase pathways and for tau hyperphosphorylation-a role in Alzheimer’s disease? J Neuroimmunol 122, 55–64.

    Article  PubMed  CAS  Google Scholar 

  22. Giovannelli, A., Limatola, C., Ragozzino, D., Mileo, A. M., Ruggieri, A., Ciotti, M. T., Mercanti, D., Santoni, A., and Eusebi, F. (1998) CXC chemokines interleukin-8 (IL-8) and growth-related gene product alpha (GROalpha) modulate Purkinje neuron activity in mouse cerebellum, J Neuroimmunol 92, 122–132.

    Article  PubMed  CAS  Google Scholar 

  23. Wang, J. G., Strong, J. A., Xie, W., Yang, R. H., Coyle, D. E., Wick, D. M., Dorsey, E. D., and Zhang, J. M. (2008) The chemokine CXCL1/growth related oncogene increases sodium currents and neuronal excitability in small diameter sensory neurons, Mol Pain 4, 38.

    Article  PubMed  Google Scholar 

  24. Felderhoff-Mueser, U., Schmidt, O. I., Oberholzer, A., Buhrer, C., and Stahel, P. F. (2005) IL-18: a key player in neuroinflammation and neurodegeneration? Trends Neurosci 28, 487–493.

    Article  PubMed  CAS  Google Scholar 

  25. Homma, Y., Brull, S. J., and Zhang, J.-M. (2002) A comparison of chronic pain behavior following local application of tumor necrosis factor alpha to the normal and mechanically compressed lumbar ganglia in the rat, Pain 95, 235–246.

    Article  Google Scholar 

  26. White, F. A., Sun, J., Waters, S. M., Ma, C., Ren, D., Ripsch, M., Steflik, J., Cortright, D. N., Lamotte, R. H., and Miller, R. J. (2005) Excitatory monocyte chemoattractant protein-1 signaling is up-regulated in sensory neurons after chronic compression of the dorsal root ganglion, Proc Natl Acad Sci USA 102, 14092–14097.

    Article  PubMed  CAS  Google Scholar 

  27. Copray, J. C., Mantingh, I., Brouwer, N., Biber, K., Kust, B. M., Liem, R. S., Huitinga, I., Tilders, F. J., Van Dam, A. M., and Boddeke, H. W. (2001) Expression of interleukin-1 beta in rat dorsal root ganglia, J Neuroimmunol 118, 203–211.

    Article  PubMed  CAS  Google Scholar 

  28. Martiney, J. A., Cuff, C., Litwak, M., Berman, J., and Brosnan, C. F. (1998) Cytokine-induced inflammation in the central nervous system revisited, Neurochem Res 23, 349–359.

    Article  PubMed  CAS  Google Scholar 

  29. Song, X. J., Zhang, J.-M., Hu, S. J., and LaMotte, R. H. (2003) Somata of nerve-injured sensory neurons exhibit enhanced responses to inflammatory mediators, Pain 104, 701–709.

    Article  PubMed  CAS  Google Scholar 

  30. Scholz, J. and Woolf, C. J. (2002) Can we conquer pain? Nat Neurosci 5 Suppl, 1062–1067.

    Article  PubMed  CAS  Google Scholar 

  31. Abdulla, F. A. and Smith, P. A. (2001) Axotomy- and autotomy-induced changes in Ca2+ and K+ channel currents of rat dorsal root ganglion neurons, J Neurophysiol 85, 644–658.

    PubMed  CAS  Google Scholar 

  32. Kim, Y. I., Na, H. S., Kim, S. H., Han, H. C., Yoon, Y. W., Sung, B., Nam, H. J., Shin, S. L., and Hong, S. K. (1998) Cell type-specific changes of the membrane properties of peripherally-axotomized dorsal root ganglion neurons in a rat model of neuropathic pain, Neuroscience 86, 301–309.

    Article  PubMed  CAS  Google Scholar 

  33. Moore, B. A., Stewart, T. M., Hill, C., and Vanner, S. J. (2002) TNBS ileitis evokes hyperexcitability and changes in ionic membrane properties of nociceptive DRG neurons, Am J Physiol Gastrointest Liver Physiol 282, G1045–G1051.

    PubMed  CAS  Google Scholar 

  34. Zhang, J.-M., Song, X. J., and LaMotte, R. H. (1999) Enhanced excitability of sensory neurons in rats with cutaneous hyperalgesia produced by chronic compression of the dorsal root ganglion, J Neurophysiol 82, 3359–3366.

    PubMed  CAS  Google Scholar 

  35. Millan, M. J. (1999) The induction of pain: an integrative review, Prog Neurobiol 57, 1–164.

    Article  PubMed  CAS  Google Scholar 

  36. Waxman, S. G., Cummins, T. R., Dib-Hajj, S., Fjell, J., and Black, J. A. (1999) Sodium channels, excitability of primary sensory neurons, and the molecular basis of pain, Muscle Nerve 22, 1177–1187.

    Article  PubMed  CAS  Google Scholar 

  37. Taskinen, H. S. and Roytta, M. (2000) Increased expression of chemokines (MCP-1, MIP-1alpha, RANTES) after peripheral nerve transection, J Peripher Nerv Syst 5, 75–81.

    Article  PubMed  CAS  Google Scholar 

  38. Tanaka, T., Minami, M., Nakagawa, T., and Satoh, M. (2004) Enhanced production of monocyte chemoattractant protein-1 in the dorsal root ganglia in a rat model of neuropathic pain: possible involvement in the development of neuropathic pain, Neurosci Res 48, 463–469.

    Article  PubMed  CAS  Google Scholar 

  39. Kleinschnitz, C., Brinkhoff, J., Zelenka, M., Sommer, C., and Stoll, G. (2004) The extent of cytokine induction in peripheral nerve lesions depends on the mode of injury and NMDA receptor signaling, J Neuroimmunol 149, 77–83.

    Article  PubMed  CAS  Google Scholar 

  40. Ji, R. R. and Strichartz, G. (2004) Cell signaling and the genesis of neuropathic pain, Sci STKE 2004, reE14.

    Article  PubMed  Google Scholar 

  41. Marchand, F., Perretti, M., and McMahon, S. B. (2005) Role of the immune system in chronic pain, Nat Rev Neurosci 6, 521–532.

    Article  PubMed  CAS  Google Scholar 

  42. Moalem, G. and Tracey, D. J. (2006) Immune and inflammatory mechanisms in neuropathic pain, Brain Res Brain Res Rev 51, 240–264.

    Article  CAS  Google Scholar 

  43. Devor, M. (2006) Response of nerves to injury in relation to neuropathic pain, in Textbook of Pain (McMahon, S. B. and Koltzenburg, M., Eds.) 5th ed., pp. 905–927, Elsevier/Churchill Livingstone, Philadelphia, PA.

    Google Scholar 

  44. Schild, J. H. and Kunze, D. L. (1997) Experimental and modeling study of Na+ current heterogeneity in rat nodose neurons and its impact on neuronal discharge, J Neurophysiol 78, 3198–3209.

    PubMed  CAS  Google Scholar 

  45. Krause, J. E., Chenard, B. L., and Cortright, D. N. (2005) Transient receptor potential ion channels as targets for the discovery of pain therapeutics, Curr Opin Investig Drugs 6, 48–57.

    PubMed  CAS  Google Scholar 

  46. Chaplan, S. R., Guo, H. Q., Lee, D. H., Luo, L., Liu, C., Kuei, C., Velumian, A. A., Butler, M. P., Brown, S. M., and Dubin, A. E. (2003) Neuronal hyperpolarization-activated pacemaker channels drive neuropathic pain, J Neurosci 23, 1169–1178.

    PubMed  CAS  Google Scholar 

  47. Cao, Y. Q. (2006) Voltage-gated calcium channels and pain, Pain 126, 5–9.

    Article  PubMed  CAS  Google Scholar 

  48. Rogers, M., Tang, L., Madge, D. J., and Stevens, E. B. (2006) The role of sodium channels in neuropathic pain, Semin Cell Dev Biol 17, 571–581.

    Article  PubMed  CAS  Google Scholar 

  49. Tanaka, M., Cummins, T. R., Ishikawa, K., Dib-Hajj, S. D., Black, J. A., and Waxman, S. G. (1998) SNS Na+ channel expression increases in dorsal root ganglion neurons in the carrageenan inflammatory pain model, Neuroreport 9, 967–972.

    Article  PubMed  CAS  Google Scholar 

  50. Black, J. A., Liu, S., Tanaka, M., Cummins, T. R., and Waxman, S. G. (2004) Changes in the expression of tetrodotoxin-sensitive sodium channels within dorsal root ganglia neurons in inflammatory pain, Pain 108, 237–247.

    Article  PubMed  CAS  Google Scholar 

  51. Beyak, M. J., Ramji, N., Krol, K. M., Kawaja, M. D., and Vanner, S. J. (2004) Two TTX-resistant Na+ currents in mouse colonic dorsal root ganglia neurons and their role in colitis-induced hyperexcitability, Am J Physiol Gastrointest Liver Physiol 287, G845–G855.

    Article  PubMed  CAS  Google Scholar 

  52. Stewart, T., Beyak, M. J., and Vanner, S. (2003) Ileitis modulates potassium and sodium currents in guinea pig dorsal root ganglia sensory neurons, J Physiol 552, 797–807.

    Article  PubMed  CAS  Google Scholar 

  53. Bielefeldt, K., Ozaki, N., and Gebhart, G. F. (2002) Mild gastritis alters voltage-sensitive sodium currents in gastric sensory neurons in rats, Gastroenterology 122, 752–761.

    Article  PubMed  CAS  Google Scholar 

  54. Molliver, D. C. and Snider, W. D. (1997) Nerve growth factor receptor TrkA is down-regulated during postnatal development by a subset of dorsal root ganglion neurons, J Comp Neurol 381, 428–438.

    Article  PubMed  CAS  Google Scholar 

  55. Ogata, N. and Ohishi, Y. (2002) Molecular diversity of structure and function of the voltage-gated Na+ channels, Jpn J Pharmacol 88, 365–377.

    Article  PubMed  CAS  Google Scholar 

  56. Wu, Z. Z. and Pan, H. L. (2004) Tetrodotoxin-sensitive and -resistant Na+ channel currents in subsets of small sensory neurons of rats, Brain Res 1029, 251–258.

    Article  PubMed  CAS  Google Scholar 

  57. Amaya, F., Decosterd, I., Samad, T. A., Plumpton, C., Tate, S., Mannion, R. J., Costigan, M., and Woolf, C. J. (2000) Diversity of expression of the sensory neuron-specific TTX-resistant voltage-gated sodium ion channels SNS and SNS2, Mol Cell Neurosci 15, 331–342.

    Article  PubMed  CAS  Google Scholar 

  58. Takeda, M., Tanimoto, T., Ikeda, M., Nasu, M., Kadoi, J., Yoshida, S., and Matsumoto, S. (2006) Enhanced excitability of rat trigeminal root ganglion neurons via decrease in A-type potassium currents following temporomandibular joint inflammation, Neuroscience 138, 621–630.

    Article  PubMed  CAS  Google Scholar 

  59. Dang, K., Bielefeldt, K., and Gebhart, G. F. (2004) Gastric ulcers reduce A-type potassium currents in rat gastric sensory ganglion neurons, Am J Physiol Gastrointest Liver Physiol 286, G573–G579.

    Article  PubMed  CAS  Google Scholar 

  60. Yoshimura, N. and de Groat, W. C. (1999) Increased excitability of afferent neurons innervating rat urinary bladder after chronic bladder inflammation, J Neurosci 19, 4644–4653.

    PubMed  CAS  Google Scholar 

  61. Yang, E. K., Takimoto, K., Hayashi, Y., de Groat, W. C., and Yoshimura, N. (2004) Altered expression of potassium channel subunit mRNA and alpha-dendrotoxin sensitivity of potassium currents in rat dorsal root ganglion neurons after axotomy, Neuroscience 123, 867–874.

    Article  PubMed  CAS  Google Scholar 

  62. Harriott, A. M., Dessem, D., and Gold, M. S. (2006) Inflammation increases the excitability of masseter muscle afferents, Neuroscience 141, 433–442.

    Article  PubMed  CAS  Google Scholar 

  63. Xu, G. Y., Winston, J. H., Shenoy, M., Yin, H., and Pasricha, P. J. (2006) Enhanced excitability and suppression of A-type K+ current of pancreas-specific afferent neurons in a rat model of chronic pancreatitis, Am J Physiol Gastrointest Liver Physiol 291, G424–G431.

    Article  PubMed  CAS  Google Scholar 

  64. Marder, E. and Prinz, A. A. (2002) Modeling stability in neuron and network function: the role of activity in homeostasis, Bioessays 24, 1145–1154.

    Article  PubMed  CAS  Google Scholar 

  65. Liu, L., Yang, T. M., Liedtke, W., and Simon, S. A. (2006) Chronic IL-1beta signaling potentiates voltage-dependent sodium currents in trigeminal nociceptive neurons, J Neurophysiol 95, 1478–1490.

    Article  PubMed  CAS  Google Scholar 

  66. Ramer, M. S., Thompson, S. W., and McMahon, S. B. (1999) Causes and consequences of sympathetic basket formation in dorsal root ganglia, Pain Suppl 6, S111–S120.

    Article  PubMed  CAS  Google Scholar 

  67. Ramer, M. S., Murphy, P. G., Richardson, P. M., and Bisby, M. A. (1998) Spinal nerve lesion-induced mechanoallodynia and adrenergic sprouting in sensory ganglia are attenuated in interleukin-6 knockout mice, Pain 78, 115–121.

    Article  PubMed  CAS  Google Scholar 

  68. Zhou, X. F., Deng, Y. S., Chie, E., Xue, Q., Zhong, J. H., McLachlan, E. M., Rush, R. A., and Xian, C. J. (1999) Satellite-cell-derived nerve growth factor and neurotrophin-3 are involved in noradrenergic sprouting in the dorsal root ganglia following peripheral nerve injury in the rat, Eur J Neurosci 11, 1711–1722.

    Article  PubMed  CAS  Google Scholar 

  69. Nakamura, M. and Ferreira, S. H. (1987) A peripheral sympathetic component in inflammatory hyperalgesia, Eur J Pharmacol 135, 145–153.

    Article  PubMed  CAS  Google Scholar 

  70. Chacur, M., Milligan, E. D., Gazda, L. S., Armstrong, C., Wang, H., Tracey, K. J., Maier, S. F., and Watkins, L. R. (2001) A new model of sciatic inflammatory neuritis (SIN): induction of unilateral and bilateral mechanical allodynia following acute unilateral peri-sciatic immune activation in rats, Pain 94, 231–244.

    Article  PubMed  CAS  Google Scholar 

  71. Milligan, E. D., Twining, C., Chacur, M., Biedenkapp, J., O’Connor, K., Poole, S., Tracey, K., Martin, D., Maier, S. F., and Watkins, L. R. (2003) Spinal glia and proinflammatory cytokines mediate mirror-image neuropathic pain in rats, J Neurosci 23, 1026–1040.

    PubMed  CAS  Google Scholar 

  72. Dobretsov, M., Hastings, S. L., Stimers, J. R., and Zhang, J.-M. (2001) Mechanical hyperalgesia in rats with chronic perfusion of lumbar dorsal root ganglion with hyperglycemic solution, J Neurosci Methods 110, 9–15.

    Article  PubMed  CAS  Google Scholar 

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Acknowledgements

This work was supported in part by NIH grants NS55860 and NS45594, and the University of Cincinnati Millennium Fund.

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Zhang, JM. (2011). Localized Inflammatory Irritation of the Lumbar Ganglia: An Animal Model of Chemogenic Low Back Pain and Radiculopathy. In: Ma, C., Zhang, JM. (eds) Animal Models of Pain. Neuromethods, vol 49. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-880-5_6

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  • DOI: https://doi.org/10.1007/978-1-60761-880-5_6

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