Elsevier

Brain Research

Volume 1513, 4 June 2013, Pages 17-25
Brain Research

Research Report
mTOR and its downstream pathway are activated in the dorsal root ganglion and spinal cord after peripheral inflammation, but not after nerve injury

https://doi.org/10.1016/j.brainres.2013.04.003Get rights and content

Highlights

  • Inflammatory pain and neuropathic pain have distinct characteristics in mechanisms.

  • Inflammation, but not nerve injury, activates mTOR and S6K1 in spinal cord and DRG.

  • Intrathecal mTOR inhibitor attenuates inflammatory pain.

  • mTOR and its downstream pathway contribute to inflammatory pain development.

Abstract

Protein translation controlled through activation of mammalian target of rapamycin (mTOR) participates in many physiological and pathological processes. However, whether such activation is required for chronic pain is still unknown. Here, we examined activation of the mTOR signaling pathway during complete Freund's adjuvant (CFA)-induced chronic inflammatory pain and L5 spinal nerve ligation (SNL)-induced neuropathic pain in rats. Western blot analysis showed significantly increased levels of phosphorylated mTOR (p-mTOR) and phosphorylated p70S6 kinase 1 (p-S6K1, a downstream effector of mTOR) in the ipsilateral L4/5 spinal cord 2 h, 1 day, 3 days, and 7 days after intraplantar CFA injection and in the ipsilateral L4/5 dorsal root ganglions (DRGs) 1 and 3 days after CFA injection. Immunohistochemistry also demonstrated increases in number of p-mTOR-labeled neurons in the ipsilateral L4/5 DRGs and in density of p-mTOR-labeled immunoreactivity in the ipsilateral L4/5 superficial dorsal horn 1 day after CFA injection. Moreover, intrathecal administration of rapamycin, a selective inhibitor of mTOR, significantly blocked CFA-induced mechanical allodynia and thermal hyperalgesia 1 day post-CFA injection. Interestingly, expression of neither p-mTOR nor p-S6K1 was markedly altered on days 3, 7, or 14 after L5 SNL in L5 spinal cord or DRG. These findings indicate that in DRG and spinal cord, mTOR and S6K1 are activated during chronic inflammatory pain, but not during neuropathic pain. Our results strongly suggest that mTOR and its downstream pathway contribute to the development of chronic inflammatory pain.

Introduction

Chronic pain, usually caused by inflammation or tissue or nerve injury, is a major public health problem worldwide. The development of optimal treatments for such pain has remained elusive because of the complex pathologic mechanisms involved. Changes in neuronal plasticity in spinal cord and dorsal root ganglion (DRG) are thought to contribute to the development and maintenance of chronic pain. Understanding the molecular mechanisms that underlie these changes could be helpful for developing novel pain treatments.

Mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase expressed in the mammalian nervous system. Once mTOR binds to Raptor protein, it forms mTOR complex 1 and phosphorylates downstream effectors, such as p70 ribosomal S6 protein kinase 1 (S6K1), to govern translation of mRNA into protein (Swiech et al., 2008, Jaworski and Sheng, 2006, Hay and Sonenberg, 2004). The activity of mTOR can be modulated in response to a variety of stimuli, such as neurotransmitters, trophic factors, mitogens, hormones, cell energy status and cellular stress, ischemia, and heat shock (Swiech et al., 2008, Jaworski and Sheng, 2006). In turn, the activation of mTOR and its downstream effectors can influence numerous physiologic and pathologic processes in the nervous system (Swiech et al., 2008, Jaworski and Sheng, 2006). Recent studies showed that mTOR and its downstream effectors might be implicated in neuropathic pain, inflammatory pain, and cancer pain. Systemic or local administration of rapamycin, a specific inhibitor of mTOR, has been shown to alleviate mechanical hypersensitivity induced by intraplantar injection of carrageenan or capsaicin (inflammatory pain), peripheral nerve injury (neuropathic pain), and injection of prostate cancer cells into tibia (bone cancer pain) (Shih et al., 2012, Xu et al., 2011, Obara et al., 2011, Norsted et al., 2010, Geranton et al., 2009, Jimenez-Diaz et al., 2008, Price et al., 2007). Although mTOR and S6K1 are highly expressed in small DRG neurons and spinal cord dorsal horn neurons, their active (phosphorylated) forms are expressed at only very low levels under normal conditions (Xu et al., 2010). We and others have shown that peripheral noxious insults caused by intraplantar carrageenan or bone cancer lead to increases in phosphorylation of mTOR (p-mTOR) and S6K1 (p-S6K1) in rat spinal dorsal horn but not in DRGs (Shih et al., 2012, Xu et al., 2011). However, little is known about the role of mTOR in chronic inflammatory pain. Moreover, whether mTOR and its downstream effectors are activated in spinal cord and DRG after peripheral nerve injury is controversial (Melemedjian et al., 2011, Asante et al., 2010, Geranton et al., 2009).

In the present study, we compared the expression and activity of mTOR and S6K1 in DRG and spinal cord of rats during chronic inflammatory pain induced by intraplantar injection of complete Freund's adjuvant (CFA) and during chronic neuropathic pain caused by spinal nerve ligation (SNL).

Section snippets

mTOR and S6K1 activity in DRG and spinal cord under chronic inflammatory pain conditions

Consistent with previous studies (Yaster et al., 2011, Park et al., 2009, Zhang et al., 2003), CFA injection produced mechanical hypersensitivity, as evidenced by a reduction in paw withdrawal threshold (PWT), and thermal pain hypersensitivity, as evidenced by a reduction in paw withdrawal latency (PWL). Hypersensitivity developed only on the ipsilateral side, was present by 2 h after CFA injection, reached a peak level around day 1, and lasted for at least 7 days (Figs. 1a and b).

Discussion

Peripheral nerve injury and inflammation produce chronic pain hypersensitivities in rodent animal models that mimic clinical chronic neuropathic pain and chronic inflammatory pain, respectively. Understanding the mechanisms that cause pain hypersensitivity may lead to novel therapeutic strategies for its prevention and/or treatment. Although chronic neuropathic pain and inflammatory pain share some intracellular signaling pathways (Latremoliere and Woolf, 2010), each type of pain may also have

Animal preparation

Adult, male Sprague Dawley rats (250–300 g) were housed in cages on a standard 12:12 h light/dark cycle. Water and food were available ad libitum until rats were transported to the laboratory for experiments. The animals were used in protocols approved by the Animal Care and Use Committee at the Johns Hopkins University. Animal procedures were consistent with the ethical guidelines of the National Institutes of Health, the International Association for the Study of Pain, and the ethical

Acknowledgments

The authors thank Claire F. Levine, MS, for her editorial assistance. The authors do not have any conflicts of interest.

References (27)

  • J.A. Garcia et al.

    Mammalian target of rapamycin inhibition as a therapeutic strategy in the management of urologic malignancies

    Mol. Cancer Ther.

    (2008)
  • S.M. Geranton et al.

    A rapamycin-sensitive signaling pathway is essential for the full expression of persistent pain states

    J. Neurosci.

    (2009)
  • X. Guan et al.

    Peripheral nerve injury up-regulates expression of interactor protein for cytohesin exchange factor 1 (IPCEF1) mRNA in rat dorsal root ganglion

    Naunyn Schmiedebergs Arch. Pharmacol.

    (2009)
  • Cited by (53)

    • Lysine-specific demethylase 1 in primary sensory neurons participates in chronic compression of dorsal root ganglion–induced neuropathic pain

      2022, Brain Research Bulletin
      Citation Excerpt :

      The mice in the sham control group underwent identical procedures but without any ligation of the L4 spinal nerve. The CFA–induced inflammatory pain mouse model was established by injecting 20 μL of 50% CFA (Mycobacterium tuberculosis; Sigma-Aldrich, St. Louis, MO, USA), an oil-saline (1:1) emulsion, subcutaneously into the plantar side of a hind paw (Liang et al., 2013a, 2013b). The control mice were injected with an equal volume of sterile normal saline instead of 50% CFA.

    • ErbB1-dependent signalling and vesicular trafficking in primary afferent nociceptors associated with hypersensitivity in neuropathic pain

      2020, Neurobiology of Disease
      Citation Excerpt :

      Responses to PGE2 were unaltered 5 days after EGF administration, which would align with our observations that concurrent nerve injury is required to reveal a sensitising effect of ErbB1 activation and support the hypothesis that intracellular signalling plays a key role in this connection. A number of reports have described the activation of Akt in DRG neurons in chronic pain hypersensitivity induced by nerve injury (Xu et al., 2007; Shi et al., 2009), chemotherapeutic-evoked neuropathy (Jiang et al., 2016; Li et al., 2016), inflammation (Liang et al., 2013), bone cancer (Guan et al., 2015) and intradermal injection of capsaicin, ephrin or formalin (Sun et al., 2007; Guan et al., 2010; Martin et al., 2017). In many cases, administration of PI 3-kinase or Akt inhibitors attenuated pain hypersensitivity, matching our observations here with the second generation highly selective Akt inhibitors, ipatasertib and afuresertib.

    View all citing articles on Scopus

    This work was supported by Grants (NS072206, NS058886, and DA033390) from the National Institutes of Health; Mr. David Koch and the Patrick C. Walsh Prostate Cancer Research Fund; the Rita Allen Foundation; and the Blaustein Pain Research Fund.

    View full text