Elsevier

Brain, Behavior, and Immunity

Volume 72, August 2018, Pages 45-50
Brain, Behavior, and Immunity

Full-length Article
Protraction of neuropathic pain by morphine is mediated by spinal damage associated molecular patterns (DAMPs) in male rats

https://doi.org/10.1016/j.bbi.2017.08.018Get rights and content

Highlights

  • Morphine administration after peripheral nerve injury increases spinal DAMP levels.

  • Elevation of DAMPs persists through at least 5 weeks after morphine cessation.

  • Increased levels of DAMPs are dependent on TLR4, P2X7R, and caspase-1.

  • These DAMPs are responsible for maintaining persistent allodynia.

Abstract

We have recently reported that a short course of morphine, starting 10 days after sciatic chronic constriction injury (CCI), prolonged the duration of mechanical allodynia for months after morphine ceased. Maintenance of this morphine-induced persistent sensitization was dependent on spinal NOD-like receptor protein 3 (NLRP3) inflammasomes—protein complexes that proteolytically activate interleukin-1β (IL-1β) via caspase-1. However, it is still unclear how NLRP3 inflammasome signaling is maintained long after morphine is cleared. Here, we demonstrate that spinal levels of the damage associated molecular patterns (DAMPs) high mobility group box 1 (HMGB1) and biglycan are elevated during morphine-induced persistent sensitization in male rats; that is, 5 weeks after cessation of morphine dosing. We also show that HMGB1 and biglycan levels are at least partly dependent on the initial activation of caspase-1, as well as Toll like receptor 4 (TLR4) and the purinergic receptor P2X7R—receptors responsible for priming and activation of NLRP3 inflammasomes. Finally, pharmacological attenuation of the DAMPs HMGB1, biglycan, heat shock protein 90 and fibronectin persistently reversed morphine-prolonged allodynia. We conclude that after peripheral nerve injury, morphine treatment results in persistent DAMP release via TLR4, P2X7R and caspase-1, which are involved in formation/activation of NLRP3 inflammasomes. These DAMPs are responsible for maintaining persistent allodynia, which may be due to engagement of a positive feedback loop, in which NLRP3 inflammasomes are persistently activated by DAMPs signaling at TLR4 and P2X7R.

Introduction

Opioids remain a gold-standard analgesic for moderate-to-severe chronic pain. Despite the recent escalation in opioid prescriptions for chronic pain, there are no clinical studies that have rigorously evaluated the long-term consequences of opioid use (Chou et al., 2015). Beyond simply an absence of benefit, there is growing evidence of harm among adults prescribed long-term opioid therapy (Chou et al., 2015, Frank et al., 2017, Hoffman et al., 2017, Sommer, 2017). We have discovered an additional negative consequence for pain in rats: a 5-day course of a moderate morphine dose, starting 10 days after sciatic chronic constriction injury (CCI), prolonged the duration of CCI-allodynia for months after morphine ceased (Grace et al., 2016). Maintenance of this morphine-induced persistent sensitization was dependent on spinal NOD-like receptor protein 3 (NLRP3) inflammasomes—protein complexes that proteolytically activate interleukin-1β (IL-1β) via caspase-1 (Grace et al., 2016). However, an unresolved question from our study is how NLRP3 inflammasome signaling is maintained long after morphine is cleared (Grace et al., 2016).

NLRP3 inflammasomes can be primed and activated by signaling through Toll like receptor 4 (TLR4) and the purinergic receptor P2X7, and both receptors are essential for morphine-induced persistent sensitization (Grace et al., 2016). TLR4 and P2X7R are activated after peripheral nerve injury by endogenous damage associated molecular patterns (DAMPs) that are released by stressed cells in the spinal cord (Grace et al., 2014). As a consequence of inflammasome activation, IL-1β may exacerbate cellular DAMP release (e.g. by disrupting glutamate homeostasis). As morphine-induced persistent sensitization was associated with persistent downregulation of the spinal astrocyte glutamate transporter GLT-1 (Grace et al., 2016), exacerbated DAMP release could be a consequence that leads to continued activation of NLRP3 inflammasomes in a positive feedback loop.

The first of three goals of this study was to determine whether the spinal DAMPs high mobility group box 1 (HMGB1) and biglycan were elevated in the lumbar dorsal spinal cord in our model of morphine-induced persistent sensitization. HMGB1 is a nuclear protein that is expressed by most cells. HMGB1 is released principally by neurons upon peripheral nerve injury, where it can induce an inflammatory response in surrounding cells (Agalave and Svensson, 2015). Biglycan is also expressed in the nucleus of spinal neurons, and in cultured astrocytes, but its function in these cells is not understood (Koops et al., 1996, Liang et al., 1997). Biglycan is upregulated after CNS injury, where it can be expressed by macrophages/microglia, and neurons (Stichel et al., 1995). A potential role for biglycan in pain has not been previously investigated. These representative DAMPs were selected due to their potential to activate NLRP3 inflammasomes: HMGB1 is a TLR4 agonist, while biglycan is an agonist of both TLR4 and P2X7R (Babelova et al., 2009, Frank et al., 2015, Grace et al., 2014). The second goal was to determine whether levels of HMGB1 and biglycan were dependent on TLR4, P2X7R or caspase-1. Finally, we aimed to identify a causal role for spinal DAMPS in the maintenance of morphine-induced persistent sensitization.

Section snippets

Subjects

Pathogen-free adult male Fischer 344 (F344) rats (n = 5–7 rats/group for each experiment; 10–12 wks old on arrival; Harlan Labs, Indianapolis, IN, USA) were used. All procedures were approved by the Institutional Animal Care and Use Committee of the University of Colorado Boulder.

Drugs

Morphine was obtained from the National Institute on Drug Abuse (Research Triangle Park, NC and Bethesda, MD, USA). (+)-Naloxone was synthesized by Dr. Kenner Rice. BoxA (HMGBiotech, Milan, Italy), fibronectin

DAMPs are elevated with morphine-induced persistent sensitization

We first tested whether levels of the spinal DAMPs HMGB1 and biglycan were elevated by the CCI and morphine treatment. As illustrated in Fig. S1, CCI or sham surgery was performed, followed ten days later—when neuropathic pain was fully established—by a 5-day course of morphine (5 mg/kg b.i.d., s.c.) or saline vehicle. We have previously shown that CCI-allodynia resolves 5 weeks after saline treatment, while CCI-allodynia persists for 11 weeks after morphine treatment (Grace et al., 2016).

Discussion

We recently discovered that morphine treatment after CCI induced allodynia that persisted for months after dosing had concluded, and was dependent on activation of spinal NLRP3 inflammasomes (Grace et al., 2016). We postulated that this was due to enduring spinal release of DAMPs that initiate TLR4 and P2X7R signaling (Grace et al., 2014), together with nonstereoselective opioid activation of TLR4 (Grace et al., 2015). However, an outstanding question from our prior work related to the

Acknowledgments

This work was supported by the American Pain Society Future Leaders in Pain Research Grants Program (P.M.G.); National Health and Medical Research Council CJ Martin Fellowship ID 1054091 (to P.M.G.); American Australian Association Sir Keith Murdoch Fellowship (P.M.G.); and, NIH Grants DE021966, DA023132 (to L.R.W.). The work of the Drug Design and Synthesis Section was supported by the NIH Intramural Research Programs of the National Institute on Drug Abuse and the National Institute of

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