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

Abstract

We have recently reported that a short course of morphine, starting 10days 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, 5weeks 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.

Keywords: Danger signals; Glia; Opioid-induced hyperalgesia; Priming.

Figure 1. Morphine-induced persistent sensitization is associated with increased DAMP levels, which are dependent on TLR4, P2X7R and caspase-1

(a, b) CCI or sham surgery was performed, followed ten days later by a 5-day course of morphine (5 mg/kg b.i.d., s.c.) or saline vehicle. (a) HMGB1 and (b) biglycan levels were elevated in ipsilateral lumbar dorsal spinal cord quadrants, 5 weeks after morphine/saline administration. (c–h) Inhibitors were continuously intrathecally infused during morphine administration, and DAMP levels was assessed in the ipsilateral lumbar dorsal quadrant of the spinal cord at 5 weeks after morphine/inhibitor treatment. HMGB1 and biglycan levels were decreased by (c, d) the TLR4 antagonist (+)-naloxone (60 µg/h), (e, f) the P2X7R antagonist A438079 (30 ng/h), and (g, h) the caspase-1 inhibitor ac-YVAD-cmk (1 µg/h). (a, b) *P < 0.05, **P < 0.01, relative to Sham+Saline. (c–h) *P < 0.05, **P < 0.01, ***P < 0.001. N = 6/group.

Figure 2. Spinal DAMPs maintain morphine-induced persistent sensitization

A DAMP inhibitor cocktail against HMGB1 (BoxA; 5 µg in 5 µl), biglycan (siRNA; 1.2 µg in 10 µl), HSP90 (17-DMAG; 10 µg in 1 µl) and fibronectin (fibronectin tetrapeptide; 10 µg in 10 µl) was administered daily for 7 days via intrathecal injection (pink hatch; 7 days). DAMP inhibitor treatment began 5 weeks after morphine administration (5 days, 5 mg/kg b.i.d. administered 10 days after CCI; shaded panel) and absolute thresholds for mechanical allodynia quantified. Saline vehicle treatment (5 days, administered 10 days after CCI; shaded panel) is included for comparison (BL: Baseline). *P < 0.05, **P < 0.01, ***P < 0.001, inhibitor vs. vehicle control. N = 6/group.