Spinal protein kinase M ζ underlies the maintenance mechanism of persistent nociceptive sensitization

Abstract

Sensitization of the pain pathway is believed to promote clinical pain disorders. We hypothesized that the persistence of a sensitized state in the spinal dorsal horn might depend on the activity of protein kinase M ζ (PKMζ), an essential mechanism of late long-term potentiation (LTP). To test this hypothesis, we used intraplantar injections of interleukin-6 (IL-6) in mice to elicit a transient allodynic state that endured ∼3 d. After the resolution of IL-6-induced allodynia, a subsequent intraplantar injection of prostaglandin E(2) (PGE(2)) or intrathecal injection of the metabotropic glutamate receptor 1/5 (mGluR1/5) agonist DHPG (dihydroxyphenylglycol) precipitated allodynia and/or nocifensive responses. Intraplantar injection of IL-6 followed immediately by intrathecal injection of a PKMζ inhibitor prevented the expression of subsequent PGE(2)-induced allodynia. Inhibitors of protein translation were effective in preventing PGE(2)-induced allodynia when given immediately after IL-6, but not after the initial allodynia had resolved. In contrast, spinal PKMζ inhibition completely abolished both prolonged allodynia to hindpaw PGE(2) and enhanced nocifensive behaviors evoked by intrathecal mGluR1/5 agonist injection after the resolution of IL-6-induced allodynia. Moreover, spinal PKMζ inhibition prevented the enhanced response to subsequent stimuli following resolution of hypersensitivity induced by plantar incision. The present findings demonstrate that the spinal cord encodes an engram for persistent nociceptive sensitization that is analogous to molecular mechanisms of late LTP and suggest that spinally directed PKMζ inhibitors may offer therapeutic benefit for injury-induced pain states.

Figure 1.

IL-6-induced persistent nociceptive sensitization initiation but not maintenance depends on local translation within the hindpaw at the time of IL-6 administration. A, Experimental design for initiation of persistent sensitization testing. B–D, IL-6 was administered intraplantarly (i.pl.) with or without inhibitors of MEK (U0126, B), general translation (anisomycin, C), or eIF4F complex formation (4EGI-1, D). Six days following IL-6 and inhibitor coadministration, PGE₂ (100 ng) was injected into the same hindpaw to assay persistent sensitization. PGE₂-induced allodynia was abrogated by U0126 (B), anisomycin (C), and 4EGI-1 (D). E, Moreover, 4EGI-1 was also able to block persistent sensitization caused by coinjection of IL-6 and NGF. F, G, Acute allodynia induced by IL-6 injection was decreased in fmr1^−/y mice (F), and persistent sensitization to PGE₂ injection on day 6 was also decreased in fmr1^−/y mice (G). H, Experimental design for maintenance of persistent sensitization testing. I, J, Anisomycin (I) or 4EGI-1 (J) injected into the hindpaw 4 d following IL-6 injection into the same hindpaw failed to attenuate PGE₂-precipitated persistent sensitization. N = 6 for all experiments. Colored stars denote significant effects compared with vehicle groups, and black stars denote significance between IL-6-treated groups. *p < 0.05, **p < 0.01, and ***p < 0.001. VEH, Vehicle.

Figure 2.

Persistent nociceptive sensitization depends on early, spinal translation and PKMζ activity: intrathecal injection of the mTOR inhibitor temsirlimus at the time of intraplantar (i.pl.) injection. A–F, IL-6 injection blocks allodynia (A) and persistent sensitization to PGE₂ (100 ng, B). Allodynia (C) and persistent (D) sensitization is also blocked by the eIF4F complex formation inhibitor 4EGI1 and by the PKMζ inhibitor ZIP (E, F). N = 6 for all experiments. Colored stars denote significant effects compared with vehicle groups, and black stars denote significance between IL-6-treated groups. *p < 0.05, **p < 0.01, and ***p < 0.001. VEH, Vehicle.

Figure 3.

A, B, PKMζ inhibition before IL-6-induced priming does not influence persistent nociceptive sensitization: intrathecal injection of ZIP or Scr ZIP 2 d before intraplantar (i.pl.) injection of IL-6 has no effect on IL-6-induced allodynia (A) or on persistent nociceptive sensitization precipitated by i.pl. injection of PGE₂ (100 ng) on day 6 (B). N = 6 per group. ***p < 0.001. VEH, Vehicle.

Figure 4.

Persistent nociceptive sensitization is maintained by spinal PKMζ. A, Intrathecal injection of the PKMζ inhibitor ZIP on day 4 reverses IL-6-induced persistent sensitization precipitated by intraplantar (i.pl.) PGE₂ injection (100 ng). Moreover, intrathecal ZIP fully reverses persistent sensitization to PGE₂ injection when given up to 16 d following IL-6 injection while Scr ZIP had no effect (B). C, Intrathecal injection of the PKMζ inhibitor ZIP on day 4 also reverses IL-6-induced persistent sensitization precipitated by intrathecal injection of DHPG on day 6. D, Intrathecal injection of pep2m on day 4 inhibits persistent sensitization to i.pl. PGE₂ administration on day 6. N = 6 for all experiments. Colored stars denote significant effects compared with vehicle groups, and black stars denote significance between IL-6-treated groups. *p < 0.05, **p < 0.01, and ***p < 0.001. VEH, Vehicle.

Figure 5.

Spinal translation inhibition after the resolution of IL-6-induced allodynia fails to reverse persistent nociceptive sensitization. A–C, Intrathecal injection of the mTOR inhibitor temsirolimus (A), the eIF4F complex formation inhibitor 4EGI-1 (B), or the general protein synthesis inhibitor anisomycin (C) on day 4 does not reverses persistent sensitization caused by intraplantar (i.pl.) IL-6 injection into the paw on day 1 when the persistent sensitization is induced by i.pl. PGE₂ (100 ng) administration. D, Intrathecal injection of staurosporine on day 5 following intraplantar IL-6 injection does not influence PGE₂-precipitated persistent sensitization on day 7. N = 6 for all experiments. Colored stars denote significant effects compared with vehicle groups. ***p < 0.001. VEH, Vehicle.

Figure 6.

Plantar incision produces a spinal PKMζ-dependent persistent nociceptive sensitization: Plantar incision was done on day 1 (BL) and mechanical allodynia was measured on postoperative day (POD) 1, 9 and 13. A, Mice with plantar incision developed allodynia lasting for 9 d and fully resolved by POD 13. ZIP or Scr ZIP was injected intrathecally (i.t.) on POD 13 and PGE₂ was injected intraplantarly on day 15. B, Intrathecal ZIP abolished persistent nociceptive sensitization induced by plantar incision. Colored stars denote significant effects compared with naive groups that also received intraplantar PGE₂, and black stars denote significance between ZIP- and Scr ZIP-treated groups. N = 6 per group. **p < 0.01, ***p < 0.001.

Figure 7.

Spinal expression of constitutively active PKCζ recapitulates persistent sensitization. A, mCherry expression in dorsal horn neurons, 31 d following intrathecal (i.t.) injection of lentiviral construct bicistronically expressing constitutively active PKCζ and mCherry. B, Neuronal viral transduction was measured with NeuN colabeling with mCherry. Prominent mCherry expression was noted in the cytoplasm of neurons (nucleus labeled with NeuN antibody) throughout the substantia gelatinosa. C, Mice receiving intrathecal injection of the active virus developed mechanical allodynia lasting until at least 12 d postinjection. N = 6 per group. D, Expression of constitutively active PKCζ increased nocifensive behaviors induced by intrathecal injection of DHPG on day 31 after virus injection. N = 5 per group. *p < 0.05, ***p < 0.001.