Acute inhibition of Ca2+/calmodulin-dependent protein kinase II reverses experimental neuropathic pain in mice

Abstract

The limited data that currently exist for the role of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) in neuropathic pain are conflicting. In the present study, we tested the hypothesis that CaMKII is required for the maintenance of neuropathic pain in a rodent model of experimental mononeuropathy. Spinal nerve L(5)/L(6) ligation (SNL) was found to increase the spinal activity of CaMKII (pCaMKII) on the ipsilateral (but not contralateral) side. This effect was blocked by 2-[N-(2-hydroxyethyl)-N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine) (KN93) (intrathecal injection), a CaMKII inhibitor. Acute treatment with KN93 dose-dependently reversed SNL-induced thermal hyperalgesia and mechanical allodynia. The action of KN93 lasted for at least 2 to 4 h. 2-[N-(4-Methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine (KN92) (45 nmol i.t.), an inactive analog of KN93, showed no effect on SNL-induced CaMKII activation, allodynia, or hyperalgesia. We further examined the pharmacologic action of trifluoperazine, a clinically used antipsychotic drug that we found to be a potent CaMKII inhibitor in these assays. Trifluoperazine (administered intraperitoneally or by mouth) dose-dependently reversed SNL-induced mechanical allodynia, thermal hyperalgesia, and CaMKII activation without causing locomotor impairment in mice at the highest doses used. In conclusion, our findings support a critical role of CaMKII in neuropathic pain. Blocking CaMKII or CaMKII-mediated signaling may offer a novel therapeutic target for the treatment of neuropathic pain.

Fig. 1.

Reversal of spinal nerve ligation-induced mechanical allodynia (A) and thermal hyperalgesia (B) by KN93, a CaMKII inhibitor. The tight ligation of L₅ and L₆ spinal nerves (SNL) induced mechanical allodynia and thermal hyperalgesia in mice, which was absent in sham-operated mice. KN93, KN92, or saline was administered by intrathecal injection 2 h before mechanical and thermal sensitivity testing on day 5 after SNL. KN93 (30 and 45 nmol) significantly reversed SNL-induced mechanical allodynia and thermal hyperalgesia. KN93 (15 nmol) or KN92 (45 nmol) did not change the level of mechanical allodynia or thermal hyperalgesia. Data are expressed in mean ± S.E.M. ***, p < 0.001, compared with the sham group; †††, p < 0.001, compared with the SNL group, n = 8 for each group. Arrows indicate the approximate time when KN93, KN92, or saline was injected.

Fig. 2.

Suppression of spinal nerve ligation-induced spinal CaMKII activation by KN93. Ipsilateral CaMKII activity (A), CaMKII expression (B), and contralateral CaMKII activity (C) in the lumbar sections of the spinal cord were determined by Western blotting. L₅/L₆ spinal nerve ligation significantly increased CaMKII activity (pCaMKII) on the ipsilateral side, but not on the contralateral side, and CaMKII expression was not altered significantly. Acute treatment with KN93 (45 and 30 nmol i.t., but not 15 nmol i.t.) reduced SNL-induced spinal CaMKII activity, but KN92 (45 nmol i.t.) did not change CaMKII activity. Histogram data, expressed in mean ± S.E.M., were constructed from the representative figure shown and three other experiments. *, p < 0.05; ***, p < 0.001 compared with the sham group; †††, p < 0.001 compared with the SNL group. OD, optical density.

Fig. 3.

Time course of reversing spinal nerve ligation-induced mechanical allodynia (A) and thermal hyperalgesia (B) by KN93. After mechanical allodynia and thermal hyperalgesia were established 5 days after L₅/L₆ SNL, mice were administered 15 to 45 nmol i.t. KN93 or saline. Mechanical and thermal sensitivities were tested at 0, 0.5, 2, 4, 8, and 24 h after the KN93 or saline injection. KN93 reversed the established SNL-induced mechanical allodynia and thermal hyperalgesia in a dose- and time-dependent manner. Data are expressed in mean ± S.E.M. *, p < 0.05; ***, p < 0.001, compared with the sham group; †, p < 0.05, †††, p < 0.001, compared with the SNL group, n = 8 for each group.

Fig. 4.

Reversal of spinal nerve ligation-induced mechanical allodynia (A) and thermal hyperalgesia (B) by trifluoperazine. After mechanical allodynia and thermal hyperalgesia were established 5 days after L₅/L₆ SNL, mice were treated with trifluoperazine (0.1–0.5 mg/kg i.p.) or saline 2 h before the mechanical and thermal sensitivity tests. Trifluoperazine at 0.25 to 0.5 mg/kg, but not 0.1 mg/kg, reversed the established SNL-induced mechanical allodynia and thermal hyperalgesia. Data are expressed in mean ± S.E.M. ***, p < 0.001, compared with the sham group; †††, p < 0.001 compared with the SNL group, n = 8 for each group. Postsurgery mechanical or thermal sensitivity was tested immediately before the treatment with trifluoperazine on postsurgery (SNL or sham operation) day 5.

Fig. 5.

Suppression of spinal nerve ligation-induced spinal CaMKII activity by trifluoperazine. The Western blotting method was used to detect ipsilateral CaMKII activity (A), CaMKII expression (B), and contralateral CaMKII activity (C) in the lumbar sections of the spinal cord. L₅/L₆ SNL significantly increased CaMKII activity (pCaMKII) in the lumbar spinal cord on the ipsilateral, but not contralateral, side compared with the sham group. CaMKII expression was not significantly altered by SNL or KN93. Acute treatment (intraperitoneal) with trifluoperazine dose-dependently reduced SNL-induced spinal CaMKII activity. Histogram data, expressed in mean ± S.E.M., were constructed from the representative figure shown and three other experiments. *, p < 0.05; ***, p < 0.001 compared with the sham group; †, p < 0.05; †††, p < 0.001 compared with the SNL group.

Fig. 6.

Time course of reversing spinal nerve ligation-induced mechanical allodynia (A) and thermal hyperalgesia (B) by trifluoperazine (administered intraperitoneally). After mechanical allodynia and thermal hyperalgesia were established 5 days after L₅/L₆ SNL, mice were given trifluoperazine (0.1–0.5 mg/kg i.p.) or normal saline. Mechanical and thermal sensitivities were tested at 0, 0.5, 2, 4, 8, and 24 h after the trifluoperazine or saline injection. Trifluoperazine dose- and time-dependently reversed established SNL-induced mechanical allodynia and thermal hyperalgesia. Data are expressed in mean ± S.E.M. ***, p < 0.001, compared with the sham group; †††, p < 0.001, compared with the SNL group, n = 8 for each group.

Fig. 7.

Reversal of spinal nerve ligation-induced mechanical allodynia (A) and thermal hyperalgesia (B) by orally administered trifluoperazine. After mechanical allodynia and thermal hyperalgesia were established 5 days after L₅/L₆ spinal nerve ligation (SNL), mice were treated with trifluoperazine (0.1–1.0 mg/kg, gastric gavage) or saline 2 h before the thermal and mechanical sensitivity tests. Trifluoperazine dose-dependently reversed the established SNL-induced mechanical allodynia and thermal hyperalgesia. Postsurgery mechanical or thermal sensitivity was tested immediately before the treatment with trifluoperazine on day 5 after surgery (SNL or sham operation). Data are expressed in mean ± S.E.M. ***, p < 0.001, compared with the sham group; †††, p < 0.001 compared with the SNL group, n = 8 for each group.

Fig. 8.

Time course of reversing spinal nerve ligation-induced mechanical allodynia (A) and thermal hyperalgesia (B) by orally administered trifluoperazine. Mice were administered trifluoperazine (0.1–1.0 mg/kg gastric gavage) or normal saline. Mechanical and thermal sensitivities were tested at 0, 0.5, 2, 4, 8, and 24 h after the trifluoperazine or saline injection. Trifluoperazine reversed the established SNL-induced mechanical allodynia and thermal hyperalgesia in a dose- and time-dependent manner. Data are expressed in mean ± S.E.M. *, p < 0.05; ***, p < 0.001, compared with the sham group; †, p < 0.05; †††, p < 0.001, compared with the SNL group, n = 8 for each group.