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mTOR and Its Downstream Pathway Are Activated in the Dorsal Root Ganglion and Spinal Cord After Peripheral Inflammation, but Not After Nerve Injury

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mTOR and Its Downstream Pathway Are Activated in the Dorsal Root Ganglion and Spinal Cord After Peripheral Inflammation, but Not After Nerve Injury

Lingli Liang et al. Brain Res.

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 2h, 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.

Figures

Fig. 1
Fig. 1
Time-dependent pain hypersensitivity and activation of mTOR and S6K1 in spinal cord after intraplantar CFA injection. a and b Intraplantar injection of CFA produced mechanical allodynia (a) and thermal hyperalgesia (b) on the ipsilateral, but not contralateral, side. n = 5. *P < 0.05, **P < 0.01 vs contralateral side, two-way ANOVA followed by the Tukey post hoc test. c and d The levels of phosphorylated (p-) mTOR (c) and p-S6K1 (d) were significantly increased in the ipsilateral L4/5 spinal cord at 2 h, 1 day, 3 days, and 7 days after CFA injection. CFA injection did not alter the expression of total mTOR (c) or S6K1 (d) in the ipsilateral L4/5 spinal cord at these time points. (e) The levels of neither p-mTOR nor p-S6K1 were altered in the contralateral L4/5 spinal cord after CFA injection during the observation period. For c–e, the upper panels depict representative Western blots, and the lower panels show the statistical summary of the densitometric analysis expressed relative to basal level (0 h) after normalization to the corresponding β-actin. n = 3/time point. *P < 0.05 vs the corresponding naïve rats (0 h), one-way ANOVA followed by the Tukey post hoc test.
Fig. 2
Fig. 2
Time-dependent activation of mTOR in dorsal root ganglion (DRG) after intraplantar CFA injection. (a) The level of p-mTOR was significantly increased in the ipsilateral L4/5 DRGs on days 1 and 3, but not at 2 h or on day 7, after CFA injection. The amount of total mTOR was not changed in the ipsilateral L4/5 DRGs during the observation period. (b) p-mTOR expression did not change in the contralateral L4/5 DRGs after CFA injection during the observation period. In a and b, the upper panels depict representative Western blots, and the lower panels show the statistical summary of the densitometric analysis expressed relative to basal level (0 h) after normalization to the corresponding β-actin. n = 3/time point. *P < 0.05 vs the corresponding naïve rats (0 h), one-way ANOVA followed by the Tukey post hoc test.
Fig. 3
Fig. 3
p-mTOR immunoreactivity increases in L4/5 DRGs and L4/5 spinal cord after intraplantar CFA injection. (a) Representative images for p-mTOR-labeled neurons in the ipsilateral (Ipsi) and contralateral (Contra) L5 DRG 1 day after CFA injection. The number of p-mTOR-labeled neurons was significantly greater in the ipsilateral L4/5 DRGs than in the contralateral L4/5 DRGs. Scale bar: 50 μm. (b) Statistical summary of the percentages of p-mTOR-labeled neurons in the ipsilateral and contralateral L4/5 DRGs. n = 3. *P < 0.05, **P < 0.01 vs the corresponding contralateral side by Student's t-test. (c) Representative images depict the distribution of p-mTOR immunoreactivity in ipsilateral and contralateral L5 dorsal horn 1 day after CFA injection. Scale bar: 30 μm.
Fig. 4
Fig. 4
Effect of mTOR inhibition on CFA-induced mechanical and thermal pain hypersensitivities. (a) Intrathecal injection of rapamycin (Rapa; 20 μg dissolved in 50% DMSO) markedly reversed the CFA-induced decrease in paw withdrawal threshold on day 1 after intraplantar CFA injection. Intrathecal injection of rapamycin in the absence of CFA injection had no effect on basal paw withdrawal threshold. (b) Intrathecal injection of rapamycin significantly reversed the CFA-induced reduction in paw withdrawal latency on day 1 after CFA injection. Intrathecal injection of rapamycin in the absence of CFA injection did not alter basal paw withdrawal latency. For a and b, n = 5/group; **P < 0.01 vs the corresponding baseline; #P < 0.05, ##P < 0.01 vs the group injected with CFA and treated with vehicle (50% DMSO), two-way ANOVA followed by the Tukey post hoc test.
Fig. 5
Fig. 5
Time course of pain hypersensitivity and expression of p-mTOR and p-S6K1 in the ipsilateral spinal cord and DRG after spinal nerve ligation (SNL). a and b L5 spinal nerve ligation produced mechanical allodynia (a) and thermal hyperalgesia (b) on the ipsilateral, but not contralateral, side. n = 5. *P < 0.05, **P < 0.01 vs contralateral side, two-way ANOVA followed by the Tukey post hoc test. (c) The levels of p-mTOR and p-S6K1 in the ipsilateral L5 spinal cord were not significantly altered on days 3, 7, or 14 post-SNL. n = 3/time point. (d) The level of p-mTOR in the ipsilateral L5 DRG was not significantly altered on days 3, 7, or 14 post-SNL. n = 3/time point. For c and d, the upper panels depict representative Western blots, and the lower panels show the statistical summary of the densitometric analysis expressed relative to basal level (0 d) after normalization to the corresponding β-actin by one-way ANOVA followed by the Tukey post hoc test.

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