Mammalian target of rapamycin in spinal cord neurons mediates hypersensitivity induced by peripheral inflammation

Abstract

mTOR, the mammalian target of rapamycin, is a serine-threonine kinase known to regulate cell proliferation and growth. mTOR has also been implicated in neuronal synaptic plasticity as well as in pain transmission in models of chemically induced and neuropathic pain. To date, the role of mTOR as a modulator of inflammatory pain has not been examined. In this study, we investigated the role of mTOR in Sprague-Dawley rats using the carrageenan model of inflammatory pain. mRNA of Ras homolog enriched in brain (Rheb), a GTPase that positively regulates mTOR activation, was significantly increased 2 h following carrageenan injection. Four hours after induction of inflammation phosphorylation (p) of p70S6 kinase (S6K), ribosomal protein S6 (S6) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) was increased, indicating mTOR activation. Inhibition of spinal mTOR with intrathecal (i.t.) injection of rapamycin (0.1-3 microg) led to a dose-dependent decrease in carrageenan-induced thermal hyperalgesia and a reduction of mechanical allodynia. In vitro studies confirmed rapamycin inhibition of the mTOR pathway. Carrageenan-induced activation of the mTOR pathway in rats was localized predominantly to dorsal horn neurons in the superficial lamina. Taken together, these data show that the mTOR pathway is activated in dorsal horn neurons during inflammatory pain, and that inhibition of spinal mTOR attenuates inflammation-induced thermal and tactile hypersensitivity. Hence, our study indicates that spinal mTOR is an important regulator of spinal sensitization and suggests that targeting mTOR may provide a new avenue for pain therapy.

Figure 1

Peripheral inflammation activates spinal Rheb, S6 kinase, S6 and 4E-BP1. Bar graphs display % change from naïve control (t = 0h) for Rheb (A), phoshpo (p)-S6 kinase (B), p-S6 (C) and p-4E-BP1 (D) in the spinal cord at different time points following carrageenan-induced inflammation. Representative Western blots of p-S6K, p-S6 and p-4E-BP1 are shown below each bar graph. Data are reported as mean ± SEM, n = 4. * represents p < 0.05 compared to naïve control.

Figure 2

Intrathecal (i.t.) injection of rapamycin attenuates thermal hyperalgesia and tactile allodynia induced by peripheral inflammation. Graphs display (A) thermal withdrawal thresholds in seconds (s) and (B) tactile thresholds in grams (g) before (t = 0h) and after injection of carrageenan to the paw and i.t. injection of vehicle or different doses of rapamycin (15 min pretreatment). The histograms represent the hyperalgesic index calculated as area under the curve (AUC, 0–240 min) for thermal hyperalgesia (C) and tactile allodynia (D) for each group shown in panel A and B. Data are reported as mean ± SEM, n = 6–7 rats per group. * represents p < 0.05 compared to vehicle control.

Figure 3

Intraplantar injection of carrageenan had no effect on S6 or 4E-BP1 phosphorylation in the DRGs. Bar graphs display % change from naïve controls (t = 0h) for (A) phospho (p)-S6 and (B) p-4E-BP1 in the ipsilateral DRGs (L4-L6) at different time points following carrageenan-induced inflammation. Representative Western blots of p-S6 and p-4E-BP1 are shown below each bar graphs. Data is shown as % change from naïve control and expressed as means ± SEM, n = 4–6.

Figure 4

Peripheral inflammation induces phosphorylation of S6 in dorsal horn neurons. (A) Representative confocal microscopy images depicting S6 phosphorylation in the contralateral (contra) and ipsilateral (ipsi) superfical laminae and deep dorsal horn 4 hours after injection of carrageenan to the paw. p-S6 immunoreactivity in the naïve dorsal horn (B) compared to 4 hours after carreageenan injection (C) demonstrates an increase in phospho (p)-S6 following induction of inflammation. The induced p-S6 (red) co-localized with NeuN, a marker for neurons (D), but not with GFAP (E) or OX-42 (F) markers for astrocytes and microglia, respectively. Rheb immunoreactivity colocalized with NeuN (G). DAPI was used as nuclear stain in panels D-F. The scale bar represents 50 μm.

Fig 5

Rapamycin treatment of PC12 cells attenuates serum-induced phosphorylation of S6 kinase, S6 and 4E-BP1. Bar graphs display % change from serum for phospho (p)-S6K (A), p-S6 (B) and p-4E-BP1 (C). Representative blots of p-S6K, p-S6 and p-4E-BP1 are shown below each bar graph. Data are reported as mean ± SEM, n = 3. * represents p < 0.05 compared to serum.