Korean J Physiol Pharmacol.  2021 Jul;25(4):365-374. 10.4196/kjpp.2021.25.4.365.

Rapamycin reduces orofacial nociceptive responses and microglial p38 mitogen-activated protein kinase phosphorylation in trigeminal nucleus caudalis in mouse orofacial formalin model

• Yeo JH
• Kim SJ
• Roh DH

• KMID: 2516875
• DOI: http://doi.org/10.4196/kjpp.2021.25.4.365

Abstract

The mammalian target of rapamycin (mTOR) plays a role in various cellular phenomena, including autophagy, cell proliferation, and differentiation. Although recent studies have reported its involvement in nociceptive responses in several pain models, whether mTOR is involved in orofacial pain processing is currently unexplored. This study determined whether rapamycin, an mTOR inhibitor, reduces nociceptive responses and the number of Fos-immunoreactive (Fos-ir) cells in the trigeminal nucleus caudalis (TNC) in a mouse orofacial formalin model. We also examined whether the glial cell expression and phosphorylated p38 (p-p38) mitogen-activated protein kinases (MAPKs) in the TNC are affected by rapamycin. Mice were intraperitoneally given rapamycin (0.1, 0.3, or 1.0 mg/kg); then, 30 min after, 5% formalin (10 l) was subcutaneously injected into the right upper lip. The rubbing responses with the ipsilateral forepaw or hindpaw were counted for 45 min. High-dose rapamycin (1.0 mg/kg) produced significant antinociceptive effects in both the first and second phases of formalin test. The number of Fos-ir cells in the ipsilateral TNC was also reduced by high-dose rapamycin compared with vehicle-treated animals. Furthermore, the number of p-p38-ir cells the in ipsilateral TNC was significantly decreased in animals treated with high-dose rapamycin; p-p38 expression was co-localized in microglia, but not neurons and astrocytes. Therefore, the mTOR inhibitor, rapamycin, reduces orofacial nociception and Fos expression in the TNC, and its antinociceptive action on orofacial pain may be associated with the inhibition of p-p38 MAPK in the microglia.

Keyword

Glia; mTOR; Orofacial pain; p38 mitogen-activated protein kinases; Rapamycin

Figure

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  Fig. 1 Effect of rapamycin in the orofacial formalin test in mice. Low- and medium-dose rapamycin (0.1 and 0.3 mg/kg, respectively) did not suppress orofacial formalin-induced nociceptive responses. On the other hand, high-dose rapamycin (1.0 mg/kg) significantly reduced nociceptive response at 3, 18–30, and 42 min after formalin injection (A). High-dose rapamycin (1.0 mg/kg) showed potent antinociceptive effects in the first and second phases (B)compared with the vehicle-treated (VEH) group (*p < 0.005, **p < 0.01, and ***p < 0.001, compared with the VEH group, n = 10–12 per group). i.p., intraperitoneally.

• 

  Fig. 2 Effect of rapamycin on the increase of Fos-immunoreactive (Fos-ir) cells in the trigeminal nucleus caudalis (TNC). The number of Fos-ir cells increased in the TNC of the vehicle-treated (VEH) group (A, E); low- and medium-dose rapamycin (0.1 and 0.3 mg/kg) did not suppress this increase of Fos-ir cells (B, C, E). On the other hand, high-dose rapamycin (1.0 mg/kg) significantly reduced the increase of Fos-ir cells in the ipsilateral TNC (D, E, ***p < 0.001, compared with the VEH group, n = 5). The boundaries of the TNC are outlined by the dotted line. Scale bar = 200 µm.

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  Fig. 3 Effect of rapamycin on glial fibrillary acidic protein (GFAP) and ionized calcium-binding adapter molecule-1 (Iba-1) expression in the trigeminal nucleus caudalis (TNC). The pixel threshold area of either GFAP (A–E) or Iba-1 (F–J) in the TNC remained unchanged in the vehicle-treated (VEH) group (A, F) and after all doses of rapamycin treatment (0.1, 0.3, and 1.0 mg/kg) (B–D, G–I). The boundaries of the TNC are outlined by the dotted line. Scale bar = 200 µm.

• 

  Fig. 4 Effect of rapamycin on phosphorylated p38 (p-p38) mitogen-activated protein kinase (MAPK) in the trigeminal nucleus caudalis (TNC). The expression of p-p38 in the TNC remained unchanged after treatment with low- and medium-dose rapamycin (0.1 and 0.3 mg/kg, respectively) (A–C, E; n = 5). On the other hand, the number of p-p38-ir cells significantly decreased after treatment with high-dose rapamycin (1.0 mg/kg) compared with the vehicle-treated (VEH) group (D, E; **p < 0.01, compared with the VEH group, n = 5). The boundaries of the TNC are outlined by the dotted line. The arrows indicate representative p-p38-ir cells. Scale bar = 200 and 100 µm (magnified panel).

• 

  Fig. 5 Co-localization of astrocyte (glial fibrillary acidic protein, GFAP), neuron (neuronal nuclear protein, NeuN), or microglia (ionized calcium-binding adapter molecule-1, Iba-1) with phosphorylated p38 (p-p38) MAPK in the trigeminal nucleus caudalis (TNC). Neither GFAP (A–C) nor NeuN (G–I) co-localized with p-p38 in the TNC. In contrast, Iba-1 immunoreactivity was co-expressed in p-p38-positive cells in the TNC in orofacial formalin-injected mice (D–F). The rectangular areas are magnified in panels (C, F, I), whereas arrows indicate representative p-p38-ir cells double-stained with Iba-1. Scale bar = 200 and 100 µm (magnified panel).

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