NMDAR and JNK Activation in the Spinal Trigeminal Nucleus Caudalis Contributes to Masseter Hyperalgesia Induced by Stress

Abstract

It is commonly accepted that psychological stress is closely associated with the occurrence and development of chronic orofacial pain. However, the pathogenesis underlying this process has not been fully elucidated. In the present study, we explored the role of N-methyl-D-aspartate receptors (NMDARs) and Jun N-terminal kinase (JNK) mediated intercellular communication between neurons and astrocytes in the spinal trigeminal nucleus caudalis (Vc) in the induction of masseter hyperalgesia by psychological stress in rats. We found that subjecting rats to 14 days of restraint stress (8 h/d) caused a significant decrease in body weight gain, behavioral changes and marked masseter hyperalgesia in the rats. We also found that exposure to restraint stress for 14 days caused the expression of pJNK in astrocytes in the Vc to significantly increase, and intrathecally infusing a JNK inhibitor significantly prevented restraint stress-induced masseter hyperalgesia in the rats. In addition, after exposure to restraint stress for 14 days, the stressed group exhibited a noticeably increased expression level of pNR2B in neurons in the Vc. Then, we intrathecally injected MK-801 (an NMDAR inhibitor) and ifenprodil (a selective NR2B subunit antagonist) and observed that the two types of inhibitors not only alleviated masseter hyperalgesia but also significantly inhibited the phosphorylation of JNK in the Vc after restraint stress; this indicates that the effect of NMDAR antagonists may influence the activation of astrocytic JNK. Furthermore, inhibitors of neuronal nitric oxide synthase (nNOS) activation and guanylate cyclase (GC) inhibitor could not only inhibit the expression of pJNK in the Vc, but also effectively alleviate masseter hyperalgesia induced by restraint stress. Taken together, our results suggest that NMDAR activation could increase JNK phosphorylation in astrocytes after restraint stress, which may depend on the nNOS-GC pathway. The intercellular communication between neurons and astrocytes in the Vc may play a key role in the induction of masseter muscle hyperalgesia by psychological stress in rats.

Keywords: N-methyl-D-aspartate receptor; c-Jun N- terminal kinase; chronic masseter pain; intercellular communication; psychological stress; spinal trigeminal nucleus caudalis.

Figure 1

Effect of chronic restraint stress on body weight gain (A), behavior (B–E) and the head withdrawal threshold (F) in rats. In the open field test, the total distance traveled (B) and the distance traveled in the center (C) were measured, and in the elevated plus-maze test, the percentage of open-arm entries (D) and the open-arm retention time (E) were measured. All data were calculated as the mean ± SD (n = 8/group). *P < 0.05, **P < 0.01 vs. the matched control group.

Figure 2

Restraint stress increases the expression of the astrocytic specific marker GFAP (A,B) and phosphorylated JNK (pJNK, C,D) in the Vc after exposure to restraint stress for 14 days. Panels (E–J) shows the higher magnification of double immunofluorescence in the control and stress group. The arrows indicate some typical cells double-labeled with pJNK and GFAP. Scale bars = 100 μm in A (applies to A–D) and 50 μm in E (applies to E–J). (K) Red square frame refers to the region of staining in Vc. (L) Western blot showing that JNK1 is phosphorylated in the Vc after exposure to restraint stress for 14 days. *P < 0.05 vs. the control group, n = 4 in each group. (M) Effects of the intrathecal infusion of the JNK inhibitor SP600125 on masseter hyperalgesia. *P < 0.05 vs. the saline group, n = 4 in each group.

Figure 3

Panels (A–F) show the double immunofluorescence of pNR2B with NeuN or GFAP. The arrow indicates the typical double-labeled cell. Scale bars = 50 μm. (G) The impression of pNR2B in control and stress group. **P < 0.01 vs. the control group, n = 4 in each group.

Figure 4

The effects of an N-methyl-D-aspartate (NMDA) receptor antagonist on restraint stress-induced masseter hyperalgesia (A) and JNK activation (B). *P < 0.05, **P < 0.01 vs. the saline group, n = 4 in each group.

Figure 5

The effects of neuronal nitric oxide synthase (nNOS) selective inhibitor 7-NINA and guanylate cyclase (GC) inhibitor ODQ on restraint stress-induced masseter hyperalgesia (A) and JNK activation (B). *P < 0.05, **P < 0.01 vs. the saline group, n = 4 in each group.