The Up-regulation of TNF-α Maintains Trigeminal Neuralgia by Modulating MAPKs Phosphorylation and BKCa Channels in Trigeminal Nucleus Caudalis

Abstract

Trigeminal neuralgia (TN) is a severe chronic neuropathic pain. Despite numerous available medical interventions, the therapeutic effects are not ideal. To control the pain attacks, the need for more contemporary drugs continues to be a real challenge. Our previous study reported that Ca²⁺-activated K⁺ channels (BK _Ca ) channels modulated by mitogen-activated protein kinases (MAPKs) in the trigeminal ganglia (TG) neurons play crucial roles in regulating TN, and some research studies demonstrated that inflammatory cytokine tumor necrosis factor alpha (TNF-α) could promote neuropathic pain. Meanwhile, the trigeminal nucleus caudalis (TNC), the first central site of the trigeminal nociceptive pathway, is responsible for processing sensory and pain signals from the peripheral orofacial area. Thus, this study is aimed to further investigate whether TNF-α and MAPKs phosphorylation in the TNC could mediate the pathogenesis of TN by modulating BK _Ca channels. The results showed that TNF-α of the TNC region is upregulated significantly in the chronic constriction injury of infraorbital nerve (ION-CCI) rats model, which displayed persistent facial mechanical allodynia. The normal rats with target injection of exogenous TNF-α to the fourth brain ventricle behaved just like the ION-CCI model rats, the orofacial mechanical pain threshold decreased clearly. Meanwhile, the exogenous TNF-α increased the action potential frequency and reduced the BK _Ca currents of TNC neurons significantly, which could be reversed by U0126 and SB203580, the inhibitors of MAPK. In addition, U0126, SB203580, and another MAPK inhibitor SP600125 could relieve the facial mechanical allodynia by being injected into the fourth brain ventricle of ION-CCI model rats, respectively. Taken together, our work suggests that the upregulation of TNF-α in the TNC region would cause the increase of MAPKs phosphorylation and then the negative regulation of BK _Ca channels, resulting in the TN.

Keywords: BKCa channel; MAPKs phosphorylation; TNF-α; trigeminal neuralgia; trigeminal nucleus caudalis.

FIGURE 1

The expressions of some cytokines and chemokines are increased in the TNC region of ION-CCI rats and exogenous TNF-α could induce similar responses like the ION-CCI model and increase the excitability of TNC neurons. (A) TNF-α, but not IL-1β, IL-6, IL-10, or IL-18, is increased significantly in the TNC region of ION-CCI rats on the postoperative day 15 compared with the sham group rats, *p < 0.05, n = 8. (B) Both of CXCL1 and CCL2 are increased significantly in the TNC of ION-CCI rats, *p < 0.05, **p < 0.01, n = 8. (C) The facial mechanical pain threshold is reduced significantly from 40th to 220th min after injection of exogenous TNF-α (20 ng and 10 μl) to the fourth brain ventricle of normal rats, *p < 0.05, **p < 0.01, n = 6. (D) At the 150th min after exogenous TNF-α injection, the expression of CCL2 is increased in the TNC, whereas CXCL1 is relatively unchanged, **p < 0.01, n = 6. (E,F) TNF-α (10 ng/ml) pre-incubated for 1 min could increase the frequency of action potential of TNC neurons significantly with the stimulation of double threshold currents, **p < 0.01, n = 10. TNF-α, tumor necrosis factor alpha; TNC, trigeminal nucleus caudalis; ION-CCI, chronic constriction injury of infraorbital nerve; CCL2, C–C motif chemokine ligand 2.

FIGURE 2

The phosphorylation of ERK is upregulated in the TNC neurons of ION-CCI rats. (A) The level of p-ERK in the TNC is increased significantly on the 15th day after ION-CCI operation compared with the sham group,*p < 0.05, n = 6. (B) The p-ERK is mainly co-localized with NeuN, but not Iba1 or GFAP. (C,D) The ratio of TNC neurons of ION-CCI rats that express p-ERK is increased significantly compared with the sham group (77.99 ± 3.34%, n = 940 neurons from four rats vs. 49.95 ± 5.43%, n = 1,194 neurons from four rats), **p < 0.01. ERK, extracellular signal-regulated kinase; TNC, trigeminal nucleus caudalis; ION-CCI, chronic constriction injury of infraorbital nerve.

FIGURE 3

The phosphorylation of p38 is upregulated in the TNC microglia of ION-CCI rats. (A) The level of p-p38 in the TNC is increased significantly on the 15th day after the ION-CCI model compared with the sham group, *p < 0.05, n = 6. (B) The p-p38 is mainly co-localized with Iba1, but not NeuN or GFAP. (C,D) The ratio of TNC microglia of ION-CCI rats that express p-p38 is increased significantly compared with the sham group (93.55 ± 0.38%, n = 1,424 microglia from 3 rats vs. 77.19 ± 1.54%, n = 1,046 microglia from three rats), **p < 0.01. TNC, trigeminal nucleus caudalis; ION-CCI, chronic constriction injury of infraorbital nerve.

FIGURE 4

The phosphorylation of JNK is upregulated in the TNC microglia of ION-CCI rats. (A) The level of p-JNK in the TNC is increased significantly on the 15th day after the ION-CCI model compared with the sham group, *p < 0.05, n = 6. (B) The p-JNK is mainly co-localized with Iba1, but not NeuN or GFAP. (C,D) The ratio of TNC microglia of ION-CCI rats that express p-JNK is increased significantly compared with the sham group (88.48 ± 1.60%, n = 1,337 microglias from three rats vs. 74.35 ± 0.91%, n = 1,201 microglia from three rats), **p < 0.01. TNC, trigeminal nucleus caudalis; ION-CCI, chronic constriction injury of infraorbital nerve; jnk, c-Jun N-terminal kinase.

FIGURE 5

The inhibitors of MAPKs could relieve the facial mechanical pain induced by the ION-CCI model and the activity of MAPKs affects the excitability of TNC neurons induced by TNF-α. (A–C) The injection of U0126 (10 μg and 10 μl), SB203580 (10 μg and 10 μl), or SP600125 (10 μg and 10 μl) to the fourth brain ventricle of ION-CCI rats could increase the facial mechanical pain threshold significantly compared with the vehicle (10 μl), *p < 0.05, **p < 0.01, n = 6. The increase of facial mechanical pain threshold reaches the maximum at the 50th min after injection of U0126 (34.52 ± 9.82 g), the 40th min after injection of SB203580 (24.74 ± 6.32 g) and SP600125 (24.74 ± 6.32 g). (D,E) U0126 (1 μM) and SB203580 (10 nM) pre-incubated for 2 min, respectively, could significantly reduce the frequency of action potential in TNC neurons induced by TNF-α under the double threshold currents stimulation, but SP600125 (10 nM) could not with the same current stimulation and drug-delivery way. *p < 0.05, n = 6. TNC, trigeminal nucleus caudalis; ION-CCI, chronic constriction injury of infraorbital nerve; MATKs, mitogen-activated protein kinases.

FIGURE 6

The expression of BK_Ca channels is reduced in the TNC neurons of ION-CCI rats. (A,B) The expression of BK_Ca channels in TNC of ION-CCI rats is reduced compared with the sham rats, *p < 0.05, n = 6. (C,D) The BK_Ca channels are co-localized with the NeuN in the TNC region, and the expression ratio is also reduced significantly in the TNC neurons of ION-CCI rats compared with the sham group (8.25 ± 2.77%, n = 1,121 neurons from three rats vs. 59.59 ± 5.34%, n = 976 neurons from three rats), **p < 0.01. TNC, trigeminal nucleus caudalis; ION-CCI, chronic constriction injury of infraorbital nerve; BK_Ca, Ca²⁺-activated K⁺ channels.

FIGURE 7

The effects of MAPKs on BK_Ca currents in TNC neurons of normal rats. (A) TNF-α (10 ng/ml) pre-incubated for 1 min, reduces the BK_Ca peak currents of TNC neurons significantly (**p < 0.01, n ≥ 10). U0126 (1 μM) and SB203580 (10 nM) pre-incubated for 2 min, respectively, could largely reverse the effect of TNF-α (^##p < 0.01, n ≥ 10), but SP600125 (10 nM) could not. (B–D) The statistical analysis of the data of each group. TNC, trigeminal nucleus caudalis; ION-CCI, chronic constriction injury of infraorbital nerve; BK_Ca, Ca²⁺-activated K⁺ channels.

FIGURE 8

The I-V and G-V curves of BK_Ca currents in TNC neurons of normal rats. (A–C) The current density-voltage curves of BK_Ca induced by TNF-α could be reversed by U0126 (1 μM) and SB203580 (10 nM), but not SP600125 (10 nM). I–V curve is the currents follows a pre-pulse (0 mV, 100 ms) and a followed 400 ms test pulses from −80 to +80 mV with 10 mV increments. (D–F) The G-V curve of BK_Ca induced by TNF-α is not changed in the presence of U0126, SB203580 or SP600125. G-V curve is the G/Gmax follows a pre-pulse (0 mV and 100 ms) and a followed 400 ms test pulses from −80 to +80 mV with 10 mV increments. TNF-α, tumor necrosis factor alpha; BK_Ca, Ca²⁺-activated K⁺ channels.