α7 Nicotinic acetylcholine receptor-mediated anti-inflammatory effect in a chronic migraine rat model via the attenuation of glial cell activation

Abstract

Background: Evidence suggests that the activation of α7 nicotinic acetylcholine receptor (α7nAChR) can greatly decrease the neuroinflammation response. Neuroinflammation plays a pivotal role in the pathogenesis of chronic migraine (CM). Clinical observations also show that nicotine gum induces analgesic effects in migraine patients. However, whether α7nAChR is involved in CM is unclear.

Objective: To investigate the role of α7nAChR in CM and provide a new therapeutic target for CM.

Materials and methods: Thirty-six male Sprague-Dawley rats were distributed randomly into control, CM, PNU-282987, and α-bungarotoxin groups (n=9 rats in each group). The CM model was established by the recurrent daily administration of inflammatory soup on the dura over the course of 1 week. The hind paw threshold and facial allodynia were assessed by the von Frey test. The expression levels of α7nAChR, tumor necrosis factor-alpha, and interleukin-1 beta were analyzed by Western blot and real-time fluorescence quantitative polymerase chain reaction. The location of α7nAChR in the hippocampus was quantified by immunofluorescence, as well as the microglial and astrocyte alterations. Changes in the calcitonin gene-related peptide and the phosphorylated JNK protein among different groups were measured by Western blot.

Results: We found that the expression of α7nAChR was reduced after repeated inflammatory soup administration. The increased expression of tumor necrosis factor-alpha, interleukin-1 beta, and calcitonin gene-related peptide in CM group were significantly decreased by PNU-282987 and aggravated by α-bungarotoxin. Moreover, PNU-282987 decreased the numbers of astrocytes and microglia compared with the numbers in the CM group in both hippocampal CA1 and CA3 regions. In contrast, α-bungarotoxin activated the astrocytes and microglia, but the differences with respect to the CM group were not significant. Activated c-Jun N-terminal kinase signaling was observed in CM rats and was also blocked by PNU-282987.

Conclusion: The activation of α7nAChR increased the mechanical threshold and alleviated pain in the CM rat model. α7nAChR activation also decreased the upregulation of astrocytes and microglia through the p-c-Jun N-terminal kinase-mitogen-activated protein kinase signaling pathway.

Keywords: analgesia; chronic migraine; glial activation; neuroinflammation; nociception; α7nAChR.

Figure 1

Schematic of the experimental design. Notes: Cannulations were performed, and after at least 1 week of recovery, PBS or IS stimulation was applied to the dura through the affixed cannula. After the last stimulation (on the eighth day), the vehicle, PNU-282987 or α-bungarotoxin was administered by intracerebroventricular injection to the different groups as illustrated. Twenty-four hours later (on the ninth day), the rats were decapitated. Abbreviations: α-B, α-bungarotoxin; CM, chronic migraine; Con, control; I.C.V, intracerebroventricular; IS, inflammatory soup; PNU, PNU-282987.

Figure 2

The decreased mechanical threshold in the CM group (relative to the control) was mitigated by PNU-282987 but aggravated by α-bungarotoxin. Notes: (A) The mechanical threshold of allodynia decreased in the CM group in hind paw stimulation (*p<0.05, **p<0.01 versus control group). (B) The hind paw withdrawal and periorbital threshold were somewhat decreased after the intracerebroventricular injection of α-bungarotoxin. The periorbital threshold was increased by PNU-282987. An increasing trend, but no significant difference, was observed between the CM and PNU-282987 groups (*p>0.05 versus CM group) (n=9 per group). Abbreviations: α-B, α-bungarotoxin; CM, chronic migraine; Con, control; PNU, PNU-282987.

Figure 3

The expression of α7nAChR decreased in the hippocampus of CM rats. Notes: The level of α7nAChR in the hippocampus was determined by WB and PCR. The recurrent injection of IS decreased α7nAChR expression significantly at both the protein (A) and the mRNA levels (B) (**p<0.01, n=6 per group). Abbreviations: α7nAChR, α7 nicotinic acetylcholine receptor; CM, chronic migraine; IS, inflammatory soup; PCR, polymerase chain reaction; WB, Western blot.

Figure 4

The distribution of α7nAChR in the hippocampus in all groups. Notes: (A) Schematic diagram of the hippocampus. (B) The expression of α7nAChR in hippocampal CA1, CA3, and DG areas. (C) The average numbers of α7nAChR were significantly decreased in hippocampal CA1, CA3, and DG areas in the CM group. PNU-282987 activated the α7nAChR in both CA1 and CA3 regions, but not in DG region. There were no significant differences between the CM and α-bungarotoxin groups in all the 3 regions (⁺⁺p<0.01 versus control, **p<0.01 versus CM). All data were expressed as the mean ± SEM, n=3 per group, bar: 250 μm of (A), 25 μm in CA1 and CA3 of (B), 100 μm in DG of (B). Abbreviations: α7nAChR, α7 nicotinic acetylcholine receptor; α-B, α-bungarotoxin; CM, chronic migraine; Con, control; DG, dentate gyrus; PNU, PNU-282987; SEM, standard error of the mean.

Figure 5

The morphology of astrocytes and microglia in the hippocampus CA1 and CA3 areas in all groups. Notes: (A) In the CM and α-bungarotoxin groups, the astrocytes and microglia exhibited swollen cell bodies and synaptic coarsening, while the morphology of the control group was small cell bodies. In the PNU-282987 group, the morphology of the labeled-activated cells showed less activation, with fewer numbers and smaller cell bodies. (B) The average numbers of astrocytes and microglia were significantly increased in the CM group and were both suppressed by PNU-282987. The GFAP-labeled cells and Iba1-positive cells showed no significant differences between the CM and α-bungarotoxin groups (⁺p<0.05 and ⁺⁺p<0.01 versus control, **p<0.01 versus CM). All data were expressed as the mean ± SEM, n=3 per group, bar: 25 μm. Abbreviations: α-B, α-bungarotoxin; CM, chronic migraine; Con, control; GFAP, goat anti-rat glial fibrillary acidic protein; Iba1, ionized calcium binding adaptor molecule 1; PNU, PNU-282987; SEM, standard error of the mean.

Figure 6

WB and PCR data showed dynamic changes in TNF-α, IL-1β, and CGRP. Notes: (A) The IS-induced increases in TNF-α and IL-1β were both significantly inhibited by PNU-282987 (⁺⁺p<0.01 versus control, **p<0.01 versus CM). In the α-bungarotoxin-treated group, TNF-α showed a tendency to increase compared with the value in the CM group (p>0.05), while IL-1β showed a significant increase (p<0.01). (B) mRNA data for TNF-α and IL-1β (⁺p<0.05 and ⁺⁺p<0.01 versus control; *p<0.05 and **p<0.01 versus CM). (C) The IS-induced increase in CGRP protein expression was significantly inhibited after PNU-282987 administration. In contrast, α-bungarotoxin stimulated a further increase in CGRP expression (⁺⁺p<0.01 versus control, **p<0.01 versus CM). All data are expressed as the mean ± SEM, n=6 per group. Abbreviations: α-B, α-bungarotoxin; CGRP, calcitonin gene-related peptide; CM, chronic migraine; Con, control; IS, inflammatory soup; PCR, polymerase chain reaction; PNU, PNU-282987; WB, Western blot.

Figure 7

Gel panels of hippocampus tissue in control, CM, PNU-282987, and α-bungarotoxin groups using WB. β-Actin was used as a loading control. Notes: The relative protein level of p-JNK was increased in CM rats and was suppressed by PNU-282987. A higher protein level of p-JNK was also observed after the administration of α-bungarotoxin, but the difference from the level in the CM group was not statistically significant (⁺⁺p<0.01 versus control, **p<0.01 versus CM). All data are expressed as the mean ± SEM, n=6 per group). Abbreviations: α-B, α-bungarotoxin; CM, chronic migraine; Con, control; p-JNK, phosphorylated c-Jun N-terminal kinase; PNU, PNU-282987; WB, Western blot; SEM, standard error of the mean.

Figure 8

The theme of this article. Notes: Microglia and astrocytes in the CNS mediated the development of CM. Microglia and astrocytes were activated in CM, they released TNF-α and IL-1β through the downstream p-JNK-MAPK signaling pathway to further aggravate CM. The activation of α7nAChR alleviated the activated glia and ameliorated the pain. Abbreviations: α7nAChR, α7 nicotinic acetylcholine receptor; CM, chronic migraine; CNS, central nervous system; IL, interleukin; MAPK, mitogen-activated protein kinase; p-JNK, phosphorylated c-Jun N-terminal kinase.; TNF, tumor necrosis factor.