A c-Fos activation map in nitroglycerin/levcromakalim-induced models of migraine

Abstract

Background: Chronic migraine is a common and highly disabling disorder. Functional MRI has indicated that abnormal brain region activation is linked with chronic migraine. Drugs targeting the calcitonin gene-related peptide (CGRP) or its receptor have been reported to be efficient for treating chronic migraine. The CGRP signaling was also shared in two types of chronic migraine models (CMMs). However, it remains unclear whether the activation of specific brain regions could contribute to persistent behavioral sensitization, and CGRP receptor antagonists relieve migraine-like pain in CMMs by altering specific brain region activation. Therefore, it's of great interest to investigate brain activation pattern and the effect of olcegepant (a CGRP receptor-specific antagonist) treatment on alleviating hyperalgesia by altering brain activation in two CMMs, and provide a reference for future research on neural circuits.

Methods: Repeated administration of nitroglycerin (NTG) or levcromakalim (LEV) was conducted to stimulate human migraine-like pain and establish two types of CMMs in mice. Mechanical hypersensitivity was evaluated by using the von Frey filament test. Then, we evaluated the activation of different brain regions with c-Fos and NeuN staining. Olcegepant was administered to explore its effect on mechanical hyperalgesia and brain region activation.

Results: In two CMMs, acute and basal mechanical hyperalgesia was observed, and olcegepant alleviated mechanical hyperalgesia. In the NTG-induced CMM, the medial prefrontal cortex (mPFC), anterior cingulate cortex (ACC), and the caudal part of the spinal trigeminal nucleus (Sp5c) showed a significant increase of c-Fos expression in the NTG group (p < 0.05), while pre-treatment with olcegepant reduced c-Fos expression compared with NTG group (p < 0.05). No significant difference of c-Fos expression was found in the paraventricular thalamic nucleus (PVT) and ventrolateral periaqueductal gray (vlPAG) between the vehicle control and NTG group (p > 0.05). In the LEV-induced CMM, mPFC, PVT, and Sp5c showed a significant increase of c-Fos expression between vehicle control and LEV group, and olcegepant reduced c-Fos expression (p < 0.05). No significant difference in c-Fos expression was found in vlPAG and ACC (p > 0.05).

Conclusions: Our study demonstrated the activation of mPFC and Sp5c in two CMMs. Olcegepant may alleviate hyperalgesia of the hind paw and periorbital area by attenuating brain activation in CMMs.

Keywords: CGRP; Chronic migraine; Migraine-like pain; c-Fos.

Fig. 1

CGRP receptor antagonist (olcegepant) alleviated acute hyperalgesia in the NTG-induced chronic migraine model. A. Representative schematic diagrams and procedures for the behavioral tests. (Created with BioRender.com) B-C. Repeated NTG administration induced mechanical hyperalgesia of periorbital area (B) and hindpaw (C), alleviated by OLC. Two-way ANOVA with the Tukey post hoc tests; * P < 0.05, **P < 0.01, NTG group compared with the VEH group, n = 8/group; #P < 0.05, ## P < 0.01, NTG group compared with the NTG+OLC group, n = 8/group. Abbreviations: VEH, vehicle; NTG, nitroglycerin; OLC, olcegepant

Fig. 2

c-Fos activation map of cortical structures in the NTG-induced chronic migraine model. A. Representative pictures of c-Fos (red) and NeuN (green) immunofluorescence labeling in the mPFC. Scale bars =100 μm. B-C. mPFC showed a significant increase of c-Fos⁺ cells density (B) and the percentage of c-Fos + cells in NeuN ⁺ cells (C), alleviated by OLC, n = 5/group. D. Representative pictures of c-Fos (red) and NeuN (green) immunofluorescence labeling in the ACC. Scale bars =100 μm. E–F. ACC showed a significant increase of c-Fos⁺ cells density (E) and the percentage of c-Fos⁺ cells in NeuN cells (F), alleviated by OLC, n = 5/group. One-way ANOVA with the Tukey post hoc tests, * P < 0.05, **P < 0.01. Abbreviations: mPFC, medial prefrontal cortex; ACC, anterior cingulate cortex; OLC, olcegepant; NTG, nitroglycerin

Fig. 3

c-Fos activation map of thalamic structure in the NTG-induced chronic migraine model. A. Representative pictures of c-Fos (red) and NeuN (green) immunofluorescence labeling in the PVT. Scale bars =50 μm. B-C. In PVT, no significant differences in c-Fos⁺ cell density (B) and the percentage of c-Fos⁺ cells in NeuN⁺ cells (C) were found. n = 5/group. One-way ANOVA with the Tukey post hoc tests, * P < 0.05, **P < 0.01. Abbreviations: PVT, paraventricular thalamic nucleus. NTG, nitroglycerin

Fig. 4

c-Fos activation map of brain stem structures in the NTG-induced chronic migraine model. A. Representative pictures of c-Fos (red) and NeuN (green) immunofluorescence labeling in the vlPAG. Scale bars =100 μm. B-C. In vlPAG, no significant differences in c-Fos⁺ cell density (B) and the percentage of c-Fos⁺ cells in NeuN cells (C) were found, alleviated by OLC, n = 5/group. D. Representative pictures of c-Fos (red) and NeuN (green) immunofluorescence labeling in the Sp5c. Scale bars =50 μm. E–F. Sp5c showed a significant increase of c-Fos⁺ cell density (E) and the percentage of c-Fos⁺ cells in NeuN⁺ cells (F). alleviated by OLC, n = 5/group. One-way ANOVA with the Tukey post hoc tests, * P < 0.05, **P < 0.01. Abbreviations: vlPAG, ventrolateral periaqueductal gray; OLC, olcegepant; Sp5c, caudal part of the spinal trigeminal nucleus

Fig. 5

CGRP receptor antagonist (olcegepant) alleviated acute hyperalgesia in the LEV-induced chronic migraine model. A. Representative schematic diagrams and procedures for the behavioral tests (Created with BioRender.com). B-C. Repeated LEV administration induced mechanical hyperalgesia of periorbital area (B) and hindpaw (C), alleviated by OLC. Two-way ANOVA with the Tukey post hoc tests, *P < 0.05, **P < 0.01, LEV group compared with the VEH group, n = 8/group; #P < 0.05, # # P < 0.01, LEV group compared with the LEV+OLC group, n = 8/group. Abbreviations: VEH, vehicle; OLC, olcegepant; LEV, levcromakalim

Fig. 6

c-Fos activation map of cortical structures in the LEV-induced chronic migraine model. A. Representative pictures of c-Fos (red) and NeuN (green) immunofluorescence labeling in the mPFC. Scale bars =100 μm. B-C. mPFC showed a significant increase of c-Fos⁺ cell density (B) and the percentage of c-Fos⁺ cells in NeuN⁺ cells (C). alleviated by olcegepant. n = 5/group. D. Representative pictures of c-Fos (red) and NeuN (green) immunofluorescence labeling in the ACC. Scale bars =100 μm. E–F. In the ACC, no significant differences in c-Fos⁺ cell density (E) and the percentage of c-Fos⁺ cells in NeuN cells (F) were found. n = 5/group. One-way ANOVA with the Tukey post hoc tests, * P < 0.05, **P < 0.01. Abbreviations: mPFC, medial prefrontal cortex; ACC, anterior cingulate cortex; OLC, olcegepant; LEV, levcromakalim

Fig. 7

c-Fos activation map of thalamic structure in the LEV-induced chronic migraine model. A. Representative pictures of c-Fos (red) and NeuN (green) immunofluorescence labeling in the PVT. Scale bars =50 μm. B-C. PVT showed a significant increase of c-Fos⁺ cell density (B) and the percentage of c-Fos⁺ cells in NeuN⁺ cells (C), alleviated by OLC. n = 5/group. One-way ANOVA with the Tukey post hoc tests, * P < 0.05, **P < 0.01. Abbreviations: PVT, paraventricular thalamic nucleus; OLC, olcegepant. LEV, levcromakalim

Fig. 8

c-Fos activation map of brain stem structures in the LEV-induced chronic migraine model. A. Representative pictures of c-Fos (red) and NeuN (green) immunofluorescence labeling in the vlPAG. Scale bars =100 μm. B-C. In vlPAG, no significant differences in c-Fos⁺ cell density (B) and the percentage of c-Fos + cells in NeuN⁺ cells (C) were found. n = 5/group. D. Representative pictures of c-Fos (red) and NeuN (green) immunofluorescence labeling in the Sp5c. Scale bars =50 μm. E–F. Sp5c showed a significant increase of c-Fos⁺ cell density (E) and the percentage of c-Fos⁺ cells in NeuN⁺ cells (F), alleviated by OLC. n = 5/group. One-way ANOVA with the Tukey post hoc tests, * P < 0.05, **P < 0.01. Abbreviations: vlPAG, ventrolateral periaqueductal gray; OLC, olcegepant; Sp5c, caudal part of the spinal trigeminal nucleus; LEV, levcromakalim

Fig.9

Brain activation in chronic migraine models. A-B. The relative c-Fos⁺ neuron density in several brain regions (A, red) and the relative c-Fos⁺ neuron density with OLC treatment (B, green) in the NTG-induced chronic migraine model or LEV-induced chronic migraine model. C. The brain activation and alteration of brain activation with OLC treatment in the NTG-induced chronic migraine model (dark red) and LEV-induced chronic migraine model (pink); No activation (gray); CGRP receptor (green); (Created with BioRender.com). Abbreviations: VEH, vehicle; NTG, nitroglycerin; LEV, levcromakalim; OLC, olcegepant; mPFC, medial prefrontal cortex; ACC, anterior cingulate cortex; PVT, paraventricular thalamic nucleus; vlPAG, ventrolateral periaqueductal gray; Sp5c, caudal part of the spinal trigeminal nucleus