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. 2024 Mar 26;25(7):3706.
doi: 10.3390/ijms25073706.

Differences in Neuropathology between Nitroglycerin-Induced Mouse Models of Episodic and Chronic Migraine

Songyi Park  1 , Harry Jung  1 , Sang-Won Han  1   2 , Sang-Hwa Lee  1   2 , Jong-Hee Sohn  1   2
Affiliations

Differences in Neuropathology between Nitroglycerin-Induced Mouse Models of Episodic and Chronic Migraine

Songyi Park et al. Int J Mol Sci. .

Abstract

Multiple animal models of migraine have been used to develop new therapies. Understanding the transition from episodic (EM) to chronic migraine (CM) is crucial. We established models mimicking EM and CM pain and assessed neuropathological differences. EM and CM models were induced with single NTG or multiple injections over 9 days. Mechanical hypersensitivity was assessed. Immunofluorescence utilized c-Fos, NeuN, and Iba1. Proinflammatory and anti-inflammatory markers were analyzed. Neuropeptides (CGRP, VIP, PACAP, and substance P) were assessed. Mechanical thresholds were similar. Notable neuropathological distinctions were observed in Sp5C and ACC. ACC showed increased c-Fos and NeuN expression in CM (p < 0.001) and unchanged in EM. Sp5C had higher c-Fos and NeuN expression in EM (p < 0.001). Iba1 was upregulated in Sp5C of EM and ACC of CM (p < 0.001). Proinflammatory markers were strongly expressed in Sp5C of EM and ACC of CM. CGRP expression was elevated in both regions and was higher in CM. VIP exhibited higher levels in the Sp5C of EM and ACC of CM, whereas PACAP and substance P were expressed in the Sp5C in both models. Despite similar thresholds, distinctive neuropathological differences in Sp5C and ACC between EM and CM models suggest a role in the EM to CM transformation.

Keywords: anterior cingulate cortex; calcitonin gene-related peptide; chronic migraine; episodic migraine; nitroglycerin; pituitary adenylate cyclase-activating peptide; substance P; trigeminal spinal subnucleus caudalis; vasoactive intestinal peptide.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 3
Figure 3
Iba1 expression patterns in mouse models of episodic and chronic NTG-induced migraine (EM and CM, respectively). (A) Representative images of Iba1 (green) immunofluorescence staining in the Sp5C of EM and CM mouse models. Scale bars = 20 μm. (B). Numbers of Iba1+ cells/mm2 in the Sp5C of EM and CM mouse models. (C) Representative images of Iba1 (green) immunofluorescence staining in the ACC of EM and CM mouse models. Scale bars = 20 μm. In the merged results, DAPI was used along with the Iba1 marker (A,C). (D) Numbers of Iba1+ cells/mm2 in the ACC of EM and CM mouse models. One-way ANOVA with post hoc Tukey test: ** p < 0.01; **** p < 0.0001; ns, not significant. Abbreviations: ACC, anterior cingulate cortex; NTG, nitroglycerin; Sp5C, spinal trigeminal nucleus caudalis; and VEH, vehicle control.
Figure 1
Figure 1
(A) Experimental protocols to establish mouse models of episodic and chronic NTG-induced migraine (EM and CM, respectively). (B–D) Experimental measurement of mechanical hypersensitivity using the von Frey filament test. (C) Non-parametric analysis (Mann–Whitney test) and (D) Parametric analysis (Unpaired t-test): *** p < 0.001. Abbreviations: NTG, nitroglycerin; VEH, vehicle control.
Figure 2
Figure 2
c-Fos and NeuN expression patterns in mouse models of episodic and chronic NTG-induced migraine (EM and CM, respectively). (A) Representative images of c-Fos (red) and NeuN (green) immunofluorescence staining in the Sp5C of EM and CM models. Scale bars = 10 μm. (B) Numbers of c-Fos+ cells/mm2 in the Sp5C of EM and CM mouse models. (C) Numbers of c-Fos+NeuN+ cells/mm2 in the Sp5C of EM and CM mouse models. (D) Representative images of c-Fos (red) and NeuN (green) immunofluorescence staining in the ACC of EM and CM mouse models. Scale bars = 10 μm. (E) Numbers of c-Fos+ cells/mm2 in the ACC of EM and CM mouse models. (F) Numbers of c-Fos+NeuN+ cells/mm2 in the ACC of EM and CM mouse models. One-way ANOVA with post hoc Tukey test: * p < 0.05; ** p < 0.01; **** p < 0.0001; ns, not significant. Abbreviations: ACC, anterior cingulate cortex; NTG, nitroglycerin; Sp5C, spinal trigeminal nucleus caudalis; and VEH, vehicle control.
Figure 4
Figure 4
Inflammatory marker expression patterns in mouse models of episodic and chronic NTG-induced migraine (EM and CM, respectively). (A) Cytokine expression in the Sp5C of EM and CM mouse models, as determined using qRT-PCR. (B) Cytokine expression in the ACC of EM and CM mouse models, as determined using qRT-PCR. (C). TNF-α, Iba1, NF-κB, and β-actin (control) protein bands and relative expression, as determined using immunoblotting. Statistical analysis was conducted using the Kruskal–Wallis test with Dunn’s multiple comparisons test and one-way ANOVA with the post hoc Tukey test. * p < 0.05; ** p < 0.01; **** p < 0.0001; ns, not significant. Abbreviations: ACC, anterior cingulate cortex; NTG, nitroglycerin; Sp5C, spinal trigeminal nucleus caudalis; VEH, vehicle control.
Figure 5
Figure 5
Neuropeptide expression patterns in the Sp5C in mouse models of episodic and chronic NTG-induced migraine (EM and CM, respectively). (A) Representative images of VIP, CGRP, PACAP, and substance P (green) immunofluorescence staining in the Sp5C of EM and CM mouse models. In the merged results, DAPI was added along with the VIP, CGRP, PACAP, and substance P markers. Scale bars = 20 μm. (B) Fluorescence intensities of VIP, CGRP, PACAP, and substance P in the Sp5C of EM and CM mouse models. One-way ANOVA with post hoc Tukey test: * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. ns, not significant.
Figure 6
Figure 6
Neuropeptide expression patterns in the ACC in mouse models of episodic and chronic NTG-induced migraine (EM and CM, respectively). (A) Representative images of VIP, CGRP, PACAP, and substance P (green) immunofluorescence staining in the ACC of EM and CM mouse models. In the merged results, DAPI was added along with the VIP, CGRP, PACAP, and substance P markers. Scale bars = 20 μm. (B) Fluorescence intensities of VIP, CGRP, PACAP, and substance P in the ACC of EM and CM mouse models. Statistical analysis was conducted using the Kruskal–Wallis test with Dunn’s multiple comparisons test and one-way ANOVA with post hoc Tukey test: * p < 0.05; ** p < 0.01; **** p < 0.0001; ns, not significant. Abbreviations: ACC, anterior cingulate cortex; CGRP, calcitonin gene-related peptide; NTG, nitroglycerin; PACAP, pituitary adenylate cyclase-activating peptide; VEH, vehicle control; VIP, vasoactive intestinal peptide.
Figure 7
Figure 7
Summary of differences in neuropathology in the Sp5C and ACC between mouse models of episodic and chronic NTG-induced migraine (EM and CM, respectively). (-); ns, not significant, White triangle; slightly increase, Red triangle; significantly increase.
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