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. 2024 May 10;25(1):74.
doi: 10.1186/s10194-024-01779-2.

Environmental enrichment alleviates hyperalgesia by modulating central sensitization in a nitroglycerin-induced chronic migraine model of mice

Lei Wang  1 , Xiaoming Liu  2 , Chenlu Zhu  1 , Shouyi Wu  1 , Zhilei Li  1 , Lipeng Jing  2 , Zhenchang Zhang  1 , Yuhong Jing  3 , Yonggang Wang  4   5
Affiliations

Environmental enrichment alleviates hyperalgesia by modulating central sensitization in a nitroglycerin-induced chronic migraine model of mice

Lei Wang et al. J Headache Pain. .

Abstract

Background: Chronic migraine (CM) is a debilitating neurofunctional disorder primarily affecting females, characterized by central sensitization. Central sensitization refers to the enhanced response to sensory stimulation, which involves changes in neuronal excitability, synaptic plasticity, and neurotransmitter release. Environmental enrichment (EE) can increase the movement, exploration, socialization and other behaviors of mice. EE has shown promising effects in various neurological disorders, but its impact on CM and the underlying mechanism remains poorly understood. Therefore, the purpose of this study was to determine whether EE has the potential to serve as a cost-effective intervention strategy for CM.

Methods: A mouse CM model was successfully established by repeated administration of nitroglycerin (NTG). We selected adult female mice around 8 weeks old, exposed them to EE for 2 months, and then induced the CM model. Nociceptive threshold tests were measured using Von Frey filaments and a hot plate. The expression of c-Fos, calcitonin gene-related peptide (CGRP) and inflammatory response were measured using WB and immunofluorescence to evaluate central sensitization. RNA sequencing was used to find differentially expressed genes and signaling pathways. Finally, the expression of the target differential gene was investigated.

Results: Repeated administration of NTG can induce hyperalgesia in female mice and increase the expression of c-Fos and CGRP in the trigeminal nucleus caudalis (TNC). Early exposure of mice to EE reduced NTG-induced hyperalgesia in CM mice. WB and immunofluorescence revealed that EE inhibited the overexpression of c-Fos and CGRP in the TNC of CM mice and alleviated the inflammatory response of microglia activation. RNA sequencing analysis identified that several central sensitization-related signaling pathways were altered by EE. VGluT1, a key gene involved in behavior, internal stimulus response, and ion channel activity, was found to be downregulated in mice exposed to EE.

Conclusion: EE can significantly ameliorate hyperalgesia in the NTG-induced CM model. The mechanisms may be to modulate central sensitization by reducing the expression of CGRP, attenuating the inflammatory response, and downregulating the expression of VGluT1, etc., suggesting that EE can serve as an effective preventive strategy for CM.

Keywords: Central sensitization; Chronic migraine; Environmental enrichment; TNC; VGluT1.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Repeated NTG administration causes hyperalgesia in female mice. A. The schematic diagram illustrates the establishment of the CM mouse model and behavioral assessments through repeated NTG administration. B. In the NTG mice, the mechanical thresholds in the hind paw were significantly decreased compared to the VEH mice both before and after NTG administration. C. The thermal thresholds in the hind paw of NTG mice were significantly decreased compared to the VEH mice both before and after NTG administration. D. On the 9th day, after NTG administration, the mechanical threshold in the periorbital region of NTG mice showed a significant decrease compared to the VEH mice. Two-way ANOVA with the Tukey post hoc tests, * p < 0.05, **p < 0.01, n = 10 /group, Abbreviations: NTG, nitroglycerin; CM, chronic migraine; VEH, vehicle
Fig. 2
Fig. 2
Repeated NTG administration resulted in increased expression of c-Fos and CGRP in the TNC. A, B. Representative fluorescent images showed significantly increased number of c-Fos positive cells (A) and CGRP immunoreactive fibers density (B) in the TNC of the NTG mice compared to the VEH mice. C, D. Quantification of c-Fos and CGRP expression. E-G. WB analysis showed that the protein expression of c-Fos (F) and CGRP (G) in the NTG mice was significantly higher than that in the VEH mice. The irregular white boxes show the TNC tissue. The square white boxes show higher-magnification views. Two-tailed Student’s t-test, * p < 0.05, **p < 0.01, ***p < 0.001, n = 5/group, Abbreviations: NTG, nitroglycerin; VEH, vehicle; TNC, trigeminal nucleus caudalis
Fig. 3
Fig. 3
EE ameliorated hyperalgesia induced by NTG administration. A. The schematic diagram illustrates the establishment of the EE and CM mouse model through NTG administration. B. In the EE + NTG mice, the mechanical thresholds in the hind paw were significantly increased compared to the SD + NTG mice both before and after NTG administration. C. In the EE + NTG mice, the thermal thresholds in the hind paw were significantly increased compared to the SD + NTG mice both before and after NTG administration. D. On the 9th day, after NTG administration, the mechanical thresholds in the periorbital region in the EE + NTG mice showed a significant increase compared to the SD + NTG mice. Two-way ANOVA with the Tukey post hoc tests, * p < 0.05, **p < 0.01, ***p < 0.001, n = 15/group, Abbreviations: NTG, nitroglycerin; EE, environmental enrichment; CM, chronic migraine; SD, standard environment
Fig. 4
Fig. 4
EE decreased the expression of c-Fos and CGRP in the TNC. A, C. Representative fluorescent images showed a significantly decreased number of c-Fos positive cells (A) and CGRP immunoreactive fibers density (C) in the TNC of the EE + NTG mice compared to the SD + NTG mice. B, D. Quantification of c-Fos and CGRP expression. E-G. WB analysis and quantification showed that the protein expression of c-Fos (F) and CGRP (G) in the EE + NTG mice was significantly lower than that in the SD + NTG mice. The irregular white boxes show the TNC tissue. The square white boxes show higher-magnification views. Two-tailed Student’s t-test, * p < 0.05, **p < 0.01, n = 4/group, Abbreviations: TNC, trigeminal nucleus caudalis; NTG, nitroglycerin; EE, environmental enrichment; SD, standard environment
Fig. 5
Fig. 5
EE decreased the hyperactivation of microglia but not astrocytes in the TNC. A, B. Representative fluorescent images (A) and quantification (B) showed that the number of activated microglia was significantly reduced in the TNC of the EE + NTG mice compared to SD + NTG mice. C, D. Representative fluorescent images (C) and quantification (D) showed that the change in activated astrocytes was not obvious in the TNC of the EE + NTG mice compared to SD + NTG mice. E. qRT-PCR analysis showed that the pro-inflammatory factor IL-1β was significantly reduced, and the anti-inflammatory factor IL10 was significantly increased in the EE + NTG mice compared to SD + NTG mice. The irregular white boxes show the TNC structure. The square white boxes show higher-magnification views. Two-tailed Student’s t-test, * p < 0.05, **p < 0.01, n = 4/group. Abbreviations: TNC, trigeminal nucleus caudalis; NTG, nitroglycerin; EE, environmental enrichment; SD, standard environment; Iba1: ionized calcium-binding adapter molecule 1; GFAP: glial fibrillary acidic protein
Fig. 6
Fig. 6
EE regulated central sensitization-related signaling pathways in the TNC. A. Volcano plot of DEGs distribution: including 209 upregulated genes and 118 downregulated genes. Scattered points on the plot represent each gene, gray dots represent genes with no significant difference, red dots represent upregulated genes, and blue dots represent downregulated genes. B. DEGs clustering heatmap: each column represents a sample and each row represents a gene. Red represents relatively highly expressed genes, and blue represents relatively lowly expressed genes. C. GO enrichment histogram of DEGs. The three major categories of GO are represented by columns of different colors. Green represents biological processes (BP), blue represents molecular functions (MF), and red represents cellular components (CC). D. KEGG enrichment histogram of DEGs. The size of the rich factor is represented by the color of the column. E. A Venn diagram was generated at the intersection of enriched genes between behavior, ligand-gated ion channel activity, and response to endogenous stimulus. Slc17a7 was a common gene in these crossover sets. n = 3/group. Abbreviations: EE, environmental enrichment; TNC, trigeminal nucleus caudalis; DEGs, differential expression genes; GO, gene ontology; KEGG, Kyoto encyclopedia of genes and genomes; Slc17a7, solute carrier family 17 member 7
Fig. 7
Fig. 7
EE decreased expression of VGluT1 (Slc17a7 gene) in the TNC. A. Clustering heatmap of partially significantly differential genes: the red marks the Slc17a7 gene. B, C. WB analysis (B) and quantification (C) showed that the protein expression of VGluT1 in the TNC of EE + NTG mice was significantly lower than that in the SD + NTG mice. D, E. Representative fluorescent images (D) and quantification (E) showed significantly decreased expression of VGluT1 in the TNC of the EE + NTG mice compared to the SD + NTG mice. The irregular white boxes show the TNC tissue. The square white boxes show higher-magnification views. Two-tailed Student’s t-test, **p < 0.01, n = 4/group, Abbreviations: EE, environmental enrichment; VGluT1, vesicular glutamate transporter 1; Slc17a7, solute carrier family 17 member 7; TNC, trigeminal nucleus caudalis; NTG, nitroglycerin; SD, standard environment
Fig. 8
Fig. 8
The schematic diagram illustrates EE alleviates hyperalgesia by modulating central sensitization in the NTG-induced CM mouse model. A. Mice were reared in SD or EE for 2 months, followed by the establishment of an NTG-induced CM model. B. Mice were examined for mechanical and thermal hyperalgesia in the hind paw and mechanical hyperalgesia in the periorbital region. C. TNC tissues were collected for molecular biological analyses. D. EE reduces activation of microglia and its mediated inflammatory response, as well as the expression of VGLuT1 and CGRP, which are expressed in different neurons of the TNC. VGLuT1 primarily affects the release of glutamate, which acts on excitatory receptors NMDAR or AMPAR to induce central sensitization of neurons. Meanwhile, CGRP regulates cAMP signaling by activating its receptor CGRPR, thereby contributing to central sensitization. Abbreviations: EE, environmental enrichment; NTG, nitroglycerin; VEH, Vehicle; CM, chronic migraine; SD, standard environment; TNC, trigeminal nucleus caudalis; VGluT1, vesicular glutamate transporter 1; CGRP, Calcitonin gene-related peptide; NMDAR, N-methyl-D-aspartate receptor; AMPAR, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor
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