Different Gut-Brain System Problems in Episodic and Chronic Migraine Seen in Mouse Models

Migraine is a prevalent neurological disorder that causes substantial episodic disability and reduced productivity [1,2,3,4]. It is marked by recurrent episodes of moderate to severe headaches, frequently accompanied by classical symptoms including nausea and vomiting. It is also often complicated by gastrointestinal (GI) conditions [5,6,7]. GI symptoms have been included in the migraine diagnostic criteria based on the International Classification Headache Disorders, and the American Headache Society recommends non-oral therapies for patients with GI symptoms [8]. Previous studies showed that migraine is associated with GI disorders as comorbidities, as well as overlapping symptomatology, including Helicobacter pylori infection, gastroesophageal reflux disorder, irritable bowel syndrome, gastroparesis, hepatobiliary disorders, and celiac disease [9,10]. Although the pathophysiology of migraine is not fully understood, this association suggests a relation with the GI tract, suggesting a pathophysiology involving the gut–brain axis. While rates of migraine headache comorbidities generally increase with increases in migraine headache frequency, the rate of stomach/intestinal ulcers is approximately double in cases of chronic migraine (CM) compared to episodic migraine (EM) [11,12].

Among the various migraine experimental models available, the nitric oxide (NO)-induced migraine model, particularly nitroglycerin (NTG)-induced migraine, has been widely utilized to mimic the clinical features of both EM and CM [13,14]. NTG, an NO donor, induces migraines by triggering vasodilation and activating inflammatory pathways, leading to prolonged sensitization of pain-processing structures [15,16].

Clinical and experimental data suggest that migraines are frequently associated with GI disturbances, including altered gut motility, inflammation, and changes in the composition of the gut microbiota [17]. However, the underlying mechanisms linking migraines to GI dysfunction or disorders, particularly the differential pathways distinguishing EM and CM, remain unclear [18,19]. While previous studies have indicated acute inflammatory gut alterations following NTG administration, a comprehensive and integrated analysis of the full spectrum of morphological, neuropeptidergic, and cellular immunological changes in both EM and CM models is lacking [20,21]. Given that inflammation plays a crucial role in the pathophysiology of migraine, investigating the stage-dependent inflammatory and cellular responses in the GI tract may provide new insights into migraine-associated systemic effects [22,23].

In this study, NTG-induced EM and CM mouse models were established to explore the impact of migraine on the gastrointestinal tract through an integrated, multi-parametric analysis of cytokine expression, calcitonin gene-related peptide (CGRP) levels, and immune cell redistribution, thereby defining the distinct gut–brain axis signatures associated with EM and CM. By identifying molecular and cellular changes in the GI tract under migraine conditions, this study enhanced our understanding of the role of the gut–brain connection in the pathophysiology of migraine.

In this study, we investigated gut-related morphological, molecular, and immunological alterations in NTG-induced EM and CM mouse models, with a particular focus on the colon as a representative site of gut–brain axis involvement. CM mice showed greater GI alterations than EM mice, with the most prominent changes being observed in the colon of the CM model, including morphological shortening, altered cytokine profiles, increased CGRP expression, and immune cell redistribution. These findings support the suggestion that CM is associated with a profound disruption of gut–brain axis homeostasis (Figure 6).

Previous reports suggest a close association between migraine and GI disorders, with individuals with migraine showing a higher likelihood of developing conditions such as constipation, diarrhea, dyspepsia, irritable bowel syndrome, and gastroesophageal reflux disease [24,25,26]. Therefore, it has been postulated that migraine may affect the gut–brain axis, but the precise mechanism underlying this relation remains unclear. A variety of factors have been suggested to influence the gut–brain interaction, including inflammatory mediators (IL-1β, IL-6, IL-8, TNF-α), the gut microbiota profile, neuropeptide and serotonin pathways, stress hormones, and dietary components [27]. Previous studies showed increased immune cell infiltration and inflammation in the colonic tissue 4 h after a single injection of NTG [20,21]. NTG-induced migraine models are widely used to study both EM and CM due to their ability to replicate clinical features [28,29,30]. In this study, to examine changes in the GI tract induced by migraine, we established EM and CM mouse models by administering NTG according to different schedules. Phenotypic differences in the GI tract that were dependent on the duration of migraine induction by NTG were detected. A morphological comparison of the stomach, small intestine, and colon tissues revealed specific shortening of the colon in the CM model. In addition, indirect analysis to assess the impact of the migraine model on GI motility revealed significant decreases in food intake and body weight in the NTG-induced model compared to controls [31,32,33,34]. The phenotypic changes observed in our study, such as the shortening of the intestinal length and decreased GI motility, were consistent with clinical observations. Mechanistically, the NO donor NTG initiates central sensitization and inflammation in the trigeminovascular system, but its peripheral effects on the gut are significant. NO, which is released directly in the brain in response to excitation and activation of trigeminal neurons, can affect the GI tract through an indirect mechanism when it crosses the blood–brain barrier (BBB) and reaches the gut. Furthermore, this central NO signaling triggers the systemic release of inflammatory mediators and neuropeptides, which then modulate the enteric nervous system (ENS) and mucosal immunity, influencing GI hormones, motility, and appetite regulation [35].

Previous reports, demonstrated acute NTG-induced mast cell inflammation and increases in proinflammatory cytokines like TNF-α and IL-1β in the colon, providing critical groundwork for GI involvement in migraine [21,28,36]. Our study significantly advances these findings by providing the first comprehensive comparison of EM and CM gut–brain axis signatures, using a uniquely integrated, multi-parametric approach. To perform a more complete and stage-dependent analysis of GI tissues than previous studies, we examined morphological shortening, CGRP expression, and detailed immune cell subsets via FACS, in addition to region-specific qRT-PCR. From a molecular perspective, differential patterns of inflammatory cytokine expression were observed throughout the GI tract, with the EM model showing predominantly proinflammatory responses (e.g., IL-6, IL-1β, and IL-8), while the CM model was characterized by increased expression of anti-inflammatory mediators (IL-4, IL-10, and TGF-β), particularly in the colon. We acknowledge that classifying IL-10 and TGF-β as purely "anti-inflammatory" may oversimplify their complex, context-dependent roles; TGF-β, for example, is critical for tissue remodeling and can have pro-fibrotic effects, while IL-10's function varies based on cellular context. However, the increase in these cytokines alongside the elevation of Treg cells and macrophages in the CM distal colon strongly suggests the activation of immune regulatory circuits or an adaptive immunomodulatory response following prolonged or repeated neurogenic inflammation, which is consistent with known mechanisms of tolerance or exhaustion pathways often mediated by IL-10 and TGF-β under chronic stimulation. This CM-specific gut immune remodeling may result from a potential causal pathway: repeated NTG exposure leads to sustained peripheral CGRP release, which, combined with systemic inflammation, continuously challenges the enteric immune system. This chronic challenge then drives the ENS and mucosal immune cells to switch from an acute proinflammatory state to a state of sustained immune tolerance and tissue preservation, characterized by the enrichment of regulatory cells. This finding significantly extends beyond the acute inflammatory changes previously reported [20,21].

CGRP is widely expressed in various organ systems, including the nervous, cardiovascular, digestive, respiratory, genitourinary, and musculoskeletal systems, where it is primarily involved in vasodilation and nociceptive modulation [37,38,39]. In particular, CGRP plays a pivotal role in the nervous, cardiovascular, and digestive systems, and is critically involved in the regulation of inflammatory processes [40,41,42]. Elevated CGRP expression in the GI tract, particularly in the colon of CM mice, further highlights the involvement of this neuropeptide not only in central trigeminovascular pathways but also in the peripheral enteric system. CGRP is known to influence gut motility, mucosal immunity, and vascular tone [43], and its persistent elevation in CM may contribute to altered GI function and immune signaling, thereby reinforcing gut–brain axis dysregulation Crucially, this robust peripheral CGRP signal provides strong experimental support for the observed clinical link between CM severity and GI comorbidities, as clinical evidence indicates that CM patients often report more severe and frequent gastrointestinal symptoms compared to EM patients. Furthermore, human studies have demonstrated associations between altered gut microbiota composition and headache frequency, as well as distinct immune activation patterns in people with migraines, providing clinical context for the immune and neuropeptide shifts observed in our model [8,27].

To validate these findings, the distribution of immune cells was examined via FACS analysis of colonic tissues, which showed significant differences in immune cell responses between the EM and CM models. FACS analysis revealed divergent immune cell distributions in the colon. In the EM model, increased Th17 and B cell populations suggested an active mucosal immune response. In contrast, the CM model showed increased T cells, Treg cells, and macrophages, particularly in the distal colon, an area increasingly recognized as a site of intense neuroimmune interaction. These differences in immune landscape between EM and CM may reflect stage-dependent immunomodulatory responses to repeated NTG stimulation, and may be relevant to the clinical transition from EM to CM [13].

The findings of the present study lend experimental support to this bidirectional model, in which persistent central sensitization and neurogenic inflammation modulate gut immunity and motility, while gut-derived inflammatory or neuropeptidergic signals may, in turn, affect trigeminal system excitability. From the perspective of translational research, these results highlight the potential of targeting gut-derived pathways in the management of migraine. Microbiome-based therapies, dietary modulation, probiotics, and mucosal immunotherapy are emerging areas of interest, particularly given recent evidence linking altered gut microbiota composition to headache frequency and severity [27,44]. The significant peripheral CGRP upregulation in CM strongly supports the efficacy of systemic CGRP-targeting therapeutics (e.g., monoclonal antibodies or gepants) that are known to act on peripheral sites [45]. Furthermore, the CM-specific shift toward an adaptive immune state observed in our model raises an intriguing translational question: Could this unique chronic gut immune environment locally influence the efficacy or pharmacodynamics of systemic CGRP-targeting drugs? The local cytokine milieu might alter CGRP receptor expression or downstream signaling in the enteric nervous system, thereby modulating the therapeutic outcome. Given the importance of the gut–brain connection in migraine pathogenesis, the observed immune cell changes and our findings also advocate for gut-targeted interventions, such as dietary modulation or microbiota-based therapies [8], as crucial adjunct strategies to complement pharmacological treatments.

Nonetheless, this study had several limitations. First, while the NTG model recapitulates certain aspects of migraine pathophysiology, it may not fully represent the heterogeneity of human migraine, particularly with respect to underlying genetic or environmental factors. We also acknowledge that the absence of parallel behavioral or pain endpoints (such as grimace scores or assessments of photophobia/phonophobia) is a limitation. This lack of concurrent data weakens the ability to directly correlate the magnitude of the observed GI morphological, molecular, and immune changes with the severity of the migraine-like phenotype, thus reducing direct translational relevance. Further studies integrating comprehensive GI analysis with detailed pain and behavioral assessments are required to clarify these direct correlations. Second, the observed cytokine and immune cell changes were limited to bulk tissue analysis and flow cytometry. Future studies using single-cell transcriptomics or spatial profiling could offer higher-resolution insights into cell type-specific alterations. Third, gut microbiota composition was not evaluated in this study, despite its known influence on immune tone and neuropeptide expression. We acknowledge that the omission of gut microbiota profiling (16S rRNA or metagenomics) is a major limitation of this study, especially since disruption of the gut–brain axis is a central claim. While we focused on host immunity and neuropeptide signaling, the pronounced alterations we observed—including the differential activation of Th17/B cells in EM versus Treg/macrophages in CM, alongside changes in anti-inflammatory cytokines—strongly suggest an underlying dysbiosis. Given the known link between the microbiome, immune tone, and neuropeptide expression (such as CGRP), integrating microbiome profiling with our host immune and neuronal signaling analyses in future work is essential to fully contextualize these findings and is a crucial next step. Finally, a total of fifteen mice were used per group. However, to accommodate methodological requirements and ensure assay-specific precision, animals were subdivided into subsets of five for qRT-PCR, FACS, and immunofluorescence analyses, respectively. This design enabled the collection of high-quality tissue and cell samples from defined intestinal regions optimized for each technique, thereby minimizing technical variability across assays. Although the effective sample size per assay was limited, preliminary experiments confirmed consistent and reproducible results under standardized conditions. We recognize that the relatively small sample size may reduce statistical power and increase the likelihood of type II errors; therefore, future studies with larger cohorts are planned to validate and extend these findings.

Overall, our findings provide experimental evidence that CM is associated with more extensive gut–brain axis disruption than EM, characterized by GI inflammation, CGRP expression, immune cell alterations, and morphological changes in the colon. These results highlight the gut as a potential therapeutic target in migraine, especially CM, and encourage further translational research to explore microbiota-based therapies, mucosal immune modulators, and peripheral CGRP-targeting interventions as disease-modifying strategies.

In conclusion, this study provided preclinical evidence that migraine, especially CM, is associated with pronounced gut–brain axis dysregulation, particularly affecting the colon through GI inflammation, CGRP expression, immune cell alterations, and morphological changes. These results underscore the value of integrating GI and neurological perspectives in migraine research and support the development of gut-targeted interventions as potential disease-modifying strategies in migraine management.