Distinct Gut–Brain Axis Dysregulation in Episodic Versus Chronic Migraine: Insights from NTG-Induced Mouse Models

The gut-brain axis regulates brain functions and maintains central nervous system homeostasis and intestinal balance. Migraine patients often present with gastrointestinal (GI) comorbidities, with stronger associations observed in chronic migraine (CM) than in episodic migraine (EM). To investigate migraine-related GI alterations, nitroglycerin (NTG)-induced mouse models of EM (N = 15) and CM (N = 15) were established using single or repeated NTG injections (10 mg/kg). EM mice were euthanized 4 h after a single injection, whereas CM mice received NTG every other day for 9 days and were euthanized after the fifth injection. On the day of sacrifice, GI tissues were analyzed for morphological changes, cytokine expression, calcitonin gene-related peptide (CGRP) levels, and immune cell profiles. NTG-treated groups exhibited significant reductions in both food intake and body weight compared with controls. In addition, colon length was markedly shortened in the chronic migraine (CM) model (<i>p</i> < 0.01). Molecular analyses revealed distinct cytokine expression profiles between models: the episodic migraine (EM) model showed increased levels of proinflammatory cytokines (IL-1β, IL-6, and IL-8; <i>p</i> < 0.01), whereas the CM model displayed elevated anti-inflammatory cytokines (IL-4, IL-10, and TGF-β; <i>p</i> < 0.01), particularly in the colon. CGRP expression was also markedly upregulated throughout the gastrointestinal tract, with the highest expression observed in the colon of CM mice (<i>p</i> < 0.01). Flow cytometric immune profiling further demonstrated divergent immune cell patterns, with increased Th17 (<i>p</i> = 0.0085) and B cell (<i>p</i> = 0.0199) populations in EM, while CM was characterized by enrichment of T cells (<i>p</i> = 0.0221), regulatory T cells (Tregs) (<i>p</i> = 0.0114), and macrophages (<i>p</i> = 0.0062), indicating more pronounced immune alterations in the distal colon. These findings indicate that CM involves more severe gut-brain axis dysregulation than EM, supporting the potential of gut-targeted therapies as adjunct strategies in chronic migraine.