Sex differences in behaviors, neuronal activation, and gut-microbiota-metabolic axis in a repeated nitroglycerin-induced chronic migraine model

Migraine is a debilitating primary headache disorder characterized by significant sex differences in epidemiology, clinical features, comorbidities, and treatment response, yet the underlying mechanisms remain obscure. This study aims to dissect the sex difference map in chronic migraine models and explore the underlying mechanisms driving these specific phenotypes. A mouse model of chronic migraine was established by intraperitoneal injection of nitroglycerin (NTG, 10 mg/kg, 5 times in 9 days). Both male and female mice were included. Mechanical withdrawal thresholds (MWTs) were measured to assess migraine-related hyperalgesia. Anxiety-like behaviors and acute malaise were evaluated using the Open Field Test (OFT), Elevated Plus Maze (EPM), and Light-Dark Box (LDB). Neuronal activation was mapped via c-Fos immunofluorescence. Medullary TNF-α levels were quantified by ELISA. 16S rRNA sequencing and targeted metabolomics characterized the gut microbiota composition and fecal metabolites, respectively. Although male and female mice showed comparable responses in acute and chronic mechanical pain hyperalgesia, significant sex differences were observed in other migraine-like behaviors: only male mice exhibited significant acute-phase motor inhibition and anxiety-like behavior in the chronic phase. These phenotypic differences coincided with a male-restricted elevation of medullary TNF-α following NTG administration. In terms of regional brain activation, the AP stood out in males for its markedly elevated c-Fos cell. This sexual dimorphism extended to the gut-brain axis: while the female NTG group microbiome was enriched with g-Akkermansia, the male profile was dominated by g-Parabacteroides. Metabolomic profiling identified 29 differential metabolites (NTG vs. VEH) in males but only 3 in females. In males, these alterations were primarily enriched in the pentose and glucuronate interconversions and biosynthesis of unsaturated fatty acids pathways that are involved in neuropsychiatric disorders. The NTG-induced chronic migraine model exhibits significant sexual dimorphism, with male mice showing greater sensitivity to acute discomfort and chronic emotional comorbidities. This exploratory study identifies the specific activation of the AP brain region, medullary neuroinflammation, and more severe disturbances in the gut microbiota-metabolic axis as potential contributors to this sex-dependent phenotype.