Sex-Specific Circadian Dysregulation in the Choroid Plexus of a Nitroglycerin Rat Model of Chronic Migraine: An RNA-Seq Analysis

Background Migraine patients show choroid plexus (CP) changes, impairing the blood-CSF barrier. The CP regulates circadian rhythms, but links between CP circadian genes and migraine are unexplored. Objective This study examined changes in the CP circadian gene transcriptome in a chronic migraine rat model compared with controls to identify migraine-related pathways. Design Chronic migraine model: Sprague-Dawley rats (3 females, 3 males) received nitroglycerin (NTG) every other day for 9 days; controls (3 females, 3 males) got saline. CP from the 4 th ventricle was collected 2 hours after the final injection for RNA-seq. Methods Migraine Behavior: Von Frey hair tests on days 1, 5, and 9, pre- and 2 hours post-NTG/saline injection, assessed basal and NTG-induced pain thresholds. RNA-seq & Analysis: Differentially expressed genes (p < 0.05, fold change > 1) were identified. GO, KEGG, and Reactome enrichment analyses were performed to assess changes in circadian gene expression. Results The NTG group showed reduced basal and NTG-induced pain thresholds on days 1, 5, and 9. Females had a greater number of upregulated genes ( MT2A , SLC7A11 ); males had upregulated ZBTB16 and S100A8 . SLC7A11 , SCG2 , and GRIA1 showed inverse regulation (up in females, down in males). Circadian gene expression altered: 10 genes upregulated (e.g., SERPINE1, MAPK9, ATF4 ), 13 downregulated (e.g., PER2, DBP, EZH2 ). Sex-specific differences: females ( FBXL12, GPR157 ), males ( NKX2-1, ATF4, CLOCK ). GO/KEGG analyses revealed significant enrichment of circadian rhythm-related pathways, insulin resistance, and inflammatory response processes, with sex-specific differences: females showed enrichment in HIF-1 signaling and hemoglobin-related pathways, while males exhibited enrichment in arachidonic acid and leukotriene production. Conclusion CP transcriptomics in the rat migraine model revealed sex-specific gene regulation, with females upregulating antioxidant genes ( MT2A , SLC7A11 ) and males upregulating inflammatory factors ( ZBTB16 , S100A8 ), alongside circadian disruption (e.g., SERPINE1 upregulated, PER2 downregulated). Pathway analyses indicate enriched circadian rhythms, HIF-1 signaling (females), inflammatory processes (males), lipid metabolism (PPARα), and heme signaling, highlighting sex-specific and circadian targets for migraine therapy.