Macrophages and iNOS contribute to the effects of dural prolactin and repeated stress in mouse migraine models.

BACKGROUND: Migraine impacts 15% of the global population, predominantly women. Previous studies have shown a role for prolactin in animal migraine models induced by either stimulation of the dura mater or repeated stress exposure. However, the site of prolactin action is not fully known nor are its downstream mechanisms. This study investigated the potential downstream mechanisms and the cell types involved in prolactin- and repeated stress-induced migraine-like responses. METHODS: Two preclinical migraine models were used in this study, dural stimulation and repeated restraint stress. Dural injections in mice enabled drug delivery to the dura mater through the intersection of the lambdoidal and sagittal sutures. Additionally, a model of repeated stress-induced periorbital hypersensitivity and priming to a subthreshold nitric oxide donor was used. Von Frey filaments were used to measure periorbital mechanical thresholds before and after dural administration of prolactin or stress. RESULTS: Conditional knockout of prolactin receptors in Nav1.8-expressing sensory neurons partially but significantly blocked the periorbital hypersensitivity caused by dural application of prolactin (0.5 µg) to female mice. Depletion of macrophages using clodronate liposome injections before dural prolactin significantly blocked the prolactin responses. The inducible nitric-oxide synthase (iNOS) inhibitor AR-C102222 (ARC; 15 mg/kg) significantly blocked the dural prolactin-induced responses. To determine whether macrophages and iNOS contribute to repetitive stress-induced periorbital hypersensitivity and priming to SNP, clodronate liposomes or ARC were given before or after repetitive stress exposure. Macrophage depletion prior to or following stress significantly inhibited stress-induced periorbital hypersensitivity in both males and females. However, ARC only blocked stress-induced migraine-like behaviors in females. CONCLUSION: This study demonstrates that dural prolactin acts through both neuronal and immune cell mechanisms and is dependent on iNOS activity. In response to repeated stress, macrophages contribute to behavioral responses in both sexes while iNOS is only required in females. These findings suggest that interactions between the immune and nervous systems are important for the effects of prolactin and stress on migraine-relevant mechanisms and demonstrate further sex differences in specific pathways.