High spatiotemporal resolution in vivo imaging reveals preferential PACAP38 action on dural vasculature and a functional therapeutic readout

PACAP38-induced cephalic vasodilation is used to assess functional PACAP pathway inhibition in humans, but the vascular populations underlying this response are not fully resolved. Defining these compartments in a preclinical model could help connect clinical pharmacodynamic responses with the underlying vascular mechanisms and support further development of PACAP-targeted therapies. Using chronic cranial windows and head-mounted microscopy, we tracked meningeal vascular responses in freely moving mice. Nitroglycerin, levcromakalim, and PACAP38 each produced net vasodilation, yet the underlying responses were markedly heterogeneous and distributed differently across the vascular populations. Notably, PACAP38 evoked greater early vasodilation in dural than pial vessels, and anti-PACAP pretreatment attenuated PACAP38-induced vasodilation. These findings show that net vasodilation can conceal vessel-level response patterns and identify preferential early dural vasodilation as a spatially defined, pharmacologically modifiable feature of the systemic PACAP38 response. This phenotype provides a translational platform for mechanistic interrogation and evaluation of functional pathway inhibition by migraine-targeted therapies.