Altered brain-wide hemodynamic recovery and network dynamics in a repeated-nitroglycerin model of chronic migraine

Migraine attacks represent dynamic transitions between distinct brain states, involving coordinated changes in neuronal activity, trigeminovascular signaling, and cerebrovascular regulation. Although migraine chronification is associated with persistent sensory sensitization and altered brain network organization, whether chronic migraine alters brain-wide hemodynamic responses underlying a subsequent migraine-like attack remains unknown. Nitroglycerin (NTG) is a clinically relevant translational model that reproduces migraine-like attacks and sensitization; however, previous studies have primarily examined regional cortical, meningeal, or large-vessel responses, leaving the temporal organization of whole-brain hemodynamic responses unresolved. In this study, we investigated whether repeated NTG exposure alters the baseline brain state and the subsequent temporal evolution and recovery of brain-wide responses to an NTG challenge. A repeated NTG administration paradigm was used to establish a chronic migraine (CM) model, with vehicle-pretreated mice serving as the vehicle (Veh) group. Migraine-related behaviors were evaluated using the mechanical withdrawal threshold, light–dark box testing, and phenol red thread assessment. Separate cohorts underwent continuous whole-brain four-dimensional functional ultrasound (4D fUS) imaging or electroencephalographic (EEG) recordings. Following a 15-min baseline period, NTG was administered and recording continued for 45 min. Whole-brain cerebral blood volume (CBV) dynamics, hemodynamic functional connectivity, and cortical electrophysiological responses were analyzed independently in the respective cohorts. Repeated NTG exposure induced mechanical hypersensitivity, light avoidance, and increased light-evoked lacrimation. Four-dimensional functional ultrasound imaging revealed that repeated NTG exposure altered both the baseline hemodynamic functional connectivity pattern and the subsequent evolution of cerebral hemodynamic responses to an NTG challenge. The initial negative CBV response was broadly comparable between groups, whereas the major divergence emerged during the subsequent rebound and recovery phases. CM mice exhibited an attenuated hyperemic rebound and a more prolonged cortex-predominant relative CBV reduction. Functional connectivity analysis revealed an altered baseline functional connectivity pattern in CM mice, with significantly increased global, within-system, and between-system connectivity. Following NTG challenge, this baseline configuration underwent time-dependent reconfiguration, including a transient reduction in connectivity during the 30–45-min period; however, no individual group-by-time connection survived FDR correction. Repeated NTG exposure altered both the baseline functional state of the brain and the subsequent physiological response to an NTG challenge. Repeatedly treated mice showed an altered baseline hemodynamic functional network configuration, while the clearest differences after NTG emerged during recovery, including prolonged cortex-predominant relative CBV reduction and delayed cortical γ-band recovery. Rather than simply amplifying the initial response, repeated NTG exposure primarily altered the trajectory of physiological recovery. These findings support a dynamic brain-state framework in which dynamic recovery may represent a complementary systems-level dimension of migraine chronification and a potential direction for future translational investigation. Not applicable.