Safe focused ultrasound-mediated blood-brain barrier opening is driven primarily by transient reorganization of tight junctions.

Focused ultrasound (FUS) with microbubbles opens the blood-brain barrier (BBB) for targeted drug delivery into the brain. How brain endothelial cells (BECs) respond to either low acoustic pressures known to open the BBB transiently, or high pressures that cause brain damage, is incompletely characterized. Here, we apply FUS at low (450 kPa) and high (750 kPa) pressures in mice where BBB tight junctions are labelled with eGFP and find that arteriole and capillary BECs respond to low pressure by a transient reorganization of tight junctions associated with BBB opening. Moreover, this process does not depend on caveolae. In contrast, BBB opening at high pressure is associated with tight junction obliteration even after 72 hours, allowing persistent fibrinogen passage and microglial activation. Single-cell RNA-sequencing of BECs from FUS-BBBO mice shows that the transcriptomic responses of BECs exposed to high pressure are dominated by the stress response and cell junction disassembly, whereas lower pressure induces primarily genes responsible for intracellular repair. Therefore, transient reorganization of tight junctions and repair responses mediate safe BBB opening for therapeutic delivery.