A bottom-up approach to understanding ultrasound mediated blood–brain barrier opening using long and short pulses

Ultrasound-mediated blood-brain barrier (BBB) opening is promising for non-invasive, localized, and reversible drug delivery to the brain. However, the underlying mechanisms remain unclear, particularly at microscopic level in real time. Recently developed short-pulse ultrasound enhances safety by reducing side effects compared to conventional long-pulse ultrasound. Using vessel-mimicking microchannels cultured with brain endothelial monolayers, we directly observed bubble dynamics and cellular bioeffects under flow conditions. Intriguingly, cyclic jetting during stable cavitation occurs at long-pulse mode, accompanied by a few localized cell detachments and extensive sonoporation. In contrast, short-pulse ultrasound induced milder, uniform bubble dynamics, reversible sonoporation and calcium signaling, promoting safer BBB modulation. In vivo two-photon imaging and histology in mice confirmed these findings, showing that long-pulse ultrasound enabled higher drug delivery efficiency but caused localized endothelial damage, while short-pulse ultrasound facilitated uniform delivery and faster BBB recovery. The differential bubble dynamics and cellular responses correlated well between in vitro and in vivo models. This study establishes a cross-scale framework for real-time analysis of ultrasound-mediated BBB opening, revealing key biophysical factors governing safety and efficacy. The findings provide guidance for the optimization of ultrasound protocols for vascular drug delivery, with potential applications in treating brain tumors, neurodegenerative diseases, and other disorders.