Multi-modality safety assessment of blood-brain barrier opening using focused ultrasound and definity microbubbles: a...

As a potentially viable method of brain drug delivery, the safety profile of blood-brain barrier (BBB) opening using focused ultrasound (FUS) and ultrasound contrast agents (UCA) needs to be established. In this study, we provide a short-term (30-min or 5-h survival) histological assessment of murine brains undergoing FUS-induced BBB opening. Forty-nine mice were intravenously injected with Definity microbubbles (0.05 microL/kg) and sonicated under the following parameters: frequency of 1.525 MHz, pulse length of 20 ms, pulse repetition frequency of 10 Hz, peak rarefactional acoustic pressures of 0.15-0.98 MPa and two 30-s sonication intervals with an intermittent 30-s delay. The BBB opening threshold was found to be 0.15-0.3 MPa based on fluorescence and magnetic resonance imaging of systemically injected tracers. Analysis of three histological measures in hematoxylin and eosin-stained sections revealed the safest acoustic pressure to be within the range of 0.3-0.46 MPa in all examined time periods post sonication. Across different pressure amplitudes, only the samples 30 min post opening showed significant difference (p < 0.05) in the average number of distinct damaged sites, microvacuolated sites, dark neurons and sites with extravasated erythrocytes. Enhanced fluorescence around severed microvessels was also noted and found to be associated with the largest tissue effects, whereas mildly diffuse BBB opening with uniform fluorescence in the parenchyma was associated with no or mild tissue injury. Region-specific areas of the sonicated brain (thalamus, hippocampal fissure, dentate gyrus and CA3 area of hippocampus) exhibited variation in fluorescence intensity based on the position, orientation and size of affected vessels. The results of this short-term histological analysis demonstrated the feasibility of a safe FUS-UCA-induced BBB opening under a specific set of sonication parameters and provided new insights on the mechanism of BBB opening.