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

Authors: Baseri B, Choi JJ, Tung YS, Konofagou EE

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.

Introduction

Purpose Drug delivery with BBB opening

Study Objective To evaluate the short-term histological safety of focused ultrasound with microbubbles for inducing blood–brain barrier opening in mice.

Animal model / Human subject Mouse (C57BL/6) (Mus musculus); strain: not specified; age: not specified; sex: male

MRI or image guidance method Ultrasound pulse-echo-based grid targeting

Targeted brain region(s) hippocampal fissure

Target coordinates 2.25 mm lateral from sagittal suture; 2 mm from lambdoid suture, depth 3 mm. below skull surface

Outcomes and Safety

Summary of Outcomes FUS with Definity microbubbles (0.05 μl/kg) at 1.525 MHz, 20 ms bursts, 10 Hz PRF and two 30-s sonications separated by 30 s produced safe short-term BBB opening with minimal histological damage at peak rarefactional pressures of ~0.3–0.46 MPa (BBB opening threshold 0.15–0.3 MPa).

Duration of biological effect 30 min, 5 h

Safety-related matter Short-term histology showed a BBB opening threshold of 0.15-0.3 MPa and identified the safest acoustic pressure range as 0.3-0.46 MPa, indicating FUS-UCA can produce safe BBB opening under specific sonication parameters. However, at some pressure amplitudes—especially 30 min post-sonication—significant adverse effects were observed (distinct damaged sites, microvacuolation, dark neurons, extravasated erythrocytes), with enhanced fluorescence around severed microvessels associated with larger tissue injury, while mild diffuse opening correlated with no or mild injury.

Brain Region

Ultrasound Parameters

Ultrasound instrument single-element circular aperture focused ultrasound transducer

FUS Frequency 1.525 MHz

FUS Pressure 0.15-0.98 MPa (peak rarefactional acoustic pressures); BBB opening threshold: 0.15-0.3 MPa; safest acoustic pressure range: 0.3-0.46 MPa

FUS Mode pulsed

Pulse duration 20 ms

Duration of a single FUS session 90 seconds

Focal Characteristics focal depth: 90 mm; focal length: None; aperture size: None

Treatment frequency single

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