Multi-Modality Safety Assessment of Blood-Brain Barrier Opening Using Focused Ultrasound and Definity Microbubbles: A Short-Term Study

Abstract

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 5h survival) histological assessment of murine brains undergoing FUS-induced BBB opening. Forty-nine mice were intravenously injected with Definity® microbubbles (0.05 μl/kg) and sonicated under the following parameters: frequency of 1.525 MHz, burst length of 20 ms, PRF 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 MR imaging of systemically-injected tracers. Analysis of three histological measures in H&E-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 while 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.

Noninvasive and localized neuronal delivery using short ultrasonic pulses and microbubbles

“…Previous work by our group and others typically optimized a few ultrasonic parameters, namely the PRP, frequency, PRF, and PL. However, increasing the delivered dose by modifying one of these parameters was also associated with either poorer distribution characteristics (17) or the onset of cellular damage (11). This paper explored a wider range of parameters that, to our knowledge, has not been used for drug delivery.…”

“…FUSinduced BBB disruption has been previously demonstrated to successfully deliver large molecular agents such as 70-kDa dextrans (17), Herceptin (24), and Doxorubicin (25). Nevertheless, concerns remain with the dose and distribution of the agents delivered (14,17) and the safety associated with enhancing these characteristics (11). For instance, increased dose can be achieved with higher PRPs and PLs, but higher PRPs have been associated with cellular damage (11) and longer PLs have been associated with increased molecular accumulation at or near larger vessels, thus indicating off-target and inhomogeneous distribution (16,17).…”

“…Each type and magnitude of cavitation activity results in distinct vascular bioeffects and are dictated by the ultrasonic pulse shape and sequence, the microbubble composition and distribution (9), and the in vivo environment the microbubbles circulate (8,10). Selection of the exposure parameters is critical for effective drug delivery while minimizing side effects (11). Not accounting for these parameters may result in adverse effects even when a therapeutic effect is not intended, such as a previously observed increase in permeability with a diagnostic array (12) and, in severe situations, hemorrhage (13).…”

“…Molecular delivery requires a minimum PRP known as the cavitation threshold, which is typically lower than 1 MPa (2,14) and drops with the center frequency (15). Exposure of tissues above, but near, the threshold has so far yielded the most promising results with molecular delivery without any associated damage, as assessed using histological analysis (11). Further increasing the PRP enhances the dose delivered, but is also associated with a highly heterogeneous distribution within the focal volume and the onset of erythrocyte extravasations, hemorrhage, and neuronal damage (11,16).…”

“…Exposure of tissues above, but near, the threshold has so far yielded the most promising results with molecular delivery without any associated damage, as assessed using histological analysis (11). Further increasing the PRP enhances the dose delivered, but is also associated with a highly heterogeneous distribution within the focal volume and the onset of erythrocyte extravasations, hemorrhage, and neuronal damage (11,16). Less understood is the effect different PLs have on delivery characteristics.…”