The therapeutic efficacy of neurological agents is severely limited, because large compounds do not cross the blood–brain barrier (BBB). Focused ultrasound (FUS) sonication in the presence of microbubbles has been shown to temporarily open the BBB, allowing systemically administered agents into the brain. Until now, polydispersed microbubbles (1–10 μm in diameter) were used, and, therefore, the bubble sizes better suited for inducing the opening remain unknown. Here, the FUS-induced BBB opening dependence on microbubble size is investigated. Bubbles at 1–2 and 4–5 μm in diameter were separately size-isolated using differential centrifugation before being systemically administered in mice (n = 28). The BBB opening pressure threshold was identified by varying the peak-rarefactional pressure amplitude. BBB opening was determined by fluorescence enhancement due to systemically administered, fluorescent-tagged, 3-kDa dextran. The identified threshold fell between 0.30 and 0.46 MPa in the case of 1–2 μm bubbles and between 0.15 and 0.30 MPa in the 4–5 μm case. At every pressure studied, the fluorescence was greater with the 4–5 μm than with the 1–2 μm bubbles. At 0.61 MPa, in the 1–2 μm bubble case, the fluorescence amount and area were greater in the thalamus than in the hippocampus. In conclusion, it was determined that the FUS-induced BBB opening was dependent on both the size distribution in the injected microbubble volume and the brain region targeted.

The activation of bubbles by an acoustic field has been shown to temporarily open the blood-brain barrier (BBB), but the trigger cause responsible for the physiological effects involved in the process of BBB opening remains unknown. Here, the trigger cause (i.e., physical mechanism) of the focused ultrasound-induced BBB opening with monodispersed microbubbles is identified. Sixtyseven mice were injected intravenously with bubbles of 1-2, 4-5, or 6-8 lm in diameter and the concentration of 10 7 numbers/ml. The right hippocampus of each mouse was then sonicated using focused ultrasound (1.5 MHz frequency, 100 cycles pulse length, 10 Hz pulse repetition frequency, 1 min duration). Peak-rarefactional pressures of 0.15, 0.30, 0.45, or 0.60 MPa were applied to identify the threshold of BBB opening and inertial cavitation (IC). Our results suggest that the BBB opens with nonlinear bubble oscillation when the bubble diameter is similar to the capillary diameter and with inertial cavitation when it is not. The bubble may thus have to be in contact with the capillary wall to induce BBB opening without IC. BBB opening was shown capable of being induced safely with nonlinear bubble oscillation at the pressure threshold and its volume was highly dependent on both the acoustic pressure and bubble diameter.

Focused ultrasound (FUS) is hereby shown to noninvasively and selectively deliver compounds at pharmacologically relevant molecular weights through the opened blood-brain barrier (BBB). A complete examination on the size of the FUS-induced BBB opening, the spatial distribution of the delivered agents and its dependence on the agent's molecular weight were imaged and quantified using fluorescence microscopy. BBB opening in mice (n=13) was achieved in vivo after systemic administration of microbubbles and subsequent application of pulsed FUS (frequency: 1.525 MHz, peak-rarefactional pressure in situ: 569 kPa) to the left murine hippocampus through the intact skin and skull. BBB-impermeant, fluorescent-tagged dextrans at three distinct molecular weights spanning over several orders of magnitude were systemically administered and acted as model therapeutic compounds. First, dextrans of 3 and 70 kDa were delivered trans-BBB while 2000 kDa dextran was not. Second, compared to 70 kDa dextran, a higher concentration of 3 kDa dextran was delivered through the opened BBB. Third, the 3 and 70 kDa dextrans were both diffusely distributed throughout the targeted brain region. However, high concentrations of 70 kDa dextran appeared more punctated throughout the targeted region. In conclusion, FUS combined with microbubbles opened the BBB sufficiently to allow passage of compounds of at least 70 kDa, but not greater than 2000 kDa, into the brain parenchyma. This noninvasive and localized BBB opening technique could thus provide a unique means for the delivery of compounds of several magnitudes of kDa that include agents with shown therapeutic promise in vitro, but whose in vivo translation has been hampered by their associated BBB impermeability.

The in vivo cavitation response associated with blood–brain barrier (BBB) opening as induced by transcranial focused ultrasound (FUS) in conjunction with microbubbles was studied in order to better identify the underlying mechanism in its noninvasive application. A cylindrically focused hydrophone, confocal with the FUS transducer, was used as a passive cavitation detector (PCD) to identify the threshold of inertial cavitation (IC) in the presence of Definity® microbubbles (mean diameter range: 1.1–3.3 μm, Lantheus Medical Imaging, MA, USA). A vessel phantom was first used to determine the reliability of the PCD prior to in vivo use. A cerebral blood vessel was simulated by generating a cylindrical channel of 610 μm in diameter inside a polyacrylamide gel and by saturating its volume with microbubbles. The microbubbles were sonicated through an excised mouse skull. Second, the same PCD setup was employed for in vivo noninvasive (i.e. transdermal and transcranial) cavitation detection during BBB opening. After the intravenous administration of Definity® microbubbles, pulsed FUS was applied (frequency: 1.525 or 1.5 MHz, peak-rarefactional pressure: 0.15–0.60 MPa, duty cycle: 20%, PRF: 10 Hz, duration: 1 min with a 30 s interval) to the right hippocampus of twenty-six (n = 26) mice in vivo through intact scalp and skull. T1 and T2-weighted MR images were used to verify the BBB opening. A spectrogram was generated at each pressure in order to detect the IC onset and duration. The threshold of BBB opening was found to be at a 0.30 MPa peak-rarefactional pressure in vivo. Both the phantom and in vivo studies indicated that the IC pressure threshold had a peak-rarefactional amplitude of 0.45 MPa. This indicated that BBB opening may not require IC at or near the threshold. Histological analysis showed that BBB opening could be induced without any cellular damage at 0.30 and 0.45 MPa. In conclusion, the cavitation response could be detected without craniotomy in mice and IC may not be required for BBB opening at relatively low pressures.

Focused ultrasound in conjunction with the systemic administration of microbubbles has been shown to open the bloodbrain barrier (BBB) selectively, noninvasively and reversibly. In this study, we investigate the dependence of the BBB opening's reversibility on the peak-rarefactional pressure (0.30-0.60 MPa) as well as the microbubble size (diameters of 1-2, 4-5, or 6-8 mm) in mice using contrast-enhanced T 1 -weighted (CE-T 1 ) MR images (9.4 T). Volumetric measurements of the diffusion of Gd-DTPA-BMA into the brain parenchyma were used for the quantification of the BBB-opened region on the day of sonication and up to 5 days thereafter. The volume of opening was found to increase with both pressure and microbubble diameter. The duration required for closing was found to be proportional to the volume of opening on the day of opening, and ranged from 24 h, for the smaller microbubbles, to 5 days at high peak-rarefactional pressures. Overall, larger bubbles did not show significant differences. Also, the extent of BBB opening decreased radially towards the focal region until the BBB's integrity was restored. In the cases where histological damage was detected, it was found to be highly correlated with hyperintensity on the precontrast T 1 images. Magn Reson Med 67:769-777,