Microbubble dynamics in brain microvessels

James H. Bezer; Paul Prentice; William Lim Kee Chang; Sophie V. Morse; Kirsten Christensen-Jeffries; Christopher J. Rowlands; Andriy S. Kozlov; James J. Choi

doi:10.1371/journal.pone.0310425

Abstract

Focused ultrasound stimulation of microbubbles is being tested in clinical trials for its ability to deliver drugs across the blood-brain barrier (BBB). This technique has the potential to treat neurological diseases by preferentially delivering drugs to targeted regions. Yet despite its potential, the physical mechanisms by which microbubbles alter the BBB permeability remain unclear, as direct observations of microbubbles oscillating in brain microvessels have never been previously recorded. The purpose of this study was to reveal how microbubbles respond to ultrasound when within the microvessels of living brain tissue. Microbubbles in acute brain slices acquired from juvenile rats perfused with a concentrated solution of SonoVue® and dye were exposed to ultrasound pulses typically used in BBB disruption (center frequency: 1 MHz, peak-negative pressure: 0.2–1 MPa, pulse length: up to 10 ms) and observed using high-speed microscopy at up to 10 million frames per second. We observed that microbubbles can exert mechanical stresses on a wide region of tissue beyond their initial location and immediate surroundings. A single microbubble can apply mechanical stress to parenchymal tissues several micrometers away from the vessel. Microbubbles can travel at high velocities within the microvessels, extending their influence across tens of micrometers during a single pulse. With longer pulses and higher pressures, microbubbles could penetrate the vessel wall and move through the parenchyma. The probability of extravasation scales approximately with mechanical index, being rare at low pressures, but much more common at a mechanical index ≥ 0.6. These results present the first direct observations of ultrasound-driven microbubbles within brain tissue, and illustrate a range of microbubble behaviors that have the potential to lead to safe drug delivery or tissue damage.

Citation: Bezer JH, Prentice P, Lim Kee Chang W, Morse SV, Christensen-Jeffries K, Rowlands CJ, et al. (2025) Microbubble dynamics in brain microvessels. PLoS ONE 20(2): e0310425. https://doi.org/10.1371/journal.pone.0310425

Editor: Girdhari Rijal, Tarleton State University, UNITED STATES OF AMERICA

Received: February 8, 2024; Accepted: August 31, 2024; Published: February 5, 2025

Copyright: © 2025 Bezer et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the manuscript and its Supporting Information Files.

Funding: his research was supported by Alzheimer's Research UK (ARUK-IRG2017A7). J.H.B. would like to acknowledge funding from the EPSRC Centre for Doctoral Training in Medical Imaging (EP/L015226/1). W.L.K.C. would like to acknowledge funding from the EPSRC Centre for Doctoral Training in Smart Medical Imaging (EP/S022104/1). S.V.M. would like to acknowledge support from the EPSRC Doctoral Prize Fellowship (EP/T51780X/1). The Shimadzu camera was supported by an ERC Starting Grant (336189). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

On-going clinical trials are demonstrating that focused ultrasound and microbubbles can disrupt the blood-brain barrier (BBB) and deliver drugs in patients with Alzheimer’s disease [1, 2], brain cancers [3–5], and other neurological conditions [6]. In these patients, pre-formed microbubbles with a diameter between 1 and 10 μm are systemically administered. Clinical trials most commonly use lipid-shelled bubbles, such as Definity® [2, 5] or SonoVue® [7], although protein-shelled bubbles are also available [8]. Focused pulses of ultrasound are then applied to a target brain region, driving the microbubbles circulating within the vessels to expand and contract, somehow delivering drugs across the BBB [9].

Despite the promise of this method, the mechanisms through which oscillating microbubbles lead to drug delivery remain poorly understood. Microbubbles exposed to ultrasound are known to exhibit a wide range of behaviors [10]. At low peak-negative pressure, bubbles expand and contract in response to the rarefactional and compressional phases of the acoustic wave. At high-peak-negative pressures, the bubble expands uncontrollably and then rapidly collapses inwards due to the inertia of the surrounding fluid. This is known as inertial cavitation [11]. Such oscillations can be stable (i.e., recurrent) across many acoustic cycles, or transient in nature. The oscillations can also be spherical or asymmetric [12]. At higher pressures, asymmetric collapse of bubbles can lead to the formation of high-speed jets of fluid [13, 14]. Microbubbles also experience radiation forces which can cause translational motion typically in the direction of wave propagation [15, 16].

Biological mechanisms have been investigated experimentally through post-mortem analysis and in vivo imaging of dye extravasation, which have shown drug delivery to be associated with disruption of tight junctions between endothelial cells [17, 18], increased numbers of transcytotic vesicles [19, 20], and vessel rupture [21, 22].

However, these biological observations cannot link the drug delivery event to the microbubble behaviors which occur in the brain during ultrasound exposure [23]. Direct observations of oscillating microbubbles within vessels have only been made within other tissue types, such as the chorioallontoic membrane of a chick embryo [24], cheek pouch of a hamster, and thin sections of the liver or muscle of rats [25]. In rat ceca, vascular confinement produced significant damping of the oscillations, as well as asymmetric bubble behaviors and vessel wall motion [26]. In the microvessels of rat mesentery, microbubble-induced vessel invagination and distension have been observed, as well as microjetting [27].

Many of these studies used short (microsecond), relatively high-amplitude pulses (0.8 – 7.2 MPa_pk-neg at a center frequency of 1 MHz), with few consecutive frames captured per pulse. In contrast, drug delivery across the BBB is typically performed using lower amplitudes (<0.8 MPa_pk-neg) and much longer pulses (typically around 10 ms) [28–30], in which the bubbles likely behave very differently. While these studies provide important insight into bubble dynamics in tissues, their relevance to drug delivery across the BBB is therefore limited.

Acute brain slices are a common tool in neurophysiology research, enabling direct measurements of cellular activity within an intact live tissue environment. Juvenile slices are especially valuable due to their increased tissue viability in vitro and increased transparency [31, 32].

Here, we use acute brain slices from juvenile rats perfused with SonoVue® microbubbles to present direct optical observations of microbubbles in brain microvessels. Individual microbubbles exposed to therapeutically-relevant ultrasound pulses (center frequency of 1 MHz; peak-negative pressures between 0.2 and 1 MPa) were imaged at up to 10 million frames per second (fps). The volumetric oscillations, translational motion of the bubbles, and displacement of surrounding tissues are presented.

Methods

Slice preparation

Slices were prepared from Wistar wild-type juvenile rats (postnatal day 5-15, Charles River). Animals were culled via intraperitoneal injection of pentobarbital, followed by femoral exsanguination, in accordance with Schedule 1 of the Animals (Scientific Procedures) Act 1986. Shortly (<15 minutes) after cessation of circulation, the carcass was transcardially perfused with a single concentrated solution of SonoVue (40% v/v, Bracco), heparin (0.05 mg/mL), and either Evans Blue (4 mg/mL) or Blue India Ink (40% v/v) diluted together in 0.9% saline. A total fluid volume of around 3-5 mL was slowly perfused over around 1 minute using a syringe pump (PHD Ultra, Harvard Apparatus, Holliston, MA, USA).

The corpus callosum was transected and live cortical slices were obtained by sectioning the frontal lobes under ice-cold artificial cerebrospinal fluid (aCSF) using a vibratome (Campden Instruments 7000smz). Between sectioning and ultrasound experiments, slices were kept in aCSF at room temperature. Slices were selected for observation if they were grossly intact following sectioning, and if microbubbles could be imaged clearly within the vessels.

Ultrasound

Brain slices were placed inside a Perspex box (depth: 2 cm, width: 5 cm) which had mylar walls on its front and back to let ultrasound propagate into and out of the box with minimal attenuation (Fig 1). The top of the box was open, allowing objective lenses to be placed within it. A 5-mm-thick layer of 0.5% agarose was placed at the bottom of the box, on which the brain slice was placed. Agarose allowed a free acoustic path, while remaining transparent enough to allow light from underneath. A 100-μm-thick glass cover slip was placed over each brain slice to keep it in place during sonication and to avoid effects from the motion of the surrounding fluid. The box was filled with aCSF at room temperature and placed within a tank of deionized water. The apparatus was set on a vibration isolation table (Vision Isostation, Newport).

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Fig 1. Experimental setup.

(Left) A 250-μm-thick brain slice from a juvenile rat was immersed in artificial cerebrospinal fluid and placed between a light source and an objective (40X or 60X). A focused ultrasound transducer emitted sound onto the brain slice while videos were captured with either a color camera (1 frame per second (fps)), a high-speed camera (5,581 fps), or an ultra-high-speed camera (5 and 10 million fps). (Right) This experimental setup was able to capture images of microbubbles in brain microvessels.

https://doi.org/10.1371/journal.pone.0310425.g001

Ultrasound pulses were emitted from an immersion transducer (A303 S-SU, diameter: 13 mm, focal distance: 15.2 mm, center frequency: 1 MHz; Olympus), driven by a function generator (33500 B Series, Agilent Technologies, Santa Clara, CA, USA) through a 50-dB amplifier (E&I) (S1 Fig). Ultrasound pulses were calibrated in situ before the start of each experiment using a needle hydrophone (diameter: 0.2 mm; Precision Acoustics), which was also used to align the focus of the transducer with the focal plane of the objective lens.

Ultrasound parameters were chosen to match those commonly used in in vivo experiments and clinical trials. A center frequency close to 1 MHz is often used in in vivo studies [5, 33–35]. This frequency is also high enough to enable a small enough focal region from a single element to avoid interactions with the objective lens. Peak Negative Pressures between 0.2 and 1 MPa were used as effective BBB disruption has been demonstrated at these pressures in previous studies [28, 33, 36, 37]. In the long pulse experiments, pulse lengths of 10 ms were used, as millisecond pulse lengths are typical in in vivo studies [28–30]. For the ultra-high frame rate images, the pulse lengths were limited by the exposure time of the camera.

Image acquisition

For the lower frame rate images (Figs 2C, 4, 5A, 5B, 5E, 5F), slices were imaged with a Chronos 1.4 camera (Kron Technologies Inc) connected to a 40x water dipping lens (LUMPLFLN, numerical aperture: 0.8, working distance: 3.3 mm; Olympus), illuminated from below with an LED light source (KL 2500, Schott) (S1 Fig left). Videos were acquired at a frame rate of 5,581 fps, with a field of view of 608x400 pixels, and an exposure time of 174 μs. The pixel pitch was 0.16 μm. Images were simultaneously acquired using a color camera (ThorLabs DCC1645C CMOS, or IDS U3-3070CP-C-HQ; pixel pitch 0.08 μm) at 1 fps over a period of up to 2 minutes. This enabled clear images of the dye within the vessels to be obtained both before and after sonication.

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Fig 2. Microbubble dynamics during confinement in brain microvessels.

(A) A microbubble’s oscillation can displace the vessel wall at the same rate. A microbubble in a microvessel was exposed to an ultrasonic pulse (center frequency (f_c): 1 MHz, peak-rarefactional pressure (P_neg): 0.8 MPa, pulse length (PL): 15 μs) and imaged at 10 million fps. (Top) The microbubble experienced a large expansion phase, followed by a sudden collapse. At 16.1 μs, an asymmetric collapse was observed in the shape of a ‘fig 8’. (Bottom) A streak of the same microbubble’s cross-section (dotted white line in Top-Left) reveals a microbubble whose maximum expansion gradually increased with the number of exposure cycles. The vessel wall distended at the same rate as the microbubble, expanding with the bubble, but not invaginating as much when the bubble collapsed into itself. (B) A microbubble’s oscillations can be asymmetric. Two microbubbles were exposed to an ultrasonic pulse (f_c: 1 MHz, P_neg: 0.8 MPa, PL: 15 μs). The axial radius of one of the microbubbles was always larger than the radial radius. (C) Microbubbles can coalesce in confinement. Two microbubbles in a microvessel were exposed to an ultrasonic pulse (f_c: 1 MHz, P_neg: 0.8 MPa, PL: 15 cycles) and imaged at ten million frames per second. (Top) Images of the microbubble were obtained before and during sonication. The bubbles were at first observed to be spatially separated, but had coalesced into a single bubble at a time point between 5.2 and 6.0 μs. (Bottom) A streak of the microbubble cross-sections (dotted white line in Top-Left) revealed microbubbles with spatially-separated oscillations that moved closer to each other. The two bubbles then merged and oscillated as a single bubble, but with larger-amplitude oscillations. A single bubble was observed after the ultrasound pulse.

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Ultra-high frame rate videos were acquired using an HPV-X camera (Shimadzu Corp) linked to a 60x water dipping lens (S1 Fig right). The pixel pitch was 0.61 μm. Images were acquired at 10 Mfps, with ultrasound pulses of 15 cycles (Fig 2A, 2B), or at 5 Mfps with 60 cycle ultrasound pulses (Fig 5C), although only approximately the first 50 were captured in the recording time in the latter instance. Illumination was provided by 10-ns synchronous laser pulses (CAVILUX Smart, Cavitar).

Microbubble selection

Microbubbles were identified within each slice. A microbubble was chosen for imaging if it could be clearly imaged on the camera. Microbubbles had to be clearly within microvessels, which were defined based on the internal vessel diameter of <30 μm, so as to avoid large arteries or veins, which do not participate in substance transport into the brain. Microbubbles close to the surface or edges of the slice were avoided to ensure they were deep within the tissue and not affected by the sectioning. This also reduced any potential effects from the motion of the surrounding fluid due to the ultrasound.

Image processing

Videos were first motion-corrected in Matlab using a 2D cross-correlation algorithm to remove residual background motion caused by environmental vibrations or the direct effects of ultrasound on the tissue. The tissue deformation images in Fig 4 were acquired by comparing selected frames before and during the pulse. A region of interest was chosen manually and compared using Digital Image Correlation software (Ncorr) [38]. Bubble radii were measured by applying an intensity threshold to the images and fitting a circle using a Hough transform [39]. This resulted in an uncertainty in the bubble diameter of approximately 0.2 μm, estimated from the variation in radius with chosen intensity threshold; this is close to the pixel pitch. The tracks of bubbles shown in Figs 2C and 4B were acquired using the same algorithm, where the centroids of each circle in the image were also calculated.

Image analysis

Measurements of vessel diameter (Fig 2A) were made manually in Fiji by three independent observers [40]. The errors calculated were the larger value out of the pixel pitch or the range of the independently measured values. Assessment of whether a bubble extravasated from a vessel was performed by two independent assessors who were blind to the ultrasound pressures applied, based on bubble motion, dye extravasation, and vessel shape changes.

Results

Microsecond time-scale observations

Microbubbles in brain microvessels exposed to ultrasound expanded and contracted during the pulse (Fig 2A and S1 Video). These oscillations caused the vascular wall to move at the same rate as the bubble oscillation, at 1 million cycles per second. In the example shown in Fig 2A, a microbubble with a diameter of approximately 5 μm in a microvessel with a diameter of 16.7 ± 0.6 μm was exposed to a 0.8-MPa_pk-neg pulse. The maximum and minimum vessel diameters over 10 cycles of oscillations were 19.4 ± 0.6 μm and 16.2 ± 0.6 μm respectively, showing that distension was greater than invagination. Interestingly, the bubble’s collapse did not result in invagination of the vessel, which contrasted with previous studies in larger vessels [27, 41]. The vessel diameter at the end of the recording, shortly after the end of the pulse, was 16.9 ± 0.9 μm, which was not significantly different to the initial diameter. The uncertainty in these measurements was the standard deviation of the three values measured by three independent observers. However, if this range was smaller than the pixel pitch, then the pixel pitch was used as the stated uncertainty.

Several instances of microbubbles undergoing asymmetric oscillations were observed. In one example depicted in Fig 2B (S2 Video), the bubble’s expansion was ellipsoidal, experiencing a maximum to minimum diameter ratio of around 1.3 (parallel and perpendicular to the central axis of the vessel), at a P_neg of 0.8 MPa.

Microbubbles also experienced periodic hourglass-shaped oscillations, not associated with fragmentation. The axis of minimum contraction coincided with the central axis of the vessel lumen (Fig 2A). These asymmetric shapes were not due to jetting, which can result in the penetration of one side of the bubble through the other [42]. These results have also confirmed experimentally that microbubbles can coalesce within small brain microvessels. As an example, two microbubbles were attracted to each other and subsequently coalesced over a short period of around 5 to 7 acoustic cycles when exposed to an 0.8 MPa ultrasound pulse (Fig 2C and S3 Video).

Millisecond-time scale observations

Microbubbles were also imaged at 5,581 fps to observe longer-timescale dynamics over the entire 10-ms pulse. During the 10-ms pulse, microbubbles could be driven tens of micrometers within the vessels (Fig 3). Bubbles typically moved along the vessels in the direction of ultrasound propagation, although this motion was often erratic (Middle of Fig 3 and S4 Video). Even when confined within vessels, microbubbles exposed to a 0.6 MPa_pk-neg pulse could achieve speeds of up to around 50 mm/s, as measured by the distance travelled between successive frames of the video.

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Fig 3. A microbubble can move several microns within the microvessel.

A microbubble was exposed to peak-rarefactional pressures of (Top) 0.6 MPa, (Middle) 0.4 MPa, and (Bottom) 0.6 MPa. The color represents the bubble’s location over the duration of the pulse. In all images, ultrasound propagated from left to right.

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In addition to the vessel wall oscillations observed at microsecond timescales, significant tissue displacements were also observed over millisecond timescales (Fig 4 and S5 Video), at PNPs of 0.2, 0.4 and 0.6 MPa. Tissue displacements were quantified using digital image correlation [38], comparing the points in the tissue at their maximum displacement with an image from before the ultrasound pulse. Microbubbles could displace tissue located over 5 μm from the vessel lumen by up to around 0.5 μm.

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Fig 4. Tissue deformation in response to acoustic cavitation in brain microvessels.

Microbubbles exposed to ultrasonic pulses (f_c: 1 MHz, PL: 10 ms) produced tissue deformations well beyond the vessel wall on the longer, 10-ms timescale. The bubbles were exposed to (Top) 0.2, (Middle) 0.6, and (Bottom) 0.4 MPa_pk-neg. The overlaid color maps show the maximum deformation of the tissue in micrometers compared to the initial state before the pulse. Black arrows show the direction of positive displacement axis. Green indicates no displacement, red indicates displacement in the direction of the black arrow, and blue indicates displacement in the opposite direction. White arrows point to the vessel wall. Images were acquired at 5,581 frames per second. (Middle) The red line shows the motion of the microbubble during the pulse; in B and C, the images of the tissue displacement are around the bubble’s initial location. In all images, ultrasound propagated from left to right.

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While microbubbles have been shown to enhance extravasation of molecules from brain microvessels in vivo, we observed that, when exposed to ms-long pulses, microbubbles themselves can extravasate by passing through the microvessel walls. This can be associated with extravasation of the dye within the vessels (Fig 5A, P_neg 0.6 MPa, PL: 10 ms), clearly indicating a puncture to the vessel. As shown in Fig 5B (S6 Video), after leaving the vessel, microbubbles can penetrate quite large distances (tens of micrometers) into the brain tissue, when exposed to a P_neg 0.4 MPa, 10 ms pulse.

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Fig 5. Extravasation of microbubbles due to the primary radiation force.

Microbubbles in microvessels exposed to an ultrasonic pulse (f_c: 1 MHz) extravasated in the direction of wave propagation. (A) A microbubble that extravasates can cause molecular delivery (video frame rate: 5,581 fps, P_neg 0.6 MPa, PL: 10 ms). The color images show the Evans Blue distribution before and after ultrasound exposure. (B) A microbubble can move within the vessel before extravasating and travel several tens of microns into the brain parenchyma (video frame rate: 5,581 fps, P_neg 0.4 MPa, PL: 10 ms). (C) A microbubble’s oscillation in confinement can evolve over time, while modifying the structure of the vessel itself, before extravasating (video frame rate: 5 Mfps, P_neg 1.0 MPa, PL: 50 μs). (D) Radius-time curve for the bubble in (C), showing low-amplitude oscillations while confined within the vessel, increasing gradually before it leaves the vessel at around 38.2 μs. (E) Proportion of bubbles which extravasated (red), remained within the vessel (green), or whose behavior was unclear (orange) during exposure to a 10-ms pulse as a function of peak-rarefactional pressure. Extravasation did not occur at 0.2 MPa, was rare at 0.4 MPa, but becomes the dominant behavior at 0.8 MPa. (F) The initial radii of microbubbles counted may have influenced the likelihood of extravasation. In general, larger bubbles were more likely to extravasate at a given pressure, although this did not occur with very large bubbles (>10 μm diameter).

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Microbubble extravasation

Extravasation of microbubbles often imparted structural changes to the microvessels that persisted after the microbubble left the vessel (Fig 5C and S7 Video). Fig 5D shows the radius-time curve of the bubble, which was exposed to a 1 MPa_pk-neg ultrasound pulse and imaged at 5 Mfps. Towards the end of the pulse, this bubble left the confines of the vessel. Over the first 15-20 cycles, the bubble initially oscillated with stable, moderate-amplitude oscillations (R_max/R₀ of around 3), before gradually increasing in amplitude (up to R_max/R₀ of around 5) as the vessel was deformed. The bubble then completely broke free of the confines of the vessel. At around the point at which the bubble left the vessel (approximately 35 μs after the start of the pulse), its oscillations became asymmetric (Fig 5C). The bubble fragments at this point, with the fragments continuing to move into the tissue.

We investigated the situations in which bubble extravasation occurs by observing the probability of extravasation at various ultrasound pressures. All microvessels from which bubbles extravasated were very small (around 5-10 μm in diameter). At mechanical indices (MI) that are generally considered safe and are close to the threshold for delivering drugs across the BBB (0.2-0.4), bubble extravasation was rare, with no instances observed at 0.2 and only one observed at 0.4 (Fig 5E). However, the probability of observing this increased significantly with increasing pressure, with bubble extravasation being the dominant response observed in slices exposed to an MI of 0.8. At an MI of 0.4 and 0.6, extravasation tended to occur in larger bubbles (Fig 5F). At 0.6, extravasating bubbles had diameters between 4.8 and 8.4 μm. At 0.4, the only bubble which extravasated was 6.1 μm in diameter. Very large bubbles, in excess of 10 μm, were rare, and did not extravasate.

Discussion

Drug delivery across the BBB using ultrasound and microbubbles has shown encouraging results in several clinical trials, yet the fundamental mechanisms behind the technique remain poorly understood due to a lack of direct observations of microbubbles within the microvessels of brain tissue. Here, we used acute brain slices from juvenile rats to directly observe microbubbles in microvessels when exposed to typical therapeutic ultrasound pulses, using high speed microscopy. We demonstrated that microbubbles can displace tissue beyond the wall of the vessel, extravasate from small microvessels, and oscillate asymmetrically under these conditions. Our study focused on small microvessels, such as capillaries, because this is where the BBB lines. Other microbubble dynamics were observed in tissue, such as microbubble-driven microstreaming, but were primarily in larger microvessels (S5 Fig and S8 Video).

Microbubbles exposed to 10-ms ultrasound pulses can displace their surroundings on both microsecond and millisecond timescales (Figs 2 and 4). In contrast to previous studies in mesentery which demonstrated vessel invagination was the dominant behavior on microsecond scales [27, 41], we showed the majority of vessels exhibited more significant distension in response to microbubbles. This may be due to the smaller vessels, different tissue type, and longer pulse lengths used here.

At such high frequencies, the mechanical properties of vessels are unknown, but these results demonstrate that the vessel wall was sufficiently elastic to oscillate periodically over microsecond timescales (Fig 2). This force may stimulate mechanotransduction pathways, as has been shown directly in isolated cell cultures [43]. Imaging at thousands of frames per second revealed displacement of tissues well beyond the vessel boundaries (Fig 4). Thus, while the focus on mechanisms have previously been on the vessel wall, one cannot ignore the mechanical stimuli that may be elicited in glial cells and neurons [44].

Microbubbles can extravasate from small microvessels under ultrasound parameters that have been used to deliver drugs across the BBB in animal and clinical studies [7, 45]. Microbubbles exited vessels and were pushed deep into the tissue by the primary radiation force (Fig 5). Vessel rupture has previously been described only at much higher pressures than those used here [46, 47]. Penetration of microbubbles deep into tissue has previously been observed in agarose gels, but only at higher PNPs in excess of 1 MPa_pk-neg at 1 MHz [48].

Microbubble extravasation could plausibly lead to localized red blood cell (RBC) extravasation. RBC extravasation has been observed in histological examinations of animals exposed to BBB opening treatments. The rate at which extravasation of microbubbles occurs in the slices correlates quite well with the relative rate of erythrocyte extravasation observed in in vivo studies of animals sonicated at these parameters, with a mechanical index of 0.2 and 0.4 being generally safe, but damage increasing at higher pressures [33, 47, 49–51].

Asymmetric oscillations of bubbles confined in small microvessels have been predicted theoretically and observed experimentally in artificial vessels. Our observations support those predictions and demonstrate this behavior can occur within real microvessels (Fig 2). This asymmetry in oscillations could alter the bubbles’ acoustic signatures, which may be significant in real-time therapy monitoring [52].

This study has several limitations when considering the relevance of its findings to a clinical or in vivo setting. Firstly, there was no blood flow or pressure in the vessels, key drivers of fluid extravasation, limiting our ability to observe dye leakage. The blood was replaced by SonoVue and dye; the presence of red blood cells in an in vivo setting may influence bubble dynamics [53]. While we attempted to avoid bubble-bubble interactions, bubbles may have been present outside the focal plane.

Because the tissue was kept in vitro, cellular activity was likely lower than in an in vivo setting. However, the viability of acute slices is typically around 6-12 hours [31], considerably longer than the <2 hours these experiments took place over (see S1 File).

Neonate animals were used due to the greater transparency and viability of their brain tissue over that of adults. While the tight junctions of the BBB are mostly developed in utero, many active transport mechanisms only develop in the weeks after birth [54]. While neonatal brain tissue provides a significantly more relevant environment to other tissue types used in previous studies, this may limit the potential of juvenile slices to fully investigate biological mechanisms of BBB disruption. Future studies may be able to refine this approach using techniques to reduce the depth of field such as pinholes or an increased numerical aperture.

This study presents direct observations of microbubble dynamics in locally-intact brain tissue, when exposed to ultrasound pulses typical of those used to deliver drugs across the BBB. We observed asymmetric bubble oscillations, and tissue deformations that extend well beyond the endothelial wall. Bubbles are not stationary in an ultrasound field and can instead travel long distances due to the primary radiation force, applying mechanical forces to its surroundings as it moves. Single bubbles were also shown to puncture the vessel wall and apply mechanical forces throughout the brain parenchyma. These results enhance our understanding of the potential mechanisms of BBB disruption in vivo, which is critical to better interpreting the findings of ongoing clinical trials and to building a new generation of safer and more robust drug delivery technologies.

Supporting information

S1 Fig. Experimental setups with optical and camera components.

A 250-μm-thick brain slice from a juvenile rat was immersed in artificial cerebrospinal fluid and placed between a light source and an objective. A focused ultrasound transducer emitted sound onto the brain slice, while videos were captured with one of two camera setups. (Left) In order to capture videos on the milliseconds timescale, light from an LED source was transmitted through a 40X objective and guided to a color camera at 1 frame per second (fps) and a high-speed camera at 5,581 fps. (Right) In order to capture videos on the microseconds time scale, light from a Cavitar 10-ns pulsed laser was transmitted through a 60X objective and guided into a Shimadzu camera at 5 or 10 million frames per second.

https://doi.org/10.1371/journal.pone.0310425.s001

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S2 Fig. Shape of a 30-cycle ultrasound pulse.

Ultrasound pulses were measured using a needle hydrophone (Top, Blue) without a box present and (Bottom, Red) with the box present. The pressure amplitudes were very similar with or without the box. The ramp-up phase was short and only very low-amplitude ultrasound was present beyond the expected 30-cycle pulse waveform.

https://doi.org/10.1371/journal.pone.0310425.s002

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S3 Fig. Triphenyltetrazolium chloride (TTC) staining of the brain slices.

The tissue is stained red when the tissue is undergoing aerobic respiration. The brain slices were stained with TTC (Left) at 0 hours, (Middle) 2 hours in aCSF, and (Right) 2 hours in PBS. The 0-hour time point refers to the time at which a typical experiment started, while the 2-hour time point refers to the time at which a typical experiment ended.

https://doi.org/10.1371/journal.pone.0310425.s003

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S4 Fig. Mean TTC intensity for the cortex.

Error bars are standard deviations between slices. * p=0.04, *** p=8.4x10^-5.

https://doi.org/10.1371/journal.pone.0310425.s004

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S5 Fig. Microstreaming in a brain microvessel.

The brain slice was exposed to a 0.4-MPa_pk-neg, 10-ms pulse and imaged at 5,581 fps. Streaming patterns surrounding the microbubbles were observed during sonication. The region of dye around the bubble was affected by streaming, resulting in a clearly-defined boundary.

https://doi.org/10.1371/journal.pone.0310425.s005

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S1 Video. Acoustic cavitation in a brain microvessel.

A microbubble in a microvessel was exposed to an ultrasonic pulse (center frequency: 1 MHz, peak-rarefactional pressure: 0.8 MPa_pk-neg, pulse length: 15 cycles) and imaged at ten million frames per second. The vessel wall distended at the same rate as the microbubble, expanding with the bubble but not invaginating as much when the bubble collapsed into itself. At 16.1 μs, an asymmetric collapse was observed in the shape of a ’figure-eight’. Frames and a streak image of this video are presented in Fig 2A.

https://doi.org/10.1371/journal.pone.0310425.s006

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S2 Video. Asymmetric oscillations in a brain microvessel.

Two microbubbles were exposed to an ultrasonic pulse (center frequency: 1 MHz, peak-rarefactional pressure: 0.8 MPa_pk-neg, pulse length: 15 cycles). The axial radius of one of the microbubbles was always larger than the radial radius. Frames of this video are presented in Fig 2B.

https://doi.org/10.1371/journal.pone.0310425.s007

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S3 Video. Microbubble coalescence in a brain microvessel.

Two microbubbles in a microvessel were exposed to an ultrasonic pulse (center frequency: 1 MHz, peak-rarefactional pressure: 0.8 MPa_pk-neg, pulse length: 15 cycles) and imaged at ten million frames per second. The bubbles were first observed spatially separated, but then coalesced into a single bubble at a time point between 5.2 and 6.0 μs. Frames of this video are presented in Fig 3.

https://doi.org/10.1371/journal.pone.0310425.s008

(AVI)

S4 Video. Microbubble movement within a brain microvessel.

A microbubble in a microvessel was exposed to a 0.6-MPa_pk-neg, 10-ms pulse. The microbubble generally moved in the direction of wave propagation (left to right), but its motion was very erratic. A frame of this video and the bubble’s travel path are presented in Fig 2C.

https://doi.org/10.1371/journal.pone.0310425.s009

(AVI)

S5 Video. A microbubble applying mechanical stress beyond the brain microvessel.

A microbubble exposed to ultrasonic pulses (center frequency: 1 MHz, pulse length: 10,000 cycles, PNP: 0.2 MPa_pk-neg) produced tissue deformations well beyond the vessel wall. The video was acquired at 5,581 frames per second. Ultrasound propagated from left to right. A frame and deformation maps for this video are presented in Fig 4.

https://doi.org/10.1371/journal.pone.0310425.s010

(AVI)

S6 Video. Microbubble extravasation on the millisecond time scale.

Extravasation of a microbubble during exposure to a 0.4-MPa_pk-neg, 10-ms pulse, imaged at 5,581 fps. Here, the bubble traveled up the vessel before exiting the vessel and traveling through the brain parenchyma. Frames of this video are presented in Fig 5B.

https://doi.org/10.1371/journal.pone.0310425.s011

(AVI)

S7 Video. Microbubble extravasation on the microsecond time scale.

Video showing extravasation of a microbubble exposed to a 1 MPa_pk-neg, 50-cycle pulse, imaged at 5 Mfps. Initially, the vessel became significantly distended and the bubble oscillation amplitude increased. The bubble then began transitioning out of the vessel and experienced highly asymmetric oscillations. The bubble then continued to penetrate into the tissue. There was a second bubble nearby which did not extravasate. Frames of this video are presented in Fig 5C.

https://doi.org/10.1371/journal.pone.0310425.s012

(AVI)

S8 Video. Microstreaming in a brain microvessel.

Microbubbles in a microvessel were exposed to an ultrasonic pulse (center frequency: 1 MHz, peak-rarefactional pressure: 0.4 MPa_pk-neg, pulse length: 10 ms) and imaged at 5,581 fps. Streaming was observed around both microbubbles in the vessel. The region of dye around the bubbles were affected by the streaming and had a clearly-defined boundary. Frames of this video are presented in S5 Fig.

https://doi.org/10.1371/journal.pone.0310425.s013

(AVI)

S1 Table. Record of microbubbles that did or did not extravasate from microbubbles.

This data set was used in Fig 5.

https://doi.org/10.1371/journal.pone.0310425.s014

(DOCX)

S1 File.

https://doi.org/10.1371/journal.pone.0310425.s015

(PDF)

References

1. Lipsman N, Meng Y, Bethune AJ, Huang Y, Lam B, Masellis M, et al. Blood–brain barrier opening in Alzheimer’s disease using MR-guided focused ultrasound. Nat Commun. 2018;9: 2336. pmid:30046032
- View Article
- PubMed/NCBI
- Google Scholar
2. Rezai AR, D’Haese P-F, Finomore V, Carpenter J, Ranjan M, Wilhelmsen K, et al. Ultrasound Blood–Brain Barrier Opening and Aducanumab in Alzheimer’s Disease. N Engl J Med. 2024;390: 55–62. pmid:38169490
- View Article
- PubMed/NCBI
- Google Scholar
3. Mainprize T, Lipsman N, Huang Y, Meng Y, Bethune A, Ironside S, et al. Blood-Brain Barrier Opening in Primary Brain Tumors with Non-invasive MR-Guided Focused Ultrasound: A Clinical Safety and Feasibility Study. Sci Rep. 2019;9: 1–7. pmid:30674905
- View Article
- PubMed/NCBI
- Google Scholar
4. Meng Y, Reilly RM, Pezo RC, Trudeau M, Sahgal A, Singnurkar A, et al. MR-guided focused ultrasound enhances delivery of trastuzumab to Her2-positive brain metastases. Sci Transl Med. 2021;13: 1–9. pmid:34644145
- View Article
- PubMed/NCBI
- Google Scholar
5. Carpentier A, Stupp R, Sonabend AM, Dufour H, Chinot O, Mathon B, et al. Repeated blood–brain barrier opening with a nine-emitter implantable ultrasound device in combination with carboplatin in recurrent glioblastoma: a phase I/II clinical trial. Nat Commun. 2024;15: 1–12. pmid:38396134
- View Article
- PubMed/NCBI
- Google Scholar
6. Abrahao A, Meng Y, Llinas M, Huang Y, Hamani C, Mainprize T, et al. First-in-human trial of blood–brain barrier opening in amyotrophic lateral sclerosis using MR-guided focused ultrasound. Nat Commun. 2019;10: 1–9. pmid:31558719
- View Article
- PubMed/NCBI
- Google Scholar
7. Carpentier A, Canney M, Vignot A, Reina V, Beccaria K, Horodyckid C, et al. Clinical trial of blood-brain barrier disruption by pulsed ultrasound. Sci Transl Med. 2016;8. pmid:27306666
- View Article
- PubMed/NCBI
- Google Scholar
8. Frinking P, Segers T, Luan Y, Tranquart F. Three Decades of Ultrasound Contrast Agents: A Review of the Past, Present and Future Improvements. Ultrasound Med Biol. 2020;46: 892–908. pmid:31941587
- View Article
- PubMed/NCBI
- Google Scholar
9. Hynynen K, McDannold N, Vykhodtseva N, Jolesz FA. Noninvasive MR Imaging–guided Focal Opening of the Blood-Brain Barrier in Rabbits. Radiology. 2001;220: 640–646. pmid:11526261
- View Article
- PubMed/NCBI
- Google Scholar
10. Kooiman K, Vos HJ, Versluis M, De Jong N. Acoustic behavior of microbubbles and implications for drug delivery. Adv Drug Deliv Rev. 2014;72: 28–48. pmid:24667643
- View Article
- PubMed/NCBI
- Google Scholar
11. Kokhuis TJA, Luan Y, Mastik F, Beurskens RHSH, Versluis M, De Jong N. Segmented high speed imaging of vibrating microbubbles during long ultrasound pulses. IEEE Int Ultrason Symp IUS. 2012; 1343–1346.
- View Article
- Google Scholar
12. Dollet B, van der Meer SM, Garbin V, de Jong N, Lohse D, Versluis M. Nonspherical Oscillations of Ultrasound Contrast Agent Microbubbles. Ultrasound Med Biol. 2008;34: 1465–1473. pmid:18450362
- View Article
- PubMed/NCBI
- Google Scholar
13. Prentice P, Cuschieri A, Dholakia K, Prausnitz M, Campbell P. Membrane disruption by optically controlled microbubble cavitation. Nat Phys. 2005;1: 107–110.
- View Article
- Google Scholar
14. Postema M, Van Wamel A, Lancée CT, De Jong N. Ultrasound-induced encapsulated microbubble phenomena. Ultrasound Med Biol. 2004;30: 827–840. pmid:15219962
- View Article
- PubMed/NCBI
- Google Scholar
15. Bezer JH, Koruk H, Rowlands CJ, Choi JJ. Elastic Deformation of Soft Tissue-Mimicking Materials Using a Single Microbubble and Acoustic Radiation Force. Ultrasound Med Biol. 2020;46: 3327–3338. pmid:32919812
- View Article
- PubMed/NCBI
- Google Scholar
16. Dayton PA, Allen JS, Ferrara KW. The magnitude of radiation force on ultrasound contrast agents. J Acoust Soc Am. 2002;112: 2183–2192. pmid:12430830
- View Article
- PubMed/NCBI
- Google Scholar
17. Sheikov N, McDannold N, Sharma S, Hynynen K. Effect of focused ultrasound applied with an ultrasound contrast agent on the tight junctional integrity of the brain microvascular endothelium. Ultrasound Med Biol. 2008;34: 1093–1104. pmid:18378064
- View Article
- PubMed/NCBI
- Google Scholar
18. Hynynen K, McDannold N, Sheikov NA, Jolesz FA, Vykhodtseva N. Local and reversible blood-brain barrier disruption by noninvasive focused ultrasound at frequencies suitable for trans-skull sonications. Neuroimage. 2005;24: 12–20. pmid:15588592
- View Article
- PubMed/NCBI
- Google Scholar
19. Sheikov N, McDannold N, Vykhodtseva N, Jolesz F, Hynynen K. Cellular mechanisms of the blood-brain barrier opening induced by ultrasound in presence of microbubbles. Ultrasound Med Biol. 2004;30: 979–989. pmid:15313330
- View Article
- PubMed/NCBI
- Google Scholar
20. Pandit R, Koh WK, Sullivan RKP, Palliyaguru T, Parton RG, Götz J. Role for caveolin-mediated transcytosis in facilitating transport of large cargoes into the brain via ultrasound. J Control Release. 2020;327: 667–675. pmid:32918963
- View Article
- PubMed/NCBI
- Google Scholar
21. McDannold N, Zhang Y, Vykhodtseva N. Blood-Brain Barrier Disruption and Vascular Damage Induced by Ultrasound Bursts Combined with Microbubbles can be Influenced by Choice of Anesthesia Protocol. Ultrasound Med Biol. 2011;37: 1259–1270. pmid:21645965
- View Article
- PubMed/NCBI
- Google Scholar
22. Burgess A, Shah K, Hough O, Hynynen K. Focused ultrasound-mediated drug delivery through the blood-brain barrier. Expert Rev Neurother. 2015;15: 477–491. pmid:25936845
- View Article
- PubMed/NCBI
- Google Scholar
23. Helfield B. A Review of Phospholipid Encapsulated Ultrasound Contrast Agent Microbubble Physics. Ultrasound Med Biol. 2019;45: 282–300. pmid:30413335
- View Article
- PubMed/NCBI
- Google Scholar
24. Faez T, Skachkov I, Versluis M, Kooiman K, de Jong N. In Vivo Characterization of Ultrasound Contrast Agents: Microbubble Spectroscopy in a Chicken Embryo. Ultrasound Med Biol. 2012;38: 1608–1617. pmid:22766113
- View Article
- PubMed/NCBI
- Google Scholar
25. Schneider M, Broillet A, Tardy I, Pochon S, Bussat P, Bettinger T, et al. Use of Intravital Microscopy to Study the Microvascular Behavior of Microbubble-Based Ultrasound Contrast Agents. Microcirculation. 2012;19: 245–259. pmid:22211713
- View Article
- PubMed/NCBI
- Google Scholar
26. Caskey CF, Stieger SM, Qin S, Dayton PA, Ferrara KW. Direct observations of ultrasound microbubble contrast agent interaction with the microvessel wall. J Acoust Soc Am. 2007;122: 1191–1200. pmid:17672665
- View Article
- PubMed/NCBI
- Google Scholar
27. Chen H, Kreider W, Brayman AA, Bailey MR, Matula TJ. Blood vessel deformations on microsecond time scales by ultrasonic cavitation. Phys Rev Lett. 2011;106: 1–4. pmid:21405276
- View Article
- PubMed/NCBI
- Google Scholar
28. McDannold N, Vykhodtseva N, Hynynen K. Targeted disruption of the blood-brain barrier with focused ultrasound: Association with cavitation activity. Phys Med Biol. 2006;51: 793–807. pmid:16467579
- View Article
- PubMed/NCBI
- Google Scholar
29. Liu HL, Fan CH, Ting CY, Yeh CK. Combining microbubbles and ultrasound for drug delivery to brain tumors: Current progress and overview. Theranostics. 2014;4: 432–444. pmid:24578726
- View Article
- PubMed/NCBI
- Google Scholar
30. Kinoshita M, McDannold N, Jolesz FA, Hynynen K. Noninvasive localized delivery of Herceptin to the mouse brain by MRI-guided focused ultrasound-induced blood-brain barrier disruption. Proc Natl Acad Sci U S A. 2006;103: 11719–11723. pmid:16868082
- View Article
- PubMed/NCBI
- Google Scholar
31. Buskila Y, Breen PP, Tapson J, Van Schaik A, Barton M, Morley JW. Extending the viability of acute brain slices. Sci Rep. 2014;4: 4–10. pmid:24930889
- View Article
- PubMed/NCBI
- Google Scholar
32. Kozlov AS, Angulo MC, Audinat E, Charpak S. Target cell-specific modulation of neuronal activity by astrocytes. Proc Natl Acad Sci. 2006;103. pmid:16782808
- View Article
- PubMed/NCBI
- Google Scholar
33. Morse S V, Pouliopoulos AN, Chan TG, Copping MMJ, Lin J, Long MNJ, et al. Rapid Short-pulse Ultrasound Delivers Drugs Uniformly across the Murine Blood-Brain Barrier with Negligible Disruption. Radiology. 2019. pmid:30912718
- View Article
- PubMed/NCBI
- Google Scholar
34. Manuel TJ, Sigona MK, Phipps MA, Kusunose J, Luo H, Yang PF, et al. Small volume blood-brain barrier opening in macaques with a 1 MHz ultrasound phased array. J Control Release. 2023;363: 707–720. pmid:37827222
- View Article
- PubMed/NCBI
- Google Scholar
35. Yang FY, Fu WM, Yang R Sen, Liou HC, Kang KH, Lin WL. Quantitative Evaluation of Focused Ultrasound with a Contrast Agent on Blood-Brain Barrier Disruption. Ultrasound Med Biol. 2007;33: 1421–1427. pmid:17561334
- View Article
- PubMed/NCBI
- Google Scholar
36. Lapin NA, Gill K, Shah BR, Chopra R. Consistent opening of the blood brain barrier using focused ultrasound with constant intravenous infusion of microbubble agent. Sci Rep. 2020;10: 1–11. pmid:33024157
- View Article
- PubMed/NCBI
- Google Scholar
37. Chu PC, Chai WY, Tsai CH, Kang ST, Yeh CK, Liu HL. Focused Ultrasound-Induced Blood-Brain Barrier Opening: Association with Mechanical Index and Cavitation Index Analyzed by Dynamic Contrast-Enhanced Magnetic-Resonance Imaging. Sci Rep. 2016;6: 1–13. pmid:27630037
- View Article
- PubMed/NCBI
- Google Scholar
38. Blaber J, Adair B, Antoniou A. Ncorr: Open-Source 2D Digital Image Correlation Matlab Software. Exp Mech. 2015;55: 1105–1122.
- View Article
- Google Scholar
39. Duda RO, Hart PE. Use of the Hough Transformation to Detect Lines and Curves in Pictures. Commun ACM. 1972;15: 11–15.
- View Article
- Google Scholar
40. Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, et al. Fiji: An open-source platform for biological-image analysis. Nat Methods. 2012;9: 676–682. pmid:22743772
- View Article
- PubMed/NCBI
- Google Scholar
41. Chen H, Brayman AA, Matula TJ. Characteristic microvessel relaxation timescales associated with ultrasound-activated microbubbles. Appl Phys Lett. 2012;101. pmid:23152641
- View Article
- PubMed/NCBI
- Google Scholar
42. Cleve S, Inserra C, Prentice P. Contrast Agent Microbubble Jetting during Initial Interaction with 200-kHz Focused Ultrasound. Ultrasound Med Biol. 2019;45: 3075–3080. pmid:31477370
- View Article
- PubMed/NCBI
- Google Scholar
43. De Cock I, Zagato E, Braeckmans K, Luan Y, de Jong N, De Smedt SC, et al. Ultrasound and microbubble mediated drug delivery: acoustic pressure as determinant for uptake via membrane pores or endocytosis. J Control Release. 2015;197: 20–28. pmid:25449801
- View Article
- PubMed/NCBI
- Google Scholar
44. Tyler WJ. The mechanobiology of brain function. Nat Rev Neurosci. 2012;13: 867–878. pmid:23165263
- View Article
- PubMed/NCBI
- Google Scholar
45. Chen H, Konofagou EE. The size of blood-brain barrier opening induced by focused ultrasound is dictated by the acoustic pressure. J Cereb Blood Flow Metab. 2014;34: 1197–1204. pmid:24780905
- View Article
- PubMed/NCBI
- Google Scholar
46. Chen H, Brayman AA, Bailey MR, Matula TJ. Blood vessel rupture by cavitation. Urol Res. 2010;38: 321–326. pmid:20680255
- View Article
- PubMed/NCBI
- Google Scholar
47. Price RJ, Skyba DM, Kaul S, Skalak TC. Delivery of colloidal particles and red blood cells to tissue through microvessel ruptures created by targeted microbubble destruction with ultrasound. Circulation. 1998;98: 1264–1267. pmid:9751673
- View Article
- PubMed/NCBI
- Google Scholar
48. Caskey CF, Qin S, Dayton PA, Ferrara KW. Microbubble tunneling in gel phantoms. J Acoust Soc Am. 2009;125: EL183–EL189. pmid:19425620
- View Article
- PubMed/NCBI
- Google Scholar
49. Shin J, Kong C, Cho JS, Lee J, Koh CS, Yoon MS, et al. Focused ultrasound-mediated noninvasive blood-brain barrier modulation: Preclinical examination of efficacy and safety in various sonication parameters. Neurosurg Focus. 2018;44: 1–10. pmid:29385915
- View Article
- PubMed/NCBI
- Google Scholar
50. Fan CH, Chang EL, Ting CY, Lin YC, Liao EC, Huang CY, et al. Folate-conjugated gene-carrying microbubbles with focused ultrasound for concurrent blood-brain barrier opening and local gene delivery. Biomaterials. 2016;106: 46–57. pmid:27544926
- View Article
- PubMed/NCBI
- Google Scholar
51. Skyba DM, Price RJ, Linka AZ, Skalak TC, Kaul S. Direct in vivo visualization of intravascular destruction of microbubbles by ultrasound and its local effects on tissue. Circulation. 1998;98: 290–293. pmid:9711932
- View Article
- PubMed/NCBI
- Google Scholar
52. Hosseinkhah N, Goertz DE, Hynynen K. Microbubbles and blood-brain barrier opening: A numerical study on acoustic emissions and wall stress predictions. IEEE Trans Biomed Eng. 2015;62: 1293–1304. pmid:25546853
- View Article
- PubMed/NCBI
- Google Scholar
53. Masuda K, Nakamoto R, Watarai N, Ren K, Taguchi Y, Kozuka T, et al. Effect of existence of red blood cells in trapping performance of microbubbles by acoustic radiation force. Jpn J Appl Phys. 2011;50.
- View Article
- Google Scholar
54. Schmitt G, Parrott N, Prinssen E, Barrow P. The great barrier belief: The blood – brain barrier and considerations for juvenile toxicity studies. Reprod Toxicol. 2017;72: 129–135. pmid:28627392
- View Article
- PubMed/NCBI
- Google Scholar

[ref1] 1. Lipsman N, Meng Y, Bethune AJ, Huang Y, Lam B, Masellis M, et al. Blood–brain barrier opening in Alzheimer’s disease using MR-guided focused ultrasound. Nat Commun. 2018;9: 2336. pmid:30046032
View Article
PubMed/NCBI
Google Scholar

[2] View Article

[3] PubMed/NCBI

[4] Google Scholar

[ref2] 2. Rezai AR, D’Haese P-F, Finomore V, Carpenter J, Ranjan M, Wilhelmsen K, et al. Ultrasound Blood–Brain Barrier Opening and Aducanumab in Alzheimer’s Disease. N Engl J Med. 2024;390: 55–62. pmid:38169490
View Article
PubMed/NCBI
Google Scholar

[6] View Article

[7] PubMed/NCBI

[8] Google Scholar

[ref3] 3. Mainprize T, Lipsman N, Huang Y, Meng Y, Bethune A, Ironside S, et al. Blood-Brain Barrier Opening in Primary Brain Tumors with Non-invasive MR-Guided Focused Ultrasound: A Clinical Safety and Feasibility Study. Sci Rep. 2019;9: 1–7. pmid:30674905
View Article
PubMed/NCBI
Google Scholar

[10] View Article

[11] PubMed/NCBI

[12] Google Scholar

[ref4] 4. Meng Y, Reilly RM, Pezo RC, Trudeau M, Sahgal A, Singnurkar A, et al. MR-guided focused ultrasound enhances delivery of trastuzumab to Her2-positive brain metastases. Sci Transl Med. 2021;13: 1–9. pmid:34644145
View Article
PubMed/NCBI
Google Scholar

[14] View Article

[15] PubMed/NCBI

[16] Google Scholar

[ref5] 5. Carpentier A, Stupp R, Sonabend AM, Dufour H, Chinot O, Mathon B, et al. Repeated blood–brain barrier opening with a nine-emitter implantable ultrasound device in combination with carboplatin in recurrent glioblastoma: a phase I/II clinical trial. Nat Commun. 2024;15: 1–12. pmid:38396134
View Article
PubMed/NCBI
Google Scholar

[18] View Article

[19] PubMed/NCBI

[20] Google Scholar

[ref6] 6. Abrahao A, Meng Y, Llinas M, Huang Y, Hamani C, Mainprize T, et al. First-in-human trial of blood–brain barrier opening in amyotrophic lateral sclerosis using MR-guided focused ultrasound. Nat Commun. 2019;10: 1–9. pmid:31558719
View Article
PubMed/NCBI
Google Scholar

[22] View Article

[23] PubMed/NCBI

[24] Google Scholar

[ref7] 7. Carpentier A, Canney M, Vignot A, Reina V, Beccaria K, Horodyckid C, et al. Clinical trial of blood-brain barrier disruption by pulsed ultrasound. Sci Transl Med. 2016;8. pmid:27306666
View Article
PubMed/NCBI
Google Scholar

[26] View Article

[27] PubMed/NCBI

[28] Google Scholar

[ref8] 8. Frinking P, Segers T, Luan Y, Tranquart F. Three Decades of Ultrasound Contrast Agents: A Review of the Past, Present and Future Improvements. Ultrasound Med Biol. 2020;46: 892–908. pmid:31941587
View Article
PubMed/NCBI
Google Scholar

[30] View Article

[31] PubMed/NCBI

[32] Google Scholar

[ref9] 9. Hynynen K, McDannold N, Vykhodtseva N, Jolesz FA. Noninvasive MR Imaging–guided Focal Opening of the Blood-Brain Barrier in Rabbits. Radiology. 2001;220: 640–646. pmid:11526261
View Article
PubMed/NCBI
Google Scholar

[34] View Article

[35] PubMed/NCBI

[36] Google Scholar

[ref10] 10. Kooiman K, Vos HJ, Versluis M, De Jong N. Acoustic behavior of microbubbles and implications for drug delivery. Adv Drug Deliv Rev. 2014;72: 28–48. pmid:24667643
View Article
PubMed/NCBI
Google Scholar

[38] View Article

[39] PubMed/NCBI

[40] Google Scholar

[ref11] 11. Kokhuis TJA, Luan Y, Mastik F, Beurskens RHSH, Versluis M, De Jong N. Segmented high speed imaging of vibrating microbubbles during long ultrasound pulses. IEEE Int Ultrason Symp IUS. 2012; 1343–1346.
View Article
Google Scholar

[42] View Article

[43] Google Scholar

[ref12] 12. Dollet B, van der Meer SM, Garbin V, de Jong N, Lohse D, Versluis M. Nonspherical Oscillations of Ultrasound Contrast Agent Microbubbles. Ultrasound Med Biol. 2008;34: 1465–1473. pmid:18450362
View Article
PubMed/NCBI
Google Scholar

[45] View Article

[46] PubMed/NCBI

[47] Google Scholar

[ref13] 13. Prentice P, Cuschieri A, Dholakia K, Prausnitz M, Campbell P. Membrane disruption by optically controlled microbubble cavitation. Nat Phys. 2005;1: 107–110.
View Article
Google Scholar

[49] View Article

[50] Google Scholar

[ref14] 14. Postema M, Van Wamel A, Lancée CT, De Jong N. Ultrasound-induced encapsulated microbubble phenomena. Ultrasound Med Biol. 2004;30: 827–840. pmid:15219962
View Article
PubMed/NCBI
Google Scholar

[52] View Article

[53] PubMed/NCBI

[54] Google Scholar

[ref15] 15. Bezer JH, Koruk H, Rowlands CJ, Choi JJ. Elastic Deformation of Soft Tissue-Mimicking Materials Using a Single Microbubble and Acoustic Radiation Force. Ultrasound Med Biol. 2020;46: 3327–3338. pmid:32919812
View Article
PubMed/NCBI
Google Scholar

[56] View Article

[57] PubMed/NCBI

[58] Google Scholar

[ref16] 16. Dayton PA, Allen JS, Ferrara KW. The magnitude of radiation force on ultrasound contrast agents. J Acoust Soc Am. 2002;112: 2183–2192. pmid:12430830
View Article
PubMed/NCBI
Google Scholar

[60] View Article

[61] PubMed/NCBI

[62] Google Scholar

[ref17] 17. Sheikov N, McDannold N, Sharma S, Hynynen K. Effect of focused ultrasound applied with an ultrasound contrast agent on the tight junctional integrity of the brain microvascular endothelium. Ultrasound Med Biol. 2008;34: 1093–1104. pmid:18378064
View Article
PubMed/NCBI
Google Scholar

[64] View Article

[65] PubMed/NCBI

[66] Google Scholar

[ref18] 18. Hynynen K, McDannold N, Sheikov NA, Jolesz FA, Vykhodtseva N. Local and reversible blood-brain barrier disruption by noninvasive focused ultrasound at frequencies suitable for trans-skull sonications. Neuroimage. 2005;24: 12–20. pmid:15588592
View Article
PubMed/NCBI
Google Scholar

[68] View Article

[69] PubMed/NCBI

[70] Google Scholar

[ref19] 19. Sheikov N, McDannold N, Vykhodtseva N, Jolesz F, Hynynen K. Cellular mechanisms of the blood-brain barrier opening induced by ultrasound in presence of microbubbles. Ultrasound Med Biol. 2004;30: 979–989. pmid:15313330
View Article
PubMed/NCBI
Google Scholar

[72] View Article

[73] PubMed/NCBI

[74] Google Scholar

[ref20] 20. Pandit R, Koh WK, Sullivan RKP, Palliyaguru T, Parton RG, Götz J. Role for caveolin-mediated transcytosis in facilitating transport of large cargoes into the brain via ultrasound. J Control Release. 2020;327: 667–675. pmid:32918963
View Article
PubMed/NCBI
Google Scholar

[76] View Article

[77] PubMed/NCBI

[78] Google Scholar

[ref21] 21. McDannold N, Zhang Y, Vykhodtseva N. Blood-Brain Barrier Disruption and Vascular Damage Induced by Ultrasound Bursts Combined with Microbubbles can be Influenced by Choice of Anesthesia Protocol. Ultrasound Med Biol. 2011;37: 1259–1270. pmid:21645965
View Article
PubMed/NCBI
Google Scholar

[80] View Article

[81] PubMed/NCBI

[82] Google Scholar

[ref22] 22. Burgess A, Shah K, Hough O, Hynynen K. Focused ultrasound-mediated drug delivery through the blood-brain barrier. Expert Rev Neurother. 2015;15: 477–491. pmid:25936845
View Article
PubMed/NCBI
Google Scholar

[84] View Article

[85] PubMed/NCBI

[86] Google Scholar

[ref23] 23. Helfield B. A Review of Phospholipid Encapsulated Ultrasound Contrast Agent Microbubble Physics. Ultrasound Med Biol. 2019;45: 282–300. pmid:30413335
View Article
PubMed/NCBI
Google Scholar

[88] View Article

[89] PubMed/NCBI

[90] Google Scholar

[ref24] 24. Faez T, Skachkov I, Versluis M, Kooiman K, de Jong N. In Vivo Characterization of Ultrasound Contrast Agents: Microbubble Spectroscopy in a Chicken Embryo. Ultrasound Med Biol. 2012;38: 1608–1617. pmid:22766113
View Article
PubMed/NCBI
Google Scholar

[92] View Article

[93] PubMed/NCBI

[94] Google Scholar

[ref25] 25. Schneider M, Broillet A, Tardy I, Pochon S, Bussat P, Bettinger T, et al. Use of Intravital Microscopy to Study the Microvascular Behavior of Microbubble-Based Ultrasound Contrast Agents. Microcirculation. 2012;19: 245–259. pmid:22211713
View Article
PubMed/NCBI
Google Scholar

[96] View Article

[97] PubMed/NCBI

[98] Google Scholar

[ref26] 26. Caskey CF, Stieger SM, Qin S, Dayton PA, Ferrara KW. Direct observations of ultrasound microbubble contrast agent interaction with the microvessel wall. J Acoust Soc Am. 2007;122: 1191–1200. pmid:17672665
View Article
PubMed/NCBI
Google Scholar

[100] View Article

[101] PubMed/NCBI

[102] Google Scholar

[ref27] 27. Chen H, Kreider W, Brayman AA, Bailey MR, Matula TJ. Blood vessel deformations on microsecond time scales by ultrasonic cavitation. Phys Rev Lett. 2011;106: 1–4. pmid:21405276
View Article
PubMed/NCBI
Google Scholar

[104] View Article

[105] PubMed/NCBI

[106] Google Scholar

[ref28] 28. McDannold N, Vykhodtseva N, Hynynen K. Targeted disruption of the blood-brain barrier with focused ultrasound: Association with cavitation activity. Phys Med Biol. 2006;51: 793–807. pmid:16467579
View Article
PubMed/NCBI
Google Scholar

[108] View Article

[109] PubMed/NCBI

[110] Google Scholar

[ref29] 29. Liu HL, Fan CH, Ting CY, Yeh CK. Combining microbubbles and ultrasound for drug delivery to brain tumors: Current progress and overview. Theranostics. 2014;4: 432–444. pmid:24578726
View Article
PubMed/NCBI
Google Scholar

[112] View Article

[113] PubMed/NCBI

[114] Google Scholar

[ref30] 30. Kinoshita M, McDannold N, Jolesz FA, Hynynen K. Noninvasive localized delivery of Herceptin to the mouse brain by MRI-guided focused ultrasound-induced blood-brain barrier disruption. Proc Natl Acad Sci U S A. 2006;103: 11719–11723. pmid:16868082
View Article
PubMed/NCBI
Google Scholar

[116] View Article

[117] PubMed/NCBI

[118] Google Scholar

[ref31] 31. Buskila Y, Breen PP, Tapson J, Van Schaik A, Barton M, Morley JW. Extending the viability of acute brain slices. Sci Rep. 2014;4: 4–10. pmid:24930889
View Article
PubMed/NCBI
Google Scholar

[120] View Article

[121] PubMed/NCBI

[122] Google Scholar

[ref32] 32. Kozlov AS, Angulo MC, Audinat E, Charpak S. Target cell-specific modulation of neuronal activity by astrocytes. Proc Natl Acad Sci. 2006;103. pmid:16782808
View Article
PubMed/NCBI
Google Scholar

[124] View Article

[125] PubMed/NCBI

[126] Google Scholar

[ref33] 33. Morse S V, Pouliopoulos AN, Chan TG, Copping MMJ, Lin J, Long MNJ, et al. Rapid Short-pulse Ultrasound Delivers Drugs Uniformly across the Murine Blood-Brain Barrier with Negligible Disruption. Radiology. 2019. pmid:30912718
View Article
PubMed/NCBI
Google Scholar

[128] View Article

[129] PubMed/NCBI

[130] Google Scholar

[ref34] 34. Manuel TJ, Sigona MK, Phipps MA, Kusunose J, Luo H, Yang PF, et al. Small volume blood-brain barrier opening in macaques with a 1 MHz ultrasound phased array. J Control Release. 2023;363: 707–720. pmid:37827222
View Article
PubMed/NCBI
Google Scholar

[132] View Article

[133] PubMed/NCBI

[134] Google Scholar

[ref35] 35. Yang FY, Fu WM, Yang R Sen, Liou HC, Kang KH, Lin WL. Quantitative Evaluation of Focused Ultrasound with a Contrast Agent on Blood-Brain Barrier Disruption. Ultrasound Med Biol. 2007;33: 1421–1427. pmid:17561334
View Article
PubMed/NCBI
Google Scholar

[136] View Article

[137] PubMed/NCBI

[138] Google Scholar

[ref36] 36. Lapin NA, Gill K, Shah BR, Chopra R. Consistent opening of the blood brain barrier using focused ultrasound with constant intravenous infusion of microbubble agent. Sci Rep. 2020;10: 1–11. pmid:33024157
View Article
PubMed/NCBI
Google Scholar

[140] View Article

[141] PubMed/NCBI

[142] Google Scholar

[ref37] 37. Chu PC, Chai WY, Tsai CH, Kang ST, Yeh CK, Liu HL. Focused Ultrasound-Induced Blood-Brain Barrier Opening: Association with Mechanical Index and Cavitation Index Analyzed by Dynamic Contrast-Enhanced Magnetic-Resonance Imaging. Sci Rep. 2016;6: 1–13. pmid:27630037
View Article
PubMed/NCBI
Google Scholar

[144] View Article

[145] PubMed/NCBI

[146] Google Scholar

[ref38] 38. Blaber J, Adair B, Antoniou A. Ncorr: Open-Source 2D Digital Image Correlation Matlab Software. Exp Mech. 2015;55: 1105–1122.
View Article
Google Scholar

[148] View Article

[149] Google Scholar

[ref39] 39. Duda RO, Hart PE. Use of the Hough Transformation to Detect Lines and Curves in Pictures. Commun ACM. 1972;15: 11–15.
View Article
Google Scholar

[151] View Article

[152] Google Scholar

[ref40] 40. Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, et al. Fiji: An open-source platform for biological-image analysis. Nat Methods. 2012;9: 676–682. pmid:22743772
View Article
PubMed/NCBI
Google Scholar

[154] View Article

[155] PubMed/NCBI

[156] Google Scholar

[ref41] 41. Chen H, Brayman AA, Matula TJ. Characteristic microvessel relaxation timescales associated with ultrasound-activated microbubbles. Appl Phys Lett. 2012;101. pmid:23152641
View Article
PubMed/NCBI
Google Scholar

[158] View Article

[159] PubMed/NCBI

[160] Google Scholar

[ref42] 42. Cleve S, Inserra C, Prentice P. Contrast Agent Microbubble Jetting during Initial Interaction with 200-kHz Focused Ultrasound. Ultrasound Med Biol. 2019;45: 3075–3080. pmid:31477370
View Article
PubMed/NCBI
Google Scholar

[162] View Article

[163] PubMed/NCBI

[164] Google Scholar

[ref43] 43. De Cock I, Zagato E, Braeckmans K, Luan Y, de Jong N, De Smedt SC, et al. Ultrasound and microbubble mediated drug delivery: acoustic pressure as determinant for uptake via membrane pores or endocytosis. J Control Release. 2015;197: 20–28. pmid:25449801
View Article
PubMed/NCBI
Google Scholar

[166] View Article

[167] PubMed/NCBI

[168] Google Scholar

[ref44] 44. Tyler WJ. The mechanobiology of brain function. Nat Rev Neurosci. 2012;13: 867–878. pmid:23165263
View Article
PubMed/NCBI
Google Scholar

[170] View Article

[171] PubMed/NCBI

[172] Google Scholar

[ref45] 45. Chen H, Konofagou EE. The size of blood-brain barrier opening induced by focused ultrasound is dictated by the acoustic pressure. J Cereb Blood Flow Metab. 2014;34: 1197–1204. pmid:24780905
View Article
PubMed/NCBI
Google Scholar

[174] View Article

[175] PubMed/NCBI

[176] Google Scholar

[ref46] 46. Chen H, Brayman AA, Bailey MR, Matula TJ. Blood vessel rupture by cavitation. Urol Res. 2010;38: 321–326. pmid:20680255
View Article
PubMed/NCBI
Google Scholar

[178] View Article

[179] PubMed/NCBI

[180] Google Scholar

[ref47] 47. Price RJ, Skyba DM, Kaul S, Skalak TC. Delivery of colloidal particles and red blood cells to tissue through microvessel ruptures created by targeted microbubble destruction with ultrasound. Circulation. 1998;98: 1264–1267. pmid:9751673
View Article
PubMed/NCBI
Google Scholar

[182] View Article

[183] PubMed/NCBI

[184] Google Scholar

[ref48] 48. Caskey CF, Qin S, Dayton PA, Ferrara KW. Microbubble tunneling in gel phantoms. J Acoust Soc Am. 2009;125: EL183–EL189. pmid:19425620
View Article
PubMed/NCBI
Google Scholar

[186] View Article

[187] PubMed/NCBI

[188] Google Scholar

[ref49] 49. Shin J, Kong C, Cho JS, Lee J, Koh CS, Yoon MS, et al. Focused ultrasound-mediated noninvasive blood-brain barrier modulation: Preclinical examination of efficacy and safety in various sonication parameters. Neurosurg Focus. 2018;44: 1–10. pmid:29385915
View Article
PubMed/NCBI
Google Scholar

[190] View Article

[191] PubMed/NCBI

[192] Google Scholar

[ref50] 50. Fan CH, Chang EL, Ting CY, Lin YC, Liao EC, Huang CY, et al. Folate-conjugated gene-carrying microbubbles with focused ultrasound for concurrent blood-brain barrier opening and local gene delivery. Biomaterials. 2016;106: 46–57. pmid:27544926
View Article
PubMed/NCBI
Google Scholar

[194] View Article

[195] PubMed/NCBI

[196] Google Scholar

[ref51] 51. Skyba DM, Price RJ, Linka AZ, Skalak TC, Kaul S. Direct in vivo visualization of intravascular destruction of microbubbles by ultrasound and its local effects on tissue. Circulation. 1998;98: 290–293. pmid:9711932
View Article
PubMed/NCBI
Google Scholar

[198] View Article

[199] PubMed/NCBI

[200] Google Scholar

[ref52] 52. Hosseinkhah N, Goertz DE, Hynynen K. Microbubbles and blood-brain barrier opening: A numerical study on acoustic emissions and wall stress predictions. IEEE Trans Biomed Eng. 2015;62: 1293–1304. pmid:25546853
View Article
PubMed/NCBI
Google Scholar

[202] View Article

[203] PubMed/NCBI

[204] Google Scholar

[ref53] 53. Masuda K, Nakamoto R, Watarai N, Ren K, Taguchi Y, Kozuka T, et al. Effect of existence of red blood cells in trapping performance of microbubbles by acoustic radiation force. Jpn J Appl Phys. 2011;50.
View Article
Google Scholar

[206] View Article

[207] Google Scholar

[ref54] 54. Schmitt G, Parrott N, Prinssen E, Barrow P. The great barrier belief: The blood – brain barrier and considerations for juvenile toxicity studies. Reprod Toxicol. 2017;72: 129–135. pmid:28627392
View Article
PubMed/NCBI
Google Scholar

[209] View Article

[210] PubMed/NCBI

[211] Google Scholar

Figures

Abstract

Introduction

Methods

Slice preparation

Ultrasound

Image acquisition

Microbubble selection

Image processing

Image analysis

Results

Microsecond time-scale observations

Millisecond-time scale observations

Microbubble extravasation

Discussion

Supporting information

S1 Fig. Experimental setups with optical and camera components.

S2 Fig. Shape of a 30-cycle ultrasound pulse.

S3 Fig. Triphenyltetrazolium chloride (TTC) staining of the brain slices.

S4 Fig. Mean TTC intensity for the cortex.

S5 Fig. Microstreaming in a brain microvessel.

S1 Video. Acoustic cavitation in a brain microvessel.

S2 Video. Asymmetric oscillations in a brain microvessel.

S3 Video. Microbubble coalescence in a brain microvessel.

S4 Video. Microbubble movement within a brain microvessel.

S5 Video. A microbubble applying mechanical stress beyond the brain microvessel.

S6 Video. Microbubble extravasation on the millisecond time scale.

S7 Video. Microbubble extravasation on the microsecond time scale.

S8 Video. Microstreaming in a brain microvessel.

S1 Table. Record of microbubbles that did or did not extravasate from microbubbles.

S1 File.

References