Shougang Wang

Microbubble-Size Dependence of Focused Ultrasound-Induced Blood–Brain Barrier Opening in Mice In Vivo

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 ¿m 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 ¿m 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 ¿m bubbles and between 0.15 and 0.30 MPa in the 4-5 ¿m case. At every pressure studied, the fluorescence was greater with the 4-5 ¿m than with the 1-2 ¿m bubbles. At 0.61 MPa, in the 1-2 ¿m 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.

Molecules of Various Pharmacologically-Relevant Sizes Can Cross the Ultrasound-Induced Blood-Brain Barrier Opening in vivo

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 agents 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: 1.525MHz, peak-rarefactional pressure in situ: 570 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 with 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. (E-mail: ek2191@columbia.edu).

A composite high-frame-rate system for clinical cardiovascular imaging

High frame-rate ultrasound RF data acquisition has been proved to be critical for novel cardiovascular imaging techniques, such as high-precision myocardial elastography, pulse wave imaging (PWI), and electromechanical wave imaging (EWI). To overcome the frame-rate limitations on standard clinical ultrasound systems, we developed an automated method for multi-sector ultrasound imaging through retrospective electrocardiogram (ECG) gating on a clinically used open architecture system. The method achieved both high spatial (64 beam density) and high temporal resolution (frame rate of 481 Hz) at an imaging depth up to 11 cm and a 100% field of view in a single breath-hold duration. Full-view imaging of the left ventricle and the abdominal aorta of healthy human subjects was performed using the proposed technique in vivo. ECG and ultrasound RF signals were simultaneously acquired on a personal computer (PC). Composite, full-view frames both in RF- and B-mode were reconstructed through retrospective combination of seven small (20%) juxtaposed sectors using an ECG-gating technique. The axial displacement of the left ventricle, in both long-axis and short-axis views, and that of the abdominal aorta, in a long-axis view, were estimated using a RF-based speckle tracking technique. The electromechanical wave and the pulse wave propagation were imaged in a cineloop using the proposed imaging technique. Abnormal patterns of such wave propagation can serve as indicators of early cardiovascular disease. This clinical system could thus expand the range of applications in cardiovascular elasticity imaging for quantitative, noninvasive diagnosis of myocardial ischemia or infarction, arrhythmia, abdominal aortic aneurysms, and early-stage atherosclerosis.

Noninvasive and Transient Blood-Brain Barrier Opening in the Hippocampus of Alzheimer's Double Transgenic Mice Using Focused Ultrasound

The spatio-temporal nature of focused ultrasound-induced blood-brain barrier (BBB) opening as a brain drug delivery method was investigated in Alzheimers disease model mice. The left hippocampus of transgenic (APP/PS1, n = 3) and nontransgenic (n = 3) mice was sonicated (frequency: 1.525 MHz, peak-negative pressure: 600 kPa, pulse length: 20 ms, duty cycle: 20%, duration: 1 min) in vivo, through their intact skin and skull, after intravenous injection of microbubbles (SonoVue®5; 25 μl). Sequential, high-field MR images (9.4 Tesla) were acquired before and after injection of gadolinium (Omniscan™ 0.75 ml, molecular weight: 573.7 Da) on two separate days for each mouse. Gadolinium deposits through the ultrasound-induced BBB opening in the left hippocampus revealed significant contrast-enhancement in the MRI. On the following day, MRI revealed significant BBB closure within the same region. However, the BBB opening extent and BBB closing timeline varied in different regions within the same sonicated location. This indicates that opening and closing were dependent on the brain region targeted. No significant difference in BBB opening or closing behaviors was observed between the APP/PS1 and the nontransgenic mice. In conclusion, a BBB-impermeable molecule was noninvasively, transiently and reproducibly delivered to the hippocampus of Alzheimers APP/PS1 mice.

10B-6 A Composite Imaging Technique for High Frame-Rate and Full-View Cardiovascular Ultrasound and Elasticity Imaging

The frame-rate of ultrasound radio-frequency (RF) data acquisition is critical for imaging of the pulse wave and electromechanical wave propagation in cardiovascular tissues as well as improving the strain estimation. Therefore, an automated method had been developed to overcome the frame-rate limitations on standard systems by retrospective multi-sector signal acquisition through an electrocardiogram (ECG) gating technique. The method achieved a frame rate of 481 Hz at a 100% field of view, 64 line densities and an imaging depth of 11 cm. The composite full-view images were reconstructed by retrospectively combining seven small-sector RF frames using the ECG-gating technique. The axial displacements of both long-axis and short-axis views of a human left ventricle and a long-axis view of the abdominal aorta were calculated using an RF based speckle-tracking technique comprising ID cross-correlation methods in a 2D search (window size of 6.9 mm and overlap of 80%). Several sequences of electromechanical waves propagating in a left ventricular long-axis and short-axis view, and long-axis view of abdominal aorta were imaged at high frame rates. Currently, the method was implemented on an Ultrasonix RP system (Ultrasonix Medical Corp. Richmond, Canada) and could be potentially implemented on other clinical systems.

Pulse wave imaging of human abdominal aortas in vivo

Vascular diseases (e.g., abdominal aortic aneurysm or, AAA) lead to changes in the regional aortic wall mechanical properties. Pulse-Wave Imaging (PWI) was previously developed by our group to map the pulse-wave propagation along the abdominal aorta of mice in vivo. In this study, the feasibility of PWI with real-time scanning is shown in human abdominal aortas in vivo. The abdominal aortas of five normal subjects and one AAA subject were scanned. A Sonix RP system (Ultrasonix Medical Corp., Burnaby, Canada) was employed with a phased array at 3.3 MHz. The beam density of the 2-D echograms was reduced to 32 beams in order to obtain a high frame rate of 180-260 Hz. The real-time scanning reduces the artifacts from respiration and transducer motion. The velocities of the aortic wall were estimated using RF-based speckle tracking. The sequences of PWI images visually depicted the propagation of the pulse wave along the aortic wall. The regional pulse-wave velocity (PWV) was measured and used to estimate the Youngs modulus of the aortic wall. In healthy volunteers (n=5), the propagation was relatively uniform, with a correlation coefficient of 0.97 plusmn 0.01 and a PWV of 3.73 plusmn 0.19 m/s. The Youngs modulus of the aortic wall was 79 plusmn 10 kPa. In the aneurysmal aorta, the propagation of the pulse wave was relatively nonuniform with lower correlation coefficients (r=0.65). The PWV and Youngs modulus of the aneurysmal aorta were both found to be higher than in the normal case. The PWI technique was successfully implemented in both normal and aneurysmal human abdominal aortas and shown to provide regional information on the mechanical properties of the aortic wall in vivo.

Chirp- and random-based coded ultrasonic excitation for localized blood-brain barrier opening

Chirp- and random-based coded excitation methods have been proposed to reduce standing wave formation and improve focusing of transcranial ultrasound. However, no clear evidence has been shown to support the benefits of these ultrasonic excitation sequences in vivo. This study evaluates the chirp and periodic selection of random frequency (PSRF) coded-excitation methods for opening the blood-brain barrier (BBB) in mice. Three groups of mice (n  =  15) were injected with polydisperse microbubbles and sonicated in the caudate putamen using the chirp/PSRF coded (bandwidth: 1.5–1.9 MHz, peak negative pressure: 0.52 MPa, duration: 30 s) or standard ultrasound (frequency: 1.5 MHz, pressure: 0.52 MPa, burst duration: 20 ms, duration: 5 min) sequences. T1-weighted contrast-enhanced MRI scans were performed to quantitatively analyze focused ultrasound induced BBB opening. The mean opening volumes evaluated from the MRI were mm3, mm3and mm3 for the chirp, random and regular sonications, respectively. The mean cavitation levels were V.s, V.s and V.s for the chirp, random and regular sonications, respectively. The chirp and PSRF coded pulsing sequences improved the BBB opening localization by inducing lower cavitation levels and smaller opening volumes compared to results of the regular sonication technique. Larger bandwidths were associated with more focused targeting but were limited by the frequency response of the transducer, the skull attenuation and the microbubbles optimal frequency range. The coded methods could therefore facilitate highly localized drug delivery as well as benefit other transcranial ultrasound techniques that use higher pressure levels and higher precision to induce the necessary bioeffects in a brain region while avoiding damage to the surrounding healthy tissue.

P4A-2 An In-Vivo Study of Frame Rate Optimization for Myocardial Elastography

In this paper, the requirement and optimization of the frame rate for myocardial elastography was investigated in normal mice and humans in vivo. Using a retrospective electrocardiogram (ECG) gating technique, the highest frame rate was 8 kHz and 481 Hz, respectively. Axial displacement and strain of myocardium were estimated using an RF speckle tracking method consisting of a 1-D kernel in a 2-D search. The frame rate was then decimated to study its effects on the image quality of myocardial elastography, in terms of elastographic signal-to-noise (SNRe) and correlation coefficient. Trade-offs between SNRe and effective frame rate were identified in the murine case. The optimum range of frame rate was found to be between 2000 and 2700 Hz, or equivalently, 250-350 frames per cardiac cycle (fpc). In the human case, the image quality increased monotonously with the frame rate. A frame rate higher than 480 Hz (i.e., 350 fpc) was thus required for both systole and diastole.

Identifying the inertial cavitation threshold in a vessel phantom using focused ultrasound and microbubbles.

Unveiling the mechanism behind the blood brain barrier using focused ultrasound (FUS) and microbubbles is essential for brain molecular delivery. Here, B‐mode imaging and rf signals were acquired to pinpoint the threshold for inertial cavitation during FUS with microbubbles. A cylindrical hole of 800 μm in diameter was generated inside a polyacrylamide gel to simulate a brain arterial vessel. Definity® (Lantheus Medical Imaging, USA) microbubbles with a 1.1–3.3 μm‐diameter and (1,2‐distearoyl‐sn‐glycero‐3‐phosphocholine) (DSPC) shelled microbubbles with a 1–2‐μm‐diameter were injected prior to sonication (frequency: 1.525 MHz; pulse length: 100 cycles; PRF: 1 kHz; pulse duration: 40 ms). The cavitation response was passively detected using a 7.5‐MHz single‐element transducer, confocal with the FUS transducer, and a one‐dimensional linear array transducer placed perpendicular to the FUS beam. The broadband spectral response of the acquired rf signals and the B‐mode images detected the occurrence and locati...

Pupil dilation and motor response elicitation by ultrasound neuromodulation

Focused ultrasound (FUS) neuromodulation has been previously proposed as a promising technique to drive neuronal activity. Here, we explored motor- and cognitive-related brain regions of mice by targeting specific brain structures using FUS neuromodulation in the mega-Hz range under a specific type of anesthesia. Contralateral motor responses were observed showing successful target specificity of the FUS neuromodulation achieved with 1.9 MHz. Higher acoustic pressures increased the success rate from 20% (at the threshold, 1.45 MPa) to 70% (1.79 MPa). The estimated latency measured by electromyography was 266 ± 37 ms. Pupil dilation was observed when neuromodulating regions in the brain covering the superior colliculus and other anxiety-related structures such as hippocampus and locus coeruleus. This study demonstrated the capability of FUS to modulate target specific regions in the brain including pupil dilation induced by FUS for the first time. Furthermore, evoked responses by cognitive regions demonstrated the capability of FUS to modulate deeper structures in the brain.