Natalia Vykhodtseva

Local and reversible blood–brain barrier disruption by noninvasive focused ultrasound at frequencies suitable for trans-skull sonications

The purpose of this study was to test the hypothesis that burst ultrasound in the presence of an ultrasound contrast agent can disrupt the blood-brain barrier (BBB) with acoustic parameters suitable for completely noninvasive exposure through the skull. The 10-ms exposures were targeted in the brains of 22 rabbits with a frequency of 690 kHz, a repetition frequency of 1 Hz, and peak rarefactional pressure amplitudes up to 3.1 MPa. The total exposure (sonication) time was 20 s. Prior to each sonication, a bolus of ultrasound contrast agent was injected intravenously. Contrast-enhanced MR images were obtained after the sonications to detect localized BBB disruption via local enhancement in the brain. Brain sections were stained with H&E, TUNEL, and vanadium acid fuchsin (VAF)-toluidine blue staining. In addition, horseradish peroxidase (HRP) was injected into four rabbits prior to sonications and transmission electron microscopy was performed. The MRI contrast enhancement demonstrated BBB disruption at pressure amplitudes starting at 0.4 MPa with approximately 50%; at 0.8 MPa, 90%; and at 1.4 MPa, 100% of the sonicated locations showed enhancement. The histology findings following 4 h survival indicated that brain tissue necrosis was induced in approximately 70-80% of the sonicated locations at a pressure amplitude level of 2.3 MPa or higher. At lower pressure amplitudes, however, small areas of erythrocyte extravasation were seen. The electron microscopy findings demonstrated HRP passage through vessel walls via both transendothelial and paraendothelial routes. These results demonstrate that completely noninvasive focal disruption of the BBB is possible.

Targeted delivery of doxorubicin to the rat brain at therapeutic levels using MRI-guided focused ultrasound.

The clinical application of chemotherapy to brain tumors has been severely limited because antitumor agents are typically unable to penetrate an intact blood‐brain barrier (BBB). Although doxorubicin (DOX) has been named as a strong candidate for chemotherapy of the central nervous system (CNS), the BBB often prevents cytotoxic levels from being achieved. In this study, we demonstrate a noninvasive method for the targeted delivery of DOX through the BBB, such that drug levels shown to be therapeutic in human tumors are achieved in the normal rat brain. Using MRI‐guided focused ultrasound with preformed microbubbles (Optison) to locally disrupt the BBB and systemic administration of DOX, we achieved DOX concentrations of 886 ± 327 ng/g tissue in the brain with minimal tissue effects. Tissue DOX concentrations of up to 5,366 ± 659 ng/g tissue were achieved with higher Optison doses, but with more significant tissue damage. In contrast, DOX accumulation in nontargeted contralateral brain tissue remained significantly lower for all paired samples (p < 0.001). These results suggest that targeted delivery by focused ultrasound may render DOX chemotherapy a viable treatment option against CNS tumors, despite previous accessibility limitations. In addition, MRI signal enhancement in the sonicated region correlated strongly with tissue DOX concentration (r = 0.87), suggesting that contrast‐enhanced MRI could perhaps indicate drug penetration during image‐guided interventions. Our technique using MRI‐guided focused ultrasound to achieve therapeutic levels of DOX in the brain offers a large step forward in the use of chemotherapy to treat patients with CNS malignancies.

Targeted disruption of the blood–brain barrier with focused ultrasound: association with cavitation activity

Acoustic emission was monitored during focused ultrasound exposures in conjunction with an ultrasound contrast agent (Optison) in order to determine if cavitation activity is associated with the induction of blood-brain barrier disruption (BBBD). Thirty-four locations were sonicated (frequency: 260 kHz) at targets 10 mm deep in rabbit brain (N = 9). The sonications were applied at peak pressure amplitudes ranging from 0.11 to 0.57 MPa (burst length: 10 ms; repetition frequency of 1 Hz; duration: 20 s). Acoustic emission was recorded with a focused passive cavitation detector. This emission was recorded at each location during sonications with and without Optison. Detectable wideband acoustic emission was observed only at 0.40 and 0.57 MPa. BBBD was observed in contrast MRI after sonication at 0.29-0.57 MPa. The appearance of small regions of extravasated erythrocytes appeared to be associated with this wideband emission signal. The results thus suggest that BBBD resulting from focused ultrasound pulses in the presence of Optison can occur without indicators for inertial cavitation in vivo, wideband emission and extravasation. If inertial cavitation is not responsible for the BBBD, other ultrasound/microbubble interactions are likely the source. A significant increase in the emission signal due to Optison at the second and third harmonics of the ultrasound driving frequency was found to correlate with BBBD and might be useful as an online method to indicate when the disruption occurs.

500-Element Ultrasound Phased Array System for Noninvasive Focal Surgery of the Brain: A Preliminary Rabbit Study With Ex Vivo Human Skulls

The aim of this study was to test a prototype MRI‐compatible focused ultrasound phased array system for trans‐skull brain tissue ablation. Rabbit thigh muscle and brain were sonicated with a prototype, hemispherical 500‐element ultrasound phased array operating at frequencies of 700–800 kHz. An ex vivo human skull sample was placed between the array and the animal tissue. The temperature elevation during 20–30‐sec sonications was monitored using MRI thermometry. The induced focal lesions were observed in T2 and contrast‐enhanced T1‐weighted fast spin echo images. Whole brain histology evaluation was performed after the sonications. The results showed that sharp temperature elevations can be produced both in the thigh muscle and in the brain. High‐power sonications (600–1080 W) produced peak temperatures up to 55°C and focal lesions that were consistent with thermal tissue damage. The lesion size was found to increase with increasing peak temperature. The device was then modified to operate in the orientation that will be used in the clinic and successfully tested in phantom experiments. As a conclusion, this study demonstrates that it is possible to create ultrasound‐induced lesions in vivo through a human skull under MRI guidance with this large‐scale phased array. Magn Reson Med 52:100–107, 2004.

Temporary Disruption of the Blood–Brain Barrier by Use of Ultrasound and Microbubbles: Safety and Efficacy Evaluation in Rhesus Macaques

The blood-brain barrier (BBB) prevents entry of most drugs into the brain and is a major hurdle to the use of drugs for brain tumors and other central nervous system disorders. Work in small animals has shown that ultrasound combined with an intravenously circulating microbubble agent can temporarily permeabilize the BBB. Here, we evaluated whether this targeted drug delivery method can be applied safely, reliably, and in a controlled manner on rhesus macaques using a focused ultrasound system. We identified a clear safety window during which BBB disruption could be produced without evident tissue damage, and the acoustic pressure amplitude where the probability for BBB disruption was 50% and was found to be half of the value that would produce tissue damage. Acoustic emission measurements seem promising for predicting BBB disruption and damage. In addition, we conducted repeated BBB disruption to central visual field targets over several weeks in animals trained to conduct complex visual acuity tasks. All animals recovered from each session without behavioral deficits, visual deficits, or loss in visual acuity. Together, our findings show that BBB disruption can be reliably and repeatedly produced without evident histologic or functional damage in a clinically relevant animal model using a clinical device. These results therefore support clinical testing of this noninvasive-targeted drug delivery method.

BLOOD-BRAIN BARRIER DISRUPTION INDUCED BY FOCUSED ULTRASOUND AND CIRCULATING PREFORMED MICROBUBBLES APPEARS TO BE CHARACTERIZED BY THE MECHANICAL INDEX

This work investigated the effect of ultrasonic frequency on the threshold for blood-brain barrier (BBB) disruption induced by ultrasound pulses combined with an ultrasound contrast agent. Experiments were performed in rabbits using pulsed sonications at 2.04 MHz with peak pressure amplitudes ranging from 0.3 to 2.3 MPa. BBB disruption was evaluated using contrast-enhanced magnetic resonance imaging. The threshold for BBB disruption was estimated using probit regression. Representative samples with similar amounts of contrast enhancement were examined in light microscopy. Results from these experiments were compared with data from previous studies that used ultrasound frequencies between 0.26 and 1.63 MHz. We found that the BBB disruption threshold (value where the probability for disruption was estimated to be 50%) expressed in terms of the peak negative pressure amplitude increased as a function of the frequency. It appeared to be constant, however, when the exposures were expressed as a function of the mechanical index (peak negative pressure amplitude estimated in situ divided by square root of frequency). Regression of data from all frequencies resulted in an estimated mechanical index threshold of 0.46 (95% confidence intervals: 0.42 to 0.50). Histologic examination of representative samples with similar amounts of blood-brain barrier disruption found that the number of regions containing extravasated red blood cells per unit area was substantially lower on average for lower ultrasound frequencies. This data suggests that the mechanical index is a meaningful metric for ultrasound-induced blood-brain barrier disruption, at least for when other parameters that are not taken into account by the mechanical index are not varied. It also suggests that lower frequency sonication produces less red blood cell extravasation per unit area.

Histologic effects of high intensity pulsed ultrasound exposure with subharmonic emission in rabbit brain in vivo

In this study, the threshold for subharmonic emission during in vivo sonication of rabbit brain was investigated. In addition, the histologic effects of pulsed sonication above this threshold were studied. Two spherically curved focused ultrasound transducers with a diameter of 80 mm and a radius of curvature of 70 mm were used in the sonications. The operating frequencies of the transducers were 0.936 and 1.72 MHz. The sonication duration was varied between 0.001 and 1 s and the repetition frequency between 0.1 and 5 Hz. The threshold for subharmonic emission at the frequency of 0.936 MHz was found to be approximately 2000 W cm-2 and 3600 W cm-2 for pulse durations of 1 s and 0.001 s, respectively. The threshold was approximately 1.5-fold as high at a frequency of 1.72 MHz. However, there was considerable variation from experiment to experiment. The multiple pulse experiments at a frequency of 1.72 MHz and an intensity of 7000 W cm-2 showed that the histologic effects ranged from no observable damage of the tissue, to blood-brain barrier breakage, to local haemorrhagia, to local destruction of the tissue, to gross hemorrhage resulting in the death of the animal. The severity of the tissue damage increased as the pulse duration, number of pulses and their repetition frequency increased. The results indicate that the end point of the tissue damage may be controlled by selecting the sonication parameters. Such control over tissue effects can have several different applications when brain disorders are treated.

PROGRESS AND PROBLEMS IN THE APPLICATION OF FOCUSED ULTRASOUND FOR BLOOD-BRAIN BARRIER DISRUPTION

Advances in neuroscience have resulted in the development of new diagnostic and therapeutic agents for potential use in the central nervous system (CNS). However, the ability to deliver the majority of these agents to the brain is limited by the blood-brain barrier (BBB), a specialized structure of the blood vessel wall that hampers transport and diffusion from the blood to the brain. Many CNS disorders could be treated with drugs, enzymes, genes, or large-molecule biotechnological products such as recombinant proteins, if they could cross the BBB. This article reviews the problems of the BBB presence in treating the vast majority of CNS diseases and the efforts to circumvent the BBB through the design of new drugs and the development of more sophisticated delivery methods. Recent advances in the development of noninvasive, targeted drug delivery by MRI-guided ultrasound-induced BBB disruption are also summarized.

Effects of Acoustic Parameters and Ultrasound Contrast Agent Dose on Focused-Ultrasound Induced Blood-Brain Barrier Disruption

Previously, it was shown that low-intensity focused ultrasound pulses applied along with an ultrasound contrast agent results in temporary blood-brain barrier (BBB) disruption. This effect could be used for targeted drug delivery in the central nervous system. This study examined the effects of burst length, pulse repetition frequency (PRF), and ultrasound contrast agent dose on the resulting BBB disruption. One hundred nonoverlapping brain locations were sonicated through a craniotomy in experiments in 26 rabbits (ultrasound frequency: 0.69 MHz, burst: 0.1, 1, 10 ms, PRF: 0.5, 1, 2, 5 Hz, duration: 20 s, peak negative pressure amplitude: 0.1 to 1.5 MPa, Optison dosage 50, 100, 250 microl/kg). For each sonication, BBB disruption was evaluated using contrast-enhanced magnetic resonance imaging. The BBB disruption threshold (the pressure amplitude yielding a 50% probability for BBB disruption) was determined using probit regression for the three burst lengths tested. Tissue effects were examined in light microscopy for representative locations with similar amounts of contrast enhancement from each group. While changing the PRF or the Optison dosage did not result in a significant difference in the magnitude of the BBB disruption (p > 0.05), reducing the burst length resulted in significantly less contrast enhancement (p < 0.01). The BBB disruption thresholds were estimated to be 0.69, 0.47 and 0.36 MPa for 0.1, 1 and 10 ms bursts, respectively. No difference was detected in histology between any experimental groups. This data suggests that over the range of parameters tested, BBB disruption is not affected by PRF or ultrasound contrast agent dose. However, both the BBB disruption magnitude and its threshold depend on the burst length.

MRI investigation of the threshold for thermally induced blood-brain barrier disruption and brain tissue damage in the rabbit brain.

The ability of MRI‐derived thermometry to predict thermally induced tissue changes in the brain was tested, and the thermal thresholds for blood–brain barrier (BBB) disruption and brain tissue damage were estimated. In addition, the ability of standard MRI to detect threshold‐level effects was confirmed. These safety thresholds are being investigated to provide guidelines for clinical thermal ablation studies in the brain. MRI‐monitored focused ultrasound heating was delivered to 63 locations in 26 rabbits. Tissue changes were detected in T2‐weighted imaging and T1‐weighted imaging (with and without contrast) and with light microscopy. The probability for tissue damage as a function of the accumulated thermal dose, the peak temperature achieved, the applied acoustic energy, and the peak acoustic power was estimated with probit regression. The discriminative abilities of these parameters were compared using the areas under the receiver operator characteristic (ROC) curves. In MRI, BBB disruption was observed in contrast‐enhanced T1‐weighted imaging shortly after the ultrasound exposures, sometimes accompanied by changes in T2‐weighted imaging. Two days later, changes in T2‐weighted imaging were observed, sometimes accompanied by changes in T1‐weighted imaging. In histology, tissue damage was seen at every location where MRI changes were observed, ranging from small (diameter <1.0 mm) areas of tissue necrosis to severe vascular damage and associated hemorrhagic infarct. In one location, small (diameter: 0.8 mm) damage was not detected in MRI. The thermal dose and peak temperature thresholds were between 12.3–40.1 equivalent min at 43°C and 48.0–50.8°C, respectively, and values of 17.5 equivalent min at 43°C and 48.4°C were estimated to result in tissue damage with 50% probability. Thermal dose and peak temperature were significantly better predictors than the applied acoustic energy and peak acoustic power (P < 0.01). BBB disruption was always accompanied by tissue damage. The temperature information was better than the applied acoustic power or energy for predicting the damage than the ultrasound parameters. MRI was sensitive in detecting threshold‐level damage. Magn Reson Med 51:913–923, 2004.