Babak Baseri

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.

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

As a potentially viable method of brain drug delivery, the safety profile of blood-brain barrier (BBB) opening using focused ultrasound (FUS) and ultrasound contrast agents (UCA) needs to be established. In this study, we provide a short-term (30-min or 5-h survival) histological assessment of murine brains undergoing FUS-induced BBB opening. Forty-nine mice were intravenously injected with Definity microbubbles (0.05 microL/kg) and sonicated under the following parameters: frequency of 1.525 MHz, pulse length of 20 ms, pulse repetition frequency of 10 Hz, peak rarefactional acoustic pressures of 0.15-0.98 MPa and two 30-s sonication intervals with an intermittent 30-s delay. The BBB opening threshold was found to be 0.15-0.3 MPa based on fluorescence and magnetic resonance imaging of systemically injected tracers. Analysis of three histological measures in hematoxylin and eosin-stained sections revealed the safest acoustic pressure to be within the range of 0.3-0.46 MPa in all examined time periods post sonication. Across different pressure amplitudes, only the samples 30 min post opening showed significant difference (p < 0.05) in the average number of distinct damaged sites, microvacuolated sites, dark neurons and sites with extravasated erythrocytes. Enhanced fluorescence around severed microvessels was also noted and found to be associated with the largest tissue effects, whereas mildly diffuse BBB opening with uniform fluorescence in the parenchyma was associated with no or mild tissue injury. Region-specific areas of the sonicated brain (thalamus, hippocampal fissure, dentate gyrus and CA3 area of hippocampus) exhibited variation in fluorescence intensity based on the position, orientation and size of affected vessels. The results of this short-term histological analysis demonstrated the feasibility of a safe FUS-UCA-induced BBB opening under a specific set of sonication parameters and provided new insights on the mechanism of BBB opening.

Noninvasive and Localized Blood—Brain Barrier Disruption using Focused Ultrasound can be Achieved at Short Pulse Lengths and Low Pulse Repetition Frequencies

Ultrasound methods in conjunction with microbubbles have been used for brain drug delivery, treatment of stroke, and imaging of cerebral blood flow. Despite advances in these areas, questions remain regarding the range of ultrasound parameters that disrupt the blood–brain barrier (BBB). In this study, several conditions were investigated to either enhance or reduce the likelihood of BBB disruption. Pulsed focused ultrasound (frequency: 1.5 MHz, pressure: 0.46 MPa, pulse repetition frequency (PRF): 0.1 to 25 Hz, pulse length (PL): 0.03 to 30 milliseconds) was noninvasively and locally administered to a predetermined region in the left hemisphere in the presence of circulating preformed microbubbles (Definity, Lantheus Medical Imaging, N. Billerica, MA, USA; 0.01, 0.05, 0.25 μL/g). Trans-BBB delivery of 3-kDa dextran was observed at PRFs as low as 1 Hz, whereas consistent delivery was observed at 5 Hz and above. Delivery was demonstrated at a PL as low as 33 microseconds. Although the delivered dextran concentration increased with the PL, this also increased the heterogeneity of the resulting distribution. In conclusion, key parameters that disrupt the BBB were identified out of a wide range of conditions. Reducing the total number of emitted acoustic cycles by shortening the PL, or decreasing the PRF, was also found to facilitate a more spatially uniform distribution of delivered dextran.

Activation of signaling pathways following localized delivery of systemically administered neurotrophic factors across the blood–brain barrier using focused ultrasound and microbubbles

The brain-derived neurotrophic factor (BDNF) has been shown to have broad neuroprotective effects in addition to its therapeutic role in neurodegenerative disease. In this study, the efficacy of delivering exogenous BDNF to the left hippocampus is demonstrated in wild-type mice (n = 7) through the noninvasively disrupted blood-brain barrier (BBB) using focused ultrasound (FUS). The BDNF bioactivity was found to be preserved following delivery as assessed quantitatively by immunohistochemical detection of the pTrkB receptor and activated pAkt, pMAPK, and pCREB in the hippocampal neurons. It was therefore shown for the first time that systemically administered neurotrophic factors can cross the noninvasively disrupted BBB and trigger neuronal downstream signaling effects in a highly localized region in the brain. This is the first time that the administered molecule is tracked through the BBB and localized in the neuron triggering molecular effects. Additional preliminary findings are shown in wild-type mice with two additional neurotrophic factors such as the glia-derived neurotrophic factor (n = 12) and neurturin (n = 2). This further demonstrates the impact of FUS for the early treatment of CNS diseases at the cellular and molecular level and strengthens its premise for FUS-assisted drug delivery and efficacy.

Identifying the Inertial Cavitation Threshold and Skull Effects in a Vessel Phantom Using Focused Ultrasound and Microbubbles

Focused ultrasound (FUS) in combination with microbubbles has been shown capable of delivering large molecules to the brain parenchyma through opening of the blood-brain barrier (BBB). However, the mechanism behind the opening remains unknown. To investigate the pressure threshold for inertial cavitation of preformed microbubbles during sonication, passive cavitation detection in conjunction with B-mode imaging was used. A cerebral vessel was simulated by generating a cylindrical hole of 610 microm in diameter inside a polyacrylamide gel and saturating its volume with microbubbles. Definity microbubbles (Mean diameter range: 1.1-3.3 microm, Lantheus Medical Imaging, N. Billerica, MA, USA) were injected prior to sonication (frequency: 1.525 MHz; pulse length: 100 cycles; PRF: 10 Hz; sonication duration: 2 s) through an excised mouse skull. The acoustic emissions due to the cavitation response were passively detected using a cylindrically focused hydrophone, confocal with the FUS transducer and a linear-array transducer with the field of view perpendicular to the FUS beam. The broadband spectral response acquired at the passive cavitation detector (PCD) and the B-mode images identified the occurrence and location of the inertial cavitation, respectively. Findings indicated that the peak-rarefactional pressure threshold was approximately equal to 0.45 MPa, with or without the skull present. Mouse skulls did not affect the threshold of inertial cavitation but resulted in a lower inertial cavitation dose. The broadband response could be captured through the murine skull, so the same PCD set-up can be used in future in vivo applications.

Ultrasound-induced blood-brain barrier opening.

Over 4 million U.S. men and women suffer from Alzheimers disease; 1 million from Parkinsons disease; 350,000 from multiple sclerosis (MS); and 20,000 from amyotrophic lateral sclerosis (ALS). Worldwide, these four diseases account for more than 20 million patients. In addition, aging greatly increases the risk of neurodegenerative disease. Although great progress has been made in recent years toward understanding of these diseases, few effective treatments and no cures are currently available. This is mainly due to the impermeability of the blood-brain barrier (BBB) that allows only 5% of the 7000 small-molecule drugs available to treat only a tiny fraction of these diseases. On the other hand, safe and localized opening of the BBB has been proven to present a significant challenge. Of the methods used for BBB disruption shown to be effective, Focused Ultrasound (FUS), in conjunction with microbubbles, is the only technique that can induce localized BBB opening noninvasively and regionally. FUS may thus have a huge impact in trans-BBB brain drug delivery. The primary objective in this paper is to elucidate the interactions between ultrasound, microbubbles and the local microenvironment during BBB opening with FUS, which are responsible for inducing the BBB disruption. The mechanism of the BBB opening in vivo is monitored through the MRI and passive cavitation detection (PCD), and the safety of BBB disruption is assessed using H&E histology at distinct pressures, pulse lengths and microbubble diameters. It is hereby shown that the BBB can be disrupted safely and transiently under specific acoustic pressures (under 0.45 MPa) and microbubble (diameter under 8 μm) conditions.

Automatic detection of blood versus non-blood regions on intravascular ultrasound (IVUS) images using wavelet packet signatures

Intravascular ultrasound (IVUS) has been proven a reliable imaging modality that is widely employed in cardiac interventional procedures. It can provide morphologic as well as pathologic information on the occluded plaques in the coronary arteries. In this paper, we present a new technique using wavelet packet analysis that differentiates between blood and non-blood regions on the IVUS images. We utilized the multi-channel texture segmentation algorithm based on the discrete wavelet packet frames (DWPF). A k-mean clustering algorithm was deployed to partition the extracted textural features into blood and non-blood in an unsupervised fashion. Finally, the geometric and statistical information of the segmented regions was used to estimate the closest set of pixels to the lumen border and a spline curve was fitted to the set. The presented algorithm may be helpful in delineating the lumen border automatically and more reliably prior to the process of plaque characterization, especially with 40 MHz transducers, where appearance of the red blood cells renders the border detection more challenging, even manually. Experimental results are shown and they are quantitatively compared with manually traced borders by an expert. It is concluded that our two dimensional (2-D) algorithm, which is independent of the cardiac and catheter motions performs well in both in-vivo and in-vitro cases.

Iterative Self-Organizing Atherosclerotic Tissue Labeling in Intravascular Ultrasound Images and Comparison With Virtual Histology

Intravascular ultrasound (IVUS) is the predominant imaging modality in the field of interventional cardiology that provides real-time cross-sectional images of coronary arteries and the extent of atherosclerosis. Due to heterogeneity of lesions and stringent spatial/spectral behavior of tissues, atherosclerotic plaque characterization has always been a challenge and still is an open problem. In this paper, we present a systematic framework from in vitro data collection, histology preparation, IVUS-histology registration along with matching procedure, and finally a robust texture-derived unsupervised atherosclerotic plaque labeling. We have performed our algorithm on in vitro and in vivo images acquired with single-element 40 MHz and 64-elements phased array 20 MHz transducers, respectively. In former case, we have quantified results by local contrasting of constructed tissue colormaps with corresponding histology images employing an independent expert and in the latter case, virtual histology images have been utilized for comparison. We tackle one of the main challenges in the field that is the reliability of tissues behind arc of calcified plaques and validate the results through a novel random walks framework by incorporating underlying physics of ultrasound imaging. We conclude that proposed framework is a formidable approach for retrieving imperative information regarding tissues and building a reliable training dataset for supervised classification and its extension for in vivo applications.

Texture-driven coronary artery plaque characterization using wavelet packet signatures

High-frequency ultrasound transducers are being widely used to generate high resolution, real time, cross-sectional images of the coronary arteries. In this paper, we present a robust unsupervised texture-derived technique based on multi-channel wavelet frames to delineate atherosclerotic plaque compositions. The intravascular ultrasound (IVUS) signals were acquired from coronary arteries dissected from 32 diseased cadaver hearts employing 40 MHz mechanically rotating, single-element transducers. The wavelet packet representations were classified using a K- means clustering algorithm to generate IVUS-histology color maps (IV-HCMs) and categorize tissues in lipidic, fibrotic and calcified. Finally, two independent observers evaluated the results contrasting the histology images corresponding to the IV-HCMs. Our results show that the proposed algorithm may have great potential as an alternative to existing spectrum-based classification techniques.

An Alternative Approach to Spectrum-Based Atherosclerotic Plaque Characterization Techniques Using Intravascular Ultrasound (IVUS) Backscattered Signals

In this paper, the discrete wavelet packet frames are used to delineate the atherosclerotic plaque components using intravascular ultrasound (IVUS) backscattered signals. The frames are classified in an unsupervised fashion deploying K-means clustering technique and the generated prognosis histology (PH) images are quantified using relative histology images. While existing tissue characterization algorithms fail to differentiate between blood and plaque signals, the proposed algorithm can be used to estimate the lumen border at higher levels of wavelet expansion. We will demonstrate the in-vitro and invivo tissue characterization as well as lumen border detection results employing 40 MHz rotating unfocused single-element transducers. It is concluded that our two dimensional (2-D) algorithm, which is independent of the cardiac and catheter motions, performs well in both in-vivo and in-vitro cases.