Fotios Vlachos

In vivo transcranial cavitation threshold detection during ultrasound-induced blood–brain barrier opening in mice

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

A quantitative pressure and microbubble-size dependence study of focused ultrasound-induced blood-brain barrier opening reversibility in vivo using MRI

Focused ultrasound in conjunction with the systemic administration of microbubbles has been shown to open the blood‐brain barrier (BBB) selectively, noninvasively and reversibly. In this study, we investigate the dependence of the BBB openings reversibility on the peak‐rarefactional pressure (0.30–0.60 MPa) as well as the microbubble size (diameters of 1–2, 4–5, or 6–8 μm) in mice using contrast‐enhanced T1‐weighted (CE‐T1) MR images (9.4 T). Volumetric measurements of the diffusion of Gd‐DTPA‐BMA into the brain parenchyma were used for the quantification of the BBB‐opened region on the day of sonication and up to 5 days thereafter. The volume of opening was found to increase with both pressure and microbubble diameter. The duration required for closing was found to be proportional to the volume of opening on the day of opening, and ranged from 24 h, for the smaller microbubbles, to 5 days at high peak‐rarefactional pressures. Overall, larger bubbles did not show significant differences. Also, the extent of BBB opening decreased radially towards the focal region until the BBBs integrity was restored. In the cases where histological damage was detected, it was found to be highly correlated with hyperintensity on the precontrast T1 images. Magn Reson Med, 2012.

The mechanism of interaction between focused ultrasound and microbubbles in blood-brain barrier opening in mice

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

Noninvasive and localized neuronal delivery using short ultrasonic pulses and microbubbles

Focused ultrasound activation of systemically administered microbubbles is a noninvasive and localized drug delivery method that can increase vascular permeability to large molecular agents. Yet the range of acoustic parameters responsible for drug delivery remains unknown, and, thus, enhancing the delivery characteristics without compromising safety has proven to be difficult. We propose a new basis for ultrasonic pulse design in drug delivery through the blood–brain barrier (BBB) that uses principles of probability of occurrence and spatial distribution of cavitation in contrast to the conventionally applied magnitude of cavitation. The efficacy of using extremely short (2.3 μs) pulses was evaluated in 27 distinct acoustic parameter sets at low peak-rarefactional pressures (0.51 MPa or lower). The left hippocampus and lateral thalamus were noninvasively sonicated after administration of Definity microbubbles. Disruption of the BBB was confirmed by delivery of fluorescently tagged 3-, 10-, or 70-kDa dextrans. Under some conditions, dextrans were distributed homogeneously throughout the targeted region and accumulated at specific hippocampal landmarks and neuronal cells and axons. No histological damage was observed at the most effective parameter set. Our results have broadened the design space of parameters toward a wider safety window that may also increase vascular permeability. The study also uncovered a set of parameters that enhances the dose and distribution of molecular delivery, overcoming standard trade-offs in avoiding associated damage. Given the short pulses used similar to diagnostic ultrasound, new critical parameters were also elucidated to clearly separate therapeutic ultrasound from disruption-free diagnostic ultrasound.

Permeability dependence study of the focused ultrasound‐induced blood–brain barrier opening at distinct pressures and microbubble diameters using DCE‐MRI

Blood–brain barrier opening using focused ultrasound and microbubbles has been experimentally established as a noninvasive and localized brain drug delivery technique. In this study, the permeability of the opening is assessed in the murine hippocampus after the application of focused ultrasound at three different acoustic pressures and microbubble sizes. Using dynamic contrast‐enhanced MRI, the transfer rates were estimated, yielding permeability maps and quantitative Ktrans values for a predefined region of interest. The volume of blood–brain barrier opening according to the Ktrans maps was proportional to both the pressure and the microbubble diameter. A Ktrans plateau of ∼0.05 min−1 was reached at higher pressures (0.45 and 0.60 MPa) for the larger sized bubbles (4–5 and 6–8 μm), which was on the same order as the Ktrans of the epicranial muscle (no barrier). Smaller bubbles (1–2 μm) yielded significantly lower permeability values. A small percentage (7.5%) of mice showed signs of damage under histological examination, but no correlation with permeability was established. The assessment of the blood–brain barrier permeability properties and their dependence on both the pressure and the microbubble diameter suggests that Ktrans maps may constitute an in vivo tool for the quantification of the efficacy of the focused ultrasound‐induced blood–brain barrier opening. Magn Reson Med, 2011.

Permeability assessment of the focused ultrasound-induced blood–brain barrier opening using dynamic contrast-enhanced MRI

Focused ultrasound (FUS) in conjunction with microbubbles has been shown to successfully open the blood-brain barrier (BBB) in the mouse brain. In this study, we compute the BBB permeability after opening in vivo. The spatial permeability of the BBB-opened region was assessed using dynamic contrast-enhanced MRI (DCE-MRI). The DCE-MR images were post-processed using the general kinetic model (GKM) and the reference region model (RRM). Permeability maps were generated and the K(trans) values were calculated for a predefined volume of interest in the sonicated and the control area for each mouse. The results demonstrated that K(trans) in the BBB-opened region (0.02 +/- 0.0123 for GKM and 0.03 +/- 0.0167 min(-1) for RRM) was at least two orders of magnitude higher when compared to the contra-lateral (control) side (0 and 8.5 x 10(-4) +/- 12 x 10(-4) min(-1), respectively). The permeability values obtained with the two models showed statistically significant agreement and excellent correlation (R(2) = 0.97). At histological examination, it was concluded that no macroscopic damage was induced. This study thus constitutes the first permeability assessment of FUS-induced BBB opening using DCE-MRI, supporting the fact that the aforementioned technique may constitute a safe, non-invasive and efficacious drug delivery method.

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.

Monitoring early tumor response to drug therapy with diffuse optical tomography

Although anti-angiogenic agents have shown promise as cancer therapeutics, their efficacy varies between tumor types and individual patients. Providing patient-specific metrics through rapid noninvasive imaging can help tailor drug treatment by optimizing dosages, timing of drug cycles, and duration of therapy-thereby reducing toxicity and cost and improving patient outcome. Diffuse optical tomography (DOT) is a noninvasive three-dimensional imaging modality that has been shown to capture physiologic changes in tumors through visualization of oxygenated, deoxygenated, and total hemoglobin concentrations, using non-ionizing radiation with near-infrared light. We employed a small animal model to ascertain if tumor response to bevacizumab (BV), an anti-angiogenic agent that targets vascular endothelial growth factor (VEGF), could be detected at early time points using DOT. We detected a significant decrease in total hemoglobin levels as soon as one day after BV treatment in responder xenograft tumors (SK-NEP-1), but not in SK-NEP-1 control tumors or in non-responder control or BV-treated NGP tumors. These results are confirmed by magnetic resonance imaging T2 relaxometry and lectin perfusion studies. Noninvasive DOT imaging may allow for earlier and more effective control of anti-angiogenic therapy.

In vivo transcranial cavitation detection during ultrasound-induced blood-brain barrier opening

The blood-brain barrier (BBB) has been shown capable being opened noninvasively through the combined application of focused ultrasound (FUS) and microbubbles. In order to better identify the underlying mechanism responsible for BBB opening as well as associated safety, the in vivo noninvasive and transcranial cavitation detection associated with FUS-induced BBB opening was studied. A cylindrically focused hydrophone, confocal with the FUS transducer, was used as a passive cavitation detector (PCD) to identify the threshold of inertial cavitation (IC) in the presence of Definity® microbubbles. After Definity® were injected intravenously, pulsed FUS was applied (frequency: 1.525 and 1.5 MHz, peak-negative pressure: 0.15–0.60 MPa, duty cycle: 20%, PRF: 10 Hz, duration: 1 min with 30s interval) on the right hippocampus in twenty-six mice in vivo through their intact scalp and skull. T1-weighted MRI was used to verify the BBB opening. A spectrogram was generated at each pressure in order to detect the inertial cavitation onset and duration. The bubble behavior was shown detectable in vivo through the intact scalp and skull. We demonstrated that: 1) the inertial cavitation response could be detected transcranially during BBB opening; 2) the inertial cavitation pressure threshold lied at 0.45 MPa but the BBB was opened at 0.30 MPa so the BBB can be opened without requiring inertial cavitation; 3) the BBB could be opened without any cellular damage.

Pressure and microbubble size dependence study of focused ultrasound-induced blood-brain barrier opening reversibility in vivo

Most currently available therapeutic compounds cannot cross the blood-brain barrier (BBB), and their delivery to the brain remains a critical impediment. Focused Ultrasound (FUS) in conjunction with systemically administered microbubbles has been shown to open the BBB locally, non-invasively and reversibly. In this study, we investigated the dependence of BBB openings reversibility timeline on the peak-rarefactional pressure (PRP) varied from 0.30 MPa to 0.60 MPa and the microbubble size in mice in vivo. The microbubbles used were monodispersed with diameters of 1-2, 4-5 or 6-8 microns. The contrast agents (Gd) diffusion was used to quantify the opening, in T1-weighted high resolution MR images acquired on the day of sonication and up to five days thereafter. The volume of opening was found to increase with both pressure and microbubble diameter. The duration required for closing was found to be proportional to the volume of opening on the day of opening, and ranged from 24 hours, for the 1-2 um and 0.4...