Focal disruption of the blood–brain barrier due to 260-kHz ultrasound bursts: a method for molecular imaging and targeted drug delivery

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Object

The goal of this study was to explore the feasibility of using low-frequency magnetic resonance (MR) image–guided focused ultrasound as a noninvasive method for the temporary disruption of the blood–brain barrier (BBB) at targeted locations.

Methods

Rabbits were placed inside a clinical 1.5-tesla MR imaging unit, and sites in their brains were targeted for 20-second burst sonications (frequency 260 kHz). The peak pressure amplitude during the burst varied between 0.1 and 0.9 MPa. Each sonication was performed after an intravenous injection of an ultrasound contrast agent (Optison). The disruption of the BBB was evaluated with the aid of an injection of an MR imaging contrast agent (MAG-NEVIST). Additional tests involving the use of MION-47, a 20-nm magnetic nanoparticle contrast agent, were also performed. The animals were killed at different time points between 3 minutes and 5 weeks postsonication, after which light or electron microscopic evaluation was performed.

The threshold for BBB disruption was approximately 0.2 MPa. More than 80% of the brain sites sonicated showed BBB disruption when the pressure amplitude was 0.3 MPa; at 0.4 MPa, this percentage was greater than 90%. Tissue necrosis, ischemia, and apoptosis were not found in tissue in which the pressure amplitude was less than 0.4 MPa; however, in a few areas of brain tissue erythrocytes were identified outside blood vessels following exposures of 0.4 MPa or higher. Survival experiments did not show any long-term adverse events.

Conclusions

These results demonstrate that low-frequency ultrasound bursts can induce local, reversible disruption of the BBB without undesired long-term effects. This technique offers a potential noninvasive method for targeted drug delivery in the brain aided by a relatively simple low-frequency device.

Abbreviations used in this paper: BBB = blood-brain barrier; EC = endothelial cell; HMW = high molecular weight; HRP = horseradish peroxidase; MR = magnetic resonance; SD = standard deviation; TUNEL = terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling.

Article Information

Address reprint requests to: Kullervo Hynynen, Ph.D., Department of Medical Biophysics, University of Toronto, Sunnybrook Health Sciences Centre, Room S6 65B, 2075 Bayview Avenue, Toronto, Ontario, Canada M4N 3M5. email: khynynen@sri.utoronto.ca.

© AANS, except where prohibited by US copyright law.

Headings

Figures

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    A: An MR image obtained immediately following sonication, revealing focal contrast enhancement in a site (left hemisphere) sonicated at a pressure amplitude of 0.4 MPa. B: Photomicrograph showing a corresponding tissue section excised 4 weeks after sonication. C and D: High-magnification views of the boxed regions in B revealing the undamaged sonicated region (C) and brain tissue appearing similar to that of the control region (D). E and F: Higher-magnification views of the boxed regions in C and D, respectively. B–F: H & E, bars = 1 cm (A & B) and 100 μm (C–F).

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    Graphs showing normalized signal intensity changes ([signal intensity before injection of contrast agent — signal intensity after injection of contrast agent]/signal intensity before injection of contrast agent), in which the signal intensity is that observed in the sonicated tissue volume and in a control site of the brain measured on T1-weighted MR images as a function of time after the injection of a bolus of MR imaging contrast agent. Upper: Graph showing tissue sites during the 30-minute time period commencing 2 hours 41 minutes after the sonication. Lower: Graph showing the same tissue sites during the 30-minute time period commencing 5 hours after the sonication. *p <0.05. The peak negative (rarefactional) pressure amplitude was 0.4 MPa.

  • View in gallery

    Graphs. Upper: Normalized signal intensity changes (means SDs) for all sonicated sites as a function of the rarefactional pressure amplitude for intact-skull and skull-window sonications. Lower: Percentages of sites with BBB disruption as a function of the rarefactional pressure amplitude in the brain for both intact-skull and skull-window sonications. In each graph the results for sonications without Optison are also shown. *p 0.05.

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    Photomicrographs showing the frontal section of a rabbit brain with minimal tissue effects 4 hours after ultrasound exposure through a bone window and intact dura mater. The pressure amplitude was 0.3 MPa. The tissue appears healthy and the vasculature is uncompromised, as evident in higher-magnification images (B and C). Image in panel C shows the same tissue displayed in the box in panel B. H & E, bars = 1 cm (A), 0.5 mm (B), and 100 m (C).

  • View in gallery

    A: Photomicrograph of the tissue section that exhibited the greatest effects 4 hours after ultrasound exposure through a bone window and intact dura. The pressure amplitude was 0.4 MPa. Multiple extravasations of erythrocytes (plus signs) are scattered throughout the sonicated region. The boundary of the area that displayed contrast enhancement on MR images is superimposed on the photomicrograph (dotted line). B: Higher-magnification view of the boxed region shown in panel A. Arrows indicate sites of petechial hemorrhage. C: Higher magnification of the boxed region shown in panel B, providing a detailed view of extravasation and minor parenchymal damage in perivascular tissue. D: The dark-stained structure is an apoptotic cell immediately adjacent to the extravasated erythrocytes. Only one such cell was found in this section, and it is most likely a glial cell. E: Neurons and neuropil surrounding the site of extravasation (arrow) appear unaffected. F: Higher-magnification view of the neurons shown in panel E demonstrating that they are lightly stained and appear normal. H & E (A–C), TUNEL staining (D), and vanadium acid fuchsin–toluidine (E and F); bars = 1 cm (A) and 100 m (B–F).

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    Graphs demonstrating normalized signal intensity changes in both the sonicated site and the control site in the contralateral hemisphere as a function of time when the large-particle MR imaging contrast agent MION-47 was injected into the blood stream. *p < 0.05.

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    Photomicrographs showing a dark cluster of hemosiderin-laden macrophages (A, arrow) and a higher-magnification view of such cells found in a rabbit brain 4 weeks after sonication at 0.4 MPa (B). H & E, bars = 100 m.

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    Electron micrographs. A: Cross-section of a brain capillary from a nonsonicated region. The darkly stained HRP reaction product is localized in several endosome-like vacuoles (arrows) in the cytoplasm of ECs. No reaction product is seen beyond the endothelial lining, in the basement membrane (b), pericyte (P), or neuropil (NP). A red blood cell (RBC) in the lumen (L) of the vessel also displays a dark stain due to the peroxidase activity of the hemoglobin. B: Another microvessel profile in a specimen obtained from the sonicated location 60 minutes after ultrasound exposure. The reaction product is again seen in EC vacuoles and also outside the endothelium in the basement membrane, which looks heavily infiltrated with HRP (some indicated with arrowheads), and everywhere in the interstitial spaces of neuropil (the dark zones, some of which are indicated by arrows). C: A portion of capillary wall obtained from the same location as the tissue shown in the above panels, at a higher magnification. Peroxidase has entered a cleft (black arrows) between two ECs, passed to the basement membrane, and infiltrated the interstitial space (white arrows) among myelinated axons (ax). Caveolae (arrowheads) filled with HRP are seen in the ECs and pericyte at the luminal, middle, and basal parts of the cell, suggesting their possible involvement in the transcellular transport of the tracer. a = astrocytic end feet; ax = cross-sections and longitudinal sections of myelinated axons; N = cytoplasm of an adjacent neuron.

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