Opening of the blood-brain barrier with an unfocused ultrasound device in rabbits

Laboratory investigation

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Object

The blood-brain barrier (BBB) is a major impediment to the intracerebral diffusion of drugs used in the treatment of gliomas. Previous studies have demonstrated that pulsed focused ultrasound (US) in conjunction with a microbubble contrast agent can be used to open the BBB. To apply the US-induced opening of the BBB in clinical practice, the authors designed an innovative unfocused US device that can be implanted in the skull and used to transiently and repeatedly open the BBB during a standard chemotherapy protocol. The goal of this preliminary work was to study the opening of the BBB induced by the authors' small unfocused US transducer and to evaluate the effects of the sonications on brain parenchyma.

Methods

Craniectomy was performed in 16 healthy New Zealand White rabbits; epidural application of a single-element planar ultrasonic transducer operating at 1 MHz was then used with a pulse-repetition frequency of 1 Hz, pulse lengths of 10–35 msec, in situ acoustic pressure levels of 0.3–0.8 MPa, and sonication for 60–120 seconds. SonoVue was intravenously injected during the US applications, and opening of the BBB was determined by detecting extravasation of Evans blue dye (EBD) in brain tissues, quantitative measurement of EBD with UV-visible spectrophotometry, and contrast enhancement after Gd injection in 4.7-T MRI. A histological study was performed to determine adverse effects.

Results

An opening of the BBB was observed over a large extent of the US beam in the brain corresponding to in situ pressures of greater than 0.2 MPa. The BBB opening observed was highly significant for both EBD (p < 0.01) and MRI Gd enhancement (p < 0.0001). The BBB opening was associated with minor adverse effects that included perivascular red blood cell extravasations that were less than 150 μm in size and not visible on MR images. Moderate edema was visible on FLAIR sequences and limited to the extent of the sonication field.

Conclusions

The results demonstrate that the BBB can be opened in large areas of the brain in rabbits with lowpower, pulsed, and unfocused US with limited damage to healthy tissue.

Abbreviations used in this paper:BBB = blood-brain barrier; EBD = Evans blue dye; ROI = region of interest; US = ultrasound.

Article Information

Drs. Beccaria and Canney contributed equally to this work.

Address correspondence to: Alexandre Carpentier, M.D., Ph.D., Department of Neurosurgery, Pitié-Salpêtrière Hospital, Assistance Publique Hôpitaux de Paris, Sorbonne University, Paris 6 School of Medicine, Paris 75013, France. email: alexandre.carpentier@psl.aphp.fr.

Please include this information when citing this paper: published online June 21, 2013; DOI: 10.3171/2013.5.JNS122374.

© AANS, except where prohibited by US copyright law.

Headings

Figures

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    Acoustic pressure measured in water generated by the 1-MHz flat 10-mm piston transducer used in this study for opening of the BBB. The pressure field was mapped in a plane perpendicular to the transducer. The pressure field shown was normalized to the acoustic pressure at a distance of 12 mm, indicated by the small “x” in the graph.

  • View in gallery

    Brain tissue sample depicting the method used for segmenting the brain for UV-visible spectrophotometric quantification of EBD extravasation. A coronal slice of brain was first cut and 3 samples were cut in each hemisphere at 3 different locations: 1 and 4, cortical; 2 and 5, subcortical; and 3 and 6, deep-brain regions.

  • View in gallery

    Representative example of EBD extravasation observed in rabbit brain after sonication. In situ acoustic pressures and pulse lengths of 0.8 MPa and 10 msec (left) and 0.5 MPa and 25 m sec (right), respectively, were used with a pulse repetition frequency of 1 Hz for 120 seconds. The sonicated cortex is colored by the blue dye (left, black circle). The coloration is visible at depth in coronal slices of brain; it is unilateral and spatially limited to the extent of the ultrasound field (right, dashed lines). Some very small areas of petechial bleeding are visible in the proximal part of the pressure field (right, arrows).

  • View in gallery

    Brain tissue sonicated with a 0.5-MPa acoustic pressure and 35-msec pulse length using a pulse repetition frequency of 1 Hz and 120 seconds of sonication. Note that coloration with EBD is more intense in the gray matter (asterisks) than in the white matter (daggers). Very small areas of petechial hemorrhages are predominantly situated in the cortical gray matter (arrows).

  • View in gallery

    Brain slices from control animals. Left: Rabbit with right craniectomy (C) without sonication. Right: Rabbit with right sonication (US) and without microbubble contrast agent. No significant blue dye coloration of the parenchyma is present in either sample: craniectomy alone and US without microbubbles do not cause significant opening of the BBB. Both controls do not show any signs of macroscopic petechial hemorrhages.

  • View in gallery

    Sections of brain parenchyma (3–4 μm thin) stained with H & E (×10). A: Grade 0, no lesion observed; asterisks indicate cortical capillaries. Grade 0 is observed in the periphery of the sonication field and in the control rabbits. B: Grade 1, a few extravasated red blood cells (arrows) are visible around a capillary (asterisk). Extravasated red blood cells are observed in the periphery of the sonication field, and are generally located in the cortical and subcortical tissues. C: Grade 2, multiple areas of petechial hemorrhages (arrows) are visible in the sonicated area. It was the histological grade that was most frequently observed in our experiments. Petechial hemorrhages generally center on a blood vessel and the largest lesion is up to 150 μm wide. D: Grade 3, large and multiple hemorrhagic lesions (arrows). This grade was observed in the brain of Rabbit 1 (Table 3) sonicated with the highest acoustic parameters (0.8 MPa and 25 msec).

  • View in gallery

    A–E: Coronal T1-weighted MRI sequences after Gd injection showing contrast enhancement in Rabbits 12–16. Acoustic parameters were the following: Rabbit 12, 0.4 MPa and 25 msec; Rabbit 13, 0.7 MPa and 25 msec; and Rabbits 14–16, 0.6 MPa and 25 msec. Contrast enhancement, indicating opening of the BBB, is limited to the extent of the sonication field. F: No hemorrhagic lesion is visible on the T2* sequence. G: An edema is limited to the extent of the sonication field on the FLAIR sequence (white dotted lines; Rabbit 15 exposed to 0.6 MPa and 25 msec). No mass effect or brain herniation is observed. H: Representation of the regions of interest (white circles) used to statistically compare signal enhancement in the cortex and in the deep-brain structures.

  • View in gallery

    Signal intensity change on T1-weighted MR images as a function of time for Rabbits 14–16 (treated with 0.6 MPa and 25 msec; Table 3) in the cortex and in the deep-brain structures (each data point represents the difference, in percentage, between the signal in the sonicated hemisphere and the signal in the nonsonicated contralateral hemisphere). Before Gd injection (circled points), negative values in the cortex signify a spontaneous hypointense signal in the MRI due to sonication. This effect was not observed in the deep-brain structures. After Gd injection, all values were positive in the cortex and in the basal ganglia, indicating a significant contrast enhancement due to significant opening of the BBB.

  • View in gallery

    The ultrasound device. A: Inferior view of the 1-cm-diameter prototype US transducer used in the experiments of this study. B: A US transducer placed on the dura mater after craniotomy in a rabbit; acoustic coupling was performed by applying gel. C: Schematic representation of the envisioned clinical application of this technique. The small transducer (white) is implanted into the skull bone and an electrical connection is established with a transcutaneous needle (gray). The sonication field is unfocused but allows a relatively targeted opening of the BBB near the site of tumor infiltration (red).

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