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1Imaging Research, Sunnybrook Research Institute, 2Department of Medical Biophysics, University of Toronto, 3Department of Medical Biophysics, and Institute of Biomaterials & Biomedical Engineering (IBBME), University of Toronto
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Microbubble-mediated focused ultrasound disruption of the blood-brain barrier (BBB) is a promising technique for non-invasive targeted drug delivery in the brain1-3. This protocol outlines the experimental procedure for MRI-guided transcranial BBB disruption in a rat model.
O'Reilly, M. A., Waspe, A. C., Chopra, R., Hynynen, K. MRI-guided Disruption of the Blood-brain Barrier using Transcranial Focused Ultrasound in a Rat Model. J. Vis. Exp. (61), e3555, doi:10.3791/3555 (2012).
Focused ultrasound (FUS) disruption of the blood-brain barrier (BBB) is an increasingly investigated technique for circumventing the BBB1-5. The BBB is a significant obstacle to pharmaceutical treatments of brain disorders as it limits the passage of molecules from the vasculature into the brain tissue to molecules less than approximately 500 Da in size6. FUS induced BBB disruption (BBBD) is temporary and reversible4 and has an advantage over chemical means of inducing BBBD by being highly localized. FUS induced BBBD provides a means for investigating the effects of a wide range of therapeutic agents on the brain, which would not otherwise be deliverable to the tissue in sufficient concentration While a wide range of ultrasound parameters have proven successful at disrupting the BBB2,5,7, there are several critical steps in the experimental procedure to ensure successful disruption with accurate targeting. This protocol outlines how to achieve MRI-guided FUS induced BBBD in a rat model, with a focus on the critical animal preparation and microbubble handling steps of the experiment.
1. Ultrasound and MRI Setup
An MRI-compatible three-axis focused ultrasound system was used in this study (FUS Instruments, Inc., Toronto, Ontario, Canada). Two different ultrasound transducers were used: an in-house constructed 551.5 kHz spherically-focused transducer (radius of curvature = 60 mm, external diameter = 75 mm, internal diameter = 20 mm), and a 1.503 MHz, 8-sector array with integrated PZT hydrophone (Imasonic Inc., Voray-sur-L'Orgnon, France) driven as a single element spherically focused transducer (FN = 0.8, aperture = 10 cm). An MRI-compatible (PVDF) receiver8 was used to record acoustic emissions when the 551.5 kHz transducer was used. If different equipment is used, the following are suggested:
2. Animal Preparation
3. Target Selection
4. Microbubble Preparation
Definity microbubbles (Lantheus Medical Imaging, MA, USA) are used by several groups for microbubble mediated FUS induced BBBD2,5,7. Appropriate dosages for other microbubble types can be found in the literature11,12.
5. Ultrasound Delivery
Appropriate pressures at different frequencies can be estimated using a mechanical index of 0.46 13.
6. MRI Evaluation of Treatment Outcome
7. Representative Results
MRI contrast agents can be successfully delivered through the BBB using focused ultrasound and circulating microbubbles. Figure 2 shows typical pre and post-FUS T1-w images. Figure 2B shows a contrast enhanced (CE) T1-w image with distinct focal openings in four sonicated locations. Sonication locations 1 and 2 show particularly bright enhancement. In Fig.3 locations 1 and 2 can also be seen to correspond with T2-w high signal, indicating edema.
The extent of T2-w edema can sometimes be more easily visualized on sagittal slices. Figure 4 shows CE-T1-w and T2-w sagittal slices through sonication locations 1 and 3. Edema is visible at location 1 but not location 3.
Spectral analysis of captured acoustic emissions data (Fig.5) may show harmonic emissions and/or sub/ultra harmonic emissions when stable cavitation is occurring. Harmonics can also arise from tissue non-linearities, while sub and ultraharmonic emissions can only occur as a result of bubble activity14. At higher pressures wideband emissions indicating inertial cavitation may also be detected. However these have been associated with greater amounts of red blood cell extravasations and microdamage than sonications without inertial cavitation11.
The use of higher sonication frequencies results in more localized openings due to the smaller focal spot size. Figure 6 shows that higher frequencies can be used to create smaller regions of opening. This allows investigation of effects mid-brain with fewer near-skull effects.
Figure 1. Experimental setup.
Figure 2. Pre (left) and post (right) treatment T1-w images of a rat brain showing enhancement at four sonication locations.
Figure 3. Pre (left) and post (right) treatment T2-w images of a rat brain (same animal as Fig.2) showing T2-w edema at sonication locations 1 and 2.
Figure 4. Post treatment sagittal T1-w (left) and T2-w (right) images from the same rat brain as Figs. 2 and 3. The opening at location 1 (left) correlates with T2-w edema (right). Location 3 shows opening (left) but no T2-w edema.
Figure 5. Frequency spectrum from data captured during a single 10 ms burst at 551.5 kHz. The fundamental frequency ( f0) as well as harmonics (2f0) and sub/ultraharmonics (0.5 f0, 1.5f0) are visible.
Figure 6. Post treatment CE-T1-w axial (left) and sagittal (right) images from a rat brain sonicated in four locations at 1.503 MHz. BBB openings at this frequency are seen to be more localized.
Preparation of the animals and microbubbles are the most critical aspects of this procedure. The hair on the animal's head must be entirely removed to avoid attenuating the ultrasound beam. The BBB can be disrupted under isofluorane anesthetic, however, it becomes more difficult to achieve consistent opening.
The microbubbles should always be handled with care and small gauge, large diameter needles must be used when drawing up, in order to avoid breaking them. Similarly, the smallest gauge catheter which can be reasonably used in the tail vein should be employed (22-gauge is recommended). If a smaller catheter is required to achieve proper placement in the vein then extra care must be taken during the microbubble injection. The microbubble injections should always be done slowly.
Burst mode sonications should always be employed. If continuous wave sonications are used the microbubbles will not replenish into the vessels at the transducer focus and BBBD will not be achieved. If CE-T1-w images post-treatment do not show disruption, the treatment can be repeated checking that the water level is topped up so that the animals head is in the water and that there are no air bubbles trapped on the skin surface.
Higher frequencies provide better localization in small animal models but require higher in situ pressures to induce opening. It is also important to consider that losses due to the skull are higher at higher pressure and must be accounted for when estimating in situ pressures. At 0.5 MHz transmission through rat skull is approximately 73%8, but drops to approximately 50%15 at 1.5 MHz. Attenuation can be assumed to be 5 Np m-1 MHz-1 in brain tissue4. Higher frequencies are better suited for work in small animal models but are less clinically relevant.
This MRI-guided approach provides advantages over ultrasound-guided techniques by allowing very precise targeting as well as treatment outcome assessment immediately post-treatment.
K. Hynynen and R. Chopra are co-founders of FUS Instruments, Inc. R. Chopra, A. Waspe and K. Hynynen are shareholders in FUS Instruments, Inc. K. Hynynen receives research support from FUS instruments, Inc.
The authors would like to thank Shawna Rideout-Gros and Alexandra Garces for their help with the animal care, and Ping Wu for her technical assistance. Support for this work was provided by the National Institutes of Health under grant no. EB003268, and the Canada Research Chairs Program.
|Small Animal Focused Ultrasound System||FUS Instruments, Inc.||RK-100|
|Definity||Lantheus Medical Imaging|
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