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DOI: 10.1148/radiol.2202001804
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(Radiology. 2001;220:640-646.)
© RSNA, 2001

Experimental Studies

Noninvasive MR Imaging–guided Focal Opening of the Blood-Brain Barrier in Rabbits1

Kullervo Hynynen, PhD, Nathan McDannold, BS, Natalia Vykhodtseva, PhD and Ferenc A. Jolesz, MD

1 From the Department of Radiology, Brigham and Women’s Hospital, Harvard Medical School, 75 Francis St, Boston, MA 02115. Received November 15, 2000; revision requested December 26; revision received February 16, 2001; accepted March 23. Supported by NCI research grant CA76550. Address correspondence to K.H. (e-mail: kullervo@bwh.harvard.edu).


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
PURPOSE: To determine if focused ultrasound beams can be used to locally open the blood-brain barrier without damage to surrounding brain tissue and if magnetic resonance (MR) imaging can be used to monitor this procedure.

MATERIALS AND METHODS: The brains of 18 rabbits were sonicated (pulsed sonication) in four to six locations, with temporal peak acoustic power ranging from 0.2 to 11.5 W. Prior to each sonication, a bolus of ultrasonographic (US) contrast agent was injected into the ear vein of the rabbit. A series of fast or spoiled gradient-echo MR images were obtained during the sonications to monitor the temperature elevation and potential tissue changes. Contrast material–enhanced MR images obtained minutes after sonications and repeated 1–48 hours later were used to depict blood-brain barrier opening. Whole brain histologic evaluation was performed.

RESULTS: Opening of the blood-brain barrier was confirmed with detection of MR imaging contrast agent at the targeted locations. The lowest power levels used produced blood-brain barrier opening without damage to the surrounding neurons. Contrast enhancement correlated with the focal signal intensity changes in the magnitude fast spoiled gradient-echo MR images.

CONCLUSION: The blood-brain barrier can be consistently opened with focused ultrasound exposures in the presence of a US contrast agent. MR imaging signal intensity changes may be useful in the detection of blood-brain barrier opening during sonication.

Index terms: Blood-brain barrier • Experimental study • Magnetic resonance (MR), guidance • Magnetic resonance (MR), temperature monitoring • Ultrasound (US), therapeutic


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Despite the large number of potent neurologically active substances and drugs, many central nervous system diseases are difficult to treat due to the inability of large-molecular-size agents to penetrate the blood-brain barrier (1). The intact blood-brain barrier is a major limitation in using neuropeptides, proteins (enzymes, antibodies), chemotherapeutic agents, and genes for therapy (2). Targeted delivery of these substances is preferred because after nonlocalized diffuse opening of the blood-brain barrier, agents systemically administered or injected into the bloodstream have undesired often dose-limiting side effects due to their spread within the central nervous system. Most of the neurologically active substances could have a more definitive therapeutic effect if their release in the brain could be well localized. A localized, transient, and reversible opening of the blood-brain barrier could provide anatomically selective targeted drug delivery.

In addition to the physiologic barrier at the level of basal lamina (3), the blood-brain barrier is formed by the endothelial cells of the cerebral microvessels that connect to each other by means of intracellular attachments known as tight junctions (1,4). The factors that determine penetration of substances from the blood to the central nervous system are lipid solubility, molecular size, and charge. The blood-brain barrier prevents penetration of ionized water-soluble materials with a molecular weight greater than 180 d (4). Chemical modification of drugs to make them lipophilic or the use of other carriers, such as amino acid and peptide carriers, are two ways to aid propagation through the barrier. Another option is to diffusely alter the function of the blood-brain barrier by temporarily opening the tight junctions, which is now possible with an increasing number of chemicals.

Most of the clinical experience, however, has been confined to opening the blood-brain barrier by using intraarterial injection of hyperosmotic solutions such as mannitol. This causes the endothelial cells to shrink which results in an opening of the tight junctions that lasts for a few hours (5). Both osmotic and chemical methods require an invasive intraarterial catheterization and produce diffuse, nonfocal, transient blood-brain barrier opening within the entire tissue volume supplied by the injected artery branch (1,4). A more localized drug delivery method can be accomplished only by injecting through a needle or catheter directly into the targeted brain area (4). Such direct injections are invasive and require opening the skull, penetrate nontargeted brain tissue, and carry the risk of neurologic damage, bleeding, and infection.

It has been known for some time that pulsed ultrasound can sometimes induce focal blood-brain barrier opening without damaging neurons in the sonicated area (6). However, to our knowledge, it has yet to be demonstrated that reversible local opening of the blood-brain barrier can be accomplished in a practical, controlled, and reproducible manner.

The purpose of our study was to determine if focused ultrasound beams can be used to locally open the blood-brain barrier without damage to the surrounding brain tissue and that magnetic resonance (MR) imaging can be used to monitor this procedure.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Ultrasound Equipment
Ultrasound fields were generated by a 16-sector, focused, piezoelectric transducer with a 100-mm diameter, 80-mm radius of curvature, and a resonant frequency of 1.63 MHz (manufactured in our laboratory; for the manufacturing techniques refer to reference 7). Each sector was driven with separate identical radio-frequency signals generated by a multichannel driving system with the control of a computer similar to one reported previously (8). For these experiments, all of the radio-frequency signals were in phase and equal in amplitude, which resulted in a single focus close to the geometric focus. The acoustic power output and the focal pressure amplitude, as a function of applied radio-frequency power, was measured as described previously (9). Pressure measurements were obtained in water at the two lowest pressure amplitude values and then extrapolated based on the measured acoustic power. The reported values are estimates for the pressure amplitude in the brain obtained by decreasing the measured water values by a factor of 0.92. This value was based on Ultrasound attenuation through 10 mm of brain, with a mean attenuation coefficient of 5 Np/m/MHz at 1.63 MHz.

The sonications were performed with MR imaging guidance and monitoring. The transducer array was mounted on an experimental positioning device (TxSonics, Haifa, Israel) integrated in the MR imaging table. The basic principle of this system was described previously (10). For these experiments, the system was used only to move the transducer. All of the sonication-related aspects were executed by an external personal computer that controlled the sonications by the multichannel amplifier system.

Animals
Eighteen male New Zealand white rabbits (Millbrook Farm, Amherst, Mass; approximately 4 kg), which included three control rabbits, were anesthetized by using a mix of ketamine (Aveco, Fort Dodge, Iowa; 40 mg per kg of body weight) and xylazine (Lloyd Laboratories, Shenandoah, Iowa; 10 mg/kg). (Note: There is some evidence that ketamine may be a neuroprotector [11,12] and thus could influence our results.) A piece of skull (approximately 20 x 20 mm) was removed, and the skin was replaced over the bone window. The sonications were executed after the wound healed and any air under the skin dissolved (a minimum of 10 days after the surgery). The animals were placed on their backs on a water blanket through which temperature controlled water was circulated to maintain the body temperature of the animal. The head was fixed in the treatment position by means of an acrylic holder. The skin (hair removed) on the top of the head was coupled to the waterbath with a degassed-water bag. The free water surface of the bag coupled the sound into the skin. Figure 1 shows a diagram of animal positioning for the experiment. Experiments were approved by our institutional animal committee.



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  		Figure 1. Diagram shows the sonication arrangement. RF = radio frequency

 
Sonications
T2-weighted MR images (the parameters will be described later) were obtained to localize the bone opening and the target depth in the brain. Before the experimental sonications were performed, a low-power (acoustic power, 1.1 W), continuous-wave, 10-second sonication was delivered while MR thermometry was performed. This was about 60% of the power that did not produce brain damage in the same experimental conditions (9). Focal temperature elevation was visible in all cases. Often a small (on the order of 1 mm) correction was required to make the target location and the focus overlap. After alignment, sonications were delivered (K.H. and N.M.) at four to six locations, 10-mm deep in the brain. The acoustic power levels ranged between 0.2–11.5 W, which translated to an acoustic temporal and spatial peak intensity range of 16–690 W/cm2. Sonications were pulsed with a burst length of 10 or 100 msec and a repetition frequency of 1 Hz. The duration of the whole sonication was 20 seconds. The number of sonications for each of the power settings is outlined in the Table.


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  		Time of Sacrifice after Sonication in 15 Rabbits Included in Histologic Examination

 
Approximately 10 seconds prior to the start of each sonication, a bolus of ultrasonographic (US) contrast agent (Optison; Mallinckrodt, St Louis, Mo) that contained microbubbles (mean diameter, 2.0–4.5 µm; concentration, 5–8 x 108 bubbles per milliliter) was injected in an ear vein. The injected volume was 0.05 mL/kg, which was in the range (0.5–5.0 mL) recommended for human use by the manufacturer. The bolus was flushed from tubing that extended out of the magnet by injecting approximately 1 mL of saline (Fig 2). A 5- to 10-minute delay between sonications allowed the bubbles to clear from the circulation.



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  		Figure 2. Diagram of experiment time line. FSE = fast spin echo, IV = intravenous, SPGR = spoiled gradient echo.

 
Three control rabbits were sonicated in exactly the same way, except that the US contrast agent was not injected into the animals, and all of the locations (13) were sonicated at the highest power level.

MR Imaging
The MR imager was a 1.5-T system (Signa; GE Medical Systems, Milwaukee, Wis). A 7.5-cm diameter surface coil was placed under the head to improve the signal-to-noise ratio. The changes in temperature-dependent proton resonant frequency shift (13) were mapped with a fast spoiled gradient-echo or a fast gradient-echo sequence (40.9/19.9 [repetition time msec/echo time msec]; flip angle, 30°; bandwidth, 3.57 kHz; resolution, 256 x 128; field of view, 12 x 9 cm; section thickness, 3 mm). The bandwidth allowed us to control the echo time in the fast spoiled gradient-echo sequence. It was selected such that adequate temporal sampling of the temperature elevation during the sonication could be achieved. Twenty images were obtained in a series with the total acquisition time of 80 seconds. The first image was triggered 4 seconds prior to the start the injection of contrast medium. The imager was programmed to reconstruct the magnitude, real, and imaginary images for each of these time points. The real and imaginary parts were used to calculate the phase difference between the two time points as described in reference 14. The imaging plane was located across the focus (perpendicular to the beam axis) at the expected focal depth.

After the sonications, T1-weighted fast spin-echo MR images were obtained (500/17 msec; echo train length, four; three signals acquired; field of view, 10 cm; matrix size, 256 x 256; section thickness, 1.5-mm interleaved; bandwith, 16 kHz). This imaging was repeated after a bolus of gadopentetate dimeglumine (Magnevist; Berlex Laboratories, Wayne, NJ; molecular weight, 928 d) was injected into ear veins of the rabbits (a dose of 0.125 mmol/kg) to depict the blood-brain barrier opening. Preliminary information of the time dependence of the opening was obtained in one brain (six separate locations), with additional bolus injections at 2.0, 3.5, and 5.5 hours after the sonications. Such long-term follow-up was not possible in the other rabbits due to the MR imaging time allocated for this study. A T2-weighted fast spin-echo sequence (2,000/85; echo train length, 8; two signals acquired; field of view, 10 cm; section thickness, 1.5 mm) was used to depict edema (increased signal intensity) in the brain tissue (N.M. and K.H.). Depending on the survival time of the animal, the imaging was repeated 48 hours after the sonications. Due to the observation that the blood-brain barrier was closed at 48 hours, additional images (six locations) were acquired at 24 hours in one case. To establish if long-term effects were induced, the imaging was repeated (in 10 locations) in two cases 7 days after the sonications.

Signal Intensity Analysis
The amount of contrast enhancement was evaluated (K.H. and N.M.) in the focal spot (location of the ultrasound beam focus) by averaging the signal intensity across 3 x 3 voxels (1.17 x 1.17 mm) after normalizing to the baseline value before the contrast injection. The contrast enhancement between two groups was compared by using a two-tailed t test.

Animal Sacrifice
The animals were sacrificed between 2 hours and 7 days after the sonications. Animals that were sacrificed on day 1 typically occurred at 4 hours after the sonications. However, one died at 2 hours and one died at 7 hours, presumably due to complications from the anesthesia. The brains were then immediately fixed in formalin, embedded in paraffin, and serially sectioned at 6 µm (across the beam direction; parallel to the MR image sections). Every 50th section (interval, 0.3 mm) was stained with hematoxylin-eosin for histologic examination. The purpose of the microscopic examination was to look for areas with damaged neurons (similar to those presented in reference 15). The histologic examination was performed (N.V.) without the knowledge of the number, location, or power of the sonications.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
 REFERENCES
 
For the main set of experiments (burst length of 0.1 second and duty cycle of 10%), the mean maximum temperature elevations measured for peak acoustic powers 11.5, 5.8, and 2.7 W were 4.8°C ± 1.7 (mean ± SD), 3.4°C ± 0.9, and 2.3°C ± 0.8, respectively. In most cases, sonications produced focal blood-brain barrier opening without visible damage to the tissue, as depicted with MR imaging, in the beam path (Fig 3). The focal increase in the signal intensity change on the T1-weighted images caused by leakage of the contrast agent into the brain was dependent on the applied acoustic pressure amplitude (Fig 4). The contrast enhancement at the sonicated locations was significantly higher than the enhancement at the control locations at all pressure amplitude values (P < .001). However, the opening was not dependent on burst length or average acoustic power with the parameters studied; that is, the sonications with 100-msec and 10-msec bursts (ie, 10% and 1% duty cycle) did not produce a statistically significant difference in enhancement (P = .79). The sonications without the injection of the US contrast agent did result in a slight increase in the contrast enhancement (0.05 ± 0.03) when compared with nonsonicated brain locations (0.02 ± 0.02) (P < .005).



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  		Figure 3. Left, T1-weighted fast spin-echo (500/17) MR image of rabbit brain after sonication shows contrast enhancement at the four locations (arrows). Images were obtained across the focal plane (coronal). Pressure amplitude values were 4.7 MPa at location 1, 2.3 MPa at location 2, 3.3 MPa at location 3, and 1.0 MPa at location 4. Right, Same image obtained along the beam axis (transverse).

 


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  		Figure 4. Graph shows normalized signal intensity change (mean ± SD) at the focal volumes after injection of MR imaging contrast agent as a function of the pressure amplitude. Contrast agent was injected after locations in the brain were sonicated. Local signal intensity increases after injection indicate opening of the blood-brain barrier. Signal intensity change was proportional to the applied focal pressure amplitude.

 
Contrast material–enhanced imaging repeated up to 6 hours after the sonications showed continuous but declining opening of the blood-brain barrier (Fig 5). However, no contrast enhancement was evident in the rabbit imaged on the next day or in the rabbits imaged 2 days after the low-power sonications. (Three of 17 of the high-power sonications that produced brain tissue damage also induced long-lasting breakdown of the blood-brain barrier.) In addition, the contrast enhancement was not present at 7 days.



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  		Figure 5. Graph shows normalized signal intensity change immediately after injection of MR imaging contrast agent as a function of time after the sonication for two pressure amplitude values in one rabbit. Each time point was after a new bolus injection. Signal intensity change was the largest immediately after the sonications and rapidly decreased as a function of time. A similar result was observed in other sonicated locations in the same brain.

 
The magnitude images acquired during the sonications showed a reduction in the signal intensity at the focal locations (Fig 6). This reduction, which remained after the sonications, was dependent on pressure amplitude and correlated with the signal intensity change at the same location observed after an injection of a bolus of MR imaging contrast agent (Fig 7). This magnitude change was more pronounced with the fast spoiled gradient-echo MR sequence compared with the fast gradient-echo sequence. These signal intensity changes remained on the images acquired for the subsequent sonication locations (up to 60 minutes).



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  		Figure 6. Magnitude image from fast spoiled gradient-echo MR sequence (coronal, no contrast agent, 49.9/19.9) obtained during the last sonication of the same rabbit brain as in Arrows indicate locations of sonications. Pressure amplitude values were 4.7 MPa at location 1, 2.3 MPa at location 2, 3.3 MPa at location 3, 1.0 MPa at location 4. Image shows focal enhancement at sonicated locations and indicates opening of blood-brain barrier.

 


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  		Figure 7. Graph shows normalized signal intensity (SI) increase at sonicated locations after injection of MR imaging contrast agent versus normalized signal intensity in the fast spoiled gradient-echo MR image acquired during the sonications. The signal intensity decrease on fast spoiled gradient-echo images acquired during sonications decreased proportionally to the signal intensity increase after injections. Thus, the signal intensity change in the fast spoiled gradient echo image correlated with the blood-brain barrier opening.

 
The histologic evaluation showed damage to brain tissue in approximately 70% (seven of 10) of the sonicated locations at the highest power level used (Fig 8). At pressure amplitudes between 2–3 MPa, the percentage of locations that showed tissue damage was approximately 25% (two of eight, three of 13). There was no neuronal damage at the lowest three exposure levels. However, in many cases, the sonicated locations showed red blood cells in the brain tissue outside of the blood vessels, indicating vascular damage (Fig 9). The rabbits that survived 7 days did not show any neurologic symptoms.



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  		Figure 8. Graph shows percentage of focal locations that showed neuronal damage as a function of the pressure amplitude of the sonication. The three lowest pressure amplitude levels (0.7 MPa and two duty cycle exposures at 1 MPa) did not show any neuronal damage although they showed opening of the blood-brain barrier (Fig 4).

 


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  		Figure 9. Histologic results in rabbit brain 48 hours after sonications in the presence of cavitation bubbles. (Hematoxylin-eosin stain.) Two sonication locations are shown. In the first (a, b), the pressure amplitude was 1.0 MPa, and no neuronal damage was observed. In the second, the pressure amplitude was 2.3 MPa, and neuron loss was observed (c, d). a, Perivascular extravasations (microhemorrhages, indicated by arrows) in the sonicated region suggest the blood-brain barrier disturbance (Original magnification, x25). b, Detail of a shows intact neurons (arrows) in the hemorrhagic area. (Original magnification, x250.) c, Extensive hemorrhages (red stained areas, such as those indicated by the arrows) associated with acute degeneration of the neuropil (cerebral infarct). (Original magnification, x25.) d, Detail of c demonstrates the lack of neurons in the infarcted area. (Original magnification, x250.)

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
REFERENCES
 
This study was a continuation of our earlier work that explored various ultrasound exposures and found subsequent unpredictable, occasional blood-brain barrier opening (6). This study demonstrates for the first time, to our knowledge, that low-power focused ultrasound applied after intravenous administration of US contrast agent can be used to induce reproducible, consistent, focal blood-brain barrier opening without obvious permanent damage to the brain tissue. The targeting and monitoring of the procedure was accomplished with MR imaging guidance. Low-power focused ultrasound was able to consistently produce transient blood-brain barrier opening by using intravascular gas bubbles as cavitation sites. The induction of these bubbles allowed us to reduce the temporal average spatial peak powers to approximately two orders of magnitude below the level required for thermal damage of the brain tissue. These power levels can be easily reached through the skull by using phased-array applicators that compensate for the skull induced distortions (16). The technology described here has implications for the feasibility of local, noninvasive, targeted drug delivery or gene therapy in the brain.

The blood-brain barrier opening allowed an MR imaging contrast agent with a molecular weight of 928 d to enter into the brain. Hence, many of the chemotherapeutic agents for potential for brain tumor treatments (molecular weight between 200 and 1,200 d) (4) could enter into the brain through this ultrasound-induced deficit. The presence of red blood cells in the extravascular space demonstrates more pronounced temporary damage of the capillary walls. However, we have yet to establish how large of a molecule can penetrate the opening or its relative dependence on the ultrasound exposure parameters. These variables, along with the duration of the opening and potential vascular damage related to the sonication conditions need to be explored.

The observation that the change in the magnitude signal intensity of the fast spoiled gradient-echo MR sequence during the sonication correlated with the amount of contrast enhancement may provide a way for online monitoring of the exposures. Since the localized signal intensity changes depicted at the first sonication location remained visible for up to 60 minutes, we are confident that they were not caused by temperature elevation. The source of this signal intensity change is not clear, but a possible explanation is a change in tissue susceptibility induced by the extravasation of some red blood cells. This phenomenon requires more study to explain its origin and to optimize the pulse sequences for its detection.

The low pressure amplitude sonications did not produce any MR imaging–detectable tissue effects or histologic effects on the neurons. In our previous work with ultrasound-induced tissue damage and apoptosis in brain (15), we used hematoxylin-eosin staining together with TUNEL and cresyl violet stains and never observed cell death if it was not also found on hematoxylin-eosin stained sections. For this reason, only hematoxylin-eosin staining was used in the current study. However, there may be some effects that would require more advanced methods for identification. This may be a topic of further study.

The maximum temperature elevation from the pulsed sonication at the highest power level was approximately 5°C with the US contrast agent. The lowest temporal average acoustic power level that consistently produced blood-brain barrier opening was 200 times lower than the highest power used in this study. Since the temperature elevation should be linearly proportional to the applied acoustic power, we can assume that the temperature elevation was approximately 0.025°C at this power level. Therefore, blood-brain barrier opening is most likely a result of mechanical stresses associated with the interaction of the ultrasound with the gas bubbles.

The lowest pressure amplitude level that induced the blood-brain barrier opening was less than 0.8 MPa, which indicates that similar events may be detected during diagnostic ultrasound exposure. Although the burst length and frequency used in this study were not the same as used with diagnostic US examinations, there may be a safety concern when brain imaging is performed with US contrast agents containing microbubbles.

Our results are in good agreement with a growing body of literature (17–19) that demonstrates the various biologic effects induced by ultrasound in the presence of US contrast agents. US contrast agents have been shown to have potential therapeutic use in enhancing tissue damage (20), accelerating thrombolysis (21), increasing blood vessel permeability in muscle (22), and enhancing gene therapy (23). However, our current observation, that it can aid in focal image controlled blood-brain barrier opening, could have even greater clinical importance.

Practical application: The sonications in this study were delivered through a craniotomy in the rabbit brain. This could be performed in humans completely noninvasively by sonicating through the skull bone. Sharp focal spots with high energy concentrations can be introduced deep in the brain with large low-frequency phased arrays designed to compensate for distortions in the ultrasound beam propagation induced by the skull bone (16,24). By combining such arrays with an MR imaging system, accurate well-controlled targeting of brain tissue should be possible (9). A larger target volume could be treated by sonicating at multiple locations. However, since we injected a bolus of US contrast agent prior to each sonication, the maximum limit of the manufacturer would be reached with three sonications if the same concentration were used in humans. It may be possible to increase the limit for therapeutic use, reduce the bubble concentration, or use an agent that has bubbles that last longer in the circulation. It may also be possible to sonicate multiple focal spots by using a phased array during a single bolus injection.

In conclusion, this study demonstrates a potential method for noninvasive image-guided focal blood-brain barrier opening. This method, combined with recent advances in US technology that permit sonications through the intact skull (16,25), may allow new approaches for targeted brain therapy to be possible. It could permit noninvasive methods of treatment for central nervous system diseases such as brain tumors, seizure, and movement disorders. Specifically, it would provide targeted access for chemotherapy and gene therapy, and allow the use of recombinant proteins, monoclonal antibodies, or antisense oligonucleotides as pharmaceuticals for the brain. It could even provide a vascular route for implanting cells in the brain (26).


   ACKNOWLEDGMENTS
 
The authors are grateful to TxSonics (Haifa, Israel) for providing the MR imaging–compatible transducer positioning system.


   FOOTNOTES
 
Author contributions: Guarantor of integrity of entire study, K.H.; study concepts, K.H., N.M.; study design, K.H.; literature research, K.H.; experimental studies, K.H., N.M., N.V.; data acquisition and analysis/interpretation, K.H., N.M., N.V.; statistical analysis, K.H., N.M., N.V.; manuscript preparation, K.H., N.M.; manuscript definition of intellectual content, K.H.; manuscript editing, revision/review, and final version approval, all authors.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 

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