Pressure and microbubble size dependence study of focused ultrasound-induced blood-brain barrier...

A Quantitative Pressure and Microbubble-Size DependenceStudy of Focused Ultrasound-Induced Blood-Brain BarrierOpening Reversibility in vivo Using MRI

Gesthimani Samiotaki1, Fotios Vlachos1, Yao-Sheng Tung1, and Elisa E. Konofagou1,2

1Department of Biomedical Engineering, Columbia University, New York, USA2Department of Radiology, Columbia University, New York, USA

AbstractFocused Ultrasound (FUS) in conjunction with the systemic administration of microbubbles hasbeen shown to open the blood-brain barrier (BBB) selectively, non-invasively and reversibly. Inthis study, we investigate the dependence of the BBB opening’s reversibility on the peak-rarefactional pressure (PRP) (0.30 MPa - 0.60 MPa) as well as the microbubble size (diameters of1-2, 4-5 or 6-8 microns) in mice using contrast-enhanced T1-weighted (CE-T1) MR images (9.4T). Volumetric measurements of the diffusion of Gd-DTPA-BMA into the brain parenchyma wereused for the quantification of the BBB-opened region on the day of sonication and up to five daysthereafter. The volume of opening was found to increase with both pressure and microbubblediameter. The duration required for closing was found to be proportional to the volume of openingon the day of opening, and ranged from 24 hours, for the smaller microbubbles, to 5 days for highPRPs. Overall, larger bubbles did not show significant differences. Also, the extent of BBBopening decreased radially towards the focal region until BBB’s integrity was restored. In thecases where histological damage was detected, it was found to be highly correlated withhyperintensity in the pre-contrast T1 images.

KeywordsBlood-brain barrier; Focused ultrasound; Gd-DTPA-BMA; CE-T1

INTRODUCTIONThe blood-brain barrier (BBB) is a specialized vascular system consisting of endothelialcells lining cerebral microvessels, with highly selective membranes, connected together bytight junctions (TJ) (1). The structure of the brain endothelium together with pericytes andastrocyte feet, tightly regulates the flux of molecules between the blood and the brainparenchyma through the capillary lumen, forcing a trancellular route, rather than aparacellular route as in most endothelia (2), albeit, with a much lower degree of trancytosisactivity than in the peripheral endothelium. The BBB acts as a physical and metabolicbarrier, with a range of passive and active features, which restricts the penetration ofmolecules, except if they are both lipid soluble and of a molecular weight (MW) <400 Da(3). The BBB, among the rest of biological membranes, has uniquely restrictive permeabilityproperties and effectively excludes 98% (4) of small-molecule drugs and 100% of all large

Address Correspondence to: Dr. Elisa E. Konofagou, Department of Biomedical Engineering, Columbia University 351 EngineeringTerrace, mail code 8904 1210 Amsterdam Avenue New York, NY 10027 USA [email protected] Tel:212-854-9661/212-342-0863.

NIH Public AccessAuthor ManuscriptMagn Reson Med. Author manuscript; available in PMC 2013 May 20.

Published in final edited form as:Magn Reson Med. 2012 March ; 67(3): 769–777. doi:10.1002/mrm.23063.

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molecule drugs and neurotherapeutic agents, including recombinant proteins and enzymes,antibodies, etc. (3).

The use of focused ultrasound (FUS) in conjunction with microbubbles has beendemonstrated by several groups (5),(6),(7),(8) to successfully induce BBB opening non-invasively, locally and reversibly. The FUS–induced BBB opening has been shown by ourgroup to allow molecules of various molecular weights into the murine brain, such as Gd-DTPA-BMA (MW 574 Da) (5), and fluorescent dextrans (MW 3kDa and 70kDa) (9).Several particles within a wide size range have also been shown to pass through thedisrupted BBB, e.g.,MRI contrast agents such as Magnevist ® (MW: 938 Da) and MION(MW: 10,000 Da) (10), trypan blue (MW: 961), horseradish peroxidase (MW: 40,000 Da)(11) and antibodies (MW: 150,000 Da) (12). In this study, the MRI contrast agent was usedas a tracer to depict the area of opening quantitatively.

Reversibility of the BBB opening in several animal models has previously been shown, butthe timeline varied among different studies, together with the variation of parameters used.Using Optison (mean diameter of 2-4.5 μm) and a 260-kHz transducer at 0.40 MPa, or a0.69 MHz transducer at pressure amplitudes of 0.8 MPa and 1 MPa (11), contrast-enhancedfast spin-echo T1-weighted images using intravenously administered Magnevist ® revealedthat the BBB was closed 5h after sonication in rabbits (10). Using Sonovue (mean diameterof 2.5 μm) at 1.1 MHz, T1-weighted fast spin-echo images (1.5 T) showed that the BBBsignal enhancement in the opened region, following Magnevist® injection, returned in itsinitial state 8 h after sonication in rabbits (13), whereas using a 1.63 MHz transducer andvarying the acoustic pressures from 1 MPa up to 4.7 MPa, the BBB closed within 24-48 h(6). In rats, with the use of Optison, at 1.5 MHz and 1.1 MPa, immunoelectron microscopyshowed that the BBB was shown permeable to Horseradish Peroxidase Passage (HRP) andLanthanum Passage up to 6 hrs after sonication, and remained impermeable 24h later, whileimmunoelectron microscopy showed that tight junctions appeared disintegrated only up to4h after FUS and were later restored (14). Using Definity (mean diameter 1.1-3.3 μm) andan unfocused single-element transducer at 2.15 MHz, T1 spin-echo images (7 T), the BBBwas shown to recover within 4-27 h (15). In Alzheimer’s disease-model mice, usingSonovue at 1.525 MHz, and PRP of 0.50 MPa, the enhancement due to Gd-DTPA-BMA(Omniscan) in CE-T1 images (9.4 T) revealed that the BBB opening was a transientphenomenon, closing within 24 h (16). In large animals, i.e., pigs, using lipid or albumincoated microbubbles at 2 MHz transducer and 2.0 W/cm2, Evans blue colorspectromorphometry as well as contrast-enhanced T1-weighted spin-echo echo planarimages (1.5 T) revealed differences between the treated and untreated sides of the brainindicating altered BBB permeability, lasting up to 3h after sonication (17).

Even though the mechanism of FUS-induced BBB opening is not yet known, overall it hasbeen shown to be affected by acoustic parameters of the ultrasound beam, such as theapplied peak rarefactional pressure (5),(6),(7),(18),(19),(20), which we further investigatedin this study by longitudinally monitoring the BBB self-repairing characteristics. Theinteraction of intravenous administered microbubbles with the ultrasound beam generates arange of biological effects (21). At low acoustic pressures, stable cavitation of the gas-filledmicrobubbles, microstreaming and acoustic radiation force may lead to shear stress exertedby the microbubbles onto the vessel endothelial cells and these effects can activate themechanosensitive ion channels (22) or deforms the vascular endothelium (23) yielding BBBopening. At higher acoustic pressure levels, or when oscillating near a boundary,microbubbles can collapse and produce shock waves and high-velocity jets (24),(25),(26), aphenomenon referred to as inertial cavitation. The BBB has been shown to open in thepresence of inertial cavitation, but without it being a necessary condition (20),(27).

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Most studies so far have been utilizing poly-dispersed ultrasound contrast agents (UCAs)(i.e. Definity, Sonovue and Optison). The duration of the microbbubles interaction with thevessel wall has been found to increase with the microbubble diameter (28). It has been wellestablished that the microbubble diameter dictates its resonance frequency (29),(30),expansion ratio (31), lifetime of stable cavitation (28),(32), pressure threshold for inertialcavitation (28),(32), and damage to the brain parenchyma (33). Choi et al. (19) used mono-dispersed microbubbles of 1-2μm and 4-5μm in diameter with a sorting method developedby Feshitan et al. (34) and 3-kDa fluorescent dextran. They showed that the FUS-inducedBBB opening was dependent on both the size distribution and injected microbubble volume,together with different pressure dependence for the opening threshold at each diameter size.Vlachos et al. 2011(35) (in press) also showed that the exchange rate between the bloodplasma and the brain tissue is proportional to the microbubble size and PRP, and that itincreases and then reaches a plateau, for larger mono-dispersed microbubbles (4-5 μm and6-8 μm) and higher PRPs, but is significantly lower for smaller bubbles (1-2 μm).

In this study, we investigated the dependence of both the spatial extent and the duration ofFUS-induced BBB opening in vivo with different microbubble sizes and PRPs. Gd-DTPA-BMA retention in the brain parenchyma was used as a signature of the area of BBB opening.Volumetric quantification of the region of BBB opening was assessed by measuring thediffusion volume of Gd-DTPA-BMA in the sonicated region of the brain, i.e., the righthippocampus, detected by the longitudinal signal enhancement. Starting with the day ofsonication and continuing up to five days following sonication, pre- and post- contrastenhancement T1-weighted high resolution MR images were consecutively acquired at eachtime-point. The objective of this study was to provide a more thorough insight into, as wellas a control over, the self repairing characteristic of the BBB after FUS opening and itsclosing timeline, when different PRPs and microbubble sizes are used. It could alsocontribute to the optimization of the parameters used, and to a more efficient drug delivery,i.e., repeated drug administration over an extended time period.

MATERIALS AND METHODSUltrasound Setup

The acoustic waves used were generated by a single-element, spherical-segment FUStransducer (center frequency: 1.525 MHz, focal depth: 90mm, radius: 30mm; Imasonic,France), which was driven by a function generator ( Agilent, Palo Alto, CA, USA) through a50-dB power amplifier (E&I, Rochester, NY, USA) (Fig.1(a)). A central-void (radius:11.2mm) of the therapeutic transducer held a pulse-echo ultrasound transducer (centerfrequency: 7.5MHz, focal length: 60mm), which was used for imaging, with their two focialigned. The imaging transducer was driven by a pulser-receiver (Olympus, Waltham, MA,USA) connected to a digitizer (Gage Applied Technologies, Inc., Lachine, QC, Canada). Acone filled with degassed and distilled water was mounted onto the transducer system andwas fitted with a polyurethane membrane (Trojan; Church & Dwight Co., Inc., Princeton,NJ, USA). The transducers were attached to a computer-controlled 3D positioning system(Velmex Inc., Lachine, QC, Canada). The targeting procedure has been described elsewhere(5). Briefly, the FUS transducer was moved 3 mm laterally of the sagittal suture and 2 mmanterior of the lambdoid suture (5). A needle hydrophone (Precision Acoustics Ltd.,Dorchester, Dorset, UK, needle diameter: 0.2 mm) was used to measure the three-dimensional pressure field in a degassed water-tank prior to the in vivo application. TheFUS focal spot overlapped with the right hippocampus and the latter portion of the thalamus,since the axial and lateral full-width at half-maximum intensities of the beam were 7.5 mmand 1 mm respectively. The left hippocampus fissure was used as a control, and was notsonicated. Pulsed FUS was emitted for 60 s, with a burst rate of 10 Hz, 100 burst cycles, atacoustic pressures adjusted to correspond to 0.30 MPa, 0.45 MPa and 0.60 MPa (peak-

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rarefractional), after accounting for 18% murine skull attenuation. These pressures wereobtained experimentally in degassed water (5). The mice were anesthetized using1.25-2.50% isoflurane (SurgiVet, Smiths Medical PM, Inc., Winsconsin, USA) mixed withoxygen during FUS.

Size Isolated MicrobubblesThe microbubbles used for this study were manufactured in-house, and they were size-isolated from a polydispersed microbubble distribution using differential centrifugation (34).The bubbles had a 1,2-disearoyl-sn-glycero-3-phosphocholine (DSPC) andpolyoxyethylene-40 stearate (PEG40S) lipid shell and perfluorobutane (PFB) core. After thecentrifugation and resuspension processes were repeated several times, three desired rangesof 1-2 μm, 4-5 μm and 6-8 μm in diameter were isolated. A bolus of 1μl/g at aconcentration of 107/ml was injected intravenously through the tail vein immediatelypreceding the sonication.

Magnetic Resonance ImagingBBB opening in the murine hippocampus was confirmed using a 9.4T system (BrukerMedical; Boston, MA, USA). All mice were anesthetized orally using 1-2% of isofluranemixed with oxygen and were placed inside the vertical-bore, having a fixed position in aplastic tube with a 3.0 cm diameter birdcage coil. Vital signals were monitored andrespiration rate was approximately 55 breaths/min. Each MRI session included a pre- and apost-contrast enhancement, T1-weighted 2D FLASH acquisition (TR/TE: 230/3.3ms, flipangle: 70°, NEX: 18, resolution 86μm × 86μm, slice thickness: 500μm, 23 slices, F.O.V.:22 mm × 16.5 mm, matrix size 256 × 192, receiver bandwidth: 50 kHz), obtainedrespectively 15 min after sonication and 55 min after injection of the MRI contrast agent(Gd-DTPA-BMA). Based on prior experience, signal enhancement in the area of thesonicated hippocampus reaches a peak approximately 1 hour after IP injection. Therefore,the CE-T1 images were acquired 55 min following the injection. Omniscan™ (Gd-DTPA-BMA) was used to enhance the MR contrast in the murine brain, and as a tracer for the BBBopening since it diffuses into the brain parenchyma following BBB’s altered permeability inthe targeted area. Gd-DTPA-BMA was admninistered intraperitoneally (IP) at a dose of 6mmol/kg. As previously reported, an IP bolus provides more temporally consistent andsustained MR enhancement as an IV bolus and is logistically simpler (15). Preliminary workon the IP bolus dosage indicated that the 6 mmol/ kg low enough so as to avoid the signaldecrease which is observed when, due to excessive Gd-DTPA-BMA, the T2 relaxivitydominates the T1 relaxivity. The same MRI session was repeated daily starting from the dayof the BBB opening (day 0) and lasting up to five days. In the five cases where significantsignal enhancement was detected on Day 5, MRI sessions were repeated on Day 7.

Animal preparationAll procedures used in this study involving animals were approved by the ColumbiaUniversity Institutional Animal Care and Use Committee. A total of forty-two (n=42) wild-type mice, (strain C57BL/6, mass: 20-25g, sex: male, Harlan, Indianapolis, IN, USA) wasused for the purposes of this study, separated into ten groups. Each mouse in the first ninegroups was sonicated with a different set of PRP, i.e., 0.30 MPa, 0.45 MPa or 0.60 MPa, anda microbubble diameter, i.e., 1-2 μm, 4-5 μm or 6-8 μm. These mice underwent MRI forseveral days after FUS. The sham group had three (n=3) mice, for which the wholeprocedure was repeated without FUS, i.e., anesthesia, injection of microbubbles, and MRIsessions for up to 5 days.

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Histology and ImagingOn day 7 after FUS-induced BBB opening, all mice were euthanized and transcardiallyperfused with 30mL PBS and 60mL 4% paraformaldehyde. Brains were soaked inparaformaldehyde for 24h. Skulls were removed, and the brains were fixed again in 4%paraformaldehyde for 6 days, followed by conventional post-fixation procedures. Theparaffin-embedded specimens were sectioned horizontally at a thickness of 6 μm. 24sections were stained and examined for each brain. Sections were stained with hematoxylinand eosin and then examined for red blood cell extravasations into the brain parenchyma aswell as cell loss.

Image Processing and Volumetric measurementsThe enhancement in CE-T1 MR images was quantified in elliptic cylindrical VOIsencompassing the hippocampal formation, at two contralateral regions, in the right(sonicated) and the left (control) hemisphere, on each brain for each day. The majordiameter of the elliptic cylinders was 4.3 mm, the minor diameter 3.4 mm, and the height 4.5mm, covering an area of approximately 14,000 voxels from a total of 9 consecutivehorizontal slices, on each side. Therefore, the total volume on each cylinder wasapproximately 52 mm3. In order to quantify the BBB opening volume at the sonicated side,an intensity threshold was determined and the contrast-enhancened pixels in the vessels andventricles were excluded. To address these issues, the signal intensity was averaged over asmall circular region of 1mm in diameter, centered around the non-sonicated contralateralside close to the hippocampus and used as a reference. The number of voxels in the right(sonicated) and left (control) VOI at an intensity of 2.5 standard deviations (S.D.) or abovethe reference were counted. The total number of these voxels in the left VOI was thensubtracted from the respective total number of voxels in the right VOI, to exclude volumecontrast-enhancement in the vessels and ventricles (Fig.1(b)). Pre-contrast images were usedfor the detection of any hyperintense areas before Gd-DTPA-BMA injection, and were notco-registered with the post-contrast.

Statistical analysisThe measurements in the sham group were used as the baseline, denoting that the integrityof the BBB was fully restored. Following the calculation of the mean and standard deviation(S.D.) of the volumetric measurements for each group, i.e., mice sonicated at the same PRPand microbubble size, a two-tailed Student’s t-test was performed, and if no statisticallysignificant difference compared to the control baseline was observed (p>0.05), then the BBBwas considered to have closed. In other words, the closing criterion was tested if met oneach day by each group and not by each measurement individually. All groups met theclosing criterion by day 5. For the five individual cases where signal enhancement wassignificant on day 5, MRI was repeated on day 7. These cases however, did not belong all inthe same group, and the groups they belonged to could still meet the closing criterion basedon the statistical analysis.

In order to investigate the differences in the timeline for closing between microbubble sizesand pressures, a statistical analysis was performed on the volume of diffusion of Gd-DTPA-BMA on Day 0. To evaluate the effect of the microbubble size, a two-tailed Student’s t-testwas performed for each PRP, i.e., between 1-2 μm and 4-5 μm, between 1-2 μm and 6-8μm, as well as between 4-5 μm and 6-8 μm. The purpose was to examine any statisticallysignificant differences between the different groups regarding the volume of BBB openingand the time required for the opened BBB to be reinstated.

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RESULTSAn example of the volume quantification is shown in Fig. 2. The VOIs were manually tracedto overlap with the right and left hippocampi. On both sides, voxels with an intensity 2.5S.D. or above the reference intensity are overlaid in red. As shown in these images, whenthere is no diffusion of Gd-DPTA-BMA from the vasculature to the brain parenchyma, e.g.,on the control side, only the vessels and the ventricles have CE-T1 intensity above thethreshold. However, when the permeability of the BBB is altered on the right hippocampusas a result of the FUS, Gd-DTPA-BMA diffuses in that region and the area of opening isoverlaid in red. The example in Fig. 2 shows the same brain in multiple 2D slices where 6-8-μm bubbles at a PRP of 0.30 MPa were used. The volume of opening was reduced radiallytowards the focal region over several subsequent days, until no trans-BBB diffusion wasdetected on day 3, signifying that the BBB was successfully reinstated.

For all cases of microbubble sizes and pressures studied, the BBB was found to be reinstatedby day 5, and the duration of opening depended on the microbubble diameter and PRP used.In Fig. 3, there are reconstructions of the horizontal planes, sliced coronally at the level ofthe hippocampal formation, at all pressures and microbubble sizes. In Fig. 3, it is also shownthat the area of Gd-DTPA-BMA diffusion in the brain and its spatial characteristics dependon the microbubble size and pressure used. The BBB permeability to Gd-DTPA-BMAappears to occur dorsally in the brain only in the cases when larger microbubbles were used,i.e., not in the 1-2 μm case, where diffusion of the contrast agent Gd-DTPA-BMA wasobserved mainly ventrally in the brain near the vasculature. This effect becomes moreapparent in higher PRPs, i.e., 0.60 MPa.

Volumetric measurements are shown in Figs. 4 (a), 4 (b) and 4 (c) for the 1-2 μm, 4-5 μmand 6-8 μm microbubble cases. At 0.30 MPa with the 1-2 μm microbubbles no BBBopening was detected. Depending on the microbubble and pressure used, the BBB closingoccurred within 24 hours and 5 days after sonication. More specifically, with the 1-2 μmmicrobubbles (Fig. 4(a)), closing was found to be closed on Day 1 and Day 2 at 0.45 MPaand 0.60 MPa, respectively. With the 4-5 μm and 6-8 μm microbubbles, the BBB was foundto be closed on Day 2 at 0.30 MPa, day 3 at 0.45 MPa and day 5 at 0.60 MPa. Aproportional relationship between the volume and the duration of the BBB openingregardless of the PPR and microbubble size used to induce the opening was thus established(Fig. 5). Linear regression showed a correlation of R2 = 0.72. Excluding Day 0 a logarithmicfit showed a correlation of R2 = 0.78.

The results of the statistical analysis on day 0 are shown in Table 1. At 0.30 MPa, openingwas induced only in the 4-5 μm and 6-8 μm cases, with no statistically significant difference(p>0.05) between these two diameter ranges (Table 1), and the BBB was restored by day 2in both cases. At 0.45 MPa, the volume of BBB opening induced with 1-2μm microbubbles,was statistically different (p<0.01) than that with larger microbubbles (Table 1), and also itoccurred within 24 hours, compared to 2-3 days needed in the cases of the largermicrobubbles. Finally, in Table 1, it is shown that at 0.60 MPa, there was a statisticallysignificant difference between the 1-2-μm and 6-8-μm bubbles (p< 0.05), and at least 2 dayswere required for the BBB to be reinstated in the 1-2 μm case, as opposed to 4-5 daysrequired for the larger microbubbles.

Good correlation was also found between all histological damage cases and hyperintensityin the pre-contrast T1-w images. An example is shown in Fig. 6, where hyperintensity wasdetected at the sonicated region in the pre-contrast image, and cell loss was detected on theH&E stained slices at the level of the hippocampus. Damage was observed upon histological

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examination only in five animals, approximately 13% of the total number of animals used,all of which were at higher PRPs.

DISCUSSIONIn this longitudinal study, the reversibility of BBB opening was investigated using Gd-DTPA-BMA that cannot cross the BBB when the BBB is reinstated or closed. Spatial (i.e.,volume) and temporal (i.e., duration) characteristics of the intact or BBB’s altered functionwere studied, while opening was induced using three different microbubble sizes and threedifferent PRPs. BBB opening induced by FUS has been shown in several studies to betransient but with differing reports on the duration which has indicated the dependence onthe acoustic parameters and bubbles used. Also, several studies have shown that the BBBopening is dependent on the acoustic pressure used as well as the microbubble size. Thefeatures of the BBB self-repairing characteristic were studied for the first time here under acombination of different acoustic parameters, and a range of mono-dispersed microbubbles.Our study indicated a proportional relationship between the BBB opening volume and thetime required for closing. Therefore, both the BBB opening volume and duration wereshown to be dependent on the acoustic pressure and the microbubble size used.

The spatial characteristics of the BBB opening and its reversibility were unveiled as follows.Firstly, as seen in the examples of Figs. 2 and 3, the BBB function was reinstated in areverse direction to that of the diffusion after opening, i.e., closing starts from the outeropened regions and ends at the focal region while also being dependent on the hippocampalvasculature. It has also been shown (34) that the permeability values towards the center ofthe focal region were higher. A first explanation is that the peak of the acoustic pressuredistribution lies at the center of the focal spot which may result in larger BBB openings,hence taking longer to close. Another explanation could be that where the vasculature isdenser, there is a higher number of opening sites, requiring thus longer timelines for closingto be completed.

Overall, even though the volume of opening induced by the 6-8-μm bubbles was greaterthan the volume induced by the 4-5 μm microbubbles, these two microbubble sizes did notdiffer significantly in this study (Table 1) and the days required for closing were also similar( Fig. 4(b) and (c). According to these observations, several conclusions can be drawn. First,it could be assumed that their differences were not significant because both 4-5-μm and 6-8-μm bubbles are within the diameter size range of the capillary, i.e., is 4-8μm, and thereforethey are both in contact with the capillary wall, exerting forces on it while being acousticallydriven. However, the mechanical stress on the capillary walls due to the acoustically drivenmicrobubbles for a specific pressure when induced by the 6-8-μm is larger than wheninduced by the 4-5-μm bubbles, because the latter would require higher PRP to reach thesame size expansion as the 6-8-μm and have the same effect (28), hence the BBB openingvolume slightly increased.

Another interesting observation is that the BBB opening volume using 1-2-μm bubbles wassignificantly lower than for the larger microbubbles (Table 1) and the BBB closedsignificantly faster. Therefore, for more efficient BBB recovery at high pressures, smallermicrobubbles may be preferable. The relative expansion of a microbubble is inverselyproportional to its resting diameter (31). 1-2-μm bubbles induced no opening at 0.30 MPa,where only stable cavitation is expected (20). This is probably due to the fact that stableoscillations of smaller microbubbles may not be sufficient to induce the needed repeatedstress against the capillary wall at 0.30 MPa, but are adequate at 0.45 MPa, and even moreso at 0.60 MPa. It has been also shown that fragmentation occurs more frequently formicrobubbles with a small rather than a large resting diameter at a threshold size of 2.5 μm.

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Below that level, fragmentation occurs (31) and bubbles smaller than 2 μm can befragmented prior to reaching the endothelium and not interact with the vessel wall (28).Therefore, it could be concluded that the smaller microbubbles were not capable of inducingopening in sites away from the bigger vessels, because they may have undergonefragmentation at the beginning of the FUS pulse before perfusing the microvasculature.Thereby, the therapeutic efficacy of 1-2 μm bubbles in the capillaries might be decreased, aneffect noted in previous studies (36),(37).

In this study, the timeline required for closing is longer than what has been reported byprevious studies, i.e., 3-24h ((6),(10),(11),(13),(14),(15),(16),(17)) as previously explained.The discrepancy with the findings presented here could be due to multiple factors. First ofall, the microbubble formulation, manufactured in-house, used in this study is different thanthe commercially available contrast agents used in the other BBB restoration studies, interms of the combination of shell properties, gas core and diameter size; therefore, thismicrobubble formulation used here is a parameter that could also induce variations.However, the size range of the commercially available microbubbles used in those studies iscloser to the case of the 1-2-μm bubbles used in our study, which are also shown here toinduce BBB opening that can close within 24 h. At lower PRPs, we showed that the BBBcloses faster, and in most of these studies, the PRPs used were below 0.50 MPa. Moreover,the FUS frequency (1.5 MHz) used in this study might also introduce differences comparedto other studies. The resonance frequency decreases with the microbubble radius, and alsodecreases with decreasing microvessel radius (30). Therefore, in this study, with the use of alower frequency for example, which would be closer to the resonance frequency of themicrobubbles within the microvessles and capillaries, and the aforementioned effects couldbe further enhanced. Finally, in other timeline studies, the agents used to cross the BBB forthe detection of opening, had larger molecular weights (Magnevist ®: 938Da, HRP: 40 kDa,Evans Blue: 961 Da) while it has been shown that the BBB becomes less permeable athigher molecular weights ((9),(38)). Since we administered Gd-DTPA-BMA , which is arelatively smaller (574 Da) agent and only above the size threshold to cross the BBB(400Da), our method may have an increased sensitivity regarding the detection of BBBopening, which could contribute to the longer times shown here.

In the five cases where cell loss was detected in the H&E stained brain sections on thesonicated region, hyperintensity was also detected on the pre-contrast MRI images at thecorresponding regions. The signal enhancement detected in these areas in the pre-contrastT1-w images could be therefore due to permanent damage, blood present in the brain orarrested Gd-DTPA-BMA in possibly damaged vasculature, and BBB could not becompletely restored. These cases however, did not belong all in the same group, and thegroups they belonged to could still meet the closing criterion based on the statisticalanalysis.

Another interesting finding in this study is that the BBB remained opened over several daysin some cases. It was thereafter restored and 87% of the cases showed no detectable damage,while the remaining 13% showed minimal cell loss. In the past, more significant damage hasbeen reported (39) including microvacuolated sites, dark neurons and sites with extravasatederythrocytes, when the BBB opening was induced in mice at similar PRPs, with Definitymicrobubbles (mean diameter: 1.1 – 3.3 μm) and the same PRF (10 Hz) but longer burstlengths (20ms) than the one used here. Also, in that study (39), survival time periods were30 min and 5 h, whereas in this study it was 7 days. It could therefore be concluded that,first, at shorter burst lengths opening is induced (40) with less damage, and second, that theself-repairing mechanism of the BBB could restore certain types of injury induced to thebrain after FUS provided sufficient time is allowed.

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There are certain limitations in this study that should be acknowledged and addressed.Firstly, BBB may have been disrupted longer than what could be detected by the sensitivitythe MRI system used and the resolution of the images acquired. Another limitation is thatthe first acquisition or post-contrast T1-w images was 1 h after FUS, and within this time itis possible that some cases with very small BBB opening might have not been detected.Finally, it was assumed that the circulation times and persistence of all the different sizes ofmicrobubbles were similar for the 10-20 sec interval between injection and FUS, however, itmust be taken into consideration that any differences could have had an impact on ourresults.

In this longitudinal study we showed dependence of the volume of BBB opening and thetime required for the BBB to be reinstated on the microbubble size and the acousticpressure. In addition, the time required for closing was found to be proportional to thevolume of opening induced by FUS, and BBB was shown to recover its functionalitybetween 24 h and 5 days after. The BBB opening volume was shown to decrease radiallytowards the center of the focal spot over time. At lower acoustic pressures, the microbubblesize becomes more important, with smaller microbubble diameters inducing a lower volumeof BBB opening and the closing timeline being significantly different than with largermicrobubbles. As the PRP increases, the differences in BBB opening and closing betweenthe different microbubble sizes become less significant. Overall, the BBB closes faster whensmall microbubbles are used, while for the 4-5 μm and 6-8 μm the same duration for closingwas required. Finally, hyperintensity in the area of BBB opening was detected in the pre-contrast MR images only in the cases where damage was seen in histology. In conclusion,this study may offer some further insight in the understanding of the FUS-opened BBB self-repairing characteristics, spatially and temporally, and the methodology may be adjusted tofit the pharmacokinetic needs of the administered CNS drugs.

AcknowledgmentsThis study was supported in part by NIH R01EB009041 and NSF CAREER 0644713. The authors wish to thankJameel Feshitan, MS (Chemical Engineering) for manufacturing the microbubbles, Kirsten Selert, BS (BiomedicalEngineering) for the histological specimen preparation as well as Anna Wong, BS (Biomedical Engineering) for thehistological specimen analysis at Columbia University.

REFERENCES1. Rubin LL, Staddon JM. THE CELL BIOLOGY OF THE BLOOD-BRAIN BARRIER. Annu. Rev.

Neurosci. 1999; 22(1):11–28. [PubMed: 10202530]

2. Abbott NJ, Rönnbäck L, Hansson E. Astrocyte-endothelial interactions at the blood-brain barrier.Nat. Rev. Neurosci. 2006; 7(1):41–53. [PubMed: 16371949]

3. Pardridge WM. Drug Targeting to the Brain. Pharm Res. 2007; 24(9):1733–1744. [PubMed:17554607]

4. Pardridge WM. The blood-brain barrier: bottleneck in brain drug development. NeuroRx. 2005;2(1):3–14. [PubMed: 15717053]

5. Choi JJ, Pernot M, Small SA, Konofagou EE. Noninvasive, transcranial and localized opening ofthe blood-brain barrier using focused ultrasound in mice. Ultrasound Med Biol. 2007; 33(1):95–104. [PubMed: 17189051]

6. Hynynen K, McDannold N, Vykhodtseva N, Jolesz FA. Noninvasive MR imaging-guided focalopening of the blood-brain barrier in rabbits. Radiology. 2001; 220(3):640–646. [PubMed:11526261]

7. Yang F-Y, Fu W-M, Yang R-S, Liou H-C, Kang K-H, Lin W-L. Quantitative evaluation of focusedultrasound with a contrast agent on blood-brain barrier disruption. Ultrasound Med Biol. 2007;33(9):1421–1427. [PubMed: 17561334]

Samiotaki et al.


NIH


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8. Liu H, Wai Y, Chen W, Chen J, Hsu P, Wu X, Huang W, Yen T, Wang J. Hemorrhage detectionduring focused-ultrasound induced blood-brain-barrier opening by using susceptibility-weightedmagnetic resonance imaging. Ultrasound Med Biol. 2008; 34(4):598–606. [PubMed: 18313204]

9. Choi JJ, Wang S, Tung Y-S, Morrison B, Konofagou EE. Molecules of various pharmacologically-relevant sizes can cross the ultrasound-induced blood-brain barrier opening in vivo. Ultrasound MedBiol. 2010; 36(1):58–67. [PubMed: 19900750]

10. Hynynen K, McDannold N, Vykhodtseva N, Raymond S, Weissleder R, Jolesz FA, Sheikov N.Focal disruption of the blood-brain barrier due to 260-kHz ultrasound bursts: a method formolecular imaging and targeted drug delivery. J. Neurosurg. 2006; 105(3):445–454. [PubMed:16961141]

11. Hynynen K, McDannold N, Sheikov NA, Jolesz FA, Vykhodtseva N. Local and reversible blood-brain barrier disruption by noninvasive focused ultrasound at frequencies suitable for trans-skullsonications. Neuroimage. 2005; 24(1):12–20. [PubMed: 15588592]

12. Kinoshita M, McDannold N, Jolesz FA, Hynynen K. Noninvasive localized delivery of Herceptinto the mouse brain by MRI-guided focused ultrasound-induced blood-brain barrier disruption.Proc. Natl. Acad. Sci. U.S.A. 2006; 103(31):11719–11723. [PubMed: 16868082]

13. Wang F, Cheng Y, Mei J, Song Y, Yang Y, Liu Y, Wang Z. Focused ultrasound microbubbledestruction-mediated changes in blood-brain barrier permeability assessed by contrast-enhancedmagnetic resonance imaging. J Ultrasound Med. 2009; 28(11):1501–1509. [PubMed: 19854965]

14. Sheikov N, McDannold N, Sharma S, Hynynen K. Effect of Focused Ultrasound Applied With anUltrasound Contrast Agent on the Tight Junctional Integrity of the Brain MicrovascularEndothelium. Ultrasound in Medicine & Biology. 2008; 34(7):1093–1104. [PubMed: 18378064]

15. Howles GP, Bing KF, Qi Y, Rosenzweig SJ, Nightingale KR, Johnson GA. Contrast-enhanced invivo magnetic resonance microscopy of the mouse brain enabled by noninvasive opening of theblood-brain barrier with ultrasound. Magn Reson Med. 2010; 64(4):995–1004. [PubMed:20740666]

16. Choi JJ, Wang S, Brown TR, Small SA, Duff KEK, Konofagou EE. Noninvasive and transientblood-brain barrier opening in the hippocampus of Alzheimer’s double transgenic mice usingfocused ultrasound. Ultrason Imaging. 2008; 30(3):189–200. [PubMed: 19149463]

17. Xie F, Boska MD, Lof J, Uberti MG, Tsutsui JM, Porter TR. Effects of Transcranial Ultrasoundand Intravenous Microbubbles on Blood Brain Barrier Permeability in a Large Animal Model.Ultrasound in Medicine & Biology. 2008; 34(12):2028–2034. [PubMed: 18692294]

18. McDannold N, Vykhodtseva N, Hynynen K. Effects of acoustic parameters and ultrasound contrastagent dose on focused-ultrasound induced blood-brain barrier disruption. Ultrasound Med Biol.2008; 34(6):930–937. [PubMed: 18294757]

19. Choi JJ, Feshitan JA, Baseri B, Wang S, Tung Y, Borden M, Konofagou EE. Microbubble-sizedependence of focused ultrasound-induced blood-brain barrier opening in mice in vivo. IEEETrans Biomed Eng. 2010; 57(1):145–154. [PubMed: 19846365]

20. Tung Y-S, Vlachos F, Choi JJ, Deffieux T, Selert K, Konofagou EE. In vivo transcranial cavitationthreshold detection during ultrasound-induced blood-brain barrier opening in mice. Phys MedBiol. 2010; 55(20):6141–6155. [PubMed: 20876972]

21. Nyborg WL. Biological effects of ultrasound: development of safety guidelines. Part II: generalreview. Ultrasound Med Biol. 2001; 27(3):301–333. [PubMed: 11369117]

22. Hamill OP. Twenty odd years of stretch-sensitive channels. Pflugers Arch. 2006; 453(3):333–351.[PubMed: 17021800]

23. Mihran RT, Barnes FS, Wachtel H. Transient modification of nerve excitability in vitro by singleultrasound pulses. Biomed Sci Instrum. 1990:26235–246.

24. Prentice P, Cuschieri A, Dholakia K, Prausnitz M, Campbell P. Membrane disruption by opticallycontrolled microbubble cavitation. Nat Phys. 2005; 1(2):107–110.

25. Kodama T, Tomita Y. Cavitation bubble behavior and bubble-shock wave interaction near agelatin surface as a study of in vivo bubble dynamics. Applied Physics B: Lasers and Optics. 2000;70(1):139–149.

26. Qin S, Ferrara KW. Acoustic response of compliable microvessels containing ultrasound contrastagents. Phys Med Biol. 2006; 51(20):5065–5088. [PubMed: 17019026]

Samiotaki et al.


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27. McDannold N, Vykhodtseva N, Hynynen K. Targeted disruption of the blood–brain barrier withfocused ultrasound: association with cavitation activity. Phys. Med. Biol. 2006; 51(4):793–807.[PubMed: 16467579]

28. Caskey CF, Stieger SM, Qin S, Dayton PA, Ferrara KW. Direct observations of ultrasoundmicrobubble contrast agent interaction with the microvessel wall. J. Acoust. Soc. Am. 2007;122(2):1191–1200. [PubMed: 17672665]

29. Qin S, Ferrara KW. The natural frequency of nonlinear oscillation of ultrasound contrast agents inmicrovessels. Ultrasound Med Biol. 2007; 33(7):1140–1148. [PubMed: 17478030]

30. Sassaroli E, Hynynen K. Resonance frequency of microbubbles in small blood vessels: a numericalstudy. Phys Med Biol. 2005; 50(22):5293–5305. [PubMed: 16264254]

31. Chomas JE, Dayton P, May D, Ferrara K. Threshold of fragmentation for ultrasonic contrastagents. J Biomed Opt. 2001; 6(2):141–150. [PubMed: 11375723]

32. Sassaroli E, Hynynen K. Cavitation threshold of microbubbles in gel tunnels by focusedultrasound. Ultrasound Med Biol. 2007; 33(10):1651–1660. [PubMed: 17590501]

33. Hynynen K, McDannold N, Martin H, Jolesz FA, Vykhodtseva N. The threshold for brain damagein rabbits induced by bursts of ultrasound in the presence of an ultrasound contrast agent(Optison). Ultrasound Med Biol. 2003; 29(3):473–481. [PubMed: 12706199]

34. Feshitan JA, Chen CC, Kwan JJ, Borden MA. Microbubble size isolation by differentialcentrifugation. Journal of Colloid and Interface Science. 2009; 329(2):316–324. [PubMed:18950786]

35. Vlachos F, Tung Y-S, Konofagou EE. Permeability assessment of the focused ultrasound-inducedblood-brain barrier opening using dynamic contrast-enhanced MRI. Phys Med Biol. 2010; 55(18):5451–5466. [PubMed: 20736501]

36. Samuel S, Cooper MA, Bull JL, Fowlkes JB, Miller DL. An ex vivo study of the correlationbetween acoustic emission and microvascular damage. Ultrasound Med Biol. 2009; 35(9):1574–1586. [PubMed: 19560856]

37. Chen S, Shohet RV, Bekeredjian R, Frenkel P, Grayburn PA. Optimization of ultrasoundparameters for cardiac gene delivery of adenoviral or plasmid deoxyribonucleic acid byultrasound-targeted microbubble destruction. J. Am. Coll. Cardiol. 2003; 42(2):301–308.[PubMed: 12875768]

38. Lemasson B, Serduc R, Maisin C, Bouchet A, Coquery N, Robert P, Le Duc G, Tropres I, Remy C,Barbier EL. Monitoring blood-brain barrier status in a rat model of glioma receiving therapy: dualinjection of low-molecular-weight and macromolecular MR contrast media. Radiology. 2010;257(2):342–352. [PubMed: 20829544]

39. Baseri B, Choi JJ, Tung Y-S, Konofagou EE. Multi-modality safety assessment of blood-brainbarrier opening using focused ultrasound and definity microbubbles: a short-term study.Ultrasound Med Biol. 2010; 36(9):1445–1459. [PubMed: 20800172]

40. Choi JJ, Selert K, Gao Z, Samiotaki G, Baseri B, Konofagou EE. Noninvasive and localized blood-brain barrier disruption using focused ultrasound can be achieved at short pulse lengths and lowpulse repetition frequencies. J. Cereb. Blood Flow Metab. 2011; 31(2):725–737. [PubMed:20842160]

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FIG.1.(a) Experimental Setup. (b) Horizontal CE-T1 image, where Gd-DTPA-BMA has diffusedand signal enhancement is shown on the right hippocampus. (c) Thresholded voxels at theleft and right VOIs of the original image in Fig.1 (b) are overlaid in red. On the lefthemisphere (control side) only the voxels of the ventricles and the vasculature are above thethreshold. On the right (sonicated) side voxels of the BBB opened area are also above thethreshold. By subtracting the voxels of the left from the right, the effect of the vasculatureand ventricles is reduced.

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FIG. 2.Horizontal consecutive CE-T1 images (500 μm thickness, dorsal on top, ventral on bottom)from Day 0 up to Day 3 for a 6-8 μm /0.30 MPa case. The BBB opening reduced radiallytowards the center of the FUS beam over time, until closing was detected in Day 3, when noGd-DTPA-BMA diffused from the vasculature to the brain parenchyma.

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FIG. 3.Coronal reconstructions from horizontal CE-T1 images with one example provided in eachcase of PRP and microbubble size.

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FIG. 4.Volume of diffusion of Gd-DTPA-BMA area, depicting BBB opening, for PRP of 0.30MPa, 0.45 MPa, and 0.60MPa and the sham group with microbubbles of (a) 1-2μm, (b)4-5μm and (c) 6-8μm in diameter. Error bars correspond to standard deviation and (*)denotes closing.

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FIG. 5.Volume of BBB opening on day 0 versus time to closing, showing that the duration of BBBopening increased monotonically with the volume of opening on Day 0.

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FIG. 6.Example of one out of the five cases where damage was detected. (a) and (b) are horizontalpre-contrast T1-weighted images, acquired on Day 0 and Day 7, respectively.Hyperintensity is shown (white arrows) on the sonicated side, more enhanced on Day 7. (c)and (d) are the H&E stained slices, magnified (10x) at the left (control) hippocampus and atthe right (sonicated) hippocampus respectively. In (c) cell loss is detected at the dentategyrus and CA1 area (white arrows).

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Table 1

P values from Statistical Analysis on the volume of diffusion of Gd-DTPA-BMA on day 0, comparingdifferent cases of microbubble diameters, per PRP.

Day 0 1-2 μm 4-5 μm

0.30 MPa 6- 8 μm - p > 0.05

0.45 MPa 6- 8 μm p < 0.01 p > 0.05

4-5 μm p < 0.01 -

0.60 MPa 6- 8 μm p < 0.05 p > 0.05

4-5 μm p > 0.05 -


Pressure and microbubble size dependence study of focused ultrasound-induced blood-brain barrier...

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