Trans-cranial opening of the blood-brain barrier in targeted regions using astereotaxic brain atlas and focused ultrasound energy
© Bing et al.; licensee BioMed Central Ltd. 2014
Received: 21 May 2014
Accepted: 11 July 2014
Published: 4 August 2014
The blood-brain barrier (BBB) protects the brain by preventing the entry of largemolecules; this poses a major obstacle for the delivery of drugs to the brain. Anovel technique using focused ultrasound (FUS) energy combined with microbubblecontrast agents has been widely used for non-invasive trans-cranial BBB opening.Traditionally, FUS research is conducted with magnetic resonance imaging (MRI)guidance, which is expensive and poses physical limitations due to the magneticfield. A system that could allow researchers to test brain therapies without MRintervention could facilitate and accelerate translational research.
In this study, we present a novel FUS system that uses a custom-built FUSgenerator mounted on a motorized stereotaxic apparatus with embedded brain atlasto locally open the BBB in rodents. The system was initially characterized using atissue-mimicking phantom. Rodent studies were also performed to evaluate whethernon-invasive, localized BBB opening could be achieved using brain atlas-basedtargeting. Brains were exposed to pulsed focused ultrasound energy at1.06 MHz in rats and 3.23 MHz in mice, with the focal pressure estimatedto be 0.5–0.6 MPa through the skull. BBB opening was confirmed in grosstissue sections by the presence of Evans blue leakage in the exposed region of thebrain and by histological assessment.
The targeting accuracy of the stereotaxic system was better than 0.5 mm inthe tissue-mimicking phantom. Reproducible localized BBB opening was verified withEvans blue dye leakage in 32/33 rats and had a targeting accuracy of±0.3 mm. The use of higher frequency exposures in mice enabled a similarprecision of localized BBB opening as was observed with the low frequency in therat model.
With this dedicated small-animal motorized stereotaxic-FUS system, we achievedaccurate targeting of focused ultrasound exposures in the brain for non-invasiveopening of the BBB. This system can be used as an alternative to MR-guided FUS andoffers researchers the ability to perform efficient studies (30 min perexperiment including preparation) at a reduced cost in a conventional laboratoryenvironment.
For most therapeutic agents aimed at treating central nervous system (CNS) diseases anddisorders, the blood-brain barrier (BBB) is a primary physiological barrier limitingdrug delivery into the brain parenchyma. The BBB is a separation present along allcapillaries in the CNS that controls the molecular transport and diffusion across theseblood vessels. The barrier is formed by layers of cells that are coupled by tightjunctions [1, 2]. Only small-molecule drugs with high lipid solubility and a low molecularmass under 400–500 Da can cross the BBB in pharmacologically significantamounts, hence excluding most current therapeutic and imaged agents from being used inthe brain [3, 4].
Non-invasive localized opening of the BBB has been demonstrated using focused ultrasound(FUS) exposures combined with circulating intravascular microbubble ultrasound contrastagents. The exact physical mechanisms governing the interactions between themicrobubbles and endothelial cells are not known, but it is likely that when stimulatedby ultrasound energy, oscillation of microbubbles produces mechanical effects induced byradiation force and/or shear stress on the blood vessel walls, temporarily opening theBBB without tissue damage –. This combination of FUS and intravascular microbubbles offers a uniquemethod for remotely actuating mechanical energy at the site of small vessels throughoutthe brain. Meanwhile, this opening occurs at lower acoustic power levels than waspreviously used without intravenous microbubbles, which makes this method substantiallyeasier to apply through the intact skull . As early as 2001, Hynynen et al. demonstrated that focused ultrasoundcombined with gas bubbles can open the BBB transiently in rabbits . In 2002, Mesiwala et al. confirmed that high-intensity focused ultrasound iscapable of a selective and non-destructive disruption of the BBB in rats . Subsequent studies confirmed the feasibility of non-invasive localized BBBopening in rodents as models of human disease [11, 12, 1, 13]–. However, the small size of rodent brains makes FUS experiments challenging,necessitating the development of dedicated small-animal exposure systems .
In the majority of studies to date, confirmation and visualization of BBB opening hasbeen achieved using magnetic resonance imaging (MRI). T1-weighted imaging is often usedto confirm successful delivery of gadolinium-based MR contrast agents across the BBB,and T2-weighted imaging can evaluate the presence of tissue damage [18, 19]. The major drawbacks of MR-guided FUS are the need for MRI-compatiblesystems, lack of availability, limited throughput of experiments, and expense. MR-guidedfocused ultrasound systems are not usually accessible to neuroscientists or otherresearchers outside of the imaging research field. A method for performing trans-cranialBBB opening without MR guidance would be desirable in order to lower the barrier toentry in this field and to achieve broader penetration of FUS technology inneuroscience.
Previous studies have incorporated systems for BBB opening outside the MRI environment.One method for targeting specific regions in the brain is the use of stereotaxy incombination with an anatomical atlas. This method is widely used in neuroscience to theinjection of agents or implantation of electrodes in specific regions of the rodentbrain. Liu et al.'s group developed a pinhole-assisted mechanical scanning device usinga stereotaxic apparatus for BBB opening , and Konofagou et al. applied a stereotaxic frame for localized BBB openingin rodents . Another method for imaging BBB opening is multi-photon fluorescencemicroscopy. The two-photon microscopy allows in vivo visualization of thecerebral vasculature and neurons at the subcellular level . Besides, cerebrovascular dynamics and kinetics of dye leakage after FUSsonication can also be imaging with multi-photon microscopy . However, in order to enable targeting of the ultrasound focus to a specificfunctional area in the brain, MR images are still required in these systems.
In this study, we present a compact stereotaxic-FUS system to perform trans-craniallocalized BBB opening in rodent models using FUS energy and a stereotaxic system with abuilt-in rodent brain atlas. The goal of this system is to provide a tool forneuroscientists to achieve non-invasive targeted BBB opening in rodents for behavioraland functional research. Using the rodent brain atlas, researchers can easily target aspecific functional area of the brain. In this paper, we introduce our stereotaxic-FUSsystem, followed by initial accuracy characterization in tissue-mimicking phantoms androdents. Successful BBB opening was confirmed through gross imaging, histopathology, andMR imaging. By all forms of measure, repeatable non-invasive localized BBB opening byour rodent stereotaxic-FUS system was verified.
Methods and materials
Focused ultrasound system and stereotaxic apparatus
Ultrasound was transmitted into the brain using a focused transducer with a 25-mmdiameter and a 20-mm radius of curvature. The fundamental frequency of the transducerwas 1.06 MHz, and the third harmonic frequency was 3.23 MHz, as measuredwith an impedance analyzer (Via Bravo, AEA Technology Inc., Carlsbad, CA, USA). Thedimensions of the ultrasound focus and the pressure output of the transducer werecharacterized using an acoustic hydrophone tank. A needle hydrophone with a 0.2-mmactive area (HGL-0200, Onda Corporation, Sunnyvale, CA, USA) was scanned in threedimensions to measure the spatial pressure distribution of the ultrasound beam at thefocus for each transmission frequency.
The transducers were connected to a custom-built compact driving system comprised ofan arbitrary waveform generator (LMS271D, Vanuix Corp., Haverhill, MA, USA), an RFamplifier (NP Technologies, Newbury Park, CA, USA), high-power low-pass filters, anda dual-frequency matching circuit that enabled efficient transmission of power to thetransducer at each operating frequency. This self-contained driving system wascontrolled from a laptop computer via USB. One of the main goals was to produce aportable system for focused ultrasound that could be easily transported forexperiments.
A standard stereotaxic apparatus (51730 M, Stoelting Co., Wood Dale, IL, USA)was used to achieve targeting of ultrasound energy within the brain with anattachment capable of registering the rat brain atlas (Neurostar, Tubingen, Germany).These systems are normally used to achieve precise insertion of electrodes orinjection of materials directly into target regions of the brain. The method ofregistering the brain atlas to the rodent involves locating the bregma and lambda onthe rodent skull sutures with a metal pointer. Once the x, y, andz coordinates of these two locations are identified, the brain atlas(which is referenced to these points) is immediately registered to the animal.Tilting correction was performed automatically based on the current animal to takethe difference in brain position and size into consideration.
Spatial accuracy characterization with the phantom
0.5 ± 0.01
1 ± 0.01
0.5 ± 0.01
1 ± 0.01
0.5 ± 0.01
0.9 ± 0.01
-2 ~ 0
1.5 ± 0.01
2 ± 0.01
1.5 ± 0.01
2 ± 0.01
1.4 ± 0.01
-1.3 ~ -0.6
1.9 ± 0.01
-1 ~ -0.5
The spatial accuracy of ultrasound delivery was evaluated by exposing targets withdifferent spacing (0.5, 1, 1.5, and 2 mm) in all three directions (x,y, z). The tissue-mimicking phantom was placed in the positionwhere the rodent head would be located, and the ultrasound transducer was coupled toit using degassed water. After the exposures, the distance between the regions ofcoagulation in the gel were measured and compared with the desired separation. Testswere completed in two planes (x-y and x-z). Foreach plane, one reference target (A) and four testing targets (B, C, D, E) wereselected to apply the sonication.
As an important next step, ultrasound exposures were delivered to thetissue-mimicking phantom in a three-dimensional (3D) pattern based on the rat brainatlas in order to cover a spatial region equivalent to the right motor cortex of thebrain. The geometric fidelity of this ultrasound exposure was evaluated in comparisonto the brain atlas.
Female 200–270-g Sprague Dawley rats (n = 56) andfemale 20–25-g Swiss Webster mice (n = 14) were used inthis study. All procedures were approved by UT Southwestern Institutional AnimalCare and Use Committee and followed guidelines set forth by the Guide. Animalswere anesthetized with a mixture of 2%–3.5% isoflurane and1–2 l/min of 100% oxygen. A 24G I.V. catheter was placed in the lateraltail vein. A pulse oximeter was attached to the animal's paw to monitor heart rateand oxygen saturation, and a rectal temperature probe (attached to a homeothermiccontrol blanket) was used to record and maintain core body temperature(PhysioSuite, Kent Scientific Corp., Torrington, CT, USA). Hair over the cranialsurface of the skull was removed using an animal trimmer and depilatory cream(VEET sensitive formula, Reckitt Benckiser, Parsippany, NJ, USA). Afterpreparation, the animal was transferred to the stereotaxic apparatus andstabilized using ear bars and bite bar. A custom-built nose cone was placed overthe animal's nose to deliver inhalant anesthetic.
Evans blue (2%; 3 ml/kg rats, 4 ml/kg mice) was injected via the tailvein catheter and allowed to circulate for a minimum of 1–3 min. Abolus injection of microbubbles (30 μl/kg rats, 60 μl/kg mice,Optison, GE Healthcare, Milwaukee, WI, USA) was delivered via the tail veincatheter, with simultaneous start of ultrasound sonication. When multiplesonications were performed (in the same animal), 5 min of wait time wasapplied to allow microbubbles to clear from circulation.
All brain targets were selected in the right hemisphere (center of caudoputamen)while the left hemisphere was kept as a control. Twenty-three rats were utilizedto optimize FUS parameters (frequency, microbubble dosage, focal pressure, etc.)on BBB opening results. After optimization, 33 rats were utilized to verify thereliability of the system and to characterize spatial accuracy. Among those rats,5 were utilized for histology. Nineteen mice were utilized to optimize ultrasoundfrequencies specific to mouse cranial space and parietal bone thickness (7 for lowfrequency, 1.06 MHz, and 12 for high frequency, 3.23 MHz).
Animals were sacrificed and perfused 5–10 min after sonication withsterile saline and 10% buffered formalin, followed by immediate harvesting of thebrain. Brain samples were then placed in 10% buffered formalin to allow for24–48 h of fixation time. Brains were sliced using a rat or mouse brainmatrix (World Precision Instrument, Sarasota, FL, USA) to confirm localization ofEvans blue leakage following BBB opening. Photographic record of Evans blueleakage was made, and a subset of subgross slices was worked up by histology.
Subgross coronal brain-matrix slices from perfusion-fixed animals were paraffinprocessed, embedded, and sectioned according to standard procedures [24, 25]. Serial paraffin sections were prepared at the epicenter ofFUS-mediated hemorrhage, seen by dark-field microscopy . Resulting sections were stained by hematoxylin and eosin (H&E) andterminal deoxynucleotidyltransferase-mediated UTP end labeling (TUNEL).H&E-stained sections were analyzed for histopathologic hallmarks of braininjury (hemorrhage, edema, nuclear condensation, inflammatory cell infiltrate,rarefaction). Histologic analysis for possible ultrasound damage to neuronal andglial cell populations in the FUS target area was performed via TUNEL; nuclei ofapoptotic and necrotic cells were labeled with fluorescein according to methods ofthe first report . Sections subjected to TUNEL were counterstained with propidiumiodide.
Single-frequency pulsed exposures (10-ms burst duration) were transmitted into thebrain with a repetition frequency of 1 Hz and a total exposure duration of120 s. In rats, the intracranial peak negative pressure at the focal pointunder low frequency (1.06 MHz) was adjusted to be between 0.5 and0.6 MPa, based on the hydrophone calibration of the transducer and publishedmeasurements of the insertion loss through rat parietal bone . Similar opening is expected with lower pressure. The threshold for theBBB opening was estimated to be inversely proportional to the square root offrequency . Prior studies have investigated extensively the effects of differentsonication parameters on the threshold of BBB opening : frequency , pulse width [31, 32], pulse repetition time , total sonication duration , microbubble size , and contrast agent dose [35, 36]. The ultrasound exposure parameters selected for this study have beenshown to achieve consistent opening of the BBB in rodent models . For the mouse exposures, a 3.23-MHz frequency was used instead of1.06 MHz in order to achieve a relatively localized ablation within the mousebrain. Skull insertion loss under high frequency is still unknown, so it wasassumed to be approximately 50% based on the skull thickness. The focusedultrasound energy was adjusted to achieve a focal pressure of0.5–0.6 MPa accordingly.
Characterization of in vivo targeting accuracy
Evans blue dye has been used frequently as an indicator of BBB opening. Evans bluebinds to albumin which cannot cross the BBB; therefore, neural tissue remainsunstained after intravenous administration of the dye . In these experiments, BBB opening was confirmed on gross tissuesections based on the leakage of Evans blue dye in the targeted area of thebrain.
As a secondary validation, two rats that received ultrasound exposures to open theBBB in a targeted region were administered an intravenous injection of gadodiamide(0.02 mmol/kg, Omniscan, GE Healthcare, Milwaukee, WI, USA) and were imagedin a 9.4 T animal imaging and spectroscopy system (Avance, Varian MedicalSystems, Palo Alto, CA, USA). Contrast-enhanced T1-weighted spin-echo MR imageswere obtained to visualize BBB opening through diffusion of the contrast agentinto the brain parenchyma (field of view 128 × 128, TR/TE151/10 ms, slice thickness 1 mm, gap 0 mm, echo number 1).
The spatial targeting accuracy of the system was evaluated through localization ofthe region of Evans blue staining on gross sections. Two specific targets weremanually selected using the brain atlas, and the spacing between targets wasmeasured on the gross sections. Atlas spacing was compared with measured spacingin three axes: anterior-posterior (AP), medial-lateral (ML), and superior-inferior(SI). Reference in ML and SI axes is the midline of the brain and top surface ofthe brain, respectively. For characterization of the AP axis, a reference pointwas selected as coordinates 0.00, 3.32, and 5.55 mm (AP, ML, and SI) relativeto bregma since the cranial landmarks disappear after removal of the skull. Fiverats were utilized to characterize targeting accuracy along each axis.
Focused ultrasound transducer characterization
Spatial accuracy characterization—phantom
Spatial accuracy characterization with the rat model
Atlas spacing (mm)
Measured spacing (mm)
Spatial accuracy characterization — animal
As further validation of the rat model, detailed histologic analysis was performedon coronal-matrix slices collected immediately following FUS and 72 hpost-FUS exposure. Figure 7 shows ultrasound-inducedBBB opening by routine histopathology. H&Es of acute and 72-h brains both showevidence of perivascular hemorrhage in the target region. Hemorrhage was limitedto the target area in both lateral-medial and anterior-posterior axes. Mildperivascular edema and condensation of adjacent neuronal nuclei was sparselyevident. Low-magnification images illustrate the contralateral hemisphere withouthemorrhage.
This study presented a novel system that interfaces a stereotaxic brain atlas and a FUSenergy transducer to achieve non-invasive localized BBB opening in rodents, without MRintervention. The spatial accuracy of the system was estimated to be ±2% with thetissue-mimicking phantom and ±5% with the rodent models. Evans blue dye leakage wasused as a subgross indicator of localized BBB opening, which was subsequently validatedby intact animal MR imaging of gadolinium contrast agent. Applying low frequency(1.06 MHz) and high frequency (3.23 MHz) on rats and mice, respectively, atight three-dimensional focus of ultrasound was achieved. This portable system can beused for rodents' BBB opening in a traditional laboratory environment.
The primary research application we envision for this system is for the targeting ofregions within the rodent brain to facilitate functional neuroscience studies inrodents. Since the majority of these investigations are conducted in conventionallaboratory environments in close proximity to behavioral testing facilities, a portableFUS system that does not require MRI targeting and guidance is desirable. Furthermore,the brain atlas is an important targeting tool for functional regions of the brain inrodents.
To our knowledge, this is the first study to explore the use of high-frequency FUSexposures in mice for BBB opening. Exposure of the mouse brain at 1.06 MHz resultedin a very large region of BBB opening in the brain encompassing the entire AP directionwithin a hemisphere. This is due to the thinner parietal bone and smaller brain size inmice. Exposures at 3.23 MHz achieved a localized region of BBB opening similar inproportion to the brain as was achieved using 1.06 MHz in rats. However, unlikeexperiments with the rat model, the skull insertion loss is still unknown at this highfrequency, and the focal pressure inside the mouse brain was estimated for this studybased on prior measurements at a lower frequency in the rat skull . Experimental measurement of the insertion loss through the mouse skull boneat 3 MHz is necessary to obtain more accurate estimates of the intracranialpressure amplitudes during these exposures.
The system described in this manuscript still suffers from a few limitations. In orderto achieve BBB opening within a larger region of the brain, rapid mechanical  or electronic  steering of the ultrasound focus is desirable. The stereotaxic systemdescribed in this study is amenable to these technical improvements, and this additionwould enable regional opening of target structures within the brain. The incorporationof acoustic feedback has been demonstrated to achieve more consistent stable cavitationduring ultrasound exposures for BBB opening [40, 41]. Due to the lack of imaging confirmation of BBB opening with the stereotaxicsystem, this type of feedback would be an important improvement to the FUS system.Another limitation of this approach is that an incision is needed to identify bregma andlambda. Potential strategies to overcome this would be to use the external ear bars as areference point or to use a technique like high-frequency ultrasound imaging tonon-invasively locate the appropriate stereotactic reference point on the skull. Theseapproaches will be the subject of future investigations. Nonetheless, while theseimprovements would expand the capabilities of the existing system, the currentconfiguration is still capable of consistent opening of the BBB in rodents.
A novel system that interfaces a stereotaxic brain atlas and a FUS transducer fornon-invasive targeted BBB opening in rodents is presented in this study. The spatialtargeting accuracy of the system was estimated to be ±2% in tissue-mimickingphantoms and ±5% in the rat brain. Localized opening of the BBB was achieved at1.06 MHz in rats and 3.23 MHz in mice. Consistent opening of the BBB wasverified through Evans blue staining in gross brain sections and histopathology inH&E-stained tissue sections. TUNEL staining confirmed that there was minimal to noapoptosis nor necrosis in the exposed regions of the brain.
This study was conducted with the financial support from the Cancer Prevention &Research Initiative of Texas (Grant No. R1308) and a generous donation from the M.R.& Evelyn Hudson Foundation. The authors would also like to acknowledge theefforts provided by Forrest Johnson in the design and fabrication of the ultrasoundtransducers used in this study, Trevor Hinshaw for conducting initial feasibilitystudies and development of the tissue-mimicking phantom, and Dr. Dawen Zhao and hisgroup for assisting with the acquisition of MR images.
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