ALZHE IMER S D I S EASE

Scanning ultrasound removes ame

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not impair the ability of the animals to perform a complex visual acuity

, injected intravenouslyye Evans blue, and thenmaging probe system). Subsequent brain dis-se resulted in a 1-mm-rating focused openingultrasound beam wasforebrain of the mousethroughout the brain,ans blue dye as early asis was also illustrated byreatment (Fig. 1C). Weed that a 0.7-MPa peakn frequency, 10% dutyoptimal. These settingsneration, as revealed byrythrocyte extravasation

as shown by hematoxylin and eosin (H&E) staining (fig. S1, E to H). To

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m Clem Jones Centre for Ageing Dementia Research, Queensland Brain Institute, TheUniversity of Queensland, St Lucia Campus, Brisbane, Queensland 4072, Australia.across the BBB (13). In assessing ultrasound-induced BBB opening,previous studies reported no difference in BBB opening or closing be-tween Ab plaqueforming APP/PS1 mice and nontransgenic (non-Tg)littermate controls (14).

fluorescence imaging 30 min to 1 hour after toptimized the ultrasound settings and establishrarefactional pressure, 10-Hz pulse repetitiocycle, and 6-s sonication time per spot weredid not cause dark neurons, reflecting degeNissl staining (fig. S1, C and D), or edema or eculature transiently opens tight junctions and facilitates transporton the principle that biologically inert and preformed microbubblescomprising either a lipid or polymer shell, a stabilized gas core, and adiameter of less than 10 mm are systemically administered and subse-quently exposed to noninvasively delivered focused ultrasound pulses(10). Microbubbles within the target volume become acoustically acti-vated by what is known as acoustic cavitation. In this process, the mi-crobubbles expand and contract with acoustic pressure rarefaction andcompression over several cycles (10). This activity has been associatedwith a range of effects, including the displacement of the vessel wallthrough dilation and contraction (11, 12). More specifically, the me-chanical interaction between ultrasound, microbubbles, and the vas-

points (as is conventionally done) or by usinbrain (Fig. 1, A to C). Mice were anesthetizedwithmicrobubbles together with the indicator dplaced under the focus of a TIPS (therapy iultrasound transducer (Philips Research) (16)section revealed that a single ultrasound pulwide blue column of Evans blue dye, demonstof the BBB (Fig. 1B). When the focus of themoved in 1.5-mm increments until the entirewas sonicated with SUS, the BBB was openedas evidenced by prevalent extravasation of Ev30min after the treatment (fig. S1, A and B). Thare used as ultrasound contrast agents (9). Ultrasound delivery is based can be opened repeatedly by ultrasound, either by using single entryg SUS across the entirememory in an Alzheimers diseasGerhard Leinenga and Jrgen Gtz*

Amyloid-b (Ab) peptide has been implicated in the pathogenesis ofpharmacological approach for removing Ab and restoringmemory fudeposited in the brain. We used repeated scanning ultrasound (SUS)without the need for any additional therapeutic agent such as anti-Aband high-resolution three-dimensional reconstruction revealed extenof activated microglia in mouse brains subjected to SUS, with no conmicroglia. Plaque burden was reduced in SUS-treated AD mice complaques were observed in 75% of SUS-treated mice. Treated AD mthreememory tasks: the Y-maze, the novel object recognition test, ansuggest that repeated SUS is useful for removing Ab in themouse brabe explored further as a noninvasive method with therapeutic poten

INTRODUCTION

Alzheimers disease (AD) is characterized by the presence of soluble oligo-mers of amyloid-b (Ab) peptide that aggregate into extracellular fibril-lar deposits known as amyloid plaques (13). Ab is elevated in the ADbrain because of the increased production of this peptide and its im-paired removal (4, 5). Recent therapeutic strategies have targeted bothprocesses (6), including the inhibition of secretase enzymes to reduceAb production, as well as active and, in particular, passive immuniza-tion approaches for boosting Ab clearance. These strategies, however,have side effects. Inhibition of secretases affects additional substrateswith potential off-target effects (7), and passive immunization may becostly once effectiveness is demonstrated in clinical trials (8).

Here, we aim to establish whether a transient opening of the blood-brain barrier (BBB) using repeated scanning ultrasound (SUS) couldassist inAb clearance. Only onemethod has been demonstrated to openthe BBB noninvasively and repeatedly, that is, nonthermal focusedultrasound coupled with intravenous injection of microbubbles, which*Corresponding author. E-mail: j.goetz@uq.edu.au

www.Scientask inwhich they had been trained.Devices that emit ultrasound capableof penetrating the human brain are currently in clinical trials. Recently, aproof-of-concept study of using magnetic resonanceguided focusedultrasound to treat tremor and chronic pain has been successfully com-pleted (15). Here, we investigate the use of SUS to remove Ab from theAD mouse brain and to improve cognition and memory.

RESULTS

Scanningultrasound isa safemethod to transientlyopen theBBBWe first established in C57BL/6 non-Tg wild-type mice that the BBByloid-b and restoresmouse model

lzheimers disease (AD). We present a non-ction in a mousemodel of AD in which Ab iseatments of the mouse brain to remove Ab,ntibody. Spinning disk confocal microscopyive internalization of Ab into the lysosomesomitant increase observed in the number ofared to sham-treated animals, and clearede also displayed improved performance onthe active place avoidance task. Our findingswithout causing overt damage, and shouldial in AD.

Focused ultrasound allows for a transient opening of the BBB in theabsence of tissue damage, as demonstrated inmany experimental species,including rhesus macaques (13). In these primates, repeated opening ofthe BBB in the region of the visual cortex using focused ultrasound diddeterminewhether SUS caused immediate damage, we analyzed non-Tg

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Sham Single entry point

CLow

Highand spatial memory deficits (17). Age-matched APP23 mice in the

rexttiespfle6

APP C-terminal fragmen

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m This test is based on the preference of miceto alternate between the arms of the maze.The analysis revealed that spontaneousalternation (calculated by the number ofcomplete alternation sequences divided bythe number of alternation opportunities)in APP23 mice treated with SUS, but notin sham-treatedanimals,was restored towild-type levels [P < 0.05, one-way analysis ofvariance (ANOVA) followed by Dunnettsmultiple comparison] (Fig. 1E). Total en-tries into the Y-maze arms did not differbetween groups (Fig. 1F). The mice receivedone additional ultrasound treatment andwere sacrificed 3 days later for histologicaland biochemical analysis.

We next used Campbell-Switzer silverstaining to distinguish the compact core ofmature amyloid plaques frommore dispersedAb deposits (Fig. 2, A and B). By analyzingevery eighth section from 0.8 to 2.8 mmfrom bregma for each mouse (total of 8 to10 sectionspermouse),we found that the cor-tical area occupied by plaques was reduced by56% (P = 0.014, unpaired t test) (Fig. 2C) andthat the average number of plaques per sec-tionwas reduced by 52% (P=0.017, unpairedt test) (Fig. 2D) in SUS-treated compared tosham-treated mice. Thioflavin-S staining (Fig.2E) and immunohistochemistry with the Ab-specific antibody 4G8 (Fig. 2F) were used toconfirm the specificity of the silver staining.We also plotted plaque load, as determinedin Fig. 2C, as a function of age and includeduntreated mice to demonstrate the baselineof plaque load at the onset of treatment (Fig.2G). It remains tobedeterminedhowourpro-tocol would need to be modified to reveal ef-ficacy in inducible models of AD, such astetO-APPswe/ind mice (18).ight hemisphere from 10 SUS-treated and 10and used these tissues to obtain two lysates,tracellular proteins and aTriton-soluble frac-ing with antibodies against Ab, we were ables of the peptide (Fig. 3, A and B). The con-ecies were quantified, and reductions wereraction for SUS-treated compared to sham-cular weight species (HMW; 58% reduction),; 38% reduction) and the trimeric Ab/toxiccontrol group (n = 10) receivedmicrobub-ble injections and were placed under theultrasound transducer, but no ultrasoundwas emitted. After the 4-week sham or SUStreatment period, the mice underwent be-havioral testing for a 2-week period inwhichthey were not treated. We analyzed spatialworking memory functions in the Y-maze.

LI-CORBright-fieldmousebrain tissue 4hours and1dayafter SUS treatmentusingacid fuchsinstain and found no evidence of ischemic damage (fig. S1, I and J).

SUS reduces Ab and amyloid plaque load in plaque-formingAPP23 transgenic miceHaving confirmed the viability of our protocol, we treated an initial co-hort of 10 male Ab plaqueforming APP23 transgenic mice five timeswith SUS over a period of 6 weeks (Fig. 1D, study design). At the ageof 12 to 13 months, APP23 mice have a substantial plaque burden

We then extracted thesham-treated APP23 micone fraction enriched in etion (19). ByWestern bloto identify different speccentrations of these Abfound in the extracellulartreated mice for high mo*56 oligomeric Ab (Ab*5

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Fig. 1. EstablishingSUS in anADmousemodel. (A) Setupof SUS equipment. (B andC) BBBopeningbyultrasound was monitored by injecting wild-type mice with Evans blue dye that binds to albumin, a pro-tein that is normally excluded from thebrain. (B) A single entry point revealed a focal openingof the BBB inresponse to ultrasound treatment,with Evans blue dye able to enter the brain at this point. (C)Widespreadopening of the BBB 1 hour after SUS was demonstrated with an Odyssey fluorescence LI-COR scanner ofbrain slices usingnitrocellulose dottedwith increasing concentrations of bluedye as a control. (D) Treatmentscheme for the first cohort of hemizygousmale Ab plaqueforming APP23mice (median age, 12.8months).The mice received SUS or sham treatment for a total duration of the experiment of 6 weeks. Mice wererandomly assigned to treatment groups. Using histological methods, Western blotting, enzyme-linked im-munosorbent assay (ELISA), and confocal microscopy, wemeasured the effect of SUS treatment on amyloidpathology in mouse brain. Before the last SUS treatment, all mice were tested in the Y-maze. (E) Thesequence of arm entries in the Y-maze was used to obtain a measure of alternation, reflecting spatialworking memory. The percentage of alternation was calculated by the number of complete alternationsequences (that is, ABC, BCA, and CAB) divided by the number of alternation opportunities. Spontaneousalternation was restored in SUS-treated compared to sham-treated APP23mice using non-Tg littermates ascontrols (n = 10 per group; one-way ANOVA followed by Dunnetts posttest, P < 0.05). (F) Total number ofarm entries did not differ between groups.D SUS

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/Fig. 2. SUS reduces Ab plaques in an AD mousemodel. (A and B) Representative images of free-floating coronal sections from APP23 transgenic

Fig. 3. SUS treatment reduces different Ab spe-

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A*56 HMW *56 3mer/CTFcies. (A to D) Western blotting of extracellular-enriched (A) and Triton-soluble (B) fractions of thebrains of the first cohort of APP23 mice with 6E10and 4G8 anti-Ab antibodies revealed a reduction indistinct Ab species in both fractions in SUS-treatedcompared to sham-treated mice. These data arequantified in (C) and (D), respectively. The Westernblots show significant reductions of HMW spe-cies, the 56-kD oligomeric Ab*56 (*56) and trimericAb (3-mer)/CTFb, in the extracellular-enrichedfraction and of *56 and 3-mer/CTFb in the Triton-soluble fraction (unpaired t tests, P < 0.05). GAPDH(glyceraldehyde 3-phosphate dehydrogenase) wasused for normalization. MWM, molecular weightmarker. (E) ELISA for Ab42 in the Triton-soluble frac-tion revealed a significant reduction in SUS-treatedcompared to sham-treatedmousebrains (unpairedt test, P < 0.05; n = 10 per group).mice (first cohort) with and without SUS treatment.Campbell-Switzer silver staining revealed compact,mature plaques (amber) and more diffuse Ab de-posits (black). A stained section at a higher magnifi-cation is shown in panel (B). (C andD) Quantificationof amyloid plaques revealed a 56% reduction in thearea of cortex occupied by plaques (unpaired t test,P = 0.017) and a 52% reduction in plaque numberper section (t test, P = 0.014) in SUS-treated com-pared to sham-treatedAPP23mice (n=10per group).(E and F) Representative sections of SUS-treatedbrains versus control brains stained with ThioflavinS (E) and4G8 (F). (G) Plaque loadplotted as a functionof age confirmed that the SUS-treated group hadsignificantly lower plaque load than the sham-treatedgroup. Baseline plaque load at the onset of treatmentis indicated by open circles. Scale bars, 1 mm (panelA) and 200 mm (panel B).HMW 3mer/CTF

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day of training (F3,84 = 5.49, P = 0.002) a

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rgender-matched APP23 mice and 10 non-Tg littermates to determine the functionaloutcomeof theSUStreatmentprotocol inmorerobust behavioral tests. The mice were ana-lyzed in the APA task, a test of hippocampus-dependent spatial learning in which micelearned to avoid a shock zone in a rotating are-na. After the APA test, the APP23 mice weredivided into two groups with matchingperformance and received weekly SUS orsham treatment for 7weeks. Thiswas followedby an APA retest and a novel object recogni-tion (NOR) test. One day after the final SUStreatment, mice were sacrificed and brainextracts were analyzed by Western blottingand ELISA. (B) Twenty APP23 mice and10 non-Tg littermates tested in the APA test,with a habituation session (labeled H)followed by four training sessions (labeledD1 to D4). (C) In the APA retest, SUS-treatedA

A

Fig. 4. SUS treatment rescues memorydeficits in an AD mouse model. (A) Treat-ment scheme of a second cohort of 20nd genotype (F1,28 = 5.41, P =

www.ScienA test

Allocation into matching groups basedon APA test:10/10 APP23 APA retestck zone in a rotating arena-Tg littermates underwentwere significant effects of

0.028, two-wayANOVA),withday as thewithin-subjects factor (Fig. 4B).APP23 mice were divided into two groups with matching performanceon the APA test and received weekly SUS or sham treatment for 7 weeks.

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Novel objectN(Fig. 4A, study design). APP23mice and4 days of training after habituation. Thand in the Triton-soluble fraction for *56(50%) and trimeric Ab/CTFb (27%) (P 99% bound to albumin in the blood and the BBBwas impermeable before treatment. After 30min,mice were deeply anes-thetized, transcardially perfused with phosphate-buffered saline (PBS)followed by 4% paraformaldehyde (PFA), and photographed under astereo microscope (Carl Zeiss). To determine damage, sections fromSUS-treated mice were stained with H&E to assess erythrocyte extrava-sation and tissue damage as well as with cresyl violet (Nissl staining) toassess neuronal damage.We furtherused the acid fuchsin stain (SantaCruzBiotechnology) to detect ischemic neurons as described (31). Additionalmarkers assessed were NF-kB and GFAP.

Tissue processingMice were deeply anesthetized with pentobarbitone before being per-fused with 30 ml of ice-cold PBS. The brains were dissected from theskull and cut along themidline. The left hemispherewas fixed in 4% (w/v)PFA for 24 hours, cryoprotected in 30% sucrose, and sectioned coronallyat 40-mmthickness on a freezing-slidingmicrotome.Aone-in-eight seriesof sectionswas stored in PBSwith sodium azide at 4C until staining. Theright hemisphere of the brain was frozen in dry ice or ethanol slurry andstored at 80C until used for biochemical analysis.

Assessment of amyloid plaque loadA one-in-eight series of coronal brain sections were cut at 40-mm thick-ness on a microtome. An entire series of sections was processed forCampbell-Switzer silver staining (36) using a protocol available onlineat http://neuroscienceassociates.com/Documents/Publications/campbell-switzer_protocol.htm. For plaque counting, an entire one-in-eight seriesof sections was stained using the Campbell-Switzer method, and allsections0.85mmto2.8mmfrombregmawereanalyzed (8 to10sectionsper mouse) after being photographed at 16magnification on a bright-field slide scanner. Plaque load in the cortexwas obtained by the ParticleAnalysis plugin of ImageJ on coded images of sections using the areafraction method.

Skeleton analysisA skeleton analysis to obtain the most accurate tree geometry possiblewas applied to quantifymicroglialmorphology in images obtained fromfixed brains as described (37). In brief, 40-mmsections were stainedwithIba1 using the nickel-diaminobenzidine method. Two images from theauditory cortex overlying the dorsal hippocampus (an area rich in pla-ques) were each converted to binary images and then skeletonized using

the Analyze Skeleton plugin by ImageJ. This plugin tags all pixel/voxels

www.Scienin a skeleton image and then counts all its junctions, triple points, andbranches and measures their average and maximum length. The num-ber of summed microglial process endpoints and summed processlength normalized to the number of microglia were determined.

Spinning disk confocal microscopy and 3D renderingConfocal imaging was conducted using a spinning disk confocal head(CSU-W1; Yokogawa Electric) coupled to a motorized inverted ZeissAxio Observer Z1 microscope equipped with a 20/0.8 Plan-Apochromatair objective and a 100/1.4 Plan-Apochromat oil objective (Carl Zeiss).Slidebook (version 5.5, Intelligent Imaging Innovations Inc.) was usedto control the instrument and acquire optically sectioned images on anORCA-Flash4.0 V2 sCMOS camera (Hamamatsu) with a pixel size of6.5 mm 6.5 mm (2048 2048 total pixels).

The imaging configuration described above achieves an XY pixelresolution of 0.31 mmand0.1 mmfor the 20 and 63 objectives, respec-tively. For analysis, image Z-stacks were acquired, with a Z-step size of1.2 mmand 0.4 mm for the 20 and 63 objectives, respectively. Exposuretimes (100 to 800 ms) were maintained consistently for each markeracross all experiments, and care was taken to avoid any incidence of pixelsaturation.

Resulting3D imagedata setswere analyzedusing Imaris 7.4 (Bitplane).Microglia (CD68-positive) were identified using an automatic surfacesegmentation tool. These surfaces were subsequently used to mask Ablabeling. The volume of thismicroglia-internalized portion of Ab labelingwasmeasuredusing the surface segmentation tool. 3Drenderingof plaqueswas achieved using contouring and manual surface creation tools. Forevaluation of the proportion ofAb containedwithinmicroglial lysosomes,five sham-treated and five SUS-treated mice were analyzed, and differ-ences were tested with a t test.

Protein extractionWeperformeda serial extractionof brains to obtain fractions enriched forextracellular and Triton-soluble fraction proteins as described elsewhere(19). The forebrain of the right hemisphere was placed in 4 (w/v) ofbuffer containing 50mMtris-HCl (pH7.6), 0.01%NP-40, 150mMNaCl,2mMEDTA, 0.1%SDS, 1mMphenylmethanesulfonyl fluoride (PMSF),and complete protease inhibitors (Roche). The tissue was dissociatedusing a syringe and a 19-gauge needle, and the solution was centri-fuged at 800g for 10min to extract soluble extracellular proteins. Triton-soluble and intracellular proteins were obtained by homogenizing theintact cell pellet in 4 volumes of 50 mM tris-HCl, 150 mMNaCl, and1% Triton X-100 and centrifuging for 90 min at 16,000g. To obtain theguanidine-insoluble fraction, the pellet was extracted in 5 M guanidineHCl followed by two centrifugations at 16,000g for 30 min each. Totalprotein concentration was determined by BCA assay (Pierce). All ex-traction steps took place at 4C, and aliquots of the samples were storedat 80C until use.

Western blottingForty micrograms each of extracellular-enriched and Triton-solubleproteins was separated on 10 to 20% tris-tricine gels (Bio-Rad) and weretransferred onto 0.45-mmnitrocellulosemembranes, usingN-cyclohexyl-3-aminopropanesulfonic acid buffer (Bio-Rad) together with 20%meth-anol. A second membrane was used to capture the Ab monomer. Forantigen retrieval, the membranes were microwaved on a high settingfor 30 s and stained briefly in Ponceau S to check transfer and equal

loading. To visualize Ab species, the membranes were then blocked in

ceTranslationalMedicine.org 11 March 2015 Vol 7 Issue 278 278ra33 9

Statistics

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m Statistical analyses were conducted with Prism 6 software (GraphPad).Values were always reported as means SEM. One-way ANOVA withDunnetts post hoc test was used for three groups, two-way ANOVAwas used for APA, and unpaired t test was used to compare two groups.Where there was significant difference in variance between groups, weapplied Welchs correction.

SUPPLEMENTARY MATERIALS

www.sciencetranslationalmedicine.org/cgi/content/full/7/278/278ra33/DC1MethodsFig. S1. Absence of brain damage after either repeated or short-term SUS treatment.Fig. S2. Skeleton analysis of microglia.Fig. S3. Increased Ab uptake by microglial cells in the presence of albumin.Fig. S4. Analysis of IDE and tau phosphorylation after SUS treatment in AD mice.Fig. S5. Analysis of SUS-treated mice for inflammatory markers.Fig. S6. Absence of astrogliosis but activation of microglia after acute ultrasound treatment inwild-type mice.Movie S1 (mp4 format). High-resolution 3D reconstruction of a plaque imaged in a 40-mmsection of a SUS-treated mouse.

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Acknowledgments: We thank M. Staufenbiel (Novartis) for the APP23 mice; T. Palliyaguru fortissue extraction and Western blot analysis; N. Cummins, S. Ellis, and H. Evans for help with dataanalysis; D. Blackmore and J. Vukovic for advice on behavioral tests; J. Ellis and R. Sullivan for his-

tological tests; L. Hammond for expert confocal analysis and image processing; L. Wernbacher,T. Hitchcock, and the animal care team for animal maintenance; E. Konofagou (Columbia Uni-versity) and R. Seip (Philips Research) for advice on ultrasound; and R. Tweedale for reading ofthe manuscript. Funding: This study was supported by the Estate of Dr. Clem Jones AO as wellas grants from the Australian Research Council (ARC; DP130101932) and the National Healthand Medical Research Council of Australia (APP1037746 and APP1003150) to J.G. Funding formicroscopes was through the ARC Linkage Infrastructure, Equipment, and Facilities scheme[LE130100078]. Author contributions: J.G. provided funding and conceived the study, J.G.and G.L. designed the experiments, G.L. performed the experiments, and J.G. and G.L. analyzedthe data and wrote the paper. Competing interests: A provisional patent entitled Neuro-degenerative disease treatment has been filed. Application number: 2014902366. Filing date:20 June 2014. Data and materials availability: Materials are available upon request.

Submitted 7 November 2014Accepted 20 February 2015Published 11 March 201510.1126/scitranslmed.aaa2512

Citation: G. Leinenga, J. Gtz, Scanning ultrasound removes amyloid-b and restores memoryin an Alzheimers disease mouse model. Sci. Transl. Med. 7, 278ra33 (2015).

R E S EARCH ART I C L E

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DOI: 10.1126/scitranslmed.aaa2512, 278ra33 (2015);7 Sci Transl Med

Gerhard Leinenga and Jrgen GtzAlzheimer's disease mouse model

and restores memory in anScanning ultrasound removes amyloid-

Editor's Summary

Alzheimer's disease.findings suggest that repeated scanning ultrasound may be a noninvasive method with potential for treating

into their lysosomes. Theseshowed that scanning ultrasound activated resident microglial cells that took up Aand the scanning ultrasound treatment did not induce any apparent damage to the mouse brain. The authors thenimprovement in three memory tasks. These improvements were achieved without the use of any therapeutic agent,

is removed and memory is restored as revealed byultrasound, Leinenga and Gtz now demonstrate that A-forming mice with scanning deposition and memory impairment. By repeatedly treating these Aincluding A

) production show several aspects of Alzheimer's disease, (ATransgenic mice with increased amyloid-Can ultrasound restore memory?

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