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Brian Bacskai, Massachusetts General Hospital, Charlestown, MA, USA

I N T R O D U C T I O NAlzheimer’s disease is an illness that can onlybe diagnosed with certainty with post-mortemexamination of brain tissue. Tissue samplesfrom afflicted patients show neuronal loss,neurofibrillary tangles (NFT) and amyloid-�plaques [1-4]. An imaging technique that per-mits in-vivo detection of NFTs or amyloid-�plaques is extremely valuable. We describetwo methods to study this: the use of fluores-cent histological dyes and in-vivo immunoflu-orescence using labeled antibodies, andadapting the technology of multiphotonmicroscopy to the challenge of imaging senileplaques in vivo in transgenic models ofAlzheimer’s disease.

M AT E R I A L S A N D M E T H O D SAnimalsTransgenic mice (PDAPP) which expressmutant human amyloid precursor protein andaccumulate amyloid-� deposits were used forin-vivo imaging of plaques [5,6]. Animals wereanesthetized with Avertin (Tribromoethanol,

B I O G R A P H YDr. Bacskai earned hisdoctorate at DartmouthCollege, NH, with adegree in biomedicalengineering. A postdoc-toral fellowship at theUniversity of California,San Diego, strengthenedhis skills at the develop-ment and application of microscopy tech-niques while focusing his interests in thefield of neuroscience. He is currently usingmultiphoton microscopy and other imagingtechniques to study Alzheimer’s and otherneurodegenerative diseases.

A B S T R A C TAlzheimer's disease is characterized by thepresence of senile plaques in the brain thatare comprised primarily of the amyloid-betapeptide. Transgenic mouse models are avail-able that develop senile plaques as they age,and are amenable for development of diag-nostic techniques as well as evaluating anti-amyloid therapies that may be clinically effec-tive. We used multiphoton microscopy toimage senile plaques in living transgenic micechronically and tested the ability ofimmunotherapy to target and remove indi-vidual senile plaques. Direct plaque imaging allowed us to quantitatively evaluate theeffect of this treatment on single, existingsenile plaques in living animals. We demon-strated that direct application of anti-amy-loid-beta antibodies to the brain is highlyeffective at clearing plaques within 3 days.These results support immunotherapy as apossible treatment for Alzheimer's disease.

K E Y W O R D SAlzheimer's, amyloid plaques, multiphotonmicroscopy, optical sections, immunofluo-rescence, in-vivo imaging

A C K N O W L E D G E M E N T SSupported by NIH grants AG08487, AG15453,a Pioneer award from the Alzheimer Associa-tion, and the Walters Family Foundation.

A U T H O R D E TA I L SDr. Brian J. Bacskai, Alzheimer’s DiseaseResearch Unit, Massachusetts GeneralHospital, Charlestown, MA 02129, USA.Tel: +1 617 724 5306 Fax: +1 617 724 1480Email: bbacskai@partners.org

Microscopy and Analysis (UK), 96, 25-27, 2003.©2003 Rolston Gordon Communications.

250 mg/kg intraperitoneally). A small skullwindow (about 1 mm in diameter) was createdusing a high-speed drill (Fine Science Tools,Foster City, CA). The site was moistened withartificial cerebrospinal fluid (ACSF; 125 mMNaCl; 26 mM NaHCO3; 1.25 mM NaH2PO4; 2.5mM KCl; 1 mM MgCl2; 1 mM CaCl2; 25 mM glu-cose) and the dura gently peeled from the sur-face of the brain. See Fig 1 for preparation ofskull for in-vivo imaging [7].

StainingThe monoclonal antibodies 10D5 and 3D6,directed against epitopes in the amino termi-nus of amyloid-� [8], were labeled with fluo-rescein (Molecular Probes, Eugene, OR).Twenty minutes after adding ~1 mg/ml solu-tion of antibody and 0.005% thioflavine S inACSF, the solution was washed off with ACSF,and the animal was prepared for imaging.

ImagingThe animal was placed in a head holder on asmall, custom-modified stage insert of an

Multiphoton Microscopy of Clearance ofMouse Amyloid-� Plaques In Vivo

Figure 1: Preparation of skull for in vivo imaging. (a) Gross appearance of skull through dissecting microscope before imaging. (b) Schematic diagram of themicroscope objective during imaging. The thinned area of skull is bathed in a pool of ACSF (light gray) that is retained by a ring of bone wax (dark gray).A small break is made in the lateral wall of the thinned area to allow for thioflavine-S entry. (c) In-vivo visualisation of thioflavine S-positive amyloid ina 15-month-old mouse. A single optical section near the surface of the skull is shown. Thioflavine S-positive amyloid angiopathy is visible ringing thepial arteriole in this image. The fainter autofluorescence of the skull bone is visible in the bottom right corner; the fibrous autofluorescence of the durais visible as a band at bottom right. (d) Another optical section from the same z-series as (c), but 50 µm deeper into the brain, showing a thioflavineS-positive amyloid deposit in layer 1 of the mouse cortex. (e) Perpendicular volume rendering of the entire stack of images, with the skull visible at thetop, the amyloid-encrusted pial vessel just beneath, and the thioflavine S-positive plaque deep in the living brain. The autofluorescent dura can also beseen as a faint layer between the vessel and the skull. The approximate levels of optical sections shown in (c) and (d) are represented by dotted lines.Scale bars = 25 µm.

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Olympus BX-50 microscope, and the siteimaged using a Bio-Rad 1024 MP system (Bio-Rad Cell Science Division, Hemel Hempstead,UK). The stage was equipped with X-Yencoders (Boeckeler, Tuscon, AZ), and the loca-tion of the initial imaging recorded. A fluores-cent angiogram was obtained using TexasRed-labeled 70,000 MW dextran (MolecularProbes) injected into the tail vein at the timeof imaging. The angiogram provided addi-tional local landmarks to ensure that the sameimaging volume was obtained at each session.Multiphoton fluorescence was generated with750-nm excitation from a mode-locked Ti:Sap-phire laser (Tsunami, Spectra Physics, Moun-tain View, CA; 10W Millenium pump laser).Custom-built external detectors (HamamatsuPhotonics, Bridgewater, NJ) collected emittedlight in the range of 380-480 nm (thioflavineS), 500-540 nm (fluorescein) and 560-650 nm(Texas Red).

R E S U LT SIn-vivo imaging of senile plaquesThioflavine S is a standard fluorescent stainthat specifically binds to amyloid proteindeposits. Dense-core thioflavine S-positiveplaques and amyloid angiopathy up to 150 µmdeep to the surface of the brain, into layers IIand III of the mouse cortex (Fig 2, a and d),were observed [7,9]. In-vivo immunofluores-cence using fluorescein-labeled monoclonalantibody 10D5 revealed numerous amyloid-�deposits, some of which appeared to be dif-fuse amyloid and others of which had discretecores (Fig 2, b and d). The diffuse deposits hada fine morphology with frequent extensions,irregular shapes and clusters identical to thoseobserved by conventional histologicalimmunostaining [9]. Amyloid angiopathy onvessels of the pia mater was revealed by stain-ing with thioflavine S and antibodies againstamyloid-� (Fig 2d). Thus, fluorescently labeledanti-amyloid-� antibodies diffused into thecortex and specifically labeled amyloid-�deposits, allowing imaging by multi-photonmicroscopy. Simultaneous injection of TexasRed-labeled dextran into a tail vein helpedvisualise capillaries and larger vessels, provid-ing a ‘road map’ for re-imaging (Fig 2, c and d).Thus, the combined techniques demonstratesimultaneous in-vivo histology, angiographyand immunofluorescence (Fig 2d)

Clearance of amyloid-� deposits byimmunotherapyTherapeutic strategies for Alzheimer's diseasemust not only decrease new amyloid-� pro-duction and deposition, but they must reversedeposits that already exist. These experimentsfollowed the observation that immunizationof PDAPP mice with amyloid-� leads to theprevention of new amyloid-� deposits [10]. Totest if the in-vivo interaction of an anti-amy-loid-� antibody with a plaque would lead to itsclearance, amyloid-� deposits were imagedusing thioflavine S in living mice before andafter therapeutic intervention (Fig 3). Micewere anesthetized, and a 1-1.5-mm cran-iotomy was performed. Thioflavine S and a

Figure 3: Clearance of dense-core amyloid-� deposits after immunotherapy. (a) and (b) A thioflavine S-positive plaque in the first imaging session (a), and 3 daysafter application of 10D5 (b). (c) and (d) A thioflavine S-positive plaque (c) in a 16B5- treated animal does not change 3 days later (d) Scale bar = 20µm.

Figure 2: Imaging of amyloid-� deposits in the live mouse: 3D reconstructions. (a) Thioflavine-S localization shown in red with 4 dense-core plaques in this field,as well as amyloid angiopathy on the blood vessel in the top left corner. (b) In-vivo immunofluorescence with labeled anti-amyloid-b antibodies, whichstain both fibrillar and diffuse amyloid-� deposits (blue). (c) Fluorescent angiography (green) provides 3-D fiduciary points to allow lining up imagingvolumes within the same animal over time. (d) Panels (a-c) merged into one 24-bit image, where the thioflavine S-positive plaques and amyloid angiopa-thy are seen as pink, surrounded by difuse amyloid-b deposits in blue. Scale bar = 100 µm.

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Figure 4: In-vivo imaging of amyloid-� deposits in 20-month-old homozygous PDAPP mice. (a) and (b) Reconstructions of stacks of Z-series images taken at 5-µm steps with a 20X objective, or (c) and (d) 2-µm steps with a 60X objective starting from just below the cortical surface to approximately 150 µmbelow the surface. Initial imaging session (a) and (c) shows numerous 10D5 immunoreactive amyloid-� plaques in the neuropil and associated withvessels in one representative animal. 3 days later (b) and (d) amyloid-� is visualized with fluorescein-labeled monoclonal antibody 3D6. Very little ofthe amyloid-� remains, showing reversal of previously existing amyloid-� deposits. Note that the vessel-associated amyloid-� remains intact and is read-ily immunostained. Scale bars: (a, b) = 50 µm; (c, d) = 25 µm

solution of antibody (1 mg/ml) were applied tothe cranial window. The animal was imagedand allowed to recover. Three days later theanimal was re-anesthetized and the same vol-ume was imaged. Texas-Red angiography andstage location assured that the exact same vol-ume was being imaged. Mice were randomlyassigned to groups treated with either 10D5 or16B5 (16B5 is a monoclonal antibody directedagainst an intracellular epitope of human tau,which does not cross react with rodent tau).

After treatment with 10D5, 70% of plaqueswere cleared 3 days after initial imaging (Fig 3,a and b). In the 16B5 group, only 20% ofplaques could not be found (Fig 3, c and d).This result demonstrated that dense-core amy-loid-� deposits were reversed by administra-tion of 10D5. The next step was to examinewhether all immunodetectable forms of amy-loid-� deposits could be cleared byimmunotherapy by repeating the experimentusing labeled 10D5 as the imaging agent atthe initial imaging session. As expected, thelabeled 10D5 revealed innumerable diffuseand dense-core amyloid-� deposits (Fig 4, aand c). The animals recovered and three dayslater were re-imaged.

Very little or no detectable fluorescenceremained from the application of fluorescein-labeled 10D5 that had been administeredthree days before [9]; labeled 3D6 was thenapplied directly to the cortex in both treat-ment groups. Few or none of the amyloid-�deposits that were present at the initial imag-ing remained (Fig 4, b and d). Amyloidangiopathy was still detected (Fig 4).

D I S C U S S I O NMultiphoton microscopy uses relativelybenign, long-wavelength light to excite stan-dard fluorophores [11,12]. An advantage ofmultiphoton microscopy is that excitationoccurs only in the focal volume of the objec-tive lens that focuses the laser. Optical imagingpermits a resolution in the order of onemicron, two orders of magnitude higher thanconventional in-vivo imaging techniques, suchas positron-emission tomography or mag-netic-resonance imaging. With multiphotonmicroscopy, tightly focused images of micro-scopic structures or lesions can be obtainedseveral hundred micrometers below the sur-face of the brain in a live animal. Here wedescribe a powerful in-vivo multiphoton imag-ing technology that allows visualisation of dis-tinct brain structures, in a living anesthetizedmouse, with a resolution of approximately1µm. This provides extraordinary in-vivoimages of individual cells or pathological struc-tures, with a resolution that far exceeds otherin-vivo technologies. In principle, any extracel-lular epitope could be visualised by in-vivoimmunofluorescence. Repeat imaging of thesame site hours or days later can be readilyobtained, and this can be extended to weekswith modifications in the protocol. This abilityto chronically image the same site in a livingmouse makes this experimental approach suit-

able for studies of diverse therapeutic inter-ventions. Using this novel technique, weshowed for the first time reversal of existingamyloid-� deposits in the brain due to anexperimental intervention [9].

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©2003 Rolston Gordon Communications.

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