Inside Alzheimer brain with CLARITY: senile plaques, neurofibrillary tangles and axons in 3-D

• DOI: 10.1007/s00401-014-1322-y
• Publication Date: 2014-07-28T05:36:53Z
• Authors: Kunie Ando; Quentin Laborde; Adina Lazar; David Godefroy; Ihsen Youssef; Majid Amar; Amy Pooler; Marie-Claude Potier; Benoit Delatour; Charles Duyckaerts
• Keywords: Male; 0301 basic medicine; Databases, Factual; Clinical Neurology; Intermediate Filaments; Neuroimaging; Plaque, Amyloid; Pathology and Forensic Medicine; Cellular and Molecular Neuroscience; 03 medical and health sciences; Imaging, Three-Dimensional; Alzheimer Disease; 616; Correspondence; Humans; [SDV.NEU] Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]; Amyloid beta-Peptides; Brain; Neurofibrillary Tangles; Sciences bio-médicales et agricoles; 3. Good health; [SDV] Life Sciences [q-bio]; Postmortem Changes; [SDV.NEU]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]; Female

Alzheimer’s disease (AD) is characterized by extracellular deposits of amyloid β (Aβ) peptide and intracellular tau aggregates. Examination these lesions in post-mortem brain tissue routinely provides a two-dimensional picture the pathology with limited depth field. Several tissue-clearing protocols have been recently published [1, 4–6, 8, 10, 11, 16]. We compared quality immunostaining on human tissues using several clearing techniques including CLARITY [4, 5], Scale [8], SeeDB [9], 3DISCO [6]. Results were best CLARITY. In that method, acrylamide hydrogel links protein maintains its structure. Removing lipids makes block transparent facilitates antibody diffusion 5]. noticed results improved if ScaleA2 [8] was used to mount after CLARITY.

Formalin-fixed frontal cortex samples from two controls five AD individuals (Braak VI Thal 5) obtained Brain Bank GIE NeuroCEB legal consent. cut at thickness 500 μm vibratome processed technique [5]. Embedded an constituted 4 % paraformaldehyde (PFA), acrylamide, 0.25 % temperature-triggering initiator VA-044, PBS, passively clarified 37 °C for 2 weeks solution (200 mM boric acid, w/v SDS, pH 8.5). The blocks immunostained rabbit polyclonal anti-tau B19 [3] mouse monoclonal anti-Aβ 4G8 (Covance). Alexa Fluor® 488 goat anti-rabbit 568 anti-mouse antibodies (Life technologies) as secondary antibodies. For Aβ neurofilament double immunohistochemistry, first incubated anti-neurofilament (M0762 Dako) then antibody. After blocking stage (10 % normal serum), biotin-labelled (Covance), revealed DyLight 488-labelled streptavidin (KPL, Eurobio, France). Triple staining (Aβ, neurofilament) also performed: M0762 third (Covance) streptavidin-Alexa 405 technology). mounting protocol modified original method: we added fixation step (4 % PFA PBS 15 min) end procedure improve stability immunostaining, incubation (0.2 M glycine quench autofluorescence. medium instead 80 % glycerol or FocusClear described method increased transparency glycerol, lowered cost experiment shortened time (overnight 2 days). immunolabellings analysed upright confocal microscope (Olympus Fluoview Fv1000). Z-stack images re-constructed Imaris software (Bitplane).

The volume “clarified” approximately 40 % passive removal lipids. While restored technique, further it but kept morphological technique. combination not appropriate stereological measures because expansion. Despite increase volume, axons (shown immunostaining) remained continuous (Supplementary 10.1007/s00401-014-1322-y). fully transparent. video, microscopy, 3-D view immunohistochemistry (10.1007/s00401-014-1322-y). Amyloid appeared diverse structure brains: some dense focal; diffuse hollow Accumulation regularly spaced clumps throughout Tau accumulated segmented discontinuous neuritic processes. Many dystrophic neurites associated dense, focal, presumably “mature” focal [2].

Plaque-induced distorted previously reported vivo observation transgenic animal [7, 12, 13, 15] affected areas brains [10, 14]. Our video showed persistence large number axons, one them (traced yellow) deflected (10.1007/s00401-014-1322-y).

Finally, triple (blue), (green) (red) tangle bearing neuron identified had no visible axonal projection region deposit contained few axons. immunoreactivity (revealed avidin biotin amplification step) excellent through whole block. Neurofilament immunoreactivities stronger 150-µm-thick layer top was, however, homogeneous. high detected bottom block: related to defect penetration laser beam during imaging process better near surface. Longer time, higher concentration detergent buffer various systems will be tested future homogeneity signal sample.

These uncover architecture demonstrate utility studying distribution 3-D, even tissue.