Questo lavoro è stato sostenuto dal Canadian Institutes of Health Research (CIHR), la Famiglia Townsend, e Jack Brown e Famiglia Alzheimer Research Foundation (a WAS). WS è titolare del Canada Research Chair nella malattia di Alzheimer. PTTL è supportato dal Scienze Naturali e Direttore di Ricerca di Ingegneria e del Michael Smith Fondazione per la borsa di studio Health Research.

1. Fissazione dei cervelli del mouse e criosezionamento <ol><li> Topi transgenici sono sacrificati con anidride carbonica, seguita dalla decapitazione e il cervello estratto. </li><li> Il cervello è sezionato longitudinalmente al centro. La metà del cervello saranno istantaneamente congelati in ghiaccio secco per l&#39;analisi biochimica. L&#39;altro sarà sommersa nel 4% paraformaldeide (PFA) per almeno 48 ore a 4 ° C. </li><li> Dopo la fissazione in PFA 4% per almeno 48 ore, il cervello emi sono trasferiti direttamente nella soluzione di saccarosio al 30% e posto a 4 ° C. Al punto, il cervello dovrebbe essere emi galleggiante nella soluzione di saccarosio. Quando il cervello Hemi affondò sul fondo, di solito richiede circa 48 ore, è pronto per essere incorporato in ottobre per criosezionamento. </li><li> Prima criosezionamento, montare un sottile strato di ottobre sulla manopola e far congelare a -20 ° C. Rimuovere il cervelletto dal resto del cervello e emi posto sullo strato di gelato ottobre Coprire immediatamente il cervello emi con OCT e permettono di congelare lentamente a -20 ° C. Una volta che l&#39;embedding ottobre il cervello si è trasformato opaco, è pronto per essere sezionato. </li><li> Impostare lo spessore di ogni sezione a 30 micron. Raccogliere ogni fetta in un contenitore con la soluzione D&#39;Olomos. </li></ol> 2. Procedura di immunoistochimica per placche neuritiche (gratuito sezioni flottante) <ol><li> Trattare ogni sezione in 88% di acido formico per 15 minuti. Dopo il trattamento con acido formico, la sezione sarà temporaneamente volta opaco e può apparire avvizzire. </li><li> Rimuovere ogni sezione in un piatto e lavare con Tx-PBS (0,3% Triton X-100 in PBS) per 5 min x 3 agitando delicatamente. </li><li> Perossido di blocco endogeno blocco dello 0,5% H 2 O 2 in Tx-PBS, RT 30 minuti agitando delicatamente. </li><li> Lavare con Tx-PBS per 10 min x 3 agitando delicatamente. </li><li> Sezione di blocco con 5% senza grassi del latte scremato sciolto in Tx-PBS a temperatura ambiente per 1 ora agitando delicatamente. </li><li> Incubare le sezioni in anticorpo primario (biotinilato 4g8, 1:500-1:2000, Sigillo) diluito in Tx-PBS contenente 5% del latte a 4 ° C per una notte agitando delicatamente. </li><li> Lavare con Tx-PBS per 10 min x 3 agitando delicatamente. </li><li> Preparare protocollo del produttore soluzione ABC successivo (Elite ABC, Vector Laboratories). Incubare le sezioni in miscela ABC per 30 minuti agitando delicatamente. </li><li> Lavare con Tx-PBS per 10 min x 3 agitando delicatamente. </li><li> Preparare 3,3 &#39;-diaminobenzidina tetraidrocloruro (DAB) (Vector Laboratories) protocollo del produttore successivo. Incubare le sezioni in miscela DAB per 5-10 min. A questo punto, si potrà osservare un colore brunastro svilupparsi su ogni sezione. </li><li> Lavare con Tx-PBS per 10 min x 3 agitando delicatamente. </li><li> Free-floating sezioni cervello sono montate su vetrini rivestiti di gelatina, per aiutare con l&#39;adesione dei tessuti. Le sezioni sono poi essiccate all&#39;aria seguita da compensazione con una serie di xilene e montati in Entellen. </li><li> Placche sono visualizzati al microscopio ottico con ingrandimento 40X e sono quantificati valutando la media della conta targa per fetta per ogni mouse. </li></ol> 3. Thioflavin S colorazione procedura per l&#39;individuazione delle placche neuritiche <ol><li> Sacrificio del mouse e procedure di estrazione del cervello sono quelli indicati in punti da 1.1 a 1.5. </li><li> Montare 4-5 sezioni del cervello emi su un vetrino e lasciare asciugare completamente prima della colorazione. </li><li> Lavare i vetrini con etanolo al 70% per 1 minuto. </li><li> Lavare i vetrini con 80% etanolo per 1 minuto. </li><li> Incubare i vetrini in soluzione filtrata (0,2 micron filtro) thioflavin S (1% a 80% di etanolo) per 15 minuti. (Soluzione Thioflavin S deve essere filtrata prima di ogni utilizzo). Nota: thioflavin S proteggere dalla luce e proteggere i vetrini colorati dalla luce. </li><li> Lavare i vetrini con 80% etanolo per 1 minuto. </li><li> Lavare i vetrini con il 70% etanolo 1 minuto. </li><li> Lavare con acqua distillata due volte. </li><li> Diapositive montare in acquosi di montaggio e permettere che i vetrini ad asciugare al buio per almeno 2 ore, seguita da coprioggetto tenuta con smalto trasparente. Sezioni colorate devono essere conservati al buio a 4 ° C. </li><li> La fluorescenza verde placche colorate possono essere visualizzate con microscropy fluorescenti. Analizzare le diapositive entro una settimana, perché la colorazione svanirà con il tempo. </li></ol> 4. Rappresentante Risultati Nel nostro recente studio sulla efficacia del farmaco anti-epilettico e stabilizzatore dell&#39;umore acido valproico (VPA) per inibire la formazione di placche neuritiche, abbiamo usato il immunocolorazione sopra descritto e thioflavin S colorazione procedure per identificare le placche neuritiche nei topi modello dC (3). La figura 1 rappresenta un tipico 9 mesi APP23 topo transgenico, colorate con 4g8 con anti-Aβ biotinilato e rilevati con il metodo ABC. Placche neuritiche sono chiaramente etichettati con l&#39;anticorpo e sono indicati dalle frecce bianche. Mediante colorazione thioflavin S, abbiamo rilevato deposizione neuritiche targa 2 mesi vecchia APP23xPS traditopi nsgenic (Figura 2). Thioflavin S legato Aβ contenenti placche neuritiche appare verde al microscopio a fluorescenza (indicato da frecce bianche). Abbiamo dimostrato che il trattamento con VPA, la formazione di placche neuritiche è significativamente ridotto (3). In un altro studio, abbiamo studiato l&#39;ipossia molecolare collegamento sottostante e patogenesi dell&#39;Alzheimer. Ancora una volta, utilizzando sia 4g8 anti-Aβ colorazione immunoistochimica e colorazione thioflavin S, abbiamo scoperto che l&#39;ipossia favorito la formazione di placche contenenti Aβ neuritiche (4). <img src="/files/ftp_upload/2831/2831fig1.jpg" alt="Figura 1"/> Figura 1. Analisi immunoistochimica della formazione di placche neuritiche in topi transgenici dC. APP23 topi all&#39;età di 9 mesi sono stati sacrificati e sono stati sezionati, fissa e sezionato. Placche neuritiche nel dell&#39;ippocampo sono state rilevate con un anti-Aβ 4g8 anticorpo e sono stati sviluppati utilizzando i metodi ABC e DAB. Le placche sono state visualizzate mediante microscopia ottica con 40X. Frecce bianche indicano placche neuritiche Aβ. <img src="/files/ftp_upload/2831/2831fig2.jpg" alt="Figura 2"/> Figura 2. Colorazione Thioflavin S di placche neuritiche nei topi dC modellato. APP23xPS45 doppio topi transgenici a 8 settimane di vita sono stati sacrificati e sono stati sezionati, fissa e sezionato. Piaghe neuritiche nel dell&#39;ippocampo sono state confermate mediante colorazione fluorescente thioflavin S e visualizzati al microscopio con ingrandimento 40x. Frecce bianche indicano thioflavin S placche neuritiche macchiato.

<ol>
	<li>Glenner, G. G., Wong, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alzheimer's+disease:+initial+report+of+the+purification+and+characterization+of+a+novel+cerebrovascular+amyloid+protein.">Alzheimer's disease: initial report of the purification and characterization of a novel cerebrovascular amyloid protein.</a> Biochem Biophys Res Commun. 120, 885-890 (1984).</li><li>Hardy, J., Selkoe, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+amyloid+hypothesis+of+Alzheimer's+disease:+progress+and+problems+on+the+road+to+therapeutics.">The amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics.</a> Science. 297, 353-356 (2002).</li><li>Qing, H., He, G., Ly, P. T., Fox, C. J., Staufenbiel, M., Cai, F., Zhang, Z., Wei, S., Sun, X., Chen, C. H., Zhou, W., Wang, K., Song, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Valproic+acid+inhibits+Abeta+production,+neuritic+plaque+formation,+and+behavioral+deficits+in+Alzheimer's+disease+mouse+models.">Valproic acid inhibits Abeta production, neuritic plaque formation, and behavioral deficits in Alzheimer's disease mouse models.</a> J Exp Med. 205, 2781-2789 (2008).</li><li>Sun, X., He, G., Qing, H., Zhou, W., Dobie, F., Cai, F., Staufenbiel, M., Huang, L. E., Song, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+facilitates+Alzheimer's+disease+pathogenesis+by+up-regulating+BACE1+gene+expression.">Hypoxia facilitates Alzheimer's disease pathogenesis by up-regulating BACE1 gene expression.</a> Proc Natl Acad Sci U S A. 103, 18727-18732 (2006).</li></ol>

Tutti gli esperimenti sono stati condotti in conformità con l&#39;Università di cura degli animali Columbia britannica e il comitato Usa e le linee guida CIHR.

Immunoistochimica utilizzando il biotinilato macchie anticorpo marcato 4g8 placche neuritiche nel AD modellato topi con specificità. Il risultato colorazione consente la quantificazione e il confronto del carico di placca tra i diversi gruppi di trattamento. Ci sono diversi passi cruciali che potrebbero influenzare l&#39;esito. Poiché il cervello emi non sono perfusi prima dell&#39;estrazione dal cranio, la cura deve essere usato durante il processo di estrazione al fine di prevenire danni al cervello emi. Inoltre, poiché il cervello emi è passivamente perfuso, si consiglia di incubazione in 4% PFA per almeno 48 ore a 4 ° C prima immersione nella soluzione di saccarosio al 30%. In alternativa, la perfusione transcardial poteva essere fatto prima di estrarre il cervello. Se il cervello è fissata correttamente, dovrebbe avere una consistenza gommosa. Nel caso in cui il cervello non è fissato bene, le sezioni sarà facile strappare nella soluzione D&#39;Olomos o durante le procedure di colorazione. L&#39;anticorpo monoclonale 4g8 è reattivo ai residui di aminoacidi 17-24 di Aβ e ha riconosciuto l&#39;epitopo nella sequenza core (VFFAE). Dal momento che 4g8 è derivato da specie del mouse, si tende a dare una colorazione di fondo più alto. Così, abbiamo scelto di incubare le sezioni alla diluizione indicata con considerazioni a raggiungere un vero segnale, minimizzando la colorazione di fondo. Oltre a immunocolorazione per neuritiche rilevamento targhe con l&#39;anticorpo 4g8, metodo di colorazione uno thioflavin S è anche utilizzato per identificare le placche. Thioflavin S è una miscela omogenea colorante che risulta dalla metilazione di dehydrothiotoluidine con acido solfonico. Thioflavin S non si lega selettivamente i contenuti foglietto beta di proteine, come quelle in oligomeri amiloide. Su vincolante, thioflavin subisce un cambiamento caratteristico blu del suo spettro di emissione. Al contrario vincolante thioflavin S alle forme monomeriche non suscitare un spostamento verso il blu e non poteva essere rilevata con il microscopio a fluorescenza. Colorazione Thioflavin S offre una valida alternativa per lo screening rapido per amiloide come l&#39;intensità della fluorescenza permette una buona visualizzazione di piccole quantità di depositi di amiloide. Tuttavia, uno svantaggio di colorazione thioflavin S è la mancanza di specificità. Molti altri componenti del tessuto tra cui fogli contenenti beta estese, quali fibrinoids, ialina, cheratina, ecc, hanno un po affinità con questo colorante.

<table border="1"><tbody><tr><th> Nome del reagente e </th><th> Azienda </th><th> Numero di catalogo </th></tr><tr><td> Paraformaldeide </td><td> Sigma-Aldrich </td><td> P6148-1KG </td></tr><tr><td> Polyvinylpyrrolidene </td><td> Sigma-Aldrich </td><td> PVP40-100G </td></tr><tr><td> Saccarosio </td><td> Fisher Scientific </td><td> S5-3 </td></tr><tr><td> Glicole etilenico </td><td> Sigma-Aldrich </td><td> 324558-2L </td></tr><tr><td> Tissue-Tek ottobre Compound </td><td> Sakura </td><td> 4583 </td></tr><tr><td> 88% di acido formico </td><td> Fisher Scientific </td><td> A118P-500 </td></tr><tr><td> Triton X-100 </td><td> ICN Biomedicals </td><td> 807426 </td></tr><tr><td> H2O2 </td><td> Sigma-Aldrich </td><td> H-1009 </td></tr><tr><td> Biotina etichetta 4g8 anticorpi </td><td> Cedarlane </td><td> SIG-39240-500 </td></tr><tr><td> Elite ABC kit </td><td> Vettore </td><td> PK-6100 </td></tr><tr><td> Imm PATTO DAB </td><td> Vettore </td><td> SK-4105 </td></tr><tr><td> Xilene </td><td> Fisher Scientific </td><td> X4-4 </td></tr><tr><td> Entellan </td><td> EM scienza </td><td> 65037-71 </td></tr><tr><td> SuperFrost * Plus preparati microscopici </td><td> Fisher Scientific </td><td> 12-550-15 </td></tr><tr><td> Criostato </td><td> Leica </td><td> CM-3050-S </td></tr><tr><td> MX35 Premier microtomo lama </td><td> Thermo Scientific </td><td> 3052835 </td></tr><tr><td> Thioflavin S </td><td> Sigma-Aldrich </td><td> T1892-25G </td></tr></tbody></table>

detection of neuritic plaques in alzheimer's disease mouse model

Malattia di Alzheimer (AD) è il disturbo più comune neurodegenerativa che porta alla demenza. La formazione di placche neuritiche è una delle caratteristiche patologiche della malattia di Alzheimer. Il componente centrale di placche neuritiche è una piccola proteina chiamata proteina filamentosa β amiloide (Aβ) 1, che è derivata da sequenziale taglio proteolitico della proteina beta-amiloide precursore (APP) da β-secretasi e γ-secretasi. L&#39;ipotesi amiloide comporta che Aγ contenenti placche come il meccanismo sottostante tossici in AD patologia 2. L&#39;analisi post-mortem della presenza di placca neuritiche conferma la diagnosi di Alzheimer. Per ulteriormente la nostra comprensione della neurobiologia Aγ nella patogenesi dell&#39;Alzheimer, vari ceppi di topi che esprimono AD-correlati mutazioni nei geni APP umani sono stati generati. A seconda della gravità della malattia, questi topi si svilupperà placche neuritiche in età diverse. Questi topi servire come preziosi strumenti per lo studio della patogenesi e sviluppo di droghe che potrebbero influenzare il percorso di elaborazione APP e la formazione della placca neuritiche. In questo protocollo, utilizziamo un metodo di immunoistochimica per l&#39;individuazione specifica delle placche neuritiche nei topi modello dC. Noi specificamente discutere della preparazione di estrarre il cervello e mezzo, la fissazione paraformaldeide, criosezionamento, e due metodi per rilevare placche nevrotico nel AD topi transgenici: rilevazione immunoistochimica utilizzando il metodo ABC e DAB e la rilevazione con metodo di colorazione fluorescente thiofalvin S.

Watch this Scientific Journal Video about Detection of Neuritic Plaques in Alzheimer's Disease Mouse Model at JoVE.com

Detection of Neuritic Plaques in Alzheimer's Disease Mouse Model

Una delle caratteristiche patologiche di Alzheimer è la formazione di placche di amiloide β neuritiche proteina positivo. In questo protocollo si descrivono due metodi per rilevare placche neuritiche in topi transgenici modello AD: rilevazione immunoistochimica utilizzando il metodo ABC e DAB e la rilevazione con metodo di colorazione fluorescente thioflavin S.

Rilevazione di placche neuritiche nel modello di malattia di Alzheimer mouse

Alzheimer's disease (AD) is the most common neurodegenerative disorder leading to dementia. Neuritic plaque formation is one of the pathological hallmarks ...

detection-of-neuritic-plaques-in-alzheimer-s-disease-mouse-model

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

36.5K Views.  The University of British Columbia. Una delle caratteristiche patologiche di Alzheimer è la formazione di placche di amiloide β neuritiche proteina positivo. In questo protocollo si descrivono due metodi per rilevare placche neuritiche in topi transgenici modello AD: rilevazione immunoistochimica utilizzando il metodo ABC e DAB e la rilevazione con metodo di colorazione fluorescente thioflavin S.

Video: Detection of Neuritic Plaques in Alzheimer's Disease Mouse Model

Establishment of a Valuable Mimic of Alzheimer's Disease in Rat Animal Model by Intracerebroventricular Injection of Composited Amyloid Beta Protein

Alzheimer&#39;s disease (AD) is an irreversible, progressive brain disease that slowly destroys memory and is accompanied by neuron loss and structure change. With the increase of AD patients worldwide, the pathology and treatment of the disease has become a focus in the International Pharmaceutical Industry. Thus, the establishment of the animal model to mimic AD in the laboratory is of great importance.
Here, we describe a detailed protocol for establishing a mimic of AD in a rat animal model though intracerebroventricular injection of amyloid beta protein 25-35 (A&#946;25-35) combined with aluminum trichloride (AlCl3) and anterodorsal thalamic nucleus injection of recombinant human transforming growth factor-&#946;1 (RHTGF-&#946;1) to rats. The related markers of AD were measured, including: spatial memory, neuronal structure and substructure, neuronal A&#946;, and neurofibrillary tangles (NFT) production. This rat model demonstrates spatial memory impairment, neuronal structure and substructure pathological changes, neuron intracellular A&#946; burden, and NFT aggregation, and provides a close mimic of the neuronal structure and function disorder to that of clinical AD patients. Thus, the presented AD rat modelprovides a valuable in vivo tool for exploring neuronal function, neuronal pathology, and drug screening of AD.

This is a protocol to mimic Alzheimer&#39;s Disease in rats by evaluation of spatial memory impairment, neuronal pathological changes, neuronal amyloid beta protein (A&#946;) burden, and neurofibrillary tangles aggregation, induced by the injection of A&#946;25-35 combined with aluminum trichloride and recombinant human transforming growth factor-&#946;1.

Istituzione di un Mimic prezioso del morbo di Alzheimer nel modello animale del ratto tramite iniezione di Intracerebroventricular di proteina Beta amiloide composte

Alzheimer&#39;s disease (AD) is an irreversible, progressive brain disease that slowly destroys memory and is accompanied by neuron loss and structure ...

Ricerca

Comportamento

A Technique for Serial Collection of Cerebrospinal Fluid from the Cisterna Magna in Mouse 

Alzheimer's disease (AD) is a progressive neurodegenerative disease that is pathologically characterized by extracellular deposition of &#946;-amyloid peptide (A&#946;) and intraneuronal accumulation of hyperphosphorylated tau protein. Because cerebrospinal fluid (CSF) is in direct contact with the extracellular space of the brain, it provides a reflection of the biochemical changes in the brain in response to pathological processes. CSF from AD patients shows a decrease in the 42 amino-acid form of A&#946; (A&#946;42), and increases in total tau and hyperphosphorylated tau, though the mechanisms responsible for these changes are still not fully understood. Transgenic (Tg) mouse models of AD provide an excellent opportunity to investigate how and why A&#946; or tau levels in CSF change as the disease progresses. Here, we demonstrate a refined cisterna magna puncture technique for CSF sampling from the mouse. This extremely gentle sampling technique allows serial CSF samples to be obtained from the same mouse at 2-3 month intervals which greatly minimizes the confounding effect of between-mouse variability in A&#946; or tau levels, making it possible to detect subtle alterations over time. In combination with A&#946; and tau ELISA, this technique will be useful for studies designed to investigate the relationship between the levels of CSF A&#946;42 and tau, and their metabolism in the brain in AD mouse models. Studies in Tg mice could provide important validation as to the potential of CSF A&#946; or tau levels to be used as biological markers for monitoring disease progression, and to monitor the effect of therapeutic interventions. As the mice can be sacrificed and the brains can be examined for biochemical or histological changes, the mechanisms underlying the CSF changes can be better assessed. These data are likely to be informative for interpretation of human AD CSF changes.

A Technique for Serial Collection of Cerebrospinal Fluid from the Cisterna Magna in Mouse

Transgenic (Tg) mouse models of AD provide an excellent opportunity to investigate how and why A&#946; or tau levels in CSF change as the disease progresses in human patients. Here, we demonstrate a refined cisterna magna puncture technique for serial CSF sampling from the mouse.

Una tecnica per la raccolta seriale del liquido cerebrospinale dalla Magna Cisterna in Mouse

Alzheimer's disease (AD) is a progressive neurodegenerative disease that is pathologically characterized by extracellular deposition of &#946;-amyloid ...

Biologia

Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the over-production of amyloid-beta and the deposition of amyloid-beta plaques in brain regions of the afflicted individuals. Throughout the years scientists have generated numerous Alzheimer&#8217;s disease mouse models that attempt to replicate the amyloid-beta pathology. Unfortunately, the mouse models only selectively mimic the disease features. Neuronal death, a prominent effect in the brains of Alzheimer&#8217;s disease patients, is noticeably lacking in these mice. Hence, we and others have employed a method of directly infusing soluble oligomeric species of amyloid-beta - forms of amyloid-beta that have been proven to be most toxic to neurons - stereotaxically into the brain. In this report we utilize male C57BL/6J mice to document this surgical technique of increasing amyloid-beta levels in a select brain region. The infusion target is the dentate gyrus of the hippocampus because this brain structure, along with the basal forebrain that is connected by the cholinergic circuit, represents one of the areas of degeneration in the disease. The results of elevating amyloid-beta in the dentate gyrus via stereotaxic infusion reveal increases in neuron loss in the dentate gyrus within 1 week, while there is a concomitant increase in cell death and cholinergic neuron loss in the vertical limb of the diagonal band of Broca of the basal forebrain. These effects are observed up to 2 weeks. Our data suggests that the current amyloid-beta infusion model provides an alternative mouse model to address region specific neuron death in a short-term basis. The advantage of this model is that amyloid-beta can be elevated in a spatial and temporal manner.

Here, we present a protocol for direct stereotaxic brain infusion of amyloid-beta. This methodology provides an alternative in vivo mouse model to address the short-term effects of amyloid-beta on brain neurons.

Infuso stereotassico di Oligomeric amiloide-beta nelle Hippocampus mouse

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the ...

Neuroscienze

Detection of Disease-associated &#945;-synuclein by Enhanced ELISA in the Brain of Transgenic Mice Overexpressing Human A53T Mutated &#945;-synuclein

In addition to established methods like Western blot, new methods are needed to quickly and easily quantify disease-associated &#945;-synuclein (&#945;SD) in experimental models of synucleopathies. A transgenic mouse line (M83) over-expressing the human A53T &#945;S and spontaneously developing a dramatic clinical phenotype between eight and 22 months of age, characterized by symptoms including weight loss, prostration, and severe motor impairment, was used in this study. For molecular analyses of &#945;SD (disease-associated &#945;S) in these mice, an ELISA was designed to specifically quantify &#945;SD in sick mice. Analysis of the central nervous system in this mouse model showed the presence of &#945;SD mainly in the caudal brain regions and the spinal cord. There were no differences in &#945;SD distribution between different experimental conditions leading to clinical disease, i.e., in uninoculated and normally aging transgenic mice and in mice inoculated with brain extracts from sick mice. The specific detection of &#945;SD immunoreactivity using an antibody against Ser129 phosphorylated &#945;S by ELISA essentially correlated with that obtained by Western blot and immunohistochemistry. Unexpectedly, similar results were observed with several other antibodies against the C-terminal part of &#945;S. The propagation of &#945;SD, suggesting the involvement of a &#8220;prion-like&#8221; mechanism, can thus be easily monitored and quantified in this mouse model using an ELISA approach.

An ELISA offering a novel quantitative approach is described. It specifically detects disease-associated &#945;-synuclein (&#945;SD) in a transgenic mouse model (M83) of synucleinopathy using several antibodies against either the Ser129 phosphorylated &#945;S form or the C-terminal part of the protein.

Il rilevamento della malattia associata α-sinucleina da avanzato ELISA nel cervello di topi transgenici A53T umano Mutato α-sinucleina

In addition to established methods like Western blot, new methods are needed to quickly and easily quantify disease-associated &#945;-synuclein (&#945;SD) ...

Medicina

Intracerebroventricular Injection of Amyloid-&#946; Peptides in Normal Mice to Acutely Induce Alzheimer-like Cognitive Deficits

Amyloid-&#946; (A&#946;) is a major pathological mediator of both familial and sporadic Alzheimer's disease (AD). In the brains of AD patients, progressive accumulation of A&#946; oligomers and plaques is observed. Such A&#946; abnormalities are believed to block long-term potentiation, impair synaptic function, and induce cognitive deficits. Clinical and experimental evidences have revealed that the acute increase of A&#946; levels in the brain allows development of Alzheimer-like phenotypes. Hence, a detailed protocol describing how to acutely generate an AD mouse model via the intracerebroventricular (ICV) injection of A&#946; is necessary in many cases. In this protocol, the steps of the experiment with an A&#946;-injected mouse are included, from the preparation of peptides to the testing of behavioral abnormalities. The process of preparing the tools and animal subjects before the injection, of injecting the A&#946; into the mouse brain via ICV injection, and of assessing the degree of cognitive impairment are easily explained throughout the protocol, with an emphasis on tips for effective ICV injection of A&#946;. By mimicking certain aspects of AD with a designated injection of A&#946;, researchers can bypass the aging process and focus on the downstream pathology of A&#946; abnormalities.

The amyloid-&#946; (A&#946;)-injected animal model enables the administration of a defined quantity and species of A&#946; fragments and reduces individual differences within each study group. This protocol describes the intracerebroventricular (ICV) injection of A&#946; without stereotactic instruments, enabling the production of Alzheimer-like behavioral abnormalities in normal mice.

Iniezione intracerebroventricular dei peptidi beta-amiloide in topi normali per indurre Acutamente Alzheimer-like Deficit cognitivi

Amyloid-&#946; (A&#946;) is a major pathological mediator of both familial and sporadic Alzheimer's disease (AD). In the brains of AD patients, ...

Correlative Light and Electron Microscopy to Study Microglial Interactions with &#946;-Amyloid Plaques

A detailed protocol is provided here to identify amyloid A&#946; plaques in brain sections from Alzheimer&#39;s disease mouse models before pre-embedding immunostaining (specifically for ionized calcium-binding adapter molecule 1 (IBA1), a calcium binding protein expressed by microglia) and tissue processing for electron microscopy (EM). Methoxy-X04 is a fluorescent dye that crosses the blood-brain barrier and selectively binds to &#946;-pleated sheets found in dense core A&#946; plaques. Injection of the animals with methoxy-X04 prior to sacrifice and brain fixation allows pre-screening and selection of the plaque-containing brain sections for further processing with time-consuming manipulations. This is particularly helpful when studying early AD pathology within specific brain regions or layers that may contain very few plaques, present in only a small fraction of the sections. Post-mortem processing of tissue sections with Congo Red, Thioflavin S, and Thioflavin T (or even with methoxy-X04) can label &#946;-pleated sheets, but requires extensive clearing with ethanol to remove excess dye and these procedures are incompatible with ultrastructural preservation. It would also be inefficient to perform labeling for A&#946; (and other cellular markers such as IBA1) on all brain sections from the regions of interest, only to yield a small fraction containing A&#946; plaques at the right location. Importantly, A&#946; plaques are still visible after tissue processing for EM, allowing for a precise identification of the areas (generally down to a few square millimeters) to examine with the electron microscope.

This article describes a protocol for visualizing amyloid A&#946; plaques in Alzheimer&#39;s disease mouse models using methoxy-X04, which crosses the blood-brain barrier and selectively binds to &#946;-pleated sheets found in dense core A&#946; plaques. It allows pre-screening of plaque-containing tissue sections prior to immunostaining and processing for electron microscopy.

Correlativo luce e microscopia elettronica allo Studio microgliali Interazioni con placche beta-amiloide

A detailed protocol is provided here to identify amyloid A&#946; plaques in brain sections from Alzheimer&#39;s disease mouse models before pre-embedding ...

Using Enzyme-based Biosensors to Measure Tonic and Phasic Glutamate in Alzheimer's Mouse Models

Neurotransmitter disruption is often a key component of diseases of the central nervous system (CNS), playing a role in the pathology underlying Alzheimer's disease, Parkinson's disease, depression, and anxiety. Traditionally, microdialysis has been the most common (lauded) technique to examine neurotransmitter changes that occur in these disorders. But because microdialysis has the ability to measure slow 1-20 minute changes across large areas of tissue, it has the disadvantage of invasiveness, potentially destroying intrinsic connections within the brain and a slow sampling capability. A relatively newer technique, the microelectrode array (MEA), has numerous advantages for measuring specific neurotransmitter changes within discrete brain regions as they occur, making for a spatially and temporally precise approach. In addition, using MEAs is minimally invasive, allowing for measurement of neurotransmitter alterations in vivo. In our laboratory, we have been specifically interested in changes in the neurotransmitter, glutamate, related to Alzheimer's disease pathology. As such, the method described here has been used to assess potential hippocampal disruptions in glutamate in a transgenic mouse model of Alzheimer's disease. Briefly, the method used involves coating a multi-site microelectrode with an enzyme very selective for the neurotransmitter of interest and using self-referencing sites to subtract out background noise and interferents. After plating and calibration, the MEA can be constructed with a micropipette and lowered into the brain region of interest using a stereotaxic device. Here, the method described involves anesthetizing rTg(TauP301L)4510 mice and using a stereotaxic device to precisely target sub-regions (DG, CA1, and CA3) of the hippocampus.

Here, we describe the setup, software navigation, and data analysis for a spatially and temporally precise method of measuring tonic and phasic extracellular glutamate changes in vivo using enzyme-linked microelectrode arrays (MEA).

Utilizzando biosensori a base di enzimi misurare Tonic e Phasic glutammato in modelli murini di Alzheimer

Neurotransmitter disruption is often a key component of diseases of the central nervous system (CNS), playing a role in the pathology underlying ...

Quantitative 3D In Silico Modeling (q3DISM) of Cerebral Amyloid-beta Phagocytosis in Rodent Models of Alzheimer's Disease

Neuroinflammation is now recognized as a major etiological factor in neurodegenerative disease. Mononuclear phagocytes are innate immune cells responsible for phagocytosis and clearance of debris and detritus. These cells include CNS-resident macrophages known as microglia, and mononuclear phagocytes infiltrating from the periphery. Light microscopy has generally been used to visualize phagocytosis in rodent or human brain specimens. However, qualitative methods have not provided definitive evidence of in vivo phagocytosis. Here, we describe quantitative 3D in silico modeling (q3DISM), a robust method allowing for true 3D quantitation of amyloid-&#946; (A&#946;) phagocytosis by mononuclear phagocytes in rodent Alzheimer&#39;s Disease (AD) models. The method involves fluorescently visualizing A&#946; encapsulated within phagolysosomes in rodent brain sections. Large z-dimensional confocal datasets are then 3D reconstructed for quantitation of A&#946; spatially colocalized within the phagolysosome. We demonstrate the successful application of q3DISM to mouse and rat brains, but this methodology can be extended to virtually any phagocytic event in any tissue.

Quantitative 3D In Silico Modeling q3DISM of Cerebral Amyloid-beta Phagocytosis in Rodent Models of Alzheimer's Disease

We developed a methodology for quantitative 3D in silico modeling (q3DISM) of cerebral amyloid-&#946; (A&#946;) phagocytosis by mononuclear phagocytes in rodent models of Alzheimer&#39;s disease. This method can be generalized for the quantitation of virtually any phagocytic event in vivo.

3D Quantitative<em&gt; In Silico</em&gt; Modeling (q3DISM) di cerebrale amiloide-beta fagocitosi in roditori modelli di malattia di Alzheimer

Neuroinflammation is now recognized as a major etiological factor in neurodegenerative disease. Mononuclear phagocytes are innate immune cells responsible ...

Assessment of Spontaneous Alternation, Novel Object Recognition and Limb Clasping in Transgenic Mouse Models of Amyloid-&#946; and Tau Neuropathology

Here we describe a staged, behavioral testing approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional motor behaviors in transgenic mouse models of &#946;-amyloidosis and tauopathy. The paradigm includes tests for spontaneous alternation in a Y-maze, novel object recognition, and limb clasping. These tests were selected because they: 1) interrogate function of cognitive or motor domains and the correlate neural circuitry relevant to the human disease state, 2) have clearly defined endpoints, 3) have easily implementable quality control checks, 4) can be run in a moderate throughput format, and 5) require little intervention by the investigator. These methods are designed for investigators looking to screen compounds for activity in short-term and working memory tasks, or functional motor behaviors associated with Alzheimer's disease mouse models. The methods described here use behavioral tests that engage a number of different brain regions including hippocampus and various cortical areas. Investigators that desire cognitive tests that specifically assess cognition mediated by a single brain region could use these techniques to supplement other behavioral tests.

Described here is a staged, behavioral screening approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional motor behaviors in transgenic mouse models of &#946;-amyloidosis and tauopathy. These methods are optimized to screen compounds for activity in short-term and working memory tasks.

Valutazione di alternanza spontanea, riconoscimento novello di oggetti e attacco degli arti nei modelli di topo transgenico Modelli di neuropathologia di Amiloide-β e Tau

Here we describe a staged, behavioral testing approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional ...

Detecting Amyloid-&#946; Accumulation via Immunofluorescent Staining in a Mouse Model of Alzheimer's Disease

Alzheimer's disease (AD) is a neurodegenerative disease that contributes to 60-70% dementia around the world. One of the hallmarks of AD undoubtedly lies on accumulation of amyloid-&#946; (A&#946;) in the brain. A&#946; is produced from the proteolytic cleavage of the beta-amyloid precursor protein (APP) by &#946;-secretase and &#947;-secretase. In pathological circumstances, the increased &#946;-cleavage of APP leads to overproduction of A&#946;, which aggregates into A&#946; plaques. Since A&#946; plaques are a characteristic of AD pathology, detecting the amount of A&#946; is very important in AD research. In this protocol, we introduce the immunofluorescent staining method to visualize A&#946; deposition. The mouse model used in our experiments is 5&#215;FAD, which carries five mutations found in human familial AD. The neuropathological and behavioral deficits of 5xFAD mice are well-documented, which makes it a good animal model to study A&#946; pathology. We will introduce the procedure including transcardial perfusion, cryosectioning, immunofluorescent staining and quantification to detect A&#946; accumulation in 5&#215;FAD mice. With this protocol, researchers can investigate A&#946; pathology in an AD mouse model.

In the neuropathology of Alzheimer's disease, one of the most crucial characteristics is the deposition of amyloid-&#946;. In this protocol, we describe the method of immunofluorescent staining in 5&#215;FAD transgenic mouse to detect amyloid-&#946; accumulation in plaques. The process of perfusion, cryosectioning, staining and quantification will be described in detail.

Alzheimer's disease (AD) is a neurodegenerative disease that contributes to 60-70% dementia around the world. One of the hallmarks of AD undoubtedly lies ...

Rilevazione di placche neuritiche nel modello di malattia di Alzheimer mouse

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