M-V.G-S. is supported by a BrightFocus Foundation Alzheimer's Disease Research Fellowship Award (A2015309F) and an Alzheimer's Association, California Southland Chapter Young Investigator Award. T.M.W. is supported by an ARCS Foundation and John Douglas French Alzheimer's Foundation Maggie McKnight Russell-JDFAF Memorial Postdoctoral Fellowship. This work was supported by the National Institute on Neurologic Disorders and Stroke (1R01NS076794-01, to T.T.), an Alzheimer's Association Zenith Fellows Award (ZEN-10-174633, to T.T.), and an American Federation of Aging Research/Ellison Medical Foundation Julie Martin Mid-Career Award in Aging Research (M11472, to T.T.). We are grateful for startup funds from the Zilkha Neurogenetic Institute, which helped to make this work possible. &#8195;

Dichiarazione di etica della ricerca: Tutti gli esperimenti che coinvolgono animali descritti nel presente documento sono stati approvati dalla University of Southern California Istituzionale cura degli animali e del Comitato uso (IACUC) ed eseguito in stretta conformità con National Institutes of linee guida per la salute e le raccomandazioni della Associazione per la valutazione e l&#39;accreditamento del laboratorio Animal Care International. 1. roditori Cervello Isolamento e preparazione per Immunocolorazione GIORNO 1: <ol><li> Posizionare ratti TgF344-AD di età (14 mesi) o APP / PS1 topi (12 mesi) in continuo anestesia isoflurano profonda (4%). Valutare la profondità di anestesia pinch punta e l&#39;assenza di recesso reflex. </li><li> Tagliare i due lati della gabbia toracica e sollevare per esporre il cuore. Inserire un ago G 23 nel ventricolo sinistro del cuore e fare una piccola incisione nell&#39;atrio destro. Procedere alla dissanguamento da perfusione transcardial con PBS ghiacciata(PBS) utilizzando una pompa peristaltica (30 mL per topi; 150 - 200 ml per i ratti). </li><li> Effettuare una incisione mediana caudale nella pelle e spostare la pelle ei muscoli da parte. Tagliare attraverso la parte superiore del cranio lungo la linea mediana e tra gli occhi. Rimuovere le piastre ossee e isolare l&#39;intero cervello dal cranio. </li><li> Mettere il cervello in una matrice roditore cervello coronale e si taglia in quarti. Incubare quarti posteriori O / N (16 h) in paraformaldeide fissativo (4% PFA in PBS) a 4 ° C. Lavare 3x in PBS, e poi il trasferimento al 70% di etanolo. Attenzione: PFA è tossico e deve essere manipolato sotto una cappa chimica con opportuni dispositivi di protezione individuale. </li></ol> GIORNO 2: <ol start="5"><li> Posizionare quarti cerebrali in cassette incorporamento e progressivamente disidratano tessuto in bagni di etanolo 1 h in successione più concentrate (70%, 80%, 95% e 100% x3). </li><li> Cancella l&#39;etanolo dal tessuto con tre bagni successivi xilene 100% (1 ora ciascuno). Attenzione: xilene è tossico e should essere trattati sotto una cappa chimica con opportuni dispositivi di protezione individuale. </li><li> Incorpora tessuti in blocchi di paraffina dopo due bagni fusi paraffina (56-58 ° C, 90 min ciascuna). </li></ol> 3 ° GIORNO: <ol start="8"><li> Tagliare 10 sezioni micron di spessore di cervelli in paraffina utilizzando un microtomo. sezioni Dip in un bagno d&#39;acqua (50 ° C per 1 min) e si applicano a vetrini da microscopio. Lasciare le diapositive asciugare O / N, garantendo l&#39;adesione dei tessuti alla diapositiva. </li></ol> 2. immunocolorazione Nota: differenti combinazioni di anticorpi possono essere utilizzati per la procedura di colorazione descritta di seguito. Cocktail di anticorpi compatibili con il tessuto cerebrale di ratti e topi sono elencati nella tabella 1. 4 ° GIORNO: <ol><li> Deparaffinare sezioni del cervello che utilizzano 2x 100% bagni xilene (12 min ciascuna). </li><li> Reidratare sezioni di cervello in bagni etanolo successive - 100% per 10 min, 95% per 5 min, 80% per 10 min, ed infine 70% per 15 min - followed da lavaggi PBS 3x [5 minuti a temperatura ambiente, con l&#39;agitazione luce]. Nel frattempo, il calore soluzione antigene di recupero di 95-97 ° C su una piastra calda con magnetica barra di agitazione. </li><li> Incubare sezioni di cervello in soluzione recupero dell&#39;antigene a 95 - 97 ° C per 30 min. Poi, lavare 3x in PBS (5 minuti a temperatura ambiente, con l&#39;agitazione della luce). </li><li> Rapidamente diapositive a secco che utilizzano delicati tergicristalli attività per evitare l&#39;essiccamento dei tessuti, e disegnare una barriera idrofobica intorno alla zona di tessuto con una penna barriera idrofoba. Riempire la regione tessuto circondato con tampone di bloccaggio [PBS contenente 0,3% Triton X-100 e il 10% Normal Donkey Serum (NDS)], ed incubare a temperatura ambiente per 1 h in una camera umidificata. </li><li> Sostituire tampone di bloccaggio con Iba1 anticorpo primario (diluito in tampone bloccante) per etichettare fagociti mononucleari, e incubare O / N a 4 ° C in una camera umidificata. Per gli host di anticorpi e diluizioni di lavoro, vedi Tabella 1 e la Tabella dei Materiali. </li></ol> GIORNO 5: <ol start="6"><li> Sciacquareanticorpo primario con i bagni 3x PBS (5 minuti a temperatura ambiente con agitazione luce). Incubare con immunofluorescenza secondario (coniugato con un fluorocromo di emissione 594 nm) per 1 h (in tampone di bloccaggio a RT al buio) seguita da bagni 3x PBS (5 min a RT agitazione luce). In questo momento, mantenere sezioni al buio per evitare fluorescente sbiancamento segnale. </li></ol> Giorno 6 - 7: <ol start="7"><li> Ripetere i punti 2.5 e 2.6 con CD68 (cervello dei topi) o LAMP1 (tessuto mouse) anticorpi e appropriati anticorpi secondari (accoppiato con un fluoroforo emissione di 488 nm) per etichettare phagolysosomes. </li></ol> Giorno 7 - 8: <ol start="8"><li> Ripetere i punti 2.5 e 2.6 con OC (tessuto di ratto) o 4G8 (cervello di topo) anticorpi e appropriati anticorpi secondari (accoppiato ad un fluoroforo 647 nm) per etichettare depositi Ap. NOTA: In alternativa, 6E10 anticorpo può essere utilizzato con successo sia sul topo e ratto tessuti. Per opportune combinazioni di anticorpi, vedere la tabella dei materiali </strong&gt;. </li><li> Lasciare sezioni completamente asciutta O / N a RT al buio. Poi, la copertura campioni con un vetrino sigillato da mezzi di montaggio fluorescenza contenenti DAPI. </li></ol> 3. Acquisizione di grande Z-stack confocale Dataset Nota: Questo protocollo richiede una scansione laser confocale completamente automatizzato dotato di un obiettivo 60X e 405 nm, 488 nm, 594 nm e 647 nm laser. Tutto l&#39;equipaggiamento è controllato da imaging e controllo laser software per computer. Prima di iniziare la procedura di imaging, accendere il computer, lampada epifluorescente, microscopio, laser e macchina fotografica. 9 ° giorno: <ol><li> Selezionare l&#39;obiettivo microscopio 60X. Aggiungere l&#39;olio di immersione alla lente, e posizionare il campione sul supporto del microscopio fase di diapositive. Sollevare l&#39;obiettivo finché l&#39;olio entra in contatto con la slitta. Regolare il piano focale per individuare placche amiloidi nell&#39;ippocampo o corteccia cerebrale che utilizzano illuminazione epifluorescente attraverso gli oculari. </li><li> Acquisire im confocaleetà compresa tra i fagociti mononucleari attivate circostanti depositi di amiloide nell&#39;ippocampo o corteccia di roditori mediante microscopia confocale (ingrandimento 60X, passi z-stack: 0.25 micron &lt;z &lt;0.40 micron, il numero di passi 25 &lt;n &lt;35). </li></ol> 4. q3DISM Nota: Al fine di produrre risultati significativi, si consiglia l&#39;analisi di un minimo di 3 immagini per animale / regione di interesse. Per ogni immagine, l&#39;abbondanza di cellule da analizzare può variare a seconda paradigmi sperimentali. Nei risultati rappresentativi riportati nella presente relazione, abbiamo analizzato 3 cellule / condizione (per esempio, fagociti mononucleari distanti da o associati a placche, vedi figure 1C - D e 2C - D). GIORNO 10: <ol><li> Analizzare i set di dati confocale con colocalization elaborazione delle immagini 3D e analisi scientifiche software (coloc) modulo per la vicinanza spaziale di Iba1 / CD68 (tessuto di ratto) o Iba1 / LAMP1 (tessuto mouse) colorazione in tutte le z-aerei simultaneamente.Creare Iba + / CD68 + o canali Iba + / + LAMPADA1 colocalizzazione che corrispondono a phagolysosomes all&#39;interno di fagociti mononucleari attivate. <ol><li> Selezionare TRITC per il canale A (corrispondente al Iba1 colorazione accoppiato con 594 nm fluoroforo) e FITC per il canale B (corrispondente al CD68 o colorazione LAMPADA1 accoppiato con 488 nm fluoroforo). Sul lato destro della finestra del software per il &#39;controllo modalità,&#39; selezionare &#39;soglia&#39; e per &#39;intensità coloc,&#39; Select &#39;canali sorgente. </li><li> Fare clic su &#39;Modifica&#39; per selezionare &#39;colore coloc&#39; sul lato destro della finestra del software. </li><li> Per ogni canale in modo indipendente, regolare le soglie per includere colorazione specifica ed escludere sfondo / segnali non specifici. Una volta regolato, non modificare le soglie tra le immagini al fine di garantire un&#39;analisi imparziale. I voxel colocalized (pixel da tutti gli z-stack) apparirà nel colore selezionato al punto 4.1.2 in tutte le z-stack SIMultaneously. </li><li> Fare clic su &#39;costruire canale Coloc&#39;. Il canale co-localizzazione creato apparirà nella finestra di regolazione del display. </li><li> Fare clic sul canale coloc per aprire le statistiche del canale. Il &#39;% del volume / materiale A di sopra della soglia colocalized&#39; rappresenta la% di segnale Iba1 (voxel corrispondente al fluoroforo 594 nm) colocalizzava con LAMPADA1 o il segnale CD68 (voxel corrispondente al fluoroforo 488 nm). Più semplicemente, questo è il volume occupato da monociti phagolysosomes (vedi figure 1C e 2C). NOTA: &#39;% del volume / materiale B di sopra della soglia colocalized&#39; corrisponde ad% di LAMPADA1 o CD68 segnale colocalized con Iba1. Questo dovrebbe essere vicino al 100%, come phagolysosomes sono strutture intracellulari. I valori sono basati sul rapporto tra segnale da tutte z-stack sopra soglia colocalized. </li></ol></li><li> Utilizzando il modulo coloc, analizzare il canale coloc creato nel passo 4.1 per la vicinanza spaziale con OC (ti rattoSSUE) o 4G8 (tessuto mouse) segnali Ap. Ciò consente la quantificazione di Ap incapsulati all&#39;interno phagolysosomes. <ol><li> Selezionare il canale A dataset coloc (corrispondente al Iba1 / CD68 o canale Iba1 / LAMP1 coloc creato nel passo 4.1) e Cy5 per il canale B (corrispondente a OC o 4G8 colorazione accoppiato con 647 nm fluoroforo). </li><li> Costruire un canale coloc come descritto ai punti 4.1.2 a 4.1.5. </li><li> Fare clic sul canale coloc per aprire le statistiche del canale. Il &#39;% del volume / materiale A di sopra della soglia colocalized&#39; rappresenta la% di Iba1 / LAMPADA1 o Iba1 / CD68 (voxel corrispondente al canale coloc costruito nel punto 4.1.5.) Colocalized con OC o il segnale 4G8 (voxel corrispondente al 647 nm fluoroforo). Questo è il volume occupato da fagolisosomale Ap (vedi figure 1D e 2D). NOTA: &#39;% del volume / materiale B di sopra della soglia colocalized&#39; corrisponde% del segnale Ap totale colocalized con phagolysosomes.Questo può essere utilizzato per valutare la frazione dei depositi totali Ap incapsulati all&#39;interno phagolysosomes (non mostrato nelle presenti risultati rappresentativi). </li></ol></li><li> Utilizzare il modulo superare per ricostruire le pile di immagini confocale e generare modelli 3D di A? Incapsulati all&#39;interno phagolysosomes monociti. <ol><li> Nella finestra &#39;di regolazione del display&#39;, selezionare TRITC (per mostrare colorazione solo Iba1). Nella finestra &#39;Proprietà volume&#39;, clicca su &#39;aggiungi nuova superficie&#39;. </li><li> Nel passo &#39;1/5 algoritmo&#39;, in &#39;Impostazioni&#39;, selezionare &#39;superficiale.&#39; Inoltre, in &#39;colore&#39;, selezionare il tipo di colore nella tavolozza o RGB e regolare la trasparenza (rosso è fissato al 60% di trasparenza nelle figure 1B e 2B). Casella &#39;di selezionare la regione di interesse&#39;. Fare clic su Avanti. </li><li> Nel passaggio &#39;2/5 regione di interesse,&#39; disegnare una finestra attorno alla cella di interesse regolando x, y, z coordinate (vedi caselle bianche in figure 1A </strong&gt; e 2A). Fare clic su Avanti. </li><li> Nel passaggio &#39;3/5 canale sorgente&#39;, selezionare il canale di origine TRITC. Selezionare la casella &#39;liscia&#39;, e impostare il livello di dettaglio superficie di 0,4 micron. Per &#39;soglia&#39;, selezionare l&#39;intensità assoluta. Fare clic su Avanti. </li><li> Nel passaggio &#39;4/5 soglia,&#39; regolare soglia in modo che il volume creato sovrappone perfettamente con il segnale del canale TRITC. Fare clic su Avanti. </li><li> Nel passaggio &#39;5/5 classificare superfici,&#39; nella sezione &#39;filtro&#39;, selezionare gli oggetti in base alla loro dimensione per includere o escludere dai volumi da creare. Fare clic su Fine, eseguire tutte le fasi di creazione, e uscire dalla procedura guidata. </li><li> Ripetere i punti da 4.3.1 a 4.3.6 per creare una superficie 3D per il canale FITC (LAMPADA1 + o CD68 + phagolysosomes). </li><li> Ripetere i punti da 4.3.1 a 4.3.6 per creare una superficie 3D per il canale Cy5 (OC + o depositi 4G8 + Ap). </li></ol></li></ol>

<ol>
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Chem. 285 (47), 36945-36957 (2010).</li><li>Blurton-Jones, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+stem+cells+improve+cognition+via+BDNF+in+a+transgenic+model+of+Alzheimer+disease.">Neural stem cells improve cognition via BDNF in a transgenic model of Alzheimer disease.</a> Proc Natl Acad Sci U S A. 106 (32), 13594-13599 (2009).</li><li>Stalder, M., Deller, T., Staufenbiel, M., Jucker, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D-Reconstruction+of+microglia+and+amyloid+in+APP23+transgenic+mice:+no+evidence+of+intracellular+amyloid.">3D-Reconstruction of microglia and amyloid in APP23 transgenic mice: no evidence of intracellular amyloid.</a> Neurobiol Aging. 22 (3), 427-434 (2001).</li><li>Leinenga, G., G&#246;tz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scanning+ultrasound+removes+amyloid-&#946;+and+restores+memory+in+an+Alzheimer's+disease+mouse+model.">Scanning ultrasound removes amyloid-&#946; and restores memory in an Alzheimer's disease mouse model.</a> Sci Transl Med. 7 (278), 33 (2015).</li><li>Liarski, V. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+distance+mapping+identifies+functional+T+follicular+helper+cells+in+inflamed+human+renal+tissue.">Cell distance mapping identifies functional T follicular helper cells in inflamed human renal tissue.</a> Sci Transl Med. 6 (230), 46 (2014).</li><li>Nichols, L., Pike, V. W., Cai, L., Innis, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+and+in+vivo+quantitation+of+beta-amyloid:+an+exemplary+biomarker+for+Alzheimer's+disease.">Imaging and in vivo quantitation of beta-amyloid: an exemplary biomarker for Alzheimer's disease.</a> Biol Psychiatry. 59 (10), 940-947 (2006).</li><li>Skovronsky, D. M., Zhang, B., Kung, M. P., Kung, H. F., Trojanowski, J. Q., Lee, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+detection+of+amyloid+plaques+in+a+mouse+model+of+Alzheimer's+disease.">In vivo detection of amyloid plaques in a mouse model of Alzheimer's disease.</a> Proc Natl Acad Sci U S A. 97 (13), 7609-7614 (2000).</li><li>Lian, H., Litvinchuk, A., Chiang, A. C. -. A., Aithmitti, N., Jankowsky, J. L., Zheng, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocyte-Microglia+Cross+Talk+through+Complement+Activation+Modulates+Amyloid+Pathology+in+Mouse+Models+of+Alzheimer's+Disease.">Astrocyte-Microglia Cross Talk through Complement Activation Modulates Amyloid Pathology in Mouse Models of Alzheimer's Disease.</a> J Neurosci. 36 (2), 577-589 (2016).</li><li>Novotny, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conversion+of+Synthetic+A&#946;+to+In+Vivo+Active+Seeds+and+Amyloid+Plaque+Formation+in+a+Hippocampal+Slice+Culture+Model.">Conversion of Synthetic A&#946; to In Vivo Active Seeds and Amyloid Plaque Formation in a Hippocampal Slice Culture Model.</a> J Neurosci. 36 (18), 5084-5093 (2016).</li><li>Tartaro, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+fluorescence-based+in+vivo+phagocytosis+assay+to+measure+mononuclear+phagocyte+system+function+in+the+rat.">Development of a fluorescence-based in vivo phagocytosis assay to measure mononuclear phagocyte system function in the rat.</a> J Immunotoxicol. 12 (3), 239-246 (2015).</li></ol>

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Il protocollo che descriviamo in questa relazione per una vera quantificazione 3D di Ap fagocitosi in vivo dai fagociti mononucleari si basa sulla etichettatura specifica dei compartimenti cellulari e subcellulari così come depositi di Ap. In particolare, usiamo Iba1 (ionizzata-calcio Binding Adaptor molecola 1), una proteina che è coinvolta nella arruffarsi membrana e fagocitosi delle cellule dopo l&#39;attivazione 18, 19, a macchia fagociti mononucleari cerebrali. Mentre le ...

La malattia di Alzheimer (AD), la demenza legata all&#39;età più comune 1, è caratterizzata da accumulo cerebrale amiloide-β (Ap) come placche &quot;senili&quot; beta-amiloide, neuroinfiammazione cronica di basso livello, tauopathy, perdita neuronale, e disturbi cognitivi 2 . In AD cervelli di pazienti, neuroinflammation è destinato dagli astrociti e fagociti mononucleari reattiva (indicato come microglia, anche se le loro centrali vs. origine periferica rimane poco chiaro) che circonda depositi Ap 3. Come le sentinelle del sistema immunitario innato del sistema nervoso centrale, microglia sono posizionati centralmente per cancellare il cervello Ap. Tuttavia, il reclutamento della microglia placche Ap è accompagnata da molto piccolo, all&#39;occorrenza, Ap fagocitosi 4,5. Una ipotesi è che la microglia sono inizialmente neuroprotettivi da phagocytozing piccole assemblee di Ap. Tuttavia, alla fine queste cellule diventano neurotossico come opprimente fardello Ap e / o legate all&#39;età d funzionaleecline, provoca microglia in un fenotipo proinfiammatoria disfunzionale, contribuendo alla neurotossicità e cognitivo declino 6. Recenti studi genome-wide (GWAS Association) hanno identificato un gruppo di alleli di rischio AD appartenenti al nucleo innate percorsi immunitario 7 che modulano la fagocitosi 8-11. Di conseguenza, la risposta immunitaria alla deposizione di amiloide cerebrale è diventato un importante area di interesse, sia in termini di comprensione AD eziologia e per lo sviluppo di nuovi approcci terapeutici 12-14. Tuttavia, vi è la necessità vitale per metodologia per valutare fagocitosi Ap in vivo. Per rispondere a questa necessità insoddisfatte, abbiamo sviluppato 3D quantitativa in silico di modellazione (q3DISM) per consentire una vera quantificazione 3D cerebrale fagocitosi Ap dai fagociti mononucleari in modelli di roditori della malattia di Alzheimer-like. Limitata solo dalla misura in cui esse ricapitolare malattia, modelli animali hannodimostrato prezioso per la comprensione AD pathoetiology e per la valutazione delle terapie sperimentali. A causa del fatto che le mutazioni nei geni presenilina (PS) e della proteina precursore dell&#39;amiloide (APP) causano indipendentemente autosomica dominante dC, questi transgeni mutanti sono stati ampiamente utilizzati per generare modelli di roditori transgenici. Topi transgenici APP / PS1 coexpressing simultaneamente &quot;svedese&quot; mutante umano APP (APP SWE) e Δ esone 9 mutante presenilina umana 1 (PS1ΔE9) presente con accelerata amiloidosi cerebrale e neuroinfiammazione 15,16. Inoltre, abbiamo generato topi bi-transgenici coinjected con APP SWE e costrutti PS1ΔE9 (linea TgF344-AD, su uno sfondo di Fischer 344). A differenza dei modelli di topi transgenici di amiloidosi cerebrale, ratti TgF344-AD sviluppano amiloide cerebrale che precede tauopathy, perdita apoptotica di neuroni, e compromissione del comportamento 17. In questo rapporto, si descrive un protocollo per immunostaining microglia, phagolysosomes e depositi Ap in sezioni del cervello di topi APP / PS1 e ratti TgF344-AD, e l&#39;acquisizione di immagini di grandi dimensioni confocale z-dimensionale. Noi dettaglio nella generazione e l&#39;analisi di veri ricostruzioni 3D di set di dati confocale che consentono la quantificazione di Ap assorbimento in phagolysosomes microgliali silico. Più in generale, la metodologia che we dettaglio qui può essere utilizzato per quantificare praticamente qualsiasi forma di fagocitosi in vivo.

<table><tbody><tr><td>Isoflurane</td><td>Abbott</td><td>NDC 0044-5260-05</td><td></td></tr><tr><td>Dissecting scissors</td><td>VWR</td><td>82027-582</td><td></td></tr><tr><td>Dissecting scissors Blunt tip</td><td>VWR</td><td>82027-588</td><td></td></tr><tr><td>Tweezers</td><td>VWR</td><td>94024-408</td><td></td></tr><tr><td>23 G needle</td><td>VWR</td><td>BD305145</td><td></td></tr><tr><td>peristaltic pump FH10</td><td>Thermo Scientific</td><td>72-310-010</td><td></td></tr><tr><td>PBS 10x</td><td>Bioland Scientific</td><td>PBS01-02</td><td>Phosphate-buffered Saline; Working concentration 1x</td></tr><tr><td>Adult Mouse Brain Matrix, Coronal slices, Stainless Steel 1 mm&amp;nbsp;</td><td>Kent Scientific</td><td>RBMS-200C</td><td></td></tr><tr><td>Adult Rat Brain Matrix, Coronal slices, Stainless Steel 1 mm&amp;nbsp;</td><td>Kent Scientific</td><td>RBMS-305C&amp;nbsp;</td><td></td></tr><tr><td>32% Paraformaldehyde aqueous solution (PFA)</td><td>EMS</td><td>15714-S</td><td>Caution: Toxic. Working concentration: 4% in PBS</td></tr><tr><td>Ethanol</td><td>VWR</td><td>89125-188</td><td>Various concentrations, see protocol</td></tr><tr><td>Tissue-Tek Uni-cassettes Sakura</td><td>VWR</td><td>25608-774</td><td></td></tr><tr><td>Embedding and Infiltration Paraffin</td><td>VWR</td><td>15147-839</td><td></td></tr><tr><td>Microtome Leica RM2125</td><td>Leica Biosystems</td><td></td><td></td></tr><tr><td>Disposable Microtome Blades&amp;nbsp;</td><td>VWR</td><td>25608-964</td><td></td></tr><tr><td>Water bath Leica HI 1210</td><td>Leica Biosystems</td><td></td><td></td></tr><tr><td>Micro slide Superfrost plus</td><td>VWR</td><td>48311-703</td><td></td></tr><tr><td>Xylene</td><td>Sigma-Aldrich</td><td>534056-4X4L</td><td>Caution: Toxic&amp;nbsp;</td></tr><tr><td>Target Retrieval Solution 10x</td><td>DAKO</td><td>S1699</td><td>Working concentration 1x</td></tr><tr><td>KimWipes</td><td>VWR</td><td>21905-026</td><td></td></tr><tr><td>Hydrophobic PAP pen</td><td>VWR</td><td>95025-252</td><td></td></tr><tr><td>Triton X-100</td><td>VWR</td><td>97062-208</td><td></td></tr><tr><td>Normal Donkey Serum (NDS)</td><td>Jackson Immuno</td><td>017-000-121</td><td></td></tr><tr><td>Coverslips</td><td>VWR</td><td>48393081</td><td></td></tr><tr><td>Prolong Gold antifade reagent with DAPI</td><td>Life Technologies</td><td>P36935</td><td></td></tr><tr><td>Glass Slide Rack</td><td>VWR</td><td>100492-942</td><td></td></tr><tr><td>Iba1 antibody (polyclonal, rabbit)</td><td>Wako</td><td>019-19741&amp;nbsp;</td><td>Working concentration 1:200</td></tr><tr><td>Iba1 antibody (polyclonal, goat)</td><td>LifeSpan Bioscience</td><td>LS-B2645</td><td>Working concentration 1:200</td></tr><tr><td>rat CD68 [KP1] antibody (monoclonal, mouse)</td><td>Abcam</td><td>ab955</td><td>Working concentration 1:200</td></tr><tr><td>mouse CD68 [FA-11] antibody (monoclonal, rat)</td><td>Abcam</td><td>ab53444</td><td>Working concentration 1:200</td></tr><tr><td>mouse CD107a (LAMP1) antibody (monoclonal, rat)</td><td>Affymetrix</td><td>14-1071</td><td>Working concentration 1:100</td></tr><tr><td>Beta-Amyloid, 17 - 24 (4G8) antibody (monoclonal, mouse)</td><td>Covance</td><td>SIG-39220</td><td>Working concentration 1:200</td></tr><tr><td>Beta-Amyloid, 1 - 16 (6E10) antibody (monoclonal, mouse)</td><td>Covance</td><td>SIG-39320</td><td>Working concentration 1:200</td></tr><tr><td>OC antibody (polyclonal, rabbit)</td><td>Gifted by D. H. Cribbs and C. G. Glabe (UC Irvine)</td><td>Working concentration 1:200</td><td></td></tr><tr><td>Alexa Fluor 488 mouse secondary antibody</td><td>Invitrogen</td><td>A-11001</td><td>Working concentration 1:1,000</td></tr><tr><td>Alexa Fluor 488 rat secondary antibody</td><td>Invitrogen</td><td>A-11006</td><td>Working concentration 1:1,000</td></tr><tr><td>Alexa Fluor 594 rabbit secondary antibody</td><td>Invitrogen</td><td>A-11037</td><td>Working concentration 1:1,000</td></tr><tr><td>Alexa Fluor 594 goat secondary antibody</td><td>Invitrogen</td><td>A-11080</td><td>Working concentration 1:1,000</td></tr><tr><td>Alexa Fluor 647 mouse secondary antibody</td><td>Invitrogen</td><td>A-21235</td><td>Working concentration 1:1,000</td></tr><tr><td>Alexa Fluor 647 rabbit secondary antibody</td><td>Invitrogen</td><td>A-21443</td><td>Working concentration 1:1,000</td></tr><tr><td>Immersion oil</td><td>Nikon&amp;nbsp;</td><td></td><td></td></tr><tr><td>A1 Confocal microscope</td><td>Nikon&amp;nbsp;</td><td></td><td></td></tr><tr><td>NIS Elements Advanced Research software</td><td>Nikon&amp;nbsp;</td><td></td><td></td></tr><tr><td>Imaris:Bitplane software version 7.6</td><td>Bitplane</td><td></td><td>&quot;coloc&quot; and &quot;supass&quot; modules are used.&amp;nbsp;Alternatively, the open-source freeware&amp;nbsp;ImageJ can be used for colocalization&amp;nbsp;analysis of confocal z-stacks datasets.</td></tr></tbody></table>

quantitative 3d in silico modeling (q3dism) of cerebral amyloid-beta phagocytosis in rodent models of alzheimer's disease

Neuroinfiammazione è ormai riconosciuto come un importante fattore eziologico nella malattia neurodegenerativa. fagociti mononucleari sono cellule immunitarie innate responsabili della fagocitosi e la bonifica di detriti e detriti. Queste cellule sono macrofagi CNS residenti note come microglia, e fagociti mononucleari infiltrati dalla periferia. La microscopia ottica è stata generalmente utilizzati per la visualizzazione fagocitosi in roditori o campioni di cervello umano. Tuttavia, i metodi qualitativi non hanno fornito la prova definitiva di fagocitosi in vivo. Qui, descriviamo 3D quantitativa nella modellazione in silico (q3DISM), un metodo affidabile che permette una reale quantificazione 3D di amiloide-β (Ap) fagocitosi da parte dei fagociti mononucleari in modelli di roditori malattia di Alzheimer (AD). Il metodo consiste nel visualizzare fluorescente Ap incapsulato all&#39;interno phagolysosomes nelle sezioni roditore cervello. Grandi serie di dati confocale z-dimensionale vengono poi ricostruite in 3D per la quantificazione di A &amp;# 946; spazialmente colocalized all&#39;interno del fagolisosoma. Dimostriamo l&#39;applicazione di successo di q3DISM di topo e ratto cervello, ma questa metodologia può essere esteso a qualsiasi evento di fagocitosi in qualsiasi tessuto.

Utilizzando la metodologia più stadi per q3DISM dettagliato sopra, siamo in grado di quantificare l&#39;assorbimento Ap in phagolysosomes monociti nel cervello di APP / PS1 topi (figura 1) e ratti TgF344-AD (Figura 2). Pertanto, la metodologia q3DISM ha permesso l&#39;analisi dei fagociti mononucleari in modelli di topo e ratto di AD. È interessante notare che il volume occupato da CD68 + phagolysosomes è significativamente aumentata in fag...

Watch this Scientific Journal Video about Quantitative 3D In Silico Modeling q3DISM of Cerebral Amyloid-beta Phagocytosis in Rodent Models of Alzheimer's Disease at JoVE.com

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

Abbiamo sviluppato una metodologia per il 3D quantitativa in silico di modellazione (q3DISM) di cerebrale amiloide-β (Ap) fagocitosi da parte dei fagociti mononucleari in modelli di roditori della malattia di Alzheimer. Questo metodo può essere generalizzato per la quantificazione di praticamente qualsiasi evento fagocitaria 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 ...

quantitative-3d-silico-modeling-q3dism-cerebral-amyloid-beta

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

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

7.9K Views.  University of Southern California (USC).  Abbiamo sviluppato una metodologia per il 3D quantitativa in silico di modellazione (q3DISM) di cerebrale amiloide-β (Ap) fagocitosi da parte dei fagociti mononucleari in modelli di roditori della malattia di Alzheimer. Questo metodo può essere generalizzato per la quantificazione di praticamente qualsiasi evento fagocitaria in vivo. 

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

Detection of Neuritic Plaques in Alzheimer's Disease Mouse Model

Alzheimer's disease (AD) is the most common neurodegenerative disorder leading to dementia. Neuritic plaque formation is one of the pathological hallmarks of Alzheimer's disease. The central component of neuritic plaques is a small filamentous protein called amyloid &beta; protein (A&beta;)1, which is derived from sequential proteolytic cleavage of the beta-amyloid precursor protein (APP) by &beta;-secretase and &gamma;-secretase. The amyloid hypothesis entails that A&gamma;-containing plaques as the underlying toxic mechanism in AD pathology2. The postmortem analysis of the presence of neuritic plaque confirms the diagnosis of AD. To further our understanding of A&gamma; neurobiology in AD pathogenesis, various mouse strains expressing AD-related mutations in the human APP genes were generated. Depending on the severity of the disease, these mice will develop neuritic plaques at different ages. These mice serve as invaluable tools for studying the pathogenesis and drug development that could affect the APP processing pathway and neuritic plaque formation. In this protocol, we employ an immunohistochemical method for specific detection of neuritic plaques in AD model mice. We will specifically discuss the preparation from extracting the half brain, paraformaldehyde fixation, cryosectioning, and two methods to detect neurotic plaques in AD transgenic mice: immunohistochemical detection using the ABC and DAB method and fluorescent detection using thiofalvin S staining method.

One of the pathological characteristics of AD is the formation of Amyloid &#x03B2; protein positive neuritic plaques. In this protocol we describe two methods to detect neuritic plaques in transgenic AD model mice: immunohistochemical detection using the ABC and DAB method and fluorescent detection using thioflavin S staining method.

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 ...

Ricerca

Neuroscienze

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 ...

Comportamento

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 ...

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, ...

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 ...

Medicina

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 ...

Full- versus Sub-Regional Quantification of Amyloid-Beta Load on Mouse Brain Sections

Extracellular accumulation of amyloid-beta (A&#946;) plaques is one of the major pathological hallmarks of Alzheimer's disease (AD), and is the target of the only FDA-approved disease-modifying treatment for AD. Accordingly, the use of transgenic mouse models that overexpress the amyloid precursor protein and thereby accumulate cerebral A&#946; plaques are widely used to model human AD in mice. Therefore, immunoassays, including enzyme-linked immunosorbent assay (ELISA) and immunostaining, commonly measure the A&#946; load in brain tissues derived from AD transgenic mice. Though the methods for A&#946; detection and quantification have been well established and documented, the impact of the size of the region of interest selected in the brain tissue on A&#946; load measurements following immunostaining has not been reported. Therefore, the current protocol aimed to compare the A&#946; load measurements across the full- and sub-regions of interest using an image analysis software. The steps involved in brain tissue preparation, free-floating brain section immunostaining, imaging, and quantification of A&#946; load in full- versus sub-regions of interest are described using brain sections derived from 13-month-old APP/PS1 double transgenic male mice. The current protocol and the results provide valuable information about the impact of the size of the region of interest on A&#946;-positive area quantification, and show a strong correlation between the A&#946;-positive area obtained using the full- and sub-regions of interest analyses for brain sections derived from 13-month-old male APP/PS1 mice that show widespread A&#946; deposition.

The present protocol describes and compares the procedure to perform a full-region or sub-region of interest analysis of sagittal mouse brain sections to quantify amyloid-beta load in the APP/PS1 transgenic mouse model of Alzheimer's disease.

Quantificazione completa rispetto a quella subregionale del carico di amiloide-beta sulle sezioni cerebrali del topo

Extracellular accumulation of amyloid-beta (A&#946;) plaques is one of the major pathological hallmarks of Alzheimer's disease (AD), and is the target of ...

Fabrication of Amyloid-&#946;-Secreting Alginate Microbeads for Use in Modelling Alzheimer's Disease

According to the amyloid cascade hypothesis, the earliest trigger in the development of Alzheimer&#8217;s disease (AD) is the accumulation of toxic amyloid-&#946; (A&#946;) fragments, eventually leading to the classical features of the disease: amyloid plaques, neurofibrillary tangles and synaptic and neuronal loss. The lack of relevant non-transgenic preclinical models reflective of disease progression is one of the main factors hindering the discovery of effective drug treatments. To this end, we have developed a protocol for the fabrication of alginate microbeads containing amyloid-secreting cells useful for the study of the effects of chronic A&#946; production.
Chinese hamster ovary cells previously transfected with a human APP gene, secreting A&#946; (i.e., 7PA2 cells), were used in this study. A three-dimensional (3D) in vitro model for the sustained release of A&#946; was fabricated by encapsulation of 7PA2 cells in alginate. The process was optimized to target a bead diameter of 500-600 &#956;m for further in vivo studies. Optimization of 7PA2 cell encapsulation in alginate was performed altering fabrication parameters, e.g., alginate concentration, gel flow rate, electrostatic potential, head vibration frequency, gelling solution. Levels of secreted A&#946; were analyzed over time and compared between alginate beads and standard cell culture methods (up to 96 h).
A concentration of 1.5 x 106 7PA2 cells/mL and an alginate concentration of 2% (w/v) buffered with HEPES and subsequent gelation in 0.5 M calcium chloride for 5 min were found to fabricate the most stable microbeads. Fabricated microbeads were 1) of uniform size, 2) with an average diameter of 550 &#956;m, 3) containing about 100-150 cells per microbead and 4) able to secrete A&#946;.
In conclusion, our optimized method for the production of stable alginate microbeads containing amyloid-producing 7PA2 cells might enable the modeling of important aspects of AD both in vitro and in vivo.

This protocol illustrates a cell encapsulation method by rapid physical gelation of alginate to immobilize cells. Obtained microbeads allow controlled and sustained secretion of amyloid-&#946; over time and can be used to study the effects of secreted amyloid-&#946; in in vitro and in vivo models.

Fabbricazione di microsfere algerine amiloidi -z-Secreting per l'uso nella modellazione del morbo di Alzheimer

According to the amyloid cascade hypothesis, the earliest trigger in the development of Alzheimer&#8217;s disease (AD) is the accumulation of toxic ...

Biologia

Establishing an Alzheimer's Disease Rat Model Through an Amyloid-Beta Peptide Injection

Source: Xiaoguang, W., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/56157">Establishment of a Valuable Mimic of Alzheimer&#39;s Disease in Rat Animal Model by Intracerebroventricular Injection of Composited Amyloid Beta Protein.</a> J. Vis. Exp. (2018).
This video demonstrates the procedure for injecting amyloid-beta peptides, aluminum chloride, and a neuroinflammatory growth factor, which collectively aids in toxic amyloid plaque formation in the hippocampus region. This causes memory impairment in the rat, characteristic of Alzheimer&#39;s disease.

Stabilire un modello di ratto con malattia di Alzheimer attraverso un'iniezione di peptide beta-amiloide

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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JoVE Encyclopedia of Experiments

Neuroscience

Neuropathology

Neurodegenerative Diseases

Direct Stereotaxic Brain Infusion of Amyloid-Beta Proteins for an Alzheimer's Disease Model

Source: Jean, Y. Y. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/52805">Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus.</a> J. Vis. Exp. (2015)
This video demonstrates the precise stereotaxic infusion of amyloid beta into the dentate gyrus to establish a mouse model of Alzheimer&#39;s disease. Accurate coordinate determination and controlled delivery of amyloid beta proteins result in neuronal degradation, disrupting memory processing and contributing to Alzheimer&#39;s disease development.

3D Quantitative

Riepilogo

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