Diese Arbeit wurde von den Canadian Institutes of Health Research (CIHR), die Townsend Familie und Jack Brown und Familie Alzheimer Research Foundation (WAS) unterstützt. WAS ist der Inhaber des Canada Research Chair in der Alzheimer-Krankheit. PTTL wird vom kanadischen Natural Sciences and Engineering Research Counsel und Michael Smith Foundation for Health Research Scholarship unterstützt.

1. Fixation der Maus Gehirne und Kryoschneiden <ol><li> Transgene Mäuse werden getötet mit Kohlendioxid-Gas, durch Enthauptung und das Gehirn entnommen gefolgt. </li><li> Das Gehirn ist in Längsrichtung in der Mitte durchtrennt. Die Hälfte des Gehirns zu blinken auf Trockeneis für die biochemische Analyse eingefroren werden. Der andere wird in 4% Paraformaldehyd (PFA) für mindestens 48 Stunden bei 4 ° C getaucht </li><li> Nach der Fixierung in 4% PFA für mindestens 48 Stunden wird die hemi Gehirn direkt in 30% Saccharose-Lösung überführt und bei 4 ° C. An dem Punkt, sollte die hemi Gehirn in der Saccharose-Lösung zu schweben. Wenn die hemi Gehirn auf den Grund sank, in der Regel erfordert etwa 48 Stunden ist es bereit, in OCT für Kryoschneiden eingebettet werden. </li><li> Vor Kryoschneiden, montieren Sie eine dünne Schicht der OCT auf den Knopf und lassen Sie sie bei -20 ° C einfrieren Entfernen Sie das Kleinhirn aus dem Rest der hemi Gehirn und Ort auf die Schicht aus gefrorenem Oktober Unmittelbar decken die hemi Gehirn mit OCT und lassen Sie sie langsam bei -20 ° C einfrieren Sobald der OCT Einbettung des Gehirns hat sich deckend, ist es bereit zu sein geschnitten. </li><li> Stellen Sie die Stärke eines jeden Abschnitts bis 30 um. Sammeln Sie jede Scheibe in einen Behälter mit D&#39;Olomos Lösung. </li></ol> 2. Immunhistochemie Verfahren für neuritischen Plaques (frei schwebenden Abschnitte) <ol><li> Behandle jeden Abschnitt in 88% iger Ameisensäure für 15 Minuten. Nach Ameisensäure Behandlung wird der Abschnitt vorübergehend auszuschalten undurchsichtig und erscheinen zu schrumpfen. </li><li> Nehmen Sie jedes Teil in einen Teller geben und gründlich mit Tx-PBS (0,3% Triton X-100 in PBS) für 5 min X 3 unter leichtem Schütteln. </li><li> Block Endogene Peroxid blockieren 0,5% H 2 O 2 in Tx-PBS, RT 30 min unter leichtem Schütteln. </li><li> Wash Abschnitt mit Tx-PBS für 10 min X 3 unter leichtem Schütteln. </li><li> Block Abschnitt mit 5% fettfreier Magermilch in Tx-PBS bei Raumtemperatur gelöst für 1 Stunde unter leichtem Schütteln. </li><li> Inkubieren Abschnitte in primärer Antikörper (biotinylierter 4G8, 1:500-1:2000, Signet) in Tx-PBS mit 5% Milch bei 4 ° C über Nacht verdünnt unter leichtem Schütteln. </li><li> Wash Abschnitt mit Tx-PBS für 10 min X 3 unter leichtem Schütteln. </li><li> Bereiten Sie ABC-Lösung folgende Protokoll des Herstellers (Elite ABC, Vector Laboratories,). Inkubieren Abschnitte in ABC-Gemisch für 30 min unter leichtem Schütteln. </li><li> Wash Abschnitt mit Tx-PBS für 10 min X 3 unter leichtem Schütteln. </li><li> Bereiten 3,3 &#39;-Diaminobenzidintetrahydrochlorid (DAB) (Vector Laboratories) nach Protokoll des Herstellers. Inkubieren Abschnitte in DAB-Gemisch für 5-10 min. An dieser Stelle werden Sie feststellen, eine bräunliche Farbe auf jedem Abschnitt zu entwickeln. </li><li> Wash Abschnitt mit Tx-PBS für 10 min X 3 unter leichtem Schütteln. </li><li> Free-Floating-Hirn-Abschnitte werden auf Gelatine beschichtete Objektträger aufgebracht, mit Gewebeadhäsion helfen. Die Schnitte werden dann an der Luft, indem Sie mit einer Reihe von Xylol getrocknet gefolgt und montiert in Entellen. </li><li> Plaques sind visualisiert unter Lichtmikroskopie bei 40-facher Vergrößerung und werden von der Beurteilung der mittleren Plaque-Zahl pro Stück für jede Maus quantifiziert. </li></ol> 3. Thioflavin S Färbung zum Nachweis von neuritischen Plaques <ol><li> Maus zu opfern und das Gehirn Extraktionsverfahren sind in Abschnitte von 1,1 bis 1,5 angegeben. </li><li> Montieren Sie 4 bis 5 hemi Hirnschnitten auf einen Objektträger aus Glas und lassen Sie sie vollständig trocknen vor dem Färben. </li><li> Waschen Sie die Objektträger mit 70% Ethanol für 1 Minute. </li><li> Waschen Sie die Objektträger mit 80% Ethanol für 1 Minute. </li><li> Inkubieren in filtriert (0,2 &amp;mgr; m-Filter) Thioflavin S-Lösung (1% in 80% Ethanol) für 15 Minuten. (Thioflavin S-Lösung muss vor jedem Gebrauch gefiltert werden). Hinweis: Schutz Thioflavin S von Licht und schützen gefärbten Folien aus Licht. </li><li> Waschen Sie die Objektträger mit 80% Ethanol für 1 Minute. </li><li> Waschen Sie die Objektträger mit 70% Ethanol 1 Minute. </li><li> Waschen mit destilliertem Wasser zweimal. </li><li> Berg rutscht in wässrigen Medien und Montage ermöglichen Dias im Dunkeln für mindestens 2 Stunden, durch Abdichten Deckglas mit klarem Nagellack gefolgt trocken. Stained Abschnitte sollten im Dunkeln bei 4 ° C gelagert werden </li><li> Die grüne Fluoreszenz gefärbten Plaques konnte visualisiert werden mit fluoreszierenden microscropy. Analysieren gleitet innerhalb einer Woche, weil die Färbung mit der Zeit verblassen wird. </li></ol> 4. Repräsentative Ergebnisse In unserer jüngsten Studie über die Wirksamkeit des anti-epileptischen Medikamenten und Stimmungsstabilisator Valproinsäure (VPA), um neuritischen Plaquebildung hemmen, verwendeten wir die oben beschriebenen Immunfärbung und Thioflavin S Färbeverfahren zu neuritischen Plaques in AD-Modell-Mäusen (3) zu identifizieren. Abbildung 1 stellt eine typische 9 Monate alten APP23 transgene Maus, mit Anti-Aß mit biotinylierten 4G8 angefärbt und mittels der ABC-Methode. Neuritischen Plaques sind deutlich mit dem Antikörper markiert und werden durch die weißen Pfeile angedeutet. Mit Thioflavin S-Färbung, entdeckten wir neuritischen Plaqueablagerung 2 Monate alt APP23xPS transgenic Mäusen (Abbildung 2). Thioflavin S gebunden Aß-haltigen neuritischen Plaques erscheint unter Fluoreszenz-Mikroskopie (angezeigt durch weiße Pfeile) grün. Wir zeigten, dass mit VPA behandelt, neuritischen Plaquebildung deutlich reduziert wird (3). In einer anderen Studie untersuchten wir die molekulare Verbindung zugrunde liegenden Hypoxie und AD Pathogenese. Wieder mit beiden 4G8 anti-Aß Immunhistochemie-Färbung und Thioflavin S-Färbung, fanden wir, dass Hypoxie erleichtert die Bildung von Aß mit neuritischen Plaques (4). <img src="/files/ftp_upload/2831/2831fig1.jpg" alt="Abbildung 1"/> Abbildung 1. Immunhistochemische Analyse der neuritischen Plaquebildung in AD transgenen Mäusen. APP23 Mäuse im Alter von 9 Monaten wurden getötet und seziert, fixiert und geschnitten. Neuritischen Plaques im Hippocampus nachgewiesen wurden mit einem Anti-Aß-Antikörper 4G8 und wurden mit Hilfe der ABC-und DAB-Verfahren. Die Plaques wurden unter dem Lichtmikroskop sichtbar gemacht mit 40X. Weiße Pfeile zeigen auf Aß neuritischen Plaques. <img src="/files/ftp_upload/2831/2831fig2.jpg" alt="Abbildung 2"/> Abbildung 2. Thioflavin S-Färbung von neuritischen Plaques in AD modelliert Mäusen. APP23xPS45 doppelt transgenen Mäusen 8 Wochen alt, wurden getötet und präpariert, fixiert und geschnitten. Neuritischen Plagen in den Hippocampus sind bestätigt wurden mit Thioflavin S Fluoreszenzfärbung und visualisiert durch Mikroskopie bei 40-facher Vergrößerung. Weiße Pfeile zeigen Thioflavin S gefärbt neuritischen Plaques.

<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>

Alle Experimente wurden in Übereinstimmung mit der University of British Columbia Animal Care und Verwenden Ausschuss und CIHR Richtlinien durchgeführt.

Immunhistochemie mit dem biotinylierten gekennzeichnet 4G8 Antikörper Flecken neuritischen Plaques in AD modelliert Mäuse mit Spezifität. Die Färbung Ergebnis ermöglicht die Quantifizierung und Vergleich der Plaque-Last zwischen den verschiedenen Behandlungsgruppen. Es gibt mehrere wichtige Schritte, die das Ergebnis beeinflussen könnten. Da die hemi Gehirne nicht vor der Extraktion aus dem Schädel perfundiert, sollte darauf bei der Extraktion verwendet werden, um Schäden an der hemi Gehirn zu verhindern. Da darüber hinaus die hemi Gehirn passiv durchströmt wird, empfehlen wir Inkubation in 4% PFA für mindestens 48 Stunden bei 4 ° C vor dem in der 30% Saccharose-Lösung eingetaucht. Alternativ könnte transcardial Perfusion vor der Extraktion des Gehirns durchgeführt werden. Wenn das Gehirn richtig befestigt ist, sollte es eine gummiartige Konsistenz. In dem Fall, dass das Gehirn noch nicht behoben ist richtig, werden die Abschnitte leicht in die D&#39;Olomos Lösung oder während der Färbeverfahren reißen. Die 4G8 monoklonalen Antikörper reaktiv Aminosäurereste 17-24 von Aß und erkannte die Epitop in der Core-Sequenz (VFFAE). Da 4G8 aus Maus Arten abgeleitet wird, neigt sie dazu, eine höhere Hintergrundfärbung zu geben. So entschieden wir uns, um die Abschnitte zu den angegebenen Verdünnung mit Überlegungen zur Erreichung eines echten Signals bei gleichzeitiger Minimierung der Hintergrundfärbung inkubieren. Zusätzlich zur neuritischen Plaques Detektion mit dem 4G8 Antikörper Immunfärbung ist ein Thioflavin S Färbemethode auch verwendet, um Plaques zu identifizieren. Thioflavin S ist eine homogene Mischung Farbstoff, der sich aus Methylierung von Dehydrothiotoluidin mit Sulfonsäuregruppen. Thioflavin S nicht selektiv bindet Beta-Faltblatt-Inhalte von Proteinen, wie sie in Amyloid-Oligomere. Bei der Bindung erfährt Thioflavin eine charakteristische blaue Verschiebung seines Emissionsspektrums. Umgekehrt Thioflavin S Bindung an die monomeren Formen nicht zu entlocken eine Blauverschiebung und konnte nicht mit Fluoreszenz-Mikroskop detektiert werden. Thioflavin S-Färbung ist eine schnelle Alternative zum Bildschirm für die Amyloid als die Intensität der Fluoreszenz ermöglicht eine gute Darstellung von kleinen Mengen von Amyloid-Ablagerungen. Allerdings ist ein Nachteil zu Thioflavin S-Färbung der mangelnden Spezifität. Viele andere Gewebe Komponenten, einschließlich mit umfangreichen Beta-Faltblätter, wie fibrinoids, hyaline, Keratin, etc, haben eine eher Affinität zu diesem Farbstoff.

<table border="1"><tbody><tr><th> Name des Reagenzes und Ausrüstung </th><th> Firma </th><th> Katalog-Nummer </th></tr><tr><td> Paraformaldehyd </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> Sucrose </td><td> Fisher Scientific </td><td> S5-3 </td></tr><tr><td> Ethylenglykol </td><td> Sigma-Aldrich </td><td> 324558-2L </td></tr><tr><td> Tissue-Tek OCT Compound </td><td> Sakura </td><td> 4583 </td></tr><tr><td> 88% iger Ameisensäure </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> Biotin markiert 4G8 Antikörper </td><td> Cedarlane </td><td> SIG-39240-500 </td></tr><tr><td> Elite ABC Kit </td><td> Vektor </td><td> PK-6100 </td></tr><tr><td> Imm PACT DAB </td><td> Vektor </td><td> SK-4105 </td></tr><tr><td> Xylol </td><td> Fisher Scientific </td><td> X4-4 </td></tr><tr><td> Entellan </td><td> EM Wissenschaft </td><td> 65037-71 </td></tr><tr><td> Superfrost * Plus Mikropräparate </td><td> Fisher Scientific </td><td> 12-550-15 </td></tr><tr><td> Kryostat </td><td> Leica </td><td> CM-3050-S </td></tr><tr><td> MX35 Premier Mikrotomklinge </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

Alzheimer-Krankheit (AD) ist die häufigste neurodegenerative Erkrankung, die zu Demenz. Neuritischen Plaquebildung ist einer der pathologischen Kennzeichen der Alzheimer-Krankheit. Die zentrale Komponente des neuritischen Plaques ist ein kleiner faserigen Protein namens Amyloid β Protein (Aß) 1, die aus der sequenziellen proteolytische Spaltung des beta-Amyloid-Vorläuferprotein (APP) durch β-Sekretase und γ-Sekretase abgeleitet ist. Die Amyloid-Hypothese zur Folge, dass Aγ-haltigen Plaques als die zugrunde liegende toxische Mechanismus in AD-Pathologie 2. Die Obduktion Analyse der Präsenz von neuritischen Plaques bestätigt die Diagnose von AD. Um unser Verständnis von Aγ Neurobiologie in AD Pathogenese wurden verschiedene Maus-Stämme, die AD-bezogenen Mutationen im menschlichen APP-Gene erzeugt. Je nach Schwere der Erkrankung, werden diese Mäuse neuritischen Plaques in den verschiedenen Altersstufen zu entwickeln. Diese Mäuse dienen als unschätzbare Werkzeuge für das Studium der Pathogenese und Entwicklung von Medikamenten, die die APP-Prozessierung Weg und neuritischen Plaquebildung beeinflussen könnten. In diesem Protokoll, beschäftigen wir eine immunhistochemische Methode zum spezifischen Nachweis von neuritischen Plaques in AD-Modell-Mäusen. Wir werden insbesondere über die Vorbereitung von der Gewinnung der Hälfte des Gehirns, Paraformaldehyd Fixierung Kryoschneiden und zwei Methoden zur neurotischen Plaques in AD transgenen Mäusen zu erkennen: immunhistochemischen Nachweis über die ABC-und DAB-Methode und Fluoreszenzdetektion mit thiofalvin S-Färbung.

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&#039;s Disease Mouse Model

Einer der pathologischen Merkmale von AD ist die Bildung von Amyloid β Protein positive neuritischen Plaques. In diesem Protokoll beschreiben wir zwei Methoden, um neuritischen Plaques in transgenen AD-Modell-Mäusen zu erkennen: immunhistochemischen Nachweis über die ABC-und DAB-Methode und Fluoreszenzdetektion mit Thioflavin S-Färbung.

Nachweis von neuritischen Plaques bei Alzheimer-Maus-Modell

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

Research

JoVE Journal

Neuroscience

Detection of Neuritic Plaques in Alzheimer's Disease Mouse Model

36.4K Aufrufe.  The University of British Columbia. Einer der pathologischen Merkmale von AD ist die Bildung von Amyloid β Protein positive neuritischen Plaques. In diesem Protokoll beschreiben wir zwei Methoden, um neuritischen Plaques in transgenen AD-Modell-Mäusen zu erkennen: immunhistochemischen Nachweis über die ABC-und DAB-Methode und Fluoreszenzdetektion mit Thioflavin S-Färbung.

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

Preparation of Oligomeric &beta;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

Impairment of synaptic connections is likely to underlie the subtle amnesic changes occurring at the early stages of Alzheimer s Disease (AD). &beta;-amyloid (A&beta;), a peptide produced in high amounts in AD, is known to reduce Long-Term Potentiation (LTP), a cellular correlate of learning and memory. Indeed, LTP impairment caused by A&beta; is a useful experimental paradigm for studying synaptic dysfunctions in AD models and for screening drugs capable of mitigating or reverting such synaptic impairments. Studies have shown that A&beta; produces the LTP disruption preferentially via its oligomeric form. Here we provide a detailed protocol for impairing LTP by perfusion of oligomerized synthetic A&beta;1-42 peptide onto acute hippocampal slices. In this video, we outline a step-by-step procedure for the preparation of oligomeric A&beta;1-42. Then, we follow an individual experiment in which LTP is reduced in hippocampal slices exposed to oligomerized A&beta;1-42 compared to slices in a control experiment where no A&beta;1-42 exposure had occurred.

Preparation of Oligomeric &amp;#946;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

One feature of Alzheimer's Disease is the elevation of A&beta;1-42 peptide. Here we provide a protocol for preparing synthetic A&beta;1-42 oligomers, which impairs hippocampal Long-Term Potentiation, a cellular correlate of memory. This procedure is useful for investigating mechanisms of A&beta;-induced pathology and drug screening.

Herstellung von oligomeren β-Amyloid 1-42 Und Induktion der synaptischen Plastizität Impairment auf Schnitten des Hippocampus

Impairment of synaptic connections is likely to underlie the subtle amnesic changes occurring at the early stages of Alzheimer s Disease (AD). ...

Forschung

Neurowissenschaften

Selection of Aptamers for Amyloid &beta;-Protein, the Causative Agent of Alzheimer&#39;s Disease

Alzheimer's disease (AD) is a progressive, age-dependent, neurodegenerative disorder with an insidious course that renders its presymptomatic diagnosis difficult1. Definite AD diagnosis is achieved only postmortem, thus establishing presymptomatic, early diagnosis of AD is crucial for developing and administering effective therapies2,3.
Amyloid &beta;-protein (A&beta;) is central to AD pathogenesis. Soluble, oligomeric A&beta; assemblies are believed to affect neurotoxicity underlying synaptic dysfunction and neuron loss in AD4,5. Various forms of soluble A&beta; assemblies have been described, however, their interrelationships and relevance to AD etiology and pathogenesis are complex and not well understood6. Specific molecular recognition tools may unravel the relationships amongst A&beta; assemblies and facilitate detection and characterization of these assemblies early in the disease course before symptoms emerge. Molecular recognition commonly relies on antibodies. However, an alternative class of molecular recognition tools, aptamers, offers important advantages relative to antibodies7,8. Aptamers are oligonucleotides generated by in-vitro selection: systematic evolution of ligands by exponential enrichment (SELEX)9,10. SELEX is an iterative process that, similar to Darwinian evolution, allows selection, amplification, enrichment, and perpetuation of a property, e.g., avid, specific, ligand binding (aptamers) or catalytic activity (ribozymes and DNAzymes).
Despite emergence of aptamers as tools in modern biotechnology and medicine11, they have been underutilized in the amyloid field. Few RNA or ssDNA aptamers have been selected against various forms of prion proteins (PrP)12-16. An RNA aptamer generated against recombinant bovine PrP was shown to recognize bovine PrP-&beta;17, a soluble, oligomeric, &beta;-sheet-rich conformational variant of full-length PrP that forms amyloid fibrils18. Aptamers generated using monomeric and several forms of fibrillar &beta;2-microglobulin (&beta;2m) were found to bind fibrils of certain other amyloidogenic proteins besides &beta;2m fibrils19. Ylera et al. described RNA aptamers selected against immobilized monomeric A&beta;4020. Unexpectedly, these aptamers bound fibrillar A&beta;40. Altogether, these data raise several important questions. Why did aptamers selected against monomeric proteins recognize their polymeric forms? Could aptamers against monomeric and/or oligomeric forms of amyloidogenic proteins be obtained? To address these questions, we attempted to select aptamers for covalently-stabilized oligomeric A&beta;4021 generated using photo-induced cross-linking of unmodified proteins (PICUP)22,23. Similar to previous findings17,19,20, these aptamers reacted with fibrils of A&beta; and several other amyloidogenic proteins likely recognizing a potentially common amyloid structural aptatope21. Here, we present the SELEX methodology used in production of these aptamers21.

Selection of Aptamers for Amyloid &amp;#946;-Protein, the Causative Agent of Alzheimer&#039;s Disease

Aptamers are short ribo-/deoxyribo-oligonucleotides selected by in-vitro evolution methods based on affinity for a specific target. Aptamers are molecular recognition tools with versatile therapeutic, diagnostic, and research applications. We demonstrate methods for selection of aptamers for amyloid &beta;-protein, the causative agent of Alzheimer's disease.

Selektion von Aptameren für Amyloid β-Protein, dem Erreger der Alzheimer-Krankheit

Alzheimer's disease (AD) is a progressive, age-dependent, neurodegenerative disorder with an insidious course that renders its presymptomatic diagnosis ...

Morris Water Maze Test for Learning and Memory Deficits in Alzheimer&#39;s Disease Model Mice

The Morris Water Maze (MWM) was first established by neuroscientist Richard G. Morris in 1981 in order to test hippocampal-dependent learning, including acquisition of spatial memoryand long-term spatial memory 1. The MWM is a relatively simple procedure typically consisting of six day trials, the main advantage being the differentiation between the spatial (hidden-platform) and non-spatial (visible platform) conditions 2-4. In addition, the MWM testing environment reduces odor trail interference 5. This has led the task to be used extensively in the study of the neurobiology and neuropharmacology of spatial learning and memory. The MWM plays an important role in the validation of rodent models for neurocognitive disorders such as Alzheimer&#x2019;s Disease 6, 7. In this protocol we discussed the typical procedure of MWM for testing learning and memory and data analysis commonly used in Alzheimer&#x2019;s disease transgenic model mice.

Morris Water Maze Test for Learning and Memory Deficits in Alzheimer&#039;s Disease Model Mice

The Morris Water Maze is a behavioral task to test hippocampal-dependent learning and memory. It has been widely used in the study of neurobiology, neuropharmacology and neurocognitive disorders in rodent models.

Morris Water Maze-Test für Lern-und Gedächtnisstörungen bei Alzheimer-Modell Mäuse

The Morris Water Maze (MWM) was first established by neuroscientist Richard G. Morris in 1981 in order to test hippocampal-dependent learning, including ...

Detection of Microregional Hypoxia in Mouse Cerebral Cortex by Two-photon Imaging of Endogenous NADH Fluorescence

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. Here we present a visualized experimental and methodological protocol to directly visualize microregional tissue hypoxia and to infer perivascular oxygen gradients in the mouse cortex. It is based on the non-linear relationship between nicotinamide adenine dinucleotide (NADH) endogenous fluorescence intensity and oxygen partial pressure in the tissue, where observed tissue NADH fluorescence abruptly increases at tissue oxygen levels below 10 mmHg1. We use two-photon excitation at 740 nm which allows for concurrent excitation of intrinsic NADH tissue fluorescence and blood plasma contrasted with Texas-Red dextran. The advantages of this method over existing approaches include the following: it takes advantage of an intrinsic tissue signal and can be performed using standard two-photon in vivo imaging equipment; it permits continuous monitoring in the whole field of view with a depth resolution of ~50 &mu;m. We demonstrate that brain tissue areas furthest from cerebral blood vessels correspond to vulnerable watershed areas which are the first to become functionally hypoxic following a decline in vascular oxygen supply. This method allows one to image microregional cortical oxygenation and is therefore useful for examining the role of inadequate or restricted tissue oxygen supply in neurovascular diseases and stroke.

Here we describe a method to directly visualize microregional tissue hypoxia in the mouse cortex in vivo. It is based on concurrent two-photon imaging of nicotinamide adenine dinucleotide (NADH) and the cortical microcirculation. This method is useful for high resolution analysis of tissue oxygen supply.

Erkennung von mikroregionale Hypoxie in Maus-Großhirnrinde Zwei-Photonen-Imaging von endogenen NADH Fluoreszenz

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. ...

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.

Stereotaktische Infusion von oligomeren Amyloid-beta in die Maus-Hippocampus

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

Generalized Psychophysiological Interaction (PPI) Analysis of Memory Related Connectivity in Individuals at Genetic Risk for Alzheimer's Disease

In neuroimaging, functional magnetic resonance imaging (fMRI) measures the blood-oxygenation-level dependent (BOLD) signal in the brain. The degree of correlation of the BOLD signal in spatially independent regions of the brain defines the functional connectivity of those regions. During a cognitive fMRI task, a psychophysiological interaction (PPI) analysis can be used to examine changes in the functional connectivity during specific contexts defined by the cognitive task. An example of such a task is one that engages the memory system, asking participants to learn pairs of unrelated words (encoding) and recall the second word in a pair when presented with the first word (retrieval). In the present study, we used this type of associative memory task and a generalized PPI (gPPI) analysis to compare changes in hippocampal connectivity in older adults who are carriers of the Alzheimer's disease (AD) genetic risk factor apolipoprotein-E epsilon-4 (APOE&#949;4). Specifically, we show that the functional connectivity of subregions of the hippocampus changes during encoding and retrieval, the two active phases of the associative memory task. Context-dependent changes in functional connectivity of the hippocampus were significantly different in carriers of APOE&#949;4 compared to non-carriers. PPI analyses make it possible to examine changes in functional connectivity, distinct from univariate main effects, and to compare these changes across groups. Thus, a PPI analysis may reveal complex task effects in specific cohorts that traditional univariate methods do not capture. PPI analyses cannot, however, determine directionality or causality between functionally connected regions. Nevertheless, PPI analyses provide powerful means for generating specific hypotheses regarding functional relationships, which can be tested using causal models. As the brain is increasingly described in terms of connectivity and networks, PPI is an important method for analyzing fMRI task data that is in line with the current conception of the human brain.

Generalized Psychophysiological Interaction PPI Analysis of Memory Related Connectivity in Individuals at Genetic Risk for Alzheimer&#039;s Disease

This manuscript describes how to implement a psychophysiological interaction analysis to reveal task-dependent changes in functional connectivity between a selected seed region and voxels in other regions of the brain. Psychophysiological interaction analysis is a popular method to examine task effects on brain connectivity, distinct from traditional univariate activation effects.

Generalisierte psychophysiologische (PPI) Interaktionsanalyse des Speichers im Zusammenhang mit Konnektivität bei Personen auf genetischen Risiko für die Alzheimer-Krankheit

In neuroimaging, functional magnetic resonance imaging (fMRI) measures the blood-oxygenation-level dependent (BOLD) signal in the brain. The degree of ...

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.

Using Enzyme-based Biosensors to Measure Tonic and Phasic Glutamate in Alzheimer&#039;s Mouse Models

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).

Enzym-basierte Biosensoren Mit Tonic und Phasic Glutamate in der Alzheimer-Maus-Modellen zu messen

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

Neurobehavioral Assessments in a Mouse Model of Neonatal Hypoxic-ischemic Brain Injury

We performed unilateral carotid artery occlusion on CD-1 mice to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of neonatal HI brain injury by studying neurobehavioral functions in these mice compared to non-operated (i.e., normal) mice. During the study, Rice-Vannucci&#39;s method was used to induce neonatal HI brain damage in postnatal day 7-10 (P7-10) mice. The HI operation was performed on the pups by unilateral carotid artery ligation and exposure to hypoxia (8% O2 and 92% N2 for 90 min). One week after the operation, the damaged brains were evaluated with the naked eye through the semi-transparent skull and were categorized into subgroups based on the absence (&#34;no cortical injury&#34; group) or presence (&#34;cortical injury&#34; group) of cortical injury, such as a lesion in the right hemisphere. On week 6, the following neurobehavioral tests were performed to evaluate the cognitive and motor functions: passive avoidance task (PAT), ladder walking test, and grip strength test. These behavioral tests are helpful in determining the effects of neonatal HI brain injury and are used in other mouse models of neurodegenerative diseases. In this study, neonatal HI brain injury mice showed motor deficits that corresponded to right hemisphere damage. The behavioral test results are relevant to the deficits observed in human neonatal HI patients, such as cerebral palsy or neonatal stroke patients. In this study, a mouse model of neonatal HI brain injury was established and showed different degrees of motor deficits and cognitive impairment compared to non-operated mice. This work provides basic information on the HI mouse model. MRI images demonstrate the different phenotypes, separated according to the severity of brain damage by motor and cognitive tests.

We performed unilateral carotid artery occlusion on postnatal day 7-10 CD-1 mouse pups to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of HI brain injury. We studied neurobehavioral functions in these mice compared to non-operated normal mice.

Neurobehavioral Bewertungen in einem Mausmodell der Neugeborenen hypoxisch-ischämische Hirnschädigung

We performed unilateral carotid artery occlusion on CD-1 mice to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of neonatal HI ...

Labeling and Imaging of Amyloid Plaques in Brain Tissue Using the Natural Polyphenol Curcumin

Deposition of amyloid beta protein (A&#946;) in extra- and intracellular spaces is one of the hallmark pathologies of Alzheimer&#39;s disease (AD). Therefore, detection of the presence of A&#946; in AD brain tissue is a valuable tool for developing new treatments to prevent the progression of AD. Several classical amyloid binding dyes, fluorochrome, imaging probes, and A&#946;-specific antibodies have been used to detect A&#946; histochemically in AD brain tissue. Use of these compounds for A&#946; detection is costly and time consuming. However, because of its intense fluorescent activity, high-affinity, and specificity for A&#946;, as well as structural similarities with traditional amyloid binding dyes, curcumin (Cur) is a promising candidate for labeling and imaging of A&#946; plaques in postmortem brain tissue. It is a natural polyphenol from the herb Curcuma longa. In the present study, Cur was used to histochemically label A&#946; plaques from both a genetic mouse model of 5x familial Alzheimer&#39;s disease (5xFAD) and from human AD tissue within a minute. The labeling capability of Cur was compared to conventional amyloid binding dyes, such as thioflavin-S (Thio-S), Congo red (CR), and Fluoro-jade C (FJC), as well as A&#946;-specific antibodies (6E10 and A11). We observed that Cur is the most inexpensive and quickest way to label and image A&#946; plaques when compared to these conventional dyes and is comparable to A&#946;-specific antibodies. In addition, Cur binds with most A&#946; species, such as oligomers and fibrils. Therefore, Cur could be used as the most cost-effective, simple, and quick fluorochrome detection agent for A&#946; plaques.

Curcumin is an ideal fluorophore for labeling and imaging of amyloid beta protein plaques in brain tissue due to its preferential binding to amyloid beta protein as well as its structural similarities with other traditional amyloid binding dyes. It can be used to label and image amyloid beta protein plaques more efficiently and inexpensively than traditional methods.

Etikettierung und Bildgebung von Amyloid-Plaques im Gehirngewebe mit dem natürlichen Polyphenol Curcumin

Deposition of amyloid beta protein (A&#946;) in extra- and intracellular spaces is one of the hallmark pathologies of Alzheimer&#39;s disease (AD). ...

Stereological Estimation of Cholinergic Fiber Length in the Nucleus Basalis of Meynert of the Mouse Brain

The length of cholinergic or other neuronal axons in various brain regions are often correlated with the specific function of the region. Stereology is a useful method to quantify neuronal profiles of various brain structures. Here we provide a software-based stereology protocol to estimate the total length of cholinergic fibers in the nucleus basalis of Meynert (NBM) of the basal forebrain. The method uses a space ball probe for length estimates. The cholinergic fibers are visualized by choline acetyltransferase (ChAT) immunostaining with the horseradish peroxidase-diaminobenzidine (HRP-DAB) detection system. The staining protocol is also valid for fiber and cell number estimation in various brain regions using stereology software. The stereology protocol can be used for estimation of any linear profiles such as cholinoceptive fibers, dopaminergic/catecholaminergic fibers, serotonergic fibers, astrocyte processes, or even vascular profiles.

Neuronal fiber length within a three-dimensional structure of a brain region is a reliable parameter to quantify specific neuronal structural integrity or degeneration. This article details a stereological quantification method to measure cholinergic fiber length within the nucleus basalis of Meynert in mice as an example.

Stereologische Abschätzung der cholinergen Faserlänge im Nukleus Basalis von Meynert des Maushirns

The length of cholinergic or other neuronal axons in various brain regions are often correlated with the specific function of the region. Stereology is a ...