この作品は、カナダ衛生研究所（CIHR）、タウンゼント家族、そしてジャックブラウンと家族のアルツハイマー病研究財団（WSする）によってサポートされていました。 WASは、アルツハイマー病におけるカナダリサーチチェアのホルダーです。 PTTLは、自然科学と工学研究の弁護士と健康研究奨学金のためのマイケルスミス財団によってサポートされています。

1。マウスの脳の固定およびcryosectioning <ol><li>トランスジェニックマウスを断頭し、抽出された脳が続く、二酸化炭素のガスを使用して犠牲にされています。 </li><li>脳は、中央に縦方向に切開される。脳の半分は、生化学分析のためのドライアイスで凍結フラッシュになります。他は4℃、少なくとも48時間、4％パラホルムアルデヒド（PFA）℃で水没されます。 </li><li>少なくとも48時間、4％PFAで固定後、半球の脳を30％ショ糖溶液に直接転送され、4に配置℃に時点では、半球の脳は、ショ糖溶液に浮かんでされるべきである。半球の脳は通常約48時間を必要とする、底に沈んだときに、それはcryosectioning用OCTで埋め込むことができるようになります。 </li><li> cryosectioningする前に、ノブにOCTの薄層をマウントし、-20℃で凍結することができます冷凍OCTの層の上に半球の脳と場所の他の部分から小脳を削除します。直ちにOCTで半球の脳をカバーし、徐々に-20℃で凍結することができます脳が不透明になっているのOCT包埋後は、区分する準備ができました。 </li><li> 30μm以下の各セクションの厚さを設定します。 D&#39; Olomosソリューションとコンテナに各スライスを収集する。 </li></ol> 2。神経突起プラークの免疫組織化学手順（フリーフローティングセクション） <ol><li> 15分間88％ギ酸中の各セクションを扱います。ギ酸処理した後、セクションでは、一時的に不透明に変わり、しぼむように見えることがあります。 </li><li>静かに撹拌しながら5分間、X 3はTx - PBS（0.3％トリトンX - 100 PBS中）を有するプレートと洗浄に各セクションを削除します。 </li><li>ブロック内因性過酸化物は、0.5％のTx - PBS、室温でのH 2 O 2穏やかに振盪しながら30分をブロック。 </li><li>静かに振とうしながら10分X 3のTX - PBSでセクションを洗ってください。 </li><li>穏やかに振盪しながら1時間室温でのTx - PBSに溶解した5％無脂肪脱脂乳を持つブロックのセクション。 </li><li>のTx - PBSで穏やかに振盪しながら℃で一晩4℃、5％の牛乳を含むで希釈した一次抗体のセクション（4G8をビオチン、1:500-1:2000、シグネット）をインキュベートする。 </li><li>静かに振とうしながら10分X 3のTX - PBSでセクションを洗ってください。 </li><li> ABCのソリューションは、次の製造者のプロトコル（エリートABC、ベクターラボラトリーズ社を、）準備する。穏やかに振盪しながら30分間ABC混合物のセクションをインキュベートする。 </li><li>静かに振とうしながら10分X 3のTX - PBSでセクションを洗ってください。 </li><li> 3,3&#39; - ジアミノベンジジン四塩酸塩（DAB）（ベクターラボラトリーズ）以下の製造業者のプロトコールを準備します。 5〜10分間DABの混合物のセクションをインキュベートする。この時点で、茶色がかった色の各セクションでの開発を遵守します。 </li><li>静かに振とうしながら10分X 3のTX - PBSでセクションを洗ってください。 </li><li>フリーフローティング脳切片は、組織の癒着を支援するため、ゼラチンコートしたスライド上にマウントされています。セクションは、その後、空気はキシレンのシリーズでクリアすることで、続くとEntellenにマウントされている乾燥させる。 </li><li>プラークは、40X倍率で光学顕微鏡下で可視化し、各マウスのスライス当たりの平均プラーク数を評価することによって定量化されています。 </li></ol> 3。神経突起プラークの検出のためのチオフラビンS染色手順<ol><li>マウスの犠牲と脳の抽出手順は、セクション1.1から1.5に示されている。 </li><li>スライドガラス上に4から5半の脳切片をマウントし、完全に染色する前に空気乾燥することができます。 </li><li> 1分間70％エタノールでスライドを洗浄します。 </li><li> 1分間80％エタノールでスライドを洗浄します。 </li><li> 15分のためにフィルター（0.2μmのフィルター）チオフラビンS溶液（エタノールの80％で1％）でスライドをインキュベートする。 （チオフラビンS溶液は使用前にフィルタリングする必要がある）。注意：光からチオフラビンSを保護し、光から染色したスライドを保護する。 </li><li> 1分間80％エタノールでスライドを洗浄します。 </li><li> 70％エタノール1分でスライドを洗浄します。 </li><li>二回蒸留水で洗ってください。 </li><li>水性マウントメディアのマウントのスライドとスライドの明確なマニキュアでカバーガラスをシールに続いて、少なくとも2時間、暗所で乾燥させます。染色した切片を4℃で暗所に保管してください</li><li>緑色蛍光染色されたプラークには、蛍光microscropyで可視化することができます。染色が時間とともにフェードアウトするので、一週間以内にスライドを分析する。 </li></ol> 4。代表的な結果老人斑の形成を抑制する抗てんかん薬や気分安定剤バルプロ酸（VPA）の有効性に関する我々の最近の研究では、我々は、ADのモデルマウス（3）の神経突起プラークを識別するために、上述の免疫染色し、チオフラビンS染色の手順を使用していました。図1は、抗Aβ使用してビオチン化4G8で染色し、ABCのメソッドを介して検出された、典型的な9ヶ月古いAPP23トランスジェニックマウスを表します。老人斑は明確抗体で標識され、白い矢印で示されている。チオフラビンS染色を使用して、我々は2ヶ月古いAPP23xPS TRA老人斑の沈着を検出nsgenicマウス（図2）。チオフラビンSバウンドAβ含有神経突起斑（白い矢印で示される）、蛍光顕微鏡下で緑色に表示されます。我々は、VPAの治療で、老人斑の形成が有意に（3）減少することを示した。別の研究では、我々は低酸素症およびADの病因の根底にある分子のリンクを学んだ。再び、4G8抗Aβ免疫組織化学染色およびチオフラビンS染色の両方を使用して、我々は低酸素症は老人斑を（4）を含むAβの形成を容易に発見。 <img src="/files/ftp_upload/2831/2831fig1.jpg" alt="図1"/> 図1。 ADトランスジェニックマウスにおける老人斑の形成の免疫組織化学的分析。9ヶ月歳の時にAPP23マウスは屠殺し、解剖した、固定し、切片。海馬での神経突起プラークは、抗Aβ抗体4G8を用いて検出したとABCとDABの方法を使用して開発されました。プラークは、40Xと光学顕微鏡により可視化した。白い矢印は、Aβ神経突起斑を指す。 <img src="/files/ftp_upload/2831/2831fig2.jpg" alt="図2"/> 図2。 ADモデル化されたマウスにおける神経突起斑のチオフラビンS染色。8週齢でAPP23xPS45ダブルトランスジェニックマウスを屠殺し、解剖した、固定し、切片。海馬での神経突起の災害は、チオフラビンS蛍光染色を用いて確認して40Xの倍率で顕微鏡により可視化されたされています。白い矢印は、チオフラビンS染色された神経突起斑を示している。

<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>

全ての実験は、ブリティッシュコロンビア州の動物ケアの大学に準拠して行われ、委員会とCIHRのガイドラインを使用していた。

ビオチン標識された4G8抗体の染色特異性を有するADモデル化されたマウスでは老人斑を用いて免疫組織化学。染色の結果は、定量化し、異なる治療群間でプラークの負荷の比較が可能になります。結果に影響を与える可能性があるいくつかの重要なステップがあります。半球の脳が頭蓋骨から抽出する前に潅流されていないため、注意が半球の脳への損傷を防止するために、抽出プロセス中に使用する必要があります。半球の脳が受動的に灌流されているのでまた、私たちは4で、少なくとも48時間を4％PFAでそれをインキュベート勧め° C前の30％ショ糖溶液中で水没する。また、transcardial灌流は脳を抽出する前に行うことができる。脳が正しく固定されている場合、それはゴムのような質感を持つ必要があります。脳が正しく固定されていない場合には、セクションでは、簡単にD&#39; Olomosの溶液中でまたは染色手順の間に引き裂くだろう。 4G8モノクローナル抗体は、アミノ酸残基Aβの17から24への反応性であり、コア配列（VFFAE）のエピトープを認識した。 4G8はマウスの種から派生しているので、それはより高いバックグラウンド染色を与える傾向にある。従って、我々は、バックグラウンド染色を最小限に抑えながら、考慮事項は真の信号を達成するために指示された希釈でセクションをインキュベートすることを選んだ。 4G8抗体を用いて神経突起プラーク検出のための免疫染色に加えて、チオフラビンS染色法は、プラークを識別するために使用されます。チオフラビンSは、均質な染料の混合物であるスルホン酸とdehydrothiotoluidineのメチル化の結果である。チオフラビンSの非選択的にそのようなアミロイドのオリゴマーにあるような蛋白質のβシートの内容を、結合する。結合すると、チオフラビンはその発光スペクトルの特徴的なブルーシフトを受ける。逆に単量体の形態にチオフラビンS結合はブルーシフトを誘発せず、蛍光顕微鏡で検出することができませんでした。蛍光の強度は、アミロイド沈着物の少量の良好な可視化を可能にするとしてチオフラビンS染色ではアミロイドのための画面への迅速な代替手段を提供します。しかし、チオフラビンS染色に関する一つの欠点は、特異性の欠如です。このようなfibrinoids、硝子、ケラチン、などなどを含む広範なβシート、を含む他の多くの組織成分には、この色素のためではなく、親和性を持っている。

<table border="1"><tbody><tr><th>試薬と機器の名称</th><th>会社</th><th>カタログ番号</th></tr><tr><td>パラホルムアルデヒド</td><td>シグマアルドリッチ</td><td> P6148 - 1KG </td></tr><tr><td> Polyvinylpyrrolidene </td><td>シグマアルドリッチ</td><td> PVP40 - 100G </td></tr><tr><td>スクロース</td><td>フィッシャーサイエンティフィック</td><td> S5 - 3 </td></tr><tr><td>エチレングリコール</td><td>シグマアルドリッチ</td><td> 324558 - 2L </td></tr><tr><td>組織- Tek社のOCTコンパウンド</td><td>桜</td><td> 4583 </td></tr><tr><td> 88％ギ酸</td><td>フィッシャーサイエンティフィック</td><td> A118P - 500 </td></tr><tr><td>トリトンX - 100 </td><td> ICNのバイオメディカル</td><td> 807426 </td></tr><tr><td> H2O2 </td><td>シグマアルドリッチ</td><td> H - 1009 </td></tr><tr><td>ビオチンは、4G8抗体を標識</td><td> Cedarlane </td><td> SIG - 39240 - 500 </td></tr><tr><td>エリートABCキット</td><td>ベクトル</td><td> PK - 6100 </td></tr><tr><td>イムPACT DAB </td><td>ベクトル</td><td> SK - 4105 </td></tr><tr><td>キシレン</td><td>フィッシャーサイエンティフィック</td><td> X4 - 4 </td></tr><tr><td> Entellan </td><td> EM科学</td><td> 65037〜71 </td></tr><tr><td> Superfrost * Plusの顕微鏡スライド</td><td>フィッシャーサイエンティフィック</td><td> 12-550-15 </td></tr><tr><td>クライオスタット</td><td>ライカ</td><td> CM - 3050 - S </td></tr><tr><td> MX35プレミアミクロトーム刃</td><td>サーモサイエンティフィック</td><td> 3052835 </td></tr><tr><td>チオフラビンS </td><td>シグマアルドリッチ</td><td> T1892 - 25G </td></tr></tbody></table>

detection of neuritic plaques in alzheimer's disease mouse model

アルツハイマー病（AD）は認知症につながる最も一般的な神経変性疾患である。老人斑の形成は、アルツハイマー病の病理学的特徴の一つです。老人斑の中心的なコンポーネントは、β-セクレターゼとγ-セクレターゼによるβ-アミロイド前駆体タンパク質（APP）の連続したタンパク質分解切断に由来する小さな糸状のタンパク質と呼ばれるアミロイドβタンパク質（Aβ）1、です。アミロイド仮説はAD病理2の基本的な毒性機序としてプラークをAγ含有することが伴います。老人斑の存在の事後分析では、ADの診断が確定する。 ADの病因におけるAγ神経生物学の理解を促進するために、ヒトAPP遺伝子のAD関連変異を表現する様々なマウス系統を作製した。疾患の重症度に応じて、これらのマウスは、年齢別の老人斑を開発します。これらのマウスは、APPのプロセシング経路と老人斑の形成に影響を与える可能性のある病態と薬剤開発を研究するための貴重なツールとして機能します。このプロトコルでは、我々は、ADのモデルマウスの老人斑を特異的に検出する免疫​​組織化学的方法を採用しています。 ABCとDAB法とthiofalvin S染色法を用いて蛍光検出を用いて免疫組織化学的検出：我々は、特に半脳、パラホルムアルデヒド固定、cryosectioning、およびADトランスジェニックマウスに神経症プラークを検出するための2つのメソッドを抽出するから準備を説明します。

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

ADの病理学的特徴の一つは、アミロイドβ蛋白質の正の神経突起斑の形成である。 ABCとDAB法とチオフラビンS染色法を用いて蛍光検出を用いて免疫組織化学的検出：このプロトコルでは、トランスジェニックADのモデルマウスの老人斑を検出するために2つの方法について説明します。

アルツハイマー病のマウスモデルにおける神経突起斑の検出

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 ビュー.  The University of British Columbia. ADの病理学的特徴の一つは、アミロイドβ蛋白質の正の神経突起斑の形成である。 ABCとDAB法とチオフラビンS染色法を用いて蛍光検出を用いて免疫組織化学的検出：このプロトコルでは、トランスジェニックADのモデルマウスの老人斑を検出するために2つの方法について説明します。

ビデオ: 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.

β-アミロイドオリゴマーの調製 1から42まで</sub海馬スライスでのシナプス可塑性の減損の>と誘導

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

神経科学

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.

アミロイドβ-蛋白質、アルツハイマー病の原因物質のためのアプタマーの選択

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.

アルツハイマー病モデルマウスにおける学習記憶障害のためのモリス水迷路試験

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.

内在性NADH蛍光の二光子イメージングによるマウス大脳皮質におけるMicroregional低酸素の検出

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.

マウス海馬にオリゴマーアミロイドベータの定位注入

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.

アルツハイマー病の遺伝的危険で個人の接続関連のメモリの一般化された精神生理学的相互作用 (PPI) 解析

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).

アルツハイマー病のマウスモデルにおけるトニックと相性グルタミン酸を測定するための酵素ベースのバイオセンサーを使用して

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.

新生児低酸素性虚血性脳損傷のマウス モデルにおける神経行動学的評価

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.

天然ポリフェノールクルクミンを用いた脳組織におけるアミロイドプラークの標識とイメージング

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.

マウス脳のマイナート核基底部におけるコリン作動性繊維長の立体的推定

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 ...