Name: quantitative 3d in silico modeling (q3dism) of cerebral amyloid-beta phagocytosis in rodent models of alzheimer's disease
Uploaded: 2016-12-26T13:00:00
Description: 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

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;

研究倫理の声明：本明細書に詳述動物に関わる全ての実験は、南カリフォルニア制度動物実験委員会（IACUC）の大学によって承認され、研究室の評価および認定協会からの国民健康ガイドラインの研究所や勧告に厳密に従って行きましたアニマルケア国際。 1.齧歯類の脳の単離と免疫染色のための準備 1日目： <ol><li>連続深いイソフルラン麻酔（4％）の下で（12ヶ月?...

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我々は、単核食細胞によりin vivoでのAβ食作用の真の3D定量化のために、この報告書に記載したプロトコルは、特定の細胞と細胞内区画の標識だけでなく、Aβ沈着に依存しています。具体的には、脳単核食細胞を染色するためにIBA1（イオン化カルシウムの結合アダプター分子1）、細胞の活性化18、19時に細胞膜の波打ち現象および食作用に関与するタンパク質を?...

アルツハイマー病（AD）、最も一般的な加齢に関連する認知症1は 、「老人」βアミロイド斑、慢性低レベルの神経炎症、タウオパチー、神経細胞の喪失、および認知障害2などの脳アミロイドβ（Aβ）の蓄積によって特徴付けられます。 （末梢起源対それらの中心は依然として不明であるものの、ミクログリアと呼ばれる）は、AD患者の脳では、神経炎症がAβ沈着物3を取り囲む反応性星状細胞および単核食細胞によって計上されています。 CNSの自然免疫番人として、ミクログリアは中心部の脳Aβをクリアするように配置されています。しかし、Aβプラークのミクログリアの募集は、もしあれば、非常に少ないのAβ貪食4,5を伴っています。 1つの仮説は、ミクログリアがAβの小さなアセンブリをphagocytozingことで、最初は神経保護的であるということです。しかし、最終的にこれらの細胞は圧倒的なAβ負荷および/または年齢関連機能dと神経毒性になりますecline、神経毒性および認知機能の低下6に貢献し、機能不全の前炎症性表現型へのミクログリアを引き起こします。 最近のゲノムワイド関連研究（GWAS）貪食8-11を調節コア先天性免疫経路7に属するADのリスク対立遺伝子のクラスターを同定しました。その結果、脳アミロイド沈着に対する免疫応答は、ADの病因を理解するという点で、および12月14日 、新しい治療法を開発するために、両方の、関心の主要なエリアとなっています。しかし、 生体内でのAβ食作用を評価するための方法論のための重要な必要性があります。この満たされていないニーズに対処するために、我々は、アルツハイマー様疾患のげっ歯類モデルにおいて単核食細胞によって脳のAβ食作用の真の3D定量を可能にするためにシリコモデリング（q3DISM） で定量的な3Dを開発しました。 それらは、動物モデルは、疾患を有する再現する程度によってのみ制限ADのpathoetiologyを理解するために、実験治療法を評価するための貴重な実証済み。プレセニリン（PS）及びアミロイド前駆体タンパク質（APP）遺伝子における変異は、独立して、常染色体優性ADを引き起こすという事実のため、これらの変異導入遺伝子は、広範ジェニック齧歯類モデルを生成するために使用されてきました。同時に「スウェーデン」変異ヒトAPP（APPのSWE）と加速脳アミロイドーシス及び神経炎症15,16を備えた本Δエクソン9変異ヒトプレセニリン1（PS1ΔE9）を共発現するトランスジェニックAPP / PS1マウス。さらに、我々は、（フィッシャー344背景にラインTgF344-AD）のAPP SWEとPS1ΔE9構築物で同時注入二重トランスジェニックラットを生成しました。脳アミロイドーシスのトランスジェニックマウスモデルとは異なり、TgF344-ADラットはタウオパチー、神経細胞のアポトーシス損失、および行動障害17の前脳アミロイドを開発します。 本稿では、IMMのためのプロトコルを記述しますミクログリア、APP / PS1マウスとTgF344-ADラットからの脳切片におけるファゴリソソームおよびAβ沈着物、および大のz次元共焦点画像の取得をunostaining。 シリコ世代とミクログリアのファゴリソソームにAβの取り込みの定量化を可能にする共焦点データセットから真の3D再構成の分析で私たちは、詳細。より広義には、ここでは詳細を、我々の方法は、 インビボでの食作用の実質的に任意の形状を定量化するために使用することができます。

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			<td height="40" style="height:40px;width:384px;">Isoflurane</td>
			<td style="width:152px;">Abbott</td>
			<td style="width:127px;">NDC 0044-5260-05</td>
			<td style="width:249px;"></td>
		</tr>
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			<td height="20" style="height:20px;width:384px;">Dissecting scissors</td>
			<td style="width:152px;">VWR</td>
			<td style="width:127px;">82027-582</td>
			<td></td>
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			<td height="20" style="height:20px;width:384px;">Dissecting scissors Blunt tip</td>
			<td style="width:152px;">VWR</td>
			<td style="width:127px;">82027-588</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Tweezers</td>
			<td style="width:152px;">VWR</td>
			<td>94024-408</td>
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			<td height="20" style="height:20px;width:384px;">23G needle</td>
			<td style="width:152px;">VWR</td>
			<td>BD305145</td>
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			<td height="20" style="height:20px;width:384px;">peristaltic pump FH10</td>
			<td style="width:152px;">Thermo Scientific</td>
			<td>72-310-010</td>
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			<td height="20" style="height:20px;width:384px;">PBS 10X</td>
			<td style="width:152px;">Bioland Scientific</td>
			<td style="width:127px;">PBS01-02</td>
			<td>Working concentration 1X</td>
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			<td height="20" style="height:20px;width:384px;">Adult Mouse Brain Matrix, Coronal slices, Stainless Steel 1mm&#160;</td>
			<td style="width:152px;">Kent Scientific</td>
			<td>RBMS-200C</td>
			<td></td>
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			<td height="20" style="height:20px;width:384px;">Adult Rat Brain Matrix, Coronal slices, Stainless Steel 1mm&#160;</td>
			<td style="width:152px;">Kent Scientific</td>
			<td>RBMS-305C&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">32% Paraformaldehyde aqueous solution</td>
			<td style="width:152px;">EMS</td>
			<td style="width:127px;">15714-S</td>
			<td>Caution: Toxic. Working concentration 4% in PBS</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Ethanol</td>
			<td style="width:152px;">VWR</td>
			<td>89125-188</td>
			<td>Various concentrations, see protocol</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Tissue-Tek Uni-cassettes Sakura</td>
			<td style="width:152px;">VWR</td>
			<td>25608-774</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Embedding and Infiltration Paraffin</td>
			<td style="width:152px;">VWR</td>
			<td>15147-839</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Microtome Leica RM2125</td>
			<td style="width:152px;">Leica Biosystems</td>
			<td style="width:127px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Disposable Microtome Blades&#160;</td>
			<td style="width:152px;">VWR</td>
			<td style="width:127px;">25608-964</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Water bath Leica HI 1210</td>
			<td style="width:152px;">Leica Biosystems</td>
			<td style="width:127px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Micro slide Superfrost plus</td>
			<td style="width:152px;">VWR</td>
			<td style="width:127px;">48311-703</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Xylene</td>
			<td style="width:152px;">Sigma-Aldrich</td>
			<td style="width:127px;">534056-4X4L</td>
			<td style="width:249px;">Caution: Toxic&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Target Retrieval Solution 10X</td>
			<td style="width:152px;">DAKO</td>
			<td style="width:127px;">S1699</td>
			<td>Working concentration 1X</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KimWipes</td>
			<td style="width:152px;">VWR</td>
			<td>21905-026</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Hydrophobic PAP pen</td>
			<td style="width:152px;">VWR</td>
			<td style="width:127px;">95025-252</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Triton X-100</td>
			<td style="width:152px;">VWR</td>
			<td style="width:127px;">97062-208</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Normal Donkey Serum</td>
			<td style="width:152px;">Jackson Immuno</td>
			<td style="width:127px;">017-000-121</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Coverslips</td>
			<td style="width:152px;">VWR</td>
			<td style="width:127px;">48393081</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Prolong Gold antifade reagent with DAPI</td>
			<td style="width:152px;">Life Technologies</td>
			<td style="width:127px;">P36935</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Glass Slide Rack</td>
			<td style="width:152px;">VWR</td>
			<td style="width:127px;">100492-942</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Iba1 antibody (polyclonal, rabbit)</td>
			<td style="width:152px;">Wako</td>
			<td style="width:127px;">019-19741&#160;</td>
			<td>Working concentration 1:200</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Iba1 antibody (polyclonal, goat)</td>
			<td style="width:152px;">LifeSpan Bioscience</td>
			<td style="width:127px;">LS-B2645</td>
			<td>Working concentration 1:200</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">rat CD68 [KP1] antibody (monoclonal, mouse)</td>
			<td style="width:152px;">Abcam</td>
			<td style="width:127px;">ab955</td>
			<td>Working concentration 1:200</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">mouse CD68 [FA-11] antibody (monoclonal, rat)</td>
			<td style="width:152px;">Abcam</td>
			<td style="width:127px;">ab53444</td>
			<td>Working concentration 1:200</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">mouse CD107a (LAMP1) antibody (monoclonal, rat)</td>
			<td style="width:152px;">Affymetrix</td>
			<td style="width:127px;">14-1071</td>
			<td>Working concentration 1:100</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Beta-Amyloid, 17-24 (4G8) antibody (monoclonal, mouse)</td>
			<td style="width:152px;">Covance</td>
			<td style="width:127px;">SIG-39220</td>
			<td>Working concentration 1:200</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Beta-Amyloid, 1-16 (6E10) antibody (monoclonal, mouse)</td>
			<td style="width:152px;">Covance</td>
			<td style="width:127px;">SIG-39320</td>
			<td>Working concentration 1:200</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">OC antibody (polyclonal, rabbit)</td>
			<td colspan="2">Gifted by D. H. Cribbs and C. G. Glabe (UC Irvine)</td>
			<td>Working concentration 1:200</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Alexa Fluor 488&#160; mouse secondary antibody</td>
			<td style="width:152px;">Invitrogen</td>
			<td style="width:127px;">A-11001</td>
			<td>Working concentration 1:1000</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Alexa Fluor 488&#160; rat secondary antibody</td>
			<td style="width:152px;">Invitrogen</td>
			<td>A-11006</td>
			<td>Working concentration 1:1000</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Alexa Fluor 594 rabbit secondary antibody</td>
			<td style="width:152px;">Invitrogen</td>
			<td>A-11037</td>
			<td>Working concentration 1:1000</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Alexa Fluor 594 goat secondary antibody</td>
			<td style="width:152px;">Invitrogen</td>
			<td>A-11080</td>
			<td>Working concentration 1:1000</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Alexa Fluor 647 mouse secondary antibody</td>
			<td style="width:152px;">Invitrogen</td>
			<td>A-21235</td>
			<td>Working concentration 1:1000</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Alexa Fluor 647 rabbit secondary antibody</td>
			<td style="width:152px;">Invitrogen</td>
			<td>A-21443</td>
			<td>Working concentration 1:1000</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Immersion oil</td>
			<td style="width:152px;">Nikon&#160;</td>
			<td style="width:127px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">A1 Confocal microscope</td>
			<td style="width:152px;">Nikon&#160;</td>
			<td style="width:127px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">NIS Elements Advanced Research software</td>
			<td style="width:152px;">Nikon&#160;</td>
			<td style="width:127px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Imaris:Bitplane software version 7.6</td>
			<td style="width:152px;">Bitplane</td>
			<td style="width:127px;"></td>
			<td>&#34;coloc&#34; and &#34;supass&#34; modules are used.&#160;Alternatively, the open-source freeware&#160;ImageJ can be used for colocalization&#160;analysis of confocal z-stacks datasets.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td style="width:152px;"></td>
			<td style="width:127px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;"></td>
			<td style="width:152px;"></td>
			<td style="width:127px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;"></td>
			<td style="width:152px;"></td>
			<td style="width:127px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

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

神経炎症は現在、神経変性疾患の主要な病因因子として認識されています。単核食細胞は、破片や残骸の食作用およびクリアランスを担当する自然免疫細胞です。これらの細胞は、CNS常駐ミクログリアとして知られるマクロファージ、および周囲から浸潤性単核食細胞が含まれます。光学顕微鏡は、一般に、齧歯類またはヒトの脳標本で食作用を視覚化するために使用されています。しかし、定性的な方法は、in vivo食作用の決定的な証拠を提供していません。ここでは、 シリコモデリング（q3DISM）、げっ歯類アルツハイマー病（AD）モデルにおける単核食細胞によるアミロイドβ（Aβ）食作用の真の3D定量化を可能にする堅牢な方法で定量的な3Dを説明します。この方法は、蛍光齧歯類の脳切片におけるファゴリソソーム内にカプセル化Aβを可視化することを含みます。大型のz次元共焦点のデータセットは、その後、3Dは、A＆の定量のために再構築されます＃946;空間的にファゴリソソーム内で共局在。我々は、マウスおよびラットの脳にq3DISMの適用が成功を実証したが、この方法論は、任意の組織で事実上すべての貪食イベントに拡張することができます。

上記で詳述しq3DISMのための多段階方法論を使用して、我々はAPP / PS1マウス（ 図1）とTgF344-ADラットの脳における単球のファゴリソソーム（ 図2）にAβの取り込みを定量化することができます。したがって、q3DISMの方法論は、ADのマウスおよびラットモデルにおける単核食細胞の解析を可能にしました。興味深いことに、CD68 +ファゴリソ?...

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

私たちは、アルツハイマー病のげっ歯類モデルにおいて単核食細胞によって脳アミロイドβ（Aβ）食作用のインシリコモデリング （q3DISM） で定量的な3Dのための方法論を開発しました。この方法は、 インビボで実質的に任意の食イベントの定量のために一般化することができます。

定量的3D<em&gt;インシリコ</emアルツハイマー病の齧歯類モデルにおける脳アミロイドベータ食作用の&gt;モデリング（q3DISM）

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

Research

JoVE Journal

Medicine

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

Examining the Characteristics of Episodic Memory using Event-related Potentials in Patients with Alzheimer's Disease

アルツハイマー病患者における事象関連電位を用いたエピソード記憶の特性を調べる

Our laboratory uses event-related EEG potentials (ERPs) to understand and support behavioral investigations of episodic memory in patients with amnestic ...

Quantitative Analysis of Chromatin Proteomes in Disease

病におけるクロマチンプロテオームの定量的解析

In the nucleus reside the proteomes whose functions are most intimately linked with gene regulation. Adult mammalian cardiomyocyte nuclei are unique due ...

Gene-environment Interaction Models to Unmask Susceptibility Mechanisms in Parkinson's Disease

パーキンソン病における感受性メカニズムのマスク解除を行う遺伝子環境相互作用モデル

Lipoxygenase (LOX) activity has been implicated in neurodegenerative disorders such as Alzheimer&#39;s disease, but its effects in Parkinson&#39;s disease ...

Modeling Stroke in Mice: Permanent Coagulation of the Distal Middle Cerebral Artery

マウスにおけるモデリングのストローク：遠位中大脳動脈の永久的な凝固

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical ...

Multi-electrode Array Recordings of Human Epileptic Postoperative Cortical Tissue

人間のてんかん術後皮質組織の多電極アレイ録音

Epilepsy, affecting about 1% of the population, comprises a group of neurological disorders characterized by the periodic occurrence of seizures, which ...

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

のための多発性嚢胞腎疾患の小齧歯類モデルにおける超高磁場MRIの使用<em&gt;インビボ</em&gt;表現型と薬物モニタリング

Several in vivo pre-clinical studies in Polycystic Kidney Disease (PKD) utilize orthologous rodent models to identify and study the genetic and molecular ...

Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease

Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease

ex vivoで腸嚢は、胃腸疾患のモデルに粘膜透過性を評価するために

The epithelial barrier is the first innate defense of the gastrointestinal tract and selectively regulates transport from the lumen to the underlying ...

Hemodynamic Characterization of Rodent Models of Pulmonary Arterial Hypertension

肺動脈性肺高血圧症の齧歯類モデルの血行動態特性評価

Pulmonary arterial hypertension (PAH) is a rare disease of the pulmonary vasculature characterized by endothelial cell apoptosis, smooth muscle ...

Neurodegeneration in an Animal Model of Chronic Amyloid-beta Oligomer Infusion Is Counteracted by Antibody Treatment Infused with Osmotic Pumps

慢性アミロイドβオリゴマー輸液の動物モデルにおける神経変性は、浸透圧ポンプを用いて抗体治療フューズによって打ち消されます

Decline in hippocampal-dependent explicit memory (memory for facts and events) is one of the earliest clinical symptom of Alzheimer&#39;s disease (AD). It ...

Application of Granger Causality Analysis of the Directed Functional Connection in Alzheimer's Disease and Mild Cognitive Impairment

アルツハイマー病と軽度認知障害の監督機能接続のグレンジャー因果関係分析の応用

Impaired functional connectivity in the Default Mode Network (DMN) may be involved in the progression of Alzheimer&#39;s Disease (AD). The Posterior ...