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;

研究伦理的声明：所有涉及此详述动物实验是由美国南加州机构动物护理和使用委员会（IACUC）的大学批准，并严格按照从协会实验室评估和认可的健康指导国家机构和建议进行动物保护国际。 1.脑鼠类分离和制备免疫染色 DAY 1： <ol><li>将年龄TgF344-AD大鼠（14月龄）或APP / PS1小鼠（12月龄）下连续深异氟醚麻醉（4％）。评估麻醉通过脚趾捏的深度和无撤退反射。 </li><li>通...

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我们通过单核吞噬细胞本报告Aβ吞噬的真3D定量描述中体内协议依赖于细胞和亚细胞的特异性标记以及Aβ存款。具体地，我们使用Iba1（离子化钙结合适配器分子1），即在细胞活化18,19参与膜皱裂和吞噬的蛋白质，以染色脑单核吞噬细胞。而Iba1 +细胞通常被视为脑驻留小胶质细胞，它仍然可能外周浸润性单核吞噬细胞包括免疫标记的细胞的子集。细胞内吞噬?...

阿尔茨海默氏病（AD），最常见的与年龄有关的痴呆1中 ，其特征在于，脑淀粉样蛋白β（Aβ）的积累“老年”β淀粉样斑块，慢性低水平神经炎症，τ病变，神经元损失和认知紊乱2 。 ，在AD患者的大脑，神经炎症是由活性星形细胞和单核吞噬细胞专用（称为小胶质细胞，虽然它们的中心与外围原点仍不清楚）周围的Aβ沉积物3。由于中枢神经系统的先天免疫哨兵，小胶质细胞都集中定位于清脑Aβ。然而，小胶质细胞招募到Aβ斑块伴随很少，如果有的话，Aβ吞噬4,5。一个假设是，小胶质细胞是由phagocytozingAβ的小型组件最初神经保护作用。然而，最终这些细胞成为神经毒性为压倒性的Aβ负担和/或年龄相关的功能ðecline，挑起小胶质细胞变成一个不正常的促炎表型，促进神经毒性和认知能力下降6。 最近有全基因组关联研究（GWAS）确定属于该调节吞噬8-11核心的先天免疫途径7 AD风险等位基因的集群。因此，脑淀粉样蛋白沉积的免疫反应已成为关注的主要领域，无论是在了解AD的病因条款和开发新的治疗方法12-14。然而，有一个重要的需要的方法在体内进行评估Aβ吞噬作用。为了解决这一尚未满足的需求，我们开发了在硅片建模（q3DISM）定量3D，以使在阿尔茨海默样疾病的啮齿动物模型单核吞噬大脑Aβ吞噬真3D定量。 只能通过其所概括的疾病，动物模型有程度上限制价值无法估量了解AD pathoetiology和评估实验治疗。由于这样的事实，在早老（PS）和淀粉状蛋白前体蛋白（APP）的基因的突变独立地引起的常染色体显性AD，这些突变体的转基因已被广泛使用，以产生转基因啮齿动物模型。转基因APP / PS1小鼠共表达同时“瑞典”突变人APP（APP SWE）和Δ外显子突变9人类早老1（PS1ΔE9）表现为加速脑淀粉样变性及神经发炎15,16。此外，我们已经产生与APP SWE和PS1ΔE9构建体共注射（线TgF344-AD上的费344背景）的双转基因大鼠。与脑淀粉样变性的转基因小鼠模型，TgF344-AD老鼠患脑淀粉样那之前τ病变，神经细胞凋亡的损失和行为障碍17。 在这份报告中，我们描述了IMM协议unostaining小胶质细胞，吞噬溶酶体和Aβ存款从APP / PS1小鼠和TgF344-AD老鼠，收购大Z三维共聚焦图像的大脑切片。我们详细的硅片产生和共聚焦数据集，允许Aβ摄取定量为小胶质细胞吞噬溶酶体真正的3D重建的分析。更广泛地说，在这里我们详细的方法可以用来量化几乎任何形式在体内吞噬。

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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>
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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>
			<td></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>
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			<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

神经炎症是目前公认的神经退行性疾病的主要病因。单核吞噬细胞负责吞噬和碎片和碎屑清除先天免疫细胞。这些细胞包括被称为小胶质细胞的CNS驻留巨噬细胞和单核吞噬细胞从周边渗入。光镜已普遍应用在啮齿类动物或人类的大脑标本可视化吞噬。然而，定性方法没有提供在体内吞噬确切的证据。这里，我们描述在计算机芯片建模（q3DISM），一个强大的方法允许通过在啮齿类阿尔茨海默氏病（AD）模型单核吞噬细胞淀粉样蛋白β（Aβ）吞噬作用的真正三维定量定量3D。该方法包括荧光可视化Aβ在啮齿动物脑切片吞噬溶酶体内封装。大Z三维数据集焦然后三维重建为A＆定量＃946;吞噬溶酶体内共定位空间。我们证明q3DISM的小鼠和大鼠脑的成功应用，但这种方法可以扩展到以任何组织几乎任何吞噬事件。

使用q3DISM上面详述的多级方法，我们能够量化Aβ摄取到在APP / PS1小鼠（ 图1）和TgF344-AD大鼠（ 图2）的脑中单核吞噬溶酶体。因此，q3DISM方法，使在AD小鼠和大鼠模型单核吞噬细胞的分析。有趣的是，由CD68 +吞噬溶酶体所占据的体积中相比，无论在APP / PS1小鼠（ 图1B，C）和TgF344-AD大鼠（ 图2B，C）的远离斑块相关Iba1...

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

我们开发了在硅片建模（q3DISM）脑淀粉样蛋白β（Aβ）吞噬在阿尔茨海默氏病的啮齿动物模型单核吞噬细胞的定量三维的方法。该方法可推广用于体内几乎任何吞噬活动的定量。

定量3D<em&gt;在硅片</em&gt;建模阿尔茨海默病的啮齿动物模型脑淀粉样蛋白-β吞噬（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

情节记忆的特点，采用事件相关电位研究在阿尔茨海默氏症患者

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

科研

医学

Quantitative Analysis of Chromatin Proteomes in Disease

在疾病的染色质蛋白质组的定量分析

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

基因-环境相互作用模型揭开帕金森病的隐形易感机制

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

造型中风小鼠：在远端大脑中动脉永久性混凝

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

人类癫痫术后皮层组织的多电极阵列录音

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

在多囊肾小型啮齿动物模型采用超高场磁共振成像<em&gt;在体内</em&gt;表型和药物监测

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

体外肠囊评估黏膜通透性在胃肠道疾病模型

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

肺动脉高压的啮齿动物模型的血流动力学特性

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

神经变性慢性淀粉样蛋白-β低聚物输注的动物模型是由抗体治疗灌注抵消与渗透泵

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

在阿尔茨海默病和轻度认知功能损害的定向功能连接格兰杰因果关系分析中的应用

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