Name: quantitative 3d in silico modeling (q3dism) of cerebral amyloid-beta phagocytosis in rodent models of alzheimer's disease
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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>通过肋骨的两侧切开并抬起以暴露心脏。插入一个23政针插入心脏的左心室和做一个小切口进入右心房。用冰冷的磷酸盐缓冲的盐水进行到放血由穿心灌注（PBS）中使用蠕动泵（30毫升小鼠; 150 - 200毫升为只大鼠）。 </li><li>使尾部中线切口进入皮肤并移动皮肤和肌肉一边。通过沿中线的头骨顶部和眼睛之间切割。去除骨板和从头骨隔离全脑。 </li><li>将脑入冠状啮齿动物的大脑矩阵切片到宿舍。在4℃孵育的多聚甲醛固定液（4％PFA的PBS）后宿舍O / N（16小时）。洗3倍的PBS，然后转移到70％乙醇。注意:PFA是有毒的，应在化学罩配备适当的个人防护装备进行处理。 </li></ol>第2天: <ol start="5"><li>脑宿舍放入嵌入盒并逐步在依次更浓缩1小时乙醇浴脱水组织（70％，80％，95％和100％×3）。 </li><li>从组织清除乙醇与三个连续100％二甲苯浴（各1小时）。注意:二甲苯是有毒的和sh下一个化学罩配备适当的个人防护装备乌尔德处理。 </li><li> （ - 58°C，每90分钟56）经过两次熔融石蜡浴嵌入石蜡块组织。 </li></ol>第3天: <ol start="8"><li>切使用切片机石蜡包埋脑10微米厚的切片。浸部分进入水浴（50℃，1分钟），并适用于显微镜载玻片。离开载片干燥O / N，从而确保在滑组织粘连。 </li></ol> 2.免疫染色注:抗体的不同组合可以用于下面描述的染色方法。从大鼠和小鼠脑组织相容抗体的鸡尾酒列于表1。 第4天: <ol><li>采用2个100％二甲苯浴Deparaffinize脑切片（各12分钟）。 </li><li>再水合脑切片在连续的乙醇浴 - 100％10分钟，95％5分钟，10分钟的80％，最后70％的15分钟 - followe通过3倍PBS洗涤[5分钟，在室温，用轻搅动] D。同时，热抗原修复溶液到95 - 97℃的热板上用磁棒搅拌。 </li><li>孵育在抗原修复溶液的脑切片，在95 - 97℃30分钟。然后，洗3倍的PBS（5分钟，RT，用轻轻搅拌条件）。 </li><li>快速干片用微妙的任务雨刷以避免组织干燥，并绘制组织周围地区的疏水屏障疏水屏障笔。用封闭缓冲液[PBS含有0.3％的Triton X-100和10％正常驴血清（NDS）]，并在室温在加湿室中孵育1小时填满包围组织区域。 </li><li>替换阻断与Iba1初级抗体缓冲液（封闭缓冲液稀释）在湿润的腔室来标记单核吞噬细胞，并孵育O / N在4℃。对于抗体的主机和工作稀释液， 见表1和材料的表 。 </li></ol>第五天: <ol start="6"><li>冲洗与3倍PBS浴初级抗体（室温下5分钟，轻轻搅拌条件）。与随后的3倍的PBS浴1小时的荧光第二抗体（用594 nm发射荧光团共轭）（以阻止在黑暗中在RT缓冲液）（在RT 5分钟，光搅动）孵育。此时，保持部分在黑暗中以避免荧光信号漂白。 </li></ol>第6天 - 7: <ol start="7"><li>重复步骤2.5 2.6 CD68（鼠脑）或LAMP1（小鼠组织）抗体和适当的二级抗体（再加上488 nm发射荧光）标记吞噬溶酶体。 </li></ol>第七天 - 8: <ol start="8"><li>重复步骤2.5 2.6 OC（大鼠组织）或4G8（老鼠大脑）抗体和适当的二级抗体（连接到647纳米荧光）标记Aβ存款。 注意:可替换地，6E10抗体可以成功地既用于对小鼠和大鼠的组织。对于相应的抗体组合，见材料的表 </str翁&gt;。 </li><li>允许部分完全干透，O / N在室温在黑暗中。然后，盖标本含有荧光DAPI安装介质密封盖玻片。 </li></ol> 3.大Z堆栈共聚焦数据集收购注:此协议需要配备一个60X的目标和405纳米，488纳米，594纳米和647纳米激光器的全自动激光扫描共聚焦显微镜。所有的设备是由计算机成像和激光控制软件控制。在此之前开始的成像协议，打开计算机电源，啶灯，显微镜，激光器和摄像头。 第9天: <ol><li>选择60X显微镜物镜。浸油添加到镜头，并放置到样本在显微镜阶段幻灯片固定器。提高目标，待油，使与滑动接触。调整焦平面定位通过目镜使用表面荧光照明在海马或大脑皮层淀粉样蛋白斑。 </li><li>收购焦IM周围的淀粉样蛋白沉积在海马或通过共聚焦显微镜的啮齿动物皮质活化单核吞噬细胞的年龄（60X放大倍率，Z堆叠步骤:为0.25μm&lt;Z &lt;0.40微米，步数25的&lt;n &lt;35）。 </li></ol> 4. q3DISM 注:为了产生显著的效果，我们建议分析至少为每个感兴趣的动物/地区3图像。对于每个图像，将细胞的丰度来分析可以根据实验范式而变化。在这份报告中显示的代表性的结果，我们分析了3个细胞/条件（ 例如 ，单核吞噬细胞从遥远或斑块有关;参见图1C - D和2C - D）。 第10天: <ol><li>分析用科学的3D图像处理和分析软件共定位（coloc）模块，用于在同时所有的z平面Iba1 / CD68（大鼠组织）或Iba1 / LAMP1（小鼠组织）染色的空间接近共焦数据集。创建伊巴+ / CD68 +或对应吞噬溶酶体激活单核吞噬细胞内伊巴+ / LAMP1 +共定位通道。 <ol><li>选择TRITC通道A（相应于加上594毫微米的荧光团Iba1染色）和FITC通道B（对应于CD68或加上488纳米的荧光团LAMP1染色）。在软件窗口中的"模式检查"，选择"门槛"，并为"coloc强度"，选择"源通道"的右侧。 </li><li>点击"编辑"来选择在软件窗口的右侧"coloc颜色"。 </li><li>对于每个通道独立地调整阈值，以包括特定染色和排除背景/非特异性信号。一旦调整，不改变图像之间的阈值，以确保公正的分析。在共定位体素（来自所有Z-堆栈像素），将出现在所有的Z-SIM卡堆在步骤4.1.2选择的颜色ultaneously。 </li><li>点击"建立coloc通道"。创建的共定位通道将出现在显示调整窗口。 </li><li>点击coloc通道打开通道的统计数据。在"高于共定位阈值体积/材料A的％"代表具有LAMP1或CD68的信号（对应于488纳米荧光体素）共定位Iba1信号（对应于594毫微米的荧光团体素）的％。更简单地说，这是由吞噬溶酶体（参见图1C和2C）占据的单核细胞的体积。 注:"共定位高于阈值音量/材料B％&#39;对应于Iba1 LAMP1共定位或CD68信号％。这应该是接近100％，成为吞噬溶酶体是细胞内的结构。值是基于所有的z栈高于阈值共定位信号的比值。 </li></ol></li><li>使用coloc模块，分析步骤4.1创造了空间接近与OC（大鼠TI的coloc通道ssue）或4G8（小鼠组织）Aβ的信号。这允许吞噬溶酶体内包封的Aβ的定量。 <ol><li>选择通道A coloc数据集（对应于Iba1 / CD68或在步骤4.1中创建Iba1 / LAMP1 coloc信道）和Cy5通道B（相应于OC或4G8染色加上647纳米的荧光团）。 </li><li>如步骤4.1.2描述4.1.5建立coloc通道。 </li><li>点击coloc通道打开通道的统计数据。在"高于共定位阈值体积/材料A的％"表示Iba1 / LAMP1或Iba1 / CD68以OC或4G8信号（对应于647体素共定位（对应于内置在步骤4.1.5的coloc通道体素）的％纳米荧光团）。这是Aβ（ 见图1D和2D）占据了phagolysosomal音量。 注:"共定位高于阈值音量/材料B％&#39;与吞噬溶酶体共定位总Aβ信号％对应。这可以用来评价吞噬溶酶体（在本代表结果未示出）封装在总的Aβ沉积的分数。 </li></ol></li><li>使用超过模块重建共焦图像栈和产生单核吞噬溶酶体内包封的Aβ3D模型。 <ol><li>在"显示调整"窗口中，选择TRITC（只显示Iba1染色）。在"卷属性"窗口中，单击"添加新的表面"。 </li><li>在步骤"1/5算法"，在"设置"，选择"面"。此外，在"色彩"，选择在调色板或RGB颜色类型和调整透明度（红是在图1B和2B设定为60％的透明度）。复选框"选择感兴趣的地区。点击下一步。 </li><li>在步骤"的兴趣2/5区域，"通过调节的x，y绘制的感兴趣的细胞周围的一个窗口和z坐标（参见图1A白框</stroNG&gt;和2A）。点击下一步。 </li><li>在步骤"3/5源通道"，选择TRITC源通道。勾选「顺利"，并设置面积细节水平至0.4微米。对于"阈值"，选择绝对强度。点击下一步。 </li><li>在步骤"4/5门槛"，调整门槛，使创建的卷与TRITC通道信号完全重叠。点击下一步。 </li><li>在步骤"5/5分类表面，&#39;节&#39;过滤器类型，"选择对象取决于它们的大小要包含或将要创建的卷排除在外。点击完成，执行所有的创建步骤，并退出向导。 </li><li>重复步骤4.3.1至4.3.6为FITC通道（LAMP1 + CD68或吞噬溶酶体+）创建一个三维表面。 </li><li>重复步骤4.3.1至4.3.6，为Cy5的通道建立3D表面（OC +或4G8 +Aβ存款）。 </li></ol></li></ol>

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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+cloning+of+CD68,+a+human+macrophage+marker+related+to+lysosomal+glycoproteins.">Molecular cloning of CD68, a human macrophage marker related to lysosomal glycoproteins.</a> Blood. 81 (6), 1607-1613 (1993).</li><li>Connor, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorylation+of+the+translation+initiation+factor+eIF2alpha+increases+BACE1+levels+and+promotes+amyloidogenesis.">Phosphorylation of the translation initiation factor eIF2alpha increases BACE1 levels and promotes amyloidogenesis.</a> Neuron. 60 (6), 988-1009 (2008).</li><li>Cai, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phospholipase+D1+corrects+impaired+betaAPP+trafficking+and+neurite+outgrowth+in+familial+Alzheimer's+disease-linked+presenilin-1+mutant+neurons.">Phospholipase D1 corrects impaired betaAPP trafficking and neurite outgrowth in familial Alzheimer's disease-linked presenilin-1 mutant neurons.</a> Proc Natl Acad Sci U S A. 103 (6), 1936-1940 (2006).</li><li>Marsh, S. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+adaptive+immune+system+restrains+Alzheimer's+disease+pathogenesis+by+modulating+microglial+function.">The adaptive immune system restrains Alzheimer's disease pathogenesis by modulating microglial function.</a> Proc Natl Acad Sci U S A. 113 (9), 1316-1325 (2016).</li><li>Lefterov, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apolipoprotein+A-I+deficiency+increases+cerebral+amyloid+angiopathy+and+cognitive+deficits+in+APP/PS1DeltaE9+mice.">Apolipoprotein A-I deficiency increases cerebral amyloid angiopathy and cognitive deficits in APP/PS1DeltaE9 mice.</a> J Biol. Chem. 285 (47), 36945-36957 (2010).</li><li>Blurton-Jones, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+stem+cells+improve+cognition+via+BDNF+in+a+transgenic+model+of+Alzheimer+disease.">Neural stem cells improve cognition via BDNF in a transgenic model of Alzheimer disease.</a> Proc Natl Acad Sci U S A. 106 (32), 13594-13599 (2009).</li><li>Stalder, M., Deller, T., Staufenbiel, M., Jucker, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D-Reconstruction+of+microglia+and+amyloid+in+APP23+transgenic+mice:+no+evidence+of+intracellular+amyloid.">3D-Reconstruction of microglia and amyloid in APP23 transgenic mice: no evidence of intracellular amyloid.</a> Neurobiol Aging. 22 (3), 427-434 (2001).</li><li>Leinenga, G., G&#246;tz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scanning+ultrasound+removes+amyloid-&#946;+and+restores+memory+in+an+Alzheimer's+disease+mouse+model.">Scanning ultrasound removes amyloid-&#946; and restores memory in an Alzheimer's disease mouse model.</a> Sci Transl Med. 7 (278), 33 (2015).</li><li>Liarski, V. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+distance+mapping+identifies+functional+T+follicular+helper+cells+in+inflamed+human+renal+tissue.">Cell distance mapping identifies functional T follicular helper cells in inflamed human renal tissue.</a> Sci Transl Med. 6 (230), 46 (2014).</li><li>Nichols, L., Pike, V. W., Cai, L., Innis, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+and+in+vivo+quantitation+of+beta-amyloid:+an+exemplary+biomarker+for+Alzheimer's+disease.">Imaging and in vivo quantitation of beta-amyloid: an exemplary biomarker for Alzheimer's disease.</a> Biol Psychiatry. 59 (10), 940-947 (2006).</li><li>Skovronsky, D. M., Zhang, B., Kung, M. P., Kung, H. F., Trojanowski, J. Q., Lee, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+detection+of+amyloid+plaques+in+a+mouse+model+of+Alzheimer's+disease.">In vivo detection of amyloid plaques in a mouse model of Alzheimer's disease.</a> Proc Natl Acad Sci U S A. 97 (13), 7609-7614 (2000).</li><li>Lian, H., Litvinchuk, A., Chiang, A. C. -. A., Aithmitti, N., Jankowsky, J. L., Zheng, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocyte-Microglia+Cross+Talk+through+Complement+Activation+Modulates+Amyloid+Pathology+in+Mouse+Models+of+Alzheimer's+Disease.">Astrocyte-Microglia Cross Talk through Complement Activation Modulates Amyloid Pathology in Mouse Models of Alzheimer's Disease.</a> J Neurosci. 36 (2), 577-589 (2016).</li><li>Novotny, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conversion+of+Synthetic+A&#946;+to+In+Vivo+Active+Seeds+and+Amyloid+Plaque+Formation+in+a+Hippocampal+Slice+Culture+Model.">Conversion of Synthetic A&#946; to In Vivo Active Seeds and Amyloid Plaque Formation in a Hippocampal Slice Culture Model.</a> J Neurosci. 36 (18), 5084-5093 (2016).</li><li>Tartaro, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+fluorescence-based+in+vivo+phagocytosis+assay+to+measure+mononuclear+phagocyte+system+function+in+the+rat.">Development of a fluorescence-based in vivo phagocytosis assay to measure mononuclear phagocyte system function in the rat.</a> J Immunotoxicol. 12 (3), 239-246 (2015).</li></ol>

作者什么都没有透露。

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。

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

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

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 ...

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

Research

JoVE Journal

Medicine

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

7.9K 次观看.  University of Southern California (USC). 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。 

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

Detection of Neuritic Plaques in Alzheimer's Disease Mouse Model

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

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

在阿尔茨海默氏症小鼠模型神经炎斑检测

Alzheimer's disease (AD) is the most common neurodegenerative disorder leading to dementia. Neuritic plaque formation is one of the pathological hallmarks ...

科研

神经科学

Establishment of a Valuable Mimic of Alzheimer's Disease in Rat Animal Model by Intracerebroventricular Injection of Composited Amyloid Beta Protein

Alzheimer&#39;s disease (AD) is an irreversible, progressive brain disease that slowly destroys memory and is accompanied by neuron loss and structure change. With the increase of AD patients worldwide, the pathology and treatment of the disease has become a focus in the International Pharmaceutical Industry. Thus, the establishment of the animal model to mimic AD in the laboratory is of great importance.
Here, we describe a detailed protocol for establishing a mimic of AD in a rat animal model though intracerebroventricular injection of amyloid beta protein 25-35 (A&#946;25-35) combined with aluminum trichloride (AlCl3) and anterodorsal thalamic nucleus injection of recombinant human transforming growth factor-&#946;1 (RHTGF-&#946;1) to rats. The related markers of AD were measured, including: spatial memory, neuronal structure and substructure, neuronal A&#946;, and neurofibrillary tangles (NFT) production. This rat model demonstrates spatial memory impairment, neuronal structure and substructure pathological changes, neuron intracellular A&#946; burden, and NFT aggregation, and provides a close mimic of the neuronal structure and function disorder to that of clinical AD patients. Thus, the presented AD rat modelprovides a valuable in vivo tool for exploring neuronal function, neuronal pathology, and drug screening of AD.

This is a protocol to mimic Alzheimer&#39;s Disease in rats by evaluation of spatial memory impairment, neuronal pathological changes, neuronal amyloid beta protein (A&#946;) burden, and neurofibrillary tangles aggregation, induced by the injection of A&#946;25-35 combined with aluminum trichloride and recombinant human transforming growth factor-&#946;1.

脑室注射复合淀粉样蛋白β模型建立大鼠阿尔茨海默病的价值模拟

Alzheimer&#39;s disease (AD) is an irreversible, progressive brain disease that slowly destroys memory and is accompanied by neuron loss and structure ...

行为学

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.

低聚淀粉样蛋白-β的立体灌注到小鼠海马

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

Intracerebroventricular Injection of Amyloid-&#946; Peptides in Normal Mice to Acutely Induce Alzheimer-like Cognitive Deficits

Amyloid-&#946; (A&#946;) is a major pathological mediator of both familial and sporadic Alzheimer's disease (AD). In the brains of AD patients, progressive accumulation of A&#946; oligomers and plaques is observed. Such A&#946; abnormalities are believed to block long-term potentiation, impair synaptic function, and induce cognitive deficits. Clinical and experimental evidences have revealed that the acute increase of A&#946; levels in the brain allows development of Alzheimer-like phenotypes. Hence, a detailed protocol describing how to acutely generate an AD mouse model via the intracerebroventricular (ICV) injection of A&#946; is necessary in many cases. In this protocol, the steps of the experiment with an A&#946;-injected mouse are included, from the preparation of peptides to the testing of behavioral abnormalities. The process of preparing the tools and animal subjects before the injection, of injecting the A&#946; into the mouse brain via ICV injection, and of assessing the degree of cognitive impairment are easily explained throughout the protocol, with an emphasis on tips for effective ICV injection of A&#946;. By mimicking certain aspects of AD with a designated injection of A&#946;, researchers can bypass the aging process and focus on the downstream pathology of A&#946; abnormalities.

The amyloid-&#946; (A&#946;)-injected animal model enables the administration of a defined quantity and species of A&#946; fragments and reduces individual differences within each study group. This protocol describes the intracerebroventricular (ICV) injection of A&#946; without stereotactic instruments, enabling the production of Alzheimer-like behavioral abnormalities in normal mice.

正常小鼠β淀粉样肽侧脑室注射敏锐地诱导老年痴呆样认知缺陷

Amyloid-&#946; (A&#946;) is a major pathological mediator of both familial and sporadic Alzheimer's disease (AD). In the brains of AD patients, ...

Assessment of Spontaneous Alternation, Novel Object Recognition and Limb Clasping in Transgenic Mouse Models of Amyloid-&#946; and Tau Neuropathology

Here we describe a staged, behavioral testing approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional motor behaviors in transgenic mouse models of &#946;-amyloidosis and tauopathy. The paradigm includes tests for spontaneous alternation in a Y-maze, novel object recognition, and limb clasping. These tests were selected because they: 1) interrogate function of cognitive or motor domains and the correlate neural circuitry relevant to the human disease state, 2) have clearly defined endpoints, 3) have easily implementable quality control checks, 4) can be run in a moderate throughput format, and 5) require little intervention by the investigator. These methods are designed for investigators looking to screen compounds for activity in short-term and working memory tasks, or functional motor behaviors associated with Alzheimer's disease mouse models. The methods described here use behavioral tests that engage a number of different brain regions including hippocampus and various cortical areas. Investigators that desire cognitive tests that specifically assess cognition mediated by a single brain region could use these techniques to supplement other behavioral tests.

Described here is a staged, behavioral screening approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional motor behaviors in transgenic mouse models of &#946;-amyloidosis and tauopathy. These methods are optimized to screen compounds for activity in short-term and working memory tasks.

自发交替评估，淀粉样蛋白β和脑神经病理学转基因小鼠模型中的新物体识别和肢体紧扣

Here we describe a staged, behavioral testing approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional ...

医学

Detecting Amyloid-&#946; Accumulation via Immunofluorescent Staining in a Mouse Model of Alzheimer's Disease

Alzheimer's disease (AD) is a neurodegenerative disease that contributes to 60-70% dementia around the world. One of the hallmarks of AD undoubtedly lies on accumulation of amyloid-&#946; (A&#946;) in the brain. A&#946; is produced from the proteolytic cleavage of the beta-amyloid precursor protein (APP) by &#946;-secretase and &#947;-secretase. In pathological circumstances, the increased &#946;-cleavage of APP leads to overproduction of A&#946;, which aggregates into A&#946; plaques. Since A&#946; plaques are a characteristic of AD pathology, detecting the amount of A&#946; is very important in AD research. In this protocol, we introduce the immunofluorescent staining method to visualize A&#946; deposition. The mouse model used in our experiments is 5&#215;FAD, which carries five mutations found in human familial AD. The neuropathological and behavioral deficits of 5xFAD mice are well-documented, which makes it a good animal model to study A&#946; pathology. We will introduce the procedure including transcardial perfusion, cryosectioning, immunofluorescent staining and quantification to detect A&#946; accumulation in 5&#215;FAD mice. With this protocol, researchers can investigate A&#946; pathology in an AD mouse model.

In the neuropathology of Alzheimer's disease, one of the most crucial characteristics is the deposition of amyloid-&#946;. In this protocol, we describe the method of immunofluorescent staining in 5&#215;FAD transgenic mouse to detect amyloid-&#946; accumulation in plaques. The process of perfusion, cryosectioning, staining and quantification will be described in detail.

Alzheimer's disease (AD) is a neurodegenerative disease that contributes to 60-70% dementia around the world. One of the hallmarks of AD undoubtedly lies ...

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

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

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

小鼠脑切片上淀粉样蛋白-β负荷的全区域与亚区域定量

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

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

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

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

制造淀粉样蛋白-β-分泌阿尔金酸微珠,用于模拟阿尔茨海默氏病

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

生物学

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

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

通过淀粉样蛋白-β 肽注射建立阿尔茨海默病大鼠模型

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

JoVE Encyclopedia of Experiments

Neuroscience

Neuropathology

Neurodegenerative Diseases

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

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

定量3D

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