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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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。细胞内吞噬溶酶体都透露出与抗体识别溶酶体/内体相关的膜糖蛋白（LAMP）家族的成员:LAMP1 20或21 CD68。后者主要定位于溶酶体和内涵体，用较小的级分循环到该单元Surface。 LAMP1和CD68抗体都在大鼠和小鼠组织被成功地使用，并在其上使用该决定主要取决于用于共染色（ 见表1）的其他抗体的主机上。要知道，各种抗体可用于检测Aβ的是重要的。在小鼠组织，4G8（识别人和小鼠的氨基酸残基17 - Aβ肽22， 图1的24）和6E10（识别人氨基酸残基1 - 17 23，数据未示出）已经被成功地使用，允许检测和定量在LAMP1 + CD68或+目前在Iba1 +单核吞噬细胞吞噬溶酶体Aβ的内容。 / - -使用我们q3DISM方法，我们在与APPPS1 / IL-10的大脑Aβ负荷的全球降低相关Iba1 +细胞中观察到增加的Aβ负荷小鼠16，而其他表现出降低Aβ的摄取到Iba1 + &lt;<html> / SUP&gt;伴随RAG-5xfAD小鼠24的大脑增加负荷Aβ和斑块体积的细胞。这些结果表明，所观察到的现象是一个活跃的Aβ吞噬的"快照"，虽然我们的方法不能用来预测如果吞噬材料被有效地消化和清除。 在大鼠组织中，我们发现使用OCAβ沉积物（识别可溶性Aβ42寡聚体/纤维25,26， 图2）或6E10 17（未示出）。有趣的是，很少OC +原纤维存在于TgF344-AD鼠脑单核吞噬溶酶体。这种现象以前已报道在使用免疫染色及三维重建27 APP23转基因小鼠。注意:为检测不同Aβ种类/构象的多种抗体可用，并可以潜在地测试，并在用户决定使用。 &gt;我们q3DISM技术已通过我们和其他核苷酸"量化公元16,24,28的上下文中的Aβ吞噬作用。然而，该方法可以适于评估几乎任何形式的吞噬作用，它也可以被应用到组织以外的脑，以及其他动物物种（包括人类）。在这个协议中所描述的程序依赖于标准的免疫染色，用荧光检测，并用共聚焦显微镜成像。在本报告中，我们根据准则已经使用石蜡包埋的组织通过将抗体生产商提供的。如果需要的话，本协议也可以为新鲜组织（嵌入在PBS 2％琼脂糖）进行了优化。该协议还可以被优化以检测coaggregating蛋白的吞噬作用，这是通过染色和获取成为可能图像组成的4个以上的荧光色29，在这种情况下，第一共定位通道（单核细胞/吞噬溶酶体）的创建之后将创建第二共存通道（聚集蛋白1 /共聚集蛋白2）。然后，这两个通道将用于三维分析和建模。 该q3DISM技术由特异性和免疫染色，抗体的质量的灵敏度，和共定位的方法，主要的限制。这个多步过程具有可以影响染色的质量，因此，Aβ摄取三维定量几个潜在的隐患。该过程的第一个关键步骤是啮齿动物的大脑的隔离。事实上，细心的组织处理是从根本上避免脑损伤，并保证切片时，大脑结构保持不变。第二，组织固定时间是非常重要的，因为过度的固定（在4％PFA超过16小时）限制的亚细胞吞噬溶酶体和Aβ含量的检测。重要的是，1）单核细胞，2）吞噬溶酶体连续染色和3）将Aβ是用于该方法的成功是至关重要的。这是埃森特IAL使用抗体consecutively-不伴随。另外，采集的z堆共焦图象是非常重要的，因为细胞，吞噬溶酶体和Aβ肽3D建模以及共定位分析依赖于细胞和亚细胞结构的成像的穿过组织部分的厚度的质量。最后，在软件的阈值的调整是用于3D建模和共定位分析至关重要。事实上，需要仔细选择，因为它们将确定被包括哪些信号（特定的）或排除（背景）从分析对不同信道的阈值。它也是至关重要的是，所选择的阈值在不同的动物/标本的比较的情况下，样本之间保持一致。到目前为止，古典immunohistostaining，磁共振成像和正电子发射断层摄影术已经首选的Aβ 体内可视化和定量的成像方法30。虽然对于在啮齿类动物或AD患者脑中淀粉样蛋白负荷的大型定量非常有用的，这些方法是低效的细胞内的Aβ可视化。使用共聚焦显微镜，荧光光谱和流动的其他技术术存在并已被用于我们和其他人以鉴别和体外 16,31,32量化细胞内的Aβ内容。再加三维重建免疫染色以前用于突出的小胶质细胞内体内 APP23转基因小鼠27不存在淀粉样蛋白原纤维的，和另一项研究中成功地使用小胶质细胞相关与β淀粉样斑块的共焦成像和三维表面重建显示有限的Aβ互动和摄取33。然而，这些技术不允许帧内phagolysosomalAβ种类的定量。更为最近开发的基于荧光体内测定允许测量在大鼠34由外周巨噬细胞的吞噬活性。此方法巧妙允许在体内吞噬溶酶体荧光生物探针的定量，但是，现有的数据迄今被限制在外围。 据我们所知，q3DISM是能提供三维可视化和Aβ吞噬定量啮齿动物的AD模型的大脑的第一个方法。这个强大的工具无疑将铺平道路脑Aβ吞噬有了更深的了解，也可以证明在其他情况下非常有用。

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>
		</tr>
		<tr height="20">
			<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>
		</tr>
		<tr height="20">
			<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>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Tweezers</td>
			<td style="width:152px;">VWR</td>
			<td>94024-408</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">23G needle</td>
			<td style="width:152px;">VWR</td>
			<td>BD305145</td>
			<td></td>
		</tr>
		<tr height="20">
			<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>
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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>
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			<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>
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		<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>
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			<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>
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			<td height="20" style="height:20px;width:384px;">Microtome Leica RM2125</td>
			<td style="width:152px;">Leica Biosystems</td>
			<td style="width:127px;"></td>
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			<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>
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			<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>
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		<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 +单核吞噬细胞是显著增加。这些数据表明，靠近斑块单核吞噬细胞被蓄势吞噬作用相比距离斑块远离细胞。为了说明从染色不同的Aβ构象数据的范围，我们使用各种抗体在小鼠和大鼠组织（在钽详述竹叶提取1和讨论） -我们不寻求直接比较广告的小鼠和大鼠模型的问候Aβ吞噬。尽管如此，这是值得注意的斑块相关的单核吞噬细胞已在APP / PS1小鼠相比远离斑块（ 图1D）细胞增多Aβ摄取。 TgF344-AD大鼠具有Aβ原纤维的非常温和的摄取整体，并在Aβ原纤维吞噬作为距离的函数从斑没有变化（ 图2D）。 <img alt="表格1" src="/files/ftp_upload/54868/54868table1.jpg" /> 表1:免疫组化染色单核吞噬细胞，吞噬溶酶体和β淀粉样从APP / PS1小鼠或TgF344-AD大鼠脑组织。大鼠或小鼠特异性抗体鸡尾酒列出并在括号中各自的主机。颜色指示用于第二抗体的荧光团。红色:的Alexa Fluor 594;绿色:的Alexa Fluor 488;品红:的Alexa Fluor 647。 <img alt="图1" src="/files/ftp_upload/54868/54868fig1.jpg" /> 图1:可视化，并在APP / PS1小鼠脑内β淀粉样吞噬的定量。 （A）从APP / PS1小鼠大脑切片显示Iba1 +细胞（红色），LAMP1 +吞噬溶酶体（绿色）和4G8 +淀粉样蛋白沉积（品红色）的共聚焦图像。插图1突出了单核吞噬细胞远离斑块，而插图2显示了与斑块相关的单核吞噬细胞。比例尺表示50微米。右边较小的面板是Iba1，LAMP1的放大和插图1和Iba1，LAMP1 2.（B）三维重建中4G8信号和4G8信号插图1和2比例尺代表3微米。通过LAMP1 +吞噬溶酶体占用Iba1 +单核吞噬细胞体积（C）的定量。 （ <str翁&gt; D）通过4G8 +Aβ占据细胞内LAMP1 + phagolysosomal量的定量。数值基于上述阈值的信号（像素）的比例被共定位。数据显示为平均值±平均值（N = 6个细胞）为远离斑块单核吞噬细胞的标准误差（1），或用斑有关（2）。 ** P &lt;0.01，通过Student t检验。 <a href="http://ecsource.jove.com/files/ftp_upload/54868/54868fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54868/54868fig2.jpg" /> 图2:可视化和β淀粉样吞噬的定量在TgF344-AD鼠脑。 （A）从TgF344-AD鼠脑切片显示Iba1 +细胞的共聚焦图像（红色），CD68 +吞噬溶酶体（绿色）和OC +可溶性原纤维＆＃<html> 946; （品红）。插图1突出了单核吞噬细胞远离斑块，而插图2显示了与斑块相关的单核吞噬细胞。比例尺表示50微米。右边较小的面板插图1和Iba1，CD68的2（B）三维重建和OC信号插图1和2比例尺内Iba1，CD68的放大和OC信号表示3微米。通过CD68 +吞噬溶酶体占用Iba1 +单核吞噬细胞体积（C）的定量。通过OC +Aβ占据细胞内CD68 + phagolysosomal量（D）定量。数值基于上述阈值的信号（像素）的比例被共定位。数据显示为平均值±平均值（N = 6个细胞）为远离斑块单核吞噬细胞的标准误差（1），或用斑有关（2）。 ** P &lt;0.01，通过Student t检验。rge.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。

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

Nanyang Technological University

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

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

Our laboratory uses event-related EEG potentials (ERPs) to understand and support behavioral investigations of episodic memory in patients with amnestic mild cognitive impairment (aMCI) and Alzheimer's disease (AD). Whereas behavioral data inform us about the patients' performance, ERPs allow us to record discrete changes in brain activity. Further, ERPs can give us insight into the onset, duration, and interaction of independent cognitive processes associated with memory retrieval. In patient populations, these types of studies are used to examine which aspects of memory are impaired and which remain relatively intact compared to a control population. The methodology for collecting ERP data from a vulnerable patient population while these participants perform a recognition memory task is reviewed. This protocol includes participant preparation, quality assurance, data acquisition, and data analysis. In addition to basic setup and acquisition, we will also demonstrate localization techniques to obtain greater spatial resolution and source localization using high-density (128 channel) electrode arrays.

The methodology for collecting high-density event-related potential data while patients with Alzheimer's disease perform a recognition memory task is reviewed. This protocol will include subject preparation, quality assurance, data acquisition, and data analysis.

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

In the nucleus reside the proteomes whose functions are most intimately linked with gene regulation. Adult mammalian cardiomyocyte nuclei are unique due to the high percentage of binucleated cells,1 the predominantly heterochromatic state of the DNA, and the non-dividing nature of the cardiomyocyte which renders adult nuclei in a permanent state of interphase.2 Transcriptional regulation during development and disease have been well studied in this organ,3-5 but what remains relatively unexplored is the role played by the nuclear proteins responsible for DNA packaging and expression, and how these proteins control changes in transcriptional programs that occur during disease.6 In the developed world, heart disease is the number one cause of mortality for both men and women.7 Insight on how nuclear proteins cooperate to regulate the progression of this disease is critical for advancing the current treatment options.
Mass spectrometry is the ideal tool for addressing these questions as it allows for an unbiased annotation of the nuclear proteome and relative quantification for how the abundance of these proteins changes with disease. While there have been several proteomic studies for mammalian nuclear protein complexes,8-13 until recently14 there has been only one study examining the cardiac nuclear proteome, and it considered the entire nucleus, rather than exploring the proteome at the level of nuclear sub compartments.15 In large part, this shortage of work is due to the difficulty of isolating cardiac nuclei. Cardiac nuclei occur within a rigid and dense actin-myosin apparatus to which they are connected via multiple extensions from the endoplasmic reticulum, to the extent that myocyte contraction alters their overall shape.16 Additionally, cardiomyocytes are 40% mitochondria by volume17 which necessitates enrichment of the nucleus apart from the other organelles. Here we describe a protocol for cardiac nuclear enrichment and further fractionation into biologically-relevant compartments. Furthermore, we detail methods for label-free quantitative mass spectrometric dissection of these fractions-techniques amenable to in vivo experimentation in various animal models and organ systems where metabolic labeling is not feasible.

Advances in mass spectrometry have allowed the high throughput analysis of protein expression and modification in a host of tissues. Combined with subcellular fractionation and disease models, quantitative mass spectrometry and bioinformatics can reveal new properties in biological systems. The method described herein analyzes chromatin-associated proteins in the setting of heart disease and is readily applicable to other in vivo models of human 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 ...

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

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical options are available for a small proportion of stroke patients in the acute phase. Current research is intensively searching for novel therapeutic strategies and is increasingly focusing on the sub-acute and chronic phase after stroke because more patients might be eligible for therapeutic interventions in a prolonged time window. These delayed mechanisms include important pathophysiological pathways such as post-stroke inflammation, angiogenesis, neuronal plasticity and regeneration. In order to analyze these mechanisms and to subsequently evaluate novel drug targets, experimental stroke models with clinical relevance, low mortality and high reproducibility are sought after. Moreover, mice are the smallest mammals in which a focal stroke lesion can be induced and for which a broad spectrum of transgenic models are available. Therefore, we describe here the mouse model of transcranial, permanent coagulation of the middle cerebral artery via electrocoagulation distal of the lenticulostriatal arteries, the so-called &#8220;coagulation model&#8221;. The resulting infarct in this model is located mainly in the cortex; the relative infarct volume in relation to brain size corresponds to the majority of human strokes. Moreover, the model fulfills the above-mentioned criteria of reproducibility and low mortality. In this video we demonstrate the surgical methods of stroke induction in the &#8220;coagulation model&#8221; and report histological and functional analysis tools.

Various murine models of middle cerebral artery occlusion (MCAO) are widely used in experimental brain research. Here, we demonstrate the model of transcranial permanent distal MCAO which produces consistent cortical infarction of a size corresponding to damage imposed by the majority of human ischemic strokes.

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

Epilepsy, affecting about 1% of the population, comprises a group of neurological disorders characterized by the periodic occurrence of seizures, which disrupt normal brain function. Despite treatment with currently available antiepileptic drugs targeting neuronal functions, one third of patients with epilepsy are pharmacoresistant. In this condition, surgical resection of the brain area generating seizures remains the only alternative treatment. Studying human epileptic tissues has contributed to understand new epileptogenic mechanisms during the last 10 years. Indeed, these tissues generate spontaneous interictal epileptic discharges as well as pharmacologically-induced ictal events which can be recorded with classical electrophysiology techniques. Remarkably, multi-electrode arrays (MEAs), which are microfabricated devices embedding an array of spatially arranged microelectrodes, provide the unique opportunity to simultaneously stimulate and record field potentials, as well as action potentials of multiple neurons from different areas of the tissue. Thus MEAs recordings offer an excellent approach to study the spatio-temporal patterns of spontaneous interictal and evoked seizure-like events and the mechanisms underlying seizure onset and propagation. Here we describe how to prepare human cortical slices from surgically resected tissue and to record with MEAs interictal and ictal-like events ex vivo.

We here describe how to perform multi-electrode array recordings of human epileptic cortical tissue. Epileptic tissue resection, slice preparation and multi-electrode array recordings of interictal and ictal events are demonstrated in detail.

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

Several in vivo pre-clinical studies in Polycystic Kidney Disease (PKD) utilize orthologous rodent models to identify and study the genetic and molecular mechanisms responsible for the disease, and are very convenient for rapid drug screening and testing of promising therapies. A limiting factor in these studies is often the lack of efficient non-invasive methods for sequentially analyzing the anatomical and functional changes in the kidney. Magnetic resonance imaging (MRI) is the current gold standard imaging technique to follow autosomal dominant polycystic kidney disease (ADPKD) patients, providing excellent soft tissue contrast and anatomic detail and allowing Total Kidney Volume (TKV) measurements.A major advantage of MRI in rodent models of PKD is the possibility for in vivo imaging allowing for longitudinal studies that use the same animal and therefore reducing the total number of animals required. In this manuscript, we will focus on using Ultra-high field (UHF) MRI to non-invasively acquire in vivo images of rodent models for PKD. The main goal of this work is to introduce the use of MRI as a tool for in vivo phenotypical characterization and drug monitoring in rodent models for PKD.

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

The use of ultra-high field MRI as a non-invasive way to obtain phenotypic information of rodent models for polycystic kidney disease and to monitor interventions is described. Compared with the traditional histological approach, MRI images can be acquired in vivo, allowing for longitudinal follow-up.

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 ...

Hemodynamic Characterization of Rodent Models of Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) is a rare disease of the pulmonary vasculature characterized by endothelial cell apoptosis, smooth muscle proliferation and obliteration of pulmonary arterioles. This in turn results in right ventricular (RV) failure, with significant morbidity and mortality. Rodent models of PAH, in the mouse and the rat, are important for understanding the pathophysiology underlying this rare disease. Notably, different models of PAH may be associated with different degrees of pulmonary hypertension, RV hypertrophy and RV failure. Therefore, a complete hemodynamic characterization of mice and rats with PAH is critical in determining the effects of drugs or genetic modifications on the disease. 
Here we demonstrate standard procedures for assessment of right ventricular function and hemodynamics in both rat and mouse PAH models. Echocardiography is useful in determining RV function in rats, although obtaining standard views of the right ventricle is challenging in the awake mouse. Access for right heart catheterization is obtained by the internal jugular vein in closed-chest mice and rats. Pressures can be measured using polyethylene tubing with a fluid pressure transducer or a miniature micromanometer pressure catheter. Pressure-volume loop analysis can be performed in the open chest. After obtaining hemodynamics, the rodent is euthanized. The heart can be dissected to separate the RV free wall from the left ventricle (LV) and septum, allowing an assessment of RV hypertrophy using the Fulton index (RV/(LV+S)). Then samples can be harvested from the heart, lungs and other tissues as needed.

Pulmonary arterial hypertension (PAH) is a disease of pulmonary arterioles that leads to their obliteration and the development of right ventricular failure. Rodent models of PAH are critical in understanding the pathophysiology of PAH. Here we demonstrate hemodynamic characterization, with right heart catheterization and echocardiography, in the mouse and rat.

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

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 is well established that synapse loss and ensuing neurodegeneration are the best predictors for memory impairments in AD. Latest studies have emphasized the neurotoxic role of soluble amyloid-beta oligomers (A&#946;o) that begin to accumulate in the human brain approximately 10 to 15 yr before the clinical symptoms become apparent. Many reports indicate that soluble A&#946;o correlate with memory deficits in AD models and humans. The A&#946;o-induced neurodegeneration observed in neuronal and brain slice cultures has been more challenging to reproduce in many animal models. The model of repeated A&#946;o infusions shown here overcome this issue and allow addressing two key domains for developing new disease modifying therapies: identify biological markers to diagnose early AD, and determine the molecular mechanisms underpinning A&#946;o-induced memory deficits at the onset of AD. Since soluble A&#946;o aggregate relatively fast into insoluble A&#946; fibrils that correlate poorly with the clinical state of patients, soluble A&#946;o are prepared freshly and injected once per day during six days to produce marked cell death in the hippocampus. We used cannula specially design for simultaneous infusions of A&#946;o and continuous infusion of A&#946;o antibody (6E10) in the hippocampus using osmotic pumps. This innovative in vivo method can now be used in preclinical studies to validate the efficiency of new AD therapies that might prevent the deposition and neurotoxicity of A&#946;o in pre-dementia patients.

To study the effects of A&#946;o in vivo, we developed a model based on repeated hippocampal infusions of soluble A&#946;o coupled with continuous infusion of A&#946;o antibody (6E10) in the hippocampus using osmotic pumps to counteract the neurotoxic effect of A&#946;o.

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 ...

Repeated Measurement of Respiratory Muscle Activity and Ventilation in Mouse Models of Neuromuscular Disease

Accessory respiratory muscles help to maintain ventilation when diaphragm function is impaired. The following protocol describes a method for repeated measurements over weeks or months of accessory respiratory muscle activity while simultaneously measuring ventilation in a non-anesthetized, freely behaving mouse. The technique includes the surgical implantation of a radio transmitter and the insertion of electrode leads into the scalene and trapezius muscles to measure the electromyogram activity of these inspiratory muscles. Ventilation is measured by whole-body plethysmography, and animal movement is assessed by video and is synchronized with electromyogram activity. Measurements of muscle activity and ventilation in a mouse model of amyotrophic lateral sclerosis are presented to show how this tool can be used to investigate how respiratory muscle activity changes over time and to assess the impact of muscle activity on ventilation. The described methods can easily be adapted to measure the activity of other muscles or to assess accessory respiratory muscle activity in additional mouse models of disease or injury.

This paper introduces a method for repeated measurements of ventilation and respiratory muscle activity in a freely behaving amyotrophic lateral sclerosis (ALS) mouse model throughout disease progression with whole-body plethysmography and electromyography via an implanted telemetry device.

在神经肌肉疾病的模型小鼠呼吸肌肉活动和通风的重复测量

Accessory respiratory muscles help to maintain ventilation when diaphragm function is impaired. The following protocol describes a method for repeated ...

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

Impaired functional connectivity in the Default Mode Network (DMN) may be involved in the progression of Alzheimer&#39;s Disease (AD). The Posterior Cingulate Cortex (PCC) is a potential imaging marker for monitoring the progression of AD. Previous studies did not focus on the functional connectivity between the PCC and nodes in regions outside the DMN, but our study is an effort to explore these overlooked functional connections. For collecting data, we used functional Magnetic Resonance Imaging (fMRI) and Granger Causality Analysis (GCA). fMRI provides a non-invasive method for studying the dynamic interactions between the different brain regions. GCA is a statistical hypothesis test for determining whether one-time series is useful in forecasting another. In simple terms, it is judged by comparing the &#34;Known all the information on the last moment, the distribution of the probability of X at this time&#34; and the &#34;Known all the information on the last moment except Y, the distribution of the probability of X at this time&#34;, to determine whether there is a causal relationship between Y and X. This definition is based on the complete information source and stationary chronological sequence. The main step of this analysis is to use X and Y to establish the regression equation and draw a causal relationship by a hypothetical test. Since GCA can measure causal effects, we used it to investigate the anisotropy of the functional connectivity and explore the hub function of the PCC. Here, we screened 116 participants for MRI scanning, and after preprocessing the data obtained from neuroimaging, we used GCA to derive the causal relationship of each node. Finally, we concluded that the directed connection is significantly different between the Mild Cognitive Impairment (MCI) and AD groups, both from the PCC to the whole brain and from the whole brain to the PCC.

Based on resting-state functional magnetic resonance imaging with Granger causality analysis, we investigated the alterations in the directed functional connectivity between the posterior cingulate cortex and whole brain in patients with Alzheimer&#39;s Disease (AD), patients with Mild Cognitive Impairment (MCI), and healthy controls.

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 ...

Assessment of Kidney Function in Mouse Models of Glomerular Disease

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function in diabetic nephropathy and podocyte-specific VEGF-A knock-out mice; therefore, this protocol describes the full kidney work-up used in our lab to assess these mouse models of glomerular disease, enabling a vast amount of information regarding kidney and glomerular function to be obtained from a single mouse. In comparison to alternative methods presented in the literature to assess glomerular function, the use of the method outlined in this paper enables the glomerular phenotype to be fully evaluated from multiple aspects. By using this method, the researcher can determine the kidney phenotype of the model and assess the mechanism as to why the phenotype develops. This vital information on the mechanism of disease is required when examining potential therapeutic avenues in these models. The methods allow for detailed functional assessment of the glomerular filtration barrier through measurement of the urinary albumin creatinine ratio and individual glomerular water permeability, as well as both structural and ultra-structural examination using the Periodic Acid Schiff stain and electron microscopy. Furthermore, analysis of the genes dysregulated at the mRNA and protein level enables mechanistic analysis of glomerular function. This protocol outlines the generic but adaptable methods that can be applied to all mouse models of glomerular disease.

This protocol describes a full kidney work-up that should be carried out in mouse models of glomerular disease. The methods allow for detailed functional, structural, and mechanistic analysis of glomerular function, which can be applied to all mouse models of glomerular disease.

小鼠肾小球疾病模型肾脏功能的评价

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function ...

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