Name: intracerebroventricular injection of amyloid-β peptides in normal mice to acutely induce alzheimer-like cognitive deficits
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Description: Watch this Scientific Journal Video about Intracerebroventricular Injection of Amyloid-ÃŽÂ² Peptides in Normal Mice to Acutely Induce Alzheimer-like Cognitive Deficits at JoVE.com

这项研究是由通过韩国保健产业振兴院（KHIDI），由卫生部和福利，韩国（:H14C04660000授权号）资助的韩国保健技术研发项目的资助。

所有的动物实验均按照健康指南实验动物的护理和使用的国家机构进行（NIH出版物号8023，修订1978），并与机构的动物护理和使用委员会的动物护理和使用委员会KIST（韩国首尔）。 1.动物的制备<ol><li>准备一组7周龄雄性ICR小鼠（或C57BL / 6）。 </li><li>让老鼠经过驯化过程3-7天运输后恢复动态平衡。在一般情况下，把一组4-5只在每个笼子并予以保持在22-23℃和40％湿度，用12/12小时亮/暗循环。提供的水和食物随意 。 注:驯化过程中是至关重要的，以允许从航运和适应新的住房，食物，水的压力恢复，嗅觉以及一个新的笼子和光照周期。在该实验中，小鼠被安置在位于所述的Spe独立通气笼（IVCS）cific病原（SPF）设施-grade </li><li>称量每只小鼠和排除受试者的重量从平均偏离±10％。 </li><li>划分受试者分为两组:一媒介物注射组（Aβ（ - ））和Aβ注射组（Aβ（+））。如果该实验是测试特定候选药物的疗效，划分小鼠分成四组:（1）将Aβ（ - ）/药物（ - ），（2）将Aβ（ - ）/药物（+），（3） Aβ（+）/药物（ - ），和（4）将Aβ（+）/药物（+）。 </li></ol> 2.Aβ肽制剂<ol><li>治疗的Aβ肽，通过化学合成5或从商业来源得到，与预冷的1,1,1,3,3,3-六氟-2-丙醇（HFIP）以溶解任何聚集体和使均匀6的肽。 <ol><li>超声处理在水浴超声波仪的Aβ/ HFIP溶液中5分钟（RT），轻轻涡旋，并在室温下孵育30分钟的溶液中。通过完全用氮气蒸发取出HFIP并在-80℃储存均匀的Aβ直到使用。 </li></ol></li><li>制备Aβ单体:（100μM的Aβ，10％DMSO，90％的PBS）使1毫Aβ二甲基亚砜（DMSO）储备和稀释10倍的磷酸盐缓冲盐水（PBS） </li><li>准备Aβ寡聚体。 <ol><li>在37℃下孵育的Aβ单体溶液（含有Aβ（1-42）或Aβ（1-40））（步骤2.2）。3或7天，分别。 </li><li>放置在含有湿纸巾，以提供加湿环境，以防止在潜伏期水蒸发拉链袋在密封管中的Aβ溶液。 </li></ol></li><li>确认通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE），用未修饰的蛋白6的光致交联制备的Aβ寡聚体。 </li><li>制备含有10％DMSO的用于车辆的ICV注射对照PBS溶液。 </li></ol> 3.注射器制剂</p&gt; <ol><li>准备一个微量用＆G不锈钢针为侧脑室注射。 </li><li>消毒在高压釜中的注射器与将被用于外科手术过程中的所有其他装置。将高压釜后，将装置内的UV光下20分钟。 </li><li>换用封口膜的微量的针至其长度调整到3.8毫米至使在注射的深度进入脑（ 图1A）最大的精确度。 （对于B6小鼠，调整注射器针头至3.6毫米）。 注意:封口膜的压缩将导致在注射超过3.6毫米腹侧的深度。为了防止封口膜的针插入过程中被压缩，密裹针多次。 </li><li>洗用70％乙醇在注射器的内部空间中，两次。 </li><li>超声处理在水浴超声波仪的注射器和针头，在室温30分钟。 </li><li>洗用70％乙醇在注射器的内部空间中，两次。 </li><li>冲洗注射器用蒸馏水和在通风橱中的时间超过30分钟完全干燥它。在开始Aβ注射前留下干净的注射器在UV光下20分钟。 </li></ol> 4.Aβ注入小鼠模型的制备方法注意:用消毒以保持无菌条件和消毒使用70％的乙醇和UV曝光ICV注射所有手术器械清洁操作场。 <ol><li>由甲苯噻嗪（20毫克/千克）和替来他明的混合物腹膜内注射麻醉小鼠·HCl的/唑拉西泮·HCl的（80毫克/千克）在ICV注射之前。放置在一个温暖的垫鼠标，以保持体温，而麻醉，通过评估脚捏响应确认足够的麻醉。 </li><li>应用无菌PBS下降到双眼，以防止该过程（ 图1B）中的角膜干。 </li><li>在difficu的情况下，提请他们表面上的多个直线的垂直线准备两个镜子LTY垂直注入注射器。 </li><li>放置一个反射镜（M1），在头部的前端旁边的小鼠的身体和其他（M2）。保持M1平行于鼠标的两只眼睛之间的假想中线和安排M2垂直于M1，从而使两个平面形成一个90°角（ 图1C）。放置在鼠标的左侧M1如果研究人员是右撇子。将M1鼠标的右侧，如果研究人员是左撇子。 </li><li>喷70％乙醇到鼠标的额头中间，并用干棉签擦。不要弄湿太大前额的区域的，以避免降低体温由于醇的蒸发。 </li><li>使用一块干净棉签用2％洗必泰溶液的额头上同一地区。与酒精和氯己定总量的各三次重复洗涤才能。 注意:如果需要，为了尽量减少possi剃鼠标的前额上发污染的相容性</li><li>找到前囟门。 <ol><li>用拇指和食指紧紧按住前额的皮肤，直接在上面两只眼睛到双眼突出的程度。皮肤拖回使前额绷紧，尽量减少皮肤下的头骨动作。 </li><li>通过分配三个顶点形成一种无形的三角形:鼠标的两只眼的地步拇指和食指相遇，前囟门。 （ 图 1B，红色箭头:前囟门） </li></ol></li><li>找到注射点用卷尺:-1.0±0.06毫米后到前囟，1.8±0.1毫米外侧矢状缝，和2.4毫米的深度（ 图1D，蓝色恒星:注射点在每个半球）。 （对于B6小鼠:-0.9毫米后，1.7毫米的横向和2.2毫米深） </li><li>填用10微升的Aβ溶液或车辆用过量溶液注射器，以避免意外的空气喷射。 </li><li>发生在第注射器Ë注入点（4.7协议所述）。使注射器垂直于注射点的平面。对齐反映在从一个固定的角度（ 图1C）上的反射镜画线这两个镜像注射器的图像。 </li><li>开始直到封口膜包裹到达皮肤插入针。 </li><li>保持手拿着注射器稳定，用另一只手注资50微升的Aβ溶液或车辆，慢慢地在5秒内没有暂停。确保注射器保持垂直贯穿始终。在完成注射后，对扩散除去注射器之前等待3到5秒。 </li><li>假设原三角形的位置，施加适度的压力，以保护头骨从任何多余的动作。无需倾斜取下注射器。 </li><li>放置在暖垫Aβ注入鼠标恢复并保持胸骨斜卧。不要让鼠标无人看管，直到它恢复意识，并不会返回并购乌斯含非手术小鼠，直到它已经完全康复笼。 </li><li>按照步骤3.3-3.6洗注射器。 </li><li>术后监测小鼠的流动性，并在小鼠中检查感染或疾病的任何迹象为5-7天。 </li></ol><img alt="图1" src="/files/ftp_upload/53308/53308fig1.jpg" /> 图1.Aβ注射入ICV区域 （A）封口膜包裹的注射器针头相比未改良的注射器针头。 （B）PBS滴到眼睛，防止干燥;形成了鼠标的拇指，食指额头上的三角形和鼠标的两只眼睛以及虚中线从每只眼睛的距离相等; （C）的两个反射镜（M1和M2）的表面上绘制的，以协助垂直注射器针头的注射多个垂直线; （D）的与所述注入点的三角形，印度语ated为蓝色恒星（前囟从-1.0±0.06毫米和1.8矢状位±0.1 mm）公鼠的每个半球;绿箭:封口膜，红色箭头:前囟门<a href="https://www.jove.com/files/ftp_upload/53308/53308fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 5.确认Aβ注射液的Y型迷宫，脑分析<ol><li>评估通过Y迷宫测试3,7,8Aβ注射小鼠的工作记忆<ol><li>等待3天注射记忆障碍发病后。 </li><li>执行使用黑色塑料迷宫与三个相同的臂的Y-迷宫测试标记为A，B和C，其中每个分支出来从迷宫的中心的120°的角度（宽×长×高为10厘米×40厘米×12厘米）。 </li><li>放置在启动臂（臂A）的小鼠，并允许受自由地探索迷宫8分钟。 </li><li>测量条目的数量到每个臂并计算altern每个主题3,9的通货膨胀率。 </li></ol></li><li>直接检查死后的大脑，以检查是否注入适当的目标区域ICV。 <ol><li>麻醉由相同的步骤以小鼠为4.1节所述。然后牺牲颈椎脱位鼠标。 </li><li>斩首鼠标和去除皮肤用手术剪，露出头骨。从头骨移除组织和肌肉。 </li><li>就在人字缝（之间的顶和interparietal骨）切口不损坏大脑。取出后枕部和interparietal骨头，然后是顶叶和额叶的骨头。使用镊子除去颅骨和通过抬起脑膜隔离从头骨大脑。 </li><li>洗冷却无菌盐水脑和切脑的侧脑室区域中用手术刀（ 图2）的冠方向。 </li><li>确认基于所述针插入对跟踪注射区域脑组织（ 图2）。检查针迹已达到心室之一。如果针迹不符合或穿过心室，从实验结果的分析排除受验者的数据。 </li></ol></li></ol>

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The authors have nothing to disclose.

在这个协议中最重要的步骤是Aβ的ICV注射。该协议被设计为注射的Aβ到小鼠ICV区而不立体定向仪11,12。在开始实验之前，实践注射用蓝色染料代替Aβ初步期间应该发生达到足够灵巧。注射后立即，有必要检查颜料喷射是否正确执行。小心取出大脑，并检查相应的区域是否被染成蓝色。着色线应达到，但不超过，该ICV区域的深度。 90％的成功率，这是经过50以上的ICV注射一般能达到的最低限度，建议。在ICV注射，避免通过注入矢状窦（接近前囟零点）损伤血管。 Aβ注射后，统计学显著记忆损伤观察在Aβ注射组，并使用Y型迷宫行为测试对照组D之间。不同类型的行为实验可以适用于测试认知障碍，如Morris水的程度迷宫13，目标识别和被动回避测试以及恐惧条件。 Morris水迷宫被用来检查海马空间记忆障碍，而恐惧条件用于测试海马依赖的联想学习和杏仁核，海马通信。一个特定对象的对象识别是适合的AD相关的认知缺损的测量，和被动回避可用于测试动物的记的厌恶刺激3的能力。以制备类似体重和年龄的小鼠，如果他们将经受利用电击，如恐惧条件和被动回避试验行为测试是非常重要的。建议在上述的试验是要进行在记忆障碍发病10天。由于它证实了Aβ是在海马后ICV注射14发现，进一步研究是必要的，以找到涉及与ICV注射的Aβ的亲和力其他脑区行为测试。 所述Aβ肽的制备和侧脑室注射的时间应取决于实验目的精心设计，并应考虑:（1）将Aβ（单体，低聚物，原纤维，或它们的混合物）的物种; （2）将Aβ（Aβ40，Aβ42，截断Aβ25-35，及其他）15-18的同种型; （3）将Aβ注射的时间点;和（4）的Aβ曝光后的延迟时间。例如，建议使用Aβ单体的注入，如果将小鼠进行的抑制Aβ单体，如聚合的异常活动的药物的施用。可溶性Aβ寡聚体的注射是suggested。如果研究的目的是评估神经毒性或炎症所引起的低聚物。如果调查旨在研究不溶性的Aβ和相关病理7,19建议高度综合的Aβ种类的注入。如果实验旨在测试的Aβ聚集抑制剂的效力，所述化合物一般应之前施加或与Aβ的沿，而Aβ清剂或抗炎化合物应当Aβ注射后给药。 相比于转基因或AD的慢性模型中，Aβ注入小鼠模型使研究人员能够控制Aβ的浓度，AD样表型的发作，并有大量疯狂老鼠的同步发展。这些优势提供自由相对于实验设计和广泛的机会去研究。考虑AD的发病机制更加密切相关的慢性暴露，而不是在A＆突然飙升＃946;大脑中的浓度，使用转基因模型更多的试验，建议从加强利用Aβ注射的小鼠模型中得出的结论。此外，由于tau蛋白是AD的另一个重要原因，tau蛋白和Aβ注射小鼠模型的发展可以进一步推进AD研究会。

鉴于淀粉样蛋白β（Aβ）是阿尔茨海默氏病（AD）的一个病理标志，AD动物模型的发展集中于Aβ的神经过度表达。因为在淀粉样前体蛋白（APP）或早老（PS）的突变导致Aβ的平衡的干扰，并最终以家族性AD 1的发病机制，涉及APP或PS基因突变的小鼠模型已被普遍接受。之间的宽范围的转基因小鼠的，典型的小鼠模型包括如下:的Tg2576，PDAPP，APP / PS1和APP23。在大脑中，这些小鼠通常显示出Aβ凝聚并最终老年斑;噬菌斑形成后跟显著认知障碍，使得它们显示在学习和记忆的行为测试性能不佳。利用转基因小鼠自然地模仿人类的​​AD病理的产生使我们能够监测迪的发展也由此促成了AD研究会sea​​se。但是，使用转基因小鼠是不经济的和费时的，因为它需要几个月的小鼠发展的Aβ斑块和甚至更长的时间才能显示Aβ诱导的突触或行为异常2,3。最初开发作为替代克服转基因小鼠模型的缺点，非转基因模型通常也由于其独特的优点使用。病原体诱导的AD模型可以通过直接注射的Aβ进入大脑产生，而AD样认知缺陷也可以通过其它化学和物理触发装置 - 如注射如东莨菪碱的神经毒性的化合物，病变的感应在认知相关的领域，如海马，或皮质伤害4。然而，所述非致病性的诱导的认知损害不能准确反映AD的根本病理生理学;相反，它只是模仿其症状的结果。相反，病原体诱导AD模型中，A46; -injected小鼠模型，不仅可以显示AD样行为异常也可以表现出Aβ病理，由家族性和散发AD共享的公共功能。 尽管以可视化的Aβ斑块在脑组织中的困难，这使得它对于AD调查吸引力Aβ注射模型的最大好处是它的可控制性。研究人员可以淘汰在小鼠模型中的个体差异，可能导致错误的数据与毒品有关的研究。及时的药物治疗是根据所述候选药物的机制使能;阐述，Aβ聚集抑制剂可以Aβ注射前被应用。此外，研究人员可以假定致病变换Aβ注射后产生从Aβ的曝光衍生，因为其它因素严格控制，包括个体差异。 在这个协议中，H生动的描述流诱导Aβ经脑室（ICV）注射正常小鼠的AD样表型立体定向仪呈现。减少到脑组织插入挑起损伤是至关重要的，以防止结构损坏和损伤诱导的炎症的可能性。缺乏在鼠标操作技能导致意想不到的神经元损伤。此外，技术，使相应的角度和深度的注射过程来实现是规避失误频频尤其重要。除了ICV注射的详细，生动的说明中，通过以下方案所产生的模型的可靠性在下面的章节也示出。下面的协议可能是一个可靠的，易于理解的工具，有助于AD的研究，从而提供一个跳板，可能最终导致一个有意义的发现为AD的社会。

<table><tbody><tr><td>ICR mouse</td><td>Orientbio</td><td></td><td>male, 6-8 weeks, 27-29 g of body weight</td></tr><tr><td>C57BL/6 mouse</td><td>Orientbio</td><td></td><td>male, 6-8 weeks, 21-23 g of body weight</td></tr><tr><td>Amyloid-beta1-42</td><td>in house synthesis</td><td></td><td>stock concentration: 1 mM/DMSO, injected concentration: 100 &amp;mu;M/10% DMSO and 90% PBS</td></tr><tr><td>ICV injection syringe (26s gauge)</td><td>Hamilton</td><td>80308</td><td></td></tr><tr><td>Evans blue dye (EBD)</td><td>abcamBIochemicals</td><td>ab120869</td><td>1% EBD in PBS</td></tr><tr><td>DMSO</td><td>Sigma</td><td>D2650</td><td></td></tr><tr><td>PBS</td><td>gibco</td><td>10010-023</td><td></td></tr><tr><td>Gradi-GelTM II Gradient PAGE Analysis Kit</td><td>ELPiS Biotech</td><td>EBS-1056</td><td>15% Gel</td></tr><tr><td>Precision Plus ProteinTM Dual Xtra Standards</td><td>Bio-Rad</td><td>161-0377</td><td></td></tr><tr><td>Silver-Staining Kit</td><td>GE-Healthcare</td><td>17-1150-01</td><td></td></tr><tr><td>LabDiet</td><td>Orientbio</td><td>5053</td><td></td></tr></tbody></table>

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.

此部分示出了可以由Aβ聚集和记忆障碍的Y-迷宫评估确认来得到的结果的例子。使用全长Aβ制作（1-42）的42个氨基酸，Aβ单体，低聚物的混合物，和原纤维（ 图3）的肽。通过HFIP诱导monomerization步骤，获得相对均匀的单体（泳道B）。 7天孵化后，Aβ聚集体（泳道C）的不同尺寸的发展。三聚体和四聚体Aβ的寡聚形式中的主导品种。空间工作记忆经由在Y迷宫测试交替在Aβ注射小鼠评估。而每个鼠被允许自由地探索迷宫的臂的选择顺序和总臂条目的数量的记录。更完好的认知能力，更多的鼠标应该有进入较少最近访问了手臂，阿尔特趋势rnating其手臂的选择。该Aβ注射组小鼠表现出显著低利率交替，表明认知缺陷（ 图4）的发展。 <img alt="图2" src="/files/ftp_upload/53308/53308fig2.jpg" /> 图2. ICV注射 （ 一 ）冠向脑切片较前囟门10和可接受的注射范围（从囟-1.0±0.06毫米和1.8±0.1毫米矢状位）的位置示意图的例子 。 （B - C）成功ICV注射的情况下（A:全脑，C的顶视图:冠状断面示出填充有蓝染料侧脑室）; （D - E）的不成功ICV注射（D ​​的情况:在整个脑部的顶视图中，E:冠状部分示出在第三和第四已经ntricles充满蓝色染料）蓝色圆圈和方向:注射部位，蓝星:潜在注入点<a href="https://www.jove.com/files/ftp_upload/53308/53308fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53308/53308fig3.jpg" /> 图3.制备 通过SDS-PAGE 一个 β物种 的确定 。Aβ肽通过SDS-PAGE分离与未修饰的蛋白质的光诱导的交联。肽条带通过银染色观察;车道（A）的蛋白质标记物，泳道（B）Aβ单体（孵化前）和莱恩（C）孵育Aβ种类。 <a href="https://www.jove.com/files/ftp_upload/53308/53308fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 图4. Y-迷宫行为测试。所述Aβ-百分比交替或媒介物注射的小鼠组在Y迷宫测试。白色固体图:车辆（Aβ（ - ），N = 10）。条纹图:Aβ注射（Aβ（+），N = 9）。统计分析用单向ANOVA随后的Bonferroni的事后比较进行（* P &lt;0.05，** P &lt;0.01，*** P &lt;0.001）。误差条代表了中小企业。 <a href="https://www.jove.com/files/ftp_upload/53308/53308fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Intracerebroventricular Injection of Amyloid-ÃŽÂ² Peptides in Normal Mice to Acutely Induce Alzheimer-like Cognitive Deficits at JoVE.com

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

淀粉样蛋白β（Aβ）-injected动物模型能够确定的数量和Aβ片段的物种的管理，并降低每个研究组内的个体差异。本协议描述Aβ的脑室（ICV）注射立体定向仪，使生产正常小鼠老年痴呆样行为异常。

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, ...

intracerebroventricular-injection-amyloid-peptides-normal-mice-to

Nanyang Technological University

Research

JoVE Journal

Neuroscience

42.0K 次观看.  Korea Institute of Science and Technology. 淀粉样蛋白β（Aβ）-injected动物模型能够确定的数量和Aβ片段的物种的管理，并降低每个研究组内的个体差异。本协议描述Aβ的脑室（ICV）注射立体定向仪，使生产正常小鼠老年痴呆样行为异常。 

视频: Intracerebroventricular Injection of Amyloid-β Peptides in Normal Mice to Acutely Induce Alzheimer-like Cognitive Deficits

Preparation of Oligomeric &beta;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

Impairment of synaptic connections is likely to underlie the subtle amnesic changes occurring at the early stages of Alzheimer s Disease (AD). &beta;-amyloid (A&beta;), a peptide produced in high amounts in AD, is known to reduce Long-Term Potentiation (LTP), a cellular correlate of learning and memory. Indeed, LTP impairment caused by A&beta; is a useful experimental paradigm for studying synaptic dysfunctions in AD models and for screening drugs capable of mitigating or reverting such synaptic impairments. Studies have shown that A&beta; produces the LTP disruption preferentially via its oligomeric form. Here we provide a detailed protocol for impairing LTP by perfusion of oligomerized synthetic A&beta;1-42 peptide onto acute hippocampal slices. In this video, we outline a step-by-step procedure for the preparation of oligomeric A&beta;1-42. Then, we follow an individual experiment in which LTP is reduced in hippocampal slices exposed to oligomerized A&beta;1-42 compared to slices in a control experiment where no A&beta;1-42 exposure had occurred.

Preparation of Oligomeric &#946;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

One feature of Alzheimer's Disease is the elevation of A&beta;1-42 peptide. Here we provide a protocol for preparing synthetic A&beta;1-42 oligomers, which impairs hippocampal Long-Term Potentiation, a cellular correlate of memory. This procedure is useful for investigating mechanisms of A&beta;-induced pathology and drug screening.

低聚β-淀粉样蛋白的制备 1-42和突触可塑性损伤诱导海马脑片

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

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

A Technique for Serial Collection of Cerebrospinal Fluid from the Cisterna Magna in Mouse 

Alzheimer's disease (AD) is a progressive neurodegenerative disease that is pathologically characterized by extracellular deposition of &#946;-amyloid peptide (A&#946;) and intraneuronal accumulation of hyperphosphorylated tau protein. Because cerebrospinal fluid (CSF) is in direct contact with the extracellular space of the brain, it provides a reflection of the biochemical changes in the brain in response to pathological processes. CSF from AD patients shows a decrease in the 42 amino-acid form of A&#946; (A&#946;42), and increases in total tau and hyperphosphorylated tau, though the mechanisms responsible for these changes are still not fully understood. Transgenic (Tg) mouse models of AD provide an excellent opportunity to investigate how and why A&#946; or tau levels in CSF change as the disease progresses. Here, we demonstrate a refined cisterna magna puncture technique for CSF sampling from the mouse. This extremely gentle sampling technique allows serial CSF samples to be obtained from the same mouse at 2-3 month intervals which greatly minimizes the confounding effect of between-mouse variability in A&#946; or tau levels, making it possible to detect subtle alterations over time. In combination with A&#946; and tau ELISA, this technique will be useful for studies designed to investigate the relationship between the levels of CSF A&#946;42 and tau, and their metabolism in the brain in AD mouse models. Studies in Tg mice could provide important validation as to the potential of CSF A&#946; or tau levels to be used as biological markers for monitoring disease progression, and to monitor the effect of therapeutic interventions. As the mice can be sacrificed and the brains can be examined for biochemical or histological changes, the mechanisms underlying the CSF changes can be better assessed. These data are likely to be informative for interpretation of human AD CSF changes.

A Technique for Serial Collection of Cerebrospinal Fluid from the Cisterna Magna in Mouse

Transgenic (Tg) mouse models of AD provide an excellent opportunity to investigate how and why A&#946; or tau levels in CSF change as the disease progresses in human patients. Here, we demonstrate a refined cisterna magna puncture technique for serial CSF sampling from the mouse.

一个串行收集脑脊液从池鼠标麦格纳技术

Alzheimer's disease (AD) is a progressive neurodegenerative disease that is pathologically characterized by extracellular deposition of &#946;-amyloid ...

生物学

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 ...

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 ...

医学

Modeling Amyloid-&#946;42 Toxicity and Neurodegeneration in Adult Zebrafish Brain

Alzheimer's disease (AD) is a debilitating neurodegenerative disease in which accumulation of toxic amyloid-&#946;42 (A&#946;42) peptides leads to synaptic degeneration, inflammation, neuronal death, and learning deficits. Humans cannot regenerate lost neurons in the case of AD in part due to impaired proliferative capacity of the neural stem/progenitor cells (NSPCs) and reduced neurogenesis. Therefore, efficient regenerative therapies should also enhance the proliferation and neurogenic capacity of NSPCs. Zebrafish (Danio rerio) is a regenerative organism, and we can learn the basic molecular programs with which we could design therapeutic approaches to tackle AD. For this reason, the generation of an AD-like model in zebrafish was necessary. Using our methodology, we can introduce synthetic derivatives of A&#946;42 peptide with tissue penetrating capability into the adult zebrafish brain, and analyze the disease pathology and the regenerative response. The advantage over the existing methods or animal models is that zebrafish can teach us how a vertebrate brain can naturally regenerate, and thus help us to treat human neurodegenerative diseases better by targeting endogenous NSPCs. Therefore, the amyloid-toxicity model established in the adult zebrafish brain may open new avenues for research in the field of neuroscience and clinical medicine. Additionally, the simple execution of this method allows for cost-effective and efficient experimental assessment. This manuscript describes the synthesis and injection of A&#946;42 peptides into zebrafish brain.

This protocol describes the synthesis, characterization, and injection of monomeric amyloid-&#946;42 peptides for generating amyloid toxicity in adult zebrafish to establish an Alzheimer&#39;s disease model, followed by histological analyses and detection of aggregations.

成年斑马鱼脑 Amyloid-β42毒性和神经的模型研究

Alzheimer's disease (AD) is a debilitating neurodegenerative disease in which accumulation of toxic amyloid-&#946;42 (A&#946;42) peptides leads to ...

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.

<meta charset="utf8"></head><body>通过免疫荧光染色检测阿尔茨海默病小鼠模型中的淀粉样蛋白β积累

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.

通过免疫荧光染色检测阿尔茨海默病小鼠模型中的淀粉样蛋白β积累

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 ...

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 ...

Controlled Infusion of A&beta; Peptide-Specific Antibodies and A&beta; Oligomers into the Rat Hippocampus

Source: Sajadi, A., et al. <a href="https://app.jove.com/v/54215">Neurodegeneration in an Animal Model of Chronic Amyloid-beta Oligomer Infusion Is Counteracted by Antibody Treatment Infused with Osmotic Pumps.</a> J. Vis. Exp. (2016).
This video demonstrates a technique for the continuous infusion of amyloid-beta (A&#946;) peptide-specific antibodies into the hippocampus of a rat. The study employs implanting a bilateral cannula system for a controlled infusion of A&#946; peptide-specific antibodies coupled with the infusion of soluble A&#946; oligomers (A&#946;o) into the rat hippocampus to study the interactions between the oligomers and the antibodies.

将 Aβ 肽特异性抗体和 Aβ 寡聚体受控输注到大鼠海马体中

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

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成年斑马鱼脑 Amyloid-β42毒性和神经的模型研究

通过免疫荧光染色检测阿尔茨海默病小鼠模型中的淀粉样蛋白β积累

通过免疫荧光染色检测阿尔茨海默病小鼠模型中的淀粉样蛋白β积累

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

将 Aβ 肽特异性抗体和 Aβ 寡聚体受控输注到大鼠海马体中