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>

<ol>
	<li>Kam, T. I., Gwon, Y., Jung, Y. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid+beta+receptors+responsible+for+neurotoxicity+and+cellular+defects+in+Alzheimer's+disease.">Amyloid beta receptors responsible for neurotoxicity and cellular defects in Alzheimer's disease.</a> Cell Mol Life Sci. 71, 4803-4813 (2014).</li><li>Elder, G. A., Gama Sosa, M. A., De Gasperi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+mouse+models+of+Alzheimer's+disease.">Transgenic mouse models of Alzheimer's disease.</a> Mt Sinai J Med. 77, 69-81 (2010).</li><li>Bryan, K. J., Lee, H., Perry, G., Smith, M. A., Casadesus, G., Buccafusco, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods of Behavior Analysis in Neuroscience Frontiers in Neuroscience. , (2009).</li><li>Van Dam, D., De Deyn, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+in+the+drug+discovery+pipeline+for+Alzheimer's+disease.">Animal models in the drug discovery pipeline for Alzheimer's disease.</a> Br J Pharmacol. 164, 1285-1300 (2011).</li><li>Choi, J. W., Kim, H. Y., Jeon, M., Kim, D. J., Kim, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+access+to+highly+pure+beta-amyloid+peptide+by+optimized+solid-phase+synthesis.">Efficient access to highly pure beta-amyloid peptide by optimized solid-phase synthesis.</a> Amyloid. 19, 133-137 (2012).</li><li>Rahimi, F., Maiti, P., Bitan, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photo-induced+cross-linking+of+unmodified+proteins+(PICUP)+applied+to+amyloidogenic+peptides.">Photo-induced cross-linking of unmodified proteins (PICUP) applied to amyloidogenic peptides.</a> J Vis Exp. , (2009).</li><li>Kim, H. V., 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=Amelioration+of+Alzheimer's+disease+by+neuroprotective+effect+of+sulforaphane+in+animal+model.">Amelioration of Alzheimer's disease by neuroprotective effect of sulforaphane in animal model.</a> Amyloid. 20, 7-12 (2013).</li><li>Jackson, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VTE+on+an+elevated+T-maze.">VTE on an elevated T-maze.</a> J Comp Psychol. 36, 9 (1943).</li><li>Kim, H. Y., 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=Taurine+in+drinking+water+recovers+learning+and+memory+in+the+adult+APP/PS1+mouse+model+of+Alzheimer's+disease.">Taurine in drinking water recovers learning and memory in the adult APP/PS1 mouse model of Alzheimer's disease.</a> Sci Rep. 4, 7467 (2014).</li><li>Paxinos, G., Franklin, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Mouse Brain in Stereotaxic Coordinates. 2nd edn. , (2001).</li><li>Ledo, J. H., 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=Amyloid-beta+oligomers+link+depressive-like+behavior+and+cognitive+deficits+in+mice.">Amyloid-beta oligomers link depressive-like behavior and cognitive deficits in mice.</a> Mol Psychiatry. 18, 1053-1054 (2013).</li><li>Laursen, S. E., Belknap, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracerebroventricular+injections+in+mice.+Some+methodological+refinements.">Intracerebroventricular injections in mice. Some methodological refinements.</a> J Pharmacol Methods. 16, 355-357 (1986).</li><li>Bromley-Brits, K., Deng, Y., Song, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morris+water+maze+test+for+learning+and+memory+deficits+in+Alzheimer's+disease+model+mice.">Morris water maze test for learning and memory deficits in Alzheimer's disease model mice.</a> J Vis Exp. , (2011).</li><li>Figueiredo, C. P., 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=Memantine+rescues+transient+cognitive+impairment+caused+by+high-molecular-weight+abeta+oligomers+but+not+the+persistent+impairment+induced+by+low-molecular-weight+oligomers.">Memantine rescues transient cognitive impairment caused by high-molecular-weight abeta oligomers but not the persistent impairment induced by low-molecular-weight oligomers.</a> J Neurosci. 33, 9626-9634 (2013).</li><li>Haley, T. J., McCormick, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+effects+produced+by+intracerebral+injection+of+drugs+in+the+conscious+mouse.">Pharmacological effects produced by intracerebral injection of drugs in the conscious mouse.</a> Br J Pharmacol Chemother. 12, 12-15 (1957).</li><li>Harkany, 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=Neuroprotective+approaches+in+experimental+models+of+beta-amyloid+neurotoxicity:+relevance+to+Alzheimer's+disease.">Neuroprotective approaches in experimental models of beta-amyloid neurotoxicity: relevance to Alzheimer's disease.</a> Prog Neuropsychopharmacol Biol Psychiatry. 23, 963-1008 (1999).</li><li>Wang, 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=The+allosteric+potentiation+of+nicotinic+acetylcholine+receptors+by+galantamine+ameliorates+the+cognitive+dysfunction+in+beta+amyloid25-35+i.c.v.-injected+mice:+involvement+of+dopaminergic+systems.">The allosteric potentiation of nicotinic acetylcholine receptors by galantamine ameliorates the cognitive dysfunction in beta amyloid25-35 i.c.v.-injected mice: involvement of dopaminergic systems.</a> Neuropsychopharmacology. 32, 1261-1271 (2007).</li><li>Yamada, K., Nabeshima, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+Alzheimer's+disease+and+evaluation+of+anti-dementia+drugs.">Animal models of Alzheimer's disease and evaluation of anti-dementia drugs.</a> Pharmacol Ther. 88, 93-113 (2000).</li><li>Cho, S. 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=Correlations+of+amyloid-beta+concentrations+between+CSF+and+plasma+in+acute+Alzheimer+mouse+model.">Correlations of amyloid-beta concentrations between CSF and plasma in acute Alzheimer mouse model.</a> Sci Rep. 4, 6777 (2014).</li></ol>

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?...

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 border="0" cellpadding="0" cellspacing="0" width="1106">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">ICR mouse</td>
			<td style="width:155px;">Orientbio</td>
			<td style="width:119px;"></td>
			<td style="width:532px;">male, 6~8 weeks, 27~29 g of body weight</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">C57BL/6 mouse</td>
			<td>Orientbio</td>
			<td></td>
			<td>male, 6~8 weeks, 21~23 g of body weight</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amyloid-beta1-42</td>
			<td>in house synthesis</td>
			<td>n.a.</td>
			<td>stock concentration: 1 mM/DMSO, injected concentration: 100 &#956;M/10% DMSO and 90% PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ICV injection syringe (26s gauge)</td>
			<td>Hamilton</td>
			<td>80308</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Evans blue dye (EBD)</td>
			<td>abcamBIochemicals</td>
			<td>ab120869</td>
			<td>1 % EBD in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMSO</td>
			<td>Sigma</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS</td>
			<td>gibco</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gradi-GelTM II Gradient PAGE Analysis Kit</td>
			<td>ELPiS Biotech</td>
			<td>EBS-1056</td>
			<td>15% Gel</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Precision Plus ProteinTM Dual Xtra Standards</td>
			<td>Bio-Rad</td>
			<td>161-0377</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Silver-Staining Kit</td>
			<td>GE-Healthcare</td>
			<td>17-1150-01</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?...

Watch this Scientific Journal Video about Intracerebroventricular Injection of Amyloid-Î² 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

Research

JoVE Journal

Neuroscience

41.8K 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

Autologous Blood Injection to Model Spontaneous Intracerebral Hemorrhage in Mice

Investigation of the pathophysiology of injury after intracerebral
hemorrhage (ICH) requires a reproducible animal model. While ICH
accounts for 10-15% of all strokes, there remains no specific
effective therapy. The autologous blood injection model in mice
involves the stereotaxic injection of arterial blood into the basal
ganglia mimicking a spontaneous hypertensive hemorrhage in man. The
response to hemorrhage can then be studied in vivo and the
neurobehavioral deficits quantified, allowing for description of the
ensuing pathology and the testing of potential therapeutic agents. The
procedure described in this protocol uses the double injection
technique to minimize risk of blood reflux up the needle track, no
anticoagulants in the pumping system, and eliminates all dead space
and expandable tubing in the system.

The autologous blood injection model of intracerebral hemorrhage in mice described in this protocol uses the double injection technique to minimize risk of blood reflux up the needle track, no anticoagulants in the pumping system, and eliminates all dead space and expandable tubing in the system.

自体血液的注入模型小鼠自发性脑出血

Investigation of the pathophysiology of injury after intracerebral
hemorrhage (ICH) requires a reproducible animal model.  While ICH
accounts for 10-15% ...

科研

神经科学

Intracerebroventricular Viral Injection of the Neonatal Mouse Brain for Persistent and Widespread Neuronal Transduction

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene expression in the mouse brain. Here we describe a technique first introduced by Passini and Wolfe for direct intracranial delivery of virally-encoded transgenes into the neonatal mouse brain. In its most basic form, the procedure requires only an ice bucket and a microliter syringe. However, the protocol can also be adapted for use with stereotaxic frames to improve consistency for researchers new to the technique. The method relies on the ability of adeno-associated virus (AAV) to move freely from the cerebral ventricles into the brain parenchyma while the ependymal lining is still immature during the first 12-24 hr after birth. Intraventricular injection of AAV at this age results in widespread transduction of neurons throughout the brain. Expression begins within days of injection and persists for the lifetime of the animal. Viral titer can be adjusted to control the density of transduced neurons, while co-expression of a fluorescent protein provides a vital label of transduced cells. With the rising availability of viral core facilities to provide both off-the-shelf, pre-packaged reagents and custom viral preparation, this approach offers a timely method for manipulating gene expression in the mouse brain that is fast, easy, and far less expensive than traditional germline engineering.

Here we demonstrate a technique for widespread neuronal transduction by intraventricular injection of adeno-associated virus into the neonatal mouse brain. This method provides a rapid and easy way to attain lifelong expression of virally-delivered transgenes.

新生小鼠脑侧脑室注射病毒的持续和广泛的神经元传导

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene ...

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method described here enables the investigator to measure long-lasting (from minutes to hours) high-quality intracellular responses from single Drosophila R1-R6 photoreceptors and Large Monopolar Cells (LMCs) to light stimuli. Because the recording system has low noise, it can be used to study variability among individual cells in the fly eye, and how their outputs reflect the physical properties of the visual environment. We outline all key steps in performing this technique. The basic steps in constructing an appropriate electrophysiology set-up for recording, such as design and selection of the experimental equipment are described. We also explain how to prepare for recording by making appropriate (sharp) recording and (blunt) reference electrodes. Details are given on how to fix an intact fly in a bespoke fly-holder, prepare a small window in its eye and insert a recording electrode through this hole with minimal damage. We explain how to localize the center of a cell's receptive field, dark- or light-adapt the studied cell, and to record its voltage responses to dynamic light stimuli. Finally, we describe the criteria for stable normal recordings, show characteristic high-quality voltage responses of individual cells to different light stimuli, and briefly define how to quantify their signaling performance. Many aspects of the method are technically challenging and require practice and patience to master. But once learned and optimized for the investigator's experimental objectives, it grants outstanding in vivo neurophysiological data.

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Sharp microelectrodes enable accurate electrophysiological characterization of photoreceptor and visual interneuron output in living Drosophila. Here we show how to use this method to record high-quality voltage responses of individual cells to controlled light stimulation. This method is ideal for studying neural information processing in insect compound eyes.

为记录细胞内电压响应电法<em&gt;果蝇</em&gt;光感受器和中间对光刺激<em&gt;在体内</em

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method ...

Whole-cell Currents Induced by Puff Application of GABA in Brain Slices

Pharmacological administration is commonly used when conducting whole-cell patch-clamp recording in brain slices. One of the best methods of drug application during electrophysiological recording is the puff technique, which can be used to study the effect of pharmacological reagents on neuronal activities in brain slices. The greatest advantage of puff application is that the drug concentration around the recording site increases rapidly, thus preventing desensitization of membrane receptors. Successful use of puff application involves careful attention to the following elements: the concentration of the drug, the parameters of the puff micropipette, the distance between the tip of a puff micropipette and the neuron recorded, and the duration and pressure driving the puff (pounds per square inch, psi). This article describes a step-by-step procedure for recording whole-cell currents induced by puffing gamma-aminobutyric acid (GABA) onto a neuron of a prefrontal cortical slice. Notably, the same procedure can be applied with minor modifications to other brain areas such as the hippocampus and the striatum, and to different preparations, such as cell cultures.

We describe the puff technique, by which pharmacological reagents can be administered during whole-cell patch-clamp recording, and highlight various aspects of the features that are crucial for its success.

粉扑诱导的全细胞电流在脑切片中的应用

Pharmacological administration is commonly used when conducting whole-cell patch-clamp recording in brain slices. One of the best methods of drug ...

Intracerebroventricular Treatment with Resiniferatoxin and Pain Tests in Mice

The transient receptor potential vanilloid type 1 (TRPV1), a thermosensitive cation channel, is known to trigger pain in the peripheral nerves. In addition to its peripheral function, its involvement in brain functions has also been suggested. Resiniferatoxin (RTX), an ultrapotent TRPV1 agonist, has been known to induce long-term desensitization of TRPV1, and this desensitization has been an alternative approach for investigating the physiological relevance of TRPV1-expressing cells. Here we describe a protocol for intracerebroventricular (i.c.v.) treatment with RTX in mice. Procedures are described for testing nociception to peripheral TRPV1 stimulation (RTX test) and mechanical stimulation (tail pressure test) then follow. Although the nociceptive responses of mice that had been administered RTX i.c.v. were comparable to those of the control groups, RTX-i.c.v.-administered mice were insensitive to the analgesic effect of acetaminophen, suggesting that i.c.v. RTX treatment can induce supraspinal-selective TRPV1 desensitization. This mouse model can be used as a convenient experimental system for studying the role of TRPV1 in brain/supraspinal function. These techniques can also be applied to studies of the central actions of other drugs.

The transient receptor potential vanilloid type 1 (TRPV1) in the supraspinal region has been suggested to play some roles in the brain function. Described here is a protocol for intracerebroventricular injection of resiniferatoxin for supraspinal TRPV1 desensitization in mice. Procedures for some pain tests are also presented.

小鼠用树脂叶毒素和疼痛测试进行脑血管内治疗

The transient receptor potential vanilloid type 1 (TRPV1), a thermosensitive cation channel, is known to trigger pain in the peripheral nerves. In ...

Evaluation of Hemisphere Lateralization with Bilateral Local Field Potential Recording in Secondary Motor Cortex of Mice

This article demonstrates complete, detailed procedures for both in vivo bilateral recording and analysis of local field potential (LFP) in the cortical areas of mice, which are useful for evaluating possible laterality deficits, as well as for assessing brain connectivity and coupling of neural network activities in rodents. The pathological mechanisms underlying Alzheimer&#39;s disease (AD), a common neurodegenerative disease, remain largely unknown. Altered brain laterality has been demonstrated in aging people, but whether or not abnormal lateralization is one of the early signs of AD has not been determined. To investigate this, we recorded bilateral LFPs in 3-5-month-old AD model mice, APP/PS1, together with littermate wild type (WT) controls. The LFPs of the left and right secondary motor cortex (M2), specifically in the gamma band, were more synchronized in APP/PS1 mice than in WT controls, suggesting a declined hemispheric asymmetry of bilateral M2 in this AD mouse model. Notably, the recording and data analysis processes are flexible and easy to carry out, and can also be applied to other brain pathways when conducting experiments that focus on neuronal circuits.

We present in vivo electrophysiological recording of the local field potential (LFP) in bilateral secondary motor cortex (M2) of mice, which can be applied to evaluate hemisphere lateralization. The study revealed altered levels of synchronization between the left and right M2 in APP/PS1 mice compared to WT controls.

小鼠二次运动皮质中双边局部场电位记录半球横向化评价

This article demonstrates complete, detailed procedures for both in vivo bilateral recording and analysis of local field potential (LFP) in the cortical ...

Inter-Brain Synchrony in Open-Ended Collaborative Learning: An fNIRS-Hyperscanning Study

fNIRS hyperscanning is widely used to detect the neurobiological underpinnings of social interaction. With this technique, researchers qualify the concurrent brain activity of two or more interactive individuals with a novel index called inter-brain synchrony (IBS) (i.e., phase and/or amplitude alignment of the neuronal or hemodynamic signals across time). A protocol for conducting fNIRS hyperscanning experiments on collaborative learning dyads in a naturalistic learning environment is presented here. Further, a pipeline of analyzing IBS of oxygenated hemoglobin (Oxy-Hb) signal is explained. Specifically, the experimental design, the process of NIRS data recording, data analysis methods, and future directions are all discussed. Overall, implementing a standardized fNIRS hyperscanning pipeline is a fundamental part of second-person neuroscience. Also, this is in line with the call for open-science to aid the reproducibility of research.

The protocol for conducting fNIRS hyperscanning experiments on collaborative learning dyads in a naturalistic learning environment is outlined. Further, a pipeline to analyze the Inter-Brain Synchrony (IBS) of oxygenated hemoglobin (Oxy-Hb) signals is presented.

开放式协作学习中的脑间同步:fNIRS超扫描研究

fNIRS hyperscanning is widely used to detect the neurobiological underpinnings of social interaction. With this technique, researchers qualify the ...

How to Calculate and Validate Inter-brain Synchronization in a fNIRS Hyperscanning Study

The dynamics between coupled brains of individuals have been increasingly represented by inter-brain synchronization (IBS) when they coordinate with each other, mostly using simultaneous-recording signals of brains (namely hyperscanning) with fNIRS. In fNIRS hyperscanning studies, IBS has been commonly assessed through the wavelet transform coherence (WTC) method because of its advantage on expanding time series into time-frequency space where oscillations can be seen in a highly intuitive way. The observed IBS can be further validated via the permutation-based random pairing of the trial, partner, and condition. Here, a protocol is presented to describe how to obtain brain signals via fNIRS technology, calculate IBS through the WTC method, and validate IBS by permutation in a hyperscanning study. Further, we discuss the critical issues when using the above methods, including the choice of fNIRS signals, methods of data preprocessing, and optional parameters of computations.&#160;In summary, using the WTC method and permutation is a potentially standard pipeline for analyzing IBS in fNIRS hyperscanning studies, contributing to both the reproducibility and reliability of IBS.

如何在fNIRS超扫描研究中计算和验证脑间同步

The dynamics between coupled brains of individuals have been increasingly represented by inter-brain synchronization (IBS) when they coordinate with each ...

Measurement of the Directional Information Flow in fNIRS-Hyperscanning Data using the Partial Wavelet Transform Coherence Method

Social interaction is of vital importance for human beings. While the hyperscanning approach has been extensively used to study interpersonal neural synchronization (INS) during social interactions, functional near-infrared spectroscopy (fNIRS) is one of the most popular techniques for hyperscanning naturalistic social interactions because of its relatively high spatial resolution, sound anatomical localization, and exceptionally high tolerance of motion artifacts. Previous fNIRS-based hyperscanning studies usually calculate a time-lagged INS using wavelet transform coherence (WTC) to describe the direction and temporal pattern of information flow between individuals. However, the results of this method might be confounded by the autocorrelation effect of the fNIRS signal of each individual. For addressing this issue, a method termed partial wavelet transform coherence (pWTC) was introduced, which aimed to remove the autocorrelation effect and maintain the high temporal-spectrum resolution of the fNIRS signal. In this study, a simulation experiment was performed first to show the effectiveness of the pWTC in removing the impact of autocorrelation on INS. Then, step-by-step guidance was offered on the operation of the pWTC based on the fNIRS dataset from a social interaction experiment. Additionally, a comparison between the pWTC method and the traditional WTC method and that between the pWTC method and the Granger causality (GC) method was drawn. The results showed that pWTC could be used to determine the INS difference between different experimental conditions and INS's directional and temporal pattern between individuals during naturalistic social interactions. Moreover, it provides better temporal and frequency resolution than the traditional WTC and better flexibility than the GC method. Thus, pWTC is a strong candidate for inferring the direction and temporal pattern of information flow between individuals during naturalistic social interactions.

This protocol describes partial wavelet transform coherence (pWTC) for calculating the time-lagged pattern of interpersonal neural synchronization (INS) to infer the direction and temporal pattern of information flow during social interaction. The effectiveness of pWTC in removing the confounds of signal autocorrelation on INS was proved by two experiments.

使用偏小波变换相干法测量 fNIRS-超扫描数据中的定向信息流

Social interaction is of vital importance for human beings. While the hyperscanning approach has been extensively used to study interpersonal neural ...

Intracerebroventricular Delivery of Gut-Derived Microbial Metabolites in Freely Moving Mice

The impact of gut microbiota and their metabolites on host physiology and behavior has been extensively investigated in this decade. Numerous studies have revealed that gut microbiota-derived metabolites modulate brain-mediated physiological functions through intricate gut-brain pathways in the host. Short-chain fatty acids (SCFAs) are the major bacteria-derived metabolites produced during dietary fiber fermentation by the gut microbiome. Secreted SCFAs from the gut can act at multiple sites in the periphery, affecting the immune, endocrine, and neural responses due to the vast distribution of SCFAs receptors. Therefore, it is challenging to differentiate the central and peripheral effects of SCFAs through oral and intraperitoneal administration of SCFAs. This paper presents a video-based method to interrogate the functional role of SCFAs in the brain via a guide cannula in freely moving mice. The amount and type of SCFAs in the brain can be adjusted by controlling the infusion volume and rate. This method can provide scientists with a way to appreciate the role of gut-derived metabolites in the brain.

Gut-derived microbial metabolites have multifaceted effects leading to complex behavior in animals. We aim to provide a step-by-step method to delineate the effects of gut-derived microbial metabolites in the brain via intracerebroventricular delivery via a guide cannula.

自由移动小鼠中肠道来源的微生物代谢物的脑室内递送

The impact of gut microbiota and their metabolites on host physiology and behavior has been extensively investigated in this decade. Numerous studies have ...