这项工作是由加拿大卫生研究院研究院（CIHR），汤森家庭，和杰克布朗和家庭阿尔茨海默氏症研究基金会（到WAS）的支持。 WAS是加拿大研究主席在阿尔茨海默氏病的人。 PTTL是由自然科学和工程研究的律师和迈克尔史密斯健康研究基金会奖学金的支持。

1。老鼠大脑中的固定和cryosectioning <ol><li>使用二氧化碳气体，斩首和大脑中提取的转基因小鼠牺牲。 </li><li>大脑是纵向中心解剖。大脑的一半将用于生化分析的干冰冰冻闪光。其他的将被淹没在4％多聚甲醛（PFA）至少48小时在4 ° C。 </li><li>后至少48小时在4％PFA固定，半脑是直接传送到30％的蔗糖溶液，置于4 ° C。在点，半脑应漂浮在蔗糖溶液。当半脑沉到了谷底，通常需要大约48小时，它准备在OCT包埋cryosectioning。 </li><li> cryosectioning之前，安装在旋钮华侨城的一个薄薄的一层，并允许在-20 ° C冻结小脑上一层冰冻华侨城删除从半侧大脑和地方休息。立即盖华侨城半侧大脑，让慢慢冻结在-20 ° C。一旦华侨城嵌入大脑已经变成了不透明的，它是准备切片。 </li><li>设置每节30微米的厚度。 D&#39; Olomos解决方案的容器收集到每个切片。 </li></ol> 2。免疫组织化学过程神经炎斑（自由浮动部分） <ol><li>对待每节15分钟，在88％甲酸。甲酸处理后，将暂时关闭部分不透明，并且可能会出现萎缩。 </li><li>取出到一个盘子和凭借TX - PBS（0.3％的Triton X - 100的PBS）洗5分钟× 3，轻轻摇动中的每个部分。 </li><li>阻断内源性过氧化物阻断0.5％H 2 O 2在Tx - PBS中，RT，轻轻摇动30分钟。 </li><li>洗10分钟× 3节的Tx - PBS，轻轻摇动。 </li><li>块段与非脂肪5％脱脂牛奶的Tx - PBS在室温下溶解，轻轻摇动1小时。 </li><li>孵育部分初级抗体（生物素4G8，1:500-1:2000，徽记）的Tx - PBS在4 5％的牛奶含有稀释° C轻轻摇动过夜。 </li><li>洗10分钟× 3节的Tx - PBS，轻轻摇动。 </li><li>准备ABC的解决方案制造商的协议（精英实验室，美国广播公司（ABC），矢量）。在ABC混合物孵育30分钟，轻轻摇动部分。 </li><li>洗10分钟× 3节的Tx - PBS，轻轻摇动。 </li><li>准备3,3&#39; - 二氨基tetrahydrochloride（民建联）（矢量实验室）以下制造商的协议。在民建联的混合物为5-10分钟孵育部分。在这一点上，你会发现一个棕褐色的每一节。 </li><li>洗10分钟× 3节的Tx - PBS，轻轻摇动。 </li><li>明胶涂层片安装在自由浮动的脑切片，以帮助组织粘连。部分，然后通过一系列二甲苯结算和风干之后安装在Entellen。 </li><li>斑块40X放大倍率的光学显微镜下观察，并通过量化评估每个鼠标的计数每片平均斑块。 </li></ol> 3。 Thioflavin S染色神经炎斑块的检测程序<ol><li>鼠标牺牲和脑提取过程是在第1.1至1.5表示。 </li><li>山半到4至5玻片脑切片和允许完全风干染色前。 </li><li>用70％乙醇1分钟的幻灯片。 </li><li>用80％乙醇1分钟的幻灯片。 </li><li>过滤（0.2微米的过滤器）thioflavin小号溶液（1％）80％的乙醇中孵育15分钟的幻灯片。 （Thioflavin s解决方案需要在每次使用前过滤）。注：保护光thioflavin S和避光染色的幻灯片。 </li><li>用80％乙醇1分钟的幻灯片。 </li><li>用70％乙醇1分钟的幻灯片。 </li><li>用蒸馏水洗两次。 </li><li>山在水溶液中的安装媒体，并允许幻灯片干燥至少2小时，用透明指甲油密封盖玻片，在黑暗中的幻灯片。染色切片，应存放在黑暗中，在4 ° C。 </li><li>绿色荧光染色斑块可与荧光microscropy可视化。分析一个星期内的幻灯片，因为随着时间的推移会褪色染色。 </li></ol> 4。代表性的成果在我们最近的抗癫痫药物的疗效和情绪稳定剂丙戊酸（VPA），以抑制神经炎斑块的形成研究中，我们使用上面介绍的免疫和thioflavin S染色程序，以确定在AD模型小鼠（3）神经炎斑。图1展示了一个典型的9个月大的APP23转基因小鼠，通过与抗生物素化的Aβ使用4G8和染色ABC法检测。神经炎斑是清楚的标记抗体，是由白色箭头表示。使用thioflavin S染色，我们发现2个月大的APP23xPS TRA神经炎斑块沉积nsgenic小鼠（图2）。 Thioflavin小号约束Aβ含神经炎斑出现绿色荧光显微镜下（白色箭头表示）。我们发现，丙戊酸钠治疗，神经炎斑块的形成明显减少（3）。在另一项研究中，我们研究的分子联系，底层缺氧和AD的发病机制。同样，使用都4G8抗Aβ的免疫组织化学染色和thioflavin S染色，我们发现，缺氧，促进形成含有神经炎斑的Aβ（4）。 <img src="/files/ftp_upload/2831/2831fig1.jpg" alt="图1"/> 图1。神经炎斑块的形成在AD转基因小鼠的免疫组织化学分析。APP23在9个月的年龄处死，并进行了解剖，固定和切片。在海马神经炎斑检测使用一种抗Aβ抗体4G8和使用ABC和DAB方法进行开发。 40X光镜与斑块。白色箭头指向的Aβ神经炎斑。 <img src="/files/ftp_upload/2831/2831fig2.jpg" alt="图2"/> 图2。 Thioflavin S染色在公元建模小鼠神经炎斑。APP23xPS45双转基因小鼠，8周龄处死，并进行了解剖，固定，并切片。在海马神经炎瘟疫被证实使用thioflavin小号荧光染色，在显微镜下可视化40X放大倍率。白色箭头指示thioflavin小号染色神经炎斑。

<ol>
	<li>Glenner, G. G., Wong, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alzheimer's+disease:+initial+report+of+the+purification+and+characterization+of+a+novel+cerebrovascular+amyloid+protein.">Alzheimer's disease: initial report of the purification and characterization of a novel cerebrovascular amyloid protein.</a> Biochem Biophys Res Commun. 120, 885-890 (1984).</li><li>Hardy, J., Selkoe, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+amyloid+hypothesis+of+Alzheimer's+disease:+progress+and+problems+on+the+road+to+therapeutics.">The amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics.</a> Science. 297, 353-356 (2002).</li><li>Qing, H., He, G., Ly, P. T., Fox, C. J., Staufenbiel, M., Cai, F., Zhang, Z., Wei, S., Sun, X., Chen, C. H., Zhou, W., Wang, K., 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=Valproic+acid+inhibits+Abeta+production,+neuritic+plaque+formation,+and+behavioral+deficits+in+Alzheimer's+disease+mouse+models.">Valproic acid inhibits Abeta production, neuritic plaque formation, and behavioral deficits in Alzheimer's disease mouse models.</a> J Exp Med. 205, 2781-2789 (2008).</li><li>Sun, X., He, G., Qing, H., Zhou, W., Dobie, F., Cai, F., Staufenbiel, M., Huang, L. E., 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=Hypoxia+facilitates+Alzheimer's+disease+pathogenesis+by+up-regulating+BACE1+gene+expression.">Hypoxia facilitates Alzheimer's disease pathogenesis by up-regulating BACE1 gene expression.</a> Proc Natl Acad Sci U S A. 103, 18727-18732 (2006).</li></ol>

所有实验均在与不列颠哥伦比亚省的动物护理和使用委员会和CIHR指引大学的规定进行。

采用免疫组织化学和素标记的4G8抗体污渍在公元神经炎斑为蓝本小鼠特异性。染色的结果，允许不同治疗组之间的斑块负荷的量化和比较。有几个关键步骤，可能会影响结果的。由于半侧大脑灌注前从头骨提取，应在提取过程中使用，以防止损害的半脑。此外，由于半脑是被动灌注，我们建议孵育至少48小时在4％煤灰4 ° C之前，淹没在30％的蔗糖溶液。另外，transcardial灌注可之前完成提取大脑。如果是固定的大脑，它应该有一个橡胶质地。大脑是不固定的正确的情况下，部分会很容易撕裂德Olomos解决方案或在染色程序。 4G8单克隆抗体反应的氨基酸残基的Aβ17-24和公认的核心序列（VFFAE）的抗原表位。由于4G8是从鼠种的，它往往给予了较高的背景染色。因此，我们选择了孵化表示稀释的部分，考虑到实现一个真正的信号，同时最大限度地减少背景染色。 此外免疫神经炎斑使用4G8抗体的检测，一个thioflavin S染色方法也可以用来识别斑块。 Thioflavin S是一个同质的染料混合物，从dehydrothiotoluidine甲基磺酸的结果。 Thioflavin非选择性结合蛋白质测试表的内容，如淀粉样蛋白的低聚物的。 thioflavin结合后，经历了它的发射光谱特性的蓝移。相反thioflavin小号单体形式结合不会引起蓝移，不能用荧光显微镜检测。 Thioflavin S染色提供了一个屏幕淀粉样蛋白的快速荧光强度允许少量的淀粉样蛋白沉积的良好的可视化。然而，关于thioflavin S染色的缺点是缺乏特异性。许多其他的组织成分，包括fibrinoids，透明，角蛋白，等，如含有丰富的测试版张，而对于这种染料亲和力。

<table border="1"><tbody><tr><th>试剂和设备的名称</th><th>公司</th><th>目录编号</th></tr><tr><td>多聚甲醛</td><td> Sigma - Aldrich公司</td><td> P6148 - 1KG </td></tr><tr><td> Polyvinylpyrrolidene </td><td> Sigma - Aldrich公司</td><td> PVP40 - 100G </td></tr><tr><td>蔗糖</td><td> Fisher Scientific则</td><td> S5 - 3 </td></tr><tr><td>乙二醇</td><td> Sigma - Aldrich公司</td><td> 324558 - 2L </td></tr><tr><td>组织TEK华侨城复合</td><td>樱花</td><td> 4583 </td></tr><tr><td> 88％的甲酸</td><td> Fisher Scientific则</td><td> A118P - 500 </td></tr><tr><td> TRITON X - 100 </td><td>仁川Biomedicals </td><td> 807426 </td></tr><tr><td>过氧化氢</td><td> Sigma - Aldrich公司</td><td> H - 1009 </td></tr><tr><td>生物素标记的4G8抗体</td><td> Cedarlane </td><td> SIG - 39240 - 500 </td></tr><tr><td>精英ABC试剂盒</td><td>矢量</td><td> PK - 6100 </td></tr><tr><td>入境事务处PACT民建联</td><td>矢量</td><td> SK - 4105 </td></tr><tr><td>二甲苯</td><td> Fisher Scientific则</td><td> X4 - 4 </td></tr><tr><td> Entellan </td><td> EM科学</td><td> 65037-71 </td></tr><tr><td> Superfrost * PLUS显微镜载玻片</td><td> Fisher Scientific则</td><td> 12-550-15 </td></tr><tr><td>低温恒温器</td><td>徕卡</td><td> CM - 3050 - S </td></tr><tr><td> MX35总理切片机刀片</td><td> Thermo Scientific的</td><td> 3052835 </td></tr><tr><td> Thioflavin小号</td><td> Sigma - Aldrich公司</td><td> T1892 - 25G </td></tr></tbody></table>

detection of neuritic plaques in alzheimer's disease mouse model

阿尔茨海默氏病（AD）是最常见的神经退行性疾病，导致老年痴呆症。神经炎斑块的形成是阿尔茨海默氏症的病理特点之一。神经炎斑的核心组成部分，是一个小的丝状蛋白质称为淀粉样β蛋白（Aβ）1，这是从连续的β-淀粉样蛋白前体蛋白（APP）的β-分泌和γ-分泌蛋白水解裂解所得。淀粉样蛋白假说，需要在AD 病理学 2的毒性机制作为基础，Aγ含斑块。事后分析神经炎斑块的存在，证实了AD的诊断。为了进一步Aγ神经生物学的认识，在AD的发病机制，不同的小鼠品系，在人类的APP基因表达AD相关的基因突变产生。根据疾病的严重程度，这些老鼠在不同的年龄发展神经炎斑。这些老鼠作为研究的发病机制及药物开发，可能会影响应用程序的处理途径和神经炎斑块的形成的宝贵工具。在这个协议中，我们采用免疫组织化学方法对AD模型小鼠神经炎斑的具体检测。我们将特别讨论中提取的半脑，多聚甲醛固定，cryosectioning，以及两种方法来检测神经质的斑块在AD转基因小鼠的制备方法：使用美国广播公司（ABC）和DAB法和荧光检测使用thiofalvin S染色方法免疫组化检测。

Watch this Scientific Journal Video about Detection of Neuritic Plaques in Alzheimer's Disease Mouse Model at JoVE.com

Detection of Neuritic Plaques in Alzheimer&#039;s Disease Mouse Model

AD的病理特征之一，是淀粉样β蛋白阳性神经炎斑的形成。在这个协议中，我们介绍两种方法来检测转基因AD模型小鼠神经炎斑：免疫组织化学检测使用美国广播公司（ABC）和DAB法和荧光检测使用thioflavin S染色方法。

在阿尔茨海默氏症小鼠模型神经炎斑检测

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

detection-of-neuritic-plaques-in-alzheimer-s-disease-mouse-model

Research

JoVE Journal

Neuroscience

Detection of Neuritic Plaques in Alzheimer's Disease Mouse Model

36.4K 次观看.  The University of British Columbia. AD的病理特征之一，是淀粉样β蛋白阳性神经炎斑的形成。在这个协议中，我们介绍两种方法来检测转基因AD模型小鼠神经炎斑：免疫组织化学检测使用美国广播公司（ABC）和DAB法和荧光检测使用thioflavin S染色方法。

视频: Detection of Neuritic Plaques in Alzheimer's Disease Mouse Model

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 &amp;#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

Selection of Aptamers for Amyloid &beta;-Protein, the Causative Agent of Alzheimer&#39;s Disease

Alzheimer's disease (AD) is a progressive, age-dependent, neurodegenerative disorder with an insidious course that renders its presymptomatic diagnosis difficult1. Definite AD diagnosis is achieved only postmortem, thus establishing presymptomatic, early diagnosis of AD is crucial for developing and administering effective therapies2,3.
Amyloid &beta;-protein (A&beta;) is central to AD pathogenesis. Soluble, oligomeric A&beta; assemblies are believed to affect neurotoxicity underlying synaptic dysfunction and neuron loss in AD4,5. Various forms of soluble A&beta; assemblies have been described, however, their interrelationships and relevance to AD etiology and pathogenesis are complex and not well understood6. Specific molecular recognition tools may unravel the relationships amongst A&beta; assemblies and facilitate detection and characterization of these assemblies early in the disease course before symptoms emerge. Molecular recognition commonly relies on antibodies. However, an alternative class of molecular recognition tools, aptamers, offers important advantages relative to antibodies7,8. Aptamers are oligonucleotides generated by in-vitro selection: systematic evolution of ligands by exponential enrichment (SELEX)9,10. SELEX is an iterative process that, similar to Darwinian evolution, allows selection, amplification, enrichment, and perpetuation of a property, e.g., avid, specific, ligand binding (aptamers) or catalytic activity (ribozymes and DNAzymes).
Despite emergence of aptamers as tools in modern biotechnology and medicine11, they have been underutilized in the amyloid field. Few RNA or ssDNA aptamers have been selected against various forms of prion proteins (PrP)12-16. An RNA aptamer generated against recombinant bovine PrP was shown to recognize bovine PrP-&beta;17, a soluble, oligomeric, &beta;-sheet-rich conformational variant of full-length PrP that forms amyloid fibrils18. Aptamers generated using monomeric and several forms of fibrillar &beta;2-microglobulin (&beta;2m) were found to bind fibrils of certain other amyloidogenic proteins besides &beta;2m fibrils19. Ylera et al. described RNA aptamers selected against immobilized monomeric A&beta;4020. Unexpectedly, these aptamers bound fibrillar A&beta;40. Altogether, these data raise several important questions. Why did aptamers selected against monomeric proteins recognize their polymeric forms? Could aptamers against monomeric and/or oligomeric forms of amyloidogenic proteins be obtained? To address these questions, we attempted to select aptamers for covalently-stabilized oligomeric A&beta;4021 generated using photo-induced cross-linking of unmodified proteins (PICUP)22,23. Similar to previous findings17,19,20, these aptamers reacted with fibrils of A&beta; and several other amyloidogenic proteins likely recognizing a potentially common amyloid structural aptatope21. Here, we present the SELEX methodology used in production of these aptamers21.

Selection of Aptamers for Amyloid &amp;#946;-Protein, the Causative Agent of Alzheimer&#039;s Disease

Aptamers are short ribo-/deoxyribo-oligonucleotides selected by in-vitro evolution methods based on affinity for a specific target. Aptamers are molecular recognition tools with versatile therapeutic, diagnostic, and research applications. We demonstrate methods for selection of aptamers for amyloid &beta;-protein, the causative agent of Alzheimer's disease.

淀粉样β-蛋白，阿尔茨海默氏病的病原体适配的选择

Alzheimer's disease (AD) is a progressive, age-dependent, neurodegenerative disorder with an insidious course that renders its presymptomatic diagnosis ...

Morris Water Maze Test for Learning and Memory Deficits in Alzheimer&#39;s Disease Model Mice

The Morris Water Maze (MWM) was first established by neuroscientist Richard G. Morris in 1981 in order to test hippocampal-dependent learning, including acquisition of spatial memoryand long-term spatial memory 1. The MWM is a relatively simple procedure typically consisting of six day trials, the main advantage being the differentiation between the spatial (hidden-platform) and non-spatial (visible platform) conditions 2-4. In addition, the MWM testing environment reduces odor trail interference 5. This has led the task to be used extensively in the study of the neurobiology and neuropharmacology of spatial learning and memory. The MWM plays an important role in the validation of rodent models for neurocognitive disorders such as Alzheimer&#x2019;s Disease 6, 7. In this protocol we discussed the typical procedure of MWM for testing learning and memory and data analysis commonly used in Alzheimer&#x2019;s disease transgenic model mice.

Morris Water Maze Test for Learning and Memory Deficits in Alzheimer&#039;s Disease Model Mice

The Morris Water Maze is a behavioral task to test hippocampal-dependent learning and memory. It has been widely used in the study of neurobiology, neuropharmacology and neurocognitive disorders in rodent models.

在阿尔茨海默氏病模型小鼠学习和记忆障碍的Morris水迷宫测试

The Morris Water Maze (MWM) was first established by neuroscientist Richard G. Morris in 1981 in order to test hippocampal-dependent learning, including ...

Detection of Microregional Hypoxia in Mouse Cerebral Cortex by Two-photon Imaging of Endogenous NADH Fluorescence

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. Here we present a visualized experimental and methodological protocol to directly visualize microregional tissue hypoxia and to infer perivascular oxygen gradients in the mouse cortex. It is based on the non-linear relationship between nicotinamide adenine dinucleotide (NADH) endogenous fluorescence intensity and oxygen partial pressure in the tissue, where observed tissue NADH fluorescence abruptly increases at tissue oxygen levels below 10 mmHg1. We use two-photon excitation at 740 nm which allows for concurrent excitation of intrinsic NADH tissue fluorescence and blood plasma contrasted with Texas-Red dextran. The advantages of this method over existing approaches include the following: it takes advantage of an intrinsic tissue signal and can be performed using standard two-photon in vivo imaging equipment; it permits continuous monitoring in the whole field of view with a depth resolution of ~50 &mu;m. We demonstrate that brain tissue areas furthest from cerebral blood vessels correspond to vulnerable watershed areas which are the first to become functionally hypoxic following a decline in vascular oxygen supply. This method allows one to image microregional cortical oxygenation and is therefore useful for examining the role of inadequate or restricted tissue oxygen supply in neurovascular diseases and stroke.

Here we describe a method to directly visualize microregional tissue hypoxia in the mouse cortex in vivo. It is based on concurrent two-photon imaging of nicotinamide adenine dinucleotide (NADH) and the cortical microcirculation. This method is useful for high resolution analysis of tissue oxygen supply.

检测内源NADH的荧光双光子成像的小鼠大脑皮层Microregional缺氧

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. ...

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 ...

Generalized Psychophysiological Interaction (PPI) Analysis of Memory Related Connectivity in Individuals at Genetic Risk for Alzheimer's Disease

In neuroimaging, functional magnetic resonance imaging (fMRI) measures the blood-oxygenation-level dependent (BOLD) signal in the brain. The degree of correlation of the BOLD signal in spatially independent regions of the brain defines the functional connectivity of those regions. During a cognitive fMRI task, a psychophysiological interaction (PPI) analysis can be used to examine changes in the functional connectivity during specific contexts defined by the cognitive task. An example of such a task is one that engages the memory system, asking participants to learn pairs of unrelated words (encoding) and recall the second word in a pair when presented with the first word (retrieval). In the present study, we used this type of associative memory task and a generalized PPI (gPPI) analysis to compare changes in hippocampal connectivity in older adults who are carriers of the Alzheimer's disease (AD) genetic risk factor apolipoprotein-E epsilon-4 (APOE&#949;4). Specifically, we show that the functional connectivity of subregions of the hippocampus changes during encoding and retrieval, the two active phases of the associative memory task. Context-dependent changes in functional connectivity of the hippocampus were significantly different in carriers of APOE&#949;4 compared to non-carriers. PPI analyses make it possible to examine changes in functional connectivity, distinct from univariate main effects, and to compare these changes across groups. Thus, a PPI analysis may reveal complex task effects in specific cohorts that traditional univariate methods do not capture. PPI analyses cannot, however, determine directionality or causality between functionally connected regions. Nevertheless, PPI analyses provide powerful means for generating specific hypotheses regarding functional relationships, which can be tested using causal models. As the brain is increasingly described in terms of connectivity and networks, PPI is an important method for analyzing fMRI task data that is in line with the current conception of the human brain.

Generalized Psychophysiological Interaction PPI Analysis of Memory Related Connectivity in Individuals at Genetic Risk for Alzheimer&#039;s Disease

This manuscript describes how to implement a psychophysiological interaction analysis to reveal task-dependent changes in functional connectivity between a selected seed region and voxels in other regions of the brain. Psychophysiological interaction analysis is a popular method to examine task effects on brain connectivity, distinct from traditional univariate activation effects.

广义心理相互作用 (PPI) 分析阿尔茨海默病遗传危险个体的记忆相关连通性

In neuroimaging, functional magnetic resonance imaging (fMRI) measures the blood-oxygenation-level dependent (BOLD) signal in the brain. The degree of ...

Using Enzyme-based Biosensors to Measure Tonic and Phasic Glutamate in Alzheimer's Mouse Models

Neurotransmitter disruption is often a key component of diseases of the central nervous system (CNS), playing a role in the pathology underlying Alzheimer's disease, Parkinson's disease, depression, and anxiety. Traditionally, microdialysis has been the most common (lauded) technique to examine neurotransmitter changes that occur in these disorders. But because microdialysis has the ability to measure slow 1-20 minute changes across large areas of tissue, it has the disadvantage of invasiveness, potentially destroying intrinsic connections within the brain and a slow sampling capability. A relatively newer technique, the microelectrode array (MEA), has numerous advantages for measuring specific neurotransmitter changes within discrete brain regions as they occur, making for a spatially and temporally precise approach. In addition, using MEAs is minimally invasive, allowing for measurement of neurotransmitter alterations in vivo. In our laboratory, we have been specifically interested in changes in the neurotransmitter, glutamate, related to Alzheimer's disease pathology. As such, the method described here has been used to assess potential hippocampal disruptions in glutamate in a transgenic mouse model of Alzheimer's disease. Briefly, the method used involves coating a multi-site microelectrode with an enzyme very selective for the neurotransmitter of interest and using self-referencing sites to subtract out background noise and interferents. After plating and calibration, the MEA can be constructed with a micropipette and lowered into the brain region of interest using a stereotaxic device. Here, the method described involves anesthetizing rTg(TauP301L)4510 mice and using a stereotaxic device to precisely target sub-regions (DG, CA1, and CA3) of the hippocampus.

Using Enzyme-based Biosensors to Measure Tonic and Phasic Glutamate in Alzheimer&#039;s Mouse Models

Here, we describe the setup, software navigation, and data analysis for a spatially and temporally precise method of measuring tonic and phasic extracellular glutamate changes in vivo using enzyme-linked microelectrode arrays (MEA).

使用基于酶的生物传感器来衡量进补和阶段性谷氨酸在阿尔茨海默氏症小鼠模型

Neurotransmitter disruption is often a key component of diseases of the central nervous system (CNS), playing a role in the pathology underlying ...

Neurobehavioral Assessments in a Mouse Model of Neonatal Hypoxic-ischemic Brain Injury

We performed unilateral carotid artery occlusion on CD-1 mice to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of neonatal HI brain injury by studying neurobehavioral functions in these mice compared to non-operated (i.e., normal) mice. During the study, Rice-Vannucci&#39;s method was used to induce neonatal HI brain damage in postnatal day 7-10 (P7-10) mice. The HI operation was performed on the pups by unilateral carotid artery ligation and exposure to hypoxia (8% O2 and 92% N2 for 90 min). One week after the operation, the damaged brains were evaluated with the naked eye through the semi-transparent skull and were categorized into subgroups based on the absence (&#34;no cortical injury&#34; group) or presence (&#34;cortical injury&#34; group) of cortical injury, such as a lesion in the right hemisphere. On week 6, the following neurobehavioral tests were performed to evaluate the cognitive and motor functions: passive avoidance task (PAT), ladder walking test, and grip strength test. These behavioral tests are helpful in determining the effects of neonatal HI brain injury and are used in other mouse models of neurodegenerative diseases. In this study, neonatal HI brain injury mice showed motor deficits that corresponded to right hemisphere damage. The behavioral test results are relevant to the deficits observed in human neonatal HI patients, such as cerebral palsy or neonatal stroke patients. In this study, a mouse model of neonatal HI brain injury was established and showed different degrees of motor deficits and cognitive impairment compared to non-operated mice. This work provides basic information on the HI mouse model. MRI images demonstrate the different phenotypes, separated according to the severity of brain damage by motor and cognitive tests.

We performed unilateral carotid artery occlusion on postnatal day 7-10 CD-1 mouse pups to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of HI brain injury. We studied neurobehavioral functions in these mice compared to non-operated normal mice.

小鼠新生儿缺氧缺血性脑损伤模型的神经行为评估

We performed unilateral carotid artery occlusion on CD-1 mice to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of neonatal HI ...

Labeling and Imaging of Amyloid Plaques in Brain Tissue Using the Natural Polyphenol Curcumin

Deposition of amyloid beta protein (A&#946;) in extra- and intracellular spaces is one of the hallmark pathologies of Alzheimer&#39;s disease (AD). Therefore, detection of the presence of A&#946; in AD brain tissue is a valuable tool for developing new treatments to prevent the progression of AD. Several classical amyloid binding dyes, fluorochrome, imaging probes, and A&#946;-specific antibodies have been used to detect A&#946; histochemically in AD brain tissue. Use of these compounds for A&#946; detection is costly and time consuming. However, because of its intense fluorescent activity, high-affinity, and specificity for A&#946;, as well as structural similarities with traditional amyloid binding dyes, curcumin (Cur) is a promising candidate for labeling and imaging of A&#946; plaques in postmortem brain tissue. It is a natural polyphenol from the herb Curcuma longa. In the present study, Cur was used to histochemically label A&#946; plaques from both a genetic mouse model of 5x familial Alzheimer&#39;s disease (5xFAD) and from human AD tissue within a minute. The labeling capability of Cur was compared to conventional amyloid binding dyes, such as thioflavin-S (Thio-S), Congo red (CR), and Fluoro-jade C (FJC), as well as A&#946;-specific antibodies (6E10 and A11). We observed that Cur is the most inexpensive and quickest way to label and image A&#946; plaques when compared to these conventional dyes and is comparable to A&#946;-specific antibodies. In addition, Cur binds with most A&#946; species, such as oligomers and fibrils. Therefore, Cur could be used as the most cost-effective, simple, and quick fluorochrome detection agent for A&#946; plaques.

Curcumin is an ideal fluorophore for labeling and imaging of amyloid beta protein plaques in brain tissue due to its preferential binding to amyloid beta protein as well as its structural similarities with other traditional amyloid binding dyes. It can be used to label and image amyloid beta protein plaques more efficiently and inexpensively than traditional methods.

使用天然多酚姜黄素对脑组织淀粉样斑块进行标记和成像

Deposition of amyloid beta protein (A&#946;) in extra- and intracellular spaces is one of the hallmark pathologies of Alzheimer&#39;s disease (AD). ...

Stereological Estimation of Cholinergic Fiber Length in the Nucleus Basalis of Meynert of the Mouse Brain

The length of cholinergic or other neuronal axons in various brain regions are often correlated with the specific function of the region. Stereology is a useful method to quantify neuronal profiles of various brain structures. Here we provide a software-based stereology protocol to estimate the total length of cholinergic fibers in the nucleus basalis of Meynert (NBM) of the basal forebrain. The method uses a space ball probe for length estimates. The cholinergic fibers are visualized by choline acetyltransferase (ChAT) immunostaining with the horseradish peroxidase-diaminobenzidine (HRP-DAB) detection system. The staining protocol is also valid for fiber and cell number estimation in various brain regions using stereology software. The stereology protocol can be used for estimation of any linear profiles such as cholinoceptive fibers, dopaminergic/catecholaminergic fibers, serotonergic fibers, astrocyte processes, or even vascular profiles.

Neuronal fiber length within a three-dimensional structure of a brain region is a reliable parameter to quantify specific neuronal structural integrity or degeneration. This article details a stereological quantification method to measure cholinergic fiber length within the nucleus basalis of Meynert in mice as an example.

小鼠大脑细胞核巴沙里斯中胆碱能纤维长度的立体估计

The length of cholinergic or other neuronal axons in various brain regions are often correlated with the specific function of the region. Stereology is a ...