Name: stereological estimation of cholinergic fiber length in the nucleus basalis of meynert of the mouse brain
Uploaded: 2020-02-05T12:00:00
Description: Watch this Scientific Journal Video about Stereological Estimation of Cholinergic Fiber Length in the Nucleus Basalis of Meynert of the Mouse Brain at JoVE.com

这项工作得到了退伍军人事务部医学研究与发展处（功绩审查1I01 BX001067-01A2）、阿尔茨海默氏症协会（NPSPAD-11-202149）和中西部生物医学资源对W.Z.S.的资助。研究基金会。

使用这些动物的所有程序都已获得堪萨斯城退伍军人事务医疗中心机构动物护理和使用委员会的批准。实验中，18个月大的小鼠过度表达瑞典突变β-淀粉样蛋白前体蛋白（APPswe）及其C57/BL6 WT杂物。育种和基因分型的细节在He等人8。
1. 灌注和组织处理
<ol><li>使用氯胺酮（100毫克/千克）和木拉津（10毫克/千克）注射，对小鼠进行麻醉。捏脚趾，以确认缺乏反?...

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在这里，我们演示了一种使用空间球（球）探测器估计NBM中胆碱能纤维密度的方法。此探头估计了感兴趣区域的总光纤长度。总长度可以除以区域的体积来获得光纤密度。为了估计区域的体积，使用了卡瓦利埃里点计数方法。Cavalieri 点计数方法是对任何区域的 3D 参考体积的无偏和高效的估计。该方法通过计算点（表示面积分数）计算截面上截面上的面积估计值，然后乘以分析为 11</...

大脑中神经纤维长度和/或密度的定量估计是神经病理学研究的重要参数。不同大脑区域的胆碱能、多巴胺能和血清素轴子的长度通常与该区域的特定功能相关。由于这些斧子的分布通常是异构的，因此使用基于设计的立体学来避免采样过程中的偏差。立体学的空间球探针被设计为在感兴趣的区域提供高效可靠的线状结构，如神经元纤维。探头制作一个虚拟球体，在组织中系统地施加，以测量与探头表面的线交交。由于不可能将球体探头放入组织进行分析，因此市售软件提供了一个虚拟的三维（3D）球体，基本上就是代表球体探头表面的一系列不同直径的同心圆。
选择性胆碱能神经退化是阿尔茨海默氏病（AD）2、3、4的一贯特征之一。功能失调的胆碱能传输被认为是AD认知衰退的一个致能因素。胆碱功能障碍在许多其他精神障碍中也很明显，如帕金森氏症、成瘾和精神分裂症。在动物模型中研究了胆碱能神经退化的不同方面（例如，减少乙酰胆碱5，ChAT蛋白6，淀粉样斑块6附近的胆碱能纤维神经退化，减少胆碱能纤维和突触性静脉曲张7，8）。纤维退化被认为比神经元损失更早发生，因为在研究中并不总是观察到胆碱能神经元损失。大多数胆碱能神经元在基底前脑和脑干，其轴突项目到各种大脑区域，如皮质和海马。NBM位于基础前脑，发现是AD中常见的大脑区域之一。
立体学的分馏方法基于多级组织的系统随机采样。截面采样分数（SSF）是立体学分馏法中基于计算机的节面系统采样。面积采样分数 （ASF） 是该节中感兴趣区域的分馏。厚度采样分数 （TSF） 是截面厚度的分馏。空间球探头允许我们在分数位置对 3D 球体中感兴趣的轮廓进行量化。在这里，我们使用空间球探针来估计小鼠大脑NBM中胆碱能纤维的总长度，以说明这些过程。目前的方案提供了有关组织处理的细节，立体学的采样方法，使用ChAT抗体的免疫组织化学染色，以及用于估计小鼠大脑NBM中胆碱能纤维长度和纤维密度的无偏立体学。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>ABC kit</td>
			<td>Vector Laboratories</td>
			<td>PK6100</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-ChAT Antibody</td>
			<td>Millipore, MA, USA</td>
			<td>AB144P</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine anti-goat IgG-B</td>
			<td>Santacruz Biotechnology</td>
			<td>SC-2347</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum, Adult</td>
			<td>Sigma-Aldrich, St. Louis, MO, USA</td>
			<td>B9433</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Lieca Microsystems, Buffalo Grove, IL, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate Buffered Saline</td>
			<td>Sigma-Aldrich, St. Louis, MO, USA</td>
			<td>D5652</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylene Glycol</td>
			<td>Sigma-Aldrich, St. Louis, MO, USA</td>
			<td>324558</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich, St. Louis, MO, USA</td>
			<td>G2025</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen Peroxide</td>
			<td>Sigma-Aldrich, St. Louis, MO, USA</td>
			<td>H1009</td>
			<td></td>
		</tr>
		<tr>
			<td>Immpact-DAB kit</td>
			<td>Vector Laboratories</td>
			<td>SK4105</td>
			<td>Enhanced DAB peroxidase substrate solution</td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Westward Pharmaceuticals, NJ, USA</td>
			<td>0143-9509-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Lieca Microsystems, Buffalo Grove, IL, USA</td>
			<td>AF6000</td>
			<td>Equipped with motorized stage and IMI-tech color digital camera</td>
		</tr>
		<tr>
			<td>Optimum cutting temperature (O.C.T.) embedding medium</td>
			<td>Electron Microscopy Sciences, PA, USA</td>
			<td>62550-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich, St. Louis, MO, USA</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr>
			<td>Permount mounting medium</td>
			<td>Electron Microscopy Sciences, PA, USA</td>
			<td>17986-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereologer Software</td>
			<td>Stereology Resource Center, Inc. St. Petersburg, FL, USA</td>
			<td>Stereologer2000</td>
			<td>Installed on a Dell Desktop computer.</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich, St. Louis, MO, USA</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizma Base</td>
			<td>Sigma-Aldrich, St. Louis, MO, USA</td>
			<td>T1503</td>
			<td>Tris base</td>
		</tr>
		<tr>
			<td>Trizma hydrochloride</td>
			<td>Sigma-Aldrich, St. Louis, MO, USA</td>
			<td>T5941</td>
			<td>Tris hydrochloride</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Bayer, Leverkusen, Germany</td>
			<td>Rompun</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylenes, Histological grade</td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>534056</td>
			<td></td>
		</tr>
	</tbody>
</table>

stereological estimation of cholinergic fiber length in the nucleus basalis of meynert of the mouse brain

不同大脑区域的胆碱能或其他神经元轴子的长度通常与该区域的特定功能相关。立体学是量化各种大脑结构神经元特征的有用方法。在这里，我们提供一个基于软件的立体学协议，以估计基底前脑Meynert（NBM）细胞核基底中胆碱能纤维的总长度。该方法使用空间球探头进行长度估计。胆碱能纤维通过胆碱乙酰转移酶（ChAT）免疫染色与马萝卜过氧化物酶-二氨基苯甲酸酯（HRP-DAB）检测系统可视化。使用立体学软件，染色协议对于各种大脑区域的纤维和细胞数估计也有效。立体学协议可用于估计任何线性轮廓，如胆囊状纤维、多巴胺可列菌纤维/二代胺纤维、血清素纤维、星形细胞过程，甚至血管型材。

代表性结果如图1和图5所示。被解码为APpswe群（APP）的C组，其纤维长度（图5B）和纤维长度密度（图5C）与其野生型（WILD）杂物相比，具有显著降低。结果表明，所分析的两组国家/地区NBM的体积无显著差异（图5A）。
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Watch this Scientific Journal Video about Stereological Estimation of Cholinergic Fiber Length in the Nucleus Basalis of Meynert of the Mouse Brain at JoVE.com

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

大脑区域三维结构内的神经元纤维长度是量化特定神经元结构完整性或退化的可靠参数。本文详细介绍了一种立体定量方法，以小鼠Meynert细胞核基质内胆碱能纤维长度为例。

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 ...

许多神经系统疾病的神经分裂是一个慢性过程，神经纤维损失往往发生在细胞体丧失之前。三维神经纤维的病理学定量为检测早期神经退行性变化提供了一种敏感而准确的方法。该技术利用可靠、高效的基于计算机的空球无偏分馏，测量3D中神经元纤维等线样结构的长度。演示程序将由博士后、我们的立体学专家普拉巴卡尔·辛格和实验室研究助理大卫·彭完成。在确认麻醉小鼠对踏板反射缺乏反应后，转心性循环与大约 50 毫升的冰冷 0.1 摩尔 DPBS，随后在 0.1 摩尔 PBS 中循环使用约 25 毫升 4%PFA。灌注结束时，在4摄氏度下将大脑固定在新鲜4%PFA的容器中24小时。第二天，用三到两分钟的新鲜DPBS洗净大脑，并在0.1 DPBS溶液中将大脑转移到15%蔗糖溶液中过夜。第二天早上，将大脑放入30%蔗糖溶液和0.1摩尔DPBS中，在4摄氏度下48小时。在孵育结束时，将大脑转移到预设置为零下20摄氏度的低温腔室。大脑可以保存在密封的管子里，储存在零下80摄氏度。对于剖面，将组织样品嵌入最佳切割温度嵌入介质中，并将样品安装到试样盘上。然后在低温的日冕平面上获取30微米厚的连续部分，将每个部分转移到含有低温保护溶液的24孔培养板的单个孔中，为零下20摄氏度的储存做准备。要识别每个大脑的第一个和最后一个部分，比较形态特征与标准的小鼠大脑图集。NBM 从百分之零零零零毫米开始，到负 1.6 毫米。选择每第八节共产生六到七个部分，并允许一个适当的误差系数的体积和光纤长度估计。对于组织部分的免疫组织化学标记，在三叶以下盐中 0.1% Triton X-100 中阻断任何非特异性结合后，在 4 摄氏度下用感兴趣的初级抗体标记这些部分 48 小时。在孵化结束时，每次洗涤用新鲜三酯缓冲盐水洗三次，三分钟，然后用适当的生物基化二次抗体在室温下孵育一小时。在孵化结束时，按照制造商的协议，在新鲜的Tris缓冲盐水中洗涤三次，然后使用Avidin生物素过氧化物酶复合物和DAB过氧化物酶基板溶液进行染色。洗涤后，将一个脑组织样本的所有部分安装到一个明胶化幻灯片上，让样品风干。要使部分脱水，请将样品浸入两次，每次稀释五分钟，如所示，在连续上升乙醇溶液中，然后用两次10分钟的二甲苯洗涤进行清除。然后使用适当的安装介质将盖玻片放在幻灯片上。对于立体学，在软件中打开一个新的研究。将打开一个研究初始化对话框。填写研究信息，确认选择了基于多级分数。双击音量可打开音量对话框。命名感兴趣的要素并选择区域点计数探测器，然后单击下一步。双击长度并提供功能的名称。选择球体探测并单击下一步。在"初始化"框中，若要在开始研究之前对组进行编码，请将从第一节开始包含感兴趣区域的节到包含感兴趣区域的最后一节和包含感兴趣区域的节采样间隔设置为 8 的部分总数。单击"旁边"以打开探针初始化对话框，该对话框将自动设置区域选择和体积估计的最低放大倍率，并确认设置。单击预览以检查能否在选定的较低放大率下识别感兴趣的区域。输入区域卷分数每个点 50，000 微米立方体，然后单击预览并完成。在对象高放大倍率下，将长度设置为 63 倍，双击长度以打开长度球体对话框。将球的直径设置为 10 微米，然后单击完成。为帧区域、帧高度、防护区域和帧间距设置适当的值。然后单击"下一步"，然后按照软件提供的说明进行操作。要插入第一部分，请将幻灯片放在 5X 目标下的显微镜舞台上，然后单击绿色箭头以在 NBM 周围绘制任意边界以定义感兴趣区域。单击绿色箭头并更改 63 倍目标以获取当前分数的光纤测量值。将显示节厚度对话框。使用手动 z 轴对焦旋钮设置截面的顶部和底部表面，以测量截面的实际厚度，然后单击完成。在剖面的框架高度中缓慢地将 z 轴从上到下移动，以标记虚拟球体探头表面的所有相交光纤。标记所有光纤后，单击"旁边"以前进到下一个位置。完成所有分数后，单击绿色箭头。软件将要求插入下一节。使用 5X 目标将幻灯片上的下一个样本集中到焦点上，并重复刚刚演示的光纤测量。测量所有部分后，软件将生成显示误差值系数的情况的结果。如果误差系数可以接受，请继续执行下一个情况。如果误差系数不可接受，软件将提供更改某些参数的建议。完成所有案例后，将生成每个案例和组的结果。在这个具有代表性的实验中，瑞典突变β淀粉样蛋白蛋白组表明，与野生型垃圾伴侣相比，纤维长度和纤维长度密度显著降低。结果表明，两组分析的NBM体积无显著差异。该技术要求在正确的方向和良好的染色方法与潜伏期适当的采集。立体学方案可用于估计任何线性轮廓，如其他神经元纤维、星细胞过程，甚至血管轮廓。

Research

JoVE Journal

Neuroscience

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

有丝分裂中的不同发育阶段小鼠胚胎皮层实时成像

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

科研

神经科学

Telomerase Activity in the Various Regions of Mouse Brain: Non-Radioactive Telomerase Repeat Amplification Protocol (TRAP) Assay

Telomerase Activity in the Various Regions of Mouse Brain: Non-Radioactive Telomerase Repeat Amplification Protocol TRAP Assay

在小鼠大脑的各个区域端粒酶活性：非放射性端粒酶重复序列扩增法（TRAP）分析

Telomerase, a ribonucleoprotein, is responsible for maintaining the telomere length and therefore promoting genomic integrity, proliferation, and ...

Analysis of Gene Expression Changes in the Rat Hippocampus After Deep Brain Stimulation of the Anterior Thalamic Nucleus

基因表达的变化大鼠海马后，前丘脑核脑深部刺激分析

Deep brain stimulation (DBS) surgery, targeting various regions of the brain such as the basal ganglia, thalamus, and subthalamic regions, is an effective ...

Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

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 ...

Live Imaging of the Ependymal Cilia in the Lateral Ventricles of the Mouse Brain

室管膜纤毛在小鼠脑侧脑室实时成像

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various ...

Microelectrode Guided Implantation of Electrodes into the Subthalamic Nucleus of Rats for Long-term Deep Brain Stimulation

微电极引导电极植入到大鼠丘脑底核的长期深部脑刺激

Deep brain stimulation (DBS) is a widely used and effective therapy for several neurologic disorders, such as idiopathic Parkinson&#8217;s disease, ...

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

小容积回收-灌注-浸没式室系统中维持的急性海马切片的突触可塑性记录

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...

Stereological Estimation of Dopaminergic Neuron Number in the Mouse Substantia Nigra Using the Optical Fractionator and Standard Microscopy Equipment

用光学分馏塔和标准显微镜设备对小鼠黑质多巴胺能神经元数的视学估计

In pre-clinical Parkinson&#39;s disease research, analysis of the nigrostriatal tract, including quantification of dopaminergic neuron loss within the ...