Name: stereological estimation of dopaminergic neuron number in the mouse substantia nigra using the optical fractionator and standard microscopy equipment
Uploaded: 2017-09-01T14:00:00
Description: Watch this Scientific Journal Video about Stereological Estimation of Dopaminergic Neuron Number in the Mouse Substantia Nigra Using the Optical Fractionator and Standard Microscopy Equipment at JoVE.

作者感谢 Keali hm、路易莎 Frieß和海克 Menzel 的专家技术援助;海尔 Brünner 的动物护理;和 Chistopher s 病房为 ImageJ 软件的光 disector 网格插件的世代和发行。

对动物的照顾和使用的所有适用的国际、国家和/或机构准则都得到遵守。该议定书由德国 Wuerzburg Regierung Unterfranken 的地方当局批准. 
 1. 组织处理和免疫组化 <ol> <li> 安乐与 CO 2 或任何其他已批准方法的小鼠. </li>
	<li> 灌注六12周老 C56Bl/6N 雄性小鼠 transcardially 10 毫升0.1 米的磷酸盐缓冲盐水 (pbs) 使用25克针和输液泵, 其次70毫升4% 甲醛 (粉煤灰) 在 0.1 M PBS. 
	<strong...

<ol>
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视学从组织加工开始。在视学分析过程中, 必须小心地进行 SN 组织的连续切割, 以防止节段丢失。另外, 一个重要的步骤是标记一个半球, 以便在执行视学时区分左 SN 的右侧。在脑干上部放置一个小孔, 在所提出的研究中产生了最好的结果。此外, 由于使用光学分馏塔方法, 要求组织被切割在厚的部分约30-40 µm, 而不是5-10 µm 更常用的免疫组化, 抗体和其他培养液组织渗透期间免疫组织化学染色必须?...

神经元细胞数的定量是临床前帕金森病研究的关键, 以确定黑质 (SN)1,2内神经的水平。对感兴趣区域中的单元格数的无偏视学估计被认为是金标准3、4、5。
在无偏视学的出现之前, 部分神经元的数量通过操纵计数的细胞剖面来进行评估, 以纠正神经元在一节中进入视线的可变概率。最常用的方法之一是修正由6所描述的量化单元计数。如果在相邻的薄片中发现同一单元格的碎片, 则此方法试图考虑到单元格可以多次量化。因此, 在计算单元的形状、大小和方向方面需要假设的公式7,8。但是, 这些假设通常没有实现, 因此导致了系统的错误和与实际单元格数 (即偏倚) 的背离。此外, 还不能通过附加采样3来减少偏差。
对于使用光学分馏塔的细胞数的视学估计, 应用数学原理直接在定义的3维体积中直接估计细胞数。这种方法的优点是它不涉及被计算单元的形状、大小和方向的假设。因此, 估计的单元格数更接近于真实值, 并且随着样本大小的增加 (即,无偏)3变得更接近。由于使用视学保持方法无偏时必须遵循许多规则, 因此已开发现成的商业视学系统 (供审查, 请参阅 20054)。专门的视学系统实现了设计视学方法与先验定义的探针和抽样方案的视学评估, 导致独立的形状, 大小, 空间分布, 和方向要分析的单元格4,9。然而, 商业上可用的视学系统是昂贵的;这可能会限制新研究的实施。
本研究的目的是开发一种可用的技术设计视学估计多巴胺能细胞计数在小鼠 SN, 采用光学分馏塔方法和使用标准显微镜设备 (即,光显微镜, 标准显微镜软件, 和机动的 x, y, z 阶段)。为此, 提出了如何处理小鼠脑组织和如何使用设计无偏视学估计 SN 细胞数的 step-by 步骤指南。此外, 还提供了计算估计单元数和误差系数 (CE) 的模板。
这里描述的方法不限于对 SN 的分析, 但可以适应在其他的解剖定义的老鼠或老鼠大脑区域使用。例如, 无偏视学被用来估计海马体的神经元细胞数10和轨迹斑11。此外, 还可以评估除神经元以外的其他细胞类型, 如星状细胞12和小胶质红细胞13。因此, 这种方法对于打算在研究中实现无偏视学的科学家是有用的, 但他们不愿意花很多钱购买视学系统。

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	<tbody>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">Paxinos mouse atlas</td>
			<td style="width:217px;"></td>
			<td style="width:119px;"></td>
			<td style="width:467px;">The Mouse Brain George Paxinos Keith B.J.FranklinCopyright @2001 by Academic Press CD Rom designet &#38; created by Paul Halasz</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">brain matrix slicer mouse</td>
			<td style="width:217px;">Zivic Instruments&#160;</td>
			<td style="width:119px;">BSMAS 001-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">paraformaldehyde</td>
			<td style="width:217px;">Merck</td>
			<td style="width:119px;">1040051000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">sucrose /D(+) Saccharose&#160;</td>
			<td style="width:217px;">Roth</td>
			<td style="width:119px;">4621.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">isopentane</td>
			<td style="width:217px;">Roth</td>
			<td style="width:119px;">3927.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">glycerol</td>
			<td style="width:217px;">Merck</td>
			<td style="width:119px;">1040931000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethanol</td>
			<td style="width:217px;">Sigma Aldrich</td>
			<td style="width:119px;">32205-1L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Name</td>
			<td style="width:217px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">phosphate buffered saline ingredients:</td>
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			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">sodium chloride</td>
			<td style="width:217px;">Sigma Aldrich</td>
			<td style="width:119px;">31434-1KG-R</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">potassium dihydrogen phosphate</td>
			<td style="width:217px;">Merck</td>
			<td style="width:119px;">1048731000</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">di-sodium hydrogen phosphate dihydrate</td>
			<td style="width:217px;">Merck</td>
			<td style="width:119px;">1065801000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">potassium chloride</td>
			<td style="width:217px;">Merck</td>
			<td style="width:119px;">1049360500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">normal goat serum</td>
			<td style="width:217px;">Dako</td>
			<td style="width:119px;">X0907</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">bovine serum albumin</td>
			<td style="width:217px;">Sigma&#160;</td>
			<td style="width:119px;">A4503-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Triton X-100</td>
			<td style="width:217px;">Sigma Aldrich</td>
			<td style="width:119px;">X100-100ml</td>
			<td>detergent</td>
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		<tr height="63">
			<td height="63" style="height:63px;width:216px;">3,3-Diaminobenzidine-tetrahydrochlorid/DAB tablets 10mg pH 7.0</td>
			<td style="width:217px;">Kem En Tec</td>
			<td style="width:119px;">4170</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:216px;">H2O2/ Hydrogen peroxide 30%</td>
			<td style="width:217px;">Merck</td>
			<td style="width:119px;">1072090250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">avidin/biotin reagent</td>
			<td style="width:217px;">Thermo Scientific</td>
			<td style="width:119px;">32050</td>
			<td>Standard Ultra Sensitive ABC Staining Kit, 1:100</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">rabbit anti mouse tyrosine hydroxylase antibody</td>
			<td style="width:217px;">abcam</td>
			<td style="width:119px;">Ab112</td>
			<td>1:1000</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">biotinylated goat-anti-rabbit IgG H+L</td>
			<td style="width:217px;">vector laboratories</td>
			<td style="width:119px;">BA-1000</td>
			<td>1:100</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">StereoInvestigator version 11.07</td>
			<td style="width:217px;">MBF</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BX53 microscope</td>
			<td style="width:217px;">Olympus</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Visiview</td>
			<td style="width:217px;">Visitron Systems GmbH</td>
			<td style="width:119px;">3.3.0.2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Axiophot2</td>
			<td style="width:217px;">Zeiss</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ImageJ software</td>
			<td style="width:217px;">NIH</td>
			<td style="width:119px;">Version 4.7</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tissue-TEK OCT</td>
			<td style="width:217px;">Sakura</td>
			<td style="width:119px;">4583</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">dry ice</td>
			<td style="width:217px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">grid overlay plugin</td>
			<td style="width:217px;">Wayne Rasband</td>
			<td style="width:119px;"></td>
			<td>https://imagej.nih.gov/ij/plugins/graphic-overlay.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">cell counter plugin</td>
			<td style="width:217px;">Kurt de Vos</td>
			<td style="width:119px;"></td>
			<td>https://imagej.nih.gov/ij/plugins/cell-counter.html).&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">optical disector macro</td>
			<td style="width:217px;">Christopher Ward</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">C57Bl/6N male mice</td>
			<td style="width:217px;">Charles River, Germany</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">SuperFrost Plus coated object slides</td>
			<td style="width:217px;">Langenbrinck, Germany</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">25G needle Microlance 3</td>
			<td style="width:217px;">BD&#160;</td>
			<td align="right" style="width:119px;">300400</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">REGLO Analog Infusion pump</td>
			<td style="width:217px;">Ismatec</td>
			<td style="width:119px;">ISM 829</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">StereoInvestigator system</td>
			<td style="width:217px;"></td>
			<td style="width:119px;"></td>
			<td style="width:467px;">StereoInvestigator version 11.07</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BX53 microscope</td>
			<td style="width:217px;"></td>
			<td style="width:119px;"></td>
			<td style="width:467px;">BX53 microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">self-assorted stereology</td>
			<td style="width:217px;"></td>
			<td style="width:119px;"></td>
			<td style="width:467px;">Visiview</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Axiophot2</td>
			<td style="width:217px;"></td>
			<td style="width:119px;"></td>
			<td style="width:467px;">Axiophot2</td>
		</tr>
	</tbody>
</table>

stereological estimation of dopaminergic neuron number in the mouse substantia nigra using the optical fractionator and standard microscopy equipment

在临床前帕金森病的研究中, 对体道的分析, 包括对黑质中多巴胺能神经元损失的定量, 是必不可少的。为了估计总的多巴胺能神经元数, 采用光分馏法的无偏视学目前被认为是金标准。因为光分馏塔方法的理论是复杂的, 因为视学是难以实现的, 没有专门的设备, 一些商业上可用的完整的视学系统, 包括必要的软件确实存在, 纯粹细胞计数的原因。由于购买专门的视学设置并不总是可行的, 由于许多原因, 本报告描述了使用标准显微镜设备对多巴胺能神经元细胞计数进行视学估计的方法, 包括光学显微镜、带有成像软件的机动对象表 (x、y、z 平面) 和用于分析的计算机。给出了用光学分馏塔法进行视学量化的 step-by 步骤说明, 并给出了计算估计单元数的预编程文件。为了评估这种方法的准确性, 对从商用视学仪器中获得的数据进行了比较。使用此协议和视学设备发现可比较的单元数, 从而证明了该协议对于无偏视学的精确性。

使用所提出的方法, 右 sn 中的 TH + 多巴胺能神经元的估计数介于7363和7987细胞之间, 在左 sn 中, 7446 和7904细胞之间。因此, 平均多巴胺能神经元 (± SEM) 为右 sn 为7647±83细胞, 左 sn 为7675±66。每个动物的计算 CE 值低于 0.08 (范围: 0.073-0. 079) (图 7)。为了确定此方法与商用和专用视学系统的可比性, 还使用视学设置 (图 8) 对 SN 单元数进...

Watch this Scientific Journal Video about Stereological Estimation of Dopaminergic Neuron Number in the Mouse Substantia Nigra Using the Optical Fractionator and Standard Microscopy Equipment at JoVE.

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

这项工作提出了一个 step-by 步协议的无偏视学估计多巴胺能神经元细胞数在小鼠黑质使用标准显微镜设备 (即,光显微镜, 机动对象表 (x, y, z平面) 和公有域软件进行数字图像分析。

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 ...

Research

JoVE Journal

Neuroscience

Imaging Odor-Evoked Activities in the Mouse Olfactory Bulb using Optical Reflectance and Autofluorescence Signals

在小鼠嗅球成像气味诱发的活动，利用光学反射和自体荧光信号

In the brain, sensory stimulation activates distributed populations of neurons among functional modules which participate to the coding of the stimulus. ...

科研

神经科学

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

在OVO在鸡胚中脑多巴胺神经元发育中的基因功能研究电穿孔

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain ...

Comprehensive Profiling of Dopamine Regulation in Substantia Nigra and Ventral Tegmental Area

在黑质和腹侧被盖区多巴胺调控的综合性能分析

Dopamine is a vigorously studied neurotransmitter in the CNS. Indeed, its involvement in locomotor activity and reward-related behaviour has fostered five ...

Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

从人多能干细胞定向多巴胺能神经元分化

Dopaminergic (DA) neurons in the substantia nigra pars compacta  (also known as A9 DA neurons) are the specific cell type that is lost in ...

The Neuromuscular Junction: Measuring Synapse Size, Fragmentation and Changes in Synaptic Protein Density Using Confocal Fluorescence Microscopy

神经肌肉接头处：测量突触大小，碎片和改变突触蛋白密度利用共聚焦荧光显微镜

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. ...

Optical Control of a Neuronal Protein Using a Genetically Encoded Unnatural Amino Acid in Neurons

一个神经元蛋白在神经元使用遗传编码的非天然氨基酸的光控制

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to ...

Assay to Measure Nucleocytoplasmic Transport in Real Time within Motor Neuron-like NSC-34 Cells

测定在运动神经元样NSC-34细胞中实时测量核细胞质运输

Nucleocytoplasmic transport refers to the import and export of large molecules from the cell nucleus. Recently, a number of studies have shown a ...

The Optical Fractionator Technique to Estimate Cell Numbers in a Rat Model of Electroconvulsive Therapy

用于估计电惊厥治疗大鼠模型中细胞数的光学分级技术

Stereological methods are designed to describe quantitative parameters without making assumptions about size, shape, orientation and distribution of cells ...

Live Images of GLUT4 Protein Trafficking in Mouse Primary Hypothalamic Neurons Using Deconvolution Microscopy

用反褶积显微镜观察小鼠原发性下丘脑神经元 GLUT4 蛋白的动态图像

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral ...

A Microbiomechanical System for Studying Varicosity Formation and Recovery in Central Neuron Axons

中枢神经元轴突静脉曲张形成和恢复的 Microbiomechanical 系统研究

Axonal varicosities are enlarged structures along the shafts of axons with a high degree of heterogeneity. They are present not only in brains with ...