Name: semi-quantitative determination of dopaminergic neuron density in the substantia nigra of rodent models using automated image analysis
Uploaded: 2021-02-02T12:30:00
Description: Watch this Scientific Journal Video about Semi-Quantitative Determination of Dopaminergic Neuron Density in the Substantia Nigra of Rodent Models using Automated Image Analysis at JoVE.com

作者要感谢大学健康网络高级光学显微镜设施（AOMF）的所有工作人员在制定此协议方面给予的时间和帮助。

所有程序均由大学健康网络动物护理委员会批准，并按照加拿大动物护理委员会制定的准则和条例执行。
1. 立体定向注射
<ol><li>配对屋成年雌性斯普拉格-道利大鼠（250-280克）在笼子里用木床上用品和广告图书馆获得食物和水。保持动物群落在一个正常的12小时光/暗周期（灯在06：30）与恒定的温度和湿度。</li>
	<li>按照先前描述的8、10，对大脑右侧的SNpc（...

<ol>
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B., 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=Expression+of+human+A53T+alpha-synuclein+in+the+rat+substantia+nigra+using+a+novel+AAV1/2+vector+produces+a+rapidly+evolving+pathology+with+protein+aggregation,+dystrophic+neurite+architecture+and+nigrostriatal+degeneration+with+potential+to+model+the+pathology+of+Parkinson's+disease.">Expression of human A53T alpha-synuclein in the rat substantia nigra using a novel AAV1/2 vector produces a rapidly evolving pathology with protein aggregation, dystrophic neurite architecture and nigrostriatal degeneration with potential to model the pathology of Parkinson's disease.</a> Molecular Neurodegeneration. 5, 43 (2010).</li><li>Koprich, J. B., 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=Progressive+neurodegeneration+or+endogenous+compensation+in+an+animal+model+of+Parkinson's+disease+produced+by+decreasing+doses+of+alpha-synuclein.">Progressive neurodegeneration or endogenous compensation in an animal model of Parkinson's disease produced by decreasing doses of alpha-synuclein.</a> PLoS One. 6 (3), 17698 (2011).</li><li>McKinnon, C., 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=Early-onset+impairment+of+the+ubiquitin-proteasome+system+in+dopaminergic+neurons+caused+by+alpha-synuclein.">Early-onset impairment of the ubiquitin-proteasome system in dopaminergic neurons caused by alpha-synuclein.</a> Acta Neuropathologica Communications. 8 (1), 17 (2020).</li><li>Henderson, M. X., 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=Spread+of+alpha-synuclein+pathology+through+the+brain+connectome+is+modulated+by+selective+vulnerability+and+predicted+by+network+analysis.">Spread of alpha-synuclein pathology through the brain connectome is modulated by selective vulnerability and predicted by network analysis.</a> Nature Neuroscience. 22 (8), 1248-1257 (2019).</li><li>Ip, C. W., 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=AAV1/2-induced+overexpression+of+A53T-alpha-synuclein+in+the+substantia+nigra+results+in+degeneration+of+the+nigrostriatal+system+with+Lewy-like+pathology+and+motor+impairment:+a+new+mouse+model+for+Parkinson's+disease.">AAV1/2-induced overexpression of A53T-alpha-synuclein in the substantia nigra results in degeneration of the nigrostriatal system with Lewy-like pathology and motor impairment: a new mouse model for Parkinson's disease.</a> Acta Neuropathologica Communications. 5 (1), 11 (2017).</li><li>Webster, J. D., Dunstan, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-slide+imaging+and+automated+image+analysis:+considerations+and+opportunities+in+the+practice+of+pathology.">Whole-slide imaging and automated image analysis: considerations and opportunities in the practice of pathology.</a> Veterinary Pathology. 51 (1), 211-223 (2014).</li></ol>

PD 临床前模型中多巴明药物系统的完整性的可靠评估对于确定潜在疾病改变疗法的有效性至关重要。因此，控制和减少可能降低组织病理数据可靠性和可重复性的潜在混淆非常重要。仔细的定量结果可以提供比定性或半定量描述更多的信息。同时，我们必须认识到，时间和资源的限制可能使执行公正的固态计数难以量化病理变化或细胞损失。然而，随着最近的进步，许多这些标准可以使用计算机?...

帕金森病 （PD） 是一种流行的神经退行性运动障碍，其特征是存在含有α-核素 （α-syn） 的蛋白质聚合物，以及亚坦蒂娅尼格拉解析 （SNpc）1中多巴明神经元的优先损失。多巴明神经元数的量化是PD研究的重要组成部分，因为它允许评估尼古菌系统的完整性，从而为评估潜在疾病改变疗法的有效性提供了一个重要的终点。目前，细胞编号的定量标准是公正的固态计数，它利用组织二维（2D）横截面来估计三维（3D）结构2、3、4的体积特征。现代基于设计的固态方法采用全面的随机采样程序，并应用计数协议（称为探针），以避免潜在的文物和系统错误，使可靠的检测差异只比动物间变异5略大。虽然立体学为体内组学研究提供了强大的分析工具，但它时间密集，承担统一的标本制备，需要分几个步骤进行验证，这可能会影响临床前转化调查日益需要的效率。
数字科学的最新技术进步使得在没有立体显微镜的情况下，能够采用新的应用来更有效地评估病理学，同时满足作为公正立体学代名词的需求。这些方法提高了速度，减少了人为错误，提高了固态技术6、7的可重复性。HALO是数字病理学中定量组织分析的图像分析平台之一。它包括各种不同的模块，并使用模式识别算法，逐细胞报告形态和多路复用表达数据。细胞核FL模块测量细胞核或细胞质中荧光标记的免疫荧光阳性。这允许报告每个标记的正细胞数量以及每个标记的强度分数。该模块可以调整，以提供单独的细胞大小和强度测量，虽然此功能不需要量化多巴明神经元。
这项研究的目的是验证这种方法与先前验证的病毒载体为基础的α合成大鼠模型的黑鬼神经退化8，9，10。在这个模型中，人类突变的A53T α合成通过定型注射腺相关病毒混合血清型1/2（AAV1+2）在SNpc中表达，导致在6周内显著的神经退化。在某些研究中，未弹出的对应性 SNpc 可能充当注射方的内部控制。更常见的情况是，在对照动物群中注射 AAV 空矢量 （AAV-EV） 用作负控制。我们使用自动图像分析软件（图 1）提供分步指南，以估计注射的 SNpc 中 6 周后剩余的多巴明神经元的密度。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>A-Syn Antibody</td>
			<td>ThermoFisher Scientific</td>
			<td>32-8100</td>
			<td></td>
		</tr>
		<tr>
			<td>ABC Elite</td>
			<td>Vector Labs</td>
			<td>PK-6102</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 secondary antibody</td>
			<td>ThermoFisher Scientific</td>
			<td>A-11008</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 555 secondary antibody</td>
			<td>ThermoFisher Scientific</td>
			<td>A-28180</td>
			<td></td>
		</tr>
		<tr>
			<td>Alkaline phosphatase-conjugated anti-rabbit igG</td>
			<td>Jackson Immuno</td>
			<td>111-055-144</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotinylated anti-mouse IgG</td>
			<td>Vector Labs</td>
			<td>BA-9200</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr>
			<td>DAKO fluorescent mouting medium</td>
			<td>Agilent</td>
			<td>S3023</td>
			<td></td>
		</tr>
		<tr>
			<td>HALO&#8482;</td>
			<td>Indica Labs</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Histo-Clear II</td>
			<td>Diamed</td>
			<td>HS202</td>
			<td></td>
		</tr>
		<tr>
			<td>ImmPACT DAB Peroxidase substrate</td>
			<td>Vector Labs</td>
			<td>SK-4105</td>
			<td></td>
		</tr>
		<tr>
			<td>LSM880 Confocal Microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NeuN Antibody</td>
			<td>Millipore</td>
			<td>MAB377</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Goat Serum</td>
			<td>Vector Labs</td>
			<td>S-1000-20</td>
			<td></td>
		</tr>
		<tr>
			<td>OCT</td>
			<td>Tissue-Tek</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>BioShop</td>
			<td>PAR070.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Sliding microtome</td>
			<td>Leica</td>
			<td>SM2010 R</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereo Investigator</td>
			<td>MBF Bioscience</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>BioShop</td>
			<td>SUC700</td>
			<td></td>
		</tr>
		<tr>
			<td>TH Antibody</td>
			<td>ThermoFisher Scientific</td>
			<td>P21962</td>
			<td></td>
		</tr>
		<tr>
			<td>VectaMount mounting medium</td>
			<td>Vector Labs</td>
			<td>H-5000</td>
			<td></td>
		</tr>
		<tr>
			<td>Vector Blue Alkaline Phosphatase substrate</td>
			<td>Vector Labs</td>
			<td>SK-5300</td>
			<td></td>
		</tr>
		<tr>
			<td>Zen Black Software</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Zen Blue Software</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

semi-quantitative determination of dopaminergic neuron density in the substantia nigra of rodent models using automated image analysis

估计亚坦蒂娅尼格拉多巴明神经元的数量是临床前帕金森病研究的关键方法。目前，不偏不倚的类固物计数是这些细胞的量化标准，但它仍然是一个费力和耗时的过程，这可能并非所有项目都可行。在这里，我们描述图像分析平台的使用，它可以准确估计预先定义的兴趣区域中标记的细胞的数量。我们描述了大鼠大脑中这种分析方法的分步协议，并证明它可以识别由于突变α核糖核素在亚种尼格拉中表达而显著减少酪氨酸羟基酶阳性神经元。我们通过与公正立体学获得的结果进行比较来验证这一方法。综合起来，该方法为检测多巴明神经元数的变化提供了一个时间高效、准确的过程，因此适合有效确定干预对细胞生存的影响。

通过将上述方法应用于AAV注射后6周收集的脑组织，我们证明，与注射空向量AV（AAV-EV）作为对照剂（图5A，B）相比，在大鼠大脑SNpc中对AAV表示突变A53T α-syn（AAV-A53T）的立体定向注射可显著降低多巴明神经元的密度。注射AAV-EV的SNpc中TH阳性神经元/mm2的平均数为276.2±34.7，注射AAV-A53T的大鼠SNpc为41.2±17（P = 0.0003）。 SNpc中多巴明神经元/mm2数?...

Watch this Scientific Journal Video about Semi-Quantitative Determination of Dopaminergic Neuron Density in the Substantia Nigra of Rodent Models using Automated Image Analysis at JoVE.com

Semi-Quantitative Determination of Dopaminergic Neuron Density in the Substantia Nigra of Rodent Models using Automated Image Analysis

在这里，我们提出了一个自动方法，用于半定量确定大鼠亚坦蒂娅尼格拉解析压实数中的多巴明神经元数。

使用自动图像分析对啮齿动物模型的亚斯坦蒂娅尼格拉多巴明神经元密度进行半定量测定

Estimation of the number of dopaminergic neurons in the substantia nigra is a key method in pre-clinical Parkinson's disease research. Currently, unbiased ...

估计亚坦蒂亚尼格拉的多巴明神经元数是帕金森病研究的关键成果指标。此协议显著缩短了估计神经元数量所需的工作量。该方法为检测多巴明神经元数的变化提供了一个时间高效、准确的过程，适合确定干预对细胞生存的影响。这种方法可以很容易地适应，以提供不同大脑区域的神经元的准确计数，同时保持成本和时间优势比传统的固态方法。要捕获 IHC 图像，请使用该软件与 10 倍放大倍数的对焦显微镜耦合。将针孔打开至 1.5 个区域单位，以捕获总长 1.5 微米的宽平面，并将焦点放在大脑的注射侧。在获取选项卡上检查磁贴扫描成像选项，并将尺寸设置为 10 乘 4。在采集模式面板下将变焦设置为 1.1，以避免磁贴扫描图像之间的任何缝合标记。将帧尺寸设置为 1024 x 1024 像素，平均值设置为 2，以确保高质量的图像采集。在通道面板设置轨道一到亚历克萨 488 和轨道二到亚历克萨 555 。将幻灯片加载到舞台上，并选择具有强 TH 染色的部分。点击收购面板上的实时直播。在通道面板中，将激光强度和增益设置为最大化信号和限制背景噪音的水平。使用范围指示器确保信号不会过度暴露。使用多个幻灯片重复此步骤。在收购选项卡上检查位置框。要开始成像，请使用眼片并选择显示正 TH 染色的第一部分。然后将焦点设置在兴趣点，并将舞台移到部分的中线。这样可以保存 X、Y 和 Z 轴中的位置，并将对捕获整个部分的瓷砖扫描进行成像。对于所有其他幻灯片（包括未注入的站点）重复此步骤。使用适当的软件将图像文件单独导入自动图像分析软件。通过选择笔注释工具来在基坦蒂亚尼格拉平价紧凑物周围绘制注释，从而定义感兴趣的区域。移动到分析选项卡，并从下拉分析菜单中选择实时调整。这将打开部分图像上的单独窗口，以便实时修改分析参数。在分析放大部分下选择合适的图像变焦。在细胞检测部分下选择核染料作为用于TH染色的染料。调整核对比阈值、最低核强度、核分割侵略性和核大小设置，同时仔细观察时间调整窗口。使用多个示例重复此过程。一旦采样了适当数量的图像，并相应地调整了实时调整，将"设置"中的分析设置保存在"下拉"菜单中。选择要分析的所有图像并单击分析。选择刚刚保存的分析设置，并在分析窗口区域检查注释层框，然后检查第一层并单击分析。完成导出所有部分的汇总分析数据后，选择导出对象分析数据的选项。此数据集可用于检查细胞大小的变化，以响应毒素或治疗。腺相关病毒或 AAV 注射六周后，AAV 向大鼠大脑中的亚坦蒂亚尼格拉注射表达突变 A53T 阿尔法核糖核素的立体定向注射，导致多巴明神经元的密度显著降低。与注射AAV空向量的老鼠相比，注射AAV-A35T的老鼠的亚坦蒂亚尼格拉每毫米TH正神经元的平均数量显著减少。类似的观察也使用公正的立体学。当尝试此协议时，请记住，软件只有经过培训才能使用。因此，必须花时间和照顾，以确保一个单元格被确定为单个细胞。该软件可以适应测量其他感兴趣的蛋白质的荧光强度，如α-核素，以确定治疗是否可以调节蛋白质水平。这项技术在测试多种潜在治疗方法对多巴明神经元密度的影响时，效率更高。

Research

JoVE Journal

Neuroscience

Automated Sholl Analysis of Digitized Neuronal Morphology at Multiple Scales

肖尔自动数字化的神经元形态分析，多尺度

Neuronal morphology plays a significant role in determining how neurons function and communicate1-3.  Specifically, it affects the ability of neurons to ...

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 ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

可视化和遗传操作在小鼠大脑皮层和使用海马树突和棘<em&gt;在子宫内</em&gt;电穿孔

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

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. ...

Long-term Continuous EEG Monitoring in Small Rodent Models of Human Disease Using the Epoch Wireless Transmitter System

在人类疾病中使用大纪元无线传输系统的小型啮齿动物模型的长期连续监测脑电图

Many progressive neurologic diseases in humans, such as epilepsy, require pre-clinical animal models that slowly develop the disease in order to test ...

High-density Electroencephalographic Acquisition in a Rodent Model Using Low-cost and Open-source Resources

高密度的脑电图采集在啮齿动物模型使用低成本的开源资源

Advanced electroencephalographic analysis techniques requiring high spatial resolution, including electrical source imaging and measures of network ...

Low-Density Primary Hippocampal Neuron Culture

低密度原海马神经元文化

The ability to probe the structure and physiology of individual nerve cells in culture is crucial to the study of neurobiology, and allows for flexibility ...

Assessment of Dopaminergic Homeostasis in Mice by Use of High-performance Liquid Chromatography Analysis and Synaptosomal Dopamine Uptake

高效液相色谱分析和触多巴胺摄取对小鼠多巴胺能稳态的评价

Dopamine (DA) is a modulatory neurotransmitter controlling motor activity, reward processes and cognitive function. Impairment of dopaminergic (DAergic) ...