Name: three-dimensional imaging and analysis of mitochondria within human intraepidermal nerve fibers
Uploaded: 2017-09-29T12:30:00
Description: Watch this Scientific Journal Video about Three-dimensional Imaging and Analysis of Mitochondria within Human Intraepidermal Nerve Fibers at JoVE.com

这项工作得到了国家卫生研究院的资助, K08 NS061039-01A2, 神经病学研究和 #38; 发现, 和密歇根大学 Taubman 医学研究所的 a。这项工作使用的形态学和图像分析核心的密歇根糖尿病研究中心, 由国家卫生研究院资助 5P90 DK-20572 来自国家糖尿病和消化系统和肾脏疾病研究所。作者要感谢 j. 鲁滨逊单身人士和 a. 戈登？史密斯 (犹他大学) 慷慨捐献人体皮肤样本。

在犹他州糖尿病中心 (盐湖城), 从一个大型社区初级保健网络招募的受试者中获得了皮肤穿孔活检。这项研究得到了密歇根大学机构审查委员会的批准, 并遵守了《赫尔辛基宣言》的宗旨。在测试之前, 从每个主题获得书面知情同意. 
 1. 荧光免疫组化 <ol> <li> 为 intraepidermal 神经纤维免疫组织化学制备穿孔活检: <ol> <li> 由医务人员进行3毫米皮肤活检, 并将整个活...

本议定书的目的是分离, 量化和分析的大小和分布的神经特异性线粒体在 IENFs 3D 从人体皮肤活检。协议中有几个关键步骤。自由浮动荧光免疫组织化学设计用于对每个样本中的多个信号进行着色和分析, 为探索性研究提供了一种更加通用的方法44,45。这一过程允许抗体渗入组织, 以最大限度地获得图像整个50µm 部分, 这是必要的获得共聚焦显微镜图像和3D ...

线粒体提供重要的细胞功能, 包括产生细胞能量, 缓冲钙, 调节坏死和凋亡细胞死亡1,2,3。神经系统具有很高的新陈代谢率, 与身体的4相比, 这表明神经元通过线粒体呼吸以三磷酸腺苷 (ATP) 的形式产生高度的细胞能量。许多证据证明, 神经元函数依赖于 ATP5, 特别是在突触的6。因此, 神经元内线粒体的分布是很重要的。
在过去的10年里, 大量的信息表明, 神经元线粒体的贩运和对接受到高度的管制。运动蛋白参与在神经元中分布线粒体到特定的细胞隔间。线粒体的贩运是特别重要的, 因为神经元项目轴突和树突远离躯体。驱动马达蛋白质主要直接顺 (远离体细胞) 贩运的线粒体沿微管, 而蛋白马达蛋白直接逆行 (向 soma) 运动7,8,9,10. 有细胞信号, 这样的线粒体膜电位和冲动传导, 影响线粒体贩运的存在和方向11,12,13。
除了传送线粒体外, 还有专门的蛋白质将线粒体定位到具有高能量需求的特定蜂窝室, 如郎和突触的节点8,14,17. 实际上, 轴突内的大部分线粒体是 non-motile9、13、18。专门的蛋白质像 syntaphilin 锚定线粒体的微管沿轴突, 而其他蛋白质锚定线粒体的肌动蛋白细胞骨架19-21。生长因子和钙等离子被报告支持停止线粒体运动, 将它们本地化为需要的区域21,22,23。
两者结合在一起, 线粒体的贩运和对接对神经元的正常功能至关重要。为支持这一点, 线粒体贩运的中断与包括阿尔茨海默病、肌萎缩侧索硬化症、腓骨-玛丽牙病、亨廷顿氏病、遗传性痉挛截瘫, 和视神经萎缩15,24,25,26,27。最近的研究侧重于线粒体功能障碍和病理作为糖尿病神经病变的潜在机制, 与糖尿病相关的感觉丧失28,29,30,31 ,32,33。假设是糖尿病改变了皮肤神经末端感官投射中线粒体的分布。因此, 在 intraepidermal 神经纤维 (IENFs), 背根神经节感觉传入的远端提示中, 开发了一种技术来可视化和量化线粒体。该技术将特定线粒体和神经纤维标签的荧光免疫组化与共焦显微镜相结合, 用强大的3D 图像分析软件对信号进行 z 系列采集, 测量神经特异性的分布线粒体从人体皮肤穿孔活检达到这个目的。

<table border="0" cellpadding="0" cellspacing="0" width="1027">
	<tbody>
		<tr height="69">
			<td height="69" style="height:69px;width:267px;">2% Zamboni&#39;s Fixative</td>
			<td style="width:200px;">Newcomer Supply, Middleton, WI&#160;</td>
			<td style="width:199px;">1459A</td>
			<td style="width:363px;">2% paraformaldehyde, 0.2% saturated picric acid in phosphate buffered saline (PBS), pH 7.4</td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;width:267px;">10X Phosphate Buffered Saline (PBS)&#160;</td>
			<td style="width:200px;">Fisher Scientific, Pittsburgh, PA</td>
			<td style="width:199px;">BP399-4</td>
			<td style="width:363px;">To make up 1X PBS</td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:267px;">Image-iT FX Signal Enhancer</td>
			<td style="width:200px;">ThermoFisher Scientific, Waltham, Massachusetts</td>
			<td style="width:199px;">I36933</td>
			<td style="width:363px;">enhances Alexa Fluor dye signals by reducing nonspecific binding</td>
		</tr>
		<tr height="73">
			<td height="73" style="height:73px;width:267px;">Anti-Protein Gene Product 9.5 Antibody (Rabbit Polyclonal)</td>
			<td style="width:200px;">Proteintech Group Inc. Rosemont, IL</td>
			<td style="width:199px;">14730-1-AP</td>
			<td style="width:363px;">abbreviated as PGP9.5, replaces discontinued AbD Serotec (Cat. No. 7863-0504) antibody</td>
		</tr>
		<tr height="66">
			<td height="66" style="height:66px;width:267px;">Anti-Pyruvate Dehydrogenase E2/E3bp Antibody (Mouse Monoclonal)</td>
			<td style="width:200px;">abcam, Cambridge, MA</td>
			<td style="width:199px;">ab110333</td>
			<td style="width:363px;">abbreviated as PDH</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:267px;">Goat anti-mouse Secondary antibody Alexa Fluor 594 conjugate</td>
			<td style="width:200px;">ThermoFisher Scientific, Waltham, Massachusetts</td>
			<td style="width:199px;">A-11034</td>
			<td style="width:363px;">red-fluorescent conjugated secondaryantibody</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:267px;">Goat anti-rabbit Secondary antibody Alexa Fluor 488 conjugate</td>
			<td style="width:200px;">ThermoFisher Scientific, Waltham, Massachusetts</td>
			<td style="width:199px;">A-11032</td>
			<td style="width:363px;">green-fluorescent conjugated secondaryantibody</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:267px;">Albumin, from Bovine Serum</td>
			<td style="width:200px;">Sigma-Aldrich, St. Louis, MO</td>
			<td style="width:199px;">A7906-100</td>
			<td style="width:363px;">abbreviated as BSA</td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;width:267px;">Triton X- 100</td>
			<td style="width:200px;">Sigma-Aldrich, St. Louis, MO</td>
			<td style="width:199px;">T9284</td>
			<td style="width:363px;">abbreviated as TX-100</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:267px;">0.22 &#181;m Filter</td>
			<td style="width:200px;">EMD Millipore, Billerica 
			MA</td>
			<td style="width:199px;">MILLEX GP SLGP 033NS</td>
			<td style="width:363px;">0.22 &#181;m Millipore filter</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:267px;">Parafilm M</td>
			<td style="width:200px;">Fisher Scientific, Pittsburgh, PA</td>
			<td style="width:199px;">13-374-10</td>
			<td style="width:363px;">Curwood Wisconsin LLC Parafilm M (PM-996)</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:267px;">Non-calibrated Loop</td>
			<td style="width:200px;">Fisher Scientific, Pittsburgh, PA</td>
			<td style="width:199px;">22-032092</td>
			<td style="width:363px;">inoculating Loop by Decon LeLoop (MP 199-25)</td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:267px;">96-well Assay Plate</td>
			<td style="width:200px;">Corning Incorporated, Corning, NY</td>
			<td style="width:199px;">3603</td>
			<td style="width:363px;">96-well flat bottom plate</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:267px;">Prolong Gold antifade reagent with DAPI</td>
			<td style="width:200px;">ThermoFisher Scientific, Waltham, Massachusetts</td>
			<td style="width:199px;">P-36931</td>
			<td style="width:363px;">DAPI staining of nuclei</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:267px;">Microscope Cover Glass 50 x 24 mm</td>
			<td style="width:200px;">Fisher Scientific, Pittsburgh, PA</td>
			<td style="width:199px;">12-544E</td>
			<td style="width:363px;">Coverslips</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:267px;">Superfrost Plus Microscope Slides</td>
			<td style="width:200px;">Fisher Scientific, Pittsburgh, PA</td>
			<td style="width:199px;">12-550-15</td>
			<td style="width:363px;">Microscope Slides</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:267px;">Leica SP5 Laser Scanning Confocal Microscope</td>
			<td style="width:200px;">Leica Microsystems, Buffalo Grove, IL</td>
			<td style="width:199px;">SP5</td>
			<td style="width:363px;">Confocal Microscope</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:267px;">Volocity x64 Software&#160;</td>
			<td style="width:200px;">Perkin Elmer, Waltham , MA</td>
			<td style="width:199px;">version 4.4.0</td>
			<td style="width:363px;">Volocity software is used for Steps 3.1 and 3.2 in the protocol for image processing</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:267px;">Imaris x64 3 Dimensional Analysis Software</td>
			<td style="width:200px;">Bitplane, Concord, MA</td>
			<td style="width:199px;">version 7.7.1</td>
			<td style="width:363px;">Imaris software is used for Steps 3.3 through 3.5 in the protocol for image analysis</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:267px;">Excel</td>
			<td style="width:200px;">Microsoft, Redmond, WA</td>
			<td style="width:199px;">version Office 2013</td>
			<td style="width:363px;">Excel spreadsheet software is used for Step 3.6 in the protocol to summarize morphometric features</td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:267px;">Optimum Cutting Temperature Compound</td>
			<td style="width:200px;">Sakura Finetek USA, Inc., Torrance, CA</td>
			<td style="width:199px;">4583</td>
			<td style="width:363px;">abbreviated as OCT</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">Leica Cryostat</td>
			<td style="width:200px;">Leica Biosystems, Buffalo Grove, IL</td>
			<td style="width:199px;">CM1850</td>
			<td style="width:363px;">Cryostat for cutting 50 &#181;m sections</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:267px;">CellLight Mitochondria-GFP, BacMam 2.0</td>
			<td style="width:200px;">ThermoFisher Scientific, Waltham, Massachusetts</td>
			<td style="width:199px;">C10600</td>
			<td style="width:363px;">Used as a postive control to label mitochondria with a green fluorescent signal</td>
		</tr>
	</tbody>
</table>

three-dimensional imaging and analysis of mitochondria within human intraepidermal nerve fibers

本议定书的目的是研究 intraepidermal 神经纤维内的线粒体。因此, 3D 成像和分析技术的发展, 以隔离神经特异性线粒体和评估疾病诱导的线粒体远端的感觉神经的变化。该协议结合荧光免疫组织化学、共聚焦显微镜和3D 图像分析技术来可视化和量化神经特异性线粒体。详细的参数在整个过程中定义, 以提供一个具体的例子, 如何使用这些技术来隔离神经特异性线粒体。抗体被用来标记的神经和线粒体信号的组织切片的皮肤穿孔活检, 其次是间接免疫荧光可视化的神经和线粒体与绿色和红色的荧光信号分别。利用共焦显微镜对 Z 系列图像进行采集, 并采用3D 分析软件对信号进行处理和分析。没有必要按照内部描述的确切参数, 但重要的是要与整个染色, 获取和分析步骤中选择的一致。该协议的优点是, 它适用于各种情况, 其中一个荧光信号被用来隔离其他的信号, 否则是不可能单独研究。

人 IENFs 中线粒体的可视化与量化
荧光免疫组织化学允许在人体皮肤活检中同时标记多个信号, 以可视化神经、线粒体和细胞核。96井板是组织免疫组化过程中的步骤的一种简便方法。图 1显示此配置占了多达8节, 可通过12阶段的解决方案进行处理。自由浮动的方法, 加上温和的搅拌与平摇杆, ?...

Watch this Scientific Journal Video about Three-dimensional Imaging and Analysis of Mitochondria within Human Intraepidermal Nerve Fibers at JoVE.com

Three-dimensional Imaging and Analysis of Mitochondria within Human Intraepidermal Nerve Fibers

该协议使用三维 (3D) 成像和分析技术来可视化和量化神经特异性线粒体。这些技术适用于在其他情况下, 一个荧光信号被用来隔离一个子集的数据, 从另一个荧光信号。

人 Intraepidermal 神经纤维内线粒体三维成像及分析

The goal of this protocol is to study mitochondria within intraepidermal nerve fibers. Therefore, 3D imaging and analysis techniques were developed to ...

Research

JoVE Journal

Neuroscience

Tissue Preparation and Immunostaining of Mouse Sensory Nerve Fibers Innervating Skin and Limb Bones

鼠标感觉神经支配的皮肤和四肢骨骼纤维组织的制备及免疫染色

Detection and primary processing of physical, chemical and thermal sensory stimuli by peripheral sensory nerve fibers is key to sensory perception in ...

科研

神经科学

Three Dimensional Vestibular Ocular Reflex Testing Using a Six Degrees of Freedom Motion Platform

三维前庭眼反射测试使用六度自由度运动平台

The vestibular organ is a sensor that measures angular and linear accelerations with six degrees of freedom (6DF). Complete or partial defects in the ...

Selective Tracing of Auditory Fibers in the Avian Embryonic Vestibulocochlear Nerve

禽流感胚胎听神经听觉神经纤维的选择性跟踪

The embryonic chick is a widely used model for the study of peripheral and central ganglion cell projections. In the auditory system, selective labeling ...

Three-dimensional Confocal Analysis of Microglia/macrophage Markers of Polarization in Experimental Brain Injury

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After brain stroke microglia/macrophages (M/M) undergo  rapid activation with dramatic morphological and phenotypic changes that  include expression of ...

Protocol for Three-dimensional Confocal Morphometric Analysis of Astrocytes

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As glial cells in the brain, astrocytes have diverse functional roles in the central nervous system. In the presence of harmful stimuli, astrocytes modify ...

Large-scale Three-dimensional Imaging of Cellular Organization in the Mouse Neocortex

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The mammalian neocortex is composed of many types of excitatory and inhibitory neurons, each with specific electrophysiological and biochemical ...

Three-Dimensional Shape Modeling and Analysis of Brain Structures

脑结构三维形状建模与分析

Statistical shape analysis of brain structures has been used to investigate the association between their structural changes and pathological processes. ...

Targeting Alpha Synuclein Aggregates in Cutaneous Peripheral Nerve Fibers by Free-floating Immunofluorescence Assay

通过自由浮动免疫荧光测定,在皮外神经纤维中定位阿尔法核糖核酸聚合体

To date, for most neurodegenerative diseases only a post-mortem histopathological definitive diagnosis is available. For Parkinson&#39;s disease (PD), the ...

3D Kinematic Analysis for the Functional Evaluation in the Rat Model of Sciatic Nerve Crush Injury

坐骨神经粉碎损伤大鼠模型中功能评估的3D运动学分析

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of ...

Imaging and Analysis of Neurofilament Transport in Excised Mouse Tibial Nerve

切除小鼠神经神经纤维神经丝的成像与分析

Neurofilament protein polymers move along axons in the slow component of axonal transport at average speeds of ~0.35-3.5 mm/day. Until recently the study ...