Name: live imaging of the ependymal cilia in the lateral ventricles of the mouse brain
Uploaded: 2015-06-01T15:00:00
Description: Watch this Scientific Journal Video about Live Imaging of the Ependymal Cilia in the Lateral Ventricles of the Mouse Brain at JoVE.com

Authors would like to thank Charisse Montgomery for her editing service. A. Alomran&#8217;s work partially fulfills the requirements for a master&#8217;s degree in Pharmacology.This work is funded by The University of Toledo&#8217;s intramural startup fund for W.A.A and NIH grant (DK080640) for S.M.N.

该程序使用的动物均按照该机构动物护理和使用委员会的指导方针，在卫生部和国家研究院​​的指南护理和使用经托莱多大学的机构动物护理和使用委员会（IACUC）实验动物。 1.脑提取，切片和组织准备<ol><li>通过用CO 2窒息深深安乐死5分钟牺牲野生型小鼠品系C57BL / 6。颈椎脱位保证死亡。 </li><li>清洁鼠标头，用70％的乙醇。 </li><li>由第一牵引皮肤脱落，开始与头...

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A., 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=Ciliary+polycystin-2+is+a+mechanosensitive+calcium+channel+involved+in+nitric+oxide+signaling+cascades.">Ciliary polycystin-2 is a mechanosensitive calcium channel involved in nitric oxide signaling cascades.</a> Circulation research. 104 (7), 860-869 (2009).</li><li>Jin, 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=Cilioplasm+is+a+cellular+compartment+for+calcium+signaling+in+response+to+mechanical+and+chemical+stimuli.">Cilioplasm is a cellular compartment for calcium signaling in response to mechanical and chemical stimuli.</a> Cell Mol Life Sci. 71 (11), 2165-2178 (2014).</li><li>Praetorius, H. A., Spring, K. 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这里描述的是协议用于制备鼠脑组织为活的成像和荧光显微镜，提供了脑室内的快速，灵敏密切观察室管膜纤毛。这种技术不限于仅侧脑室;它可以被用来观察在其他脑室的纤毛。这种成像技术提供的实时流类似于在离体环境的脑脊液由纤毛跳动的运动。此外，它使纤毛的定向运动的分析。这主要是促进通过使用高分辨率DIC和荧光成像系统。这个系统的一个优点是，在显微镜被封闭在一个环...

纤毛是感官微管系的细胞器，从细胞表面延伸到细胞外环境。取决于它们的微管组织，纤​​毛可分为两种类型 - “9 + 0”或“9 + 2”。在功能上，根据他们的运动，这些可被归类为运动型或非运动纤毛1。原发性纤毛是一种常用来表示“9 + 0”不动纤毛的术语。这些有九并行双峰微管（记为&#39;9&#39;）和一个中心对微管是中央鞘（表示为&#39;0&#39;）内不存在。然而，一些“9 + 0”的纤毛，如节点纤毛，从而调节胚胎偏侧是能动2。另一方面，运动纤毛的特征在于，除了九个并行微管双峰，通过附加中央对微管双峰，并与动力蛋白马达蛋白相关联，以促进蠕动。另外，一些“9+2“纤毛等纤毛的嗅觉都是非能动3。室管膜细胞衬脑室和脊髓的中央管的特征在于运动纤毛该推动脑脊液（CSF）沿脑室4。 该方法的总的目标是促进学习运动纤毛动力学和结构异常。大脑的健康和发展严重依赖于脑脊液的脑室中的高效流通。例如，正常脑脊液流量和流体平衡需要正常跳动和功能室管膜纤毛5,6，进而在调节神经元细胞的定向运动发挥关键作用和干细胞迁移7。因此，异常的室管膜纤毛功能或结构可能会导致异常脑脊液流动，这与脑积水，医疗条件，其中有脑脊液中的b的心室的异常积累雨。这可能进而使得颅内压增高和头部，抽搐，隧道视野的逐步扩大，以及精神残疾8。 该技术优于现有方法的优点是，它允许首次报道三个不同的室管膜细胞类型：I，II和III，基于其独特睫状打浆次数跳动角度。这些室管膜细胞在脑室某些区域内本地化。此外，年龄和药理学试剂如酒精和西洛他唑在改变室管膜细胞类型或它们的本地化的影响可以证明，这是不可能的这种分类室管膜细胞之前。西洛他唑是磷酸二酯酶3抑制剂，即代谢的cAMP为AMP的酶，它也调节细胞内钙9。采用高速荧光成像分析，可以成像的独特细胞内和细胞量化室管膜细胞的钙振荡特性。例如，酒精和西洛他唑显著改变室管膜纤毛跳动频率以及细胞内钙振荡性能，这反过来可能导致由室管膜纤毛10在脑脊液体积置换的变化。总之，该技术是关键，以提供三种不同类型的室管膜细胞具有不同钙振荡特性的第一个证据。 在下面的章节中，程序的详细一步一步的概述提供，密切关注组织准备和处理到。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>DMEM/HIGH GLUCOSE</td>
			<td>Cellgro Mediatech Inc.</td>
			<td>10-013-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Hyclone</td>
			<td>SH30088-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Thermo Scientific</td>
			<td>SV30010</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Thermo Scientific</td>
			<td>SH30256-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde&#160;</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710-SP</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S-2395</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton-X</td>
			<td>Sigma-Aldrich</td>
			<td>T9284</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluo-2&#160;</td>
			<td>TEF Labs</td>
			<td>#0200</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Gibco</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>VECTASHIELD HardSet Mounting Medium with DAPI</td>
			<td>Vector Labs</td>
			<td>H-1500</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-acetylated a-tubulin antibody</td>
			<td>Sigma-Aldrich</td>
			<td>T7451&#160;</td>
			<td>clone 6-11B1</td>
		</tr>
		<tr>
			<td>FITC Anti-mouse antibody</td>
			<td>Vector Labs</td>
			<td>FI-2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture plate</td>
			<td>VWR Vista Vision</td>
			<td>30-2041</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover Slip (18x18)</td>
			<td>VWR Vista Vision</td>
			<td>16004.326</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>Leica Biosystems</td>
			<td>Leica VT1200S</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Leica Biosystems</td>
			<td>Leica CM1860</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Fluorescence Microscope</td>
			<td>Nikon&#160;</td>
			<td>Nikon TE2000</td>
			<td>60X oil&#160;</td>
		</tr>
		<tr>
			<td>Microscope cover glass 24x60mm</td>
			<td>VWR Vista Vision</td>
			<td>16004-312</td>
			<td></td>
		</tr>
		<tr>
			<td>Mounting Medium with DAPI</td>
			<td>Vector Laboratories</td>
			<td>H-1500</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI filter cube</td>
			<td>Chroma Technology</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

live imaging of the ependymal cilia in the lateral ventricles of the mouse brain

Multiciliated室管膜细胞排列在成人大脑脑室。功能异常或室管膜纤毛的结构与各种神经功能障碍有关。目前能动纤毛室管膜技术的体外实时成像允许纤毛动态以下几个步骤进行详细的研究。这些步骤包括：小鼠安乐死二氧化碳根据托莱多的机构动物护理和使用委员会（IACUC）的大学协议;骨瓣其次为脑拆除和矢状脑解剖与vibratome或锋利的​​刀片通过大脑侧脑室，其中室管膜纤毛可以可视化，以获得非常薄的部分。大脑的切片在含有Dulbecco氏改良的Eagle氏培养基（DMEM）/高葡萄糖，在37℃在95％/ 5％氧气的存在下2 / CO 2混合物定制玻璃底板孵育必须保持所述组织活中实验。纤毛跳动的视频然后，使用高分辨率微分干涉显微镜记录。则视频被分析帧由帧来计算纤毛跳动频率。这允许基于其纤毛跳动频率和角度的室管膜细胞为三类或类型的不同分类。此外，这种技术允许使用高速荧光成像分析以表征室管膜细胞的独特细胞内钙振荡特性以及药理学试剂的钙振荡的效果和睫状跳动频率。此外，该技术适用于免疫荧光成像纤毛结构和纤毛蛋白定位研究。这是在疾病的诊断和表型的研究尤为重要。该技术的主要局限性是由于在活动纤毛运动的减少作为脑组织开始死亡。

在活老鼠大脑室管膜下的测量功能纤毛在这个协议中所描述的方法被用于监控在新鲜组织从小鼠脑以及监测和研究纤毛跳动频率解剖室管膜纤毛功能和结构。接着完成一个完整的实验步骤描述于一个概要流程图（图1）。强烈建议在实验很短的时间内，以保持运动纤毛尽可能积极进行。代表时间推移电影和室管膜细胞和它们的运动纤毛的图像也显示（电影1 <e...

Watch this Scientific Journal Video about Live Imaging of the Ependymal Cilia in the Lateral Ventricles of the Mouse Brain at JoVE.com

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

使用高分辨率微分干涉对比（DIC）显微镜， 离体观察位于小鼠脑室内游动室管膜纤毛的跳动的证明通过实时成像。该技术允许独特纤毛跳动频率的记录和跳动角度以及它们的细胞内钙振荡起搏属性。

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 ...

Research

JoVE Journal

Neuroscience

Neuromodulation and Mitochondrial Transport: Live Imaging in Hippocampal Neurons over Long Durations

神经调节和线粒体运输：在较长的时间里，海马神经元的实时成像

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured ...

科研

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Manual Drainage of the Zebrafish Embryonic Brain Ventricles

手动排水的斑马鱼胚胎脑室

Cerebrospinal fluid  (CSF) is a protein rich fluid contained within the brain ventricles. It is  present during early vertebrate embryonic development and ...

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 ...

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 ...

3D Modeling of the Lateral Ventricles and Histological Characterization of Periventricular Tissue in Humans and Mouse

脑室周围组织的侧脑室及组织学特性对人类和鼠标3D建模

The ventricular system carries and circulates cerebral spinal fluid (CSF) and facilitates clearance of solutes and toxins from the brain. The functional ...

Imaging Serotonergic Fibers in the Mouse Spinal Cord Using the CLARITY/CUBIC Technique

影像学检查HT能纤维在小鼠脊髓使用CLARITY / CUBIC技术

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal ...

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

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

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During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or ...

Exploring Deep Space - Uncovering the Anatomy of Periventricular Structures to Reveal the Lateral Ventricles of the Human Brain

探索深空-揭开人脑侧脑室的脑室结构解剖

Anatomy students are typically provided with two-dimensional (2D) sections and images when studying cerebral ventricular anatomy and students find this ...