Name: isolation of cerebrospinal fluid from rodent embryos for use with dissected cerebral cortical explants
Uploaded: 2013-03-11T13:00:00
Description: Watch this Scientific Journal Video about Isolation of Cerebrospinal Fluid from Rodent Embryos for use with Dissected Cerebral Cortical Explants at JoVE.com

我们是从美国国立卫生研究院（奖励R00 NS072192 MKL，HD029178到AS.L.，2 RO1 NS032457到CAW）的支持表示感谢。 MKL是波士顿​​儿童医院的职业发展奖学金/哈佛医学院岸奖学金和研究员Alfred P. Sloan基金会的收件人。 CAW是霍华德·休斯医学研究所的研究员。

1。胚胎分离/准备工作这种技术可以用于小鼠或大鼠。在这个协议中，我们展示了CSF采集技术，大脑皮质的外植体解剖小鼠胚胎脑。我们会就任何重要的差异大鼠与小鼠存在于一般技术。为胚胎的年龄分期系统，E1被归类为天大鼠插头，E0.5被归类为对小鼠的插头的天。 <ol><li>准备一个用Sylgard有机硅弹性体的显微解剖盘。 Sylgard的184有机硅弹性体提供液体成分由两部分组成，?...

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	<li>Dziegielewska, K. M., Evans, C. A., Lai, P. C., Lorscheider, F. L., Malinowska, D. H., Mollgard, K., Saunders, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteins+in+cerebrospinal+fluid+and+plasma+of+fetal+rats+during+development.">Proteins in cerebrospinal fluid and plasma of fetal rats during development.</a> Developmental Biology. 83 (1), 193-200 (1981).</li><li>Gato, A., Martin, P., Alonso, M. I., Martin, C., Pulgar, M. A., Moro, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+cerebro-spinal+fluid+protein+composition+in+early+developmental+stages+in+chick+embryos.">Analysis of cerebro-spinal fluid protein composition in early developmental stages in chick embryos.</a> Journal of Experimental Zoology. Part A, Comparative Experimental Biology. 301 (4), 280-289 (2004).</li><li>Gato, A., Moro, J. A., Alonso, M. I., Bueno, D., Mano, D. L., Martin, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+cerebrospinal+fluid+regulates+neuroepithelial+survival,+proliferation,+and+neurogenesis+in+chick+embryos.">Embryonic cerebrospinal fluid regulates neuroepithelial survival, proliferation, and neurogenesis in chick embryos.</a> The Anatomical Record. Part A, Discoveries in Molecular, Cellular, and Evolutionary Biology. 284 (1), 475-484 (2005).</li><li>Parada, C., Gato, A., Aparicio, M., Bueno, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteome+analysis+of+chick+embryonic+cerebrospinal+fluid.">Proteome analysis of chick embryonic cerebrospinal fluid.</a> Proteomics. 6 (1), 312-320 (2006).</li><li>Parada, C., Gato, A., Bueno, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+embryonic+cerebrospinal+fluid+proteome+has+greater+apolipoprotein+and+enzyme+pattern+complexity+than+the+avian+proteome.">Mammalian embryonic cerebrospinal fluid proteome has greater apolipoprotein and enzyme pattern complexity than the avian proteome.</a> Journal of Proteome Research. 4 (6), 2420-2428 (2005).</li><li>Zappaterra, M. D., Lisgo, S. N., Lindsay, S., Gygi, S. P., Walsh, C. A., Ballif, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+proteomic+analysis+of+human+and+rat+embryonic+cerebrospinal+fluid.">A comparative proteomic analysis of human and rat embryonic cerebrospinal fluid.</a> Journal of Proteome Research. 6 (9), 3537-3548 (2007).</li><li>Desmond, M. E., Jacobson, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+brain+enlargement+requires+cerebrospinal+fluid+pressure.">Embryonic brain enlargement requires cerebrospinal fluid pressure.</a> Developmental Biology. 57 (1), 188-198 (1977).</li><li>Lehtinen, M. K., Walsh, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurogenesis+at+the+brain-cerebrospinal+fluid+interface.">Neurogenesis at the brain-cerebrospinal fluid interface.</a> Annual Review of Cell and Developmental Biology. 27, 653-679 (2011).</li><li>Lehtinen, M. K., Zappaterra, M. W., Chen, X., Yang, Y. J., Hill, A. D., Lun, M., Maynard, T., Gonzalez, D., Kim, S., Ye, P., 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=The+cerebrospinal+fluid+provides+a+proliferative+niche+for+neural+progenitor+cells.">The cerebrospinal fluid provides a proliferative niche for neural progenitor cells.</a> Neuron. 69 (5), 893-905 (2011).</li><li>Martin, C., Alonso, M. I., Santiago, C., Moro, J. A., De la Mano, A., Carretero, R., Gato, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+embryonic+brain+development+in+rats+requires+the+trophic+influence+of+cerebrospinal+fluid.">Early embryonic brain development in rats requires the trophic influence of cerebrospinal fluid.</a> International Journal of Developmental Neuroscience: The Official Journal of the International Society for Developmental Neuroscience. 27 (7), 733-7340 (2009).</li><li>Martin, C., Bueno, D., Alonso, M. I., Moro, J. A., Callejo, S., Parada, C., Martin, P., Carnicero, E., Gato, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FGF2+plays+a+key+role+in+embryonic+cerebrospinal+fluid+trophic+properties+over+chick+embryo+neuroepithelial+stem+cells.">FGF2 plays a key role in embryonic cerebrospinal fluid trophic properties over chick embryo neuroepithelial stem cells.</a> Developmental Biology. 297 (2), 402-416 (2006).</li><li>Zappaterra, M. W., Lehtinen, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cerebrospinal+fluid:+regulator+of+neurogenesis,+behavior,+and+beyond.">The cerebrospinal fluid: regulator of neurogenesis, behavior, and beyond.</a> Cellular and Molecular Life Sciences: CMLS. 69 (17), 2863-2878 (2012).</li></ol>

心室CSF收集所描述的方法，已经取得了相当纯的胚胎脑脊液样品与在一些细胞分析9的稳定的蛋白质组合物，和一致的活性。有了一个良好的采集技术和产仔数的10 E14.5小鼠，可以预期的CSF收集10-15微升，并从E16鼠一窝，谁也无法预料的CSF收集约50-90微升。此收集技术，从血液和组织中的污染最小化，通过仔细观察，离心分离，和废弃的样品与细胞碎片或血色调的任何证据。我们已经分析了?...

CSF是一种复杂的流体，沐浴神经上皮1-6，并提供必要的压力7和促进生长发育中的大脑8-12线索。研究CSF在大脑发育过程中，我们开发的技术隔离室CSF来自发展中国家的大鼠或小鼠胚胎过程中各阶段的发展6,9。往前的隔离的方法包括用玻璃微针和隔离的CSF使用微喷射器1,2。我们的方法利用玻璃微毛细吸管的尖端一直拉到创建一个超细点，以提高组织的渗透。玻璃微毛细吸管被连接到一个吸气器与温和的压力变化，使心室CSF可以控制收集。为了研究干细胞的的CSF信号的影响，我们解剖大脑皮质外植体，将它们放置在聚碳酸酯膜，并把它们飘浮在适当的铜CSF样品9 lture培养基中。利用这种技术，减少体积的介质来培养的组织，是足够允许脑脊液9的有效利用。

<table border="1"><tbody><tr><td> 的试剂/材料名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> Wiretrop II毛细管针</td><td>德拉蒙德科学</td><td> ＃5-000-2020 </td><td></td></tr><tr><td> SYLGARD </td><td>安士澳粘合剂</td><td> ＃184 SIL ELAST包的</td><td></td></tr><tr><td>吸气管组件</td><td>西格玛</td><td> ＃A5177-5EA </td><td></td></tr><tr><td>一次性过滤器</td><td>文丘里或当地的药店</td><td>不可用</td><td>标准香烟过滤嘴</td></tr><tr><td> Roundstock眼科刀（15度捅一刀） </td><td>世界精密仪器公司</td><td> ＃500250 </td><td></td></tr><tr><td> 35毫米玻璃底培养皿</td><td> MatTek公司</td><td> ＃P35G-1.5-14-C </td><td&gt; </td></tr><tr><td>铂铱丝</td><td> Tritech公司研究</td><td> ＃PT-9010-010-3FT </td><td></td></tr><tr><td>核孔径迹蚀刻膜</td><td> Whatman公司</td><td> ＃09-300-57 </td><td></td></tr><tr><td> Hanks平衡盐溶液</td><td> Fisher Scientific则</td><td> ＃SH30031.FS </td><td></td></tr><tr><td>虹膜切除术剪刀</td><td>精细科学工具</td><td> ＃15000-02 </td><td></td></tr></tbody></table>

isolation of cerebrospinal fluid from rodent embryos for use with dissected cerebral cortical explants

CSF是一种复杂的流体动态变化的蛋白质组的整个开发过程，并在成年后。在胚胎发育过程中，新生的CSF区别于前神经管闭合时的羊水。 CSF卷，然后在随后的日子里，排队的神经上皮干细胞的心室和脉络膜丛产生CSF增加。胚胎CSF接触的顶端，心室发育中的大脑和脊髓神经干细胞的表面。 CSF提供了重要的流体压力发育中的大脑的扩展和分发重要的增长推动因素，神经祖细胞在时间上的具体方式。要调查的CSF的功能，重要的是不从血液或显影端脑组织污染隔离胚胎脑脊液的纯样品。在这里，我们描述了一种技术，以隔离的心室胚胎CSF可用于相对纯的样品质谱，蛋白质电泳，和细胞与初级外植体培养的实验分析，包括范围广泛的。我们演示了如何剖析和文化多孔聚碳酸酯膜的皮层外植体对以成长发展的皮层组织与媒体或CSF的量的减少。使用这种方法，可以进行实验，使用CSF从不同年龄或条件调查对靶细胞的CSF蛋白质组的生物活性。

CSF集合应得到一个明确的，透明的液体。不应该有任何从血液中的污染的证据证明，抽吸，并在Eppendorf管中的流体由一个红色或黄色带点。此外，也应该没有证据表明组织中吸出物和Eppendorf管。当CSF离心，也可以评估的CSF显微镜，以确保有没有污染。如果有污染的迹象时，CSF应该被丢弃。从E14.5鼠标乱抛垃圾，一个可以预见收集10-15微升CSF。 表1描述了从不同胚龄小鼠和大鼠的平均窝?...

Watch this Scientific Journal Video about Isolation of Cerebrospinal Fluid from Rodent Embryos for use with Dissected Cerebral Cortical Explants at JoVE.com

Isolation of Cerebrospinal Fluid from Rodent Embryos for use with Dissected Cerebral Cortical Explants

心室脑脊液（CSF）沐浴在早期的大脑发育在胚胎神经上皮和大脑皮质祖细胞。在这里，我们描述的方法，从不同年龄层的啮齿动物胚胎隔离室CSF，为了研究其生物学功能。此外，我们展示了我们的脑皮质外植体的解剖和文化的技术，可以为外植体生长的培养基或CSF以最小的体积。

脑脊液分离解剖大脑皮质外植体使用的啮齿动物胚胎

The CSF is a complex fluid with a dynamically varying proteome throughout development and in adulthood. During embryonic development, the nascent CSF ...

Research

JoVE Journal

Neuroscience

Isolation and Culture of Mouse Cortical Astrocytes

小鼠脑皮层星形胶质细胞的分离和培养

Astrocytes  are an abundant cell type in the mammalian brain, yet much remains to be  learned about their molecular and functional characteristics. In ...

科研

神经科学

Ex utero Electroporation and Whole Hemisphere Explants: A Simple Experimental Method for Studies of Early Cortical Development

Ex utero Electroporation and Whole Hemisphere Explants: A Simple Experimental Method for Studies of Early Cortical Development

前子宫穿孔和整个半球的外植体早期皮质发育的研究：一个简单的实验方法

Cortical development involves complex interactions between neurons and non-neuronal elements including precursor cells, blood vessels, meninges and ...

Isolation and Culture of Dissociated Sensory Neurons From Chick Embryos

分离和游离感觉神经元的文化活动从鸡胚

Neurons are multifaceted cells that carry information essential for a variety of functions including sensation, motor movement, learning, and memory. ...

Isolation and Cannulation of Cerebral Parenchymal Arterioles

分离及脑实质小动脉插管

Intracerebral parenchymal arterioles (PAs), which include parenchymal arterioles, penetrating arterioles and pre-capillary arterioles, are high resistance ...

Sample Preparation for Endopeptidomic Analysis in Human Cerebrospinal Fluid

人脑脊液 Endopeptidomic 分析的样品制备

This protocol describes a method developed to identify endogenous peptides in human cerebrospinal fluid (CSF). For this purpose, a previously developed ...

An Improved Method for Collection of Cerebrospinal Fluid from Anesthetized Mice

一种改进的麻醉小鼠脑脊液采集方法

The cerebrospinal fluid (CSF) is a valuable body fluid for analysis in neuroscience research. It is one of the fluids in closest contact with the central ...

Isolation of Cerebral Capillaries from Fresh Human Brain Tissue

新鲜人脑组织中毛细血管的分离

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic ...

Rat Model of Widespread Cerebral Cortical Demyelination Induced by an Intracerebral Injection of Pro-Inflammatory Cytokines

由亲炎细胞因子的内脑注射诱导的广度脑皮质脱叶的鼠模型

Multiple sclerosis (MS) is the most common immune-mediated disease of the central nervous system (CNS) and progressively leads to physical disability and ...

Effects of Blast-induced Neurotrauma on Pressurized Rodent Middle Cerebral Arteries

爆炸性神经损伤对压生啮鼠中脑动脉的影响

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral ...

In Vivo Imaging of Cerebrospinal Fluid Transport through the Intact Mouse Skull using Fluorescence Macroscopy

使用荧光宏镜检查,通过"完整小鼠头骨"进行脑脊液传输的 Vivo 成像

Cerebrospinal fluid (CSF) flow in rodents has largely been studied using ex vivo quantification of tracers. Techniques such as two-photon microscopy and ...