支持我们的工作是由瑞士国家科学基金会（民阵），瑞士在系统生物学（SystemsX.ch），美国肌肉萎缩症协会（MDA）含量，协会法国驳LES​​肌病（AFM），美国线粒体疾病基金会倡议（UMDF的），Gebert联阵基金会罕见疾病计划（GRF），瑞士肌肉疾病（SSEM / FSRMM），瑞士人寿“Jubiläumsstiftung献给Volksgesundheit和medizinische Forschung”，罗氏研究基金会和大学的研究学会巴塞尔。

1。小学人体肌肉细胞培养制备<ol><li>建立人体肌肉细胞培养根据对explantation重新explantation的技术4。首先，从非肌肉组织活检的。然后，嵌入在血浆凝块，这使得从外植体成纤维细胞出现1毫米3肌肉植。 </li><li>嵌入植体转入明胶等离子涂层菜，让他们在正常生长介质中成长（的MEM 25％，中型199，10％小牛血清[胎牛血清，1％青霉素/链霉素，10μg/ ml的胰岛素，补充10 ng / ml的重组人表皮生长因子，表皮生长因子]，12.5 ng / ml的人类成纤维细胞生长因子[成纤维细胞生长因子）在单层。出现在这些条件下，从植细胞，肌肉细胞。这些细胞外植体取出后，将继续增长单层。 </li><li>文化aneurally 0.1％明胶涂层板在正常生长介质的细胞。这些细胞可以aneurally培养和扩增的需要。最好是在3日和8之间的通道使用的肌肉细胞进行联合培养。这是至关重要的支配年轻的肌管;这一点后，共同的文化有一个失败的高风险。此外，肌管不宜过大，过厚。 </li><li> 35毫米组织培养皿密度每盘50细胞的正常生长培养基（或6孔板）支配计划的前两天，盘上的肌肉细胞。在密度，细胞在适当的汇合于翌日开始融合，一天之后，随后的支配。利用这些条件，肌管是最佳的支配和纤维收缩，因此后续在合适的阶段。 </li><li>第二天，组成一个融合的MEM培养基辅以25％，中型199，5％胎牛血清，10μg/ ml的胰岛素，1％青霉素/替换中的正常生长介质。 </li></ol> 2。 isolati<html>大鼠胚胎脊髓植在双目显微镜下进行隔离脊髓外植体的整个过程，在汉克的含10％胎牛血清（FBS），平衡盐溶液（HBSS中，GIBCO / Invitrogen公司的14170号）。 <ol><li>大鼠胚胎ED 13日和14日之间有不同的发展阶段，将危及整个过程，这是至关重要的。牺牲与CO 2的怀孕妇女，并收集在HBSS中型全胚胎链。隔离每个胚胎和安乐死斩首。 </li><li>在一块休息的身体从解剖胚胎脊髓和消除周围的肌肉和结缔组织。必须非常小心，以保持连接到脊髓，以确保功能的神经支配的背根神经节（病种付费）。 </li><li>每个脊髓横向切片的方式，每个外植体包含至少2个（1毫米3植）连接到它的背根神经节。 </li></ol><html> ove_title“&gt; 3。支配人体肌肉细胞的胚胎大鼠脊髓植<ol><li>去除肌肉细胞培养融合的媒介，留在单层的中型精细层。 </li><li>将外植体对肌肉细胞层，每35毫米盘5个均匀分布的植仔细。 </li><li>新增很缓慢，下降到每个外植体，融合媒介明智的。这是需要添加肌肉细胞的媒介生存，但不能太多，让植坚持先与细胞接触。如果媒体不加正确（太强烈或过多介质），外植体漂浮，将无法支配肌肉细胞。 </li><li>一旦植开始坚持，顶部的融合中等至2毫升每35毫米的菜，放菜，非常仔细地进入孵化器。 </li></ol> 4。异种神经肌肉文化的维护<ol><li>改变的融合中每周两次。 </li></ol>_title“&gt; 5代表结果。 只要两天后的支配，这是可以看到突起，每个外植体和新兴的按钮状结构（ 图1）所代表的肌肉细胞，建立联系。 早4-6天的支配后，个别纤维将开始合同。 宫缩持续许多个月，维持简单地通过改变培养基每周两次。 约2周后，脊髓外植体本身的退化，但保守的支配。这是正常的。罚款的神经元件层停留在表面的肌肉细胞层。这种神经网络是不足以维持电机单元的功能。 如果外植体漂浮在中期的一天支配后，他们将永远坚持。这种情况发生时肌肉细胞层没有充分融合，或当介质添加过于粗略。有时，会出现植坚持，但没有突起。这可能是由于缺乏连接到它的背根神经节。的支配，以及在这种情况下，将不能成立。操纵的菜肴，非常仔细的第一周，为外植体仍然可以容易分离。交换的融合媒体，用最小的速度增加液滴明智地避免细胞脱落。 <img alt="图1" src="/files/ftp_upload/3616/3616fig1.jpg" /> 图1。脊髓植肌细胞培养至15天从第一天的合作。

<ol>
	<li>Delaporte, C., Dautreaux, B., Fardeau, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+myotube+differentiation+in+vitro+in+different+culture+conditions.">Human myotube differentiation in vitro in different culture conditions.</a> Biol. Cell. 57, 17-22 (1986).</li><li>Kobayashi, T., Askanas, V., Engel, W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+muscle+cultured+in+monolayer+and+cocultured+with+fetal+rat+spinal+cord:+importance+of+dorsal+root+ganglia+for+achieving+successful+functional+innervation.">Human muscle cultured in monolayer and cocultured with fetal rat spinal cord: importance of dorsal root ganglia for achieving successful functional innervation.</a> J. Neurosci. 7, 3131-3141 (1987).</li><li>Braun, S., Croizat, B., Lagrange, M. C., Warter, J. 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Cell Res. 259, 204-212 (2000).</li><li>Lefebvre, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+a+spinal+muscular+atrophy-determining+gene.">Identification and characterization of a spinal muscular atrophy-determining gene.</a> Cell. 80, 155-165 (1995).</li><li>Schrank, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inactivation+of+the+survival+motor+neuron+gene,+a+candidate+gene+for+human+spinal+muscular+atrophy,+leads+to+massive+cell+death+in+early+mouse+embryos.">Inactivation of the survival motor neuron gene, a candidate gene for human spinal muscular atrophy, leads to massive cell death in early mouse embryos.</a> Proc. Natl. Acad. Sci. U.S.A. 94, 9920-9925 (1997).</li><li>Hsieh-Li, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mouse+model+for+spinal+muscular+atrophy.">A mouse model for spinal muscular atrophy.</a> Nat. Genet. 24, 66-70 (2000).</li><li>Monani, U. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+centromeric+survival+motor+neuron+gene+(SMN2)+rescues+embryonic+lethality+in+Smn(-/-)+mice+and+results+in+a+mouse+with+spinal+muscular+atrophy.">The human centromeric survival motor neuron gene (SMN2) rescues embryonic lethality in Smn(-/-) mice and results in a mouse with spinal muscular atrophy.</a> Hum. Mol. Genet. 9, 333-339 (2000).</li><li>Sleigh, J. N., Gillingwater, T. 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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscular+dystrophies+involving+the+dystrophin-glycoprotein+complex:+an+overview+of+current+mouse+models.">Muscular dystrophies involving the dystrophin-glycoprotein complex: an overview of current mouse models.</a> 12, 349-361 (2002).</li><li>Schnabel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroscience:+Standard+model.">Neuroscience: Standard model.</a> Nature. 454, 682-685 (2008).</li><li>Benatar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lost+in+translation:+treatment+trials+in+the+SOD1+mouse+and+in+human+ALS.">Lost in translation: treatment trials in the SOD1 mouse and in human ALS.</a> Neurobiol. Dis. 26, 1-13 (2007).</li><li>Dorchies, O. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+innervation+and+differentiation+of+X-linked+myotubular+myopathy+muscle+cells+in+a+nerve-muscle+coculture+system.">Normal innervation and differentiation of X-linked myotubular myopathy muscle cells in a nerve-muscle coculture system.</a> Neuromuscul. Disord. 11, 736-746 (2001).</li><li>McFerrin, J., Engel, W. K., Askanas, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+innervation+of+cultured+human+muscle+overexpressing+betaAPP+experimentally+and+genetically:+relevance+to+inclusion-body+myopathies.">Impaired innervation of cultured human muscle overexpressing betaAPP experimentally and genetically: relevance to inclusion-body myopathies.</a> Neuroreport. 9, 3201-3205 (1998).</li></ol>

我们什么都没有透露。

生理体外的工具来研究肌肉细胞的功能，在正常和病理情况下是最高的myologists的利益，因为肌肉细胞培养不重述多种细胞和细胞类型连接的重要性的。增加肌肉细胞纯化的运动神经元是不够的，因为雪旺氏细胞的存在是必需的支配有效率5，它不是拓扑（3D +特约经销）有关的协议描述这里实现功能的运动单位被成功地用于建立神经支配的肌肉细胞， 在体外和在一个完整的NMJ?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td> HBSS中</td><td> GIBCO / Invitrogen公司</td><td> 14170 </td></tr><tr><td>的MEM </td><td> GIBCO / Invitrogen公司</td><td> 31095 </td></tr><tr><td>中等199 </td><td> GIBCO / Invitrogen公司</td><td> 31153 </td></tr><tr><td>胎牛血清</td><td>胚胎克隆Perbio </td><td> SH30066.03 </td></tr><tr><td>胰岛素</td><td>西格玛</td><td> I9278 </td></tr><tr><td>人类表皮生长因子</td><td>西格玛</td><td> E9644 </td></tr><tr><td>人类FGF </td><td>硅GMA </td><td> F0291 </td></tr><tr><td>青霉素/链霉素溶液</td><td> Gibco公司</td><td> 15140 </td></tr></tbody></table>

a functional motor unit in the culture dish: co-culture of spinal cord explants and muscle cells

人类的主要肌肉细胞单层培养aneurally很少自发的，因为在一个神经组件的情况下，细胞分化是有限的和运动神经元的刺激，失踪1人的合同。这些限制妨碍在体外培养的肌肉细胞中的许多神经肌肉疾病的研究。重要的是，单层，培养肌细胞的实验限制是可以克服的功能神经支配的肌纤维与脊髓外植体，在联合培养。 在这里，我们实现了高效，正确支配人类的主要肌肉细胞所需的不同步骤，根据Askanas 2开发的方法，导致完成分化和纤维收缩。这样做，肌肉细胞共同培养大鼠胚胎脊髓植13.5海关，背根节还连着脊髓切片。几天后，肌肉FIBERS开始合同，并最终成为通过支配跨横纹肌从外植体，连接到肌肉细胞的脊髓投射功能的突起。这种结构可以维持很多个月，只需定期交流培养基。 这宝贵的工具的应用有很多，因为它代表了一种对人体肌肉的发展和支配的多学科分析的功能模型。事实上，一个完整的从头神经肌肉交界处安装发生在培养皿中，让每一步，从根本上和生理范围内，一个容易的多参数测量。 只是举出几个例子，基因和/或蛋白质的研究，可以直接进行联合培养。此外，前和突触后的效果可以明确，并分别在神经肌肉接头处评估，从不同的物种，因为这两个组件，老鼠和人，分别为。共培养神经肌肉也可以从肌肉或神经肌肉疾病患者分离出人体肌肉细胞进行，因而可以作为候选药物的筛选工具。最后，没有特殊的设备，但需要定期BSL2设施重现功能的运动单位在培养皿。因此，这种方法是有价值的肌肉以及肌肉神经肌肉功能的生理和机理研究研究社区，在正常和疾病的情况下。

Watch this Scientific Journal Video about A Functional Motor Unit in the Culture Dish: Co-culture of Spinal Cord Explants and Muscle Cells at JoVE.com

A Functional Motor Unit in the Culture Dish: Co-culture of Spinal Cord Explants and Muscle Cells

体外培养肌细胞是不够的模型来概括神经支配的肌肉<em&gt;在体内</em&gt;。可以复制功能的运动单位<em&gt;在体外</em&gt;分化人类的主要肌肉细胞用鼠胚胎脊髓植支配。本文介绍了如何建立脊髓植和肌肉细胞共培养。

一个在培养皿中的功能电机组：脊髓植和肌肉细胞共培养

Human primary muscle cells cultured  aneurally in monolayer rarely contract spontaneously because, in the absence of  a nerve component, cell ...

a-functional-motor-unit-culture-dish-co-culture-spinal-cord-explants

Research

JoVE Journal

Neuroscience

15.2K 次观看.  University of Basel. 体外培养肌细胞是不够的模型来概括神经支配的肌肉在体内。可以复制功能的运动单位在体外分化人类的主要肌肉细胞用鼠胚胎脊髓植支配。本文介绍了如何建立脊髓植和肌肉细胞共培养。

视频: A Functional Motor Unit in the Culture Dish: Co-culture of Spinal Cord Explants and Muscle Cells

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

从胚胎脊髓​​连合神经的解剖和文化

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

科研

神经科学

Dissection and Culture of Chick Statoacoustic Ganglion and Spinal Cord Explants in Collagen Gels for Neurite Outgrowth Assays

The sensory organs of the chicken inner ear are innervated by the peripheral processes of statoacoustic ganglion (SAG) neurons. Sensory organ innervation depends on a combination of axon guidance cues1 and survival factors2 located along the trajectory of growing axons and/or within their sensory organ targets. For example, functional interference with a classic axon guidance signaling pathway, semaphorin-neuropilin, generated misrouting of otic axons3. Also, several growth factors expressed in the sensory targets of the inner ear, including Neurotrophin-3 (NT-3) and Brain Derived Neurotrophic Factor (BDNF), have been manipulated in transgenic animals, again leading to misrouting of SAG axons4. These same molecules promote both survival and neurite outgrowth of chick SAG neurons in vitro5,6.
Here, we describe and demonstrate the in vitro method we are currently using to test the responsiveness of chick SAG neurites to soluble proteins, including known morphogens such as the Wnts, as well as growth factors that are important for promoting SAG neurite outgrowth and neuron survival. Using this model system, we hope to draw conclusions about the effects that secreted ligands can exert on SAG neuron survival and neurite outgrowth. 
SAG explants are dissected on embryonic day 4 (E4) and cultured in three-dimensional collagen gels under serum-free conditions for 24 hours. First, neurite responsiveness is tested by culturing explants with protein-supplemented medium. Then, to ask whether point sources of secreted ligands can have directional effects on neurite outgrowth, explants are co-cultured with protein-coated beads and assayed for the ability of the bead to locally promote or inhibit outgrowth. We also include a demonstration of the dissection (modified protocol7) and culture of E6 spinal cord explants. We routinely use spinal cord explants to confirm bioactivity of the proteins and protein-soaked beads, and to verify species cross-reactivity with chick tissue, under the same culture conditions as SAG explants. These in vitro assays are convenient for quickly screening for molecules that exert trophic (survival) or tropic (directional) effects on SAG neurons, especially before performing studies in vivo. Moreover, this method permits the testing of individual molecules under serum-free conditions, with high neuron survival8.

We demonstrate how to dissect and culture chick E4 statoacoustic ganglion and E6 spinal cord explants. Explants are cultured under serum-free conditions in 3D collagen gels for 24 hours. Neurite responsiveness is tested with growth factor-supplemented medium and with protein-coated beads.

小鸡Statoacoustic神经节和脊髓植在胶原凝胶夹层和文化突起生长实验

The sensory organs of the chicken inner ear are innervated by the peripheral processes of statoacoustic ganglion (SAG) neurons. Sensory organ innervation ...

Chicken Embryo Spinal Cord Slice Culture Protocol

Slice cultures can facilitate the manipulation of embryo development both pharmacologically and through gene manipulations. In this reduced system, potential lethal side effects due to systemic drug applications can be overcome. However, culture conditions must ensure that normal development proceeds within the reduced environment of the slice. We have focused on the development of the spinal cord, particularly that of spinal motor neurons. We systematically varied culture conditions of chicken embryo slices from the point at which most spinal motor neurons had been born. We assayed the number and type of motor neurons that survived during the culture period and the position of those motor neurons compared to that in vivo. We found that serum type and neurotrophic factors were required during the culture period and were able to keep motor neurons alive for at least 24 hr and allow those motor neurons to migrate to appropriate positions in the spinal cord. We present these culture conditions and the methodology of preparing the embryo slice cultures using eviscerated chicken embryos embedded in agarose and sliced using a vibratome.

Slice cultures facilitate the manipulation of embryo development by gene and pharmacological perturbations. However, culture conditions must ensure that normal development can proceed within the reduced environment of the slice. We illustrate a protocol that facilitates normal spinal cord development to proceed for at least 24 hr.

鸡胚胎脊髓片培养协议

Slice cultures can facilitate the manipulation of  embryo development both pharmacologically and through gene manipulations. In  this reduced system, ...

Analysis of Developing Tooth Germ Innervation Using Microfluidic Co-culture Devices

Innervation plays a key role in the development, homeostasis and regeneration of organs and tissues. However, the mechanisms underlying these phenomena are not well understood yet. In particular, the role of innervation in tooth development and regeneration is neglected.
Several in vivo studies have provided important information about the patterns of innervation of dental tissues during development and repair processes of various animal models. However, most of these approaches are not optimal to highlight the molecular basis of the interactions between nerve fibres and target organs and tissues.
Co-cultures constitute a valuable method to investigate and manipulate the interactions between nerve fibres and teeth in a controlled and isolated environment. In the last decades, conventional co-cultures using the same culture medium have been performed for very short periods (e.g., two days) to investigate the attractive or repulsive effects of developing oral and dental tissues on sensory nerve fibres. However, extension of the culture period is required to investigate the effects of innervation on tooth morphogenesis and cytodifferentiation.
Microfluidics systems allow co-cultures of neurons and different cell types in their appropriate culture media. We have recently demonstrated that trigeminal ganglia (TG) and teeth are able to survive for a long period of time when co-cultured in microfluidic devices, and that they maintain in these conditions the same innervation pattern that they show in vivo.
On this basis, we describe how to isolate and co-culture developing trigeminal ganglia and tooth germs in a microfluidic co-culture system.This protocol describes a simple and flexible way to co-culture ganglia/nerves and target tissues and to study the roles of specific molecules on such interactions in a controlled and isolated environment.

Co-cultures represent a valuable method to study the interactions between nerves and target tissues and organs. Microfluidic systems allow co-culturing ganglia and whole developing organs or tissues in different culture media, thus representing a valuable tool for the in vitro study of the crosstalk between neurons and their targets.

微流控采用共培养开发设备牙胚支配分析

Innervation plays a key role in the development, homeostasis and regeneration of organs and tissues. However, the mechanisms underlying these phenomena ...

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing organisms, combined with the embryonic optical clarity, served as initial compelling attributes of this model. Over the past two decades, the success of this model has been further propelled by its amenability to large-scale mutagenesis screens and by the ease of transgenesis. More recently, gene-editing approaches have extended the power of the model.
For neurodevelopmental studies, the zebrafish embryo and larva provide a model to which multiple methods can be applied. Here, we focus on methods that allow the study of an essential property of neurons, electrical excitability. Our preparation for the electrophysiological study of zebrafish spinal neurons involves the use of veterinarian suture glue to secure the preparation to a recording chamber. Alternative methods for recording from zebrafish embryos and larvae involve the attachment of the preparation to the chamber using a fine tungsten pin1,2,3,4,5. A tungsten pin is most often used to mount the preparation in a lateral orientation, although it has been used to mount larvae dorsal-side up4. The suture glue has been used to mount embryos and larvae in both orientations. Using the glue, a minimal dissection can be performed, allowing access to spinal neurons without the use of an enzymatic treatment, thereby avoiding any resultant damage. However, for larvae, it is necessary to apply a brief enzyme treatment to remove the muscle tissue surrounding the spinal cord. The methods described here have been used to study the intrinsic electrical properties of motor neurons, interneurons, and sensory neurons at several developmental stages6,7,8,9.

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

This manuscript describes methods for electrophysiological recordings from spinal neurons of zebrafish embryos and larvae. The preparation maintains neurons in situ and often involves minimum dissection. These methods allow for the electrophysiological study of a variety of spinal neurons, from the initial electrical excitability acquisition through the early larval stages.

斑马鱼<em&gt;原位</em&gt;脊髓制备用于脊髓感觉和运动神经元的电生理记录

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing ...

Laminectomy and Spinal Cord Window Implantation in the Mouse

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated digital vaporizer is utilized to achieve a stable plane of anesthesia at a low-flow rate of isoflurane. A single vertebral spine is removed, and a commercially available cover-glass is overlaid on a thin agarose bed. A 3D-printed plastic backplate is then affixed to the adjacent vertebral spines using tissue adhesive and dental cement. A stabilization platform is used to reduce motion artifact from respiration and heartbeat. This rapid and clamp-free method is well-suited for acute multi-photon fluorescence microscopy. Representative data are included for an application of this technique to two-photon microscopy of the spinal cord vasculature in transgenic mice expressing eGFP:Claudin-5 &#8212; a tight junction protein.

This protocol describes implantation of a glass window onto the spinal cord of a mouse to facilitate visualization by intravital microscopy.

拉米内切手术和脊髓窗口植入小鼠

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated ...

Identifying Cell Surface Markers of Primary Neural Stem and Progenitor Cells by Metabolic Labeling of Sialoglycan

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized niches in vivo and can be isolated and expanded in vitro, serving as an important resource for cell transplantation to repair brain damage. However, NSPCs are heterogeneous and not clearly defined at the molecular level or purified due to a lack of specific cell surface markers. The protocol presented, which has been previously reported, combines a neural-endothelial co-culture system with a metabolic glycan labeling method to identify the surface sialoglycoproteome of primary NSPCs. The NSPC-endothelial co-culture system allows self-renewal and expansion of primary NSPCs in vitro, generating a sufficient number of NSPCs. Sialoglycans in cultured NSPCs are labeled using an unnatural sialic acid metabolic reporter with bioorthogonal functional groups. By comparing the sialoglycoproteome from self-renewing NSPCs expanded in an endothelial co-culture with differentiating neural culture, we identify a list of membrane proteins that are enriched in NSPCs. In detail, the protocol involves: 1) set-up of an NSPC-endothelial co-culture and NSPC differentiating culture; 2) labeling with azidosugar per-O-acetylated N-azidoacetylmannosamine (Ac4ManNAz); and 3) biotin conjugation to modified sialoglycan for imaging after fixation of neural culture or protein extraction from neural culture for mass spectrometry analysis. Then, the NSPC-enriched surface marker candidates are selected by comparative analysis of mass spectrometry data from both the expanded NSPC and differentiated neural cultures. This protocol is highly sensitive for identifying membrane proteins of low abundance in the starting materials, and it can be applied to marker discovery in other systems with appropriate modifications

Presented here is a protocol that combines an in vitro neural-endothelial co-culture system and metabolic incorporation of sialoglycan with bioorthogonal functional groups to expand primary neural stem and progenitor cells and label their surface sialoglycoproteins for imaging or mass-spectrometry analysis of cell surface markers.

通过Sialoglycan的代谢标记识别主要神经干细胞和祖细胞的细胞表面标记

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized ...

Direct Injection of a Lentiviral Vector Highlights Multiple Motor Pathways in the Rat Spinal Cord

Introducing proteins of interest into cells in the nervous system is challenging due to innate biological barriers that limit access to most molecules. Injection directly into spinal cord tissue bypasses these barriers, providing access to cell bodies or synapses where molecules can be incorporated. Combining viral vector technology with this method allows for introduction of target genes into nervous tissue for the purpose of gene therapy or tract tracing. Here a virus engineered for highly efficient retrograde transport (HiRet) is introduced at the synapses of propriospinal interneurons (PNs) to encourage specific transport to neurons in the spinal cord and brainstem nuclei. Targeting PNs takes advantage of the numerous connections they receive from motor pathways such as the rubrospinal and reticulospinal tracts, as well as their interconnection with each other throughout spinal cord segments. Representative tracing using the HiRet vector with constitutively active green fluorescent protein (GFP) shows high fidelity details of cell bodies, axons and dendritic arbors in thoracic PNs and in reticulospinal neurons in the pontine reticular formation. HiRet incorporates well into brainstem pathways and PNs but shows age dependent integration into corticospinal tract neurons. In summary, spinal cord injection using viral vectors is a suitable method for introduction of proteins of interest into neurons of targeted tracts.

This protocol demonstrates injection of a retrogradely transportable viral vector into rat spinal cord tissue. The vector is taken up at the synapse and transported to the cell body of target neurons. This model is suitable for retrograde tracing of important spinal pathways or targeting cells for gene therapy applications.

直接注射慢病毒载体突出大鼠脊髓中的多种运动途径

Introducing proteins of interest into cells in the nervous system is challenging due to innate biological barriers that limit access to most molecules. ...

Conversion of Human Induced Pluripotent Stem Cells (iPSCs) into Functional Spinal and Cranial Motor Neurons Using PiggyBac Vectors

We describe here a method to obtain functional spinal and cranial motor neurons from human induced pluripotent stem cells (iPSCs). Direct conversion into motor neuron is obtained by ectopic expression of alternative modules of transcription factors, namely Ngn2, Isl1 and Lhx3 (NIL) or Ngn2, Isl1 and Phox2a (NIP). NIL and NIP specify, respectively, spinal and cranial motor neuron identity. Our protocol starts with the generation of modified iPSC lines in which NIL or NIP are stably integrated in the genome via a piggyBac transposon vector. Expression of the transgenes is then induced by doxycycline and leads, in 5 days, to the conversion of iPSCs into MN progenitors. Subsequent maturation, for 7 days, leads to homogeneous populations of spinal or cranial MNs. Our method holds several advantages over previous protocols: it is extremely rapid and simplified; it does not require viral infection or further MN isolation; it allows generating different MN subpopulations (spinal and cranial) with a remarkable degree of maturation, as demonstrated by the ability to fire trains of action potentials. Moreover, a large number of motor neurons can be obtained without purification from mixed populations. iPSC-derived spinal and cranial motor neurons can be used for in vitro modeling of Amyotrophic Lateral Sclerosis and other neurodegenerative diseases of the motor neuron. Homogeneous motor neuron populations might represent an important resource for cell type specific drug screenings.

Conversion of Human Induced Pluripotent Stem Cells iPSCs into Functional Spinal and Cranial Motor Neurons Using PiggyBac Vectors

This protocol allows rapid and efficient conversion of induced pluripotent stem cells into motor neurons with a spinal or cranial identity, by ectopic expression of transcription factors from inducible piggyBac vectors.

利用 PiggyBac 载体将人诱导的多能干细胞 (Ipsc) 转化为功能性脊柱和颅内运动神经元

We describe here a method to obtain functional spinal and cranial motor neurons from human induced pluripotent stem cells (iPSCs). Direct conversion into ...

Intra-Arterial Delivery of Neural Stem Cells to the Rat and Mouse Brain: Application to Cerebral Ischemia

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to intracranial delivery, intra-arterial administration of NSCs is less invasive and produces a more diffuse distribution of NSCs within the brain parenchyma. Further, intra-arterial delivery allows the first-pass effect in the brain circulation, lessening the potential for trapping of cells in peripheral organs, such as liver and spleen, a complication associated with peripheral injections. Here, we detail the methodology, in both mice and rats, for delivery of NSCs through the common carotid artery (mouse) or external carotid artery (rat) to the ipsilateral hemisphere after an ischemic stroke. Using GFP-labeled NSCs, we illustrate the widespread distribution achieved throughout the rodent ipsilateral hemisphere at 1 d, 1 week and 4 weeks after postischemic delivery, with a higher density in or near the ischemic injury site. In addition to long-term survival, we show evidence of differentiation of GFP-labeled cells at 4 weeks. The intra-arterial delivery approach described here for NSCs can also be used for administration of therapeutic compounds, and thus has broad applicability to varied CNS injury and disease models across multiple species.

A method for delivering neural stem cells, adaptable for injecting solutions or suspensions, through the common carotid artery (mouse) or external carotid artery (rat) after ischemic stroke is reported. Injected cells are distributed broadly throughout the brain parenchyma and can be detected up to 30 d after delivery.

神经干细胞向大鼠和小鼠大脑的动脉内传递：对脑缺血的应用

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to ...