这项研究得到了日本科学促进会（JSPS）17H05661和18K19903、核心-2核心项目和超越人工智能研究所的支持。

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在该协议的一部分中，一项专利已授权给吉克萨克生物工程公司，该公司是由川田吉罗创立的。

此协议描述了运动神经器官 （MNO） 的形成，该有机体具有从人类 iPS 细胞生成的运动神经元球体中延伸的轴束。形成的轴束厚、灵活，在单向结构中组织良好。通过解剖轴突束，可以充分获得高纯度轴蛋白和RNA进行生化分析。神经元活性可以通过钙成像在轴突束和球形中测量。西方的污点没有检测到轴突利酸盐中核蛋白和树突蛋白的污染，这表明我们的方法有效地将轴突与细胞体和树突分离出来。
此协议的优点之一是 MNO 配备轴成捆绑包的快速分化和生成，其中所有流程均可在 4 周内使用 3D 协议完成，使用 2D 协议可在 5-6 周内完成。与其他通常需要 3-4 周才能从胚胎干细胞和 iPS 细胞17 中分化成 MN 的协议相比，这很短，并且需要额外的 2-4 周才能获得轴突拉伸。与 2D 协议相比，3D 协议通常优先于 2D 协议，因为与 2D 协议相比，没有分离步骤的分化时间更短、步骤更少、技术风险更小。基于微流体的组织培养芯片的设计方式使MNS的轴突可以通过微通道向另一个腔室拉长，从而通过诱导轴突的相互作用和轴突之间的亲和力，促进轴突束的形成。由于简单的实验设置，这里描述的所有方案不仅可以由生物工程师执行，他们熟悉组织培养芯片的操作，而且生物学家和神经科学家也不熟悉微流体和微压造技术。需要注意的是，第 1 步和第 2 步也可以使用外部制造服务执行。
完成协议的关键步骤之一是文化媒介的连续变化。建议在分化过程中每一步彻底改变文化介质，使乏力介质中的因素不干扰MN分化。此协议的另一个关键点是保持未分化的 iPS 细胞质量良好。初始 iPS 细胞培养物的质量显著影响获得运动神经元和 MNO 的效率。另一点是MNS的直径应该小于芯片孔的大小（1.5毫米）。较大的球体无法进入腔室，并有可能在中心部分经历严重的缺氧坏死。MNS 的大小可以通过更改 iPS 细胞的初始播种数量（在 3D 协议中）或运动神经元（在 2D 协议中）来控制。应针对每个 iPS 细胞线优化细胞的播种密度。
带微胶和小孔滤镜的分体微流体设备已被广泛用于将轴突与细胞体和树突分离。这种技术还可以将轴突与细胞体和树突分离出来，在捆绑组织中具有超强的轴突丰度。与其他方法相比，该方法的一个主要局限性是，在组织培养芯片的当前设计中，它不能分离两种不同的培养基，这妨碍了需要两种不同介质的两种不同细胞的共生能力。另一个限制是 PDMS 芯片对组织大小施加了预先确定的限制。大于孔的球体不能进入腔室，轴束不能比微流道的宽度更厚。
这种方法可以应用于其他类型的神经元。我们小组已经显示出使用修改的方法结合脑器官技术18对脑路进行建模的能力。皮质球体被引入舱室，轴突自发地向每个球体相互拉长，随后自发形成轴突束。因此，两个皮质球体可以通过轴束连接，组织可以作为一块获得。这表明，无论神经元细胞类型如何，这种方法都具有高度的多功能性，可以形成轴突束组织。在此协议中，使用了人类iPS细胞，但是，其他干细胞，包括人类ES细胞和人类神经干细胞，可以与修改提出的协议。神经元的3D球形可以通过不同的协议19，20产生。这种用轴束制作组织的方法将来有可能与其他分化方案相结合，用于制造3D MN球形。此外，只需改变组织培养芯片微通道的宽度和高度，即可控制轴束的厚度和长度，以备将来发展之用。
我们相信，这一协议可用于药物测试和筛选，并有助于了解轴感筋膜发育和疾病背后的机制。

脊髓运动神经元 （MN） 将轴突扩展到骨骼肌肉以控制身体运动。它们的轴突轨迹在发展过程中具有高度的组织性和规范性。尽管对轴承扩展和指导1进行了许多研究，但组织轴束形成机制仍在调查中。运动神经元的轴突经常受到肌萎缩性侧索硬化症（ALS）2等神经退行性疾病的损害，但对轴突筋膜损伤的病理生理机理知之甚少。因此，需要一个生理和病理模型来回顾轴束的形成和回归。
人类干细胞源性运动神经元是了解ALS3等发育和疾病的有希望的平台。人类诱导的多能干细胞（iPS细胞）可用于使用患者衍生细胞模拟疾病。迄今为止，从多能干细胞到MN的各种分化方法已报告4，5，6。然而，二维培养中神经元的轴突是随机定向的，在发育中的神经中，轴突通过密集的轴突-轴位相互作用7进行单向组装，不能在体内微环境中重述。为了克服这个问题，我们开发了一种技术，从人类iPS细胞8中产生类似于运动神经的三维组织，并将组织命名为运动神经器官（MNO）。MNO由位于运动神经元球形（MNS）中的细胞体和从球形延伸出去的轴状筋膜组成。圆柱体中的轴突是单向方向的，类似于发展运动神经的轴突。因此，MNOs独特地提供了一种生理轴突微环境，这是任何其他以前开发的神经元培养方法所没有的。
在此协议中，我们描述了已开发芯片中组织培养芯片制造、快速运动神经元分化和运动神经器官形成的方法。我们的组织培养芯片非常简单，它只包含一个接受球形的隔间，一个用于形成轴束的微通道，以及一个用于安装轴星终端的隔间。该设备不包含复杂的结构，包括微流道或微孔过滤器，通常用于分离轴突和细胞体的大小9，10。因此，如果有光刻设置，则可以通过遵循本协议中描述的步骤轻松制造我们的设备。
通过诱导和模式因子（SB431542、LDN-193189、转录剂酸（RA）和平滑激动剂（SAG）和加速因子（SU5402 和 DAPT）的优化组合，实现了人类 iPS 细胞的快速分化。据报道，SU5402和DAPT的结合加速了外周神经元和神经峰细胞11的分化。在此协议中，我们提供了三种不同的方法来生成 MNO，以便读者可以决定最适合其需求的方法。我们建议在形成球形（3D方法）后对人体iPS细胞进行分化，因为分化的MNS可以直接转移到组织培养芯片中。或者，人类iPS细胞可以在单层（2D）培养中分化成运动神经元，然后创建成三维运动神经元球形，正如我们之前报道的8。我们更新了协议，通过本协议中描述的三维分化方法，可以避免从 2D 到 3D 的过渡，并且无需分离步骤即可以更短的分化时间、更少的步骤和更低的技术风险获得 MNO。商业上可用的神经元也可用于生成 MNS 以减少分化时间。
为了生成 MNO，我们在组织培养芯片中培养了 MNS。轴突从球体拉长，并延伸到轴突聚集和单向对齐的微通道。这有利于斧轴相互作用和在微通道中自发形成紧密组装的轴突单向束组织，这是本协议所独有的，而无论是自发捆绑形成还是引导轴突定向单独可以通过其他协议12、13、14来实现。在一个典型的实验中，很少有细胞从球体迁移到微通道，而且大多数细胞都留在球形附近。这种方法允许轴突自发地从球体中分离出来，而无需使用依赖大小的物理屏障（例如微沟或微孔过滤器）将轴突与细胞体分离。
由此产生的 MNO 可接受各种检查，包括形态学、生化和物理分析。细胞体和扩展轴束可以通过切割进行物理分离，并可单独分析以进行下游实验，例如生化检测。生物材料，包括RNA和蛋白质，可以从几个轴成束中分离，用于常规生化检测，包括RT-PCR和西方印迹。在这里，我们描述了从iPS细胞中生成运动神经器官的协议，它提供了一个有吸引力的生理和病理模型来研究轴心筋膜发育和疾病的机制。

<table>
	<tbody>
		<tr>
			<td>(Tridecafluoro-1,1,2,2-tetrahydrooctyl)-1-trichlorosilane</td>
			<td>Sigma</td>
			<td>440302</td>
			<td></td>
		</tr>
		<tr>
			<td>16% Paraformaldehyde (formaldehyde) aqueous solution</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td></td>
		</tr>
		<tr>
			<td>200&#181;l Wide Bore Pipet Tips</td>
			<td>BMBio</td>
			<td>BMT-200WRS</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plates</td>
			<td>Violamo</td>
			<td>2-8588-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>ICT</td>
			<td>AT104</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 Supplement (50X)</td>
			<td>Gibco</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma</td>
			<td>A6003</td>
			<td></td>
		</tr>
		<tr>
			<td>Brain-derived neurotrophic factor (BDNF)</td>
			<td>Wako</td>
			<td>020-12913</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 incubator</td>
			<td>Panasonic</td>
			<td>MCO-18AIC</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostor CS10</td>
			<td>Stem Cell Technologies</td>
			<td>07959</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPT</td>
			<td>Sigma</td>
			<td>D5942</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Sigma</td>
			<td>D8437</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluo-4 AM</td>
			<td>Dojindo Laboratories</td>
			<td>CS22</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX Supplement</td>
			<td>Gibco</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Growth factor reduced Matrigel (basement membrane matrix)</td>
			<td>Corning</td>
			<td>354230</td>
			<td></td>
		</tr>
		<tr>
			<td>HB9 Antibody</td>
			<td>Santa Cruz</td>
			<td>sc-22542</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Wako</td>
			<td>085-09355</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Sigma</td>
			<td>14533</td>
			<td></td>
		</tr>
		<tr>
			<td>iCell motor neuron (commercially available human iPS cell-derived motor neurons)</td>
			<td>Cellular Dynamics</td>
			<td>R1051</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropyl alcohol (IPA)</td>
			<td>Wako</td>
			<td>166-04836</td>
			<td></td>
		</tr>
		<tr>
			<td>Knock Out Serum Replacement</td>
			<td>Gibco</td>
			<td>10828028</td>
			<td></td>
		</tr>
		<tr>
			<td>LDN193189</td>
			<td>Sigma</td>
			<td>SML0559</td>
			<td></td>
		</tr>
		<tr>
			<td>MEA probe</td>
			<td>Alpha MED Scientific inc</td>
			<td>MED-P5004A</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Non-essential Amino Acid Solution (100x) (NEAA)</td>
			<td>Sigma</td>
			<td>M7145</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Glass</td>
			<td>Matsunami</td>
			<td>S9111</td>
			<td></td>
		</tr>
		<tr>
			<td>mTeSR Plus</td>
			<td>Stem Cell Technologies</td>
			<td>05825</td>
			<td></td>
		</tr>
		<tr>
			<td>N2 supplement</td>
			<td>Wako</td>
			<td>141-08941</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Gibco</td>
			<td>21103049</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Photoresist SU-8 2100</td>
			<td>Microchem</td>
			<td>#SU-8 2100</td>
			<td></td>
		</tr>
		<tr>
			<td>Prime surface 96U</td>
			<td>Sumitomo Bakelite</td>
			<td>MS-9096U</td>
			<td></td>
		</tr>
		<tr>
			<td>ReLeSR (passaging reagent)</td>
			<td>Stem Cell Technologies</td>
			<td>05872</td>
			<td></td>
		</tr>
		<tr>
			<td>Retinoic acid</td>
			<td>Wako</td>
			<td>186-01114</td>
			<td></td>
		</tr>
		<tr>
			<td>SAG</td>
			<td>Sigma</td>
			<td>SML1314</td>
			<td></td>
		</tr>
		<tr>
			<td>SB431542</td>
			<td>Wako</td>
			<td>192-16541</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicon wafer</td>
			<td>SUMCO</td>
			<td>PW-100-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Silpot 184 w/c kit</td>
			<td>Dow Toray</td>
			<td>Silpot 184 w/c kit</td>
			<td></td>
		</tr>
		<tr>
			<td>Smi32 Antibody</td>
			<td>Biolegend</td>
			<td>801701</td>
			<td></td>
		</tr>
		<tr>
			<td>SU5402</td>
			<td>Sigma</td>
			<td>SML0443</td>
			<td></td>
		</tr>
		<tr>
			<td>SU-8 Developer</td>
			<td>Microchem</td>
			<td>Y020100</td>
			<td></td>
		</tr>
		<tr>
			<td>Synapsin I Antibody</td>
			<td>Millipore</td>
			<td>Ab1543</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE Express liquid without phenol red (dissociation solution)</td>
			<td>Gibco</td>
			<td>12604-021</td>
			<td></td>
		</tr>
		<tr>
			<td>Tuj1 Antibody</td>
			<td>Biolegend</td>
			<td>801202</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>Wako</td>
			<td>030-24021</td>
			<td></td>
		</tr>
	</tbody>
</table>

three-dimensional motor nerve organoid generation

轴突的筋膜是神经系统中观察到的主要结构图案之一。轴感筋膜的破坏可能导致发育和神经退行性疾病。虽然已经对轴突进行了大量研究，但由于缺乏坚固的体外模型，我们对轴突筋膜的形成和功能障碍的理解仍然有限。在这里，我们描述了一个循步骤的协议，从微流体为基础的组织培养芯片中从人类诱导的多能干细胞（iPS）细胞中快速生成运动神经器官（MNO）。首先，描述了用于该方法的芯片的制造。从人类iPS细胞中，形成运动神经元球形（MNS）。接下来，区分的 MNS 将传输到芯片中。此后，轴突自发地从球形中长出来，在芯片中装备的微通道内组装成一个圆柱体，从而产生一个MNO组织，携带一束从球形延伸的轴突。对于下游分析，MNO 可以从芯片中取出，用于形态分析或剖析以进行生化分析，以及钙成像和多极阵列记录。通过该协议产生的多国机构可以促进药物测试和筛选，并有助于了解轴位筋膜发育和疾病的基本机制。

在3D分化程序中，运动神经元在12-14天内被分化（图4和图5）。重要的是，超过60%的细胞在分化过程中表示运动神经元标记HB9。免疫化学显示，MNS中大约80%的细胞是SMI32阳性运动神经元。HB9和SMI32是已建立的早期运动神经元标记15，16。HB9 和 SMI32 的表达是需要确认以确保运动神经元细胞识别的关键参数。在将 MNS 引入培养芯片后，轴突扩展到通道和轴突捆绑形式。由于微通道充当物理导引，轴突从 MNS 拉长，并通过轴突交互（图 6A）形成捆绑。必须通过微观观察确认轴束的形成，以确认MNO的生成。成功的 MNO 承载一个宽于 50μm 的轴突捆绑包，通道中的捆绑包中几乎没有分离的轴突。在引入球体24小时后，可以观察到轴突的初始拉长。在接下来的3-4天内，轴突到达微通道的中心，然后在10天内到达另一端（图6A）。因此，轴突在芯片的2-3周内组装并形成了一个直线和单向束，之后观察到神经元活动。
运动神经器官可以通过将 PDMS 从显微镜玻璃中分离以进行生物分析（图 6B）从芯片中收集。在显微镜下使用手术刀或钳子切割，可以解剖和分离轴束和细胞体（图6B）。这些生物材料，包括RNA和蛋白质，可用于常规生化检测，如RT-PCR和西方印迹。在多核子的轴突束中，在西方印迹（图6C）中未检测到核蛋白或对气质制造蛋白。
结合钙指标（Fluo-4 AM），神经元活性可以在组织培养芯片中捕获。在MNO中观察到球形和轴束中运动神经元的自发活动。此外，还使用多极阵列系统观察到神经活动。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61544/61544fig01.jpg"> 图1:PDMS组织培养芯片的尺寸。 （A） 组织培养芯片的光面具。（B） 组织培养芯片中微通道的尺寸。支撑运动神经元球形的基室直径为2毫米，腔体上方的PDMS孔为1.5毫米。微通道桥接两个腔室的宽度和高度均为 150 μm。 <a href="https://www.jove.com/files/ftp_upload/61544/61544fig01large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61544/61544fig02.jpg"> 图2:运动神经元分化的示意图。 （A） 分化步骤涉及神经诱导、运动神经元血统的模式以及运动神经元的成熟。（B） 从iPS细胞中创建运动神经元球形（MNS）的两种选择:3D协议和具有运动神经元分离步骤的二维协议。运动神经器官 （MNO） 可以通过这两种协议获得。<a href="https://www.jove.com/files/ftp_upload/61544/61544fig02large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61544/61544fig03.jpg"> 图3:地下室膜基质涂层和运动神经元球形引入的分步协议。 （A） 组织培养芯片通道中的地下室膜基质涂层。（B） MNS 引入芯片的孔。（C） 文化媒介因耗尽媒介的愿望而改变。 <a href="https://www.jove.com/files/ftp_upload/61544/61544fig03large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61544/61544fig04.jpg"> 图4:2D和3D运动神经元分化。 （A） 具有代表性的 3D MNS 分化（3D 协议）的时间过程。随着时间的推移，MNS 的大小逐渐增加。比例:500μm。（B） -D2、D0、D1、D6、D12（2D协议）的2D分化时间过程。比例:500μm。<a href="https://www.jove.com/files/ftp_upload/61544/61544fig04large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61544/61544fig05.jpg"> 图5:运动神经元的特征。 （A） （左） 沾有SMI-32抗体和DAPI的MNS的低温剖析。（中）在地下室膜矩阵涂层表面上重镀 MNS 的相位对比图像。观察到轴拉伸。（右）重镀 Mns 的轴染色为 Synapsin I 和 Tuj1 抗体。刻度条:500μm（左、中）和50μm（右）。（B） 二维运动神经元免疫与 Tuj 和 HB9 抗体的代表性图像。比例:200μm。 <a href="https://www.jove.com/files/ftp_upload/61544/61544fig05large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/61544/61544fig06.jpg"> 图6:培养芯片中产生的运动神经器官（MNO）的特征。 （A） D32 上轴伸长和厚轴束形成的代表性图像。比例:500μm。（B） SMI-32和DAPI对运动神经器官（MNO）进行免疫。轴突和细胞体可以通过物理切割进行分离。刻度条:1毫米（C）来自轴突和细胞体的蛋白质纯度，由西方印迹量化。MAP2，一种树突标记物，没有在轴蛋白中检测到，而轴蛋白Tau1富含轴蛋白。 <a href="https://www.jove.com/files/ftp_upload/61544/61544fig06large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>

Watch this Scientific Journal Video about Three-Dimensional Motor Nerve Organoid Generation at JoVE.com

Three-Dimensional Motor Nerve Organoid Generation

该协议提供了一个全面的程序，通过自发组装从组织培养芯片中的球形延伸的强健的轴突束来制造人类iPS细胞衍生的运动神经器官。

三维运动神经器官生成

A fascicle of axons is one of the major structural motifs observed in the nervous system. Disruption of axon fascicles could cause developmental and ...

three-dimensional-motor-nerve-organoid-generation

Research

JoVE Journal

Neuroscience

9.0K 次观看.  The University of Tokyo. 该协议提供了一个全面的程序，通过自发组装从组织培养芯片中的球形延伸的强健的轴突束来制造人类iPS细胞衍生的运动神经器官。

视频: Three-Dimensional Motor Nerve Organoid Generation

Motor Nerve Transection and Time-lapse Imaging of Glial Cell Behaviors in Live Zebrafish

The nervous system is often described as a hard-wired component of the body even though it is a considerably fluid organ system that reacts to external stimuli in a consistent, stereotyped manner, while maintaining incredible flexibility and plasticity. Unlike the central nervous system (CNS), the peripheral nervous system (PNS) is capable of significant repair, but we have only just begun to understand the cellular and molecular mechanisms that govern this phenomenon. Using zebrafish as a model system, we have the unprecedented opportunity to couple regenerative studies with in vivo imaging and genetic manipulation. Peripheral nerves are composed of axons surrounded by layers of glia and connective tissue. Axons are ensheathed by myelinating or non-myelinating Schwann cells, which are in turn wrapped into a fascicle by a cellular sheath called the perineurium. Following an injury, adult peripheral nerves have the remarkable capacity to remove damaged axonal debris and re-innervate targets. To investigate the roles of all peripheral glia in PNS regeneration, we describe here an axon transection assay that uses a commercially available nitrogen-pumped dye laser to axotomize motor nerves in live transgenic zebrafish. We further describe the methods to couple these experiments to time-lapse imaging of injured and control nerves. This experimental paradigm can be used to not only assess the role that glia play in nerve regeneration, but can also be the platform for elucidating the molecular mechanisms that govern nervous system repair.

Although the peripheral nervous system (PNS) is capable of significant repair after injury, little is known about the cellular and molecular mechanisms that govern this phenomenon. Using live, transgenic zebrafish and a reproducible nerve transection assay, we can study dynamic glial cell behaviors during nerve degeneration and regeneration.

电机神经切断和时间推移成像胶质细胞行为在现场斑马鱼

The  nervous system is often described as a hard-wired component of the body even  though it is a considerably fluid organ system that reacts to external ...

科研

神经科学

Protocol for Three-dimensional Confocal Morphometric Analysis of Astrocytes

As glial cells in the brain, astrocytes have diverse functional roles in the central nervous system. In the presence of harmful stimuli, astrocytes modify their functional and structural properties, a condition called reactive astrogliosis. Here, a protocol for assessment of the morphological properties of astrocytes is presented. This protocol includes quantification of 12 different parameters i.e. the surface area and volume of the tissue covered by an astrocyte (astrocyte territory), the entire astrocyte including branches, cell body, and nucleus, as well as total length and number of branches, the intensity of fluorescence immunoreactivity of antibodies used for astrocyte detection, and astrocyte density (number/1,000 &#181;m2). For this purpose three-dimensional (3D) confocal microscopic images were created, and 3D image analysis software such as Volocity 6.3 was used for measurements. Rat brain tissue exposed to amyloid beta1-40 (A&#946;1-40) with or without a therapeutic&#160;intervention was used to present the method. This protocol can also be used for 3D morphometric analysis of other cells from either in vivo or in vitro conditions.

Astrocytes in the CNS change their functional and structural properties in response to harmful stimuli. This report presents a protocol for assessment of three-dimensional astrocyte morphology in diseased conditions or after therapeutic interventions.

协议星形胶质细胞的三维共聚焦形态分析

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

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

The goal of this protocol is to study mitochondria within intraepidermal nerve fibers. Therefore, 3D imaging and analysis techniques were developed to isolate nerve-specific mitochondria and evaluate disease-induced alterations of mitochondria in the distal tip of sensory nerves. The protocol combines fluorescence immunohistochemistry, confocal microscopy and 3D image analysis techniques to visualize and quantify nerve-specific mitochondria. Detailed parameters are defined throughout the procedures in order to provide a concrete example of how to use these techniques to isolate nerve-specific mitochondria. Antibodies were used to label nerve and mitochondrial signals within tissue sections of skin punch biopsies, which was followed by indirect immunofluorescence to visualize nerves and mitochondria with a green and red fluorescent signal respectively. Z-series images were acquired with confocal microscopy and 3D analysis software was used to process and analyze the signals. It is not necessary to follow the exact parameters described within, but it is important to be consistent with the ones chosen throughout the staining, acquisition and analysis steps. The strength of this protocol is that it is applicable to a wide variety of circumstances where one fluorescent signal is used to isolate other signals that would otherwise be impossible to study alone.

This protocol uses three-dimensional (3D) imaging and analysis techniques to visualize and quantify nerve-specific mitochondria. The techniques are applicable to other situations where one fluorescent signal is used to isolate a subset of data from another fluorescent signal.

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 ...

Fiber Connections of the Supplementary Motor Area Revisited: Methodology of Fiber Dissection, DTI, and Three Dimensional Documentation

The purpose of this study is to show the methodology for the examination of the white matter connections of the supplementary motor area (SMA) complex (pre-SMA and SMA proper) using a combination of fiber dissection techniques on cadaveric specimens and magnetic resonance (MR) tractography. The protocol will also describe the procedure for a white matter dissection of a human brain, diffusion tensor tractography imaging, and three-dimensional documentation. The fiber dissections on human brains and the 3D documentation were performed at the University of Minnesota, Microsurgery and Neuroanatomy Laboratory, Department of Neurosurgery. Five postmortem human brain specimens and two whole heads were prepared in accordance with Klingler's method. Brain hemispheres were dissected step by step from lateral to medial and medial to lateral under an operating microscope, and 3D images were captured at every stage. All dissection results were supported by diffusion tensor imaging. Investigations on the connections in line with Meynert's fiber tract classification, including association fibers (short, superior longitudinal fasciculus I and frontal aslant tracts), projection fibers (corticospinal, claustrocortical, cingulum, and frontostriatal tracts), and commissural fibers (callosal fibers) were also conducted.

The purpose of this study is to show each step of the fiber dissection technique on human cadaveric brains, the 3D documentation of these dissections, and the diffusion tensor imaging of the anatomically dissected fiber pathways.

补充电机区的光纤连接重新审视:光纤解剖，DTI和三维文档的方法

The purpose of this study is to show the methodology for the examination of the white matter connections of the supplementary motor area (SMA) complex ...

Generation and Long-term Maintenance of Nerve-free Hydra

The interstitial cell lineage of Hydra includes multipotent stem cells, and their derivatives: gland cells, nematocytes, germ cells, and nerve cells. The interstitial cells can be eliminated through two consecutive treatments with colchicine, a plant-derived toxin that kills dividing cells, thus erasing the potential for renewal of the differentiated cells that are derived from the interstitial stem cells. This allows for the generation of Hydra that lack nerve cells. A nerve-free polyp cannot open its mouth to feed, egest, or regulate osmotic pressure. Such animals, however, can survive and be cultured indefinitely in the laboratory if regularly force-fed and burped. The lack of nerve cells allows for studies of the role of the nervous system in regulating animal behavior and regeneration. Previously published protocols for nerve-free Hydra maintenance involve outdated techniques such as mouth-pipetting with hand-pulled micropipette tips to feed and clean the Hydra. Here, an improved protocol for maintenance of nerve-free Hydra is introduced. Fine-tipped forceps are used to force open the mouth and insert freshly killed Artemia. Following force-feeding, the body cavity of the animal is flushed with fresh medium using a syringe and hypodermic needle to remove undigested material, referred to here as "burping". This new method of force-feeding and burping nerve-free Hydra through the use of forceps and syringes eliminates the need for mouth-pipetting using hand-pulled micropipette tips. It thus makes the process safer and significantly more time efficient. To ensure that the nerve cells in the hypostome have been eliminated, immunohistochemistry using anti-tyrosine-tubulin is conducted.

Generation and Long-term Maintenance of Nerve-free Hydra

Through a double treatment with colchicine, a plant-derived toxin that kills dividing cells, nerve-free Hydra vulgaris can be generated. These Hydra cannot feed or egest on their own. This paper describes an improved method for long-term maintenance of nerve free Hydra vulgaris in the laboratory.

无神经的一代和长期维持<em&gt;水润</em

The interstitial cell lineage of Hydra includes multipotent stem cells, and their derivatives: gland cells, nematocytes, germ cells, and nerve cells. The ...

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. Single-molecule imaging is gaining momentum as it provides exceptional access to the visualization of individual molecules in living cells. Here, we describe a technique that we developed to perform single-particle tracking photo-activated localization microscopy (sptPALM) in Drosophila larvae. Synaptic communication relies on key presynaptic proteins that act by docking, priming, and promoting the fusion of neurotransmitter-containing vesicles with the plasma membrane. A range of protein-protein and protein-lipid interactions tightly regulates these processes and the presynaptic proteins therefore exhibit changes in mobility associated with each of these key events. Investigating how mobility of these proteins correlates with their physiological function in an intact live animal is essential to understanding their precise mechanism of action. Extracting protein mobility with high resolution in vivo requires overcoming limitations such as optical transparency, accessibility, and penetration depth. We describe how photoconvertible fluorescent proteins tagged to the presynaptic protein Syntaxin-1A can be visualized via slight oblique illumination and tracked at the motor nerve terminal or along the motor neuron axon of the third instar Drosophila larva.

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

Here we illustrate how single molecule photo-activated localization microscopy can be carried out on the motor nerve terminal of a live Drosophila melanogaster third instar larva.

在体内果蝇前运动神经末端的单分子跟踪

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. ...

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

The mammalian neocortex is composed of many types of excitatory and inhibitory neurons, each with specific electrophysiological and biochemical properties, synaptic connections, and in vivo functions, but their basic functional and anatomical organization from cellular to network scale is poorly understood. Here we describe a method for the three-dimensional imaging of fluorescently-labeled neurons across large areas of the brain for the investigation of the cortical cellular organization. Specific types of neurons are labeled by the injection of fluorescent retrograde neuronal tracers or expression of fluorescent proteins in transgenic mice. Block brain samples, e.g., a hemisphere, are prepared after fixation, made transparent with tissue clearing methods, and subjected to fluorescent immunolabeling of the specific cell types. Large areas are scanned using confocal or two-photon microscopes equipped with large working distance objectives and motorized stages. This method can resolve the periodic organization of the cell type-specific microcolumn functional modules in the mouse neocortex. The procedure can be useful for the study of three-dimensional cellular architecture in the diverse brain areas and other complex tissues.

Here we describe a procedure for tissue clearing, fluorescent labeling, and large-scale imaging of mouse brain tissue which, thereby, enables visualization of the three-dimensional organization of cell types in the neocortex.

小鼠大脑皮层细胞组织的大尺度三维成像

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

Statistical shape analysis of brain structures has been used to investigate the association between their structural changes and pathological processes. We have developed a software package for accurate and robust shape modeling and group-wise analysis. Here, we introduce an pipeline for the shape analysis, from individual 3D shape modeling to quantitative group shape analysis. We also describe the pre-processing and segmentation steps using open software packages. This practical guide would help researchers save time and effort in 3D shape analysis on brain structures.

We introduce a semi-automatic protocol for shape analysis on brain structures, including image segmentation using open software, and further group-wise shape analysis using an automated modeling package. Here, we demonstrate each step of the 3D shape analysis protocol with hippocampal segmentation from brain MR images.

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. ...

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

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of sciatic nerve injury rodent models. In this protocol, we describe a novel kinematic analysis method that uses a three-dimensional (3D) motion capture apparatus for functional evaluations using a rat sciatic nerve crush injury model. First, the rat is familiarized with treadmill walking. Markers are then attached to the designated bone landmarks and the rat is made to walk on the treadmill at the desired speed. Meanwhile, the posterior limb movements of the rat are recorded using four cameras. Depending on the software used, marker tracings are created using both automatic and manual modes and the desired data are produced after subtle adjustments. This method of kinematic analysis, which uses a 3D motion capture apparatus, offers numerous advantages, including superior precision and accuracy. Many more parameters can be investigated during the comprehensive functional evaluations. This method has several shortcomings that require consideration: The system is expensive, can be complicated to operate, and may produce data deviations due to skin shifting. Nevertheless, kinematic analysis using a 3D motion capture apparatus is useful for performing functional anterior and posterior limb evaluations. In the future, this method may become increasingly useful for generating accurate assessments of various traumas and diseases.

We introduce a kinematic analysis method that uses a three-dimensional motion capture apparatus containing four cameras and data processing software for performing functional evaluations during fundamental research involving rodent models.

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 ...

Purification of Fibroblasts and Schwann Cells from Sensory and Motor Nerves in Vitro

The principal cells in the peripheral nervous system are the Schwann cells (SCs) and the fibroblasts. Both these cells distinctly express the sensory and motor phenotypes involved in different patterns of neurotrophic factor gene expression and other biological processes, affecting nerve regeneration. The present study has established a protocol to obtain highly purified rat sensory and motor SCs and fibroblasts more rapidly. The ventral root (motor nerve) and the dorsal root (sensory nerve) of neonatal rats (7-days-old) were dissociated and the cells were cultured for 4-5 days, followed by isolation of sensory and motor fibroblasts and SCs by combining differential digestion and differential adherence methods sequentially. The results of immunocytochemistry and flow cytometry analyses showed that the purity of the sensory and motor SCs and fibroblasts were &#62;90%. This protocol can be used to obtain a large number of sensory and motor fibroblasts/SCs more rapidly, contributing to the exploration of sensory and motor nerve regeneration.

Here, we present a method to purify fibroblasts and Schwann cells from sensory and motor nerves in vitro.

从体外感觉和运动神经中纯化成纤维细胞和施万细胞

The principal cells in the peripheral nervous system are the Schwann cells (SCs) and the fibroblasts. Both these cells distinctly express the sensory and ...