Name: in vitro time-lapse live-cell imaging to explore cell migration toward the organ of corti
Uploaded: 2020-12-04T14:45:00.000+00:00
Duration: 8 min 8 s
Description: Watch this Scientific Journal Video about In vitro Time-lapse Live-Cell Imaging to Explore Cell Migration toward the Organ of Corti at JoVE.com

这项工作得到了韩国国家研究基金会（NRF-2018-R1D1A1B07050175，HURF-2017-66）和韩国国家研究基金会（NRF）和哈莱姆大学研究基金的研究赠款的支持。

所有涉及ICR小鼠的研究协议都得到了元州医学院延世大学机构动物护理与使用委员会（IACUC）的批准。实验是根据世界医学协会的道德规范进行的。在本协议中，怀孕的ICR小鼠被保存在12/12小时的光/暗循环中，可以自由获得食物和水。
1. 科克利解剖
<ol><li>通过打开紫外线约30分钟，对层层流动组织培养罩进行消毒，并在使用前用70%乙醇喷洒所有表面。让表面干燥。</li>
	<li>将解剖仪器放在70%乙醇中10分钟，在使用前干燥。</li>
	<li>使用手术刀片斩首产后3-4天大的老鼠（图2A）。</li>
	<li>将头骨置于层压流罩的立体显微镜下，并将组织浸泡在 70% 乙醇中。</li>
	<li>快速将组织浸泡在组织解剖溶液中（1x 汉克平衡盐溶液，1 mM HEPES），以去除乙醇。</li>
	<li>用手术刀片切开头骨的中心线（图2B，C）。</li>
	<li>通过前拉下皮肤并切割耳朵的外部听觉导管（图2D）来暴露头骨。</li>
	<li>从前切至颅骨的后部穿过眼线（图2E）。</li>
	<li>打开头骨，用钝钳切除前脑、小脑和脑干（图2F，G）。</li>
	<li>使用微钳，将科克菌与时间骨分离（图2H）。</li>
	<li>将鸡毛虫转移到含有组织解剖溶液的培养皿中。</li>
	<li>仔细解剖所有的软骨胶囊，只留下内部的软骨软组织（图2I，J）。</li>
	<li>用钳子和绞蜗管用另一对钳子握住鸡毛虫的修饰，慢慢地将两个组织分开（图2K）。</li>
	<li>通过轻轻剥去纹状血管和纹状膜去除纹状血管（图2L，M）。</li>
	<li>将灭菌塑料盖片放在新的组织解剖溶液中，然后将Corti的器官放在直径为9毫米的盖片上，确保罗勒膜朝下（图2N-P）。</li>
	<li>通过用钳子（图2N-P）将里斯纳的膜和剩余的模样组织压在盖上来固定组织。</li>
	<li>将带嵌入式组织的盖片转移到直径为 35 mm 的圆盘中心。</li>
	<li>将玻璃克隆缸放在盘子上，将仙人皮去植置于菜的中心，并在菜缸内加入100微升的去植物培养介质（DMEM/F12，10%胎儿牛血清（FBS），1%N2补充剂，安培素（10微克/毫升）10在圆柱体内（图2Q）。</li>
	<li>板 5 × 103 细胞的小鼠骨髓衍生的绿色荧光蛋白 （GFP） 标签 MSC 在 2 mL 培养介质 （45% DMEM + 45% DMEM/F12， 10% FBS， 1% N2 补充， 10μg/mL 的安非他林） 玻璃缸外 （图 2R）.<ol>
<li>当MSC是80-90%的对流，通过分离他们与锥蛋白-乙二胺四乙酸。</li>
	</ol>
</li>
	<li>小心地将圆锥体菜转移到加湿的孵化器中，并在 5% 的 CO2 大气中以 37 °C 的温过夜孵化。</li>
	<li>吸气缸内外的所有介质，然后从圆盘中取出玻璃缸。</li>
	<li>在圆盘中加入 2 mL 的新鲜培养介质，并在潮湿的孵化器中孵化组织培养盘，直到准备好进行分析。</li>
</ol><img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61947/61947fig2v2.jpg"> 图2。解剖小鼠鸡皮和科尔蒂和MSC器官的共养。 （A） 斩首鼠标， （B） 和 （C） 头部的中线下垂解剖， （D）和 （E） 大脑的冠状解剖， （F）和 （G）大脑和时间骨的切除， （H）骨绞痛，（I）去除骨绞痛壁，（J）分离的绞痛，（K）从修饰品分离的古利叶管， （L） 将纹状血管 （SV） 和螺旋韧带 （SL） 与 Corti 器官分离，（M）去除地膜，（N-P） 将毛细管固定在塑料盖片上，（Q）盖片和玻璃缸的位置，（R）接种 MSC. 白色鳞片条（A-E） = 1 厘米:橙色（F， G， P）和黄色比例杆（H，I） = 1 毫米:绿色比例杆（J-O） = 0.5 mm.<a href="https://www.jove.com/files/ftp_upload/61947/61947fig2v2large.jpg" target="_blank">请点击这里查看此图的更大版本。</a>
2. 延时成像
<ol><li>对于此处介绍的实验，请使用带舞台顶部孵化器系统的共聚焦显微镜系统。</li>
	<li>打开共焦显微镜、荧光灯和计算机。</li>
	<li>将放置在共焦显微镜舞台上的舞台顶孵化器的状况设置为 37 °C 和 5% CO2 大气。</li>
	<li>将样品盘放在盘子固定容器上，用盘子固定盖盖住，用顶部加热器盖合腔盖。</li>
	<li>调整变焦和对焦，使科尔蒂和MSC的器官在视界中定位。</li>
	<li>打开图像处理软件。根据 定位 选项，选择 20 倍计划-阿波克罗马特目标（数孔径 0.8） 和 0.5 倍作物面积。</li>
	<li>在收购下，单击智能设置并选择EGFP。</li>
	<li>在获取下打开通道选项卡，并将激光功率设置为0.2%，针孔设置为44 μm，主增益为750 V，数字增益设置为1.0。</li>
	<li>在成像设置下单击ESID，并将ESID 增益设置为4，数字增益设置为7.5。</li>
	<li>点击 瓷砖 和 股份 ，生产210块瓷砖。</li>
	<li>打开 对焦策略 并选择 对焦模式。</li>
	<li>根据 时间系列，将 持续时间 设置 为24小时 ， 间隔 为 10分钟。</li>
	<li>在收购下，将帧尺寸设置为512 x 512 像素，扫描速度设置为8，方向为双向，平均为 4 倍，每像素位为16。</li>
	<li>单击 启动实验 以开始实验。</li>
</ol>3. 图像文件修改
<ol><li>在处理下，单击拼接，并将最小覆盖物设置为5%，最大换档设置为10%。</li>
	<li>点击电影出口，设置未压缩，并设置速度为7.5。</li>
</ol>4. 免疫染色
<ol><li>小心吸气介质，用磷酸盐缓冲盐水 （PBS） 清洗样品两次，时间为 5 分钟。</li>
	<li>在 PBS 中用 4% 正式素修复样品 15 分钟，用 PBS 清洗样品 3 次 5 分钟。</li>
	<li>在PBS中将样品渗透到0.1%的Triton X-100中10分钟，用PBS清洗3次5分钟。</li>
	<li>加入 250 μL 的法洛丁-iFluor 647 试剂（PBS 中稀释 1:1000），并在摇床的室温下将样品孵育 1 小时。</li>
	<li>用PBS清洗样品3次5分钟。</li>
	<li>将盖子滑移到玻璃滑梯上，并添加 2 滴安装解决方案。</li>
	<li>轻轻地在幻灯片上放置盖子。</li>
	<li>用透明指甲油密封盖片，在黑暗中储存在 4 °C，直到观察到细胞。</li>
	<li>使用带适当滤光片的对焦显微镜在兴奋/发射时（Ex/Em）=650/665 nm 用于法勒林，在 Ex/Em=488/507 nm 上为 EGFP 映像幻灯片。</li>
</ol>

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作者没有什么可透露的。

对MSC移植到受损部位，促进受损细胞再生进行了广泛的研究，治疗效果明显。据报道，MSC的移植和随后的分化，以恢复大鼠的听力损失引起的3硝基丙酸13。虽然李等人将MSC应用于人类，但他们在听力14方面并没有取得任何显著改善。直到最近，通过MSC移植，还进行了近12项实验，以恢复啮齿动物模型的听力。虽然由于异质性，结果有些不清楚，但有迹象表明，MSC可以促进听力。
与化疗相关的各种生长因子和化疗因子都涉及移植干细胞向受损区域的迁移，无论异质性或同质性如何。切莫金是组织间质、免疫反应和伤口愈合的细胞迁移的关键调节器。由于体内和体外实验条件的差异，化学诱发的体外化疗可能与体外化疗不同。虽然体外实验条件有其自身的局限性，但在体内研究中很难促进细胞16的有效迁移、招募和再生。
虽然对移植用于听力恢复的MSC的跟踪在再生医学研究中很重要，但在体内实验中，特别是涉及听力的实验中，大多数情况下都是劳动密集型的，因此很难在样本量大的情况下进行。因此，结果的同质性较低，偏差严重。因此，在体外条件下首先研究这种基于移徙的事件是有效的。大多数关于细胞迁移的体外研究都是通过使用特兰斯韦尔或伤口愈合方法进行的。
建立了一种用MSC去除绞痛的造币方法，以跟踪迁移的干细胞，这些干细胞识别损伤部位并朝它们移动。大多数研究都使用一种方法，在去除后涂有β-甲基乙醇的玻璃盖上附着科尔蒂器官。虽然这里也使用了同样的方法，但很难检查组织，因为它们经常从盖片上分离或卷起。因此，进行了新的试验，以便更容易监测组织。首先，Corti 的器官被放置在塑料盖上，然后通过使用钳子按压在解剖过程中产生的 Reissner 膜的剩余部分，组织在盖片上固定。
这使得 Corti 器官稳定地附着在盖片上，以便通过共聚焦显微镜成功获取图像。虽然这里提出的组织培养方法价格低廉且省时，但解剖熟练程度的发展确实需要一些时间。已知的组织培养方法包括通过涂上多L-ornithine和层氨酸16的盖片来附着组织:应用不需要胶粘剂或涂层材料的有机细胞培养插入物17， 从而消除对粘合物质的需求，从而减少除草剂损伤:并使用由从海洋贝类中提取的蛋白质组成的组织粘合剂10。该协议既不需要涂层材料，也不需要有机膜，也不需要组织粘合剂:便宜的塑料盖子就足够了。如果考虑 8 mm x 8 mm 的面积以及 Corti 器官组织厚度的 Z 堆栈，则可以获得更好的图像。考虑到要存储的大量数据和时间推移的适当时间，获得了整个细胞运动的图像，重点是 MSC 而不是 Corti 的器官。
图4中显示的结果似乎表明，当拍摄的图像长 2 mm 且 Z 栈与时间推移相结合时，Corti 和 MSC 的器官都可以可视化为清晰的视频图像。在这里，虽然与MSC和去剂进行了共同培养实验，但此设置可以应用于不同的细胞类型或条件，供将来研究使用。例如，它将有助于应用此协议来检查保护科克菌损害的药物或外体的影响。胚胎干细胞（ESC）或诱导多能干细胞（iPSCs）可以在功能和形态上再生成机械感性毛细胞状细胞18。因此，此协议可用于研究 iPSC 或 ESC 在听觉毛细胞或支持细胞中的分化。这种前活体方法可以帮助再生医学恢复听力。

听力损失可能是先天发生的，也可以是由几个因素逐渐引起的，包括衰老、药物和噪音。听力损失往往很难治疗，因为一旦负责听力的毛细胞受损，恢复受损的功能是非常具有挑战性的。据世界卫生组织统计，全世界估计有4.61亿人听力损失，占世界人口的6.1%。在听力损失者中，93%是成年人，7%是儿童。
已尝试采取若干方法治疗听力损失:值得注意的是，使用MSC的再生方法已成为一种很有前途的治疗方法。当组织受损时，MSC自然释放到循环系统中，并迁移到损伤部位，在那里它们分泌各种分子，形成促进再生的微环境2。因此，重要的是要开发一种方法，通过外部植入的MSC迁移来治疗受损组织的目标器官及其随后的分子分泌，导致强大的免疫调节，血管生成和抗凋亡，以加强恢复受损细胞功能3，4，5。
MSC 迁移到受损组织的回程过程可能是最重要的障碍。MSC 具有系统性定位机制，具有连续的系绳/滚动、激活、逮捕、迁移/迁移和迁移6、7、8等步骤。目前，正在努力确定改进这些步骤的方法。各种策略，包括基因改造，细胞表面工程，体外启动，磁导，已经测试了6，7。此外，还多次试图通过将MSC吊到受损的科克菌部位来促进听觉毛细胞的保护和再生。然而，在体内跟踪MSC是耗时和劳动密集型的，需要高度专业化的技能9。
为了解决这个问题，开发了一种方法，通过延时共焦显微镜观察耳蜗中MSC的生长情况，该显微镜可拍摄细胞在几个小时内的迁移（图1）。它开发于20世纪初 ，最近已成为研究特定细胞迁移的有力工具。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61947/61947fig01.jpg" style="font-size: 14px;"> 图1:图形摘要。 （A） Corti 的解剖器官使用钳子粘附在塑料盖片上后，盖片被放置在 35 毫米玻璃底的圆角微碟上，并且（B） 玻璃缸被定位。（C） 在玻璃缸内填充中等物后，在气缸外小心添加带有中型的 GFP 标签的 MSC。（E） 在一夜之间孵化后，（F） 玻璃缸被移除，图像用共聚焦显微镜拍摄。缩写:GFP = 绿色荧光蛋白;MSC=中性干细胞。<a href="https://www.jove.com/files/ftp_upload/61947/61947fig01large.jpg" style="font-size: 14px;" target="_blank">请单击此处查看此图的较大版本。</a>

<table><tbody><tr><td>10X PBS Buffer</td><td>GenDEPOT</td><td>P2100-104</td><td></td></tr><tr><td>4% Formalin</td><td>T&amp;amp;I</td><td>BPP-9004</td><td></td></tr><tr><td>Ampicillin</td><td>sigma&amp;nbsp;</td><td>A5354-10ml</td><td></td></tr><tr><td>BSA</td><td>sigma&amp;nbsp;</td><td>A4503-100G</td><td></td></tr><tr><td>confocal dish</td><td>SPL</td><td>200350</td><td></td></tr><tr><td>confocal microscope&amp;nbsp;</td><td>ZEISS</td><td>LSM800</td><td></td></tr><tr><td>coverslip</td><td>SPL</td><td>20009</td><td></td></tr><tr><td>DMEM/F12</td><td>Gibco</td><td>10565-018</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Thermo Fisher scientific</td><td>16140071</td><td></td></tr><tr><td>Fluorsheild with DAPI</td><td>sigma&amp;nbsp;</td><td>F6057</td><td></td></tr><tr><td>Forcep</td><td>Dumont</td><td>0508-L5-P0</td><td></td></tr><tr><td>HBSS</td><td>Thermo Fisher scientific</td><td>14065056</td><td></td></tr><tr><td>HEPES</td><td>Thermo Fisher scientific</td><td>15630080</td><td></td></tr><tr><td>N2 supplement</td><td>Gibco</td><td>17502-048</td><td></td></tr><tr><td>Phalloidin-iFluor 647 Reagent</td><td>abcam</td><td>ab176759</td><td></td></tr><tr><td>Stage Top Incubator</td><td>TOKAI HIT</td><td>WELSX</td><td></td></tr><tr><td>Strain C57BL/6 mouse messenchymal stem cells with GFP</td><td>cyagen</td><td>MUBMX-01101</td><td></td></tr><tr><td>Triton X-100</td><td>sigma&amp;nbsp;</td><td>T8787</td><td></td></tr></tbody></table>

in vitro time-lapse live-cell imaging to explore cell migration toward the organ of corti

为了研究间质干细胞（MSCs）对细胞再生和治疗的影响，该方法跟踪MSC迁移和与声蜗上皮共同培养后的形态变化。Corti 的器官通过按压解剖过程中产生的里斯纳膜的一部分，在塑料盖上固定。当气缸被移除时，被玻璃缸限制的 MSC 迁移到高音表皮。它们的主要定位是在科尔蒂器官的修饰中观察到的，其方向与神经纤维的方向相似。然而，一些MSC在limbus区域被本地化，并显示出水平拉长的形状。此外，进入毛细胞区的迁移增加，在卡那霉素治疗后，MSC的形态转变为各种形式。最后，这项研究的结果表明，MSC与人工增生上皮的共生将有助于通过细胞移植和细胞再生研究，可以检查各种条件和因素的治疗发展。

在三维模式下，MSC的体外迁移已通过Transwell系统或传统的伤口愈合方法进行评估，以观察二维（2D）模式11中的迁移。科尔蒂的器官是一个复杂的结构，由各种细胞组成，如博彻细胞，克劳迪斯细胞，神细胞，支柱细胞，汉森的细胞，外毛细胞，内毛细胞，神经纤维，罗勒膜，和网状拉米纳12。当MSC被移植到由这种复杂细胞组成的组织中时，使用技术确定细胞的招募和稳定位置对于了解使用MSC进行细胞治疗和再生的机制至关重要。此外，为了确定 MSC 在存在或不存在损伤的情况下如何本地化，2D 方法（如伤口愈合方法）比 Transwell 系统更适合细胞在一个方向上随机向下移动。
在这项研究中，MSC 的二维路径通过简单地使用玻璃缸作为 MSC 和 Corti 器官之间的屏障，并在 MSC 连接后移除气缸，以伤口愈合方法进行跟踪。当科尔蒂的器官被培养时，成纤维细胞从最外层的外毛细胞向外生长非常快（图3，视频1）。因此，大多数GFP标签的MSC似乎被快速增长的成纤维细胞（视频1）推出。尽管如此，一些GFP标记的MSC穿透成纤维细胞层，并成功地迁移到Corti的器官（图3，视频1）。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61947/61947fig03.jpg"> 图3。科尔蒂和 GFP 标记的 MSC 器官的对焦图像。 在取出玻璃缸和额外的4小时的孵化后，进行了固定和染色。比例杆 +100 μm。缩写:GFP = 绿色荧光蛋白;MSC:中性干细胞。<a href="https://www.jove.com/files/ftp_upload/61947/61947fig03large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
视频 1: 科尔蒂和 GFP 标记的 MSC 器官的延时共焦显微镜。在取出玻璃缸和额外的4小时的孵化后，进行了固定和染色。比例杆 +100 μm。缩写:GFP = 绿色荧光蛋白;MSC:中性干细胞。<a href="https://www.jove.com/files/ftp_upload/61947/Video%20JoVE.mp4" target="_blank">请点击这里下载此视频。</a>
72小时孵化的结果表明，MSC迁移到科尔蒂的器官，主要在流体和从流变中分离的毛利叶神经纤维聚集区域局部:MSC的形态被改变为类似于神经纤维的径向形状（图4B）。有趣的是，细胞沿着limbus线被转换成线性形状，而不是毛细胞区域（图4E，见箭头）。虽然Corti器官的肛门和基底两端结合在一起，以防止MSC移动到巴西拉膜的中间侧，但MSC通过穿透各种细胞层和物理屏障（图4E）能够在神经纤维收集区迁移和生长。当用1mM卡那霉素治疗16小时，并培养细胞72小时后，去除卡那霉素，MSC不仅在修饰符，而且在外毛细胞（图4C，D）中局部化。
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61947/61947fig4v2.jpg"> 图4。科尔蒂和 GFP 标记的 MSC 器官的对焦图像。（A） 在取出玻璃缸和另外16小时的孵化后，在固定和染色后拍摄图像。比例杆 +200μm. （B） MSC 主要分布在莫多卢斯地区，比例杆 +20μm。从盘子中取出玻璃缸后，细胞被进一步孵育1.0 mM卡那霉素16小时，培养无卡那霉素介质72小时，然后固定和染色。（C ）外毛细胞区域的图像，比例杆= 20 μm;（D） 莫多卢斯地区， 规模酒吧 = 50μm;和 （E） 整个科克伦， 规模酒吧 = 100 μm.<a href="https://www.jove.com/files/ftp_upload/61947/61947fig4v2large.jpg" target="_blank">请点击这里查看此图的更大版本。</a>
这些结果表明，本研究所展示的实验系统将是利用MSC开发细胞治疗策略的有用测试工具，可专门用于研究各种因素造成的听力损失。

Watch this Scientific Journal Video about In vitro Time-lapse Live-Cell Imaging to Explore Cell Migration toward the Organ of Corti at JoVE.com

In vitro Time-lapse Live-Cell Imaging to Explore Cell Migration toward the Organ of Corti

在这项研究中，我们提出了一种实时成像方法，使用共聚焦显微镜观察细胞通过与含有Corti器官的耳蜗上皮的外活体孵化向受损组织移动。

体外延时活细胞成像探索细胞向科尔蒂器官迁移

To study the effects of mesenchymal stem cells (MSCs) on cell regeneration and treatment, this method tracks MSC migration and morphological changes after ...

in-vitro-time-lapse-live-cell-imaging-to-explore-cell-migration

Nanyang Technological University

Research

JoVE Journal

Medicine

3.7K 次观看.  Yonsei University Wonju College of Medicine. 在这项研究中，我们提出了一种实时成像方法，使用共聚焦显微镜观察细胞通过与含有Corti器官的耳蜗上皮的外活体孵化向受损组织移动。

视频: In vitro Time-lapse Live-Cell Imaging to Explore Cell Migration toward the Organ of Corti

Live-cell Imaging of Migrating Cells Expressing Fluorescently-tagged Proteins in a Three-dimensional Matrix

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound healing, tissue regeneration, and cancer metastasis, cells must navigate through complex, three-dimensional extracellular tissue. To better understand the mechanisms behind these biological processes, it is important to examine the roles of the proteins responsible for driving cell migration. Here, we outline a protocol to study the mechanisms of cell migration using the epithelial cell line (MDCK), and a three-dimensional, fibrous, self-polymerizing matrix as a model system. This optically clear extracellular matrix is easily amenable to live-cell imaging studies and better mimics the physiological, soft tissue environment. This report demonstrates a technique for directly visualizing protein localization and dynamics, and deformation of the surrounding three-dimensional matrix. 
Examination of protein localization and dynamics during cellular processes provides key insight into protein functions. Genetically encoded fluorescent tags provide a unique method for observing protein localization and dynamics. Using this technique, we can analyze the subcellular accumulation of key, force-generating cytoskeletal components in real-time as the cell maneuvers through the matrix. In addition, using multiple fluorescent tags with different wavelengths, we can examine the localization of multiple proteins simultaneously, thus allowing us to test, for example, whether different proteins have similar or divergent roles. Furthermore, the dynamics of fluorescently tagged proteins can be quantified using Fluorescent Recovery After Photobleaching (FRAP) analysis. This measurement assays the protein mobility and how stably bound the proteins are to the cytoskeletal network.
By combining live-cell imaging with the treatment of protein function inhibitors, we can examine in real-time the changes in the distribution of proteins and morphology of migrating cells. Furthermore, we also combine live-cell imaging with the use of fluorescent tracer particles embedded within the matrix to visualize the matrix deformation during cell migration. Thus, we can visualize how a migrating cell distributes force-generating proteins, and where the traction forces are exerted to the surrounding matrix. Through these techniques, we can gain valuable insight into the roles of specific proteins and their contributions to the mechanisms of cell migration.

Cellular processes such as cell migration have traditionally been studied on two-dimensional, stiff plastic surfaces. This report describes a technique for directly visualizing protein localization and analyzing protein dynamics in cells migrating in a more physiologically relevant, three-dimensional matrix.

活细胞成像的移植细胞在一个三维矩阵表示荧光标记的蛋白质

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound ...

科研

生物工程

An Organotypic Slice Assay for High-Resolution Time-Lapse Imaging of Neuronal Migration in the Postnatal Brain

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as well as differentiation and integration of new neurons into existing neural circuits. For postnatal neurogenesis in the olfactory bulbs, these events are segregated within three anatomically distinct domains: proliferation largely occurs in the subependymal zone (SEZ) of the lateral ventricles, migrating neuroblasts traverse through the rostral migratory stream (RMS), and new neurons differentiate and integrate within the olfactory bulbs (OB). The three domains serve as ideal platforms to study the cellular, molecular, and physiological mechanisms that regulate each of the biological events distinctly. This paper describes an organotypic slice assay optimized for postnatal brain tissue, in which the extracellular conditions closely mimic the in vivo environment for migrating neuroblasts. We show that our assay provides for uniform, oriented, and speedy movement of neuroblasts within the RMS. This assay will be highly suitable for the study of cell autonomous and non-autonomous regulation of neuronal migration by utilizing cross-transplantation approaches from mice on different genetic backgrounds.

This protocol describes an organotypic slice assay optimized for the postnatal brain and high-resolution time-lapse imaging of neuroblast migration in the rostral migratory stream.

对于高分辨率延时成像神经细胞迁移的一个器官型产后脑切片检测

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as ...

神经科学

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration have been thoroughly analyzed in vitro. However, in vivo cell migration somehow differs from in vitro migration, and has proven more difficult to analyze, being less accessible to direct observation and manipulation. This protocol uses the migration of the prospective prechordal plate in the early zebrafish embryo as a model system to study the function of candidate genes in cell migration. Prechordal plate progenitors form a group of cells which, during gastrulation, undergoes a directed migration from the embryonic organizer to the animal pole of the embryo. The proposed protocol uses cell transplantation to create mosaic embryos. This offers the combined advantages of labeling isolated cells, which is key to good imaging, and of limiting gain/loss of function effects to the observed cells, hence ensuring cell-autonomous effects. We describe here how we assessed the function of the TORC2 component Sin1 in cell migration, but the protocol can be used to analyze the function of any candidate gene in controlling cell migration in vivo.

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Combining cell transplantation, cytoskeletal labeling and loss/gain of function approaches, this protocol describes how the migrating zebrafish prospective prechordal plate can be used to analyze the function of a candidate gene in in vivo cell migration.

分析<em&gt;在体内</em&gt;细胞迁移使用细胞移植和时间推移成像在斑马鱼胚胎

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration ...

发育生物学

Time-lapse Imaging of Neuroblast Migration in Acute Slices of the Adult Mouse Forebrain

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells in restricted areas of the adult mammalian brain. Newborn neuroblasts from the subventricular zone (SVZ) migrate along the rostral migratory stream (RMS) to their final destination in the olfactory bulb (OB)1. In the RMS, neuroblasts migrate tangentially in chains ensheathed by astrocytic processes2,3 using blood vessels as a structural support and a source of molecular factors required for migration4,5. In the OB, neuroblasts detach from the chains and migrate radially into the different bulbar layers where they differentiate into interneurons and integrate into the existing network1, 6.
In this manuscript we describe the procedure for monitoring cell migration in acute slices of the rodent brain. The use of acute slices allows the assessment of cell migration in the microenvironment that closely resembling to in vivo conditions and in brain regions that are difficult to access for in vivo imaging. In addition, it avoids long culturing condition as in the case of organotypic and cell cultures that may eventually alter the migration properties of the cells. Neuronal precursors in acute slices can be visualized using DIC optics or fluorescent proteins. Viral labeling of neuronal precursors in the SVZ, grafting neuroblasts from reporter mice into the SVZ of wild-type mice, and using transgenic mice that express fluorescent protein in neuroblasts are all suitable methods for visualizing neuroblasts and following their migration. The later method, however, does not allow individual cells to be tracked for long periods of time because of the high density of labeled cells. We used a wide-field fluorescent upright microscope equipped with a CCD camera to achieve a relatively rapid acquisition interval (one image every 15 or 30 sec) to reliably identify the stationary and migratory phases. A precise identification of the duration of the stationary and migratory phases is crucial for the unambiguous interpretation of results. We also performed multiple z-step acquisitions to monitor neuroblasts migration in 3D. Wide-field fluorescent imaging has been used extensively to visualize neuronal migration7-10. Here, we describe detailed protocol for labeling neuroblasts, performing real-time video-imaging of neuroblast migration in acute slices of the adult mouse forebrain, and analyzing cell migration. While the described protocol exemplified the migration of neuroblasts in the adult RMS, it can also be used to follow cell migration in embryonic and early postnatal brains.

We describe a protocol for real-time videoimaging of neuronal migration in the mouse forebrain. The migration of virally-labeled or grafted neuronal precursors was recorded in acute live slices using wide-field fluorescent imaging with a relatively rapid acquisition interval to study the different phases of cell migration, including the durations of the stationary and migration phases and the speed of migration.

急性片的成年小鼠前脑的神经细胞迁移的延时成像

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells ...

Visualization of Tangential Cell Migration in the Developing Chick Optic Tectum

Time-lapse imaging is a powerful method to analyze migrating cell behavior. After fluorescent cell labeling, the movement of the labeled cells in culture can be recorded under video microscopy. For analyzing cell migration in the developing brain, slice culture is commonly used to observe cell migration parallel to the slice section, such as radial cell migration. However, limited information can be obtained from the slice culture method to analyze cell migration perpendicular to the slice section, such as tangential cell migration. Here, we present the protocols for time-lapse imaging to visualize tangential cell migration in the developing chick optic tectum. A combination of cell labeling by electroporation in ovo and a subsequent flat-mount culture on the cell culture insert enables detection of migrating cell movement in the horizontal plane. Moreover, our method facilitates detection of both individual cell behavior and the collective action of a group of cells in the long term. This method can potentially be applied to detect the sequential change of the fluorescent-labeled micro-structure, including the axonal elongation in the neural tissue or cell displacement in the non-neural tissue.

We describe the methods for fluorescent labeling of tangentially migrating cells by electroporation, and for time-lapse imaging of the labeled cell movement in a flat-mount culture in order to visualize migrating cell behavior in the developing chick optic tectum.

发展中雏鸡光学顶盖中切向细胞迁移的可视化

Time-lapse imaging is a powerful method to analyze migrating cell behavior. After fluorescent cell labeling, the movement of the labeled cells in culture ...

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to neuroblasts able to move along the rostral migratory stream (RMS) towards the olfactory bulb (OB). This long-distance migration is required for the subsequent maturation of newborn neurons in the OB, but the molecular mechanisms regulating this process are still unclear. Investigating the signaling pathways controlling neuroblast motility may not only help understand a fundamental step in neurogenesis, but also have therapeutic regenerative potential, given the ability of these neuroblasts to target brain sites affected by injury, stroke, or degeneration.
In this manuscript we describe a detailed protocol for in vivo postnatal electroporation and subsequent time-lapse imaging of neuroblast migration in the mouse RMS. Postnatal electroporation can efficiently transfect SVZ progenitor cells, which in turn generate neuroblasts migrating along the RMS. Using confocal spinning disk time-lapse microscopy on acute brain slice cultures, neuroblast migration can be monitored in an environment closely resembling the in vivo condition. Moreover, neuroblast motility can be tracked and quantitatively analyzed. As an example, we describe how to use in vivo postnatal electroporation of a GFP-expressing plasmid to label and visualize neuroblasts migrating along the RMS. Electroporation of shRNA or CRE recombinase-expressing plasmids in conditional knockout mice employing the LoxP system can also be used to target genes of interest. Pharmacological manipulation of acute brain slice cultures can be performed to investigate the role of different signaling molecules in neuroblast migration. By coupling in vivo electroporation with time-lapse imaging, we hope to understand the molecular mechanisms controlling neuroblast motility and contribute to the development of novel approaches to promote brain repair.

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

Neuroblast migration is a fundamental event in postnatal neurogenesis. We describe a protocol for efficient labeling of neuroblasts by in vivo postnatal electroporation and subsequent visualization of their migration using time-lapse imaging of acute brain slices. We include a description for the quantitative analysis of neuroblast dynamics by video tracking.

成神经细胞迁移的小鼠急性脑片在体内电穿孔产后和时间推移成像

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to ...

Long-term Time Lapse Imaging of Mouse Cochlear Explants

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building the highly ordered, complex structure of the mammalian cochlea proceeds for around ten days. In order to study changes in gene expression over this period and their response to pharmaceutical or genetic manipulation, long-term imaging is necessary. Previously, live imaging has typically been limited by the viability of explanted tissue in a humidified chamber atop a standard microscope. Difficulty in maintaining optimal conditions for culture growth with regard to humidity and temperature has placed limits on the length of imaging experiments. A microscope integrated into a modified tissue culture incubator provides an excellent environment for long term-live imaging. In this method we demonstrate how to establish embryonic mouse cochlear explants and how to use an incubator microscope to conduct time lapse imaging using both bright field and fluorescent microscopy to examine the behavior of a typical embryonic day (E) 13 cochlear explant and Sox2, a marker of the prosensory cells of the cochlea, over 5 days.

Live imaging of the embryonic mammalian cochlea is challenging because the developmental processes at hand operate on a temporal gradient over ten days. Here we present a method for culturing and then imaging embryonic cochlear explant tissue taken from a fluorescent reporter mouse over five days.

老鼠耳蜗外植体的长期时间推移成像

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building ...

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then radial migration in the molecular layer and the Purkinje cell layer to reach the internal granular layer of the cerebellar cortex. Default in migratory processes induces either cell death or misplacement of the neurons, leading to deficits in diverse cerebellar functions. Centripetal granule cell migration involves several mechanisms, such as chemotaxis and extracellular matrix degradation, to guide the cells towards their final position, but the factors that regulate cell migration in each cortical layer are only partially known. In our method, acute cerebellar slices are prepared from P10 rats, granule cells are labeled with a fluorescent cytoplasmic marker and tissues are cultured on membrane inserts from 4 to 10 hr before starting real-time monitoring of cell migration by confocal macroscopy at 37 &#176;C in the presence of CO2. During their migration in the different cortical layers of the cerebellum, granule cells can be exposed to neuropeptide agonists or antagonists, protease inhibitors, blockers of intracellular effectors or even toxic substances such as alcohol or methylmercury to investigate their possible role in the regulation of neuronal migration.

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

During postnatal cerebellum development, immature granule cells originating from the germinal zone exhibit distinct modalities of migration to reach their final destination and to establish neuronal networks. This protocol describes the preparation of cerebellar slices and the confocal macroscopic approach used to investigate the factors that regulate neuronal migration.

产后的小脑颗粒细胞迁移利用共聚焦肉眼离体成像

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then ...

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It has helped to describe the different steps of radial migration and characterize the molecular mechanisms controlling this process. To directly and dynamically analyze migrating neurons they have to be traced over time. This protocol describes a workflow that combines in utero electroporation with organotypic slice culture and time-lapse confocal imaging, which allows for a direct examination and dynamic analysis of radially migrating cortical neurons. Furthermore, detailed characterization of migrating neurons, such as migration speed, speed profiles, as well as radial orientation changes, is possible. The method can easily be adapted to perform functional analyses of genes of interest in radially migrating cortical neurons by loss and gain of function as well as rescue experiments. Time-lapse imaging of migrating neurons is a state-of-the-art technique that once established is a potent tool to study the development of the cerebral cortex in mouse models of neuronal migration disorders.

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

This protocol provides instructions for direct observation of radially migrating cortical neurons. In utero electroporation, organotypic slice culture, and time-lapse confocal imaging are combined to directly and dynamically study the effects of overexpression or downregulation of genes of interest in migrating neurons and to analyze their differentiation during development.

延期共聚焦显像移植神经元在器官性切片培养胚胎小鼠脑使用<em&gt;在Utero</em&gt;电穿孔

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It ...

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate tangentially from their place of origin in the ventral telencephalon (including from the medial and caudal ganglionic eminences (MGE, CGE)) to the dorsal cortical plate in response to a variety of intrinsic and extrinsic cues. Different methodologies have been developed over the years to genetically manipulate specific pathways and investigate how they regulate the dynamic cytoskeletal changes required for proper IN migration. In utero electroporation has been extensively used to study the effect of gene repression or overexpression in specific IN subtypes while assessing the impact on morphology and final position. However, while this approach is readily used to modify radially migrating pyramidal cells, it is more technically challenging when targeting INs. In utero electroporation generates a low yield given the decreased survival rates of pups when electroporation is conducted before e14.5, as is customary when studying MGE-derived INs. In an alternative approach, MGE explants provide easy access to the MGE and facilitate the imaging of genetically modified INs. However, in these explants, INs migrate into an artificial matrix, devoid of endogenous guidance cues and thalamic inputs. This prompted us to optimize a method where INs can migrate in a more naturalistic environment, while circumventing the technical challenges of in utero approaches. In this paper, we describe the combination of ex utero electroporation of embryonic mouse brains followed by organotypic slice cultures to readily track, image and reconstruct genetically modified INs migrating along their natural paths in response to endogenous cues. This approach allows for both the quantification of the dynamic aspects of IN migration with time-lapse confocal imaging, as well as the detailed analysis of various morphological parameters using neuronal reconstructions on fixed immunolabeled tissue.

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

Here, we provide a low-cost and reliable method to generate electroporated brain organotypic slice cultures from mouse embryos suitable for confocal microscopy and live-imaging techniques.

前子宫内胚胎小鼠脑电穿孔和脊髓切片培养迁移 GABAergic 中间神经元的活体成像

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate ...