Name: investigating functional regeneration in organotypic spinal cord co-cultures grown on multi-electrode arrays
Uploaded: 2015-09-23T14:00:00.000+00:00
Duration: 8 min 25 s
Description: Watch this Scientific Journal Video about Investigating Functional Regeneration in Organotypic Spinal Cord Co-cultures Grown on Multi-electrode Arrays at JoVE.com

This study was supported by the Swiss National Foundation (n&#176; 3100AO-120327 and 31003A_140754/1)

动物保健是符合经瑞士地方当局（金额献给Landwirtschaft UND NATUR德冠德伯尔尼，Veterinärdienst，Sekretariat Tierversuche，批准NRS。一十一分之五十二和35/14）的指导方针。这些准则与欧盟指令六十三分之二千零十/欧盟的协议。 注意:在层流罩无菌仪器和解决方案适用于所有步骤1 - 5，包括子步骤。 1.准备多边环境协定注意:多边环境是由玻璃基板，微制造的金属电极和SU-8聚合物绝缘层的（也参见Tscherter 等 3）。本研究的目的，市售的多边环境协定进行排序，定制的电极阵列布局（ 图1 A和​​B）。的68个电极被布置在矩形网格被分成两个区域由一个300微米宽的槽自由电极，ELECTRiCal的导线和绝缘。每个电极是40×40微米的尺寸并且它们由200微米从中心到中心间隔开。四个大型接地电极定位在录制现场周围。他们从它们的大小（21毫米×21毫米）其它商用标准的多边环境协定不同，他们没有固定的录音室。 <ol><li>为在100％乙醇（2×），70％的乙醇（1x）和蒸馏水（2×）对每个至少30秒消毒冲洗多边环境。让我们干。把10 - 12多边环境协议在干净的玻璃培养皿（150毫米×25毫米），并盖上盖子。 </li><li>高压釜多边环境20分钟，在120℃。存储在RT。 </li><li>制备涂布液（见材料清单）的多边环境和存储等分在-20℃。 </li><li>把每个MEA在一个单独的无菌皮氏培养皿（35毫米×10毫米）。 </li><li>使用冷却的吸移管将在电极的顶部150微升冷却的涂布溶液，并关闭培养皿的盖子。经过约10分钟，检查是否有气泡之上电极，轻轻地删除它们用橡胶覆盖的锅铲，如果有必要的。让我们休息1小时，在室温。 </li><li>吸涂液残留，洗1个中等产前和胚胎神经细胞和2x用蒸馏水，无菌水进行了优化。如果多边环境不使用直到第二天，使他们在蒸馏水，无菌水，在37℃培养箱中O / N。否则，我们直接干在室温。 </li></ol> 2.配料所需的器官培养的制备及成长<ol><li>准备无钙洗涤液（见材料清单）。 </li><li>重建在洗涤溶液鸡等离子体（1:1，轻轻摇动，避免形成气泡），离心机以3,000×g离心约20分钟，倒出清楚内容到无菌管中。等份加入200μl到冷冻管并储存在-20℃。 </li><li>重新构造从牛血浆相应（200U / ml）和无菌过滤器（0.2微米孔径过滤器）凝血酶。分装200微升到crytotubes并储存在-20℃。 </li><li>对于30培养制备100ml营养培养基（见材料清单）。 </li></ol> 3.脊髓组织解剖注意:过程的产量，根据胚胎，脊髓切片的大约25的数目 - 35共培养物，并在无菌条件下在层流罩内制备。 <ol><li>通过肌内注射施加致死剂量戊巴比妥（0.4毫升），以母兽。通过检查踏板撤回反射确认深麻醉。剖宫产和牺牲斩首交付E14大鼠胚胎。 </li><li>胚的体转移到培养皿充满无菌，冷冻，洗涤溶液。 </li><li>与上面的后肢解剖刀和另一个上面的前肢执行一个完整的横向切口和从体内取出的肢体。接下来，在额面切到内脏含有脊柱合作后片分开RD。 </li><li>对切​​片，转移含有脊髓1背面片在一个时间的安装盘上，并切成厚度为225 - 250微米用纸巾斩波器。放一滴洗涤溶液对切碎组织和35毫米×10毫米充满无菌，冷冻，洗涤溶液培养皿转移切片用刮刀。 </li><li>对于每片单独，解剖脊髓远离剩余组织。离开附加背根神经节。 </li><li>让片休息1小时，4℃。 </li></ol> 4.安装在多边环境协定的脊髓组织切片<ol><li>热身无菌营养培养基中的孵化器（37℃，轻轻拧开盖子氧合）。 </li><li>位置的MEA用无菌镊子在立体显微镜在焦距上的电极阵列下培养皿橡胶被覆提示在RT。居中鸡血浆的6微升液滴清洁，无尘，无菌和电极阵列上。使用小铲，小心地将2脊髓切片与腹侧彼此面对的两侧到等离子体液滴。请勿触摸电极用锅铲。 </li><li>添加8微升凝血酶周围的鸡血浆滴。使用凝血酶的枪头，精心搭配和周围的两片蔓延鸡等离子/凝血酶混合物。再次，不要直接触摸脆性电极阵列。就在凝固之​​前，吸出多余的鸡血浆/凝血酶。 </li><li>盖上培养皿保持高湿度，而MEA /文化装配在37℃坐约1小时，在孵化器内湿盒。 </li><li>小心加入10微升营养培养基来培养室，盖上培养皿并放回培养箱中约30分钟。 </li><li>将每个MEA /文化组装无菌，橡胶覆盖的提示成卷管，加3毫升营养培养基，并紧紧盖上盖子。放置在辊筒的辊筒在1旋转 - 2的转速，在5％CO的培养箱<suB&gt; 2含气氛下在37℃。 </li><li>每周2次- 7天后在体外 （DIV）和事后1改变半营养培养基中的。 </li></ol> 5.机械病变<ol><li>采取在MEA /培养装配用无菌，橡胶被覆提示出卷管并置于培养皿中没有立体显微镜在焦距上的组织下营养培养基。目视确认两片融合。 </li><li>按住MEA /文化通过将镊子在MEA橡胶覆盖的提示组装稳定。 </li><li>放置手术刀刀片在MEA靠近组织切片的槽。握住手术刀相当水平。 </li><li>抬起解剖刀手柄向上而是让手术刀刀片留在MEA的凹槽以这样的方式，从基地刀片"辊"到尖端，并由此穿过组织覆盖的槽切割。 </li><li>与严重将25g针头，如果necess任何残留组织连接元。仅在凹槽内的区域工作，不要哪壶边。 </li><li>把MEA /文化组件装回辊筒。提供3毫升新鲜培养基向培养并把它们放回辊筒在孵化器。 </li></ol>自发活动6.电录音<ol><li>为了研究DIV的选定号码后两脊髓切片中再生功能，挂载MEA /文化装配在显微镜一个录音室，并应用约500微升细胞外液（见材料清单）。 </li><li>等待10分钟的第一记录之前，使系统稳定。 </li><li>记录基本的自发活动2X从MEA在室温下的各项活动，检测电极约10分钟。 </li><li>为了确保稳定的细胞外的条件下，每个记录会议结束后交换细胞外液。 </li><li>到disinhibit网络，应用细胞外溶液共ntaining士的宁（1μM）和gabazine（10μM）。等待至少2分钟开始记录之前。 </li></ol> 7.数据分析注意:对于外记录动作电位的检测用于每个电极基于标准差和随后的鉴别器的检测器。此过程中详细Tscherter 等人 3所述。 <ol><li>根据标准程序3（ 图1F）显示在一个光栅情节每个电极检测到的神经元活动。 </li><li>确定并通过总结在10毫秒的滑动窗口中的，根据信道的事件，由1毫秒步骤移位的数量显示为每个切片的总的网络活动（图1G和看到Tscherter 等3）。 </li><li>在检测每片单独的突发（活动的集群上出现的数个电极）。故，设置相应的总网络活动最小活动高峰阈值，并确定每个突发开始和突发结束。例如，一个适当的阈值是平均突发幅度的25％和脉冲串的开始可以由是在至少5毫秒，一个脉冲串结束的一个时间窗口中的第一事件被定义由是在在一个时间窗口中的最后的事件至少25毫秒（参见海德曼等人 10）。 </li><li>量化官能再生计算这两个片之间的同步脉冲串的百分比。要做到这一点，计算一个切片一个突发和在其他切片以下突发之间的延迟。 注意:一对突发被称为"同步"时的延迟比一般的突发长度要小。特别是在共培养了大量的活动，也存在双方不约而同地开始在选定的等待时间窗口一阵。这一事实，必须考虑到的SY的百分比量化nchronized阵阵。用于计算的详细描述见海德曼等人 10。 </li></ol> 8.固定的文化和免疫组化<ol><li>对于包括洗涤或孵化的所有步骤，确保把文化上的倾斜表，以确保通过组织液的正常扩散。 </li><li>直接在记录会话结束后采取MEA /文化组装出了设置的，把它放在一个皮氏培养皿（35毫米×10毫米），并增加大约细胞外液2毫升</li><li>要在体外的时候已经形成了周围的细胞层分隔片，取 10微升的吸头和圆切片。注意不要损坏文化。它可以省略此步骤，但后来的嵌入是容易当它被执行。 </li><li>使用薄非金属注射器针头（见材料清单）结合1ml注射器填充有细胞外soluti上轻轻一击的文化断MEA。 </li><li>除去巴斯德吸管的尖端和配合上slivered端的橡胶球。使用后端到培养（用磷酸盐缓冲盐水（PBS，0.1M）4％多聚甲醛）转移到固定剂。不要使用巴斯德吸管的小费标准为转移，因为它太小了，文化也可以折叠，可能损坏。修正了1 - 2小时，在4℃。用蒸馏水冲洗的MEA和消除组织残留，用指尖但不要用指甲划伤。商店多边环境在蒸馏水中，在4℃。 </li><li>在PBS中的每个的至少10分钟洗3次培养。存储在PBS在4℃或直接启动与染色步骤。 </li><li>孵育培养物在封闭溶液（5％山羊血清，1％牛血清白蛋白，在PBS中0.3％的洗涤剂）至少90分钟。 </li><li>添加稀释于封闭溶液中的第一抗体孵育3晚于4℃。 </li><li>洗3次在PBS在升东各30分钟。 </li><li>添加第二抗体在PBS中稀释为2小时，在室温。 </li><li>洗3次在PBS中的每个的至少30分钟。 </li><li>转移用巴斯德吸管上的对象滑动移除尖端片。去除多余的PBS，并嵌入带安装介质。 </li></ol>

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The authors have nothing to disclose.

所呈现的过程中的一些内容是根本，准确的和可重复的实验成果。在无菌条件下在层流罩中进行制备的多边环境，溶液和生产的切片培养的过程中的所有步骤。抗生素不施加到营养培养基中，因为他们可影响自发活动14。期间在MEA涂层中，最重要的部分是，以确保外基质凝胶溶液在所有时间保持冷。解冻在冰箱并使其保持在冰上的整个过程，以确保一个最高温度接近0℃。在切片的快速分离和切片的制备，以及注意于低温使用冰冷夹层缓冲器增加健康培养物的百分比。此外，等离子/凝血酶凝结是最重要的步骤之一。首先，请确保等离子是正确混用ð有凝血酶。其次，要特别注意去除尽可能多的残留物尽量保证切片的多边环境协定的正确连接。这一步是非常时间敏感;如果时间帧太短，太多的等离子体/凝血酶混合物被除去。如果时间帧太长，凝固已经发生，增大沿与残留血浆/凝血酶除去切片的风险。 如果机械病变计划，为使用多边环境与一个精确的设计是很重要的。的病变预定的面积需要是自由的电极，导线和绝缘。否则，该MEA的电气将要损坏，因此，没有录音可以再执行。 另一个关键的一步涉及到病变的大小。它先前已表明，损伤后重新连接两个年幼的片需要两天左右10。对于所有的实验中，经验法则所使用的空间betwe恩损伤后切片应不大于MEA槽（300微米）的宽度。一个更大的病变的大小可能会导致重新连接一个较长的时间框架，或者，如果病变太大，不重新连接的。注释，前实验揭示的初始放置的两片远于1毫米导致非常低的连接速率。 录音代替培养箱37℃，至少10分钟至RT的调整时间，开始前，向细胞外溶液中，而不是营养介质的建议。在这些初始分钟密切观察活动模式的确保所测量的数据所代表的文化的记录开始后适当的破裂图案。 对于活动，数据采集和深加工的检测，自制的硬件（录音室，连接到放大器和放大器），在实验室方案VIEW，C ++和IgorPro使用。商业MEA设置和其他软件包适合以及进行这些操作。这里，看到的电极的信号被放大1001倍，然后进行数字化以6KHZ的速率。 该模型可以是一个有用的工具来检查propriospinal连接，因为在体内 ，它们很难不升降纤维束的干扰学习。在共同培养两脊髓切片可以优雅地绕过这个问题。将培养提供可以严格控制，并容易操纵的微环境。另一方面，脊髓上和感觉输入是当然的缺失。此外，将培养制备过程表示的CNS损伤的情况。胶质细胞样星形细胞和小胶质细胞是已知的病灶具有增加的增殖，因此响应，组织是在一定程度上进行了重组。关于所呈现的模式没有观察到升执行机械损伤后一胶质瘢痕的形成。一种解释可能是，星形胶质细胞的适应机制在制备过程中已经触发了培养物的病变不导致进一步的响应。此外，也没有证据表明髓鞘相关抑制剂的参与， 例如 ，的Nogo-A，在文化损毁在一个旧时代的低再生潜力。然而，已显示与磷酸二酯酶4抑制剂咯利普兰，切片10之间执行病变在23格增加官能再生后直接开始治疗。这些结果表明，功能恢复可以在旧培养物增加。值得注意的是，计算轴突穿越病变部位的数量时，文化损毁在年轻的时候和文化的损毁，在老年间无差异被发现。这些结果表明，缺乏轴突再生的不是主要原因˚F或回收的晚期损伤培养后的损失，因此强调需要一个模型，它允许再生的功能分析。突触的形成和突触可塑性可以发挥在恢复过程中10了重要作用。这些机制得到了很多的关注最近几年，这是时下普遍认为他们有脊髓损伤后对结果产生重大影响。因此，一个模型，提供稳定的条件下，以调查这些进程隔离和非常详细当然可以帮助，以获取有关脊髓功能恢复的洞察力。

中枢神经系统（CNS）的器官切片文化已被广泛用于研究各种生理和病理现象达到从神经发育神经退行性病变。相比解离的细胞培养物，器官切片提供的更多的复杂性的优点具有完整细胞结构和本地突触电路。同时，可将组织在一个孤立的方式，而不需要牵连整体的复杂体内上下文进行分析。此外，容易和反复获取所选择的细胞是必要的，细胞外环境，可以精确地控制，通常， 在体外模型比其在体内的对应成本更低。近年来，各种各样的研究提出了长期培养物随相应活体动物1,2的发育轮廓之间惊人的相似。它已被示出的各种中枢神经系统区域选择性的那个神经元回路在开发过程中NS表达自发的网络活动和器官切片部分保持这一现象3-7。分离的脊髓制剂已被特别用于研究节奏产生6和神经元回路1的形成。 一种方法来记录器官切片的自发活动是培养他们多电极阵列（多边环境协定）的顶部。这些设备包含多个电极，可以监控通过动作电位从众多的细胞同时（多单位记录）所产生的外势。在MEA记录的高时间分辨率可用于在给定的神经元回路重构精确活性动力学。此外，相对于膜片钳技术是非侵入性的，这允许在一个事实，即神经元不受在他们的行为长期测量和结果。 F或本研究的目的，器官脊髓共培养物和多站点记录的组合被用来研究在脊髓内的功能再生。由于成人高等脊椎动物具有有限电位从脊髓的损伤中恢复过来，不同的策略进行了研究，以促进脊髓损伤后再生。到目前为止，皮质脊髓束处往往是首选的模型系统这样的调查。这些实验通常是费时的，昂贵的，并需要大的动物同伙由于单个组内的高变异性。此外，有证据表明propriospinal连接（即完全在脊髓内的神经元限制），可以起到恢复过程以下脊髓病变8举足轻重的作用。这些纤维难以在体内研究无升降纤维束的干扰。邦尼奇和Kapfhammer 9用纵向脊髓出生后的小鼠作为一种替代办法切片文化。执行机械损伤后，他们分析了半定量脊髓节段之间的轴突的形态再生。他们观察到的较少，但不是没有轴突穿越病变部位在文化切成一个成熟的年龄。损坏后7天 - 与此相反，损伤在年轻的阶段，文化5显示的大量轴突再生的。然而，证明功能的连接并没有出现。 因此 ，在propriospinal纤维体外模型的代表的发展，使功能再生的调查可以是一个有价值的工具来扩展有关在脊髓再生过程我们的知识。

<table><tbody><tr><td>Planar multielectrode array</td><td>Qwane Biosciences</td><td></td><td>custom-made according to the design of our lab</td></tr><tr><td>Nutrient medium</td><td></td><td></td><td>For 100 ml</td></tr><tr><td>Dulbeccos modified Eagle&#039;s medium</td><td>Gibco</td><td>31966-021</td><td>80 ml</td></tr><tr><td>Horse serum</td><td>Gibco</td><td>26050-070</td><td>10 ml</td></tr><tr><td>distilled, sterile water</td><td></td><td></td><td>10 ml&amp;nbsp;</td></tr><tr><td>Nerve growth factor-7S [5 ng/ml]</td><td>Sigma-Aldrich</td><td>N0513</td><td>200 &amp;micro;l</td></tr><tr><td colspan="4">reconstituted in wash solution with 1% BSA</td></tr><tr><td>Coating solution</td><td></td><td></td><td></td></tr><tr><td>Extracellular matrix gel</td><td>BD Biosciences</td><td>356230</td><td>Must stay cold at all times; dilute 1:50 with medium optimized for prenatal and embryonic neurons</td></tr><tr><td>medium optimized for prenatal and embryonic neurons</td><td>Gibco</td><td>21103-049</td><td></td></tr><tr><td>Wash solution</td><td></td><td></td><td>[mg/L]</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;-6H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td>100</td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;-7H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td>100</td></tr><tr><td>KCl</td><td></td><td></td><td>400</td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td></td><td></td><td>60</td></tr><tr><td>NaCl</td><td></td><td></td><td>8,000</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt; anhydrous</td><td></td><td></td><td>48</td></tr><tr><td>D-Glucose (Dextrose)</td><td></td><td></td><td>1,000</td></tr><tr><td colspan="4">According to the protocol on http://www.lifetechnologies.com/ch/en/home/technical-resources/media-formulation.152.html</td></tr><tr><td>Extracellular solution (pH 7.4)</td><td></td><td></td><td>[mM]</td></tr><tr><td>NaCl</td><td></td><td></td><td>145</td></tr><tr><td>KCl</td><td></td><td></td><td>4</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td></td><td></td><td>1</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td></td><td></td><td>2</td></tr><tr><td>HEPES</td><td></td><td></td><td>5</td></tr><tr><td>Na-pyruvate</td><td></td><td></td><td>2</td></tr><tr><td>Glucose</td><td></td><td></td><td>5</td></tr><tr><td>Blocking Solution</td><td></td><td></td><td></td></tr><tr><td>Detergent: Triton X-100</td><td></td><td></td><td>0.3%, harmful if swallowed</td></tr><tr><td>Bovine serum albumin</td><td>Sigma-Aldrich</td><td>A9418</td><td>1%</td></tr><tr><td>Goat serum</td><td>Gibco</td><td>16210-064</td><td>5%</td></tr><tr><td>Chicken Plasma</td><td>Sigma-Aldrich</td><td>P2366</td><td>Lyophilized, reconstitute with wash solution</td></tr><tr><td>Gabazine</td><td>Sigma-Aldrich</td><td>SR-95531</td><td></td></tr><tr><td>Mecamylamine hydrochloride</td><td>Tocris</td><td>2843</td><td>toxic if swallowed</td></tr><tr><td>Micropipette</td><td>World Precision Instruments, Inc.</td><td>MF28G-5</td><td></td></tr><tr><td>Paraformaldehyde</td><td></td><td></td><td>harmful, 4%</td></tr><tr><td>SMI-31</td><td>Covance</td><td>SMI-31P</td><td></td></tr><tr><td>SMI-32</td><td>Covance</td><td>SMI-32R-500</td><td></td></tr><tr><td>Strychnine</td><td>Sigma-Aldrich</td><td>S0532</td><td>toxic</td></tr><tr><td>Thrombin</td><td>Merck Millipore</td><td>1123740001</td><td>irritant, sensitizing, reconstitute with wash solution</td></tr><tr><td>Tissue culture flat tube 10 (= roller tube)</td><td>Techno Plastic Products AG</td><td>91243</td><td></td></tr></tbody></table>

investigating functional regeneration in organotypic spinal cord co-cultures grown on multi-electrode arrays

Adult higher vertebrates have a limited potential to recover from spinal cord injury. Recently, evidence emerged that propriospinal connections are a promising target for intervention to improve functional regeneration. So far, no in vitro model exists that grants the possibility to examine functional recovery of propriospinal fibers. Therefore, a representative model that is based on two organotypic spinal cord sections of embryonic rat, cultured next to each other on multi-electrode arrays (MEAs) was developed. These slices grow and, within a few days in vitro, fuse along the sides facing each other. The design of the used MEAs permits the performance of lesions with a scalpel blade through this fusion site without inflicting damage on the MEAs. The slices show spontaneous activity, usually organized in network activity bursts, and spatial and temporal activity parameters such as the location of burst origins, speed and direction of their propagation and latencies between bursts can be characterized. Using these features, it is also possible to assess functional connection of the slices by calculating the amount of synchronized bursts between the two sides. Furthermore, the slices can be morphologically analyzed by performing immunohistochemical stainings after the recordings. 
Several advantages of the used techniques are combined in this model: the slices largely preserve the original tissue architecture with intact local synaptic circuitry, the tissue is easily and repeatedly accessible and neuronal activity can be detected simultaneously and non-invasively in a large number of spots at high temporal resolution. These features allow the investigation of functional regeneration of intraspinal connections in isolation in vitro in a sophisticated and efficient way.

调查从E14大鼠胚胎的脊髓得到的共培养物的功能恢复的潜力，病变的8的时间窗进行，以28 DIV。 2至3周后，自发的神经元活动的记录与多边环境（图1 C＆D）。所述外记录动作电位脱机确定了每个单独的电极（图1E）。从这些数据中生成光栅打印（图1F）中，随后的网络活动曲线到每一侧（ 图1G）单独可视的总活性。在每个片的自发活动通常安排突发。如果切片在功能上连接，这些脉冲串可以传播从一个切片到另一个。因此，在两片之间的同步脉冲串的量经计算为定量测量功能再生。对于如何变换int的详细描述澳不同重复执行，以及如何计算公式同步活性的百分数，推导，请参见海德曼等人 10。 在所有实验中，第一次在标准条件下记录的活性。在第二步骤中，突触抑制被拦截通过加入士的宁（1μM）和gabazine（10μM）至细胞外溶液浴中。此去抑制通常导致与长期突发和脉冲间隔更经常活动模式，这有利于突发的定义，因此切片之间脉冲串同步的确定。 文化损伤在年轻的时候（7 - 9 DIV）病变2-3周后显示高量的同步活动，而文化损毁在稍后阶段（&gt; 19 DIV）表现出明显降低的再生能力（ 图2 A - F）。这些结果表明，再生能力在脊髓切片培养官能连接减小从一个高的水平，表示在体内的胚胎状态，为低电平，表示进一步发展状态的体内 ，在三周内中培养。 实际参与两片之间的突发同步什么类型的连接是什么？此外propriospinal连接，纤维也可能来自运动神经元或背根神经节的神经元。先前已经表明，背根神经节神经元是不相关的功能连接之间的脊髓切片10。然而，运动神经元的参与保持的可能性。由于运动神经元是公知，以形成通过烟碱受体13胆碱连接到脊髓神经元，脊髓共培养物的活动的记录，在8 DIV中，当培养物显示出高度同步的活动 10一个时间，和所述烟碱拮抗剂Mecam基胺（MEC，100μM）加入到细胞外溶液浴中。运动神经元中的两片之间的连接部分A的参与将导致同步活性的降低百分比。然而，这种情况并非如此（MEC:91.0±4.5％，N = 7;控制:93.6±4.6％，N = 7，P&gt; 0.05，魏氏配对检验）。这些结果表明，胆碱能突触没有贡献切片之间的功能性连接。然而，由于运动神经元也被证明至CNS 11,15内释放的谷氨酸突触，这些实验不完全排除它们的参与。 以确定运动神经元对非磷酸化神经丝ħ免疫染色带SMI-32进行。他们发现几个标记大细胞体典型为运动神经元，分布在切片（ 图3A）。的SMI-32抗体已报道标记motoneuro的细胞体NS 12这一发现持有同样适用于所用培养13。此外，神经元细胞体在一般情况下， 例如，针对B-Ⅲ微管蛋白或的NeuN以及神经胶质细胞体，例如，对染色，星形胶质细胞用GFAP抗体在与其他报告线和匹配这些细胞类型的形态描述（ 图3B - D）。然而，轴突的SMI-32抗体的标记是关于（图3E）。反对的期望，使用这种抗体时，将出现一个巨大的光纤网络，它看起来类似于标签磷酸化的神经丝H（ 图3F）的SMI-31染色。这一结果的一种解释可能是神经丝磷酸/去磷酸化的发育调控并不像紧在用于培养相比， 在体内情况 。混合发生磷酸化和非磷酸化神经丝在相同的轴突可以EXPL艾因这一发现。另一种可能性是，所述SMI-32标记的轴突源自投射神经元。曾荫权和他的同事16显示， 例如，克拉克的列，中间外侧细胞柱神经元通过SMI-32除了运动神经元染色。这种神经元的轴突明显的扩散也可以解释SMI-32阳性纤维的丰度。 <img alt="图1" src="/files/ftp_upload/53121/53121fig1.jpg" /> 图1显示和自发活动的分析。（A）一MEA的示意图。铂焊条描绘于黑色，红色透明导线及在黄MEA的中间槽。 （B）的特写位于MEA的中心的电极阵列。该图是由M. Heuschkel博士惠赠。 （C）的8格老文化性亮场图像即切片已经长大，并沿彼此面对的侧面融合。黄色栏中表示MEA的electrode-和绝缘的无槽。比例尺= 400微米（D）时间轴的实验。 E14大鼠胚胎的两个脊髓切片被并排放置彼此上多边环境。在几天内，切片生长和保险丝沿彼此面对的两侧。在8时限 - 28 DIV，完整的病变是通过融合部位进行。两到三个星期后的自发活动记录和文化是固定的免疫组化染色。 （E）23 DIV旧文化的每个电极的自发活动的痕迹。为了更清晰的可视化，只有每秒跟踪说明。橙色痕迹描绘从左侧活动已记录从切片的右侧，蓝色痕迹。大部分的脉冲串是在两者之间保持同步。在左切片而仅为P发生的箭头指向一个突发artially传播到合适的切片。在右切片的活性但是没有达到的，根据侧的平均最大峰活性的至少25％的所选择的阈值，因此，不被检测为一个突发。右边倍率描绘上次同步爆对。 （F）光栅（E）中所示的活动的阴谋。 （G）的网络活动曲线图与（E）中所示的活动的定义的突发（下面基线柱）。从海德曼等 10改编。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53121/53121fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53121/53121fig2.jpg" /> 图2.同步与不同步的活动。（A，B）光栅图文化的多巴胺ð在年轻的时候（在7 - 9格），显示出标准条件（A），并在去抑制（B）的同步活动。 （C，D）光栅图损毁的老年（在&gt; 19 DIV）文化，显示出标准条件（C），并在去抑制（D）下不同步活动。同步活动（E，F）的平均比例绘制的标准条件（E），并在去抑制（F）记录下每个个体病变一天。录音被送往2 - 损害之日起3周。从海德曼等 10改编。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53121/53121fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53121/53121fig3.jpg" /> Figure 3.免疫组化特征。motoneuronal细胞体（A）染色与SMI-32。 （二）成熟的神经元细胞体和他们的过程中β-III微管蛋白标签。 （三）确定的星形胶质细胞GFAP的。成熟的神经元细胞体（D）的标记用的NeuN。 （E）SMI-32染色非磷酸化神经丝H.（F）磷酸化神经丝^ h的确定SMI-31。从海德曼等 10改编。需要注意的是与两个，SMI-31和SMI-32，纤维的大网络是在培养物中可见。酒吧AD = 20微米，H＆F = 100微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53121/53121fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Investigating Functional Regeneration in Organotypic Spinal Cord Co-cultures Grown on Multi-electrode Arrays at JoVE.com

Investigating Functional Regeneration in Organotypic Spinal Cord Co-cultures Grown on Multi-electrode Arrays

在这里，我们提出了一个协议，基于多电极阵列培养来研究在体外 propriospinal连接官能再生脊髓切片。

调查器官脊髓联合培养物中生长多电极阵列功能再生

Adult higher vertebrates have a limited potential to recover from spinal cord injury. Recently, evidence emerged that propriospinal connections are a ...

investigating-functional-regeneration-organotypic-spinal-cord-co

Nanyang Technological University

Research

JoVE Journal

Neuroscience

9.0K 次观看.  University of Bern. 在这里，我们提出了一个协议，基于多电极阵列培养来研究在体外 propriospinal连接官能再生脊髓切片。 

视频: Investigating Functional Regeneration in Organotypic Spinal Cord Co-cultures Grown on Multi-electrode Arrays

Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the central nervous system1,2. These endogenous EFs are generated by cellular regulation of ionic transport combined with the electrical resistance of cells and tissues. It has been reported that applied EF treatment can promote functional repair of spinal cord injuries in animals and humans3,4. In particular, EF-directed cell migration has been demonstrated in a wide variety of cell types5,6, including neural progenitor cells (NPCs)7,8. Application of direct current (DC) EFs is not a commonly available technique in most laboratories. We have described detailed protocols for the application of DC EFs to cell and tissue cultures previously5,11. Here we present a video demonstration of standard methods based on a calculated field strength to set up 2D and 3D environments for NPCs, and to investigate cellular responses to EF stimulation in both single cell growth conditions in 2D, and the organotypic spinal cord slice in 3D. The spinal cordslice is an ideal recipient tissue for studying NPC ex vivo behaviours, post-transplantation, because the cytoarchitectonic tissue organization is well preserved within these cultures9,10. Additionally, this ex vivo model also allows procedures that are not technically feasible to track cells in vivo using time-lapse recording at the single cell level. It is critically essential to evaluate cell behaviours in not only a 2D environment, but also in a 3D organotypic condition which mimicks the in vivo environment. This system will allow high-resolution imaging using cover glass-based dishes in tissue or organ culture with 3D tracking of single cell migration in vitro and ex vivo and can be an intermediate step before moving onto in vivo paradigms.

This protocol demonstrates methods used to establish 2D and 3D environments in custom-designed electrotactic chambers, which can track cells in vivo/ex vivo using time-lapse recording at the single cell level, in order to investigate galvanotaxis/electrotaxis and other cellular responses to direct current (DC) electric fields (EFs).

电场控制的二维和三维环境中的神经祖细胞定向迁移

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the ...

科研

生物工程

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate a method to fabricate suction electrodes that are used to examine CPG activity, or fictive locomotion, in dissected rodent spinal cords. The rodent spinal cords are placed in artificial cerebrospinal fluid and the ventral roots are drawn into the suction electrode. The electrode is constructed by modifying a commercially available suction electrode. A heavier silver wire is used instead of the standard wire given by the commercially available electrode. The glass tip on the commercial electrode is replaced with a plastic tip for increased durability. We prepare hand drawn electrodes and electrodes made from specific sizes of tubing, allowing consistency and reproducibility. Data is collected using an amplifier and neurogram acquisition software. Recordings are performed on an air table within a Faraday cage to prevent mechanical and electrical interference, respectively.

A demonstration of the fabrication and use of an extracellular suction electrode used to measure electrophysiological recordings of neonatal rodent spinal cords in vitro

脊髓电二：外吸电极的制作

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate ...

神经科学

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

Human primary muscle cells cultured aneurally in monolayer rarely contract spontaneously because, in the absence of a nerve component, cell differentiation is limited and motor neuron stimulation is missing1. These limitations hamper the in vitro study of many neuromuscular diseases in cultured muscle cells. Importantly, the experimental constraints of monolayered, cultured muscle cells can be overcome by functional innervation of myofibers with spinal cord explants in co-cultures.
Here, we show the different steps required to achieve an efficient, proper innervation of human primary muscle cells, leading to complete differentiation and fiber contraction according to the method developed by Askanas2. To do so, muscle cells are co-cultured with spinal cord explants of rat embryos at ED 13.5, with the dorsal root ganglia still attached to the spinal cord slices. After a few days, the muscle fibers start to contract and eventually become cross-striated through innervation by functional neurites projecting from the spinal cord explants that connecting to the muscle cells. This structure can be maintained for many months, simply by regular exchange of the culture medium.
The applications of this invaluable tool are numerous, as it represents a functional model for multidisciplinary analyses of human muscle development and innervation. In fact, a complete de novo neuromuscular junction installation occurs in a culture dish, allowing an easy measurement of many parameters at each step, in a fundamental and physiological context.
Just to cite a few examples, genomic and/or proteomic studies can be performed directly on the co-cultures. Furthermore, pre- and post-synaptic effects can be specifically and separately assessed at the neuromuscular junction, because both components come from different species, rat and human, respectively. The nerve-muscle co-culture can also be performed with human muscle cells isolated from patients suffering from muscle or neuromuscular diseases3, and thus can be used as a screening tool for candidate drugs. Finally, no special equipment but a regular BSL2 facility is needed to reproduce a functional motor unit in a culture dish. This method thus is valuable for both the muscle as well as the neuromuscular research communities for physiological and mechanistic studies of neuromuscular function, in a normal and disease context.

Cultured muscle cells are an inadequate model to recapitulate innervated muscle in vivo. A functional motor unit can be reproduced in vitro by innervation of differentiated human primary muscle cells using rat embryo spinal cord explants. This article describes how co-cultures of spinal cord explants and muscle cells are established.

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 ...

Spiral Ganglion Neuron Explant Culture and Electrophysiology on Multi Electrode Arrays

Spiral ganglion neurons (SGNs) participate in the physiological process of hearing by relaying signals from sensory hair cells to the cochlear nucleus in the brain stem. Loss of hair cells is a major cause of sensory hearing loss. Prosthetic devices such as cochlear implants function by bypassing lost hair cells and directly stimulating SGNs electrically, allowing for restoration of hearing in deaf patients. The performance of these devices depends on the functionality of SGNs, the implantation procedure and on the distance between the electrodes and the auditory neurons. 
We hypothesized, that reducing the distance between the SGNs and the electrode array of the implant would allow for improved stimulation and frequency resolution, with the best results in a gapless position. Currently we lack in vitro culture systems to study, modify and optimize the interaction between auditory neurons and electrode arrays and characterize their electrophysiological response. To address these issues, we developed an in vitro bioassay using SGN cultures on a planar multi electrode array (MEA). With this method we were able to perform extracellular recording of the basal and electrically induced activity of a population of spiral ganglion neurons. We were also able to optimize stimulation protocols and analyze the response to electrical stimuli as a function of the electrode distance. This platform could also be used to optimize electrode features such as surface coatings.

We present a protocol to culture primary murine spiral ganglion neuron explants on multi electrode arrays to study neuronal response profiles and optimize stimulation parameters. Such studies aim to improve the neuron-electrode interface of cochlear implants to benefit hearing in patients as well as the energy consumption of the device.

螺旋神经节神经元外植体培养及电多电极阵列

Spiral ganglion neurons (SGNs) participate in the physiological process of hearing by relaying signals from sensory hair cells to the cochlear nucleus in ...

Coculture of Axotomized Rat Retinal Ganglion Neurons with Olfactory Ensheathing Glia, as an In Vitro Model of Adult Axonal Regeneration

Olfactory ensheathing glia (OEG) cells are localized all the way from the olfactory mucosa to and into the olfactory nerve layer (ONL) of the olfactory bulb. Throughout adult life, they are key for axonal growing of newly generated olfactory neurons, from the lamina propria to the ONL. Due to their pro-regenerative properties, these cells have been used to foster axonal regeneration in spinal cord or optic nerve injury models.
We present an in vitro model to assay and measure OEG neuroregenerative capacity after neural injury. In this model, reversibly immortalized human OEG (ihOEG) is cultured as a monolayer, retinas are extracted from adult rats and retinal ganglion neurons (RGN) are cocultured onto the OEG monolayer. After 96 h, axonal and somatodendritic markers in RGNs are analyzed by immunofluorescence and the number of RGNs with axon and the mean axonal length/neuron are quantified.
This protocol has the advantage over other in vitro assays that rely on embryonic or postnatal neurons, that it evaluates OEG neuroregenerative properties in adult tissue. Also, it is not only useful for assessing the neuroregenerative potential of ihOEG but can be extended to different sources of OEG or other glial cells.

We present an in vitro model to assess olfactory ensheathing glia (OEG) neuroregenerative capacity, after neural injury. It is based on a coculture of axotomized adult retinal ganglion neurons (RGN) on OEG monolayers and subsequent study of axonal regeneration, by analyzing RGN axonal and somatodendritic markers.

与嗅觉恩希廷格利亚合体培养的 Axotom 化大鼠视网膜甘利翁神经元，作为成人轴再生体外模型

Olfactory ensheathing glia (OEG) cells are localized all the way from the olfactory mucosa to and into the olfactory nerve layer (ONL) of the olfactory ...

Establishment of an Electrophysiological Platform for Modeling ALS with Regionally-Specific Human Pluripotent Stem Cell-Derived Astrocytes and Neurons

Human pluripotent stem cell-derived astrocytes (hiPSC-A) and neurons (hiPSC-N) provide a powerful tool for modeling Amyotrophic Lateral Sclerosis (ALS) pathophysiology in vitro. Multi-electrode array (MEA) recordings are a means to record electrical field potentials from large populations of neurons and analyze network activity over time. It was previously demonstrated that the presence of hiPSC-A that are differentiated using techniques to promote a spinal cord astrocyte phenotype improved maturation and electrophysiological activity of regionally specific spinal cord hiPSC-motor neurons (MN) when compared to those cultured without hiPSC-A or in the presence of rodent astrocytes. Described here is a method to co-culture spinal cord hiPSC-A with hiPSC-MN and record electrophysiological activity using MEA recordings. While the differentiation protocols described here are particular to astrocytes and neurons that are regionally specific to the spinal cord, the co-culturing platform can be applied to astrocytes and neurons differentiated with techniques specific to other fates, including cortical hiPSC-A and hiPSC-N. These protocols aim to provide an electrophysiological assay to inform about glia-neuron interactions and provide a platform for testing drugs with therapeutic potential in ALS.

We describe a method for differentiating spinal cord human induced pluripotent-derived astrocytes and neurons and their co-culture for electrophysiological recording.

建立区域特异性人多能干细胞衍生星形胶质细胞和神经元模拟ALS的电生理平台

Human pluripotent stem cell-derived astrocytes (hiPSC-A) and neurons (hiPSC-N) provide a powerful tool for modeling Amyotrophic Lateral Sclerosis (ALS) ...

<meta charset="utf8"></head><body>记录大脑组件中的网络活动：多电极阵列方法

A Multi-Electrode Array Platform for Modeling Epilepsy Using Human Pluripotent Stem Cell-Derived Brain Assembloids

Human brain organoids are three-dimensional (3D) structures derived from human pluripotent stem cells (hPSCs) that recapitulate&#160;aspects of fetal brain development. The fusion of dorsal with ventral regionally specified brain organoids in vitro generates assembloids, which have functionally integrated microcircuits with excitatory and inhibitory neurons. Due to their structural complexity and diverse population of neurons, assembloids have become a useful in vitro tool for studying aberrant network activity. Multi-electrode array (MEA) recordings serve as a method for capturing electrical field potentials, spikes, and longitudinal network dynamics from a population of neurons without compromising cell membrane integrity. However, adhering assembloids onto the electrodes for long-term recordings can be challenging due to their large size and limited contact surface area with the electrodes. Here, we demonstrate a method to plate assembloids onto MEA plates for recording electrophysiological activity over a 2-month span. Although the current protocol utilizes human cortical organoids, it can be broadly adapted to organoids differentiated to model other brain regions. This protocol establishes a robust, longitudinal, electrophysiological assay for studying the development of a neuronal network, and this platform has the potential to be used in drug screening for therapeutic development in epilepsy.

<meta charset="utf8"></head><body>作者聚焦：通过基于多电极阵列的人脑组件长期电生理监测推进遗传性癫痫研究

This protocol aims to stably plate dorsal-ventral fused assembloids on multi-electrode arrays for modeling epilepsy in vitro.

使用人类多能干细胞来源的脑组件对癫痫进行建模的多电极阵列平台

Human brain organoids are three-dimensional (3D) structures derived from human pluripotent stem cells (hPSCs) that recapitulate&#160;aspects of fetal ...

Studying the Regeneration of Functional Connections between Spinal Cord Slices Using a Multi-Electrode Array

Source: Heidemann, M., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/53121">Investigating Functional Regeneration in Organotypic Spinal Cord Co-cultures Grown on Multi-electrode Arrays.</a> J. Vis. Exp. (2015).
The video demonstrates the functional regeneration of propriospinal connections using a multi-electrode array (MEA)-based assay. The spinal cord slices are placed on the MEA and are allowed to fuse. The fused slices are mechanically separated by creating a lesion, inducing propriospinal neurons to grow over the lesion and connect the slices. The electrical activity of the slices is assessed to identify the regeneration of functional connections.

使用多电极阵列研究脊髓切片之间功能连接的再生

1. Preparation of MEAs
Note: MEAs are composed of a glass substrate, micro-fabricated metal electrodes and a SU-8 polymer insulation layer. For the ...

调查器官脊髓联合培养物中生长多电极阵列功能再生

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电场控制的二维和三维环境中的神经祖细胞定向迁移

脊髓电二：外吸电极的制作

一个在培养皿中的功能电机组：脊髓植和肌肉细胞共培养

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与嗅觉恩希廷格利亚合体培养的 Axotom 化大鼠视网膜甘利翁神经元，作为成人轴再生体外模型

建立区域特异性人多能干细胞衍生星形胶质细胞和神经元模拟ALS的电生理平台

使用人类多能干细胞来源的脑组件对癫痫进行建模的多电极阵列平台

使用人类多能干细胞来源的脑组件对癫痫进行建模的多电极阵列平台

使用人类多能干细胞来源的脑组件对癫痫进行建模的多电极阵列平台

使用多电极阵列研究脊髓切片之间功能连接的再生