这项工作是由国家卫生部批准RO1DC002756和普渡大学研究基金会。我们感谢与实验的意见和罗德尼帮助数字McPhail多丽丝吴和维泽张。

食谱小鸡林格<table border="1"><tbody><tr><td> 7.2摹</td><td>氯化钠</td></tr><tr><td> 0.23摹</td><td>氯化钙2 + 2H 2 O </td></tr><tr><td> 0.37摹</td><td>氯化钾</td></tr><tr><td> 0.115摹</td><td> NA 2 HPO 4 </td></tr><tr><td> 900毫升</td><td>水（组织文化品位） </td></tr></tbody></table>注：调整pH值至7.4。使终体积为1000毫升水。 10X PBS <table border="1"><tbody><tr><td> 40克</td><td>氯化钠</td></tr><tr><td> 1克</td><td>氯化钾</td></tr><tr><td> 7克</td><td> NA 2 HPO 4 </td></tr><tr><td> 1.2摹</td><td> KH 2 PO 4 </td></tr><tr><td> 450毫升</td><td> DEPC水</td></tr></tbody></table>注：调整pH值至7.4。带来最终体积500毫升水。工作浓度（1X）是由9部分，如用微孔紫外线照射系统生成水18MΩ- cm电导率获得的组织培养级水，稀释1部分10X股票。 凹陷外植体举行的中期<ul><li> L -谷氨酰胺的DMEM/F12，10毫米的HEPES </li><li> 10％的胰岛素，转移，硒（及其1） </li><li> 1％钢笔链球菌</li></ul>植文化介质<ul><li>凹陷外植体举行的中期</li><li> 10 ng / ml的神经营养因子3（NT - 3） </li><li> 10毫微克/毫升睫状神经营养因子（CNTF） </li></ul>脊髓的解剖和控股中等<ul><li> L - 15 </li><li> 10％胎牛血清</li><li> 1％钢笔链球菌</li></ul>孵育的鸡蛋，在37-38 ° C。 70％乙醇消毒解剖工具和Sylgard解剖盘。解剖菜和工具，也可以在一夜之间紫外线消毒。 <ol><li>与1毫升无菌PBS，混合，并等待珠定居管底部微管珠。 </li><li>珠洗净，取出上清液后珠相继落户，并在PBS悬浮几次。 </li><li>孵育珠纯化蛋白（PBS稀释）或PBS单独（管制）在室温下1小时。 </li><li>在24孔板用1ml PBS冲洗，PBS和存储珠，直到胶原蛋白。 </li></ol> 2。 Statoacoustic神经节清扫<ol><li>在E4中删除，从卵胚胎，并将其放置在一碟鸡林格氏液删除胚胎膜。 </li><li>将胚胎在Sylgard ©内衬培养皿菜充满了冷的HBSS和位置的胚胎，它与奥迪面临的囊泡的一面。使用解剖引脚引脚胚胎的菜。 </li><li>使用两个解剖引脚，立即通过皮肤水平切腹侧运河袋，约符合蜗管。垂直切割前部和后部凹陷。使用镊子或解剖针，解除和删除三个削减接壤的皮肤瓣。 注：凹陷的otocyst anteroventral边背袋，这是很容易与明基地照明可见，腹蜗管项目ventromedially和咽弓遮挡之间的中途，位于。耳蜗管拉长，所以它的背腹层面将逐步增加E4汉堡 - 汉密尔顿阶段23日至25日9之间。 </li><li>在一个单独从凹陷的后方向拉otocyst。置换周围组织清扫引脚凹陷，小心取出的胚胎，使用＃55钳凹陷。删除从凹陷的间叶组织和神经束凸出大块。 </li><li>使用宽口吸管尖，外植体转移到24孔板0.5毫升凹陷持有介质的外植体。植在冰上或在室温下保持长达4小时。 </li></ol> 3。脊髓夹层<ol><li> E6的胚胎从鸡蛋中取出，并放置在含有鸡林格氏液的解决方案的菜。删除头部和胚胎膜。 </li><li>将在Sylgard ©解剖菜含有冷脊髓夹层介质的身体。位置和针胚胎“腹侧向下”和尾鳍面临实验者。将通过每个肢体和解剖引脚通过前结束。 </li><li>使用镊子和/或解剖引脚，小心地取出皮肤和组织，在腹侧方向移动，直到脊髓背表面可见。 </li><li>切割用解剖针脊髓背中线，在后前方向，沿整个长度的脊髓。这创建了一个脊髓单独的左，右两侧的“开卷”。 </li><li&gt;删除周围组织，横向隔离脊髓和暴露的背根神经节（DRG）。卸下背根神经节，脊髓和背根神经节之间的摩擦在解剖针或镊子和脑膜。 </li><li>从身体中取出，用镊子尾鳍最末端的脊髓和解除实验者和远离脊髓。删除剩余的背根神经节和脑膜。 </li><li>按住钳（或针菜）脊髓的一端，并用Vannas剪刀切沿腹正中线，平分脊髓。 </li><li>按住钳固定的组织，并用Vannas剪刀切沿小外植体组织的长度（长度为100-500微米）的外植体的一端。底板可以被视为一个明确沿着一个外植体边缘增厚组织。 </li><li>使用宽口吸管尖0.5毫升冷夹层介质转移到24孔板植。保持在冰上植，以保持组织活力，F长达4小时。 </li></ol> 4。外植体与纯化蛋白质的文化测试突起响应<ol><li>准备在一个15毫升的锥形管的1.5毫克/毫升的胶原溶液，在冰上，根据制造商的指示。 </li><li>验证胶原溶液的pH值7-7.4使用pH试纸。加入1μL体积的NaOH调整pH值。 </li><li>植转移到24孔板中，用一个宽口吸管尖和吸出多余的液体。多植，可培养一个孔，但应放在彼此相距至少500微米。 </li><li>加入0.5毫升的胶原蛋白的以及含有外植体。井底表面上的外植体的位置。请务必设置与未来以及出发前，每一种文化，完全是因为胶原蛋白会开始在室温下聚合。 </li><li>将培养板在37 ° C幻灯片30-45分钟的温暖聚合胶原蛋白。当胶原蛋白已polymerized将类似于凝胶。 </li><li>加入0.5毫升的温暖外植体培养基中，无论是纯化的蛋白质（PBS稀释）或PBS（管制）补充，并在37 ° C，5％的CO 2。突起生长的影响应该是24小时可见。 注：我们使用相同的协议，以文化凹陷植，达42小时。 </li></ol> 5。外植体与蛋白包被的磁珠共培养测试定向突起生长<ol><li>开始与植文化的步骤4.1 - 4.4。 </li><li>使用吸管，转让1-5珠从PBS以及含胶原蛋白溶液和外植体。 </li><li>使用镊子，从外植体的边缘位置珠50-500微米。 </li><li>放在37 ° C幻灯片30-45分钟的温暖聚合胶原蛋白的板块。组织或珠可能成为流离失所的胶原蛋白的聚合。在第5分钟，检查文化和重新定位，组织和珠。 </li><li>添加0.5米升凹陷每口井的媒体和孵育24小时。 </li></ol> 6。通过免疫组化突起生长的可视化<ol><li>在PBS冲洗的文化，并在4％多聚甲醛的PBS在室温下1小时修复。 </li><li>从井的墙壁跟踪周围的凝胶边缘圆润的聚四氟乙烯微型抹刀结束发行的凝胶。执行井凝胶的免疫染色。在PBS冲洗数次。 </li><li>培育基地0.5毫升封闭液（10％小牛血清，0.1％的Triton X - 100的，0.1％的PBS叠氮化钠）4℃过夜° C。 </li><li>在0.5毫升的主要抗体（抗β-微管蛋白抗体稀释于封闭液1:500）孵育4℃过夜° C。 </li><li>在PBS冲洗数次。在0.5毫升的二抗（1:500稀释的Alexa Fluor 488山羊抗鼠IgG 2B抗体）孵育4℃过夜° C。从这一步，保持在黑暗或铝箔包装板，由于对光线的敏感度二抗。 </li><li>在4 ° C的PBS长达一周冲洗多次在PBS和存储。 </li></ol> 7。代表性的成果<img alt="图1" src="/files/ftp_upload/3600/3600fig1.jpg" /> 图1。代表纯化的NT - 3和E4鸡凹陷植NT - 3涂层珠突起促进作用结果 。 凹陷的神经元细胞体和免疫染色与β-微管蛋白和共聚焦显微镜10倍的目标与成像突起。 体外培养 24小时后，凹陷植显示在100毫微克/毫升纯化的人NT - 3添加到细胞培养液（1B）存在较大的突起生长，控制（1A） 。凹陷神经元共培养浸泡in100 ng / ml的NT - 3的证明，NT - 3的点源，可以在本地促进轴突生长的珠子。植显示时间和致密突起生长的一面植面临珠相比，对面（1D）和珠浸泡在PBS（1C）与对照组比较文化。比例尺= 200微米。 <img alt="图2" src="/files/ftp_upload/3600/3600fig2.jpg" /> 图2。代表E6的小鸡脊髓纯化Wnt5a和Wnt5a涂珠的突起促进影响结果。 脊髓外植体与β-微管蛋白的免疫染色和共聚焦显微镜成像。经过24小时的纯化鼠标Wnt5a（400ng/ml），从鸡胚脊髓外植体的轴突生长的增长增加（2B）相比没有Wnt5a（2A）培养的控制。珠，浸泡在500毫微克/毫升Wnt5a放置在离边缘的脊髓植300-500微米，也促进突起生长（2D）与对照组比较文化（2C）。类似的合作培养结果察看Wnt5a的表达细胞8。比例尺= 200微米。

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没有利益冲突的声明。

我们提出了一个方法来剖析和文化的E4凹陷和E6脊髓植，从小鸡无血清条件下，。目前，这个过程是在我们的实验室研究SAG神经元的存活和突起生长分泌各种配体的影响。本议定书的小说方面，包括使用的无血清培养的外植体浸泡在生长因子的研究凹陷突起生长的影响的珠。珠的方法类似我们已经描述鸡胚脊髓 10 。传统上，珠已被用于研究与经典的轴突导向因子和morphogens。在这里，我们?...

<table border="1"><tbody><tr><td> 试剂 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> 设备 </td><td></td><td></td><td></td></tr><tr><td>解剖镜下</td><td></td><td></td><td>我们用明基地照明的Leica M80体视显微镜</td></tr><tr><td>滑入温暖</td><td> CS＆E </td><td></td><td>胶原蛋白的聚合</td></tr><tr><td>鸡蛋孵化器</td><td></td><td></td><td></td></tr><tr><td> 37 ° C / 5％CO 2培养箱细胞培养孵化器</td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> 夹层材料 </td><td></td><td></td><td></td></tr><tr><td> Sylgard＆副本;涂层的培养皿</td><td></td><td></td><td></td></tr><tr><td> 24孔细胞培养板</td><td>康宁</td><td> 3526 </td><td></td></tr><tr><td> ＃5杜蒙钳</td><td>精细科学工具</td><td> 11252-30 </td><td></td></tr><tr><td> ＃55杜蒙钳</td><td>精细科学工具</td><td> 11295-51 </td><td></td></tr><tr><td>不锈钢解剖引脚</td><td>精细科学工具</td><td> 26002-10 </td><td>胚胎穿针，精细解剖</td></tr><tr><td>莫里亚穿孔勺</td><td>精细科学工具</td><td> 10370-17 </td><td>胚胎收获/转让</td></tr><tr><td> 200μL宽口吸管提示</td><td>点科学公司</td><td> 118 - 96R </td><td>外植体转移</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> E4和E6鸡蛋</td><td></td><td></td><td> ＆NBSP; </td></tr><tr><td>林格小鸡的解决方案</td><td></td><td></td><td>胚胎收获</td></tr><tr><td> HBSS </td><td>西格玛</td><td> H8264 </td><td>凹陷清扫</td></tr><tr><td> L15的介质</td><td>西格玛</td><td> L5520 </td><td>脊髓夹层介质</td></tr><tr><td>胎牛血清</td><td>亚特兰大生物</td><td> S11150 </td><td>脊髓夹层介质</td></tr><tr><td> Vannas剪刀</td><td>世界精密仪器</td><td> 501778 </td><td>脊髓夹层</td></tr><tr><td>&nbsp;</td><td></td><td></td><td></td></tr><tr><td> 胶原蛋白的准备 </td><td></td><td></td><td></td></tr><tr><td>鼠尾I型胶原</td><td> BD公司</td><td> 354249 </td><td>植文化</td></tr><tr><td> 10X PBS（无菌） </td><td></td><td></td><td>为神经tralize胶原蛋白</td></tr><tr><td> 1N氢氧化钠（无菌） </td><td></td><td></td><td>为了抵消胶原蛋白</td></tr><tr><td>蒸馏水（无菌） </td><td></td><td></td><td>为了抵消胶原蛋白</td></tr><tr><td> pH值指标文件（6.0-8.1） </td><td>瓦特曼</td><td> 2629-990 </td><td>要检查胶原蛋白的pH值</td></tr><tr><td> 15 ml锥形管</td><td>点科学公司</td><td> 818 - PG </td><td></td></tr><tr><td> 500μL宽口吸管提示</td><td>点科学公司</td><td> 119 - R100 </td><td>吸管胶原蛋白</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> 植文化 </td><td></td><td></td><td></td></tr><tr><td> DMEM/F12培养基</td><td>西格玛</td><td> D8437 </td><td>植文化介质</td></tr><tr><td>及其1 </td><td>西格玛</td><td> I2521 </td><td>中等补充</tD&gt; </tr><tr><td>青霉素链霉素</td><td> Invitrogen公司</td><td> 15140-122 </td><td>中等补充</td></tr><tr><td>睫状神经营养因子（大鼠） </td><td>西格玛</td><td> C3835 </td><td>中等补充</td></tr><tr><td> NT - 3（人） </td><td>西格玛</td><td> N1905 </td><td>中等补充</td></tr><tr><td>纯净鼠标Wnt5a </td><td> R＆D系统</td><td> 645 - WN - 010 </td><td></td></tr><tr><td>阿菲 - 凝胶蓝胶珠</td><td> Bio - Rad公司</td><td> 153-7302 </td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> 免疫组化 </td><td></td><td></td><td></td></tr><tr><td>多聚甲醛</td><td>西格玛</td><td> P6148 </td><td>固定</td></tr><tr><td> PBS </td><td></td><td></td><td>冲洗</td></tr><tr><td> TRITON X - 100 </td><td>西格玛</td><td> T9284 </td><td>封闭液</td></tr><tr><td>钠叠氮化物</td><td>西格玛</td><td> S8032 </td><td>封闭液</td></tr><tr><td>小牛血清</td><td> Invitrogen公司</td><td> 16170-078 </td><td>封闭液</td></tr><tr><td>鼠单克隆IgG2b抗β微管蛋白1 +第二抗体</td><td>西格玛</td><td> T8535 </td><td>标签胞体和突起</td></tr><tr><td>的Alexa Fluor 488山羊抗鼠IgG2的B抗体</td><td> Invitrogen公司</td><td> A21141 </td><td>检测β微管蛋白的抗体</td></tr><tr><td>聚四氟乙烯微型抹刀</td><td> VWR </td><td> 57949-033 </td><td>以及推出胶原凝胶</td></tr></tbody></table>

dissection and culture of chick statoacoustic ganglion and spinal cord explants in collagen gels for neurite outgrowth assays

鸡内耳感觉器官外围过程statoacoustic节（SAG）的神经元支配。感觉器官支配取决于轴突的指导线索和生存因素位于沿轨迹的轴突生长和/或在其感觉器官目标2的组合。例如，一个典型的轴突导向信号通路，semaphorin neuropilin，功能产生的干扰misrouting 3奥迪轴突。此外，多种生长因子在内耳的感官指标，包括神经营养因子3（NT - 3）和脑源性神经营养因子（BDNF），表示已在转基因动物的操纵，再次领先misrouting凹陷轴突 4 。同样是这些分子促进体外 5,6和突起生长的小鸡凹陷神经元的生存。 在这里，我们描述和证明在体外的方法，我们目前正在ü唱鸡凹陷突起的可溶性蛋白，其中包括知名morphogens如Wnts，以及促进SAG突起生长和神经元的存活重要的生长因子的响应测试。利用该模型系统，我们希望得出结论分泌的配体可以发挥SAG神经元的存活和突起生长的影响。 凹陷外植体解剖胚胎第4天（E4），并在三维胶原凝胶中培养24小时无血清条件下。首先，突起的响应测试与蛋白培养基中培养的外植体。然后，询问是否分泌配体的点源可以对突起生长的方向影响，外植体共同培养蛋白包被的磁珠和珠本地促进或抑制生长的能力检测。我们还包括一个解剖示范（修改后的协议7）E6脊髓植文化。我们经常使用脊髓植确认生物活性的蛋白质和蛋白质浸泡珠，来验证物种小鸡组织凹陷植相同的培养条件下，交叉反应。这些方便快速筛选分子施加营养（生存）或热带（定向）凹陷神经元的影响，特别是在执行体内研究，在体外实验。此外，这种方法允许单个分子的无血清条件下的测试，具有较高的神经元的存活8。

Watch this Scientific Journal Video about Dissection and Culture of Chick Statoacoustic Ganglion and Spinal Cord Explants in Collagen Gels for Neurite Outgrowth Assays at JoVE.com

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

我们演示了如何解剖和文化小鸡E4 statoacoustic节和E6脊髓植。外植体无血清条件下24小时的三维胶原凝胶培养。突起的响应测试与生长因子补充介质与蛋白包被的磁珠。

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

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

dissection-culture-chick-statoacoustic-ganglion-spinal-cord-explants

Research

JoVE Journal

Neuroscience

12.0K 次观看.  Purdue University. 我们演示了如何解剖和文化小鸡E4 statoacoustic节和E6脊髓植。外植体无血清条件下24小时的三维胶原凝胶培养。突起的响应测试与生长因子补充介质与蛋白包被的磁珠。

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

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

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

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

从胚胎脊髓​​连合神经的解剖和文化

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

科研

神经科学

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 ...

Dissection and Culture of Mouse Dopaminergic and Striatal Explants in Three-Dimensional Collagen Matrix Assays

Midbrain dopamine (mdDA) neurons project via the medial forebrain bundle towards several areas in the telencephalon, including the striatum1. Reciprocally, medium spiny neurons in the striatum that give rise to the striatonigral (direct) pathway innervate the substantia nigra2. The development of these axon tracts is dependent upon the combinatorial actions of a plethora of axon growth and guidance cues including molecules that are released by neurites or by (intermediate) target regions3,4. These soluble factors can be studied in vitro by culturing mdDA and/or striatal explants in a collagen matrix which provides a three-dimensional substrate for the axons mimicking the extracellular environment. In addition, the collagen matrix allows for the formation of relatively stable gradients of proteins released by other explants or cells placed in the vicinity (e.g. see references 5 and 6). Here we describe methods for the purification of rat tail collagen, microdissection of dopaminergic and striatal explants, their culture in collagen gels and subsequent immunohistochemical and quantitative analysis. First, the brains of E14.5 mouse embryos are isolated and dopaminergic and striatal explants are microdissected. These explants are then (co)cultured in collagen gels on coverslips for 48 to 72 hours in vitro. Subsequently, axonal projections are visualized using neuronal markers (e.g. tyrosine hydroxylase, DARPP32, or &beta;III tubulin) and axon growth and attractive or repulsive axon responses are quantified. This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of mesostriatal and striatonigral axon growth and guidance during development. Using this assay, it is also possible to assess other (intermediate) targets for dopaminergic and striatal axons or to test specific molecular cues.

Explants from the midbrain dopamine system and striatum are used in a collagen matrix assay for the in vitro analysis of mesostriatal and striatonigral pathway development. In this assay axonal outgrowth and guidance can be manipulated and quantified. It can also be modified for assessing other regions or molecular cues.

小鼠多巴胺和三维胶原基质检测纹状体外植体的解剖和文化

Midbrain dopamine (mdDA) neurons project via the medial forebrain bundle towards several areas in the telencephalon, including the striatum1. ...

Chicken Embryo Spinal Cord Slice Culture Protocol

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

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

鸡胚胎脊髓片培养协议

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

Dissection and Lateral Mounting of Zebrafish Embryos: Analysis of Spinal Cord Development

The zebrafish spinal cord is an effective investigative model for nervous system research for several reasons. First, genetic, transgenic and gene knockdown approaches can be utilized to examine the molecular mechanisms underlying nervous system development. Second, large clutches of developmentally synchronized embryos provide large experimental sample sizes. Third, the optical clarity of the zebrafish embryo permits researchers to visualize progenitor, glial, and neuronal populations. Although zebrafish embryos are transparent, specimen thickness can impede effective microscopic visualization. One reason for this is the tandem development of the spinal cord and overlying somite tissue. Another reason is the large yolk ball, which is still present during periods of early neurogenesis. In this article, we demonstrate microdissection and removal of the yolk in fixed embryos, which allows microscopic visualization while preserving surrounding somite tissue. We also demonstrate semipermanent mounting of zebrafish embryos. This permits observation of neurodevelopment in the dorso-ventral and anterior-posterior axes, as it preserves the three-dimensionality of the tissue.

Developmental processes such as proliferation, patterning, differentiation, and axon guidance can be readily modeled in the zebrafish spinal cord. In this article, we describe a mounting procedure for zebrafish embryos, which optimizes visualization of these events.

解剖和斑马鱼的胚胎横向安装：脊髓发展分析

The zebrafish spinal cord is an effective investigative model for nervous system research for several reasons. First, genetic, transgenic and gene ...

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several weeks, and this is accompanied by short distance regeneration (a few mm) of propriospinal axons and spinal-projecting axons from the brainstem. Among the 36 large identifiable spinal-projecting neurons, some are good regenerators and others are bad regenerators. These neurons can most easily be identified in wholemount CNS preparations. In order to understand the neuron-intrinsic mechanisms that favor or inhibit axon regeneration after injury in the vertebrates CNS, we determine differences in gene expression between the good and bad regenerators, and how expression is influenced by spinal cord transection. This paper illustrates the techniques for housing larval and recently transformed adult sea lampreys in fresh water tanks, producing complete spinal cord transections under microscopic vision, and preparing brain and spinal cord wholemounts for in situ hybridization. Briefly, animals are kept at 16&#160;&#176;C and anesthetized in 1% Benzocaine in lamprey Ringer. The spinal cord is transected with iridectomy scissors via a dorsal approach and the animal is allowed to recover in fresh water tanks at 23 &#176;C. For in situ hybridization, animals are reanesthetized and the brain and cord removed via a dorsal approach.

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

Lampreys recover locomotion after a complete spinal cord injury. However, some spinal-projecting neurons are good regenerators and others are not. This paper illustrates the techniques for housing sea lamprey larvae (and recently transformed adults), producing complete spinal cord transections and preparing wholemount brains and spinal cords for in situ hybridization.

完全性脊髓损伤和脑解剖协议后续Wholemount<em&gt;原位</em&gt;杂交苗种海七鳃鳗

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several ...

RNA-Seq Analysis of Differential Gene Expression in Electroporated Chick Embryonic Spinal Cord

In ovo electroporation of the chick neural tube is a fast and inexpensive method for identification of gene function during neural development. Genome wide analysis of differentially expressed transcripts after such an experimental manipulation has the potential to uncover an almost complete picture of the downstream effects caused by the transfected construct. This work describes a simple method for comparing transcriptomes from samples of transfected embryonic spinal cords comprising all steps between electroporation and identification of differentially expressed transcripts. The first stage consists of guidelines for electroporation and instructions for dissection of transfected spinal cord halves from HH23 embryos in ribonuclease-free environment and extraction of high-quality RNA samples suitable for transcriptome sequencing. The next stage is that of bioinformatic analysis with general guidelines for filtering and comparison of RNA-Seq datasets in the Galaxy public server, which eliminates the need of a local computational structure for small to medium scale experiments. The representative results show that the dissection methods generate high quality RNA samples and that the transcriptomes obtained from two control samples are essentially the same, an important requirement for detection of differential expression genes in experimental samples. Furthermore, one example is provided where experimental overexpression of a DNA construct can be visually verified after comparison with control samples. The application of this method may be a powerful tool to facilitate new discoveries on the function of neural factors involved in spinal cord early development.

This video shows the basic steps for performing whole transcriptome analysis on dissected chick embryonic spinal cord samples after transfection with in ovo electroporation.

对基因差异表达的RNA序列中电穿孔鸡胚脊髓分析

In ovo electroporation of the chick neural tube is a fast and inexpensive method for identification of gene function during neural development. Genome ...

Spinal Cord Neurons Isolation and Culture from Neonatal Mice

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on postnatal day 1-3. A mouse litter, usually 4-10 pups born from one breeding pair, is gathered for one experiment, and spinal cords are collected individually from each mouse after euthanasia with isoflurane. The spinal column is dissected out and then the spinal cord is released from the column. The spinal cords are then minced to increase the surface area of delivery for an enzymatic protease that allows for the neurons and other cells to be released from the tissue. Trituration is then used to release the cells into solution. This solution is subsequently fractionated in a density gradient to separate the various cells in solution, allowing for neurons to be isolated. Approximately 1-2.5 x 106 neurons can be isolated from one litter group. The neurons are then seeded onto wells coated with adhesive factors that allow for proper growth and maturation. The neurons take approximately 7 days to reach maturity in the growth and culture medium and can be used thereafter for treatment and analysis.

This study presents a technique for the isolation of neurons from WT neonatal mice. It requires the careful dissection of the spinal cord from the neonatal mouse, followed by the separation of neurons from the spinal cord tissue through mechanical and enzymatic cleavage.

脊髓神经元从新生小鼠中分离和培养

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on ...

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

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

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

神经干细胞向大鼠和小鼠大脑的动脉内传递：对脑缺血的应用

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

Induction of Complete Transection-Type Spinal Cord Injury in Mice

Spinal cord injury (SCI) largely leads to irreversible and permanent loss of function, most commonly as a result of trauma. Several treatment options, such as cell transplantation methods, are being researched to overcome the debilitating disabilities arising from SCI. Most pre-clinical animal trials are conducted in rodent models of SCI. While rat models of SCI have been widely used, mouse models have received less attention, even though mouse models can have significant advantages over rat models. The small size of mice equates to lower animal maintenance costs than for rats, and the availability of numerous transgenic mouse models is advantageous for many types of studies. Inducing repeatable and precise injury in the animals is the primary challenge for SCI research, which in small rodents requires high-precision surgery. The transection-type injury model has been a commonly used injury model over the last decade for transplantation-based therapeutic research, however a standardized method for inducing a complete transection-type injury in mice does not exist. We have developed a surgical protocol for inducing a complete transection type injury in C57BL/6 mice at thoracic vertebral level 10 (T10). The procedure uses a small tip drill instead of rongeurs to precisely remove the lamina, after which a thin blade with rounded cutting edge is used to induce the spinal cord transection. This method leads to reproducible transection-type injury in small rodents with minimal collateral muscle and bone damage and therefore minimizes confounding factors, specifically where behavioral functional outcomes are analyzed.

This protocol describes how to create a precise laminectomy for induction of stable transection-type spinal cord injury in the mouse model, with minimal collateral damage for spinal cord injury research.

小鼠完全转型脊髓损伤的诱导

Spinal cord injury (SCI) largely leads to irreversible and permanent loss of function, most commonly as a result of trauma. Several treatment options, ...