Name: a procedure for implanting a spinal chamber for longitudinal in vivo imaging of the mouse spinal cord
Uploaded: 2014-12-03T15:00:00.000+00:00
Duration: 11 min 20 s
Description: Watch this Scientific Journal Video about A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord at JoVE.com

We thank Dr. Joseph R. Fetcho for his input throughout the development of the procedure.We would like to acknowledge funding from the US National Institutes of Health (R01 EB002019 to C.B.S and DP OD006411 to Joseph R. Fetcho) and the National Science and Research Council of Canada (to M.J.F.) for financial support.

注:护理和实验操作本文所有小鼠被批准康奈尔大学机构动物护理和使用委员会。 1.设置外科手术<ol><li>消毒用高压锅或批准的消毒程序所需的所有工具。至少，包括解剖刀，剪刀，万纳剪刀，镊子，珠宝商的螺丝刀，棉签，一套拉钩的，植入物（ 图1A）和它的传送系统（ 图1D，E）。 </li><li>擦拭所有表面，包括任何立体镜旋钮，用70％的乙醇和/或合适的消毒剂。 </li><li>通过叠置定制stereotax（ 图1D）和围绕台式用无菌窗帘创建一个无菌区。加热垫（优选反馈控制）应放置在stereotax的顶部和下无菌布帘。 </li></ol> 2.准备鼠标外科手术<ol><li>麻醉下的5％异氟醚/氧气鼠标的感应室，直到呼吸减慢速度约1呼吸/秒。 </li><li>转印鼠标到分段区域远离无菌区，用鼻锥，继续异氟醚暴露。减少异氟烷至2％。适用眼药，以防止角膜干燥。 </li><li>在0.05毫克/ 100g的鼠施用格隆（抗胆碱能药物）肌内成使用29后肢 - 地下30针和3/10毫升注射器，每肢注射量的递送约50％。 注:格隆溴铵用于防止流体积聚在肺中，而鼠标是在麻醉下。 </li><li>使用电剪，2厘米宽，并集中在胸主动脉弓剃鼠标的背侧，大约3厘米长的部分。清洁任何修剪头发远离剃的区域。 </li><li>管理下列注射皮下和远端的剃贴片使用29 - 地下30的针头和3/10立方厘米注射器1毫升/ 100克鼠标5％葡萄糖盐水中（对于水合），5mg / kg的小鼠的酮洛芬（一种NSAID镇痛），0.2毫克/千克小鼠塞米松（抗炎类固醇，以减轻炎症）。 </li><li>使用棉签，执行碘和70％乙醇的三个交替洗涤，等待1分钟在洗涤之间。 </li><li>施用100μl的0.125％布比卡因（一外周神经阻滞）皮下使用29 - 地下30针和3/10毫升注射器的剃贴片的中心，以减少疼痛，在切口部位。 </li><li>传输鼠标自定义stereotax（ 图1D），插入直肠温度计，并等待大约10分钟的布比卡因生效。 </li></ol> 3.揭露和清理的3个相邻胸椎的背纹层<ol><li>使用手术刀刀片，创建一个小的（5毫米或更小）的切口的兴趣（ 图2A）在椎骨上方。 </li><li&gt;使用或者镊子或通过插入外科剪刀的尖端和扩展的把手，增加通过钝器解剖中的开口的皮肤的大小。 </li><li>使用手术刀或手术剪，轻轻地分解皮肤和下面的肌肉之间的结缔组织筋膜。 </li><li>用拉钩忍住皮肤，允许尽可能大的工作区域越好（ 图2B）。小心不要增加负载的胸部，从而阻碍呼吸。 </li><li>用一对镊子，把手喙最椎骨将通过上覆的肌肉被暴露。使用手术刀，切离组织上背进程从所有3椎骨（ 图2c）两侧。 </li><li>使用的手术刀刀片，棉签，和/或骨刮刀任何组合，除去从背薄片（ 图2D）的所有组织覆盖。 </li><li>用手术剪刀，小心切掉连接到脊椎骨上都拉泰拉筋脊柱（ 图2E）的升边沿。 </li><li>使用手术刀，轻轻地从脊柱尽可能的横向边缘，以便提供一个清洁的夹紧表面除去尽可能多的组织。 </li><li>轻轻清创使用骨刮刀和/或棉签任何剩余的软组织的椎骨。剪掉其他地方使用手术剪（ 图2F）任何不协调的组织。 注意:这是既夹紧和执行所述背侧椎板的椎骨被干净地暴露必不可少的。一个cauterizer可能在随后的控制出血有用。 </li><li>移拉钩忍住周围除了皮肤（ 图2F）的脊柱组织。 </li><li>附着在交付职位植入侧栏的插脚，并加载这些后入者（ 如图1E）。 </li><li>采用侧杆夹住3椎体，运用足够的压力来保存securelY（ 图2G，H）。使用镊子，以帮助确保3椎骨的电平钳位。需要注意的是多次尝试可能是必要的。确保该侧杆都是水平和平行之前执行椎板切除术。 </li><li>仔细清洁和干燥用棉签夹紧的椎骨的背水面。 </li></ol> 4.执行背椎板切除术<ol><li>插入vannas剪刀的前端到内侧夹紧椎骨的硬膜外腔和挤压手柄轻轻;如果骨是干的，它应当沿背薄片的长度开裂。重复在对侧此过程释放层。 </li><li>轻轻地握住松层的背过程钳，小心切掉任何结缔组织的椎板的喙和尾两端，抬起它拿走。 </li><li>用0.9％的盐水和/或人造脑脊髓液（ACSF），彻底洗去任何血液overlyi纳克脊髓。用棉签吸收多余的液体。 </li><li>使用vannas剪刀，小心地修剪骨的横向边缘后面的植入物的侧杆。再次，洗去和控制用棉签和盐水/学联出血。 </li><li>地下30针 - 倘骨膜的一部分已经脱落，覆盖脊髓，用棉签和/或29轻轻取出这个组织。小心不要弄伤脊髓在程序的这一部分。不要混淆宽松与骨膜包裹严实硬脑膜。 </li><li>仔细，密封骨的剩余暴露边缘的一个或两个的牙科丙烯酸和氰基丙烯酸酯（ 图2I）的。请特别小心不要让胶水流到暴露脊髓。 </li></ol> 5.密封商会<ol><li>小心地定位在顶板，使得4个插槽排列起来的4个孔上的侧杆和开口覆盖的露出旋人线（ 图1B和2J）。 </li><li>使用珠宝商的螺丝刀，小心启动第一螺丝，稳定螺丝刀的轴与另一对钳。不拧紧螺丝在这个阶段，但将其插入，使最初的几个螺纹接合。对于其他3个螺丝重复此过程。 注:这是非常有用的使用钛或其他非磁化钳帮位置的螺丝在这个阶段。 </li><li>有了所有的4个螺丝，拧紧小的增量每个螺丝交替相同的量，直到螺丝紧固（ 图2K）。需要注意的是它是不寻常的顶板到在此过程中弯曲。小心不要将螺丝拧得太多，因为螺钉头可​​断和过度下行压力将打跑钳和/或导致脊髓损伤。 </li><li>使用有机硅弹性体以填充脊髓和玻璃窗之间的空间。因为它母鹿在固化过程中不是产生醋酸，使用KwikSil品牌弹性体和混合的技巧。适用的有机硅的自由部分，以暴露脊髓。照顾摆脱含气泡从混合头之前施加到脊髓的任何硅氧烷。 </li><li>工作迅速，轻轻按下一个5毫米＃0盖玻片到顶板的插图。施加足够的压力，以逐渐挤压液态弹性体以包围脊髓，但不会导致脊髓压迫（ 图2L）。设置时间弹性大约为90秒，所以要确保盖玻片迅速应用。 </li><li>在一个密封失败的情况下，等到弹性体已成立，轻轻地从用钳子取出植入物，重复5.4 - 5.5。 </li><li>覆盖渗出弹性体的牙科丙烯酸/腈基丙烯酸酯的边缘并附着到周围骨尽可能防止弹性体从移在脊髓的表面上。 </li><li>除去拉钩和拉扯皮肤围绕所述植入物的边缘。使用氰基丙烯酸酯，附着在皮肤到植入物（ 图2M，N）的边缘。 </li><li>插入＃0-80 - 1/4"设置螺钉插入顶板的翼（ 图1C和图2 0）。 </li><li>使用牙科丙烯酸，密封在顶板（ 图2P）的螺旋槽的缝隙。 </li></ol> 6.术后护理<ol><li>在一个大笼子里干净去除麻醉和地方的动物。确保笼足够大，使得该腔室将不会在任何抽丝（如V形浸一水瓶和/或食物）的正常运动的过程中在整个笼中。 注:一个标准的鼠笼是理想的。 </li><li>将笼上加热表面，而动物恢复。不鼠标返回到其他动物的公司，直到它已完全恢复。 </li><li>检查动物BEHAvior在1 - 2小时术后疼痛或痛苦，并植入完整的迹象。 注意:在正常条件下，将动物应该能够走动具有适度扰动运动。 </li><li>继续检查动物，每8 - 12小时，第72小时，体重测量和监控行为。检查动物是在夜间活跃和移动。在地面覆盖的形式湿的食物周星驰与水或营养凝胶，州城颗粒混合，饮水应在地面提供。通过皮下或intraperiotoneal注射额外水合应提供必要的。 </li><li>辖5毫克/千克小鼠酮洛芬和0.2mg / kg的地塞米松小鼠皮下，在24和48小时的时间点，术后。此外，0.005-0.010毫克/ 100g的鼠应当施用每8小时皮下注射72小时。 </li></ol> 7. 体内成像<ol><li>麻醉鼠标下5％异氟烷/氧在感应腔直到呼吸速率减慢到大约1呼吸/秒。 </li><li>鼠标转移到自定义stereotax（ 图1D）与反馈控制的加热垫，插入直肠温度计。减少异氟烷至2％。 </li><li>辖格隆0.05毫克/ 100g的鼠肌内成使用29后肢 - 地下30针和3/10毫升注射器，每肢注射量的递送约50％。 </li><li>辖1毫升/ 100克小鼠的5％葡萄糖（水合）皮下盐水一次每小时。 </li><li>插入螺纹室持有者为用于手术（ 图1F）后持有者。调整高度和扭支柱上的固定螺钉在顶板。 </li><li>与这两个人连接，提高在后持有人持有，直至胸部可以根据灵感自由膨胀。 注:持有人无论是坚定执着和胸部significan的自由扩张TLY减少因呼吸运动伪影。 </li><li>根据需要进行成像。 </li><li>当成像完成后，较低的职位，拧开室支架，移除直肠体温计，放鼠标在加热表面恢复。不鼠标返回到其他动物的公司，直到它已完全恢复。 </li></ol>

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The authors having nothing to disclose, and no competing financial interests.

这里展示的技术允许重复，时间推移， 体内成像背侧鼠标SC出许多周后可操作，而不需要后续的外科手术。此过程是一项重大改进，重复手术成像研究或以上portmortem组织学方法，其中，对细胞动力学信息丢失。我们先前30展示了这种技术的价值为研究SCI病理体内 。 成像的最大纵向范围是由致密，纤维组织在SC的背表面上的生长确定。随着时间的推移，这种增长导致了图象对比度和分辨率的损失。这种增长也被视为在备选外科制剂31。有趣的是，我们已观察到，这种增长可以最小化通过仔细洗涤该背侧的SC表面，以去除血液制品，密封的表面切骨与腈基丙烯酸酯，密封腔室的边缘以及与硅氧烷弹性体，和最小化背SC和盖玻璃之间的间隙空间。 最近，另一种方法类似于我们自己使用聚碳酸酯室已经证明32。使用的聚碳酸酯是有利的，因为它是用X射线和声学成像方式，这是不针对我们的不锈钢零件的情况相兼容。然而，随着三维打印机的现在普遍存在的技术，腔室部分可以从各种各样的材料制成，以适应特定的需要。最近，我们在印刷清晰感光树脂我们所有的零部件。 一个封闭的腔室中的一个关键的缺点是不能施用药物或适于成像的SC 34的外源染料重复剂量。然而，通过利用我们现有系统的机械稳定性，我们已经使用了我们目前室成功LY锚鞘内导管连接到皮下植入注射端口，其中，即使在一个封闭的腔室系统（未发表的作品）允许用于药物递送在多个时间点。另外，由于顶板的模块化性质，该制剂的未来版本将包括一个可重新密封的腔室，以允许两者的治疗剂和荧光标记的重复应用。另外，也可以设想顶板与支架用于记录电极，光学镶件，和端口用于药物递送。这种增加很可能会更具挑战性，以实现使用Fenrich 等 31多个极简系统。总之，我们的腔室提供一个网关平台在其上不同的实验，可以根据。

时间推移在体内显微镜在完整有机体使复杂的生物过程是不可访问的，以传统的单时间点分析，如免疫组织化学的直接可视化。具体地，多光子显微镜（MPM）的1允许成像散射组织，如啮齿动物新皮层，其中，成像高达2,3和超过41毫米已经实现。当与外科制剂5-7，其中单个程序允许光进入大脑数周至数月合并，这些显微镜方法已被用于研究在大脑中的健康和疾病状态8-11的动态过程。此外，协议已经开发12,13提供用于体内成像在清醒（ 即非麻醉）的动物，从而允许在行为分析蜂窝分辨率功能成像。这些协议已被用于comparis相关神经元活动的14项，星形胶质细胞中的钙和麻醉清醒动物信令15，该识别特定任务的神经元集群16，和神经元的能力，以在晶须刺激17辨别对象的位置。 给出了这种方法的潜在澄清健康和病理机制，时间推移的体内成像施加到小鼠脊髓（SC），允许对急性轴突变性的识别（AAD），为疾病机制18。随后的研究调查了周围性病变对背根神经节（DRG）轴突再生19，血管在轴突再生20的作用，趋化胶质响应损伤21，在实验性自身免疫性脑脊髓炎的T细胞迁移（EAE）22，活动的效果小胶质细胞在响应于amyotroph 23,24和25的星形胶质细胞IC侧索硬化症（ALS），STAT-3在SC损伤（SCI）26后的轴突出芽的作用，以及轴突丢失和恢复在EAE 27的一个机构。尽管这些方法的成功，所有这些研究仅限于任一单一成像会话，从而限制了研究，以短期动态，否则需要在每个成像时间点重复该动物的手术开口，限制访问的时间点的数目并增加混杂实验文物的可能性。协议，这些手术已经预先28,29出版。 最近，我们公布的技术30为一种慢性脊髓室，其使能在小鼠的SC在多个周的时间推移的MPM成像而不需要重复手术植入。简单地说，这个手术准备包括执行椎板在较低的胸椎和四部分腔着床。包括室3定制加工的不锈钢​​零件夹紧围绕椎板椎骨和玻璃盖玻片放置在SC和固定用硅氧烷弹性体。这种技术允许用于常规成像出在健康和受伤状态术后多于5周而不需要重复手术。的成像时间点的数目仅由在该动物能耐受麻醉诱导的频率的限制。成像寿命是由致密，纤维组织在SC的表面生长的限制。此外，我们验证了外科植入对运动功能没有长期的效果。 自从我们最初发布，其他方法还使长期成像在SC已经在其他地方31-33描述。该协议证明了我们的程序植入我们开发了脊髓腔。

<table><tbody><tr><td>Vannas scissors</td><td>Fine Science Tools</td><td>15000-04</td><td></td></tr><tr><td>forceps, scissors, scalpel, etc.</td><td>Fine Science Tools</td><td>multiple&amp;nbsp;</td><td></td></tr><tr><td>retractor kit -magnetic fixator</td><td>Fine Science Tools</td><td>18200-02</td><td></td></tr><tr><td>retractor kit -retractor</td><td>Fine Science Tools</td><td>18200-09</td><td>other retractors may also be used</td></tr><tr><td>retractor kit -elastomer</td><td>Fine Science Tools</td><td>18200-07</td><td></td></tr><tr><td>feedback controlled heating blanket</td><td>CWE Inc.</td><td>Model: TC-1000 Mouse&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Part No. 08-13000</td><td></td></tr><tr><td>stereotax</td><td></td><td>see Farrar et al, Nat. Meth., 9, 297, 2012, Supplement (online)</td><td></td></tr><tr><td>spinal chamber</td><td></td><td>see Farrar et al, Nat. Meth., 9, 297, 2012, Supplement (online)</td><td></td></tr><tr><td>spinal chamber holders</td><td></td><td>see Farrar et al, Nat. Meth., 9, 297, 2012, Supplement (online)</td><td></td></tr><tr><td>Cyanoacrylate glue</td><td>Loctite</td><td>Loctite 495</td><td>multiple suppliers</td></tr><tr><td>Teets Cold Cure Coral Powder (dental acrylic powder)</td><td>Teets</td><td>Mfg. Part: 8101</td><td>multiple suppliers</td></tr><tr><td>Teets Cold Cure Liquid (dental acrylic liquid)</td><td>Teets</td><td>Mfg. Part: 8501</td><td>multiple suppliers</td></tr><tr><td>Glycopyrrolate</td><td>MWI Veterinary Supply</td><td>MWI SKU: 29706 (Baxter 1001901602)</td><td></td></tr><tr><td>D-(+)-Glucose</td><td>Sigma-Aldrich</td><td>G5767</td><td></td></tr><tr><td>Bupivacaine</td><td>MWI Veterinary Supply</td><td>MWI SKU: 029841 (Hospira 116301)</td><td></td></tr><tr><td>Ketoprofen</td><td>MWI Veterinary Supply</td><td>MWI SKU: 002800 (Pfizer 2193)</td><td></td></tr><tr><td>Dexamethasone</td><td>MWI Veterinary Supply</td><td>MWI SKU: 501012 (VetOne 1DEX032)</td><td></td></tr><tr><td>KwikSil Elastomer</td><td>World Precision Inc.</td><td>KWIK-SIL</td><td></td></tr><tr><td>KwikSil Mixing Tips</td><td>World Precision Inc.</td><td>KWIKMIX</td><td></td></tr></tbody></table>

a procedure for implanting a spinal chamber for longitudinal in vivo imaging of the mouse spinal cord

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by repeated, time-lapse in vivo imaging in live rodents. These studies were made possible by straightforward surgical procedures, which enabled optical access for a prolonged period of time without repeat surgical procedures. In contrast, analogous studies of the spinal cord have been previously limited to only a few imaging sessions, each of which required an invasive surgery. As previously described, we have developed a spinal chamber that enables continuous optical access for upwards of 8 weeks, preserves mechanical stability of the spinal column, is easily stabilized externally during imaging, and requires only a single surgery. Here, the design of the spinal chamber with its associated surgical implements is reviewed and the surgical procedure is demonstrated in detail. Briefly, this video will demonstrate the preparation of the surgical area and mouse for surgery, exposure of the spinal vertebra and appropriate tissue debridement, the delivery of the implant and vertebral clamping, the completion of the chamber, the removal of the delivery system, sealing of the skin, and finally, post-operative care. The procedure for chronic in vivo imaging using nonlinear microscopy will also be demonstrated. Finally, outcomes, limitations, typical variability, and a guide for troubleshooting are discussed.

按照上述步骤，在手术中的每个阶段应模仿结果在图2中所概述的手术的每个阶段。施加有机硅弹性体，以消除在玻璃下气泡时特别必须小心，或至少确保它们位于感兴趣（ 图2L）的区域的外周。对于有经验的外科医生，手术并发症需要安乐死无论在手术或前24个小时的术后内发生在操作的约20％。这些并发症过度软组织出血或失败到室牢固地固定到脊柱最常出现。 在成功植入室中，该窗口通常保持光学透明的多周（ 图3）。尽管最好的努力，不透明，纤维状，肿瘤生长经常形成在几天内，造成局部obscuri窗口纳克（ 图3C - 女 ，黑色虚线）。荧光结构是很难看到使用2PEF显微镜此纤维组织下方。我们发现结果的双峰分布，具有成功植入窗户大约50％的剩余明确超过5周术后，和大约50％的范围内变得模糊处理1 - 3周。对于保持在视窗的经验只是一个温和的程度的新生血管或硬脑膜（ 图3D - 楼绿星号）上面清晰，大多数地方的窗口，这并不妨碍2PEF成像。我们过去的研究显示，30窗口和相邻椎骨下增加小胶质细胞，但不是星形胶质细胞密度，说明轻度炎症类似于见于颅窗口准备5。 在背根gangl的一个子集表达黄色荧光蛋白（YFP）的转基因小鼠IA（DRG）神经元（YFPH线;杰克逊实验室），轴突分别清晰可辨了几个星期术后（ 图4A和B），并且只要在一个动物4个月（数据未示出）。血管（ 图4A和B）可见通过眼眶注入荧光德克萨斯红标记的葡聚糖进入血浆。小胶质细胞也显现在小鼠中CX3CR1敲入小鼠表达GFP（CX3CR1-GFP;杰克逊实验室）杂交，以该YFPH线（ 图4C）。对比度和分辨率适度恶化随着时间的推移（ 图4C）由于先前30描述的纤维过度生长的形成。我们经常拍摄3个小时在一个会话中没有死亡和频率高达每12小时。会话的持续时间和频率是由动物麻醉诱导，伴随的伦理考虑伴随的疼痛和痛苦到实验动物的耐受性的限制。 &lt;<html> P类="j​​ove_content"FO:保持together.within页="总是"&gt; <img alt="图1" src="/files/ftp_upload/52196/52196fig1highres.jpg" /> 图1。定制手术套件有助于室分娩植入过程中，并在随后的成像会议稳定室，两个边的酒吧和顶板（A）是组装（B）用小螺丝进入椎管腔（C）。一种外科表可扩展的帖子（D）允许侧酒吧（E）采用控股引脚椎骨的交付和夹紧。随后的成像会议改用内螺纹的帖子通过对植入物（F）的机翼固定螺丝连接到室内。面板这个数字B已经被修改法拉等人 ， 自然方法 ，2012 30与自然出版集团的许可。96 / 52196fig1large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图2" src="/files/ftp_upload/52196/52196fig2highres.jpg" /> 图2.背椎板执行和慢性成像室被植入以提供光接入到脊髓。该过程在蒙太奇说明。首先，皮肤（板A，B，协议部分3.1 - 3.4）和肌肉（C; 3.5）缩回，露出三个基本椎骨。所述组织覆盖这三个椎骨被除去（D，E，F; 3.6 - 3.10）和横向进程刮清洁它们将被锁定在两侧（G，H; 3.12）之前。小心，中央椎骨的背薄片是去除与骨的边缘密封（ 我 </strong&gt;的; 4. 6）。顶板应用（J; 5.1）和螺钉固定到位（K; 5.3）用密封硅橡胶和玻璃盖（L; 5.5）室之前，确保室。后输送销被除去（M; 5.8），皮肤被粘在腔室的侧面（M，N; 5.8）和固定螺钉插入到翼（O; 5.9）。最后，螺丝槽密封用牙科丙烯酸（P 5.10），和鼠标被放置在一个干净的笼子里恢复<a href="https://www.jove.com/files/ftp_upload/52196/52196fig2large.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/52196/52196fig3highres.jpg" /> 图3.慢性脊髓腔保持光学透明OV呃多个星期。白色光图像中的脊髓的，范围从几分钟（A）至术后3周（F）中 。血管标志是可识别的各时间点（黄色箭头）。几乎没有变化被认为在1天术后（B）中 。致密的纤维增生（C - 女 ，围边虚线上右下）的存在早三天结果在成像区域的局部模糊处理。轻度新生血管（D，E，F，绿星号）也存在，但并不能掩盖在白光或2PEF成像原有血管。 <a href="https://www.jove.com/files/ftp_upload/52196/52196fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/52196/52196fig4highres.jpg" /><p类="jove_content"&gt; 图4.慢性脊髓腔允许重复多光子成像，而不需要重复手术。在脊髓的背funiculi表达YFP的背根神经节轴突（绿色）的一个子集的转基因小鼠反复成像线和血管（红色）标记血管内注射染料（A，B）。小胶质细胞（紫红色）的活性，也可以随时间跟踪Thy1肾炎-YFP / CX3CR1-GFP的双转基因小鼠（C）的 。虽然轴突和小胶质细胞保持可见，对比度和分辨率小幅下降时间（C）。 <a href="https://www.jove.com/files/ftp_upload/52196/52196fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord at JoVE.com

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

在这段视频中，我们描述了植入一种慢性的光学成像室在活老鼠的脊髓背侧的过程。室，外科手术，慢性成像的审查和论证。

一个程序，植入脊柱商会纵<em&gt;在体内</em&gt;鼠标脊髓成像

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by ...

a-procedure-for-implanting-spinal-chamber-for-longitudinal-vivo

Nanyang Technological University

Research

JoVE Journal

Neuroscience

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

14.1K 次观看.  Cornell University. 在这段视频中，我们描述了植入一种慢性的光学成像室在活老鼠的脊髓背侧的过程。室，外科手术，慢性成像的审查和论证。 

视频: A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has significantly broadened our knowledge of physiological and pathological processes in numerous tissues in vivo 4-7. In studies of the central nervous system (CNS), there has been a broad application of in vivo imaging in the brain, which has produced a plethora of novel and often unexpected findings about the behavior of cells such as neurons, astrocytes, microglia, under physiological or pathological conditions 8-17. However, mostly technical complications have limited the implementation of in vivo imaging in studies of the living mouse spinal cord. In particular, the anatomical proximity of the spinal cord to the lungs and heart generates significant movement artifact that makes imaging the living spinal cord a challenging task.
We developed a novel method that overcomes the inherent limitations of spinal cord imaging by stabilizing the spinal column, reducing respiratory-induced movements and thereby facilitating the use of two-photon microscopy to image the mouse spinal cord in vivo. This is achieved by combining a customized spinal stabilization device with a method of deep anesthesia, resulting in a significant reduction of respiratory-induced movements. This video protocol shows how to expose a small area of the living spinal cord that can be maintained under stable physiological conditions over extended periods of time by keeping tissue injury and bleeding to a minimum. Representative raw images acquired in vivo detail in high resolution the close relationship between microglia and the vasculature. A timelapse sequence shows the dynamic behavior of microglial processes in the living mouse spinal cord. Moreover, a continuous scan of the same z-frame demonstrates the outstanding stability that this method can achieve to generate stacks of images and/or timelapse movies that do not require image alignment post-acquisition. Finally, we show how this method can be used to revisit and reimage the same area of the spinal cord at later timepoints, allowing for longitudinal studies of ongoing physiological or pathological processes in vivo.

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

A minimally invasive protocol to stabilize the mouse spinal column and perform repetitive in vivo spinal cord imaging using two-photon microscopy is described. This method combines a spinal stabilization device and an anesthetic regimen to minimize respiratory-induced movements and produce raw imaging data that require no alignment or other post-processing.

在体内脊髓的鼠标线使用双光子显微镜成像

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has ...

科研

神经科学

Surgical Technique for Spinal Cord Delivery of Therapies: Demonstration of Procedure in Gottingen Minipigs

This is a compact visual description of a combination of surgical technique and device for the delivery of (gene and cell) therapies into the spinal cord. While the technique is demonstrated in the animal, the procedure is FDA-approved and currently being used for stem cell transplantation into the spinal cords of patients with ALS. While the FDA has recognized proof-of-principle data on therapeutic efficacy in highly characterized rodent models, the use of large animals is considered critical for validating the combination of a surgical procedure, a device, and the safety of a final therapy for human use. The size, anatomy, and general vulnerability of the spine and spinal cord of the swine are recognized to better model the human. Moreover, the surgical process of exposing and manipulating the spinal cord as well as closing the wound in the pig is virtually indistinguishable from the human. We believe that the healthy pig model represents a critical first step in the study of procedural safety.

Short visual description of the surgical technique and device used for the delivery of (gene and cell) therapies into the spinal cord. The technique is demonstrated in the animal but is entirely translatable and currently being used for human application.

外科技术治疗脊髓交付：在哥廷根小型猪演示程序

This is a  compact visual description of a combination of surgical technique and device  for the  delivery of (gene and cell) therapies into the spinal ...

医学

Implanting Glass Spinal Cord Windows in Adult Mice with Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) in adult rodents is the standard experimental model for studying autonomic demyelinating diseases such as multiple sclerosis. Here we present a low-cost and reproducible glass window implantation protocol that is suitable for intravital microscopy and studying the dynamics of spinal cord cytoarchitecture with subcellular resolution in live adult mice with EAE. Briefly, we surgically expose the vertebrae T12-L2 and construct a chamber around the exposed vertebrae using a combination of cyanoacrylate and dental cement. A laminectomy is performed from T13 to L1, and a thin layer of transparent silicone elastomer is applied to the dorsal surface of the exposed spinal cord. A modified glass cover slip is implanted over the exposed spinal cord taking care that the glass does not directly contact the spinal cord. To reduce the infiltration of inflammatory cells between the window and spinal cord, anti-inflammatory treatment is administered every 2 days (as recommended by ethics committee) for the first 10 days after implantation. EAE is induced only 2-3 weeks after the cessation of anti-inflammatory treatment.
Using this approach we successfully induced EAE in 87% of animals with implanted windows and, using Thy1-CFP-23 mice (blue axons in dorsal spinal cord), quantified axonal loss throughout EAE progression. Taken together, this protocol may be useful for studying the recruitment of various cell populations as well as their interaction dynamics, with subcellular resolution and for extended periods of time. This intravital imaging modality represents a valuable tool for developing therapeutic strategies to treat autoimmune demyelinating diseases such as EAE.

We describe a method for implanting and maintaining glass windows over the exposed spinal cords of adult mice for studying experimental autoimmune encephalomyelitis. These windows provide chronic optical access to the spinal cord for monitoring cell dynamics at the subcellular level in live animals using two-photon microscopy.

玻璃植入脊髓的Windows在成年小鼠实验性自身免疫性脑脊髓炎

Experimental autoimmune encephalomyelitis (EAE) in adult rodents is the standard experimental model for studying autonomic demyelinating diseases such as ...

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability.

一个<em&gt;离体</em&gt;激光诱导脊髓损伤模型，以评估轴索变性的机制在实时

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

Two-photon Imaging of Cellular Dynamics in the Mouse Spinal Cord

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for live-cell imaging in the murine spinal cord. This technique is uniquely suited to analyze neural precursor cell (NPC) dynamics following transplantation into spinal cords undergoing neuroinflammatory demyelinating disorders. NPCs migrate to sites of axonal damage, proliferate, differentiate into oligodendrocytes, and participate in direct remyelination. NPCs are thereby a promising therapeutic treatment to ameliorate chronic demyelinating diseases. Because transplanted NPCs migrate to the damaged areas on the ventral side of the spinal cord, traditional intravital 2P imaging is impossible, and only information on static interactions was previously available using histochemical staining approaches. Although this method was generated to image transplanted NPCs in the ventral spinal cord, it can be applied to numerous studies of transplanted and endogenous cells throughout the entire spinal cord. In this article, we demonstrate the preparation and imaging of a spinal cord with enhanced yellow fluorescent protein-expressing axons and enhanced green fluorescent protein-expressing transplanted NPCs.

A new ex vivo preparation for imaging the mouse spinal cord. This protocol allows for two-photon imaging of live cellular interactions throughout the spinal cord.

细胞动力学在小鼠脊髓双光子成像

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for ...

Experimental Demyelination and Remyelination of Murine Spinal Cord by Focal Injection of Lysolecithin

Multiple sclerosis is an inflammatory demyelinating disease of the central nervous system characterized by plaque formation containing lost oligodendrocytes, myelin, axons, and neurons. Remyelination is an endogenous repair mechanism whereby new myelin is produced subsequent to proliferation, recruitment, and differentiation of oligodendrocyte precursor cells into myelin-forming oligodendrocytes, and is necessary to protect axons from further damage. Currently, all therapeutics for the treatment of multiple sclerosis target the aberrant immune component of the disease, which reduce inflammatory relapses but do not prevent progression to irreversible neurological decline. It is therefore imperative that remyelination-promoting strategies be developed which may delay disease progression and perhaps reverse neurological symptoms. Several animal models of demyelination exist, including experimental autoimmune encephalomyelitis and curprizone; however, there are limitations in their use for studying remyelination. A more robust approach is the focal injection of toxins into the central nervous system, including the detergent lysolecithin into the spinal cord white matter of rodents. In this protocol, we demonstrate that the surgical procedure involved in injecting lysolecithin into the ventral white matter of mice is fast, cost-effective, and requires no additional materials than those commercially available. This procedure is important not only for studying the normal events involved in the remyelination process, but also as a pre-clinical tool for screening candidate remyelination-promoting therapeutics.

Demyelinating diseases can be modeled in animals by focal application of lysolecithin into the CNS. A single injection of lysolecithin into mouse spinal cord produces a lesion that spontaneously repairs over time. The goal is to study factors involved in de- and remyelination, and to test agents for enhancing repair.

实验脱髓鞘和小鼠脊髓髓鞘再生由溶血卵磷脂的焦点注射

Multiple sclerosis is an inflammatory demyelinating disease of the central nervous system characterized by plaque formation containing lost ...

Laminectomy and Spinal Cord Window Implantation in the Mouse

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

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

拉米内切手术和脊髓窗口植入小鼠

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

In vivo Imaging of Biological Tissues with Combined Two-Photon Fluorescence and Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy enables label-free imaging of the biological tissues in its natural microenvironment based on intrinsic molecular vibration, thus providing a perfect tool for in vivo study of biological processes at subcellular resolution. By integrating two-photon excited fluorescence (TPEF) imaging into the SRS microscope, the dual-modal in vivo imaging of tissues can acquire critical biochemical and biophysical information from multiple perspectives which helps understand the dynamic processes involved in cellular metabolism, immune response and tissue remodeling, etc. In this video protocol, the setup of a TPEF-SRS microscope system as well as the in vivo imaging method of the animal spinal cord is introduced. The spinal cord, as part of the central nervous system, plays a critical role in the communication between the brain and peripheral nervous system. Myelin sheath, abundant in phospholipids, surrounds and insulates the axon to permit saltatory conduction of action potentials. In vivo imaging of myelin sheaths in the spinal cord is important to study the progression of neurodegenerative diseases and spinal cord injury. The protocol also describes animal preparation and in vivo TPEF-SRS imaging methods to acquire high-resolution biological images.

In vivo Imaging of Biological Tissues with Combined Two-Photon Fluorescence and Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy allows label-free imaging of biomolecules based on their intrinsic vibration of specific chemical bonds. In this protocol, the instrumental setup of an integrated SRS and two-photon fluorescence microscope is described to visualize cellular structures in the spinal cord of live mice.

体内 使用双光子荧光和受激拉曼散射显微镜对生物组织进行成像

Stimulated Raman scattering (SRS) microscopy enables label-free imaging of the biological tissues in its natural microenvironment based on intrinsic ...

生物工程

In Vivo Mouse Model of Spinal Implant Infection

Spine implant infections portend poor outcomes as diagnosis is challenging and surgical eradication is at odds with mechanical spinal stability. The purpose of this method is to describe a novel mouse model of spinal implant infection (SII) that was created to provide an inexpensive, rapid, and accurate in vivo tool to test potential therapeutics and treatment strategies for spinal implant infections.
In this method, we present a model of posterior-approach spinal surgery in which a stainless-steel k-wire is transfixed into the L4 spinous process of 12-week old C57BL/6J wild-type mice and inoculated with 1 x 103 CFU of a bioluminescent strain of Staphylococcus aureus Xen36 bacteria. Mice are then longitudinally imaged for bioluminescence in vivo on post-operative days 0, 1, 3, 5, 7, 10, 14, 18, 21, 25, 28, and 35. Bioluminescence imaging (BLI) signals from a standardized field of view are quantified to measure in vivo bacterial burden.
To quantify bacteria adhering to implants and peri-implant tissue, mice are euthanized and the implant and surrounding soft tissue are harvested. Bacteria are detached from the implant by sonication, cultured overnight and then colony forming units (CFUs) are counted. The results acquired from this method include longitudinal bacterial counts as measured by in vivo S. aureus bioluminescence (mean maximum flux) and CFU counts following euthanasia.
While prior animal models of instrumented spine infection have involved invasive, ex vivo tissue analysis, the mouse model of SII presented in this paper leverages noninvasive, real time in vivo optical imaging of bioluminescent bacteria to replace static tissue study. Applications of the model are broad and may include utilizing alternative bioluminescent bacterial strains, incorporating other types of genetically engineered mice to contemporaneously study host immune response, and evaluating current or investigating new diagnostic and therapeutic modalities such as antibiotics or implant coatings.

The protocol describes a novel in vivo mouse model of spinal implant infection where a stainless-steel k-wire implant is infected with bioluminescent Staphylococcus aureus Xen36. Bacterial burden is monitored longitudinally with bioluminescent imaging and confirmed with colony forming unit counts after euthanasia.

脊柱植入物感染的体内小鼠模型

Spine implant infections portend poor outcomes as diagnosis is challenging and surgical eradication is at odds with mechanical spinal stability. The ...

啮齿动物脊髓手术和光学柄植入术

Implantation of Optoelectronic Devices in the Rodent Spinal Cord

Neuromodulation can provide diagnostic, modulatory, and therapeutic applications. While extensive work has been conducted in the brain, modulation of the spinal cord remains relatively unexplored. The inherently delicate and mobile spinal cord tissue imposes constraints that make the precise implantation of neural probes challenging. Despite recent advances in neuromodulation devices, particularly flexible bioelectronics, opportunities to expand their use in the spinal cord have been limited by the surgical complexities of device implantation. Here, we provide a series of surgical protocols tailored specifically for the implantation of a custom-made optoelectronic device that interfaces with the spinal cord in rodents. The steps to place and anchor an optical shank on a specific segment of the spinal cord via two different surgical implantation methods are detailed here. These methods are optimized for a diverse range of devices and applications, which may or may not require direct contact with the spinal cord for optical stimulation. To elucidate the methodology, the vertebral anatomy is referenced first to identify prominent landmarks before making a skin incision. The surgical steps to secure an optical shank over the cervical spine in rodents are demonstrated. Procedures are then outlined for securing the optoelectronic device connected to the optical shank in a subcutaneous space away from the spinal cord, minimizing unnecessary direct contact. Behavioral studies comparing animals receiving the implants to those undergoing sham surgeries indicate that the optical shanks did not adversely affect hindlimb or forelimb function seven days post-implantation. The present work broadens the neuromodulation toolkit for use in future studies aimed at investigating various spinal cord interventions.

作者聚焦：在啮齿动物脊髓中植入和保护神经探针的创新方法

This protocol details a surgical procedure for performing spinal cord surgery and for implanting and securing an optical shank over the spinal cord in rodents.

在啮齿动物脊髓中植入光电器件

Neuromodulation can provide diagnostic, modulatory, and therapeutic applications. While extensive work has been conducted in the brain, modulation of the ...

一个程序，植入脊柱商会纵

摘要

探索更多视频

此视频中的章节

在体内脊髓的鼠标线使用双光子显微镜成像

外科技术治疗脊髓交付：在哥廷根小型猪演示程序

玻璃植入脊髓的Windows在成年小鼠实验性自身免疫性脑脊髓炎

一个

细胞动力学在小鼠脊髓双光子成像

实验脱髓鞘和小鼠脊髓髓鞘再生由溶血卵磷脂的焦点注射

拉米内切手术和脊髓窗口植入小鼠

体内使用双光子荧光和受激拉曼散射显微镜对生物组织进行成像

脊柱植入物感染的体内小鼠模型

在啮齿动物脊髓中植入光电器件

一个程序，植入脊柱商会纵

摘要

探索更多视频

此视频中的章节

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