S.S. 部分由不列颠哥伦比亚大学的四年博士奖学金资助。AM 部分由加拿大健康研究所 （CIHR） 的加拿大研究生奖学金 - 硕士学位支持。D.S. 感谢 Michael Smith Health Research British Columbia Scholar Award 的资助。这项工作由加拿大政府的新前沿研究基金 - 转型 （NFRFT-2020-00238） 部分资助。 图 1 中的原理图是使用 Biorender.com 生成的，3D 模型是在 sketchfab.com 的许可下获得的。

所有程序均根据加拿大动物护理委员会的指导方针进行，并由不列颠哥伦比亚大学动物护理委员会监督。将体重 350-450 g、年龄 6-8 个月的雌性 Long-Evans 大鼠分组饲养 （21 °C;12 h：12 h 光周期），并在手术前后 随意 给予标准啮齿动物饮食。用于本研究的试剂和设备的详细信息列在 材料表中。
1. 术前准备
<ol><li>使用高压灭菌器对所有手术工具进行消毒。</li>
	<li>用异氟醚（5% 用于诱导和 2% 用于维持）以 1 L/min 的流速输送在氧气中麻醉动物。</li>
	<li>将动物从感应室转移到加热垫上，并立即连接异氟醚鼻锥。通过确保双腿完全失去脚趾捏反射，验证动物是否处于手术麻醉平面下。</li>
	<li>从耳朵底部开始剃掉大鼠的背部，如图 2A 所示。</li>
	<li>在眼睛上涂抹大量无菌眼药膏，皮下注射丁丙诺啡（稀释至 0.03 mg/kg）和 10 mL 乳酸林格（加热至体温）。</li>
	<li>用抗菌手术擦洗膏（洗必泰），然后用异丙醇以圆周运动擦拭剃光区域，从剃光区域的中心开始，加宽圆直径。再重复肥皂/酒精过程两次。</li>
	<li>将动物固定在立体定位框架中，使用润滑的耳杆定位头部以保持稳定性（图 2A）。 注意：在整个过程中，始终如一地提供热支持，通过脚趾捏反射验证麻醉深度，并监测生命体征。</li>
	<li>将无菌手术单放在动物顶部。</li>
</ol>2. 颈脊髓暴露
<ol><li>使用无菌镊子，首先触诊颅底。感觉有一个突出的棘突，向颅底附近尾部延伸;这是 C214 （图 1B 和 图 2B）。</li>
	<li>继续对 C2 尾部进行触诊，发现一个明显尖锐的棘突，可识别为 T215 （图 1B 和 图 2C）。</li>
	<li>使用手术刀在皮肤上切开一个切口，从 C2 开始，向尾部延伸约 1.5 厘米（图 2D）。 注意：不同大小的动物的切口大小可能会有所不同。确保提前确定解剖标志并相应地进行。</li>
	<li>用手术刀小心地切开皮下脂肪层，露出完整的底层背肌组织。</li>
	<li>一旦背肌暴露出来，使用两个 Adson 镊子将它们从中线拉开进行钝解剖（图 2E）。 注意：重要的是进行钝性解剖（将肌肉纤维拉开）而不是切割肌肉以尽量减少出血。背侧肌肉组织的充分暴露应露出球形肌肉（图 2G）。这块肌肉完全覆盖 C2 部分覆盖 C3。</li>
	<li>使用咬合钳和无菌镊子，立即从尾部提起一块皮肤，直至切口区域。使用 rongeurs 创建一个小的皮下袋 - 这将是设备的位置（图 2F）。 注意：皮下袋应大于设备本身。建议外科医生重新定位自己以面向动物前方，以提高打开皮下袋时的控制和可见性。</li>
	<li>放置一个牵开器以露出脊柱（图 2G）。</li>
	<li>使用 rongeurs，去除覆盖椎骨的任何剩余肌肉或组织，并开始识别脊髓节段。紧邻球形肌肉尾部的是 C4，其次是 C5 和 C6（图 2G-I）。完成后，用无菌盐水冲洗手术区域并用无菌纱布擦干。 注意：球形肌肉完全包裹 C2 并部分延伸到 C3 上。直接位于其尾部的脊髓段，接触最少，称为 C4。</li>
	<li>根据探针的预期目的进行椎板切除术。<ol><li>对于椎板下的探针，在 C5 和 C6 进行横向椎板切除术，在椎板中创建一个侧向开口，以备将来放置探针（图 2H， 补充图 1A）。</li>
		<li>对于椎板上的探针，进行 C5 的内侧椎板切除术，确保不去除棘突的外侧 - 仅暴露用于探针放置的内侧通路（图 2I， 补充图 1B）。</li>
		<li>椎板切除术后，用无菌盐水冲洗该区域并用无菌纱布擦干以去除任何骨碎屑。 注意：进行椎板切除术时，将咬骨向上推到骨骼上并避免任何向下运动以防止损伤脊髓至关重要。</li>
	</ol></li>
</ol>3. 设备的硬膜外放置
<ol><li>用塑料尖端无菌镊子握住无菌装置（图 3A），并将其驱动到步骤 2.9 前面制作的皮下开口内。 注意：为避免使用非无菌手套/工具接触设备以保持设备无菌至关重要。使用指定的无菌镊子在皮下放置和定位装置。</li>
	<li>将设备缝合或粘合到相邻的肌肉层以确保其安全（图 3B）。 注意： 如果将设备缝合到肌肉上，请使用不可吸收的缝合线。否则，该装置在体内缝合吸收后容易移动。如果使用胶水，请确保胶水/胶粘剂的长期稳定性和生物相容性。避免将设备固定在皮下脂肪/脂肪层，以确保可靠的锚定点。<ol><li>在继续执行此步骤之前，请规划设备主体位置。由于该装置将被永久固定，因此如果装置距离脊髓所需水平太近或太远，则可能会出现探针放置问题（步骤 3.4）。</li>
	</ol></li>
	<li>将牵开器放在脊髓周围，并打开一个合适的窗口，将探头放在脊髓上。</li>
	<li>探头放置：<ol><li>探针放置在叶片下：使用塑料尖端镊子小心地将探针滑过步骤 2.9.1 中制作的侧向通道，将其插入（C5 和 C6）叶片下方（图 3D）。</li>
		<li>探头放置在脊髓上：<ol><li>使用塑料尖端镊子调整探针以对齐并将探针尖端放在步骤 2.9.2 中在 C5 创建的内侧窗口的顶部（图 3C）。</li>
		</ol></li>
	</ol></li>
	<li>在一个无菌的、小的、最好是陶瓷的容器中，混合一勺牙科水泥粉、3 滴高科技润滑脂和一滴催化剂来制备水泥。与无菌牙签混合，直到达到粘稠度。 注意：强烈建议一名不进行手术的人准备粘接剂，以便外科医生可以在涂抹粘接剂之前立即将探头固定到位并干燥所需的区域。此步骤对时间敏感。确保外科医生在准备粘接剂之前定位探头。一旦使用催化剂，如果延迟，水泥会变得太稠，无法有效地与骨骼结合。手术前使用粘接剂进行练习，以确保在将其放置在探针/骨骼上之前达到适当的稠度。</li>
	<li>将椎骨上预期的粘合区域完全干燥，以形成可靠的锚点。在探针尖端涂抹 1-2 滴牙科水泥，应将其放置在预期的椎骨水平顶部，在这种情况下为 C4（图 3C，D）。 注意：在粘合之前，椎骨必须尽可能干燥。否则，水泥将无法粘附，探头将无法固定。</li>
	<li>暂停 30 秒并轻轻触摸水泥以验证其是否已固化。</li>
	<li>如果水泥没有完全固化，请再等待 20 秒，然后重新涂抹新鲜制成的水泥，直到探针牢固地固定在骨头上。 注意：当粘接剂摸起来变硬时，它已经正确粘合。 注意： 在粘接剂太稠时涂抹水泥可能会导致其渗入椎骨之间的脊髓组织。如果发生这种情况，请等待大约 10 秒，让水泥变稠至更像牙龈的稠度，然后轻轻使用镊子去除与脊髓接触的水泥。</li>
</ol>4. 术后程序
<ol><li>手术完成后，使用 5-0 维克缝合线缝合切口部位。轻轻地将动物从立体定位装置中取出，并将其转移到加热的恢复室中。在手术后的最初 3-5 天内，为动物提供柔软、湿润的食物、零食和水凝胶水。</li>
	<li>在手术后第一周每天两次彻底监测动物。在接下来的 2 天内，每天两次给予丁丙诺啡和温热的乳酸林格氏注射液，如果疼痛迹象持续存在，则延长注射时间。继续每日检查，直到动物没有临床健康问题。此后，每周至少检查一次动物。</li>
	<li>（可选）在手术和切口闭合后勾勒出设备在皮肤上的周边，以直观地指示设备在体内的位置（图 4A）。 注意：当设备需要外部访问时，此步骤特别有用，例如需要通过天线耦合至关重要的外部发射器进行无线供电的设备，或必须通过皮肤重新填充的给药泵 16,17,18。</li>
</ol>

啮齿动物脊髓手术和光学柄植入术

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作者没有利益冲突。

脊髓的神经调控和治疗干预通常需要将探针放置在精确的目标节段3、4、7、13 中。鉴于脊髓固有的活动性，必须可靠地固定探针才能进行慢性研究。根据具体应用，控制探针是否与脊髓进行物理接触，或者是否可以减少接触以尽可能减轻炎症组织反应可能很重要。因此，描述了两种方法中每一种的手术步骤。该协议特别详细说明了如何在 C5 的脊髓颈段放置探针。尽管如此，使用所描述的脊髓 T2 或 T10 标志，探针可以类似地放置在胸部或腰椎区域的精确位置，方法是分别从 T2 或 T10 开始计数椎骨，一旦它们暴露。此外，为了最大限度地减少脊髓组织损伤，我们将设备主体固定在远离脊髓的皮下空间，与连接的探针相比，它通常更大、更坚硬。
植入与探头耦合的设备有一些关键点。首先，在固结探头之前确定设备主体的位置至关重要。这确保了探针尖端和设备主体之间的距离得到优化，以减少探针上的张力，并避免额外的探针长度，例如，这会导致探针扭曲或位移。从本质上讲，目标是确保探针的长度与从放置设备主体的皮下空间到探针粘合的目标脊髓区域的距离相似。通过进行测试各种探针长度的终端手术程序，可以确定目标段的最佳尺寸。
为了保持无菌，应小心处理装置，以防止在插入皮下袋时与皮肤外层接触。这种接触会损害设备的无菌性，可能导致术后感染。此外，重要的是在用镊子握住设备时尽量减少施加在设备上的力，以防止损坏其涂层，该涂层通常是一层薄的保护、绝缘和无菌层20,21。去除涂层可能会大大缩短设备的使用寿命，例如，缩短电路、对动物造成电击和/或引起体内炎症反应。用塑料镊子处理装置可能有助于减少此类并发症。
将装置缝合到软组织时，重要的是要避免缝合到皮下脂肪组织。正如在初步试验中观察到的那样，脂肪层不是缝合线的可靠锚定点，因为它们容易破裂。相反，使用不可吸收的缝合线将装置主体缝合到皮下间隙的相邻肌肉层上，以将装置永久放置在体内。另一方面，当将探针固定到棘突上时，重要的是要确保在涂抹水泥之前将探针固定到的部位是干燥的。湿骨/探针会延长固化时间，并可能导致工艺完全失败。
在植入手术之前，需要仔细解决一些与植入式装置相关的关键考虑因素。（1） 器件的电活性部分必须由绝缘钝化层封装。钝化层中的任何剥夺都可能导致器件功能故障。（2） 植入式设备必须根据设施动物协议进行彻底消毒。（3） 设备与神经探针或刺激柄之间的连接处必须牢固形成。由于动物的不断运动，连接将承受可重复的机械应力。（4） 连接到设备的神经探针或刺激柄必须足够灵活且可拉伸，以避免在不同点卡住。
所描述的方案可以扩展到不同大小的动物模型中的植入装置。在确定解剖标志后，可以系统地定制所描述的手术方法，以将任何神经探针或刺激小腿固定在脊髓的目标节段，并植入其相关的控制模块。但是，根据应用的不同，不同的设备可能具有与本文中植入的设备不同的尺寸、材料和厚度;例如，连接到外部控制模块的设备需要额外的考虑。此外，必须注意的是，虽然该协议是为光遗传学刺激量身定制的，但其他神经调节应用，例如药物输送或电刺激/记录，需要略有不同的外科手术。具体来说，这些应用需要硬膜下植入，以确保与硬脑膜下方的脊髓直接接触7。然而，对于光遗传学来说，与组织的亲密接触通常是不必要的，因为啮齿动物硬脑膜不会显着阻碍光的穿透，这使得光源能够硬膜外放置10。

脊髓促进一系列基本的中枢神经系统功能，从协调运动行为到调节稳态过程，如呼吸 1,2。阐明跨脊髓的复杂电路网络的作用需要接口，无论是电刺激、记录、药物输送还是对目标区域的光刺激 3,4,5,6。尽管已经开发了能够进行此类审讯的设备 7,8,9,10,11，但它们在脊髓中的慢性植入需要专门的手术技术 4。特别是，脊髓和相关椎骨对自然运动（如伸展和弯曲）引起的机械变形的敏感性增加 8,12,13。脊髓的这些独特特性使得确保植入的探针在很长一段时间内保持稳定、功能和固定在特定节段本质上具有挑战性。
在此，描述了一种手术方案，用于将光学柄插入和固定脊髓的目标段（图 1A）。由于与颈部区域接触尤其被证明会带来独特的挑战9，因此在 C5 颈部区域专门展示了植入步骤。据推测，颈椎的复杂性源于其更深的位置和丰富的肌肉组织，这一特征在脊髓的其余部分并不那么突出。无论如何，本协议中概述的程序旨在适用于各种脊髓区域的手术。提供了逐步说明，以使用可从皮肤上识别的明显解剖“标志”来定位和识别脊髓节段（图 1B）。然后，该方案阐明了两种外科植入技术：一种是为需要与脊髓直接接触的探针量身定制的，另一种是为可能不需要直接接触的探针量身定制的。所描述的步骤旨在由任何受过啮齿动物生存手术培训的研究人员复制。
该协议包括在 C5 颈椎水平上植入带有柔性光学柄的光电设备 （18 mm x 13 mm） 的分步说明。植入式装置固定在 C5 尾部的皮下，由一个微型发光二极管 （μLED） 指示器组成，当脊髓光刺激发生时，该指示器会亮起，提供设备功能的实时反馈。在接受植入物的啮齿动物身上评估植入的光学柄对自然运动功能的影响，并与接受假手术的啮齿动物进行比较。结果表明，探针在植入后 7 天不会对动物的自然后肢和前肢功能产生不利影响。

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			<td>Provided at 5% for induction and 2% for mainentance through precision vaporizer&#160;</td>
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			<td>11.38</td>
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			<td>Radial Magnets, Inc.</td>
			<td>0.53</td>
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			<td>761036</td>
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			<td>Fine Science Tools</td>
			<td>10015-00</td>
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implantation of optoelectronic devices in the rodent spinal cord

神经调控可以提供诊断、调节和治疗应用。虽然已经在大脑中进行了广泛的工作，但脊髓的调节仍然相对未得到探索。脊髓组织本身脆弱且可移动，因此存在一些限制，这使得神经探针的精确植入具有挑战性。尽管神经调控装置，特别是柔性生物电子学最近取得了进展，但由于装置植入的手术复杂性，扩大它们在脊髓中的使用的机会受到限制。在这里，我们提供了一系列专为植入与啮齿动物脊髓接口的定制光电设备量身定制的手术方案。此处详细介绍了通过两种不同的手术植入方法将光学柄放置和锚定在脊髓的特定节段上的步骤。这些方法针对各种设备和应用进行了优化，这些设备和应用可能需要也可能不需要直接接触脊髓进行光学刺激。为了阐明该方法，首先参考椎体解剖结构以确定突出的标志，然后再进行皮肤切口。演示了将光学柄固定在啮齿动物颈椎上的手术步骤。然后概述了将连接到光学柄的光电器件固定在远离脊髓的皮下空间的程序，从而最大限度地减少不必要的直接接触。将接受植入物的动物与接受假手术的动物进行比较的行为研究表明，光学柄在植入后 7 天不会对后肢或前肢功能产生不利影响。目前的工作拓宽了神经调控工具包，用于未来旨在调查各种脊髓干预的研究。

将 补充图 2 中所示的具有详细功能图的光电器件植入四只 Long Evans 大鼠体内。 补充图 3 显示了准备植入的最终光电器件。其他 3 只动物接受了假手术，其中涉及 C5 的内侧椎板切除术，无需植入装置。光电器件由一个尖端带有嵌入式 μLED 的柔性探针组成，该探针由集成的 LED 驱动器激活。LED 驱动器由具有可编程固件的微控制器控制。它还包括一个装置主体，该装置主体被缝合到皮肤下的肌肉层。使用化学气相沉积 （CVD） 在整个器件上沉积一层聚对二甲苯 C （~10 μm）。第二层聚二甲基硅氧烷 （PDMS） （~800 μm） 覆盖光电器件主体（补充图 3），与组织形成柔软的界面。探针尖端固定在 C4 处，μLED 悬停在 C5 上。器件上使用了 μLED 指示灯（从皮肤下可以看到其光线），该指示灯与光学柄的 μLED 同时亮起，用于实时验证器件功能。手术后对动物进行了 7 天的监测，以确认它们的性能随时间推移的持续可靠性（图 4B）。
使用 Martinez 旷场运动评定量表19 评估动物的运动功能。为了评估旷场行为，两名不了解治疗组的训练有素的观察员在手术前以及手术后第 3 天、第 5 天和第 7 天进行了测试。数据收集后，进行 Mann-Whitney U 检验以确定植入物组和假手术组之间前肢和后肢评分在每个时间点的差异。我们的分析表明，到第 7 天，种植体组和假手术组的前肢功能评分相似（图 5A）。同样，在所有时间点的后肢评分方面，两组之间没有统计学上的显着差异（图 5B）。
植入后 7 天进行尸检，以确认探针和设备主体是否保持在原位。未发现缝合线或装置的明显脱落。此外，拉动设备主体不会导致其与组织分离（补充图 4A）。然后将先前解剖和缝合的肌肉暴露在脊髓上，并确认探针仍然牢固地粘合在脊髓上（补充 图 4B）。与设备本体类似，探针头连续向后拉靠着胶合点，以评估其与探针层机械接头的附着。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66992/66992fig01.jpg"> 图 1：装置植入和解剖标志的示意图概述。 （A） 演示探针在脊髓上的位置和设备在皮下的位置。（B） 3D 模型，指示用于确定脊髓水平的标志。显示了 C2、T2 和 T10 棘突以供参考。较深的颜色表示相应的级别。 <a href="https://www.jove.com/files/ftp_upload/66992/66992fig01large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66992/66992fig02.jpg"> 图 2：暴露脊髓并准备皮下袋。 （A） 立体定位装置位于动物身上。（B） C2 棘突和 （C） T2 棘突 通过 触诊确定。（D） 穿过皮肤和皮下脂肪层切开，以暴露颈椎水平感兴趣点的背肌组织。（E） 通过钝器解剖背侧肌肉组织，暴露颈椎。（F） 创建一个皮下袋以将植入式装置尾部固定到切口部位。（G） 充分解剖后放置牵开器，露出颈椎和球形肌肉，完全覆盖 C2 并部分掩盖 C3。虚线表示球形肌肉。一旦颈脊髓暴露，（H） 在 C5 和 C6 进行两次外侧椎板切除术以在椎骨下放置探针，或 （I） 在 C5 进行内侧椎板切除术以将探针放置在椎骨上。星号表示外侧椎板切除术的部位。比例尺 = 3 毫米。 <a href="https://www.jove.com/files/ftp_upload/66992/66992fig02large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66992/66992fig03.jpg"> 图 3：设备植入和探针放置。 （A） 将装置放置在皮下袋中。（B） 将装置缝合到肌肉组织上。（C） 探针固定在已接受内侧椎板切除术的 C5 椎板顶部。（D） 该装置放置在 C5 和 C6 椎板下方，它们都接受了外侧椎板切除术。在 （C） 和 （D） 中，探针尖端都粘合在完整的 C4 上。比例尺 = 3 毫米。 <a href="https://www.jove.com/files/ftp_upload/66992/66992fig03large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66992/66992fig04.jpg"> 图 4：术后标记设备和验证功能。 （A） 缝合后可以选择在皮肤上标记设备的位置，以便于术后识别。（B） 该图描绘了动物植入后的情况。通过观察皮肤下可见的 μLED 指示剂来验证设备的功能，确认设备成功运行（动物右侧的凸起是设备主体植入的位置）。 <a href="https://www.jove.com/files/ftp_upload/66992/66992fig04large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/66992/66992fig05.jpg"> 图 5：假手术组和种植体组的前肢和后肢性能随时间变化的 Martinez 旷场行为评分。 这些图说明了 （A） 前肢和 （B） 后肢旷场评估在四个时间点的平均行为评分：0（基线）、植入后 3 天、5 天和 7 天 （DPI）。误差线表示均值 （SEM） 的标准误差。假手术组和种植体组之间的显着差异 （p < 0.05） 在特定时间点用星号 （*） 表示。图例用虚线表示假手术组，而种植体组用实线表示。假样本量为 n = 3，植入物为 n = 4。采用非参数 Mann-Whitney U 检验评价各时间点组间差异的显著性。 <a href="https://www.jove.com/files/ftp_upload/66992/66992fig05large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
补充图 1：椎板切除术的插图。 虚线表示 （A） 两个外侧椎板切除术用于在椎骨下放置探针，以及 （B） 内侧椎板切除术用于探针放置在椎骨上。 <a href="https://www.jove.com/files/ftp_upload/66992/Supplementary%20Figure%201.zip" target="_blank">请点击此处下载此文件。</a>
补充图 2：光电器件的示意图。 显示了设备的详细框图。左上角的块描绘了无线电源接收器天线谐振 LC 谐振电路。接收的功率经过整流并馈入低压差稳压器 （LDO）。微控制器单元根据编程参数自动激活器件，LED 驱动器为探头中嵌入的任何 μLED 供电。 <a href="https://www.jove.com/files/ftp_upload/66992/Supplementary%20Figure%202.zip" target="_blank">请点击此处下载此文件。</a>
补充图 3：光电器件。 具有生物相容性封装的最终光电器件连接到尖端包含 1 μLED 的光学柄。虚线矩形描述了 μLED 的位置。 <a href="https://www.jove.com/files/ftp_upload/66992/Supplementary%20Figure%203.zip" target="_blank">请点击此处下载此文件。</a>
补充图 4：设备稳定性的事后验证。 植入后 7 天，（A） 装置主体在植入时的位置保持与肌肉组织缝合，并且 （B） 胶合探针仍固定在 C4 椎板的顶部。 <a href="https://www.jove.com/files/ftp_upload/66992/Supplementary%20Figure%204.zip" target="_blank">请点击此处下载此文件。</a>

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

该协议详细介绍了用于进行脊髓手术以及在啮齿动物脊髓上植入和固定光学柄的外科手术。

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 ...

implantation-of-optoelectronic-devices-in-the-rodent-spinal-cord

Research

JoVE Journal

Neuroscience

Implantation of Optoelectronic Devices in the Rodent Spinal Cord

1.1K 次观看.  University of British Columbia. 我们引入了一种创新且易于执行的方法，用于在啮齿动物脊髓中植入和固定神经探针。植入方案适用于需要与脊髓直接接触的神经探针，并且可以为不需要直接接触的探针量身定制。柔性生物电子学的最新进展极大地改进了植入式神经调节装置。虽然这些设备已广泛用于大脑研究，但关于它们在脊髓脆弱和可移动组织中的应用报道有限。此外，...

视频: 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

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

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 ...

Spinal Cord Transection in the Larval Zebrafish

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability to reinitiate spinal neurogenesis. However, some anamniotes including the zebrafish Danio rerio exhibit both sensory and functional recovery even after complete transection of the spinal cord. The adult zebrafish is an established model organism for studying regeneration following spinal cord injury, with sensory and motor recovery by 6&#160;weeks post-injury. To take advantage of in vivo analysis of the regenerative process available in the transparent larval zebrafish as well as genetic tools not accessible in the adult, we use the larval zebrafish to study regeneration after spinal cord transection. Here we demonstrate a method for reproducibly and verifiably transecting the larval spinal cord. After transection, our data shows sensory recovery beginning at 2&#160;days post-injury (dpi), with the C-bend movement detectable by 3 dpi and resumption of free swimming by 5 dpi. Thus we propose the larval zebrafish as a companion tool to the adult zebrafish for the study of recovery after spinal cord injury.

After spinal transection, adult zebrafish have functional recovery by six weeks post-injury. To take advantage of larval transparency and faster recovery, we present a method for transecting the larval spinal cord. After transection, we observe sensory recovery beginning at 2&#160;days post-injury, and C-bend movement by 3 days post-injury.

在斑马鱼幼虫脊髓横断

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability ...

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 ...

Diffusion Imaging in the Rat Cervical Spinal Cord

Magnetic resonance imaging (MRI) is the state of the art approach for assessing the status of the spinal cord noninvasively, and can be used as a diagnostic and prognostic tool in cases of disease or injury. Diffusion weighted imaging (DWI), is sensitive to the thermal motion of water molecules and allows for inferences of tissue microstructure. This report describes a protocol to acquire and analyze DWI of the rat cervical spinal cord on a small-bore animal system. It demonstrates an imaging setup for the live anesthetized animal and recommends a DWI acquisition protocol for high-quality imaging, which includes stabilization of the cord and control of respiratory motion. Measurements with diffusion weighting along different directions and magnitudes (b-values) are used. Finally, several mathematical models of the resulting signal are used to derive maps of the diffusion processes within the spinal cord tissue that provide insight into the normal cord and can be used to monitor injury or disease processes noninvasively.

The goal of this protocol is to obtain high-quality diffusion weighted magnetic resonance imaging (DWI) of the rat spinal cord for noninvasive characterization of tissue microstructure. This protocol describes optimizations of the MRI sequence, radiofrequency coil, and analysis methods to enable DWI images free from artifacts.

在大鼠颈脊髓弥散成像

Magnetic resonance imaging (MRI) is the state of the art approach for assessing the status of the spinal cord noninvasively, and can be used as a ...

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 ...

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in adult mammalian animals remains elusive. In this study, we describe an experimental procedure for measuring calcium dynamics through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy. This method enables recordings and quantifications of neural activity in bilateral mouse brain regions one at a time in the same experiment for a prolonged period in vivo. Key aspects of this method, which can be completed within an hour, include minimally invasive surgery procedures for creating dual optical windows, and the use of 2P imaging. Although we only demonstrate the technique in the S1 area, the method can be applied to other regions of the living brain facilitating the elucidation of structural and functional complexities of brain neural networks.

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

We describe an experimental procedure for measuring neuronal activity through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy in vivo.

胸腺1-gmcmp6 转基因小鼠在原代体血源皮质中的成像神经活性

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...

Novel Passive Clearing Methods for the Rapid Production of Optical Transparency in Whole CNS Tissue

Since the development of CLARITY, a bioelectrochemical clearing technique that allows for three-dimensional phenotype mapping within transparent tissues, a multitude of novel clearing methodologies including CUBIC (clear, unobstructed brain imaging cocktails and computational analysis), SWITCH (system-wide control of interaction time and kinetics of chemicals), MAP (magnified analysis of the proteome), and PACT (passive clarity technique), have been established to further expand the existing toolkit for the microscopic analysis of biological tissues. The present study aims to improve upon and optimize the original PACT procedure for an array of intact rodent tissues, including the whole central nervous system (CNS), kidneys, spleen, and whole mouse embryos. Termed psPACT (process-separate PACT) and mPACT (modified PACT), these novel techniques provide highly efficacious means of mapping cell circuitry and visualizing subcellular structures in intact normal and pathological tissues. In the following protocol, we provide a detailed, step-by-step outline on how to achieve maximal tissue clearance with minimal invasion of their structural integrity via psPACT and mPACT.

Here, we present two novel methodologies, psPACT and mPACT, for achieving maximal optical transparency and subsequent microscopic analysis of tissue vasculature in the intact rodent whole CNS.

一种新的用于全中枢神经系统光透明快速生成的无源清除方法

Since the development of CLARITY, a bioelectrochemical clearing technique that allows for three-dimensional phenotype mapping within transparent tissues, ...

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 ...

Preparation of Rhythmically-active In Vitro Neonatal Rodent Brainstem-spinal Cord and Thin Slice

Mammalian inspiratory rhythm is generated from a neuronal network in a region of the medulla called the preB&#246;tzinger complex (pBC), which produces a signal driving the rhythmic contraction of inspiratory muscles. Rhythmic neural activity generated in the pBC and carried to other neuronal pools to drive the musculature of breathing may be studied using various approaches, including en bloc nerve recordings and transverse slice recordings. However, previously published methods have not extensively described the brainstem-spinal cord dissection process in a transparent and reproducible manner for future studies. Here, we present a comprehensive overview of a method used to reproducibly cut rhythmically-active brainstem slices containing the necessary and sufficient neuronal circuitry for generating and transmitting inspiratory drive. This work builds upon previous brainstem-spinal cord electrophysiology protocols to enhance the likelihood of reliably obtaining viable and rhythmically-active slices for recording neuronal output from the pBC, hypoglossal premotor neurons (XII pMN), and hypoglossal motor neurons (XII MN). The work presented expands upon previous published methods by providing detailed, step-by-step illustrations of the dissection, from whole rat pup, to in vitro slice containing the XII rootlets.

This protocol both visually communicates the brainstem-spinal cord preparation and clarifies the preparation of brainstem transverse slices in a comprehensive step-by-step manner. It was designed to increase reproducibility and enhance the likelihood of obtaining viable, long lasting, rhythmically-active slices for recording neural output from the respiratory regions of the brainstem.

头脊髓和薄片体外制备心律失常活性

Mammalian inspiratory rhythm is generated from a neuronal network in a region of the medulla called the preB&#246;tzinger complex (pBC), which produces a ...