Name: implantation of optoelectronic devices in the rodent spinal cord
Uploaded: 2024-07-12T14:40:00.000+00:00
Duration: 4 min 35 s
Description: Neuromodulation can provide diagnostic, modulatory, and therapeutic applications. While extensive work has been conducted in the brain, modulation of the ...

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。

<table><tbody><tr><td>Adson Forceps&amp;nbsp;</td><td>Fine Science Tools</td><td>11027-12</td><td></td></tr><tr><td>Alm 3 Point Retractor</td><td>Fine Science Tools</td><td>17010-10</td><td></td></tr><tr><td>Buprenorphine / Vetergesic&amp;nbsp;</td><td>CDMV</td><td>124918</td><td>Manufacturer provides at 0.3 mg/mL but must be diluted to 0.03 mg/kg for use in rats</td></tr><tr><td>Chlorhexidine 2% Solution</td><td>Partnar</td><td>PCH-020&amp;nbsp;</td><td></td></tr><tr><td>Curved Long Hemostat Forceps</td><td>KaamKaaj Tools</td><td>14.5</td><td>Curved Long Hemostat Forceps with A Stainless Steel Ratchet Locking Tweezer</td></tr><tr><td>CVD Parylene Machine: SCS Labcoter 2</td><td>Specialty Coating Systems</td><td>PDS 2010</td><td></td></tr><tr><td>Dental Cement - Catalyst&amp;nbsp;</td><td>Parkell, Inc</td><td>S371</td><td></td></tr><tr><td>Dental Cement - Metabond</td><td>Parkell, Inc</td><td>S398</td><td></td></tr><tr><td>Dental Cement - Powder&amp;nbsp;</td><td>Parkell, Inc</td><td>S396</td><td></td></tr><tr><td>Forceps with Replaceable Plastic Tips</td><td>Fine Science Tools</td><td>11980-13</td><td></td></tr><tr><td>Friedman-Pearson Rongeurs&amp;nbsp;</td><td>Fine Science Tools</td><td>16121-14</td><td></td></tr><tr><td>Isoflurane USP</td><td>Fresenius Kabi&amp;nbsp;</td><td>CP0406V2</td><td>Provided at 5% for induction and 2% for mainentance through precision vaporizer&amp;nbsp;</td></tr><tr><td>Isopropyl Alcohol 70%</td><td>McKesson</td><td>350600</td><td></td></tr><tr><td>Lacri-Lube Sterile Eye Ointment&amp;nbsp;</td><td>Refresh&amp;nbsp;</td><td></td><td></td></tr><tr><td>Long Evans Rats</td><td>Charles River Laboratories</td><td>6</td><td></td></tr><tr><td>Low temperature solder paste</td><td>Chip Quik Inc.</td><td>11.38</td><td></td></tr><tr><td>Magnets</td><td>Radial Magnets, Inc.</td><td>0.53</td><td>Magnet Neodymium Iron Boron (NdFeB) N35 (3.00 mm x 1.00 mm)</td></tr><tr><td>Olsen-Hegar Needle Holders with Suture Cutters&amp;nbsp;</td><td>Fine Science Tools</td><td>12002-12</td><td></td></tr><tr><td>PDMS: SYLGARD&amp;nbsp;184</td><td>Sigma Aldrich</td><td>761036</td><td></td></tr><tr><td>Scalpel Blades - #15</td><td>Fine Science Tools</td><td>10015-00</td><td></td></tr><tr><td>Scalpel Handle - #3</td><td>Fine Science Tools</td><td>10003-12</td><td></td></tr><tr><td>Solder flux</td><td>Chip Quik Inc.</td><td>14.25</td><td></td></tr><tr><td>Stereotaxic Frame&amp;nbsp;</td><td>David Kopf Instruments</td><td>Model 900</td><td></td></tr><tr><td>Sterile Kwik-Sil Adhesive</td><td>World Precision Instruments</td><td>KWIK-SIL-S</td><td></td></tr><tr><td>UV Flashlight</td><td>Vansky</td><td>19.99</td><td></td></tr><tr><td>Wireless Charger</td><td>Nilkin</td><td>NKT06</td><td></td></tr><tr><td>Wireless Charging coil</td><td>TDK Corporation</td><td>WT202012-15F2-ID</td><td></td></tr></tbody></table>

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://jove.unicartagenaproxy.elogim.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://jove.unicartagenaproxy.elogim.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://jove.unicartagenaproxy.elogim.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://jove.unicartagenaproxy.elogim.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://jove.unicartagenaproxy.elogim.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://jove.unicartagenaproxy.elogim.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://jove.unicartagenaproxy.elogim.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://jove.unicartagenaproxy.elogim.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://jove.unicartagenaproxy.elogim.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

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

Implantation of Optoelectronic Devices in the Rodent Spinal Cord

1.2K 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

Implantation and Recording of Wireless Electroretinogram and Visual Evoked Potential in Conscious Rats

The full-field electroretinogram (ERG) and visual evoked potential (VEP) are useful tools to assess retinal and visual pathway integrity in both laboratory and clinical settings. Currently, preclinical ERG and VEP measurements are performed with anesthesia to ensure stable electrode placements. However, the very presence of anesthesia has been shown to contaminate normal physiological responses. To overcome these anesthesia confounds, we develop a novel platform to assay ERG and VEP in conscious rats. Electrodes are surgically implanted sub-conjunctivally on the eye to assay the ERG and epidurally over the visual cortex to measure the VEP. A range of amplitude and sensitivity/timing parameters are assayed for both the ERG and VEP at increasing luminous energies. The ERG and VEP signals are shown to be stable and repeatable for at least 4 weeks post surgical implantation. This ability to record ERG and VEP signals without anesthesia confounds in the preclinical setting should provide superior translation to clinical data.

We show surgical implantation and Recording procedures to measure visual electrophysiological signals from the eye (electroretinogram) and brain (visual evoked potential) in conscious rats, which is more analogous to the human condition where recordings are conducted without anesthesia confounds.

植入和无线电图记录和视觉诱发电位清醒大鼠

The full-field electroretinogram (ERG) and visual evoked potential (VEP) are useful tools to assess retinal and visual pathway integrity in both ...

科研

行为学

Simultaneous Electroencephalography, Real-time Measurement of Lactate Concentration and Optogenetic Manipulation of Neuronal Activity in the Rodent Cerebral Cortex

Although the brain represents less than 5% of the body by mass, it utilizes approximately one quarter of the glucose used by the body at rest1. The function of non rapid eye movement sleep (NREMS), the largest portion of sleep by time, is uncertain. However, one salient feature of NREMS is a significant reduction in the rate of cerebral glucose utilization relative to wakefulness2-4. This and other findings have led to the widely held belief that sleep serves a function related to cerebral metabolism. Yet, the mechanisms underlying the reduction in cerebral glucose metabolism during NREMS remain to be elucidated.
	One phenomenon associated with NREMS that might impact cerebral metabolic rate is the occurrence of slow waves, oscillations at frequencies less than 4 Hz, in the electroencephalogram5,6. These slow waves detected at the level of the skull or cerebral cortical surface reflect the oscillations of underlying neurons between a depolarized/up state and a hyperpolarized/down state7. During the down state, cells do not undergo action potentials for intervals of up to several hundred milliseconds. Restoration of ionic concentration gradients subsequent to action potentials represents a significant metabolic load on the cell8; absence of action potentials during down states associated with NREMS may contribute to reduced metabolism relative to wake.
	Two technical challenges had to be addressed in order for this hypothetical relationship to be tested. First, it was necessary to measure cerebral glycolytic metabolism with a temporal resolution reflective of the dynamics of the cerebral EEG (that is, over seconds rather than minutes). To do so, we measured the concentration of lactate, the product of aerobic glycolysis, and therefore a readout of the rate of glucose metabolism in the brains of mice. Lactate was measured using a lactate oxidase based real time sensor embedded in the frontal cortex. The sensing mechanism consists of a platinum-iridium electrode surrounded by a layer of lactate oxidase molecules. Metabolism of lactate by lactate oxidase produces hydrogen peroxide, which produces a current in the platinum-iridium electrode. So a ramping up of cerebral glycolysis provides an increase in the concentration of substrate for lactate oxidase, which then is reflected in increased current at the sensing electrode. It was additionally necessary to measure these variables while manipulating the excitability of the cerebral cortex, in order to isolate this variable from other facets of NREMS.
	We devised an experimental system for simultaneous measurement of neuronal activity via the elecetroencephalogram, measurement of glycolytic flux via a lactate biosensor, and manipulation of cerebral cortical neuronal activity via optogenetic activation of pyramidal neurons. We have utilized this system to document the relationship between sleep-related electroencephalographic waveforms and the moment-to-moment dynamics of lactate concentration in the cerebral cortex. The protocol may be useful for any individual interested in studying, in freely behaving rodents, the relationship between neuronal activity measured at the electroencephalographic level and cellular energetics within the brain.

A procedure is described for manipulating the activity of cerebral cortical pyramidal neurons optogenetically while the electroencephalogram, electromyogram, and cerebral lactate concentration are monitored. Experimental recordings are performed on cable-tethered mice while they undergo spontaneous sleep/wake cycles. Optogenetic equipment is assembled in our laboratory; recording equipment is commercially available.

同时脑电图，实时测量的啮齿动物的大脑皮质中的神经元活动的乳酸浓度与Optogenetic的操作

Although the brain represents less than 5% of the body  by mass, it utilizes approximately one quarter of the glucose used by the body  at rest1. The ...

神经科学

Fiber-optic Implantation for Chronic Optogenetic Stimulation of Brain Tissue

Elucidating patterns of neuronal connectivity has been a challenge for both clinical and basic neuroscience. Electrophysiology has been the gold standard for analyzing patterns of synaptic connectivity, but paired electrophysiological recordings can be both cumbersome and experimentally limiting. The development of optogenetics has introduced an elegant method to stimulate neurons and circuits, both in vitro1 and in vivo2,3. By exploiting cell-type specific promoter activity to drive opsin expression in discrete neuronal populations, one can precisely stimulate genetically defined neuronal subtypes in distinct circuits4-6. Well described methods to stimulate neurons, including electrical stimulation and/or pharmacological manipulations, are often cell-type indiscriminate, invasive, and can damage surrounding tissues. These limitations could alter normal synaptic function and/or circuit behavior. In addition, due to the nature of the manipulation, the current methods are often acute and terminal. Optogenetics affords the ability to stimulate neurons in a relatively innocuous manner, and in genetically targeted neurons. The majority of studies involving in vivo optogenetics currently use a optical fiber guided through an implanted cannula6,7; however, limitations of this method include damaged brain tissue with repeated insertion of an optical fiber, and potential breakage of the fiber inside the cannula. Given the burgeoning field of optogenetics, a more reliable method of chronic stimulation is necessary to facilitate long-term studies with minimal collateral tissue damage. Here we provide our modified protocol as a video article to complement the method effectively and elegantly described in Sparta et al.8 for the fabrication of a fiber optic implant and its permanent fixation onto the cranium of anesthetized mice, as well as the assembly of the fiber optic coupler connecting the implant to a light source. The implant, connected with optical fibers to a solid-state laser, allows for an efficient method to chronically photostimulate functional neuronal circuitry with less tissue damage9 using small, detachable, tethers. Permanent fixation of the fiber optic implants provides consistent, long-term in vivo optogenetic studies of neuronal circuits in awake, behaving mice10 with minimal tissue damage.

The development of optogenetics now provides the means to precisely stimulate genetically defined neurons and circuits, both in vitro and in vivo. Here we describe the assembly and implantation of a fiber optic for chronic photostimulation of brain tissue.

光纤植入慢性Optogenetic刺激脑组织

Elucidating patterns of neuronal connectivity has been a challenge for both clinical and basic neuroscience. Electrophysiology has been the gold standard ...

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 ...

医学

Optogenetic Manipulation of Neuronal Activity to Modulate Behavior in Freely Moving Mice

Optogenetic modulation of neuronal circuits in freely moving mice affects acute and long-term behavior. This method is able to perform manipulations of single neurons and region-specific transmitter release, up to whole neuronal circuitries in the central nervous system, and allows the direct measurement of behavioral outcomes. Neurons express optogenetic tools via an injection of viral vectors carrying the DNA of choice, such as Channelrhodopsin2 (ChR2). Light is brought into specific brain regions via chronic optical implants that terminate directly above the target region. After two weeks of recovery and proper tool-expression, mice can be repeatedly used for behavioral tests with optogenetic stimulation of the neurons of interest.
Optogenetic modulation has a high temporal and spatial resolution that can be accomplished with high cell specificity, compared to the commonly used methods such as chemical or electrical stimulation. The light does not harm neuronal tissue and can therefore be used for long-term experiments as well as for multiple behavioral experiments in one mouse. The possibilities of optogenetic tools are nearly unlimited and enable the activation or silencing of whole neurons, or even the manipulation of a specific receptor type by light.
The results of such behavioral experiments with integrated optogenetic stimulation directly visualizes changes in behavior caused by the manipulation. The behavior of the same animal without light stimulation as a baseline is a good control for induced changes. This allows a detailed overview of neuronal types or neurotransmitter systems involved in specific behaviors, such as anxiety. The plasticity of neuronal networks can also be investigated in great detail through long-term stimulation or behavioral observations after optical stimulation. Optogenetics will help to enlighten neuronal signaling in several kinds of neurological diseases.

With optogenetic manipulation of specific neuronal populations or brain regions, behavior can be modified with high temporal and spatial resolution in freely moving animals. By using different optogenetic tools in combination with chronically implanted optical fibers, a variety of neuronal modulations and behavioral testing can be performed.

神经活性的光遗传学操作，调节自由移动小鼠的行为

Optogenetic modulation of neuronal circuits in freely moving mice affects acute and long-term behavior. This method is able to perform manipulations of ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

在啮齿动物中枢神经系统的体内光遗传学刺激

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

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.

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

In this video, we describe a procedure for implanting a chronic optical imaging chamber over the dorsal spinal cord of a live mouse. The chamber, surgical procedure, and chronic imaging are reviewed and demonstrated.

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 ...

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 ...

Optrode Array for Simultaneous Optogenetic Modulation and Electrical Neural Recording

During the last decade, optogenetics has become an essential tool for the investigation of neural signaling due to its unique capability of selective neural modulation or monitoring. As specific types of neuronal cells can be genetically modified to express opsin proteins, optogenetics enables optical stimulation or inhibition of the selected neurons. There have been several technological advances in the optical system for optogenetics. Recently, it was proposed to combine the optical waveguide for light delivery with electrophysiological recording to simultaneously monitor the neural responses to optogenetic stimulation or inhibition. In this study, an implantable optrode array (2x2 optical fibers) was developed with embedded multichannel electrodes. 
A light-emitting diode (LED) was employed as a light source, and a microfabricated microlens array was integrated to provide sufficient light power at the tip of the optical fibers. The optrode array system comprises the disposable part and the reusable part. The disposable part has optical fibers and electrodes, while the reusable part has the LED and electronic circuitry for light control and neural signal processing. The novel design of the implantable optrode array system is introduced in the accompanying video in addition to the procedure of the optrode implantation surgery, optogenetic light stimulation, and the electrophysiological neural recording. The results of in vivo experiments successfully showed time-locked neural spikes evoked by the light stimuli from hippocampal excitatory neurons of mice.

Here, we present the fabrication method of an optrode system with optical fibers for light delivery and an electrode array for neural recording. In vivo experiments with transgenic mice expressing channelrhodopsin-2 show the feasibility of the system for simultaneous optogenetic stimulation and electrophysiological recording.

用于同步光遗传学调制和电神经记录的光驱阵列

During the last decade, optogenetics has become an essential tool for the investigation of neural signaling due to its unique capability of selective ...

Development of a Surgical Technique for Subretinal Implants in Rats

Retinal degeneration, such as age-related macular degeneration (AMD), is a leading cause of blindness worldwide. A myriad of approaches have been undertaken to develop regenerative medicine-based therapies for AMD, including stem cell-based therapies. Rodents as animal models for retinal degeneration are a foundation for translational research, due to the broad spectrum of strains that develop retinal degeneration diseases at different stages. However, mimicking human therapeutic delivery of subretinal implants in rodents is challenging, due to anatomical differences such as lens size and vitreous volume. This surgical protocol aims to provide a guided method for transplanting implants into the subretinal space in rats. A user-friendly comprehensive description of the critical steps has been included. This protocol has been developed as a cost-efficient surgical procedure for reproducibility across different preclinical studies in rats. Proper miniaturization of a human-sized implant is required prior to conducting the surgical experiment, which includes adjustments to the dimensions of the implant. An external approach is used instead of an intravitreal procedure to deliver the implant to the subretinal space. Using a small sharp needle, a scleral incision is performed in the temporal superior quadrant, followed by paracentesis to reduce intraocular pressure, thereby minimizing resistance during the surgical implantation. Next, a balanced salt solution (BSS) injection through the incision is carried out to achieve focal retinal detachment (RD). Lastly, insertion and visualization of the implant into the subretinal space are conducted. Post-operative assessment of the subretinal placement of the implant includes imaging by spectral domain optical coherence tomography (SD-OCT). Imaging follow-ups ascertain the subretinal stability of the implant, before the eyes are harvested and fixated for histological analysis.

The present protocol describes the scleral approach for subretinal device implantation, a feasible surgical technique for implementation in animal models of retinal diseases in research.