Name: direct injection of a lentiviral vector highlights multiple motor pathways in the rat spinal cord
Uploaded: 2019-03-15T14:30:00
Description: Watch this Scientific Journal Video about Direct Injection of a Lentiviral Vector Highlights Multiple Motor Pathways in the Rat Spinal Cord at JoVE.com

这项工作的资金来自国家神经疾病研究所和中风 R01 R01 R01NS103481 和 shriners 儿科研究医院的赠款, 该医院提供 SHC 84051 和 SHC 86000 的赠款, 以及国防部 (SC140089) 的赠款。

以下所有手术和动物护理程序都已获得坦普尔大学动物护理和使用委员会的批准。
1. 手术前制剂
<ol><li>在手术前几天使用3.5 纳米管玻璃毛细管移液器准备用于病毒注射的拉拉玻璃针。根据制造商的说明, 将每个移液器拉上两步针拉拔器, 以创建两个针头模板。</li>
	<li>用微型剪刀切割约1-2 毫米的多余玻璃, 以细化针头模板的尖端。使用显微镜校准幻灯片在显微镜下测量近似?...

基因操纵的神经元在大脑和脊髓已有助于突出感觉, 运动和自主途径通过荧光追踪, 并探索损伤 27, 28 后神经元束的再生潜力,29,30,31,32,33. 将具有逆行可运输的病毒载体直接注入脊髓, 可以通过神经元的突触连接针对神经元群...

病毒载体是重要的生物工具, 可以将遗传物质引入细胞, 以补偿有缺陷的基因, 增强重要的生长蛋白或制造标记蛋白, 突出的结构和突触连接他们的目标。本文的重点是直接注射一个高效的逆行可转移性慢病毒载体到大鼠脊髓, 以突出主要运动途径与荧光追踪。 这种方法也是高度适用于轴突再生和再生研究, 以引入感兴趣的蛋白质的不同群体的神经元, 并已被用来沉默神经元的功能映射研究1,2。
通过 bda 和氟金 3、4、5、6、7等经典示踪剂的直接注射研究, 阐明了脊柱运动通路的许多解剖细节。,8. 这些示踪剂被认为是金本位, 但可能有某些缺点, 如受损的轴突吸收, 或在注射部位9、10、11周围的白质中的轴突吸收.这可能会导致对途径连接的不正确解释, 并可能是再生研究中的一个缺点, 在后来的分析12中, 受损或被切断的轴突吸收染料可能会被误认为是再生纤维。
慢病毒载体在基因治疗研究中很受欢迎, 因为它们在13、14、15、16、17、18的神经元群中提供稳定、长期的表达 ,19。然而, 传统上包装的慢病毒载体可能有有限的逆行运输, 并可能触发免疫系统反应时, 在体内使用4,20, 21。加藤等人通过使用狂犬病毒糖蛋白修饰病毒包络, 以创建一种混合载体, 改善逆行运输 22,23,从而生产出一种名为 hiret 的高效逆行运输载体。
逆行追踪将一个矢量引入目标神经元的突触空间, 使其被该细胞的轴突占据并输送到细胞体。hiraet 的成功运输已经被证明从神经元突触到小鼠和灵长类动物的大脑 23, 24 和从肌肉到运动神经元22。该方案演示注射到腰椎脊髓, 特别是针对突触端子的内脊髓间神经元和脑干神经元。Pn 接受来自许多不同脊髓通路的连接, 因此可以用来针对脊髓和脑干中不同的神经元群。这项研究中的标记神经元代表了与后肢运动功能相关的刺激运动神经元池的电路。坚固的标记可以在脊髓和脑干中看到, 包括树突乔木和轴突末端的高保真细节。我们在以前的颈髓研究中也使用了这种方法来标记本体和脑干网状脊髓通路25。
该方案演示了在大鼠腰椎脊髓注射病毒载体的方法。如电影 1所示, 切口的目标是识别位于最后一根肋骨的 l1 椎体。这被用作一个月额地标为3-4 厘米的切口, 暴露在 L1-l4 脊髓肌肉组织。对 T11-t13 椎骨背侧的椎体进行层压, 将斜玻璃针从中线侧向0.8 毫米, 降低1.5 毫米深到灰质中注入病毒。

<table>
	<tbody>
		<tr>
			<td>#10 Scalpel Blades</td>
			<td>Roboz</td>
			<td>RS-9801-10</td>
			<td>For use with the scalpel.</td>
		</tr>
		<tr>
			<td>1 mL Syringes</td>
			<td>Becton, Dickinson and Company</td>
			<td>309659</td>
			<td>For anesthetic IP injection, potential anesthetic booster shots, and antibiotic injections.</td>
		</tr>
		<tr>
			<td>10mL Syringes</td>
			<td>Becton, Dickinson and Company</td>
			<td>309604</td>
			<td>For injecting saline into the animal, post-surgery.</td>
		</tr>
		<tr>
			<td>4.0 Chromic Catgut Suture</td>
			<td>DemeTECH</td>
			<td>NN374-16</td>
			<td>To re-bind muscle during closing.</td>
		</tr>
		<tr>
			<td>48000 Micropipette Beveler</td>
			<td>World Precision Instruments</td>
			<td>32416</td>
			<td>Used to bevel the tips of the pulled glass capillary tubes to form functional glass needles.</td>
		</tr>
		<tr>
			<td>5% Iodine Solution</td>
			<td>Purdue Products L.P.</td>
			<td>L01020-08</td>
			<td>For use in sterilzation of the surgical site.</td>
		</tr>
		<tr>
			<td>70% Ethanol</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>For sterilization of newly prepared glass needles, animal models during surgical preparation, and surgeon&#39;s hands during surgery, as well as all other minor maintainances of sterility.</td>
		</tr>
		<tr>
			<td>Anesthetic (Ketamine/Xylazine Solution)</td>
			<td>Zoetis</td>
			<td>240048</td>
			<td>For keeping the animal in the correct plane of consciousness during surgery.</td>
		</tr>
		<tr>
			<td>Antibiotic (Cefazolin)</td>
			<td>West-Ward Pharmaceuticals</td>
			<td>NPC 0143-9924-90</td>
			<td>To be injected subcutaneously to prevent infection post-surgery.</td>
		</tr>
		<tr>
			<td>Bead Sterilizer</td>
			<td>CellPoint</td>
			<td>5-1450</td>
			<td>To heat sterilize surgical instruments.</td>
		</tr>
		<tr>
			<td>Bonewax</td>
			<td>Fine Science Tools</td>
			<td>19009-00</td>
			<td>To seal up bone in the case of bone bleeding.</td>
		</tr>
		<tr>
			<td>Cauterizer</td>
			<td>Fine Science Tools</td>
			<td>18010-00</td>
			<td>To seal any arteries or veins severed during surgery to prevent excessive blood loss.</td>
		</tr>
		<tr>
			<td>Digital Scale</td>
			<td>Okaus</td>
			<td>REV.005</td>
			<td>For weighing the animal during surgical preparation.</td>
		</tr>
		<tr>
			<td>Flexible Needle Attachment</td>
			<td>World Precision Instruments</td>
			<td>MF34G-5</td>
			<td>For cleaning glass needles and loading red oil into glass needles.</td>
		</tr>
		<tr>
			<td>Gelfoam</td>
			<td>Pfizer</td>
			<td>H68079</td>
			<td>To seal up bone in the case of bone bleeding.</td>
		</tr>
		<tr>
			<td>Glass Capillary Tubes</td>
			<td>World Precision Instruments</td>
			<td>4878</td>
			<td>For pulled glass needles - should be designed for nanoliter injectors.</td>
		</tr>
		<tr>
			<td>Hair Clippers</td>
			<td>Oster</td>
			<td>111038-060-000</td>
			<td>For clearing the surgical site of hair.</td>
		</tr>
		<tr>
			<td>Hemostats</td>
			<td>Roboz</td>
			<td>RS-7231</td>
			<td>For general use in surgery.</td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>Kimtech</td>
			<td>34155</td>
			<td>For general use in surgery.</td>
		</tr>
		<tr>
			<td>Medium Point Curved Forceps</td>
			<td>Roboz</td>
			<td>RS-5136</td>
			<td>For general use in surgery.</td>
		</tr>
		<tr>
			<td>Micromanipulator with a Vernier Scale</td>
			<td>Kanetec</td>
			<td>N/A</td>
			<td>For precise targeting during surgery.</td>
		</tr>
		<tr>
			<td>Microscissors</td>
			<td>Roboz</td>
			<td>RS-5621</td>
			<td>For cutting glass whisps off of freshly pulled glass capillary tubes.</td>
		</tr>
		<tr>
			<td>Microscope with Light and Vernier Scale Ocular</td>
			<td>Leitz Wetzlar</td>
			<td>N/A</td>
			<td>Used to visualize and measure beveling of pulled glass capillary tubes into functional glass needles.</td>
		</tr>
		<tr>
			<td>MicroSyringe Pump Controller</td>
			<td>World Precision Instruments</td>
			<td>62403</td>
			<td>To control the rate of injection.</td>
		</tr>
		<tr>
			<td>Nanoliter 2000 Pump Head Injector</td>
			<td>World Precision Instruments</td>
			<td>500150</td>
			<td>To load and inject virus in a controlled fashion.</td>
		</tr>
		<tr>
			<td>Needle Puller</td>
			<td>Narishige</td>
			<td>PC-100</td>
			<td>To heat and pull apart glass capillary tubes to form glass needles.</td>
		</tr>
		<tr>
			<td>Ophthalamic Ointment</td>
			<td>Dechra Veterinary Products</td>
			<td>RAC 0119</td>
			<td>To protect the animal&#39;s eyes during surgery.</td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Bemis</td>
			<td>PM-996</td>
			<td>To assist with loading virus into the nanoinjector.</td>
		</tr>
		<tr>
			<td>PrecisionGlide Needles (25G x 5/8)</td>
			<td>Becton, Dickinson and Company</td>
			<td>305122</td>
			<td>For use with the 1mL and 10 mL syringes to allow injection of the animal model.</td>
		</tr>
		<tr>
			<td>Rat Tooth Forceps</td>
			<td>Roboz</td>
			<td>RS-5152</td>
			<td>For griping spinous processes.</td>
		</tr>
		<tr>
			<td>Red Oil</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>To provide a front for visualization of virus entering tissue during injection.</td>
		</tr>
		<tr>
			<td>Retractors</td>
			<td>Roboz</td>
			<td>RS-6510</td>
			<td>To hold open the surgical wound.</td>
		</tr>
		<tr>
			<td>Rimadyl Tablets</td>
			<td>Bio Serv</td>
			<td>MP275-050</td>
			<td>For pain management post-surgery.</td>
		</tr>
		<tr>
			<td>Rongeurs</td>
			<td>Roboz</td>
			<td>RS-8300</td>
			<td>To remove muscle from the spinal column during surgery.</td>
		</tr>
		<tr>
			<td>Scalpel Blade Handle</td>
			<td>Roboz</td>
			<td>RS-9843</td>
			<td>To slice open skin and fat pad of animal model during surgery.</td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Roboz</td>
			<td>RS-5980</td>
			<td>For general use in surgery.</td>
		</tr>
		<tr>
			<td>Stainless Steal Wound Clips</td>
			<td>CellPoint</td>
			<td>201-1000</td>
			<td>To bind the skin of the surgical wound during closing.</td>
		</tr>
		<tr>
			<td>Staple Removing Forceps</td>
			<td>Kent Scientific</td>
			<td>INS750347</td>
			<td>To remove the staples, should they be applied incorrectly.</td>
		</tr>
		<tr>
			<td>Sterile Cloth</td>
			<td>Phenix Research Products</td>
			<td>BP-989</td>
			<td>To provide a sterile surface for the operation.</td>
		</tr>
		<tr>
			<td>Sterile Cotton-Tipped Applicators</td>
			<td>Puritan</td>
			<td>806-WC</td>
			<td>To soak up blood in the surgical wound while maintaining sterility.</td>
		</tr>
		<tr>
			<td>Sterile Gauze</td>
			<td>Covidien</td>
			<td>2146</td>
			<td>To clean the surgical area and surgical tools while maintaining sterility.</td>
		</tr>
		<tr>
			<td>Sterile Saline</td>
			<td>Baxter Healthcare Corporation</td>
			<td>281324</td>
			<td>For use in blood clearing, and for replacing fluids post-surgery.</td>
		</tr>
		<tr>
			<td>Surgical Gloves</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>For use by the surgeon to maintain sterile field during surgery.</td>
		</tr>
		<tr>
			<td>Surgical Heating Pad</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>For maintaining the body temperature of the animal model during surgery.</td>
		</tr>
		<tr>
			<td>Surgical Microscope</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>For enhanced visualization of the surgical wound.</td>
		</tr>
		<tr>
			<td>Surgical Stapler</td>
			<td>Kent Scientific</td>
			<td>INS750546</td>
			<td>To apply the staples.</td>
		</tr>
		<tr>
			<td>T/Pump Heat Therapy Water Pump</td>
			<td>Gaymar</td>
			<td>TP500C</td>
			<td>To pump warm water into the water convection warming pad.</td>
		</tr>
		<tr>
			<td>Water Convection Warming Pad</td>
			<td>Baxter Healthcare Corporation</td>
			<td>L1K018</td>
			<td>For use in the post-operational recovery area to maintain the body temperature of the unconscious animal.</td>
		</tr>
		<tr>
			<td>Weighted Hooks</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>To hold open the surgical wound.</td>
		</tr>
	</tbody>
</table>

direct injection of a lentiviral vector highlights multiple motor pathways in the rat spinal cord

将感兴趣的蛋白质引入神经系统的细胞是具有挑战性的, 因为固有的生物屏障限制了对大多数分子的访问。直接注射到脊髓组织绕过这些障碍, 提供进入细胞体或突触的机会, 在那里可以纳入分子。将病毒载体技术与这种方法相结合, 可以将目标基因引入神经组织, 用于基因治疗或肠道追踪。在这里, 一种为高效逆行转运 (Hirat) 而设计的病毒被引入到外源间神经元 (Pn) 突触上, 以鼓励向脊髓和脑干核的神经元进行特定的迁移。瞄准 Pn 利用了他们从运动通路 (如脊椎和网状脊椎通道) 获得的众多连接, 以及它们在整个脊髓段之间的相互连接。使用 HiRet 矢量和本构活性绿色荧光蛋白 (GFP) 进行代表性追踪, 显示胸椎 Pn 和网状网状结构中网状脊髓神经元的细胞体、轴突和树突状乔木的高保真度细节。Hiraet 很好地融入了脑干通路和 Pn, 但显示年龄依赖性整合到皮质脊髓神经元中。总之, 脊髓注射使用病毒载体是一个合适的方法, 将感兴趣的蛋白质引入目标区域的神经元。

病毒载体的成功注射和运输应导致脊髓和某些脑干核中的单侧神经元的强群的传导。图 1显示了胸腔脊髓和脑桥网状脑干在注射后四周形成的神经元和轴突的定型标记。在注射侧同侧的胸脊髓灰质神经元中可见显著的 GFP 表达 (图 1 a, 盒装区域)。在对侧也观察到一些神经元, 特别是在中线附近。在白质中, GFP 的表达是在同侧脐带 (图 1a?...

Watch this Scientific Journal Video about Direct Injection of a Lentiviral Vector Highlights Multiple Motor Pathways in the Rat Spinal Cord at JoVE.com

Direct Injection of a Lentiviral Vector Highlights Multiple Motor Pathways in the Rat Spinal Cord

该协议演示了在大鼠脊髓组织中注射一种具有可追溯性的可转移性病毒载体。载体在突触处被占据, 并被输送到目标神经元的细胞体。该模型适用于重要脊柱通路的逆行追踪或基因治疗应用中的靶向细胞。

直接注射慢病毒载体突出大鼠脊髓中的多种运动途径

Introducing proteins of interest into cells in the nervous system is challenging due to innate biological barriers that limit access to most molecules. ...

病毒载体可以将遗传物质引入细胞，以补偿有缺陷的基因，提高重要的生长蛋白，或制造可以追踪神经回路的标记物。由于与生俱来的生物学障碍，将病毒载体引入神经系统是具有挑战性的。直接注射到脊髓组织中绕过这些屏障，允许进入细胞体或突触。正确定位 L1 到 L4 脊髓水平可能很困难。使用地标来验证目标并记住这些段线到 T11 到 T13 椎骨非常重要。在手术当天，将喷油器插入微泵，然后将微泵放入具有更薄度的微操纵器中。使用带柔性针头的注射器小心地将彩色染料装入其中一个玻璃针中，并将玻璃针插入喷油器中。然后确认针头已正确播种到垫圈中，喷油器盖拧紧，并且钢喷油器针头的延伸距离约为玻璃针长度的四分之三。在开始手术之前，解冻至少一微升，加上从冷冻室注射少量额外病毒，并确认麻醉大鼠的针头趾缺乏反应。沿着后侧中线从臀部剃到动物肩骨的劣质角度，拉紧皮肤，进行更轻松、更精确的剃须，并连续用 5% 碘和 70% 乙醇磨砂对裸露的皮肤进行消毒。将眼科软膏涂抹在两只眼睛上。然后将动物放在无菌布上，将纱布放在膀胱下方以收集尿液。要识别皮肤切口的部位，请轻轻按压最后一根肋骨的手指以定位 L1 椎骨。使用这个椎骨作为一个里程碑，并举行皮肤紧绷的轻轻蔓延，同时用手术刀用力按，使用数字10手术刀片，使三到四厘米的皮肤切口结束只是低于L1暴露肌肉。使用钳子和剪刀来感受旋转过程。做小的滚切，让空间牢牢地抓住与大鼠牙钳的上部过程。并尽可能接近流程，进行两次长而深的切割。固定横向肌肉到位后，清除周围的肌肉，以确定其头部的形状。然后可视化旋转过程头部的形状，以识别拉明切除术位点。T11 和连接 T12 和 T13 过程将是拉明切除术的站点。一旦目标区域被正确识别，轻轻展开椎骨以露出椎间韧带，并使用旋颈小咬椎的旋转过程和椎背的侧部。清除中线外骨骼，以便观察中线血管，留下一个窗口，清楚地显示脊髓组织，并且没有碎屑。然后将稳定钳紧到旋转过程，将胸腺和胸膜固定到拉明切除术窗口，将动物固定在脊柱架中。提高动物的腹部，以抵消呼吸运动的影响。要将病毒加载到喷油器中，将解冻库存的大约五微升移液器插入一块 Parafilm 上，并放置玻璃针，使尖端位于液滴内。使用微泵以 20 到 100 纳米升/秒的速度提取多达 4 微升的病毒，并设置控制器注射，然后从针尖释放少量病毒，以确保病毒不会被阻塞。用实验室擦拭清除任何多余的病毒，并放置微操纵器，使可见比额。将针头放在脊髓的中线，并使用微操纵器上的测光量表横向引导针头 0.8 毫米。然后将针头降到脊柱和弦，直到它缩进，但不是穿刺，杜拉，并使用快速扭曲运动刺穿杜拉与针，直到针尖已下降到1.5毫米的深度。一旦针头就位，编程喷油器以每分钟400纳米光的速度传递病毒，并通过观察染料前部的进度来确认病毒正在进入脊髓和弦。注射完成后，让针头在脊髓中休息两到五分钟，以方便病毒的传播，然后慢慢提取针头，以便定位到下一个注射部位。最后一次注射后，将动物从脊柱架上取出，以便取出用于传播横向肌肉的任何装置。然后用 4 . 0 缝合线关闭肌肉，用 9 毫米伤口夹固定皮肤。成功注射四周后，在侧胸脊髓的灰质内的神经元中观察到显著的GP表达，对注射进行刺激。在白质中，在轴线中的轴子中观察到GSP表达，特别是在自体轴体的典型区域。灰质神经元的放大率越高，就会发现神经元体和树突中典型信号表达的例子。神经元中的GP表达也可以在脑干核中观察到，例如在浮核内柱形成。成功脊髓注射程序的关键因素是正确的椎骨靶向，并确保病毒进入组织如果在手术前进行肩骨脊髓损伤，直接注射可用于追踪在损伤周围重新建立连接的神经元。

Research

Education

JoVE Core

JoVE Journal

Anatomy and Physiology

Neuroscience

Unit 1

Functions of the Central and Peripheral Nervous System

SCIENTISTS IN ACTION

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

一个在培养皿中的功能电机组：脊髓植和肌肉细胞共培养

Human primary muscle cells cultured  aneurally in monolayer rarely contract spontaneously because, in the absence of  a nerve component, cell ...

科研

神经科学

Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord

立体定向注射病毒载体的基因操作在小鼠脊髓条件

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central ...

Production of RNA for Transcriptomic Analysis from Mouse Spinal Cord Motor Neuron Cell Bodies by Laser Capture Microdissection

通过激光捕获微分路，从小鼠脊髓运动神经细胞体中生成RNA进行转录分析

Preparation of high-quality RNA from cells of interest is critical to precise and meaningful analysis of transcriptional differences among cell types or ...

Spinal Cord Transection in the Larval Zebrafish

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 ...

Diffusion Imaging in the Rat Cervical Spinal Cord

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 ...

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

斑马鱼<em&gt;原位</em&gt;脊髓制备用于脊髓感觉和运动神经元的电生理记录

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing ...

Laminectomy and Spinal Cord Window Implantation in the Mouse

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 ...

Thoracic Spinal Cord Hemisection Surgery and Open-Field Locomotor Assessment in the Rat

胸脊髓截断手术和大鼠开放场运动评估

Spinal cord injury (SCI) causes disturbances in motor, sensory, and autonomic function below the level of the lesion. Experimental animal models are ...

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

神经干细胞向大鼠和小鼠大脑的动脉内传递：对脑缺血的应用

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

In Vivo Intracellular Recording of Type-Identified Rat Spinal Motoneurons During Trans-Spinal Direct Current Stimulation

在跨脊柱直流刺激期间类型识别大鼠脊柱腺瘤的 Vivo 细胞内记录

Intracellular recording of spinal motoneurons in vivo provides a &#8220;gold standard&#8221; for determining the cells&#8217; electrophysiological ...