直接注射慢病毒载体突出大鼠脊髓中的多种运动途径
Summary
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。
Abstract
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。
Introduction
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。
Protocol
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 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。 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?…
Representative Results
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?…
Discussion
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…
Divulgations
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| #10 Scalpel Blades | Roboz | RS-9801-10 | For use with the scalpel. |
| 1 mL Syringes | Becton, Dickinson and Company | 309659 | For anesthetic IP injection, potential anesthetic booster shots, and antibiotic injections. |
| 10mL Syringes | Becton, Dickinson and Company | 309604 | For injecting saline into the animal, post-surgery. |
| 4.0 Chromic Catgut Suture | DemeTECH | NN374-16 | To re-bind muscle during closing. |
| 48000 Micropipette Beveler | World Precision Instruments | 32416 | Used to bevel the tips of the pulled glass capillary tubes to form functional glass needles. |
| 5% Iodine Solution | Purdue Products L.P. | L01020-08 | For use in sterilzation of the surgical site. |
| 70% Ethanol | N/A | N/A | For sterilization of newly prepared glass needles, animal models during surgical preparation, and surgeon's hands during surgery, as well as all other minor maintainances of sterility. |
| Anesthetic (Ketamine/Xylazine Solution) | Zoetis | 240048 | For keeping the animal in the correct plane of consciousness during surgery. |
| Antibiotic (Cefazolin) | West-Ward Pharmaceuticals | NPC 0143-9924-90 | To be injected subcutaneously to prevent infection post-surgery. |
| Bead Sterilizer | CellPoint | 5-1450 | To heat sterilize surgical instruments. |
| Bonewax | Fine Science Tools | 19009-00 | To seal up bone in the case of bone bleeding. |
| Cauterizer | Fine Science Tools | 18010-00 | To seal any arteries or veins severed during surgery to prevent excessive blood loss. |
| Digital Scale | Okaus | REV.005 | For weighing the animal during surgical preparation. |
| Flexible Needle Attachment | World Precision Instruments | MF34G-5 | For cleaning glass needles and loading red oil into glass needles. |
| Gelfoam | Pfizer | H68079 | To seal up bone in the case of bone bleeding. |
| Glass Capillary Tubes | World Precision Instruments | 4878 | For pulled glass needles – should be designed for nanoliter injectors. |
| Hair Clippers | Oster | 111038-060-000 | For clearing the surgical site of hair. |
| Hemostats | Roboz | RS-7231 | For general use in surgery. |
| Kimwipes | Kimtech | 34155 | For general use in surgery. |
| Medium Point Curved Forceps | Roboz | RS-5136 | For general use in surgery. |
| Micromanipulator with a Vernier Scale | Kanetec | N/A | For precise targeting during surgery. |
| Microscissors | Roboz | RS-5621 | For cutting glass whisps off of freshly pulled glass capillary tubes. |
| Microscope with Light and Vernier Scale Ocular | Leitz Wetzlar | N/A | Used to visualize and measure beveling of pulled glass capillary tubes into functional glass needles. |
| MicroSyringe Pump Controller | World Precision Instruments | 62403 | To control the rate of injection. |
| Nanoliter 2000 Pump Head Injector | World Precision Instruments | 500150 | To load and inject virus in a controlled fashion. |
| Needle Puller | Narishige | PC-100 | To heat and pull apart glass capillary tubes to form glass needles. |
| Ophthalamic Ointment | Dechra Veterinary Products | RAC 0119 | To protect the animal's eyes during surgery. |
| Parafilm | Bemis | PM-996 | To assist with loading virus into the nanoinjector. |
| PrecisionGlide Needles (25G x 5/8) | Becton, Dickinson and Company | 305122 | For use with the 1mL and 10 mL syringes to allow injection of the animal model. |
| Rat Tooth Forceps | Roboz | RS-5152 | For griping spinous processes. |
| Red Oil | N/A | N/A | To provide a front for visualization of virus entering tissue during injection. |
| Retractors | Roboz | RS-6510 | To hold open the surgical wound. |
| Rimadyl Tablets | Bio Serv | MP275-050 | For pain management post-surgery. |
| Rongeurs | Roboz | RS-8300 | To remove muscle from the spinal column during surgery. |
| Scalpel Blade Handle | Roboz | RS-9843 | To slice open skin and fat pad of animal model during surgery. |
| Scissors | Roboz | RS-5980 | For general use in surgery. |
| Stainless Steal Wound Clips | CellPoint | 201-1000 | To bind the skin of the surgical wound during closing. |
| Staple Removing Forceps | Kent Scientific | INS750347 | To remove the staples, should they be applied incorrectly. |
| Sterile Cloth | Phenix Research Products | BP-989 | To provide a sterile surface for the operation. |
| Sterile Cotton-Tipped Applicators | Puritan | 806-WC | To soak up blood in the surgical wound while maintaining sterility. |
| Sterile Gauze | Covidien | 2146 | To clean the surgical area and surgical tools while maintaining sterility. |
| Sterile Saline | Baxter Healthcare Corporation | 281324 | For use in blood clearing, and for replacing fluids post-surgery. |
| Surgical Gloves | N/A | N/A | For use by the surgeon to maintain sterile field during surgery. |
| Surgical Heating Pad | N/A | N/A | For maintaining the body temperature of the animal model during surgery. |
| Surgical Microscope | N/A | N/A | For enhanced visualization of the surgical wound. |
| Surgical Stapler | Kent Scientific | INS750546 | To apply the staples. |
| T/Pump Heat Therapy Water Pump | Gaymar | TP500C | To pump warm water into the water convection warming pad. |
| Water Convection Warming Pad | Baxter Healthcare Corporation | L1K018 | For use in the post-operational recovery area to maintain the body temperature of the unconscious animal. |
| Weighted Hooks | N/A | N/A | To hold open the surgical wound. |
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Citer Cet Article
Keefe, K. M., Junker, I. P., Sheikh, I. S., Campion, T. J., Smith, G. M. Direct Injection of a Lentiviral Vector Highlights Multiple Motor Pathways in the Rat Spinal Cord. J. Vis. Exp. (145), e59160, doi:10.3791/59160 (2019).