使用脑和脊柱联合手术用双病毒载体靶向新生大鼠的皮质脊髓束
Summary
该协议展示了一种通过将顺行性化学遗传修饰剂注射到体细胞运动皮层和逆行可运输的Cre重组酶到颈脊髓中，将基因疗法应用于出生后5-10天新生大鼠细胞亚群的新方法。
Abstract
成功解决限制新生大鼠研究的障碍对于研究小儿脊髓损伤（SCI）与成年SCIs相比结果的差异非常重要。此外，将疗法可靠地引入中枢神经系统（CNS）的靶细胞可能具有挑战性，不准确可能会损害研究或治疗的疗效。该方案将病毒载体技术与一种新的手术技术相结合，以在出生后第5天将基因疗法准确地引入新生大鼠。在这里，一种设计用于Cre逆行运输（retroAAV2）的病毒被引入脊髓中皮质脊髓神经元的轴突末梢，随后被运送到细胞体。然后将由设计药物（DREADD）病毒专门激活的双絮体倒定向（DIO）设计受体注射到大脑的躯体运动皮层中。这种双重感染技术仅在共同感染的皮质脊髓束（CST）神经元中促进DREADD的表达。因此，同时共注射躯体运动皮层和宫颈CST末端是研究新生大鼠宫颈SCI模型后恢复的化学遗传调控的有效方法。
Introduction
虽然SCI在儿科人群中相对罕见，但它特别具有创伤性，并导致永久性残疾，需要巨大的后勤远见。此外，与成人人群相比，被归类为宫颈和完全性的儿童 SCI 的比例更高1，2。哺乳动物物种的一个标志是新生儿从SCI中恢复明显优于成人，这为评估年轻种群恢复的驱动机制提供了机会3，4，5。尽管如此，针对新生儿和婴儿啮齿动物研究的多模态研究较少，部分原因是在更严格的年轻动物解剖标志中准确靶向选定的神经元群体增加了难度6。本文重点研究高效顺行和逆行腺相关载体直接注射到大鼠脊髓中，以应用Cre依赖性DREADDs调节主要运动通路，扩大多模态再生研究的范围。
病毒载体是重要的生物工具，应用广泛，包括引入遗传物质以替代靶基因、上调生长蛋白以及追踪中枢神经系统7，8，9 的解剖景观。脊髓运动通路的许多解剖学细节已经使用经典示踪剂（即生物素化的葡聚糖胺）进行了研究。虽然传统的示踪剂在挖掘神经解剖学方面发挥了重要作用，但它们并非没有缺点：即使正确注射，它们也会不分青红皂白地标记通路，研究发现它们被受损的轴突吸收10，11，12。因此，这可能导致再生研究中的错误解释，其中切断的轴突可能会被误认为再生纤维。
以下方法利用最近在调制研究中推广的双病毒载体系统，在同一神经元13，14的两个独立区域中使用两种不同的病毒载体。第一种是局部感染投射神经元细胞体的载体。另一个是从投影神经元的轴突末端运输的逆行向量（图1）。逆行载体携带Cre重组酶，局部载体包含“Cre-On”双絮凝序列，其中编码荧光蛋白（mCherry）。表达hM3Dq和mCherry的天然转基因相对于启动子倒置，两侧有两个LoxP位点（图2）。因此，mCherry仅在双转导的投射神经元中表达，其中Cre重组酶诱导LoxP位点之间的重组事件，将转基因的方向翻转到适当的阅读框并允许DREADD和荧光蛋白的表达。一旦病毒转基因处于正确的方向，并且在适用的情况下，DREADDs可以通过单独注射的配体（即氯氮平-N-氧化物）瞬时诱导神经调控。该协议旨在验证新生儿的诱导神经调控研究，其中注射DREADDS以选择性地调节CST。双病毒系统充当保险单，确保每个DREADD阳性细胞在荧光下以高保真度进行追踪，以验证注射。
这种方法也有助于弥合新生儿研究的差距。儿科SCI提出了挑战，分析再生，发芽或可塑性的研究应强调新生儿和成人之间的差异3，15，16，17。通过优化手术程序并使用Nissl染色进行先前的解剖学研究，验证了颅脑和脊柱注射的坐标。目的是提供一种对新生大鼠进行双重注射的方法，以提高保真度和生存能力。
对于当前模型，使用bregma作为参考18，19将顺行载体注射到躯体运动皮层的细胞体中。在脊柱注射方面，逆行载体被注射到CST轴突末端所在的叶片V-VII中20，21。有许多基本问题隐藏在某些病变模型如何以不同的方式影响年幼的动物，以及随后的恢复如何与老年动物不同。这项研究展示了研究新生儿啮齿动物宫颈损伤和前肢功能恢复性的可靠方法。相比之下，以前的大多数研究都涉及腰椎或胸部损伤后的恢复运动5，22，23，24。通过将双病毒载体与此处描述的新型注射技术配对，该方案有助于减轻可能困扰新生儿啮齿动物研究的某些问题（即生存能力）。这种方法稳健、实用且用途广泛：技术的微小变化将允许针对不同的途径，即腹侧 CST、背侧 CST 和上升背侧通路。
对于该系统，将一种局部作用的病毒（例如AAV2）注射到感兴趣的神经元细胞体区域。控制局部病毒表达的第二种逆行运输病毒在该神经元群体的轴突末端注射。因此，根据定义，只有皮质脊髓神经元被标记。选择具有组成活性的CMV启动子的retroAAV-Cre病毒，因为穿梭质粒用于在几种细胞类型中产生几种AAV血清型，用于Cre依赖性表达。对于皮质注射，选择AAV2与由突触素-1启动子驱动的转基因一起，以限制对神经元的任何表达。由于2病毒系统更多地依赖于感兴趣的神经元群体的起源和终止，因此可以使用几种不同的启动子，如果它们可以驱动感兴趣的神经元群体中目的基因的表达。例如，兴奋性神经元启动子CamKII可以替代突触素-1。除了使用这些AAV血清型外，还可以使用高逆行可运输慢病毒（HiRet）来实现逆行转运到未成熟，并且在较小程度上，成人皮质脊髓运动神经元也可以实现。HiRet慢病毒使用嵌合狂犬病/VSV糖蛋白靶向突触摄取以进行逆行转运。结合Tet-On启动子，该2病毒系统以逆行依赖性方式支持诱导表达26，27。
逆行病毒将载体插入目标神经元的突触空间，使其被该细胞的轴突吸收并运输到细胞体。虽然慢病毒载体以前取得了巨大的成功，在基因治疗研究中提供了长期表达，但这种方法转向腺相关病毒载体有几个简单的原因26，28： AAV更经济，同样有效，并且后勤负担更少，因为它具有较低的生物安全水平名称29，30，31，32.虽然最常用的血清型AAV2显示出CST轴突的稳健转染，但未来的研究人员可能会注意到AAV1在跨标记时提供了一些多功能性，因此在未来的研究中提出了几种可能的迭代33。最后的适应是用Cre重组酶对逆行病毒进行编码，以便可以同时引入多个顺行载体，从而减少不必要的内部病毒浪费，并最大限度地提高DREADD以正确方向表达的可能性。
最终，该方案展示了同时注射到皮质和颈椎中，分别针对皮质脊髓束的细胞体和轴突末梢。高保真转染可见于大脑皮层和脊髓。虽然所描述的方案对于 Sprague Dawley 大鼠 5 天大鼠来说是完善的，但它适用于出生后 4-10 天，对麻醉和立体定向坐标进行细微调整。
Protocol
以下所有外科和动物护理程序均已获得天普大学动物护理和使用委员会的批准。所描述的方案是一种生存手术，动物最终在完成时间点时通过腹膜内注射100mg / kg戊巴比妥钠被安乐死。 1. 术前准备 使用3.5nL玻璃毛细管移液器准备至少两个用于病毒注射的拉动玻璃针;一根针用于DREADD，一根针用于rCre。作为预防措施，准备4-5根针，以防它们在术中断裂。 使用微型?…
Representative Results
病毒载体的成功注射和运输应导致脊髓和运动皮层中单侧神经元的转导。 图 4 显示了表达 Cre-依赖性 DREADDs-mCherry 与对侧脊柱注射 rCre 共同注射的脑冠切片运动皮层中 V 层 CST 神经元的标记。切片用dsRed抗体染色。 图 1：演示本协议中使用…
Discussion
使用可注射的化学遗传修饰剂对大脑活动的诱导遗传调节是研究从SCI恢复的各种机制的有力工具。当考虑到荧光示踪验证了组织学中的解剖学精度时，诱导性G蛋白偶联受体（DREADDs）的靶向准确性进一步提高。本文讨论了一种可靠的方法来探索抑制或刺激选定的神经元通路（具有兴奋性或抑制性DREADDS）是否会导致增强的轴突再生或发芽34，35。将逆行可?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作由Shriners儿童医院SHC-84706的奖学金资助。
Materials
| #11 scalpel blades | Roboz | RS-9801-11 | For use with the scalpel. |
| #10 Scalpel Blades | Roboz | RS-9801-10 | For use with the scalpel. |
| 1 mL Syringes | Becton, Dickinson and Company | 309659 | For anesthetic SC injection and fluid bolus |
| 4.0 silk suture | Ethicon | 771-683G | For skin closure |
| 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 |
| Ketamine (Ketaset) | Zoetis | 240048 | For keeping the animal in the correct plane of consciousness during surgery. |
| Bead Sterilizer | CellPoint | 5-1450 | To heat sterilize surgical instruments. |
| 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. |
| Glass Capillary Tubes | World Precision Instruments | 4878 | For pulled glass needles – should be designed for nanoliter injectors. |
| Hemostats | Roboz | RS-7231 | 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. |
| Lab Standard Stereotaxic Instrument | Stoelting | 51600 | To hold the neonatal sterotaxic holder in place |
| Lab Standard with Mouse & Neonates Adaptor | 51615 | For neonatal skull fixation during cranial surgery and spinal injections | |
| 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. |
| pAAV-CMV-scCre | Wu lab | Cre plasmid | |
| pAAV-hSyn-DIO-hM3Dq-mCherry (plasmid #44361) | Bryan Roth’s lab through Addgene | DREADD plasmid | |
| 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. |
| 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. |
| 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. |
| 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. |
| Liquid bandage | NewSkin | 985838 | To apply along sutures following surgery and encourage wound healing |
| Wire Cage Lamp | ZooMed | LF10EC | To help animals recover from anesthesia and retain warm body temperature naturally |
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Cite This Article
Smit, R. D., Campion III, T. J., Stingel, R. L., Shah, P. H., Chen, J., Smith, G. M. Targeting the Corticospinal Tract in Neonatal Rats with a Double-Viral Vector using Combined Brain and Spine Surgery. J. Vis. Exp. (172), e62698, doi:10.3791/62698 (2021).