小鼠脑脊髓子宫转导后超微结构神经可塑性参数的评价
Summary
透射电子显微镜和子宫转导的结合是研究神经系统发育过程中精细超微结构形态变化的有力方法。这种组合方法可以深入了解神经可塑性相对于其地形表现的结构细节的变化。
Abstract
本研究结合子宫转导和透射电子显微镜 (tem), 旨在精确地对地形结构中的超微结构参数进行形态测量分析, 这是受感兴趣的蛋白质影响的通过病毒转移引入生物体。这种组合方法允许从宏观结构到超微结构识别的顺利过渡, 通过遵循组织地图集中的地形导航地图。子宫内转染组织的高分辨率电子显微镜揭示了神经 pil 的精细超微结构及其可塑性参数, 如横截面突触 bouton 区、内突触囊和线粒体的数量。bouton 剖面、突触线长度、横截面轴突区、髓鞘厚度、髓鞘层数和线粒体剖面横截面区。对这些参数的分析揭示了对神经系统区域超微结构可塑性变化的重要洞察, 这些变化受到基因结构病毒转移的影响。这种组合方法不仅可以用来研究基因工程生物分子和药物对神经元可塑性的直接影响, 而且还为研究子宫内神经元可塑性的抢救提供了可能性 (例如, 在神经退行性疾病)。
Introduction
任何光子都不能穿透电子深度等级的超薄组织标本。与光学显微镜技术相比, 这在捕获精细结构的纳米分辨率图像方面具有宝贵的优势。例如, tem 允许细胞内细胞器的可视化, 如线粒体、黑色素体和各种类型的分泌颗粒、微管、微丝、纤毛、微绒毛和细胞间连接 (细胞表面)(专业), 特别是突触在神经系统1,2,3,4。本方法研究的总体目标是结合子宫内的最先进技术, 对产前干预后发育过程中神经可塑性变化的超微结构识别。病毒编码的感兴趣的蛋白质在子宫中被转化为中枢神经系统5,6,7, 包括脊髓6。例如, 在子宫转导联合 tem 已被用来研究细胞粘附分子 l1 对 l1 缺乏小鼠运动学习可塑性的影响, 特别是在 l1 和核受体蛋白之间的相互作用在小脑神经元7。
神经可塑性参数的分析需要关于神经系统中最小区域定位的精确信息。因此, 它是足够的描述超微结构细节和他们的确切地形方向相对于其他结构。在本研究中, 提出了一种基于光镜和电子显微镜的具体准备方法, 旨在对不同的形态区域进行详细的研究。这种方法结合了组织操作的几种技术, 从小鼠大脑和脊髓的子宫转导开始, 然后是灌注固定, 霉菌包埋, 并处理组织的 tem。在透射电镜组织的嵌入和处理之间的一个重要步骤是组织的记录, 使用干涉光反射技术, 允许精确的微照片和低放大倍率的记录组织标本8,9,10。在目前的方法中, 这项技术使研究人员能够在准备 tem 之前检查神经组织表面和标本切片剖面的地形和结构细节。
一个特殊的框架, 用于分割整个大脑对应于立体定向坐标。该框架有利于神经组织中区域的形态三维重建, 可用于形态分析。可视化剖面的宏观图被指定为地形坐标, 按顺序编号的部分在组织地图集中生成地图。
根据上述组织地图集的地图, 在树脂处理后, 将嵌入组织切割成含有选定区域的超薄切片 (& lt;70 nm)。超薄切片采用透射电镜, 以获得其内容物的可塑性参数 (例如突触波或轴突纤维的横截面剖面区域) 的高分辨率图像, 以及与复合物内相邻结构的接触的图像神经皮。
通过本文所述的方法, 从可视化宏观结构到微观和纳米结构的平稳过渡, 使发育中神经的子宫传导后的形态神经元可塑性进行比较深入的研究系统。
Protocol
关于动物问题的所有程序都得到了德国联邦各州汉堡和北莱茵-韦斯特法伦的动物伦理机构委员会的批准。 在整个外科过程中使用无菌仪器、防护手套和无菌外套。 1. 子宫传导 在 ph 值7.4 的磷酸盐缓冲盐水 (pbs) 中, 为所需的靶标 (4 x10 11 病毒凝集物μμμl) 准备与腺病毒相关的 1 (aav1) 编码。加入 0.1 mgμμl 快速绿色, 并将 aav1-fast-绿色混合物保持在37°c。 ?…
Representative Results
为了对小鼠进行可靠、快速的麻醉, 考虑了许多安全参数, 并且麻醉单元的优化工作空间被证明是足够的 (图 1a)。该装置旨在精确控制液体异氟烷和环境空气的混合物, 并对小动物 (如小鼠和大鼠) 进行成功的手术。空气和异氟醚根据所需的设置混合在蒸发器中, 并通过面罩或通过面罩传送到动物体内 (图 1a</stron…
Discussion
在子宫转导的一个关键的步骤是注射程序。精确注射到脑室或其他感兴趣的领域需要经验和动手技巧。微毛细管尖端越薄, 可能发生的组织损伤越少;然而, 这是以增加注射压力为代价的。与子宫电穿孔19、20、21、22 相比, 在子宫转导后注射胚胎的存活率非常高。所有子宫角的胚胎都可以注射, 即使胚胎?…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
作者感谢鲁尔大学波鸿医学院动物设施的同事们的支持和动物护理。
Materials
| 2,4,6-Tris(dimethyl-aminomethyl)phenol | Serva | 36975 | |
| 26Gx 1'' needle | Henke-Sass, Wolf GmbH | ||
| 410 Anaesthesia Unit for air pump | Biomedical Instruments (Univentor) | 8323102 | |
| Adeno-associated virus serotype 1 (AAV1) | UKE (Viral Core Facility) | – | For references and target areas of AAV1 see: https://www.addgene.org/viral-vectors/aav/aav-guide/ and also: Designer gene delivery vectors: molecular engineering and evolution of adeno-associated viral vectors for enhanced gene transfer. Kwon I, Schaffer DV. Pharm Res. 2008 Mar;25(3):489-99. Recombinant AAV viral vectors pseudotyped with viral capsids from serotypes 1, 2, and 5 display differential efficiency and cell tropism after delivery to different regions of the central nervous system. Burger C, Gorbatyuk OS, Velardo MJ, Peden CS, Williams P, Zolotukhin S, Reier PJ, Mandel RJ, Muzyczka N. Mol. Ther. 2004 Aug;10(2):302-17. Self-complementary recombinant adeno-associated virus (scAAV) vectors promote efficient transduction independently of DNA synthesis. McCarty DM, Monahan PE, Samulski RJ. Gene Ther. 2001 Aug;8(16):1248-54. |
| Agarose | Sigma-Aldrich | A9414 | low gelling agarose |
| Air Pump | Biomedical Instruments (Univentor) | Eheim 100 | |
| Araldite | CIBA-GEIGY | 23857.9 | resin for embedding of tissue |
| aspirator tune assemblies | Sigma-Aldrich | A5177-5EA | |
| Breathing Mask Mouse Anodized Aluminium | Biomedical Instruments (Univentor) | – | |
| buprenorphine | Temgesic | ampules | painkiller |
| capillaries | Science-Products | GB100TF-10 | with fillament |
| Dodecenylsuccinic anhydride | Fluka | 44160 | |
| Dumont tweezers (#3, 12 cm, straight, 0.2 x 0.12 mm) | FST | 11203-23 | |
| electric shaver | Phillips | – | |
| Ethicon sutures (Ethilon, 6-0 and 3-0) | Ethicon | – | polyamide |
| eye lubricant | Bepanthene | – | |
| Fast Green | Sigma-Aldrich | F7252 | for visualization of injected liquids |
| Gas Routing Switch 4/2 connectors | Biomedical Instruments (Univentor) | 8433020 | |
| halsted Mosquito hemostatic forceps (12.5 cm, straight) | FST | 13011-12 | |
| Heparin-Natrium | Ratiopharm | 25 000 I.E./5 ml | |
| Induction box for mice with horizontally moving lid. Inner dimensions: LxBxH: 155x115x130 mm. Wall thickness: 6 mm |
Biomedical Instruments (Univentor) | – | |
| iris forceps (10cm, curved, serrated) | FST | 14007-14 | |
| iris scissors (11cm, straight, tungsten carbide) | FST | 14501-14 | |
| Isofluran OP Tisch, electrically heated, sm Outer dimensions: 257x110x18 mm. Heating area: 190×90 mm The removal of the isoflurane escaping the breathing mask is downwards in compliance with the regulations |
Biomedical Instruments (Univentor) | – | |
| isoflurane (Attane) | JD medical | inhalation anesthesia | |
| LED RGB lights | Cameo | CLQS15RGBW | LEDs 2 x 15 W |
| Light microscope Basic DM E | Leica | – | 4x (N.A. 0.1 ∞/-), 10x (N.A. 0.22 ∞/0.17), 40x (N.A. 0.65 ∞/0.17), 100x (N.A. 1.25 ∞/0.17) objectives |
| micropipette puller | Science-Products | P-97 | |
| Mosquito hemostatic forceps (12.5cm, curved) | FST | 13010-12 | |
| Nickel grids, 200 mesh | Ted Pella | 1GC200 | |
| Osmium (VIII)-oxid | Degussa | 73219 | |
| Propylene oxide | Fluka | 82320 | |
| razor blades | Schick | 87-10489 | |
| Sodium pentobarbital (Narcoren) | Merial GmbH | – | |
| TC01mR 1-Channal temperature controller with feedback | Biomedical Instruments (Univentor) | – | |
| Technovit 4004 two components glue | Kulzer | ||
| Telemacrodevice | Canon | – | Canon Spiegelreflex Kamera EOS2000D, EF-S 18-55 mm f/3.5-5.6 IS STM Objective, Extension below 150 mm, Manual Extension Tube 7 mm ring, 14 mm ring, 28 mm ring, Macro reverse ring (58 mm), Canon copy stand. |
| Thermopuller P-97 | Sutter Instruments | – | |
| thin vibrating razor blade device | Krup | – | with Szabo thin blades |
| toluidine blue | Sigma-Aldrich | 89640 | |
| Transmission electron microscope C20 | Phillips | – | up to 200 kV |
| Tygon 6/4 Tubing material for connection of all parts Outer diameter: 6mm Inner diameter: 4mm Wa ll thickness: 1mm |
Biomedical Instruments (Univentor) | – | |
| Ultracut E | Reichert-Jung | – | ultramicrotome |
| Univentor Scavenger | Biomedical Instruments (Univentor) | 8338001 | |
| Vannas scissors (8 cm, straight) | FST | 15009-08 |
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Lutz, D., von Düring, M., Corvace, F., Augustinowski, L., Trampe, A., Nowak, M., Förster, E. Assessment of Ultrastructural Neuroplasticity Parameters After In Utero Transduction of the Developing Mouse Brain and Spinal Cord. J. Vis. Exp. (144), e59084, doi:10.3791/59084 (2019).