氟 TrFE 导管内许旺细胞移植桥断大鼠脊髓残肢促进轴突再生跨越间隙
1The Miami Project to Cure Paralysis,University of Miami Miller School of Medicine, 2Department of Materials Science and Engineering,New Jersey Institute of Technology, 3Department of Biomedical Engineering,New Jersey Institute of Technology, 4Department of Cell Biology,University of Miami Miller School of Medicine, 5Department of Neurological Surgery,University of Miami Miller School of Medicine
Summary
本文介绍了一项技术, 插入一个中空导管之间的脊髓残肢完全横断面和填充雪旺细胞 (SCs) 和可注射基底膜基质, 以桥梁和促进轴突再生跨越的差距。
Abstract
在大鼠脊髓损伤的各种模型中, 钝挫伤模型是最常用的, 因为它是最常见的类型的人脊髓损伤。完整的横断面模型, 虽然与钝挫伤模型没有临床相关性, 但却是评价轴突再生最严格的方法。在钝挫伤模型中, 由于存在残余的组织损伤, 很难区分再生与发芽或幸免的轴突。在完整的横断面模型中, 需要一个桥接方法来填补间隙, 并从侧到尾端以建立连续性, 以评估治疗效果。一个可靠的搭桥手术是必要的测试结果的措施, 减少因手术方法的变异性。此处所述的协议用于在移植前制备雪旺细胞 (scs) 和导管, 在胸椎8级 (T8) 上完全横断脊髓, 插入导管, 并将 SCs 移植到导管中。这种方法也使用原位胶凝剂基底膜基质与 SC 移植, 允许改善轴突生长横跨侧和骶管接口与宿主组织。
Introduction
脊髓损伤修复是一个复杂而富有挑战性的问题, 需要一个组合治疗策略, 例如, 使用细胞和生物材料为移植细胞功能和轴突提供有利的微环境损伤部位再生。半 1,2,3,4,,5,6,7,8 和完全横断9 ,11,12,131415161718 、20、21、22模型经常用于评估 biomaterial-based 桥接疗法的效果。使用半模型的优点是它为桥接结构提供了更稳定的比完全横断面。然而, 在半模型中, 由于无组织的存在, 很难证明轴突再生是应用治疗方法的结果。完全横断面模型是显示轴突再生的最严格的方法。
各种天然和合成材料已被研究用于注射凝胶, 预先凝胶放置在挫伤或半模型, 或作为一个结构的管道到半或完整的横断面模型 (详细的审查23,24,25).原位可注射基质/sc 合剂的凝胶性在移植物和轴突交叉的主绳之间创建一个更宽松的接口26,27与 pre-gelled 基质/sc 种植体相比5,18,19,28.原位胶凝允许矩阵在不规则的主机接口周围进行轮廓, 而更刚性和结构化的导管或较不模压的预先凝胶不能。与注射基质相比, 结构化导管通常提供接触引导和植入稳定性。这里介绍的协议描述了一个手术过程, 既利用了可注射基底膜基质的优势 (例如,基质, 参见材料表,称为可注射基质), 以及结构化管道评价轴突再生的最严格的脊髓损伤模型。
纺聚 vinylidenedifluoride-乙烯 (PVDF-TrFE) 对齐的纤维空心导管用于我们的实验方法。PVDF-TrFE 是一种压电聚合物, 它在机械变形时产生瞬态电荷, 并被证明可以促进突起扩展和轴突再生,无论是体外29、30还是在体内31. 静电纺丝是一种常用的脚手架制作方法, 可以使用各种可控性能的聚合物快速生成可靠的纤维支架, 如纤维对齐、纤维直径和脚手架的厚度神经和其他应用程序32,33,34。
大量研究大鼠 SCs 移植到脊髓损伤部位已显示治疗效果5,9,18,19,20,,21 ,26。这些移植是神经保护的组织周围的病变, 减少病变腔大小, 并促进轴突再生到病变/移植部位和鞘再生轴突。人体 SCs 可以 autologously 移植, 比其他大多数神经相关细胞24的优势。经外周神经活检, SCs 可以分离和纯化, 并将激增到预期的数量, 移植到人类。自体 SC 移植治疗脊髓损伤患者已被证明是安全的在伊朗35,36,37,38, 中国39,40, 以及美国41,42。众所周知, SCs 分泌大量的神经营养因子和胞外基质蛋白对轴突生长起重要作用, 并在周围神经损伤后的轴突再生中起着必不可少的功能。我们的目标是描述一种方法, 可以研究导管设计, 以改善大鼠脊髓横断模型中 SC 移植的结果。
Protocol
根据 NIH 和美国农业部的指导原则, 女性成年休鼠 (180-200 和 #160; g 体重) 被安置。迈阿密大学机构动物保育和使用委员会 (IACUC) 批准了所有的动物程序. 1. 移植前准备 导管准备。 在解剖显微镜下使用 #10 刀片将管道长度削减到5毫米. 注: 导管内径介于 2.4-2.7 毫米之间;外径介于 2.5-2.8 mm 之间. 沿纵向侧轻轻折叠导管 ( 图 1A…
Representative Results
使用这种手术技术的目的是评估使用的结构导管和注射基质, 最大限度地发挥 SC 功能后移植到完成断脊髓。移植后三周, 动物被灌入4% 甲醛, 脊柱被严重解剖, 并固定在同一定影液中, 用于另24小时。然后解剖脊髓, 将低温矢状切面的样品放入30% 蔗糖溶液中冻。从另一组解剖脊髓的 SC 桥中间分离出来的1毫米厚的横断面被放进戊二醛固定剂中, 用于塑料切片。样品被服从于电?…
Discussion
建立有效的横断面模型的最关键的一步是在一个或两个切口切断脊髓。2-2. 5 毫米之间的间隙之间的侧和尾部脊髓残肢应存在于横断现场。三最可能的原因, 这样的差距没有出现的是 (1) 背部/腹根没有被正确删除, (2) 腹侧硬脑膜没有被充分删除, 和/或 (3) 该动物没有正确放置在她下面的轧辊。
在树桩之间进行有效的导管插入: (1) 导管的直径应根据实验中所用动物的特定种类和年…
Divulgations
The authors have nothing to disclose.
Acknowledgements
我们要感谢在迈阿密项目的病毒载体和动物核心, 以治疗瘫痪的生产 lenti-GFP 病毒和提供动物护理, 分别和组织学和成像核心的使用低温, 共焦显微镜,荧光显微镜与立体侦探。资金由一 (09923), 国防部 (W81XWH-14-1-0482) 和 NSF (DMR-1006510) 提供。鹰是克里斯汀·林恩杰出的神经科学教授。
Materials
| Cryogenic vials | ThermoFisher Scientific | 5000-0020 | |
| 10 cm Petri dish | VWR | 25382-428 | |
| Dulbecco's modified Eagle's medium: nutrient mixture F-12 | ThermoFisher Scientific | 11039-021 | "DMEM/F12" in protocol. |
| Penicillin-streptomycin | ThermoFisher Scientific | 15140-122 | "Pen/Strep" in protcol. |
| Fetal bovine serum | Hyclone | SH300-70-03 | "FBS" in protocol. |
| Pituitary extract | Biomedical Technologies | BT-215 | |
| Forskolin | Sigma-Aldrich | F6886 | |
| Heregulin | R&D Systems | 396-HB/CF | |
| Poly L-lysine | Sigma-Aldrich | P2636 | "PLL" in protocol. |
| Dulbecco's modified Eagle's medium | ThermoFisher Scientific | 11965-092 | "DMEM" in protocol. |
| Hank's balanced salt solution | ThermoFisher Scientific | 14170-112 | "HBSS" in protocol. |
| Tryspin-EDTA | ThermoFisher Scientific | 15400-054 | |
| Female Fischer rat (160-180g) | Envigo | ||
| Vannas scissor, straight | FST | 15018-10 | |
| Ketamine | Vedco Inc | 5098976106 | 100 mg/ml |
| Xylazine | Lloyd Inc | AnaSed | 20 mg/ml |
| Gentamycin | APP Pharmaceuticals | NDC 63323-010-02 | Can be any brand of choice. |
| Micro Spatula | FST | 10089-11 | Can be any brand of choice. |
| Curved scissors with blunt end | FST | 14017-18 | Can be any brand of choice. |
| Blunt forceps | FST | 11006-12 | Can be any brand of choice. |
| rongeur | FST | 16121-14 | Can be any brand of choice. |
| Angled spring scissors | FST | 15006-09 | Can be any brand of choice. |
| Absorption triangles | FST | 18105-03 | Can be any brand of choice. |
| Gelfoam | Henry Schein | 9083300 | "Compressed foam" in protocol. |
| #10 blades | Sklar | 06-3010 | Can be any brand of choice. |
| Matrigel | Corning | 354234 | "Injectable matrix" in protocol. |
| Chicken anti-green fluorescent protein antibody | Millipore | AB16901 | |
| Mouse RT97 hybridoma antibody | DSHB | RT97 | |
| Rabbit anti-neurofilament antibody | Encor Biotechnology, Inc | PRCA-NF-H | |
| Polyclonal Rabbit anti-Glial Fibrillary Acidic Protein antibody | Dako | Z033401 | |
| Alexa Fluor 488 goat anti-chicken IgG (H+L) | ThermoFisher Scientific | A-11039 | |
| Alexa Fluor 546 goat anti-rabbit IgG (H+L) | ThermoFisher Scientific | A-11035 | |
| Alexa Fluor 647 goat anti-rabbit IgG (H+L) | ThermoFisher Scientific | A-21244 | |
| Alexa Fluor 647 goat anti-mouse IgG (H+L) | ThermoFisher Scientific | A-21236 | |
| Confocal Microscopy | Nikon | clsi |
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Citer Cet Article
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