在脊髓损伤的实验挫伤型号神经干细胞移植
Summary
脊髓损伤是一种创伤性条件，导致严重的发病率和死亡率高。在这项工作中，我们详细描述脊髓损伤的小鼠，随后通过神经干细胞的移植挫伤模型。
Abstract
脊髓损伤是一种破坏性的临床状况，其特征在于神经机能障碍的复合体。脊髓损伤的动物模型，可以同时使用，调查的生物反应损伤和测试潜在疗法。挫伤或压迫损伤传递到外科手术暴露脊髓是最广泛使用的模型的病理状况。在此报告的实验挫伤通过使用无穷水平（1H）撞击的设备，它允许创建一个可再现损伤动物模型中，通过具体的损伤参数定义进行。干细胞移植通常被认为用于固化本衰弱病症的潜在有用的策略。大量的研究已经评估移植了各种干细胞的影响。在这里，我们证明了脊髓损伤后尾静脉注射细胞在CD1小鼠的适应方法。总之，我们提供的程序：一）细胞标记无线网络第一个重要的示踪物，ⅱ）手术前护理小鼠，ⅲ）执行的挫伤性脊髓损伤，和验尸神经前体的四）静脉内给药。这个挫伤模型可以用于评估在再生医学的方法的有效性和干细胞移植的安全性。
Introduction
脊髓损伤（SCI）是由高能量创伤样机动车事故中最常见的损伤，跌伤，运动和暴力1。在严重的SCI，伤害力摧毁或损害的神经组织，引起神经功能突然丧失。外伤性脊髓损伤经常发生在10和40岁之间的青壮年。这极大地影响了患者的精神和身体状况，并导致巨大的经济影响，社会2。在急性期的治疗方法往往仅限于高剂量糖皮质激素，手术稳定和减压，以减轻可能进一步损害3-4，但是SCI后，这些方法在运动恢复中的作用仍存在争议。除了 急性组织丢失，跌打损伤和退行性变引起脱髓鞘多种细胞类型5-6和死亡继发性机制的激活。功能恢复程度可以了10被关联到幸免脑白质的损伤部位7的程度。
脊髓损伤的动物模型，可以使用这两种，调查组织损伤的生物反应，并测试潜在的治疗方法。此外，人体病理学的有用的动物模型不仅具有重现该条件的某些方面，但还必须提供超过直接的临床观察和实验的优点。脊髓损伤的最广泛使用的模型包括挫伤或压迫损伤传递到外科手术暴露脊髓8。受控体重下降挫伤的发展代表了SCI研究史上的一个重要里程碑。俄亥俄州立大学脊髓研究中心一直追求可用于诱导脊髓与冲击的由计算机9控制参数的特定压缩设备的技术挑战。这原本是专为使用无线网络日大鼠;后来它被修改申请对小鼠10。这种方法的优点是，损伤的生物力学能在深入研究以上和损伤的参数可以在一个更完整的方式，以获得一个重现的实验模型来限定，因此，允许的效果更精确的评估测试治疗对功能恢复的过程。
许多研究已评估了移植的效果在脊髓损伤模型11的各种干细胞。我们最近分离出的子脑室区（SVZ）几个小时成人神经干细胞的老鼠捐助12-13死后。此过程提供了神经干细胞，称为后验神经前体（PM-NPC的），这似乎是在再生医学的方法用于治疗脊髓损伤有利的群体。在本文中，我们将证明：i）在细胞标记协议的重要示踪剂PKH26，II）的外科学的iCal过程在外伤性脊髓损伤执行，以及iii）在静脉内（IV）标记的细胞的施用。另外，在本工作中，我们表明，移植的细胞移植到脊髓损伤部位并分化大多成微管相关蛋白（MAP）2阳性的细胞。此外，该分化伴有促进后肢功能的稳定的恢复。
Protocol
Representative Results
Discussion
在本文中，我们描述了在70克迪涅（重度）的力的方法来获得外伤性脊髓损伤的可重复使用的模型是无限的地平线撞击。使用较大的力的范例（80克迪涅），我们可能会导致不幸的是具有较高的小鼠的死亡率相关联的更严重的伤害。为了避免这个问题，我们一般选择温和的力量范式（70克迪涅），使用功能和较低的死亡率逐渐复苏关联到一个可重复的病变。为了产生这样的稳定损伤是非常重要?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
The Authors acknowledge the economic support by FAIP (Federazione Associazioni Italiane Paraplegici), “Neurogel-en-Marche” Foundation (France), Fondazione “La Colonna”.
Materials
| Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
| PKH26GL-1KT | Sigma | 091M0973 | |
| Infinite horizon (IH) Impactor device | Precision Systems and Instrumentation, LLC | Model 0400 Serial 0171 | |
| Gentamycin 10mg/ml | Euroclone | ECM0011B | 1mg/ml in sterile saline solution |
| Isoflurane-Vet 250ml | Merial | B142J12A | |
| Blefarolin POM OFT 10g | |||
| Slide Warmer | 2Biological Instruments | HB101-sm-402 | |
| Scalpel, size 10 | Lance Paragon | 26920 | |
| Small Graefe Forceps | 2Biological Instruments | 11023-14 | |
| Rongeur | Medicon Instruments | 07 60 07 | |
| Micro scissors | 2Biological Instruments | 15000-00 | |
| Absorbable sutures (4/0) | Safil Quick | C0046203 | |
| Hemostat | 2Biological Instruments | 13014-14 | |
| Reflex 7 wound clip applicator | 2Biological Instruments | 12031-07 | |
| 7mm Reflex wound clips | 2Biological Instruments | 12032-07 | |
| NGS | Euroclone | ECS0200D | |
| Triton X 100 | Merck Millipore | 1086431000 | |
| Anti Microtubule Assocoated Protein (MAP) 2 | Millipore | AB5622 | |
| Alexa Fluor 488 | Invitrogen | A11008 | |
| FluorSave Reagent | Calbiochem | 345789 | |
| Neural stem cells medium | DMEM-F12 medium (Euroclone) containing 2 mm l-glutamine (Euroclone), 0.6% glucose (Sigma-Aldrich), 9.6 gm/ml putrescine (Sigma-Aldrich), 6.3 ng/ml progesterone (Sigma-Aldrich), 5.2 ng/ml sodium selenite (Sigma-Aldrich), 0.025 mg/ml insulin (Sigma-Aldrich), 0.1 mg/ml transferrin (Sigma-Aldrich), and 2 μg/ml heparin (sodium salt, grade II; Sigma-Aldrich), bFGF (human recombinant, 10 ng/mL; Life Technologies) and EGF (human recombinant, 20 ng/mL; Life Technologies) | ||
| DMEM-F12 | Euroclone | ASM5002 | |
| l-glutamine | Euroclone | ECB3000D | |
| glucose | Sigma-Aldrich | G8270-100G | |
| putrescine | Sigma-Aldrich | P5780-25G | |
| progesterone | Sigma-Aldrich | P6149-1MG | |
| Sodium-selenite | Sigma-Aldrich | S9133-1MG | |
| transferrin | Sigma-Aldrich | T 5391 | |
| Insulin | Sigma-Aldrich | I1882 | |
| Heparin sodium-salt | Sigma-Aldrich | H0200000 | |
| bFGF | Life Technology | PHG0024 | |
| h-EGF | Life Technology | PHG6045 | |
| Syringe 0.33cc 29G | Terumo | MYJECTOR | |
| buprenorphine | Schering Plough SpA | TEMGESIC | |
| eye gel | Bausch & Lomb | LIPOSIC |
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