椎管内针对肌萎缩性脊髓侧索硬化症和外伤性脊髓损伤颈腹角细胞移植
Summary
神经前体移植是一种很有前途的战略，保护和/或更换丢失/功能失调颈椎膈运动神经元在脊髓损伤（SCI）和运动神经元症，肌萎缩边音硬化症（ALS）。我们提供了一个细胞传递到脊髓型颈椎病ALS和SCI的啮齿类动物模型腹角的协议。
Abstract
呼吸妥协由于膈运动神经元的损失是一个衰弱的后果了大量人力外伤性脊髓损伤（SCI）的例1的比例，是与运动神经元症，肌萎缩硬化症（ALS）2支管患者死亡的最终原因。
ALS是一种破坏性的神经系统疾病的特点是较快的上下运动神经元的变性。屈服于平均2-5年的疾病的患者最终因呼吸麻痹由于膈运动神经元膜片3 innnervation损失的诊断。绝大多数情况下是零星的，而10％的家族形式。大约二十％的家族性病例是在21号染色体 4铜/锌超氧化物歧化酶1（SOD1）基因点突变。转基因小鼠4,5和6大鼠携带突变人类的SOD1基因（G93A，G37R，G86R，G85R）已经产生，并且，尽管存在着其他的动物模型运动神经元的损失，目前该疾病的最高度使用的模型。
脊髓损伤（SCI）是一个异构的条件，造成身体创伤，脊髓功能的结果，根据不同的类型，位置和严重程度的伤害 7 。然而，大约一半人类脊髓损伤病例的影响颈椎的地区，导致呼吸功能障碍衰弱由于膈运动神经元的损失和伤害的降bulbospinal呼吸轴突1。已开发的脊髓损伤动物模型，最常用的和临床相关挫伤8。
各种类的神经前体细胞（NPC的）移植是治疗外伤性中枢神经系统损伤和神经退行性疾病，包括ALS和SCI的一种很有前途的治疗策略，因为更换丢失或功能失调性中枢神经系统的细胞类型，提供神经保护作用的能力，并提供基因因素9利益。
ALS和SCI的动物模型，可以模拟许多这些疾病的临床相关的各个方面，包括膈运动神经元的损失和由此产生的呼吸妥协10,11。为了评估全国人民代表大会为基础的战略，在ALS和SCI这些动物模型的呼吸功能的疗效，必须专门针对蜂窝干预含有治疗膈运动神经元等有关目标的地区。我们提供一个详细的协议为多节段椎管内的NPC移植到颈椎脊髓腹侧灰质神经退行性型号如SOD1的G93A小鼠和大鼠，以及脊髓损伤大鼠和小鼠11。
Protocol
方法 1。细胞制备作为一个例子，我们将介绍的程序编制胶质祖细胞移植，因为我们的经验与此类型的细胞12。然而，该协议的细节，包括中型和胰蛋白酶的使用，例如，将依赖于特定的细胞用于移植的类型。 所有的解决方案前温37.0 ° C的水洗澡。 的HBSS冲洗烧瓶2X。添加5.0 0.05％胰蛋白酶/ EDTA mL/T-75烧瓶。在37.0 ° C培养箱中孵育3分钟烧…
Discussion
研究涉及的SOD1 G93A小鼠和大鼠，年龄和性别匹配的一组内的动物，和分发不同群体的同一窝内的动物。最好是使用ALS和SCI模型相同性别的所有动物，因为疾病的进程可能男性和女性之间有所不同，但是，它也可能是有用的男女双方有足够的动物，以检测出可能特定性别的影响，这一现象已报道G93A SOD1的老鼠13和G93A SOD1的老鼠14,15。考虑细胞移植到动物的年?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
我想感谢：莱波雷的所有成员，Maragakis和罗斯坦有益的讨论实验室;美国和克雷格H.尼尔森基金会拨款瘫痪退伍军人。
Materials
| Name of reagent | Company | Catalog number |
| HBSS | Gibco | 14170 |
| 0.05% Trypsin | Gibco | 25300 |
| Soybean Trypsin Inhibitor (optional) | Sigma | T-6522 |
| Acepromazine maleate (0.7 mg/kg) | Fermenta Animal Health | |
| Ketamine (95 mg/kg) | Fort Dodge Animal Health | |
| Xylazine (10 mg/kg) | Bayer | |
| #11 Feather surgical blade | Electron Microscopy Sciences | 72044-11 |
| Cotton-tipped applicators (6 inch) | Fisher | 23-400-101 |
| Rat-toothed forceps | Fine Science Tools | Rat: 11023-15; Mouse: 11042-08 |
| Medium-sized spring scissors | Fine Science Tools | 15012-12 |
| Mini spring scissors | Fine Science Tools | 15000-10 |
| Rongeur | Fine Science Tools | Rat: 16121-14; Mouse: 16221-14 |
| Microknife | Fine Science Tools | 10056-12 |
| Needle holders | Fine Science Tools | 12502-14 |
| Suture: 4-0 | Vicryl | S-183 |
| Staples: 9 mm | Autoclip | 427631 |
| Stapler: 9 mm (Reflex #203-1000) | World Precision Instruments | 5000344 |
| Warm water pump (T/Pump) | Gaymar | P/N 07999-000 |
| Cyclosporin A: 250.0 mg/5.0 mL ampules | Novartis/Sandimmune | NDC 0078-0109-01 |
| FK-506 | LC Laboratories | F-4900 |
| Rapamycin | LC Laboratories | R-5000 |
| Injector | World Precision Instruments | UMP2 |
| Micro 4 Microsyringe Pump Controller | World Precision Instruments | UMC4 |
| Micromanipulator | World Precision Instruments | Kite-R |
| 10.0 μL Hamilton syringe | Hamilton | 80030 |
| Hamilton needles: 33-gauge, 45° bevel, 1 inch | Hamilton | 7803-05 |
| Glass 20.0 μL microcapillary pipettes (optional) | Kimble | 71900-20 |
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Tags
Intraspinal Cell TransplantationTargeting Cervical Ventral HornAmyotrophic Lateral SclerosisTraumatic Spinal Cord InjuryRespiratory CompromisePhrenic Motor Neuron LossMotor Neuron DisorderRapid DegenerationUpper And Lower Motor NeuronsRespiratory ParalysisDiaphragm InnervationFamilial CasesCu/Zn Superoxide Dismutase 1 GeneSOD1 Gene MutationsTransgenic Mice And RatsAnimal ModelsSpinal Cord InjuryHeterogeneous Conditions
Citazione di questo articolo
Lepore, A. C. Intraspinal Cell Transplantation for Targeting Cervical Ventral Horn in Amyotrophic Lateral Sclerosis and Traumatic Spinal Cord Injury. J. Vis. Exp. (55), e3069, doi:10.3791/3069 (2011).