重复性小鼠坐骨神经挤压和再生的后续评估整装肌肉分析
Summary
在这份报告中，我们描述了一种方法来粉碎小鼠坐骨神经。这种方法使用现成的止血钳和轻松，重现产生完整的坐骨神经。此外，我们描述了一种方法来准备适合分析坐骨神经后神经再生肌肉整个坐骑。
Abstract
再生周围神经系统（PNS）被广泛研究人类疾病的相关了解PNS的神经元，从而有可能阐明中枢神经系统再生1失败安装强大的再生反应。坐骨神经（axonotmesis）的是在啮齿类动物2周围神经损伤的最常见的车型之一。粉碎中断所有轴突，但雪旺氏细胞基部叶片，使再生的最佳3,4保留。这使得研究者，研究正是越来越轴突的互动能力与雪旺氏细胞和基底椎板4。大鼠一般都首选的动物模型实验神经压榨。它们被广泛使用，其损毁坐骨神经提供了一个合理的近似人类神经病变5,4。虽然在规模小于大鼠神经，鼠神经有许多相似的特质。但最重要的，谅解备忘录E型是由于转基因株系的广泛的可用性日益成为宝贵的，现在允许一个关键的单个分子神经再生6，7的详细解剖。此前研究者已经使用了多种方法来产生神经挤压或损伤，包括简单的直角钳，冷藏镊子，止血钳，血管钳，研究者设计的夹8,9,10,11,12。研究者还利用各种方法标记的损伤部位，包括缝合，碳粒子和荧光珠13,14,1。我们描述的方法来获取准确和持久性标志的使用罚款的止血钳和随后的碳爱恋标志着网站的爱恋现场1再现完整的坐骨神经。作为我们描述的坐骨神经过程的一部分，我们还包括肌肉整体的一个比较简单的方法安装，我们使用到随后量化再生。
Protocol
1。动物主题 1.1。治疗所有动物的程序应与本地机构的动物护理和伦理委员会的批准，并按照使用和卫生准则委员会和国家机构，采取措施以减少疼痛和不适。 我们的小鼠饲养温度控制在12小时的反向光明与黑暗周期条件下，而美联储鼠标议员和水随意。 对于成人再生的研究，老鼠应该是年龄至少6周时，坐骨神经进行。这个年龄段是超出修剪polyneural神经?…
Discussion
我们已经提出了一个方法来获取可靠完整的坐骨神经与精确标记的美眉现场。如前所述，坐骨神经是一种常见的周围神经损伤的小鼠和大鼠模型。虽然每个美眉的方法都有其优点和缺点，我们发现，这种方法产生一个完整的挤压，很容易被一个特殊的设备（如特殊夹具等）的最低标记。
粉碎方法
仪器用于小鼠坐骨神经的最常见的是5号直角钳。虽然它有可能…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这项工作是由国家卫生研究院资助K08NS065157（TAF），此外支持，宾夕法尼亚肌肉劳损，关节炎研究所奖号码P30AR050950，肌肉骨骼及皮肤疾病（TAF和史蒂文与舍雷尔）支持这项工作。最后，施赖纳斯儿科研究中心种子资金（TAF），支持这项工作。我们要感谢年轻的博士，金的儿子，初步证明她的援助，在整个安装过程和Amy A.金生产的草图图1。
Materials
| Name (description/quantity) | Supplier | Catalogue Number |
| Mini Clipper with No. 0000 Blade | Roboz | RC-5903 |
| Nair Hair Remover (9 oz.) | Church and Dwight Co., Inc. | N/A |
| Betadine Surgical Scrub (1 gallon) | Fisher Scientific | 19066452 |
| Ophthalmic Ointment (1 oz.) | Fisher Scientific | 19082795 |
Surgical Tools:
| Name (description/quantity) | Supplier | Catalogue Number |
| FST 250 Hot bead sterilizer | Fine Science Tools | 18000-45 |
| Iris Scissors (11 cm long) | World Precision Instruments | 500216 |
| Potts-Smith Forceps (Straight; 18cm; 1×2 Teeth) | Fine Science Tools | 11024-18 |
| McPherson-Vannas Scissors (5 mm blades) | World Precision Instruments | 14124-G |
| Dumont #5 Forceps – Dumoxel Standard Tip | Fine Science Tools | 11252-30 |
| Dumont #5/45 Forceps – Dumoxel Standard Tip | Fine Science Tools | 11251-35 |
| Ultra Fine Hemostats (Straight; Smooth Inside Surfaces) | Fine Science Tools | 13020-12 |
| Powdered Activated Carbon (500 g) | Fisher Scientific | C272-500 |
| Size 6-0 Sutures with C-1 Needle (Sterile, Silk, Black, Braided , Non-absorbable; 18” Length; Box of 36) | Roboz | SUT-1073-11 |
| Reflex Clip Applier (for 9 mm clips) | World Precision Instruments | 500345 |
| 9mm Stainless Steel Reflex Clips (100/box) | World Precision Instruments | 500346 |
Animal Care:
| Name (description/quantity) | Supplier | Catalogue Number |
| 0.9% Sodium Chloride Injection (Preservative Free; 20 mL) | Hospira | 0409-4888-20 |
| Complete Homeothermic Blanket System with Flexible Probe (Medium, 115 VAC, 60 Hz) | Harvard Apparatus | 507222F |
Surgical Platform and Retractors:
(purchased at local hardware store)
| Name (description/) | Quantity |
| Stainless Steel Platform (Milled; ~12”x12”x1/8”) | 1 |
| Button Magnets (Example: Eclipse E825) | 3 |
| Stainless Steel Bolts (3” Long; Diameter Determined by Button Magnets) | 3 |
| Stainless Steel Nuts (Sized to Fit Bolts) | 9 |
| Rubber Bands (Light Tension) | 3 |
| Insect Pins (Both Ends Bent to Form Hooks) | 3 |
Semi-Thins:
| Name (description/quantity) | Supplier | Catalogue Number |
| Paraformaldehyde (1 kg) | Sigma-Aldrich | P6148-1KG |
| Sodium Phosphate Dibasic Anhydrous (500 g; Used to Prepare Phosphate Buffer) | Fisher Scientific | S375-500 |
| Sodium Phosphate Monobasic Anhydrous (1 kg; Used to Prepare Phosphate Buffer) | Fisher Scientific | AC38987-0010 |
| Glutaraldehyde (50%; 10 x 10 mL) | Ted Pella, Inc. | 18431 |
| Osmium Tetroxide (4% Aqueous, 10 x 10 mL) | Ted Pella, Inc. | 18465 |
| Propylene Oxide (450 mL) | Ted Pella, Inc. | 18601 |
| Embed 812 (Kit, for hard blocks/high image contrast) | Electron Microscopy Sciences | 14120 |
| Toluidine Blue (25 g) | Ted Pella, Inc. | 19451 |
Whole Mount Muscle Preparation and Immunohistochemistry:
| Name (description/quantity) | Supplier | Catalogue Number |
| Paraformaldehyde (1 kg) | Sigma-Aldrich | P6148-1KG |
| Sodium Phosphate Dibasic Anhydrous (500 g; Used to Prepare Phosphate Buffer) | Fisher Scientific | S375-500 |
| Sodium Phosphate Monobasic Anhydrous (1 kg; Used to Prepare Phosphate Buffer) | Fisher Scientific | AC38987-0010 |
| 10% BSA Diluent/Blocking Solution (200 mL) | Kirkegaard & Perry Laboratories, Inc. | 50-61-00 |
| Triton X-100 (100 mL) | Dot Scientific Incorporated | 9002-93-1 |
| Glycine, 98% (1 kg) | Fisher Scientific | AC12007-0010 |
| Tissue-Tek Cryo-OCT Compound (Case of 12; 4 oz bottles) | Fisher Scientific | 14-373-65 |
| Sylgard DOW 170 (2 lb. Kit) | Fisher Scientific | NC9492579 |
| Stainless Steel Insect Pins, Size 1 (100/pkg) | Fine Science Tools | 26001-40 |
| Tetramethylrhodamine-A-Bungarotoxin (0.5 mg) | Sigma-Aldrich | T0195-.5MG |
| Mouse Monoclonal Antibody Against SMI-312 (0.1 mL) | Covance | SMI-312R |
| Mouse Monoclonal Antibody Against SV2 (0.1 mL) | Developmental Studies Hybridoma Bank (DSHB) | SV2 |
| Rabbit Polyclonal Antibody Against GAP-43 | Novus Biologicals | NB300-143 |
| Flourescein Conjugated Goat Anti-Mouse IgG, Fcγ Subclass 1 Specific | Jackson ImmunoResearch | 115-095-205 |
| DyLight 649 Conjugated Donkey Anti-Rabbit IgG | Jackson ImmunoResearch | 711-495-152 |
| 4′,6-diamidino-2-phenylindole (DAPI, dilactate; 10 mg) | Invitrogen | D3571 |
| Vectashield Mounting Medium (10 mL) | Vector Laboratories | H-1000 |
| Superfrost Plus Microscope Slides (White; Size: 75 x 25 mm; Pack of 144) | Fisher Scientific | 12-550-15 |
| Fisherfinest Premium Cover Glasses (Size: 40 x 22 mm; Pack of 1 oz.) | Fisher Scientific | 12-548-5C |
Riferimenti
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- Hossain-Ibrahim, M. K., Rezajooi, K., Stallcup, W. B., Lieberman, A. R., Anderson, P. N. Analysis of axonal regeneration in the central and peripheral nervous systems of the NG2-deficient mouse. BMC Neurosci. 8, 80-80 (2007).
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Tags
ReproducibleMouse Sciatic Nerve CrushRegenerationWhole Mount Muscle AnalysisPeripheral Nervous SystemPNS NeuronsCNS RegenerationAxonotmesisPeripheral Nerve InjurySchwann Cell Basal LaminaeGrowing AxonSchwann Cell InteractionBasal LaminaeRat ModelsExperimental Nerve CrushMouse ModelsTransgenic LinesNerve RegenerationNerve Crush Methods
Citazione di questo articolo
Bauder, A. R., Ferguson, T. A. Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis. J. Vis. Exp. (60), e3606, doi:10.3791/3606 (2012).