面神经干切断小鼠：模型，研究运动神经元损伤反应
Summary
We present a surgical protocol detailing how to perform a cut or crush axotomy on the facial nerve in the mouse. The facial nerve axotomy can be employed to study the physiological response to nerve injury and test therapeutic techniques.
Abstract
这种手术协议的目标是从茎乳孔露出面神经，它支配的面部肌肉，在其出口处，要么切割或粉碎它来诱导外周神经损伤。这种手术的优点是它的简单性，高再现性，以及缺乏对从后续面神经麻痹生命功能或流动性的效果，因此导致相对温和的手术结果相比其他神经损伤的模型。使用颅神经损伤模型的一个主要优点是，运动神经元驻留在一个相对同质种群的面部运动核在脑桥，简化了运动神经元细胞体的研究。因为面部神经支配的对称性质和缺乏面部运动核之间的串扰的，操作可以单方面与unaxotomized侧作为配对的内部控制进行的。各种分析术后可以进行以驴S中的生理反应，其中的细节超出了本文的范围。例如，肌肉功能的恢复可以作为行为标记神经支配，或运动神经元可以被量化以测量细胞的存活。此外，运动神经元能够精确地使用激光显微切割进行分子分析捕获。因为面部神经切断是微创和良好的耐受性，它可以用于在多种转基因小鼠。另外，该手术模型可用于分析的外周神经损伤的治疗的有效性。面神经损伤提供了一种装置，用于调查不仅运动神经元，但中枢和外周神经胶质的微环境，免疫系统，和目标肌肉组织也响应。面神经损伤模型是被广泛接受的外周神经损伤模型充当一个强有力的工具用于研究神经损伤和再生。
Introduction
许多周围神经损伤模型存在，但一个突出的运动神经元的研究是面神经干切断模型。面神经，也被称为脑神经七，起源于脑桥和支配的面部表情1,2的肌肉。在此手术协议，面神经在其出口处从茎乳孔露出，或者切断或粉碎。神经损伤的严重性可以分类以下桑德兰3的分类，其中区分基于所述轴突，神经内膜，神经束膜和外膜的完整性的损伤，这是结缔组织层，其依次绕轴突束包裹。在挤压伤（axonotmesis），轴突被切断，但神经束膜和外膜被保留。从面神经压榨完整的功能恢复发生在11天左右，因为完整的神经鞘作为管道内其轴突继续生长4,5。在另一方面，在该切伤（神经断裂），轴突和所有3结缔组织层被切断，并且整个远端神经必须重新生长，恢复肌肉的神经支配。往往是在人类患者神经切断伤进行神经外膜手术重新连接，但是复苏的结果是很少最佳。进一步的研究是需要理解为什么神经未能重新生长到它的目标和什么疗法可用于改善和加速再生处理。
有许多优势，使用面神经干切断模型研究神经损伤。一，面神经干切断过程快速，简便，具有很强的可重复性;和面部肌肉的合成瘫痪不会影响重要功能，是深受动物的耐受性。因为这是一个脑神经损伤模型，研究了运动神经元细胞体被简化，因为运动神经元驻留在一个相对同质种群在第Ë面部运动核在脑桥。人口不基于面部运动核中的亚核图案不同，因为有七个亚核每个特定于支配肌肉的特定组，因此响应于干切断亚核的差异可能会影响结果-2,6,7-。
面神经损伤模型的一个主要好处是，unaxotomized方可以作为一个配对的内部控制，因为神经支配是非常对称的，还有就是面部运动核8之间没有串扰。使用这种手术方法的另一个优点是，缺乏直接创伤至CNS或血脑屏障9中断。并发症，如出血过多和感染是罕见的这一程序。
各种分析可以被执行以评估对神经损伤的生理反应。眼眨眼反射和晶须活性的恢复可以作为一个行为测量功能恢复10,11。触须活动的录像是目前最有效的方法检测面神经支配12,13的恢复。安乐死后，可以在运动神经元胞体的面部运动核内进行脑干组织学分析。面部运动核被细分成七个亚核，每个特定的某些面部肌肉，从而为应对不同检查损伤2,6。面部运动神经元进行计数来定量细胞存活，或免疫组织化学可用于鉴定生物标志物和特定细胞群14。面部运动核可以利用激光捕获到神经损伤15,16的细胞反应的分子分析准确显微切割。面神经干切断的影响，可以在运动皮层17,18进行分析。此外，神经可以解剖研究沃勒变性或19轴突再生20和肌肉能够被去除以研究神经肌肉接头21。面神经切断，也可以用来研究所附中枢和外周神经胶质细胞22，目标肌肉21，以及免疫系统反应23。虽然取得了显着的研究面神经切断模型24，是必需的外周神经损伤的进一步研究，因为神经损伤是为患者一个显著问题和目前的治疗不能产生最佳的效果。该模型是一种强大的工具，用于检查到神经损伤的生理反应和分析神经再生疗法的有效性。
Protocol
Representative Results
Discussion
The critical step for this protocol is positioning the mouse properly before surgery is begun. If the mouse is not lying flat on its side, the ear is not taped at the correct angle, or the incision is made in an incorrect location, then finding the facial nerve becomes much more difficult. When this technique is mastered, surgeries will take only minutes per mouse.
Either sutures, glue, or wound clips can be used to close the wound. Wound clips are preferred because of the small size of incis…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work is funded by NIH RO1 NS 40433 (K.J.J.).
Materials
| Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
| Stereo Microscope | Leica | M60 | |
| Labeling tape | Fisher Scientific | 15-952 | |
| Vannas-Tübingen Spring Scissors – Straight/Sharp/8.5cm/5mm cutting edge | Fine Science Tools | 15003-08 | Sterilize before use |
| Dumont #5/45 Forceps – Standard tips/Angled 45°/Dumoxel/11cm | Fine Science Tools | 11251-35 | Sterilize before use |
| Michel Suture Clips – 7.5mm x 1.75 mm | Fine Science Tools | 12040-01 | Described as "wound clip" in protocol, sterilize before use |
| Hagenbarth Cross Action Wound Clip Applier 5" | George Tiemann & Co | 160-910 | Used to apply wound clip, sterilize before use |
| Michel Suture Clip Applicator & Remover – For 7.5 mm Clips | Fine Science Tools | 12029-12 | Used to remove wound clip |
| 0.9% Sodium Chloride Injection, USP | Hospira | 0409-4888-10 | |
| Betadine, 16 oz, with dispenser | Fisher Scientific | 19-027132 | |
| 70% Ethanol | |||
| Glass Bead Sterilizer |
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