腓神经损伤方法:可靠检测，以确定和测试因素修复神经肌肉接头

摘要

We have developed a nerve injury method to reliably examine muscle reinnervation, and thus regeneration of neuromuscular junctions in mice. This technique involves injuring the common fibular nerve via a simple and highly reproducible surgery. Muscle reinnervation in then assessed by whole-mounting the extensor digitorum longus muscle.

摘要

神经肌肉接头（NMJ）发生有害的结构和功能改变的衰老，伤害和疾病的结果。因此，必须了解涉及维护和修理NMJs的细胞和分子变化。为了这个目的，我们已开发了一种方法，以可靠地和始终如一地检查小鼠再生NMJs。这种神经损伤的方法包括，因为它经过了膝盖附近的腓肠肌肌腱外侧头粉碎腓总神经。用70天的雌性小鼠，我们证明了运动神经元轴突开始于7天粉碎后reinnervate以前的突触后目标。他们12日后完全再用他们以前的突触领域。为了确定这种损伤方法的可靠性，我们比较个人70天岁的雌性小鼠神经支配之间的比率。我们发现，神经再支配突触后的网站数量是老鼠相似的7,9，和12天粉碎后。以确定是否这种损伤测定也可用于分子变化在肌肉比较，我们检查了肌肉烟碱样受体（γ-乙酰胆碱受体）和肌肉特异性激酶（麝香）的伽玛亚基水平。伽玛乙酰胆碱受体亚单位和麝香高度上调下面去神经，并返回到以下NMJs的神经支配恢复正常水平。我们发现这些基因和肌肉神经支配的地位转录水平有密切的关系。我们认为，这种方法将加速我们的参与修复的NMJ和其它突触的细胞和分子变化的理解。

引言

In young adult and healthy animals, the neuromuscular junction (NMJ) is a highly stable connection between the presynapse, the nerve ending of an α-motor axon, and the postsynapse, the specialized region of an extrafusal muscle fiber where nicotinic acetylcholine receptors (AChRs) selectively aggregate¹. The nearly perfect apposition of the pre- and post-synaptic apparatuses is necessary for proper neurotransmission, survival of α-motor neurons and muscle fibers and motor function. Unfortunately, the function of the NMJ is adversely affected by aging, diseases such as amyotrophic lateral sclerosis (ALS), autoimmune diseases and injury to muscles and peripheral nerves^2-5. These insults often result in degeneration of presynaptic nerve endings, leaving muscles denervated and significantly altering motor skills. For this reason, the identification of molecules that function to maintain and repair the NMJ has become a priority. Because peripheral nerves regenerate and reinnervate targets, peripheral nerve injury models have been used to identify molecular changes associated with regenerating NMJs.

Peripheral nerve injury models often involve either completely cutting or crushing specific nerve branches⁶. Following a cut, the endoneurial tube has to be reformed, delaying axonal regeneration and reinnervation of target cells and tissues. The severity of this type of injury also causes axons to meander away from their original path, resulting in their failure to reach original targets. This is in contrast to nerves injured via crush where the endoneurium remains contiguous, providing a path for efficient and proper regrowth of regenerating axons. It also allows axons to find and reinnervate their original muscle fiber partners. Irrespective of injury model, there are a number of cellular and molecular changes that must occur for axons to regenerate and reinnervate targets. After an injury, the nerve segment proximal to the target is broken down and removed via a process termed Wallerian Degeneration⁷. This process involves reprogramming and de-differentiation of Schwann cells into non-myelinating cells that secrete regenerative factors, clear myelin, and recruit macrophages to the site of injury⁸. Macrophages in turn complete the clearance of myelin and axonal debris, which would otherwise impede growth of the regenerating axon⁹. In parallel, motor and sensory neurons activate mechanisms needed to promote regeneration of their severed axons. Once the regenerating axon reaches the target, it must transform from a growth cone to a nerve ending capable of properly transmitting (for motor axons) or receiving (for sensory axons) information¹⁰. In this regard, alpha-motor axons undergo a series of well-orchestrated changes that culminate in their growth cone differentiating into a fully functional presynaptic nerve ending that nearly perfectly opposes the post-synaptic site on the target muscle fiber¹¹.

The sciatic, tibial and accessory nerves have been the primary choices for studying axonal and NMJ regeneration^12-14. However, there are a number of drawbacks when using these models to examine cellular and molecular changes associated with regenerating NMJs between animals and under different conditions. Firstly, the sciatic nerve supplies the majority of the muscles of the hind limb, with injury significantly limiting both movement and sensation. It is therefore not possible to use this method to study the impact of exercise alone or in combination with other factors. Additionally, the sciatic nerve is a rather thick structure and thus requires a large amount of compressive force to fully injure all axons. This in turn may result in complete transection of the more superficial axons while leaving the endoneurial tube of deeper lying axons intact, introducing significant variability in the rate and fidelity of regeneration among these axons. Complete transection of this nerve is even less desirable given that many axons will fail to reconnect with the same muscle fibers. Complicating matters, the sciatic nerve possesses intrinsic anatomic variability, both in the number and site of origin of its terminal nerve branches. It is therefore very difficult to lesion the same site. While the tibial nerve is smaller and more amenable to crush injuries, there is also no readily available landmark to serve as a lesion site for this nerve branch.

The accessory nerve branch (part of cranial nerve XI) that supplies the sternocleidomastoid muscle has also been used to study regeneration of NMJs¹⁵. This nerve is particularly attractive because NMJs in the sternocleidomastoid muscle can be more readily imaged in live animals compared to NMJs in other muscles. But similar to the sciatic and tibial nerves, there is no specific landmark that can be used to injure this nerve in the same location, limiting it as a model for comparing regeneration of NMJs among individual animals of an experimental cohort. An inconsistent lesion site introduces variability in the rates of NMJ reinnervation. Due to these shortcomings, the procedure presented here utilizes the injury of a different peripheral nerve branch to examine regenerating NMJs.

The common fibular nerve, also called the common peroneal nerve, contains many features that make it a reliable nerve to examine regeneration of NMJs between animals and across different treatments. The common fibular nerve has a predictable anatomic course as it runs over the tendon of the lateral head of the gastrocnemius muscle in the knee, the intersection serving as a stable landmark for lesions. The nerve is accessed through a small and minimally invasive incision near but anatomically segregated from the muscles of interest. The findings presented here demonstrate that regenerating motor axons begin to reform NMJs in the extensor digitorum longus (EDL) muscle 8 days after crushing the fibular nerve in 70 days old young adult female mice. Importantly, the pattern and rate of reinnervation is consistent among animals of the same age and sex and therefore provide a reliable injury model that will significantly hasten our understanding of the cellular and molecular changes required to maintain and repair NMJs.

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研究方案

所有实验均在由弗吉尼亚理工大学机构动物护理和使用委员会批准NIH的指导方针和动物的协议进行的。

1.准备动物外科手术

1. 麻醉小鼠用氯胺酮（90毫克/公斤），并通过皮下注射腹股沟注射甲苯噻嗪（10毫克/千克）的用无菌1毫升胰岛素注射器的混合物中。载体溶液含有0.9％的盐水，17.4毫克/毫升氯胺酮，和2.6毫克/毫升甲苯噻嗪的混合物。将动物放回笼子在等待的药物才能生效。
   注意:如果该负荷剂量不会对过程的持续时间提供了足够的麻醉，负荷剂量的额外的25％可以被注入。
2. 监控动物注射后，检查呼吸平稳率和觉醒水平适当的抑郁症。请与一个后足捏，这应该引起无反应时，充分麻醉唤醒水平。
   注:这通常需要3-5分对于一个年轻的成年小鼠平均25至30g。如果动物仍10分钟后注射后响应，麻醉负荷剂量的额外的25％可以被注入。
3. 应用凡士林和轻矿物油眼药膏的动物的眼睛，以防止干燥。在干净，平坦的表面上取下笼和地方的动物。剃从足所需的后肢使用的电动毛发修剪器骨盆，只露出肢的外侧面。
4. 应用化学毛发去除的剃光部位1分钟。使用手动实验室抹布去除毛。干净，实验室面积脱毛浸抹在乙醇中。

2.手术过程

1. 通过消毒高压灭菌器或其他适当的方法手术器械。清洁外科手术部位和手术板用80％的乙醇/ H ₂ O。消毒手术部位与proviodine。放置在手术板上的鼠标和肢体约束对齐。保持与膝关节解剖学自然位置目标后肢稍微延伸而不内部或外部的转动。
2. 将动物和板手术显微镜下。东方通过肤浅的标志性建筑，特别是骨性膝关节和胫骨前肌和腓肠肌之间的山脊触诊适当的切口部位。
3. 使通过皮肤使用解剖刀或弹簧剪刀，而使用一般的镊子用于夹持一个大约3厘米的切口。使切口在垂直于共同腓骨神经的基本过程。
4. 通过浅筋膜继续切口，暴露股二头肌和股外侧肌肉。通过连接深筋膜切割分开这些肌肉。 1-2厘米的切口就足够了。
5. 缩回二头肌尾部采用机械拉钩，揭示了腓总神经肌肉股四头肌。
6. 近端跟踪神经，直到它的内部与腓肠肌外侧头的腱部分被找到。注意:曝光可能需要缩回皮肤和肌肉的额外的操作。该交点被用作神经损伤的稳定的地标。
7. 抓住神经用细镊子，对准以并行的方式提示腓肠肌肌腱外侧缘。通过施加持续的，硬压5秒粉碎常见的腓骨神经。
8. 通过手术范围证实通过目测的神经完全粉碎。它将在损伤部位出现半透明的。如果使用的是表达在轴突末梢荧光蛋白的小鼠，荧光就会消失，从受伤的部位。
9. 除去拉钩并在他们的解剖位置重新调整肌肉。关闭切口部位用6-0丝线缝合。 1-3简单间断缝合就足够了。放在一个干净的笼子里加热垫鼠标休养。
10. 监控所有的动物2小时后戏重刑检查呼吸和麻醉任何不良反应。经由皮下腹股沟注射给药的丁丙诺啡0.05-0.10毫克/千克的初始剂量立即从手术恢复以下。给另外3个剂量，每12小时在接下来的48小时。完全恢复后，返回小鼠动物护理设施。

3.隔离和伸肌染色趾长肌（EDL）肌肉

1. 使用异氟醚牺牲动物。分配0.5毫升液体异氟烷到50毫升管中填充有吸收性labwipes。放置不封顶管在一个密封2500厘米³室中的动物。曝光中的至少4分钟就足够了。测试双边眼睑，脚趾捏，和尾掐反射的损失，以确保每个动物灌注继续之前不省人事。
2. Transcardially第一用10ml 0.1M的PBS灌注¹⁶动物，则25毫升4-％多聚甲醛的0.1M PBS（pH 7.4）中。肝素（30单位/ 20克动物体重）可以与PBS被加入（10单位/ ml），以防止血液凝固的小毛细血管床，改进灌注的结果。
3. 通过使用剪刀通过腹部周围的周长皮肤横向切割除去覆盖后肢皮肤。过去的后肢和脚使用镊子剥离下来的皮肤。
4. 抓住并用钳子取出剥皮后肢浅筋膜。如果使用荧光表达蛋白的轴突末梢小鼠，一夜之间在4％PFA 50毫升管后修复整个小鼠。用PBS冲洗三次。
   注:固定的小鼠可在4℃储存于PBS中。如果没有，请跳过此步骤并继续执行步骤无3.6定影后的动物。
5. 从解剖小鼠后肢肌肉EDL ^18，是一定要保持近端和远端肌腱作为完整越好。
6. 孵育在封闭缓冲液EDL肌肉（含有0.5％的Triton X-100，3％BSA和5％山羊血清1×PBS中）至少1小时。
7. 为了显现运动轴突和它们的神经末梢，地点的肌肉在含有神经丝（1:1,000）管和突触结合蛋白2（1:250）在3天阻断缓冲液中稀释抗体。洗肌肉用1×PBS清洗3次，每次10分钟。注意:如果跳过这一步使用表达末梢神经轴突荧光蛋白（XFP）小鼠。
8. 染色没有受伤EDL为完整的NMJ神经支配的阳性对照对侧。阴性对照应包括以4天损伤后，一时间点，其中NMJ是完全失神经获得的EDL，以及与只用二抗染色一个EDL。
9. 孵育肌肉用适当的荧光标记的第二抗体来检测神经丝和突触结合蛋白2 1天。洗肌肉用1×PBS清洗3次，每次10分钟。注意:此步骤可以与步骤3.10进行。如果使用的表达在轴突末梢荧光蛋白的小鼠跳过此步骤。
10. 以可视化的postsy所述NMJ的naptic区，孵化肌肉用5微克/毫升的Alexa-555缀合的α-银环蛇毒素在至少2小时的封闭缓冲液中稀释。洗肌肉用1×PBS清洗3次，每次10分钟。
11. 为安装在带正电荷的载玻片整个肌肉，直接放置在肌肉上滑动，添加基于安装在滑动介质甘油几滴并用盖玻片覆盖。按盖玻片对幻灯片拼合肌肉。从幻灯片和盖玻片使用实验室湿巾的周边浸泡过的安装介质。应用指甲油密封盖玻片和幻灯片之间的边缘。

4.成像和数据分析

1. 分析NMJs，图像使用装有激发488，555和633纳米的光，用20X和40X目标捕获所发射的光的共焦激光扫描显微镜的EDL肌肉的结构。
2. 为了形象化整个NMJs，创造光秒的最大强度投影图像系统蒸发散间隔1至2微米从最低NMJ来最高可见区域外扩展。创建使用市售的图像处理软件的最大强度的预测。
3. 为了确定神经再生率，分类NMJs为:1）完全失神经突触后=站点是完全没有轴突和乙酰胆碱受体之间有轴突的接触，不到5％的共定位。 2）部分支配=轴突部分重叠轴突和乙酰胆碱受体之间的postsynapse，5-95％的共定位。 3）全支配=前和后突触之间近乎完美的同位语，轴突和乙酰胆碱受体之间的大于95％的共定位。排除位于垂直于成像平面或没有在图象中完全显现NMJs。注:在所有这些实验中，进行了检查，至少3只动物和动物每50 NMJs。结果使用学生t检验具有小于0.05的P值视为显著。
4. 盲操作，独立的个体可以执行手术和图像分析。未经治疗组的知识，分析仪可与NMJ得分目标。可替代地，图像可以被随机化，并呈现给操作者用于分析没有源动物的知识。

5.定量PCR

1. 用牺牲异氟醚和颈椎脱位动物。去除皮肤和浅筋膜覆盖按照步骤3.3腿部肌肉。解剖前胫骨和根据步骤3.4 EDL肌肉。
2. 闪光灯冻结在1.5ml管上液氮整个胫骨前和EDL肌肉。在预冷的研钵在液氮部分淹没移除管和地方组织。冷冻研磨成肌肉用迫击炮和杵细粉。
3. 溶解冷冻肌肉粉末到市售的RNA提取试剂和根据制造商的我市售的试剂盒进行RNA提取和基因组DNA去除nstructions。
4. 利用根据制造商的说明可商购的逆转录酶混合进行逆转录。
5. 执行使用市售的试剂盒，使用适当的看家基因的qPCR（见材料表）。使用市售的定量PCR仪进行PCR（见材料表）。
6. 设置退火温度至58℃。调整附加循环参数到Taq聚合酶/ SYBR绿色混合物的制造商的规范。包括在由以0.5℃逐渐增加从65℃至95℃，以测试引物的特异性和引物二聚体形成的热循环仪程序最终熔解曲线的步骤。
7. 确定由2相对mRNA表达水平^-使用18S RNA作为对照基因^ΔΔCT方法^21。

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结果

共同腓骨神经，也称为腓总神经，从坐骨神经腘窝上面，在那里它摆动围绕腓骨头到腿部（ 图1A）的前方面就产生了。在那里，它分支到浅层和深层腓骨神经，同时供给的脚和脚趾的背伸（胫前肌，趾长伸肌和短，和伸halluces长肌的肌肉），而脚的everters（腓骨肌）。这种神经还携带投射到脚和腿的下半部的外侧面的背部感觉纤维。它是运动和感觉轴突构成的?...

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讨论

在这个手稿提出的方法提供了鉴定参与修复神经肌肉接头（NMJ）机制的独特的机会。此方法涉及，因为它经过了膝盖附近的腓肠肌腱破碎的腓总神经。我们表明，只有5用镊子神经压迫秒钟后，完成变性的伤害后4天指出。在年轻的成年小鼠，α-运动神经元轴突开始以7天reinnervate在趾长伸肌（EDL）以前的突触部位损伤后，突触前部位是由12天那些未受伤的小鼠没有区别的改革高潮。此外，我们证明了...

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材料

Name	Company	Catalog Number	Comments
Ketamine	VetOne	501072
Xylazine	Lloyd Inc.	003437
Buprenorphine	Zoopharm	1Z-73000-150910
Nair	Nair
Kim-wipes	Kimtech	34155
Electric Razor	Braintree Scientific	CLP-64800
80% EtOH/H20
10% Proviodine
1 ml Insulin Syringe
Spring Scissors	Vannas	91500-09
No. 15 scalpel	Braintree Scientific	SSS 15
#5 Forceps	Dumont	11252-00
6-0 silk suture on reverse cutting needle	Suture Express	752B
Rodent Heating Pad	Braintree Scientific	AP-R-18.5
Alexa 555 conjugated alpha-BTX	Molecular Probes	B35451
Vectashield	Vector Labs	H-1000
Olympus Stereo Zoom Microscope	Olympus	562037192
Zeiss 700 Confocal Microscope	Zeiss
Variable-flow peristaltic perfusion pump	Fisher Scientific	13-876-3
Aurum Total RNA Mini Kit	Bio-Rad	7326820
Bio-Rad iScript RT Supermix	Bio-Rad	1708840
SsoFast Evagreen Supermix	Bio-Rad	1725200
Bio-Rad CFX96	Bio-Rad	1855196
Puralube Vet ointment	Puralube	1621
Synaptotagmin-2 antibody	Antibodies-Online	ABIN401605
Neurofilament antibody	Antibodies-Online	ABIN2475842