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
Summary
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.
Abstract
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。
Introduction
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 nerves2-5. 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 regeneration12-14. 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.
Protocol
Representative Results
Discussion
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…
Declarações
The authors have nothing to disclose.
Acknowledgements
The authors thank members of the Valdez laboratory for intellectual input on experiments and comments on the manuscript.
Materials
| 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 |
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