Малоберцового нерва Метод Травма: Надежный Анализ для идентификации и испытания факторов, которые Ремонт нервно-мышечных синапсах
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
Нервно-мышечного соединения (НМС) претерпевает пагубное структурные и функциональные изменения в результате старения, травм и болезней. Таким образом, крайне важно понять, клеточные и молекулярные изменения, связанные с поддержанием и ремонтом NMJs. Для этой цели мы разработали метод, позволяющий надежно и последовательно исследовать регенерации NMJs у мышей. Этот метод включает в себя повреждение нерва дробления общий малоберцовый нерв, как она проходит по боковой головки икроножной мышцы сухожилия возле колена. Использование 70 день старых самок мышей, мы показали, что моторные аксоны начинают reinnervate предыдущие постсинаптические цели в течение 7 дней после давки. Они полностью оккупировать свои прежние синаптические области на 12 дней. Для того, чтобы определить надежность этого метода травмы, мы сравнили цены реиннервация между отдельными 70-дневных самок мышей. Мы обнаружили, что количество reinnervated постсинаптических участков была одинаковой у мышей на 7, 9 и 12 дней после давки. Для того, чтобы определить, является лиэта травма анализ также может быть использован для сравнения молекулярных изменений в мышцах, мы исследовали уровни гамма-субъединицы мышечного никотиновых рецепторов (гамма-АХР) и мышечно-специфической киназы (салицилаты). Субъединица гамма-AChR и мускус, чтобы высоко активируются следующие денервации и вернуться к нормальному уровню после реиннервации НМС. Мы обнаружили тесную взаимосвязь между уровнями транскриптов этих генов и состояния иннервации мышц. Мы считаем, что этот метод ускорит наше понимание клеточных и молекулярных изменений, участвующих в восстановлении НМС и других синапсов.
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
Метод, представленный в этой рукописи предоставляет уникальные возможности для выявления механизмов, участвующих в восстановлении нервно-мышечных синапсах (НМС). Этот метод включает в себя дробление общий малоберцовый нерв, как она проходит через икроножной сухожилия возле колена. П?…
Divulgazioni
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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