Fibular Nerve Injury Metod: En tillförlitlig analys för att identifiera och testa faktorer som Reparation neuromuskulära korsningar
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
Den neuromuskulära förbindelsen (NMJ) genomgår skadliga strukturella och funktionella förändringar som en följd av åldrande, skador och sjukdom. Således är det viktigt att förstå de cellulära och molekylära förändringar som är involverade i underhåll och reparationer NMJs. För detta ändamål har vi utvecklat en metod för att tillförlitligt och konsekvent undersöka regenere NMJs i möss. Detta nervskada Metoden innebär att krossa den gemensamma fibular nerv när den passerar över den laterala huvudet av gastrocnemius senan nära knäet. Använda 70 dagar gamla honmöss, visar vi att motor axoner börjar reinnervate tidigare postsynaptiska mål inom 7 dagar efter krossa. De återta helt sina tidigare synaptiska områden med 12 dagar. För att bestämma tillförlitligheten hos denna skada metod jämförde vi reinnervation priser mellan enskilda 70 dagar gamla honmöss. Vi fann att antalet reinnervated postsynaptiska platser var liknande mellan möss vid 7, 9, och 12 dagar post-krossa. För att bestämma omdenna skada Analysen kan också användas för att jämföra molekylära förändringar i muskler, undersökte vi nivåer av gamma-underenheten av muskeln nikotinreceptorn (gamma-AChR) och muskelspecifikt kinas (MuSK). Gamma-AChR subenhet och mysk till är mycket uppreglerade efter denervering och återgå till normala nivåer efter reinnervation av NMJs. Vi hittade en nära relation mellan transkriptnivåer för dessa gener och innervation status av muskler. Vi tror att denna metod kommer att påskynda vår förståelse av de cellulära och molekylära förändringar som är involverade i att reparera NMJ och andra synapser.
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
Den metod som presenteras i detta manuskript ger unika möjligheter att identifiera mekanismer som är involverade i att reparera neuromuskulära korsningar (NMJ). Denna metod innefattar krossning av det gemensamma fibular nerven då den passerar över gastrocnemius senan nära knät. Vi visar att efter endast fem sekunder av nervkompression med en pincett, är klar degeneration noterats av 4 dagar efter skada. Hos unga vuxna möss, alfa-motoriska axoner börjar reinnervate tidigare synaptiska platser i extensor digitor…
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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