Name: the fibular nerve injury method: a reliable assay to identify and test factors that repair neuromuscular junctions
Uploaded: 2016-08-11T15:00:00
Description: Watch this Scientific Journal Video about The Fibular Nerve Injury Method: A Reliable Assay to Identify and Test Factors That Repair Neuromuscular Junctions at JoVE.com

The authors thank members of the Valdez laboratory for intellectual input on experiments and comments on the manuscript.

Tüm deneyler Virginia Tech Kurumsal Hayvan Bakım ve Kullanım Komitesi tarafından onaylanmış NIH kurallar ve hayvan protokol çerçevesinde yürütülmüştür. Cerrahi 1. hazırlanması Hayvanlar <ol><li> steril 1 ml insülin şırınga ile ketamin (90 mg / kg) ve deri altı enjeksiyon yoluyla inguinal ksilazin (10 mg / kg) karışımı ile fareler anestezi. Taşıyıcı çözeltisi% 0.9 tuzlu su, 17.4 mg / ml ketamin ve 2.6 mg / ml ksilazin içeren bir karışım içerir. yürürlü...

<ol>
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J., Alton, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+age+impairs+the+response+of+the+neuromuscular+junction+to+nerve+transection+and+repair:+An+experimental+study+in+rats.">How age impairs the response of the neuromuscular junction to nerve transection and repair: An experimental study in rats.</a> J Orthop Res. 27 (3), 385-393 (2009).</li><li>Balice-Gordon, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+changes+in+neuromuscular+innervation.">Age-related changes in neuromuscular innervation.</a> Muscle Nerve Suppl. 5, S83-S87 (1997).</li><li>Valdez, G., Tapia, J. C., Lichtman, J. W., Fox, M. A., Sanes, J. 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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+cellular+interactions+in+peripheral+nerve+regeneration.">Molecular mechanisms of cellular interactions in peripheral nerve regeneration.</a> Curr Opin Neurol. 14 (5), 635-639 (2001).</li><li>Gaudet, A. D., Popovich, P. G., Ramer, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wallerian+degeneration:+gaining+perspective+on+inflammatory+events+after+peripheral+nerve+injury.">Wallerian degeneration: gaining perspective on inflammatory events after peripheral nerve injury.</a> J Neuroinflammation. 8, 110 (2011).</li><li>Chen, P., Piao, X., Bonaldo, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+macrophages+in+Wallerian+degeneration+and+axonal+regeneration+after+peripheral+nerve+injury.">Role of macrophages in Wallerian degeneration and axonal regeneration after peripheral nerve injury.</a> Acta Neuropathol. 130 (5), 605-618 (2015).</li><li>Chen, Z. -. L., Yu, W. -. M., Strickland, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+regeneration.">Peripheral regeneration.</a> Annu Rev Neurosci. 30, 209-233 (2007).</li><li>Darabid, H., Perez-Gonzalez, A. P., Robitaille, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuromuscular+synaptogenesis:+coordinating+partners+with+multiple+functions.">Neuromuscular synaptogenesis: coordinating partners with multiple functions.</a> Nat. Rev. Neurosci. 15 (11), 703-718 (2014).</li><li>Geuna, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+sciatic+nerve+injury+model+in+pre-clinical+research.">The sciatic nerve injury model in pre-clinical research.</a> J. Neurosci. Methods. 243, 39-46 (2015).</li><li>Batt, J. A. E., Bain, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tibial+nerve+transection+-+a+standardized+model+for+denervation-induced+skeletal+muscle+atrophy+in+mice.">Tibial nerve transection - a standardized model for denervation-induced skeletal muscle atrophy in mice.</a> J. Vis. Exp. (81), e50657 (2013).</li><li>Savastano, L. E., Laurito, S. R., Fitt, M. R., Rasmussen, J. A., Gonzalez Polo, V., Patterson, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sciatic+nerve+injury:+a+simple+and+subtle+model+for+investigating+many+aspects+of+nervous+system+damage+and+recovery.">Sciatic nerve injury: a simple and subtle model for investigating many aspects of nervous system damage and recovery.</a> J. Neurosci. Methods. 227, 166-180 (2014).</li><li>Kang, H., Lichtman, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motor+axon+regeneration+and+muscle+reinnervation+in+young+adult+and+aged+animals.">Motor axon regeneration and muscle reinnervation in young adult and aged animals.</a> J Neurosci. 33 (50), 19480-19491 (2013).</li><li>Gage, G. J., Kipke, D. R., Shain, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+animal+perfusion+fixation+for+rodents.">Whole animal perfusion fixation for rodents.</a> J. Vis. Exp. (65), e3564 (2012).</li><li>Feng, G., Mellor, R. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+Neuronal+Subsets+in+Transgenic+Mice+Expressing+Multiple+Spectral+Variants+of+GFP.">Imaging Neuronal Subsets in Transgenic Mice Expressing Multiple Spectral Variants of GFP.</a> Neuron. 28 (1), 41-51 (2000).</li><li>Sanes, J. R., Lichtman, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+vertebrate+neuromuscular+junction.">Development of the vertebrate neuromuscular junction.</a> Annu Rev Neurosci. 22, 389-442 (1999).</li><li>Bowen, D. C., Park, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+and+regulation+of+MuSK+at+the+neuromuscular+junction.">Localization and regulation of MuSK at the neuromuscular junction.</a> Dev Biol. 199 (2), 309-319 (1998).</li><li>Gay, S., Jublanc, E., Bonnieu, A., Bacou, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myostatin+deficiency+is+associated+with+an+increase+in+number+of+total+axons+and+motor+axons+innervating+mouse+tibialis+anterior+muscle.">Myostatin deficiency is associated with an increase in number of total axons and motor axons innervating mouse tibialis anterior muscle.</a> Muscle Nerve. 45 (5), 698-704 (2012).</li><li>Livak, K. J., Schmittgen, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+relative+gene+expression+data+using+real-time+quantitative+PCR+and+the+2(-Delta+Delta+C(T))+Method.">Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.</a> Methods. 25 (4), 402-408 (2001).</li></ol>

Bu yazıda sunulan yöntem nöromüsküler kavşaklar (NMJ) tamir katılan mekanizmaları tanımlamak için benzersiz fırsatlar sunar. o diz yakın gastroknemius tendonu üzerinden geçerken Bu yöntem yaygın fibula siniri ezme içerir. Bir forseps ile sinir sıkışmasının sadece 5 saniye sonra, tam bir dejenerasyonu yaralanması 4 gün sonra belirtilen olduğunu göstermektedir. genç yetişkin farelerde, alfa-motor aksonlar 12 gün yaralanmamış farelere olanlardan ayırt edilemez presinaptik sitelerin Reformasy...

In young adult and healthy animals, the neuromuscular junction (NMJ) is a highly stable connection between the presynapse, the nerve ending of an &#945;-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 &#945;-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.

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			<td>10% Proviodine</td>
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			<td>1 mL Insulin Syringe</td>
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			<td>Dumont</td>
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			<td>Alexa 555 conjugated alpha-BTX </td>
			<td>Molecular Probes</td>
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			<td>Vectashield</td>
			<td>Vector Labs</td>
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			<td>Antibodies-Online</td>
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the fibular nerve injury method: a reliable assay to identify and test factors that repair neuromuscular junctions

nöromüsküler bileşke (NMJ) yaşlanma, yaralanma ve hastalık sonucu zararlı yapısal ve fonksiyonel değişikliklere uğrar. Nedenle, bakım ve onarım NMJs katılan hücresel ve moleküler değişiklikleri anlamak için şarttır. Bu amaçla, güvenilir ve sürekli olarak farelerde NMJs rejenere incelemek için bir yöntem geliştirdik. o diz yakın gastroknemius kası tendonun lateral başının üzerinden geçerken Bu sinir yaralanması yöntemi yaygın fibula siniri ezme içerir. 70 günlük dişi fareler kullanarak, motor aksonlar 7 gün sonrası ezmek içinde önceki postsinaptik hedefleri reinnervate başlar göstermektedir. Bunlar tamamen 12 gün önceki sinaptik alanları yeniden işgal. Bu yaralanma yöntemin güvenilirliğini belirlemek için, bireysel 70 günlük dişi fareler arasında reinnervation oranları karşılaştırıldı. Biz tekrar innerve olmuş postsinaptik sitelerin sayısı 7, 9 fareler arasında benzer olduğunu buldu ve 12 gün sonrası crush. olmadığını belirlemek içinBu yaralanma deneyi, kas moleküler değişiklikleri karşılaştırmak için kullanılabilir biz kas nikotinik reseptör (y-AChR) ve kas özel kinaz (MuSK) gama alt-biriminin seviyeleri araştırılmıştır. gama-AChR alt birim ve MuSK yüksek aşağıdaki denervasyon regüle ve NMJs reinnervation aşağıdaki normal seviyelere geri oluştururlar. Biz bu genlerin ve kasların innervasyonu durumu transkript seviyeleri arasında yakın bir ilişki bulundu. Biz bu yöntem NMJ ve diğer sinaps tamir katılan hücresel ve moleküler değişiklikler anlayışımız hızlandırmak inanıyoruz.

Ortak fibula sinir, aynı zamanda, peroneal sinir olarak adlandırılan bacak (Şekil 1A) ön yönüne fibula başının etrafında sallanır popliteal fossada yukarıdaki siyatik sinirin, doğar. yüzeysel ve derin fibula sinir içine dalları birbirine ayak ve ayak dorsifleksör tedarik (anterior tibialis, extensor digitorum longus ve brevis ve ekstansör halluces longus kasları) ve ayağın everters (peroneus kaslar) Orada olmuyor. Bu sinir de ayak ve bacağın alt y...

Watch this Scientific Journal Video about The Fibular Nerve Injury Method: A Reliable Assay to Identify and Test Factors That Repair Neuromuscular Junctions at JoVE.com

The Fibular Nerve Injury Method: A Reliable Assay to Identify and Test Factors That Repair Neuromuscular Junctions

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.

Fibula Sinir Yaralanması Yöntem: Güvenilir Testi tanımak ve Test O Tamir Nöromüsküler Junctions Faktörleri

The neuromuscular junction (NMJ) undergoes deleterious structural and functional changes as a result of aging, injury and disease. Thus, it is imperative ...

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Retina Ganglion Hücre Survival Eğitim Bir Optik Sinir Crush Yaralanma murin Modeli

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Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

Tüm Dağı Muscle Analizi ile tekrarlanabilir Fare Siyatik Sinir Ezilme ve Rejenerasyon daha sonraki Değerlendirme

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Loading a Calcium Dye into Frog Nerve Endings Through the Nerve Stump: Calcium Transient Registration in the Frog Neuromuscular Junction

Kurbağa Sinir Uçlarına Sinir Kütüğü Vasıtasıyla Kalsiyum Boyası Yükleme: Kurbağa Nöromüsküler Kavşağında Kalsiyum Geçici Kayıt

One of the most feasible methods of measuring presynaptic calcium levels in presynaptic nerve terminals is optical recording. It is based on using ...

Dorsal Root Ganglion Injection and Dorsal Root Crush Injury as a Model for Sensory Axon Regeneration

Sırt kök ganglion Enjeksiyon ve Dorsal Kök Duyusal Axon Rejenerasyon Modeli Olarak Ezilme Yaralanması

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each ...

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

Sinirsel aktivite Thy1kullanarak birincil somatosensor kortekste Imaging-GCaMP6s transgenik fareler

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...