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.

כל הניסויים בוצעו על פי הנחיות NIH ופרוטוקולי חיה אושרו על ידי ועדת הטיפול בבעלי החיים המוסדית וירג&#39;יניה טק ושימוש. 1. בעלי חיים הכנות לקראת ניתוח <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> להרדים עכברים בתערובת של קטמין (90...

<ol>
	<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=Induction,+assembly,+maturation+and+maintenance+of+a+postsynaptic+apparatus.">Induction, assembly, maturation and maintenance of a postsynaptic apparatus.</a> Nat. Rev. Neurosci. 2 (11), 791-805 (2001).</li><li>Moloney, E. B., de Winter, F., Verhaagen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ALS+as+a+distal+axonopathy:+molecular+mechanisms+affecting+neuromuscular+junction+stability+in+the+presymptomatic+stages+of+the+disease.">ALS as a distal axonopathy: molecular mechanisms affecting neuromuscular junction stability in the presymptomatic stages of the disease.</a> Front. Neurosci. 8, 252 (2014).</li><li>Apel, P. 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>

השיטה המוצגת בכתב היד הזה מספקת הזדמנויות ייחודיות לזהות מנגנונים המעורבים בתיקון צמתים תוקפות (NMJ). שיטה זו כרוכה ריסוק העצב שׁוֹקִיתִי נפוץ כפי שהיא עוברת מעל גיד הגסטרוקנמיוס ליד הברך. אנו מראים כי רק אחרי 5 שניות של לחץ בעצבים עם מלקחיים, ניוון מוחלט יצוין ב -4 ימים ...

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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the fibular nerve injury method: a reliable assay to identify and test factors that repair neuromuscular junctions

צומת עצב-שריר (NMJ) עובר שינויים מבניים ותפקודיים מזיקות כתוצאה הזדקנות, פציעה ומחלה. לכן, זה הכרחי כדי להבין את השינויים התאיים ומולקולריים מעורבים בשמירה ותיקון NMJs. לשם כך, פיתחנו שיטה אמין ועקבי לבחון התחדשות NMJs בעכברים. שיטת עצב פגיעה זו כרוכה ריסוק העצב שׁוֹקִיתִי נפוץ כפי שהיא עוברת מעל הראש הלטרלי של גיד שריר הסובך ליד הברך. שימוש עכברות בן 70 יום, אנו מראים כי אקסונים מוטוריים מתחילים reinnervate מטרות postsynaptic קודמים תוך 7 ימים לאחר למעוך. הם לגמרי לכבוש מחדש שטחים הסינפטי הקודם שלהם ב -12 ימים. כדי לקבוע את האמינות של שיטת פגיעה זו, השווינו שיעורי reinnervation בין עכברות ישנות פרט 70 יום. מצאנו כי מספר אתרי postsynaptic reinnervated היה דומה בין עכברים בבית 7, 9, ו -12 ימים למחוץ-פוסט. כדי לקבוע אםassay פגיעה זו יכולה לשמש גם כדי להשוות השינויים המולקולריים בשרירים, בדקנו רמות של למקטע-גמא של קולטן ניקוטינית שריר (גמא-AChR) ואת קינאז ספציפי-שריר (מושק). למקטע גמא AChR ומושק לדוחות אלו שהוגברו מאוד denervation הבאה ולחזור לרמות נורמליות לאחר reinnervation של NMJs. מצאנו קשר הדוק בין רמות תמליל עבור גנים אלה ומצב עצבוב השרירים. אנו מאמינים כי שיטה זו תאיץ את הבנתנו את השינויים התאיים ומולקולריים מעורבים בתיקון NMJ וסינפסות אחרות.

העצב שׁוֹקִיתִי משותף, גם הנקרא העצב peroneal משותף, נובעת גיד הנשה מעל fossa popliteal, שם הוא נדנדות סביב ראשו של שוקית אל ההיבט הקדמי של הרגל (איור 1 א). יש לה סניפים לתוך עצבים שטחיים ועמוקים שׁוֹקִיתִי, יחד לספק את dorsiflexors של כף הרגל ועל בהונות (הקדמי t...

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.

עצב שׁוֹקִיתִי השיטה פגיעה: A Assay אמין כדי לזהות מבחן הגורמים תיקון Neuromuscular הצומת

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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ממשק אדם-מכונה-שילוב חיישנים בעלות נמוכה עם מערכת גירוי חשמלי Neuromuscular לשיקום מאזן פעימות הודעה

A stroke is caused when an artery carrying blood from heart to an area in the brain bursts or a clot obstructs the blood flow to brain thereby preventing ...

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

גב שורש הגרעינים עצב ותאי גזע שמקורם בשומן Differentiated:<em&gt; במבחנה</em&gt; משותף תרבות דגם לחקר התחדשות של עצבים היקפיים

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the ...

A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila

A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila

שיטה לגרום סגור ראש הפגיעה מוחית טראומטית ב<em&gt; דרוזופילה</em

Traumatic brain injury (TBI) affects millions of people each year, causing impairment of physical, cognitive, and behavioral functions and death. Studies ...

Assessment of Neuromuscular Function Using Percutaneous Electrical Nerve Stimulation

הערכה של תפקוד העצבי באמצעות גירוי עצבי חשמלי Percutaneous

Percutaneous electrical nerve stimulation is a non-invasive method commonly used to evaluate neuromuscular function from brain to muscle (supra-spinal, ...

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

טעינת צבע סידן לתוך צפרדע עצב צפרדע דרך גדם עצב: סידן חולף הרשמה צפרדע Neuromuscular צפרדע

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

הזרקת גנגליון שורש הגבה ו שורש הגבה Crush פגיעה כמודל להתחדשות החושית אקסון

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

הדמיה פעילות עצבית בתוך קליפת המגע הראשי באמצעות Thy1-העכברים הטרנסגניים GCaMP6s

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