Name: the fibular nerve injury method: a reliable assay to identify and test factors that repair neuromuscular junctions
Uploaded: 2016-08-11T15:00:00.000+00:00
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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.

Alle Experimente wurden unter NIH-Richtlinien und Tier Protokolle, die von der Virginia Tech Institutional Animal Care und Use Committee genehmigt durchgeführt. 1. Vorbereiten Tiere für Chirurgie <ol><li> Anästhesieren Mäuse mit einem Gemisch aus Ketamin (90 mg / kg) und Xylazin (10 mg / kg) über eine subkutane Injektion inguinal mit einer sterilen 1 ml Insulinspritze. Trägerlösung enthält eine Mischung aus 0,9% Kochsalzlösung, 17,4 mg / ml Ketamin und 2,6 mg / ml Xylazin. Legen Sie Tiere zurück in Käfigen, während für Medikamente warten zu übernehmen. HINWEIS: Wenn die Ladungsdosis nicht ausreichend Anästhesie für die Dauer des Verfahrens vorsieht, eine zusätzliche 25% der Initialdosis injiziert werden kann. </li><li> Monitor-Tiere der Injektion für den stationären Atemfrequenz und entsprechende Depression von Erregungsniveau zu überprüfen. Überprüfen Sie Erregungsniveau mit einem Hinterfuß Prise, die keine Antwort auslösen sollte, wenn ausreichend narkotisiert. HINWEIS: Dies dauert in der Regel 3-5min für einen jungen Erwachsenen Maus 25 bis 30 g im Durchschnitt. Wenn das Tier immer noch anspricht nach 10 min nach der Injektion ist eine zusätzliche 25% des Anästhetikums Dosis kann injiziert werden. </li><li> Bewerben Petrolatum und leichtes Mineralöl Augensalbe auf die Augen des Tieres Trockenheit zu verhindern. Entfernen Sie Tiere aus dem Käfig nehmen und auf eine saubere, ebene Fläche. Shave die gewünschte Hinterbein vom Fuß bis zum Becken einen elektrischen Haarschneider verwenden, werden nur die lateralen Seite des Gliedes auszusetzen. </li><li> Tragen Sie einen chemischen Haarentferner auf die rasierte Stelle für 1 min. Entfernen Sie manuell die Haare Labortücher verwenden. Reinigen Sie die enthaarte Bereich mit Labor wipe in Ethanol getränkt. </li></ol> 2. Chirurgisches Verfahren <ol><li> Sterilisieren von chirurgischen Instrumenten über Autoklaven oder andere geeignete Verfahren. Reinigen Sie die Operationsstelle und chirurgische Board mit 80% Ethanol / H 2 0. Desinfizieren Sie die Operationsstelle mit proviodine. Platzieren Sie die Maus auf chirurgische Bord und richten mit Gliedmaßen Stützen. Behaltendas Ziel der hinteren Gliedmaßen in einer anatomisch natürlichen Position mit dem Kniegelenk verlängert leicht ohne interne oder externe Rotation. </li><li> Platzieren Sie Tier- und Brett unter dem Operationsmikroskop. Orient auf den richtigen Einschnittstelle über die Palpation von oberflächlichen Sehenswürdigkeiten, speziell der knöchernen Kniegelenk und dem Grat zwischen der tibialis anterior und gastrocnemius Muskeln. </li><li> Machen Sie einen ca. 3 cm Schnitt durch die Haut mit einem Skalpell oder Feder Schere, während allgemeine Pinzette zum Greifen. Machen Sie den Einschnitt senkrecht zur zugrunde liegenden Verlauf des N. fibularis communis. </li><li> Fortsetzung der Einschnitt durch die oberflächliche Faszie, Freilegung der Bizeps femoris und vastus lateralis Muskeln. Trennen Sie diese Muskeln, indem sie durch die Verbindungs ​​tiefe Faszie zu schneiden. Ein 1-2 cm geschnitten, sollte ausreichend sein. </li><li> Fahren Sie die Bizeps femoris kaudal mechanischen Retraktoren mit der N. fibularis communis enthüllt. </li><li> Verfolgen Sie die Nerven proximal, bis seine interAbschnitt mit der Sehne des lateralen Kopf des M. gastrocnemius gefunden wird. Hinweis: Die Belichtung kann zusätzliche Manipulation der eingezogenen Haut und Muskeln. Dieser Schnittpunkt wird als stabil Meilenstein für die Nervenverletzung verwendet. </li><li> Fassen Sie den Nerv mit einer feinen Pinzette, die Spitzen in einer parallelen Weise an den seitlichen Rand der gastrocnemius Sehne ausrichten. Crush the N. fibularis communis durch die Anwendung stabil, harten Druck für 5 Sek. </li><li> Corroborate voll verknallt des Nerven durch visuelle Inspektion durch den chirurgischen Umfang. Es wird transluzente an der Stelle der Verletzung auftreten. Wenn Mäuse, die Fluoreszenzproteine ​​in peripheren Axonen verwenden, wird die Fluoreszenz von der Stelle der Verletzung verschwinden. </li><li> Entfernen Sie Retraktoren und neu auszurichten Muskeln in ihren anatomischen Positionen. Schließen Sie die Inzisionsstelle mit 6-0 Seidennähten. 1-3 einfache Nähten ist ausreichend. Platzieren Maus recuperating auf einem Heizkissen in einen sauberen Käfig. </li><li> Überwachen Sie alle Tiere für 2 Stunden nach der Opertion für die Atmung und keine negativen Reaktionen auf die Anästhesie zu überprüfen. Verwalten Sie eine anfängliche Dosis von Buprenorphin 0,05-0,10 mg / kg über eine subkutane Injektion Leisten- sofort von der Operation folgenden Erholung. Geben Sie 3 zusätzliche Dosen alle 12 Stunden über die nächsten 48 Stunden. Nach der vollständigen Genesung, Rückkehr Mäuse auf die Tierstation. </li></ol> 3. Isolierung und Färbung der Extensor communis digitorum (EDL) Muskeln <ol><li> Sacrifice Tiere mit Isofluran. Dispense 0,5 ml Flüssigkeit Isofluran in einen 50-ml-Röhrchen mit einem saugfähigen labwipes verpackt. Setzen Sie den unverschlossenen Röhrchen mit dem Tier in einem verschlossenen 2.500 cm 3 Kammer. Mindestens 4 min Expositions ausreichend. Test für den Verlust von bilateralen palpebral, toe-Pinch und Schwanz-Pinch Reflexe, um sicherzustellen, dass jedes Tier, bevor Sie mit Perfusion bewusstlos ist. </li><li> Transkardial perfundiert 16 Tiere zuerst mit 10 ml 0,1 M PBS, dann wurden 25 ml 4% Paraformaldehyd in 0,1 M PBS (pH 7,4). Heparin (30 Einheiten / 20g Tiergewicht) mit dem PBS (10 Einheiten / ml) zu verhindern Blutgerinnung in kleinen kapillaren Betten, die Verbesserung der Perfusionsergebnisse hinzugefügt werden. </li><li> Entfernen Sie die Haut bedeckt Hinterbeine durch Schere quer durch die Haut um den Umfang des Bauches zu schneiden. Ziehen Sie die Haut nach unten vorbei an den hinteren Gliedmaßen und Füße einer Pinzette. </li><li> Entfernen Sie Oberflächenfaszie von Hintergliedmaßen durch Greifen und Peeling mit einer Pinzette. Wenn Mäuse, die Fluoreszenz-Proteine ​​in peripheren Axonen mit, post-fix ganze Mäuse über Nacht in 4% PFA in 50-ml-Röhrchen. Spülen dreimal mit PBS. HINWEIS: Fest Mäuse können in PBS bei 4 ° C gelagert werden. Wenn nicht, überspringen Sie diesen Schritt und fahren 3.6 ohne Post-fixierenden Tiere zu treten. </li><li> Präparieren aus EDL Muskeln 18 aus Maus Hinterextremitäten, sicher zu sein , proximale und distale Sehnen zu halten so intakt wie möglich. </li><li> Inkubieren EDL Muskeln in Blocking-Puffer für mindestens 1 Stunde (1x PBS 0,5% Triton X-100, 3% BSA und 5% Ziegenserum enthielt). </li><liund synaptotagmin-2 (1: 250) Antikörper verdünnt in Blockierungspuffer für 3 Tage:&gt; Um Motoraxonen und ihre Nervenenden, statt Muskeln in Röhrchen, die Neurofilament (1000 1) zu visualisieren. Waschen Sie Muskeln dreimal mit 1x PBS und je 10 min. Hinweis: Überspringen Sie diesen Schritt, wenn Mäuse, die Fluoreszenzproteine ​​(XFP) in peripheren Axonen verwenden. </li><li> Stain die kontralaterale heil EDL als positive Kontrolle für die komplette NMJ Innervation. Negative Kontrollen sollten eine EDL erhalten bei 4 Tage nach der Verletzung, ein Zeitpunkt enthalten, in dem die NMJ vollständig denervierten ist, sowie eine EDL mit sekundären Antikörper gefärbt nur. </li><li> Inkubieren Muskeln mit entsprechenden fluoreszierend markierten Sekundärantikörper Neurofilament und synaptotagmin-2 für 1 Tag zu erfassen. Waschen Sie Muskeln dreimal mit 1x PBS und je 10 min. Hinweis: Dieser Schritt kann durchgeführt werden, zusammen mit Schritt 3.10. Überspringen Sie diesen Schritt, wenn Mäuse, die Fluoreszenz-Proteine ​​in peripheren Axonen verwenden. </li><li> Zur Visualisierung der postsynaptic Bereich des NMJ inkubieren Muskeln mit 5 ug / ml Alexa 555-konjugierten alpha-Bungarotoxin verdünnten Puffer für mindestens 2 h in blockieren. Waschen Sie Muskeln dreimal mit 1x PBS und je 10 min. </li><li> So montieren ganze Muskeln auf positiv geladene Objektträger aus Glas, legen Sie den Muskel direkt auf der Folie, ein paar Tropfen Glycerin hinzufügen basierten Montagemedium auf dem Objektträger geben und mit einem Deckglas. Drücken Sie die Deck gegen die Folie, um die Muskeln zu glätten. Tränken weg Eindeckmedien vom Umfang der Rutsche und Deckglas mit Labor wischt. Bewerben Nagellack die Kanten zwischen dem Deckglas und Folie zu versiegeln. </li></ol> 4. Imaging und Datenanalyse <ol><li> Zur Analyse der Struktur von NMJs, Bild der EDL Muskel eines konfokalen Laser-Scanning-Mikroskop ausgerüstet 488, 555 und 633 nm Licht zur Anregung und Erfassung des emittierten Lichts mit 20X und 40X Ziele. </li><li> Um ganze NMJs sichtbar zu machen, schaffen Maximum Intensity Projection Bilder von optischen secnen Abstand von 1 bis 2 um abgesehen von der niedrigsten zur höchsten erstreckt sichtbaren Bereich des NMJ. Erstellen Sie maximale Intensität Projektionen mit handelsüblichen Bildbearbeitungssoftware. </li><li> Um die Preise von reinnervation bestimmen, zu kategorisieren NMJs als: 1) vollständig denervierten = postsynaptischen Seite völlig frei von Kontakt mit Axon ist, weniger als 5% Kolokalisation zwischen dem Axon und AChRs. 2) Teilweise innervated = das Axon teilweise überlappt die Postsynapse, 5-95% Kolokalisation zwischen dem Axon und AChRs. 3) Voll Innervation = nahezu perfekt Apposition zwischen Pre- und Post-Synapse, mehr als 95% Kolokalisation zwischen dem Axon und AChRs. Ausschließen NMJs, die senkrecht zu der Abbildungsebene liegen oder nicht vollständig in dem Bild sichtbar gemacht. Anmerkung: In all diesen Versuchen mindestens 3 Tieren und 50 NMJs pro Tier untersucht. Die Ergebnisse wurden mit einem P-Wert von weniger als 0,05 signifikant unter Verwendung eines Student-t-Test angesehen. </li><li> Um blenden den Bediener können separate Individuen durchführendie Operation und Bildanalyse. Ohne Kenntnis der Behandlungsgruppen kann der Analysator mit NMJ scoring Ziel sein. Alternativ können Bilder randomisiert und den Operator für die Analyse ohne Kenntnis der Quelle Tier präsentiert. </li></ol> 5. Quantitative PCR <ol><li> Sacrifice Tiere mit Isofluran und Genickbruch. Entfernen Sie die Haut und oberflächliche Faszie bedeckt Beinmuskulatur nach 3.3 zu treten. Präparieren tibialis anterior und EDL Muskeln nach 3.4 zu treten. </li><li> Blitzeis die gesamte tibialis anterior und EDL Muskeln in einem 1,5-ml-Röhrchen über flüssigem Stickstoff. Entfernen Sie Gewebe aus Rohr und in einem vorgekühlten Mörser teilweise in flüssigem Stickstoff eingetaucht. Grind gefrorenen Muskel in ein feines Pulver mit einem Mörser und Stößel. </li><li> Man löst gefrorene Muskel Pulver in einen kommerziell erhältlichen RNA-Extraktion Reagenz und führen die RNA-Extraktion und genomische DNA Entfernung mit einem im Handel erhältlichen Kits des Herstellers i nachnweise. </li><li> Führen der reversen Transkription mit einer im Handel erhältlichen reversen Transkriptase Mischung gemäß den Anweisungen des Herstellers. </li><li> Führen Sie qPCR eines kommerziell erhältlichen Kits mit geeigneten Housekeeping-Gene (siehe Tabelle von Materialien). Verwenden Sie eine im Handel erhältliche quantitative PCR-Thermocycler zur Durchführung von PCR (siehe Tabelle der Materialien). </li><li> Eingestellt Annealingtemperatur auf 58 ° C. Passen Sie zusätzliche Zyklierungsparameter den Spezifikationen des Herstellers von Taq-Polymerase / SYBR grün-Mix. Umfassen eine endgültige Schmelzkurve Schritt in der Programm Thermocycler bestehend aus 0,5 ° C inkremental erhöht sich von 65 ° C bis 95 ° C für die Primer-Spezifität zu testen und Dimerbildung Primers. </li><li> Bestimmen Sie relativen mRNA - Expressionsniveaus durch die 2 - ΔΔCT Methode 21 unter Verwendung von 18S RNA als Kontroll - Gen. </li></ol>

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The authors have nothing to disclose.

Das Verfahren in diesem Manuskript präsentiert bietet einzigartige Möglichkeiten Mechanismen bei der Reparatur von neuromuskulären Verbindungen (NMJ) beteiligt zu identifizieren. Dieses Verfahren beinhaltet das Brechen des N. fibularis communis, wie es gastrocnemius Sehne in der Nähe des Knies verläuft. Wir zeigen, dass nach nur 5 Sekunden von Nervenkompression mit einer Zange, vollständige Degeneration um 4 Tage nach der Verletzung festgestellt wird. Bei jungen erwachsenen Mäusen, beginnen alpha-Motoraxonen vorh...

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.

<table><tbody><tr><td>Ketamine</td><td>VetOne</td><td>501072</td><td></td></tr><tr><td>Xylazine</td><td>Lloyd Inc.&amp;nbsp;</td><td>003437&amp;nbsp;</td><td></td></tr><tr><td>Buprenorphine</td><td>&amp;nbsp;Zoopharm</td><td>1Z-73000-150910&amp;nbsp;</td><td></td></tr><tr><td>Nair</td><td>Nair</td><td></td><td></td></tr><tr><td>Kim-wipes</td><td>Kimtech</td><td>34155</td><td></td></tr><tr><td>Electric Razor</td><td>Braintree Scientific</td><td>CLP-64800</td><td></td></tr><tr><td>80% EtOH/H&lt;sub&gt;2&lt;/sub&gt;0</td><td></td><td></td><td></td></tr><tr><td>10% Proviodine</td><td></td><td></td><td></td></tr><tr><td>1 ml Insulin Syringe</td><td></td><td></td><td></td></tr><tr><td>Spring Scissors</td><td>Vannas</td><td>91500-09</td><td></td></tr><tr><td>No. 15 scalpel</td><td>Braintree Scientific</td><td>SSS 15</td><td></td></tr><tr><td>#5 Forceps</td><td>Dumont</td><td>11252-00</td><td></td></tr><tr><td>6-0 silk suture on reverse cutting needle</td><td>&amp;nbsp;Suture Express</td><td>752B&amp;nbsp;</td><td></td></tr><tr><td>Rodent Heating Pad</td><td>Braintree Scientific</td><td>AP-R-18.5</td><td></td></tr><tr><td>Alexa 555 conjugated alpha-BTX</td><td>Molecular Probes</td><td>B35451</td><td></td></tr><tr><td>Vectashield</td><td>Vector Labs</td><td>H-1000</td><td></td></tr><tr><td>Olympus Stereo Zoom Microscope</td><td>Olympus</td><td>562037192</td><td></td></tr><tr><td>Zeiss 700 Confocal Microscope</td><td>Zeiss</td><td></td><td></td></tr><tr><td>Variable-flow peristaltic perfusion pump</td><td>Fisher Scientific</td><td>13-876-3</td><td></td></tr><tr><td>Aurum Total RNA Mini Kit</td><td>Bio-Rad</td><td>7326820</td><td></td></tr><tr><td>Bio-Rad iScript RT Supermix</td><td>Bio-Rad</td><td>1708840</td><td></td></tr><tr><td>SsoFast Evagreen Supermix</td><td>Bio-Rad</td><td>1725200</td><td></td></tr><tr><td>Bio-Rad CFX96</td><td>Bio-Rad</td><td>1855196</td><td></td></tr><tr><td>Puralube Vet ointment</td><td>Puralube</td><td>1621</td><td></td></tr><tr><td>Synaptotagmin-2 antibody</td><td>Antibodies-Online</td><td>ABIN401605</td><td></td></tr><tr><td>Neurofilament antibody</td><td>Antibodies-Online</td><td>ABIN2475842</td><td></td></tr></tbody></table>

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

Die neuromuskulären Synapse (NMJ) erfährt schädliche strukturelle und funktionelle Veränderungen als Folge des Alterns, Verletzungen und Krankheiten. Daher ist es zwingend notwendig, um die zellulären und molekularen Veränderungen bei der Aufrechterhaltung und der Reparatur NMJs beteiligt zu verstehen. Zu diesem Zweck haben wir eine Methode entwickelt, um zuverlässig und konsistent untersuchen NMJs in Mäusen zu regenerieren. Diese Nervenverletzung Verfahren umfasst das Zerkleinern des N. fibularis communis, wie es der lateralen Kopf des M. gastrocnemius Sehne in der Nähe des Knies verläuft. vorherige postsynaptischen Ziele innerhalb von 7 Tagen nach reinnervate Post-crush unter Verwendung von 70 Tage alten weiblichen Mäusen zeigen wir, dass Motoraxonen beginnen. Sie reoccupy vollständig ihre vorherigen synaptischen Bereiche von 12 Tagen. Um die Zuverlässigkeit dieser Verletzung Methode bestimmen, verglichen wir reinnervation Raten zwischen den einzelnen 70 Tage alten weiblichen Mäusen. Wir fanden, dass die Anzahl der postsynaptischen reinnervated Stellen zwischen Mäusen ähnlich war bei 7, 9 und 12 Tage nach-crush. Um festzustellen, obDiese Verletzungs Assay kann auch zum Vergleich molekularen Veränderungen in Muskeln verwendet werden, untersuchten wir Ebenen der gamma-Untereinheit des nikotinischen Rezeptors Muskel (gamma-AChR) und der muskelspezifische Kinase (MuSK). Die Gamma-AChR-Untereinheit und MuSK zu folgen Denervierung hoch oben reguliert und zurück auf ein normales Niveau folgende reinnervation von NMJs. Wir fanden eine enge Beziehung zwischen Transkriptniveaus für diese Gene und Innervation Zustand der Muskeln. Wir glauben, dass diese Methode das Verständnis der zellulären und molekularen Veränderungen bei der Reparatur der NMJ und anderen Synapsen beteiligt beschleunigen wird.

Der N. fibularis communis, auch genannt der N. peronaeus, ergibt sich aus dem Ischiasnerv oberhalb der Kniekehle, wo es um den Kopf der Fibula zur Vorderseite des Beins (1A) schwingt. Dort zweigt in die oberflächlichen und tiefen fibular Nerven, zusammen, um die Dorsalflexoren des Fußes und der Zehen Versorgung (Tibialis anterior, extensor digitorum longus und brevis und extensor Zehen longus) und die everters des Fußes (Peronaeus Muskeln). Dieser Nerv trägt auch sen...

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.

Die Fibula Nervenverletzung Methode: Ein zuverlässiger Test zur Identifizierung und Test Faktoren, die Reparatur neuromuskulären Synapsen

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

the-fibular-nerve-injury-method-reliable-assay-to-identify-test

Research

JoVE Journal

Neuroscience

15.6K Aufrufe.  Virginia Tech. 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. 

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

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

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative response mounted by PNS neurons thereby possibly illuminating the failures of CNS regeneration1. Sciatic nerve crush (axonotmesis) is one of the most common models of peripheral nerve injury in rodents2. Crushing interrupts all axons but Schwann cell basal laminae are preserved so that regeneration is optimal3,4. This allows the investigator to study precisely the ability of a growing axon to interact with both the Schwann cell and basal laminae4. Rats have generally been the preferred animal models for experimental nerve crush. They are widely available and their lesioned sciatic nerve provides a reasonable approximation of human nerve lesions5,4. Though smaller in size than rat nerve, the mouse nerve has many similar qualities. Most importantly though, mouse models are increasingly valuable because of the wide availability of transgenic lines now allows for a detailed dissection of the individual molecules critical for nerve regeneration6, 7. Prior investigators have used multiple methods to produce a nerve crush or injury including simple angled forceps, chilled forceps, hemostatic forceps, vascular clamps, and investigator-designed clamps8,9,10,11,12. Investigators have also used various methods of marking the injury site including suture, carbon particles and fluorescent beads13,14,1. We describe our method to obtain a reproducibly complete sciatic nerve crush with accurate and persistent marking of the crush-site using a fine hemostatic forceps and subsequent carbon crush-site marking. As part of our description of the sciatic nerve crush procedure we have also included a relatively simple method of muscle whole mount we use to subsequently quantify regeneration.

In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.

Reproduzierbare Maus Ischiasnerv Crush und anschließende Bewertung der Regeneration von Whole mount Muskelanalyse

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

Forschung

Neurowissenschaften

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from traumatic injury, disease and various congenital, genetic and acquired conditions are quite common. Tissue engineering and regenerative medicine technologies have enormous potential to provide a therapeutic solution. However, utilization of biologically relevant animal models in combination with longitudinal assessments of pertinent functional measures are critical to the development of improved regenerative therapeutics for treatment of VML-like injuries. In that regard, a commercial muscle lever system can be used to measure length, tension, force and velocity parameters in skeletal muscle. We used this system, in conjunction with a high power, bi-phase stimulator, to measure in vivo force production in response to activation of the anterior crural compartment of the rat hindlimb. We have previously used this equipment to assess the functional impact of VML injury on the tibialis anterior (TA) muscle, as well as the extent of functional recovery following treatment of the injured TA muscle with our tissue engineered muscle repair (TEMR) technology. For such studies, the left foot of an anaesthetized rat is securely anchored to a footplate linked to a servomotor, and the common peroneal nerve is stimulated by two percutaneous needle electrodes to elicit muscle contraction and dorsiflexion of the foot. The peroneal nerve stimulation-induced muscle contraction is measured over a range of stimulation frequencies (1-200 Hz), to ensure an eventual plateau in force production that allows for an accurate determination of peak tetanic force. In addition to evaluation of the extent of VML injury as well as the degree of functional recovery following treatment, this methodology can be easily applied to study diverse aspects of muscle physiology and pathophysiology. Such an approach should assist with the more rational development of improved therapeutics for muscle repair and regeneration.

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

We describe an in vivo protocol to measure dorsiflexion of the foot following stimulation of the peroneal nerve and contraction of the anterior crural compartment of the rat hindlimb. Such measurements are an indispensable translational tool for evaluating skeletal muscle pathology and tissue engineering approaches to muscle repair and regeneration.

Anwendungen<em&gt; In Vivo</em&gt; Funktionstest der Ratte Vorderer Schienbeingewebe Skeletal Muscle Repair Engineered für die Bewertung

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from ...

Biotechnik

Assessment of Neuromuscular Function Using Percutaneous Electrical Nerve Stimulation

Percutaneous electrical nerve stimulation is a non-invasive method commonly used to evaluate neuromuscular function from brain to muscle (supra-spinal, spinal and peripheral levels). The present protocol describes how this method can be used to stimulate the posterior tibial nerve that activates plantar flexor muscles. Percutaneous electrical nerve stimulation consists of inducing an electrical stimulus to a motor nerve to evoke a muscular response. Direct (M-wave) and/or indirect (H-reflex) electrophysiological responses can be recorded at rest using surface electromyography. Mechanical (twitch torque) responses can be quantified with a force/torque ergometer. M-wave and twitch torque reflect neuromuscular transmission and excitation-contraction coupling, whereas H-reflex provides an index of spinal excitability. EMG activity and mechanical (superimposed twitch) responses can also be recorded during maximal voluntary contractions to evaluate voluntary activation level. Percutaneous nerve stimulation provides an assessment of neuromuscular function in humans, and is highly beneficial especially for studies evaluating neuromuscular plasticity following acute (fatigue) or chronic (training/detraining) exercise.

We present a protocol to assess changes in neuromuscular function. Percutaneous electrical nerve stimulation is a non-invasive method that evokes muscular responses. Electrophysiological and mechanical properties of these responses permit the evaluation of neuromuscular function from brain to muscle (supra-spinal, spinal and peripheral levels).

Beurteilung der neuromuskulären Funktion Mit Perkutane elektrische Nervenstimulation

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

Measuring Neuromuscular Junction Functionality

Neuromuscular junction (NMJ) functionality plays a pivotal role when studying diseases in which the communication between motor neuron and muscle is impaired, such as aging and amyotrophic lateral sclerosis (ALS). Here we describe an experimental protocol that can be used to measure NMJ functionality by combining two types of electrical stimulation: direct muscle membrane stimulation and the stimulation through the nerve. The comparison of the muscle response to these two different stimulations can help to define, at the functional level, potential alterations in the NMJ that lead to functional decline in muscle.
Ex vivo preparations are suited to well-controlled studies. Here we describe an intensive protocol to measure several parameters of muscle and NMJ functionality for the soleus-sciatic nerve preparation and for the diaphragm-phrenic nerve preparation. The protocol lasts approximately 60 min and is conducted uninterruptedly by means of a custom-made software that measures the twitch kinetics properties, the force-frequency relationship for both muscle and nerve stimulations, and two parameters specific to NMJ functionality, i.e. neurotransmission failure and intratetanic fatigue. This methodology was used to detect damages in soleus and diaphragm muscle-nerve preparations by using SOD1G93A transgenic mouse, an experimental model of ALS that ubiquitously overexpresses the mutant antioxidant enzyme superoxide dismutase 1 (SOD1).

A functional assessment of the neuromuscular junction (NMJ) can provide essential information on the communication between muscle and nerve. Here we describe a protocol to comprehensively evaluate both the NMJ and muscle functionality using two different muscle-nerve preparations, i.e. soleus-sciatic and diaphragm-phrenic.

Neuromuskuläre Synapse Messfunktionalität

Neuromuscular junction (NMJ) functionality plays a pivotal role when studying diseases in which the communication between motor neuron and muscle is ...

Murine Flexor Tendon Injury and Repair Surgery

Tendon connects skeletal muscle and bone, facilitating movement of nearly the entire body. In the hand, flexor tendons (FTs) enable flexion of the fingers and general hand function. Injuries to the FTs are common, and satisfactory healing is often impaired due to excess scar tissue and adhesions between the tendon and surrounding tissue. However, little is known about the molecular and cellular components of FT repair. To that end, a murine model of FT repair that recapitulates many aspects of healing in humans, including impaired range of motion and decreased mechanical properties, has been developed and previously described. Here an in-depth demonstration of this surgical procedure is provided, involving transection and subsequent repair of the flexor digitorum longus (FDL) tendon in the murine hind paw.&#160;This technique can be used to conduct lineage analysis of different cell types, assess the effects of gene gain or loss-of-function, and to test the efficacy of pharmacological interventions in the healing process. However, there are two primary limitations to this model: i) the FDL tendon in the mid-portion of the murine hind paw, where the transection and repair occur, is not surrounded by a synovial sheath. Therefore this model does not account for the potential contribution of the sheath to the scar formation process. ii) To protect the integrity of the repair site, the FT is released at the myotendinous junction, decreasing the mechanical forces of the tendon, likely contributing to increased scar formation. Isolation of sufficient cells from the granulation tissue of the FT during the healing process for flow cytometric analysis has proved challenging; cytology centrifugation to concentrate these cells is an alternate method used, and allows for generation of cell preparations on which immunofluorescent labeling can be performed. With this method, quantification of cells or proteins of interest during FT healing becomes possible.

Flexor tendons in the hand are commonly injured, leading to impaired hand function. However, the scar-tissue healing response is not well characterized. A murine model of flexor tendon healing is demonstrated here. This model can enhance overall understanding of the healing process and assess therapeutic approaches to improve healing.

Murine Beugersehne Verletzung und Reparatur Chirurgie

Tendon connects skeletal muscle and bone, facilitating movement of nearly the entire body. In the hand, flexor tendons (FTs) enable flexion of the fingers ...

Medizin

Functional and Physiological Methods of Evaluating Median Nerve Regeneration in the Rat

The main goal of this investigation is to show how to create and repair different types of median nerve (MN) lesions in the rat. Moreover, different methods of simulating postoperative physiotherapy are presented. Multiple standardized strategies are used to assess motor and sensory recovery using an MN model of peripheral nerve lesion and repair, thus permitting easy comparison of the results. Several options are included for providing a postoperative physiotherapy-like environment to rats that have undergone MN injuries. Finally, the paper provides a method to evaluate the recovery of the MN using several noninvasive tests (i.e., grasping test, pin prick test, ladder rung walking test, rope climbing test, and walking track analysis), and physiological measurements (infrared thermography, electroneuromyography, flexion strength evaluation, and flexor carpi radialis muscle weight determination). Hence, this model seems particularly appropriate to replicate a clinical scenario, facilitating extrapolation of results to the human species.
Although the sciatic nerve is the most studied nerve in peripheral nerve research, analysis of the rat MN presents various advantages. For example, there is a reduced incidence of joint contractures and automutilation of the affected limb in MN lesion studies. Furthermore, the MN is not covered by muscle masses, making its dissection easier than that of the sciatic nerve. In addition, MN recovery is observed sooner, because the MN is shorter than the sciatic nerve. Also, the MN has a parallel path to the ulnar nerve in the arm. Hence, the ulnar nerve can be easily used as the nerve graft for repairing MN injuries. Finally, the MN in rats is located in the forelimb, akin to the human upper limb; in humans, the upper limb is the site of most peripheral nerve lesions.

Presented is a protocol to produce different types of median nerve (MN) lesions and repair in the rat. Additionally, the protocol shows how to evaluate the functional recovery of the nerve using several noninvasive behavioral tests and physiological measurements.

Funktionelle und physiologische Methoden zur Beurteilung der medianen Nervenregeneration in der Ratte

The main goal of this investigation is to show how to create and repair different types of median nerve (MN) lesions in the rat. Moreover, different ...

Maximum Isometric Tetanic Force Measurement of the Tibialis Anterior Muscle in the Rat

Traumatic nerve injuries result in substantial functional loss and segmental nerve defects often necessitate the use of autologous interposition nerve grafts. Due to their limited availability and associated donor side morbidity, many studies in the field of nerve regeneration focus on alternative techniques to bridge a segmental nerve gap. In order to investigate the outcomes of surgical or pharmacological experimental treatment options, the rat sciatic nerve model is often used as a bioassay. There are a variety of outcome measurements used in rat models to determine the extent of nerve regeneration. The maximum output force of the target muscle remains the most relevant outcome for clinical translation of experimental therapies. Isometric force measurement of tetanic muscle contraction has previously been described as a reproducible and valid technique for evaluating motor recovery after nerve injury or repair in both rat and rabbit models. In this video, we will provide a step-by-step instruction of this invaluable procedure for assessment of functional recovery of the tibialis anterior muscle in a rat sciatic nerve defect model using optimized parameters. We will describe the necessary pre-surgical preparations in addition to the surgical approach and dissection of the common peroneal nerve and tibialis anterior muscle tendon. The isometric tetanic force measurement technique will be detailed. Determining the optimal muscle length and stimulus pulse frequency is explained and measuring the maximum tetanic muscle contraction is demonstrated.

Evaluation of motor recovery remains the benchmark outcome measure in experimental peripheral nerve studies. The isometric tetanic force measurement of the tibialis anterior muscle in the rat is an invaluable tool to assess functional outcomes after reconstruction of sciatic nerve defects. The methods and nuances are detailed in this article.

Maximale isometrische tetanische Kraftmessung des Tibialis-Vordermuskels bei ratten

Traumatic nerve injuries result in substantial functional loss and segmental nerve defects often necessitate the use of autologous interposition nerve ...

Implantation and Control of Wireless, Battery-free Systems for Peripheral Nerve Interfacing

Peripheral nerve interfaces are frequently used in experimental neuroscience and regenerative medicine for a wide variety of applications. Such interfaces can be sensors, actuators, or both. Traditional methods of peripheral nerve interfacing must either tether to an external system or rely on battery power that limits the time frame for operation. With recent developments of wireless, battery-free, and fully implantable peripheral nerve interfaces, a new class of devices can offer capabilities that match or exceed those of their wired or battery-powered precursors. This paper describes methods to (i) surgically implant and (ii) wirelessly power and control this system in adult rats. The sciatic and phrenic nerve models were selected as examples to highlight the versatility of this approach. The paper shows how the peripheral nerve interface can evoke compound muscle action potentials (CMAPs), deliver a therapeutic electrical stimulation protocol, and incorporate a conduit for the repair of peripheral nerve injury. Such devices offer expanded treatment options for single-dose or repeated dose therapeutic stimulation and can be adapted to a variety of nerve locations.

This is a protocol for the surgical implantation and operation of a wirelessly powered interface for peripheral nerves. We demonstrate the utility of this approach with examples from nerve stimulators placed on either the rat sciatic or phrenic nerve.

Implantation und Steuerung von drahtlosen, batterielosen Systemen für periphere Nervenschnittstellen

Peripheral nerve interfaces are frequently used in experimental neuroscience and regenerative medicine for a wide variety of applications. Such interfaces ...

Modified Spared Nerve Injury Surgery Model of Neuropathic Pain in Mice

Spared nerve injury (SNI) is an animal model that mimics the cardinal symptoms of peripheral nerve injury for studying the molecular and cellular mechanism of neuropathic pain in mice and rats. Currently, there are two types of SNI model, one to cut and ligate the common peroneal and the tibial nerves with intact sural nerve, which is defined as SNIs in this study, and another to cut and ligate the common peroneal and the sural nerves with intact tibial nerve, which is defined as SNIt in this study. Because the sural nerve is purely sensory whereas the tibial nerve contains both motor and sensory fibers, the SNIt model has much less motor deficit than the SNIs model. In the traditional SNIt mouse model, the common peroneal and the sural nerves are cut and ligated separately. Here a modified SNIt surgery method is described to damage both common peroneal and sural nerves with only one ligation and one cut with a shorter procedure time, which is easier to perform and reduces the potential risk of stretching the sciatic or tibial nerves, and produces similar mechanical hypersensitivity as the traditional SNIt model.

<meta charset="utf8"></head><body>Modifiziertes Modell der Chirurgie der Nervenverletzung bei neuropathischen Schmerzen bei Mäusen

The modified surgery is a simplified method for mouse or rat spared nerve injury model that requires only one ligation and one cut to injure both common peroneal and sural nerves.

Die Fibula Nervenverletzung Methode: Ein zuverlässiger Test zur Identifizierung und Test Faktoren, die Reparatur neuromuskulären Synapsen

Zusammenfassung

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