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.

Tutti gli esperimenti sono stati eseguiti secondo le linee guida NIH e protocolli animali approvati dal Comitato di Virginia Tech Istituzionale Animal Care e Usa. 1. Gli animali prepara per la chirurgia <ol><li> Anestetizzare topi con una miscela di ketamina (90 mg / kg) e xilazina (10 mg / kg) mediante iniezione sottocutanea con inguinale sterile 1 ml siringa da insulina. soluzione Carrier contiene una miscela di soluzione fisiologica 0,9%, 17,4 mg / ml ketamina e 2,6 mg / ml xilazina. Mettere gli animali di nuovo in gabbia in attesa che il farmaco abbia effetto. NOTA: Se la dose di carico non fornisce anestesia sufficienti per la durata del procedimento, un ulteriore 25% della dose di carico può essere iniettato. </li><li> animali Monitor dopo l&#39;iniezione per verificare la frequenza respiratoria e la depressione costante appropriato di livelli di eccitazione. Controllare il livello di eccitazione con un pizzico zampa posteriore, che dovrebbe suscitare alcuna risposta quando sufficientemente anestetizzati. NOTA: Questo richiede di solito 3-5min per un giovane topo adulto in media da 25 a 30 g. Se l&#39;animale è ancora reattivo dopo la post iniezione 10 min, un ulteriore 25% della dose di carico anestetico può essere iniettato. </li><li> Applicare vaselina e olio minerale leggero pomata oftalmica per gli occhi degli animali per prevenire la secchezza. Rimuovere gli animali da gabbia e posto su una superficie piana e pulita. Radere l&#39;arto posteriore desiderato dal piede al bacino tramite un trimmer capelli elettrici, esponendo solo l&#39;aspetto laterale dell&#39;arto. </li><li> Applicare un dispositivo di rimozione dei capelli chimica al sito rasato per 1 min. Rimuovere manualmente i capelli con salviette di laboratorio. Pulire la zona depilata con laboratorio wipe imbevuto di etanolo. </li></ol> 2. Procedura chirurgica <ol><li> Sterilizzare gli strumenti chirurgici tramite autoclave o altro metodo appropriato. Pulire il sito chirurgico e bordo chirurgica con l&#39;80% di etanolo / H 2 0. Disinfettare il sito chirurgico con proviodine. Posizionare il mouse sul bordo chirurgico e allinearsi con restrizioni degli arti. Tenerel&#39;arto destinazione posteriori in posizione anatomica naturale con il ginocchio leggermente esteso senza rotazione interna o esterna. </li><li> Mettere animale e tavola sotto il microscopio operatorio. Orientare al corretto sito di incisione attraverso la palpazione dei punti di riferimento superficiali, in particolare il ginocchio ossuto e il crinale tra il tibiale anteriore e muscoli gastrocnemio. </li><li> Fare un&#39;incisione di circa 3 centimetri attraverso la pelle con un bisturi o forbici primavera durante l&#39;utilizzo di pinze generali per la presa. Rendere l&#39;incisione perpendicolare al corso di fondo del nervo peroneo comune. </li><li> Continuare l&#39;incisione attraverso la fascia superficiale, esponendo il bicipite femorale e vasto laterale muscoli. Separare questi muscoli tagliando attraverso la fascia profonda di collegamento. Un taglio 1-2 cm dovrebbe essere sufficiente. </li><li> Ritrarre muscolo bicipite femorale caudale utilizzando divaricatori meccanici, rivelando il nervo peroneo comune. </li><li> Tracciare il nervo prossimalmente fino alla sua trasezione con il tendine del capo laterale del muscolo gastrocnemio è trovato. Nota: L&#39;esposizione può richiedere ulteriori manipolazione della pelle ritratta e muscoli. Questa intersezione viene utilizzato come punto di riferimento stabile per la lesione del nervo. </li><li> Afferrare il nervo con una pinza sottile, allineando le punte in modo parallelo al bordo laterale del tendine gastrocnemio. Schiacciare il nervo peroneale comune applicando costante, pressione dura per 5 sec. </li><li> Corroborare pieno schiacciamento del nervo mediante ispezione visiva attraverso l&#39;ambito chirurgico. Apparirà traslucida presso il sito di lesione. Se si utilizza topi che esprimono proteine ​​fluorescenti in assoni periferici, la fluorescenza scompare dal sito di lesione. </li><li> Rimuovere divaricatori e riallineare i muscoli nelle loro posizioni anatomiche. Chiudere il sito di incisione con 6-0 punti di sutura in seta. 1-3 punti staccati semplici è sufficiente. Mettere recupero del mouse su una piastra elettrica in una gabbia pulita. </li><li> Monitorare tutti gli animali per 2 ore post-operazione per controllare la respirazione e le eventuali reazioni avverse all&#39;anestesia. Somministrare una dose iniziale di buprenorfina 0,05-0,10 mg / kg tramite iniezione sottocutanea inguinale subito dopo il recupero da un intervento chirurgico. Dare 3 ulteriori dosi ogni 12 hr per il prossimo 48 hr. Dopo il recupero completo, tornare topi per la casa di cura degli animali. </li></ol> 3. Isolamento e colorazione di estensore lungo delle dita Muscoli (EDL) <ol><li> Sacrificare animali con isoflurano. Dispensare 0,5 ml isoflurano liquido in un tubo da 50 ml piena di labwipes assorbenti. Posizionare il tubo non livellata con l&#39;animale in una camera stagna 2500 centimetri 3. Almeno 4 minuti di esposizione è sufficiente. Test per la perdita di palpebrale bilaterale, toe-pinch, e riflessi coda pizzico per garantire che ogni animale è incosciente prima di procedere con la perfusione. </li><li> Transcardially profumato 16 animali prima con 10 ml di 0,1 M PBS, poi 25 ml di 4% paraformaldeide in PBS 0,1 M (pH 7,4). Eparina (30 unità / 20g di peso animale) può essere aggiunto con il PBS (10 unità / ml) per prevenire la coagulazione del sangue nei piccoli capillari, migliorando i risultati di perfusione. </li><li> Rimuovere la pelle che copre arti posteriori utilizzando forbici per tagliare trasversalmente attraverso la cute intorno alla circonferenza dell&#39;addome. Sbucciare giù la pelle oltre le arti e le zampe posteriori con pinze. </li><li> Rimuovere fascia superficiale degli arti posteriori afferrando e peeling con una pinza. Se si utilizza topi che esprimono proteine ​​fluorescenti negli assoni periferici, post-risolvere tutto topi durante la notte nel 4% PFA in provette da 50 ml. Risciacquare tre volte con PBS. NOTA: fissa i topi possono essere memorizzati in PBS a 4 ° C. In caso contrario, saltare questo passaggio e passare al punto 3.6, senza animali pubblicare truccate. </li><li> Sezionare i muscoli EDL 18 da topo arti posteriori, essendo sicuri di mantenere il più possibile intatto prossimale e distale tendini. </li><li> Incubare muscoli EDL in tampone bloccante (1x PBS contenente 0,5% Triton X-100, 3% BSA e 5% siero di capra) per almeno 1 ora. </li><li&gt; Per visualizzare gli assoni a motore e dei loro terminali nervosi, posto muscoli in provette contenenti neurofilament (1: 1.000) e synaptotagmin-2 (1: 250) anticorpi diluito in tampone di bloccaggio per 3 giorni. Lavare i muscoli tre volte con PBS 1x e 10 minuti ogni volta. Nota: Ignorare questo passaggio se si utilizzano topi che esprimono proteine ​​fluorescenti (XFP) in assoni periferici. </li><li> Colorare il controlaterale indenne EDL come controllo positivo per la completa innervazione NMJ. I controlli negativi dovrebbero comprendere l&#39;EDL ottenuto a 4 giorni post-lesione, un punto temporale in cui il NMJ è completamente denervato, nonché un EDL colorate con solo anticorpo secondario. </li><li> Incubare i muscoli con opportuni anticorpi secondari fluorescente con tag per rilevare neurofilament e synaptotagmin-2 per 1 giorno. Lavare i muscoli tre volte con PBS 1x e 10 minuti ogni volta. Nota: Questa operazione può essere effettuata con passo 3.10. Ignorare questo passaggio se si utilizzano topi che esprimono proteine ​​fluorescenti in assoni periferici. </li><li> Per visualizzare il postsyregione naptic del NMJ, incubare i muscoli con 5 mg / ml di Alexa-555 coniugato Alfa Bungarotossina diluito in tampone di bloccaggio per almeno 2 ore. Lavare i muscoli tre volte con PBS 1x e 10 minuti ogni volta. </li><li> Per montare i muscoli interi su vetrini con carica positiva, posizionare il muscolo direttamente sulla diapositiva, aggiungere qualche goccia di glicerolo media sulla slitta di montaggio basati e coprire con un vetrino. Premere il vetrino contro il vetrino per appiattire il muscolo. Impregna fuori dal mezzo di montaggio dal perimetro della diapositiva e coprioggetto utilizzando salviette di laboratorio. Applicare lo smalto per sigillare i bordi tra il vetrino e scivolo. </li></ol> 4. Imaging e analisi dei dati <ol><li> Per analizzare la struttura di NMJs, l&#39;immagine del muscolo EDL utilizzando un microscopio confocale a scansione laser attrezzato per eccitare 488, 555 e 633 nm di luce e catturare la luce emessa con 20X e 40X obiettivi. </li><li> Per visualizzare tutta NMJs, creare immagini di proiezione massima intensità del sec otticazioni distanziate da 1 a 2 micron che si estende a parte il più basso al più alto regioni visibili della NMJ. Crea proiezioni di massima intensità utilizzando software di imaging disponibili in commercio. </li><li> Per determinare i tassi di reinnervazione, catalogare NMJs come: 1) completamente denervato = sito post-sinaptica è completamente privo di contatto con gli assoni, meno del 5% colocalizzazione tra l&#39;assone e AChRs. 2) parzialmente innervato = l&#39;assone si sovrappone parzialmente il postsynapse, 5-95% colocalizzazione tra l&#39;assone e AChRs. 3) innervazione completa = quasi perfetta apposizione tra il pre e post-sinapsi, superiore al 95% colocalizzazione tra l&#39;assone e AChRs. Esclusione NMJs che giacciono perpendicolare al piano di imaging e non pienamente visualizzabili nell&#39;immagine. Nota: In tutti questi esperimenti, di almeno 3 animali e 50 NMJs per animale sono stati esaminati. I risultati sono stati ritenuti significativi utilizzando un t-test di Student con un valore di P inferiore a 0,05. </li><li> Per accecare l&#39;operatore, individui separati possono eseguirel&#39;intervento chirurgico e l&#39;immagine di analisi. Senza la conoscenza dei gruppi di trattamento, l&#39;analizzatore può essere obiettivo con NMJ punteggio. In alternativa, le immagini possono essere randomizzati e presentati all&#39;operatore per analisi senza la conoscenza dell&#39;animale sorgente. </li></ol> 5. PCR quantitativa <ol><li> Sacrificare animali utilizzando isoflurano e dislocazione cervicale. Togliere la pelle e fascia superficiale che copre i muscoli delle gambe in base al punto 3.3. Sezionare tibiale anteriore e muscoli EDL in base al punto 3.4. </li><li> Flash congelare l&#39;intera tibiale anteriore e muscoli EDL in una provetta da 1,5 ml over azoto liquido. Rimuovere il tessuto dal tubo e posto in un mortaio di pre-raffreddata parzialmente immerso in azoto liquido. Macinare muscolo congelato in una polvere fine con un mortaio e pestello. </li><li> Sciogliere in polvere muscolare congelati in un reagente di estrazione dell&#39;RNA disponibili in commercio e procedere all&#39;estrazione del RNA e la rimozione DNA genomico con un kit disponibile in commercio secondo i produttoreSTRUZIONI. </li><li> Eseguire la trascrizione inversa con un mix trascrittasi inversa disponibili in commercio in base alle istruzioni del produttore. </li><li> Eseguire qPCR utilizzando un kit disponibile in commercio utilizzando opportuni geni housekeeping (vedi tabella dei materiali). Utilizzare un quantitativo termociclatore PCR disponibile in commercio per eseguire la PCR (vedi tabella dei materiali). </li><li> Impostare la temperatura di ricottura a 58 ° C. Regolare i parametri di ciclismo aggiuntive alle specifiche del costruttore di Taq polimerasi / SYBR mix verde. Includere una fase curva di fusione finale in programma termociclatore comprensivi di 0,5 ° aumenti incrementali C da 65 ° C a 95 ° C per testare primer specificità PRIMER formazione dimero. </li><li> Determinare relativi livelli di espressione dell&#39;mRNA per il 2 - metodo di ΔΔCT 21 utilizzando 18S RNA come il gene di controllo. </li></ol>

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The authors have nothing to disclose.

Il metodo presentato in questo manoscritto fornisce opportunità uniche per identificare i meccanismi coinvolti nella riparazione di giunzioni neuromuscolari (NMJ). Questo metodo comporta la frantumazione del nervo peroneo comune che passa sopra il gastrocnemio tendine vicino al ginocchio. Abbiamo dimostrato che dopo soli 5 secondi di compressione del nervo con una pinza, completa degenerazione è notato da 4 giorni dopo l&#39;infortunio. Nei topi giovani adulti, gli assoni alfa-motori cominciano a Reinnervate precedent...

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

La giunzione neuromuscolare (NMJ) subisce cambiamenti strutturali e funzionali deleteri a causa di invecchiamento, lesioni e malattie. Pertanto, è indispensabile per capire i cambiamenti cellulari e molecolari coinvolti nella manutenzione e riparazione NMJs. A questo scopo, abbiamo sviluppato un metodo per affidabile e coerente esaminare rigenerante NMJs nei topi. Questo metodo comporta la lesione del nervo schiacciamento del nervo peroneo comune che passa sopra la testa laterale del tendine del muscolo gastrocnemio vicino al ginocchio. Utilizzando vecchi topi di sesso femminile di 70 giorni, dimostriamo che assoni motori cominciano a Reinnervate precedenti obiettivi post-sinaptici entro 7 giorni post-cotta. Essi rioccupano completamente le loro precedenti zone sinaptiche da 12 giorni. Per determinare l&#39;affidabilità di questo metodo di infortunio, abbiamo confrontato i tassi di reinnervazione tra i singoli vecchi topi femmina 70 al giorno. Abbiamo scoperto che il numero di siti post-sinaptici reinnervated era simile tra i topi a 7, 9, e 12 giorni post-cotta. Per determinare sequesto test lesione può essere utilizzato anche per confrontare cambiamenti molecolari nei muscoli, abbiamo esaminato livelli di gamma-subunità del recettore nicotinico muscolare (gamma-AChR) e la chinasi muscolo-specifica (MuSK). La subunità gamma-AChR e muschio di sono altamente sovraregolati dopo denervazione e tornare a livelli normali seguenti reinnervazione di NMJs. Abbiamo trovato una stretta relazione tra i livelli di trascrizione di questi geni e lo stato innervazione dei muscoli. Noi crediamo che questo metodo accelererà la nostra comprensione dei cambiamenti cellulari e molecolari coinvolti nella riparazione del NMJ e altre sinapsi.

Il nervo peroneale comune, chiamato anche il nervo peroneo comune, nasce dal nervo sciatico di sopra della fossa poplitea, dove si oscilla intorno alla testa del perone alla faccia anteriore della gamba (Figura 1A). Ci si dirama nei nervi peroneale superficiali e profondi, insieme fornendo i flessori dorsali del piede e dita del piede (tibiale anteriore, estensore lungo delle dita e brevis, e alluci estensori muscoli longus), e le everters del piede (muscoli peronei). Qu...

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.

Il nervo del perone Metodo Injury: Un test affidabile di individuare e testare i fattori che Repair neuromuscolari giunzioni

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

15.6K Views.  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. 

Video: 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.

Riproducibile nervo sciatico Crush mouse e successiva valutazione da parte della Rigenerazione intera analisi Muscle Monte

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

Ricerca

Neuroscienze

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.

applicazioni di<em&gt; In Vivo</em&gt; Test funzionale del Topo tibiale anteriore per la valutazione di ingegneria tessutale muscolo scheletrico di riparazione

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

Bioingegneria

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).

Valutazione della Neuromuscolare Funzione Con percutanea stimolazione elettrica del nervo

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.

Funzionalità di misurazione della giunzione neuromuscolare

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.

Murino tendine flessore Lesioni e Chirurgia riparazione

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

Medicina

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.

Metodi funzionali e fisiologici per valutare la rigenerazione mediana del nervo nel ratto

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.

Misurazione massima della forza tetanica isometrica del muscolo anteriore tibiale nel ratto

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.

Impianto e controllo di sistemi wireless senza batteria per l'interfacciamento dei nervi periferici

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>Modello modificato di chirurgia delle lesioni nervose risparmiate del dolore neuropatico nei topi

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.

Il nervo del perone Metodo Injury: Un test affidabile di individuare e testare i fattori che Repair neuromuscolari giunzioni

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