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.

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			<td>Braintree Scientific</td>
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			<td>Dumont</td>
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			<td>Alexa 555 conjugated alpha-BTX </td>
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			<td>Vector Labs</td>
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			<td>Olympus</td>
			<td>562037192</td>
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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

An Optic Nerve Crush Injury Murine Model to Study Retinal Ganglion Cell Survival

Injury to the optic nerve can lead to axonal degeneration, followed by a gradual death of retinal ganglion cells (RGCs), which results in irreversible vision loss. Examples of such diseases in human include traumatic optic neuropathy and optic nerve degeneration in glaucoma. It is characterized by typical changes in the optic nerve head, progressive optic nerve degeneration, and loss of retinal ganglion cells, if uncontrolled, leading to vision loss and blindness.
The optic nerve crush (ONC) injury mouse model is an important experimental disease model for traumatic optic neuropathy, glaucoma, etc. In this model, the crush injury to the optic nerve leads to gradual retinal ganglion cells apoptosis. This disease model can be used to study the general processes and mechanisms of neuronal death and survival, which is essential for the development of therapeutic measures. In addition, pharmacological and molecular approaches can be used in this model to identify and test potential therapeutic reagents to treat different types of optic neuropathy.
Here, we provide a step by step demonstration of (I) Baseline retrograde labeling of retinal ganglion cells (RGCs) at day 1, (II) Optic nerve crush injury at day 4, (III) Harvest the retinae and analyze RGC survival at day 11, and (IV) Representative result.

This protocol shows how to retrogradely label retinal ganglion cells, and how to subsequently make an optic nerve crush injury in order to analyze retinal ganglion cell survival and apoptosis. It is an experimental disease model for different types of optic neuropathy, including glaucoma.

Ein Optic Nerve Crush Injury Mausmodell zur retinalen Ganglienzellen Survival Study

Injury to the optic nerve can lead to axonal degeneration, followed by a gradual death of retinal ganglion cells (RGCs), which results in irreversible ...

Forschung

Neurowissenschaften

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 ...

An Injury Paradigm to Investigate Central Nervous System Repair in Drosophila

An experimental method has been developed to investigate the cellular responses to central nervous system (CNS) injury using the fruit-fly Drosophila. Understanding repair and regeneration in animals is a key question in biology. The damaged human CNS does not regenerate, and understanding how to promote the regeneration is one of main goals of medical neuroscience. The powerful genetic toolkit of Drosophila can be used to tackle the problem of CNS regeneration.
A lesion to the CNS ventral nerve cord (VNC, equivalent to the vertebrate spinal cord) is applied manually with a tungsten needle. The VNC can subsequently be filmed in time-lapse using laser scanning confocal microscopy for up to 24 hr to follow the development of the lesion over time. Alternatively, it can be cultured, then fixed and stained using immunofluorescence to visualize neuron and glial cells with confocal microscopy. Using appropriate markers, changes in cell morphology and cell state as a result of injury can be visualized. With ImageJ and purposely developed plug-ins, quantitative and statistical analyses can be carried out to measure changes in wound size over time and the effects of injury in cell proliferation and cell death. These methods allow the analysis of large sample sizes. They can be combined with the powerful genetics of Drosophila to investigate the molecular mechanisms underlying CNS regeneration and repair.

An Injury Paradigm to Investigate Central Nervous System Repair in Drosophila

An injury paradigm using the Drosophila larval ventral nerve cord to investigate central nervous system regeneration and repair is described. Stabbing followed by laser scanning confocal microscopy in time-lapse and fixed specimens, combined with quantitative analysis with purposefully developed software and genetics, are used to investigate the molecular mechanisms of CNS regeneration and repair.

Eine Verletzung Paradigm Central Nervous System Repair in Untersuchen<em&gt; Drosophila</em

An experimental method has been developed to investigate the cellular responses to central nervous system (CNS) injury using the fruit-fly Drosophila. ...

A Human-machine-interface Integrating Low-cost Sensors with a Neuromuscular Electrical Stimulation System for Post-stroke Balance Rehabilitation

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 delivery of oxygen and nutrients. About half of the stroke survivors are left with some degree of disability. Innovative methodologies for restorative neurorehabilitation are urgently required to reduce long-term disability. The ability of the nervous system to reorganize its structure, function and connections as a response to intrinsic or extrinsic stimuli is called neuroplasticity. Neuroplasticity is involved in post-stroke functional disturbances, but also in rehabilitation. Beneficial neuroplastic changes may be facilitated with non-invasive electrotherapy, such as neuromuscular electrical stimulation (NMES) and sensory electrical stimulation (SES). NMES involves coordinated electrical stimulation of motor nerves and muscles to activate them with continuous short pulses of electrical current while SES involves stimulation of sensory nerves with electrical current resulting in sensations that vary from barely perceivable to highly unpleasant. Here, active cortical participation in rehabilitation procedures may be facilitated by driving the non-invasive electrotherapy with biosignals (electromyogram (EMG), electroencephalogram (EEG), electrooculogram (EOG)) that represent simultaneous active perception and volitional effort. To achieve this in a resource-poor setting, e.g., in low- and middle-income countries, we present a low-cost human-machine-interface (HMI) by leveraging recent advances in off-the-shelf video game sensor technology. In this paper, we discuss the open-source software interface that integrates low-cost off-the-shelf sensors for visual-auditory biofeedback with non-invasive electrotherapy to assist postural control during balance rehabilitation. We demonstrate the proof-of-concept on healthy volunteers.

A novel low-cost human-machine interface for interactive post-stroke balance rehabilitation system is presented in this article. The system integrates off-the-shelf low-cost sensors towards volitionally driven electrotherapy paradigm. The proof-of-concept software interface is demonstrated on healthy volunteers.

Eine Mensch-Maschine-Schnittstelle Integration von Low-Cost-Sensoren mit einem Neuromuskuläre Elektrostimulation System zur Post-Stroke Balance Rehabilitation

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 (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the study of nerve regeneration and myelination. The glial cells of the peripheral nervous system, Schwann cells (SC), are key facilitators of these processes; it is therefore crucial that the interactions of these cellular components are studied together. Direct contact between DRG neurons and glial cells provides additional stimuli sensed by specific membrane receptors, further improving the neuronal response. SC release growth factors and proteins in the culture medium, which enhance neuron survival and stimulate neurite sprouting and extension. However, SC require long proliferation time to be used for tissue engineering applications and the sacrifice of an healthy nerve for their sourcing. Adipose-derived stem cells (ASC) differentiated into SC phenotype are a valid alternative to SC for the set-up of a co-culture model with DRG neurons to study nerve regeneration. The present work presents a detailed and reproducible step-by-step protocol to harvest both DRG neurons and ASC from adult rats; to differentiate ASC towards a SC phenotype; and combines the two cell types in a direct co-culture system to investigate the interplay between neurons and SC in the peripheral nervous system. This tool has great potential in the optimization of tissue-engineered constructs for peripheral nerve repair.

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

Dorsal root ganglia (DRG) are structures containing the sensory neurons of the peripheral nervous system. When dissociated, they can be co-cultured with SC-like adipose-derived stem cells (ASC), providing a valuable model to study in vitro nerve regeneration and myelination, mimicking the in vivo environment at the injury site.

Dorsalwurzelganglien Neuronen und differenzierten Fettgewebe gewonnene Stammzellen: Eine<em&gt; In-vitro-</em&gt; Co-Kultur Modell zur Regeneration peripherer Nerven Studieren

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

Traumatic brain injury (TBI) affects millions of people each year, causing impairment of physical, cognitive, and behavioral functions and death. Studies using Drosophila have contributed important breakthroughs in understanding neurological processes. Thus, with the goal of understanding the cellular and molecular basis of TBI pathologies in humans, we developed the High Impact Trauma (HIT) device to inflict closed head TBI in flies. Flies subjected to the HIT device display phenotypes consistent with human TBI such as temporary incapacitation and progressive neurodegeneration. The HIT device uses a spring-based mechanism to propel flies against the wall of a vial, causing mechanical damage to the fly brain. The device is inexpensive and easy to construct, its operation is simple and rapid, and it produces reproducible results. Consequently, the HIT device can be combined with existing experimental tools and techniques for flies to address fundamental questions about TBI that can lead to the development of diagnostics and treatments for TBI. In particular, the HIT device can be used to perform large-scale genetic screens to understand the genetic basis of TBI pathologies.

A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila

Here we describe a method to inflict closed head traumatic brain injury (TBI) in Drosophila. This method provides a gateway to investigate the cellular and molecular mechanisms that underlie TBI pathologies using the vast array of experimental tools and techniques available for flies.

Eine Methode, um geschlossene Kopftraumatische Hirnverletzung in Verursacht<em&gt; Drosophila</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

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, ...

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

One of the most feasible methods of measuring presynaptic calcium levels in presynaptic nerve terminals is optical recording. It is based on using calcium-sensitive fluorescent dyes that change their emission intensity or wavelength depending on the concentration of free calcium in the cell. There are several methods used to stain cells with calcium dyes. Most common are the processes of loading the dyes through a micropipette or pre-incubating with the acetoxymethyl ester forms of the dyes. However, these methods are not quite applicable to neuromuscular junctions (NMJs) due to methodological issues that arise. In this article, we present a method for loading a calcium-sensitive dye through the frog nerve stump of the frog nerve into the nerve endings. Since entry of external calcium into nerve terminals and the subsequent binding to the calcium dye occur within the millisecond time-scale, it is necessary to use a fast imaging system to record these interactions. Here, we describe a protocol for recording the calcium transient with a fast CCD camera.

Here, we describe a method for loading a calcium-sensitive dye through the frog nerve stump into the nerve endings. We also present a protocol for the recording and analysis of fast calcium transients in the peripheral nerve endings.

Laden eines Kalziumfarbstoffs in Froschnervenendungen durch den Nervenstumpf: Calciumübergangsregistrierung im Frosch Neuromuscular Junction

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

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each other anatomically, it is important to identify an appropriate model to use for the study of axon regeneration. By using a suitable model, we can formulate a specific treatment based on the severity of injury, the neuronal cell type of interest, and the desired spinal tract for assessing regeneration. Within the sensory pathway, DRG neurons are responsible for relaying sensory information from the periphery to the CNS. We present here a protocol that uses a DRG injection with a viral vector and a concurrent dorsal root crush injury in the lower cervical spinal cord of an adult rat as a model to study sensory axon regeneration. As demonstrated using a control virus, AAV5-GFP, we show the effectiveness of a direct DRG injection in transducing DRG neurons and tracing sensory axons into the spinal cord. We also show the effectiveness of the dorsal root crush injury in denervating the forepaw as an injury model for evaluating axon regeneration. Despite the requirement for specialized training to perform this invasive surgical procedure, the protocol is flexible, and potential users can modify many parts to accommodate their experimental requirements. Importantly, it can serve as a foundation for those in search of a suitable animal model for their studies. We believe that this article will help new users to learn the procedure in a very efficient and effective manner.

This protocol presents the use of a dorsal root ganglion (DRG) injection with a viral vector and a concurrent dorsal root crush injury in an adult rat as a model to study sensory axon regeneration. This model is suitable for investigating the use of gene therapy to promote sensory axon regeneration.

Dorsalwurzelganglion Injection und dorsale Wurzel Quetschverletzungen als Modell für Sensory Axonregeneration

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

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in adult mammalian animals remains elusive. In this study, we describe an experimental procedure for measuring calcium dynamics through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy. This method enables recordings and quantifications of neural activity in bilateral mouse brain regions one at a time in the same experiment for a prolonged period in vivo. Key aspects of this method, which can be completed within an hour, include minimally invasive surgery procedures for creating dual optical windows, and the use of 2P imaging. Although we only demonstrate the technique in the S1 area, the method can be applied to other regions of the living brain facilitating the elucidation of structural and functional complexities of brain neural networks.

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

We describe an experimental procedure for measuring neuronal activity through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy in vivo.

Neuronalen Aktivität im primären somatosensorischen Cortex mit Thy1Imaging-GCaMP6s transgene Mäuse

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

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

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