Name: subcutaneous neurotrophin 4 infusion using osmotic pumps or direct muscular injection enhances aging rat laryngeal muscles
Uploaded: 2017-06-13T15:00:00.000+00:00
Duration: 5 min 50 s
Description: Watch this Scientific Journal Video about Subcutaneous Neurotrophin 4 Infusion Using Osmotic Pumps or Direct Muscular Injection Enhances Aging Rat Laryngeal Muscles at JoVE.com

Diese Arbeit wurde unterstützt durch Stipendien des Nationalen Instituts für Taubheit und andere Kommunikationsstörungen (R21DC010806 bis CAM und JCS und R01DC011285 bis CAM).

Verwenden Sie männliche Fischer 344-Brown Norwegen Ratten in 6 und 30 Monate alt für dieses Protokoll. Ratten wurden von der National Institute of Aging Nagetier Kolonie erhalten. Wir haben Ratten für diese Studie verwendet, weil die Struktur des Rattenkehlkopfes ähnlich der des Menschen ist, funktionell für den Atemwegsschutz und Spezies-spezifische Vokalisationen dient Diese Studie wurde in Übereinstimmung mit der PHS-Politik auf humane Pflege und Verwendung von Labor-Tiere durchgeführt, Der NIH-Leitfaden für die Pflege und Verwendung von Labortieren und das Tierschutzgesetz (7 USC ff.); Wurde das Tiernutzungsprotokoll vom Institutional Animal Care and Use Committee (IACUC) der University of Kentucky genehmigt. 1. Anästhesie der Ratten <ol><li> Bereiten Sie die Anästhetika vor, indem Sie Ketaminhydrochlorid (dissoziatives Anästhetikum) und Xylazinhydrochlorid (Beruhigungsmittel und Analgetikum) in gepufferter Kochsalzlösung mischen. Die Konzentrationen von Ketamin und Xylazin in der Endlösung betragen 100 mg / 8 mg pro kgKörpergewicht. </li><li> Injektion der Anästhetika in die Ratte durch intraperitoneale Verabreichung unter Verwendung einer Spritze mit einer 25 G-Nadel. </li><li> Bestimmen Sie, dass die Ratte genügend betäubt ist, indem Sie den Zeh oder den Fuß mit einer Pinzette einklemmen. Wenn die Ratte nicht auf die Prise reagiert, dann kann die Operation beginnen. Wenn die Ratte auf die Zehen-Prise mit Reflex- oder Muskelkontraktionen reagiert, dann warten Sie 1-2 min und wiederholen Sie den Prise Test. Wenn die Ratte wieder reagiert, ersetzen Sie die Ratte durch ein neues Tier und wiederholen Sie das Verfahren ab Schritt 1.2. </li><li> Tragen Sie ophthalmische Salbe auf die Augen der Ratte, nachdem die Ratte unbeweglich ist, um zu verhindern, dass die Hornhäute austrocknen. </li></ol> 2. Osmotische Pumpenimplantation <ol><li> Lege die Ratte ventral auf den aseptischen chirurgischen Bereich. Meloxicam als präanästhetische Medikamente verabreichen. Intraperitoneal in einer Dosierung von 1-4 mg / kg Körpergewicht unter Verwendung einer Spritze mit einer 25 G-Nadel verabreichen. </li><li> Verwenden Sie Clippers, um eine approxima zu entfernen1 &quot;x 1&quot; Quadrat des Pelzes von der Rückseite des Halses, und ungefähr 1 &quot;kaudal des Raumes zwischen den Schultern. Rasieren Sie so nah an der Haut wie möglich. </li><li> Den Rücken und den Nacken mit deminfizierendem Ethanol (70%) benetzen. </li><li> Nach der Rasur, schrubben die dorsalen Aspekt des Halses mit 3 Scrubs in Folge von Jod-Alkohol Finishing mit Alkohol. </li><li> Halten Sie die Körpertemperatur der Ratte, indem Sie sie auf einen Heizkissen legen, der auf 34 ° C eingestellt ist. </li><li> Füllen Sie aseptisch hergestellte osmotische Pumpen mit entweder 50 μl NTF4 oder Kochsalzlösung für die systemische NTF4-Behandlung (Abbildung 1 ). <ol><li> Benutzen Sie ein Skalpell, um einen horizontalen Schnitt ungefähr 2 cm breit durch die Haut zu bilden, gerade kranial zum Raum zwischen den scapulae. Heben Sie die hintere Kante des Schnittes mit einer Pinzette mit einer Hand an, während Sie die Spitze der Hämostaten einsetzen und vorsichtig nach hinten in den Einschnitt schieben. </li><li> Nach der Spitze der Hämostaten ist ca. 2 cm cRanial zum Einschnitt, öffnen Sie die Griffe auf den Hämostaten, erweitern die Spitzen, um eine hohle &quot;Tasche&quot; subkutan zu der Inzisionsstelle zu bilden. Dies ist die Platzierungsstelle für die Pumpe. </li></ol></li><li> Richten Sie das Pumpenförderportal zuerst nach der Einfügung aus, um jegliche Wechselwirkung des NTF4 und die Heilung der Pocket Inzisionsstelle zu minimieren. </li><li> 50 μl NTF4 Kochsalzlösung für 7 14 Tage geben. Die 7-tägige Gruppe erhielt 6,72 mg / Tag NTF4 für eine Gesamtdosis von 47,04 mg. Die 14-tägige Gruppe erhielt 6,72 mg / Tag für eine Gesamtdosis von 94,08 mg NTF4 25 . </li><li> Verwenden Sie 5-0 Nylon Naht Faden, Hämostaten und Pinzette, um den Einschnitt für Pumpe Platzierung zu schließen. </li><li> Beobachten Sie die Ratten für mindestens 30 min, wie sie sich von der Anästhesie erholen. Kriterien für die Vollendung der Überwachung gehören das Tier aktiv, bewegt sich über den Käfig, Trinkwasser, und beginnen andere normale Aktivitäten wie Pflege. </li><li> Tiere täglich überwachenFür die erste Woche durch Beobachtung der Heilung der chirurgischen Stelle, normale Futtermittel und Wasserverbrauch und Verabschiedung von Urin / Kot und irgendwelche abnorme Verhaltenszeichen von Stress, Schmerzen oder anderen postoperativen Komplikationen. </li><li> Wenn die Ratte in Schmerzen oder Not zu sein scheint, versorgen Sie die Ratte mit einer 5 mg / kg subkutanen Injektion von Carprofen einmal alle 24 Stunden für bis zu 5 Tage, um Schmerzen zu lindern. </li><li> Wenn es scheint, eine Infektion zu sein, konsultieren Sie einen Tierarzt, um zu folgen, dass die Wunde richtig heilt. </li><li> Je nachdem, welche experimentelle Gruppe die Ratte in innen, entfernen Sie die 5-0 Nylon Naht 7-10 Tage nach der Operation, um Reizungen aus dem Faden zu verhindern. </li></ol> 3. Anästhesie der Ratten für die direkte Injektion <ol><li> Verzehr von den Ratten in der Nacht vor dem Eingriff. Dadurch wird sichergestellt, dass es keine Nahrung gibt, um das Endoskop oder die Injektionsnadel zu blockieren. </li><li> Wiegen Sie Ratten und bereiten Sie Acepromazin 1-2 mg / kg Körpergewicht vor. Inaktiv intramuskulär (der IM - Standort ist derLinks thyroarytenoid muskel). </li><li> Legen Sie die Ratte in die Induktionsbox. Anästhesie im Induktionskasten mit 5% Isofluran und 1 LO 2 induzieren. </li><li> Bewegen Sie die Ratte auf einen Nasenkonus mit 2% Isofluran und 600 ml O 2 . </li><li> Bestimmen Sie, dass die Ratte genügend betäubt ist, indem Sie den Zeh oder den Fuß mit einer Pinzette einklemmen. Wenn die Ratte nicht auf die Prise reagiert, kann das Injektions-Protokoll beginnen. Wenn die Ratte auf die Zehen-Prise mit Reflex- oder Muskelkontraktionen reagiert, dann warten Sie 1-2 min und wiederholen Sie den Prise Test. Wenn die Ratte wieder reagiert, ersetzen Sie die Ratte durch ein neues Tier und wiederholen Sie das Verfahren ab Schritt 3.4. </li></ol> 4. Direkteinspritzung und Visualisierung <ol><li> Platzieren Sie aseptisch hergestellte 50-μL-Dosierungen, die NTF4 oder Kochsalzlösung enthalten, in einem H 2 O-Bad, das auf 25 ° C für 30 min vor der Injektion eingestellt wurde. </li><li> Legen Sie die Ratte in eine Rückenlage und verstellbare Position auf einer Plexiglas-Plattform (Abbildung 2 ). SusHängen Sie die Ratte in die zurückgelehnte Haltung von ihren vorderen oberen Schneidezähnen über einen Führungsdraht, der über die Oberseite der Plattform geschleudert wird. </li><li> Befestigen Sie eine 50 mm, 30 gauge, 100 μl Spritze auf ein 1,9 mm, 30 ° Sinus Endoskop (Abbildung 3 ). HINWEIS: Die Spritzenanordnung wird über eine Lehre befestigt, die die Kanüle fest an der Außenwand des Endoskops hält. Das Endoskop ermöglicht die Visualisierung der Stimmlippen und die Injektion der Spritze. Die Position der Kanülenspitze wird vor jedem Tier eingestellt, um sicherzustellen, dass die Spitze über die endoskopische Ansicht vollständig sichtbar ist (Abbildung 4 ). </li><li> Verwenden Sie eine gummi-pippige Pinzette, um die Zunge zu verlängern und verschieben Sie sie seitlich. Danach legen Sie ein Plastikspekulum ein, um die mündliche Durchgängigkeit zu bewahren. Machen Sie das Spekulum aus einem 5 ml Plastikspritzenfaß, der auf eine Länge von 1,5 bis 2 cm geschnitten ist, wobei die Schnittkanten entgratet und poliert sind. </li><li> Schalte die Lichter ausM und befestige eine Halogen-Lichtquelle zum Endoskop. Schalten Sie den Videorecorder ein, um die Prozedur zu erfassen. </li><li> Tauchen Sie das distale Ende des Endoskops für einige Sekunden in warmes Wasser ein, um die Entwicklung der Kondensation auf der Glasspitze zu minimieren, wenn sie in den Mund der Ratte eingeführt wird. </li><li> Mit der visuellen Rückmeldung vom Monitor führen Sie die Nadel sorgfältig in den Bereich der linken Stimmlippe. </li><li> Zeit die Injektion der Lösung mit der Inspirationsphase des Atmungszyklus der Tiere, um vollständig auf die Stimmfalte zuzugreifen. Während der inspiratorischen Phase der Atmung ist die Stimmfalte vollständig ausgesetzt. <ol><li> Sobald die Stimmfalte vollständig sichtbar ist, stecke die Nadel in das linke Thyreoheldoid ein, das seitlich zur weißen medialen Kante der Stimmlippe gefunden wird. Mit der Nadel an Ort und Stelle, liefern die Injektion durch Depression der Spritze. </li></ol></li><li> Schalten Sie die Halogen-Lichtquelle auf dem Endoskop und dem Videoplayer aus und drehen Sie den Rack wieder anOm leuchtet </li><li> Bringt die Ratte in den Hauskäfig zurück und platziere auf einem Heizkissen. </li><li> Lassen Sie die Ratte vor dem Entfernen aus dem Heizkissen zurückgewinnen. Ersetzen Sie Nahrung und Wasser im Käfig. </li><li> Überwachen Sie Ratten für 7 Tage nach der Injektion und dann euthanisieren. Entfernen Sie die Larynges für die Kryoschaltung 24 . </li></ol> 5. Euthanisierung von Ratten <ol><li> Anästhesieren von Ratten mit Ketaminhydrochlorid und Xylazinhydrochlorid (100 mg / 8 mg pro kg Körpergewicht injiziert intraperitoneale Injektion). </li><li> Euthanisieren durch Exsanguination nach einer medialen Thorakotomie. </li></ol>

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Die Autoren haben nichts zu offenbaren.

Larynxmuskulatur ist anfällig für die ungünstigen Auswirkungen des Alterns. Frühere Studien haben Veränderungen in der Alterung der Kehlkopfmuskulatur gezeigt, die Veränderungen in der Fasergröße, die Gesamtzahl der Fasern, die Regenerationsfähigkeit, die NMJ-Größe und die Mengenänderungen zusätzlich zu den Variationen der kontraktilen Funktion und der Myosin-Isoformverschiebungen 4 , 11 , 27 , <sup clas...

Larynx Muskeln kontrahieren schnell und konsequent und sind anfällig für die nachteiligen Auswirkungen des Alterns. Diese ständige Aktivität wird dazu gedacht, zu Stimmproblemen oder Dysphagie bei Personen über 65 Jahre im Alter von 1 , 2 , 3 , 4 , 5 , 6 , 7 zu helfen. Mehrere molekulare und pathophysiologische Mechanismen tragen zu dieser altersbedingten Dysfunktion bei. Diese Mechanismen können die Umgestaltung von Kehlkopfschleimhaut, Muskelfaseratrophie oder -verlust, Mangel an Muskelfaser-Regeneration oder Atrophie, die eine Verbeugung der Stimmlippen und Unfähigkeit des Glottis-Verschlusses 8 , 9 , 10 , 11 bewirken , umfassen . Es gibt keine bewährte medizinische Therapie zu diesem Zeitpunkt, die comDiese altersbedingten Veränderungen in diesen Muskeln zu verhindern oder zu rehabilitieren. Die Modulation der Wirksamkeit der neuromuskulären Transmission kann die Neuromotorleistung stark beeinflussen. Die Familie der Neurotrophine umfasst den Nervenwachstumsfaktor (NGF), den Gehirn-abgeleiteten Nervenwachstumsfaktor (BDNF), Neurotrophin 3 (NTF3) und NTF4 12 , 13 . Es wurde gezeigt, dass Neurotrophine die synaptische Wirksamkeit 1 , 4 modulieren. Hepatocytenwachstumsfaktor, transformierende Wachstumsfaktor Beta und Fibroblastenwachstumsfaktor wurden vor kurzem beim Menschen für die Behandlung von Stimmfalten Narben 15 - 17 verwendet. NTF4 reguliert auch die NMJ-Wirksamkeit; Mäuse ohne NTF4 zeigen zerlegt NMJs 11 , 18 , 19 . Diese Studien führen zu vielversprechenden Wirkungen von BehandlungenT der alternden Larynxmuskelstörungen und der Denervation mit Wachstumsfaktoren. Direkte Injektionstherapeutika zu den Geweben der Stimmlippen sind beim Menschen nicht beispiellos. Zum Beispiel werden lokale Injektionen von Botulinumtoxin derzeit als wirksame Behandlung für neurologische Bewegungsstörungen verwendet, die die Muskeln im Kehlkopf beeinflussen, wie krampfhafte Dysphonie und bilaterale rezidivierende Larynxnervenlähmung 20 , 21 . Hyaluronsäure-Hydrogel ist eine weitere injizierbare, die verwendet wird, um Stimmfalten Angst und Glottal Insuffizienz 22 , 23 zu behandeln. Injektion Laryngoplastie kann verwendet werden, um eine Vielzahl von Kommunikationsstörungen zu behandeln 24 . Diese Direktinjektionsmethoden versprechen, die Stimmfunktion und das Schlucken in alternden Populationen zu verbessern.

<table><tbody><tr><td>Neurotrophin 4</td><td>Pepro Tech</td><td>450-04</td><td>200 ng in 50 &amp;mu;L</td></tr><tr><td>Alzet Osmotic Pump</td><td>DURECT Corporation</td><td>2001D</td><td></td></tr><tr><td>30 &amp;deg; endoscope</td><td>Stoltz</td><td>61029D</td><td></td></tr><tr><td>50 mm 30 gauge 100-&amp;mu;L syringe</td><td>Hamilton</td><td>84850 and 201812</td><td></td></tr><tr><td>saline (sodium chloride solution)</td><td>Sigma-Aldrich</td><td>S8776</td><td></td></tr><tr><td>ketamine hydrochloride</td><td>Henry Schein</td><td>56344</td><td></td></tr><tr><td>xylazine hydrochloride</td><td>Henry Schein</td><td>33198</td><td></td></tr><tr><td>25 G 5/8 needle</td><td>Becton-Dickinson</td><td>305901</td><td></td></tr><tr><td>1 mL syringe</td><td>Becton-Dickinson</td><td>309659</td><td></td></tr><tr><td>ophthalmic ointment</td><td>Henry Schein</td><td>8897</td><td></td></tr><tr><td>clippers</td><td>Oster</td><td>44-018</td><td></td></tr><tr><td>ethanol</td><td>Decon</td><td>2716</td><td></td></tr><tr><td>iodine (Betadine)</td><td>Purdue Pharma L.P.</td><td>606404</td><td></td></tr><tr><td>heating pad</td><td>Sunbeam</td><td>731-5</td><td></td></tr><tr><td>5-0 nylon suture thread</td><td>AD Surgical</td><td>PMN-518R6</td><td></td></tr><tr><td>crile hemostat</td><td>Fine Science Tools</td><td>13005-14</td><td></td></tr><tr><td>delicate suture tying forceps</td><td>Fine Science Tools</td><td>11063-07</td><td></td></tr><tr><td>meloxicam</td><td>Henry Schein</td><td>49756</td><td></td></tr><tr><td>carprofen</td><td>Merritt Veterinary Supplies</td><td>148700</td><td></td></tr><tr><td>antibiotic ointment</td><td>Henry Schein</td><td>57110</td><td></td></tr><tr><td>acepromizine Aceproject</td><td>Henry Schein</td><td>3845</td><td></td></tr><tr><td>isoflurane Isothesia</td><td>Henry Schein</td><td>50033</td><td></td></tr><tr><td>induction box (anesthetizing box)</td><td>Harvard Apparatus</td><td>50-0116</td><td></td></tr><tr><td>oxygen compressed tank</td><td>Scott Gross</td><td>UN1072</td><td></td></tr><tr><td>plexiglas platform</td><td>Small Parts Inc (Amazon)</td><td></td><td></td></tr><tr><td>rubber tipped forceps</td><td>Fine science tools rubber</td><td>11075-00</td><td></td></tr><tr><td>liquid rubber for forceps above</td><td>Lowe&#039;s</td><td>42518</td><td></td></tr><tr><td>plastic spectula (BD syringe cut to length)</td><td>Becton-Dickinson</td><td>309659</td><td></td></tr><tr><td>halogen light source rhino-laryngeal stroboscope</td><td>Kay-Pentax</td><td>RLS 9100 B</td><td></td></tr><tr><td>video recorder</td><td>Kay-Pentax</td><td></td><td></td></tr><tr><td>sucrose</td><td>Sigma-Aldrich</td><td>S0389-500G</td><td></td></tr><tr><td>phosphate buffered saline</td><td>Sigma-Aldrich</td><td>P4417-100TAB</td><td></td></tr><tr><td>cryostat Mictotom HM525</td><td>Thermo Scientific</td><td>HM 525</td><td></td></tr><tr><td>Gill 1 hematoxylin</td><td>VWR</td><td>10143-142</td><td></td></tr><tr><td>Shandon eosin-Y alcoholic</td><td>Thermo Fisher Scientific</td><td>6766007</td><td></td></tr><tr><td>anti-sodium channel Nav1.5 antibody produced in rabbit</td><td>Sigma-Aldrich</td><td>S0819</td><td></td></tr><tr><td>Texas red-X phalloidin</td><td>Sigma-Aldrich</td><td>T7471</td><td></td></tr><tr><td>alpha- bungarotoxin alexa fluor 488 conjugate</td><td>Thermo Fisher Scientific</td><td>B-13422</td><td></td></tr><tr><td>Small animal anaesthesia machine</td><td>Smiths Medical</td><td>CDS 9000</td><td></td></tr></tbody></table>

subcutaneous neurotrophin 4 infusion using osmotic pumps or direct muscular injection enhances aging rat laryngeal muscles

Kehlkopfdysfunktion bei älteren Menschen ist eine Hauptursache für Behinderung, von Stimmungsstörungen bis hin zu Dysphagie und Verlust von Atemwegsschutzreflexen. Nur wenige, wenn überhaupt, gibt es Therapien, die eine altersbedingte Larynxmuskel-Dysfunktion erreichen. Neurotrophine sind an der Muskelinnervation und Differenzierung von neuromuskulären Übergängen (NMJs) beteiligt. Es wird angenommen, dass Neurotrophine die neuromuskuläre Transmission durch Erhöhung der Neurotransmitterfreisetzung verstärken. Die neuromuskulären Kreuzungen (NMJs) werden kleiner und weniger reich an alternden Ratten-Larynxmuskeln, mit Anzeichen einer funktionalen Denervation. Wir untersuchten die Auswirkungen von NTF4 auf zukünftige klinische Anwendung als Therapeutikum zur Verbesserung der Funktion bei der Alterung menschlicher Larynxmuskulatur. Hier stellen wir das detaillierte Protokoll für die systemische Anwendung und die direkte Injektion von NTF4 zur Verfügung, um die Fähigkeit des alternden Ratten-Larynxmuskels zu untersuchen, um in Reaktion auf NTF4-Applikation umzuwandeln. Bei dieser Methode erhielten Ratten entweder NTF4 entweder systemisch über oSmotische Pumpe oder durch direkte Injektion durch die Stimmlippen. Larynxmuskeln wurden dann seziert und für die histologische Untersuchung der Morphologie und der altersbedingten Denervation verwendet.

Die Ratten wurden nach 2 Wochen osmotischer Pumpeninfusion oder 1 Woche nach Direkteinspritzung von NTF4 euthanasiert. Larynge wurden geerntet, in Kryoschutzmittel (30% Saccharose und 70% phosphatgepufferte Kochsalzlösung) gegeben und dann seriell in 10 &amp; mgr; m Breite mit einem Kryostat geschnitten. Alterung der Larynxmuskulatur wird durch die Verabreichung von NTF4 25 beeinflusst. Neben der jungen und alten Ratte verglichen wir die injizierte und nicht inji...

Watch this Scientific Journal Video about Subcutaneous Neurotrophin 4 Infusion Using Osmotic Pumps or Direct Muscular Injection Enhances Aging Rat Laryngeal Muscles at JoVE.com

Subcutaneous Neurotrophin 4 Infusion Using Osmotic Pumps or Direct Muscular Injection Enhances Aging Rat Laryngeal Muscles

Hier stellen wir ein Protokoll vor, um die Verwendung von Neurotrophin 4 (NTF4) systemisch und direkt zu modulieren Rattenalterung Larynx Muskeln zu beschreiben.

Subkutane Neurotrophin-4-Infusion unter Verwendung von Osmotik-Pumpen oder direkter Muskel-Injektion verbessert die Ratten-Larynx-Muskeln

Laryngeal dysfunction in the elderly is a major cause of disability, from voice disorders to dysphagia and loss of airway protective reflexes. Few, if ...

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Research

JoVE Journal

Developmental Biology

8.4K Aufrufe.  University of Kentucky.  Hier stellen wir ein Protokoll vor, um die Verwendung von Neurotrophin 4 (NTF4) systemisch und direkt zu modulieren Rattenalterung Larynx Muskeln zu beschreiben. 

Serie: Subcutaneous Neurotrophin 4 Infusion Using Osmotic Pumps or Direct Muscular Injection Enhances Aging Rat Laryngeal Muscles

Intraspinal Cell Transplantation for Targeting Cervical Ventral Horn in Amyotrophic Lateral Sclerosis and Traumatic Spinal Cord Injury

Respiratory compromise due to phrenic motor neuron loss is a debilitating consequence of a large proportion of human traumatic spinal cord injury (SCI) cases 1 and is the ultimate cause of death in patients with the motor neuron disorder, amyotrophic laterals sclerosis (ALS) 2.
ALS is a devastating neurological disorder that is characterized by relatively rapid degeneration of upper and lower motor neurons. Patients ultimately succumb to the disease on average 2-5 years following diagnosis because of respiratory paralysis due to loss of phrenic motor neuron innnervation of the diaphragm 3. The vast majority of cases are sporadic, while 10% are of the familial form. Approximately twenty percent of familial cases are linked to various point mutations in the Cu/Zn superoxide dismutase 1 (SOD1) gene on chromosome 21 4. Transgenic mice 4,5 and rats 6 carrying mutant human SOD1 genes (G93A, G37R, G86R, G85R) have been generated, and, despite the existence of other animal models of motor neuron loss, are currently the most highly used models of the disease.
Spinal cord injury (SCI) is a heterogeneous set of conditions resulting from physical trauma to the spinal cord, with functional outcome varying according to the type, location and severity of the injury 7. Nevertheless, approximately half of human SCI cases affect cervical regions, resulting in debilitating respiratory dysfunction due to phrenic motor neuron loss and injury to descending bulbospinal respiratory axons 1. A number of animal models of SCI have been developed, with the most commonly used and clinically-relevant being the contusion 8.
Transplantation of various classes of neural precursor cells (NPCs) is a promising therapeutic strategy for treatment of traumatic CNS injuries and neurodegeneration, including ALS and SCI, because of the ability to replace lost or dysfunctional CNS cell types, provide neuroprotection, and deliver gene factors of interest 9.
Animal models of both ALS and SCI can model many clinically-relevant aspects of these diseases, including phrenic motor neuron loss and consequent respiratory compromise 10,11. In order to evaluate the efficacy of NPC-based strategies on respiratory function in these animal models of ALS and SCI, cellular interventions must be specifically directed to regions containing therapeutically relevant targets such as phrenic motor neurons. We provide a detailed protocol for multi-segmental, intraspinal transplantation of NPCs into the cervical spinal cord ventral gray matter of neurodegenerative models such as SOD1G93A mice and rats, as well as spinal cord injured rats and mice 11.

Neural precursor transplantation is a promising strategy for protecting and/or replacing lost/dysfunctional cervical phrenic motor neurons in spinal cord injury (SCI) and the motor neuron disorder, amyotrophic laterals sclerosis (ALS). We provide a protocol for cell delivery to cervical spinal cord ventral horn in rodent models of ALS and SCI.

Intraspinale Cell Transplantation für Targeting Cervical Vorderhorn in Amyotrophe Lateralsklerose und traumatische Rückenmarksverletzungen

Respiratory compromise due to phrenic motor neuron loss is a debilitating consequence of a large proportion of human traumatic spinal cord injury (SCI) ...

Forschung

Medizin

A Murine Model of Muscle Training by Neuromuscular Electrical Stimulation

Neuromuscular electrical stimulation (NMES) is a common clinical modality that is widely used to restore1, maintain2 or enhance3-5 muscle functional capacity. Transcutaneous surface stimulation of skeletal muscle involves a current flow between a cathode and an anode, thereby inducing excitement of the motor unit and the surrounding muscle fibers. 
NMES is an attractive modality to evaluate skeletal muscle adaptive responses for several reasons. First, it provides a reproducible experimental model in which physiological adaptations, such as myofiber hypertophy and muscle strengthening6, angiogenesis7-9, growth factor secretion9-11, and muscle precursor cell activation12 are well documented. Such physiological responses may be carefully titrated using different parameters of stimulation (for Cochrane review, see 13). In addition, NMES recruits motor units non-selectively, and in a spatially fixed and temporally synchronous manner14, offering the advantage of exerting a treatment effect on all fibers, regardless of fiber type. Although there are specified contraindications to NMES in clinical populations, including peripheral venous disorders or malignancy, for example, NMES is safe and feasible, even for those who are ill and/or bedridden and for populations in which rigorous exercise may be challenging. 
Here, we demonstrate the protocol for adapting commercially available electrodes and performing a NMES protocol using a murine model. This animal model has the advantage of utilizing a clinically available device and providing instant feedback regarding positioning of the electrode to elicit the desired muscle contractile effect. For the purpose of this manuscript, we will describe the protocol for muscle stimulation of the anterior compartment muscles of a mouse hindlimb.

A murine model of neuromuscular electrical stimulation (NMES), a safe and inexpensive clinical modality, to the anterior compartment muscles is described. This model has the advantage of modifying a readily available clinical device for the purpose of eliciting targeted and specific muscle contractions in mice.

Einem Mausmodell der Muscle Training durch neuromuskuläre Elektrostimulation

Neuromuscular electrical stimulation (NMES) is a common clinical modality that is widely used to restore1, maintain2 or enhance3-5 muscle functional ...

Subcutaneous Administration of Muscarinic Antagonists and Triple-Immunostaining of the Levator Auris Longus Muscle in Mice

Hind limb muscles of rodents, such as gastrocnemius and tibialis anterior, are frequently used for in vivo pharmacological studies of the signals essential for the formation and maintenance of mammalian NMJs. However, drug penetration into these muscles after subcutaneous or intramuscular administration is often incomplete or uneven and many NMJs can remain unaffected. Although systemic administration with devices such as mini-pumps can improve the spatiotemporal effects, the invasive nature of this approach can cause confounding inflammatory responses and/or direct muscle damage. Moreover, complete analysis of the NMJs in a hind limb muscle is challenging because it requires time-consuming serial sectioning and extensive immunostaining. 
The mouse LAL is a thin, flat sheet of muscle located superficially on the dorsum of the neck. It is a fast-twitch muscle that functions to move the pinna. It contains rostral and caudal portions that originate from the midline of the cranium and extend laterally to the cartilaginous portion of each pinna. The muscle is supplied by a branch of the facial nerve that projects caudally as it exits the stylomastoid foramen. We and others have found LAL to be a convenient preparation that offers advantages for the investigation of both short and long-term in vivo effects of drugs on NMJs and muscles. First, its superficial location facilitates multiple local applications of drugs under light anesthesia. Second, its thinness (2-3 layers of muscle fibers) permits visualization and analysis of almost all the NMJs within the muscle. Third, the ease of dissecting it with its nerve intact together with the pattern of its innervation permits supplementary electrophysiological analysis in vitro9,5. Last, and perhaps most importantly, a small applied volume (&tilde;50&mu;l) easily covers the entire muscle surface, provides a uniform and prolonged exposure of all its NMJs to the drug and eliminates the need for a systemic approach1,8.

We describe procedures for repeated administration of inhibitors of muscarinic signaling to the levator auris longus (LAL) muscle of young adult mice and for subsequent immunostaining of its neuromuscular junctions (NMJs) in wholemounts. The LAL muscle has unique advantages for revealing in vivo pharmacological effects on NMJs.

Subkutane Verabreichung von Muscarinic Antagonisten und Triple-Immunfärbung der Levator Auris longus in Mäuse

Hind limb muscles of rodents, such as gastrocnemius and tibialis anterior, are frequently used for in vivo  pharmacological studies of the signals ...

Neurowissenschaften

Tibial Nerve Transection - A Standardized Model for Denervation-induced Skeletal Muscle Atrophy in Mice

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of denervation-induced skeletal muscle atrophy in rodents. Although originally developed and used extensively in the rat due to its larger size, the tibial nerve in mice is big enough that it can be easily manipulated with either crush or transection, leaving the peroneal and sural nerve branches of the sciatic nerve intact and thereby preserving their target muscles. Thus, this model offers the advantages of inducing less morbidity and impediment of ambulation than the sciatic nerve transection model and also allows investigators to study the physiologic, cellular and molecular biologic mechanisms regulating the process of muscle atrophy in genetically engineered mice. The tibial nerve supplies the gastrocnemius, soleus and plantaris muscles, so its transection permits the study of denervated skeletal muscle composed of fast twitch type II fibers and/or slow twitch type I fibers. Here we demonstrate the tibial nerve transection model in the C57Black6 mouse. We assess the atrophy of the gastrocnemius muscle, as a representative muscle, at 1, 2, and 4 weeks post-denervation by measuring muscle weights and fiber type specific cross-sectional area on paraffin-embedded histologic sections immunostained for fast twitch myosin.

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of skeletal muscle atrophy. The model surgical protocol is described and demonstrated in C57Black6 mice. &#160;

Tibialisnerv Durchtrennung - ein standardisiertes Modell für Denervation-induzierte Skelettmuskelatrophie in Mice

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of denervation-induced skeletal muscle atrophy in rodents. ...

Transplantation of Olfactory Ensheathing Cells to Evaluate Functional Recovery after Peripheral Nerve Injury

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory system is characterized by its ability to give rise to new neurons even in adult animals. This particular ability is partly due to the presence of OECs which create a favorable microenvironment for neurogenesis. This property of OECs has been used for cellular transplantation such as in spinal cord injury models. Although the peripheral nervous system has a greater capacity to regenerate after nerve injury than the central nervous system, complete sections induce misrouting during axonal regrowth in particular after facial of laryngeal nerve transection. Specifically, full sectioning of the recurrent laryngeal nerve (RLN) induces aberrant axonal regrowth resulting in synkinesis of the vocal cords. In this specific model, we showed that OECs transplantation efficiently increases axonal regrowth.
OECs are constituted of several subpopulations present in both the olfactory mucosa (OM-OECs) and the olfactory bulbs (OB-OECs). We present here a model of cellular transplantation based on the use of these different subpopulations of OECs in a RLN injury model. Using this paradigm, primary cultures of OB-OECs and OM-OECs were transplanted in Matrigel after section and anastomosis of the RLN. Two months after surgery, we evaluated transplanted animals by complementary analyses based on videolaryngoscopy, electromyography (EMG), and histological studies. First, videolaryngoscopy allowed us to evaluate laryngeal functions, in particular muscular cocontractions phenomena. Then, EMG analyses demonstrated richness and synchronization of muscular activities. Finally, histological studies based on toluidine blue staining allowed the quantification of the number and profile of myelinated fibers.
All together, we describe here how to isolate, culture, identify and transplant OECs from OM and OB after RLN section-anastomosis and how to evaluate and analyze the efficiency of these transplanted cells on axonal regrowth and laryngeal functions.

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth of the primary olfactory neurons. This specific property can be used for cellular transplantation. We present here a model of cellular transplantation based on the use of OECs in a laryngeal nerve injury model.

Transplantation von olfaktorischen Zellen ensheathing zu Funktionswiederherstellung nach peripheren Nervenschäden Bewerten

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory ...

Intramuscular Injections Along the Motor End Plates: A Minimally Invasive Approach to Shuttle Tracers Directly into Motor Neurons

Diseases affecting the integrity of spinal cord motor neurons are amongst the most debilitating neurological conditions. Over the last decades, the development of several animal models of these neuromuscular disorders has provided the scientific community with different therapeutic scenarios aimed at delaying or reversing the progression of these conditions. By taking advantage of the retrograde machinery of neurons, one of these approaches has been to target skeletal muscles in order to shuttle therapeutic genes into corresponding spinal cord motor neurons. Although once promising, the success of such gene delivery approach has been hampered by the sub-optimal number of transduced motor neurons it has so far shown to yield. Motor end plates (MEPs) are highly specialized regions on the skeletal musculature that are in direct synaptic contact to the spinal cord &#945; motor neurons. In this regard, it is important to note that, so far, the efforts to retrogradely transfer genes into motor neurons were made without reference to the location of the MEP region in the targeted muscles. Here, we describe a simple protocol 1) to reveal the exact location of the MEPs on the surface of skeletal muscles and 2) to use this information to guide the intramuscular delivery and subsequent optimal retrograde transport of retrograde tracers into motor neurons. We hope to utilize the results from these tracing experiments in further studies into investigating retrograde transport of therapeutic genes to spinal cord motor neurons through the targeting of MEPs.

The efficacy of intramuscular uptake and retrograde transport of molecules to corresponding motor neurons depends on the location of the injection sites with respect to the motor end plates (MEPs). Here, we describe how to locate MEPs on skeletal muscles to optimise retrograde transport of tracers into motor neurons.

Intramuskuläre Injektionen Entlang der motorischen Endplatten: eine minimal-invasive Ansatz zur Shuttle Tracers Direkt in Motoneuronen

Diseases affecting the integrity of spinal cord motor neurons are amongst the most debilitating neurological conditions. Over the last decades, the ...

Nerve Stimulator-guided Injection of Autologous Stem Cells Near the Equine Left Recurrent Laryngeal Nerve

Recurrent laryngeal neuropathy (RLN) commonly affects horses and is characterized by abnormal respiratory sounds and exercise intolerance. The recurrent laryngeal nerve shows lesions of demyelination. The benefit of applying stem cells to demyelinated nerves has been demonstrated in various animal models. The aim of the study was to test the feasibility and safety of a peri-neuronal injection of autologous muscle-derived mesenchymal stem cells to the left recurrent laryngeal nerve in healthy horses by using an electrical nerve stimulator.
Muscle-derived stems cell are obtained from five healthy Standardbred horses by sampling 20 mg of muscle tissue with a semi-automatic 14 G biopsy needle from the triceps muscle. Movements of the larynx are monitored via upper-airway video endoscopy. The left recurrent laryngeal nerve is approached with an insulated nerve block needle. Nerve stimulation is applied, starting at 2 mA, and the successful abduction of the left arytenoid is monitored. The stimulation intensity is reduced progressively. When a loss of the motor response is observed at 0.5 mA, 107 autologous muscle-derived stem cells are injected. Two examiners, who are blinded to the time point, score the laryngeal function of the horses prior to the treatment and at day 1, day 7, and day 28 after the injection of the cells. In a sixth horse, 1 mL of 2% lidocaine is injected to further confirm the correct positioning of the needle. This leads to a temporary paralysis of the left arytenoid cartilage.
This study proves that the recurrent laryngeal nerve can be approached with the help of an electrical nerve stimulator and that the electrical stimulation of the nerve is well tolerated by the horses. No modification of the laryngeal function was observed in any of the horses after the injection of the stem cells. Further studies should be conducted to describe the effects of a peri-neuronal injection of autologous muscle-derived mesenchymal stem cells to horses suffering from RLN.

Here, we present a protocol to inject autologous muscle-derived stem cells in the proximity to the recurrent laryngeal nerve with the help of an electrical nerve stimulator. This novel technique may become useful for the treatment of equine recurrent laryngeal neuropathy.

Nerv Stimulator-gesteuerte Injektion von autologen Stammzellen in der Nähe der Pferde Links wiederkehrende Laryngeal Nerv

Recurrent laryngeal neuropathy (RLN) commonly affects horses and is characterized by abnormal respiratory sounds and exercise intolerance. The recurrent ...

An Implantable System For Chronic In Vivo Electromyography

Electromyography (EMG) measures the muscle response to electrical stimulation or spontaneous activity of motor units and plays an important role in assessing neuromuscular function. Chronic recording of EMG activity reflecting a muscle&#8217;s reinnervation status after nerve injury has been limited, due to the invasive nature of traditional EMG recording techniques. In this regard, an implantable system is designed for long-term, in vivo EMG recording and nerve stimulation. It has been applied and tested in a study on reinnervation of laryngeal muscles. This system consists of 1) two bipolar electrode nerve cuffs and leads for stimulating each of two nerves: the recurrent laryngeal nerve (RLN) and internal branch of the superior laryngeal nerve (SLN); 2) two EMG recording electrodes and leads for each of the two laryngeal muscles: posterior cricoarytenoid (PCA) muscle and thyroarytenoid-lateral cricoarytenoid (TA-LCA) muscle complex; and 3) a skin receptacle interfacing all implanted lead terminals to an external recording preamplifier and stimulator using a connection cable. The wire leads are Teflon-coated, multi-filament, type 316 stainless steel. They are coiled and can stretch during body movement of the awake animal to prevent lead breakage and electrode migration. This system is implanted during an aseptic surgery. Afterwards, baseline EMG recordings are performed before the RLN is transected in the second surgery to study muscle reinnervation. Throughout the study, multiple physiological sessions are conducted in the anesthetized animal to obtain evoked and spontaneous EMG activity that reflects the reinnervation status of laryngeal muscles. The system is compact, free of infection over the course of the study, and highly durable. This implantable system can provide a reliable platform for research in which long-term recording or nerve stimulation is required in an anesthetized or freely moving animal.&#160;

Presented here is a protocol for the manufacturing of an implantable system for in vivo chronological recording of evoked and spontaneous electromyographic potentials. The system is applied to the investigation of reinnervation of laryngeal muscles following nerve injury. &#160;&#160;

Ein implantierbares System für die chronische In-Vivo-Elektromyographie

Electromyography (EMG) measures the muscle response to electrical stimulation or spontaneous activity of motor units and plays an important role in ...

Preparation of the Rat Vocal Fold for Neuromuscular Analyses

The purpose of this tutorial is to describe the preparation of the rat vocal fold for histochemical neuromuscular study. This protocol outlines procedures for rat laryngeal dissection, flash-freezing, and cryosectioning of the vocal folds. This study describes how to cryosection vocal folds in both longitudinal and cross-sectional planes. A novelty of this protocol is the laryngeal tracking during cryosectioning that ensures accurate identification of the intrinsic laryngeal muscles and reduces the chance of tissue loss. Figures demonstrate the progressive cryosectioning in both planes. Twenty-nine rat hemi-larynges were cryosectioned and tracked from the emergence of the thyroid cartilage to the appearance of the first section that included the full vocal fold. The full vocal fold was visualized for all animals in both planes. There was high variability in the distance from the appearance of the thyroid cartilage to the appearance of the full vocal fold in both planes. Weight was not correlated to depth of laryngeal landmarks, suggesting individual variability and other factors related to tissue preparation may be responsible for the high variability in the appearance of landmarks during sectioning. This study details a methodology and presents morphological data for preparing the rat vocal fold for histochemical neuromuscular investigation. Due to high individual variability, laryngeal landmarks should be closely tracked during cryosectioning to prevent oversectioning tissue and tissue loss. The use of a consistent methodology, including adequate tissue preparation and awareness of landmarks within the rat larynx, will assist with consistent results across studies and aid new researchers interested in using the rat vocal fold as a model to investigate laryngeal neuromuscular mechanisms.

This protocol describes methods used to prepare rat vocal folds for histochemical neuromuscular study.

Vorbereitung der Stimmlippe der Ratte für neuromuskuläre Analysen

The purpose of this tutorial is to describe the preparation of the rat vocal fold for histochemical neuromuscular study. This protocol outlines procedures ...

<meta charset="utf8"></head><body>Elektroporation und Nervenverletzung im Levator Auris longus-Muskel der Maus

Combined In Vivo Electroporation and Short&#45;Term Reinnervation of the Cranial Levator Auris Longus Skeletal Muscle

The neuromuscular junction (NMJ) is the peripheral synapse controlling the contraction of skeletal muscle fibers to allow the coordinated movement of many organisms. At the NMJ, a presynaptic motor axon terminal contacts a muscle postsynaptic domain and is covered by terminal Schwann cells. The integrity and function of the NMJ is compromised under several conditions, including aging, neuromuscular diseases, and traumatic injuries. To analyze the potential contribution of muscle-derived proteins to NMJ maintenance and regeneration, an in vivo gene transfer strategy has been combined with the denervation of the cranial levator auris longus (LAL) muscle after mechanical nerve injury. Previous findings showed that the forced expression of control fluorescent proteins does not alter NMJ organization or neurotransmission. This procedure aims to describe a detailed method of in vivo electroporation of the LAL muscle followed by transection or crushing of the specific branch of the facial nerve innervating cranial muscles, leading to NMJ denervation and reinnervation, respectively. The combination of these experimental strategies in the convenient LAL muscle constitutes an efficient method to study the potential contribution of muscle protein overexpression or silencing in the context of short-term NMJ reinnervation.

<meta charset="utf8"></head><body>Autor im Rampenlicht: Regenerative Rollen von Muskelproteinen an der neuromuskulären Verbindung nach Nervenverletzung

Here, we present a protocol that combines in vivo electroporation and denervation of the cranial levator auris longus (LAL) muscle. This procedure enables the study of the potential role of muscle-derived proteins in the regeneration of the neuromuscular synapse.

Kombinierte In vivo Elektroporation und kurzfristige Reinnervation des kranialen Levator Auris Longus Skelettmuskel

The neuromuscular junction (NMJ) is the peripheral synapse controlling the contraction of skeletal muscle fibers to allow the coordinated movement of many ...