Questo lavoro è stato sostenuto da sovvenzioni dell&#39;Istituto Nazionale sulla Sordità e altri disturbi della comunicazione (R21DC010806 per CAM e JCS e R01DC011285 a CAM).

Usa i topi maschi Fischer 344-Brown Norvegia a 6 e 30 mesi di età per questo protocollo. I ratti sono stati ottenuti dall&#39;Istituto Nazionale di Colonia di Roditore di Aging. Abbiamo utilizzato i ratti per questo studio perché la struttura della laringa del ratto è simile a quella dell&#39;uomo, funzionalmente funzionale alla protezione delle vie aeree e alle vocalizzazioni specifiche per specie. Questo studio è stato eseguito in conformità con la politica PHS sulla cura umana e l&#39;uso di animali da laboratorio, La Guida NIH per la cura e l&#39;uso degli animali da laboratorio e la legge sulla protezione degli animali (7 USC et seq.); Il protocollo sull&#39;utilizzo degli animali è stato approvato dal Comitato Istituzionale per la cura e l&#39;uso degli animali (IACUC) dell&#39;Università del Kentucky. 1. Anestesia dei ratti <ol><li> Preparare l&#39;anestetico mescolando la cloridrato di ketamine (anestetico dissociativo) e cloridrato xilazine (sedativo e analgesico) in soluzione salina tamponata. Le concentrazioni di ketamina e xilazina nella soluzione finale sono 100 mg / 8 mg per kgPeso corporeo rispettivamente. </li><li> Iniettare le anestetiche nel ratto mediante somministrazione intraperitoneale usando una siringa con un ago da 25 G. </li><li> Determinare che il ratto è sufficientemente anestetizzato pinzando la punta o il piede con pinze. Se il topo non reagisce al pizzico, allora la chirurgia può iniziare. Se il topo reagisce al pizzico della punta con riflessi o contrazioni muscolari, attendere 1-2 minuti e ripetere il test di pizzicotto. Se il ratto reagisce di nuovo, sostituire il ratto con una nuova animale e ripetere la procedura a partire dal punto 1.2. </li><li> Applicare unguento oculare agli occhi del ratto, dopo che il ratto è immobile, per evitare che le cornee si asciugano. </li></ol> 2. Impianto osmotico della pompa <ol><li> Posizionare il ventrale del ratto sulla zona chirurgica asettica. Amministrare meloxicam come farmaco preanestetico. Amministrare intraperitonealmente ad un dosaggio di 1-4 mg / kg di peso corporeo usando una siringa con un ago da 25 G. </li><li> Utilizzare le taglierine per rimuovere un approssimaCon uno spessore di 1 &quot;x 1&quot; di pelliccia dalla parte posteriore del collo e circa 1 &quot;caudale di spalla tra le spalle. Rasare il più vicino possibile alla pelle. </li><li> Bagnare la schiena e il collo con l&#39;etanolo disinfettante (70%). </li><li> Dopo la rasatura, strofinare l&#39;aspetto dorsale del collo con 3 scrubs in successione di alcool iodio-alcool finitura con alcool. </li><li> Mantenere la temperatura corporea del ratto inserendola su un pannello di riscaldamento impostato a 34 ° C. </li><li> Riempire pompe osmotiche preparate asetticamente con 50 μL di NTF4 o salina per il trattamento sistemico di NTF4 ( Figura 1 ). <ol><li> Utilizzare un bisturi per realizzare un&#39;incisione orizzontale di circa 2 cm di larghezza attraverso la pelle, solo cranica nello spazio tra la scapola. Sollevare il bordo posteriore dell&#39;incisione con le pinze con una mano mentre inserisci la punta degli emostatici e spingete delicatamente alla parte posteriore all&#39;incisione. </li><li> Dopo che la punta degli hemostats è di circa 2 cm cRaniale all&#39;incisione, aprire le maniglie sugli emostatici, espandendo le punte per formare una &quot;tasca&quot; vuota sottocutanea al sito di incisione. Questo sarà il sito di posizionamento per la pompa. </li></ol></li><li> Orientare il portale di erogazione della pompa prima sull&#39;inserimento per ridurre al minimo l&#39;interazione del NTF4 e la guarigione del sito di incisione della tasca. </li><li> Consegnare 50 μL di soluzione salina NTF4 per 7-14 giorni. Il gruppo di 7 giorni ha ricevuto 6,72 mg / die di NTF4 per una dose totale di 47,04 mg. Il gruppo di 14 giorni ha ricevuto 6,72 mg / die per una dose totale di 94,08 mg di NTF4 25 . </li><li> Usare filo di sutura da 5-0 nylon, hemostati e pinze per chiudere l&#39;incisione fatta per il posizionamento della pompa. </li><li> Osservare i ratti per almeno 30 minuti mentre recuperano da anestesia. I criteri per il completamento del monitoraggio includono l&#39;animale che diventa attivo, muove in giro per la gabbia, l&#39;acqua potabile, e inizia altre attività normali come la cura. </li><li> Monitorare gli animali quotidianamentePer la prima settimana osservando la guarigione del sito chirurgico, il consumo normale dell&#39;alimentazione e dell&#39;acqua e il passaggio di urine / feci e qualsiasi segno abnormale comportamentale di stress, dolore o altre complicazioni postoperatorie. </li><li> Se il ratto sembra essere in dolore o disturbo, fornire al ratto una iniezione sottocutanea di carprofen da 5 mg / kg ogni 24 ore per un massimo di 5 giorni per alleviare il dolore. </li><li> Se sembra che ci sia un&#39;infezione, consultare un veterinario per far sì che la ferita si guari correttamente. </li><li> A seconda di quale gruppo sperimentale il ratto è dentro, rimuove la sutura in nylon da 5-0 7-10 giorni dopo la chirurgia per evitare irritazioni dal filo. </li></ol> 3. Anestesia di ratti per iniezione diretta <ol><li> Disattiva il cibo dai ratti la notte prima della procedura. Ciò assicura che non ci sia cibo per bloccare l&#39;endoscopio o l&#39;ago di iniezione. </li><li> Pesare i ratti e preparare acepromazina 1-2 mg / kg di peso corporeo. Iniettare in modo intramuscolare (la posizione IM è laSinistra il muscolo tiroarytenoide). </li><li> Posizionare il ratto nella scatola di induzione. Indurre l&#39;anestesia nella scatola di induzione con 5% isoflurano e 1 LO 2 . </li><li> Spostare il ratto in un cono a naso con 2% isoflurano e 600 ml O 2 . </li><li> Determinare che il ratto è sufficientemente anestetizzato pinzando la punta o il piede con pinze. Se il topo non reagisce al pizzico, il protocollo di iniezione può iniziare. Se il topo reagisce al pizzico della punta con riflessi o contrazioni muscolari, attendere 1-2 minuti e ripetere il test di pizzicotto. Se il ratto reagisce di nuovo, sostituire il ratto con una nuova animale e ripetere la procedura a partire dal punto 3.4. </li></ol> 4. Iniezione diretta e visualizzazione <ol><li> Posizionare i dosaggi di 50 μl preparati asetticamente contenenti NTF4 o saline in un bagno di H 2 O impostati a 25 ° C per 30 minuti prima dell&#39;iniezione. </li><li> Posizionare il ratto in posizione supina e reclinata su una piattaforma di plexiglass ( figura 2 ). SusPend il ratto nella posizione reclinata dai loro incisivi frontali superiore attraverso un filo di guida strinse sulla parte superiore della piattaforma. </li><li> Fissare una siringa da 50 μm, 30 gauge, da 100 μL ad un endoscopio sinusale da 1,9 mm, 30 ° ( figura 3 ). NOTA: L&#39;assieme della siringa è collegato tramite un jig che tiene saldamente la cannula alla parete esterna dell&#39;endoscopio. L&#39;endoscopio consente la visualizzazione delle pieghe vocali e la guida della siringa intraorale. La posizione della punta della cannula viene regolata prima di ogni animale per assicurare che la punta sia completamente visibile attraverso la vista endoscopica ( Figura 4 ). </li><li> Utilizzare un paio di pinze in gomma per estendere la linguetta e spostarla lateralmente. Successivamente, inserire un speculum di plastica per mantenere la penetrazione orale. Fai speculum da un barile da 5 ml di siringa in plastica tagliato a una lunghezza di 1,5 a 2 cm, con i bordi tagliati sbiaditi e lucidati lisci. </li><li> Spegnere le luci nel vanoM e collegare una sorgente di luce alogena all&#39;endoscopio. Accendere il videoregistratore per catturare la procedura. </li><li> Immergere l&#39;estremità distale dell&#39;endoscopio in acqua calda per alcuni secondi per ridurre al minimo lo sviluppo della condensa sulla punta di vetro quando viene inserito nella bocca del ratto. </li><li> Usando feedback visivo dal monitor, guidare con cura l&#39;ago nella zona della piega vocale sinistra. </li><li> Tempo l&#39;iniezione della soluzione con la fase inspiratoria del ciclo respiratorio degli animali per accedere completamente alla piega vocale. Durante la fase inspiratoria della respirazione, la piega vocale è completamente esposta. <ol><li> Una volta che la piega vocale è completamente visibile, inserire l&#39;ago nel tireoarotenoide sinistro, trovato lateralmente al bordo bianco mediale della piega vocale. Con l&#39;ago posto, trasportare l&#39;iniettore attraverso la depressione della siringa. </li></ol></li><li> Spegnere la sorgente di luce alogena sull&#39;endoscopio e sul lettore video e tornare indietro sul roLuci om. </li><li> Rimettere il ratto alla sua gabbia di casa e mettere su un tappetino di riscaldamento. </li><li> Lasciare recuperare il ratto prima di rimuovere dal tampone di riscaldamento. Sostituire il cibo e l&#39;acqua nella gabbia. </li><li> Monitorare i ratti per 7 giorni dopo l&#39;iniezione e poi eutanizzare. Rimuovere le laringe per la criocondutturazione 24 . </li></ol> 5. Eutanizzazione dei ratti <ol><li> Anestetizzare i ratti con cloridrato di ketamina e cloridrato di xilazina (100 mg / 8 mg per kg di iniezione intraperitoneale iniettabile). </li><li> Eutanizzare da exsanguination dopo una toracotomia mediale. </li></ol>

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I muscoli della laringe sono vulnerabili agli effetti sfavorevoli dell&#39;invecchiamento. Studi precedenti hanno dimostrato variazioni nei muscoli linfari che prevedono cambiamenti nella dimensione della fibra, nel numero totale di fibre, nella capacità rigenerativa, nella dimensione e nella modifica della quantità di NMJ, oltre alle variazioni della funzione contrattile e dei cambi di isoenzima di miosina 4 , 11 , 27 ,...

I muscoli laringei contratti rapidamente e costantemente e sono vulnerabili agli effetti negativi dell&#39;invecchiamento. Questa attività costante è pensata per contribuire a problemi vocali o disfagia osservati in persone di età superiore ai 65 anni 1 , 2 , 3 , 4 , 5 , 6 , 7 . Molti meccanismi molecolari e patofisiologici contribuiscono a questa disfunzione legata all&#39;età. Questi meccanismi possono includere il rimodellamento della mucosa laringea, l&#39;atrofia muscolare o la perdita, la mancanza di rigenerazione delle fibre muscolari o atrofia che provoca l&#39;arricciamento delle pieghe vocali e l&#39;incapacità della chiusura glottica 8 , 9 , 10 , 11 . Non esiste una sperimentata terapia medica in questo momento che può comPrevenire completamente o riabilitare questi cambiamenti legati all&#39;età in questi muscoli. La modulazione dell&#39;efficacia della trasmissione neuromuscolare può influenzare notevolmente la prestazione neuromotoria. La famiglia delle neurotrofine comprende il fattore di crescita del nervo (NGF), il fattore di crescita del nervo derivato dal cervello (BDNF), la neurotrofina 3 (NTF3) e la NTF4 12 , 13 . Le neurotrofine hanno dimostrato di modulare l&#39;efficacia sinaptica 1 , 4 . Il fattore di crescita dell&#39;epatocyte, il fattore di crescita trasformante beta e il fattore di crescita dei fibroblasti sono stati recentemente impiegati negli esseri umani per il trattamento delle cicatrici vocali 15 - 17 . NTF4 regola anche l&#39;efficacia della NMJ; I topi che mancano di NTF4 mostrano NMJ disassemblati 11 , 18 , 19 . Questi studi portano ad effetti promettenti dei trattamentiT dei disturbi muscolari linfatici e denervazione con fattori di crescita. Le terapie di iniezione diretta ai tessuti delle pieghe vocali non sono senza precedenti negli esseri umani. Ad esempio, le iniezioni locali della tossina botulinica sono attualmente utilizzate come un trattamento efficace per i disturbi neurologici che influenzano i muscoli della laringa, come la disfonia spasmodica e la paralisi nervosa laringe ricorrente bilaterale 20 , 21 . L&#39;idrogel acido ialuronico è un altro iniettabile, usato per trattare la scocciatura vocale e l&#39;insufficienza glottale 22 , 23 . La laringoscopia d&#39;iniezione può essere usata per trattare una serie di disturbi della comunicazione 24 . Questi metodi di iniezione diretta sono promettenti per migliorare la funzione vocale e deglutire nelle popolazioni che invecchiano.

<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

La disfunzione della laringe negli anziani è una delle principali cause di disabilità, dai disturbi vocali alla disfagia e dalla perdita di riflessi protettivi delle vie aeree. Pochi, se esistono, esistono terapie che mirano a disfunzione muscolare laringea legata all&#39;età. Le neurotrofine sono coinvolte nell&#39;innervazione muscolare e nella differenziazione delle giunzioni neuromuscolari (NMJs). Si pensa che le neurotrofine aumentino la trasmissione neuromuscolare aumentando il rilascio del neurotrasmettitore. Le giunzioni neuromuscolari (NMJ) diventano più piccole e meno abbondanti nei muscoli laringei del ratto di invecchiamento, con prove di denervazione funzionale. Abbiamo esplorato gli effetti di NTF4 per l&#39;uso clinico futuro come terapeutico per migliorare la funzione nei muscoli laringei umani di invecchiamento. Qui forniamo il protocollo dettagliato per l&#39;applicazione sistemica e l&#39;iniezione diretta di NTF4 per esaminare la capacità del muscolo laringeo ratto di invecchiare di rimodellare in risposta all&#39;applicazione NTF4. In questo metodo, i ratti ricevevano NTF4 sistematicamente via oPompa smotica o mediante iniezione diretta attraverso le pieghe vocali. I muscoli della laringe furono poi disseccati e utilizzati per l&#39;esame istologico della morfologia e della denervazione legata all&#39;età.

I ratti sono stati eutanizzati dopo 2 settimane di infusione osmotica o una settimana dopo l&#39;iniezione diretta di NTF4. Le laringe sono state raccolte, poste in cryoprotectant (30% saccarosio e 70% saline fosfato tamponato) e poi sezionate in serie in larghezza 10 μm con un criostato. L&#39;invecchiamento dei muscoli laringei è influenzato dalla somministrazione di NTF4 25 . Oltre al ratto giovane e vecchio, abbiamo confrontato il lato iniettato e non iniett...

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

Qui presentiamo un protocollo per descrivere sistemicamente e direttamente l&#39;uso della neurotrofina 4 (NTF4) per rimodellare i muscoli laringei dell&#39;invecchiamento del ratto.

Infusione sottocutanea di neurotrofina 4 usando le pompe osmotiche o l&#39;iniezione muscolare diretta migliora i muscoli del laringe del ratto di invecchiamento

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

subcutaneous-neurotrophin-4-infusion-using-osmotic-pumps-or-direct

Research

JoVE Journal

Developmental Biology

8.3K Views.  University of Kentucky.  Qui presentiamo un protocollo per descrivere sistemicamente e direttamente l'uso della neurotrofina 4 (NTF4) per rimodellare i muscoli laringei dell'invecchiamento del ratto. 

Video: 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 trapianto di cellule cervicali targeting per corno ventrale nella Sclerosi Laterale Amiotrofica e le lesioni traumatiche del midollo spinale

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

Ricerca

Medicina

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.

Un modello murino della formazione muscolare da stimolazione elettrica neuromuscolare

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.

Somministrazione sottocutanea di antagonisti muscarinici e Triple-immunocolorazione del lungo muscolo elevatore Auris in topi

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

Neuroscienze

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;

Resezione del nervo tibiale - Un modello standardizzato per Denervation indotta scheletrico atrofia muscolare nei topi

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.

Il trapianto di cellule olfattive ensheathing per valutare il recupero funzionale dopo 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 ...

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.

Iniezioni intramuscolari Lungo le piastre Motor finale: un approccio mini-invasivo di navetta Tracers Direttamente nella Motor Neurons

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.

Nervo stimolatore-guida iniezione di cellule staminali autologhe vicino il nervo laringeo ricorrente di sinistra equina

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;

Un sistema impiantabile per l'elettromiografia cronica in vivo

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.

Preparazione della piega vocale del ratto per le analisi neuromuscolari

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>Elettroporazione e lesione nervosa nel muscolo elevatore lungo del topo

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>Autore in primo piano: Ruoli rigenerativi delle proteine muscolari nella giunzione neuromuscolare post-lesione nervosa

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.

Elettroporazione combinata in vivo e reinnervazione a breve termine del muscolo scheletrico elevatore cranico Auris Longus

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

Infusione sottocutanea di neurotrofina 4 usando le pompe osmotiche o l'iniezione muscolare diretta migliora i muscoli del laringe del ratto di invecchiamento

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