Questo lavoro è stato finanziato dal National Institutes of Health di Grant RO1DC002756 e la Purdue Research Foundation. Ringraziamo Wu Doris e Wiese Chang per la consulenza con esperimenti e McPhail Rodney aiuto con figure.

Ricette Chick Ringers <table border="1"><tbody><tr><td> 7,2 g </td><td> NaCl </td></tr><tr><td> 0,23 g </td><td> CaCl 2 + 2H 2 O </td></tr><tr><td> 0,37 g </td><td> KCl </td></tr><tr><td> 0,115 g </td><td> Na 2 HPO 4 </td></tr><tr><td> 900 ml </td><td> Acqua (cultura grado Tissue) </td></tr></tbody></table> Nota: Regolare il pH a 7,4. Portare il volume finale a 1000 ml con acqua. PBS 10X <table border="1"><tbody><tr><td> 40 g </td><td> NaCl </td></tr><tr><td> 1 g </td><td> KCl </td></tr><tr><td> 7 g </td><td> Na 2 HPO 4 </td></tr><tr><td> 1,2 g </td><td> KH 2 PO 4 </td></tr><tr><td> 450 ml </td><td> DEPC acqua </td></tr></tbody></table> Nota: Regolare il pH a 7,4. Portare finalevolume di 500 ml con acqua. Concentrazione di lavoro (1X) è fatto diluendo 1 magazzino 10X parte con 9 parti di tessuto-cultura dell&#39;acqua grado, come quella ottenuta con un sistema Millipore irraggiamento UV per generare acqua con 18 MΩ-cm di conducibilità. SAG medio espianto tenendo <ul><li> DMEM/F12 con L-glutammina, 10 mM Hepes </li><li> 10% di insulina, Trasferimento, Selenio (ITS +1) </li><li> 1% pen-streptococco </li></ul> Espianto terreno di coltura <ul><li> SAG medio espianto tenendo </li><li> 10 ng / ml neurotrofina-3 (NT-3) </li><li> 10 ng / ml di fattore neurotrofico ciliare (CNTF) </li></ul> Dissezione del midollo spinale e medie tenendo <ul><li> L-15 </li><li> 10% di siero fetale bovino </li><li> 1% pen-streptococco </li></ul> Uova incubare a 37-38 ° C. Sterilizzare strumenti di dissezione e piatto Sylgard dissezione nel 70% di etanolo. Dissecting piatti e strumenti possono anche essere sterilizzati ai raggi UV durante la notte. <ol><li> Perle Mettere in una microtubo con 1 ml di PBS sterile, mescolare e attendere che le perline di depositarsi sul fondo della provetta. </li><li> Lavare le perle più volte rimuovendo il surnatante dopo le perline sono stabiliti e risospendere in PBS. </li><li> Incubare le perline in proteine ​​purificate (diluito in PBS) o solo PBS (controllo) per 1 ora a temperatura ambiente. </li><li> Sciacquare le perline in PBS e conservare in un 24-pozzetti con 1 ml di PBS fino al momento in collagene. </li></ol> 2. Ganglio dissezione Statoacoustic <ol><li> Sulla E4 rimuovere l&#39;embrione da l&#39;uovo e mettetelo in un piatto con la soluzione di Ringer pulcino per rimuovere membrane embrionali. </li><li> Posto l&#39;embrione in una Sylgard © allineati capsula di Petri riempite con HBSS freddo e la posizione l&#39;embrione su un lato con la vescicola otica rivolto verso l&#39;alto. Perno l&#39;embrione al piatto con perni dissezione. </li><li> Utilizzando due pin dissezione, fare un taglio orizzontale attraverso la pelle immediatamente ventrale alsacchetti canale, circa dove si incontra il dotto cocleare. Fare un taglio verticale anteriore e posteriore per il SAG. Usare pinze o una spilla dissezione per sollevare e rimuovere il lembo di pelle delimitato dai tre tagli. Nota: Il SAG è situato ai margini del anteroventral otocyst, circa a metà strada tra la sacca dorsale, che è facilmente visibile con l&#39;illuminazione di base chiaro, e il dotto cocleare ventrale che proietta ventromedially ed è oscurato dagli archi della faringe. Il dotto cocleare allunga così la sua dimensione dorsoventrale aumenterà progressivamente su E4 tra Hamburger-Hamilton fasi 23-25 ​​settembre. </li><li> Tirare la otocyst in direzione posteriore per separarlo dalla SAG. Spostare il tessuto che circonda la SAG con perni dissezione e rimuovere con attenzione il SAG dall&#39;embrione utilizzando # 55 pinze. Rimuovere grossi pezzi di tessuto mesenchimale e fasci nervosi che sporge dalla SAG. </li><li> Usando una bocca larga punta della pipetta, trasferire l&#39;espianto ad un24-pozzetti con 0,5 ml di espianto SAG tenendo media. Tenere espianti su ghiaccio oa temperatura ambiente per un massimo di 4 ore. </li></ol> 3. Dissezione del midollo spinale <ol><li> Rimuovere la E6 dell&#39;embrione dall&#39;uovo e mettetelo in un piatto contenente la soluzione di Ringer pulcino. Togliere la testa e le membrane embrionali. </li><li> Mettere il corpo in un Sylgard © piatto contenente la dissezione a freddo medio dissezione del midollo spinale. Posizione e il pin del &quot;giù ventrale&quot; embrione e caudale di fronte al sperimentatore. Luogo pin dissezione attraverso ogni arto e fino alla fine anteriore. </li><li> Con pinze e / o dissezione pin, rimuovere con attenzione la pelle e tessuto, muovendosi in una direzione ventrale, fino a quando la superficie dorsale del midollo spinale è visibile. </li><li> Tagliare la linea mediana dorsale del midollo spinale con un perno dissezione, in un posteriore in direzione anteriore, lungo l&#39;intera lunghezza del midollo spinale. Questo crea un &quot;libro aperto&quot;, come i lati sinistro e destro del midollo spinale separati. </li><li&gt; Eliminare il tessuto circostante, lateralmente, per isolare il midollo spinale ed esporre i gangli della radice dorsale (DRG). Rimuovere il DRG e meningi per sfregamento un perno dissezione o pinze tra il midollo spinale e DRG. </li><li> Togliere il midollo spinale dal corpo tenendo il più a fine caudale del midollo spinale con una pinza e di sollevamento in alto e lontano dal sperimentatore. Rimuovere DRG rimanente e meningi. </li><li> Tenere un capo del midollo spinale con una pinza (o pin al piatto) e Vännäs usare le forbici per tagliare lungo la linea mediana ventrale, a bisecare il midollo spinale. </li><li> Tenere un capo del espianto con una pinza per immobilizzare il tessuto e usare le forbici per tagliare Vännäs espianti piccolo (100-500 um di lunghezza) per tutta la lunghezza del tessuto. La lastra di fondo può essere riconosciuto come un chiaro ispessimento del tessuto lungo un bordo espianto. </li><li> Utilizzare una bocca larga punta della pipetta per trasferire espianti di un piatto ben 24 con 0,5 ml di mezzo di dissezione a freddo. Tenere espianti su ghiaccio per mantenere la vitalità dei tessuti, fo fino a 4 ore. </li></ol> 4. Espianto cultura con le proteine ​​purificate di verificare le risposte dei neuriti <ol><li> Preparare un 1,5 mg / ml soluzione di collagene in una provetta da 15 ml conica, sul ghiaccio, secondo le istruzioni del produttore. </li><li> Verificare la soluzione di collagene ha un pH di 7-7,4 carta pH utilizzando l&#39;indicatore. Regolare il pH con l&#39;aggiunta di NaOH in 1 volumi microlitri. </li><li> Trasferimento espianti a un 24-pozzetti utilizzando una pipetta punta larga bocca e aspirare il liquido in eccesso. Espianti multiple possono essere coltivate in un pozzo, ma devono essere collocati ad almeno 500 micron di distanza l&#39;uno dall&#39;altro. </li><li> Aggiungere 0,5 ml di collagene per il pozzetto contenente l&#39;espianto. Posizionare l&#39;espianto sulla superficie inferiore del pozzo. Assicurarsi di impostare ogni cultura completamente prima di procedere con il pozzo successivo perché il collagene comincia a polimerizzare a temperatura ambiente. </li><li> Posizionare la piastra di coltura in A ° C più calda scivolo per 30-45 minuti per polimerizzare il collagene 37. Quando il collagene ha polimerasirizzato la renderanno simile ad un gel. </li><li> Aggiungere 0,5 ml di caldo terreno di coltura espianto integrato sia con proteine ​​purificate (diluito in PBS) o PBS (Control) e incubare a 37 ° C, 5% di CO 2. Effetti sulla crescita dei neuriti dovrebbe essere visibile da 24 ore. NOTA: Abbiamo utilizzato lo stesso protocollo per espianti SAG cultura fino a 42 ore. </li></ol> 5. Espianto co-coltura con le proteine ​​rivestite di perline prova escrescenza direzionale dei neuriti <ol><li> Inizia con passi cultura espianto 4,1-4,4. </li><li> Utilizzando una pipetta, trasferire 1-5 perle da PBS al pozzo contenente la soluzione di collagene ed espianto. </li><li> Utilizzare pinze per posizionare perline 50-500 micron dal bordo l&#39;espianto. </li><li> Posizionare la piastra su A ° C più calda scivolo per 30-45 minuti per polimerizzare il collagene 37. Il tessuto o perla può diventare sfollati come il collagene polimerizza. Controllare le culture e riposizionare il tessuto e perline durante i primi 5 min. </li><li> Aggiungere 0,5 ml SAG media per ogni pozzetto e incubare per 24 ore. </li></ol> 6. Visualizzazione di crescita dei neuriti di immunoistochimica <ol><li> Sciacquare le culture in PBS e fissare per 1 ora a temperatura ambiente in 4% paraformaldeide in PBS. </li><li> Rilasciare il gel dalle pareti dei pozzi tracciando intorno al bordo del gel con la punta arrotondata di una spatola in teflon micro. Eseguire la immunostain con il gel nei pozzetti. Sciacquare più volte in PBS. </li><li> Incubare in 0,5 ml di soluzione di bloccaggio (10% di siero bovino, 0,1% Triton X-100, sodio azide allo 0,1% in PBS) notte a 4 ° C. </li><li> Incubare in 0,5 ml di anticorpo primario (anti-β-tubulina anticorpo diluito 1:500 in blocco soluzione) una notte a 4 ° C. </li><li> Sciacquare più volte in PBS. Incubare in 0,5 ml di anticorpo secondario (Alexa Fluor 488 capra anti-topo IgG 2b diluito 1:500) notte a 4 ° C. Da questo passo in avanti, tenere le piastre al buio o avvolgerlo in un foglio, a causa della sensibilità alla luce dianticorpi secondari. </li><li> Sciacquare più volte in PBS e conservare a 4 ° C in PBS per un massimo di una settimana. </li></ol> 7. Rappresentante Risultati <img alt="Figura 1" src="/files/ftp_upload/3600/3600fig1.jpg" /> Figura 1. Risultati rappresentativi dei neuriti con gli effetti di purificazione e di NT-3 NT-3-rivestito perline su E4 espianti SAG pulcino. SAG corpi cellulari dei neuroni e neuriti erano immunostained con β-tubulina e ripreso con un obiettivo 10x su un microscopio confocale. Dopo 24 ore in vitro, gli espianti SAG visualizzata crescita dei neuriti maggiore in presenza di 100 ng / ml umano purificato NT-3 aggiunti al mezzo di coltura cellulare (1B), rispetto ai controlli (1A). SAG neuroni sono stati co-coltura con perline impregnato IN100 ng / ml, NT-3 per dimostrare che una sorgente puntiforme di NT-3 può localmente promuovere crescita dei neuriti. Espianti visualizzati più lunga e più densa dei neuriti escrescenza sul lato dellaespianto di fronte al tallone rispetto al lato opposto (1D) e rispetto al controllo culture con perline imbevute di PBS (1C). Barra di scala = 200 micron. <img alt="Figura 2" src="/files/ftp_upload/3600/3600fig2.jpg" /> Figura 2. Risultati rappresentativi dei neuriti con gli effetti di purificazione e di Wnt5a Wnt5a perle rivestite in E6 pulcino midollo spinale. Espianti di midollo spinale sono state immunostained con β-tubulina e ripreso con un microscopio confocale. Dopo 24 ore di crescita in presenza di Wnt5a purificato del mouse (400ng/ml), crescita dei neuriti da espianti pulcino midollo spinale è stato aumentato (2B), rispetto ai controlli colta senza Wnt5a (2A). Perline imbevuto di 500 ng / ml Wnt5a, posto 300-500 micron dal bordo di espianti midollo spinale, anche promosso crescita dei neuriti (2D) rispetto al controllo culture (2C). Simili co-coltura risultati sono stati precedentemente pubblicato con Wnt5a cellule che esprimono 8. Barra di scala = 200micron.

<ol>
	<li>Fekete, D. M., Campero, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axon+guidance+in+the+inner+ear.">Axon guidance in the inner ear.</a> Int. J. Dev. Biol. 51 (6-7), 549-549 (2007).</li><li>Fritzsch, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+inner+ear+afferent+connections:+forming+primary+neurons+and+connecting+them+to+the+developing+sensory+epithelia.">Development of inner ear afferent connections: forming primary neurons and connecting them to the developing sensory epithelia.</a> Brain Res. Bull. 60 (5-6), 423-423 (2003).</li><li>Gu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuropilin-1+conveys+semaphorin+and+VEGF+signaling+during+neural+and+cardiovascular+development.">Neuropilin-1 conveys semaphorin and VEGF signaling during neural and cardiovascular development.</a> Dev. Cell. 5 (1), 45-45 (2003).</li><li>Tessarollo, L., Coppola, V., Fritzsch, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NT-3+replacement+with+brain-derived+neurotrophic+factor+redirects+vestibular+nerve+fibers+to+the+cochlea.">NT-3 replacement with brain-derived neurotrophic factor redirects vestibular nerve fibers to the cochlea.</a> J. Neurosci. 24 (10), 2575-2575 (2004).</li><li>Avila, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain-derived+neurotrophic+factor+and+neurotrophin-3+support+the+survival+and+neuritogenesis+response+of+developing+cochleovestibular+ganglion+neurons.">Brain-derived neurotrophic factor and neurotrophin-3 support the survival and neuritogenesis response of developing cochleovestibular ganglion neurons.</a> Dev. Biol. 159 (1), 266-266 (1993).</li><li>Bianchi, L. M., Cohan, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+the+neurotrophins+and+CNTF+on+developing+statoacoustic+neurons:+comparison+with+an+otocyst-derived+factor.">Effects of the neurotrophins and CNTF on developing statoacoustic neurons: comparison with an otocyst-derived factor.</a> Dev. Biol. 159 (1), 353-353 (1993).</li><li>Juurlink, B. e. r. n. h. a. r. d. H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chick+Spinal+Somatic+Motoneurons+in+Culture.">Chick Spinal Somatic Motoneurons in Culture.</a> Protocols for Neural Cell Culture.. 59, (2001).</li><li>Fantetti, K. N., Zou, Y., Fekete, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnts+and+Wnt+inhibitors+do+not+influence+axon+outgrowth+from+chicken+statoacoustic+ganglion+neurons.">Wnts and Wnt inhibitors do not influence axon outgrowth from chicken statoacoustic ganglion neurons.</a> Hear. Res. , (2011).</li><li>Hamburger, V., Hamilton, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+series+of+normal+stages+in+the+development+of+the+chick+embryo.">A series of normal stages in the development of the chick embryo.</a> Journal of Morphology. 88, 49-49 (1951).</li><li>Bourikas, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sonic+hedgehog+guides+commissural+axons+along+the+longitudinal+axis+of+the+spinal+cord.">Sonic hedgehog guides commissural axons along the longitudinal axis of the spinal cord.</a> Nat. Neurosci. 8 (3), 297-297 (2005).</li><li>Charron, F., Tessier-Lavigne, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+brain+wiring+functions+for+classical+morphogens:+a+role+as+graded+positional+cues+in+axon+guidance.">Novel brain wiring functions for classical morphogens: a role as graded positional cues in axon guidance.</a> Development. 132 (10), 2251-2251 (2005).</li><li>Augsburger, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BMPs+as+mediators+of+roof+plate+repulsion+of+commissural+neurons.">BMPs as mediators of roof plate repulsion of commissural neurons.</a> Neuron. 24 (1), 127-127 (1999).</li><li>Lyuksyutova, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anterior-posterior+guidance+of+commissural+axons+by+Wnt-frizzled+signaling.">Anterior-posterior guidance of commissural axons by Wnt-frizzled signaling.</a> Science. 302 (5652), 1984-1984 (2003).</li></ol>

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Vi presentiamo un metodo per sezionare e cultura SAG E4 ed E6 espianti midollo spinale, da ragazza, sotto siero senza condizioni. Questa procedura è attualmente utilizzato nel nostro laboratorio per studiare gli effetti dei vari ligandi secreto SAG sulla sopravvivenza dei neuroni e crescita dei neuriti. Nuovi aspetti di questo protocollo includono l&#39;uso di un siero privo di espianti di sistema per la coltura e l&#39;uso di perline imbevute di fattori di crescita per studiare gli effetti sulla escrescenza SAG neurit...

<table border="1"><tbody><tr><td> Reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commento </td></tr><tr><td> Attrezzatura </td><td></td><td></td><td></td></tr><tr><td> Dissezione microscopio </td><td></td><td></td><td> Usiamo un stereomicroscopio Leica M80 con campo chiaro di base di illuminazione </td></tr><tr><td> Far scorrere più caldi </td><td> CS &amp; E. </td><td></td><td> Collagene polimerizzazione </td></tr><tr><td> Pollo uovo incubatore </td><td></td><td></td><td></td></tr><tr><td> 37 ° C / 5% CO 2 umidificata coltura cellulare incubatore </td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> Dissezione Materiali </td><td></td><td></td><td></td></tr><tr><td> Sylgard &amp;copia; rivestito capsula di Petri </td><td></td><td></td><td></td></tr><tr><td> 24-oltre piastra di coltura cellulare </td><td> Corning </td><td> 3526 </td><td></td></tr><tr><td> # 5 Dumont forcipe </td><td> Strumenti Scienza multa </td><td> 11252-30 </td><td></td></tr><tr><td> # 55 Dumont forcipe </td><td> Strumenti Scienza multa </td><td> 11295-51 </td><td></td></tr><tr><td> Perni in acciaio inox dissezione </td><td> Strumenti Scienza multa </td><td> 26002-10 </td><td> Embrione pinning, dissezione multa </td></tr><tr><td> Moria mestolo forato </td><td> Strumenti Scienza multa </td><td> 10370-17 </td><td> Embrione raccolta / trasferimento </td></tr><tr><td> 200 ul larga bocca pipetta suggerimenti </td><td> Dot Scientific Inc </td><td> 118-96R </td><td> Espianto trasferimento </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> E4 e uova di gallina E6 </td><td></td><td></td><td> &amp; Nbsp; </td></tr><tr><td> Soluzione pulcino Ringer </td><td></td><td></td><td> Embrione vendemmia </td></tr><tr><td> HBSS </td><td> Sigma </td><td> H8264 </td><td> SAG dissezione </td></tr><tr><td> L15 media </td><td> Sigma </td><td> L5520 </td><td> Midollo spinale medio dissezione </td></tr><tr><td> Siero di vitello fetale </td><td> Atlanta Biologicals </td><td> S11150 </td><td> Midollo spinale medio dissezione </td></tr><tr><td> Vännäs Forbici </td><td> Mondo Strumenti di precisione </td><td> 501778 </td><td> Dissezione del midollo spinale </td></tr><tr><td>&nbsp;</td><td></td><td></td><td></td></tr><tr><td> Collagene preparazione </td><td></td><td></td><td></td></tr><tr><td> Rat-tail collagene di tipo I </td><td> BD Biosciences </td><td> 354249 </td><td> Espianto cultura </td></tr><tr><td> 10X PBS (sterili) </td><td></td><td></td><td> Per neutralize collagene </td></tr><tr><td> NaOH 1N (sterili) </td><td></td><td></td><td> Per neutralizzare collagene </td></tr><tr><td> Acqua distillata (sterile) </td><td></td><td></td><td> Per neutralizzare collagene </td></tr><tr><td> pH carte indicatore (6,0-8,1) </td><td> Whatman </td><td> 2629-990 </td><td> Per controllare il pH collagene </td></tr><tr><td> Provette da 15 ml </td><td> Dot Scientific Inc </td><td> 818-PG </td><td></td></tr><tr><td> 500 microlitri larga bocca pipetta suggerimenti </td><td> Dot Scientific Inc </td><td> 119-R100 </td><td> Al collagene pipetta </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> Espianto cultura </td><td></td><td></td><td></td></tr><tr><td> DMEM/F12 media </td><td> Sigma </td><td> D8437 </td><td> Espianto terreno di coltura </td></tr><tr><td> IL SUO 1 </td><td> Sigma </td><td> I2521 </td><td> Supplemento media </td&gt; </tr><tr><td> Penicillina-Streptomicina </td><td> Invitrogen </td><td> 15140-122 </td><td> Supplemento media </td></tr><tr><td> CNTF (ratto) </td><td> Sigma </td><td> C3835 </td><td> Supplemento media </td></tr><tr><td> NT-3 (umano) </td><td> Sigma </td><td> N1905 </td><td> Supplemento media </td></tr><tr><td> Purificato del mouse Wnt5a </td><td> R &amp; D Systems </td><td> 645-WN-010 </td><td></td></tr><tr><td> Affi-gel blu gel perline </td><td> Bio-Rad </td><td> 153-7302 </td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> Immunoistochimica </td><td></td><td></td><td></td></tr><tr><td> Paraformaldeide </td><td> Sigma </td><td> P6148 </td><td> Fissazione </td></tr><tr><td> PBS </td><td></td><td></td><td> Risciacqui </td></tr><tr><td> Triton X-100 </td><td> Sigma </td><td> T9284 </td><td> Soluzione bloccante </td></tr><tr><td> Sodioazide </td><td> Sigma </td><td> S8032 </td><td> Soluzione bloccante </td></tr><tr><td> Siero di vitello </td><td> Invitrogen </td><td> 16170-078 </td><td> Soluzione bloccante </td></tr><tr><td> Monoclonale IgG2b anti-tubulina β 1 ​​+ II anticorpi </td><td> Sigma </td><td> T8535 </td><td> Corpi cellulari etichette e neuriti </td></tr><tr><td> Alexa fluor 488 capra anti-topo IgG2 anticorpi b </td><td> Invitrogen </td><td> A21141 </td><td> Rileva anticorpi β tubulina </td></tr><tr><td> Micro teflon spatola </td><td> VWR </td><td> 57949-033 </td><td> Gel di collagene rilascio da ben </td></tr></tbody></table>

dissection and culture of chick statoacoustic ganglion and spinal cord explants in collagen gels for neurite outgrowth assays

Gli organi sensoriali dell&#39;orecchio interno di pollo sono innervate dai processi periferici del ganglio statoacoustic (SAG) neuroni. Innervazione organo sensoriale dipende da una combinazione di segnali guida degli assoni 1 e fattori di sopravvivenza 2 situata lungo la traiettoria degli assoni in crescita e / o all&#39;interno degli stessi obiettivi degli organi sensoriali. Per esempio, l&#39;interferenza funzionale con un percorso classico di guida degli assoni segnalazione, semaforina-Neuropilin, generato misrouting di assoni otic 3. Inoltre, diversi fattori di crescita espresse negli obiettivi sensoriali dell&#39;orecchio interno, compresi neurotrofina-3 (NT-3) e del fattore neurotrofico cervello derivato (BDNF), sono stati manipolati in animali transgenici, ancora una volta porta a misrouting di assoni SAG 4. Queste stesse molecole promuovere sia la sopravvivenza e la crescita dei neuriti dei neuroni SAG pulcino in vitro 5,6. Qui, descriviamo e dimostrare il metodo in vitro stiamo ucantare per testare la reattività dei neuriti SAG pulcino alle proteine ​​solubili, tra cui morfogeni noto come il Wnts, così come fattori di crescita che sono importanti per la promozione dei neuriti escrescenza SAG e la sopravvivenza dei neuroni. Usando questo sistema modello, speriamo di trarre conclusioni circa gli effetti che ligandi secreto può esercitare sulla sopravvivenza dei neuroni e SAG crescita dei neuriti. Espianti SAG sono sezionato il giorno embrionale 4 (E4) e coltivate in gel tridimensionale di collagene sotto siero senza condizioni per 24 ore. In primo luogo, la reattività dei neuriti è testato da espianti coltura integrata con proteine ​​di media. Poi, per chiedere se fonti puntuali di ligandi secreto può avere effetti direzionali sulla crescita dei neuriti, gli espianti sono co-coltura con le proteine ​​rivestite perline e analizzati per la capacità del cordone di promuovere a livello locale o inibire escrescenza. Abbiamo anche una dimostrazione della dissezione (modificato protocollo 7) e la cultura della E6 espianti midollo spinale. Noiabitualmente uso espianti midollo spinale per confermare bioattività delle proteine ​​e proteine-impregnati perline, e per verificare le specie reattività crociata con i tessuti pulcino, alle condizioni stessa cultura espianti SAG. Questi test in vitro sono utili per lo screening rapido per le molecole che esercitano trofico (sopravvivenza) o tropico (direzionale) effetti sui neuroni SAG, soprattutto prima di effettuare studi in vivo. Inoltre, questo metodo permette la sperimentazione di singole molecole sotto siero senza condizioni, con alta sopravvivenza dei neuroni 8.

Watch this Scientific Journal Video about Dissection and Culture of Chick Statoacoustic Ganglion and Spinal Cord Explants in Collagen Gels for Neurite Outgrowth Assays at JoVE.com

Dissection and Culture of Chick Statoacoustic Ganglion and Spinal Cord Explants in Collagen Gels for Neurite Outgrowth Assays

Dimostriamo come sezionare e cultura pulcino ganglio statoacoustic E4 ed E6 espianti midollo spinale. Espianti sono coltivate sotto siero senza condizioni in gel di collagene 3D per 24 ore. Reattività dei neuriti è testato con fattore di crescita integrata medie e con le proteine ​​rivestite perline.

Dissezione e Cultura della gangliari Chick Statoacoustic Espianti e del midollo spinale in gel di collagene per saggi crescita dei neuriti

The sensory organs of the chicken inner ear are innervated by the peripheral processes of statoacoustic ganglion (SAG) neurons. Sensory organ innervation ...

dissection-culture-chick-statoacoustic-ganglion-spinal-cord-explants

Research

JoVE Journal

Neuroscience

12.0K Views.  Purdue University. Dimostriamo come sezionare e cultura pulcino ganglio statoacoustic E4 ed E6 espianti midollo spinale. Espianti sono coltivate sotto siero senza condizioni in gel di collagene 3D per 24 ore. Reattività dei neuriti è testato con fattore di crescita integrata medie e con le proteine ​​rivestite perline.

Video: Dissection and Culture of Chick Statoacoustic Ganglion and Spinal Cord Explants in Collagen Gels for Neurite Outgrowth Assays

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

Dissezione e la cultura di neuroni embrionali commissurali da midollo spinale

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

Ricerca

Neuroscienze

A Functional Motor Unit in the Culture Dish: Co-culture of Spinal Cord Explants and Muscle Cells

Human primary muscle cells cultured aneurally in monolayer rarely contract spontaneously because, in the absence of a nerve component, cell differentiation is limited and motor neuron stimulation is missing1. These limitations hamper the in vitro study of many neuromuscular diseases in cultured muscle cells. Importantly, the experimental constraints of monolayered, cultured muscle cells can be overcome by functional innervation of myofibers with spinal cord explants in co-cultures.
Here, we show the different steps required to achieve an efficient, proper innervation of human primary muscle cells, leading to complete differentiation and fiber contraction according to the method developed by Askanas2. To do so, muscle cells are co-cultured with spinal cord explants of rat embryos at ED 13.5, with the dorsal root ganglia still attached to the spinal cord slices. After a few days, the muscle fibers start to contract and eventually become cross-striated through innervation by functional neurites projecting from the spinal cord explants that connecting to the muscle cells. This structure can be maintained for many months, simply by regular exchange of the culture medium.
The applications of this invaluable tool are numerous, as it represents a functional model for multidisciplinary analyses of human muscle development and innervation. In fact, a complete de novo neuromuscular junction installation occurs in a culture dish, allowing an easy measurement of many parameters at each step, in a fundamental and physiological context.
Just to cite a few examples, genomic and/or proteomic studies can be performed directly on the co-cultures. Furthermore, pre- and post-synaptic effects can be specifically and separately assessed at the neuromuscular junction, because both components come from different species, rat and human, respectively. The nerve-muscle co-culture can also be performed with human muscle cells isolated from patients suffering from muscle or neuromuscular diseases3, and thus can be used as a screening tool for candidate drugs. Finally, no special equipment but a regular BSL2 facility is needed to reproduce a functional motor unit in a culture dish. This method thus is valuable for both the muscle as well as the neuromuscular research communities for physiological and mechanistic studies of neuromuscular function, in a normal and disease context.

Cultured muscle cells are an inadequate model to recapitulate innervated muscle in vivo. A functional motor unit can be reproduced in vitro by innervation of differentiated human primary muscle cells using rat embryo spinal cord explants. This article describes how co-cultures of spinal cord explants and muscle cells are established.

Un gruppo motore funzionale nella piastra di coltura: Co-coltura degli espianti midollo spinale e le cellule muscolari

Human primary muscle cells cultured  aneurally in monolayer rarely contract spontaneously because, in the absence of  a nerve component, cell ...

Dissection and Culture of Mouse Dopaminergic and Striatal Explants in Three-Dimensional Collagen Matrix Assays

Midbrain dopamine (mdDA) neurons project via the medial forebrain bundle towards several areas in the telencephalon, including the striatum1. Reciprocally, medium spiny neurons in the striatum that give rise to the striatonigral (direct) pathway innervate the substantia nigra2. The development of these axon tracts is dependent upon the combinatorial actions of a plethora of axon growth and guidance cues including molecules that are released by neurites or by (intermediate) target regions3,4. These soluble factors can be studied in vitro by culturing mdDA and/or striatal explants in a collagen matrix which provides a three-dimensional substrate for the axons mimicking the extracellular environment. In addition, the collagen matrix allows for the formation of relatively stable gradients of proteins released by other explants or cells placed in the vicinity (e.g. see references 5 and 6). Here we describe methods for the purification of rat tail collagen, microdissection of dopaminergic and striatal explants, their culture in collagen gels and subsequent immunohistochemical and quantitative analysis. First, the brains of E14.5 mouse embryos are isolated and dopaminergic and striatal explants are microdissected. These explants are then (co)cultured in collagen gels on coverslips for 48 to 72 hours in vitro. Subsequently, axonal projections are visualized using neuronal markers (e.g. tyrosine hydroxylase, DARPP32, or &beta;III tubulin) and axon growth and attractive or repulsive axon responses are quantified. This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of mesostriatal and striatonigral axon growth and guidance during development. Using this assay, it is also possible to assess other (intermediate) targets for dopaminergic and striatal axons or to test specific molecular cues.

Explants from the midbrain dopamine system and striatum are used in a collagen matrix assay for the in vitro analysis of mesostriatal and striatonigral pathway development. In this assay axonal outgrowth and guidance can be manipulated and quantified. It can also be modified for assessing other regions or molecular cues.

Dissezione e Cultura del mouse dopaminergici e striatali Espianti di saggi di collagene matrice tridimensionale

Midbrain dopamine (mdDA) neurons project via the medial forebrain bundle towards several areas in the telencephalon, including the striatum1. ...

Chicken Embryo Spinal Cord Slice Culture Protocol

Slice cultures can facilitate the manipulation of embryo development both pharmacologically and through gene manipulations. In this reduced system, potential lethal side effects due to systemic drug applications can be overcome. However, culture conditions must ensure that normal development proceeds within the reduced environment of the slice. We have focused on the development of the spinal cord, particularly that of spinal motor neurons. We systematically varied culture conditions of chicken embryo slices from the point at which most spinal motor neurons had been born. We assayed the number and type of motor neurons that survived during the culture period and the position of those motor neurons compared to that in vivo. We found that serum type and neurotrophic factors were required during the culture period and were able to keep motor neurons alive for at least 24 hr and allow those motor neurons to migrate to appropriate positions in the spinal cord. We present these culture conditions and the methodology of preparing the embryo slice cultures using eviscerated chicken embryos embedded in agarose and sliced using a vibratome.

Slice cultures facilitate the manipulation of embryo development by gene and pharmacological perturbations. However, culture conditions must ensure that normal development can proceed within the reduced environment of the slice. We illustrate a protocol that facilitates normal spinal cord development to proceed for at least 24 hr.

Embrione di pollo midollo spinale Fetta protocollo Cultura

Slice cultures can facilitate the manipulation of  embryo development both pharmacologically and through gene manipulations. In  this reduced system, ...

Dissection and Lateral Mounting of Zebrafish Embryos: Analysis of Spinal Cord Development

The zebrafish spinal cord is an effective investigative model for nervous system research for several reasons. First, genetic, transgenic and gene knockdown approaches can be utilized to examine the molecular mechanisms underlying nervous system development. Second, large clutches of developmentally synchronized embryos provide large experimental sample sizes. Third, the optical clarity of the zebrafish embryo permits researchers to visualize progenitor, glial, and neuronal populations. Although zebrafish embryos are transparent, specimen thickness can impede effective microscopic visualization. One reason for this is the tandem development of the spinal cord and overlying somite tissue. Another reason is the large yolk ball, which is still present during periods of early neurogenesis. In this article, we demonstrate microdissection and removal of the yolk in fixed embryos, which allows microscopic visualization while preserving surrounding somite tissue. We also demonstrate semipermanent mounting of zebrafish embryos. This permits observation of neurodevelopment in the dorso-ventral and anterior-posterior axes, as it preserves the three-dimensionality of the tissue.

Developmental processes such as proliferation, patterning, differentiation, and axon guidance can be readily modeled in the zebrafish spinal cord. In this article, we describe a mounting procedure for zebrafish embryos, which optimizes visualization of these events.

Dissezione e laterale Montaggio di embrioni di zebrafish: analisi dello sviluppo del midollo spinale

The zebrafish spinal cord is an effective investigative model for nervous system research for several reasons. First, genetic, transgenic and gene ...

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several weeks, and this is accompanied by short distance regeneration (a few mm) of propriospinal axons and spinal-projecting axons from the brainstem. Among the 36 large identifiable spinal-projecting neurons, some are good regenerators and others are bad regenerators. These neurons can most easily be identified in wholemount CNS preparations. In order to understand the neuron-intrinsic mechanisms that favor or inhibit axon regeneration after injury in the vertebrates CNS, we determine differences in gene expression between the good and bad regenerators, and how expression is influenced by spinal cord transection. This paper illustrates the techniques for housing larval and recently transformed adult sea lampreys in fresh water tanks, producing complete spinal cord transections under microscopic vision, and preparing brain and spinal cord wholemounts for in situ hybridization. Briefly, animals are kept at 16&#160;&#176;C and anesthetized in 1% Benzocaine in lamprey Ringer. The spinal cord is transected with iridectomy scissors via a dorsal approach and the animal is allowed to recover in fresh water tanks at 23 &#176;C. For in situ hybridization, animals are reanesthetized and the brain and cord removed via a dorsal approach.

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

Lampreys recover locomotion after a complete spinal cord injury. However, some spinal-projecting neurons are good regenerators and others are not. This paper illustrates the techniques for housing sea lamprey larvae (and recently transformed adults), producing complete spinal cord transections and preparing wholemount brains and spinal cords for in situ hybridization.

Completa lesioni del midollo spinale e del cervello dissezione Protocollo per Wholemount successiva<em&gt; In Situ</em&gt; Ibridazione in larvale Sea Lamprey

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several ...

RNA-Seq Analysis of Differential Gene Expression in Electroporated Chick Embryonic Spinal Cord

In ovo electroporation of the chick neural tube is a fast and inexpensive method for identification of gene function during neural development. Genome wide analysis of differentially expressed transcripts after such an experimental manipulation has the potential to uncover an almost complete picture of the downstream effects caused by the transfected construct. This work describes a simple method for comparing transcriptomes from samples of transfected embryonic spinal cords comprising all steps between electroporation and identification of differentially expressed transcripts. The first stage consists of guidelines for electroporation and instructions for dissection of transfected spinal cord halves from HH23 embryos in ribonuclease-free environment and extraction of high-quality RNA samples suitable for transcriptome sequencing. The next stage is that of bioinformatic analysis with general guidelines for filtering and comparison of RNA-Seq datasets in the Galaxy public server, which eliminates the need of a local computational structure for small to medium scale experiments. The representative results show that the dissection methods generate high quality RNA samples and that the transcriptomes obtained from two control samples are essentially the same, an important requirement for detection of differential expression genes in experimental samples. Furthermore, one example is provided where experimental overexpression of a DNA construct can be visually verified after comparison with control samples. The application of this method may be a powerful tool to facilitate new discoveries on the function of neural factors involved in spinal cord early development.

This video shows the basic steps for performing whole transcriptome analysis on dissected chick embryonic spinal cord samples after transfection with in ovo electroporation.

RNA-Seq Analisi di espressione genica differenziale in elettroporate Chick embrionali del midollo spinale

In ovo electroporation of the chick neural tube is a fast and inexpensive method for identification of gene function during neural development. Genome ...

Spinal Cord Neurons Isolation and Culture from Neonatal Mice

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on postnatal day 1-3. A mouse litter, usually 4-10 pups born from one breeding pair, is gathered for one experiment, and spinal cords are collected individually from each mouse after euthanasia with isoflurane. The spinal column is dissected out and then the spinal cord is released from the column. The spinal cords are then minced to increase the surface area of delivery for an enzymatic protease that allows for the neurons and other cells to be released from the tissue. Trituration is then used to release the cells into solution. This solution is subsequently fractionated in a density gradient to separate the various cells in solution, allowing for neurons to be isolated. Approximately 1-2.5 x 106 neurons can be isolated from one litter group. The neurons are then seeded onto wells coated with adhesive factors that allow for proper growth and maturation. The neurons take approximately 7 days to reach maturity in the growth and culture medium and can be used thereafter for treatment and analysis.

This study presents a technique for the isolation of neurons from WT neonatal mice. It requires the careful dissection of the spinal cord from the neonatal mouse, followed by the separation of neurons from the spinal cord tissue through mechanical and enzymatic cleavage.

Isolamento e cultura dei neuroni del midollo spinale da topi neonatali

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on ...

Intra-Arterial Delivery of Neural Stem Cells to the Rat and Mouse Brain: Application to Cerebral Ischemia

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to intracranial delivery, intra-arterial administration of NSCs is less invasive and produces a more diffuse distribution of NSCs within the brain parenchyma. Further, intra-arterial delivery allows the first-pass effect in the brain circulation, lessening the potential for trapping of cells in peripheral organs, such as liver and spleen, a complication associated with peripheral injections. Here, we detail the methodology, in both mice and rats, for delivery of NSCs through the common carotid artery (mouse) or external carotid artery (rat) to the ipsilateral hemisphere after an ischemic stroke. Using GFP-labeled NSCs, we illustrate the widespread distribution achieved throughout the rodent ipsilateral hemisphere at 1 d, 1 week and 4 weeks after postischemic delivery, with a higher density in or near the ischemic injury site. In addition to long-term survival, we show evidence of differentiation of GFP-labeled cells at 4 weeks. The intra-arterial delivery approach described here for NSCs can also be used for administration of therapeutic compounds, and thus has broad applicability to varied CNS injury and disease models across multiple species.

A method for delivering neural stem cells, adaptable for injecting solutions or suspensions, through the common carotid artery (mouse) or external carotid artery (rat) after ischemic stroke is reported. Injected cells are distributed broadly throughout the brain parenchyma and can be detected up to 30 d after delivery.

Consegna intra-arteriosa di cellule staminali neurali al cervello di ratto e topo: applicazione all'ischemia cerebrale

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to ...

Induction of Complete Transection-Type Spinal Cord Injury in Mice

Spinal cord injury (SCI) largely leads to irreversible and permanent loss of function, most commonly as a result of trauma. Several treatment options, such as cell transplantation methods, are being researched to overcome the debilitating disabilities arising from SCI. Most pre-clinical animal trials are conducted in rodent models of SCI. While rat models of SCI have been widely used, mouse models have received less attention, even though mouse models can have significant advantages over rat models. The small size of mice equates to lower animal maintenance costs than for rats, and the availability of numerous transgenic mouse models is advantageous for many types of studies. Inducing repeatable and precise injury in the animals is the primary challenge for SCI research, which in small rodents requires high-precision surgery. The transection-type injury model has been a commonly used injury model over the last decade for transplantation-based therapeutic research, however a standardized method for inducing a complete transection-type injury in mice does not exist. We have developed a surgical protocol for inducing a complete transection type injury in C57BL/6 mice at thoracic vertebral level 10 (T10). The procedure uses a small tip drill instead of rongeurs to precisely remove the lamina, after which a thin blade with rounded cutting edge is used to induce the spinal cord transection. This method leads to reproducible transection-type injury in small rodents with minimal collateral muscle and bone damage and therefore minimizes confounding factors, specifically where behavioral functional outcomes are analyzed.

This protocol describes how to create a precise laminectomy for induction of stable transection-type spinal cord injury in the mouse model, with minimal collateral damage for spinal cord injury research.

Induzione della lesione completa del midollo spinale di tipo transezione nei topi

Spinal cord injury (SCI) largely leads to irreversible and permanent loss of function, most commonly as a result of trauma. Several treatment options, ...