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La cura e il trattamento degli animali in questa procedura sono state condotte in conformità con le linee guida del Comitato istituzionale di cura e uso degli animali presso l&#39;Università del Colorado. 1. Preparazione delle soluzioni <ol><li> Preparare e conservare tutte le soluzioni a temperature adeguate, come mostrato nella Tabella 1 . </li></ol> 2. Rivestimenti di pozzetti e diapositive NOTA: i neuroni non aderiscono bene a superfici in plastica o vetro. <ol><li> Un giorno prima dell&#39;isolamento dei neuroni, coprire i pozzetti di una piastra di coltura a 24 pozzetti con 0,5 ml di poli-D-lisina (PDL, Tabella 2 ) e lasciarla in un cappuccio di flusso laminare O / N. NOTA: Il metodo preferito è quello di utilizzare piastre a 24 pozzetti e di rivestire solo i pozzetti centrali, in quanto l&#39;evaporazione dai pozzetti periferici tende ad essere più accelerata e portare a risultati incoerenti. Inoltre, le diapositive in vetro possono essere collocate all&#39;interno dei pozzetti precedentiO rivestimento. I neuroni si attaccheranno alle vetrate rivestite e possono essere rimosse in seguito per ulteriori immagini microscopiche . </li><li> Nel giorno dell&#39;isolamento, rimuovere il PDL dai pozzetti. Lavare con acqua sterile per alcuni minuti prima di rimuovere l&#39;acqua rimanente e lasciarla asciugare per 1 ora (vedi sotto). </li><li> Dopo l&#39;essiccazione, coprire le diapositive con laminina ( Tabella 2 ). NOTA: Circa 10 μL di laminina (10 μg / μL) è sufficiente a coprire ogni pozzetto di una piastra sterile a 24 pozzetti. Questo viene mescolato con 340 μL di mezzo (volume totale: 350 μL) per coprire l&#39;intero pozzetto. Lasciate riposare per 2 ore a RT in un cofano di cultura e aspirare prima di ricoprire le celle. </li></ol> 3. Raccogliere i cavi spinali NOTA: Tutti gli strumenti devono essere autoclavati (135 ° C e 30 psi per cicli di 4 x 7 min) per la sterilità. <ol><li> Eutanizzare cuccioli di topo C57BL / 6 di 1-3 giorni in una camera con isoflurano. Aspettare30 s dopo la cessazione del movimento e pizzicare la gamba per confermare la mancanza di risposta. </li><li> Separare la testa dal corpo usando forbici, con cucciolo nella posizione predisposta. </li><li> Stabilizzare le gambe posteriori o coda e braccia sulla tabella di procedura, con il lato dorsale rivolto verso l&#39;utente. </li><li> Tagliare la pelle usando le forbici curve dell&#39;iride. </li><li> Tagliare il midollo spinale dalla regione lombare appena sopra le anche e procedere a tagliare entrambi i lati del torace per separarlo dal corpo. NOTA: Ciò richiede un&#39;attenta dissezione del midollo spinale da organi viscerali per evitare danni involontari ad altri organi ( Figura 1a ). </li><li> Lavare in sequenza per 10 s in 3 x 10 cm di piatti Petri contenenti 5 mL di 0,2 μm filtrato-sterilizzato fosfato-tamponato salina (PBS) per rimuovere il tessuto in eccesso. </li><li> Inserire un ago e una siringa da 22 G riempiti di 5 mL di PBS sterilizzato con filtro nell&#39;estremità caudale della colonna vertebrale e scorrere cranialmente, lasciando il cavo tO uscire in un quarto piatto di Petri ( figura 1b ). </li><li> Raccogliere il midollo spinale in un tubo da 15 mL con 5 mL di HABG ( Tabella 1 ) sul ghiaccio. Usate attenzione per evitare di schiacciare il midollo spinale. </li><li> Ripetere i passaggi 3.1-3.8 per ogni cucciolo nella stiva. NOTA: Idealmente, questo processo dovrebbe richiedere meno di 30 minuti per il midollo spinale per garantire l&#39;isolamento sano del neurone. </li></ol> 4. Neuroni isolanti NOTA: La fase seguente deve essere eseguita in un cappuccio di flusso laminare. Si prevede la conoscenza della tecnica base sterile. <ol><li> Tissue Mincing <ol><li> Prendete il tubo contenente le corde spinali e agitate leggermente per sospendere il tessuto. </li><li> Versare il tessuto dal tubo in un piatto da 60 mm di vetro Petri e dadi con una lama di rasoio per creare pezzi fini di circa 0,5 mm. </li><li> Trasferire il tessuto con una pipetta largo-bore in un tubo da 15 mL contenente 5 mL di HABG. </li><li> Posizionarlo in un30 ° C per 30 minuti per consentire alle cellule di equilibrare a questa temperatura. Mantenere le cellule su un agitatore appena sufficiente per consentire loro di essere sospesi nel fluido. NOTA: Questo passaggio è fatto per evitare di sconvolgere le cellule dopo il trasferimento dal ghiaccio al mezzo di digestione. Mantenere le cellule a 30 ° C contribuisce a ridurre la morte cellulare associata ad un metabolismo altrimenti aumentato a 37 ° C. </li></ol></li><li> Preparare il mezzo di digestione ( Tabella 1 ). </li><li> Preparare il gradiente di densità (Tabella 1). <ol><li> Preparare ciascuno dei 4 strati in 4 tubi separati da 15 ml, come descritto nella tabella 1 . </li><li> Aggiungere 1 ml da ogni strato in un nuovo tubo da 15 ml. Inizia con lo strato 1 in basso e aggiungi successivamente fino a raggiungere il livello 4 in alto. Evitare di disturbare gli strati durante l&#39;aggiunta. </li></ol></li><li> Lavare le piastre rivestite con PDL dalla fase 2.2. Lavare con acqua sterile per alcuni minuti prima di rimuovere qualsiasi acqua rimanente eConsentendo loro di asciugare per 1 h. </li><li> Trasferire il tessuto al mezzo di digestione. <ol><li> Rimuovere il tubo contenente i tessuti dal bagno dell&#39;acqua shaker a 30 ° C e lasciarlo accomodare per alcuni minuti. </li><li> Rimuovere il tubo mediano di digestione dal bagno d&#39;acqua di 37 ° C e aspirarlo in una siringa di chiusura. </li><li> Aspirare l&#39;eccesso di HABG dal tubo contenente i tessuti. </li><li> Utilizzare un filtro da 0.2 μm a sbirciamento sulla siringa per aggiungere il mezzo di digestione al tubo contenente i tessuti. </li><li> Mettere il tubo in un bagno d&#39;acqua di 30 ° C per 30 minuti. Tenere le cellule agitate abbastanza da permettere loro di essere sospese nel fluido. NOTA: È importante non mantenere troppo lungamente le cellule nel mezzo di digestione o lasciare che la temperatura sia troppo elevata, che potrebbe portare ad un&#39;eccessiva digestione e provocare il sospensione del tessuto in una miscela gelatinosa. </li></ol></li><li> Durante questo periodo, rivestire la laminina come nel punto 2.3. </li> &lt;Li&gt; Esegui la triturazione ( cioè la separazione delle cellule dal tessuto). <ol><li> Rimuovere il tubo dal bagno di agitazione a 30 ° C e lasciarlo accomodare per qualche minuto. </li><li> Aspirare un mezzo di digestione in eccesso. </li><li> Sospendere il tessuto in 2 ml di HABG. </li><li> Usando una pipetta stretta, triturare 10 volte per 45 s. NOTA: Questo è probabilmente il passo singolo più cruciale e può ridurre in modo significativo il rendimento se non viene eseguito correttamente. <ol><li> Aspirare il tessuto nella pipetta e vuotare immediatamente il contenuto. </li><li> Evitate l&#39;introduzione dell&#39;aria, poiché diminuirà significativamente la resa vitale. NOTA: la pipetta ideale è una pipetta di vetro da 9 &quot;, la punta della pipetta deve essere lucidata a fuoco per lisciare le superfici ruvide e quindi essere siliconizzata posizionandosi in una soluzione 1:20 di diclorodimetilsilano (DMDCS) in cloroformio e lasciata O La pipetta deve quindi essere rimossa e lasciata asciugare in ariaDeve essere autoclavato per la sterilizzazione. Attenzione: DMDCS e cloroformio sono altamente infiammabili, e la siliconizzazione deve essere effettuata in una cappa di fumo. </li></ol></li><li> Aspirare i primi 2 ml di supernatante e inserirlo in un nuovo tubo da 15 ml denominato &quot;raccolta&quot;. </li><li> Ripetere i passaggi 4.7.3-4.7.5 due volte supplementari (il tubo di raccolta delle cellule dovrebbe avere 6 ml alla fine). </li><li> Trasferire lentamente il contenuto del tubo di raccolta nel tubo di gradiente preparato al punto 4.2, evitando la rottura del gradiente. </li></ol></li><li> A questo punto, rimuovere il mezzo neurobasale già preparato ( Tabella 1 ) dal frigorifero e lasciarlo riscaldare in un bagno a 37 ° C. </li><li> Purificare i neuroni. <ol><li> Centrifugare il tubo di gradiente per 15 minuti a 800 xg e 22 ° C. </li><li> Raccogliere lo strato desiderato con una pipetta ( figura 2 ) e collocare in un nuovo tubo da 15 ml. Per la più alta purezza della neuroIsolare ( cioè &gt; 90%), raccogliere lo strato 3. Per ottenere una maggiore resa con meno purezza ( es. &gt; 70-80%), raccogliete i livelli 2 e 3. </li><li> Diluire il gradiente di densità aggiungendo 5 mL di HABG ai nuovi strati raccolti. </li><li> Centrifugare a 200 xg per 2 min a 22 ° C. </li><li> Scartare il surnatante, ri-sospendere in 5 mL di HABG e far scorrere il pellet per sospendere le cellule. </li><li> Centrifugare per 2 min 200 xg a 22 ° C. </li><li> Scartare il surnatante, risospendere in 3 ml di mezzo neurobasale e far scattare il pellet per riposizionare le cellule. </li></ol></li><li> Conte le celle. <ol><li> Prendere 10 μL della soluzione, ora con le cellule in mezzo neurobasale, e mescolare con 10 μL di Trypan blu. </li><li> Mettere 10 μL di questa miscela in una camera di conteggio del vetro. </li><li> Utilizzando una camera standard di conteggio del vetro conteggiate il numero di celle in ciascuno dei quattro quadranti 4 x 4. Aggiungere tutte le celle contate (n), multiPelo per 2 (fattore di diluizione), divide per 4 (numero di quadranti contati), moltiplica per 3 (volume di media neurobasale) e moltiplica per 10 4 per ottenere la concentrazione di cellule in cellule / ml. <img alt="Equazione" src="/files/ftp_upload/55856/55856eq0.jpg" /></li></ol></li><li> Seme le cellule sulle piastre di coltura <ol><li> Diluire la sospensione cellulare a 300.000 cellule in 1 mL di mezzo neurobasale. NOTA: Sulla base della concentrazione delle cellule ottenute nelle fasi precedenti, viene aggiunto un supporto neurobasale supplementare per ottenere una concentrazione finale di 3 x 10 5 cellule / mL. L&#39;equazione è C 1 V 1 = C 2 V 2 , dove C 1 è la concentrazione iniziale delle cellule ottenute dal raccolto; V 1 è 3 mL; E C 2 è 3 * 10 5 cellule / ml, come sopra discusso. L&#39;equazione viene risolta per V 2 . Aggiungere il volume del mezzo neurobasale necessario per rendere uguale il volume totale delle cellule in soluzioneV 2 . </li><li> Agitare delicatamente per distribuire le cellule in soluzione e aggiungere 1 mL ad ogni pozzetto nelle piastre ricoperte a 24 pozzetti. </li></ol></li></ol>

<ol>
	<li>Qayumi, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+model+for+investigation+of+spinal+cord+injury+caused+by+aortic+cross-clamping.">Animal model for investigation of spinal cord injury caused by aortic cross-clamping.</a> J Invest Surg. 10 (1-2), 47-52 (1997).</li><li>Fang, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ischemic+preconditioning+protects+against+spinal+cord+ischemia-reperfusion+injury+in+rabbits+by+attenuating+blood+spinal+cord+barrier+disruption.">Ischemic preconditioning protects against spinal cord ischemia-reperfusion injury in rabbits by attenuating blood spinal cord barrier disruption.</a> Int J Mol Sci. 14 (5), 10343-10354 (2013).</li><li>Taira, Y., Marsala, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+proximal+arterial+perfusion+pressure+on+function,+spinal+cord+blood+flow,+and+histopathologic+changes+after+increasing+intervals+of+aortic+occlusion+in+the+rat.">Effect of proximal arterial perfusion pressure on function, spinal cord blood flow, and histopathologic changes after increasing intervals of aortic occlusion in the rat.</a> Stroke. 27 (10), 1850-1858 (1996).</li><li>Stoppini, L., Buchs, P. -. A., Muller, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+organotypic+cultures+of+nervous+tissue.">A simple method for organotypic cultures of nervous tissue.</a> J Neurosci Methods. 37 (2), 173-182 (1991).</li><li>Ahlemeyer, B., Baumgart-Vogt, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+protocols+for+the+simultaneous+preparation+of+primary+neuronal+cultures+of+the+neocortex,+hippocampus+and+cerebellum+from+individual+newborn+(P0.+5)+C57Bl/6J+mice.">Optimized protocols for the simultaneous preparation of primary neuronal cultures of the neocortex, hippocampus and cerebellum from individual newborn (P0. 5) C57Bl/6J mice.</a> J Neurosci Methods. 149 (2), 110-120 (2005).</li><li>Brewer, G. J., Torricelli, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+adult+neurons+and+neurospheres.">Isolation and culture of adult neurons and neurospheres.</a> Nat Protoc. 2 (6), 1490-1498 (2007).</li><li>Banker, G. A., Cowan, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+hippocampal+neurons+in+dispersed+cell+culture.">Rat hippocampal neurons in dispersed cell culture.</a> Brain Res. 126 (3), 397-425 (1977).</li><li>Graber, D. J., Harris, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+culture+of+spinal+motor+neurons+from+rat+embryos.">Purification and culture of spinal motor neurons from rat embryos.</a> Cold Spring Harb Protoc. 2013 (4), 319-326 (2013).</li><li>Anderson, K. N., Potter, A. C., Piccenna, L. G., Quah, A. K., Davies, K. E., Cheema, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+motor+neurons+from+the+newborn+mouse+spinal+cord.">Isolation and culture of motor neurons from the newborn mouse spinal cord.</a> Brain Res Prot. 12 (3), 132-136 (2004).</li><li>Gingras, M., Gagnon, V., Minotti, S., Durham, H. D., Berthod, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+protocols+for+isolation+of+primary+motor+neurons,+astrocytes+and+microglia+from+embryonic+mouse+spinal+cord.">Optimized protocols for isolation of primary motor neurons, astrocytes and microglia from embryonic mouse spinal cord.</a> J Neurosci Methods. 163 (1), 111-118 (2007).</li><li>Brewer, G. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+survival+of+hippocampal+neurons+in+B27-supplemented+Neurobasal,+a+new+serum-free+medium+combination.">Optimized survival of hippocampal neurons in B27-supplemented Neurobasal, a new serum-free medium combination.</a> J Neurosci Res. 35 (5), 567-576 (1993).</li><li>Fang, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ischemic+preconditioning+protects+against+spinal+cord+ischemia-reperfusion+injury+in+rabbits+by+attenuating+blood+spinal+cord+barrier+disruption.">Ischemic preconditioning protects against spinal cord ischemia-reperfusion injury in rabbits by attenuating blood spinal cord barrier disruption.</a> Int J Mol Sci. 14 (5), 10343-10354 (2013).</li><li>Su, M., Zhong, W., Ren, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dose-dependent+protection+of+reseveratrol+against+spinal+cord+ischemic-reperfusion+injury+in+rats.">Dose-dependent protection of reseveratrol against spinal cord ischemic-reperfusion injury in rats.</a> Trop J Pharm Res. 15 (6), 1225-1233 (2016).</li><li>Haapanen, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remote+ischemic+preconditioning+protects+the+spinal+cord+against+ischemic+insult:+An+experimental+study+in+a+porcine+model.">Remote ischemic preconditioning protects the spinal cord against ischemic insult: An experimental study in a porcine model.</a> J Thorac Cardiovasc Surg. 151 (3), 777-785 (2016).</li><li>Conrad, M. F., Ye, J. Y., Chung, T. K., Davison, J. K., Cambria, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spinal+cord+complications+after+thoracic+aortic+surgery:+long-term+survival+and+functional+status+varies+with+deficit+severity.">Spinal cord complications after thoracic aortic surgery: long-term survival and functional status varies with deficit severity.</a> J Vasc Surg. 48 (1), 47-53 (2008).</li><li>Wong, D. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delayed+spinal+cord+deficits+after+thoracoabdominal+aortic+aneurysm+repair.">Delayed spinal cord deficits after thoracoabdominal aortic aneurysm repair.</a> Ann Thorac Surg. 83 (4), 1345-1355 (2007).</li><li>Freeman, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alpha-2+agonist+attenuates+ischemic+injury+in+spinal+cord+neurons.">Alpha-2 agonist attenuates ischemic injury in spinal cord neurons.</a> J Surg Res. 195 (1), 21-28 (2015).</li><li>Freeman, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spinal+cord+protection+via+alpha-2+agonist-mediated+increase+in+glial+cell-line-derived+neurotrophic+factor.">Spinal cord protection via alpha-2 agonist-mediated increase in glial cell-line-derived neurotrophic factor.</a> J Thorac Cardiovasc Surg. 149 (2), 578-586 (2015).</li></ol>

Gli autori non hanno alcuna divulgazione.

Questa tecnica consente la cultura affidabile dei neuroni del midollo spinale. Una volta raggiunta la conoscenza della tecnica, ci vogliono circa 3,5 ore per completare. Siamo stati in grado di effettuare l&#39;isolamento dei neuroni da 2 cucciolate separate (16 topi totali) in circa 4 h. Il passo fondamentale della fattibilità è poter estrarre efficacemente i cavi spinali dai topi. La resa consente di plating diversi pozzetti e per la capacità di testare i neuroni in varie condizioni. Siamo stati in grado di trattar...

<html> La comprensione della patologia del midollo spinale richiede l&#39;uso di vari modelli, sia a livello macroscopico che microscopico. I modelli animali grandi e piccoli 1 , 2 , 3 sono usati per le indagini in vivo di malattie del midollo spinale e lesioni. Mentre studia questi problemi in vivo ha i suoi meriti, l&#39;analisi del midollo spinale è limitata all&#39;intero homogenato del midollo spinale o alle sezioni del tessuto 4 . Ciò crea qualche ambiguità quando si cerca di isolare risposte e obiettivi specifici nel midollo spinale tra i neuroni residenti e la loro regione circostante. La crescente disponibilità di topi manipolati geneticamente permette di effettuare approfondite indagini sulla biologia a livello cellulare e molecolare. Pertanto, qui viene utilizzato un modello del topo neonatale, che consente lo studio delle proprietà uniche e della biologia dei neuroni del midollo spinale in vitro . L&#39;isolamento e la manutenzione dei neuroni in vitro non è particolarmente diretto. Esiste una abbondanza relativa di tecniche per l&#39;isolamento dei neuroni dal tessuto corticale dei roditori adulti che sembrano provocare un numero considerevole di neuroni isolati ( ossia milioni) 5 , 6 , 7. Al contrario, la resa dei neuroni dal tessuto del midollo spinale è inferiore a 8 , 9 , 10 , in parte dovuta alla più piccola massa di tessuto. Inoltre, nei topi c&#39;è una relativa poca tecnica di isolamento dei neonatali I neuroni del midollo spinale ei metodi esistenti sono limitati da rese inferiori ai neuroni ( cioè centinaia) 9 o tecniche laboriose e pesanti che richiedono l&#39;isolamento dei topi embrionali 10 . In questo protocollo, noiUtilizzare una tecnica che consente l&#39;isolamento costoso ed efficace di un numero considerevole di neuroni dai cavi spinali di topi neonatali. Come è comune nelle tecniche precedentemente pubblicate, utilizziamo il papain come una proteasi enzimatica, che consente il rilascio di neuroni dal tessuto del midollo spinale 5 , 6 . Inoltre, utilizziamo un gradiente di densità per la separazione delle cellule raffinate, che è stato precedentemente dimostrato essere efficace 6 , 10 . Mentre il mezzo in cui le cellule sono incubate può variare, nella nostra esperienza e come pubblicato in precedenza 11 , l&#39;integrazione con supplemento di terreno di coltura fresco B27 è risultato essere critico per la longevità del neurone. I neuroni sono tipicamente vitali per un massimo di 10 giorni, permettendo di eseguire il trattamento.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Hibernate A Medium - 500 mL</td>
			<td style="width:116px;">Thermo-Fisher</td>
			<td style="width:107px;">A1247501</td>
			<td style="width:805px;">https://www.thermofisher.com/order/catalog/product/A1247501</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hibernate A Minus Calcium - 500 mL</td>
			<td style="width:116px;">Brainbits</td>
			<td>HA-Ca</td>
			<td>http://www.brainbitsllc.com/hibernate-a-minus-calcium/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glutamax 100X - 100 mL</td>
			<td style="width:116px;">Thermo-Fisher</td>
			<td>35050061</td>
			<td>https://www.thermofisher.com/order/catalog/product/35050079</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">B27 Supplement 50X - 10 mL</td>
			<td style="width:116px;">Thermo-Fisher</td>
			<td>17504044</td>
			<td>https://www.thermofisher.com/order/catalog/product/17504044</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Papain, Lyophilized - 100 mg</td>
			<td style="width:116px;">Worthington</td>
			<td>LS003119</td>
			<td>http://www.worthington-biochem.com/pap/cat.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Neurobasal A Medium - 500 mL</td>
			<td style="width:116px;">Thermo-Fisher</td>
			<td>10888022</td>
			<td>https://www.thermofisher.com/order/catalog/product/10888022</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td style="width:116px;">Thermo-Fisher</td>
			<td>15140122</td>
			<td>https://www.thermofisher.com/order/catalog/product/15140122</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Poly-D-lysine hydrobromide - 5 mg</td>
			<td style="width:116px;">Sigma-Aldrich</td>
			<td>P6407-5MG</td>
			<td>http://www.sigmaaldrich.com/catalog/product/sigma/p6407?lang=en&#38;region=US</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse Laminin - 1 mg</td>
			<td style="width:116px;">Thermo-Fisher</td>
			<td>23017015</td>
			<td>https://www.thermofisher.com/order/catalog/product/23017015</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypan Blue - 20 mL</td>
			<td style="width:116px;">Sigma-Aldrich</td>
			<td>T8154-20ML</td>
			<td>http://www.sigmaaldrich.com/catalog/product/sigma/t8154?lang=en&#38;region=US</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">OptiPrep Density Gradient Medium - 250 mL</td>
			<td style="width:116px;">Sigma-Aldrich</td>
			<td>D1556-250ML</td>
			<td>http://www.sigmaaldrich.com/catalog/product/sigma/d1556?lang=en&#38;region=US</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dichlorodimethylsilane (DMDCS, Sigma Silicoat)</td>
			<td style="width:116px;">Sigma-Aldrich</td>
			<td>440272-100ML</td>
			<td>http://www.sigmaaldrich.com/catalog/product/aldrich/440272?lang=en&#38;region=US</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chloroform</td>
			<td style="width:116px;">Sigma-Aldrich</td>
			<td>288306-1L</td>
			<td>http://www.sigmaaldrich.com/catalog/product/sial/288306?lang=en&#38;region=US</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Glass Pippette - 9&#34;</td>
			<td style="width:116px;">Sigma-Aldrich</td>
			<td style="width:107px;">13-678-20C</td>
			<td>http://www.sigmaaldrich.com/catalog/product/sigma/cls7095d9?lang=en&#38;region=US</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pipette bulb - 5 mL</td>
			<td style="width:116px;">Sigma-Aldrich</td>
			<td>Z186678-3EA</td>
			<td>http://www.sigmaaldrich.com/catalog/product/aldrich/z186678?lang=en&#38;region=US&#38;cm_sp=Insite-_-prodRecCold_xviews-_-prodRecCold10-1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BRAND&#174;&#160;Petri dish, glass - 60x15 mm</td>
			<td style="width:116px;">Sigma-Aldrich</td>
			<td>BR455717-10EA</td>
			<td>http://www.sigmaaldrich.com/catalog/product/aldrich/br455717?lang=en&#38;region=US</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile 24 Well Cell Culture Plate</td>
			<td style="width:116px;">Sigma-Aldrich</td>
			<td>M8812-100EA</td>
			<td>http://www.sigmaaldrich.com/catalog/product/sigma/m8812?lang=en&#38;region=US</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Hausser Hemacytometer (glass counting chamber)</td>
			<td style="width:116px;">Fischer Scientific</td>
			<td>02-671-6</td>
			<td>https://www.fishersci.com/shop/products/hausser-bright-line-phase-hemacytometer-hemacytometer/026716</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glass Slides - 12 mm sterile cover glass - uncoated</td>
			<td>Neuvitro</td>
			<td>GG-12-1.5-Pre</td>
			<td>http://www.neuvitro.com/german-coverslip-12mm-diameter.htm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NeuN Rabbit Monoclonal Antibody - 100 &#181;L</td>
			<td style="width:116px;">Abcam</td>
			<td>ab177487</td>
			<td>After fixing in paraformaldehyde and blocking with 5% BSA, cells on a 12 mm coverslip were incubated in the antibody diluded to 1:200 for 18 hours in 4 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MAP-2 Mouse Monoclonal Antibody - 50 &#181;L</td>
			<td style="width:116px;">Abcam</td>
			<td>ab11267</td>
			<td>After fixing in paraformaldehyde and blocking with 5% BSA, cells on a 12 mm coverslip were incubated in the antibody diluded to 1:500 for 18 hours in 4 &#176;C</td>
		</tr>
	</tbody>
</table>

spinal cord neurons isolation and culture from neonatal mice

Presentiamo un protocollo per l&#39;isolamento e la cultura dei neuroni del midollo spinale. I neuroni sono ottenuti da topi neonatali C57BL / 6 e sono isolati nel giorno postnatalo 1-3. Un cucciolo di topo, di solito 4-10 cuccioli nati da una coppia di allevamento, viene raccolto per un esperimento e le corde spinali vengono raccolte individualmente da ciascun mouse dopo eutanasia con isoflurano. La colonna vertebrale viene disseccata e poi il midollo spinale viene rilasciato dalla colonna. I cavi spinali vengono quindi macinati per aumentare la superficie di consegna per una proteasi enzimatica che consente di liberare i neuroni e altre cellule dal tessuto. La triturazione viene poi usata per liberare le cellule in soluzione. Questa soluzione viene successivamente frazionata in un gradiente di densità per separare le varie cellule in soluzione, consentendo il isolamento dei neuroni. Circa 1-2,5 x 10 6 neuroni possono essere isolati da un gruppo di letti. I neuroni vengono poi seminati su pozzetti rivestiti di facto adesivoR che permettono una corretta crescita e maturazione. I neuroni richiedono circa 7 giorni per raggiungere la maturità nel mezzo di crescita e coltura e possono poi essere utilizzati per il trattamento e l&#39;analisi.

Utilizzando questa tecnica, una singola lettiera (4-10 cuccioli) consente l&#39;isolamento di 1-2,5 10 neuroni adatti per l&#39;invaso su piastre di coltura. Tipicamente, 4-8 pozzetti vengono seminati alla concentrazione menzionata sopra ( cioè 300.000 cellule / ml). La Figura 3 mostra l&#39;apparenza dei neuroni a questa concentrazione dopo una settimana di coltura a microscopia leggera di ingrandimento ( a ) e ad alta ( b...

Watch this Scientific Journal Video about Spinal Cord Neurons Isolation and Culture from Neonatal Mice at JoVE.com

Spinal Cord Neurons Isolation and Culture from Neonatal Mice

Questo studio presenta una tecnica per l&#39;isolamento dei neuroni da topi neonatali WT. Richiede l&#39;attenta disezione del midollo spinale dal topo neonatale, seguita dalla separazione dei neuroni dal tessuto del midollo spinale attraverso la scissione meccanica ed enzimatica.

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 ...

spinal-cord-neurons-isolation-and-culture-from-neonatal-mice

Research

JoVE Journal

Neuroscience

19.0K Views.  University of Colorado Anschutz Medical Campus.  Questo studio presenta una tecnica per l'isolamento dei neuroni da topi neonatali WT. Richiede l'attenta disezione del midollo spinale dal topo neonatale, seguita dalla separazione dei neuroni dal tessuto del midollo spinale attraverso la scissione meccanica ed enzimatica. 

Video: Spinal Cord Neurons Isolation and Culture from Neonatal Mice

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

Lateral Fluid Percussion: Model of Traumatic Brain Injury in Mice

Traumatic brain injury (TBI) research has attained renewed momentum due to the increasing awareness of head injuries, which result in morbidity and mortality. Based on the nature of primary injury following TBI, complex and heterogeneous secondary consequences result, which are followed by regenerative processes 1,2. Primary injury can be induced by a direct contusion to the brain from skull fracture or from shearing and stretching of tissue causing displacement of brain due to movement 3,4. The resulting hematomas and lacerations cause a vascular response 3,5, and the morphological and functional damage of the white matter leads to diffuse axonal injury 6-8. Additional secondary changes commonly seen in the brain are edema and increased intracranial pressure 9. Following TBI there are microscopic alterations in biochemical and physiological pathways involving the release of excitotoxic neurotransmitters, immune mediators and oxygen radicals 10-12, which ultimately result in long-term neurological disabilities 13,14. Thus choosing appropriate animal models of TBI that present similar cellular and molecular events in human and rodent TBI is critical for studying the mechanisms underlying injury and repair.
Various experimental models of TBI have been developed to reproduce aspects of TBI observed in humans, among them three specific models are widely adapted for rodents: fluid percussion, cortical impact and weight drop/impact acceleration 1. The fluid percussion device produces an injury through a craniectomy by applying a brief fluid pressure pulse on to the intact dura. The pulse is created by a pendulum striking the piston of a reservoir of fluid. The percussion produces brief displacement and deformation of neural tissue 1,15. Conversely, cortical impact injury delivers mechanical energy to the intact dura via a rigid impactor under pneumatic pressure 16,17. The weight drop/impact model is characterized by the fall of a rod with a specific mass on the closed skull 18. Among the TBI models, LFP is the most established and commonly used model to evaluate mixed focal and diffuse brain injury 19. It is reproducible and is standardized to allow for the manipulation of injury parameters. LFP recapitulates injuries observed in humans, thus rendering it clinically relevant, and allows for exploration of novel therapeutics for clinical translation 20.
We describe the detailed protocol to perform LFP procedure in mice. The injury inflicted is mild to moderate, with brain regions such as cortex, hippocampus and corpus callosum being most vulnerable. Hippocampal and motor learning tasks are explored following LFP.

Lateral fluid percussion (LFP), an established model of traumatic brain injury in mice, is demonstrated. LFP fulfills three major criteria for animal models: validity, reliability and clinical relevance. The procedure, consisting of surgical craniotomy, fixation of hub followed by induction of injury, resulting in focal and diffuse injuries, is described.

Fluido laterale Percussioni: Modello delle lesioni cerebrali traumatiche nei topi

Traumatic brain injury (TBI) research has attained renewed momentum due to the increasing awareness of head injuries, which result in morbidity and ...

Isolation and Culture of Hippocampal Neurons from Prenatal Mice

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual neurons, researchers are able to analyze properties related to cellular trafficking, cellular structure and individual protein localization using a variety of biochemical techniques. Results from such experiments are critical for testing theories addressing the neural basis of memory and learning. However, unambiguous results from these forms of experiments are predicated on the ability to grow neuronal cultures with minimum contamination by other brain cell types. In this protocol, we use specific media designed for neuron growth and careful dissection of embryonic hippocampal tissue to optimize growth of healthy neurons while minimizing contaminating cell types (i.e. astrocytes). Embryonic mouse hippocampal tissue can be more difficult to isolate than similar rodent tissue due to the size of the sample for dissection. We show detailed dissection techniques of hippocampus from embryonic day 19 (E19) mouse pups. Once hippocampal tissue is isolated, gentle dissociation of neuronal cells is achieved with a dilute concentration of trypsin and mechanical disruption designed to separate cells from connective tissue while providing minimum damage to individual cells. A detailed description of how to prepare pipettes to be used in the disruption is included. Optimal plating densities are provided for immuno-fluorescence protocols to maximize successful cell culture. The protocol provides a fast (approximately 2 hr) and efficient technique for the culture of neuronal cells from mouse hippocampal tissue.

We provide a protocol for the culture of highly purified hippocampal neurons from prenatal mouse brains without the use of a feeder glial cell layer.

Isolamento e la coltura di neuroni ippocampali da topi prenatali

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual ...

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, ...

Isolation and Culture of Dissociated Sensory Neurons From Chick Embryos

Neurons are multifaceted cells that carry information essential for a variety of functions including sensation, motor movement, learning, and memory. Studying neurons in vivo can be challenging due to their complexity, their varied and dynamic environments, and technical limitations. For these reasons, studying neurons in vitro can prove beneficial to unravel the complex mysteries of neurons. The well-defined nature of cell culture models provides detailed control over environmental conditions and variables. Here we describe how to isolate, dissociate, and culture primary neurons from chick embryos. This technique is rapid, inexpensive, and generates robustly growing sensory neurons. The procedure consistently produces cultures that are highly enriched for neurons and has very few non-neuronal cells (less than 5%). Primary neurons do not adhere well to untreated glass or tissue culture plastic, therefore detailed procedures to create two distinct, well-defined laminin-containing substrata for neuronal plating are described. Cultured neurons are highly amenable to multiple cellular and molecular techniques, including co-immunoprecipitation, live cell imagining, RNAi, and immunocytochemistry. Procedures for double immunocytochemistry on these cultured neurons have been optimized and described here.

Cell culture models provide detailed control over environmental conditions and thus provide a powerful platform to elucidate numerous aspects of neuronal cell biology. We describe a rapid, inexpensive, and reliable method to isolate, dissociate, and culture sensory neurons from chick embryos. Details of substrata preparation and immunocytochemistry are also provided.

Isolamento e Cultura della dissociato neuroni sensoriali da embrioni di gallina

Neurons are multifaceted cells that carry information essential for a variety of functions including sensation, motor movement, learning, and memory. ...

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing organisms, combined with the embryonic optical clarity, served as initial compelling attributes of this model. Over the past two decades, the success of this model has been further propelled by its amenability to large-scale mutagenesis screens and by the ease of transgenesis. More recently, gene-editing approaches have extended the power of the model.
For neurodevelopmental studies, the zebrafish embryo and larva provide a model to which multiple methods can be applied. Here, we focus on methods that allow the study of an essential property of neurons, electrical excitability. Our preparation for the electrophysiological study of zebrafish spinal neurons involves the use of veterinarian suture glue to secure the preparation to a recording chamber. Alternative methods for recording from zebrafish embryos and larvae involve the attachment of the preparation to the chamber using a fine tungsten pin1,2,3,4,5. A tungsten pin is most often used to mount the preparation in a lateral orientation, although it has been used to mount larvae dorsal-side up4. The suture glue has been used to mount embryos and larvae in both orientations. Using the glue, a minimal dissection can be performed, allowing access to spinal neurons without the use of an enzymatic treatment, thereby avoiding any resultant damage. However, for larvae, it is necessary to apply a brief enzyme treatment to remove the muscle tissue surrounding the spinal cord. The methods described here have been used to study the intrinsic electrical properties of motor neurons, interneurons, and sensory neurons at several developmental stages6,7,8,9.

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

This manuscript describes methods for electrophysiological recordings from spinal neurons of zebrafish embryos and larvae. The preparation maintains neurons in situ and often involves minimum dissection. These methods allow for the electrophysiological study of a variety of spinal neurons, from the initial electrical excitability acquisition through the early larval stages.

zebrafish<em&gt; In Situ</em&gt; Spinal Cord Preparazione per elettrofisiologiche Le registrazioni da spinale sensoriali e motori neuroni

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing ...

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

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

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

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

Imaging di attività neurale nella corteccia somatosensoriale primaria utilizzando Thy1-topi transgenici GCaMP6s

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

Preparation of Rhythmically-active In Vitro Neonatal Rodent Brainstem-spinal Cord and Thin Slice

Mammalian inspiratory rhythm is generated from a neuronal network in a region of the medulla called the preB&#246;tzinger complex (pBC), which produces a signal driving the rhythmic contraction of inspiratory muscles. Rhythmic neural activity generated in the pBC and carried to other neuronal pools to drive the musculature of breathing may be studied using various approaches, including en bloc nerve recordings and transverse slice recordings. However, previously published methods have not extensively described the brainstem-spinal cord dissection process in a transparent and reproducible manner for future studies. Here, we present a comprehensive overview of a method used to reproducibly cut rhythmically-active brainstem slices containing the necessary and sufficient neuronal circuitry for generating and transmitting inspiratory drive. This work builds upon previous brainstem-spinal cord electrophysiology protocols to enhance the likelihood of reliably obtaining viable and rhythmically-active slices for recording neuronal output from the pBC, hypoglossal premotor neurons (XII pMN), and hypoglossal motor neurons (XII MN). The work presented expands upon previous published methods by providing detailed, step-by-step illustrations of the dissection, from whole rat pup, to in vitro slice containing the XII rootlets.

This protocol both visually communicates the brainstem-spinal cord preparation and clarifies the preparation of brainstem transverse slices in a comprehensive step-by-step manner. It was designed to increase reproducibility and enhance the likelihood of obtaining viable, long lasting, rhythmically-active slices for recording neural output from the respiratory regions of the brainstem.

Preparazione di ritmicamente-attivo In Vitro neonatale del roditore del tronco cerebrale-spinale e fetta sottile

Mammalian inspiratory rhythm is generated from a neuronal network in a region of the medulla called the preB&#246;tzinger complex (pBC), which produces a ...

Isolation and Culture of Oculomotor, Trochlear, and Spinal Motor Neurons from Prenatal Islmn:GFP Transgenic Mice

Oculomotor neurons (CN3s) and trochlear neurons (CN4s) exhibit remarkable resistance to degenerative motor neuron diseases such as amyotrophic lateral sclerosis (ALS) when compared to spinal motor neurons (SMNs). The ability to isolate and culture primary mouse CN3s, CN4s, and SMNs would provide an approach to study mechanisms underlying this selective vulnerability. To date, most protocols use heterogeneous cell cultures, which can confound the interpretation of experimental outcomes. To minimize the problems associated with mixed-cell populations, pure cultures are indispensable. Here, the first protocol describes in detail how to efficiently purify and cultivate CN3s/CN4s alongside SMNs counterparts from the same embryos using embryonic day 11.5 (E11.5) IslMN:GFP transgenic mouse embryos. The protocol provides details on the tissue dissection and dissociation, FACS-based cell isolation, and in vitro cultivation of cells from CN3/CN4 and SMN nuclei. This protocol adds a novel in vitro CN3/CN4 culture system to existing protocols and simultaneously provides a pure species- and age-matched SMN culture for comparison. Analyses focusing on the morphological, cellular, molecular, and electrophysiological characteristics of motor neurons are feasible in this culture system. This protocol will enable research into the mechanisms that define motor neuron development, selective vulnerability, and disease.

Isolation and Culture of Oculomotor, Trochlear, and Spinal Motor Neurons from Prenatal Islmn:GFP Transgenic Mice

This work presents a protocol to yield homogeneous cell cultures of primary oculomotor, trochlear, and spinal motor neurons. These cultures can be used for comparative analyses of the morphological, cellular, molecular, and electrophysiological characteristics of ocular and spinal motor neurons.

Isolamento e coltura di Oculomotor, Trochlear, e MotoNeuroni Spinali da Prenatal Islmn:GFP Topi Transgenici

Oculomotor neurons (CN3s) and trochlear neurons (CN4s) exhibit remarkable resistance to degenerative motor neuron diseases such as amyotrophic lateral ...

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 ...