Questo lavoro è finanziato dalla scienze naturali e ingegneria Research Council del Canada (sovvenzione # 249669, # 482986 e) e Heart and Stroke Foundation of Canada (sovvenzione # 485508). Gli autori ringraziano Youssef El-Hayek e il dottor Qi Wan per la loro assistenza nello sviluppo dei protocolli sperimentali.

Tutte le procedure che comportano il trattamento degli animali sono state approvate dal Comitato Università di Toronto Animal Care in conformità con le linee guida istituzionali (protocollo n. 20009387). I metodi seguenti devono essere effettuate utilizzando strumenti sterili e tecniche, in una cappa a flusso laminare dove applicabile. Nel testo del protocollo sottostante, la frase &quot;EFH-SFM&quot; si riferisce a mezzi di informazione liberi siero integrato con il fattore di crescita epidermico, fondamentale fattore di crescita dei fibroblasti e eparina. EFH-SFM viene utilizzato quando studiando la galvanotaxis di NPCs indifferenziate perché questi mitogeni mantenere NPC nel loro stato indifferenziato 4. Quando si verifica il galvanotaxis di NPC indotte a differenziarsi in tipi di cellule mature, &quot;FBS-SFM&quot; si riferisce a mezzi di informazione liberi siero addizionato con 1% di siero fetale bovino. FBS promuove la differenziazione dei PNG in mature fenotipi neurali 5. 1. Isolamento e la coltura dei precursori neurali (nonmostrato nel video) <ol><li> Anestetizzare un topo CD1 (6-8 settimane) con isofluorano e sacrificio tramite dislocazione cervicale. </li><li> Spegnere la testa in etanolo al 70% e decapitare l&#39;animale con le forbici affilate dissezione. </li><li> Mentre si tiene la testa con una pinza chirurgica, togliere la pelle sulla superficie dorsale per esporre il cranio. </li><li> Utilizzando un bisturi e non. 11 lame, segnare il cranio al seno frontale lungo l&#39;asse medio-laterale, e anche lungo la sutura sagittale in direzione rostrocaudale. </li><li> Sbucciare le ossa parietali di distanza dalla testa senza. 7 pinze curve, facendo attenzione a non perforare il tessuto cerebrale. </li><li> Inserire una spatola sottile sotto il cervello a partire da sotto il cervelletto e avanzando verso i bulbi olfattivi. Tenendo il cranio in posizione con le pinze, tirare delicatamente il cervello dal cranio e subito mettere in ghiaccio freddo liquido cerebrospinale artificiale (vedi le ricette sotto). </li><li> Sotto un microscopio di dissezione, utilizzando steriledissezione forbici e pinze tagliare il cervello a metà lungo la linea mediana. Ruotare ciascun emisfero in modo che il mediale (taglio) di superficie rivolta verso l&#39;alto. </li><li> Selezionare un emisfero, e con la superficie mediale rivolta verso l&#39;alto, individuare il splenio del corpo calloso (regione posteriore del corpo calloso). </li><li> Effettuare un&#39;incisione dalla superficie della corteccia alla splenio del corpo calloso lungo l&#39;asse dorsoventrale. </li><li> Sbucciare la corteccia incisa verso il bulbo olfattivo per esporre le pareti mediale e laterale del ventricolo laterale. </li><li> Ruotare l&#39;emisfero in modo che la superficie dorsale rivolta verso l&#39;alto, e usare microforbici curve per tagliare e raccogliere le pareti esposte mediale e laterale, che contengono la regione periventricolare dove PNG risiedono 6. </li><li> Ripetere i passaggi 1,8-1,11 per l&#39;altro emisfero. </li><li> Pipettare il tessuto isolato in 7 ml di soluzione di tripsina (vedi ricetta sotto) in un tubo da 15 cc, e posizionare il tubo su un agitatore a 37 ° Cincubatore per 25 min. </li><li> Centrifugare la provetta a 1500 rpm per 5 minuti, aspirare il surnatante, risospendere il tessuto e in 2 ml di soluzione di tripsina (vedi ricetta sotto). </li><li> Triturare delicatamente il tessuto con un piccolo pozzo pipetta Pasteur 30-50 volte con attenzione per evitare bolle d&#39;aria. </li><li> Centrifugare la provetta a 1500 rpm per 5 minuti, aspirare il supernatante, e risospendere in 1-2 ml di SFM (vedi ricetta sotto) triturando il pellet 3-5 volte. </li><li> Centrifugare la provetta a 1.500 rpm per 3 minuti, aspirare il surnatante e risospendere in 1 ml di SFM + EFH. </li><li> Contare vivere densità delle cellule con un emocitometro e piastra le cellule in una beuta T25 coltura ad una densità di 10 cellule per microlitri di SFM + EFH. </li><li> Lasciare che la cultura di crescere per 7 giorni per ottenere indisturbati neurosfere primarie liberamente fluttuanti composti da NPC. </li></ol> 2. Galvanotaxis camera di preparazione <ol><li> Mettere 3 vetro quadrato no. 1 vetrini coprioggetto (22 x 22 x 0.17 mm) ina bottiglia di acido cloridrico 6N overnight. </li><li> Il giorno successivo, utilizzare una punta di diamante di vetro taglierina per tagliare sei strisce rettangolari (22 x 5 x 0.17 mm) di vetro da nessuna piazza. 1 coperchio scivola. </li><li> Trasferire i tagliandi quadrati lavata con acido e marze rettangolari in una cappa a flusso laminare. Lavare le strisce rettangolari e quadrate prima con etanolo al 70%, poi con coltura tissutale acqua di grado autoclavato, e lasciare asciugare su un Kim Wipe (per sterilità aggiunto, il vetro può essere consentito di aria secca). </li><li> Applicare grasso per vuoto al perimetro di una superficie dei vetrini quadrati, e sigillare a base di piatti di plastica 60 millimetri Petri. </li><li> Applicare grasso per vuoto lungo l&#39;asse di una superficie delle strisce rettangolari di vetro, e sigillare a bordi opposti dei vetrini quadrati (in modo che siano paralleli tra loro) in modo da creare una depressione centrale. </li><li> UV-sterilizzare le camere per almeno 15 min in cappa a flusso laminare. </li><li> Pipettare 250-300 microlitri di poli-L-lizzane sul canale centrale delle camere e incubare a temperatura ambiente per 2 h. </li><li> Circa 15 minuti prima della fine del periodo di incubazione, preparare la soluzione Matrigel (vedi ricetta sotto). </li><li> Aspirare il poli-L-lisina, lavare gli avvallamenti centrali con 1 ml di acqua autoclavato, e pipetta 250-300 microlitri di soluzione di Matrigel sulle depressioni centrali. </li><li> Incubare le camere a 37 ° C per 1 ora. </li><li> Aspirare la soluzione Matrigel e lavare delicatamente i trogoli centrali con 1-2 ml di SFM. </li><li> Pipettare 100 ul di EFH-SFM o FBS-SFM sulle depressioni centrali e trasferire le camere galvanotaxis sul palco di un microscopio di conteggio. </li><li> Pipetta 3-4 ml di neurosfere contenente coltura in piastre da 60 mm Petri e trasferire il piatto Petri alla fase del microscopio conteggio. </li><li> Ad un obiettivo di visualizzazione 5x, utilizzare una pipetta P10 trasferire 5-8 neurosfere interi (fino a quattro alla volta) sul canale centrale di ogni galvanotaxiscamera senza dissociare, e con cura diffondere le neurosfere di tutto il canale centrale, senza interrompere il substrato Matrigel. </li><li> Aggiungi un ulteriore 150-200 ml di EFH-SFM o FBS-SFM sulle depressioni centrali. </li><li> Trasferire le camere galvanotaxis in un 37 ° C, 5% di CO 2, 100% incubatore umidificato per 17-20 hr (se analizzando NPCs indifferenziate) per consentire la neurosfere di aderire al substrato Matrigel e dissociarsi in singole cellule, come mostrato in Figura 1 . Se l&#39;analisi di NPC differenziati, il periodo di incubazione dovrebbe essere esteso per 69-72 ore per permettere la differenziazione delle cellule. </li></ol> 3. Live cellulare Time-Lapse Imaging <ol><li> Permettono il sistema live cell imaging equilibrare a 37 ° C, 5% di CO 2 per un minimo di 30 minuti prima dell&#39;inizio del time-lapse. </li><li> Tagliate due 12 pezzi cm di 1 mm di diametro del filo d&#39;argento, l&#39;avvolgimento di bobine da un capo, e metterli in Clorox blisciviazione per 20 minuti per formare Ag / AgCl elettrodi. </li><li> Trasferire le camere galvanotaxis sul palco di un microscopio di conteggio e scegliere quale camera sarà utilizzato per live-cella di analisi di immagini di migrazione sulla base dei seguenti criteri: i) le neurosfere dovrebbe essere quasi completamente dissociato in singole cellule e ii) le cellule devono possedere morfologie rotondi con piccole-to-nessun processo si estende dai corpi cellulari. </li><li> Trasferire la camera selezionata galvanotaxis in una cappa a flusso laminare, con un no quadrato separato. 1 coperchio in vetro antiscivolo e grasso per vuoto. </li><li> Lavare il coperchio scivolare prima con etanolo 70%, poi con acqua autoclavata, e applicare una striscia di grasso per vuoto su due bordi paralleli del vetrino. </li><li> Aspirare il mezzo di coltura dalla vasca centrale della camera, quindi rapidamente posizionare il vetrino (grasso lato rivolto verso il basso) sulla camera di modo che il resto del grasso strisce sulle due strisce parallele rettangolari di vetro, creando un tettoalla camera. </li><li> Pipettare 100 ul di fresco EFH-SFM o FBS-SFM nella vasca centrale tramite azione capillare. </li><li> Utilizzare grasso per vuoto per creare bordi per piscine di terreni di coltura su ciascuna estremità del canale centrale, come mostrato in Figura 2. </li><li> Tagliate due 15 pezzi cm di tubo in PVC, e utilizzare una siringa da 10 cc con un ago da 18 gauge per iniettare con attenzione la soluzione di agarosio nel tubo, assicurando che nessun bolle si formano nei tubi, e permettere al gel di solidificare per 5 minuti. </li><li> Trasferire la camera galvanotaxis al sistema live imaging cellulare, insieme al gel di agarosio tubi, Ag / AgCl elettrodi, e un paio di piatti vuoti 60 millimetri Petri che verranno utilizzati come mezzi di coltura serbatoi e conterrà il Ag / AgCl elettrodi. Lasciare la camera galvanotaxis ad arrestarsi entro i 37 ° C, 5% di CO 2 ambiente per 20-30 min. </li><li> Durante questo tempo, preparare i coperchi dei 2 piatti vuoti Petri e il coperchio della scatola di Petri della camera galvanotaxis da parte di foriin loro con un utensile Dremel o simile come mostrato in Figura 3. </li><li> Pipettare 1-1,5 ml di EFH-SFM o FBS-SFM su entrambi i lati del canale centrale, e 7-8 ml di SFM in ciascuna piastra di Petri vuota. Mettere una piastra di Petri su ciascun lato del canale centrale della camera galvanotaxis e mettere un Ag / AgCl in ogni piatto. Colmano il divario tra la camera galvanotaxis e le piastre di Petri di stabilire una continuità elettrica con i ponti gel agarosio, come mostrato in Figura 4. </li><li> Collegare le Ag / AgCl elettrodi a un alimentatore esterno, con un amperometro in serie per misurare la corrente elettrica, e accendere l&#39;alimentazione. Utilizzare un voltmetro per misurare la forza del campo elettrico direttamente attraverso il trogolo centrale, e regolare l&#39;uscita dell&#39;alimentatore finché la forza desiderata campo elettrico viene ottenuto (i saggi effettuati in questo laboratorio utilizzano una forza dcEF di 250 mV / mm con corrente elettrica tra 1 e 1,5 mA). </li><li> Iniziativate il time-lapse modulo sul sistema live imaging cellulare, e che l&#39;esperimento possa funzionare per il tempo desiderato. Dopo il completamento del test, fissare le cellule in paraformaldeide al 4% per l&#39;analisi immunoistochimica standard. </li></ol>

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Nessun conflitto di interessi dichiarati.

Questo protocollo è stato adattato da ben consolidati metodi di studi precedenti 7-9. Camere Galvanotactic può essere costruito utilizzando una varietà di tecniche diverse, compresa la costruzione di un bicchiere ben separata per il confinamento di semina cellulare, o mediante ablazione laser CO 2 per microfabbricazione del trogolo centrale 10,11. Alcune tecniche possono essere più laborioso o costose di altre. Abbiamo descritto un test semplice e conveniente per la costruzione di un...

<table border="1"><tbody><tr><td height="44"> Nome della voce </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti </td></tr><tr><td></td><td></td><td></td><td> Neural Precursore isolamento cellulare </td></tr><tr><td> 2M NaCl </td><td> Sigma </td><td> S5886 </td><td> 11,688 g sciolti in 100 ml di dH 2 O </td></tr><tr><td> KCl 1M </td><td> Sigma </td><td> P5405 </td><td> 7,456 g sciolti in 100 ml di dH 2 O </td></tr><tr><td> 1M MgCl 2 </td><td> Sigma </td><td> M2393 </td><td> 20,33 g sciolti in 100 ml di dH 2 O </td></tr><tr><td> 155 mm NaHCO 3 </td><td> Sigma </td><td> S5761 </td><td> 1,302 g sciolti in 100 ml di dH 2 O </td></tr><tr><td> Glucosio 0.5M </td><td> Sigma </td><td> G6152 </td><td> 9,01 g sciolti in 100ml di dH 2 O </td></tr><tr><td> 108 mM CaCl 2 </td><td> Sigma </td><td> C7902 </td><td> 1,59 g sciolti in 100 ml di dH 2 O </td></tr><tr><td> Penicillina-streptomicina </td><td> Gibco </td><td> 15070 </td><td></td></tr><tr><td> Pancreas tripsina bovina </td><td> Sigma </td><td> T1005 </td><td></td></tr><tr><td> Pecora testicoli ialuronidasi </td><td> Sigma </td><td> H6254 </td><td></td></tr><tr><td> Acido Kynurenic </td><td> Sigma </td><td> K3375 </td><td></td></tr><tr><td> Ovomucoid inibitore della tripsina </td><td> Worthington </td><td> LS003086 </td><td></td></tr><tr><td> DMEM </td><td> Invitrogen </td><td> 12100046 </td><td></td></tr><tr><td> F12 </td><td> Invitrogen </td><td> 21700075 </td><td></td></tr><tr><td> 30% Glucosio </td><td> Sigma </td><td> G6152 </td><td></td></tr><tr><td> 7,5% NaHCO 3 </td><td> Sigma </td><td> S5761 </td><td></td></tr><tr><td> HEPES 1M </td><td> Sigma </td><td> H3375 </td><td> 23,83 g sciolti in 100 ml di dH 2 O </td></tr><tr><td> L-glutammina </td><td> Gibco </td><td> 25030 </td><td></td></tr><tr><td> FEG </td><td> Invitrogen </td><td> PMG8041 </td><td> Ricostituire in 1 ml di miscela di ormoni e aliquotare in 20 unità pl. </td></tr><tr><td> FGF </td><td> Invitrogen </td><td> PHG0226 </td><td> Ricostituire in 0,5 ml di miscela di ormoni e aliquotare in 20 unità pl. </td></tr><tr><td> Eparina </td><td> Sigma </td><td> H3149 </td><td></td></tr><tr><td> Apo-transferrina </td><td> R &amp; D Systems </td><td> 3188-A </td><td> 0,1 g sciolti in 4 ml di dH 2 0 </td></tr><tr><td> Putrescina </td><td> Sigma </td><td> P7505 </td><td> Sciogliere 9,61 mg in Apo-transferrina cosìluzione </td></tr><tr><td> Insulina </td><td> Sigma </td><td> I5500 </td><td> Sciogliere 25 mg in 0,5 ml di HCl 0,1 N e aggiungere 3,5 ml di dH 2 0 </td></tr><tr><td> Selenio </td><td> Sigma </td><td> S9133 </td><td></td></tr><tr><td> Progesterone </td><td> Sigma </td><td> P6149 </td><td></td></tr><tr><td> Strumenti di dissezione standard </td><td> Strumenti Scienza Belle </td><td></td><td></td></tr><tr><td> Dissezione microscopio </td><td> Zeiss </td><td> Stemi 2000 </td><td></td></tr><tr><td></td><td></td><td></td><td> Galvanotaxis camera di preparazione </td></tr><tr><td> Quadrati di vetro di copertura diapositive </td><td> VWR </td><td> 16004 </td><td></td></tr><tr><td> Acido cloridrico 6N </td><td> VWR </td><td> BDH3204-1 </td><td></td></tr><tr><td> Vuoto Grasso per alte </td><td> Dow Corning </td><td></td><td></td></tr><tr><td> 60 millimetri Petri piatti </td><td> Fisher Scientific </td><td> 0875713A </td><td></td></tr><tr><td> Poli-L-lisina </td><td> Sigma </td><td> P4707 </td><td></td></tr><tr><td> Matrigel </td><td> BD Biosciences </td><td> 354234 </td><td> Scongelare e aliquotare in 150 unità pl </td></tr><tr><td> FBS </td><td> Invitrogen </td><td> 10082139 </td><td> Utilizzare solo se indurre il differenziamento NPC, altrimenti utilizzare SFM + terreni di coltura EFH come sopra indicato </td></tr><tr><td> Conteggio microscopio </td><td> Olimpo </td><td> CKX41 </td><td></td></tr><tr><td></td><td></td><td></td><td> Live cellulare Time-Lapse Imaging </td></tr><tr><td> Filo di argento </td><td> Alfa Aesar </td><td> 11434 </td><td></td></tr><tr><td> Ultrapura agarosio </td><td> Invitrogen </td><td> 15510-027 </td><td></td></tr><tr><td> Calore Inactivated FBS </td><td> Sigma </td><td> 16140071 </td><td></td></tr><tr><td> Tubi in PVC </td><td> Fisher Scientific </td><td> 80000006 </td><td> 3/32 &quot;ID x 5/32&quot; OD </td></tr><tr><td> Candeggina </td><td> Clorox </td><td></td><td></td></tr><tr><td> Siringa da 10 cc </td><td> BD </td><td> 309604 </td><td></td></tr><tr><td> 18 gauge </td><td> BD </td><td> 305195 </td><td></td></tr><tr><td> Dremel trapano </td><td> Dremel </td><td> Modello 750 </td><td></td></tr><tr><td> Microscopio invertito dotato di umidificata, camera incubata </td><td> Zeiss </td><td> Axiovert-200M </td><td></td></tr><tr><td></td><td></td><td></td><td> Ricette <table border="1"><tbody><tr><td> Voce </td><td> Volume </td></tr><tr><td> 2M NaCl </td><td> 6,2 ml </td></tr><tr><td>KCl 1M </td><td> 0,5 ml </td></tr><tr><td> 1M MgCl 2 </td><td> 0,32 ml </td></tr><tr><td> 155mm NaHCO 3 </td><td> 16,9 ml </td></tr><tr><td> Glucosio 1M </td><td> 1 ml </td></tr><tr><td> 108 mM CaCl 2 </td><td> 0,09256 ml </td></tr><tr><td> Penicillina-streptomicina </td><td> 1 ml </td></tr><tr><td> Acqua autoclavata </td><td> 74 ml </td></tr></tbody></table> Artificiale liquido cerebrospinale <table border="1"><tbody><tr><td> Voce </td><td> Volume o massa </td></tr><tr><td> Artificiale liquido cerebrospinale </td><td> 30 ml </td></tr><tr><td> Pancreas tripsina bovina </td><td> 40 mg </td></tr><tr><td> Pecora testicoli ialuronidasi </td><td> 22,8 mg </td></tr><tr><td> Acido Kynurenic </td><td> 5 mg </td></tr></tbody></table> &lt;strong&gt; Tripsina Solution <table border="1"><tbody><tr><td> Voce </td><td> Volume o massa </td></tr><tr><td> SFM </td><td> 15 ml </td></tr><tr><td> Ovomucoid inibitore della tripsina </td><td> 10 mg </td></tr></tbody></table> Inibitore della tripsina Solution <table border="1"><tbody><tr><td> Voce </td><td> Volume </td></tr><tr><td> Acqua autoclavata </td><td> 37 ml </td></tr><tr><td> 10X DMEM/F12 </td><td> 10 ml </td></tr><tr><td> 30% Glucosio </td><td> 2 ml </td></tr><tr><td> 7,5% NaHCO 3 </td><td> 1,5 ml </td></tr><tr><td> HEPES 1M </td><td> 0,5 ml </td></tr><tr><td> Transferrina, putrescina soluzione </td><td> 4 ml </td></tr><tr><td> 25 mg soluzione di insulina </td><td> 4 ml </td></tr><tr><td> Selenio </td><td> 100 &amp; mu; L </td></tr><tr><td> Progesterone </td><td> 100 pl </td></tr></tbody></table> Ormone Mix (100 ml totali, conservare a -20 ° C) <table border="1"><tbody><tr><td> Voce </td><td> Volume </td></tr><tr><td> Acqua autoclavata </td><td> 37,5 ml </td></tr><tr><td> DMEM/F12 10X (3:1) </td><td> 5 ml </td></tr><tr><td> 30% Glucosio </td><td> 1 ml </td></tr><tr><td> 7,5% NaHCO 3 </td><td> 0,75 ml </td></tr><tr><td> HEPES 1M </td><td> 0,25 ml </td></tr><tr><td> Ormone mix </td><td> 5 ml </td></tr><tr><td> L-glutammina </td><td> 0,5 ml </td></tr><tr><td> Penicillina-streptomicina </td><td> 0,5 ml </td></tr></tbody></table> Siero Free Media EFH-SFM: aggiungere 10 ml di EGF, 10 pl di FGF, e 3,66 ml di eparina FBS-SFM: aggiungere 0,5 ml di FBS <tabile confine = &quot;1&quot;&gt; <tbody><tr><td> Voce </td><td> Volume </td></tr><tr><td> Matrigel </td><td> 150 pl </td></tr><tr><td> SFM </td><td> 3,6 ml </td></tr></tbody></table> Matrigel Soluzione aliquota Matrigel deve essere collocato in una scatola di ghiaccio e lasciata scongelare lentamente 4-5 ore per formare un liquido viscoso prima della miscelazione con SFM. Ciò garantirà la formazione di uno strato di substrato liscio Matrigel. Se non scongelato lentamente, il substrato risultante conterrà ciuffi di Matrigel, eventualmente ostacolare la migrazione cellulare. <table border="1"><tbody><tr><td> Voce </td><td> Volume o massa </td></tr><tr><td> Ultrapura agarosio </td><td> 300 mg in 10 ml DDH 2 0 </td></tr><tr><td> SFM Calore FBS inattivato </td><td> 8 ml 2 ml </td></tr></tbody></table> MatrigelSoluzione Mix 8 ml di SFM con 2 ml di FBS inattivato al calore in un tubo 15 cc falcon. Mix agarosio con 10 ml DDH 2 0 in un matraccio di Erlenmeyer e riscaldare in un forno a microonde per 30 sec a 10 sec-intervalli, garantendo per rimuovere la soluzione dalla microonde dopo ogni intervallo di 10 secondi e mescolare. Dopo la finale di 10 secondi periodo di forno a microonde, mescolare la soluzione di agarosio con il SFM / soluzione di FBS e conservare in un bagno d&#39;acqua ° 57 C. </td></tr></tbody></table>

a galvanotaxis assay for analysis of neural precursor cell migration kinetics in an externally applied direct current electric field

La scoperta di staminali e cellule progenitrici (collettivamente chiamati cellule precursori neurali) (NPC) nel cervello dei mammiferi adulti ha portato ad un corpo di ricerca finalizzata ad utilizzare le proprietà multipotenti e proliferativi di queste cellule per lo sviluppo di strategie neurorigenerativo. Un punto critico per il successo di tali strategie è la mobilitazione di NPC verso un sito di lesione dopo trapianto esogena o per migliorare la risposta dei precursori endogeni che si trovano nella regione periventricolare del SNC. Di conseguenza, è essenziale per comprendere i meccanismi che promuovono, guidare, e migliorare la migrazione NPC. Il nostro lavoro si concentra su l&#39;utilizzo di campi elettrici a corrente diretta (dcEFs) per la promozione e la migrazione diretta NPC - un fenomeno noto come galvanotaxis. Campi elettrici endogeni fisiologici operato come spunti critici per la migrazione delle cellule durante il normale sviluppo e la riparazione delle ferite. Interruzione farmacologica dellatrans-neurale potenziale tubo in embrioni axolotl causa gravi malformazioni dello sviluppo 1. Nel contesto della guarigione delle ferite, il tasso di riparazione di cornea feriti è direttamente correlata con la grandezza del potenziale lesione epiteliale che sorge dopo la lesione, come mostrato dal miglioramento farmacologico o interruzione di questo dcEF 2-3. Abbiamo dimostrato che adulti NPC subependimali incontro a rapida e diretta migrazione catodiche in vitro quando esposti ad un dcEF applicata esternamente. In questo protocollo si descrivono le tecniche del nostro laboratorio per la creazione di un saggio galvanotaxis semplice ed efficace ad alta risoluzione, osservazione a lungo termine della traslocazione cellula diretto del corpo (migrazione) su una singola cellula. Questo saggio sarebbe adatto per indagare i meccanismi che regolano la trasduzione dcEF in motilità cellulare attraverso l&#39;uso di topi transgenici o knockout, brevi RNA interferenti, o agonisti dei recettori specifici / antagonisti.

Analisi cinematica rivela che in presenza di un mV / mm 250 dcEF, NPCs indifferenziate esporre galvanotaxis altamente orientati e rapido verso il catodo (figura 5A, Movie 1). In assenza di un dcEF, movimento casuale delle cellule si osserva (figura 5B, Movie 2). A questa forza di campo,&gt; 98% di NPCs indifferenziate migrare per l&#39;intero 6-8 hr per cui essi vengono esposte, e poiché le cellule morte non migrano questo suggerisce che essi rimangono vitali durante questo periodo, in...

Watch this Scientific Journal Video about A Galvanotaxis Assay for Analysis of Neural Precursor Cell Migration Kinetics in an Externally Applied Direct Current Electric Field at JoVE.com

A Galvanotaxis Assay for Analysis of Neural Precursor Cell Migration Kinetics in an Externally Applied Direct Current Electric Field

In questo protocollo si dimostra come costruire camere personalizzate che consentono l&#39;applicazione di un campo di corrente elettrica diretta a consentire time-lapse imaging del cervello adulto derivato neurale traslocazione cellula precursore durante galvanotaxis.

Un test Galvanotaxis Analisi della Cinetica precursori neurali migrazione cellulare in un campo applicato esternamente diretto Corrente elettrica

The discovery of neural stem and progenitor  cells (collectively termed neural precursor cells) (NPCs) in the adult  mammalian brain has led to a body of ...

a-galvanotaxis-assay-for-analysis-neural-precursor-cell-migration

Research

JoVE Journal

Neuroscience

12.4K Views.  University of Toronto. In questo protocollo si dimostra come costruire camere personalizzate che consentono l'applicazione di un campo di corrente elettrica diretta a consentire time-lapse imaging del cervello adulto derivato neurale traslocazione cellula precursore durante galvanotaxis.

Video: A Galvanotaxis Assay for Analysis of Neural Precursor Cell Migration Kinetics in an Externally Applied Direct Current Electric Field

Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). Furthermore, this serum-free culture system has also been employed to expand stem cells and determine their frequency from a variety of tumors and normal tissues. It has been shown recently that a one-to-one relationship does not exist between neurosphere formation and NSCs. This suggests that the NSA as currently applied, overestimates the frequency of NSCs in a mixed population of neural precursor cells isolated from both the embryonic and adult mammalian brain. This video practically demonstrates a novel collagen based semi- solid assay, the neural-colony forming cell assay (N-CFCA), which has the ability to discriminate stem from progenitor cells based on their long-term proliferative potential, and thus provides a method to enumerate NSC frequency. In the N-CFCA, colonies &ge;2 mm in diameter are derived from cells that meet all the functional criteria of a NSC, while colonies &lt; 2mm are derived from progenitors. The N-CFCA procedure can be used for cells prepared from different sources including primary and cultured adult or embryonic mouse CNS cells. Here we use cells prepared from passage one neurospheres generated from embryonic day 14 mice brain to perform N-CFCA. The cultures are replenished with proliferation medium every seven days for three weeks to allow the plated cells to exhibit their full proliferative potential and then the frequency of neural progenitor and bona fide neural stem cells is calculated respectively by counting the number of colonies that are &lt; 2mm and the ones that are &ge;2mm in reference to the number of cells that were initially plated.

This video protocol demonstrates how to discriminate and enumerate bona fide neural stem cells in a mixed population of neural precursor cells using the neural colony-forming cell assay.

Neural-formanti colonia saggio Cell: un test per discriminare Bona Fide cellule staminali neurali da cellule progenitrici neurali

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). ...

Ricerca

Neuroscienze

Analysis of Trunk Neural Crest Cell Migration using a Modified Zigmond Chamber Assay

Neural crest cells (NCCs) are a transient population of cells present in vertebrate development that emigrate from the dorsal neural tube (NT) after undergoing an epithelial-mesenchymal transition 1,2. Following EMT, NCCs migrate large distances along stereotypic pathways until they reach their targets. NCCs differentiate into a vast array of cell types including neurons, glia, melanocytes, and chromaffin cells 1-3. The ability of NCCs to reach and recognize their proper target locations is foundational for the appropriate formation of all structures containing trunk NCC-derived components 3. Elucidating the mechanisms of guidance for trunk NCC migration has therefore been a matter of great significance. Numerous molecules have been demonstrated to guide NCC migration 4. For instance, trunk NCCs are known to be repelled by negative guidance cues such as Semaphorin, Ephrin, and Slit ligands 5-8. However, not until recently have any chemoattractants of trunk NCCs been identified 9. 
Conventional in vitro approaches to studying the chemotactic behavior of adherent cells work best with immortalized, homogenously distributed cells, but are more challenging to apply to certain primary stem cell cultures that initially lack a homogenous distribution and rapidly differentiate (such as NCCs). One approach to homogenize the distribution of trunk NCCs for chemotaxis studies is to isolate trunk NCCs from primary NT explant cultures, then lift and replate them to be almost 100% confluent. However, this plating approach requires substantial amounts of time and effort to explant enough cells, is harsh, and distributes trunk NCCs in a dissimilar manner to that found in in vivo conditions. 
Here, we report an in vitro approach that is able to evaluate chemotaxis and other migratory responses of trunk NCCs without requiring a homogenous cell distribution. This technique utilizes time-lapse imaging of primary, unperturbed trunk NCCs inside a modified Zigmond chamber (a standard Zigmond chamber is described elsewhere10). By exposing trunk NCCs at the periphery of the culture to a chemotactant gradient that is perpendicular to their predicted natural directionality, alterations in migratory polarity induced by the applied chemotactant gradient can be detected. This technique is inexpensive, requires the culturing of only two NT explants per replicate treatment, avoids harsh cell lifting (such as trypsinization), leaves trunk NCCs in a more similar distribution to in vivo conditions, cuts down the amount of time between explantation and experimentation (which likely reduces the risk of differentiation), and allows time-lapse evaluation of numerous migratory characteristics.

An approach to analyze the migration of explanted cells (trunk neural crest cells) is described. This method is inexpensive, gentle, and capable of distinguishing chemotaxis from both chemokinesis and other influences on migratory polarity such as those derived from cell-cell interactions within the primary trunk neural crest cell culture.

Analisi della migrazione cresta neurale cellulare Trunk con un Modified Zigmond Assay Camera

Neural crest cells (NCCs) are a transient population of cells present in vertebrate development that emigrate from the dorsal neural tube (NT) after ...

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative response mounted by PNS neurons thereby possibly illuminating the failures of CNS regeneration1. Sciatic nerve crush (axonotmesis) is one of the most common models of peripheral nerve injury in rodents2. Crushing interrupts all axons but Schwann cell basal laminae are preserved so that regeneration is optimal3,4. This allows the investigator to study precisely the ability of a growing axon to interact with both the Schwann cell and basal laminae4. Rats have generally been the preferred animal models for experimental nerve crush. They are widely available and their lesioned sciatic nerve provides a reasonable approximation of human nerve lesions5,4. Though smaller in size than rat nerve, the mouse nerve has many similar qualities. Most importantly though, mouse models are increasingly valuable because of the wide availability of transgenic lines now allows for a detailed dissection of the individual molecules critical for nerve regeneration6, 7. Prior investigators have used multiple methods to produce a nerve crush or injury including simple angled forceps, chilled forceps, hemostatic forceps, vascular clamps, and investigator-designed clamps8,9,10,11,12. Investigators have also used various methods of marking the injury site including suture, carbon particles and fluorescent beads13,14,1. We describe our method to obtain a reproducibly complete sciatic nerve crush with accurate and persistent marking of the crush-site using a fine hemostatic forceps and subsequent carbon crush-site marking. As part of our description of the sciatic nerve crush procedure we have also included a relatively simple method of muscle whole mount we use to subsequently quantify regeneration.

In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.

Riproducibile nervo sciatico Crush mouse e successiva valutazione da parte della Rigenerazione intera analisi Muscle Monte

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

Analysis of Neural Crest Migration and Differentiation by Cross-species Transplantation

Avian embryos provide a unique platform for studying many vertebrate developmental processes, due to the easy access of the embryos within the egg. Chimeric avian embryos, in which quail donor tissue is transplanted into a chick embryo in ovo, combine the power of indelible genetic labeling of cell populations with the ease of manipulation presented by the avian embryo.
Quail-chick chimeras are a classical tool for tracing migratory neural crest cells (NCCs)1-3. NCCs are a transient migratory population of cells in the embryo, which originate in the dorsal region of the developing neural tube4. They undergo an epithelial to mesenchymal transition and subsequently migrate to other regions of the embryo, where they differentiate into various cell types including cartilage5-13, melanocytes11,14-20, neurons and glia21-32. NCCs are multipotent, and their ultimate fate is influenced by 1) the region of the neural tube in which they originate along the rostro-caudal axis of the embryo11,33-37, 2) signals from neighboring cells as they migrate38-44, and 3) the microenvironment of their ultimate destination within the embryo45,46. Tracing these cells from their point of origin at the neural tube, to their final position and fate within the embryo, provides important insight into the developmental processes that regulate patterning and organogenesis.
Transplantation of complementary regions of donor neural tube (homotopic grafting) or different regions of donor neural tube (heterotopic grafting) can reveal differences in pre-specification of NCCs along the rostro-caudal axis2,47. This technique can be further adapted to transplant a unilateral compartment of the neural tube, such that one side is derived from donor tissue, and the contralateral side remains unperturbed in the host embryo, yielding an internal control within the same sample2,47. It can also be adapted for transplantation of brain segments in later embryos, after HH10, when the anterior neural tube has closed47.
Here we report techniques for generating quail-chick chimeras via neural tube transplantation, which allow for tracing of migratory NCCs derived from a discrete segment of the neural tube. Species-specific labeling of the donor-derived cells with the quail-specific QCPN antibody48-56 allows the researcher to distinguish donor and host cells at the experimental end point. This technique is straightforward, inexpensive, and has many applications, including fate-mapping, cell lineage tracing, and identifying pre-patterning events along the rostro-caudal axis45. Because of the ease of access to the avian embryo, the quail-chick graft technique may be combined with other manipulations, including but not limited to lens ablation40, injection of inhibitory molecules57,58, or genetic manipulation via electroporation of expression plasmids59-61, to identify the response of particular migratory streams of NCCs to perturbations in the embryo's developmental program. Furthermore, this grafting technique may also be used to generate other interspecific chimeric embryos such as quail-duck chimeras to study NCC contribution to craniofacial morphogenesis, or mouse-chick chimeras to combine the power of mouse genetics with the ease of manipulation of the avian embryo.62

An approach for analyzing migration and eventual fate of avian neural crest cells in quail-chick chimeric embryos is described. This method is a simple and straightforward technique for tracing neural crest cells during migration and differentiation that are otherwise difficult to distinguish within an unmanipulated chick embryo.

Analisi della migrazione cresta neurale e la differenziazione delle specie da Cross-trapianto

Avian embryos provide a unique platform for studying many vertebrate developmental processes, due to the easy access of the embryos within the egg. ...

The Neuroblast Assay: An Assay for the Generation and Enrichment of Neuronal Progenitor Cells from Differentiating Neural Stem Cell Progeny Using Flow Cytometry

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of the central nervous system (CNS); namely, astrocytes, oligodendrocytes and neurons. These characteristics make neural stem and progenitor cells an invaluable renewable source of cells for in vitro studies such as drug screening, neurotoxicology and electrophysiology and also for cell replacement therapy in many neurological diseases. In practice, however, heterogeneity of NSC progeny, low production of neurons and oligodendrocytes, and predominance of astrocytes following differentiation limit their clinical applications. Here, we describe a novel methodology for the generation and subsequent purification of immature neurons from murine NSC progeny using fluorescence activated cell sorting (FACS) technology. Using this methodology, a highly enriched neuronal progenitor cell population can be achieved without any noticeable astrocyte and bona fide NSC contamination. The procedure includes differentiation of NSC progeny isolated and expanded from E14 mouse ganglionic eminences using the neurosphere assay, followed by isolation and enrichment of immature neuronal cells based on their physical (size and internal complexity) and fluorescent properties using flow cytometry technology. Overall, it takes 5-7 days to generate neurospheres and 6-8 days to differentiate NSC progeny and isolate highly purified immature neuronal cells.

This video protocol demonstrates a novel method for the generation and subsequent purification of neuronal progenitor cells from a renewable source of neural stem cells (NSCs) based on their physical (size and internal granularity) and fluorescent properties using flow cytometry technology.

Il saggio neuroblasti: un test per la generazione e arricchimento delle cellule progenitrici neuronali di differenziare i Progeny cellule staminali neurali in citometria a flusso

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

Isolamento e la coltura delle cellule della cresta neurale da Embryonic Murine tubo neurale

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors

Neural crest cells (NCC) are a transient and multipotent cell population that originates from the dorsal neural tube and migrates extensively throughout the developing vertebrate embryo. In addition to providing peripheral glia and neurons, NCC generate melanocytes as well as most of the cranio-facial skeleton. NCC migration and differentiation is controlled by a combination of their axial origin along the neural tube and their exposure to regionally distinct extracellular cues. Such contribution of extracellular ligands is especially evident during the formation of the enteric nervous system (ENS), a complex interconnected network of neural ganglia that locally controls (among other things) gut muscle movement and intestinal motility. Most of the ENS is derived from a small initial pool of NCC that undertake a long journey in order to colonize - in a rostral to caudal fashion - the entire length of the prospective gut. Among several signaling pathways known to influence enteric NCC colonization, GDNF/RET signaling is recognized as the most important. Indeed, spatiotemporally controlled secretion of the RET ligand GDNF by the gut mesenchyme is chiefly responsible for the attraction and guidance of RET-expressing enteric NCC to and within the embryonic gut. Here, we describe an ex vivo cell migration assay, making use of a transgenic mouse line possessing fluorescently labeled NCC, which allows precise quantification of enteric NCC migration potential in the presence of various growth factors, including GDNF.

We present an ex vivo cell migration assay that allows precise quantification of enteric neural crest cell migration potential in the presence of various growth factors.

Un saggio di migrazione cellulare quantitativa per murini enterici neurali Progenitori

Neural crest cells (NCC) are a transient and multipotent cell population that originates from the dorsal neural tube and migrates extensively throughout ...

Simultaneous Recordings of Cortical Local Field Potentials, Electrocardiogram, Electromyogram, and Breathing Rhythm from a Freely Moving Rat

Monitoring the physiological dynamics of the brain and peripheral tissues is necessary for addressing a number of questions about how the brain controls body functions and internal organ rhythms when animals are exposed to emotional challenges and changes in their living environments. In general experiments, signals from different organs, such as the brain and the heart, are recorded by independent recording systems that require multiple recording devices and different procedures for processing the data files. This study describes a new method that can simultaneously monitor electrical biosignals, including tens of local field potentials in multiple brain regions, electrocardiograms that represent the cardiac rhythm, electromyograms that represent awake/sleep-related muscle contraction, and breathing signals, in a freely moving rat. The recording configuration of this method is based on a conventional micro-drive array for cortical local field potential recordings in which tens of electrodes are accommodated, and the signals obtained from these electrodes are integrated into a single electrical board mounted on the animal's head. Here, this recording system was improved so that signals from the peripheral organs are also transferred to an electrical interface board. In a single surgery, electrodes are first separately implanted into the appropriate body parts and the target brain areas. The open ends of all of these electrodes are then soldered to individual channels of the electrical board above the animal's head so that all of the signals can be integrated into the single electrical board. Connecting this board to a recording device allows for the collection of all of the signals into a single device, which reduces experimental costs and simplifies data processing, because all data can be handled in the same data file. This technique will aid the understanding of the neurophysiological correlates of the associations between central and peripheral organs.

This study introduces a method for the simultaneous recording of local field potentials in the brain, electrocardiograms, electromyograms, and breathing signals of a freely moving rat. This technique, which reduces experimental costs and simplifies data analysis, will contribute to the understanding of the interactions between the brain and peripheral organs.

Registrazioni simultanee di potenziali di campo locale corticale, elettrocardiogramma, elettromiogramma e respirazione ritmo da un ratto liberamente commovente

Monitoring the physiological dynamics of the brain and peripheral tissues is necessary for addressing a number of questions about how the brain controls ...

Wide-field Single-photon Optical Recording in Brain Slices Using Voltage-sensitive Dye

Wide-field single photon voltage-sensitive dye (VSD) imaging of brain slice preparations is a useful tool to assess the functional connectivity in neural circuits. Due to the fractional change in the light signal, it has been difficult to use this method as a quantitative assay. This article describes special optics and slice handling systems, which render this technique stable and reliable. The present article demonstrates the slice handling, staining, and recording of the VSD-stained hippocampal slices in detail. The system maintains physiological conditions for a long time, with good staining, and prevents mechanical movements of the slice during the recordings. Moreover, it enables staining of slices with a small amount of the dye. The optics achieve high numerical aperture at low magnification, which allows recording of the VSD signal at the maximum frame rate of 10 kHz, with 100 pixel x 100-pixel spatial resolution. Due to the high frame rate and spatial resolution, this technique allows application of the post-recording filters that provide sufficient signal-to-noise ratio to assess the changes in neural circuits.

We introduce a reproducible and stable optical recording method for brain slices using voltage-sensitive dye. The article describes voltage-sensitive dye staining and recording of optical signals using conventional hippocampal slice preparations.

Registrazione ottica a singolo fotone a campo largo nelle sezioni cerebrali utilizzando Colorazione sensibile alla tensione

Wide-field single photon voltage-sensitive dye (VSD) imaging of brain slice preparations is a useful tool to assess the functional connectivity in neural ...

High-Content Screening Differentiation and Maturation Analysis of Fetal and Adult Neural Stem Cell-Derived Oligodendrocyte Precursor Cell Cultures

The main hurdle in developing drug screening techniques for assessing the efficacy of therapeutic strategies in complex diseases is striking a balance between in vitro simplification and recreating the complex in vivo environment, along with the main aim, shared by all screening strategies, of obtaining robust and reliable data, highly predictive for in vivo translation.
In the field of demyelinating diseases, the majority of drug screening strategies are based on immortalized cell lines or pure cultures of isolated primary oligodendrocyte precursor cells (OPCs) from newborn animals, leading to strong biases due to the lack of age-related differences and of any real pathological condition or complexity.
Here we show the setup of an in vitro system aimed at modeling the physiological differentiation/maturation of neural stem cell (NSC)-derived OPCs, easily manipulated to mimic pathological conditions typical of demyelinating diseases. Moreover, the method includes isolation from fetal and adult brains, giving a system which dynamically differentiates from OPCs to mature oligodendrocytes (OLs) in a spontaneous co-culture which also includes astrocytes. This model physiologically resembles the thyroid hormone-mediated myelination and myelin repair process, allowing the addition of pathological interferents which model disease mechanisms. We show how to mimic the two main components of demyelinating diseases (i.e., hypoxia/ischemia and inflammation), recreating their effect on developmental myelination and adult myelin repair and taking all the cell components of the system into account throughout, while focusing on differentiating OPCs.
This spontaneous mixed model, coupled with cell-based high-content screening technologies, allows the development of a robust and reliable drug screening system for therapeutic strategies aimed at combating the pathological processes involved in demyelination and at inducing remyelination.

We describe the production of mixed cultures of astrocytes and oligodendrocyte precursor cells derived from fetal or adult neural stem cells differentiating into mature oligodendrocytes, and in vitro modeling of noxious stimuli. The coupling with a cell-based high-content screening technique builds a reliable and robust drug screening system.

Selezione ad alto contenuto di differenziazione e analisi della maturazione di colture cellulari precursori di oligodendrociti derivati da cellule staminali neurali fetali e adulte

The main hurdle in developing drug screening techniques for assessing the efficacy of therapeutic strategies in complex diseases is striking a balance ...