Ringraziamo il Resource Rat e Research Center, University of Missouri, Columbia, Missouri, per fornire topi GFP; Neuralstem Inc. per la fornitura di cellule staminali neurali umane. Questo lavoro è stato sostenuto dalla Veterans Administration, NIH (NS09881), Canadian Spinal Research Organization, la Fondazione Craig H. Neilsen, e Bernard e Anne Spitzer Charitable Trust.

Tutti i protocolli animali sono approvati da VA-San Diego Institutional Animal Care ed uso commissione (IACUC). Linee guida NIH da laboratorio la cura e la sicurezza degli animali sono rigorosamente seguite. Gli animali hanno libero accesso a cibo e acqua durante lo studio e sono adeguatamente trattati per ridurre al minimo il dolore e il disagio. 1. Preparazione di fibrina componenti contenenti fattore di crescita Cocktails <ol><li> Sciogliere 25 mg di ratto fibrinogeno in 0,5 ml di PBS per ottenere 50 mg / ml 2x soluzione stock (vedi tabella Materials). Il fibrinogeno è difficile da sciogliere e deve essere collocato in un incubatore a 37 ° C o bagnomaria e agitato ogni 5-10 minuti per 1-2 ore. </li><li> Sciogliere 100 ratto trombina delle Nazioni Unite in 2 ml di 10 mM sterile CaCl 2 per ottenere 50 U / ml 2x soluzione stock. </li><li> Sciogliere fattori di crescita in PBS alle seguenti concentrazioni di ottenere 100 microlitri soluzione stock: 1 mg / ml: BDNF; NT-3 (alta concentrazione magazzino in infusione intratecale in vivo <sup&gt; 19); 200 mg / ml: GDNF; IGF; bFGF; EGF; PDGF; aFGF; HGF (circa 1.000 x superiore a quello utilizzato per la coltura cellulare). </li><li> Sciogliere 25 mg MDL28170 (inibitore della calpaina) in 1.3 ml di DMSO avere 50 soluzione stock DMSO mM, e poi diluire 50x in PBS per ottenere 1 mM soluzione stock. </li><li> Mescolare 100 ml di ciascun fattore componente di crescita e 100 ml di MDL28170 (soluzione 1 mM) per produrre 1.000 ml soluzione fattore di crescita cocktail (Figura 1). </li><li> Mescolare 500 ml di fibrinogeno 2x soluzione stock con 500 microlitri di crescita fattore di cocktail per ottenere una soluzione 25 mg / ml di fibrinogeno di lavoro contenente i fattori di crescita e le aliquote in 10 o 20 microlitri per la conservazione a -70 ° C. La soluzione è stabile fino a 12 mesi a -70 ° C. </li><li> Allo stesso modo, mescolare 500 ml di trombina 2x soluzione stock con 500 microlitri fattore di crescita cocktail avere 25 soluzione di lavoro U / ml contenente trombina fattori di crescita e aliquota in simili volumi di 10 o 20 microlitri per la conservazione di unt -70 ° C fino a 12 mesi. </li><li> Il giorno di innesto, collocare cocktail-fibrinogeno e fattore di crescita-trombina contenente in ghiaccio fino mescolato con le cellule. </li></ol> 2. T3 Spinal Cord transezione <ol><li> Anestetizzare femmina adulta Fischer 344 ratti o topi nudi atimici con metodi accettabili. I soggetti sono profondamente anestetizzati, quando la risposta alla coda e zampa pizzico sono completamente assenti. Nota: Usiamo tipicamente 150-200 g ratti Fischer o 180-220 g topi atimici, e un cocktail anestesia (2 ml / kg) di ketamina (25 mg / ml), xilazina (1,3 mg / ml) e acepromazina (0,25 mg / ml). </li><li> Applicare una pomata oculare per prevenire la secchezza o lesioni degli occhi, mentre gli animali sono sotto anestesia. </li><li> Radere la zona toracica superiore e pulire la pelle con Betadine. </li><li> Tagliare la pelle e muscolo utilizzando # 15 Lama, esporre T3 vertebra con un divaricatore chirurgico, ed eseguire una laminectomia a T3 vertebra per esporre T3 / 4 midollo spinale tramite un rongeur. </li><li> Fare un longitudinale incisione mediana nella durata di circa 2 mm di lunghezza con una lama # 11. </li><li> Posizionare iridectomia forbici sotto la dura tagliare il laterale destro intera midollo spinale. Fare un altro taglio sullo stesso lato 1,5 millimetri più cadually. </li><li> Aspirare il tessuto del midollo spinale tra i due segmenti di taglio con un ottuso 23 G ago collegato al vuoto intensità moderata. Utilizzare verifica visiva per garantire la completa resezione ventralmente e lateralmente. Questo completerà un emisezione laterale. </li><li> Per estendere la lesione ad una resezione del midollo spinale a tutta larghezza, posizionare incisioni specchio nel hemicord sinistra. Tentativo di lasciare la dura intatta, diverso dal primo taglio, per mantenere l&#39;impresa matrice innesto / fibrina nel sito della lesione quando innestato due settimane più tardi. </li><li> Suturare il muscolo, applicare la polvere antibiotica e fiocco la pelle. </li><li> Iniettare lattato (3 ml) di Ringer, Bannamine (2,5-5 mg / kg) e ampicillina (80-100 mg / kg) (vedi tabella Materials) immediatamente dopo l&#39;intervento chirurgico e mairatti ntain in un incubatore caldo fino a quando viene ristabilita termoregolazione. </li><li> Svuotare la vescica e iniettare la soluzione di Ringer e ampicillina due volte al giorno per circa due settimane fino all&#39;istituzione della vescica reflex svuotamento (Figura 1B). </li></ol> 3. Preparazione di recente dissociata embrionali Giorno 14 Spinal Cord Neural Stem Cells <ol><li> Ottenere Inbreed transgenici GFP ratti F344 (F344-Tg (UBC-EGFP) F455Rrrc) dal Ratto delle risorse e Research Center, Università del Missouri. </li><li> Iniettare 40 mcg des-Gly 10, [D-Ala 6]-luteinizante ormone rilasciante ormone Ethylamide (LHRH) diluito in PBS ad una concentrazione di 200 ug / ml per via intraperitoneale (IP) per maturo ratto femmina (8 settimane o più) dopo 2 induzione luce -3 ore per la sincronizzazione di raccolta degli embrioni. </li><li> Accoppiarsi con un maturo GFP omozigote o eterozigote notte ratto maschio 4 giorni dopo l&#39;iniezione. </li><li> Raccogliere embrioni a giorni 13,5-14,5 postcoitusIn tampone HBSS freddo (pc), esaminare l&#39;espressione GFP sotto un &quot;Nightsea&quot; torcia elettrica e filtri bicchieri (BLS1 - BlueStar torcia elettrica e filtro barriera modello VG1) o di un microscopio a fluorescenza. </li><li> Sezionare midollo spinale in condizioni asettiche da ciascun feto GFP-positive e rimuovere le meningi sovrastanti e gangli spinali attaccato con le forbici iridectomia e gioiellieri pinza sottile. </li><li> Raccogliere le midollo spinale sezionato in 2 ml di freddo tampone HBSS in un tubo cornical 15 ml e tenere in ghiaccio. </li><li> Seguire riferimento # 20 di dissociare il midollo spinale sezionato: <ol><li> Brevemente, aggiungere 2 ml di 0,25% tripsina-EDTA alla provetta contenente midolli spinali sezionati (vari cordoni possono essere digeriti insieme, 1 cavo può generare cellule sufficiente per innesto di un soggetto) e digerire a 37 ° C per 10-12 min. </li><li> Arrestare la reazione tripsina aggiungendo 10 ml di DMEM contenente 10% FBS al tubo e centrifugare il tessuto a 2.500 rpm per 2 min. </li><li> Risospendere tessuto in 2 ml Neurobasal medio containing 2% B27, e triturare il tessuto del midollo spinale utilizzando sempre più piccoli fuoco lucido Pasteur pipette. </li><li> Centrifugare le cellule a 2.500 rpm per 2 min, risospendere in 2 ml di terreno contenente Neurobasal B27, e celle filtranti con filtro cella filtro 40 micron. </li></ol></li><li> Contare le cellule con un emocitometro e dividere le celle equamente in due provette Eppendorf. </li><li> Centrifugare le cellule e rimuovere completamente il surnatante (1-2 min sono tenuti a ritirare tutto il surnatante con una punta di vuoto basso) in modo che il cocktail di fattori di crescita non saranno diluiti rimanendo surnatante dopo cellula ri-sospensione. </li><li> Risospendere ogni metà delle cellule del fibrinogeno o soluzione di lavoro trombina contenente fattori di crescita, rispettivamente, ad una concentrazione di 250.000 cellule / ml (conteggi pellet cellulare per circa ⅓ volume finale) e disporli sul ghiaccio prima trapianto (Figura 1A). Si noti che il fibrinogeno può spontaneamente gel quando miscelato con la cellulas prima di combinare con trombina presso il sito di lesione / innesto; per evitare gelificazione precoce, mescolare celle fibrinogeno immediatamente prima dell&#39;innesto cellule in vivo. </li></ol> 4. Trapianto <ol><li> Reexpose la lesione T3 / 4 midollo spinale due settimane dopo resezione del midollo spinale. </li><li> 5 microlitri fibrinogeno-cellule e 5 trombina-cellule microlitri potrebbero essere iniettate direttamente in nove punti del sito della lesione attraverso il tessuto cicatriziale sigillato e dura intatto senza riaprire il sito di lesione. </li><li> Utilizzare un ago G 27 per creare 9 fori sulla superficie del tessuto cicatriziale e dura intatta 1-2 settimane dopo la lesione: 3 in centro (una nel punto centrale e due laterali in, e distanziati 0,5 millimetri a parte) e 3 in entrambi rostrale e 3 interfaccia caudale (circa 0,5 millimetri rostrale o caudale al centro della lesione). </li><li> Poi iniettare metà volume di soluzione di cellule fibrinogeno in tre punti centrali del sito della lesione e un volume quarto in 3 punti dell&#39;interfaccia rostrale e aqVolume uarter in 3 punti dell&#39;interfaccia caudale. </li><li> Poi, iniettare immediatamente le cellule throbmbin negli stessi punti. La miscela di cellule fibrinogeno e trombina cellule all&#39;interno del sito della lesione formerà fibrina gel per contenere celle nel sito di lesione e cocktail fattore di crescita sosterrà la sopravvivenza delle cellule. Le cellule vengono iniettate con pipette di vetro tirati collegati ad una PicoSpritzer (General Valve, Inc.). </li><li> Trapianto di cellule staminali neurali umane in coltura 21 nella stessa procedura sopra descritta, e utilizzare fibrina e fattori di crescita cocktail a base di reagenti umani. </li></ol>

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Uno dei principali ostacoli per NSC trapianto nel midollo spinale danneggiato è scarsa sopravvivenza al centro della lesione. Eventuali lacune o cavità nel sito della lesione potrebbe potenzialmente ridurre o indebolire la formazione di relè neuronali funzionali tra assoni sovraspinali e separati segmenti del midollo spinale al di sotto della lesione. Inoltre, la scarsa sopravvivenza delle cellule staminali neurali trapiantate può influenzare la loro integrazione con il tessuto ospite, e quindi ridurre la connettivi...

Lesioni del midollo spinale (SCI), spesso danni non solo tratti di sostanza bianca che trasportano i segnali da e verso il cervello, ma anche la materia grigia centrale, causando la perdita segmentale degli interneuroni e motoneuroni. La conseguenza di SCI è perdita sia funzioni motorie e sensoriali sotto della lesione. Purtroppo, il sistema nervoso centrale adulto (CNS) non rigenera spontaneamente, con conseguente deficit funzionali permanenti 1. Pertanto, la ricostruzione dei feriti midollo spinale adulto e il miglioramento in motoria, sensoriale e della funzione autonomica è un obiettivo importante della ricerca SCI. Cellule staminali neurali (NSC), sia direttamente isolata da embrionale o adulto CNS, sono cellule candidati validi per sostituire i neuroni perduti e glia. Inoltre, queste cellule hanno il potenziale di formare nuovi relè funzionali per ripristinare conduzione assonale attraverso un 2,3 sito della lesione. Ad oggi, non c&#39;è stata una spiegazione dettagliata del anatomica, electrophysiological ed effetti comportamentali della formazione relè neuronale da cellule staminali neurali trapiantate dopo il grave SCI. Ci sono diverse ragioni per questo: primo, NSC trapiantati o fetale del tessuto del sistema nervoso centrale sopravvivere male quando innestati in grandi cavità della lesione. Precedenti studi mostrano sostanziale perdita di cellule presto, lasciando grandi cavità lesione cistica vuoti 4,5. In alcuni studi le cellule innestate dovessero successivamente dividono e riempire la cavità lesione 4,5, ma questo potrebbe verificarsi dopo un ritardo di giorni o settimane, e successivo riempimento sito della lesione non sia completa o coerente. In secondo luogo, un sistema di monitoraggio efficiente che fornisce dati attendibili sulla sopravvivenza cellulare, differenziamento / maturazione, e di conseguenza trapiantato cellule staminali neurali era carente. La maggior parte dei primi studi utilizzati anterograda e retrograda etichettatura per tracciare le proiezioni assonali di trapianti di 2,3. Tuttavia, queste tecniche solo parzialmente e spesso proiezioni assonale poco chiaro etichettati derivanti da cellule innestate e metodi traccianti sono soggettivit di artefatti di perdite di colorante oltre le cellule impiantate. Altri gruppi utilizzati marcatori neuronali specifici umane per etichettare proiezioni assonale dopo il trapianto di cellule staminali neurali fetali umani in roditori feriti midollo spinale 5,6. Tuttavia, in questi studi, gli xenotrapianti non hanno sempre sopravvivono bene. Recentemente, la consegna virale del gene reporter GFP è stato usato per etichettare le cellule staminali neurali in coltura 7,8. Tuttavia, l&#39;espressione della GFP era spesso incoerente e può essere down-regolato 7. Recentemente, l&#39;uso di topi o ratti donatori transgenici che esprimono stabilmente il gene reporter GFP, o la fosfatasi alcalina placentare umano, ha notevolmente migliorato l&#39;inseguimento di trapiantate neurali cellule staminali / progenitori in vivo 9,11. In terzo luogo, diversi studi indicano che le cellule staminali neurali di ratto coltivate in vitro derivate da embrioni o adulto CNS differenziano esclusivamente in linee gliali quando trapiantati in ambienti della cor midollo intatto o feriti adultid 7,12,13, nonostante il fatto che queste cellule staminali neurali sono capaci di differenziarsi in neuroni e glia sia in vitro, indicando che ambienti locali possono dettare il destino delle cellule staminali. In alternativa, le cellule staminali neurali in coltura, in particolare quelli derivati ​​da SNC adulto, possono avere intrinseche proprietà defaulty di differenziarsi in linee gliali in vivo 13. A causa delle limitazioni di cui sopra, il nostro gruppo ha recentemente sviluppato un nuovo protocollo per migliorare embrionale inseguimento NSC, la sopravvivenza e la differenziazione / maturazione nel midollo spinale adulto gravemente ferito. In breve, abbiamo iniziato con una linea transgenica Fischer 344 ratti Inbreed stabile che esprime un gene reporter GFP che sostiene espressione GFP dopo il trapianto in vivo 14. Successivamente, abbiamo utilizzato NSC appena isolati dal giorno embrionale 14 Fischer 344 midollo spinale, una fase di sviluppo che mantiene il potenziale per generare sia i neuroni e glia. Infine, si conficcaronoNSC appena dissociate in una matrice di fibrina che contiene fattori di crescita 15-17 per mantenere le cellule in modo uniforme e distribuirli all&#39;interno di una grande cavità della lesione, con l&#39;obiettivo di sostenere la sopravvivenza delle cellule del trapianto, differenziazione e integrazione. Gli innesti sono stati collocati in luoghi di T3 resezione completa, due settimane dopo la lesione del midollo spinale. Queste cellule innestate costantemente riempite siti transezione complete e differenziate in neuroni abbondanti che si estendevano gran numero di assoni in molte midollo spinale su lunghe distanze 18. Risultati simili sono stati ottenuti utilizzando colture di innesti di cellule staminali neurali umane immunodeficienza ratti 18.

<table border="1">
 <tbody>
 <tr>
 <td>Reagents</td>
 <td>Company</td>
 <td>Catalogue</td>
 <td>Comment</td>
 </tr>
 <tr>
 <td>Fibrinogen(rat)</td>
 <td>Sigma</td>
 <td>F6755-25MG</td>
 <td>2 hr at 37 &deg;C to dissovle 
 Stock Concentration: 50 mg/ml 
 Final Concentration: 25 mg/ml</td>
 </tr>
 <tr>
 <td>Thrombin (rat)</td>
 <td>Sigma</td>
 <td>T5772-100UN</td>
 <td>Dissovle in 10 mM CaCl2 
 Stock Concentration: 50 U/ml 
 Final Concentration: 25 mg/ml</td>
 </tr>
 <tr>
 <td>bFGF (human)</td>
 <td>Sigma</td>
 <td>F0291 (25 &mu;g)</td>
 <td>Stock Concentration: 200 ng/&mu;l 
 Final Concentration: 10 ng/&mu;l</td>
 </tr>
 <tr>
 <td>EGF (murine)</td>
 <td>Sigma</td>
 <td>E1257 (0.1 mg)</td>
 <td>Stock Concentration: 200 ng/&mu;l 
 Final Concentration: 10 ng/&mu;l</td>
 </tr>
 <tr>
 <td>BDNF(human)</td>
 <td>Peprotech</td>
 <td>452-02 (1 mg)</td>
 <td>Stock Concentration: 1000 ng/&mu;l 
 Final Concentration: 50 ng/&mu;l</td>
 </tr>
 <tr>
 <td>NT-3 (human)</td>
 <td>Peprotech</td>
 <td>452-03 (1 mg)</td>
 <td>Stock Concentration: 1000 ng/&mu;l 
 Final Concentration: 50 ng/&mu;l</td>
 </tr>
 <tr>
 <td>GDNF (rat)</td>
 <td>Sigma</td>
 <td>G1401 (10 &mu;g)</td>
 <td>Stock Concentration: 200 ng/&mu;l 
 Final Concentration: 10 ng/&mu;l</td>
 </tr>
 <tr>
 <td>IGF-1(mouse)</td>
 <td>Sigma</td>
 <td>I8779 (50 &mu;g)</td>
 <td>Stock Concentration: 200 ng/&mu;l 
 Final Concentration: 10 ng/&mu;l</td>
 </tr>
 <tr>
 <td>aFGF (human)</td>
 <td>Sigma</td>
 <td>F5542 (25 &mu;g)</td>
 <td>Stock Concentration: 200 ng/&mu;l 
 Final Concentration: 10 ng/&mu;l </td>
 </tr>
 <tr>
 <td>PDGF-AA (human)</td>
 <td>Sigma</td>
 <td>P3076 (10 &mu;g)</td>
 <td>Stock Concentration: 200 ng/&mu;l 
 Final Concentration: 10 ng/&mu;l</td>
 </tr>
 <tr>
 <td>HGF (human)</td>
 <td>Sigma</td>
 <td>H9661 (5 &mu;g)</td>
 <td>Stock Concentration: 200 ng/&mu;l 
 Final Concentration: 10 ng/&mu;l</td>
 </tr>
 <tr>
 <td>MDL28170</td>
 <td>Sigma</td>
 <td>M6690 (25 mg)</td>
 <td>Stock in DMSO 
 Stock Concentration: 1 mM 
 Final Concentration: 50 &mu;M</td>
 </tr>
 <tr>
 <td>PBS</td>
 <td>Millipore</td>
 <td>BSS-1005-B</td>
 <td>Stock Concentration: 1X 
 Final Concentration: 1X</td>
 </tr>
 <tr>
 <td>DMSO</td>
 <td>Sigma</td>
 <td>D2650</td>
 <td></td>
 </tr>
 <tr>
 <td>Ketamine</td>
 <td>Putney</td>
 <td>26637-411-01</td>
 <td>40-80 mg/kg 
 Stock Concentration: 100 mg/ml 
 Final Concentration: 25 mg/ml</td>
 </tr>
 <tr>
 <td>Xylazine</td>
 <td>Lloyd</td>
 <td>0410,</td>
 <td>2.5-8 mg/ml 
 Stock Concentration: 100 mg/ml 
 Final Concentration: 1.3 mg/ml</td>
 </tr>
 <tr>
 <td>Acepromazine</td>
 <td>Butler</td>
 <td>003845</td>
 <td>0.5-4 mg/ml 
 Stock Concentration: 10 mg/ml 
 Final Concentration: 0.25 mg/ml</td>
 </tr>
 <tr>
 <td>Betadine</td>
 <td>Healthpets</td>
 <td>BET16OZ</td>
 <td></td>
 </tr>
 <tr>
 <td>Ringers</td>
 <td>Abbott</td>
 <td>04860-04-10</td>
 <td>2-3 ml/inj</td>
 </tr>
 <tr>
 <td>Banamine</td>
 <td>Schering-Plough</td>
 <td>0061-0851-03</td>
 <td>2.5 -5 mg/kg 
 Stock Concentration: 50 mg/ml 
 Final Concentration: 0.5 mg/ml</td>
 </tr>
 <tr>
 <td>Ampicillin</td>
 <td>Sandoz</td>
 <td>0781-3404-85</td>
 <td>80-100 mg/kg 
 Final Concentration: 50 mg/ml</td>
 </tr>
 <tr>
 <td>LH-RH</td>
 <td>Sigma</td>
 <td>L4513</td>
 <td>200 &mu;g/kg 
 Final Concentration: 200 &mu;g/ml</td>
 </tr>
 <tr>
 <td>HBSS</td>
 <td>Gibco</td>
 <td>14175-096</td>
 <td></td>
 </tr>
 <tr>
 <td>Trypsin</td>
 <td>Gibco</td>
 <td>25200056 </td>
 <td>Stock Concentration: 0.25 % 
 Final Concentration: 0.125 %</td>
 </tr>
 <tr>
 <td>DMEM</td>
 <td>Gibco</td>
 <td>11995073 </td>
 <td></td>
 </tr>
 <tr>
 <td>FBS</td>
 <td>Gibco</td>
 <td>16000044 </td>
 <td>Stock Concentration: 100 % 
 Final Concentration: 10 %</td>
 </tr>
 <tr>
 <td>Neurobasal Medium</td>
 <td>Gibco</td>
 <td>21103049 </td>
 <td></td>
 </tr>
 <tr>
 <td>B27</td>
 <td>Gibco</td>
 <td>17504044</td>
 <td>Stock Concentration: 100x 
 Final Concentration: 1x</td>
 </tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Note, use human reagents for grafts of human NSCs</td>
 </tbody>
</table>

promotion of survival and differentiation of neural stem cells with fibrin and growth factor cocktails after severe spinal cord injury

Cellule staminali neurali (NSC) possono auto-rinnovarsi e differenziarsi in neuroni e glia. NSC trapiantate possono sostituire i neuroni persi e glia dopo la lesione del midollo spinale (SCI), e possono formare relè funzionali di ri-collegare segmenti del midollo spinale sopra e sotto una lesione. Precedenti studi innesto cellule staminali neurali sono stati limitati dalla sopravvivenza dell&#39;innesto incomplete all&#39;interno del midollo spinale cavità lesione. Inoltre, il monitoraggio della sopravvivenza cellulare dell&#39;innesto, differenziazione, e l&#39;estensione processo non era stata ottimizzata. Infine, in studi precedenti, NSC ratto coltivate erano tipicamente riferite a differenziarsi in cellule gliali quando innestato al midollo spinale danneggiato, piuttosto che neuroni, salvo destino è stato guidato per un tipo cellulare specifico. Per affrontare questi problemi, abbiamo sviluppato nuovi metodi per migliorare la sopravvivenza, l&#39;integrazione e la differenziazione delle cellule staminali neurali ai siti di ancora grave SCI. NSC sono stati appena isolati da embrionale giorno 14 del midollo spinale (E14) da un transgenico Fischer 344 linea di ratto stabile esprimere fl verdeuorescente proteina (GFP) e sono stati incorporati in una matrice di fibrina contenente fattori di crescita; questa formulazione mirava a conservare cellule innestate nella cavità lesione e supportare la sopravvivenza cellulare. NSC in fibrina / fattore di crescita cocktail sono stati impiantati due settimane dopo toracico livello 3 (T3) completi transections midollo spinale, evitando periodi di picco di infiammazione. Innesti risultanti completamente riempito la cavità della lesione e differenziate in entrambi i neuroni, che si estendevano assoni nell&#39;ospite midollo spinale sopra straordinariamente lunghe distanze, e glia. Innesti di cellule staminali neurali umane in coltura che esprimono GFP portato a risultati simili. Così, i metodi sono definiti per migliorare neurale innesto di cellule staminali, la sopravvivenza e l&#39;analisi dei risultati in vivo.

GFP etichettatura immunoistologico dimostra che le cellule staminali neurali ratto innestato risospese in tampone fosfato (PBS) (manca una matrice di fibrina e fattori di crescita) sono sopravvissuti male nel sito resezione T3, solo attaccarsi al margine della lesione / host e lasciando la maggior parte del sito della lesione vuota senza cellule innestate (Figura 2A). La sopravvivenza di NSC migliorata quando co-innestato con matrici di fibrina solo, ma l&#39;innesto non completamente compili una grande...

Watch this Scientific Journal Video about Promotion of Survival and Differentiation of Neural Stem Cells with Fibrin and Growth Factor Cocktails after Severe Spinal Cord Injury at JoVE.com

Promotion of Survival and Differentiation of Neural Stem Cells with Fibrin and Growth Factor Cocktails after Severe Spinal Cord Injury

Matrici di fibrina che contengono fattori di crescita sono stati usati per mantenere le cellule staminali neurali innestate nei siti di completo recisione del midollo spinale. Cellule trapiantate completamente riempite la cavità della lesione e differenziate in diversi tipi di cellule neurali, tra cui i neuroni che si estendevano assoni nel midollo spinale ospite su lunghe distanze.

Promozione della sopravvivenza e la differenziazione delle cellule staminali neurali con fibrina e fattore di crescita Cocktails dopo gravi lesioni del midollo spinale

Neural stem cells (NSCs) can self-renew and differentiate into neurons and glia. Transplanted NSCs can replace lost neurons and glia after spinal cord ...

promotion-survival-differentiation-neural-stem-cells-with-fibrin

Research

JoVE Journal

Neuroscience

12.3K Views.  Veterans Administration Medical Center, San Diego.  Matrici di fibrina che contengono fattori di crescita sono stati usati per mantenere le cellule staminali neurali innestate nei siti di completo recisione del midollo spinale. Cellule trapiantate completamente riempite la cavità della lesione e differenziate in diversi tipi di cellule neurali, tra cui i neuroni che si estendevano assoni nel midollo spinale ospite su lunghe distanze. 

Video: Promotion of Survival and Differentiation of Neural Stem Cells with Fibrin and Growth Factor Cocktails after Severe Spinal Cord Injury

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

Growth and Differentiation of Adult Hippocampal Arctic Ground Squirrel Neural Stem Cells

Arctic ground squirrels (Urocitellus parryii, AGS) are unique in their ability to hibernate with a core body temperature near or below freezing 1. These animals also resist ischemic injury to the brain in vivo 2,3 and oxygen-glucose deprivation in vitro 4,5. These unique qualities provided the impetus to isolate AGS neurons to examine inherent neuronal characteristics that could account for the capacity of AGS neurons to resist injury and cell death caused by ischemia and extremely cold temperatures. Identifying proteins or gene targets that allow for the distinctive properties of these cells could aid in the discovery of effective therapies for a number of ischemic indications and for the study of cold tolerance. Adult AGS hippocampus contains neural stem cells that continue to proliferate, allowing for easy expansion of these stem cells in culture. We describe here methods by which researchers can utilize these stem cells and differentiated neurons for any number of purposes. By closely following these steps the AGS neural stem cells can be expanded through two passages or more and then differentiated to a culture high in TUJ1-positive neurons (~50%) without utilizing toxic chemicals to minimize the number of dividing cells. Ischemia induces neurogenesis 6 and neurogenesis which proceeds via MEK/ERK and PI3K/Akt survival signaling pathways contributes to ischemia resistance in vivo7 and in vitro 8 (Kelleher-Anderson, Drew et al., in preparation). Further characterization of these unique neural cells can advance on many fronts, using some or all of these methods.

Neural stem cells were prepared from the hippocampus of adult non-hibernating yearling Arctic ground squirrels (AGS). These neural stem cells can be expanded through numerous passages, differentiated and maintained as a nearly 50:50 neuron to glial culture.

Crescita e la differenziazione degli adulti ippocampale suolo artico Cellule Staminali neurali Squirrel

Arctic ground squirrels (Urocitellus parryii, AGS) are unique in their ability to hibernate with a core body temperature near or below freezing 1. These ...

Surgical Transplantation of Mouse Neural Stem Cells into the Spinal Cords of Mice Infected with Neurotropic Mouse Hepatitis Virus

Mice infected with the neurotropic JHM strain of mouse hepatitis virus (MHV) develop pathological and clinical outcomes similar to patients with the demyelinating disease Multiple Sclerosis (MS). We have shown that transplantation of NSCs into the spinal cords of sick mice results in a significant improvement in both remyelination and in clinical outcome. Cell replacement therapies for the treatment of chronic neurologic diseases are now a reality and in vivo models are vital in understanding the interactions between the engrafted cells and host tissue microenvironment. This presentation provides an adapted method for transplanting cells into the spinal cord of JHMV-infected mice. In brief, we provide a procedure for i) preparation of NSCs prior to transplant, ii) pre-operative care of mice, iii) exposure of the spinal cord via laminectomy, iv) stereotactic injection of NSCs, and iv) post-operative care.

The transplantation of mouse neural stem cells (NSCs) into the spinal cords of mice with established demyelination is detailed. The preparation of NSCs, the laminectomy of thoracic vertebra 9 (T9), and transplantation of NSCs is outlined along with the pre- and post-operative care of the mice.

Il trapianto chirurgico di cellule staminali neurali del mouse nel midollo spinale di topi infettati con virus dell&#39;epatite neurotropi mouse

Mice infected with the neurotropic JHM strain of mouse hepatitis virus (MHV) develop pathological and clinical outcomes similar to patients with the ...

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 ...

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

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

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

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

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

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

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability.

Un<em&gt; Ex Vivo</em&gt; Laser-indotta Spinal Cord Injury modello per valutare i meccanismi di degenerazione assonale in tempo reale

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

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 ...

A Neurosphere Assay to Evaluate Endogenous Neural Stem Cell Activation in a Mouse Model of Minimal Spinal Cord Injury

Neural stem cells (NSCs) in the adult mammalian spinal cord are a relatively mitotically quiescent population of periventricular cells that can be studied in vitro using the neurosphere assay. This colony-forming assay is a powerful tool to study the response of NSCs to exogenous factors in a dish; however, this can also be used to study the effect of in vivo manipulations with the proper understanding of the strengths and limitations of the assay. One manipulation of the clinical interest is the effect of injury on endogenous NSC activation. Current models of spinal cord injury provide a challenge to study this as the severity of common contusion, compression, and transection models cause the destruction of the NSC niche at the site of the injury where the stem cells reside. Here, we describe a minimal injury model that causes localized damage at the superficial dorsolateral surface of the lower thoracic level (T7/8) of the adult mouse spinal cord. This injury model spares the central canal at the level of injury and permits analysis of the NSCs that reside at the level of the lesion at various time points following injury. Here, we show how the neurosphere assay can be utilized to study the activation of the two distinct, lineally-related, populations of NSCs that reside in the spinal cord periventricular region - primitive and definitive NSCs (pNSCs and dNSCs, respectively). We demonstrate how to isolate and culture these NSCs from the periventricular region at the level of injury and the white matter injury site. Our post-surgical spinal cord dissections show increased numbers of pNSC and dNSC-derived neurospheres from the periventricular region of injured cords compared to controls, speaking to their activation via injury. Furthermore, following injury, dNSC-derived neurospheres can be isolated from the injury site &#8212; demonstrating the ability of NSCs to migrate from their periventricular niche to sites of injury.

Here, we demonstrate the performance of a minimal spinal cord injury model in an adult mouse that spares the central canal niche housing endogenous neural stem cells (NSCs). We show how the neurosphere assay can be used to quantify activation and migration of definitive and primitive NSCs following injury.

Un saggio di Neurosphere per valutare l'attivazione delle cellule staminali neurali endogene in un modello murino di ferita del midollo spinale Minimal

Neural stem cells (NSCs) in the adult mammalian spinal cord are a relatively mitotically quiescent population of periventricular cells that can be studied ...

Identifying Cell Surface Markers of Primary Neural Stem and Progenitor Cells by Metabolic Labeling of Sialoglycan

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized niches in vivo and can be isolated and expanded in vitro, serving as an important resource for cell transplantation to repair brain damage. However, NSPCs are heterogeneous and not clearly defined at the molecular level or purified due to a lack of specific cell surface markers. The protocol presented, which has been previously reported, combines a neural-endothelial co-culture system with a metabolic glycan labeling method to identify the surface sialoglycoproteome of primary NSPCs. The NSPC-endothelial co-culture system allows self-renewal and expansion of primary NSPCs in vitro, generating a sufficient number of NSPCs. Sialoglycans in cultured NSPCs are labeled using an unnatural sialic acid metabolic reporter with bioorthogonal functional groups. By comparing the sialoglycoproteome from self-renewing NSPCs expanded in an endothelial co-culture with differentiating neural culture, we identify a list of membrane proteins that are enriched in NSPCs. In detail, the protocol involves: 1) set-up of an NSPC-endothelial co-culture and NSPC differentiating culture; 2) labeling with azidosugar per-O-acetylated N-azidoacetylmannosamine (Ac4ManNAz); and 3) biotin conjugation to modified sialoglycan for imaging after fixation of neural culture or protein extraction from neural culture for mass spectrometry analysis. Then, the NSPC-enriched surface marker candidates are selected by comparative analysis of mass spectrometry data from both the expanded NSPC and differentiated neural cultures. This protocol is highly sensitive for identifying membrane proteins of low abundance in the starting materials, and it can be applied to marker discovery in other systems with appropriate modifications

Presented here is a protocol that combines an in vitro neural-endothelial co-culture system and metabolic incorporation of sialoglycan with bioorthogonal functional groups to expand primary neural stem and progenitor cells and label their surface sialoglycoproteins for imaging or mass-spectrometry analysis of cell surface markers.

Identificazione dei marcatori di superficie cellulare delle cellule staminali primarie e delle cellule progenitrici mediante l'etichettatura metabolica di Sialoglycan

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized ...

Activity-based Training on a Treadmill with Spinal Cord Injured Wistar Rats

Spinal cord injury (SCI) results in lasting deficits that include both mobility and a multitude of autonomic-related dysfunctions. Locomotor training (LT) on a treadmill is widely used as a rehabilitation tool in the SCI population with many benefits and improvements to daily life. We utilize this method of activity-based task-specific training (ABT) in rodents after SCI to both elucidate the mechanisms behind such improvements and to enhance and improve upon existing clinical rehabilitation protocols. Our current goal is to determine the mechanisms underlying ABT-induced improvements in urinary, bowel, and sexual function in SCI rats after a moderate to severe level of contusion. After securing each individual animal in a custom-made adjustable vest, they are secured to a versatile body weight support mechanism, lowered to a modified three-lane treadmill and assisted in step-training for 58 minutes, once a day for 10 weeks. This setup allows for the training of both quadrupedal and forelimb-only animals, alongside two different non-trained groups. Quadrupedal-trained animals with body weight support are aided by a technician present to assist in stepping with proper hind limb placement as necessary, while forelimb-only trained animals are raised at the caudal end to ensure no hind limb contact with the treadmill and no weight-bearing. One non-trained SCI group of animals is placed in a harness and rests next to the treadmill, while the other control SCI group remains in its home cage in the training room nearby. This paradigm allows for the training of multiple SCI animals at once, thus making it more time-efficient in addition to ensuring that our pre-clinical animal model mimics the clinical representation as close as possible, particularly with respect to the body weight support with manual assistance.

This protocol demonstrates our model of activity-based locomotor treadmill training for rats with spinal cord injury (SCI). Included is both quadrupedal and forelimb-only groups, in addition to two distinct types of non-trained control groups. Investigators are able to assess training effects on SCI rats using this protocol.

Corsi di formazione basati su attività su un tapis roulant con midollo spinale feriti ratti Wistar

Spinal cord injury (SCI) results in lasting deficits that include both mobility and a multitude of autonomic-related dysfunctions. Locomotor training (LT) ...