Agradecemos ao Resource Rat e Centro de Pesquisa da Universidade de Missouri, Columbia, Missouri, para a prestação de ratos GFP; Neuralstem Inc. para fornecer células-tronco neurais. Este trabalho foi apoiado pela Administração dos Veteranos, NIH (NS09881), da Organização de Pesquisa Spinal canadense, a Fundação Craig H. Nielsen, eo Bernard e Anne Spitzer Charitable Trust.

Todos os protocolos de animais são aprovados pela VA-San Diego Institutional Animal Care e do Comitê Use (IACUC). Diretrizes do NIH para cuidados com animais de laboratório e segurança sejam rigorosamente seguidas. Os animais têm livre acesso a comida e água ao longo do estudo e são tratados de forma adequada para minimizar a dor eo desconforto. 1. Preparação de Fibrina componentes contendo Fator de Crescimento Cocktails <ol><li> Dissolver 25 mg de fibrinogênio rato em 0,5 ml de PBS para obter / ml 2x solução estoque de 50 mg (ver tabela de materiais). O fibrinogénio é difícil de dissolver e deve ser colocado numa incubadora a 37 ° C ou banho de água e agitou-se a cada 5-10 min durante 1-2 horas. </li><li> Dissolver 100 trombina rato ONU em 2 ml de 10 mM estéril CaCl2 obter 50 U ml de solução / 2x. </li><li> Dissolve-se os factores de crescimento em PBS a concentrações que se seguem para se obter uma solução de 100 ul de estoque de 1 mg / ml: BDNF; NT-3 (concentração estoque elevado como infusão intratecal in vivo <sup&gt; 19); 200 ug / ml: GDNF; IGF; bFGF; EGF; PDGF; aFGF; HGF (cerca de 1000 x mais elevada do que utilizado para cultura de células). </li><li> Dissolve-se 25 mg de MDL28170 (inibidor da calpaína) em 1,3 mL de DMSO para se obter uma solução estoque de 50 mM de DMSO e, em seguida, dilua 50 vezes em PBS para obter a solução estoque 1 mM. </li><li> Misturar 100 ml de cada componente do factor de crescimento e de 100 ul de MDL28170 (solução de reserva 1 mM) para produzir 1,000 ul solução de factor de crescimento de cocktail (Figura 1). </li><li> Misturar 500 mL de solução stock de fibrinogénio 2x com 500 ul de coquetel de factor de crescimento para se obter uma solução a 25 mg / ml de fibrinogénio contendo os factores de crescimento de trabalho e alíquota em 10 ou 20 ul de armazenamento a -70 ° C. A solução é estável durante até 12 meses a -70 ° C. </li><li> Do mesmo modo, misturar 500 ul de trombina solução estoque 2x com 500 ul de factor de crescimento de coquetel para obter a solução de trabalho de 25 U / ml de trombina, contendo factores de crescimento e alíquota em volumes de 10 ul ou 20 semelhantes, para armazenamento de umt -70 ° C durante até 12 meses. </li><li> No dia do enxerto, colocar cocktails fibrinogénio e do factor de crescimento contendo trombina em gelo até misturados com as células. </li></ol> 2. T3 Spinal Cord transecção <ol><li> Anestesiar fêmea adulta Fischer 344 ratos ou ratos pelados atímicos usando métodos aceitáveis. Os assuntos são profundamente anestesiado, quando a capacidade de resposta à cauda e pata pitada estão completamente ausentes. Nota: normalmente usam 150-200 g ratos Fischer ou 180-220 g ratos atímicos, e um cocktail de anestesia (2 ml / kg) de cetamina (25 mg / ml), xilazina (1,3 mg / mL) e acepromazina (0,25 mg / ml). </li><li> Aplicar pomada para evitar ressecamento ou lesão de olhos, enquanto os animais estão sob anestesia. </li><li> Raspar a área torácica superior e limpar a pele com Betadine. </li><li> Cortar a pele eo músculo usando # 15 blade, expor T3 vértebra usando um afastador cirúrgico e realizar uma laminectomia em T3 vértebra para expor T3 / 4 medula espinhal usando um rongeur. </li><li> Faça um longitudinal incisão na linha média na dura de aproximadamente 2 mm de comprimento utilizando uma lâmina de # 11. </li><li> Coloque Iridectomia tesoura debaixo da dura para cortar toda a medula espinhal lateral direito. Faça outro corte no mesmo lado 1,5 milímetros mais cadually. </li><li> Aspirar o tecido da medula espinal entre os dois segmentos de corte com um romba 23 L agulha conectada a vácuo intensidade moderada. Use a verificação visual para garantir a transecção completa ventral e lateralmente. Isto irá completar a hemissecção lateral. </li><li> Para estender a lesão a um transecção da medula espinhal de largura total, coloque incisões espelho no hemicord esquerda. Tente deixar a dura intacto, diferente do primeiro corte, para manter a matriz de enxerto / fibrina firme no local da lesão, quando enxertados duas semanas depois. </li><li> Suturar o músculo, aplique pó antibiótico e grampo da pele. </li><li> Injetar de Ringer com lactato (3 ml), Bannamine (2,5-5 mg / kg) e Ampicilina (80-100 mg / kg) (ver tabela de Materiais), imediatamente após a cirurgia e mairatos ntain em uma incubadora quente até termorregulação é re-estabelecida. </li><li> Esvazie a bexiga e injetar a solução de Ringer e Ampicilina duas vezes por dia por cerca de duas semanas, até o estabelecimento de bexiga reflexo esvaziamento (Figura 1B). </li></ol> 3. Preparação de Freshly Dissociated dia embrionário 14 Medula Espinhal Neural Stem Cells <ol><li> Obter consanguinidade transgênicos GFP ratos F344 (F344-Tg (UBC-EGFP) F455Rrrc) de rato de Recursos e Centro de Pesquisa da Universidade de Missouri. </li><li> Injectar 40 ug des-Gli 10, [D-Ala 6]-Leuteinizing Hormone Releasing Hormone etilamida (LHRH) diluído em PBS a uma concentração de 200 ug / ml, por via intraperitoneal (IP) por rato fêmea madura (8 semanas ou mais), após 2 -3 hr luz indução para a sincronização da colheita de embriões. </li><li> Companheiro com um maduro GFP overnight rato macho homozigoto ou heterozigoto 4 dias após a injecção. </li><li> Coletar embriões nos dias pós-coito 13,5-14,5(Pc) em tampão HBSS frio, examinar a expressão GFP sob um &quot;NightSea&quot; lanterna e filtro óculos (BLS1 - BlueStar Lanterna e filtro barreira Modelo VG1) ou um microscópio fluorescente. </li><li> Dissecar medula espinhal em condições assépticas de cada feto GFP-positivo e remover sobrejacentes meninges e gânglios espinhais anexado com uma tesoura Iridectomia e joalheiros pinça fina. </li><li> Recolha as medulas espinhais dissecadas em 2 ml de tampão HBSS frio em um tubo de 15 ml cornical e manter em gelo. </li><li> Siga referência n º 20 dissociar medulas espinhais dissecados: <ol><li> Resumidamente, adicionar 2 ml de 0,25% de tripsina-EDTA para o tubo contendo medulas dissecadas (vários cordões pode ser digerido em conjunto, um cabo pode gerar células suficientes para enxerto de um sujeito) e digestão a 37 ° C durante 10-12 min. </li><li> Parar a reacção de tripsina por adição de 10 ml de DMEM contendo FBS a 10% para o tubo centrifugador e o tecido a 2500 rpm durante 2 min. </li><li> Ressuspender o tecido em 2 ml de meio neurobasal containing 2% B27, e triturar o tecido da medula espinhal usando progressivamente menores pipetas Pasteur polido-fogo. </li><li> Centrifugar as células a 2.500 rpm durante 2 min, ressuspender em 2 ml de meio neurobasal contendo B27, e as células do filtro com um filtro de 40 um filtro de células. </li></ol></li><li> Contar as células com um hemocitómetro e as células dividem igualmente em dois tubos Eppendorf. </li><li> Centrifugar as células e remover completamente o sobrenadante (1-2 min são obrigados a retirar todo o sobrenadante com uma ponta de baixo vácuo) para que os coquetéis de fatores de crescimento não será diluída pela sobrenadante remanescente após célula re-suspensão. </li><li> Ressuspender cada metade das células, em que o fibrinogénio ou a solução de trabalho de trombina contendo factores de crescimento, respectivamente, a uma concentração de 250.000 células / mL (contagens da pelota celular durante cerca de ⅓ volume final) e colocá-los em gelo antes do transplante (Figura 1A). Note-se que o fibrinogénio pode gel espontaneamente quando misturado com celulars antes de combinar com trombina no local da lesão / enxerto; para evitar a gelificação prematura, misture pilhas em fibrinogênio imediatamente antes de enxerto de células in vivo. </li></ol> 4. Transplantation <ol><li> Reexpose a lesionada T3 / 4 medula espinal de duas semanas após a transecção da medula espinal. </li><li> 5 fibrinogénio células ul e 5 ul de trombina células pode ser injectado directamente nove pontos do local da lesão, através de tecido da cicatriz e selado dura intacta, sem re-abertura do local da lesão. </li><li> Use uma agulha de 27 G para criar 9 buracos na superfície do tecido da cicatriz e dura intacta 1-2 semanas após lesão: 3 no centro (uma no ponto médio e duas em laterais, e espaçadas 0,5 milímetros de intervalo) e 3 em ambos rostral e 3 na interface de caudal (cerca de 0,5 mm rostral caudal ou para o centro da lesão). </li><li> Em seguida, metade do volume de injectar a solução da célula de fibrinogénio em três pontos centrais do local da lesão e um quarto do volume em três pontos da interface rostral e aqvolume de uarter em três pontos da interface do caudal. </li><li> Em seguida, injetar imediatamente células throbmbin nos mesmos pontos. A mistura de células de fibrinogénio e de trombina de células dentro do local da lesão irá formar um gel de fibrina para manter as células no local da lesão e os cocktails de factor de crescimento que suporta a sobrevivência de células. As células são injectadas usando pipetas de vidro puxado ligados a um Picospritzer (Geral válvula, Inc.). </li><li> Transplant cultivadas NSC humanos 21 no mesmo procedimento descrito acima, e utilizam de fibrina e do factor de crescimento cocktails feitos a partir de reagentes humanos. </li></ol>

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Um dos principais obstáculos para o transplante de NSC na lesão medular é pobre sobrevivência no centro da lesão. Quaisquer lacunas ou cavidades no local da lesão pode potencialmente reduzir ou enfraquecer a formação de relés neuronais funcionais entre axônios supraespinhais e separados segmentos da medula espinhal abaixo da lesão. Além disso, a baixa sobrevivência de NSCs enxertadas pode afectar a sua integração com o tecido hospedeiro, e, portanto, reduzir a conectividade dos neurônios enxertadas com n...

A lesão medular (LM), muitas vezes danos não só tratos da substância branca que levam os sinais de e para o cérebro, mas também a matéria cinzenta central, causando perda segmentar de interneurônios e neurônios motores. A conseqüência da SCI é a perda de ambos função motora e sensorial abaixo da lesão. Infelizmente, o sistema nervoso central adulto (CNS) não regenerar-se espontaneamente, resultando em défices funcionais permanentes 1. Portanto, a reconstrução do adulto ferido medula espinhal e melhoria no motor, sensorial e função autonômica é uma meta importante da pesquisa SCI. Células-tronco neurais (NCCC), seja diretamente isolado do embrionário ou adulto CNS, são células candidatos atraentes para substituir neurônios perdidos e glia. Além disso, estas células têm o potencial para formar novos centros funcionais para restaurar a condução axonal, através de um local de lesão de 2,3. Até o momento, não houve um esclarecimento detalhado da anatomia, electrophysiological e efeitos comportamentais da formação relé neuronal por NSC transplantadas após grave SCI. Há várias razões para isso: primeiro, NSCs ou tecido transplantado fetal CNS sobreviver mal quando enxertados em grandes cavidades lesão. Estudos anteriores mostram perda de células no início substancial, deixando grandes cavidades vazias lesão cística 4,5. Em alguns estudos, as células enxertadas seria posteriormente dividir e preencher a cavidade lesão 4,5, mas isso pode ocorrer após um atraso de dias ou semanas, e posterior enchimento local da lesão pode não ser completa ou consistente. Em segundo lugar, um sistema de rastreamento eficiente que fornece dados de som na sobrevivência celular, diferenciação / maturação, e conseqüência do transplantado NSCs faltava. A maioria dos estudos iniciais utilizados anterógrada e marcação retrógrada traçar projeções axonais de transplantes 2,3. No entanto, essas técnicas apenas parcialmente e, muitas vezes, as projeções axonais unclearly rotulados decorrentes de células enxertadas e métodos de rastreamento são subjetividadet às interferências provocadas por fugas de corante para além das células implantadas. Outros grupos utilizaram marcadores neuronais humanas específicas para rotular projeções axonal após o transplante de NSCs fetais humanos em roedor feridos medula espinhal 5,6. No entanto, nesses estudos, os xenotransplantes não consistentemente sobreviver bem. Recentemente, a entrega viral do gene GFP repórter foi utilizada para rotular NSC cultivadas 7,8. No entanto, a expressão da GFP foi muitas vezes inconsistentes e pode ser regulada para baixo 7. Recentemente, a utilização de ratinhos transgénicos ou ratos doadores de forma estável que expressam o gene repórter, GFP, ou a fosfatase alcalina da placenta humana, melhorou dramaticamente o rastreamento de transplantadas células estaminais neurais / progenitoras in vivo 9,11. Em terceiro lugar, muitos estudos indicam que, in vitro NSC rato cultivados derivados a partir de qualquer embrionário ou SNC adulto exclusivamente diferenciar em linhagens gliais quando transplantadas para o meio do adulto intacta ou lesada cor espinald 7,12,13, apesar do facto de essas células estaminais neurais são capazes de se diferenciar em ambos os neurónios e células da glia in vitro, indicando que os ambientes locais podem ditar o destino das células estaminais. Alternativamente, NSC cultivadas, especialmente os derivados de SNC adulto, pode ter propriedades defaulty intrínseca de se diferenciar em linhagens da glia in vivo 13. Por causa das limitações discutidas acima, o nosso grupo desenvolveu recentemente um novo protocolo para melhorar embrionário rastreamento NSC, sobrevivência e diferenciação / maturação no adulto medula espinhal gravemente ferido. Resumidamente, começamos com um transgênico linha Fischer 344 consanguinidade rato estável expressando um gene repórter GFP que sustenta expressão GFP após o transplante in vivo 14. Em seguida, utilizamos NSCs recém-isoladas de dia embrionário 14 Fischer 344 medula espinhal, um estágio de desenvolvimento que mantém o potencial de gerar neurônios e células gliais. Finalmente, incorporadoNSC recém-dissociada em uma matriz de fibrina contendo fatores de crescimento 15-17 para reter as células e uniformemente distribuí-los dentro de uma grande cavidade da lesão, com o objetivo de apoiar a sobrevivência das células do enxerto, diferenciação e integração. Os enxertos foram colocados em locais de T3 transecção completa, duas semanas após a lesão medular. Estas células enxertadas preenchido de forma consistente locais transecção completa e diferenciada em neurônios abundantes que estenderam um grande número de axônios em acolhimento medula espinhal através de longas distâncias 18. Resultados semelhantes foram obtidos com enxertos de células-tronco neurais humanas cultivadas para imunodeficientes ratos 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

Células-tronco neurais (NCCC) pode se auto-renovar e diferenciar em neurônios e células gliais. NSC transplantados pode substituir neurônios perdidos e glia após lesão medular (LM), e podem formar relés funcionais para re-conectar segmentos da medula espinhal acima e abaixo de uma lesão. Estudos anteriores de enxerto de células estaminais neurais têm sido limitados pela sobrevivência do enxerto incompleto no interior da cavidade da lesão da medula espinal. Além disso, o controle de sobrevivência do enxerto de células, diferenciação, e extensão do processo não tinha sido optimizada. Finalmente, em estudos anteriores, NSC de rato em cultura foram tipicamente relatada para se diferenciarem em células da glia, quando enxertado na medula espinal ferida, em vez de neurónios, a menos que o destino foi conduzido para um tipo de célula específico. Para tratar dessas questões, desenvolvemos novos métodos para melhorar a sobrevivência, integração e diferenciação de NSC para os locais de mesmo grave SCI. NSC foram recentemente isoladas de embriões dia 14 de medula espinhal (E14) de um transgênico Fischer 344 linhagem de ratos estável expressando fl verdeproteína uorescent (GFP) e foram incorporados numa matriz de fibrina contendo factores de crescimento; esta formulação destinada a reter as células enxertadas na cavidade lesão e apoiar a sobrevivência da célula. NSC no fator de fibrina / crescimento cocktail foram implantadas duas semanas após torácica nível 3 (T3) completos transecções da medula espinhal, evitando os períodos de pico de inflamação. Enxertos resultantes completamente preenchida a cavidade lesão e diferenciado em ambos os neurônios, que se estenderam axônios para o acolhimento da medula espinhal mais notavelmente longas distâncias, e glia. Enxertos de NSC humanas cultivadas que expressam GFP revelou resultados semelhantes. Assim, os métodos são definidos para melhorar o enxerto de células estaminais neuronais, de sobrevivência e de análise de resultados in vivo.

Rotulagem imuno GFP demonstra que NSC rato enxertado ressuspensas em tampão fosfato salino (PBS) (falta de uma matriz de fibrina e os factores de crescimento) sobreviveram mal no local transecção T3, apenas inerente à margem da lesão / hospedeiro e deixando a maior parte do local da lesão, sem vazios As células enxertadas (Figura 2A). A sobrevivência de NSC melhorada quando co-enxertados com matrizes de fibrina por si só, mas o enxerto não encher completamente uma cavidade grande lesão (dados...

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

Matrizes de fibrina que contêm factores de crescimento foram utilizados para reter as células estaminais neurais enxertados em locais de completa a transecção da medula espinal. Células enxertadas encheu completamente a cavidade lesão e diferenciado em vários tipos de células neurais, incluindo neurônios que se estenderam axônios em acolhimento medula espinhal através de longas distâncias.

Promoção da sobrevivência e diferenciação de células-tronco neurais com fibrina e Fator de Crescimento Cocktails após Spinal Cord Injury grave

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 visualizações.  Veterans Administration Medical Center, San Diego.  Matrizes de fibrina que contêm factores de crescimento foram utilizados para reter as células estaminais neurais enxertados em locais de completa a transecção da medula espinal. Células enxertadas encheu completamente a cavidade lesão e diferenciado em vários tipos de células neurais, incluindo neurônios que se estenderam axônios em acolhimento medula espinhal através de longas distâncias. 

Vídeo: 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.

Dissecção e Cultura de Neurônios da Medula Espinhal comissural embrionárias

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

Neurociência

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.

Crescimento e Diferenciação de Adultos Hippocampal terra ártico Squirrel células-tronco neurais

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.

Transplante de Células-Tronco cirúrgica do rato Neural na medula espinhal de camundongos infectados com o vírus da Hepatite neurotrófico rato

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.

Percussão Fluid lateral: Modelo de Lesão em Ratos

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.

Spinal Cord Injury completo e Cérebro Dissection Protocolo para Wholemount Subsequente<em&gt; In Situ</em&gt; Hibridização em larval da lampreia de mar

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.

Um<em&gt; Ex Vivo</em&gt; Induzida por laser Lesão Medular modelo para avaliar mecanismos de axonal Degeneration em tempo real

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.

Imagem de atividade Neural no córtex somatossensorial primário usando Thy1-GCaMP6s de ratos transgênicos

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.

Um ensaio de Neurosphere para avaliar a ativação de células-tronco neurais endógena em um modelo do rato da lesão medular mínima

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.

Identificando marcadores de superfície celular da haste neural primária e células progenitoras por rotulagem metabólica de 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.

Treinamento baseado em atividade em uma esteira com medula espinhal ferido de ratos Wistar

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